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Metabolism of 2-ketogluconate by Pseudomonas aeruginosa Kay, William Wayne 1965

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THE METABOLISM OF • 2-KETOGLUCONATE BY.PSEUDOMONAS AERUGINOSA by WILLIAM W.> KAY B.•S. A . U n i v e r s i t y of B r i t i s h Columbia, 1963  A THESIS SUBMITTED IN PARTIAL FULFILMENT'OF THE REQUIREMENTS FOR THE 'DEGREE OP 'Master >of 'Science In A g r i c u l t u r a l M i c r o b i o l o g y i n t h e D i v i s i o n of Animal S c i e n c e  We a c c e p t t h i s t h e s i s as conforming t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA November, 1965  In p r e s e n t i n g t h i s the  requirements  British  mission  his  I agree that the L i b r a r y s h a l l  f o r r e f e r e n c e and s t u d y .  f o r extensive copying  p u r p o s e s may  be g r a n t e d  representatives^,  cation of this without  I t i s understood  Date  D e p a r t m e n t o r by  that copying  gain  shall  permission.  A g r i c u l t u r a l Microbiology Columbia  December 6, 1965  per-  thesis forscholarly  by t h e Head o f my  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  make i t f r e e l y  I f u r t h e r agree that  of this  thesis for financial  my w r i t t e n  Department o f  fulfilment of  f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f  Columbia,  available  thesis in partial  or publi-  n o t be  allowed  ii ABSTRACT  The n o n - p h o s p h o r y l a t e d o x i d a t i v e pathway of g l u c o s e dissimilation  has been c o n f i r m e d i n Pseudomonas a e r u g i n o s a  u s i n g whole c e l l s and c e l l - f r e e e x t r a c t s . g l u c o s e t o 2 - k e t o g l u c o n a t e was  The o x i d a t i o n of  complete and  stoichiometric  i n c e l l - f r e e e x t r a c t s and c e l l - f r e e e x t r a c t s of g l u c o s e grown c e l l s were shown t o be i n c a p a b l e of m e t a b o l i z i n g 2-ketogluconate. I t was  shown t h a t whole c e l l s c o m p l e t e l y degraded  2 - k e t o g l u c o n a t e and q u a n t i t a t i v e l y accumulated p y r u v i c a c i d i n the presence of s p e c i f i c  inhibitors.  The i n i t i a l  i n v o l v e d i n 2-ketogluconate d i s s i m i l a t i o n  step  was found t o be  e x c e p t i o n a l l y l a b i l e t o the e f f e c t s of a v a r i e t y of metabolic  inhibitors.  The m e t a b o l i s m of 2 - k e t o g l u c o n a t e was demonstrated i n v o l v e the i n i t i a l p h o s p h o r y l a t i o n w i t h adenosine phosphate  tri-  donor.  The  resultant  i n t e r m e d i a t e , 2-keto-6-phosphogluconate,  was  identified  was  (ATP) as the phosphate  to  and  shown t o undergo r e d u c t i o n by a n i c o t i n a m i d e adenine  d i n u c l e o t i d e phosphate  l i n k e d reductase to  6-phosphogluconate  w h i c h , i n t u r n , was m e t a b o l i z e d t o p y r u v a t e by enzymes of the E n t n e r - D o u d o r o f f pathway. 14  R a d i o a c t i v i t y from 2-ketogluconate-C  was  rapidly  incorporated into c e l l u l a r constituents, primarily protein, by washed c e l l s u s p e n s i o n s of _P. a e r u g i n o s a , but  oxidation  of 2 - k e t o g l u c o n a t e keto-acid The  d i d not i n v o l v e the a c c u m u l a t i o n  o  intermediates. r o l e of 2 - k e t o g l u c o n i c  a c i d as- a key  inter-  mediate f o r the c o n s e r v a t i o n of excess carbon under c o n d i t i o n s where n i t r o g e n i s l i m i t i n g was , d i s c u s s e d .  iv Table of Contents • •• •  •  Page  INTRODUCTION . . . . . . . . . . ......  . . . .  • r.ix 1  LITERATURE REVIEW" . . . . . . . .1.  The Non-phosphorylated O x i d a t i o n o f Glucose  II.  Pathways o f 2-Ketogluconat'e D i s s i m i l a t i o n  III.  II. III.  IV. V.  7  Oxidative A s s i m i l a t i o n . . . . . . . . . . . . . .  13  VII.  15  . . . . . . . . . . . . . . . . . . . .... . . .  15  . P r e p a r a t i o n of Washed C e l l . Suspensions . . . . . .  16  Organism and Media  ........  P r e p a r a t i o n of C e l l - f r e e E x t r a c t s  16  . . . . . . .  1.  Sonic o s c i l l a t i o n  16  2.  French press  . . . . . . . . . . . . . . .  16  3.  Hughes* p r e s s  . . . . . . . . . . . . ..... .  17  Manometric  18  Measurements  19  S p e c t r o p h o t o m e t r y Enzyme Assays 1.  2-keto-6-phosphogluconate  2.  2-ketogluconokinase  3.  2-keto-3-deoxy-6-phosph6gluconate  4 . ., 6-phosphogluconate VI.  1  ...  MATERIALS AND METHODS I.  . .  reductase  . . .  ....  19  .  20  aldolase  22  dehydrase  22  A s s i m i l a t i o n Experiments .1.'  I n c u b a t i o n w i t h 2-KG . . .  2.  F r a c t i o n a t i o n of whole c e l l s  E l e c t r o p h o r e t i c and chromatographic methods  20  . 22. 22 . .  23  1.  E l e c t r o p h o r e s i s - . . . .'. .-. ... . ;.. . ; • ^24  2.  Chromatography  24  V  .Table of •'Contents  (continued) Page  a. anion•exchange column 24  chromatography ... . ... . ... . . . b. paper chromatography  24  c. d e t e c t i o n r e a g e n t s VIII. IX.  I s o t o p i c Methods  . . . . .  :  A n a l y t i c a l Methods.  ......  '.  25 26 28  1.  Ammonia d e t e r m i n a t i o n s •  28  2.  Glucose  28  3.  Chemical  4.  Enzymatic  determinations d e t e r m i n a t i o n o f 2-KG  ....... *  d e t e r m i n a t i o n of k e t o - a c i d s . . .  a. l a c t i c dehydrogenase  28 29  assay  f o r pyruvate  . .  29  b. g l u t a m i c dehydrogenase assay f o r CK•-ketoglutarate 5.  .  29  Protein determinations  EXPERIMENTAL RESULTS AND DISCUSSION . ....... ....... ... I.  29  30  The Non-phosphorylated O x i d a t i v e Pathway of . . - 30  'Glucose D i s s i m i l a t i o n 1.  I d e n t i f i c a t i o n , o f products  of-glucose, 30  oxidation 14  2. II. III.  Preparation.and  i d e n t i f i c a t i o n of -2-KG-C  O x i d a t i v e A s s i m i l a t i o n of 2 - K e t o g l u c o n a t e  .  . . . .  I n t e r m e d i a t e s of 2-KG M e t a b o l i s m 1.  35 39 52  I d e n t i f i c a t i o n of i n t e r m e d i a t e s o f 2-KG d i s s i m i l a t i o n w i t h whole c e l l s  ......  54  . Table of C o n t e n t s / ( c o n t i n u e d ) 2.  . I d e n t i f i c a t i o n of i n t e r m e d i a t e s o f 2-KG dissimilation inhibitors  • '•' •  3.  i n t h e presence of  a.  sodium a r s e n i t e  b.  bromopyruvic a c i d  c.  sodium s e l e n i t e  d.  sodium f l u o r i d e and iodoa'cetamide  e.  ethylenediaminetetracetic'• acid  Inhibitors  56  ..................... . . . . . ... . . .  58 58  ... ..... . . .  and-2-KG-U-C ^' 1  60 63  ...  ... . . .  66  . 6 8  . . .  4 . ' I d e n t i f i c a t i o n , o f i n t e r m e d i a t e s of-2-KG d i s s i m i l a t i o n by " c a r r i e r " experimentation,.  5.  . ... . . ... . . ...... .  a.  non-phosphorylated c a r r i e r s  b.  phosphbrylated c a r r i e r s  75  . . . . . .  . . . . . . . .'. .79  D e m o n s t r a t i o n . o f 2-KG.metabolism i n c e l l free' e x t r a c t s . . . . a.  6.  74  enzyme a s s a y s  86  •-•  v ........... .  O x i d a t i o n o f 2-KG by phosphate, s t a r v e d c e l l s  .  86  93 98  GENERAL DISCUSSION . . . . . . . .  SUMMARY . . ... ... ........ ...... ... . . . . ' . . . '. . . . 102 ' BIBLIOGRAPHY •. ... . . ... . ......  '.  103  L i s t of- F i g u r e s Figure 1.  2. 3. • 4.  Page Oxygen uptake by P. a e r u g i n o s a w i t h 5;u.M of g l u c o s e , g l u c o n i c a c i d o r 2-ketogluconic acid . ... . ... ...  32  Disappearance of g l u c o s e and t h e f o r m a t i o n o f •2-KG.in 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  36  .  • 14  E l u t i o n sequence o f C compounds and U.V. a b s o r b i n g m a t e r i a l from a. Dowex 1-formate a n i o n exchange column -  -38  Time course of 2-KG d i s a p p e a r a n c e , oxygen uptake, and NR3 r e l e a s e o r d i s a p p e a r a n c e w i t h r e s t i n g c e l l s of P. a e r u g i n o s a . . . . . . . .  42  Time course o f t h e d i s t r i b u t i o n .of C ^ added t o •washed c e l l -suspensions as 2-KG-U-C 4 . . . . . .  45  6A.  Oxygen consumption, by washed c e l l s u s p e n s i o n s i n .the presence of a r s e n i t e and 5 A M 2-KG ... .  59  6B.  P y r u v a t e a c c u m u l a t i o n from 5J^M 2-KG by washed c e l l s u s p e n s i o n s i n t h e presence of '.various c o n c e n t r a t i o n s of a r s e n i t e .. ... ..... .......  59  Oxygen uptake by w a s h e d . c e l l s u s p e n s i o n s i n the presence of bromopyruvate and 5A-M 2-KG . ..-  .61  P y r u v a t e a c c u m u l a t i o n from. 2-KG by washed c e l l s u s p e n s i o n s i n t h e presence of v a r i o u s conc e n t r a t i o n s o f bromopyruvate .............  6l  8A.  Oxygen, uptake by washed c e l l s u s p e n s i o n s i n t h e presence of s e l e n i t e and 5JU.M 2-KG -  62  8B.  P y r u v a t e a c c u m u l a t i o n from 2-KG by washed c e l l . s u s p e n s i o n s i n t h e presence of v a r i o u s conc e n t r a t i o n s of selenite-  5.  1  1  7A. 7B.  9. 10. 11.  .62  Oxygen uptake by washed c e l l s u s p e n s i o n s i n t h e presence o f sodium f l u o r i d e and 5JU.M 2-KG . . .  64  Oxygen uptake by washed c e l l s u s p e n s i o n s i n t h e presence o f iodoacetamide and 5/U.M 2-KG ......  65  Oxygen uptake by washed c e l l s u s p e n s i o n s i n the presence of'EDTA and 5 J I M 2-KG . . . . . . .  67  vi i i L i s t of F i g u r e s ( c o n t i n u e d ) Figure 12.  Page E l u t i o n from a Dowex 1-formate a n i o n exchange column o f compounds from g l u c o s e - U - C ^ o x i d a t i o n by P. a e r u g i n o s a . .. 1  13. 14.  15.  16.  Oxygen uptake a t 20 C by w a s h e d - c e l l s u s p e n s i o n s o f P. a e r u g i n o s a . E l u t i o n sequence o f compounds from a Dowex-1-formate a n i o n exchange column a f t e r 2-KG-Cl^v o x i d a t i o n i n t h e presence of 2-K-3-D-6-PG . Demonstration o f 2-ketogluconokinase a c t i v i t y and 2-ketogluconoreductase a c t i v i t y i n c e l l - f r e e e x t r a c t s o f 2-KG grown c e l l s . Demonstration of 6-PG dehydrase a c t i v i t y i n c e l l - f r e e e x t r a c t s o f g l u c o s e grown c e l l s and 2-K-3-D-6-PG a l d o l a s e i n c e l l - f r e e e x t r a c t s of 2-KG grown c e l l s ......  82 84  85 88  89  ACKNOWLEDGMENT  I would l i k e t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o Dr. A. Gronlund f o r h e r i n t e r e s t and h e l p f u l c r i t i c i s m d u r i n g t h e course of t h e experimental  work and the w r i t i n g of t h i s  • I w i s h f u r t h e r -to acknowledge t h e t e c h n i c a l h e l p of Miss C a r o l i n e P i n c h and M i s s Myrna  Mattice.  thesis.  INTRODUCTION  The n o n - p h o s p h o r y l a t e d o x i d a t i o n of g l u c o s e by Pseudomonas a e r u g i n o s a has been w e l l documented ( N o r r i s and Campbell, 19^9; Stokes and Campbell, 1 9 5 1 ;  Claridge  and Werkman, 1953)* b u t t h e m e t a b o l i c • r o l e of 2 - k e t o g l u c o n a t e , t h e apparent end p r o d u c t of these o x i d a t i v e r e a c t i o n s , has remained o b s c u r e .  A l l p r e v i o u s attempts  t o e l u c i d a t e t h e d i s s i m i l a t o r y . scheme i n v o l v e d i n 2 - k e t o g l u c o n a t e metabolism, i n t h i s , organism have been u n s u c c e s s ful. • Pseudomonas f l u o r e s c e n s , a l s o o x i d i z e s g l u c o s e by the n o n - p h o s p h o r y l a t e d r o u t e , but w i t h t h i s organism t h e d e g r a d a t i o n o f - 2 - k e t o g l u c o n a t e has been shown t o proceed s o l e l y v i a t h e E n t n e r - D o u d o r o f f pathway.  ( K o v a c h e v i c h and  Wood, 1 9 5 5 ; Narrod and Wood, 1 9 5 6 ) . I t was t h e o b j e c t of .this endeavor t o study t h e m e t a b o l i s m of t h i s organism, e s t a b l i s h t h e d e g r a d a t i v e r o u t e of ' 2 - k e t o g l u c o n a t e c a t a b o l i s m and t o , i f p o s s i b l e , why t h e key enzymes have r e s i s t e d d e m o n s t r a t i o n so tenaciously.  determine  LITERATURE REVIEW  The f o r m a t i o n o f - 2 - k e t o g l u c o n a t e (2-KG) d u r i n g t h e o x i d a t i o n of g l u c o s e was i n i t i a l l y observed i n growing c u l t u r e s of Acetobacter gluconicum. G o r l i c k , 1935).  (Bernhauer and  The o x i d a t i o n of 2-KG was found t o be  i n h i b i t e d by e i t h e r - h i g h a e r a t i o n or by i n c u b a t i n g t h e c e l l s a t r e l a t i v e l y low t e m p e r a t u r e s f o r 3 0 - t o 60 days. I.  The Non-phosphorylated  O x i d a t i o n o f Glucose  The f o r m a t i o n o f g l u c o n a t e and 2-KG as t r a n s i e n t i n t e r m e d i a t e s d u r i n g g l u c o s e d i s s i m i l a t i o n by Pseudomonas a e r u g i n o s a was demonstrated  q u a l i t a t i v e l y by  N o r r i s and Campbell ( l 9 5 l ) > by the i s o l a t i o n of p r o d u c t s from c u l t u r e s growing i n a glucose-ammonium-phosphate medium-. Prom t h i s e v i d e n c e and q u a n t i t a t i v e manometric data  observed  d u r i n g t h e o x i d a t i o n of g l u c o s e b o t h by whole c e l l s and by d r i e d c e l l p r e p a r a t i o n s i t was c o n c l u d e d t h a t g l u c o n a t e and 2-KG were i n t e r m e d i a t e s , and t h a t most of t h e g l u c o s e , i f not a l l , was m e t a b o l i z e d v i a the n o n - p h o s p h o r y l a t e d r o u t e . U s i n g c e l l - f r e e e x t r a c t s of P.•aeruginosa  Claridge  and Werkman (1953)> s u b s t a n t i a t e d t h e q u a n t i t a t i v e  observ-  a t i o n s of Stokes and Campbell and assumed t h a t hexose phosphates were n o t I n t e r m e d i a t e s i n these r e a c t i o n s .  This  o x i d a t i v e system proved t o be s e n s i t i v e t o c y a n i d e and i t was c o n c l u d e d t h a t metal l i n k e d c a r r i e r s were operative" i n 1  2  i n the transport of e l e c t r o n s . Non-phosphorylated hexose o x i d a t i o n has been r e c o g nized i n a great v a r i e t y of microorganisms, p r i n c i p a l l y i n bacteria. gluconate  K o e p s a l l ( 1 9 5 © ) , demonstrated t h a t g l u c o s e and were r a p i d l y o x i d i z e d t o 2-KG and t h a t  i n t e r m e d i a t e was u t i l i z e d 'Pseudomonas f l u o r e s c e n s . corroborated  this  s l o w l y i n growing c u l t u r e s o f Wood and Schwert  (1952),  these o b s e r v a t i o n s u s i n g c e l l - f r e e e x t r a c t s  of T. f l u o r e s c e n s w h i c h o x i d i z e d g l u c o s e  and g l u c o n a t e  q u a n t i t a t i v e l y t o 2-KG and a t t e m p t s t o demonstrate phosp h o r y l a t e d hexose o x i d a t i o n were u n s u c c e s s f u l . o x i d a t i o n of, g l u c o s e , g l u c o n a t e  The  and 2-KG by S e r r a t i a  marscesens r e q u i r e d 3 . 0 , 2 . 5 , and 2^Cm of oxygen p e r ^ M of s u b s t r a t e r e s p e c t i v e l y , and 2-KG accumulated as a t r a n sitory, intermediate Katznelson  (Wasserman, Hopkins, and S e i b l e s , 1 9 5 6 ) .  ( 1 9 5 9 ) * surveyed 19 s p e c i e s b e l o n g i n g t o  Xanthomonas, Pseudomonas, A g r o b a c t e r i u m , E r w i n i a and C o r y n e b a c t e r i u m genera f o r t h e a b i l i t y t o o x i d i z e gluconate, gluconate  and 2-KG.  glucose,  A l l s p e c i e s o x i d i z e d g l u c o s e and  b u t o n l y two s p e c i e s were capable of o x i d i z i n g  2-KG. • C e r t a i n Aerobacter  s p e c i e s have been shown t o  u t i l i z e t h e n o n - p h o s p h o r y l a t e d r o u t e of g l u c o s e  oxidation  s i m i l a r t o t h e pathway e s t a b l i s h e d i n Pseudomonas. (DeLey, 1 9 5 5 ) .  However, • DeLey .and Cornut (19.51) demon-  s t r a t e d t h a t t h i s enzyme system was n o t formed p r e f e r e n t i a l l y  under a e r o b i c growth c o n d i t i o n s but was b i c a l l y cultured bacteria.  a c t i v e i n anaero-  They p o s t u l a t e d t h a t the  a e r o b i c a l l y grown c e l l s had cytochrome systems w h i c h a l l o w e d a l t e r n a t e pathways of g l u c o s e d i s s i m i l a t i o n t o be utilized  selectively.  In g l u c o s e grown c e l l s and c e l l - f r e e e x t r a c t s of the a c e t i c a c i d bacterium, Gluconobacterium o x i d a t i o n of g l u c o s e proceeded t o 2-KG  liquefaciens,  not o n l y t h r o u g h  gluconate  but a l s o t o the more o x i d i z e d i n t e r m e d i a t e  d i k e t o g l u c o n a t e (2,5-KG) v i a 5 - k e t o g l u c o n a t e (Stouthamer,  196l).  1957).  2,5-  (5-KG)  The A c e t o b a c t e r were found t o o x i d i z e  g l u c o s e i n a s i m i l a r manner t h r o u g h g l u c o n a t e t o 25-KG.  the  (Pewester, 1956;  Schramm, Gromet, and  and  Hestrin,  K a t z n e l s o n , • Tanenbaum, and Tatum ( 1 9 5 3 ) > have  r e p o r t e d that- A c e t o b a c t e r melanogenum produces 2,5-KG from g l u c o s e and Schramm and Backer  (1957)> have r e p o r t e d a'  r a p i d o x i d a t i o n of g l u c o s e t o 2-KG  i n Acetobacter-xylinum  w i t h o n l y a minor p r o p o r t i o n of g l u c o s e b e i n g p h o s p h o r y l a t e d directly. D i r e c t non-phosphorylated  o x i d a t i o n of g l u c o s e  a l s o been found i n Waring-blended the fungus C a l d a r i o m y c e s  has  m y c e l i a l suspensions  fumago w h i c h n o r m a l l y does not  u t i l i z e 2-KG--the o x i d a t i o n end product--due  to a toxic  a c c u m u l a t i o n of'RVjOg* however, t h i s i n h i b i t i o n  was  e l i m i n a t e d by the subsequent a d d i t i o n of c a t a l a s e (Ramachandran and G o t t l i e b ,  1963).  of  4  N i c o t i n a m i d e adenine amide adenine  d i n u c l e o t i d e (NAD) and n i c o t i n -  d i n u c l e o t i d e p h o s p h a t e (NADP) have been shown  t o a c t as hydrogen a c c e p t o r s i n t h e o x i d a t i o n of g l u c o s e - 6 phosphate (G-6P) t o 6-phosphogluconate (6-PG) by y e a s t s , b a c t e r i a , and a n i m a l t i s s u e s .  However, a number of b a c t e r i a  have been r e p o r t e d t o o x i d i z e g l u c o s e t o g l u c o n i c a c i d w i t h an.enzyme system l i n k e d t o p a r t i c u l a t e s t r u c t u r e s of t h e cell-free extract.  These b a c t e r i a i n c l u d e • A c e t o b a c t e r  suboxydans ( K i n g and C h e l d e l i n , 1 9 5 7 ) * ? . f l o u r e s c e n s (Wood and Schwert,  1 9 5 3 ) * Pseudomonas  (Dowling and L e v i n e , 1 9 5 6 ) , pyrogallica  pseudomonallei  Pseudomonas q u e r c i t o -  ( B e n t l e y and S c h l e c t a , i 9 6 0 ) ,  Aerobacter  aerogenes (Dalby and Blackwood, 1 9 5 5 ) , A z o t o b a c t e r  vinelandii  ( B r o d i e and Lipmann, 1 9 5 5 ) * and B a c t e r i u m a n i t r a t u m (Hauge, i960).  No e v i d e n c e has been found t o i n d i c a t e e i t h e r NAD  o r NADP p a r t i c i p a t i o n i n t h e d e h y d r o g e n a t i o n  process.  It  has been h y p o t h e s i z e d t h a t these o x i d a t i v e enzymes a r e l i n k e d t o the r e s p i r a t o r y c h a i n o f t h e microorganisms and, as such, they appear bound t o t h e p a r t i c u l a t e of a l l c e l l - f r e e e x t r a c t s . dehydrogenase systems, role  I n some s o l u b l e a l d o s e  NAD and NADP do p l a y an i m p o r t a n t  ( H a r r i s o n 1 9 3 1 ; Wiemberg, 1 9 6 1 ) .  o f Corynebacterium  fraction  Cell-free extracts  c r e a t i n o v o r a n s were found t o d i f f e r  from  whole c e l l s i n t h e i r a b i l i t y t o o x i d i z e g l u c o s e t o 2-KG, but t h e a d d i t i o n of f l a v i n e adenine  d i n u c l e o t i d e (FAD)  t o the c e l l - f r e e e x t r a c t s p e r m i t t e d q u a n t i t a t i v e o x i d a t i o n  of g l u c o s e t o g l u c o n i c a c i d ( G h i r e t t i and 1954).  