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A study of the incorporation of inorganic phosphate by Pseudomonas aeruginosa Campbell, James N. 1957

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A STUDY OF THE INCORPORATION OF INORGANIC PHOSPHATE BY PSEUDOMONAS AERUGINOSA  by  James N. Campbell B.A.  A T h e s i s Submitted i n P a r t i a l F u l f i l m e n t of the Requirements f o r the Degree of Master of Science i n 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 the D i v i s i o n of Animal S c i e n c e .  We accept t h i s t h e s i s as conforming t o the s t a n dard r e q u i r e d from candidates f o r the degree of Master of Science  The U n i v e r s i t y of B r i t i s h J u l y 1957  Columbia  i ABSTRACT  The  o x i d a t i o n of glucose by Pseudomonas aeruginosa  i s known to f o l l o w the sequence:  glucose  ^gluconic acid  k e t o g l u c o n i c a c i d —«—»pyruvic a c i d and thence cycle.  Two  i n t o the  a s p e c t s of t h i s p i c t u r e which arouse  Krebs'  interest are,  the missing steps i n the pathway and the f a c t t h a t the steps of t h i s pathway do not i n v o l v e phosphorylated Phosphate undoubtedly plays an important  initial  intermediates.  p a r t i n the metabolism  of t h i s organism hence the q u e s t i o n i s immediately  asked  where phosphorous does become i n v o l v e d .  questions  y i e l d two  *2  These two  p o s s i b l e r o u t e s of i n v e s t i g a t i o n f o r t h e i r own  as to  so-  l u t i o n ; one t o f o l l o w the f a t e of glucose as i t i s metabolized past the 2 ketogluconate  l e v e l , or two,  t o f o l l o w the f a t e of  i n o r g a n i c phosphate as i t i s i n c o r p o r a t e d .  The  hope i n each  case i s t h a t the i n f o r m a t i o n gleaned w i l l h e l p e l u c i d a t e how two  the  phenomena of glucose breakdown and u t i l i z a t i o n of phosphorous  are c o r r e l a t e d . Both these r o u t e s of i n v e s t i g a t i o n were attempted. The  i n v e s t i g a t i o n s along e i t h e r l i n e a r e , however, f r a u g h t w i t h  difficulties. To study the breakdown of g l u c o s e , attempts were made to f i n d a c e l l p r e p a r a t i o n , or an i n h i b i t o r , or a combination  of  both, which would a l l o w the breakdown of glucose past the 2 ketogluconate  l e v e l , but not a l l o w i t s complete o x i d a t i o n t o CO2  and H2O, thus accumulating which c o u l d be i d e n t i f i e d . a c t i n g on 2 ketogluconate  a h e r e t o f o r e unknown i n t e r m e d i a t e I t appears,  however, t h a t the system  as a s u b s t r a t e i s the most l a b i l e  ii i n t h i s c h a i n , f o r i f i n h i b i t i o n was  obtained,  the 2 ketogluconate l e v e l , r e g a r d l e s s The the  were found t o i n c o r p o r a t e  curve obtained  phosphate.  inorganic  C e l l free  accumulation of the  preparations  phosphate when incubated with  p a r t i a l l y s e n s i t i v e to d i n i t r o p h e n o l . phosphate  not a simple r e a c t i o n w i t h r e s u l t a n t  organic acceptor  but r a t h e r was  with  succinate,  on measurement of l o s s of i n o r g a n i c  i n d i c a t e d t h a t t h i s was  acceptor,  of the technique employed.  t o a l e s s e r extent when incubated  such i n c o r p o r a t i o n being The  at  i n v e s t i g a t i o n t h e n moved t o an attempt t o f o l l o w  i n c o r p o r a t i o n of i n o r g a n i c  glucose and  i t occurred  or a s i n g l e product of  this  a complex procedure i n v o l v i n g s e v e r a l  steps. The  f a c t that c e l l free preparations  took up  inorganic  phosphate i n the absence of added s u b s t r a t e , coupled w i t h the growing o p i n i o n t h a t the endogenous r e s p i r a t i o n i n t h i s  organism  i s not a simple r e a c t i o n , independent of added s u b s t r a t e ,  showed  the n e c e s s i t y of a s c e r t a i n i n g f i r s t what t h i s organism does w i t h i n o r g a n i c phosphate i n the absence of added s u b s t r a t e , so t h a t when s u b s t r a t e are and  i s added, i t w i l l be  p o s s i b l e t o know what phenomena  what are not, a r e s u l t of i t s presence. The  a d d i t i o n of i n o r g a n i c  phosphate l a b e l l e d w i t h P  t o a c e l l f r e e system i n the absence of added s u b s t r a t e i n the accumulation of two with u l t r a v i o l e t absorption spectively. t h a t they may  32  resulted  a l k a l i n e l a b i l e , acid stable f r a c t i o n s peaks a t 258  - 262  mjj and  263  T h e i r a c i d - a l k a l i n e behaviour leads t o the be nucleoside-polyphosphate-sugar  irresuggestion  complexes.  In p r e s e n t i n g the  this thesis in partial fulfilment  requirements f o r an advanced degree at the  of  University  of B r i t i s h Columbia, I agree t h a t  the  L i b r a r y s h a l l make  it  and  study.  f r e e l y a v a i l a b l e f o r reference  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may  be granted by the  Department o r by h i s r e p r e s e n t a t i v e .  Head o f  my  I t i s understood  that  copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l  gain  s h a l l not  be allowed without my  The U n i v e r s i t y of B r i t i s h Vancouver 8, Canada.  Date  <So  Columbia,  written  permission.  ACKNOWLEDGEMENTS  I would l i k e t o express my s i n c e r e thanks t o P r o f e s s o r J.J.R. Campbell f o r h i s d i r e c t i o n , encouragement and a s s i s t a n c e throughout the course of these i n v e s t i g a t i o n s . I would a l s o l i k e t o thank Dr. H.G. Khorana of the B r i t i s h Columbia Research C o u n c i l f o r h i s g e n e r o s i t y i n a l l o w i n g me the use of some of t h e i r equipment, and t o D r . W.E. R a z z e l l f o r h i s advice and a s s i s t a n c e i n the p a r t s of t h i s i n v e s t i g a t i o n  ^2 i n v o l v i n g the use of Phosphorous  .  The f i n a n c i a l a s s i s t a n c e g i v e n me by the B r i t i s h Columbia E l e c t r i c Railway Company and the N a t i o n a l Research C o u n c i l of Canada i s g r a t e f u l l y acknowledged.  J.N.C.  15/7/57  TABLE OF CONTENTS Page INTRODUCTION HISTORICAL REVIEW I II III  h  .V  Carbohydrate Metabolism  k  Oxidative Phosphorylation  h  R a d i o a c t i v e Isotopes  MATERIALS AND I  1  .  29 32  METHODS  32  Organism S t u d i e s With C e l l P r e p a r a t i o n s  32  1. 2.  Manometric s t u d i e s Chromatography of r e a c t i o n mixtures (a) P r e p a r a t i o n of s o l u t i o n s (b) S o l v e n t systems (c) Sprays •  32 33 33 3*+ 3*+  3.  Q u a n t i t a t i v e e s t i m a t i o n of pentose  k.  P r o t e i n determination  36  I I I S t u d i e s With Inorganic Phosphate  37  II  1. 2.  P r e p a r a t i o n of the c e l l f r e e e x t r a c t Manometric procedures  3.  Measurement of i n o r g a n i c phosphate  IV S t u d i e s With P h o s p h o r o u s 1. 2. 3'. k.  32  I n c u b a t i o n procedure Counting procedure P r e p a r a t i o n of the Dowex column Charging and e l u t i o n of column (a) A l k a l i n e e l u t i o n (b) A c i d i c e l u t i o n s 5. Acetone p r e c i p i t a t i o n of n u c l e o t i d e s EXPERIMENTAL AND DISCUSSION I Studies With C e l l Preparations 1. Manometric S t u d i e s 2. Chromatography S t u d i e s  •  3&  37 39 '  kO ^2 }+2 M-3 kk kk k$ k? kQ ^9 ^9 50 51  I I S t u d i e s With Inorganic Phosphate 1. C e l l p r e p a r a t i o n s and c h o i c e of an assay . 2. Experimental r e s u l t s Ill S t u d i e s W i t h Phosphorous32 . 1. With A T P 2. With i n o r g a n i c P32 (a) A l k a l i n e e l u t l o n (b) C h a r c o a l a d s o r p t i o n (c) A c i d e l u t l o n (d) P r e c i p i t a t i o n and a b s o r p t i o n spectrum 3 2  SUMMARY BIBLIOGRAPHY  1  INTRODUCTION  The  o x i d a t i o n of glucose by Pseudomonas  i s known t o f o l l o w the sequence:  Two  i n t o the K r e b s  aspects of t h i s p i c t u r e which arouse  interest  the missing steps i n the pathway and the f a c t t h a t the steps of t h i s pathway do not i n v o l v e phosphorylated Phosphate undoubtedly plays an important  where phosphorous does become i n v o l v e d .  .  1  are,  initial  intermediates.  p a r t i n the metabolism  of t h i s organism hence the q u e s t i o n i s Immediately asked  y i e l d two  »2  g l u c o s e - — • g l u c o n i c acid  k e t o g l u c o n i c acid--•-•pyruvic a c i d and thence cycle.  aeruginosa  These two  as t o  questions  p o s s i b l e r o u t e s of i n v e s t i g a t i o n f o r t h e i r own  so-  l u t i o n ; one t o f o l l o w the f a t e of glucose as i t i s metabolized past the 2 ketogluconate  l e v e l , or two,  t o f o l l o w the f a t e  i n o r g a n i c phosphate as i t i s i n c o r p o r a t e d .  The  of  hope i n each  case i s t h a t the i n f o r m a t i o n gleaned w i l l h e l p e l u c i d a t e how two  the  phenomena of glucose breakdown and u t i l i z a t i o n of phosphorous  are c o r r e l a t e d . Both these r o u t e s of i n v e s t i g a t i o n were attempted. The  i n v e s t i g a t i o n s along e i t h e r l i n e a r e , however, f r a u g h t w i t h  difficulties. To study the breakdown of g l u c o s e , attempts were made t o f i n d a c e l l p r e p a r a t i o n , or an i n h i b i t o r , or a combination both, which would a l l o w the breakdown of glucose past the ketogluconate and H2O,  2  l e v e l , but not a l l o w i t s complete o x i d a t i o n t o  thus accumulating  of  a h e r e t o f o r e unknown i n t e r m e d i a t e  C0  2  which could be i d e n t i f i e d .  I t appears, however, that the system  a c t i n g on 2 ketogluconate as a substrate i s the most l a b i l e i n t h i s chain, f o r i f i n h i b i t i o n was obtained, i t occurred a t the 2 ketogluconate l e v e l , regardless of the technique employed. The i n v e s t i g a t i o n then moved t o an attempt t o f o l l o w the i n c o r p o r a t i o n of inorganic phosphate.  C e l l free  preparations  were found t o incorporate inorganic phosphate when incubated  with  glucose and t o a l e s s e r extent when incubated w i t h s u c c i n a t e , such i n c o r p o r a t i o n being p a r t i a l l y s e n s i t i v e t o d i n i t r o p h e n o l . The curve obtained  on measurement of l o s s of inorganic phosphate  i n d i c a t e d that t h i s was not a simple r e a c t i o n w i t h r e s u l t a n t accumulation of the organic acceptor  or a s i n g l e product "of t h i s  acceptor, but r a t h e r was a complex procedure i n v o l v i n g s e v e r a l steps. The f a c t that c e l l f r e e preparations took up inorganic phosphate i n the absence of added s u b s t r a t e , coupled w i t h the growing opinion that the endogenous r e s p i r a t i o n i n t h i s organism i s not a simple r e a c t i o n , independent of added s u b s t r a t e , showed the n e c e s s i t y of a s c e r t a i n i n g f i r s t what t h i s organism does w i t h inorganic phosphate i n the absence of added s u b s t r a t e , so that when substrate i s added, i t w i l l be possible t o know what phenomena are and what are not, a r e s u l t of I t s presence. The a d d i t i o n of inorganic phosphate l a b e l l e d w i t h P^  2  to a c e l l f r e e system i n the absence of added substrate r e s u l t e d  i n the accumulation of two a l k a l i n e l a b i l e , a c i d s t a b l e w i t h u l t r a v i o l e t a b s o r p t i o n peaks a t spectively.  258 - 262 mp  fractions and  2 6 3 m | j re-  T h e i r a c i d - a l k a l i n e behaviour l e a d s t o the s u g g e s t i o n  that they may be nucleoside-polyphosphate-sugar complexes.  3  HISTORICAL REVIEW  I.  Carbohydrate Metabolism The  t o p i c of carbohydrate  metabolism i n microorganisms  has been the s u b j e c t of s e v e r a l r e c e n t r e v i e w s .  No attempt  has  been made t o review the l i t e r a t u r e on t h i s s u b j e c t i n t h i s presentation.  For such i n f o r m a t i o n , the r e a d e r ' s  drawn t o a r t i c l e s by O'Kane and Wood II.  (1955) (5*0  Oxidative The  and  (1956), (117)  Campbell  (195*0  attention i s  Gunsalus, Horecker  (2*0.  Phosphorylation p u b l i s h e d l i t e r a t u r e on the t o p i c of o x i d a t i v e  phosphorylation,  e s p e c i a l l y s i n c e 1939  i s so voluminous t h a t a  complete resume of a l l the c o n t r i b u t i o n s i s not p o s s i b l e w i t h i n the l i m i t s of a p r e s e n t a t i o n such as t h i s .  An attempt has "been  made t o t r a c e our i n c r e a s e i n knowledge of t h i s s u b j e c t , and assemble the a v a i l a b l e data which have c o n t r i b u t e d t o bur understanding.  The  present  In an e f f o r t t o improve the c o n t i n u i t y of the  d i s c u s s i o n , the t o p i c has been subdivided sections.  to  i n t o s e v e r a l major  l i t e r a t u r e on the s u b j e c t has not, however, been  p u b l i s h e d under s i m i l a r groupings,  so t h i s d i v i s i o n w i l l  perforce  s u f f e r from some o v e r l a p p i n g . The  phenomenon of o x i d a t i v e p h o s p h o r y l a t i o n  mechanism whereby i n o r g a n i c phosphate i s converted e s t e r s or anhydrides  w i t h concurrent  put i t very s u c c i n c t l y : -  oxidation.  i s the  i n t o phosphate  Hunter (66)  has  " I t i s c l e a r t h a t the e n t r y of phosphate c o n s t i t u t e s a mechanism whereby most of the energy change due i s not l o s t as heat but i s conserved organic phosphate compound  t o the o x i d a t i o n  i n the form of an  ... the energy r i c h phosphate  l i n k i s t r a n s f e r r e d t o the adenylate  system w i t h  little  l o s s i n energy and t h a t from t h i s p o i n t i t f u r n i s h e s energy f o r a number of c e l l u l a r p r o c e s s e s . " The  phosphate method of c o u p l i n g the energy from  o x i d a t i o n of s u b s t r a t e s t o u n r e l a t e d energy r e q u i r i n g processes i s a p p a r e n t l y present  in a l l tissue.  I t i s the most common, but  not the o n l y known type of energy c o u p l i n g system. Some of the f i r s t  o b s e r v a t i o n s l i n k i n g phosphorous and  o x i d a t i v e r e a c t i o n s i n g l y c o l y s i s were made by Warburg C h r i s t i a n (150)  and N e g e l e i n and Bromel (109), who  organic phosphate was  converted  (h2)  that i n -  E a r l i e r , i n the p e r i o d 1927  the  t o It 3 d i p h o s p h o g l y c e r l c t o 1930,  E g g l e t o n and  Eggleton  and F i s k e and Subbarow (*f7), among others were a b l e t o  demonstrate the r e g e n e r a t i o n of phosphocreatine r e c o v e r y of muscle t i s s u e a f t e r c o n t r a c t i o n . t h i s and the l a t e r work was Naehmansohn (107)  during oxidative  The  analogy  between  not q u i t e complete however f o r  and Lundsgaard (101)  the same phenomenon d u r i n g anaerobic (*+5)  noted  t o organic phosphate d u r i n g  o x i d a t i o n of 3 phositoglyceraldehyde acid.  and  were a b l e t o demonstrate glycolysis.  u s i n g pigeon and mammalian red blood c e l l s ,  In 1932 i n the  Engelhardt  presence  6 of f l u o r i d e t o prevent pyruvate f o r m a t i o n , and w i t h methylene blue and  oxygen now  necessary as e l e c t r o n a c c e p t o r s , was  able  t o demonstrate r e s p i r a t i o n dependent p h o s p h o r o l y s i s .  Working w i t h k i d n e y e x t r a c t s , K a l c k a r (76)  in  1937  observed r e s p i r a t i o n dependent c o n v e r s i o n of i n o r g a n i c phosphate t o hexose diphosphate.  Two  years l a t e r  a d d i t i o n of c i t r a t e , glutamate,  (77)  he found t h a t the  fumarate or malate i n c r e a s e d the  oxygen consumption of these e x t r a c t s w i t h c o r r e s p o n d i n g i n c r e a s e s i n the p h o s p h o r y l a t i o n of a d e n y l i c a c i d , glucose or g l y c e r o l . I n h i b i t i o n of r e s p i r a t i o n by cyanide r e s u l t e d of p h o s p h o r y l a t i o n .  The f o r m a t i o n of ATP-- d u r i n g 1  o x i d a t i o n i n b r a i n t i s s u e was (5),  w h i l e Lipmann (95)  inhibition  pyruvate  noted by Banga, Ochoa and  observed  t h a t the o x i d a t i o n of  by b a c t e r i a l p r e p a r a t i o n s r e s u l t e d phosphate t o  i n an  Peters pyruvate  i n the c o n v e r s i o n of i n o r g a n i c  ATP.  1 the f o l l o w i n g a b b r e v i a t i o n s are used  i n this presentation:  AMP—adenosine monophosphate; A D P — a d e n o s i n e diphosphate; A T P — a d e n o s i n e t r i p h o s p h a t e ; GMP—guanosine monophosphate; GDP—guanosine diphosphate; GTP—guanosine triphosphate; D M P — u r i d i n e monophosphate; U D P — u r i d i n e diphosphate; D T P — u r i d i n e t r i p h o s p h a t e ; F A D — - f l a v i n adenine d i n u c l e o t i d e ; DPN~diphosphopyridine n u c l e o t i d e ; DPNH—diphosphopyridine n u c l e o t i d e (reduced form); TPN—triphosphopyridine nucleotide; T P H N — t r i p h o s p h o p y r i d i n e . n u c l e o t i d e (reduced form); DNP—2:*+ d i n i t r o p h e n o l ; BAL—2:3 dimercaptopropanol; UDPG—uridine diphosphate g l u c o s e ; T C A — t r i c h l o r a c e t i c a c i d ; P:0—the r a t i o of moles of i n o r g a n i c phosphate e s t e r i f i e d per atom of oxygen taken up.  7 T h i s same year (1939) saw quantitative estimations (7)  the f i r s t  data l e a d i n g  presented, when B e l i t z e r and  to  Tsibakowa  s k e l e t a l muscle, n e a r l y 2 moles  noted t h a t w i t h heart and  of phosphocreatine were formed per atom of oxygen consumed. They then suggested that the i n t e r m e d i a t e fer  as w e l l as s u b s t r a t e  phosphorylation.  