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The effect of Semliki Forest virus infection on phosphatidylcholine biosynthesis in baby hamster kidney-21… Whitehead, Frederick William 1979

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THE EFFECT OF SEMLIKI FOREST VIRUS INFECTION ON PHOSPHATIDYLCHOLINE BIOSYNTHESIS IN BABY HAMSTER KIDNEY-21 CELLS by  FREDERICK WILLIAM WHITEHEAD B.Sc,  U n i v e r s i t y o f B r i t i s h Columbia, 1973  A THESIS SUBMITTED  IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department o f B i o c h e m i s t r y ) We accept t h i s  t h e s i s as conforming  to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA March, 1979 (©Frederick W i l l i a m Whitehead, 1979  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  fulfiIment  o f the requ irements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, the I  L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e  f u r t h e r agree t h a t p e r m i s s i o n  for  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  that  study. thesis  s c h o l a r l y purposes may be granted by the Head of my Department or  by h i s of  for  I agree  this  representatives. thesis  It  is understood that copying or p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  written permission.  Department of The  Biochemistry  U n i v e r s i t y of B r i t i s h  2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  March  1?.  iq7q  Columbia  not be allowed without my  ABSTRACT Semliki Forest i n c o r p o r a t i o n of  (SF) v i r u s caused an  Q H~J c h o l i n e i n t o p h o s p h a t i d y l c h o l i n e 3  of Baby Hamster Kidney -21 infection  (p.i.).  (BHK)  cells,  ^ mg  take  a K  c e l l p r o t e i n ^"' i n mock-irifected  is inhibited  and  pool and  synthesis.  of 17 yM arid V _ o f 381 max  m  (PC)  the mechanism of i n h i b i t i o n  to understand the r e g u l a t i o n of PC C h o l i n e uptake has .  in  a t 6*5-7% hours post  C h o l i n e uptake, enzyme a c t i v i t i e s ,  s i z e s were measured to c l a r i f y  min  i n h i b i t i o n of 77%  in infected c e l l s ,  pmoles  (control) c e l l s .  although  such  Up-  inhibition  o n l y p a r t l y accounts f o r i n c o r p o r a t i o n i n h i b i t i o n . Maximal v e l o c i t i e s of the enzymes of de novo PC i n nmoles min  ^ g cells  ^, from c o n t r o l c e l l s , were:  kinase - 7.3;  c y t o s o l i c phosphocholine  - 1 7 . 3 ; microsomal CT - 14.6;  photransferase  (CPT)  activities  were l e s s :  5.2,  and  In i n f e c t e d c e l l s , 12.1,  4.2,  choline  cytidylyltransferase  ( c y t o s o l i c CT)  - 47.6.  synthesis  and  19.8,  cholinephosthe r e s p e c t i v e at 7 hours  p.i.. C h o l i n e , phosphocholine, and CDP-choline were separated ion  exchange and  c h a r c o a l chromatography.  CDP-choline were hydrolyzed enzymically.  to c h o l i n e , which was  D i g l y c e r i d e was  measured e n z y m i c a l l y .  CTP  was  hydrolyzed  liquid  ATP  and  measured e.. •  to g l y c e r o l , which  measured by a new  nique which uses r a t l i v e r CT. bance a f t e r high pressure  Phosphocholine  by  was  enzymic t e c h -  was'measured by i t s absor-  chromatography.  PC was  measured  -  by  lipid  from  phosphorUSO a n a l y s i s .  control  (and  47  -  infected)  - 34  phosphocholine ide  i i i -  ( 4 3 ) ; CTP  6*1-7% h o u r s  (120); 149  -  p.i..  Pool sizes  cells  ( 7 9 ) ; and  i n CTP  and  The  pool size  o f PC,  i n ymoles/g  and  similarly,  lar  In  compared  cells  which  i s not In  of  the  time  little  phocholine virus  p.i..  was  The  very  simi-  Q H~J  choline,  the  of choline,  compared  to  with  3  pool of choline  Q H~]  choline,  3  turned over  cells,  g r e a t e r , compared  ;  exists,  to c o n t r o l  Thus,  s y n t h e s i s ) was  per  fraction  unit  the  not  cells.  of  of  Conse-  (k x p o o l  1 . 5 1 in infected cells.  the  y e t the p o o l s i z e  of phosphocholine  ^, was  o f PC  hours  cells,  cells,  size) and  a  turnover of  inhibited  phos-  by S F  infection. In  likely  BHK  cells,  catalyze  because: flux.  (or r a t e  with  p o o l which  1 . 0 5 , in control  less,  (p<0.05).  cells.  a large  experiment  g cells  CDP-choline,  synthesis.  turnover rate  nmoles min  and  a l l at  in control  diglyceride  smaller in infected  the  3.4  was  PC  that  3  was  and  a t 1-lh  half-life  Q H~j p h o s p h o c h o l i n e  quently, in  and  i n PC  a pulse-chase  phosphocholine  (1080),  cells,  to i n f e c t e d  suggested  active  (k) was  cells,  w h i c h were l a b e l l e d  radioactivity  phosphocholine,  1800  diglycer-  were a l l s i g n i f i c a n t  c o m p o s i t i o n o f both  BHK  specific  ATP,  3.0 in infected  in control  -  (15.7);  Increases i n phosphocholine  decreases  acid  ATP  cells,  - 146 ( 6 8 ) ;  choline - 6.1  CDP-choline  and  fatty  were:  i n nmoles/g  1. V  m  a  the  t h r e e enzymes o f de  novo s y n t h e s i s o f  n e a r - e q u i l i b r i u m (not r a t e - l i m i t i n g ) x  o f each  enzyme  2 . P o o l s o f t h e enzyme  i s much g r e a t e r t h a n  PC  reactions the  s u b s t r a t e s of .choline k i n a s e  pathway and  -  iv -  •CT do n o t a p p e a r t o be g r e a t enough t o s a t u r a t e t h e enzymes. 3.  A l l t h r e e enzymes  yet  t h e pathway  equilibrium  flux  are reduced  i s not reduced.  reaction,  o v e r t h e CT s t e p .  this  flux.  increase of  I f CT  by v i r u s catalyzes  Similarly, step  Phosphocoline  a  near-  CDP-choline (which was  accumulation would  a c c u m u l a t i o n may possibly  i n amount o f t h e p r o d u c t , CMP).  restore  restore the  l e s s e n e d by an I f indeed, the  enzymes  de novo s y n t h e s i s o f PC c a t a l y z e n e a r - e q u i l i b r i u m r e a c t i o n s ,  t h e n a change i n any s u b s t r a t e o r p r o d u c t w i l l way  infection,  t h e n a s m a l l e r CTP p o o l w o u l d l e s s e n t h e  flux  f l u x o v e r t h e CPT  in activity  flux,  balances  unless a response the i n i t i a l  change.  i n another  change t h e p a t h -  substrate or product  -  V  -  TABLE OF CONTENTS Page, INTRODUCTION. . . . . . (a) P h o s p h a t i d y l c h o l i n e - I t ' s R e l e v a n c e and Biosynthesis . . . . . . . . . (b) S t e p s o f De Novo PC S y n t h e s i s (i) Choline Transport (ii) C h o l i n e Kinase . (iii) Cytidylyl.tr-an.sf erase (iv) Cholinephosphotransferase (c) A l t e r n a t e F o r m a t i o n o f PC (i) P h o s p h a t i d y l e thanolamine M e t h y l t r a n s f e r a s e . . ( i i ) Base E x c h a n g e (d) P o t e n t i a l S i t e s o f R e g u l a t i o n o f De Novo S y n t h e s i s (i) Choline Transport . . . . . . . . (ii) Choline Kinase (iii) Cytidylyltransferase (iv) Cholinephosphotransferase . . . . . . . . . (e) Regulation of Phosphatidylcholine Biosynthesis (i) Liver 1. A v a i l a b i l i t y o f S u b s t r a t e s . . . . . . . . 2. R a t e - L i m i t i n g Step . . . . . 3. E x p e r i m e n t a l l y A l t e r e d PC S y n t h e s i s . . . 4. Phosphatidate . . . . . . . . (ii) Lung . . . . . . . . . . . . (iii) Brain . . . . . (f) E f f e c t s o f V i r u s e s on C e l l C u l t u r e B i o s y n t h e s i s o f PC (g) E f f e c t o f S e m l i k i F o r e s t V i r u s on BHK C e l l PC Biosynthesis MATERIALS AND METHODS (a) C h e m i c a l s and I s o t o p e s (b) G e n e r a l Methods (i) T h i n - L a y e r Chromatography < . (ii) S c i n t i l l a t i o n Counting . . . . (iii) Protein . . . . . . . . . . (iv) Statistics (c) C e l l Culture . . . . . . . . . . (d) Propagation of Semliki Forest V i r u s . . . . . . . (e) I n f e c t i o n of C e l l s with Semliki Forest V i r u s . . . (f) Preparation of Subcellular Fractions . . . . . . . (g) Enzyme A s s a y s (i) C h o l i n e Kinase (ii) Cytidylyltransferase (iii) Cholinephosphotransferase . . . . (iv) Phosphatidylethanolamine Methyltransferase . (v) Choline Oxidase  1 1 3 8 9 10 14 16 17 18 18 19 20 22 23 26 27 30 31 34 37 38 39 39 39 39 40 40 40 41 42 43 44 44  - v i -  Page  (h) (i)  Lipid Activation of Cytidylyltransferase . . . . Taurocholate A c t i v a t i o n of Cholinephosphotransferase . . . . . . (j) Choline Transport . . . . . . . . . ll) . P o o l S i z e M e a s u r e m e n t s (i) C h o l i n e - C o n t a i n i n g Compounds . (ii) PC . . . . . . . (iii) Diglyceride . . . . . . (iv) P h o s p h o c h o l i n e and CTP . . . . . . . . . . (v) Nucleotides (m) F a t t y A c i d A n a l y s i s . . . (n) Q H~jCholine Pulse-Chase . . . . . . . . . . . . . 3  .  45  . .  45 45  47 52 56 . 57 - 59 63 . 64  RESULTS (a) C e l l W e i g h t and P r o t e i n C o n c e n t r a t i o n 65 (b) Choline Transport . . 67 (c) Enzyme A c t i v i t i e s . . . . . . 69 (d) S e p a r a t i o n and I d e n t i f i c a t i o n o f C h o l i n e C o n t a i n i n g Compounds 83 (e) I n c o r p o r a t i o n o f Q HJJ C h o l i n e . . . . . . . . . . 86 (f) P o o l S i z e M e a s u r e m e n t s - 1 . PC and PC P r e c u r s o r s . 88 (g) P o o l S i z e Measurements - 2. N u c l e o t i d e s . . . . . 94 (h) Time S e q u e n c e o f N u c l e o t i d e P o o l S i z e s D u r i n g SF V i r u s I n f e c t i o n . . . . . . . . . . . . . . . . 1 0 1 (i) F a t t y A c i d A n a l y s i s o f L i p i d s f r o m BHK C e l l s I n f e c t e d w i t h SF V i r u s . . . . . . . . . . . . . . 1 0 5 (j) P u l s e - C h a s e o f Q H~] C h o l i n e i n BHK C e l l s I n f e c t e d w i t h SF V i r u s . . . . . . . . . . . . . . 1 0 8 3  3  DISCUSSION (a) Does SF V i r u s I n f e c t i o n A f f e c t t h e R a t e o f PC S y n t h e s i s i n BHK C e l l s ? . . . . . . . . . . . . . 113 (b) Why I s t h e I n c o r p o r a t i o n o f Q H^] C h o l i n e i n t o PC I n h i b i t e d by SF V i r u s I n f e c t i o n o f BHK C e l l s ? . . . . 113 (c) What C a u s e s t h e E f f e c t s o f SF V i r u s I n f e c t i o n on E a c h S t e p o f PC S y n t h e s i s ? (i) Choline Transport . . . . . . . . . . . . . . 114 (ii) Choline Kinase . . . . .116 (iii) Cytidylyltransferase . . . . . . . . . . . 116 (iv) Cholinephpsphotransferase . . . . . . . . . 117 (d) Do ATP and CTP I n h i b i t CK and CT I n V i v o ? . . . . 118 (e) A r e P o o l S i z e M e a s u r e m e n t s an A c c u r a t e I n d i c a t i o n of In Vivo Concentrations? (i) I s t h e P o o l o f C h o l i n e i n BHK C e l l s Unevenly D i s t r i b u t e d i n S u b c e l l u l a r Compartments? . . . . . . . . . . . . . . . . 119 (ii) Why A r e P h o s p h o c h o l i n e P o o l S i z e R e s u l t s Variable? . . . . . . . . . . . . . . . . . 121 3  - vii-  Pag o  (f)  (g)  (h) (i) (j)  How Does SF V i r u s I n f e c t i o n Cause Changes in the Pool S i z e o f PC P r e c u r s o r s ? (i) N u c l e o t i d e s 122 (ii) Phosphocholine 122 (iii) CDP-choline 123 What C o n t r o l s the Rate o f PC S y n t h e s i s i n BHK C e l l s ? (i) Some G e n e r a l i z a t i o n s About C o n t r o l o f Flux 124 (ii) What C o n t r o l s the Flux i n BHK C e l l s ? . . . 126 (iii) Prediction 128 Are Routes o f PC S y n t h e s i s Other than the De Novo Pathway o f Importance i n BHK C e l l s ? . . 129 Some Unanswered Questions . 129 Concluding Remarks . . . . . . . 131  - viii  -  LIST OF TABLES Page Table 1 Table 2 Table 3 Table 4 Table 5  E f f e c t o f V i r u s I n f e c t i o n s on Animal PC and PE S y n t h e s i s  33  E f f e c t s of Semliki Forest Virus Infection on BHK C e l l Pool S i z e s  35  Wet Weight o f BHK C e l l s I n f e c t e d with SF V i r u s  65  P r o t e i n C o n c e n t r a t i o n s i n BHK I n f e c t e d with SF V i r u s  66  Cells  Enzymes o f De Novo S y n t h e s i s o f PC i n BHK C e l l s I n f e c t e d with SF V i r u s  81  I n h i b i t i o n o f Microsomal CT and CPT by SF V i r u s I n f e c t i o n o f BHK C e l l s  81  Table 7  S e p a r a t i o n o f [~ C^] C h o l i n e - C o n t a i n i n g Compounds . . . . . . . . . .  84  Table 8  I n c o r p o r a t i o n o f Q *C] C h o l i n e i n t o PC and i t s P r e c u r s o r s i n BHK C e l l s I n f e c t e d with SF V i r u s . . . .  87  S p e c i f i c R a d i o a c t i v i t y and Pool S i z e o f PC and i t s P r e c u r s o r s i n BHK C e l l s I n f e c t e d with SF V i r u s  91  Table 6  Table 9  llf  3  Table 10 Pool S i z e s o f C y t i d y l y l t r a n s f e r a s e Subs t r a t e s i n BHK C e l l s I n f e c t e d with SF V i r u s . Table 11 P o o l S i z e s o f N u c l e o t i d e s i n BHK C e l l s I n f e c t e d with SF V i r u s . . . . . . Table 12 Absorbance o f the Medium o f BHK C e l l s I n f e c t e d with SF V i r u s . Table 13 Comparison o f N u c l e o t i d e R e s u l t s by Two Procedures  95 99  . . 100 104  Table 14 Percent D i s t r i b u t i o n o f F a t t y A c i d s i n PC and PE from BHK C e l l s I n f e c t e d with SF V i r u s  106  Table 15 Percent D i s t r i b u t i o n o f F a t t y A c i d s i n D i g l y c e r i d e from BHK C e l l s I n f e c t e d with SF V i r u s  107  Table 16 F r a c t i o n a l Turnover Rates o f PC P r e c u r s o r s i n BHK C e l l s I n f e c t e d with SF V i r u s . . . . . 112 Table 17 Comparison o f Values o f V and Flux f o r Mock-Infected BHK C e l l s 127 m a x  - ix -  LIST OF FIGURES Page  Figure 1  Phosphoglyceride  Figure 2  B i o s y n t h e s i s of PC, triglyceride  structure . . . PE,  2  and  in rat l i v e r  4-5  .  6  Figure 3  De  Figure 4  Standard curve f o r c h o l i n e determination Standard curves f o r l i p i d phosphorus and d i g l y c e r i d e d e t e r m i n a t i o n s . . . . . . Time course f o r the c o n v e r s i o n of CTP i n t o CDP-choline . . . . . . . . . . . . .  Figure 5 Figure 6  novo b i o s y n t h e s i s o f PC  . .  Figure 7  Standard curve  f o r CTP  Figure 8  Uptake of c h o l i n e by BHK  . . . . . . .  determination  . . .  53 55 60 61  cells  i n f e c t e d with SF v i r u s . . . . . . . . . .  68  Figure 9  CK  a c t i v i t y vs.. p r o t e i n and  71  F i g u r e 10  CK  a c t i v i t y vs. choline  F i g u r e 11  CK  a c t i v i t y v s . ATP  F i g u r e 12  C y t o s o l i c CT a c t i v i t y v s . p r o t e i n and  time . . . . . .  .  72 73  time  75  F i g u r e 13  C y t o s o l i c CT  .  76  F i g u r e 14 F i g u r e 15  C y t o s o l i c CT a c t i v i t y v s . CTP Microsomal CT a c t i v i t y v s . phosphocholine . . . . . . . . . . . . . . . . . A c t i v a t i o n of c y t o s o l i c CT by r a t liver lipid .  77  F i g u r e 16 F i g u r e 17  a c t i v i t y v s . phosphocholine  of  . . . . . . . . . . . .  Q H~J c h o l i n e - l a b e l l e d l i p i d  F i g u r e 18  TLC  F i g u r e 19  Pool s i z e of PC and i t s p r e c u r s o r s i n BHK c e l l s i n f e c t e d with SF v i r u s S e p a r a t i o n of n u c l e o t i d e s from mocki n f e c t e d BHK c e l l s  F i g u r e 21  80  I d e n t i f i c a t i o n of the h y d r o l y t i c product of phosphocholine  F i g u r e 20  79  3  S e p a r a t i o n of n u c l e o t i d e s from SF v i r u s - i n f e c t e d BHK c e l l s  . . .  85 89 93 96 97  -  X  Page  Figure Figure Figure  0  22 23 24  E f f e c t o f d u r a t i o n o f i n f e c t i o n on CTP, UTP, and GTP p o o l s i z e s . . . E f f e c t of duration ATP p o o l s i z e Pulse cells  of  infection  c h a s e o f [^ EQ choline i n f e c t e d w i t h SF v i r u s 3  . . . .  102  on 103  i n BHK . . . . . . .  109  LIST OF ABBREVIATIONS A - absorbance ADP - adenosine diphosphate AMP - adenosine monophosphate ATP - adenosine t r i p h o s p h a t e [jATP-Mg^' - 1 s 1 complex of ATP and Mg, the p u t a t i v e s u b s t r a t e of CK B - pool s i z e BHK c e l l s - Baby Hamster Kidney -21 c e l l s BSS-2%DCS - E a r l e ' s Balanced S a l t S o l u t i o n , and 2% d i a l y z e d c a l f serum b u f f e r A - 0. IM N H 4 H C O 3 b u f f e r , pH 8.9: b u f f e r B - I M N H 4 H C O 3 b u f f e r , pH 10.0 cc - c u b i c centimeter Ci - curie CK - c h o l i n e kinase CMP - c y t i d i n e monophosphate CoA - coenzyme A cone. - concentrated cpm - counts per minute CPT - c h o l i n e p h o s p h o t r a n s f e r a s e CT - phosphocholine c y t i d y l y l t r a n s f e r a s e CTP - c y t i d i n e t r i p h o s p h a t e [^CTP-Mg]] - 1:1 complex of CTP and Mg, the p u t a t i v e s u b s t r a t e of CT DEAE - d i e t h y l a m i n o e t h y l DNA - d e o x y r i b o n u c l e i c a c i d dpm - d i s i n t e g r a t i o n s per minute EDTA - e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d , EGTA - e t h y l e n e g l y c o l - b i s (3-aminoethyl ether) N, N~ tetraacetic acid EK - ethanolamine kinase EPT - e t h a n o l a m i n e p f r ^ ET - phosphoethanolamine c y t i d y l y l t r a n s f e r a s e Fig. - figure g - gram g_ - g r a v i t y GDP - guanosine diphosphate <GTP - guanosine t r i p h o s p h a t e h - hour ITP - i n o s i n e t r i p h o s p h a t e k - f r a c t i o n a l turnover r a t e ( a l s o ; a b b r e v i a t i o n f o r rate constant) K^ - d i s s o c i a t i o n constant of an enzyme-inhibitor complex K - Michaelis-Menten constant m  I, 1 - l i t e r LPC - l y s o p h o s p h a t i d y l c h o l i n e LPE lysophbsphatidylethanolamine m - meter M - molar r~Mg-ATP~J -.1:1 complex of ATP and Mg, the p u t a t i v e s u b s t r a t e o f CK min - minute N - normal nd - not d e t e c t a b l e nmol - nanomole NS - not s i g n i f i c a n t p - statistical probability PBS - phosphate-buffered s a l i n e , pH 7.4 PC - p h o s p h a t i d y l c h o l i n e PE -phosphatidylethanolamine PEMT - phosphatidylethanolamine methyltransferase PFU - plaque forming u n i t p . i . - post i n f e c t i o n P^ - i n o r g a n i c phosphate pk - negative l o g a r i t h m of the a c i d d i s s o c i a t i o n constant q - turnover r a t e or f l u x R - radioactivity ref. - reference Rf - r a t i o o f d i s t a n c e moved by a s o l u t e to t h a t moved by the solvent front RNA - r i b o n u c l e i c a c i d S - s u b s t r a t e c o n c e n t r a t i o n ( a l s o a b b r e v i a t i o n f o r Svedberg unit) s.d. - standard d e v i a t i o n SF v i r u s - S e m l i k i F o r e s t v i r u s t - time TLC - t h i n l a y e r chromatography T r i s - t r i s (hydroxymethyl) aminomethane UTP - u r i d i n e t r i p h o s p h a t e UV - u l t r a v i o l e t v - volume V - velocity Vf - forward v e l o c i t y (of an enzyme r e a c t i o n ) V - maximal v e l o c i t y (of an enzyme r e a c t i o n ) V - reverse v e l o c i t y (of an enzyme r e a c t i o n ) w - weight a  m a x  r  p - disequilibrium ratio Notes -2 -3 1. S t a n d a r d g p r e f i x e s are£„c ( c e n t i ) - 1 0 _ , i m ( m i l l i ) - 1 0 ; u(micro)-10~ ; "ri(naho)-10. ; p ( p i c o ) - 1 0 2. A b b r e v i a t i o n s f o r c e l l types other than BHK are not l i s t e d here.  ACKNOWLEDGEMENTS  I extend the utmost a p p r e c i a t i o n to my s u p e r v i s o r , Dr. Dennis E. Vance f o r h i s continued guidance, which kept t h i s t h e s i s p r o j e c t on a s t r a i g h t l i n e of r e s e a r c h . I am very g r a t e f u l to Dr. P a t r i c k Choy f o r h i s work on the measurement of CTP and phosphocholine with c y t i d y l y l t r a n s f e r a s e and f o r h i s many h e l p f u l d i s c u s s i o n s . I owe Mr. Harry Paddon many thanks for h i s g r e a t a s s i s t a n c e i n some of the longer and more t e d i o u s experiments. I am indebted to Dr. A l l e n Delaney f o r h i s sugg e s t i o n to use c h a r c o a l as a means of s e p a r a t i o n of phosphoc h o l i n e and CDP-choline, to Dr. G.M. Tener f o r h i s u s e f u l sugg e s t i o n s and comments, and to Dr. C a r o l i n e A s t e l l f o r her encouragement. Thanks are due to Dr. Everard T r i p and Dr. Michae Smith f o r t h e i r tremendous a s s i s t a n c e i n high pressure l i q u i d chromatography. I am o b l i g e d to Miss Karen Catherwood, Mrs. Jenny Toone, and Mr. Harry Paddon, who a l l a s s i s t e d i n the t i s s u e c u l t u r e of BHK-21 c e l l s .  XIV  DEDICATION  -Dedicated  to the C r e a t o r o f a l l l i f e  -  1 -  INTRODUCTION  (a)  Phosphatidylcholine  - I t ' s R e l e v a n c e and  Glycerophospholipids ture  shown i n F i g u r e  (PC),  i s of  importance  most a n i m a l in  brain  (except  The' p h o s p h o l i p i d , since  i t i s the major p h o s p h o l i p i d  in  I t predominates  w h i c h have more  phosphatidyl-  (PE)), heart,  liver,  kidney,  lung,  i n the  (1).  struc-  phosphatidylcholine  thymus, p i t u i t a r y ,  spleen,  adrenals,  p i g e r y t h r o c y t e s w h i c h have more PE,  pholipid  general  pigeon  e r y t h r o c y t e s which  brane  the  human and  muscle, pancreas,  goat  with  t i s s u e s w h i c h have been a n a l y z e d .  ethanolamine  (except  1.  are molecules  Biosynthesis  l a c k PC)  subcellular  In BHK  cell  (1).  fractions  PC  and  and  skeletal  erythrocytes .  s h e e p , cow,  i s a l s o the major  t e s t e d , except  and phos-  p l a s m a mem-  p l a s m a membranes however, PC  i s the  major  phospholipid ( 2 ) . In b i o l o g i c a l since  they  membranes, p h o s p h o l i p i d s  are primary  proteins  are  tributed  asymmetrically  to  three-quarters  t o be for  thought  s t r u c t u r e s , forming  i n the  an  lipid's  outer  of  across  the  half  asymmetry o f PC accessibility  PC of  the  across  asymmetry.  d i f f e r e n c e was  and  PE  by  e_t a l  a bilayer  rat liver  For  membrane  (6,  2  (8) , c o n f l i c t  with  between t h e  microsomes d i s p e r s e d with  two-thirds  (5).  appear Evidence  However, the  detergent  '• ; .  be.dis-  i s b a s e d on  7).  i n c r e a s i n g amounts o f p h o s p h o l i p a s e ,  m i c r o s o m e s and  may  example,  microsomes A  i n which  rat erythrocytes  cytoplasmic  observed  importance  Phospholipids  a membrane.  to phospholipase  Sundler  PC  4).  i n human and  e x p e r i m e n t s by No  (3,  to f l o a t  are of  notion  the  recent of  h y d r o l y s i s of between (8).  intact  -  2  -  Figure 1  PHOSPHOGLYCERIDE STRUCTURE  0  0  II  C H  II  2  - 0 - C - R  I  R - C - 0 - CH  I  O  I I L  C H 2 - 0 - P - 0 - X 0  Phosphatidic  acid  X = H  Phosphatidyl  choline  Phosphatidyl  ethanolamine  X = CH CH S(CH ) 2  2  3  X = CH CH NH 2  2  C00~  Phosphatidyl  serine  I  X = CH CH 2  ^NH Phosphatidyl  glycerol  3  3  X = CH CHCH OH 2  2  I OH  3  -  The  r e g u l a t i o n o f PC s y n t h e s i s ,  metabolic For  c o n t r o l , may  may  availability.  physical isolation a l s o be l i m i t e d  formational,  active The  may  Finally, control  across  be  unavailable  a s u b c e l l u l a r membrane.  Substrates  however, may  by t h e c o n c e n t r a t i o n species  a l s o be l i m i t e d  of s p e c i f i c  of a given  required  con-  by t h e enzyme.  En-  i t was shown t h a t  3 2  into phospholipids,  be  The p r e s e n t  inert.  PC b i o s y n t h e s i s  Figure  alter  enzyme  and d e g r a d a t i o n  kinetics.  o f an enzyme  may  i t sa c t i v i t y (9).  corporated  2.  In  the f o l l o w i n g  t h e s e compounds were t h o u g h t t o  o f PC i s shown  introductory  S t e p s o f De Novo PC Choline  features  m  cilitated Similarly,  3.  s e c t i o n s , PC s y n t h e s i s i s  of i t s r e g u l a t i o n are o u t l i n e d .  Synthesis  Transport  two a p p a r e n t K ' s. uptake  i s outlined in  i n Figure  In E h r l i c h - L e t t r e * - A s c i t e s tumor c e l l s ,  affinity  i s rapidly i n -  from g l y c e r o l - 3 - p h o s p h a t e  and t h e known  (i)  P-orthophosphate  u n d e r s t a n d i n g o f the dynamics o f r a t  De novo s y n t h e s i s  described  has  ionic,  by t h e e q u i l i b r i u m between  e f f e c t o r s may  the r a t e s o f s y n t h e s i s  Before  (b)  mechanisms o f  and i n a c t i v e f o r m s , o r t h e r a t e o f c l e a v a g e o f p r o e n z y m e s .  binding  liver  other  r e a c t i o n s c a n be c o n t r o l l e d by  Substrates,  or tautomeric  zyme a c t i v i t y  like  be c o n t r o l l e d by a v a r i e t y o f f a c t o r s .  example, e n z y m e - c a t a l y z e d  substrate by  3 -  H i g h - a f f i n i t y uptake  ( K , 0.22 mM)  diffusion choline  choline  m  and s i m p l e  are probably diffusion,  i s transported  across  transport  ( K , ..59 uM) m  and low-  carrier-mediated f a r e s p e c t i v e l y (10).  the g u i n e a p i g jejunum  -  F i g u r e 2. B i o s y n t h e s i s  4  -  o f PC, PE, and t r i g l y c e r i d e  in rat liver  CH OH 2  HOCH  0  (Glycerol-3-phosphate)  II I  CH OP - OH 2  I. O A c y l CoA <  y  Fatty acid  ^  CoA  AMP ATP + CoA + pyrophosphate Acylglycerol-3-phosphate A c y l CoA CoA CH2OR1  I 0 I I I CH 0P  (Phosphatidic a c i d ;  R OCH 2  - OH  2  L  0  4 . \ | r ^ - phosphate CH2OR1  R OCH 2  !•  I'  CH OH  \  { 1 , 2 - d i a c y l g l y c e r o l , or d i g l y c e r i d e )  V  2  CDPethanolamine,  CDPcholine  CMP -J/  CH2OR1  I R OCH I 2  3 S-adenosyl-'"'^3 S-adenosylmethionine homocysteine  (Triglyceride)  CH2OR3  - 5 -  Figure  2  continued Enzyme Commission Number  Enzyme Name 1.  Acyl-CoA  2.  Glycerophosphate  3.  Acylglycerol-3-phosphate  4.  PhOsphatidate  5.  Ethanolaminephosphotransferase  6.  Choiinephosphotransferase  7.  Diacylglycerol acyltransferase  8.  synthetase  6.2.1.3  acyltransferase  2.3.1.15  acyltransferase  phosphatase  3.1.3.4 (EPT)  (CPT)  2.7.8.1 2.7.8.2 2.3.1.20  Phosphatidylethanolamine methyltransferase  Note:  (PEMT)  A l l o f t h e above enzymes o c c u r  ' 2.1.1.17 in rat.liver  microsomes.  -  Figure  3. De n o v o b i o s y n t h e s i s  (CH -) NCH CH 0H 3  j3  6 -  2  o f PC  (choline)  2  0 ATP (CH ) NCH CH 3  o  ADP  (CH ) NCH CH OP 3  3  2  (betaine-aldehyde)  2  5  -  2  3  '  0  OH  I. ( p h o s p h o c h o l i n e()C H ) N C H C|_ 3  3  T  P  N 2.  NH -  Pyrophosphate 0  M (CH ) ^CH CH 0.r 3  (betaine)  0  0 C  2  3  2  2  J  -  P-O-P- 0- C H 0  1,2-diacylglycerol  0  IN  2  0  (CDP-choline)  0 OH  OH  3. CMP  N PC  Enzyme Name 1. 2. 3. 4. 5.  C h o l i n e K i n a s e (CK) •• Phosphocholine c y t i d y l y l t r a n s f e r a s e (CT) Cholinephosphot r a n s f e r a s e (CPT) C h o l i n e dehydrogenase Betaine-aldehyde dehydrogenase  Enzyme Commission Number 2.7.1.32  Enzymes o f  2 . 7.7*15  de novo  2.7.8.2 1.1.99.1  synthesis  -  mucosa by a s a t u r a b l e affinity port  process,  of choline  toma c e l l s . value  h i g h - a f f i n i t y process  as a l o w -  p r o b a b l y due t o d i f f u s i o n ( 1 1 ) . The  has a l s o  been s t u d i e d  In t h e s e c e l l s ,  using  the t r a n s p o r t  Novikoff process  h i g h - a f f i n i t y u p t a k e by r a t b r a i n  0.83 yM)  i s proportional  t o t h e _in v i v o  n e u r o n s and t o a c e t y l c h o l i n e uptake  e x c l u s i v e l y found cells'  Many l i n e s limiting  r a t hepa-  has a K  m  turnover  synaptosomes ( K , m  activity  of cholinergic  ( 1 3 ) . The h i g h - a f f i n i t y  i n c h o l i n e r g i c nerve  transport  of evidence  dissociated  terminals.  ( 1 0 ) , i t i s an a c t i v e  show t h a t  and r e g u l a t o r y  In c o n t r a s t ,  this  in acetylcholine  cells  of phosphocholine  of  i n t h e medium, t h e added c h o l i n e  choline  recovered portion  m  was  a smaller  measured was n o t t h e same p r o c e s s w h i c h o c c u r s  Very h i g h - a f f i n i t y c h o l i n e  Also,  i n support of t h i s  (of g u i n e a p i g s )  high  pro-  ( 1 5 ) . The h i g h - a f f i n i t y t r a n s p o r t i n  port  late  a  increasingly  c a s e was n o t an a c t i v e p r o c e s s .  until  with  concentrations  this  velop  i s rate-  ( K , 16 yM)  At higher  as a c e t y l c h o l i n e and f r e e c h o l i n e , w i t h  of phosphocholine  system.  formation (14).  i s associated  formation.  Unlike  :  system  h i g h - a f f i n i t y uptake o f c h o l i n e  r a t embryo b r a i n  I t may  transport  transport  proportion  over  trans-  i s s o d i u m - , p o t a s s i u m - , and c h l o r i d e - d e p e n d a n t .  ascites  by  as w e l l  o f 4-7 yM ( 1 2 ) . The  be  7 -  uptake  i n embryogenesis,  a c e t y l c h o l i n e at a high  the embryonic  ( K , approx as o c c u r s  trans-  in adults.  