The  p a r t i c u l a t e a l d o s e dehydrogenase of  Rhodopseudomonas s p h e r o i d e s has  been shown t o be  upon an u n i d e n t i f i e d , h e a t - s t a b l e ,  dependent  soluble cofactor f o r .its-  (Szymona and D o u d o r o f f , i 9 6 0 ) .  activity  Guzman-Barron,  Niederpruem  and  B o u d o r o f f ( 1 9 6 5 ) * have shown.that t h i s c o f a c t o r i s p r e s e n t i n . the p a r t i c u l a t e f r a c t i o n of a e r o b i c a l l y grown c e l l s a l s o i n the s o l u b l e f r a c t i o n , but the c e s s a t i o n of e x p o n e n t i a l  E s c h e r i c h i a c o l i , yeast,  only i n the l a t t e r a f t e r  growth.  f r o m a n a e r o b i c a l l y grown c e l l s and  The  c o f a c t o r was  from.P.  absent  flourescens,  and mouse l i v e r .  • Glucose o x i d a t i o n i n B a c i l l u s s u b t i l i s was r e q u i r e NAD  and  and hydrogen p e r o x i d e was  formed.  shown t o  In a d d i t i o n ,  the o x i d a t i o n of g l u c o s e by spore e x t r a c t s of B a c i l l u s c e r e u s was system.  found t o be c a t a l y z e d by a s o l u b l e NAD  Thus the g l u c o s e ,  and p o s s i b l y the  requiring  gluconate,  dehydrogenases of G r a m - p o s i t i v e organisms appeared t o •from those of Gram-negative organisms.  (Halvorson  differ  and  Church, 1 9 5 5 ) . In P. a e r u g i n o s a , the o x i d a t i o n of g l u c o s e t o - a c i d was  gluconic  shown t o proceed v i a an apparent non-NAD or -NADP  r e q u i r i n g o x i d a t i v e s t e p t o g l u c o n o l a c t o n e and enzymic h y d r o l y s i s of the l a c t o n e  the  t o g l u c o n a t e was  be magnesium dependent (Gr'onlund, 1 9 6 1 ) .  Gluconic  subsequent found t o dehydro-  genase, w h i c h c a t a l y z e s the o x i d a t i o n of -gluconic a c i d t o 2-KG  without concurrent phosphorylation,  was  partially  p u r i f i e d by Ramakrishnan and Campbell (1955)* and f o r s u b s t r a t e s p e c i f i c i t y and f o r an . e l e c t r o n  examined  acceptor  r e q u i r e m e n t , but the r e a c t i o n had no p y r i d i n e n u c l e o t i d e r e q u i r e m e n t and pyocyanine f u n c t i o n e d o p t i m a l l y as  an  electron acceptor.  DeLey and Stouthamer • (1959)> i n v e s t i -  gated the g l u c o n a t e  dehydrogenases of A. suboxydans  A. melanogenum and  showed tha,t separate  and  s o l u b l e NADP  s p e c i f i c dehydrogenases e x i s t e d for-2-KG and 5-KG  formation  and t h a t a p a r t i c u l a t e , p o s s i b l y cytochrome l i n k e d , gluconate  oxidase  yielding•2-KG.existed  i n these organisms.  Campbell, L i n n e s , and E a g l e s (1954), concluded from molar growth y i e l d e x p e r i m e n t s , u s i n g amounts of g l u c o s e ,  gluconate,  source,  t h a t no energy was  glucose  t o 2-KG  or 2-KG  limiting-equimolar as the s o l e carbon  g a i n e d from the o x i d a t i o n of  s i n c e almost i d e n t i c a l amounts of growth of  P*?' a e r u g i n o s a , a s measured by t u r b i d i t y and p r o t e i n were o b t a i n e d .  content,  L a t e r , however, S t r a s d i n e , Campbell,  'Hogenkamp, and Campbell (1961), f o l l o w e d - l a b e l l e d I n o r g a n i c phosphorous (P  radioactively  ) incorporation into  r e s t i n g c e l l s and found, an I n v e r s e r e l a t i o n between the 32 degree. of s u b s t r a t e o x i d a t i o n and 1  P  Incorporation  i n d i c a t i n g that phosphorylation.was. c o i n c i d e n t w i t h  thereby the  passage of e l e c t r o n s t o oxygen t h r o u g h the e l e c t r o n t r a n s p o r t chain. The  enzymic d e g r a d a t i o n  of 2-KG  i n microorganisms  i s , i n almost a l l c a s e s , - r e a d i l y i n h i b i t e d by  intense  a e r a t i o n w h i c h a l l o w s n e a r l y q u a n t i t a t i v e a c c u m u l a t i o n of  2-KG from g l u c o s e and t h e r e f o r e t h i s procedure has v a l u e as a commercial source of the compound.  R e c o v e r i e s of-75-85$  have been a c h i e v e d w i t h shake c u l t u r e s o f P. a e r u g i n o s a ( H i l l , .1952), P. f l u o r e s c e n s ( A s a i and Ikeda, 1948), and Gluconobacter  s p e c i e s ( i k e d a , 1960); 90-97$ y i e l d s have  been r e p o r t e d w i t h P. p u t i d a (DeLey and Cornut, 1951)* •Cyanococcus chromospirans ( B r i t i s h p a t e n t , 1951)* " d a  r e c e n t l y w i t h S e r r a t i a marscesens (Misenheimer and Anderson, 1964). II.  Pathways of 2-Ketogluconate D i s s i m i l a t i o n DeLey (1953)* demonstrated an enzyme, 2-keto-  g l u c o n o k i n a s e , i n alumina  ground or Hughes' p r e s s  e x t r a c t s of 2-KG adapted c e l l s of A e r o b a c t e r  cell-free  cloacae.  T h i s new enzyme a f f e c t s the t r a n s p h o s p h o r y l a t i o n . of 2-KG i n the presence of adenosine t r i p h o s p h a t e (ATP) magnesium i o n s .  and.  Subsequent p a r t i a l p u r i f i c a t i o n of the  enzyme p e r m i t t e d the i s o l a t i o n o f ' t h e  phosphorylated.acid  2-keto-6-phosphogluconate (2-K-6-PG), w h i c h was  identified  by the s p e c t r a l c h a r a c t e r i s t i c s of the f r e e a c i d and i t s quinoxaline derivative.  Aerobacter. aerogenes, grown on  2-KG, c o n t a i n e d a s p e c i f i c , i n d u c i b l e , k i n a s e and the enzyme was  purified.124 f o l d . . . (Frampt on - and Wood, 1961).  G h i r e t t i and Guzmann-Barron (1954), showed t h a t the p h o s p h o r y l a t i o n of 2-KG by c e l l - f r e e e x t r a c t s o f C. c r e a t i n o v o r a n s was  a s t e p i n the pathway -of g l u c o s e  8  d i s s i m i l a t i o n i n t h i s organism;, t h e end p r o d u c t of • t h e t r a n s p h o s p h o r y l a t i o n with.ATP was not i s o l a t e d h u t was assumed t o be 2-K-6-PG. demonstrated,  Narrod and Wood  (1956),  and s e p a r a t e d t h e i n d u c e d enzyme  g l u c o n o k i n a s e from t h e c o n s t i t u t i v e enzyme  2-keto-  gluconokiha.se  u s i n g c e l l - f r e e e x t r a c t s of P. f l u o r e s c e n s and t h e phosp h o r y l a t e d i n t e r m e d i a t e 2-K-6-PG was i d e n t i f i e d . and Vandamme ( 1 9 5 5 ) *  extended  DeLey  t h e i r e a r l y o b s e r v a t i o n s on  i n d u c i b l e kinases i n Aerobacter species t o a wider of organisms genera  and demonstrated  spectrum  2-ketogluconokinase i n the  Pseudomonas, Xanthomonas, E s c h e r i c h i a , .Aerobacter,  P a r a c o l o b a c t e r u m , S e r r a t i a , E r w i n i a , and B a c i l l u s . :  Pewster  (1957)*  2 - k e t o g l u c o n o k i n a s e and  demonstrated  a new enzyme, 5 - k e t o g l u c o n o k i n a s e , i n • s o n i c e x t r a c t s of A. suboxydans by manometric measurements of a c i d p r o d u c t i o n d u r i n g t r a n s p h o s p h o r y l a t i o n r e a c t i o n s and p o s t u l a t e d , and l a t e r • c o n f i r m e d , t h a t 2-K-6-PG was d e c a r b o x y l a t e d t o arabonic a c i d . DeLey and Stouthamer ( 1 9 5 9 ) * 5-ketogluconoklnase  were unable t o d e t e c t  i n t h e i r s t r a i n of A. .suboxydans.  C e l l - f r e e p r e p a r a t i o n s of A. melanogenum grown w i t h g l u c o s e as t h e carbon.source  r e a d i l y p h o s p h o r y l a t e d 2-KG as measured  m a n o m e t r i c a l l y by t h e r e l e a s e of C 0 b u f f e r (Pewster, 1 9 5 7 ) .  2  from a b i c a r b o n a t e  DeLey's s t r a i n of t h i s  organism  c o u l d n o t be shown t o p h o s p h o r y l a t e e i t h e r 2-KG or 5-KG, but r a t h e r t h e metabolism, of b o t h k e t o g l u c o n a t e s was shown  9  t o c o n s i s t of a r e d u c t i o n t o g l u c o n a t e w h i c h was sequently phosphorylated  t o 6-PG  (DeLey and  sub-  Stouthamer,  1959).  A decarboxylase product  of 2-KG  f o r 2,5-diketogluconate  (2, 5-KG), a  d i s s i m i l a t i o n i n A. melanogenum,  was  p u r i f i e d from the organism and the d e c a r b o x y l a t i o n p r o d u c t the r e a c t i o n was  i s o l a t e d as the  o-nitrophenylhydrazone.  The  a p p a r e n t i n a b i l i t y t o . i d e n t i f y the r e a c t i o n product  due  t o the extreme l a b i l i t y of the f r e e i n t e r m e d i a t e  and t o a l e s s e r • d e g r e e the hydrazone d e r i v a t i v e Hochster,  and K a t z n e l s o n , 1 9 5 8 ) .  of  The  was  (Datta,  i d e n t i t y of t h i s  i n t e r m e d i a t e has not been r e p o r t e d t o d a t e . Fermentation  p r o d u c t a n a l y s e s and i s o t o p e t r a c e r data  f r o m s p e c i f i c a l l y l a b e l l e d s u b s t r a t e s have been used t o e l l u c i d a t e 2-KG  d e g r a d a t i v e pathways i n c e r t a i n l a c t i c  b a c t e r i a . -Blackwood and B l a k l e y concluded  t h a t the  f e r m e n t a t i o n s of g l u c o s e , g l u c o n a t e , and 2-KG mesenteroides  were s i m i l a r  A s p e c i f i c a d a p t i v e 2-KG c e l l s when grown on 2-KG. f i e d and was  acid  by Leuconostoc  (Blackwood and B l a k l e y , 1 9 5 6 ) .  k i n a s e was The  i s o l a t e d from these  enzyme was  partially-puri-  a c t i v e i n the p r e s e n c e of magnesium i o n s w i t h  e i t h e r ATP,  ITP, or GTP  The p r o d u c t  of t h e k i n a s e r e a c t i o n , 2-K-6-PG, was  and i d e n t i f i e d .  a c t i n g as the phosphate donor.  C i f f e r i and B l a k l e y ( 1 9 5 9 ) *  demonstrated  t h a t the metabolism•of•2-KG i n L. mesenteroides a c c o r d i n g t o the f o l l o w i n g pathway :  isolated  proceeded  10  2-KG  - 2-K-6-PG  5-phosphate + COg a c e t y l phosphate  «- 6-PG  »- r i b u l o s e -  xylulose-5~phosphate  .  + glyceraldehyde-3-phosphate.  of 2-K-6-PG t o 6-PG  The  reduction  r e q u i r e d e i t h e r NADHg or NADPHg.  Goddard and Sokatch (1963) showed t h a t S t r e p t o c o c c u s f a e c a l i s c o n v e r t e d 2-KG  t o pentose phosphate,  decarboxylation.  Pentose-phosphate  but by a p r i m a r y was  then fermented  to  p y r u v a t e t h r o u g h a s e d o h e p t u l o s e d i p h o s p h a t e v a r i a t i o n of the pentose phosphate  pathway found i n t h i s  DeLey and D e F l o o r ( 1 9 5 9 ) demonstrated  organism. and  i z e d ' 2 - k e t o g l u c o n o r e d u c t a s e a c t i v i t y i n the 2-KG  characteradapted  b a c t e r i u m Corynebacterium h e l v o l u m , the y e a s t Debaromyces h a n s e n i i and the mold A s p e r g i l l u s n i d u l a n s , and i t was  shown t h a t the enzyme was  w i t h NADPHg than w i t h NADHg.  20 t i m e s more a c t i v e  S o n i c e x t r a c t s of P. f l u o r e s c e r i s  have been shown t o have r e d u c t a s e a c t i v i t y i n b o t h g l u c o s e and 2-KG  grown c e l l s  (Frampton  and Wood, 1 9 6 1 ) . 14  M e t a b o l i c i n h i b i t o r and C  tracer studies with  i n t a c t c e l l s and crude enzyme p r e p a r a t i o n s of Pseudomonas' s a c c h a r o p h i l a proved t o E n t n e r and Doudoroff  (1952)*  that  g l u c o s e and g l u c o n i c a c i d were u t i l i z e d i n such a manner as t o y i e l d 2 m o l e c u l e s of p y r u v i c a c i d p e r m o l e c u l e of substrate.  The  c a r b o x y l groups of o n e - h a l f of the p y r u v a t e  m o l e c u l e s were d e r i v e d from carbon atom one of g l u c o s e i n s t e a d of carbon atoms 3 and 4 w h i c h would have been the r e s u l t i f the Embden-Meyerhof pathway was  operative.  These  11 workers t h e r e f o r e proposed  an o x i d a t i v e pathway i n v o l v i n g  a 3=3 s p l i t by t h e cleavage o f 6-PG, o r an isomer o f t h e compound,.to p r o v i d e t r i o s e phosphate and p y r u v i c a c i d . I t was h y p o t h e s i z e d t h a t 6-PG underwent an enzymic d e h y d r a t i o n and rearrangement  t o 2-keto-3-deoxy-6-phosphogluconate  (2-K-3-D-6-PG), which was s u b s e q u e n t l y s p l i t by an a l d o l a s e type o f r e a c t i o n .  McGee and Doudoroff  (1954), s e p a r a t e d two  enzymatic a c t i v i t i e s from c e l l - f r e e e x t r a c t s o f P. s a c c h a r o p h i l a ; one c o n v e r t e d 6-PG t o t h e p h o s p h o r y l a t e d k e t o - a e i d i d e n t i f i e d as t h e p r e v i o u s l y p o s t u l a t e d 2-K-3-D-6-PG; and one s p l i t t h i s k e t o - a c i d i n t o p y r u v i c a c i d and g l y c e r a l d e hyde-3-phosphate. E x t r a c t s o f P. f l u o r e s c e n s were shown t o c a r r y out a r a p i d o x i d a t i o n o f G-6-P and 6-PG b u t l a c k e d t h e complete spectrum  o f enzymes n e c e s s a r y f o r g l y c o l y s i s (Wood and  Schwert,.1954).  The enzyme, 6-PG dehydrase, was s u b s e q u e n t l y  p u r i f i e d from d r i e d c e l l s and 2-K-3-D-6-PG was i s o l a t e d as the p r o d u c t o f t h e r e a c t i o n o f the p u r i f i e d enzyme w i t h 6-PG ( K o v a c h e v i c h and Wood, 1955).  A second enzyme was o b t a i n e d  which had no a c t i v i t y w i t h 6-PG, but which c a t a l y z e d t h e c l e a v a g e o f 2-K-3-D-6-PG ( K o v a c h e v i c h and Wood, 1955yaj. S p e c i f i c k i n a s e s f o r t h e non-phosphorylated  analogue  2-keto-3-deoxygluconic a c i d (2-K-3-DG-) have been demons t r a t e d i n E. c o l i grown w i t h g l u c u r o n i c o r g a l a c t u r o n i c a c i d s (Cynkin and A s h w e l l , i960), and i n Rhodopseudomonas  12  i960.).  s p h e r o i d e s grown i n g l u c o s e (Szymona and D o u d o r o f f ,  HugheS'-', p r e s s 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 were demonstrated  t o c o n t a i n a weak 2-K-6-PG  reductase  ( R e i d , 1 9 5 9 ) * and a v e r y p o t e n t 2-K-3-D-6-PG a l d o l a s e (Gronlund, 1961),  s u g g e s t i n g - t h a t t h i s organism  possesses,  a t l e a s t i n p a r t , pathways of g l u c o s e d i s s i m i l a t i o n  similar  t o those e s t a b l i s h e d i n o t h e r Pseudomonas s p e c i e s . C l a r i d g e and Werkman ( 1 9 5 3 ) * c o n c l u d e d t h a t 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 do not m e t a b o l i z e 2-KG, but r a d i o a u t o g r a p h i c a n a l y s e s of whole c e l l i n c u b a t i o n m i x t u r e s w i t h 2-KG showed t h e presence  of a i t r i c a r b o x y l i c a c i d  cycle  and of f u n c t i o n i n g a s s i m i l a t i o n mechanisms. S i n c e t h e e s t a b l i s h m e n t of the E n t n e r - D o u d o r o f f pathway i n 195^,  t h e r e has been growing evidence t h a t the  f o r m a t i o n of 2 - k e t o - 3 - d e o x y sugar a c i d s r e p r e s e n t s a u b i q u i t o u s m e t a b o l i c mechanism i n t h e b a c t e r i a l of carbohydrates.  Thus, the metabolism  compounds as D - g a l a c t o s e D.-arabinose Doudoroff,  of such d i v e r s e 1  (DeLey and D o u d o r o f f ,  ( A s h w e l l , 1957)*  utilization  L-arabinose  1957)*  ( P a l l e r o n i and  1 9 5 6 ) * L g l u c o s a m i c a c i d ( M e r r i c k and Roseman,  1 9 5 8 ) , D-ribose-5-phosphate  (Weissbach  and H u r w i t z , 1 9 5 9 ) *  D - a r a b i n o s e - 5 - p h o s p h a t e ( L e v i n and Racker,  1959)*  D - g l u c u r o n l c and mannonic a c i d s ( A s h w e l l , Wahba, and Hickman, 1 9 5 0 ; Hickman and A s h w e l l , i 9 6 0 ; i960;  C y n k i n and A s h w e l l , i 9 6 0 ) ,  Smiley and A s h w e l l ,  and a l g i n i c a c i d  ( P r e i s s and  A s h w e l l , 1 9 6 2 ) , have been shown t o i n v o l v e the f o r m a t i o n of  analogous i n t e r m e d i a t e s , a l l p o s s e s s i n g t h i s common s t r u c t u r a l feature. In two s e p a r a t e i n s t a n c e s e v i d e n c e has been produced that 2-keto-3-deoxy-sugar  a c i d s p r o v i d e an . i n t e r m e d i a t e  a v a i l a b l e f o r - - a l t e r n a t e b i o s y n t h e t i c pathways.  Shikimic  a c i d has been:shown t o r e s u l t f r o m t h e c y c l i z a t i o n o f 2 - k e t o 3 - d e o x y - h e p t o n i c a c i d (Weissbach and H u r w i t z , 1 9 5 9 ) * s i m i l a r l y , c* - k e t o g l u t a r a t e has been found t o a r i s e L-arabinose through the Intermediate arabonic acid III.  from  2-keto-3-deoxy-L-  (Weimberg, 1 9 5 9 ) .  Oxidative Assimilation The e a r l y concept o f the phenomenon o f o x i d a t i v e  a s s i m i l a t i o n was c o n s i d e r e d t o be t h e i n c o r p o r a t i o n o f s t r i c t l y n o n - n i t r o g e n o u s o r g a n i c compounds i n t o endogenous s t o r a g e p r o d u c t s d u r i n g t h e o x i d a t i o n o f carbon compounds ( B a r k e r , - 1 9 3 6 ; ' Macrae and W i l k i n s o n , 1 9 5 8 ) .  However, the  r e s t r i c t i o n o f t h i s concept t o t h e i n c o r p o r a t i o n o f nonn i t r o g e n o u s s u b s t a n c e s was shown t o be l i m i t i n g when i t was found t h a t ammonia was a p r o d u c t o f e n d o g e n o u s , r e s p i r a t i o n i n P. a e r u g i n o s a and t h a t r e i n c o r p o r a t i o n o f t h i s p r o d u c t i n t o - c e l l u l a r m a t e r i a l was i n d u c e d by t h e a d d i t i o n o f an o x i d i z a b l e c a r b o h y d r a t e (Warren, E l l s and Campbell,  i960).  T h i s o b s e r v a t i o n was extended t o o t h e r m i c r o o r g a n i s m s (Gronlund and Campbell, 1961)  and was suggested as a g e n e r a l  phenomenon o c c u r r i n g w i t h a l l b a c t e r i a t h a t have the a b i l i t y  t o u t i l i z e ammonia as a n i t r o g e n  source.  R a t h e r than.accumulate carbonaceous m a t e r i a l cellular^,  P. a e r u g i n o s a  <*-ketoglutarate  was  intra-  shown t o accumulate  e x t r a c e l l u l a r l y d u r i n g the o x i d a t i o n of  14  g l u c o s e - U-C  by r e s t i n g c e l l s .  The  u t i l i z a t i o n of  e x t r a c e l l u l a r <X - k e t o - a c i d c o i n c i d e d w i t h the  the  Incorporation  of ammonia and r a d i o a c t i v i t y i n t o c e l l u l a r c o n s t i t u e n t s — p r i n c i p a l l y p r o t e i n (Duncan and  Campbell, . 1 9 6 2 ) .  This  work has been extended t o i n c l u d e a v a r i e t y of b a c t e r i a , and  although  o<-ketoglutarate  accumulation during  glucose  o x i d a t i o n would seem t o be a w i d e s p r e a d phenomenon ( A s i a , A i d a , S u g i s a k i and Y a k e i s h i , 1955;  P e r r y and Evans, i 9 6 0 ) .  Tomlinson and Campbell ( 1 9 6 3 ) * have shown t h a t the g e n e r a l a s s i m i l a t i o n p a t t e r n may be s i m i l a r but the. a c c u m u l a t i n g i n t e r m e d i a t e need not be < x - k e t o g l u t a r a t e , and the f a i l u r e t o accumulate an o x i d i z a b l e i n t e r m e d i a t e  r e s u l t s i n low  14 l e v e l s of C and  . a s s i m i l a t i o n i n c e r t a i n s t r a i n s of  Azotobacter•studied The  little  Acetobacter  (Tomlinson and Campbell, 1 9 6 3 ) ,  a s s i m i l a t i o n of h e x o n i c a c i d s has  a t t e n t i o n although  received  C l a r i d g e and Werkman ( 1 9 5 3 ) * have  acknowledged the e x i s t e n c e of a s s i m i l a t i o n mechanisms i n P. a e r u g i n o s a  o x i d i z i n g 2-KG-u-C  14  1.5  MATERIALS AND METHODS  I.  Organism and Media Pseudomonas a e r u g i n o s a  :  ATCC 9 0 2 7 , an o b l i g a t e  aerobe,.was t h e o r g a n i s m used t h r o u g h o u t t h i s S t o c k c u l t u r e s were m a i n t a i n e d  study.  i n a glucose-ammonium  s a l t s medium a t 6 C a f t e r a t w e n t y - f o u r  hour growth p e r i o d  and these were s t r e a k e d a t r e g u l a r i n t e r v a l s onto g l y c e r o l peptone agar and examined f o r t y p i c a l c o l o n i a l and c e l l u l a r morphology as w e l l as f o r t h e a b i l i t y  t o produce t h i s  organism's c h a r a c t e r i s t i c • p i g m e n t , p y o c y a n i n . C e l l s r e q u i r e d f o r • e x p e r i m e n t a l p r o c e d u r e s were r o u t i n e l y grown i n Roux f l a s k s u s i n g a 1$ f r e s h twentyf o u r hour i n o c u l u m 0.2$ to  i n a medium c o n t a i n i n g 0 . 3 $ NH^H^PO^,  KgHPB^, 0 . 