role of the citric acid cycle in the metabolism of

ROLE OF THE CITRIC ACID CYCLE IN THE METABOLISM OF STREPTOMYCES SPECIES

by

EARL McKENZIE BUTTERWORTH

A THESIS

submitted to

OREGON STATE COLLEGE

in partial fUlfillment ot the requirements for the

degree ot

DOCTOR OF PHILOSOPHY

June 1955

IPPBOYEDS

Redacted for Privacylrtlrtent roflrror of

In Cbmgr of lrJor Redacted for Privacy

Gbrlrren of DrprtrHmt of Drottrlology

Redacted for Privacy

0helrnm of Sshool Orrirtrtr

Redacted for PrivacyDcen of Oreamplrtc 8ohool

Deto thmfi 1r pncrmtod

Ipt by Yrme laglutm

ACKNOWLEDGMENl

My sincere appreciation is extended to Dr c M

Gilmour for his guidance and cooperation in the researoh

and in the preparation of this thasJs

To Dr c H Wang ror his counsel during the radioshy

active tracer research

To the National Institute of Health United States

Public Health Service for partial financial assistance

To the members of the Bacteriology Department for

their cooperation durins the course of the investigation

To these friends and others are due the authors

sincere gratitude

bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull

bull bull bull bull bull bull

bull bull

bull bull bull bull bull bull bull bull


bull bull bull bull bull bull bull bull bull bull bull

bull bull bull bull bull bull bull


bull bull bull bull bull bull bull bull bull

TABLE OF CONTENTS

Page

1 Introduction bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull 1

2 His torical bullbull bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull

3 Experimental Methods bull bull bull bull bull bull bull bull bull bull bull bull 9

Manometric Studies bull bull bull bull bull bull bull bull bull bull bull bull 9

Cultures 9

Preparation of cells bull bull bull bull bull bull bull bull bull bull 9

Manometry bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull 10

Chemical bullbullbull bull bull bull bull bull bull bull bull bull bull bull bull bull 11

Radioactive Tracer Studies bull bull 12

Acetate Incorporation bull bull bull bull bull bull bull bull bull 12

Treatment of cells and apparatus bull 12

Carbon dioxide fix a t1 on 15

Treatment of cells and apparatus bull bull bull 15

Chemical 17

4 Experimental Results bull 19

I Manometric Studies bull bull bull bull bull bull bull 19

Oxidative patterns of selected Streptomycesmiddot species bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull 19

Oxidative behavior of 2bull grise~ bull bull bull 21

Endogenous respiration bull bull bull bull bull bull bull bull 21

Mole ratio studies bullbull bull 23

Respiratory quotient bull 26

Substrate oxidat ion 27



TABLE OF CONTENTS

(continued)

Page

Permeability effects bull bull bull bull bull bull bull bull bull 30

pH effects bull bull bull bull bull bull bull bull bull bull bull bull bull bull

Inhibition studies bull

32

Malonic acid bull bull bull bull bull bull bull bull bull bull bull bull

Pyrophosphate bull bull bull bull bull bull bull bull bull bull bull 36

F1uoroacetate 39bull bull bull bull 0 bull bull bull bull bull bull

II RadioactiveJTracer Studies 41

Incorporation of acetate-l-cl4 bynon-proliferating cells bull bull bull bull bull bull bull bull 41

Incorporation of acetate-l-c14 bygrowing cells bull bull bull bull bull bull bull bull bull bull bull bull bull 42

Incorporation or cl4o2 by growing cells 44

s Discussion bull 49

6 Summary bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull 56

7 Bibliography bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull 57

LIST OF FIGURES

Figure Page

1 The effect ot age ot cells on respirationof s griseus bull bull bull bull bull bull bull bull bull bull bull bull bull 22

2 The effect of middotstarvat1on on respirationof abull griseus bull bull bull bull bull bull bull bull bull bull bull bull bull 24

3 Observed respiration of intact cells of ~middot gri seus bull middotbull bull bull bull bull bull bull bull bull bull bull bull bull 28

4 The oxidation of selected amino acids by cells of ~middot griseus bull bull bull bull bull bull bull bull bull bull 29

5 The oxidation of 0167 M succinate and alpha ketoglutarate by intact cells of ~middot griseus bull bull bull bull bull bull bull bull bull bull bull bull bull 31

6 The oxidation of alpha ketoglutarate at various substrate concentrations by s griseus bull bull bull bull bull bull bull bull bull bull bull bull bull bull 33

7 The oxidation or 0167 Mmalate and fushymarate by s griseus cells after a 120 minute preincUbation period bull bull bull bull bull bull 35

8 The oxidation of 0167 M citrate by s friseus cells after a 550 minute preshyncubation period bull bull bull bull bull bull bull bull bull bull bull 37

9 The stimulation of the endogenous respirashytion of ~middot griseus by 004 pyrophosshyphate bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull bull 38

10 The inhibition of 0003 M oxalacetate oxishydation with 0003 M fluoroacetate and the stimulation of acetate oxidation byoxalacetate bull bull bull bull bull bull bull - bull bull bull bull bull bull 40

11 Radioautograph of a two dimensional chrobull matogram demonstrattng the incorporationof radioactivity into amino acids duringfixat1 on of cl4o2 bull bull bull bull bull bull bull bull bull bull bull 45

LIST OF TABLES

Table Page

1 The oxidation of selected substrates by various Streptomyces species bull ~ bull 20

2 Stoichiometric ratios for the dissimilashytion of various compounds bull bull bull bull bull bull 25

Dist~ibution of cl4 in glutamic acid from non-proliferating cells of bull griseusutilizing CHBcl4ooH bullbullbullbullbullbullbullbullbull 43

4 Distribution of cl4 in aspartic acid from growing cells or s sriseua utilizingcl4o2 and nonbullradioactve pyruvate bullbull 47

5 Distribution of cl4 in glutamic acid from growing cells of s griseus utilizing cl4~ and non-radioactive pyruvate bullbull 48

ROLE OF THE CITRIC ACID CYCLE

IN THE METABOLISM OF STREPTOMYCES SPECIES

INTRODUCTION

The tricarboxylic acid cycle has received general

acceptance as the principal pathway for the oxidation of

acetic acid by animal tissues However data obtained

in studies with bacteria do not always satisfy the crishy

teria on which the cycle was baaed in animal tissues (20

pl66) Factors such as limiting cell permeability and

the existance of other known metabolic pathways are mainshy

ly responsible tor the ditticul t1 es encountered

Until quite recently research on the physiology ot

the actinomyceteamp dealt largely with general nutritional

requirements of the group Few studies of a fundamental

nature have been conducted on the metabolism ot these

organisms

This investigation was designed to study the mechanshy

ism(amp) involved in the oxidative dissimilation of various

substrates by intact cella ot ~middot griseus with special

reference to the citric acid cole Techniques adapted

to the Warburg constant volume respirometer and to

2

radioactive tracer exper1men ts were us d

HISTORICAL

Since the announcement of the isolation of the antibull

biotic streptomycin (34 pp66-69) an extensive literashy

ture has accumulated on the general nutrition of Streptoshy

myces griseus For the most part these studies dealt

with streptomycin titers and not with the accompanying

physiology of the organism However some aspects of the

physiology of 1ih1s organism have been elucidated by tragshy~

mentary data accumulated during the studies on streptoshy

mycin production

Studies conducted by Dulaney and Perlman (12 pp

504-511) indicated that there were two phases of metashy

bolic activity During the growth phase the production

ot mycelium was accompanied by a reduction in the soluble

constituents of the medium (N-0-P) fermentation of the

available carbohydrate a high oxygen demand and little

production of streptomycin During the autolytic phase

the mycelium weight decreased markedly inorganic phosshy

phorus and soluble nitrogen were released into the medhlm

the oxygen demand dropped and considerable quantities or

streptomycin were produced The pH rose gradually

throughout the period of the fermentation

bull griseus has been reported to utilize various orshy

ganic acids some of which are directly or indirectly

4

associated with the tricarboxylic acid cycle Pridham

and Gottlieb (33 pl09) found that acetate succinate

and citrate were utilized for growth by various strains

of~middot griseus Dulaney (13 plO) observed that acetate

and malonate would not support growth However citrate

fumarate gluconate lactate malate pyruvate and sueshy

cinate were utilized to varying degrees In a related

study Hubbard and Thornberry (19 ppSl-74) noted that

acetate citrate lactate glutarate malonate malate

and succinate were utilized by ~middot griseus It should be

mentioned however that in the aforementioned investigashy

tion glucose was a constituent of the basal medium The

investigations of Perlman and Wagman (31 p257) revealed

that cells grown on lard oil appeared to be more active

metabolically than cells grown on a medium containing

glucose (Q02 was 50 as compared with 27 on glucose) The

addition of inorganic phosphate speeded the metabolism of

glucose and glycerol by both types of cells (as measured

by oxygen demand) but did not affect the rate of metaboshy

liam of stearate acetate or lard oil by the cells

The utilization of amino acids such as glutamate

aspartate and alanine commonly associated with Krebs

cycle intermediates was observed by Dulaney (14 p313)

