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A study of the oxidation of 2-Ketogluconate using cell preparations of pseudomonas aeruginosa Campbell, Lorne Arthur 1954

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A Study of the Oxidation of 2-Ketogluconate Using Cell Preparations of Pseudomonas aeruginosa L'ORHE A. CAMPBELL A THESIS SUBMITTED IN PARTIAL FULFILMEI\TT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Dairying We accept this thesis as conforming to the Standard required from candidates for the degree of MASTER OF SCIENCE Memberf/s of the Department of Dairying THE UNIVERSITY OF BRITISH COLUMBIA October 1 '95^ A B S T R A C T P s e u d o m o n a s a e r u g i n o s a i s k n o w n t o d i s s i m i - l a t e g l u c o s e b y way o f a p a t h w a y w h i c h ' d o e s n o t i n  v o l v e p h o s p h o r y l a t i o n a t t h e h e x o s e l e v e l . The e s t a b l i s h e d i n t e r m e d i a t e s i n t h i s p a t h w a y a r e g l u  c o n a t e , 2 - k e t o g I u c o n a t e , p y r u v a t e a n d f i n a l l y t h e c o m p o u n d s o f a n u n c o n v e n t i o n a l t r i c a r b o x y l i c a c i d c y c l e . The m a j o r g a p i n o u r k n o w l e d g e c o n c e r n s t h e f a t e o f 2 - k e t o g l u c o n a t e . ' The e n z y m e s r e s p o n  s i b l e f o r t h e d e g r a d a t i o n o f t h i s c o m p o u n d h a v e p r o v e n t o be v e r y u n s t a b l e a n d p r e v i o u s a t t e m p t s t o o b t a i n a n a c t i v e c e l l p r e p a r a t i o n o r c e l l f r e e e x t r a c t h a v e me t w i t h l i t t l e s u c c e s s . T h e r e i s some e v i d e n c e , t h a t d r y i n g c e l l s i n a n a t m o s p h e r e o f c a r b o n m o n o x i d e p r e s e r v e s t h e e n z y m e i n c u e s t i o n w h i l e i n t e r f e r i n g w i t h t h e c o m p l e t e o x i d a t i o n o f 2 - k e t o g l u c o n a t e . T h i s s h o u l d r e s u l t i n t h e a c c u m u - l a t i o n o f a n i n t e r m e d i a t e p r o d u c t a n d t h u s w o u l d make p o s s i b l e t h e e l u c i d a t i o n o f o n e m o r e s t e p i n t h e s e q u e n c e o f r e a c t i o n s . F o r t h i s r e a s o n t h e w o r k o n m o n o x i d e - d r i e d c e l l s was c o n t i n u e d i n t h e h o p e , t h a t t h e y w o u l d s e r v e a s a s o u r c e o f t h e 2 - k e t o g l u c o n a t e e n z y m e . » T h i s . t e c h n i q u e p r o d u c e d a p r e p a r a t i o n w i t h a g o o d a b i l i t y t o o x i d i z e 2 - k e t o g l u c o n a t e . H o w e v e r , t h e v i s c o u s n a t u r e o f t h e p r e p a r a t i o n made c e n t r i f u g a l s e p a r a t i o n o f t h e r e m a i n i n g l i v e c e l l s a l m o s t i m  p o s s i b l e „ G l u c o s e a n d g l u c o n a t e g r o w n c e l l s when s u s  p e n d e d i n a ^5% s o l u t i o n o f s u c r o s e a n d s u b j e c t e d t o s o n i c v i b r a t i o n s p r o d u c e d a r e p r o d u c i b l e c e l l f r e e p r e p a r a t i o n w i t h a g o o d a b i l i t y t o o x i d i z e 2 - k e t o  g l u c o n a t e . T h i s p r e p a r a t i o n h a d a n o p t i m u m p H o f 7«- a n d a r e s p i r a t o r y q u o t i e n t o f 3» The m e c h a n i s m o f t h i s o x i d a t i o n r e m a i n s u n e x p l o r e d . P y r u v i c a c i d was i d e n t i f i e d a s a p r o d u c t o f t h i s r e a c t i o n . T h e c r u d e s u c r o s e s o n i c a t e was n o t s t i m u l a t e d b y A T P a n d n o p h o s p h o r y l a t i o n c o u l d be d e t e c t e d a e r o - b i c a l l y o r a n a e r o b i c a l l y b y m e a s u r i n g a c i d s t a b l e ( e s t e r ) p h o s p h a t e . The p r e p a r a t i o n was n o t i n h i b i t e d b y 2.5 x 10~ 2 s o d i u m f l u o r i d e . M o r e o v e r , a c r u d e s o n i c a t e o f g l u c o n a t e g r o w n c e l l s i n t h e p r e s e n c e o f A T P a n d m a g n e s i u m , s h o w e d no p h o s p h o r y l a t e d c o m p o u n d s , b y t h e c h r o m a t o g r a p h i c m e t h o d s e m p l o y e d . The s t u d y f o r d e t e c t i o n o f p h o s p h o r y l a t e d c o m p o u n d s was c a r r i e d o u t u n d e r s t r i c t a n a e r o b i c c o n d i t i o n s . No new i n t e r m e d i a t e s w e r e i s o l a t e d i n t h e p a t h w a y o f 2 - k e t o g l u c o n a t e b r e a k d o w n a n d t h i s p a t h w a y s t i l l r e  m a i n s u n k n o w n . H o w e v e r , , t h i s w o r k h a s p r o v i d e d s e v e r a l m e t h o d s o f o b t a i n i n g a c t i v e c e l l p r e p a r a t i o n s . I ACKNOWLEDGMENTS I w i s h t o e x p r e s s ray s i n c e r e t h a n k s t o D r . J . J . R . C a m p b e l l f o r h i s e n c o u r a g e , m e n t a n d i n v a l u a b l e a s s i s t a n c e t h r o u g h  o u t t h e c o u r s e o f t h i s v / o r k , a n d t o D e a n 3. A. E a g l e s f o r h i s h e l p f u l s u g  g e s t i o n s a n d a s s i s t a n c e t h r o u g h o u t t h i s s t u d y . . I a l s o w i s h t o t h a n k t h e N a t i o n a l R e  s e a r c h C o u n c i l f o r a g r a n t t o c a r r y o u t p a r t o f t h i s w o r k . TABLE OF CONTENTS Page Introduction 1 Methods Ba c t e r i o l o g i c a l 11 Experimental 1*+ (a) Methods employed to re duce endogenous r e s p i r a t  ion 15 Cb) Methods 16 Cc) Attempts to obtain repro ducible results with carbon monoxide dried c e l l s 18 Figure I - 2 0 Figure II 21 (a) Attempts to obtain repro ducible r e s u l t s from f u e l gas dried c e l l s 26 Figure I I I 27 Table I 28 Table II 30 Figure IV 3 ^ Table III 36 (b) D i a l y s i s work with whole c e l l s 37 (c) Work with Streptomycin .38 Figure V kO (a) Adapting the pressure crushing technique to enzyme work k-2 Figure VI F i g u r e V i i F i g u r e V I I I T a b l e I V T a b l e V ( b ) S o n i c p r e p a r a t i o n s ( c ) R e s p i r a t o r y q u o t i e n t s F i g u r e I X F i g u r e X F i g u r e X I T a b l e V I ( d ) O p t i m u m pH a c t i v i t y ( e ) A t t e m p t s t o d e m o n s t r a t e p h o s p h o r y l a t i o n F i g u r e X I I ( f ) A n a e r o b i c s t u d i e s w i t h 2 - k e t o g l u c o n a t e F i g u r e X I I I T a b l e V I I I D i s c u s s i o n Summary B i b l i o g r a p h y 1 . INTRODUCTION The universal existence and acceptance of the Embden- Meyerhof scheme as a mechanism for the anaerobic dissimi lation of carbohydrates and the Warburg-Dicken1s system r plus the tricarboxylic acid cycle as mechanisms for the aerobic dissimilation of carbohydrates has caused many workers to view any new metabolic pathway with suspicion, This attitude i s particularly true with the pathway de scribed for Pseudomonas aeruginosa ATCC 9027 ( 2 9 ) . In contrast to the established pathways this one does not i n  volve phosphorylation at the hexose level and therefore seems to contradict our accepted ideas on energy generation and transfer. It has been shown that this organism oxidizes glucose to carbon dioxide and water by way of gluconic, 2-ketogluconic (29) and pyruvic acids (3*0 » The pyruvic acid in turn i s oxidized by way of an unconventional t r i  carboxylic acid cycle ( 8 ) , Studies on oxidation of hexoses are relatively new, the i n i t i a l work being done by Dickens ( 1 3 ) . In a pre liminary note in 1936 he outlined a scheme of carbohydrate oxidation which consisted of the esterification of the hexose to hexosemonophosphate, oxidation of this compound to the phosphohexonate, further oxidation to 2-ketophosphohexonate, decarboxylation to a pentose phosphoric acid. These oxi dations and decarboxylations were pictured, as continuing 2* u n t i l p y r u v a t e was f o r m e d . A t t h a t t i m e e v i d e n c e i n f a v o u r o f t h i s scheme was s l i g h t * No- p r o o f o f t h e f o r m a t i o n o f p e n t o s e p h o s p h o r i c a c i d s b y o x i d a t i o n o f h e x o s e m o n o p h o s p h a t e o r i f p h o s p h o - h e x o n a t e e x i s t e d . The f i r s t d a t a s u p p o r t i n g t h e f o r m a t i o n o f 2 - k e t o p h o s p h o h e x o n i c a c i d a s a n i n t e r m e d i a t e was a d  v a n c e d b y L i p m a n n (22) o n t h e b a s i s o f o x y g e n u p t a k e e x  p e r i m e n t s . L i p m a n n f o u n d t h a t p h o s p h o h e x o n i c a c i d was o x i  d i z e d b y g r o u n d y e a s t , a n d t h a t f o r e a c h m o l e o f p h o s p h o - h e x o n a t e u s e d one a t o m o f o x y g e n was c o n s u m e d , a n d 1 t o 1 . 5 m o l e s o f C 0 2 a p p e a r e d . The a m o u n t o f C0 2 was r e d u c e d t o a b o u t 1 m o l e i n t h e p r e s e n c e o f m o n o b r o m o a c e t a t e . L i p  mann t h e n a d v a n c e d t h e t h e o r y t h a t 2 - k e t o - 6 - p h o s p h o g l u c o n i c a c i d was f i r s t f o r m e d (§02 c o n s u m e d ) , , t h i s was t h e n d e c a r b o x y - l a t e d (ICO2 l i b e r a t e d ) a n d t h e f u r t h e r p r o c e s s c o n s i s t e d o f t h e f e r m e n t a t i o n o f t h e t h e o r e t i c a l l y e x p e c t e d d - a r a b i n o s e p h o s p h a t e . T h i s l a s t s t a g e was t h o u g h t t o be i n h i b i t e d b y b romo a c e t a t e . Some e v i d e n c e t e n d i n g t o sho\j k e t o - a c i d f o r m a t i o n was p r o v i d e d b y e x p e r i m e n t s ( D i c k e n s . 1 9 3 & ( 1 3 ) ) i n w h i c h K C N a p p e a r e d t o a c t a s a k e t o n e f i x a t i v e . I n o t h e r e x p e r i m e n t s a d d i t i o n o f c a r b o x y l a s e t o a c a r b o x y l a s e f r e e enzyme p r e p a r a t i o n c a u s e d a f u r t h e r o x i d a t i o n a n d d e c a r b o x y  l a t i o n o f p h o s p h o h e x o n a t e . W a r b u r g a n d C h r i s t i a n ( 1 9 3 6 ( 3 5 ) ) s h o w e d t h a t t h e " e n d p o i n t " i n L i p m a n n ' s e x p e r i m e n t s was o n l y a p p a r e n t and was i n 3 . i reality due to a gradual destruction of enzyme activity. Dicken's experiments with HCN and carboxylase (13) apparent ly were too indirect, and could not be r i g i d l y interpreted. Later in 1938 Dicken's ( 12) established two c r i t i c a l points: 1 . Pentose phosphoric acids arose from the oxidation and decarboxylation of hexose phosphoric acids. 2, Pentose phosphoric acids were readily oxidized and fermented by yeast extracts. In the f i r s t part of this important paper the oxi dation of phosphohexonic acid was described and the occurence of five carbon phosphoric esters giving the pentose reaction (Warburg & Christian 1937) was confirmed. In addition further oxidation of the phosphohexonic acid to a h carbon phosphoric ester was shown to occur. In the second part of the same paper i t was shown that ribose pentose phosphoric acid was oxidized by yeast extract in the.presence of co enzyme two. In a subsequent paper (Dickens 1938) i t was shown that pentose phosphoric acid was readily fermentable by yeast en zymes in the presence of coenzyme one and inorganic phosphate. The important point which arose during this work was that the pentose phosphoric acid most, readily attacked both oxida- tively and by fermentation was not arabinose - 5 phosphate which would be expected to arise from simple two carbon oxidation and decarboxylation of glucose but was the stereo isomer, ribose-5-phosphoric acid* Ribose i t s e l f was not fermented either by yeast cells or yeast extract, nor was i t oxidized by brain slices or by yeast extract. Dickens advanced the theory that ribose—5-phosphate originated from hexoses by a process of phosphorylation,, oxidation and de carboxylation* Although Dickens' work gave the f i e l d of aerobic met abolism a tremendous impetus i t remained for Smyrniotis and Seigmuller to carry this line of thought to a f r u i t f u l stage. Horecker and Smyrniotis in 1950 (18) purified from yeast the enzyme which catalyzed the oxidation of 6-phosphogluconate. They found that pentose phosphate was produced quantitatively, according to the equation: 6-phosphogluconate 4- TPW ^pentose phosphate + CO2 •*• TPNH2 In addition 85. per cent of this pentose phosphate corres ponded to ribose-5-phosphate on a paper shromatogranu Various other procedures such as the enzymatic removal of phosphate and paper chromatography with four solvent systems confirmed the presence of ribose. Their results placed ribose-5-phosphate in the direct pathway of phosphogluconate oxidation. Finally i n 1951 Smyrniotis and Seigmuller (17) employing a purified yeast preparation showed that glucose was oxidized by way of glucose-6-phosphate and 6-phosphogluconate, They then postulated that the next intermediate i n the sequence was 3-keto-6-phosphogluconate which was decarboxylated to ribu- lose—5-phosphate» Ribulose-5-phosphate was identified as the 5. product of 6-phosphogluconate oxidation hut the postulated intermediate s t i l l remains hypothetical never having been confirmed by experiment,. It was a year later that Etner and Doudoroff (1*0 showed a variation to the previously described scheme of Warburg and Dickens as completed by Smyrniotis and Seig muller.. These workers, using saccarophilia. shoved that the pathway was identical to that previously described with the'exception that 6-phosphogluconic acid was sp l i t to yield 3-phosphoglyceraldelhyde and pyruvic acid... The 3-phospho- glyceraldelhyde was then oxidized to pyruvate. As previously stated the intermediate 3-^eto-6-phospho- gluconate i s s t i l l hypothetical. But, recently in Belgium, J. De Ley (10) employing Aerobacter cloacae concluded that 2-keto-d-gluconic acid i s phosphorylated by a specific kinase when employing adenosine triphosphate and magnesium. This worker concluded that his preparation contained a mixture of enzymes which decomposed the primary phosphorylated pro duct, into unidentified phosphate esters and hence he was un able to i l l u s t r a t e the presence of 2-keto-d-gluconate-6-phosphate. In addition De Ley claims that 2-keto-d-gluconokinase acts only when the bacteria are previously cultivated on 2-keto gluconate or, in other words, that the enzyme i s adaptive in nature. .Basing his argument on the premise that 2-keto-d- gluconate i s formed during the reaction of this kinase, he he f e l t that this compound was an intermediate in the direct oxidation of glucose. Harrod and Wood ( 2 7 ) found that fluorescens A^12 contained a magnesium dependent 2-ketogluconate phosphory- lating enzyme. They used the formation of acid-stable (ester) phosphate and acid formation as a c r i t e r i a for phosphorylation. With crude preparations they found that a 2,l+-dinitrophenylhydrazine reactive keto acid was formed in the presence of both 2-ketogluconate and ATP but not 2-ketogluconate alone» More support for a phosphorylated route comes from Claridge and Werkman ( 9 ) while working with aeruginosa  ^ 0 2 7 . These workers presented data to indicate that two glucose dissimilating mechanisms are operative in this organism. Cell free extracts of this organism were r e  ported to phosphorylate glucose and the presence of triose- phosphate dehydrogenase and aldolase was shown. The aldo lase had only weak activity and had i t s point of optimum activity at pH8.3. This would indicate that glycolysis i s not important i n this organism.