A STUDY OF THE LACTIC AND MALIC OXIDATIVE ENZYMES OF TOBACCO SEEDLINGS GROWN IN ASEPTIC CULTURE

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The Pennsylvania State College The Graduate School Department of Agricultural and Biological Chemistry

A Study of the Lactic and Malic Oxidative Enzymes of Tobacco Seedlings Grown in Aseptic Culture

A Dissertation by Joseph R. Riden, Jr.

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 19^1

Approved: ^

^ PrnfesS'orf of Phytochemistry

Head, Department of Agricultural and Biological Chemistry

AC KN 0VVLEDGEMENT The writer wishes to express his appreciation to Dr. C. 0. Jensen for having suggested this problem and for his advice and criticism during the course of this thesis and its preparation; and to Dr. R. A. Steinberg, Department of Agriculture, Beltsville, Maryland, for demonstrating the technique of growing tobacco plants in aseptic culture.

TABLE OF CONTENTS Page I INTRODUCTION ...................................... II REVIEW OF

1

L I T E R A T U R E .....................

3

A.Plant Oxidative Enzymes ......................

3

B. Methods for the Determination of Oxidative Systems ...........................

10

III EXPERIMENTAL A. Development of Method 1.Sterile Culture of Tobacco Plants

. .

13

2. Preparation of Plant Tissue for Enzyme S t u d i e s ................« . . .

15

3. Details of Methods Used to Determine Oxidative Enzymes

21

..........

B. Measurement of Plant Enzymes 1. The Lactic Enzyme a. Cofactors Necessary for A c t i v a t i o n ...................

21j.

b. pH S t u d i e s ...................

26

c. Determination of Carbon Dioxide P r o d u c e d .....................

28

d.

Cyanide Inhibition Study

• . • •

29

e.

Optimum

Substrate Studies • • . •

32

f. Triphenyltetrazolium Chloride Studies . . • • • • •

32

g. Methylene Blue Studies on Acetone Extracted Tissue . . . .

36

h. The Effect of Sucrose Upon ................ Lactic Oxidase

38

Page i. Lactic Oxidase Content of Etiolated Tissue ............... j. Purification of Lactic Oxidase

39 .

k. Identification of an End Product

I4J. L^2

2. The Malic E n z y m e ......................

2+3

a. Cofactors Necessary for A c t i v a t i o n ......................

lj.3

b. pH S t u d i e s ......................

2+7

c. Determination of Carbon Dioxide P r o d u c e d ...............

2+7

d. Triphenyltetrazolium Chloride S t u d i e s ..........................

5>0

e. Methylene Blue Studies on Acetone Extracted T i s s u e ............. .

51

IV DISCUSSION OP R E S U L T S .......................... V SUMMARY AND CONCLUSIONS

$$

........................

60

VI B I B L I O G R A P H Y .....................................

62

A

I INTRODUCTION "When distressed by the thought that in your own particular branch of biochemistry progress is slow, significant facts are few, and confusion is rampant, you will be consoled to learn that some other fields of biochemistry are in an even more primitive state of development*" " ....

In view of the present state of knowledge

of plant respiratory enzymes it would be unwarranted to accept such an assumption without proof." The first statement was made by Lardy (27) in 1951 while reviewing the latest in a series of volumes on enzymology.

Maxwell (3 6 ) was speaking about the presence

of cytochrome oxidase in corn embryos when he published the latter statement in 19^0.

This thesis was undertaken

in hopes of making a contribution to the now meager knowledge of plant enzymes and the methods used to study them. When a method appeared in the literature for growing tobacco plants in aseptic culture, the technique was adapted for this study of tobacco enzymes.

By using

sterile tobacco plants, the contaminating enzymes of bacteria, molds, fungi and other living matter found on tobacco grown under field conditions can be eliminated. By the proper handling of sterile plant tissue, we may be certain that we are studying only the enzymes which are

2 found in the green tobacco plant. Cigar leaf tobacco is an important crop in Pennsylvania agriculture.

The tobacco must be cured or

fermented before it is usable, being caused by enzymes. or plant origin.

such processes probably

These enzymes may be of bacterial

Jensen and Parmele (23) have studied the

fermentation of cigar-type tobacco and have isolated and identified seven species of bacteria from fermenting tobacco.

Reid, McKinstry,

and Haley (5l, 5>2) have also

made a study of the bacterial flora of fermenting tobacco, and have shown that more than one billion bacteria per gram may exist in sweating tobacco.