5 ppm i r o n as FeSO^.  The pH was a d j u s t e d  7 . 4 and g l u c o s e and MgS0^7Hg0 were added a f t e r  z a t i o n , from 10$ s t o c k s o l u t i o n s , t o g i v e f i n a l  sterili-  concen-  t r a t i o n s o f 0 . 2 $ and 0.1$ r e s p e c t i v e l y . When r e q u i r e d I n the medium 2-KG,  a t f i n a l c o n c e n t r a t i o n s of .0.2  •was s u b s t i t u t e d f o r g l u c o s e .  The k e t o - a c i d was s t e r i l i z e d  by p a s s i n g a 10$ s t o c k s o l u t i o n t h r o u g h Millipore  filter.  or 0 . 6 $ ,  a 0.3  type PH  16 II.  P r e p a r a t i o n of Washed C e l l  Suspensions  C e l l s were h a r v e s t e d by c e n t r i f u g a t i o n a t 3000xg a t 6 C and washed t w i c e w i t h 0.85$ s a l i n e pH 7.4. and the washed c e l l s u s p e n s i o n s were resuspended t o e x a c t l y t e n times the  growth c o n c e n t r a t i o n i n .'tris-(hydroxymethyl)aminomethane  b u f f e r ( T r i s ) pH 7.4.  T h i s corresponded t o an o p t i c a l  d e n s i t y (O.D.) r e a d i n g o f 13.5/ml- a t 650mjLC  i n a Beckman  model B spectrophotometer and a p p r o x i m a t e l y 5ragd r y weight of  cells/ml.  I I I . • Preparation of C e l l - f r e e Extracts 1.  Sonic  oscillation M  H a r v e s t e d washed c e l l s were resuspended  in  Tris  b u f f e r (pH 7.4) t o a f i n a l c o n c e n t r a t i o n o f 40 times growth ( a p p r o x i m a t e l y 2 0 mg,. of' d r y weight o f c e l l s p e r m l ) .  This  c o n c e n t r a t e d c e l l s u s p e n s i o n , o f not l e s s than 1 0 ml, was s u b j e c t e d t o 5 minutes o f s o n i c o s c i l l a t i o n  i n a 1 0 Kc.  Raytheon o s c i l l a t o r w h i c h was f o l l o w e d by c e n t r i f u g a t i o n a t 6 C f o r 1 0 minutes a t 3000xg t o remove whole c e l l s . The. r e s u l t a n t s u p e r n a t a n t f l u i d was removed and kept on i c e . 2 . French press C e l l s were h a r v e s t e d and washed as d e s c r i b e d p r e v i o u s l y and resuspended a t 40 times growth c o n c e n t r a t i o n i n ^ T r i s b u f f e r (pH 7.4). m e r c i a l deoxynuclease  Three t o . f o u r drops o f a com-  (DNase)„ s o l u t i o n ( l mg/ml) were  mixed w i t h t h e c e l l s and t h e c y l i n d e r o f t h e F r e n c h p r e s s u r e cell  ( M i l n e r , Lawrence, and F r e n c h , 1950) was charged w i t h  the c e l l s u s p e n s i o n .  An a p p l i e d p r e s s u r e o f 15-18000 pounds  p. s. i . was a f f e c t e d w i t h a C a r v e r hand-operated press. for  hydraulic  The r e s u l t i n g p r e s s a t e was c e n t r i f u g e d a t 3000xg  10 m i n u t e s . t o remove whole c e l l s and t h e s u p e r n a t a n t  f l u i d was removed and s t o r e d on i c e . 3. Hughes' p r e s s Washed c e l l p e l l e t s were packed i n t o open-ended p y r e x v i a l s o f 11 mm .in d i a m e t e r .  The v i a l s were t i g h t l y  s e a l e d w i t h r u b b e r s t o p p e r s and-these  c e l l s were q u i c k -  f r o z e n i n an e t h a n o l d r y - i c e b a t h f o r 30 minutes.  The  c e l l - f r e e e x t r a c t s were p r e p a r e d i m m e d i a t e l y by c r u s h i n g . the c e l l s i n a Hughes' p r e s s (Hughes, 1951)* p r e v i o u s l y c o o l e d t o -22 C, by a p p l y i n g a p r e s s u r e o f a p p r o x i m a t e l y 12,000.pounds p . . s . i . w i t h a C a r v e r h y d r a u l i c p r e s s . The p r e s s a t e was d i l u t e d w i t h c o l d |r T r i s b u f f e r (pH 7.4) a t a r a t i o o f 1:6 v/v t o y i e l d an approximate 200 mg wet weight o f c e l l s / m l .  concentration of  A p p r o x i m a t e l y 3-4 drops o f  DNase were added t o each p r e p a r a t i o n p r i o r t o homogenizing i n a p r e - c h i l l e d Potter-homogenizer.  A f t e r -homogenizing,  the s u s p e n s i o n was s u b j e c t e d t o c e n t r i f u g a t i o n a t 300oxg for  10 minutes a t 5 C and t h e r e s u l t i n g s u p e r n a t a n t  was kept on i c e .  fluid  18 A l l enzyme assays were- c a r r i e d out immediately a f t e r preparation of the c e l l - f r e e IV.  extracts.  Manometric Measurements R e s p i r o m e t r y was c a r r i e d out on washed c e l l  suspensions  and p r e s s a t e s i n | j — T r i s b u f f e r (pH 7.4) a t 30 C'by means of a c o n v e n t i o n a l Warburg a p p a r a t u s .  Experiments a t 20 C  were performed w i t h a r e f r i g e r a t e d Warburg r e s p i r o m e t e r . A r e p r e s e n t a t i v e Warburg r e a c t i o n m i x t u r e i s p r e s e n t e d as follows: Endogenous C e l l s u s p e n s i o n (lOx growth) M/20  T r i s b u f f e r (pH 7.4)  S u b s t r a t e (25;b(M/ml)  Test  1.00.ml .1.00  ml  1.00  ml  1.00  ml  0.20 ml  -  D i s t i l l e d H20  1.00  ml  0.80 ml  20$ KOH  .0.15 ml  0.15 ml  '  V a r i a t i o n s i n p r o c e d u r e , such as t h e a d d i t i o n o f c o - f a c t o r s or m e t a b o l i c i n h i b i t o r s , were accompanied by an a p p r o p r i a t e decrease i n t h e volume.of t h e d i s t i l l e d w a t e r .  In e x p e r i -  ments i n v o l v i n g m e t a b o l i c i n h i b i t o r s , 'a volume of water e q u a l t o t h e s u b s t r a t e volume was added t o t h e s l d e a r m o f the endogenous c o n t r o l cup so t h a t i n h i b i t o r ' c o n c e n t r a t i o n s i n t h e cups p r i o r t o s u b s t r a t e o x i d a t i o n would be i d e n t i c a l . A l l s u b s t r a t e s and i n h i b i t o r s were p r e p a r e d a t c o n c e n t r a t i o n s of 25/JCM/ml o r 50>^;M/ml and were a d j u s t e d t o pH 7.  Calcium  was removed from g l u c o n a t e o r 2-ketogluconate w i t h Dowex 50 H prior to neutralization.  +  V.  S p e c t r o p h o t o m e t r y Enzyme A s s a y s ' Enzyme a s s a y s i n v o l v i n g the o x i d a t i o n and r e d u c t i o n  of p y r i d i n e n u c l e o t i d e s were f o l l o w e d Toy measuring changes in optical  d e n s i t y a t 3^0  mju, w i t h a Beckman model DU  s p e c t r o p h o t o m e t e r or a Beckman model DB spectrophotometer equipped w i t h a V a r i c o r d l i n e a r / l o g  recorder.  W i t h l i m i t i n g p y r i d i n e n u c l e o t i d e c o n c e n t r a t i o n s and excess s u b s t r a t e the r e a c t i o n s were i n i t i a l l y zero  order,  the r a t e o f 'the r e a c t i o n b e i n g dependent on enzyme concentration.  A l l enzyme a c t i v i t i e s were e x p r e s s e d as change i n  O.D. p e r minute p"er mg p r o t e i n of the c e l l - f r e e  extract  p  xlO . R e a c t i o n m i x t u r e s were p r e p a r e d i n q u a r t z  cuvettes  w i t h a 1 cm l i g h t p a t h and a t o t a l volume o f 1 m l . 1. 2-Keto-6-phosphogluconate  reductase  2-K-6-PG~reductase a c t i v i t y was determined by measuring NADPH^ o x i d a t i o n d u r i n g the c o n v e r s i o n o f  •2-K-6-PG t o 6-PG.. The method of R e i d (1959) was used 1  as m o d i f i e d by Gronlund (1961). A t y p i c a l assay was as f o l l o w s :  2-K-6-PG was k i n d l y s u p p l i e d by Dr. F . J . Simpson, P r a i r i e R e g i o n a l L a b o r a t o r y , Saskatoon, Saskatchewan.  20 enzyme control  substrate control  Test  M 5 T r i s - b u f f e r pH 7.4  0.20 ml  0.20 ml  0.20 ml  MgClg (l00>cM/ml)  0.10 ml  0.10 ml  0.10 ml  2K6PG (25,U,M/ml)  -  0.05 ml  0.05 ml  Pressate  0.01 ml  -  D i s t i l l e d HgO  0.67  0.63 ml  0.62 ml  TPNH 0.005 M.  0.02 ml  0.02 ml  0.02 ml  0.01 ml  A l l c o n s t i t u e n t s , w i t h t h e e x c e p t i o n o f t h e s u b s t r a t e , were added t o t h e c u v e t t e and t h e i n s t r u m e n t r e a c t i o n m i x t u r e a t an O.D. o f 0.500.  "zeroed" on t h e The s u b s t r a t e ,  2-K-6-PG, was added a t z e r o t i m e , t h e r e a c t i o n m i x t u r e shaken, and t h e decrease 2.  i n O.D. r e c o r d e d ,  2-ketogluconokinase The procedure used t o measure t h i s enzyme was  dependent on t h e presence o f 2-K-6-PG r e d u c t a s e .  The  s u b s t r a t e 2-KG r e p l a c e d 2-K-6-PG i n t h e 2-K-6-PG-reductase assay and 0.01 ml o f adenosine t r i p h o s p h a t e (ATP) (25ju.M/ml) was i n c l u d e d i n t h e r e a c t i o n m i x t u r e .  The ATP dependent  o x i d a t i o n o f NADPHg a t a r a t e s i g n i f i c a n t l y g r e a t e r - than t h a t o f t h e c o n t r o l would i n d i c a t e a 2-KG k i n a s e a c t i v i t y . 3. 2-keto-3-deoxy-6-phosphogluconate a l d o l a s e T h i s enzyme was measured i n d i r e c t l y by t h e c o n v e r s i o n of p y r u v i c a c i d , a product o f t h e a l d o l a s e r e a c t i o n , t o  21 l a c t i c a c i d accompanied by NADHg o x i d a t i o n i n the of an excess- o f commercial and Wood,-1954).  l a c t i c dehydrogenase  presence  (Kovachevich  The r a t e o f . a c t i v i t y was dependent on  2 f o r m a t i o n o f p y r u v i c a c i d from 2-K-3-D-6-PG i n t e r f e r e n c e by g l y c e r a l d e h y d e - 3 - p h o s p h a t e was  inhibited with iodoacetate.  and any dehydrogenase  The f o l l o w i n g r e a c t i o n  m i x t u r e s were used: enzyme control  substrate control  Test  M/5 T r i s b u f f e r pH 7.4  0.20 ml  0.20 ml 0.20 ml  MgClg (50jLLM/ml)  0.05 ml  0.05 ml 0.05 ml  2-K-3-D-6-PG  0.05 ml 0.05 ml  (25jLlM/ml)  I o d o a c e t a t e ( 1 0 mg/ml)  0.10 ml  0.10 ml 0.10 ml  L a c t i c dehydrogenase ( 1 0 0 mg/ml)  0.02 ml  0.02 ml 0.02 ml  NADHg 0.005 M  0.02 ml  0.02 ml 0.02 ml  Pressate  0.01 ml  Distilled  water  0.60 ml  0.01 ml 0.56 ml 0.55 ml  A l l components, w i t h the e x c e p t i o n o f 2-K-3-D-6-PG, were . added t o the c u v e t t e and the i n s t r u m e n t was "zeroed" a t an O.D. r e a d i n g o f 0.500,  The r e c o r d i n g o f t h e • r a t e o f 0.D,  decrease began a t z e r o time i m m e d i a t e l y f o l l o w i n g r e a c t i o n i n i t i a t i o n by t h e a d d i t i o n o f the s u b s t r a t e .  - 2K3D6PG was-the generous g i f t o f Dr. W. A. Wood, Dept. of A g r i c u l t u r a l C h e m i s t r y , M i c h i g a n S t a t e U n i v e r s i t y .  22 6-phosphogluconate dehydrase  4.  Enzyme a c t i v i t y was measured by u s i n g the  previously  d e s c r i b e d assay for•2-K-6-PG a l d o l a s e w i t h 6-PG as t h e s u b s t r a t e and 0.05 ml o f p r e s s a t e . was  The r a t e o f a c t i v i t y  found t o be dependent on t h e i n i t i a l d e h y d r a t i o n o f  6-PG t o 2-K-3-D-6-PG. VI.  A s s i m i l a t i o n Experiments 1.  I n c u b a t i o n w i t h 2-KG E x p e r i m e n t s i n v o l v i n g the o x i d a t i v e a s s i m i l a t i o n o f  14 C  l a b e l l e d 2-KG were c a r r i e d out i n Warburg  respirometer  v e s s e l s i n order t h a t oxygen consumption c o u l d be f o l l o w e d . A t y p i c a l manometric r e a c t i o n m i x t u r e has been d e s c r i b e d , and a l l experiments were c a r r i e d out at'30°C. were t e r m i n a t e d contents  Reactions  a t s p e c i f i c time i n t e r v a l s by p i p e t t i n g t h e  o f t h e v e s s e l s i n t o i c e c o l d Pyrex c e n t r i f u g e  tubes and t h e tubes were i m m e d i a t e l y c e n t r i f u g e d a t 6 C. The  s u p e r n a t a n t s were removed and kept on i c e or f r o z e n  at -22 C. C e l l p e l l e t s were f r a c t i o n a t e d i m m e d i a t e l y . 2.  F r a c t i o n a t i o n of whole c e l l s The  c h e m i c a l f r a c t i o n a t i o n procedure used was essen-  t i a l l y , t h a t of R o b e r t s e t a l (1955)* w i t h the  modification  by C l i f t o n and Spbek (1961) i n c o r p o r a t e d and t h e h o t t r i c h l o r a c e t i c a c i d (TCA)  s o l u b l e f r a c t i o n was p r e p a r e d by  h e a t i n g the sample i n 5$ TCA f o r 20 minutes a t 90°C r a t h e r  23 t h a n . a t 100°C f o r • 3 0 ' m i n u t e s . represents  The c o l d TCA s o l u b l e  the t r a n s i e n t c e l l Intermediates,  and p o s s i b l y c a r b o h y d r a t e m e t a b o l i s m acid-ethanol  f r a c t i o n represents  fraction  amino-acids,  intermediates;.the  l i p i d s , phospholipids  and  a l c o h o l s o l u b l e p r o t e i n ; the hot TCA. s o l u b l e f r a c t i o n cont a i n s n u c l e i c a c i d s ; and t h e hot T C A , i n s o l u b l e m a t e r i a l contains VII.  t h e b u l k of t h e  cellular protein.  E l e c t r o p h o r e t i c .and chromatographic methods 1.  Electrophoresis Paper • e l e c t r o p h o r e s i s was c a r r i e d out. w i t h .a. water-  c o o l e d e l e c t r o p h o r e s i s apparatus s i m i l a r t o a Resco model E-800-2B equipped w i t h a Resco model 1911 power The  supply.  b u f f e r system r o u t i n e l y used was 0.1 M ammonium  carbonate (NH^HCO^•NHgCOONH^-Analar) pH 8.6 and samples were s p o t t e d  on Whatman #4 f i l t e r paper.  C u r r e n t was a p p l i e d  f o r 1.5 - 2 hours a t maximum v o l t a g e w h i c h v a r i e d between 700 and 750 v o l t s depending on the a r e a o f the paper involved. 2.  •  .  Chromatography a. anion.exchange'column chromatography Formic acid-ammonium formate e l u t i o n from Dowex 1  formate columns .was used t o s e p a r a t e r a d i o a c t i v e m e t a b o l i c intermediate's. .The column was p r e p a r e d i n t h e f o l l o w i n g • manner:  24 the f i n e s were decanted from an aqueous suspension  of the  r e s i n ; t h e r e s i n was packed i n t o a 1 x 15 cm column and washed w i t h 500 ml o f d i s t i l l e d w a t e r ; t h e packed column was washed w i t h s e v e r a l l i t e r s o f 0.5 N NaOH u n t i l o n l y . weakly p o s i t i v e for-CI"", then washed w i t h w a t e r ; t h e column was repacked i n , and washed w i t h , 2.0 N f o r m i c a c i d u n t i l t h e c o n c e n t r a t i o n o f t h e e l u a t e was t h e same as t h e wash a c i d ; t h e column was washed w i t h d i s t i l l e d w a t e r - u n t i l the pH was c o n s t a n t regenerated  (pH 4.5).  A f t e r use, t h e columns were  with 4 N formic a c i d followed with  distilled  w a t e r - u n t i l t h e e l u a t e was pH 4.5. • Reaction  supernatants  o r p e r c h l o r i c a c i d (PCA)  e x t r a c t s were a d j u s t e d t o pH 5.0 p r i o r t o c h a r g i n g t h e column.  The m i x t u r e was washed i n t o t h e column w i t h 10 ml  of d i s t i l l e d water and then e l u t i o n commenced.  Gradient  e l u t i o n s from 0.0 - 1.0 N f o r m i c a c i d ( B a r t l e t t , .1958), and w i t h 4 N f o r m i c a c i d and 0.4 N ammonium formate ( H u r l b e r t , Schmitz, to  separate  Brumm, and P o t t e r , 1954), were used,  intermediates.  Flow r a t e s o f a p p r o x i m a t e l y  1 ml p e r minute were a f f e c t e d and a p p r o x i m a t e l y  10 ml  f r a c t i o n s were c o l l e c t e d w i t h a G i l s o n f r a c t i o n  collector  model T-10. b. paper - chromatography Paper chromatography was r o u t i n e l y performed on r e a c t i o n mixtures  and supernatant  f l u i d s by s p o t t i n g  samples on Whatman No. 4 f i l t e r paper and r u n n i n g the d e s c e n d i n g t e c h n i q u e .  them by  The s o l v e n t systems commonly  employed f o r c a r b o h y d r a t e s were e t h y l a c e t a t e - p y r i d i n e s a t u r a t e d aqueous b o r i c a c i d (60:25.:20 v/v) B a l l o u , 1961), and a c e t o n e - p y r i d i n e - w a t e r .(Szymona? and D o u d o r o f f , i960).  (Grado and (2:1:1 v/v)  Amino a c i d s were s e p a r a t e d  w i t h b u t a n o l - a c e t i c a c i d - w a t e r (60:15"-25 v/v) phenol-ammonia (200:1 v/v) (Smith, . Keto-acid  and w i t h  i960).  2 , 4 - d i n i t r o p h e n y l h y d r a z o n e s were p r e p a r e d I n 2 N HC1  by r e a c t i o n w i t h 2,4-dinitrophenylhydrazine  (2 mg/ml) a t 37 C f o r 30 minutes and t h e d e r i v a t i v e s were e x t r a c t e d i n t o s e v e r a l volumes o f e t h y l a c e t a t e .  These  hydrazones were t h e n e x t r a c t e d i n t o 1 M T r i s pH 11, made a c i d i c , and e x t r a c t e d back I n t o e t h y l a c e t a t e f o r chromato•graphy. The  removal o f phosphate f r o m p h o s p h o r y l a t e d  mediates w a s e c a r r i e d  inter-  out by i n c u b a t i o n w i t h a commercial  b a c t e r i a l a l k a l i n e phosphatase i n pH 8 ^  Tris buffer at  37- C c. d e t e c t i o n r e a g e n t s C a r b o h y d r a t e s were d e t e c t e d  by d i p p i n g chromato-  grams i n 0.12$ m-periodate i n acetone and s p r a y i n g 0.18$ b e n z i d i n e  i n acetone ( C i f o n e l l i ,  sugars were d e t e c t e d  1954).  with  Reducing  by d i p p i n g chromatograms i n 0.5$  AgNO^ i n acetone and d e v e l o p i n g  i n 0.5 N NaOH i n 70$  ethano.l and t h e background c o l o u r was removed w i t h 5$  aqueous NagSgQ^HgO  (Smith, i960).  Organic  d e t e c t e d by s p r a y i n g w i t h 0.1$ b r o m - c r e s o l t o 1/5 c o n c e n t r a t i o n i n a c e t o n e .  diluted  a c i d s were green i n e t h a n o l  K e t o - a c i d s were  d e t e c t e d by s p r a y i n g t h e chromatograms w i t h 2$  orthophenylene-  diamine i n 0.05 N HC1, h e a t i n g t h e chromatograms a t 70-80°C, and o b s e r v i n g t h e q u i n o x a l i n e d e r i v a t i v e s as c h a r a c t e r i s t i c c o l o u r e d compounds and by t h e i r f l u o r e s c e n c e when I r r a d i a t e d w i t h u l t r a - v i o l e t l i g h t (Lanning and Cohen, 1951)• The f o l l o w i n g c o l o u r s were observed: visible  ultra  pyruvate  pink  fluorescent  tf-ketoglutarate  yellow  fluorescent  2 KG  yellow  fluorescent  2K6PG  purple  no f l u o r e s c e n c e  2K3D6PG  yellow-green  fluorescent  standards  violet  Keto-acid 2,4-dinitrophenylhydrazone d e r i v a t i v e s were observed as u l t r a - v i o l e t a b s o r b i n g spots o r as c o l o u r s o b t a i n e d on s p r a y i n g t h e chromatograms w i t h 0.5 N NaOH i n e t h a n o l (Smith, i960).  Phosphate e s t e r s were  d e t e c t e d by s p r a y i n g w i t h 1$ ammonium molybdate I n acetone and d e v e l o p i n g t h e c o l o u r under u l t r a - v i o l e t l i g h t  (Smith,  I960). VIII.  I s o t o p i c Methods U n i f o r m l y - l a b e l l e d glucose-C  14 , g l u c o s e - l - C 14,  1I1  and g l u c o s e - 6 - C  were o b t a i n e d from Merck  and Co. L t d . and were d i l u t e d w i t h n o n - r a d i o a c t i v e s o l u t i o n s so t h a t 5jU'M o f g l u c o s e w i t h a s p e c i f i c of 3.5JX c u r i e p e r 5JU.M was o b t a i n e d 1962).  glucose activity  (Duncan and Campbell,  The p r e p a r a t i o n o f v a r i o u s l y l a b e l l e d 2-KG. i s des-  cribed i n the t e x t . Aliquots of r e a c t i o n mixtures, c e l l  suspensions,  c e l l f r a c t i o n s , and chromatographic e l u a t e s were p l a t e d , i n d u p l i c a t e , o h . s t a i n l e s s s t e e l p l a n c h e t s and d r i e d under an i n f r a - r e d lamp.  R a d i o a c t i v e C0 was determined by q u a n t i 2  t a t i v e l y removing t h e f i l t e r paper and KOH from t h e Warburg r e a c t i o n v e s s e l and r i n s i n g the, c e n t e r w e l l w i t h s e v e r a l volumes o f d i s t i l l e d water- (Gronlund and Campbell, 1964).. The  combined r i n s i n g s , . KOH, and paper were made up t o 5 ml  in a volumetric flask.  The paper and r i n s i n g s were then  mixed i n a t e s t tube w i t h a g l a s s r o d by means o f a v o r t e x mixer.  A l i q u o t s were p l a t e d i n q u a d r u p l i c a t e and d r i e d  as above.  P l a n c h e t s were counted a t i n f i n i t e  a Nuclear-Chicago automatic tube.  thinness using  s c a l e r model l 8 l A equipped w i t h an  gas-flow.counter- h a v i n g a thin-end-window  C o r r e c t i o n s were made f o r background.  