Concise encyclopedia of biochemistry 9783111710990, 9783110078602

Table of contents :
Preface
Using this book
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z

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Concise Encyclopedia of Biochemistry

Concise Encyclopedia of Biochemistry

w DE

G

Walter de Gruyter Berlin • New York 1983

Title of the Original, German Language Edition Brockhaus ABC Biochemie edited by H.-D. Jakubke, Prof. Dr. rer. nat. habil. H. Jeschkeit, Dr. rer. nat. habil. Copyright © 1976, 1981 VEB F. A. Brockhaus Verlag Leipzig

Translated into English, revised and enlarged by Thomas Scott, Ph. D. Department of Biochemistry University of Leeds, England Mary Brewer, Ph. D. Menlo Park California 94025, USA

CIP-Kurztitelaufnahme der Deutschen Bibliothek Concise encyclopedia of biochemistry / [transl. into Engl., rev. and enl. by Thomas Scott; Mary Brewer]. - Berlin; New York: de Gruyter, 1983. Einheitssacht.: Brockhaus -ABC Biochemie 'engl.' ISBN 3-11-007860-0 NE: Scott, Thomas [Bearb.]; EST Library of Congress Cataloging in Publication Data Brockhaus ABC Biochemie. English. Concise encyclopedia of biochemistry. Translation of: Brockhaus ABC Biochemie. I. Scott, 1. Biological chemistry-Dictionaries. Thomas, 1935II. Brewer, Mary. III. Title. [DNLM: 1. Biochemistry-Dictionaries.QU 13 C744] 82-22148 QD415.B713 1983 574.19'2'0321 ISBN 3-11-007860-0

English Language Edition Copyright © 1983 by Walter de Gruyter & Co. Berlin 30. All rights reserved. No part of this book may be reproduced in any form - by photoprint, microfilm, or any other means - nor transmitted nor translated into a machine language without written permission from the publishers. Typesetting: Dörlemann-Satz, Lemförde. - Printing: Karl Gerike, Berlin Mikolai, Berlin. - Coverdesign: Rudolf Hübler, Berlin. Printed in Germany.

Binding: Dieter

Preface

The "Brockhaus ABC Biochemie" was first published in 1976 in Leipzig, the second edition followed in 1981. When we undertook to translate this book, based on the second German edition, it was clear that our work would also involve considerable updating of existing entries and the introduction of new material. Such a task can, of course, never be complete. It is a rare and fortunate author or editor in the life sciences, and particularly in biochemistry, whose material is still completely up to date at the time of publication; progress in this field is so rapid and shows no sign of abating. Therein, however, lies the excitement and challenge of this venture. Already we have started collecting, classifying and revising in preparation for a subsequent edition. We have departed from the style of the German edition by quoting a few literature references. These have been included with some of the new material, and we hope they will be useful to readers who want more information than can be fitted into a work of this sort. Where possible, we have also given each enzyme its EC (Enzyme Commission) Number, according to the Recommendations (1978) of the Nomenclature Committee of the International Union of Biochemistry (published in "Enzyme Nomenclature" Academic Press, 1979). We apologize to any biochemist whose pet compound, mechanism or pathway has been overlooked, and we should be grateful to receive suggestions for new entries. It is also recognized that a reference work should reach into the past, defining terms no longer used, but encountered when using the older literature. In this respect, suggestions from our more "senior" readers would be most welcome. Finally, thanks are due to Dr. Rudolf Weber of de Gruyter Publishers for his guidance and encouragement in the preparation of the manuscript and the production of this book. January 1983

Mary Brewer, Menlo Park, California, USA. Thomas Scott, Leeds, Yorkshire, England.

Using this book

Cross referencing is indicated by the word "see", and the subject of the cross reference starts with a high case letter, e . g . . . . in the Posttranslational modification of proteins (see), o r . . . see Enzyme induction. Numbers, Greek letters and configurational letters at the beginning of names are ignored in the allocation of alphabetical order, e.g. fi-Galactosidase is listed unter G ; L-Histidine unter H ; JV-2-Hydroxyethylpiperazine . . . under H. The main entry title is printed in bold type, followed by synonyms in bold italics. The remaining text uses only two further types, normal and italics. Abbreviations: (The standard biochemical abbreviations, e.g. ATP, NAD, etc. are found as entries in the appropriate alphabetical positions). abb. [a] b. p. c °C (d.) p IP M m. p. MT n syn.

abbreviation specific optical rotation boiling point concentration degrees Celsius with decomposition sensity isoelectric point molar melting point relative molecular mass refractive index synonym

A

A: abb. for adenine; abb. for absorbance. A: Angstrom unit. AAR : abb. for Antigen-antibody reaction (see). A band: a transverse dark band, consisting of thick and thin filaments, seen in electron microscope preparations of myofibrils from striated muscle. Abietic acid: a diterpene carboxylic acid, M t 302.46, m.p. 173 to 175 °C, b. p. 9 5 248-250 °C,[a] D — 106 °C (alcohol). A.a. and the isomeric neoabietic acid, m.p. 171 to 173 °C, [a] D + 161° (alcohol) can easily be interconverted. These two resin acids are the main components of rosin (up to 90%), from which they are obtained by treatment with heat or acids, possibly as products of the rearrangement of other diterpene carboxylic acids. Amber contains derivatives of A.a.

dry matter). Its structure was determined in 1965. It exists in two stereoisomeric forms, depending on the cis or trans orientation of the A2»-' double bond. The eis-isomer is the predominant form in all plants; small amounts of the irans-isomer are occasionally found. The fra/w-isomer is active only in bioassays performed in the light, presumably because it undergoes light-induced isomerization to the cu-form. Both stereoisomers can exist in optically active forms (asymmetric Catom at CI'), but only the ( + )-form is found naturally.

(S)-(+ )-Abscisic acid

Abrln: see Ricin. Abscislc acid, abb. ABA, abscisin, dormin: (S)( + )-5-( 1 '-hydroxy-4'-oxo-2',6',6'-trimethyl-2-cyclohexen-l-yl)-3-methyl-as, trans-2,4-pentadienoic acid, a widely occurring, sesquiterpene plant hormone. Its action is mainly inhibitory. M r 264.3, m.p. 160 to 162 °C,[a] D + 430°, X max 260 nm. ABA appears to be ubiquitous in plants and acts as antagonist to the auxins, gibberellins and cytok i n e s . It inhibits growth and the germination of seeds. It induces dormancy in seeds and promotes the falling of leaves and fruits. It is thus present in relatively large quantities in fruits, dormant seeds, buds and wilting leaves. The p-D-glucose ester of ABA has been found in the yellow lupine (Lupinus luteus), rose (Rosa), beans (Phaseolus) and maple (Acer pseudoplatanus). It is assayed both spectroscopically and by biological tests based on its growth-inhibiting properties. The biosynthesis of ABA is still unknown. A direct path from isopentenyl pyrophosphate via geranyl and farnesyl pyrophosphate, or formation from carotenoids by photochemical cleavage of violaxanthin via xanthoxin have both been proposed. It was first isolated in 1963 from cotton bolls (9 mg/75 kg dry matter) by Addicot and Lyon and by Wareing from maple leaves (0.27 mg/27 kg

Absolute oils: see Essential oils. Absorbance, extinction, optical density: a measure of the quantity of light obsorbed by a solution. It is equal to log I 0 /1, where I 0 is the intensity of the incident light, and I is the intensity of the transmitted light. Absorbance Index: see Absorptivity. Absorption coefficient: see Absorptivity. Absorptivity: the proportionality constant e, in Beer's law for light absorption : A = elc, where A is absorbance, I is the length of the light path, and c is the concentration. If concentration is expressed on a molar basis, e becomes the molar absorptivity, molar absorption coefficient, or molar extinction coefficient, i.e. E = A/lc, where I is the length of the light path in centimeters, and c is the molar concentration. Acceptor RNA: see Transfer RNA. Acceptor site: the ribosomal binding site for the aminoacyl-tRNA during protein biosynthesis. Accumulation of metabolic intermediates: see Mutant technique. Acetaldehyde, etbanal: CH,-CHO, important intermediate in the degradation of carbohydrates, m.p. - 123 °C, b.p. 20.1 °C. In its activated form (see Thiamine pyrophosphate), it is involved in a number of reactions (see Alcoholic fermentation). Two molecules A. can undergo acyloin condensation to form Acetoin(see). 3'-Acetamido-3'-deoxyadenosine: see 3'-Amino-3'-deoxyadenosine. Acetate kinase, acetokinase (EC 2.7.2.1): see Acetyl phosphate and Phosphoroclastic pyruvate cleavage. Acetic acid, ethanoic acid: CH 3 COOH, a very common monocarboxylic acid. m.p. 16.7 °C, b.p. 118 °C. A.a. occurs in the free form as the end

2

Acetogenins product of fermentation a n d oxidation reactions in some organisms. Acetate is f o r m e d metabolically by dehydration of acetaldehyde, catalysed either by aldehyde oxidase (EC 1.2.3.1) or a N A D ( P ) + -dependent aldehyde dehydrogenase (EC 1.2.1.3). The activated form of A.a., Acetylcoenzyme A (see) is a key substance in intermediary metabolism. Acetogenins: see Polyketides. Acetoin, 3-hydroxy-2-butaaone, acetyl methyl carbinol: C H 3 - C O - C H O H - C H 3 , a reduction product of diacetyl which arises u n d e r certain conditions as a side product of the pyruvate decarboxylase (EC 4.1.1.1) reaction. A. is also formed by decarboxylation of acetolactate by acetolactate decarboxylase (EC 4.1.1.5). It is oxidized in a reversible reaction to diacetyl by acetoin dehydrogenase (EC 1.1.1.5), and in some microorganisms it is converted to 2,3-butanediol by D( — )butanediol dehydrogenase (EC 1.1.1.4). Acetylcarnltine: see Carnitine. Acetylcholine: a biogenic amine which is biologically highly active. MT 163.2. Phylogenetically, A. is a very ancient h o r m o n e which appears even in protists. It could be a predecessor of the neurohormones. A. acts as a cholinergic neurotransmitter in nerves a n d neuromuscular synapses; it induces a muscle contraction by changing the permeability of the sarcolemma. It is degraded by acetylcholinesterase ( E C 3.1.1.7). Drugs which block the acetylcholine receptors (succinoylbischoline) cause muscles to relax (muscle relaxant for surgical operations). A. is f o u n d at the synapses in the central nervous system. It dilates blood vessels, causes a d r o p in blood pressure, a n d induces contractions in the smooth musculature of the bronchia a n d the gastrointestinal tract. It therefore promotes peristalsis in the latter.

CH,

0

I®

II

CH3-N - C H 2 - C H j - 0 - C - C H 3 CH 3 Acetylcholine Acetylcholinesterase (EC 3.1.1.7): "true cholinesterase", catalyses the hydrolysis of acetylcholine into choline and acetate. Due to the high turnover n u m b e r of A. (0.5 to 3.0.10 6 molecules substrate per molecule enzyme per min), the acetylcholine released at a synapse is hydrolysed within 0.1 ms. This enzyme is f o u n d in the central nervous system, particularly in the postsynaptic membranes of the striated muscles, the parasympathetic ganglia, the erythrocytes and the electric organs of fish. Crystalline A. (M r 330000) has been isolated from the electric organ of the electric eel (Electrophorus electricus). It consists of 4 identical inactive subunits of M r 82 500; the halfmolecules consisting of 2 covalently b o u n d subunits ( M t 165000) are enzymatically active. Proteolytic attack on the subunits produces two fragments of M r 60000 and 22500. The active center of A. has two parts, the anionic binding site for the quaternary nitrogen, which is

Acid plants responsible for the alcohol specificity, a n d the esterase center, where a catalytic serine a n d histidine lyse the ester bond. The enzyme is inactivated by blockage of either the serine hydroxyl (by organic p h o s p h a t e esters, such as diisopropylf l u o r o p h o s p h a t e or diethyl p-nitrophenylphosphate), or the anionic center by trimethylammonium derivatives. If the enzyme has been blocked by organophosphates, it can be reactivated by pralidoxime salts, which are therefore used as antidotes to organophosphate poisoning. Acetyl-coenzyme A, acetyl-CoA, active acetate: C H 3 C O ~ S C o A , a derivative of acetic acid in which the acetyl residue is b o u n d by a high-energy b o n d to the free SH-group of coenzyme A. M t 809.6, ^nax = 260 nm. The very reactive thioester has a high potential for transfer of the acetyl group, a n d is therefore a universal intermediate which provides the C 2 fragment for numerous syntheses. The free energy of the b o n d (34.3 k J / mol = 8.2 kcal/mol), however, has n o significance as a form of energy storage. In the transfer reactions mediated by acetyl-CoA, either the carboxyl group (electrophilic reaction) or the methyl group (nucleophilic reaction) can react. By far the most important pathways for the synthesis of acetyl-CoA (Table) are 1) the oxidative decarboxylation of pyruvate, 2) the degradation of fatty acids and 3) the degradation of certain a m i n o acids. The formation of acetyl-CoA involves either 1) the transfer of an acetyl residue f r o m a suitable donor, such as pyruvate, a n d simultaneous reduction of N A D + , or 2)the activation of free acetate in a one or two-step process which requires ATP a n d free coenzyme A. Acetyl-CoA is the hub of carbohydrate metabolism a n d has a central position in overall metabolism. The products of carbohydrate, fat a n d protein metabolism are channeled via acetyl-CoA into oxidative degradation in the tricarboxylic acid cycle. The acetyl residue is used in the synthesis of esters and amides (e.g. acetylcholine, Nacetylglucosamine, JV-acetylglutamate). AcetylC o A is also the starting point for isoprenoid synthesis via mevalonic acid and for fatty acid synthesis. The latter path is especially important in the transformation of carbohydrates into fat, and was elucidated in 1951 by Lynen and Lipman. /V-Acetylglutamic acid, N-acetylgiutamate, abb. Ac-GIu: HOOC-CH(NHCOCH3)-CH2-CH2 C O O H , the acetylated form of glutamic acid, is the cofactor of carbamoyl p h o s p h a t e synthetase (ammonia) (EC 6.3.4.16) a n d allosterically activates this enzyme. See Carbamoyl phosphate. Acetyl methyl carbinol: see Acetoin. Acetyl phosphate: C H 3 - C O O P O ( O H ) 2 , an energy-rich acyl phosphate. It is the product of acetate activation in some organisms: Acetate + ATP = A.p. + A D P ; the reaction is catalysed by acetate kinase (EC 2.7.2.1). The back reaction can be used for A T P synthesis, for example in the phosphoroclastic cleavage of pyruvate. Acid amides: see Carboxylic acids. Acidic ci|-glycoprotein: see Orosomucoid. Acid plants, ammonium plants: plants which ac-

Aconitate hydratase

Actinomycins

3

Table. Reactions in which acetyl-coenzyme

A is

synthesized.

Enzyme

Reaction

Occurrence/ Significance

Acetyl-CoA synthetase (EC 6.2.1.1)

C H , C O O - + ATP + Co A

Yeasts, Animals, Higher plants

Acyl-CoA synthetase (GDP-forming) (EC 6.2.1.10) Acetate kinase

Phosphate acetyltransferase (EC 2.3.1.8) ATP citrate (pro-35)-lyase (EC 4.1.3.8)

+ GTP + CoA

Liver

CH3COO- + ATP

Microorganisms

^ C H 3 C 0 - 0 - P 0 3 H 2 + ADP (Acetyl phosphate) C H , C 0 - 0 - P 0 , H , + CoA

Microorganisms

Citrate + ATP + C o A

Outside the mitochondria

+ A D P + P; Pyruvate dehydrogenase complex (EC 1.2.4.1, 2.3.1.12 and 1.6.4.3)

CH3COCOO- + N A D + + C o A

Acetyl-CoA transacetylase (EC 2.3.1.9)

C H 3 C O C H 2 C O - C O A + CoA (Acetoacetyl-CoA)

(Pyruvate)

Mitochondrial particles

TPP, LipS 2

CH3-CO-C0A + C0 2 + N A D H + H + Fatty acid degradation

Abb. T P P = thiamine p y r o p h o s p h a t e ; LipS 2 = Lipoamide cumulate organic acids in their leaf cells, which are neutralized by a m m o n i u m ions. Aconitate hydratase, aconitase, (EC 4.2.1.3): a hydratase which catalyses one stage of the tricarboxylic acid cycle, the reversible interconversion of citrate a n d isocitrate. The reaction proceeds via the enzyme-bound intermediate, as-aconitate. At equilibrium, the relative abundances are 90% citrate, 4 % cw-aconitate a n d 6 % isocitrate. Thus citrate is favored at equilibrium, but in respiring tissues the reaction proceeds from citrate to isocitrate, as isocitrate is oxidized by isocitrate dehydrogenase. The enzyme contains Fe(II) a n d requires a thiol such as cysteine or reduced glutathione. The Fe(II) ion forms a stable chelate with citric acid. X-ray analysis of Fe(II) complexes of tricarboxylic acids suggested the "ferrous wheel" hypothesis of aconitase action. According to this mechanism, three points on the m - a c o n i t a t e molecule are b o u n d at separate sites on the enzyme surface; in addition the molecule is also complexed with the Fe(II) atom at the active center. The stereospecific trans addition of water to cis-aconitate to form either citrate or isocitrate is achieved by rotation of the ferrous wheel, which can a d d O H to either side of the molecule. Aconitase is inhibited by fluorocitrate. Two isoenzymes are present in animal tissues, one in the cytosol a n d o n e in the mitochondria. Ref: Glusker, J. P., in Boyer, P. D. (ed.), The Enzymes, 5, 434, Academic Press Inc., 1971. Aconitic acid: an unsaturated tricarboxylic acid, usually occurring in the cis form, but sometimes in the trans, cis-A.a., m.p. 130 °C., transA.a., m.p. 194 to 195 °C. A.a. was discovered in free f o r m in aconite, Aconitum napellus. The anionic f o r m of m - A . a . (propen-cj's-l,2,3-trioic acid) is important as an intermediate in the isom-

erization of citrate to isocitrate in the Tricarboxylic acid cycle (see). Aconitine: an Aconitum alkaloid (see Terpene alkaloids) f r o m the roots of aconite (Aconitum napellus) a n d other Aconitum a n d Delphinium species, m.p. 197 to 198 °C,[a] D 2 0 - 36° (benzene).A. is an esterified alkaloid. It is extremely poisonous a n d can cause death in adults at a dose of 1 to 2 mg by paralysing the heart and respiration. Its hydrolysis products are only slightly toxic. In spite of useful physiological properties, A. is rarely used in medicine, d u e to its toxicity. It is sometimes used internally as tincture for rheumatism a n d neuralgias a n d externally as a pain-killing salve. In antiquity, aconitine preparations were used as arrow poisons by the Greeks a n d (East) Indians. Aconitum alkaloids: a group of terpene alkaloids, some of them very poisonous, f r o m various aconite (Aconitum) species. The best-known representative is aconitine. ACP: abb. for acyl carrier protein. ACTH: abb. for adrenocorticotropic h o r m o n e . See Corticotropin. Actin: see Muscle proteins. Actinldine: a widely occurring terpene alkaloid. See Valeriana alkaloids. Actinomycins: a large group of peptide lactone antibiotics produced by various strains of Streptomyces. These highly toxic red c o m p o u n d s contain a chromophore, 2-amino-4,6-dimethyl-3-ketophenoxazine-l,9-dioic acid (actinocin), which is linked to two 5-membered peptide lactones by the a m i n o groups of two threonine residues. The various A. differ only in the a m i n o acid sequence of the lactone rings. In vivo, A. inhibit the D N A d e p e n d e n t R N A synthesis at the level of transcription by interacting with the D N A . T h e concentration required for inhibition d e p e n d s on the

Activated amino acids base composition of the DNA; more is required for DNA with a low guanine content. A. are pharmacologically very important due to their bacteriostatic and cytostatic effect. Actinomycin D (Fig.) is one of the most widespread A. Its spatial structure has been elucidated by N M R studies, and the specificity of its interaction with deoxyguanosine was demonstrated by X-ray analysis. Actinomycin D is used as a cytostatic, e.g. in the treatment of Hodgkin's disease.

Activated amino acids: see Aminoacyl adenylate. Activated carbon dioxide: see Biotin enzymes. Activated carboxylic acids: derivatives of carboxylic acids which are very reactive, and thus capable of reactions which the free acids do not undergo. The biochemically important A.c.a. are either anhydrides or thioesters. Activated choline: see Cytidine diphosphocholine. Activated fatty acids: fatty acyl coenzyme A thioesters which, as high energy compounds, have a large potential for group transfer. They are formed during fatty acid biosynthesis, or by the activation of free fatty acids. Acyl CoA synthetases catalyse formation of the CoA derivatives according to the reaction: CH 3 (CH 2 )„COO+ ATP + HS-CoA — C H 3 ( C H 5 n C O ~ S C o A + AMP + PP, The reaction involves acyladenylate as an intermediate, which is cleaved by coenzyme A to form acyl-CoA and AMP. Several such enzymes are known, and they are named according to the length of carbon chain that shows optimal activity, e.g. acetyl CoA synthetase converts C 2 and C 3 fatty acids, octanoyl CoA synthetase (C 4 to C 12 ) and dodecanoyl CoA synthetase (C 10 to C, 8 ). Mitochondria also contain an acyl CoA synthetase that cleaves GTP to GDP and Pj. Acyl CoA derivatives of short chain fatty acids may also be formed in a transfer reaction involving succinylCoA, catalysed by thiophorases: SuccinylSCoA + R-COOH succinic acid + R-COSCoA Activated fatty acids are in equilibrium with acylcarnitine in the organism. They are the starting point for fatty acid degradation. Activated glucose: see Nucleoside diphosphate sugars. Activated glycol aldehyde: 2-(l,2-dihydroxyethyl)-thiamine pyrophosphate, abb. DETPP, glycol aldehyde bound to the C-2 atom of the thiazole ring of thiamine pyrophosphate. It is formed in carbohydrate metabolism by cleavage of a ke-

4

Active formate tose and is transferred as C-2 group to an aldose in a transketolation reaction. Activated amino acids: see Aminoacyl adenylate. Activated fatty acids: derivatives of carboxylic acids which are very reactive, and thus capable of reactions that free acids do not undergo. Biochemically important A.f.a. are either anhydrides or thioesters; see, e.g. Acetyl-coenzyme A. Activation hormone: see Insect hormones. Activator protein: see Calmodulin. Active acetaldehyde: a-hydroxyethylthiamine pyrophosphate, abb. HETPP, the activated form of acetaldehyde formed by decarboxylation of active pyruvate. The aldehyde is bound to the C-2 atom of the thiazole ring of thiamine pyrophosphate. HETPP is an intermediate in alcoholic fermentation. Active acetate: see Acetyl-coenzyme A. Active aldehyde: see Thiamine pyrophosphate. Active center: that part of an enzyme or other protein which binds the specific substrate and converts it to product (enzymes) or otherwise interacts with it (heme proteins, various carrier and receptor proteins). Tlie A.c. of an enzyme thus consists of the actual catalytic center, which is relatively unspecific, and the substrate-binding site, which is responsible for the specificity of the enzyme. The A.c. may lie on the surface (in chymotrypsin, for example) or in a cleft (in lysozyme, papain, carboanhydrase or ribonuclease) in the enzyme molecule. It involves only a limited number of amino acid residues. The A.c. must be particularly flexible in order to bind its substrate and carry out catalysis. It therefore lacks regular structures, such as a-helix. The amino acids involved in catalysis may lie at a considerable distance from each other in the absence of a substrate; they are brought into play by conformational changes induced by the substrate when it binds (induced fit model suggested by Koshland) (see Chymotrypsin and Serine proteases). For example, the amino acids involved in the catalysis step in the serine proteases (including trypsin, chymotrypsin and elastase) are serine 195, histidine 57 and aspartate 102. The amino acids responsible for binding the substrate are serine 189 and glycine 216 in chymotrypsin, aspartate 189 and glycine 216 in trypsin, and serine 189 and valine 216 in elastase. Information on the amino acids involved in the A.c. is obtained by specific marking with coenzyme, inhibitors or reagents specific for particular side chains. Some widely used irreversible inhibitors for the catalytic center of the serine proteases are tosyllysine chloromethyl ketone (TLCK), which selectively blocks the imidazole group of the histidine 57 in trypsin, diisopropylfluorophosphate (DFP), and phenylmethane sulfonyl fluoride (PMSF), which form stable esters with serine 195 of all serine proteases and many carboxyesterases. Active C0 2 : see Biotin enzymes. Active formaldehyde: see Active one-carbon units; Thiamine pyrophosphate. Active formate: see Active one-carbon units.

5

Active glucose Active glucose: see Nucleoside diphosphate sugars. Active glycolaldehyde: 2-(l,2-dihydroxyethyl)-thiamine pyrophosphate, abb DETPP, glycolaldehyde bound to C-2 of the thiazole ring of thiamine pyrophosphate. It is formed in carbohydrate metabolism by cleavage of a ketose, and is transferred as a 2C group to an aldose in a transketolation reaction. Active methionine: see S-Adenosyl-L-methionine. Active methyl groups: see S-Adenosyl-L-methionine. Active one-carbon units, abb. Ct units: C, fragments which are activated by binding to tetrahydrofolic acid, or less commonly, to thiamine pyrophosphate. The active ethylenediamine group of

Active transport succinyl-coenzyme A. It is important as an intermediate in the tricarboxylic acid cycle. Active sulfate: see Phosphoadenosine phosphosulfate. Active transport: a process in which solute molecules or ions move across a biomembrane from lower to higher concentration, i.e. against the concentration gradient. Since thermodynamic work is involved, A.t. must be coupled to an exergonic reaction. In primary A.t., the coupling is direct. The transport of N a + and K + ions across a cell membrane by the N a + , K + - A T P a s e system, for example, requires the simultaneous hydrolysis of ATP. Secondary A.t. utilizes the energy of an electrochemical gradient established for a second solute to transport the first. One form of secondary A.t. is cotransport, in which the transport of

Table. Formation and uses of active one-carbon units. Type of C, unit

Biogenesis

Use

JV 10 -Formyl-THF

From formate + ATP by formyl-THF synthetase (EC 6.3.4.3); from AT 5l0 -methylene-THF From L-serine and THF by serine hydroxymethyltransferase (EC 2.1.2.1); from glycine and T H F directly or via glyoxylate and formate By anaerobic purine degradation via formiminoglycine; by histidine degradation via formiminoglutamate From JV 510 -methylene-THF

Purine synthesis after conversion to JV 510 -methylenyl-THF and JV 5,0 -methylene-THF Purine synthesis ; formation of the 5-methyl group of thymine and the methoxyl group of hydroxymethyl cytosine

AP.io_methylene-THF

JV 5 -Formimino-THF

Af 5 '-methyl-THF

tetrahydrofolic acid serves as a carrier for the metabolic transfer of a formyl or methyl group. Fig. 1 shows the active forms of tetrahydrofolic acid (THF). The various C, units can be interconverted while attached to T H F (Fig. 2). The main source of C, units is the hydroxymethyl group of serine, which is transferred to THF by serine hydroxymethyltransferase (EC 2.1.2.1), forming hydroxy methyl-THF (activated formaldehyde). The formation of C, units in the course of histidine catabolism or the anaerobic degradation of purines is of particular importance. C, units are used in purine biosynthesis and as the donors of the 5-methyl group of thymine. The formation and uses of the C, units are given on the table. The most important are 1) active formaldehyde (Afr'O-methylene-THF, hydroxymethyl-TMF), 2) active formate (JV^-formyl-THF), 3) ^ - m e t h y l T H F (see L-Methionine). Active pyruvate: a-lactyl-thiamine pyrophosphate. The lactyl is bound to the C-2 atom of the thiazole ring of the thiamine pyrophosphate. A.P. is an intermediate in the oxidative decarboxylation of pyruvate to acetyl-coenzyme A and in its decarboxylation to acetaldehyde in alcoholic fermentation. Active succinate: the high-energy thioester

After conversion to N s ' 1 °-methylenyl-TH F and JV 510 -methylene-THF

Methionine synthesis.

one solute drives that of the other. An example is the Na+-dependent transport of certain sugars and amino acids in animal cells: the concentration of N a + in the cell is maintained at a level far below the intercellular concentration by the Na + , K + pump. A specific transport protein (carrier) binds both glucose and N a + outside the cell and releases them on the inside. The process is energetically favorable because the N a + is moving from a region of higher concentration to lower concentration. In other cases, the membrane potential generated by electron flow along the respiratory chain drives the active transport of sugars or amino acids. A third form of A.t. is called group translocation because the solute is changed in the course of transport. An example is the phosphotransferase system in some bacteria, in which sugars are phosphorylated in the course of transport. An interesting feature of this system is that phosphoeno/pyruvate rather than ATP is the phosphate donor. A.t. processes are highly specific, and they are saturable. This implies that enzyme-like proteins, or carriers, mediate the transport. (The term "carriers" also applies to the mechanism of Facilitated diffusion [see]). The bacterial transport systems called permeases

6

Active transport

OH

„ A

H,N

N

Active

form

N10

H I . N

H -CH2T

N I H

Active transport

/ //

\

V

COOö 0 " C - N H - C H — C H 2 — C H j — COO®

Tetrahydrofolic a c i d ( F H J

Reactive part of F H 4

Group t r a n s f e r r e d

O I II N-CH

— Formyl—FH¿

-CH

(Formyl-)

© CH^ -N^ N-

N

HN=CH I -N

—Formimino-

CH 2

/V5-,0-Methylene-

F i g . 1. Structure

H I N-

/ CH2 (Formaldehyde-)

N'"

-Hydroxymethyl-

-N

/ N-CH2OH

; C H 2 (Formaldehyde-)

W5

— Methyl-

CH, I -N

N-

- C H j (Methyl-

of tetrahydrofolic

acid and active

HOOCH ATP -

ADP+P• SAM + PPi + Pt. The adenosine residue of the ATP is transferred to the methionine.

COOH

S-Adenosyl-L-methionine The transmethylation reaction produces, in addi-

Stimulation + Inhibition — +

Liver + +

Salivary glands, pancreas Bones Thymocytes Liver Liver Liver Liver Various glands Liver Heart muscle Fat tissue Kidneys Liver Stomach epithelium Thrombocytes Slime mold

+ + + —

+ + + + + + + + —

+ + -

tion to the methylated product, S-adenosyl-Lhomocysteine: systematic name S-(5'-deoxyadenosine-5')-homocysteine. It may be reconverted to SAM after cleavage into adenosine and L-homocysteine. L-Homocysteine is the substrate of dimethylthetin-homocysteine methyltransferase (EC 2.1.1.3). See Methionine. Adenylate cyclase (EC 4.6.1.1): see Adenosine phosphates. Adenylate kinase, myokinase(EC 2.7.4.3): a trimeric enzyme found in the mitochondria of muscles and other tissues. It is resistant to heat and acid. M r 68 000, subunit M r 23 000. It catalyses the conversion of two molecules of ADP into ATP + AMP, thus making available the energy of the ADP. At equilibrium, the concentrations of the three adenosine phosphates are nearly equal. In many energy-requiring reactions ATP is converted into pyrophosphate and A M P (see Adenosine phosphates). A.k. is important because it catalyses the first stage (AMP to ADP) in the conversion of this AMP into ATP. Adenylic acid: see Adenosine phosphates. Adenylosuccinate, N-snccinyladenylate, abb. sAMP: 5-aminoimidazole-4- Af-succinocarboxamide ribonucleotide, an intermediate in purine biosynthesis. MT 463.31. Adenylylsulfate reductases: enzymes of sulfur metabolism which reduce either phosphoadenylylsulfate (APS reductase) or adenylylsulfate. Adenylylsulfate reductase (EC 1.8.99.2) is identical with one component of the sulfate reductase in sulfate assimilation, since adenylylsulfate is the donor of the sulfate group. The table shows the properties of some of these reductases. The re-

12

Adermine ductase is in every case a complex of three components, an adenylylsulfate transferase (see sulfate assimilation, Fig. 1), a low-molecular-weight carrier and the actual adenylylsulfate reductase.

Adrenal corticosteroids Phosphoadenylylsulfate reductase from Saccharomyces cerevisiae requires NADPH and has been partly purified and fractionated.

Table. Properties of adenylylsulfate reductases from rarious organisms Organism

pH optimum

Mr

Desulfovibrio

7.4

220000

Thiobacillus thioparus1

7.4

170000

Thiocapsa roseopersicina1

8.0

180000

1

Comments Contains 1 molecule FAD and 6 to 8 atoms nonheme iron Contains 1 molecule FAD and 8 to 10 atoms nonheme iron Contains 1 molecule FAD, 4 atoms non-heme iron and 2 atoms heme iron 60 to 80 fold enrichment to a homogeneous preparation in the ultracentrifuge Partly purified enzyme

Chlorella pyrenoidosa2 330000 1. With F e ( C N ) 6 3 - ; 2. a thiol as electron donor; the enzyme from Chlorella is active with phosphoade nylylsulfate only in the presence of 3'-nucleotidase. Adermine: vitamin B6. See Vitamins. ADH: abb. for Antidiuretic hormone. See Vasopressin. Adluretin: see Vasopressin. Adjuvant: a mixture of oils, emulsifiers, killed bacteria and other components which serves to intensify unspecifically the immune response. The A., which is not (supposed to be) itself antigenic, is injected several times, intramuscularly or subcutaneously, together with an antigen into an animal to produce the maximal amount of antibodies. In experimental immunology, Freund's incomplete A., an emulsion of paraffin oils which protects the antigen from too rapid degradation, and Freund's complete A., which contains in addition killed mycobacteria or tuberculosis bacteria, are most commonly used. A used in the production of vaccines are aluminum hydroxide and calcium phosphate gels. They are thought to activate the phospholipase A of the macrophages, so that more lecithin is converted to lysolecithin. The latter can itself act as an A., and this may be the reason that it sometimes leads to oversensitivity (allergy). Ado: abb. for Adenosine. ADP: abb. for Adenosine 5'-diphosphate. ADP-rlbosylatlon of proteins: attachment of monomelic or polymeric ADP-ribosyl groups to a protein by transfer from N A D + : Adenine Nicotinamide I 1+ (ribose-(?)-(p)-ribose)n + Protein —>• Adenine I Protein-(ribose-(p)-(p)-ribose)n + Nicotinamide + + H , where n can vary from 1 to 50. Poly ADP-ribosyl groups represent a novel homopolymer of repeating ADP-ribose units linked l'-2' between respective ribose moieties: Ir Adenine-ribose-(p)-(p)-ribose ir Adenine-ribose-(p)-(p)-ribose

I

The free energy of hydrolysis of the p-N-glycosidic linkage of NAD + is - 3 4 . 3 5 kjoules ( - 8 . 2 kcal)/mole at pH 7 and 25 °C; it is therefore a so-called high energy bond, and N A D + can act as an ADP-ribosyl transferring agent. The transfer of one ADP-ribosyl group ( n = 1 in above equation) is catalysed by ADP-ribosyl transferase. Formation and concomitant transfer of poly ADP-ribose to an acceptor is catalysed by poly(ADP-ribose) synthetase (n is greater than one in the above equation). Diphtheria toxin, produced by strains of Corynebacterium diphtheriae that carry P-phage, inhibits protein synthesis in eukaryotic cells by catalysing the transfer of an ADP-ribose moiety from N A D + to elongation factor 2. Pseudomonas toxin catalyses a similar reaction. T4 phage catalyses the monomelic ADP-ribosylation of RNA polymerase and other proteins in Escherichia coli. Choleragen activates adenylate cyclase by catalysing transfer of ADP-ribose from NAD + to the enzyme. Poly ADP-ribose groups are found in eukaryotic chromosomal proteins, mitochondrial protein and histones. The biological function of the ADP-ribosylation of proteins in eukaryotic cells is not known, but the occurrence of poly ADP-ribosyl groups in nuclear proteins, particularly in association with chromatin, suggests a regulatory role in nuclear function. The nature of the linkage to protein is not known, but it appears to involve attachment to basic amino acids. In the choleragen-activated ADPribosylation of adenylate cyclase, an arginine residue appears to be the chief receptor for ADP-ribose. Ref: Hayaishi, O. and Ueda, K. Ann. Rev. Biochem. (1977) 46, 95-116. "ADP-ribosylation of nuclear proteins" by Purnell, M.R., Stone, P.R. and Whish, W.J.D., Biochemical Society Transactions (1980) 8; 215-227. Adrenal corticosteroids, adrenocorticoids, corticosteroids, corticoids, cortins: an important

Adrenal gland group of steroid hormones, formed in the adrenal cortex in response to adrenocorticotropic hormone. A.c. are structurally related to pregnane (see Steroids); they contain a carboxyl group with a neighboring «.¡¡-unsaturated bond in ring A, a ketol side chain in position 17, and other oxygen functions, particularly in positions 11, 17, 21 and 18. More than 30 steroids have been found in the adrenal cortex; the following show marked A.c. activity: Cortisol (see), Cortisone (see), Cortexolone (see), 11 -Dehydrocorticosterone (see), Corticosterone (see), and Aldosterone (see). Quantitatively the most significant members of this group are Cortisol, corticosterone and aldosterone, which are secreted daily into the blood in quantities of 15, 3 and 0.3 mg, respectively. The production of A.c. is increased in physical a n d / o r psychological stress. Deficiency of A.c., e.g. caused by pathological changes in the adrenal glands, results in Addison's disease (Morbus Addison)', this condition is characterized by tiredness, emaciation, decrease of blood sugar, and dark pigmentation of those areas of the skin exposed to light. Adrenalectomy of experimental animals leads to rapid death, unless exogenous A.c. are given. A.c. control mineral metabolism by causing the retention of N a + , C I " and water, with a simultaneous K + diuresis (mineralocorticoidal or mineralotropic action). They also regulate carbohydrate metabolism, in particular glycogen synthesis in the liver (glucocorticoidal or glucotropic action). Depending on the type of activity that predominates, the A.c. are classified as mineralocorticoids (aldosterone, cortexone, cortexolone), and glucocorticoids (Cortisol, cortisone, corticosterone, 11-dehydro-corticosterone). For the therapy of adrenal insufficiency, extracts of adrenal cortex are no longer used and have been replaced by pure A.c. High doses reveal other pharmacological properties, especially anti-inflammatory and antiallergic activities. This discovery led to the development of highly active, synthetic derivatives, which are now used widely for the treatment of rheumatism, asthma, allergies, eczema, etc., e.g. prednisone (see Prednisolone), Dexamethasone (see) and Triamcinolone (see). A.c. are biosynthesized from cholesterol via progesterone; the latter is converted into A.c. by stepwise hydroxylation in positions 17, 21 and 11. Adrenal gland, suprarenal gland, Gianduia suprarenalis: a heavily vasculated, vertebrate endocrine gland, weighing about 15 g in the adult human. There are two A.g., one just above each kidney. The A.g. consists of two developmentally and functionally distinct parts: the mesodermal adrenal cortex (AC) and the ectodermal adrenal medulla (AM). The AC, which contains three histologically distinct zones, produces and exports glucocorticoids (see Cortisol) and mineralocorticoids (see Aldosterone) in response to the action of the pituitary hormones, corticotropin and renin/angiotensin II, respectively. The AC also produces sex steroids (see Androgens).

13

Adrenosterone The AM (Paraganglion suprarenale) is the largest (but not the only) ganglion of the sympathetic nervous system. It produces the hormones Adrenalin (see) and Noradrenalin (see). The AM is a model example of the close association of the sympathetic nervous system with an endocrine system. The secretory cells are richly innervated by cholinergic, preganglionic, sympathetic nerve fibers. No nerve supply to the AC has been demonstrated. Adrenalin, epinephrine: 4-[l-hydroxy-2-(methylamino)ethyl]-1,2-benzenediol, M r 183.20, a catecholamine hormone and drug. The L-form is physiologically active, affecting carbohydrate metabolism and the circulatory system. A. is synthesized in the adrenal cortex and sympathetic nervous system from tyrosine (via dopa, dopamine and noradrenalin), stored in the chromaffin granules and released into the blood stream upon nervous stimulation by the nervus splanchnicus. It is an adrenergic neurotransmitter. A. activates, via the adenylate cyclase system, the liver and muscle phosphorylases (EC 2.4.1.1) (glycogenolysis) and the lipase of adipose tissue, leading to higher blood concentrations of glucose (hyperglycemia), lactate and free fatty acids. The latter undergo oxidation, resulting in a higher oxygen consumption. A. is degraded after O-methylation and oxidative deamination by a monoamine oxidase. It is excreted in the urine as 3-methoxy-4-hydroxymandelic acid (vanillinemandelic acid). Analogs of A. are used to control blood pressure, counteract depression, stimulate the appetite and relieve asthma. OH

Adrenocorticotropic hormone: see Corticotropin. Adrenocortlcotropln: see Corticotropin. Adrenosterone: androst-4-ene-3,l 1,17-trione, a steroid derived from androstane. M r 300.9, m.p. 224 °C, [a]D +262 (alcohol). A. is synthesized in the adrenal cortex and is considered one of the male gonadal hormones, due to its weak androgenic effect (see Androgens).

14

Adsorption chromatography Adsorption chromatography: see Chromatography. Affinity chromatography: see Proteins. Anatoxins: microbial products belonging to the group of mycotoxins. They are natural carcinogens, causing liver cancers, and they are 100 times as active as previously known liver carcinogens. A. are produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus oryzae, as well as some Penicillium strains. A. are coumarin difuran derivatives. They have been found in a number of foodstuffs. The molecular mechanism of their toxicity may be an inhibition of RNA synthesis, either by formation of a toxin-DNA complex which inhibits the RNA polymerase, or by direct interaction with the enzyme. 0

0 2 3

Aflatoxin Bi

Aflotoxins Bt and G,. In aflotoxins B, and G 2 the ring labelled with an asterisk does not contain a double bond. AGA: abb. for N-Acetylglutamate. Agar-agar: a polysaccharide plant slime from various red algae. It consists of about 70% polygalactan, which is about 70% agarose and 30% agaropectin. Agarose is a linear polymer of alternating D-galactose and 3,6-anhydrogalactose. Agaropectin consists of D-galactose units, linked P-1,3 glycosidically. In some of them, position 6 is esterified with sulfate. A. K obtained by hotwater extraction of bleached algae, which may contain up to 40% A. It is sold in blocks, strands or powder, and is used as a gelling agent in the pharmaceutical and food industries. It is used by bacteriologists to make solid medium for microorganisms.

galactose V

Agarose

Alanine Agglutination: the clumping of insoluble antigens bound to particles, such as bacteria, viruses, erythrocytes, by the appropriate antibodies. These must be at least bivalent, in order to bind the antigen-carrying particles together. A. is much more sensitive than precipitation, because the antigen-antibody reaction takes place on the surface of larger particles. The lower limit of recognition for precipitation is about 10 (ig/m/ serum, but with A. the limit is 0.01 |ig/m/. Passive hemagglutination has a lower limit of detection of 3 to 6 ng antibodies/m/ serum. In this technique, soluble antigens are bound to the surface of erythrocytes,which are agglutinated when the antigen-antibody reaction occurs. This method is 1000 times as sensitive as precipitation. Agglutinins: see Lectins. Aglycons: see Glycosides. Agnosterol: 5a-lanosta-7,9(l l),24-trien-3(i-ol, a tetracyclic triterpene alcohol derived structurally from 5a-lanostane (see Lanosterol). M r 424.7, m.p. 165 °C, [a] D +66°. A. is a zoosterol (see Sterols) present in the sebacious oil of sheep's wool.

Agnosterol a^AGp: see Orosomucoid. AICAR: abb. for 5(4)-Aminoimidazole-4(5)-carboxamide ribotide. (See Purine biosynthesis). AIR: abb. for 5-Aminoimidazole ribotide. (See Purine biosynthesis). AJmallne: a Rauwolfia alkaloid, m.p. 205 to 207 °C, [a] D 20 = + 144 ° (c = 0.8 in chloroform). A. is used medicinally to normalize heart rhythm. In high doses it has the tranquilizing effect of Rauwolfia alkaloids. AKNF: see Cooperativity model. Alanine, aminopropionic acid: MT 89.1; 1. L-aatanine, abb. Ala, CH 3 -CH(NH 2 )-COOH, a proteogenic amino acid. m.p. 297 °C (d.), [a] D 2 5 = + 1.8 (c = 2.0, water). Ala is glucogenic and is closely involved in the metabolism of sugars and organic acids. It is one of the main components of silk fibroin. Free Ala, together with glycine, occurs in relatively large amounts in human blood plasma. It is produced from pyruvate by transamination, and in some microorganisms, for example bacilli, by reductive amination, catalysed by alanine dehydrogenase (EC 1.4.1.1). This enzyme has been reported to be a protomer of the oligomeric glutamate dehydrogenase (EC 1.4.1.2). Ala is degraded to pyruvate and ammonia by alanine dehydrogenase (syn. alanine oxidase; see Flavin enzymes), or it can be converted into pyruvate by Transamination (see).

Alar 85

15

2. (¡-Alanine, H 2 N-CH 2 -CH 2 -COOH, a nonproteogenic amino acid. m.p. 196 °C (d.). P-A. occurs in free form, for example in the human brain, and is a component of the dipeptides carnosine and anserine, and of coenzyme A. It is not usually formed by decarboxylation of L-aspartate, but rather in the course of reductive pyridine degradation. It can be further metabolized to acetate by deamination, decarboxylation and oxidation. Alar 85: see AT-Dimethylsuccinamide. Alblzzfin, 2-amino-3-ureidopropionic acid: H 2 NCONHCH ? CH(NH 2 )-COOH, a nonproteogenic amino acid, occurring primarily in species of the genus Albizzia. It is presumably formed

Alcohol dehydrogenase 62 % of the serum protein, and is one of the few carbohydrate-free proteins in blood plasma, or the serum obtained from it by clotting. Due to its relatively low M r of 67 500 and high net charge (IP 4.9), serum albumin has a good binding capacity for water, Ca 2 + , Na + , K + , fatty acids, bilirubin, hormones and drugs. Its main function is the regulation of the colloidal osmotic pressure of the blood. Bovine and human serum albumins contain 16% nitrogen and are used as standard proteins for calibration, due to the ease of obtaining them in crystalline, highly purified form. Human serum albumin consists of a single polypeptide chain of 584 amino acids, which are sta-

H,C—C—N

Albomycin from carbamylphosphate and 2,3-diaminopropionic acid by transcarbamylation. It is an antagonist of glutamine. Albomycin: an antibiotic synthesized by Actinomyces subtropicus. It is a cyclic polypeptide with a pyrimidine base (cytosine) (Fig.). It contains 4.16% iron in the form of a hydroxamate iron(III) complex. A. is one of the sideromycins and interferes with iron metabolism as an antimetabolite of the sideramines. It is similar to or identical to grisein. It is effective against both Gram-positive and Gram-negative bacteria, and inhibits the aerobic metabolism of Staphylococcus aureus and Escherichia colt Albumins: a group of simple proteins. They are found in body fluids and tissues and in some plant seeds. In contrast to the globulins, they are of low molecular weight, water-soluble and easily crystallizable, and they contain an excess of acidic amino acids. A. can only be precipitated by high concentrations of neutral salts. They are rich in glutamate and aspartate (20 to 25%) and leucine and isoleucine (up to 16%), but contain little glycine (1%). Important representatives of this group are serum albumin,a-lactalbumin (milk proteins) and ovalbumin from animals, and the poisonous ricin (from Rizinus seeds), leucosin (from seeds of wheat, rye and barley), and legumelin (from legumes). Serum albumin (plasma albumin) makes up 55 to

bilized by 17 disulfide bridges. In contrast, a-lactalbumin and ovalbumin (M r 44000) contain one oligosaccharide chain each, coupled to the peptide chain via an aspartate residue (3.2% carbohydrate of Mt 1550 in ovalbumin). In ovalbumin, one serine residue is esterified with phosphate. Alcohol dehydrogenase, abb. ADH (EC 1.1.1.1): a zinc-containing oxidoreductase which, in the presence of N A D + , reversibly oxidizes primary and secondary alcohols to the corresponding aldehydes and ketones. ADH occurs in bacteria, yeasts, plants and the liver and retina of animals. The ADH from yeast, which is distinguished by its high affinity for ethanol, is of practical significance as the last enzyme in alcoholic fermentation. In the liver, ADH acts in concert with other mechanisms to clear the blood of ethanol. The ADH in the retina, however, serves to convert the vitamin A aldehyde, all-irans-retinal, to retinol. The aldehyde is generated in the visual process, and its conversion to retinol is a prerequisite for regeneration of the visual pigment. ADH from animal organs and yeast has low substrate specificity, since it dehydrogenates both short (C 2 to C 6 ) and long-chain alcohols (e.g. retinol), and linear and cyclic alcohols. Yeast ADH (M r 145000) consists of four catalytically active, zinc-containing subunits (Af r 35000) with four N A D + or NADH binding sites per molecule of A D H ; the dimeric horse liver enzyme (M r

16

Alcoholic fermentation 80000) contains two zinc atoms (one is essential for catalysis) and one coenzyme binding site per subunit (Mr 40000, 374 amino acids, sequence known; Cys 46 is the site of binding and catalysis). In the dehydrogenation process, a ternary complex among ADH, N A D + and ethanol is formed in which both the coenzyme and the substrate are bound to the reactive SH group of Cys 46 via a zinc atom (enzyme-metal bridge complex). Due to the fact that there are two very similar polypeptide chains E and S, there are three types of liver ADH: the two isoenzymes EE (preferentially dehydrogenates ethanol) and SS (active with sterols) and a hybrid with a M r of 60000. It also differs from other A. in its subunit structure (8 subunits of M r 7400). Alcoholic fermentation: the anaerobic (occuring in the absence of oxygen) formation of ethanol and carbon dioxide from glucose. The most important fermenting organisms are yeasts and other microorganisms, but A.f. can also be carried out by the tissues of higher plants, e.g. carrots and maize roots. For lack of pyruvate decarboxylase (see below), there is no A.f. in animals. The process produces energy under anaerobic conditions: the fermentation of 1 mol glucose yields 2 mols ATP. The starting point for A. f. is glucose 6-phosphate, which is converted by the glycolysis reactions to pyruvate. Pyruvate is the branching point

Aldonic acids mation of fusel oils is considered a side reaction of A.f. Historical. The simple equation for A.f., C 6 H l 2 0 6 = 2 C 0 2 + 2 CH 3 CH 2 OH was established in 1815 by Gay-Lussac (Gay-Lussac's equation). In 1857, Pasteur proposed that A.f. could only be carried out by living organisms (vitalistic theory of fermentation). This was disproved in 1897 by Buchner, who established that a cell-free filtrate of disrupted yeast cells was capable of A.f. This discovery was the beginning of modern enzymology. The enzyme system responsible for A.f., which was originally thought to be one enzyme, was called zymase. In 1905 the role of phosphate in A.f. was described by Harden and Young. In 1912, Neuberg proposed the first fermentation scheme, which was revised in 1933 by Embden and Meyerhof. Alcohols: hydrocarbon derivatives carrying one or more hydroxyl (-OH) group. There are primary (RCH,OH), secondary (R'R 2 CHOH) and tertiary (R'R 2 R 3 COH) A. In multiple A. there are more than one hydroxyl group; in glycerol, for example, there are three. In nature, the A. in the form of esters are important components of the essential oils, fats and waxes. A number of lower A., ethanol for example, are formed by fermentative processes from carbohydrates and proteins. Aldehyde oxidase (EC 1.2.3.1): see Molybdenum enzymes. C02

Glycolysis •if

CH3—CO—COO •

Mg

Mg

R,—N.

Pyruvate decarboxylase

Pyruvate

R, R, - N

CHj-C-OH I

COO" Active pyruvate

Thiamine pyrophosphate NADH+H+

R,

V

R,—Nf

CH.-C —OH I H Active

Formation

NAD+

V

-=• Acetaldehyde v = : Alcohol dehydrogenase

CH3-CH20H I Ethanol I

acetaldehyde

of etbanol from

pyruvate.

for the last step of carbohydrate degradation and is decarboxylated by pyruvate decarboxylase (EC 4.1.1.1) to acetaldehyde, which is then reduced to ethanol by alcohol dehydrogenase (EC 1.1.1.1) (Fig.). Balance: C 6 H 1 2 0 6 + 2 P, + 2 ADP — 2 CH 3 CH 2 OH + 2C0 2 + 2ATP (glucose) + 2 H 2 0 . A.f. is the longest known and technologically most important form of fermentation. The most important substrates are the monosaccharides D-glucose, D-fructose, D-mannose, and sometimes D-galactose. In some cases the disaccharides sucrose and maltose and the polysaccharide starch can serve as substrates. The for-

Aldoketomutase: see Lactoyl-glutathione lyase. Aldolase: see Fructose-bisphosphate aldolase (EC 4.1.2.13). Aldonic acids: monocarboxylic acids derived from aldoses by oxidation of the aldehyde group. The names of these acids are formed by replacing the "-ose" of the parent aldose with "-onic acid". A.a. may form a 1,4-lactone ring (y-lactones) or the more stable 1,5-lactone (ô-lactones). Some important A.a. are L-arabonic acid, xylonic acid, D-gluconic acid, D-mannonic acid and galactonic acid.

Aldoses Aldoses: polyhydroxyaldehydes, one of the two main subdivisions of monosaccharides. (The other is the ketoses. See Carbohydrates). A. are characterized by their terminal aldehyde group -CHO, which is always given the number 1 in systematic nomenclature. The A. are formally derived from their simplest representative, glyceraldehyde, by chain extension. They are classified according to the number of carbon atoms in their chain as trioses, tetroses, etc. The pentoses and hexoses are particularly important in biochemistry. Aldosterone: 1 ip,21-dihydroxy-3,20-dioxopregn-4-en-18-al-18 11 -hemiacetal, a highly active mineralocorticoid hormone from the adrenal cortex. M r 360.45, m.p. 112 °C (hydrated crystals), 164 °C (nonhydrated), [a] D 2 3 +152.2° (anhydr; c = 2 in acetone). In contrast to other adrenal cortex hormones, A. has a carbonyl group on C-18, which forms a hemiacetal with the 11 (3-hydroxyl group. It is the most important mineralocorticoid, regulating NaCl resorption and potassium excretion. It also has a certain degree of glucocorticoid activity. It is synthesized in the liver from progesterone via cortexone and corticosterone, which is oxidized at C-18. A. was first isolated in 1953 from the bovine adrenal cortex by several groups; the total synthesis was achieved in 1957 by Schmidlin et al.

Aldosterone ALG: abb. for Antilymphocyte globulin. See Antilymphocyte sera. Alglnlc acid: a polyuronic acid extracted from seaweeds. It is composed of varying proportions of D-mannuronic and L-guluronic acids, linked P-1,4. The Afr is about 120000. The polymer replaces pectin in the brown algae, from which it is extracted by sodium hydroxide treatment. It can absorb up to 300 times its weight of water. Because it is easily digested, A.a. is widely used in the food industry, in surgery as resorbable sutures, and in the pharmaceutical and cosmetic industries. The salts and esters of A.a. are called alginates. The salts with alkaline earth anions are exceptionally good gelling agents, exceeding starch by a factor of ten. Alizarin: 1,2-dihydroxyanthraquinone, a red dye. m.p. 290 ° C. It occurs in the root of the madder plant (Rubia tinctorum L.) and other Rubiaceae in combination with 2 moles glucose, forming the compound ruberythric acid. A. was an important natural dye. It has been made synthetically since 1871, so the production from madder

17

Alkaloids

D-Mannuronic I acid

y

Alginic

L-Guluronic acid

/

acid

Algiaic acid has died out. A. and several of its derivatives are widely used as alizarin dyes. A. was isolated in 1826 by Colin and Robiquet, and its structure was determined in 1868 by Graebe and Liebermann. The first technical syntheses were developed independently by Caro and W.H. Perkin.

Alizarin

Alkaloids: basic natural products occurring primarily in plants. They contain one or more heterocyclic nitrogen atoms and are generally found in the form of salts with organic acids. At present, several thousand A. are known, and the structures of many have been determined. They usually have trivial names based on those of the plants in which they were discovered. Classification. It is difficult to define A. in such a way as to exclude other nitrogen-containing plant products. If they were classified according to occurrence and function, those of animal and microbial origin would be excluded, and in addition, some A. (for example nicotine) are found so widely that a strict classification by botanical origin is not possible. If the A. are classified chemically, on the basis of the structure of their skeletons, the colchicum alkaloids would not be included because they lack the heterocyclic nitrogen. Recently the A. have been divided on the basis of their biogenesis into the protoalkaloids (see Biogenic amines), pseudoalkaloids and the A. in the narrower sense. The protoalkaloids include, for example, the decarboxylation products of amino acids, and the pseudoalkaloids include compounds which are structurally related to other classes of natural products (for example, the terpenes). The A. in the narrower sense can be further subdivided, depending on their biogenetic precursors, into derivatives of ornithine, lysine, phenylalanine, tryptophan and anthranilic acid. In the classification given in the table, both structural and biogenetic features are taken into consideration. Occurrence. Probably 10 to 20% of all higher plants contain A.; they are particularly frequent in some families of dicotyledons. Closely related

18

Alkaloids families often produce similar A. A plant usually contains a mixture of A. of similar structure (primary and secondary A.) in the form of hydrophilic salts dissolved in the vacuolar sap. A. are found in all parts of the plants, but are particularly abundant in the seeds, bark and roots. Heterocyclic compounds similar to the A. are also found in many microorganisms and a few animals (for example, salamander alkaloids). Isolation and determination of structure. The free A. are displaced from the genuine salts by treatment with alkaline solutions and can be extracted with organic solvents. They can be further separated and purified by means of salt formation (picrates, Reinecke salts) and by chromatography. The Dragendorff reagent can detect A. in concentrations of a few ng. In recent years, the classical techniques of structure determination have been largely replaced by physical methods such as UV, IR, NMR and mass spectroscopy. Most A. are optically active, and almost all are levorotatory. Biosynthesis. A. are the end products of secondary metabolism, and are not subject to significant degradation. They are accumulated because the plant has no excretory organs. Most A. are derivatives of amino acids, especially ornithine, lysine, phenylalanine or tyrosine, tryptophan, or of anthranilic acid, which provide the heterocyclic nitrogen (Table). In addition, acetic acid, mevalonic acid and one-carbon units may be involved in the synthesis. The connection between amino acid metabolism and A. synthesis was discovered around the turn of the century. A new era was opened by experiments attempting to synthesize A. under physiological conditions, i.e. with native reactants, without high pressure or temperature, and at neutral pH. The biosynthesis of A. in vivo could be followed by administration of amino acids labelled with 13C, 14C, l 5 N or 3 H. It was found that plants create the very large number of structures from only a few components, using only a few mechanisms of cyclization: ^-heterocyclic rings are made by Mannich condensation, or by the formation of amide or azomethine bonds. Secondary

Alkaloids cyclizations, i.e. ring closures not involving nitrogen, are the result of oxidative coupling (phenol oxidation). The biosyntheses of many A. have been studied by isotope techniques, but little is known about the enzyme systems involved. The A. which are built up entirely from acetic or mevalonic acid (conium and terpene alkaloids) occupy a special position: in their case, neither the source of the nitrogen nor the form of ring closure is known. Synthesis. The first synthesis of an A. was reported in 1886 by Ladenburg, who generated coniine from a-picoline. On the hypothesis that A. are synthesized in the plant as amino acid derivatives, Robinson and Schöpf developed corresponding synthetic pathways and tested them under physiological conditions (see Tropinone). These studies of biosynthetic pathways were also fruitful for chemical syntheses in the laboratory, and some compounds were synthesized for the first time by routes similar to the biosyntheses. The technical preparation of A. for medical purposes are based mainly on plant materials as starting points. Biological and economic significance. A. general explanation of the biological significance of A. cannot yet be given. Their protective function against consumption by animals has been proven in only a few cases, but the fact that most insects are limited to one or a few plant species may be due to their alkaloid contents. Many alkaloids have a strong and very specific effect on certain centers of the nervous system. Therefore A. are widely used therapeutically, as combinations of pure compounds, as extracts of total alkaloids or as synthetic analogs. However, their use is often accompanied by side effects, primarily toxicity and narcosis. Historical. Most of the alkaloid plants were known very early in folk medicine for their toxicity or useful pharmacological properties. Morphine was first isolated from poppies as the "sleep-inducing principle" by F.W. Sertürner in 1806. The term A. was coined in 1819 by C.F.W. Meißner.