The data obtained by Eiser and McFarlane (15 ppl64-173) I

suggested that the utilization of aspartic aoid involving

--

5

the aspartamiddotsebulltransamination reaction brought about inbull

creased growth at 1he cost ot streptomycin production

These workers were able to replace aspartic acid with

glutamic acid or even with succinate and nitrate and

obtain similar results

Several investigations have indio a ted that carbon

dioxide is the main product of carbohydrate utilization

Detailed analysis of the various growth media used has

revealed no large accumulation of volatile or non-wlatlle

acids The minor accumulation of lactic acid has been

reported by several research groups (12 ppSl0-511 7

p729 31 pp254-260) Cochrane and Dimmick (7 p729)

also reported that small amounts ot suceinate were pro

duoed but did not appear until a full day after the glushy

cose had essentially disappeared from the medium The

accumulation of acetate during the utilization of both

glucose and lipids was observed by Perlman and Wagman

(31 pp254bull260) While investigating the biosynthesis

ot streptomycin Numeror et al (27 pl342) noted the

presence of free alanine and glutamic acid in the fermenshy

tation medium

trntil recently 11ttle emphasis was placed on the

actual mechanisms by which middot sriseus derived from orshy

ganic substrates the energy required by the cell for

growth and other cell processes middotThe incomplete oxidation

6

of g1 ucos e was reported by Sevcik (35 p bull 303) according

to the following equation

Lactic and acetic acids were isolated and identified as

intermediates in the above oxidation Scevola and Valshy

curane (36 p624) also working on the intermediates of

carbohydrate metabolism by~middot griseus were able to inshy

tercept triose phosphate and pyruvic acid in the respirashy

tion of living cells of this organism As acetaldehyde

was found in only one instance it did not seem probable

that pyruvate was oxidized by simple decarboxylation

Preliminary evidence against the existance of a tribull

carboxylic acid cycle in ~middot griseus was reported by

Garner and Koffler (16 pl39) The respiration of cells

grown in liquid media cm taining single carbon sources

led to the conclusion that only the enzymes responsible

for the oxidation of succinate and pyruvate were constishy

tutive Enzymes enabling cells to use other intermediates

of the cycle were thought to be adaptive and hence did

not appear to be components of a main terminal oxidative

pathway However a subsequent investigation by Coty

lbull (11 p74) demonstrated that cell free extracts

of ~middot griseus prepared from alumina-ground cells had the

ability to perform nearly all the critical reactions of

the tricarboxylic acid cycle as shown by the following

7

1 Oxalacetate + acetylphosphate ----lbull~ citrate

Scevola and Zorzoli (37 p552) have reported the

formati on (and isolation as the acetyl phenylhydrazine) of an actibulle acetyl radical during the respiration of~middot

griseus

2 Citrate or cisaconitate --~bull~ alpha ketoglutarate

This reaction was reversible as evidenced by the

reoxidation of reduced triphosphopyridine nucleotide

(TPN) if sodium bicarbonate was added to the reaction

3 Malate oxidation was reversed by the addition of

oxalacetate Manganous ions were found to be essential

Reactions 2 and 3 were TPN specific

4 Succinate------~ fumarate-----)~middot malate

Labeled fumarate and malate were form9d from cl4

labeled succinate Succinic dehydrogenase activity was

demonstrated by the Thunberg technique

Preliminary work carried out in this laboratory (6

p94) indicated that several members of the tricarboxylic

acid cycle were oxidized by non-proliferating cells No

oxygen uptake was observed with alpha ketoglutarate

succinate fumarate and malate unless substrate concenshy

trations approaching Ol6M were employed Citrate was

not oxidized even in the presence of high concentrations

of substrate

8

Oxidative mechanisms have been postulated for other

members of the genus Streptomyces Cochrane al (10

ppl7-23) concluded that the hexose monophosphate shunt

was preseh t in sect scabies and bull coelicolor That the

shunt sequence might be mainly or exclusively responsible

tor the initial steps in the metabolism of glucose was

suggested by the results with labeled glucose by the

failure to locate the early steps of the Embden-Meyerhotshy

Parnas system and by the observation that glucose-6bull

phosphate was 80 to 90 per cent metabolized in a system

containing only TPN as coenzyme

Cochrane and Peck demonstrated that sect_ scabies (8

p5) and sectbull coelicolor (9 pp37-44) were able to carry

out the reactions of the tricarboxylic acid cycle The

use of cell free extracts was essential with both organshy

isms primarily because or the low permeability or the

cells to certain key substrates et alMatsuoka -- (26

pl69) have suggested that a tricarboxylic acid cycle is

operative 1n ~middot venezuelae They were able to detect

most ot the intermediates free in the medium They also

proposed that glutamic acid was the most important amino

acid probably linking nitrogen metabolism with carbohyshy

drate metabolism in S venezuelae Glutamic acid was-readily detected free in the medium

- -

9

EXPERIMmTAL METHODS

Two approaches were used in studying the metabolism

of~middot sriseus The first phase of the work centered on

the investi gation of terminal ox~ations as evidenced by

manometric techniques The second phase entailed the use

of radioactive acetate and radioactive carbon dioxide

with intact cells Each of these approaches is treated

separately in the following section on experimental

methods

Manometric Studies

Cultures

All the cultures used were obtained from the Amaribull

can Type Culture Collection ~middot sriseus (3475) was used

throughout all manometric studies s albus (3004) s Yiridochromogenus 3356) and ~middot willmorii (6867) were

used in preliminary comparative studies Stocks were

maintained in sterile soil and successive transfers on

laboratory media were limited to insure physiological

eons tancy or the organisms

Preparation 2 cells

Stock cultures were prepared by adding spore suspenshy

sions to tubes of sterile soil Cultures for inocula

were obtained by spreading spores from a soil tube over

10

Blake bottle slants of modified nutrient agar (20 agar

lO glucose os sodium chloride) After 4 to 5 days

incubation at 2aoc the spores were harvested by adding

10 ml of sterile broth to each Blake bottle The spores

were teased off the agar with a sterile wire loop~ One

ml of this spore preparation served -as inoculum Pitty

ml of nutrient broth supplemented with 01 per cent glushy

cose and 05 per cent sodium chloride contained in 250

ml Erlenmeyer flasks were inoculated and mounted on a

rotary shaker set at a speed of 230 RPM The temperashy

ture of incubation was 2aoc for a period of 24 hours

The resulting cell crop was harvested by centrifugation

washed four times in phosphate buffer (006 N pH 69)