Using pyruvate as the  the phosphate a c c e p t o r , ratio  o x i d a t i o n may  ATP  1  ase and  eliminated  other  glucose  demonstrated a  for disrupted  undoubtedly low  even w i t h f l u o r i d e .  but  and  (cell  P.O  I t was  of  completely  then accepted t h a t phosphate  only w i t h the  o x i d a t i o n of  the  a l s o w i t h the passage of e l e c t r o n s through the Ochoa (115)  electron transport  system.  at  r a t i o by t a k i n g i n t o account l o s s e s by A T P  an accurate  type r e a c t i o n s .  P!0 He  obtained  made an attempt t o a r r i v e  phosphorylation  of  diphosphoglyceric  From these experiments he concluded t h a t the  r a t i o f o r pyruvate o x i d a t i o n would be 3»°  average of 3 phosphorylations ,yin  ase  from r e a c t i o n s known t o produce a s i n g l e phos-  p h o r y l a t i o n (e.g. 3 phosphoglyceraldehyde t o 153  P:0  1  a q u a n t i t a t i v e p i c t u r e of such  l o s s e s by examining under i d e n t i c a l c o n d i t i o n s , the y i e l d  acid).  as  free) brain  as a r e s u l t  s i d e r e a c t i o n s which could not be  uptake must be a s s o c i a t e d not substrate  substrate  for  phosphate e s t e r i f l e d per atom of  oxygen consumed) approaching 2.0 T h i s r a t i o was  i n electron trans-  be r e s p o n s i b l e  Ochoa (11*+) i n 19^1  ( i . e . moles of i n o r g a n i c  preparations.  steps  t o 1, t h a t i s an  w i t h each of the  the metabolism of pyruvate v i a the c i t r i c  corrected  5 oxidative  acid cycle.  steps This  8 set many people t o work t o a s c e r t a i n the r a t i o s a t each step i n the c y c l e .  individual  That the o x i d a t i v e steps i n the c i t r i c  acid  c y c l e do indeed e x h i b i t coupled p h o s p h o r y l a t i o n was confirmed i n 19^9  (36).  hy C r o s s , Taggert, Covo and Green  The f o l l o w i n g  g i v e s a summary of the f i n d i n g s of these i n v e s t i g a t i o n s .  table  No  attempt i s made here t o acknowledge a l l the c o n t r i b u t i o n s made: For such i n f o r m a t i o n the reader i s r e f e r r e d t o Hunter  (66)~ p. 302 f f .  P:0 R a t i o s f o r O x i d a t i v e Steps i n Pyruvate O x i d a t i o n and Kreb's C y c l e P:0 r a t i o s Observed Probable  Reaction Pyruvate + 2 ^ 0 —>3C0 +2H 0 2  2  Pyruvate + £ 0 — Acetate '+ C 0 2  2.5  2  3i0 k,0  2  Pyruvate + J 02+ oxalacetate-»citrate +  C0 + 2  3»0  H0 2  I s o c i t r a t e + £ 0 ->oxalosucclnate + H 0  3*0  2  2  c<keto glutarate + £ 0 — s u c c i n a t e + C 0 2  Succinate + J 0 - * f umarate '+ H2O 2  Malate + \ O2-»oxalacetate  2  3*0  h..O  1.6  2.0  + H0  3.0  2  Although Ochoa found P:0 r a t i o s which i n d i c a t e d  that  p h o s p h o r y l a t i o n must be a l s o coupled w i t h the e l e c t r o n t r a n s p o r t system, he was unable t o demonstrate DPNH as s u b s t r a t e .  such p h o s p h o r y l a t i o n u s i n g  The c o n t r i b u t i o n s of Cross (36)  and h i s  a s s o c i a t e s , F r i e d k i n and Lehhinger ( W Lehninger and Kennedy (88) and Hunter  (6*0  wherein they showed t h a t the mechanisms r e s p o n s i b l e  f o r o x i d a t i v e p h o s p h o r y l a t i o n were l o c a t e d w i t h i n the p a r t i c u l a t e f r a c t i o n of the t i s s u e i n v o l v e d , l e d t o the use of a c t i v e washed  9 p a r t i c u l a t e p r e p a r a t i o n s , thereby e l i m i n a t i n g many of the c o n taminating  side r e a c t i o n s .  These " c l e a n e r " systems, arid the  a v a i l a b i l i t y by 19^6 of a p u r i f i e d yeast hexokinase which, w i t h added glucose would form glucose-6-phosphate from any ATP which was  generated,  abetted the i n v e s t i g a t i o n s a t t h i s s t a g e .  c e s s f u l demonstration  A suc-  of the i n c o r p o r a t i o n of i n o r g a n i c P ^  2  u s i n g DPNH as a s u b s t r a t e was made by F r i e d k i n and Lehninger  (^8)  i n 19^-9 but t h e i r work was hampered by h i g h c o n c e n t r a t i o n s of DPN  i n t h e i r DPNH p r e p a r a t i o n s .  DPNH o x i d a t i o n .  The presence of DPN i n h i b i t s  That same year Lehninger  (89) extended  this  work w i t h washed p a r t i c l e s u s i n g the^3 hydroxybutyrate—>aeetoacetate s i n g l e step DPN l i n k e d  o x i d a t i o n as h i s s u b s t r a t e r e a c t i o n ,  a g a i n showing t h a t i n o r g a n i c phosphate was taken up as a r e s u l t of DPNH o x i d a t i o n .  By 1951 (90) he had obtained  highly purified  DPNH, which, when used as a s u b s t r a t e , f o r h i s r a t l i v e r e x h i b i t e d coupled  phosphorylation.  particles,  T h i s p h o s p h o r y l a t i o n was not  demonstratable under an atmosphere of n i t r o g e n , nor i n the absence of magnesium.  I t r e q u i r e d a l s o an a d d i t i o n of cytochrome c or an  a l t e r i n g of the m i t o c h o n d r i a l membrane, by exposure t o h y p e r t o n i c solutions.  Thus the p i c t u r e of j u s t where the p h o s p h o r y l a t i o n s  occur began t o open up.  Although  many of the steps were not  demonstrated e x p e r i m e n t a l l y u n t i l l a t e r , Lipmann (97) (98) and B a l l (3) by 1 9 ^ 6 , u s i n g o x i d a t i o n r e d u c t i o n p o t e n t i a l s as a b a s i s , suggested  the f o l l o w i n g p l a c e s f o r the g e n e r a t i o n of energy r i c h  phosphate bonds.  1.  O x i d a t i o n of the s u b s t r a t e by DPN,  TPN  or another  2.  O x i d a t i o n of DPNH or TPNH by a f l a v i n (e.g.  3.  O x i d a t i o n of reduced  FAD  *f.  O x i d a t i o n of reduced  cytochrome c by cytochrome  5.  R e o x i d a t i o n of cytochrome oxidase by oxygen.  carrier.  FAD).  e t c . by cytochrome c. oxidase.  That the s u b s t r a t e o x i d a t i o n i t s e l f produces phosp h o r y l a t i o n has been shown by Bergstrom Sudduth and 7  (9)  and by Copenhaver and Lardy  (35)  Lehninger  u s i n g p hydroxybutyrate  as  s u b s t r a t e and f e r r i c y t o c h r o m e c as e l e c t r o n a c c e p t o r , i n the presence  of cyanide t o prevent r e - o x i d a t i o n of the cytochrome. Many attempts were made p r i o r t o 1951  coupled  t o demonstrate  p h o s p h o r y l a t i o n i n the cytochrome r e g i o n , but  e f f o r t s met w i t h l i t t l e  success.  these  Since e a r l i e r q u a n t i t a t i v e  data i n d i c a t e d t h a t i t d i d occur, the i n a b i l i t y t o demonstrate i t was  l a i d t o such t h i n g s as the l a b i l i t y of the system, unknown  d i f f e r e n c e s between added cytochrome c and intact mitochondrial unit etc. and Lardy  (103),  (9)  195^ 1955'Maley (111) (110),  However, i n  N i e l s e n and Lehninger  U l Hassan and Sudduth (92)  t h a t e x i s t i n g i n the and  Lehninger,  and Bergstrom^Sudduth and  Lehninger  were a b l e t o demonstrate e x p e r i m e n t a l l y t h a t the o x i d a t i o n  of ferrocytochrome r a t i o being c i r c a  c e x h i b i t e d coupled 1.0.  p h o s p h o r y l a t i o n , the  P:0  S i m i l a r data f o r the p h o s p h o r y l a t i o n s t e p  coupled w i t h the o x i d a t i o n of the reduced  f l a v i n compounds" by  the cytochromes or a t the step wherein oxygen, a c t i v a t e d by c y t o chrome oxidase, o x i d i z e s the l a s t cytochrome l i n k has not been presented  as y e t .  11 I n t i s s u e s where such p a r t i c l e s e x i s t , the a b i l i t y t o c a r r y out the o x i d a t i v e p h o s p h o r y l a t i o n r e a c t i o n seems t o be a s s o c i a t e d p r i m a r i l y w i t h the m i t o c h o n d r i a .  I t has a l r e a d y been  noted t h a t washed m i t o c h o n d r i a l p r e p a r a t i o n s e x h i b i t  this  phenomenon and many u n d e s i r a b l e s i d e r e a c t i o n s such as t h a t o f ATP 'ase, a s s o c i a t e d l a r g e l y w i t h the s o l u b l e f r a c t i o n , are done away w i t h .  O x i d a t i v e p h o s p h o r y l a t i o n was demonstrated i n a sub-  m i t o c h o n d r i a l p a r t i c u l a t e p r e p a r a t i o n of p i g h e a r t by Green and Beinert (32)  (52)  (37)  i n 1951.  I n 1956  made d i g i t o n i n e x t r a c t s of r a t l i v e r m i t o c h o n d r i a and  s t u d i e d the a c t i v i t y on s e v e r a l s u b s t r a t e s . p r e p a r a t i o n a c t i v e on j3 hydroxybutyrate  They found  their  and s u c c i n a t e and found  the o n l y phosphate acceptor t o be adenosine 5'  (30)  Lehninger Cooper and Delven  5'  phosphate.  The  phosphates of i n o s i n e , u r i d i n e c y t i d i n e and thymidine were  essentially inactive. (15*0  That same year Z i e g l e r , L e s t e r and Green  broke up beef h e a r t m i t o c h o n d r i a by exposure t o a phosphate  b u f f e r e d s o l u t i o n of 1% types of fragments,  ethanol.  T h i s treatment  yielded  two  heavy, presumably c o n t a i n i n g some i n t a c t  mitochondria and l i g h t , which were separated by d i f f e r e n t i a l centrifugation.  A combination  of these p a r t i c l e s maintained a t  pH 8 i n a sucrose s o l u t i o n e x h i b i t e d o x i d a t i v e a c t i v i t y toward a wider  spectrum  of s u b s t r a t e s than d i d Lehninger's  digitonin  e x t r a c t s , which may i n d i c a t e t h a t t h i s i s somewhat of a g e n t l e r technique.  These p a r t i c l e s w i t h s t o o d  lesser extent, f r e e z i n g .  storage a t 10°C and t o a  Sonic d i s r u p t i o n f o r 1 minute y i e l d e d  an a c t i v e p r e p a r a t i o n from r a t l i v e r mitochondria by K i e l l e y and Bronk (85) never e x h i b i t e d a P:0  i n 1957.  r a t i o i n excess of one.  a d d i t i o n of  They p r o f f e r the  t h a t the a d d i t i o n of cytochrome c t o the  disrupted  i n t e r f e r e s w i t h the normal sequence, the added  chrome c a c c e p t i n g intermediate The  The  however,  r e s u l t e d i n an i n c r e a s e i n  oxygen uptake, but not of p h o s p h o r y l a t i o n .  mitochondria  reported  These p r e p a r a t i o n s ,  cytochrome c t o these p r e p a r a t i o n s  suggestion  as  cyto-  i t s e l e c t r o n s out of t u r n , thus bypassing  t r a n s f e r s where p h o s p h o r y l a t i o n  normally  advantage of the p a r t i c u l a t e p r e p a r a t i o n s  i n t a c t mitochondria  and  the  takes  place.  e x t r a c t s over  seems t o be t h a t they e x h i b i t l e s s ATP  'ase  activity. The  use  of P  32 and  obvious technique,  and  otherwise undetectable t h a t the use  of 0  0"^  i n these i n v e s t i g a t i o n s was  t h e i r u t i l i z a t i o n brought t o l i g h t some phenomena.  I n 195l> Cohn (27)  phosphatases showed t h a t the cleavage occurred phorous and  of an organic  i n o r g a n i c phosphate, the cleavage occurred In 1953  phate r a p i d l y had coupled  oxygen, but  in  (28)  he noted that 0 ^  phosphate compound from between carbon and  l a b e l l e d inorganic  phos-  the isotope r e p l a c e d by normal oxygen d u r i n g  o x i d a t i o n of &C ketoglutarate,y<3 hydroxybutyrate  succinate.  phos-  r e a c t i o n s c a t a l y z e d by muscle phosphorylase,  which i n v o l v e s the f o r m a t i o n  oxygen.  the  between the  oxygen r a t h e r t h a n between carbon and  the p h o s p h o r o l y s l s  reported  i n h y d r o l y s i s r e a c t i o n s c a t a l y z e d by  A  an  and  T h i s exchange of l a b e l l e d oxygen f o l l o w e d c l o s e l y the  e s t e r i f i c a t i o n of phosphate but the number of phosphate molecules participating  i n t h i s exchange f a r exceeded the number of h i g h  energy phosphate bonds formed.  He r e p o r t e d t h a t t h i s  exchange  occurred a t each s t e p of the e l e c t r o n t r a n s p o r t c h a i n but not a t the In  s u b s t r a t e o x i d a t i o n , a t l e a s t i n the <=*^ k e t o g l u t a r a t e system. Conn's evidence of oxygen exchange was  confirmed by  Boyer, Falcone and H a r r i s o n ( 1 3 ) , but they showed t h i s  exchange  to be independent of oxygen uptake and net change of i n o r g a n i c phosphate and ATP c o n c e n t r a t i o n .  They were a l s o a b l e t o demon-  s t r a t e an exchange of P  3 2  between i n o r g a n i c phosphate and ATP i n the absence of any d e t e c t a b l e m i t o c h o n d r i a l o x i d a t i o n . Using 0  1  (29)  ft  l a b e l l e d water and 0  18  labelled  phosphate, Cohn and D r y s d a l e  i n 1 9 5 5 showed t h a t the presence or absence of o x i d i z a b l e  s u b s t r a t e had no e f f e c t upon r a t e of oxygen replacement i n the i n o r g a n i c phosphate but the presence of s u b s t r a t e had a marked s t i m u l a t o r y e f f e c t on the r a t e of oxygen replacement i n ATP.  They  suggested t h a t both oxygen and an unknown oxygen donor are i n v o l v e d i n o x i d a t i v e p h o s p h o r y l a t i o n and t h a t the mechanism of oxygen replacement might be d i f f e r e n t a t v a r i o u s p o i n t s i n the e l e c t r o n t r a n s p o r t system when phosphate was  esterifled.  Luchsinger and Falcone (lk) - demonstrated an oxygen  exchange  between phosphate and water and a phosphorous exchange ADP, ATP and i n o r g a n i c phosphate.  between  T h i s phosphorous exchange  s p e c i f i c f o r the adenine base compounds. p i g k i d n e y c o r t e x mitochondria was  Boyer,  was  Data obtained u s i n g  presented by Gibson,Ayengar  l»f and Sanadi (*+9)  which showed t r a n s p h o s p h o r y l a t i o n s o c c u r r i n g  between GTP-AMP, CTP-AMP, ATP-GMP, GTP-UDP, UTP-ITP, when these r e a c t i o n s are coupled t o the o < k e t o g l u t a r a t e o x i d a t i o n system. More d e s c r i p t i v e r e s u l t s of i n v e s t i g a t i o n s u s i n g 0 ^  and  were presented i n 1956 by Boyer, E r n s t e r and L i n d b e r g When i n o r g a n i c p 3  2  was added t o an o x i d a t i v e  P^  2  (15).  phosphorylation  system, w i t h i n 30 t o *+5 seconds the t e r m i n a l group of the m i t o c h o n d r i a l ATP had a c q u i r e d 8 times the s p e c i f i c a c t i v i t y of the  18 middle group.  W i t h H2O  the c o n c e n t r a t i o n s of the i s o t o p e  appeared as f o l l o w s , i n descending order of c o n c e n t r a t i o n : intramitoehondrial inorganic  phosphate.  t e r m i n a l phosphate group ATP. middle phosphate group of ATP. l a b i l e phosphate group of The r a p i d exchange  ADP.  of phosphate of ADP  i n the medium  w i t h i n o r g a n i c phosphate i n v o l v e s the t e r m i n a l phosphate of ATP, the "bridge oxygen" being s u p p l i e d by ADP. pyrophosphate versus ATP was l a b i l e groups was  evidenced.  I f doubly l a b e l l e d  t r i e d , equal l a b e l l i n g  of b o t h the  They conclude from t h i s t h a t the  primary a c t i v a t i o n of o x i d a t i v e p h o s p h o r y l a t i o n i s p r i n c i p a l l y or e n t i r e l y t h a t of the i n o r g a n i c phosphate group which forms an i n t e r m e d i a t e which subsequently donates a phosphate group t o  ADP.  R e v e r s a l of the primary a c t i v a t i o n r e a c t i o n s accounts f o r observed r a p i d exchange  of oxygen between phosphate and water.  This  phenomenon was  s t u d i e d i n d i g i t o n i n e x t r a c t s of mammalian  exchange  15 mitochondria by Cooper and Lehninger (31)  (33)  wherein they found  exchange phenomena s i m i l a r t o those observed by Boyer and h i s collaborators. p3  2  Cooper and Lehninger, n o t i n g an i n c o r p o r a t i o n of  from i n o r g a n i c  phosphate i n t o ADP and ATP, concluded t h a t a  l a b i l e r e v e r s i b l e e q u i l i b r i u m e x i s t s between the t h r e e .  Their  p r e p a r a t i o n was s p e c i f i c f o r t h e adenosine phosphates and would not a f f e c t i n o s i n e , c y t i d i n e , u r i d i n e or guanidine phosphates. Some i n t e r e s t i n g d a t a has been put forward by DeMoss and  N o v e l l i ( 3 6 ) which may, or may not be u n r e l a t e d  t o t h i s exchange  They were able t o demonstrate an 1-amino a c i d de-  phenomenon.  pendent exchange mechanism between l a b e l l e d i n o r g a n i c  pyrophosphate  and ADP w i t h e x t r a c t s of a wide v a r i e t y of microorganisms.  This  exchange r e a c t i o n r e q u i r e d t h e presence of l e u c i n e , i s o l e u c i n e tryptophane, v a l i n e , t y r o s i n e , p h e n y l a l a n i n e , The  f a c t o r s which a f f e c t the phenomenon of o x i d a t i v e  phosphorylation organic  h i s t i d i n e or methionine.  a r e many and v a r i e d .  phosphate i t s e l f .  L e t us f i r s t c o n s i d e r  in-  I t i s g e n e r a l l y assumed t h a t phosphate  i s an o b l i g a t o r y component of the s u b s t r a t e  oxidation reaction.  T h i s does not c o n t r a d i c t observed "uncoupling" of o x i d a t i o n and phosphorylation,  which w i l l be d i s c u s s e d  l a t e r , f o r t h i s uncoupling  does not appear t o remove the requirement f o r phosphate.  Demon-  s t r a t i o n s of phosphate requirements i n o x i d a t i v e r e a c t i o n s were made by Ochoa (116)  i n the f o l l o w i n g : -  3 phosphoglyceraldehyde 1 step pyruvate ketoglutarate  oxidation  oxidation oxidation.  ' In  the f i r s t  two i n s t a n c e s , and w i t h somewhat l e s s  success i n the t h i r d , he was phosphate. or  16  able t o s u b s t i t u t e arsenate f o r  He suggested t h a t the arsenate may  organic phosphate t o i n o r g a n i c phosphate.  