Table. The most important classes of alkaloids and their precursors C l a s s of alkaloid

Structural type (main precursor emphasized]

Ornithine Acetate

Pyrrolidine

Pyrrolidine

Tropane

Biogenetic precursors

u/ ~A

-j

N —

\

V-OR

r

and

Ornithine

Ornithine Acetate

and

19

Alkannin

C l a s s of alkaloid

Allergy

Structural type {main precursor emphasized)

Biogenetic precursors

Pipe ridine Conium

Acetate

Punica, S e d u m and Lobelia

Lysine, Acetate or P h e n y l a l a n i n e

1

Quinolizidine

Isoquinoline

CÒ QÇ1

Lysine

Phenylalanine or T y r o s i n e

Indole

Rutaceae

Terpene

Tryptophan

©Cu • w

Anthranilic

Mevalonic

acid

acid

N

1 Alkannin: see N a p t h o q u i n o n e s (table). Alkaptonuria: see L-Tyrosine. Alkylating agents: chemical c o m p o u n d s which can d o n a t e alkyl groups, usually methyl or ethyl. M o n o f u n c t i o n a l A.a., like dimethylsulfate or ethylmethanesulfonate, can transfer only a single functional group, while bifunctional A.A., like mustard gas, nitrogen mustard gas or cyclophosphamide, can react with several molecules or parts of a macromolecule, thus cross-linking them. A.A. are frequently carcinogenic a n d mutagenic, but some of them are nevertheless used in chemotherapy of cancer. See Mitomycin C. Allantoic acid: diureidoacetate, a degradation product of allantoin in aerobic purine degradation a n d in anaerobic allantoin degradation. M r 176.14, m.p. 173 ° C (d.). Allantoin: S-ureidohydantoin, glyoxyldiureide, an intermediate in aerobic purine degradation. M r 158.13, m.p. 238 ° C (d.), [ct] D 22 + 93°. A. was discovered in 1799 in the allantoic fluid of the cow. It is the end product of purine metabolism in most mammals and some reptiles, a n d is excreted in their urine. A. is also f o u n d widely in plants. In a n u m b e r of plant families known as

ureide plants, A. is a b u n d a n t in the soluble nitrogen pool. In certain species of bacteria (Arthrobacter allanloicus a n d Streptococcus allantoicus), A. can serve as C, N a n d energy source under anaerobic conditions. It is first converted to allantoic acid by allantoinase (EC 3.5.2.5), a n d the allantoic acid is degraded by allantoate deiminase ( E C 3.5.3.9) to ureidoglycine, N H 3 a n d C O , (anaerobic allantoin degradation).

-NH H, N — 2

C—NH—CH

N H

O

Allantoin Allantoinase ( E C 3.5.2.5): see Purine degradation (aerobic). Allelochemicals: see Pheromones. Allen-Dolsy test: see Estrogens. Allergy: a hypersensitivity of the i m m u n e apparatus, a pathological i m m u n e reaction induced either by antibodies (immediate hypersensitivity) or by living lymphoid cells (delayed type A.). U n -

Allocholane like the delayed type, immediate hypersensitivity can be passively transmitted in the serum. The symptoms begin shortly after contact a n d decay rapidly, but the delayed type symptoms d o not reach their m a x i m u m for 24 to 48 hours, and decline slowly over a period of days or weeks. Examples of this type of A. are anaphylaxis, the Arthus reaction a n d serum sickness. The bestk n o w n A., anaphylaxia, can occur as a local (cutaneous) reaction (for example a rash with blisters) or as a systemic reaction (anaphylactic shock). Asthma, hay fever a n d nettle rashes are also examples of local anaphylactic reactions which are induced by reagins (see Immunoglobulins). Only primates can be sensitized by injection with h u m a n reagins. An example of the delayed type A. is the tuberculin reaction, which is based on a cellular immune response. Allocholane: outdated term for Sa-cholane, see Steroids. Allodeoxycholic acid: 3a,12ot-dihydroxy-5acholan-24-oic acid, one of the bile acids. A dihydroxy steroid carboxylic acid. M r 392.58, m.p. 214 ° C , [a] D + 42°. Unlike most other bile acids, A. has an A / B - t r a n s ring coupling. It was isolated f r o m the bile a n d feces of rabbits. Alloglbberelllc acid: see Gibberellins. Allomones: see Pheromones. Allophanate hydrolase: see Urea amidolyase. Allophanlc acid: see Urea amidolyase. Allopregnane: outdated term for Sa-pregnane, see Steroids. Allosterlc enzymes: see Cooperative oligomeric enzymes. All-or-nothing model: see Cooperativity model. Allostery: the p h e n o m e n o n of changes in conformation of proteins with quaternary structure u p o n binding to certain low-molecular-weight ligands. A. has an important role in enzyme regulation (see Effectors) and in the uptake of oxygen by hemoglobin. Alnulln: see Taraxerol. ALS: abb. for Antilymphocyte serum. Amanltln: see Amatoxins. Amaranthin: a red dye belonging to the betacyanin group. In place of the glucose residue f o u n d in betanin, A. has a glycosidically linked disaccharide [5- 0(P-D-glucopyranosyluronic acid)-5-0-f)-D-glucopyranoside]. It is f o u n d in Amaranthus species, for example the foxtail Celosia argentea. Amaryllldaceae alkaloids: a group of complicated alkaloids f o u n d only in the plant family Amaryllidaceae. They can be classified as phenylisoquinoline alkaloids (see Isoquinoline alkaloids), because their biosynthesis (Fig.) is similar to that of the isoquinoline alkaloids, beginning with phenylethylamine or tyramine and a carbonyl c o m p o u n d . The final structures are generated by secondary ring cleavage a n d cyclization. The last step of the biosynthesis is catalysed by a phenol oxidase. The main alkaloid galanthamine is isolated f r o m Caucasian snowdrops, Galanthus woronowii, a n d is used therapeutically as an inhibitor of acetylcholinesterase. Amatoxins: a group of bicyclic octapeptides

20

A m b e r mutants

HO' OH Protocatechualdehyde

HO

Tyramine

Schiff's

base

OH

-CH3

HO. HO

CH30 Gelanthamine

Belladine

Biosynthesis loids.

of belladine and galanthamine

alka-

which, together with the phallatoxins, are the most important poisons in the death cap fungus, Amanita phalloides (fig.). These poisons inhibit the n u c l e o p l a s m ^ R N A polymerase II (EC 2.7.7.6) of eukaryotic cells, which leads to necrosis of liver and kidney cells. The poisonous effects of the A. and phallatoxins can be inhibited by a simultaneous application of a n t a m a n i d . More than 90% of fatal m u s h r o o m poisonings are d u e to consumption of Amanita phalloides a n d related species. The structure of the amanitins has been established by Th. Wieland. h3c-ch-ch-ch2-r2 HN-CH-CO-NH-CH-CO-NH-CH 2 -CO I 0C

¿h'

1

X7

H. ^ HOo V — til H

o=s N I H CH2

I NH

/CH3

H-C—CH I \ CO CH2CH3

OC—CH—NH—CO—CH—NH—CO—CH2—NH I H i - •CORj a-Amanrtin ß -Amanitin -y-Amanitin Amanin Amanullin

R, OH OH OH OH H

R2

OH

OH H

OH H

R*

R3 nh2

OH

OH

OH

nh2 OH nh2

OH H OH

Amatoxins Amber codon, nonsense codon: the sequence U A G in a m R N A . It does not code for any of the 20 proteogenic amino acids, a n d it results in the premature termination of protein synthesis. It may be f o r m e d by mutation of a sense c o d o n : potential precursors are the codons U C G (serine), U A U and U A C (tyrosine) a n d C A G (glutamine). Amber mutants: mutant bacteria in which the m R N A contains the codon U A G because of a point mutation (see Amber codon). The mutation

21

Amicetins

Amino acids: aminocarboxylic acids, organic acids carrying amino groups, usually not more than two. A.a. are classified as a-, ()-, y-..., depending on the position of the carbon bearing the — NH 2 group with respect to the — C O O H (Fig. 1). The a-A.a., as the components of proteins and peptides, but also in their free form, are one of the most important classes of organic substances in the cell. There are about 20 A.a. which are normally components of proteins. They are called proteogenic A.a., or proteinogenic A.a. (Table 1).

is not necessarily lethal, because a compensatory suppressor mutation in a t R N A may enable the protein synthesizing system to recognize the amber codon as a sense codon. The term "amber" was arbitrarily chosen by the discoverer of the mutants. Amicetins: pyrimidine antibiotics (see Nucleoside antibiotics) synthesized by various Streptomyces species. Amicetin A is formed by Streptomyces fasciculatus and St. vinaceusdrappus. Mt 618.67, m.p. (anhydride) 243 to 244 °C, [a] D 2 4 + 116.5° (c = 0.5 in 0.1 N HC1). It is primarily bacch3\ H

I

Amino acids

y—-N NH-

- o -

Cytosine

p-Aminobenzoic acid

\N

NH2 0 II I NH- •c— c—CH 2 0H I CH,

H0CH: H H Amosamine

1

/

a-Methyl-D-serine

- Amicetin B -Amicetin A Structures of amicetins A and B teriostatic, especially against Gram-positive bacteria. 0.5 |ig amicetin A/ml inhibits the growth of Mycobacterium tuberculosis. Amicetin B (plicacetin) was isolated from Streptomyces plicatus by Sensi (1957) and Haskell (1958). It is presumed to be the precursor of amicetin A. M r 517.57, m.p. 160 to 163 "C (colorless needles, ethyl acetate) [a] D 2 6 + 181° (c = 2.7, methanol). Its spectrum isthe same as that of amicetin A, but its effect is weaker. Cytimidine, M r 331.33, m.p. 197 to 199 ° C (d.), [a] D 2 1 + 25.6° (water), is a degradation product of A. It consists of cytosine, 4-aminobenzoic acid and 2-methyl-D-serine. Amldinotransferases, transamidinases, (EC 2.1.4): a group of enzymes catalysing transamidination. They are involved in the biosynthesis of creatine. An A. from Streptomyces griseus and St. baikiniensis is involved in the biosynthesis of streptidine. In addition, the presence of A. of unexplored substrate specificity in the biosynthesis of guanidine derivatives has been reported. The transfer of the intact amidine group by A. was demonstrated by double labelling with 14 C and 15 N of the amidine group of L-arginine. This A. has both transferase and hydrolase activities, and is in this sense a possible arginase. Amination: the introduction of the amino group ( —NH 2 ) into an organic carbon compound. It may be accomplished either by reductive A. or transamination. Reductive A. requires a reduced pyridine nucleotide as reducing agent. The most common reductive A. of 2-oxoglutarate by L-glutamate dehydrogenase (EC 1.4.1.4) requires NADPH. Aminoacetlc acid: see Glycine.

Table 1. Proteogenic amino acids Amino acid

abb.

L-Alanine L-Arginine L-Asparagine L-Aspartic acid L-Cysteine L-Glutamic acid L-Glutamine Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Valine

Ala Arg Asn Asp Cys Glu Gin Gly His lie Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

Amino acids occurring only in special

proteins

Amino acid

Occurrence

8- Hydroxy- L-lysine L-3,5-Dibromotyrosine L-3,5- Diiodotyrosine L-3,5,3'-triiodothyronine L-Thyroxin Hydroxy-L-proline a-Aminoadipic acid

Fish collagen Skeleton of Primnoa lepadifera (coral) Skeleton of Gorgonia cavolinii (coral) Thyreoglobulin (tissue protein in the thyroid gland) Thyreoglobulin Collagen, gelatins Maize protein

22

Amino acids H ?es'.du® , R - C - C O O H (variable) |

a -Carboxyl '

g r o u Kp 3

NH 2 a-Amino

group

Fig. 1. Structure of an a-amino acid. The amino acids are classified as acidic or basic, depending on their isoelectric points, or, depending on the nature or their side chain, they are divided into four groups: 1. A.a. with neutral, hydrophobic (non-polar) side chains, glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline and methionine; 2. A.a. with neutral and hydrophilic (polar) side chains, serine, threonine, tyrosine, cysteine, asparagine and glutamine; 3. A.a. with acid and hydrophilic

Amino acids isoleucine) asymmetric C-atoms (except for glycine), they occur in nature as optically active compounds. With a few exceptions, natural A.a. have the L configuration. D-A.a. occur in the cell walls, capsules and culture media of some microorganisms, and in many antibiotics. Besides the a-A.a., there are rare natural A.a. in the f)-, y- and other forms. These occur as the free acids or as components of natural products. Properties. A.a. are amphoteric, since they carry both an — NH 2 and a - C O O H group,and their solutions are ampholytes. In the solid state and in strongly polar solvents, they are zwitterions, H 3 + N-CHR-COO-. With a few exceptions, they are highly soluble in water, ammonia and other polar solvents, but barely so in nonpolar and less polar solvents such as ethanol, methanol and ace-

Table 2. Minimal requirements of human beings for essential aminoacids in mg per kg and day Leu

Lys

90 10.4

lie

9.9

90 8.8

Woman

5.2

7.1

WHO Norms

3.0

3.4

Child Man

Phe

Met

Cys

3.3

901 4.32 13.33 3.14

85 1.5 13.2 4.7

11.6 0 0.5

3.0

2.05

1.6

1.4

2

' i n the presence of L-tyrosine; Tyr 15.9 mg/ kg: Tyr/Trp =5.5; 3 in the absence of L-tyrosine; 4 Tyr 15.6 mg/kg: Tyr/Trp = 7.4; 5 Tyr 5.0 mg/kg: Tyr/Trp = 2.0. WHO is the World Health Organization. (polar) side chains, aspartic acid and glutamic acid; 4. A.a. with basic and hydrophilic (polar) side chains, lysine, arginine and histidine. In addition to this chemical classification, A.a. can be divided according to their degradation products into glucogenic and ketogenic A.a. Glucogenic A.a. can be degraded to C4-dicarboxylic acids or pyruvate, while ketogenic A.a. are degraded to ketone bodies, in particular to acetoacetic acid. Finally, the A.a. can be classified as essential and nonessential, depending on whether the organism in question is able to synthesize them in amounts adequate for its needs. These must be taken up in the food, as an inadequate supply leads to negative nitrogen balances, and to inhibition of growth and protein biosynthesis (Table 2). Half-essential A.a. are in a special class, because the need for them depends on the physiological state of the organism. They include A.a. which the young animal cannot synthesize in adequate amounts to insure normal growth, or to supply a particular need. Glycine, for example, is halfessential for chicks because they, like all sauropsids, use glycine in the synthesis of the uric acid, which is their chief end product of nitrogen excretion. Since the A.a. contain one or two (threonine and

Thr

Trp

Val

60 6.5

30 2.9

85 8.8

3.5

2.1

9.2

2.0

3.0

tone. A.a. with hydrophilic side chains are more soluble in water. The water solubility of the A.a. is lowest at their isoelectric points, because the dominating zwitterionic form reduces the hydrophilic property of the amino and carboxyl groups. The dissociation of the A.a. is strongly dependent on the pH value of the solution, and the zwitterionic form is only present between pH 4 and 9. In the more acid range, the A.a. are present as cations, H3 + N-CHR-COOH, and in more alkaline solutions, as anions H 2 N-CHR-COO~. The titration curves of the A.a. therefore show two buffering zones, and are also affected by the dissociation behavior of the side chains, especially the acidic and basic ones. The acid-base behavior of the A.a. is a model for that of peptides and proteins and also the basis for separation by electrophoresis and ion-exchange chromatography. The UV absorption of A.a. with chromophoric side chain functions (e.g. tryptophan, tyrosine and phenylalanine) makes it possible to determine them quantitatively, both as the free A.a. and in proteins and peptides. The proteogenic A.a. in the cell make up the amino acid pool (or pools in the case of compartmentation), into which the A.a. from nutritional sources, proteolysis and de novo synthesis mix. This pool also includes nitrogen-containing precursors and intermediates of the proteogenic A.a. and the nonproteogenic A.a. The proteogenic A.a. can be grouped into families according to the biosynthetic sources of their carbon skeletons: 1. the serine family includes the A.a. derived from trióse phosphate: serine, gly-

Amino acids

23

Amino acids

Alanine

Valine

+ (NH 3 2-Oxoisovaleric acid

-Acetyl-CoA

Pyruvate

2-Aminoadipic acid pathway

s» Lysine

HC02) Oxaloacetate

Leucine

MNH3) Aspartate

2- Oxobutyrate-"

-Homoserine -

isoteucine

Diaminopimelic acid

-^-Lysine

-^-Threonine

+ (Cysteine-S) +( Serine )

+ (Pyruvate}

F i g . 2 . The pyruvate

+(Pyruvate)

Methionine

family

of amino

acids.

cine, cysteine and cystine; 2. The ketoglutarate family is made up of those A.a. whose skeletons are derived from oxoglutarate supplied by the tricarboxylic acid cycle: glutamate, glutamine, ornithine, citrulline, arginine (see Urea cycle) proline and hydroxyproline; 3. The pyruvate family is derived from pyruvate and oxaloacetate (Fig. 2); 4. The pentose family includes histidine and the three aromatic A.a. (see Aromatic biosynthesis) phenylalanine, tyrosine and tryptophan. The biosynthetic pathways of the individual A.a. include various nonproteogenic A.a. as intermediates. The microbial synthesis of A.a. is technically exploited. The A.a. L-lysine, L-glutamic acid, L-valine, L-isoleucine, L-tryptophan and L-tyrosine are produced by mutants of Corynebacterium glutamicum and other bacteria. L-Lysine and L-glutamic acid are produced on a large scale. The yield of glutamic acid reaches 50 kg for each 100 kg glucose supplied. Up to 50 g L-glutamate/1 medium is accumulated. This overproduction of A.a. is primarily due to defects in the regulation of the mutants used (see Production strains). The A.a. in the cell's amino acid pool either are used for the synthesis of new proteins, or they are degraded in A.a. metabolism. The major metabolic pathways are: 1. the transamination to 2-oxoacids; 2. decarboxylation; 3. transformation of the side chain; and 4. oxidative deamination to 2-oxoacids. (Table 3). Nonproteogenic A.a. (nonprotein A.a.) are usually not incorporated into proteins. They include A.a. which are intermediates in the biosynthesis of proteogenic A.a., for example a-aminoadipic acid, diaminopimelic acid and cystathionine. At present about 200 nonprotein A.a. are

known, most of them occurring in plants and limited in each case to certain taxonomic groups. The majority can be grouped according to their biosyntheses into the four groups of biogenetically related A.a. A few nonprotein A.a. have also, in exceptional cases, been detected in proteins, for example L-citrulline in the protein of hair follicles and a-aminoadipic acid in maize protein. The occurrence of these rare natural A.a. can be used in chemotaxonomy. The terms "rare" or "unusual" refer only to their sporadic occurrence and their structural differences from proteogenic A.a. However, they are structurally related to the proteogenic A.a. More than 20 nonprotein A.a. are known which differ from alanine by substitution of one hydrogen atom of the methyl group. Many other rare, naturally occurring A.a. are structurally related to proteogenic A.a. Nonprotein A.a. may also act as A.a. antagonists, e.g. azetidine-2-carboxylic acid, a toxic constituent of lily of the valley, is a structural analog of proline, in which the ring is contracted by one C-atom. In lily of the valley, uncontrolled incorporation of azetidine-2-carboxylic acid into the plant's own protein is avoided by the highly specific synthesis of prolyl-tRNA, but in other organisms azetidine-2-carboxylic acid becomes incorporated in place of proline, leading to marked alterations in the tertiary structure and biological activity of proteins. Nonprotein A.a. are particularly common in certain plant families, e.g. the Mimosaceae contain 2-diaminopropionic acid and its derivatives, thioether derivatives of L-cysteine, and derivatives of lysine and glutamic acid. Some nonprotein A.a. are biologically active, e.g. Albizzin (see), Lathyrogenic A.a. (see) and indospicine

24

A m i n o acid activating enzymes

A m i n o acid reagents

Table 3. Metabolic reactions of amino acids Type of reaction

Equation Transamination

RCHCOOH + R'CCOOH ^ I II NH, O

RCCOOH + R'CHCOOH II I O NH,

Decarboxylation NH2 RCCOOH + N H , + NAD(P)H2 -

Animation

Deamination Modification of side chain Hydroxy! group a - A m i n o group a-Carboxyl group Peptide formation

•RCHCOOH I + NAD(P) NH, +H,0 • RCCOOH II 4- N H O

- 2(H) RCHCOOH • RCCOOH I II NH2 NH ATP R-OH • R — O - P 0 2 H 2 (phosphorylation) R — NH2 R — N H — C O C H j (acetylation) R - C O O H ATP C O N H J (amide synthesis) NH RCHCOOH R'CHCOOH RCHCO — NHCHCOOH ).-H,0 I I NH2 R

Amino acid activation (Protein biosynthesis 1)

R C H C - O H + AMP ~ P ~ P O I Enz. II NHj Enz. A M P - C C H R + HF'

A m i n o acid transfer (Protein biosynthesis 2) (Synthesis of aminoacyl-tRNA)

Enz. A M P ~ C C H R + t R N S - O H • AMP I O NH, II + Enz. + t R N S - O - C C H R I 0 1 NH, Enz. A M P ~ C C H R + fc-'H

O

Gramicidin S synthesis

NH2

NH,

Enz. - Enzyme; E - Protein II of gramicidin S-synthetase

Amino acid analyser: see Proteins. Amino acid oxidases: see Flavin enzymes. Amino acid reagents: reagents for the colorimetric identification a n d quantitation of a m i n o acids. O n e of the most important is the ninhydrin reaction, in which a blue-violet dye, called Ruhem a n n ' s Purple (absorbance m a x i m u m at 570 nm, for proline at 440 nm) is formed by the reaction of 2,2-dihydroxy-lH-indene-l,3(2H)-dione (ninhydrin) with the amino acid.

(L-2-amino-6-amidinocaproic acid) f r o m Indigofera spicata, which acts as a liver toxin a n d causes growth deformities. N e w A.a. are usually discovered by chromatography, on the basis of their unusual Revalues or unusual color reactions, e.g. with ninhydrin. Amino acid activating enzymes: see Aminoacyl-tRNA synthetases. Amino acid activation: see A m i n o acids (Table 3).

S

A M P + Enz. + E-s ~ C O C H R I NH2

.0

OH

-CO,

+H2N-CHR-COOH

-2Hj0

OH

r s

( ¿ > - 0

0

Amino acid

Ninhydrin

Ozi-

„ '=NrCHR

Ketimine

CHR

OH

Aldimine

Ninhydrin

0

0

Ruhemann's purple

x

c = o

25

Aminoacyladenylate In the fluorescamine technique, the amino acids are converted by reaction with 4-phehyl[furan2H(3H)-r-phthalane]-3,3'-dione (fluorescamine) into strongly fluorescing c o m p o u n d s which can be detected even in n a n o m o l e quantities at 336 nm. The reagent itself is not fluorescent, and in contrast to ninhydrin, it is not sensitive to ammonia (Udenfriend, 1972). Other highly sensitive reagents are 2,4,6-trinitrobenzosulfonic acid, l,2-naphthoquinone-4-sulfonic acid (Folin's reagent) a n d 4,4'tetramethyldiaminodiphenylmethane (TDM). Intensely fluorescing amino acid derivatives are f o r m e d by reaction with o-phthalaldehyde in the presence of reducing agents; with pyridoxal and zinc2+ ions; and with dansyl chloride (5-dimethylaminonaphthalene sulfonyl chloride. Aminoacyladenylate, activated amino acid: the product of the first enzymatic reaction of protein biosynthesis. It consists of an amino acid linked by a n acid anhydride b o n d to the phosphate of A M P . In the cell, these c o m p o u n d s are always associated with aminoacyl-tRNA synthetases, which also catalyse a further reaction, the transfer of the amino acyl residue of the A. to a specific t R N A . A M P is released in this second step. See Aminoacyl-tRNA Synthetases. Amlnoacyl-tRNA: a transfer R N A charged with a specific a m i n o acid; the transport f o r m in which the amino acid is brought to the specific acceptor site on the ribosome. The carboxyl g r o u p of the amino acid is esterified to either the

2-Aminoadipic acid 2' or the 3' O H - g r o u p of the ribose of the terminal adenosine of the t R N A . The free energy of hydrolysis of this ester b o n d is 29.0 k J / m o l see Energy-rich bonds, see Aminoacyl-tRNA Synthetases. Amlnoacyl-tRNA synthetases, amino-acid activating enzymes (EC 6.1.1.): a group of enzymes which activate amino acids and transfer them to specific t R N A molecules as the first step in protein biosynthesis. The process consists of two steps, illustrated here with leucine: 1) Leu + A T P + leucyl-tRNA synthetase ^ [LeuAMPEnzyme] + PP, 2) [LeuAMPEnzyme] + t R N A ^ " — l e u c y l - t R N A ^ " + A M P + enzyme. 1) a n d 2) Leu + ATP + t R N A 1 ^ " — Leucylt R N A ^ " + A M P + PPf The A. are highly specific with respect to the a m i n o acid they activate, and they also recognize the t R N A with great precision. The mechanism by which the enzyme recognizes the appropriate t R N A is still unclear. A. may consist either of one polypeptide chain or of two or four homologous or heterologous subunits. Eukaryotic cells contain more than 20 different A., because the mitochondria a n d plastids have their own amino-acid specific A., which differ in their specificity toward homologous t R N A f r o m those of the cytoplasm. Some A. are able to load several amino-acid-specific t R N A s , e.g. the leucyl-tRNA synthetase of Escherichia coli, which can load 5 t R N A £ co/ ,- Leu . 2-Amlnoadlpic acid, abb. Aad: H O O C - C H , C H 2 - C H 2 - C H ( N H 2 ) - C O O H , a n amino acid which

"OOC-CH2-CH2-CO-COO" I

2-Qxogtutarate

[«£

— NH3 - NAD (P) H+H+

Glutamate dehydrogenase (EC U.I.3)

•^-NAD(P)+

'OOC-CH2-CHrCH (NH2)-COO~ Glutamate Glutamate decarboxylase (EC 4.1.1.15)

COj V "OOC-CH2-CH2-CH2-NH2 4—Aminobutyrate

Aminobutyrate aminotransferase (EC 2.6.1.19)

£

Glutamate

"OOC-CH,-CH,-CHO

Succinate-se mi aldehyde dehydrogenase (EC 1.2.1.24) "OOC-CH2-CH2-COO" Succinate 4-Aminobutyrate

pathway

[

2-Oxoglutarate

2-Aminoadipic acid pathway is proteogenic only in maize. Afj. 161.1, m.p. 206 °C (d.). Aad is an intermediate in the biosynthesis of L-lysine by the Aad-pathway. The free acid cyclizes in boiling water to piperidone carboxylic acid. 2-Aminoadiplc acid pathway: see L-Lysine. 4-Aminobutyrate pathway, y-aminobutyrate pathway: see 4-Aminobutyric acid. 4-Amlnobutyrlc acid, abb. 4-Abu, y-aminobutyric acid\ abb. GAB A: H 2 N-CH 2 -CH 2 -CH 2 COOH, a nonproteogenic amino acid. M r 103.12, m.p. 202 °C (d.). Formation of GAB A from L-glutamic acid, by the action of glutamate decarboxylase (EC 4.1.1.15), has been demonstrated in brain, various microorganisms (e.g. Clostridium welchii, Escherichia coli), higher plants (e.g. spinach, barley) and other animal tissues (liver and muscle). It can also be formed from 4-guanidobutyric acid (see Guanidine derivatives) by removal of urea in higher fungi (Basidiomycetes) and Streptomycetes. Degradation of GABA proceeds by transamination to succinic semialdehyde and subsequent oxidation to succinic acid, which is oxidized in the tricarboxylic acid cycle. Synthesis of GABA is particularly important in the brain, where it functions as an inhibitory neurotransmitter. On account of its neural activity, GABA is used for the treatment of epilepsy, cerebral hemorrhage, etc. The 4-aminobutyrate pathway (Fig.) represents a bypass of the oxidative decarboxylation of 2-oxoglutarate in the tricarboxylic acid cycle. Only some brain cells make GABA, and only approximately 25 % of 2-oxoglutarate produced in these cells is converted to GABA. In total, the 4-aminobutyrate pathway accounts for less than 10% of the total oxidative metabolism of the brain. Amlnocarboxylic acids: see Amino acids. Amino citric acid: CH(NH 2 )-C(OH)-CH 2 I I I COOH COOH COOH, an amino acid identified in acid hydrolysates of ribonucleoproteins from calf thymus, bovine and human spleen, Escherichia coli and Salmonella typhi. It is an acidic amino acid, elutes before cysteic acid from the amino acid analyser, and gives a characteristic yellow color with ninhydrin. Ref: Wilhelm, G. and Kupka, K. D. (1981) FEBS Letters, 123, 141-144. 3'-Amino-3'-deoxyadenosine: a purine antibiotic synthesized by Cordyceps militaris and Helminthosporium species (see Nucleoside antibiotics). m.p. 271 to 273 °C (d.), [a] D 25 - 3 7 ° (c = 2, 0.1N HC1). A. has antitumor activity. The acetylated derivative 3'-acetamido-3'-deoxyadenosine has also been isolated from Helminthosporium species. 2-Amino-2-deoxy-D-galactose: see D-Galactosamine. Aminoethanol: see Ethanolamine. Amlnoethanol phosphoglycerldes: see Phosphatides. L-a-Amlnoglutaric acid: see L-Glutamic acid. 5(4)-Aminoimidazole-4(5)-carboxamide ribonucleotide, abb. AICAR: 5-amino-l-ribofuranosylimidazole-4-carboxamide 5'-phosphate, an intermediate in purine biosynthesis. M r 337.21.

26

Amino sugars Nn2 N

N^Sl

R-H

w H0CH2 / ° \

s

3'-Amino-3'deoxyadenosine R—CO —CH3 3' Acetamido-3'deoxyadenosine

h Y NH OH I R 5(4)-Amlnoimidazole-4(5)-carboxyrlbonucleotlde: an intermediate in purine biosynthesis. 5-Amlnolmldazole ribonucleotide, abb. AIR: an intermediate in purine biosynthesis and in the formation of thiamine. M r 295.2. In certain microbial mutants lacking purine synthesis, AIR can polymerize to a red pigment. 5-Aminoimidazole-4-jV-succlnocarboxamlde ribonucleotide: an intermediate in purine biosynthesis. Amlnoisobutyrlc acid: in the p-form (2-methylP-alanine), H 2 N-CH 2 -CH(CH 3 )-COOH, a product of the reductive degradation of thymine (see pyrimidine degradation). The a-form, (2-methylalanine) H 2 N-C(CH 3 ) 2 -COOH, does not occur in nature. Neither A.a. is incorporated in proteins. Since a-A.a. is not metabolized (or only to a negligible extent), it is used as a model substance to study transport and cytokinin effects. 5-Aminolevulinlc acid, S-amiaolerulinic acid: H 0 0 C - C H 2 - C H 2 - C 0 - C 0 2 - N H 2 , an amino acid. M r 167.6 (hydrochloride), m.p. 118 to 119 °C (free base). A.a. is an intermediate in the biosynthesis of the porphyrins and part of the Shemin cycle (see Succinate-glycine cycle). Aminopeptldases: exopeptidases, EC 3.4.11, usually containing metal ions, which shorten proteins and peptides from the Af-terminal end of the chain, removing one amino acid residue per step. The best-known A. is leucine aminopeptidase, which can be obtained from the intestinal mucosa, kidneys and lens of the eye in highly purified or crystalline form. The best synthetic substrates for this enzyme are leucinamide, leucine />-nitroanilide, and leucine hydrazide. Its effectors are bivalent metal ions. A characteristic of all A. is their inability to hydrolyse prolylpeptide bonds. A. are large proteins (M r 230000 to 330000). The liver A. consists of two subunits, the kidney enzyme of four, and the lens A. of six. A. is used in peptide sequencing. Aminoproplonic acid: see Alanine. Amino sugars: monosaccharides in which an hydroxyl group has been replaced by an amino group ( —NH 2 ). The amino group is often acetylated. The 2-amino-2-deoxyaldoses are particularly important as: components of bacterial cell walls; in some antibiotics, e.g. streptomycin; in blood group stubstances; in milk oligosaccharides; and in high molecular weight natural products, such as chitin. Examples are D-galactosamine, D-glucosamine, D-mannosamine, neuraminic acid and muramic acid.

27

Ammonia

In the synthesis of amino sugars, the amino group is supplied by transamination from glutamine. Fructose 6-phosphate is aminated to D-glucosamine 6-phosphate by a hexose-phosphate transaminase. The latter is then converted by a transacetylase into the N-acetyl derivative. This is isomerized to the 1-phosphate, then activated by coupling to UTP, to form UDP-JV-acetylglucosamine. The latter can be isomerized to UDP-Nacetylgalactosamine. Neuraminic acid is often found as cytidine monophosphate- JV-acetylneuraminate or as N-glycoloylneuraminate. It is synthesized in the form of the JV-acetyl derivative from mannosamine and phosphoeno/pyruvate. Muramic acid is synthesized from UDP- JV-acetylglucosamine, which is condensed with phosphoeno/pyruvate. This compound is then reduced to UDP-JV-acetylglucosamine lactate (UDP-muramate). Ammonia, M f } : a colorless gas with a sharp smell. It can be compressed at 20 °C and 8.6atm. to a colorless liquid with low viscosity. At normal pressure, the condensation temperature is about — 40 °C. Solid N H j forms colorless, transparent crystals (m.p. —77.7 °C). NH 3 is very soluble in cold water, but it is completely driven off by boiling. The aqueous solution is weakly basic, due to the ability of NH 3 to take up protons, forming ammonium: NH 3 + H 2 O ^ N H 4 + + O H " . The reaction equilibrium lies far to the left,so that NH 3 can be displaced from ammonium compounds by bases. The toxicity of NH 3 is related to the high permeation rate of the nonprotonated form and its tendency to become protonated. Occurrence. NH 3 is the end product of the degradation of nitrogen-containing organic matter. It is therefore to be found in the form of ammonium salts in the soil.The concentration of ammonium ions in the body fluids of animals or in any cells is relatively low, since 1. NH 3 is eliminated, by detoxification reactions (see Ammonia detoxification), and 2. NH 3 , as the starting material for nitrogen metabolism, is rapidly consumed by various reactions of ammonia assimilation. Metabolism. NH 3 is the product of nitrate reduction, biological nitrogen fixation, and deamina-

Ammonia assimilation tion of amino acids and various catabolic pathways, for example oxidative purine degradation and reductive pyrimidine degradation. In this sense, NH 3 is the nitrogen-containing, inorganic end product of the degradation of proteins and nucleic acids. It is taken up into the pool of organic nitrogen by various reactions of primary nitrogen assimilation and is further distributed by reactions transferring nitrogen-containing groups (see Group transfer). Through these reactions, NH 3 is used for the synthesis of JV-containing body substances. Plants in particular have a well developed metabolism of inorganic nitrogen. Like many microorganisms, they can grow with ammonium salts as their only source of nitrogen. Carbamyl phosphate and glutamine can be regarded as "activated NH 3 " (metabolically active NH 3 ), since the biosynthesis of many nitrogencontaining compounds depends on them. Ammonia assimilation: the utilization of ammonia in the net synthesis of the nitrogen-containing groups of nitrogenous cell constituents, e.g. amino acids, amides, carbamyl and guanido compounds. Incorporation of ammonia into the amide group of glutamine, catalysed by glutamine synthetase (EC 6.3.1.2), is of central importance: L-glutamate + NH 3 + ATP ->• L-glutamine + ADP + P;. The amide nitrogen of L-glutamine is then used in various syntheses: 1. L-glutamine + a-ketoglutarate + 2H + + 2e~— 2 L-glutamate (Glutamate synthase). The glutamate takes part in the synthesis of other amino acids by transamination; thus a series of coupled reactions result in the net assimilation of ammonia into the amino groups of amino acids (Fig.). Reducing power for bacterial glutamate synthase (EC 1.4.1.13) is provided by NADPH, whereas chloroplast glutamate synthase (EC 1.4.7.1) utilizes reduced ferredoxin. Glutamine synthetase and glutamate synthase occur in plant chloroplasts, where ATP and reduced ferredoxin are supplied directly by the light reaction of photosynthesis. Animals lack glutamate synthase, and they cannot achieve the net synthesis of amino groups from ammonia. • 2. L-glutamine + HCO-3 + 2ATP + H 2 0 -* NO; a j NOS" X

|a-keto-acid

transamination amino acid

|glutamate|^

— |glutamate|

glutamate synthase

a-ketoglutarate|

N2 J

c x 1 N H 3 | + ATP

glutamine synthetase

|glutamine

"^-ADP + Pi

2H + + 2e~ F i g . Assimilation

c, nitrogenase

of ammonia

into amino groups of amino acids, a, n i t r a t e r e d u c t a s e , b , n i t r i t e r e d u c t a s e ,

28

Ammonification of nitrate carbamyl phosphate + L-glutamate + 2 A D P + Pj. (see carbamoyl phosphate). N-Acetyl-glutamate is an essential positive allosteric effector for this enzyme (carbamoyl-phosphate synthetase, EC 6.3.5.5). In eukaryotes, the enzyme is located in the cytoplasm. This carbamoyl phosphate provides C-2 and N-3 in the synthesis of pyrimidines and it contributes to the synthesis of the guanido group of arginine in plants and bacteria. 3. The amide group of glutamine is used in purine synthesis, where it provides N-3 and N-9 of the purine ring, and the 2-NH 2 group of guanine. 4. In several syntheses, nitrogen is derived directly from the amide group of L-glutamine, e.g. histidine synthesis; conversion of chorismate into anthranilate (see tryptophan synthesis); the synthesis of amino sugars; amination of UTP to CTP. 5. In some organisms, the amide group of glutamine is transferred to aspartate by the action of asparagine synthetase (glutamine hydrolysing) (EC 6.3.5.4): L-glutamine + L-aspartate + ATP - 106, acting together with F. IX a to activate F. X. Its absence causes hemophilia A. MR 55000 (bovine), single-chain glycoprotein. Its absence causes hemophilia B. Mr 55 000, glycoprotein composed of a light and a heavy chain. Activated by a mixture of F. IX a and VIII a + C a 2 + or VII a -(-tissue factor + Ca 2 + . Mr 124000, a glycoprotein composed of two similar or identical polypeptides joined by a disulfide bond(s). Activates F. IX. XI a is inhibited by antithrombin III, trypsin inhibitors, aj-trypsin inhibitor and CI inhibitor. Mr 76000 (human), single-chained glycoprotein. Activated by plasmin, kallikrein and XII a . Inhibited by antithrombin III, CI esterase inhibitor and lima bean trypsin inhibitor. Inhibition by antithrombin III accelerated by heparin. Activation of F. XII initiated by contact with abnormal surfaces. It is the first factor in the intrinsic pathway. MT 350000, 4-chained c^-globulin. XIII a is the transpeptidase responsible for cross-linking precipitated fibrin monomers.

Blood group antigens No.

Intrinsic

Name

Properties and functions

Prekallikrein (Fletcher factor) HMW kininogen (high molecular weight kininogen,contact activation cofactor, Fitzgerald factor, Williams factor, Flaujeac factor)

Activated to kallikrein, a serine protease which activates F. XII. Activated to a kinin involved in activation of F. XII, at least in vitro.

pathway

a c t i v a t e d by s u r f a c e contact

Extrinsic

pathway

a c t i v a t e d by t i s s u e

damage

Tissue factor (Lipoprotein) (inactive t i s s u e t h r o m b o k i n a s e )

Fibrinogen

Fibrin

monomer - fibrinopeptides J, (A2+ BÏ)

Fibrin p o l y m e r

( u r e a - soluble)

Fibrin p o l y m e r (clot) (cross-(inked, urea - insoluble)

Blood group antigens: s p e c i f i c o l i g o s a c c h a r i d e

structures attached to glycoproteins in the membranes of blood cells which are recognized as antigens by the immune systems of other individuals or organisms. The antigens are attached to the protein glycophorin in erythrocytes and to both proteins and lipids in other parts of the body. In humans, five systems of antigens have been identified, the ABO system, the MN, P, rhesus and Lutheran systems. Only the ABO and rhesus systems affect blood transfusions between humans; the other systems have been identified using animal antibodies against human blood. The structural differences between the ABO oligosaccharides are shown in Fig.l. The genetic basis for these groups is the existence of three alleles of a gene coding for the synthesis of a glyco-

63

Blood sugar syltransferase. In A type individuals, the enzyme transfers ^-acetylgalactosamine onto the terminal positions of the oligosaccharide chains, while in B type individuals, the enzyme is specific for galactose. The O gene appears to produce an inactive enzyme. Another gene, the H gene, codes for a fucosyltransferase which places an L-fucose on the oligosaccharide. When the H gene is inactive, the individual has the rare type I blood, or, if he has an active Le gene, which codes for an enzyme adding fucose to the JV-acetylglucosamine, he has type Lea. Individuals with both active H and Le genes have type Le b . (Le is for Lewis factor.) About 80% of the population has an active Se (secretion) gene, so that they secrete glycoproteins bearing the blood group substance into saliva and other body fluids. The structure of the carbohydrate portion of such a glycoprotein is shown in Fig.2.

a-Fuc

Bombykol

Type A terminal. Type B has a - G a l here; Type 0 h a s nothing

r

a - G a l N A c (1-

•3) ß-Gal (1-

a - L - Fuc

-3,4)

p-GlcNAc-

ku a-L-Fuc P r e s e n t in Types Le b a n d Le".

Fig. 1. Ends of oligosaccharide chains in individuals with different blood groups.

a-Fuc CI—»21 (l-»3) -l-'1"*4' »ß-Gal »0-GlcNAc

{1-»-3NySa| (,_»,) (,-M, ( 1 - * f t ( l - H ) a-Fuc - — ^ fl-Gal——p-GlcNAc (J-GlcNAc tfl"»3) ot_l uc " p-Sal

Linkage is 1-»-3 in type 1 chains and 1—>-4 in type 2 chains

Protein

p-GlcNAc

i l l ^ i GalMAc - 0 - CH2-Serine îtl-'ô) ?[ p-GlcNAc Th reonme ÎCl-^3) j P-Gal Protein

Fig. 2. Structure of the carbohydrate component of the Leb glycoprotein of human blood. GlcNAc = N-acetyl-D-glucosaminyl ; GalNAc = N-acetyl-D-galactosaminyl; Gal = D-galactosyl ; Fuc = L-fucosyl. Blood sugar: see D-Glucose. Blue-green bacteria, cyanobacteria, blue-green algae, Cyanophyta, Cyanophyceae: a group of photosynthetic prokaryotic organisms using H 2 0 as hydrogen donor (see Photosynthesis). Many are also able to fix atmospheric nitrogen (see Nitrogen fixation). Since they are prokaryotes, they have no nucleus, mitochondria or chloroplasts (see Cell, 1.). They were originally classified as plants on the basis of their capacity for photosynthesis, but they are phylogenetically unrelated to the green algae, which are eukaryotes. There are many similarities between B.g.b. and chloroplasts, and the endosymbiotic theory of evolution proposes that chloroplasts are descended from symbiotic B.g.b. In the B.g.b. photosynthesis takes place on the thylakoids or thylakoid stacks, which are derived from the cell membrane. The photosynthetic pigments are chlorophyll a, carotenoids and biliproteins (phycocyanin and phycoerythrin). The cytoplasm contains prokaryotic-type ribosomes. Typical of most B.g.b. are cyanophycin granules; these are colorless and spherical or polyhedral, and visible under the light microscope. Cyanophycin granules contain storage material consisting of a copolymer of arginine and aspartic acid. The storage carbohydrate of B.g.b. is a polymer of glucose with a degree of branching intermediate between that of glycogen and amylopectin.

Cytochemically it resembles glycogen: it stains brown with iodine and occurs as discrete granules of diameter 25-30 nm located between the thylakoids. Polyphosphate granules (syn. metachromatin, volutin, metachromatic granules) are also present in the cytoplasm of B.g.b.; these are spherical and vary in size from sub-light microscopic to several microns in diameter; they are concretions of the potassium salts of high M r linear polyphosphates. The inner cell wall, like that of bacteria, consists of murein anchored in the cell membrane. It is attacked by lysozyme. Outside the murein layer, there is a plasmatic layer, and beyond that there may be a slime capsule. There are two major classes of B.g.b.: the chroococcals, in which the cells are solitary (e.g. Anacystis) or colonial, held together by mucoid hulls; and the hormogonals, which grow in filaments (trichomas), often enclosed in a sheath. The cells of hormogonal B.g.b. communicate with each other and form a physiological unit. In the trichomas there is a certain degree of specialization: the heterocysts, characterized by thick, highly refractory cell walls, are the site of nitrogen fixation. BOD: see Biochemical oxygen demand. Bombykol, 10-trans-12-cis-hexadecadienol-(l): a pheromone exuded by female silk moths (Bombyx mori) to attract males. B. is an oil, nD20 1.4835. TTie first determination of structure was

Bongkrekic acid

64

made on 15 mg B. isolated from the abdominal glands of 500000 female moths. The configuration was established by Butenandt by comparison of the biological activities of synthetic compounds with the natural product.

Bombykol Bongkrekic acid: 3-carboxymethyl-17-methoxy-6,18,21 -trimethyldocosa-2,4,8,12,14,18,20heptaenedioic acid, M r 486.61. One of two toxic antibiotics produced by Pseudomonas cocovenenans in spoiled bongkrek (a coconut product consumed in Indonesia). It is an inhibitor of adenine nucleoside translocation and affects carbohydrate metabolism. Bornane: see Monoterpenes, Fig. Boron: an element essential for growth of higher plants. It is absorbed by the roots in the form of borate. Lack of boron causes heart rot of sugar beet and other roots. Botulln: see Toxic proteins. Bowman-Birk Inhibitor: see Soybean trypsin inhibitor. Bradykinin, kallidin I, kinin 9: Arg-Pro-ProGly-Phe-Ser-Pro-Phe-Arg, one of a group of plasma hormones called kinins. Like the other kinins, it is produced from a plasma precursor by the action of Kallikrein (see), Trypsin (see) or Plasmin (see). It causes dilation of blood vessels, and thus a reduction of blood pressure, causes the smooth muscles of the bronchia, intestines and uterus to contract, and is a potent pain-producing agent. Lysylbradykinin has similar activity. Brassicasterol, ergosta-5,22-dien-3$-ol: a plant sterol (see Sterols). Mx 398.69, m.p. 148 °C, [a] D — 64°. B. was first isolated from rapeseed (Brassica campestris) oil.

Buxus alkaloids peptidase and is used in protein chemistry to hydrolyse polypeptide chains into large fragments. Brimacombe fragments: see Ribosomal proteins. Bruclne: 2,3-dimethoxystrychnine. M r 394.47, m.p. 105 ° C (tetrahydrate), m.p. 178 °C (anhydrous). It is used in preparative chemistry to separate racemic acids into optic antipodes. B. is highly poisonous, but 10 times less so than strychnine, from which it is derived. The main physiological effect is a paralysis of the smooth musculature. For formula and biosynthesis, see Strychnos alkaloids. Bryoklnin: see N 6 -(y, y-dimethylallyl)Adenosine. Bufadlenolides: see Toad poisons, Cardiac glycosides. Bufogenlns: see Cardiac glycosides. Bufotenine, 5-hydroxy-N-dimetbyItryptamine: a Toad poison (see). It is also found in toadstools from which it was first isolated by Wieland. Bufotoxin: the main toxin in the venom of the European toad Bufo vulgaris. M r 757. It is a steroid derivative. The minimum lethal dose (in cats) is 390 |ig/kg. Buoyant density: see Density gradient centrifugation. 2,3-Butanediol: see Fermentation products. 2,3-Butylene glycol: see Fermentation products. n-Butyrlc acid, butanoic acid: CH3-(CH2)2COOH, the simplest fatty acid. M r 88.1, m.p. - 5 °C. B. a. accounts for 3 to 5% of the fatty acids esterified to glycerol in butterfat. When butter becomes rancid, it is the free B. a. produced by hydrolysis which is responsible for the unpleasant odor. B. a. is found in free form in many plants and fungi, and in traces in sweat. Esters of B. a. are found in many essential oils. Buxus alkaloids: a group of steroid alkaloids which are characteristic of plants in the boxwood genus (Buxus). The B. a. are derived structurally from pregnane (see Steroids), having additional methyl groups on positions 4 and 14, amino or methylated amino functions at positions 3 and 20, and usually a 16a-hydroxyl group and a 9,10-cyclopropane ring. A typical example is cyclobuxamine H (Fig.). The B. a. are biosynthesized via cycloartenol or a similar triterpenoid precursor.

HO

NHCHo

Brassicasterol. Bromelain: a thiol enzyme (EC 3.4.22.4) from the stems and fruits of the pineapple plant. The stem enzyme is a basic glycoprotein (M r 33000; IP 9.55) structurally and catalytically similar to papain. B. is activated by mercaptoethanol and other SH compounds. It is irreversibly inhibited by agents which block SH groups. It is an endo-

—OH

H,N Cyclobuxamine

H

c

C: see Cytosine. C 4 -acid cycle: see Hatch-Slack-Kortschack cycle. Cactus alkaloids: see Anhalonium alkaloids. Cadalin precursor: see P-Cadinene. Cadaverlne, 1,5-diaminopentane: a biogenic amine produced enzymatically by decarboxylation of lysine. It is a precursor of a few alkaloids. C. is one of the compounds responsible for the odor of decaying meat and fecal matter, and is poisonous. It is a preferred substrate of the amine oxidase (EC 1.4.3.6). Cadlnane: see Sesquiterpenes, Fig. P-Cadinene: an optically active sesquiterpene found in the essential oils of junipers and cedars (-)-P-C., MT 204.36, b.p. 124 °C, [cx]$ - 2 5 1 ° . Together with its isomers and their hydroxyl derivatives, C. is representative of the cadinolenes, which are among the best known and most widespread sesquiterpenes. They are called cadaline precursors, because they can be dehydrated to this aromatic compound. For formula and biosynthesis, see Sesquiterpenes. Cadinols: see fl-Cadinene. Caffeine, 1,3,7-trimethy¡xanthine: a purine derivative (Fig., see Methylated xanthines), m.p. 238 °C. C. is found in coffee beans and leaves, tea leaves and cola nuts. It is usually produced from tea leaves (1.5 to 3.5% caffeine content) and as a byproduct from the production of caffeine-free coffee. Due to its stimulatory effects on the central nervous system, C. and C.-containing beverages are used to stimulate the heart and circulation. Its effects are chiefly due to the inhibition of the phosphodiesterase which degrades cyclic AMP to AMP, which prolongs the adrenalinproducing effects of cAMP. Calciferol: same as vitamin D. see Vitamins. Calcitonin, thyreocaicitonin: a polypeptide hormone containing 32 amino acids. M r (human) 3420. It is formed in the parafollicular cells of the thyroid in mammals, and in the ultimobranchial gland of nonmammalian species. (In both cases, the gland is derived from the 5th gill pocket of the embryonic gut). C. causes a rapid but shortlived drop in the level of calcium and phosphate in the blood by promoting the incorporation of these ions in the bones. C. is released in response to a rising C a 2 + level and is antagonized by Parathormone (see). It is detected by radioimmunological methods. The total synthesis of C. from various species has been accomplished. Calcium-dependent regulator protein: see Calmodulin. C-alkaloids: see Curare alkaloids.

Callistephin: see Pelargonidin. Calmodulin: a ubiquitous calcium-binding protein, which mediates the function of Ca 2 + in eukaryotes. Many effects of Ca 2 + are exerted through calmodulin-regulated enzymes: (calmodulin) , n a c „ v e + C a 2 + ^ (calmodulin^2+)a(,„V(, (E)/ OH , activity + ( c a l m o d u l i n ^ + ) ^

(E.calmo-

activity

Some enzymes and processes thought to be activated in this way by calmodulin are: adenylate cyclase, phosphodiesterase, phospholipase A 2 , Ca 2 + -ATPase, microtubule disassembly, membrane phosphorylation, neurotransmitter release, NAD kinase, Ca 2 + -dependent protein kinase, guanylate cyclase, phosphorylase kinase, myosin light chain kinase. C. shows no tissue or species specificity. It closely resembles troponin C in its physicochemical properties, and about 70% of the amino acid sequence of C. is homologous with that of troponin C; in fact bovine C. substitutes for troponin C in the activation of muscle actomyosin ATPase. Bovine calmodulin contains 148 amino acid residues of known primary sequence. Ref: Wai Yiu Cheung, Science (1980) 207, 19-27. Calvin cycle, photosynthesis cycle, reductive pentose phosphate cycle: a series of at least 15 enzymatic reactions which, taken together, generate 1 molecule of hexose phosphate from 6 molecules of C 0 2 . In the process, which does not require light (dark reactions), 12 molecules NADPH and 18 molecules of ATP are consumed per molecule of hexose phosphate. The C. can be divided into 4 phases (Fig.): 1) In the carboxylation phase, 1 molecule of ribulose 1,5-bisphosphate and 1 molecule of C O j produce 2 molecules 3-phosphoglyceric acid in a reaction catalysed by ribulose-bisphosphate carboxylase (see Photosynthetic carboxylation). 2) In the reduction phase, the carboxyl group of 3-phosphoglyceric acid is reduced to the aldehyde group of glyceraldehyde 3-phosphate. In this reaction, the carboxyl group must be activated by ATP before it can be reduced by NADPH. In principle, the reaction is the reverse of the oxidation of glyceraldehyde 3-phosphate by triose-phosphate dehydrogenase in the course of glycolysis. The reduction consumes the products of the light reaction, ATP and NADPH, and is thus the point at which the light and dark reactions are coupled. 3) In the regeneration phase, the acceptor of CO2, ribulose 1,5-bisphosphate, is regenerated by a series of steps (Table). Two molecules of triose phosphate are con-

66

Calvin cycle densed to fructose 1,6-bisphosphate, which, after removal of one phosphate, enters the 4) synthetic phase as fructose 6-phosphate. In this phase, suerose or starch is generated. The C. can be sum-

Calvin cycle marized as follows: 6 C 0 2 + 12 NADPH + 18 ATP + 6 H 2 0 hexose phosphate + 18 ADP + 17 Pj + 12 N A D P + .

R e a c t i o n 1: r e a c t i o n of r i b u l o s e b i s p h o s p h a t e c a r b o x y l a s e ( E C 4.1.1.3) ( c a r b o x y d i s m u t a s e ) ; the i n t e r m e d i a t e s h o w n in b r a c k e t s is e n z y m e - b o u n d c h

2

C0

©OOC-C-OH

I

2

>

CHj-O-

coos I

hUO

C = 0

2

I

H - C - O H

I

H-C-OH

H - C - O H

I

o - ®

CH2

- O - ®

I c=o I H-C-OH I

(!)

CH,

c h

_

- o - ®

- o - ®

2 - Carboxy - 3 ketoribitol 1,5-bisphosphate

Ribulose 1,5-bisphosphate

2

3-Phosphoglycerate

3 - PhosphoglycerQte ATP

Phosphoglycerate kinase ( E C 2.7.2.3)

ADP

3-Phosphoglyceroyl phosphate NADPH+ H+ NADP*"

Glyceraldehyde-phosphate ^ - P / dehydrogenase (EC 1.2.1.13)

Glyceraldehyde 3 - p h o s p h a t e

Table. Reactions regenerating ribulose 1,5-bisphosphate in the Cairin cycle. Reaction

Enzyme

Reaction

Enzyme

Glyceraldehyde-P Dihydroxyacetone-P

Triosephosphate isomerase (EC 5.3.1.1) Fructose-bisphosphate aldolase (EC 4.1.2.13)

Sedoheptulose-l,7-P 2 ^ Sedoheptulose-7-P + P ;

Sedoheptulose-bisphosphatase (EC 3.1.3.37) Transketolase (EC

Glyceraldehyde-P + Dihydroxyacetone-P ^ Fructose-1,6-P 2 Fructose-1,6-P2 ^ Fructose-6-P + Pi Fructose-6-P + Glyceraldehyde-P ^ Xylulose-5-P + Erythrose-4-P Erythrose-4-P + Dihydroxyacetone-P ^ Sedoheptulose-l,7-P 2

Fructose-bisphosphatase (EC 3.1.3.11) Transketolase (EC 2.2.1.1)

Aldolase (EC 4.1.2.13)

Sedoheptulose-7-P + Glyceraldehyde-P ^ Ribose-5-P + Xylulose-5-P Ribose-5-P ^ Ribulose-5-P Xylulose-5-P ^ Ribulose-5-P

2.2.1.1)

Ribosephosphate isomerase (EC 5.3.1.6) Ribulosephosphate 3epimerase (EC 5.1.3.1) Ribulose-5-P + ATP ^ Phosphoribulokinase Ribulose-1,5-P2 + ADP (EC 2.7.1.19).

Sum: 5 Triose-P ->• 3 Pentose-P

Sucrose I Fruclose bisphosphate |

Cairin cycle reactions

Fructose-P

' Glucose-P

Starch

Calvin plants

67

Calvin plants: see C 3 Plants. cAMP: abb. for cyclic adenosine 3',5'-monophosphate. See Adenosine phosphates. cAMP receptor protein: see Adenosine phosphates, Table 2. Campesterol: (24R)-ergost-5-en-3$-oI: a plant sterol (see Sterols). Mr 400.68, m.p. 158 °C, — 33° (22.5 mg in 5 ml chloroform). C. is found in the oils of rapeseed (Brassica campestris), soybean and wheat germ and in some molluscs.

Campesterol Camphor: a bicyclic monoterpene ketone found widely in plants. Both optical isomers occur naturally: ( + ) — C. (Japan camphor), m.p. 180 °C, b.p. 204 °C, [ct]$ +43.8° (c = 7.5 in abs. ethanol), and ( — ) - C . (Matricaria camphor), m.p. 178.6 °C, b.p. 204 °C, - 4 4 . 2 ° (ethanol). C. is obtained commercially from camphor trees (Cinnamomum camphora) native to the coastal areas of Eastern Asia. The partial synthesis from pinene is also important, although the product is a racemic mixture used mostly in plastics. Natural C. is used pharmaceutical^ in salves. For formula and biosynthesis, see Monoterpenes. Camptothecln: the main alkaloid from the wood and bark of the Chinese tree Camptotheca acuminata. MT 348, m.p. 264-267 °C (d.), [ a ] ^ + 31.3° (in chloroform/methanol, 8:2). Its total

L-Canavanine synthesis from an indole compound has been reported. C. is one of the most active natural substances against leukemia and tumors.