and then resuspended at growth volume The colonial agshy

gregates of ~ viridoehromoeen~~ were subjected to 15

seconds in a Waring blendor The final growth of the

other organisms was finely dispersed and did not require

the blendmg proc~dure In some instances the cells were

subjected to a starvation procedure Usually 50 ml of

a washed cell suspension contained 1n sterile 250 ml

Erlenmeyer flasks were returned to the rotary shaker

Samples were taken at various time intervals for manoshy

metric determinations

Manometry

Measurements of the gaseous exchange of resting cell

11

suspensions were made by the use of conventional manoshy

metric techniques as detailed by Umbreit Burris and

Stauffer (40 ppl-16) and will not be repeated except

Where modifications in method were made A standard Warbull

burg constant volume microrespirometer was used throughshy

out the work The water bath was maintained at 301degC

Flasks were shaken at the rate ot 60 complete strokes per

minute through a distance of 32 em ln a typical exshy

periment to determine the rate ot oxygen consumption

each flask contained 25 ml ot oell suspension (06 to

oa mgms of N) the sidearm contained 05 ml bull of substrate

in o067M buffer and the center well contained 02 ml of

20 per cent potassium hydroxide to give a total flask

content of 32 ml All experiments were carried out in

an air atmosphere Substrate concentrations were 0003 M

unless otherwise stated Carbon dioxide evolution was

determined by replacing the potassium hydroxide with

butter in one pair of flasks and calculating carbon dioxshy

ide by difference (40 ppl7bull20) Allowance was made for

the retention of carbon dioxide in the buffer at the pH

of the reaction

Chemical

Total oell nitrogen was determined by the mioroshy

Kjeldahl method (25 pp280middot282) The one dimensional

paper chromat ographic method of Lugg and Overall (23bull

12

pp98-l04) using a waterbutanolformic acid 541) solshy

vent was employed tor the detection of non-volatile car

boxylic acids~ The method is sensitive to from 2 to 8

gamma of acid In some iDS tances the phosphate buffer

used for suspending the cells was replaced w1 th distilled

water as phosphate salts proved troublesome dlring chroshy

matographic analysis shy

Radioactive Tracer Studies

Acetate incorporation

Studies on the incorporation of labeled acetate by

intact cells were made using both growing and nonbull

proliferating cells of ampbull griseus 3475) The nonshy

proliferating cells were suspended in phosphate butter

0067M pH 69) Growing cells were suspended in a synbull

thetic medium This medium was prepared as tollGws t

NaCl -- 5 grams MgS041H20 -- 05 grams

K2HP04 -- 2 grams FeS041H20 -- 0002 grams

(NH4)2HP04 -- 4 grams ZnS04e7H20 -- 0001 grams

Distilled water was added to 1000 ml The pH of the

medium after autoclaving was sa

Treatment of cells ~ apparatus

The apparatus consisted of a 250 ml three necked

~

13

flask with attachments as follows entering through one

side neck was an aeration tube with a sintered tip middotexbull

tending to the bottom ot the flask Air was passed

through a soda-lime tower a sulfuric acid trap and

finally through a sterilized eotton-f1lled tube before

entering the culture flask

Carbon dioxide l9av1ng tne flask was dispersed

through a sintered glass tube into 25 ml of 3 N NaOH middot

Provision was made to permit sampling through the center

neck of the flask The temperature for all experiments

was 2aoc Forty-five mgs of aeetate-lbullC14 with a total

activity of 728 x 106 counts per minute were mixed thorbull

oughly with the 100 ml ot cells in the culture flask

Samples were taken periodically Activity of the medium

the cells and the carbon dioxide was counted and exshy

pressed as counts per minute These were deposited and

counted directly in oupped metal planchets after evaporabull

tion Appropriate aliquots were used Although the conbull

tent of inorganic salts of the samples of media was relabull

t1vely high and the resulting activities were thereby

reduced owing to increased self-absorption preliminary

experiments indiea ted that the method was s u1table for

control and comparative purposes The experiments were

terminated by adding 6 N HOl The cells separated from

the medium by cen tritugati on and repeated washing (twice

--

14

~

with distilled water once with alcohol) were hydrolysed

1n 6 N HCl for 20 hours under reflux The hydrolysate

was freed or HCl by VampCUUm desiccation then iesuspend ed

in a minimum ot water for analysis The f1n81 medium and

the alcohol extract were chromatographed The descending

method was employed in preparing both one and two dimen

sional paper ehromatograms (22 pp396-399) One dimenbull

sional chromatographs were developed in a waterbutanol

acetic acid (541) solvent system The seconi solvent

system was water saturated phenol containing a trace of

8 hydroxy quinoline Amino acids were detected by sprayshy

ing dried chromatograms with a 02 per cent solution of

ninhydrin in butanol saturated w1th water The method

ot Lugg and Overall (23 pp98-l04) was used to detect

other nonbullvelatile organic acids Radi oautographamp were

prepared after the method of Benson et al (4 pbull l716)

For kinetic studies 5 ml of boiling alcohol ware

added to 5 ml aliquots trom the reaction vessel The

cells were washed twice with distilled water once with

ether then were dried over F205 Weighed amounts of the

dried cells were placed in vials to which were added 1 ml

ot 6 N HCl The vials were sealed then autoclaved at

l21degC for 6 hours The hydrolysates were dried and freed

of HCl by vacuum dess1eat1on over P2o5 and KOH After

resuspension 1n a small amount of dis tilled water

15

appropriate aliquots ere taken for ehromatographic

analysis

Glutamic acid was isolated from the alcohol extract

of the non-proliferating cells in the form ot the free

acid after dilution with non-isotopic lbullglutamic acid

The purity of the isolated glutamic acid was checked with

paper chromatography It was then subjected to the folshy

lowing degradation procedures

(a Combustion provided a mea ure of the total

activity

(b) N1nhydrin middot decarboxylation (42 p253) removed

the alpha carboxyl

(c) Schmidts reaction 2 ppl564-1568) yielded

alpha gamma diaminobutyrio acid and the gamma

carboxyl ~s carbon dioxideJ

Radioactivities werbulle detbullrmined middot as barium carbonate in

the convent onal manner counting data were corrected foi

background and Smiddotelf-absorption

Carbon dioxide fixation

Treatment 2f cells and apparatus

The apparatus ccnsisted of a three neck flask with

attachments as follows One neck was equipped with a

rubber hood The carbon dioxide oxygen and pyruvate

were admitted into the vessel with the appropriate

16

attaChments on a middothypodermic needle Provision was made

on one neck of the flask for attaChment to a water aashy

piratar The third neck was connected to a mercury

manometer

100 ml of sectbull -ariseus cells suspended 1n the inorshy

ganic basal medium were put in the three neck flask The

system was evacuated and checked for leaks Two millibull

moles of sodium pyruvate in aqueous solution was added

Then labeled carbon dioxide generated from sodium carshy

bonate with a total activity of 5 4 x 106 counts per minshy

ute was passed in Finally oxygen was introduced The

experimm t was conducted under a negative pressure The

culblre flask was ag1 tated 1n a specially constructed

water bath at a temperature of 2aoc for a period of 8

hours At the end of the experiment 10 ml of 6 N HCl

were added The Ct9lls were separated from the growth

medium by centrif~g~tio~ washed twice with water once

with alcohol and finally once with ether The resultshy

ing cella were dried over P205bull weighed then acid hybull

drolysed Aliquots of the resulting hydrolysate were

analysed with two dimensional paper chromatography Subshy

sequent preparation of radioautographamp revealed the exshy

tent and degree ot incorporation of radioactivity in the

amino acids or the hydrolysate

17

Chemical

The following scheme outlines the procedures empl~d

for the separation of glutamic acid and aspartic acid

from the protein hydrolysate

Protein hydrolysate HClbullfree

Dowex 3

Effluent Eluate (with lN HCl)

Iall amino acids

I vacuum distill

except aspartate and I glutamate aspartic auid

glutamic aci~

add CuC03 (5 p318)

CU aspartate glutamic acid

HCl gas

aspartic acid glutanrlc acid HCl

Controls were maintained throughout the separation operashy

tions with ninhydrin spot tests Microbiological assays

(18 ppl-12) were used to determine the amount of glushy

tamate and aspartate present

The aspartic acid was subjected to the following

18

degradation procedures

(a) Combustion provided a measure middot o~ the total

activity

(b) Ninhydrin decarboxylation (42 p253) removed

both carboxyls

(c) Deamination with silver nitrite produced malic

acid which was subjected to decarbonylation

with concentrated sulturic acid (41 p581)

