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Phosphate metabolism of Pseudomonas aeruginosa Hogenkamp, Harry P. C. 1958

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PHOSPHATE METABOLISM OP PSEUDOMONAS AERUGINOSA by H a r r y P. C. Hogenkamp, B. S. A, A T h e s i s S u b m i t t e d i n P a r t i a l F u l f i l l m e n t o f th e Requirements f o r t h e Degree o f MASTER OP SCIENCE IN AGRICULTURAL MICROBIOLOGY We ac c e p t t h i s t h e s i s as co n f o r m i n g t o t h e s t a n d a r d r e q u i r e d from c a n d i d a t e s f o r the degree o f Master o f S c i e n c e The U n i v e r s i t y o f B r i t i s h Columbia J u l y , 1958 ABSTRACT The o x i d a t i o n o f g l u c o s e by Pseudomonas a e r u g i n o s a i s known t o f o l l o w t h e sequence: g l u c o s e — • g l u c o n i c a c i d — • 2 - k e t o g l u c o n i c a c i d mpyruvic a c i d and thence i n t o the t r i c a r b o x y l i c a c i d c y c l e . The most s t r i k i n g a s p e c t o f t h i s pathway i s t h a t t h e f i r s t two o x i d a t i v e s t e p s do not i n v o l v e p h o s p h o r y l a t e d i n t e r m e d i a t e s at t h e s u b s t r a t e l e v e l . I n t h e p r e s e n t s t u d y r a d i o a c t i v e phosphorus was u s e d i n an attempt t o e l u c i d a t e t h e c a r b o h y d r 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 . C e l l f r e e p r e p a r a t i o n s o f P. a e r u g i n o s a , o b t a i n e d by c r u s h i n g a c e l l p a s t e i n t h e Hughes p r e s s , i n c u b a t e d w i t h added c o f a c t o r s , ADP and p32 r e s u l t e d i n the f o r m a t i o n of l a b e l l e d ADP and ATP. The presence o f g l u c o s e or s u c c i n a t e i n the r e a c t i o n m i x t u r e g r e a t l y d e p r e s s e d t h e amount o f ATP found. The c e l l f r e e p r e p a r a t i o n s were f o u n d to y i e l d ATP as measured i n t h e h e x o k i n a s e t r a p , but t h e f o r m a t i o n of ATP was not i n c r e a s e d by the a d d i t i o n o f g l u c o s e , g l u c o n i c a c i d , i i i 2 - k e t o g l u c o n i c a c i d o r s u c c i n i c a c i d . These r e s u l t s s u g g e s t e d t h a t no net energy was g a i n e d b y t h e e x t r a c t by the o x i d a t i o n glucose—» g l u c o n i c a c i d — > 2 - k e t o g l u c o n i c a c i d . f r e e p r e p a r a t i o n d i d not o x i d i z e g l u c o s e - 6 - p h o s p h a t e , r i b o s e -5-phosphate, O C - k e t o g l u t a r a t e , c i t r a t e and i s o c i t r a t e . G l u c o s e was o x i d i z e d w i t h t h e uptake o f two atoms o f oxygen per mole o f s u b s t r a t e . I n t h e presence o f ATP, g l u c o s e was o x i d i z e d w i t h the uptake o f o n l y one atom o f oxygen. G l u c o n i c a c i d and g l u c o n o l a c t o n e were o x i d i z e d w i t h the u p t a k e o f one atom o f oxygen; ATP had no e f f e c t on t h e s e l a s t two o x i d a t i o n s . Prom t h e s e d a t a two r e a c t i o n s beyond 2-ketogluconate have been p o s t u l a t e d . I n manometric e x p e r i m e n t s i t was found t h a t the c e l l g l u c o s e c a r r i e r r e d u c e d c a r r i e r g l u c o n i c a c i d -2H 2 - k e t o g l u c o n i c a c i d ATP 2-keto-6-phosphogluconic a c i d r e d u c e d c a r r i e r + 2H ( * ^ c 6 - p h o s p h o g l u c o n i c a c i d In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representative. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed, without my written permission. The University of B r i t i s h Columbia, Vancouver 8, Canada. TABLE OF CONTENTS Page INTRODUCTION 1 HISTORICAL REVIEW It MATERIALS AND METHODS 20 I Organism . 20 I I P r e p a r a t i o n o f t h e C e l l Free E x t r a c t s 20 a) Hughes P r e s s 20 b) S o n i c O s c i l l a t i o n . . . . 21 I I I D e t e c t i o n o f ATP G e n e r a t i n g Systems 22 IV S t u d i e s w i t h P h o s p h o r u s ^ 2 22 V Chromatography o f t h e R e a c t i o n M i x t u r e s . . . . 2l\. a) Dowex-l-Cl Chromatography 21+ b) Dowex-l-formate Chromatography 25 VI Acetone P r e c i p i t a t i o n o f N u c l e o t i d e s 26 V I I I d e n t i f i c a t i o n o f N u c l e o t i d e s 26 a) A b s o r p t i o n S p e c t r a 26 b) Paper chromatography 27 c) P a p e r s t r i p C o u n t i n g 27 V I I I Paper chromatography o f O r g a n i c A c i d s 28 I X D e t e r m i n a t i o n o f t h e Presence o f U n s a t u r a t e d Bonds 29 X D e t e r m i n a t i o n o f I n o r g a n i c Orthophosphate and Pyrophosphate 29 XI Manometric S t u d i e s 30 EXPERIMENTAL RESULTS 31 DISCUSSION 55 i i i i v Page SUMMARY 60 BIBLIOGRAPHY 62 ACKNOWLEDGEMENTS I would l i k e t o e x p r e s s my s i n c e r e thanks t o P r o f e s s o r J . J . R. Campbell f o r h i s d i r e c t i o n , encouragement and a s s i s t a n c e t h r o u g h o u t the course of t h i s i n v e s t i g a t i o n . I would a l s o l i k e t o thank Dr. H. G. Khorana of the B r i t i s h Columbia Research C o u n c i l f o r h i s g e n e r o s i t y i n a l l o w i n g me the use of t h e i r equipment and Dr. G. Tener f o r h i s a d v i c e and a s s i s t a n c e . I am " g r a t e f u l a l s o t o the B r i t i s h Columbia E l e c t r i c Company L i m i t e d under whose F e l l o w s h i p i n A g r i c u l t u r e t h i s work was u n d e r t a k e n . H a r r y P. C. Hogenkamp, B. S. A. v PHOSPHATE METABOLISM OP PSEUDOMONAS AERUGINOSA INTRODUCTION P r e v i o u s work has shown t h a t Pseudomonas a e r u g i n o s a A.T.C.C. 9027 d i f f e r s from f a c u l t a t i v e a e r o b i c organisms i n t h a t g l u c o s e i s o x i d i z e d by way o f g l u c o n i c a c i d and 2 - k e t o g l u c o n i c a c i d to p y r u v i c a c i d and thence by way o f t h e t r i c a r b o x y l i c a c i d c y c l e t o carbon d i o x i d e and w a t e r (68). I t has a l s o been shown t h a t e q u i m o l a r amounts o f t h e s e s i x -carbon compounds s u p p o r t e d e q u a l growth (15). I t was, t h e r e f o r e , c o n c l u d e d t h a t growing c e l l s o f P. a e r u g i n o s a do not g a i n energy d u r i n g the o x i d a t i o n s t e p s g l u c o s e — ^ g l u c o n i c a c i d — » 2 - k e t o g l u c o n i c a c i d . I n the same paper the a u t h o r s 1 2 showed t h a t d i p h o s p h o p y r i d i n e n u c l e o t i d e " * (DPN) o r TPN d i d not act as hydrogen a c c e p t o r s d u r i n g t h e o x i d a t i o n o f g l u c o s e o r g l u c o n i c a c i d by s o n i c e x t r a c t s o f P. a e r u g i n o s a and t h a t t h e s e e x t r a c t s d i d not c o n t a i n e i t h e r a DPNH or TPNH o x i d a s e . T h e i r c o n c l u s i o n , however, t h a t d u r i n g t h e o x i d a t i o n o f g l u c o s e t o 2 - k e t o g l u c o n a t e no ATP was g e n e r a t e d was i n v a l i d because AMP was used as t h e h i g h energy phosphate a c c e p t o r . Chance (18) has su g g e s t e d t h a t r a t h e r t h a n AMP, ADP i s t h e energy a c c e p t o r and t h a t t h e l a t t e r i s c o n v e r t e d t o ATP, w h i c h i s t h e l i m i t i n g f a c t o r i n t h e h e x o k i n a s e energy t r a p . The a d d i t i o n o f P32 l a b e l l e d o r t h o p h o s p h a t e t o a c e l l f r e e e x t r a c t o f P. a e r u g i n o s a A.T.C.C. 9027 w i t h added c o f a c t o r s i n t he absence o f s u b t r a t e r e s u l t e d i n t h e i n c o r p o r a t i o n o f t h e r a d i o a c t i v e phosphate i n t o o r g a n i c phosphate (16). I n t h e p r e s e n t s t u d y the i n c o r p o r a t i o n o f r a d i o phosphorus i n t o ADP and ATP was found i n c e l l f r e e e x t r a c t s o f P. a e r u g i n o s a A.T.C.C. 9027 w i t h and w i t h o u t added s u b s t r a t e . The a d d i t i o n o f g l u c o s e o r s u c c i n a t e as s u b s t r a t e r e s u l t e d i n a . the f o l l o w i n g a b b r e v i a t i o n s are u s e d i n t h i s p r e s e n t a t i o n : AMP - adenosine monophosphate; ADP - adenosine d i p h o s p h a t e ; ATP - adenosine t r i p h o s p h a t e ; GMP - guanosine monophosphate; GDP - guanosine d i p h o s p h a t e ; GTP - guanosine t r i p h o s p h a t e ; UMP - u r i d i n e monophosphate; TJDP - u r i d i n e d i p h o s p h a t e ; TJTP - u r i d i n e t r i p h o s p h a t e ; PAD - f l a v i n adenine d i n u c l e o t i d e ; DPN - d i p h o s p h o p y r i d i n e n u c l e o t i d e ; DPNH - r e d u c e d d i p h o s p h o p y r i d i n e n u c l e o t i d e ; TPN - t r i p h o s p h o p y r i d i n e n u c l e o t i d e ; TPNH - re d u c e d t r i p h o s p h o p y r i d i n e n u c l e o t i d e ; DNP - 2, i L - d i n i t r o p h e n o l ; TCA - t r i c h l o r o a c e t i c a c i d . 3 decrease i n the amount of ATP found. Since any oxidation re s u l t s i n the l i b e r a t i o n of energy i t seems l o g i c a l to assume that the energy gained i n the oxidation of glucose or gluconate to 2-ketogluconate i s not available for c e l l growth. HISTORICAL REVIEW Tremendous developments have o c c u r r e d i n t h e f i e l d s o f b i o l o g i c a l thermodynamics o r e n e r g e t i c s i n t h e p a s t f i f t e e n y e a r s and o n l y a v e r y l i m i t e d d i s c u s s i o n o f t h e s e events w i l l be attempted h e r e . I n b i o l o g i c a l o x i d a t i o n s c h e m i c a l energy i s s e t f r e e and t r a n s f o r m e d i n t o a s p e c i a l form b e f o r e i t can be c o n v e r t e d t o o t h e r k i n d s o f energy. T h i s s p e c i a l f o rm o f energy i s t h a t w h i c h i s s t o r e d i n t h e pyrophosphate bond o f ATP. T h i s compound i s t h e p r i m a r y c a r r i e r between e x e r g o n i c or energy y i e l d i n g r e a c t i o n s and e n d e r g o n i c or energy r e q u i r i n g f u n c t i o n s o f a l l c e l l s . I n 1929 Lohman i s o l a t e d ATP and i n subsequent y e a r s p r o v e d i t s s t r u c t u r e and demonstrated i t s s t e p w i s e d e g r a d a t i o n t o ADP and AMP ( 6 l ) . The ATP formed i n t h e s e e x e r g o n i c r e a c t i o n s i s not s t o r e d as such but i s p l a c e d i n a r e s e r v e p o o l o f energy r i c h compounds, t h e "phosphagens." I n v e r t e b r a t e s t h i s energy i s s t o r e d as p h o s p h o c r e a t i n e ; i n i n v e r t e b r a t e s as 1+ 5 p h o s p h o a r g i n i n e , g l y c y a m i n e p h o s p h a t e , t aurocyaminephosphate o r l o m b r i c i n e p h o s p h a t e ( 3 8 , 69) and i n m i c r o o r g a n i s m s p o l y p h o s p h a t e may s e r v e as a phosphagen ( 6 0 , 6 5 ) . These compounds are c a l l e d h i g h energy compounds because t h e y l i b e r a t e r e l a t i v e l y l a r g e amounts o f f r e e energy on h y d r o l y s i s , compared t o t h e "normal" phosphate e s t e r s . The e x t r a o r d i n a r y f e a t u r e o f t h e s e compounds does not seem t o be t h e i r h i g h energy c o n t e n t but r a t h e r t h e i r r e m a r k a b l e s t a b i l i t y i n s p i t e o f t h i s h i g h bond energy ( 5 3 ) . A r s e n a t e competes w i t h i n o r g a n i c phosphate i n t h e p r i m a r y p h o s p h o r y l a t i n g r e a c t i o n , , t h e a r s e n a t e e s t e r s h y d r o l y z e i n s t a n t a n e o u s l y i n c o n t r a s t t o t h e r e l a t i v e s t a b i l i t y o f t h e i r phosphate c o u n t e r p a r t s . T h i s phenomenon was c a l l e d " a r s e n o l y s i s . " Crane and Lipraann (25) found t h a t a r s e n a t e i n c r e a s e d t h e r e s p i r a t i o n o f washed m i t o c h o n d r i a I n t h e absence o f i n o r g a n i c phosphate. I t was c o n c l u d e d t h a t a r s e n a t e d i s r u p t s a e r o b i c p h o s p h o r y l a t i o n by s u b s t i t u t i n g f o r phosphate. A comparison o f a c e t i c a n h y d r i d e , a c e t y l p h o s p h a t e and p y r o p h o s p h a t e , a l l o f w h i c h y i e l d a p p r o x i m a t e l y e q u a l amounts o f energy on h y d r o l y s i s , shows t h a t t h e f i r s t compound has a l i f e t i m e i n water o f o n l y a few seconds w h i l e t h e pyrophosphate l i n k a g e has almost i n f i n i t e s t a b i l i t y under t h e same c o n d i t i o n s ( 5 l ) . Kay (l+l) showed i n 1928 t h a t t h e energy r e l e a s e d on the h y d r o l y s i s o f a s t a b l e phosphate e s t e r i s l a r g e l y a r e s u l t o f t h e " d r i v i n g f o r c e o f w a t e r . " ( A P = 200 c a l . , c o r r e c t e d 6 f o r the aqueous medium A F = - 2 2 8 0 c a l . ) . The energy i s not s t o r e d i n the phosphate e s t e r but r e p r e s e n t s t h e " d r i v i n g a c t i o n o f w a t e r . " The h i g h e r energy bonds are t h e r e s u l t o f s e v e r a l s e p a r a t e e f f e c t s , t h e major one i s t h e g r e a t e r resonance s t a b i l i z a t i o n o f the h y d r o l y s i s p r o d u c t s . The guanidium i o n has t h r e e resonance s t r u c t u r e s w h i l e guanidium-phosphate has o n l y tx%ro resonance s t r u c t u r e s . NH"2 +NH? N H p + 1 II I + H 2N = 0 - NH-2 •« •> - C - ™ 2 " m 2 - G = ^ 2 H i l l and M o r a l e s (37) p o i n t out t h a t t h e i n s t a b i l i t y o f the pyrophosphates may a l s o be due t o t h e e l e c t r o s t a t i c r e p u l s i o n o f the n e g a t i v e l y charged a c i d g roups. The r e l e a s e o f energy by t h e combustion i n b i o l o g i c a l o x i d a t i o n s t a k e s p l a c e i n t h r e e major s t e p s ( I 4 . 