L-Canalin: 2-amino-4-aminooxy-butyric acid, M r 131.4, m.p. 214 °C (d.). It is a hydrolysis product of L-Canavanine (see), and is found in a few canavanine-containing legumes. L-Canavanlne, 2-amino-4-guaaidiaohydroxybutyric acid: H 2 N-C(NH) 2 -0-(CH2) 2 -CH(NH 2 )COOH a structural analog of Arginine (see). M r +7.9° (c = 3.2 in water) found only 176.2, in certain legumes. The presence or absence of C. is a useful trait in the Chemical taxonomy (see) of the legumes. In the seeds of jack beans and some other legumes, it accounts for up to 4% of the dry weight and is a soluble nitrogen reserve substance for these plants (see Ammonia detoxification). It is synthesized in the leaves opposite the fruits of the plant and in the immature pods, and stored in the seeds, presumably in water-insoluble form. When the seed germinates, C. is transported to the embryo and there hydrolysed by arginase (EC 3.5.3.1) to urea and L-canalin, which is converted via homoserine to L-aspartate. The aspartate is deaminated to oxaloacetic acid, which is metabolized in the tricarboxylic acid cycle. The nitrogen in the C. is completely mobilized, as the urea is hydrolysed to ammonia and C 0 2 by the urease present in these seeds. (Fig.).

Cancer research Other degradation pathways for C. have been found in microorganisms. In Streptococcus it is enzymatically hydrolysed to guanidine and Lhomoserine, while Pseudomonads hydrolyse it to hydroxyguanidine and homoserine. Because it is a structural analog of arginine, C. is a competitive inhibitor of the metabolic reactions of the latter. However, its toxicity to cattle and to C.-free plants grafted onto C.-containing plants cannot be explained by this competitive inhibition. C. was first isolated from the seeds of jack beans Canavalia ensiformis in 1929 by Kitawa and Tomiyama. Cancer research, scientific study of 1) the factors which lead to the formation of the various malignant tumors, 2) the continuous growth of the malignant tumors, and 3) of their inhibition by anti-cancer agents, in order to find effective methods of therapy. A tumor is an abnormal new tissue growth (neoplasm), which may start in any tissue and is not, or only partly, under the physiological control of the organism. A benign tumor generally grows slowly and does not leave the site of its formation to invade neighboring tissue. It may spontaneously regress and disappear; sometimes it is encapsulated by connective tissue. A cancer or malignant tumor may grow slowly or rapidly, but its tendency to grow is irreversible. It invades neighboring tissue and destroys it. It often, but not always, has the tendency to metastasize, that is, to shed cells or shreds of tissue which are carried by the blood or lymph into other parts of the body. Benign tumors may become malignant, so that in this case they represent an intermediate stage in the malignant transformation of normal cells. Chemical substances which induce cancer are called carcinogens. They include inorganic ions of various chemical elements and organic compounds, among them various synthetic aromatic and aliphatic compounds and some natural products. The aromatic carcinogens include 1) condensed polycyclic aromatic hydrocarbons, e.g. benzanthracene, 3,4-benzpyrene and methylcholanthrene, 2) aromatic amines, e.g. />dimethylaminoazobenzene, 2-acetamidofluorene and Narylhydroxylamines, 3) aminoazo dyes and diarylazo compounds, and 4) aminostilbenes and stilbene analogs of sex hormones. The aliphatic carcinogens include 1) alkylating agents (dialkylnitrosoamines, ethionenes, etc.) 2) lipophilic agents, detergents and substances which interfere with hydrogen bonds (chlorinated hydrocarbons, bile acids, certain phenols and urethane, etc.) and 3) naturally occurring carcinogens from plants and fungi (safrole and capsaicin from chili pepper, cycasin from Cycas, pyrrolizidene alkaloids from plants of the genus Senecio, the microbial products patulin, griseofulvin, penicillin G, anatoxins and actinomycin. Carcinogens are non-specific cell poisons which cause structural changes, and thus the loss of physiological activities of a large number of receptors for metabolic regulators and metabolic control points. The carcinogen is apparently ac-

68

Cantharidin cumulated in subcellular structures, covalently bound to biopolymers, and it denatures proteins (changes their biologically active tertiary structures) by interfering which the hydrogen bonds, hydrophobic interactions, etc. The affected cells always escape from the physiological control of the organism, and lose their differentiation - they become simpler, less specialized cells. Cells of this type respond to continuously available nutrients with continuous growth. Uncontrolled, unlimited growth is an essential characteristic of the cancer cell. The basic problem in understanding cancer is to understand the mechanisms by which growth, cell replication and differentiation, and, in multicellular organisms, the subordination of the individual cell to the whole organism, the interaction of its parts and their integration into the organism are regulated. The causes of dedifferentiation can only be deduced from a thorough understanding of the processes of differentiation. Carcinogenesis (development of cancer) has many different causes: chemical or physical factors, and oncogenic (tumor-inducing) viruses. A change in the structure of a gene is presumably always the actual mechanism of carcinogenesis. Since hormonal regulation is an important form of regulation in multicellular organisms, it is not surprising that hormones may play an important part in the development and growth of neoplasms. Cancer cells resemble embryonal cells in a number of ways. They preempt essential nutrients from other tissues in the organism. Many scientists believe the malignant transformation to be irreversible. However, changes in the gene structure do not always occur in just one direction. In the course of the search for anti-cancer agents, chemotherapeutics, antibiotics, plant alkaloids, and hormonal and immunological control mechanisms have been studied. As yet, chemotherapy of cancer has not had resounding general success, partly because the biochemical differences between tumor and normal cells are less marked than the biochemical differences between pathogenic bacteria or parasites and the host cells which are exploited by chemotherapy. Alkaloids from the periwinkle, Vinca rosea have been used with a rather high rate of success against various types of leukemia. The use of antimetabolites has been tried, but it has been found that the neoplasms often inactivate the inhibitor more rapidly than the normal cells do. Candlclne, N,N,N-trimethyltyramiae: a biogenic amine found especially in grasses and cacti. Cane sugar: see Sucrose. Cantharidin: toxic agent produced by the beetle Cantharis vesicatoria (Spanish fly, blistering beetle), which is native to Southern and Central Eu-

Cantharidin

Caoutchouc rope M r 196.21, m.p. 218 °C. It is biosynthesized from mevalonic acid. Its use as an aphrodisiac has caused fatalities. Caoutchouc: elastic, high-polymer hydrocarbons (elastomers) which become rubber on vulcanization. 1) Natural caoutchouc, (C 5 H 8 ) n , is the most important representative of the polyterpenes. It is a mixture of polyisoprenes with varying molecular weights, which usually range from 300000 to 700000. X-ray and IR data have shown that its double bonds are cis oriented, while in the caoutchouc-like polyterpenes, gutta and balata, they are trans (see Polyterpenes, Fig.). As an unsaturated hydrocarbon, C. reacts easily with oxidizing agents. Complete ozonolysis yields up to 90% levulinic acid derivatives. C. is biosynthesized from mevalonic acid and this process can be carried out with isolated enzymes. Occurrence. There are hundreds of species of plants which contain C. in their latex, but it can only be obtained on a large scale from a few representatives of the spurge (Euphorbiaceae), dogbane (Apocyznaceae) and milkweed (Asclepiadaceae) families. The following types of C. are utilized: C. from the rubber tree Hevea brasiliensis, which is native to the Amazon region but is also grown in India and Indonesia, and which is most important with regard to the amounts produced. Guayule C. is similar to that from Hevea and differs from it only in its lower molecular weight. It is obtained from Parthenium argentatum in Mexico and California. Kok-saghys is a C. from the roots of the dandelion species Taraxacum kok-saghys, which has been cultivated since 1935 in the USSR. C. is harvested by making cuts in the bark of the trees, without injuring the cambium. Latex flows from the cut latex channels, in amounts of 40 to 80 ml per tree and harvest. On the average, latex contains 25 to 35% dry weight of C., 60 to 75% water, 2% protein, 2% resin, 1.5% sugar and 1% ash. The C. is in the form of fine droplets which are prevented from coagulating by protein. It is precipitated with dilute acid or sodium fluorosilicate, pressed out in sheets to remove the water, and sometimes smoked.

Capsorubin

69

Capon test: see Androgens. n-Caproic acid, decaaoic acid: a fatty acid, CH 3 -(CH 2 ) 8 -COOH. M r 172.3, m.p. 31.3 °C, b.p. 268 °C. Occurs in milk fat (2%), coconut oil ( < 1 %) and various other seed and essential oils. n-Capronlc acid, hexanoic acid: CH 3 -(CH 2 )4COOH, a fatty acid. M, 116.16, m.p. - 1 . 5 °C, b.p. 207 °C. Found in milk fats (2%) and in small amounts in coconut oil and other palm oils. It also occurs in essential oils from plants and plant fats. n-Capryllc acid, octanoic acid: CH 3 -(CH 2 )6COOH, a fatty acid. Mt 144.2, m.p. 16.5 °C, b.p. 237 °C. It is found in various glycerides, e.g. 1 to 2% in milk fat, and as 6 to 8% of the coconut oil fats. It is also found in other plant fats. Capsaicin: a pungent principle in the fruits of some peppers (Capsicum). Mr 305.42, m.p. 64 to 65 °C. The aromatic part is biosynthesized from phenylalanine. It is occasionally used as a counter-irritant.

Capsaicin Capsanthin: a carotenoid pigment isolated from paprika (Capsicum anauum). Mt 584.85, m.p. 176 °C, [ a ] c d = +36° (chloroform). It is characterized by a terminal five-membered ring. The secondary hydroxyl groups have the R configuration on C 3 and the S-configuration on C 3'. C. is the main red pigment in paprika, where it is accompanied by Capsorubin (see), cryptocapsin and capsanthin-5,6-epoxide. Ripe fruits contain 9.6 mg C. per 100 g fresh weight. C. was isolated and its structure was elucidated by Zechmeister and von Cholnoky. Capsld: see Viral coat protein. Capsorubin: a carotenoid pigment found in paprika (Capsicum annuurri). Mr 600.85, m.p. 218 °C. C. contains two identical cyclopentanol rings. The hydroxyl groups have the S-configuration on OH

2) Synthetic C. is similar in structure and properties to natural C. It is produced by technical polymerization of unsaturated monomers. The most important starting material is butadiene. Capon-comb unit: see Androgens.

C-atom 3 and the R-configuration on C-atom 3'. C. is usually present in paprika fruits in the esterified form. Ripe fruits contain about 1.5 mg per 100 g fresh weight. C. was isolated in 1943 by Zechmeister.

C a 2 + pump

70

6-Carbamoylthreonyl purine nucleoside OH

Capsorubin Ca 2 + pump: see Membrane transport. Caran: see Monoterpenes, Fig. Carbamate: see Carbamoyl phosphate. Carbamic acid: see Carbamoyl phosphate. Carbamide: see Urea. 6-Carbamoylglycyl purine nucleoside: see 6Carbamoylthreonyl purine nucleoside. Carbamoyl phosphate: H 2 N - C 0 0 ~ P 0 3 H 2 , an energy-rich phosphorylated carbamate which is an important metabolic intermediate. Carbamic acid, NH2COOH, is unstable in free form. Carbamate removed hydrolytically from carbamyl compounds, for example ureidopropionic acid (see Pyrimidine degradation), decomposes immediately into C 0 2 and NH 3 . C. p. is also a relatively labile compound. It is the starting point for the biosynthesis of arginine and urea (see Urea cycle), and of pyrimidines via the orotic acid pathway (see Pyrimidine biosynthesis).

Nucleic •cids

Pyrimidines

Carbamoyl phosphate

•-L-Arginine -

Metabolism: C. p. is synthesized de novo or generated by phosphorolysis of ureido compounds. De novo synthesis involves three different enzymes: 1) carbamoyl-phosphate synthetase (ammonia) (EC 6.3.4.16), which is found in the vertebrate liver, catalyses the formation of C. p. from ammonium hydrogencarbonate at the expense of 2 ATP. It requires JV-acetyl-L-glutamate (AGA) as cofactor: ,Mg2 + N H 4 H C 0 3 + 2 ATP H 2 N-COO-PO 3 H 2 AGA + 2 ADP + PJ. The reaction is irreversible. AGA is required for the active enzyme conformation, not for the activation of the carbon dioxide. The enzyme is localized in the mitochondria and is used for the synthesis of arginine and urea. 2) Carbamoyl-phosphate synthetase (glutaminehydrolysing) (EC 6.3.5.5) requires the amide

group of L-glutamine as an N donor: H C 0 3 ~ ME2

+

+ L-glutamine + ATP + H 2 0 —-—> C. p. + L-glutamate + ADP. The overall reaction is irreversible because it involves an hydrolytic step. This enzyme is located in the cytoplasm and is used for pyrimidine synthesis. Free ammonia ions can replace the glutamine, but only at higher than physiological concentrations. The enzyme was first discovered in the cultivated mushroom, but it occurs widely. 3) Carbamate kinase (carbamyl phosphokinase) (EC 2.7.2.2) catalyses the phosphorylation of carbamate by ATP: NH 2 -COC>- + ATP ^ N H 2 - C 0 0 - P 0 3 H 2 + ADP. It is found in various microorganisms (Streptococcus, Neurospora, etc.). Thermodynamically, the formation of ATP is favored, so the enzyme is thought to be used for the generation of ATP rather than the synthesis of C. p. There are various microorganisms which catalyse the phosphorolysis of allantoin and citrulline, forming C. p. which can then be utilized for ATP formation. Phosphorolysis of citrulline produces ornithine and C. p. In the course of allantoin fermentation by Streptococcus allantoicus and Arthrobacter allantoicus, carbamyloxamic acid (oxaluric acid) is formed and then phosphorolysed: N H r C O - N H - C O - C O O H + Pj (Oxaluric acid) NH 2 CO-COOH + NH 2 -COO-PO 3 H 2 (Oxamic acid) The formation of ATP from C. p. obtained from citrulline or allantoin is an example of Substrate phosphorylation (see). All transcarbamylation reactions require C. p. as donor of the carbamyl group, i.e. transcarbamylation directly from an ureido compound is not possible. C. p. is probably also the carbamyl donor in the biosynthesis of O- and JV-carbamyl derivatives, such as Albizziin (see). C. p. is a metabolically active form of ammonia used as the starting material for the synthesis of other nitrogen compounds. Carbamoyl-phosphate synthetase: see Carbamoyl phosphate. 6-Carbamoylthreonyl purine nucleoside, N (nebularin-6-ylcarbamoyl)-threomae: one of the rare nucleic acid bases. It has so far been de-

71

Carbamyl Phosphokinase tected in six specific transfer RNAs. The analogous compound 6-carbamoylglycyl purine nucleoside has been isolated from yeast tRNA. See Fig. HN—C—R

R- — N H — C H - C O O H

6-Carbamoylglycyl purine nucleoside

CH 3 HCOH R=—NH—CH—COOH

6-Carbamoylthreonyl p u r i n e nucleoside

Carbohydrate metabolism Under certain conditions, carbohydrates may be resynthesized from the degradation products of C.m. The starting materials for this Gluconeogenesis (see) are lactate and glucogenic amino acids. There are three different phases of C.m., 1. mobilization, in which poly-, oligo- and disaccharides are cleaved and phosphorylated to hexose phosphates, particularly to glucose 6-phosphate. In digestion, the cleavage is achieved by hydrolysis. 2. Interconversions: the mutual transformations of monosaccharides involve the following types of reactions: a) epimerization, the reversal of the steric arrangement on a C-atom by epimerases (e.g. in galactose metabolism); b) isomerization, the reversible transformation by isomerases of aldoses into ketoses (e.g. glyceraldehyde 3-phosphate into dihydroxyacetone phosphate); c) transfer of C 3 (see Transaldolation) and C 2 (see Transketolation) fragments in the form of a dihydroxyacetone phosphate residue or "active glycolaldehyde" ; d) oxidation of an aldose to an acid and its subsequent decarboxylation. In this second phase of C.m., the intermediary products of the first phase are incompletely degraded. The main products are triose phosphates. In these

Glycogen

Glycogen

CO,

Glycolysis 25%

Glucose 6-phosphate

Pentose phosphate cycle 2%

55% I Glucose

CO,

Glycolysis 19%

2%T Glucose 6-phoshate

Pentose phosphate cycle

TZ—>C02

73%| Y Glucose

Fig. 1. Turnover of glucose 6-phosphate under normal (left) and disease (diabetes meiiitus) conditions (right), as percent of total.

Carbamyl phosphoklnase: see carbamoyl phosphate. Carbohydrases: see Glycosidases. Carbohydrate metabolism: the constant formation, transformation and degradation of the carbohydrates in the organism. The most important reactions in C.m. are 1. Interconversions of the polymeric storage forms (glycogen and starch) and the monomelic transport and substrate form (glucose), 2. reactions of carbohydrate degradation and interconversion, and 3. reactions for the synthesis of glucose from noncarbohydrate substances (glucogenic amino acids, fats). Glucose 6-phosphate has a central position in the entire C.m. Aside from the minor pathways (see Glucuronate pathway; Entner-Doudoroff pathway; Phosphoketolase pathway) there are four main pathways for glucose 6-phosphate: 1. Glycolysis (see), 2. Glycogen synthesis (see Glycogen metabolism), 3. Pentose phosphate cycle (see), and 4. enzymatic hydrolysis to free glucose. The effectiveness of these pathways depends, in the animal organism, on the function of the tissue in question. A change in the activity of the tissue, as in illness, has a large effect on its C.m. (Fig. 1).

processes, about a third of the total free energy potential is released and partly used for the synthesis of the energy-rich adenosine triphosphate. 3. Amphibolic reaction chains. The degradation products of C.m. flow into the general metabolism as pyruvate and acetyl-CoA (Fig. 2). Biosyntheses in C.m. Plants are the main producers of carbohydrates in nature. In photosynthesis, a series of enzymatic reactions produces phosphorylated monosaccharide derivatives, which can be hydrolysed to free sugars or converted to Nucleoside diphosphate sugars (see). In the biosynthesis of polysaccharides like starch, glycogen and cellulose, the monosaccharide units are activated by the formation of nucleotide derivatives, and they are transferred in this form by the appropriate enzymes to the non-activated, growing end of the polysaccharide chain. The nucleotide is split off in the coupling reaction. The biosynthesis depends on the presence of a highly polymerized starter molecule. Uridine diphosphate glucose (UDPG) and adenosine diphosphate glucose (ADPG) play the most important role in the biosynthesis of the oligosaccharide sucrose and the polysaccharide starch (Fig. 3).

72

Carbohydrate metabolism

Carbohydrate metabolism

Oligosaccharides

1

ATP A D P Hexoses

T T P,-

1

N T P P,

Hexose phosphates (Glucose • 6-phosphate)

AA

Polysaccharides

H2O

Interconversions: Isomerases Epimerases Decarboxylases Transaldolases Transketolases

Entner Doudoroffpathway

Glycolysis

Pentose phosphate cycle

| Pentose

(atp)^ »>| Triose

Glucuronate pathway

phosphates

Phosphoketolase pathway

phosphates

|

1 J Pyruvate

Other & products / £ y s Lipids Amino g v acids

Fig. 2. The three phases of carbohydrate metabolism. NTP is nucleotide triphosphate.

Guanosine diphosphate glucose can also serve as a precursor for the synthesis of the 1,4-glucosyl chain of cellulose. Fructose 6-phosphate + UDP-glucose

? UDP+ Sucrose

Fructose + | UDP-glucose |

^

phosphate

: U D P + I Sucrose I

UTP • Glucose ATP P P P ';

I

1-phosphote

\]|

"Starter molecule" I e - 9- s t a r c h o r l.i-oligosacca-

|ADP~glucose|

1 Vide)

i • ADP +

Starch

Fig. 3. Reactions of starch and sucrose synthesis.

The oligo- and polysaccharides are degraded in organisms by specific enzymatic hydrolysis (hydrolases) or phosphorolysis (phosphorylases). Regulation ofC.m. is characterized by tight interactions between the individual metabolic pathways mediated by the metabolic products. Glycolysis is controlled by allosteric regulation of the enzymes phosphofructokinase and pyruvate kinase. The controlling factor is the ATP/ADP ratio. Increased production of ATP in the respiratory chain leads to inhibition of phosphofructokinase, while an increased consumption of ATP stimulates the glycolytic turnover of glucose 6phosphate via the increased level of ADP, which stimulates the phosphofructokinase. This in turn causes increased ATP formation by substrate chain phosphorylation. Another regulatory par-

73

Carbohydrates ameter of this system is the amount of available oxygen (Pasteur effect). Glucose 6-phosphate activates glycogen synthetase. Therefore, a high concentration of glucose 6-phosphate leads to increased production of the storage carbohydrate glycogen. Intermediary products of the pentose phosphate cycle inhibit the first enzyme of glycolysis, phosphoglucose isomerase, which reduces the amount of glycolytic degradation. The N A D P H produced in larger amounts by an active pentose phosphate cycle can be used in fatty acid synthesis. Excessive fat synthesis is prevented by the inhibitory action of long-chain acyl-coenzyme A compounds on the key enzyme of the pentose phosphate cycle, glucose-6-phosphate dehydrogenase. Carbohydrates: a large class of natural substances, structurally the polyhydroxycarbonyl compounds and their derivatives. In general they correspond to the composition (C) n (H 2 0) n . They were originally characterized as hydrated forms of carbon and were named C. in 1844 by K. Schmidt. The name has been retained, although it is inaccurate from a chemical point of view and now compounds are included in the C. which have other elemental compositions, e.g. the aldonic acids, uronic acids and deoxysugars, or

Carbohydrates which contain additional elements, e.g. aminosugars, mucopolysaccharides. Mono- and oligosaccharides are also called sugars. Individual C. have trivial names or systematic names derived from them which end in -ose, e.g. glucose, fructose. The IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature published tentative rules for carbohydrate nomenclature in 1969. C. are present in every plant or animal cell and make up the largest portion, in terms of mass, of organic compounds present on earth. They are formed in plants in the course of assimilation processes. Together with fats and proteins, they are the organic nutrients for humans and animals. The C. are subdivided on the basis of their molecular size. 1. Monosaccharides (simple C.) cannot be further hydrolysed into simpler types of C. They can be regarded as the primary oxidation products of aliphatic polyalcohols, usually with unbranched carbon chains. If the oxidation occurs at the terminal primary alcohol group, the resulting polyhydroxy-aldehyde is called an aldose (Fig. 1). Oxidation of a secondary hydroxyl, usually the C-atom 2, produces a polyhydroxyketone, or ketose.

,

y 9

A

D-(+)-Xylose


. max 520 to 535, and 3) the x or Soret band, X m a x 400 to 415 nm. Hemosiderin: an iron storage protein of the mammalian organism, functionally related to Ferritin (see).H. is deposited in the liver and spleen (hemosiderosis), particularly in diseases associated with increased blood destruction, such as pernicious anemia, or with increased iron resorption (hemochromatosis), or even in hemorrhages. Most of the deposits are located in the liver, which may contain up to 50 g H., compared to the normal content of 120 to 300 mg H. H. from horse spleen consists of 26 to 34% iron(III), and up to 35% protein (aposiderin). The rest is made u p of octasubstituted porphyrin, mucopolysaccharides and fatty acid esters. Heparin: an acid mucopolysaccharide from animal tissues which prevents blood clotting. M t about 16000. H. consists of equal amounts of Dglucosamine and D-glucuronic acid, a-l,4-glycosidically linked, and contains O- and ^-sulfate residues. It is dextrorotatory. Due to its acid character it can form salts; the effective protein component in blood is called heparin complement. H. prevents the clotting of blood by preventing the conversion of prothrombin to thrombin and of fibrinogen to fibrin. Clinically, it is applied parenteral^ for the treatment of thrombosis, phlebitis and embolism. CHjOH

NHSO3H

C00H

n R=H or SOjH

Heparin

Heparitln sulfate, heparan s u l f a t e : a monosulfate ester of an acetylated (N-acetyl) heparin. As isolatedfrom animal tissues (liver), H.s. is probably a mixture of mucopolysaccharides with varying degrees of sulfatation or amino group acetylation. Hepes: see A f -2-Hydroxyethylpiperazine-A'-2ethanesulfonic acid. Heptoses: monosaccharides containing 7 C atoms. The 7-phosphates of D-mannoheptulose and D-sedoheptulose are important in carbohydrate metabolism.

Heterophagy Heroin, diacetylmorphine, diamorphine: one of the most dangerous narcotics, m.p. 173°C, [a] D 2 5 —166° (methanol). H. is not very stable and decomposes in boiling water. It is synthesized by acetylation of the two hydroxyl groups of morphine with acetyl chloride. This increases the analgesic effect by a factor of six. Due to the extreme danger of addiction, the therapeutic use of H. is forbidden in most countries. In New York alone, the number of heroin addicts is estimated to be 300000. CH3COO.

CH3COO Heroin

Hershberg test: see Anabolic steroids. Hesperldin: a flavone glycoside, constituting 8 % of the dry weight of orange peel (see Vitamin P)O-Glucose I Rhamnose OH Hesperidin

Heteroauxln: see Auxins Heterogeneity: in proteins, a term for differences in the structure of a species of protein, which cause no change in the biological activity. H. can be either genetically controlled or can arise through partial chemical or enzymatic modification. Chemical changes include, for example, phosphorylation of serine and threonine residues in casein, or the covalent attachment of carbohydrate chains in the glycoproteins. The most common cause of enzymatic modification is limited proteolysis, which can produce artefacts. The pseudoisoenzymes p- (single chain) and a (double chain) trypsin and the erroneously high number of subunits, e.g. of yeast hexokinase (4 instead of 2), are examples of H. The latter are caused during the isolation procedure by proteinase contaminants which are co-purified with the desired protein. Microheterogeneities are slight differences in the primary structure, which are limited to a few unimportant residues in the peptide chain, or, in glycoproteins, slight differences in the number, length and composition of the carbohydrate chains. Heteroglycans: polysaccharides composed of two or more different carbohydrate residues, for example, pectins, plant mucilages, plant gums and mucopolysaccharides. Heterophagy: see Intracellular digestion.

203

Heteropolar bond

Heteropolar bond: see Noncovalent bonds. Heteropolypeptldes: see Proteinoids. Heteroslde: a compound of one or several carbohydrate residues and a component belonging to a different class of substance, the aglycon or genin. E.g. glycosides are H. Herotrophy, heterotrophic

nutrition:

a nutri-

tional dependence on organic compounds. In carbon heterotrophy, organic carbon compounds serve as sources of carbon and energy for the synthesis of body substituents and ATP. The degree of H. varies widely among the various heterotrophic organisms (all animals, including humans, and most microorganisms), and may include a dependence on externally supplied essential amino acids, fatty acids and vitamins. Auxotrophic mutants (see) have special nutritional requirements. Parasitism (see), Saprophytism (see) and Symbiosis (see) are special forms of heterotrophic feeding. The terms autotrophy and heterotrophy do not suffice to describe the widely varying forms of microbial nutrition, since here the nature of the carbon source and the energy source as well as the chemical nature of the reducing agent used for reductive syntheses must be taken into account. The converse of H. is Autotrophy (see). HETPP: see Active acetaldehyde. Hexestrol: meso-hexestrol, a synthetic compound with estrogenic activity. It is not a steroid, but is used therapeutically in the same way as natural estrogens.

Hexestrol

Hexitols: sugar alcohols with 6 C-atoms. Of the 10 possible isomers, D-sorbitol, dulcitol, D-mannitol, iditol and allitol are found in nature. Hexoklnase: see Kinases Hexosans: high-molecular-weight plant polysaccharides, belonging to the group of homoglycans, and composed of hexoses. Examples are glucans, fructans, mannans and galactans.

Hill plot rally occurring monosaccharides D-fructose and L-sorbose. Hexuloses: see Hexoses.

Hlbbert's ketones: see Lignin. High energy bonds, energy-rich bonds: chemi-

cal bonds which release more than 25 kJ/mol on hydrolysis. They are usually esters (enol, thio and phosphate esters), acid anhydrides, or amidine phosphates. In biological systems, the energy released is used to transfer the hydrolysed residue to other metabolic compounds (group transfer). The H.e.b. are symbolized by ~ instead of the usual hyphen between groups (e.g. CHjCO ~ SCoA). The free energy of hydrolysis of H.e.b. is at the same time a measure of the potential for group transfer. The table summarizes the standard values of the free energy of hydrolysis for some important compounds. See Energy-rich phosphates. Table. AGC of hydrolysis. Compound

Creatine phosphate 42.7 Phospho cnolpyruvate 53.2 Acetylcoenzyme A 34.3 Aminoacyl-tRNA 29.0 A T P - ADP + Pj 30.5 ATP— AMP + P ~ P 36.0 P ~ P - 2Pi 28.0

(kcal/mol) 10.2 12.7 8.2 7.0 7.3 8.6 6.7

High-yielding strains: see Production strains. Hill plot: a graphic method for the determination of the degree of cooperativity of an enzyme (see Cooperativity). The plot of log K s /(l-y s ) versus log a is a curve with a slope of 1 for large or small a and a finite energy of interaction between the substrate-binding sites. ys is the saturation function, that is, the fraction of the enzyme in the enzyme-substrate complex, and a = S/ Km. For the values of a usually obtained experimentally, an approximate straight line is obtained with the maximal slope h (Fig.), the Hill coefficient. This serves as a measure of the cooperativity, and is not usually identical to the number n of subunits of an enzyme, but is only a minimal estimate of n.

Hexose monophosphate pathway: see Pentose

phosphate cycle. Hexoses: aldoses containing 6 C-atoms; one of the important groups of monosaccharides (see Carbohydrates). All possible stereoisomeric aldohexoses (there are four asymmetric C atoms) have been isolated or synthesized. D-Glucose, D-mannose, D-galactose and L- and D-talose are widespread in nature, both as free sugars and in bound form. Some of the phosphorylated H. are particularly important. The two 6-deoxy-sugars L-rhamnose and L-fucose are also H. The ketohexoses corresponding to the aldohexoses are called hextiloses. They include the natu-

AG 0 (kJ/ mol)

Hill-Plot

204

Hill reaction

If the energy of interaction between the substrate-binding sites is infinite, the Hill plot degenerates into a straight line with a slope equal to the number of subunits. The segment of the ordinate AB, constructed as shown in the Fig., can be used to determine the total energy of interaction A G w between substrate or effector-binding sites: A D w = 2.303 « r - A B . Here R is the general gas constant and Tis the absolute temperature. The saturation functions for effectors can also be determined. y E is the fraction of the enzyme in the enzyme-effector complex. For enzymes which are in equilibrium with their enzyme-substrate complexes, the saturation function can be replaced by the kinetic saturation v/ Vm, so that log v(Vm v) can be plotted against log a. Correspondingly, for an effector, log (v - vQ)/( V - v) must be plotted against log a. v is the measured rate of reaction at constant S, v0 is the rate in the absence of effector at the same S, and Kis the rate at a saturating concentration of effector for the chosen S; S is the substrate concentration. Hill reaction: light-dependent production of oxygen by the photosynthetic system in the presence of an artificial oxidizing agent (electron acceptor). R. Hill first observed this reaction in illuminated isolated chloroplasts, in the absence of C 0 2 , and using iron(III) oxalate as oxidizing agent. Iron(III) oxalate (Fe 3+ is reduced to Fe 2+ in the reaction) can be replaced by potassium ferricyanide, quinone and other compounds (Hill reagents). Spinach chloroplasts catalyse the following H.: 4K 3 Fe(CN) 6 + 2HzO + 4K + — 4K 4 Fe(CN) 6 + 4 H + + 0 2 . The "natural" Hill reaction is the photolysis of water, and the "natural" Hill reagent is oxidized NADP. Hlppurlc acid: C 6 H 5 - C O - N H - C H r C O O H , the JV-benzoyl derivative of glycine. Mammalian herbivores detoxify benzoic acid by converting it to H.a. His: abb. for L-Histidine. Histamine: ¡)-imidazol-4(5)ethylamine, a biogenic amine. M r 114.14. H. is formed by enzy-

L Histidine matic decarboxylation of L-histidine. It stimulates the glands in the fundus of the stomach to secrete digestive juices, dilates the blood capillaries (important for increasing blood flow and decreasing blood pressure), increases the permeability (urtication and reddening after local application of histamine), and causes contraction of the smooth muscles of the digestive tract, the uterus and the bronchia (in bronchial asthma). H. is catabolized by diamine oxidases and aldehyde oxidases to imidazolylacetic acid. H. is widely distributed in the plant and animal kingdoms, occurring for example in stinging nettles, ergot, bee venom and the salivary secretions of biting insects. As a tissue hormone, H. is present in the liver, lungs, spleen, striated muscles, mucus membranes of stomach and intestine, and it is stored with heparin in mast cells. The amounts found in tissue are on the order of (ig/g fresh weight. CH2—ch2—nh2 IN

H

Histamine

L-Hlstldlne, abb. His: imidazolylalanine, a half-essential amino acid used in protein synthesis. Mt 155.2, m.p. 277° C (d.), [ a ] g = + 11.8 (c = 2 in 5N HC1) or -38 • 5 (c = 2, water). The proportion of His in hemoglobin is especially high. It is also a component of carnosine and anserine. His is part of the active centers of many enzymes, and is an important buffer in the physiological pH range. His is weakly glucoplastic. In the absence of dietary His, the adult animal can maintain its nitrogen balance for a short time, but it is absolutely necessary for the growing animal. The imidazole ring cannot be synthesized by mammals. His is formed via imidazole glycerophosphate in the last part (Fig. 1) of the ATP-imidazole cycle (Fig. 2).

Imidazole nucleus

-N R HCOH I

R

I

Imidazole glycerol CH2 phosphate dehydratase

HCOH

5>

^

CH20® Imidazole glycerol phosphate

Imidazole acetol phosphate transaminase

7

c=o

1 CHjO®

^ a-KG

Imidazole acetol phosphate

R Histidinol phosphate phosphatase |

I~(p)

Glu

R 2

hc-nh2 ch2OH

Histidinol dehydrogenase 2NAD

L-Histidinol

2NADH

I

CH2 *HC—NH2 COOH L-Histidine

Fig. 1. Terminal reactions in histidine biosynthesis

R I

CH,

^ I

HC-NH CH20® L-Histidinol phosphate

205

Histones L-Histidine A

Holarrhena alkaloids

De novo purine synthesis

I

Imidazoleglycerophosphate I

i

| Aminoimidazole carboxamidoribotide

[X]

Inosinic

L-Glu-NHj--^

\

Phosphoribulosyl f o r m i m i n o a m i n o imidazolecarboxamidoribotide [Amadori

acid

Adenylosuccinate

rearrangement]^

Phosphoribosyl formiminoaminoimidazolecarboxamidoribotide [Cyclohydrolase] Phosphoribosyl-AMP PPi

Fig. 2. ATP-imidazole

cycle

Phosphoribosyl-ATP

The starting compound in the ATP-imidazole cycle is 5-aminoimidazole-4-carboxamidoribotide, abb. AICAR (see Purine biosynthesis). The intermediate products are inosinic acid, AMP, ATP, phosphoribosyl-ATP, phosphoribosyl-AMP, phosphoribosyl-formimino-aminoimidazole carboxamidoribotide. The latter compound is synthesized by an Amadori rearrangement, which is relatively rare in cellular metabolism. It is significant that ATP is the substrate. Only the C-2 atom and the N-l atom of the purine ring (Fig. 3) are incorporated into the His molecule. His is catabolized by histidase to urocanic acid (imidazoleacrylic acid) and then, via imidazolonepropionic acid and formiminoglutamic acid, to glutamic acid. The formimino group is used for the synthesis of Active one-carbon units (see). His is used in the treatment of allergies and anemias. Phosphoribosylpyrophosphate

ATP Phosphoribosyl-PP

classes: 1. very lysine-rich (27% Lys) 212 amino acids, Afr 21000; 2. moderately lysine-rich (16% Lys), 125 amino acids, M t 13775; 3. leucine-rich (11% Leu, 12% Lys), 140 amino acids, M r 15000; 4. arginine-rich (15% Arg), 102 amino acids, A/r 11300; 5. arginine-and glutamate-rich (13% Arg, 12% Glu), 101 amino acids, MT 11200. Hlstoplne: see D-Octopine. hMG: abb. for human menopausal gonadotropin. HMTPP: see Thiamine pyrophosphate. Holarrhena alkaloids, kurchialkaioids: a group of steroid alkaloids which are the characteristic active substances in plants of the dogbane (Apocynaceae) genus Holarrhena. The representatives so far isolated (about 50) are formal derivatives of the hydrocarbon pregnane (see Steroids) which is substituted in the 3 and 20 positions with amino or methylamino groups, e.g. holarrhimine and conessine (Fig.) In the most widespread com-

HC=C—CH 2 -C-COOH I I L-Glutamate P u r i n e - N , — - N H - ^ G l u t a m i n e (Amide N) (ATP) CH t Purine C2(ATP) Fig. 3. Scheme of histidine biosynthesis Histones: a group of simple, basic proteins of the cell nucleus. They are not tissue specific, and occur in all eukaryotic organisms. They have low molecular weights. H. form reversible complexes with DNA, called nucleohistones. H. are the most important component of the chromatin-associated proteins and are thought to act less as specific regulators of gene expression than as nonspecific repressors of transcription, which, by changing the conformation of the chromosomes, limit the availability of the DNA. The primary structures of the H. are for the most part known. They are divided into the following 5 main

pound, conessine, and its 12(}-hydroxy derivative holarrhenine, the 20 amino group is bound to Catom IB to form a pyrrolidine ring. Conessine is important as a starting material for the synthesis of aldosterone. H.a. have blood-pressure-reducing, curare-like, diuretic and narcotic properties. The alkaloid-rich bark of the shrub Holarrhena antidyserenterica is used for treating dysentery in

206

Holarrhenine

India. The H.a. are biosynthesized via cholesterol and pregnenolone. Holarrhenine: see Holarrhena alkaloids. Holarrhlmlne: see Holarrhena alkaloids. Holoenzyme: see Coenzyme. Holosldes: compounds consisting of glycosidically linked sugar residues, e.g. oligo- and polysaccharides. Holothurlnes: a group of highly toxic compounds from sea cucumbers (Holothurioidea). H. are triterpene saponins (see Saponins), which contain holothurinogenins as aglycons and Dglucose, D-xylose, 3(0)-methylglucose and Dquinose as sugars. An important representative is holothurine A from the sea cucumber Actinopyga agassizi, which also contains bound sulfuric acid, and which has 22,25-oxidoholothurinogenin as aglycon. Holothurinogenins: see Holothurines. Homoarginine, abb. Har: a higher homologue of arginine with an additional methylene group in the side chain. Homocysteine, abb. Hey: a higher homologue of cysteine with an additional methylene group in the side chain. Homoglycans: straight or branched chain polysaccharides containing only one kind of monosaccharide residue. H. are widespread in the vegetable kingdom. They include arabans, xylans, glucans, fructans, mannans, galactans, the starch

Homologous proteins components amylose and amylopectin, cellulose and glycogen. Homologous proteins: proteins which have arisen through divergent evolution from a common ancestor. They usually have very similar primary and tertiary structures. Examples are the cytochromes, hemoglobin and myoglobin, the ferredoxins (non-heme iron proteins), fibrin peptides, immunoglobulins, peptide hormones (e.g. insulin and hypophyseal hormones), snake venom toxins and enzymes like the serine proteases of the pancreas (trypsin, chymotrypsin, elastase) or the blood-clotting enzymes (e.g. plasmin, thrombin) and lactate dehydrogenase. As examples of homologies in primary structure, the partial sequences around the catalytically important acid residues of the serine proteases are given in the table. A comparison of the primary structures and the location of the disulfide bridges of trypsin, chymotrypsin, and elastase is shown in Fig. 1. Fig. 2 shows that the structural homologies are also reflected in the conformation of the polypeptide chains, taking chymotrypsin and elastase as an example. Although only about 40% of the amino acids of the two proteins are homologous, that is, identical, their spatial folding is similar. NH 2 and COOH indicate the beginning and end, respectively, of the chain. Positions 57, 102 and 195 are occupied by the important amino acids histidine, aspartic acid and serine.

Table. Partial sequences of homologous proteolytic (A) and esterolytic (B) enzymes from the region about the active serine residue*. Enzyme

Sequence

ATrypsin (beef, sheep, pig, dogfish, shark, shrimp) Chymotrypsin A and B (beef) Elastase (pig) Thrombin (beef) "Trypsin" (Streptomyces griseus) B Acetylcholinester-

Asp

Ser

Cys

Glu

Gly

Asp

Ser*

Gly

Gly

Pro

Ser

Ser

Cys

Met

Gly

Asp

Ser*

Gly

Gly

Leu

Ser Asp Asp

Gly Ala Thr

Cys Cys Cys

Glu Glu Glu

Gly Gly Gly

Asp Asp Asp

Ser* Ser* Ser*

Gly Gly Gly

Gly Gly Gly

Pro Pro Pro

Phe

Gly

Glu

Ser*

Ser

Glu

Gly

Gly

Glu

Ser*

Ala

Gly

Gly

Gly

Glu

Ser*

Ala

Gly

Gly

Ser*

Gly

His

Ser*

Ala

Ala

Pseudocholinesterase (horse) Liver esterase (pig, horse, sheep, chicken) Pancreatic lipase (Pig) Alkaline phosphatase (Escherichia coli)

Leu Asp

Tyr

Val

Thr

Asp

Ser

Homopolymer

Hormones

207 g - Chymotrypsin

Odifferent amino acids

Trypsin

• i d e n t i c a l amino a c i d s

Fig. 1. Comparison of the primary structures and the positions of the disulfide bridges of four serine proteases. A to G indicate the homologous disulfide bridges. Fig. 2. see page 208 Homopolymer: a polymer built up of identical monomeric units, for example, amylose and polyphenylalanine. In a narrower sense, H. are synthetic polynucleotides in which all the nucleotides contain the same base, for example polyadenylic acid, polyuridylic acid, polydeoxyadenylic acid. H. in the narrower sense are synthesized in vitro from nucleoside di- or triphosphates using the appropriate polymerases without a matrix. An oligonucleotide is needed as a primer. The diphosphates can be polymerized by polynucleotide phosphorylase. H. are usually singlestranded, but are occasionally double-stranded. H., especially poly(A) sequences, occur naturally in some of the RNA of eukaryotes (see Messenger RNA). Homosteroids: see Steroids Hordein: see Prolamin. Hordenine, anhaline: N, 7^-dimethyltyramine, one of the biogenic amines, widely distributed in nature, m.p. 117-118°C, b.p. 173-174°C. As a derivative of phenylethylamine, H. is one of the amines which increase blood pressure, but it has a low physiological activity.

Hormones: organic compounds in the plant and animal kingdoms. H. are synthesized in cells and glands specialized for this function. Very low concentrations of H. usually produce large metabolic responses in another tissue of the same organism, or in another organism. As a rule, they are not species-specific. Together with the nervous system, they serve to transmit information between cells. In contrast to the nervous system, however, the endocrine system cannot store information. Phylogenetically, the H. and nervous systems arose simultaneously, as is shown by acetylcholine, which stimulates unicellular organisms and serves as a neurotransmitter in multicellular organisms, where it carries information across the synapses (nerve endings). The next step in development, which is realized in worms and arthropods, is neurosecretion. Here a nervous signal is transformed in one cell into a hormonal signal, a Neurohormone (see). Steroid and polypeptide hormones evolved in parallel with neurosecretion. A complete hormone system with neurosecretion, glandular hormones and a coordinating

Hormones

208

Hormones

Hormones

209

hormone center in the central nervous system is found only in the vertebrates. H. are classified, according to their chemical structures, into steroids (see Steroid hormones), amino acid derivatives (e.g. see Adrenalin), peptides (see Peptide hormones), proteins (see Protein hormones) or fatty acid derivatives (see Prostaglandins); according to where they are produced as glandular, tissue or neurohormones; and according to the organism producing them as phytohormones (plant hormones), invertebrate and vertebrate hormones. To understand the biological action of a H., one must remember that the effect of the H. on the regulation of molecular biological processes at the cellular level cannot be considered in isolation, and that a H., like every other chemical compound in the organism, is subject to metabolism. Every H. is synthesized in a specific way, may be stored, is secreted on demand and is transported in various ways (blood, lymph, intracellular fluids) as a first messenger from the site of synthesis to the site of action. It is bound in the target organ by a specific receptor on the target cell. The mechanism triggered by the interaction of the hormone and receptor may form cAMP via the adenylate cyclase system. The cAMP then acts as a second messenger to induce the hormone effect in the cell. Thereafter the H. is usually subject to rapid inactivation a n d / o r degradation and excretion. Biosynthesis, storage and secretion: There are two different types of specialized cells for the synthesis of H., those which synthesize the peptide and protein hormones, and those which synthesize steroid hormones. In the case of protein and peptide hormones, the cells are stimulated to protein synthesis by neurotransmitters, H., metabolic products or dietary substances. The H. or their precursors (for example proinsulin) synthesized on the ribosomes enter the Golgi apparatus of the cell and are packed there into small vesicles and stored in the cytosol. On demand, which is signaled by various stimuli (for example, an increase in blood sugar produces a glucose stimulus, which causes the B-cells in the islets of Langerhans of the pancreas to secrete insulin), the vesicles move to the cell membrane, where the H. is secreted into the blood stream. The enzymes required for steroid hormone biosynthesis must be synthesized or activated when the cell is stimulated; for example, corticotropin causes cholesterol to be converted into glucocorticoids in the adrenals. Steroid hormones are not stored, since the appropriate enzyme system can be rapidly activated and deactivated. Cells which produce steroid hormones are characterized by a large Golgi apparatus, much smooth endoplasmic reticulum, lipid droplets and lysosomes. H. are transported in free form or bound to specific or unspecific proteins. Oxytocin or vasopressin, for example, is bound to neurophysin, transported within the axon of a nerve from the hypothalamus to the posterior lobe of the hypophysis, and stored there. This binding is loose, non-covalent and easily dissociated. The same is true of H.

Hormones transport in the blood stream. For some hormones, the dissociation constants of the H.-transporter complex can be determined. The albumins carry somatotropin; transcortin transports steroid hormones from the site of synthesis to the target cell. The H. is partially inactivated by this binding, but also stabilized and protected against enzymatic attack. Any method for H. determination in the blood must take into account the fact that the H. is present in both bound and free forms. Mode of action: Due to the heterogeneity in the types of substances which are H., they have more than one mode of action. At the goal, the target cell, the H. is bound by a specific receptor (a protein macromolecule) which is either on the cell surface (peptide and protein hormones) or in the cytosol (steroid H.). In the absence of its receptor, the H. does not find its goal and has no effect. The H.-receptor interaction on the cell membrane can activate, via a modulator which has not yet been characterized, the adenylate cyclase on the inner surface of the cell membrane. This enzyme produces cAMP from ATP, and the cAMP, as a second messenger, specifically influences the cell metabolism by activation of protein a n d / o r histone kinases. An immediate reaction is a change in enzyme activities and cell permeability. A classical example of this type of H. effect is the mobilization of glycogen by glucagon and adrenalin. A slower reaction is the regulation of transcription by the histone kinases in the nucleus and of translation by the protein kinases, which act on the ribosomes. This is the way in which somatotropin, for example, stimulates general growth. The modulator, which communicates between the receptor and adenylate cyclase, can be affected by prostaglandins or cGMP. Steroid hormones are bound by a receptor in the cytosol. This H.-receptor complex is transported to the cell nucleus, where it reacts with a nuclear acceptor, which is released in the process from the chromatin. This process corresponds to gene activation, which induces RNA and protein biosynthesis. In insects, the action of the H. ecdysone can be observed morphologically as the formation of puffs in the giant chromosomes of the salivary glands. The enzymes synthesized as a result of these processes then effect changes in metabolism. The role of cAMP in the process has not been completely elucidated. Inactivation of the H.: The action of a H. can be stopped immediately in one of two ways. Either the second messenger (cAMP) is converted to 5'AMP by a specific phosphodiesterase, and thus cannot serve as a messenger, or the H. is enzymatically degraded, in which case one must presume that the enzymes are located near to the receptors. Peptide and protein H. are inactivated by proteolytic enzymes, catecholamines by monoamine oxidases, and steroid H. by reduction of the unsaturated ketone groups and of the A ring as well as by elimination of the side chain. After conversion to a glucuronide or sulfate in the liver, they are excreted by the kidneys into the urine. In every H. effect, two points must be kept in mind: 1. there is no such thing as an isolated H.

Hp

210

effect. H. have a definite, but not the dominant role in the regulation of intracellular metabolism a n d of total metabolism. 2. H. effects are usually recognizable in the form of a change in enzyme activity. Regulation of H. effects: Every h o r m o n e effect is subject to finely tuned regulation. The H. themselves, the metabolic products dependent on them and the nerve system interact in this feedback mechanism. In most cases the synthesis a n d secretion of the first h o r m o n e in a system is inhibited, in negative feedback, by the H. whose production it stimulates. A classic example of the coordination of various regulatory circuits is the system composed of hypothalamus (releasing hormones), anterior lobe of the hypophysis (hypophyseal H.) a n d target organ, in which there is feedback f r o m every level. Pathobiochemistry: D u e to the close coordination of the H. and nervous systems with metabolism, any disturbance in the synthesis, secretion or transport of H., or the lack of receptors, or a disturbance of H. catabolism is reflected by a disturbance of the entire metabolism. E.g. the lack of mineralocorticoids leads to a disruption of the mineral a n d water balance; too much or too little somatotropin, the growth hormone, produces gigantism or d w a r f i s m ; a n d disturbances in the thyroid h o r m o n e system (see Thyroxin and Triiodothyronin) upset the energy metabolism a n d are reflected in a hyper- or hypothyreosis. Sexual functions a n d the normal course of pregnancy can be severely disturbed when G o n a d o t r o p i n s (see) a n d G o n a d a l h o r m o n e s (see) are under- or overactive. Methods of determination: Qualitative a n d quantitative methods of H. determination which are specific a n d sensitive a n d can be carried out with a reasonable a m o u n t of effort are absolutely necessary for studies of the physiological a n d pathological effects of H. Since H. are present in biological fluids and organs only in n a n o g r a m (ng) quantities, a certain a m o u n t of analytical work cannot be avoided. The usual methods of determination are chemical, biological, immunological a n d radiochemical, or a combination of these. G a s chromatography is suitable for the detection of steroid H. a n d prostaglandins. An example of a biological method is the biological pregnancy test, in which the chorionic gonadotropins formed in the placenta and excreted in the urine are administered to a mouse, which is later examined for ripe follicles in the ovary. The same H. can also be detected immunologically in an immunological pregnancy test. At present, however, the most important determination m e t h o d s are combinations of two principles. The m e t h o d of choice is radioimmunological determination of H., which can be used for nearly all H., although it must be emphasized that the a m o u n t s of H. f o u n d by this method are often very different f r o m those f o u n d by biological methods. For radioimmunological H. determination, guinea pigs or rabbits are injected at particular intervals with the H. to be determined in as pure f o r m as possible, so that they will form antibodies against

Hyaluronic acid it. A preparation of the same H. is also labelled with radioactive 1 2 5 iodine. In the test itself, the ,2 ' l - h o r m o n e is mixed with t h e H. antibodies, a n d a labelled H.-antibody complex is formed. If one then adds the body fluid to be tested, the unl a b e l e d H. present in the fluid interacts with the complex and displaces some of the labelled H. The free a n d complexed H. are then separated by electrophoresis, chromatography or precipitation, a n d the radioactivity of the free and b o u n d H. is measured. The desired H. concentration can be calculated f r o m these values, because the a m o u n t of free radioactive H. depends on the a m o u n t displaced f r o m the complex, i.e. on the a m o u n t of unlabelled H. in the solution tested. The h o r m o n e receptor m e t h o d (radioligand-hormone receptor method) is a combination of a biological with a radiochemical m e t h o d : H. receptors can be isolated form animal organs, and these are allowed to react with the H. to be measured as the first step. The H. can be isolated in this way (discrimination). In a second step, ' ^ i o dine-labelled H. is a d d e d and it occupies all unoccupied sites on the receptors. The radioactivity of the resulting complex is related inversely to the concentration of native H. in the fluid tested. Hp: abb. for Haptoglobin. HSK cycle: abb. for Hatch-Slack-Kortschak cycle. Human chorionic gonadotropin: see Choriogonadotropin. Human lactogen: see Placenta lactogen. Human menopausal gonadotropin, HMG, castration gonadotropin: a glycoprotein of the anterior lobe of the pituitary. M r 31000. C a r b o h y d r a t e content 30 %. H M G has a similar action to follicle stimulating hormone. Its primary structure is not yet known. Increased quantities of H M G are f o r m e d in the pituitary of women in the m e n o p a u s e , or women who have been ovarectomized. The increased synthesis (accompanied by an increased excretion) of H M G is explained by the absence of the negative feedback by sex h o r m o n e s f r o m t h e ovary on the hypothalamus a n d the pituitary. Humulane type: see Sesquiterpenes (Fig.). Humulene: isomeric monocyclic sesquiterpene hydrocarbons f o u n d in various aromatic oils. M t 204.36. a - H u m u l e n e , a-caryophyllene, b.p. l 0 123°C, p 4 0.8905, n D 1.5508. p-Humulene, p 4 0.8907, n D 1.5012, has the same eleven-membered ring system as the a-isomer, but one of the three double b o n d s is exocyclic. The H. are f o u n d primarily in h o p oil, together with oxygen-containing derivatives a n d P-caryophyllene. For the formulas a n d biosynthesis, see Sesquiterpenes. Hyaloplasm: see Cell 2) Hyaluronic acid: an u n b r a n c h e d mucopolysaccharide. M r 200000 to 400000. T h e basic subunit is a disaccharide, A'-acetyl-D-glucosamine glycosidically linked p-1,4 to D-glucuronic acid. Thelatter is (i-1,3 linked to the next disaccharide unit. H.a. occurs in various animal tissues and joint fluids. Aqueous solutions of it have high viscosity, which explains its biological function as a lubricant. It is synthesized f r o m D-glucose in the fibroblasts.