This was followed by oxidation of the carbon

monoxide to carbon dioxide to provide a measure

of the alpha carboxyl

The glutamic hydrochloride was degraded by the combustion

and ninhydrin reactions used in a previous section for

glutamic acid

19

EXPERIMENTAL RESULTS

I Manometric Studies

Oxidative patterns ~ selected Streptomices species

The cultures used in the 1n1 tial stages of this

study were selected on the basis of species differences

obser~ed during phage ~ping work An investigation

of the oxidative behavior of these cultures (Table l)

showed considerable variation in respect to the number

and kind of substrates oxidized It should be noted that

glucose was the only substrate commonly utilized and that

alpha ketoglutarate was the only substrate not oxidized

by any of the species It is interesting that citrate

was not used by any of the test cultures except ~middot albus

This eulture also oxidized all of the other compounds

employed with the exception of the alpha keto acids s-viridochromosenus oxidized malate succinate and fumarate

but oddly enough would not utilize acetate ~middot sriseus

differed from the other species in oxidizing pyruvate and

oxalacetate and in showing no oxygen uptake on the reshy

maining Krebs cycle intermediates The oxidation of pyshy

ruvate and oxalacetate by intact cells of ~middot griseus has

been reported by Oginsky bull (29 p640) In the

case of ~middot willmorii only acetate and glucose were utilshy

ized

20

TABLE 1

The oxidation of selected substrates by various StreEtamzces species

Substrate s virido- s albus it- s willbull s griseus chromosenus - -morir

Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)

Oxalaeetate No No No 180(82)

Alpha ketobull glutarate Nmiddoto No No No

Malate 150(87) 190(132) No No

Fumarate 13587) 190(132) No No

Succinate 130(87) 172(132) No No

Citrate No 164(132) No No

Pyruvate No No No 160(82)

~middot albus-bulloxygen consumption 1n microlitres tor 220 minutes All others are based on a 60 minute period

Endogenous respiration values are in parenthesis

21

Oxidative behavior of s griseus--------- -------- -- = Since bull griseus is presently being used as the host

organism in the study of phage multiplication it was deshy

cided to use this culture in all subsequent physiological

studies

Endogenous respira t1 on

It may be noted in Table 1 that the endogenous resshy

piration of sect_ griseus and of the other species is very

high This high endogenous respiration is characteristic

of actinomycetes and fungi (45 p315 39 p267) Since

marginal rates of respiration are difficult to ascertain

in the presence of a high endogenous experiments deshy

signed to reduce the endogenous were carried out The

influence of age of cells on the endogenous respiration

of sectbull griseus is soown in Figure 1

No signif loan t differences were observed in the enshy

dogenous rates of respiration between 24 and 48 hour old

cells However the rate of oxygen uptake of 24 hour

cells in the presence of glucose was twice that of the

48 hour old cells Gottleib and Anderson (17 ppl72shy

173) have reported a similar decrease in ~middot sriaeus resshy

piration during the growth period

The procedure of incubating cells in the absence ot

substrate for a three hour period served to reduce the

200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1

The effect of age of cells on respiration of s sriseus ro ro

23

endogenous (Figure 2) but subsequent substrate oxidation

was proportionally reduced Similar effects were obshy

served with starved 48 hour cells Periods of starvation

longer than 3 hours decreased the rates of substrate and

endogenous respirations but these reductions were not so

definite as were those of the 3 hour starvation periods

Since no reduction 1n endogenous respiration was obtained

without a concommitant reduction in substrate respiration

24 hour unstarved cells were used for all subsequent

manometric determinations

2 -ra-t-io- studies

The amount of oxygen used by cells in the presence

of a known amount of a specific substrate may be used to

determine the extent ot oxidation of the substrate by the

cella The oxygen consumption per mole of substrate

metabolized is compared with the theoretical values for

various stages of oxidation Such inpoundormation can often

be used to identity the produc~)ot oxidation of the subshy

strate by the cella Manometric determinations showed

(Table 2) that resting cells of ~middot griseus consumed less

than one third of the theoretical oxygen required for the

complete oxidation of these substrates Apparently glushy

cose and glutamate were oxidized to the level of succinshy

ate Similarly acetate if oxidized via the Krebs cycle

24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2

stoich1ome trio ratios for the dissimilation of varioua compounds

Moles ot oxygen consumedSubstrate per mole ot substrate Actual Theoretical gt ot Theoretical

Glucose 208 middot 60 35

Acetate 0 62 20 31

Glutamic 120 45 27

26

was oxidized to t h e level of succinate It was recogshy

nized however that oxidative assimilation could account

for the observed low oxygen consumption Although it has

been reported that sect_ griseua does not form succmate

until late in the growth period 7 p725) an analysis

of the acetate medium was carried out using paper chromshy

atography No succinate was detected However a slow

moving spot with an Rf lower than any of the acid conshy

trols employed was shown to give a positive ninhydrin

test Subsequent investigations proved the compound to

be glutamic acid The relation between acetate oxidation

and the production of glutamic acid is discussed in more

detail in a following section

Respiratory quotient

The respiratory quotient can often be used advanshy

tageously to interpret results and to buttress results

obtained by other methods of 1nr eatigation The results

of the mole ratio studies (Table 2) indicated incomplete

oxidation cpound glucose If succinate were the product of

oxidation for example 2 moles of carbon dioxide would

be evolved for every 2 12 moles d oxygen consumedbull The

R Q of this theoretical system would be oso The obshy

served R Q for tta oxidation of glucose by ~middot griseus

was very close to 10 In a typical experiment the folshy

lowing R Q values were obtained endogenous 100

27

glucose 107 endogenous plus substrate 103

The results of mole ratio and R Q studies using

glucose as t he substrate attests to the combined effects

of incomplete oxidation combined with considerable assimishy

lation

Substrate oxidation

The results of experiments already tabulated in

Table 1 have been represented in graphical form in Figure

3 The data illustrated in Figure 3 discloses that glushy

cose acetate pyruvate lactate and oxalacetate were

oxidized at significant rates by Amiddot griseus at a final

substrate concentration of 0003M No significant inshy

crease in the respiration of ~middot griseus was noted with

succinate citrate alpha ketoglutarate fumarate and

malate However as shown in Figure 4 the amino anashy

logues of pyruvate oxalacetate and alpha ketoglutarate

were rspidly oxidized It is apparent from a comparison

of these data that cells of this organism show some inshy

consistency between the relative rates of oxidation of

amino acids and related organic acids Compounds such

as alpha ketoglutarate and succinate that can be preshy

dicted as intermediates in the dissimilation of glutamic

acid showed no oxygen uptake However glutamic acid was

oxidized at a rate closely approaching that of glucose

In the same sense aspartate (after a short lag) and

200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60

Observed respiration of in tact cells of s griseus

0 10 20 30 TIME MINUTES

FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120

w ~ lt( t-CL gt eo z w ()

gtshyX 0

40

TIME MINUTES FIGURE 4

The oxidation of selected amino acids by cells of s griseus

30

asparagine were oxidized more rapidly than oxalaoetate

The lag in oxidation shown by glutamate and proline (Figshy

ure 4) is worthy ot note A lag in proline utilization

has been reported by Woodruff and Ruger (46 pp315middot321)

