A function of iron in the metabolism of Clostridium perfringens

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A FUNCTION OF IRON IN THE METABOLISM OF CLOSTRIDIUM PERFRINGENS

BY RAYMOND C. BARD Bachelor cf S cien ce, 1933 College of th e C ity of New York N kster cf A rts , 1947 Indiana U n iv e rsity

Submitted to th e F aculty of th e Graduate School in p a r t i a l f u lf illm e n t of the requirem ents f o r th e degree of Doctor of Philosophy in th e Department of B acterio lo g y , Indiana U n iv e rsity , September, 1949

ProQuest Number: 10295203

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▲GKBDBXSDGUBHTS The au th o r tak e s t h i s o p p o rtu n ity t o express h is g r a titu d e to P ro fe sso r I* C. Gunsalus fo r th e guidance and a s s is ta n c e so r e a d ily extended by him.

His c o n stan t i n t e r e s t and v aluable c ritic is m s con­

tr ib u te d g r e a tly to th e d ire c tio n and execution of t h i s work* In a d d itio n , th e a u th o r expresses h is a p p re c ia tio n fo r the award of an A ll-U n iv e rsity Fellow ship, Indiana U n iv e rsity , fo r the p erio d 1947-49*

VITA

Raymond Camille Bard was born on August Connecticut*

2 6

, 1918 in Hew B r ita in ,

He a tte n d e d th e p u b lic schools of the C ity o f New York,

g rad u atin g from James Monroe High School in 1934*

He rec eiv e d th e

degree o f Bachelor of Science i n 1938 from th e College o f the C ity of New York and p ra c tic e d m edical technology in se v e ra l h o s p ita ls from 1938 to 1942.

In March, 1942 he was ordered t o extended a c tiv e duty

in th e Amy of th e United S ta te s , serving in th e In fa n try and S ignal Corps.

He was re lie v e d from d uty i n January, 1946 and began a tte n d ­

ance a t Indiana U n iv e rsity as a graduate stu d e n t i n b a c te rio lo g y in February, 1946. ology i n 1947*

He receiv ed th e degree o f ^fester o f A rts in B a c te ri­ During t h i s p e rio d , he h e ld an E li L illy and Company

Research Fellow ship i n B acterio lo g y , and subsequently was a h older of an A ll-U n iv e rsity Fellow ship from Indiana U n iv e rsity from 1947 to 1949* In February, 1949 he was appointed In s tru c to r in B acterio lo g y .

He i s

a member of the S o c iety o f American B a c te rio lo g is ts , American Chemical S o ciety and Sigma X i.

iii

To RITA

iv

TABLE OF CONTENTS Page INTRODUCTION

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

1

METHODS , * . . ............................................................................................................

7

Bacte r i o l o g i c a l ........................................................................... . . •

7

Chemical

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

9

M a n o m s tr ic .................................................................................. * ♦ . •

13

EXPERIMENTAL RESULTS.................................................................................................... 14 PART I .

THE EXISTENCE OF A METALLO-ALDOIASB......................... 14

G lycolysis

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

15

Rates of g ly c o ly s is by Fe-f and Fe- c e l l s .............15 I n h ib itio n by m etal-eomplexing ag en ts

. ............. 15 .

A l d o l a s e ...................................................................................... P ro p e rtie s •

17

Requirement f o r m etals and reducing agent PART I I .

. . . . * •

20

FURTHER EVIDENCE FOR THE EXISTENCE OF THE IvEYERHQF-EMBDEN SCHEME..............................................24

Isom erase . • ....................

24

Glyceraldehyde Phosphate Dehydrogenase

. . . . . . . . . .

E thanol Dehydrogenase . . . . . . . . . . . PART I I I .

17

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

25 26

STUDIES OF HYDROGEN METABOLISM...................................................27

Hydrogenase . . . . . . . . . . . . . . . . . . . . . . . .

27

L a c tic D e h y d ro g e n a s e ....................

29

Pyruvate D iss im ila tio n

32

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

v

Page RESPIRATION . ......................................................................

PART IV.

Glucose o x id atio n

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

34 34

Phosphorus exchange

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

35

E ffe c t o f in h ib ito r s . .

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

36

G lycerol o x i d a t i o n ........................ DISCUSSION.............................................

41 42

SUMMARY................................................................................................................................. 46 REFERENCES

........................................................................................................ 48

vi

INTRODUCTION Iro n is known to p la y an im portant ro le i n th e m etabolism o f members of th e genus C lo strid iu m .

Numerous s tu d ie s ha ye d e a lt w ith the

im portance o f iro n f o r growth (H astings and McCoy, 1932; L erner and P ic k e tt, 1945; Pappenheimer and Shaskan, 1944)* fo r v itam in p roduction (H ick^r, 1945* le v ito n , 1946; Rodgers, Henika and Hansen, 1946), and fo r to x in p ro d u ctio n (Bernheimer, 1944; Feeney, M ueller and M ille r, 1943 S c o tt, 1926; Tamura, e t a l . . 1941)*

These r e s u l ts e s ta b lis h the e ssen ­

t i a l i t y o f ir o n to th e s e organisms but do not in d ic a te the fu n c tio n o f ir o n in t h e i r m etabolic p ro c e sse s. A d i r e c t approach to the fu n c tio n of ir o n in the m etabolism of C lo strid iu m p e rfrin g e n s was made by Pappenheimer and Shaskan (1944) who found t h a t c e l ls harv ested frcm a medium co n tain in g l e s s th an 0 .1 mg ir o n per l i t e r c a rrie d out a hom olactic ferm en tatio n of g lu c o s e .

C ells

from a medium containing 0 .6 mg or more iro n per l i t e r y ie ld e d the u s u a l products o f g ly c o ly sis fo r t h i s organism, namely, hydrogen, carbon d io x id e, e th a n o l, and a c e tic and b u ty ric a c id s with only sm all amounts of l a c t i c a c id . of ir o n .

The organism did not grow in media deprived com pletely

A ddition o f

'- d i p y i id y l , a d iv a le n t m e ta l-tra p p in g a g en t,

in h ib ite d l a c t i c a c id production by ir o n - d e f ic ie n t c e l l s , gas p roduction by i r o n - s u f f ic i e n t c e l l s , and growth in d efin ed media.

From these ob­

s e rv a tio n s , the a u th o rs con eluded " th a t two sep arate mechanisms e x is t fo r the breakdown o f g lu c o se .

One o f th ese would re q u ire the presence

of a considerable amount of an iro n -c o n ta in in g e n ^ m e ."

I t was sug­

g ested t h a t an iro n -c o n ta in in g enzyme c a ta ly z e d the breakdown of an

in term ed iate compound of glypo l y s i s , p o ss ib ly pyruvic a c id o r a d e riv a ­ t iv e th e r e o f , to y ie ld hydrogen, carbon dio x id e, e t c . Hanson and Rodgers (1946) re p o rte d a sim ila r e f f e c t of iro n on the products of glucose ferm en tatio n of C lostridium acetobutvlic»m t Ferm entation an aly ses were perform ed on c u ltu r e s grown 38 hours a t 37 C in a re n n e t whey medium c o n tain in g about 0 .2 mg iro n per l i t e r supple­ mented w ith p-am inobenzoic a c id , zinc s u l f a te , manganese s u l f a t e , c a l­ cium c arb o n ate, tric a lc iu m phosphate and v arying amounts of fe rro u s s u lfa te • As th e iro n co n ce n tra tio n was in c re a se d from 0*4 rag to 4 mg iro n per l i t e r the y ie ld s of l a c t a t e , form ate and e th a n o l, per u n it of sugar ferm ented, dim inished whereas th e y ie ld s of hydrogen, carbon d io x id e , a c e to n e , a c e ty lm e th y lc a rb in o l, a c e ta te and b u ty ra te in c re a s e d . Butanol p ro d u ctio n was not a ffe c te d by iro n c o n c e n tra tio n .

The normal

c u ltu re was passed through 50 tr a n s f e r s a t 24 hour in te r v a ls in the same medium co n tain in g 1 mg iro n per l i t e r to y ie ld an a tte n u a te d s t r a i n which ferm ented glucose in com pletely w ith l a c t a t e as the p r in c ip a l p ro d u ct. L erner and P ic k e tt (1945) re p o rte d g r e a te r g ly c o ly tic a c t i v i t y of C lo stridium t e t a n i suspensions h arv ested from media co n ta in in g in c re a se d q u a n titie s o f ir o n .