The contributions of

bacterial enzymes to the changes taking place in fermenting tobacco are undoubtedly important.

However there is not

sufficient evidence to indicate that plant enzymes play no part in the process. The relation of enzymes of the tobacco plant and enzymes of microorganisms to the curing of tobacco is largely unknown.

Before such knowledge is produced more

information concerning the enzymes of tobacco itself and a study of the microflora must be available.

3

II REVIEW OP THE LITERATURE A.

Plant Oxidative Enzymes.

The oxidative enzymes of

higher plants have not had the thorough investigation that has been given to the oxidative systems of animals and bacteria.

A partial explanation for this may be the lack

of a common oxidative system such as that found in all animals.

The literature on plant enzymes is composed of

many small pieces of information on the variation of oxidative systems between species of plants between differ­ ent parts of the same plant, and even between the same plant at various stages of growth.

For example, Tolbert and

Burris (61) reported that the leaves, but not the roots, of the mature tobacco plant contain glycolic oxidase, which is not present in etiolated tissue and which is not stable in young plants.

Also, Albaum and Eichel (2) found that

respiration in oat seedlings is mediated by the cytochrome system for the first 72 hours, but after that period is not inhibited by any of the cytochrome system inhibitors.

As

a result of this variation in the enzyme composition of plants, a review of the literature provides few general­ izations and many inconclusive statements regarding plant oxidative enzymes. The oxidative enzymes in plants may be classified either as dehydrogenases, which remove hydrogen atoms from substrates and pass them along a chain of carriers to atmospheric oxygen, or as oxidases which directly unite the

1* substrate's hydrogen or the substrate itself with oxygen. A terminal oxidase is an oxidase which accepts electrons from the dehydrogenase carrier chain, and reduces molecular oxygen, thereby completing the system.

In a study of the

electron carriers of plant dehydrogenase systems, Whatley (6 8 ) found only traces of diphosphopyridine nucleotide (DPN, Coenzyme I) in green leaves, although 20 ;ug. of triphosphopyridine nucleotide (TPN, Coenzyme II) per gram of dry tissue weight was present.

Lockhart (31) found flavoprotein

in peas, beans and white potatoes, although it was stable only four days.

Bach (4) reported that garden vegetables

were rich in dehydrogenases, but contained very little DPN. In studying the dehydrogenases of plant tissues, it was necessary to add DPN to activate the dehydrogenases. Berger and Avery (7) have made a study of malic dehydrogenase in the avena coleoptile, using Thunberg techniques with thionin as the hydrogen acceptor.

The

enzyme was purified by ammonium sulfate fractionation and activity was obtained only after DPN and flavoprotein were added to the system.

An accumulation of the end-product,

oxaloacetic acid, inhibited the system, but could be removed by binding it with cyanide.

The addition of various

non-heavy metallic ions had no effect upon the system. Thionin was used in this study and prevented gaining any knowledge of the nature of the terminal oxidase.

I'alic

dehydrogenase has been found in corn embryos (2lj.), spinach leaves (6 9 ), pollen (5»5>) and parsley roots (6 5 ).

$

Succinic dehydrogenase has been round in wheat term by Goddard (20) who reported that it was destroyed by acetone precipitation of the tissue.

In spinach leaves,

Bonner and Wildman (11) found it rapidly destroyed after injury to the leaves.

This instability of succinic dehydro­

genase may well be the reason that Berger and Avery (7) were not able to detect it in oat seedlings.

This dehydro­

genase has been found in corn embryos by Jensen, Sacks and Baldauski (2lj.).

A detailed discussion of this enzyme has

been given by Stauffer (55). Mathews and Vennesland (33) examined the action of formic dehydrogenase which they isolated from green pea seeds.

They found DPN was necessary for the reaction and

followed its reduction spectcphotometrically.

The formic

dehydrogenase reduced the DPN, while another enzyme catalyzed the oxidation of DPN by methylene blue.

The plant

formic dehydrogenase received no direct stimulation from the addition of the adenosine phosphates; this being in contrast to animal formic dehydrogenase.

Formic dehydrogenase has

been found in many plant seeds. Eerger and Avery (8 ) have studied the glutamic dehydrogenase of avena coleoptiles.

They have shown that

it requires DPN for its action and had an optimum pH from 6.5 to 8.7.

The enzyme was inhibited by 0.001 molar copper -

and cobalt solutions.

Glutamic dehydrogenase also appears

in spinach leaves (1 1 ). Isocitric dehydrogenase has been found in the avena

6 coleoptile and is unusual among plant enzymes as it requires TFN for activity (8 ).