These workers showed that cells could not be adapted to

proline which indicated that the lag was not one of

adaptation but one of permeability

Permeability effects

During the course of investigations on the oxidation

of Krebs cycle intermediates by species of the genus

Streptomyces (8 p5 9 pp57-44) several key substrates

were not oxidized by intact cells These workers resorted

to cell free extracts to overcome the inability of some

substrates to penetrate the cell The data to be preshy

sented indicates that high substrate concentrations can

be used to overcome similar permeability barriers with

~middot griseus cells

In early experiments glutamic acid was rapidly oxishy

dized by intact cells of sectbull griseus Subsequent mole

ratio studies indicated that the product of glutamate

oxidation went beyond the alpha ketoglutarate stage

This observation suggested that permeability might be

responsible for the lack of oxidation of 0003M alpha

ketoglutarate and other key intermediates This premise

was tested by a number of indirect methods ha~ing the

2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES

FIGURE 5 The oxidation of 0167 M succinate and alpha ketoglutarate by intact cells of s griseus

32

common effect of influencing cellular pe~eability

In the present study when the ccn oentration of subshy

strate in the reaction flasks was increased considerably

in excess of that normally used two compounds that were

not metabolized were now oxidized rapidly In both cases

optimal substrate oxidation was attained at the 0167 M

level (Figure 5 As shown in Figure 6 successive in~

creases in oxygen consumption occurred with concommi tant

increases in the concentration of alpha ketoglutarate

The results with succinate though not as pronoWlced

proved to be quite ccnclusive Succinate was oxidized

at concentrations ranging from 003M (Q02N =113) to

ol67M (Qo2N bull 201)

E effects

If the permeability of the cell wall to various subbull

strates is a f unction of the unionized acid concentration

then an increase in the hydrogen ion concentration should

have the same effect as an increase in the substrate conbull

centration Lowering the pH to 6 1n the presence of

0003M substrate slightly but significantly increased the

rate of oxygen ccnsumption ot succinate and alpha ketobull

glutarate over endogenous The activity of the organism

was reduced at pH 55 as was evidenced by lo1Jer rates of

ox~dation of substrates The presence of malate fumarshy

ate or citrate at 0003M concentration inhibited the

200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0


The oxidation of alpha ketoglutarate at various s griseus

60

substrate concentrations by

4 0 50

34

endogenous respiration between pH 55 and 65 Although

the oxidation ot citrate fumarate and malate were not

observed with these procedures it can be assumed that

permeability barriers inhibited the oxidation of 0003M

succinate snd alpha ketoglutarate

Recent publications by Stone and Wilson (38 pp605shy

617) Willi~s and Wilson (44 pp353-360) and particushy

larly Barrett and Kallio (3 pp517-524) showed that

factors other than enzyme compliment may be responsible

for a slow initial rate of utilization or a negative

utilization of substrate These workers and others have

demonstrated conclusively that apparent adaptive lags or

negative utilization can often be attributed to the inbull

ability of a substrate to oross the cell wall

It has been shown in the present study that the

permeability barriers imposed by cells of ~middot griseus may

be overcome by increasing the concentration of substrate

in the oase of succinate and alpha ketoglutarate Alshy

though the oxidation of malate fumarate and citrate were

not obtained even with high concentrations of substrate

it was possible to obtain oxidation of these acids by

preincubating the cells for several hours with a specific

substrate Reference to Figure 7 discloses that malate

and fumarate were oxidized after 4 hours 1n contact with

the cells The oxidation of citrate was observed after

4

_ 300 ~

z

~ ~ 100

120 200 250 300


The oxidation of 0167 M malate and fumarate by s griseus cells after a 120 minute preincubation period

36

a 10 hour preincubation period (Figure 8) This acid

slightly inhibited the endogenous respiration Wltil oxishy

dation occurred The possib1li ty of bacterial contaminashy

tion being responsible for the observed oxygen uptake was

negated by repeatin tne experiments under aseptic condibull

tions

Inhib1 tion studies

Malonic ~middot Garner and Koffler (16 pl39) found

that the respiration of ~middot geiseus on succinate was inshy

hibited by malonate In the present investigation it was

not possible to demonstrate inhibition unless the malonbull

ate was used at concentrations much higher than those

recommended by Pardee and Potter 30 pp241-250) It

was presumed that effective diffusion barriers reduced

the amount or malonate penetrating the cells

Pyrophosphate This 1nh1b1 tor has also been reshy

ported to competitively inhibit the oxidation of succinshy

ate (21 ppSl-88) In the present work pyrophosphate

stimulated the endogenous respiration of ~middot griseus The

degree ot a thuulation increased with concentration of

pyrophosphate from OOlM to 004M As shown in Figure 9

the stimulation of endogenous by 004 pyrophosphate was

very definite The rates of glucose oxidation with and

without added pyrophosphate were identical Similarly

4 00

The oxidation of 0167 M citratamp by s griseua cells after a 550 minute preincub a ti on perio d shy

J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120


The stimulation of the endogenous respiration of s sriseus by 004 M ~

pyrophosphate (X)

39

the rate of 0003M glutamate oxidation was not inhibited

by pyrophosphate Complete inhibttion was observed with

00006 M 2 uM) glutamate However the evaluation of

pyrophosphate inhibition was complicated by the marked

s timula t1on of the endo sen ous It appeared that the

stimulation was responsible to a reaction peculiar to

the endogenous since the rates of 0003M glucose or glushy

tamate oxidation were identical with and without the adshy

dition or pyrophosphate

Fluoroacetate It was shown Figure 8) that citrate

is oxidized by intact cells of~middot griseus Further proof

of tb1 s oxidation was obtained by indirectly 1nhibiting

the oxidation of citrate with fluoroacetate Fluoroaceshy

tate has been shown to inhibit aconitase (32 pp310middot315)

through the format1 on of a fluorotricarboxylic acid which

competes w1 th citric acid for the enz~e site The inshy

hibition by 0003M fluoroacetate of cells oxidizing 0003M

oxalacetate is shown 1n Figure 10 Attempts to detect

citrate 1n the fermentation medium were not successful

This might be expected however since the cell wall was

not freely permeable to citrate Glutamic acid was deshy

tected free 1n the medium after the inhibi t1on of the oxishy

dation of either pyruvate or oxalacetate with fluoroshy

acetate

4

~

UJ

a

Lu

~

The inhibition of 0003 M oxalacetate oxidation with 0003 M fluoroacetate and the stimulation of acetate oxidation by oxalacetate

~

0 l OXALACITATI + AOt TATt

2 ACpoundTATE

I OX ALACITATE

300 bull OXALACCTATE + LUOROAGpoundTATE

200

100

ENDOGtHOU8

30 60 9 0

TIME MINUTES FI GURE 10

0

41

The stimulatimiddoton 1n acetate oxidation noted with

oxalacetate was not regarded as a sparking effect This

observation was included in Figure 10 for comparison with

the fluoroacetate data

II Radiegtaotive Tracer Studies

The initial experiment was designed to demonstrate

any existing relationship between the oxidation of

acetate by non-proliferating cells and the presence of

free glutamic acid 1n the medium Since acetate carbon

was incorporated into cell protein by non-proliferating

cells it appeared logical that a more extensive incorshy

poration would result if a growth supporting medium were

used The extent of this incorporation and the sequence

ot the ~-ynthesis of amino acids from labeled acetate was

detet-mined with glowing cells The probabli ty of carbon

dioxide fixation by growing cells was also investigated

using cl4o2

Incorporation of acetate-middotCl4 ~ non-proliferating ~middot

Radioactive glutamic acid was detected in middotthe medium

throughout the experiment No other nonbullvolatile comshy

pounds were observed The incorporation ot cl4 into

cellular protein was demonstrated with radioautographs

The main activity on the radioautographamp or the protein

42

hydrolysate was found in glutamic and aspartic acids

These acids were identified by their respective Rt valbull

ues No other amino acids present showed significant

actiVi ty

It is interesting to note that the alcohol extract

of the cells contained free glutam1o acid as well as

lipid material Glu tamie acid was isolated as the free

acid and its purity was established by paper chromatograbull

phy The intramolecular distribut1 on of cl4 in the glushy

tamic acid is given in Table 3 The conversion of

acetate to glutamic acid resulted in isotopic labeling

exolusi vely in the carboxyl groups The ratio of activishy

ties 1n the g~a and alpha carboxyls approached 2 to 1

respectively

Incorporation pound acetatebullmiddotCmiddotl4 ~ growing cells

The salient qualitative results were the extensive

incorporation and the apparent sequence of incorporation

of cl4 into the amino acids The number of amino acids

derived from acetate carbon was much greater in the proshy

tein of growing cells bull As witn non-proliferating cells

glutamic and aspartic acids became radioaet1ve early in

the experiment Whereas no other amino acids were

labeled in the case or non-proll1erat1ng cells growing

cells were shown to 1n corporate abullct1vity into other amino

acids 1n the following sequence glycine serine and the

43

TABLE 3

Distribution of cl4 in glutamic acid from non-proliferat~ cells of sectbull e1seus utilizing CH3ol4ooH