1 yM) may n o t d e -  m  conjecture,  were f o u n d  Perhaps  i n chick  postnatal  brain  r e t i n a (16). synaptosomes  t o form p h o s p h o c o l i n e p r e f e r e n t i a l l y choline  concentration (17).  - 8 -  Free c h o l i n e of  adult  rats  t r a n s p o r t may ability  by a p r o c e s s w i t h a s i n g l e be  n e c e s s a r y because  to s y n t h e s i z e c h o l i n e  (ii) An  Choline  the p r e s e n c e  in  y e a s t , and (22).  formed  of M g  i n acetone Kornberg  lipid  a liver  100,000 x £ et  er  v i a PE m e t h y l a t i o n  Pricer and  3 2  showed 1 4  C)  from  (CK)  activity  choline  i n 1952  i s almost  of r a t l i v e r which  electrophoresis  different At 26).  and  times  least  CK  and  and  and  soy beans the  ation  first into  choline ganism  CK  has  appear  and  and  double-  exclusively  brain  ethanolamine  (24, 2 6 ) .  Two  sensitivity  to a  was  a  Upreti  microsomal  (24,  kinase  i n the  (24).  26,  (EK)  and  the  rat spinal  s e p a r a t e d from  i n the p r o t o z o a n , E n t o d i n i u m does n o t a f f e c t  i s intimately associated  In r a t l i v e r ,  in  however, c h o l i n e  m  at  (28).  EK  (24,  germinat-  caudatum  ethanolamine  ethanolamine  K 's  appear  cord  sites  liv-  non-de-  with d i f f e r e n t  t o have s e p a r a t e a c t i v e  In the s e c o n d ,  which  CK's  27).  in rat  to hemicholinium  been p h y s i c a l l y  case, choline  lipids.  (31).  Kornberg  transferred  homogenates  d u r i n g development of  (29)  uptake,  ATP  intestine, that  lacks.corroboration  and  different  one EK  no  (19-21).  brain,  c o u l d be  supernatant of • r a t l i v e r  choline  Such  or  have been shown by D E A E - c e l l u l o s e c h r o m a t o g r a p h y and  for  In  little  d i s c o v e r e d by W i t t e n b e r g  ( P  forms o f b o t h CK  naturing  ing  kinase  (25), a f i n d i n g  Multiple  has  (18) .  p r e p a r a t i o n (23).  a L . r e p o r t e d t h a t CK  enzyme  rat brain  powders o f l i v e r ,  and  phosphocholine  Choline  \iM)  (440  m  phosphocholine  was  + +  labelled by  K  Kinase  enzyme w h i c h  in  kidney  i s t r a n s p o r t e d a c r o s s the b l o o d - b r a i n b a r r i e r  (30).  incorpor-  does n o t a f f e c t  w i t h CK  in this  i s a highly  -  or-  effective  -  inhibitor hibitor with  o f a t l e a s t one EK, whereas e t h a n o l a m i n e  o f CK  (24, 26, 3 2 ) . R a t l i v e r  an e x c e s s o f M g  + +  , or v i c e  presence o f equimolar M g Choline to  betaine.  choline,  9 -  i t has been  CK i s i n h i b i t e d  versa;  by ATP  b u t n o t by ATP i n t h e  (24).  + +  h a s an a l t e r n a t e In f a c t ,  i s a poor i n -  fate  in liver.  by i n t r a p o r t a l  I t may be  injections  shown t h a t , c h o l i n e  oxidized  o f Q "*C 1  oxidation  i s at least  as f a s t as p h o s p h o r y l a t i o n ( 3 3 ) . (iii) The  Cytidylyltransferase  pathway o f PC b i o s y n t h e s i s was f u r t h e r  Kennedy and W e i s s stimulated They  They  the i n c o r p o r a t i o n  found a s o l u b l e  sible  discovered  that  enzyme w h i c h  catalyzed  + phosphocholine<  the f o l l o w i n g  rever-  cytidylyltransferase  CDP-choline + Pyrophosphate  (CT) was f o u n d  to require  CT e x i s t s  weight  2 x 10 and o v e r 1 x 10 i n r a t l i v e r  i n two i n t e r c o n v e r t i b l e  5  6  enzyme has been  Choy e t a l . ( 3 5 ) .  purified  supernatant  The  has been  by S u n d l e r  reversible  960-fold  (37).  reactions  purified  adex G-200.  co-chromatographed ( 3 8 ) .  + +  and l u n g  (35, 36) . cytosol  by  phosphoethanolamine 1 1 0 0 - f o l d from  rat liver cata-  37).  from r a t l i v e r  and m o n o m e t h y l e t h a n o l a m i n e  or Mn  Both c y t i d y l y l t r a n s f e r a s e s  (35,  S e p a r a t e d from  + +  forms o f m o l e c u l a r  p h o s p h o c h o l i n e and d i m e t h y l e t h a n o l a m i n e  cytidylyltransferases  Mg  from r a t l i v e r  The a n a l a g o u s enzyme,  cytidylyltransferase  lyze  CTP s p e c i f i c a l l y  o f l a b e l l e d p h o s p h o c h o l i n e i n t o PC.  (34).  The  by  reaction: CTP  The  (34).  elucidated  phosphate  co-chromatograph  t h i s peak,  on Seph-  the phosphoethanolamine  phosphate c y t i d y l y l t r a n s f e r a s e s  also  - 10  (iv)  Cholinephosphotransferase  Kennedy and W e i s s d i s c o v e r e d t h a t CDP c h o l i n e verted This  t o PC by t h e 18,000 x cj p e l l e t  reaction  in  a rat liver  was s t i m u l a t e d by d i g l y c e r i d e  photransferase The  from  :(CPT) . from  rat liver  c o u l d be c o n -  (34).  h a s been w e l l  homogenate.  Cholinephosstudied.  s e p a r a t e n a t u r e o f t h e enzymes c a t a l y z i n g the  final  de novo s y n t h e s i s o f PC and PE h a s been c o n t r o v e r s i a l .  c o n t r o v e r s y was l a r g e l y solubilized rat  liver  resolved  microsomes  (39).  was s e p a r a t e d  dependant from  forms.  EPT (39) .  CDP-ethanolamine castor that CPT  into: + +  bean endosperm  and EPT r e s p o n d (43).  treatment  + +  (40).  Lack  and 2. M n  + +  X-100.  or M g  + +  -  o f CPT was n o t s e p a r a t e d  phosphotransferase occurs i n o f such  competition  i n the protozoan  differently  Indirect  form  with T r i t o n  c o m p e t i t i o n between C D P - c h o l i n e and  a single  s e p a r a t e enzymes o c c u r  acids  after  The M n - r e q u i r i n g  suggest  (EPT) from  T h i s s e p a r a t i o n was a c h i e v e d by s u c -  1. M n - d e p e n d a n t  In c o n t r a s t ,  The  separation of  CPT and e t h a n o l a m i n e p h o s p h o t r a n s f e r a s e  rose g r a d i e n t c e n t r i f u g a t i o n CPT  by t h e p a r t i a l  step  evidence  caudatum ( 4 1 ) .  to deoxycholate shows t h a t  suggests  ( 4 2 ) , and f a t t y  r a t adipose c e l l s  have  s e p a r a t e enzymes ( 4 4 ) . CPT K  1 m  s  may o c c u r  i n more t h a n one f o r m .  for CDP-choline  brain  (45).  Chick  appear  brain  glial  and n e u r o n a l C P T s 1  (46).  are  by f a t t y  affected  differently  different  d u r i n g development o f the c h i c k  ent K ' s for CDP-choline m  CPT's w i t h  Also,  rat brain  acids (43).  have  differ-  and l i v e r  CPT's  -  The  remaining d i s c u s s i o n  zyme, u n l e s s o t h e r w i s e Since labelled  Bremer p r o p o s e d step  and c o - w o r k e r s  of CDP-choline  injection,  equilibration  from  methyl-  B j ^ r n s t a d and  took p l a c e o v e r  radioactivity  t h e CPT  was n o t c o n -  using methyl-labelled  o f the t i s s u e  storage of l i v e r five-fold  have s i n c e  i s much s m a l l e r  freeze-clamping  obtained  of CDP-choline  methionine  i n t r a p o r t a l l y (48).  Sundler  shown  that  than p r e v i o u s l y i s performed  before extraction  raised  the p o o l  earlier  the C D P - c h o l i n e times  caused  after  ( 3 3 ) . One m i n u t e o f the CDP-choline  a low e s t i m a t e o f s p e c i f i c  injection  specific  of labelled  radioactivity  size  estimated i f  ( 4 9 ) . The h i g h e r e s t i m a t e s o f t h e C D P - c h o l i n e  of CDP-choline  As  rapid  by S a l e r n o and B e e l e r ,  injected  line,  intraperitoneal  that  en-  e q u a l t o t h a t o f PC, o f r a t l i v e r ,  ( 4 7 ) . However, e q u a l s p e c i f i c  firmed  four  radioactivity  m e t h i o n i n e was a b o u t after  o f CPT r e g a r d s t h e r a t l i v e r  stated.  the s p e c i f i c  20-80 m i n u t e s  11 -  choline.  in liver  was  level pool  radioactivity F o r example, reported  as  t h a t o f PC, b u t o n l y o n e - t w e l f t h t h a t o f p h o s p h o c h o -  a t twenty  minutes  a consequence,  after  intraperitoneal  the importance  i n j e c t i o n (47).  o f t h e CPT back  reaction  was  overestimated. A current back  e s t i m a t e o f the r a t i o  v e l o c i t y jLn v i v o  velocity  calculations  that d i f f e r e n t thesized  species  at different  i s between 1.2 over  this  o f the forward v e l o c i t y t o and 2.3  However,  s t e p a r e c o m p l i c a t e d by t h e f a c t  and s u b c e l l u l a r rates  (49) .  (50) .  p o o l s o f PC may  be s y n -  -  Still, since  the  (51). vary  are  Since at  the  reaction  cies  of  a l l equally  no  marked  Lack o f  degrades  1-palmitoyl, The position  glycerides,  the  CPT  lein  The cificity or  of  and  for  then  the  reaction  PC  are  had  (56)  r e a c t i o n has of  the  diglyceride.  acids  at  this  1-palmitoyl  transferase  fatty  (53,  affected  preferred  than  spe-  (52). trans-  1-myristoyl,  by  fatty  acids  at  over  1-stearoyl diglycerides, for  dio-  (44).  been shown t o have l i t t l e  2-position  of  However, the  1-stearoyl  endogenous degree of the  (at a  spe-  (53,  54)  unsatura-  degree  diglyceride  40%  substrate  2-  the  a marked p r e f e r e n c e  inhibits  i s the  since  ethanol-dispersed  of ide  the  W i t h exogenous d i -  t h a t CDP-e.thanolamine  CPT  back  54).  demonstrated  uM)  which  a n a l y s i s of  p o s i t i o n does m o d i f y  over  at  of preference,  H o l u b has 24  acids by  (diglyceride).  r a t adipose c e l l s  acids  the  reaction  p r o d u c e PC's  1-position,  is also  species  back  product, d i g l y c e r i d e  S i m i l a r l y , using  fatty  the  1-acylly-  the  1-stearoyl  substrate  for  of  unlikely,  verified  order  forward  preference  be  was  in decreasing  CPT  fatty  may  synthesis  l-stearoyl-2-oleyl glycerol  exogenous  tion  PC,  for  that  i n the  (55).  from  over  i t appears  1-palmitoyl  glycerides  CPT  pathways o f  selectivity  forward  1 of  by  i s observed  and  CPT  used  selectivity  substrate,  Selectivity ferase  three  2-position,  the  o f PC  l a b e l l e d from c h o l i n e , m e t h i o n i n e , or  these  has  position.  -  p h y s i c a l l y separate pools  pools  solecithin  12  (55).  of Also,  concentration  more i f exogenous h e x a e n o i c d i g l y c e r i f exogenous monoenoic d i g l y c e r i d e i s  used  (57).  nation  This  against  Assuming PC  and  phate  e f f e c t may  PC  synthesis  that  [_ 1  i s incorporated to c a l c u l a t e  ide  i n PC  acid.  By  respectively,  in  synthesis  PE  These  methylated  Cycling  PE w h i c h  tetraenoic  possibly In  by  in v i v o  and  PC's.  and  back  tetraenoic  the CPT  and  Thus,  PC may was  into phos-  be  back  reaction  M o r i y a and  reaction,  phosphatidic and  oneused  acid  (58).  reaction  (58).  are p r e f e r e n t i a l l y a c y c l i n g of  occur  in v i v o  initially  transferred  diglycer-  diglycerides  polyenoic (53, 58) .  proposed  l a b e l l i n g indicated  that  from PC  by  methyl  to CDP-choline,  (63).  Kanoh o b s e r v e d an e q u i l i b r a t i o n o f  However, the r e a c y l a t i o n back  P ~J  one-third  from p h o s p h a t i d i c  (54, 5 9 - 6 1 ) .  could  that  from  s p e c i f i c r a d i o a c t i v i t i e s of tetraenoic  the CPT  32  d i s t r i b u t i o n of  h e x a e n o i c PE's  In. v i v o  the CPT  r a t lung,  the  than  and  of hexaenoic d i g l y c e r i d e  of methionine  ( F i g . 2 ) , and Q  calculated  a r i s e by  between PE  T i n o c o e_t a l (62) . groups  i t was  could  units  (57).  i s incorporated  d i d not o r i g i n a t e  of hexaenoic  t o form PC  diglyceride  glycerol  acid only  do n o t o r i g i n a t e  diglycerides  Once f o r m e d ,  H ~J  discrimi-  hexaenoic d i g l y c e r i d e  the f a t t y a c i d  t h i s method,  half,  3  the i n v i v o  v i a any CDP-base pathway, S u n d l e r e_t a l  were a b l e  and  from  (3) -  PE v i a p h o s p h a t i d i c  units  partly explain  could  be  diglycerides  of 1 - a c y l l y s o l e c i t h i n , responsible  rather  f o r the e f f e c t  (64). One operating reaction  other  line  _in v i v o . i s ImM  of evidence points The  t o the back  of CDP-choline  reaction  f o r t h e CPT  f o r the microsome-bound enzyme  (51).  reverse CDP-choline  -  has  a concentration  inhibition of  the  should  back  of  not  14  -  a b o u t 10 yM occur.  The  reaction is s t i l l ,  as  in rat liver  oriented in rat liver  accessible  .to  Coleman and  trypsin  Bell  y e t , not  1-7  sible,or  cytoplasmic  acid  (c)  in Figure  phosphatase  teolysis  intraperitoneal lipids  donor  and  (67).  (68).  (68).  of  t h a t the  first  Furthermore, pool  -  1  '*c]]  was  size  found  totally (65).  novo enzymes protease-accesfor  phosphatidic  resistant  to  that rats given  methionine  methylation  formed  f u n c t i o n as  pro-  step  very  three orders  liver and  microsomes  little  supporting  in  the  is rate-limiting  e x p e r i m e n t s have shown t h a t the  are  an  a methyl  in rat liver  phospholipids,  ethanolamine  (69).  are  (66).  i n c h o l i n e , with  m o n o m e t h y l e t h a n o l a m i n e and  in rat liver  the  was  asymmet-  di-methylethanolamine,  phosphatidyl  PC  on  reported  mono- and  mono-and d i - m e t h y l e t h a n o l a m i n e clusion  Q Me  enzyme s y s t e m was  Most r a d i o a c t i v i t y  are  Methyltransferase  A d e n o s y l m e t h i o n i n e would The  understood.  PC  Greenberg  injection  that  microsomes  de  d i s r u p t e d microsomes)  containing labelled  choline  the  phosphatase a c t i v i t y  and  Bremer  CPT  one-sided  definitely  Phosphatidylethanolamine  In 1959,  and  s i d e of microsomes, except  (The  in intact  2 are  clearly  microsomes, s i n c e both  in intact,  A l t e r n a t e Formation of (i)  CT  have shown t h a t a l l o f  numbered  so  physiological significance  Vance e t a l . e s t a b l i s h e d t h a t b o t h rically  (49)  con-  (68).  levels  phosphatidylf.dimethyl-  o f magnitude, s m a l l e r  than  PE  and  of  - 15 -  Recently, tion  s e p a r a t e enzymes w h i c h c a t a l y z e :  o f PE t o t h e monomethyl p r o d u c t  t o form  PC, have been d e m o n s t r a t e d  e r y t h r o c y t e membranes enzyme is  i s located  the methyla-  further  i n bovine  methylation  a d r e n a l m e d u l l a and  In t h e e r y t h r o c y t e , t h e f i r s t  on t h e c y t o p l a s m i c s u r f a c e , whereas t h e s e c o n d  on t h e e x t e r n a l membrane f a c e ( 7 1 ) . Rat.liver  PC  (70, 7 1 ) .  and 2.  1.  phosphocholine_„and_.GDPreh61ine„are  i s labelled,  methionine  after  (48).  an i n j e c t i o n  Consequently,  3  of  H~J  labelled  ethanolamine or  S a l e r n o and B e e l e r p r o p o s e d  phosphoethanolamine  and C D P - e t h a n o l a m i n e were d i r e c t l y  in  However, t h i s  ratliver  [~_ 3 H ~ J  (48).  ethanolamine  activity  t i m e , when compared Methionine ionine than  level,  N-methylation  sis  [_ H~J  tion  from  3  glycerol  [~_ m e t h y l -  inhibits  nosylethionine tentially  i n these c e l l s  r a tliver  t h e amount  N-methylation N-methyl bases.  ^CjJ  methionine  (72) .  ET.  Feedback  S-adenosylhomocysteine  (73) .  Inhibition  o f PE a v a i l a b l e  radiowith  o f methhigher  PE c o n v e r s i o n  may c o n t r o l N - m e t h y l a t i o n  have no e f f e c t  reduce  1  *Q  3  forms PC a t 20-40% o f t h e r a t e  by S - a d e n o s y l m e t h i o n i n e  compound  Addition  a doubling of Q  after  i n l i v e r (50).  at a concentration slightly  At a saturating  tration,  because  i s v e r y low and does n o t change  caused  that  methylated  the s p e c i f i c  t o t h a t o f phosphoethanolamine  to r a thepatocytes  (72).  i s unlikely,  s t i m u l a t e s . PE N - m e t h y l a t i o n .  the plasma  t o PC  idea  i s administered to rats,  o f phosphocholine  before  conceno f syntheinhibisince  this  and S-ade-  o f ET c o u l d p o -  f o r methylation.  c o u l d be r e g u l a t e d by t h e a v a i l a b i l i t y  N-mono o r N , N - d i m e t h y l e t h a n o l a m i n e  cause  o f the a reduced  -  conversion Q^C  of  |_. H U PE  of  3] m e t h i o n i n e  suggests  that  (ii)  the  Base  Choline  16  t o PC,  i n t o PC  b a s e s may  but  increase  in hepatocytes  enter  the  into rat liver  m i c r o s o m a l enzyme + +  (74-76).  (77,  change o f p h o s p h o l i p i d (78). and  K 's,  A  result  pathway,  b r a i n PC  by  catalyzed is  a  by  a  synthesized in rat  s i n g l e enzyme c a t a l y z i n g the  ethanolamine  and  s e r i n e was  e f f e c t s o f pH  suggest  m  have s e p a r a t e  This  e n e r g y - i n d e p e n d e n t exchange  78).  However, d i f f e r e n t  differing  Phosphotidylserine  -requiring  mitochondria  and  process,  + +  liver  (72).  Exchange  i s incorporated  a similar Ca  incorporation  methylation  Ca -dependant, nucleotide-independent  by  the  that  and  postulated  various  rat liver  (79)  ex-  inhibitors,  and  brain  exchange enzymes f o r c h o l i n e , e t h a n o l a m i n e ,  (76) and  serine. The pulse tion  labelling  also lack  (80) .  Rat  ine 3-6%  by  of  has  with total  o f PC  U  C  J  base w i t h  a second  hepatoma  choline  choline did PC's  of  rat  reliver,  (33).  a v a i l a b l e f o r exchange w h i c h  p r e l a b e l l i n g PC the  [  Iri v i v o  incorpora-  Novikoff  lipid-bound  into polyunsaturated  a pool  removal of  the  with  choline  (48).  f r e e and  labelling  been d e b a t e d .  f o r any  base exchange  a base e x c h a n g e p r o c e s s  brain  labelled  evidence  exchange between  c a l c u l a t e d by  petitive  no  incorporation  p r e s u m a b l y by  be  PC  However, _in v i v o rapid  base exchange has  provided  into rat liver  cells  veal  s i g n i f i c a n c e of  with  choline,  followed  by  com-  c h o l i n e , e t h a n o l a m i n e , or  isotope.  b r a i n m i c r o s o m a l PC  This  pool  (81).  represents  can  ser-  only  -  In v i v o  experiments  change o c c u r s choline (82),  (82,  and  i n the 83).  choline  brain  choline  and  explain  such  i n the  r e s u l t of  results  (84).  in g l i a l  (d)  P o t e n t i a l S i t e s of Choline  biosynthesis  mammalian c e l l s .  the  cellular  port  20  yM,  tion  (87).  At  transport  for  t o be  example, but  Base exchange  e t h a n o l a m i n e and  serine  localized  of  De  Novo  be  regulated  K  (80).  incorporation i n the  by  would  exchange  in neuronal  cells,  Synthesis  f r a c t i o n derived  At  f o r PC  same means i n  (86).  almost a l l of  from l a b e l l e d c h o l i n e  The  K  m  of  choline  o f m a g n i t u d e below the  i s equal  m  the  hepatoma c e l l s ,  medium c o n c e n t r a t i o n s  choline,  (80).  (83).  (85) .  order  limiting  limiting  rather  phosphorylation,  into l i p i d .  In N o v i k o f f  parently  centration  i n t o PC  cerebellum,  choline  appear  not  acid-soluble  into l i p i d  biphasic  base exchange enzyme i n r a t  Regulation  may  i s more than one  the  is  minutes a f t e r i n j e c t i o n  the  medium, i s p h o s p h o c h o l i n e  these c e l l s  into l i p i d  Transport  all  the  In  The  cells  ex-  i n t r a c e r e b r a l i n j e c t i o n s with  incorporation  little  in  few  causes diminished  with  PC  first  for a choline  enzymes from r a b b i t b r a i n  (i)  incorporation  base  i n j e c t i o n of l a b e l l e d  i s p r e f e r e n t i a l l y incorporated  the  choline  indicate that  3 minutes a f t e r  hemicholinium-3.  hemicholinium-3 increased  first  support  comes from  -  in rat brain  Choline  than p h o s p h o c h o l i n e Further  17  to  the  K  m  of  into l i p i d  choline  o f CK  another  is  choline  step  than  incorpora-  However, above 100  that  in  less  transport  i s independent of  medium, s u g g e s t i n g  m  of c h o l i n e  these c o n c e n t r a t i o n s , biosynthesis.  K  trans-  ap-  yM con-  becomes  - 18  (ii)  Choline Kinase  In c o n t r a s t  t o hepatoma c e l l s ,  t o t h e medium o f E h r l i c h phosphocholine) experiments The for  inside  suggest  Mg  + + _  ascites  spermine  c o n c e n t r a t i o n , spermine  (88).  Polyamines  amines c o u l d the e f f e c t s (iii)  on CT  and  and  160  times  c o n c e i v a b l y be  might  two  rat liver  during rapid  by CT  the i n c o r p o r a t i o n  by  CK  tissue  the  K  (88).  CK,  m  At  nine-fold  growth.  stimulating  is limiting regulated  of  o f HeLa c e l l s  activity  Poly-  but  cannot  until be made.  phatidylcholine  (LPC).  forms  (LPE)  activates.  of r a t l i v e r  molecular  weight  more t h a n  99%  3  H  which  (33).  -  J  choline  that  suggests  of  that  Phosphocholine  the a c t i o n  phosphatase  of a  activity  phos-  does  not  into phospholipids  (89).  i s stimulated PC  However, Choy and  ified  Q  by  i s 6 times  i n acetone-butanol-extracted post-microsomal  supernatant of r a t l i v e r  (90).  purified  However, p h o s p h o c h o l i n e  strains  CT  spermidine decrease  that of CDP-choline,  levels  of  and  the p o o l o f p h o s p h o c h o l i n e  catalyzed  parallel  (not  (10).  a r e known, a c o n c l u s i o n  the r e a c t i o n  phatase.  as c h o l i n e  In t h e s e c e l l s , pulse-^chase  synthesis  CPT  added  Cytidylyltransferase  In r a t l i v e r , choline,  PC  remains  choline  increases kinase a c t i v i t y  accumulate  initiate  cells  is limiting  of p a r t i a l l y  J  most l a b e l l e d  the c e l l s .  t h a t CK  polyamines,  [~_ATP -  2 mM  -  d i d not s t i m u l a t e  Vance CT,  found  by  lysophos-  the enzyme  t h a t LPC  inhibits  activity the  pur-  whereas l y s o p h o s p h a t i d y l e t h a n o l a m i n e  A t lmg/ml.,.. p i g l i v e r form  five-fold  eighteen-fold,  inhibition (9l).  LPE  stimulates  the  low  whereas p i g l i v e r  LPC  causes  -  Opposite The  enzyme  fect  is  i s stimulated  (92).  clear  by  The  the  deprived  tinal  i n CT  are  g r e a t l y by  bile  by  a fistula, are  unaffected  (93).  restores protein  (93).  Chylomicron  inhibited  by  fistula,  the  lung  tidylglycerol;.7 Of  the  cubation roughly PC  three  mixtures linear  Fatty  ides.  acids  54,  release  intestinal  protein  or and  by  PE  LPC  and  most a c t i v e  amounts o f  that contain  result  the  from  added  to i n t e s -  greatly stimulates which  PC was  (93).  phospholipid,  phospha-  protozoan,  supernatant  ** C  suggests  Ehtodinium  144,000 x g_ s u p e r n a t a n t  substrates 1  mucosa  (36).  i n the  to  ef-  triglyceride  mucosal c e l l s ,  by  no  labelling  i s also partly restored  is restricted  are  are  and  added  cell  to i n -  membranes, a  choline  incorporation  t h a t CT  is  known t o have a v a r i e t y o f  (43)  However, the  95) .  Choline  has  a c t i v a t i o n seems  while  O l e i c a c i d , f o r example,  rat liver  ceride)  and  mucosa.  into  limiting.  Cholinephosphotransferase  r o s o m a l CPT. or  I f the  PC  d_e novo enzymes  This  whereas LPE  f o r LPC  labelling  stimulation of  (31).  (iv)  (94)  CT  the  If various  occurs  LPC,  i s also stimulated  being  caudatum, o n l y fraction.  CT  rat intestinal  diminished,  labelling  Fetal  from  p h y s i o l o g i c a l reason  precursors  cells  -  following experiments. of  radioactive labelling  e f f e c t s occur  19  in rat liver  CPT,  i n the  is inhibited  stimulates  presence of  endogenous f o r w a r d or  e f f e c t s on  reaction  unaffected  chicken  micliver  exogenous d i g l y c e r (no by  added oleate  digly(43,  -  ATP  and  conversion formation CoA  had  late al  of  added  labelled  o f PC,  similar  the  but  t h a t ATP  CPT,but not  inhibiting  be  will  largely  may  The  M n - d e p e n d a n t EPT  reversibly  d a n t EPT  is inhibited  plus  CoA  (98).  (e)  Regulation  of  1.  Availability  of  the  and/or  given  in l i v e r .  increased Fatty slices  human l i v e r  inhibit  rat  likely  the  5  I t was  1  -(aB-methylene)  suggested  that  enzyme p h o s p h o r y l a t i o n inhibited,  while  pantetheine,  but  (97).  Mg -depen+ +  not  by  ATP  the  regulated  least  and  by  e i t h e r the  active, limiting  phospholipid  Liver  places  availability  enzyme.  The  c o n t r o l a t some p o i n t  the  (101),  synthesis  triglyceride  Fatty acids  slices  Biosynthesis  pathway.  stimulate  (100).  S r i b n e y ejt  i s not  adenosine  regu-  Substrates  r e s p e c t i v e l y , by  acids  since  plus  Palmitoyl  (96).  pantetheine  a c y l CoA  regulation effectively  Triglyceride control  of  b i o s y n t h e s i s must be  type  An  Phosphatidylcholine  Liver  substrates  plus  r e p l a c e ATP.  ATP  reduced  "  (i)  outside  or ATP  and  formation.  glycerolipids  is similarly by  PE  increased  t h a t a c y l CoA's m i g h t  i n a c t i v a t e d by  + +  first  CoA  triglyceride  on  suggesting  irreversibly.  CPT  of  effect  agent, p a r t i c u l a r l y  triphosphate  PC  no  different  plus  microsomes caused  d i g l y c e r i d e to  had  of  -  to r a t l i v e r  effects,  synthesis  found  liver  CoA  20  fasting  stimulate and  labelling  and  labelling  are  of  under  i s decreased  refeeding  of  rats  neutral lipids  triglyceride  r a t hepatocytes  independent  (99).  in rat  labelling  (102).  and  In a l l  liver in  -  cases, As  phospholipid  C ''C I]  phospholipids glyceride  "The  (103).  data  glycerol  so  be  the  synthesis.  In  line  Specific  thesis  substrates,  labelling  o f PC  and  in isolated  olamine occurs choline  higher  Sundler  need be  this  met,  tri-  concentrations increased s i g -  have s t a t e d  (49): in  then a d d i t i o n a l d i a c y l -  triacylglycerol." availability  pool  pool  concerns  15%  of  the  of c h o l i n e might e x i s t in rat liver  results  used  suggest  f o r PC  and  choline.  available for  represents  is oxidized  are  cells,  for d i a c y l g l y c e r o l  i s c a l c u l a t e d as  choline PE,  PC  total  in mito-  mitochondria  that separate  sphingomyelin  (approx  ethanolamine  ethanolamine  r e s p e c t i v e l y , from  yM  cho-  syn-  (72).  r e l e v a n t b a s e d on  (17 0.24  yM) mM)  in liver  (72),  and  (33).  the  stimulate  [] H^] 3  the  glycerol,  mea-  Maximum s t i m u l a t i o n by  concentration,  i s m a x i m a l a t 1 mM  ethanolamine  and  r a t hepatocytes.  a t 40  physiologically  liver  into  radioactivity  in rat heart  rat liver  (105) .  The  sured  since choline  pools  first  choline pool  P o s s i b l y a separate  (104).  the  (99-102).  f a s h i o n , whereas  until  A k e s s o n and  converted  rat liver  medium o f  Triglyceride labelling  factor in substrate  a l l of  chondria,  increase  synthesis w i l l  will  affected  in a hyperbolic  d i d not  T h u s , as  t o the  far indicate that  Another  (33).  labelled  -  i s much l e s s  i s added  were r e a c h e d .  phospholipid  Not  are  labelling  of palmitate moidally  synthesis  palmitate  1  21  ethan-  whereas s t i m u l a t i o n  by  These s t i m u l a t i o n s c o u l d  be  the  serum c o n c e n t r a t i o n  concentration,of  However, the  i s approximately  0.1  of  choline  in  concentration  of  mM  which  (49,  106),  -  would a p p e a r liver no  22 -  s a t u r a t i n g f o r s t i m u l a t i o n by t h e f r e e b a s e .  concentrations  are only  estimates,  compartmentation,  and t h a t  the d e n s i t y o f l i v e r  2.  Rate-Limiting  The  CK s t e p  assuming  that  These  there i s  i s 1 g/cc.  Step  i n r a tliver  i s c a l c u l a t e d , according  to Infante  (107) , t o be f u r t h e r from e q u i l i b r i u m t h a n  t h e CT s t e p .  disequilibrium  -f t h e e q u i l i b r i u m  ratio, p  (mass a c t i o n r a t i o  constant)  i s 49 t i m e s s m a l l e r  step,  i t i s t r u e however, t h a t o n l y  pool for  CK, making  active substrate  with  I t would be e x p e c t e d  vice  versa.  fect  on t h e k i n a s e  Over ibrium,  (109). strate  activity  o f the c h o l i n e  become  + +  since:  increases  t h a t changes  i n flux  greater  mass a c t i o n r a t i o  any enzyme s t e p , will  (107).  CK and CT s h o u l d and p r o d u c t  forms  through a r a t e -  c a u s e s an e f -  However, c h o l i n e  response  by a change  (see below).  insensitive  CPT i s p r o b a b l y  (107) , and s h o u l d  equil-  to a proportion-  i n t h e mass a c t i o n  t h u s be r e l a t i v e l y  concentrations.  concentrations.  + +  t h e enzyme  t h e f u r t h e r a r e a c t i o n i s from  be t h e f l u x  i n p.;, as c a u s e d  1. M g  i n t h e mass a c t i o n r a t i o , and  i s u n l i k e l y t o have a s i n g l e e f f e c t  e q u i l i b r i u m _in v i v o bolite  would  Dietary choline d e f i c i e n c y probably  the smaller  change  then  t h e CT  (108).  s t e p would c a u s e c h a n g e s  deficiency  a fraction  ATP and 2. M g  f o r [[ Mg-ATP H *  limiting  than over  t h e CK r e a c t i o n c l o s e r t o e q u i l i b r i u m .  might c o n t r o l the kinase  + +  affinity  ate  t h e CK s t e p  i s a v a i l a b l e f o r PC s y n t h e s i s ,  Mg the  over  The  ratio  t o sub-  closer to  be more s e n s i t i v e t o meta-  -  The are  3.1  maximal v e l o c i t i e s  from  rate  represents  plus  PC,  tion.)  the  or s u b s t r a t e  i s estimated flux  would  Thus,  be  and  The  cytosolic  1.3  of phosphocholine  two  steps  insensitive uncertain  (50).  (This  i n t o CDP-choline back  reac-  b i o s y n t h e s i s cannot  enzymes  than V  liver  in rat  t o the CPT  o f PC  independent  availability  in rat liver  y moles/min/10 g,  the i n f l u e n c e due  rather  CT  n e t s y n t h e s i s o f PC  t o be much l e s s  i t remains  substrate  and  y mole /min/10 g, l i v e r  conversion  Hence, the f i r s t  vity. or  a t 0.2  thus e x c l u d i n g  saturated flux  r e f . 110).  i s estimated  -  o f CK  y moles/min/10 g,_, l i v e r  (calculated liver  23  m  a  (109) .  f o r both  x  t o changes  the  steps,  i n enzyme  as t o whether  c o n t r o l the f l u x  Since  be  enzyme  t h r o u g h de  acti-  activity novo  PC  biosynthesis. By c o m p a r i s o n , cyte  PE  synthesis  ling  o f PE,  phosphate,  and  acids cyte  o f PE  labelling  i n c r e a s e , and  t o be  levels.  vitro  was  On  a c i d , and  d i r e c t e d a t EPT.  for  minutes  ethanolamine  the o t h e r  hand,  inhibition  O l e i c and  affect  incubation.  label-  lauric  The  phosphoethanolamine  by  or  hepato-  fatty CDP-  (72).  Experimentally  A l t e r e d PC  Bygrave  inhibited  (72).  by o l e i c  15-30  a c i d s do n o t a p p r e c i a b l y  R o b e r t s and  f o r hepato-  d e c r e a s e , r e s p e c t i v e l y , the amount o f  d i g l y c e r i d e during  3.  rate-limiting  c a u s e s an a c c u m u l a t i o n o f  not CDP-ethanolamine  a c i d , appear  ethanolamine  be  since, at saturating concentrations  ethanolamine  stimulation lauric  ET c a n c l e a r l y  by C a  found + +  ,  that  which  Synthesis rat liver could  be  PE  synthesis  reversed  by  Mg  in + +  .  -  Mitochondria,  which  authors proposed the  Mg  + +  /Ca  t a k e up Ca  Mitochondria  stimulate  the  higher  CPT, is  which  (112).  Ca  treme i n h i b i t i o n  with  uptake  regard  sibly  that M g  + +  + +  effect i s  inhibition  + +  and M n  substrate,  such  + +  than  (51) .  It  form a m e t a l i o n  CDP-choline, but  m i g h t be on CK o r CT, e x c e p t a t e x -  microsomes  (114).  i s inversely correlated  S i m i l a r l y , a d d i t i o n o f the C a  the l a b e l l i n g  of triglyceride  the a c t i o n o f a C a of choline  i n r a tl i v e r  + +  -sensitive  dictated  ion-  pos-  lipase.  synthesis  The l e v e l o f  40-70% when c a l c u l a t e d p e r  gram body w e i g h t , by a b o u t 1 week o f c h o l i n e However, l i p i d - b o u n d  triglyceride  t h e r a t e o f PC  C~J  (115).  was i n c r e a s e d ,  dietary choline.  i s reduced  + +  1 1 +  and p h o s p h o -  i n lymphocytes  case, d i g l y c e r i d e l a b e l l i n g  a u s e f u l model m i g h t be t o l i m i t free choline  for this  f o r t h e back r e a c t i o n  3  the l e v e l  EGTA, m i m i c s  EPT t o C a  C H ^ ] g l y c e r o l , when t e s t e d  through If  chelator,  o f t r i g l y c e r i d e s and d i g l y c e r i d e s from Q  p h o r e , A23187, r e d u c e s  However, i n t h i s  times  A l t e r n a t i v e l y , i f CPT i s n o t a l i m i t i n g  in rat liver  the l a b e l l i n g  from  five  o f CPT.  glycerol-3-phosphate  lipid  i s ' c o n t r o l l e d by  PE s y n t h e s i s  explanation  of rat liver  enzyme, t h e e f f e c t s o f C a  + +  + +  the p h o s p h o t r a n s f e r a s e  does n o t ( 1 1 3 ) .  Ca  The C a  i s known to/.be t r u e  interesting i n this  + +  The  e f f e c t i s c y a n i d e - s e n s i t i v e , and  A possible  sensitivity  complex w i t h  synthesis  rat liver  This  due t o ATP s y n t h e s i s .  stimulation  PE s y n t h e s i s .  (111).  more than PC s y n t h e s i s . not  , stimulated  that phospholipid  ratio  + +  24 -  l e v e l s a r e n o t changed  deficiency (116).  (116).  -  Choline  synthesis.  i s increased  cient and  reported  rats  Vance  (118,  tration, must be  CK  The  this  Q Me-  stimulation  of  lipid-bound  choline  for a lack of The  fatty  1 k  deficiency could  tion  of  or  CDP-choline could  synthesis.  The of  pool the  measured by  choline  choline  proportion  of  CT  •• l i v e r .  d e f i c i e n c y i n rats;.  (110) .  d e f i c i e n c y c a u s e s an _in v i v o incorporation  Increased  methylation  into  liver  might  compen-  observed  i n cho-  be  explained  i f d e f i c i e n c y impairs  necessary  lipid  for synthesis  i n turn  are  responsible (but  c h o l i n e or  in rat liver (123).  levels  forma-  transport  for  of  hepatic  phosphocholine  for defective l i p o p r o t e i n  in a rat liver  phosphate  phosphocholine  not  or  necessary  A l t e r n a t i v e l y , a lack of be  decreases  antibody t i -  blood  of phosphocholine  deficiency,  Schneider  whereas c y t o s o l i c CT  Thus, a higher  by  by  defi-  decreased  synthesis  water-soluble  in choline  and  choline.  Phosphocholine  doubles p r o t e i n  Skurdal  and  o f PC  (121).  CPT  choline-deficient rat  plasma l i p o p r o t e i n s , which release  (117).  substantiated  Cmethionine  free  line  lipid  not  (120).  (116).  liver  a species  cholinephosphotransferase  amount o f CT,  result,  enzymes  whereas deficiency  PEMT i s s t i m u l a t e d  of  diglyceride acyltransfer-  i s unaffected,  altered  to  activities  liver  w h i c h was  i n a c t i v e i n the  Corresponding  the  a decrease of hepatic  (110).  i s not  Liver  sate  choline  119),  (110).  significantly  Rat  in activity,  i s ' unchanged, d u r i n g Cornatzer  -  deficiency also alters  of g l y c e r o l i p i d ase  25  CDP-choline)  cell-free  system  (122).  i s l a r g e , comprising At  two  days of  (u m o l e s / l i v e r / g r a m  9%  choline body w e i g h t )  -  are  70%  lower  it  has  of  rat liver  than  been n o t e d  that CDP-choline  ratio  rosomal p r o t e i n  but  not  the  injections of  (126,  female  (127,  larger  rats,  129). PC  causes p r o l i f e r a t i o n  in  CPT  and  brane s y n t h e s i s 4.  (Fig.  3)  CTP,  or  to a r i s e like  phospholipid  surprisingly, treatment  accounted  hours a f t e r  f o r by  by  PE  i n male  (128).  The  a larger  pool  phenobarbital  injection,  N-methylation  phenobarbital  (127,  130).  injection,  smooth e n d o p l a s m i c  been shown t o p r o d u c e  be  regulated  i n the m e t h y l a t i o n  would  be may  r e g u l a t i o n by be  converted  c a t a l y z e d by CTP  diglyceride  reticulum.  significant  increases  f o r i n c r e a s e d mem-  i n the de  o f PE the  A  reactivity  of  of  pos-  phosphatidate. by  a reaction  cytidylyltransferase;  the a c i d i c  phosphatidyl  third  phosphohydrolase.  CDP-  phospholipids,  s e r i n e , and p h o s p h a t i d y l i -  Diglyceride i s a precursor  (Fig. 2).  (Fig.. 2).  phosphatidate  i s the p r e c u r s o r  novo pathways  to CDP-diglyceride  by p h o s p h a t i d a t e  phosphatidylglycerol,  PE  increase in l i v e r  phenobarbital  of h e p a t i c  an i n -  (131).  to d i g l y c e r i d e  nositol.  glycosylation  microsomal p h o s p h o l i p i d to mic-  PEMT, w h i c h m i g h t a c c o u n t  s y n t h e s i s may  Phosphatidate  or  Also,  Phosphatidate  sibility  with  s t i m u l a t e s the  been o b s e r v e d ,  to r a t s ,  i n g e s t i o n has  liver  PC  12  appears  ethanol  An  i s mainly  At  pool  has  after  increase  Feeding  Ethanol  (124).  of p h e n o b a r b i t a l cause  rat liver 127).  radioactivity  phospholipid o f PC  rats  3 - l i p o p r o t e i n (125) .  i n the  specific  -  in choline-supplemented  Intraperitoneal crease  26  of  triglyceride,  PC  and  The  ratio  phosphatase  of  -  27  -  activities  of  the  i s constant  in rat l i v e r  range of p h o s p h o l i p a s e - g e n e r a t e d (132),  i n d i c a t i n g that  c y t i d y l y l t r a n s f e r a s e and microsomes over  membrane-bound  phosphatidate  does n o t  a wide  phosphatidate  control  this  ratio. The tivity Mg  p h o s p h a t a s e may  c o r r e l a t e s with  and  + +  amphiphilic  be  triglyceride  of phosphatidate  ceride,  probably  However, the  is  only  somes  (ii).. The studied  tress of  deserves  l e s s to d i g l y -  i n t e r a c t i o n s of  r o l e of  amount o f  that  the  cations  phosphatidate i t s substrate,  phophatase phosphatidate,  of d i g l y c e r i d e i n r a t l i v e r  substrate  lung  micro-  b u i l d s up  at a regulatory  PC  synthesis  has  some comment h e r e .  material  i s composed o f  called 85%  d e f i c i e n c y i s thought  expansion).  and  lipid,  of which  to cause  Dipalmitoyl  species  sheep l u n g ,  of  PC  been  step,  This 75%  by  i s an  decreasing  not  cow  lung  contains  material  i s PC.  atelectasis  lung  (in  Sur-  infant respiratory  dis-  (failure  e s s e n t i a l component surface  i s p r e d o m i n a n t among PC's but  extensively  Mammalian  surfactant.  is characterized  iiii t s function  dipalmitoyl pig  more c o n -  Lung  and  surfactant  bit,  the  of  syndrome, w h i c h  lung  The  since  r e g u l a t i o n of  lung)  factant  regulatory  Often,  a surface-active dog  (133).  (134-136).  one-quarter  (137).  i t s ac-  in liver  relatively  d i g l y c e r i d e , and  through p h y s i c a l  with phosphatidate  questionable  t o CDP  enzyme, s i n c e  biosynthesis  c a t i o n i c drugs cause  version  is  a regulatory  (139) .  tension of  rat,  of  (138). rab-  -  Surfactant lung  pared CPT  i s synthesized  (Type 1 e p i t h e l i a l  alveolar  wall)  (140).  to s y n t h e s i s  selectivity  of  PC  somes tive  for  (141). with The  l l t  C~J  increase  LPC  incorporation  that  change acid  a c y l a t i o n of  birth, These  acyltransferase) lung  enzymes On rapid  before (144).  rabbit  during  ( F i g . 3) the  saturated  LPC  of  a c t i o n of  of  two  PC  observed  PC. PC  was  hand, the lung  PC  only around  or  i n r a t lung of Q  H ~J  3  PC's  located  LPC:LPC  was  finding  de-  indi-  acyltransferase, (143).  increases  (and  Simi-.  dramadoes  not  lysophosphatidic  i n c r e a s i n g PC the  content  three (145,  known p r i m a r y  route  time o f  pri-  during  declined  the  ac-  constant  This  development, w h i l e constant  micro-  i s poorly  a c y l CoA:LPC a c y l t r a n s f e r a s e  correlate"with  sub-  In c o n t r a s t ,  glycerol. by  PC  in incorporation  disaturated  occurs  en-  (142).  acyltransferases  fetal  the  this  i n mouse l u n g  LPC;LPC a c y l t r a n s f e r a s e but  com-  poor  a c y l CoA:LPC a c y l t r a n s f e r a s e  remained  other  synthesis  C-2  the  However,  r a t lung  increase  R a d i o a c t i v i t y i n the  i n mouse l u n g ,  tically  reactions  enzyme from  an  of  i n lung  d i p a l m i t o y l g l y c e r o l are  into saturated  a t C - l and  an  of  Endogenous d i p a l m i t o y l  into saturated  d i r e c t a c y l a t i o n by  larly,  to r u l e out  forward  of  cells  structural cells  (141).  i s p a r a l l e l e d by  about e q u a l l y  not  the  synthesis.  back and  palmitoyl  velopment.  cates  are  v i a CDP-choline  S i m i l a r l y , the  large  glycerol  Type 2 e p i t h e l i a l  exogenous d i p a l m i t o y l g l y c e r o l  to b i r t h  ;[]l-  by  i s minor  would a p p e a r  the  -  N-methylation  phospholipase-generated  strates  or  cells PE  zyme i n d i p a l m i t o y l PC and  28  birth  de  of  novo  146). for  the  i s by  de  novo  -  synthesis. 1.  For  creases ing  The  and  is s t i l l  r a t lung, lack of  (145,  P e r h a p s none o f  synthesis.  open  A l t h o u g h CPT  of  since: f i n d i n g s have shown b o t h i n -  a l l three  are  this  result  under o p t i m a l  the  de  3.  The  thesis.  has  the  conditions  lowest  ceride, late  increases  gestation The  CT  gesting lipid,  increasing this  r a t lung but  dramatically  activity  67%  after  reaction  likely  proof  met  PC  of  the lung  rates  _in v i v o .  a limiting  d e t e r m i n e PC  i n r a b b i t lung  700%  day  synthesis  slices  f i v e - f o l d within  low  one  Phosphatidylglycerol  day  (148).  just  step.  syn-  diglyduring  lipid  (148),  before  into l i p i d  sug-  birth,  CT  with  increases  birth  i n lung  four  activating  Correlating  before  (150). activity  since:  increases one  lung  Without  i s implicated at b i r t h  by  afterbirth  incorporation  11  i n r a t lung  birth  Phosphatidylglycerol by  one  is s t i l l  Q *C ^] c h o l i n e  p o s s i b l y PC  2.  not  enzyme a c t i v i t y .  enzyme a c t i v i t y  levels  surfactant  in fetal rat  ijn v i t r o  not  i s stimulated  only  Phosphatidylglycerol  1.  and  for  a c t i v i t y of  a c i d phosphatase, which produces  in specific  birth,  result,  to a d u l t  (147)  which are  a c t i v i t i e s are  a r e g u l a t i o n of the  dur-  (149).  from  days before  specific  a v a i l a b i l i t y o f d i g l y c e r i d e may  Phosphatidic  novo enzymes  novo enzymes i s l i m i t i n g  i s inconclusive since  T h u s , enzyme s p e c i f i c  de  147).  novo enzymes i n f e t a l monkey l u n g  (148),  and  -  conflicting  increases  f e t a l development 2.  de  issue  29  day  i n amount i n r a b b i t '  after  accounts s o l e l y  birth  (151) .  f o r the  stimulation  -  of  fetal  CT  3.  r a t lung  Cortisol  which  I t has  been p r o p o s e d  to the b r a i n since,  not s t i m u l a t e the enzyme  from  present  in fetal  lung  sphingomyelin pronovo  (154).  i s transported (155).  form  i n the  This  idea i s  i n the r a t , i n t r a p e r i t o n e a l i n j e c t i o n s o f  will  l a b e l plasma (156)  PC  and  LPC,  1  but u n e s t e r i f i e d  ( Q C ~Jethanolamine  would  14  by N - m e t h y l a t i o n o f PE, w h i c h  [~ ''C  could  3  cho-  label  t h e n be e x p o r t e d  the l i v e r ) . The  Brain  origin  cannot  of choline  significantly by m e t h y l a t i o n  in rat brain  remains  convert  ethanolamine  (157) .  After  line  lipids  tion  o f r a d i o a c t i v e e t h a n o l a m i n e , A n s e l l and  detect  labelled free choline  Kewitz  and  In  effluent  (or enzymes) o f de  that choline  in a lipid-bound  i s not l a b e l l e d PC  o f PC  r e s u l t s have been r e p o r t e d  Brain  hepatic  tive  (36).  responds, t o c o r t i c o s t e r o i d s i s c o n t r o v e r s i a l  (iii)  ethanolamine  from  i n lung  the p r o d u c t i o n  At present,  conflicting  tenable  line  stimulates  (153).  synthesis  blood  i s absent  lipid  (152).  infants  i n r a b b i t s , b u t does  duction  since  r a t lung  i n f a n t s w i t h r e s p i r a t o r y d i s t r e s s syndrome and  newborn c o n t r o l  lavage  -  by a d u l t  Phosphatidylglycerol  newborn in  from  30  P l e u l found  ethanolamine  fact,  brain and  caused  choline  PC.  Kewitz  ated  in rat brain,  that  significant  had  a higher  P l e u l proposed  that  forming c h o l i n e  lipids  to  cho-  intraperitoneal injec-  i n the b r a i n intravenous  uncertain.  Spanner  (155).  By  injections of  labelling specific  contrast, radioac-  of brain  choline.  r a d i o a c t i v i t y than  f r e e ethanolamine (158).  d i d not  i s methyl-  - 31 -  In n e u r o b l a s t o m a c e l l s , by  separate  pathways.  PE and PC a p p e a r  synthesis  This proposal  enzyme a c t i v i t i e s . exogenous c h o l i n e in  embryonic  glial  cells  out,  (161).  This  i n PC  i s generally labelled  from  than c h o l i n e o r C D P - c h o l i n e  ( 1 5 ) , and i n r a b b i t n e u r o n a l also tentatively  substrate  The l a r g e p o o l  t h a t CT i s l i m i t i n g . the expected  during  (159).  points  and  t o the  i n PC s y n t h e s i s .  t h e main c o n t r o l p o i n t  gests  t h e PC pathway  as r a t e - l i m i t i n g  extent  finding  summary, a l t h o u g h  the CT s t e p .  with  to a higher  t h e PE  was however b a s e d on in v i t r o  Phosphocholine  rat cerebral cells  CT as l i m i t i n g In  enters  b r a i n CT was p r o p o s e d  (160).  synthesized  Monomethylethanolamine e n t e r s  pathway and d i m e t h y l e t h a n o l a m i n e Mammalian  t o be  availability  c a n n o t be r u l e d  f o r de novo PC s y n t h e s i s may be a t of phosphocholine  in rat liver  sug-  As m e n t i o n e d , CT a c t i v i t y c o r r e l a t e s  r a t e o f PC s y n t h e s i s ,  c h o l i n e d e f i c i e n c y , and r i s i n g  falling  i n lung  i n the l i v e r  t i s s u e about the  time o f b i r t h . (f)  Effects of Viruses The  several  use o f c e l l  on C e l l  Culture  c u l t u r e to study  Biosynthesis  lipid  synthesis  a d v a n t a g e s o v e r whole a n i m a l o r o r g a n  1.  Homogenous p o p u l a t i o n s  2.  Small-scale  3.  Labelled precursors  of c e l l s  o f PC provides  studies:  are e a s i l y  obtained.  e x p e r i m e n t s c a n be e f f i c i e n t l y may be added d i r e c t l y  performed. t o the c e l l  medium. Gallaher has  and B l o u g h have r e p o r t e d  a half-life  of less  than  four  hours  t h a t PC i n BHK-21 (162).  This  cells  result  was  -  b a s e d on p u l s e - c h a s e  that  les during Synthesis  synthesis  o f PC i s h a l v e d  density-dependant was e s t i m a t e d  P-labelled  tivity  by l a b e l l i n g  (164).  into phospholipids  since  by c h a n g i n g  Older  c u l t u r e s , however, r e q u i r e d  cellular  different  r a d i o a c t i v i t y of  Specific  radioac-  much l e s s .  This e f -  t h e PC p o o l  serum p r o t e i n  i n f e c t i o n s o f animal c e l l s  s i z e i s not  of Q ^C J 1  ace-  i s stimu-  to display  this  i n f e c t i o n has been shown c o n c l u s i v e l y  e f f e c t on PC s y n t h e s i s  incorporation  The  inhibition  (Table  1)  mutants.  proteins  a r e known t o p e r t u r b  (Table I ) .  data  compared  in cells  e a s e v i r u s s u g g e s t s s u c h an e f f e c t  able  (163).  t h e medium i n t h e a b s e n c e o f serum.  PC and PE s y n t h e s i s  No v i r u s  sitive  growth.  (165) .  Many v i r u s  virus  doub-  glycerol  o f e a r l y p a s s a g e WI-38 c e l l s  five-fold  though  3  The i n c o r p o r a t i o n  lated  effect  L7 2- H~J  with  ATP i s s t i m u l a t e d  apparently  increased  the i n t e r e s t i n g  i n the s p e c i f i c  on t u r n o v e r ,  radioactive  o f BHK c e l l s  PC o f E h r l i c h a s c i t e s c e l l s .  o f the precursor  with  w h i l e PE s y n t h e s i s  inhibition  f e c t on PC i s l i k e l y  tate  labelling  The same a u t h o r s made  Serum c a u s e s an i n c r e a s e 3 2  -  and e q u i l i b r i u m  g l y c e r o l or choline. finding  32  authors proposed  incorporation  Mutants with d e f e c t i v e  to synthesize  viral  that  virus  RNA d i d n o t c a u s e  by  Sindbis  temperature-sen-  nucleocapsid  incorporation.  inhibition  dis-  (175).  (172) was examined u s i n g  inhibited choline  t o PE s y n t h e s i s , a l -  i n f e c t e d with Newcastle  o f Q C ~J c h o l i n e 1h  t o have a  o r membrane  However, m u t a n t s u n inhibition.  o f h o s t RNA a n d / o r  The  protein  Table 1  Effect of virus infections on animal PC and PE synthesis: Effect on: Cell Line (or Animal)  Virus Family  Virus  Picornaviridae  Polio virus (166).  Picornaviridae  Mengovirus (167)  Adenoviridae  Time of Infection  PC PE Synthesis Synthes  Measurement  + 70%  HeLa  3  +120%  6h  i *+PC rL~ -3 2P] -]p p (sa)  L  9h  r_ "C]choline -» PC (sa)  Adenovirus type 5 (168)  HEK  8h  jj*c] acetate  Herpetoviridae  Pseudorabies virus (169)  RK  9h  r_H[] choline •* l i p i d  t 20%  Retroviridae  Friend virus (170)  BALB/c mice  14 days  [^"•C^ choline •* PC (sa)  + 90%  + 60%  + 70%  + 70%  + 60%  + 40%  + 40%  + 40%  32  p  p  1  i  E( s a )  + 60%  l  •> PC ( s a )  5 , 6  f_"*C]acetate •<• PE (sa)  + 60%  t 60%  6  3  ["C] ethanolamine •* l i p i d minus PC (sa) Togaviridae  Togaviridae  Togaviridae  Japanese encephalitis virus (171)  BHK-21  Sindbis virus (172)  Chick embryo fibroblasts  10 h  Semliki Forest virus (173, 174)  BHK-21  7h  24 h  [^'"C^oleate + PE  4  [/"C^oleate •* PC  L~P>i 2 3  PC PE  £?H] choline •» PC [J'^C] ethanolamine ->• PE  1 '-•' represents 'incorporation into 2 (sa) specific activity 3 represents 'increase' 4 'i' represents 'decrease' 5 PC plus sphingomyelin 6 specific activity = % l i p i d cpm in PC or PE ; pg protein The numbers in brackets refer to references. 1  -  synthesis  was r e s p o n s i b l e  port of this RNA  f o r the i n c o r p o r a t i o n e f f e c t .  idea, actinomycin  synthesis)  thesis)  34 -  D  and c y c l o h e x i m i d e  inhibit  choline  actinomycin  i m i d e has o n l y  inhibitory  tion  (172).  Sindbis  cause a s m a l l BHK c e l l s  a slow  inhibits  DNA-dependant  (which  inhibits  protein  i n chick  embryo  D has no e f f e c t effect  virus infection  reproducible  (172).  (which  incorporation  However, i n BHK c e l l s  In s u p -  decrease  The c a u s e o f t h i s  cells.  and  on c h o l i n e  cyclohex-  incorpora-  d o e s , on t h e o t h e r in choline decrease  syn-  hand,  i n c o r p o r a t i o n by  i s consequently  obscure. (g)  Effect  of Semliki  Semliki the  Forest  virus  genus A l p h a v i r u s ,  liki  Forest  lipid the  similar  (178).  family  nucleocapsid  6  (177) .  The n u c l e o c a p s i d  core  and s i n g l e - s t r a n d e d Richardson  that envelope p r o t e i n s  tion  from  move  Forest  o f the t o t a l  virus infection  and s p e c i f i c  i s composed o f a  RNA o f  from e n d o p l a s m i c  by b u d d i n g , a p r o c e s s  pro-  molecular  and Vance have p r o v i d e d  p l a s m a membrane o f i n f e c t e d BHK c e l l s  Semliki  originates  (179).  activities,  evi-  reticulum to The v i r u s  w h i c h may e x e r t  caused  Sem-  outer  glycosylated  4 .5 x 1 0  synthesis.  and an  three  weight  PC  (176, 1 7 7 ) .  The e n v e l o p e c o n t a i n s  protein species  on  i s a member o f  compositions are  single  the c e l l  Biosynthesis  lipid  3  exits  PC  their  and E .  2  virus)  envelope apparently  p l a s m a membrane, s i n c e  Ei, E  the  Sindbis  i n the T o g a v i r i d a e  The l i p i d  teins,  dence  (like  on BHK C e l l  v i r u s c o n s i s t s o f an i n n e r  envelope.  host  Forest Virus  some  50% and 40%  effect  inhibi-  r e s p e c t i v e l y , o f BHK  -  cell  CPT,  It  RNA  or  do  not  mimic  this  protein synthesis  embryo c e l l s  CDP-choline  (Table  synthesis. chondria fection  reduces  Also,  II). Ca  i s elevated (182).  Ca  chondrial  release,  synthesis  of  the  pool  of  uptake  + +  may  not  that  likely virus  size  and  regulate  2.5  PC  D  and  inhibition  causes  o f ATP,  ADP,  could  embryo  of  (173) .  infection  o f BHK  or  CTP,  and  control  PC  fibroblast  mito-  h o u r s a f t e r SF v i r u s - i n -  synthesis  u p t a k e , would be  although  expected  to  Infection  on  mito-  inhibit  lipid.  of Semliki BHK  C e l l Type  Actinomycin  these pools  immediately  + +  so  into chick  Table Effects  not  Forest  the  One  (173).  effect,  are  i s a l s o known t h a t S e m l i k i  chick  -  7 hours a f t e r i n o c u l a t i o n  cycloheximide host  35  W%  Forest  Cell  Virus  Pool  Sizes  Time of infection  PFU/cell  Infected/ Mock-infected Pool Size Ratio  Pool  Reference  CEF  5  5h  ATP  0.68  (180)  CEF >> 1 BHK/CEF  5  5h  ADP  1.25  (180)  10-50  4h  CTP  0.15  (181)  BHK/CEF  10-50  4h  CDP-choline  0.30  (181)  l v  1  1.  date  Cell  t y p e was aim  the  p r e c i s e mechanism o f  thesis  the  clearly  The  choline  of  not  i n the  original  t h e s i s p r o j e c t o u t l i n e d h e r e was  incorporation outlined  stated  i n t o PC.  in this  Semliki The  Forest  four  reference to  eluci-  virus inhibition  steps  o f de  i n t r o d u c t i o n were examined  novo  of  syn-  in mock-infected  36  -  and  virus-infected  i n vierQcenzyme ' in vivo sitive  cells.  activities  activities, enzyme  However, s i n c e  in  b o t h mock- and v i r u s - i n f e c t e d  line.  As a r e s u l t ,  phosphocholine lel  turnover  that  in infected  cells  was  was  Since the p o o l the p o i n t  as a r e s u l t  cause  was  of  size  inhibition  with  Q H^]  the d i s a p p e a r a n c e inhibited  not reduced  a transient  o f Q H^j 3  However,  in infected  paral-  because  cells,  by v i r u s  cho-  3  in a fashion  f o r an e n l a r g e d p h o s p h o c h o l i n e  but the l i k e l y  CDP-choline,  experiments  enlarged was  Sen-  so t h a t m o d i f i c a t i o n s  o f i n c o r p o r a t i o n . i n t o PC.  of phosphocholine  The r e a s o n  certain, tivity,  rate  pulse-chase shown  the p o o l o f p h o s p h o c h o l i n e  w i t h the  t h e PC p o o l was measured  cells.  i t was  t o the i n h i b i t i o n  tion.  Also,  d i d not unambiguously d e l i n e a t e to perform  that  Much o f t h e c u r r e n t metho-  insufficient,  new methods were n e c e s s a r y .  was d e c i d e d  correspond  phosphocholine,  were p e r f o r m e d .  or  it  realized  measurements were r e q u i r e d .  assays of c h o l i n e ,  f o r t h e s e a s s a y s was  results  i t was  do n o t n e c e s s a r i l y  pool size  CTP, and d i g l y c e r i d e dology  -  the  infec-  p o o l remains  un-  r e d u c t i o n i n CT a c -  o f the d i m i n i s h e d p o o l s i z e  o f CTP.  - 37  -  ' MATERIALS AND (a)  Chemicals Choline  PC  and  palmitoleyl  Lipids  LPC,  P h e n o l Reagent  oleyl  LPE,  Solution  acid  was  from M a l l i n c k r o d t . & Bell. Fetal  acid,  PC  and  calf  Solution  Enzymes  were  dipalmitoyl  dipalmitin,  and  diolein)  Laboratories.  2N  (for protein  Norit-A  The  supplied  by Flow  E a g l e Medium, Medium  phosphatase,  and  inorganic  anion exchanger,  50W-X8 were p u r c h a s e d r~5- H~] C y t i d i n e 3  L-methionine , Q3H~J  Laboratories.  199,  and  Island  Balanced  Biological  Company.  1-X10  and  were bought  the c a t i o n  3  r~_l, 2 - 1 ' • C J J  [_y- P~2 32  exchanger,  choline  S-adenosyl-  chloride,  d i p h o s p h o c h o l i n e , [^Methyl- ^ C j J 1  -  Adenosine  from The  from  Laboratories.  5 ' - t r iphosphate , [[Methyl- H~J  []3H3] Hexadecane was  Dul-  phosphodiesterase, choline kinase,  from B i o - R a d  toluene,  was  Earle's  [ [ ^ C ] t o l u e n e were o b t a i n e d from New  sham.  Tri-  grade.  choline, clear.  sup-  by M a t h e s o n , C o l e -  cytidine  and  were  charcoal,  ( j Y l e t h y l - *C - J 11  PC,  Scientific.  pyrophosphatase) AG  P-L  d e t e r m i n a t i o n ) , and  produced  were p r o d u c t s o f Grand  glycerokinase,  purchased  from Amachem, w h i l e p e r c h l o r i c ; . a c i d  serum was  yolk  Sigma o r  PE,  c h e m i c a l s were o f r e a g e n t  (alkaline  Sigma.  Tris  and  3-Heptanone was  Other  becco's M o d i f i e d  AG  p h o s p h o c h o l i n e , egg  d e o y x c h o l a t e were p r o d u c t s o f F i s h e r  chloroacetic  Salt  (pig l i v e r  by S e r d a r y R e s e a r c h  sodium  iodide,  N u c l e o t i d e s were o b t a i n e d from e i t h e r  Biochemicals.  man  choline  and 1T-E) , t a u r o c h o l i c  from Sigma.  plied  Isotopes  chloride,  (Type V-E  METHODS  phospho-  England  Nu-  R a d i o c h e m i c a l C e n t r e , Amer-  5 ' - t r i p h o s p h a t e was  from  Amersham/  - 38 -  Searle.  