7 ) . I n t h e f i r s t s t e p t h e l a r g e m o l e c u l e s a re broke n down t o s m a l l u n i t s , c a r b o h y d r a t e s are c o n v e r t e d t o h e x o s e s , p r o t e i n s t o amino a c i d s and f a t s t o g l y c e r o l and f a t t y a c i d s , the energy l i b e r a t e d i n t h i s s t e p b e i n g r a t h e r s m a l l . I n the second s t e p the p r o d u c t s are i n c o m p l e t e l y combusted t o one o f t h r e e s u b s t a n c e s , a c e t y l CoA, o c - k e t o g l u t a r i c a c i d and o x a l o - a c e t i c a c i d , b e s i d e s some C O 2 and H 2 0 . These t h r e e end p r o d u c t s t a k e p a r t i n t h e t h i r d s t e p where t h e y a re c o m p l e t e l y o x i d i z e d t o C O 2 and H 2 O i n t h e t r i c a r b o x y l i c a c i d c y c l e . A p p r o x i m a t e l y o n e - t h i r d o f t h e t o t a l energy o f combustion i s l i b e r a t e d i n s t e p 2 and t w o - t h i r d s i n 7 s t e p 3. The mechanism o f t h e f o r m a t i o n o f ATP i n a n a e r o b i c g l y c o l y s i s i s w e l l e s t a b l i s h e d . F o r the l a s t two s t e p s i n t h e r e a c t i o n sequence the e q u i l i b r i u m c o n s t a n t approaches one s i n c e the energy c o n t a i n e d i n t h e a c y l - m e r c a p t a n i s p r e s e r v e d i n t h e ATP formed. I n s i m i l a r ways h i g h energy bonds are formed i n p h o s p h o e n o l p y r u v i c a c i d i n a n a e r o b i c g l y c o l y s i s and s u c c i n y l l i p o i c a c i d i n the o x i d a t i o n o f cx - k e t o g l u t a r i c a c i d . These t h r e e r e a c t i o n s are r e f e r r e d t o as " a n a e r o b i c p h o s p h o r y l a t i o n s at t h e s u b s t r a t e l e v e l , " because no m o l e c u l a r oxygen i s i n v o l v e d i n t h e s e p h o s p h o r y l a t i o n s . Independence from m o l e c u l a r oxygen i s a l s o t y p i c a l f o r p h o t o s y n t h e t i c p h o s p h o r y l a t i o n s t a k i n g p l a c e i n g r e e n p l a n t s and p h o t o s y n t h e t i c b a c t e r i a . D u r i n g p h o t o s y n t h e s i s a p o r t i o n o f t h e l i g h t e nergy i s t r a n s f o r m e d i n t o ATP w i t h o u t f i r s t b e i n g s t o r e d i n some p r o d u c t s o f C 0 2 a s s i m i l a t i o n ( 3 ) . Arnon and coworkers ( I 4 . ) found t h a t o n l y p a r t o f t h e l i g h t energy absorbed by c h l o r o p h y l l i s t r a p p e d i n t h e pyrophosphate bond o f ATP, t h e remainder i s used f o r t h e f o r m a t i o n o f TPNH+H+ from TPN +, a c c o r d i n g t o t h e o v e r a l l r e a c t i o n : 2ADP + 2P + 2TPN + I+H20 - 2ATP + 0 2 + 2TPNH 2 + 2H 20 The re d u c e d t r i p h o s p h o p y r i d i n e n u c l e o t i d e i s t h e n o x i d i z e d by a 6 carbon compound, formed from a pentose and C 0 2 , r e s u l t i n g i n sugarphosphates and TPN*. 8 The t h i r d mechanism by which energy i s generated i n the form of ATP i s oxidative phosphorylation by which inorganic phosphate i s converted into phosphate esters or anhydrides with concurrent oxidation. Whereas anaerobic phosphorylation at the substrate l e v e l occurs i n homogenous solutions the mechanisms responsible for oxidative phosphorylation and photosynthetic phosphorylation are associated with insoluble p a r t i c l e s , mitochondria and chloroplasts respectively. The mechanism by which ATP i s formed i n oxidative phosphorylation i s not known. Lehninger et a l . (73) proposed the following hypothetical scheme. The reduced c a r r i e r AH 2 reacts with the next c a r r i e r i n the respiratory chain, B, not d i r e c t l y but after i n t e r a c t i o n with an enzyme C. During t h i s oxidation-reduction reaction the low energy linkage of AH-2-C i s converted to the high energy linkage of A ^ C . The next reaction i s the coupled phosphorylation by revers i b l e phosphorolysis of C and reversible transfer of the phosphate from P<—C to ADP. AH 2 + C ^ ' AH2 - C AH 2 - C + B .. ^ A ^ G + BH 2 A ~~ C + Pi . " A + P ~~ C P ~ C + ADP "ATP + C Oxidative Phosphorylation by Mitochondrial Fragments It has been generally observed that oxidative phosphorylation with high e f f i c i e n c y may be demonstrated with mitochondria i s o l a t e d from animal tissue only i f they are 9 r e l a t i v e l y i n t a c t m o r p h o l o g i c a l u n i t s . D i s r u p t i o n o f the m i t o c h o n d r i a l s t r u c t u r e r e s u l t s i n the l o s s o f the ph o s p h o r y l a t i n g a c t i o n s although the e l e c t r o n t r a n s p o r t system may s u r v i v e such treatment. Cooper and Lehninger (20) were able to demonstrate o x i d a t i v e p h o s p h o r y l a t i o n s i n l i p o p r o t e i n r i c h f r a c t i o n s o f r e l a t i v e l y low p a r t i c l e weight separated from d i g i t o n i n e x t r a c t s o f r a t l i v e r m i t o c h o n d r i a . E x t r a c t s o f mi t o c h o n d r i a o b t a i n e d by v i b r a t i o n , exposure to butanol-x^ater mixtures, d r y i n g w i t h acetone, exposure to hypotonic media, g r i n d i n g and treatment w i t h c h o l a t e or desoxycholate were found to be t o t a l l y i n a c t i v e . T h e i r p r e p a r a t i o n d i d not r e q u i r e M g + + f o r maximal P:0 r a t e s and C a + + d i d not uncouple p h o s p h o r y l a t i o n even i n q u i t e h i g h c o n c e n t r a t i o n s . The 5 -diphosphates o f i n o s i n e , u r i d i n e , c y t i d i n e , thymidine and guanosine were e s s e n t i a l l y i n a c t i v e as phosphate a c c e p t o r s . In the same s e r i e s o f papers D e v l i n and Lehninger (27) observed t h a t when e x t e r n a l DPN + was added to a system c o n t a i n i n g enzyme p r e p a r a t i o n , / 3-hydroxybutyrate, cytochrome c and cyanide to prevent r e o x i d a t i o n o f cytochrome c, the r a t e o f r e d u c t i o n o f cytochrome c was i n c r e a s e d but the P:2e r a t e s decreased. Antimycin A d i d not completely i n h i b i t the r e d u c t i o n o f cytochrome c when DPN + was added, although the r e d u c t i o n was completely i n h i b i t e d by antimycin A i n the absence o f e x t e r n a l DPNH . T h i s suggested that t h e r e may be an a l t e r n a t e pathway f o r e l e c t r o n t r a n s f e r which i s not s e n s i t i v e to antimycin A. The r e d u c t i o n o f cytochrome c by c h e m i c a l l y prepared DPNH 10 u s u a l l y proceeded at a h i g h e r r a t e t h a n r e d u c t i o n by /3-hydro x y b u t y r a t e . I t was much l e s s s e n s i t i v e t o a n t i m y c i n A and no p h o s p h o r y l a t i o n o c c u r r e d . The a u t h o r s c o n c l u d e d t h a t the m i t o c h o n d r i a l p r e p a r a t i o n i s capable o f c a t a l i z i n g two pathways f o r t h e r e d u c t i o n o f cytochrome c; one o p e r a t e s t h r o u g h t h e ^3-hydro x y b u t y r ate dehydrogenase enzyme and t h e bound DPN + p r e s e n t i n t h e complex. T h i s pathway i s c o m p l e t e l y b l o c k e d by a n t i m y c i n A and i s p h o s p h o r y l a t i n g . The second pathway i n t e r v e n e s when f r e e DPNH i s added o r when i t i s formed from f r e e DPW+ added t o t h e medium. T h i s pathway i s l e s s s e n s i t i v e t o a n t i m y c i n A and i s n o n - p h o s p h o r y l a t i n g . I t appears v e r y p r o b a b l e t h a t the ATPase a c t i v i t y o f the enzyme complex o b t a i n e d from d i g i t o n i n e x t r a c t s o f m i t o c h o n d r i a has a f u n c t i o n a l r e l a t i o n t o the enzyme mechanism by w h i c h p h o s p h o r y l a t i o n o f ADP i s c o u p l e d t o e l e c t r o n t r a n s p o r t . The ATPase a c t i v i t y i s a r e f l e c t i o n o f t h e h y d r o l y t i c breakdown o f an i n t e r m e d i a t e h i g h energy phosphate e s t e r n o r m a l l y formed d u r i n g o x i d a t i v e p h o s p h o r y l a t i o n . T h i s h y p o t h e t i c a l i n t e r m e d i a t e P <~ C seems t o be more exposed t o t h e h y d r o l y t i c a c t i o n o f w a t e r i n t h e d i g i t o n i n e x t r a c t , s i n c e the ATPase a c t i v i t y i s h i g h e r i n t h i s e x t r a c t t h a n i t i s i n i n t a c t m i t o c h o n d r i a ( 2 2 ) . A s t r i k i n g c o n t r a s t t o o b s e r v a t i o n s on the r a t e o f ATP-Pi exchange r e a c t i o n s i n i n t a c t m i t o c h o n d r i a , r e l a t i v e t o r a t e s o f P i upt a k e d u r i n g c o u p l e d o x i d a t i o n , was found by the same w o r k e r s . The r a t e o f exchange r e a c t i o n i n i n t a c t m i t o c h o n d r i a was t w i c e as g r e a t as i s t h e r a t e o f P i uptake d u r i n g t h e o x i d a t i o n o f p - h y d r o x y b u t y r a t e , whereas i n d i g i t o n i n e x t r a c t s t h e ATP-P-^2 exchange was o n l y a 11 v e r y s m a l l f r a c t i o n o f the maximal r a t e o f net upta k e o f P i . Cooper and L e h n i n g er a s c r i b e t h i s d i f f e r e n c e t o t a) the p o s s i b l e o c c u r r e n c e o f D N P - s e n s i t i v e exchange r e a c t i o n s i n i n t a c t m i t o c h o n d r i a w h i c h are ext r a n e o u s t o o x i d a t i v e phosphory-l a t i o n and whi c h do not o c c u r i n t h e i s o l a t e d enzyme complex; b) the p o s s i b l e r e q u i r e m e n t o f the exchange i n t h e i s o l a t e d enzyme complex f o r o t h e r as y e t u n i d e n t i f i e d f a c t o r s ; and c) the l o s s o f a l a r g e p a r t o f t h e t r a n s f e r a s e a c t i v i t y d u r i n g t h e i s o l a t i o n o f t h e d i g i t o n i n complex. A m i t o c h o n d r i a l p r e p a r a t i o n d i f f e r i n g from t h o s e p r e v i o u s l y mentioned was o b t a i n e d by K i e l l e y and Bronk (ii3) from r a t l i v e r . S o n i c e x t r a c t s o f r a t l i v e r m i t o c h o n d r i a e x h i b i t e d p h o s p h o r y l a t i o n c o u p l e d t o the o x i d a t i o n o f s u c c i n a t e o r DPNH w i t h P:0 r a t i o somewhat l e s s t h a n u n i t y . T h i s p r e p a r a t i o n a l s o showed a d e f i n i t e M g + + r e q u i r e m e n t f o r the co u p l e d p h o s p h o r y l a t i o n and e x t e r n a l DPN + d i d not decrease t h e P:0 r a t i o . Abood and A l e x a n d e r (1) d e s c r i b e d a multienzyme p r e p a r a t i o n from r a t b r a i n m i t o c h o n d r i a by t r e a t m e n t w i t h t h e d e t e r g e n t T r i t o n , a n o n - i o n i c d i s p e r s i n g a g e n t . The sub-m i t o c h o n d r i a l f r a c t i o n thus o b t a i n e d was cap a b l e o f c a r r y i n g on o x i d a t i v e p h o s p h o r y l a t i o n w i t h most o f the t r i c a r b o x y l i c a c i d i n t e r m e d i a t e s i n c o n t r a d i s t i n c t i o n t o L e h n i n g e r ' s l i v e r f r a c t i o n where the o n l y o x i d a s e s o f a p p r e c i a b l e a c t i v i t y were t h o s e o f s u c c i n i c a c i d and - h y d r o x y b u t y r i c a c i d . A p p a r e n t l y t h e d i g i t o n i n t r e a t m e n t i s more d e s t r u c t i v e t h a n t h e t r e a t m e n t w i t h T r i t o n . 12 A l t h o u g h L e h n i n g e r ' s l i v e r f r a c t i o n r e q u i r e s no M g + + , the b r a i n s u b m i t o c h o n d r i a l u n i t appeared t o have an a b s o l u t e r e q u i r e m e n t f o r M g + + and ADP f o r o x i d a t i v e p h o s p h o r y l a t i o n . Other n u c l e o t i d e s c o u l d n o t r e p l a c e ADP as phosphate a c c e p t o r . The a u t h o r s suggested t h a t t h e a c t i o n o f CDP and ITDP i s m e d i a t e d t h r o u g h ADP and s i n c e the endogenous ADP i s removed from t h e i n t a c t m i t o c h o n d r i a t h r o u g h d i s r u p t i o n , t h e s e n u c l e o t i d e s are no l o n g e r a c t i v e . A s u b m i t o c h o n d r i a l p a r t i c u l a t e u n i t has been s t u d i e d e x t e n s i v e l y by Green et a l . ( 3 5 ) . T h i s e l e c t r o n t r a n s p o r t p a r t i c l e (E.T.P.) c o n t a i n e d t h e complete e l e c t r o n system f o r the a e r o b i c o x i d a t i o n o f s u c c i n a t e and DPNH. T h e i r w o r k i n g h y p o t h e s i s was t h a t E.T.P. i s a s i n g l e enzymic u n i t i n w h i c h a c o n s i d e r a b l e number o f i n d i v i d u a l p r o t e i n s are l i n k e d t o g e t h e r t o form a c o n t i n u o u s e l e c t r o n t r a n s f e r c h a i n . T h i s p a r t i c l e , however, d i d not c a r r y o x i d a t i v e p h o s p h o r y l a t i o n . I n a subsequent paper Z i e g l e r e t a l . (76) r e p o r t e d t h e p r e p a r a t i o n o f an e l e c t r o n t r a n s p o r t p a r t i c l e from b e e f h e a r t m i t o c h o n d r i a t h a t c o u l d c a r r y on o x i d a t i v e p h o s p h o r y l a t i o n w i t h the same e f f i c i e n c y as t h e i n t a c t m i t o c h o n d r i a . T h i s p h o s p h o r y l a t i n g e l e c t r o n t r a n s p o r t p a r t i c l e (P.E.T.P.) showed a P : 0 r a t i o o f it f o r the o x i d a t i o n o f « - k e t o g l u t a r a t e t o s u c c i n a t e , 3 f o r the one s t e p o x i d a t i o n s o f m a l a t e and p y r u v a t e and 0 f o r the o x i d a t i o n o f s u c c i n a t e . When DPNH was added t o P.E.T.P. i t was r a p i d l y o x i d i z e d but no p h o s p h o r y l a t i o n o c c u r r e d ; the a u t h o r s c o n c l u d e d t h a t o n l y o x i d a t i o n s c a t a l y z e d by t h e bound p y r i d i n o - p r o t e i