Carbon atom Radioactivity

cpm x 107 Per cent of total

Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E

Speeiti c activity (total) is expressed as counts per middot minute per millimole or glutamic acid the activities or 1nd1Vidual carbon atoms are counts per minute per millimole of carbon

44

threonine-alanine group followed by valine proline and

the leucine-isoleucine-phenylalanine group

A radioautograph of the protein hydrolysate further

resolved with two dimensional chromatography demonstrated

that glutamic acid aspartic acid md serine contained

the highest activity at the end of 5 hours These were

followed in order by alanine glycine and threonine

The faster moving group of amit)o acids was not further

resolved Although th~ sequence of synthesis of amino

acids was apparent the separaticn was not complete

enough to permit the formulation of definite conclusions

Incorporation pound cl4o2 2z srowing cells I

The demonstration of cl4 in the protein fraction ot

bull sriseus indicated the participa t1 on of carbon dioxide

in protein synthesis The protein hydrolysate was found

to have a total activity ot 432 x 106 counts per minute~

Since the total activity of the initial radioactive carbull

bon dioxide was 57 x lOi counts per minute this figure

represented a carbon dioxide fixation of B per cent

A radioautograph prepared from a two dimensional

chromatogram (Figlire 11) served to demonstrate the relashy

tive activities of some of the amino acids Aspartic

acid glutamic acid serine glycine and threonine were

radioactive

45

1 bullbullc

FIGURE 11

Radioautograph of a two dimensional chromatogrwm demonstrating the incorporation of radioactivity into amino acids during fixation of cl4o2bull The spots were identified as follows (1) threonine (2) glycine(3) serine (4) glutamate and (5) aspartate

46

Glutamic middot acid and aspartic acid were separated from

the protein hydrolysate Sulsequent degradation prooebull

dures gave the intramoleculax- distribution of x-adio-I

actiVity Only the carboxyl carbons- of aspartic acid

were labeled (Table 4 As shown in Table 51 the alpha

carboxyl carbon of glutamic ~cid contained all ot the

radioactiVity

47

TABLE 4

Distribution or cl4 1n as~trtic acid from growing cells of ~middot griseus utilizing C 02 and non-radioactive pyruvate



Total 610 100

COOH 130 213

CHNH2 oo oo CH2 oo oo COOH 487 797

Specific acivity (total) is expressed as counts perminute per millimole or glutamic acid the activities of individual carbon atoms are counts per minute permillimole of carbon

48

TABLE 5

Distribution of cl4 1n ftutamic acid from growing cells ot 2bull s riseus utilizing C 02 and non-radioactive pyruvate

Carbon atom Radioactiv1 ty

Per cent of total

Total 279 100

Alpha carboxyl 268 96

Gamma carboxyl 011 4

Specific activity (total) is expressed as counts perminute per millimole of glutamic acid the activi tiea ot individual carbon atoms are counts per minute permilltmole of carbon

49

DISCUSSION

The ci trio acid cycle as finally proposed by Krebs

(20 ppl65-l99) for animal tissue has been the subject

of intensive study and of considerable controversy in the

field of microbial physiology The question of the role

of the aforementioned cycle in the metabolism of Streptoshy

mzces cultures has been investigated in the present

study Lines of eYidence in favor of the citric acid

cycle representing the principal oxidative pathway have

been presented

The reliability of the first method of proof is deshy

pendent upcn the validity of the concept that a demonshy

stration of substrate oxidation by intact cells is indicshy

ative of the organization of the enz-mic complem~nt of

the oells If this concept be valid there can be little

doubt that ~middot griseus cells possess constitutive enz-mes

enabling them to utilize acetate pyruvate oxalacetate

alpha ketoglutarate and succinate Only one known oxishy

dative pathway requires the active participation of the

five acids mentioned above the citric aci~ cycle It

should be noted that in the case of succinate and alpha

ketoglutarate recourse was made to the accepted method

(24 p31middot) of using high substrate concentration to overbull

come a limiting cellular permeability The positive rebull

sults obtained through the use of this method were

50

substantiat~d by the observation that glutamic acid is

oxidized beyond the stage of alpha ketoglutarate The

slight inhibition of succinate oxidation observed with

malonate requires further study

The constitutive nature of the enzymes necessary for

the oxidation of malate fumarate and citrate is hoween

not beyond question even though it has been shown that

the presence ot any one of these acids will cause an inshy

crease in oxygen consumption by cells of 2bull griseus The

question or the nature and the effects or the processes

causing a limiting permeability becomes a subject ot

detinite importance In the ease of all three or the

acids oxidation was observed only after long periods ot

complete inactivity Such data are commonly interpreted

as a definite indication of adaptive processes within the

cell and not as manifestations of permeability Whether

or rot critical significance can be attributed to the obshy

servation of adaptive lags in oxidation has been the subshy

ject of several recent publications Barret and Kallio

(5 pp517bull524) demonstrated conclusively that Krebs

cycle intermediates were oxidized by way of the convenshy

tional Krebs cycle despite the tact that intact cells

showed lags in oxidative acti v1 ty towards the same intershy

mediates In the present investigation inhibition

stud es were employed to demonstrate that at least the

51

enzymes required for the synthesis and the oxidation ot

citric acid are part of the normal enzyme complement of

2bull sriseus cells Fluoroacetate itself is not toxic to

oxidative processes The inhibitory action or this acid

is manifested through the fluorotricarboxylic acid formed

by the condensation of oxalacetate w1 th tluoroacetate

(32 pp310middot315) The condensation product inhibits the

oxidation or citric acid by competing tor the enzyme

aconitase The observed inhibition of oxidation by

tluoroacetate inters that citric acid is formed and that

citric acid is further oxidized The stimulatory effect

ot oxalacetate on acetate oxidation though open to sevshy

eral explanations is assumed by the writer to be an exshy

pression ot the requirement for a 4 carbon intermediate

to overcame a rate limiting reaction

Although the large part of the data presented thus

tar is strongly suggestive or the participation or the

citric acid cycle in sane phase ot the oxidative proshy

cesses one observation in particular detracts from the

finality or such a conclusion The demonstration of glubull

tam1c acid accumulation is not a point in favor of a combull

plete cyclic oxidation Such an accumulation could indishy

cate that there is a block in the cycle in the sense or

a rate limiting reaction

On the other hand since no quantitative recovery

52

ot glutamate was attempted no detin1te relationship beshy

tween acetate oxidation and glutamate accumulation can be

inferred The latter point was clarified when it was

shown that radioactive glutamate accumulated during the

oxidation of acetate-l-cl4

Since the exchange reactions resulting in the inbull

corporation of radioactive carbon into cellular protein

were localized in glutamic and aspartic acids the inv~veshy

ment of oxalacetate and alpha ketoglutarate in the oxishy

dation of acetate is apparent Further trom the intrashy

molecular distribution of cl4 in the isolated glutamic

acid (Table 3) it can be seen that the conversion of

acetate to glutamate results in isotopic labeling exclushy

sively in the carboxyl groups Pertinent to th1 s obsershy

vation is the report of Wang bull (43a pp683-68B)

