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Terminal respiration in pseudomonas aeruginosa Smith, Roberts Angus 1953

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TERMINAL RESPIRATION IN PSEUDOMONAS AERUGINOSA By ROBERTS A. SMITH, B.S.A* A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURAL MICROBIOLOGY i n the Department of Dairying We accept t h i s thesis as conforming to the standard required from candidates f o r the degree of MASTER OF SCIENCE IN AGRICULTURAL MICROBIOLOGY. Members of the Department of THE UNIVERSITY OF BRITISH COLUMBIA October, 1955© ABSTRACT The conventional tricarboxylic acid cycle is generally accepted as the sole means of terminal respiration in aerobic micro-organisms» Cell-free extracts of Pseudomonas aeruginosa were found to con-tain the condensing enzyme and were able to oxidize a l l the intermediates of the conventional tricar-boxylic acid cycle. In spite of this evidence in favour of the conventional tricarboxylic acid cycle some deviations from the normal scheme were found* Even though an ability to oxidize isocitrate was noted the cell-free extracts had no ability to equilibrate isocitrate with citrate, indicating possession of an impaired aconitase system. Further* more, when citrate was used as substrate a l l attempts to isolate alpha-Ice toglutarate in the fermentation liquor failed* Moreover, the 2,4, dinitrophenylhydra-zone of glyoxylate was easily isolated in relatively large quantities when either citrate or eis->aeonitate were used as substrateso Although glyoxylate was never isolated when isocitrate was used as substrate i t was produced from citrate or cis-aconitate under either aerobic or anaerobic conditions* Since the re-action proceeded in the presence or absence of oxygen i t was assumed to be a hydrolytic cleavage of cis-aconitate© In addition to glyoxylate, succinate was found as a product of the anaerobic degradation of citrate or cis-aconitate and in the presence of the ce l l - extract citrate was readily formed by synthe-i sis from glyoxylate and succinates Succinate was then shown to be oxidized by P.aeruginosa through fumarate and 1-malate to oxalacetate, indicating a similarity to the tricarboxylic acid cycle* These results represent a deviation from the conventional tricarboxylic acid cycle and show that the fragmentary evidence normally accepted is not sufficient to prove the presence of a convention-al tricarboxylic acid cycle© A C K H O W L E D G M E JS T S I wish to thank Dr» J . J.» R. Campbell f o r his kind guidance, keen i n t e r e s t , and enthusiastic co-operation i n carrying out t h i s i n v e s t i g a t i o n . I also wish to thank the nation a l Research Council of Canada f o r the f i n a n c i a l assistance granted to me throughout the course of my graduate work* TABLE OF CONTENTS Page Introduction . 1 H i s t o r i c a l «... 3 Experimental Methods 20 Ba c t e r i o l o g i c a l ., 30 Chemical 22 (a) Citrate Determination ...... 22 (b) Chromatography of Keto Acias 24 (o) Chromatography of Organic Acids ...................... 27 (d) Succinic Acid Determination 28 (e) Chromatography of Amino Acids ••••••• 29 Experimental 31 Discussion • .. • 48 Summary and Conclusions . . . . . . . . . . . . . . . . . o 53 Bibliography ••••<> 54 -1-INTROPUCTION In 1937 Krebs and Johnson (35) proposed the essential reactions of the t r i c a r b o x y l i c acid cycle and established this cycle as the mechanism for t e r -minal r e s p i r a t i o n of carbohydrates i n animal tissue. Since that time, the o r i g i n a l reactions.put fort h by Krebs and Johnson have been added to, and the t r i -carboxylic acid cycle has been accepted almost un-i v e r s a l l y as the terminal respiratory mechanism f o r carbohydrate degradation i n animal and many bacter-i a l t issues. (12) (71) (16) (30.) Campbell and Stokes (12) were the f i r s t to conclude the existence of the t r i c a r b o x y l i c acid cyole i n b a c t e r i a l tissue. Working with P.aeruginosa, the presence of the cycle was established on the evidence that c e l l - f r e e extracts of the organism were able to oxidize the intermediates of the cycle, thereby i n d i c -ating;;, the presence of the necessary enzymes f o r the operation of the cycle. Since only manometric data had been used as the c r i t e r i a f o r testing the existence of the cycle i n P.aeruginosa, i t appeared es s e n t i a l to obtain other supporting data i n order to be able to establish un-equivocally the presence or absence of a t r i c a r b o x y l i c acid cycle i n this micro-organism. -2-To t h i s end work was undertaken to establis h the aotual presence of as many i n t e r -mediates as possible a r i s i n g from the degradation of carbohydrate i n the metabolism of P«,aeruginosa» -3-HISTORICAL Although by 1937 considerable detailed knowledge of the anaerobic degradation of carbo-hydrate was available, our knowledge of the aerobic degradation was fragmentary, Szent Gyorgi (75) had shown that succinate, fumarate, and oxalacetate play-ed some role in the oxidative degradation of carbo-hydrate, but the details of the mechanism were obscure* Szent Gyorgi observed that pigeon breastmuscle tissue when fresh had a high rate of respiration but that the tissue soon became fatigued or the rate of respiration decreased© He was able to show, that the tissue fat-igue was coincident with sucoinate disappearance and that succinate, fumarate, 1-malate or oxalacetate when added in very small amounts rejuvenated the fat« igued tissue® Martius (40) observed that citrate and alpha-ketoglu-tarate had a similar ability to rejuvenate the fatigued tissue when added in catalytic amounts* (Fig# 1 ) and reasoned that citrate and alpha-ketoglu-tarate served only as sources of succinate* In addition to Szent Gyorgi's observations, Szent Gyorgi postulated the Szent Gyorgi shunt, oxalacetic SbH2 > Sb CoIHg 1-malate ^ >Fumarate Sucoinate iy to chromes Fig* 1 SZENT GYORGI SHTJHT -4-Krebs and Johnson (35) investigated the c a t a l y t i c ; action of c i t r a t e i n the oxidative degrad-ation of carbohydrate, and from t h e i r experiments proposed the skeleton of the o i t r i o acid or t r i * oarboxylic acid; (TCAj c y c l e . Since c i t r a t e acted cat-a l y t i o a l l y and none of i t ' s breakdown products aceum-ulated, Krebs reasoned that i t must be removed by a primary reaction and resynthesized. by some subsequent reaotion* With the use of i n h i b i t o r s Krebs was able to show the accumulation of some of the breakdown products of c i t r a t e . In the presence of arsenite, which i n h i b i t s the oxidation of alpha-keto a d d s , alpha-keto»glutarate accumulated during the primary removal of citrate© Therefore Krebs reasoned that the resynthesis of c i t r a t e involved alpha«keto-glutar«i ate© In further work, malonio acid was used as a s t r u c t u r a l i n h i b i t o r of succinic dehydrogenase, and succinate accumulated coincident with o i t r a t e disap-pearance© Again i t was reasoned that succinate had some part i n the proposed resynthesis of c i t r a t e * From his own and Szent Gyorgi*s work Krebs knew the e s s e n t i a l breakdown products of c i t r a t e and he reasoned that from one or more of these breakdown products