n s o f P.E.T.P. i n v o l v e e s t e r i f i c a t i o n o f i n o r g a n i c phosphate. Racker (1±8) showed a s i m i l a r r e l a t i o n s h i p between " i n t e r n a l " 13 and " e x t e r n a l " systems. Large oxygen uptakes were o b s e r v e d i n a m i t o c h o n d r i a l system c o n t a i n i n g e t h a n o l , DPN, c r y s t a l l i n e a l c o h o l dehydrogenase and t h e u s u a l supplements f o r the d e m o n s t r a t i o n o f p h o s p h o r y l a t i o n . The oxygen uptake c o r r e s p o n d e d t o t he o x i d a t i o n o f e t h a n o l by t h e " e x t e r n a l " a l c o h o l dehydrogenase system v i a the m i t o c h o n d r i a ; however, no p h o s p h o r y l a t i o n was o b s e r v e d . I t was co n c l u d e d t h a t o n l y " i n t e r n a l " DPNH c o u l d be o x i d i z e d by the m i t o c h o n d r i a i n such a way t h a t p h o s p h o r y l a t i o n s a l s o o c c u r r e d . A p p a r e n t l y a l c o h o l dehydrogenase cannot p e n e t r a t e t o t h e s i t e o f t h e i n t e r n a l DPNH and e x t e r n a l DPNH cannot r e a c h t h e i n t e r n a l cytochrome c. R e c e n t l y Pearce e t a l . (6Lj_) r e p o r t e d a c y t o c h e m i c a l d e m o n s t r a t i o n o f the r e l a t i o n s h i p s between t h e DPN-diaphorase (DPND) and t h e s u c c i n i c dehydrogenase (SD) systems. Prom o b s e r v a t i o n s o f the d i f f e r e n t c e l l s t h e y c o n c l u d e t h a t t h r e e t y p e s o f m i t o c h o n d r i a are p r e s e n t : a) o n l y SD c o n t a i n i n g ; b) o n l y DPND c o n t a i n i n g ; c) SD- and DPND c o n t a i n i n g . There i s some evi d e n c e t h a t t y p e s a) and b) can c o - e x i s t i n a s i n g l e c e l l . The cytochrome c o x i d a s e a c t i v i t y o f s u s p e n s i o n s o f p l a n t m i t o c h o n d r i a was i n c r e a s e d from 2.8 t o £2 t i m e s by i n c u b a t i n g them w i t h a d i g i t o n i n s o l u t i o n f o r 30 seconds. Simon (66) s u g g e s t e d t h a t d i g i t o n i n a c t s by d i s p e r s i n g l i p i d l i n k s between p r o t e i n m o l e c u l e s i n t h e m i t o c h o n d r i a i n such a way as to r e n d e r the cytochrome o x i d a s e m o l e c u l e s more a c c e s s i b l e t o t h e i r exogenous s u b s t r a t e , reduced cytochrome c. Ik O x i d a t i v e P h o s p h o r y l a t i o n by B a c t e r i a The l i t e r a t u r e on t h e o x i d a t i v e p h o s p h o r y l a t i o n by b a c t e r i a has been r e v i e w e d by Campbell ( 1 6 ) and M a h l e r ( 5 6 ) . More r e c e n t l y T i s s i e r e s e t a l . ( 7 0 ) have r e p o r t e d the f r a c t i o n a t i o n o f an e x t r a c t from A z o t o b a c t e r v i n e l a n d i i i n t o a l a r g e p a r t i c l e f r a c t i o n and a s m a l l p a r t i c l e f r a c t i o n . The s m a l l p a r t i c l e s showed a g r e a t e r r e s p i r a t o r y a c t i v i t y t h a n t h e l a r g e p a r t i c l e s and a l s o a h i g h e r P : 0 r a t i o w i t h s u c c i n a t e and DPNH as s u b s t r a t e s . The c o n c l u d e d t h a t t h e r e s p i r a t o r y c h a i n i n t h e s e e x t r a c t s i s l o c a l i z e d i n t h e s m a l l p a r t i c l e s . The DPNH o x i d a s e a c t i v i t y o f the s m a l l p a r t i c l e f r a c t i o n x-jas s i m i l a r t o t h a t o f t h e e l e c t r o n - t r a n s p o r t i n g p a r t i c l e d e s c r i b e d by Bruemmer et a l . ( 1 1 ) . C e n t r i f u g a t i o n o f s o n i c e x t r a c t s o f A z o t o b a c t e r v i n e l a n d i i y i e l d e d a " f l u f f y l a y e r " and a r e d d i s h s u p e r n a t a n t ( 3 6 ) . The s u p e r n a t a n t was found t o e x h i b i t b o t h o x i d a t i v e and c o u p l e d p h o s p h o r y l a t i v e a c t i v i t y , whereas no d e t e c t a b l e o x i d a t i o n o f ot - k e t o g l u t a r a t e o r d i s a p p e a r a n c e o f ort h o p h o s p h a t e c o u l d be demonstrated u s i n g the " f l u f f y l a y e r . " The o x i d a t i v e p h o s p h o r y l a t i o n system was e x t r e m e l y l a b i l e , but c o u l d be s t a b i l i z e d by t h e a d d i t i o n o f M g + + and ATP. P : 0 r a t i o s g r e a t e r t h a n u n i t y were r o u t i n e l y o b t a i n e d w i t h p y r u v a t e and u s u a l l y w i t h o c - k e t o g l u t a r a t e . The a u t h o r s o b s e r v e d an i n c o m p l e t e c y a n i d e i n h i b i t i o n f o r t h e o x i d a t i o n o f s u c c i n a t e , m a l a t e and fumarate and concluded t h a t a p o r t i o n o f t h e e l e c t r o n s were t r a n s f e r r e d to oxygen by a s o l u b l e n o n - p h o s p h o r y l a t i v e pathway. U n d i a l y z e d p r e p a r a t i o n s were found t o e s t e r i f y i n -o r g a n i c phosphate t o a l i m i t e d e x t e n t i n t h e absence o f an 15 a c c e p t o r system; d i a l y z e d e x t r a c t s r e q u i r e d the a d d i t i o n o f a phosphate a c c e p t o r system. Both ATPase and myokinase a c t i v i t i e s were found i n t h e s e e x t r a c t s . O x i d a t i v e p h o s p h o r y l a t i o n i n I n t a c t A c e t o b a c t e r  suboxydans has been s t u d i e d by K l u n g s o y r e t a l . (kk), who showed p h o s p h o r y l a t i o n c o u p l e d t o t h e o x i d a t i o n o f g l u c o s e , f r u c t o s e and g l y c e r o l w i t h low P:0 r a t i o s o f 015 o r l e s s . D e s p i t e i t s a e r o b i c c h a r a c t e r i s t i c s A. suboxydans was found t o be e s s e n t i a l l y d e v o i d o f t h e TCA c y c l e . I n o r g a n i c pyrophosphate may serve as an i n t e r m e d i a t e i n p h o s p h o r y l a t i o n i n t h i s o r g a n i s m . Polymeta-phosphate was found i n r e l a t i v e l y l a r g e amounts and t h e a u t h o r s s u g g e s t e d t h a t t h i s m a t e r i a l may p a r t i c i p a t e i n the f o r m a t i o n o f p y r o p h o s p h a t e . The f o l l o w i n g scheme was s u g g e s t e d f o r ATP f o r m a t i o n s : A - R - PP + p 3 2 - p32 m A - R - P - p32 _ P 3 2 + p U t t e r e t a l . ( 72 ) r e p o r t e d t h e p r e p a r a t i o n o f a s u b c e l l u l a r p a r t i c l e from y e a s t , w h i c h showed p h o s p h o r y l a t i o n c o u p l e d t o t h e o x i d a t i o n o f s u c c i n a t e and l a c t a t e . The p h o s p h o r y l a t i o n was dependent on the p r e s ence o f a ATP t r a p p i n g system, w h i l e ADP was the o n l y phosphate a c c e p t o r . They c o n c l u d e d t h a t , because t h e y were a b l e t o c a r r y out o x i d a t i v e p h o s p h o r y l a t i o n w i t h o u t s u p p l e m e n t a t i o n by s o l u b l e c e l l components, the y e a s t p a r t i c l e s r e s e m b l e d a n i m a l m i t o c h o n d r i a more t h a n d i d m i c r o b i a l systems. 16 ATP G e n e r a t i n g S i t e s The f r e e energy o f h y d r o l y s i s o f ATP t o ADP has been c a l c u l a t e d by Meyerhof (57) t o be - 1 2 , 0 0 0 c a l . and l a t e r c o r r e c t e d t o - 1 1 , 5 0 0 c a l . ( 5 $ ) . More r e c e n t c a l c u l a t i o n s a r r i v e d at much l o w e r v a l u e s and some as low as - 7 , 0 0 0 c a l . have now been e s t a b l i s h e d ( 5 6 ) . U s i n g the e q u a t i o n ^ F = nPE, 7,000 c a l xirould c o r r e s p o n d t o an e l e c t r o n p o t e n t i a l o f a p p r o x i m a t e l y 0 . 1 5 v o l t s . The sequence o f t h e enzymes i n t h e r e s p i r a t o r y c h a i n f o r o x i d a t i v e p h o s p h o r y l a t i o n has been d e f i n i t e l y e s t a b l i s h e d by Chance and W i l l i a m s ( l 8 ) . A n t i m y c i n A t r e a t m e n t c l e a r l y d i v i d e d them I n t o two groups: cytochromes a-3, a and c and cytochrome b, f l a v o p r o t e i n and DPNH. The f o l l o w i n g r e s p i r a t o r y c h a i n f o r s u b s t r a t e o x i d a t i o n was f o r m u l a t e d b y t h e a u t h o r s : 0 2 — • a - j — • a — - c — » b — » f p — » D P N — • > s u b s t r a t e Working w i t h m i t o c h o n d r i a l f r a c t i o n s o f r a t l i v e r F r i e d k i n and L e h n i n g e r (32) showed p h o s p h o r y l a t i o n c o u p l e d t o the o x i d a t i o n o f DPNH by oxygen. Subsequent s t u d i e s by Leh n i n g e r (50) r e s u l t e d i n t h e e s t a b l i s h m e n t o f P : 0 r a t i o s o f a p p r o x i m a t e l y 2 .6 for- t h i s o x i d a t i o n . A s i m i l a r P : 0 r a t i o was o b t a i n e d when - h y d r o x y b u t y r a t e was used as an e l e c t r o n donor. I t i s now g e n e r a l l y a c c e p t e d t h a t t h r e e p h o s p h o r y l a t i o n s are co u p l e d t o t h e o x i d a t i o n o f DPNH t o oxygen. 1 7 Measurements o f t h e p h o s p h o r y l a t i o n c o u p l e d t o e l e c t r o n t r a n s p o r t between / 3 - h y d r o x y b u t y r a t e and added f e r r i -cytochrome c were made by Borg s t r o m e t a l . ( 8 ) . The cytochrome o x i d a s e system was b l o c k e d by c y a n i d e and the P : 0 r a t i o was found t o be c o n s i s t e n t w i t h t h e e x i s t e n c e o f 2 p h o s p h o r y l a t i o n s . S i m i l a r r e s u l t s were o b t a i n e d by D e v l i n and L e h n i n g e r w i t h d i g i t o n i n e x t r a c t s o f r a t l i v e r m i t o c h o n d r i a ( 2 7 ) . The o x i d a t i o n o f a s c o r b i c a c i d t o d i h y d r o a s c o r b i e a c i d by s u s p e n s i o n s o f r a t l i v e r m i t o c h o n d r i a supplemented w i t h f e r r i c y t o c h r o m e c and c o f a c t o r s caused a c o u p l e d p h o s p h o r y l a t i o n w i t h P:0 r a t i o s a p p r o a c h i n g u n i t y (1+9). N i e l s e n and L e h n i n g e r ( 6 2 ) c o n c l u d e d t h a t one o f the t h r e e p h o s p h o r y l a t i o n s c o u p l e d t o t h e t r a n s p o r t o f a p a i r o f e l e c t r o n s from DPNH t o oxygen o c c u r r e d i n t h e span between cytochrome c and oxygen. The d a t a i n t h e i r s t u d y d i d not p e r m i t c o n c l u s i o n s r e g a r d i n g the mechanism o f t h i s p h o s p h o r y l a t i o n ; however, i t appeared u n l i k e l y t h a t the p h o s p h o r y l a t i o n i s c o u p l e d t o t h e r e d u c t i o n o f f e r r i c y t o c h r o m e a by f e r r i c y t o c h r o m e c, s i n c e t h e s t a n d a r d o x i d a t i o n r e d u c t i o n p o t e n t i a l o f cytochrome c i s +0.26 v o l t and t h a t o f cytochrome a +0.29 v o l t . I t appeared, t h e r e f o r e , more l i k e l y t h a t i t i s co u p l e d t o t h e span f e r r i c y t o c h r o m e a to oxygen. Work done by Copenhaven and Lardy (2lt) shoxjed t h a t the o x i d a t i o n o f s u c c i n a t e produced an average P : 0 r a t i o o f 1 . 7 ; t h i s s u g g e sted o n l y two s i t e s o f o x i d a t i v e p h o s p h o r y l a t i o n . S i n c e t h e en z y m a t i c d e h y d r o g e n a t i o n o f s u c c i n a t e does n ot i n v o l v e a p y r i d i n e n e u c l e o t i d e system, i t c o u l d be i n f e r r e d t h a t one o f t h e ATP g e n e r a t i n g s i t e s i s l o c a t e d between DPHH and PAD. 18 Hulsmann and S l a t e r (39) r e p o r t e d t h a t l i v e r and h e a r t m i t o c h o n d r i a c o n t a i n f o u r d i f f e r e n t enzyme systems w h i c h b r i n g about h y d r o l y s i s o f added ATP. Three o f t h e s e systems, d e s i g n a t e d by t h e pH o p t i m a 6 .3 , 7.4 and 8.5 were s t i m u l a t e d by DNP. The a u t h o r s suggested t h a t t h e s e t h r e e d i f f e r e n t adenosine t r i p h o s p h a t a s e s might be r e l a t e d t o t h e t h r e e DNP s e n s i t i v e p h o s p h o r y l a t i v e steps i n t h e r e s p i r a t o r y c h a i n . peaks w i t h g l u t a m a t e , two w i t h s u c c i n a t e and one w i t h a s c o r b a t e . The number o f peaks c o r r e s p o n d e d t o the number o f p h o s p h o r y l a t i v e s t e p s a s s o c i a t e d w i t h t h e s e t h r e e s u b s t r a t e s . The r e s u l t s i n d i c a t e d t h a t t h e 6.3 enzyme common t o a l l t h r e e s u b s t r a t e s itfas a s s o c i a t e d w i t h the cytochrome o x i d a s e end o f the r e s p i r a t o r y c h a i n . The 8.5 enzyme was found o n l y w i t h g l u t a m a t e p h o s p h o r y l a t i o n o c c u r r i n g i n the r e g i o n o f DPN w h i l e t h e 7.4 enzyme w h i c h was found w i t h g l u t a m a t e and s u c c i n a t e , but not w i t h a s c o r b a t e , was supposed t o l i e i n t h e m i d d l e o f t h e r e s p i r a t o r y c h a i n . The a u t h o r s p r e s e n t e d t h e f o l l o w i n g scheme: The pH a c t i v i t y c u r v e s o f the P:0 r a t i o showed t h r e e s u c c i n a t e I g l u t a m a t e P p I I R e s p i r a t o r y c h a i n - k e t o - l i p . g l u t a r a t e — a c i d - * D P N - * F p I — c y t b - * c y t c — • c y t a - ~ c y t &3~®z> Energy c o n s e r v a t i o n P h o s p h o r y l a t i o n ATP P enz. ATP 8.5 enz. I ATP 7.4- enz. ATP 6.3 enz. 19 T h i s i s i n a c c o r d w i t h t h e o x i d a t i o n r e d u c t i o n p o t e n t i a l s o f the members o f t h e r e s p i r a t o r y c h a i n . The spans between cytochrome c and cytochrome a ( - 0 . 0 3 v o l t ) and between PAD and cytochrome b ( - 0 . 