cause a breakdown (66)  Hunter  that even when phosphate i s o b l i g a t o r y , the o x i d a t i o n may first,  and the phosphate be e s s e n t i a l i n s p l i t t i n g  product from the enzyme.  suggested occur  o f f an o x i d i z e d  I n the e l e c t r o n t r a n s p o r t  system,  phosphate appears t o be s t i m u l a t o r y but an a b s o l u t e requirement has not yet been shown.  The enhancing e f f e c t  of phosphate accep-  t o r s s u c h as glucose plus hexokinase, on the .coupled o x i d a t i o n ?  p h o s p h o r y l a t i o n mechanisms was I t was  shown as f a r back as 1951  suggested by S i e k e v i t z and P o t t e r  c h o n d r i a l o x i d a t i o n s may phate a c c e p t o r .  be l i m i t e d  (66).  ( 1 3 1 ) ( 1 3 2 ) that m i t o -  by the a v a i l a b i l i t y of phos-  The s t i m u l a t o r y e f f e c t of the presence of phos-  phate a c c e p t o r r e c e i v e d roundabout c o n f i r m a t i o n as a r e s u l t of work by P o l i s , P o l l s , K e r r i g a n and J e d e i k i n (122) (10)(11).  and B h a t t a c h a r y a  They both noted that i n s u l i n p r e p a r a t i o n s  increased  o x i d a t i v e p h o s p h o r y l a t i o n i n k i d n e y t i s s u e , whereas c r y s t a l l i n e i n s u l i n would n o t . Bhattacharya was  (11)  The s t i m u l a t o r y e f f e c t was  traced  by  t o the g l y c e r o l i n the i n s u l i n p r e p a r a t i o n which  a c t i n g as a phosphate  acceptor.  There i s s t i l l some disagreement as t o the  immediate  acceptor of the h i g h energy phosphates generated by o x i d a t i v e phosphorylation.  Data presented by B a r k u l i s and Lehninger  Krebs, Johnson, E g g l e s t o n and Hems (87) i n d i c a t e s that ADP r a t h e r than AMP  (6),  and K i e l l e y and K i e l l e y  i s the immediate  (8U-)  a c c e p t o r , the  presence  of myokinase type systems being h e l d r e s p o n s i b l e f o r  the involvement  of AMP.  (133)  Slater  data t o be i n d i c a t i v e , maintained a c t i n g as a primary acceptor may (6)  B a r k u l i s and Lehninger Cooper and Lehninger i n d i c a t e d t h a t ADP furthermore,that  t h a t the p o s s i b i l i t y of not be e x c l u d e d .  and  on d i g i t o n i n e x t r a c t s of  the primary a c c e p t o r .  AMP  Work done by  on i n t a c t mitochondria  (3D  was  while acknowledging t h i s  later  by  mitochondria  I t i s considered,  the primary a c c e p t o r i s an adenine d e r i v a t i v e .  Phosphate which i s i n c o r p o r a t e d does show up i n the other t i d e s causing such c o n v e r s i o n s as UMP-DDP, UDP-UTP, such  nucleofact  having been o f t e n r e p o r t e d but i s c o n s i d e r e d t h a t i n most cases at was to  l e a s t , i t i s a r e s u l t of a t r a n s f e r mechanism from ATP, the product  of the i n i t i a l  phosphorylation.  The  exception  t h i s g e n e r a l i z a t i o n seems t o be the work of Sanadi,  Ayengar, and O u e l l e t (2)  028)  (129)  who  which  Gibson,  working w i t h heart P  enzyme showed the f o l l o w i n g r e a c t i o n s to occur: Succinyl^S  - CoA  + GDP GTP  In t h i s case GDP  + i n o r g a n i c P — ^ s u c c i n a t e + CoA-SH +GTP  + ADP—* GDP  (or IDP)  formed through a secondary  +  ATP  i s the i n i t i a l a c c e p t o r , and ATP  is  t r a n s p h o s p h o r y l a t i o n , c a t a l y z e d by a  separate enzyme. P h o s p h o r y l a t i o n of the deoxy r i b o s e compounds has a l s o been observed. observed  For example S a b l e , Wilber,Cohen and Kane  p h o s p h o r y l a t i o n of 2 deoxyadenosine 5'  P h o s p h o r y l a t i o n of deoxyribose  phosphate.  c y t i d y l i c a c i d was  shown by  (126)  18 Hecht P o t t e r and Herbert  (56)  and Herbert  P o t t e r and Takagi  I n a d d i t i o n t o showing p h o s p h o r y l a t i o n of d e o x y r i b o s i d e phosphates, C a r t e r (26)  *  5'  demonstrated t h a t a l l the deoxy mononucleo-  t i d e s , e s p e c i a l l y deoxy AMP,were s t r o n g i n h i b i t o r s , and i n h i b i t i o n was  (57).  r e v e r s e d by AMP,  ADP  or  that t h i s  ATP.  Magnesium i s e s s e n t i a l t o the proper f u n c t i o n i n g of  the o v e r a l l o x i d a t i v e p h o s p h o r y l a t i o n system, but  A requirement f b r magnesium i o n  f u n c t i o n s i s somewhat d o u b t f u l . i n the pyruvic and Lipmann (95)  and  and Green (138)  o<Cketoglutaric  Ochoa (116) obtained  j u s t where i t  dehydrogenases "was  shown by  r e s p e c t i v e l y . Stumpf ', Zarudnaya  p r e p a r a t i o n s t h a t had no magnesium r e -  quirement f o r o x i d a t i o n but these a l s o had no demonstrable phosphate requirement.  T h i s suggested  t h a t magnesium may  be  i n the p h o s p h o r y l a t i n g mechanism, e i t h e r f o r phosphate t o c i p a t e or f o r t r a n s p h o s p h o r y l a t i o n t o the adenylate 1956  B a i t s c h e f f s k y (h)  of mitochondria  quotes Lehninger  necessary parti-  system.  In  as having a p r e p a r a t i o n  which would f u n c t i o n i n a magnesium f r e e medium,  and r e p o r t s the s u c c e s s f u l p r e p a r a t i o n of such a system h i m s e l f . Conceivably,  however, there could be a c e r t a i n amount of endogenous  magnesium p r e s e n t . T h i s coupled  o x i d a t i o n - p h o s p h o r y l a t i b n process may  uncoupled or d i s s o c i a t e d by two p r e p a r a t i o n s and  g e n e r a l p r o c e s s e s : ageing  the a d d i t i o n of uncoupling  process w i l l be considered  first.  agents.  The  be  of the ageing  19 I t was  noted  and Hunter and Hixon  i n 19^9,  (63)  by Lehninger  (89),  (51)  Green  t h a t i f washed p a r t i c u l a t e ( m i t o c h o n d r i a l )  p r e p a r a t i o n s were allowed t o age, the l o s s of a b i l i t y t o phosphorylate was I t was age,  much q u i c k e r than the l o s s of a b i l i t y to o x i d i z e .  r e p o r t e d by Pullman and Racker (123)  something (s) was  t h a t as  mitochondria  r e l e a s e d i n t o the medium which, i f concen-  trated,would i n h i b i t p h o s p h o r y l a t i o n a s s o c i a t e d w i t h the o x i d a t i o n  ofy3 hydroxybutyrate,  without a f f e c t i n g oxygen uptake.  pound seemed t o enhance the a c t i o n of 2,*+ known uncoupling agents. serum albumin.  T h i s com-  d i n i t r o p h e n o l and  I t s e f f e c t i s c o u n t e r a c t e d by  other  bovine  M i t o c h o n d r i a , whose p e r m e a b i l i t y has been a f f e c t e d  by exposure t o hypotonic s o l u t i o n s or ice-water  (so-called  s w o l l e n mitochondria) were r e p o r t e d by Hunter and Ford (68) r e l e a s e cytochrome c, DPN rounding medium.  and unknown n u c l e o t i d e s i n t o the s u r -  Since the a d d i t i o n of DPN  Hunter and Ford suggested  to  r e v e r s e d the u n c o u p l i n g ,  t h a t the uncoupling e f f e c t  of ageing  D i a n z a n i (39)  verified  might be c h i e f l y due t o a l o s s of DPN. these r e s u l t s but suggested  t h a t i t might not be the whole s t o r y  and t h a t f a c t o r s other than DPN  were a l s o i n v o l v e d f o r he showed  t h a t the a d d i t i o n of ATP and manganese a l s o provided f o r some r e v e r s a l , whereas the a d d i t i o n of a m i t o c h o n d r i a l e x t r a c t provided the g r e a t e s t r e v e r s a l of a l l . haeme p r o t e i n was Shmulker (123) uncouple  An e l e c t r o p h o r e t i c a l l y homogeneous  i s o l a t e d from l i v e r mitochondria by P o l l s  and  which l i k e the compound of Pullman and Racker would  p h o s p h o r y l a t i o n and  o x i d a t i o n , and whose e f f e c t  was  20 reversed  by bovine serum albumin.  They suggested t h a t  this  f a c t o r could p a r t i c i p a t e i n the process of p h o s p h o r y l a t i o n acceptor  of h i g h energy phosphates, and  as  an  a c t as an i n h i b i t o r when  released. Agents which uncouple o x i d a t i o n and  phosphorylation  ( t h a t i s which prevent the uptake of phosphorous without a f f e c t i n g , or a f f e c t i n g t o a l e s s e r degree, oxygen uptake) are many and v a r i e d but as y e t there  seems t o be no c l e a r c o r r e l a t i o n of  the  data on such agents which would a l l o w g e n e r a l i z a t i o n s t o be made. P r o b a b l y the b e s t known and  most w i d e l y used u n c o u p l i n g agent i s  2s> d i n i t r o p h e n o l which was  used f i r s t by Cross and  orators  (36)  and  Loomis and  Llpmann ( 9 9 ) .  malonate, g r a m i c i d i n , aureomycin and agents whose e f f e c t s were r e p o r t e d erature.  The  mode of a c t i o n of DNP  e s t a b l i s h e d but One  suggestion  requirement & r  Arsenite,  a t an e a r l y date i n t h e has not  lit-  been-definitely  there are s e v e r a l t h e o r i e s as t o i t s f u n c t i o n . i s t h a t , r a t h e r t h a n doing away w i t h the  phosphate  o x i d a t i o n , i t causes an immediate breakdown of  i n the absence of mitochondria but  DNP  has  •ase in>preparations  but i t i s f e l t  noted by Hunter (66)  no e f f e c t upon  ATP  i n t h e i r presence i t i n c r e a s e s  the l i b e r a t i o n of i n o r g a n i c phosphate ( 6 6 ) .  I t was  arsenate,  heat are a l s o good u n c o u p l i n g  primary phosphorylated i n t e r m e d i a t e s .  story.  his c o l l a b -  DNP  stimulates  ATP  t h a t t h i s i s n ' t the whole and  Spector  (65)  that  DNP  would not c o m p l e t e l y i n h i b i t phosphate uptake i n the case of c>< ketoglutarate  oxidation.  I f the r e a c t i o n was  run  anaerobically,  21 the loss of inorganic phosphate dropped to a lower level, but this lower level was unaffected by the addition of DNP. They concluded therefore, that the DNP resistant phosphorylation was associated with substrate phosphorylation and not with the phosphorylation resultant from the oxidation of DPNH.  Hunter  offered the following scheme:«=-iketoglutarate + H POi 3  f  H^POij. + electron transfer ^ — — from DPNH —»0 •Y'^ PO^.'^H^PO^  A  T  P  1  ase  2  (66) 316.  DNP  X^POlf is the substrate level intermediate which is insensitive to DNP. Y ' V P O L , is a l l the primary phosphorylated intermediates or prosthetic groups i n the electron transport system. The proposal of this 2 stage process of a substrate level phosphorylation and an electron transport phosphorylation, only the latter of which is DNP sensitive, was supported by Anfinsen and Kielley (1). Cooper and Lehninger (31)  In digitonin extracts of mitochondria, (3*0 observed that the addition of  magnesium or DNP stimulated ATP ase activity, dicbumarol, gramicidin 1  pentachlorophenol and DNP caused uncoupling of phosphorylation, while calcium and thyroxin had no uncoupling effect. not evoke the hydrolysis of any of the nucleoside except ATP.  DNP did  triphosphates  22 An apparent uncoupling  e f f e c t e x h i b i t e d by t h y r o x i n  on m i t o c h o n d r i a l p r e p a r a t i o n s was f i r s t and Hess (10*0  i n 1951.  demonstrated by M a r t i u s  I n i t i a l l y some o p p o s i t i o n was met but  c o n f i r m a t o r y data appeared from such sources as Hock and Lipmann (59)  and subsequently  ments (*fl).  I n 1956,  the uncoupling  effect  t h i s reagent  has been used i n many e x p e r i -  however, Tapley and Cooper (1 1) L  of t h y r o x i n and noted  e x h i b i t e d o n l y on i n t a c t mitochondria From t h i s he assumed t h a t the e f f e c t p h y s i o l o g i c a l halophenol,  studied  t h a t i t s e f f e c t was  and not on the e x t r a c t s . of t h y r o x i n , which i s a  i s l i k e l y an i n d i r e c t  r e v e r s e d by the a d d i t i o n of magnesium.  one.  This i s  S i n c e DNP w i l l i n h i b i t  p h o s p h o r y l a t i o n i n e x t r a c t s , Tapley and Cooper ( l * f l ) c o n s i d e r t h a t the uncoupling compounds.  mechanism o f DNP d i f f e r s from t h a t of the other  F u r t h e r s t u d i e s on the e f f e c t of t h y r o x i n on o x i d a t i v e  p h o s p h o r y l a t i o n i n normal and tumor mitochondria Brombacher (¥f)  i n d i c a t e d t h a t the uncoupling  somewhat v a r i e d seemed stronger i n the case chondria, i n d i c a t i n g  by Emmelot and  effect  although  of the tumor  mito-  t h a t some d i f f e r e n c e may occur between these  and normal m i t o c h o n d r i a .  T h i s seems t o d i s a g r e e w i t h e a r l i e r work  by Wenner and Weinhouse (15D  i n which DNP a c t e d i n a s i m i l a r  f a s h i o n on both normal and tumor t i s s u e .  As d i s c u s s e d e a r l i e r ,  however, the a c t i o n o f the two i n h i b i t o r s seems t o be d i f f e r e n t .  It  has a l r e a d y been mentioned t h a t magnesium or manganese  are e s s e n t i a l f o r t h i s r e a c t i o n . t r a t i o n they a r e i n h i b i t o r y .  However, i n t o o great a concen-  Preincubation of preparations  with  23 c a l c i u m or z i n c was  shown by Hunter Davies and C a r l a t " (69)  i n h i b i t both oxygen uptake and Jacob, Sanadi and  (71)  Bradley  p h o s p h o r y l a t i o n while r e p o r t e d t h a t low  to  Jacobs,  concentrations  6 of the cadmium i o n (5 x 10"°M) e f f e c t i v e l y uncoupled the r e a c t i o n . Cadmium uncoupling  was  r e v e r s e d by versene, BAL,  extent by manganese, c o b a l t or n i c k e l . experiments was  The  and t o a l e s s e r  cadmium i n these  shown t o be f i r m l y bound f o r i t c o u l d n ' t be r e -  moved by washing.  Versene and  g l u t a t h i o n e were shown by Park,  Meriwether, Park, Mudd and Lipmann (118)  to reverse  inhibition  by thyroxin, t r i i o d o t h y r o n i n e and adrenochrome but they would not r e v e r s e uncoupling I t has  by  DNP.  o f t e n been r e p o r t e d t h a t i n t h y r o t o x i c t i s s u e  or i n f a t t y l i v e r t i s s u e , the o x i d a t i v e p h o s p h o r y l a t i o n mechanism seems uncoupled.  T h i s has r e c e n t l y p r e c i p i t a t e d a c o n s i d e r a b l e  amount of work on the e f f e c t s of v a r i o u s substances phenomenon. to 2.5:1  PsO  r a t i o s of 3 . 5 * 1  i n hypothyroid  r a t s as opposed  i n normal c o n t r o l r a t s were r e p o r t e d by S p r i t e s and  Andose ( 1 3 6 ) .  Such substances  as chlorpromazine  (1^9)  0.2M  urethane (*f3)  (135)  rogen mustard) e x h i b i t uncoupling oxidizing c^ketbglutarate  (8)  17°*hydroxy  (but not t e s t o s t e r o n e p r e g n a n e d i o l and ( l W  upon t h i s  progesterone progesterone)  (but not c o l c h i c i n e or n i t -  a c t i v i t y on  or s u c c i n a t e .  preparations  Numbers of n a r c o t i c s (1^3)  and a l c o h o l s were t e s t e d by T r u i t t , Wolpert B e l l and Krantz who  observed  uncoupling w i t h pentanol  of a c t i v i t y but was  (which gave only 17$  reduction  the only a l i p h a t i c a l c o h o l e x h i b i t i n g any  2k uncoupling e f f e c t ) acetaldehyde,  sodium p h e n o b a r b i t a l and  calcium.  B i l i r u b i n has a l s o been shown t o have an uncoupling e f f e c t  (153).  C o n f l i c t i n g r e p o r t s concerning the e f f e c t of f l u o r o a c e t a t e are i n the l i t e r a t u r e , Judah and Rees (88)  s t a t i n g t h e y c o u l d demon-  s t r a t e no uncoupling e f f e c t upon o x i d a t i v e p h o s p h o r y l a t i o n w h i l e F a i r h u r s t , S m i t h and G a l (*f6)  r e p o r t e d t h a t f l u o r o a c e t a t e , and  i n a d d i t i o n f l u o r o c i t r a t e abet the r e l e a s e or i n o r g a n i c phosphate or the s u p p r e s s i o n of i t s uptake.  A r s e n i t e and a z i d e , i n a  s l i g h t l y h y p e r t o n i c medium abet ATP'ase r e a c t i o n s w h i l e  inhibiting  the phosphorous exchange r e a c t i o n a c c o r d i n g t o Swanson ( l U O ) . The e f f e c t of i n j e c t i o n of DNP  intraperitoneally  into  r a t s upon the o x i d a t i v e p h o s p h o r y l a t i o n a c t i v i t i e s of t h e i r mitochondria was  s t u d i e d by D i a n z a n i and Scuro  were i s o l a t e d a t 20 hours and coupling.  they r e p o r t e d evidence  They r e p o r t e d f u r t h e r t h a t repeated  i n t o r a t s l e d t o t h e i r l i v e r s becoming f a t t y . however, was  unable  1956  (86)  Kodicek  (^O).  liver  Mitochondria of some un-  i n j e c t i o n s of Parker  DNP  (119)»  t o d u p l i c a t e these i n v i v o experiments.  In  r e p o r t e d t h a t a n t i c o a g u l a n t s , d e r i v a t i v e s of  V i t a m i n K, i n c r e a s e d the o x i d a t i v e p h o s p h o r y l a t i o n l e v e l s of Vitamin K d e f i c i e n t chicks. d i r e c t involvement  He  suggests t h a t t h e r e may  not be a  of V i t a m i n K i n the r e s p i r a t o r y c h a i n .  O x i d a t i v e p h o s p h o r y l a t i o n has been demonstrated t o occur i n b a c t e r i a and a l t h o u g h b a c t e r i a do not e x h i b i t mitochondria such, the r e a c t i o n appears  t o be a s s o c i a t e d w i t h the  m a t e r i a l w i t h i n the c e l l .  Evidence  as  particulate  of o x i d a t i v e p h o s p h o r y l a t i o n  25 a s s o c i a t e d w i t h DPNH o x i d a t i o n was (120)  found  by Pinchot and Racker  using soniced p r e p a r a t i o n s of E s c h e r i c h i a c o l i .  demonstrations  were made by Lipmann (96)  d e l b r u e c k i i and  i n 1939,  on L a c t o b a c i l l u s  i n 19*+2 by Vogler and Umbreit (IV7)  o x i d a t i o n of sulphur by T h i o b a c i l l u s t h i o x i d a n s .  