c i t r a t e must be resynthesized. Each of the known intermediates was then incubated with minced mus* ole tissue under anaerobic conditions and analysis for citrate was carried out. Oxalacetate was the on-ly known product of citrate wMoh reoondensed anaer-obically to yield citrate and this reaction was en* hanoed i f hexose di-phosphate, alpha«*glyoerophosphate, pyruvate or glycogen were present* On the basis of the preceding work, Krebs postulated a cycle (Fig* 2) whioh had the net effect of oxidizing an unknown intermediate, termed a "triose*! for each completion of the cycle* ° 2 oxalacetate 1— citrate <-"triose1* (pyruvate or acetate) FIG* 2 THE CITRATE CYCLE Other investigators suggested further inter-mediate compounds in the oxidation of citrate. Wagner* Jauregg and Rauen (78) found that isocitrate had a re« juvinating effect on fatigued tissue equivalent to that of citrate© Martius (40) showed that cis-aconitate was oxidized as readily as citrate and that muscle tissue was able to convert i t to citrate© Also, Martius sug-gested oxalosuooinate as the oxidation product of iso-citrate since i t was known that this acid produces alpha»keto-glutarate spontaneously in acid solution* Thus, with this information Krebs proposed the t r i * carboxylic acid cycle (Fig* 3) as the terminal respir-atory mechanism for carbohydrate breakdown in animal tissue* " t r i o s e " oxalacetate L • ^ c i t r a t e 1-malate 2 c i s _ a c o n l t a t e fumarate i s o c i t r a t e It * H | -»-HOO ^ succinate > ' alpha-keto-glutarate+CO + ccr " ~ ~ 2 2 -2H Fig* 3 The Tricarboxylic cycle 1937* The r e v e r s i b i l i t y of the reactions invol» ving succinate, fumarate, malate and oxalacetate was shown by the anaerobic production of succinate from oxalacetate* This then showed that succinate arose oxidatively from oxalacetate or reduotively from oxalacetate through 1-malate© The t r i c a r b o x y l i c acids, c i t r a t e , iso» c i t r a t e , and cis^aconitate were placed i n the cycle i n what was considered a l o g i o a l order though no experimental evidenoe was at f i r s t known to est~ abligih t h i s order. (30)» In any tissue synthesizing the t r i c a r b o x y l i c acids the three acids, c i t r a t e , i s o * c i t r a t e and cis«?aconitate are always present. Since t h i s i s the ease i t was not decided whether c i t r a t e was the primary condensation product or only a man-i f e s t a t i o n of a side reaction from eis«*aeonitate© Isocitrate is formed at about nine fold the rate that citrate is formed from cis-aconitate* Therefore Krebs assumed that citrate would be pre-sent only in small amounts i f i t were a side reaction product. Wood, Werkman, Hemingway and Hier (82) using isotopic oarbon dioxide and pyruvate prepared labelled oxalacetate which was further condensed with pyruvate to yield citrate. The citrate was metabolized to the alpha»keto-glutarate level and then the position of the labelling was traced* Labelling was found only in the alpha carboxyl of alpha-keto-glutarate, yet i f citrate were the primary condensation product,, beoausec of i t j ; symmetry, labelling would be expected equally in both the alpha and gamma carboxyl of alpha«keto-glutarate (Pig. 4 ) -8-9 H 3 boon c*Oc C*00H fe H C^ COOH I 2 COOH (fOOH CHOH H^ -COOH COOH COOH C=0 6H9 I 2 +- COo C H 2 8 C O O H COOH C=0 h COOH 1 + (fOOH fa oxalaeetic acid CH.COCOOH o I 2 HO-C-COOH •+-CH„ f 2 COOH COc COOH I CH„ b -COOH • f e I COOH (fOOH I CHg H-C-COOH CHOH COOH COOH CH„ » 2 CHg -4 c=o COOH c i t r i c acid cis-aoonitio acid i s o o i t r i c acid CO, alpha-keto-glutario acid Fig.4. THE THEORETICAL DISTRIBUTION OF LABELLING FROM C0 2 IN THE TCA CYCLE* C*indicates the radioactive isotope G 9-From t h e i r r e s u l t s , Wood, Werkman, Hemingway and Uier concluded that c i t r a t e arose from a side re-action of cis-aconitate and that cis-aconitate was the primary condensation product. On the basis of th i s work c i t r a t e was removed from the scheme for seven or eight years. Ogston (46) did not agree with t h i s i n t e r -pretation and reasoned that c i t r a t e could be the prim-ary condensation product. His- conditions were that an * asymmetric enzyme attacking a symmetrical molecule could di s t i n g u i s h between the i d e n t i c a l groups of the substrate i f : (a) attachment of symmetrical substrate to the asymmetric enzyme occurred at three points. (b) the three s i t e s of attachment on the asymmetric enzyme d i f f e r i n c a t a l y t i c properties. On this basis, c i t r a t e could act as an asym-metric molecule and therefore be the primary condens-ation product. The enzyme aconitase ca t a l y s i s the reaction y i e l d i n g cis-aconitate fandn i s o c i t r a t e from c i t r a t e . (31). At equilibrium there i s 90.9% c i t r a t e , 6.20% i s o c i t r a t e and 2.9% cis-aconitate. However, i n some experiments with muscle tissue pyruvate and oxalacet-ate yielded 51% c i t r a t e (33) and therefore i t was assumed that c i t r a t e could not be the primary con-densation product. -10* Stern and Oohoa (66) using aconitase-free muscle tissue, showed that oxalacetate and acetate yielded c i t r a t e when incubated with t h e i r prepar-ation. This strengthened Ogston's hypothesis and settled the arguement regarding the p o s i t i o n of c i t -rate i n the TCA cycle. The order, c i t r a t e — > cis-aoonitate H> i s o c i t r a t e i s now accepted almost u n i v e r s a l l y . The next s i g n i f i c a n t advance which strength-q ened the evidence f o r the TCA cycle was the elucidation of the mechanism of the condensation reaction. This reaction i s the condensation of a three carbon un i t , pyruvate, and a four carbon un i t , oxal-acetate, to y i e l d a s i x carbon un i t , c i t r a t e , plus one mol of C0 g. At f i r s t i t was believed that a seven carbon unit, oxalocitramalic acid, was the condens-ation product. Martius (41) succeeded i n synthesiz-ing the seven carbon compound but found, however, that i t . was metabolically i n a c t i v e . Stern and Ochoa ( 6 6 ) observed that besides pyruvate, two-carbon units derived from acetoaeetate, f a t t y acids or acetate could condense with oxalacetate to y i e l d c i t r a t e i n pigeon l i v e r preparations. Coenzyme A (CoA) but not adenosinetriphosphate (ATP) was shown to be necessary for this reaction. TTovelli and L i p -mann (45) reported s i m i l a r results using preparations from yeast and Escherichia o o l i . Stern and Ochoa -11-(65) further showed that c i t r a t e synthesis proceeded thro-/iugh two steps: (a) acetate-f- ATP >"active acetate" (b) "active acetate"-*- oxalacetate—-^citrate The condensing enzyme, which catalyses step (b) was found i n many animal and b a c t e r i a l tissues including Azotobacter a g i l i s o Reaction (a) was found to be catalysed by the enzyme phosphotransaoetylase (61) (63)* The reaction involves the CoA-dependent interchange of acetyl-bound and inorganic phosphate© . This f i n d i n g led to the pos-tulate that phpsphotransacetylase catalyses the form-ation of aoetylc- CoA and t h i s compound i s the immedi-ate precursor of the two carbon residues used i n aoe-t y l a t i o n reactions© Direct proof that "active acetate" or acetyl