The s t r a i n used c a r r ie d out an e th a n o l-

carbon d io xide fe rm e n ta tio n with the form ation of sm all amounts of b u ta n o l and tr a c e s of v o la tile and l a c t i c a c id s ; only 74 per cent o f tfce t h e o r e tic a l carbcn recovery was ob tain ed . b a la n c e s, hydrogen production was re p o rte d .

In some ferm en tatio n C e lls h a rv e ste d from

medium co ntaining no added iron showed n e g lig ib le g ly c o ly tic a c t i v i t y , as Jieasured by carbon dioxide pro d u ctio n from g lu c o se , whereas c e l ls h arv e ste d from media with in cre a se d iro n concentrations e x h ib ite d

3 in c re a s in g r a te s o f carbon dioxide evolution*

The

va^ues reached

a maximum o f about 10 w ith iro n c o n ce n tra tio n s o f 1*0 or 10 mg iro n p e r l i t e r o f growth medium.

No in d ic a tio n was g iven of th e type of fermen­

t a t i o n c a rrie d out i n th e absence of added iro n and i t may be concluded t h a t only th e r a t e of g ly c o ly sis i s a ffe c te d by iro n sin c e ir o n - d e f ic ie n t c e lls had a

= 1*

Other attem p ts have been made to determ ine the fu n c tio n of iro n in g ly c o ly sis by the use of m etal-com plexing agents such as carbon monoxide, cyanide, * , * * -d ip y rid y l, e tc .

Using c e l l suspensions of

C lostridium butyricum which c a rry out a ty p ic a l b u ty ric a c id ferm enta­ t i o n , Kempner (1933)* Kempner and Kubowitz (1933)^ and Kubowitz (1934)

v

found t h a t 0*01 M cyanide in h ib ite d gas prod u ctio n by 93 per c e n t; glucose breakdown was in h ib ite d 56 per cent and l a c t i c acid was th e m ajor en d -product•

Lower c o n cen tratio n s of cyanide in h ib ite d gas produc­

tio n somewhat and th e s h i f t toward a hom olactic ferm en tatio n was c o rre s­ pondingly le s s marksd*

S im ila r in h ib itio n of gas production was obtained

w ith carbon monoxide; th e in h ib itio n by carbcn men oxide was rev e rse d by exposure to in te n se lig h t*

Accordir?g to Y/arburg (1943), in h ib itio n by

cyanide or carbon monoxide in d ic a te s the e x iste n c e of heme-type iro n systems*

However, c o n cen tratio n s of cyanide lower th a n 0*001 M i n h ib i t

such systems s tro n g ly , and th u s use of 0*01 M cyanide r a is e s the p o s s i­ b i l i t y of e f f e c ts o th e r than in h ib itio n of a heme-type c a t a l y s t . In s tu d ie s of C., acetobutvlicum , Simon (1947) found t h a t growth \ in a com mash medium was in h ib ite d by bubbling a steam of carbon monoxide through the nash*

S im ilar tre a tm e n t of gLycolyzing c e l l su s­

pensions re s u lte d in a s h i f t from an a ceto n e-b u tan o l type ferm en tatio n to a hom olactic ferm entation*

4 Schlayer (1935) rep o rte d the in h ib itio n o f gas p ro d u ctio n by .£• t e t a n i and the ^gas gangrene b a c i ll u s 11 (presumably CJ. p e rfrin g e n s ) w ith carbon monoxide.

From th e d a ta p resen te d , i t appears th a t th e gases

produced were evolved d uring endogenous m etabolism sin c e no mention was made of the su b stra te used, an im pression supported by th e low le v e ls of g ly c o ly tic a c t i v i t y , L em er and P ic k e tt (1945) did not find a carbon monoxide e f f e c t v

on g lu co se ferm en tatio n by C. t e t a n i whereas 0 ,0 2 M cyanide in h ib ite d the ra te of carbcn d ioxide p ro d u ctio n by 5& per cent*

Decreased con­

c e n tra tio n s of cyanide r e s u lte d in correspondingly l e s s in h ib itio n o f g ly c o ly s is .

The d iffe re n c e in r e s u l ts obtained w ith carbon monoxide

by th e s e workers and by Schlayer (1935) may be a t t r i b u t e d to s t r a i n v a ria tio n s or to th e f a c t th a t the measurements o f th e l a t t e r worker a p p a re n tly p e rta in e d to endogenous m etabolism , • According to Stephenson (1949) 9 Bacon re p o rte d th a t glucose ferm en tatio n by C. p e rfrin g en s in an atmosphere of carbon monoxide r e ­ s u lte d in a non-gaseous ferm e n ta tio n with l a c t i c a c id accounting f o r 70 p e r cent of the glucose used.

This fin d in g with C, p e rfrin g e n s i s

in agreement with the observations of Schlayer (1935)* The in h ib itio n o f g ly co ly sis of C, p e rfrin g e n s by has been d escrib ed above,

1- d ip y rid y l

Lerner and P ic k e tt (1945) re p o rte d a s im ila r

o b se rv atio n fo r the g ly c o ly sis o f C, t e t a n i . From th ese data i t appears t h a t iro n fu n ctio n s in more than one p o s itio n in the g ly c o ly tic scheme of c l o s t i i d i a .

Not only a re th e e x a c t

s i t e s unknown but l i t t l e inform ation i s a v a ila b le reg ard in g the glyco­ l y t i c system of th e s e organism s.

On th e b a sis of analogy, i t has been

t a c i t l y assumed t l a t a system s im ila r to the Meyerhof-Embden scheme

5 e x is ts i n th ese organisms (P re s c o tt and Dunn, 1940; Stephenson, 1949),



b u t the a c tu a l d a ta are n o t s p e c if ic nor s u f f i c i e n tl y d e ta ile d to w arran t such an assum ption. The a v a ila b le in fo rm atio n concerning g ly c o ly tic mechanisms in c l o s t r i d i a i s contained i n a few r e p o r ts .

Using Cl acetobutylicpra

P e tt and Wynne (1932) re p o rte d th a t ferm en tatio n o f fru c to se diphosphate y ie ld e d m ethylglyoxal and tra c e s of p y ru v ate .

L erner and P ic k e tt (1945)

re p o rte d complete in h ib itio n of the g ly c o ly sis of C. t e t a n i by 0.0001 M m onoiodoaeetate, suggesting the e x iste n ce o f trio sep h o sp h ate dehydrogenase. I t was a ls o foind th a t 0 .0 1 M potassium a r s e n ite in h ib ite d g ly c o ly sis com pletely, suggesting the form ation of pyruvate as an in term ed iate in glucose breakdown.

However, Stone and Werkman (1937) were unable to

id e n tif y phosphoglyceric a c id a s a product of th e breakdown of fru c to se diphosphate by C. butylicum . C. h isto lv tic u m and G. sporogenes; in s te a d , an u n id e n tifie d yellow product was o b tain ed , which th e se workers re p o rte d was a ls o found in y e a st ferm e n ta tio n .

The accum ulation of th e yellow

product d uring y e a st g ly c o ly s is , which has been amply dem onstrated to proceed according to the Meyerhof-Embden scheme, does n o t c o n tra d ic t th e p o s s i b i l i t y t h a t such a g ly c o ly tic system occurs in c l o s t r i d i a . Thus, alth o u g h th e d ata a re lim ite d , the in d ic a tio n s a re th a t a g ly c o ly tic scheme s im ila r , a t l e a s t in p a r t, to th e Meyerhof-Embden scheme e x is ts in c l o s t r i d i a .

The r e a l d i f f i c u l t y i n deciding the is s u e

a r i s e s from the p a u c ity o f data concerning c l o s t r i d i a l g ly c o ly s is . The p resen t study concerns th e iro n system which appeared to be o b lig a to ry f o r any type o f glucose metabolism by C. p e rfrin g e n s .