Early  d u r i n g the (121)  Pinchot  l a t e r demonstrated t h i s phenomenon i n soniced A l c a l i g e n e s f a e c a l i s . He obtained 2 enzyme f r a c t i o n s and a heat s t a b l e n o n - d i a l y z a b l e component.  One  enzyme system, which he c a l l e d the oxidase  factor,  e x h i b i t e d oxygen uptake plus some p h o s p h o r y l a t i n g a c t i v i t y .  The  a d d i t i o n of e i t h e r the other enzyme f r a c t i o n or the heat s t a b l e f r a c t i o n s t i m u l a t e d oxygen uptake but not p h o s p h o r y l a t i o n . both were added t o g e t h e r , both o x i d a t i o n and stimulated.  If  p h o s p h o r y l a t i o n were  Other microorganisms i n which o x i d a t i v e p h o s p h o r y l a t i o n  has been demonstrated are Mycobacteria and Gray ( 1 6 ) ( 1 7 ) , A z o t o b a c t e r  ( T i s s i e r e s and S l a t e r  Ochoa)(125), yeasts (Nossal,Keech (Bovarnik)(12).  and C o r y n e b a c t e r i a (1^2)  (Brodie Rose and  and U t t e r ) ( 1 1 3 ) and R i c k e t t s e a e  Brodie and Gray (18),among others^have  shown  t h a t b a c t e r i a l systems can be fractionated i n t o a p a r t i c u l a t e oxidase f r a c t i o n and  supernatant  f r a c t i o n s which don't e x h i b i t  coupled  o x i d a t i o n and  bined.  B a c t e r i a l systems e x h i b i t a high degree of s e n s i t i v i t y  to  p h o s p h o r y l a t i o n s e p a r a t e l y , but do when com-  sonic o s c i l l a t i o n , presumably because of d i s r u p t i o n of p a r t i c -  ulate material.  B r o d i e and Gray (18)  r e p o r t a maximum t o l e r a n c e  of h minutes sonic o s c i l l a t i o n f o r t h e i r p r e p a r a t i o n s , a f t e r which a c t i v i t y dropped o f f markedly.  Four minute s o n i c i n g l e f t n e a r l y  a l l the dehydrogenase a c t i v i t y as w e l l as cytochromes a, b and c a s s o c i a t e d w i t h the p a r t i c u l a t e m a t e r i a l .  A c t i v e washed p a r t i c u l a t e  26 p r e p a r a t i o n s of Azotobacter Ochoa (125)  v i n e l a n d i i were obtained by Rose  and Bruemmer, Wilson,Glenn and Crane (21)  and Gray (18)  (19)  component was  needed.  B r o d i e and Gray observed  p r e s e r v a t i o n of the a c t i v i t y .  u a t i o n observed  t h a t w i t h crude  w i t h mammalian mitochondria  n a t i v e p r o t e i n and  the a c t i v i t y . treatments The  lipid  sit-  where c o n t r o l of i s o Probably  the  presence  They subjected t h e i r p r e p a r a t i o n s t o v a r i o u s  and found  a picture very l i k e that i n  mitochondria.  the most l a b i l e , s u f f e r i n g i n -  a c t i v a t i o n as a r e s u l t of f r e e z i n g , too prolonged  sonic  oscillation  T h i s seems t o i n d i c a t e t h a t s p a t i a l c h a r a c t e r i s t i c s , t h a t  i s , i n t a c t n e s s of c o n f i g u r a t i o n s i s an important t e r i a l oxidative phosphorylation. of  necessary  i n the crude p r e p a r a t i o n s p r o t e c t s  p h o s p h o r y l a t i o n mechanism was  etc.  not  T h i s i s c o n t r a r y t o the  t o n i c i t y during preparation i s e s s e n t i a l . of  Brodie  showed t h a t w i t h Mycobacterium p h l e i a s o l u b l e  b a c t e r i a l p r e p a r a t i o n s the presence of sucrose was for  but  and  N o s s a l and  c o l l a b o r a t o r s (113)  f a c t o r i n bac-  The y e a s t granule  preparations  e x h i b i t e d an even more l a b i l e  p h o s p h o r y l a t i o n system than t h a t of the M y c o b a c t e r i a .  Sonic  o s c i l l a t i o n f o r 5 seconds y i e l d e d a p r e p a r a t i o n which e x h i b i t e d a P:0 r a t i o i n excess was  of 1.  I f , however, the time of s o n i c i n g  i n c r e a s e d t o 30 seconds, the P:0 r a t i o f e l l  off sharply.  T h e i r system r e q u i r e d the a d d i t i o n of versene and They a l s o presented  data which i n d i c a t e s t h a t the  f r a c t i o n contains a f a c t o r from o x i d a t i o n .  (s) which uncouples  magnesium. supernatant  phosphorylation  The work on Azotobacter (125)  p a r t i c l e s by Rose and Ochoa  yielded several i n t e r e s t i n g observations.  were only p a r t i a l l y s e n s i t i v e t o the uncoupling  These  particles  a c t i o n of DNP.  Whereas i n p r e p a r a t i o n s of mammalian mitochondria  1 x 10  M  DNP completely uncouples p h o s p h o r y l a t i o n from o x i d a t i o n , with the b a c t e r i a l p r e p a r a t i o n 1 x 1 0 ~ ^ M DNP caused o n l y 80%  uncoupling.  T h e i r p a r t i c u l a t e p r e p a r a t i o n was e s s e n t i a l l y f r e e of myokinase a c t i v i t y and ADP was the only f u n c t i o n a l phosphate acceptor t h i s system.  with  The p r e p a r a t i o n s c o u l d be s t o r e d s a t i s f a c t o r i l y  with  only s l i g h t l o s s i n a c t i v i t y as f o l l o w s : F r o z e n whole c e l l s  survived  (-18°C)  1  month.  P a r t i c u l a t e p r e p a r a t i o n ( 0 C ) s u r v i v e d 2 weeks. G  Frozen p a r t i c u l a t e p r e p a r a t i o n s u r v i v e d n i l . In 1 9 5 3 S l a t e r and C l e l a n d ( 1 3 ^ ) r e p o r t e d t h a t versene would help p r o t e c t m i t o c h o n d r i a l p r e p a r a t i o n s a g a i n s t l o s s of a c t i v i t y , the same p r o t e c t i v e a c t i o n being claimed f o r bovine serum albumin i n l a t e r work by Lewis and S l a t e r ( 1 2 7 ) .  (123)  These o b s e r v a t i o n s were confirmed  ( 9 * 0 and Saktor  by Pullman and Racker  who went f u r t h e r t o demonstrate t h a t bovine  would b r i n g about r e c o u p l i n g i n aged p r e p a r a t i o n s .  serum albumin The f o l l o w i n g  p r o t e i n s were t e s t e d f o r r e c o u p l i n g a c t i v i t y : Bovine serum albumin e x h i b i t e d r e c o u p l i n g a c t i v i t y of 1 0 0  it  2 0  Gelatin  ti  0  Casein  it  0  Insulin  it  0  lactoglobulin  Bovine g l o b u l i n  0  28 T h i s s e c t i o n on o x i d a t i v e p h o s p h o r y l a t i o n may be w e l l concluded  w i t h a few words concerning  perhaps  the c o u p l i n g of  p h o s p h o r y l a t i o n d u r i n g the o x i d a t i o n of compounds other  than  those which c o n s t i t u t e i n t e r m e d i a t e s i n the carbohydrate  cycles.  Coupled p h o s p h o r y l a t i o n was  observed  i n 1939  (77)  by K a l c k a r  d u r i n g the o x i d a t i o n of glutamate by mammalian m i t o c h o n d r i a l preparations.  Glutamate o x i d a t i o n by p r e p a r a t i o n s of R i c k e t t s e a e  evidenced  coupled  p h o s p h o r y l a t i o n , as r e p o r t e d independently  Bovarnick  (12)  i n 1956.  That same y e a r , Jacobs (70)  and Hopps, Hahn, Wisseman, Jackson  when r a t l i v e r mitochondria  and Smadel  coupled  (61)  r e p o r t e d i n a note, t h a t  o x i d i z e s i l i c o m o l y b d a t e and  cyanide, the e l e c t r o n s go t o the haeme chains of the p a r t i c l e s and  by  p h o s p h o r y l a t i o n i s observed.  ferro-  mitochondrial Kennedy and  Weiss ( 8 0 ) ( 8 1 ) ( 8 2 ) have r e p o r t e d t h a t the f o r m a t i o n of  lecithin  i n mammalian l i v e r by e i t h e r r e a c t i o n . (a)  choline  >liver l e c i t h i n  (b)  phosphorylcholine  >liver l e c i t h i n  r e q u i r e s concurrent  oxidative phosphorylation.  I n these r e a c t i o n s of course how  (via interaction w i t h CTP)  there i s the q u e s t i o n of  much p h o s p h o r y l a t i o n i s a t t r i b u t a b l e , t o the f i r s t o x i d a t i o n  of the s u b s t r a t e and  how  much t o subsequent o x i d a t i o n of  or hydrogen a c c e p t o r s reduced  by such o x i d a t i o n s .  however, l i t t l e doubt t h a t there i s a c o u p l i n g of w i t h e l e c t r o n t r a n s f e r wherever DPN acceptor.  or TPN  products  There i s , phosphorylation  i s the primary e l e c t r o n  29 III.  Radioactive Isotopes Since the present i n v e s t i g a t i o n s involve the use of  Phosphorous  i t i s considered worthwhile t o include i n t h i s  3  s e c t i o n a few b r i e f points concerning the substance.  Unless  otherwise noted the source of m a t e r i a l f o r t h i s s e c t i o n were the books by Hevesy (58) and Hodgman, Weast and Selby ( 6 0 ) . P  3 2  i s prepared by the bombardment of carbon d i s u l f i d e  w i t h f a s t neutrons according t o the equation. 32 16  1  o  H  The neutron source may be obtained by bombarding b e r y l l i u m w i t h accelerated heavy p a r t i c l e s i n a c y c l o t r o n or a p i l e , but i n the case of radiophosphorous a mixture of powdered b e r y l l i u m w i t h radium c h l o r i d e or radium s u l f a t e (1 gram radium s a l t w i t h 5 grams of b e r y l l i u m ) i s used.  The neutron source i s placed on  the centre of a f l a s k containing about 10 l i t r e s of carbon d i sulfide.  Bombardment f o r lH-,3 days y i e l d s \ maximum amount of P . 3 2  The l i q u i d i s l a t e r treated w i t h water or d i l u t e a c i d s i n which P  32 i s soluble w h i l e carbon d i s u l f i d e i s n o t . The d i l u t e a c i d  or water used t o e x t r a c t the radiophosphorous contains P  3 2  maybe some P31 present i n the i r r a d i a t e d carbon d i s u l f i d e .  and The P  can a l s o be c o l l e c t e d by subjecting the i r r a d i a t e d s o l u t i o n t o 32 an e l e c t r i c c u r r e n t .  The P  32  migrates t o the e l e c t r o d e s , about  one and a h a l f times more on the anode than the cathode. recovery of P32 by these methods i s about 9 5 $ .  Total  The copper e l e c t r o d e s  30 are l a t e r d i s s o l v e d and the copper p r e c i p i t a t e d  as copper  sulfide  32 w h i l e the P  remains i n s o l u t i o n .  Decay of t h e radioelements law, the h a l f l i f e 5700 y e a r s .  of P  C, another  i s much more d i f f i c u l t source of neutrons  3 2  being lh.3 days w h i l e t h a t of C ^ i s 1  commonly used i s o t o p e i n metabolic work t o prepare, r e q u i r i n g a much more i n t e n s e  ( a c t i o n of a p i l e  i s formed by a complex mixture  1  Ih N  7  f o l l o w s the e x p o n e n t i a l  on b e r y l l i u m ) .  of r e a c t i o n s o n l y one of which i s  1  lk n  0  •  C  H  +  6  1  R a d i o a c t i v e compounds emit one or more of from t h e i r  Usually i t  ,J3 °- ^ p a r t i c l e s  nuclei.  p a r t i c l e s a r e helium n u c l e i , i . e . , helium atoms which have l o s t 2 e l e c t r o n s and hence bear  a double  p o s i t i v e charge.  o^C r a y s  are s t r o n g l y i o n i z i n g and weakly p e n e t r a t i n g . y<3 p a r t i c l e s are n e g a t i v e l y charged yO  p a r t i c l e s or e l e c t r o n s .  The  c^-rays.  r a y s a r e more p e n e t r a t i n g but l e s s i o n i z i n g than  B o t h c < a n d y 3 r a y s a r e d e f l e c t e d by e l e c t r i c and magnetic  fields.  ^ r a y s a r e h i g h l y p e n e t r a t i n g r a d i a t i o n s which are u n a f f e c t e d by e l e c t r i c and magnetic f i e l d s .  They have the same  nature but a h i g h e r frequency than X r a y s .  o< c*£ r a y s a r e emitted by such substances y S  r a y s are emitted by such substances  as P  3 2  as Na  and C **. 1  J  22 and Mg V  rays  31 are emitted by such substances as Na Both C*" and P 1  4  3 2  , Na ^, Co 2  , etc •  are purej3 emitters but C ^ 1  radiations are softer than those of P  3 2  .  The following comparisons  w i l l illustrate this: Energy of Radiation ( i n M i l l i o n cele'ctf o.ri. v o l t s )  1.718 Mev. 0.155 Mev. Although p3  2  emits no X  r a d i a t i ons, i n concentrated  solutions the p a r t i c l e s are retarded by absorption of the sample (a) i n the immediate v i c i n i t y of the atomic nucleus, such r e tardation gives r i s e t o X rays (called i n t e r n a l  Bremsstrahlung)  (b) loss of v e l o c i t y i n the surrounding atoms likewise leads to X r a d i a t i o n (external Bremsstrahlung).  This Bremsstrahlung  released by the l3 rays can amount to 1 Y> quantum per 25 particles. emissions make preparations incorporating t h i s compound more succeptible to autoabsorption so preparations of i n f i n i t e thinness are necessary. In use of photographic techniques, of the order of ,6  p a r t i c l e s must s t r i k e each square cm. of the emulsion  to produce a s a t i s f a c t o r y image.  32  MATERIALS AND METHODS  I.  Organism The  organism used throughout  Pseudomonas aeruginosa ATCC 9 0 2 7 .  these i n v e s t i g a t i o n s was  Stock c u l t u r e s were maintained  by l y o p h i l i z a t i o n of c e l l s i n a s t e r i l e menstruum of three p a r t s normal beef serum and one p a r t 7*5% glucose d i s s o l v e d i n peptone broth.  When r e q u i r e d , t h e d r i e d  organisms were suspended i n  s t e r i l e d i s t i l l e d water, p l a t e d on Standard Methods agar and an i s o l a t e d t y p i c a l c o l o n y i n o c u l a t e d i n t o the glucose medium o f N o r r i s and Campbell ( 1 1 2 ) , plus 0 . 1 $ yeast e x t r a c t .  Stock c u l t u r e s  f o r day to, day work were grown on l i v e r g e l a t i n s t o c k agar aid h e l d at 2°C u n t i l used.  C e l l s f o r h a r v e s t i n g were grown i n 100 ml. of  the glucose medium p l u s yeast e x t r a c t i n Roux f l a s k s , the c e l l s being c o l l e c t e d by c e n t r i f u g a t i o n a t 18 t o 20 hours. I I . Studies with C e l l Preparations 1.  Manometric S t u d i e s Oxygen uptake by c e l l - f r e e p r e p a r a t i o n s was s t u d i e d  u s i n g 3 . 0 ml. volume of r e a c t i o n mixture arm Warburg f l a s k .  side-  I n c u b a t i o n was c a r r i e d out i n a standard  G i l s o n c i r c u l a r Warburg apparatus was  i n a 15 ml. s i n g l e  a t 30°C.  An endogenous cup  r u n and a l l v a l u e s of oxygen uptake expressed have the c o r -  responding endogenous values s u b t r a c t e d . t y p i c a l p r o t o c o l f o r a Warburg r u n .  The f o l l o w i n g i s a  33 Endogenous M/15 Phosphate b u f f e r pH 7.0  Test  1.5 ml.  1.5 ml.  Glucose ( 5 ^ M / 0 . 2 ml.)  0.2 ml.  ( i n sidearm)  ATP (neutralized) (5^1/0.3 ml.) .  0.3 ml.  0.3 ml.  C e l l preparation (200 mg (wet weight)/ml.)  1.0 ml.  1.0 ml.  20% KOH  0.15 m l .  0.15ml.  D i s t i l l e d Water  t o t o t a l volume of 3.15 ml.  2.  ( i n centre well)  Chromatography of R e a c t i o n Mixtures (a)  Preparation i.  of s o l u t i o n s  For chromatographic s t u d i e s , l a r g e Warburg  cups containing a t o t a l volume of 10 ml. of r e a c t i o n mixture were used.  The cup contents were t r e a t e d as f o l l o w s :  at the completion of the r e a c t i o n 10% (1 ml.) of M A  Acetate  b u f f e r , pH 3.8 was added t o the cup contents which were then held o at 60 C f o r 5 minutes. the r e a c t i o n s .  This  p r e c i p i t a t e d the p r o t e i n and stopped  The p r o t e i n was removed by c e n t r i f u g a t i o n , the  supernatant n e u t r a l i z e d w i t h NaOH and concentrated i n a C r a i g f l a s h evaporator t o 2 ml. T h i s s o l u t i o n was t h e n used f o r f u r t h e r s t u d i e s w i t h paper chromatography f o r phosphate compounds. ii. strips  For attempts a t sugar  identifications,  of the phosphate paper chromatograms were c u t out and  e l u t e d w i t h 5 ml. d i s t i l l e d water.  T h i s e u l a t e was brought t o N/1  3^ b y a d d i t i o n o f HC1, h e l d a t 100°C f o r 3 0 m i n u t e s phosphate  and t h i s  t o remove  s o l u t i o n used f o r f u r t h e r c h r o m a t o g r a p h i c  studies. In a l l cases, #+ p a p e r were u s e d . off  d e s c e n d i n g chromatograms, u s i n g  I f t h e s o l v e n t was t o be a l l o w e d  Whatman  torun  t h e p a p e r , t h e l o w e r edge o f e a c h c h r o m a t o g r a m s h e e t  was  serrated.  (b)  Solvent i.  systems  For separating  phosphates - 7  Isopropanol Ammonium Distilled ii.  parts  hydroxide - 1 part - 2  water  For separating  parts  Sugars. - h  Butanol  parts  G l a c i a l a c e t i c a c i d - 1 part Distilled This  m i x t u r e was s h a k e n i n a s e p a r a t o r y  allowed used  - 5 parts  water  t o stand  f o r 2 hours.  funnel  The b u t a n o l  f o r 2 m i n u t e s and  ( u p p e r ) f r a c t i o n was  i n t h e t r o u g h a s t h e moving s o l v e n t , t h e w a t e r  was p l a c e d  i n t h e bottom  (c)  of the tank t o saturate  (lower) f r a c t i o n  the atmosphere.  Snravs i.  D e m o n s t r a t i o n o f phosphate  - Hanes and Isherwood  (w/w) p e r c h l o r i c a c i d N/1 h y d r o c h l o r i c  acid  (55)  - 1 part - 2  parts  h% (w/v) ammonium m o l y b d a t e  - 5 parts  Distilled  - 21 p a r t s  water  35 The  sheets were d r i e d a t room temperature,  sprayed w i t h the above  s o l u t i o n , suspended i n a 70 C oven f o r 10 minutes,  steamed w i t h  l i v e steam f o r 1 minute (without e x c e s s i v e w e t t i n g of the then exposed t o u l t r a v i o l e t l i g h t u n t i l the c o l o u r  paper),  developed.  Phosphate compounds y i e l d a c h a r a c t e r i s t i c b l u e c o l o u r . ii.  Demonstration  of adjacent h y d r o x y l groups -  V i s c o n t i n i , Hoch and K a r r e r D r i e d sheets were sprayed w i t h 0.h% metaperiodate minutes.  ( n e u t r a l i z e d w i t h NaOH) and l e f t t o stand  Care was  (1^6) sodium 10  taken t o j u s t cover the s u r f a c e w i t h the  and not t o a p p l y i t i n excess.  The  sheet was  then sprayed  spray with  a s o l u t i o n of 350 mg b e n z i d i n e i n 20 ml. g l a c i a l a c e t i c a c i d 80 ml. absolute a l c o h o l .  