0 2 3 ) are not l a r g e enough, t o a l l o w t h e g e n e r a t i o n o f a h i g h energy bond. MATERIALS AND METHODS I Organism The o r g a n i s m used t h r o u g h o u t t h e s e s t u d i e s was Pseudomonas a e r u g i n o s a A.T.C.C. 9027 . L y o p h i l i z e d s t o c k c u l t u r e s were suspended i n s t e r i l e d i s t i l l e d w a t e r , p l a t e d on S t a n d a r d Methods agar and an i s o l a t e d t y p i c a l c o l o n y i n o c u l a t e d i n t o t h e g l u c o s e medium o f N o r r i s and Campbell ( 6 3 ) , p l u s 0.1°/o y e a s t e x t r a c t . C e l l s f o r h a r v e s t i n g were grown i n 100 ml p o r t i o n s o f t h i s g l u c o s e - y e a s t e x t r a c t medium i n Roux f l a s k s . A f t e r 18 t o 20 hours i n c u b a t i o n t h e c e l l s were c o l l e c t e d by c e n t r i f u g a t i o n i n a S e r v a l l r e f r i g e r a t e d c e n t r i f u g e a t 11 ,000 x g. f o r 10 m i n u t e s . I I P r e p a r a t i o n o f t h e C e l l F r e e E x t r a c t s a) F r e s h l y h a r v e s t e d c e l l s were washed once w i t h d i s t i l l e d w a t e r and once w i t h a 0 . 2 mg. p e r c e n t g l u t a t h i o n e s o l u t i o n . The s u p e r n a t a n t was d i s c a r d e d and t h e wet c e l l p a s t e was packed i n t o open ended g l a s s tubes o f an i n s i d e d i a m e t e r about 1 mm. l e s s t h a n t h a t o f t h e l a r g e h o l e i n t h e Hughes p r e s s . 20 21 The tubes were c o r k e d and immersed i n t o an a l c o h o l - d r y - i c e b a t h f o r a p p r o x i m a t e l y 20 m i n u t e s . The q u i c k f r o z e n c y l i n d e r s were s t o r e d at - 1 8 ° u n t i l u s e d . When needed t h e c e l l s were crushed i n the Hughes p r e s s by a p p l y i n g 10 t o 12 thousand p . s . i . p r e s s u r e w i t h a C a r v e r h a n d - o p e r a t e d h y d r a u l i c p r e s s . A f t e r c r u s h i n g t h e b l o c k was q u i c k l y opened and t h e f r o z e n p r e p a r a t i o n was put i n t o an a p p r o p r i a t e volume o f c h i l l e d d i l u e n t (5°) t o y i e l d a f i n a l p r e p a r a t i o n o f a p p r o x i m a t e l y 200 mg. wet w e i g h t o f c e l l s / m - 0 . The c o m p o s i t i o n o f t h e d i l u e n t was: 0 . 0 5 M. g l y c y l g l y c i n e 0 . 2 5 M. s u c r o s e 500 mg. p e r c e n t egg a l b u m i n (16) The c r u s h e d c e l l s were mixed w i t h t h i s d i l u e n t i n a P o t t e r homogenizer. T h i s t r e a t m e n t r e s u l t e d i n an e x t r e m e l y v i s c o u s p r e p a r a t i o n w h i c h was d i s i n t e g r a t e d i n a 10 Kc. s o n i c o s c i l l a t o r f o r 15 seconds. The p r e p a r a t i o n was c e n t r i f u g e d a t 3 ,000 x g. i n the c o l d f o r 7 minutes a f t e r w h i c h the s u p e r n a t a n t was drawn o f f v e r y c a r e f u l l y and used i m m e d i a t e l y . b) F r e s h l y h a r v e s t e d c e l l s were x^ashed once w i t h a 0.2°/6 K C l s o l u t i o n , t h e s u p e r n a t a n t was d i s c a r d e d and the wet c e l l p a s t e suspended i n t h e same s o l u t i o n t o y i e l d a p p r o x i m a t e l y 200 mg. wet c e l l s / m ^ . These c e l l s were b r o k e n up on a 10 Kc. s o n i c o s c i l l a t o r f o r 12 m i n u t e s ; t h e r e s u l t i n g c e l l p r e p a r a t i o n was c e n t r i f u g e d i n t h e c o l d f o r one hour at 28 ,000 x g. The s u p e r n a t a n t was drawn o f f and s t o r e d i n i c e u n t i l u s e d . 22 I I I D e t e c t i o n o f ATP G e n e r a t i n g Systems The p r o d u c t i o n o f ATP was measured by t h e method o f Kornberg (1+5). A t y p i c a l r e a c t i o n m i x t u r e i n a c u v e t t e c o n t a i n e d : T r i s ( h y d r o x y m e t h y l ) aminomethane M// 0.05" raZ. M g C l 2 200 mg./mZ. 0.02 mZ. H e x o k i n a s e 5 mg./m^. 0 .05 m-£. Gl u c o s e - 6 - p h o s p h a t e dehydrogenase 5 mg./ r a^ 0.0^ . G l u c o s e 2 5 " M M . / W . 0 .10 m i . TPN 5 A i M . / W . 0.02 m£. Water up t o 1 mZ. A f t e r 2 minutes 0 .01 ra£. ATP (12 .5 yuM . / W . ) o r 0 .01 m£. o f the ATP g e n e r a t i n g system was added and the r e d u c t i o n o f the TPN was r e a d at 31x0 mu. on a model DU Beckman s p e c t r o p h o t o m e t e r . I V S t u d i e s w i t h P h o s p h o r u s ^ 2 a) The i n c u b a t i o n m i x t u r e was a s l i g h t l y m o d i f i e d v e r s i o n o f t h a t used by Campbell (16) and was s e t up as f o l l o w s : Endogenous T e s t G l y c y l g l y c i n e (50 ;uM . /0.3 . ) l5.0mZ. 15*.0 m*. p l u s o rthophosphate 2/iM . / 0 . 3 m^. Sucrose (0.05" M. ) t o y i e l d a f i n a l volume o f 50 . 0 m ^ . NaP (200 AjM./ml. ) 2.5" raZ. 2.5 mZ. MgCl 2 . 7 H 2 0 (100 joM./mZ.) 2.5 mZ. 2.5>mZ. 23 ADP (lj.0 AiM./mi. ) 2 . 5 m i . 2 . 5 m i . Cytochrome c ( 0 . 5 ;uM./mi. ) P^ 2 s o l u t i o n ( 0 . 1 m c . / W . ) 1 . 0 m i . 1 .0 m i . 1 . 0 m i . 1.0 m i . C e l l f r e e e x t r a c t 1 5 . 0 m i . 1 5 . 0 mi. G-lucose (100 ;uM./mi. ) 5 . 0 mi. ( S u c c i n a t e 100 pM./mt.) The i n c u b a t i o n m i x t u r e was measured i n t o a 250 m i . Erl e n m e y e r and i n c u b a t e d at 3 0 ° ; w i t h s h a k i n g , on a B u r r e l l w r i s t a c t i o n s h a k e r . A f t e r 30 m i n u t e s ' i n c u b a t i o n the r e a c t i o n was st o p p e d by t h e a d d i t i o n o f 250 m i . c o l d e t h a n o l ( - 1 8 ° ) ; the m i x t u r e was shaken a g a i n f o r 30 minutes and h e l d at - 1 8 ° f o r 10-12 h o u r s . P r e c i p i t a t e d p r o t e i n was removed by c e n t r i f u g a t i o n , and a g a i n e x t r a c t e d w i t h 250 m i . c o l d e t h a n o l f o r 30 m i n u t e s and c e n t r i f u g e d . B oth s u p e r n a t a n t s were combined and t h e a l c o h o l was removed by e v a p o r a t i o n under vacuum i n t h e f l a s h e v a p o r a t o r . I n subsequent e x p e r i m e n t s t h e p r o t e i n p r e c i p i t a t e a f t e r two a l c o h o l t r e a t m e n t s was made up t o 50 m i . w i t h d i s t i l l e d w a t e r and e x t r a c t e d w i t h 250 m i . o f a O . 2 I 4 . N HCIO^ s o l u t i o n f o r 30 minutes at 5 ° 5 n e u t r a l i z e d w i t h K0H and t h e p r o t e i n p r e c i p i t a t e removed by c e n t r i f u g a t i o n . The p e r c h l o r i c a c i d e x t r a c t was ev a p o r a t e d t o s m a l l volume i n t h e f l a s h e v a p o r a t o r . p i p e t t e d on an u n l a c q u e r e d , u n c o r k e d s t e e l p r e s s u r e type b o t t l e cap and d r i e d under an i n f r a - r e d lamp. Counts were made u s i n g a T r a c e r l a b model SC9D manual changer and a B e r k e l e y D e c i m a l S c a l e r Model 2 1 0 5 . R e s u l t s were e x p r e s s e d as counts per m i n u t e . b) The samples ( u s u a l l y 0 . 2 mi.) to be counted were 2k Each sample was counted at l e a s t k m i n u t e s . Prom t h e s e v a l u e s the background count, o b t a i n e d by c o u n t i n g a c l e a n p l a n c h e t , was s u b t r a c t e d t o y i e l d t h e net count. V Chromatography o f the R e a c t i o n M i x t u r e s a) D o w e x - 1 - c h l o r i d e chromatography Dowex 1, C l ~ x l O r e s i n , 200 t o l+OO mesh was t r e a t e d i n t h e u s u a l manner by removing th e f i n e s , t r e a t i n g w i t h 109o (v/v) HC1 and w a s h i n g w i t h d i s t i l l e d w a t e r u n t i l t h e w a s h i n g gave a n e g a t i v e AgNO^ t e s t . A s l u r r y o f the r e s i n was poured i n t o a 1 cm. g l a s s column and a l l o w e d t o s e t t l e . The h e i g h t o f t h e r e s i n i n the column was a p p r o x i m a t e l y 20 cm. The c o n c e n t r a t e d r e a c t i o n m i x t u r e was brought t o pH 8 , added to t h e column and was a l l o w e d t o f l o w t h r o u g h at about 1 m i . per m i n u t e . F i v e column volumes o f d i s t i l l e d w a t e r were p a s s e d t h r o u g h a f t e r t h e column had almost gone d r y and the e f f l u e n t was checked f o r t h e p r e s ence o f r a d i o a c t i v i t y , t o ensure t h a t a l l t h e r a d i o a c t i v e m a t e r i a l was h e l d by the r e s i n . The adsorbed m a t e r i a l was removed by g r a d i e n t e l u t i o n i n t h e f o l l o w i n g manner. The r e s e r v o i r a t t a c h e d t o t h e column c o n t a i n e d 5 0 0 m i . 0.003 N HC1, w h i l e t h e o t h e r r e s e r v o i r c o n t a i n e d 5 0 0 m i . 0 . 3 N LIC1 i n 0.003 N HC1; t h e column was s t r i p p e d w i t h 5 0 0 m i . 0 . 5 N M C I i n 0 . 0 0 3 N HC1. I n some expe r i m e n t s th e e l u t i o n was s t a r t e d w i t h a d i s t i l l e d w a t e r - 0 . 0 0 3 N HC1 g r a d i e n t , f o l l o w e d by a 0 . 0 0 3 N HC1 .- 0 . 3 N L i C l i n 0 . 0 0 3 N HC1 g r a d i e n t ; t h i s t r e a t m e n t r e s u l t e d i n s e p a r a t i o n o f t h e f i r s t peak i n t o two components. I n a l l c a s e s , the e l u e n t from t h e column was c o l l e c t e d at a r a t e o f 1 mZ. per minute ( l p . s . i . N 2 was a p p l i e d t o i n s u r e t h e p r o p e r r a t e ) i n 10 mZ. f r a c t i o n s by a G-.M.E. f r a c t i o n c o l l e c t o r . C o u n t i n g p r o c e d u r e s were c a r r i e d out on 0 . 2 mZ. a l i q u o t s . 260 mu a b s o r p t i o n was checked on 1 mZ. a l i q u o t s i n the model DU Beckman s p e c t r o p h o t o m e t e r . b) Dowex - 1 -formate Chromatography S i n c e H u r l b e r t e t a l . (liO) o b t a i n e d e x c e l l e n t s e p a r a t i o n o f the a c i d s o l u b l e n u c l e o t i d e s from r a t l i v e r u s i n g Dowex - 1 -formate columns and e l u t i o n w i t h formate systems, i t seemed p r o m i s i n g to use t h i s method. Dowex - 1 -formate was p r e p a r e d from Dowex-l-Cl i n t h e f o l l o w i n g manner. Dox^rex-l-Cl x 10 200-liOO mesh was washed w i t h 2 l i t r e s 3 N sodium formate and t h e n w i t h 3 l i t r e s d i s t i l l e d w a t e r . The r e s i n s l u r r y was poured i n t o a column 20 x 2 . l i cm., t r e a t e d w i t h 2lx0 raZ. o f a m i x t u r e o f 6 N f o r m i c a c i d and 0 . 1 N sodium f o r m a t e , next w i t h 100 mZ. 88°/6 v/v f o r m i c a c i d and f i n a l l y wa'ahed w i t h a p p r o x i m a t e l y 2 l i t r e s d i s t i l l e d w a t e r . The column was charged w i t h t h e r e a c t i o n m i x t u r e as d e s c r i b e d p r e v i o u s l y . A f t e r c h a r g i n g t h e column was washed w i t h a p p r o x i m a t e l y 50 mZ. w a t e r . F i v e mZ. f r a c t i o n s were c o l l e c t e d at a f l o w r a t e o f 1 mZ. per m i n u t e . The e l u t i o n was s t a r t e d w i t h 300 raZ. I N f o r m i c a c i d f o l l o w e d by 350 m.Z. l i N f o r m i c a c i d , it00 mZ. 0 . 2 N ammonium formate i n l i N f o r m i c a c i d and f i n a l l y 200 m]S. O.li N ammonium formate i n i i N f o r m i c a c i d . 26 VI Acetone P r e c i p i t a t i o n o f N u c l e o t i d e s The samples c o n s t i t u t i n g t h e peak areas o f the e l u t i o n c u r v e s , as d e t e r m i n e d hy 260 mu r e a d i n g s and r a d i o a c t i v i t y measurements were p o o l e d , n e u t r a l i z e d and c o n c e n t r a t e d t o s m a l l volume i n the f l a s h e v a p o r a t o r . The c o n c e n t r a t e s were t r a n s f e r r e d t o l y o p h i l i z i n g tubes and l y o p h i l i z e d t o d r y n e s s . The d r y m a t e r i a l was d i s s o l v e d and suspended i n a s m a l l volume o f c o l d ( - 1 8 ° ) m e t h a n o l , u s u a l l y 1-3 m i . E i g h t t i m e s t h e volume o f c o l d ( - 1 8 ° ) acetone was added and p r e c i p i t a t i o n was a l l o w e d t o c o n t i n u e at - 1 8 ° f o r 1 h o u r . I n subsequent e x p e r i m e n t s anhydrous e t h e r was added t o d e c r e a s e the s o l u b i l i t y o f t h e n u c l e o t i d e s . The w h i t e p r e c i p i t a t e x^as i s o l a t e d by c e n t r i f u g a t i o n and washed w i t h c o l d ( - 1 8 ° ) a c e t o n e . The r e s u l t i n g m a t e r i a l was d r i e d o ver D r i e r i t e and u s e d f o r i d e n t i f i c a t i o n on paper chromatograms. V I I I d e n t i f i c a t i o n o f N u c l e o t i d e s a) A b s o r p t i o n S p e c t r a A s m a l l sample o f the l y o p h i l i z e d m a t e r i a l was d i s s o l v e d i n d i s t i l l e d w a t e r and t h e spectrum between 210 and 310 mu d e t e r m i n e d . The s p e c t r a t h u s o b t a i n e d were compared w i t h t h o s e shown i n t h e Pabst L a b o r a t o r i e s C i r c u l a r OR-10 ( 7 1 ) . The s p e c t r a combined w i t h the e l u t i o n p o s i t i o n viere u s e d as a t e n t a t i v e i d e n t i f i c a t i o n o f t h e n u c l e o t i d e s . 27 b) Paper Chromatography I n a l l cases d e s c e n d i n g chromatograms, u s i n g Whatman No. 1 paper, were used. S o l v e n t Systems • a) i s o b u t y r i c a c i d - ammonia s o l v e n t (1+2) i s o b u t y r i c a c i d 100 m i . 1 N ammonium h y d r o x i d e 60 m i . 0.1 M d i s o d i u m v e r s e n a t e 1.6 m i . b) i s o a m y l a l c o h o l - d i s o d i u m phosphate s o l v e n t (17) i s o a m y l a l c o h o l $0 m i . Na 2HP0^ 5 % 50 m i . ,'c) s a t u r a t e d ammonium s u l f a t e - i s o p r o p a n o l -w a t e r s o l v e n t (29) s a t u r a t e d (NH^JgSO^ 79 p a r t s i s o p r o p a n o l 2 p a r t s H 2 0 19 p a r t s c) P a p e r s t r i p C o u n t i n g I n some cases t h e c o n c e n t r a t i o n o f t h e 260 mu a b s o r b i n g m a t e r i a l was so low t h a t u l t r a v i o l e t l i g h t d i d not r e v e a l any s p o t s . The paper chromatograms were cut l e n g h t w i s e i n 3 . 