Qyiethyl- H^] C h o l i n e c h l o r i d e 3  England line  Nuclear  was s u p p l i e d by New  C ^ ^ ] Glycerophosphocho-  o r Amer sham/Sear l e .  1  was a p r o d u c t o f ICN P h a r m a c e u t i c a l s .  [jyiethyl- H^]  Phos-  3  p h o c h o l i n e was s y n t h e s i z e d by t h e t e c h n i q u e  o f Paddon and Vance  (89). (b)  General  0.25  (i)  Thin-Layer  TLC  was p e r f o r m e d  mm  G-25  Methods Chromatography on 20 cm x 20 cm l a y e r s  i n t h i c k n e s s , supported  by g l a s s p l a t e s .  7GF ( M a l l i n c k r o d t ) p l a t e s were u s e d .  solvent  s y s t e m s were:  A. This  CH OH - 0.6% N a C l  system  taine  i s useful (R  f  f  sprayed  B.  To s e e t h e s e 1  Routinely  used  f  (R^ 0-0.13),  0.53-0.63) and b e -  compounds,  reagent  phos-  t h e TLC p l a t e  (183) and a molybdenum  *C^] G l y c e r o p h o s p h o c h o l i n e ,  d e t e c t e d by a u t o -  has an R^ v a l u e o f 0.50.  i s useful  diacylglycerols CHCI3  system  (R  l  Benzene - C H C 1  system  C.  f o rseparating choline  with Dragendorff s  (184).  radiography,  This  Either S i l  (50/50/5;v/v/v) ( 7 2 ) .  NH.4OH  0.25-0.38), C D P - c h o l i n e  (R: 0.56-0.66).  reagent  -  3  phocholine  This  gel,  ( M a c h e r y - N a g e l & Co., s u p p l i e d by Brinkmann I n s t r u m e n t s ) o r  Chromar  was  of s i l i c a  3  - CH OH 3  in isolating  are separated - CH OH - H 0 3  2  (80/15/5;v/v/v). diglyceride.  by t h i s  system  1,2- and 1,3-  (185).  (70/30/4;v/v/v) .  separates phospholipids.  (See s e c t i o n  (I)  (ii).)  - 39 -  (ii)  Scintillation  Lipid  s a m p l e s were c o u n t e d  azole  (PPO, 4g/jl)  (POPOP,  50mg/jl) .  Counting  ter 1!  cluding nal  c o u n t i n g was done  C.  Counting  standards  tained dards each  ratio  Q *Q- or 3  except  using  ' C~J 4  set of  samples.  (iii)  Protein  serum a l b u m i n  3  P  and  3 2  P  w h i l e ex-  by t h e e x t e r -  s t a n d a r d s which hexadecane.  fluid  con-  Stan-  were c o u n t e d  with  by t h e method o f Lowry e t a l . (187) as a s t a n d a r d .  Statistics  Tests of s i g n i f i c a n c e  were  t h o s e d e s c r i b e d by W o o l f  (188).  Cell Culture BHK-21 c l o n e 13 c e l l s  Cells  Q Hj  3 2  coun-  scintillation  was d e t e r m i n e d  toluene, or  was d e t e r m i n e d  bovine (iv)  (c)  to count  of chloroform-quenched  Scin-  scintillation  in a Tri-Carb  efficiency  (Aqueous  (186).  f o r samples c o n t a i n i n g  i n the a p p r o p r i a t e s c i n t i l l a t i o n  Protein  were o b t a i n e d  were grown a t 37° as m o n o l a y e r s  LUX S c i e n t i f i c )  o r on medium  dishes.  were grown  (and  i n an ISOCAP/300  ( P a c k a r d ) , w h i c h has a window llf  i n ACS  Amersham) o r i n T r i t o s o l  'These samples were c o u n t e d  counter  2,5-diphenylox-  Aqueous s a m p l e s were c o u n t e d  (Nuclear-Chicago)  *C.  i n toluene with  and 1, 4 - b i s - [ J 2 - ( 5 - p h e n y l o x a z o l y l ) ~J -benzene  Scintillant,  tillation  Counting  Cells  5% f e t a l  calf  serum).  c o n f l u e n t when u s e d ,  (100 mm  x 15 mm,  By v i s u a l  Flow L a b o r a t o r i e s .  on l a r g e  i n Dulbecco's  unless noted  from  (150 mm  Falcon)  x 15 plastic  Modified Eagle  inspection,  otherwise.  mm,  cells  Medium were  -  (d)  Propagation The  Semliki  SF  plicity  of  (and  fetal  v i r u s was  a b o u t 0.2 calf  removed and tored  by  until  used  plaque  as  2%  fetal  calf  medium d e s c r i b e d  After  30  minutes  forth.  infected not  (f)  cells  Preparation  cells  PBS  (189).  PBS  each  £  The  time.  f o r 10-15  above  (and  was  40  For  multi-  o f Medium  37°,  40  the  ml  199  of  the  medium  was  Infectivity  was  were s t o r e d  2%  Forest  washed  moniat  -70°  Virus  was  PFU/cell,  at  37°,  37°,  fetal  the  the  calf  Virus  twice with  a l s o adding  dishes  medium was  incubated  as  cell  applies  to 4 l a r g e  twice with  C e l l s were p a c k e d 4°.  The  back Ten  Mock-  that  virus  desired.  Fractions  m o n o l a y e r was  minutes at  of  ml.  serum) were a d d e d .  C e l l s were f u r t h e r  were washed  3  removed.  except  procedure  and  were t i p p e d  same way,  Subcellular  Medium  enough  volume o f  i n the  of  3 ml  r a p i d l y thawed  t o make a t o t a l  1 hour a t  following  Confluent  of  were t r e a t e d  added.  The  Semliki  serum).  incubation  After  o f Medium 199  was  ml  37°  preparations  d i s h of c e l l s  added a t a m u l t i p l i c i t y  ml  10  hours a t  virus.  (173).  b o t t l e s at a  1 hour a t  24  a source of  I n f e c t i o n of C e l l s with  (and  and  After  Virus  roller  a l s o adding  After  assay.  Each l a r g e  the  to  described  use.  (e)  199  added  serum).  Virus  previously  PFU/cell,  same medium were added.  -  Forest  v i r u s o r i g i n a t e d as  propagation,  2%  of  40  a b o u t 3 ml  removed by  dishes  was  cells.  per  dish  of  twice with  3 ml  of  c e n t r i f u g a t i o n at  pellet  of  k e p t on  ice  900 and  x  -  resuspended KC1 a  and  15  i n 2.5 mM  ml  of  50%  suspension  organelles The  4°.  at  100,000 x  g for  the  pellet  CK,  CT,  and  (g)  CPT  (i)  as  (unless  and  (either bation  Rethy  and  1 or at  mersing  exchange  was  The  The  at  retained  9,000 x £  for  and  centrifuged (cell  as  cytosol)  sources  p e l l e t s were r e s u s p e n d e d mM  added The  were u s e d  i n 10  in  buffer.  supernatant  Tris-HCl  of  using  (pH  7.4)  otherwise).  (24).  100  MgCl  20  reaction  2  optimal  from BHK  , and  0.25  tubes  (OH  the  i n b o i l i n g water x 4 cm  of  M Tris-HCl  with  1 ml  o f w a t e r and  t h i s r i n s e was  Water  15  ml)  (pH  8.0).  was  to a  choline  ymoles chloride incu-  by  im-  2 minutes.  column o f AG  previously  also drained  1.-X10  been washed  t u b e s were  through  of  reaction  After  stopped  for  Assay  forced  7.5  1 ml.  was  method  procedure  8.0),  3  reaction  f o r m ) , w h i c h had  the  [~_HTJ  volume o f  i n t o a 0.5  the  added (pH  pinole . o f  1 ml  (about  was  Tris-HCl  in a f i n a l  minutes,  activity,  cells  ymoles of  drained  resin 0.1  For  with  columns.  mM  strokes  mentioned  obtained  10  homogenate was  centrifuged  4°.  20  measured e s s e n t i a l l y by  Cytosol  for  E a c h m i x t u r e was ion  at  noted  5 Ci/mole)  37°  the  the  with  Kinase  follows.  b o t h ATP  7.4),  Assays  mixture containing of  To  s u p e r n a t a n t was  1 hour  Enzyme a c t i v i t y  was  was  Dounce h o m o g e n i z e r ,  Choline  Weinhold  (pH  previously  activities.  EDTA  Enzyme  Tris-HCl  (microsomes) so  a loose-fitting 1 mM  i n the  cell  minutes at  and  mM  C e l l s were h o m o g e n i z e d by  sucrose  of  10  -  Dounce h o m o g e n i z e r .  10  with  of  MgCl2•  tight-fitting  2.5  ml  41  into  the  rinsed the  columns  to  - 42 -  elute ml  choline.  Q3H[]  3  Phosphocholine  f Q  was e l u t e d  o f IN NaOH f o l l o w e d by 1.5 ml o f 0.IN NaOH.  acid  (about 70 uft) was,added  nescence  above  the  reaction  the  results  i o n exchange method  mixture  (pH 8 . 0 ) , Q HJ  line  incubation  chemilumi-  o f the mixture  reagent  method,  and 0.025 ymole'."  volume o f 100 y £ . A f -  (50 yjl) , w i t h 40 yg o f c h o as c a r r i e r s ,  were s p o t t e d  (183), and p h o s p h o c h o l i n e ,  reagent  in sol-  (184).  Phosphocholine  The enzyme a c t i v i t y  with  by s p r a y i n g was e l u t e d o b t a i n e d was  40 m i n u t e s .  Cytidylyltransferase  Cytidylyltransferase that of A n s e l l  was a s s a y e d  and C h o j n a c k i  a d s o r p t i o n , was u s e d  were added  verified  10 ymoles o f T r i s - H C l  The p l a t e was d e v e l o p e d  and c o u n t e d .  to at least  choline.  to ions i n  C h o l i n e was d e t e c t e d by s p r a y i n g t h e p l a t e  the s i l i c a  (ii)  method  In t h e s e c o n d  in a final  g e l TLC p l a t e .  w i t h a molybdenum  coal  acetic  a t 3 7 ° , t h e a s s a y was t e r m i n a t e d as d e s c r i b e d  A.  Dragendorff's  linear  A second  2  and 200 yg o f p h o s p h o c h o l i n e  vent system  sensitive  o f ATP, 1 ymole o f M g C l ,  (50 C i / m o l e )  An a l i q u o t  onto a s i l i c a  from  (see R e s u l t s ) .  to a mixture containing  0.75 ymole choline  3  above.  Glacial  t o reduce  i s quite  o b t a i n e d by i o n e x c h a n g e .  c y t o s o l was added  ter  t o the e l u a t e  w i t h 0.5  before counting.  The  of  Q  (190), e x c e p t  BHK c e l l  containing  s i m i l a r to  t h a t TLC, n o t c h a r -  to separate phosphocholine  For optimal a c t i v i t y , to a mixture  by a p r o c e d u r e  from CDP-  c y t o s o l o r microsomes  2 ymoles o f T r i s - s u c c i n a t e  (pH 6 . 0 ) , 1.2 ymoles o f Mg a c e t a t e , 0.30 ymole;') o f CTP ( f o r t h e  - 43 -  cytosolic  enzyme) o r 0.20 ymole; ; o f CTP ( f o r t h e m i c r o s o m a l  enzyme), and 0.16 umoleQ o f ["Methyl- H~J - o r [ j M e t h y l - *C~J 3  phosphocholine 0.20  (except f o r m o c k - i n f e c t e d microsomes, t o which  ymole^/ were added) i n a f i n a l  cific  radioactivity  After  20 m i n u t e s  as  for choline  on  a silica  rier.  used  the  silica  NaOH.  volume o f 100 y i U  of phosphocholine  incubation  kinase.  An a l i q u o t  0.6 mg o f C D P - c h o l i n e  i n solvent  CDP-choline.  was s t o p p e d  o f t h e m i x t u r e was s p o t t e d  g e l TLC p l a t e w i t h a b o u t  to visualize  The s p e -  was 12.8 o r 7.5 C i / m o l e .  a t 37°, the r e a c t i o n  The p l a t e was d e v e l o p e d  was  1l  system  CDP-choline  A.  UV  car-  light  was e l u t e d  from  w i t h 0.5 ml o f IN NaOH, f o l l o w e d by 1.5 ml o f 0.IN  Glacial  acetic  acid  (about 70 y£) was added  t o the e l u a t e  before counting. (iii) CPT Burke  Cholinephosphotransferase  was a s s a y e d  (173), e x c e p t  CDP-choline minutes, The  that  the s p e c i f i c  was 0.3 C i / m o l e ,  and t h e e x t r a c t e d  diglyceride  digestion  by t h e p r o t o c o l d e s c r i b e d by Vance and  the assay  Q *^] 1 1  radioactivity incubation  (191).  u n h y d r o l y z e d PC by s i l i c i c  acid  chromatography.  ether,  t h e h y d r o l y z e d sample was l o a d e d o n t o  gen.  -  t i m e was 15  D i g l y c e r i d e was s e p a r a t e d  (18 g o f s i l i c a )  was e l u t e d  ether.  *C J  PC was blown d r y w i t h a i r .  column  roleum  1  s u b s t r a t e was p r e p a r e d by a p h o s p h o l i p a s e C  o f egg y o l k PC  diglyceride  of  A silicic  acid  was washed w i t h 100 ml o f p e t r o l e u m t h e c o l u m n , and  w i t h 200 ml o f 25% e t h y l  ether i n pet-  The s o l v e n t was removed w i t h a s t r e a m  The i d e n t i t y  from  of diglyceride  was v e r i f i e d  of n i t r o -  by TLC i n  - 44 -  comparison ide  with  was p r e p a r e d  solution  1,2-diolein.  f o r t h e CPT a s s a y  Water  added.  as f o l l o w s .  (50 y& a t 40 mg/ml i n c h l o r o f o r m )  5 o r 10 ml b e a k e r . gen.  (See (£)(iii) below;)  A diglyceride  was p i p e t t e d  The c h l o r o f o r m was e v a p o r a t e d  of diglyceride  was formed  w i t h a S o n i c Dismembrator  ter,  80.  Inc.) a t s e t t i n g  The e m u l s i o n  (Quigley-Roches-  was c o o l e d on i c e a f t e r  Phosphatidylethanolamine Methyltransferase as d e s c r i b e d by R e h b i n d e r and  Greenberg  (192).  a mixture  c o n t a i n i n g 110 o r 148 ymoles o f T r i s - H C l  in  and 0.15 ymole:  a final  2 minutes  30 m i n u t e s  without  Q  The l i p i d  A solution  After  mixing  Choline  lated  60.  was s t o p p e d  After with  (2.5 ml) and n - b u t a -  and c e n t r i f u g i n g , an.:  f o r counting.  Oxidase  three times with as u s u a l  (25 C i / m o l e )  by s o n i c a t i o n  at setting  o f 0.9% N a C l  To p r e p a r e m i t o c h o n d r i a , e a c h washed  400 yg o f d e o x y c h o -  s u b s t r a t e (1.12 mg) had  w i t h a S o n i c Dismembrator  HC1.  (pH 9 2 2 ) ,  S-adenosylmethionine  l Q  o f t h e t o p phase was removed  (v)  EDTA were added t o  i n 1.5 ml o f 0.4% d e o x y c h o l a t e  (2.5 ml) were added.  aliquot  3  i n c u b a t i o n a t 37°, the r e a c t i o n  0.2 ml c o n e . nol  of  volume o f 0.5 m l .  been e m u l s i f i e d for  Microsomes prepared  yg o f p h o s p h a t i d y l m o n o m e t h y l e t h a n o l a m i n e ,  late,  then  sonication.  PEMT was measured e s s e n t i a l l y  75  nitro-  by two 3 m i n u t e  periods of sonication  (iv)  under  into a  (2.8 ml) and 001% Tween 20 (v/v) (1.2 ml) were  An e m u l s i o n  the f i r s t  Diglycer-  (section  large d i s h o f  3 ml o f i c e - c o l d  PBS.  ( f ) ) and homogenized  BHK c e l l s  Cells i n 50 mM  were  was iso-  Tris-HCl  -  buffer  (pH  7.0)  lated  procedure (h)  Activation  In t h i s  by  activity  of Schneider  Lipid  was  and of  v e n t was the a s s a y disperse (i)  the  liver  two  a stream  days o f  was  was  within 5  assayed  From 0 t o 250 yg o f to assay  of nitrogen.)  hours  acetone-  tubes. Before  (The  sol-  assaying,  on  The  tubes  centration  7.4)  the  lh  CDP-choline.  y  £  ice.  (pH At  a t 20 mM,  Each  7.4).  the end  The  o f t a u r o c h o l a t e was (Section  from  tube  0.3 Ci/mole)  0 t o 2%.  included  mixture  of t h i s  were i n c u b a t e d a t 3 7 ° f o r 20 m i n u t e s .  as u s u a l  (pH  i c e , w h i c h c o n t a i n e d m i c r o s o m e s and [_ C^]  solution  Tris-HCl  was  f o r 30 m i n u t e s on (20  ...i.,  of taurocholate  cubated 1 4  i n 50 mM  dissolved  o f 50 ymoles o f T r i s - H C l  Q C^] CDP-choline  to  of Cholinephos.photraBsferase  a total  (j)  the  •  i t s use.  components e x c e p t  extracted  CT  lipids.  acid  added t o a m i x t u r e  the  by  components minus enzyme were v i g o r o u s l y v o r t e x e d  Taurocholic  assay  (193).  Slater  in mitochondria  were added  Taurocholate A c t i v a t i o n  within  iso-  (110).  cytosolic  lipids  removed w i t h  M i t o c h o n d r i a were  measured  Vance  10 s t r o k e s '  by  Cytidylyltransferase•  experiment,  rat liver  cells)  the method o f Myers and  o f p r e p a r a t i o n o f the c y t o s o l . extracted  (14 ml/g  Dounce h o m o g e n i z e r .  i n the same b u f f e r  Choline oxidase  -  0.25 M sucrose  with  of a t i g h t - f i t t i n g  45  was  in-  time,  was  added  The  final  ^CJ  PC  and con-  was  (g)(iii)).  Choline Transport Medium-size d i s h e s of c e l l s  ments.  Infection  was  were used  for transport experi-  a t 40 P F U / c e l l as u s u a l .  A t 6h  hours  -  post-infection, Earle's  Balanced  neutralized without the  the  with  5%  bath  at  3  1,  (incubation amount o f  choline since  medium was  known.  three  scraped acetic  the  the  binding 30  to  by  to  cells  time w i t h  with  placing  them  l e s s water  addition  i n 30  1.5  ml  time w i t h  of  of  in a than  of  The  minutes could choline the  cold  2 ml  0.9%  the  medium  for counting.  incubation,  i n 5 ml  or  float.  removed  minutes  uCi)  i n 2 ml  a little  of  serum)  cells  r a d i o a c t i v i t y of  Aliquots  PBS,  10%  NaCl  in a  be  the  cells  (4°)  of  in  were and  trichloroby  cen-  tight-fitting  homogenate were t a k e n  c o n t r o l experiments, the  external  washed  described  surface  u p t a k e , as twice,  above,  same s o l u t i o n c o n t a i n i n g  at.37°  37°  the  ml  calf  a c i d - i n s o l u b l e p e l l e t s were o b t a i n e d  minutes of  cells.were  the  30  homogenized  separate  solution  of  to  (6.3  1.5  for  determination.  In  After  added  the  specific  Dounce h o m o g e n i z e r .  line  by  times, each  The and  dialyzed  m i n u t e s a f t e r the  up  After  times with  choline  dishes  a l i q u o t was  times, each  acid.  protein  30  taken  o f f three  trifugation  the  and  a 5%  calculated  washed  2%  temperature, with  20,  1),  G  two  D i s h e s were k e p t a t  this  10,  3 l  i o d i d e , was  t h a t w h i c h would c a u s e At  (and  E  NaHC0 .  cold choline  -  were washed  Salt Solution  same medium.  water  cells  46  of  followed  by  choline  2).  The  percent  1.5  Q H^]  of  cells  above  time w i t h  medium were removed a f t e r 1 and (incubation  extent  the  described  each  5 mM  the  was  measured.  (incubation ml  of  the  addition  of  iodide.  Aliquots  30  2 ml  minutes of  release  was  cho-  3  of of  1), salt the 5%  incubation  calculated  as:  -  cpm, Cpm  0 minutes;  cpm, 30 m i n u t e s ; i n c u b a t i o n 2 i n c u b a t i o n 1 - cpm, 30 m i n u t e s ;  a t 0 m i n u t e s were o b t a i n e d  (k)  L a b e l l i n g of C e l l s with  (6% h o u r s p . i . ) . neutralized calf  Next,  Balanced  cessed (£)  Salt Solution  Q  3  tQ  in section  several  (and 2%  ml o f  dialyzed  (3 ml o f t h e same  medium  choline)  a t 37°, the c e l l s  Choline-Containing each p r e p a r a t i o n ,  were u s e d .  Each d i s h  i c e - c o l d PBS.  the  cells  tube  was  added.  were c h i l l e d  meAfter  and p r o -  (£)(i).  from  cells  each d i s h . a cold  and  5 dishes  dishes  were s c r a p e d  l t  t h e l o w e r p h a s e was  counting,  2  and  centrifuge  C[]-choline,  at 4°.  extracted  (1/1; v / v ) .  TLC, and p h o s p h o l i p i d  v/v).  added t o  p o l i c e m a n was  t o the c e n t r i f u g e  by c e n t r i f u g a t i o n  3  t o each d i s h  2 ml o f water  a rubber  Standard  20 ml o f CH OH-H 0  3 ml  20 ml o f C H C I 3 - C H 3 O H (1/1;  with  cells  times with  into a plastic  for calculation of recoveries.  colder,  three  (about 2 ml)  were removed i n a n o t h e r  room a t 4 ° .  of confluent  was washed  added  Removal o f c e l l s  were s e p a r a t e d  with  5 large  Water was  C D P - c h o l i n e were added  samples  Compounds  of c e l l s  (or t u b e s ) c o n t a i n i n g  Residual  for  twice with  of virus  P o o l S i z e Measurements  For  or  Choline  3  10 y C i o f  incubation  as d e s c r i b e d  (i)  in  Q H~J  the l a b e l l i n g  dium w h i c h c o n t a i n e d  1  extrapolation.  C e l l s were washed  Earle's  serum).  30 m i n u t e s  by  x 100 incubation  were l a b e l l e d a t 6% h o u r s a f t e r a d d i t i o n  Cells  of  47 -  done  phosphocholine,  tubes o f s e l e c t e d  After  mixing,  Keeping three  the phases  t h e t u b e s a t 4°  times  further  Lower p h a s e s were r e t a i n e d . phosphorus.;  analysis.  -  The  upper  by g r a v i t y form). 20  phases  into  This  a 30 cm x 1 cm column o f AG 1-X10 r e s i n  s t e p took 3  flash  several  umn was a t room During  occurs  temperature,  this  procedure  o f t h e column was r i n s e d  with  phases a r e  contained choline.  pump. Next,  the column.  phosphocholine  e l u a t e s were e v a p o r a t e d  The c o l -  i n the r e s i n but  The c o n t a i n e r a t t h e  s i x times with  These  column w i t h a p e r i s t a l t i c  contained  developed  the column's performance.  and once w i t h w a t e r .  through  i f t h e upper  bubbles  top  pumped  was mixed  w h i l e e l u a t e s were c o l l e c t e d on  not a f f e c t  far,  The r e s i n  b e f o r e i o n exchange c h r o m a t o g r a p h y .  did  v/v)  hours.  (OH  (1/1; v/v) b e f o r e p a c k i n g t h e c o l u m n .  2  o f phosphocholine  evaporated  ice.  -  were c o m b i n e d , p l a c e d on i c e , and d r i p p e d  volumes o f CH OH-H 0  Hydrolysis  48  CH3OH-H2O  r i n s e s were f o r c e d  (1/1;  through the  The column e l u a t e  o b t a i n e d so  0.2 M N H H C O ( a b o u t  500 ml) was  4  The s e c o n d  3  eluate  and C D P - c h o l i n e . a t 3 7 ° - 4 0 ° under  thus o b t a i n e d ,  The f i r s t reduced  and s e c o n d  p r e s s u r e un-  t i l dry. The plied  second  eluate  to a charcoal  packed  column  in a Millipore  filter.  The f l a s k  was d i s s o l v e d  ethanol. The  eluate  These which  containing  apparatus  the second  resulted,  and a p -  The c h a r c o a l was  over  a glass  fiber  e l u a t e was r i n s e d s i x  and t h r e e t i m e s w i t h  r i n s e s were p a s s e d  e v a p o r a t e d as d e s c r i b e d CDP-choline  (1.3 cm x 1.6 cm).  filtration  t i m e s w i t h 10 ml o f water  i n 10 ml o f water  through  10 ml o f 2%  the c h a r c o a l  column.  c o n t a i n e d p h o s p h o c h o l i n e , and was  above.  was o b t a i n e d by e l u t i o n  60 ml 40% e t h a n o l / 1 % NH 0H. 4  To a v o i d  o f the c h a r c o a l  violent  boiling  with  of ethanol,  - 49 -  the.CDP-choline perature  fraction  and s u b s e q u e n t l y warmed  Phosphocholine  7 ml o f water  choline added  fraction  t o which  about  was added  was d i s s o l v e d  10.  Alkaline  phosphodiesterase prepared  (Sigma,  The f i n a l  3 units,  three times  i n 150 y£) was added  Phosphatase unitQ) line  (about 2 u n i t s )  in a total  sample.  per minute. Since  Company as h y d r o l y s i s  hydrolysis  case  (about sample.  (about 0.06  t o each  i s denoted  CDP-choby t h e  o f 1 ymole o f s u b s t r a t e  o f TLC i n s o l v e n t  were d i a l y z e d  A) and 1 M  tively.  Dialysis  choline  phosphocholine  was n o t c o m p l e t e d  was n e c e s s a r y .  (buffer  volumes  a g a i n s t 200  I n c u b a t i o n s were a t 3 7 ° o v e r n i g h t .  zyme d i g e s t i o n  200  each  Phosphatase  volume o f 200 y£ were added  shown by t h e r e s u l t s  phosphatase  adamanteus venom) were  (pH 9.0).  in this  sample was  111-S, from E . c o l i ) and  f o r 2 hours  t o each  was  (100 y£) and  and p h o s p h o d i e s t e r a s e  An enzyme u n i t  Sigma C h e m i c a l  (Sigma  The CDP-  t o which  pH o f e a c h  from C r o t a l u s  volumes o f 0.1 M b o r a t e b u f f e r  tem-  was d i s s o l v e d  3 ml o f IN NaOH.  i n 5 ml o f water  phosphatase  by d i a l y z i n g  fraction  (In one c a s e , IN NaOH  (40 y£) were added.)  a t room  were h y d r o l y z e d t o c h o l i n e  The p h o s p h o c h o l i n e  60 y£ o f IN NaOH.  IN HC1  evaporated  t o 37°-40°.  and C D P - c h o l i n e  by enzyme d i g e s t i o n . in  was i n i t i a l l y  NH4HCO3  system  against  0.1 M  buffer,  for at least  a portion  t r e a t m e n t , as  A, f u r t h e r e n -  P h o s p h o d i e s t e r a s e and a l k a l i n e NH4HCO3  buffer,  pH 10.0 ( b u f f e r  was done t h r e e t i m e s a g a i n s t  of buffer  samples,  by t h i s  2 hours.  pH 8.9  B), respec-  approximately F o r the phospho-  (1 ml) o f t h e p r e v i o u s m i x t u r e was  -  mixed w i t h and  75-300  0.7-0.925  samples,  ml o f b u f f e r  an a l i q u o t  phosphatase A.  separated  counted region  B, t o make 2 m l .  For the CDP-choline  (1.5 ml) o f t h e p r e v i o u s m i x t u r e was mixed  hydrolysis,  (about 0.12 u n i t ) ,  0.3 ml o f w a t e r ,  by TLC i n s o l v e n t  (choline  system  time w i t h  e_t a l . ( 1 9 4 ) .  on t h e i o n - p a i r  boron  i n heptanone,  phosphorylation  measured by t h e c o l o r i m e t r i c  from  BHK c e l l s . dissolved  the r e s u l t s  extraction  of choline  isolated  by Q Y -  into 3 2  P^]  0-0.7,  were  hydrolyzed phosphocholine c a s e , samples  i n 5 ml o f w a t e r ,  method  method  eventually T h i s method i s  with  tetraphenyl-  a solution  o f HC1, and  ATP w i t h c h o l i n e  on t h e f o l l o w i n g :  from BHK c e l l s ,  In t h e f i r s t  (195).  of choline  re-extraction  by t h i s  The method  and McCaman  E x t r a c t i o n s were p e r f o r m e d  derived  cpm f r o m R^  The h y d r o l y z e d samples  However,  was t h a t o f G o l d b e r g  choline  The e l u a n t s were  85% f o r h y d r o l y z e d p h o s p h o c h o l i n e , and  (see R e s u l t s s e c t i o n ) .  based  free  the s i l i -  The p e r c e n t o f cpm i n t h e Rf 0-0.2  was i n i t i a l l y  were v a r i a b l e chosen  from  were  by l y o p h i l i z a t i o n .  Choline Hayashi  compounds  A, and e l u t e d  2 ml I N HC1.  f o r hydrolyzed CDP-choline.  concentrated  and 1 ml o f b u f -  r e g i o n ) compared t o t h e t o t a l  92% f o r c h o l i n e ,  50 u£ o f  4 hours.  the c h o l i n e - c o n t a i n i n g  for radioactivity.  averaged  of  (about 15-60 u n i t s )  (about 10 u n i t s ) ,  two t i m e s , e a c h  94%  phosphatase  I n c u b a t i o n was a t 3 7 ° f o r a t l e a s t  After  ca  uJ, o f a l k a l i n e  150. \iZ o f p h o s p h o d i e s t e r a s e  with  fer  50 -  kinase.  firstly,  on  and s e c o n d l y , on c h o l i n e and C D P - c h o l i n e of isolated  and e x t r a c t e d  pools o f '  choline  twice with  were  5 ml o f  - 51  -  tetraphenylboron  i n heptanone  were r e e x t r a c t e d  w i t h 10 ml o f 0.4  IN HC1.  The  combined  HC1  (10 mg/ml).  phases  The  N HC1,  and  heptanone  layers  then w i t h 4  were e x t r a c t e d  ml  w i t h 10 ml  of  heptanone. Choline derived purified from  phosphocholine  in a s l i g h t l y different  the l y o p h i l i z e d  choline  from  w i t h 70%  were done.  The  (33).  precipitate  centrifuge.  combined  s u p e r n a t a n t s to about  followed  by  C h o l i n e was  70%  lyophilization.  extractions  0.4  e q u a l volume o f h e p t a n o n e .  37°  The  choline  The  under  removed by warming a stream  and  extracted  phase was HC1  extracted  p h a s e was  c o n d i t i o n s g i v e n h e r e were c h o s e n  the  y i e l d of  3  suggested duced  choline.  that  CDPonly  The  lyophilized  t o maximize  samples  were r e -  before assaying. was  done i n a t o t a l volume o f 50 y£ .  i s more c o n v e n i e n t t h a n  (195), a l t h o u g h  by an  an  t e t r a p h e n y l b o r o n i n h e p t a n o n e were done.  assay of c h o l i n e  volume  The  with  i d e n t i c a l l y , except  extraction  This  times  lyophilized.  The  The  three  w i t h an e q u a l volume  e x t r a c t i o n s with  i n water  nitrogen,  (10 mg/ml).  two  dissolved  of  the  phosphocholine hydrolysates  h y d r o l y s a t e s were t r e a t e d  [] H~]  CDP-  in a  e t h a n o l was  the HC1  and  removed by c e n t r i f u g a t i o n  i n 2 ml o f water  Finally,  extracted  each  h e p t a n o n e l a y e r s were r e e x t r a c t e d  N HC1.  was  o f 1 ml  2 ml o f t e t r a p h e n y l b o r o n i n h e p t a n o n e  combined of  The  Three  was  desk-top  with  manner.  CDP-choline  hydrolysates of phosphocholine  ethanol  were t h e n d i s s o l v e d  and  increase  10  y&,  which  i s the  the s e n s i t i v i t y o f t h e a s s a y  i n volume.  The  volume is re-  published procedure  (195)  -  was  followed,  (25  u&) o f b u f f e r - s u b s t r a t e  ATP,  except  and 100 mM  "*") .  25°,  not 37°.  incubation  0.3 M b a r i u m 75 y £ .  acetate  columns  used  say  of a mixture  of c h o l i n e cells  form.  inhibition  iodide.  3  2  p  J  (about  P  to  iodide  chloride  effect  was  was (196).  tested  by a s -  a known amount  isolated  which depressed  Part  ( n o t 1-X8)  from  BHK  the assay  However, by d i l u t i n g  t h e sample,  and  samples a s s a y e d d i d n o t i n h i b i t  as  T  ( F i g . 4) was  Choline  choline  One sample o f c h o l i n e  a l l free choline  A  increased  curve  sample w i t h  this  pro-  The amount o f  by t h e samples was  d i d c o n t a i n •. an i n h i b i t o r  mg  contaminant  t o AG 1-X10  iodide.  of h a l f of a given  (Sigma,  f o r 1 minute a t 4 ° .  The s t a n d a r d  like  with  by 15 y£, o f s a m p l e .  t o p r e c i p i t a t e ATP was  i t i s not hydroscopic  Lack o f enzyme  C  to phosphocholine.)  t o 2.5 nmoles o f c h o l i n e  since  kinase  any c h o l i n e  (75 yfi,) was a p p l i e d  i n the formate  2 mM  2  f o r 15 m i n u t e s a t  f o r 15 m i n u t e s a t 2 5 ° .  used  MgCl >  (0.8 ymole min  converts  Samples were t h e n c e n t r i f u g e d  the s u p e r n a t a n t  linear  amount o f c h o l i n e  then added, f o l l o w e d  was  10 mM  o f t h i s m i x t u r e was  (Preincubation  An a l i q u o t  b u f f e r , pH 8.0) was mixed  6 yg o f p r o t e i n  Preincubation  y C l , i n 5 y£) was  Further  (which c o n t a i n s  This  t h e enzyme p r e p a r a t i o n  0.5  of  kinase.  