The optimum pH was 6,5 and the

enzyme was found to be activated by magnesium,nanganese or cobalt ions, although Ochoa (1|3) stated that manganese was not an activator,

Isocitric dehydrogenase has also

been found in spinach leaves (1 1 ), parsley roots (6L|.) and corn embryos (2JLf.)* Alcohol dehydrogenase has been reported found in tobacco leaves (6 3 ), as well as in oat seedlings (7 ) and corn embryos (2i+).

There seems to be little character!- ~

zation of the enzyme, Watanabae (6 7 ) claimed the presence of lactic dehydrogenase in seaweed, and studied it using Thunberg tubes and methylene blue.

He found that DPN and flavo­

protein were necessary for activity and that urethan but not 0*01 molar cyanide would inhibit the system. optimum pH was 9.1*

The

Watanabae claimed that the enzyme

was tied through the cytochrome system.

These findings

are very similar to those reported for the animal lactic dehydrogenase.

Jensen, Sacks and Baldauski (214.) also

reported a lactic dehydrogenase in corn embryos*

This

enzyme required DPN and reduced triphenyl tetrazolium chloride. Miyake and Kurausa (J4.O) reported finding glucose, xylose, galactose, and arabinose dehydrogenase in plant tissue.

Watanabae (6 7 ) found malic, 1-alanine,

charic dehydrogenases in seaweed.

and sac­

Matsusina (3U-) stated

7 that the dehydrogenase activity of fresh tobacco leaves was very weak.

I.Iikhlin and Kolesnikov (39) found cyto­

chrome oxidase and, to a lesser extent, polyphenol oxidase in fresh tobacco leaves. Clagett, Tolbert and Burris (II4.) reported an dl -hydroxyacid oxidase which was present in a large number of, but not in all, the plant orders studied.

It has been

concentrated by ssilting out with ammonium sulfate and remained stable for a short time when held at l4.°C.

The

enzyme was capable to oxidizing lactic, glycolic and 1hydroxymonocarboxylic acids.

-

The enzyme appeared to have

no cofactors (6 0 ), to be insensitive to sodium azide and cyanide, and to have an optimum pH about 8*0

Later,

Tolbert and Burris (61) showed that the enzyme fraction responsible for glycolic acid oxidation was not active until the plant had been photosynthesifcing

a

while, but

that the lactic oxidase was present in etiolated tissue. DuBuy, Woods, and Lackey (17) found that lactic acid utilization by tobacco plant tissue was not affected by 0.0001 molar cyanide. Although the terminal oxidase for animals is known to be cytochrome oxidase, the terminal oxidase or terminal oxidases for plants is still a matter of speculation. Evidence has accumulated to favor three enzymes as terminal oxidases in plants:

ascorbic acid oxidase, polyphenol

oxidase, and cytochrome oxidase.

It appears that flavo-

proteins nay also act as terminal oxidases since cyanide,

8 which inhibits the three metallic enzymes just mentioned, fails to inhibit the oxygen uptake of many plants. In 1930 Szent Gyorgyi (£l) found in the leaves of cabbage an enzyme capable of oxidizing ascorbic acid. Since that time, the enzyme has been reported as occurring in many plants and vegetables, but there is no evidence of it in animals or fruits (1|.5>).

Ascorbic acid oxidase

contains about 0.2lj. per cent copper (Lj.8), has its optimum pH at 5,6 (I4.9 ), and is inhibited by cyanide and diethy1dithiocarbamate (9).

Many studies have been made on this

enzyme in view of its ability to destroy ascorbic acid and thus deprive the human body of this vitamin.

The function

of this oxidase in the plant has been examined, and while James and Cragg (22) believed that it acted as the terminal oxidase in barley respiration, this viewpoint has not been held by many other workers in the field. Polyphenol oxidase (tyrosinase) is another coppercontaining enzyme which uses a number of mono- and poly­ phenols as its substrate, including tyrosine.

Polyphenol

oxidase is inhibited by cyanide, by the nitro analogs of its normal substrates, and by compounds which bind the copper complex (16).

Nelson and Dawson (U2) reviewed the

literature on the enzyme and believed that it acted as a terminal oxidase in plant respiration.

This viewpoint is

held by many as an explanation for the wide occurrence of polyphenol oxidase in plant tissue.

Baker and Nelson (5>)

have shown that at least 8£ per cent of the oxygen uptake

9 in respiring potato tubers is catalyzed by polyphenol oxidase.

