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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, University of 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 of Biochemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1979 (©Frederick William Whitehead, 1979 In presenting th i s thes i s in p a r t i a l fu l f i Iment of the requ irements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f ree l y ava i l ab le for reference and study. I further agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of this thesis for f inanc ia l gain sha l l not be allowed without my written permission. Department of B i o c h e m i s t r y The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e M a r c h 1 ? . i q 7 q ABSTRACT Semliki Forest (SF) virus caused an i n h i b i t i o n of 77% in incorporation of Q3H~J choline into phosphatidylcholine (PC) of Baby Hamster Kidney -21 (BHK) c e l l s , at 6*5-7% hours post infection ( p . i . ) . Choline uptake, enzyme a c t i v i t i e s , and pool sizes were measured to c l a r i f y the mechanism of i n h i b i t i o n and to understand the regulation of PC synthesis. Choline uptake has a K of 17 yM arid V _ of 381 pmoles . m max min ^ mg c e l l protein "^' in mock-irifected (control) c e l l s . Up-take i s inhibited in infected c e l l s , although such i n h i b i t i o n only p a r t l y accounts for incorporation 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 synthesis in nmoles min ^ g c e l l s ^, from control c e l l s , were: choline kinase - 7.3; c y t o s o l i c phosphocholine c y t i d y l y l t r a n s f e r a s e (cytosolic CT) -17.3; microsomal CT - 14.6; and cholinephos-photransferase (CPT) - 47.6. In infected c e l l s , the respective a c t i v i t i e s were l e s s : 5.2, 12.1, 4.2, and 19.8, at 7 hours p . i . . Choline, phosphocholine, and CDP-choline were separated by ion exchange and charcoal chromatography. Phosphocholine and CDP-choline were hydrolyzed to choline, which was measured e.. • enzymically. Diglyceride was hydrolyzed to g l y c e r o l , which was measured enzymically. CTP was measured by a new enzymic tech-nique which uses rat l i v e r CT. ATP was'measured by i t s absor-bance after high pressure l i q u i d chromatography. PC was measured - i i i -by l i p i d phosphorUSO a n a l y s i s . Pool s i z e s i n nmoles/g c e l l s , from c o n t r o l (and i n f e c t e d ) c e l l s were: c h o l i n e - 1 4 6 ( 6 8 ) ; phosphocholine - 34 ( 1 2 0 ) ; CDP-choline - 6 . 1 ( 1 5 . 7 ) ; d i g l y c e r -ide - 4 7 ( 4 3 ) ; CTP - 1 4 9 ( 7 9 ) ; and ATP - 1 8 0 0 ( 1 0 8 0 ) , a l l at 6*1-7% hours p . i . . Increases i n phosphocholine and CDP-choline, and decreases i n CTP and ATP, were a l l s i g n i f i c a n t ( p < 0 . 0 5 ) . The p o o l s i z e of PC, i n ymoles/g c e l l s , was 3 . 4 i n c o n t r o l c e l l s , and s i m i l a r l y , 3 . 0 i n i n f e c t e d c e l l s , a t 1-lh hours p . i . . The f a t t y a c i d composition of both PC and d i g l y c e r i d e was very s i m i -l a r i n c o n t r o l compared to i n f e c t e d c e l l s . In BHK c e l l s which were l a b e l l e d with Q3H~J c h o l i n e , the s p e c i f i c r a d i o a c t i v i t y and h a l f - l i f e of c h o l i n e , compared t o ; phosphocholine, suggested that a l a r g e p o o l of c h o l i n e e x i s t s , which i s not a c t i v e i n PC s y n t h e s i s . In a pulse-chase experiment with Q3H~] c h o l i n e , the f r a c t i o n of the Q3H~j phosphocholine p o o l which turned over per u n i t of time (k) was smaller i n i n f e c t e d c e l l s , y e t the pool s i z e of phosphocholine was g r e a t e r , compared to c o n t r o l c e l l s . Conse-qu e n t l y , the turnover rate o f phosphocholine (k x p o o l s i z e ) i n nmoles min g c e l l s ^, was 1 . 5 1 i n i n f e c t e d c e l l s , and a l i t t l e l e s s , 1 . 0 5 , i n c o n t r o l c e l l s . Thus, the turnover of phos-phocholine (or rate of PC s y n t h e s i s ) was not i n h i b i t e d by S F v i r u s i n f e c t i o n . In BHK c e l l s , the three enzymes of de novo s y n t h e s i s o f PC l i k e l y c a t a l y z e 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 ) r e a c t i o n s because: 1 . V m a x of each enzyme i s much g r e a t e r than the pathway f l u x . 2 . Pools of the enzyme s u b s t r a t e s of .choline kinase and - i v -•CT do not appear to be gr e a t enough to s a t u r a t e the enzymes. 3 . A l l three enzymes are reduced i n a c t i v i t y by v i r u s i n f e c t i o n , y e t the pathway f l u x i s not reduced. I f CT c a t a l y z e s a near-e q u i l i b r i u m r e a c t i o n , then a s m a l l e r CTP p o o l would l e s s e n the f l u x over the CT s t e p . Phosphocoline accumulation would r e s t o r e t h i s f l u x . S i m i l a r l y , CDP-choline accumulation may r e s t o r e the f l u x over the CPT step (which was p o s s i b l y l e s s e n e d by an i n c r e a s e i n amount of the product, CMP). I f indeed, the enzymes of de novo s y n t h e s i s of 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 , then a change i n any s u b s t r a t e or product w i l l change the path-way f l u x , unless a response i n another s u b s t r a t e or product balances the i n i t i a l change. - V -TABLE OF CONTENTS Page, INTRODUCTION. . . . . . 1 (a) P h o s p h a t i d y l c h o l i n e - I t ' s Relevance and B i o s y n t h e s i s . . . . . . . . . 1 (b) Steps of De Novo PC Synt h e s i s (i) C h o l i n e T r a n s p o r t 3 ( i i ) C h o l i n e Kinase . 8 ( i i i ) Cytidylyl.tr-an.sf erase 9 (iv) C h o l inephosphotransferase 10 (c) A l t e r n a t e Formation of PC (i) Phosphatidyle thanolamine M e t h y l t r a n s f e r a s e . . 14 ( i i ) Base Exchange 16 (d) P o t e n t i a l S i t e s o f Re g u l a t i o n of De Novo Sy n t h e s i s (i) C h o line T r a n s p o r t . . . . . . . . 