statistical  Geiger  To reduce  d e v i a t i o n a t l e a s t 1000 counts were  recorded  when p o s s i b l e . R a d i o a c t i v e chromatograms were scanned by r u n n i n g approximately  one-inch  s t r i p s through a  Nuclear-Chicago  Model C 100 B A c t i g r a p h I I w i t h t h e gas f l o w c o u n t e r and a Model 1620 B A n a l y t i c a l Count r a t e m e t e r chart recorder.  equipped w i t h a  28 IX.  A n a l y t i c a l Methods 1.  Ammonia d e t e r m i n a t i o n s The ammonia i n the s u p e r n a t a n t f l u i d s from r e a c t i o n  m i x t u r e s was determined-by t h e Conway m i c r o d i f f u s i o n method (Conway, 1950).,  Under t h e c o n d i t i o n s employed,  v a l u e s from 0.0 - 0.75_>U.M p e r sample c o u l d be measured. 2.  Glucose  determinations  Glucose c o n c e n t r a t i o n s i n r e a c t i o n m i x t u r e s were measured by t h e " g l u c o s t a t " method o f 'Worthington Corp.  Biochemical  The enzymatic r e a c t i o n o f added g l u c o s e o x i d a s e w i t h  substrate g l u c o s e ' r e s u l t e d i n peroxide production which was measured s p e c t r o p h o t o m e t r i c a l l y i n t h e presence o f o r t h o - d i a n i s i d i n e and p e r o x i d a s e . the.range 3.  Glucose was measured i n  0.1 - 1.0JAM p e r sample. Chemical d e t e r m i n a t i o n o f 2-KG  2-KG was measured i n r e a c t i o n s u p e r n a t a n t s by t h e method o f I a n n i n g and Cohen (1951), which i s based on t h e f o r m a t i o n o f t h e g u i n o x a l i n e d e r i v a t i v e on r e a c t i o n w i t h o-phenylenediamine.  The assay p e r m i t s a measurement o f  10 - 100JU gs p e r sample.  Corrections f o r glucose,  p y r u v a t e , and c< - k e t o g l u t a r a t e i n t e r f e r e n c e were made when these compounds were p r e s e n t .  29 4. Enzymatic d e t e r m i n a t i o n s o f k e t o - a c i d s a. L a c t i c dehydrogenase  assay f o r p y r u v a t e  Pyruvate was measured d i r e c t l y by t h e q u a n t i t a t i v e decrease i n O.D. a t 340 my,  caused by t h e o x i d a t i o n o f  'NADHg i n t h e presence of commercial l a c t i c dehydrogenase. . Under t h e c o n d i t i o n s , employed 0.02  - O.lOjUM o f p y r u v i c  a c i d p e r sample c o u l d be measured. B. G l u t a m i c dehydrogenase G l u t a m i c dehydrogenase photometry  assay f o r ©(-ketoglutarate  was used as a s p e c t r o -  assay f o r ©(-ketoglutarate i n t h e presence o f  •excess ammonia, l i m i t i n g  ©(-ketoglutarate and NADHg.  The  enzyme was purchased from N u t r i t i o n a l B i o c h e m i c a l Co.-and. was found t o be r e a d i l y i n a c t i v a t e d i n d i l u t e s o l u t i o n so t h a t 0.005 ml o f c o n c e n t r a t e d enzyme was used. • The s e n s i t i v i t y of t h e assay was t h e same as t h a t of t h e l a c t i c • dehydrogenase very  assay f o r p y r u v a t e and b o t h methods were  specific. 5.  Protein determinations P r o t e i n was determined w i t h t h e P o l i n - p h e n o l r e a g e n t  as d e s c r i b e d by Lowry- e t a l (1951), and i t i s s p e c i f i c f o r • s u b s t i t u t e d a r o m a t i c r i n g s , hence f o r t y r o s i n e , p h e n y l a l a n i n e , and t r y p t o p h a n e r e s i d u e s of p r o t e i n . s t a n d a r d used was egg a l b u m i n .  The p r o t e i n  30 EXPERIMENTAL RESULTS AND DISCUSSION  I.  The  Non-phosphorylated O x i d a t i v e  Pathway of Glucose  Dissimilation ' 1.  I d e n t i f i c a t i o n of p r o d u c t s of g l u c o s e o x i d a t i o n The  a c c u m u l a t i o n of 2-KG from g l u c o s e by a e r o b i c  organisms such as P. a e r u g i n o s a under c o n d i t i o n s o f i n t e n s e a e r a t i o n (Lockwood, Tabenkin, and  Stodola,  194l; Ney, 1948;  H i l l , .1951)* demonstrated, t h a t a n o n - p h o s p h o r y l a t e d o x i d a t i v e mechanism was  a v a i l a b l e t o the c e l l s .  However, as 2-KG I s  an endproduct under these a d v e r s e , s p e c i f i c c o n d i t i o n s i t does not n e c e s s a r i l y f o l l o w t h a t .the k e t o - a c i d i s an i n t e r mediate o f g l u c o s e d i s s i m i l a t i o n under normal c o n d i t i o n s . N o r r i s and  Campbell (1951)* demonstrated t h a t P. a e r u g i n o s a  o x i d i z e d g l u c o s e by way  o f - b o t h g l u c o n a t e and 2-KG  growing under normal p h y s i o l o g i c a l conditions'.  when  These i n t e r -  mediates accumulated i n the growth medium over an e i g h t hour growth p e r i o d .  R e s t i n g c e l l s o f P. a e r u g i n o s a were shown t o  have a s t r o n g c o n s t i t u t i v e a b i l i t y t o o x i d i z e gluconate, The  glucose,  and 2-KG. e v i d e n c e t h a t ' 2-KG  i s a transient  intermediate  d u r i n g g l u c o s e d i s s i m i l a t i o n by growing c u l t u r e s was  corro-  borated i n t h i s i n v e s t i g a t i o n .  the  As an a l t e r n a t i v e t o  s a m p l i n g of growing c u l t u r e s over a p e r i o d o f t i m e , 20 hour carbon l i m i t e d c u l t u r e s o f P. a e r u g i n o s a ( s t a t i o n a r y phase c e l l s ) were p u l s e d w i t h 0.2$ g l u c o s e ,  g l u c o n i c , or 2-KG  and permitted- t o o x i d i z e t h e carbon source f o r 1 hour.  At .  the end of t h i s t i m e , t h e . c u l t u r e s were c e n t r i f u g e d , • t h e s u p e r n a t a n t f l u i d s t r e a t e d b a t c h w i s e w i t h Dowex 50 H  +  and  reduced to. a s m a l l .volume ( a p p r o x i m a t e l y 10 mis) by f l a s h e v a p o r a t i o n a t 40 C.  The s u p e r n a t a n t f l u i d s were a n a l y z e d  by paper e l e c t r o p h o r e s i s and paper chromatography.  Gluconate  and 2-KG were shown t o be i n t e r m e d i a t e s i n g l u c o s e  dissimi-  l a t i o n and 2-KG was i d e n t i f i e d as a n • i n t e r m e d i a t e i n g l u c o n i c a c i d d i s s i m i l a t i o n ; no o t h e r compounds were found t o accumul a t e w i t h any of t h e t h r e e carbon sources (Table 1).  Table 1.  Substrate  Washed  I n t e r m e d i a t e s of g l u c o s e and g l u c o n i c a c i d d i s s i m i l a t i o n by growing c u l t u r e s .  Paper chromatography  Paper e l e c t r o p h o r e s i s  glucose  2-KG,  gluconate.  only gluconate or 2-KG were  Gluconate.  2-KG,  gluconate.  d e t e c t e d by e l e c t r o phoresis  2 -KG  2-KG  s u s p e n s i o n s of g l u c o s e grown c e l l s a c t i v e l y m e t a b o l i z e three substrates ( F i g . l ) .  these  Glucose and g l u c o n i c a c i d were  m e t a b o l i z e d by w a s h e d - c e l l s u s p e n s i o n s a t t h e same r a t e but 2-KG was i n i t i a l l y o x i d i z e d more s l o w l y p r i m a r i l y due t o a 5^10  minute l a g p e r i o d .  600  •A  O  A — •  Glucose  O—o  Gluconate  • — • 2-KG  30  P i g . 1.  60  90 Minutes  120  150  Oxygen uptake by P. a e r u g i n o s a w i t h 5/lM of g l u c o s e , .gluconic a c i d o r 2 - k e t o g l u c o n i c acid.  The  n o n - p h o s p h o r y l a t e d o x i d a t i o n o f g l u c o s e t o 2-KG  by d r i e d c e l l - f r e e p r e p a r a t i o n s P. a e r u g i n o s a by Stokes and  was  f i r s t demonstrated i n  Campbell i n 1951• These c e l l  p r e p a r a t i o n s • w e r e shown t o o x i d i z e g l u c o s e w i t h the uptake of '2 JU. atoms o f oxygen, and  g l u c o n i c a c i d w i t h the uptake o f In each case, 2-KG  lJUatom of oxygen p e r j l i M of s u b s t r a t e . was  i d e n t i f i e d , by paper chromatography as the o n l y  p r o d u c t of the r e a c t i o n s thus s u g g e s t i n g a c c u m u l a t i o n under these c o n d i t i o n s .  end-  quantitative  I d e n t i c a l manometric  d a t a has been obtained, u s i n g c e l l - f r e e e x t r a c t s p r e p a r e d by s o n i c a t i o n ( C l a r i d g e and Werkman, 1963)* and a l s o u s i n g c e l l - f r e e e x t r a c t s p r e p a r e d w i t h a Hughes' p r e s s (Hogenkamp  1956). Hogenkamp a l s o showed t h a t the o x i d a t i o n of gluconol a c t o n e t o 2-KG  was  accompanied - by l^Uatom of oxygen uptake  per jUM of s u b s t r a t e and t h i s was  s u b s t a n t i a t e d by Gronlund  (1961), who demonstrated t h a t the enzymie c o n v e r s i o n l a c t o n e t o the a c i d was observations  magnesium dependent.  of  the  These l a t t e r  agreed w i t h the f i n d i n g s of B r o d i e  and  Lipmann  (1954), who showed t h a t s o n i c a t e d c e l l - f r e e 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 o x i d i z e d g l u c o s e t o g l u c o n i c a c i d by two  d i s t i n c t enzymie r e a c t i o n s ;  aldehyde t o the l a c t o n e ; and l y s i s t o the f r e e acid-.  the o x i d a t i o n of  i t s subsequent enzymie hydro-  Ramakrishnan and  Campbell (1955)*  p a r t i a l l y p u r i f i e d g l u c o n i c dehydrogenase from c e l l s and  the enzyme was  the  sonicated  found t o c a t a l y z e the c o n v e r s i o n •  of g l u c o n i c a c i d t o 2-KG w i t h o u t c o n c u r r e n t  phosphorylation.  N e i t h e r NAD n o r NADP f u n c t i o n e d i n t h i s o x i d a t i o n . The f o r m a t i o n o f 2-KG from g l u c o s e and g l u c o n i c a c i d has been s u b s t a n t i a t e d d u r i n g t h e course  of t h i s  investi-  g a t i o n d u r i n g v a r i o u s attempts t o d e t e c t i n t e r m e d i a t e s o f 2-KG m e t a b o l i s m and t o c o r r e l a t e these w i t h g l u c o s e and g l u c o n a t e metabolism.  The r e a c t i o n s were c a r r i e d out i n  l a r g e Warburg v e s s e l s u s i n g c e l l - f r e e e x t r a c t s , 25^LIM o f g l u c o s e , g l u c o n a t e , o r 2-KG, and 5QZIM o f Mg- . One ++  r e a c t i o n m i x t u r e , w i t h g l u c o s e as t h e s u b s t r a t e , was i n c u bated w i t h o u t added magnesium. The r e a c t i o n s were a l l o w e d t o proceed f o r 90 minutes w i t h , the e x c e p t i o n o f t h e magnesium d e f i c i e n t m i x t u r e was stopped a t 20 m i n u t e s .  which  A l l r e a c t i o n s were t e r m i n a t e d  w i t h PCA and f o l l o w i n g t h e removal of p r o t e i n ^ n u c l e i c a c i d s and excess PCA t h e r e s u l t i n g c l e a r supernatant t r e a t e d batchwise  w i t h Dowex 50 H  +  f l u i d s were  and a n a l y z e d by e l e c t r o -  p h o r e s i s and paper chromatography.  From t h e r e s u l t s  (Table 2) i t can be seen t h a t g l u c o n o l a c t o n e , g l u c o n i c a c i d , and 2-KG were formed from glucose-by  a cell-free  pressate  and t h a t 2-KG was t h e end-product o f t h e r e a c t i o n .  These  r e s u l t s a r e analogous t o those o b t a i n e d by Stokes and Campbell (1951), and by Gronlund (1961).  35 Table 2.  P r o d u c t s o f g l u c o s e , g l u c o n a t e and 2-KG d i s s i m i l a t i o n by c e l l - f r e e e x t r a c t s  Substrate  Paper electrophoresis  Paper chromatography  Glucose  2-KG  Glucose (no magnesium)  2-KG,•gluconolactone, glueonate  Gluconate  2-KG  d e t e c t e d by  2-KG  2-KG  electrophoresis  2.  only  or 2-KG were  P r e p a r a t i o n and i d e n t i f i c a t i o n o f 2-KG-C Consequently,  gluconate  14  an i n v e s t i g a t i o n i n t o t h e time  course  and q u a n t i t a t i o n o f t h e c o n v e r s i o n o f g l u c o s e t o 2-KG by c e l l - f r e e p r e s s a t e s o f P.. a e r u g i n o s a was c a r r i e d out p r i o r t o t h e enzymic p r e p a r a t i o n o f 2-KG-C  14 from  A c e l l - f r e e p r e s s a t e was i n c u b a t e d on t h e Warburg w i t h 5>iM g l u c o s e , T r i s b u f f e r (pH 7.4), i n each o f s e v e r a l Warburg v e s s e l s .  14  glucose-C  apparatus  and lOflM o f MgClg  R e a c t i o n s were t e r m i n -  a t e d a t v a r i o u s time i n t e r v a l s on* t h e a d d i t i o n o f 0.2 ml o f — 1.4N.'1TPCA t o t h e i n c u b a t i o n m i x t u r e , , and a n a l y s e s f o r g l u c o s e , 2-KG, p y r u v a t e  a n d t f - k e t o g l u t a r a t e were performed on t h e  n e u t r a l i z e d supernatant  fluids.  The r e s u l t s a r e g r a p h i c a l l y  demonstrated ( F i g . 2) and i t can be seen t h a t under these c o n d i t i o n s t h e c o n v e r s i o n o f g l u c o s e t o 2-KG was r a p i d and e s s e n t i a l l y q u a n t i t a t i v e thus p r o v i d i n g an e x c e l l e n t means f o r t h e p r e p a r a t i o n o f r a d i o a c t i v e 2-KG f r o m r a d i o a c t i v e glucose.  36  F i g . 2.  Disappearance .of g l u c o s e and f o r m a t i o n of i n a c e l l f r e e ' e x t r a c t of P. a e r u g i n o s a  2-KG  37 Frampton and Wood (1961), used a p a r t i c u l a t e  fraction  o b t a i n e d from a c e l l - f r e e p r e p a r a t i o n o f P. f l u o r e s c e n s t o p r e p a r e a 2-KG-.1-C r  14 and 2-KG-6-C /- 14 from  glucose-1-C  l4  lb  and glucose-o-C  r e s p e c t i v e l y . T h e i r r e a c t i o n s were t e r -  minated a f t e r ljU.M o f oxygen/uM o f s u b s t r a t e had been consumed, and the p a r t i c u l a t e m a t t e r was removed by c e n t r i fugation.  The c l e a r s u p e r n a t a n t l i q u i d was heated and used  d i r e c t l y as the source of l a b e l l e d 2-KG and no attempt a t c o n f i r m a t o r y i d e n t i f i c a t i o n was mentioned. The procedure used i n t h i s work employed  a cell-free  p r e s s a t e o f P. a e r u g i n o s a and r e a c t i o n m i x t u r e s were p r e p a r e d and t r e a t e d as d e s c r i b e d p r e v i o u s l y .  The r e s u l t a n t  p r o t e i n - f r e e n e u t r a l i z e d s u p e r n a t a n t f l u i d was a p p l i e d t o a Dowex 1-formate column w h i c h was e l u t e d by the l i n e a r •gradient t e c h n i q u e w i t h 500 ml o f d i s t i l l e d water and 500 ml of f o r m i c a c i d . for  Ten ml a l i q u o t s were c o l l e c t e d . a n d assayed  r a d i o a c t i v i t y and UV a b s o r b i n g m a t e r i a l ( F i g . 3)* and  the a p p r o p r i a t e t u b e s , . w h i c h i n almost a l l cases c o n s t i t u t e d the o n l y major r a d i o a c t i v e peak, were p o o l e d and e v a p o r a t e d t o d r y n e s s s e v e r a l t i m e s a t 40 C w i t h a f l a s h e v a p o r a t o r t o remove the f o r m i c a c i d .  The r e s i d u e was resuspended i n  w a t e r , e x t r a c t e d w i t h s e v e r a l volumes o f e t h y l - e t h e r t o remove any t r a c e s o f f o r m i c a c i d , e v a p o r a t e d t o d r y n e s s , resuspended —  i n d i s t i l l e d w a t e r , and d i l u ^ t e d t o the d e s i r e d s p e c i f i c • a c t i v i t y w i t h n o n - r a d i o a c t i v e 2-KG.  N e g l i g i b l e amounts o f  r a d i o a c t i v i t y were e x t r a c t e d i n t o the e t h e r .  Radioactivity  12,5  14 O--O-0.D. at 26 0  2.0 E O  1.5  iO  CM  1.0  /  Q5  o  90 40 50 60 70 80 FRACTION No. a4 compounds and U.V. a b s o r b i n g m a t e r i a l sequence of. 30  P i g . 3.  SlulslGn^ from"a Dowex 1-formate a n i o n exchange  ad  '00  r  column'  oo co  r e c o v e r i e s were 95-100$ and  2-KG-C  y i e l d s v a r i e d from  85-95$ depending l a r g e l y upon the p r e s s a t e  u s e d . The  r a d i o a c t i v e p r o d u c t was i d e n t i f i e d c o n c l u s i v e l y as 2-KG by i t s p o s i t i o n of e l u t i o n from the column, e l e c t r o p h o r e t i c m o b i l i t y a t pH 8, and by paper chromatography o f b o t h the f r e e a c i d and i t s 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e d e r i v a t i v e (Table 3). II.  Oxidative- A s s i m i l a t i o n o f 2-Ketogluconate The  o x i d a t i v e a s s i m i l a t i o n o f g l u c o s e by  P. a e r u g i n o s a has undergone thorough i n v e s t i g a t i o n by Duncan and Campbell (1962), who.showed, u s i n g  glucose-U-C ^, 1  14 that a.large  proportion  of assimilated C  was i n c o r p o r a t e d  i n t o p r o t e i n a c e o u s m a t e r i a l v i a the r e i n c o r p o r a t i o n o f accumulated ( X - k e t o g l u t a r a t e ammonia.  and endogenously  supplied  I n the presence o f added ammonia i t was found  t h a t a s s i m i l a t i o n was g r e a t l y I n c r e a s e d , no ctf-ketoglutarate  14 accumulated i n the s u p e r n a t a n t and the time course o f C i n c o r p o r a t i o n was markedly s h o r t e n e d . these observations  The t e n a b i l i t y o f  was r e i n f o r c e d by the e x t e n s i o n  work t o o t h e r s p e c i e s  of t h i s  o f Pseudomonas and a l s o Achromobacter  s p e c i e s but a t t e m p t s t o r e v e a l o t h e r key  intermediate  sub-  s t a n c e s t h a t were "pacemakers" f o r g l u c o s e o x i d a t i o n and a s s i m i l a t i o n i n P. a e r u g i n o s a were V o n - T i g e r s t r o m and  unsuccessful.  Campbell (1965)* have demonstrated t h a t  p y r u v a t e a l s o accumulated i n . t h e Warburg s u p e r n a t a n t a l t h o u g h a t a much e a r l i e r time than  rX-ketoglutarate.  fluid  40 T a b l e 3.  Compound  14 I d e n t i f i c a t i o n of• 2-KG-U-C as t h e end-product of t h e enzymie o x i d a t i o n of g l u c o s e - U - C l 4 Paper chromatography Electrophoresis R g . B . P . O.P.D. . 2,4-DNPH(Rf) ' (Re)  Glucose  1.00  + absorbing  2-KG  0.51  + fluorescent  Gluconate  0.31  +  0.19  Gluconolactone  0.36  +  0.19  G-6-P  0.12  + absorbing  0.23  6-P-G  0.09  +  0.33  Ribose-5phosphate  0.10  +  Pyruvate  1.00  oh k e t o g l u t a r a t e 0.53 Reaction product  B.P.  . 0.51  0  0.41  -  - fluorescent - fluorescent  0.19  0.26  0.72,0.56 0.23  + fluorescent  0.41  0.19  . - Benzidene-periodate  O.P.D.  - Orthophenylenediamine  2,4-DNPH  - 2,4-Dintrophenylhydrazone  Rg  - r e l a t i v e t o glucose  Re  - r e l a t i v e t o the length of the electrophoretogram  41 P y r u v a t e , u n l i k e o < - k e t o g l u t a r a t e , d i d not appear t o be i m p o r t a n t as a p r i m a r y a s s i m i l a t i o n compound.because i t a l s o accumulated i n the presence o f added.ammonia and d u r i n g growth i n non-nitrogen limiting.-media. S i n c e 2-KG,  p y r u v a t e and o<-ketoglutarate a l l have a  common s t r u c t u r a l f e a t u r e , i t was  o f i n t e r e s t t o determine  whether or not 2-KG c o u l d s e r v e i n a c a p a c i t y s i m i l a r t o o<-ketoglutarate d u r i n g o x i d a t i v e a s s i m i l a t i o n .  I t was  also  14 of i n t e r e s t t o compare the a s s i m i l a t i o n of 2-KG-U-C  with  14 the w e l l e s t a b l i s h e d p a t t e r n o b t a i n e d w i t h glucose-U-C •  in  an attempt t o demonstrate p o s s i b l e d i v e r g e n c e s i n g l u c o s e m e t a b o l i s m r e l a t i v e t o 2-KG metabolism.  The r a t e of 2-KG  d i s a p p e a r a n c e , oxygen u p t a k e , and the appearance  o f endo-  g e n o u s l y produced ammonia d u r i n g the o x i d a t i o n of '2-KG by washed c e l l s u s p e n s i o n s were measured ( F i g . 4a).  Pyruvate  and o c - k e t o g l u t a r a t e were measured e n z y m i c a l l y but n e g l i g i b l e amounts accumulated i n the s u p e r n a t a n t f l u i d s . son w i t h the p a t t e r n e s t a b l i s h e d ' b y Duncan and  I n compariCampbell  (1962), i t may be seen t h a t the d i s a p p e a r a n c e of 2-KG was not complete u n t i l a p p r o x i m a t e l y 60 m i n u t e s , whereas g l u c o s e was  found t o d i s a p p e a r by 30 m i n u t e s .  Of more importance i s  the apparent i n a b i l i t y o f the organism t o accumulate k e t o a c i d s , s p e c i f i c a l l y o<-ketoglutarate, under t h e s e c o n d i t i o n s . T h i s o b s e r v a t i o n s h a r p l y c o n t r a s t s the f i n d i n g s of VonT i g e r s t r o m and Campbell 1.5>i.M of  tf-ketoglutarate  (1963), who found t h a t a p p r o x i m a t e l y and 1.0^u.M of p y r u v a t e accumulated  MINUTES Fig..4.  