and  T h i s r e s u l t s i n a blue c o l o u r on the  sheet except where the p e r i o d i c a c i d i s used up i n o x i d i z i n g a c i s or t r a n s h y d r o x y l or a keto h y d r o x y l c o n f i g u r a t i o n . spots the blue c o l o u r w i l l not iii.  of Sugars.  Aniline  - 0.93  grams  P h t h a l i c a c i d - 1.66  grams  Water s a t u r a t e d b u t a n o l - 100  Macek and Tadra  suspended  I f present, the f o l l o w i n g substances  y i e l d these c h a r a c t e r i s t i c c o l o u r s : - brown  Ketohexose - brownish purple Pentose  (102)  ml.  The d r i e d sheets were sprayed and  Hexose  these  develop.  Demonstration  i n a 105°C oven f o r 5 minutes.  At  - ruddy red t o maroon.  36 3.  Q u a n t i t a t i v e E s t i m a t i o n of Pentose - 3 ml.  Stock s o l u t i o n 0.1$  Meijbaum (105)  F e C l ^ i n c HC1  + 10 mg/ml o r c i n o l ( o r c i n o l i s added j u s t p r i o r t o use) S o l u t i o n t o be t e s t e d - x ml ] y D i s t i l l e d water -{3-x)mlJ  T o t a l volume 3 ml. x i s adjusted t o f a l l w i t h i n the range of s e n s i t i v i t y of the t e s t .  The whole i s heated a t 100°C f o r 30 minutes and o p t i c a l d e n s i t y i s read a t 650 mjj i n a F i s h e r E l e c t r o p h o t o m e t e r .  The  machine i s zeroed on a blank c o n t a i n i n g 3 ml. s t o c k s o l u t i o n and 3 ml. water.  The o p t i c a l d e n s i t y i s converted t o micrograms of  pentose by a p p l i c a t i o n of the O.D. v a l u e s t o a standard  curve  prepared w i t h known d i l u t i o n s of a r a b i n o s e . if.  P r o t e i n D e t e r m i n a t i o n S u t h e r l a n d , C o r i , Haynes and Olsen (339) (a modified Kabat and Mayer technique  (75) To 0 . 5 ml. of a p p r o p r i a t e l y d i l u t e d p r o t e i n was added 3 . 0 mis. of a l k a l i n e copper l a t t e r c o n s i s t s of 2% CuSOk  2  CO3  solution.  The  ^  k% Na K t a r t r a t e I h% Wa  solution containing  made up j u s t p r i o r t o use.  J  T h i s mixture i s incubated 15 minutes a t U-5°C. F o l i n - C i o c a l t e a u phenol reagent the s o l u t i o n mixed immediately  Then 0 . 3 ml. of  ( d i l u t e d 1:3 w i t h water) i s added, by i n v e r s i o n and incubated a t room  37 temperature  f o r 15 minutes.  Readings were made a t 650 mJJ  the F i s h e r e l e c t r o p h o t o m e t e r .  in  The o p t i c a l d e n s i t y r e a d i n g s thus  obtained were a p p l i e d t o a standard curve and the corresponding values i n mg. p r o t e i n o b t a i n e d . t e s t i s 0 t o 150 % per tube  The range of s e n s i t i v i t y of t h i s  ( i . e . per 0 . 5 ml. d i l u t e d  protein  solution). III.  S t u d i e s w i t h Inorganic Phosphate 1.  P r e p a r a t i o n of the C e l l Free E x t r a c t F r e s h l y harvested c e l l s were washed once w i t h d i s t i l l e d  water and once w i t h d i s t i l l e d water c o n t a i n i n g 0 . 2 mg percent glutathione.  The supernatant was d i s c a r d e d and the wet c e l l  paste was packed i n t o open ended g l a s s tubes of an i n s i d e about 1mm. l e s s than the diameter One  diameter  of the hole i n the Hughes p r e s s .  end of these tubes was plugged w i t h a c o r k .  The c e l l s were  packed i n t o a h e i g h t equal t o between £ and ^ the depth of the hole i n the Hughes p r e s s .  The tube p l u s cork was weighed b e f o r e  and a f t e r packing w i t h c e l l s , thus determining the wet weight of the c e l l s .  A f t e r weighing, the c y l i n d e r s were corked a t the  top, and immersed i n an a l c o h o l - d r y i c e bath f o r q u i c k f r e e z i n g . The c y l i n d e r s were l e f t i n the bath 20 minutes t o i n s u r e a thorough f r e e z i n g and then s t o r e d a t -18°C u n t i l used.  C e l l s were always  used w i t h i n 21 days of h a r v e s t i n g . The Hughes press and p i s t o n were assembled and h e l d a t l e a s t 2h hours a t -18°C, t o come t o temperature.  After a f i n a l  38 t i g h t e n i n g of the Hughes press b l o c k , the end  corks were removed  from the tube of f r o z e n c e l l s , the o u t s i d e of the tube warmed byhand j u s t enough t o f r e e the c y l i n d e r of c e l l s from the g l a s s , and  the c y l i n d e r of f r o z e n c e l l s was  appropriate p i s t o n was  hole of the c h i l l e d  pushed i n t a c t i n t o the  Hughes press b l o c k .  The  cold  i n s e r t e d i n t o the hole on top of the c e l l s and  c e l l s crushed  by a p p l y i n g pressure  w i t h a Carver  hand operated  p i s t o n was  10 t o 12 thousand p . s . i . )  (about  hydraulic press.  d r i v e n t o the l i m i t  the  As soon as  of i t s path and  the  the f r o z e n  cells  had been f o r c e d through the i n t e r f a c e s i n t o the r e s e r v o i r 30 seconds) the b l o c k was was  the f r o z e n p r e p a r a t i o n  put i n t o an a p p r o p r i a t e volume of c h i l l e d d i l u e n t (2°C)  yield The  q u i c k l y opened and  (about  a f i n a l p r e p a r a t i o n of 200  crushed  mg.  wet  to  weight of c e l l s / m l .  c e l l s were mixed w i t h the d i l u e n t as g e n t l y as p o s s i b l e  w i t h a t e s t tube homogenizer. The  composition M/20 0.25M  of the d i l u e n t wassg l y c y l glycine sucrose  Bovine serum albumin (500  mg.  T h i s r e s u l t e d i n an extremely v i s c o u s presumably as a r e s u l t of DNA (113)  i n the 10 KC  sonic o s c i l l a t o r  broke up the The  (123)  preparation,  p o l y m e r i z a t i o n , however 15  yielding a centrifugable preparation. was  percent)  crushed  seconds  viscidity, cell  suspension  c e n t r i f u g e d at 3000 x g i n the c o l d f o r 7 minutes t o remove  39 s u r v i v i n g whole c e l l s and d e b r i s , and the supernatant (the subsequent c e l l f r e e p r e p a r a t i o n ) was c a r e f u l l y drawn o f f w i t h a s y r i n g e and h e l d i n i c e u n t i l use. were always made immediately 2.  Manometric  C e l l free  preparations  prior t o use.  Procedures  7 ml. Warburg cups were s e t up a c c o r d i n g t o the f o l lowing p r o t o c o l : (67)  1  Endogenous 50 p M/0.3 6 p M/0.3 M/20  ml. g l y c y l g l y c i n e \ „ „, ml. i n o r g a n i c p h o s p h a t e ) p  sucrose  diluent  0.3 ml. to b r i n g yolune  Test  0.3 m l . 1.0 ml.  Sodium f l u o r i d e 10^ M/0.05 ml.  0.05 ml.  0.05 ml.  Ma gnesium c h l o r i d e 5j/M/P.Q5 ml,  0.05 ml..  0.05 ml.  O.05 ml.  0.05 ml.  0.01 ml.  0.01 ml.  APP  ( n e u t r a l i z e d ) 2^ M/0.05 ml.  Cytochrome c 0.01y M/0.01 m l . Glue ose 10 y M/0.1  0.1  ml,  (sidearm)  C e l l free preparation  0.39 ml.  0.39 ml.  20% KOH  0.1  0.1  ml.  (centre well)  Cytochrome c i s claimed by Hunter (67) t o be necessary when crude mammalian t i s s u e p r e p a r a t i o n s a r e used, so i t was  1+0 I t i s noted, however, t h a t Kamen and Takeda (78)  i n c l u d e d here.  have r e p o r t e d t h a t the cytochrome c from P. a e r u g i n o s a w i l l n o t f u n c t i o n i n mammalian systems and e x h i b i t s a d i f f e r e n t amino a c i d spectrum  than i t s mammalian c o u n t e r p a r t .  Oxygen uptake was measured on each i n d i v i d u a l cup and a t the a p p r o p r i a t e time the cup and i t s corresponding endogenous cup were removed, and contents p i p e t t e d i n t o 0.1 m l . i c e c o l d h0% t r i c h l o r a c e t i c a c i d t o p r e c i p i t a t e p r o t e i n and stop the reaction.  0.2^  ml. N/1 sodium a c e t a t e was added t o b r i n g the pH  t o h-.O ( t o preserve l a b i l e e s t e r s ) and the supernatatant t e s t e d f o r the presence  of i n o r g a n i c phosphate.  In cases where something l i k e DNP was i n c l u d e d i n t h e r e a c t i o n mixture, corresponding adjustments  were made i n t h e  sucrose d i l u e n t t o m a i n t a i n a f i n a l volume of 1.0 ml. 3«  Measurement of I n o r g a n i c Phosphate  Lowry and Lopez  (100)  Bruemmer and O ' D e l l  (20)  The r e a c t i o n i s s e t up as f o l l o w s , and I n the order shown, i n a 1 ml. quartz spectrophotometer  cuvette.  Test s o l u t i o n ( a p p r o p r i a t e l y d i l u t e d  - 1 ml.  w i t h a c e t a t e b u f f e r , pH ^-.0) 1% Ammonium molybdate i n 0.05  N H  2  1% a s c o r b i c a c i d Copper sulphate (0.5 /> M/0.1  SO^ - 0.1 ml. - 0.1 ml.  ml.)  - 0.1 ml.  Bruemmer and O ' D e l l (20) r e p o r t e d t h a t i n t h i s the presence  technique  of s u l f h y d r y l compounds w i l l i n h i b i t c o l o u r development.  The  presence of 0 . 5  p M CuSOk prevents t h i s  inhibition.  Timing began w i t h the a d d i t i o n of the a s c o r b i c a c i d . Readings were made at 660 m i l l i m i c r o n s i n the Beckman DU photometer at 5 and 10 minutes. back t o zero time.  These v a l u e s were e x t r a p o l a t e d  Each t e s t was  run i n p a r a l l e l w i t h a t e s t  a standard s o l u t i o n c o n t a i n i n g 0 . 0 5 ml. was  spectro-  on  y M i n o r g a n i c phosphate per  Any f l u c t u a t i o n i n the e x t r a p o l a t e d value of the a p p l i e d t o t h a t corresponding t e s t v a l u e .  standard  The e x t r a p o l a t e d  o p t i c a l d e n s i t y v a l u e s were a p p l i e d t o a standard curve and corresponding v a l u e in^/M i n o r g a n i c phosphate o b t a i n e d .  the  Values  of i n o r g a n i c phosphate present per 1 ml. o r i g i n a l r e a c t i o n mixture were obtained as f o l l o w s : Volume of cup contents Volume of added TCA and acetate T o t a l volume c o n t a i n i n g 6 y M i n o r g a n i c phosphate less precipitate  found t h a t 0.13  buffer  (pH h,0)  was  -1.3 ml. -0.0*f ml. k  ml.  ml. of t e s t s o l u t i o n p l u s 1.87  ml. a c e t a t e  gave a p r e p a r a t i o n which y i e l d e d v a l u e s w i t h i n  the l i m i t s of the standard c u r v e . (x 2 x 10)  -0.3^m]»  1.3  Test s o l u t i o n I t was  - 1 ml.  Therefore a f a c t o r of  20  a p p l i e d t o the v a l u e s obtained from the standard  curve t o y i e l d ^ M phosphate per ml. r e a c t i o n mixture. are the v a l u e s expressed  i n the  curves.  These  h2 IV.  Studies w i t h P h o s p h o r o u s 1.  32  I n c u b a t i o n Procedure The  i n c u b a t i o n mixtures were s e t up as f o l l o w s : Endogenous  G l y c y l g l y c i n e (50 + 6  M inorganic  M/20  M/0.3  ml)  phosphate/0.3 ml.  sucrose d i l u e n t  Glucose  3.0  Test  3.0  ml.  t o y i e l d a f i n a l volume of  (10y^M/0.1 ml.)  ml.  10.0 ml. 1.0 ml.  NaF (10/u M/0.05 ml.)  0.5 m l .  0.5 ml.  (5 fj M/0.05 ml.)  0.5 ml.  0.5 ml.  0.5 m l .  0.5 ml.  0.1 ml.  0.1 ml.  0.5 ml.  0.5 ml.  0.1 m l .  0.1 ml.  extract  3.9  3.9  I n subsequent experiments, 0.2  ml. i n o r g a n i c  MgCl  2  ADP (2 yt/ M/0.05 ml.) Cytochrome C DNP ATP  (1 3  M/0.1 ml. ) 2  C e l l free  was  (0.01 p M/0.01 ml.)  s u b s t i t u t e d f o r the A T P The  3 2  ml.  ml.  phosphate  0  and DNP was o m i t t e d .  i n c u b a t i o n mixture was measured i n t o a chromate  cleaned 50 ml. Erlenmeyer f l a s k and i n c u b a t e d , w i t h shaking, on a B u r r e l l w r i s t a c t i o n shaker.  From t h i s f l a s k , a l i q u o t s (1 or 2 ml.)  k  were removed a t i n t e r v a l s , or i n l a t e r experiments the e n t i r e ml. was  p r e c i p i t a t e d a t 30  3  10  minutes.  P r e c i p i t a t i o n of p r o t e i n was  e f f e c t e d i n one of  two  ways:i.  Acid  Precipitation  10% of i c e c o l d h0% TCA was r e a c t i o n mixture then the pH was  added t o the  quickly raised to . 0 k  w i t h the  a d d i t i o n of 2h% M / l sodium a c e t a t e . ii.  Alcohol Precipitation To each volume of r e a c t i o n mixture was  5 volumes of c o l d e t h a n o l .  The whole was  added  then held a t -17°C f o r  8 t o 12 hours.  Precipitated  The a l c o h o l was  removed from the supernatant by e v a p o r a t i o n under  p r o t e i n was  removed by c e n t r i f u g a t i o n .  vacuum i n the f l a s h e v a p o r a t o r . 2.  Counting Procedure The m a t e r i a l ( u s u a l l y 0 . 2  t o an unlacquered, uncorked  suspended  pipetted  s t e e l pressure type b o t t l e cap and  d r i e d under an i n f r a r e d lamp. counted, i t was  ml.) t o be counted was  I f a c h a r c o a l r e s i d u e was  t o be  i n e t h a n o l and poured i n t o a p l a n c h e t .  R e s i d u a l c h a r c o a l was washed from the tube w i t h more ethanol. Counts were made u s i n g a T r a c e r l a b model changer and a B e r k e l e y Decimal S c a l e r Model 2 1 0 5 . expressed i n counts per minute  i n each c a s e .  v a l u e s the number of counts per h minutes was by h t o o b t a i n a b e t t e r average v a l u e .  SC9D  manual  Results are  To o b t a i n these t a k e n and d i v i d e d  In the case of extremely  a c t i v e m a t e r i a l , the time r e q u i r e d f o r a minimum of 1000 counts was taken and converted t o counts per minute.  From the v a l u e s so  obtained the counts per minute obtained w i t h a c l e a n p l a n c h e t , and termed h e r e i n a f t e r "background", were s u b t r a c t e d .  Such back-  ground was determined  procedures  each time a s e r i e s of c o u n t i n g  were c a r r i e d o u t . 3»  P r e p a r a t i o n of the Dowex Column Dowex 1, x 10 r e s i n , 200 t o kOO mesh was washed r e -  p e a t e d l y and t h e f i n e s removed by s e t t l i n g .  The r e s i n was  shaken w i t h N/1 HC1, the r e s i n allowed t o s e t t l e , and the supernatant a c i d removed.  The r e s i n was washed r e p e a t e d l y w i t h  d i s t i l l e d water u n t i l the washings gave no d e t e c t a b l e p r e c i p i t a t e upon the a d d i t i o n of %  aqueous s i l v e r n i t r a t e .  A suspension of  the r e s i n was placed i n a 1 cm. standard g l a s s column and allowed to s e t t l e .  The h e i g h t of the r e s i n i n the column was 2.5 cms.  At no time was the column allowed t o r u n d r y , a minimum head of \ i n c h of l i q u i d being kept over the r e s i n .  The r a t e of the flow  from the column was r e g u l a t e d by a d j u s t i n g the stopcock. •  *+.  Charging and E l u t l o n of the Column The  concentrated i n c u b a t i o n mixture was brought  and c a r e f u l l y i n s e r t e d  i n t o t h e column.  through at about 3 ml. per minute. r e s i n would be uncovered  I t was allowed t o flow  J u s t before the top of t h e  water was added and allowed t o flow  through t o remove any unadsorbed m a t e r i a l . When checking t o ensure  t o pH 8.0  effluents  t h a t the r a d i o a c t i v e m a t e r i a l was being h e l d up by the  h$ r e s i n , 2 ml. samples were d r i e d and counted.  When e l u t i n g mix-  t u r e s , e t c . were added t o the column, care was taken t h a t the r e s i n was not d i s t u r b e d . (a)  Alkaline Elutlon The  and  Khym and Cohn  (83)  e l u t i n g s o l u t i o n s were made up i n the volumes  sequence shown i n the f o l l o w i n g  chart.  Included a l s o a r e  the compounds Khym and Cohn r e c o v e r e d w i t h each e l u t i n g s o l u t i o n . The  s o l u t i o n s were placed  i n a r e s e r v o i r over the column and  allowed t o flow by g r a v i t y through the r e s i n a t about 1 ml. per minute.  The e l u e n t  was c o l l e c t e d i n 5 ml. q u a n t i t i e s by a G i l s o n  volumetric f r a c t i o n c o l l e c t o r .  0 . 2 ml. a l i q u o t s of the tube  contents were d r i e d and counted.  k6 Composition. Volume and Sequence Eluting  0.001  1.  Solution  Volume used  M  50 ml.  NHLOH  of E l u t i n g S o l u t i o n s Substance Recovered free glucose  0.025 M NHkCl  2 . 0 . 0 0 1 M NHkOH  200 ml  glucose 1 phosphate i n o r g a n i c phosphate  300 ml  glucose 6 phosphate  250 ml.  f r u c t o s e 6 phosphate r i b p s e 5 phosphate  5. 0.03 M NHkCl  150 m l .  r i b o s e 5 phosphate  6. 0.005 M HCl  200 ml.  AMP  7. 0 . 0 1  200 ml  2 phosphoglyceric a c i d  0.01  M Na Bk07 2  0.025 M NH^Cl 3. 0.0025 H NHkOH  0.001 M Na Bi .0y 2  4  0.025 M N H L C I h. 0.0025 M NHkOH 0.00001 M N a B 0 2  8.  M HCl  k  7  ADP  HCl KCl  150 ml.  f r u c t o s e 1:6 diphosphate  0.02 M H C l 0.2 M K C l  150 m l .  ATP  0.02 0.02  M M  (b)  Acidic Elutions Gradient  the f o l l o w i n g diagram  A  e l u t i o n apparatus was s e t up as shown i n (12 ). k  -  ll  1 lb- air pressure  \  Flask 1  AK  -  \  1  i  to column  Flask 2 magnetic stirrer  i  In a l l cases, the e l u e n t from the column was c o l l e c t e d a t the r a t e of 1 ml./minute, i n 1G ml. f r a c t i o n s by a Technicon fraction collector.  Counting procedures were c a r r i e d  previously described.  260 m f  a b s o r p t i o n was checked  out as on 1 ml.  a l i q u o t s e i t h e r i n the Beckman model DU or the Carey r e c o r d i n g spectrophotometer.  The g r a d i e n t e l u t i n g systems used were as  follows: i.  Acid Lithium Chloride F l a s k 1 contained  (12 ) k  500 ml. 0 . 5 M l i t h i u m  chloride i n 0.003 N HCl. F l a s k 2 contained  500 ml. 0 . 0 0 3 N H C l .  I n a l a t e r experiment, the c o n c e n t r a t i o n of l i t h i u m c h l o r i d e was c u t t o 0.3  M.  I n t h i s g r a d i e n t , i n o r g a n i c phosphate comes o f f v e r y q u i c k l y and DDPG comes o f f about 0.06 M l i t h i u m c h l o r i d e . ii.  D i s t i l l e d Water-Acid F l a s k 1 c o n t a i n e d 0.01 N H C 1 . F l a s k 2 c o n t a i n e d d i s t i l l e d water.  5.  Acetone P r e c i p i t a t i o n of N u c l e o t i d e s  (106)  The samples c o n s t i t u t i n g the peak a r e a s of the curve were pooled and c o n c e n t r a t e d t o c i r c a 1 ml. i n the f l a s k evaporator.  