17 ( i i ) C h o l i n e Kinase 18 ( i i i ) C y t i d y l y l t r a n s f e r a s e 18 (iv) C h o l inephosphotransferase . . . . . . . . . 19 (e) Reg u l a t i o n of P h o s p h a t i d y l c h o l i n e B i o s y n t h e s i s (i) L i v e r 1. A v a i l a b i l i t y of Su b s t r a t e s . . . . . . . . 20 2. R a t e - L i m i t i n g Step . . . . . 22 3. E x p e r i m e n t a l l y A l t e r e d PC Sy n t h e s i s . . . 23 4. Phosphatidate . . . . . . . . 26 ( i i ) Lung . . . . . . . . . . . . 27 ( i i i ) B r a i n . . . . . 30 (f) E f f e c t s of 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 31 (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 B i o s y n t h e s i s 34 MATERIALS AND METHODS (a) Chemicals and Isotopes 37 (b) General Methods (i) Thin-Layer Chromatography < . 38 ( i i ) S c i n t i l l a t i o n Counting . . . . 39 ( i i i ) P r o t e i n . . . . . . . . . . 39 (iv) S t a t i s t i c s 39 (c) C e l l C u l t u r e . . . . . . . . . . 39 (d) Propagation o f S e m l i k i F o r e s t V i r u s . . . . . . . 40 (e) I n f e c t i o n of C e l l s with S e m l i k i F o r e s t V i r u s . . . 40 (f) P r e p a r a t i o n o f S u b c e l l u l a r F r a c t i o n s . . . . . . . 40 (g) Enzyme Assays (i) C h o l i n e Kinase 41 ( i i ) C y t i d y l y l t r a n s f e r a s e 42 ( i i i ) C h o l i nephosphotransferase . . . . 43 (iv) Phosphatidylethanolamine M e t h y l t r a n s f e r a s e . 44 (v) Choline Oxidase 44 - v i -Page (h) L i p i d A c t i v a t i o n o f C y t i d y l y l t r a n s f e r a s e . . . . . 45 ( i ) T a u r o c h o l a t e A c t i v a t i o n o f 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 . . . . . . . 45 ( j ) C h o l i n e T r a n s p o r t . . . . . . . . . . 45 ll) . P o o l S i z e Measurements ( i ) C h o l i n e - C o n t a i n i n g Compounds . 47 ( i i ) PC . . . . . . . 52 ( i i i ) D i g l y c e r i d e . . . . . . 56 ( i v ) P h o s p h o c h o l i n e and CTP . . . . . . . . . . . 57 (v) N u c l e o t i d e s - 59 (m) F a t t y A c i d A n a l y s i s - . . . 63 (n) Q 3 H ~ j C h o l i n e Pulse-Chase . . . . . . . . . . . . . . 64 RESULTS (a) C e l l Weight and P r o t e i n C o n c e n t r a t i o n 65 (b) C h o l i n e T r a n s p o r t . . 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 3 HJJ C h o l i n e . . . . . . . . . . 86 (f) P o o l S i z e Measurements - 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 Sequence 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 . . . . . . . . . . . . . . . . 101 ( 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 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 . . . . . . . . . . . . . . 105 ( j ) Pulse-Chase o f Q3H~] 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 . . . . . . . . . . . . . . 108 DISCUSSION (a) Does SF V i r u s I n f e c t i o n A f f e c t the Rate 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 Q3H^] 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 Causes the 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 Each Step o f PC S y n t h e s i s ? ( i ) C h o l i n e T r a n s p o r t . . . . . . . . . . . . . . 114 ( i i ) C h o l i n e K i n a s e . . . . . 1 1 6 ( i i i ) C y t i d y l y l t r a n s f e r a s e . . . . . . . . . . . 116 ( i v ) C h o l i n e p h p s p h o t r a n s f e r a s e . . . . . . . . . 117 (d) Do ATP and CTP I n h i b i t CK and CT In V i v o ? . . . . 118 (e) Are P o o l 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 V i v o C o n c e n t r a t i o n s ? ( i ) I s the 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 ( i i ) Why Are 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 V a r i a b l e ? . . . . . . . . . . . . . . . . . 121 - v i i -Pag o (f) How Does SF Virus Infection Cause Changes in the Pool Size of PC Precursors? (i) Nucleotides 122 ( i i ) Phosphocholine 122 ( i i i ) CDP-choline 123 (g) What Controls the Rate of PC Synthesis in BHK Cells? (i) Some Generalizations About Control of Flux 124 ( i i ) What Controls the Flux in BHK Cells? . . . 126 ( i i i ) Prediction 128 (h) Are Routes of PC Synthesis Other than the De Novo Pathway of Importance in BHK Cells? . . 129 (i) Some Unanswered Questions . 129 (j) Concluding Remarks . . . . . . . 131 - v i i i -LIST OF TABLES Page Table 1 E f f e c t of Virus Infections on Animal PC and PE Synthesis 33 Table 2 Eff e c t s of Semliki Forest Virus Infection on BHK C e l l Pool Sizes 35 Table 3 Wet Weight of BHK C e l l s Infected with SF Virus 65 Table 4 Protein Concentrations in BHK C e l l s Infected with SF Virus 66 Table 5 Enzymes of De Novo Synthesis of PC in BHK C e l l s Infected with SF Virus 81 Table 6 Inhibition of Microsomal CT and CPT by SF Virus Infection of BHK C e l l s 81 Table 7 Separation of [~llfC^] Choline-Containing Compounds . . . . . . . . . . 84 Table 8 Incorporation of Q 3*C] Choline into PC and i t s Precursors in BHK C e l l s Infected with SF Virus . . . . 87 Table 9 S p e c i f i c Radioactivity and Pool Size of PC and i t s Precursors in BHK C e l l s Infected with SF Virus 91 Table 10 Pool Sizes of Cytidylyltransferase Sub-strates in BHK C e l l s Infected with SF Virus . 95 Table 11 Pool Sizes of Nucleotides in BHK C e l l s Infected with SF Virus . . . . . . 99 Table 12 Absorbance of the Medium of BHK C e l l s Infected with SF Virus . . . 100 Table 13 Comparison of Nucleotide Results by Two Procedures 104 Table 14 Percent D i s t r i b u t i o n of Fatty Acids in PC and PE from BHK C e l l s Infected with SF Virus 106 Table 15 Percent D i s t r i b u t i o n of Fatty Acids in Diglyceride from BHK C e l l s Infected with SF Virus 107 Table 16 Fractional Turnover Rates of PC Precursors in BHK C e l l s Infected with SF Virus . . . . . 112 Table 17 Comparison of Values of V m a x and Flux for Mock-Infected BHK C e l l s 127 - ix -LIST OF FIGURES Page Figure 1 Phosphoglyceride structure . . . 2 Figure 2 Biosynthesis of PC, PE, and t r i g l y c e r i d e in rat l i v e r . . 