Time c o u r s e ' o f 2-KG disappearance,, oxygen u p t a k e , and NHo r e l e a s e or disappearance w i t h r e s t i n g c e l l s o f P. a e r u g i n o s a . (A) w i t h o u t added NHo (B) w i t h added  W7.  :  ,  43 i n t h e Warburg•supernate glucose.  during the r a p i d o x i d a t i o n of  The f a i l u r e t o accumulate k e t o - a c i d s from 2-KG  would suggest t h a t t h e r e was a s u p p l y of ammonia a v a i l a b l e w h i c h would p e r m i t immediate  c e l l u l a r u t i l i z a t i o n of  O f - k e t o g l u t a r a t e as i t was formed.  E x p e r i m e n t s w i t h added  ammonia appeared t o i n c r e a s e o x i d a t i v e a s s i m i l a t i o n a s evidenced, by a decrease i n Og uptake b u t d i d n o t enhance 2-KG d i s a p p e a r a n c e , and a g a i n , n e i t h e r o<-ketoglutarate nor p y r u v a t e were found t o accumulate i n t h e Warburg s u p e r n a t a n t f l u i d s ( F i g . 4B) .  The f a i l u r e t o accumulate o c - k e t o g l u t a r a t e  i n t h e presence o f ammonia i s analogous t o t h e s i t u a t i o n d u r i n g g l u c o s e o x i d a t i o n (Duncan and Campbell, 1962), b u t the r e a s o n f o r t h e i n a b i l i t y t o accumulate p y r u v a t e i s n o t o b v i o u s s i n c e p y r u v a t e a c c u m u l a t i o n I s u n a f f e c t e d by added ammonia d u r i n g g l u c o s e o x i d a t i o n by t h i s organism .(Von T i g e r s t r o m and Campbell, 1965). Analogous e x p e r i m e n t s were c a r r i e d out w i t h 2-KG-U-C""^ 1  and t h e r a d i o a c t i v i t y was found i n a l l major c e l l ( T a b l e 4).  fractions  The c o l d TCA s o l u b l e f r a c t i o n appeared t o be  i m p o r t a n t d u r i n g e a r l y s t a g e s o f a s s i m i l a t i o n , and most o f the s u b s t r a t e appeared t o be a s s i m i l a t e d i n t o n i t r o g e n o u s components b u t c o n s i d e r a b l y e a r l i e r than was observed w i t h (Duncan and Campbell, 1962).  glucose-U-C ^ 1  A f t e r 60  minutes i n c u b a t i o n , t h e r a d i o a c t i v i t y i n t h e c e l l s d e c r e a s e d  14/ w i t h a concomitant i n c r e a s e i n C  \  0^ ( F i g . 5A).  T h e decrease  14 in cellular C  was, f o r t h e most p a r t , due t o a . l o s s o f  r a d i o a c t i v i t y from the p r o t e i n f r a c t i o n .  T h i r t y - f i v e percent  of t h e r a d i o a c t i v i t y o f t h i s f r a c t i o n was l o s t d u r i n g t h e  .44  T a b l e 4.  14  Incorporation of C i n t o washed c e l l s  14  from 54M o f 2-KG-U-C  fo o ft o t a l C 14 Hot TCA soluble  Residual protein  Total i n fractions  -  -  13.50  6.20  4.25  11.90  •27.25  5.45  8.00  4.64  15.20  33.29  3.87.  8.44  3.19  11.71  27.16  2.64  6.28  2.78  Time min.  C o l d TCA soluble  20  •3.10  30  4.90  60 90 120  % of t o t a l  Acid alcoholsoluble  -  9.80  20.50  14 C  incorporated into c e l l f r a c t i o n s  . 20  23.0  -  -  -  100  30  18.0  22.8  15.6  43.6  100  6o  . 16.4  24.0  13.9  45.7  100  90  13.5  29.4  ll.l  45.0  . 100  120  12.9  30.7  13.9  48.9  100  100  ^-Supernatant fluid  30  F i g . 5.  60  90  (A)  A - S u p e r n a t ant (B) fluid  120 MINUTES  30  14  60  90  Time c o u r s e o f t h e d i s t r i b u t i o n o f C added t o washed c e l l s u s p e n s i o n s a s 2-KG-U-G 4 (A) I n t h e absence o f added. NH^. (B) I n t h e p r e s e n c e o f 5MM added NH^. 1  >  120  46 60-120 min i n t e r v a l .  .  The c o l d TCA s o l u b l e f r a c t i o n and, t o  a l e s s e r -extent, t h e h o t TCA s o l u b l e f r a c t i o n a l s o decreased In r a d i o a c t i v i t y a t t h i s t i m e .  Duncan and Campbell (1962),  observed s i m i l a r • l o s s e s from the p r o t e i n and c o l d TCA f r a c t i o n s b u t not from .the h o t TCA f r a c t i o n .  I n g e n e r a l , a much  g r e a t e r i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o c e l l u l a r c o n s t i -  •' 14 than w i t h glucose-U-C14  t u e n t s was observed w i t h 2-KG-U-C  In f a c t , t h e data o b t a i n e d by Duncan and Campbell (1962), concerning  the e f f e c t s o f  of added ammonia on g l u c o s e -  14 U-C  a s s i m i l a t i o n c l o s e l y corresponds t o that  achieved  14 w i t h 2-KG-U-C  i n t h e absence of added ammonia.  T h i s i s con-  s i d e r e d t o be f u r t h e r evidence f o r the a v a i l a b i l i t y o f ammonia d u r i n g the o x i d a t i o n o f 2-KG.by P. a e r u g i n o s a .  The q u a n t i t y o f  r a d i o a c t i v i t y i n c o r p o r a t e d i n t o the c e l l f r a c t i o n s was t w i c e as g r e a t a t 60 minutes as the maximum a c h i e v e d by Duncan and Campbell (1963)* a t 120 minutes, and t h i s i n c r e a s e was manifested  as a g e n e r a l i n c r e a s e i n r a d i o a c t i v i t y o f a l l  cellular fractions.  The i n c o r p o r a t i o n o f ' r a d i o a c t i v i t y i n t o  the a c i d - a l c o h o l s o l u b l e f r a c t i o n ( l i p i d , p h o s p h o l i p i d , and a l c o h o l . s o l u b l e p r o t e i n ) was a c c u m u l a t i v e  whereas w i t h  g l u c o s e - U - C ^ e x p e r i m e n t s (Duncan and Campbell, 1962), 1  the r a d i o a c t i v i t y was s l o w l y r e d u c e d . When 5jU.M o f ammonia were added t o the r e a c t i o n v e s s e l s , t h e r e was an i n c r e a s e of'36$ i n the t o t a l q u a n t i t y of a s s i m i l a t e d ' r a d i o a c t i v i t y and almost 100$ of t h i s i n c r e a s e appeared i n the p r o t e i n f r a c t i o n .  An even more  pronounced e f f e c t o f exogenous ammonia was t h e r a p i d i t y of C ^ I n c o r p o r a t i o n (Table 5). Duncan and Campbell (1962), 1  47 demonstrated a 110$ i n c r e a s e i n a s s i m i l a t i o n o f r a d i o a c t i v i t y  14 from glucose-U-C  i n t h e presence o f  5juM  exogenous ammonia.  The lower i n c r e a s e o f a s s i m i l a t e d r a d i o a c t i v i t y f r o m  14 2-KG-U-C  i n t h e presence o f  added ammonia emphasizes  the p r o b a b i l i t y o f t h e g r e a t e r a v a i l a b i l i t y o f ammonia i n the presence o f 2-KG. These o b s e r v a t i o n s may be e x p l a i n e d , i n p a r t , by t h e f a i l u r e t o accumulate c x - k e t o g l u t a r a t e under t h e s e c o n d i t i o n s . I f «-ketoglutarate  had been i m m e d i a t e l y i n c o r p o r a t e d  into  c e l l u l a r m a t e r i a l then t h e p r o b a b i l i t y o f complete s u b s t r a t e o x i d a t i o n v i a t h e t r i c a r b o x y l i c c y c l e would have been r e d u c e d . Duncan and Campbell (1962), have demonstrated t h a t chloram-  14 p h e n i c o l markedly reduced C  incorporation into cellular  • m a t e r i a l , and e x p l a i n e d t h i s as a m a n i f e s t a t i o n o f t h e i n a b i l i t y o f t h e c e l l t o s y n t h e s i z e a s p e c i f i c - permease n e c e s s a r y f o r t h e r e i n c o r p o r a t i o n o f accumulated (^-ketoglutarate.  However,.chloramphenicol had a much l e s s e r e f f e c t  on oxygen uptake w i t h 2-KG t h e r e b y s u p p o r t i n g t h e o b s e r v a t i o n t h a t c * - k e t o g l u t a r a t e does n o t accumulate d u r i n g t h e o x i d a t i o n of 2-KG (Table 6).  The u n c o u p l i n g  agent,.sodium a z i d e ,  had more than t w i c e t h e s t i m u l a t o r y e f f e c t on oxygen uptake w i t h 2-KG than w i t h g l u c o s e .  This substantiates the greater  14 q u a n t i t a t i v e a s s i m i l a t i o n observed w i t h 2-KG-U-C  relative  14 to  glucose-U-C The p o s t u l a t i o n t h a t more ammonia i s a v a i l a b l e  d u r i n g 2-KG o x i d a t i o n than g l u c o s e o x i d a t i o n by washed c e l l s u s p e n s i o n s has been s u p p o r t e d by i n d i r e c t e v i d e n c e .  48  Table 5.  I n c o r p o r a t i o n of" C from 5jHM;<2-KG-U-C into washed . c e l l s . .In the presence, of. 5_HM NH^Cl. % of t o t a l c  Time  C o l d TCA soluble  15  5.93  8.52  60  4.64  . 90  4.16  io o f t o t a l  A c i d a l c o h o l - Hot TCA soluble soluble  Residual protein  Total i n fractions  2.91  13.20  30.56  8.85  4.54  27.55  45.30  9.32  4.09  22.05  39.62  14 cell fractions C incorporated into  15  19.4  27.9  9.5  43.2  100  60  10.3  19.5  10.0  60.7  • 100  90  10.4  23.5  10.30  55.7  100  49  Table 6.  The e f f e c t of c h l o r a m p h e n i c o l and sodium a z i d e on t h e oxygen consumption, by washed c e l l suspensions i n t h e presence of 5>iM g l u c o s e o r •2-KG  fo change i n 0 uptake based on 2-KG  Substrate 5JUM  Assimilation inhibitor  Glucose  chloramphenicol 6.2 x lO"* M  -14.5  2-KG  chloramphenicol  -7.8  Glucose  NaN3 5 x 10-3 M  + 4.8  2-KG  NaN  5 x 10" M  +1=2.2  p  4  6.2  x 10-4'M  3  3  * T h i s c a l c u l a t i o n was based on t h e assumption t h a t a l l g l u c o s e was m e t a b o l i z e d v i a 2-KG and t h e r e f o r e 2 atoms of oxygen/UM o f g l u c o s e was s u b t r a c t e d p r i o r t o c a l c u l a t i o n o f % change.  P r o t e i n and RNA have been e s t a b l i s h e d as s u b s t r a t e s o f endogenous r e s p i r a t i o n i n P. a e r u g i n o s a  (Gronlund and  Campbell, 1963), and when'these c e l l u l a r components were  14  s p e c i f i c a l l y l a b e l l e d the l a b e l was shown t o appear as C  0^  d u r i n g i n c u b a t i o n o f the c e l l s i n the absence o f exogenous substrate.  The e f f e c t s o f v a r i o u s exogenous s u b s t r a t e s on  14 the r e l e a s e of'C  Og f r o m s p e c i f i c a l l y l a b e l l e d c e l l s was  determined (.Gronlund and Campbell, 1965).  I t was .found  t h a t 2-KG, a s an exogenous s u b s t r a t e , s t i m u l a t e d the endo14 ^ genous C Og e v o l u t i o n by 150% over the v a l u e o b t a i n e d w i t h exogenous g l u c o s e available.  and p r e s u m a b l y - 1 . 5 5 t i m e s more NH^ was made  However, a l l s u b s t r a t e s i n c l u d i n g g l u c o s e ,  o ( - k e t o g l u t a r a t e , a s p a r t a t e , 2-KG, and adenosine suppressed o f c e l l u l a r RNA' t o v a r i o u s degrees a t 30, 60, 90,  degradation and  120 m i n u t e s , but 2-KG i n c r e a s e d RNA o x i d a t i o n above the  endogenous l e v e l a t 10 minutes - the i n t e r v a l t o the 2-KG o x i d a t i o n l a g .  corresponding  A t t h i s time from 35-123$ more  14 Og was e v o l v e d w i t h 2-KG as the s u b s t r a t e r e l a t i v e t o C 0  other substrates t e s t e d .  These data were i n t e r p r e t e d as  meaning t h a t 2-KG s t i m u l a t e d RNA d e g r a d a t i o n  as w e l l as •  p r o t e i n but the RNA d e g r a d a t i o n  associated  was p r o b a b l y  w i t h the l a g i n oxygen u p t a k e . The  depression  o f oxygen consumption by exogenous  ammonia was more pronounced w i t h 2-KG than w i t h  glucose  as can be seen f r o m the manometric data i n Table 7. Approximately  300$ more oxygen consumption  depression  Table 7.  I n h i b i t o r y e f f e c t of ammonia on oxygen consumption by washed c e l l s u s p e n s i o n s i n t h e ' p r e s e n c e of v a r i o u s s u b s t r a t e s  Substrate . 5,11M  Time minutes  $ Inhibition based on 0 uptake of •2-KG 2  60  16.6  18.0  120.  12.5  12.0  180  3.8  Glucose  2-KG  $ Inhibition  . 6 0  23.8  120  27.0  180  13.5  -  4.0  o c c u r r e d w i t h 2-KG  than w i t h g l u c o s e a t 180. m i n u t e s .  These  14 d a t a support the g r e a t e r C  . incorporation into  cellular  • 14 c o n s t i t u e n t s observed w i t h 2-KG-U-C presence o f added ammonia.  r e a c t i o n s , i n the •  Trace amounts of p y r u v a t e  and  <x-ketoglutarate were d e t e c t e d by chromatography of t h e i r ' 2,4-dinitrophenyl-hydrazones 2-KG cose.  i s , i n f a c t , degraded  thus d e m o n s t r a t i n g t h a t  v i a TCA  i n t e r m e d i a t e s as i s g l u -  No o t h e r d e t e c t a b l e i n t e r m e d i a t e s , such as g l u t a m i c  or g l u c o s a m i c a c i d w h i c h would.be expected as a r e s u l t of the a m i n a t i o n of o < - k e t o g l u t a r a t e o r 2-KG  respectively,  were p r e s e n t i n the s u p e r n a t a n t f l u i d s a f t e r  hydrazone  extraction. III.  I n t e r m e d i a t e s of 2-KG  Metabolism.  A study of the pathways of metabolism of a compound must,be made w i t h extreme c a r e and w i t h as many d i f f e r e n t t e c h n i q u e s as i s n e c e s s a r y t o c o n c l u s i v e l y s u b s t a n t i a t e the v a l i d i t y o f " i n t e r m e d i a t e s and/or of f u n c t i o n i n g enzyme systems.  The f a c t t h a t a l i v i n g  cell  does p o s s e s s and u t i l i z e a p a r t i c u l a r pathway s h o u l d be demonstrated  by the i s o l a t i o n and i d e n t i f i c a t i o n of  i n t e r m e d i a t e s , and t h i s approach may  be f a c i l i t a t e d by the  use of v a r i o u s a v a i l a b l e m e t a b o l i c i n h i b i t o r s . r e q u i s i t e enzymes or enzyme systems s h o u l d be  The demonstrated  i n c e l l - f r e e e x t r a c t s o r , I f p o s s i b l e , i n whole c e l l s and  the  r e l a t i v e a c t i v i t i e s measured. . . F i n a l l y , l a b e l l e d s u b s t r a t e s  and t h e i s o l a t i o n of key i n t e r m e d i a t e s may be used t o o b t a i n i n f o r m a t i o n on t h e r o u t e t h r o u g h w h i c h t h e d i f f e r e n t i n t e r mediates have p a s s e d .  The use o f o n l y one o f these  approaches may g i v e erroneous answers as Racker (1954), has emphasized.' The p r e v i o u s a p p l i c a t i o n  o f these b a s i c p r i n -  c i p l e s t o t h e problem o f 2-KG m e t a b o l i s m i n P. a e r u g i n o s a has met w i t h r e l a t i v e l y l i t t l e c e l l - f r e e e x t r a c t s prepared  success.  R e i d (1959)* u s i n g  w i t h a Hughes p r e s s , was 1  u n s u c c e s s f u l i n . f i n d i n g i n t e r m e d i a t e s and key'enzymes i n v o l v e d c o u l d n o t be demonstrated, w i t h t h e e x c e p t i o n o f a r e l a t i v e l y weak 2-K-6-PG r e d u c t a s e . (1953)* concluded  C l a r i d g e and Werkman  t h a t c e l l - f r e e e x t r a c t s o f t h i s organism  were i n c a p a b l e o f • m e t a b o l i z i n g 2-KG and t h e r e f o r e i n v e s t i -  •14 gated t h i s p r o b l e m w i t h whole c e l l s , and 2-KG-U- C However, no key s i x carbon i n t e r m e d i a t e s were i d e n t i f i e d b u t v a r i o u s TCA i n t e r m e d i a t e s and amino a c i d s were i s o l a t e d and identified.  G l u c o s e - 6 - p h o s p h a t e was i s o l a t e d and i t s '  e x i s t e n c e was j u s t i f i e d by t h e p r e s u m p t i o n o f e i t h e r a reversal  of d i r e c t o x i d a t i o n , which I s very u n l i k e l y , or  by a r e v e r s a l  of g l y c o l y s i s .  Gronlund (1961), c o n f i r m e d t h e  presence of t h e enzyme 2-K-6-PG r e d u c t a s e , and i s o l a t e d g l u c o s e - 6 ~ p h o s p h a t e from a n a e r o b i c  2-KG r e a c t i o n s w i t h  c e l l - f r e e e x t r a c t s and ATP, and suggested t h a t t h e s u b s t r a t e has been d i s s i m i l a t e d v i a t h e pentose phosphate pathway. Two o t h e r compounds were i s o l a t e d and were t e n t a t i v e l y . i d e n t i f i e d as.3-phosphoglyceraldehyde and  3-phosphoglyceric  a c i d from t h e i r m o b i l i t i e s on e l e c t r o p h o r e s i s . The  5  4  e x i s t e n c e o f these t r i o s e s as i n t e r m e d i a t e s of 2-KG metabolism was c o n s i d e r e d f e a s i b l e i n l i g h t o f t h e i d e n t i f i c a t i o n o f G-6-P and t h e e x i s t e n c e o f t h e pentose phosphate pathway. 1.  I d e n t i f i c a t i o n of i n t e r m e d i a t e s of 2-KG d i s s i m i l a t i o n w i t h whole c e l l s S i n c e c e l l - f r e e e x t r a c t s have proved t o be r e l a t i v e l y  i n e r t toward 2-KG i t was d e c i d e d t o c o n t i n u e t h e I n v e s t i g a t i o n u s i n g whole c e l l s u s p e n s i o n s .  R e a c t i o n s were c a r r i e d  out i n .large Warburg' cups w i t h 25/lM o f 2-KG as t h e s u b s t r a t e . Cups were removed a t v a r i o u s time i n t e r v a l s and t h e r e a c t i o n s were t e r m i n a t e d by p i p e t t i n g t h e cup c o n t e n t s i n t o i c e - c o l d c e n t r i f u g e tubes and t h e Warburg s u p e r n a t a n t f l u i d s were o b t a i n e d by c e n t r i f u g a t i o n .  Hydrazones were p r e p a r e d and t h e  s u p e r n a t a n t f l u i d s were c o n c e n t r a t e d and s u b j e c t e d t o chromatographic experiments  and e l e c t r o p h o r e t i c a n a l y s i s .  Anaerobic  w e r e . c a r r i e d out s i m i l a r l y , under an atmos-  phere o f Ng.  I n one s e t o f r e a c t i o n m i x t u r e s t h e r e a c t i o n  was c a r r i e d out i n a n e u t r a l i z e d h y d r a z i n e s u l f a t e b u f f e r i n o r d e r t o t r a p any s h o r t - l i v e d t r a n s i e n t ketone o r aldehyde Intermediates  (Meyerhof and J u n o w i c z - K o c h o l a t y ,  the 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e and analyzed..  19^3)* and  d e r i v a t i v e s were p r e p a r e d  The r e s u l t s a r e t a b u l a t e d i n Table 8.  No  i n t e r m e d i a t e s o t h e r than p y r u v a t e and «-ketoglutarate c o u l d be demonstrated and, i n t h e case of r e a c t i o n s c o n t a i n i n g the t r a p p i n g agent, h y d r a z i n e , l a r g e amounts o f , t h e s e  55 Table 8.  P r o d u c t s of 2-KG d i s s i m i l a t i o n by washed c e l l s u s p e n s i o n s of P. a e r u g i n o s a  R e a c t i o n time minutes  Paper Chromatography Supernatant Hydrazone  Paper Electrophoresis  Endogenous  10  2-KG  pyruvate  2-KG  or g l u c o n a t e  2-KG  or g l u c o n a t e  2-KG  or g l u c o n a t e  20 30  pyruvate, <X-ketoglutarate  40  pyruvate, rx-ketoglutarate  Anaerobic 10  2-KG  -  "  ' t r a c e of pyruvate  10  2-KG  pyruvate  20  •"  pyruvate, cx-ketoglutarate  20  •.  Hydrazine  Gluconate and 2-KG have the same e l e c t r o p h o r e t i c m o b i l i t y u n d e r - t h e c o n d i t i o n s employed.  56 k e t o - a c i d s accumulated.  Traces o f p y r u v a t e were d e t e c t -  a b l e I n the a n a e r o b i c r e a c t i o n m i x t u r e s but i t c o u l d not u n e q u i v o c a l l y be concluded t h a t a s e r i e s o f n o n - o x i d a t i v e s t e p s were f u n c t i o n i n g t o g i v e r i s e t o these i n t e r m e d i a t e s due t o the p o s s i b i l i t y o f s m a l l amounts of 'Og c o n t a m i n a t i o n . Prom these experiments mediates  i t was concluded t h a t any i n t e r -  t h a t form d u r i n g the d i s s i m i l a t i o n o f 2-KG must  be e x t r e m e l y t r a n s i e n t and do not accumulate cellularly.  extra-  T h e r e f o r e s p e c i a l t e c h n i q u e s had t o be employed  t o promote the a c c u m u l a t i o n o f i n t e r m e d i a t e s . 2.  I d e n t i f i c a t i o n o f i n t e r m e d i a t e s o f 2-KG d i s s i m i l a t i o n i n the presence  • A l t h o u g h 'the assumption  of i n h i b i t o r s t h a t one can prove o r  d i s p r o v e the e x i s t e n c e o f o p e r a b l e m e t a b o l i c pathways by the use o f i n h i b i t o r s has been open t o much c r i t i c i s m (Racker, 1954), the use o f these agents has found u s e f u l a p p l i c a t i o n I n carbohydrate b i o c h e m i s t r y . Doudoroff  E n t n e r and  (l95l)> f o u n d . t h a t the s u b s t r a t e a s s i m i l a t i o n  mechanisms o f P . - s a c c h a r o p h i l a c o u l d be i n h i b i t e d by r e l a t i v e l y weak C o n c e n t r a t i o n s ' o f i o d o a c e t a t e (2 x 10 M), d i n i t r o p h e n o l (2.5 x. 10"  M), and 'arsenite. (2 x 10  M)  and, i n the cases o f the l a t t e r two p o i s o n s , q u a n t i t a t i v e a c c u m u l a t i o n o f p y r u v i c a c i d from g l u c o s e and g l u c o n i c a c i d s u b s t r a t e s was a c h i e v e d .  