The

re p o rt of Pappenheimer and Shaskan (1944) t h a t (K$ n

o

o

A

5

c 7)

m

o

o CP >

jo “n pi +

0 ■n pi 1

Z o p

o

(/> CD

-
z o

? o

n

r r MINUTES

co

o

c 2) m

(P CD

16 f o r th e form ation o f an enzyme(s) causing gaseous ferm en tatio n o r w hether ir o n is involved in the fu n c tio n of an enzyme(s) causing gas p ro d u c tio n , perhaps a s an a c tiv a to r o r i n a p r o s th e tic group* Beyond the fu n c tio n of iro n in gas p ro d u ctio n , the complete in h ib itio n of ferm en tatio n —even of l a c t i c a c id production (see e x p e ri­ ments of Pappenheimer and Sbaskan, 1944), in d ic a te d a second fu n c tio n of ir o n ; i * e . , in e it h e r enzyme form ation or fu n c tio n .

The s i t e o f

t h i s a c tio n has been s tu d ie d i n th e follow ing experim ents*

For t h i s

stu d y , s o - c a lle d ir o n - d e f ic ie n t c e lls which la c k the gas p ro d u ctio n mechanism were used* Reagents which form complexes w ith fe rro u s ir o n such as 0C9 0( * -d ip y rid y l, o-phenanth ro lin e and sodium pyrophosphate were found to i n h i b i t the g ly c o ly sis of iro n d e f ic ie n t c e l l s , in t h i s case a hom olactic ferm en tatio n (ta b le 3) * D ipyridyl and phenanthroline i n h ib i t io n of g ly c o ly sis was r e lie v e d by F e ^ o r Co’*”*’ but not by Fe'*”*"*', Zn4+, Cu44, Ni44, Mg’*”*’, nor Mi"*"*.

R e lie f of pyrophosphate in h ib itio n

could not be determ ined m anom etrically in the b icarb o n ate b u ffe r because a d d itio n of m etal ions to pyrophosphate-bicarbonate s o lu tio n s caused l i b e r a t i o n of carbon d io x id e, probably due to h y d ro ly sis of the pyrophospha t e « In the absence of in h ib i t o r s , 0*001M Fe444, Zn44 and Cu’*"* in ­ h ib ite d g ly c o ly sis com pletely; Ni44 in h ib ite d about 60 per c e n t; Mg44 and Mi44 caused no s ig n i f ic a n t changes in the g ly c o ly tic r a t e s . Cysteine (0.01M to 0.02M) stim u la te d the g ly c o ly tic r a t e s l i g h t l y . These r e s u lts in d ic a te d a fu n c tio n of fe rro u s i r o n , and a p p a re n tly c o b a lt, in th e g ly c o ly tic scheme.

The fin d in g s of la rb u rg and C h ris tia n

Table 3 In h ib itio n o f g ly c o ly s is and r e s to r a tio n

%Hyc o ly s is No a d d itio n s

With 0.001 M Fe**

With 0.001 M Co4*

115-13$

145

130

C e lls + 0.001 M d ip y rid y l

0

90

135

C e lls + 0*001 M phenanthroline

3

140

165





C e lls

C e lls + 0 .0 1 M pyrophosphate

45

^ G ly co ly sis = pL CC> 2 formed per mg d ry wt per hour Per Warburg fla s k s NaHC03 (0.0168 M) C ell su sp en sio n (2 t o 3

2 .0 ml d ry wt Fe- c e l l s )

0.3

Glucose (0.03 M), in sidearm

0 .2

I n h ib ito r s or m etal s o lu tio n s

0.5

T o ta l volume

3 *0

Atmosphere:

30 per cent COg, 70 per cent

pH 6 .5

17 (1943) t h a t y e a s t a ld o la s e is a m e ta llo -p ro te in suggested t h a t a ld o la s e might be the s i t e of iro n a c tio n . I n h ib itio n o f a ld o la s e .

C e ll-f re e e x tr a c ts were prepared and t e s te d

f o r a ld o la s e a c t i v i t y as d e scrib e d under Methods in the absence and presence of fe rro u s iro n i n h ib i t o r s .

The r e s u l t s (ta b le 4) in d ic a te

t h a t d ip y rid y l, phenanthroline and pyrophosphate in h ib ite d markedly a ld o la s e a c t i v i t y .

The a d d itio n of Fe* 4 and Co4 4 to th e in h ib ite d

enzyme re s to re d a c t i v i t y .

A ddition of Fe4 4 4 , 2n44, Cu44, lig4 4 and Mi4 4

did n o t re lie v e d ip y r id y l in h ib itio n o f a ld o la se a c t i v i t y .

In the

case of pyrophosphate, r e l i e f w ith Fe++ or Co++ was not s a t i s f a c t o r y . Warburg and C h ris tia n (1943) found th a t r e l i e f of p y ro p h o sp h a te-in h ib ited y e a s t a ld o la s e is a tim e r e a c tio n : i n h i b i t o r , enzyme a c t i v i t y follow ed.

i f th e m etal was added before th e This phenomenon i s probably due

to th e i r r e v e r s ib le binding of th e enzyme p ro te in by pyrophosphate, a re a c tio n ta k in g about fiv e m inutes.

Thus, i f the pyrophosphate was

added f i r s t and a f t e r fiv e m inutes the m etal was added, th e n r e l i e f of i n h ib i t io n was not adequate f o r enzym atic a c t i v i t y .

The l a t t e r procedure

was used in the in h ib itio n experim ents. From ta b le 4 i t may be noted t h a t a d d itio n of e it h e r Fe4 4 o r Co++ to th e enzyme e x tr a c t stim u la te d a ld o la se a c t i v i t y about f o u r - f o ld , in d ic a tin g a p a r t i a l r e s o lu tio n of a ld o la s e w ith re s p e c t to m e ta l. n e ta l d e fic ie n c y allow s a stu d y of the s p e c i f i c i t y of v arious m etals f o r a c t iv a t io n . Proper t i e s of a ld o la s e .

The c e l l - f r e e enzyme e x tr a c t was d ilu te d

a p p ro p ria te ly w ith in the range of r e a c tio n c o lo r and a l i n e a r i t y of

The

Table 4 I n h ib itio n and r e s to r a tio n of a ld o la s e a c t i v i t y

A lk a li- la b ile phosphorus formed*

Enzyme e x tr a c t *

+ G.QQ5M d ip y rid y l

n + 0.005M p h enanthroline n + 0.01M pyrophosphate

No a d d itio n s

With 0.005M Fe4 4

jig

PS

With 0.005M Co4 4 PS 231

69

2 3 8

1 2

103

72

1 2 0

73

6

27

4

1 2

* A lk a li- la b ile phosphorus formed per h r per mg p ro te in

18 response was obtained w ith enzyme c o n ce n tra tio n (fig u re

4

)*

The time, course of a c t i v i t y was found to give a s t r a ig h t l in e In g e n e ra l, a ssa y s were run

from 15 m inutes to one hour (fig u re 5 ) .

15 m inutes and the r a te per hour c a lc u la te d . t i o n and r a te are lin e a r (fig u re s

4

and

5

Since both enzyme d ilu ­

)> the u n i ts of a c t i v i t y :

jug p e r hour per mg p r o te in , may be c a lc u la te d . The r e la tio n s h ip between s u b s tra te c o n c e n tra tio n and t r i o s e phosphate fo rm atio n is shown i n fig u re

6

.

The M ichaelis-M enten c o n stan t

i s approxim ately 0.001 M f o r the c l o s t r i d i a l enzyme.

This i s i n good

agreement w ith the fig u re re p o rte d by H e rb ert, et, a l . (1940) f o r r a b b it a ld o la s e and S ib le y and Lehninger (1949) Tor a ld o la s e -c o n ta in in g e x tr a c ts whose o r ig in were not d e sc rib e d . The optimum pH f a r a ld o la s e a c t i v i t y i s about 7*5 (fig u re 7)> w ith a sharp slope on th e a c id s id e of pH 7*5 b u t w ith a grad u al slo p e on th e a lk a lin e s id e .

This pH i s lower th an the optimum of 8.5 t o 9*0

re p o rte d by S ib ley and Lehninger (1949) who used phosphate in the range up to 7*4* tris(hydroxym ethyl)am inom ethane from pH 7*4 to 10.5 and b o rate a t pH

1 1

.0 .