CoA exists was obtained by Lynem and Reiohert (38) who is o l a t e d i t from yeast juice© They also show-ed a c e t y l - Co*l could effect the aoetylation of sulfan-ilamide i n crude pigeon l i v e r extracts with no added acetyl donor© Stern (et al) (68) have since shown that acetyl i - CoA can replace the phosphotransacetylase acetyl phosphate system as an acetyl donor i n the syn-thesis of c i t r a t e by the condensing enzyme© Stadtman (62) presented evidence that CoA i s sulfhydryl compound© Ochoa and Lynen then showed that the acetyl sulfhydryl bond i s energy r i c h (67) thereby f a c i l i t a t i n g acetyl transfers without an added energy source© Bacterial and animal tissue differ in their acetate activating mechanisms (5). When ATP and ace-tate are incubated with a fresh pigeon liver extract, Lipmann and Tuttle (37) have shown that acetyl CoA is formed without the intermediate formation of ace-tyl Phosphate© In bacterial tissue on the other hand, i t has been shown that acetyl phosplate is formed from ATP and acetate by extracts of E©ooli©(65) (24), This compound has also been shown to react with formate in a reversal of the phosphoroolastie reaction to yield pyruvate (72)© Thus pyruvate is an acetyl donor for the condensing reaotion© There are two mechanisms existing for the synthesis of acetyl CoA from ATP, acetate and CoA in mioro-organisms (64)© These are: (a) (1) ATP + CoA =r^CoA - PP AMP (2) CoA - PP+ acetate =^ N acetylv CoA PP Sum: ATP+CoA+acetate ^ acetylvCoA+ AMP-f-PP and (b) (1) ATP+ acetate acetyl-P -t- ADP (2) Aeetyl-P-hCoA^acetylxCoA+Pi Sum: ATP+acetate4-CoAacetylCoA + ADP+Pi Abbreviations used: (1) PP....pyrophosphate (2) AMP...adenosinemonophosphate (3) ADP...adenosinediphosphate (4) Acetyl-P....acetyl phosphate (5) Pi....inorganic phosphate -13-Lipmann et al (7) (36) have shown (a) to ooour both in animal and bacterial tissue* Re-action (a) (2) is implied in that CoA is decomposed by pyrophosphate (PP) and in that the overall re-action is stoichiometric* The enzymes oatalysing reaction (b)(1) and (b) (2) are widely distributed in bacteria (64). Korey has isolated the enzyme for reaction (b) (1) from Streptococcus hemolytious and has shown that the reaotion does not require CoA. Stadtman et al, have purified the enzyme phosphotransaoetylase whioh catalyses reaction (b) (2) and shown it's wide ooour-renoe in bacteria (48)* iBhe net effect of both re-actions (a) and (b) is the formation of acetyl CoA which serves as the acetyl donor in aoetylating re-actions including the oondensing reaction* Krebs (35) was in i t i a l l y confused when he found citrate resulting from the condensation of pyruvate and oxalacetate. Pyruvate,however, has been shown to be an acetyl donor. Gunsalus et al (48) have shown that the pyruvate oxidase factor (PDF) is need* ed specifically for the oxidation of pyruvate (21) by cell suspensions of organisms grown on a complete-ly synthetic medium with glucose as the sole carbon source. POF exists in several forms, one of which has been isolated and characterized as alpha-lipoic aoid. (51). Hg — C - CHg - C - (CHg)4 - COOH S (23) (52) (10) (11) Purified alpha lipoic acid is capable of supporting the growth of Lactobacillus oasei in the absence of -14-acetate (60), Tetrahymena g l e i l i n the absence of protogen (58) and Butryibacterium r e t t g e r i i i n the absence of the B.R. factor (27). < The large number of naturally occuring forms of alpha-lipoic acid such as protogen and B.R. factor are explained by the existence of various d i -s u l f i d e and thiamine derivatives (53) (49) (50) (28) (21). I t has now been shown with E. c o l i extracts together with phosphotransaeetylase and Lactic dehy-drogenase that the coenzymes, CoA, diphosphopyridine-nucleotide (DPU), thiamine pyrophosphate (TPP) alpha l i p o i c acid and Mg are required f o r the d i s -mutation of pyruvate to acetyl phosphate and l a c t i o acid. Thus the condensation of pyruvate and oxalaoet-ate to y i e l d c i t r a t e i s explained through the form-ation of acetyl phosphate or acetyl-CoA i n b a c t e r i a l tissue. There has been evidence for and against the existence of the TCA cycle i n bacteria. Karlsson and Barker (25) working with Azotobacter a g i l i s concluded that no TCA cycle existed i n this organism. This con-clusion was based on the technique of simultaneous adaptation. They grew the organism on aoetate and found that i t did not attack the di-carboxylie -15 acids* Stern and Ochoa (66) i s o l a t e d the condensing enzyme from this organism and Cohen (16 ) along with most other workers concluded that i t must have a TCA oyole* I t was thought that the i n a b i l i t y to attack the t r i - c a r b o x y l i c acids was a permeability effect© Stone and Wilson (71) concluded that Azotobacter  v i n e l a n d i i possesses a TCA cycle since l a b e l l e d ace-tate was incorporated into c i t r a t e , alpha-keto-glutarate and succinate by cell-ifree extracts of this organism* Brewer and Workman (8) working with Aerobaoter aerogenes, which normally attacks c i t r a t e , found c i t r a t e was s p l i t , presumably through cis-aconitate, to y i e l d oxalacetate and acetate* A pathway i n whioh the TCA cycle reaotions are not involved has been suggested by Foulkes (20) f o r the degradation of c i t r a t e by yeas to In the pre-sence of semicarbozide he found no increase i n alpha or beta keto a d d s * Foulkes did f i n d one mol of carbon dioxide was produced per mol of c i t r a t e used* Campbell and Stokes (12) concluded that a TCA cycle functions i n P» aeruginosa since c e l l - f r e e ex-; tracts of this organism grown on either glucose or ace-tate could oxidize the known intermediates of the TCA cycle* In 1920 Thunberg (76) suggested the dioarbox« y l i o acid cycle as a means of terminal r e s p i r a t i o n * This - 1 6 -eycle involves the condensation of two molecules of acetate to yield one molecule of succinate, (Fig.5) CH,C00H O T r CGOH CHgCOOH 2 mols of acetate 2H COOH I C H 3 CO acetate -2H COOH I H -COOH •2H +HOH CH HO0C -HOH succinate fumarate C O O H I C - O ^ I co2 pyruvate COOH CHOH OHg I-COOH malate 2H -2H COOH I _ C=0 - C H 2 COOH oxalacetate Fig. 5 THE THUNBERG-^ KNO OP CONDENSATION OR DI-CARB02YLIC ACID CYCLE. Ajl and Wong ( 4 ) have suggested that the dicarboxylie acid cycle is operative in Aerobacter 14 aerogenes since they found that when methyl C labelled acetate was oxidized by these cells, in the presence of unlabelled TCA carrier compounds, the largest part of the labelling was recovered in the respired G0 2 and not in the carrier compounds, Krebs ( 3 4 ) reported similar results with bakers' yeast and A j l l et al ( 3 ) poportod thcoo re--17-ported these results with Mloroooocus lysodeiktious • Saz and Krampitz (56) emphasize that isotope experiments used with the carrier technique can be interpreted to establish or exclude a given metabol-ic pathway only i f each of the added carriers is in complete equilibrium with the enzymatieally produced intermediates. Saz and Krampitz repeated Ajl's earlier work with M. lysodeiktious and could find no activity in the addedoarrier compounds. However, in another experiment, using enough enzyme prepar-ation to permit isolation of endogenous TCA