When g l u c o n a t e -  14 o r g l u c o n a t e - 6 - Cl4 were used as a s u b s t r a t e the  1-C  l a b e l was  found In the c a r b o x y l and methyl carbons r e s a 3:3 s p l i t  p e c t i v e l y of p y r u v a t e , t h e r e b y s u g g e s t i n g mechanism, c o m p l e t e l y  d i f f e r e n t from the  Embden-Meyerhof pathway.  established  A s i m i l a r approach l e d Prampton  and Wood (1961), t o the c o n c l u s i o n t h a t under c o n d i t i o n s of a r s e n i t e p o i s o n i n g  a crude c e l l - f r e e e x t r a c t o f  P. f l u o r e s c e n s c o u l d c o n v e r t  v a r i o u s l y l a b e l l e d 2-KG  q u a n t i t a t i v e l y t o p y r u v a t e , and  subsequent  degradation  s t u d i e s a l s o I n d i c a t e d the presence of a S O ' ^ s p l i t mechanism.  Katznelson  (1958), s t u d y i n g hexose phosphate meta-  b o l i s m i n A. melanogenum, found t h a t a r s e n i t e and f l u o r i d e treated sonicates allowed l a t i o n when 6-PG  was  q u a n t i t a t i v e pyruvate'accumu-  used as a s u b s t r a t e , but  relatively  l i t t l e p y r u v a t e accumulated when a pentose phosphate was used as a s u b s t r a t e . t h a t 6-PG  was  Prom t h i s experiment they c o n c l u d e d  more l i k e l y t o be degraded by the E n t n e r -  D o u d o r o f f r o u t e r a t h e r than by the pentose-phosphate cycle. S e v e r a l i n h i b i t o r s were i n v e s t i g a t e d i n the  course  of t h i s work f o r two r e a s o n s : f i r s t l y t h a t an enzyme i n v o l v e d i n 2-KG d e g r a d a t i o n  might be s e l e c t i v e l y i n h i b i t e d  t h e r e b y a l l o w i n g the a c c u m u l a t i o n of a t l e a s t chromatog r a p h i c amounts of a key i n t e r m e d i a t e ,  and  secondly that  perhaps enough p y r u v a t e c o u l d be accumulated t o a l l o w i n s i g h t I n t o the mechanism of 2-KG d e g r a d a t i o n . e f f e c t of v a r i o u s c o n c e n t r a t i o n s  The  of each i n h i b i t o r was  f o l l o w e d m a n o m e t r i c a l l y and p y r u v a t e a c c u m u l a t i o n was  58determined q u a n t i t a t i v e l y a t each  concentration.employed.  a) sodium a r s e n i t e A r s e n i t e proved- t o be a p o t e n t i n h i b i t o r o f 2-KG o x i d a t i o n w i t h maximum e f f e c t a t c o n c e n t r a t i o n s g r e a t e r than 10JUM/cup as can be seen from F i g u r e 6A. The maximum y i e l d o f p y r u v a t e , 6jLlM, ( F i g . 6B) was a c h i e v e d w i t h 3>IM of was  a r s e n i t e / c u p and a l t h o u g h q u a n t i t a t i v e a c c u m u l a t i o n not a c h i e v e d , subsequent  a n a l y s i s of the supernatant  f l u i d showed t h a t some 2-KG s t i l l remained u n r e a c t e d and . was n o t l i k e l y "to r e a c t under these i n h i b i t e d . c o n d i t i o n s as oxygen uptake had ceased.  These data suggested t h e  e x i s t e n c e o f some type of. a 3 = 3 s p l i t mechanism. :  Of c o n s i d e r a b l e importance wa.s t h e o b s e r v a t i o n t h a t no oxygen consumption  occurred w i t h concentrations of -  i n h i b i t o r e x c e e d i n g 6..QKM b u t some p y r u v a t e was s t i l l found.  These r e s u l t s v e r y s t r o n g l y suggested t h a t a  mechanism f o r t h e d e g r a d a t i o n o f 2-KG t o p y r u v a t e w i t h o u t o x i d a t i o n must be o p e r a t i v e . b) bromopyruvic  acid  Meloche (1965), s t u d i e d t h e e f f e c t o f t h e p r o d u c t analogue, bromopyruvate,  on t h e enzyme 2-K-3-D-6-PG  a l d o l a s e and found i t t o be a p o t e n t i n h i b i t o r .  From  k i n e t i c s t u d i e s , i t was seen t h a t ' a r e v e r s i b l e enzymebromopyruvate complex was formed p r i o r t o enzyme i n a c t i vation.  400  IA)  o Z 300  (/) O U ^  0  0  o  100  50 Fig. Fig.  6A.  100  150 MINUTES  5  10  40 ^m ARSENITE i n t h e AM presence  Oxygen consumption by washed c e l l s u s p e n s i o n s of a r s e n i t e and 2-KG. Endogenous v a l u e s have .been subtracted. 6B. Pyruvate a c c u m u l a t i o n from 5./UM -2-KG by washed c e l l suspensions i n the presence of v a r i o u s c o n c e n t r a t i o n s o f a r s e n i t e  6o Because  o f Meloche's work bromopryuvate was  thought t o be an i d e a l i n h i b i t o r of • 2-K-3-D-6-PG a l d o l a s e i n whole c e l l s o f P. a e r u g i n o s a due- t o i t s p r o b a b l e specificity.  I n h i b i t o r y c o n c e n t r a t i o n s were determined  ( F i g . 7A) and p y r u v a t e a c c u m u l a t i o n was measured ( P i g . 7B) .• A l t h o u g h t h e p y r u v a t e d e t e r m i n a t i o n s were h i n d e r e d somewhat by t h e r e a c t i v i t y of t h e analogue, w i t h l a c t i c dehydrogenase, was  c a r r i e d out.  bromopyruvate,  a f a i r l y accurate determination  A t t h e c o n c e n t r a t i o n s o f bromopyruvate  w h i c h a l l o w e d ' s u f f i c i e n t • o x y g e n consumption.to  account  f o r t h e o x i d a t i o n o f t r i o s e p h o s p h a t e t o p y r u v a t e , almost q u a n t i t a t i v e pyruvate accumulation, detected.  2>lM/ilM  2-KG, was  These r e s u l t s may be e x p l a i n e d by t h e f u n c t i o n -  i n g o f an a c t i v e E n t n e r - D o u d o r o f f pathway as has been demonstrated i n t h i s organism ( G r o n l u n d , 196l). c) sodium  selenite  Enzymes s u s c e p t i b l e t o t h e i n h i b i t o r y e f f e c t s o f s e l e n i t e have b e e n . w i d e l y - i n v e s t i g a t e d ( R o s e n f e l d and B e a t h , 1964)  and i t has been suggested t h a t s e l e n i t e  f u n c t i o n s . b y combining w i t h an a c t i v e group i n t h e enzyme, presumably a s u l f h y d r y l group. 2-KG  I n t h i s i n v e s t i g a t i o n of  metabolism, - s e l e n i t e was found t o be a s t r o n g i n h i -  b i t o r o f 2-KG o x i d a t i o n ( P i g . 8A), e x h i b i t i n g c o n s i d e r a b l e i n h i b i t i o n a t l e v e l s g r e a t e r than 2.5MM/cup and complete i n h i b i t i o n o f oxygen consumption a t 20juM.  However, as i n  the case of a r s e n i t e i n h i b i t i o n , p y r u v a t e was produced  400  12  Br © m o - (A) pyruvate  (B)  UJ  &- _ < 9  >  fit >•  a.  6  3  800 INUTES F i g . 7A. P i g . 7B.  150  15  30 45 BROMOPYRUVATE  Oxygen uptake by washed c e l l - s u s p e n s i o n s i n t h e presence of. s e l e n i t e and 5xM 2-KG. Endogenous v a l u e s have been s u b t r a c t e d . P y r u v a t e a c c u m u l a t i o n f r o m 2-KG by washed c e l l s u s p e n s i o n s i n the p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f bromopyruvate.  100 MINUTES  150  10  20 30 SELENITE  F i g . 8A„; Oxygen uptake by washed c e l l suspensions i n t h e presence o f s e l e n i t e and 5jUM 2-KG. Endogenous v a l u e s have been subtracted. F i g . 8 B . Pyruvate a c c u m u l a t i o n from 2-KG by washed c e l l s u s p e n s i o n s i n t h e presence of v a r i o u s c o n c e n t r a t i o n s o f s e l e n i t e .  63 w i t h o u t concomitant oxygen consumption s u b s t a n t i a t i n g t h e e x i s t e n c e o f an no'n-oxidative m e t a b o l i c r o u t e from 2-KG to pyruvate. d) sodium f l u o r i d e  and iodoacetamide  Much i n f o r m a t i o n has been made a v a i l a b l e by t h e j u d i c i o u s use o f sodium f l u o r i d e  and iodoacetamide from t h e  p o i n t o f view o f I n t e r m e d i a t e a c c u m u l a t i o n i n t h e presence of these substances and as a guide t o an assessment o f pathway i n v o l v e m e n t , p a r t i c u l a r l y g l y c o l y s i s and Q u a s t e l , 1963). fluoride  In t h i s study, n e i t h e r  (Hochster sodium  nor iodoacetamide caused a s i g n i f i c a n t  accumula-  t i o n of p y r u v i c a c i d , s u g g e s t i n g • i n h i b i t i o n o f 2-KG metab o l i s m a t an e a r l i e r s t a g e .  C o n c e n t r a t i o n s o f 900/iM  .sodium f l u o r i d e were - n e c e s s a r y t o c o m p l e t e l y oxygen uptake from 2-KG ( F i g . 9)•  inhibit  Conversely, iodoace-  tamide proved t o be a p o t e n t i n h i b i t o r i n t h a t 10/lM p e r cup reduced oxygen consumption t o 14$ o f t h e c o n t r o l v a l u e ( F i g . 10).  Complete  i n h i b i t i o n o f oxygen  uptake  w i t h iodoacetamide was a d i f f i c u l t t a s k r e q u i r i n g h i g h concentrations of the i n h i b i t o r .  Oxygen uptake v a l u e s  between 50 and 60/1(1 were c o n s t a n t l y encountered w i t h c o n c e n t r a t i o n s up t o 20^M/cup s u g g e s t i n g t h a t perhaps no i n h i b i t i o n a t t h e l e v e l of t r i o s e p h o s p h a t e s t o o k p l a c e .  400  >\M  NaF  o UJ  30  Pig.  9.  60 90 MINUTES  120  15  Oxygen;uptake by washed, ' c e l l suspensions i n the presence of sodium f l u o r i d e and, 5xM 2-KG.. • Endogenous.values have been s u b t r a c t e d .  65  }\M Iodoacetamide  30  F i g . 10.  60  SO  120  150  Oxygen uptake by washed c e l l s u s p e n s i o n s i n the presence o f iodoacetamide and 5><M 2-KG. Endogenous values' have been s u b t r a c t e d .  66 e) e t h y l e n e d i a m i n e t e t r a c e t i c a c i d (EDTA) EDTA was a l s o used as a m e t a b o l i c i n h i b i t o r In an attempt t o i s o l a t e m e t a b o l i c  intermediates.  The  i n h i b i t o r y a c t i o n o f 'EDTA on oxygen consumption was c o n s i s t e n t l y unusual  i n that the i n h i b i t i o n • i n c r e a s e d  r a p i d l y w i t h i n c r e a s i n g EDTA c o n c e n t r a t i o n s between 5 and 20>iM/cup, b u t then a r e v e r s a l o f i n h i b i t i o n a t h i g h e r c o n c e n t r a t i o n s ( P i g . 11).  occurred  No e x p l a n a t i o n has  been found f o r t h i s observed phenomenon. During the i n v e s t i g a t i o n of the e f f e c t of metabolic i n h i b i t o r s on 2-KG metabolism, I t was observed t h a t w i t h every i n h i b i t o r used, w i t h t h e e x c e p t i o n o f EDTA, t h e oxygen uptake a c h i e v e d w i t h g l u c o s e as t h e s u b s t r a t e was always 1 .OjuM/ftM- s u b s t r a t e g r e a t e r - than when 2-KG was t h e substrate.  T h i s suggested t h a t , under i n h i b i t e d  condi-  t i o n s , g l u c o s e was o x i d i z e d q u a n t i t a t i v e l y t o 2-KG.  Sub-  sequent a n a l y s e s demonstrated t h a t 2-KG was an end p r o d u c t of t h e i n h i b i t e d r e a c t i o n s w i t h g l u c o s e . p a r a t i v e a n a l y s e s of p y r u v a t e  accumulation  However, com(Table 8 ( a ) )  showed t h a t c o n s i s t e n t l y g r e a t e r p y r u v a t e y i e l d s were a c h i e v e d when g l u c o s e was used as t h e s u b s t r a t e . demonstrates t h a t a l t e r n a t e r o u t e s o f g l u c o s e  This  metabolism  o t h e r than t h e n o n - p h o s p h o r y l a t e d o x i d a t i v e pathway a r e u t i l i z e d by 'P. a e r u g i n o s a .  These pathways a r e p r o b a b l y  e i t h e r v i a p h o s p h o r y l a t i o n of g l u c o s e by h e x o k i n a s e , p h o s p h o r y l a t i o n of g l u c o n a t e by t h e g l u c o n o k i n a s e , both pathways (Gronlund,  1961).  by  o r by  67  400  EDTA  60 90 MINUTES  P i g . 11.  120  150  Oxygen uptake by washed c e l l s u s p e n s i o n s i n the. presence o f v a r i o u s c o n c e n t r a t i o n s o f 'EDTA and ^JIM. 2-KG. Endogenous v a l u e s have been s u b t r a c t e d . '  68 The  i d e n t i f i c a t i o n of accumulating  metabolic  i n t e r m e d i a t e s was attempted u s i n g l a r g e Warburg cups, . 50/iM 2-KG, and c o n c e n t r a t i o n s o f the v a r i o u s  inhibitors  w h i c h would reduce oxygen consumption t o l e s s than lOOyKJL. Cups were removed a t v a r i o u s time i n t e r v a l s and chromatography was performed as p r e v i o u s l y d e s c r i b e d f o r t h e u n i n h i b i t e d r e a c t i o n ..mixtures.  Pyocyanine, a p h y s i o l o g i c a l  hydrogen a c c e p t o r s y n t h e s i z e d by t h i s organism M a c Q u i l l a n , E a g l e s and Smith, metabolic  (Campbell,  1957)* was a l s o used as a  i n h i b i t o r • (Table 9).  With'the f o u r i n h i b i t o r s  employed o n l y p y r u v a t e , o < - k e t o g l u t a r a t e ,  and the  compound were i s o l a t e d . •In each case, w i t h the  starting exception  o f ' t h e iodoacetamide i n h i b i t e d r e a c t i o n s , p y r u v a t e was p r e s e n t , as expected, tems.  and 2-KG was d e t e c t e d i n a l l s y s -  I t appeared l i k e l y then, t h a t a l t h o u g h t h e  d l s s i m i l a t o r y r e a c t i o n s f o r - 2 - K G u t i l i z a t i o n were f u n c t i o n i n g , t h e observed i n h i b i t i o n was a g e n e r a l r e t a r d a t i o n o f the r a t e o f 2-KG breakdown o r perhaps i n h i b i t i o n , of t h e i n i t i a l s t e p .  .'Either e x p l a n a t i o n  would r e s u l t i n much of t h e 2-KG r e m a i n i n g unreacted.  essentially  Since no o t h e r i n t e r m e d i a r y compounds were  d e t e c t e d by the methods used i t was f e l t t h a t compounds may  be p r e s e n t  o n l y i n minute amounts. 1 4  3. I n h i b i t o r s and 2-KG-U-C  14 V a r i o u s i n h i b i t e d r e a c t i o n s u s i n g 2-KG-U-C the s u b s t r a t e were r u n .  as  Paper chromatography of t h e  69  Table 8(a). P y r u v a t e a c c u m u l a t i o n by washed c e l l s u s p e n s i o n s i n t h e presence of m e t a b o l i c i n h i b i t o r s  Inhibitor  Glucose  Substrate  2-KG  pyruvate a r s e n i t e 3>(M* iodoacetamide selenite  mixture.  20JUM  10JUM  7.32  5.63  2.40  0.00  3.66  1.50  C o n c e n t r a t i o n o f i n h i b i t o r p e r 3 ml r e a c t i o n ,  ;  1  70 Table 9.  P r o d u c t s of 2-KG d i s s i m i l a t i o n by washed c e l l s u s p e n s i o n s i n the presence of v a r i o u s metabolic i n h i b i t o r s Paper chromatography Supernatant Hydrazone ... ° fluid  Inhibitor  . , minutes  Arsenite  20  2-KG  pyruvate  60  2-KG  pyruvate, cx-ketoglutarate  20  2-KG  60  .2-KG  20  2-KG  pyruvate  60  2-KG  pyruvate  20  2-KG  pyruvate  60  2-KG  pyruvate  Iodoacetamide  Pyocyanine  Selenite  1  ' Paper Electrophoresis 2-KG or •gluconic  71 hydrazones  a n d of- t h e u n r e a c t e d s u p e r n a t a n t s was p e r f o r m e d .  P r o m -Table 10 - i t c a n be s e e n t h a t no new i n t e r m e d i a t e s w e r e d e t e c t e d by " t h i s a p p r o a c h . activity  F r o m t h e amount o f r a d i o -  incorporated into the c e l l s  i t was o b s e r v e d  i n most c a s e s a s s i m i l a t i o n h a d b e e n i n t e r r u p t e d c o m p l e t e l y by t h e c o n c e n t r a t i o n s o f i n h i b i t o r s  that  almost used.  The i n h i b i t o r y e f f e c t o f i o d o a c e t a m i d e was u n u s u a l due t o t h e f a c t t h a t no p r o d u c t o f a c c u m u l a t i o n was d e t e c t e d y e t 50JU.L o f o x y g e n was s t i l l radioactivity  consumed.  The  l o s s f r o m t h e s u p e r n a t a n t f l u i d was a c c o u n t e d  • 14 for  as C  Og.  I t i s perhaps u n l i k e l y t h a t a l l of the u t i -  14  l i z e d . s u b s t r a t e was o x i d i z e d c o m p l e t e l y t o C Og a n d w a t e r , u n l e s s perhaps a p r i m a r y . d e c a r b o x y l a t i o n t o o k p l a c e and  14 the  r e s u l t i n g p e n t o s e was c o m p l e t e l y o x i d i z e d t o C  Og  via  r e c y c l i n g - t h r o u g h the pentose-phosphate  To  gain further insight to this p e c u l i a r i t y ,  shunt.  iodoacetamide  r e a c t i o n m i x t u r e s were r e p e a t e d b u t 2 - K G - l - C ^ , 1  2-KC-6-C ^, 1  14 and 2-KG-U-C  were u s e d a s s u b s t r a t e s .  The r e a c t i o n s were  a l l o w e d t o p r o c e e d f o r one h o u r a t w h i c h t i m e t h e c e l l s w e r e f r a c t i o n a t e d a n d t h e COg a n a l y z e d f o r r a d i o a c t i v i t y (Table l l ) .  I f the assumption  that triose-phosphate  d e h y d r o g e n a s e i s g r e a t l y i n h i b i t e d by i o d o a c e t a m i d e i s c o r r e c t , t h e n t h e s e d a t a s u g g e s t t h a t a 3=3 s p l i t six  c a r b o n compound  occurs.  of a  I f the Entner-Doudoroff  14 p a t h w a y was o p e r a t i v e , t h e n one w o u l d  expect p y r u v a t e - l - C  14 f r o m 2-KG-l-C  , and t h e r a d i o a c t i v i t y would  l a r g e l y be  72  T a b l e 10.  14  • P r o d u c t s o f 2-KG-U-C d i s s i m i l a t i o n by washed c e l l s u s p e n s i o n s i n t h e p r e s e n c e of various metabolic i n h i b i t o r s Paper chromatography  Inhibitor  Arsenite  >UM/cup  6  supernatant  2-KG  hydrazones  pyruvate  * c cells  1 4  2.10  Bromopyruvate  80  0.19  Iodoacetamide  10  0.13  750  2.26  Sodium fluoride Pyocyanine  3.6  Selenite  5  Anaerobic  pyruvate  0.15 4.54  (Trace)  0.82  73  T a b l e 11.  14 C Cu r e l e a s e f r o m v a r i o u s l y l a b e l l e d 2-KG b y washed c e l l s u s p e n s i o n s i n h i b i t e d w i t h 1Q/LJM o f • i o d o a c e t a m i d e  14  Substrate  C  Og  2-KG-l-C ^  18.0  2-KG-6-C  .11.6  1  14  2-KG-U-C ^ 1  1  Expressed as a percentage i n t h e Warburg v e s s e l .  *  4.8  of the t o t a l  radioactivity  74 l o s t b y d e c a r b o x y l a t i o n on c o n v e r s i o n t o a c e t y l - C o A . r  14  However, • 2-KG-o-C  w o u l d n o t be c o n v e r t e d t o p y r u v a t e due  t o t h e assumed i n h i b i t i o n dehydrogenase  of 3-phosphoglyceraldehyde  a n d w o u l d be e x p e c t e d t o a c c u m u l a t e .  Since  no t r i o s e - p h o s p h a t e s were d e t e c t e d i n .the p r e s e n c e o f i o d o a c e t a m i d e i t i s s u g g e s t e d t h a t a c o n d e n s a t i o n .of two triose-phosphates, a r e v e r s a l o f the aldolase-, r e a c t i o n , a dephosphorylation of fructose-1,6-diphosphate t o f r u c t o s e - 6 - p h o s p h a t e a n d hence 6-PG,  t o G-6-P, an o x i d a t i o n t o  and d i s s i m i l a t i o n p r i m a r i l y v i a the Entner-  14 D o u d o r o f f pathway, f r o m 2-KG-6-C  lZ|  would r e s u l t  i na lesser C  r e l a t i v e t o 2-KG-l-C '-. li|  Og y i e l d  T h i s c o u l d be  p r i m a r i l y due t o t h e r e t a r d a t i o n o f t h i s c o m p l e x sequence  reaction  b y ^ t h e i n h i b i t o r a n d p o s s i b l y by a m i n o r  parti-  c i p a t i o n o f t h e p e n t o s e phosphate pathway r e s u l t i n g i n a  14 much s l o w e r r e l e a s e o f C  Og w h i c h c o u l d r e s u l t f r o m a  d e c a r b o x y l a t i o n o f 6-PG t o r i b u l o s e - 5 - p h o s p h a t e . 4.  I d e n t i f i c a t i o n o f i n t e r m e d i a t e s o f 2-KG d i s s i m i l a t i o n by " c a r r i e r " e x p e r i m e n t a t i o n The  failure  t o accumulate o r detect key i n t e r -  m e d i a t e s i n a n y i n v e s t i g a t i o n o f t h i s n a t u r e may be due t o one o r b o t h o f t h e f o l l o w i n g :  (a) that the i n t e r -  mediates a r e t r a n s i e n t and never accumulate i n d e t e c t a b l e amounts e v e n u n d e r  inhibited  conditions;  (b) t h a t t h e  i n t e r m e d i a t e s a r e a l w a y s bound t o t h e r e q u i s i t e concerned w i t h substrate d i s s i m i l a t i o n .  enzymes  One a v a i l a b l e  75 method t o c i r c u m v e n t  these  p r o b l e m s i s t o f l o o d a n enzyme  system w i t h an u n l a b e l l e d p o s s i b l e i n t e r m e d i a t e  during the  course  I f the  cell will,  of degradation  of a labelled  substrate.  