H erb ert, e t a l . (1940) re p o rte d an optimum pH

about 9 b u t d id not in d ic a te the b u ffe r u se d . optimum pH a t

8 .5

Stumpf (1948) found an

f o r pea a ld o la s e , using veronal b u f f e r from pH

to 10.5 and a c e ta te b u ffe r a t pH 5*4*

6

Bounce and Beyer (1948) re p o rte d

an optimum pH cf 6.7 w ith c r y s ta llin e rabD it a ld o la s e ; no statem en t of the b u ffe r used was made. a s fo llo w s:

The pH optimum as re p o rte d here was determ ined

the HDP and hydrazine so lu tio n s were a d ju s te d to th e de­

s ir e d pH values and the pH of th e re a c tio n m ixtures determ ined w ith a Beclanan pH m eter during the course o f th e re a c tio n i n d u p lic a te r e a c tio n

[uG/HR]

FIGURE 4 ALDOI^ASE A the enzyme l o s t a c t i v i t y on stan d in g a t 37 C with o r w ithout added m etals; a ls o , a d d itio n o f both Fe’*"*' and Co** d id not a c tiv a te the enzyme to a g re a te r e x te n t th a n e i t h e r a lo re .

In f a c t , Fe** and Co** ( a t 0.Q05M and Q.QQQ5M re s p e c tiv e ly )

a c tiv a te d th e enzyve le s s th a n Fe** alone and approxim ated th e value

Table 5 B fTect o f Fe** and Co** com binations on a ld o la s e a c t i v i t y

A lk a li L abile Phosphorus (ug p e r h r p e r mg p ro te in ) Combinations

Zero Time

30 min/37C

60 min/37C

Enzyme e x tr a c t

115

23

13

Enzyme + O.OupM Fe* 4

7 2 8

4 6 2

168

Enzyme + 0.0005M Co**

550

188

1 7 0

Enzyme + 0.005M Fe** + 0.QQ05M Co**

5 0 6

307

99

21 f o r Co

a lone •

The in a c tiv a tio n of the enzyme during in c u b a tio n a t

37 C appears to be due to oxidation* and t h i s is in d ic a te d by th e ex­ perim en ts d e scrib e d below. Iynen and H ofton-W albeck (194BJ re p o rte d t h a t the a ld o la s e a c t i v i t y of c e l l —fre e e x tr a c ts of F e n ic illiu m n o t a t e mycelium were com pletely in a c tiv a te d by in c u b a tio n f o r t io n was o lie r e d .

4

hours a t 18 Cj no explana­

The s i t u a t i o n w ith jP. not a turn a ld o la s e appears to be

analogous to t h a t of C. p e rfrin g e n s a ld o la s e . E f fe c t o f reducing a g e n ts on a ld o la s e a c t i v i t y .

Since nany enzymes*

e s p e c ia lly m etal-co n tain in g systems* a re a c tiv a te d or ^protected** by reducing ag en ts s e v e ra l compounds were t e s te d a s a c tiv a to r s of the c e l l f r e e a ld o la s e enzyme.

Weil* K ocholaty and Smith (1939) have re p o rte d

th e p ro te in a s e of J3. p e rfrin g e n s to be stim u la te d by c y ste in e o r Fe** and t h a t com bination of cy stein e and Fe** extended the p ro te in a s e a c t i v i t y to in clu d e the breakdown o f a d d itio n a l p r o te in s .

Ascorbate*

c y stein e* g lu ta th io n e and th io g ly c o lla te in c o n c e n tra tio n s of 1 0

” % were te s te d a s a c t i v a t o r s .

1 0

* % to

The r e s u l t s in fig u re 9 in d ic a te t h a t

the re a g e n ts were p a r t i a l l y e ffe c tiv e as a ld o la s e a c tiv a to rs * th e de­ g ree of a c tiv a tio n depending on the c o n c e n tra tio n .

Since th e r a tu r e

o f th e reducing a g e n t d id not seem to be s p e c if ic , cysteine* a t 0.001M* was used f o r subsequent experim ents. M etal a c t iv a t io n of a ld o la se i n oresence of c y s te in e . ta b le

6

The r e s u l t s in

in d ic a te t h a t ( l ) cy stein e s ta b iliz e s enzyme a c t i v i t y during

in c u b a tio n a t 37 C* (2) the a d d itio n o f Fe+* i n th e presence o f c y s te ire a c tiv a te d a ld o la s e to a g r e a te r degree than e ith e r reag en t

FIGURE 0

PROTEIN]

250 —

[~G/HR/MG

ALKALI LABILE

PHOSPHORUS

EFFECT OF REDUCING AGENTS ON ALDOLASE ACTIVITY

200

•CYSTEINE

GLUTATHIONE

150 W/O AOONS A ASCORBATE

100 ° THIOGLYCOLLATE

I0"5

I0“4 I0“3 I0"2 M REDUCING AGENT

Table 6 E f fe c t of combinations o f F e ^ t C o ^ and cy stein e on a ld o la s e a c t i v i t y

A lk a li L abile Phosphorus (pg p e r h r per mg p ro te in ) Zero Time

30 m in/370

Enzyme e x tr a c t

115

23

13

Enzyme + Q.005M Fe**

728

462

168

" + 0.0Q05M Co++

550

188

170

" + 0.005K Fe++ + 0.0005M So"1^

50b

307

99

213

219

213

1225

1045

1030

+ 0.001M e y stein e

60 min/37C

» 4

n + 0*005M Fe**

« 4

» 4 0.0005M Co**

506

417

422

«

« 4 0.005M Fe** + O.OQQ5M Co**

980

815

670

4

a lo n e , (3 ) w ith Co** a s the a c t i v a t o r , eysteir*s did not in c re a s e th e degree of s tim u la tio n but d id prevent lo s s of a c t i v i t y during incuba­ t i o n , ( 4 ) Fe** i s a more e f f e c tiv e a c t iv a t o r of a ld o la s e th an is Co**, a s a lre a d y shown i n f ig u r e 8 and t a b le 5, The in c re a s e d a c tiv a tio n by Fe** in th e presence of c y s te in e , a n o n -s p e c ific reducing a g e n t, in d ic a te s t h a t lo s s o f enzyme a c t i v i t y on stan d in g i s due to o x id a tio n .

Ifeintenance of the high le v e l o f a c t i v i t y

under th e se c o n d itio n s suggests the e x iste n c e of an a c tiv e s i t e ( s ) on th e enzyme which fu n c tio n s o p tim a lly in th e reduced s t a t e , p o ss ib ly s u lfh y d ry l groups re q u ire d fo r binding Fe**. To determ ine th e optimum m etal c o n c e n tra tio n fo r a ld o la s e a c t i v i t y , the e f f e c t of Fe

j*

and Co

was r e te s te d i n the presence o f c y stein e*

I f th e lo s s of a ld o la s e a c t i v i t y cn stan d in g were due to o x id a tio n , a p a r t i a l l y in a c tiv e p re p a ra tio n , a s fo r example in experim ent 8

2

, fig u re

, sh o u ld be g r e a tly stim u la te d by the a d d itio n of m e ta llic ions in the

p resence of c y s te in e . 13 days a t 3 5

4

This e f f e c t was observed w ith an e x tr a c t sto re d

C as shown i n ta b le

6

.

This e x tr a c t was r e - t e s t e d a f t e r

days* sto ra g e a t 4 C w ith the r e s u l ts shown in fig u re 10. As compared to the 13-day-old e x tr a c t (ta b le

3 5

6

) th e n o n -a c tiv a te d

-d ay -o ld enzyme showed about the same a ld o lase a c t i v i t y ; a d d itio n of

Fe** gave a tw o -fo ld s tim u la tio n compared to the sev en -fo ld s tim u la tio n a t 13 d a y s.

However, in th e presence of c y stein e and Fe** th e enzyme

was a c tiv a te d about tw e lv e -fo ld , or to approxim ately the same e x te n t a s a t 13 d ay s.

From th e se d a ta i t appears th a t th e enzyme was n e a rly re ­

so lv ed m th re sp e c t to iro n and had undergone in a c tiv a tio n during aging which could be r e s to re d by q y s te in e , presumably by r e v e r s a l of o x id a tiv e

ALKALI U

g / hr/ mg

cn o o

o o

PHOSPHORUS

PROTEIN] (O o

o

r\) o o

0 — 0

I \

\

OF ALDOLASE

N

M C o* +

\

\

IN PRESENCE

\

FIGURE 10

/

ACTIVATION

M F e ++

METAL



LABILE

OF

23 e f f e c t s o ccu rrin g during s to r a g e .