These workers showed that bakers yeast grown 1n the presbull

ence of carboxyl labeled acetate yielded glutamic acid

with activity in the carboxyl carbons but with twice as

much aotivi ty 1n the gamma as 1n the alpha carboxyl It

was made clear that th1 s was the ratio to be expected

if glutamat~ was derived from alpha ketoglutarate formed

tram extensive operation of the tricarboxylic acid cycle

If the cycle stopped at alpha ketoglutarate the glutamate

would have been labeled only in the gamma carboxyl The

extent of labeling in the alpha carboxyl indicated that

53

essentially all of the acetate reacted with bullreeycledt

oxalacetate such extensi ve recycling showed that the

cycle represented a major oxidative pathway rather than

just a means of synthesis of glute-nie acid In the

present work the gamma carboxyl contained 22 times the

activity or the alpha carboxyl Such distribution is

almost in accord with expectations it the citric acid

cycle is assumed to operate extensively The glutamic

acid usedmiddot for the degradation procedures accumulated in

the cell during the oxidation of acetate Therefore not

all of the labeled acetate would react with trecycled

oxalacetate The cyclic activity of same or the endogenshy

ous oxalacetate would be reduced to less than one commiddot

plete turn or the eycle This is reflected in the d1tshy

ference between the cl4 content of the alpha and gamma

carboxyl groups or the glu tamio acid Thus the observashy

tion that the gamma carboxyl cmtained 22 times tbe

activity of the alpha carboxyl is 1n accord with the

operatt on of the tricarboxylic acid cycle

It is significant that the tricarboxylic acid cycle

participates extensively in the s~thesis of the carbon

skelmiddotetons ot some or the amino acids during the growth

or poundbull sriseus on acetate Most or the amino acids deshy

tected in the protein hydrolysate were radioactive It

appears significant that glutamate end aspartate should

54

be the fi~st amino acids to become radioactive during the

growth of ~middot sriseus on labeled acetate Abelson (1 PPbull

335-343) has postulated that many of the other aliphatic

amino acids are synthesized through glutamic and aspartic

acids during the growth or middot ~middot Although it appears

possible that this could also be true in the case or ~middot

sriseus turther work or a more quampltitative nature is

necessary to establish these relationships

The incorporation of radioactivity into alanine

from carboxyl labeled acetate suggests that reactions

similar to the malic enzyme (28a p979) or the Wood

Werkman reaction (43b pl35) could be operative The

removal ot the beta carboxyl group from alpha beta carshy

boxyl labeled oxalacetate would result in carboxyl

labeled pyruvate Since the source ot the tour carbon

condensing partner necessary tor the oxidation of acetate

is not lmown the possibility or a C3-C1 condensation was

investigated The demonstration ot the incorporation of

cl4o2 into the protein fraction of middot griseus leaves no

doubt that this organ1 sm possesses a reaction for the

fixation of carbon d1 oxide As shown in Figure 11 asshy

partic aeid glutamic acid serine glycine and threonine

were radioactive Alanine was not radioactive The

intramolecular dismiddottribution or cl4 in aspartic acid

(Table 4) and in glutamic acid (Table 5) was in accord

55

with a c3-c1 condensation The total activity of asparshy

tic acid was found in the carboxyl carbons The higher

activity in the beta carboxyl carbon is in agreement with

the occurrence of a Cs-Cl condensation A partial equishy

librium with a symetrical four carbon acid such as tum~c

would account for the activity on the alpha carboxyl ot

oxalacetate or aspartic acid (47 p205)

The activity of glutamic acid was located almost exshy

clusiv~ly in the alpha carboxyl carbon This labeling is

in agreement with the existence of an extensive cycling

through an asymetrical Cs compound (43a p687) The low

actvi ty on the gamma carboxyl carbon indicates that the

reversal or a Thunberg type reaction is not occurring to

any extent This data shows that carbon d1 oxide enters

the carbon skeletons or glutamic and aspartic aci ds via

carbon dioxide fixati on and the tricarboxylic acid cycle

The carboxyla1fion of pyruvate leading to malate or oxalbull

acetate (28 pp56-l06) appears to represent a major

pathway of carbon dioxide utilization in ~middot griseus

56

SUMMARY

The oxidation or most or the tricarboxylic acid inmiddot

termediates was demonstrated with intact cells or s-griseus Limiting cellular permeability proved responshy

sible for the lack or oxygen consumption encountered

during the investigations with several intermediates or

the tricarboxylic acid cycle The constitutive nature

of the enzymes required for the formation and the oxishy

dation of citric acid was demonstrated by the inhibition

or oxalacetate oxidation with tluoroacetate

The sequence or the tricarboxylic acid cycle reshy

actions was shom by determining the intramolecular disshy

tribution or cl4 in glutamic acid formed during the oxishy

dation or acetatebulll-cl4

It bas been smwn that a c3cl condens at on coupled

with tricarboxylic acid cycle activity is the major pathbull

way or carbon dioxide fixation by ~middot griseus

The intramolecular distribution of cl4 in glutamic

acid arising from the incorporation or labeled carbon

dioxide further confirmed that the tricarboxylic acid

cycle operates extensively in the terminal respiration

or sectmiddot griseus

57

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2 Adamson D W New synthesis or basic amino acids and glycine Journal of the chemical society 1564-1568 1939

Barrett J T and R E Kallio Terminal respirashytion in Pseudomonas fluorescens component enzymes of the trlc~arboxyilc acid cole Jmiddotoumal of bacteriology 66517-524 1953

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58

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15 Elser H M and w D McFarlane Studies on the metabolism of Streptamtces griseus in relation to the production of s reptomycn Canadian journal of research 26164-173 1947

16 Garner H R and H Koffler Preliminary evidence against the existence of a Krebs cycle in Streptomyces ar1seus Proceedings of the society Of American bacteriologists 51139 1951

17 Gottlieb D and H w Anderson The respiration of Streptomyces sriseus Science 107172-173 1948

18 Horn M J 1 bull Me thods for m1crob1o1og1oal and chemical determinations of essential amino acids in proteins and foods Washington us Govt Publishing office 1950 l2p

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59

21 Leloir L F and M Dixon The action of cyanideand pyrophosphate on dehydrogenaseamp Enzymo1oshygia 281-88 1937

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25 Ma T s and G Zuazaga M1cro-Kjeldahl determinashytion of nitrogen Industrial and engineeringchemistry analytical edition 14280middot282 1942

26 Matsuoka M K Yagishita and H Umezawa Studies on the intermediate ma tabolism of chloramphenishycol production II On the carbohydra~ metabshyolism of Streitomtces venezuelae Japan Jourshynal of medica sc 7 enoe and biology 6161-169 1953

27 Numeroft P et al Biosynthesis of streptomycinI Studieswith cl4 labeled glycine and acetate Journal of American chemical society 761341shy1344 1954

28a Ochoa s A H Mehler and A J Kornberg Biosynshythesis of dicarboxylic acids by carbon dioxide fixa tion Journal of biological chemistry 174 979-1000 1948

28b Ochoa S Biological mechanisms or carboxylationand decarboxylation Physiological reviews 31 56-106 1951

29 Oginsky E L P H Smith and W W Umbreit The action of streptomycin on the respiration of Streptomyces fsectiseus Journal of bacteriology61639-642 95l

60

30

31

32

33

34

35

36

37

38

39

Pardee A bull B and V R Potterbull Malonate inhibitions of oxidations in the Krebs tricarboxylic acid cycle middot Journal of biological chemistry 178241middot 250 1949

Perlman D and G bull H Wagman Studies on the utilibull zation of lipids by Stretomres riseus Jourshynal of bacteriology 632 3-2 2 952

Peters R A and T H Wilson A turther study of the inhibition of aconitase by inhibitor fracbull tion isolated rrom tissues poisoned with fluoroacetate Biochemistry and biophysi~s acta 9310~315 1952

Pridham T G and D Gottlieb The utilization of carbon ccnpounds by some actinomJces as an aid for species determination Joumal of bacteribull ology 56107-114 1948

Schatz A E Bug1e m~d s A W~ksman Streptobull mycm a substance exhibiting antibiotic activbull ity against gram positive and middotgram negative bacteria Proceedings of the society of exshypemiddotr1mental biological medicine 5566-69 1944