inter-mediates, Saz and Krampitz found added labelled ace-tate was in complete equilibrium with a l l the inter-mediates of the TCA oyole. These results are a sig-nificant criticism of the carrier technique. AJ1 and Kamen (2) using acetate adapted E.ooli reported further oarrier studies with methyl 14 C labelled acetate. In this work they reoovered the activity in the methylene groups of succinate and found no activity in alpha-keto-glutarate. They concluded that a THunberg type condensation was op-erative in this organism. In other isotope studies Swim and Krampitz (74) have shown that a Thunberg oyole does not func-tion in E.ooli to a significant extent. They found the following reaction occurred anaerobically: -18-1 acetate+ 4 fumarate-t-2H 0 -—> 4 succinate 2C0-i t was founa (73)- wl-tsa earboxyl C 1 4 labelled aoetate that the succinate contained nearly a l l the activity and l i t t l e or none was found in the C0g. In order to determine whether the conversion of acetate to suc-cinate occurred by a TCA oyole or a Thuaberg oonden* sation an experiment using methyl labelled G 1 3 ace-tate was performed. The labelled succinate formed was oonverted to ethylene and its' mass determined. A mixture of ethylenes of mass 29 and 28 was founda If a Thunberg condensation were operative one would expect ethylene of mass 30 only« therefore i t was concluded that acetate was incorporated into succinate by a TCA cycle© In the metabolism of moulds a TCA cycle and a Thunberg condensation have been shown (1) to exist and in addition to these pathways methyl group oxid-ation of acetate has been reported by Hord and Vit-uoci (43) Working with Fomes annosus lord demonstrat-ed the formation of oxalio acid from acetate or gly-oolate. He then suggested the mechanism of methyl group oxidation of aoetate. (Fig.7) CH3 - 2H CHgOH »2E CHO +i|0 2 COOH COOH + H0H> COOH C^OOH COOH acetate glycolate glyoxylate oxalate Methyl group oxidation of acetate. Figure^7 o -19-Both glycolate and glyoxylate have been found in cultures of Aspergillus niger utilizing acetate (6) (15)• Foster (19) does not believe that this mechanism provides for much of the oxidation of acetate* Rat liver has been shown to oxidize gly-colate through glyoxylate and glyoxylate has been shown to transaminate to glycine. (79) Thus pro-tein and oarbohydrate metabolism at least in ani-mal tissue are linked through two carbon units not involving acetyl CoA. Krebs (30) postulated that the intern-mediates in the TCA cycle could serve as synthetic building blocks. Sanadi and LiStlefield (55) (54) and Kaufman (26) have shown the oxidation of alpha-keto- glutarate i s DPN and CoA dependent giving rise to sucoinyl CoA. Shemin et al (81) (57) obtained evidence for the incorporation of succinyl CoA into the porphyrin molecule* Thus Kreb's hypothesis has been greatly strengthened© J -20-Experimental Methods Baoteriologioal: The organism used throughout t h i s work was a strongly pigmented s t r a i n of Pseudomonas  aeruginosa A.T.C.C. 9027* g e l a t i n agar of the following composition: (44) 1% tryptone 0.5% KgHPC-4 0.1% glucose 0.3% g l y c e r o l ; 10.0% l i v e r extract 0.5% agar 2.0% gelatine pH 7.2 After growth was i n i t i a t e d at 30°C, the cultures were ref r i g e r a t e d . Day to day transfers were made on glucose-acetate agar slants of the following com-posi t i o n : used i n manometric studies was of the following composition: I t was maintained i n a l i v e r extract 0.3% glucose 0.1% sodium acetate made to volume with tap water. The medium used for growing c e l l s to be -21-0*6% sodium acetate (anhydrous) 0.3% (KH4)H2P04 0.3% K2HP04 5.0 p$.m. Fe as FeS04o4HgO pH 7.2 distilled water to volume* The medium was sterilized after dispensing in 100 ml. quantities in 500ml.Florence flasks. After cooling 1 ml* of a 10% solution of MgS04 #7H90 was added aseptioally to each flask. One per cent of a 24 hour culture grown in a medium of the same composition was used as inoeulum and the resulting cells were harvested after 22 to 26 hrs. incubation on a Gump shaker at 28°C. The eelIswere washed in one quarter the original volume in M/30 phosphate buffer (pH7.0) and resuspended in a 0.2% solution of KC1(300 mg.wet wt. cells per ml. of 0.2% KC1) for disintegration in a Raytheon 10KG Magnelorestrio-tive Oscillator. 50 mis. of a oell suspension were disintegrated at one time and the duration of os-cillation was 20 mins. Cell-free extraots prepared in this way were centrifuged at 13,000 r.p.m. to remove any residual cells* A conventional Warburg apparatus (77) was used to follow oxygen uptake and COg evolution by the oell free extraots* -22-Chemioal: (a) Citrate Determination Mioro analyses for citrate were carried out by a modification of Ettinger's (18) method. The reagents required are: 1* Sulfuric aeid,9it 2. Metaphosphorio acid, 40% 3. Potassium bromide, 2M 4. Potassium permanganate, saturated solution 5. Hydrogen peroxide, 6fo (kept at 5°C ) 6. Heptane, practical grade 7. Potassium hydroxide, 30% 8. Pyridine, analytical grade 9. Citric acid, stock standard, lOOmgs. anhydrous citric acid in lOOmls.l UH2S04 10* Zinc sulphate (ZnS04«7 HgO), (6 gms.) and copper sulphate (CuSO^HgO), (0.1 gm.) made to lOOmls. note: the pyridine must be redistilled i f i t does o not remain eolourless when heated to 80 C with strong alkali. This method is sensitive to the range from 0 to 40 micrograms of oitrate and therefore only small quantities of a reaction mixture were needed* -23-TTsually an experiment was carried out i n a normal 15 ml. Warburg cup and c i t r a t e determined on the de-proteinized contents. Two mis. (except i n the case of cups containing c i t r a t e as substrate, when only 1 ml. was used) of cup contents were added to a 10 ml. volumetric f l a s k containing 1 ml. of zinc s u l -phate-copper sulphate deproteinizing reagent. (42). The volume was then brought to 10 mis. and the whole mixture f i l t e r e d through Mo. 1 Whatmant f i l t e r s . Four mis. of the f i l t r a t e were used f o r a c i t r a t e deter-mination. To a standard taper ground glass test tube was added H 2S0 4(1 ml., 9M), metaphosphoric acid (0.3 ml., 40%) and test f i l t r a t e (4 mis.). The tubes were then immersed i n anice bath and kept below 15°C throughout the rest of the procedure. One blank tube (containing a l l reagents, but no ci t r a t e ) and four standard tubes containing 10, 20, 30 and 40 ugms. of c i t r a t e respect-i v e l y ( i . e . , 1,2,3 and 4 mis. of a 1:100 d i l u t i o n of standard c i t r a t e ) were run simultaneously with each set of determinations. To each tube was then added KBr (2M, 0.5 ml.) and KMn04 (satBd spl'n, 1.5 mis.). The tubes were then shaken and allowed to stand f or 10 mins. After this 10 min. period decolourization of the excess KMnO. was brought about by the dropwise -24-adaition of H 20 2(6%). Care was taken not to add any excess. Heptane (8 mis.) was then added to each t tube and the tubes shaken for one minute at top speed on a reciprocating shaker. The tubes were quickly replaced i n the ice bath. To another set of pyrex test tubes were added KOH (2 mis. 30%) and pyridine (4 mis.). These tubes were also kept i n the ice bath. Five mis. of the heptane layer were then pipetted to the tubes containing pyridine and these were vigor-ously shaken f o r 30 seconds. The tubes containing pyridine were then placed i n a water bath at 80°C. for 4 mins. and quickly replaced i n the ice bath. Colour development occurs at 80°C. The heptane layer was removed by suotion and 3 to 3.5 mis. of the py-ri d i n e layer were transferred to a micro-colourimeter tube and centrifuged at 2,500 r.p.m. for 10 mins. Colour develpment was then read i n a Fisher c o l o u r i -meter using the 525 mu f i l t e r and the micro-attachment, (b) Chromatography of Keto-Acids The chromatography of keto-aeids was car?