The com binations o f Co'*”*' and O.OOIM

c y ste in e ga-ve about th e same low l e v e l p re v io u s ly found (ta b le 6) and were 34 p e r cent a s e f f e c tiv e a s th e b e s t Fe

lj,

and c y stin e combinations*

24 PART I I FURTHER EVIDENCE FOR THE EXISTENCE OF THE MIERHOF-EMBDEN SCHEIE IN CLOSTRIDIUM PBRFRINGENS The e x iste n c e of a

he ta llo - a ld o la s e

in c e l l —fr e e e x tr a c ts o f

£ • P-grfringens has been d escrib ed in P a rt I .

In the co arse of t h i s

in v e s tig a tio n , evidence was o b tain ed fo r the e x iste n c e of insom erase, g lyeeraldehyde phosphate dehydrogenase and e th an o l dehydrogenase in e x tr a c ts o f t h is organism* Isom erase.

The method of S ib ley and Lehninger (1949) f o r the determ in­

a tio n of a ld o la s e a c t i v i t y can a ls o be used to dem onstrate insomerase a c t i v i t y by using an enzyme e x tr a c t d ia ly z e d to remove diphosphopyridine n u c le o tid e (DPN), the coenzyme of 1 , 3-diphosphoglyceraldehyde dehydro­ g e n ase .

Under th e se c o n d itio n s, i f hydrazine i s om itted from th e system

th e n th e phosphorylated t r i o s e s formed from the breakdown o f hexose diphosphate w ill n o t be dehydrogenated and the t r i o s e s w i l l come to e q u ilib riu m as a r e s u l t of isom erase a c tio n .

Thus, the t r i o s e s w i l l

be p re s e n t i n the r a t i o of 95 m olecules of dihydroxyacetone phosphate to 5 m olecules of 3-phosphoglyceraldehyde.

Since i t was shown by

S ib le y and Lehninger t h a t i n t h e i r method most of the c o lo r i s produced by th e 2 ,

4

-dinitrophenylhydrazone of dihydroxyacetone, the s h i f t of

the 50:50 r a t i o occurring i n the presence of hydrazine to the 95:5 r a t i o o c c u rrin g in th e absence of hydrazine should r e s u l t in approxi­ m ately tw ice as much c o lo r form ation and can be used as a measure o f insom erase a c t i v i t y . To demonstrate th e presence of isom erase in the c e l l- f r e e e x tr a c t

25 P Q rfring& is* th e e x tr a c t was d ia ly z e d f o r t a t e d d i s t i l l e d water*

13

hours a g a in s t a g i­

Using 0*2 ml of the d ia ly z e d e x tr a c t , a ld o la s e

a c t i v i t y was assayed i n the absence and presence of hydrazine*

A fte r

a d d itio n o f 10 per cent TGA, hydrazine was added to the tu b es not con­ ta in in g i t d uring the r e a c tio n . a l k a l i - l a b i l e phosphorus

In th e presence of hydrazine 73 pg

was formed per hour per ml e x t r a c t , whereas

i n th e absence o f h y d razin e, 250 pg a l k a l i - l a b i l e phosphorus ml e x tr a c t were found.

p er hour p e r

This in c re a s e i s in te r p r e te d a s being due to th e

form ation of la rg e r amounts of dihydroxyacetone phosphate a s a r e s u l t of isom erase a c t i v i t y during the breakdown of HDP*

I t i s n o ted , however,

t h a t th e equivalence of a l k a l i - l a b i l e phosphorus

found i n the absence

o f hydrazine was approxim ately th re e tim es g re a te r th a n th e equivalence found i n th e presence o f h y d razin e.

This fin d in g in d ic a te s t h a t th e

r a te o f a ld o la s e a c t i v i t y i s lower in th e presence of hy d razin e.

In an

addendum t o t h e i r p a p er, S ib ley and Lehninger (1949) rep o rte d t h a t th e ty p e of c arb o n y l-trap p in g re a g e n t appeared to have some e f f e c t on th e r e a c tio n r a t e s , although the fin d in g s were c o rre c t on a r e l a ti v e b asis* G lvceraldehyde Phosphate Dehydrogenase * Warburg and C h ristia n (1939) is o l a t e d and c r y s ta lliz e d from y e a s t the enzyme which dehydrogenates 1,

3

-diphosphoglyceraldehyde to y ie ld 1 , 3-diphosphoglyceric a c id .

The

a c t i v i t y of the enzyme was determ ined by measuring sp e c tro p h o to m e tric a lly a t 340 mp the re d u c tio n of i t s coenzyme, diphosphopyridine n u c le o tid e (DPN).

L a te r, Warburg and C h ris tia n (1943) is o la te d and c r y s ta lliz e d

a ld o la s e from r a t muscle a s w e ll a s obtain in g y e a s t a ld o la se i n a h ig h ly p u r if ie d form .

To a s s a y a ld o la s e a c t i v i t y , use was made by

26 th e s e workers of sp ectro p h o to m e tric issthod to msasure DPN re d u c tio n a t 340 mp i n a system c o n ta in in g fru c to s e - 1 6-diphosphate (pH 7*4) > g ly c in e , sodium a r s e n a te , DPN and glyceraldehyde phosphate dehydro­ genase*

The HDP served a s a b u ffe r w hile glycine was included to

n e u tr a liz e t r a c e s o f copper which in h ib ite d the dehydrogenase* In th e p re s e n t stu d y , th e a ssa y system o f Warburg and C h ris tia n was used*

Since c e l l - f r e e enzyme e x tr a c ts of C* p e rfrin g e n s had been

shown to c o n ta in a ld o la s e , th e a s s a y system could be used to dem onstrate the presence of glyceraldehyde phosphate dehydrogenase*

The d a ta in

f ig u r e 11 c le a r l y in d ic a te the re d u c tio n o f DPN a t a ra p id r a t e which i s evidence f o r th e occurrence of glyceraldehyde phosphate dehydrogenase i n the c e l l - f r e e e x t r a c t . E th an o l Dehydrogenase*

The i s o l a t i o n and c r y s t a l l i z a t i o n o f e th a n o l

dehydrogenase from brew er1s y e a s t were accom plished by N egelein and W ulff (1937 a , b ) . n u c le o tid e .

The coenzyme was shown to be diphosphopyridine

The enzyme c a ta ly z e s the o x id atio n of e th a n o l a s fo llo w s: CHjCH GH + DPN —> CH^CHO + DPN.2H

I t can be seen from the d a ta (fig u re 11) th a t the c e l l - f r e e e x tr a c t c a n ta in sd an enzyme which o xidized DFN.2H a t a r a p id ra te when approxim ately 5 pM CH^CHO was added.

This o b serv atio n c o n s titu te s

evidence f o r the e x is te n c e of e th a n o l dehydrogenase in t h i s organism*

LEGSND FOR FIGURE 11

Per a b so rp tio n c e l l : Na^HAsC^ * 7* ^

^

^ r

0.30 ml

Glycine (20 icg per ml)

0 .3 0

NaF (0 .2 M)

0.75

DPN (1.2 mg per ml)

0.20

Cysteine (0.01 M)

0.30

H2 °

0.65

Enzyme e x tra c t

0.20

HDP ( 0.03 M, pH 7 .4 )

0 .3 0

T otal volume

3.00

Beckman spectrophotom eter* 3W nip.} room tem perature

FIGURE II

GLYCERALDEHYDE PHOSPHATE AND ETHANOL DEHYDROGENASES # 0* o -o -o -

o*

I

0 ..

/

I

o

[3 4 0 DENSITY OPTICAL

0

\

0.4

0.3

0.2 HOP

0.1

4

8 12 TIME [MIN]

16

27 PART I I I STUDIES OF THE HYDROGEN METABOLISM OF CLOSTRIDIUM PERFRINGENS During th e course o f in v e s tig a tio n of the in term ed iary m etabolism —• E g rfrin g e n s * c e r ta in o b se rv atio n s o f the hydrogen metabolism of t h i s organism were made.