4-5 Figure 3 De novo biosynthesis of PC . . . . . . . . 6 Figure 4 Standard curve for choline determination 53 Figure 5 Standard curves for l i p i d phosphorus and diglyceride determinations . . . . . . 55 Figure 6 Time course for the conversion of CTP into CDP-choline . . . . . . . . . . . . . 60 Figure 7 Standard curve for CTP determination . . . 61 Figure 8 Uptake of choline by BHK c e l l s infected with SF virus . . . . . . . . . . 68 Figure 9 CK a c t i v i t y vs.. protein and time . . . . . . 71 Figure 10 CK a c t i v i t y vs. choline . 72 Figure 11 CK a c t i v i t y vs. ATP 73 Figure 12 Cytosolic CT a c t i v i t y vs. protein and time 75 Figure 13 Cytosolic CT a c t i v i t y vs. phosphocholine . 76 Figure 14 Cytosolic CT a c t i v i t y vs. CTP 77 Figure 15 Microsomal CT a c t i v i t y vs. phospho-choline . . . . . . . . . . . . . . . . . 79 Figure 16 Activation of c y t o s o l i c CT by rat l i v e r l i p i d . 80 Figure 17 I d e n t i f i c a t i o n of the hydrolytic product of phosphocholine . . . . . . . . . . . . 85 Figure 18 TLC of Q3H~J choline-labelled l i p i d . . . 89 Figure 19 Pool size of PC and i t s precursors in BHK c e l l s infected with SF virus 93 Figure 20 Separation of nucleotides from mock-infected BHK c e l l s 96 Figure 21 Separation of nucleotides from SF virus-infected BHK c e l l s 97 - X Page F i g u r e 22 E f f e c t of 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 . . . . . . . 102 F i g u r e 23 E f f e c t of d u r a t i o n of i n f e c t i o n on ATP p o o l s i z e 103 F i g u r e 24 Pulse chase of [^3EQ c h o l i n e i n BHK c e l l s i n f e c t e d with SF v i r u s . . . . . . . 109 0 LIST OF ABBREVIATIONS A - absorbance ADP - adenosine diphosphate AMP - adenosine monophosphate ATP - adenosine triphosphate [jATP-Mg^' - 1 s 1 complex of ATP and Mg, the putative substrate of CK B - pool size BHK c e l l s - Baby Hamster Kidney -21 c e l l s BSS-2%DCS - Earle's Balanced Salt Solution, and 2% dialyzed c a l f serum buffer A - 0. IM N H 4 H C O 3 buffer, pH 8.9: buffer B - I M N H 4 H C O 3 buffer, pH 10.0 cc - cubic centimeter Ci - curie CK - choline kinase CMP - cytidine monophosphate CoA - coenzyme A cone. - concentrated cpm - counts per minute CPT - cholinephosphotransferase CT - phosphocholine c y t i d y l y l t r a n s f e r a s e CTP - cytidine triphosphate [^CTP-Mg]] - 1:1 complex of CTP and Mg, the putative substrate of CT DEAE - diethylaminoethyl DNA - deoxyribonucleic acid dpm - disintegrations per minute EDTA - ethylenediaminetetraacetic acid , EGTA - ethyleneglycol-bis (3-aminoethyl ether) N, N~ -tetraacetic acid EK - ethanolamine kinase EPT - ethanolaminepfr^ ET - phosphoethanolamine cy t i d y l y l t r a n s f e r a s e F i g . - figure g - gram g_ - gravity GDP - guanosine diphosphate <GTP - guanosine triphosphate h - hour ITP - inosine triphosphate k - f r a c t i o n a l turnover rate (also;abbreviation for rate constant) K^  - d i s s o c i a t i o n constant of an enzyme-inhibitor complex K m - Michaelis-Menten constant I, 1 - l i t e r LPC - lysophosphatidylcholine LPE - lysophbsphatidylethanolamine m - meter M - molar r~Mg-ATP~J -.1:1 complex of ATP and Mg, the putative substrate of CK min - minute N - normal nd - not detectable nmol - nanomole NS - not s i g n i f i c a n t p - s t a t i s t i c a l p r o b a b i l i t y PBS - phosphate-buffered s a l i n e , pH 7.4 PC - phosphatidylcholine PE -phosphatidylethanolamine PEMT - phosphatidylethanolamine methyltransferase PFU - plaque forming unit p . i . - post i n f e c t i o n P^ - inorganic phosphate pk a - negative logarithm of the acid d i s s o c i a t i o n constant q - turnover rate or flux R - r a d i o a c t i v i t y ref. - reference Rf - r a t i o of distance moved by a solute to that moved by the solvent front RNA - ribonucleic acid S - substrate concentration (also abbreviation for Svedberg unit) s.d. - standard deviation SF virus - Semliki Forest virus t - time TLC - thin layer chromatography T r i s - t r i s (hydroxymethyl) aminomethane UTP - uridine triphosphate UV - u l t r a v i o l e t v - volume V - v e l o c i t y Vf - forward v e l o c i t y (of an enzyme reaction) V m a x - maximal v e l o c i t y (of an enzyme reaction) V r - reverse v e l o c i t y (of an enzyme reaction) w - weight p - disequilibrium r a t i o Notes -2 -3 1. Standardgprefixes are£„c (centi)-10_,i m(milli)-10 ; u(micro)-10~ ; "ri(naho)-10. ; p(pico)-10 2. Abbreviations for c e l l types other than BHK are not l i s t e d here. ACKNOWLEDGEMENTS I extend the utmost appreciation to my supervisor, Dr. Dennis E. Vance for his continued guidance, which kept this thesis project on a straight l i n e of research. I am very grat e f u l to Dr. Patrick Choy for his 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 for his many helpful discussions. I owe Mr. Harry Paddon many thanks for his great assistance in some of the longer and more tedious experiments. I am indebted to Dr. Allen Delaney for his sug-gestion to use charcoal as a means of separation of phospho-choline and CDP-choline, to Dr. G.M. Tener for his useful sug-gestions and comments, and to Dr. Caroline A s t e l l for her en-couragement. Thanks are due to Dr. Everard Trip and Dr. Michae Smith for their tremendous assistance in high pressure l i q u i d chromatography. I am obliged to Miss Karen Catherwood, Mrs. Jenny Toone, and Mr. Harry Paddon, who a l l assisted in the tissue culture of BHK-21 c e l l s . X I V DEDICATION - D e d i c a t e d t o the C r e a t o r o f a l l l i f e - 1 -INTRODUCTION (a) P h o s p h a t i d y l c h o l i n e - I t ' s Relevance and B i o s y n t h e s i s G l y c e r o p h o s p h o l i p i d s are molecules with the g e n e r a l s t r u c -ture shown i n F i g u r e 1. The' p h o s p h o l i p i d , p h o s p h a t i d y l c h o l i n e (PC), i s of importance s i n c e i t i s the major