The c o n c e n t r a t e was t r a n s f e r r e d t o a g l a s s  centrifuge  tube, a vacuum p u l l e d on the tubB through a d r y i c e condenser and the samples evaporated t o d r y n e s s .  The m a t e r i a l was  solved and suspended i n 0.5 ml. c o l d methanol,  dis-  h ml. c o l d  acetone was added and the whole kept a t -17°C f o r 1 hour t o a l l o w p r e c i p i t a t i o n t o complete.  A white c r y s t a l l i n e  precipitate  was formed which upon i s o l a t i o n by c e n t r i f u g a t i o n , a n d washing w i t h acetone was  found t o be water s o l u b l e .  Presumably here the  l i t h i u m s a l t s of sugar phosphates are s o l u b l e i n acetone but the l i t h i u m s a l t of the n u c l e o t i d e s are n o t .  A,water i n s o l u b l e  grey p o r t i o n of the p r e c i p i t a t e was a l s o noted.  dirty  EXPERIMENTAL AND DISCUSSION  I.  Studies with C e l l Preparations E a r l i e r work i n t h i s l a b o r a t o r y by Campbell and N o r r i s  ( 1 1 2 ) ( 2 2 ) , subsequently supported by Wood and Schwerdt's (152) s t u d i e s of Pseudomonas f l u o r e s c e n s . has shown t h a t Pseudomonas aeruginosa,  s t r a i n A.T.C.C. 9027 o x i d i z e s glucose by way of a  pathway i n v o l v i n g g l u c o n i c and 2 k e t o g l u c o n i c a c i d s . c e l l s o f t h i s organism  Resting  o x i d i z e glucose t o C O 2 and water as deter'  mined by manometric measurements.  I f , however, the c e l l s a r e  d i s r u p t e d , f o r example by s o n i c o s c i l l a t i o n the uptake of oxygen stops a t two atoms, and 2 ketogluconate accumulates (Stokes and Campbell) ( 1 3 7 ) .  Campbell, Ramakrlshnan, Linnesand  Eagles  (25)  were unable t o demonstrate concurrent p h o s p h o r y l a t i o n i n these reactions.  I n s p i t e of t h e i r d a t a , i t i s obvious t h a t phosphory  l a t i o n must p l a y a r o l e i n the i n t e r m e d i a t e metabolism o f t h i s organism. The g e n e r a l aim of the present i n v e s t i g a t i o n was t o es t a b l i s h the i n c o r p o r a t i o n of phosphorous i n t o t h e metabolic systems.  I n the i n i t i a l s t a g e s , attempts  were made t o study t h i ;  problem using s u b s t r a t e s such as 2 ketogluconate and g l u c o s e . I t became apparent  however, t h a t the i n c o r p o r a t i o n of phosphate  i n t o the metabolic pathways of the organism t a b l i s h e d i n the absence of added substrate..  should f i r s t be e s -  50 1.  Manometric  Studies  With t h i s problem i n mind attempts were made t o o b t a i n enzyme p r e p a r a t i o n s which would ketogluconate l e v e l .  o x i d i z e g l u c o s e beyond the 2  I f s u c c e s s f u l , t h i s approach would  s e v e r a l advantages.  have  F i r s t l y , i t would presumably be p o s s i b l e t o  i d e n t i f y the end product and e s t a b l i s h the pathway of o x i d a t i o n . Secondly, t h i s would mean t h a t i t would be p o s s i b l e t o a s s o c i a t e phosphate uptake w i t h known m e t a b o l i c r e a c t i o n s .  F i n a l l y the  use of a system which i s capable of more complete o x i d a t i o n of the  s u b s t r a t e would  o f f e r a b e t t e r chance of demonstrating  p h o s p h o r y l a t i o n than Campbell, Ramakrishnan and co-workers had. Many techniques are a v a i l a b l e f o r the d i s i n t e g r a t i o n of b a c t e r i a l cells  (50)  but o n l y a few of what would appear t o be the more  promising ones were used h e r e . 1.  Sonic o s c i l l a t i o n  B r i e f l y , these were:-  Whole c e l l suspensions of Pseudomonas  aeruginosa were made and subjected t o d i s i n t e g r a t i o n i n the 10 KC sonic  oscillator. (a)  200 mg.  (wet weight) b a c t e r i a per ml M/30  phosphate  b u f f e r pH 7.0 were exposed t o o s c i l l a t i o n f o r 20 minutes. (b)  100 mg  (wet weight) b a c t e r i a per ml. b u f f e r plus  alumina f o r 5 minutes. (c)  Techniques (a) and  ( b ) , the c e l l s being soniced i n the  presence of 5 nig percent g l u t a t h i o n e plus 7 percent sucrose. 2.  S a t u r a t i o n of a c e l l suspension w i t h an atmosphere  of N , 2  N0  ?  r  51 or CO under 100 and 200  pounds p r e s s u r e , and subsequent i n s t a n -  taneous r e l e a s e of t h i s  pressure.  3*  Drying of c e l l p r e p a r a t i o n s over a d e s i c c a n t i n an  atmosphere of N 0  2  or  CO.  L y o p h i l i z a t i o n of c e l l s t o complete or p a r t i a l Techniques of the c e l l w a l l .  evacuated  dryness.  3 and h were aimed at a l t e r i n g the p e r m e a b i l i t y  That t h i s was  accomplished  was  evidenced  by  the f a c t t h a t the d r i e d c e l l s would o x i d i z e sodium c i t r a t e when f r e s h whole c e l l s would not.• T h i s organism c y c l e (Campbell  exhibits a  Krebs  1  and S t o k e s ) ( 2 3 ) hence c i t r a t e i s i n i t s c o n s t i -  t u t i v e metabolic pathway but because of the i n a b i l i t y of t h i s s u b s t r a t e t o penetrate the c e l l membrane, whole c e l l s do not oxidize i t .  F i g u r e 1, which i s a composite  curve of two e x p e r i -  ments, shows the i n f l u e n c e of d r y i n g on the a b i l i t y of a suspension t o metabolize  cell  citrate.  The methods of d i s r u p t i n g or a l t e r i n g the p e r m e a b i l i t y of the c e l l s d i d not, however, prove f r u i t f u l , and no  satisfactory  p r e p a r a t i o n s which would take up more than 2 atoms of oxygen on glucose were o b t a i n e d .  Attempts t o i n h i b i t metabolism w i t h  pyocyanin a l s o r e s u l t e d i n i n h i b i t i o n o n l y at the 2  ketogluconate  i  level.  .4 '• 2.  Chromatography S t u d i e s The  c o n c l u s i o n t h a t the pathway of glucose o x i d a t i o n  i n t h i s organism two  does not i n v o l v e p h o s p h o r y l a t i o n d u r i n g the  o x i d a t i v e steps d i f f e r e n t i a t e s i t from w i d e l y d i s t r i b u t e d  first  250  Figure  1:  The i n f l u e n c e of d r y i n g upon the a b i l i t y of P. aeruginosa t o o x i d i z e added x  x  whole  o  o  lyophilized  citrate.  cells cells  52 pathways passing through glucose-6-phosphate.  This observation  a l s o leads one t o wonder where the phosphate does become i n v o l v e d i n i t s pathway, s i n c e high energy phosphate t r a n s f e r i s the mechanism of energy t r a n s f e r , and c a t a b o l i s m of i s the organises c h i e f energy source.  I n 1955  carbohydrates (79)  Katznelson  presented some data which i n d i c a t e d t h a t the a d d i t i o n of a phosphate donor (ATP)  plus TPN  to a c e l l f r e e system of Pseud;omonas  a n g u l a t a would r e s u l t i n g r e a t e r oxygen uptake on glucose  and  gluconate.  prepar-  Attempts t o d u p l i c a t e these r e s u l t s w i t h c e l l  a t i o n s of 9027 were u n s u c c e s s f u l but the q u e s t i o n was  posed as t o  whether there might p o s s i b l y be p h o s p h o r y l a t i o n o c c u r i n g without further oxidation.  In an attempt  t o determine  compound accumulated, paper chromatography was  if a  phosphorylated  used.  Large  Warburg cups c o n t a i n i n g a t o t a l volume of 10 ml»were used  as  r e a c t i o n v e s s e l s and were incubated by shaking i n a 30°C water bath u n t i l oxygen uptake, measured i n a p a r a l l e l standard 15 cup c o n t a i n i n g 3 ml. of r e a c t i o n mixture, l e v e l l e d at  2 atoms).  ml.  o f f (as always  I n a l l cases r e a c t i o n s w i t h glucose ( t e s t ) and  the absence of glucose (endogenous) were r u n i n p a r a l l e l .  In  The  contents were a c i d p r e c i p i t a t e d , the supernatant n e u t r a l i z e d concentrated as d e s c r i b e d i n M a t e r i a l s and Methods.  The  and  resultant  t e s t s o l u t i o n s and a stock s o l u t i o n of ATP were spotted upon Whatman # f f i l t e r  paper.  Descending  chromatograms were run u s i n g  the isopropanol-ammonium hydroxide-water s o l v e n t was  s o l v e n t system.  The  allowed t o r u n o f f the paper f o r a p e r i o d of 2h  to  32 hours i n order to o b t a i n maximum s e p a r a t i o n of the slow moving  53 p h o s p h a t e compounds, R f v a l u e s w e r e t h e r e f o r e u n o b t a i n a b l e . The v a l u e s e x p r e s s e d a r e t h e d i s t a n c e s i n ems. t h a t t h e l e a d i n g ( f r o n t ) edge o f e a c h s p o t t r a v e l l e d chromatograms were d e v e l o p e d  obtained using these techniques.  m a t e r i a l as i t i s developed characteristic  line.  using the acid-molybdate  F i g u r e 2 i s a diagram  the p e r i o d a t e spray. picture  from the s t a r t i n g  s p r a y and  of the chromatographic  The b e h a v i o u r  using the acid-molybdate  of i t s n a t u r e and t h i s t e c h n i q u e w i l l  of a  spray i s often yield  v e r y v a l u a b l e c l u e s as t o t h e n a t u r e o f m a t e r i a l s b e i n g with.  dealt  T h e s e c h a r a c t e r i s t i c s a n d what t h e y may i n d i c a t e a r e h e r e  b r i e f l y described i ntabular  Behaviour  Spot Colour  The  form.  Table 1 o f C e r t a i n Compounds When S p r a y e d Acid-Molybdate Spray  with  When i t F i r s t A p p e a r s  Behaviour  i sCharacteristic of:  yellow  i m m e d i a t e l y upon spraying  inorganic  phosphate  yellow  u p o n h e a t i n g t h e p a p e r i n o r g a n i c p h o s p h a t e o r maybe a l a b i l e (1) p h o s p h a t e  yellow  upon  blue  upon u l t r a v i o l e t irradiation  steaming  In t h i s  weakly  bound  phosphate  s t r o n g l y bound  phosphate  case, a f a i n t y e l l o w c o l o u r appeared  upon s p r a y i n g t h e s h e e t s .  immediately  T h i s c o l o u r was g r e a t l y i n t e n s i f i e d b y  h e a t i n g and steaming  and hence, a l a b i l e phosphate contaminated  i n o r g a n i c phosphate,  was s u s p e c t e d .  by  Acld-Molybdofe Endogenous  Sproy ATP  Test o  •o .  Note:  3 l/2"  excessive /( t r a i l i n g  f  I  I Y e l l o w blue Spots  ^unknown *  Periodote-Benzidine Endogenous o  jest o  Spray  ATP  0  .5'  •7"  B f u e background . ' White, S p o t s ' ;  Figure 2.  •  ._'  .*unkriaw.n"* . •  ._  .J  ^_  ' . '_  Chromatograms of i n c u b a t i o n mixtures of P^ a e r u g i n o s a i n the presence  of g l u c o s e and ATP.  The  p e r i o d a t e spray, which upon s p r a y i n g w i t h  benzidene  y i e l d s a blue c o l o u r , except where the p e r i o d i c a c i d has  been  used up i n o x i d i z i n g c i s hydroxyls (or t r a n s a t a slower r a t e ) i n d i c a t e d t h a t there i s a compound, e x h i b i t i n g a d j a c e n t h y d r o x y l groups, e x a c t l y corresponding t o the phosphate s p o t .  T h i s spot  i s designated i n F i g u r e 2 and h e r e i n a f t e r as "unknown". i n the diagram, t h i s spot appeared  As shown  both i n endogenous and  test  reactions. The problem now  was  t o i s o l a t e , and  i f possible,  identify  t h i s unknown m a t e r i a l and then t o o b t a i n something of a q u a n t i t a t i v e p i c t u r e on i t ,  i n order t o a s c e r t a i n whether or not glucose  c o n t r i b u t i n g t o the amount of t h i s m a t e r i a l which I n other words, was  was  accumulated.  i t a product of glucose metabolism.  u n s u c c e s s f u l attempts were made t o separate the crude  Several  reaction  mixture by column chromatography, u s i n g c h a r c o a l and Dowex 1 ( c h l o r i d e form).  I t was  c o n s i d e r e d b e t t e r t o t r y and e l u t e the  compound from a paper chromatogram and thence  obtain further i n -  f o r m a t i o n as t o i t s n a t u r e . R e a c t i o n mixture of Whatman  filter  ( u s u a l l y 0.5 ml) was  c o n c e n t r a t e s were streaked a c r o s s sheets  paper r e p e a t e d l y u n t i l a measured amount d i s t r i b u t e d i n one f i n e l i n e .  The  sheets  :  were r u n as p r e v i o u s l y d e s c r i b e d and upon d r y i n g , a s t r i p , approximately the s h e e t s .  one q u a r t e r i n c h i n width was These s t r i p s were developed  cut from each s i d e of  u s i n g the a c i d molybdate  spray and the p o s i t i o n of the spots marked.  The  s t r i p s were placed  55 a l o n g s i d e the parent sheets, which were marked a t the p o s i t i o n of the spots on the s t r i p s a t each s i d e , the a p p r o p r i a t e a r e a cut out of the sheet and e l u t e d w i t h d i s t i l l e d water. was brought  t o IN w i t h h y d r o c h l o r i c a c i d and h e l d a t  30 minutes,  t o hydrolyze o f f the phosphate.  was spotted on Whatman #+ f i l t e r  The e l u a t e  100°C for*  The r e s u l t i n g  solution  paper and r u n w i t h the b u t a n o l -  a c e t i c a c i d - water s o l v e n t a g a i n s t known s u g a r s . systems were t r i e d but the butanol-acetate-water  Several solvent gave the best  s e p a r a t i o n of the known compounds and was the one subsequently used.  The chromatograms were developed  a c i d spray.  u s i n g the a n i l i n e - p h t h a l i c  T h i s spray g i v e s a d i f f e r e n t i a l c o l o u r r e a c t i o n  w i t h hexoses y i e l d i n g a brown c o l o u r , k e t o h e x o s i s a p u r p l i s h c o l o u r , and pentoses a maroon c o l o u r . chromatograms a r e g i v e n i n Table  T y p i c a l r e s u l t s of these  2.  Table 2 R e s u l t s of Chromatography of A c i d Treated Unknown Spot Colour o f Spot  Compound  RF Value  Glucose  brown  29.5A7 = 0.628  2 ketogluconate  purple brown  2V.OA7.5 = 0.505  Etibose  maroon  25.lA2.00 = 0.597  Arabinose  maroon  22.3A2.5 = 0.525  maroon  2 2 A A 2 . 5 =. 9.527  Test s o l u t i o n  0  56 Other  pentoses  t e s t e d i n t h i s s o l v e n t system ( x y l o s e ,  r i b u l o s e ) e x h i b i t e d a higher R f value than r i b o s e . of these experiments  the hexoses and pentoses,  As a r e s u l t  other than r i b o s e  and arabinose which were c l o s e s t t o the unknown were d i s r e g a r d e d . The next step was t o mix together samples of (a) r i b o s e p l u s unknown and (b) arabinose p l u s unknown.  These two mixtures were  spotted on paper and r u n as p r e v i o u s l y d e s c r i b e d . the 2 compounds was e f f e c t e d  o n l y i n the r i b o s e - unknown  the arabinose - unknown mixture  t r a v e l l i n g as one s p o t .  were comparable t o those d e s c r i b e d i n Table 2 . i n d i c a t i v e of a pentose  S e p a r a t i o n of mixture, Rf values  The r e d d i s h c o l o u r ,  of the spot obtained from the unknown,  coupled w i t h i t s s i m i l a r i t y of R f , i n t h i s s o l v e n t , t o arabinose l e d t o the assumption, that the sugar moiety  f o r purposes  of f u r t h e r  of the unknown spot was  experimentation, arabinose.  To o b t a i n a q u a n t i t a t i v e p i c t u r e of t h i s pentose  apparent  f r a c t i o n , e l u a t e s from the q u a n t i t a t i v e " s t r i p " chromato-  grams d e s c r i b e d e a r l i e r were subjected t o the o r c i n o l - f e r r i c c h l o r i d e method of pentose  determination.  Q u a n t i t a t i v e measure-"  ments were made by measuring the o p t i c a l d e n s i t y of an a l i q u o t of the r e a c t i o n mixture a t 650 m i l l i m i c r o n s i n a F i s h e r e l e c t r o p h o t o meter, and comparing these readings w i t h a standard curve from fenown d i l u t i o n s of a r a b i n o s e . g a t i o n i t was found  prepared  I n t h i s f a c e t of the i n v e s t i -  impossible t o o b t a i n s a t i s f a c t o r y d u p l i c a t e  quantitative r e s u l t s .  57 The r e a s o n f o r the f a i l u r e of the q u a n t i t a t i v e chromatography t o y i e l d reproducable r e s u l t s may s e p a r a t i o n that was was  be l a i d t o the poor  obtained when the i n i t i a l r e a c t i o n mixture  chromatographed.  As has been pointed out i n F i g u r e 2 ,  presence of i n o r g a n i c phosphate i n excessive t r a i l i n g .  the  i n the crude p r e p a r a t i o n r e s u l t e d  Moreover the s h o r t d i s t a n c e s of t r a v e l  of the compounds, even a f t e r long p e r i o d s of r u n M n g , decreased the p o s s i b i l i t y of s e p a r a t i o n .  Consequently, when the s t r i p  cut from the sheet, i n s t e a d of c o n t a i n i n g a r e l a t i v e l y  was  pure  compound, i t undoubtedly contained a mixture of i n c o m p l e t e l y separated substances.  T h i s would decrease the accuracy of the  q u a n t i t a t i v e measurements. I t became suspect t h a t t h i s pentose may i n the metabolic p i c t u r e as had been thought. see i f i t was  present as a contaminant  a c t i v i t y an experiment was  or was  s e t up as f o l l o w s :  f o l l o w i n g s o l u t i o n s were brought t o N/1  not be  I n an attempt  to  a r e s u l t of m e t a b o l i c Samples of the  with hydrochloric  and placed a t 100°C f o r the times i n d i c a t e d .  involved  acid  They were n e u t r a l i z e d ,  spotted on paper and r u n as before w i t h b u t a n o l - a c e t i c acid-water s o l v e n t a g a i n s t r i b o s e and arabinose and developed w i t h the a n i l i n e phthallc acid spray. The a c t i o n of adenosine, a d e n y l i c a c i d , ATP and the unknown were compared i n the absence  of a c i d a t zero time, and  i n the presence of the a c i d a t zero time, 5 and 30  minutes.  