-r i e d out by a modification of C a v a l l i n i ' s method (13 (14). The reagents required were: 1. 2,4 dinitrophenylhydrazine (2% i n 2KHC1) 2. Ethyl Acetate (reagent grade) 3. Sodium Carbonate (10%) 4. Hydrochloric acid (concentrated) - 2 5 -5. Ethyl aloohol(95%) 6. Phosphate buffer (pH 7 ©2, E/5) 7© Butyl alcohol (reagent grade) For the chromatographic analysis of keto-acids the reaotions were carried out in large War-burg cups© Total volume of reactants was 15 mis© and usually 25 micromols of substrate were used* The reaction was stopped at the desired time either by the addition of sufficient HgS04( 1© H) to lower the pH to 3.5 or 4©0 and by heating at 80°C for 5 mins© or by the addition of 7©5 mis© of the ZnS04e7HgO, CuSO ©5H 0 deproteinizing reagent© The deproteinized reaction mixture was then eentrifuged at 5,000 r©p©m© for 15 mins© and filtered© The filtrate was placed in a 125 ml© separatory funnel along with 15 mis© of the 2,4, dinitrophenylhydrazine aeagent© This was then placed at 37°G for 1 hr© or allowed to react overnight at room temperature© The aqueous reaotion mixture was then extracted several times with ethyl acetate and the final volume of the extract was 50 mis© The ethyl aoetate layer was then extracted with 50 mis© of 10% sodium carbonate© The sodium oarbonate layer was extracted with 5 mis© of ethyl acetate to remove any free 2,4, dinitrophenylhydrazine© After this extraction the sodium oarbonate -26-layer was brought to a strongly acid reaction with concentrated HCl. The a c i d i f i e d 2,4 dinitrophenyl-hydrazones were extracted with 25 mis. of ethyl-acetate. The f i n a l ethyl acetate extract was evapor-ated to dryness at 50°C. under vacuum. The residue was dissolved i n 0.5 ml. of ethyl alcohol (95%) plus 0.5 ml. of M/5 (pH7.2) phosphate buffer s o l u t i o n . Chromatographic standards were prepared i n the same way using 15 mis. of a solution containing 2.5 mgs. of a xnown compound per ml. of standard. The chromatograms were prepared using No. 3 Whatman f i l t e r paper. Descending type chromatographic chambers were used throughout. Drops of the alcohol-buffer solutions were placed on a l i n e 8 cms. from the edge of the paper with a very fine c a p i l l a r y tube. The spots were placed 2.5 to 3 cms. apart. The chrom-atograms were run for 12 to 16 hours i n a 30°C incuba-tor. After allowing the solvent to dry, the spots were traced over an ultra-violet lamp and the Rf values recorded. Several solvents were used i n separating the 2,4 dinitrophenylhydrazones prepared i n this way, but the most eonsidbent solvent system was n-butanftl 50, water 40, and ethanol (95%) 10. For very rapid separ-ations at room temperature a 1% solution of sodium carbonate was found useful (22). -27-(c) Chromatography of Acids. For t h i s technique the reagents required are ether, M/15, pH7.4, phosphate buffer, and the standard acids prepared i n aqueous solution (5 mgs. acid per ml.). The solvents used here were n-butanol saturated with 4 TS formic acid and ethanol (95%) 86, water 16, and concentrated NH^ OH 4, The chromatograms were prepared i n the same manner as f o r the keto-acids, with the exception that either Whatman f i l t e r paper No. 4 or No. 1 was used. For the development of these chromatograms a spray made up of 200 mgs. of chloro-phenol-red i n 100 mis. of water at pH7.0 was used. The ohromatograms were run from 12 to 16 hours at 30°C or for 5 hours at 37°C and the solvent removed by steam d i s t i l l a t i o n . (59). The steam d i s t i l l a t i o n was carried out by hanging the dry papers f o r 20 to 30 mins. i n a hood i n which water was continuously b o i l i n g . After the papers were dry they were sprayed with chlonphenol-red spray which gave yellow spots for the aoids and a purple background. The spots were outlined immediately i n pencil since they soon faded. The reactions on which this type of chroma-tography were carried out were performed as previous-l y described i n large Warburg cups. The cup contents -28-were deproteinized with the ZnSQ^HgO, CUSO4.5H2O reagent. ( 7 . 5 mis. added to 15 mis. of reaction mixture). The f i l t r a t e was made alkaline and the volume reduced i n an open dish over a steam-bath. This oonentrate was then brought to pH 2.0 with concentrated H2SO4 and then extracted with ether i n a conventional l i q u i d - l i q u i d extraction apparatus for 8 hours. The ether was then evaporated and the acids taken up i n 1 ml. of M/15, pH 7 .4 phosphate buffer solut i o n . The phosphate buffer solution con-tained a l l the acids whioh had been ether extracted and this solution was used f o r chromatography or for the enzymatic succinic acid determination. (d) Succinic Acid Determination The technique for determining succinic acid involves the use of a s p e c i f i c succinic de-hydrogenase extracted from pig heart muscle. ( 4 7 ) . The succinic ddehydrogenase was extracted from pig heart by grinding the pig heart i n a Van Potter homogenizer (1 gm. pig heart to 9 mls.M/15 pH7.4 phosphate buffer) u n t i l an even suspension resulted. The manometric method f o r the determination of succinic acid as outlined by Krebs (32) was used except that the deproteinization and ether extraction was carried out as previously described i n the sec-t i o n on acid chromatography. -29-The ether used must be kept over sodium wire and freshly d i s t i l l e d as needed. For the man-ometric determination 0.2 and 0.5 mis. of the buffer solution were used. As a control, to determine the percent recovery, 50 umols of succinate were added to a reaction mixture made up exactly as that f o r the endogenous test reaction, but the ZnSO^^HgO CUSO4.5H2O deproteinizing reagent were added at once. Usually succinic acid determinations were carried out on reaction performed anaerobically. (e) Chromatography of Amino Acids The analysis for amino acids was carried out on the undiluted c e l l - f r e e extract or on the deproteinized o e l l - f r e e extraot as previously de-scribed. The descending method of paper chroma-tography, developed by Consden et a l (17) was used. A large sheet of No. 1 Whatman f i l t e r paper (22" x 18,f) was used. A pen c i l l i n e was drawn across the sheet about 5 cms. from one end. The solutions to be analysed were applied as spots along t h i s l i n e from the t i p of a Pasteur pipette. Spots were usually 3 cms. apart. Standard solutions of amino acids were made up using 2 to 3 mgs. of amino acid per ml. of water. The solvent used was butanol acetic acid and was made up as follows: n-butanol 40, water 50, g l a o i a l acetic acid 10. -30-These chromatograms were run f o r 12 to 16 hours, dried i n