- As previously stated the work from our laboratory does not support the phosphorylated mechanisms and favors a scheme which i s operative without phosphorylation at the hexose level., The starting point of this work might be 7.. traced back to Lockwood in 19^0 ( .23) who reported that large quantities of gluconic and 2-ketogluconic acids accumulated during the growth of fluoresceins on a glucose medium.. Barron and Friedmann ( 2 ) later showed that the oxidation of glu cose by aeruginosa was not affected by sodium fluoride and therefore did not involve phosphorylation. Again in 1 9 ^ 6 Lockwood and Stodola (2*+) showed that fluorescens NRRL--B-6 gave alpha ketoglutaric acid as a major product. Also in the same year these workers obtained a preparation which could oxidize pentoses to the corresponding pentonic acids ( 2 5 K Four years later in 1 9 5 0 Koepsell ( 2 0 ) found that fluorescens formed alpha ketoglutarate and pyruvate from gluconate during growth. The accumulation of these acids was Influenced by the iron content of the medium. The above mentioned were interested only in the commercial production of organic acids and were not interested in pathways of metabolism. Koepsell, however,, did mention that 2-keto gluconate undoubtedly was formed by the dephosphorylation of 6-phospho-2-ketogluconate, Norris and Campbell ( 6 ) found detectable amounts of gluconic and 2-ketogluconic acids i n sixteen and twenty- four hour cultures of aeruginosa when glucose was used as the growth substrate. Since the oxidation systems for these compounds remained strong over this period of time they concluded that the compounds must be formed and oxi dized continuously thus being intermediates in a pathway of 8. glucose oxidation. The absence of inhibition by sodium fluoride, the lack of activity under anaerobic con ditions, the absence of phosphorylated hexoses and the quantitative accumulation of 2-ketogluconate with dried c e l l preparations led Campbell and Norris (6) to conclude that, a conventional glycolytic scheme was not present in aeruginosa. Warburton, Eagles and Campbell (3*0 in 1951 demon strated that at the end of sixteen, twenty-eight and forty hours there was an accumulation of pyruvic acid in glucose grown cultures of aeruginosa. In addition, these cultures had a strong a b i l i t y for oxidizing pyru vate. In the same year Campbell and Stokes (7) offered data to show that resting cells had no a b i l i t y to oxi dize citrate cis-aconitate, isocitrate, alpha keto- glutarate, succinate or fumarate, whereas dried cells had the a b i l i t y to oxidize the foregoing compounds and malate, acetate, oxalacetate and pyruvate in addition. These workers concluded that this organism must possess a conventional tricarboxylic acid cycle. Many arguments have been offered for and against this non-phosphorylated scheme. For example, i t has been stated that these compounds are produced as side reactions by-members of the-Warburg^Dickens1 -pathway-. Wood and Schwerdt (36 and 37) have shown that c e l l free 9. extracts of fluorescens have many of the enzymes of the Warburg Dickens pathway but lack hexokinase and there fore are unable to oxidize glucose by way of a conven tional Warburg Dickens. The work of Claridge and Werk- man (9) would appear to refute the data since they have shown that aeruginosa can phosphorylate glucose,. No evidence to date can refute the data that aerug  inosa can oxidize glucose by way of gluconic, 2-keto gluconic and pyruvic acids,- However, no data are avail able on the fate of 2-ketogluconate during oxidation. It was in 1935 that Bernaauer and Gorlick (3) f i r s t demonstrated that 2-ketogluconic acid was produced during bacterial dissimilation. Later Bernhauer and Knoblock (h and 5) showed that when Acetobacter suboxydans was grown on calcium gluconate or glucose plus calcium car bonate, 2-ketogluconate and 5-ketogluconate accumulated. These data were c r i t i c i z e d on the grounds that an impure culture must have been employed since no known organism produces both intermediates. Twelve years after this work Katznelson and Tanen- baum (19) found that Acetobacter melanogenum oxidized glucose, gluconate and 2-ketogluconate to a common end product. This end product was tentatively identified as 2,5-diketogluconic acid. It was characterized chemically but unfortunately i t s ins t a b i l i t y at room temperature and 10. physiological pH prevented i t s being employed for mano- metric studies, McDonald (26) has been able to show that when cells of aeruginosa were dried in vacuo over calcium chloride in the presence of carbon monoxide and the absence of light they retained a limited a b i l i t y to oxidize 2-ketogluconate and for a l l purposes behaved as a c e l l free preparation,, being freely permeable to a l l substrates. Although this work revealed no new intermediates i t was f e l t that continutation of this method would make possible the identification of intermediates between 2-ketogluconate and pyruvate. It was the purpose of this thesis to continue using gas dried cells and also to dis cover any other readily available means for securing a preparation which would have a limited a b i l i t y to oxidize 2-ketogluconate and thus accumulate identifiable inter mediates. Such information might serve as a key to esta blishing the mechanisms whereby 2-ketogluconate i s de graded to pyruvic acid. 1 1 . METHODS B a c t e r i o l o g i c a l T h e o r g a n i s m u s e d t h r o u g h o u t t h i s s t u d y was P s e u d o   monas a e r u g i n o s a A T C C 9 0 2 7 a n d was m a i n t a i n e d o n s t o c k c u l t u r e m e d i a c o n s i s t i n g o f : The s t o c k c u l t u r e s w e r e t r a n s f e r r e d q u a r t e r l y a n d e x a m i n e d m i c r o s c o p i c a l l y a t e a c h t r a n s f e r . S t o c k c u l  t u r e s w e r e s t o r e d a t a p p r o x i m a t e l y 6 ° G . The m e d i u m u s e d f o r o b t a i n i n g a c t i v e c e l l s w i t h a l o w r a t e o f e n d o g e n o u s r e s p i r a t i o n was t h a t o f C a m p b e l l e t * a l . ( 6 ) . T h e med ium was d i s p e n s e d i n 1 0 0 m l q u a n t i t i e s i n R o u x f l a s k s . A f t e r i n o c u l a t i o n t h e s e w e r e i n c u b a t e d a t 3 0 ° C . f o r a p p r o x i m a t e l y 1 8 h o u r s . T h e i n o c u l u m was p r e p a r e d f r o m a s t o c k c u l t u r e b y t r a n s f e r r i n g t o a g l u c o s e a g a r s l a n t a t l e a s t t h r e e t i m e s a t 2k h o u r i n t e r v a l s » When g l u c o n i c a c i d was t h e c a r b o n s o u r c e t h e g r o w t h f r o m a g l u  c o s e a g a r s l a n t was w a s h e d i n t o a R o u x f l a s k c o n t a i n i n g t h e p r e v i o u s l y d e s c r i b e d m e d i u m p l u s g l u c o n a t e . T h i s f l a s k a f t e r i n c u b a t i o n f o r 1 8 t o 2k h o u r s s e r v e d a s t h e s o u r c e o f t h e one p e r c e n t i n o c u l u m f o r t h e l a r g e r v o l u m e o f m e d i a .- G l u c o s e G l y c e r o l T r y o t o n e KpHPOif 0.5? 0.1% 2.0% Y e a s t E x t r a c t A g a r G e l a t i n 12 Sonic disruption of c e l l s Gluconate grown c e l l s f o r c e l l free studies were washed once with M/30 pH 7 . 0 phosphate buffer after harvesting. The resultant c e l l paste was resuspended i n a ^ 5 per cent solution of commercial sucrose at a rate of 2 0 0 to 2 5 0 milligrams of wet c e l l s per ml of sucrose s o l u t i o n . The c e l l suspension was subjected to sonic o s c i l l a t i o n f o r f i f t e e n minutes i n a Raytheon 1 0 k i l o c y c l e o s c i l l a t o r . The resu l t i n g mixture known as the sonicate was placed i n previously c h i l l e d p l a s t i c centrifuge cups and car r i e d i n a chipped i c e bath to a Serval SS-1 centrifuge maintained at approximately - 1 0 ° C . The sonicate was then centrifuged f o r exactly f i v e minutes at 2 5 0 0 0 times gravity and used immediately for mano- metric studies. The unused portion was kept frozen for future use. The c e l l s used f o r studies of the e f f e c t of f u e l gas and varying l e v e l s of i r o n were treated as for desic cation studies except that all-glassware employed was • chemically cleaned. Chemically clean glassware was not used i n the plating procedure. Crushing of c e l l s at low temperatures: Glucose grown c e l l s were harvested as previously de scribed for- treatment by sonic o s c i l l a t i o n . The cylinder of a Carver laboratory press was previously c h i l l e d i n a mixture of dry ice, and alcohol, dry ice and acetone or 1 3 j u s t d r y i c e . The w a s h e d c e l l p a s t e was p i p e t t e d i n t o t h e c h i l l e d c y l i n d e r w h e r e i t i m m e d i a t e l y f r o z e . T h i s was t h e n s u b j e c t e d t o a p r e s s u r e o f 1 2 , 0 0 0 p o u n d s o r u n t i l t h e f r o z e n m a s s m e l t e d w i t h t h e e x t r e m e p r e s s u r e . The r e s u l t i n g p r o t e i n a c e o u s m i x t u r e was t h e n c e n t r i f u g e d a t 5 0 0 0 r e v o l u t i o n s p e r m i n u t e a t a p p r o x i m a t e l y 10°C f o r 1 5 t o 2 0 m i n u t e s . A f t e r t h i s t r e a t m e n t i t was i m m e d i a t e  l y u s e d f o r m a n o m e t r i c s t u d i e s . Ik. EXPERIMENTAL METHODS Previous work with resting cells of Pseudomonas  aeruginosa 9027 has shown that this organism has a strong a b i l i t y to oxidize glucose, gluconate and 2,-keto- gluconate. In addition cells of this organism dried under vacuum with phosphorous pentoxide as a desiccant, retained their a b i l i t y to oxidize glucose or gluconate quantitatively to 2-ketogluconate (32), C e l l free ex tracts also w i l l convert glucose or gluconate to 2-keto gluconate. This quantitative conversion i s possible be cause dried c e l l preparations have no synthetic a b i l i t i e s and therefore do not exhibit oxidative assimilation. In addition they have lost their a b i l i t y to oxidize 2-keto gluconate. Since dried cells are freely permeable and do not oxidatively assimilate, a block in the catabolic pathway w i l l result in the quantitative accumulation of an intermediate. This in turn w i l l make possible the isolation and identification of this intermediate com pound and w i l l aid in plotting the general direction of the pathway. H i l l worked for several years on various modifica tions of this drying technique but found that his treat ments,did not preserve the a b i l i t y of the organism to oxidize 2-ketogluconate. However, McDonald obtained c e l l preparations with some activity towards 2-ketogluconate.(26). These active preparations were obtained by drying resting cells i n vacuo over calcium chloride in an atmosphere of fuel gas or carbon monoxide. Despite the fact that McDonald obtained a prepara tion with activity toward 2-ketogluconate, many major problems remained outstanding. Firstly,, the method of desiccating cells in an atmosphere of carbon monoxide produced a very high rate of endogenous respiration which made a quantitative interpretation of results impossible. Secondly, desiccation over phosphorous pentoxide in the presence of a partial vacuum produced cells, with no activity on 2-ketogluconate suggesting inactivation of this enzyme by oxidation. Finally no available methods of rupturing bacterial cells had yielded a c e l l free preparation cap able of oxidizing 2-ketogluconate* Monoxide Dried Cells The previously described mechanism whereby dried c e l l preparations accumulated intermediates was applicable to McDonald's monoxide drying method. It was f e l t that con tinued use of this method \rould reveal any intermediates between 2-ketogluconate and pyruvate and point to the path way of oxidation of the former compound. The f i r s t problem which had to be approached was the reduction of the high rate of endogenous respiration while maintaining activity against 2-ketogluconate. (a) Methods employed to reduce endogenous respiration. As 16. pointed out by Norris (28) the rate of endogenous r e  spiration i s of particular importance in a study of aerobic microorganisms. The importance may be at tributed to the fact that the endogenous respiration i s normally high and may be completely suppressed in the presence of a readily oxidizable substrate and therefore may be disregarded. More often, however,, i t is only partially inhibited by substrate.. On the other hand a stimulation of the endogenous respiration may result in the presence of oxidizable substrate * Norris and Campbell (28) have shown that the endogenous respira tion of aeruginosa Is unaffected by siibstra.te when this substrate i s glucose., However, i t was later found that this situation differed with different substrates. It seems probable that i f the substrate is dissimilated by a pathway that has nothing in common with the pathway of storage product breakdown, then the rates of the two reactions w i l l be independent. However, i f a single set of enzymes is being competed for one might expect the endogenous to be' suppressed in the presence of oxidiz> able substrate... Methods. The most usual approach to the problem of en dogenous respiration i s to work with cultures whose en dogenous a b i l i t