p h o s p h o l i p i d i n most animal t i s s u e s which have been analyzed. I t predominates i n b r a i n (except human and pigeon which have more p h o s p h a t i d y l -ethanolamine (PE)), heart, l i v e r , kidney, s p l e e n , lung, s k e l e t a l muscle, pancreas, thymus, p i t u i t a r y , a d r e n a l s , and e r y t h r o c y t e s . (except p i g e r y t h r o c y t e s which have more PE, and sheep, cow, and goat e r y t h r o c y t e s which lack PC) ( 1 ) . PC i s a l s o the major phos-p h o l i p i d i n the s u b c e l l u l a r f r a c t i o n s t e s t e d , except plasma mem-brane ( 1 ) . In BHK c e l l plasma membranes however, PC i s the major p h o s p h o l i p i d ( 2 ) . In b i o l o g i c a l membranes, p h o s p h o l i p i d s are of importance s i n c e they are primary s t r u c t u r e s , forming a b i l a y e r i n which ' • ; . p r o t e i n s are thought to f l o a t (3, 4 ) . P h o s p h o l i p i d s may b e . d i s -t r i b u t e d asymmetrically across a membrane. For example, two-thirds to t h r e e - q u a r t e r s of the PC i n human and r a t e r y t h r o c y t e s appear to be i n the outer h a l f of the c y t o p l a s m i c membrane (5 ). Evidence f o r an asymmetry of PC across r a t l i v e r microsomes i s based on the l i p i d ' s a c c e s s i b i l i t y to phospholipase A 2 (6, 7 ) . However, recent experiments by Sundler e_t a l (8) , c o n f l i c t with the n o t i o n of asymmetry. No d i f f e r e n c e was observed between the h y d r o l y s i s of PC and PE by i n c r e a s i n g amounts of phospholipase, between i n t a c t microsomes and microsomes d i s p e r s e d with detergent ( 8 ) . - 2 -F i g u r e 1 PHOSPHOGLYCERIDE STRUCTURE 0 I I R - C - 0 - CH 0 I C H 2 - 0 - C - R O I I C H 2 - 0 - P - 0 - X L 0 P h o s p h a t i d i c a c i d X = H Ph o s p h a t i d y l c h o l i n e X = CH 2CH 2S(CH 3) 3 P h o s p h a t i d y l ethanolamine X = CH 2CH 2NH 3 C 0 0 ~ I P h o s p h a t i d y l s e r i n e X = CH 2CH ^NH 3 P h o s p h a t i d y l g l y c e r o l X = CH 2CHCH 2OH I OH - 3 -The r e g u l a t i o n of PC s y n t h e s i s , l i k e other mechanisms of metabolic c o n t r o l , may be c o n t r o l l e d by a v a r i e t y of f a c t o r s . For example, enzyme-catalyzed r e a c t i o n s can be c o n t r o l l e d by s u b s t r a t e a v a i l a b i l i t y . S u b s t r a t e s , however, may be u n a v a i l a b l e by p h y s i c a l i s o l a t i o n across a s u b c e l l u l a r membrane. Su b s t r a t e s may a l s o be l i m i t e d by the c o n c e n t r a t i o n of a given i o n i c , con-f o r m a t i o n a l , or tautomeric s p e c i e s r e q u i r e d by the enzyme. En-zyme a c t i v i t y may a l s o be l i m i t e d by the e q u i l i b r i u m between a c t i v e and i n a c t i v e forms, or the r a t e of cleavage of proenzymes. The b i n d i n g of s p e c i f i c e f f e c t o r s may a l t e r enzyme k i n e t i c s . F i n a l l y , the r a t e s of s y n t h e s i s and degradation of an enzyme may c o n t r o l i t s a c t i v i t y (9). Before i t was shown t h a t 3 2P-orthophosphate i s r a p i d l y i n -corporated i n t o p h o s p h o l i p i d s , these compounds were thought to be i n e r t . The present understanding of the dynamics of r a t l i v e r PC b i o s y n t h e s i s from glycerol-3-phosphate i s o u t l i n e d i n F i g u r e 2. De novo s y n t h e s i s of PC i s shown i n F i g u r e 3. In the f o l l o w i n g i n t r o d u c t o r y s e c t i o n s , PC s y n t h e s i s i s d e s c r i b e d and the known f e a t u r e s of i t s r e g u l a t i o n are o u t l i n e d . (b) Steps of De Novo PC Synt h e s i s (i) C h o l i n e T r a n s p o r t 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 , c h o l i n e t r a n s p o r t has two apparent K m' s. H i g h - a f f i n i t y uptake (K m, ..59 uM) and low-a f f i n i t y uptake (K m, 0.22 mM) are probably c a r r i e r - m e d i a t e d f a -c i l i t a t e d d i f f u s i o n and simple d i f f u s i o n , r e s p e c t i v e l y (10). S i m i l a r l y , c h o l i n e i s t r a n s p o r t e d across the guinea p i g jejunum - 4 -Figure 2. Biosynthesis of PC, PE, and t r i g l y c e r i d e in rat l i v e r CH2 OH HOCH 0 (Glycerol-3-phosphate) I I CH2OP - OH I. O Fatty acid Acyl CoA < y ^ CoA AMP ATP + CoA + pyrophosphate Acylglycerol-3-phosphate Acyl CoA CoA (Phosphatidic acid; C H 2 O R 1 I R2OCH 0 I I CH 20P - OH L 0 4 . \|r^- phosphate C H 2 O R 1 R2OCH !• \ {1,2-diacylglycerol, or diglyceride) I ' V CH2OH CDP-choline CDP-ethanolamine, CMP -J/ 3 S-adenosyl-'"'^3 S-adenosyl-methionine homocysteine C H 2 O R 1 I R2OCH (Triglyceride) I C H 2 O R 3 - 5 -F i g u r e 2 continued Enzyme Name Enzyme Commission Number 1. Acyl-CoA synthetase 6.2.1.3 2. G l y c e r o p h o s p h a t e a c y l t r a n s f e r a s e 2.3.1.15 3. A c y l g l y c e r o l - 3 - p h o s p h a t e a c y l t r a n s f e r a s e 4. PhOsphatidate phosphatase 3.1.3.4 5. Ethanolaminephosphotransferase (EPT) 2.7.8.1 6. C h o i i n e p h o s p h o t r a n s f e r a s e (CPT) 2.7.8.2 7. D i a c y l g l y c e r o l a c y l t r a n s f e r a s e 2.3.1.20 8. Phosphatidylethanolamine m e t h y l t r a n s f e r a s e (PEMT) ' 2.1.1.17 Note: A l l of the above enzymes occur i n r a t . l i v e r microsomes. - 6 -F i g u r e 3. De novo b i o s y n t h e s i s of PC (CH 3-) j 3 NCH2CH 20H (choline) ATP ADP 0 o (CH 3) 3NCH 2CH 2OP I. 0 (CH 3) 3NCH 2CH (betaine-aldehyde) 5 ' 0 - OH C T P N Pyrophosphate M (CH 3) 3NCH 2C (betaine) (phosphocholine) |_ 0 2. 0 0 (CH 3 ) 3^CH 2CH 20.r J - P-O-P- 0- CH 2 0 NH -1,2-diacyl-g l y c e r o l CMP 0 0 (CDP-choline) IN OH OH 3. N PC Enzyme Name 1. Ch o l i n e Kinase (CK) •• 2. Phosphocholine c y t i d y l y l t r a n s f e r a s e (CT) 3. Cholinephospho-t r a n s f e r a s e (CPT) 4. Ch o l i n e dehydrogenase 5. Betaine-aldehyde dehydrogenase Enzyme Commission Number 2.7.1.32 Enzymes of 2 . 