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211

Hybridization: H. of nucleic acid molecules is a molecular biological technique for comparison of the nucleotide sequences and characterization of the information contents of differing nucleic acids. The filter, or gel technique is important. Here the D N A is thermally denatured a n d quickly cooled, a n d the single-stranded D N A so obtained is e m b e d d e d in agar or polyacrylamide gel, or it is adsorbed on cellulose nitrate. The D N A to be tested, radioactively labelled a n d also in singlestranded form, is put on the gel or cellulose nitrate filter, where complementary strands of radioactive and non-radioactive D N A bind (hybridize) after a suitable incubation time. After removal of the u n b o u n d radiactive D N A , the hybridization rate can be determined. This gives information on the extent of complementary nucleotide sequences in the two forms of D N A . The same technique is also used for hybridization of D N A a n d R N A . Here unlabelled denatured D N A is b o u n d to a solid phase a n d radioactive R N A is a d d e d in solution. The R N A forms ribonuclease-resistant hybrids with complementary segments of the codogenic strand. This technique permits the estimation of the information content of the D N A . In cytological H., radioactive R N A is hybridized with chromosome-bound D N A in a histological preparation. This yields information on the site of synthesis of the R N A in the chromosome. Hybridoma: see Immunoglobulins. Hydroazulene: see Proazulene. Hydrocarbon degradation, microbial hydrocarbon degradation: Some microorganisms are able to degrade hydrocarbons a n d use them as their sole source of carbon and energy. This ability is important for the microbial production of protein and for the elimination of environmental contamination by mineral oils. The degradation of hydrocarbons depends highly on their structure. The u n b r a n c h e d paraffins (alkanes) of chain length C 1 0 to C 1 8 are most readily degraded. The most important pathway is the oxidation of o n e end to the corresponding fatty acid via the alcohol a n d aldehyde as intermediates. Further degradation of the fatty acids is achieved by p-oxidation. Aromatic hydrocarbons are less readily degraded than aliphatic structures. Ring cleavage is always preceded by the formation of a phenol by an oxygenase. Further degradation proceeds via pyrocatechol, cis.cis muconic acid a n d a-ketoadipic acid to the components of the tricarboxylic acid cycle, acetic acid a n d succinic acid. Hydrocyanic acid, hydrogen cyanide, HCN: a highly poisonous c o m p o u n d f o u n d widely in nature in the f o r m of Cyanogenic glycosides (see). It is released f r o m these by ft-glucosidases (such as emulsin) a n d oxinitrilases. A n u m b e r of plants,

Hydrogen metabolism especially those which contain cyanogenic glycosides, can metabolize H C N , usually by binding it to serine or cysteine to form cyanoalanine. Addition of water converts the latter to asparagine. Hydrogen: see Bioelements. Hydrogenase: see Hydrogen metabolism. Hydrogenatlon: see Reduction. Hydrogen bonding: see Noncovalent bonds. Hydrogen metabolism: 1. metabolic redox reactions, involving pyridine nucleotide a n d flavin coenzymes; 2. all metabolic reactions involving hydrogen, i.e. hydrogenation, dehydrogenation, transhydrogenation, activation a n d formation of molecular hydrogen. In anaerobic a n d aerobic respiration, substrates are oxidized by removal of hydrogen (dehydrogenation), a process that does not involve transfer of oxygen. Dehydrogenations are catalysed by dehydrogenases ( N A D + and N A D P * act as coenzymes) a n d by oxidases ( F A D and less frequently F M N act as redox prosthetic groups or cofactors of the flavoenzymes). Hydrogen atoms removed from a substrate (H = H + + e~) are transferred to the active group of the dehydrogenase or oxidase. Hydrogen is transferred to N A D + a n d N A D P * as a hydride ion, i.e. one proton ( H + ) a n d an electron pair (2e") are transferred f r o m the substrate hydrogen (2[H]), leaving one p r o t o n free (see Nicotinamide-adenine-dinucleotide). In anaerobic carbohydrate degradation, the reduced coenzyme ( N A D H ) is reoxidized by coupling its oxidation to the reduction of an e n d p r o d u c t of glycolysis; this is an internal oxidoreduction system in which molecular oxygen plays no part. In the complete, aerobic oxidation of glucose via glycolysis, the TCA-cycle a n d the respiratory chain, N A D H is reoxidized by the respiratory chain with the uptake of molecular oxygen. N A D a n d N A D P serve different metabolic functions. In aerobic metabolism, most of the N A D H produced by the cell is reoxidized by the respiratory chain for the purposes of energy p r o d u c t i o n (see Oxidative phosphorylation). Oxidation a n d production of N A D H is controlled in such a way that cytoplasmic N A D exists predominantly in the oxidized form, i.e. N A D V N A D H > 1. O n the other h a n d , in the mitochondria, this ratio is about 100 times lower than in the cytoplasm, i.e. N A D exists predominantly in the reduced form, despite the fact that mitochondria are the site of oxidation of N A D H to N A D + . Evidently, the high activity of the TCA-cycle a n d the (i-oxidation of fatty acids maintain a rapid production and constant high level of N A D H . In addition, the P-hydroxybutyrate - acetoacetate oxidoreduction system has a low redox potential which helps to buffer the N A D + - N A D H system in the reduced state. In contrast, the ratio N A D P V N A D P H in the cytoplasm is low. N A D P H does not transfer hydrogen to the respiratory chain, a n d it functions as a reducing agent in reductive biosynthesis. Many reductive syntheses require N A D P H , e.g. reduction of glyceric acid 3-phosphate to glyceraldehyde 3-phosphate in the photosynthetic assimilation of C O , (see Calvin cycle); reductive syn-

Hydrogen metabolism

212

thesis of glutamate by glutamate synthase (see Ammonia assimilation); Fatty acid biosynthesis (see). In aerobic respiration, the cytoplasmic N A D H produced in glycolysis is oxidized by transfer of its reducing equivalents to the respiratory chain. However, the inner mitochondrial membrane is impermeable to N A D + and N A D H in both directions. Reducing power from cytoplasmic N A D H is therefore transported into the mitochondria by shuttle systems: 1. P-Hydroxybutyrate/acetoacetate shuttle. Acetoacetate in the cytoplasm is reduced to p-hydroxybutyrate by an NADH-dependent reductase (EC 1.1.1.30). p-Hydroxybutyrate enters the mitochondrion, where it is oxidized to acetoacetate by an NAD-dependent dehydrogenase. The resulting N A D H is oxidized by the respiratory chain, and the acetoacetate leaves the mitochondrion to be reduced by more cytoplasmic NADH. The reality of this system is in some doubt. Certainly it could not operate in mitochondria that lack p-hydroxybutyrate dehydrogenase, i.e. hepatic mitochondria of ruminants. Vertebrate red muscle contains high levels of mitochondrial P-hydroxybutyrate dehydrogenase, so the shuttle might operate in this tissue. 2. Dihydroxyacetone phosphate/a-glycerolphosphate shuttle. Dihydroxyacetone phosphate is reduced in the cytoplasm by glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) and NADH. The resulting a-glycerolphosphate is oxidized in the mitochondria by a different glycerol 3-phosphate dehydrogenase (an FAD-flavoprotein, EC 1.1.99.5), and dihydroxyacetone phosphate returns to the cytoplasm. Whereas the oxidation of N A D H by the respiratory chain gives rise to 3ATP, this shuttle system produces 2ATP per N A D H oxidized, because the mitochondrial dehydrogenase is FAD-dependent. This is an active shuttle system in blowfly flight muscle. The component enzymes are also present in mammalian muscle in amounts compatible with the operation of the shuttle, but it seems doubtful that this particular shuttle is operative in liver. Malate/oxaloacetate shuttle. Oxaloacetate is reduced at the expense of N A D H by the action of a cytoplasmic malate dehydrogenase (EC 1.1.1.37). Malate enters the mitochondria, where it is dehydrogenated by mitochondrial malate dehydrogenase. Oxaloacetate does not leave the mitochondria, but is transaminated with glutamate to form aspartate and 2-oxoglutarate. Aspartate crosses the mitochondrial membrane and is transaminated to oxaloacetate in the cytoplasm. This shuttle does not cause an imbalance of charge across the mitochondrial membrane, because the entry of malate is coupled with the exit of 2-oxoglutarate (both dicarboxylic acids) and the entry of glutamate is coupled with the exit of aspartate (both acidic amino acids). Many other shuttles can be contrived theoretically, but they lack experimental support. Liver cells are probably served by several shuttles, rather than one major system. In heterotrophic organisms, N A D P H is formed

Hydrogen metabolism in the oxidative phase of the Pentose phosphate cycle (see), and in photosynthetic organisms (except photosynthetic bacteria) in the light reaction of Photosynthesis (see). Another important source is the cytoplasmic oxidative decarboxylation of malate by NADP + -linked malate dehydrogenase (EC 1.1.1.40). This enzyme is very active and operates near to equilibrium in the cytoplasm. A cycle exists, in which pyruvate enters the mitochondria and undergoes ATP-dependent carboxylation to oxaloacetate. Hydrogenation of the oxaloacetate by malate dehydrogenase and N A D H produces NAD* and malate. Malate leaves the mitochondria and is decarboxylated to pyruvate by the cytoplasmic N A D P M i n k e d malate dehydrogenase (EC 1.1.1.40) i.e. there is an export of reducing equivalents from the mitochondria. In contrast to the oxidation of N A D H , there is no mechanism for the direct oxidation of N A D P H by the respiratory chain. In fact the physiological problem faced by animal cells in particular is how to maintain a sufficiently plentiful supply of N A D P H for reductive biosynthesis, and how to supplement this by exploiting the reducing equivalents of N A D H , e.g. by the malate-pyruvate cycle described above. Nevertheless, some of the hydrogen of N A D P H probably does become oxidized by the respiratory chain. For example, N A D H and N A D P H might become equilibrated by any enzyme that can use both, and such an equilibration has been shown to occur. Enzymes that can use both cofactors with more or less equal facility are glycerol dehydrogenase (pig liver, rat liver, Escherichia coli), glutamate dehydrogenase (muscle, liver, yeast) and 3P-hydroxysteroid dehydrogenase (liver). Liver and heart mitochondria perform a transhydrogenation in which hydrogen is transferred from N A D H to N A D P + : N A D H + NADP* NAD+ + NADPH. The process is energy-dependent (ATP) and is catalysed by an enzyme in the inner mitochondrial membrane. The equilibrium is so strongly in favor of the formation of N A D P H , that the reverse process is not easily measured. Clearly this reaction will not lead to the net production of N A D H from N A D P H ; rather it serves to supply N A D P H required for intramitochondrial reductive biosynthesis. Both the donor (NADH) and acceptor (NADP*) interact with transhydrogenase on the M-side of the mitochondrial membrane. During transhydrogenation, there is no exchange of hydrogen with protons of water. The hydrogen atom is transferred from the A side of N A D H to the B side of N A D P + , indicating that the planes of the nicotinamide rings of these two cofactors must become closely associated on the enzyme surface. Cytoplasmic transhydrogenases have also been reported, which catalyse transhydrogenation with an equilibrium constant of about unity. In the majority of living organisms, molecular hydrogen has no significant metabolic role. However, certain enzymes called hydrogenases have been found in a wide range of living organisms, including bacteria, plants and animals. It has

Hydrogen transfer

213

been suggested (Krebs) that hydrogenase serves to release H 2 from excess NADH, when biosyntheses have a prevalence of oxidative steps and therefore upset the normal redox balance of the cell, e.g. in microorganisms growing anaerobically, excess reducing power may be used to synthesize reduced products that are excreted, or it may simply be released as molecular hydrogen by the agency of hydrogenase. Some hydrogenases are membrane-bound, and they are often linked to formate dehydrogenase (HCOOH + NAD + - C 0 2 + NADH + H + ; NADH + H + - NAD + + Hj). In strictly anaerobic bacteria, hydrogenases are linked to ferredoxin, and they catalyse an oxidoreduction reaction between hydrogen and ferredoxin: H 2 + 2Fd ox ^ 2H* + 2Fd red , where Fd ox and Fd red represent oxidized and reduced ferredoxin, respectively. Certain bacteria, e.g. Hydrogemonas, Pseudomonas and Alcaligenes, can oxidize H 2 with 0 2 ; a normal electron transport chain operates and 3 molecules of ATP are generated. Ferredoxin-dependent hydrogen production catalysed by hydrogenase, e.g. in Clostridium, is inhibited by carbon monoxide, and is independent of ATP. It therefore differs from the ATP-dependent, CO-sensitive hydrogen production by Nitrogenase (see). Evolution of hydrogen gas during nitrogen fixation is due to competition between nitrogen and protons for electrons at the reducing center of the enzyme. In addition to ferredoxin, the Redoxins (see) are important electron transferring agents in hydrogen metabolism. Hydrogen transfer: see Hydrogen metabolism; Pyridine nucleotide coenzymes. Hydrolases: see Enzymes, table 1. Hydrophobic bonds: see Noncovalent bonds. Hydroxamic acids: derivatives of carbonic acid containing the tautomeric group R - C - NHOH ^ R - C = N - O H II I O OH H.a. can form stable, five-membered rings with metal ions. They are especially important in the iron metabolism of many organisms. Well-known examples of H.a. are aspergillic acid (Fig.), synthesized from the amino acids leucine and isoleucine by Aspergillus flavus, and the Siderochromes(see).

Aspergillic acid Hydroxyacetlc acid: see Glycolic acid. Hydroxyacld: a carboxylic acid, in which one or more hydrogen atoms of the alkyl moiety is replaced by an hydroxyl group. The position of the OH-group in the alkyl chain is indicated by a,f),Y,8, etc., or by 2,3,4,5, where the C-atom of the COOH is No. 1; thus lactic acid is 2-hydroxypropionic, or a-hydroxypropionic acid. Impor-

Hypochromic effect tant biological H. are glyceric, malic, lactic and citric acids. 3-Hydroxy-2-butanone: see Acetoin. o-Hydroxyclnnamlc acid lactone: see Coumarin. jV-2-Hydroxyethylplperazlne-jV-2-ethanesulfonicacid, Hepes: M r 238.3, used for the preparation of buffers in the pH range 6.8-8.2. Hydroxylases: see Oxygenases. Hydroxylatlon: see Oxygenases. 5-Hydroxymethylcytoslne: a pyrimidine compound, one of the rare nucleic acid bases. M t 141.12, d. above 200°C without melting. H. is not synthesized as a modification of cytosine already incorporated in the nucleic acid but is formed de novo in the course of pyrimidine biosynthesis as 5-hydroxymethyldeoxcytidylic acid. H. was isolated in 1952 from DNA. It is found in the DNA of bacteriophages of the T2, T4, T6 series in place of cytosine. Hydroxymethyl glutarate cycle: see Ketogenesis. Hydroxynervon: see Glycolipids. Hydroxynervonlc acid: A 15 -2-hydroxytetracosanoic acid, CH 3 -(CH 2 ) 7 -CH = CH-(CH 2 ) 12 CHOH-COOH, an hydroxylated, unsaturated fatty acid. M r 382.5, m.p. 65°C. An important component of cerebrosides. 3a-Hydroxy-5a-pregnan-20-one: a catabolite of progesterone. M r 318.4, m.p. 175°C,[a] D +96° (alcohol). Like its stereoisomers, 3(5-hydroxy-5apregnan-20-one, m.p. 194°C, [a] D +91° (alcohol), and 3a-hydroxy-5fi-pregnan-20-one, m.p. 149° C, [a] D + 106° (alcohol), 3a-H. appears in the urine of pregnant women. 9-Hydroxy-trans-2-decenoic acid: see Queen substance Hygrine: see Pyrrolidine alkaloids. Hyocholic acid: 3a, 6a, 7a-trihydroxy-5P-cholan-24-oic acid, one of the bile acids, a trihydroxylated steroid carboxylic acid. M r 408.58, m.p.l97°C, [a]D +5.5° (alcohol). H. is a component of pig and rat bile and, like the main component of pig bile, hyodeoxycholic acid (3a, 6a-dihydroxy-5f)-cholan-24-oic acid), M r 392.58, m.p.l97°C, [a] D + 8 ° (methanol), it is important as a starting material for the synthesis of steroid hormones. Hyodeoxycholic acid: see Hyocholic acid. Hyosclne: see Scopolamine. Hyoscyamine: see Atropine Hyp: abb. for hypoxanthine and hydroxyproline (see L-Proline). Hyperchromlc effect: increase in the extinction of a solution at a particular wavelength due to structural changes in the solute molecules. H.e. is important in the denaturation of DNA when the double helix held together by hydrogen bonds is transformed into a disordered random coil. Hypertensln: see Angiotensin. Hypervltamlnosls: see Vitamins. Hypochromes: see Pigment colors. Hypochromic effect: optical phenomenon in molecules with several chromophores, in which the sum of the optical densities of the individual components is greater than the optical density of

Hypophysis

214

the whole molecule. The extinction of the nucleic acids at 260 nm, for example, is less than the calculated sum of the extinctions of the component bases. The H. depends on the content of adenine and thymine and is therefore greater in DNA than in RNA. Double-stranded polynucleotides have a greater H. than single-stranded, because the effect is intensified by hydrogen bonds. Hypophysis, pituitary gland: a vertebrate organ for hormone production. In humans, it weighs 0.7 g and lies at the base of the brain. It is connected to the midbrain by the hypophyseal stalk. The H. is composed of two parts with different ontogenies. The adenohypophysis, which includes the anterior and middle parts, arises from the roof of the embryonic mouth. The neurohypophysis, or posterior lobe, is formed from an outgrowth of the base of the midbrain. In the anterior lobe, releasing hormones from the hypothalamus stimulate the formation of somatotropin and prolactin in the acidophilic (a-)cells, follicle-stimulating hormone, luteinizing hormone and thyreotropin in the basophilic (P-)cells; and corticotropin in the chromophobic (y-)cells. Melanotropin is synthesized in the middle part of the H. The neurohormones, oxytocin and vasopressin, are stored in the posterior lobe and released on demand into the blood stream. (See separate entries for each of the above-mentioned hormones). Hypothalamus: the lowest part of the midbrain, which also includes the thalamus and epithalamus. The hypophyseal stalk with the neurohypo-

Hypoxanthinosine physis arises from the underside of the H. As part of the limbic system, the H. is a "gateway to consciousness". Afferent (incoming) stimuli from the breast and abdominal areas and from the circulatory system are processed by the thalamus and passed on to the cerebrum. Conversely, efferent stimuli from the cerebrum flow to the thalamus and H. A series of nuclear regions controlling important vegetative functions, such as blood pressure, temperature, sweat formation, water balance, motions of the digestive tract organs, and sexual function are located in the H. The entire metabolism can be greatly influenced by the appetite and eating center of the H. In addition, in certain nuclear areas nervous excitation is transformed into hormonal signals (releasing hormones and neurohypophyseal hormones), and conversely, hormones from the hypophyseal hormone glandular system exert negative feedback reactions on the formation of hormones in the nerve cells. Hypovltaminosis: see Vitamins. Hypoxanthine, abb. Hyp: 6-hydroxypurine, a purine derivative (formula, see Inosine). Air 136.11, m.p. 150°C (d.). Hyp occurs in the course of aerobic Purine catabolism (see) through deamination of adenine compounds or through hydrolysis of inosine compounds. It is found as a rare base in certain transfer RNAs. It is widely distributed in the plant and animal kingdoms. Hypoxanthinosine: see Inosine.

I

IAA: see Auxins. Ibotenlc acid: a-amino-3-hydroxy-5-isoxazoleacetic acid. MT 158, m.p.l45°C (d.) I.a. is a psychotropic, weakly insecticidal substance which, like its decarboxylation product, Muscimol (see), belongs to the group of amanita poisons. It is found only in a few species of Amanita (fly agaric), at concentrations averaging 0.05% of the fresh weight. I.a. is pharmacologically very active, but less so than muscimol in most tests. Another derivative of I.a. is the erythro-dihydroibotenic acid, Tricholomic acid (see). ICSH: see Luteinizing hormone. Idaelne: see Cyanidin. IDP: abb. for Inosine 5'-diphosphate. I.E.P.: abb. for Isoelectric point. Ig: abb. for Immunoglobulins. lie: abb. for L-Isoleucine. Imidazole alkaloids: alkaloids of sporadic occurrence, possessing the imidazole ring system. Their most important representative is Pilocarpine (see). The biosynthesis of the I.a. is coupled to histidine metabolism. Immobilized enzymes: soluble enzymes bound to an insoluble organic or inorganic (e.g. porous glass) matrix, or encapsulated within a membrane in order to increase their stability and make possible their repeated or continual use. The four most commonly used methods of enzyme immobilization are: 1. Adsorption onto an inert or electrically charged carrier (cross-linked dextrans); 2. Covalent binding to a carrier polysaccharide, e.g. Sepharose; 3. Entrapment in a three dimensional cross linked carrier, e.g. polyacrylamide gel; 4. Cross linkage, i.e. condensation of several en-

—OH 1. Activation: — OH

BrCN pH 10.5

zyme molecules with bi- or polyfunctional agents, like glutaraldehyde and epichlorhydrin; 5. Microencapsulation. Covalent binding to a carrier is widely used. Since the enzyme is chemically fixed, it is not washed out and can be used repeatedly. Of the organic carriers, Sepharose and large pore cross-linked dextrans come close to meeting all the requirements of an enzyme carrier, i.e. minimal solubility, high mechanical strength, suitable particle size and shape, high binding capacity for the enzyme, but no adsorption of substrates and products, and resistance to attack by microorganisms. Being organic, however, they are subject to microbial attack, and they swell and shrink depending on pH and other environmental conditions. Since 1969, silanized porous glass, which has neither of these disadvantages, has been used increasingly for both adsorptive and covalent attachment of enzymes. For covalent attachment, functional groups must be present on the carrier. A frequently used activation procedure, especially with dextran gels, consists of reaction with cyanogen bromide (CNBr), which produces reactive iminocarbonate groups (Fig. 1). At pH 7-9, these react with the free amino groups of the enzyme protein, with the formation of substituted imidocarbonates ( = C = NProtein). Many other coupling techniques have been reported for the covalent attachment of enzymes to agar, agarose and Sephadex supports, and to the silanized surface of porous glass. Details of these techniques and other aspects of immobilized enzymes are comprehensively treated in Methods in Enzymology, volume XLIV, 1976, edit. Klaus Mosbach, Academic Press.

—0, C=NH —0

C=NH+ H 2 N-1Enzyme]-C00H •

2. Coupling: —0

pH 8-9

C = N-|Enzyme|-C00H

Fig. 1. Covalent binding of an enzyme to an unsubstituted polysaccharide (e.g. Sepharose), by the cyanogen bromide method.

216

Immune response

The quantities of covalently bound enzymes are generally low (1-5%); in exceptional cases, especially when carrier and protein are oppositely charged, 10% or more may be bound, e.g. 12% catalase bound to cellulose derivatives. Properties. In general, the free and immobilized enzyme catalyse the same reaction, but depending on the supporting material and nature of the binding, there may be changes of pH and temperature optima (the latter is usually increased), Revalue and specific und maximal activities (the latter is usually decreased) (see Enzyme kinetics). Chief reasons for these alterations are the decreased flexibility and mobility of the coupled enzyme, and steric factors which interfere with access of the substrate to, and diffusion of product from, the active center. These changes are usually more than compensated by increased stability of the enzyme. They can be avoided or reduced by attaching the enzyme to the support by a side chain, or spacer, which allows greater mobility and unhindered contact with substrates. Enzymes may also be immobilized by microencapsulation. In this technique, which has medical applications, enzymes are enclosed by various forms of semipermeable membranes, e.g. polyamide, polyurethane, polyphenyl esters and phospholipids. Microcapsules of phospholipids are also called liposomes. The microencapsulated enzymes and proteins outside the microcapsule cannot pass the membrane envelope, but low MT substrates can pass into, and products can leave, the microcapsule. Such encapsulated proteins do not elicit an antigenic response, and they are not attacked by proteases outside the microcapsule. They are therefore suitable for the delivery of enzymes for therapeutic purposes. This area of application is still at an early stage of development, but positive results have been reported from animal experiments and clinical studies, e.g. treatment of inherited catalase deficiency with encapsulated catalase, and treatment of asparagine-de-

Urea

o—

Urease ,

Other uremic products (uric acid, creatinine)

Fig. 2. Schematic representation of an artificial ceil, containing urease and albumin-coated active charcoal as an adsorbent for uric acid, ammonia and creatinine. A 10 ml suspension of these 20 nm diameter urease capsules corresponds to a surface area of 20000 cm2, which is larger than that of the conventional artificial kidney.

Immunization pendent lymphosarcoma with encapsulated asparaginase. There are various methods of administration, e.g. injection intramuscularly, subcutaneously and intraperitoneally. However, their major area of application is outside the body, e.g. microencapsulated urease can be employed as an artificial kidney in hemodiffusion (Fig.2). A future possible development is the immobilization on one carrier of several enzymes of a reaction chain for use as enzyme reactors, e.g. protein degradation by immobilized proteolytic enzymes. I.e. can also serve as models for cell organelles, e.g. a model for mitochondrial oxaloacetate production and utilization has been constructed from immobilized malate and lactate dehydrogenase and citrate synthase. Immune response: a specific protective or defense reaction against foreign substances. These foreign substances, known as antigens, release from the surface of the lymphocytes the signal for cellular or humoral I.r. The T-lymphocytes are responsible for the cellular I.r.; the antibody-producing B-lymphocytes (as plasma cells) for the humoral I.r. The cellular I.r. plays an important role in rejection of transplants, autoimmune diseases, chronic bacterial infections and viral and fungal infections. Protein antigen-antibody complexes can, in the presence of excess antibody, induce the cellular I.r., as can lower antigen concentrations or intracutaneous application of the antigens. The Lymphokines (see) mediate the cellular I.r. The humoral I.r. occurs in the extracellular space, and it is mediated by the immunoglobins secreted by the plasma cells. IgM is responsible for the primary I.r., while IgG (see Immunoglobins) maintains long-lasting immunity. The immunoglobins are especially active in acute bacterial infections, by binding the antigens as antigen-antibody complexes, thus inactivating them and making them susceptible to phagocytosis. Immune serum: see Antiserum. Immune suppression: the unspecific suppression of the Immune response (see) by corticosteroids, antimetabolites (purine analogs, folic acid antagonists), alkylating substances like cyclophosphamide (see), ionization radiation or antilymphocyte serum (see). Although the latter is directed against the cells of the immune system, the other agents have an antiproliferative effect, i.e. they hinder the multiplication of the plasma cells. At the molecular level, these agents directly or indirectly inhibit the synthesis of DNA and RNA. I.s. is an important form of therapy for autoimmune diseases and for preventing the rejection of transplanted organs. However, the suppression of antibody formation results in higher susceptibility to infection and there is the danger of increased tumor formation. A true alternative to I.s. is specific I.s. This can be achieved by means of antigen-specific immune sera or by the induction of an immune tolerance. In the latter, there is an antigen-specific suppression of the immune response, without a reduction of the general resistance. Immunization: the artifical stimulation of antibody production for protection against pathogens

Immunochemistry

217

and other antigens. There are two kinds of I., 1) active, produced by injection of live, weakened pathogens (Sabin poliomyelitis vaccine, smallpox vaccine) or killed pathogens or purified fractions of them, containing the most important determinants and 2) passive, produced by injection of antiserum or antibodies which have been formed against the pathogen in another organism and extracted from it. While repeated active I. makes life-long immunity possible, passive I. is effective for only a few weeks and cannot be repeated with antiserum or antibodies from the same species of animal, because the foreign antibodies act as antigens. Passive I. therefore is used for short-term prophylaxis or therapy, to bridge the time until the body can produce its own antibodies, (for example in tetanus simultaneous immunization). Immunochemistry: see Immunology. Immunoelectrophoresis: see Plasma proteins. Immunofluorescence: a sensitive technique for detection of antigens or antibodies in which the specific antibody (direct I.) or the anti-antibody (indirect I.) is labeled with fluorochromes, for example fluorescein isothiocyanate. After the antigen-antibody reaction has occurred, the complex can be located by fluorescence microscopy. Immunoglobulins, abb. Ig., antibodies: specific defense proteins found in blood plasma, lymph and many body secretions of all vertebrates. Each individual has a unique collection of Ig. The most salient features of the system are its ability to respond to the presence of any foreign antigen, and its ability under normal circumstances not to respond to self antigens (self-nonself discrimination). The phylogenetic precursors of the Ig., the cell-bound hemagglutinins, are also found in invertebrates including annelids, crustaceans, spiders and molluses. Lymphocytes derived from bone marrow in mammals or the Bursa of Fabricius in birds (B-cells) display Ig. on their surfaces. Upon exposure to antigen, those cells whose surface Ig. can bind the antigen are stimulated to proliferate and differentiate into plasma cells which secrete antibodies of the same specificity as the ancestor of the clone. The process of stimulation is complex and involves cooperation with other lymphocytes (T-cells) and macrophages; but B-cells can also be stimulated to proliferate by polyclonal activators such as concanavalin A (see Lectins) or bacterial lipopolysaccharide. When bound to antigen, some Ig. can fix the Clq component of the Complement system (see), thus facilitating the lysis of the foreign cell to which they are bound. Because they are multivalent, Ig. also cross-link soluble antigens and facilitate their clearance from the blood or lymph by macrophages. Structure: The basic structure of Ig. is a tetramer of two light (Mr 22000 to 24000) and two heavy, carbohydrate-containing chains (Mr 50000 to 73000) (Fig. 1). The heavy (H-) chains are always linked to each other by one or more disulfide bonds; the light (L-) chains are usually linked to the heavy chains by disulfide bonds; and the structure is also stabilized by strong non-covalent bonds. Papain treatment of Ig. releases monova-

Immunoglobulins lent antigen-binding fragments, Fab (M r 50000) and a complement-binding, Fc (Mr 60000) fragment. Pepsin cleavage, on the other hand, releases a bivalent antigen-binding F(ab)2 fragment and a somewhat smaller complement-binding fragment, Fc'. The F(ab)2 can be dissociated by thiol reagents into monovalent fragments, indicating that the two H-chains in the intact molecule are held together by a disulfide bond between the sites of pepsin and papain cleavage (Fig. 2). The Y-shape of the Ig. molecule has been confirmed by electron microscopy and X-ray diffraction. The former shows that they are flexible at the "hinge" region, as was postulated from the susceptibility of this region to proteolytic attack. Kappa-Type

Lamda-Type

x

I j

i

lgG

I

; i

K

x / IgA

oi ;

I

(Monomer intracellular)

/ ;

a I

Secretory IgA: Oi-,Tri- a n d Tetramer

x

% :

IgM

I*

;

j j

(Monomer intracellular)

K_

H| I

Serum-IgM: Pentamer

^ ;

lgD

X 6

i

;

j

6 j

x

k I

igE

y

! i

£ i

j

£ :'

! Disulfide b r i d g e s

Fig. 1. Chain structure of the fire classes of human immunoglobulins: the two L-chain types k and X, and the five H-chain types y, a, (i, 8, and e. There are five known classes of Ig., IgA, IgD, IgE, IgG and IgM. These can be distinguished electrophoretically and serologically. In addition, most mammals appear to have several subclasses of IgG, which can be distinguished immunologically, and humans are known to have subclasses of IgA as well. The classes and subclasses (or isotypes) are determined by the constant regions of the heavy chains, which are given the corresponding Greek letters (a, 8, e, |±, and y l , y2, etc.) Each

Immunoglobulins

218

Table. Properties of human immunoglobias IgG

Immunoglobulins

(Ig).

IgM

IgA IgD Secretions

Serum Sedimentation constant Mr, of which the L-chain represents 23 000 H-chain type and M r Chain formula (L = K or X) Carbohydrate Fraction of the serum Ig Serum concentration mg/100 ml Valence of binding Biological halflife (days) Complement binding

6.5 . . . 7S

19S

7S

IIS

6.8 . . . 7.9S

8.2S

155000

940000 (Pentamer)

170000

380000 (dimer)

185000

196000

yl . . . 4 50 000 to 60000

H 71000

5 60 000 to 70000

e

L

(L2M5

L282

L2E2

12.7% 0.03 . . . 1 %

1 0 . . .12% 0.05%

2T2

2 . . . 3% 70 . . .75%

140 (60 . . . 280) 5(10)

(IgG 3) or 21 5.1

yes

yes

t

region" FC

CH2 1

L2a2

Pepsin

~

CH3 H-Chain

Papain

Fig. 2. Scheme of the structure of ao IgG molecule. Variable (VL, VH) and constant (CL, CH) parts (domains) of the light and heavy chains, respectively: KH = carbohydrate chain. VL and VH form the antigen binding site. Papain cleavage produces two Fab and one Fc fragment; pepsin cleavage forms a (Fab') 2 fragment and a low molecular weight cleavage peptide of the Fc fragment. PC = pyrrolidone carboxylic acid.

(L2a2)2 8 . . . 10% 1 0 . . . 22%

10...12%

1300 (800 . . . 1800) 2

"Hinge

a

64000

7 . . .10%

type of heavy chain can be associated with either of the two types of light chain, k or X, but in a given molecule, both light chains are of the same class.

L-Chain

IgE

210 (100-450)

7

0.03 0.01 to 0.14) 2

2.8

2...3

(1 . . . 40)

1 5.8

75000

no

Because of the extreme heterogeneity of normal Ig., it is impractical to attempt to purify them for amino acid sequencing. However, it was found that Paraproteins (see) associated with some pathological conditions are the homogeneous products of single clones of transformed plasma cells. The early work on sequence and structure was done on Bence-Jones proteins (see), which are L-chains, and myeloma proteins, which are whole Ig. molecules. More recently, the technique of cell fusion has been applied to produce antibody-secreting hybrids between normal pancreatic or peripheral lymphocytes and myeloma lines maintained in tissue culture. The resultant hybridoma clones can be selected for production of Ig. of the desired antigen specificity and class; with them, comparisons of sequence and function in homogeneous Ig. have become very refined. In addition, Ig genes from the hybridomas and normal cells have been cloned (using gene-splicing techniques) and compared. These studies have shown that in the course of differentiation of plasma cells, genes for the constant region (see below), a joining region (or two) and the variable region have been spliced together; the intervening D N A sequences are lost. (For an introduction to this work, see M.M. Davis et al. [1980] Nature 283, 733-739.)

219

Immunoglobulins

Immunoglobulins -iValX

vfGlnl(ThiT(MetXGIriXlieX 5—N (GluT (Gin) 160 (Sen

(Sen W

(Vol) 150

IPheL

SD £r)20

(GW 100

/90

(Trp) (Gin)

fflr)

fLeu)

© Maj (Glu) (¿¡ni

Variable

ft 140

Constant

(Phé) (Asn) (Asn) (Leu) (Leu)

M)110 (Ala)

(W (Phe)

Saccharopine

acid

acid

N-Succinyl-2-oxo-L-6aminopimelic

acid

acid COOH

I

H - C — NH, ^ N-Succinyl- L-2,6 diaminopimeUc

acid

I

^ L-2,6 - DiaminopimeUc

(CH,)3

acid

H - C

I

L - L y s in e

—NH,

COOH Meso-2,6 - Diaminopimelic

Fig. 5. Diaminopimelic acid pathway of

acid

L-lysinebiosynthesis.

Lysocephalins: see Phosphatides. Lysogeny: see Phage development. Lysoleclthins: see Phosphatides. Lysoplne: see D-Octopine. Lysosomes: organelles, 0.2-2 nm diameter, found in the cytoplasm of eukaryotic cells. L. are bounded by a single lipoprotein membrane, but

otherwise show no fine structure. Under the light or electron microscope, L. are markedly polymorphic. They can be characterized biochemically or histochemically, but not morphologically. L. are sites of Intracellular digestion (see), particularly of biological macromolecules, such as proteins, polynucleotides, polysaccharides, lipids, glyco-

Lysozyme

260

proteins, glycolipids, etc. Approximately 40 different lysosomal hydrolases are responsible for this degradative activity; they all show optimal activity at acidic pH values. Marker enzyme for L. is acid phosphatase. Under anaerobic conditions, L. are destroyed, and the lysosomal enzymes are released into the cytoplasm; subsequent degradation of the cell contents by the lysosomal enzymes is known as autolysis. Autolysis is a characteristic post mortem process. Primary L. are formed in the Golgi apparatus of the cell. Fusion of L. with phagocytosing and pinacytosing vacuoles (phagosomes) produces digestive vacuoles, known as secondary L. Excess or old cell parts, including mitochondria, may be digested by L. (phagolysosomes). In ameba, L. apparently provide the digestive enzymes. L. were discovered in 1959 by De Duve (Nobel prize 1974). Lysozyme, endofysin, muramidase, N-acetylmuramide glycanobydrolase (EC 3.2.1.17): a widely occurring hydrolase, found in phages, bacteria, plants, invertebrates and vertebrates. In the latter, it is found particularly in egg white, saliva, tears and mucosas. L. acts as a bacteriolytic enzyme by hydrolysing the ¡3-1,4 linkage between ^-acetylglucosamine and JV-acetylmuraminic acid in the proteoglycan of the bacterial cell wall. L. there-

Lysylbradykinin fore affords protection against bacterial invasion. All animal L. consist of a single chain of 129 amino acid residues, with homologous sequence (A/r 14200-14600). There are four disulfide bridges, and the chain is folded into a known tertiary structure (42% a-helix, with hydrophobic tryptophan residues on the outer surface), with prominent hydrogen bonding between the side chains of Ser, Thr, Asn and Gin. Like other hydrolases (e.g. papain, ribonuclease), the molecule of L. has a cleft which houses the active center of the enzyme and serves for the attachment of the substrate, in this case a hexasaccharide unit of the proteoglycan molecule. There is a remarkable correspondence between the primary and tertiary structures of L. and a-lactalbumin (123 residues). It is thought that both proteins arose from a common precursor protein with lysozyme activity, an example of divergent evolution by gene duplication. On the other hand, there is no structural relationship between animal L. and bacteriophage L. The latter contain 157 residues (X, phage endolysin, M r 17 873), or 164 residues (T4- and T2phage L., M r 18720), and they are either endoacetylmuramidases (T4, T2), or endoacetylglucosaminidases (Streptococcal L.) Lysylbradykinin: see Bradykinin.

M

Macdougallin: 14a-methyl-5a-cholest-8(9)-en3(5, 6a-diol, a phytosterol (see Sterols), M t 416.69, m.p. 173°C, [a] D +72° (chloroform). M. was isolated from the cacti, Peniocerius fosteriunus and Peniocerius macdougalli. It has the structure of a tetracyclic triterpene with a 30,31-bisdemethy 11anostane skeleton, and it represents an intermediate structure between lanosterol and the sterols derived from lanosterol. Macroelements: see Mineral nutrients aj-Macroglobulin, Oj-antiplasmin: an a 2 plasma protein. The first reported M r determination by sedimentation diffusion gave a value of 820000. Later determination by sedimentation equilibrium gave values of 725 000. These results, together with studies of subunit composition, indicate that the true MT lies in the range 650000-725000. a 2 -M. is a glycoprotein containing 8.2% carbohydrate. The carbohydrate moiety contains mannose, fucose, JV-acetylglucosamine and sialic acid. Electron microscope studies of the protein reveal a structure resembling two beans facing each other; these two identical subunits are bound noncovalently. Each subunit consists of two peptide chains linked covalently by a disulfide bridge. a 2 -M. binds tightly and inhibits a number of proteases of varying specificity and origin, e.g. trypsin, plasmin, thrombin, kallikrein and chymotrypsin. It is therefore a natural inhibitor of plasmin. Unlike other protease inhibitors, it does not block the active centers of the enzymes, so that 1! - H ^ „ ^ E. Reactions 1, 2, 3 n are enzyme-catalysed conversions, leading to the endproduct, according to the principle of "organization by specificity" (Dixon). E may be accumulated or excreted. Per unit time, the quantity of S converted is proportional to the quantity of E produced (it is assumed that the quantity of available S is so large that its decrease per unit time can be ignored). The steady state concentrations of the intermediates I,, I 2 ,1 3 I n are constant, i.e. the rate of change ofconcentrationiszero:

Metalloproteins _ OS = OE. Q 1 „ M „= Q dt dt' dt The quantity of I 2 formed per unit time is equal to the quantity of I, converted, and is proportional to theconcentrationofl, :

—

= k1\1 (k = rate constant)

At equilibrium, Arj I, = k 2 \ 2 ,

or

T1 = I2

This leads to the following conclusions: 1. Concentrations of intermediates depend on the rate constants; the larger the rate constant of a reaction, the smaller the concentration of the intermediate. 2. The rate of the conversion S E is determined by the rate of the slowest step; Krebs called this reaction the pacemaker reaction. The situation is usefully illustrated by analogy with a sluice: above the sluice gate (pacemaker), the water level is high (intermediates are present in high saturating concentrations). The sluice gate determines the rate of water flow (substrate conversion). A sudden increase in the concentration of an intermediate would be self-regulated by an overflow and diversion of the intermediate into alternative metabolic channels, which occur earlier than the pacemaker reaction. After the pacemaker reaction, such an increase leads to increased synthesis of endproduct, which is stored or excreted. The endproduct may also regulate its own synthesis by actually inhibiting the synthetic process, so that the rate of production of endproduct is finely adjusted to its rate of utilization by the cell (i.e. endproduct inhibition, see Metabolic control). See also Primary metabolism; Secondary metabolism, Metabolic shunt; Intermediary metabolism; Energy metabolism, Carbon dioxide assimilation; Respiration. Metallollavoprotelns: see Flavin enzymes. Metabolite: a substance produced or consumed by metabolism. Biopolymers are not included in this definition. The precursors and degradation products of biopolymers are, however, true M. All small molecules produced or converted by enzymes during metabolism are M. According to the nomenclature of enzymology, substances (including biopolymers) that are attacked by enzymes are called substrates. Metalloproteins: proteins containing complexed metals. In the metalloenzymes, the metals are functional components. In the metal-transporting M. (e.g. the blood proteins, transferrin and coeruloplasmin) and the metal-storage depot proteins (e.g. ferritin), the metal binding is reversible and the metal is a temporary component. Important iron-containing M. are the cytochromes, respiratory pigments (e.g. hemoglobin) and enzymes (e.g. catalase). Zinc is present in insulin and in carbonic anhydrase. Some glycolytic enzymes and proteases contain Manganese (see). Cu is present in certain oxidoreductases (see Copper proteins). The catalytic M. also include enzymes that require specific cations for activity,

273

Metallothioneins

e.g. the sodium-dependent membrane ATPases. See also Molybdoenzymes. Metallothioneins: cytoplasmic metalloproteins, inducible in mammalian liver and kidney in response to Zn, Cu, Hg and especially Cd. From any single source (e.g. horse kidney), there appears to be only one apoprotein (i.e. thionein), and the M. from that source differ only with respect to the bound metal. M. may function in the storage and regulation of Zn and Cu, and they are thought to be involved in the detoxication of heavy metals. Thionein from horse kidney cortex contains no aromatic amino acids, and rather large amounts of lysine (11.1%), serine (11.0%), glycine (10.0%), proline (4.3%) and cysteine (about 32%); there are no disulfide bridges, and all the thiol groups are involved in cation binding. Methemoglobin: a hemoglobin in which the iron is trivalent (Fe III). M. are unable to transport oxygen. L-Methionine, Met: a-amino-y-methylmercaptobutyric acid, a sulfur-containing, essential proteogenic amino acid. Afr 149.2, m.p. 281°C (d.),[a] D 25 +23.2° (c=0.5 - 2.0, 5M HC1), or - 1 0 . 0 ° (c = 0.5 - 2.0, water). The nutritional value of many plant proteins is limited by their low content of Met. In the first stage of Met biosynthesis in Escherichia coli, cysteine and homoserine condense to form cystathionine. The latter is cleaved to give homocysteine, which is methylated to Met by the transfer of a methyl group from A^-methyl-tetrahydrofolic acid (Fig.). Vitamin B 12 is a coenzyme in this methyl group transfer. The active form of Met., i.e. S-Adenosyl-L-methionine (see), is the methyl group donor in Transmethylation (see). Degradation of Met proceeds via L-Cysteine (see).

CH,OH

SH

CH,

CH,

HCNH,

HCNH2

I I

I

CH,—

I

I

I

-

Homoserine

Cysteine

HCNH;-

I

HCNH;

COOH

COOH

— CHj

CH2

COOH Cystathionine

CHJSH • CH;

+

CH3COCOOH +NHJ

HCNH; COOH Homocysteine (+Vitamin B,;l

N-Methyltetrahydrofolic

acid

v

CH;-S-CH

I

CH; I HCNH;

+

3

Tetrahydrotolic

acid

COOH Methionine

Methionine biosynthesis in Escherichia coli

Methods of biochemistry DL-Met is prepared on the industrial scale (about 100000 tons in 1977) by a Strecker synthesis, using p-methylmercaptopropionaldehyde prepared from acrolein and methylmercaptan. DL-Met is used to supplement poultry feed. Both D- and Informs are effective, so that no prior separation of enantiomers is necessary. Methods of biochemistry: usually methods for the study of metabolic processes, but in the widest sense including isolation, identification and characterization of natural substances. Methods for the study of metabolism are classified into 3 types: 1. In vivo methods employ whole organisms, their organs or cells, or populations of cells of microorganisms. In balance studies, substances are administered to the organism and the time course of their conversion to various products is determined by analysis of body materials or excretory products (feces, urine, expired gases) (see, e.g. Nitrogen balance). Balance studies are also performed on isolated organs, e.g. perfused liver. The load test is also a form of balance study, in which excess of a substance is administered to the organism to test the ability of an organ or organ system to deal with the substance in question. Such tests are used clinically to investigate organ function, e.g. to test kidney function by measurement of the rate of urinary excretion of injected phenol red. The most important in vivo methods are the indicator methods. Classical amongst these is the use, by Dakin and Knoop, of "noncombustible" aromatic residues on fatty acids, which led to the discovery of the P-oxidation of fatty acids (see Fatty acid degradation). This and similar chemical labelling methods have the drawback that the administered material is different from the natural material under investigation. These difficulties are now overcome by the use of isotopes; a compound in which one or more of the constituent atoms is present as a stable or radioactive isotope, is chemically and biochemically identical with the unlabelled compound (see Isotope technique). Thus isotopically labelled natural metabolites can be administered and their natural fate within the organism can be determined by isotopic tracing. A further important in vivo method is the Mutant technique (see). A combination of isotope and mutant techniques has made a considerable contribution to the exponential growth of biochemical knowledge. 2. In vitro methods are performed outside the whole organism. They are essentially "test t u b e " methods, employing crude cell homogenates, subcellular fractions thereof, or purified enzymes. For methods of cell disruption, subcellular fractionation and enzyme purification see Proteins and Density gradient centrifugation. The techniques of histochemistry and cytochemistry are also in vitro methods: the sites of metabolites, enzymes or metabolic reactions are identified in organ or tissue slices and in cells by characteristic chemical reactions, e.g. specific color reactions. A large proportion of in vitro studies is concerned with the activity and behavior of enzymes.

Methyl-accepting Chemotaxis proteins coenzymes and substrates. Such studies require enriched or purified enzymes. The degree of purity necessary for the reliable determination of kinetic parameters varies according to the nature of the enzyme and the possible impurities, but all such investigations are preferably performed with a homogeneous enzyme protein. The completely pure enzyme is necessary for the further investigation of physical and chemical properties, such as photometric measurements and determination of amino acid composition. Ultimately, the crystalline enzyme is required for the study of the detailed mechanism of enzyme catalysis, and for the determination of the three dimensional structure of the enzyme and enzyme-substrate complexes. 3. Synthetic methods include the construction and reconstruction of e.g. multienzyme complexes and biochemical systems; and modelling and simulation, e.g. Synzymes (see) and computer simulation of glycolysis. Methyl-accepting chemotaxls proteins,

Methylglyoxal

274

MCP:

cell membrane proteins in Escherichia coli involved in the iniation and control of chemotactic behavior. There are at least 3 different MCP in E. coli, each responsive to stimuli from different types of chemoreceptor, including both attractants and repellants. Attractants elicit counterclockwise rotation of the fiagella, which results in smooth swimming. Repellants elicit clockwise rotation, which changes the bacterium's direction of motion. Methylation of MCPs results in adaptation, or lessened responsiveness to stimulation. D. Sherris & J. S. Parkinson (1981) Proc. Natl. Acad. Sci. USA 78, 6051-6055. Methylated xanthines: N-methyl derivatives of xanthine, biosynthesized by the enzymatic methylation of free xanthine (N-1,3 and 7) with •S-adenosyl-L-methionine. Caffeine, theobromine and theophylline (Fig.) occur in certain plants and are known as purine alkaloids. Caffeine (syn. thein, coffeine, guarine) acts as a central stimulant, and is present in tea, coffee, maté leaves, guaraña paste and cola nuts. Theobromine is the principal alkaloid of cacao beans (1.5-3%) and it is also present in cola nuts and tea. Theobromine is usually prepared from cacao bean hulls, which contain 0.7-1.2%. It acts as a diuretic, smooth muscle relaxant, cardiac stimulant and vasodilator. Theophylline is present in small amounts in tea. It has similar pharmaceutical properties to theobromine. The water solubility of M.x. can be increased by formation of molecular compounds with diethanolamine or isopropanolamine, and their solubility is also greatly increased by the presence of alkali benzoates, cinnamates, citrates or salicylates. For therapeutic purposes synthetic derivatives of M.x. are often used; these have improved water solubility compared with the natural M.x., e.g. 7-theophyllineacetic acid (l,2,3,6-tetrahydro-l,3-dimethyl-2,6-dioxopurine-7-acetic acid) and 1-theobromineacetic acid (2,3,6,7-tetrahydro-3,7-dimethyl-2,6-dioxo-lH-purine-1-acetic acid), which have the same therapeutic properties as theophylline and theobromine. The salt of 1-theobromineacetic acid with bromocholine phosphate is used as an

antihypertensive. The 1-hexyl derivative of theobromine, called pentifylline, is used as a vasodilator; it has increased lipid solubility, which favors absorption.

0 Ri—líí^SÍ

R

Xanthine Theophylline Theobromine Caffeine Structures

i H CH 3 H CH 3

of methylated

r3 H CH 3 CH 3 CHj

R, H H CH 3 CH 3

xanthines

O-Methylbufotenln: a toxin from the toad, Bufo alvarius (see Toad poisons), which has also been found in plants. In addition to its general properties as a toad poison, M. also has a psychotropic effect. The lowest fatal dose for mice is 75 mg per kg. Methylglyoxal: an intermediate of carbohydrate degradation in certain organisms. In some bacteria (Pseudomonas spp.) glyceraldehyde 3phosphate does not enter the normal glycolytic pathway; instead it is dephosphorylated to glyceraldehyde, followed by dehydration to M., which is converted to lactate (precursor of pyruvate) by a catalytic cycle involving glutathione (Fig.). Fig. see page 275

N 6 -cis-y-Methyl-Y-hydroxymethylallyl-adenosine 2 7 5

Microbiological preservation Glyceraldehyde phosphatase

Glucose -

• Glyceraldehyde

3-phosphate

3-phosphate

Glyceraldehyde

p, Pyruvate Lactate dehydrogenase (EC 1.1.1.27) Lactate

Glyceraldehyde dehydrase

^

H20

•• G l u t a t h i o n e C H j — CO — C H O -S-Lactoylglutathione

Hydroxyacylgiutathione ( G l y o x y l a s e H) (EC 3.1.2.6]

hydrolase H20

Metabolism of glyceraldehyde 3-phospbate in Pseudomonas.

N6-cis-y-Methyl-y-hydroxymethylallyl-adenoslne: 6-(4-hydroxy-3-methyl-but-ra-2-enyl)-aminopurine, an adenine derivative, a n d o n e of the rare nucleic acid components occurring in certain t R N A . Biosynthesis occurs by modification of an adenosine residue in the nucleic acid. It is the cis isomer of Zeatin (see), and the free c o m p o u n d , like zeatin, shows cytokinin activity. Methyltestosterone: 17a-methyltestosterone, 17a-methyl-17P-hydroxyandrost-4-ene-3-one, a synthetic androgen. M. shows high biological activity when administered orally, and it is used especially for the therapy of hypogenitalism, hormonal impotence, and peripheral circulatory disturbances. It is the 17a-derivative of Testosterone (see). Mevaldic acid: an intermediate in Terpene (see) biosynthesis. Mevalonic acid: an intermediate in Terpene (see) biosynthesis. Meyerhof quotient: see Pasteur effect. MF: abb. of maize factor (see Zeatin). MH: abb. of Maleic hydrazide. Michaelis constant: see Michaelis-Menten kinetics. Michaelis-Menten kinetics: 1. The MichaelisMenten stoichiometric model shows the relationship between free enzyme (E), substrate (S), enzyme-substrate complex (Michaelis complex, ES) and product (P): E + S^ ES - ^ - E + P where fc(~ind k^ are rate constants (fcj is also known as the catalytic constant, or fccat). (k_ j + kq)/k\ is a kinetic constant, known as the Michaelis constant and represented by Km. If k^• Siroheme — Nitrite. Assimilatory ferredoxin-dependent N.r. (EC 1.7.7.1) f r o m Spinach has A/r 61000, contains one molecule of siroheme and one inorganic (Fe 2 S 2 )-center. In vivo, reducing power is supplied by reduced ferredoxin f r o m the light reaction of photosynthesis. The sequence of electron transfer is: Ferredoxin —• (Fe 2 S 2 ) —>• Siroheme —»-Nitrite. Nitrogen: see Bioelements. Nitrogenase (EC 1.18.2.1): the enzyme system responsible for biological nitrogen fixation. N. consists of two proteins, both of which are required for activity. One of these proteins contains iron, m o l y b d e n u m and acid-labile sulfur (Mo-Fe protein, component I ; molybdoferredoxin, abb. azofermo) and the other contains iron a n d labile sulfur (Fe protein; component II; azoferredoxin abb. azofer.) The two components are present in the ratio I Mo-Fe protein: 2 Fe-proteins. Both protein components have been isolated a n d characterized f r o m various nitrogen fixing organisms. The separated components can be reconstituted to active N . The Mo-Fe c o m p o n e n t has molecular weight in the range 200000-270000, and is tetrameric. In addition to the four subunits, the Mo-Fe protein also contains a low molecular weight c o m p o n e n t which carries the molybdenum. This same molybdenum " c o f a c t o r " may be present in other molybdenum-containing enzymes. The Fe protein has molecular weight of about 60000, a n d is dimeric. N. f r o m different sources are very similar. Fe protein f r o m o n e organism (e.g. Azotobacter) will often cross react with Fe-Mo protein f r o m another organism (e.g. Klebsiella) to give a functional enzyme. On the other h a n d , heterologous N. components may generate catalytically inactive complexes, e.g. N. of Clostridium is inhibited by Mo-Fe protein of Azotobacter, because Clostridium Fe protein forms tight, inactive complex with Azotobacter Mo-Fe protein, even in the presence of excess Clostridium Mo-Fe protein. Synthesis of N. is determined by the aif operon, which has been m a p p e d in Klebsiella by phage P,-mediated transduction. Three structural genes for N., a n d for the molybdenum cofactor a n d electron transport to N. have been identified. Another gene appears to have a regulatory product, but its precise role is still unclear. Three further genes necessary for the full expression of the aif operon have been m a p p e d , but their role is unknown. The aif operon maps very close to the operon for histidine biosynthesis. The aif operon a n d the ability to fix nitrogen have been transferred f r o m Klebsiella pneumoniae to Escherichia coli by R-factor mediated conjugation; this represents the first case of the transfer of nitrogen fixing ability to a non-nitrogen fixing organism.

Nitrogenase

Klebsiella aif genes have been inserted into a plasmid (RP4, first isolated from Pseudomonas aeruginosa), which is t r a n s f e r a b l e to a wide variety of Gram-negative bacteria. When Klebsiella aif genes are transferred on RP4 to mutant aif Azotobacter, nitrogen fixation is restored despite the different physiology of the d o n o r a n d recipient organisms. N . is inoperative when nitrate is available as a nitrogen source, probably because N. is powerfully inhibited by traces of nitrite p r o d u c e d during nitrate reduction. Transcription of the aif operon is p r o m o t e d by active (non-adenylylated) glutamine synthetase. N. synthesis is repressed by ammonia, because an excess of a m m o n i a initiates a control mechanism, which leads to adenylylation (inactivation) of glutamine synthetase. N. catalyses the ATP-dependent reduction of several substrates that are of similar molecular size to, and are isoelectronic with molecular nitrogen, e.g. azide, nitrous oxide, cyanide, acetylene. Attempts to p r o d u c e models f o r the substrate activating center of N. have resulted in the preparation of organic complexes of transition metals. These are of potential interest in chemical industry, where more efficient catalysts are sought for industrial a m m o n i a synthesis. Such catalysts might also be exploited in the synthesis of hydrazine, which is a high energy and environmentally clean fuel, but prohibitively expensive. Combustion of hydrazine produces nitrogen and water, a n d the heat yield is about 544 k J / m o l (130 kcal/mol). N. is regarded as having two active centers: the electron-activating, a n d the substrate-activating center. The electron-activating center is on the Fe protein, which has the unique property of being able to transfer electrons to the Mo-Fe protein. The ATP consumed by N. is largely required to increase the reducing potential of the electrons carried by the Fe proteins, thereby producing an extremely p o w e r f u l reducing agent. Thus a ferredoxin (or flavodoxin, or rubredoxin)-dependent ATPase activates a n d reduces a metal-containing group X to X * e d , which represents a metal hydride. The Mo-Fe protein contains the substrate-activating center. With the aid of molybdenum, the substrate is b o u n d in such a way that it can be reduced by the electrons received f r o m X ' e d on the Fe protein. The Mo-Fe protein contains 2Mo, 28-32 Fe a n d a b o u t 28 acid-labile sulfurs per tetrameric protein. U p to four Fe 4 S 4 centers are present. Two other centers appear to contain MoFe 8 S 6 , part of which may exist as a cubane structure similar to Fe 4 S 4 , but with one Fe replaced by M o (i.e. M o F e j S J . There may also be an Fe 2 S 2 center. The multinuclear Mo-containing center appears to be b o u n d by two symmetrical polypeptide chains. Electrons are transferred singly from the ferredoxin-like Feprotein, and stored in the Mo-Fe center; acting rather like an electrical capacitor, this center is then able to transfer several electrons in o n e stage (Fig.); in the case of the natural substrate, N 2 , 6 electrons are transferred. Electrons may also be transferred to protons, producing molecular hy-

302

Nitrogen balance

Nitrogen fixation ammonia of the synthesis of various enzymes of nitrogen metabolism. See Ammonia assimilation. Nitrogen cycle: see Nitrogen fixation.

drogen. This ATP-dependent hydrogen evolution competes with ammonia synthesis. It is quite distinct from bacterial hydrogenase, which does not Nitrogenase

E l e c t r o n - a c t i v a t i n g centerj

Na2S204 \

/

N, 2e-

T

Mg

ATP

Ferredoxin-2e

H2

T

x

red—f

ADP+P; (ATPase)

Hydrogenase

Pyruvate

Substrate-binding

H2

ATP-dependent

H2

+ 6 e •

center

2NH,

N,0 + 2 e •

N2+H20

C2H2 + 2 e - *

C2H4

HCN + 4 e

CHJNH2

HCN + 6 e — »

CH^+NH3

evolution

require ATP. In Azotobacter and in Rhizobium bacteroids, hydrogen produced by N. is taken up again by hydrogenase, then oxidized with consequent synthesis of ATP. The activity of cell-free preparations of N. is usually measured by colorimetric assay of ammonia, following incubation with molecular nitrogen. Nitrogen fixation in general can be determined from the incorporation of 15 N 2 into cell material, but the method is relatively expensive and tedious. The use of acetylene (ethyne) as an alternative substrate of N. has revolutionized studies of nitrogen fixation. Assays are performed in a closed system. The product, ethylene (ethene), is not assimilated and it can be easily assayed by gas chromatography. Nitrogen balance: difference between the total nitrogen intake of an organism and its total nitrogen loss. Young growing animals are in positive N.b., i.e. they retain more nitrogen (as protein added during growth) than they excrete. Mature, healthy adults show a zero N.b., i.e. nitrogen intake is exactly balanced by nitrogen excretion. A negative N.b. results from a deficiency of an essential amino acid; a decrease in the concentration of any proteogenic amino acid in the body's amino acid pool impairs total protein synthesis, so that the concentration of all other free amino acids in the pool is increased; this leads to an exaggeration of degradative pathways and an increase in urea formation. The classical deletion method for the determination of the essential (indispensable) amino acid requirements of an animal involves the measurement of N.b. in adult animals receiving a complete diet, except for the omission of the amino acid under test. The daily food intake is determined, and the nitrogen content of an identical food sample is measured. Strictly, in determining the daily loss of nitrogen, dropped hair, sloughed skin and perspiration should be taken into account, but it is usually sufficient to omit these minor contributions, and use only the nitrogen content of feces and urine in the determination of N.b. Nitrogen catabollte repression: repression by

Nitrogen excretion: see Ammonia detoxification. Nitrogen fixation: a process in which atmospheric nitrogen is converted into ammonia. Activation of molecular nitrogen and its reduction to ammonia depend upon the catalytic activity of the enzyme Nitrogenase (see). The ammonia is then incorporated into the various nitrogenous compounds of the cell by the processes of Ammonia assimilation (see). Nitrogenase is a very unstable enzyme, especially in anaerobic organisms. N.f. is fundamentally important for the nitrogen economy of soils and waters, and it forms an essential stage in the nitrogen cycle of the biosphere. Certain free living soil microorganisms, especially of the genera Clostridium and Azotobacter, are capable of N.f. Other microorganisms fix nitrogen in symbiosis with higher plants, notably the Leguminosae. Many instances of N.f. by symbiotic associations between microorganisms and nonleguminous plants are also known, e.g. an Actinomycete has been isolated from the nitrogen fixing root nodules of Alder (Alnus). In water, especially in the ocean, the most important nitrogen fixers are the blue green bacteria (also commonly known as blue green algae, a term that is preferably avoided, because these nitrogen fixing organisms are prokaryotes, whereas the algae are eukaryotes). N.f. by blue green bacteria is of practical importance for the cultivation of rice in the tropics. The lichens (symbiotic associations between a blue-green bacterium and a fungus), and the symbiotic system Nostoc-Gunnera (i.e.symbiosis between a blue green bacterium and an angiosperm) are ecologically very important, since they are able to colonize habitats that have extreme climates or are poor in nutrients. The carbon and nitrogen requirement of the lichens are met by photosynthesis and N.f. Such symbiotic systems are therefore sustained largely by the atmosphere, and their nutritional demands on the remaining environment are relatively small. Lichens pioneer the exploitation of barren environments and pave the way for later colonization by plants with more exacting nutritional re-

Nitrogen storage

303

quirements. In poor soils, the Nostoc-Gunnera system can fix about 70 g atmospheric nitrogen per m 2 per year. The ability to perform N.f. is a special characteristic of relatively few prokaryotic organisms; it has never been detected in a eukaryote. In the bacterium, Clostridium pasteurianum, which contributes to the nitrogen enrichment of agricultural soils, both the reducing power and the ATP required for N.f. are derived from phosphoroclastic pyruvate cleavage. In cell-free enzyme preparations pyruvate can be replaced by ATP or an ATP generating system, and a reducing agent (hydrogen or electron doner). Suitable reducing agents include sodium dithionite and potassium borohydride. Nitrogenase will also catalyse the transfer of electrons from molecular hydrogen to nitrogen in the presence of a ferredoxin-dependent hydrogenase. In most nitrogen-fixing systems, the natural electron donor is a ferredoxin; in certain cases, this is replaced by other electron transferring proteins, e.g. flavodoxin, or rubredoxin. Four molecules of ATP are required for the transfer of each pair of electrons. The stepwise reduction of nitrogen on the surface of the nitrogenase may take place via enzyme-bound intermediates, but free intermediates between ammonia (the product) and N 2 (substrate of N.f.) have not been observed. So far, the most extensively studied N.f. system is the symbiotic association between members of the Leguminosae and Rhizobium; the legume often chosen for these studies is Glycine max (Soybean). Infection of the plant roots by virulent Rhizobia leads to the formation of root nodules, which have the capacity to fix nitrogen. The Rhizobia living free in the soil do not fix nitrogen. Under laboratory conditions, however, pure cultures of Rhizobium will fix nitrogen, providing a pentose (e.g. arabinose) and a dicarboxylic acid (fumarate or succinate) are present in the culture medium. During the infection process, the Rhizobium cells lose their rod-like shape and eventually become globular-shaped bacteroids. Reduction of nitrogen to ammonia and the assimilation of the ammonia occur in these bacteroids; carbon compounds for the respiration of the bacteroids and for the assimilation of the ammonia are provided by the plant; amino acids are exported to the host plant tissues. The natural electron donor for the symbiotic N.f. has not been identified. Leghemoglobin is necessary for N.f. by legume root nodules, but it is not required by nonlegume N.f. systems. The concentration of leghemoglobin in root nodules is an index of the nitrogen fixing capacity. The bacteroids are bathed in a solution of leghemoglobin, which is enclosed by a membrane envelope. The rate of oxygen transport through an unstirred solution of leghemoglobin is eight times higher than its rate of diffusion through water. This facilitated diffusion of oxygen to the bacteroids permits a high respiration rate, which is necessary to produce the relatively large quantities of ATP required by the nitrogenase. In contrast, oxygen interferes during the laboratory preparation of active bacte-

Nonhistone chromatin proteins roids, due to the presence of phenols and polyphenol oxidases from the host plant tissue. These can be inactivated by adsorption onto polyvinyl pyrrolidone in the presence of ascorbic acid. Nitrogen fixing bacteroid suspensions can therefore be isolated from homogenized root nodules by using strictly anaerobic conditions, e.g. centrifugation of the homogenate under argon, or by abolishing polyphenol oxidase activity. The bacteroids can then be treated like any other bacterial source of nitrogenase. Subsequent disruption of the cells and purification of the enzyme by selective precipitation and column chromatography must be performed under strictly anaerobic conditions, because nitrogenase is irreversibly inactivated by oxygen; this is especially critical at later stages of purification, as the oxygen sensitivity of nitrogenase increases with purification. The separate protein components of nitrogenase are both inactivated by oxygen, and the Fe-protein is the more sensitive. Nitrogen storage: see Ammonia detoxification. nm: abb. for nanometer. Nodule bacteria: see Rhizobia. Noncompetitive Inhibition: see Effectors. Noncovalent bonds: various types of noncovalent bond are responsible for maintaining chain conformation and quarternary structure of proteins, and they are also important in the structure and function of nucleic acids. 1. Hydrogen bonds are formed between neighboring peptide bonds (separation distance 0.28 nm), between tyrosyl and carboxyl or imidazole, and between seryl and threonyl residues. Hydrogen bonding in particular is an important factor in nucleic acid structure, and in template recognition during replication, transcription and translation (see Base pairing; Ribonucleic acid; Protein biosynthesis; Deoxyribonucleic acid). 2. Heteropolar (electrostatic) bonds in proteins are formed between residues of opposite charge, e.g. lysyl and glutamyl. 3. Apolar (hydrophobic) bonds in proteins are formed between very close, uncharged groups, e.g. - C H 3 and — CH 2 OH, or between more widely separated, uncharged groups, e.g. phenyl and leucyl. The effective strength of these hydrophobic bonds is increased by the entropy effect of the repulsion of the surrounding water, and they contribute to the stability of protein conformation especially at elevated temperatures. 4. Van der Waals forces act at only very short distances, and they represent the weak attraction between the positively charged nucleus of one atom and the negatively charged electrons of another. They are important in base stacking in the double helix of DNA (see Deoxyribonucleic acid). Nonheme Iron proteins, NHI-proteins: proteins containing iron that is not bound in a heme system. In these proteins, the iron is bound by the sulfur of cyteine residues, and it is often also associated with inorganic sulfur. They are also called iron-sulfur proteins. See Ferredoxin; Rubredoxin. Nonhistone chromatin proteins: "acidic chromatin proteins", a highly heterogeneous group of

N o n o r d e r e d conformation

304

tissue-specific proteins, which are b o u n d to certain D N A sequences. Their M r in detergents is 30000-70000, i.e. markedly higher t h a n the M r of Histones (see). As gene derepressors, they play a part in the regulation of gene expression in mammalian cells, especially in cell proliferation. Nonordered conformation: see Proteins. Nonsense codon: an Amber codon (see), or Ochre codon (see). Nopallne: see D-Octopine. Nopallnic acid: see D-Octopine. Noradrenalln, norepinephrine, arterenol: dihydroxyphenylethanolamine, a h o r m o n e with activity on the nervous system a n d the vascular system. Mt 169.2. M. is a catecholamine a n d a biogenic amine. Together with Adrenalin (see), N. is synthesized f r o m L-tyrosine via D o p a (see Dopamine) in the adrenal medulla and in the nervous system. In the sympathetic nervous system, N . acts as an adrenergic Neurotransmitter (see). N. causes contraction of blood vessels, with the exception of the coronary vessels; it therefore causes an increase in the peripheral vascular resistance, with a consequent rise in blood pressure. The effect of N. on carbohydrate metabolism and blood sugar is similar to, but weaker than that of adrenalin. N. is deactivated by O-methylation; oxidative deamination by a m o n o a m i n e oxidase then produces the urinary excretory product, 3methoxy-4-hydroxymandelic acid (vanillylmandelic acid). Vanillymandelic acid (VMA) is the major metabolite of N. in the peripheral parts of the b o d y ; it is measured in the urine as an index of parasympathetic nervous function, and for the diagnosis of tumors that produce N. or adrenalin, e.g. pheochromocytomas and neuroblastomas. h2n—ch2 HO—CH

Noradrenalin Norepinephrine: see Noradrenalin. Norgestrel: a synthetic gestagen. N. is used inoral contraceptives, and it is the most potent of the orally active gestagens. OH

Norgestrel

Nucleic acids Norlaudanoslne: see Benzylisoquinoline alkaloids. Nornicotlne: see Nicotine. Norsterolds: see Steroids. Notatin: see Mycotoxins. /V-terminal amino acid: see Peptides. Nu bodies, nucleosomes: subunits of Chromatin (see) containing 180 to 200 base pairs of D N A and approximately an equal weight of histone. They are released by partial digestion of chromatin by staphylococcal nuclease. Nucleases: a group of hydrolytic enzymes, which cleave nucleic acids. Exonucleases attack the nucleic acid molecule at its terminus, whereas endonucleases are able to catalyse a hydrolytic cleavage within the polynucleotide chain. Deoxyribonucleases (DNAases) are specific for D N A , and ribonucleases (RNAases) for R N A . All N. are Phosphodiesterases (see); they catalyse the hydrolysis of either the 3' or 5' bond of the 3',5'phosphodiester linkage. Ribonuclease (see) has been extensively studied. The /V-glycosidic bond of nucleic acids is cleaved by Nucleosidases (see). Nucleic acid bases: constituent bases of nucleic acids. N.a.b. are f u n d a m e n t a l to the function of nucleic acids in the storage a n d transfer of genetic information. They are Adenine (see), G u a n i n e (see), Cytosine (see), Thymidine (see), Uracil (see), a n d others that occur less frequently (see Rare nucleic acid components). See also Nucleic acids; Genetic code; Base pairing. Nucleic acids: polymerized nucleotides; a m o n g the most essential components of all living cells, viruses and bacteriophages. N.a. were first isolated in 1869 from the white blood cells of pus by Miescher, w h o called the material nuclein. The term N.a. was introduced in 1889 by Altmann, in recognition of their acidic properties. There are two main classes of N.a., distinguished by their carbohydrate c o m p o n e n t : Ribonucleic acid ( R N A ) (see), which contains ribose, and Deoxyribonucleic acid ( D N A ) (see), which contains 2-deoxy-D-ribose. Both types have certain c o m m o n structural features, but they have different biological functions. D N A stores genetic information; it is replicated during cell division, so that each daughter cell receives D N A that is identical in structure a n d informational content (see Genetic code). R N A is intimately involved in protein synthesis, and is primarily responsible for translating the information of the D N A into the primary structure of specific proteins (see Protein biosynthesis). N.a. are Polynucleotides (see) with M r between 20000 a n d several million. They contain three characteristic structural components: 1. the purine a n d pyrimidine bases, adenine, guanine, cytosine a n d uracil (in RNA), thymine (in D N A ) ; in addition, there are over 30 other Rare nucleic acid components (see), which occur in various N.a. These rare components are formed by modification of existing structures within the N.a., e.g. by methylation, hydrogenation or rearrangement of normal bases; 2. the pentose monosaccharide, D-ribose (in RNA), or 2-deoxy-D-ribose (in D N A ) ; 3. phosphoric acid.