58 Table 3 R e s u l t s of Chromatography of R e a c t i o n A d d i t i v e s Treatment  Adenosine  No a c i d  Adenylic  nil  Acid 0 time  acid  nil  as f o r 0 time  from the added ATP  chromatographic  The  nil  a r a b i n o s e spots a r a b i n o s e p r e s e n t , r i b o s e spots spots appear present r i b o s e spots appear  A p p a r e n t l y then, t h i s pentose was  1.  nil  As f o r zero t i me  30 minutes  sprays.  Unknown  one spot one spot one spot one spot corresponding c o r r e s p o n d i n g corresponding corresponding t o r i b o s e one t o a r a b i n o s e to arabinose t o a r a b i n o s e spot c o r r e s ponding t o arabinose  5 minutes  taminant  ATP  Two  As f o r 0  i n t r o d u c e d as a  time  con-  and cannot be c o r r e l a t e d w i t h the  data obtained w i t h the phosphate and  periodate  p o s s i b l e e x p l a n a t i o n s of the problem a r e : -  pentose  contaminant  phosphate, t h i s combination  moves f o r t u i t o u s l y w i t h i n o r g a n i c producing the "unknown" spot  and  j u x t a p o s i t i o n of the p e r i o d a t e and molybdate r e a c t i o n s r e s p e c t i v e l y . T h i s s u g g e s t i o n however does not answer the d a t a of the p e r i o d a t e sprayed  sheets where i t i s seen t h a t there i s no  "unknown" spot i n the ATP demonstratably  present.  column wherein  corresponding  t h i s contaminant  is  2.  The pentose contaminant moves in the  isopropanol-ammonium  hydroxide s o l v e n t a t the same r a t e or near i t ,  as does ATP and  hence i s i n d i s t i n g u i s h a b l e from i t w i t h the p e r i o d a t e s p r a y . The "unknown" spot i s a complex  of some s o r t , c o n t a i n i n g  and a c i s h y d r o x y l s t r u c t u r a l component. the complex  phosphate  I f t h i s i s a sugar,  i s a c i d s t a b l e and hence u n d e t e c t a b l e on subsequent  chromatography.  The a r a b i n o s e - l i k e compound was  the contaminant  introduced w i t h the ATP and o v e r l a p p i n g i n t o the a r e a cut out of the chromatograms. The d a t a w i t h phosphate spray a l t h o u g h throwing no l i g h t upon p o s s i b l e mechanisms, d i d seem t o i n d i c a t e t h a t  phos-  phorous was  This  i n v o l v e d i n the mechanisms of t h i s organism.  data does not, however, n e c e s s a r i l y suggest t h a t t h i s involvement, i f i t occurs, i s a r e s u l t of glucose o x i d a t i o n . II.  S t u d i e s With Inorganic I t was  Phosphate  obvious a t t h i s stage t h a t the methods of  approach h e r e t o f o r e used l e d t o too many u n c o n t r o l l a b l e i n the experiments so another approach was  facets  instituted.  Since involvement of phosphate was  i n d i c a t e d , i t was  l o g i c a l t o f o l l o w the f a t e of i n o r g a n i c phosphate when t h i s o r ganism i s placed i n the presence of a s u b s t r a t e .  Furthermore,  since the mechanism f o r the t r a n s f o r m a t i o n of low energy i n o r g a n i c phosphate t o h i g h energy organic phosphate s u i t a b l e t o a c t as a phosphate donors f o r other r e a c t i o n s i s o x i d a t i v e  phosphorylation,  60 i t was considered f e a s i b l e t h a t the f i r s t  step should be a n  attempt t o demonstrate t h a t phosphate was being taken up i n t o the system i n the presence of s u b s t r a t e . was  The second p o s t u l a t e d  step  t o determine where t h i s phosphate was going i n the hope t h a t  t h i s i n f o r m a t i o n would shed l i g h t on the problem of where phosp h o r y l a t i o n occurs d u r i n g glucose r o u t e of t h i s metabolic path of d e g r a d a t i o n  I n other words, the  i n v e s t i g a t i o n s h i f t s from f o l l o w i n g the  of glucose  i n o r g a n i c phosphate.  oxidation.  t o f o l l o w i n g the i n c o r p o r a t i o n of  Nossal,Keech and U t t e r (113) noted  that  (30 seconds) i n the s o n i c o s c i l l a t o r were  even s h o r t p e r i o d s  enough t o uncouple the p h o s p h o r y l a t i o n mechanism i n a p a r t i c u l a t e preparation of yeast.  W i t h t h i s i n mind, a Hughes press which  would have l e s s chance of d i s r u p t i n g p a r t i c u l a t e m a t e r i a l was chosen.  For a photograph of the type of press used i n these ex-  periments the reader I t i s noted  i s r e f e r r e d t o W.B. Hugo (62) (195*+) p. 9 1 .  here t h a t throughout these experiments p e r i o d i c p r o t e i n  determinations were made by the modified Kabat and Meyer t  The  technique.  v a l u e s of the c e l l f r e e p r e p a r a t i o n s always f e l l w i t h i n the  12-20  mg- per ml- range.  Having s e t t l e d on a method o f o b t a i n i n g enzyme p r e p a r a t i o n s , the next  reasonable  s t e p was t o choose a method of assay  f o r the e x i s t e n c e of an o x i d a t i v e p h o s p h o r y l a t i o n  system.  61 Reaction 1  Reaction 2 X  oxidized  Substrate  inorganic phosphate  oxidative phosphorylation  v  ADP  Glucose  ATP  Hexokinase Glueose-6-Phosphate  TPNH  TPN G-6-P  dehydrogenase  6 Phosphogluconate I n the above r e a c t i o n , i f one supplies, an excess the components except  of a l l  a source o f high energy phosphate, t h e r e  should be no r e d u c t i o n of TPN.  However, i f one adds a system  f o r the p r o d u c t i o n o f high energy phosphate the r a t e and amount of TPN r e d u c t i o n w i l l be a measure of the a b i l i t y of the system t o convert i n o r g a n i c phosphate t o h i g h energy phosphate. c o n c e n t r a t i o n was measured by determining 3*+0  m i l l i m i c r o n s i n the  TPNH  i t s optical density at  spectrophotometer.  I t i s noted t h a t i f the above R e a c t i o n s  1 and 2 were  t o be c a r r i e d out s i m u l t a n e o u s l y i n a r e a c t i o n v e s s e l (e.g. a spectrophotometer  cuvette) the presence  of TPNH oxidase  activity  i n the c e l l f r e e p r e p a r a t i o n would l e a d t o erroneous  results.  To check on t h i s p o s s i b i l i t y a sample of the crushed  suspension  was incubated w i t h b u f f e r and TPNH.  The r e s u l t s showed t h a t the  p r e p a r a t i o n does c o n t a i n TPNH oxidase, the r a t e of a c t i o n of which a p p a r e n t l y i n c r e a s e s w i t h s u b s t r a t e c o n c e n t r a t i o n .  Since  the r a t e of TPNH o x i d a t i o n i s not constant but w i l l i n c r e a s e as  62 the c o n c e n t r a t i o n of TPNH i n c r e a s e s , one could not a c c u r a t e l y c o n t r o l t h i s l o s s and hence t h i s method of assay i s u n s a t i s f a c t o r y . I n an attempt t o circumvent t h i s d i f f i c u l t y , c e l l  free  e x t r a c t and s u b s t r a t e were i n c u b a t e d and a l l o w i n g , e i t h e r ATP or, a l t e r n a t i v e l y , i n the presence of hexokinase and g l u c o s e , glucose-6-phosphate t o accumulate.  The r e a c t i o n was stopped  w i t h a c i d or heat, thus d e s t r o y i n g t h e TPNH oxidase a c t i v i t y . a l i q u o t of t h i s r e a c t i o n mixture was added t o the i n d i c a t o r  An  system  as a sou*rce of ATP or glucose-6-phosphate, and TPNH a c c u m u l a t i o n measured. The a d d i t i o n of heat p r e c i p i t a t e d  c e l l free extract t o  the i n d i c a t o r system as a s o l e source of ATP p e r m i t t e d the gene r a t i o n of TPNH.  There i s t h e r e f o r e some contaminating ATP i n  the p r e p a r a t i o n . I f the c e l l f r e e e x t r a c t was incubated w i t h ATP f o r 0,  15,  and 50 minutes and a l i q u o t s used as s o l e ATP source i n the  i n d i c a t o r system, the c o n c e n t r a t i o n of ATP was seen t o f a l l o f f w i t h time, i n d i c a t i n g t h a t these p r e p a r a t i o n s e x h i b i t marked ATP'ase a c t i v i t y .  Attempts t o i n h i b i t t h i s a c t i v i t y w i t h sodium  f l u o r i d e were not v e r y s u c c e s s f u l , only p a r t i a l i n h i b i t i o n was ever o b t a i n e d , even w i t h c o n c e n t r a t i o n s as h i g h as 10^M/ml, which was the one f i n a l l y used. I f c e l l f r e e e x t r a c t was added t o t h e i n d i c a t o r as s o l e source of glucose-6-phosphate dehydrogenase,  system  positive  63 r e s u l t s were o b t a i n e d . present  There i s glucose-6-phosphate dehydrogenase  ( 1 0 8 ) , t h e r e f o r e , glucose-6-phosphate would not accumulate. These experiments showed t h a t w i t h t h i s crude  p r e p a r a t i o n i t was  not p o s s i b l e t o q u a n t i t a t i v e l y measure o x i -  d a t i v e p h o s p h o r y l a t i o n by the accumulation glueose-6-phosphate. c u l t t o c o n t r o l and was  cell  Since these i t was  not  of TPNH, ATP  e r r o r s were present  or  and  diffi-  sure that the adenylic a c i d system  the system of energy t r a n s f e r i n t h i s organism, t h i s method  of assay was  d i s c a r d e d i n favour  of d i r e c t measurement of i n o r g a n i c  phosphate uptake. I n a l l subsequent experiments c e l l f r e e were prepared  u s i n g the Hughes press technique  M a t e r i a l s and Methods.  preparations  described i n  As d i s c u s s e d i n the h i s t o r i c a l s e c t i o n ,  some workers (e.g. Rose and  0 c h o a ) ( 1 2 5 ) have shown o x i d a t i v e  phosphorylative  a c t i v i t y a s s o c i a t e d w i t h p a r t i c u l a t e matter i n  the c e l l s .  s o l u b l e f r a c t i o n , although  The  c o n t a i n i n g most of  the myokinase and ATP ase a c t i v i t y r e p o r t e d l y does a l s o c o n t a i n 1  something (s) which abets o x i d a t i v e p h o s p h o r y l a t i o n . and Gray ( l 8 ) ( 1 9 ) .  P a r t i c u l a t e preparations  by d i f f e r e n t i a l c e n t r i f u g a t i o n , but these  (Brodie  of 9027 were made  preparations exhibited  g r e a t l y reduced oxygen uptake on s u b s t r a t e s , compared w i t h crude p r e p a r a t i o n s from which o n l y the whole c e l l s and had  been removed.  The  l a t t e r p r e p a r a t i o n was  t r i e d f o r o x i d a t i v e a c t i v i t y on glucose, butyrate.  the  cell  chosen f o r use  sodium  Glucose and  walls and  succinate,<*ketosodium s u c c i n a t e  6h were the most s u c c e s s f u l s u b s t r a t e s as evidenced by oxygen uptake and were subsequently  used.  Although sodium s u c c i n a t e d i d cause  oxygen uptake  some l o s s of i n o r g a n i c phosphate i t e x h i b i t e d much slower tivity.  Moreover the glucose system was  presence  of i n o r g a n i c phosphate was  Lowry-Lopez technique was  (100)(17).  The  ac-  the i n i t i a l i n t e r e s t  so f u r t h e r experiments were concerned w i t h glucose The  and  and  oxidation.  measured by the m o d i f i e d  o p t i c a l d e n s i t y a t 660  my  a p p l i e d t o a standard curve and the v a l u e s i n p M phosphate  obtained. When 10  JJ M glucose was  i n o r g a n i c phosphate was Figure 3.  added t o an i n c u b a t i o n m i x t u r e ,  i n c o r p o r a t e d as i s shown i n Table k and  In t h i s experiment,  7 ml«Warburg r e a c t i o n v e s s e l s were  used, each c o n t a i n i n g 1 ml-of r e a c t i o n mixture. was  Oxygen uptake  measured manometrically f o r each v e s s e l , and at the  i n d i c a t e d i n Table  the d e s i g n a t e d cup was  times  removed a^ong w i t h  i t s corresponding endogenous cup, the r e a c t i o n s stopped  with  a c i d and the supernatants analyzed f o r i n o r g a n i c phosphorous.  60 Minutes  F i g u r e 3»  l o s s of i n o r g a n i c phosphate as a r e s u l t of the a c t i o n of a c e l l f r e e preparation^ on glucose M phosphate--present i n endogenous cups x - -x  M phosphate present i n t e s t cups  o  M  o  phosphate t a k e n up  (2-1)  65 Microlitres Time i n Minutes  Table k of Oxygen Uptake  1  on lOyuM  CUP Number  2  3  h  5  52  51  58  30  110  108  119  h5  152  150  160  167  189  15  53  56  60 75  196  90  192  atoms of Oxygen taken up  0.k7  1.0  P: 0 r a t i o  0.68  0  x  Glucose  1.36  0  l. 9  1.66  0.21  0.h2  k  Endogenous i s s u b t r a c t e d . A similar  experiment was r u n , t h i s time i n c o r p o r a t i n g  a cup c o n t a i n i n g 1 y Mole 2th  d i n i t r o p h e n o l ( w i t h and without  s u b s t r a t e ) per ml. of r e a c t i o n mixture, endogenous and s u b s t r a t e cups. are shown i n Table the r e s u l t s  along w i t h the u s u a l  The r e s u l t s  5 and F i g u r e h.  of t h i s experiment  Table 6 and F i g u r e 5 show  of an i d e n t i c a l experiment, w i t h the r e a c t i o n s  at s h o r t e r time  intervals.  stopped  a.  3  Time  F i g u r e k.  in  Minutes  The e f f e c t of DNP on phosphorous uptake by a c e l l system on glucose •  •  no DNP  o  o  plus  DNP  free  20  0  F i g u r e 5.  Time  in  30  Minutes  The e f f e c t of DNP on Phosphorous uptake by a c e l l free system on glucose •  •  o- - -o  no DNP plus  DNP  Microlitres Time i n Minutes  Table 5 of Oxygen Taken Up on 10 u M Glucose  1  l+DNP  50  15  Cuo Number 2+DNP 2  kk  53  h5  0.58  P:0 r a t i o s  0.1+7  1.37  0.08  0.03  3+DNP  51  39  39  161  1^3  li+8  168  173  90 Atoms of Oxygen taken up  3  1.^3  0  1.5  0.36  0.23  Endogenous i s s u b t r a c t e d . Table 6 Microlitres  Time i n Minutes  10  of Oxygen Taken Up on 10 y M Glucose  1  l+DNP  26  31  20  2  Cup Number 2+DNP  3  3+DNP  19  25  22  22.  62  71  60  63  96  97  30 Atoms of Oxygen taken up  0.23  1+.28  0.55  0.63  0.86  0.87  PsO r a t i o  1.3  1.0  1.02  o.i+V  0.21  0.25  x  Endogenous i s s u b t r a c t e d . These r e s u l t s show t h a t phosphate i s i n c o r p o r a t e d and  t h a t some i n h i b i t i o n i s e x h i b i t e d by DNP. however, f a r from complete.  This i n h i b i t i o n i s ,  The p e c u l i a r shape of the curves,  both endogenous and t e s t , i n d i c a t e s t h a t more than one process i s involved.  I n v e s t i g a t i o n s upon these r e a c t i o n mixtures would be  difficult  s i n c e there i s no p r i o r knowledge as t o what i t might  67 be t h a t was u t i l i z i n g  the phosphate, and t h i s would make the  c h oi ce of an assay method a l a b o r i o u s one w i t h s m a l l of success w i t h i n a reasonable  time.  possibilities  T h i s l e d t o the obvious  thought of u s i n g l a b e l l e d phosphorous which, i f i n c o r p o r a t e d could be f o l l o w e d r e a d i l y and, i n subsequent f r a c t i o n a t i o n s of r e a c t i o n mixtures,  i t s presence or absence i n v a r i o u s f r a c t i o n s  would be r e a d i l y d e s c e r n i b l e .  III.  Op  S t u d i e s w i t h Phosphorous-^  One i n i t i a l attempt was made using A T P  (1  3 2  pM/ml.  stock s o l u t i o n ) s u p p l i e d by the organic chemistry d i v i s i o n of the B r i t i s h Columbia Research C o u n c i l .  The i n c u b a t i o n mixture  was s e t up as d e s c r i b e d i n M a t e r i a l s and Methods, and 1.0 m l , a l i q u o t s were removed as f o l l o w s ; I n c u b a t i o n Mixture i ii iii iv  Endogenous (no glucose) Test (plus glucose) Endogenous plus DNP Test plus DNP  A l i q u o t s of Each Removed A t 0 , 5 , 15> 3 0 ,  k  5 , 6 0 , 90  120 and 150 minutes  In each sample the r e a c t i o n was stopped w i t h O . l ml.h-0% t r i c h l o r a c e t i c a c i d , and 0 . 3 ml. N/1 sodium a c e t a t e added immediately. supernatant  P r o t e i n was removed by c e n t r i f u g a t i o n and the (1 ml.) was mixed f o r 5 minutes w i t h $0 mg. a c i d  washed N o r i t e A + 0 . 0 1 ml.ethanol.  The c h a r c o a l was removed by  c e n t r i f u g a t i o n , and washed t h r e e times w i t h d i s t i l l e d water. supernatant  and c h a r c o a l f r a c t i o n s were counted.  Both  No counts 32  appeared on the c h a r c o a l even a t 0 time. being i n s t a n t a n e o u s l y broken down.  Hence the ATP  T h i s i s reasonable  was since  0.1  68 ml of a 1:100 d i l u t i o n of ATP i s 1 millimicromole ATP of approximately  32  was used per f l a s k .  per 10 ml.  J  That i's^ there  A l s o present i s 3»9 ml.  a 10 mg/ml, p r o t e i n s o l u t i o n , which equals  mg. p r o t e i n per 10 ml.  39  Now i f a system i s present i n the endogenous  which w i l l u t i l i z e ATP a t the r a t e of 1 ^ M per hour per mg. p r o t e i n , which i s not an e x c e s s i v e f i g u r e , t h i s p r e p a r a t i o n would use 39 y M per hour o r , i t would take 60/39000. = 1/600 hour = 1/10 :  second  to u t i l i z e the i n i t i a l c o n c e n t r a t i o n of ATP. .When i t i s c o n s i d e r e d t h a t there i s probably more than one system i n a crude p r e p a r a t i o n which would u t i l i z e added ATP i t i s reasonable t r a t i o n of A T P  3 2  that t h i s  concen-  c o u l d be dropped below the l i m i t of s e n s i t i v i t y  of the d e t e c t i o n technique r e q u i r e d was e i t h e r A T P  3 2  almost i n s t a n t a n e o u s l y .  What was  of a higher s p e c i f i c a c t i v i t y , or b e t t e r ,  inorganic P  32 of a h i g h s p e c i f i c a c t i v i t y .  The l a t t e r i s the more  a d v i s a b l e because as has been mentioned, there i s nothing t o say t h a t ADP i s the normal acceptor  of i n o r g a n i c phosphate.  An experiment was s e t up as p r e v i o u s l y d e s c r i b e d but i n c o r p o r a t i n g two changes: P  3 2  0.2 ml.of a p r e p a r a t i o n of i n o r g a n i c  (17 m i c r o c u r i e s per ml. s u p p l i e d by D r . D.H. Copp) was sub-  stituted  f o r the A T P  3 2  and the c o n c e n t r a t i o n of c o l d  i n the g l y c y l g l y c i n e b u f f e r was reduced  phosphate  t o 2 p Moles per 0 . 3 ml.  