Arnon (3) found it present in the chloroplast

fraction of spinach leaves.

Recently, llelson (L|_l) stated

that more evidence is needed before a definite conclusion about the role of phenol oxidases as terminal oxidases in plant tissues may be reached. Cytochrome oxidase, an iron enzyme, which acts as the terminal oxidase in animal tissue, has also been found in plant tissue.

Goddard (12, 20) made a thorough study of

this enzyme and isolated cytochrome C from wheat embryos. Recently, Maxwell (3 6 ) has done the same with corn embryos. In a review article, James (21) stated that cytochrome oxidase has been found in legumes, roots of dandelions, turnips, onions, pollen and in other scattered plant tissues. Cytochrome oxidase acts through a cytochrome system in which various heme-like carriers have been shown to exist between DFN and cytochrome oxidase.

The cyto­

chrome system is heat labile and inhibited by cyanide, azide, and carbon monoxide (12).

The carbon monoxide

inhibition may be demonstrated only In the dark as light dissociates the enzyme-carbon monoxide complex, whereas the inhibition of copper enzymes occurs in the light as well as in the dark.

This fact has often been used to distinguish

between the two types of terminal oxidases. Levy and Schade (29» 30) have examined potato tuber respiration and state that there are two terminal oxidases present, polyphenol oxidase and cytochrome oxidase.

10 They believe the cytochrome system is the principle one, although Baker and Nelson (£) disagree.

liosenberg and Ducet

(£3 ) found polyphenol oxidase and cytochrome oxidase in spinach leaves, in contrast to Bonner and Y/ildman (11) who stated that they could only find polyphenol oxidase there. Maxwell (3&) felt that the copper enzymes, ascorbic acid oxidase and polyphenol oxidase, were not involved in seed corn at the germinating stage, but replace cytochrome oxidase as the plant grows older. Finally, mention must be made of the non-metallic enzymes which have been described in the literature.

Marsh

and Goddard (32) have shown that cyanide will inhibit respiration in the young carrot, but has little effect upon the mature leaf. same way.

Inhibition with sodium azide proceeds the

Similar phenomena have been reported with oat

seedlings (2 ), avocado fruit (3 0 ) and the chloroplasts of spinach leaves (20).

These inhibition studies indicated

the presence of a terminal oxidase that does not contain iron or copper.

This cyanide-insensitive terminal oxidase

has been described as an auto-oxidizable flavoprotein (1 0 ). B.

Methods for the Determination of Oxidative Systems.

The general method of determining enzymatic activity quantitatively is to follow the progress of the reaction that the enzyme catalyzes.

In the case of oxidative enzymes,

this may be accomplished by observing the consumption of oxygen by the reaction, or by measuring the reduction of

11 an electron carrier involved in the oxidative system.

The -

former of these two methods is usually accomplished by using the Warburg apparatus. The Warburg apparatus was first described by Warburg (6 6 ), although the basic instrument was built by Bancroft and Haldane (6 ) to measure the small amounts of gases present in blood.

The Warburg apparatus consists of

a number of small reaction flasks, each of which is connected to a mannometer.

When these flasks are placed in a constant

temperature bath and shaken, the difference between the pressure inside the flask, due to the reaction taking place, and atmospheric pressure may be measured.

Prom these dif­

ferences, the oxygen taken up or the carbon dioxide evolved by the system may be measured.

Although the accuracy of

measurements made with the Warburg manometers is only

5

per

cent, the great advantage of the instrument is a result of the fact that no contaminating compound need be added to an enzyme system in order to measure its oxidizing ability. Another application of the YJarburg apparatus is to 3tudy systems in which the terminal oxidase is inactivated or purposely destroyed.

Here the uptake of oxygen is

channeled through an auto-oxidizable dye such as methylene blue or throxigh a ferro-ferricyanide reaction as described by Q,uastel and Wheatley (50).

These methods may be used to

great advantage in separating the components of a multi­ enzyme oxidative system. Thunberg (59) designed a special tube from which the

12 atmosphere could be evacuated when working with methylene blue.

The reduced (leuco) methylene blue, which is color­

less, is easily re-oxidized by atmospheric oxygen.

Thus,

with the evacuated Thunberg tube, the disappearance of the blue color may be followed colorimetrically to give a quantitative measurement of enzymatic activity.

Other dyes

that may be used instead of methylene blue include thionin, anthraquinone, 2,6 dichlorophenol indophenol,

and meta-

cresol red. Triphenyltetrazolium chloride is a dye which has recently found favor for dehydrogenase determinations because it is not re-oxidizable by atmospheric oxygen.