Nucleic acids

Nucleic acids

305

The intensity of UV-absorption depends upon the conformation, i.e. the secondary structure of the N.a. Thus optical methods can also be used in the structural elucidation of N.a., and for detecting and monitoring structural changes. At high temperatures, or with very large changes of ionic concentration, the secondary structure of N.a. is destroyed (denaturation). Thus, DNA loses its double helix structure and becomes a single stranded random coil (helix-coil transition). Denaturation results in an increase of absorption at 260 nm. (see Hyperchromic effect). From the course of this so-called melting point curve, it is possible to assess the helical content of a N.a., and to determine the GC-content of a

Each mononucleotide unit is linked to its neighbor by a phosphate group, which forms an ester linkage with position 3' of one sugar and 5' of the neighboring sugar. This 3',5' linkage results in a linear chain of phosphate-linked sugar residues; a base is attached to C-1 of each sugar by an Nglycosidic linkage. The linear order of bases is statistically irregular, representing the informational code of the N.a. Determination of the base sequence of N.a. is therefore of great interest and importance. The complete primary structure of some tRNA (see Transfer RNA) species has been determined by chromatographic and electrophoretic identification of components during step-

- 0—ch2

h n ^ Y ™

3

cAn^

r0 î ^ {Oi 0n

Pî — o — c h 2

o Polynucleotide structure wise, specific hydrolysis with nucleases. The reactive NH 2 -, OH- and NH-groups of purine and pyrimidine bases are responsible for certain properties of N.a., e.g. formation of specific hydrogen bonds between purines and pyrimidines, leading to secondary structures. Thus, complementary linear chains can form a double helix (see DNA), or a linear strand can fold on itself, forming alternate linear and helical regions (RNA). Other forces involved in the spatial conformation of N.a. (see Noncovalent bonds) are homopolar cohesive forces (Van der Waals forces), hydrophobic interactions between bases and solvent, and electrostatic interactions (ionic bonds). Physical properties and analytical methods: The conjugated double bonds in the heterocyclic rings of the bases absorb UV-light in the region of 260 nm. UV-spectrophotometric analysis is therefore used for the characterization and quantitative determination of N.a. and lower M r , related compounds, such as mono- and oligonucleotides.

p—0-— I

e

3'

DNA sample (see T m -value). If the heat-denatured N.a. is cooled slowly, the original structure is largely reformed (renaturation). Thus, heat-denatured, single stranded DNA, consisting of a mixture of complementary strands, will form a double helix if the solution is cooled very slowly below the melting point. Renaturation is not, however, always complete. Bacterial D N A renatures more extensively than nuclear D N A from higher organisms. Heat-denatured RNA can also be largely renatured to its original form. Other physical methods used in the investigation of N.a. structure are: Hybridization (see), electron microscopy, analytical ultracentrifugation, CsCl-density gradient centrifugation, X-ray diffraction analysis, infrared spectroscopy, optical rotatory dispersion, light scattering photometry, and viscosity measurements. N.a. are purified by column chromatography (e.g. methylalbumin-silicic acid columns), by electrophoresis (e.g. polyacrylamide gels) and by density gradient centrifugation (e.g. sucrose or CsCl).

306

Nucleocidin N.a. may be determined quantitatively from the UV-absorption due to the bases, by determination of the phosphate content, or by specific color reactions for ribose or deoxyribose ( Dische reagent for DNA; Dische-Schwarz reagent for RNA). The Feulgen reaction is used for the histochemical detection of DNA. Nucleocidin: a purine antibiotic synthesized by Streptomyces calms (see Nucleoside antibiotics). M t 392, [a]g —33.3°. N. is active against bacteria and fungi, and it is used therapeutically against trypanosomes. It inhibits protein synthesis.

Nucleocidin Nucleolus (plur. Nucleoli): a compartment of the nucleus. The N. contains the N. organizer (see). The number of N. per nucleus varies widely. The main components of the N. are proteins (over 80% of the dry weight), RNA (over 5%) and DNA. The N. contains the following recognizable structures: a ground material of amorphous protein, ribonucleoprotein granules, ribonucleoprotein fibrils, and the chromatin fibrils of the N. organizer. The N. is the site of biosynthesis of ribosomal RNA (see Ribosomes). During nuclear division, the N. temporarily disappears as a visible structure. Nucleolus organizer: a specific region on one or more eukaryotic chromosomes, where the nucleolus is formed. The DNA in this region contains genetic information for the synthesis of ribosomal RNA. Nucleoplasm: see Nucleus. Nucleoproteln: heteropolar complexes of nucleic acids (in particular, nuclear DNA) with basic, acid-soluble proteins (histones or protamines),

Nucleoside antibiotics and with acidic, base- or detergent-soluble nonhistone-chromatin proteins. N. occur mainly in the chromatin of the cell nucleus in its quiescent state, and in the chromosomes when the nucleus is active, i.e. dividing. Many viruses consist entirely of N., but N. are absent from bacteria. N. are concerned in DNA replication, and in the control of gene function during protein biosynthesis. The protein-RNA complexes of the ribosome are also N. Nucleosidases: enzymes that catalyse the cleavage of the bond between the sugar residue and the base of a Nucleoside (see). The reaction is usually a phosphorolysis (not a hydrolysis), involving orthophosphate. Nucleoside antibiotics: purine or pyrimidine nucleosides with antibiotic activity. They act as antimetabolites of natural substrates, and inhibit the growth of microorganisms by blocking the metabolism of purines, pyrimidines and proteins. Some N.a. (e.g. showdomycin) contain an analog base, others (e.g. gougerotin) contain an analog sugar, or both moieties may be modified (e.g. puromycin) (see Table). The analog components are formed by the modification of primary metabolites. The sugars or sugar derivatives, such as cordycepose, psicose, angustose and glucuronamide are derived from Dglucose or D-ribose by various reactions, e.g. epimerization, isomerization, oxidation, reduction and decarboxylation. Methyl groups occur frequently in N.a., and are derived by transmethylation. A normal nucleoside may be modified to a N.a. without prior cleavage of the JV-glycosidic bond, e.g. the synthesis of tubercidin. Alternatively, the free base may combine with the analog sugar, as in the synthesis of psicofuranin. Unusual amino acids occur in some N.a., e.g. amicetin contains a-methyl-D-serine. Some N.a. contain unusual bond types, such as the aza bond in 5-azacytidine, or unusual functional groups, such as the CN-group in toyocamycin. The same N.a. may be formed by systematically unrelated organisms, e.g. cordycepin from Cordyceps and Aspergillus. One organism may produce several N.a., e.g. psicofuranin and decoyinin from Streptomyces hygroscopicus.

Nucleoside antibiotics. Naturally occurring nucleoside analoges with antibiotic acirity. For mode of action, see separate entry for each compound. Antibiotic

Produced by

Base

Helminthosporium spec. Adenine 3' Acetoamido3'-deoxyadenosine Amicetin A Streptomyces fascinila- Cytosine tus, Streptomyces vinaceits-drappus Cytosine Amicetin B (Plica- Streptomyces plicatus cetin) 3'-Amino-3'-deoxy- Cordyceps militaris Hel- Adenine adenosine minthosporium spec. Angustmycin A Streptomyces hygroscop-Adenine (Decoyinin)

Sugar

Antimetabolite of

3-Acetamido-3-de- Adenosine oxyribose Amicetose Cytidine Amicetose

Cytidine

3-Amino-3-deoxy- Adenosine ribose L-2-Ketofucopyra- Adenosine, Guanonose sine

307

Nucleoside antibiotics Antibiotic

Produced by

Angustmycin C (Psicofuranin) Arabinofuranosyladenine 5-Azacytidine

Streptomyces hygrosco- Adenine picus Streptomyces antibioti- Adenine cus Streptoverticillius /afajd-Azacytosine amus Streptomyces plicatus Cytosine Streptomyces gríseoCytosine chromogenes

Bamicetin Blasticidin S Cordycepin

Cordyceps militaris Aspergillus nidulans Nocardia interforma

Formycin

Nucleoside antibiotics Sugar

Base

Antimetabolite of

Psicose (Psicofura- Adenosine, Guanonose) sine Arabinofuranose Adenosine

Adenine

Ribose

Cytidine

Amicetose 4-Deoxy-4-amino2,3-hexenuronic acid Cordycepose (3-Deoxyribose) Ribose

Cytidine Cytidine, AcyltRNA Adenosine

7-Aminopyrazolopyrimidine 7-Hydroxypyrazol- Ribose Nocardia interforma Streptomyces lavendu- opyrimidine lae, Streptomyces roseochromogenes Streptomyces gougeroti Cytosine 4-Dideoxyglucopyranuronamide Agaricus (Clitocybe) Purine Ribose nebularis, Streptomyces spec. Streptomyces calms Adenine Hexose of unknown structure 3-Amino-3-deoxyStreptomyces albo-niger Dimethylaminopurine ribose Streptomyces spec. 4-Amino-5-carbox- Ribose amide-7-pyrrolopyrimidine Streptomyces showdoen-Ma\eimide Ribose sis Streptomyces toyocaen- 4-Amino-5-cyano- Ribose sis, Streptomyces rimo- 7-pyrrolopyrimisus dine Streptomyces tubercidi- 4-Amino-7-pyrrolo- Ribose cus pyrimidine

Formycin B

Gougerotin Nebularin Nucleocidin Puromycin Sangivamycin Showdomycin Toyocamycin Tubercidin

Adenosine Adenosine

Cytidine, AcyltRNA Adenosine Adenosine Acyl-tRNA Adenosine

Uridine Adenosine

Adenosine

NH, 'r

n

Oy

N

R

r

= JO HOCH^O

H

Cordycepin

R-=

CHoOH OH OH

OH

HOCHj n

L^ \ l

CH3

Psicofuranin

R=

Angustmycin A

01 OH OH

Decoyinin

OH OH

Fig. 1. Structures of cordycepin, psicofuranin, decoyinin and angustmycin A.

Nucleoside diphosphate compounds

308

NH2

Nucleosides uridine diphosphate glucose, and it is a precursor of glucuronides (see Glucuronate pathway).

k

R=H

Tubercidin

R = C=N

Toyocamycin

HOCH^O^

R=C0—NH 2 Sangivamycin

hY^H OH OH

Fig. 2. Structures of tubercidia, toyocamycin and sangiramycin. Nucleoside diphosphate compounds: compounds containing a nucleoside diphosphate grouping. This grouping has an activating effect, so that the molecule has a high group transfer potential. Examples of N.d.c. are Nucleoside diphosphate sugars (see) and Cytidine diphosphate choline (see). Nucleoside diphosphate sugars, nucleotide sugars: energy-rich nucleotide derivatives of monosaccharides. The activating group is a nucleoside diphosphate. Uridine diphosphate glucose (UDP-glucose, UDPG, "active glucose") is of widespread general importance in carbohydrate metabolism. It is synthesized from glucose 1-phosphate by reaction with uridine triphosphate (Fig.). Other nucleoside diphosphate groups found in N.d.s. are listed in the table.

Glucose

Activated molecule

Function

Uridine diphosphate

Glucose

Involved generally in carbohydrate metabolism. Glycogen (see) synthesis. Murein (see) synthesis. Galactose (see) metabolism. Glucuronate pathway (see). Glucuronate synthesis. Metabolism of amino sugars. Chitin synthesis. Starch (see) synthesis. Synthesis of L-fucose, D-rhamnose and 6-deoxyhexoses. Synthesis of Lrhamnose.

Galactose Glucuronate

JV-Acetylglucosamine Adenosine di- Glucose phosphate Guanosine di- Mannose phosphate

6-phosphate

Phosphoglucom u t a s e ( E C 2.7.5.1) Glucose

Table. Nucleoside diphosphate sugars. Activating nucleoside diphosphate group

r

Glucose 1,6 - diphosphate

1-phosphate-

Glucose 1 - p h o s p h a t e uridylyltransferase ( E C 2.7.7.9)

Uridine t r i p h o s p h a t e ( U T P )

Deoxythymidine diphosphate Cytidine diphosphate

Glucose

Ribitol Glycerol

Synthesis of Teichoic acids (see).

Pyrophosphate

OH'

N CHjOH

w

CHrO-®-®-5\

)H

OH OH

Uridinediphosphate

glucose

Synthesis of uridinediphosphate

glucose

The activated sugar can take part in various metabolic reactions. Of particular importance is the transfer of the sugar moiety to the OH-group of another molecule, e.g. in the synthesis of oligoand polysaccharides, (see Carbohydrate metabolism). Nucleoside diphosphate derivatives of uronic acids are also metabolically important, e.g. uridine diphosphate glucuronic acid is formed from

Nucleosides: JV-glycosides of heterocyclic nitrogenous bases. The JV-glycosides of purines and pyrimidines with pentoses are of particular biological importance. The sugar component is either D-ribose or D-2-deoxyribose, both in the furanose form (Table). C-l of the pentose residue is linked to N-9 of the purine or N-l of the pyrimidine by an N-glycosidic linkage (C-N bond). To distinguish between the numbering systems of the base and sugar, the numbers of the sugar atoms are characterized by a prime, i.e. C-atoms 1' to 5'. Deoxynucleosides contain D-2-deoxyribose,whereas ribonucleosides contain D-ribose. N. have trivial names derived from the component base. Pyrimidine N. end in -idine, purine N. in -osine. The Rare nucleic acid components (see) represent nucleoside moieties in which the base or sugar is chemically modified. N. and deoxynucleosides can be synthesized via a Salvage pathway (see). They are also produced by the hydrolysis of nucleic acids and nucleotides.

Nucleosides

309

Nucleosides

Nucleoside phosphorylases and deoxynucleoside phosphorylases catalyse the reversible, phosphate-dependent cleavage of N. and deoxyribonucleosides, forming ribose 1-phosphate or deoxyribose 1-phosphate and the free base. N. and deoxyribonucleosides can be converted into

Purine

base

R'= H Nucleoside or Deoxynucleoside

their corresponding nucleotides by the action of specific kinases. Strictly speaking, the term nucleoside is reserved for base-sugar combinations present in nucleic acids, but the term is often applied to any basesugar compound.

Pyrimidine

R}= H Nucleoside or Deoxynucleoside

base

R2= H

Adenosine Deoxyadenosine

R4 = 0H

R, = NH, R 6 = OH G u a n i n e

Guanosine Deoxyguanosine

r 4 = nh 2 „

Cytidine Deoxycytidine

R2= H HypoR 6 = OH xanthine

Inosine (Deoxyinosine)

R t =0H R5=CH3Thym,ne

Ribothymidine Thymidine*

R'=

R'=

R'=

R6 = N H 2 A d e n i n e

Nucleotides :

of purine

®

Adenosine Guanosine y monophosInosine phate Uridine Cytidine Thymidine.

*The sugar component

T a b l e . Structure

p _ j_l

of t h y m i d i n e

and pyrimidine

is

Uridine Deoxyuridine

Uracil Cytosine

® ~ ®

Adenosine Guanosine diphosInosine " phate Uridine Cytidine Tymidine .

Adenosine Guanosine .triphosInosine phate Uridine Cytidine Thymidine -

2'-deoxyribose

bases, nucleosides,

deoxynucleosides

1

R = H Nucleoside or Deoxynucleoside

Purine base

R^ R, R^ R2 R,;

= NH2 = NH, =OH = H =OH

Adenine

„ Guanlne

Hypoxan thine

Adenosine Deoxyadenosine Guanosine Deoxyguanosine Inosine (Deoxyinosine)

®~®~®

= = = =

OH H NH, H

R 4 = CH

..

nucleotides

R> = H Nucleoside or Deoxynucleoside

Pyrimidine base R, R* R4 R5

and

.,

Uracl1

Thy" 1 ' 116

R>=® Ri = ® ~ © Adenosine Adenosine Guanosine Guanosine . Inosine mono-phos- Inosine Nucleotides : diphosphate Uridine phate Uridine Cytidine Cytidine Thymidine Thymidine ' The sugar component of thymidine is 2'-deoxvribose

Uridine Deoxyuridine Cytidine Deoxycytidine Ribothymidine Thymidine* R1 = ® ~ ® ~ ® Adenosine Guanosine Inosine Uridjne

Cytidine Thymidine

Nucleotide coenzyme

310

Nucleotide coenzyme: a coenzyme containing a nucleotide structure. N.c. are Pyridine nucleotide coenzymes (see), the nucleoside diphosphate moieties of Nucleoside diphosphate sugars (see) and Coenzyme A (see). The Flavin nucleotides (see) are also N.c. Strictly speaking FMN is not a nucleotide, but the term nucleotide can be generally applied to any base-sugar-phosphate group. Nucleotides, nucleoside phosphates: phosphoric acid esters of Nucleosides (see), o-Phosphoric acid is esterified with a free OH-group of the sugar. If the sugar is D-ribose, the N. is called a ribonucleotide or ribotide. If the sugar is D-2deoxyribose, the N. is a deoxyribonucleotide, deoxynucleotide or deoxyribotide. The phosphate may be present on position 2', 3' or 5' (in deoxyribonucleotides, only the 3'- and 5'-phosphate are possible). The 5'-nucleoside phosphates are metabolically very important; they may be mono-, dior triphosphorylated, e.g. guanosine 5'-monophosphate, cytidine 5'-diphosphate and adenosine 5'-triphosphate. The cyclic N. (cyclic 3', 5'-monophosphates) have important regulatory properties (see Adenosine phosphates; Guanosine phosphates; Inosine phosphates; Uridine phosphates). Nucleoside monophosphates are synthesized de novo in the course of Purine biosynthesis (see) and Pyrimidine biosynthesis (see). They are then phosphorylated stepwise by the action of kinases, to produce nucleoside di- and triphosphates. The 2-deoxyribose moiety is formed by reduction of the ribose in ribonucleotides (see Ribonucleotide reductase). Reduction of free ribose to 2-deoxyribose does not occur in vivo. N. and deoxynucleotides are the monomelic components of Oligonucleotides (see) and Polynucleotides (see). Enzymatic degradation of oligo- and polyribonucleotides (but not the corresponding deoxyribonucleotides) produces cyclic 2', 3'-nucleoside phosphates. N. are cleaved hydrolytically to nucleosides by the action of 5'or 3'-nucleotidases, which function as phosphomonoesterases. N. are cleaved to free bases and phosphoribosylpyrophosphate in a pyrophosphate-dependent reaction catalysed by N. pyrophosphorylases. Certain coenzymes contain nucleotide structures (see Nucleotide coenzymes); thus NADP contains the structure of adenosine 2', 5'-diphosphate; and coenzyme A the structure of adenosine 3', 5'-diphosphate. In all living cells, N. (especially adenosine 5'-triphosphate) act as so-called high energy compounds in the storage and transfer of chemical energy. Nucleotide sugar: see Nucleoside diphosphate sugars. Nucleus: a large structure ( ~ 5 |im diam.) in eukaryotic cells, containing the bulk of the cellular DNA, and representing the chief site for the storage, replication and expression of genetic information. In the period between cell divisions, i.e. at interphase, the nucleus is densely and uniformly packed with DNA and shows few distinct structures, even under the electron microscope.

Nutrient medium Distinguishable features are Chromatin (see), nucleoplasm, nuclear membrane and the Nucleoli (see). At nuclear division, the highly structured Chromosomes (see) are formed from the chromatin. Chromatin and chromosomes are composed mainly of DNA, RNA and numerous proteins. Important enzyme proteins are DNA-polymerase for DNA-replication, and RNA-polymerase for transcription. Other chromosomal proteins (histones, protamines and acidic proteins) are engaged in the regulation of replication and transcription (see Chromosomes). The molecular structure of the nuclear DNA-protein complexes is largely unclear. Other important metabolic processes occur in the nucleoplasm, e.g. glycolysis and tricarboxylic acid-cycle. NAD is synthesized only in the nucleus. The nuclear N a + concentration is ten times higher than that of the cytoplasm. The nucleus also contains a complete protein synthesizing system, but it is still largely unknown which nucleoproteins are synthesized in the nucleus. The nuclear membrane is important in the transfer of high and low M r compounds between nucleus and cytoplasm. Isolated nuclei are highly permeable to histones, protamines and other biological macromolecules, whereas ATP and N a + become tightly bound. The chief components of isolated and disrupted nuclei are DNA-histone complexes (nucleohistones), ribonucleic acids and poorly soluble acidic proteins (residual proteins) Nuclei also contain high concentrations of an arginase and an adenosine 5'-phosphatase of unknown function. Nuphara alkaloids: a group of alkaloids possessing a piperidine or quinolizidine ring system, which are found in various species of water lily (Nuphar spp.). All N.a. contain a sesquiterpene skeleton, which is cyclized by the inclusion of other atoms (nitrogen, oxygen or sulfur). The chief N.a. are nupharidine, nupharamine and thiobinupharidine. Castoreum or castor, the secretion from the preputial follicles of the beaver, contains castoramine (M r 247, m.p. 65-66°C) which is related to the N.a.; it is not known whether it is synthesized by the animal, or derived from water lilies in its diet. For the biosynthesis of N.a., see Terpene alkaloids.

Nupharamine

Nupharidine

Nutrient medium, growth medium: a medium, liquid or solid, for the cultivation of microorganisms, cells, tissues or organs. Solid media are

Nutritional physiology of microorganisms

311

prepared f r o m liquid media by the addition of a gelling agent, e.g. gelatine (nowdays used only in special cases), silicic acid (when exclusion of organic compounds is necessary), or Agar-agar (see) (used widely in bacteriology, usually at a concentration of 1.5-2%). N.m. contain fairly large amounts of mineral elements, together with trace elements. Sufficient quantities of certain trace elements are often already present as impurities in the other components of the medium. The mineral constituents, or inorganic nutrients, must be correctly balanced in order to avoid competitive effects of ions, to achieve an appropriate p H value, and to establish the correct oxido-reduction status of the N.m. The composition of a complex N.m. is more or less ill defined. This may arise when the nutritional requirements of the culture are not exactly known, and the requirements can only be satisfied by the inclusion of, e.g. yeast extract, yeast autolysate, meat extract, peptone, coconut milk, or other complicated natural mixtures. The constant aim in the use of N.m. is the definition of the minimal growth requirements for the system in question; this enables the use of synthetic N.m. (i.e. made by mixing defined chemicals of known purity), or minimal N.m. (synthetic media of exactly known composition, containing only those components that are required). Some growth systems have rather complicated and exacting growth requirements, so that it is necessary to add Growth factors (see). Auxotrophic organisms also show requirements for growth factors, which are not required by the parent, wild-type prototrophic organism (see Mutant technique). Glucose often serves as the source of carbon and energy in synthetic N.m. In the presence of glucose, the utilization of other, less efficient carbon sources is usually prevented by catabolite repression. The source of nitrogen depends on the growth system; it may be inorganic, e.g. nitrate or a m m o n i u m ; or organic, e.g. urea. Table 1 shows the composition of a simple bacteriological growth medium. Tables 2 and 3 give the composition of a vitamin solution and a trace element solution used in the preparation of some N.m.

Table 1. A simple synthetic culture medium microorganisms (after Schlegel) Glucose NH 4 C1 K2HP04 MgS04. 7 H 2 0 FeS04. 7HjO CaClj Trace element solution Water

10.0 g 1.0 g 0.5 g 0.2 g 0.01 g 0.01 g 1 ml 1000 ml

for

Nutritional physiology of microorganisms Table 2. Vitamin solution used for the preparation of culture media for soil and water bacteria (after Schlegel). 2 - 3 ml of this solution are added to 1000 ml of culture medium. Biotin Vitamin B 12 Nicotinic acid p-Aminobenzoic acid Thiamine Pantothenic acid Pyridoxamine Distilled water

0.2 mg 2.0mg 2.0 mg 1.0 mg 1.0 mg 0.5 mg 5.0 mg 1000 mi

Table 3. The A-Z solution, or trace element solution of Hoagland. A1 2 (S0 4 ) 3 K.I KBr Ti02 SnClj. 2 H 2 0 LiCl MnClj. 4 H 2 0 B(OH) 3 ZnS04 CuS04. 5H20 NiS04. 7H20) CO(N0 3 ). 6 H 2 0 Distilled water

0.055 0.028 0.028 0.055 0.028 0.028 0.389 0.614 0.055 0.055 0.059 0.055 1000 ml

g g g g g g g g g g g g

Nutritional physiology of microorganisms: The terms autotrophy and heterotrophy are too broad to distinguish between all the different forms of microbial nutrition. Different types of nutritional physiology are classified according to: 1. the nature of the carbon source; 2. the source and mechanism of formation of ATP; 3. the source of reducing power for the synthesis of cell constituents. Phototrophs use light energy (see Photosynthesis), and if they obtain all their energy from light and all their carbon from C 0 2 , they are called photoautotrophs. Some phototrophs can use organic carbon sources, obtaining all or some energy from light; these are called photoheterotrophs (i.e. they grow under mixotrophic conditions, see below). Chemotrophs obtain ATP by the oxidation of inorganic or organic substrates, and assimilate C 0 2 at the expense of the resulting oxidation energy (see Chemosynthesis). Bacteria that obtain energy from the oxidation of inorganic compounds (i.e. inorganic hydrogen donors) are called lithotrophs or chemolithotrophs, e.g. Nitrosomonas, which oxidizes ammonium to nitrite and nitrate; Hydrogemonas, which oxidizes gaseous hydrogen with oxygen; Thiobacillus, which oxidizes sulfide, elemental sulfur, thiosulfate or sulfite to sulfate. If all of their carbon is derived from C 0 2 , they are called chemoautotrophs. Most of these bacteria possess an electron transport chain, which is similar to that in other bacteria and mitochondria, and ATP is synthesized by "oxidative" phosphorylation during oxi-

Nutritional physiology of microorganisms

312

dation of the inorganic source of reducing power. The potentials of the reactions involved may, however, be lower than in the aerobic respiration of organic substrates, so that P / O ratios are also lower. The use of organic sources of reducing power is known as organotrophy. Most microorganisms, like animals, are chemoorganotrophs. Cyanobacteria and purple sulfur bacteria, which carry out photosynthesis, are photolithotrophs. Some autotrophs can also grow heterotrophically (facultative autotrophs), using organic energy sources. Obligate autotrophs are unable to grow heterotrophically, e.g. cyanobacteria, some species of Thiobacillus, and Nitrosomonas. On the other hand, most obligate autotrophs can assimilate organic compounds as carbon sources, but not as energy sources. This ability makes growth

Nutritional physiology of microorganisms possible under conditions that are best described as mixotrophic. Under these conditions, the energy source is needed only for the generation of ATP; the organic compound provides a source of reduced carbon and, if necessary, reducing power. Beggiatoa, a sulfur bacterium, is unable to grow on a completely inorganic medium (reduced sufur compounds and C0 2 ), and in order to utilize reduced sulfur compounds, it requires an organic carbon source such as acetate, i.e. it exhibits mixotrophy. Similarly, in the mixotrophic (photoheterotrophic) culture of green algae or euglenoids, the growth medium contains an organic energy and carbon source (e.g. glucose) and the culture is illuminated; the cells become green, and growth is supported at least in part by photosynthesis.

o

Oat coleoptile test, Arena test: a biotest for the quantitative determination of auxins, which is carried out as follows: The tip of the coleoptile is cut off, and the cotyledon within it is removed by pulling on its base. An agar block with the test substance is set on one side of the coleoptile stump. The auxin diffuses into the side of the coleoptile covered by the agar and, due to the one-sided stimulation of growth, causes it to bend. The angle of the bend is a function of the auxin concentration. Ochratoxlns mycotoxins produced by Aspergillus ochraceus during food spoilage. In rats and mice O. cause pronounced liver damage. The three known O. are designated A, B and C.

Ochratoxin Ochre codon: the UAA sequence in mRNA. Like the amber codon, it signals the end of protein biosynthesis. The synthesized polypeptide chain is released after the incorporation of the amino acid encoded immediately before the O.c. Ochre is probably the natural termination codon, and the one most widely employed by all living systems. See Ochre mutants. Octopine family: R-CH-COOH I NH I CH,- CH-COOH

Ochre mutants: bacterial mutants, which, as a result of a point mutation, possess a UAA codon in their mRNA (see Ochre codon). There are specific Suppressors (see) of ochre mutations. Oclmene: a triply unsaturated monoterpene hydrocarbon. O. is an oily liquid. M r 136.24, b.p. 176-178°C, p 1 5 0.8031, n j | 1.4857. It is a double bond positional isomer of Myrcene (see), and a component of many essential oils. D-Octopine: N-a-(l-carboxyethyl)-arginine, N 2 -(D-1 -carboxyethyl)-L-arginine. Mr 246.3, m.p.262-263°C (d.), [a]g> + 20.6 (c = 1, water). It is found in the muscles of certain invertebrates, e.g. Octopus, Pecten maximus, Sipunculus nudus, where it serves as a functional analog of lactic acid, i.e. the NADH produced by glycolysis is oxidized to NAD during the synthesis of D-O., and NAD can be reduced to NADH by the reversal of the same process. D-O. is biosynthesized from pyruvate and arginine by a reductive condensation, catalysed by an unspecific NADH-dependent dehydrogenase. D-O. is also found in certain plant tumors induced by Agrobaclerium tumefaciens. This bacterium induces tumors in dicotyledenous plants by transferring a large bacterial plasmid (called the T; plasmid) to the eukaryotic cell. In the transformed tissue, the T, plasmid determines the synthesis of novel amino acids, which serve as specific substrates for the bacterium. These may be D-O. and related compounds (the "octopine family"), or nopaline and nopalinic acid (the "nopaline family"), but not both.

NH II R = NH 2 -C-NH(CH 2 ) 3 -,

Octopine.

R = NH 2 -(CH 2 ) 3 -,

Octopinic acid.

R = NH 2 -(CH 2 ) 4 -,

Lysopine.

. CH 2 -,

R = N

Histopine.

NH \\/

Nopaline family: R-CH-COOH I NH I H O O C - (CH 2 ) 2 -CH-COOH

NH II R = N H 2 - C - N H ( C H 2 ) 3 - , Nopaline.

R = NH 2 -(CH 2 ) 3 -,

Nopalinic acid or Ornaline.

314

Octopinic acid

Octopinic acid: see D-Octopine. Oenin: see Malvidin. Oestradiol: see Estradiol. Oestriol: see Estriol. Oestrogen: see Estrogen. Oestrone: see Estrone. Oils: water-insoluble, liquid organic compounds. They are combustible, lighter than water, and soluble in ether, benzene and other organic solvents. Naturally occurring O. may be glycerides, e.g. the O. stored in certain seeds, or fish liver oils (see Fats); or they may be nonsaponifiable lipids, e.g. Essential oils (see). Oiticica oil: the seed fat of Licania rigida, a tree native to the semiarid areas of northeast Brazil. Licanic acid (see) represents 50-80% of the total esterified fatty acids of O.o. In addition, O.o. contains esterified palmitic, stearic, oleic and eleostearic acids. Okazaki fragments: see Deoxyribonucleic acid. 5a-Oleane: see Amyrin. Oleandomycin: a Macrolide (see) antibiotic. Oleanolic acid: a monounsaturated, pentacyclic Triterpene (see), with a carboxylic acid group. M r 456.71, m.p. 310°C, [a]D +80° (methanol). It differs structurally from p-amyrin by the presence of a carboxyl group in place of the 28-methyl group (see Amyrin). O.a. occurs free, esterified with acetic acid, or as the aglycon of triterpene saponins (see Saponins) in many plants, e.g. sugar beet, bilberry, mistletoe, cloves and cacti. Oleic acid: A 9 -octadecenoic acid, CH3-(CH2)7-CH = CH-(CH)7-COOH, the most widely distributed naturally occurring unsaturated fatty acid. M r 282.45, m.p. 13°C, b.p. 10 223°C. The double bond has the cis conformation. The trans isomer is called elaidic acid. O.a. is present in practically all the glycerides of depot

Ommochromes

and milk fats, and it is a component of phospholipids. Oleoresins: see Balsams. Ollgo-1,6-giucosldase: see Dextrin 6-a-D-glucanohydrolase. Oligonucleotides: linear sequences of up to 20 nucleotides, joined by phosphodiester bonds. Position 3' of each nucleotide unit is linked via a phosphate group to position 5' of the next unit. In the terminal units, the respective 3' and 5' positions may be free (i.e.-OH groups) or phosphorylated. O. are named according to chain length, i.e. di-, tri-, tetra-, pentanucleotides, etc. Linear sequences of more than 20 nucleotide units are called Polynucleotides (see and compare). Oligopeptides: see Peptides. Oligosaccharides: see Carbohydrates. Ommatins: see Ommochromes. Ommins: see Ommochromes. Ommochromes: a class of natural pigments, which contain the phenoxazone ring system. Their colors range from yellow through red to violet. They are especially common in, but not limited to the Arthropoda, and were named from their occurrence in the ommatidia of the insect eye. They are divided into two groups: the low M r , alkali-labile, dialysable ommatins; and the high M r , alkali-stable ommins. In the organism they are often bound to protein as chromoprotein granules. Crystalline dihydroxanthommatin was first prepared from the meconium (post pupal secretion) of the small tortoiseshell butterfly (Vanessa urticae). It is found universally in insects as an eye pigment, accompanied in most orders by a greater amount of ommin. In the eyes of many Diptera (Calliphora erythrocephala, Syrphus pyrastri, Musca domestica, Drosophila melanogaster), xan-

COOH I CHNH, I CHj

Xanthommatin

COOH I CHNHj

(yellow-brown)

R = H, D i h y d r o x a n t h o m m a t i n R = S 0 3 H , Ommatin

R = 1-glucosyl, Rhodommatin

¿cicd R =C0 — CH2— CH(NHj)-COOH O m m i n A (violet - black)

(red).

D (red). (red).

OMP

315

thommatin is the only O. Rhodommatin (the Oglucoside of dihydroxanthommatin) and ommatin D (the sulfate ester of dihydroxanthommatin) have only been found in the Lepidoptera; they are present in the wings of the Nymphalidae, where they contribute greatly to pigmentation, but they are absent from the eyes and all other ectodermal structures. They are also present in meconium as their water soluble ammonium salts, and the first isolates were made from this source. The large amounts of xanthommatin found in meconium were probably derived from ommatin D, and it doubtful whether xanthommatin occurs in fresh secretions. Xanthommatin has also been identified in the eggs of the marine worm Urechis caupo (Echiuridae), and very small amounts, together with ommin, have been found in some crustaceans. Ommin has been found in all investigated orders of the Insecta and Crustaceae. It is an especially common eye pigment in crabs, spiders, insects and cephalopods, and it is also present in the epidermis of Cragnon, Limulus and Grytlus, but not Carcinus and Portunus. Ommin is present in the eyes and skin, but not in the ink, of Sepia officianalis (Cephalopoda). About 75% of the pigment known as ommin contains a violet-black component called ommin A. O. are derived biosynthetically from 3-hydroxykynurenine, an intermediate of L-Tryptophan (see) metabolism. The phenoxazine ring system is formed by the oxidative coupling of two molecules of 3-hydroxykynurenine; it is a general reaction of o-aminophenols that they can be oxidized, chemically or enzymatically, to phenoxazones. Further cyclization of one side chain produces the quinoline ring system that is also present in the ommatins. Mutants of many insects are known, in which the capacity for O. synthesis is impaired. Such mutations are usually recognized from the abnormal eye color. Mutation may affect the conversion of L-tryptophan to A^formylkynurenine (e.g. white eye mutation of Periplaneta americana), JV-formylkynurenine to kynurenine (e.g. a mutation of Ephestia kuhniella), kynurenine to 3-hydroxykynurenine (e.g. cinnabar mutation of Drosophila melanogaster), 3-hydroxykynurenine to O. (e.g. white-2 mutation of Bombyx mori), or the synthesis of the protein which binds the O. may be impaired (e.g. wa mutation of Ephestia kiihmella). Our knowledge of the structure and biochemistry of O. is due largely to the work of Butenandt et al. OMP: abb. of Orotidine 5'-monophosphate. Oncogenic virus: see Cancer research. Oncovin: see Vincristin. Onlc acids: see Aldonic acids. Open system: 1. a system in dynamic equilibrium (see Steady state) with its surroundings, i.e. there is a continual exchange of material, energy and information with the enviroment. Application of the theory of O.s. to living systems (by Bertalanffy) involves the thermodynamics of irreversible processes. 2. In biology, plants are considered to be O.s.,

Operon

whereas animals are closed systems. The plant is theoretically capable of unlimited growth; certain cells remain embryonal and able to divide and differentiate, so that growth occurs from vegetative sites, such as the meristematic regions of the shoot and root tips, intercalary meristems, etc. In the animal, however, differentiation is essentially complete after the conclusion of embryonal development. Operator: see Operon. Operon: a group of neighboring genes, which represent a functional unit. An operon contains, 1. structural genes (S, to S4 in the Fig.), which code for the primary structures of enzyme proteins catalysing successive steps in a metabolic pathway, e.g. the enzymes in the biosynthesis of an amino acid. The primary transcription product of this group of genes is a polycistronic mRNA; therefore, during the control of transcription, all the structural genes are affected equally. 2. The promotor (P in the Fig.) is the starting point of transcription. This section of the DNA is "recognized" by RNA polymerase with the aid of the sigma factor. The affinity of the promotor for RNA polymerase (apparently determined by the structure of the promotor) is one of the factors that regulate the transcriptional frequency of the operon. When the RNA polymerase has bound to the promotor, it must then pass through the operator region in order to reach the structural genes. Operon

Schematic

representation

of an operon

3. The operator (O in the Fig.) is the control gene for the function (i.e. the transcription) of the structural genes. It is able to bind a repressor protein, which is the product of the regulator gene (R in the Fig.). R. is not a part of the operon, and it is located in a different region of the chromosome. If the specific repressor protein binds to the operator, transcription of the structural genes is blocked, i.e. the RNA polymerase cannot pass to the structural genes. If the operator is unoccupied, transcription of the structural genes can proceed. The details of these control mechanisms are described under Enzyme induction (see) and Enzyme repression (see). The nucleotide sequence of the lactose operator was elucidated in 1973 by Gilbert and Maxam; this operator is double stranded and consists of the following base pairs: 5 ' T G G A A T T G T G A G C G G A T A A C A A T T 3 ' 3 ' A C C T T A A C A C T C G C C T A T T G T T A A S '

The operon model was developed by Jacob and Monod, and it has only been demonstrated in prokaryotic systems. It is the basis for the expla-

Opium

316

nation of Enzyme induction (see) a n d Enzyme repression (see). See also Attenuation. Opium: the congealed or dried latex of Papaver somniferum. The chief active constituents of O. are the O p i u m alkaloids (see). O. is smoked, or preparations f r o m it may be ingested or injected. Uncontrolled use leads to addiction a n d deterioration of personality. The action of O. is essentially similar to, but weaker t h a n that of its constituent M o r p h i n e (see). Opium alkaloids: the Papaveraceae alkaloids (see) that occur in O p i u m (see). There are about 40 different O.a.; the chief representatives are M o r p h i n e (see), Codeine (see) a n d Papaverine (see). M o r e than 1000 tons of m o r p h i n e are produced annually. Nowadays the straw of the p o p p y plant is also used for the isolation of O.a. This new source accounts for about one third of the a n n u a l production of morphine. Opsin: see Vitamins (Vitamin A). Opsonin: the n a m e given by Wright and Douglas in 1903 to the thermostable material present in serum, which stimulates phagocytosis of bacteria. It was shown to act directly on bacteria and not on the phagocytes. It is probably identical to C3b of the complement system (see Opsonization), although other complement components may also be active to a lesser extent. In addition to its role in the opsonization of i m m u n e complexes, C3b can bind to various structures, such as foreign erythrocytes and bacterial cells, a n d render them more readily phagocytosed by i m m u n e adherence to phagocytes, Opsonization: the ability of serum to render an immune complex more readily phagocytosed. O. is a property of the Complement system (see). Although immune complexes are subject to phagocytosis, interaction with complement greatly increases the rate. C o m p o n e n t C3b (activated C3 of the complement system) has a labile binding site(s), which permits it to bind tightly to antigen-antibody complexes (opsonic adherence). Other stable binding sites on C3b enable this opsonized immune complex to bind to polymorphonuclear leucocytes, monocytes and macrophages (immune adherence); this binding results in enhanced phagocytosis. The phagocytosis can be abolished by metabolic inhibitors, whereas the immune a n d opsonic adherence (due to the physical chemical interaction of receptor and binding sites) are unaffected. Optical density: see Absorbance. Optical test: a method introduced in 1936 by Otto Warburg for the determination of the enzymatic activity of N A D and N A D P - d e p e n d e n t dehydrogenases. Absorbance at 340 nm (or another suitable wavelength in that region) is measured as an index of the degree of reduction of N A D or N A D P (see Nicotinamide-adenine-dinucleotide). The principle of this method is now widely used for measuring enzyme activities a n d the concentration of metabolites. A coupled enzyme system may be used, in which the reaction of interest does not produce or consume N A D ( P ) H , but is linked to one that does, e.g. the concentration of glucose can be determined f r o m the increase in

L-Ornithine

absorbance at 340 nm, when t h e u n k n o w n glucose concentration is incubated in the presence of excess A T P and N A D P + a n d t h e enzymes hexokinase a n d glucose 6-phosphate dehydrogenase: Glucose + A T P —>• Glucose 6-phosphate + ADP. Glucose 6-phosphate + N A D P + —• 6-Phosphogluconate + N A D P H + H + . Determinations of dehydrogenase activities by the O.t. are now conveniently p e r f o r m e d in an automatic recording spectrophotometer, so that the change in absorbance at 340 n m is monitored continuously with time, e.g. the activity of malate dehydrogenase is measured f r o m the rate of increase of absorbance at 340 n m d u e to the production of N A D H in the reaction: N A D + + Malate —• Oxaloacetate + N A D H + H + . Orchlnol: 9,10-dihydro-2,4-dimethoxy-7-phenanthrol, a phytoalexin, m.p. 168-170° C. O. is formed by the orchid, Orchis militaris, as a defense against infection by the f u n g u s , Rhizoctonia repens. The structurally related hircinol (9,10-dihydro-4-methoxyphenanthrene-2,5-diol, m.p. 162.5°C) is f o r m e d by the orchid, Himantoglossum (Loroglossum) hircinum, against infection by Rhizoctonia. RI

R2

R3

OCH3

H

OCH3

OH

OH

H

I = Onchinol H= Hircinol

I R3

Ord: abb. of Orotidine. ORD: abb. of Optical Rotatory Dispersion. Organotrophy: see Nutritional physiology of microorganisms. Orn: abb. of L-Ornithine. Ornallne: see D-Octopine. L-Ornlthlne, abb. Orn: a,S-diaminovaleric acid, 2,5-diaminovaleric acid, H2N-CH2-CH2C H 2 - C H ( N H 2 ) - C O O H , a nonproteogenic amino acid. In mammals, Orn. is an intermediate in the Urea cycle (see), a n d it is an intermediate in the biosynthesis of Arginine (see) in all arginine-synthesizing organisms. In a few microorganisms, Orn. can be formed f r o m citrulline by the action of citrulline phosphorylase. Various antibiotics contain Orn., e.g. the peptide antibiotic gramicidin S. Orn. is decarboxylated to putrescine (tetramethylenediamine) by microorganisms, a-JV-Acetyl-L-ornithine (2-iV-acetyl-L-ornithine) is an intermediate in the biosynthesis of arginine from glutamic acid in microorganisms, a n d it is an important allosteric effector of carbamoyl phosphate synthetase (see Urea cycle; Arginine). 8- Acetyl-L-ornithine (5- JV-acetyl-L-onithine), f o u n d in various plants, is a nonproteogenic amino acid, and a structural analog of citrulline.

Ornithine cycle

317

Ovulation inhibitors

Ornithine cycle: see Urea cycle. Oro: abb. of Orotic acid. Orosomucoid, a ¡-seromucoid, a ¡-acid glycoprotein, abb. U f A G p : a plasma protein of mammals and birds. O. contains about 38% carbohydrate, and is the most carbohydrate rich a n d water soluble of all the plasma proteins. The seromucoid fraction of blood consists of O. together with two other carbohydrate rich proteins (Cl-inactivator and hemopexin). Increased blood levels of O. a n d of the total seromucoid fraction are associated with inflammation, pregnancy a n d various disease states, such as cancer, pneumonia, rheumatoid arthritis. After major surgery, increased levels of O. are produced until the w o u n d is healed. O. also binds certain steroids, especially progesterone, but the affinity of binding is lower than that for corticosteroid binding globulin. Membranes of h u m a n platelets contain considerable quantities of tightly b o u n d O. H u m a n O. is a single chain glycoprotein ( M t 39000 by sedimentation equilibrium, 41600 by sedimentation diffusion; isoionic p H 3.5; O. has p r o n o u n c e d anion binding capacity so that the isoelectric point dep e n d s on the type a n d concentration of anions). The protein chain of O. contains 181 a m i n o acid residues (primary sequence: Schmid, K. et al. Biochemistry (1973) 12, 2711-2724). Five oligosaccharide units are attached to the P-carboxyl groups of asparaginyl residues at positions 15, 38, 54, 75 a n d 85. The native protein contains two disulfide bridges, located between cysteine residues at 5 and 147, and at 164 a n d 72. The amino terminus is pyroglutaminyl, and the carboxyl terminus is serine. The C-terminal half of O. shows great similarities of sequence with the a chain of haptoglobin and the H-chain of immunoglobulin G. Analysis of the carbohydrate of O. (Schwarzmann, O.H.G. et al. J. Biol. Chem. (1978) 253, 6983-6987) shows 14 residues of Nacetyineuraminic acid (sialic acid), about 34 residues of neutral hexoses ( D - g a l a c t o s e / D - m a n n o s e in an average ratio of 1.4); 31 residues of N-acetylglucosamine; 2 residues of L-fucose. N-Acetylneuraminic acid is always located terminally via a 2-ketosidic b o n d ; the galactose occupies a penultimate position, linked (3-glycosidically to the third sugar, Af-acetylglucosamine. O. is synthesized in the liver. Synthesis of the carbohydrate moiety is initiated by transfer of an /V-acetylglucosaminyl residue to an asparaginyl residue when the nascent polypeptide chain is still attached to the ribosome (see Post translational modification of proteins).

metabolism, but it is p r o d u c e d by mutants of Neurospora crassa. Orotidine 5'-monophosphate, abb. OMP: a nucleotide of orotic acid. M r 368.2. O M P is an intermediate in Pyrimidine biosynthesis (see). Orotidine 5'-phosphate pyrophosphorylase catalyses the synthesis of O M P f r o m orotic acid and 5phosphoribosyl 1-pyrophosphate. Orthonil, PRB 8: 2-(p-chloro-P-cyanoethyl)-6chlorotoluene, a synthetic, stimulatory growth regulator. It is claimed that O. causes an increase in the sugar content of sugar beet.

Orotic acid, abb. Oro: uracil 4-carboxylic acid. M r 156.1, m.p. 344-347°C (d.). O.a. is an intermediate in Pyrimidine biosynthesis (see), a n d it accumulates in large quantities during growth of mutants of Neurospora crassa, which require uridine, cytidine or uracil. Orotidine, abb. Ord: orotic acid-3-|3-D-ribofuranoside, a (5-glycosidic Nucleoside (see) of D-ribose and the pyrimidine, orotic acid. M r 288.21, m.p. > 400°C. Cyclohexylamine salt: m.p. 184°C, [ a ] g +14.3° (c= 1, water). O. does not appear to be a normal intermediate of pyrimidine

Ostreasterol: see Chalinasterol. Ouabagenin: see Strophanthins. Ouabain: see Strophanthins. Ouchterlony technique: see Precipitation. Ovalbumin: see Albumins. Ovosiston: an oral contraceptive (see Ovulation inhibitors), consisting of a mixture of the progestin, C h l o r m a d i n o n e acetate (see), a n d the estrogen, Mestranol (see). Ovulation inhibitors: a group of steroids, which inhibit ovulation by feedback inhibition of the production of Luteinizing h o r m o n e (see) a n d / o r

Orthonil Orycenin: see Glutelins. Osmosis: a p h e n o m e n o n associated with semi-permeable membranes, especially Biomembranes (see). When two solutions are separated by a m e m b r a n e which is permeable only to selected components of the solution (e.g. water), the component which can cross the m e m b r a n e will flow from the side on which its partial pressure is higher to the side on which its partial pressure is lower. Cytoplasm is a concentrated solution of salts, sugars a n d other small molecules, most of which cannot diffuse across the plasma membrane, in water, which can cross the membrane. As a result, the cells of fresh-water organisms, a n d of plant root hairs, experience an inflow of water until the pressure within the cell increases to the point where the partial pressure of water is equal inside and outside the cell. Fresh-water unicellular animals expell the excess water by means of contractile vacuoles; multicellular animals excrete it through their kidneys. Plant cells are surrounded by rigid Cell walls (see), which enable them to withstand the high internal pressure caused by O. without bursting. Higher land plants utilize the pressure gradient created by O. to help drive their sap u p f r o m the roots. Salt-water organisms live in an environment in which the salt concentration outside the plasma membranes is higher than inside. O. thus tends to dehydrate the cell rather than to cause it to burst, and the organism must expend metabolic energy to excrete salt in order to maintain the lower internal concentration.

318

Oxalic acid

Follicular stimulating hormone (see). They are used extensively as oral contraceptives (otherwise known as "the pill"). Most commercially available O.i. preparations contain a combination of a progestin and an estrogen. The progestin is the actual O.i., whereas the estrogen is included to prevent breakthrough (midcycle) bleeding. Combination O.i. consist of a progestin-estrogen combination, which is taken daily throughout the cycle. A sequential O.i. was introduced in 1965 to mimic more closely the normal rise and fall of estrogen and progestin in a woman's monthly cycle; the first IS pills contain estrogen only, and the last 5 contain a progestin-estrogen combination. Since these were less effective than the combination products, and showed undesirable side effects, they were removed from the U.S. market in 1976. The "minipill" contains a low dose of progestin (0.075 mg norgestrel, or 0.35 mg norethindrone) and no estrogen; it is free from many side effects, but users sometimes have irregular bleeding. Trials are also being conducted on the use of once-a-month and once-a-week O.i., and on injectable depot O.i. Since the natural progestin and estrogens show poor gut absorption, all the progestins and estrogens used in oral O.i. are synthetic products, which show efficient gut absorption. Synthetic progestins used as O.i. are norethindone, norgestrel, norethynodrel, ethynodiol diacetate, dimethisterone. Synthetic estrogens used in O.i. preparations are Ethinylestradiol (see), Mestranol (see), ethinylestradiol 3-cyclopentyl ether (quingestanol). The quantities used are 0.5-5 mg progestin, and 0.05-0.1 mg estrogen

Oxalate

Oxalate metabolism in Pseudomonas

oxalaticus

Oxalic acid

per tablet. There are several different commercial oral contraceptives, containing various combinations of the above synthetic progestins and estrogens. See also Chlormadinone acetate. O.i. are also used for the treatment of dysmenorrea, endometrioses, cycle-dependent migraines and sterility. Oxalic acid: HOOC-COOH, m.p. 189.5°C (anhydrous). O.a. occurs widely in plants as its calcium, magnesium and potassium salts. Owing to its ability to bind calcium, large amounts of O.a. are poisonous. By forming insoluble calcium oxalate in the intestine, O.a. hinders the absorption of calcium. Animals cannot metabolize O.a. The normal daily O.a. excretion in the human is 10-30 mg. Higher levels may lead to kidney damage (formation of oxalate kidney stones). The O.a. content of fruits and vegetables is usually less than 10 mg/100 g fresh weight. Rhubarb leaves contain 700 mg, rhubarb stems 300 mg, celery stems up to 600 mg, and spinach 60-200 mg/100 g fresh weight. O.a. was first prepared in 1773 from wood sorrel (Oxalis acetosella). It is synthesized in plants by the oxidation of excess glyoxylate. It is also produced by the oxidative cleavage of phenylpyruvate to benzaldehyde and O.a. (a reaction in the conversion of phenylalanine to hippuric acid). In the bacterium, Pseudomonas oxalaticus, O.a. is metabolized (via oxalyl-CoA) by an oxidative and a reductive pathway, which are coupled and operate simultaneously (Fig.); the balance is: 2-OOC-COO2CO, + CHO-COO" H,0.