5 cm. s t r i p s and each s t r i p was passed t h r o u g h the T r a c e r l a b model S C 9 D manual changer; areas 1 cm. i n l e n g t h were counted f o r two minute s ; a f t e r t h i s time the paper s t r i p was moved e x a c t l y 1 cm. i n t o t h e changer and counted a g a i n . T h i s p r o c e d u r e was r e p e a t e d u n t i l t h e s t r i p was c o m p l e t e l y scanned. The Rf o f a r a d i o a c t i v e spot was t h e n compared w i t h t h e Rf o f a r e f e r e n c e compound. 28 S o l v e n t c was not v e r y u s e f u l ; as a r e s u l t o f t h e l a r g e amounts o f s a l t p r e s e n t e l u t i o n o f the s p o t s was d i f f i c u l t . I l l u m i n a t i o n o f t h e paper w i t h u l t r a v i o l e t l i g h t r e v e a l e d the n u c l e o t i d e s as d a r k s p o t s a g a i n s t a l i g h t o r sometimes f l u o r e s c e n t background. To remove i n t e r f e r i n g c o n c e n t r a t i o n s o f s a l t s a few beads o f Dowex 50 hydrogen form were added b e f o r e s p o t t i n g on the paper. The unknown compounds were always r u n a l o n g w i t h r e f e r e n c e compounds. I n t h e f i n a l s t a g e o f i d e n t i f i c a t i o n t h e unknown and r e f e r e n c e compounds were s p o t t e d s i m u l t a n e o u s l y . V I I I Paper Chromatography o f O r g a n i c A c i d s Descending chromatograms on Whatman No. 1 were r u n u s i n g the f o l l o w i n g s o l v e n t system: B u t a n o l It p a r t s A c e t i c a c i d 1 p a r t D i s t i l l e d w a t e r 5 p a r t s The m i x t u r e was shaken i n a s e p a r a t o r y f u n n e l f o r 2 m i n u t e s and a l l o w e d t o s t a n d f o r 2 h o u r s . The b u t a n o l (upper) f r a c t i o n was used i n t h e t r o u g h as t h e moving s o l v e n t ; t h e w a t e r ( l o w e r ) f r a c t i o n was p l a c e d i n t h e bottom o f t h e t a n k t o s a t u r a t e t h e atmosphere. The chromatograms were d e v e l o p e d by s p r a y i n g w i t h an a l c o h o l i c c h l o r o p h e n o l r e d s o l u t i o n (0.0)4.°/)) a f t e r the papers had been steamed f o r 15> minutes t o remove the a c e t i c a c i d o f t h e s o l v e n t . 29 I X D e t e r m i n a t i o n o f t h e Presence o f U n s a t u r a t e d Bonds ( 2 8 ) To 0 . 1 mZ. o f a sample on a c u v e t t e were added 0 . 1 ml. 1 0 % m e t a p h o s p h o r i c a c i d , 0 . 3 mZ. d i s t i l l e d w a t e r and 1 . 0 mZ. O.OOii N KMnO^. A c o n t r o l was r u n c o n t a i n i n g 0 . 1 mZ. d i s t i l l e d w ater i n s t e a d o f t h e sample. The r e a c t i o n m i x t u r e was a l l o w e d t o s t a n d i n t h e d a r k f o r 20 - 50 m i n u t e s , and r e a d at 530 mu. a g a i n s t a d i s t i l l e d w a t e r b l a n k i n a Beckman DU s p e c t r o p h o t o m e t e r . X D e t e r m i n a t i o n o f I n o r g a n i c Orthophosphate and Pyrophosphate I n o r g a n i c o r t h o p h o s p h a t e and pyrophosphate were determined by t h e c o l o r i m e t r i c method d e s c r i b e d by P l y n n e t a l . (31). A t y p i c a l r e a c t i o n m i x t u r e was s e t up as f o l l o w s : 0 . 5 mZ• sample t o be a n a l y z e d 3.6 mZ. d i s t i l l e d w a t e r 0.5 mZ. molybdate r e a g e n t (2.5°/> (NHi. )/Mo 7 0 p ) l .ILHOO i n 5W H 2 S 0 i , ) ^ 5 ' 4 0 . 2 mZ. c y s t e i n e HC1 s o l u t i o n [175 pM./mZ.) 0 . 2 ml. e i k o n o g e n s o l u t i o n ( 6 . 6 5 gm. N a 2S 2 0 £ and 0 . 2 5 gm. Na-SOo d i s s o l v e d i n 50 ml. w a t e r . W i t h a l i t t l e o f t h i s s o l u t i o n 0 . 1 2 5 g r . l - a m i n o - 2 - n a p h t h o l - i i - s u l f o n i c a c i d was ground i n a m o r t a r , washed w i t h t h e remainder o f th e s o l u t i o n and f i l t e r e d ) E x a c t l y 7 minutes a f t e r t h e e i k o n o g e n s o l u t i o n was added, t h e o p t i c a l d e n s i t y o f t h e s o l u t i o n was r e a d a t 660 mu. i n a Beckman DU s p e c t r o p h o t o m e t e r . A second r e a d i n g was made a f t e r e x a c t l y 90 m i n u t e s . The 7 minute r e a d i n g d e t e r m i n e d t h e o r t h o p h o s p h a t e w h i l e the 90 minute r e a d i n g d e t e r m i n e d t h e t o t a l phosphate 30 p r e s e n t as o r t h o - and pyrophosphate. XI Manometric S t u d i e s Oxygen u p t a k e by j c e l l f r e e p r e p a r a t i o n s was s t u d i e d u s i n g a 3 . 0 ml. volume o f r e a c t i o n m i x t u r e i n a s i n g l e s i d e a r m Warburg f l a s k . I n c u b a t i o n was c a r r i e d out i n a s t a n d a r d G i l s o n c i r c u l a r Warburg apparatus a t 3 0 ° . A t y p i c a l r e a c t i o n m i x t u r e c o n s i s t e d o f t h e f o l l o w i n g components: G l y c y l g l y c i n e ( 50;uM . / 0 . 3 ml.) p l u s o r t h o p h o s p h a t e (2 ;uM . /0.3 ml. ) 0 .90 ml. Sucrose (0.05 M) t o b r i n g volume t o 3 .00 ml. NaP (200 uM./m-g. ) 0.15" ml. MgCl2.7H20 (100 pM./ml.) ADP (I4.0 MM./mi. ) 0 . 1 5 ml. 0 . 1 5 ml. Cytochrome c ( 0 . 5 }M./ml.) C e l l f r e e e x t r a c t (200 mgm/mi.) Gluco s e (100/oM./mi. ) 0.99 ml. 0 .30 ml. 0.06 ml. EXPERIMENTAL RESULTS I Manometric S t u d i e s The o x i d a t i o n o f g l u c o s e by Pseudomonas a e r u g i n o s a s t r a i n A.T.C.C. 9027 by way o f g l u c o n i c a c i d and 2 - k e t o g l u c o n i c a c i d has been e s t a b l i s h e d i n t h i s l a b o r a t o r y (63). Whole r e s t i n g c e l l s o f t h i s o r g a n i s m o x i d i z e g l u c o s e t o CO^ and w a t e r , whereas c e l l s d i s r u p t e d by s o n i c o s c i l l a t i o n o x i d i z e g l u c o s e t o 2 - k e t o g l u c o n i c a c i d w i t h t h e up t a k e o f 2 atoms o f oxygen (68). No e v i d e n c e f o r c o n c u r r e n t p h o s p h o r y l a t i o n d u r i n g t h e o x i d a t i o n o f g l u c o s e t o 2 - k e t o g l u c o n a t e by s o n i c e x t r a c t s o f P. a e r u g i n o s a was o b t a i n e d (68). However, p h o s p h o r y l a t i n g a c t i v i t y has been shown i n c e l l f r e e e x t r a c t s p r e p a r e d i t f i t h t h e Hughes p r e s s . S i n c e t h e e a r l i e r work d e t e r m i n i n g the a b i l i t y o f e x t r a c t s o f P. a e r u g i n o s a t o o x i d i z e v a r i o u s s u b s t r a t e s was c a r r i e d out w i t h s o n i c e x t r a c t s i t seemed d e s i r a b l e t o o b t a i n comparable d a t a f o r the e x t r a c t s o b t a i n e d w i t h t h e Hughes p r e s s . I n the r e a c t i o n m i x t u r e shown i n T a b l e I g l u c o s e was o x i d i z e d w i t h the u p t a k e o f 1 atom o f oxygen. 31 32 TABLE I Endogenous Test G l y c y l g l y c i n e * " " phosphate b u f f e r 0.90 ml. 0.90 ml. Sucrose 0 .60 ml. 0.1x0 ml. NaF 0.15 0.15 m-g. M g C l 2 . 7 H 2 0 0.15 ral. 0.15 ADP 0.15 ral. 0.15 Cytochrome c 0 .06 ml. 0.06 ml. C.F.X. 0 .99 0 .99 m^. Glu c o s e (2$pM./ml.) 0 .20 ml. KOH 0.15 ral. 0.15 G l u c o s e - 6 - p h o s p h a t e , r i b o s e - 5 - p h o s p h a t e , < * _ k e t o g l u t a r a t e , c i t r a t e and i s o c i t r a t e were n ot o x i d i z e d ; s u c c i n a t e , however, was r e a d i l y a t t a c k e d by t h i s system. Manometric e x p e r i m e n t s w i t h the r e a c t i o n m i x t u r e g i v e n i n T a b l e I I shox^ed t h a t g l u c o s e was o x i d i z e d w i t h t h e n e t up t a k e o f 2 atoms o f oxygen. see Methods and M a t e r i a l s FIG.I. OXIDATION of SUBSTRATES in absence of ATP 120 MINUTES 33 TABLE II Endogenous Test T r i s (hydroxymethyl) pH 7.1J. aminomethane M/10 1 . 5 mi. 1 . 5 mi. to bring volume to 3 . 0 mi. C.F.X. 200 mgm./mi. 1 .0 mi. 1 .0 mi. Substrate 2£;uM./mi. 0 . 2 mi. KOH 20°y6 ( i n centre well) 0 . 1 5 mi. 0 . 1 5 mi. I f 0 .3 mi. ATP (l£;uM./rai.) were added to the above reaction mixture glucose was oxidized with the net uptake of only 1 atom of oxygen. These data show that the addition of ATP causes a decrease i n oxygen uptake when glucose Is substrate. To determine the range of substrates oxidized by the system as well as the effect of ATP on the oxidations the .. following substrates were used: glucose, gluconolactone, gluconate and 2-ketogluconate. The r e s u l t s are shown i n Figures I and 2 . Gluconolactone and gluconic acid were oxidized with the uptake of one atom of oxygen and the addition of ATP did not effect these oxidations. 2-ketogluconate was not oxidized under any conditions tested. II Studies with Phosphorus32 E a r l i e r work i n t h i s laboratory (16) had shown that 30 minutes' incubation of the reaction mixture yielded the maximum amount of charcoal absorbable l a b e l l e d material. As a r e s u l t i t was decided to carry out a l l the reactions for t h i s 34 i n t e r v a l . P r e l i m i n a r y e x p e r i m e n t s u s i n g ItO p.c P-^2 i n a - 1 0 ml. r e a c t i o n m i x t u r e showed t h a t a f t e r t h e f i r s t a l c o h o l t r e a t m e n t a p p r o x i m a t e l y 75></6 o f the r a d i o p h o s p h o r u s was r e t a i n e d i n t h e p r o t e i n p r e c i p i t a t e . The f i r s t a l c o h o l e x t r a c t was chroraatographed on a Dowex 1 C l column and e l u t e d w i t h a H C l - L i C l g r a d i e n t ( F i g u r e 3 ) . The p r o t e i n p r e c i p i t a t e was a g a i n e x t r a c t e d w i t h 5>0 ml. c o l d e t h a n o l ; a f t e r t h i s second e x t r a c t i o n th e p r o t e i n r e t a i n e d a p p r o x i m a t e l y 22°/6 o f the i n i t i a l p 3 2 a c t i v i t y . I n subsequent e x p e r i m e n t s two washings w i t h c o l d e t h a n o l were r o u t i n e l y u s e d . T h i s second e x t r a c t was a l s o chromatographed on a Dowex 1 C l column and d e v e l o p e d i n a s i m i l a r manner ( F i g u r e From t h e s e r e s u l t s i t can be seen t h a t a p p a r e n t l y t h e m a t e r i a l w i t h t h e g r e a t e r n e g a t i v e charge was bound more t i g h t l y t o the p r o t e i n because t h i s m a t e r i a l was o n l y p r e s e n t i n t h e second e t h a n o l e x t r a c t i o n . S i n c e t h e p r i m a r y i n t e r e s t o f t h i s f i r s t e xperiment was t o determine t h e i n c o r p o r a t i o n o f i n o r g a n i c r a d i o p h o s p h a t e i n t o o r g a n i c phosphates o n l y tubes 17 t o 31 i n c l u s i v e o f column 1 (peak I 2) and t u b e s 13 t o 25 i n c l u s i v e (peak I I 2) and 26 t o $1 i n c l u s i v e (peak I I 3) o f column 2 were p o o l e d , e v a p o r a t e d t o s m a l l volume, l y o p h i l i z e d and acetone p r e c i p i t a t e d as o u t l i n e d i n Methods and M a t e r i a l s . Campbell (16) s u g g e s t e d the p o s s i b i l i t y o f t h e presence o f a n u c l e o s i d e p o l y p h o s p h a t e sugar complex from t h e a l k a l i n e l a b i l i t y , a c i d s t a b i l i t y and u l t r a v i o l e t a b s o r p t i o n s p e c t r a o f the peaks found a f t e r chromatography on Dowex 1 C l . Samples o f each peak ( 1 2 , I I 2 and I I 3) were a n a l y z e d i n a Legend: Optical Density at 260 mu counts per minute per 0.1 ml FIG.4. ELUTION CURVE of SECOND ETHANOL EXTRACT. 10 2 0 3 0 4 0 5 0 6 0 F R A C T I O N NUMBER 35 Carey r e c o r d i n g s p e c t r o p h o t o m e t e r , y i e l d i n g t h e f o l l o w i n g r e s u l t s : TABLE 1 peak ' Xmax. acid R a t i o s at 250 n e u t r a l 250/260 , 2 6 0 , 280 mu 2 8 0 / 2 6 0 Xmin. a c i d n e u t r a l I 2 258 0.320 = . 9 5 0 . 3 3 6 0 . 1 7 4 = . 5 2 0 . 3 3 6 243 I I 2 259 0 . 5 6 0 = . 8 5 0 . 6 6 2 0 . 2 3 1 = . 3 5 0.662 229 I I 3 256 260 a c i d 0 . 5 5 0 = .89 0 . 6 1 7 n e u t r a l 0 . 5 2 1 = . 8 5 0 . 6 1 0 0 . 2 8 8 = .47 0 . 6 1 7 0 .318 = . 5 2 0.610 231 230 Comparison o f t h e s e v a l u e s w i t h S p e c t r o p h o t o m e t r y C o n s t a n t s o f 5 - r i b o n u c l e o t i d e s (71) d i d not g i v e any c l u e s as t o the i d e n t i t y of t h e s e peaks. I t appeared, however, v e r y p r o b a b l e t h a t t h e peaks were c o n t a m i n a t e d and t h a t t h e c o n t a m i n a n t ( s ) would mask t r u e v a l u e s . The A max. and Xmin. would suggest adenosine d e r i v a t i v e s f o r a l l t h r e e peaks. I d e n t i f i c a t i o n o f t h e unknown compound(s) i n t h e peaks on paper chromatograms u s i n g t h e s o l v e n t systems d e s c r i b e d i n Methods and M a t e r i a l s were not v e r y s u c c e s s f u l because o f t h e e x t r e m e l y low c o n c e n t r a t i o n s o f t h e s e compounds. However, t h e Rf v a l u e s found s u g g e s t e d peak I 2 and peak I I 2 t o be ADP and peak I I 3 t o be ATP. H u r l b e r t e t a l . (40) and Beyer e t a l . (6) o b t a i n e d e x c e l l e n t s e p a r a t i o n o f the a c i d s o l u b l e n u c l e o t i d e s u s i n g a Dowex 1 formate r e s i n and a f o r m i c a c i d , ammonium formate g r a d i e n t . I t was c o n s i d e r e d w o r t h w h i l e t o use t h e i r t e c h n i q u e t o o b t a i n b e t t e r s e p a r a t i o n because the r a d i o p h o s p h a t e peak c o i n c i d e d i n p a r t w i t h t h e peak 36 t e n t a t i v e l y i d e n t i f i e d as AMP. An experiment was s e t up as p r e v i o u s l y d e s c r i b e d but i n c o r p o r a t i n g two changes: 132 p.c ¥^2. w a s u s e ( j i n s t e a d o f I4 .0 p.c and a l l the o t h e r c o n s t i t u e n t s o f t h e r e a c t i o n m i x t u r e were i n c r e a s e d f i v e f o l d t o y i e l d a f i n a l volume o f 50 m i . A f t e r i n c u b a t i o n f o r 30 minutes a t 30°, the r e a c t i o n was stopped by the a d d i t i o n o f 250 m i . c o l d e t h a n o l ( - 1 8 ° ) , t h e p r o t e i n was spun down as b e f o r e and the p r o t e i n p r e c i p i t a t e a g a i n washed w i t h 250 m i . c o l d e t h a n o l . The two e t h a n o l e x t r a c t s were combined and e v a p o r a t e d t o s m a l l volume. The p r o t e i n p r e c i p i t a t e , a f t e r the two w a s h i n g s , c o n t a i n e d 57.5°/° o f the i n i t i a l r a d i o p h o s p h o r u s a c t i v i t y , w h i l e the s u p e r n a t a n t showed • I 4 J 4 - . 