an oven at 90°C fo r 5 mins., spray-ed with ninhydrin, (0.1% i n n-butanol) and again dried at 90°G fo r 5 mins. In order to avoid d i f f i -c u l t i e s a r i s i n g from the fading of the spots, they were outlined i n p e n c i l . -31-Experimental In order to follow the reactions of the TCA cycle i n P. aeruginosa, i t was thought best to follow the u t i l i z a t i o n of acetate by c e l l - f r e e ex-tracts of this organism. Two methods of preparing the c e l l - f r e e extracts were employed; di s i n t e g r a t i o n i n a sonic o s c i l l a t o r , and alumina grinding by the method of Mollwain (39) as recommended by Stone and Wilson (70). Although whole c e l l s were found to oxidize the acetate readily* neither type of c e l l - f r e e extract showed any appreciable a c t i v i t y on th i s substrate. I t was thought that necessary cofactors f o r the oxid-ation of acetate were being diluted out i n the prepar-ation of the c e l l - f r e e extract.. For th i s reason additions of the known cofactors, ATP, DPN, Mn, Mg, Cysteine, CoA, and i n some cases yeast extract, were made to the Warburg vessels along with the enzyme preparation. Even these additions did not increase the a b i l i t y of the c e l l - f r e e extract to oxidize acetate. Sparking the reaction mixture with 1 jimol of fumarate according to the technique of Stone and Wilson (70) gave a s l i g h t increase i n oxygen uptake over the endogenous controls after 2-§- hrs. However, this small a c t i v i t y was not considered as in d i c a t i v e of the u t i l --32 i z a t i o n of acetate. I t was then decided that oxygen consump-tion was not a good c r i t e r i o n f o r acetate u t i l i z -ation, since acetate could be incorporated into the c i t r a t e molecule by a non-oxidative reaction and pos-s i b l y c i t r a t e was not being used by this enzyme pre-paration. Two experiments were then set up to check the non-oxidative u t i l i z a t i o n of acetate. Monofluro-acetate and acetate were used as substrates i n these experiments. Buffa (9) has shown that monfluroacetate w i l l accumulate a f l u r o c i t r a t e molecule which i s not acted upon by aconitase. In these experiments 5 umols of either substrate were incubated f o r a t o t a l of 60 mins. with the c e l l - f r e e extract at pH 7.4 i n a normal Warburg vessel at 30 C. Micro - c i t r a t e determinations were then made on the reaction mixtures. The results are recorded i n Table I . -33-Table I Colorimeter readings f or the c i t r a t e determination* Test Solution 30 mins* 60 mins. acetate cup 98.5 % 98 f0 monofluroacetate cup 99 % 97*5 fo endogenous control 98 % 98 % 15 ugms c i t r a t e 82 % 82 $ reagent blank f 100 % 100% Warburg vessels contained: 5 jamols of substrate 1 ml* c e l l - f r e e extract, 1*5 mis©M/15 pH 7.4 phosphate buffer, w^ter to 3 mis* I t was decided to use pyruvate as an acetyl donor, rather than acetate, since i t was known that c e l l - f r e e extract?, prepared from ? P.aeruginosa as previously described, would take up considerable oxygen. The oxygen consumption was taken as a c r i t e r i o n that either the pyruvate was being d i r e c t l y oxidized or was condensing to y i e l d c i t r a t e and that oxidation of the c i t r a t e was taking place. Pyruvate was incubated with the c e l l -free extract and miciio c i t r a t e analyses were carried out at various time i n t e r v a l s * The accumulation and the u t i l i z a t i o n of c i t r a t e are shown i n Pig.8 I t was FIGURE 8 0 20 40 60 80 10 0 TIME (MINS.) - 3 5 -assumed that c i t r a t e was being formed i n i t i a l l y at a greater rate than i t was being removed. As the condensing reaction augmented the amount of c i t r a t e present, the rate of the reaction removing c i t r a t e was increased. Since i t was established that c i t r a t e was u t i l i z e d by a c e l l - f r e e preparation of P. aerug-inosa, i t was decided to compare the rates of ox-idation of the three t r i c a r b o x y l i c acids, c i t r a t e , cis-aconitate and i s o c i t r a t e . A l l three of these acids were readily oxidized by the preparation, though F i g . 9 shows that c i t r a t e required the leas t oxygen, the other acids were found chromatographio-a l l y to be s l i g h t l y contaminated with succinic acid. In l a t e r experiments, pure i s o c i t r a t e , k i n d l y sup-p l i e d by Dr. K l e i n , was shown to be only s l i g h t l y oxidized by a s i m i l a r preparation. Since a c t i v i t y was found on c i t r a t e , c i s -aeonitate, and i s o c i t r a t e , i t was decided to invest-igate whether alph-keto-glutarate occurred as an intermediate i n the oxidation of these compounds. The method chosen was the formation of the 2,4 d i n i -trophenylhydrazone derivatives of the carbonyl com-pounds which were reaction products. These deriva-FIGURE 9 THE OXIDATION of THE TRICARBOXYLIC ACIDS /Isocitrate TIME (Mins.) Warburg v e s s e l s contained: 5 jumols su b s t r a t e , 1 ml. c e l l -f r e e e x t r a c t , 1.5 mis. M/ PH 7.4 phosphate b u f f e r , water to 3 mis'. -37-tives were formed and chromatographed as describ-ed nnder the section on methods* The results ob-tained from these experiments were not quantitative but did show the f i r s t proof of deviation from, the conventional TCA oyole* Alpha-Ice to-glutarate could not be isolated from the reaction products of either citrate, cis-aconitate or isocitrate. However gly-oxylate and pyruvate were found in rather large quantities from either citrate or cis-aconitate* Moreover, only pyruvate could be isolated as a de-gradation product of isocitrate* This was confus-ing, since i t would appear more logical to write glyoxylate as a product of the cleavage of iso-citrate rather than citrate* The hypothesized cleavage is as follows: COOH I CHOH / H-C-COOH I COOH COOH C^ -H glyoxylate COOH ! CH I 2CH I 2COOH succinate isocitrate Since isooitrate did not yield glyoxylate under aerobic conditions two postulates were formed* A* Isocitrate could be the precursor of gly-oxylate by an anaerobic oleavage* 38-Bo Cis-aconitate could be the precursor of glyoxylate by a hydrolytic cleavage which could occur under aerobic or anaerobic conditions. In order to bear out one or the other of these postulates, experiments were set up i n which the three t r i c a r b o x y l i c acids were tested as pre-cursors of glyoxylate both aerobically and anaerobic-a l l y . For the anaerobic experiments, an atmosphere of nitrogen was used and a small amount of white phosphorous was placed i n the centre w e l l of the Warburg vessel. Several repetitions of these exper-iments were made and the r e s u l t s , given i n Table I I , were always s i m i l a r . -39-Table I I Rf Values of 2,4, Dinitrophenylhydrazones of the reaction products of P •aeruginosa STANDARDS Rf VALUES RECORDED Oxalacetate Pyruvate Glyoxolate Alpha-Jke t oglutara te Acetaldehyde Phenyl&ydrazine 0.31 0.50 0.65 0.41 0.57 0.35 0.90 0.91 Reaction Tested Citra t e (aerobic) 0.51 0.67 0.42 0.57 0.91 Citrate (anaerobic) 0.50 0.66 0.43 0.56 0.90 Cis^Aconitate (aerobic) 0.51 0.65 0.41 0.57 0.89 Cis-Aconitate (anaerobic) 0.50 0.66 0.42 0.56 0.91 I s o c i t r a t e (aerobic) 0.50 0.65 0.89 I s o c i t r a t