y has been reduced to a point where i t is not considered a serious factory 17. However, i t i s not always possible to achieve t h i s i d e a l and attempts must be made to reduce storage pro ducts., that have accumulated. The f i r s t technique to accomplish t h i s was that used by Quastel and Whetham on resting c e l l s of E.. c o l i . This i s known as the starvation technique ( 3 8 ) . Resting c e l l s of aeruginosa were desiccated according to McDonald's technique. The c e l l s were then suspended i n one hundred to one hundred and f i f t y mis of d i s t i l l e d water and vigorously aerated h- - 6 hours thus forcing the organism to" oxidize i t s stored products. After t h i s treatment the c e l l s were harvested and again treated as previously with monoxide gas. The dried c e l l s were used i n manometric studies and although the endogenous a c t i v i t y was lowered the a c t i v i t y on 2-ketogluconate had disappeared. Several weeks of study using varying aeration times produced negative r e s u l t s . Aging at low temperatures i s a second method used to lower endogenous r e s p i r a t i o n . Monoxide dried c e l l s were placed i n a small test tube which was inserted i n a 125 ml Buchner f l a s k containing a shallow layer of an hydrous calcium chloride. Nitrogen was bubbled through the f l a s k as well as the test tube containing the dried c e l l s , for f i v e minutes. The f l a s k was then sealed shut with screw clamps and stored at -10°C. 1 8 . •, The nitrogen atmosphere was employed i n order to prevent i n a c t i v a t i o n by oxidation.. This procedure did r e s u l t i n the production of dried c e l l s with a low rate of endogenous a c t i v i t y but i t also inactivated the 2-ketogluconate en zyme. Numerous attempts with variations of from 2 - 2h hours storage always gave the same results., A t h i r d procedure c a l l e d for storing t h e ' c e l l s as a wet pack i n the r e f r i g e r a t o r at 1 Q 0 C . Samples were re moved at i n t e r v a l s from k - 2h hours and dried under mon oxide gas. Results show l i t t l e or no change i n endogenous a c t i v i t y and again the c e l l s were inactive against 2-keto gluconate. The next attempt was to vary the time required for drying the c e l l s . The normal procedure produced dried c e l l s i n frora *+8 - 7 2 hours. In order to dry c e l l s quickly they were harvested and then spread over a porous plate with a minimum amount of d i s t i l l e d water. The c e l l s were then scraped from the plate and dried i n the usual fashion. Again as i n the previous methods the r e s u l t s were negative. I t was then f e l t that a high endogenous r e s p i r a t i o n was essen t i a l i f 2-ketogluconate a c t i v i t y was to be maintained with monoxide dried cells., (c) Attempts to obtain reproducible results with carbon monoxide- dried c e l l s . Resting c e l l s have a strong a b i l i t y to oxidize 2-ketogluconate and there i s no accumulation of intermediate • compounds . I t i s t h e r e f o r e e s s e n t i a l t h a t n o l i v i n g c e l l s be i n t r o d u c e d w i t h t h e m o n o x i d e d r i e d c e l l s o r a p r e p a r a t i o n c a p a b l e o f o x i d i z i n g a l l i n t e r m e d i a t e s w o u l d r e s u l t . A l s o t h e o x y g e n u p t a k e d a t a w o u l d a l  w a y s be p o s i t i v e a n d w o u l d be a s s u m e d t o be due t o t h e d r i e d c e l l s . R e s t i n g c e l l s a r e i m p e r m e a b l e t o c i t  r a t e , a n d t h e r e f o r e c a n n o t o x i d i z e i t , w h e r e a s c e l l e x t r a c t s o r d r i e d c e l l s a r e a b l e t o o x i d i z e c i t r a t e . T h e r e f o r e t h e a b i l i t y t o o x i d i z e c i t r a t e c a n be t a k e n a s a c h a r a c t e r i s t i c o f a c e l l p r e p a r a t i o n . C e l l e x  t r a c t s o r d r i e d c e l l p r e p a r a t i o n s h a v e n o s y n t h e t i c a b i l i t i e s a n d t h e r e f o r e c a n n o t f o r m a d a p t i v e e n z y m e s . M a l o n a t e i s o x i d i z e d b y a d a p t i v e e n z y m e s a n d so t h i s s u b s t r a t e c a n be u s e d f o r d e t e r m i n i n g t h e p r e s e n c e o f n o r m a l l i v i n g c e l l s . I n a d d i t i o n p l a t i n g c e l l s o n n u t r i e n t a g a r a n d c o u n t i n g t h e c o l o n i e s c o u l d be u s e d a s a c h e c k o n a n u m b e r o f l i v i n g c e l l s i n t h e d r i e d c e l l p r e p a r a t i o n * P l a t e c o u n t s showed o n l y a f e w c o l o n i e s f o r a 1:100 d i l u t i o n o f 3 m g s . o f t h e c e l l p r e p a r a t i o n a n d ( f i g u r e I ) s h o w s t h e c e l l p r e p a r a t i o n was o v e r w h e l m i n g l y a c e l l f r e e e x t r a c t . The c u r v e s i n ( f i g u r e ^ r e p r e  s e n t d a t a t h a t w e r e o b t a i n e d a t i n f r e q u e n t i n t e r v a l s a n d w h i c h w e r e o f l i t t l e u s e t o t h i s s t u d y b e c a u s e o f t h e i r n o n - r e p r o d u c i b i l i t y . The f o l l o w i n g m e t h o d s , w h i c h 20: T I M E IN M I N U T E S F i g u r e I — T e s t i n g c a r b o n m o n o x i d e d r i e d c e l l s f o r t h e p r e  s e n c e o f l i v i n g - c e l l s b y t h e 1 - I a l o n a t e - C i t r a t e t e c h n i q u e . The o x y g e n u p t a k e i s s h o w n u s i n g 5 m i c r o m o l e s o f s u b s t r a t e . The r e a c t i o n was c a r r i e d o u t a t 3 1 ° C . a n d pH 7.k.. E a c h W a r b u r g c u p c o n t a i n e d 20 mgs o f d r i e d c e l l s , A - G l u c o s e B - C i t r a t e C - 2 - k e t o g l u c o n a t e D - E n d o g e n o u s a n d m a l o n a t e 21. 100 140 I 80 T I M E IN M l N U T E S F i g u r e II - T e s t i n g t h e a c t i v i t y o f c a r b o n " m o n o x i d e d r i e d c e l l s o n v a r i o u s s u b s t r a t e s . The o x y g e n u p t a k e i s shown u s i n g 5 m i c r o m o l e s o f s u b s t r a t e . i 'he r e a c t i o n was c a r r i e d o u t a t 3 1 ° C . a n d p H E a c h W a r b u r g c u p c o n t a i n e d 20 mgs o f d r i e d c e l l s . A - G l u c o s e B - G l u c o n a t e C - 2 - k e t o g l u c o n a t e D - E n d o g e n o u s • 2 2 . w e r e u s e d o v e r a p e r i o d o f s e v e r a l m o n t h s , w e r e e m  p l o y e d i n a n e f f o r t t o l i m i t t h e v a r i a b l e s e n c o u n t e r e d d u r i n g t h e g r o w t h a n d d r y i n g a n d t h e r e f o r e g i v e a p r e  p a r a t i o n t h a t c o u l d be r e p r o d u c e d o n a n y o c c a s i o n . B y d r y i n g c e l l s u n d e r a p a r t i a l v a c u u m o f h y  d r o g e n M c D o n a l d h a d p r o d u c e d a c e l l p r e p a r a t i o n w i t h a weak a b i l i t y t o o x i d i z e 2 - k e t o g l u c o n a t e ( 2 6 ) . I t was f e l t t h a t a m i x t u r e o f c a r b o n m o n o x i d e a n d h y  d r o g e n g a s w o u l d be m o r e e f f e c t i v e t h a n c a r b o n m o n  o x i d e a l o n e s i n c e h y d r o g e n w o u l d e x e r t a r e d u c i n g e f f e c t o n a n y s u l f h y d r y l e n z y m e s . A l t h o u g h t h e c o m  b i n a t i o n o f g a s e s w h i c h c o u l d be u s e d a r e i n n u m e r a b l e i t was f e l t t h a t a m i x t u r e o f h y d r o g e n a n d c a r b o n m o n o x i d e w o u l d b e s t s e r v e t h e p u r p o s e . H y d r o g e n was m a n u f a c t u r e d b y d r o p p i n g 6 N HC1 o n  t o m o s s y z i n c a n d p a s s i n g t h e r e s u l t i n g g a s t h r o u g h w a t e r t o a b s o r b a l l H C l f u m e s . The c a r b o n m o n o x i d e was m a n u f a c t u r e d a s d e s c r i b e d p r e v i o u s l y . . B o t h g a s e s w e r e p e r m i t t e d t o m i x f r e e l y i n a B u c h n e r f l a s k b e f o r e e n t e r i n g t h e d e s i c c a t o r . The r e s u l t s w e r e a g a i n d i s  c o u r a g i n g a n d t h e r e was l i t t l e d i f f e r e n c e b e t w e e n m o n  o x i d e d r i e d c e l l s a s o p p o s e d t o m o n o x i d e p l u s h y d r o g e n d r i e d c e l l s « The n e x t s t e p was t o go b a c k t o t h e t h e o r y t h a t a p a r t i a l v a c u u m a l l o w e d o x i d a t i o n a n d h e n c e i n a c t i v a t i o n o f t h e 2 - k e t o g l u c o n a t e e n z y m e . I t was d e c i d e d t h a t , a p e t r i d i s h f i l l e d w i t h s m a l l p i e c e s o f w h i t e p h o s  p h o r o u s c o u l d be p l a c e d i n t h e d e s i c c a t o r d u r i n g t h e e v a c u a t i o n a n d t h u s a b s o r b a l l t h e o x y g e n p r e s e n t . T h e s e p r e p a r a t i o n s w e r e a l s o i n a c t i v e . M a n y v a r i a  t i o n s o f t h i s theme w e r e a t t e m p t e d , s u c h a s , c a r b o n m o n o x i d e p l u s h y d r o g e n d r i e d i n t h e p r e s e n c e o f w h i t e p h o s p h o r o u s , o r h y d r o g e n o r c a r b o n m o n o x i d e a l o n e w i t h w h i t e p h o s p h o r o u s i n t h e d e s i c c a t o r . B u t a l l o f many d u p l i c a t e o r e v e n q u i n t u p l i c a t e t r i a l s p r o d u c e d n e  g a t i v e r e s u l t s . M o s t o f t h e p o s i t i v e r e s u l t s w e r e o b t a i n e d w i t h p r e p a r a t i o n s w h i c h h a d b e e n d r i e d i n t h e a b s e n c e o f l i g h t . I t was f e l t t h a t 20-30 m i n u t e s r e q u i r e d f o r the 1 " g a s s i n g p r o c e d u r e m i g h t h a v e b e e n a s u f f i c i e n t e x p o s u r e t o l i g h t t o i n a c t i v a t e t h e c e l l s . D u r i n g d r y i n g t h e d e s i c c a t o r w a s s t o r e d i n t h e d a r k . . T o c h e c k o n t h i s p o s s i b l e s o u r c e o f t r o u b l e t h e d e s i c c a t o r was e n c a s e d i n a . b o x c o m p l e t e l y s e a l e d o f f f r o m a n y l i g h t , s u b j e c t e d t o g a s s i n g a n d l e f t t o d r y w i t h o u t e v e r l e a v i n g t h e b o x . S e v e r a l a t t e m p t s a t t h i s p r o c e d u r e g a v e r e s u l t s c o m p a r a b l e t o t h e n o r m a l d r y i n g p r o c e d u r e u n d e r m o n  o x i d e a n d w e r e a b a n d o n e d . A c o m b i n a t i o n o f c a r b o n m o n  o x i d e , h y d r o g e n a n d w h i t e p h o s p h o r o u s w i t h c e l l s d r i e d 2k. i n c o m p l e t e d a r k n e s s a l s o p r o v e d f u t i l e a n d a g a i n p r o d u c e d a n i n a c t i v e p r e p a r a t i o n . I n a d d i t i o n a n enzyme i s m u c h m o r e s t a b l e i n t h e p r e s e n c e o f i t s s u b s t r a t e so t h e . a b o v e p r o c e d u r e was a p p l i e d t o c e l l s d r i e d i n t h e p r e s e n c e o f 2 - k e t o g l u c o n a t e . A g a i n , a f t e r n u m e r o u s a t t e m p t s r e s u l t s w e r e n e g a t i v e o r n o t r e p r o d u c i b l e . S a l t s o f b i l e s u c h a s g l y c o c h o l a t e h a v e b e e n shown t o h a v e a s o l u b i l i z i n g e f f e c t o n v a r i o u s e n z y m e s a n d h a v e b e e n p a r t i c u l a r l y s u c c e s s  f u l i n t h i s l a b o r a t o r y when u s e d t o s o l u b i l i z e t h e g l u c o n a t e o x i d i z i n g e n z y m e . I t was f e l t t h a t t h e s e s a l t s m i g h t e x e r t a s o l u b i l i z i n g a c t i o n o n d r i e d c e l l s a n d t h e 2 - k e t o g l u c o n a t e a c t i v i t y m i g h t be p r e s e r v e d i n t h e s u p e r n a t a n t f l u i d . D r i e d c e l l s a t 20 m g s / m l w e r e s u s p e n d e d i n 1 g m , o f g l y c o c h o l a t e a t a f i n a l c o n  c e n t r a t i o n o f 10^ a n d l e f t a t room t e m p e r a t u r e f o r v a r y i n g p e r i o d s . T h e c e l l s w e r e t h e n c e n t r i f u g e d a n d t h e a b i l i t y o f t h e s u p e r n a t a n t t o o x i d i z e 2 - k e t o g l u  c o n a t e t e s t e d . The r e s u l t s w e r e n e g a t i v e . The n e x t c o u r s e t h a t r e m a i n e d w a s t o s e e i f d r y  i n g h a d c a u s e d t h e p r e p a r a t i o n t o l o s e i t s a b i l i t y t o s y n t h e s i z e c o e n z y m e s t h u s a c c o u n t i n g f o r t h e l a c k o f a c t i v i t y . The c o - f a c t o r s w e r e made u p a s f o l l o w s a n d w e r e u s e d a t t h e s e c o n c e n t r a t i o n s f o r m a n o m e t r i c s t u d i e s : -2 5 . C o e n z y m e A". - 2 0 . 0 0 u n i t s / ' . l m l D P N - 0 . 0 2 m i c r o m o l e s / . 1 m l G l u t a t h i o n e - 5 0 . 0 0 m i c r o m o l e s / . 1 m l C y s t e i n e - 0.30 m i s o f a 20% s o l u t i o n D e s p i t e v a r i o u s c o m b i n a t i o n s o f t h e s e c o - f a c t o r s n o s t i m u l a t i o n was d e t e c t e d . M a n y w e e k s w e r e s p e n t t r y  i n g t o s t a b i l i z e t h e d r i e d c e l l p r e p a r a t i o n so t h a t a r e p r o d u c i b l e p r e p a r a t i o n c o u l d be e m p l o y e d f o r f u r t h e r s t u d i e s . C o u n t l e s s m a n o m e t r i c s t u d i e s p r o d u c e d n e g a  t i v e r e s u l t s a n d t h e e x h a u s t i v e s t u d y w i t h t h e M c D o n a l d t e c h n i q u e o f m o n o x i d e d r i e d c e l l s w o u l d n o t a p p e a r t o p r o d u c e a s t a b l e w o r k a b l e p r e p a r a t i o n . . F u e l Gas Dried C e l l s F u e l g a s h a d t h e f o l l o w i n g c o m p o s i t i o n : : hi.7% h y d r o g e n 27 .h% c a r b o n m o n o x i d e lh.h% m e t h a n e 7*7% n i t r o g e n 3«Q% u n s a t u r a t e d g a s e s e t h y l e n e , p r o p y l e n e , a c e t y l e n e 3.6^ c a r b o n m o n o x i d e O.h% o x y g e n A l t h o u g h M c D o n a l d d r i e d c e l l s i n a n a t m o s p h e r e - o f f u e l g a s a n d o b t a i n e d n o a c t i v i t y w i t h them i t was f e l t t h a t t h i s g a s s h o u l d be a g o o d a t m o s p h e r e i n w h i c h t o d r y t h e c e l l s p r o v i d e d c h e m i c a l t r a p s c o u l d be i n t r o d u c e d t o r e  move a n y t o x i c e l e m e n t s . F u e l g a s p r o v i d e d a n a t m o s p h e r e c o n t a i n i n g t h e h i g h p e r c e n t a g e o f c a r b o n m o n o x i d e a n d h y d r o g e n , b o t h o f w h i c h p r e v i o u s l y p r o v e d s u c c e s s f u l f o r d r y i n g c e l l s , i n a c o n v e n i e n t a n d i n e x p e n s i v e s t a t e . . 