0 ^ 6 o f t h e i n i t i a l a c t i v i t y . The s u p e r n a t a n t was poured on a Dowex-1- formate column 7 ' x 3/8" and d e v e l o p e d as d e s c r i b e d i n Methods and M a t e r i a l s . F i g u r e 5 p r e s e n t s t h e superimposed r a d i o a c t i v i t y and E260 r e a d i n g s f o r each 5 m i . f r a c t i o n . The b e t t e r s e p a r a t i o n compared t o F i g u r e s 1 and 2 i s o b v i o u s . I t i s i n t e r e s t i n g t o note t h a t e x t r a c t s p r e p a r e d i n t h i s f a s h i o n are r e l a t i v e l y low i n f r e e n u c l e o t i d e s , o n l y 3 peaks l a r g e enough t o be i d e n t i f i e d . Peaks a, b, 1|. and 5 (b and 5 not shown i n f i g u r e ) are p r e s e n t i n o n l y v e r y low c o n c e n t r a t i o n . T h i s may be due t o t h e e t h a n o l t r e a t m e n t because TCA e x t r a c t i o n o f whole c e l l s o f P. a e r u g i n o s a showed a l a r g e number o f f r e e n u c l e o t i d e s ( 1 2 ) . S e c o n d l y , H u r l b e r t et a l . (1+.0) u s e d the e x t r a c t s - o f 75 gm. o f r a t l i v e r , w h i l e e x t r a c t s o f o n l y 3 gm. Optical Density at 260 mu counts per minute per 0.1 m-t tube 116=12939 c/m FRACTION NUMBER FIG. 5. ELUTION CURVE of DOWEX - hformate COLUMN. 37 o f P. a e r u g i n o s a were u s e d i n t h i s i n v e s t i g a t i o n . The tubes c o r r e s p o n d i n g t o the peak areas were p o o l e d , c o n c e n t r a t e d and l y o p h i l i z e d . Peak 1 Peak a Peak b Peak 2 Peak 3 Peak 4 Peak 5 tube s 1 tubes 70 t u b e s 95 tubes 119 t u b e s 222 tubes 2 8 l tubes 308 liO i n c l u s i v e 94 i n c l u s i v e 118 i n c l u s i v e 156 i n c l u s i v e 26J4. i n c l u s i v e 289 i n c l u s i v e 322 i n c l u s i v e " " L y o p h i l i z a t i o n o f t h e peaks 4 and 5 was v e r y d i f f i c u l t because o f the e x t r e m e l y h i g h s a l t c o n c e n t r a t i o n . Treatment o f t h e s e peaks w i t h A m b e r l i t e 1 RC - 50 (H) b e f o r e l y o p h i l i z a t i o n p r o v e d v e r y h e l p f u l . Chromatography on Whatman No. 1 f i l t e r p a p e r , u s i n g . the s o l v e n t s a, b and c, gave the r e s u l t s shown i n T a b l e 2 . TABLE 2 R f V a l u e s i n S o l v e n t s a and b Sample S o l v e n t System Sample S o l v e n t System a b a b Peak 1 0 . 4 5 0 . 6 1 0 . 7 6 0 . 5 9 ATP 0 . 2 2 0 . 8 5 Peak 2 0 . 3 2 0.82 ADP 0 . 3 1 0 . 8 0 Peak 3 0 . 7 4 AMP 0 . 4 5 0 . 7 4 Peak l i 0 . 7 6 0 . 9 1 Adenosine 0 . 8 0 0 . 5 5 Peak 5 0 . 7 7 tubes 314 , - 1 5 , - 1 6 , - 1 7 , -18 were l o s t FI6.6. SPECTRA of PEAK I, HYPOXANTHINE and FUMARIC ACID. 20J 220 230 240 250 260 270 280 WAVELENGTH m*j o — o peaKI x x hypoxanthine • • fumaric acid 38 Prom t h e s e r e s u l t s i t can be seen t h a t peak 1 c o n s i s t s o f two components, one o f w h i c h i s AMP; peak 2 c o n s i s t s o f ADP. The s p o t s from peaks 3, i | and £ under u l t r a v i o l e t l i g h t a l l showed f l u o r e s c e n t a r e a s due t o t h e h i g h s a l t c o n c e n t r a t i o n . The R f v a l u e s o f the second component i n peak 1 c o u l d i n d i c a t e C y t i d i n e o r h y p o x a n t h i n e . Subsequent chromatograms e l i m i n a t e d t h e p o s s i b i l i t y o f c y t i d i n e , but the Rf v a l u e c o r r e s p o n d e d v e r y w e l l t o h y p o x a n t h i n e . Because i t appeared v e r y u n l i k e l y t h a t the c e l l f r e e e x t r a c t would deaminate adenine t o y i e l d h y p o x a n t h i n e , s p e c t r a were r u n f o r b o t h h y p o x a n t h i n e and i t s c o r r e s p o n d i n g s p o t . The s p e c t r a shown i n F i g u r e 6 i n d i c a t e w i t h o u t any doubt t h a t t h e y are d i s s i m i l a r . The a b s o r p t i o n spectrum o f t h e second component o f peak 1 i n d i c a t e s t h a t i t i s not h y p o x a n t h i n e but some s i m i l a r i t y t o the f u m a r i c a c i d spectrum was n o t i c e d . Because t h e components o f peak 1 were h e l d on a Dowex-1-formate column t h e y must p o s s e s s at l e a s t one n e g a t i v e charge at an a l k a l i n e pH. S e c o n d l y , s i n c e t h e a b s o r p t i o n spectrum i s s i m i l a r t o t h a t o f f u m a r i c a c i d t h e p o s s i b i l i t y o f an u n s a t u r a t e d bond had to be c o n s i d e r e d . The d e t e r m i n a t i o n o f t h e pr e s e n c e o f u n s a t u r a t e d bonds has been d e s c r i b e d p r e v i o u s l y (Methods and M a t e r i a l s I X ) . O p t i c a l d e n s i t y due t o t h e permanganate d e c r e a s e d from 0.^20 t o 0.387 on the a d d i t i o n o f t h e sample. From t h e s e r e a d i n g s i t c o u l d be co n c l u d e d t h a t t h e compound(s) c o n t a i n e d a double bond o r an e a s i l y o x i d i z e d group. Paperchromatography o f peak 1 u s i n g a b u t a n o l , a c e t i c 39 a c i d , water s o l v e n t and s p r a y i n g the d e v e l o p e d chromatograms w i t h 0 . 0 1L% c h l o r o p h e n o l r e d r e s u l t e d i n t h r e e compounds w i t h R f ' s 0 . 0 9 3 , 0 . 1 3 5 and 0 . 1 6 0 . The f i r s t two components were d i s t i n c t l y a c i d , w h i l e t h e l a s t one was n e u t r a l . None of t h e t h r e e s p o t s c orresponded t o one o f the f o l l o w i n g a c i d s : f u m a r i c , c i s a c o n i t i c o r o C - k e t o g l u t a r i c a c i d . Because peak 1 d i d n o t c o n t a i n any r a d i o a c t i v e phosphate no f u r t h e r i d e n t i f i c a t i o n o f thes e c a r b o h y d r a t e - l i k e compounds was a t t e m p t e d . I d e n t i f i c a t i o n o f peak 3 was h i n d e r e d b y i t s h i g h s a l t c o n c e n t r a t i o n . An attempt was made t o remove t h e s e s a l t s by a d s o r b i n g the n u c l e o t i d e on c h a r c o a l ; 0 . 3 m i . o f peak 3 c o n t a i n i n g 1h,,li3>^> counts per minute were t r e a t e d w i t h 15 mgm. a c i d x^ashed N o r i t e A. f o r 5 m i n u t e s . The c h a r c o a l was removed by c e n t r i f u g a t i o n and washed t h r e e times xtfith d i s t i l l e d w a t e r . Both s u p e r n a t a n t and c h a r c o a l f r a c t i o n s were co u n t e d . The s u p e r n a t a n t c o n t a i n i n g 5,14.36 counts p e r minute xKras decanted and t h e c h a r c o a l was t r e a t e d w i t h 0 . I4. m i . 5 0 % e t h a n o l . R e c o v e r y o f t h e absorbed m a t e r i a l was v e r y poor and s u s p e n d i n g the c h a r c o a l i n 0 .I4. m i . 5 % NH^OH i n 9 5 % e t h a n o l gave a s l i g h t l y b e t t e r r e c o v e r y . The e l u t e d m a t e r i a l gave t h e f o l l o w i n g o p t i c a l d e n s i t y r a t i o s : 280/260 2 5 0 / 2 6 0 Peak 3 0 . 2 3 0 . 8 9 Adenosine 0 . 2 2 0 . 8 6 AMP 0 . 2 2 O.8I4. ADP 0 . 2 1 0 . 8 5 ATP 0 . 2 2 0 . 8 5 These r a t i o s w ould i n d i c a t e t h a t peak 3 i s an adenosine d e r i v a t i v e ; t h i s i n f o r m a t i o n c o u p l e d t o t h e e l u t i o n p o s i t i o n from t h e Dowex 1 formate column would i n d i c a t e t h a t peak 3 i s i d e n t i c a l w i t h ATP. Paper chromatography o f t h i s peak as w e l l as peaks i i and 5> p r o v e d u n s u c c e s s f u l because o f t h e e x t r e m e l y low c o n c e n t r a t i o n o f t h e s e compounds. A l t h o u g h H u r l b e r t ' s p r o c e d u r e r e s u l t e d i n a much b e t t e r s e p a r a t i o n o f t h e n u c l e o t i d e s t h a n the method used i n the f i r s t 'experiment, th e h i g h s a l t c o n c e n t r a t i o n made i t almost i m p o s s i b l e to o b t a i n r e l a t i v e l y pure p r e p a r a t i o n s . The i n s o l u b i l i t y o f t h e l i t h i u m s a l t s o f the n u c l e o t i d e s i n acetone made t h e method f i r s t u s e d s u p e r i o r . Prom the d a t a thus f a r o b t a i n e d i t was c o n c l u d e d t h a t i n o r g a n i c phosphorus i s c o n v e r t e d t o o r g a n i c phosphorus and t h a t ATP was formed as a r e s u l t o f the a c t i o n o f t h e c e l l f r e e e x t r a c t . A l t h o u g h t h i s n u c l e o t i d e had o n l y been i d e n t i f i e d by i t s 280/260, 250/260 r a t i o s and column p o s i t i o n and n o t on paper chromatograms i t was f e l t t h a t the n e x t l o g i c a l s t e p i n t h i s i n v e s t i g a t i o n , namely, t h e s t u d y o f the i n f l u e n c e o f s u b s t r a t e s , c o u l d be u n d e r t a k e n . At t h i s stage o f the i n v e s t i g a t i o n t h e p o s s i b i l i t y o f t h e f o r m a t i o n o f ATP-^2 hy mechanisms, o t h e r t h a n o x i d a t i v e p h o s p h o r y l a t i o n h a d t o be c o n s i d e r e d . DeMoss and N o v e l l ! (26) were a b l e t o demonstrate an 1-aminoacid dependent exchange r e a c t i o n between l a b e l l e d i n o r g a n i c pyrophosphate and ATP w i t h e x t r a c t s o f a wide v a r i e t y o f m i c r o o r g a n i s m s . E g g l e s t o n (30) r e p o r t e d t h a t u n l e s s ATP was added no f o r m a t i o n o f r a d i o a c t i v e ATP from p32 o r t h o p h o s p h a t e , 41 AMP and i n o r g a n i c pyrophosphate c o u l d be d e t e c t e d . A s i m i l a r exchange between p32 and ATP was found by Cooper and L e h n i n g e r ( 2 3 ) . T h i s exchange r e a c t i o n o c c u r r e d i n t h e presence o f ATP but 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 ADP i n h i b i t e d t h e r a t e o f ATP-P32 exchange. S i n c e i n t h i s i n v e s t i g a t i o n the ADP c o n c e n t r a t i o n i n t h e r e a c t i o n m i x t u r e i s v e r y h i g h , an exchange s i m i l a r t o t h a t o b t a i n e d by Cooper e t a l . (23) can be n e g l e c t e d . An exchange between ATP and P^ 2 i s v e r y u n l i k e l y , because o f th e r e l a t i v e l y low ATP c o n c e n t r a t i o n . L o w e n s t e i n (52) r e p o r t e d the c h e m i c a l p r e p a r a t i o n o f P 3 2 l a b e l l e d adenosine p o l y p h o s p h a t e s u s i n g the r e a c t i o n o f ortho p h o s p h a t e w i t h adenosine £ 1-monophosphate, - d i p h o s p h a t e and - t r i p h o s p h a t e i n the presence o f N N ' - d i c y c l o h e x y l c a r b o d i i m i d e . W i t h o u t t h e a d d i t i o n o f t h i s c a t a l y s t no l a b e l l e d p o l y p h o s p h a t e s were formed. I t can be c o n c l u d e d from t h e s e r e p o r t s t h a t t h e f o r m a t i o n o f adenosine P-P-p32 from ADP and p32 does n o t o c c u r c h e m i c a l l y w i t h o u t t h e presence o f a c a t a l y s t and does not o c c u r e n z y m a t i c a l l y by s t r a i g h t a d d i t i o n . I t was f e l t t h a t the a b i l i t y o f t h e c e l l f r e e e x t r a c t t o g e n e rate ATP c o u l d be d e t e r m i n e d u s i n g t h e h e x o k i n a s e r e a c t i o n , G lucose ATP Mg"*""1"^  g l u c o s e - 6 - phosphate h e x o k i n a s e / TPN G - 6 - P dehydrogenase ( *TPNH 6 - phosphogluconate T h i s r e a c t i o n can be f o l l o w e d by t h e r e d u c t i o n o f t h e TPN t o TPNH as d e s c r i b e d b e f o r e . I n the absence o f ATP no r e d u c t i o n 4-2 o f TPN o c c u r s , because no g l u c o s e - 6 - p h o s p h a t e i s formed.