7.7*15 de novo 2.7.8.2 s y n t h e s i s 1.1.99.1 - 7 -mucosa by a s a t u r a b l e h i g h - a f f i n i t y process as w e l l as a low-a f f i n i t y p r o c e s s , probably due to d i f f u s i o n (11). The t r a n s -p o r t of c h o l i n e has a l s o been s t u d i e d using N o v i k o f f r a t hepa-toma c e l l s . In these c e l l s , the t r a n s p o r t process has a K m value of 4-7 yM (12). The h i g h - a f f i n i t y uptake by r a t b r a i n synaptosomes ( K m , 0.83 yM) i s p r o p o r t i o n a l to the _in v i v o a c t i v i t y o f c h o l i n e r g i c neurons and to a c e t y l c h o l i n e turnover (13). The h i g h - a f f i n i t y uptake i s sodium-, potassium-, and chloride-dependant. I t may be e x c l u s i v e l y found i n c h o l i n e r g i c nerve terminals. : U n l i k e a s c i t e s c e l l s ' t r a n s p o r t (10), i t i s an a c t i v e t r a n s p o r t system. Many l i n e s of evidence show that t h i s t r a n s p o r t system i s r a t e -l i m i t i n g and r e g u l a t o r y i n a c e t y l c h o l i n e formation (14). In c o n t r a s t , h i g h - a f f i n i t y uptake of c h o l i n e (K m, 16 yM) by d i s s o c i a t e d r a t embryo b r a i n c e l l s i s a s s o c i a t e d with a high p r o p o r t i o n of phosphocholine formation. At higher c o n c e n t r a t i o n s of c h o l i n e i n the medium, the added c h o l i n e was i n c r e a s i n g l y recovered 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 , with a smal l e r pro-p o r t i o n of phosphocholine (15). The h i g h - a f f i n i t y t r a n s p o r t i n t h i s case was not an a c t i v e p r o c e s s . Perhaps the embryonic t r a n s -p o r t measured was not the same process which occurs i n a d u l t s . Very h i g h - a f f i n i t y c h o l i n e uptake (K m, approx 1 yM) may not de-velop u n t i l l a t e i n embryogenesis, as occurs i n c h i c k r e t i n a (16). A l s o , i n support of t h i s c o n j e c t u r e , p o s t n a t a l b r a i n synaptosomes (of guinea pigs) were found to form phosphocoline p r e f e r e n t i a l l y over a c e t y l c h o l i n e at a high c h o l i n e c o n c e n t r a t i o n (17). - 8 -Free c h o l i n e i s t r a n s p o r t e d across the b l o o d - b r a i n b a r r i e r of a d u l t r a t s by a process with a s i n g l e K m (440 \iM) (18) . Such t r a n s p o r t may be necessary because r a t b r a i n has l i t t l e or no a b i l i t y to s y n t h e s i z e c h o l i n e v i a PE m e t h y l a t i o n (19-21). ( i i ) C h o l i n e Kinase An enzyme which formed phosphocholine from c h o l i n e and ATP i n the presence of Mg + + was d i s c o v e r e d by Wittenberg and Kornberg i n y e a st, and i n acetone powders of l i v e r , b r a i n , i n t e s t i n e , and kidney (22). Kornberg and P r i c e r showed i n 1952 that double-l a b e l l e d phosphocholine ( 3 2 P and 1 4C) c o u l d be t r a n s f e r r e d to a l i p i d by a l i v e r p r e p a r a t i o n (23). C h o l i n e kinase (CK) a c t i v i t y i s almost e x c l u s i v e l y i n the 100,000 x £ supernatant of • r a t l i v e r homogenates (24). U p r e t i et aL. r e p o r t e d that CK of r a t l i v e r and b r a i n was a microsomal enzyme (25), a f i n d i n g which l a c k s . c o r r o b o r a t i o n (24, 26, 27). M u l t i p l e forms of both CK and ethanolamine kinase (EK) i n r a t l i v -er have been shown by D E A E - c e l l u l o s e chromatography and non-de-n a t u r i n g e l e c t r o p h o r e s i s (24, 26). Two CK's with d i f f e r e n t K m's fo r c h o l i n e and d i f f e r e n t s e n s i t i v i t y to hemicholinium appear at d i f f e r e n t times during development of the r a t s p i n a l cord (28). At l e a s t one CK has been p h y s i c a l l y separated from EK (24, 26). CK and EK appear to have separate a c t i v e s i t e s i n germinat-ing soy beans (29) and i n the protozoan, Entodinium caudatum (30). In the f i r s t case, c h o l i n e does not a f f e c t ethanolamine i n c o r p o r -a t i o n i n t o l i p i d s . In the second, ethanolamine does not a f f e c t -c h o l i n e uptake, which i s i n t i m a t e l y a s s o c i a t e d with CK i n t h i s o r -ganism (31). In r a t l i v e r , however, c h o l i n e i s a h i g h l y e f f e c t i v e - 9 -i n h i b i t o r of at l e a s t one EK, whereas ethanolamine i s a poor i n -h i b i t o r of CK (24, 26, 32). Rat l i v e r CK i s i n h i b i t e d by ATP with an excess of Mg + +, or v i c e v e r s a ; but not by ATP i n the presence of equimolar Mg + + (24). Chol i n e has an a l t e r n a t e f a t e i n l i v e r . I t may be o x i d i z e d to b e t a i n e . In f a c t , by i n t r a p o r t a l i n j e c t i o n s of Q 1 "*C c h o l i n e , i t has been shown t h a t , c h o l i n e o x i d a t i o n i s at l e a s t as f a s t as p h o s p h o r y l a t i o n (33). ( i i i ) C y t i d y l y l t r a n s f e r a s e The pathway of PC b i o s y n t h e s i s was f u r t h e r e l u c i d a t e d by Kennedy and Weiss (34). They d i s c o v e r e d that CTP s p e c i f i c a l l y s t i m u l a t e d the i n c o r p o r a t i o n of l a b e l l e d phosphocholine i n t o PC. They found a s o l u b l e enzyme which c a t a l y z e d the f o l l o w i n g r e v e r -s i b l e r e a c t i o n : CTP + phosphocholine< CDP-choline + Pyrophosphate The c y t i d y l y l t r a n s f e r a s e (CT) was found to r e q u i r e M g + + or Mn + + (34). CT e x i s t s i n two i n t e r c o n v e r t i b l e forms of molecular weight 2 x 105 and over 1 x 106 i n r a t l i v e r and lung (35, 36) . The enzyme has been p u r i f i e d 960-fold from r a t l i v e r c y t o s o l by Choy et a l . (35). The analagous enzyme, phosphoethanolamine c y t i d y l y l t r a n s f e r a s e has been p u r i f i e d 1100-fold from r a t l i v e r supernatant by Sundler (37). Both c y t i d y l y l t r a n s f e r a s e s c a t a -l y z e r e v e r s i b l e r e a c t i o n s (35, 37). The phosphocholine and dimethylethanolamine phosphate c y t i d y l y l t r a n s f e r a s e s from r a t l i v e r co-chromatograph on Seph-adex G-200. Separated from t h i s peak, the phosphoethanolamine and monomethylethanolamine phosphate c y t i d y l y l t r a n s f e r a s e s a l s o co-chromatographed (38). - 10 (iv) 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 Kennedy and Weiss d i s c o v e r e d that CDP c h o l i n e c o u l d be con-v e r t e d to PC by the 18,000 x cj p e l l e t from a r a t l i v e r homogenate. T h i s r e a c t i o n was s t i m u l a t e d by d i g l y c e r i d e (34). Cholinephos-p h o t r a n s f e r a s e :(CPT) . from r a t l i v e r has been w e l l s t u d i e d . The separate nature of the enzymes c a t a l y z i n g the f i n a l step i n de novo s y n t h e s i s of PC and PE has been c o n t r o v e r s i a l . The con t r o v e r s y was l a r g e l y r e s o l v e d by the p a r t i a l s e p a r a t i o n of s o l u b i l i z e d CPT and ethanolaminephosphotransferase (EPT) from r a t l i v e r microsomes (39). T h i s s e p a r a t i o n was achieved by suc-rose g r a d i e n t c e n t r i f u g a t i o n a f t e r treatment with T r i t o n X-100. CPT was separated i n t o : 1. Mn + +-dependant and 2. Mn + + or Mg + +-dependant forms. The Mn + +-requiring form of CPT was not separated from EPT (39) . In c o n t r a s t , c o mpetition between CDP-choline and CDP-ethanolamine suggest a s i n g l e phosphotransferase occurs i n ca s t o r bean endosperm (40). Lack of such c o m p e t i t i o n suggests that separate enzymes occur i n the protozoan caudatum (41). CPT and EPT respond d i f f e r e n t l y to deoxycholate (42), and f a t t y a c i d s (43). I n d i r e c t evidence shows that r a t adipose c e l l s have separate enzymes (44). CPT may occur i n more than one form. CPT's with d i f f e r e n t K m 1 s f o r CDP-choline appear d u r i n g development of the chi c k b r a i n (45). Chick b r a i n g l i a l and neuronal CPT 1s have d i f f e r -ent K m's for CDP-choline (46). A l s o , r a t b r a i n and l i v e r CPT's are a f f e c t e d d i f f e r e n t l y by f a t t y a c i d s (43). - 11 -The remaining d i s c u s s i o n of CPT regards the r a t l i v e r en-zyme, unless otherwise s t a t e d . Since the s p e c i f i c r a d i o a c t i v i t y of CDP-choline from methyl-l a b e l l e d methionine was about equal to that of PC, of r a t l i v e r , 20-80 minutes a f t e r i n t r a p e r i t o n e a l i n j e c t i o n , B j ^ r n s t a d and Bremer proposed that r a p i d e q u i l i b r a t i o n took p l a c e over the CPT step (47). However, equal s p e c i f i c r a d i o a c t i v i t y was not con-firmed by Salerno and B e e l e r , using m e t h y l - l a b e l l e d methionine i n j e c t e d i n t r a p o r t a l l y (48). Sundler and co-workers have s i n c e shown that the pool s i z e of CDP-choline i s much smaller than p r e v i o u s l y estimated i f freeze-clamping of the t i s s u e i s performed (33). One minute of storage of l i v e r before e x t r a c t i o n r a i s e d the CDP-choline l e v e l f i v e - f o l d (49). The higher estimates of the CDP-choline p o o l obtained e a r l i e r caused a low estimate of s p e c i f i c r a d i o a c t i v i t y of CDP-choline a f t e r i n j e c t i o n of l a b e l l e d c h o l i n e . For example, the CDP-choline s p e c i f i c r a d i o a c t i v i t y i n l i v e r was repo r t e d as four times that of PC, but onl y o n e - t w e l f t h that of phosphocho-l i n e , at twenty minutes a f t e r i n t r a p e r i t o n e a l i n j e c t i o n (47). As a consequence, the importance of the CPT back r e a c t i o n was overestimated. A c u r r e n t estimate of the r a t i o of the forward v e l o c i t y to back v e l o c i t y jLn v i v o i s between 1.2 and 2.3 (49) . However, v e l o c i t y c a l c u l a t i o n s over t h i s step are complicated by the f a c t that d i f f e r e n t s p e c i e s and s u b c e l l u l a r pools of PC may be syn-t h e s i z e d a t d i f f e r e n t r a t e s (50) . - 12 -S t i l l , p h y s i c a l l y separate pools of PC may be u n l i k e l y , s i n c e the pools l a b e l l e d from c h o l i n e , methionine, or 1 - a c y l l y -s o l e c i t h i n are a l l e q u a l l y used by CPT f o r the back r e a c t i o n (51). Since these three pathways of s y n t h e s i s produce PC's which vary at the 2 - p o s i t i o n , i t appears that the t r a n s f e r a s e back r e a c t i o n has no marked s e l e c t i v i t y f o r f a t t y a c i d s at the 2-p o s i t i o n . Lack of s e l e c t i v i t y was v e r i f i e d by a n a l y s i s of spe-c i e s of the s u b s t r a t e , PC, and the product, d i g l y c e r i d e (52). S e l e c t i v i t y i s observed i n the 1 - p o s i t i o n , s i n c e the t r a n s -f e r a s e degrades i n d e c r e a s i n g order of p r e f e r e n c e , 1 - m y r i s t o y l , 1 - p a l m i t o y l , and then 1 - s t e a r o y l PC (53, 54). The CPT forward r e a c t i o n i s a l s o a f f e c t e d by f a t t y a c i d s at p o s i t i o n 1 of the s u b s t r a t e ( d i g l y c e r i d e ) . With exogenous d i -g l y c e r i d e s , 1 - p a l m i t o y l s p e c i e s are p r e f e r r e d over 1 - s t e a r o y l g l y c e r i d e s (55). S i m i l a r l y , using e t h a n o l - d i s p e r s e d d i g l y c e r i d e s , the CPT from r a t adipose c e l l s had a marked pr e f e r e n c e f o r d i o -l e i n over l - s t e a r o y l - 2 - o l e y l g l y c e r o l (44). The CPT forward r e a c t i o n has been shown to have l i t t l e spe-c i f i c i t y f o r f a t t y a c i d s of the 2 - p o s i t i o n of endogenous (53, 54) or exogenous (56) d i g l y c e r i d e . However, the degree of unsatura-t i o n of f a t t y a c i d s at t h i s p o s i t i o n does modify the degree of preference of 1 - p a l m i t o y l over 1 - s t e a r o y l d i g l y c e r i d e (55). A l s o , Holub has demonstrated that CDP-e.thanolamine (at a c o n c e n t r a t i o n of 24 uM) i n h i b i t s CPT 40% more i f exogenous hexaenoic d i g l y c e r -ide i s the s u b s t r a t e than i f exogenous monoenoic d i g l y c e r i d e i s used (57). T h i s e f f e c t may p a r t l y e x p l a i n the i n v i v o d i s c r i m i -n a t i o n a g a i n s t PC s y n t h e s i s from hexaenoic d i g l y c e r i d e (57). Assuming t h a t [_ 1 (3) - 3 H ~J g l y c e r o l i s i n c o r p o r a t e d i n t o PC and PE v i a p h o s p h a t i d i c a c i d o n l y ( F i g . 