The r e s u l t s a re of a p re lim in a ry n a tu re but serve to

in d ic a te th e o u tlin e o f fu tu re stu d y of th e s e problem s.

Among the meta­

b o lic p ro ce sses stu d ie d a re those involving hydrogenase, l a c t i c dehydro­ genase and pyruvate d is s im ila tio n , Hydrogenase.

In t h i s s e c tio n refe re n c e w ill be made to c e rta in e x p e ri­

ments involving m olecular hydrogen.

For t h i s purpose, hydrogenase i s

d e fin e d according to Stephenson (1949) a s : H + A »AH 2 T 2 where A i s a s u ita b le a c c e p to r of hydrogen.

In these experim ents only

i r o n - s u f f ic i e n t c e lls h a rv e ste d from Medium A were used. To determ ine i f oxygen a c ts a s a hydrogen a c c e p to r, c e l ls placed i n 0,033 M phosphate b u ffe r, pH 6 .5 , d id not cause any s ig n if ic a n t change in na none t r i e p re s su re s when p laced in an atm osphere of one p a r t

and

th re e p a r ts 02j a d d itio n cf 15 mg Difco y e a st e x tr a c t did not a l t e r t h is re s u lt.

This fin d in g in d ic a te s t h a t m olecular H2 i s n o t oxidized by

m olecular 02 under th e c o n d itio n s te s te d . Study of g ly c o ly sis in a n atmosphere of hydrogen y ie ld e d the r e s u l t s p lo tte d in f ig u re 12.

In the absence of a d d itio n s , hydrogen

p ro d u ctio n from glucose reached a maximum in 30 m inutes with subsequent hydrogen uptake during the n e x t 60 m inutes amounting to 3 . 4pm H2 .

In

th e presence of 15 mg D ifco y e a st e x tr a c t, hydrogen p ro d u ctio n reached

LEGEND FOR FIGURE 12

Phosphate b u f fe r, 0.05 M, pH 6*5

2 .0 ml

C e ll suspension (5*4

0.3

dry wt)

Glucose, 0*05 M (10 uM), in sidearm.

0 .2

Yeast e x tr a c t , D ifco, or HO * 2 KCN, 0*001 M, or HO

0.3

KOH or KOH-KCN (in cen ter w ell)

0 .2

T o ta l volume

3 .2 ml

Atmosphere, Hgj 37 C

0.2

700

/

(n

vl KCN

[

.

500

00N

< O zo

1

**

EVOLUTION

o-^.


\

300 /


^+15 M 2 from p y ru v a te , even when y e a s t e x tr a c t or l i v e r e x tr a c t was added. I r o n - d e f ic ie n t c e lls showed p r a c t i c a l l y no a c t i v i t y on pyruvate under a n a e ro b ic c o n d itio n s.

The

a**i ^002 aiaourite d to 1 to 2 .

This

fin d in g i s not s u ip ris in g sin c e Fe- c e lls produce only sm all q u a n titie s of H„ and C0o from g lu c o se . ^

I f p y ru v ate , or a d e riv a tiv e th e re o f, i s

0mm

th e in te rm e d iate whose breakdown y ie ld s th e se g a se s , then Fe- c e l ls la c k th e enzyme system re q u ire d fo r pyruvate breakdown.

Thus, a t t n is p o in t,

th e optimum co n d itio n s fo r pyruvate d is s im ila tio n have not been a sc e r­ t a in e d .

34 PAST IV RESPIRATION OF CLOSTRIDIUM FERFRIMGENS In the course of experim ents involving g ly c o ly s is w ith washed c e l l su sp en sio n s o f J3. p e rfrin g e n s . i t was found th a t c e l ls grown in Medium A to o k up oxygen and re le a s e d carbon dioxide w ith glucose a s su b stra te * Aubel and co-w orkers (Aubel and Houget, 1939; Aubel and Perdigon, 1940; 1945) had re p o rte d a s im ila r fin d in g with C lo strid iu m saccharobutyricum . Using glu cose a s s u b s tr a te , th ese workers found th a t C. saccharobutyricum consumed oxygen and furtherm ore t h a t exposure to a i r d id not k i l l the c e lls n o r im pair the g ly c o ly tic a c t i v i t y of the organism when conditions were nade an aero b ic a g a in .

Aubel and co-w orkers concluded t h a t th e

oxygen ta k e n up o xidized the hydrogen a s w e ll a s o th er in te rm e d ia te s formed during g ly c o ly s is .

In the presence cf oxygen the C. compounds, 4 b u ty r ic a c id and b u ta n o l, norm ally formed during g ly c o ly sis occurred only i n sm a ll q u a n titie s . Glucose o x id a tio n .

Using 10 pM glucose a s s u b s tra te and 3 to 5 nag dry

weight of c e l l s , the r a t e s of

0 2

uptake (Qq2) and C0 2 p ro d u ctio n ( Q ^ ) ,

a s w e ll a s the r e s p ir a to r y q u o tie n t (RQ) and e x te n t of 02 consumed and CO^ produced, showed con sid erab le v a r ia tio n (ta b le 7 ); no sim ple explana­ t io n i s a v a ila b le f o r th is v a ria tio n #

During the course of manometric

ex p erim en ts, i t was observed on numerous occasions th a t 5 to 15 m inutes a f t e r a d d itio n of g lu c o s e , a change in th e r a te of

(>2

uptake occurred.

This change was in te r p r e te d a s being due to gas production oth er than CO

probably H , sin c e KOH was p resen t i n th e Warburg f l a s k .

Thus, i t

T able 7 B e sp ira tio n o f C,* p e rfr in g e n s :

ranges o f r a te s and e x te n t o f gas changes

Bange

Average

2

UD - 180

135

0(302

190 - 310

255

%

^ 2

consumed:

uM

7 .6

-

1 0 .2

C0 ^ produced: juM 14.5 - 17.9 BQ

1 .6

-

2 .0

S.5 16.1

1 .8

Hates were c a lc u la te d on the b a s is of gas changes during the f i r s t 15 m inutes a f t e r a d d itio n of su b stra te * s ig n if ic a n t*

Endogenous gas changes were in ­

T o ta l gas changes were g e n e ra lly completed i n 20 to 30

m in u tes, depending upon the q u a n tity of c e l ls used (3 to 5 *ag d ry w t). P er Warburg f l a s k t Phosptate b u ffe r (0.05 M), pH 6.5

2*0 ml

C e ll suspension

0*3

Glucose (0.05 M), i n sidearm

0 .2

K0H

0 .2

(2 0

per cen t) o r H^O, in c e n te r w ell

H 0 to t o t a l volume 2 Atmosphere* a i r } 37 C

3*2

35 appears t h a t the "values obtained f o r 02 consumption a re n o t s t r i c t l y q u a n ti t a t iv e , and a method m ust be devised to overcome t h i s te c h n ic a l d iffic u lty .

The d ata on gas exchanges have n o t suggested an o v e ra ll

r e a c tio n which might re p re s e n t the in term ed iary s te p s inv o lv ed . Two attem p ts were made to d e te c t hydrogen peroxide during r e s p ira ­ tio n *

An e x tr a c t of p o ta to was prepared according to th e method of Main

and bhinn (1939) &s a source o f peroxidase and an a c tiv e p re p a ra tio n was o b tain ed .

An a ero b ic manometrie experiment with 10 pM glucose was p e r­

formed and a t th e tim e t h a t

uptake was about o n e -h a lf complete

0 2

(1 5

m in u te s), two Warburg f l a s k co n te n ts (w ith and w ithout KQH) were te s te d f o r H^O^•

Another p a i r o f f la s k c o n te n ts were t e s te d a f t e r 60 m inutes.

Hydrogen peroxide was not d e te c te d under th e se c o n d itio n s.

In a d d itio n ,

th e e f f e c t of c a ta la s e on r e s p ir a tio n in the presence o f 10 pM glucose was t e s t e d .

An a c t i v e , c o n cen trated s o lu tio n of c a ta la s e was obtained

from b e e f l i v e r a c c o rd ir^ to th e method of Sumner and Somers (1947* P* 2 1 1 ).