-S u b s t i t u t i n g ATP w i t h ADP and the c e l l f r e e e x t r a c t s h o u l d y i e l d r e d u c t i o n o f TPN i f the c e l l f r e e e x t r a c t i s c a p a b l e o f g e n e r a t i n g ATP. Campbell et a l . (15) r e p o r t e d t h a t no ATP was g e n e r a t e d but t h e i r system c o n t a i n e d AMP i n s t e a d o f ADP as the phosphate a c c e p t o r . T h e i r experiment was r e p e a t e d u s i n g ADP as t h e a c c e p t o r and the r e s u l t s ( F i g u r e 7) show t h a t o n l y i n t h e p r e s e nce o f ADP and c e l l f r e e e x t r a c t p r e p a r e d by s o n i c d i s r u p t i o n was ATP g e n e r a t e d . A s i m i l a r experiment u s i n g a c e l l f r e e e x t r a c t p r e p a r e d by t r e a t i n g t h e c e l l s i n a Hughes p r e s s i n s t e a d o f s o n i c o s c i l l a t i o n gave the same r e s u l t s as • shown i n F i g u r e 7 . I f the c e l l f r e e e x t r a c t was h e a t e d at 100° C. f o r 3 minutes no r e d u c t i o n o f TPN c o u l d be shown. I t can be c o n c l u d e d from t h e s e r e s u l t s t h a t ATP i s formed o n l y i f ADP and c e l l f r e e e x t r a c t are i n c l u d e d i n the r e a c t i o n m i x t u r e . S i n c e the o x i d a t i o n of a s u b s t r a t e y i e l d s energy and t h i s energy i s t r a p p e d as ATP i n o x i d a t i v e p h o s p h o r y l a t i o n , t h e h e x o k i n a s e system was c o n s i d e r e d as a means t o e s t i m a t e t h e ATP formed on a d d i t i o n o f a s u b s t r a t e . However, g l u c o s e i s o x i d i z e d by c e l l f r e e e x t r a c t s o f P. a e r u g i n o s a and f o r t h a t r e a s o n cannot be used i n t h e h e x o k i n a s e r e a c t i o n . G l u c o s e c o u l d be r e p l a c e d by f r u c t o s e f o r t h e l a t t e r I s not o x i d i z e d by t h e c e l l f r e e p r e p a r a t i o n s ; mannose d i d n o t r e p l a c e g l u c o s e , a p p a r e n t l y mannose - 6-phosphate cannot be o x i d i z e d by g l u c o s e - 6 - p h o s p h a t e dehydrogenase. I n subsequent e x p e r i m e n t s 0 .1 m i . f r u c t o s e FIG. 7 D E T E R M I N A T I O N of A T P by the H E X O K I N A S E T R A P . O.I5i SECONDS 43 2% pM./mi. was used and 0.1 ml. o f a s u b s t r a t e (25 vM./ml.) was added to t h i s f r u c t o k i n a s e r e a c t i o n . G l u c o s e , g l u c o n i c a c i d , 2 - k e t o g l u c o n i c a c i d and s u c c i n i c a c i d were used as s u b s t r a t e s . Each r e a c t i o n m i x t u r e c o n t a i n e d 0.01 ml. CPX, 0.01 ml. ADP 50 frVi./ml. and 0.1 ml. s u b s t r a t e 25 )xM./ml. F i g u r e 8 and T a b l e 3 show t h a t a p p a r e n t l y no ATP i s ge n e r a t e d on t h e a d d i t i o n o f t h e s u b s t r a t e s t e s t e d . TABLE 3 E f f e c t o f Added S u b s t r a t e on the G e n e r a t i o n o f ATP O p t i c a l D e n s i t y at 34 0 mix. time seconds ATP ADP CFX £ ADP CFX . lUCcinate ADP CFX g l u c o n a t e ADP CFX 2-ketogl'cnte ADP CFX g l u c o s e 15 0.050 0.123 0.097 0.101 0.063 0.107 30 0.089 0.134 0.100 0.107 0.075 0.112 45" 0.118 0.145 0.105 0.114 0.088 0.121 6o 0.136 0.160 0.110 0.123 0.095 0.131 90 0.154 0.187 0.125 0.138 0.112 0.153 120 0.163 0.206 0.136 0.147 0.126 0.171 180 0.170 0.236 0.147 0.158 0.140 0.196 240 0.174 0.252 0.164 0.165 0.147 0.207 ^ ATP 0.124 0.129 O .O67 0.064 0.084 0.100 Less ATP i s formed i f a s u b s t r a t e i s p r e s e n t i n the r e a c t i o n m i x t u r e ; t h i s w o u l d suggest some k i n d o f a c t i v a t i n g r e a c t i o n , u t i l i z i n g ATP, b e f o r e t h e s u b s t r a t e can be broken down. T h i s f i n d i n g i s i n a c c o r d w i t h p r e v i o u s r e s u l t s o b t a i n e d i n t h i s FIG.8. E F F E C T of S U B S T R A T E on A T P F O R M A T I O N . 0 . 3 Q 30 120 SECONDS 180 210 "24*0 l a b o r a t o r y (l£); a p p a r e n t l y no net ATP i s g a i n e d i n the o x i d a t i o n o f t h e s e s u b s t r a t e s by c e l l f r e e e x t r a c t s o f P. a e r u g i n o s a under the c o n d i t i o n s u s e d . The n e x t l o g i c a l s t e p i n o r d e r t o e l u c i d a t e t h i s p e c u l i a r energy r e l a t i o n s h i p seemed t o be t o f o l l o w the f a t e o f r a d i o a c t i v e o r t h o p h o s p h a t e i n a r e a c t i o n m i x t u r e t o w h i c h a s u b s t r a t e was added. To a r e a c t i o n m i x t u r e , as d e s c r i b e d b e f o r e (Methods and M a t e r i a l s IV) were added $00 pM. o f g l u c o s e , and a t t h e same time a r e a c t i o n m i x t u r e w i t h o u t added g l u c o s e was r u n as a c o n t r o l . T h i s c o n t r o l w i l l be r e f e r r e d t o as "endogenous." Because the e t h a n o l p r e c i p i t a t i o n and e x t r a c t i o n removed o n l y a p p r o x i m a t e l y 5>0°/6 o f the r a d i o a c t i v e m a t e r i a l i t was d e c i d e d to f o l l o w t h i s t r e a t m e n t by e x t r a c t i o n w i t h 2$0 m i . o f 0.21+. N HCIO^ g i v i n g a f i n a l a c i d i t y of 0.20 11. The HCIO^ e x t r a c t i o n was c o n t i n u e d f o r 30 m i n u t e s at 5°, n e u t r a l i z e d w i t h KOH £ N.. and c e n t r i f u g e d at 1 6,000 x g f o r l£ minutes t o remove t h e p r o t e i n and KC10^_ p r e c i p i t a t e s . The f o u r e x t r a c t s are d e s i g n a t e d h e r e a f t e r as: endogenous e t h a n o l e x t r a c t endogenous HCIO^ e x t r a c t g l u c o s e e t h a n o l e x t r a c t g l u c o s e HC10[|_ e x t r a c t . The d i s t r i b u t i o n o f the r a d i o a c t i v i t y i s shown i n T able It. Legend: —• Optical Density at 260 mu counts per minute per 0.1 mi I 4 0 Q 1 2 0 0 ^ Q 1 0 0 0 >-cn UJ 80CH o »-0. o 6 0 0 UJ i 400| cr UJ CL 10 H Z o o 20d t u b e 10= 2 9 9 8 c / n O.D. o o ii n • i • i i i PEAK 3 8 10 20 3b" 40" 55 6 0 ~ 7 0 80" F R A C T I O N N U M B E R 240(3 ro 0 200$ X > cn z UJ <=>I60Q| _ i < o t-o. o I 2 0 d UJ i -Z 3 ct 80 d Ul O L to r-3 O o 4 0 d tubel2 = 22492 c/m O.D. 0 0 P E A K I 10 20 30 40 50 F R A C T I O N N U M B E R R G , 10. E L U T I O N C U R V E of G L U C O S E E T H A N O L E X T R A C T . 14001 \ tube 217 O.D. 1.90 180 FRACTION NUMBER FIG. II. E L U T I O N C U R V E of E N D O G E N O U S HCIO„ E X T R A C T 260 tube 125 O.D. 1.60 127 1741 c/m tube 212 O.D. 1.49 1200 F R A C T I O N N U M B E R FIG. 12. ELUT ION C U R V E of G L U C O S E H C I O A E X T R A C T . 45 TABLE Jx D i s t r i b u t i o n o f the R a d i o a c t i v e Phosphate endogenous count s'""/min. p e r c e n t of i n i t . a c t i v i t y g l u c o s e counts/min. p e r c n t .of i n i t . a c t . i n i t i a l a c t i v i t y 596 x IO*1 100 638 x 10^ 100 p r o t e i n p r e c i p i t a t e 332 x lcA 55.7 330 x 10^ 51.7 e t h a n o l e x t r a c t 269 x 1(A 45.1 314 x l O ^ 49.2 HClOj^ e x t r a c t 258 x io4 43.3 284 x 1014- 44.5 The e f f i c i e n c y o f t h e HCIO^ e x t r a c t i o n i s r e a d i l y n o t i c e a b l e s i n c e the p r o t e i n p r e c i p i t a t e s a f t e r the a c i d e x t r a c t i o n r e t a i n e d o n l y 12.4% and 7.2^0 r e s p e c t i v e l y of the i n i t i a l a c t i v i t y ( c a l c u l a t e d ) . The f o u r e x t r a c t s were e v a p o r a t e d t o s m a l l volume and chromatographed on Dowex-1-C1 column u s i n g the H C l - L i C l g r a d i e n t s mentioned b e f o r e . The e l u t i o n c u r v e s are shown i n F i g u r e s 9, 10, 11 and 12. Each of these f i g u r e s p r e s e n t s th e superimposed r a d i o a c t i v i t y and E260 r e a d i n g s f o r each 10 ml. f r a c t i o n c o l l e c t e d . The most s t r i k i n g d i f f e r e n c e between F i g u r e s 9 and 10 i s the complete absence o f peaks 2 and 3 i n f i g u r e 10.The e x t r e m e l y h i g h i n o r g a n i c o r t h o p h o s p h a t e peak does not c o i n c i d e w i t h the h i g h e s t c o n c e n t r a t i o n of the 260 mu a b s o r b i n g m a t e r i a l . The tubes c o n s t i t u t i n g t h e peaks were p o o l e d , e v a p o r a t e d t o s m a l l volume, l y o p h i l i z e d t o dryness and the l i t h i u m s a l t s o f the n u c l e o t i d e s p r e c i p i t a t e d i n acetone. S p e c t r a f o r t h e s e peaks were r u n on a model DU Beckman s p e c t r o p h o t o m e t e r between 210 and 310 mu. no c o r r e c t i o n s were made f o r r a d i o a c t i v e decay. 1+6 TABLE 5 S p e c t r o p h o t o m e t r y C o n s t a n t s o f the I s o l a t e d Peaks Sample max. mLn. R a t i o s at 250 . 260 . 280 mu (mu) (mu) 2^0/260 280/260 Endogenous e t h a n o l e x t . l 260 230 0.79 0.20 2 * 260 232 0 .94 0.1+1 3 260 228 0.78 0 .23 HCIO^ e x t . 1 2^8 228 0.79 0 . 2 2 2 260 228 0.77 0 .19 3 260 228 0.79 0.21 G l u c o s e e t h a n o l e x t . l 260 230 0.79 0.20 HCIO^ e x t . 1 259 230 0.81 0.31+ 2* 260 0.1+2 0.08 3 260 228 0 .77 0.36 adenosine (71) 259 227 0.79 0 . 1 5 c y t i d i ne 271 21+9 0.86 0.91+ u r i d i n e 262 230 0 . 7 5 0 .36 guanosine 253 223 1.18 0.68 The r e s u l t s shown i n T a b l e 5 i n d i c a t e the presence of adenosine o r u r i d i n e d e r i v a t i v e s ; however, t h e l a t t e r i s not v e r y l i k e l y because ADP i n s u b s t r a t e amounts was added t o the r e a c t i o n m i x t u r e s . R f v a l u e s of the unknown compounds chromatographed e x t r e m e l y low c o n c e n t r a t i o n 1+7 on paper, u s i n g s o l v e n t s a and b, are shown i n T a b l e 6 . TABLE1 6 Rf V a l u e s o f the I s o l a t e d Peaks Sample S o l v e n t System Ide n t i t y a b Endogenous e t h a n o l e x t . l 0.1+14- 0 .72 AMP c 3 0 .23 0 . 7 3 ATP HCIO^ e x t . 1 0.1+3 0 . 7 5 AMP 2 0.28 0 . 8 3 ADP 3 0.21+ 0 .86 ATP Gl u c o s e e t h a n o l e x t . l o.l+5 0 . 7 2 AMP HCIO^ e x t . 1 o.l+5 0.71+ AMP 2 * 0.81 ADP 3 0 .23 0 . 8 5 ATP AMP 0.1+6 0.71+ ADP 0.31 0 .80 ATP 0 . 2 2 0 . 8 5 I n the h e x o k i n a s e r e a c t i o n the endogenous HCIO^ e x t r a c t 3 c o u l d r e p l a c e ATP t o r e s u l t i n the r e d u c t i o n o f TPN. The g l u c o s e HClOj^ e x t r a c t 3 gave a s l i g h t r e d u c t i o n of TPN i f 0 .1 m i . o f c o n c e n t r a t i o n v e r y low 48 this solution was used and i f the 0.05 m>3. TPN was used instead of 0.02 m£. The results shown in T a b l e 6 prove the identity of most of the peaks. Even i f Rf values of the endogenous ethanol extract 3 correspond t o AfP in solvent a and t o AMP in solvent b, the spectrum of this compound combined with the column position prove i t t o be ATP. The concentrations of the compounds marked with an asterisk were extremely low, E2£,o readings of 0.28ii and 0.188 being obtained in concentrated solution. However, i t was felt that their column position, A max. andAmin. suggested that they were identical with ADP. C o n s i d e r i n g figures 9, 10, 11 and 12, the complete absence of the d i - and tri-phosphates i n F i g u r e 10 x^ould suggest that the addition o f glucose t o the reaction mixture renders these di- and triphosphates non-extractable with cold ethanol. The possibility that they are "protein- or particle-bound" has t o be considered. The most striking difference occurs between F i g u r e s 11 and 12. F i g u r e 11 shows a high ATP peak, E 2 6 Q = 1-90, ADP peak E 2 6o = 1 « 0 0 and an AMP peak E 2 £ , 0 = 1.10; the ATP concentration in F i g u r e 12 i s lower, E2&o = 1»50, the ADP concentration has decreased approximately 2.5 times to O.I4.2 while the AMP concentration increased to 1.60. The greatly decreased ADP concentration suggests that on the addition o f 4-9 g l u c o s e more ADP i s u s e d , p r o b a b l y t o form ATP; however, the ATP c o n c e n t r a t i o n i s l o w e r t h a n t h a t i n the endogenous t e s t . The l a r g e r AMP c o n c e n t r a t i o n i n t h e g l u c o s e t e s t , c o u p l e d t o the evidence shown above seems t o suggest a mechanism whereby ADP i s c o n v e r t e d t o ATP i n o x i d a t i v e p h o s p h o r y l a t i o n . On a d d i t i o n of an energy sou r c e more ADP i s u s e d t o be c o n v e r t e d t o ATP; the ATP formed, however, i s u t i l i z e d i m m e d i a t e l y i n r e a c t i o n s where ATP i s c o n v e r t e d t o AMP. I n an e f f o r t t o determine whether t h i s a pparent t r a n s p h o s p h o r y l a t i o n and p y r o p h o s p h o r o l y s i s i s a s s o c i a t e d , i n p a r t i c u l a r w i t h the d e g r a d a t i o n o f g l u c o s e , or whether t h e phenomenon w o u l d be e v i d e n t w i t h o t h e r s u b s t r a t e s , an experiment was c a r r i e d out w i t h s u c c i n a t e r e p l a c i n g g l u c o s e as s u b s t r a t e . T h i s TCA i n t e r m e d i a t e was u s e d because i t i s o x i d i z e d by c e l l f r e e e x t r a c t s o f P. a e r u g i n o s a whereas c x - k e t o g l u t a r a t e i s n o t . Because the main purpose o f t h i s e xperiment was t o d e t e r m i n e whether o r not t h e a d d i t i o n o f s u c c i n a t e r e s u l t e d I n a s i m i l a r n u c l e o t i d e d i s t r i b u t i o n , the e t h a n o l e x t r a c t i o n was o m i t t e d and the r e a c t i o n was stopped w i t h 2^ 0 m i . 0.21+ N HCIO^. A f t e r n e u t r a l i z a t i o n w i t h ^ N KOH and c e n t r i f u g a t i o n t h e s u p e r n a t a n t was t r e a t e d as d e s c r i b e d b e f o r e . The d i s t r i b u t i o n of t h e r a d i o a c t i v e m a t e r i a l i s shown i n Table 7. 5o TABLE 7 D i s t r i b u t i o n o f the R a d i o a c t i v e Orthophosphate when S u c c i n a t e i s S u b s t r a t e count s/min. °/6 o f i n i t i a l a c t i v i t y i n i t i a l a c t i v i t y HCIO^ e x t r a c t p r o t e i n pre dp.** 19.0 x 10 6 15.8 x 10 6 3.2 x 10 6 100 83.2 16.8 F i g u r e 13 shows the e l u t i o n curve o f t h e chromatography o f the s u p e r n a t a n t on a D o wex-l-Cl column; h e r e a g a i n r a d i o a c t i v i t y and E260 r e a d i n g s are p l o t t e d f o r each 10 mi. f r a c t i o n . The c o n c e n t r a t i o n o f fee f i r s t peak was t o o h i g h t o be r e a d i n the s p e c t r o p h o t o m e t e r and 1:10 d i l u t i o n s were made f o r t u b e s I4.5 t o 70 i n c l u s i v e as shown. Tubes c o n s t i t u t i n g peaks 1, 2, 3 and [j. were p o o l e d and t h e n u c l e o t i d e s i s o l a t e d as d e s c r i b e d b e f o r e . U l t r a v i o l e t a b s o r p t i o n s p e c t r a were d e t e r m i n e d on the Carey r e c o r d i n g s p e c t r o p h o t o m e t e r , at a c i d and n e u t r a l pH. The s p e c t r o p h o t o -m e t r y c o n s t a n t s o f t h e s e peaks are shown i n T a b l e 8. -:c- by d i f f e r e n c e 280Q tube 47= l l686c /m 240CH ro O 2000J 16001 CO Z UJ o _i < o r-CL o UJ Z or £ 800j CO 1200 z O o 400. 20 FIG. 13. I-10 dilution 40 80 100 FRACTION NUMBER ELUTION CURVE of SUCCINATE HCI0 4 EXTRACT. 51 TABLE 8 S p e c t r o p h o t o m e t r y C o n s t a n t s o f t h e I s o l a t e d Peaks A \ Ratios at 250 , 260, 280 mu max. (mu) min . (raw) 2^ 0/260 280/260 Sample acid neutral acid neutral acid neutral acid neutral peak 1 256 258 233 234 0.87 0.82 0.33 0.24 pe ak 2 256 259 231 227 0.82 0.88 0.33 0.25 peak 3 257 258 231 227 0.82 0.83 0.45 0.31 peak 4 256 258 230 231 0.88 0.85 0.27 0.18 Comparison of the constants with those of the known 5' nucleotides (71) indicates that the peaks are adenosine derivatives; deviation from the true values i s probably due.