319

Oxaloacetic acid Oxaloacetic acid: HOOC-CO-CH r COOH, an oxodicarboxylic acid present in fairly high concentrations in plants, e.g. red clover, peas. The anion (oxaloacetate) is an intermediate in the Tricarboxylic acid cycle (see), and it is the oxoacid derived from Aspartic acid (see) by Transamination (see). The enol form of O.a. may be cis or trans: hydroxymaleic acid (cis, m.p. 152° C), and hydroxyfumaric acid (trans, m.p. 184°C [d.j). Oxalosucclnlc acid: a p-ketotricarboxylic acid (2-oxotricarboxylic acid): COOH

I

HOOC—CH2—CH —CO—COOH

The anion (oxalosuccinate) is an intermediate in the isocitrate dehydrogenase reaction of the Tricarboxylic acid cycle (see). Isocitrate dehydrogenase catalyses both the oxidation of isocitrate to oxalosuccinate, and its decarboxylation to 2-oxoglutarate. Oxidases: oxidoreductases (see Enzymes), which use molecular oxygen as an electron acceptor. See also Flavinenzymes. Oxidation: the loss of electrons. Classically, O. was defined as combination with oxygen or removal of hydrogen. The electrons are transferred to the oxidizing agent, which becomes reduced. Therefore O. is always coupled to Reduction (see), so that any O. or reduction is part of an oxidoreduction process. In metabolism there are different mechanisms of enzyme catalysed O., i.e. dehydrogenation, electron transfer, introduction of oxygen, and hydroxylation (Table).

Type of

reaction

Dehydrogenation

Oxidative phosphorylation ential E^ at pH 7.0 is taken as the reference point. E^ has a value of 0.42 volt on the E 0 scale. The E^value is only valid under standard reaction conditions, i.e. all reactants at unit activity. Such conditions do not obtain in the living cell, and the ratio of concentrations of oxidized and reduced forms of the redox pair is included in the calculation. The actual redox potential E' is therefore expressed as: E' - E ' + — log ^ (30°C). The redox potenn Red tial is related to free enthalpy (Gibb's potential) as follows: A G° = — nFE0> where n is the change of valency, and F the Faraday constant. As oxidizing potential increases E^ becomes more positive, so that strong reducing reducing agents are characterized by high negative E^ values, and strong oxidizing agents by high positive values. See Table 3 under the entry for Respiratory chain; here the members of the respiratory chain and other biological redox systems are arranged in order of their redox potentials. Each component in the table is a more powerful oxidizing agent than those above it. Oxidative metabolism: see Respiration. Oxidative phosphorylation, respiratory chain phosphorylation: formation of ATP coupled with the operation of the Respiratory chain (see). Energy available from the flow of electrons from substrate to oxygen via the respiratory chain drives the synthesis of ATP from ADP and inorganic phosphate. Oxidation of one molecule of reduced nicotinamide adenine dinucleotide (NADH + H + ) generates three molecules of ATP, while reduction of one molecule of reduced

Enzymes

General reaction

Oxidoreductases

SH2

^ D

Electron transfer

Oxygen

I* a n d 2 electron transferring oxidases transfer

Hydroxylation S= substrate,

Dioxygenases

io

2

02

S + 02

s + dh 2 + o 2

Hydroxylases D= h y d r o g e n c a r r y i n g

f or

S DH2

H20 j H202 S02

»

SOH + D + H 2 0 cofactor

Oxidation reactions in metabolism The oxidizing or reducing power of a substance is indicated by its redox (reduction-oxidation) potential. The redox potential is related to the potential of the hydrogen electrode, and it is a quantitative index of electron affinity (i.e. oxidizing power) or tendency to lose electrons (i.e. reducing power). The defined standard potential in physical chemistry, or Normal potential, E 0 (pH = O, pH 2 = 1 atmosphere) is however, inappropriate for biological purposes. Instead, the Normal pot-

flavin adenine dinucleotide (FADH 2 ) yields two ATP. Complete oxidation of one molecule of glucose yields 38 ATP, 2 from glycolysis and 36 from O.p. The mechanism of O.p., i.e. the nature of the energy transducing mechanism that converts the energy of electron flow into the chemical energy of ATP, has always been a controversial area of biochemistry. The earliest hypothesis, chemical coupling, proposes the existence of an energy-rich in-

22,25-Oxidoholothurinogenine

320

termediate generated by electron flow and consumed in the phosphorylation of ADP:

Ared + B0I + C —• Aox ~ C + Bred Ant + C + ADP + P/ *• AOJ[ + Bred + ATP + C \ e d + Box + ADP + P,— Aox + Bred + ATP

A second hypothesis, conformational coupling, proposes that the energy is stored in a protein conformational change caused by electron transfer; return to the original conformation is linked to ATP synthesis. Whatever the mechanism of O.p. eventually accepted, there seems little doubt that it will be based on the chemiosmotic theory proposed by Nobel laureate P. Mitchell. In chemiosmosis, the operation of the respiratory chain drives protons across the inner mitochondrial membrane (see Mitochondria). The resulting proton gradient then drives ATP synthesis. The F, "knobs" visible on the inner surface of the inner mitochondrial membrane, together with their associated hydrophobic membrane proteins, form an ion pump, which generates ATP at the expense of the proton gradient. The mechanism whereby proton transport is coupled to the operation of the respiratory chain is not clear, and the final mechanism may well invoke some version of the conformational coupling theory. The chemiosmotic theory differs from other theories of O.p. in that operation of the respiratory chain is not directly linked to ATP synthesis. Even in the absence of an operational respiratory chain, production of a proton gradient by any other method should, according to the chemiosmotic theory, promote ATP synthesis. This has been verified experimentally. Formation of a proton gradient, i.e. a decrease of pH outside the mitochondrial membrane, during operation of the respiratory chain has also been proved experimentally. The same theory also explains Photophosphorylation (see). O.p. is normally tightly coupled to the flow of electrons along the respiratory chain, i.e. electrons do not flow unless ADP and inorganic phosphate are available for the synthesis of ATP. This respiratory control may be absent in pathological states, or destroyed artificially by uncoupling agents. In the brown fat tissue of some mammals, respiratory control may be partly relaxed so that electron flow without ATP production can be used to generate heat. In isolated mitochondria, lack of respiratory control indicates that the organelles are damaged. According to the chemiosmotic theory, uncoupling agents, e.g. 2,4-dinitrophenol, act by rendering the inner mitochondrial membrane permeable to protons, so that the gradient collapses and is unable to drive ATP synthesis. The respiratory chain is then free of restraint, so that the respiration rate of uncoupled mitochondria is usually higher than that of optimally respiring, coupled mitochondria. 22,25-Oxidoholothurinogenine: see Holothurines. Oxidoreductases: see Enzymes (Table 1). Oxidoreduction: see Oxidation. Oxoeieostearic acid: see Licanic acid. 9-Oxo-trans-2-decenoic acid: see Queen bee substance.

Oxygenases

Oxygen: see Bioelements. Oxygenases: enzymes that catalyse the incorporation of the oxygen of molecular oxygen into their organic substrates, i.e. the oxygen atom(s) appearing in the product is (are) derived from atmospheric 0 2 , and not from water. Dioxygenases (oxygen transferases), catalyse the introduction of both atoms of molecular oxygen. Monooxygenases or hydroxylases catalyse the introduction of one atom from molecular oxygen; the other atom becomes reduced to water. Monooxygenases therefore require a second substrate, which serves as an electron donor; for this reason they are also called mixed function oxygenases. They catalyse the following type of reaction: AH + 0 2 + DH 2 -ThS CH,

I

Phalloin^ R — C — C H ,

I

OH

Pharmaceutical biology, pharmacognosy: the science of drugs a n d their active principles, a n d of drug-yielding plants a n d animals. Pharmaceutical chemistry: the study of the chemical properties, development, preparation and analysis of pharmaceuticals. Pharmacognosy: see Pharmaceutical biology. Pharmacology: in the b r o a d sense the study of all pharmaceutically active substances. In the narrow sense, the study of the action of substances foreign to the body, a n d the action of un-

340

Pharmacology

Pharmacology

(j

• ID

•o ^E co5koj O 4> g. c Ol

.O o E ^ E g

CO 10 *D N £ 1

O

^

JZ E o ••5 dJ . HC C O „ »o g0J >3 yO XO

—0— CH2 — CH2— N(CH3)3 OH R = Alkyl or Alkenyl

Fig. 4. Sphingosine phosphatides. (EC 3.1.1.32). Plasmalogens (Fig. 2) are glycerophospholipids in which the glycerol bears a 1alkenyl ether group. Plasmenic acids are compounds in which glycerol 3-phosphate has a 1alkenyl ether group and a fatty acid esterified to the second hydroxyl. In analogy to the derivatives of phosphatidic acid, these compounds are called plasmenylethanolamine, plasmenylserine, etc. In diphosphatidylglycerol, two phosphatidic acid moieties are esterified to a single molecule.

Phosphopantethelne: see Pantetheine 4'-phosphate. Phosphoproteins: conjugated proteins, containing phosphate esterified with the hydroxyl groups of serine or (less often) threonine residues. Well known P. are casein (see Milk proteins) and ovalbumin (see Albumins). The latter can contain one or two phosphate groups; this microheterogeneity (see Heterogeneity) is due to variations in the production and activity of the

350

Phosphopyruvate kinase

phosphoprotein phosphokinase in the hen oviduct. Other P. are phosphovitin and vitellin of egg yolk, and pepsin of gastric juice. Phosphopyruvate kinase: see Pyruvate kinase. 5-Phosphorlbose l-dlphosphate: see 5-Phosphoribosyl 1-pyrophosphate. 5-Phosphoribosylamine, abb. PR A: an intermediate in Purine biosynthesis (see). 5-Phosphorlbosyl l-pyrophosphate, 5-phosphoribose ¡-diphosphate, abb. PRPP: an energyrich sugar phosphate formed by the transfer of a pyrophosphoryl residue from ATP to ribose 5phosphate. MT 390.1. PRPP is concerned in various biosynthetic reactions, e.g. biosynthesis of purines, pyrimidines and histidine. Phosphoroclastlc fission of pyruvate: a special mechanism for the cleavage of pyruvate found only in saccharolytic Clostridia. It is responsible for the synthesis of ATP during nitrogen fixation. The first stage is the synthesis of acetyl phosphate : 0 II

C H r C - C 0 0 H + P,

>

Pyruvate

0 C H 3 - C - O — P 0 3 H 2 + CO2 + H 2 Acetyl phosphate

followed by synthesis of ATP from acetyl phosphate catalysed by acetokinase: Acetyl phosphate + ADP ^ ATP + Acetate. The first stage requires several enzymes (in the form of a multienzyme system analogous to pyruvate dehydrogenase, together with phosphotranssacetylase and hydrogenase) and cofactors (thiamine pyrophosphate, ferredoxin and coenzyme A). Phosphotransacetylase: see Acetyl phosphate. Phosphotransferase system: see Active transport. Phosphorus: see Bioelements. Phosphovitin: an egg yolk protein containing 10% phosphate (M r 180000). Serine constitutes 50% of the total amino acid content, and all the serine residues are phosphorylated. P. is synthesized in the liver of the laying hen and transported in the blood to the developing egg. Photocltral: a cyclization product of Citral (see). Photoheterotrophlsm: see Nutritional physiology of microorganisms. Photolysis of water: cleavage of water by the light reaction of Photosynthesis (see). P. is a property of photosystem II. It is not a simple photodissociation of water, but is the physiological counterpart of the Hill reaction (see). Electrons are withdrawn from water or O H - ions, thentransported to NADP+ via an electron transport chain; this results in the production of molecular oxygen with the formation of NADPH + H + . Since Hill reagents oxidize water by withdrawal of electrons, NADP+ is the natural Hill reagent.

Photoreactivation Photophosphorylatlon: the synthesis of ATP in Photosynthesis (see). The mechanism of P. is similar to that of Oxidative phosphorylation (see) by the respiratory chain; in both cases cytochromes are involved in electron transport. A distinction is drawn between cyclic and noncyclic P. Both forms are found in green algae and higher plants. Cyclic P. involves cyclic electron transport. Under the influence of light, electrons emitted from chlorophyll a return to chlorophyll a via an electron transport chain, thereby giving rise to ATP synthesis. TTius the positive holes left in the chlorophyll structure by the loss of electrons are refilled, and the electron excitation energy is transduced to the chemical energy of ATP. Cyclic P. involves cytochrome f , and the only product is ATP. In noncyclic P., ATP synthesis is linked to the transport of electrons from water (see Photolysis of water) to N A D P + , thus producing both ATP and a reducing agent (NADPH). The production of molecular oxygen, the byproduct of water photolysis, is characteristic of photosynthesis in green plants and algae. Noncyclic P. can be considered as a Hill reaction (see) coupled to the synthesis of ATP. Whereas cyclic P. requires only photosystem I, noncyclic P. depends upon the joint operation of both photosystems, which are connected in series; electrons are transported from O H - ions of water to N A D P + in an openchain (noncyclic) system. The two kinds of P. are functionally and structurally separate in the chloroplast, but they are closely interrelated. They can be separated experimentally, e.g. by the inhibitors o-phenanthroline or dichlorophenyldimethylurea (DCMU), which block photosystem II. One molecule of ATP is formed per electron pair in cyclic P: ADP + P, ATP (P/2e = 1), which indicates the existence of a single phosphorylation site in cyclic photosynthetic electron transport. In noncyclic P., 2 molecules of ATP are synthesized per electron pair: 2 N A D P + + 2P,—2NADPH + 2H+ + 2ATP + 0 2 , which indicates the existence of two phosphorylation sites. Photoprotelns: proteins responsible for luminescence in many light-emitting coelenterates. Light emission by P. does not involve a luciferinluciferase system (see Luciferin), and the reaction proceeds in the absence of oxygen. Aequorin, the P. of the jelly fish Aequorea, contains a substituted 2-aminopyrazine as chromophore; light production 469 nm) is activated specifically by Ca 2 + . A similar Ca 2 + -activated P., obelin, has been isolated from Obelia geniculaia (>. max of emitted light 475 nm). Both of these proteins have been employed as sensitive probes for measuring intracellular Ca 2 + concentrations, with a sensitivity of at least 10 nM C a 2 + . Ref: Campbell, A. K. and Simpson, J. S. A., Chemi- and bioluminescence as an analytical tool in biology, in Techniques in Metabolic Research, B213, pp. 1-56, Elsevier (1979). Photoreactivation: repair of biological systems damaged by UV-irradiation, in a process promoted by light of a different wavelength. Pyrimidine dimers, which result from UV-irradiation, can be monomerized by the action of UV light of

shorter wavelength or light of longer wavelength, which promotes the action of repair enzymes. The enzyme binds only to UV-damaged DNA and converts pyrimidine dimers to monomers when irradiated with light of an appropriate wavelength. Photorespiration: light enhanced respiration in photosynthetic organisms. Illumination of C3plants markedly increases the rate of oxygen utilization; this increase in respiration can be as high as 50% of the net photosynthetic rate. P. thus results in a loss of yield in the photosynthesis of C3-plants. In C4-plants, P. is either absent or extremely low. P. is largely due to the oxygenase activity of Ribulosebisphosphate carboxylase (see), which oxidatively cleaves ribulose 1,5-bisphosphate into phosphoglycolate and 3-phosphoglycerate. Glycolate (derived from the phosphoglycolate) leaves the chloroplasts and enters the peroxisomes, where it is oxidized (by a flavoprotein oxidase) to glyoxylate. Hydrogen peroxide from the action of the flavoprotein oxidase may oxidize some of the glyoxylate to formate and C0 2 , but the majority is destroyed by peroxidases and catalase. Most of the glyoxylate is transaminated to glycine, which enters the mitochondria. Glycine may be decarboxylated and/or converted into serine, some of which may reenter the peroxisomes and become oxidized to hydroxypyruvate and D-glycerate. Thus various reactions occur that result in loss of carbon as C0 2 . The process depends on light because light is required for the operation of the Calvin cycle, which supplies the ribulose 1,5-bisphosphate. Some of these reactions are illustrated in the diagram of the glycolate cycle shown under Glycine (see), but here the phosphoglycolate is shown as coming from active glycolaldehyde. See also C0 2 -compensation point; Light compensation point. Photosynthesis: 1. Any light-dependent syn-

thesis. 2. The reductive synthesis of carbohydrate in green plants and Photosynthetic bacteria (see). P. was formerly defined as the assimilation of carbon dioxide, but it is now recognized as primarily a process of energy transduction, in which light energy is converted into the chemical energy of oxidizible organic carbon compounds. P. in green plants (but not bacterial P.) can be represented by: 6C0 2

Photosynthesis

351

Photorespiration

+

6H 2 Q

C 6 H 12 G 6

+ 602. In principle, this general equation is the reverse of Respiration (see). The reaction is cata-

lysed by chlorophyll a, which is structurally bound in the Thylakoids (see). In photosynthetic bacteria, chlorophyll a is replaced by bacteriochlorophyil a (see Bacterial photosynthesis). Other Photosynthetic pigments (see) serve as auxilliary pigments for light absorption and energy transfer. P. is the most important process for the production of organic material in the biosphere. All nonphotosynthetic organisms are directly or indirectly dependent on P. of phototrophic organisms. By comparison, Chemosynthesis (see) plays a quantitatively insignificant role in the carbon cycle of the biosphere. P. is an energy-dependent (endergonic) process, in which light energy is converted into the chemical energy of ATP by Photophosphorylation (see). In addition, P. in green plants (not in photosynthetic bacteria) produces reduced NADP, a reducing agent used by the cell in reductive biosyntheses. Synthesis of ATP, NADPH and H + occurs in noncyclic photophosphorylation, which is linked to the photolysis of water. ATP and NADPH, known as the primary products of P., have a transitory existence (i.e. they are rapidly utilized) and they do not accumulate. They are the earliest relatively stable products of the light reaction of P., in which light energy, via electron excitation energy, is transduced to the chemical energy of an energy-rich compound (ATP) and a reducing agent (NADPH). Strictly speaking, the light reaction consists of purely photochemical events, and the chemical reactions of photophosphorylation in which light plays no direct part should be described as dark reactions. By convention, however, the photochemical events in structurally bound chlorophyll, together with the reduction of NADP+ and the synthesis of ATP (i.e. the production of assimilatory power, a term coined by Arnon) constitute the light reaction of P. It is characteristic of the light reaction processes (in contrast to the purely chemical processes of the dark reaction) that they are not temperature dependent. The dark reaction of P. includes the reactions responsible for the reductive synthesis of carbohydrate, e.g. synthesis of starch, sucrose, and it includes the initial reactions of C0 2 fixation in C4-plants. Thus the dark reaction includes all reactions from the initial binding of carbon dioxide to the formation of carbohydrate reserves, e.g. starch. For a description of these processes, see Calvin cycle; Hatch-Slack-Kortschack cycle; Starch.

Fig.l. Relationship between light and dark reactions of photosynthesis.

Photosynthetic bacteria

352

In green plants, P. is localized in the Chloroplasts (see). All the processes of the reaction occur in the membrane system of the thylakoids, whereas the dark reaction proceeds in the stroma of the chloroplasts, which (with the exception of the thylakoid carboxydismutase) contains the enzymes of carbon dioxide assimilation (Fig. 1). The stroma also contains the Protein biosynthesis (see) system of the chloroplast. Photosynthetic bacteria possess only one photosystem, known as photosystem I, whereas green plants possess photosystem I and II (see Photosystems). Cyclic phosphorylation requires only photosystem I. In noncyclic phosphorylation, both system are coupled together, and their interrelationship is often shown diagrammatically by the so-called "zig-zag" energy diagram (Fig. 2). Chlorophylls QJ and absorb light and emit electrons. The electrons emitted by chlorophyll a j are received by ferredoxin and transferred to N A D P + . Electrons emitted by chlorophyll a j j are transported to chlorophyll a j via an electron transport chain containing plastoquinone, plastocyanin and cytochrome / ; these electrons enter the positive holes (electron vacancies) of chlorophyll aj. Operation of the electron transport chain is coupled to the synthesis of ATP. The positive holes of chlorophyll a n are filled by electrons from the photolysis of hydroxyl ions. The joint operation of the two photosystems leads to the synthesis of ATP and NADPH. Electrons from chlorophyll ¡2j may also take part in cyclic electron transport via a cytochrome chain and back to chlorophyll aj, thereby producing ATP, but not NADPH (see Photophosphorylation). The natural electron acceptor of photosystem I has not been identified; it is referred to as ferredoxin reducing substance (FRS). The nature of electron transfer between photosystem II and plastoquinone is also unclear; the primary electron acceptor of photosystem II (which quenches the fluorescence of chlorophyll, and is therefore called substance Q) is unidentified.

Fig.2. Coupling of the two light reactions in noncyclic photophosphorylation. Chi chlorophyll, Cyt cytochrome, Fd ferredoxin, FRS ferredoxin reducing substance, Q quenching substance. Some herbicides act as artificial electron donors or acceptors which short circuit the electron transport chain of P., e.g. the bipyridylium herbi-

Photosynthetic bacteria

cides, paraquat and diquat, compete with ferredoxin for electrons from the ferredoxin reducing substance (FRS); the reduced bipyridylium system is then spontaneously oxidized, forming hydrogen peroxide which damages the plant tissues. Other herbicides block electron flow from water in photosystem II, e.g. monuron (3-(4-chlorophenyl)l,l-dimethylurea, or CMU) and diuron (dichlotophenyldimethylurea, or DCMU). Nomenclature: Photosystems I and II are responsibleforlightreactions 1 and2,orphotoevents 1 and2. On the other hand it is customary to refer to the sum of the light photoevents, including ATP synthesis and NADPreduction, as the light reaction of P. Similarly, one may speak of the dark reactions or the overall dark reaction of P. Photosystem I is so called because it arose first in evolution (i.e. in photosynthetic bacteria), whereas photosystem II and the accompanying photolysis of water were later evolutionary developments. Photosynthetic bacteria: phototrophic bacteria, e.g. green sulfur bacteria (Chlorobacteriaceae), purple sulfur bacteria (Thiorhodaceae) and nonsulfur purple bacteria (Athiorhodaceae). The Chlorobacteriaceae include Chlorochromatium consortium and species of Chlorobium. The Thiorhodaceae are represented by Chromatium okenii and Thiospirillum jenense, which are of interest in Athiorhodaceae include three genera: Rhodopseudomonas, Rhodospirillum and Rhodomicrobium. All P.b. are deeply colored, due to the presence of photosynthetic pigments. In place of chlorophyll a, P.s. contain bacteriochlorophyll a (2-devinyl-2-acetyl-3,4-dihydrochlorophyll a). Green sulfur bacteria also contain bacteriochlorophylls c and d (formerly called Chlorobium chlorophylls), which absorb between 700 and 760 nm. In addition to bacteriochlorophylls, P.b. contain Carotenoids (see), which serve as auxilliary photosynthetic pigments. Due to their different pigmentation, the various types of P.b. are able to exploit different areas of the spectrum for photosynthesis. P.b. neither produce nor consume molecular oxygen. They do not perform the photolysis of water, and they can live anaerobically, most of them being strict anaerobes. They contain one photosystem, analogous to the photosystem I of higher green plants, algae and blue-green bacteria. Green and purple sulfur bacteria perform the photolysis of hydrogen sulfide: 2H 2 S + C 0 2 [CH 2 OJ n + H 2 0 + 2S Carbohydrate Sufficient energy for this process is achieved in one photoevent (in contrast to the photolysis of water, which requires two photoevents coupled in series; see Photosynthesis). Reducing power is trapped by ferredoxin, but some of the energized electrons are recycled (see Photophosphorylation) for the synthesis of ATP. Nonsulfur purple bacteria use various substances as donors of hydrogen and electrons, e.g. isopropanol: 2CH3CHOHCH3 + m 2 light, [CH 2 0] n + Isopropanol carbohydrate 2CH3COCH3 + H 2 O Acetone

353

Photosynthetic carboxylation Photosynthetic carboxylation: the

Photosystems Photosynthetic pigments: pigments that take part in the trapping and utilization of light in Photosynthesis (see). Seed plants (Spermatophyta), ferns (Pteridophyta), mosses (Bryophyta),green algae (Chlorophyta, e.g. Chlorella), euglenoids (Euglenophyta, e.g. Euglena) and brittleworts (Characeae) contain both chlorophylls a and b, and carotenoids, but no biliproteins. The latter are found in red algae (see Rhodoplasts) und Blue-green bacteria (see). Certain algae lack chlorophyll b (Chrysophyta, Pyrrophyta and Cryptophyta). Table 1 lists the P.p. of various organisms, and table 2 shows the thylakoidal (see Thylokoids) carotenoids of the red beech (Fagus silvatica) and the green alga, Chlorella pyrenoidosa. All P.p. are either hydrophobic, or they possess a strongly hydrophobic grouping, e.g. the phytol residue of chlorophyll. A simple model, in which P.p. are associated with the lipid layer of the thylakoid membrane, is however, unsatisfactory. It is necessary to propose a certain degree of ordered

enzymatic

fixation of carbon dioxide in photosynthesis. In C-3 plants, the photosynthetic carboxylation enzyme is ribulose bisphosphate carboxylase (EC 4.1.1.39). In C-4 plants it is phosphoeno/pyruvate carboxylase (EC 4.1.1.31). P.c. is the first step of carbon dioxide assimilation in photosynthesis, and one of the dark reactions. Photosynthetic cycle: see Calvin cycle. Photosynthetic experimental organisms and

systems: for technical reasons, certain systems are preferred for the investigation of photosynthesis, e.g. green algae such as Chlorella, photosynthetic bacteria and isolated chloroplasts. Green algae and euglenoids (e.g. Euglena gracilis) can be cultured under defined conditions in an illuminated chemostat (see Fermentation techniques), and they can also be grown in Synchronous culture (see). It is relatively easy to obtain chlorophyll-deficient mutants of these organisms, which must grow heterotrophically (see Mutant technique). The production of plastids can be

Table 1. Photosynthetic pigments and their occurrence in the plant kingdom. Organism

a

Chlorophylls b c d

e

B a Bc/d

Biliproteins Carotenoids Per Pcy Carotenes Xanthophylls + + + + + + + + + + + (+) + + + + + (+) + +

Higher plants* + + Green algae + + Brown algae + + Diatoms + + Red algae + + + Blue-green bacteria + Green sulfur bacteria + + Purple sulfur bacteria + Footnotes to Table 1: * seed plants, ferns and mosses. Ba = bacteriochlorophyll a, Bc/d = bacteriochlorophylls c and d. Per = phycoerythrin, Pcy = phycocyanin, ( + ) = a trace. Table 2. Percentage compositon of thylakoidal carotenoids (from Wiessner). Red beech (Fa- Chlorella pyrengus sylvatica) oidosa me % 4 a-Carotene ß-Carotene Lyco- 34 15 pene Sum of carotenes 34 19 Lutein 45 50 Violaxanthin 14 10 Neoxanthin 7 12 72 Sum of xantho- 66 phylls Ratio xanthophylls/carotenes 1.95 (Fagus), 4.3 (Chlorella). prevented by culture in the dark; the admission of light induces the formation of the photosynthetic apparatus. Such systems are therefore especially suited to the study of the regulation of autotrophism and heterotrophism.

structure for P.p., and this is not possible if P.p. are subject to the random mobility of the lipid membrane components, as demanded by the fluid-mosaic model for membrane structure. The binding of a P.p. molecule to a protein would also be an unsatisfactory model, because the various P.p. would then be too widely separated for the efficient transfer of photons or resonance energy. A more feasible model would involve the binding of several P.p. molecules to one protein, and there is much evidence for a system of this kind; e.g. several chlorophyll-binding proteins have been isolated from thylakoid membranes, in particular P700-Chlorophyll a-protein (see) and Light-harvesting protein (see). Photosystems, pigment systems: structuralfunctional units of the light reaction of Photosynthesis (see). The quantum efficiency of photosynthesis in chloroplasts falls sharply at wavelengths longer than 680 nm, although chlorophyll still absorbs light from 680 to 700 nm. This phenomenon is known as the "red drop". However, the quantum efficiency of light above 680 nm is increased by the simultaneous presence of shorter wave-

354

Phototrophism

length light. This Emerson effect (see) led to the proposal that photosynthesis depends u p o n the interaction of two light reactions (i.e. two photosystems), b o t h driven by light less t h a n 680 nm, but only one by light of longer wavelengths. As a working hypothesis for the distribution of chlorophylls in the two photosystems (photosystems I a n d II, also called pigment systems I a n d II), it is proposed that the pigments are stacked in order of their maximum absorption wavelengths, arranged to trap light rather in the way that a rain gauge is designed to catch water. Thus in photosystem I, chlorophyll b Q^,^ 650 nm) absorbslight; by resonance transfer, quanta are then passed to chlorophyll a ( X ^ x 670 nm), f r o m there to chlorophyll a (>. max 680 nm), then to chlorophyll a (A l n a x 695 nm); finally all the collected energy is concentrated into and t r a p p e d by the relatively few molecules of P700. Light is also directly absorbed a n d transferred by each chlorophyll in the system. There are relatively few molecules of P700 (see Pigment 700), which is the ultimate acceptor or energy trap. Similarly, photosystem II contains chlorophyll b 6 i 0 , chlorophyll chlorophyll a^gQ a n d a final energy trap known as P690. Photosystem II is responsible for the photolysis of water. It has been shown that 4 quanta are required for the production of one oxygen molecule by PS II. Manganese a n d chloride ions have been strongly implicated in this process, a n d a possible mechanism at the metalloenzyme center might be represented as follows : 2[H20-Mn(II)]

2 [ H O ' -Mn(II)] —•

2 [ H 2 0 - M n + ( I I ) ] | p » 2[HO ' - M n + ( I I I ) ] —• 2[Mn(II)] + 0 2 + 2H + TTie two photosystems are coupled in series (see Photosynthesis). Phototrophism: see Nutritional physiology of microorganisms. Phrenosine: see Glycolipids. Phycoblllns: see Biliproteins. Phylloqulnone: see Vitamins; Vitamin K. Physostigmlne, eserine: an indole alkaloid from calabar beans, the ripe seeds of Physostigma venenosum (a woody vine indigenous to the west coast of Africa). P. contains a pyrrolidinoindole ring structure and a urethane group (Fig.), and exists in a stable form, m.p. 105-106°C, and an unstable form, m.p. 87° C. M r 275.35, [a] D - 8 2 ° (chloroform). It occurs with its A'-oxide, geneserine, m.p. 129°C, [a] D - 175° (acetone). H

Pbysostigmiae

Phytoalexins P. is used in opthalmic practice in the same way as pilocarpine, for pupil contraction a n d for the reduction of intraocular pressure. It is an inhibitor of acetylcholinesterase, a property shared by certain other basic urethanes, such as neostigmine (prostigmine); these urethanes presumably acylate, a n d therefore block, the enzyme. P. and neostigmine have been used in surgery to counteract the action of curare, a n d both have been used for the relief of Myasthenia gravis, a disease characterized by muscular weakness associated with a rapid breakdown of acetylcholine. These have now been largely replaced by other synthetic c o m p o u n d s . The fatal h u m a n dose of P. is about 10 mg. It was first isolated in 1864, when forty-six children in Liverpool were poisoned by eating calabar beans thrown on a rubbish h e a p f r o m a West African cargo ship. Phytln: see Myo-inositol. Phytic acid: see Myo-inositol. Phytoalexins, phytoncides, stress compounds: substances with antibiotic activity produced by plants in response to injury or stress, e.g. infection with fungi, bacteria a n d viruses, mechanical wounding, UV-irradiation, dehydration, cold, and treatment with phytotoxic chemicals (e.g. heavy metals). They function as growth inhibitors of phytopathogenic organisms, chiefly fungi. They have also been defined as novel post-infectional metabolites p r o d u c e d by plants in response to fungal infection, a n d which, because of their antifungal activity, protect the plant f r o m attack by fungi. Under certain conditions, P. may also be antibacterial. At present, there is no universally accepted definition; thus chlorogenic acid, which is f o u n d widely in plants, is considered by some authors to be a.P., when it is produced in increased quantities in response to infection or physical d a m a g e to plant tissues. P. do not belong, structurally or biosynthetically, to any one class of c o m p o u n d s . M a n y are phenols, e.g. Pisatin (see) a n d Phaseolin (see); there are also terpenes, e.g. I p o m e a m a r o n e (see); other examples are rishitin, steroid alkaloids, and unbranched, long chain, unsaturated compounds, e.g. safynol and Wyerone acid (see). For some phenolic P., derived f r o m phenylalanine, onset of infection is marked by a rapid increase in the activity of L-Phenylalanine ammonia-lyase (see) and a general increase in protein synthesis, presumably reflecting synthesis of enzymes required for P. formation. The greater n u m b e r of known P. have been isolated f r o m leguminous plants. A special group is f o u n d in orchids, which are only formed when the roots a n d shoot of the germinating orchid seed are colonized by fungi. The fungi penetrate with the plant, but they are prevented f r o m speading further by P., e.g. Orchinol (see) a n d hircinol. Activity of P. against microorganisms is expressed as E D 5 0 (see Dose). Ingham, J.L. "Phytoalexins a n d Other Natural Products as Factors in Plant disease Resistance" (1972) The Botanical Review (published by N e w York Botanical Garden), 38, 343-424.

Picrotoxin

Phytochemistry

OH

Phytochemistry: the chemistry of natural products from plants (see Natural product chemistry). It is part of plant biochemistry, and it is concerned chiefly with secondary metabolites. Phytoecdysone: see Ecdysone. Phytoene: an aliphatic, colorless, hydrocarbon carotenoid. Mt 544. P. is a polyisoprenoid. It contains six branch methyl groups, two terminal isopropylidene groups and nine double bonds, three of them conjugated. Only the A 1 5 double bond has cis configuration. Biosynthetically, P. is derived from two molecules of geranylgeranyl pyrophosphate, and it serves as a C^-starter molecule in the biosynthesis of other carotenoids; phytofluene, ^-carotene, neurosporene and lycopene are formed by the stepwise dehydrogenation of P. It is found widely in plants, and is especially plentiful in e.g. tomatoes and carrot oil. It was isolated in 1946 from tomatoes by J.W. Porter and F.P. Zscheile, and its structure was elucidated in 1956 by W.J. Rabourn and F.W. Quackenbush. For structure and biosynthesis, see Tetraterpenes. Phytolluene: an aliphatic, polyisoprenoid hydrocarbon carotenoid. Afr 548. P. contains ten double bonds (five of them conjugated), six branch methyl groups and two terminal isopropylidene groups. As in phytoene, the central double bond between C-15 and C-15 has cis configuration. P. is found widely in plants, e.g. tomatoes and carrots, and it is an intermediate in lycopene biosynthesis (see Tetraterpenes). Structural elucidationbyL.Zechmeisterin 1954. Phytohemagglutinins: see Lectins. Phytohormones, plant hormones: a group of natural (endogenous) plant growth regulators. P. are synthesized in small amounts in the plant, then transported to other plant parts, where they influence growth and differentiation. In contrast to animal hormones, P. have mulitple activities and low action specifities. The known stimulatory P. are Auxins (see), Gibberellins (see) and Cytokinins (see); those with inhibitory activity are Ab-

Wyerone acid

scissic acid (see), Flowering hormone (see) and Fruit ripening hormone (see). P. can be determined quantitatively by biological assay. The mode and site of action of P. at a molecular level are largely unknown. Phytoklnins: see Cytokinins. Phytol: see Chlorophyll. Phytoncldes: see Phytoalexins. Phytopharmacology: study of the action of synthetic and biogenic substances on plants. A knowledge of the action of biocides on plants is important for the application and development of herbicides, growth regulators and crop protection agents. Phytosterols: see Sterols. Phytotoxins: see Toxic proteins. Plcromycin: see Macrolides. Plcrotoxin, cocculin: a molecular compound of one molecule picrotoxinin and one molecule picrotin. P. is a neurotoxin, which occurs in the seeds of Anamirta coculus, and is also found in Tinomiscium phitippinense. As a specific antagonist of 4-Aminobutyric acid (see), it acts as a central and respiratory stimulant, and is an antidote to barbituates. It is extremely toxic to fish. Picrotoxinin is the active component of the molecular compound; picrotin is physiologically inactive.

Picrotoxinin, R: = CH 2 OH Picrotin, R: < C H

356

Pigment 700

Pigment 700, P700: a Chlorophyll (see) with an absorption maximum at 700 nm. P.700 is a component of photosystem I and serves as an energy sink or trapping center in the light reaction of this system. It is important in the primary energy transformation of Photosynthesis (see): the highly negative redox potential of P.700 (following its excitation by light) results in the transfer of electrons to the primary acceptor, which then transfers electrons to NADP via ferredoxin. The transiently oxidized P.700 (due to emission of electrons), is reduced again by electrons from cytochrome / The reaction center of photosystem I consists of about 500 molecules of closely packed P.700 in a quasicrystalline state, resembling the crystal of a semiconductor. Owing to the high degree of structural order, the electron excitation energy can be transferred by resonance. Pigment systems: see Photosystems. PIH: abb. for prolactin release inhibiting hormone (see Releasing hormones). Pilocarpine: an imidazole alkaloid, and the chief alkaloid from the leaves of Brazilian Pilocarpus species. Afr 208.26, m.p. 34° C, b.p. 5 260° C, [ct] — 100.5° (chloroform). P. is used therapeutically as a diaphoretic, i.e. to induce sweating, and especially in nephritis to relieve the kidneys and remove toxic metabolites. It is also used in opthalmology as an antagonist of atropine, and for regulating the intraocular pressure in glaucoma.

Plant mucilages L-Baikiain(l,2,3,6-tetrahydropyridine-acarboxylic acid), a rare nonproteogenic amino acid first isolated from the wood of Baikiaea plurijuga, is structurally related to L-P.a. Piper alkaloids: a group of alkaloids occurring in various species of Piper, especially black pepper (Piper nigrum). Structurally, P.a. consist of an aromatic carboxylic acid with an unsaturated side chain (e.g. piperic acid, sinapic acid) in amide linkage with a basic component, usually piperidine. The chief representative is Pipeline (see). Piperidine alkaloids: a group of alkaloids containing the piperidine ring system. Simple P.a. are the alkyl substituted piperidines which occur sporadically. The other P.a. are classified according to their origin, e.g. Conium alkaloids (see), Punica alkaloids (see), Sedum alkaloids (see) and Lobelia alkaloids (see). These various groups are structurally different and have different mechanisms of biosynthesis. Other P.a. are found in water lilies, and are biosynthesized from mevalonic acid (see Nuphara alkaloids). A dehydropiperidine structure is present in the Areca alkaloids (see) and the Betalains (see). Piperine: piperic acid piperidide, a Piper alkaloid (see), the chief alkaloid of black pepper (Piper nigrum) and responsible for its sharp taste.M r 285.35, m.p. 216°C. Both double bonds are trans (Fig.). The cis-cis isomer, earlier called chavicine, does not occur naturally.

Pilocarpine Plmaradlene type: see Diterpenes (Fig.). Plnane type: see Monoterpenes (Fig.). Pineal gland, pineal body, epiphysis, Corpus pineale: a small, cone-shaped, unpaired organ situated between the cerebral hemispheres on the roof of the third ventricle of the mammalian brain. Phylogenetically, the P.g. is a vestigial parietal eye, the light sensitive organ of reptiles. It produces the hormone, Melatonin (see). Ping-Pong mechanism: see Cleland notation. Pinitol: see Cyclitols. L-Plpecolic acid: piperidine-2-carboxylic acid, a nonproteogenic amino acid. It is formed from L-lysine, either by a-deamination followed by cyclization and reduction, or as a normal intermediate in the degradation of lysine to a-aminoadipic acid. The 4- and 5-hydroxy derivatives of LP.a. are found especially in mimosas and palms. H

H L-Pipecoiic acid

N H COOH H L-Baikiain

Piperine Plsatin: a Phytoalexin (see). P. is synthesized by peas (Pisum sativum) in response to infection to phytopathogenic microorganisms. M.p. 61 °C, [a]g> + 280° (c = 0.11, ethanol). P. is biosynthesized from acetate and cinnamic acid. Pituitary gland: see Hypophysis. PL: abb. for placentalactogen. Placentagonadotropin: see Choriogonadotropin. Placentalactogen: see Choriomammotropin. Plant hormones: see Phytohormones. Plant mucilages: high M r , complex, colloidal polysaccharides, which form gels and have adhesive properties. They are widely distributed in the plant kingdom, being found as secondary membrane thickening and as intercellular and intracellular material. They occur in root, bark, cortex, leaves, stalks, flowers, endosperm and seed coat. Some bulbs contain special mucilage cells. Some P.m. may function as food reserves. On account of their high affinity for water, certain P.m. may be used as water reservoirs (i.e. as antidessicants) by plants that live under very dry conditions; mucilagenous seed coatings may have a similar function. Together with the structurally related plant gums, P.m. form an ideal material for sealing da-

Plant pigments

357

maged tissue; owing to their often heterogeneous carbohydrate composition, P.m. are relatively resistant to microbial attack. P.m. can be roughly classified into three groups: 1. Neutral, containing one or more types of sugar, but no uronic acids, e.g. a linear polymer of 1,4linked m a n n o p y r a n o s e isolated f r o m the tubers of certain orchids. 2. Acidic, resembling the plant gums, but usually containing D-galacturonic acid as the acidic residue, e.g. a P.m. f r o m the bark of Ulmus fiilva (slippery elm), which contains D-galactopyranose, D-galacturonic acid, L-rhamnopyranose a n d 3-methyl-D-galactose. In this type of P.m., the ratio of uronic acid residues to neutral sugar residues is usually about 1 :3. 3. P.m. present in algae (notably seaweeds), often of highly complicated structure a n d very high M p a n d often containing esterified sulfate. This group is well exemplified by the agars f r o m seaweeds. The agars range from a neutral species, agarose (see Agar-Agar), to the highly acidic, sulfated Carrageenans (see). The primary sequence of the entire agar family is based on a repeating disaccharide unit of galactose derivatives. Other seaweed P.m. are Alginic acid (see), laminarin (p-1,3 linked glucose residues with some p-1, 6 b r a n c h points; isolated from Laminaria) a n d fucoidin (contains sulfated fucose residues; isolated f r o m Laminaria and Fucus spp.). Plant pigments: see Natural pigments. Plaque: a transparent area in a lawn of bacteria on the surface of a solidified growth medium. P. is caused by lysis of bacteria in that area by bacteriophage. U n d e r controlled conditions, each P. represents a center of infection initiated by one infective bacteriophage particle. The number of P. p r o d u c e d after evenly spreading a k n o w n volume of phage suspenison over the surface of the bacterial culture is used as a simple assay of the n u m b e r of infective phage particles. The term is also used in a general sense for an area of lysed cells in a lawn. In immunology, a lawn of red blood cells is used to detect immunoglobulin-producing cells in a P. assay. When complement is a d d e d to the system, those erythrocytes that have b o u n d to the immunoglobulin molecules are lysed, leaving a transparent P. Since most antigens can be artificially b o u n d to erythrocyte membranes, the technique can be used for practically any antibodies capable of fixing complement. Plasma albumin: see Albumins. Plasma factors: see Blood coagulation. Plasmaklnlns: physiological, highly active oligopeptides, with hormone-like properties. P. act

Plasma proteins u p o n the smooth muscle of blood vessels, gastrointestinal tract, uterus and bronchi. Important representatives are Bradykinin (see), kallidin and methionyl-lysyl-bradykinin. Plasmalemma: the cell membrane. See Biomembrane. Plasmalogens: see Phospholipids. Plasma proteins: a complex of predominantly conjugated proteins present in the b l o o d plasma of vertebrates. The number of P.p. is estimated to be more t h a n 100. In mammalian plasma the concentration of P.p. is 6 - 8 % . Serum proteins lack Fibrinogen (see) a n d Prothrombin (see), but are otherwise essentially the same as P.p. Approximately 60 P.p. have been isolated and characterized. Of these, only albumin, prealbumin, retinol binding protein a n d a few trace proteins (e.g. lysozyme) are free f r o m carbohydrate. The remaining P.p. are glycoproteins e.g. Orosomucoid (see), Hemopexin (see), Haptoglobin (see), Cl-inactivator, Immunoglobulins (see); some may also contain lipids (see Lipoproteins). P.p. help to regulate the pH-value and osmotic pressure of the b l o o d ; they transport ions, hormones, lipids, vitamins, metabolic products, etc. P.p. are also responsible for blood coagulation, for defense against foreign proteins or microorganisms (see Immunoglobulins), and for certain enzyme reactions. With the exception of immunoglobulins, the P.p. are synthesized in the liver. The 5 main groups of P.p., i.e. albumin, -toluene-sulfonic acid is nonvolatile, and must be loaded onto the analytical column with the amino acids; it emerges early and tends to interfere with the separation of the acidic amino acids. Tryptophan may also be determined from the UV-absorption of the P., after making allowance for the tyrosine content. Some P. can be hydrolysed for analytical purposes by a mixture of proteolytic enzymes, such as papain or subtilisin (both fairly nonspecific) in conjunction with leucine aminopeptidase and proline iminopeptidase, but such hydrolyses are often incomplete. The next stage in primary structure determination is the reductive (2-thioethanol) or oxidative (performic acid) cleavage of disulfide bridges, followed by enzymatic (e.g. with trypsin) and/or chemical (e.g. with cyanogen bromide) cleavage of the polypeptide chain. Peptides from this cleavage are separated by ion-exchange chromatography or by the finger print technique (a two-dimensional separation on paper or thin layer plates, in which electrophoresis is used in the first dimension and partition chromatography in the second dimension). Finally, the amino acid composition and the N- and C-terminal amino acids of each cleavage peptide are determined. In the Edman degradation, the peptide is reacted with phenylisothiocyanate at pH 8-9 at 40° C; acid treatment removes the ^-terminal amino acid (Rj) as a substituted phenylthiohydantoin (PTHamino acid), which is identified chromatographically (Fig. 6); the next amino acid (R2) becomes the JV-terminus of the new peptide. The method has been automated, and with the aid of a specially built sequenator, it is possible to determine the sequence of amino acids in a peptide chain, starting from the JV-terminus. The longest sequence so far determined by this method is 60 amino acid residues. The total operation is complete in a few days, and it requires between 10 and 50 mg of peptide. Primary sequences of many P. (e.g. p-lactoglobulin) have been determined with the aid of the automated Edman degradation. JV-terminus identification may also be performed with Sanger's reagent or dansyl chloride. Once the amino acid sequence of each peptide is known, their order in the original P. must be established. Peptides at the JV- and C-termini can be identified by comparison with the termini of the original P. Different peptide fragments are required, which overlap the cleavage sites of the first set of peptides. More than one method is therefore used to cleave the P. into peptides, e.g. proteolytic enzymes of different specificities can be used, and the e-amino group of lysine may be blocked with CS2 or by trifluoroacetylation be-

Proteins fore attack with trypsin, so that the P. is cleaved only at the arginine sites. Positions of disulfide bridges are determined as follows: after determination of the linear sequence, tryptic digestion is performed with the disulfide bridges intact; cystine-containing peptides are isolated and their disulfide bridges are cleaved; the resulting two peptides are identified by comparison with the tryptic peptides of the linear sequence; during this procedure, any free SH-groups are blocked with iodoacetate while the disulfide bridges are still intact. An early method of sequence determination employs partial hydrolysis of the P. with acid or nonspecific endopeptidases. The resulting hydrolysate contains a large number of relatively small peptides, representing many overlapping regions of the sequence. By analysis of a sufficient number of peptides, and comparison of their amino acid sequences, it is possible to construct the linear sequence of the original P. (hence the term "jig saw puzzle" method). The classical determination of the primary structure of the A chain of insulin (see) was performed in this way. Comparative studies on the primary structures of homologous P. from different species (e.g. hemoglobin from vertebrates, cytochrome c from a wide range of species, see homologous proteins), or analogous P. (e.g. subtilisin from Bacillus subtilis and mammalian trypsin) have made a valuable biochemical contribution to questions of divergent and convergent evolution. However, for an explanation of P. function and behaviour, especially the mechanism of enzyme action, the primary structure alone is insufficient and a knowledge of secondary and tertiary structure is needed. Secondary structure refers to the way in which the polypeptide chain is folded. Folding is due to the formation of hydrogen bonds between peptide bonds in close juxtaposition to one another (separated by 0.28 nm). Hydrogen bonds form between the carbonyl oxygen of one peptide bond and the amide hydrogen of the other. In helical structures, hydrogen bonds exist within the peptide chain, whereas pleated sheets or P-structures result from hydrogen bonding between two lengths of polypeptide chain (which may or may not be part of the same chain). The most common helical structure is the a-helix, with 3.6 amino acid residues per revolution (Fig. 7). Collagen (see) has a specialized structure containing interchain, hydrogen-bonded, left-handed helices. Otherwise, all known P. helices are right-handed, but the possibility remains that left-handed helices might be found in P. that have not yet been analyzed by X-ray diffraction, e.g. membrane P. In the pleated sheet structure, the polypeptide chain is more or less stretched out, and neighboring lengths of chain can be parallel or antiparallel (with respect to their N- and C-termini), e.g. the pleated sheet structure of silk fibroin (Fig. 8) consists of antiparallel polypeptide chains. Examples of P. with extreme contents of a-helix structure are myoglobin (75 % a-helix) and a-chymotrypsin

383

Proteins

• -C. © = H, hydrogen

©-R,

•N,

O-O,

bonds

Fig. 7. Secondary structure of proteins. Left, ppleated sheet structure of a fibrous protein; right, a-helix structure of a polypeptide chain

Fig. 8. fi-Pleated sheet structure; antiparaliel arrangement of two polypeptide chains (8% a-helix). Almost 100% P-structure is found in silk fibroin and in Bence-Jones P. When moist hair is stretched, the a-helical structure of the akeratin is reversibly converted to the p-structure. Most P. also contain a large number of amino acid residues that cannot be assigned to any regular structure. Only certain peptide conformations are permissible; others are impossible because they would bring neighboring unbonded atoms too close together (see Ramachandran plot). The fully denatured state of a polypeptide is referred to as a random coil; this completely disordered structure is also found in helix-coil transitions and in synthetic polyamino acids.

Proteins The percentage of a-helical structure in a P. can be estimated by measurements of optical rotatory dispersion (ORD) and circular dichroism (CD). These methods depend on the fact that the helices are themselves optically active. The slow rate of exchange of P. hydrogen with deuterium oxide or tritium oxide dependes inversely on the percentage of helical structure; it is presumably related to the greater difficulty of replacing hydrogens involved in intramolecular hydrogen bonds (i.e. helix stabilization) compared with those that are hydrogen-bonded to water. There are discrepances in the percentage helical structure indicated by the optical methods and by isotopic hydrogen exchange; this could be caused by the existence of left-handed helices (sign of optical activity is opposite to that of a right handed helix), which would put the optical methods at fault; or it could be the result of the retardation of hydrogen exchange by factors in the P. other than intrahelical hydrogen bonds, e.g. p-structures, or areas so surrounded by hydrophobic bonds that hydrogen exchange is difficult. X-ray crystallography, which is used for the determination of tertiary structure, also provides detailed information about secondary structure. Hemoglobin and myoglobin have about 75 % helical content as determined by X-ray crystallography and ORD, but these two methods show poor agreement on the a-helical content of other P., such as cytochrome c and carbonic anhydrase. This probably signifies differences in the secondary structures of dissolved and crystalline P. Tertiary structure defines the overall molecular shape and gives detailed information about the spatial arrangement of reactive amino acid residues, e.g. at the active sites of enzymes, or at the antigen-binding site of antibodies. With a knowledge of tertiary structure it is possible to visualize the three dimensional shape of a P. to a high resolution (0.2 nm or better) and to observe the changes in molecular architecture that accompany the formation of an enzyme-substrate or enzyme-inhibitor complex. Tertiary structure is determined from the X-ray diffraction of isomorphic crystalline heavy metal derivatives of the P. The resulting diffraction diagram, and the electron density map constructed from it, provide information on the type and position of amino acid residues; at higher resolution (0.15 nm) it is even possible to determine the distance between atoms in the P. molecule. Despite the very specialized and demanding nature of this technique, the spatial structures of more than 60 P., mostly enzymes, have been determined (Fig. 9) (see, e.g. Ribonuclease, Immunoglobulins, Homologous proteins). The occurrence and stabilization of the three-dimensional P. structure is the result of several forces: hydrogen bonds between tyrosyl and carboxyl or imidazole groups, and between seryl and threonyl residues; disulfide bridges, which have a primary function in the stabilization of conformation; Van-der-Waals forces; noncovalent mutual attraction between the uncharged (— CH 3 , — CHjOH) or hydrophobic (phenyl, leucyl) residues separated by about 0.3nm; electros-

384

Proteins

Proteins

Heme CT

NT

Front

Side

Fig. 9. Chain conformation (tertiary structure) of myoglobin. The molecule consists of eight stretches of a-helix, lettered A to H. NT is the ¿V-terminus, containing two nonhelical amino acids. CT is the Cterminus, containing four nonhelical amino acids. The number of amino acid residues in each helical stretch is A(16), B(16), C(7), D(7), E(20), F(10), G(19), H(26). The nonhelical regions between the helical stretches are AB(1 amino acid residue), CD(8), EF(8), FG(4), GH(5). tatic attraction between polar side groups (e.g. COO+NH 3 ), which are also involved in the solvation of the molecule; interaction of P. and aqueous solvent, which favors the formation of hydrophobic bonds (interatomic distance 0.31-0.41 nm) in the nonpolar interior of the molecule, and thus makes an important contribution to natural P. conformation. Secondary and tertiary structures are referred to jointly as chain conformation. In fact, it is sometimes difficult to make a clear distinction between secondary and tertiary structure. According to nuclear magnetic resonance (NMR) studies, the chain conformation of a protein is changeable within certain limits, so that the conformation determined by X-ray crystallography represents one of several possible states "frozen" by crystallization.

Fig. 10. Quartemary structure of hemoglobin, showing spatiai arrangement of the a- and f)chains. Black discs represent heme groups.

Aggregation or association of two or more identical or different polypeptide chains by noncovalent interaction leads to stable oligomeric (or multimeric) P. These ordered associations represent quartemary structure, and the individual polypeptide chains are called subunits (Fig. 10). In rare cases, quartemary structure may also be maintained by disulfide bridges. P. with quarternary structure are of widespread occurrence. 650 P. (including 500 enzymes) with subunit structure had been described by 1974. Most of the known multimeric P. contain either 2 or 4 similar sized subunits. Far less common are P. with uneven numbers of subunits (by 1973, 28 trimeric P., including 23 enzymes, 5 pentameric, 2 heptameric and 1 nonameric P. were known), with different sized subunits (e.g. RNA-polymerases, ATP-ases, aspartate transcarbamylase, cytochrome oxidase, tryptophan synthase, aspartate kinase), with subunits capable of independent enzymatic activity (e.g. tryptophan synthase, aldolase, transaldolase, sucrase, isomaltase, leucine aminopeptidase and alcohol dehydrogenase), or with both regulatory and catalytic subunits (aspartate transcarbamylase, protein kinase, aldose reductase). Possession of quartemary structure appears to confer a flexibility of shape and activity, which is necessary for the physiological role of the P. Monomers derived from multimeric enzymes are usually inactive. The subunit composition of oligomeric P. can be determined by dissociation of the aggregate and investigation of the separate subunits by ultracentrifugation, polyacrylamide disc electrophoresis, gel filtration, ion exchange chromatography or viscometry. Alternatively, the structure of the intact molecular aggregate can be studied by electron microscopy, or by low angle X-ray or

Proteolysis

385

Proteinoids

neutron diffraction. Aggregates are dissociated by treatment with sodium dodecyl sulfate (1%), 8M urea or 6M guanidinium chloride, by changes of p H , temperature or protein concentration, or by chemical modification (succinylation, maleylation, removal or attachment of cofactors). Detailed structures, determined by X-ray diffraction analysis, have been reported for the following oligomeric P.: hemoglobin (Fig. 10), lactate-, malate-, alcohol- and glyceraldehyde phosphate-dehydrogenases, 5 other glycolytic enzymes and aspartate transcarbamylase. Proteinoids: heteropolyamino acids; artificially prepared polypeptides ( M I > 1000) formed in 20-40% yield by heating a mixture of several amino acids for 16 h at 170°C (thermal condensation). P. show many similarities with globular proteins, e.g. relative quantities of individual amino acids, solubility, spectral properties, denaturation, degradation by proteases, catalytic activity (e.g. esterase, ATPase, decarboxylase activities) and hormone action ( M S H activity). They can be regarded as models for the first information-carrying molecules. In water, they become organized into microsystems with a typical ultrastructure (microspheres), which share a number of properties with living cells, i.e. they possess a bilayer membrane, which exhibits a certain degree of semipermeability; they can also multiply in the absence of nucleic acids, by budding. Proteoglycans: high Mt compounds of carbohydrate and protein, found in animal structural tissues, e.g. the ground substance of cartilage and bone. The ground substance and gel fluids of these tissues owe their viscosity, elasticity and resistance to infective organisms to the presence of P. Each P. contains 40-80 acidic mucopolysaccharide chains (glucosaminoglycans) bound to protein via 0-glycosidic linkages to serine or threonine. In contrast to the Glycoproteins (see), the prosthetic group of P. has MT 20000-30000, consisting of many (100-1000) unbranched, regularly repeating disaccharide units. The disaccarides are composed of N-acetylhexosamine (which may or may not be sulfated) linked to a uronic acid or to galactose. In the chondroitin

sulfates, the linkage region between polysaccharide and protein contains xylose linked O-glycosidically to serine; this is followed by two galactose residues and one glucuronic acid, to which the first repeating disaccharide is attached. In corneal keratan sulfate, the polysaccharide chain is linked to the protein via a glucosamine residue, which is joined to an asparagine side chain by a glycosylamine linkage. In cartilage keratan sulfate, most of the carbohydrate-protein linkages are O-glycosidic bonds between JV-acetylglucosamine and the hydroxyl of serine or threonine. The chondroitin sulfates, together with collagen, form the major component of cartilage. Mammalian skin contains proteodermatan sulfate, and the intestinal mucosa contains protein-bound heparin. Heterogeneity of P. is due to differences in polypeptide chain length, and to the number and distribution of the attached polysaccharide chains. Microheterogeneity also exists, due to small differences in the chain lengths of the polysaccharide chains, and the distribution of sulfate residues. P. can be extracted from cartilage under mild conditions with 4 M guanidinium chloride. The resulting P. subunit has S w = 16S (see Sedimentation coefficient), and M r 1.6 x 106. In the tissue, P. exist as giant molecular aggregates (S w = 70S und 600S), which are formed by noncovalent association of P. subunits with a glycoprotein. Proteohormones, protein hormones: proteins (often glycoproteins) with hormonal function. Like other proteins, they are synthesized by the translation of appropriate m R N A , and degraded by proteolysis. M r of P. are between 5000 and 25000 for the monomers and correspondingly higher for the dimers and polymers. See Choriogonadotropin; Follicle stimulating hormone; Luteinizing hormone; Thyreotropin. Although a close relationship exists between P. and Peptide hormones (see), a distinction is made between these two. Proteolysis, protein degradation: hydrolysis of proteins by the action of proteolytic enzymes, or nonenzymatically by acids (e.g. 6 M HC1 at 110° C for 24 h or longer) or alkalis. Ultimate pro-

Table. Formation of biologically active proteins from inactive precursors

by limited

proteolysis.