i s f r e e l y permeable t o the p o s s i b l e i n t e r m e d i a t e , i t theoretically,  intermediate, thereby  e q u i l i b r a t e with the r a d i o a c t i v e creating a " c a r r i e r " pool of the i n t e r -  m e d i a t e w h i c h t h e n w o u l d be r a d i o a c t i v e a n d more  easily  detectable. a)  non-phosphorylated."carriers"  Dickens and Glock  (l95l)> e x p l a i n e d t h e p r e s e n c e  of pentoses and pentose-phosphates i n a n i m a l . t i s s u e s by a postulated decarboxylation  o f 2-K-6-PG.  Fewester-(1957)>  d e m o n s t r a t e d t h a t 5-KG u n d e r w e n t d e c a r b o x y l a t i o n t o g i v e xylulose  I n e x t r a c t s o f A. s u b o x y d a n s , a n d D a t t a  (1958)* p u r i f i e d a' d e c a r b o x y l a s e which catalyzed the formation diketogluconic acid.  f r o m A_. melanogenum  of a pentose from  /3-OH-pyruvate a n d g l y c e r a l d e h y d e of the decarboxylation  with glyceraldehyde  2,5-  Hough a n d J o n e s (1938), p r o p o s e d  t h a t 2-KG u n d e r w e n t a n a l d o l a s e - t y p e  product  et a l .  split, t o give  with glycolaldehyde, a  of/3-OH-pyruvate,  t o give r i b o s e .  condensing  S t a f f o r d (1954),  p r o p o s e d t h a t 2 - k e t o h e x o n i c a c i d s were c l e a v e d by an a l d o l a s e - l i k e enzyme t o g i v e O H - p y r u v i c a c i d a n d g l y o x y l i c acid i nplants.  They d e m o n s t r a t e d a s p e c i f i c  dehydrogenase which would reduce hydroxypyruvic glyceric acid.  glyceric acid to  De L e y (1954), s u g g e s t e d an a l d o l a s e  split  76 o f 2-K-6-PG i n t o t r i o s e - p h o s p h a t e  and h y d r o x y - p y r u v a t e o r ,  as a n . a l t e r n a t i v e , t h e t r a n s f o r m a t i o n d i e n o l , and t h i s was  o f 2-K-6-PG t o a  i n t o 3-keto-6-phosphogluconate, which  decarboxylated  w i t h the f o r m a t i o n  of 'ribulose-5-  p h o s p h a t e w h i c h , i n t u r n , was t r a n s f o r m e d  to triose-  p h o s p h a t e i n A_. c l o a c a e . Several o f these  compounds were s e l e c t e d a s p o s s i b l e  intermediates  o f 2-KG d i s s i m i l a t i o n by P_. a e r u g i n o s a a n d  were u t i l i z e d  as " c a r r i e r s " d u r i n g t h e o x i d a t i o n o f r a d i o -  a c t i v e 2-KG.  T h e s e r e a c t i o n s were c a r r i e d o u t i n d o u b l e -  sidearm  Warburg cups.  t h e washed c e l l  T h e , l a b e l l e d 2-KG was o x i d i z e d b y  suspension  f o r 12 m i n u t e s a n d t h e n t h e  " c a r r i e r " was a d d e d f r o m a s e c o n d s i d e a r m . allowed  t o continue  O x i d a t i o n was  f o r a n a d d i t i o n a l 12 m i n u t e s , w i t h t h e  exception  of the gluconate  c a r r i e r r e a c t i o n w h i c h was  terminated  a t 5 minutes.  The r e a c t i o n s were s t o p p e d b y  p i p e t t i n g t h e cup c o n t e n t s  i n t o c o l d c e n t r i f u g e tubes  f o l l o w e d by c e n t r i f u g a t i o n . The c a r r i e r s were  isolated  by e l e c t r o p h o r e s i s a n d c h r o m a t o g r a p h y a s t h e i r  2,4-dinitro-  p h e n y l h y d r a z o n e d e r i v a t i v e o r a s t h e f r e e compound. the r e s u l t s o f these concluded  experiments (Table  i t was  t h a t n e i t h e r r i b o s e , w h i c h c o u l d have f o r m e d  from a d i r e c t decarboxylation  of'2-KG,  a c i d , w h i c h c o u l d have f o r m e d f r o m a 3:3 split  12),  Prom  hydroxypyruvic. aldolase-type  o f 2-KG, n o r g l y o x y l a t e , w h i c h c o u l d have f o r m e d  f r o m a 2:4  split  o f 2-KG o r 2-K-6-PG, were  intermediates  i n v o l v e d i n 2-KG d i s s i m i l a t i o n u n l e s s t h e y a r e v e r y  T a b l e 12.  Compounds I s o l a t e d d u r i n g 2-KG-U-G d i s s i m i l a t i o n b y washed suspensions i n the presence of " c a r r i e r " substrates  cell  Paper chromatography Carrier  •Supernatant  Ribose  2-KG Ribose unknown ( l )  14  C  + +  2-KG o r g l u c o n i c  •+  +  2-KG o r g l u c o n i c  +  -  unknown (1)  +  2-KG o r g l u c o n i c  +  unknown (1)  +  2-KG o r g l u c o n i c  +  +  CM  2-KG OH-Py^ OC-KG ' 1  unknown ( l )  +  2-KG  +  2-KG glyoxylate pyruvate of-KG 1  unknown ( l )  +  2-KG  +  2 OH-Py-  hydroxypyruvate  +  +  +  2-KG  cK-ketoglutarate.  1 4  2-KG o r g l u c o n i c glyceraldehyde unknown (1).  +  -  1 Ctf-KG-  o  -  2-KG Gluconic  Glyceralde' hyde + 5juM I o d o acetamide  Electrophoresis 2-KG o r g l u c o n i c ribose unknown (1)  Gluconic  Glyoxylate  1 4  +  2-KG Glyceraldehyde unknown ( l )  Hydroxypy.ruvate  c  —  Glyceraldehyde acid  Hydrazones  -  . +  -  + +  +  -  +  t i g h t l y h o u n d t o t h e enzymes i n v o l v e d i n t h e i r f o r m a t i o n and s u b s e q u e n t u t i l i z a t i o n .  Since c a r r i e r gluconate d i d not  become r a d i o a c t i v e i t i s most p r o b a b l e t h a t dehydrogenase  i s not f r e e l y r e v e r s i b l e .  gluconic  I t was a l s o  thought  14 possible that gluconate-C  c o u l d have been formed  6-PG-C ^ b y t h e a c t i o n o f a s p e c i f i c p h o s p h a t a s e 1  ing  t h e c o n v e r s i o n o f 2-K-6-PG t o 6-PG..  from  follow-  Similarly,  g l y c e r a l d e h y d e c o u l d have a r i s e n n o t o n l y f r o m an a l d o l a s e l i k e r e a c t i o n i n v o l v i n g n o n - p h o s p h o r y l a t e d 2-KG b u t p e r h a p s by t h e a c t i o n o f a p h o s p h a t a s e phosphate  on g l y c e r a l d e h y d e - 3 -  a p r o d u c t o f 'the E n t n e r - D o u d o r o f f p a t h w a y .  G l y c e r a l d e h y d e d i d n o t become r a d i o a c t i v e e v e n i n t h e p r e s e n c e o f 5JJLM o f i o d o a c e t a m i d e . In the  each o f t h e r a d i o a c t i v e r e a c t i o n m i x t u r e s , w i t h  e x c e p t i o n o f t h e g l u c o n a t e c a r r i e r and i o d o a c e t a m i d e  I n h i b i t e d r e a c t i o n s , an unknown compound a c c u m u l a t e d i n the  supernatant f l u i d .  est  a c c u m u l a t i o n was o b s e r v e d i n t h e h y d r o x y p y r u v i c a c i d  c a r r i e r experiment. between.the  I t was o f i n t e r e s t t h a t t h e g r e a t -  Whether t h e r e e x i s t s a c o r r e l a t i o n  apparent r e p r e s s i o n of pyruvate formation  and t h e f o r m a t i o n o f t h e unknown r a d i o a c t i v e compound i s unknown.  I t was s u s p e c t e d f r o m t h e p o s i t i o n o f t h e  compound on p a p e r  c h r o m a t o g r a m s a n d on e l u t l o n f r o m a n  a n i o n exchange r e s i n t h a t t h i s amino a c i d .  Subsequent  labelled  compound was a n  paper chromatography  and r e a c t i o n s  w i t h n i n h y d r i n showed t h a t t h i s r a d i o a c t i v e compound was, in fact,  t h r e e amino a c i d s , two o f w h i c h were  identified  79 a s a s p a r t i c a c i d and g l u t a m i c a c i d and t h e t h i r d identification. and g l u t a m a t e - C  Since pyruvate-C  14  resisted  14 , o ( - k e t o g l u t a r a t e - C14 ,  . h a v e .been i d e n t i f i e d  in this  Investiga-  • 14 l i k e l y t i o n t h e n o x i d a t i y e a s s i m i l a t i o n w i t h 2-KG-U-C 14 f o l l o w s a s i m i l a r • m e c h a n i s m as w i t h  glucose-U-C  A s p a r t a t e c o u l d h a v e a r i s e n by a d i r e c t t r a n s a m i n a t i o n of o x a l o a c e t i c a c i d .  aminationor  P h e n y l a l a n i n e and  m e t h i o n i n e were a l s o shown t o be p r e s e n t i n t h e pyruvate  hydroxy-  " c a r r i e r " r e a c t i o n m i x t u r e b u t i n much s m a l l e r  quantities.  I t i s s i g n i f i c a n t that serine, which  would  a r i s e from t r a n s a m i n a t i o n i n v o l v i n g hydroxypyruvate,  was  not found i n the r e a c t i o n m i x t u r e . b) p h o s p h o r y l a t e d c a r r i e r s C e r t a i n c l a s s e s o f c h e m i c a l compounds' a r e known n o t to  p e n e t r a t e b a c t e r i a l c e l l s under normal  N u c l e o t i d e s and h e x o s e of  s u c h compounds.  phosphates  conditions.  provide classic  ( K e p e s and Cohen, 1962).  R o b e r t s , A b e l s o n , C o w i e , - B o l t o n , and B r i t t e n (20  h a v e shown t h a t a t l o w e r e d t e m p e r a t u r e s phosphate  and f r u c t o s e - 1 : 6 - d i p h o s p h a t e  I t was  However,  (1957)* C)  suspensions of  a l s o o b s e r v e d t h a t some d i f f u s i o n  i n t r a c e l l u l a r . " p o o l " m a t e r i a l s out o f ' t h e c e l l at  glucose-1-  as w e l l a s many  amino a c i d s w e r e t a k e n up by w a s h e d c e l l E. c o l i .  examples  lower temperatures.  Lowered t e m p e r a t u r e s  seemed a m e n a b l e t o c a r r i e r e x p e r i m e n t s 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 i n 2-KG  of  occurred  therefore  involving  metabolism.  possible A.  8© preliminary  i n v e s t i g a t i o n i n t o the a p p l i c a b i l i t y  a p p r o a c h was  p e n s i o n s as a t e s t  system.  significant  by washed c e l l  I t was  sus-  f o u n d t h a t a t 20  suspensions I n d i c a t i n g that, both  p h o s p h o r y l a t e d compounds were a b l e t o e n t e r t h e • R e a c t i o n m i x t u r e s c o n t a i n i n g 5>|M 5MM 6-PG,  lOJUM ATP,  C  oxygen consumption, a l t h o u g h a t a  s l o w r a t e , by w a s h e d c e l l  or  this  conducted u s i n g the o x i d a t i o n of g l u c o s e - 6 -  p h o s p h a t e and 6 - p h o s p h o g l u c o n a t e  t h e r e was  of  glucose-6-phosphate M Tris  lQ^M M g C l g , and 0.10  w e r e a l l o w e d t o i n c u b a t e at' 20  cell.  (pH  7.4)  C for-100 m i n u t e s p r i o r t o  the  a d d i t i o n of l a b e l l e d  s u b s t r a t e and o x y g e n  consumption  was  f o l l o w e d t o e n s u r e t h a t t h e p h o s p h o r y l a t e d compounds  14 were d i f f u s i n g  i n t o the c e l l .  FiveJUJVI o f g l u c o s e - U - C  w e r e a d d e d and t h e r e a c t i o n was a d d i t i o n a l 20 of'PCA.  The  m i n u t e s and was  a l l o w e d t o p r o c e e d f o r an  t e r m i n a t e d by t h e a d d i t i o n  two s u p e r n a t a n t f l u i d s a f t e r  w e r e n e u t r a l i z e d , and a p p l i e d t o a  centrifugation  Dowex-l-formate  c o l u m n and e l u t e d w i t h f o r m i c a c i d and ammonium f o r m a t e . • The p r o d u c t s o f t h e 6-PG  "carrier" reaction with glucose-C  a s s u b s t r a t e a r e shown i n t h e e l u t i o n p r o f i l e P e a k s 1,  2 and 3 were i d e n t i f i e d a s g l u c o s e ,  a c i d and 2-KG  respectively.  of 'Figure  12.  glutamic  ' P e a k s 4 and 5 were  identified  a s g l u c o s e - 6 - p h o s p h a t e and 6-PG r e s p e c t i v e l y , by p a p e r c h r o m a t o g r a p h y b e f o r e and a f t e r p h o s p h a t e h y d r o l y s i s w i t h . b a c t e r i a l a l k a l i n e phosphatase  ( T a b l e 13).  Since  this  •14 p r o c e d u r e was  s u c c e s s f u l w i t h glucose-U-C  oxidation i t  14 was  d e c i d e d t o a p p l y i t t o 2-KG-U-C  oxidation.  1  81 T a b l e 13.  I d e n t i f i c a t i o n of p h o s p h o r y l a t e d intermediates o f g l u c o s e - U - C ^ o x i d a t i o n by p a p e r c h r o m a t o graphy 1  Compounds Rg.  *  A f t e r treatment w i t h b a c t e r i a l a l k a l i n e phosphatase . Rg. Inference  Glucose  1.00  Gluconate  0.31  2-KG  0.52  Glucosesphosphate  0.12  1.00  6-PG  0.06  0.31  I  1.00  glucose  Peak I I  0.4l  glutamic acid**  Peak I I I  0.50  2-KG  Peak IV  0.12  1.00  G-6-P  0.06  0.32  6-P.G  Peak  Peak  V  •  Rg - R e l a t i v e t o g l u c o s e ( S o l v e n t , E t h y l a c e t a t e / P y r i d i n e , B o r i c a c i d ) and d e v e l o p e d w i t h b e n z i d i n e - p e r i o d a t e .  **  - Checked w i t h B u t a n o l / A c e t i c  a c i d and  with  ninhydrin.  Fig.  12.'  E l u t i o n s e q u e n c e f r o m a Dowex 1 - f o r m a t e a n i o n • e x c h a n g e c o l u m n .of compounds f r o m . g l u c o s e - U - C ^ o x i d a t i o n b y P. a e r u g i n o s a 1  83 I d e n t i c a l r e a c t i o n m i x t u r e s were p r e p a r e d t u t i n g 2-KG-U-C ^ f o r g l u c o s e - U - C 1  li|  ' a n d 6-PG, 2-K-6-PG,  and 2-K-3-D-6-PG w e r e u s e d a s c a r r i e r s . the r e l a t i v e r a t e s o f d i f f u s i o n  substi-  F i g u r e 13 d e p i c t s  o f the v a r i o u s c a r r i e r s as  m e a s u r e d b y o x y g e n c o n s u m p t i o n . • The s u p e r n a t a n t  fluids  were t r e a t e d a s p r e v i o u s l y d e s c r i b e d and a r e p r e s e n t a t i v e elution  sequence i s demonstrated  ( F i g . , 14) .  The i n t e r m e d -  i a t e s c o n t a i n e d i n t h e f o r m i c acid-ammonium f o r m a t e were i d e n t i f i e d by paper  chromatography  eluate  (Table l4A).  In  b o t h t h e 2-K-6-PG and- 6-PG c a r r i e r e x p e r i m e n t s , 2-K-6-PG and 3 - p h o s p h o g l y c e r i c a c i d w e r e I s o l a t e d a s r a d i o a c t i v e compounds.  U n f o r t u n a t e l y , 6-PG was n o t r a d i o a c t i v e ,  t h o u g h r e i s o l a t e d . • The i d e n t i f i c a t i o n t o be a n a r t i f a c t  even  o f 2-KG I s c o n s i d e r e d '  o f t h e p u r i f i c a t i o n o f -these compounds  r e s u l t i n g f r o m a n a c i d h y d r o l y s i s o f • 2 - K - 6 - P G . t o 2-KG a n d phosphate.  T h i s i s f e a s i b l e c o n s i d e r i n g the p o s i t i o n o f  e l u t i o n f r o m t h e f o r m a t e c o l u m n ( F i g . 14), a c i d conditions encountered.  Only  and the  highly  2-K-3-D-6-PG was i s o -  l a t e d a s a l a b e l l e d p r o d u c t o f t h e " c o l d " 2-K-3-D-6-PG c a r r i e r experiment.  These d a t a demonstrate  conclusively  t h a t 2-K-6-PG, 2-K-3-D-6-PG, a n d 3 - p h o s p h o g l y c e r i c  acid  a r e i n t e r m e d i a t e compounds d u r i n g t h e o x i d a t i o n o f 2-KG by g l u c o s e grown c e l l s o f P. a e r u g i n o s a a n d t h i s r e p r e s e n t s the f i r s t cells  e v i d e n c e f o r a c o n s t i t u t i v e 2-KG-kinase i n whole  o f a n y Pseudomonas s p e c i e s a n d a l s o d e m o n s t r a t e s t h e  p a r t i c i p a t i o n o f the E n t n e r - D o u d o r o f f pathway d u r i n g t h e o x i d a t i o n o f 2-KG.  84  300  50 F i g . .13.  100 MINUTES  15 0  200  Oxygen u p t a k e a t 20°C by w a s h e d - c e l l s u s p e n s i o n s o f P.-' a e r u g i n o s a w i t h 2-KG( O - O ), 2-KGPG (ft - #), 6 P G ( A - A ) a n d 2-K-3-D-6-PG ( A —A).. 2 ~ K G - C " w a s a d d e d a n d r e a c t i o n s were s t o p p e d where i n d i c a t e d . 1  14001  — ^  0-1.ON HCOOH  4.0 N HCOOK 0.4N N^COOH  400 l 3 Z  ^300  2-K-3-D-6-P0  2-KG  CO  H z  D200  o o  100  P i g . 14,  30 FRACTION 14  40 No.  -  E l u t i o n sequence o f C compounds f r o m a Dowex-1-formate. a n i o n e x c h a n g e column a f t e r 2-KG-C14-, o x i d a t i o n i n t h e p r e s e n c e o f 2-K-3-D-6-PG.  86 5.  Demonstration  o f 2-KG m e t a b o l i s m  in' c e l l - f r e e  extracts a) Enzyme a s s a y s A l t h o u g h a 2-KG-kinase h a s been r e p o r t e d i n a variety of bacteria induced kinase. demonstrated  i t h a s , i n a l m o s t a l l c a s e s , b e e n an,  Ghiretti  a n d G u z m a n - B a r r o n (1954),  a k i n a s e f o r 2-KG u s i n g a c e l l - f r e e  extract of  C. c r e a t i n o y o r a n s b u t no p r o d u c t i s o l a t i o n  was a t t e m p t e d  and t h e k i n a s e a c t i v i t y was n o t c o n s t a n t l y  reproducible.  However, N a r r o d a n d Wood (1955)* d e m o n s t r a t e d  a relatively  weak 2-KG k i n a s e i n g l u c o s e grown c e l l s o f P. f l u o r e s c e n s , partially purified  t h e enzyme, a n d i s o l a t e d  and i d e n t i f i e d  2-K-6-PG a s t h e e n d p r o d u c t o f t h e e n z y m i e t r a n s p h o s p h o r y l a t i o n w i t h ATP. During the course o f t h i s were made t o d e m o n s t r a t e  investigation  attempts  a 2-KG k i n a s e a s a c o n s t i t u t i v e  enzyme i n g l u c o s e grown c e l l s .  Sonic e x t r a c t s ,  Hughes'  p r e s s a t e s , a n d F r e n c h c e l l p r e s s a t e s were s e e m i n g l y of  this  enzyme.  C e l l breakage  was c a r r i e d o u t i n b o v i n e -  serum a l b u m i n , g l u t a t h i o n e , c y s t e i n e , or  glycylglycine  2-mercaptoethanol,  i n an.effort t o stabilize  a l l without apparent  success.  t h e 2-KG k i n a s e -  N e i t h e r - A T P , CTP,•GTP, n o r  I T P gave a n y e v i d e n c e o f a c t i v i t y a s p h o s p h a t e attempts t o demonstrate  void  2-KG-kinase a c t i v i t y  donors and  i n pressates  f r o m 20 h o u r , 0.2$ 2-KG grown c e l l s w e r e a l s o u n s u c c e s s f u l .  8  C i f e r r i e_t aJL. (1959) activity  ?  showed t h a t t h e 2 - K G - k i n a s e  o f L . m e s e n t e r o i d e s was p o o r when c e l l s w e r e h a r -  v e s t e d from t h e s t a t i o n a r y phase o f growth and t h a t t h e p u r i f i e d enzyme was r a p i d l y i n a c t i v a t e d above 30 C. In  an attempt t o demonstrate  phase c e l l s ,  kinase a c t i v i t y  i n log  t h e c e l l s were grown on 0.5$ 2-KG o r on 0.5$  g l u c o s e a s c a r b o n s o u r c e s a n d ' h a r v e s t e d a t 20 h o u r s .  The  c e l l s w e r e b r o k e n i n ^ T r i s pH 7.4 a n d 0.025 M g l y c y l g l y c i n e b y means o f a F r e n c h p r e s s u r e c e l l . r e d u c t a s e , 6-PG d e h y d r a s e  The 2-K-6-P-G  a n d 2-K-3-D-6-PG a l d o l a s e , a s  " w e l l a s 2-KG k i n a s e a c t i v i t i e s were measured, w i t h b o t h t h e g l u c o s e grown a n d 2-KG grown c e l l s . demonstrate  F i g u r e s 15 and•16  t h e v a r i o u s enzyme a c t i v i t i e s  f o u n d i n 2-KG  grown c e l l s a n d t h e r e l a t i v e enzyme a c t i v i t i e s g l u c o s e grown c e l l s a r e shown i n T a b l e 15. of  o f 2-KG o r  The a c t i v i t i e s  t h e enzymes m e a s u r e d w e r e , w i t h t h e e x c e p t i o n o f t h e  k i n a s e , t w i c e . a s h i g h w h e n ' t h e c e l l s were grown on 2-KG rather-than glucose.  The h i g h r e l a t i v e v a l u e f o r t h e  2-KG-kina.se f r o m 2-KG grown c e l l s was o b v i o u s l y due t o t h e low—almost glucose. all  i m m e a s u r a b l e — a c t i v i t y f r o m c e l l s grown on  The 2-KG i n d u c e d k i n a s e was t h e l e a s t a c t i v e o f  t h e enzymes m e a s u r e d w i t h a n a c t i v i t y  one-tenth the a c t i v i t y  of approximately  o f t h e r e l a t i v e l y weak 6-PG-  dehydrase. . As a c o n f i r m a t i o n o f t h e s e enzymic r e a c t i o n s w e r e made t o i s o l a t e  attempts  the end-products of the v a r i o u s  88  Enzynrve  30  Control  60 90 SECONDS  120  150  F i g . '15.. • D e m o n s t r a t i o n o f 2 - k e t o g l u c o n o k i n a s e a c t i v i t y f A - A | and 2 - k e t o g l u c o n o r e d u c t a s e a c t i v i t y ( O - . o ) i n c e l l - f r e e e x t r a c t s o f 2-KG grown cells  180  89  Enzyme Control  30  Pig.  60 90 SECONDS  120  16. - D e m o n s t r a t i o n o f 6-PG d e h y d r a s e  150  (A-A.) i n c e l l - f r e e e x t r a c t s o f g l u c o s e grown c e l l s ' a n d 2-K-3-D-6-PG a l d o l a s e ( O - o ) i n cell-free e x t r a c t s o f 2-KG grown c e l l s  180  90 Table 14.  I d e n t i f i c a t i o n of•phosphorylated of 2-KG-U-C14 metabolism  standards  solventR gl  .-2-KG gluconate glycerate 2-K- 6-PG 6-PG' 2-K- 3-D- 6-PG 3-PG 4  1  1  solvent3 2  gl  2-KG-C 6-PG  +  14 2-KG-C + 2-K-3-D-6-PG  1.00 0.72 0.46 o.8o  0.85  + +  0.86  0.58 1.00'  0.94 0.72 0.86  0.58 0.18. 0.20  0.58 1.00*  0.94  0.58 0.30  o.8o  .  2-KG-C ^,ATP, 0.94 Mg ,NADPH 0.72 1  ++  2  1. • 2. 3/ 4. 5,6. here, the R  14 2KG-C 2- K-6-PG-C 14 3- P G - C 14 214  KG-C  2- K-6-PG-C 14 14 3PG-C 2- KG-C 14 . 2- K-3-D-  6-  0.72  .  