26.. T h e g a s was p a s s e d t h r o u g h a l k a l i n e p y r o g a l l o l 0*5% KOH a n d 15% p y r o g a l l i c ) t o r e m o v e o x y g e n ; w a t e r , t o c a t c h a n y w a t e r s o l u b l e compound i n t h e f u e l g a s ; a n o t h e r p y r o g a l l i c t r a p t o r e m o v e r e m a i n i n g t r a c e s o f o x y g e n , a n d c o n c e n t r a t e d s u l f u r i c a c i d t o d r y i t b e f o r e f i n a l l y e n t e r i n g t h e v a c u u m d e s i c c a t o r . T h e d e s i c c a t o r was e v a c u a t e d b y v a c u u m pump a n d a f t e r r e  f i l l i n g t h r e e t i m e s was l e f t w i t h a p a r t i a l v a c u u m i n a n a t m o s p h e r e o f f u e l g a s . A s c h e m a t i c d r a w i n g s h o w s t h e e s s e n t i a l s o f t h e s e t u p ( f i g u r e I I I ) . A g a i n , a s w i t h m o n o x i d e - d r i e d c e l l s , f u e l g a s - d r i e d c e l l s g a v e r e s u l t s w h i c h w e r e n o t r e p r o d u c i b l e . . M a l o n a t e - c i t r a t e a n d p l a t i n g t e c h n i q u e s p r e v i o u s l y d e s  c r i b e d showed t h a t a c e l l p r e p a r a t i o n h a d b e e n p r o d u c e d t h u s w a r r a n t i n g f u r t h e r e x p e r i m e n t a t i o n . ( a ) A t t e m p t s t o o b t a i n r e p r o d u c i b l e r e s u l t s f r o m f u e l g a s . d r i e d c e l l s . I n a g r e e m e n t w i t h S t e r n a n d M e l n i c k i n 19kl ( 3 1 ) i t was f e l t t h a t f u e l g a s b o u n d t h e i r o n o f c y t o c h r o m e i n t o a n i r o n - m o n o x i d e c o m p l e x . I t was f e l t . t h a t a n y e x c e s s i v e i r o n m i g h t a c t a s a c a t a l y s t f o r tfee o x i d a t i o n o f s u l f h y d r y l g r o u p s d u r i n g t h e d r y i n g p e r i o d a n d so e x p e r i m e n t s w i t h k n o w n l i m i t i n g a m o u n t s o f i r o n w e r e c a r r i e d o u t . C h e m i c a l l y c l e a n g l a s s w a r e a n d i r o n a d d e d a s f e r r o u s s u l p h a t e s e p t a h y d r a t e w e r e e m p l o y e d . 2 7 . B F U E L G A S T R A P S DIAGRAMMATIC F i g u r e I I I - E s s e n t i a l s o f s e t u p u s e d i n f u e l g a s d r y i n g t e c h n i q u e S q u a r e s A t o F r e p r e s e n t a 2 5 0 m l B u c h n e r f l a s k c o n t a i n i n g t h e f o l l o w i n g : A - S o u r c e o f f u e l g a s B - A l k a l i n e p y r o g a l l o l (*+5$ KOH a n d 15% p y r o g a l l i c a c i d C - W a t e r D - A n a d d i t i o n a l p y r o g a l l o l t r a p E - C o n c e n t r a t e d B^SOlj . F - A n e m p t y B u c h n e r t o r e c e i v e t h e d r y g a s G - V a c u u m pump H - V a c u u m g u a g e I - V a c u u m D e s i c c a t o r 28 The ' m i n e r a l m e d i u m o f C a m p b e l l e t a l . (6) w i t h t h e f o l l o w i n g v a r i a b l e s was d i s p e n s e d i n t o R o u x f l a s k s . F l a s k 1 F l a s k 2 F l a s k F l a s k F l a s k 5 F l a s k -6 F l a s k 7 No a d d e d i r o n 0 . 1 ppm i r o n a u t o c l a v e d i n m e d i u m 0 * 1 ppm i r o n a d d e d a s e p t i c - a l l y 2 . 0 ppm i r o n a u t o c l a v e d i n m e d i u m 2 . 0 ppm i r o n a d d e d a s e p t i c a l l y 5 . 0 ppm i r o n a u t o c l a v e d i n m e d i u m 5 . 0 ppm i r o n a d d e d a s e p t i c a l l y C e l l s w e r e h a r v e s t e d a n d t h e n d r i e d u n d e r f u e l g a s . T h e d r i e d c e l l s w e r e u s e d i n t h e V7arburg r e a c t i o n v e s s e l a t t h e r a t e o f 20 mgs p e r c u p . T a b l e 1 s h o w s t h e r e s u l t s o f t h e p r e l i m i n a r y m a n o m e t r i c t r i a l s * TABLE I THE E F F E C T OF A D D I N G V A R Y I N G L E V E L S OF I R O N S u b s t r a t e I r o n a d d i t i o n ..- ppm T r e a t m e n t O x y g e n U p t a k e 2 - k e t o g l u c o n a t e 0 ( O x i d a t i o n - n o t c o m p l e t e d a t 3 h o u r s ) - 1 7 T ti 0 . 1 a d d e d a s e p t i c a l l y 1 2 5 tt 0 . 1 a u t o c l a v e d I n 1 2 5 it 2 . 0 a d d e d a s e p t i c a l l y 0 u 2 . 0 . a u t o c l a v e d i n 0 l i  5 . 0 add.ed__ a s e p t i c a l l y 0 ft 5 . 0 a u t o c l a v e d i n o. 29, F r o m t a b l e I i t w o u l d a p p e a r - t h a t a c t i v i t y to w a r d s 2 - k e t o g l u c o n a t e d e c r e a s e d w i t h i n c r e a s i n g c o n  c e n t r a t i o n s o f i r o n i n t h e g r o w t h med ium, , I n a d d i t i o n , c e l l s g r o w n w i t h n o a d d e d i r o n h a d a n u n i m p a i r e d a b i l i t y t o o x i d i z e t h e s u b s t r a t e t o c o m p l e t i o n , s h o w  i n g n o e v i d e n c e o f a b l o c k i n t h e p a t h w a y o f o x i d a t i o n . The r e s u l t s w e r e i n a c c o r d w i t h t h e o r i g i n a l p r e m i s e , b u t f u r t h e r s u b s t a n t i a t i o n w i t h c h e m i c a l l y c l e a n g l a s s w a r e was n e c e s s a r y i n o r d e r t o i n s u r e t h e r e  m o v a l o f a l l r e s i d u a l i r o n . A l l g l a s s w a r e was l e f t o v e r n i g h t i n h o t 10% n i t  r i c a c i d . I t was t h e n r i n s e d 10 t i m e s w i t h t a p w a t e r , t w i c e w i t h d i s t i l l e d w a t e r , f i l l e d w i t h , o r s o a k e d i n , g l a s s d i s t i l l e d w a t e r a n d a u t o c l a v e d a t 15 p o u n d s f o r 15 m i n u t e s . A f t e r a u t o c l a v i n g , t h e f l a s k s w e r e r i n s e d t w i c e w i t h g l a s s d i s t i l l e d w a t e r . I t was a l s o n e c e s s a r y t o r e m o v e i r o n f r o m a ?>0% s o l u t i o n o f g l u  c o s e , w h i c h s e r v e d as t h e g r o w t h s u b s t r a t e i n t h e s e s t u d i e s . T h i s was a c c o m p l i s h e d b y p a s s i n g i t t h r o u g h i o n - e x c h a n g e r e s i n IRC - 50. T h e r e s i n was c o n  d i t i o n e d b y a l l o w i n g i t t o r e m a i n o v e r n i g h t i n 10% h y d r o c h l o r i c a c i d a n d t h e n w a s h i n g f r e e o f a c i d w i t h d i s t i l l e d w a t e r . The r e s i n now b e i n g i n t h e a n i o n i c f o r m was c a p a b l e o f a d s o r b i n g a l l c a t i o n s f r o m a 30% g l u c o s e s o l u t i o n . S i n c e t h e g l u c o s e was n e u t r a l i t 3 0 , w o u l d p a s s f r e e l y t h r o u g h t h e r e s i n a n d l e a v e t h e i r o n b e h i n d , . The m e d i u m p r e v i o u s l y d e s c r i b e d ( 6 ) was made u p i n g l a s s d i s t i l l e d w a t e r a n d s t e r i l i z e d a t 1 5 p o u n d s p r e s s u r e f o r 1 5 m i n u t e s . E a c h R o u x f l a s k was i n  o c u l a t e d w i t h 5 d r o p s o f a 1 / 1 0 0 d i l u t i o n o f g l u c o s e g r o w n c e l l s a n d i n c u b a t e d f o r 1 8 h o u r s a t 3 0 ° C . T h e c u l t u r e g r o w n i n t h e a b s e n c e o f i r o n h a d a v e r y l o w c o u n t a n d t h e c e l l s w e r e e x t r e m e l y c o l o r l e s s a s c o n t r a s t e d t o n o r m a l p i n k c e l l s o b t a i n e d w i t h 0 . 5 ppm i r o n . . The c e l l s w e r e t h e n d r i e d u n d e r a p a r t i a l v a c u u m o f f u e l g a s i n c h e m i c a l l y c l e a n p e t r i p l a t e s . T A B L E I I MANOMETRIC S T U D I E S OH C E L L S GROWN  I H C H E M I C A L L Y C L E A N GLASSWARE S u b s t r a t e i r o n ppm O x y g e n u p t a k e y*J' 2 - k e t o g l u c o n a t e 0 . 0 8 8 I t o.h 1 3 5 I I 0 . 8 0 f t 1 . 0 • 0 Tt 5 . 0 o The r e s u l t s f r o m t a b l e I I show t h a t c e l l s g r o w n w i t h n o a d d e d i r o n h a d a l i m i t e d a b i l i t y t o o x i d i z e 2 - k e t o  g l u c o n a t e . A l t h o u g h t h e c e l l s w e r e g r o w n a n d d r i e d i n t h e a b s e n c e o f a d d e d i r o n , u n d o u b t e d l y t r a c e s w e r e p r e s e n t a n d t h e r e s u l t s c o n f i r m t h e o r i g i n a l h y  p o t h e s i s e C e l l s g r o w n i n c h e m i c a l l y c l e a n g l a s s  w a r e i n t h e p r e s e n c e o f O . 1 * ppm i r o n w e r e s i m i l a r •to t h o s e g r o w n i n n o r m a l g l a s s w a r e w i t h 0.5 ppm i r o n . The m a r k e d o x i d a t i v e s t i m u l a t i o n b y i r o n d e  m o n s t r a t e s t h e d e p e n d e n c e o f o v e r - a l l m e t a b o l i s m o n a n a d e q u a t e h y d r o g e n t r a n s p o r t s y s t e m . F u r t h e r w o r k i n t h i s f i e l d s h o u l d p r o v e t o be v e r y f r u i t  f u l , A s e r i e s o f e x p e r i m e n t s u s i n g 2 - k e t o g l u c o n a t e a s t h e s o u r c e o f c a r b o n i n p l a c e o f g l u c o s e w e r e u n d e r t a k e n . The c e l l s w e r e d e s i c c a t e d a s p r e  v i o u s l y a n d t h e e x p e r i m e n t s w e r e i d e n t i c a l t o t h o s e c a r r i e d o u t w h e n g l u c o s e was t h e s o l e s o u r c e o f c a r  b o n . . F r o m n u m e r o u s W a r b u r g r u n s t h e r e s u l t s w e r e i d e n t i c a l t o t h o s e f o r g l u c o s e g r o w n c e l l s . A g a i n , u n r e p r o d u c i b l e r e s u l t s n e c e s s i t a t e d a new a p p r o a c h t o t h e p r o b l e m . A s e a r c h was b e g u n f o r a c h e m i c a l m e t h o d t o m e a s u r e t h e d i s a p p e a r a n c e o f t h e s u b s t r a t e a n d i n t h i s m a n n e r s u b s t a n t i a t e t h e o x y g e n u p t a k e d a t a o b  t a i n e d i n t h e p r e s e n c e o f a h i g h e n d o g e n o u s r e s  p i r a t i o n . A t t h e same t i m e , i t was f e l t t h a t a q u a n t i t a t i v e m e a s u r e o f s u b s t r a t e d i s a p p e a r a n c e w o u l d a i d i n i d e n t i f y i n g a n y i n t e r m e d i a t e c o m p o u n d s f o r m e d 32 d u r i n g t h e o x i d a t i o n o f 2 - k e t o g l u c o n a t e . F o r t h i s p u r p o s e t h e F o l i n - M a l m r o s r e d u c i n g s u g a r m e t h o d w a s e m p l o y e d . T h i s m e t h o d w a s c a r r i e d o u t a s o u t  l i n e d b y U m b r e i t e t a l . (33)• P u r e c o m p o u n d s s u c h a s 2 - k e t o g l u c o n a t e w o r k e d v e r y w e l l b u t a s e r i e s o f t r i a l s w i t h d r i e d c e l l s w e r e v e r y d i s a p p o i n t i n g . U n f o r t u n a t e l y , t h e e n d o g e n o u s o r c o n t r o l v e s s e l a l s o g a v e a b l u e c o l o r ( p o s i t i v e t e s t ) e v e n i n t h e a b s e n c e o f 2 - k e t o g l u c o n a t e . A l l t h e v a r i a b l e s w e r e a l t e r e d b u t s t i l l t h e p o s i t i v e t e s t r e m a i n e d « f o r t h e c o n  t r o l v e s s e l a n d t h e m e t h o d was a b a n d o n e d . . The m e t h o d o f F r i e d m a n a n d H a u g e n (15) w h i c h e m p l o y s 2 , ^ - d i n i t r o p h e n y l h y d r a z i n e a s a r e a g e n t f o r a l p h a k e t o a c i d s w a s a l s o t e s t e d . The h y d r a z o n e i s e x t r a c t e d w i t h s o d i u m c a r b o n a t e a n d may be m e a s u r e d q u a n t i t a t i v e l y . D e s p i t e many t r i a l s i t w o u l d a p p e a r t h a t t h e h y d r a z o n e o f 2 - k e t o g l u c o n a t e w i l l n o t f o r m u n d e r t h e c o n d i t i o n s d e s c r i b e d b y t h e s e w o r k e r s * The m e t h o d o f L a n n i n g a n d C o h e n ( 2 1 > w h i c h i s s i m p l e a n d r a p i d was t r i e d . T h i s m a k e s u s e o f t h e c o n d e n  s a t i o n o f o - p h e n y l e n e d i a m i n e w i t h t h e k e t o h e x o n i c a c i d s t o f o r m 2 - h y d r o x y q u i n o x a l i n e s . T h e r e a g e n t was f r e s h l y p r e p a r e d 2 . 5 ^ a q u e o u s s o l u t i o n o f o - p h e n y l e n e d i a m i n e d i h y d r o c h l o r i d e o r one c o n t a i n i n g 15 nigs o f f r e e a m i n e p e r m l . o f 0.25 N H C 1 . 3 3 To 2 m i s o f a n e u t r a l s o l u t i o n c o n t a i n i n g 1 0 t o 1 0 0 gammas o f 2 - k e t o h e x o n i c a c i d was a d d e d 1 m l o f r e a g e n t . The r e a c t i o n t u b e was h e a t e d i n a b o i l i n g w a t e r b a t h f o r 3 0 m i n u t e s a n d c o o l e d t o r o o m t e m p e r a  t u r e . O p t i c a l d e n s i t y w h e n m e a s u r e d a t 3 3 0 mA a n d 5 6 0 m/* g a v e a r a t i o o f 1 . 5 f o r 2 - k e t o g l u c o n a t e . A s t a n d a r d c u r v e was s e t u p u s i n g s o l u t i o n s c o n  t a i n i n g t h e f o l l o w i n g a m o u n t s o f c a l c i u m 2 - k e t o g l u  c o n a t e made t o v o l u m e i n a 2 5 m l v o l u m e t r i c f l a s k . . ( F i g u r e IV) ( 1 ) 1 2 . 1 g m s . ( 2 ) 2 H - , 2 " ( 3 ) 3 6 . 3 '-' (k) »• ( 5 ) 6 0 . 5 " ( 6 ) 7 2 . 6 " T h e r e a g e n t b l a n k c o n t a i n e d w a t e r a s a s o l v e n t , s i n c e i t b e h a v e d t h e same a s M / 1 5 p H 7 » 0 p h o s p h a t e b u f f e r i n s p e c t r o p h o t o m e t r i c s t u d i e s , • R e s p i r o m e t e r s t u d i e s w e r e c a r r i e d o u t w i t h f u e l g a s d r i e d c e l l s a t 2 0 mgs o f d r i e d c e l l s p e r c u p , PROTOCOL FOR MEASURING 2 - K E T 0 G L U C O N A T E D I S A P P E A R A N C E pH l.k M / 1 5 E n d o g e n o u s CUD R e a c t i o n Cut> Pho s p h a t e B u f f e r 1 . 5 m i s 1 . 5 n i l s • W a t e r 1 . 0 m i s 0 . 8 m i s C e l l s 0 . 5 7 m l s 0 . 5 m i s 2 - k e t o g l u c o n a t e - 0 . 2 m i s KOH 0 . 1 5 m i s 0 . 1 5 m i s 0 . . 2 m i s o f s u b s t r a t e e q u a l s 5 / V . o f 2 - k e t o g l u c o n a t e 0 1 1 L N GAMMAS 2 - k * t o ? l u e 0 0 0 t « F i g u r e IV A s t a n d a r d curve t o measure pure 2 - k e t o g l u c o n a t e . To 1 ml o f r e a g e n t ( 1 5 nigs o f o-phenylenediamine per ml o f 0 . 2 5 N HC1) was added 2 mis o f a n e u t r a l s o l u t i o n c o n t a i n i n g 2 - k e t o g l u c o n a t e i n the f o l l o w i n g c o n c e n t r a t i o n s : (a) 1 2 . 1 micrograms. (d) kQ .k micrograms .(b) 2k.2 " (e) 6 0 . 5 " (c) 3 6 . 3 . 1 1 ' ( f ) 7 2 . 6 The f o r m a t i o n ' o f h y d r o x y q u i n o x a l i r . e s , by the r e a c t i o n , was measured s p e c t r o p h o t o m e t r i c a l l y a t • The curve was i n  t e r p o l a t e d beyond 7 2 . 6 micrograms o f 2 - k e t o g l u c o n a t e . 