2), and Q 3 2 P ~J phos-phate i s i n c o r p o r a t e d v i a any CDP-base pathway, Sundler e_t a l were able to c a l c u l a t e the f a t t y a c i d d i s t r i b u t i o n of d i g l y c e r -ide u n i t s i n PC and PE which d i d not o r i g i n a t e from p h o s p h a t i d i c a c i d . By t h i s method, i t was c a l c u l a t e d that o n e - t h i r d and one-h a l f , r e s p e c t i v e l y , of hexaenoic and t e t r a e n o i c d i g l y c e r i d e s used i n PE s y n t h e s i s do not o r i g i n a t e from p h o s p h a t i d i c a c i d (58). These d i g l y c e r i d e s c o u l d a r i s e by the CPT back r e a c t i o n (58). Once formed, t e t r a e n o i c and hexaenoic PE's are p r e f e r e n t i a l l y methylated to form PC (54, 59-61). Thus, a c y c l i n g of p o l y e n o i c d i g l y c e r i d e u n i t s between PE and PC may occur in v i v o (53, 58) . C y c l i n g of hexaenoic d i g l y c e r i d e was i n i t i a l l y proposed by Tinoco e_t a l (62) . In. v i v o l a b e l l i n g i n d i c a t e d t h a t methyl groups of methionine c o u l d be t r a n s f e r r e d from PC to CDP-choline, p o s s i b l y by the CPT back r e a c t i o n (63). In r a t lung, Moriya and Kanoh observed an e q u i l i b r a t i o n of the in v i v o s p e c i f i c r a d i o a c t i v i t i e s of t e t r a e n o i c d i g l y c e r i d e s and PC's. However, the r e a c y l a t i o n of 1 - a c y l l y s o l e c i t h i n , r a t h e r than the CPT back r e a c t i o n , c o u l d be r e s p o n s i b l e f o r the e f f e c t (64). One other l i n e of evidence p o i n t s to the back r e a c t i o n o p e r a t i n g _in v i v o . The of CDP-choline f o r the CPT reverse r e a c t i o n i s ImM f o r the microsome-bound enzyme (51). CDP-choline - 14 -has a c o n c e n t r a t i o n of about 10 yM i n r a t l i v e r (49) so that i n h i b i t i o n should not occur. The p h y s i o l o g i c a l s i g n i f i c a n c e of the back r e a c t i o n i s s t i l l , as y e t , not c l e a r l y understood. Vance et a l . e s t a b l i s h e d that both CT and CPT are asymmet-r i c a l l y o r i e n t e d i n r a t l i v e r microsomes, s i n c e both are t o t a l l y a c c e s s i b l e .to t r y p s i n i n i n t a c t , one-sided microsomes (65). Coleman and B e l l have shown that a l l of the de novo enzymes numbered 1-7 i n F i g u r e 2 are d e f i n i t e l y on the p r o t e a s e - a c c e s -s i b l e , o r c y t o p l a s m i c s i d e of microsomes, except f o r p h o s p h a t i d i c a c i d phosphatase (The phosphatase a c t i v i t y was r e s i s t a n t to pro-t e o l y s i s i n i n t a c t and d i s r u p t e d microsomes) (66). (c) A l t e r n a t e Formation of PC (i) Phosphatidylethanolamine M e t h y l t r a n s f e r a s e In 1959, Bremer and Greenberg rep o r t e d that r a t s given an i n t r a p e r i t o n e a l i n j e c t i o n of Q Me - 1 ' * c ] ] methionine formed l i v e r l i p i d s c o n t a i n i n g l a b e l l e d mono- and di-methylethanolamine, and c h o l i n e (67). Adenosyl methionine would f u n c t i o n as a methyl donor (68). The enzyme system was found i n r a t l i v e r microsomes (68). Most r a d i o a c t i v i t y was i n c h o l i n e , with very l i t t l e i n mono-and di-methylethanolamine p h o s p h o l i p i d s , s u p p o r t i n g the con-c l u s i o n t h at the f i r s t m e t h y l a t i o n step i s r a t e - l i m i t i n g (68). Furthermore, pool s i z e experiments have shown that the l e v e l s of p h o s p h a t i d y l monomethylethanolamine and phosphatidylf.dimethyl-ethanolamine are three orders of magnitude, smaller than PE and PC i n r a t l i v e r (69). - 15 -Recently, separate enzymes which c a t a l y z e : 1. the methyla-t i o n of PE to the monomethyl product and 2. f u r t h e r m e t h y l a t i o n to form PC, have been demonstrated i n bovine a d r e n a l medulla and e r y t h r o c y t e membranes (70, 71). In the e r y t h r o c y t e , the f i r s t enzyme i s l o c a t e d on the cy t o p l a s m i c s u r f a c e , whereas the second i s on the e x t e r n a l membrane face (71). R a t . l i v e r phosphocholine_„and_.GDPreh61ine„are l a b e l l e d before PC i s l a b e l l e d , a f t e r an i n j e c t i o n of 3 H ~ J ethanolamine or methionine (48). Consequently, Salerno and Beeler proposed that phosphoethanolamine and CDP-ethanolamine were d i r e c t l y methylated in r a t l i v e r (48). However, t h i s idea i s u n l i k e l y , because a f t e r [~_ 3 H ~ J ethanolamine i s administered to r a t s , the s p e c i f i c r a d i o -a c t i v i t y of phosphocholine i s very low and does not change with time, when compared to that of phosphoethanolamine i n l i v e r (50). Methionine stimulates. PE N-methylation. A d d i t i o n of meth-io n i n e to r a t hepatocytes a t a c o n c e n t r a t i o n s l i g h t l y higher than the plasma l e v e l , caused a dou b l i n g of Q 3 * Q PE con v e r s i o n to PC (72). At a s a t u r a t i n g [~_ methyl- 1 ^ C j J methionine concen-t r a t i o n , N-methylation forms PC at 20-40% of the rate of synthe-s i