e (anaerobic) 0.50 0.66 0.90 Citrate ( c e l l - e x t r a c t treated with Dowexl to remove CoA) 0.42 9 0.50 0.66 Endogenous Control 0.50 0.65 0.91 Succinate (aerobic) 0.31 0.50 0.66 Note: A l l Rf values are recorded from chromatograms run at 30.0c. Solvent used was, n-butanol 50, ethanol (95%) 10, water 40. -40-From Table I I i t i s seen that either c i t -rate or cis-aconitate act as precursors of glyoxylate i n the presence or absence of oxygen. Also i t i s seen that i s o c i t r a t e did not y i e l d glyoxylate under any circumstance tested. Moreover, i t i s shown that CoA i s not required for the reaction. These results posed the questions: (a) Does P.aeruginosa have a conventional aeonitase system? (b) What other products r e s u l t i n the de-gradation of ci t r a t e ? In order to answer the f i r s t question an experiment was devised i n which cis-aoonitate or i s o -c i t r a t e were incubated with the c e l l - e x t r a c t at pH 7.4. Inoubation times of 40 and 100 minutes were used: c i t r a t e determinations were then carried out. The results of these experiments are given i n Table I I I . Table I I I The Formation of Citrate from Cis-aconitate. Test Solution 40 mins. l o o mins. Cis-aconitate 64 ugms. 135 jagms. I s o c i t r a t e 11 ugms. 11 ugms. End ogenous 11 jigms. 11 ugms. Warburg vessels contained: 5 umols (870 ngms) substrate, 1 ml. of c e l l - e x t r a c t , 1.5 mis. of M/15 pH 7.4 phosphate buffer, water to 3 mis. -41-From the reaults i n Table I I I i t i s seen that cis-aconitate, but not i s o c i t r a t e , gives r i s e to c i t r a t e . Therefore, i t would appear that P.aer-uginosa does not possess a conventional aeonitase system. This was taken as conclusive evidence that i s o c i t r a t e could not act as a precursor for glyoxylate. To answer the other question, i t was postu-lated that succinate was another product of c i t r a t e degradation. The cleavage was pictured as follows: COOH I. CH2 HD.eC-COOH • I : CHp I COOH -H20 + H 20 COOH I CH If C-COOH I 1*' COOH -H20 c i t r a t e COOH ° C H glyoxylate COOH \ CHp CHp COOH succinate cis-aconitate Twov methods were used to check whether or not succinate occurred as a product of c i t r a t e de-gradation. Citrate was incubated anaerobically with the enzyme-preparation, the reaction mixture deprot-einized, and extracted with ether as previously de-scribed. Succinate was assayed with succinic de-r hydrogenase and also i d e n t i f i e d by paper chromatography. - 4 2 -i n nearly a l l experiments succinate was recovered chromatographically, but i n only one experiment was a quantitative enzymatic assay made. I t appeared that some i n h i b i t o r of succinic dehydrogenase was also present i n the ether extract, since no a c t i v i t y was found even when succinate was added to the extract. The results of the one quantitative enzymatic assay of succinate are given i n Table IV. Table IV Succinate Recovery from Citrate and Cis-Aconitate Reaction Tested Succinate Recovered Cis-aconitate 50 umols. 39 umols. Citrate 50 umols. 32 umols. Succinate 50 umols. 40 umols. Warburg vessels contained: 50 .umols substrate, 10 mis. c e l l - e x t r a c t , 15 mi's. 1/15 pH 7.4 phos-phate buffer, water to 30 mis., white phosphorous, Mgatmosphere. From the above results i t appears that c i t r a t e and cis-aconitate y i e l d succinate almost quantitatively, since i n control experiments an 80% recovery of succinate was found. The ether extracts used i n the enzymatic assay for succinate were chromatographed and the Rf values observed are recorded i n Table V. -43-Table V The Rf values of organic acids© Acid Rf Citrate 0,35 Cis-aconitate 0.31 Fumarate 0.81 succinate 0*72 Malate 0.45 alpha-keto-glutarate 0*48 glycolio 0*51 glyoxylio 0.62 unknown (oitrate substrate) 0.71 0.86 Unknown(eis^aoonitate substrate 0.72 0.86 acetate*fumarate (substrate) 0.45 0.73 0.81 glycolate+fumarate (substrate) 0.44 0.50 0.72 0.80 Solvent: n-butanol saturated with 417 formic acid. From Table V i t is seen that succinate was identified as arising from oitrate and cis-aconitate. Moreover i t is seen that some unknown acid having an Rf value of 0*86 appeared. A l l attempts to identify the unknown acid failed. The Rf values recorded for acetate-fumarate and glycolate fumarate are a result of experiments suggested by Dr. Krampitz (29) to which reference will be made later© Since glyoxylate and succinate were identified as products of citrate de-gradation i t was decided to investigate the reversi-bility of the reaction. To this end glyoxylate and succinate were incubated together with the cell-free extract and glyoxylate and fumarate were also tested^ -44-f o r c i t r a t e production. The r e s u l t s of these ex-periments are recorded i n Table VI. Table VI Citrate from Glyoxylate and Succinate Reaction Tested ugms c i t r a t e J i n 100 mins. 370 ugms glyoxylate 295 ;ugms succinate 113 370 ugms glyoxylate 7.0 295 ugms succinate 13 370 ugms glyoxylate 290 ugms fumarate 10.0 290 jigms fumarate 12.5 endogenous control 7.9 Table VI shows that suocinate and glyoxylate incubated together under a£iFobio conditions i n the presence of the c e l l - f r e e extract of P.aeruginosa w i l l y i e l d c i t r a t e . I t now remained to check the pathway by whioh suocinate was metabolized. Hanometrie and ehromato* graphic data were used and the results given i n Table II and F i g . 10 indicate that suocinate i s oxidized through fumarate and 1-malate to oxalacetate. When succinate was incubated with the c e l l - e x t r a c t and the 2,4, dinitrophenylhydrazone derivatives of the reaction products were ohromatographed, oxalacetate - 4 5 1 FIGURE 10 THE OXIDATION of THE DICARBOXYLIC ACIDS Succinate 20 40 TIME (mins.) 60 80 100 Warburg vessels contained:* .5 jUmols" substrate, 1 ml. c e l l -free extract,-1.5 mis. M/15 pH 7.4 phosphate buffer, water to 3 mis. The c e l l - f r e e extract was found, never able to oxidize oxalacetate. 4 6 -was found. Also, when fumarate and acetate were incubated anaerobically with the c e l l - e x t r a c t Table V shows that fumarate, 1-malate and succinate were recovered. F i g . 10 shows that succinate re-quired 2 atoms of oxygen, whereas fumarate and mal-ate required only 1 atom each to reach oxalacetate. Therefore i t was concluded that succinate was meta-bolized by the conventional pathway through fumarate, and 1-malate to oxalacetate* Previously i t was shown that pyruvate condensed readily to give c i t r a t e . This was taken as s u f f i c i e n t o r i t e r i a f o r the presence of the con-densing enzyme, but the source of a four carbon acid was unknown* Some residual oxalacetate was assumed to be present i n the cell--extract, but i t was also thought that there were s u f f i c i e n t free amino acids present to act as a source of oxalacetate* Amino acid chromatography was carried out on the c e l l -free extract, and the deproteinized extract, the results are recorded i n Table V I I . 