compound  gl  0.58 0..180.20  B - cell-free extract•reactions 2-KG-C14, ATP,0.94- ' '0.58 Mg  solvent 2 a f t e r phosphatase  0.58 0.36 1.00 0.18 0.10 0.30 0.20  0.94 0.94  r e a c t i o n mixture A - whole,cell reactions 0.94 2-KG-C14 + 2-K-6-PG 0.72  14  intermediates  O.58  0.18  0.58  0.58 0.18  O.58  PG-C  14  2-K-6-PG-C 14 2-K-6-PG-C 14  0.58  acetone/pyridine/water R . . - - r e l a t i v e to. g l y c e r a t e g -L ethylacetate/pyridine/boric acid 3-P<3—3-phosphoglyceric a c i d Only a t r a c e o f r a d i o a c t i v e g l y c e r i c a c i d was found b u t not. enough t o account f o r t h e disappearance of -, ,0.18-0.20 compounds.  91 reactions.  R e a c t i o n m i x t u r e s were p r e p a r e d i n a manner  i d e n t i c a l t o those o f t h e k i n a s e assay w i t h t h e e x c e p t i o n t h a t t h r e e t i m e s t h e amount o f c e l l - f r e e e x t r a c t was used and t h a t one r e a c t i o n was w i t h o u t NADPHg.  Warburg f l a s k s  were used f o r t h e i n c u b a t i o n s which were c a r r i e d ' o u t at"30 C f o r one-hour a t which time t h e r e a c t i o n s were t e r m i n a t e d w i t h 1.4 N PCA, c e n t r i f u g e d , and t h e c l e a r f l u i d n e u t r a l i z e d w i t h I N KOH. a n a l y z e d i n a manner analogous  These  supernatant  supernatants'were  t o the phosphorylated  carrier  e x p e r i m e n t s . • From Table l 4 B i t can be seen t h a t 2-K-6-PG was  t h e i d e n t i f i e d end p r o d u c t of t h e t r a n s p h o s p h o r y l a t i b n  w i t h ATP and t h a t 6-PG was n o t ' p r e s e n t , b u t r a t h e r 2-K-6-PG was  again .isolated.  The- a c t i v e E n t n e r - D o u d o r o f f  enzymes  p r o b a b l y degraded t h e 6-PG as f a s t as i t was formed.  The  aboye o b s e r v a t i o n s then e s t a b l i s h t h e e x i s t e n c e o f a d i s s i m i l a t o r y pathway f o r 2-KG metabolism A s i m i l a r pathway has been demonstrated  i n P. a e r u g i n o s a .  I n P. f l u o r e s c e n s  (Narrod and Wood,.1955; Frampton and Wood, 196l), b u t i t :  s h o u l d be noted t h a t c e l l - f r e e e x t r a c t s o f P. f l u o r e s c e n s c o n t a i n r e l a t i v e l y a c t i v e enzymes f o r 2-KG u t i l i z a t i o n , s i n c e c e l l s grown on g l u c o s e o r 2-KG e x h i b i t 2-KG-kinase a c t i v i t y and t h e c e l l - f r e e  demonstrable  preparations•degrade  2-KG q u a n t i t a t i v e l y t o p y r u v a t e — i n t h e presence of arsenite.  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 do n o t 1  e x h i b i t t h i s apparent a c t i v i t y .  I t i s i n c o m p r e h e n s i b l e why  the presumably i n i t i a l s t e p i n v o l v e d i n 2-KG u t i l i z a t i o n s h o u l d be r e l a t i v e l y i n a c t i v e i n c e l l - f r e e e x t r a c t s and  92  Table. 15.  Comparative enzyme a c t i v i t i e s o f g l u c o s e and 2-KG grown c e l l s o f P. a e r u g i n o s a  Enzyme 2-KG-kinase 2-K-6-PG-red-  Substrate Glucose 2-KG  Ratio of a c t i v i t y 2-KG/glucose.  0.13  1.8l  12.95  45.00  84.40  1.87  8.70  17.50  '2.01  30.30  60.30  1.99  uctase 6-PG dehydrase 2-K-3-D-6-PG aldolase  Enzyme a c t i v i t i e s a r e expressed as change i n 0.- D. p e r minute p e r mg p r o t e i n x 100  93 a l s o In whole c e l l s .  Whole c e l l s do not i m m e d i a t e l y meta-  b o l i z e 2-KG as t h e y do g l u c o s e and g l u c o n a t e ( F i g u r e l ) , but r a t h e r a d i s t i n c t 5-10 minute oxidation.  l a g occurs p r i o r to r a p i d  A c o n s t i t u t i v e enzyme e x i s t s for- the o x i d a t i o n -  of 2-KG s i n c e t h i s apparent l a g p e r i o d remained when c e l l s were t r e a t e d w i t h c h l o r a m p h e n i c o l .  unchanged I t has been  observed t h a t 2-KG i n d u c e s a b r i e f d e g r a d a t i o n o f r i b o n u c l e i c a c i d (RNA)—presumably r i b o s o m a l — d u r i n g t h i s l a g p e r i o d , whereas s u b s t r a t e s such as g l u c o s e and o<-ketoglut a r a t e do not (Gronlund and Campbell,  1965).  I t i s pos-  s i b l e t h a t a n e c e s s a r y c o f a c t o r f o r 2-KG d e g r a d a t i o n i s d e r i v e d e i t h e r d i r e c t l y o r i n d i r e c t l y from the d e g r a d a t i o n p r o d u c t s o f R.N-.A.  I t was reasoned t h a t i f t h i s p o s t u l a t e  i s v a l i d then' c e l l s e x t r e m e l y  d e f i c i e n t i n ribosomal  complement would be unable t o u t i l i z e 2-KG as an energy s o u r c e . . Phosphate s t a r v e d c e l l s o f P. a e r u g i n o s a were demonstrated  t o be d e f i c i e n t i n r i b o s o m a l m a t e r i a l r e l a t i v e  to non-starved c e l l s  (Hou, 1965).  Preliminary  experiments  w i t h p h o s p h a t e - s t a r v e d c e l l s d i s c l o s e d t h a t these  cells  were i n f a c t unable t o u t i l i z e 2-KG as observed by oxygen consumption  (Table 16).  S i n c e the f i r s t  step  i n v o l v e d i n 2-KG u t i l i z a t i o n by t h i s organism i s a phosp h o r y l a t i o n i t c o u l d be argued t h a t the l i m i t i n g r e q u i r e ment was phosphate.  However, the a d d i t i o n o f i n o r g a n i c  phosphate had r e l a t i v e l y l i t t l e  stimulatory effect  (Table 16), but the a d d i t i o n o f a n u c l e o s i d e p o o l had a  94  Table 16.  The s t i m u l a t i o n of 2-KG o x i d a t i o n i n phosphate s t a r v e d c e l l s u s p e n s i o n s of P. a e r u g i n o s a by i n o r g a n i c phosphate-  Compound added  Oxygen consumption a t 150 minutes  2-KG  70  2-KG + P i *  90  * P i - i n o r g a n i c phosphate  T a b l e 17.  The s t i m u l a t i o n of 2-KG o x i d a t i o n i n phosphate s t a r v e d c e l l s of P. a e r u g i n o s a by a n u c l e o s i d e pool  Compound added  Oxygen consumption a t 150 minutes  nucleoside p o o l *  162  "  "  + 2-KG  346  "  "  + Pi + 2-KG + P i  197 .467  "  o f each of. 2 JJLM , 4 nucleosides (adenosine, c y t i d i n e , X  suanosine, and u r i d i n e )  95 marked, s t i m u l a t i o n on oxygen uptake (Table 17).  Sub-  sequent s t u d i e s using-one ^LM of i n d i v i d u a l n u c l e o s i d e s demonstrated t h a t a l l the n u c l e o s i d e s were s t i m u l a t o r y not t o the same degree (Table 18).  Guanosine was  be the b e s t s t i m u l a t o r , c y t i d i n e was . u r i d i n e , and. adenosine was f r e e base guanine was  slightly  found t o  better•than  the l e a s t s t i m u l a t o r y .  The  found t o have^ a s t i m u l a t o r y e f f e c t  s i m i l a r t o guanosine (Table 19)  and t h e r e b y e l i m i n a t e d  p o s s i b l e importance of the r i b o s e m o i e t y . F-. a e r u g i n o s a  but  the  Since  has been shown t o l i b e r a t e ammonia d u r i n g  the d e g r a d a t i o n  of p u r i n e and p y r i m i d i n e  compounds  (Campbell, Gronlund, and Duncan, 1963), and  s i n c e guanine  and guanosine b o t h had e s s e n t i a l l y -the same s t i m u l a t o r y power on 2-KG  u t i l i z a t i o n i t was  decided  ammonia.as a p o s s i b l e s t i m u l a t o r y agent.  to test inorganic I t can be  seen  f r o m Table 19 t h a t ammonia a l s o had a marked s t i m u l a t o r y effect. was  This latter- observation  i n i t i a t e d two  queries:  the d e c r e a s e d r a t e of oxygen uptake w i t h . p h o s p h a t e -  s t a r v e d c e l l s the r e s u l t of 'the a c c u m u l a t i o n of a t o x i c intermediate;  or was  ammonia r e q u i r e d f o r the f o r m a t i o n  some c o f a c t o r mandatory f o r the p h o s p h o r y l a t i o n The  former p o s t u l a t l o n was  Tomlinson (1964), who  of  of 2-KG?  f e a s i b l e i n l i g h t of the work of  demonstrated t h a t s t a r v a t i o n d e c r e a s e d  o x i d a t i v e a s s i m i l a t i o n and p e r m i t t e d of k e t o - a c i d i n t e r m e d i a t e s  greater  accumulation  i n P. a e r u g i n o s a .  However, i n  t h i s i n v e s t i g a t i o n the a n a l y s i s of r e a c t i o n s u p e r n a t a n t s gave no i n d i c a t i o n of any  keto-acid accumulation  and  96 manometric data (Table 20) demonstrated t h a t ©(-ketoglut a r a t e was o x i d i z e d t o t h e same degree whether 2-KG was p r e s e n t i n t h e r e a c t i o n o r n o t . The l a t t e r  postulation  •that ammonia was n e c e s s a r y f o r perhaps some  cofactor  synthesis  i s f e a s i b l e , b u t i s open t o c r i t i c i s m i n view o f  the l i m i t e d data a v a i l a b l e a t t h i s t i m e . enzymatic p h o s p h o r y l a t i o n  was d i s c o v e r e d  ( P u j i m o t o and Smith, i960; Smith, 1961).  A new type o f by Smith I t was found  t h a t E. c o l i e x t r a c t s c a t a l y z e d d i r e c t p h o s p h o r y l a t i o n  of '  g l u c o s e w i t h p o t a s s i u m phosphoramidate, w i t h N-phosphorylg l y c i n e o r , more s l o w l y , w i t h m o n o p h o s p h o r y l h i s t i d i n e . T h i s then would s u f f i c e as one p o s s i b l e e x p l a n a t i o n ammonia r e q u i r e m e n t for-2-KG  f o r an  phosphorylation.  T a b l e 18.' The s t i m u l a t i o n o f 2-KG o x i d a t i o n i n phosphate s t a r v e d c e l l s o f P. a e r u g i n o s a by p u r i n e and pyrimidine ribosides Compound added 2-KG + 2-KG.+ 2-KG + 2-KG:+  Pi Pi Pi Pi  _  + + + +  adenosine cytidine• guanosine uridine  Oxygen consumption a t 150 minutes JH1 above 2-KG.+ P i c o n t r o l  215 350 400 315  97  T a b l e 19.  The s t i m u l a t i o n of 2-KG o x i d a t i o n i n phosphate s t a r v e d c e l l s u s p e n s i o n s o f P. a e r u g i n o s a  Compound added  Oxygen consumption a t 140 minutes jjJL above 2-KG + P i c o n t r o l  guanine  95  guanosine  80  NH~  70  T a b l e 20.  The e f f e c t o f «-ketoglutarate on 2-KG o x i d a t i o n i n phosphate s t a r v e d c e l l s u s p e n s i o n s of P. a e r u g i n o s a  Compound added {^--ketoglutarate + P i '  Oxygen consumption a t 180 minutes  91  2-KG. + P i  226  o<-ketoglutarate + 2-KG + P i  271  98 • GENERAL DISCUSSION The, d e m o n s t r a t i o n of s e q u e n t i a l enzymes concerned i n s u b s t r a t e d i s s i m i l a t i o n a l o n e i s not n e c e s s a r i l y v a l i d evidence,for metabolic  the e x i s t e n c e  pathway. ..The  of a p a r t i c u l a r f u n c t i o n i n g  i s o l a t i o n of the r e q u i s i t e i n t e r -  mediates, however does q u a l i f y the e x i s t e n c e  of the pathway  i n c e l l - e x t r a c t s but the q u a n t i t a t i v e e s t i m a t i o n of  the  p a r t i c i p a t i o n of such a pathway cannot be a p p l i e d t o whole c e l l s where the c e l l u l a r i n t e g r i t y has altered.  The  not been d r a s t i c a l l y  a b i l i t y of a c e l l t o use a p a r t i c u l a r pathway  i s no a s s u r a n c e t h a t the organism r e a d i l y u t i l i z e s  that  r o u t e d u r i n g growth but w i l l , u n q u e s t i o n a b l y ,  accord-  ing  t o the a v a i l a b i l i t y of a l t e r n a t e pathways and  according-to  of m e t a b o l i c  Similarly,  i n h i b i t o r s i s a u s e f u l t o o l f o r the  d a t i o n of c a t a b o l i c pathways but i s l i m i t e d due general  also  the needs of .the organism-as governed by b o t h  growth c o n d i t i o n s and d e v e l o p m e n t a l s t a g e . use  vary  the  eluci-  to a  l a c k of s p e c i f i c i t y e x h i b i t e d by most i n h i b i t o r s .  Indeed, i n t h i s i n v e s t i g a t i o n the v a r i o u s i n h i b i t o r s used were t o o r e s t r i c t i v e i n a f f e c t i n g the i n i t i a l stages of 2-KG  utilization.  I t a g a i n must be emphasized t h a t  e x t r e m e l y u n p h y s i o l o g i c a l c o n d i t i o n s are imposed upon the c e l l s by the a c c u m u l a t i o n of m e t a b o l i t e s , and  conclusions  of pathway p a r t i c i p a t i o n under t h e s e c o n d i t i o n s  are  nebulous'.'' E n t n e r and Doudoroff- (1952), c o n c l u d e d t h a t P. s a c c h a r o p h i l a  m e t a b o l i z e d g l u c o s e and  gluconate v i a  one  pathway t o p y r u v i c a c i d , but the c e l l s were p o i s o n e d w i t h  99 a r s e n i t e , thereby criticism.  l e a v i n g such a c o n c l u s i o n open t o  However, t h e i r c o n c l u s i o n was s u b s t a n t i a t e d  i n , n o n - i n h i b i t e d c e l l s by "Stern, Wang, and Gilmour•(1959)> using radiorespirometry.  Prampton and Wood (1961), showed  t h a t 2-KG was q u a n t i t a t i v e l y d i s s i m i l a t e d v i a t h e EntnerDoudoroff pathway t o p y r u v a t e under c o n d i t i o n s of a r s e n i t e i n h i b i t i o n i n e x t r a c t s of P. f l u o r e s c e n s , b u t no data i s a v a i l a b l e as t o t h e r e l a t i v e p a r t i c i p a t i o n o f t h i s pathway i n a non-poisoned c e l l , a l t h o u g h S t e r n , Wang, and Gilmour (1959)* have demonstrated t h a t Pseudomonas r e p t i l o v o r a , a c l o s e l y r e l a t e d - organism, d i s s i m i l a t e s 28$ o f t h e a v a i l a b l e g l u c o s e by t h e pentose-phosphate r o u t e . i n v e s t i g a t i o n ^ P. a e r u g i n o s a  In t h i s  was shown t o degrade 2-KG  q u a n t i t a t i v e l y t o p y r u v a t e under c o n d i t i o n s of a r s e n i t e o r bromopyruvate p o i s o n i n g , but w i t h o u t  isotope  distribution  data i t cannot be c o n c l u d e d t h a t a l l t h e p y r u v a t e was a p r o d u c t o f o n l y t h e E n t n e r - D o u d o r o f f pathway.  S t e r n , Wang,  and Gilmour c a l c u l a t e d t h a t t h e pentose-phosphate pathway f u n c t i o n s t o t h e same e x t e n t i n P. a e r u g i n o s a  as i n  P. r e p t i l o v o r a . The use of lowered t e m p e r a t u r e s as an a i d t o perm e a b i l i t y i n whole c e l l s i s an e x c i t i n g c o n c e p t .  Whether  a c t i v e t r a n s p o r t mechanisms a r e r e a d i l y i n a c t i v a t e d or whether p h y s i c a l c e l l u l a r b a r r i e r s a r e broken down, thus a l l o w i n g a' g r e a t e r freedom of d i f f u s i o n , may be t h e r e a s o n f o r i n c r e a s e d p e r m e a b i l i t y i s unknown a t p r e s e n t , but t h e  100 a p p l i c a t i o n of t h i s t e c h n i q u e t o the e l u c i d a t i o n of pathways of c a r b o h y d r a t e d i s s i m i l a t i o n i s of v a l u e as demonstrated d u r i n g the course of t h i s i n v e s t i g a t i o n .  Thus,in t h i s study,  i n c u b a t i o n s of whole c e l l s w i t h the p h o s p h o r y l a t e d compounds, 6-PG, 2-K-6-PG, o r 2-K-3-D-6-PG a t 200 p e r m i t t e d o x i d a t i o n o f ' t h e s e compounds and when 2-KG-C iHgluco'se-6-phosphate,  and t h e l a t t e r two compounds were used as " c a r r i e r " s t r a t e s and from t h e a p p r o p r i a t e l a b e l l i n g w h i c h  sub-  subsequently  ensued, evidence was o b t a i n e d f o r a d i s s i m i l a t o r y pathway of 2-KG d e g r a d a t i o n i n i n t a c t c e l l s .  T h i s t e c h n i q u e and t h e  r e s u l t s w h i c h were o b t a i n e d from i t s a p p l i c a t i o n p e r m i t t e d a f u l l i n v e s t i g a t i o n o f t h e enzymes f u n c t i o n i n g i n 2-KG dissimilation. The f a i l u r e t o accumulate o ( - k e t o g l u t a r i c a c i d d u r i n g the a c t i v e d i s s i m i l a t i o n of-2-KG by P. a e r u g i n o s a i s , on f i r s t c o n s i d e r a t i o n , a d e t r i m e n t t o t h e economy of t h e c e l l as i t has o b v i o u s l y l o s t a means of c o n s e r v i n g t h e carbon compound w h i c h a c t s as t h e p o i n t of e n t r y i n t o p r o t e i n and n u c l e i c a c i d s y n t h e s i s when ammonia becomes a v a i l a b l e . -However, M a c K e l v i e  (1965)* demonstrated  t h a t c u l t u r e s grown  i n a n i t r o g e n . l i m i t i n g medium w i t h g l u c o s e as t h e s o l e source accumulate  carbon  f a r g r e a t e r q u a n t i t i e s of -2-KG r e l a t i v e t o  p y r u v a t e o r c < - k e t o g l u t a r a t e and s i n c e these c e l l s a r e analogous, a t l e a s t I n r i b o s o m a l complement, t o phosphates t a r v e d - c e l l s i t becomes obvious t h a t 2-KG a c c u m u l a t i o n i s a more e f f i c i e n t means of c o n s e r v i n g carbon compounds than i s  101 ( X - k e t o g l u t a r a t e because: u n i v e r s a l metabolite 2-KG and  o<-ketoglutarate  i s almost a  among microorganisms but -2-KG i s n o t ;  can support more c e l l u l a r s y n t h e s i s than ( X - k e t o g l u t a r a t e ; from i n d i r e c t d a t a p r e s e n t e d i n t h i s r e p o r t 2-KG may i n  f a c t be i n c o r p o r a t e d  directly into cellular  constituents.  The mechanism of ammonia s t i m u l a t i o n of 2 - K G . u t i l i z a t i o n i s as y e t a mystery but i t i s p r o b a b l e t h a t  ammonia  i s f u n c t i o n i n g e i t h e r as a c o n t r o l mechanism or t h a t i t of 2-KG and  assumes a more d i r e c t r o l e i n t h e d e g r a d a t i o n f u n c t i o n s as a p r e c u r s o r cofactor.  t o t h e s y n t h e s i s of a n e c e s s a r y and Smith, .i960;  A phosphoramidate (Pujimoto  Smith,.1961) may be such a c o f a c t o r . The u n u s u a l l a b i l i t y of 2-KG k i n a s e  i n cell-free  e x t r a c t s , of P. a e r u g i n o s a r e l a t i v e t o t h e other f u n c t i o n i n g . i n 2-KG d e g r a d a t i o n  enzymes  i s p u z z l i n g s i n c e none of  the common agents used t o s t a b i l i z e t h e enzyme were e f f e c t i v e w i t h the exception  of g l y c y l g l y c i n e .  G l y c y l g l y c i n e may"be  s e r v i n g as the s u b s t r a t e f o r a p r o t e a s e w h i c h , when l i b e r a t e d d u r i n g c e l l breakage, a t t a c k s 2-KG k i n a s e .  Another  p o s s i b i l i t y i s t h a t g l y c y l g l y c i n e i s s e r v i n g as a s u b s t r a t e the u t i l i z a t i o n of w h i c h may produce a s u p p l y s t i m u l a t e 2-KG d e g r a d a t i o n .  of ammonia and  . However, the l a t t e r p o s t u l a t l o n  i s perhaps t h e most u n l i k e l y s i n c e c e l l breakage r e s u l t i n the a v a i l a b i l i t y of a v a r i e t y  should  of•nitrogenous  compounds w h i c h would a l s o serve as sources of ammonia.  102 SUMMARY  The  non-phosphorylated  o x i d a t i o n of g l u c o s e  through  g l u c o n o l a c t o n e , g l u c o n i c a c i d and 2-KG, as w e l l as t h e r o l e o f 2-KG as a t r a n s i e n t i n t e r m e d i a t e d u r i n g g l u c o s e by P. a e r u g i n o s a have been  catabolism  confirmed.  I t was found t h a t g l u c o s e grown c e l l s  metabolized  2-KG v i a 2-K-6-PG and 2-K-3-D-6-PG,intermediates t h e r e b y e s t a b l i s h i n g t h e e x i s t e n c e of a k i n a s e f o r 2-KG and t h e p a r t i c i p a t i o n of t h e Entner-Doudoroff-pathway d u r i n g 2-KG degradation. Two-keto-6-phosphogluconoreductase, 6-phosphogluconate dehydrase, and 2-keto-3-deoxy-6-phosphogluconate a l d o l a s e were c o n f i r m e d as c o n s t i t u t i v e enzymes of g l u c o s e grown c e l l s and these t h r e e enzymes as w e l l as a  2-ketoglucono-kinase  were demonstrated s p e c t r o p h o t o m e t r i c a l l y in.2-KG grown c e l l s . A l t h o u g h 2-KG k i n a s e c o u l d n o t be demonstrated  spectrophoto-  m e t r i c a l l y i n g l u c o s e grown c e l l s t h e product o f 2-KG t r a n s p h o s p h o r y l a t i o n w i t h ATP i n t h e presence o f magnesium w i t h c e l l - f r e e e x t r a c t s was shown t o be.2-K-6-PG. The r o l e of 2-KG I n o x i d a t i v e a s s i m i l a t i o n was demonstrated t o be c o n s i d e r a b l y d i f f e r e n t from g l u c o s e e s p e c i a l l y w i t h r e s p e c t t o t h e q u a n t i t a t i o n and speed of assimilation. 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