35. C e l l s w e r e w e i g h e d o u t o n a n a n a l y t i c a l b a l a n c e a n d s u s p e n d e d i n d i s t i l l e d w a t e r a t a r a t e o f hO mgs p e r m l . The c e l l s w e r e b r o u g h t i n t o s u s p e n s i o n b y u s e o f a V a n P o t t e r H o m o g e n i z e r . . A f t e r t h e r e  a c t i o n was c o m p l e t e d t h e c u p c o n t e n t s w e r e t r e a t e d w i t h 2 m i s o f a 30% p e r c h l o r i c a c i d a n d l e f t o v e r  n i g h t t o d e p r o t e i n i z e . A f t e r d e p r o t e i n i z a t i o n t h e d e n a t u r e d p r o t e i n was r e m o v e d b y g r a v i t y f i l t r a t i o n . The s u p e r n a t a n t f l u i d was t h e n b r o u g h t t o n e u t r a l i t y w i t h 10 W NaOH a n d made t o v o l u m e i n a 25 m l v o l u  m e t r i c f l a s k . I n a d d i t i o n t o v o l u m e t r i c f l a s k s c o n  t a i n i n g t h e e n d o g e n o u s a n d t h e r e a c t e d 2 - k e t o g l u c o n a t e , a t h i r d v o l u m e t r i c f l a s k c o n t a i n i n g 1 m l o f b o i l e d . d r i e d c e l l s p l u s 1.5 m i s o f p H 7 A M/15 p h o s p h a t e b u f f e r a n d 5/Vof 2 -ke tog lucona te was made t o v o l u m e . T h e t h i r d f l a s k was e m p l o y e d i n o r d e r t o d e t e r m i n e t h e a b i l i t y t o r e c o v e r 2 -ke tog lucona te i n t h e p r e s e n c e o f d r i e d . c e l l s . A f t e r t r e a t i n g 2 m i s o f e a c h v o l u  m e t r i c f l a s k a s p r e v i o u s l y d e s c r i b e d t h e s o l u t i o n s w e r e r e a d i n a B e c k m a n M o d e l B s p e c t r o p h o t o m e t e r a n d t h e r e s u l t s a r e s h o w n i n T a b l e I I I . T h e e n d o g e n o u s f l a s k was u s e d a s a r e a g e n t b l a n k : 36. T A B L E I I I R E S U L T S OF 0 - P R E r J Y X E N E D I A M I N E TECHNIQUE S u b s t a n c e O p t i c a l D e n s i t y a t 330 O p t i c a l D e n s i t y a t 560 D V ^ R a t i o R e a c t e d 2 - k e t o g l u  c o n a t e O.36 0.55 0.65 B o i l e d c e l l s p l u s 2 - k e t o  g l u c o n a t e 0.72 0..51 l.hh. 2 - k e t o g l u c o n  a t e 0.75 0.50 1.50 F r o m t a b l e I I I i t w i l l be s e e n t h a t t h e b o i l e d c e l l s g i v e a p r o p e r r a t i o o f l . * f \4hich i s c l o s e t o t h a t o f p u r e 2 - k e t o g l u c o n a t e w h i c h i s 1.5. I n a d d i t i o n t h i s f l a s k when c a l c u l a t e d a c c o r d i n g t o t h e s t a n d a r d c u r v e p r o v e d t o c o n t a i n *+.9 m i c r o m o l e s o f 2 - k e t o g l u c o n a t e . On t h e o t h e r h a n d , t h e r e a c t e d 2 - k e t o g l u c o n a t e g a v e a r a t i o o f 0.65 w h i c h a p p e a r s t o show t h a t t h e d r i e d c e l l p r e p a r a t i o n h a d a c t e d u p o n 2 - k e t o g l u c o n a t e a n d o x i d i z e d i t t o a new c o m p o u n d . I n a d d i t i o n t h e c o m p o u n d f l u o r e s  c e d a l i g h t a m b e r c o l o r u n d e r u l t r a v i o l e t i n c o n t r a s t t o t h e u n m i s t a k a b l e g r e e n i s h - g r e y f l u o r e s c e n c e o f p u r e 2 - k e t o g l u c o n a t e . A l t h o u g h t h i s m e t h o d w o r k e d w e l l w i t h p u r e c o m p o u n d s t h e f a c t t h a t new c o m p o u n d s w e r e f o r m e d w h i c h m a s k e d a n y u n r e a c t e d 2 - k e t o g l u c o n a t e made 3 7 . i t i m p o s s i b l e t o t e l l q u a n t i t a t i v e l y t h e e x t e n t o f 2 - k e t o g l u c o n a t e o x i d a t i o n . M a n y a t t e m p t s a t v a r y  i n g d e p r o t e i n i z i n g s o l u t i o n s made n o d i f f e r e n c e u p o n t h e t e c h n i q u e w i t h r e g a r d t o t h e a p p a r e n t f o r m a t i o n o f a new c o m p o u n d . T h i s m e t h o d was a b a n d o n e d s i n c e i t was i n a u g u r a t e d a s a q u a n t i t a t i v e m e t h o d t o m e a s u r e t h e d i s a p p e a r a n c e o f 2 - k e t o g l u c o n a t e . The n e w c o m  p o u n d m a s k e d a n y r e s i d u a l 2 - k e t o g l u c o n a t e a n d s i n c e a 1 0 0 p e r c e n t c o n v e r s i o n o f t h i s s u b s t r a t e w a s h i g h l y u n l i k e l y t h e m e t h o d a p p e a r e d u n w o r k a b l e . A n o u t  s t a n d i n g f e a t u r e o f t h i s t e c h n i q u e , a f t e r many a t  t e m p t s , was t h e r e p r o d u c i b i l i t y o f t h e s t a n d a r d c u r v e when e m p l o y i n g p u r e 2 - k e t o g l u c o n a t e * T h i s r e p r o  d u c i b i l i t y w a r r a n t s f u r t h e r e x p e r i m e n t a t i o n w i t h c e l l p r e p a r a t i o n s b e f o r e i t c o u l d become a u s e f u l t o o l i n enzyme s t u d i e s , ( b ) D i a l y s i s w o r k w i t h w h o l e c e l l s . I t was d e c i d e d t h a t d i a l y s i s o f 2 - k e t o g l u c o n a t e g r o w n c e l l s m i g h t r e m o v e a n y e x c e s s i r o n . T h i s f r e e i r o n c o u l d be r e s p o n s i b l e f o r c a t a l y z i n g t h e o x i d a t i o n o f s u l f h y d r y l g r o u p s . C e l l s w e r e h a r v e s t e d a t 1 8 h o u r s a n d d i a l y z e d a g a i n s t i c e w a t e r . D u r i n g t h i s p r o c e s s t h e c e l l s w e r e s t i r r e d v i g o r o u s l y b y a m o t o r d r i v e n p r o p e l l o r f o r 8 h o u r s . A l i q u o t s w e r e t a k e n a t v a r i o u s i n t e r v a l s f r o m t h e d i  a l y s i s s a c k a n d a f t e r b e i n g made t o 57% t r a n s m i s s i o n 38. i n a F i s h e r e l e c t r o p h o t o m e t e r w e r e u s e d i n m a n o m e t r i c s t u d i e s a t t h e r a t e o f 0.5 m i s p e r c u p . I n i t i a l r e  s u l t s w e r e v e r y e n c o u r a g i n g b u t when d i a l y z e d c e l l s w e r e e m p l o y e d a g a i n s t w h o l e u n d i a l y z e d c e l l s t h e r e  s u l t s w e r e i d e n t i c a l a n d i t a p p e a r e d t h a t d i a l y s i s h a d no e f f e c t o n w h o l e c e l l s . V/o rk o n f u e l g a s d r i e d c e l l s was c o n t i n u e d i n o r d e r t o o b t a i n r e p r o d u c i b l e r e s u l t s v / i t h t h e s e p r e  p a r a t i o n s . I t was o b s e r v e d d u r i n g m a n o m e t r i c s t u d i e s t h a t w h e n t h e s u b s t r a t e was t i p p e d i n t o t h e c u p a f t e r e q u i l i b r a t i o n a w h i t e c u r d y p r e c i p i t a t e was f o r m e d . S i n c e t h e c a l c i u m s a l t . o f 2 - k e t o g l u c o n i c a c i d w a s b e i n g e m p l o y e d a s a s u b s t r a t e i t was f e l t t h a t t h e c a l c i u m was f o r m i n g a n i n s o l u b l e p r e c i p i t a t e w i t h t h e p h o s p h a t e b u f f e r . I R C -50 r e s i n was c o n d i t i o n e d a s p r e v i o u s l y d e s c r i b e d a n d t h e c a l c i u m 2 - k e t o g l u c o n i c a c i d was s h a k e n w i t h t h e r e s i n f o r s e v e r a l h o u r s t o r e m o v e t h e c a l c i u m . A l t h o u g h t h i s t r e a t m e n t p r e v e n t e d p r e c i p i t a t i o n i t h a d n o b e n e f i c i a l e f f e c t o n t h e a c t i v i t y o f t h e p r e  p a r a t i o n , ( c ) W o r k w i t h S t r e p t o m y c i n . T h e f a c t o r w h i c h was t o a l a r g e m e a s u r e r e s p o n s i b l e f o r t h e u n r e p r o d u c i b l e r e s u l t s o f f u e l g a s d r i e d c e l l s was t h e s u r v i v a l o f t h e s m a l l n u m  b e r o f l i v e c e l l s . S t r e p t o m y c i n i n h i b i t s t h e f o r m a t i o n o f a d a p t i v e e n z y m e s a n d t h e r e f o r e t h e s y n t h e s i s o f c e l l . 39 p r o t e i n b u t d o e s n o t p r e v e n t o x i d a t i o n . . I t w a s , t h e r e f o r e , f e l t t h a t when u s e d i n c o n j u n c t i o n w i t h f u e l g a s d r i e d c e l l s i t w o u l d p r o v e v e r y u s e f u l b y p r e v e n t i n g c e l l m u l t i p l i c a t i o n w i t h o u t i n h i b i t i n g o x i d a t i o n . A s o l u t i o n o f s t r e p t o m y c i n c o n t a i n i n g 37.5 m g s / m l was made u p a n d 0.1 m i s was u s e d f o r e a c h W a r b u r g c u p . C e l l s w e r e h a r v e s t e d a n d s u b j e c t e d t o f u e l g a s . A f t e r a s e r i e s o f 20 W a r b u r g t r i a l s t h e o u t  come was c o n s i s t e n t l y a s d e p i c t e d i n ( f i g u r e V . ) I t w i l l be n o t i c e d f r o m ( f i g u r e V ) t h a t t h e e n d o g e n o u s a c t i v i t y was r e d u c e d b y t h e s t r e p t o m y c i n t r e a t m e n t a n d t h a t t h e o x i d a t i v e a b i l i t y was a l s o r e d u c e d a c c o r d i n g l y . S i n c e a g r e a t n u m b e r o f u n k n o w n f a c t o r s p l a y e d a p r o m i n e n t r o l e i n t h i s t e c h n i q u e i t was f e l t t h a t i t w o u l d be p o s t p o n e d u n t i l f u r t h e r w o r k c o u l d be a c c o m p l i s h e d . The w o r k w i t h c o - f a c t o r s was a l s o c a r r i e d o u t a s f o r m o n o x i d e d r i e d c e l l s b u t a g a i n t h e r e s u l t s w e r e n e g a t i v e a n d d i d n o t a f f e c t t h e p r e p a r a t i o n s s t a b i l i t y . D e s p i t e t h i s f a c t a c h r o m a t o g r a p h i c s u r v e y was c a r r i e d o u t b u t i t f a i l e d t o r e v e a l a n y new c o m  p o u n d s i n t h e p a t h w a y o f 2 - k e t o g l u c o n a t e b r e a k d o w n . C e l l F r e e P r e p a r a t i o n s T h e l i m i t i n g f a c t o r i n t h e u t i l i z a t i o n o f many ho. i A 2 0 60 100 140 1*0 TIME IN MINUTE S F i g u r e V The e f f e c t o f s t r e p t o m y c i n o n f u e l g a s d r i e d c e l l s . Oxygen u p t a k e i s shown u s i n g 5 m i c r o m o l e s o f s u b s t r a t e . The r e a c t i o n was c a r r i e d o u t a t 31 °C . a n d j>E-7»h. E a c h W a r b u r g c u p c o n t a i n e d 20 mgs o f d r i e d c e l l s . A - 2 - k e t o g l u c o n a t e a n d f u e l g a s d r i e d c e l l s . B - E n d o g e n o u s C - 2 - k e t o g l u c o n a t e , f u e l g a s d r i e d c e l l s a n d 3.75mgs o f s t r e p t o m y c i n . D - E n d o g e n o u s p l u s 3»75 mgs o f s t r e p t o m y c i n . hi. i n t e r m e d i a t e s i s t h e p e r m e a b i l i t y o f t h e c e l l mem b r a n e . C a m p b e l l a n d S t o k e s (7) f o u n d t h a t r e s t i n g c e l l s h a r v e s t e d f r o m a g r o w t h m e d i u m c o n t a i n i n g a c e  t a t e a s t h e s o l e s o u r c e o f c a r b o n h a d n o a b i l i t y t o o x i d i z e c i t r a t e , c _ i s - a c o n i t a t e , i s o c i t r a t e , a l p h a k e t o g l u t a r a t e , s u c c i n a t e o r f u m a r a t e w i t h o u t a p e r i o d o f a d a p t a t i o n . . B u t when t h e s e c e l l s w e r e d r i e d t h e y h a d t h e a b i l i t y t o o x i d i z e t h e s e c o m p o u n d s i m m e d i a t e  l y i l l u s t r a t i n g t h e b r e a k d o w n o f t h i s p e r m e a b i l i t y b a r r i e r . M a n y t e c h n i q u e s h a v e b e e n a d v a n c e d a n d many o f t h e s e e m b r a c e t h e r u p t u r i n g o f t h e c e l l w a l l so t h a t t h e b a c t e r i a l c o n t e n t s may be r e l e a s e d i n t o a s u i t a b l e m e d i u m a n d f u r t h e r s t u d i e d . H e n c e , i n t r a c e l l u  l a r e n z y m e s may be p u r i f i e d a n d s t u d i e d w i t h o u t t h e i n t e r f e r i n g p e r m e a b i l i t y f a c t o r . , A v a l i d c r i t i c i s m o f i s o l a t e d i n t r a c e l l u l a r enzyme s y s t e m s i s t h a t e n  zymes o b t a i n e d i n t h i s m a n n e r a r e b e i n g s t u d i e d i n a n e n v i r o n m e n t w h i c h , i s c o m p l e t e l y f o r e i g n t o t hem w i t h r e g a r d t o p H e t c . D e s p i t e t h i s c r i t i c i s m , w h i c h may be a p p l i e d t o many b i o l o g i c a l s t u d i e s , a g r e a t d e a l - o f t h e w o r k t o d a y h a s a c c r u e d f r o m s t u d i e s w i t h c e l l f r e e e x t r a c t s . S u c c e s s f u l w o r k h a s b e e n c a r r i e d o u t i n t h i s l a b o r a t o r y w i t h t h e g l u c o n a t e o x i d i z i n g e n  zyme o b t a i n e d f r o m t h e c e l l b y s o n i c v i b r a t i o n a n d e m p l o y e d i n a p u r e s t a t e w i t h v a r i o u s h y d r o g e n a c c e p t  o r s . U n f o r t u n a t e l y , a l l p r e v i o u s a t t e m p t s o f a p p l y  i n g t h i s t e c h n i q u e t o t h e 2 - k e t o g l u c o n a t e enzyme h a v e b e e n u n s u c c e s s f u l . I t was f e l t t h a t a p r e p a r a t i o n o b  t a i n e d i n t h i s m a n n e r w o u l d be a b i g s t e p t o w a r d s s o l v i n g t h e m a j o r p r o b l e m o f 2 - k e t o g l u c o n a t e d e g r a  d a t i o n . The l a s t p o r t i o n o f t h i s t h e s i s w i l l be c o n  c e r n e d w i t h t h e t r i a l s a t t e m p t e d i n o r d e r t o o b t a i n a c e l l f r e e p r e p a r a t i o n o f a e r u g i n o s a w h i c h w o u l d m a i n t a i n i t s a c t i v i t y a g a i n s t 2 - k e t o g l u c o n a t e . Ca) A d a p t i n g t h e p r e s s u r e c r u s h i n g t e c h n i q u e t o enzyme w o r k . A t e c h n i n u e h a d b e e n d e v i s e d w h e r e b y f r o z e n c e l l s s u b j e c t e d t o g r e a t p r e s s u r e m e l t a n d b u r s t o p e n d u r i n g t h i s t h a w i n g p r o c e s s . T h i s t e c h n i q u e h a d b e e n e m p l o y e d f o r s t u d y i n g t h e s t r u c t u r e o f b a c t e r i a l n u  c l e i c a c i d s a n d i t a p p e a r e d t o be r e a d i l y a d a p t a b l e t o enzyme s t u d i e s . I n p a r t i c u l a r t h e 2 - k e t o g l u c o n a t e enzyme b e i n g o f a n u n s t a b l e c h a r a c t e r , m i g h t r e m a i n a c t i v e w i t h t h i s t e c h n i q u e . G l u c o s e g r o w n c e l l s w e r e h a r v e s t e d a f t e r 18 h o u r s a n d w a s h e d o n c e w i t h d i s  t i l l e d w a t e r . The c e l l s w e r e t h e n s u s p e n d e d i n d i s  t i l l e d w a t e r a t a c o n c e n t r a t i o n o f 300 m g s / m l , s l o w l y a d d e d t o a c o l d s t e e l c y l i n d e r a n d q u i c k l y f r o z e n i n a b a t h o f d r y i c e . A s t e e l p i s t o n was t h e n i n s e r t e d i n t o t h e c y l i n d e r a n d s u b j e c t