A

red formazan, insoluble in water, but soluble in organic solvents (pyridine, acetone, propyl alcohol), Is the product of triphenyltetrazolium chloride reduction.

The

use of this dye for determining biological oxidations was described by Mattson, Jensen, and Dutcher (35) in 19lj-7* Other tetrazolium salts have been prepared which yield a blue color upon reduction. Finally, the general methods used in the studies of non-oxidative enzymes, i.e., methods of following the appearance of an endproduct or the disappearance of the substrate, are also applicable In the determination of oxidative enzymes.

13

III EXPERIMENTAL A.

Development of a Method, 1.

Sterile Culture of Tobacco Plante.

The plants

used in this study were tobacco seedlings, grown In aseptic culture, following a technique suggested by Steinberg (56). Briefly, the procedure consists of sterilizing tobacco seeds by washing them in silver nitrate solution and starting them on moist filter paper in sterile petri dishes.

The seedlings are aseptically transferred to

Erlenmeyer flasks and grown in nutrient solution which has been solidified with agar-agar. The plants grown in this study were the SwarrHibshman strain of Pennsylvania cigar leaf tobacco.

Seeds

of this strain were placed in a screw-top vial and soaked with water over-night in the cold*

They were sterilized

by pouring off the water and filling the vial with a 0.1 per cent solution of silver nitrate.

After 30 minutes,

the silver nitrate solution was decanted and the seeds rinsed three times with sterile water.

The seeds and the

third rinsing were quickly poured into a previously sterilized petri dish containing several layers of moist filter paper covered with a layer of sea sand.

The seeds

were allowed to germinate 10 to 12 days in the dark at 20°C., after which time they were placed in the light for several days before transplanting. The tobacco seedlings were transplanted into the

11* flasks by means of a nichrome wire which had a slight hook in the end.

The wire was flamed, cooled in sterile water,

and a seedling picked up on the hook.

A flask was opened,

flamed, and the seedling placed on the agar; its root was gently pushed below the surface of the agar, and the flask reflamed and plugged.

The flasks were not opened again until

the plant was ready for the enzyme activity tests.

If the

seedlings were from 7 to 10 mm. long, they could be trans­ planted without injury and a 100 per cent yield could be expected. Nutrient elements for the plants were contained in a solution developed by McMurtrey (37).

This solution

was prepared by dissolving 29*16 gm. CadlO-j^*^ H 20, 2.19 gm. KNO^, 3.19 gm. Mg(N0 2 >2 #6 HgO, 5.15 gm. KH2P0^, 3»08 gm. MgS0||*7 H2O

and l.fjo gm. NH^Cl in water and bringing the

final volume to 1 liter.

Micro-elements,

also necessary

for plant growth, were obtained in a solution formulated by Eastwood (19).

This solution was made up as follows;

Nutrient Ion

Chemical Source

Iron Manganese Boron Copper Zinc

Grams per Liter

Final ppm. 3.0

FeSOu MnSOJr

3.0 O.I4.

H3BO3

0.5

0.5 0.5

CuSoi, ZnSoj£

o.olj.

o.o5

o.oij.

0.05

Five ml. of concentrated sulfuric acid were added to keep the iron in solution and the final volume was brought to 1 liter.

The nutrient stock solution was diluted 1;20 and the

1$ micro-element stock solution 1:200 for use as a plant nutriont solution. The medium for growing the seedlings was prepared by adding 90 ml. of nutrient stock solution and 9 ml. of micro-element stock solution to 1700 ml. of tap water.

The

pH of this solution was adjusted to about 6.6 to prevent hydrolysis of the agar-agar during autoclaving.

After

heating the solution, 21.6 gm.of agar-agar and 36.0 of sucrose were added, giving a 1.2 per cent solution of agaragar and a 2 per cent solution of sucrose.

This solution

was steamed for an hour to dissolve the agar-agar, and then 60 ml. of it was added to each of thirty 2$0 ml. Erlenmeyer flasks.

The flasks were plugged with cotton and autoclaved

30 minutes at 15? pounds pressure. The flasks containing the plants were set out on wire racks and grown at 20°C. under Sylvania lj.0 watt Day­ light Fluorescent lights.

A timeclock was set to turn off

the lights for two hours every night, although tobacco is not injured by continuous illumination (l£). 2.

Preparation of Plant Tissue for Enzyme Studies.

The liberation of the enzymes from the cellular plant structure was studied and various methods were used in an attempt to obtain the enzymes in a pure state.