A ddition o f c a ta la s e to f la s k s containing c e l ls and glucose in

an atm osphere of a i r had no e f f e c t upon e i t h e r the ra te s of t i o n and C02 p ro au ctio n or the e x te n t of gas exchanges. r e s u l t s , i t i s concluded th a t

0 2

consump­

From these

d id no t accumulate during r e s p ir a tio n

and p o ss ib ly i s no t formed during glucose o x id a tio n by t h is organism . Phosphorus exchange during glucose, d is s im ila tio n .

I t was shorn t h a t

02 uptake arri CC>2 form ation w ith glucose as s u b s tra te were stim u la te d by in o rg an ic phosphate ( ta b le

8

).

These data in d ic a te the p a r tic ip a tio n

of in o rg an ic phosphate i n glucose o x id a tio n . In o rder to compare phosphorus exchange during aero b ic ( r e s p ir a ­ tio n ) and anaero bic (fo m e n ta tio n ) g ly c o ly s is , a s im ila r experiment was

T able 8 S tim u la tio n o f glucose o x id a tio n by in o rg an ic phospimte

Hates o f o x id a tio n

%Q2

°2

co2

56

125

75

1 3 8

93

2 1 0

1 0 0

2 4 6

193

280

215

^ 0 2

P h th la te b u ff e r, Q.Q6 ?M, pH 6.5 n

+ 100 jag in o rg . P.

Phosphate b u f fe r, 0.06?MpH 6.5

E xtent o f o x id a tio n juL in 60 min.

80

Per Warburg f l a s k : B uffer (as above) C e ll suspension ( 2

2 .0 ml .6

mg diy wt)

0 .4

Glucose (0.05 M), in sidearm

0 .2

K0H

0 .2

(2 0

p er cen t) or H2 0, in c e n ter vuell

T o ta l volume Atmosphere:

2 .8 a ir$ 37 C

36 perform ed under a ero b ic and a n ae ro b ic c o n d itio n s w ith 10 pM glucose and p h th la te b u if e r co n ta in in g 100 jtig in o rg an ic phosphorus*

Inorganic

phosphorus and ^ 7 —phosphorus were measured a t in te r v a ls during gly ­ c o ly s is (fig u re 1 3 ).

The Qq2 was 120 and th e Qm was 2*25; o x id a tio n

proceeded to th e e x te n t o f 10*5 pM 02 consumed while 19*3 pM H2 were lib e r a te d a n a e ro b ic a lly *

Both a e ro b ic and an aero b ic g ly c o ly sis were

completed a f t e r 25 m inutes follow ing a d d itio n o f glucose* The d a ta c le a r ly in d ic a te the uptake o f in o rg an ic phosphorus and th e fo rm atio n o f A 7-phosphorus during the f i r s t 5 m inutes of g ly c o ly s is , both a e r o b ic a lly and a n a e ro b ic a lly .

Subsequent to th e in c re a se s of

A 7-phosphorus, th e se values re tu rn e d to about the same le v e ls during both ty p es of g ly c o ly s is .

However, whereas a n a e ro b ic a lly th e amount of

in o rg an ic phosphorus ro se t o a le v e l seme what h ig h er than th e o r ig in a l one, a e r o b ic a lly a co n sid erab le amount of inorganic phosphorus d id not reap p ear a s such nor a s A 7-phosphorus,

T his fin d in g suggests the ac­

cum ulation o f a phosphate compound(s) which is s ta b le during a c id h y d ro ly sis f o r 7 m inutes.

I t i s p o ssib le th a t such a compound i s not

m etab o lized or Is in h ib ito r y to oth er r e a c tio n s , a s i t u a t i o n accounting, a t l e a s t i n p a r t , fo r the f a i l u r e to o b ta in growth of t h i s organism under a e ro b ic c o n d itio n s .

I f the compound were a hexose or t r i o s e

phosphate such as i s u s u a lly foimed during g ly c o ly s is , i t i s not c le a r why i t should accum ulate glucose oxidation* E f fe c t o f -in h ib ito rs on glucose o x id a tio n * The e f f e c t o f s e v e ra l in ­ h i b it o r s on r e s p ir a tio n with glucose as su b stra te was in v e s tig a te d . In th e presence o f 0*01 M io d o a c e ta te , 0

uptake was com pletely in h ib ite d .

T his fin d in g suggests the e x iste n c e of 1,3-diphosphoglyceraldehyde

> 499-501. BACON 1949 BARKER, H. A.

Unpublished d a ta , c ite d by Stephenson, M*, 1949. 1944

On th e ro le o f carbon d ioxide in th e m etabolism

o f C lo strid iu m therm oaceticum .

P ro c. N at. Acad. S c i ., 30. SB-90.

BARKER, H. A. and KAMEN, M. D.

1945

Carbon d ioxide u t i l i z a t i o n

i n the s y n th e s is of a c e tic a c id by C lostridium therm oaceticum .

P ro c. N at.

Acad. S c i . , 31. 219,*225. BARKER, S . B. and SUMMERSON, W. H.

1941

m in atio n of l a c t i c a c id in b io lo g ic a l m a te r ia l.

The c o lo rim e tric d e te r ­ J . B io l. Chem., 138.

535-554* BERNHEIMER, A. W.

1944

N u tritio n a l requirem ents and fa c to rs

a f f e c tin g t t e p ro d u ctio n o f to x in of C lostridium septicum .

J . Exp. Med.,

80 , 321-331* BREED, R. S ., MURRAY, E. G. D. and HITCHENS, A. P . manual of d eterm in ativ e b a c te rio lo g y . more, Md.

1529 PP*

1948

Bergey»s

Williams and W ilkins Co., B a lti­

49 DICKENS, F •

1938

O xidation o f phosphohexonate and pentose

phosphoric a c id s by y e a s t enzynes.

Biochem. J . , 2 2 , 1626-1643.

FEENEY, R. E ., MUELLER, J . H. and MILLER, P. A. requirem ents of C lo strid iu m t e t a n i . th e organism#

Growth

II# F a c to rs exhausted by growth of

J* B a c t., /j6 , 559-562.

FISKE, C. H. and SUBBAROW, Y. o f phosphorus.

1943

J , Biol* Chera#,

6 6

1925

, 375-400.

FRIEDEMANN, T. E* and HAUGEN, G# E* k e to a c id s in blood and u rin e .

1943

The d eterm in atio n of

J* B io l. Chem., 147 . 415-442#

FUIJITA, A. and KODAMA, T. Garung pathogener B a k tie re n .

The c o lo rim e tric d e term in atio n

1934

Untersuchungen iiber Atcrung und

I I I . M itte ilu n g :

Uber Cytochrom und das

sau er sto ffx ibertragende Ferment, sowie die Atmungschemmung der pathogenen B a k tie re n durch CO und KCN. GRANT, W. M. form ic a c id .

1947

Biochem. Z e its c h r ., 273. 186-197* C olorim etric micromethod fo r d e term in atio n of

In d . Eng. Chem., A nal. E d ., 19. 206-207*

HANSON, A. M. and RODGERS, N. E .

1946

In flu en ce of iro n con­

c e n tra tio n and a tte n u a tio n on the m stabolisn of C lostridium acetobutylicum * J . B a c t., 51. 568. HASTINGS, E . G. and MG COY, E.

1932

th e c u lt i v a t io n of an aerobic b a c te r ia .

The use o f reduced iro n in

J . B a c t.,

2 2

., 54-56.

HERBERT, D ., GORDON, H ., SUBRAHMANYAN, V, and GREEN, D. E. Zymohexase*

1940

Biochem. J . , 34. 1108-1123.

HICKEY, R. J .

1945

The in a c tiv a tio n of ir o n by 2 , 2*-b ip y rid in e

and i t s e f f e c t on r ib o f la v in sy n th e sis by C lostridium acetobutylicum . Arch. Biochem., B, 439-447BOUNCE, A# Lo and BEYER, G. T»

1948

A new method fo r th e determ ina­

t i o n o f th e enzyme a ld o lase# J# Biol# Chem#, 175, 159-173. HOGBBOQM, G. H# and BAERY, G* T#

1948

P u r if ic a tio n o f diphosphopyr-

id in e riu c le o tid e ' by w u n te r-c u rre * ft d l s t r ib u t i o n # J# B# C#, 17#, 935-948 #

50 KGEFSELL, H. J . and JOHNSON, M. J .

1942

Dis s im ila tio n o f py­

ru v ic a c id by c e l l - f r e e p re p a ra tio n s of Clostridium , butylicum . Chem.,

J . B io l.