e d t o 1 2 0 0 0 p o u n d s p r e s s u r e ^3 f r o m a C a r v e r L a b o r a t o r y p r e s s . A f t e r c e n t r i f u g i n g a t 10 ° C . t h e p r e p a r a t i o n was e m p l o y e d a t t h e r a t e o f 1 m l / c u p i n m a n o m e t r i c s t u d i e s . ( F i g u r e V I ) s h o w s t h e p r o m i s i n g n a t u r e o f t h i s t e c h n i q u e w i t h r e g a r d t o i t s l i m i t e d a b i l i t y t o o x i d i z e g l u c o s e , g l u c o n a t e , a n d 2 - k e t o g l u c o n a t e , B u t f r o m ( F i g u r e V I I ) i t w i l l be s e e n t h a t t h e r e s u l t s a r e i n c o n s i s t e n t a n d u n - r e p r o d u c i b l e . A l s o , i t w o u l d a p p e a r t h a t l i v e c e l l s a r e m u l t i p l y i n g s i n c e t h e r e a c t i o n was n o t c o m p l e t e d e v e n a f t e r 3 h o u r s , ( F i g u r e V I I I ) shows a n e x c e l l e n t p r e p a r a t i o n w i t h a g o o d a b i l i t y t o o x i d i z e 2 - k e t o  g l u c o n a t e w i t h t h e r e a c t i o n c o m p l e t e d a f t e r ihO m i n u t e s . B u t , a g a i n , , i t was n o t p o s s i b l e t o r e p r o  d u c e t h i s . T a b l e h shows t h e v a r i o u s m e t h o d s u s e d i n a n a t t e m p t t o p r e s e r v e t h e 2 - k e t o g l u c o n a t e enzyme d u r i n g c r u s h i n g . hh. A 20 60 100 |40 180 T I M E I N M I N UTES ! J T i g i t r e V I k p r e s s u r e c r u s h e d c e l l p r e p a r a t i o n s h o w i n g a c t i v i t y o n v a r i o u s s u b s t r a t e s . O x y g e n u p t a k e i s shown u s i n g 5 m i c r o m o l e s o f s u b s t r a t e . The r e a c t i o n was c a r r i e d o u t a t 3 1 ° C a n d pH 7.h. R e s t i n g c e l l s w e r e f r o z e n 2 0 m i n u t e s b e f o r e b e i n g s u b j e c t e d t o 1 2 0 0 0 p o u n d s p r e s s u r e . A - G l u c o s e B — G l u c o n a t e C - 2 - k e t o g l u c o n a t e D - E n d o g e n o u s A 20 60 100 MO 180 T I M E I N M I N U T E S F i g u r e VII - A p r e s s u r e c r u s h e d c e l l p r e p a r a t i o n s h o w i n g a c t i  v i t y a f t e r 3 h o u r s . I n c o m p l e t e o x y g e n u p t a k e d a t a i s ' s h o w n u s i n g 5 m i c r o - m o l e s o f s u b  s t r a t e . The r e a c t i o n was c a r r i e d o u t a t 31 ° C . a n d p H 7.h, R e s t i n c e l l s w e r e f r o z e n 20 m i n u t e s b e f o r e b e i n g s u b j e c t e d t o 12000 p o u n d s p r e s s u r e . A - G l u c o s e C - 2 - k e t o g l u c o n a t e B - G l u c o n a t e D - E n d o g e n o u s ^6. e 20 60 K)0 140 180 TIME I N M J NilTE 8 F i g u r e V I I I - A p r e s s u r e c r u s h e d c e l l p r e p a r a t i o n . N e t o x y g e n u p t a k e d a t a i s shown u s i n g 5 m i c r o m o l e s o f s u b  s t r a t e . The r e a c t i o n was c a r r i e d o u t a t 3I ° C . a n d pH 7 A . A - G l u c o s e B - G l u c o n i c C - 2 - k e t o g l u c o n a t e h7. T A B L E k METHODS TO OBTAIN REPRODUCIBLE  2-KETOGLUCONATE ACTIVITY A g e o f C e l l s C o n e . P r o t e c t i v e S u b s t r a t e N a t u r e o f F r e e z i n g R e s u l t o n 2 - k e t o g l u  c o n a t e 0 1 8 a n d 24- h o u r s 3 0 0 mgs/fol 7% s u c r o s e f r o z e n h r s a n d c r u s h e d a t 1 2 0 0 0 p o u n d s No A c t i v i t y 1 8 a n d 2h h o u r s 3 0 0 m g s / m l 0 . 3 # C y s t e i n e - H y d r o  c h l o r i d e f r o z e n 2 0 m i n s , c r u s h e d a t 1 2 0 0 0 p o u n d s No A c t i v i t y 1 8 a n d 24- h o u r s 3 0 0 m g s / m l 2% l a c t - a l b u m i n f r o z e n 2 0 m i n s . c r u s h e d a t 1 2 0 0 0 p o u n d s No A c t i v i t y A n o t h e r d i f f i c u l t y w i t h t h e p r o c e d u r e was t h a t w h o l e u n c r u s h e d c e l l s a d h e r e d t o t h e gummy s u p e r n a t a n t m a t e r i a l a n d p r o d u c e d v e r y e r r a t i c r e s u l t s . M a n y m e t h o d s w e r e a t t e m p t e d t o c o r r e c t t h i s f a u l t a n d t h e w h o l e p r o c e d u r e may be summed u p i n T a b l e V, 1 T A B L E 5  R E S U L T S OF V A R I O U S T R I A L S  WITH CRUSHING TECHNIQUES C a r b o n S o u r c e A g e o f c e l l s T r e a t m e n t R e s u l t s ( 1 ) G l u c o s e 1 8 h o u r s a n d 2*f h o u r s C e n t r i f u g e d t w i c e a t 5 0 0 0 r p m 1 s © io°c . N e g l i b l e a c t i v i t y ( 2 ) G l u c o s e 1 8 h o u r s a n d 2'-f h o u r s A g i t a t e d g e n t l y w i t h c h i l l e d V a n P o t t e r p e s t l e a n d c e n t r i f u g e d t w i c e a t 5 0 0 0 r p m ' s a t 1 0 ° C . No a c t i v i t y ( 3 ) G l u c o s e 1 8 h o u r s a n d 2h h o u r s D i l u t e d p r e p a r a  t i o n w i t h o n e v o l u m e o f M / 3 0 p H 7 . 0 P h o s p h a t e B u f f e r C e n t r i  f u g e d o n c e @ 5 0 0 0 r p m ' s © io°c.. No a c t i v i t y 0*) G l u c o s e 1 8 h o u r s a n d 2h h o u r s A s a b o v e b u t i n a d d i t i o n u s e d 2 , 6 - d i c h l o r o - p n e n o l i n d o p h e n o l a s a h y d r o g e n a c c e p t o r No a c t i v i t y ( 5 ) G l u c o s e 1 8 h o u r s a n d 2 H - h o u r s C e l l s f r o z e n i n e t h y l a l c o h o l d r y i c e b a t h No a c t i v i t y ( 6 ) 2 - k e t o  g l u c o  n a t e 1 8 h o u r s a n d 2h h o u r s A s f o r m e t h o d s 1 t o 6 i n c l u  s i v e No a c t i v i t y ( b ) S o n i c P r e p a r a t i o n s . A s s t a t e d p r e v i o u s l y a l l e f f o r t s t o o b t a i n s o n i c a t e s a c t i v e o n 2 - k e t o g l u - "'• Neonate h a d m e t w i t h f a i l u r e . I n t h i s l a b o r a t o r y a R a y t h e o n s o n i c o s c i l l a t o r v i b r a t i n g a t 1 0 k i l o  c y c l e s i s e m p l o y e d . A l a m i n a t e d n i c k e l r o d i s made t o v i b r a t e a t 1 0 k c s . p e r s e c o n d a n d t h i s i n t a r n m o v e s a d i a p h r a g m u p a n d down i n a b a c t e r i a l s u s p e n s i o n a n d t h e c e l l s a r e r e p o r t e d l y c r u s h e d b y t r e m e n d o u s p r e s s u r e s due t o c a v i t a t i o n . The c e l l w a l l b u r s t s r e s u l t i n g i n w h a t i s r e f e r r e d t o a s t h e c r u d e s o n i c a t e . T h i s s o n i c a t e i s t h e n c e n t r i  f u g e d a t 2 5 0 0 0 x g f o r 3 0 - 6 0 m i n u t e s a t - 1 0 ° C . T h i s r e m o v e s a l l c e l l d e b r i s a l l o w i n g t h e u s e o f a c e l l e x t r a c t i n m a n o m e t r i c s t u d i e s . P r e l i m i n a r y s t u d i e s u s i n g o s c i l l a t i n g t i m e s o f f r o m f i v e t o e i g h t m i n u t e s o n a c h i l l e d p r e p a r a t i o n o f 2 - k e t o  g l u c o n a t e o r g l u c o s e g r o w n c e l l s showed a v e r y l i m i t e d o x i d a t i o n o f 2 - k e t o g l u c o n a t e w i t h a maximum u p t a k e o f 2 8 m i c r o l i t r e s o f o x y g e n . P o s i t i v e r e s u l t s w e r e i n f r e q u e n t , b u t w o r k .-.was c o n t i n u e d a n d v a r i o u s a g e n t s w e r e a d d e d t o t h e p r e p a r a t i o n p r i o r t o s o n i c - i n g . C y s t e i n e o r g l u t a t h i o n e was a d d e d i n v a r y i n g a m o u n t s t o p r o t e c t a n y s u l f h y d r y l e n z y m e s , b u t r e  p e a t e d t r i a l s o f d i f f e r e n t c o n c e n t r a t i o n s p r o v e d n e  g a t i v e . F o r e x a m p l e , one t r i a l i n 2 0 m i g h t g i v e a n e n c o u r a g i n g r e s u l t . V a r i a t i o n s i n t h e s u s p e n d i n g 50 m e d i u m f o r t h e c e l l s w e r e e x p l o r e d a n d v a r i o u s b u f f e r s f r o m M / 1 5 t o M / 5 0 a n d f r o m pH 7 . 0 t o 7.h- a g a i n g a v e n e g a t i v e r e s u l t s . A n o t h e r r e s o r t was t o v a r y t h e s o n i c i n g t i m e s f r o m 5 t o 8 m i n u t e s i n p l a c e o f t h e u s u a l 1 5 m i n u t e s , a n d i n t h i s m a n n e r r u p t u r e s u f f i c i e n t c e l l s t o p r o v i d e l i m i t e d a c t i v i t y o n 2 - k e t o g l u c o n a t e . The r e s u l t s v a r i e d f r o m 1 0 t o 2 8 m i c r o l i t r e s o f o x y g e n u p t a k e a n d p r o v i d e d e n c o u r a g e m e n t f o r c o n t i n u a t i o n o f t h e s e s t u d i e s . The u s e o f 8% s u c r o s e was n o t s u c c e s s  f u l b u t S a i t o ( 3 0 ) w o r k i n g o n t h e b i o c h e m i s t r y o f v i s i o n h a d u s e d \5% s u c r o s e a s h i s o n l y means o f o b t a i n i n g a p u r e p r o t e i n p r e p a r a t i o n . I n a d d i t i o n K o e p s e l l ( 2 0 ) r e p o r t e d t h a t g l u c o n a t e g r o w n c e l l s w e n t c o m p l e t e l y p a s t 2 - k e t o g l u c o n a t e t o a l p h a k e t o - g l u t a r a t e , w h e r e a s , g l u c o s e g r o w n c e l l s a c c u m u l a t e d 2 - k e t o g l u c o n a t e a n d o n l y s l o w l y u t i l i z e d i t t o a l p h a - k e t o g l u t a r a t e a f t e r a p e r i o d o f f o u r t e e n t o e i g h t e e n d a y s . - K o e p s e l l ' s w o r k a p p l i e d t o f l u o r e s c e n s T b u t i t w a s f e l t t h a t g l u c o n i c g r o w n c e l l s o f a e r u g i n o s a w a s h e d i n a b u f f e r a n d s u s p e n d e d i n h5% s u c r o s e m i g h t r e t a i n t h e i r a c t i v i t y a g a i n s t 2 - k e t o g l u c o n a t e . . F i g u r e ( I X ) shows t h e g o o d a c t i v i t y o n 2 - k e t o g l u c o n a t e w i t h t h e p r e p a r a t i o n l e v e l l i n g o f f a t a l m o s t 1 a t o m o f o x y  g e n . . T h e s e r e s u l t s w e r e e a s i l y d u p l i c a t e d a n d i n 51- a d d i t i o n l o s t l i t t l e a c t i v i t y d u r i n g s t o r a g e o v e r  n i g h t i n t h e f r o z e n s t a t e a t -10°C . ( F i g u r e X ) . T h e r e s u l t s a p p e a r e d v a l i d a n d w o r k was c o n t i n u e d o n t h i s m e t h o d . The c r u d e p r e p a r a t i o n was t e s t e d f o r a c t i v i t y o n s u c r o s e . 10 m i c r o m o l e s o f s u c r o s e w e r e e m p l o y e d a s a s u b s t r a t e a n d no a c t i v i t y c o u l d be d e t e c t e d m a n o m e t r i c a l l y , d u r i n g t h e same p e r i o d t h a t t h i s p r e p a r a t i o n h a d t a k e n up a n d l e v e l l e d o f f a t 38 m i c r o - l i t r e s o f o x y g e n , when 2 - k e t o g l u c o n a t e was u s e d a s a s u b s t r a t e . G l u c o s e g r o w n c e l l s w e r e s u b j e c t e d t o t h e same t r e a t m e n t ( F i g u r e X I ) a n d a l s o s h o w e d a c t i  v i t y o n 2 - k e t o g l u c o n a t e u p t o 1 a t o m o f o x y g e n . I n a l l c a s e s t h e e n d o g e n o u s was v e r y h i g h so i t was n e c e s s a r y t o show t h e c u r v e s w i t h o u t t h e e n d o g e n o u s r e s p i r a t i o n . ( c ) R e s p i r a t o r y Q u o t i e n t s , The r e s p i r a t o r y q u o t i e n t (RQ) i s d e f i n e d a s t h e r a t i o o f C 0 2 p r o d u c e d t o o x y g e n c o n  s u m e d . T h i s i s a v a l u a b l e t o o l i n d e t e r m i n i n g t h e e x t e n t o f d e c a r b o x y l a t i o n o f a s p e c i f i c s u b s t r a t e , f o r e x a m p l e , a n RQ o f 3 f o r 2 - k e t o g l u c o n a t e w o u l d i n d i c a t e 3 m o l e c u l e s o f C 0 2 l i b e r a t e d f o r e v e r y o x y g e n t a k e n u p . . H Q ' s w e r e c a r r i e d o u t a c c o r d i n g t o t h e m e t h o d o f U m b r e i t e t a l . (33) a n d t h e t y p i c a l p r o t o c o l f o r s u c h a p r o c e d u r e i s s e e n i n T a b l e V I , 5 2 . TIME IN MINUTE S F i g u r e iX - G l u c o n a t e g r o w n c e l l s s o n i c e d i n t h e p r e s e n c e o f hffi s u c r o s e . N e t o x y g e n u p t a k e , d a t a i s shown u s i n g 5 m i c r o m o l e s o f s u b  s t r a t e . The r e a c t i o n w a s - c a r r i e d o u t a t 3 1 ° C . a n d p H 7.-^. S o n i c e d c e l l s w e r e u s e d a t t h e r a t e o f 1 m l p e r U a r b u r g v e s s e l . A - 2 - k e t o g l u c o n a t e . 5 3 , » 0 t 4 0 .J O SO X O 2 0 1 0 TIME IN MINUTES F i g u r e X - S u c r o s e s o n i c e d p r e p a r a t i o n s t o r e d o v e r n i g h t i n t h e f r o z e n s t a t e . N e t o x y g e n d a t a i s shown u s i n g 5 m i c r o m o l e s o f s u b s t r a t e , The r e a c t i o n was c a r r i e d o u t a t 3 1 ° c « a n d p K 7.h. A 2 - k e t o g l u c o n a t e . A 10 60 100. 140 100 . T l MB IN M l N U T B t F i g u r e X l - G l u c o s e g r o w n c e l l s o n i c e d i n t h e p r e s e n c e o f k5% s u c r o s e . N e t o x y g e n u p t a k e d a t a i s shown u s i n g 5 m i c r o m o l e s o f s u b - ' s t r a t e . The* r e a c t i o n was c a r r i e d o u t a t 31°C. a n d pH 7.4-. S o n i c e d c e l l s w e r e u s e d a t a r a t e o f 1 m l ' p e r W a r b u r g v e s s e l . A - G l u c o s e 55. • T A B L E Y I PROTOCOL FOR D E T E R M I N I N G R E S P I R A T O R Y QUOTIENTS i t ) E n d o g e n o u s 2 - k e t o g l u c o n a t e E n d . 2 - k e t o g l u c o n a t e 2 - k e t o gluconate MA 5- PH7.0 P h o s p h a t e b u f f e r 1.5 m i s . 1.5 m i s . 1.5 m i s . 1.5 m i s . 1.5 m i s . W a t e r 0.5 m i s 0.3 m i s . 0.5 m i s . 0.3 m i s . l.H-5mls. S o n i c a t e 1.0 m i s . 1.0 m i s . 1.0 m i s . 1.0 m i s . - S u b s t r a t e 0.2 m i s . - 0.2 m i s . 0.2mls, KOH 0.15 m i s . 0.15 m i s . - - The l a s t c u p shows t h e a m o u n t o f C 0 2 p r e s e n t i n t h e r e a g e n t s . A f t e r t h e r u n was c o m p l e t e d a n e q u a t i o n was p r o v i d e d f o r c a l c u l a t i n g t h e R Q . XQQ2 = ( h - h !k0 2 ,/K0 2)KC0 2 h = c o r r e c t e d r e a d i n g o f C0 2 e v o l v e d h = -4-.5 h ' = r e a d i n g o f m a n o m e t e r w i t h KOH h ' = -H-0.3 k'02 = c o n s t a n t o f m a n o m e t e r w i t h KOH k'0'2= 13.74- K0 2 = c o n s t a n t o f m a n o m e t e r w i t h o u t KOH k©2 - 17 .61 KC0 2 = c o n s t a n t o f m a n o m e t e r w i t h o u t KOH KCO2 = 19.59 E n d o g e n o u s C0 2 XC0 2 = -.4-5-C-4-0.3 x 13.7V17.61) .19.59= 526.9 o f co2 C0 2 due t o 2 - k e t o . = . 785.8/"1 o f C0 2 C0 2 due t o r e a g e n t s = 50/* 1 of. C0 2 A m o u n t o f C0 2 due t o 2 - k e t o g l u c o n a t e =735.8-526..9=208.9 O2 c o n s u m e d = 80/*l R . Q . = C02 = 208.9 = 2.6 56. S i m i l a r l y , a n R . Q . w i t h a new p r e p a r a t i o n was a s f o l l o w s : C O 2 due t o e n d o g e n o u s = 338/** 1 G O 2 due t o 2 - k e t o g l u c o n a t e = 530/"! 