AA, amino acids; Chy, chymotrypsin; CP, carboxypeptidase Inactive protein Pepsinogen Prorennin Trypsinogen Chy-ogen Pro-CP A Prothrombin Proinsulin Fibrinogen

Mr

No. of A A 42500 36200 24000 25666 90000 72000 9100 340000

362 321 229 245 850 560 84 3400

Active fragment Pepsin Rennin Trypsin Chy A CPA Thrombin Insulin Fibrin monomer

Mr

N o . of A A 34500 30700 23400 25170 34300 39000 6000 327000

327 272 223 241 307 309 51 -3270

In limited P. only certain peptide bonds of a protein are hydrolysed; this results in the production of biologically active (e.g. enzymes or hormones) or inactive (e.g. para-K-casein) proteins or peptides. Limited P. occurs in digestion, blood coagulation and milk clotting; it is responsible for the activation ofzymogens and for the release of certain peptide hormones, e.g. insulin, angiotensin, vasopressin, oxytocin and various kinins (see Table).

386

Prothrombin

ducts of P. are amino acids. Dietary proteins are hydrolysed to L-amino acids by P. in the intestine. After absorption, these amino acids are used in the synthesis of new proteins specific to the organism ; these, in turn, are eventually hydrolysed to amino acids by P. as part of the continual process of synthesis and degradation of cellular constituents (see Turnover). A distinction is made between extracellular P. (e.g. digestion and blood coagulation) and intracellular P. The latter occurs at neutral and acidic pH values, and the relevant endopeptidases (called cathepsins) are localized chiefly in the lysosomes. The cathepsins from protease-rich organs, like liver, spleen and kidney, can be separated into cathepsins A, B, C, D, E and L. Cathepsins A to E have pH optima in the range 2.5-6, and (with the exception of D and E) they also hydrolyse synthetic, low Mr substrates. Other cathepsins are active only at neutral pH, and they attack only proteins. M t of cathepsins are between 25000 (cathepsin B) and 100000 (cathepsin E). Cathepsins B, B2, C and some neutral cathepsins are SH-enzymes. In the intact cell P. is controlled and it occurs in the lysosomes (autophagy). In damaged cells, the same cathep-

CHCHCH ' 1 1

H,N-Ala-

Peptide Intermediates (Mr7000) (Mr 25000)

h

H«

Protoheme 25 000). Activation is finally achieved by cleavage of the thrombin precursor into the A and B chains of thrombin (Fig.), which are joined by a disulfide bridge. The concentration of P. in human plasma is only about 6 mg/100 ml. Prothromboplastln: see Blood coagulation. Protoalkaloids: see Biogenic amines. Protocyanln: see Cyanidin. Protoheme, heme, ferroheme, ferroprotoporphyrin, protoheme IX: [7,12-diethenyl-3,8,13,17-tetramethyl-21H, 23 H-porphine-2,18-dipropanoate (2-)-N21,N22,N23,N24]-iron; or 1,3,5,8-tetramethyl-2,4-divinylporphine-6,7-dipropionic acid ferrous complex. C 34 H 32 FeN 4 0 4 . Mr 616.48. Protoheme crystallizes as fine brown needles with a violet sheen; 6572 = 5.5 x 103; in phosphate buffer pH 7.0, absorption maxima occur at 575 nm and about 550 nm. It is the prosthetic group of a number of hemoproteins, e.g. hemoglobins, erythrocruorins, myoglobins, some peroxidases, catalase and cytochromes b. The four coordinate bonds of the iron lie in the plane of the nearly planar porphyrin ring structure, while the two unoccupied sites of the iron are perpendicular to it.

CH 1 -Th

Arg,

A-chain (Mr 6 0 0 0 )

H*

B-chain (Mr 33000)

*HThrombin

^Thrombin precursor (Mr 39000)^ I n t e r m e d i a t e 1 (Mr 6 5 0 0 0 )

^

P r o t h r o m b i n (Mr 7 2 0 0 0 )

Diagrammatic representation of prothrombin structure and thrombin formation. Vertical arrows show the sites of proteolytic cleavage of the prothrombin chain (sites 1,2 and 3) during conversion into thrombin. CH carbohydrate. sins are released from the ruptured lysosomes and are responsible for autolysis, i.e. the uncontrolled total degradation of the cell. Prothrombin, factor II: an enzymatically inactive, calcium-binding, single chain c^-glycoprotein of blood plasma. Mr 72000; carbohydrate content 14.7% (bovine), or 11.8% (human). Synthesis of P. occurs in vertebrate liver and requires vitamin K. During Blood coagulation (see), P. acts as a proenzyme (zymogen): it is converted by thrombokinase (EC 3.4.21.6) (also required are active factor V, a plasma globulin, phospholipid and calcium) into the coagulation enzyme Thrombin (EC 3.4.21.5) (see). Activation occurs in three stages by proteolytic cleavage. The first intermediate (M r 65000) retains all four carbohydrate chains of the original P. This is then hydrolysed by factor Xa, to form the single chain thrombin precursor (M r 39000) and a carbohydrate-rich cleavage product (intermediate 3, Mr

CH,

CH=CH,

H3CCH,I CH, I COOH

-CH,

/

N

Fe

V

CH2 ch 2 COOH Protoheme

N

CH,

-CH = CH,

Protokaryote

Psychotropic agents

387

Protokaryote: see Cell. Protomer: see Subunit. Protopectln(s): a ground substance in plant cell walls. P. consist of insoluble Pectins (see) and are probably not pure homoglycans. They are present in the cell wall as salts of calcium and magnesium. The constituent polygalacturonic acid chains of P. are linked to one another by salt linkages, phosphate bonds and esterification with arabinose. Protoplast: see Cell, 2. Prototrophism: the property of being able to grow at the expense of usual or common nutrients (see Nutrient medium), with no special requirement for Growth factors (see). P. is shown by prototrophic organisms. Provitamin D 2 : see Ergosterol. Provitamin D 3 : see 7-Dehydrocholesterol. Provitamins: inactive precursors of Vitamins (see). P. are mostly of vegetable origin, and are converted into active vitamins after absorption from the diet. PHPP: abb. for 5-Phosphoribosyl 1-pyrophosphate. Pseudoalkalolds: a group of alkaloids earlier assigned to other groups (e.g. some were grouped with the terpenes) with which they show a close structural relationship. At the time, their nitrogen content seemed incidental. Pseudoglbberellln A,: see Gibberellins. Pseudolndlcans: an old name for Iridoids (see). Pseudo-lsoenzymes: multiple forms of an enzyme, which catalyse the same reaction. They have similar properties to isoenzymes, but do not have genetically determined differences of primary structure. Their multiplicity is the result of enzymatic or nonenzymatic modification of one original primary sequence, either in vivo or in vitro (i.e. during isolation). Examples of P. formed in vivo are the different chymotrypsins, trypsins, pepsins and carboxypeptidases, each group being derived from a single zymogen. Also in this category are the different degrees of aggregation shown by oligomeric enzymes that consist of identical subunits, e.g. glutamate dehydrogenase. P. formation in vitro is responsible for the occurrence of the 4 a-amylases, the 2 yeast phosphofructokinases, as many as 13 heart muscle lipoyl dehydrogenases, and the numerous forms of phosphoglucose isomerase. Pseudopelletlerlne, AMP

a-

©-0-CH,

a

OH OH

5- Phospho- a - D - R i b o s e 1- d i p h o s p h a t e

® Ç H 2, ©-O 0-C

5'-Phosphoribosyl-5-amino-4imidazolecarboxylate

^- -GGlluuttaam mi n e

Amidophosphoribosyltransferase (EC 2 . 4 . 2 . 1 4 )

-H,0 ^ PPP: gG l u t a m a t e 0

C02 HC

NH,

Phosphoribosylaminoimidazole carboxylase (EC 4.1.1.21) - N

OH OH 5- P h o s p h o - 0 - D-ribosylarnine Phosphoribosylglycinamide synthetase (EC 6.3.4.13) \ H,C-NH

I

« L — HzO

AIP ADP diphosphate (TDP)

Nucleoside J^ATP diphosphate kinase ^ a o p (EC 2.7.4.61

0

| Thymidine triphosphate

(TTP||

O*

Ci H\|

H, /H

OH H Deoxyuridine monophosphate (dUMP)

r

Thymidylate synthase (EC 2.1.1.45)

s .o ^ — N , N -Methylene tetrahydrofolate

H,0 ««O^

HCHO "

Purine biosynthesis

Tetrahydrofolate »Dihydrofolate

NADP + NADPH+H+

CH,

IT)

H2N"

^n-

@-0—CH,

Cr

H

OH H

|

OH

OH

Thymidine monophosphate (TMP). Thymidylic acid

Aminoimidazole ribotide

Fig. 2. Biosynthesis of thymidine nucleotides.

CHO

nh2

=NH /

-u

H\|

|/H

H j N ' ^ l j l ^

RiP

OH H Deoxycytidine

5'-monophosphate

N?N,0-Methylene

tetrahydrofolate

""-^Tetrahydrofolate

NH2 X

OHC

TRiP -C-3-Fragment?

NH2 -RiP

D-O-CH;

I y i 0.

M H\. OH

tt VH

H

5-Hydroxymethyld e o x y c y t i d i n e monophosphate

Fig. 3. Biosynthesis of dylic acid.

S-hydroxymethyl-deoxycyti-

2-MethyI-4-amino-5-fTydnoxymetlTylpyrimidine

Fig. 4. Synthesis of 2-methyI-4-amino-S-hydroxymethyl-pyrimidine from aminoimidazole ribotide (RiP = ribose 5-phosphate).

Pyrimidine degradation

397

sis of aspartate carbamoyltransferase and dihydroorotate oxidase (EC 1.3.3.1). Biosynthesis of the pyrimidine ring of thiamine (vitamin Bj) from aminoimidazoleribonucleotide. The 2-methyl-4-amino-5-hydroxymethyl-pyrimidine ring present in thiamine is synthesized from aminoimidazoleribonucleotide, which is an intermediate in purine biosynthesis (Fig. 4). Pyrimidine degradation, pyrimidine cataboiism:

reductive or (in special cases) oxidative reactions leading to the cleavage of the heterocyclic ring of natural pyrimidines. 1. Reductive P.d. (Fig.). To a certain extent, this process represents a reversal of Pyrimidine bioUMP

Pyrroles aminoisobutyrate. These endproducts are transaminated and metabolized to common metabolic intermediates (Fig.). 2. Oxidative P.d. In Corynebacterium and Mycobacterium uracil is oxidized to barbituric acid, which is cleaved hydrolytically to urea and malonic acid. Thymine is oxidized to 5-methylbarbituric acid, followed by hydrolysis to urea and methylmalonic acid. Pyrimidine dlmers: s e e D i m e r s . Pyroglutamlc acid, pyrrolidine carboxylic acid,

abb. Pyr or < Giu, S-oxoproIine: an internal cyc-

lic lactam of Glutamic acid (see) representing a condensation of the a-amino with the y-carboxyl TMP

UMP-Pyrophosphorylase

Dihydrouracildehydrogenase

H2N—CO—NH—Ofe—CH,- -COO" 3-Ureidopropionate p-Ureidopropionase NH3+ COj + H2N—CH2—CH2—COO" ß-Alanine-aminotransferase OHC—CH2—COO" Malonate-sem ¡aldehyde I Acetyl-Coenzyme A

H2N—CO—NH—CH2—CH—COO" Ureidoisobutyrate ß-Ureidopropionase CHs NH3 + C02 + H2N—CH2—CH—COO"

CH, I OHC —CH—COO" Methylmalonate - semialdehyde \ I Succinyl-Coenzyme A

Reductive degradation of uracil and thymine.

synthesis (see). The pyrimidine ring is partially hydrogenated, and the resulting dihydrocompound is cleaved hydrolytically. Cytosine is converted to uracil by deamination, and uracil is degraded to ¡3-alanine. Thymine is degraded to f3-

group. TV-Terminal residues of Pyr are found in certain peptide hormones, e.g. thyrotopin releasing hormone. Pyrroles, pyrrole derivatives: c o m p o u n d s

con-

taining the pyrrole ring. They are subdivided into

Pyrrolidine alkaloids

398

mono, di, tri and tetrapyrroles. The tetrapyrroles may be noncyclic or cyclic. Bile pigments (see) and the chromophores of Biliproteins (see) are linear, while Porphyrins (see) and Corrinoids (see) are cyclic tetrapyrroles. Pyrrolidine alkaloids: a group of Alkaloids (see) with simple structures. P.a. are either derivatives of proline (e.g. stachydrin and its diastereoisomer, betonicin), or they are derived from a Nmethyl-2-alkylpyrrolidine (e.g. hygrin and cuskhydrin). The latter occur together with the tropane alkaloids, with which they share the same biogenetic precursors, ornithine and acetate.

I

CH3

Pyruvate The necic acids (esterified with the hydroxyls of the necines) are branched aliphatic, mono or dibasic acids, containing 5—10 carbon atoms, e.g. angelic, monocrotalic, senecic and tiglic acids. The various combinations of necines and necic acids, together with amine oxides and isomers gives rise to a very large number of P.a. Free necines also occur naturally. P.a. occur chiefly in species of Senecio, the largest genus of the Compositae. They are hepatotoxic, and can cause liver cirrhosis in grazing animals. Pyrrolo qulnollne qulnone, PPQ: 2,7,9-tricarboxy-1 //-pyrrolo [2,3-/] quinoline-4,5-dione, the cofactor of the enzyme methanol dehydrogenase (EC 1.1.99.8) from Hyphomicrobium X and Methylophilus methylotrophus, and of glucose dehydrogenase (EC 1.1.99.-) from Acinebacter calcoaceticus. Ref: Duine, J.A., Frank, J., and Verwiel, P.E.J. (1980) Eur. J. Biochem. 108, 187-192.

Hygrin Pyrrolldine-2-carboxyllc acid: see L-Proline. Pyrrolldone carboxylic acid: see Pyroglutamic acid. Pyrrollzidlne alkaloids, Senecio alkaloids: a group of ester alkaloids, in which amino alcohols (necines) are esterified with necic acids. The necines are derivatives of the p y r r o l i d i n e ring system (also known as 1-azabicyclo [0,3,3] octane) (see Alkaloids, Table) and they possess one or two alcoholic hydroxyls, e.g. retronecine (Fig.).

Retronecine

COOH

Pyrrolo quinoline quinone Pyruvate: the anion of pyruvic acid. P. is an important metabolic intermediate in aerobic and anaerobic metabolism (Fig.). P. synthesis: 1. P. is synthesized from phosphoeno/pyruvate in Glycolysis (see). Phosphoeno/pyruvate is an enol ester and an energy-rich compound with a free energy of hydrolysis of 50.24 k j (12 kcal) per mol. During catalysis by pyruvate kinase, this free energy is exploited for the transfer of the phosphate group to ADP, resulting in the synthesis of ATP und P. Serine

Cysteine

CH2=C—COO" Phosphoeno/pyruvate Pyruvate ADP—V kinase ATP" — Alanine Lactate dehydrogenase

Central position of pyruvate in metabolism. TPP thiamine pyrophosphate, PAL-P pyridoxal phosphate, Lip lipoamide.

Pyruvate carboxylase 2. P. is produced in the metabolism of certain amino acids, in particular transamination of alanine, dehydration of serine, and desulfhydration of cysteine. P. metabolism: 1. P. is reduced to lactate in anaerobic glycolysis. 2. It is converted to ethanol in anaerobic Alcoholic fermentation (see). 3. By the action of the pyruvate dehydrogenase complex, under aerobic conditions, P. is oxidatively decarboxylated to acetyl coenzyme.A. The latter is an important metabolite in various other biosynthetic and biodegradative processes. Equation for oxidative decarboxylation of P.: C H 3 C O C O O - + HSCoA + N A D + — (Pyruvate) (Coenzyme A) CH3CO-SC0A + C 0 2 + NADH + H + (Acetyl coenzyme A) Complete oxidation of one molecule of P. via the TCA-cycle results in the production of 15 molecules of ATP (14 from the respiratory chain + 1 from substrate level phosphorylation in the conversion of succinyl-CoA to succinate). 4. P. is carboxylated to oxaloacetate (see Carboxylation), representing the first stage in Gluconeogenesis (see). 5. During Nitrogen fixation (see) in Clostridium, P. undergoes phosphoroclastic fission into acetyl phosphate and C 0 2 . Pyruvate carboxylase (EC 6.4.1.1): a biotin-dependent ligase, in animals and plants, which catalyses the addition of C 0 2 to pyruvate: Pyruvate + C 0 2 + ATP + H 2 0 ^SL* Oxaloacetate + ADP + Pj. The enzyme is practically inactive in the absence of its positive allosteric effector, acetyl-CoA. This reaction is an important early stage of Gluconeogenesis (see), and is an example of C 0 2 fixation in the animal organism. For the mode of attachment of the coenzyme, biotin, and the mechanism of C 0 2 transfer, see Biotin enzymes. The active form of P.c. is a tetramer, Mr 600000 (yeast), 650000 (chicken liver), or 520000 (porcine liver), which is in equilibrium with the corresponding dimers and monomers. The dimers and monomers (M r 130000, 3 chains, each of MT 47 000) of the porcine enzyme are also enzymatically active. Avian P.c. is cold sensitive, and reversibly dissociates into inactive monomers (M r 160000) at 0°C. Like all biotin enzymes, P.c. is inactivated by avidin, due the binding of the coenzyme as an avidin (biotin) 4 -complex.

399

Pythocholic acid On the basis of their structural homologies, P.c. and acetyl-coenzyme-A-carboxylase are thought to have evolved from a common precursor enzyme. Pyruvate decarboxylase, carboxylase (EC 4.1.1.1): a thiamine pyrophosphate (TPP)-dependent lyase, absent from animals, and present in high activity in yeast and wheat seedlings. P.d. is a specific enzyme of alcoholic fermentation, which catalyses the cleavage of pyruvate (via active acetaldehyde) into acetaldehyde and C 0 2 . The cofactors are Thiamine pyrophosphate (see) and magnesium ions. In the plant cell, P.d. competes with the pyruvate dehydrogenase complex for pyruvate. Mr of P.d. from yeast and Escherichia coli is 190000 (two identical subunits, M r 95000). Pyruvate dehydrogenase: see Multienzyme complex. Pyruvate kinase, phosphopyruvate kinase (EC 2.7.1.40): a widely distributed, metal ion-dependent phosphotransferase, present in yeast, muscle, liver, erythrocytes and other organs and cells. It catalyses the last reaction of glycolysis: Phosphoeno/pyruvate (PEP) + ADP ^ Pyruvate + ATP (Substrate level phosphorylation). Each subunit of P.k. forms an intermediate, cyc•• . , P.k.-Mn-ADP he, ternary metal bridge complex ^ pjip in which the PEP and ADP are bound to the enzyme via a manganese (II) ion. Tetrameric P.k. from muscle and erythrocytes (M r 230000) shows Michaelis-Menten type kinetics (plot of initial velocity against substrate concentration is a rectangular hyperbola), whereas the yeast enzyme (M r 190000, 4 or 8 subunits) is an allosteric enzyme, showing sigmoid kinetics. Pyruvate phosphate dlkinase: see HatchSlack-Kortschak cycle. Pyruvic acid: CH 3 -CO-COOH, the simplest and most important a-ketoacid (2-oxoacid), m.p. 11.8°C, b.p. 165°C (d.). For the role of P.a. in metabolism, see Pyruvate. Pythocholic acid: 3a,7a,16a-trihydroxy-5Pcholan-24-oic acid, a bile acid possessing three hydroxyl groups. Afr 408.58, m.p. 187°C. P.a. is a characteristic component of the bile of many snakes, and has been isolated from the bile of the tiger snake, python and boa-constrictor, among others.

Q

Q: abb. for coenzyme Q. See Ubiquinone. Quantasome: the smallest structural unit of photosynthesis; small elementary units of the thylakoid, measuring 18 x 15 x 10 nm, M t 2 million, containing 230 chlorophyll molecules, cytochromes, copper and iron. Q. are obtained by ultrasonic disintegration of isolated chloroplasts, and they can be visualized in the electron microscope. They may also be observed as granular units in the chloroplast lamella. The functional status of Q. is not clearly defined; they may be involved in both electron transport (see Photosynthesis) and photosynthetic ATP synthesis (see Photophosphorylation), and therefore analogous to the electron transport particles of the respiratory chain. Quantum efficiency, quantum

yield:

see Q u a n -

tum requirement. Quantum requirement: the number of light quanta required for the formation of one molecule of 0 2 in Photosynthesis (see). Two quanta are required per electron. The theoretical value of Q.r. is eight, since the production of one molecule of 0 2 proceeds according to the following equation, with transfer of four electrons from water (see Photolysis of water) to NADP+. 2 H 2 0 —• 0 2 + 4H+ + 4e~. The experimentally determined value of Q.r. lies between 8 and 10 for leaves, and between 10 and 14 for isolated chloroplasts. It is influenced by the physiological state of the experimental system. The inverse of Q.r. is the quantum yield (or quantum efficiency). The quantum yield therefore represents the number of molecules transformed (i.e. C 0 2 molecules consumed, 0 2 molecules liberated) per quantum of light absorbed. Quantum yield, quantum

efficiency:

see Q u a n -

tum requirement. Queen substance: originally a term for the entire mandibular gland secretion of the queen bee, which contains about 30 substances. It is now the trivial name for 9-oxo- irani-2-decenoic acid. This compound, together with 9-hydroxy-frans-2-decenoic acid, is very important as a pheromone for the maintenance of the division of labor within the hive. In the course of caring for the young,

R= 0

Queen substance

R-H.OH

9 - Hydroxy-trans-2-decenoic

acid

the worker bees lick the pheromone mixture off the queen. This causes their ovaries to shrink, and

they are inhibited from building queen cells. Larvae in queen cells are not fed honey, but royal jelly, a mixture of pollen and secretions. Royal jelly does not contain Q. It is recommended as a health product, but its effectiveness is disputed. Quaternary structure: s e e P r o t e i n s .

Quebrachltol: see Cyclitols. Quercetin: see Flavones (Table). Quercltrln: see Flavones (Table). Quinazollne alkaloids: a group of about 30 alkaloids, which occur in higher plants (in families which are taxonomically very distant from one another), animals and bacteria. They are derived biosynthetically from anthranilic acid. The simplest representative of the Q. is Glomerine (see) which is a very rare animal Q. Of the plant Q., Febrifugine (see) has some significance. In the wide sense, tetrodotoxin from the puffer fish can be included among the Q., as it is a zwitterionic poIyhydroxy-2-iminoperhydroquinazoline. Quinidlne: see Quinine.

Quinine: the most important of the cinchona alkaloids. Mr 324.21, m.p. 57°C (trihydrate), 174 to 175°C for dehydrated crystals, [a]^ -284.5 (0.1 N H 2 S0 4 ). In Q., a quinoline ring system is connected via a secondary hydroxyl on C4 to a quinuclidine structure (see Cinchona alkaloids, Fig.). Q. occurs in nature in association with its stereoisomers quinidine, the C-9 empimer, m.p. 172.5°C, [a]^ = +334.1 (O.IN H 2 S0 4 ), and epiquinine and epiquinidine, the C 8' epimers. Q. forms bitter-tasting salts and has many physiological effects. It is used therapeutically as a drug against malaria and bacterial influenza. By reducing the rate of tissue respiration, it has an antipyretic effect. It also acts as an analgesic and inhibits heart excitation, although quinidine surpasses it in this last respect. Q. is toxic, leading to deafness and blindness, and in quantities of 10 g it is fatal. Quinoline alkaloids: a group of alkaloids based on the quinoline skeleton. They are found both in microorganisms (see Viridicatine) and in higher plants. The most important therapeutically are the Chinchona alkaloids (see). The starting material for the biosynthesis of some Q. is anthranilic acid (see Viridicatine); for others it is tryptophan (see Cinchona alkaloids). Quinollzldine alkaloids: a group of alkaloids based on the quinolizidine (norlupinane) skeleton. The most important Q. are the lupine alkaloids, which are synthesized from lysine via cadaverine. The Nuphara alkaloids (see), in contrast, are synthesized by the terpene pathway. These alkaloids, which have a Q. skeleton, have been isolated from pond lilies.

Quinones

402

Quinones: aromatic dioxo compounds derived from benzene or multiple-ring hydrocarbons such as naphthalene, anthracene, etc. They are classified as Benzoquinones (see), Naphthoquinones (see) Anthraquinones (see), etc. on the basis of the ring system. The C = 0 groups are generally ortho or para, and form a conjugated system with at least two C = C double bonds; hence the compounds are colored, yellow, orange or red. This type of chromophore is found in many natural and synthetic pigments.

Quinones The Q. are a large and varied group of natural products found in all major groups of organisms. Those with long isoprenoid side chains, such as plastoquinone, ubiquinone and phytoquinone are involved in the basic life processes of photosynthesis and respiration. Q. are biosynthesized from acetate/malonate via shikimic acid. A few Q. are used as laxatives and worming agents, and others are used as pigments in cosmetics histology and aquarell paints.

R

Radlolmmunological determination: see Hormones. Rafflnose, melitose: a nonreducing trisaccharide. M.p. 120°C, [a]™ + 123°C (water). R. contains units of D-galactose, D-glucose and D-fructose. The galactose and glucose are linked by an a-l,6-glycosidic bond, a n d the fructose is linked to the glucose by an a , P-1,2-glycosidic b o n d . R. is easily fermented by yeasts. Yeast enzymes hydrolyse R. into D-fructose a n d melibiose; emulsin hydrolyses R. into sucrose a n d D-galactose. In plant metabolism, R. may function in place of sucrose as a transport carbohydrate. R. is f o u n d widely in many higher plants, where it is the second (the first is sucrose) most commonly occurring free sugar. Sugar beet, molasses and many seeds, e.g. cotton seeds are especially rich in R. Ramachandran plots, conformational maps: plots of rotation about the aC-carbonyl-C bond ( T ) in a peptide linkage against rotation about the a C - a m i n o - N bond ( - 2 ° (water).It can be prepared by catalytic or electrochemical reduction of the configurationally related D-glucose, D-fructose or L-sorbose. Technically, it is prepared by catalytic hydrogénation of D-glucose. D-S. is the starting material for the technical synthesis of ascorbic acid; it is used in the food industry as a preservative and as a softening agent in sweets. Since it is well tolerated, it is used as a sweetner in diabetic diets. By heating in the presence of acid catalysts, D-S. undergoes an intramolecular loss of water with the formation of internal ethers. One such ether, 1,4-sorbitan, is converted commercially into dispersants and emulsifying agents by partial esterification with fatty acids and reaction with ethylene oxide.

ch2oh

I

H—C—OH

I

HO—C—H H—¿—OH

I

H—C—OH

I

CH 2 0H

Sorbitol

i H 2 H1

H—C—OH

I

HO—C—H

I H—C I

O I

1

H —C—OH

I

CH 2 0H

1,4-Sorbitan

L-Sorbose: a monosaccharide hexulose, Afr 180.16, m.p. 162°C, [a]g> - 4 2 . 9 ° , present in certain plant juices, e.g. rowan berries, and biosynthesized from D-sorbitol. L-S. is an intermediate in the commercial synthesis of ascorbic acid. Soybean trypsin inhibitor, abb. STI: the best known plant trypsin inhibitor. With bovine trypsin, at pH 8.3, it forms a stoichiometric, enzymatically inactive, stable complex with an association constant of 5 • 10® per mol STI. It also inhibits other vertebrate and invertebrate trypsins and plasmin. Chymotrypsin is inhibited to a small extent, and other endopeptidases not at all. STI is a single polypeptide chain, M r 21100, 181 amino acid residues (of known sequence) and two disulfide bridges. The molecule is compact and has a low a-helix content due to the presence of proline residues. It is consequently resistant to proteases and to denaturation. The reactive center contains a specific peptide bond, Arg 63 - Ile M , which is hydrolysed when the trypsin-STI complex is formed. The ensuing interaction with the active site of trypsin, which also involves Arg65 of STI, results in firm binding of STI and inactivation of the trypsin. Soybean also contains the Bowman-Birk inhibitor, which consists of 78 amino acid residues (MT 8000) and is much smaller than STI. It is a "double headed" inhibitor, i.e. in addition to inhibiting trypsin, it can simultaneously inhibit a second protease, e.g. chymotrypsin. Since the nutritional value of protein-rich soybean products may be reduced by the presence of STI and other inhibitors, these are inactivated by heating the milled beans. Specific activity: see Enzyme kinetics. Specific incorporation rate: see Isotope technique. Specificity constant, physiological effectirity: a measure of the turnover of a substrate. S.c. is the ratio of the catalytic constant and the Michaelis constant: k ^ / K ^ . It is equal to the rate constant of a reaction for the rate equation:

Specific radioactivity

434

v = fccatE0S/( K m + S), where E 0 is the total enzyme concentration, and S is the substrate concentration, when S < K m . Specific radioactivity: see Isotope technique. Spectlnomycln: see Streptomycin. Spectrin: a protein which makes up about 75% of the "skeleton" of the erythrocyte membrane. It is not found in any other type of cell. S. is a heterodimer or tetramer of two polypeptides, band 1 with a MT of 240000 and band 2 with a MR of 222000. (The terminology is derived from electrophoresis patterns of membrane proteins on sodium dodecylsulfate-containing polyacrylamide gels.) Under normal conditions, S. appears to exist as a mixture of dimers and tetramers, linked into a loose network by actin. This flexible structure lies just inside the lipid bilayer of the membrane (see Biological membranes). The relative amounts of dimers and tetramers appear to be determined by the degree of phosphorylation of S., which has four phosphorylation sites at the extreme carboxy terminal end of band 2. S. is noncovalently bound to Ankyrin (see), which in turn is linked to one or more "integral" or membrane-spanning proteins. Ankyrin may be linked to the anion-transport protein and to glycophorin A (see Glycophorins), which bears the M, N blood group antigens. The physical state of the S. network is sensitive to the intracellular Ca 2 + levels and the ATP charge of the cell; when the C a 2 + level is high or ATP is depleted, it is more highly polymerized, and the erythrocyte loses its typical shape. Thus S. is probably responsible for maintaining the concave-disc shape of the erythrocyte, and for allowing the cell the extreme flexibility it needs to pass through capillaries. A number of hemolytic anemias are related to abnormalities in the S. structure which make the cells more fragile. Spermaceti: a solid animal wax, m.p. 45-50°C, obtained from the head of the sperm whale, Physeter macrocephalus. An oily, liquid, crude sperm oil is secreted in a special large cylindrical organ in the upper region of the huge jaw and above the right nostril of the whale. After capture, this cavity is emptied of its oil, which, on cooling, deposits crystalline S. This is separated by pressure and purified by remelting and washing with dilute NaOH to remove the last traces of oil. S. consists chiefly of cetyl palmitate, accompanied by smaller quantities of cetyl laurate and myristate. It is used in the pharmaceutical and cosmetic industries as a basis for creams. Spermine: H 2 N-(CH 2 ) 3 -NH(CH 2 ) 4 -NH(CH 2 ) 3 NH 2 , a biogenic, aliphatic tetraamine, m.p. 55-60° C. S. occurs in high concentrations, together with the triamine, spermidine (mono(Y-aminopropyl)-putrescine), in human sperm. It is also present in ribosomes and some viruses. Acting as a base, it probably neutralizes the negative charges on DNA; this prevents inter and intramolecular repulsion and allows closer packing and supercoiling of DNA molecules. Spheroldlne: see Tetrodotoxin. Sphingomyelins: see Phospholipids. Sphlngoslne: 2-amino-4-octadecene-l,3-diol,

Spirostane

MT 299.48, m.p. 67° C, a long-chain amino alcohol which is a component of sphingomyelins (see Phospholipids) and Glycolipids (see). It is not found in free form in plants or animals. Spider toxins: toxic substances produced in the venom glands of many spiders. They serve to paralyse and kill prey, and are dangerous to humans only in rare cases, e.g. the toxin of the South European Latrodectus tredecimguttatus, or the American black widow (Latrodectus mactans). The active principles of S.t. are proteinaceous and related to those of snake and scorpion venoms. They contain hyaluronidase and proteolytic activity, but phospholipases and hemolytic or blood clotting activities are absent. Splnasterols: a group of very similar phytosterols (see Sterols), found in higher plants. Chief representative is a-spinasterol (5a-stigmasta-7, 22diene-3P-ol; Fig ), M r 412.7, m.p. 172°C, isolated e.g. from spinach, senega root and lucerne, a-, p, y- and 5-S. differ in the position of the side chain double bond.

Splnochromes: derivatives of 1 ^-naphthoquinone (see Naphthoquinones), responsible for the red or orange color of sea urchin shells. Over 20 different hydroxylated S. are known. In the native state, they are present as calcium and magnesium salts. They differ from the echinochromes, which occur in the eggs, perivisceral fluid and internal organs of the sea urchin. Echinochrome A is a red pigment in the eggs and skeleton of the sea urchin; it is a pentahydroxy-1 ^-naphthoquinone with an ethyl group at C6, m.p. 223°C. Spinuloslne: see Benzoquinones. Spiramycin: a Macrolide (see) antibiotic. Splrographis porphyrin: see Chlorocruoroporphyrin. Splrosolane: see Solanum alkaloids. Spirostane: the oxygen-containing parent structure of the steroid saponins (see Saponins).

(25 S)-SA-Spirostane

Spirostanols

435

The S. system is formally derived from the parent hydrocarbon cholestane (see Steroids). The name S. embraces the configuration of all asymmetric centers, with the exception of positions S and 25. Spirostanols: see Saponins. Splicing see Intron. Splicing enzymes: see Intron. Split gene: see Intron. Split proteins: see Ribosomes. Spongonucleosldes: see Arabinosides. Spongoslne: 9-p-D-ribofuranosyl-2-methoxyadenine, a nucleoside with a modified base, isolated from sponges. Spongosterol: (24R)-5a-ergost-22-ene-3p-ol, a marine zoosterol (see Sterols), Mr 400.66, m.p. 153°C, [a] D + 10°C (chloroform), occurring as a typical sterol of sponges (Spongia) and isolated, e.g. from Suberitis domuncula and Suberitis compacta. Spongothymldlne: see Arabinosides. Spongourldlne: see Arabinosides. Sporldesmolldes: cyclic depsipeptides from the fungus Pithomyces chartarum. Sporidesmolide I is cyclo-(-Hyv-D-Val-D-Leu-Hyv-Val-MeLeu-); sporidesmolide II contains D-allo-isoleucine in place of D-valine, while sporidesmolide III contains L-leucine in place of L- jV-methylleucine. (Hyv represents a residue of a-hydroxyisovaleric acid). Sporopollenin: the material of the outermost cell wall layer (exine) of pollen grains and spores of pteridophytes, and also present in small amounts in fungal zygospore walls (e.g. zygospore wall of Mucor mucedo contains 1 % S.). S. is extremely resistant to physical, chemical and biological degradation. Pollen grains are therefore well preserved in geological strata, and have proved archeologically useful as quantitative and qualitative markers of previous plant life and agriculture. P. is an intimate mixture or complex of 10-15% cellulose, 10% of an ill-defined xylan fraction, 10-15% of a lignin-like material, and 55-65% of a lipid. It is now generally accepted that the lipid material is formed by the oxidative polyerization of carotenoids; a virtually indentical material can be synthesized in the laboratory by the catalytic oxidation of P-carotene. Ref: "Sporopollenin", by Shaw, G., pp. 31-58 in Phytochemical Phytogeny (Edit. Harborne, J. B.) 1970, Academic Press. S-proteln: a cleavage product of ribonuclease, representing amino acid residues 21-124 of the ribonuclease primary sequence. It is produced, together with S-peptide (positions 1-20), by the action of subtilisin on ribonuclease. Squalene: biochemically the most important aliphatic triterpene. Mr 408, b.p. 10 262-264° C, p 0.8584. For formula see Triterpenes. S. was first isolated from fish liver oils, and later found in many plant oils. It is the intermediate in the biosynthesis of all cyclic triterpenes. Cyclization of S. is catalysed by a mixed function oxygenase, and proceeds via 2,3-epoxysqualene (squalene epoxide). SRH: abb. for somatotropin releasing hormone. See Releasing hormones.

Starch

sRNA: abb. for the obsolete name, soluble RNA, now known as Transfer RNA (see) or tRNA. S R S : abb. for slow reacting substance. See Leukotrienes. Stachydrine: see Pyrrolidine alkaloids. Stachyose: a nonreducing tetrasaccharide (see Carbohydrates) found in plants. M.p. 170°C, [alg 1 + 149°C. The four sugar residues are linked in the order D-galactose-D-galactose-D-glucoseD-fructose. Starch: a high Afr polysaccharide, formula (C 6 H 10 O 5 ) n ; the chief storage carbohydrate in most higher plants, consisting of about 80% water-insoluble Amylopectin (see) and 20% watersoluble Amylose (see). In plant metabolism, S. first appears as an assimilation product in the chloroplasts. It is then degraded, the products of degradation are translocated, and S. is resynthesized as storage S. (S. grains or granules) in storage organs, e.g. roots, tubers or pith. S. grains are classified as compound, simple, centric or acentric, depending on their characteristic shapes and stratifications. On the basis of these characteristic forms, flour from different sources (e.g. maize, rice, wheat, rye) can be identified microscopically. S. is biosynthesized from adenosine-diphosphate-glucose (Fig.). During animal digestion, S. is hydrolysed by amylases. a-Amylase (EC 3.2.1.1) hydrolyses a(l—>4) linkages at random, forming a mixture of glucose and maltose. Maltose is hydrolysed to glucose by a-D-glucosidase (maltase, EC 3.2.1.20). Amylose is thus completely degraded to glucose. a-Amylase cannot hydrolyse a(l—>6) linkages at the branch points of amylopectin; the product of a-amylase action on amylopectin is therefore a large, highly branched core or limit dextrin, representing 40% of the original amylopectin. The small intestine also contains an oligo-a(l—•6)-glucosidase, which hydrolyses the 1 —* 6 linkages, and completes the total degradation of amylopectin. fi-Amylases (EC 3.2.1.2) are found especially in germinating seeds (e.g. malt); they act on the nonreducing end of the polysaccharide chain, removing successive maltose units. P-Amylase is also unable to attack a(l—>6) branch points. In plant cells, storage S. is remobilized by phosphorolysis to glucose 1-phosphate. i>. is very important in human nutrition, supplying most of the dietary carbohydrate requirement (humans require about 500 g carbohydrate per day). Potatoes, cereals and bananas are particularly rich in S. Metabolism of 1 g S. supplies 16.75 k j (4 kcal). S. is prepared commercially from plant sources, in particular potatoes, wheat rice and maize, and it has many uses in the food industry and in technology. Fig. see page 436

Start codon

Steady state

436

Adenosinediphosphateglucose (

2* Pl


CO, I —

C— CH, I OH

R

f"'

9" = |

/C—CH,

•

\

I

iC— CH,* TPP

H—C—CH, rJ

Fig. 2. Mechanism of thiamine pyrophosphate (TPP) catalysis for the decarboxylation of pyruvate.

Thin-layer chromatography

459

Thin-layer chromatography, TLC: a form of chromatography (see) in which the solid carrier material is spread in a thin layer on a glass or plastic plate. The advantages of the method are the short distances required for good separation and the correspondingly short development times, high sensitivity, separation of very small amounts of substances, and, if inorganic carrier materials are used, the possibility of using caustic detection reagents. Pre-spread thin-layer plates are available commercially, as is a spreading device with which one can spread any desired thickness of carrier from 0 to 2 mm. With appropriate equipment, the plates can be used for ascending, descending, horizontal or multiple chromatography. TLC can be used preparatively as an "open column" (see Column chromatography). Depending on the size of the plate, up to 100 g of material can be separated by preparative TLC while analytical TLC can be used for amounts between 10 ng and 10 mg. TLC was originally developed for the separation of lipophilic substances on inorganic carrier materials. However, if cellulose powder is used as carrier, hydrophilic substances, including amino acids, nucleotides and carbohydrates, can also be separated. Lipophilic substances can be separated on aluminum oxide, silica gel, acetylated cellulose and polyamide; hydrophilic substances on cellulose, cellulose ion exchangers, diatomaceous earth and polyamides. The cellulose ion exchangers used in TLC have shorter cellulose fibers than those used for column chromatography. Polyamide TLC makes use of hydrophilic or hydrophobic polyamides for separation of a wide range of substances. It depends of the reversible formation of hydrogen bonds between the substances and the amide groups of the carrier. The eluant (water, methanol, formamide, etc.) displaces these hydrogen bonds, forming its own with the carrier. The separation thus depends on the differences in the strengths of the hydrogen bonds formed by the substances to be separated. Thioblnupharldine: see Nuphara alkaloids. Thioctic acid: see Lipoic acid. Thloester, acylmercaptan: a compound of the general formula RS — CO-R,. The thioester (acylmercaptan) bond is energy-rich. All fatty acyl coenzyme A derivatives (activated fatty acids, e.g. acetyl-CoA) are T. During substrate phosphorylation on glyceraldehyde 3-phosphate dehydrogenase a thiol group of the enzyme forms an energy-rich intermediate T. Thioethers: see Sulfur compounds. Thiol enzyme, SH-enzyme: an enzyme whose activity depends upon the presence of a certain number of free thiol groups. T.e. are found amongst the hydrolases, oxidoreductases and transferases. Known T.e. are bromelain, papain, urease, various flavoenzymes, pyridine nucleotide enzymes, pyridoxal phosphate enzymes and thiolproteinases. T.e. are typically inhibited by Sulfhydryl reagents (see). Thiolesterases: see Esterases. Thiol group, sulfhydryl group, mercapto group:

Thioredoxin -SH, the functional group of thiols (mercaptans), RSH, where R is the remainder of the molecule. T.g. may be structurally important as in Thiol enzymes (see), or functionally important as in Coenzyme A (see), Pantetheine 4'-phosphate (see), Lipoic acid (see), Thioredoxin (see), etc. The functional form of lipoic acid and thioredoxin is a dithiol. Thiols: see Sulfur compounds. 2-Thlomethyl-/V ®-isopentenyladenosine: 2-methylmercapto-6-isopentenyladenosine, an adenosine derivative and one of the rare nucleic acid components found in tRNA from wheat. It is an active cytokinin. The hydroxylated derivative, 2-methylmercapto-6-(4-hydroxy-3-methyl-cis-2-en ylamino)-purine has also been found in some species of tRNA. Thioredoxin: a heat-stable, acidic, metal-free redoxin, M r 12000. T. is a component of deoxyriCTP (ribose)

dCTP (deoxyribose)

Deoxyadenosyl cobalamine - H 2

Deoxyadenosylcobalamine

Thioredoxin -S

Thioredoxin-(SH}2

Thioredoxin -

-s-NADP"1"

Fig. 1. Ribonudeoside triphosphate reductase and thioredoxin reductase in Lactobacillus leishmannii. bose synthase, in which T. and thioredoxin reductase form a hydrogen transfer system linked to the reduction of ribose or ribonudeoside phosphates by NADPH + H + . T. is a single polypeptide chain of 109 amino acid residues with Nterminal serine. The functional group of the oxidized form consists of a disulfide bridge between two cysteine residues, which are separated by 10 amino acid residues. Ribonudeoside triphosphate reductase from Lactobacillus leishmannii (Fig. 1) also requires a cobalamine coenzyme (cobamide) as a hydrogen carrier. The cobala[-SH + Enz + D B C -

•—Enz-DBCH;

"-SH Thioredoxin (reduced)

- Enz-DBH2+CTP-

+ Enz +DBC +dCTP + H , 0

-SH + NADP

+

-SH

Fig. 2. Hypothetical role of cobalamine coenzyme (DBC) in the action of ribonudeoside triphosphate reductase.

Thr

460

mine coenzyme (DBC = oxidized, DBCH 2 = reduced form) mediates an intramolecular hydrogen transfer: hydrogen from T. is transferred to the DBC-coenzyme in a ternary complex of T./reductase/DBC; hydrogen is then transferred from the complex to the ribonucleoside triphosphate, e.g. cytidine triphosphate (CTP) (Fig. 2). In its mechanism of action, T. resembles Lipoic acid (see). Thr: abb. for L-threonine. L-Threonine, abb, Thr: L-threo-a-amino-P-hydroxybutyric acid, H 3 C-CH(OH)-CH(NH 2 )COOH, a proteogenic, essential amino acid with two asymmetric C-atoms. Afr 119.1, m.p. 253°C (d.), [ a j y - 2 8 . 5 (c = 1-2, water). A useful reaction for the determination of L-T. is oxidation with periodate to acetaldehyde, glyoxylate and ammonia. Enzymatic hydrolysis of peptide bonds involving L-T. appears to be particularly difficult, which may be relevant to the nutritional physiology of this amino acid. The principal degradative reaction of L-T. in most organisms is conversion to 2-oxobutyrate and ammonia by the pyridoxal phosphate-dependent enzyme, L-threonine dehydratase (EC 4.2.1.16). This degradative enzyme (also called threonine deaminase) is distinct from biosynthetic threonine dehydratase needed for the production of 2-oxobutyrate in the biosynthesis of isoleucine; in Escherichia coli, the latter enzyme is allosterically inhibited by isoleucine. LThreonine acetaldehyde-lyase (threonine aldolase; EC 4.1.2.5) is a pyridoxal phosphate enzyme which converts L-T. into glycine and acetaldehyde. It is present in various organisms, including mammals, and appears to be a purely degradative enzyme. Oxidation of L-T. to 2-amino-3-oxobutyrate, followed by decarboxylation, produces aminoacetone, a urinary constituent. In microorganisms, aminoacetone is converted to ft-l-amino-2propanol, an intermediate in the biosynthesis of vitamin B12. Aminoacetone may also be oxidatively deaminated to methylglyoxal, which can be attacked by glyoxalase to form D-lactate. In plants and microorganisms, L-T. is biosynthesized from phosphohomoserine by a y-elimination of phosphate followed by ¡¡-replacement with an OH-group. This total reaction is catalysed by the pyridoxal phosphate enzyme, threonine synthase (EC 4.2.99.2). The phosphohomoserine is derived from aspartate via aspartyl phosphate, aspartate semialdehyde and homoserine. Thrombin: a blood coagulation enzyme, responsible for the conversion of fibrinogen to fibrin. T. is a glycoprotein (5% carbohydrate), M t 39000, produced by activation of Prothrombin (see). Degradation of bovine Prothrombin (see) (582 amino acid residues) by factor X a and by thrombin itself produces the two chains of a-T. of known primary structure (A-chain, Mr 5700, Nterminal threonine, 49 amino acids; B-chain, M r 32000, Af-terminal isoleucine, 259 amino acids, with carbohydrate attached), which are linked by one disulfide bridge. Further degradation with loss of the A-chain and cleavage of the B-chain produces fi-T. (M r 28000). A further cleavage of

Thymidylic acid

the B-chain then produces y-T. (M r 28000). Both fi- and y-T. have T. activity. T. is a typical Serine protease (see) with catalytically important residues His 58 , Asp 102 and Ser 195 in the B-chain, and considerable sequence homology with trypsin, chymotrypsin and elastase. Autolysis of T., leading to a decrease of Mr from 39000 to 26000 at the expense of the B-chain (Af r 33000 to 19500) causes no loss of activity. Thrombosthenln: see Muscle proteins. Thujane: see Monoterpenes (Fig.). Thy: abb. for thymine. Thylakoids: internal membrane structures of the chloroplast. Under the electron microscope, T. appear as disc-shaped, flattened vesicles, about 600 nm diameter. These are arranged in stacks, which are the grana observable under the light microscope. In addition to these granal T., there are also stromal T., which pass singly through the stroma of the chloroplast, joining together various stacks of granal T. The functional unit of T. is thought to be the Quantasome (see). The T. membrane is about 9 nm thick, enclosing a thin internal space or loculus; it contains approximately equal quantities of protein and lipid, and is notable for its high content of galactosyl diglyceride, digalactosyl glyceride and sulfolipid. There is probably a greater proportion of lipid molecules on the inside of the membrane and more protein on the outside, but distinct layering into protein and lipid seems to be absent. The inside lipid layer contains the chlorophylls and carotenoids, and the chlorophyll is present largely, if not entirely, in the form of protein complexes. The protein subunits of the outer layer have a diameter of 4 nm. Thymidine, more correctly deoxythymidine, Abb. dTbd, thymine deoxyriboside: a Nucleoside (see) of thymine and D-2-deoxyribose. M r 242.33, m.p. 185-186°C [a.] ¿f +32.8 (c = 1.04, lMNaOH). dThd should not be confused with Ribothymidine, which is also (and more correctly) called thymidine. For metabolic importance, see Thymidine phosphates. Thymidine phosphates, more correctly deoxythymidine phosphates: Nucleotides (see) of thymine; phosphate esters of deoxythymidine. Although T.p. contain deoxyribose, the prefix deoxy is usually omitted, because the corresponding ribose derivatives hardly ever occur naturally. Thymidine S'-monophosphate abb. TMP, thymidylic acid (more correctly deoxythymidine S'-monophosphate, abb. dTMP, deoxythymidylic acid): a component of DNA, and an intermediate in the synthesis of TPP (see Pyrimidine biosynthesis). Mt 322.2, m.p. 225-230° C. Stepwise phosphorylation of TMP leads to thymidine S'-diphosphate, abb. TDP (more correctly deoxythymidine S'-diphosphate, abb. dTDP), MT 402.2, which serves as the activating group in certain Nucleoside diphosphate sugars (see); and to thymidine 5'-triphosphate, abb. I1F(more correctly deoxythymidine S'-triphosphate, abb. dTTP), M, 482.18, a substrate of DNA synthesis. Thymidylic acid: see Ribothymidine; Thymidine phosphates.

Thymin

Tissue hormones

461

Thymln: see Thymopoetin. Thymine, abb. T or Thy: 2,6-dihydroxy-5-methylpyrimidine, 5-methyluracil, a pyrimidine base present in DNA. Mr 126.1, m.p. 321-326° C (d.). T. was first isolated in 1893 from thymonucleic acid. It is formed by the degradation of thymidine 5'-monophosphate; the methyl group of T. does not arise from methionine, but from an active one carbon unit (see Pyrimidine biosynthesis). Thymine deoxyrlboslde: s e e T h y m i d i n e . Thymine dlmer: s e e D i m e r s .

Thymonucleic acid, thymus nucleic acid: nucleic acid from the thymus gland; effectively an obsolete term for DNA. Thymopoetin, thymin, thymosin: a polypeptide hormone from the thymus. Afr 5562 (49 amino acid residues of known sequence). It is required for the general differentiation of thymocytes, but has no influence on the acquisition of the immunological repertoire. Thymosin: see Thymopoetin. Thyrocalcltonln: see Calcitonin. Thyroid gland: Glandula thyreoidea, a well vasculated gland at the front of the neck. It is paired in amphibians and birds, and unpaired in elasmobranch fish and mammals, weighing 20-60 g in the human. The T.g. synthesizes, stores (in the thyroid follicles) and secretes Thyroxin (see) and triiodothyronin, under the influence of the anterior pituitary hormone thyrotropin. It also synthesizes Calcitonin (see) in the parafollicular Ccells. Thyroid stimulating hormone: s e e T h y r o t r o p i n .

Thyrotropin, thyroid stimulating hormone, abb. TSH: a glycoprotein hormone, M r 25000 (bovine), containing 23% carbohydrate. Primary structure of some TSH molecules is known. It consists of an a - and a p-chain, and the a-chain is structurally similar to Luteinizing hormone (see). Synthesis occurs in the basophilic cells of the anterior pituitary. Both synthesis and secretion are stimulated by thyrotropin releasing hormone (see Releasing hormones) from the hypothalamus, and inhibited by thyroxin. TSH generally stimulates the thyroid gland; blood circulation of the thyroid gland is increased, uptake of iodine is promoted, the rate of synthesis of thyroglobin, triiodothyronine and thyroxin is increased, and the secretion of thyroid hormones is stimulated. Inactivation occurs in the kidney. Blood concentrations of TSH are in the order of ng/ml, and can be measured radioimmunologically. Thyrotropin releasing hormone: s e e R e l e a s i n g

hormones. Thyroxin, 3,S,3',S'-tetraiodothyronine, abb. T4: a hormone produced by the thyroid gland and absolutely essential for growth and development. Mt 776.9. T 4 and the second thyroid hormone, 3,^'-triiodothyronine (abb. T 3 , Mr 651.0) are synthesized from L-tyrosine residues in thyroglobulin, a dimeric glycoprotein (M r 670000) that constitutes the bulk of the thyroid follicle. Tyrosine residues in thyroglobulin become iodinated, so that the protein contains several mono- and diiodotyrosine residues. The nature of the subse-

quent coupling reaction is uncertain, but it is equivalent to the transfer of iodinated rings from some iodotyrosine residues to form ether linkages by reaction with the hydroxyl functions of other iodinated tyrosine residues. T 4 and T 3 are released by proteolysis of thyroglobulin. Synthesis and release of T 4 and T 3 from thyroid epithelial cells, together with a parallel increase in the uptake of iodine by the thyroid gland, are stimulated by the hormone thyrotropin from the anterior pituitary. Both hormones are carried in the blood to all body cells, partly in the free form and partly bound to prealbumin and glycoprotein, T 4 being more tightly bound than T 3 .

Thyroxin Metabolic action of T3 and T 4 : increased oxygen uptake by mitochondria, and increased heat production (calorigenic effect); in physiological concentrations both hormones increase synthesis of R N A and protein; in higher doses they act catabolically, causing negative nitrogen balance and mobilization of fat depots. Independently of their calorigenic effect, they increase the rate of cell differentiation and metamorphosis, e.g. development of tadpoles into frogs. Biological half life of T 4 is 7-12 days (based on the sustained activity of a single T 4 dose). Degradation consists of removal of iodine (reused by the thyroid gland), deamination and coupling with glucuronic acid or sulfate in the liver, followed by urinary excretion. Hyperthyroidism is caused by overactivity of the thyroid gland leading to an excess of T 4 and T 3 . Hypothyroidism results from decreased hormone production; this may be caused by iodine deficiency, administration of goitrogens, defective enzymes in hormone synthesis, autoimmune thyroiditis (antibodies are formed against the body's own thyroid tissue), etc. Prolonged hyperthyroidism may result in dwarfism, mental deficiency, goiter and myxedema. Tin, tin: a metal occurring in many tissues and dietary components. The redox potential of Sn 2 + ^ Sn 4 + is 0.13 volt, near to the redox potential of the flavin enzymes, suggesting a possible biological role. It is still not certain that Sn is biologically essential, and its presence in tissue may be environmental contamination. It has been reported that Sn is essential for the growth of rats. Tlngitanin: see Guanidine derivatives. Tissue hormones: hormones produced in specialized, single cells scattered through a tissue rather than clumped in a gland, (see Hormones). They fall into three groups: 1. Secretin (see), Gastrin (see), and Cholecystokinin (see) from the gastrointestinal tract; 2. Angiotensin (see) and Bradykinin (see), which occur as inactive precursors in the blood; and 3. Biogenic amines (see), such as Histamine (see), Serotonin (see), Tyramine (see)

TMP and Melatonin (see). This last group is an exception to the rule that hormones act at sites removed from the cells which produce them; they affect the immediately surrounding tissue. TMP: see Thymidine phosphates. T m -value, melting point: the temperature, in ° C, at which a double stranded nucleic acid becomes 50% denatured to the single stranded form. A DNA solution is heated and its absorbance at 260 nm is plotted against temperature. Transition from double to single stranded DNA occurs over a relatively narrow temperature range, and is characterized by an increase in absorbance at 260 nm. Tm is taken as the temperature at the mid point (half the final increase of absorbance at 260 nm) of the S-shaped curve. The sharpness of the transition indicates a cooperative alteration of structure throughout the molecule (T m is well above the temperature required to unstack the bases and destroy a single helix, but these are preserved by hydrogen bonding between the two helices; separation of the two strands occurs when the hydrogen bonds between base pairs finally break). Single stranded RNA, on the other hand, shows only a gradual increase of absorbance over a wide temperature range, and has no Tm. Under standard conditions of pH and ionic strength, Tm of D N A is proportional to the stability of the molecule. Since the base pair guaninecytosine (see Base pairing) has 3 hydrogen bonds, and adenine-thymine has only 2 hydrogen bonds, there is a linear relationship between the GC-content of DNA and its Tm value: Tm = 69°C + 0.41 (molar % G-C), i.e. the higher the degree of hydrogen bonding the higher the temperature required to separate the strands of the double helix. TN: abb. for troponin; see Muscle proteins.

462

a-Tomatine

Toad toxins: poisons found in the secretions of the skin glands of toads (Bufonidae) (Fig.). These are classified as 1. bufadienolides (bufogenins) with digitalis-like effects on the heart (see Cardiac glycosides), e.g. bufotoxin. They strengthen and slow the heartbeat. The bufadienolides are present in toad blood at a dilution of 1 :5000 to 1 :20 000, and they are necessary for normal heart activity. 2. Alkaline toxins which are alkaloids derived from tryptamine or indole, e.g. bufotenine, dehydrobufotenine and O-methylbufotenine. The alkaline toxins of some species of toads also contain adrenalin and similar substances. Bufotenines increase the blood pressure and have a paralyzing effect on the motor centers of the brain and spinal column. T.t. have an anesthetic effect which is several times as potent as that of cocaine. Tobacco alkaloids: see Nicotiana alkaloids. Tobacco mosaic virus: see Virus coat proteins. Tocopherol: see Vitamins (Vitamin E). Tocoqulnone: see Vitamins (Vitamin E). Tolerogens: see Immunotolerance. Tomatldenol: see fi-Solamarine. Tomatldlne: see a-Tomatine. a-Tomatine, tomatine: a Solanum alkaloid, and the chief alkaloid of the tomato (Lycopersicon esculentum), also occurring in other Lycopersicon and Solanum spp. T. is a glycoalkaloid of the aglycon tomatidine [(22S :25S)-5a-spirosolane-3pol, Mr 415.7, m.p. 210°C, [a] D +6.5° (chloroform)] and the tetrasaccharide P -lycotetraose. T. imparts a bitter taste and protects the tomato plant from attack by the Colorado potato beetle. It also has antibiotic activity against the causative agents of tomato wilt and other pathogenic fungi.