47-Table VII AMINO ACIDS PRESENT IN CELL-FREE EXTRACT OF P. AERUGINOSA Rf values recorded Amino Acid Standard C•F.E. 1.5 d i l D . ZnCu° H 2S0 4 a glycine O o l l O o l l 0*12 0.11 0.11 aspartate 0.08 0*08 0.08 0.08 methionine 0*38 0*37 0.38 mm serine 0*15 0.15 mm 0.14 mm leucine 0.52 0.53 mm 0*52 0.53 alanine 0.20 0.21 pm • 0.19 -valine 0.37 0.38 - 0*37 0.39 Solvent used: n-butanol 40, HgO 50, g l a c i a l acetic acid 10. (a) C.F.E© i s c e l l - f r e e extract. (b) a 1:5 d i l u t i o n of C.F.E. to determine which amino acid was i n highest concentration. (c) C.F.E. deproteinized with ZnSO^^HgO, CuS04.5H20 reagent. (d) C.F.E. deproteinized with heat and HgS04. I t was concluded from the results presented i n Table VII that the amino acids l i s t e d were present i n the c e l l - f r e e extract and that glycine was present to the greatest extent. I t was postulated then that aspartate could serve as a source of oxalacetate and that gly-oxylio acid derived from oitr a t e could act as a source of glycine© -48-The existence of the TCA cycle has been concluded i n various tissues and sometimes upon presentation of only fragmentary data. For example, Cohen (16) concluded that the demonstrat-ion of the condensing enzyme was froof of the pre* sence of a TCA cycle. Moreover Campbell and Stokes (12) concluded the presence of a TCA cycle i n P.aeruginosa by demonstrating the presence of the enzymes necessary to oxidize the intermediates of this cycle* The evidence presented i n the previous section would indicate that P.aeruginosa possesses an unconventional TCA cycle, i n that the steps i n -volved from cis-aconitate through i s o c i t r a t e , oxal-osuocinate, and alpha-keto-glutarate to succinate are not present i n this organism. P.Aeruginosa was shown however, to possess the condensing enzyme and the conventional pathway for the oxidation of succinate through fumarate, and 1-malate to oxal-acetate. The oyole i n P.aeruginosa i s pictured as i n F i g . 11. The r e v e r s i b i l i t y of the reaction going from c i t r a t e through cis-aconitate to succinate and glyoxylate was q u a l i t a t i v e l y demonstrated. Ko at-tempt was made to calculate the equilibrium constants -49-AcetylCoA + COOH CH2 HO-C-COOH I 2 CGOH c i t r a t e U COOH C=0 P 2 COOH < +H 20 COOH CH (J-COOH CH„ A OOH oia-aconitate COOH CH0 , COOH i 2 -f- i CHg CHO 6OOH oxalacetate -2H •I-2H COOH CHOH COOH 1-malate -E20 succinate-*- glyoxylate A 4-2H r>2K COOH I COOH fumarate F i g . 11 TEE TRICARBOXYLIC ACID CYCLE IH P.AERUGIHOSA of these reactions sinoe they were oarried out aerobically and presumably suocinate at least was always being removed from the reaction* -50-I t was not always possible to demonstrate glyoxy-la t e as a product of c i t r a t e degradation and though the procedure f o r i s o l a t i n g i t was the same i n each case, variable amounts were found« This led to the b e l i e f that some mechanism existed f o r the removal of glyoxylateo The demonstration of free glycine i n the c e l l ^ f r e e extract coupled with Weinhouse and Friedman 1s (80) evidence that glyoxylate serves as a source of glycine i n animal tissue strengthened th i s b e l i e f , Krampitz (29) suggested that glyoxylate was not a product of c i t r a t e but was an artefact a r i s i n g from a powerful dehydrogenase system. The two carbon u n i t , presumably acetate, acting as sub*-strate for this dehydrogenase system could arise from oxalacetate or c i t r a t e . I t was suggested that acetate and fumarate or glycolate and fumarate be incubated together with the c e l l - e x t r a c t under an* aerobio oonditions« The products of these reactions were the o r i g i n a l substrates plus succinate and 1-malate (Table V) Glyoxylate oould not be demon-strated among the products even when the 2,4, d i n i t r o -phenylhydrazone derivatives were prepared. It was concluded that a hydrogen donor present i n the c e l l -free extract was capable of producing 1-malate and succinate from fumarate. Since glyoxylate was not -51-found and the reaotions were carried out anaero-b i c a l l y i t was assumed that neither the acetate nor the glyoolate present served as hydrogen donors. Although i s o o i t r a t e and alpha-keto-glutarate were not shown to he intermediates i n the cycle presented i n F i g . 11, o e l l - f r e e extracts of P. aeruginosa are known to oxidize these compounds. (12). Sinoe alpha-keto-glutarate i s known to be an important compound i n transamination reactions some other mechanism for i t s formation may e x i s t i n th i s organism. On the other hand, both i s o c i t r a t e and alpha-keto-glutarate may serve as sources of sucoinate as Szent Gyorgi suggested (75). Aconitase i s defined as the enzyme which converts c i t r a t e through cis-aconitate to i s o c i t r a t e . However, P.aeruginosa appears to possess only a par-t i a l aconitase system manifest by i t s i n a b i l i t y to equilibrate i s o c i t r a t e and c i t r a t e . Perhaps then,the reaction normally ascribed to aconitase i s a re-action of two enzymes, not one as usually thought. I f the aconitase reaction i s brought about by two en-zymes, P.aeruginosa may be described as lacking that f r a c t i o n of aconitase which y i e l d s i s o c i t r a t e . This evidence may also be taken as further support to strengthen Kreb's o r i g i n a l hypothesis that c i t r a t e i s -52-the i n i t i a l condensation product of oxalacetate and "active acetate". The cycle found i n Poaeruginosa appears to be the major mechanism f o r the degradation of c i t r a t e i n this organism. Though some steps of TCA cycle were not demonstrable under the experimental conditions used the results reported do not exclude the p o s s i b i l i t y that a normal TCA cycle functions i n this organism. A TCA cycle may function to a much lesser extent than the cycle presented and i f sueh were the case, under the experimental condit-ions used, i t would not be possible to demonstrate the existence of alpha-keto-glutarate as an i n t e r -mediate i n c i t r a t e degradation. In order to strength-en t h i s postulate, a great many i n h i b i t o r studies were oarried out with the hope of i n h i b i t i n g succinic dehydrogenase or alpha-keto-glutarate oxidase* No i n h i b i t o r was found which would block these reaotions and therefore i t remained impossible to fin d the pathway by which alpha-keto-glutarate was synthesiz-ed or oxidized. Krebs (35)was able to use malonate as a s t r u c t u r a l i n h i b i t o r of succinic dehydrogenase and arsenite as an i n h i b i t o r of alpha-keto-glutarate oxidase. However even though these i n h i b i t o r s have been used successfully i n many animal tissues they would not cause the desired i n h i b i t i o n s i n a c e l l r free preparation of P.aeruginosa. -53-Summary and Conclusions 1» P <> aeruginosa possesses the condensing enzyme as evidenced by i t s a b i l i t y to y i e l d c i t r a t e when a c e l l - f r e e extract i s incubated with pyruvates 2o P»aeruginosa does not possess a conventional aconitase system since c e l l - f r e e extracts do not produce c i t r a t e from i s o c i t r a t e but do y i e l d o i t r a t e from ciSr?aconitate© -3. Glyoxylate and succinate are intermediates i n the degradation of o i t r a t e and eis«*aconitate by Peaeruginosa. 4. The reaction of c i t r a t e leading to the formation of glyoxylate and succinate has been shown to be reversible* 5. 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