represents  tein  in  i n the f o l l o w i n g d e t a i l s .  sodium p h o s p h a t e  5 y£ o f c h o l i n e from y e a s t )  52 -  cpm.  eliminated, t h e enzyme  shown by t h i s method. (i i j  PC  Lipid  samples were p r e p a r e d  except  f o r one sample  as d e s c r i b e d  in section  ( o f seven) w h i c h was o b t a i n e d  (£) ( i ) ,  by t h e  0  0  0.4  0.8  1.2  C h o l i n e , nmoles F i g u r e 4. Standard curve f o r c h o l i n e d e t e r m i n a t i o n . C h o l i n e was measured by t h e p r o c e d u r e o f G o l d b e r g and McCaman ( 1 9 5 ) , m o d i f i e d a s d e s c r i b e d i n M a t e r i a l s and Methods, s e c t i o n (1) ( i ) . The same u n i t s a p p l y t o the o r d i n a t e and a b s c i s s a o f the l a r g e f i g u r e and t h e i n s e t graph.  -  following moved an  procedure.  54 -  C e l l s were washed  i n 8-10 ml o f i c e - c o l d PBS.  e q u a l volume o f CHCI3-CH3OH  Dounce h o m o g e n i z e r twice with  t w i c e w i t h PBS, and r e -  L i p i d s were e x t r a c t e d  (2/1; v / v ) i n a  (20 s t r o k e s ) .  The l i p i d  8-10 ml o f CH OH-0..9% N a C l - C H C l 3  into  tight-fitting  p h a s e was  extracted  (48/47/3 ; v / v / v )  3  (197). Protein lipid  samples over  trated  of  fiber  filter  extracts,  combined  TLC  plates  tem  separated  in  filter.  Aliquots  and r i n s e s funnel,  with  lipid  were  (about  o f the f i l t e r e d  and r e d i s s o l v e d i n  then d r i e d  and c o u n t e d .  1 m l , CHCI3-CH3OH  (2/1;  7 ml o f CHCI3-CH3OH  i n solvent  (see R e s u l t s ) ,  from a measurement o f t h e s p e c i f i c  This  sys-  from t h e  Since  eluted  the  the r a d i o a c t i v i t y appeared pool  s i z e s were  calculated  r a d i o a c t i v i t y o f PC.  i n i s o l a t e d PC was measured This  recovered.  (Of t h e cpm  7 ml.)  the  These  PC was e l u t e d  i n the f i r s t  v/v))  to s i l i c a g e l  s y s t e m C.  (2/1; v / v ) .  dpm were known, and s i n c e  d u r e o f R a h e j a e t a_L ( 1 9 8 ) .  cpm  s a m p l e s were a p p l i e d  PC from PE, LPC, and L P E .  phosphorus:  concen-  f o r r a d i o a c t i v i t y (cpm).  w h i c h were d e v e l o p e d  i n PC  o f the  and t h e t u b e s w h i c h had c o n t a i n e d  were measured  be e x c l u s i v e l y  pholipid  Samples were  a t 35° or lower  14 m l , 85% were r e c o v e r e d  total to  fiber  cpm were an a v e r a g e o f 0.3% o f t h e t o t a l  Aliquots  silica  a glass  (2/1; v / v ) .  the f i l t r a t i o n  lipid  s o l i d s were remo.ved by f i l t r a t i o n  by f l a s h e v a p o r a t i o n  CHC13-CH3OH The  and o t h e r  by t h e p r o c e -  method gave a l i n e a r  curve  t o 10 yg o f p h o s p h o r u s :  in dipalmitoyl  cific  r a d i o a c t i v i t i e s i n dpm/yg p h o s p h o r u s  PC were  Phos-  standard  (Fig. 5). thus  Spe-  determined.  16  Phosphorus,  u g  Diglyceride,  nmoies  F i g u r e 5. S t a n d a r d c u r v e s f o r l i p i d p h o s p h o r u s ' ( l e f t ) and d i g l y c e r i d e iright) determinations. L i p i d p h o s p h o r u s was measured by the method o f R a h e j a e t a l . (198). F o r m a t i o n o f t h e c o l o u r e d complex was d e t e r m i n e d by i t s a b s o r bance a t 710 nm. D i g l y c e r i d e was measured by S c h n e i d e r ' s , p r o c e d u r e (199), m o d i f i e d as d e s c r i b e d i n M a t e r i a l s and Methods, s e c t i o n (£,) ( i i i ) . Isolated g l y c e r o l - [ [ P 2] p h o s p h a t e was c o u n t e d f o r r a d i o a c t i v i t y . 32  -  To  calculate  molecular is  based  21 c e l l s fetal  specific  weight  calf  and  ide  ("Day  composition  This  o f the l i p i d  f o r BHK  from  prepared  cell  samples  cells  the s i l i c a  Medium  from  with  palmitin  2.5%  mock- o r v i r u s -  t o 0.76  was  with  and d i o l e i n  after  was  between R:^ 0.75  visualized  B.  In a c o n t r o l  TLC and e l u t i o n  diglyceride  and 0 . 7 8 ) . vapour  had  and  eluted  (75/25/1; o f the  t o o b t a i n low b l a n k s the y i e l d  the s i l i c a  was  and  Diglycer-  Distillation  experiment,  from  Diolein  as TLC s t a n -  4 ml o f 2 - p r o p a n o l - h e x a n e - w a t e r (199).  infected  between R^ 0.66  by i o d i n e  b e f o r e use was e s s e n t i a l assay.  were u s e d  The p r e p a r e d  v / v / v ) as d e s c r i b e d by S c h n e i d e r  diglyceride  estimate  r e f . 163.),.  f o r CPT a s s a y  diglyceride.  o f 0.64  BHK  2-propanol  average  o f PC r e p o r t e d f o r BHK-  Essential  3", T a b l e V,  (with a contaminant  from  estimated.  an  were c h r o m a t o g r a p h e d on TLC i n s o l v e n t s y s t e m  an R f range 0.73  acid  i n Eagle's Minimal  diglyceride  dards  f o r PC was  i n dpm/ymole,  Diglyceride  Aliquots cells  o f 831.1  serum  (iii)  -  radioactivities  on t h e f a t t y grown  56  i n the  ofd i -  shown  t o be  quantitative. Hydrolysis of diglyceride of  glycerol  and p h o s p h o r y l a t i o n  by g l y c e r o k i n a s e were by S c h n e i d e r ' s  After  t h e enzyme  assay  tube.  reaction,  0.8  plugged  ml o f water was  The c o n t e n t s o f each  2 cm column o f c h a r c o a l - c e l i t e pipet  to g l y c e r o l  with g l a s s wool.  water were p a s s e d  through  tube  Two  added  were t h e n  (1/1; by w e i g h t ) further  the c h a r c o a l .  method  (199).  t o each  applied  over  i n a pasteur  rinses with  0.8  A i r p r e s s u r e was  ml  used  a  -  to d r a i n  the columns.  c u r v e was l i n e a r Lack  The e l u t e s were c o u n t e d .  from  original p.i., with of  Phosphocholine  of 5 large  10% p e r c h l o r i c  The  for  A t 6h  hours  three  times  (200).  was washed  PBS.  were removed w i t h  acid.  The c e l l s  The c e l l u l a r  6 ml  material in perchloric  5 strokes of a t i g h t - f i t t i n g  Dounce  room a t 4 ° .  e x t r a c t s were c e n t r i f u g e d a t 30,000 x £ f o r 10 m i n u t e s a t  1 hour,  ml  by an  The above s t e p s were done i n a c o l d  4°, n e u t r a l i z e d  The  independently  dishes of c e l l s  was h o m o g e n i z e d w i t h  homogenizer.  w h i c h when  and CTP  method w h i c h has been d e s c r i b e d  each  by m i x i n g  cpm.  and CTP were a s s a y e d  3 ml o f i c e - c o l d  acid  2 nmoles o f d i p a l m i t i n ,  gave 88% o f t h e e x p e c t e d  Phosphocholine  (Fig. 5).  by t h e s a m p l e s was c h e c k e d  o n e - h a l f o f one sample w i t h  (iv)  The s t a n d a r d  0 t o 10 nmoles o f d i p a l m i t i n  o f enzyme i n h i b i t i o n  assayed  57 -  with  KOH  and r e c e n t r i f u g e d  supernatant o f water.  ( t o pH 7 - 8 ) , h e l d on i c e f o r a t l e a s t a t 6,000 x £ f o r 10 m i n u t e s  o b t a i n e d was l y o p h i l i z e d  This solution  charcoal.  was a b s o r b e d  Phosphocholine  and r e d i s s o l v e d  i n 10  was a g a i n c e n t r i f u g e d a t 6,000 x £  10 m i n u t e s a t 4° t o remove t h e s a l t  8 ml o f s u p e r n a t a n t  (201).  remained  twice  precipitate. in a total  CTP i n  o f 0.30 g o f  i n the s u p e r n a t a n t .  The  c h a r c o a l was e x t r a c t e d fb.ur t i m e s w i t h  1.5 ml o f e t h a n o l - w a t e r -  ammonium  After  hydroxide  (50/49/1; v / v / v ) .  tion,  t h e c h a r c o a l and e l u a n t were h e a t e d  After  each  gation  extraction,  the f i r s t  t o 60° f o r 2  t h e c h a r c o a l was c o l l e c t e d  a t 6,000 x £ f o r 5-10 m i n u t e s .  extracminutes.  by c e n t r i f u -  The combined  e l u a n t s were  -  filtered  through a M i l l i p o r e  particles 37°.  of charcoal.  58  -  filter  (0.22 ym)  The s o l u t i o n  The r e s i d u e c o n t a i n i n g  This  fraction  line  were c o n c e n t r a t e d by  t o remove  was f l a s h  evaporated a t  CTP was d i s s o l v e d  and t h e s u p e r n a t a n t w h i c h  i n 2 ml o f w a t e r .  c o n t a i n e d phosphocho-  lyophilization.  To measure p h o s p h o c h o l i n e , a sample o r a s o l u t i o n dard  phosphocholine  containing  y£  (20 y£) was mixed  1 ymole o f T r i s - s u c c i n a t e  magnesium a c e t a t e ,  3  of partially  tase  (Sigma,  from y e a s t ) .  tion  to favour CDP-choline  37° f o r 45 m i n u t e s ,  tube  in boiling  fuged  water  CTP  formation.)  rat liver  20 ymoles  t h e CT  The r e a c t i o n  reac-  proceeded  the r e a c t i o n  The m i x t u r e was  centri-  and p a r t o f t h e s u p e r n a t a n t  (50 y£) was i n c u b a t e d w i t h 50 y£ o f a s o l u t i o n ymolef) o f C D P - c h o l i n e ,  CT  pyrophospha-  causes  and was s t o p p e d by p l a c i n g  a t 3,000 x £ f o r 10 m i n u t e s  solution  (5 C i / m o l e ) , 50  purified  (Pyrophosphatase  f o r 2 minutes.  of stan-  (pH 7 . 0 ) , 1.2 ymoles o f  C5-H~J  0.05 ymole:;, o f  (about 0.5 mg p r o t e i n )  w i t h 80 y& o f a  ( 2 0 0 ) , and 0.008 u n i t / ? (ymole min ^) o f i n o r g a n i c  at  fine  containing  of Tris-glycine  buffer  0.1  (pH  -1  10.5),  and 0.30 units.;.-' (ymole  (Sigma,  from E . c o l i ) .  After  was s t o p p e d as d e s c r i b e d rated  from  Q H~J  1 M ammonium CTP  3  cytidine  ) of alkaline  30 m i n u t e s  chromatography  (7/3;  was measured by an a s s a y  10 ymoles  a t 37°, the r e a c t i o n  3H  by p a p e r  was mixed  phosphatase  [ ] L ] C D P - c h o l i n e was  above.  a c e t a t e , pH 7.1  s t a n d a r d CTP s o l u t i o n taining  min  ethanol-  v/v).  i n which  w i t h 80 \il  of Tris-succinate  with  sepa-  20 \il  o f a sample o r  of a solution  con-  (pH 7 . 0 ) , 1.2 ymoles o f  -  magnesium a c e t a t e , phosphocholine and  tion. as  (7.5  0.008 u n i V ) o f  against  The  formation The the  Figure  assay  A  linear  by  the  ymole-: o f  y l o f CT  ATP  was  partially  50  of  previously  of  10  standard  added  Q HJ  values  (6%/hour a t  and  for  CT  prepara-  centrifuged  (section  (g)(ii)).  i n t o CDP-choline against from  o f C D P - c h o l i n e was (1 t o 10  time 1 to  unin  10  also  nmoles),  a in  the  CTP.  3  were c o r r e c t e d 4°)  protect  3  obtained  added p h o s p h o c h o l i n e  nmoles o f  to  [~ HCDP-choline  is plotted  Formation  3  purified  described  c u r v e was  (^Methyl- H~J  preparation,  h e a t e d , and  nmoles o f CTP  described  (Fig. 7).  function  CTP  50  t u b e s were i n c u b a t e d ,  incorporation  of  0.1  pyrophosphatase.  assay c o n d i t i o n s  presence  o f ATP,  Ci/mole),  measured as  nmoles o f CTP  acid  15  -  f o r p h o s p h o c h o l i n e measurement.  was  6.  linear  or  ymole<  h y d r o l y s i s o f CTP  described  der  0.1  59  for hydrolysis  the  by  10%  overall isolation  perchloric  yield  (61%)  (200). (v)  Nucleotides  High-pressure BHK  liquid  cell:nucleotides.  A n g e l ) was Elion  c h r o m a t o g r a p h y was  C h r o m a t o g r a p h y on  p e r f o r m e d , as  suggested  by  used  to  Partisil-10  a recent  separate SAX  (Reeve-  publication  of  e t a l . (202) . Each l a r g e  dish  of  of  i c e - c o l d PBS  by  two  s i m i l a r methods.  in  10%  p e r c h l o r i c a c i d and  lized,  before  cells  centrifuged,  and  was  washed  extraction. In  the  times with  Nucleotides  first,  extracts  three  3-5  were measured  nucleotides  were  extracted  were h o m o g e n i z e d , n e u t r a -  l y o p h i l i z e d as  described  ml  in  section  ..  - 60 -  I  o  TIME  (MIN)  F i g u r e 6. Time c o u r s e f o r the c o n v e r s i o n o f CTP i n t o CDP-choline. Ten nmoles o f CTP was a s s a y e d i n the p r e s e n c e o f 100 nmoles o f [~JH~J p h o s p h o c h o l i n e . CDP-choline format i o n was measured as d e s c r i b e d i n M a t e r i a l s and Methods s e c t i o n (£,) ( i v ) a t v a r i o u s t i m e s o f i n c u b a t i o n . 3  - 61 -  0  2  4 C T P  6  8  10  12  (NMOL)  F i g u r e 7. S t a n d a r d C u r v e f o r CTP d e t e r m i n a t i o n . CTP (0-10 nmoles) was a s s a y e d as d e s c r i b e d i n M a t e r i a l s and Methods s e c t i o n (£)(iv). The amount o f C D P - c h o l i n e f o r m a t i o n was c a l c u l a t e d from the dpm i n the p r o d u c t , w h i c h has t h e same s p e c i f i c / r a d i o a c t i v i t y as t h e s u b s t r a t e , Q H ~ J CTP. 3  -  (1) ( i v ) .  Nucleotides  equipped with Scientific nel 0.01 and  were s e p a r a t e d  operating  (pH  M KC1  3.5)  (total  triphosphates  were d e t e r m i n e d  and  Michael  Smith's  ml)  The  the  254 A  which  6 ml  sample  by  centrifugation.  impurities).  to serve twice  The  as  with was  on  chan-  gradient  of  3.5)  4  nucleotide  of  by  the  triphosphates  Chromatographic  Dr.  by  nucleotides  peak a r e a s  Everard  Trip  an  3 ml  internal  a 45  and  ITP  sepa-  in  Dr.  were added  standard  o f CH OH-H 0 3  added.  then  cm  (Model 153,  a recorder  Chromatographic e l u t i o n  mm  was  and  again  under of  reduced  water, by  Nucleotides  column e q u i p p e d w i t h Altex S c i e n t i f i c  a linear  to by  concentrated  (Linear Instruments  with  (202).  P h a s e s were s e p a r a t e d  d i s s o l v e d i n 1 ml  x 2.1  to  (1/0.8; v/v)  2  under a s t r e a m o f n i t r o g e n .  detector nm  a dual  2  u p p e r p h a s e s were e v a p o r a t e d  r e s i d u e was  t o a b o u t 40°  a t 280  Altex  M KH P0 (pH  nucleotide  t o remove a p r e c i p i t a t e ,  w a v e l e n g t h UV  and  e l u t e d the  dividing  for standard  of chloroform  were s e p a r a t e d  column  laboratory.  of c e l l s  centrifuged  nm  linear  t o 0.5  s e c o n d method, known amounts o f  The  mm  (Model 152,  concentrations  c a l c u l a t i o n s were made by  were removed  ating  M KC1  100  height.  i n each  Cells  heating  and  x 2.1  overlap.  for diphosphate  rations  pressure.  0.01  volume:  peak a r e a / n m o l e v a l u e s  every.dish  nm  cm  were c a l c u l a t e d by m u l t i p l y i n g peak h e i g h t s  peak w i d t h s a t h a l f  In  a 40  detector  a t 280  and  without  Peak a r e a s  (corrected  on  (Linear Instruments Corp.).  M KHzPC^ 0.5  -  a d u a l w a v e l e n g t h UV  Inc.)  recorder  62  a  Inc.)  single oper^.  Corp.).  gradient of  0.1  M  -  KH2P04  (pH  3.8)  to 1 M KH P0 2  4  63  -  (pH  3.8)  (total  Peak a r e a s were d e t e r m i n e d  by p l a n i m e t r y .  b o t h methods a g r e e d  well  (m)  Fatty Acid Cells  Cell  by  glyceride  was  the p r o c e d u r e  were  isolated  ized  by  isolated by TLC  Lipids  by TLC  The  blowing  (section 3  system  with  0.2%  methyl  liquid mesh Gas at  esters  180°.  18:2,  18:3;  liver  lipids.  the  PE  visual-  dichlorofluorescein  of n i t r o g e n over done by  proceeded  the  10 ml  in  o f m a n n i t o l and  a t 80°  for at least  a column o f 12%  included mixtures acids: fatty  14:0, acid  H1-EFF-2BP  A 7610A H i g h was  18:0,  esters  Peak a r e a s were measured  3  and  the 12  with  20:0,  removal hours. gas-  80/100 Efficiency t h e oven s e t  which c o n t a i n e d  16:0,  methyl  used,  CHC1 -  samples..  the method o f Vance  (Hewlett Packard)  fatty  and  were  e x t r a c t e d i n hexane were s e p a r a t e d by  Standards  e s t e r s o f the  Lipids  and  by h e a t i n g t o  Science Laboratories).  Chromatograph  PC  evaporated  c h r o m a t o g r a p h y on  (Applied  C.  B.  Di-  s o l v e n t was  the a d d i t i o n  The  system  (197).  with  Sweeley  reaction  et aL  and  the s i l i c a  was  The  (2/1; v/v)  from  a stream  (203), w i t h o u t  3  in solvent  Methanolysis of l i p i d s  o f HC1.  from  ( f ) ) at 7 hours p . i . .  in•CHC1 -CH OH  in solvent  were e l u t e d  (2/1; v / v ) .  5 2 - 5 5 ° and  results  ml).  Results).  d e s c r i b e d by F o l c h  s p r a y i n g the p l a t e s  methanol. 3  as u s u a l  were homogenized  extracted  (see  The  80  Analysis  were p r e p a r e d  pellets  CH OH  fairly  volume:  methyl  16:1,  o f r a t and  by p l a n i m e t r y .  18:1, trout  - 64 -  (n)  \2H~J 3  Pulse-Chase  6h h o u r s p . i . ,  At was  Choline  removed from e a c h  dium  t h e Medium large dish.  (7 m l o f t h e same medium  c h o l i n e ) was added were washed  minute  intervals,  as d e s c r i b e d  layer  was e x t r a c t e d  The upper  phases  ted  by i o d i n e  compound  2.25 cm TLC l a n e .  vials.  vapour.  The 3  2  taken  ( v ) . The  6 ml o f CH OH-H 0 3  A l i q u o t s were s y s t e m A.  2  The c a r r i e r  was added  eva-  then  Carrier  (0.9 mg e a c h ) were a d compounds  were d e t e c -  g e l c o r r e s p o n d i n g t o each into  scintilla-  2 m l 0.IN NaOH arid 100y£ :  acid.  l o w e r c h l o r o f o r m p h a s e s were e x t r a c t e d  6 ml CH OH-H 0  A t 20  were c o n c e n t r a t e d by f l a s h  Areas o f s i l i c a  To e a c h v i a l  acetic  To s t a r t t h e  (I)  were s c r a p e d o f f t h e TLC p l a t e d i r e c t l y  glacial  (1/0.8; v / v ) , t r a n s f e r r e d  3  dried  w i t h a second  p h o s p h o c h o l i n e , and C D P - c h o l i n e  t o each  tion  and Methods s e c t i o n  and d i s s o l v e d , i n 1 ml o f w a t e r .  ded  the c e l l s  immediately added.  f o r c o u n t i n g and f o r TLC i n s o l v e n t  choline,  3  A t 6h h o u r s p . i . ,  were  serum)  20 y C i o f [] H~J  m e t h a n o l - w a t e r - s o l u b l e e x t r a c t s were  in Materials  (1/0.8; v / v ) .  taken  contained  calf  t h e p u l s e me.-  t w i c e w i t h 5 ml o f t h e same medium.  10ml o f t h e same medium  poration  Immediately,  which  t o each d i s h .  chase,  chloroform  199 (and 2% f e t a l  by a i r , and c o u n t e d .  t w i c e more w i t h  to s c i n t i l l a t i o n  vials,  65  -  -  RESULTS'"  (a)  Cell The  in  Table  (Table virus dish  W e i g h t and  Protein  wet  w e i g h t o f BHK  3.  The  3).  The  cells  w e i g h t was  the  to r e s u l t s  per  W e i g h t o f BHK  Cells  gram  used (wet  large dishes  same a t 6% not  or  i s shown  7 hours p . i .  changed  to convert weight) of  by  results  SF per  cells.  3  Infected with  Wet 6h  five  ( T a b l e 3:); was  T h i s a v e r a g e was  Table Wet  from  about  average weight  infection. of c e l l s  Concentration  SF V i r u s  1  weight, g  hours p.i.  7 hours p.i.  Average  Mock-infected  0.70,  1.04  0.97  0.90±0.18  SF  0.72,  1.12  0.85  0.90±0.20  virus-infected  1 F i v e d i s h e s o f c e l l s were h a r v e s t e d as u s u a l ( M a t e r i a l s and Methods s e c t i o n ( f ) ) . ' C e l l p e l l e t s were w e i g h e d i n a t a r e d c e n t r i f u g e tube. 2 The v a r i a t i o n n o t e d i n t h i s and s u c c e s s i v e t a b l e s i s the standard d e v i a t i o n . Since tion  o f BHK  cells  to p r o t e i n c o n c e n t r a t i o n s , these  sured  and  enzyme a c t i v i t i e s  in mock-infected  and  were measured  in  rela-  c o n c e n t r a t i o n s were mea-  virus-infected  cells.  1 A l l methods were t h o s e t h a t a r e d e s c r i b e d M e t h o d s , e x c e p t where i t i s o t h e r w i s e noted.  Table  4 shows  in Materials  -  66 -  Table 4 P r o t e i n C o n c e n t r a t i o n s i n BHK C e l l s I n f e c t e d w i t h SF V i r u s  Protein  concentration  (mg/g c e l l s ) Significance  Total , protein" 1  Cytosol  2  Microsomes  3  Mock-infected ,  SF v i r u s - i n f e c t e d  • _ 21.7 ±1.8 (5)  18.0  of 4 difference  . ±1.8 ( 5 )  J  p<0.025  7.12±0.66(4)  5.50 ± 0 . 5 2 ( 4 )  p<0.01  2.37±0.56(5)  2.14 ± 0 . 0 7 ( 5 )  NS, p>0.25  D e t e r m i n a t i o n s were made on BHK c e l l p r o t e i n w h i c h was p r e c i p i t a t e d by t r i c h l o r o a c e t i c a c i d a t 7 h o u r s p . i . and t h e n homogenized i n 0.9% N a C l ( M a t e r i a l s and Methods ( j ) ) . ^ C y t o s o l and m i c r o s o m e s were p r e p a r e d a t 7 h o u r s p . i . . The numbers i n b r a c k e t s i n t h i s and s u c c e s s i v e tables r e f e r t o t h e number o f e x p e r i m e n t s . 4 S i g n i f i c a n c e o f d i f f e r e n c e was measured by a g r o u p comparison t test.  -  the  total  protein  infection tein  cells  was  4).  also  The  (b)  reduced  cells)  Choline The  was  by  tion.  Since  an  virus  such  cells.  The  the  of  of  tion  of choline  take  in mock-infected  of  u p t a k e by  total  cell  p l o t of ^  values  1/V '  were n o t  Since increase  measured by  the  At  6%  proBHK  amount  of  hours p . i . ,  o f mock- o r  virus-  7 hours p . i . (Table  w h i c h was could  3  incorporation  caused  be  SF  4).  i n the  17  medium was cells  yM.  The  protein vs.  1/S '  followed V  m a x  was  not  PC  infec-  inhibition in  BHK  experiments  l e s s uptake  the  of  are  occurred Inhibi-  concentra-  (Fig. 8).  The  up-  Michaelis-Menton k i n e t i c s .  value  In SF  into  to c o n t r o l c e l l s .  increased  the  virus  examined  transport  t o 7 hours p . i . . . compared  by  a r e s u l t of  p r o c e s s was  choline  to e s t a b l i s h  i n f e c t i o n became more marked as  was  381  pmoles m i n u t e  virus-infected cells  linear,  so  that K  m  and  of  Q  3 H  D  choline  a p p a r e n t amount o f competition  to  the  transport,  with u n l a b e l l e d  cell  surface  such b i n d i n g  choline.  1  however, V „ max m  obtained.  binding  the  at  cell  c y t o s o l of  4).  mg/g  choline  3  From 6%  8.  tion  was  [] H~J  []] H~J  virus-infected cells  value  (Table (2.89  transport  r e s u l t s of  shown i n F i g u r e  m  i n the  t h e s i s p r o j e c t was  inhibition  transport,  K  i n the  i n f e c t i o n , while  reduced  inhibition  choline  SF  this  inhibition  cells,  the  total  s i m i l a r to t h a t  purpose of  o f BHK  mg  reduction  Virus  Transport  mechanism o f  The  virus.  amount o f m i c r o s o m a l p r o t e i n  infected  in  i n f e c t e d w i t h SF  amount o f p r o t e i n  m i c r o s o m a l p r o t e i n was n o t the  -  caused a s i g n i f i c a n t  (Table  cells  i n BHK  67  would was  C e l l s were  -  iu io a.  ce o  68 -  0  80H  M0CKINFECTED > ^  1  0  60H  to UJ _J  o  Q.  VIRUS-  x  ^  40 H  —  INFECTED X  < 0. 3  20H  UJ  o X  o  1  5  CHOLINE CONCENTRATION  — F (pM)  F i g u r e 8. U p t a k e o f c h o l i n e by BHK c e l l s i n f e c t e d w i t h SF v i r u s . Uptake r e p r e s e n t s t h e d i s a p p e a r a n c e o f Q H~J c h o l i n e from t h e medium o f BHK c e l l s o v e r a 30 m i n u t e p e r i o d , s t a r t i n g a t 6h h j u r s p . i . . R e s u l t s a r e as pmoles o f c h o l i n e minute mg o f t o t a l c e l l p r o t e i n 3  expressed  - 69  incubated choline, choline cells cent  firstly  was  measured  r e l e a s e was  6.3%  3.3%  rated  from  choline.  (The a v e r a g e  by v i r u s  5.2%  with  5.2%  per-  f o r SF  results  ± 0.7%  infection  cells  the  0.32-0.80 yM  than  19%  f o r SF  r a t i o of t o t a l  was  0.44.  T h u s , 56%  rj rfj 3  inhibition  would be e x p e c t e d  p o o l s o f SF v i r u s - i n f e c t e d  (Fig. 8).  dpm  cells  inhibition  of  in a l l compared of  to  uptake  (See D i s c u s s i o n . )  Enzyme  Activities  enzymes o f de  t o e x p l a i n the p o s s i b l e caused  by SF v i r u s  novo PC  infection.  rate-limiting  noted.  o f de The  activities  were measured  from  I t was  enzyme would be  BHK  cells  thought inhibited  in order  novo s y n t h e s i s w h i c h  cytidylyltransferase  (CPT)  t i o n s w h i c h were p r e p a r e d i s otherwise  s y n t h e s i s were examined  inhibition  (CK), p h o s p h o c h o l i n e  linephosphotransferase  it  and  the  c h o l i n e - c o n t a i n i n g p o o l s were s e p a - i  experiments  choline-containing  kinase  cells,  these c o n c e n t r a t i o n s , l e s s  mock-infected  and  of three release  w h i c h were l a b e l l e d  However, i n t h e s e  The  cells,  3  ( j ) ) . In  choline,  3  [~JH^]  cells.)  c h o l i n e uptake  occurred.  fJ H[]  yM  unlabelled  r e l e a s e of  Methods s e c t i o n  0.61  for mock-infected  cells At  percent  for mock-infected  cells.  ± 6.2%  secondly with  The  ( M a t e r i a l s and  In t h r e e e x p e r i m e n t s ,  was  time.  w h i c h were i n c u b a t e d w i t h  virus-infected  (c)  c h o l i n e and  3  f o r 30 m i n u t e s each  virus-infected was  fJ H~J  with  -  of c h o l i n e ( C T ) , and  in subcellular  a t 7 hours  p.i.,  t h a t perhaps o n l y by  infection.  chofracunless  the  70  -  The  kinetics  BHK  cell  and  time o f a s s a y  c y t o s o l was  apparently (Fig.  o f CK  were examined  linearly  CK  ( t o 35 m i n u t e s )  activity  concentration enzyme was  was  ( F i g . 11).  n o t changed  trations,  i t was  found  separation  of choline  p e n d a n t on  the  ment, b o i l e d  Thus, a t each  activities  activity  was  of  M MgCl  In a c o n t r o l  from  tube  found  after  of  Q H~J  of  r~ATP-Mg~J  3  fect  the  resin  choline was  by  cpm  the  became more p r o n o u n c e d  recovery of  w h i c h c o n t a i n e d 0,  4  Such a d d i t i o n  C~J  5, o r  phosphocholine  25 mM  QATP-MgJJ .  made to  add  from  the  The r e d u c t i o n  the c o n c e n t r a t i o n  of MgCl from  was  This proce-  wash. as  34%.  useful  w h i c h were e l u t e d  sodium h y d r o x i d e  reduced.  com-  termination of  the  ion exchange  1  to  [~_ATP-Mg~J , a c o r r e c t i o n  cpm  [] '*C~j  [~ATP-Mg~J ,  100%  dure  choline  experi-  hydroxide  5 mM-25mM  but b e f o r e i o n exchange s e p a r a t i o n . []3H~J  de-  ( i n the assay m i x t u r e ) , a l l  linearly  assay  concen-  the o t h e r a s s a y  F u r t h e r , i t was  to each  2  cytosolic  S e p a r a t i o n was  However, from  c o n c e n t r a t i o n of  ATP  f o r the e x t e n t o f  cpm were r e c o v e r e d i n the sodium  recovery decreased  mM  9-11).  the r e a c t i o n reduced  the  of  a t v a r i o u s ATP  to c o r r e c t  r~/ATP-Mg~J  of phosphocholine.  100' y£ o f 0.1  yg)  60-180 yg o f p r o t e i n ) and  assay columns.  phosphocholine  loss  (50  ( t o 250  (Figs.  phosphocholine.  o r 5 mM  the p h o s p h o c h o l i n e  for  and  CK  activity  infection  necessary  in  as a f u n c t i o n  (10 nmoles) were mixed w i t h  A t 0 mM  from  o f CK  activity  a t a c o n c e n t r a t i o n o f 0.25  i o n s i n the a s s a y m i x t u r e .  cytosol  phosphocholine  eluant  (Fig. 9).  specific  by v i r u s  CK  to p r o t e i n  a l s o measured  The  In the c a l c u l a t i o n  first.  related  saturated with choline  10).  ponents.  -  2  d i d not a f -  assay  mixtures  -  71  -  Protein,  mg  12-^  Time,  min  F i g u r e 9. CK a c t i v i t y v s . p r o t e i n and t i m e . C y t o s o l from mock-(o) and SF v i r u s - i n f e c t e d (•) BHK c e l l s was a s s a y e d . After the a s s a y , the p r o d u c t was s e p a r a t e d by i o n e x c h a n g e . Activity was measured a t 10 mM ATP, 10 mM Mg* , and 0.25 mM. fJ H^] c h o l i n e (1 C i / m o l e ) . The upper g r a p h shows the e f f e c t o f c y t o s o l p r o t e i n on CK a c t i v i t y . The l o w e r g r a p h shows t h e e f f e c t o f time on CK a c t i v i t y ( i n 87-126 yg o f c y t o s o l p r o t e i n ) . A b l a n k v a l u e was subtracted from enzyme a c t i v i t i e s i n t h i s and s u c c e s s i v e figures and t a b l e s . B l a n k s c o n t a i n e d b o i l e d p r o t e i n o r no p r o t e i n . +  3  4  Choline, m M F i g u r e 10. CK a c t i v i t y v s . c h o l i n e . CK was a s s a y e d i n mock- (o) and SF v i r u s i n f e c t e d (•) c y t o s o l from BHK c e l l s . A f t e r the a s s a y , t h e p r o d u c t was s e p a r a t e d by i o n 3 exchange. C h o l i n e c o n c e n t r a t i o n was v a r i e d by t h e a d d i t i o n o f QH~J c h o l i n e (5 C i / m o l e ) . ATP'and M g c o n c e n t r a t i o n s were 10 mM. S e p a r a t e b l a n k s , were a s s a y e d a t e a c h c o n c e n 3 3 t r a t i o n o f rjH~J c h o l i n e . The b l a n k cpm i n c r e a s e d w i t h h i g h e r fJ H]] c h o l i n e c o n c e n t r a tions. (CK a c t i v i t i e s from v i r u s - i n f e c t e d c e l l s a t 0.75 mM and 1 mM c h o l i n e were n o t used t o p l o t the c u r v e s i n c e t h e y were a v e r a g e s o f v e r y v a r i a b l e d u p l i c a t e r e s u l t s . ) + +  -  73  -  ATP ,  mM  Figure 1 1 . CK a c t i v i t y v s . ATP. C y t o s o l from mock- (o) and SF v i r u s - i n f e c t e d (•) BHK c e l l s was a s s a y e d . A f t e r the a s s a y , the p r o d u c t was s e p a r a t e d by the i o n exchange method. ATP and M g * were added t o each a s s a y i n e q u a l amounts. Act i v i t i e s were c o r r e c t e d as d e s c r i b e d i n R e s u l t s s e c t i o n ( e ) . +  -  CK  activity  choline  and  was  verified  phosphocholine  linear  Mg  ) .  ^ mg  cells.  The tivity  from  the a s s a y  minutes)  ( F i g . 12).  concentration not a f f e c t e d cytosolic line The  appeared  ^ in cytosol  from  is similar  Table  mM  and  was  mM  ATP was  a s s a y was  infected  to the r e s u l t  0.30  and  from  con-  the i o n  5).  proportional  yg)  this  both  were examined n e x t .  t o the  Cytosolic  CT  t o the amount o f time o f a s s a y  infection  Cytosolic (Figs.  12,  CT  i n c r e a s e d by SF v i r u s  (up t o  13), except  both m o c k - i n f e c t e d  and  CT  40  at a  activity  was that  phosphocho-  infection  for cytosolic  ac-  protein  s a t u r a t e d by p h o s p h o c h o l i n e  ( F i g . 13).  kinetics  activity  i n BHK  o f CT  ( F i g . 13).  was  3 mM  SF v i r u s - i n f e c t e d  for cells  cell yg)  i n m i c r o s o m e s were a l s o e x a m i n e d . m i c r o s o m e s was  protein  (up t o 68  CT  from  SF v i r u s - i n f e c t e d  at  1.25  mM  was  7.5  14). The  CT  activity  o p t i m a l c o n c e n t r a t i o n o f CTP  (Fig.  3  at suboptimal c o n c e n t r a t i o n s of  t o be  the enzyme from  incubated with  by  by SF v i r u s  activity  C y t o s o l from  o f CK  CT  o f 1.25  was  i n which  The  was  (up t o 72  system,  phosphocholine  o f CT  cells  assay  fJH~J  ( F i g . 11,  BHK  a second  formation of  protein  kinetics  by  cells  This a c t i v i t y  exchange a s s a y  in  The  t o 30 m i n u t e s .  nmoleV min trol  + +  -  were s e p a r a t e d by TLC.  mock- o r SF v i r u s - i n f e c t e d (and 10 mM  74  and  time cells  (up t o 40 m i n u t e s ) . was  c o n c e n t r a t i o n , whereas CT  s a t u r a t e d by p h o s p h o c h o l i n e  proportional  s a t u r a t e d by  to  The  assay  Microsomal  phosphocholine  from m o c k - i n f e c t e d  cells  a t a c o n c e n t r a t i o n o f 2 mM  or  - 75 -  o  P r o t e i n , mg  Time,  min  F i g u r e 12. C y t o s o l i c CT a c t i v i t y v s . p r o t e i n and t i m e . CT was a s s a y e d a t 1.25 mM *C~J p h o s p h o c h o l i n e , 3 mM CTP and 12 mM M g . The upper f i g u r e shows CT a c t i v i t y i n r e l a t i o n t o the p r o t e i n i n t h e a s s a y . The l o w e r f i g u r e shows CT a c t i v i t y as a f u n c t i o n o f time o f a s s a y . C y t o s o l from m o c k - i n f e c t e d c e l l s , ( o ) ; from SF v i r u s - i n f e c t e d c e l l s , ( • ) . I  + +  -  76 -  E n z y m e nmoles  m i n  Activity, m g protein  F i g u r e 13. C y t o s o l i c CT a c t i v i t y - v s . p h o s p h o c h o l i n e . CT was a s s a y e d a t 3 mM CTP, 12 mM M g , and 0.31-1.6 mM C~J phosphocholine. B a c k g r o u n d ( b l a n k ) cpm were d e t e r m i n e d a t e a c h concentration of C~J phosphocholine. C y t o s o l from mocki n f e c t e d c e l l s , ( o ) ; from SF v i r u s - i n f e c t e d c e l l s , ( • ) . + +  4  4  -  7 7  -  F i g u r e 14. C y t o s o l i c CT a c t i v i t y v s . CTP. CT was a s s a y e d a t 1.25 mM [_ C~J p h o s p h o c h o l i n e , 12 mM M g , and 0 mM - 16 mM CTP. A b l a n k was used w h i c h c o n t a i n e d no CTP. T h i s b l a n k had a b o u t the same v a l u e as a b o i l e d enzyme b l a n k . C y t o s o l from m o c k - i n f e c t e d c e l l s , ( o ) ; from SF v i r u s - i n f e c t e d c e l l s , (•). l4  + +  i  - 78  higher^  ( F i g . 15).  tration  o f 2 mM  A possible incorporation of  lipid  than  fold  lipid  The BHK  Not  virus  of c y t o s o l i c cells.  one  SF v i r u s - i n f e c t e d inhibition  infection CT  was  ( a t 2.5  the v i r u s - i n f e c t e d  equally,  cells  as e x p e c t e d ,  w i t h SF v i r u s  the m i c r o s o m a l  enzyme, CPT,  vity  because  of s i g n i f i c a n t  inhibition  ties  (Table 6).  activity  Greater  i f the a c t i v i t y  cells.  cells lipid  However,  CT  2.4-  2 . 8 - f o l d i n mic-  by SF v i r u s  inhibition had  in Table  infection.  cells).  were r e d u c e d protein  The  in  o f the s p e c i f i c  of microsomal  CT  a t 2 mM  cytosolic  in total  in infected  CT  activity cells  5.  Micro-  were r e d u c e d  been measured  PC  novo s y n t h e s i s  acti-  activi-  would  be  phosphocho-  c o n c e n t r a t i o n f o r t h e enzyme from  somes o f m o c k - i n f e c t e d  in soluble  further  b u t a l l t h r e e enzymes o f de  and  decrease  level  in infected  a r e summarized  somal CT  s o l i c enzyme, CK,  the  o f the enzymes o f de novo s y n t h e s i s o f  infected  (the s a t u r a t i o n  choline  3  ( F i g . 16).  in total  line  fJ H^]  mg/ml) s t i m u l a t e d c y t o s o l i c about  concen-  microsomes.  i s that  lower  were r e d u c e d  expected  of  s t i m u l a t e CT more i n i n f e c t e d  activities  cells  g r e a t e s t a t a CTP  I f t h i s were t r u e ,  i n c o n t r o l m i c r o s o m e s and  rosomes from  in  after  i n mock-infected  liver  was  e x p l a n a t i o n f o r the  i n t o PC  would  activity  i n b o t h mock- and  activation  activation rat  CT  -  and  micro-  the c y t o -  because  of  the  (Table 4).  2 Two e x p e r i m e n t s were combined t o o b t a i n t h i s r e s u l t f o r mock-infected c e l l s ( F i g . 15). In the f i r s t , m i c r o s o m a l CT a c t i v i t i e s were d e t e r m i n e d a t up t o 1.56 mM p h o s p h o c h o l i n e . In the s e c o n d e x p e r i m e n t , the f i r s t p o i n t ( a t 1.6 mM phosphocholine) was f i t t e d o n t o t h e c u r v e from the f i r s t e x p e r i m e n t by l i n e a r r e g r e s s i o n o f a p l o t o f 1/V v s . 1/S. Further values of a c t i v i t i e s a t h i g h e r p h o s p h o c h o l i n e c o n c e n t r a t i o n s were n o t f i t t e d o n t o the c u r v e , b u t were c a l c u l a t e d as a p r o p o r t i o n o f the f i r s t p o i n t a t 1.6 mM p h o s p h o c h o l i n e .  ">  u  < ® E > N C  Phosphocholine,  mM  F i g u r e 15. M i c r o s o m a l CT a c t i v i t y v s . p h o s p h o c h o l i n e . CT was a s s a y e d a t 2 mM CTP, 3 12 mM M g , and 0-2.8 mM rj C~J- o r £_ ti~\ p h o s p h o c h o l i n e (7.4-12.8 C i / m o l e ) i n m i c r o s o m e s from m o c k - i n f e c t e d (0,0) o r v i r u s - i n f e c t e d (•) c e l l s . CT a c t i v i t i e s a t h i g h phosphoc h o l i n e c o n c e n t r a t i o n s (•) were measured i n a s e p a r a t e e x p e r i m e n t . + +  llt  - 80 -  0  > 0  1  0.5  1  1.0  1  1.5  RAT LIVER LIPID IN ASSAY  "i—  1  2.0 (MG  2.5 ML~')  F i g u r e 16. A c t i v a t i o n o f c y t o s o l i c CT by r a t l i v e r l i p i d . CT was a s s a y e d i n c y t o s o l w h i c h was p r e p a r e d f r o m mock- and SF v i r u s - i n f e c t e d BHK c e l l s a t 6h h o u r s p . i . . Enzyme a c t i v i t y was measured a t 1.6 mM [] H~] p h o s p h o c h o l i n e , .3 mM CTP, and 12 mM Mg . Of t h e 100 u£ o f a s s a y m i x t u r e , an a l i q u o t (40 y&) was s p o t t e d o n t o a TLC p l a t e f o r s e p a r a t i o n o f p h o s p h o c h o l i n e and CDP-choline. A c e t o n e - e x t r a c t e d r a t l i v e r l i p i d was added t o the f i n a l c o n c e n t r a t i o n s w h i c h a r e shown a b o v e . C y t o s o l from m o c k - i n f e c t e d c e l l s , (O); from SF v i r u s - i n f e c t e d c e l l s , ( A ) . 3  + +  -  81  -  Table  5  Enzymes o f De Novo S y n t h e s i s o f PC i n BHK C e l l s I n f e c t e d w i t h SF V i r u s Activities SF  Mock- i n f e c t e d Enzyme  Specific activity  1  Total activity  2  virus-infected  Specific activity  3  0.435  7.3  2  Total activity  3  5.2  CK  0.432  CT (cytosolic)  1.02  17.3  1.02  12.1  CT (microsomal)  1.71  14.6  0.91  4.2  CPT  5.60  47.6  2.40  19.8  1 Enzymes were a s s a y e d i n c y t o s o l o r m i c r o s o m e s from a s i n g l e p r e p a r a t i o n o f mock- and SF v i r u s - i n f e c t e d BHK c e l l s . ~ nmoles m i n mg p r o t e i n ^ nmoles m i n g cells - 1  -  - 1  Table 6 I n h i b i t i o n o f M i c r o s o m a l CT and CPT by SF V i r u s I n f e c t i o n o f BHK C e l l s Activities Enzyme  Mock-infected  1  virus-infected  SF  Significance difference  CT ( m i c r o somal) ^  1.61±0.54(5)  1 . 1 9 ± 0 . 56(5)  p<0.10  CPT  3.32±0.74(5)  2 . 3 7 ± 0 . 23(5)  p<0.025  of  3  nmoles min mg p r o t e i n CT a c t i v i t y was m e a s u r e d a t 1.25 mM p h o s p h o c h o l i n e . S i n c e t h e a c t i v i t i e s were n o t s i g n i f i c a n t l y d i f f e r e n t when t e s t e d by g r o u p c o m p a r i s o n , and s i n c e t h e a c t i v i t i e s were c o v a r i a n t , a p a i r e d c o m p a r i s o n t t e s t was made. S i g n i f i c a n c e was d e t e r m i n e d by a g r o u p c o m p a r i s o n t t e s t 1  2 3  4  1  - 82 -  The  assay c o n d i t i o n s  f o r the t h i r d  enzyme o f de novo  t h e s i s o f PC, namely CPT, have been e s t a b l i s h e d tion  o f CPT by t h e d e t e r g e n t ,  might  r e l i e v e the virus-induced  (0.4%; w/v). T h i s caused  inhibition.  i n f e c t i o n , since  fected  and v i r u s - i n f e c t e d c e l l s  (i.e.,  3.8-fold  tivated tivity 1  and 3 . 7 - f o l d ,  by 0.6% t a u r o c h o l a t e could  CPT i n m i c r o s o m e s  treatment d i d not reverse  by v i r u s  be s e d i m e n t e d  Activa-  i t was r e a s o n e d ,  a t 6h h o u r s p . i . was a c t i v a t e d  from BHK c e l l s  CPT  taurocholate,  (173).  syn-  by  taurocholate  the i n h i b i t i o n o f  t h e enzyme from  was a c t i v a t e d  respectively).  about  mock-inequally  CPT w h i c h was a c -  was n o t s o l u b i l i z e d , s i n c e  the a c -  by c e n t r i f u g a t i o n a t 100,000 x £ f o r  hour. Two  other  enzymes, p e r i p h e r a l  were e x a m i n e d . tivity  was n o t d e t e c t e d  0.19  nmole'x min  ilarly, limits  chondria  had  oxidase  .  o f d e t e c t i o n was  By c o m p a r i s o n ,  activities  o f 0.39 nmole": min  mock-infected  liver  mg p r o t e i n  The l i m i t  the a c t i v i t y i n  m i c r o s o m e s was 2.5 nmoles min ^ mg p r o t e i n  choline  cells  an a c t i v i t y  mg p r o t e i n  and 0.60 nmole;:'\min  line  oxidase a c t i v i t y  uble  r a d i o a c t i v i t y i n fJ H[]  Sim-  i n mitochondria mg p r o t e i n  from  i n mito-  b o t h a t 6h h o u r s p . i . . R a t  (which were s u p p l i e d  o f 0.66 nmoleC> min  ^.  were below t h e d e t e c t i o n  from v i r u s - i n f e c t e d c e l l s ,  mitochondria  ac-  i n m o c k - i n f e c t e d o r v i r u s - i n f e c t e d BHK  microsomes a t 7 hours p . i . .  liver  o f PC,  Phosphatidylethanolamine methyltransferase  cell  rat  t o de novo s y n t h e s i s  1  by M i s s L i n d a  mg p r o t e i n  1  .  Grieve)  Low c h o -  was expected s i n c e most o f t h e w a t e r - s o l 3  choline-labelled cells  was  identified  -  by TLC  as c h o l i n e  section (d)  (£)  83  o r an e s t e r  of choline  (Materials  and  Methods  ( i) ) .  S e p a r a t i o n and  Identification  o f C h o l i n e - C o n t a i n i n g Com-  pounds In and  the measurement o f p o o l s i z e s o f c h o l i n e ,  CDP-choline,  compounds on  the f i r s t  a preparative  pounds s h o u l d be resin  (33).  However,  was  eluted  from AG  Moreover,  to separate  the s e p a r a t i o n  with a l i n e a r in this 1  these  R e p o r t e d l y , t h e t h r e e com-  s e p a r a t e d by column c h r o m a t o g r a p h y  acid  choline  on AG  1  g r a d i e n t o f 0-0.02N  Q "*^]  laboratory,  ( f o r m a t e form) by water  of phosphocholine  and  phospho-  1  alone.  CDP-choline  line,  phosphocholine,  method was  developed  s e p a r a t e d from  t o g r a p h y on AG choline  p u b l i s h e d procedure  and  CDP-choline  for this  was  purpose.  phosphocholine  1 i o n exchange  and  resin.  their  separation  and  yield,  for separating  not  found,  a  CDP-choline  by  4  CDP-  In o r d e r  C]] ^ c h o l i n e ,  p h o s p h o c h o l i n e , or C D P - c h o l i n e  was  traction  cells  (water-methanol-chloroform,  result  o f the s e p a r a t i o n  m i x t u r e o f BHK  1/0.5/0.5; v / v / v ) .  The  phosphocholine,  CDP-choline  compound,  1>  *C  and  radioactivity  the a p p r o p r i a t e f r a c t i o n s  line)  or colums o f c h a r c o a l  added  and  charcoal.  either  choline  chroma-  Phosphocholine on  t o the p r e l i m i n a r y  of  i s shown i n T a b l e 7.  (cpm) from  was  cho-  new  In t h i s method,  were s e p a r a t e d by c h r o m a t o g r a p h y  to monitor  in  was  complete. Because a workable  was  scale.  ( f o r m a t e form) e l u t e d  formic  not  n e c e s s a r y s t e p was  phosphocholine,  recovered  columns o f AG  ( p h o s p h o c h o l i n e and  ex-  choline, For  each  quantitatively 1 resin  (cho-  CDP-choline).  - -84 -  Table 7 Separation  of Q ^ C f ] Choline-Containing  Choline  Phosphocholine  96.0%  3.5%  Phosphocholine  0.6%  97.2%  CDPcholine  1.5%  Choline  rj C"J llt  fJ C~J llf  The method (i) .  (I)  tion  After the  i s described  by  and Methods s e c t i o n  to choline  TLC.  eluted A  2*2  (2)  *6  (3)  91.9%  and Methods  was f o u n d  the h y d r o l y s i s  the hydro-  determined  (from.the h y d r o l y s a t e )  was i n a s i n g l e peak  a s a u t h e n t i c . (~JH~J c h o l i n e  s e c o n d peak, w h i c h e l u t e d  identified  experiment,  t o be 80% c o m p l e t e when  from AG 50W-X8 r e s i n ,  a t t h e same p o i n t  by TLC (Ma-  p h o s p h o c h o l i n e was In t h i s  3  before  choline,  sec-  and C D P - c h o l i n e ,  as c h o l i n e  (£,) ( i ) ) . A l s o ,  Most o f t h e r a d i o a c t i v i t y  was e l u t e d  (1)  enzyme h y d r o l y s i s o f p h o s p h o c h o l i n e  i o n exchange chromatography.  lysis  0.4%  in Materials  product of r a d i o a c t i v e l y l a b e l l e d by  CDPcholine  6. 6%  p r o d u c t o f h y d r o l y s i s was v e r i f i e d  terials  Number o f exper iments  P e r c e n t o f cpm i n e a c h f r a c t i o n  Added compound  ^CfJ  Compounds  which  which  (Fig. 17).  was assumed  t o be  - 85 -  Choline  Fraction  Number  F i g u r e 17. I d e n t i f i c a t i o n o f t h e h y d r o l y t i c p r o d u c t o f phosphocholine. H^J - L a b e l l e d p h o s p h o c h o l i n e from m o c k - i n f e c t e d BHK c e l l s was i s o l a t e d on an AG 1 column ( t h e e l u a n t was e v a p o r a t e d and t h e n d i s s o l v e d i n 5 m l o f w a t e r ) and on a c o l umn o f c h a r c o a l , as u s u a l . P h o s p h o c h o l i n e was h y d r o l y z e d w i t h a b o u t 3 u n i t s o f a l k a l i n e p h o s p h a t a s e i n a t o t a l volume o f 8.2 ml a t pH 9.2. P a r t o f t h e h y d r o l y s a t e was a p p l i e d t o a column (10 cm x 1.5 cm) o f AG 50W-X8 (ammonium f o r m ) . The column was e l u t e d w i t h a l i n e a r g r a d i e n t o f ammonium f o r m a t e (0.1M-0.5M; t o t a l volume, 400 m l ) . A l i q u o t s o f 5 ml f r a c t i o n s o f e l u a n t were c o u n t e d . H cpm were n o t c o r r e c t e d f o r C . ( T C~| p h o s p h o c h o l i n e was added t o t h e c e l l e x t r a c t . ) rj Hj Choline e l u t e d a t the p o s i t i o n o f the arrow. 3  1 4  1 4  3  -  phosphocholine,  since  choline  type of  to  this  Q H[]  Choline,  3  was  again  tested  were made on phocholine, fected  cells.. of  choline  regions,  the  infection, Thus,  i t was  strates  of  PC  choline,  doubled  by  also  poration tion. was  virus  The  PC  not  i n t o CDP-choline  reduced  the  incorporation in a c t i v i t y  SF  phos-  virus-in-  expressed  was  an  as  the  and  CDP-  Q  3  l Q  f o r 30  (10  minutes,  into  r e s u l t s supported  label  flux.  phosphothese uCi  ex-  in 3  choline  was  phosphocholine the  after virus  w h i c h were  could  s t a r t i n g at  whereas  the  the  reduced  choline  into cellular  cells  the  In  unaltered  sub-  Initially,  8.  significantly),  virus  the  into choline,  Incorporation  i n BHK  of  since  in Table  by  accumulate.  enzyme w i t h  i s shown  remained  reduced  cells.  measured  of  dish)  was  concentrations  incorporation  (but  choline,  enzyme would  3  infection.  increased  These  that  were l a b e l l e d w i t h  hours p . i . .  free  Tests  phosphocholine,  [] H]] c h o l i n e  o f medium f o r e a c h l a r g e  6^-7  of  substrate  and  s y s t e m A.  region,  i n i n f e c t e d BHK  of  extraction,  Choline  choline  i n the  than  96.6%.  t o measure  3  cells  choline  substrate  CDP-choline,  each of  a s i n g l e enzyme s t e p  Q H^]  tightly  ion-pair  in solvent  in choline,  3  incorporation  periments,  was  of  TLC pool  Q HQ  synthesis  accumulate The  the  useful  incorporation also  of  less  p o o l s o f mock- and  cpm  averaged  f l u x over then  i s o l a t e d by  i n the  combined  Incorporation If  ml  cpm  binds  (155).  from one  CDP-choline  percent  (e)  the  resin  f o r p u r i t y by  The  -  phosphocholine  w h i c h was  choline and  86  idea  incorinfec-  that  infected  CK  with  Table Incorporation Precursors  of  i n BHK  [] H^] C h o l i n e 3  Cells  Incorporation Mock-infected  co  Choiine  1.10±  Phosphocholine  4.1  CDP-choline  0.78± 0.21  Phosphatidylcholine  26.4  Total  32.4  0.18  8  (3)  ± 2.5 (3) (3)  ±13.1(7)  i n t o PC  Infected with  (dpm x 10  -6  /g  and SF  its  Virus"  cells)  SF v i r u s - i n f e c t e d  Significance of difference  2.28±0.48 (3)  p<0.05  9.5  NS,  p>0.1  0.84±0.33 (3)  NS,  p>0.5  4.8  p<0.005  ±5.4(3) ±4.4(7)  17.4  C e l l s were l a b e l l e d f o r 30 m i n u t e s , s t a r t i n g a t 6%-7 hours p . i . . C h o l i n e c o n t a i n i n g compounds were s e p a r a t e d as d e s c r i b e d i n M a t e r i a l s and Methods s e c t i o n 2 S i g n i f i c a n c e was d e t e r m i n e d by a g r o u p c o m p a r i s o n t t e s t .  (£,)(i).  -  SF  virus, since  creased. take  Over  dilution  PC  was  sult  of  virus  as  E H[]  of  (Table  into  cells.  of  77% ± 23% total  This  r e s u l t implied  SF  the  virus  not  section  Q H^]  choline  3  great  in infected an  re-  of  cells  inhibition  i n f e c t i o n , which  inhibition  a  i n c o r p o r a t i o n -of  54%  u p t a k e by  as  in-  (s.d.) as  only  e x t r a c t o f BHK  choline,  3  was  i n LPC,  g o m y e l i n was  the  than  ac-  cells  r a d i o a c t i v i t y seemed  w h i c h were l a b e l l e d t o be  exclusively  (Materials  (£)  l a b e l l e d by  (i)).  The  lipid  identified  as  LPE,  ( F i g . 18).  or  PE  PC  l a b e l l e d , since  that of  Pool/Size  LPC)  Rf  by  w h i c h was TLC  ( F i g . 18). I t i s not  in this  value  solvent  l e s s than  and  Little  likely  t h a t o f PC  Q H^] 3  or  that  system,  Meth-  no  PC  and  PC  Since  and  incorporation  s i z e of pool  the  s i z e s of  r e s u l t s may  be  (but  great-  Precursors  biased  l a b e l l e d compound, s p e c i f i c PC  and  i t s precursors  by  H  i t migrates  (204).  Measurements - 1.  3  sphin-  i  pool  of  incorporation  p h o s p h o c h o l i n e , or C D P - c h o l i n e  t o a p o s i t i o n w i t h an  (f)  However, the  a methanol-water  Q H[]  choline  er  incorporation  of  section  was  the  was  t h e s e do  (Compare R e s u l t s  average of  was  choline  r e s u l t s s u c h as  for a large part  choline,  ods  intracellular  PC. In  with  8)  choline  3  an  infection.  choline  counted  in  i n h i b i t e d by  in mock-infected  46%  of  i n t o account.  seven e x p e r i m e n t s ,  into  3  labelling  -  However, i n c o r p o r a t i o n  isotope  Q H^]  the  88  a change  in  radioactivities  were m e a s u r e d .  B  15  •  O  10 H  E a o I co  1.0  0.5  K  «f  f  F i g u r e 18. TLC o f Q H j choline-labelled l i p i d . L a b e l l e d l i p i d was i s o l a t e d as described i n M a t e r i a l s and Methods s e c t i o n (2,) ( i ) . P a r t o f the l i p i d was a p p l i e d t o a s i l i c a g e l TLC p l a t e , which was d e v e l o p e d i n s o l v e n t s y s t e m C. PC (R^ 0.13-0.18) was s e p a r a t e d from LPC (R 0.03-0.06), LPE (R 0.19-0.24) , and PE (R 0.46-0.52). T r i - and d i g l y c e r i d e s moved t o the s o l v e n t f r o n t . S e c t i o n s o f 10% o f the s i l i c a g e l l a n e were s c r a p e d o f f and e l u t e d w i t h 7 ml o f c h l o r o f o r m - m e t h a n o l (2/1; v / v ) . The e l u a n t was d r i e d and t h e n c o u n t e d f o r H. L i p i d was from mock- (A) o r SF v i r u s - i n f e c t e d ( B X l e e l l s , 3  f  f  3  f  - 90 -  The  colorimetric  determination of choline  ( 1 9 4 ) , was i n i t i a l l y sults  3  test,  chromatography,  the s p e c i f i c  (which  form  w i t h ammonium  (which  Also,  specific  which  and c h o l i n e  and C D P - c h o l i n e  and McCaman  ured  by t h e method o f R a h e j a  CDP-choline phocholine  by column contaminao f the pur-  and e v a p o r a t e d radioactivity  caused  became  an i n c r e a s e d a b choline.  o f the c h o l i n e - c o n t a i n i n g  com-  The compounds were i s o l a t e d with  which  (195).  3  was d e r i v e d  Phosphoruss  increased, while  radioactivity  from  fJ H^] c h o l i n e f o r 30 m i n -  e t a_L ( 1 9 8 ) .  d e c r e a s e d , as a r e s u l t specific  for-  a column o f AG 1 i n t h e  the s p e c i f i c formate  by  When p a r t  from  were measured e s s e n t i a l l y  o f Goldberg  of choline  purified  determination of standard  (Table 9 ) .  dure  activity  formate.  through  had been l a b e l l e d  Free choline  choline  ammonium  radioactivity  pounds was measured BHK c e l l s  form  t o 584 and 345 dpm/  t o be c a u s e d  s h o u l d remove f o r m a t e )  i n the c o l o r i m e t r i c  The  utes.  appeared  s h o u l d remove ammonia),  sorbance  on AG 50W, ammonium  radioactivity  sample was p a s s e d  dpm/nmole.  o f 1360  by TLC ( i n s o l v e n t  In t h e c a s e o f c h o l i n e  the d e c r e a s e  o f the c h o l i n e choline  radioactivity  w i t h a g r a d i e n t o f 0.IM t o 0.5M ammonium  nmole, r e s p e c t i v e l y .  hydroxyl  was separated., as •  of choline  A) o r by column c h r o m a t o g r a p h y  mate) d e c r e a s e d  672  had a s p e c i f i c  Further p u r i f i c a t i o n  (which was e l u t e d  ified  However, t h e r e -  3  dpm/nmole.  tion  C h o l i n e , which  e t a_L  fJ H^] c h o l i n e - l a b e l l e d BHK c e l l s and t h e n measured  the c o l o r i m e t r i c  system  t o measure c h o l i n e .  CH~J  were v a r i a b l e .  u s u a l , from by  used  of Hayashi  phospho-  by t h e p r o c e i n PC was meas-  The s p e c i f i c  radio-  t h a t o f p h o s p h o c h o l i n e and  of virus  infection.  would be f u r t h e r  (Phos-  decreased,  - 91 -  Table 9 Specific  Radioactivity i n BHK C e l l s  and P o o l S i z e Infected  w i t h SF  Specific  Phosphatidylcholine  Signify icance  (dpm/praole) 9.5+ 3.4(3) 38.9+ 7.5(3) 128 +48 (3) 83 +28 (3) 149 +59 (3) 59.2+ 9.2(3) (dpm/nmole) 7400+1700(7) "1700+1500(7)  Mock-infected  Phosphatidylcholine  1  SF v i r u s - i n f e c t e d  Pool  Choline Phosphocholine CDP-choline  Virus  radioactivity  Mock-infected  Choline Phosphocholine CDP-choline  o f PC and i t s P r e c u r s o r s  p<0.01 NS,p>0.10 p<0.10 p<0.005  size Signify icance  SF v i r u s - i n f e c t e d  (nmoles/g c e l l s ) 146 + 71 68 + 27 (3) + 12 34 (3) 120 + 40 6.,1 + 1 • 0(3) 15. 7+ 5.• (ymoles/g c e l l s ) 3.,4 + 1 • 4 (7) 3. 0+ 1.•  C e l l s were l a b e l l e d w i t h £ H ^ | c h o l i n e L a b e l l i n g began a t 6^-7.hours p . i . . 3  (3) (3) 6 (3)  NS,p>0.1 p<0. 025 p<0 . 05  4 (7)  NS,p>0.5  f o r 30  minutes.  2 S i g n i f i c a n c e o f d i f f e r e n c e was d e t e r m i n e d comparison t t e s t . 3 By a p a i r e d c o m p a r i s o n t t e s t , p was l e s s  by a  group  t h a n 0.1.  o  - 92  except  that  t h e r e was  into phosphocholine activity  -  a nonsignificant ( T a b l e 8).)  o f c h o l i n e was  less  increase in  Curiously,  than  incorporation  the s p e c i f i c  radio-  that of phosphocholine.  (See  Discussion.) V a l u e s o f p o o l s i z e s were c a l c u l a t e d total  3  H  dpm  separation lipid  by  sults  ( b ) , the s p e c i f i c  Pool sizes  a r e shown  i n T a b l e 9 and  t h o s e o f T a b l e 8.  line  predominated  was  phosphocholine,or  Of  was  line  were made g r e a t e r , and  choline over of  F i g u r e 19.  ( T a b l e 9,  (and C D P - c h o l i n e )  the CT  while  F i g . 19). likely  The  cells,  respectively).  Diglyceride w h i c h were used the p o o l s i z e  was  (i.e.,  of diglyceride At  from  186  i n some o f  and  the At  cho-  pool of and  of  cho-  CDP-choSF  phospho-  reduced-activity The  very s i m i l a r  f o r measurement o f PC.  (s.d.) n m o l e s / g c e l l s .  re-  u n a f f e c t e d by  (See D i s c u s s i o n . )  cells  measured  was  Accumulation  resulted  a l l t h r e e w a t e r - s o l u b l e p o o l s was virus-infected  The  phosphocholine  cells.  t h e p o o l o f PC  (and C P T ) . s t e p s .  and  com-  experiments  made s m a l l e r , the p o o l s o f p h o s p h o c h o l i n e  fected  16  of each  the same  cells  in virus-infected  line  infection  ( i n the  t h e t h r e e w a t e r - s o l u b l e compounds,  in mock-infected  the most a b u n d a n t  virus  i n T a b l e 9 and  (after  o r PC  radioactivity  F i g u r e 19 were from  ( a ) , the  CDP-choline  i o n exchange or c h a r c o a l c o l u m n s ) ,  e x t r a c t ) by  pound.  as  in choline,  by d i v i d i n g  204  total  amount  i n mock-innmoles/g  same l i p i d  extracts  7 t o 7% h o u r s p . i . ,  in mock-infected  BHK  t h e same t i m e ,  i n SF  cells  was  47±  virus-infected  -  93  -  Nanomoles / 5 15 cm. dishes of cells o  00  o  o  -J  o 7 0 3 It  o 7  0 3 9  u 7  0 (A •o  o 0 I  n  ]  ID =5 5  7 0 3 ID  si ID  1  Micromoles / 5 15 cm. dishes of cells Ul  to ©  io in  ?  I  1  u  a  7 0  at  0 7 = V 3 2 9  a  F i g u r e 19. P o o l s i z e o f PC and i t s p r e c u r s o r s i n BHK c e l l s i n f e c t e d w i t h SF v i r u s . C e l l s were l a b e l l e d and compounds were s e p a r a t e d as d e s c r i b e d i n T a b l e 8. The e r r o r b a r s r e p r e s e n t s t a n d a r d e r r o r s o f t h e mean. R e s u l t s are expressed per 5 l a r g e d i s h e s o f BHK c e l l s . C h o l i n e p h o s p h a t e i s synonymous w i t h p h o s p h o c h o line.  94  cells, two  the p o o l  values  values t  d e n t methods. (Table  The  10).  (See  results The  reduced  (p>0.5, g r o u p could  not  these  The  in size  10)  by  two  i n BHK  size  was  by  the  phosphocho-  cells  not  agree-  caused  e x t r a c t i o n of  virus infection  nucleotide pool  indepen-  determina-  were p r o b a b l y  before  SF  caused  methods were i n f a i r  p o o l o f CTP  by  comparison  have  d i f f e r e n c e s i n phosphocholine (Table  two  synthesis.  were e a c h measured of  The  was  (Table limited  sig-  10). to  CTP  (Results section (g)). Pool  S i z e Measurements - 2.  mock-infected  (v).  CTP  Discussion.)  Nucleotide  the  and  These  5 determinations.  p e r t u r b a t i o n s o f PC  However, a s m a l l e r  (g)  nmoles/g c e l l s .  different  incubations of c e l l s  nificantly  alone  average of  w h i c h were o b s e r v e d  different line.  (s.d.)  Thus, d i g l y c e r i d e c o n c e n t r a t i o n  ' Phosphocholine  tions  43±17  significantly  virus-mediated  ment  was  were e a c h an  were n o t  test).  any  size  -  first  concentrations  and  SF  extraction  were t a k e n  20  to each d i s h o f c e l l s wash, b u t  from  and  before  21.  removal of of  the  high  column was  The  recovery  The  results  the  concentration  tration.  