Tonoplast

463

Toyocamycin

Tomatiae Tonoplast: see Vacuole. Toxalbumlns: see Toxic proteins. Toxicology: the study of toxins, their physiology, biochemistry and the molecular basis of their activity. Toxic proteins: mostly low M t , single chain, nonenzymic proteins, produced especially by snakes and invertebrate animals, but also by some plants (phytotoxins) and virulent strains of bacteria. With the exception of bacterial enterotoxins and Botulinus toxins, T.p. show practically no oral activity, and are only toxic when injected, i.e. when the digestive tract is bypassed. Best studied are the Snake venoms (see) from the cobras and vipers. Scorpion venoms (see) resemble those of the cobras. Known pbytotoxins are 1. the homologous viscotoxins (M r 4840,46 amino acid residues of known sequence, 3 disulfide bridges) from leaves and branches of the European mistletoe, which have hypotensive activity and cause a slowing of the heart beat; 2. the toxalbumins, Ricin (see) and abrin, which inhibit protein biosynthesis. Best studied of the bacterial toxins are the thermolabile exotoxins of Gram positive bacteria, which are secreted into the surrounding medium: 1. Five enterotoxins are secreted by Staphylococcus aureus in the gastrointestinal tract, causing diarrhea and vomiting. Enterotoxin B is of known primary structure (M : 28370, 293 amino acid residues, one disulfide bridge). 2. The Diphtheria toxin from Corynebacterium diphtheriae is an acidic, single chain protein (M r 62 000), of high toxicity (1 ng/kg body weight is fatal). It inactivates peptidyl transferase II in eukaryotic cells, by promoting the attachment of ADP-ribose to the enzyme. 3. Tetanus toxin (Mr 150000) from Clostridium tetani is a potent neurotoxin, which occurs in two forms; filtrate toxin, consisting of two subunits (M x 95 000 and 55000), and cell toxin consisting of one chain. In the mouse, 0.01 ng/kg is fatal. 4. The five highly toxic Botulinus toxins (Type A M r 150000, Type B M t 167000) from Clostridium botulinum are SH-proteins and require the presence of one free SH-group for neurotoxic activity. Fatal dose in the mouse, 0.03 ng/kg. They are resistant to proteolytic digestive enzymes, but they are destroyed by boiling. The relatively heat-stable endotoxins are released by autolysis of the bacteria. Cholera toxins (Mt 84000-102000, composed of two functionally different subunits, type L and type H) are endotox-

ins released from the Gram negative Choleravibrio in the intestine. Toxicity is the result of their high affinity for gangliosides of the membranes of nerve cells, adipocytes, erythrocytes, etc. The type L subunit is responsible for binding to the cell surface, whereas the type H subunit is responsible for toxicity. The colicins (Af r 60000) are endotoxins produced by intestinal bacteria. Their toxicity is due to inhibition of cell division and inhibition of DNA and RNA degradation (colicin E2), or to inhibition of protein biosynthesis by inactivation of the 30S ribosomal subunit (colicin E3). Toxollavln: 3,8-dimethyl-2,4-dihydroxypyrimido(5,4-e)-as-triazine, an antibiotic from Pseudomonas coccovenans, with high antibacterial activity, but no activity against fungi. M.p. 171°C (d.). In the biosynthesis of T., C8 of a purine precursor is removed, and the as-triazine ring is formed by introduction of the aminomethyl group of glycine (Fig.). Both methyl groups are introduced by transmethylation. Xanthothricin from Streptomyces albus is identical with T. It interferes in the transport of electrons in the cytochrome system. Purine p r e c u r s o r

I

*

COOH Methionine

Biosynthesis of toxoflarin by Pseudomonas coccovenans. Toyocamycin: 4-amino-5-cyano-7-(D-ribofuranosyl)-pyrrolo-(2,3-d)-pyrimidine, 6-amino-7-cyano-9-ß-D-ribofuranosyl-7-deazapurine, a 7-deazaadenine antibiotic from Streptomyces toyocaensis and S.rimosus. M.p. 243° C. Biosynthesis is analogous to that of Tubericidin (see), i.e. the carbon atoms of the pyrrole ring are derived from 5-

464

TPN

phosphoribosyl 1-pyrophosphate. T. is particularly active against Candida albicans, Saccharomyces cerevisiae and Mycobacterium tuberculosis. TPN: abb. for triphosphopyridine nucleotide. See Nicotinamide-adenine-dinucleotide phosphate. TPP: abb. for Thiamine pyrophosphate (see). Trace elements, microelements: elements required in very small quantities by living organisms. They act catalytically, or are components of catalytic systems. A clear distinction between T.e. and other mineral nutrients is not always possible, e.g. in the case of iron. A further classification into T.e. and ultratrace elements is sometimes used. Deficiency of T.e. can lead to characteristic deficiency symptoms or diseases, thus indicating the essential nature of these nutritional factors. For example, iodine is a component of the thyroid hormones and essential for thyroid function. Iodine deficiency is responsible for endemic goitre, and certain types of cretinism; it can be avoided by addition of iodides to the drinking water. Other T.e. are chromium, copper, fluoride, magnesium, manganese, nickel, vanadium, silicon, tin, selenium, zinc (see individual entries). Trace element solution: see Nutrient medium (Table 3). Trace nutrients, micronutrients: a general term for any essential dietary component required in small quantities, like Trace elements (see) and Vitamins (see). Deficiency of T.n. leads to defiCH,0H I C=0 I HO—C—H I H—C—OH I H—C—OH I H—C—OH

Transamidation Tracer technique: see Isotope technique. Transacylases: see Transacylation. Transacylatlon: reversible transfer of acyl groups (R-CO-) from a donor to an acceptor, e.g. transfer of the acyl residue CH 3 -CO- by acetylCoA to an acceptor Y: C H 3 - C O ~ S - C o A + Y ^ C H j - C O - Y + CoA T. is catalysed by transacylases, which are important in the synthesis and degradation of fatty a cids, synthesis of conjugated bile acids via cholic acid-CoA compounds, and other reactions such as acetylation of amino acids and amines. Transaldolase (EC 2.2.1.2): see Transaldolation. Transaldolatlon: a reaction of carbohydrate metabolism, in which a C 3 -unit (equivalent to a dihydroxyacetone unit) is transferred from a ketose to an aldose. T. is catalysed by transaldolase (EC 2.2.1.2). The C 3 -unit does not exist in the free state, but remains bound to the e-group of a lysine residue in the enzyme (Fig.). Only fructose 6-phosphate and sedoheptulose 7-phosphate are cleaved by transaldolase. Acceptors for the C 3 unit are the aldose phosphates, D-glyceraldehyde 3-phosphate, D-erythrose 4-phosphate and more rarely ribose 5-phosphate. TTiere is no coenzyme and the mechanism of reaction is similar to that of aldolase (EC 4.1.2.13). Transaminase: see Transamidation. Transamidation: transfer of the amide nitrogen of Glutamine (see) as an NH 2 -group. T. is catalysed by transamidases. All glutamine transamiCH,OH I H.

+

CH 2 0® D-Sedoheptulose 7phosphate

I H—C—OH

Transaldolase

I H—C—OH I H—C—OH

+

CH 2 0®

D-Erythrose Uphosphate

H2COH I ft. C—N

Ç=£N-(CH 2 )Î-CH-CO—) K H H—Ç— R NH

H

H - J + I• +R OH

OH • Lysine

I HO—C— H I H—C—OH I H—C—OH

CH 2 0®

CH 2 0@ D-Glyceraldehyde phosphate

H2(jX)H

-Sugar -

c=o

0

S

D-Fructose 6phosphate

(CH2)„-CH—CO — NH

Enzyme protein

Transaldolase reaction (above), and binding of the ketose to the e-amino group of a lysine residue of tbe enzyme (below). ciency symptoms, e.g. vitamin deficiency diseases. T.n. act catalytically or are precursors of catalytically active substances in the organism. Essential amino acids therefore have an equivocal status in this classification. Flavoring principles are definitely not T.n.

dases so far investigated have a catalytically important thiol group in their active centers and are inhibited by the glutamine analogs, azaserine, 6diazo-5-oxonorleucine (DON) and L-2-amino-4oxo-2-chloropentanoic acid ("chloroketone"), e.g. anthranilate synthase (EC 4.1.3.27), carba-

Transamidinases

465

moyl phosphate synthetase (EC 6.3.5.5), transglutaminase (EC 2.3.2.13), 5'-phosphoribosyl-N-formylglycinamidine synthetase (EC 6.3.5.3), glutamate synthase (EC 1.4.1.13). Transamidinases, amidinotraasferases: enzymes catalysing Transamidination (see). T. catalyse transfer of the amidine group of arginine in the synthesis of creatine and other Phosphagens (see). T. from Streptomyces griseus and S. baikiniensis catalyses amidine transfer in the biosynthesis of streptidine. T. are also involved in the synthesis of certain Guanidine derivatives (see). Transfer of the intact amidine group from L-arginine has been proved by double labelling with and 15 N. T. also has hydrolytic activity and is therefore a potential Arginase (see). Transamidination: reversible enzymatic transfer of the amidine group, NH II -C-NH 2 , between guanidines. T. is a group transfer reaction of nitrogen metabolism, which occurs in two stages and involves an intermediate enzyme-amidine complex: NH

Transaminases lytic lysine residue, or another basic group is thought to act as an electron sink. Further rearrangement produces a nonquinonoid ketimine (secondary Schiffs base), which is hydrolysed to the new oxoacid and pyridoxamine 5'-phosphate. This represents one half of the transamination process. Another oxoacid condenses with the pyridoxamine 5'-phosphate, and the sequence of reactions is reversed to form a new amino acid, thus completing the amino group transfer. Interconversion of the tautomeric Schiffs bases is the rate limiting step of transamination. Practically all amino acids and oxoacids can take part in transamination. The specificity of most transaminases, however, demands that one of the reaction partners should be an acidic amino acid (i.e. glutamate or aspartate) or its corresponding oxoacid. It should be noted that transamination is freely reversible, anergonic process, i.e. no high energy compound (e.g. ATP) is produced or required, and the direction of transamination depends entirely upon a mass action effect of its substrates. Thus, in the liver, when amino acids are in excess, transamination converts them to oxNH

R-NH-C-NH 2 + Enzyme-SH ^ R-NH 2 + Enzyme-S-C-NH 2 NH NH Il II Enzyme-S-C-NH 2 + R-NH 2 ^ R-NH-C-NH 2 + Enzyme-SH In the absence of a suitable acceptor, the enzyme-amidine complex is stable; on standing in aqueous solution or on heating it releases urea. T. is catalysed by Transamidinases (see). Formamidine disulfide, a SH-blocking agent, is a powerful inhibitor of T. The most important amidine donor in T. is L-arginine; biosynthesis of L-arginine is equivalent to the de novo synthesis of the amidine group. T. is important in the biosynthesis of Phosphagens (see). Transaminases, aminotransferases (EC subsub-group 2.6.1): enzymes catalysing transamination, i.e. the reversible transfer of the amino group of a specific amino acid to a specific oxoacid, forming a new amino acid and a new oxoacid. Coenzyme of T. is pyridoxal 5'-phosphate, which becomes bound to the apoenzyme by condensation of its carboxyl group with the E-amino group of a lysine residue, forming a Schiffs base or internal aldimine. During ransamination, however, the coenzyme reacts with the incoming amino acid, which displaces the lysine and forms an external aldimine or primary Schiffs base (Fig.). Formation of a chelate ring by a bridge proton between the amino nitrogen and the phenolic oxygen of the coenzyme helps to maintain the conjugated system of the Schiffs base in a planar conformation. Rearrangement, with loss of the a-hydrogen as a proton, produces a quinonoid ketimine (transitional Schiffs base), containing a conjugated system extending from the carboxyl group to the ring nitrogen. At this stage, the cata-

oacids (which enter carbohydrate metabolism) and the amino groups appear in glutamate and aspartate (and are subsequently incorporated into urea, see Urea cycle). In plants and bacteria, most pathways of amino acid synthesis involve elaboration of the oxoacid, which is finally transaminated (usually with glutamate) to the required amino acid. At no stage in transamination is free ammonia produced; the amino nitrogen is always covalently bound in an amino acid or in the pyridoxamine phosphate coenzyme. Animal tissues, especially liver and heart muscle, contain very high activities of glutamate-oxaloacetate T. (GOT) (preferred name, aspartate aminotransferase, EC 2.6.1.1) and glutamate pyruvate T. (GPT) (preferred name, alanine aminotransferase, EC 2.6.1.2). GPT occurs in the liver as a cytosolic enzyme and shows only very low activity in heart muscle, whereas GOT is higher in heart than liver. GOT is about equally distributed between cytosol and mitochondria in both organs, M r 90000 (2 identical subunits, Mt 45 000). The primary sequence of pig heart cytoplasmic GOT is known (each subunit contains 412 amino acid residues). The serum activity of T. is very low, but increases markedly in certain illnesses associated with tissue damage. The value and ratio of GOT and GPT activities are used in the early diagnosis of, and for following the progress of treatment of, different liver diseases (greatly increased in acute liver inflammation, moderately increased in chronic cases, and

Transduction

466

Transamination

F o r m a t a t i o n of o x o a c i d ^ „ F o r m a t i o n of a m i n o

"Internal"

aldimine

acid

" External " aldimine or P r i m a r y S c h i f f ' s b a s e

Ketimine (quinonoid structure), or T r a n s i t i o n a l S c h i f f ' s b a s e

H2N-Lys

K e t i m i n e , or S e c o n d a r y S c h i f f ' s base

Pyridoxamine

phosphate

Mechanism of transamination; the solid curved line represents part of the surface of the apoenzyme, showing a catalytically important lysine residue. hardly increased in obstructive jaundice) and heart muscle infarction. Both T. are determined by coupled optical tests. GPT: a serum sample is added to a buffered mixture of L-alanine, 2-oxoglutarate, lactate dehydrogenase (excess) and NADH. As pyruvate is formed it is reduced to lactate by the action of lactate dehydrogenase and NADH. The rate of formation of NAD+, measured from the rate of decrease of absorption at 340 nm (or other suitable wavelength, e.g. 366 nm), is a measure of GPT activity. GOT: the reaction mixture contains L-aspartate, 2-oxoglutarate, malate dehydrogenase (excess) and NADH; the resulting oxaloacetate acts as the substrate of malate dehydrogenase, and the procedure is completely analogous to that described for GPT. Transamination: reversible transfer of amino groups, between two amino acids and their respective keto acids, catalysed by transaminases. T. is fundamentally important in the amino acid metabolism of all living organisms. For detailed mechanism, see Transaminases. Transcarbamylation: transfer of the carbamyl group of Carbamoyl phosphate (see). Transcarboxylatlon: see Biotin enzymes. Transcortln: see Cortisol. Transcriptase: see RNA-polymerase. Transcription: the DNA-dependent synthesis of RNA. See Ribonucleic acid. Transdeaminatlon: conversion of the amino group of an amino acid to ammonia by the combined action of a transaminase (TA) and L-glutamate dehydrogenase (GDH) (EC 1.4.1.2; 1.4.1.3):

Amino acid + 2-oxoglutarate 2-oxoacid + Glutamate; Glutamate + NAD+ + H 2 0 223. 2-oxoglutarate+ NADH + H+ + NH+ 4 . T. is an important process in ureotelic organisms, where it accounts for most of the ammonium entering the Urea cycle (see) via carbamoyl phosphate (the other nitrogen atom incorporated into urea is derived directly from aspartate, formed by transamination of amino acids with oxaloacetate). Alternatively, the ammonia may be assimilated as the amido nitrogen of L-glutamine, then used in a variety of other processes (see Ammonia assimilation; Transamidination), depending on the biosynthetic capabilities of the organism in question. Transduction: transfer of DNA from one bacterial cell to another by bacteriophage. There are two types. In generalized T. the phage infects the bacterial cell (the donor) and enters a nonlysogenic cycle leading to lysis of the cell and release of phage progeny (see Phage development). Most of the phage progeny are normal, but during the process of phage assembly within the infected bacterial cell, pieces of degraded bacterial DNA occasionally become falsely packaged into phage heads; in the case of Escherichia coli and phage Pi this falsely packaged DNA cannot be larger than 3 % of host genome, and it represents an entirely random sample of fragmented host DNA. The new phage population is then used to infect a second bacterial culture (recipient); the majority of phage particles, being normal, kill the bacterial

467

Transferases

cells that they infect. Under correctly chosen conditions (one phage particle per bacterial cell), most of the cells in the recipient culture are killed; the remaining viable cells are those that received falsely packed bacterial DNA instead of phage DNA during the infection process. This DNA is integrated into the recipient genome by genetic recombination, and is expressed by the recipient cell. In specialized T. the phage (e.g. X) becomes integrated at a specific site in the DNA of the recipient, under lysogenic conditions. The lytic cycle is then initiated (by temperature change, UV-light, etc.), and the phage DNA of some phage progeny carries small fragments of bacterial DNA from the specific integration site. Following infection of the recipient under lysogenic conditions, phage DNA and any attached bacterial DNA become integrated into the recipient DNA. The transfered fragment of bacterial DNA does not represent a random sample, and can only be derived from donor'DNA in the region of the specific integration site. Thus a Xphage is known that integrates in the region of the histidine utilization genes (hut) of Salmonella. Similarly, studies on the organization of the tryp-

Isopentenyl —

Transfer-RNA tophan synthase operon were aided by transduction with a X-phage that specifically integrates into Escherichia coli DNA in the region of the genes for tryptophan synthesis. Transferases: see Enzymes, Table 1. Transfer factors: see Elongation factors. Transferrin: see Siderophilins. Transfer-RNA, tRNA, soluble RNA, sRNA, acceptor RNA, transport RNA: The smallest known functional RNA, present in all living cells and essential for Protein biosynthesis (see). Different tRNAs contain between 70 and 85 nucleotide residues; average Mt is 25000. There is at least one specific tRNA per cell for each of the 20 protein amino acids. TTiere may be between 50 and 70 tRNA species within one cell; this multiplicity is the result of organelle specificity, and the fact that there may be two or more different but specific tRNAs for one amino acid. The source and specificity of a tRNA species is indicated by a code, e.g. tRNAValyeast is the valine-specific tRNA from yeast. [' 4 C-Val]tRNA Val yeast represents thenamed tRNA esterified with 14C-labelled valine. Function. tRNA is esterified with its specific am-

A

(serine)

Fig. 1. Clorer leaf model of a tRNA molecule (serine-specific tRNA from yeast) A, adenine; C, cytosine; G, guanine; I, inosine; U, uracil; T, thymine; T, pseudouracil.

468

Transformation ino acid by the action of Aminoacyl-tRNA synthetase (see). The resulting aminoacyl-tRNA becomes bound to the acceptor site of the SOS-subunit of a ribosome, where antiparallel base pairing occurs between the anticodon of the t R N A and the complementary codon of the associated mRNA. The specificity of this base pairing ensures that the amino acid is incorporated into the correct position in the growing polypeptide chain. During translation the deacylated t R N A is released from the ribosome and becomes available for recharging with its amino acid.

3' OH

TipC s t e m

Fig. 2. Three dimensional structure of yeast tRNApb' as determined by X-ray crystallography. Double solid lines represent hydrogen bonds between bases in double helical stems. Dotted lines represent hydrogen bonding between bases outside the helices, i.e. 8 14, 9 23, 10 45, 15 48, 18 55, 19 56, 22 46, 26 44, and 54 58. Structure. The primary structures of more than 50 different tRNAs from various organisms are known. The first t R N A structure was determined by Holley in 1965 for tRNA A l a y e a s t ; indeed, this was the first reported primary sequence of any nucleic acid. Sequence determination is facilitated by the occurrence of unusual nucleic acid components, which act as markers in the identification Of oligonucleotide fragments produced by nuclease degradation of the tRNA. Maximal base pairing of the primary structure gives rise to a secondary structure, known as the clover leaf model (Fig. 1), which contains three main loops and four stems: 1. The anticodon loop contains the Anticodon (see), a sequence of 3 nucleotides specific for the relevant amino acid. Uracil is always found next to the 5'-end of the anticodon, and a purine or purine derivative is always next to the 3'-end. In t R N A from plants, this purine derivative is often N 6 - (y,y-dimethylallyl) -ade-

Transformation nine, also known as triacanthine. 2. The dihydrouracil (diHU) loop always contains dihydrouracil. 3. TTie thymine-pseudouracil (TPC) loop, characterized by the sequence S'-GTTC-S' appears to be involved in the binding of aminoacylt R N A to the 50S ribosome. A fourth loop may be present, and it may even have an associated stem with base pairing, but it is not an invariable feature of every tRNA. The 3'-terminus of all tRNAs is 3'-ACC. The 3'terminal adenine is always separated from the first nucleoside in the T4*C loop (ribothymidine or uridine) by 21 nucleosides. TTie 5'-terminus is always phosphorylated. The aminoacyl-tRNA species that binds to the ribosome, and is active in protein biosynthesis, carries the amino acid esterified at position 3' of the 3'-terminal adenosine. The free aminoacyl-tRNA, however, represents a tautomeric mixture of 2'- and 3'-aminoacyl-tRNA. Moreover, the initial product of aminoacyl-tRNA biosynthesis is either the 2'- or the 3'-aminoacyl derivative, depending on the specificity of the Aminoacyl-tRNA synthetase (see). The highly specific recognition of a t R N A by the corresponding aminoacyl-tRNA synthetase (amino acid activating enzyme) apparently depends upon the three-dimensional structure of each tRNA. X-ray analysis of crystalline t R N A confirms the existence of four regions of base pairing and three loops, and shows how these are arranged in a three-dimensional structure (Fig. 2). The T ¥ C and diHU loops and stems are folded back, so that the molecule has a compact shape, 9.0 nm long and 2.5 nm wide. The anticodon loop and the 3'-ACC end are still widely separated as in the clover leaf model. In addition to hydrogen bonding between bases in the helical stems, the structure is stabilized by an extensive network of hydrogen bonds involving specific interactions between bases and the ribose phosphate backbone, and by some hydrogen bonding between base pairs outside the helices. Synthesis and processing. A precursor molecule of t R N A is synthesized by transcription from DNA, then processed to the functional tRNA. For example, the precursor of tRNA 7 '"' coli consists of 126 nucleotides and is 41 nucleotides longer than the mature, active molecule. The extra sequence is removed by a specific endonuclease. Certain nucleotide residues also undergo further posttranscriptional modifications, resulting in several Rare nucleic acid components (see), which are partly responsible for the specific spatial structure of each tRNA. Transformation: conceptually the simplest form of genetic transfer. " N a k e d " D N A from a donor cell enters a recipient cell and is incorporated into the recipient D N A by genetic recombination. There is no other carrier substance or structure involved; small fragments of the donor D N A simply penetrate the membrane (and wall if it is present) of the recipient cell. T. was first described in 1944 by Avery (USA) for the transformation of R (rough)-type nonpathogenic Pneumococci into S (smooth)-type pathogenic Pneumococci by treatment with killed S-type cells. The

Transglycosidation

Transketolase

469

sults in a gene mutation. It may occur spontaneously, or it may be promoted experimentally with mutagens. Transketolase (EC 2.2.1.1): an enzyme that catalyses transketolation, an important process of carbohydrate metabolism, especially in the Pentose phosphate cycle (see) and Calvin cycle (see). T. has been found in a wide variety of cells and tissues, including mammalian liver, green plants and many bacterial species. The enzyme contains divalent metal cations and the coenzyme, thiamine pyrophosphate. Transketolation involves transfer of a C 2 -unit (often called active glycolaldehyde, or a ketol moiety) from a ketose to CI of an aldose. Only ketoses with the L-configuration at C3 and preferably a trans configuration on the next carbon (i.e. CI, 2, 3 and preferably 4 as in fructose) can serve as donors of the C 2 -unit. The acceptor is always an aldose. Transketolation is reversible; a list of known reactions is shown in the Table. Details of the reaction in which xylu-

"transforming principle" was eventually shown to be DNA; this work gave the first proof that the genetic material of the cell is DNA. Frequency of T. may be low ( < 1 %) owing to rapid degradation of donor DNA before genetic recombination can occur. Transglycosidation: transfer of a glycosidically bound sugar residue to another molecule with a suitable recipient OH-group. T. is catalysed by transglycosidases, e.g. galactosidase catalyses transfer of a galactose residue from lactose to the C6-hydroxyl group of glucose, or to a further lactose molecule to form the trisaccharide, 6-galactosidolactose. T. is sometimes involved in the synthesis of oligo- and polysaccharides. Transhydrogenase: see Hydrogen metabolism. Transhydrogenatlon: see Hydrogen metabolism. Transition: replacement of a purine by a different purine, or a pyrimidine by a different pyrimidine in the polynucleotide chain of DNA. T. re-

"C" I

H- - C — O H

CH,OH ch 3 I 0=C I HO—C—H + R,- h*/ I V : H—C—OH H

CH20® Xylulose 5phosphate

Thiamine pyrophosphate

CH20®

CH,

Glyceraldehyde 3 - p h o s p h a t e

Rï-N®

CH3

s H 0 - CH0H-

I

•cI -c

CH 2 0H H

R,—N HO—C

OH

CH20®

H 2 COH + H + active

Glycolaldehyde

CHO I

H—C—OH

I

H—C—OH

Thiaminepyrophosphate

I

H—C—OH CH20® Ribose 5 phosphate CH,

CH 2 0H

^

R,

Ri—N

C=0

I

HO—C—H

H>0—C-CH20H

I

H—C—OH

HO

I

H—C—OH

I

H—C—OH

I

H — C - •OH

H—C—OH

I

H — C - •OH

CH20® Sedoheptulose

I

-C—H

I

7-phosphate

CH20®

Mechanism of action of transketolase (only tie thiazole group of thiamine pyrophosphate is shown).

470

Transketolation lose 5-phosphate serves as the donor of the C 2 unit, and ribose 5-phosphate as the acceptor are shown in the Fig.: The C 2 -unit becomes bound to the thiamine pyrophosphate as 2-(a, fi-dihydroxyethyl)-thiamine pyrophosphate, and the remainder of the molecule is released as glyceraldehyde

Transport

cules through biological membranes. Most polar molecules do not pass freely across biomembranes. Exchange of essential metabolites between a cell and its surroundings, or between cytoplasm and organelles, therefore depends on T. mechanisms within the membranes. All T. me-

Table. Transketolase reactions (P = phosphate) Donor (R-CHOHCO-CH 2 OH)

+ Acceptor (R,-CHO)

^ Donor (R,-CHOHCO-CHjOH)

L-Erythrulose D-Xylulose 5-P D-Fructose 6-P D-Sedoheptulose 7-P D-Fructose 6-P D-Sedoheptulose 7-P

+ + + + + +

^ ^ ^ ^ ^ ^

Glycolaldehyde D-Glyceraldehyde 3-P D-Erythrose 4-P D-Ribose 5-P D-Glyceraldehyde 3-P D-Glyceraldehyde 3-P

3-phosphate; transfer of the C 2 -unit to ribose 5phosphate produces sedoheptulose 7-phosphate. If the ribose 5-phosphate were replaced by erythrose 4-phosphate, the products would be glyceraldehyde 3-phosphate and fructose 6-phosphate. Transketolation: see Transketolase. Translation: in the wider sense equivalent to Protein biosynthesis (see). In the narrower sense, T. is the decoding process whereby each codon (see Genetic code) in mRNA is translated into one of 20 amino acids during protein synthesis on polysomes. Translocation: see Transport. Transmethylases: see Transmethylation. Transmethylation: transfer of a methyl group (-CHj) from a physiological methyl donor to C-, O- and N-atoms of biomolecules. T. to oxygen produces the methoxy group (-OCH 3 ). The most important methyl donor is S-Adenosyl-L-methionine (see). Thus the methyl groups in a wide variety of methylated natural products originate from the methyl group of methionine. The thioether group of methionine itself does not participate directly in T.; it must first be activated to a sulfonium group by S-adenosylation, i.e. by synthesis of 5-adenosyl-L-methionine. Betaines (see) and Thetins (see) have limited physiological significance as methyl donors. Methylated nucleic acid components are formed by methylation of the polynucleotide chains of nucleic acids by Sadenosyl-L-methionine, catalysed by specific transmethylases (methyltransferases). Exceptions are thymine and 5-hydroxymethylcytosine, where the methyl (or hydroxymethyl) group is derived from N5,10-methylenetetrahydrofolic acid (seeActive one carbon units). Transphosphatases: see Kinases. Transplantation antigens, histocompatibility antigens: antigens on the surface of nucleated cells, particularly leucocytes and to a lesser extent thrombocytes. A lack of correspondence between T.a. of donor and recipient leads to transplant rejection. To ensure optimal compatibility between donor and recipient, the T.a. of both are typed by the histocompatibility test. Transport: passage of ions and certain mole-

+ Acceptor (R-CHO)

L-Erythrulose + Glycolaldehyde D-Xylulose 5-P + D-Glyceraldehyde 3-P D-Fructose 6-P + D-Erythrose 4-P D-Sedoheptulose 7-P + D-Xylulose 5-P D-Xylulose 5-P + D-Erythrose 4-P D-Xylulose 5-P + D-Ribose 5-P chanisms catalysed by biomembranes have three characteristic properties: saturation, substrate specificity and specific inhibition. T. may be active or passive. Passive T. can only operate in the presence of an appropriate concentration gradient, which acts as the driving force in transporting the material through pores in the membrane. The commonest form of passive T. involves a carrier mechanism. According to this model, the process is capable of on-off regulation, and the carrier is a specific protein which attaches to the substance to be transported on one side of the membrane, takes it across the membrane, releases it on the other side, then returns to the starting position. Depending on the concentration gradient, passive T. is reversible and can occur in either direction. Important passive T. systems in animal tissues are, e.g. the glucose carrier in human erythrocyte membranes, and the ATP-ADP carrier of the mitochondrial membrane, which normally transports one molecule of ADP into the mitochondrial matrix, and one molecule of ATP (formed by oxidative phosphorylation) from the matrix to the cytoplasm. In active T. material is transported against a concentration gradient, and the process is linked to the cleavage of ATP. In recognition of the involvement of ATP, active T. systems ("pumps") are also called ATPases. In the model for active T., the substance in question becomes attached to a complementary binding site on the protein carrier, then transported to the other side of the membrane by diffusion, rotation, or a change of conformation. The free energy of ATP cleavage is required for release of the substrate from the carrier; this probably involves a conformational change in the carrier protein, which alters the binding affinity for the substrate. E.g. in animal tissues: the N a + and pumps, and the active transport mechanisms for glucose and other sugars, and for amino acids. The so-called Na + K+-ATPase also appears to be important for the T. of glucose and amino acids. Furthermore, ATPases play an important role in nerve transmission, muscle control and sensory perception. The Na+ and K + transporting ATPase syst e m ( N a + K + pump) transports N a + ions out of

471

Transport antibiotics

the cell against the electrochemical gradient (intracellular Na + concentration < 10 mM; extracellular conc. about 150 mM) and K.+ ions from the surrounding milieu into the cell (extracellular K.+ concentration < 4 mM; intracellular conc. 120-160 mM). The high internal K + concentration is biochemically important, being essential for protein biosynthesis and for the maximal activity of various enzymes, etc. The N a + / K + gradient across the cell membrane is necessary for the excitation response of muscle cells and for signal transmission by nerve cells. A two-stage process is postulated for the transport mechanism: ATP + Na+internal + © ^ Na-©~P^Na- A

Na- © ~ P + ADP

~P

K+external + H 2 0 + Na- A ~ P ^ © + P, + Na+external + K + internal Two different conformations (O and A) are postulated for the ATPase. The intermediate phosphorylated form of the enzyme contains an acyl phosphate group bound to an aspartic acid residue at the active center of the ATPase. N a + K + -ATPase has Mr 250000-300000 and consists of two different subunits. Kidney and brain cells use about 70% of their synthesized ATP for the exchange transport of Na + and K+ catalysed byNa + K+-ATPase. A mechanism in which glucose transport is coupled with a Na+ gradient is postulated for the active transport of glucose, against a concentration gradient, from the small intestine in the blood stream, and from the glomerular filtrate through the epithelial cell layer of the kidney tubuli into

CH2-SH

Trehalose teria can bind sugars, amino acids and inorganic ions, and therefore play an important part as carriers associated with T. systems. Especially aerobic bacteria possess true active T. systems. Group translocation is a special type of membrane T. A membrane-bound enzyme catalyses a reaction between substrates on opposite sides of the membrane, and the product accumulates on one side of the membrane. E.g. various bacteria take up glucose by phosphorylating it to glucose 6phosphate. Group translocation is also involved in the T. of amino acids. In contrast to active T., group translocation involves modification of the transported material. Transport antibiotics: see Facilitated diffusion. Transport RNA: see Transfer RNA. Transsulfuratlon: exchange of sulfur between L-homocysteine and L-cysteine, with L-cystathionine as an intermediate (Fig.). Strictly speaking, T. is not a group transfer reaction, because the sulfur bond formed in the synthesis of cystathionine is different from that broken in the formation of L-cysteine. T. operates in the biosynthesis of L-cysteine from L-methionine, and in the biosynthesis of L-methionine. The methionine precursor, L-homocysteine, is formed by T. as follows: L-Homoserine + Succinyl-CoA —• CoA + OSuccinyl-L-homoserine OSuccinyl-L-homoserine + L-Cysteine —• LCystathionine + Succinate L-Cystathionine + H 2 0 —• L-Homocysteine + Pyruvate + NH 3 In some organisms L-homoserine is initially acylated to O-acetyl-L-homoserine (by acetyl-CoA), and in plants it is converted to oxalyl-L-homoser-

+ L-Serine

I R L-Homocysteine

CH

>\ R

2

-S-CH

2

2-°*°buJ;yrate

I R,

L-Cystathionine

CH2-SH

» I R, L-Cysteine

Formation of cysteine from homocysteine (derived from methionine). the blood. Na+ ions are pumped out of the cell by Na + K + -ATPase, thus forming a higher Na+ concentration outside than inside cell. Glucose and Na+ are then transported into the cell by a passive carrier that has binding sites for both glucose and N a + (cotransport or symport). Active amino acid T. has certain similarities with the active T. of glucose, especially from the intestine into the blood. More than five different T. systems are known. In some cells amino acid T. also appears to be coupled with a Na+ gradient. The ATP-dependent, intracellular T. of C a 2 + from the sarcoplasm into the sarcoplasmic reticulum is an essential process in the initiation of muscle relaxation. This process depends on the activity of a Ca 2+ -ATPase system (Ca2 + pump).Mitochondria of animal cells are also able to accumulate Ca 2 + against a steep gradient. Specific proteins in the periplasmic space of bac-

ine by reaction with oxalyl-CoA. Transvaalin: see Scillaren A. Transversion: replacement of a pyrimidine by a purine, or a purine by a pyrimidine in the polynucleotide chain of DNA. T. results in a gene mutation. It may occur spontaneously, or it may be promoted experimentally with mutagens. Trehalose: a nonreducing disaccharide, consisting of two glucopyranoside residues. Mr 342.30. There are three forms of T., depending on the nature of the glycosidic linkage: a, a-T. (m.p. 204°C, [alg1 +197°); a, P-T. or neotrehalose ([a]g>+95°); fl, fi-T. or isotrehalose (m.p. 135°C, [ a j g ' - 4 2 ° ) , T. is present in algae, bacteria, numerous lower and higher fungi, and occurs sporadically in nonphotosynthetic tissues of higher plants. It is the "blood sugar" of insects. T. is cleaved by many fungal enzymes, and it is fermented by certain yeasts.

TRF

472

a,a-TrehaIose TRF: abb. for thyrotropin releasing factor (see Releasing hormones). TRH: abb. for thyrotropin releasing hormone (see Releasing hormones). Triamcinolone: 16a-hydroxy-9a-fluoroprednisolone; 9a-fluoro-l 1P, 16 a, 17a,21 -tetrahydroxypregna-l,4-diene-3,20-dione, a synthetic steroid prepared from Cortisol. The antiinflammatory activity of T. is 50 times greater than that of cortisone acetate (see Cortisone), and it does not cause undesirable salt retention. It is used in the treatment of arthritis, allergies, etc. Tricarboxylic acid cycle, TCA-cycle, citric acid cycle, Krebs cycle: a fundamentally important cycle of reactions in the terminal oxidation of proteins, fats and carbohydrates (Fig. 1). C 0 2 is formed by the decarboxylation of oxo-acids (oxalosuccinate, not shown in Fig. 1, is an intermedi-

Fig. 1. The tricarboxylic acid cycle (TCA-cycle)

Tricarboxylic acid cycle ate between isocitrate and 2-oxoglutarate; see Isocitrate dehydrogenase). Together with the Respiratory chain (see), operation of the TCA-cycle leads to the synthesis of the energy-rich compound ATP. In addition to energy production, the TCA-cycle provides intermediates for biosynthesis. Several important groups of substances are derived from intermediates of the TCA-cycle, and various other metabolic cycles are linked with the TCA-cycle. In eukaryotes, the TCA-cycle operates in the mitochondria, where it is structurally and functionally integrated with the respiratory chain, and with the degradation of fatty acids. In prokaryotes, the enzymes of the TCA-cycle are localized in the cell cytoplasm. The TCA-cycle is responsible for the oxidation and cleavage of the acetyl group of acetyl-CoA, with the formation of two molecules of C 0 2 . During one operation of the TCA-cycle, there are four dehydrogenations (each equivalent to 2 hydrogen atoms) in which the hydrogen is transferred to N A D + or FAD. The reduced coenzymes are then reoxidized by the respiratory chain, which catalyzes the oxidation of the hydrogen to water.

Tricarboxylic acid cycle

473

Oxidation in the TCA-cycle is achieved by the addition of water and subsequent removal of hydrogen; there is no direct involvement of oxygen: C H 3 . C O ~ S C O A + 3H 2 0 — 2COj + 8[H] + HSCoA. The initiating reaction of the TCA-cycle is the condensation of acetyl-CoA with oxaloacetate, catalysed by citrate synthase. One molecule

Tricarboxylic acid cycle three operations of the chain from N A D H ( N A D H + 0.50 2 + H + — N A D + + H 2 0 , A G ° ' = 219.4 kJ [52.4 kcal]), and one operation of the chain from F A D H 2 ( F A D H 2 + 0.502 — F A D + H 2 0 , A G ° ' = 151.6 kJ [36.2 kcal]). A portion of this energy is used in the synthesis of 12 molecules of ATP, representing about 40% of the total

"OOCCOCH2CH2COO~

HSCoA

Enzyme 2 —NH—CO—(CH2)t

Enzyme 3 FADH2

Fig. 2. Mechanism of the oxidative decarboxylation of 2-oxoglutarate by the 2-oxoglutarate dehydrogenase complex (EC 1.2.4.2).

Enzyme, = 2-oxoglutarate decarboxylase Enzyme 2 = lipoyl-reductase-transacetylase poyl reductase + transsuccinylase) Enzyme 3 = dihydrolipoyl dehydrogenase TPP = thiamine pyrophosphate HSCoA = coenzyme A

of water is consumed, and the products are citrate and coenzyme A. Citrate is then converted into oxaloacetate by 7 consecutive, enzyme-catalysed steps (Table 1). Reactions 3 and 4 involve decarboxylation. Fig. 2 shows the mechanism of action of the 2-oxoglutarate dehydrogenase complex. Energy balance of the TCA-cycle. A total of about 900 kJ (215 kcal) free chemical energy is available from the oxidation of acetyl-CoA by the TCA-cycle, with the involvement of the respiratory chain. Operation of the respiratory chain alone accounts for - 8 1 0 kJ ( - 193.4 kcal) of this free energy, i.e.

free energy: Reactions 3, 4 and 8 give 3 X 3ATP by the oxidation of 3X N A D H in the respiratory chain; Reaction 6 gives 2ATP by F A D H 2 oxidation in the respiratory chain; and the G T P produced in reaction 5 (see substrate level phosphorylation) is energetically equivalent to ATP, i.e. GTP + A D P ^ G D P + ATP has an equilibrium constant of 1.0. The overall equation, including the respiratory chain and oxidative phosphoryla- S C o A + G D P + 11 A D P + -2C< + HSCoA.

(li-

Table 1. The reactions of the tricarboxylic acid cycle. Reaction number

Equation

Name of enzyme (see also under separate entries)

Inhibitors

AG"' in k J / m o l (kcal/mol)

1

Acetyl-CoA + oxaloacetate + H 2 0 —• citrate + HSCoA + H +

Citrate fsij-synthase (EC 4.1.3.7)

none

-38.04 (-9.08)

2a

. Fe 2 + , G S H Citrate s> isocitrate

Aconitate hydratase (EC 4.2.1.3)

Fluorocitrate*, trans-Aconitate*

+ 6.66 (+1.59)

Table 1 (continued on page 474)

474

Tricarboxylic acid cycle

Tricarboxylic acid cycle

Table 1 (cont.) Reaction number

Equation

Name of enzyme (see also under separate entries)

2b

„ . , F e 2 + , GSH Citrate > cis-aconitate 2 . . Fe + , GSH cis-Aconitate >

Aconitase

+ 8.54 ( + 2.04)

Aconitase

-1.89 (-0.45)

Isocitrate + NAD + Mg 2 + (Mn 2 + ), ADP_

Isocitrate dehydrogenase (EC 1.1.1.41)

2c

2-oxoglutarate + NADH + H+ + C02 2-Oxoglutarate + HSCoA + 2-Oxoglutarate dehy2 drogenase complex NADMg +,TPP,LipS2> (EC 1.2.4.2) succinyl-CoA + C 0 2 + NADH + H + Succinyl-CoA + G D P + P, Succinyl-CoA syntheMe2+ tase (EC 6.2.1.4) + H 2 0 6 > succinate + GTP + HSCoA 2 + Succinate dehydrogeFe Succinate + FAD > nase (EC 1.3.99.1) fumarate + FADH 2 Fumarate hydratase Fumarate + H 2 0 —• (EC 4.2.1.2) L-malate L-Malate + N A D + ->• oxal- Malate dehydrogenase (EC 1.1.1.37) oacetate + NADH + H +

Inhibitors

ATP

-7.12 (-1.70)

Arsenite, Parapyruvate*

— 36.95 (-8.82)

Hydroxylamine

-8.85 (-2.12)

Malonate*, Oxaloacetate* meso-Tartrate*

TCA c

' ycle>

~0 —3.68 (-0.88)

Oxaloacetate*, Fluoromalate*

Sum of equations 1 -8, i.e. balance of the TCA-cycle without the respiratory chain : Acetyl-CoA + 3NAD+ + FAD + GDP + P , + 2 H 2 0

AG°' in k j / m o l (kcal/mol)

2C02

+

+28.02 ( + 6.69) — 60.00 14 32)

'

HSCoA + 3 NADH + H + + FADH 2 + GTP Abbr.: HSCoA = Coenzyme A; GSH = Glutathione; AM(D)(T)P = Adenosine mono(di) (triphosphate; TPP = Thiamine pyrophosphate; LipS 2 = Lipoic acid amide; GD(T)P = Guanosine di(tri)phosphate; P,- = Inorganic phosphate; FAD(H 2 ) = Enzyme-bound oxidized (reduced) Flavin-adenine-dinucleotide ; N A D + (H) = Oxidized (reduced) Nicotinamide-adenine-dinucleotide. Compounds with* are competitive inhibitors.

The TCA-cycle is linked to Gluconeogenesis (see) by the conversion of oxaloacetate to phosphoenofpyruvate. It is also the source of intermediates in the synthesis of many amino acids, especially aspartic and glutamic acid and other amino acids derived from them. Succinyl-CoA is a precursor of the porphyrins, e.g. heme, chlorophyll, vitamin B12. The function of the TCA-cycle may be modified by integration with other pathways, e.g. yAminobutyrate pathway (see), Glyoxylate cycle (see), and the Succinate-glycine cycle (see). Carboxylation of pyruvate (see Pyruvate carboxylase; see Carboxylation) serves as one stage in gluconeogenesis from pyruvate; it is also an anaplerotic reaction (see Metabolic cycle) of the TCA-cycle, i.e. it maintains the level of oxaloace-

tate, which would otherwise be depleted by removal of intermediates of the TCA-cycle for biosynthesis. In animals the net synthesis of carbohydrate from acetyl-CoA (and therefore from fatty acids) is not possible. In plants and bacteria, however, the presence of the glyoxylate cycle permits the incorporation of a second acetyl group from acetyl-CoA, resulting in the net synthesis of TCA-cycle intermediates (and therefore of carbohydrate) from two-carbon units. This is important in the utilization of oil reserves in seeds for the synthesis of carbohydrate (e.g. cellulose of cell walls) during germination, and in the growth of bacteria at the expense of simple carbon sources, such as acetate.

475

Tricarboxylic acid cycle

Tricarboxylic acid cycle

Alanine

Glycine Carbohydrates

Valin

Tyrosine

Cysteine

CO,

Phosphoenol pyruvate

Jr

^ ¡ » d ^ P a r t a t e

w L t e ^

h>

2-OxoglutarateX

j/^COt

Succinate

Aspartate

^^^semialdehyde . . Methionine

X

SuccinylCoA Glycine 2-Amino-3-oxoadipate

I

1

Lysine

Isocitrate t I

J

Fumarate v \

^

1

Glycine

Glyoxylate

I

Threonine

^ Terpenoids,

v

SteTOÌ d S

Citrate Oxaloacetate f Acetyl-

. » . . Aspartylphosphate

Diaminopimelate

Isopentenylmirnnhrtcnhn+a pyrophosphate

A c e t o a c e t y l - CoA

Tryptophan

Asparagine À

jf Malonyl-CoA

\

Erythrose - U phosphate

Phenylalanine

Fatty acid

Leucine

Serine

5-Aminolevulinate

Isoleucine

1

Porphyrins

Fig. 3. The biosynthetic functions of the tricarboxylic acid cycle

Purine nucl nucleotides

/

Glutamine A

I | Glutamate | I ^ Glutamatesemialdehyde

1 I

Proline ^Ptî

Ornithine

Citrulline

I

Arginine

Table 2. Sites of regulation of the TCA-cycle. Reaction number

Name of enzyme

Location

Requirement for

Release of

1

Citrate synthase

Mitochondria

Citrate HSCoA

3a

NAD-dependent isocitrate dehydrogenase NADP-dependent isocitrate dehydrogenase Glutamate dehydrogenase

Mitochondria

AcetylCoA, Oxaloacetate NAD +

3b

9

Activated by

NADH,

ADP

CytoNADP+ plasm and Mitochondria

NADPH, C02

Oxaloacetate?

Mitochondria

NADP+ or NAD +

ADP

Table 2 (continued on page 476)

NADPH or NADH, NH 3

co2

Inhibited by

Remarks

Longchain acyl-CoA

Control point for utilization of acetyl-CoA

ATP, NADH

For high rates of TCA-cycle see reaction 3b.

The extramitochondrial enzyme is important in the production of NADPH GDP+ NADH

Tricarboxylic acid cycle

476

Tricarboxylic acid cycle

Table 2 (cont) Reaction number

Name of enzyme

Location

Requirement for

Release of

Activated by

10

Pyruvate carboxylase AcetylCoA carboxylase Citrate lyase

Cytoplasm

ATP, co2

ADP

AcetylCoA

Cytoplasm

ATP, co2

ADP

Citrate

Cytoplasm

Citrate

Isocitrate lyase

Cytoplasm

Isocitrate

AcetylCoA, Oxaloacetate Glyoxylate, Dicarboxylic acids

11

12

13

Reactions analogous to those of the TCA-cycle are found in the biosynthesis of leucine and lysine (Fig. 4). Similarly, the sequence of reactions represented by the dehydrogenation of succinate by a flavoenzyme, followed by hydration of fu0 CH3C—SCoA

H20

Acetyl-CoA +

Inhibited by

Control of carbohydrate metabolism Longchain acyl-CoA

Phospho enol pyruvate

A

HO—C—COO-

I R

2-Substituted malate

H—C—COO'

I

CH2

c / / h2o

I H—C—COO" R NAD(P)+S

\ nh3

or T r a n s a m i n a t i o n

2 2-Aminoadipate

NAD(P)H+H+*4

0 II C I

0

• Lysine

~r

COO" CO,

CH2

R

1 Glutamate

H 2 0-j

H—C—COO"

NAD(P) H + H +

/ '

•COO" II c - •COO" RI

HC-

OH

1 Oxaloacetate R = - C H 2 — C O O " 2 2-0xoR = - C H 2 — C H 2 — COO" glutarate •CH, / 3 2-OxoisoR--CK valerate 'CH, NAD(P)+

Important for the extramitochondrial synthesis of AcetylCoA Only found in bacteria and plants

H20

C H 2 — COO"

I

NH3

Control of fat synthesis

the regulation of the allosteric enzyme isocitrate dehydrogenase. The enzyme is activated by ADP, and inhibited by ATP and NADH (Table 2). Other sites of regulation are the syntheses of acetyl-CoA, oxaloacetate and citrate. Oxaloacetate is

HSCOA

0 II c—C00I R

Remarks

c—COO"

I

H—C—COO

I R

1 2 Oxo-

glutarate 2-Oxoadipate

3 Leucine 3

2-0xoisocaproate

Fig. 4. Some metabolic reaction sequences, which are analogous to part of the TCA-cycle marate to malate, then dehydrogenation of malate by a NAD-linked dehydrogenase, finds a counterpart in the initial stages of fatty acid degradation (see). Regulation of the TCA-cycle. ADP/ATP ratios and N A D / N A D H + H + ratios have a regulatory influence on the TCA-cycle, particularly via

the catalytic intermediate of the TCA-cycle for the oxidation of acetyl-CoA to C 0 2 + H 2 0 ; it also inhibits succinate dehydrogenase and malate dehydrogenase. Since the TCA-cycle only works in conjunction with the respiratory chain, its activity is also regulated by the supply of oxygen. When an aerobically respiring cell is made anaer-

477

Tricetinidin

obic, the TCA-cycle comes to a halt with the accumulation of the reduced coenzymes NADH and FADH 2 . Asymmetrical metabolism of citrate. Although the citrate molecule has perfect bilateral symmetry, it is degraded asymmetrically (see prochirality). According to the stereochemical numbering proposed by Hirschmann, C-l is at the end of the chain occupying the pro-S position. Citrate synOH

2CH 2

I

iCOOH

Fig. 5. Formula of citrate with procbiral numbering of the carbon atoms thesized in the TCA-cycle from oxaloacetate and [l-14C]acetyl-CoA contains 14C in position 1, and is referred to as sn-[l-14C]citrate. Aconitase catalyzes the removal of the OH-group from C-3 and the H/j proton from C-4. After rehydration of the cis-aconitate (see Aconitate hydratase; see Aconitic acid), the resulting isocitrate carries its OHgroup on the carbon originating from C-4 of citrate. The C0 2 from the conversion of isocitrate into 2-oxoglutarate is therefore derived from the original oxaloacetate and not from the acetyl group of the acetyl-CoA. Subsequent decarboxylation to succinyl-CoA removes yet another carbon of the original oxaloacetate. Thus, the new "catalytic" molecule of oxaloacetate, formed after one round of the TCA-cycle, contains only two carbons of the original oxaloacetate plus both carbon atoms of the original acetyl group. Since fumarate and succinate are metabolized symmetrically, the original acetyl C-l atom becomes equally distributed between C-l and C-4, and the original acetyl C-2 between C-2 and C-3 of the new oxaloacetate. In the next round of the cycle, all of the original acetyl C-l is removed as C0 2 . The C-2 of the original acetyl group becomes distributed amongst all four carbons of the new oxaloacetate, so that it theoretically can never be entirely removed by decarboxylation in the TCA-cycle; it does not contribute to C0 2 until the third round of the cycle. Patterns of labelling from the incorporation of 14C-labelled acetylCoA are therefore complex, but the fundamental experimental observation is that some 14C from labelled acetyl-CoA is retained by intermediates of the TCA-cycle. Historical. The TCA-cycle was discovered almost simultaneously in 1937 by Krebs, and by Martius and Knoop. Green coined the term "cyclophorase" for the total multienzyme complex of the TCA-cycle and the associated respiratory chain. Early studies with tissue suspensions showed that 14 C from C-l of acetate was found only in the 4carboxyl carbon of 2-oxoglutarate, whereas the symmetrical metabolism of citrate would demand

Trichochromes the presence of label in both the 2- and 4-carboxyl groups. It was therefore suggested that citrate could not be an intermediate in the TCA-cycle. In 1948, however, Ogston proposed his three-point attachment theory, in which the active sites of citrate synthase and aconitase are asymmetrical: three functional groups of citrate must become attached to three complementary, asymmetrically arranged binding sites. With the recognition of prochirality, it is now no longer necessary to propose that the site of asymmetry lies within the enzyme. Ogston's theory is, however, not disproved, and the precise nature of citrate binding to aconitase will not be known until the active center of the enzyme has been mapped. CH,COOH

ziry

Fig. 6. Diagrammatic representation of the three point attachment theory of Ogston, where X represents a site of attachment of citrate to the active center of aconitase. Tricetinidin: 3-deoxydelphinidin, see Anthocyanins. Trichochromes: yellow-orange and violet natural pigments containing a substituted A2-2'bis(l,4-benzothiazine) ring system. Color is due to the conjugated chromophore system,-S-C = CC = N-. Biosynthetically, T. are related to the Melanins (see). Together with the phaeomelanins, T. are responsible for the red and auburn colors of human hair and bird feathers. T. B and C are yellow-orange; T. E and F are violet.

Trichochrome B: R,= H; R2 = CH2-CHNH2-COOH; R3=COOH Trichochrome C: R^CHj-CHN^-COOH, R2=H;R3=H

COO H Trichochrome E: Ri = H; R2 = CH2-CHNH2-C00H Trichochrome F: R,= CH2-CHNH2-COOH,- R2=H

478

Tricholomic acid

Tricholomic acid: erythro-dihydroibotenic acid, a compound isolated from the Basidomycete (mushroom) Tricholoma muscarium. It can also be prepared by reduction of ibotenic acid. It possesses flavor promoting activity similar to, but much more active than, that of sodium glutamate. It also has a synergistic effect on the flavor improving property of inosinic acid and guanosine 5'-phosphate.

//

H3N H—C~ooü

H

NH

Tricholomic acid Trl(hydroxymethyl)methylamlne, THIS: H 2 NC(CH 2 OH) 3 , Mr 121, a widely used buffer substance, suitable for the pH range 7-9. The required pH is usually obtained by adding HC1 to TRIS dissolved in water. TRIS buffers have a high temperature/pH gradient, e.g. 0.05 M TRIS, pH 7.05 (adjusted with HC1) at 37° C has pH 7.20 at 23°C. The pH of a TRIS buffer should therefore be established at the intended working temperature. 3,5,3'-Triiodothryonln: see Thyroxin. Trlmethylglyclne: see Betaines. Trlose phosphates: D-glyceraldehyde 3-phosphate ( P 0 3 H 2 - 0 C H 2 - C H 0 H - C H 0 ) and dihydroxyacetone phosphate ( P 0 3 H 2 - 0 C H 2 - C 0 - C H 2 OH), important intermediates in Glycolysis (see) and Alcoholic fermentation (see). The two T.p. are interconvertible via the ene-diol form, by the action of T.p. isomerase; the equilibrium mixture contains 96% ketotriose phosphate and 4% aldotriose phosphate. The T.p. hold a key position in carbohydrate metabolism, being intermediates of gluconeogenesis and photosynthetic C 0 2 fixation. Trloses: glyceraldehyde and dihydroxyacetone phosphate. TTiey contain 3 C-atoms and are the simplest monosaccharides. Their phosphates are important metabolic intermediates (see Triose phosphates). Triphosphomonoesterases: see Esterases. Triphosphopyrldine nucleotide: see Nicotinamide adenine dinucleotide phosphate. Triple helix: see Collagen. Triplet code: see Genetic code. TRIS: see Tri(hydroxymethyl)methylamine. Trisaccharldes: see Carbohydrates. Trisporic acids, fungal sex hormones: structurally similar C 18 -terpene carboxylic acids from heterothallic fungi of the Mucorales type, e.g. Blakeslea trispora or Mucor mucedo. T.a. are only produced when ( + )-strains are mixed with ( - ) strains; a prohormone from the (—)-strain is transformed into T.a. by the ( + )-strain. The T.a. then induce formation of zygospores in the ( —)cells. Trisporic acid Cis the most important member of the group, Mr 306; 20 |xg are sufficient to induce zygospore formation. Unlike other diterpenes, T.a. are biosynthesized by the cleavage of P-carotene.

Triterpenes

COOH Trisporic acid C Triterpenes: an extensive group of terpenes biosynthesized from six isoprene units. Apart from squalene, which is acyclic, most of this group are tetra or pentacyclic hydroaromatic compounds based on the parent hydrocarbon, sterane, i.e. they are steroids. Included with T. are those terpenoid natural products with fewer than 30 Catoms, which are biosynthesized via a C 30 intermediate, but with the subsequent loss of one or more C-atoms. Addition of extra C-atoms and incorporation of heteroatoms are also possible, e.g. the steroid alkaloids. Many T. have high biological activity, in particular the steroid hormones. Table. Triterpenes. Type of compound

Sex hormones Estrogens Androgens Progestins Adrenal corticosteroids Cardiac glycosides Steroid alkaloids Bile acids Sapogenins Vitamin D Molting hormones Sterols Mycosterols Zoosterols Phytosterols Cucurbitanes

No. of carbon atoms

Examples

18 19

Estradiol Androsterone Progesterone Corticosterone

21 21

21,23, 24 21,27 24.27 27 27, 28 27 27.28 27, 28, 30 29,30 30

Digitoxigenin Tomatidine Cholic acid Digitogenin Vitamin D 2 ß-Ecdysone Ergosterol Cholesterol ß-Sitosterol Cucurbitacin D

Biosynthesis. Tail-to-tail condensation of two molecules of farnesylpyrophosphate (see Terpenes) produces squalene (Fig. 1), which serves as the precursor of the cyclic T. Squalene is first oxidized to squalene 2,3-epoxide by the action of squalene monooxygenase. By loss of an electron, the 2,3-epoxide forms a 3-hydroxysqualene cation (C2 is positively charged), which cyclizes on the surface of a cyclase enzyme. The many possible types of cyclic T., e.g. steroids and sterols, are determined at this stage by various foldings of the squalene chain and migration of groups within the molecule (anionotropy). In the biosynthesis of pentacyclic T., the 2-hydroxysqualene cation cyclizes to a prosterol cation, which has not yet been identified in the free state. A Wagner-Meer-

Triterpene saponins

H.

479

Tropane alkaloids

H2 S

CH2

CH

CH2

CH

H, O—PP

Farnesylpyrophosphate

CH3 H

PP-O

~Q\ ^ ¡ 2

CH^C'fy

Farnesylpyrophosphate

£

^ 2

H2 H2 CH

CH,

CH,d I >C

CH

CH 2

Squalene

Fig. 1. Formation of squalene from two molecules of farnesylpyrophosphate. wein rearrangement and ring closure leads to the expansion of ring D (compounds I and II, Fig. 2). A further Wagner-Meerwein rearrangement results in expansion of ring E in III, to form IV

H3C

CH3

Compound I

ÇH3 H3C—L^

H3C

moiety, esterification with different acids and the occurrence of isomers and demethylated derivatives. Tropane-3P-ol-2-carboxylic acid (ecgonine, Fig. 1) is important as the base component of the

^CH 3 Compound HL

H3C' CH3 Compound H

H 3 C ^CH 3 Compound I I

Fig. 2. Formation of pentacyclic triterpenes. which is the precursor of a large number of pentacyclic T. (germanicol, friedelin, multiflorenol, taraxol, etc.). For the biosynthesis of other T., see Steroids. Triterpene saponins: see Saponins. Trltyroslne: see Resilin. tRNA: abb. for transfer-RNA. tRNA methylases: see Polynucleotide methyltransferases. Tropane alkaloids: a group of Alkaloids (see). T.a. are esters of various aminoalcohols based on a substituted tropane (Af-methyl-8-azabicyclo3,2,1-octane) ring system. This system is not optically active, because the two rings can only be linked in a cis configuration, thus resulting in a meso form. Introduction of a hydroxyl group at position 3 produces the geometric isomers, tropine (tropane-3o-ol; the OH-function is trans to the CH 3 N-group, Fig. 1) and pseudotropine (