The simplest

way, of course, is to shred or grind the whole plant tissue with water or a buffer and to use that suspension as a source of enzymes.

This has the disadvantage of leaving

most of the plant cells intact with the enzymes not released.

16 A modification of this method is the homogenize!*, of Potter and Elvehjem (I4.7 ) in which a sample of tissue is reduced to a suspension of particulate components of proto­ plasm plus a solution of the easily diffusible parts of protoplasm such as inorganic ions, coenzymes and flavoproteins.

This method is well adapted to the small amount

of tissue used in this study and excluded the undesirable effects of air, heat and long handling of tissue enzymes. In this study, the plant tissue was supplied as a homogenate, unless otherwise specified. The homogenizer consisted of a mortar and a closely-fitting power-driven pestle.

The pestle was made

by machining a length of stainless steel into a cylinder 55 mm. by 16 ram..

The bottom of this cylinder was rounded

and the sides knurled about two-thirds of its length from the bottom. pestle.

A 6 mm. rod was threaded into the top of the

The mortar was a 20 mm. by 150 mm. Corning hard

glass Ignition tube.

The tubes were checked for a close

fit by filling them with water, and if the fit was satis­ factory, the tubes fell off the pestles very slowly when not supported • In making enzymatic determinations, the size rather than the age of the tobacco plant was the criterion for harvesting the plant.

This was necessary as the seed­

lings showed great variation in their abilities to get firm rooting in the agar and consequently in the time required to reach equal size.

Figure I illustrates the size of the

17 plant used in this study.

In harvesting, the leaves were

cut from the stem and removed separately from the flask. Any tough leaves were discarded and the mid-ribs removed from the remaining leaves which were then torn into pieces about 1 cm, square.

These were placed into the tube with

the desired amount of cold buffer (3 to 10 ml,) and homo­ genized 30 seconds.

In homogenizing, the tissue was forced

between the pestle and the grinding surface of the tube byslowly raising and lowering the latter. Another method that was employed to prepare leaves for use as an enzyme sotirce was an acetone extraction process.

In working with triphenyltetrazolium chloride,

the color of the chlorophyll in the leaf tissue interferes with the color of the formazan produced, making colorimetric estimations of enzymatic activity difficult.

By homogen­

izing the tissue with cold acetone the chlorophyll is removed and a colorless tissue preparation remains.

This

method has been used by Tewfik and Stumpf (58) to prepare plant tissue for aldolase determinations.

Aldolase pre­

pared in this manner will retain its activity for months if kept at The detailed procedure employed was as follows: The plant tissue was selected as previously indicated and homogenized with 10 ml. of cold acetone.

The homogenate

was filtered through a sintered glass crucible of medium porosity, using suction, and washed twice with 10 ml. portions of cold acetone.

The powder was dried on the

Figure I

Plants B and C indicate the size of plant!

19 crucible by passing air through it.

Ethyl ether was sub­

stituted for the acetone in an attempt to cut down on the . length of the drying time.

A modification of this method

was to quick-freeze the green tissue in an acetone-dry icebath at -lj.O°C. and to repeat the above procedure keeping the temperature in that low range during the entire treat­ ment.

The results of using acetone extracted tissue as an

enzyme preparation are discussed in a later section of this thesis. The ideal situation in any enzyme study is to isolate the enzyme being studied from as many other factors as possible.

It is obvious that, in an homogenate such as

just described, conditions are the exact opposite.

An

attempt was made to purify the enzyme by using a combination of filtration and dialysis.

Green plant tissue was homo­

genized in 20 ml. of buffer at various pH levels,

and the

suspension allowed to stand in the cold to permit diffusion of the soluble enzymes from the tissue.

After a period

varying from 2 to 12 hours, the suspension was filtered through a sintered glass crucible of coarse porosity and the filtrate placed in a sausage skin.

This extract was

dialysed against cold running tap water for periods of time ranging upwards to 2J^. hours.

An insoluble powder separated

during the dialysis which, upon testing with various soluble cofactors and ions, gave no activity with lactate, either with triphenyltetrazolium chloride or the Warburg technique. Jensen, Sacks, and Baldauski,

(2lj.) purifying the enzymes of

20 c o m embryos by a similar process, were able to demon­ strate the presence of several dehydrogenases by adding triphenyltetrazolium chloride and DPN. Another procedure for the purification of enzymes is the salting out of the protienaceous enzyme by a selective saturation and half-saturation of the enzyme solution with ions.