Sevcik v Metabolism of ~middot fbiseus Ceskaslov biol 297-303 1952 ( straeted in Chemical abstracts 477596h 1953)

Scevola M E bull and G Valcurone Intermediates of carbohydrate metabolism of Streltomyces ~r iseus Pavia Farm science and techno ogy 762 -624 1952 (Abstracted in Chemical abstracts 47 4422 1953)

Scevola M E and P Zorzoli The formation of an active acetyl radical during the respiration with acetate by s sriseus Pavia Il tarmaco ed science 855~ 1953 (Abstracted in Chembull ical abstracts 48799a 1954)

Stone R w and P w Wilson Respiratory act1v1 ty of cell free extracts from Azotobacter Journal ot bacteriology 63605-617 l952

Stout H A and B Koffler Blochanis try of the tilamentoua tung I Oxidative metabolism of glucose by Penicilli~ chriso~enum Journal of bacteriology 62253-268 95 bull

61

40 Umbreit w w R H Burris and J F Stauffer Manometric techn1ques and tissue metabOliam Minneapolis Burgess Publishing Company 1949 227p

41 Utterbull bull F InterrelationShips of oxalacetic and 1-malic acids in carbon dioxide fixation Jourshynal ot biological Chemistry 188847-863 1951

42 Van Slyke D D D A MacFadyen and P B Hamilton The gaseous determination of amino acids in urine by the ninhydrin -carbon dioxide method Journal of biological Chemistry 150251middot258 1943

43a Wang c H B E Whristensen and v H Cheldelin bullbull Conversion of acetate and pyruvate to glutamicacid in yeast Journal of biological chemistry201683-688 1953

43b Werkman c H and H G Wood Heterotrophic assimishyla tion of carbon dioxide Advances 1n enzymolshyogy 2135-182 1942

44 Williams A M and P bull Wilson Adaptation of Azotobacter cells to tricarboxylic acid submiddot strates Journal of bacteriology 67353-360 1954

45 Woodruff H B and J w Foster Microbiological aspects or streptothricin I Metabolism and streptothricin formation in stationary and submerged cultures of Actinamtces lavendulae Archives of middotbiochemistry 2~0 -315 1943

46 Woodruff H B and M Ruger Studies on the physiology or streptomycin producing strain of Streptomzcea fbiaeua on a proline medium Jourshynal ot bacter ology 56315-321 1948

47 Woods H G The synthesis of liver glycogen in the rat as an indication of intermediary metabolism Cold spring harbor symposia on quantitativebiology 13201middot210 1948

IPPBOYEDS

Redacted for Privacylrtlrtent roflrror of

In Cbmgr of lrJor Redacted for Privacy

Gbrlrren of DrprtrHmt of Drottrlology

Redacted for Privacy

0helrnm of Sshool Orrirtrtr

Redacted for PrivacyDcen of Oreamplrtc 8ohool

Deto thmfi 1r pncrmtod

Ipt by Yrme laglutm

ACKNOWLEDGMENl











sincere gratitude



bull bull







TABLE OF CONTENTS

Page





Cultures 9









Chemical 17











TABLE OF CONTENTS

(continued)

Page




32











LIST OF FIGURES

Figure Page












LIST OF TABLES

Table Page








INTRODUCTION













organisms






2


HISTORICAL









mycin production















4


























--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










ACKNOWLEDGMENl











sincere gratitude



bull bull







TABLE OF CONTENTS

Page





Cultures 9









Chemical 17











TABLE OF CONTENTS

(continued)

Page




32











LIST OF FIGURES

Figure Page












LIST OF TABLES

Table Page








INTRODUCTION













organisms






2


HISTORICAL









mycin production















4


























--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61












bull bull







TABLE OF CONTENTS

Page





Cultures 9









Chemical 17











TABLE OF CONTENTS

(continued)

Page




32











LIST OF FIGURES

Figure Page












LIST OF TABLES

Table Page








INTRODUCTION













organisms






2


HISTORICAL









mycin production















4


























--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61












TABLE OF CONTENTS

(continued)

Page




32











LIST OF FIGURES

Figure Page












LIST OF TABLES

Table Page








INTRODUCTION













organisms






2


HISTORICAL









mycin production















4


























--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










LIST OF FIGURES

Figure Page












LIST OF TABLES

Table Page








INTRODUCTION













organisms






2


HISTORICAL









mycin production















4


























--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










LIST OF TABLES

Table Page








INTRODUCTION













organisms






2


HISTORICAL









mycin production















4


























--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61












INTRODUCTION













organisms






2


HISTORICAL









mycin production















4


























--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










2


HISTORICAL









mycin production















4


























--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










HISTORICAL









mycin production















4


























--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










4


























--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










--

5





















tation medium





6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










6

























7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










7




griseus



















of substrate

8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










8
























- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










- -

9

EXPERIMmTAL METHODS








methods

Manometric Studies

Cultures








Preparation 2 cells




10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










10
























Manometry


11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










11





















of the reaction

Chemical




12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










12























~

13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










13


























--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










--

14

~


























15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










15


analysis








activity


the alpha carboxyl













16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










16




manometer





















17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










17

Chemical





Dowex 3


Iall amino acids

I vacuum distill


glutamic aci~

add CuC03 (5 p318)


HCl gas







18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










18



activity


both carboxyls









glutamic acid

19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










19























ized

20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










20

TABLE 1



Glucose 213(87)+ 256(132) 145(53) 260(82)

Acetate No 190(132) 100(53) 166(82)



Malate 150(87) 190(132) No No







21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










21




studies




















200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










200-------------------------------------------

16

J ~20

w ~

~ Cl =gta z w ()

gtX 0

40

0 10 20 30 40 50 60 TIME MINUTES

FIGURE 1


23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










23











2 -ra-t-io- studies














24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










24

--

0 II)

Q

~--~--~--~--~--~--~----~--~~0

00

00

00

00

0

0 CJ)

co 1-

co II)

~

(1

1r1 3gtt1ld

fl N3~XO

FIGU

RE

2

The

effe

ct o

f sta

rvatio

n on

resp

ira io

n

of s

gris e

us

25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










25

TABLE 2




Acetate 0 62 20 31

Glutamic 120 45 27

26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










26


























27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










27





lation

Substrate oxidation




















200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










200

eo

_J

~120 LampJ ~

~ 0

J

z LampJ () ~ X 0

4

40 50 60



FIGURE 3

0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










0 10 20 40 50 60 70 80

200

160

_J

~ 120


gtshyX 0

40



30


























2

-512

yen

~ CL gt eo z ~ )( 0

4

0 10 20 40 50 60 TIME MINJTES


32














E effects












200

160

J ~

12

w ~ ltt ~ a gt 8 z w ()

gtshy~

40

0 10 20 3 0



60


4 0 50

34


























4

_ 300 ~

z

~ ~ 100

120 200 250 300



36






tions

Inhib1 tion studies


















4 00


J 300=-

LampJ ~

~ ~200

z LIJ

~ ~ 100

650 700 750


300

24

6

0 20 40 60 80 100 120



pyrophosphate (X)

39
























acetate

4

~

UJ

a

Lu

~


~


2 ACpoundTATE

I OX ALACITATE


200

100

ENDOGtHOU8

30 60 9 0


0

41

















using cl4o2







42




actiVi ty










respectively












43

TABLE 3




Total 251 100

COOH 077 307

)OHWH2 CH2 )- - - - - - - - - 002 oa )CH2

COOH 172 68E


44
























radioactive

45

1 bullbullc

FIGURE 11


46







radioactiVity

47

TABLE 4




Total 610 100

COOH 130 213



48

TABLE 5



Per cent of total

Total 279 100




49

DISCUSSION









been presented
















50


























51


























52


























53


























54


























55




















56

SUMMARY





















57

BIBLIOGRAPHY











58











59











60

30

31

32

33

34

35

36

37

38

39











61










role of the citric acid cycle in the metabolism of

Documents