145. 379-386. KOEPSELL, H. J . , JOHNSON, M. J . and MEEK, J . S .

1944 Role o f

phosphate in pyruvic a c id d is s im ila tio n by c e l l- f r e e e x tr a c ts of Clos­ trid iu m b utylicum . KREBS, H. A.

J . B io l. Chem., 154. 535-547. 1933 W eitere Untersuchungen uber den Abbau d er

Aminosauren im T ie rk o rp e r. Z. p h y s io l. Chem., 2 1 8 .157-159. KEMPNER, W. B u tte rsa u reg a ru n g .

1933

Wirkung von Blausaure und Kohlenoxyd a u f d ie

Biochem. Z e its c h r ., 257. 41-56.

KEMPNER, W. and KUBOwTTZ, F .

1933

Kohlenoxydhemmung d er B u ttersau reg aru n g .

Wirkung des L ieh tes a u f d ie Biochem. Z e its c h r ., 265.

246-252. KUBOdlTZ, F . Kohlenoxyd.

1934 N6er d ie Hemmung d er B uttersauregarung durch

Biochem. Z e its c h r ., 274 . 285-298.

Ty.tftiF.R-, E . M. and PICKETT, M. J . cose bv C lo strid iu m t e t a n i . LEVITON, A.

1946

1945

Arch. Biochem., 8 , 183-196.

The m ic ro b io lo g ic a l sy n th e sis o f rib o fla v in -

a th e o ry concerning i t s i n h ib itio n .

J . Amer. Chem. S o c ., 68 . 835-840.

LINEN, F . and HOFFMANN-WALEBCK, H. P . p ilz e n .

The ferm en tatio n o f glu­

1948 Enzyme aus Schiramel-

I . Ober e in ig e Ga'rungsfermente aus P e n ic illiu m notatum .

Ann.

chem., 559. 153-168. MAIN, E . R. and SHINN, L. E . p ero x id e i n b a c t e r i a l c u ltu re s* MICHAELIS, L. 234. 139-141.

1939

The determ in atio n of hydrogen

J . B io l. Chem*, 128. 417—423*

1931 Der A c eta t-V ero n al-P u ffer.

Biochem. Z e its c h r .,

51 NE&ELEIN, E . and WULFF, H .-J . der A cetald eh y d red u k tase.

1 9 3 7

NELSON, N.

K r i s t a l l i s a t i o n des P ro te in s

Biochem. Z e its c h r ., 289. 436-437.

NEGEXEIN, E . and WULFF, H. - J . A lkohol, A cetaldehyd.

a

1937h

D iphosphopyridinproteid

Biochem. Z e its c h r ., 293. 351-389.

1944 A photom etric a d a p ta tio n o f the Somogyi method

f o r th e d e term in atio n o f g lu c o se .

J . B io l. Chem., 153. 375-380*

NEUHEHG, C ., LUSTIG, H. and ROTHENBBRG, M. A* d ip h o sp h o ric a c id and fructose-6-m onophosphoric a c id .

1943

F ru c to se -1 ,6-

Arch. Biochem., ^

33-44* PAPPENHEBSSR, A. M., JR. and SHASKAN, E . carbohydrate m etabolism of C lo strid iu m w e lc h ii.

1944 E ffe c t of iro n on J* B io l. Chem., 155.

265-275* EETT, L . B. and WYNNE, A. M. ky C lo strid iu m aceto b u ty lic u m .

1932

The form ation of m ethylglyaxal

J . B io l. Chem.,

PRESCOTT, S . C. and DUNN, C. G.

1940

177-182.

I n d u s t r i a l m icrobiology.

McGraw-Hill Book Company, I n c . , New York, N. Y ., 541 PP* ROBINSON, H. W. and HOGDEN, C. G* 1940 the d e te rm in a tio n o f serum p r o te in s .

The b iu r e t r e a c tio n in

I . A study of the conditions neces­

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1946

R elatio n

o f s t r a i n v a ria tio n and c u ltu re h is to r y to the § y th esis of r ib o f la v in by C lo strid iu m aeetobut.vlicum in whey. ROE, J . H.

1934

A c o lo rim e tric method f o r the d eterm in atio n

o f fru c to se in blood and u r in e . SCHLAYER, C.

1935

J . B a c t., £ 1 , 569-570.

J . B io l. Chem., 107. 15-22.

Wirkung des Kohlenoxyds auf die Oarung von

Tetanus-und G asb ran d b azillen .

Biochem. Z ie ts c h r ., 27,6. 460-463*

52 SCOTT, J* P*

1926

A method of in c re a s in g the v iru len ce o f

C lo strid iu m chauvoei by th e use o f f e r r i c s a l t s .

J . I n f . D is ., 38.

511-513* SIBLEY, J . A. and IEHNIWGER, A. L* la s e i n a n im al t i s s u e s . SIMON, E .

1947

The form ation of l a c t i c a c id by C lostridium Arch. Biochem.,

SNELL, F . D. and SNELL, C. T. Volume I .

SOMOGYI, M.

D eterm ination o f aldo­

J . B io l. Chem., 177, 859-872.

aceto b u ty iicu m (Weizmann).

a n a ly s is .

1949

1936

237-243. C o lo rim etric methods of

Van Nostrand C o., New York, N. Y ., 1930

pp.

A method f o r th e p re p a ra tio n o f blood f i l t r a t e s

f o r th e d e te rm in a tio n of su g a r. STEPHENSON, M.

7 6 6

1949

J . B io l. Chem., 86, 655-663*

B a c te ria l m etabolism .

Longmans, Green and Co.,

New York, N. Y ., 398 pp. STONE, R. W. and WERKMAN, C. H.

1937

The occurrence of phospho-

g ly c e ric a c id in th e b a c t e r i a l d is s im ila tio n o f g lu co se.

Biochem. J . ,

31 , 1516-1523. STUMPF, P . K. a ld o la s e .

Carbohydrate metabolism in higher p la n ts .

J . B io l. Chem., 176 . 233-241*

SUMER, J . B. and SOMERS, G. F . enzymes*

I . Pea

1947

Chemistry and methods o f

Academic P ress I n c ., New York, N* Y ., 415 pp*

TAMURA, J , T ., TITELL, A. A ., BOYD, M. J . and LOGAN, M. A. P roduction of C l. w e lc h ii

to x in in p e p to n e -fre e medium.

1941

Proc. Soc. Exp.

B io l. Med., £2* 284-287* UMBHEIT, W. W., BURRIS, R. H. and STAUFFER, J . F .

1945

Manometrie

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Bur­

53 WARBURG, 0* Ferment en.

194$

Schwerrae ta ll© a l s Wirkungsgruppen von

V erlag Dr* Werner Saenger, B e rlin , 195 pp.

WARBURG, 0* and CHRISTIAN, W.

1937

durch T ri-p h o s p h o -p y rid in -n u c le o tid .

Abbau von R obisonester

Biochem. Z e its c h r ., 292* 287-295.

WARBURG, 0 . and CHRISTIAN, W.

1939

Is o lie ru n g und K rista l l i s a t io n

des P ro te in s des oxydierenaen G arungsferm ents.

Biochem. Z e its c h r ., 303*

40- 68. WARBURG, 0 . and CHRISTIAN, W. t io n des GArungsferments Zymobexase. WEBB, M* 1948

1943

Is o lie ru n g und K r i s t a l l i s a -

Biochem. Z e its c h r ., 314. 149-176.

The in flu e n c e of magnesium on c e l l d iv is io n .

I.

The growth of C lo strid iu m w e lc h ii i n canplex media d e fic ie n t in magnesium. J . Gen. M ic ro b io l., 2 , 275-287. WEIL, L ., KOCHOLATI, W. and SMITH, L. S.

1939

S tudies on p ro -

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Biochem. J . ,

31, 893-897. UERINGA, K. T.

1940

The form ation of a c e ti c a c id from carbon

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Ant. van

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1936

H ydrogenlyases.

Biochem. J . , 3£L* 515-527.

WILLIAMSON, S. and GREEN, D. E . from y e ast*

IV. The s y th e s is o f form ic

1940

J# Biol* Chem., 155, 345—346*

The p re p a ra tio n o f coenzyme I