Clagett, Tolbert and Burris (llj.) were

thus able to concentrate an enzyme from the sap of the tobacco plant capable of oxidizing

-hydroxy acids.

The method of Clagett, Tolbert and Burris (llj.) was employed for certain studies in this thesis.

The first

step of the method consisted in squeezing the sap of ground tobacco plants through cheesecloth.

The sap was adjusted

to a pH of 8.0 and centrifuged to remove chloroplasts, cell fragments and starch granules.

The pH was then adjusted

to 5.2 with acetic acid and the sap again centrifuged.. Five gm. of sodium sulfate were added for each 100 ml. of sap and the precipitate discarded.

Ten gm* of sodium

sulfate were added and the sap centrifuged after holding at room temperature for 30 minutes.

The precipitate which

formed contained the active enzyme and was suspended in water and its pH adjusted to 7*2 with sodium hydroxide. The addition of 5 ©a. of ammonium sulfate per 100 ml. of sap gave a precipitate that was discarded; the addition of 15 gm. per 100 ml. sap gave an active enzyme preparation which retained its activity for several days when held at 5°C.t Clagett et al. state that "no success was attained when very

21 young tobacco plants were used.11

After following their

procedure, we are in complete agreement with that statement. 3.

Details of Methods Used to Determine Oxidative

Enzymes.

The quantitative determination of activity of the

oxidative enzymes studied was mainly accomplished by measur­ ing the volume of oxygen consumed during the reaction in the Warburg instrument.

In a few cases, it was desirable to

by-pass the terminal oxidase of the system in order to study the dehydrogenase alone. hydrogen acceptor.

This was done by uaing TTC as the

The detailed procedure of these two

methods is given below. The manometers on the Warburg instrument were filled with Brodie*s solution, prepared and standardized according to directions given by Umbreit, Burris and Stauffer (62).

(The procedures, techniques and calculations

used in this study and dealing with the Warburg instrument were obtained from this source.)

The constant temperature

bath was maintained at a temperature of 29*6 C.± 0.05. The shaking mechanism was adjusted to give 1 cm. shakes at the rate of 120 per minute. The reaction flasks used were the conventional type, holding about IS mis., fitted with a center well for holding,alkali and a side arm.

These flasks were cleaned

by placing in gasoline for 30 minutes, dried, and then placed over-night in dichrornate-sulfuric acid cleaning solu­ tion.

They were rinsed at least 6 times in running distilled

22 water before using.

The volume of reactants in each flask

was held close to 3*0 ml. and in most cases, consisted of 1.0 ml. of tobacco tissue homogenate, 1.0 ml. of phosphate buffer, 0.3 nil. of substrate and 0.7 ml. water to which various cofactors, ions, or inhibitors could be added. In making a run, the center well of the flask was greased and the substrate placed in the side arm which was then fitted with the side arm plug lubricated with lanolin. The buffer and reactants were placed in the flask, the tobacco being homogenized and added to the flask at the last moment.

The center wells were filled with 0.3 ml. of

10 per cent potassium hydroxide solution and a 1 cm. square piece of filter paper added to increase the surface area of the alkali.

The reaction flasks were placed in

the constant temperature bath of the Warburg instrument and shaken to permit their coming to the temperature of the bath. After 15 minutes, the shaking was stopped and the manometers, with both sides open to the air, were adjusted to the 2£o mm. mark.

The flasks were tipped and the

substrate allowed to flow from the side arm into the center chamber to initiate the reaction.

The manometers were

closed, the shaking begun again, and the reaction followed by resetting the closed arm of the manometer to the 250 mm. mark and recording the height of Brodie*s solution in the open end. As the fall of Brodie's solution in the open end

23 of the manometer seldom exceeded three-quarters of a mm. per minute, no attempt was made to obtain manometer readings to the exact second.

The reaction periods were

timed with a stop watch and when the desired time period ended, the first manometer reading was taken, followed by the other readings as soon as possible.

At the completion

of each run, the pH of the flask's contents was determined by a Beckman model H glass electrode pH meter. In cases where TTC was used to replace the terminal oxidase, the anaerobic tests were run in evacuated Thunberg tubes and the aerobic tests in ordinary test tubes plugged with cotton.

The contents of these tubes consisted of 1

ml. each of buffer and tobacco homogenate, 0.3 ml* of substrate,

and 0.5 ml. of 0.1 per cent TTC.

The remaining

0.2 ml. necessary to bring the final volume to the desired 3.0 ml. was used to dissolve any cofactors, ions or inhibit­ ors, or added as distilled water.