incubator  i n the  was  this  cells  77%  Methods s e c t i o n  a t 6h  o f one  dish)  i n 10%  by (£)  hours p . i . f o r experiment ITP  was  after  the  are added PBS  perchloric acid.  sample w h i c h was  compared w i t h  e x t r a c t s of  were measured  experiment,  nmoles/large  The  pressure  cells  the  In  (23.5  ITP  BHK  i n M a t e r i a l s and  of n u c l e o t i d e s .  shown i n F i g u r e s  in perchloric acid  virus-infected  method d e s c r i b e d  Cells  Nucleotides  the  injected  expected  f o r e x t r a c t s from b o t h  onto  concen-  mock-  -  95  Table  -  10  Pool Sizes of C y t i d y l y l t r a n s f e r a s e Substrates i n BHK C e l l s I n f e c t e d w i t h SF V i r u s  Pool  size  Mock-infected SF v i r u s - i n f e c t e d (nmoles/g c e l l s )  Significance-^f difference  Phosphor choline^  34±12(3)  120±40(3)  p<0.025  64± 7(3)  147±37(3)  p<0.02  Phosphocholine-  3  CTP  3  149± 7(3)  79±16(3)  p<0.005  CTP  4  116±35(3)  36±13(3)  p<0.025  S i g n i f i c a n c e was d e t e r m i n e d by a g r o u p c o m p a r i s o n t t e s t . 2 P o o l s i z e s were d e t e r m i n e d by t h e method m o d i f i e d from G o l d b e r g and McCaman ( 1 9 5 ) . C e l l s were t a k e n from t h e i n c u b a t o r ( f o r e x t r a c t i o n ) a t 7-7% h o u r s p . i . . 3 P o o l s i z e s were d e t e r m i n e d by t h e method o f Choy e t a l (200). C e l l s were t a k e n a t 6-1/2 - 6-5/6 h o u r s p . i . . ^ P o o l s i z e s were d e t e r m i n e d by h i g h p r e s s u r e l i q u i d chromatography. C e l l s were t a k e n a t 6h h o u r s p . i . .  F i g u r e 21. Separation of i n u c l e o t i d e s from SF v i r u s - i n ] f e c t e d BHK c e l l s . See t e x t f o r • 'details. 1, ADP; 2, GDP; 3, CTP 4, UTP; 5, ATP; 6, ITP ( i n t e r n a l s t a n d a r d ) ; 7, GTP. j  :l=rh  SF VIRUS  -  infected  of  to the d i s h e s ,  recovery.  were  The p o o l  i n f e c t e d with  the p y r i m i d i n e  nucleotide  value.  Similarly,  value.  were  increased,  nucleotide  equally,  equally  t o about  in viral  smaller  w h i c h were t h e r e s u l t  Since cells (205), The  12).  260  was This  nm  (or 280  abolished  nm)  by t r e a t m e n t w i t h  otides  Thus, v e r y leaked  little  by v i r u s  monitored  (5% o f t h e p o o l  i n absorbance infection, acid  (or p r o t e i n s )  o f the i n t r a c e l l u l a r cells.  SF v i r u s  the absorbance a t  trichloroacetic  o u t o f the i n f e c t e d  out of  leak out.  was  The i n c r e a s e  removed v i r u s  f o r the  to leak  a l s o might  w h i c h was c a u s e d  treatment presumably  medium.  shown  of infected c e l l s  by 0 . 0 5 1 .  nucleotide  infection.  A l e a k a g e o f 10 nmoles o f A T P / d i s h  nm o f 3 ml o f medium  o f the con-  be t h e r e a s o n  would have i n c r e a s e d  ATP  diphosphates  i n f e c t e d with  that nucleotides  virus-infected cells)  260 at  thought  w h i c h were  were  o f the c o n t r o l  nucleotide  of v i r u s  which  amounts  CTP and UTP,  three-fifths  s y n t h e s i s may  a b s o r b a n c e o f t h e medium  (Table in  i t was  The  triphosphates,  l a c t a t e d e h y d r o g e n a s e has been  ( c h i c k embryo c e l l s )  cells  11.  ( T a b l e 11) > Use o f  so  triphosphates pools  RNA  i n BHK  t o about o n e - t h i r d  In c o n t r a s t , b o t h p u r i n e again  were c o r r e c t e d f o r  i n Table  triphosphates,  both p u r i n e  GTP, were r e d u c e d  e x p e r i m e n t s , ITP  and t h e r e s u l t s  SF v i r u s a r e shown  by v i r u s i n f e c t i o n  trol  In o t h e r  sizes of nucleotides  reduced  and  -  and v i r u s - i n f e c t e d c e l l s .  was n o t added 77%  98  pools  (Table  12).  i n the of nucle-  -  99 -  Table Pool  11  S i z e s o f N u c l e o t i d e s i n BHK I n f e c t e d w i t h SF V i r u s  Pool  Cells  size"*"  MockSF v i r u s infected infected (nmoles/g c e l l s )  Ratio of pool sizes (virus/mock)  Significance of difference 2  CTP  116±135  36+. 13  0.30  p<0.05  UTP  213±  76±  34  0.35  p<0 . 05  1080±320  0.60  p<0 .05  77  1800±520  ATP GTP  213±  67  122±  42  0.57  p<0 . 05  ADP  405±  99  654±100  1.65  p<0.02  GDP  61±  21  1.69  p<0.10  97±  21  R e s u l t s were an a v e r a g e o f t h r e e d e t e r m i n a t i o n s . Cells were t a k e n f o r e x t r a c t i o n a t 6h h o u r s p . i . . S i n c e measurements were c o v a r i a n t , s i g n i f i c a n c e was d e t e r m i n e d by a p a i r e d c o m p a r i s o n t t e s t . 1  2  - 100 -  Table  12  A b s o r b a n c e o f the Medium o f BHK I n f e c t e d w i t h SF V i r u s  Cells  Absorbance 280nm  BSS-2% DCS BSS-2% DCS  from m o c k - i n f e c t e d  BSS-2% DCS  from  cells  SF v i r u s - i n f e c t e d  1  cells  1  260nm  1.689  1.129  1.688  1.212  1.701  1.243  0.210  1.198  0.194  1.199  2 After  t r i c h l o r o a c e t i c acid  treatment:  Supernatant  from m o c k - i n f e c t e d  Supernatant medium  from  cell  SF v i r u s - i n f e c t e d  medium cell  L a r g e d i s h e s o f c e l l s were i n f e c t e d as u s u a l . At 6-3/4 h o u r s p . i . , the c e l l s were washed t w i c e w i t h 3 ml o f E a r l e ' s B a l a n c e d S a l t S o l u t i o n , 2% d i a l y z e d c a l f serum (BSS-2% DCS). E a c h d i s h o f c e l l s was i n c u b a t e d w i t h 3 ml o f the same medium f o r 30 m i n u t e s a t 37 . The medium was t h e n removed and c e n t r i f u g e d (6,ooo x c[, 10 m i n u t e s ) . The a b s o r b a n c e o f t h e s u p e r n a t a n t was m e a s u r e d . A f t e r c e n t r i f u g a t i o n o f the medium, an a l i q u o t (0.75 ml) was mixed w i t h 40% t r i c h l o r o a c e t i c a c i d (0.25 m l ) . The m i x t u r e was c e n t r i f u g e d (6,000 x £ , 10 m i n u t e s ) . The a b s o r b a n c e o f the s u p e r n a t a n t was m e a s u r e d .  -  (h)  101 -  Time Sequence o f N u c l e o t i d e  Pool  S i z e s During  SF V i r u s  Infection Since virus  b o t h CTP and ATP p o o l s  infection,  of nucleotide  pool  concentrations on  CT o r CK.  i t was d e c i d e d  infection.  c o n t r o l PC s y n t h e s i s  The t i m e s e q u e n c e o f Q H 2 ]  were measured  Methods s e c t i o n  by SF sequence  E i t h e r CTP o r ATP through  choline  3  i n t o PC has been examined p r e v i o u s l y trations  in size  t o i n v e s t i g a t e t h e time  sizes during  could  were d e c r e a s e d  (173).  their  effects  incorporation  Nucleotide  concen-  by t h e s e c o n d method o f M a t e r i a l s and  (£) ( v ) . The r e s u l t s  are presented  i n Figures  22 and 23. The  pools  o f CTP and UTP i n c r e a s e d  hours p . i . i n mock-infected infected time The in all  cells  (Fig. 22).  i n both mock-infected pool  size  nucleotide  r e a d y been c a u s e d of  2\  changes  over  this  (Fig. 22).  t h e same a t 2h o r 7% h o u r s p . i .  some r e d u c t i o n  by v i r u s i n f e c t i o n ,  (Fig. 23).  i n pool  size  a t the e a r l i e s t  i n any o f the n u c l e o t i d e  times p . i . ( F i g s . choline  22, 2 3 ) , do n o t match  incorporation into l i p i d  virus-infected cells.  since  in virus-  in size  and v i r u s - i n f e c t e d c e l l s  and v i r u s - i n f e c t e d c e l l s pools,  static  In  had a l time  point  hours p . i . . The  and  increased  2h t o lh  from  but remained  GTP p o o l s  o f ATP was a b o u t  both mock-infected four  cells,  in size  labelling  sizes at various  the changes f o r both  However, t h i s  does n o t r e f l e c t  (Results section ( j ) ) .  (173),  pool  the true  i n Q H^] 3  mock-infected  i s o f minor  importance  rate of synthesis  -  102 -  0 -I  1  1  1  -r-  1  r  1  2  3  4  5  6  7  T i m e , hours  post-infection  Figure 22. E f f e c t o f d u r a t i o n o f i n f e c t i o n on CTP, UTP, and GTP p o o l s i z e s . BHK c e l l s were i n f e c t e d w i t h SF v i r u s as usual. N u c l e o t i d e s were measured by t h e s e c o n d method o f M a t e r i a l s and Methods s e c t i o n (£) ( v ) . R e s u l t s a r e e x p r e s s e d i n nmoles p e r l a r g e d i s h o f c e l l s . P o o l s from m o c k - i n f e c t e d c e l l s a r e : CTP, ( o ) ; UTP, (•); GTP, ( A ) . The s o l i d symbols r e p r e s e n t t h e e q u i v a l e n t p o o l s i n SF v i r u s - i n f e c t e d c e l l s .  -  103 -  300 (0  •5 0)  a  tn 200] o  o  E  c  XL  <  100  2  3  4  5  6  Time, hours post-infection F i g u r e 23. E f f e c t o f d u r a t i o n o f i n f e c t i o n on ATP p o o l size. The e x p e r i m e n t was p e r f o r m e d and r e s u l t s a r e e x p r e s s e d as n o t e d i n F i g u r e 21. ATP from m o c k - i n f e c t e d c e l l s , ( o ) ; from SF v i r u s - i n f e c t e d c e l l s , (•).  -  The  cells  were n o t q u i t e cells then in  w h i c h were used confluent.  i n t h e time  However,  sequence  i f i t was  w e i g h e d t h e same as c o n f l u e n t  cells  fair  agreement (Table  standard  w h i c h was  used  w h i c h was  Comparison o f N u c l e o t i d e  t h e c h r o m a t o g r a p h y c o l u m n , and areas.  Results  by Two  result  Procedures  2  3  3 SF v i r u s - i n f e c t e d  CTP  129%  95%  UTP  136%  81%  ATP  112%  102%  GTP  145% ,  134%  1  tion  P r o c e d u r e s a r e d e s c r i b e d i n M a t e r i a l s and Methods s e c (v) . P e r c e n t a g e s a r e : r e s u l t s by s e c o n d method r e s u l t s by method x 100. C e l l s were t a k e n f o r e x t r a c t i o n a t 6h h o u r s p . i . .  (&) 2  first 3  i n the  13  Percent Mock-infected  i n the  nucleotides  They a l s o d i f f e r e d  t h e method o f measurement o f peak Table  dish),  and i n t h e t y p e o f  t o c a l c u l a t e amounts o f  to elute  these  a n a l y s i s were  The two methods d i f f e r e d  standards).  used  that  (0.18 g / l a r g e  f o r e x t r a c t i o n ,~pf n u c l e o t i d e s ,  ( e x t e r n a l or i n t e r n a l gradient  13).  experiment  assumed  t h e r e s u l t s by t h e two methods o f n u c l e o t i d e  procedure  in  104 -  -  (i)  Fatty  Acid  w i t h SF The after in its  rate  Lipids  tion  of  Infected  fJ H]] c h o l i n e  of  some p a r t i c u l a r  synthesis  of  PC  and  Consequently,  diglyceride to  Q H]]  incorporation  with a  the  i n t o PE  PC  change  species of  PC  from  fatty acid  com-  were m e a s u r e d .  choline  3  into  3  have been a s s o c i a t e d  (Similar  anolamine  Cells  i n f e c t i o n could  of  analyzed.  from BHK  incorporation  precursor, diglyceride.  positions  Also,  PE  incorporation,  i s i n h i b i t e d by  was  **C]]  SF  virus  eth-  infec-  (174).) For  fatty acid  analysis,  hours p . i . , at which cubator  and  section  (m)).  infected similar sition  o f PC  in  fatty acid  synthesis  and  PE, PE  cells  were i n f e c t e d  from BHK  and  cells  The  from  in Materials  and  after  PC  species of was  similar  to  of  was  virus  indicated PC  the  from d i g l y c e r i d e  p r o b a b l y not  greatly  was  that  very compo-  of  a  apparently (However,  Lack o f that  virus-  The  infection.  in diglyceride.)  composition of various  was  in-  from  cells 15).  18:2  the  7  Methods  lipids  ( T a b l e s 14,  proportion  PE  until  were removed  composition of  diglyceride  (163).  reacylation)  infection.  or  change o c c u r r e d  of  cells  (as d e s c r i b e d  fatty acid  i n b o t h PC  similar  BHK  i n comparison with mock-infected  report  no  by  The  f o r PC,  decreased  time the  processed  cells  previous  sis  of  -  Virus  i n h i b i t i o n of  virus  the  Analysis  105  a  change  relative (or  affected  by  synthevirus  - 106 -  Table  14  P e r c e n t D i s t r i b u t i o n o f F a t t y A c i d s i n PC and PE from BHK C e l l s I n f e c t e d w i t h SF V i r u s  Fatty  acid  PE •  PC Mockinfected  SF v i r u s infected  Mockinfected  SF v i r u s infected  (% o f t o t a l )  (% o f t o t a l )  nd  0.8  14:0  0.8  0.8  16:0  18.3  19.6  6.7:  7.4,;  16:1  9.1  9.8  3.1..  3.8  18 : 0  7.0  7.1  20.3  20.6  18 :1  50 . 5  51.8  51.0  47.1  18:2  4.5  2.8  5.1  2.0  Other  9.7  8.0  13 .8  18 .3  (9.1)  (8.8)  (Other>18:3)  1 2 longer  (1.4)  (nd)  not d e t e c t a b l e This line represents fatty t h a n t h a t o f 18:3.  acids with  1  r e t e n t i o n times  - 107  Table  -  15  Percent D i s t r i b u t i o n of Fatty Acids i n Diglyceride from BHK C e l l s I n f e c t e d w i t h SF V i r u s  Fatty Acid  Mock-infected  SF v i r u s - i n f e c t e d  (% o f t o t a l )  14 : 0  10.2  10.9  16:0  14 . 7  19.9  16:1  3.2  3.1  18 :0  32.7  32.0  18:1  26.5  20.5  18:2  1.1  1.3  18 : 3  0.7  0.4  20:4  0.6  nd  11.7  10.4  Other (Other>18 : 3 )  „ longer  2  (10.0)  (8.2)  not detectable This l i n e represents t h a n t h a t o f 18:3.  1  fatty  acids  with  retention  times  - 108 -  (j)  Pulse-Chase SF  of  pearance In e a c h  of  Q H~J 3  i n t o PC r e l a t e d  3  choline  from  [] H^] c h o l i n e  i n phosphocholine  the r a d i o a c t i v i t y  decreased  i s a p r e c u r s o r s : o f PC.  Such a c o n v e r s e  amount o f r a d i o a c t i v i t y  in choline  than  that of phosphocholine.  choline  and C D P - c h o l i n e -, u n l i k e  a r e shown Over choline cells,  of chase.  the chase  In e a c h  indicated  experiment,  and C D P - c h o l i n e  that  the  was much  small-  The amount o f r a d i o a c t i v i t y i n phosphocholine,  did.not vary  o f one o f t h e s e  the d i s a p p e a r a n c e  f o r the appearance of H  from  3  of H 3  i n PC; i n v i r u s - i n f e c t e d  I n c o r p o r a t i o n o f r~_H^] c h o l i n e  bited  by SF v i r u s  3  o f Q H~J  24).  3  infection.  much  experiments  experiment,  since  3  i n PC.  cells  into  from  phospho-  In m o c k - i n f e c t e d was 106% o f  i t was 148% ( F i g .  lipid  (PC) was  inhi-  C o n c u r r e n t l y , the r a t e o f d i s a p -  phosphocholine  Inhibition  of H  phosphocholine  24).  this  relationship  The r e s u l t s  period,  the disappearance  the a p p e a r a n c e  (Fig.  d u r i n g the chase  i n F i g . 24.  accounted  pearance  experiments,  i n PC i n c r e a s e d i n b o t h m o c k - i n f e c t e d and  phosphocholine  60 m i n u t e s  how  t o the d i s a p -  pulse-chase  3  cells.  over  with  t h e w a t e r - s o l u b l e p r e c u r s o r s o f PC.  virus-infected  er  Infected  were done t o show more p r e c i s e l y  o f [] Hy] c h o l i n e  of three separate  radioactivity  while  C h o l i n e i n BHK C e l l s  experiments  incorporation  the  $  Virus  Pulse-chase the  \2' lQ  o f fJ H~J 3  the t o t a l  was  reduced  choline  by v i r u s  uptake  water-soluble  infection  d i d not occur i n radioactivity  -  109  Time  -  of  Chase,  min  F i g u r e 24. P u l s e c h a s e o f Q H^[ c h o l i n e i n BHK c e l l s i n f e c t e d w i t h SF V i r u s . BHK c e l l s w h i c h were n o t q u i t e confluent were i n f e c t e d w i t h SF v i r u s as u s u a l . The c e l l s were l a b e l l e d w i t h Q H]] c h o l i n e i n a 15 m i n u t e p u l s e w h i c h began a t 6h h o u r s p.i.. B o t h p u l s e and c h a s e m e d i a were Medium 199 (and 2% f e t a l c a l f serum). The r e s u l t s a r e e x p r e s s e d i n dpm per two large dishes of c e l l s . R a d i o a c t i v i t y ( H) was measured i n : choline (A), p h o s p h o c h o l i n e (o), C D P - c h o l i n e (V), and PC (•) from m o c k - i n f e c t e d cells. The s o l i d s y m b o l s r e p r e s e n t r e s u l t s from SF v i r u s - i n fected c e l l s . 3  3  3  - 110  (choline at  plus phosphocholine  -  plus CDP-choline)  the b e g i n n i n g o f the chase,  cells  between  rate of disappearance 20  and  60 m i n u t e s  the pool size !  iment  :  between  7 hours and  in mock-infected  about  or  the  same,  virus-infected  ( F i g . 24). The  that  was  20  the h a l f - l i f e  fractional turned  of chase  60 m i n u t e s  p.i.),  then  per u n i t  o f time  c o u l d be  the  The  in this  exper-  50 m i n u t e s  -  turnover rate calculated.  fraction  (206) .)  exponential  Were i t assumed  (6 h o u r s  the f r a c t i o n a l  of phosphocholine  was  d i d not vary  o f chase  turnover r a t e denotes  over  ( F i g . 24).  of phosphocholine  and  30 m i n u t e s  of phosphocholine  (k)  (The  o f a p o o l which i s  v a l u e o f k was  calcu-  -kt lated  by  activity  the e q u a t i o n : R ^ / R Q = e ( H dpm).  R Q was  3  utes of chase) 60 m i n u t e s  and  of chase).  The  phosphocholine  two  measurements,  u t e s was (s.d.)  pool 6-5/6  42.4+4.2  in c e l l s  The size  (206) .  the i n i t i a l  The  time  interval  half-life  infected.with  SF  r e p r e s e n t e d by (206).  cells  and  q, e q u a l l e d kB,  were used  1.51  t.  Thus, f o r i n min-  67.4±3.0  where B was  I f the p o o l s i z e s o f p h o s p h o c h o l i n e  and  or  virus.  (or f l u x ) ,  rate of phosphocholine  min-  ( a t 40  of phosphocholine  i n mock-infected  p . i . ( T a b l e 10)  was  in mock-infected  cells  infected  If phosphocholine  cells.  was  radio-  ( a t 20  readioactivity  e q u a l l e d 0.693/k  the a v e r a g e (s.d.)  R represented  radioactivity  the s u b s e q u e n t  half-riife  turnover rate  hours  turnover  R ^ was  (206).  nmoles min  nmoles min was  a t 6-1/2-  as v a l u e s o f B,  1.05  g cells  the  then  the  ^ g cells 1  ^  in virus-  o n l y c o n v e r t e d j t o PC,  which  - Ill -  was  assumed to be the case, then t h i s r a t e a l s o represented  r a t e of s y n t h e s i s of PC from phosphocholine  the  i n these c e l l s .  Thus,  i n h i b i t i o n of l a b e l l i n g of PC d i d not mean that s y n t h e s i s of PC was  inhibited. I t should be noted  that the f l u x represents s y n t h e s i s of PC  and not turnover of PC, ing  s i n c e the c e l l s may  (although the c e l l s were incubated  have s t i l l  been grow-  i n a maintenance medium  r a t h e r than a growth medium, f o r galsercHase experiments) .  In  c e l l s which are growing, the r a t e of s y n t h e s i s of PC would  ex-  ceed  the rate, of degradation,. The  values of the f r a c t i o n a l turnover r a t e  phosphocholine,  and CDP-choline  of k f o r phosphocholine" in t h i s s e c t i o n . t h e s i s of PC was  considered^constant,  hours p . i . ) .  k was  fection. cells).  (as. a  e a s i l y calculated for  from the equation, k = q  B.  The  pool  were from Table 9  As can be seen i n Table 16,  c h o l i n e .and CDP-choline,  Values  (q) over each step of de novo syn-  (B) values f o r c h o l i n e and CDP-choline  (at 7-7%  i n Table 16.  were c a l c u l a t e d as d e s c r i b e d p r e v i o u s l y  Since the f l u x  c h o l i n e and CDP-choline size  are shown  (k) f o r c h o l i n e ,  the value of k i s reduced  f o r phosphoby v i r u s in^-  consequence of the l a r g e r p o o l s i z e s i n i n f e c t e d  For c h o l i n e , the value of k i s a p p a r e n t l y i n c r e a s e d by  infection.  However, not a l l of the c h o l i n e p o o l may  PC s y n t h e s i s . l i n e pool was  (See D i s c u s s i o n . ) estimated  be a c t i v e i n  A h y p o t h e t i c a l " a c t i v e " cho-  as the pool s i z e of c h o l i n e which would  have the same s p e c i f i c r a d i o a c t i v i t y as phosphocholine.  For  this  -  pool, this  112 -  t h e v a l u e o f k was a l s o pool  was l a r g e r  reduced  in infected  by v i r u s  infection  (since  cells).  T a b l e 16 F r a c t i o n a l T u r n o v e r R a t e s o f PC P r e c u r s o r s BHK C e l l s I n f e c t e d w i t h SF V i r u s  in  1  k, min Mock-infected  SF v i r u s - i n f e c t e d  0.0072  0.022  0.097  0.047  Phosphocholine  0.016  0.010  CDP-choline  0.17  0.096  Choline "Active"  choline  2  V a l u e s o f k were b a s e d on t h e r a t e o f d e c a y o f Q H^] p h o s p h o c h o l i n e ( F i g . 24) and on p o o l s i z e s ( T a b l e s 9, 10) w h i c h were b o t h measured between 6h and lh h o u r s p . i . . The p o o l s i z e o f " a c t i v e " c h o l i n e was t h e amount o f c h o l i n e w h i c h would be n e c e s s a r y t o d i l u t e t h e Q H]] c h o l i n e i n t h e c e l l t o t h e same s p e c i f i c r a d i o a c t i v i t y as p h o s p h o c h o l i n e (Table 9). T h i s p o o l o f c h o l i n e i s 11 (and 32) n m o l e s / g c e l l s i n mocki n f e c t e d c e l l s (arid v i r u s - i n f e c t e d c e l l s ) . 3  2  3  -  113  -  DISCUSSION  (a)  Does SF  Virus  i n BHK  of  SF  virus  fJ H~J  h y p o t h e s i s was  fected  Is SF  r a t e or  turnover  flux,  section  of  q,  The  equals  PC  fJ H~J  phos-  3  so than  Consequently, SF  rate of  kB.  in infected c e l l s  i n h i b i t e d by  initial  rate of  i s much h i g h e r ,  (j)).  incorpora-  ( 1 7 3 ) , our  inhibited.  infection, B  was  section  the  explained. fJ H~J  f r a c t i o n of  rjH~J 3  of  the  Although that  the  mock-inrate  v i r u s . i n f e c t i o n but  of may  3  for  i n t o PC by of  Choline  i n t o PC  SF  the  (i).)  The  inhibition  virus  infection.  rjH~J 3  I n h i b i t i o n of  phosphocholine  thesis  i s i n h i b i t e d by  uptake of  (c)  present  parallels  an  project. average  Such  choline.  inhibi(See  incorporation  can  be  an  of  dis-  ( F i g . 24).  inhibition In o t h e r  rj H]] p h o s p h o c h o l i n e w h i c h t u r n e d 3  Inhibited  Cells?  basis  (e))  inhibition  section  appearance of  the  incorporation  (Results  Discussion  the  was  I n f e c t i o n o f BHK  question  exceeds  simply  cells  to the  Incorporation  Virus  3  tion  equal  i s not  the  [] H^] C h o l i n e 77%  Synthesis  stimulated.  This  of  synthesis  turnover  (Results  o f PC  be  Why by  PC  o f BHK  r a t e : i s a c t u a l l y higher  synthesis  (b)  i n t o PC  after virus  cells  indeed  that  This  i s lower  turnover  inhibition  i s considered  phocholine. k  i n f e c t i o n c a u s e s an  choline  3  synthesis  Rate o f PC  Cells?  Since tion  I n f e c t i o n A f f e c t the  over  words, per  unit  -  of  time  pared  to  10).  (See  the  i s 2.3  control cells  Discussion  i s formed  pool  phosphocholine of rather  inhibition  of  than  (i)  the  (ii).)  cell,  of  PC  E f f e c t s of  SF  Virus  mechanism o f  inhibition  of  i n BHK  process  ( F i g . 8 ) , which  suggests  that  uptake of  that  smaller  choline  i n the  hibition  two  host  since  a K^  kinase  no  host  3  (Table  H^J  phospho-  the  larger  dilution  synthesis,  of  i s the  I n f e c t i o n on  the  cause  Each  cells i t is  is a  of  Step  r e a c t i o n , of are  not by  SF  v i r u s p a r t i c l e s are  Furthermore,  since  (178,  179,  may  cause  110  virus  the  virion  ( 2 0 8 ) , a mechanism  extruded 207), an  un-  saturable  t o a K^  yM , r  linked.  SF  is  carrier-mediated.  a choline-specific transport  infected cells  proteins  transport  17 yM,. compared  synthesis  p l a s m a membrane o r g a n i z a t i o n uptake.  of  activities  protein  amount o f  membranes o f  has  choline  these  of  natively,  The  choline  uptake  gests  Q  com-  Transport  However, c h o l i n e  choline  Thus,  i s d i l u t e d by  known.  The  hours p . i .  infected c e l l s .  inhibition  of  Synthesis? Choline  The  i n the  size  in virus-infected c e l l s  6-1/21- 6-5/6 (f)  pool  incorporation.  What C a u s e s o f PC  at  section  which  isotope,  However, the  times l a r g e r  choline  (c)  -  i s less in infected c e l l s .  phosphocholine  of  114  which  sug-  Perhaps i n results^in protein. from  a Alter-  the  plasma  d i s r u p t i o n of  inhibition  lipid  for  of  envelope  for excluding  such  the  choline  contains proteins  3 The K v a l u e was o b t a i n e d by l i n e a r r e g r e s s i o n o f 1/V as a f u n c t i o n o f 1/S. The d a t a used were from F i g . 10 (from mocki n f e c t e d and v i r u s - i n f e c t e d c e l l s ) a t c h o l i n e c o n c e n t r a t i o n s of 0.05 t o 0.5 mM and 1.25 mM.  -  is  necessary.  protein  and  possibility caused  by  Whatever  lipid  of  the of  i t too  could  p l a s m a membrane. choline  area of  could  be  surface  At  virus  cells. as  7  usually  Such  area  the  final  infected c e l l s .  i n shape t h a n m o c k - i n f e c t e d a decreased  A  transport  w h i c h were i n f e c t e d w i t h SF  reflect  disrupt  a  a result  virus infection. Transport  experiments less  than  8).  The  line  19%  higher  i n the  i n the  SF  level  incomplete  medium w i l l  higher  inhibition  inhibition  does not  occur  experiment  between  the  dialyzed  the  two  serum was  whereas E a r l e ' s  of  i n SF  pulse-chase  the  of  experiments in  dialyzed  cells.  inhibition  (Fig.  these  of  calf  choline  cho-  transport  p r e s e n t work e x p l a i n s i n t o PC  choserum  Additional  Perhaps a g r e a t e r  incorporation  here,  inhithe com-  (173).  r H^j 3  choline  uptake  virus-infected cells  (Results  section  (j)).  (Results of One  i s t h a t Medium 199  medium f o r  Balanced  inhibition  i s observed  incompletely  i n the  experiments the  which  (Fig. 8).  choline  an  incorporation  a t t r i b u t a b l e to a d d i t i o n a l  increase  to previous experiments Oddly,  in three  transport  washing of  transport of  be  from  virus-infected cells choline  from  inhibition  medium, e i t h e r  of  (b))  expected of  56%  (b)), although  e x p e r i m e n t s may  bition  pared  averages  section  m i g h t be  because of  line  inhibition  (Results  incorporation  in  mechanism,  in surface  cells  shape change c o u l d  or  -  inhibition  a reduction  were more r o u n d e d  of  the  structure  i s that  h o u r s p . i . , BHK  115  the  Salt Solution  pulse-chase  section  the difference with  un-  experiment,  with d i a l y z e d  serum  was  -  used of  i n the t r a n s p o r t  t h e media may  116 -  experiments.  explain  why  Some f a c t o r p r e s e n t  transport  inhibition  i n one  i s not  repro-  ducible . (ii)  Choline  Because  t h e amount o f c y t o s o l i c p r o t e i n  (mg/g c e l l s ) total vels  Kinase  i s diminished  CK a c t i v i t y  i s reduced  are probably  protein  reduced  (Table  manner as CK microsomes,  CT i s a f f e c t e d  (Table  5).  i s reduced  is likely  by v i r u s  The o t h e r  in specific  the  inhibition  of host  viral  e n v e l o p e and n u c l e o c a p s i d  with microsomes proteins.  of host p r o t e i n  (179), c o u l d  The  activity  synthesis. proteins,  mask l o s s e s  after virus  apparent d i f f e r e n c e s  between CT from  infected  n o t be a d i f f e r e n c e  competitive vary  reaction  CT a c t i v i t i e s  6).  Inhiafter  The a c c u m u l a t i o n o f  which  are  associated  of c e l l u l a r  the s t a t i c  i n f e c t i o n o f BHK  microsomal  amount o f  cells  4).  (Table  i n a c t i v a t i o n by p h o s p h o c h o l i n e  and m o c k - i n f e c t e d i n the p r o p e r t i e s  cells o f CT.  ( s u c h as a p h o s p h o c h o l i n e  in activity  microsomes the  (Table  o f t h e enzyme p r o t e i n  Such an h y p o t h e s i s e x p l a i n s  microsomal p r o t e i n  i n f e c t i o n i n t h e same  f o r m o f t h e enzyme, f o u n d i n  because o f t u r n o v e r  inhibition  in  4),  Cytosolic protein l e -  because o f v i r a l  viral  could  5).  (Table  Cytidylyltransferase  Cytosolic  may  infection  cells  synthesis.  (iii)  bition  by SF v i r u s  i n BHK  as a r e s u l t o f v i r u s  f o r phosphocholine  from m o c k - i n f e c t e d  ( F i g s . 13, 15) Instead,  some  phosphatase)  infection.  i s almost  However,  t h e same f o r  and v i r u s - i n f e c t e d c e l l s  •  - 117  (0.34 the  mM  and  0.35  mM,  respectively).'*  suggestion of a v a r i a t i o n  other hand, i n c y t o s o l the  -  T h i s r e s u l t makes u n l i k e l y  in a competitive reaction.  the  f o r phosphocholine i s 0.32  enzyme from mock-infected c e l l s and 4  virus-infected  cells..  higher i n a c t i v i t y  On  Thus, the  in cytosol  0.18  mM  the  mM  for that  for  of  idea of a c o m p e t i t i v e  reaction,  from mock-infected c e l l s ,  i s more  tenable i n t h i s case. CT as are  from f r e s h c y t o s o l the  r a t lung  of BHK  cells  enzymes from r a t l i v e r  (36) .  