0 2 u p t a k e = 5 7 / * l R . Q . = 1 | 2 = 3.3 The R . Q . was t h e n a v e r a g e d a n d a c c e p t e d a s 3« ( d ) O p t i m u m p H a c t i v i t y . D o u b l e s t r e n g t h v e r o n a l b u f f e r was e m p l o y e d b u t f a i l e d t o m a i n t a i n i t s p H t h r o u g h  o u t t h e W a r b u r g r u n . I n s t e a d , a 1 M T r i s b u f f e r s o l u t i o n was s u b s t i t u t e d a n d w o r k e d v e r y w e l l . F i g u r e X I I shows t h e pH c u r v e u s i n g i n c r e m e n t s f r o m 6.8, 7.3,, 7.6, 7.9, 8.3 a n d 8.7 w i t h t h e o p t i m u m a p p e a r  i n g a t p H 7.^« The pH was t e s t e d a t t h e c o m p l e t i o n o f t h e W a r b u r g r u n i n a B e c k m a n p H m e t e r . U n l i k e t h e v e r o n a l b u f f e r w h i c h s h o w e d g r e a t u t i l i z a t i o n , t h e T r i s b u f f e r m a i n t a i n e d i t s pH t h r o u g h o u t . ( e ) A t t e m p t s t o d e m o n s t r a t e p h o s p h o r y l a t i o n . A T P h a d n o s t i m u l a t o r y e f f e c t o n t h e p r e p a r a t i o n a t a f i n a l c o n c e n t r a t i o n o f 1 0 0 m i c r o m o l e s , i n t h e p r e s e n c e o r a b s e n c e o f m a g n e s i u m . A v a l u a b l e t e c h n i q u e o f B a r k e r a n d L i p m a n n ( 1 ) , w h i c h m e a s u r e s a c i d - s t a b l e ( e s t e r ) p h o s p h a t e o r a c i d f o r m a t i o n , e i t h e r a e r o b i c a l - l y o r a n a e r o b i c a l l y , was e m p l o y e d a n d p r o v e d n e g a t i v e . I n a d d i t i o n t h e r e was no i n h i b i t i o n b y 2.5 x 1 0 " M s o d i u m f l u o r i d e . O f v a r i o u s s u b s t r a t e s e m p l o y e d 57. PH F i g u r e XII - p H c u r v e The W a r b u r g c u p s c o n t a i n e d 1 ml c r u d e s t i c r o s e s o n i c a t e , 0.2 m i s 2 - k e t o g l u c o n a t e (5 m i c r o m o l e s ) , d i s t i l l e d w a t e r a n d 1.0 m i s o f 1 M T r i s b u f f e r . N e t o x y g e n u p t a k e i n 1.5 h o u r s . 53. r i b o s e - 5 - p h o s p h a t e , a r a b i n o s e a n d r i b o n i c a c i d s h o w e d n o a c t i v i t y when u s e d w i t h a s o n i c a t e w h i c h was a c t i v e o n 2 - k e t o g l u c o n a t e . . ( f ) A n a e r o b i c s t u d i e s w i t h 2 - k e t o g l u c o n a t e . T h e r e was n o s t i m u l a t i o n b y A T P d u r i n g a e r o b i c s t u d i e s a n d n o p h o s p h o r y l a t e d c o m p o u n d s h a d b e e n i d e n t i f i e d u s i n g m e t h o d s 6 a n d 7 o f T a b l e VII. B a r k e r a n d L i p m a n n T s t e c h n i q u e was n e g a t i v e b o t h a e r o b i c a l l y a n d a n a e r o b i c a l l y . I t was t h e n d e c i d e d t h a t a c r u d e s o n i c a t e i n t he p r e s e n c e o f A T P a n d m a g n e s i u m i o n s m i g h t a s f o r Wood (27) a n d D e L e y (10) p r o d u c e a p h o s p h o r y l a t e d c o m p o u n d i f a n a e r o b i c c o n d i t i o n s v/ere a d o p t e d . I n p a r t i c u l a r i t was f e l t t h a t t h i s compound c o u l d be 2 -ke to . -D-gIuc6nate~6 -phosphate a n d t h i s a v e n u e s t i l l r e m a i n e d o p e n t o f u r t h e r w o r k . G l u c o n i c g r o w n c e l l s w e r e h a r v e s t e d a t 18 h o u r s a n d w a s h e d t w i c e w i t h M/50 p H 7.0 p h o s p h a t e b u f f e r . The c e l l s w e r e s u s p e n d e d i n M/50 pH 7.0 p h o s p h a t e b u f f e r a t t h e r a t e o f 250 m g s / m l a n d g l u t a t h i o n e was a d d e d a t t h e r a t e o f 20 mgs/100 m i s . T h i s r e s u l t a n t s u s p e n s i o n was s o n i c e d f o r 15 m i n u t e s a n d t h e n c e n t r i  f u g e d f o r one h o u r a t 3 ^ ° ? . W a r b u r g s t u d i e s w e r e c a r r i e d o u t o n t he c r u d e s o n i c a t e u s i n g s t r i c t a n a e r o b i o s i s . S i x c u p s c o n t a i n  i n g M/15 pH 7,h p h o s p h a t e b u f f e r , m a g n e s i u m i o n s , A T P 59. t o a f i n a l c o n c e n t r a t i o n o f 0.03M a n d x ^ a t e r , s e r v e d a s t h e e n d o g e n o u s . S i x a d d i t i o n a l c u p s w e r e s e t u p a s p r e v i o u s l y d e s c r i b e d w i t h t h e e x c e p t i o n t h a t e a c h c o n t a i n e d h m i c r o m o l e s o f 2 - k e t o g l u c o n a t e . W h i t e p h o s p h o r o u s was p l a c e d i n t h e c e n t e r w e l l o f e a c h W a r b u r g V e s s e l i n o r d e r t o a b s o r b a l l d i s s o l v e d o x y  g e n , N i t r o g e n was a l l o w e d t o p a s s t h r o u g h e a c h c u p w h i l e ' t h e y e q u i l i b r a t e d 7 m i n u t e s a t room t e m p e r a t u r e . A f t e r b e i n g c l o s e d o f f a l l v e s s e l s w e r e a l l o w e d t o e q u i l i b r a t e i n t h e W a r b u r g b a t h . The s u b s t r a t e was t h e n t i p p e d i n t o t h e r e a c t i o n v e s s e l a n d t h e w h o l e a l l o w e d t o r e a c t f o r 2 h o u r s a t 31°c«' A s s o o n a s 1 8 0 m i n u t e s h a d e l a p s e d t h e c u p s w e r e r e m o v e d a n d a l l t h e c u p s m a k i n g u p t h e e n d o g e n  o u s w e r e p o o l e d . T h o s e c u p s c o n t a i n i n g 2 - k e t o g l u c o n  a t e w e r e t r e a t e d s i m i l a r l y . T h i s was n e c e s s a r y t o k e e p w i t h i n t h e p h o s p h a t e r a n g e o f B a n e s a n d I s h e r - w o o d ' s s p e c i f i c a t i o n s f o r t h e c h r o m a t o g r a p h y o f p h o s  p h a t e e s t e r s ( a s i n m e t h o d 6 , T a b l e 1). C a l c u l a t i o n s w e r e b a s e d o n a 20% c o n v e r s i o n o f t h e 2 - k e t o g l u c o n  a t e t o t h e p h o s p h o r y l a t e d c o m p o u n d s . I t was n e c e s s a r y t o t a k e i n t o c o n s i d e r a t i o n t h a t t h e c o m b i n e d 6 c u p s c o n t a i n i n g 2 - k e t o g l u c o n a t e be h a l v e d b e f o r e c o n c e n  t r a t i o n , s i n c e o n e p o r t i o n was t o be h y d r o l y z e d . I n a d d i t i o n i t was f e l t a c a p i l l a r y p i p e t t e w o u l d d e l i v e r 60. 1- 2 m i c r o l i t r e s . D e p r o t e i n i z a t i o n o f b o t h t h e e n d o g e n o u s a n d t h e 2 - k e t o g l u c o n a t e c u p s was c a r r i e d o u t b y a d d i n g O.h m i s o f O . H - M a c e t a t e b u f f e r a t p H 3.8 a n d h e a t i n g f o r 3 m i n u t e s i n a b o i l i n g w a t e r b a t h . . D e n a t u r e d p r o t e i n was r e m o v e d b y g r a v i t y f i l t r a t i o n . The e n d o g e n o u s was c o n c e n t r a t e d t o 2-3 d r o p s i n v a c u o a t 2 5 ° C . The o t h e r p o r t i o n c o n t a i n i n g t h e r e a c t e d 2- k e t o g l u c o n a t e was h a l v e d . One h a l f was v a c u u m e d d i s t i l l e d t o s e v e r a l d r o p s a n d t h e o t h e r b r o u g h t t o 1 N w i t h HC1. The l a t t e r h a l f was h e a t e d i n a b o i l  i n g w a t e r b a t h f o r 30 m i n u t e s . T h i s l a t t e r p o r t i o n c o n t a i n i n g a n y h y d r o y z a b l e p h o s p h a t e e s t e r s was a l s o c o n c e n t r a t e d t o s e v e r a l d r o p s . C h r o m a t o g r a p h i c a n a l y s i s u s i n g H a n e s a n d I s h e r - w o o d ' s m e t h o d a s p r e v i o u s l y d e s c r i b e d , r e v e a l e d n o p h o s p h o r y l a t e d c o m p o u n d s . F r o m a r e p r o d u c t i o n o f a c h r o m a t o g r a p h i n f i g u r e (13) i t w i l l be s e e n t h a t t h e h y d r o l y s i s was e f f e c t i v e s i n c e t h e p y r o p h o s p h a t e was c l e a v e d t o i n o r g a n i c p h o s p h a t e . The m e t h o d i s a l s o v a l i d a s e v i d e n c e d b y a d e n y l i c a c i d w h i c h was e m p l o y e d i n t h e r a n g e o f t h a t c a l c u l a t e d p r e v i o u s l y . C h r o m a t o g r a p h i c a n a l y s i s I n o r d e r t o s t u d y t h e p r o d u c t s c h r o m a t o g r a p h i c a l - l y t h e r e a c t i o n was c a r r i e d o u t i n a 125 m l c a p a c i t y Inorganic Phosphorous CHROMATOGRAPHIC ANALYSIS FOR PHOSPHORYLATED COMPOUNDS FIGURE XIII Eanes and Ishe rvood * s method. Ultra violet absorbing Reacted" Hydrolyzed Endogenous AMP 2"-keto- 2-keto- gluconate gluconate 62. T A B L E V I I I SOLVENTS AND SPRAYS EMPLOYED  DURING IDENTIFICATION OF UNKNOWN Solvent Spray and treatment Purpose (1) 0.08M Acetate buffer pH 3 . 5 i n V vols, ethanol 5% ammoniacal AgN03 Ribose-5-POLj. Ribulose-5-POi^. (2) Ethyl acetate-2 Pyridine -1 Water -2 m-phenylene- diaminedihydro- chloride i n 7&% ethanol 5 mins. © 1 0 5°C, Sugar spots es p e c i a l l y ketohexonic acids show a well defined fluorescence i n u l t r a v i o l e t . ( 3 ) Phenol (or) Butanol - 8 0 H 20 -20 Ethanol -20 Aniline hydrogen oxalate A n i l i n e - 0 . 9 3 g m s . Ethanol - 5 0 . 0 0 m l s . mix,with equal v o l . 0 . 2 M , o x a l i c aciddaq) A red color with,, aldohexoses, uronic acids, pentoses. 0+) Butanol - 8 0 H 20 -20 Ethanol -10 Anil-fnp hyd rugw 1 phthalatp A n i l i n e - 0 . 9 3 g m s . p h t h a l i C l.66gms. acid H 20 sat._ loomls. butanol 5 minutes @ 1 0 5 ° C A bright red color, aldohexoses, desoxy sugars, uronic acids give various shades of red and brown. 1 ( 5 ) Ethanol -1+5 Methanol -1+5 . H 20 -10 0.1N AgNOo i n 5N NB+OH D Hexoses, hexonic and ketohexonic acids Keto acids. 2 ( 6 ) Isopropanol -2 (NRVpSOif _ 1 (1% solution). (NHl+)2S0l f washed saner. Hanes and Isherwood spray ( 1 6 ) . Heat 7 mins.steam 1 5 mins. reduce with H2S. Separation of _ Phosphoric esters.- 5 ( 7 ) TCA - 5 g m s . propanol - 1 5 m l s. H2O - 2 5 m l s . NH3 0 . 2 5 m l s . Hanes and Isherwood spray. Heat 2 mins. U l t r a v i o l e t 7 mins. (Bandurski & Axelrod) Separation of Phosphoric esters. 1 Both these sprays gave red spots(positive)with 2-ketogluconate. 2 This spray and solvent confirmed the presence of pyruvate. 3 Sucrose gave a blue spot(positive) a f t e r spraying and heating at 7 5 - 8 5 ° C 63 Warburg reaction vessel containing 25 micromoles of 2-ketogluconate (1 ml), 7 . 5 mis of a pH 7 .h M/15 phosphate buffer, 5 mis of a sonicate from gluconate grown cel ls and k mis of d i s t i l l e d water. The rate of oxidation was followed with a conventional War burg system containing one-fifth of the above con stituents plus potassium hydroxide in the center well.. When the reaction was completed the large cup was deproteinized by the addition of 1 ml.of a solution containing 6 gms of ZnSOi+^R^O and 0 . 1 gms of CUS01+.5 H2O to 2 mis. of cup content and le f t overnight. After gravity f i l t r a t i o n to remove denatured protein the whole was concentrated to approximately 1 ml in vacuo at 25°C. •A large cup was run using the crude sucrose soni cate and chromatographic analyses was carried out as for Table VIII. Every solvent and spray in that table was employed to no a v a i l . The d i f f i c u l t y was that the 1+5J« sucrose used during the sonic process had become very concentrated during the vacuum d i s t i l l a t i o n . Hence, every attempt at chromatographic analyses was masked by the heavy concentration of sucrose which obscured any products. Acid-bsse chromatography had revealed an unknown acidic spot. If this product was acidic , then i t could be adsorbed to a basic ion-exchange resin and the sucrose being neutral could be washed free. 6k. The r e s i n could then be made basic and the r e  covered product could be chromatographed. Such was the case. Several res ins were t r i e d but held the a c i d i c compound too t i g h t l y to be eluted by d i l u t e or strong NaOH.: IR-^-B an a.mberlite ion-exchange r e s i n proved s a t i s f a c t o r y . The r e s i n was condi t ion ed 12 hours i n a 1/k so lu t ion of 'concentrated ammon ium hydroxide . The deproteinized contents were passed through the r e s i n very s lowly . The whole was washed free of sucrose with 5 l i t r e s of d i s t i l l e d water and the column was made basic with 50 mis of 0 . 0 1 N NaOH. The resu l tant eluent was concentrated to 1 ml by vacuum, d i s t i l l a t i o n . c The product was ascertained to be pyruvic ac id by acid-base chromatography. This detect ion was made possible by the use of butanol saturated with kll formic ac id as a solvent . Unknown and known com pounds at a f i n a l concentration of 5 mgs/ml were spotted on Whatman No. I f i l t e r paper-by means of a c a p i l l a r y p ipe t te . The whole was allowed to develop overnight at room temperature i n a descending chroma tographic tank 28 cms i n diameter and 6 l cms i n heighth. The tank was l e f t sealed for 2k hours p r i o r to chromatographing i n order to al low e q u i l i b r a t i o n of t h e s o l v e n t . . A f t e r t h e c h r o m a t o g r a p h h a d d e v e l o p  e d i t was s p r a y e d w i t h 0,02% c h l o r o p h e n o l r e d d i s  s o l v e d i n OoOl N N a O H . T h e p r e s e n c e o f p y r u v i c a c i d was s u b s t a n t i a t e d b y t e c h n i q u e 5 o n T a b l e V I I . DISCUSSION I n t h e p a s t , e x t r e m e i n s t a b i l i t y o f t h e 2 - k e t o  g l u c o n a t e enzyme h a s made i t i m p o s s i b l e t o c h a r t t h e r o u t e o f 2 - k e t o g l u c o n a t e d e g r a d a t i o n . The p r o p o s e d p a t h w a y o f g l u c o s e d e g r a d a t i o n b y way o f g l u c o n a t e a n d 2 - k e t o g l u c o n a t e d e p e n d s u p o n t h e f a t e o f t h e l a t t e r c o m p o u n d i f t h i s r o u t e i s t o be p r o v e n f u n c  t i o n a l i n P_j_ a e r u g i n o s a . M c D o n a l d ' s m e t h o d o f o b  t a i n i n g a d r i e d c e l l p r e p a r a t i o n w i t h a l i m i t e d a b i l i  t y t o o x i d i z e 2 - k e t o g l u c o n a t e w a s t h e f i r s t p o s i t i v e p r o g r e s s made i n 3 y e a r s . The n e c e s s i t y o f p u r s u i n g a n d c l a r i f y i n g t h i s t e c h n i q u e i n o r d e r t o s o l v e 2 - k e t o g l u c o n a t e o x i d a t i o n w a s c o n s i d e r e d o f p r i m e i m p o r t a n c e i n t h e s t u d y o f t h e i n t e r m e d i a t e m e t a  b o l i s m o f P^. a e r u g i n o s a . I n o r d e r t h a t a n y i n t e r m e