The tubes were timed,

starting with the addition of the substrate, and edning with the appearance of a positive formazan color. The tubes were placed in a bath at 37°C. and kept dark during the entire run.

The formazan produced was

estimated by visual examination and rated on a simple comparative scale between tubes in that run.

By this

method, no comparison could be made between runs, nor was it necessary.

The green color of the chlorophyll and the

turbidity of the tissue prevented a more accurate reading. The pH

of the tubes was checked at the end of each run as

24 was done in the case of tests made with the Warburg instrument.

By these two methods the data presented in

the following pages were obtained. B.

Measurement of Plant Enzymes. In preliminary studies, where tests were run on

the enzymes of sterile tobacco leaves, it was noted that, of all the substrates used (malate, lactate,

succinate,

glutamate, formate, glucose and alcohol), sodium lactate was outstanding in its oxygen consumption.

This was not

expected as lactate is not present in the Krebs cycle

(25)

nor thought to be one of the important compounds photo­ synthesized by plants

(13).

The comparatively large volume

of oxygen consumed, and the unexplained presence of the enzyme, lead to a more complete investigation of the enzyme which utilized lactate as its substrate. 1•

The Lactic Enzyme. a.

Cofactors Necessary for Activation.

shown in Section II that most of the dehydrogenases re­ quired cofactors to aid them in oxidizing their substrates. A test was made to determine the effect of cofactors upon the enzyme capable of oxidizing sodium lactate. test was run in Warburg flasks

This

the contents being 1.0 ml.

each of 0.1 M. phosphate buffer at pH 7*4 and tobacco leaf homogenate, and 0*3 ml. of 0.5 M. sodium lactate.

The co­

factors were dissolved in the remaining 0.7 ml. of distilled water necessary to bring the final volume to 3*0 ml.,

It was

25

Potassiura hydroxide was added to the center well to absorb any carbon dioxide produced.

The tests were run for 120

minutes in the dark and the pH was at the end of the run.

in all the flasks

The nature of the added cofactors

and their effect upon the system is given in Table I*

TABLE I THE EFFECT OF ADDED COFACTORS UPON TIIE LACTIC ENZYME

No.

Cofactors Added Cytochrome C DPN mg. mg.

microliters

Run $b 1;3 0.2

U6 b7 51

55

— 0.2 0.2 0.2 0.2 0.2 (No tissue added) 0.2 0.2 (No substrate added)

119 109 116 109 Q 9

Run bS 14-6 1*7 51

0.2

..

0.2

0.2 0.2

106 98 ioU 99

From Table I it may be seen that the lactic enzyme is not dependent upon added DPN or Cytochrome C for its activity.

As seen in later studies with malic dehydrogenase,

tobacco plant tissue does not have sufficient DPN or flavoprotein to activate that system.

Therefore it may be con­

cluded that the lactic enzyme does not need DPN, flavoproteln or cytochrome C for its activity.

26 b.

pH Studies.

As two lactic dehydrogenases are

reported in the literature (54) > the yeast lactic dehydro­ genase with an optimum pH of £.2 and the animal lactic dehydrogenase with its optimum pH at 9*3* a study was made to determine the effect of pH upon the activity of tobacco leaf lactic enzyme. The tobacco leaf was homogenized in 0*01 M. phos­ phate buffer and 1 ml. added to the Warburg flask.

One ml*

of 0.1 M. phosphate buffers at various pHs, 0.3 nil. of 0.5 M. sodium lactate and 0.7 nil. of distilled water completed the flask contents.

The run was made for 120 minutes, carbon

dioxide absorbed by potassium hydroxide in the center well, and the pHs read at the end of each run.

The volumes of

oxygen taken up at various pH levels are shown in Table II.

TABLE II THE EFFECT OF pH UPON THE ACTIVITY OF THE LACTIC ENZYME Run 86_______ Upt ake pH 02 mm. 3 5.90

40.2

6.00

27.5 34.§ 54.8 70.6 90.0 43.5

6.15 6.65

7.10 7.50 9.10

pH

Run 86______ Uptake 02 mm. 3

5.55 5.8o 5.90

6.18 6.65

7.20

9.5 34.2 32.9 36.3 51.4 58.5 84.5

PH

Run 90 Uptake 02 5 m m .J

7.1 7.3 7.6 7.8

8.0 10.2

63

62

78 79 99 7

The data from Table II were evaluated and a composite pH curve was drawn which is given in Figure II.

This

> h £ HO