-t.o s l i g h t contamination. The A max., A min. and the s h i f t of the spectrum at low pH. are t y p i c a l for adenosine derivatives. Behaviour i n paper chromatography using solvent systems a and b i s shown i n Table 9 . ' TABLE 9 Rf V a l u e s o f the I s o l a t e d Peaks Sample Solvent System a b Identity pe ak 1 o.5o 0.76 AMP peak 2 0.31 0.20 0.81, 0.87 ADP peak 3 0.23 0.86 ATP peak 4- 0.51 0.76 AMP AMP 0.50 0.76 ADP 0.34 0.83 ATP 0.23 0.86 52 The c o n c e n t r a t i o n o f the n u c l e o t i d e i n peak 3 was v e r y low; however, i t c o n t a i n e d s u f f i c i e n t r a d i o a c t i v i t y (184 c/m p e r 10/pl) to d etermine i t s R f v a l u e s by t h e " s t r i p c o u n t i n g t e c h n i q u e " d e s c r i b e d b e f o r e . The g r e a t l y i n c r e a s e d f o r m a t i o n of t h e a d e n o s i n e - 5 ' -phosphate i s e v i d e n t i f one compares the e f f e c t s o f t h e a d d i t i o n o f g l u c o s e and s u c c i n a t e t o t h e r e a c t i o n m i x t u r e . The added ADP i s u t i l i z e d by t h e c e l l f r e e e x t r a c t but the amount o f ATP formed i s l e s s i n t h e p r e s e n c e o f t h e s u b s t r a t e s t e s t e d t h a n i n the endogenous systems. I n e a c h r e a c t i o n m i x t u r e 500 pM o f NaF were added t o i n h i b i t t h e a c t i o n o f ATPase and myokinase. To check the e f f i c i e n c y o f t h i s i n h i b i t i o n and t o d etermine t h e p r e s ence o f a pyrophosphatase i n the c e l l f r e e e x t r a c t , t h e c e l l f r e e e x t r a c t was i n c u b a t e d w i t h ATP and w i t h ATP and g l u c o s e i n the p r e s e n ce o f NaF. The r e a c t i o n m i x t u r e s were s e t up as f o l l o w s : T e s t i T e s t I I T e s t I I I G l y c y l g l y c i n e b u f f e r M/20 pH 7.2 C e l l f r e e e x t r a c t 200 mgm/mZ. 3.0 me. 4.0 mZ. 3.0 me. 4.0 mZ. 3.0 mZ. 4.0 mZ. NaF 200 /uM/mZ. ' 0.5 raZ. 0.5 me. 0.5 me. ATP 25 ;M/mZ. 0.8 mZ. 0.8 mZ. G l u c o s e 100 fM/mZ. 1.0 mZ. Sucrose M/20 1.7 me. 0.7 me. 1.5 Pyrophosphate 20/uM/m£. 1.0 mZ. 53 The r e a c t i o n m i x t u r e s were measured i n 25 m i . Erlenraeyers i n c u b a t e d at 3 0 ° , w i t h s h a k i n g , on a B u r r e l l w r i s t a c t i o n s h a k e r . One m i . samples were w i t h d r a w n a f t e r 0, 2, 5 , 10, 20 and 30 minutes i n t o 1 m i . Q% t r i c h l o r o a c e t i c a c i d . The p r o t e i n p r e c i p i t a t e was removed by c e n t r i f u g i n g a t 5,000 x g f o r 10 m i n u t e s . H a l f m i l l i l i t e r a l i q u o t s were u s e d f o r t h e d e t e r m i n a t i o n o f o r t h o p h o s p h a t e and pyrophosphate a c c o r d i n g t o t h e method d e s c r i b e d by F l y n n et' a l . ( 3 1 ) . The r e s u l t s g i v e n i n Table 10 show t h a t t h e c e l l f r e e e x t r a c t s t i l l c o n t a i n s c o n s i d e r a b l e ATPase a c t i v i t y i n the presence o f NaF, 28°/6 o f t h e added ATP b e i n g b r o k e n down i n 30 m i n u t e s . The pyrophosphatase a c t i v i t y i s v e r y s m a l l f o r o n l y lt.5°/6 °f t h e pyrophosphate was h y d r o l i z e d i n I4 .0 m i n u t e s . TABLE 10 ATPase and Pyrophosphatase A c t i v i t y o f C e l l Free E x t r a c t time yx Mole s of Pho s phate mm. I I I I I I 0 3.2 3.0 6.2 2 3.2 Ij-.O 6.8 5 4 .0 6. It 6.8 10 6.14. 6.it 6.6 20 . 7.2 8.0 7.2 30 8.8 8.0 7.8 ko 8.8 8.0 £>Pi 5.6 5.8 1.8 54 The addition of ATP to the reaction mixture did not re s u l t i n the formation of detectable amounts of pyrophosphate, determined as 90 minute phosphate. DISCUSSION The p r e s e n t e x p e r i m e n t s p r o v i d e e v i d e n c e f o r t h r e e d i f f e r e n t b u t c l o s e l y r e l a t e d phenomena: F i r s t l y t h e a d d i t i o n o f ATP t o a c e l l f r e e e x t r a c t o f P. a e r u g i n o s a w h i c h n o r m a l l y o x i d i z e s g l u c o s e w i t h the up t a k e o f 2 atoms o f oxygen, r e s u l t e d i n t h e upt a k e o f o n l y one atom o f oxygen. ATP c o u l d be r e p l a c e d by ADP and c o f a c t o r s r e q u i r e d f o r o x i d a t i v e p h o s p h o r y l a t i o n . S e c o n d l y , o x i d a t i v e p h o s p h o r y l a t i o n o c c u r r e d i n c e l l f r e e e x t r a c t s o f P. a e r u g i n o s a , b u t t h e a d d i t i o n o f the two s u b s t r a t e s t e s t e d l o w e r e d the net amount o f ATP formed. F i n a l l y , ATP was formed by the c e l l f r e e e x t r a c t as i s e v i d e n t from the h e x o k i n a s e r e a c t i o n . The a d d i t i o n o f t h e s u b s t r a t e s t e s t e d d i d not y i e l d a d d i t i o n a l r e d u c t i o n o f TPN and c o n s e q u e n t l y no a d d i t i o n a l ATP. These f i n d i n g s are i n a c c o r d w i t h t h o s e o f Campbell et a l . ( 1 5 ) who c o n c l u d e d t h a t the same amount o f energy was o b t a i n e d from e q u i m o l a r amounts o f g l u c o s e , g l u c o n a t e , o r 2 - k e t o g l u c o n a t e . Bauchop ( 5 ) i n t r o d u c e d the M o l a r 5 5 56 Growth Y i e l d (M.G.Y.) o f an or g a n i s m . He d e f i n e d the M.G.Y. as the ugm. d r y weight o f c e l l s per /a-mole o f s u b s t r a t e u t i l i z e d . The M.G.Y.'s o f S t r e p t o c o c c u s f a e c a l i s and o f Saccharomyces  c e r e v i s i a e grown a n a e r o b i c a l l y on g l u c o s e as energy source were 22 and 21 ug/u mole r e s p e c t i v e l y . S i n c e b o t h t h e s e organisms m e t a b o l i z e g l u c o s e a n a e r o b i c a l l y by t h e Embden-Meyerhof pathway, two moles o f ATP are formed d u r i n g t h e s e f e r m e n t a t i o n s . One ATP t h e r e f o r e c o r r e s p o n d s t o 10 - 11 ug d r y w e i g h t . The same r e l a t i o n s h i p was shown w i t h S . f a e c a l i s grown on a g l u c o s e -c o n t a i n i n g medium supplemented w i t h L - a r g i n i n e . The M.G.Y. from a r g e n i n e was 10 yug/n mole. The r e s u l t s o b t a i n e d w i t h P. a e r u g i n o s a would suggest t h a t t h e M.G.Y.'s o f g l u c o s e , g l u c o n i c a c i d and 2 - k e t o g l u c o n i c a c i d are e q u a l . Campbell e t a l . (11+-) showed t h a t t h e pathway o f g l u c o s e o x i d a t i o n does not i n v o l v e p h o s p h o r y l a t i o n d u r i n g t h e f i r s t two o x i d a t i v e s t e p s . 2 - k e t o g l u c o n a t e was i s o l a t e d and i d e n t i f i e d by t h e s e w o r k e r s . I n s p i t e o f t h i s , i t i s o b v i o u s t h a t p h o s p h o r y l a t i o n must p l a y a r o l e i n the i n t e r m e d i a t e m e t a b o l i s m o f t h i s o r g a n i s m . The d a t a p r e s e n t e d i n t h i s t h e s i s suggest t h a t g l u c o s e o x i d a t i o n s t o p s at 2 - k e t o g l u c o n i c a c i d o n l y i f no h i g h energy phosphate o r no h i g h energy phosphate a c c e p t o r i s p r e s e n t . I f , however, h i g h energy phosphate i s a v a i l a b l e 2 - k e t o g l u c o n i c a c i d may be p h o s p h o r y l a t e d t o 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 , w h i c h i n t u r n may be red u c e d t o 6 - p h o s p h o g l u c o n i c a c i d by a system s i m i l a r t o t h a t p r o p o s e d by Blackwood and B l a k l e y ( 7 ) . 57 D e h y d r a t i o n o f t h e l a t t e r compound would y i e l d 2-keto-3 deoxy-6-phosphogluconic a c i d . The e n o l form of "this compound would e x h i b i t a b s o r p t i o n at 260 mu and would g i v e a p o s i t i v e permanganate r e a c t i o n . One o f t h e two o x i d a t i v e s t e p s may y i e l d a r e d u c e d c a r r i e r and t h i s c a r r i e r w o u l d be r e o x i d i z e d w i t h t h e c o n c u r r e n t r e d u c t i o n o f 2-keto-6-phosphogluconate t o 6-phosphogluconate. Campbell e t a l . (15) found t h a t n e i t h e r DPN or TPN are r e d u c e d w i t h g l u c o s e as a s u b s t r a t e ; t h e y suggest t h a t t h i s system i s p o s s i b l y f l a v i n - o r c y t o c h r o m e - l i n k e d . S i n c e glucose-6-phosphate i s n o t o x i d i z e d by t h e c e l l f r e e e x t r a c t t h e f o r m a t i o n of 6-phosphogluconate by d i r e c t o x i d a t i o n o f glucose-6-phosphate i s not p o s s i b l e . The f o l l o w i n g pathway f o r t h e o x i d a t i o n of g l u c o s e by P. a e r u g i n o s a i s suggested: g l u c o s e ( c a r r i e r r e d u c e d c a r r i e r g l u c o n i c a c i d -2H 2 - k e t o g l u c o n i c a c i d ATP 2-keto-6-phosphogluconic a c i d ( r e d u c e d c a r r i e r c a r r i e r 6 -phosphogluconic a c i d " U n f o r t u n a t e l y t h e f i n d i n g t h a t AMP i s formed at v e r y h i g h c o n c e n t r a t i o n i n t h e o x i d a t i v e p h o s p h o r y l a t i o n m i x t u r e i s not e x p l a i n e d by the proposed breakdown o f g l u c o s e . The assumption 58 can be made t h a t t h e AMP i s d e r i v e d f rom ATP, t h e l a t t e r b e i n g formed by o x i d a t i v e p h o s p h o r y l a t i o n . T h i s w o u l d suggest a p y r o p h o s p h o r y l a t i o n r e a c t i o n , s i m i l a r t o t h a t proposed by Lynen and Ochoa (51}-) f o r the a c t i v a t i o n o f f a t t y a c i d s a c c o r d i n g to the r e a c t i o n : enzyme <d RCOOH + ATP + Co ASH > RC*>"»-' SCoA + AMP + PP A s i m i l a r a c t i v a t i o n i s needed i n t h e b i o s y n t h e s i s o f Coenzyme A ( 5 3 ) . However, no peak c o r r e s p o n d i n g t o pyrophosphate i s found and the pyrophosphatase a c t i v i t y o f t h e c e l l f r e e e x t r a c t i s n e g l i g i b l e . These two f i n d i n g s e l i m i n a t e a mechanism s i m i l a r t o t he a c t i v a t i o n o f f a t t y a c i d s . As mentioned i n the H i s t o r i c a l Review i n o r g a n i c p o l y p h o s p h a t e has been s u g g e s t e d t o serv e as a phosphagen i n b a c t e r i a . The p o s s i b i l i t y t h a t t he ATP formed as a r e s u l t o f o x i d a t i v e p h o s p h o r y l a t i o n i n P. a e r u g i n o s a i s s t o r e d as polymetaphosphate has been c o n s i d e r e d . K o r n b e r g e t a l . ( I 4 . 6 ) p u r i f i e d an enzyme from E. c o l i w h i c h c a t a l y s e s t h e r e a c t i o n : xATP + [ ( P 0 3 " ) n ] -xADP + ( P 0 3 " ) n + x p r i m e r The f o l l o w i n g r e a c t i o n scheme has been proposed by Mudd e t a l . ( 6 0 ) DPN i n h i b i t i o n . X o r t h o P A T P ^ = = ± p o l y -pPolyphosphatase o r t h o p ADP ADP ( o r AMP) t I 4— g l u c o s e a z a s e r i n e X amino a c i d s i n h i b i t i o n 1 RNA, DNA 59 Winder and Denneny (75) found t h a t c e l l f r e e e x t r a c t s o f Mycobacterium smegmatis c o n t a i n an i n o r g a n i c p o l y p h o s p h a t a s e , w h i c h a t t a c k s p o l y p h o s p h a t e w i t h t h e f o r m a t i o n o f o r t h o p h o s p h a t e . R e a c t i o n s s i m i l a r t o t h e s e c o u l d e x p l a i n the l a r g e AMP peaks found i n t h i s i n v e s t i g a t i o n . The ATP formed d u r i n g o x i d a t i v e p h o s p h o r y l a t i o n i s c o n v e r t e d t o i n o r g a n i c p o l y p h o s p h a t e and AMP. Polyphosphate i s r a p i d l y and r e v e r s i b l y t r a n s f o r m e d t o orthophosphate ( 7 4 ) • As shown i n the e x p e r i m e n t a l r e s u l t s , a f t e r e t h a n o l e x t r a c t i o n a p p r o x i m a t e l y %0°/o o f the r a d i o a c t i v e phosphorus i s a s s o c i a t e d w i t h the p r o t e i n p r e c i p i t a t e . The p e r c h l o r i c a c i d e x t r a c t i o n i s much more e f f e c t i v e . I t seems p r o b a b l e t h a t the ATP formed i s s t o r e d as p o l y p h o s p h a t e i n c e l l f r e e e x t r a c t s of P. a e r u g i n o s a ; t h i s phosphagen i s t h e n broken down e n z y m a t i c a l l y by a p o l y p h o s p h a t a s e o r c h e m i c a l l y by t h e HCTO[|_ t r e a t m e n t . SUMMARY 1. Incubation of a c e l l f r e e p r e p a r a t i o n of Pseudomonas aeruginosa w i t h i n o r g a n i c r a d i o a c t i v e orthophosphate, ADP and added c o f a c t o r s , r e s u l t e d i n the formation of r a d i o a c t i v e ATP. A d d i t i o n of glucose or succinate to t h i s i n c u b a t i o n mixture decreased the amount of ATP found. 2 . The c e l l f r e e preparations were found to be able to form ATP from added ADP as determined by the kexokinase t r a p . The formation of ATP was not increased by the a d d i t i o n of glucose, gluconic a c i d , 2-ketogluconic a c i d or s u c c i n i c a c i d to t h i s r e a c t i o n mixture. 3 . The c e l l f r e e p r e p a r a t i o n d i d not o x i d i z e glucose-6-phosphate, ribose-5 -phosphate, e x - k e t o g l u t a r a t e , c i t r a t e and i s o c i t r a t e . Glucose, g l u c o n i c a c i d and s u c c i n i c a c i d were o x i d i z e d . In the presence of ATP or an ATP generating system glucose was o x i d i z e d w i t h the uptake of only one atom of oxygen. I f no ATP was added the o x i d a t i o n of glucose r e q u i r e d 2 atoms of oxygen. 60 6 l l i . Two r e a c t i o n s beyond the 2-ketogluconate l e v e l have been proposed: a p h o s p h o r y l a t i o n and a r e d u c t i o n by a c a r r i e r . T h i s c a r r i e r was not TPNH. 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