d i a t e s may be i d e n t i  f i e d i n m e t a b o l i c s t u d i e s , i t i s n e c e s s a r y t o o b t a i n a s t a b l e , r e p r o d u c i b l e c e l l p r e p a r a t i o n . S u c h a p r e p a r a t i o n m u s t be o b t a i n e d i n l a r g e a m o u n t s a n d o n d i f f e r e n t o c c a s i o n s . A n i n a c t i v e o r u n s t a b l e p r e  p a r a t i o n w i l l w a s t e s e v e r a l w e e k s o r m o n t h s o f p r e  l i m i n a r y w o r k . Thus- a g r e a t d e a l o f t i m e w a s s p e n t i n a t t e m p t s t o o b t a i n c o n s i s t e n t l y r e p r o d u c i b l e p r e  p a r a t i o n s . A d i f f i c u l t y e n c o u n t e r e d when d r y i n g c e l l s u n d e r 67. c a r b o n m o n o x i d e v/as t h e m e t h o d o f p r o d u c t i o n o f t h e g a s . The l a r g e number o f t r a p s made t h i s w o r k h a z a r  d o u s a n d t h e m e t h o d s u s e d t o p r o d u c e t h e g a s made i t somewhat e x p e n s i v e . T h u s , w h e n f u e l g a s was f o u n d t o s u b s t i t u t e s u c c e s s f u l l y f o r c a r b o n m o n o x i d e t h e r e s u l t s w e r e v e r y e n c o u r a g i n g . U n f o r t u n a t e l y , - h o w e v e r a l l a t t e m p t s t o a c q u i r e a r e p r o d u c i b l e p r e p a r a t i o n g a v e d i s c o u r a g i n g r e s u l t s . D e s p i t e t h e s e r e s u l t s f u e l g a s c o u l d be a v e r y i m p o r t a n t m e d i u m i n f u t u r e w o r k w i t h t h e 2 - k e t o g l u c o n a t e e n z y m e . The p r e s s u r e c r u s h i n g t e c h n i q u e was new t o e n  zyme w o r k b u t t h e r e s u l t s w e r e v e r y e n c o u r a g i n g . M a n y v a r i a b l e s s t i l l r e m a i n e d t o be e x p l o r e d , e . g . , v a r y i n g t h e f r e e z i n g t i m e a s w e l l a s t h e f r e e z i n g m e d i u m a n d e m p l o y i n g p r o t a m i n e s u l p h a t e t o p r e c i  p i t a t e a l l n u c l e i c a c i d s . V/hen t h e n u c l e i c a c i d s h a v e b e e n r e m o v e d i t i s f e l t t h a t l i v e c e l l s w i l l be e a s i l y r e m o v e d b y c e n t r i f u g a t i o n . The r e s u l t s • w i t h s o n i c p r e p a r a t i o n s w e r e t h e m o s t e n c o u r a g i n g . The f a c t t h a t a r e p r o d u c i b l e p r e  p a r a t i o n was o b t a i n e d w i t h a g o o d a b i l i t y , t o o x i d i z e 2 - k e t o g l u c o n a t e i s a s t e p t o w a r d t h e s o l u t i o n o f t h e p r o b l e m o n h a n d . A l t h o u g h t h e r e s p i r a t o r y q u o t i e n t o f t h r e e was s u b s t a n t i a t e d b y t h e p r e s e n c e o f p y  r u v a t e i n t h e r e a c t i o n m i x t u r e t h e p r o b l e m i s b y n o means s o l v e d . F o r we do n o t u n d e r s t a n d t h e m e c h a n i s m 68. whereby the t h r e e c a r b o n atoms are l o s t . No o t h e r compounds were d e t e c t e d by the ch r o m a t o g r a p h i c methods employed. An o u t s t a n d i n g problem i s the h i g h endogenous r e s p i r a t i o n . I t appears t h a t the optimum pH f o r endogenous a c t i v i t y i s i d e n t i c a l t o t h a t f o r 2 - k e t o  g l u c o n a t e o x i d a t i o n , namely, 7»h. I n a d d i t i o n , any at t e m p t s t o reduce the h i g h endogenous a c t i v i t y r e  duces the a c t i v i t y on 2 - k e t o g l u c o n a t e a c c o r d i n g l y . When g l u c o s e o r g l u c o n a t e grown c e l l s were d i s  i n t e g r a t e d , the endogenous o f the e x t r a c t was ex t r e m e l y low. But when s o n i c a t e s o f 2 - k e t o g l u c o n a t e grown c e l l s were employed the endogenous was j u s t as h i g h as f u e l gas or monoxide d r i e d c e l l s . I t remains f o r t r a c e r t e c h n i q u e s t o c l a r i f y t h i s p i c t u r e . L a s t l y , the absence o f p h o s p h o r y l a t i o n e i t h e r a e r o b i c a l l y o r a n a e r o b i c a l l y does n o t conform to the work o f D e l e y (10) or Wood ( 2 7 ) , A g a i n , i t i s i m p o s s i b l e t o g e n e r a l i z e and s t a t e t h a t P. a e r u g i n o s a i s un i q u e I n i t s m e t a b o l i s m s i n c e p h o s p h o r y l a t i o n does n o t o c c u r a t the 2 - k e t o g l u c o n a t e • l e v e l . I t may w e l l be t h a t the p h o s p h o r y l a t e d p r o d u c t i s e x t r e m e l y l a b i l e or i s decomposed i n t o u n i d e n t i f i a b l e 6 9 . p h o s p h a t e e s t e r s . The d a t a h a v e p r o v i d e d s e v e r a l means o f o b  t a i n i n g p r e p a r a t i o n s a c t i v e a g a i n s t 2 - k e t o g l u c o n a t e . I n a d d i t i o n s e v e r a l new t e c h n i q u e s h a v e b e e n i n t r o  d u c e d . 70. SUMMARY M c D o n a l d h a d o b t a i n e d a n a c t i v e c e l l p r e p a r a t i o n o f P. a e r u g i n o s a ( 2 6 ) . T h i s p r e p a r a t i o n w a s o b  t a i n e d b y d r y i n g r e s t i n g c e l l s i n a p a r t i a l v a c u u m o v e r c a l c i u m c h l o r i d e , i n t h e p r e s e n c e o f c a r b o n m o n o x i d e , a n d i n t h e a b s e n c e o f l i g h t . W o r k was c o n t i n u e d o n t h i s p r e p a r a t i o n . T h e p r e p a r a t i o n h a d a l i m i t e d a b i l i t y t o o x i d i z e 2 - k e t o g l u c o n a t e b u t r e s u l t s "were n o t r e p r o d u c i b l e . The m o n o x i d e - d r i e d p r e p a r a t i o n h a d a n e x t r e m e l y h i g h e n d o g e n o u s r e s p i r a t i o n . I t w o u l d a p p e a r t h a t e n d o g e n o u s a c t i v i t y c o u l d n o t be r e d u c e d " w i t h  o u t d e s t r o y i n g 2 - k e t o g l u c o n a t e o x i d i z i n g a c t i v i t y . The f o l l o w i n g a t t e m p t s t o p r o v i d e m o n o x i d e - d r i e d c e l l s , w h i c h w o u l d p r o d u c e a s t a b l e p r e p a r a t i o n a c t i v e a g a i n s t 2 - k e t o g l u c o n a t e , w e r e u n s u c c e s s f u l . ( a ) C e l l s w e r e d r i e d i n t h e p r e s e n c e o f h y d r o g e n a n d m o n o x i d e g a s . ( b ) C e l l s w e r e d r i e d i n t h e p r e s e n c e o f w h i t e p h o s p h o r o u s i n o r d e r t o p r e v e n t i n a c t i v a t i o n o f t h e 2 - k e t o g l u c o n a t e enzyme b y o x i d a t i o n . ( c ) C e l l s w e r e d r i e d i n t h e p r e s e n c e o f v a r y  i n g a m o u n t s o f s u b s t r a t e o n t h e p r e m i s e t h a t c n enzyme i s more s t a b l e i n t h e p r e s e n c e o f i t s s u b s t r a t e . 71. ( d ) C e l l s w e r e t r e a t e d w i t h b i l e s a l t s i n a n a t t e m p t a t s o l u b i l i z i n g t h e 2 - k e t o g l u c o n a t e e n z y m e . ( e ) V a r i o u s c o - f a c t o r s w e r e a d d e d t o t h e c e l l p r e p a r a t i o n i n t h e e v e n t t h a t i t h a d l o s t i t s a b i l i t y t o s y n t h e s i z e c o e n z y m e s d u r i n g t h e d r y i n g p r o c e s s . ( f ) C e l l s w e r e g a s s e d w i t h c a r b o n m o n o x i d e i n t h e c o m p l e t e a b s e n c e o f l i g h t , t o e n s u r e t h a t i n a c t i v a t i o n o f t h e e n z y m e b y l i g h t , d i d n o t o c c u r d u r i n g t h e g a s s i n g p r o c e s s . F u e l g a s was s u c c e s s f u l l y s u b s t i t u t e d f o r c a r  b o n m o n o x i d e b u t , a g a i n t h e p r e p a r a t i o n was n o t r e p r o d u c i b l e . I t was h y p o t h e s i z e d t h a t f u e l g a s b o u n d t h e i r o n o f c y t o c h r o m e i n t o a n i r o n - m o n o x i d e c o m p l e x r e  v e r s i b l e b y l i g h t . A n y o f t h e e x c e s s i v e i r o n t h e n a c t e d a s a c a t a l y s t f o r t h e o x i d a t i o n o f s u l f h y d r y l g r o u p s d u r i n g t h e d r y i n g p r o c e s s . V/ork w i t h c h e m i c a l l y c l e a n g l a s s w a r e a n d v a r y  i n g l e v e l s o f i r o n s u p p o r t e d t h i s h y p o t h e s i s . The f o l l o w i n g c h e m i c a l m e t h o d s w e r e a d o p t e d t o m e a s u r e t h e d i s a p p e a r a n c e o f 2 - k e t o g l u c o n a t e a n d . s u b s t a n t i a t e o x y g e n u p t a k e d a t a . 72. ( a ) F o l i n M a l m r o s r e d u c i n g s u g a r m e t h o d ( 3 3 ) . ( b ) F r i e d m a n a n d H a u g e n m o d i f i e d 2 , ^ - d i n i t r o - p h e n y l h y d r a z i n e m e t h o d ( 1 5 ) • ( c ) L a n n i n g a n d C o h e n ' s m e t h o d w h i c h m a k e s u s e o f t h e c o n d e n s a t i o n o f o - p h e n y l e n e - d i a n i n e w i t h k e t o h e x o n i c a c i d s t o f o r m 2 - h y d r o x y q u i n o x a l i n e ( 2 1 ) . 8. When 2 - k e t o g l u c o n a t e was s u b s t i t u t e d f o r g l u c o s e a s t h e g r o w t h s u b s t r a t e , t h e r e s u l t s w i t h f u e l g a s d r i e d c e l l s , w e r e i d e n t i c a l t o t h o s e o b  t a i n e d when g l u c o s e g r o w n c e l l s w e r e e m p l o y e d , 9 . R e s t i n g c e l l s w e r e d i a l y z e d t o r e m o v e a n y e x  c e s s i r o n i n t h e e v e n t t h a t t h i s i r o n c o u l d be r e s p o n s i b l e f o r c a t a l y z i n g t h e o x i d a t i o n o f s u l f h y d r y l g r o u p s . The r e s u l t s w e r e i d e n t i c a l t o t h o s e o b t a i n e d w i t h u n d i a l y z e d c e l l s . 1 0 . S t r e p t o m y c i n was e m p l o y e d i n o r d e r t o r e d u c e t h e h a z a r d o f l i v e c e l l s a p p e a r i n g i n t he f u e l g a s p r e p a r a t i o n . A l t h o u g h t h e e n d o g e n o u s r e s  p i r a t i o n was r e d u c e d t h e 2 - k e t o g l u c o n a t e a c t i  v i t y was r e d u c e d a c c o r d i n g l y . 1 1 . A t e c h n i q u e was e m p l o y e d w h e r e b y f r o z e n c e l l s , s u b j e c t e d t o g r e a t p r e s s u r e , b u r s t o p e n d u r i n g t h e t h a w i n g p r o c e s s . The p r e s e n c e o f some l i v e c e l l s made a l l a t t e m p t s t o o b t a i n a w o r k a b l e p r e p a r a t i o n i m p o s s i b l e . 73. 12o R e s t i n g c e l l s s u s p e n d e d i n s u c r o s e w e r e s o n i c e d b y a R a y t h e o n 10 K C . s o n i c o s c i l l a t o r . T h i s p r o d u c e d a s o n i c a t e w i t h a good a b i l i t y t o o x i d i z e 2 - k e t o g l u c o n a t e , t h e r e a c t i o n b e i n g c o m p l e t e d a t 1 t o 2 a t o m s o f o x y g e n . 13. p H 7v*+ was f o u n d t o be t h e o p t i m u m pH f o r t h i s s o n i c p r e p a r a t i o n . , I n - . The p r e p a r a t i o n h a d a r e s p i r a t o r y q u o t i e n t o f 3 . 0 . 15. The p r e p a r a t i o n was n o t s t i m u l a t e d b y A T F a n d showed no i n h i b i t i o n b y 2.5 x I O " 2 M s o d i u m f l u o r i d e . 16. No p h o s p h o r y l a t i o n c o u l d be d e t e c t e d e i t h e r a e r o b i c a l l y o r a n a e r o b i c a l l y b y m e a s u r i n g a c i d s t a b l e ( e s t e r ) p h o s p h a t e o r a c i d f o r  m a t i o n . 17. C h r o m a t o g r a p h i c s t u d i e s w i t h a c r u d e s o n i c a t e i n t h e p r e s e n c e o f A T P a n d m a g n e s i u m u n d e r a n a e r o b i c c o n d i t i o n s r e v e a l e d n o p h o s p h o r y - l a t e d p r o d u c t s . 13. The r e a c t i o n p r o d u c t o f 2 - k e t o g l u c o n a t e o x i  d a t i o n was d e t e r m i n e d t o be a c i d i c b y a c i d - b a s e c h r o m a t o g r a p h y a n d was f i n a l l y i d e n t i  f i e d c h r o m a t o g r a p h i c a l l y a s p y r u v i c a c i d . 7k. B I B L I O G R A P H Y B a r k e r , H . A . , a n d L i p m a n n , F . , The r o l e o f p h o s p h a t e i n t h e m e t a b o l i s m o f P r o p i o n i b a c t e r - i u m p e n t o s a c e u m . . J . B i o l , C h e m . , 1 7 9 . 217 B a r r o n , E . S . G . , a n d F r i e d m a n n , T . E . , S t u  d i e s o f b i o l o g i c a l o x i d a t i o n s : O x i d a t i o n b y m i c r o o r g a n i s m s w h i c h do n o t f e r m e n t g l u  c o s e . J . . B i o l . C h e m . , IT7_, 593 (19^1). B e r n h a u e r , K . , a n d G o r l i c k , B . , O x i d a t i o n s w i t h a c e t i c a c i d b a c t e r i a I V F o r m a t i o n o f 2 - k e t o g l u c o n i c a c i d 3 ^ g l u c o n i c u m . B i o c h e m . , r Z . . , a21> 308 - 315, ( 1 9 ^ 0 ) . B e r n h a u e r , K . , a n d K n o b l o c k , H . , O x i d a t i o n s w i t h a c e t i c a c i d b a c t e r i a . V I C o m p a r a t i v e s t u d y o n t h e f o r m a t i o n o f r e d u c i n g s u g a r c a r - b o x y l i c a c i d s a n d t h e p r e p a r a t i o n o f 2 - k e t o  g l u c o n i c a c i d . , B i o c h e m . , Z . , 280. 367 - 37^, (1935). B e r n h a u e r , K . , a n d K n o b l o c k , H . , D e c o m p o s i t i o n o f g l u c o s e b y A c e t o b a c t e r s u b o x i d a n s . 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H . , E v i d e n c e f o r a l t e r n a t e p a t h w a y s f o r t h e o x i d a t i o n o f g l u c o s e b y P s e u d o m o n a s a e r u g i n o s a . J o u r . B a c t . , 68, 77, • (195*0. 10. D e L e y , J . , The p h o s p h o r y l a t i o n o f some c a r b o h y  d r a t e s c o n n e c t e d w i t h t h e d i r e c t o x i d a t i o n b y A e r o b a c t e r c l o a c a e . E n z y m p l o g i a , , 16,, 99 (1953) * 11. Pickens, F . , O x i d a t i o n o f p h o . s p h o h e x o n a t e a n d p e n t o s e p h o s p h o r i c a c i d s b y y e a s t e n z y m e s , I ' o x i  d a t i o n o f p h o s p h o h e x o n a t e . I I o x i d a t i o n o f p e n t o s e p h o s p h o r i c a c i d s . B i o c h e m . J o u r . , "32, 1626 - 16H5, (1938). 12,. D i c k e n s , F . ' , M c l l w a i n , H . , P h e n a z i n e c o m p o u n d s a s c a r r i e r s i n t h e h e x o s e m o n o p h o s p h a t e s y s t e m . B i o c h e m . J o u r . , . 32, l 6 l 5 , (1938). 13. 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F . , C a r b o h y d r a t e o x i d a t i o n b y P s e u d o m o n a s f l u o r e s c e n s . I I M e c h a n i s m o f h e x o s e . p h o s p h a t e d e g r a d a t i o n . J o u r . B i o l . C h e m . , ; 2 0 6 , 6 2 5 , ( 1 9 5 * 0 . 

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