T h i s l a s t step was t o ensure a high s p e c i f i c a c t i v i t y i n the phosphate p o o l . Endogenous (no glucose)  The f l a s k s , which were Test Endogenous (glucose) + DNP  Test + DNP were  incubated  69 and  2 m l , a l i q u o t s removed at 0 ,  Each a l i q u o t was was  30,  a c i d p r e c i p i t a t e d and  t r e a t e d w i t h 80 mg.  the a d s o r b i n g ,  10,  60 and  120  minutes.  1 ml.of each  supernatant  N o r i t e A as p r e v i o u s l y d e s c r i b e d .  c e n t r i f u g i n g and washing was  carried  All  out i n 1  ml.  Misco c e n t r i f u g e tubes t o e l i m i n a t e l o s s of c h a r c o a l i n t r a n s f e r from tube t o tube.  The washed c h a r c o a l of each a l i q u o t was  on a planchet, d r i e d , and  put  counted.  F i g u r e 6 shows the r e s u l t s of t h i s experiment.  Since  i n o r g a n i c phosphate i s not adsorbed t o c h a r c o a l , some of i t i s o b v i o u s l y being i n c o r p o r a t e d  i n t o the organic f r a c t i o n .  Nucleo-  t i d e m a t e r i a l i s s e l e c t i v e l y adsorbed t o c h a r c o a l i f the i s not present r e s u l t s was corporated  i n excess so a p o s s i b l e e x p l a n a t i o n of  t h a t the l a b e l l e d i n o r g a n i c phosphate was i n t o a n u c l e o t i d e i n the case of the  whereas i f s u b s t r a t e was  DNP  being i n -  s u p p l i e d as w i t h g l u c o s e , i t was  e x h i b i t s , i f anything, a s l i g h t  e f f e c t which doesn't c o r r e l a t e w i t h the data.  these  endogenous,  being donated to some phosphate acceptor which was on c h a r c o a l .  However at the present  stage  charcoal  oxidative  not  then  adsorbed  stimulatory phosphorylation  of the i n v e s t i g a t i o n no  explanation i s possible. The  supernatant  fractions ( i . e . after charcoal  s o r p t i o n ) of the t e s t i n c u b a t i o n mixtures ( p l u s glucose) contained  the r e s i d u a l i n o r g a n i c phosphate and  l a b e l l e d compounds were analyzed supernatants  were adjusted  as f o l l o w s :  t o pH 8 . 0  adwhich  p o s s i b l y other The  pooled  substrate  w i t h ammonium hydroxide  and  32 F i g u r e 6.  The a d s o r p t i o n of compounds l a b e l l e d w i t h P endogenous x_ _ _x • a_  • _ -a  endogenous plus test t e s t plus  DNP  DNP  t o N o r i t e A.  70 passed  through a 2% x 1 cm column of Dowex 1 ( c h l o r i d e form)  resin.  The e f f l u e n t was  t o be n e g l i g i b l e . column.  then, held on the  e l u t e d a c c o r d i n g t o the method of Khym  ml«aliquots of every t h i r d  on planchets and counted. checked  found  5 ml-samples were c o l l e c t e d by a G i l s o n f r a c t i o n  c o l l e c t o r and 0.2  tube was  f o r a c t i v i t y which was  The l a b e l l e d m a t e r i a l was,  The column was  and Conn ( 6 2 ) .  checked  sample were d r i e d  I f a peak appeared, every n e i g h b o r i n g  for a c t i v i t y .  F i g u r e 7 shows the r e s u l t s of t h i s e l u t i o n .  The  peak which came o f f i n the area of e l u t i o n of Glueose-1-phosphate and  i n o r g a n i c phosphate was  column was  the o n l y peak found.  c o l d a f t e r t h i s peak came o f f , so a l l the a c t i v e  t e r i a l came o f f i n t h i s a r e a . 7 t o 10 i n c l u s i v e ) was presence  The Dowex  The  shoulder of the curve  ma-  (tubes  concentrated t o 1 ml«and t e s t e d f o r the  of glue ose-1-phosphate by combining phosphoglucomutase  w i t h glucose-6-phosphate  dehydrogenase and TPN.  t e s t were: O p t i c a l d e n s i t y of 0.1 y M TPNH/ml, = 0.2  O.63.  ml-of the concentrate added t o the r e a c t i o n mixture  produced an i n c r e a s e i n O.D.  of 0 . 0 0 5 .  T h e r e f o r e there i s 5/360 x 0.1 0.001 p M glucose-l^-phosphate The  The r e s u l t s of t h i s  techniques u t i l i z e d  = 1/3200JJM = l e s s  than  p r e s e n t , which i s n e g l i g i b l e .  i n experiments  of t h i s  nature  are somewhat l e n g t h y and demand the use of much equipment. f a c t , coupled w i t h the f a c t t h a t the decay r a t e of P  3 2  This  i s high  ( h a l f l i f e l*+.3 days) makes the concurrent a n a l y s i s of more than  Figure 7. Alkaline elution of a Dowex 1 Cl~ column charged with charcoal supernatant fraction.  71 one r e a c t i o n mixture d i f f i c u l t . was  then t o l e a r n f i r s t what was occuring  reaction.  i n the endogenous  T h i s i n f o r m a t i o n would be necessary f o r l a t e r  with substrates the  The most l o g i c a l mode of a t t a c k  i n order  studies  t o determine which r e s u l t s a r e due t o  presence o f the s u b s t r a t e . Although c h a r c o a l p r e f e r e n t i a l l y adsorbs  nucleotide  m a t e r i a l under r i g i d c o n d i t i o n s , i t has been observed (106) t h a t an excess of c h a r c o a l w i l l p i c k up other example, sugar phosphates.  compounds a s , f o r  Furthermore, compounds may be  i r r e v e r s i b l y adsorbed, e s p e c i a l l y i f an excess of c h a r c o a l i s . used. Experiments were r u n using a c e l l f r e e e x t r a c t , d i l u t e d as i t would be i n a r e a c t i o n mixture, and exposing i t t o v a r y i n g  con-  c e n t r a t i o n s o f c h a r c o a l t o a s c e r t a i n how much of the n u c l e o t i d e m a t e r i a l (as evidenced by a b s o r p t i o n taken up.  of l i g h t a t 260 my)  The r e s u l t s show t h a t 90$ of the 260 m j J  was  absorbing  m a t e r i a l i n a 1 ml. a l i q u o t i s adsorbed on 2h mg. c h a r c o a l  (Figure  8). The second experiment was t o check t h e e f f i c i e n c y of removing t h i s m a t e r i a l from the c h a r c o a l by using and 5% ammonium hydroxide i n 95% e t h a n o l .  In this  10 mg.charcoal per ml. d i l u t e d e x t r a c t was used. about 70$ of the 260 absorbing was  material.  a l i q u o t of the e l u t i n g m a t e r i a l . Table 7.  experiment, T h i s removed  The adisorption procedure  i d e n t i c a l t o that previously described,  by 15 minute exposure, w i t h s t i r r i n g  50$ e t h a n o l ,  e l u t i o n s being made  of the c h a r c o a l , t o a 1 ml.  The r e s u l t s a r e summarized i n  F i g u r e 8.  Adsorption  of n u c l e o t i d e m a t e r i a l t o N o r i t e  72 Table 7 E l u t l o n of N u c l e o t i d e s from C h a r c o a l Amount of m a t e r i a l Present % Recovered (O.D. a t 260 mp ) from C h a r c o a l  1. Charcoal  0  0.902  2 . 50$ E t h a n o l  0 . 5 0 5 / 0 . 9 0 2 x 100 = 56$  0.5057  supernatant  5$ Ammoniacal a l c o h o l 3 . supernatant a p p l i e d t o 1 a f t e r 50$ E t h a n o l treatment  0.230  25$  I n c r e a s i n g time of c o n t a c t t o 30 minutes o f f e r e d no worthwhile i n c r e a s e i n r e c o v e r y .  I t can be seen t h a t the r e c o v e r y  was poor, about *+5$ o f the m a t e r i a l being l o s t , which l o s s c o u l d be i l l  a f f o r d e d i f i t contained  Even subsequent treatment  some l a b e l l e d compound ( s ) .  of the a l c o h o l e l u t e d c h a r c o a l w i t h  ammoniacol e t h a n o l s t i l l l e f t 25$ on the c h a r c o a l .  ;  Charcoal  was then d i s c a r d e d as a good choice f o r the i n i t i a l s e p a r a t i n g system. From F i g u r e 6 i t i s seen t h a t 30 minutes i n c u b a t i o n of the r e a c t i o n mixture,  i n the absence of added s u b s t r a t e , y i e l d s  the.maximum amount of c h a r c o a l absorbable  labelled material.  Fear that the severe a c i d i c c o n d i t i o n s p r e v i o u s l y used t o p r e c i p i t a t e p r o t e i n may be damaging t o some phosphate complex l e d to the a d o p t i o n of the a l c o h o l p r e c i p i t a t i o n t e c h n i q u e . r e a c t i o n mixture, a f t e r removal of p r o t e i n , was reduced  The t o 5 ml.  volume, the pH a d j u s t e d t o 8 . 0 and put d i r e c t l y onto a Dowex 1 C l ~  73 column as before. before  A check of an a l i q u o t of t h i s  p u t t i n g i t on the column showed t h a t a g a i n ,  of a c t i v i t y were adsorbed t o the c h a r c o a l .  concentrate l a r g e amounts  As before, the  l a b e l l e d m a t e r i a l was held up by the Dowex column, and as b e f o r e , when the column was e l u t e d by the method  of Khym and Cohn, a l l  the  l a b e l l e d m a t e r i a l came o f f the column i n the one peak w i t h  the  i n o r g a n i c phosphate ( F i g u r e 7).  the  peak r e v e a l e d  Further  i n v e s t i g a t i o n s of  that i n samples on the ascending s i d e of the  peak some of the l a b e l l e d m a t e r i a l present  was c h a r c o a l adsorbable  whereas t h i s was not true f o r samples from the apex and descending s i d e s of the peak. inorganic  Obviously  phosphate present The reasoning  follows:  1.  i n that  peak.  on the problem a t t h i s stage r a n as  Since a c t i v i t y i s being inorganic organic  2.  then, there was more than j u s t  taken up by the c h a r c o a l , the  phosphate i s being  incorporated  i n t o an  molecule.  I f one assumes t h a t i t i s an a l k a l i n e l a b i l e compound l i k e u r i d i n e diphosphate glucose (UDPG) t h a t i s being  formed the compound could t h e n  conceivably  be being broken down by the a l k a l i n e e l u t i n g s o l u t i o n s on the column. For example, UDPG a l k a l i n e U r i d y l i c a c i d + g l u e o s e >  conditions  1-phosphate  The u r i d y l i c a c i d would not be l a b e l l e d and hence would be undetected, and the g l u c o s e - l - p h o s p h a t e , a l t h o u g h l a b e l l e d , would come o f f under the i n o r g a n i c  phosphate peak.  7k The experiment was then repeated, as described above, up to and including the precipitation of the protein.  The r e -  action mixture was then concentrated, the pH adjusted to 8.0, put on the Dowex 1 CI column and quickly washed with water until the pH of the effluent was that of d i s t i l l e d water (circa 6 . 3 ) . Each time, the period from adjustment of pH to 8.0 to the f i n a l washing of the column, took less than 5 minutes.  This time the  acidic gradient elution system of lithium chloride to hydrochloric acid was employed.  10 ml fractions were collected in a Technicon  fraction collector and 0 . 2 ml aliquots of the fractions were counted as before. The results of this elution are shown i n Figure 9 . Here, there is a division of the labelled material into three. Peak 1, presumably inorganic phosphate or non-nucleotide phosphate, Peaks 2 and 3 unknown compounds.  A check on each peak tube was  made for 260 my absorption.  A l l tubes, including Peak 1 exhibited  good absorption at 260 my .  Since Peak 1 was so very sharp,  i t was put back on a Dowex 1 Cl~ column as before and eluted with the HC1 to d i s t i l l e d water gradient, i n an attempt to spread the peak.  Measurements of radioactivity and 260 my absorption were  done on the eluted fractions.  Figure 10 is a graph representing  the results obtained, which shows that there is no correlation between the 260 absorbing material and the radioactive material. An expansion of the above experiment was run on another incubation mixture.  This experiment was carried out exactly as the  one  shown i n F i g u r e 9 except t h a t 0.3N l i t h i u m c h l o r i d e was used  i n s t e a d of 0 . 5 N , i n the g r a d i e n t e l u t i o n t o spread more.  Samples were checked f o r r a d i o a c t i v i t y and 2 6 0 myj abThe r e s u l t s a r e shown i n F i g u r e 1 1 .  sorption. was  the peaks out  Here, the p i c t u r e  e s s e n t i a l l y the same, the most noteworthy p o i n t s being (a)  a c o n f i r m a t i o n of the previous  experiment wherein the 2 6 0 ab-  s o r b i n g m a t e r i a l i n Peak 1 i s shown t o be l a r g e l y c o l d , a d i s t i n c t c o r r e l a t i o n between 2 6 0 absorbing  (b)  a b i l i t y and s p e c i f i c  a c t i v i t y i n Peaks 2 and 3 . Tubes 15 t o 3 0 , i n c l u s i v e and tubes kO t o 6 0 i n c l u s i v e were pooled concentrates  and concentrated  t o a volume of 2 ml.each.  These  are r e f e r r e d t o as Peak 2 and Peak 3 r e s p e c t i v e l y .  Samples of each were r u n i n a Carey r e c o r d i n g y i e l d i n g the data shown i n F i g u r e 1 2 . i s noted, y i e l d a peak at  2 +0 l  spectrophotometer,  These p r e p a r a t i o n s , i t  m i l l i m i c r o n s f o r Peak  both a t  2  pH 3 . 0 and pH 6 . 3 and a peak a t 2h0 f o r Peak 3 a t pH 3 . 0 and pH 11.0.  V a r i a t i o n of pH produced no s h i f t i n the curve.  not correspond  w i t h known n u c l e o t i d e a b s o r p t i o n curves  T h i s does so a p u r i -  f i c a t i o n step on the Peaks was c a r r i e d out t o see i f the presence of l i t h i u m s a l t s e t c . could be a f f e c t i n g the c u r v e . In each peak the suspect w i t h acetone as d e s c r i b e d  n u c l e o t i d e was p r e c i p i t a t e d  i n M a t e r i a l s and Methods.  An aqueous  s o l u t i o n was made of each p r e c i p i t a t e and i t s s p e c i f i c checked and i t was a g a i n scanned i n the Carey r e c o r d i n g photometer.  activity spectro-  i 1  Figure 11.  A c i d e l u t i o n of Dowex 1 C l  column charged  w i t h i n c u b a t i o n mixture, o- - -o  o p t i c a l d e n s i t y at 260  •  counts per minute per 0.2  •  m ml.  F i g u r e 12.  Scan of peak f r a c t i o n s . • o- - -o  •  Peak 2 Peak 3  76 The p r e c i p i t a t e i n both cases was s t r o n g l y l a b e l l e d and upon scanning  y i e l d e d the curves as i l l u s t r a t e d  i n Figures  13 and Ih. Peak 2 - pH 2.0  e x h i b i t s a h i g h a t 258 my  pH 11.0  e x h i b i t s a h i g h a t 262 m^ , a low a t 220 my  Peak 3 - PH 3-O^j  e  x  h  l  D  i t  Table 8 i s a l i s t  a h i g h a t 263 and a low a t 216/my  s  of approximate v a l u e s f o r the occurrence  of peaks obtained when known 5  1  Ribonucleotides  •  Wave Length a t Which V a l l e y Occurs  Wave Length a t Which High Peak Occurs pH 2  PH 7  Adenosine (AMP, ADP, ATP)  257  259  Sytidine . (CMP, CDP, CTP)  280  272  [Jridine (UMP, UDP, UTP)  _  262  Juanosine (GMP, GDP, GTP)  pH 1.0  256 pH 2.0  Peak 2  258 3  pH 3.0  eak 3  263  252  pH 11  -  262 257-267 plateau  -  (lMf).  r i b o n u c l e o t i d e s are scanned  Table 8 A b s o r p t i o n Spectra of 5'  Derivatives  , a low a t 223  262 213  pH 2  pH 7  230  227  2+1  250  —  _  230  239  227  223  230  223  -  -  pH 1.0  PH $A 216  pH 11  -  216  Comparison of the curves obtained w i t h the above t a b l e i n d i c a t e s t h a t Adenosine d e r i v a t i v e s or U r i d i n e d e r i v a t i v e s may be involved.  P r e l i m i n a r y attempts a t i d e n t i f i c a t i o n by paper chroma-  tography both .of the n u c l e o t i d e moiety and the sugar moiety i f i t e x i s t s were made.  No d e f i n i t e r e s u l t s , other than the f a c t t h a t i t  280  320  240  200  Wavelength  Figure  13.  Scan of acetone p r e c i p i t a t e •  •  pH 2.0  o  o  pH  11.0  of Peak 2.  0-8 •  F i g u r e lh.  Scan of acetone p r e c i p i t a t e .  .  o  o  P  H  pH  3.0 11.0  of Peak 3 .  77 moves a t a slower r a t e  on paper than UDPG were o b t a i n e d .  d i d not permit an e x t e n s i o n of these  investigations.  Time  78 SUMMARY  1.  An attempt was made t o study the degradation of glucose  by Pseudomonas aeruginosa  by making use of various types of  9027  c e l l preparations alone, and i n combination w i t h pyocyanine i n the hope of i n h i b i t i n g the metabolism somewhere between the 2 ketogluconate and pyruvate Dsrels thus accumulating an intermediate which could then be i d e n t i f i e d .  Apparently, however, the enzyme  system responsible f o r the breakdown of 2 ketogluconic a c i d i s as l a b i l e as any i n the system,since i f i n h i b i t i o n was obtained, i t was a t the 2 ketogluconate 2.  level.  Incubation of c e l l free preparations of the organism  w i t h ATP and glucose y i e l d e d , on paper chromatography, a spot which acted as a l a b i l e phosphate and yielded a p o s i t i v e periodate reaction.  T e c h n i c a l d i f f i c u l t i e s , which are d i s c u s s e d , rendered  c o n t i n u a t i o n of these techniques u n p r o f i t a b l e . 3.  Incubation of a c e l l f r e e preparation w i t h i n o r g a n i c  phosphate r e s u l t e d i n an increased uptake of i n o r g a n i c phosphate by preparations incubated w i t h glucose.  This uptake was only  p a r t i a l l y i n h i b i t e d by DNP, which e x h i b i t e d no i n h i b i t o r y e f f e c t on oxygen uptake.  The crude c e l l preparations used were shown  to c o n t a i n ATP, ATP'ase, TPNH oxidase, and glucose-6-phosphate dehydrogenase.  79 I n c u b a t i o n of a c e l l f r e e  preparation with inorganic  32 phosphate l a b e l l e d w i t h P acid stable,  resulted  i n the accumulation of  a l k a l i n e l a b i l e f r a c t i o n s which c o u l d be  by column chromatography.  activity,  and when scanned i n the spectrophotometer  p i c t u r e approaching that  separated  The l i t h i u m s a l t s of these  which were p r e c i p i t a t e d from a c e t o n e , e x h i b i t e d h i g h  of adenine  fractions specific  yielded a  or u r i d i n e d e r i v a t i v e s .  T h e i r apparent a l k a l i n e l a b i l i t y and a c i d s t a b i l i t y  differen-  t i a t e s them from compounds such as ATP and the p o s s i b i l i t y a nucleoside  two  polyphosphate sugar complex i s  suggested.  of  80 BIBLIOGRAPHY  1.  A n f i n s e n , C.B. and K i e l l e y W.W. 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