While the  of l i p i d  a c t i v i t y of CPT  6), probably for the A preliminary t h e s i s of Ca  Ca  agent i n p h o s p h o l i p i d b i o s y n t h e s i s m a r i l y by  i n h i b i t i n g CPT  somes were t r e a t e d minutes at 37°)  and  treatment does not  infection  ;  (afid2EPT) .  with the Ca  (111). In the  The  ion may  microsomes of v i r u s - i n f e c t e d gent, t a u r o c h o l a t e , a c t i v a t e s  cells. CPT,  control act  pri-  experiment, microEGTA.(20 mM  then c o l l e c t e d by c e n t r i f u g a t i o n . the  (Table  hypo-  been proposed as a  chelator,  however, lessen  not  is inhibited.  performed to t e s t the has  CT  16).  same reason that microsomal CT  inhibition.  (92) , and  i n f e c t i o n does  i s i n h i b i t e d by v i r u s  experiment was  lipids,  a c t i v a t i o n of  that SF v i r u s  markedly a f f e c t t h i s a c t i v a t i o n ( F i g . (iv) Choliriephosphotransferase The  by  (91) , r a t i n t e s t i n e  function  remains u n c l e a r , i t i s c e r t a i n  is activated  for 5 Such  i n h i b i t i o n of CPT  from  S i m i l a r l y , while the i t does not  relieve  deter-  the  4  K values were obtained by f u n c t i o n of 1/S.  linear regression  of 1/V  as a  -  virus-induced The least  reasons.  c e r i d e w h i c h was  BHK  derived  strate  i n 0.03%  nullify pared '(d)  high  the  relative  CTP  the  I n h i b i t CK  11,  14).  Such  system  from E.  mM  Q CTP-Mg ~\  and  choline  has  (31).  However, i n c o n t r a s t , bited  by  CT  by  [_ CTP-Mg  it  could  present  [] ATP-Mg []  be  has  argued  (24). not  that,  could  merely  In  and  this  one  by  sub-  however, com-  Vivo? CT  are  the  "2  QATP-Mg ]]  kinase mM)  rat liver  i t seems t h a t  represent  an  2.5  of  from  rat  (24). inhi-  inhibition  enzyme a c t i v i t i e s  in crude  CTP  above  i s />jv  been p r e v i o u s l y o b s e r v e d . the  and  incorporation  (above 16  from  ATP  by  in a reconstituted  ethanolamine  CK  inhibited  have p h y s i o l o g i c a l r e l e -  system,  Also,  since  of  form _in v i v o .  substrates,  inhibit  purified  that  dispersed  does n o t  reported  [_ ATP-Mg  work were measured  inhibition  that  Also,  i s ;;^^rnh^b±tedM? by  CT  could  been  above 1 mM  from  in infected c e l l s  nucleotide  caudatum. In  i n t o PC  and  inhibition  A similar effect  digly-  substrate  was  the  at  cells.  i s i n t e r e s t i n g t h a t b o t h CK the  from  o f CPT  It  of  substrate  activities  inhibition control  This  p h y s i c a l form o f  ATP  concentrations  PC.  different  for  assayed with  quite different  The  about a b s o l u t e  and  yolk  Do  vance.  liver  f r o m egg  radically  i s questionable  enzyme was  Secondly,  to mock-infected  (Figs.  1  the  Tween 20.  i s probably  uncertainty  activities  a c i d composition  diglyceride.  sonication  Any  First,  have a f a t t y  cell  -  inhibition.(Results section (c)).  s i g n i f i c a n c e o f CPT  two  could  118  of  Indeed, of  the  s u b c e l l u l a r f r a c t i o n s , such a c t i o n by  nucleotides  on  -  some o t h e r of  such  protein.  inhibition  centration otides (e)  Vivo  is  true  the s i g n i f i c a n c e con-  and t h e e f f e c t s o f t h e n u c l e -  enzymes.  S i z e Measurements an A c c u r a t e  I n d i c a t i o n o f In  Concentrations? Is the Pool  of Choline  Distributed  in Subcellular  A curious pool  assess  knowledge o f t h e c y t o p l a s m i c  o f both n u c l e o t i d e s ,  Are Pool  line  To r e a l i s t i c a l l y requires  on p u r i f i e d  (i)  119 -  feature  i s larger  explanation  o f BHK c e l l s  than  in rat liver  Compartments? i s the f a c t , t h a t the cho-  that of phosphocholine.  (33) and-HeLa c e l l s  for this  The  (209).  A  reverse  possible  r e s u l t would be i f e t h a n o l a m i n e was  v e r t a n t l y measured w i t h c h o l i n e . than a t h i r d  i n BHK C e l l s U n e v e n l y  This  inad-  i s unlikely since  o f t h e e t h a n o l a m i n e , w h i c h has a pK  less  o f 9.5  (210),  a in  the f r e e c h o l i n e  f r a c t i o n w h i c h was  (pH>10), would be i o n i z e d . e t h a n o l a m i n e would phenylboron. interfere or  sibleiexplanatioh  line  choline. in  since neither  interfere greatly  i s that  the c h o l i n e  pool  (195).  1.  Another pos-  so t h a t  districho-  one compartment e q u i l i b r a t e s w i t h e x t r a c e l l u l a r  Evidence  PC s y n t h e s i s  tetra-  monomethyl-  i s unevenly  i n more t h a n one s u b c e l l u l a r compartment, from o n l y  o f the  e t h a n o l a m i n e p r o b a b l y does n o t  the assay o f c h o l i n e ,  dimethylethanolamine  buted  Thus, l e s s than a t h i r d  cells  be a v a i l a b l e t o form an i o n p a i r w i t h  In any c a s e ,  with  i s o l a t e d from BHK  f o r a small  pool  of choline  which  i s active  i s as f o l l o w s :  The s p e c i f i c  r a d i o a c t i v i t y of choline  ( i n both  -  mock-infected than  and v i r u s - i n f e c t e d  that o f phosphocholine  expected pared  t o have a h i g h e r  line  choline  the pulse  intracellular fractional fast.  In o t h e r  flux  16).  should  (choline  the c e l l  choline  be c o n s t a n t o v e r  In line  each  for rat liver  chondrion, Q H]]  which  choline.  3  Q E~2 3  phosphocholine  t h e same, t h e n  linear  the p o o l  that of  size  phos-  (B) o f  phosphocholine.  (105).  incoming  Q lT] 3  choline. effect.  o f a c h o l i n e p o o l , f o r example  i s not available  choline while  pathway  one p o o l , o f c h o l i n e has been  (33) and r a t . h e a r t  A second  ( j ) ) . The  f o r c h o l i n e than  two p o s s i b l e e x p l a n a t i o n s f o r t h i s isolation  i s very  turnover  (Results section  step of this  an  t h a t the  pool  c o n c l u s i o n , ^ l a r g e amount o f t h e c e l l u l a r  physical  of  f o r c h o l i n e than  be much s m a l l e r t h a n  does n o t mix w i t h  least  indicates  an i n t r a c e l l u l a r  kB  i n cho-  t o go t h r o u g h  result  Thus,, i f k i s g r e a t e r  Similarly/,-/more than indicated  remains  3  i s expected  (q) e q u a l s  and q r e m a i n s  com-  _. a l l o f t h e [~ l T ]  Since  r a t e o f such  The f l u x  should  little  words, t h e v a l u e o f t h e f r a c t i o n a l  to PC).  phocholine,  radioactivity  most o f t h e r a d i o -  and v e r y  (k) would be much g r e a t e r  (Table  specific  pool of choline, this  turnover  lower  A / p r e c u r s o r ..would be  experiment,  (Fig. 24).  which e n t e r s  i s unexpectedly  product.  i s i n phosphocholine,  after  rate  or equal  In t h e p u l s e - c h a s e  activity  cells)  (Table 9 ) .  to the corresponding 2.  120 -  p o o l o f choThere are a t One i s t h e i n the.mito-  f o r s y n t h e s i s o f PC  possibility  from  i s the p h o s p h o r y l a t i o n  choline i s transported into  the c e l l .  -  In t h i s  case,  the  inactive  of phosphocholine bility.one  to t h i s  The p.i.  Why  Are  hours  p . i . (Table 10).  cells  were i n c u b a t e d calf  these c e l l s  t o be  Pool Size  6-5/6  hours  tions  a r e made.  s m a l l e r than  remains  Pool size  First, cells,  assumed partly  For  from  depleted.  a t 6-1/2  6%  t o 7%  measurement,  Solution  that  time,  These v a l u e s are s i m i l a r  (and  but  The  the 30 m i n u t e s  pool of  the 2%  calculation,  (and  2%  p.i..  two  -  phospho-  virus-infected,  fetal  calf  Second,  serum)  i t is  the p o o l o f p h o s p h o c h o l i n e  cells  a r e d e r i v e d by  phosphocholine and  102  .  assump-  the p o o l o f or  size  starva-  i n c u b a t i o n ( a t 6-1/2  Thus, p o o l . s i z e s . o f  cells  6-5/6  the  this  hours  -  over  mock-infected  gram o f m o c k - i n f e c t e d  of v i r u s - i n f e c t e d  10).  Salt  i t i s assumed t h a t  t h a t q d o e s n o t v a r y as  nmoles p e r  that  hours  Thus,  after  w h i c h were i n c u b a t e d i n Medium 199 does n o t change  pool  before processing.  without  either  the  Results Variable?  former  in E a r l e ' s Balanced  p.i.) - q(30).  i n BHK  since  p o o l would c o n t i n u e t o t u r n o v e r .  would e q u a l :  possi-  reason  p o o l s a t 7 - lh  However, f o r the  which  product  synthesis.  were s t a r v e d o f c h o l i n e  of phosphocholine  choline  i n PC  the  i s f o r some  pool of choline,  serum) f o r 30 m i n u t e s  phosphocholine  tion  t h a t CK  Phosphocholine  appears  c o u l d be  However, i n t h i s  measurement o f p h o s p h o c h o l i n e  ( T a b l e 9)  dialyzed  degradation.  liberated  a p p a r e n t l y not a c t i v e (ii)  pool of choline  would have t o i m a g i n e  inaccessible is  (or PC)  -  121  of  32  nmoles p e r  the above  is  gram  calculation.  to the e x p e r i m e n t a l r e s u l t s  (Table  -  ( f ) How  Does SF V i r u s I n f e c t i o n  o f PC (i)  i s caused  in pool  by  SF  s i z e s of nucleotide  virus  infection  difference  probably  of purine  virus  RNA  just  over  26S,  and  half  base c o m p o s i t i o n  of  the  intracellular  of  to purine  is  ATP  t h a t GTP  and  cells,  are  sis.  The  unused p u r i n e  until  new  steady  :(ii)  sizes pool  11,  of  Figs.  22,  23)  purines  is  in  comprise  RNA,  and  22S,  cells  and  pools  pyrimidine  of pyrimidine Another  the  The  virus-infected  inhibition  less  of i n -  nucleotide  residues  biosyn-  possibility  for protein synthesis in i n -  inhibition  of host  n u c l e o t i d e s would  then  s t a t e c o n c e n t r a t i o n s are  protein  synthe-  accumulate  reached.  Phosphocholine c a t a l y z e s a Type 1 r e a c t i o n ( D i s c u s s i o n s e c t i o n ( g ) ) ,  i t becomes e a s y o f CTP size  of  result  synthesis.  of v i r a l  biosynthesis.  used  as a r e s u l t  RNA  guanyl-  RNAs o f SF  increased  t h e s i s compared  then  and  d i f f e r e n c e between p u r i n e  a result  the  o f p y r i m i d i n e s and  Adenyl-  The  I f CT  use  be  (Table  synthesis.  42S  be  fected  Size  triphosphates  and.pyrimidine  infection  because of p r e f e r e n t i a l  could  Pool  t o i n c r e a s e d use  because o f v i r a l  between p o o l s  triphosphates after  (211).  may  o f n u c l e o t i d e s y n t h e s i s or due  triphosphates,  viral  Cause Changes i n the  Precursors?  decrease  hibition  not  -  Nucleotides  The which  122  and  o f CTP  t o e x p l a i n the o p p o s i t e  phosphocholine becomes s m a l l e r  after  virus  in infected  changes  in pool  infection. cells  The  (compared  to  - 123  mock-infected liminary choline  cells)  early  experiment, i n t o PC,  the  which  -  in infection  inhibition  depends on  of  an  ( F i g . 22). incorporation  increase  o f p h o s p h o c h o l i n e , does n o t become n o t i c e a b l e hours  p.i..  first.  Thus,  T h i s change would  the r e a c t i o n other tion  the change  (since  would d i m i n i s h  be  (since  velocity).  the  However, an  (iii) The easy  could  be  to e x p l a i n  the C D P - c h o l i n e  pool apparently occurs  the f l u x  through  and  i n the p o o l s i z e  as  that of phosphocholine.  with phosphocholine  17%  f o r the 2 . 6 - f o l d infection  were t o i n c r e a s e  minus kinase accu-  increase  t h u s , the  flux  ( T a b l e 7) which  flux  would  i f the p o o l s i z e cells,  be m a i n t a i n e d .  could  as o f -i:.  would  pool of mock-infected  However, by  pool.size  i s not  Contamination  cells,  i n the p o o l s i z e  in infected  i n the C D P - c h o l i n e  constant  increase  (Table 9 ) .  t o t h a t p r e s e n t e d above,  crease  the r e a c -  would  of CDP-choline  o f the p o o l o f v i r u s - i n f e c t e d  CMP,  In  restored.  cells  after  through  of  ).  the c h o l i n e  o f the apparent CDP-choline  line  (V^)  equals forward v e l o c i t y  amount t o 12%  account  6h  after  CDP-choline  increase  and  until  [^phosphocholine  reaction  rfJ  size  i n phosphocholine  o f the CT  3  i n the p o o l  p h o s p h o c h o l i n e must o f n e c e s s i t y  increase  forward v e l o c i t y  t h r o u g h CT  flux  S i n c e the f l u x  step c o n t i n u e s unabated, mulate.  inhibited;  rj  of  the forward v e l o c i t y  e q u a l s k [jZTP  words, CT would  reverse  i n the CTP  lessen  In a p r e -  does  not  o f CDP-cho-  reasoning  o f the CPT  similar  product,  then a subsequent result.  Similar  in-  Thus, a  to the case  f o r CT,  -  the of  initial  pool  f l u x over  (g)  size  t h e CPT  What C o n t r o l s  change would c a u s e a t r a n s i e n t  the  Rate o f PC  Synthesis  i n BHK  Two  general  parameters,  in flux  over  these  and  i f v a r i e d , may  a given  two  parameters are  two  occur  in a metabolic  pathway.  substrate-dependant  types  reactions  Cells?  Flux  produce a  enzyme r e a c t i o n .  enzyme c o n c e n t r a t i o n s .  non-equilibrium,  inhibition  step.  Some G e n e r a l i z a t i o n s A b o u t C o n t r o l o f  substrate  may  -  (i)  response are  124  coordinate  These f a c t o r s  Corresponding  to  o f enzyme r e a c t i o n s w h i c h They a r e  near-equilibrium,  (hereafter called  Type 1)  s u b s t r a t e - independent r e a c t i o n s  and  (Type  2)  (212). In a Type 1 r e a c t i o n , the In o t h e r ration,  words, p, of  i t i s not  this  enzymatic  step w i l l  t o the  reverse  velocity  vided  by  forward  velocity  to  1,  t h e g r e a t e r must be  to  be m a i n t a i n e d ,  ratio  s i n c e the  It follows  of  the  (Vf)  both  a p p r o a c h e s u n i t y , V^  flux.  i s not  rate-limiting.  equal  the  enzyme  Vf  be  V , r  f l u x .is equal and  V  t h a t t h e V_~£  f  The d i s e q u i l i b r i u m close  t o 1.  enzyme r e a c t i o n  (109). and  flux-generating.  The  (V )  di-  r  c l o s e r V.^/V^ i s  i f a constant  flux  t o V f - V . . As  the  r  become much g r e a t e r  value  p is  than  o f a Type 1 r e a c t i o n  is  the will  Ilia 2\  be much g r e a t e r action, will  than  s m a l l changes  have a g r e a t  the  flux  in substrate  effect  on  flux  Type 2 r e a c t i o n s g e n e r a t e given  pathway  (212).  (212).  For  this  (or p r o d u c t )  o f enzyme  re-  concentrations  (109).  (or c o n t r o l ) the  A criterion  type  flux  for a controlling  through enzyme  a  step  -  is  t o have a V  step  (213).  value  m a x  Thus,  If V  ratio  i s very  saturated small.  indicators, separately,  controlling herein  enzyme s t e p  modulator, which a f f e c t s the V the f l u x  To  illustrate  reaction. be  through these  substrate(s)  However, e i t h e r  and t h e d i s -  o f these  latter  a r e t h o u g h t n o t t o be c r i t e r i a  (213).  that  that  to the f l u x , i t i s i m p l i e d with  A Type 2 r e a c t i o n  as a r a t e - l i m i t i n g r e a c t i o n .  affect  t o the f l u x over  t h e enzyme o p e r a t e s a t maximal  i s equal  m a x  t h e enzyme i s h i g h l y  equilibrium two  approximately equal  i n t h i s case  v e l o c i t y _in v i v o . that  125 -  m a x  identically  step.  two t y p e s o f r e a c t i o n ,  c o n t r o l l e d by t h e mass a c t i o n  termed  F o r s u c h a r e a c t i o n , any  o f t h e enzyme, w i l l  enzyme  In a Type 1 r e a c t i o n ,  i s also  for a  the f l u x over  consider  t h e CT  t h e CT s t e p  would  ratio:  [_ C D P - c h o l i n e ] ] [ P y r o p h o s p h a t e ] ] .QCTP]] -In a Type 2 r e a c t i o n , tration  t h e f l u x would  be c o n t r o l l e d by t h e c o n c e n -  o f enzyme.  The  extremes o f these:two types o f r e a c t i o n s  zyme r e a c t i o n flux^over in  [[Phosphocholinej  which  the step  i s completely  (Type 2 ) .  Both extremes are improbable,  biological  system  are  midway between  the  mass a c t i o n  could  be c l a s s e d  change  i f not impossible,  The f l u x t h r o u g h enzyme s t e p s  t h e two e x t r e m e s would  ratio  f o r w h i c h a change  would c a u s e a p r o p o r t i o n a l  (213).  type.  in flux in a  which  be c o n t r o l l e d by b o t h  and enzyme c o n c e n t r a t i o n .  as a t h i r d  an e n -  a t e q u i l i b r i u m , w i t h no n e t  (Type 1) and a r e a c t i o n  enzyme c o n c e n t r a t i o n  are:  Such enzymes  - 126 -  (ii) To  What Controls.;, t h e F l u x i n BHK  answer  formed.  Perhaps  experiment of  fJ HJ 3  t h e most c o n s p i c u o u s  phosphocholine  9).  which  at a constant flux,  a v e r y deep s e c t i o n down.(the  water v e l o c i t y cells,  These  PC  As d i s c u s s e d  much s m a l l e r t h a n  the H  label,  3  reservoir which  remains:  Three  lines  the f l u x  support  I f t h e maximal v e l o c i t i e s  a r e compared  with  the f l u x  then one c a n o b s e r v e at a velocity  that  appears  the idea  In BHK  t h u s a c t s as a s i n k out.  Still,  of choline  enzymes o f de novo PC s y n t h e s i s i n BHK c e l l s 1.  river  pool i s  the flow  i s constant).  i s only slowly flushed  o f evidence  to a  i s s m a l l e r ) but the  of phosphocholine  What c o n t r o l s  (Table  t o PC, w h i c h  i s analogous  turnover rate  that  t h e p o o l o f CDP-  i n which  (the f l u x  24).  r e s u l t s may be e x -  The p h o s p h o c h o l i n e  o f the r i v e r ,  fractional  (Fig.  that of phosphocholine  the s i t u a t i o n  does n o t change  the l a r g e  question  cell  3  t o a much s m a l l e r e x -  to choline,  flows a t a constant v e l o c i t y .  to slow  for  rjH~J  I f one c o n s i d e r s the' f l o w o f c h o l i n e m o l e c u l e s  occurs  like  of  pool i s l i k e l y  i s a l s o much s m a l l e r t h a n  per-  the disappearance  o f t h e p r e c u r s o r s o f PC.  In a d d i t i o n  were  o f the p u l s e - c h a s e  between  are l a b e l l e d  ( F i g . 24).  ( e ) , the c h o l i n e  of phosphocholine. choline  relationship  and C D P - c h o l i n e  by t h e p o o l s i z e s  section  result  and t h e a p p e a r a n c e  than p h o s p h o c h o l i n e  plained in  q u e s t i o n , pulse-chase experiments  i s the converse  Free c h o l i n e tent  this  Cells?  that  the  t o PC?  the three:  a r e o f Type 1.  o f CK, CT, and CPT in v i t r o  iri v i v o ,  f o r mock-infected  t h e enzyme a c t i v i t i e s  below t h e maximum  BHK  operate  (Table 17).  cells,  i n the  The enzyme  -  127 -  Table  17  Comparison o f V a l u e s o f V M o c k - I n f e c t e d BHK  m a x  and F l u x f o r Cells  Velocity, nmoles min g cells  In v i t r o  -  V ^  1  CK  7  CT  ( M i c r o s o m a l and C y t o s o l i c )  CPT In v i• v o  may  likely  compete  should  be u n d e r e s t i m a t e s s i n c e  contain  other  f o r the s u b s t r a t e s  flux  in — v i—v o  be r a t h e r  true Table 2.  i s much  less  than  5, R e s u l t s  prepara-  cells  since  o f V n i c i x•, t h e f l u x m  i n enzyme This  which  concentration.  conclusion  i n f e c t e d with  SF  also  virus.  section(j).)  yM c o n c e n t r a t i o n s , i n BHK  In c o n c l u s i o n ,  the values  t o changes  i n BHK  I f i t i s assumed  under  (Table  5  t h e c r u d e enzyme  and p r o d u c t s .  insensitive  f o r the f l u x  phosphocholine are  0  enzymes, s u c h as p h o s p h a t a s e s ,  Thus, the: r e a c t i o n s c a n n o t be o f Type 2.  present  6  M a x i m a l v e l o c i t i e s a r e " from T a b l e 5. F l u x i s from R e s u l t s s e c t i o n ( j ) .  activities  (See  -  I -  2  holds  7  2  3=  the  3  31.9 4  Flux  tions  -  that pool then  cells,  s i z e s i n nmoles/g c e l l s r e -  the c o n c e n t r a t i o n s  e i t h e r i n f e c t e d or  12% o f t h e c o n c e n t r a t i o n  10, F i g s . 13-15, R e s u l t s  o f CTP and  mock-infected,  f o r maximal v e l o c i t y  section  (c)).  ATP p o o l s  o f CT would  )  represent and  24%  and  pools,  infected  of  son  as  the  trations  and Type  are  not  mock-infected  11,  Fig.  represent  is.r:saturated  c h o l i n e may  58%  In  be  saturated  11). (mock-  concentration  (Table  9,  Fig.  a v a i l a b l e f o r PC  addition, this  kind  of  provides  synthe-  concen-  Compartmenta-  further evidence  i n v i v o , and  10).  compari-  w h i c h would c a u s e d i f f e r e n t  in differentccompartments.  comparison  that  thus these r e a c t i o n s  CK are  of  1. The  total  virus infection rate)  i s not  enzymes a r e respond  a l l three  5), yet  inhibited  a l l three  the  (Results  flux  i n the  flux  enzymes a r e  reduced  (phosphocholine  section  then a d e c r e a s e  (j)).  in a c t i v i t y  of choline  to  I f any should  turnof  the  cor-  PC.  this  i n the  also varies  enzyme r e a c t i o n s o f d_e novo PC  near-equilibrium  t o the  of  mass a c t i o n concept  (Fig. 8),  i n the  levels  uptake,  would a l s o  (Type 1 ) , e a c h one  then  same way,  follows.  cells the at  I f the  is varied  so  intracellular least  i t is predicted  increase.  synthesis  should  r a t i o ofireach r e a c t i o n .  i s as  medium o f BHK  also vary of  of  Prediction  t o be  sensitive diction  (Table  to a d e c r e a s e  Since appear  activities  o f Type 2,  (iii)  tion  from  (Table  hand, would  a t w h i c h CK  a l l cellular  this  f o r CK  ( v i r u s - i n f e c t e d • c e l l s ) o f the  of metabolites  3.  over  optimum  compartmentation  aside, CT  -  respectively  other  27%  mM)  128  previously discussed.  ignores  tion  the  and  (0.25  However, n o t sis,  on  cells)  of choline  PC  14%  virus-infected cells,  Choline  by  -  transiently.  At  rate of  very pre-  concentra-  that choline of  the  One  choline  pool  that  be  uptake  choline  would  increased  synthesis  of  -  (h)  129 -  A r e R o u t e s o f PC S y n t h e s i s Pathway o f I m p o r t a n c e  De  novo s y n t h e s i s  synthesis CPT  Other  In s u p p o r t  of this  i n BHK m i c r o s o m e s has a s p e c i f i c  activity liver  direct  section  (c)).  Also,  and PC  the r a t i o s  pool  t o 1.  of substrate  should  competitive The  identified fractions  warrants  that of r a t rela-  ( F i g . 24) i s e v i d e n c e  that  and PC.  additional evidence i n BHK c e l l s .  as t o t h e  F o r Type 1  and p r o d u c t c o n c e n t r a t i o n s  (absolute  Also, with  f o r CPT, t h e e q u i l i b r i u m  a purified  of cellular  further study.  by m o n i t o r i n g from c e l l s  specific  en&yme, s o t h a t  are eliminated.  compartmentation  certainly  PEMT  knowledge  be d e t e r m i n e d  reactions  110),  one-third  The measurement r e q u i r e s  sizes are not necessary).  constant-  o f about  Questions  n a t u r e o f t h e c o n t r o l o f PC s y n t h e s i s  of  while  between c h o l i n e  Measurement o f p would p r o v i d e  r e a c t i o n s , -p- i s c l o s e  conclusion,  the p r e c u r s o r - p r o d u c t  base e x c h a n g e d o e s n o t o c c u r Some Unanswered  route o f  i s l e s s than o n e - t e n t h  between p h o s p h o c h o l i n e  (i)  important  activity  6 and r e f e r e n c e  i n BHK m i c r o s o m e s  (Results  tionship  (Table  t h e De Novo  i n BHK C e l l s ?  o f PC may be t h e o n l y  i n BHK c e l l s .  that of r a t l i v e r  than  of choline  Subcellular pools  the s p e c i f i c  which  pools  radioactivity  are l a b e l l e d  with  and C;TP  could  be  in subcellular  exogenous c h o l i n e or  cytosine. Changes caused  in cellular  CTP c o n t e n t  by c h a n g e s o c c u r r i n g  in infected cells  e x c l u s i v e l y i n the nucleus.  may be However,  - 130 -  the pool of CTP which i s important f o r PC s y n t h e s i s that of the c y t o s o l .  In HeLa c e l l s  the cytoplasmic  concentration  s y n t h e s i s of PC  (214).  i s presumably  i n f e c t e d with p o l i o v i r u s ,  of CTP c o r r e l a t e s with  the r a t e of  I t would be i n t e r e s t i n g to see whether  the apparent time l a g between CTP pool s i z e changes and phosphoc h o l i n e pool s i z e changes i s indeed,  real.  t i o n , phosphocholine pool s i z e s should times p . i . .  To answer  be measured  Such a time l a g could represent  t h i s ques-  at various  the time r e q u i r e d  for changes i n the CTP pool of the nucleus to be r e f l e c t e d i n the cytoplasm. The c h o l i n e t r a n s p o r t process The s p e c i f i c i t y of the process, would a c t as competitive  a l s o deserves f u r t h e r study.  and the kinds of compounds t h a t  and noncompetitive i n h i b i t o r s , would  help to c h a r a c t e r i z e the p u t a t i v e c h o l i n e c a r r i e r p r o t e i n . I t would a l s o be of i n t e r e s t to repeat f l u x to see i f the r a t e of PC s y n t h e s i s virus-infected c e l l s .  PC s y n t h e s i s 1. kinase  increased i n  unanswered questions  by v i r u s  infection.  about the r e g u l a t i o n of  include:  What i s the r e l a t i o n s h i p of c h o l i n e t r a n s p o r t to c h o l i n e activity?  2.  What are the s i g n i f i c a n c e of m u l t i p l e forms of CK  26, 28), CT 3. in PC  is truly  T h i s r e s u l t seems u n l i k e l y , s i n c e i n no  experiment was c h o l i n e uptake s t i m u l a t e d Other g e n e r a l  the measurement of  (35, 36)", and CPT  What r o l e does l i p i d  synthesis?  (24,  (39, 43, 45, 46)? a c t i v a t i o n of CT  (36, 90-93) p l a y  -  4. to  the  loose  Mg  + +  If  What i s the  131  physical  -  r e l a t i o n s h i p of  m i c r o s o m a l enzymes o f de  novo PC  the  soluble  enzymes  Is  there  a  such  as  synthesis?  association? 5.  What i s the  and  Ca  6.  Are  so,  + +  ,  s i g n i f i c a n c e of d i v a l e n t c a t i o n s ,  i n the  c o n t r o l o f PC  multiple  what  i s the  pools  of  source of  synthesis?  choline  the  found  in other  i n a c t i v e pool?  cell  types?  What i s i t s  function? 7. act  How  does an  to s p e c i f i c a l l y (j)  external stimulate  Concluding  The  r e s u l t s of  interpreting  are  PC  low  choline dence  activity  this  t h e s i s demonstrate  activity  the  known.  synthesis  activities (10,  68, It  ues,  (Table  activity  3  Such a c o n c l u s i o n PC  o f CK  Q H]]  of  5)  o f CK  the and  choline  i s not  146,  the  vivo  the 8)  necessity until  and  149,  flux.  specific  be  are  For  accumulation could  of  Q H]]  of  3  t a k e n as  synthesis  and  an  inhibition  on  Yet, the  160),  evi-  that  of  this ac-  synthesis.  c o n t r o l l i n g steps  basis or  o f PC  more  example,  i n f e c t i o n , w h i c h c a u s e s an  justified.  147,  in  c o n t r o l s PC  have been p r o p o s e d  (142,  (153)),  S i m i l a r l y , enzyme a c t i v i t i e s  is inhibited after virus  cumulation  Cortisol  r e s u l t s with caution  a f t e r v i r u s i n f e c t i o n (Table  that  as  synthesis?  u n d e r s t a n d a b l e when compared w i t h the  (such  Remarks'  incorporation  radioactivities  agent  in vitro  incorporation  of  enzyme  results  143) . i s c l e a r by  that  comparison  of  f l u x and  i n v i t r o enzyme a c t i v i t i e s  in this  maximal v e l o c i t y system, a s s a y e d  valr-.z.  under  -  optimal  c o n d i t i o n s , are not  tivities. ties  of  that  the  of  Since  the  t h r e e de  three  ratios,  understanding chemists the  and of  living  a reliable  than  is controlled Information  equilibrium constants this  guide  novo enzymes from BHK  products.  cell  pathway.  into  interact  t o the  by  the  about  will  add  the  veloci-  i t is clear concentrations  g r e a t l y to  acour  a challenge'to bioof  the  microcosm  framework; a  the h o r i z o n s o f our  to maintain  ac-  f l u x e s , mass  of events  theoretical  in vivo  the maximal cells,  It is truly  interplay  a broad  h o p e f u l l y extend  f o r c e s which  -  i s much l e s s  t o f i t t h e dynamic  which w i l l the  flux  s t e p pathway  s u b s t r a t e s and  tion  of  the  132  living  challenge  understanding system.  of  -  133  -  1.  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