s from [_ 3H~J g l y c e r o l i n these c e l l s (72) . Feedback i n h i b i -t i o n by S-adenosylmethionine may c o n t r o l N-methylation s i n c e t h i s compound i n h i b i t s r a t l i v e r ET. S-adenosylhomocysteine and S-ade-n o s y l e t h i o n i n e have no e f f e c t (73) . I n h i b i t i o n of ET could po-t e n t i a l l y reduce the amount of PE a v a i l a b l e f o r m e t h y l a t i o n . N-methylation could be r e g u l a t e d by the a v a i l a b i l i t y of the N-methyl bases. N-mono or N,N-dimethylethanolamine cause a reduced - 16 c o n v e r s i o n of |_. H U PE to PC, but i n c r e a s e the i n c o r p o r a t i o n of Q ^ C 3] methionine i n t o PC i n hepatocytes (72). T h i s r e s u l t suggests t h a t the bases may enter the m e t h y l a t i o n pathway, ( i i ) Base Exchange Ch o l i n e i s i n c o r p o r a t e d i n t o r a t l i v e r and b r a i n PC by a Ca + +-dependant, nucl e o t i d e - i n d e p e n d e n t process, c a t a l y z e d by a microsomal enzyme (74-76). P h o s p h o t i d y l s e r i n e i s s y n t h e s i z e d by a s i m i l a r C a + + - r e q u i r i n g energy-independent exchange i n r a t l i v e r mitochondria (77, 78). A s i n g l e enzyme c a t a l y z i n g the ex-change of p h o s p h o l i p i d ethanolamine and s e r i n e was p o s t u l a t e d (78). However, d i f f e r e n t e f f e c t s of pH and v a r i o u s i n h i b i t o r s , and d i f f e r i n g K m's, suggest that r a t l i v e r (79) and b r a i n (76) have separate exchange enzymes f o r c h o l i n e , ethanolamine, and s e r i n e . The s i g n i f i c a n c e of base exchange has been debated. Iri v i v o pulse l a b e l l i n g p rovided no evidence f o r any c h o l i n e i n c o r p o r a -t i o n i n t o r a t l i v e r PC by base exchange (48). N o v i k o f f hepatoma c e l l s a l s o l a c k exchange between f r e e and l i p i d - b o u n d c h o l i n e (80) . However, _in v i v o l a b e l l i n g with [ U C J c h o l i n e d i d r e -v e a l r a p i d i n c o r p o r a t i o n i n t o p o l y u n s a t u r a t e d PC's of r a t l i v e r , presumably by a base exchange process (33). Rat b r a i n has a pool of PC a v a i l a b l e f o r exchange which can be c a l c u l a t e d by p r e l a b e l l i n g PC with c h o l i n e , followed by com-p e t i t i v e removal of the base with c h o l i n e , ethanolamine, or s e r -ine l a b e l l e d with a second i s o t o p e . T h i s pool r e p r e s e n t s o n l y 3-6% of the t o t a l b r a i n microsomal PC (81). - 17 -In v i v o experiments i n r a t b r a i n i n d i c a t e t h a t base ex-change occurs i n the f i r s t 3 minutes a f t e r i n j e c t i o n of l a b e l l e d c h o l i n e (82, 83). C h o l i n e i n c o r p o r a t i o n i n t o l i p i d i s b i p h a s i c (82), and c h o l i n e i s p r e f e r e n t i a l l y i n c o r p o r a t e d i n t o PC r a t h e r than phosphocholine i n the f i r s t few minutes a f t e r i n j e c t i o n (83). Further support f o r a c h o l i n e base exchange enzyme i n r a t b r a i n comes from the r e s u l t of i n t r a c e r e b r a l i n j e c t i o n s with c h o l i n e and hemicholinium-3. In the cerebellum, f o r example, hemicholinium-3 causes dim i n i s h e d c h o l i n e p h o s p h o r y l a t i o n , but i n c r e a s e d c h o l i n e i n c o r p o r a t i o n i n t o l i p i d . Base exchange would e x p l a i n such r e s u l t s (84). The ethanolamine and s e r i n e exchange enzymes from r a b b i t b r a i n appear to be l o c a l i z e d i n neuronal c e l l s , with l i t t l e i n g l i a l c e l l s (85) . (d) P o t e n t i a l S i t e s of R e g u l a t i o n of De Novo Sy n t h e s i s (i) C h o line Transport PC b i o s y n t h e s i s may not be r e g u l a t e d by the same means i n a l l mammalian c e l l s . In N o v i k o f f hepatoma c e l l s , almost a l l of the c e l l u l a r a c i d - s o l u b l e f r a c t i o n d e r i v e d from l a b e l l e d c h o l i n e i n the medium, i s phosphocholine (86). The K m of c h o l i n e t r a n s -p o r t i s more than one order of magnitude below the K m of CK i n these c e l l s (87). At medium c o n c e n t r a t i o n s of c h o l i n e l e s s than 20 yM, the t r a n s p o r t K m i s equal to the K m of c h o l i n e i n c o r p o r a -t i o n i n t o l i p i d (80). At these c o n c e n t r a t i o n s , t r a n s p o r t i s ap-p a r e n t l y l i m i t i n g f o r PC b i o s y n t h e s i s . However, above 100 yM c h o l i n e , i n c o r p o r a t i o n i n t o l i p i d i s independent of c h o l i n e con-c e n t r a t i o n i n the medium, suggesting that another step becomes l i m i t i n g (80). - 18 -( i i ) C h o l i n e Kinase In c o n t r a s t to hepatoma c e l l s , most l a b e l l e d c h o l i n e added to the medium of E h r l i c h a s c i t e s c e l l s remains as c h o l i n e (not phosphocholine) i n s i d e the c e l l s . In these c e l l s , pulse-^chase experiments suggest t h a t CK i s l i m i t i n g (10). The polyamines, spermine and spermidine decrease the K m f o r [~_ATP - M g + + _ J of p a r t i a l l y p u r i f i e d r a t l i v e r CK (88). At 2 mM c o n c e n t r a t i o n , spermine i n c r e a s e s kinase a c t i v i t y n i n e - f o l d (88). Polyamines accumulate d u r i n g r a p i d t i s s u e growth. P o l y -amines co u l d i n i t i a t e PC s y n t h e s i s by s t i m u l a t i n g CK, but u n t i l the e f f e c t s on CT and CPT are known, a c o n c l u s i o n cannot be made. ( i i i ) C y t i d y l y l t r a n s f e r a s e In r a t l i v e r , the p o o l of phosphocholine i s 6 times that of c h o l i n e , and 160 times that of CDP-choline, which suggests that the r e a c t i o n c a t a l y z e d by CT i s l i m i t i n g (33). Phosphocholine l e v e l s might c o n c e i v a b l y be r e g u l a t e d by the a c t i o n of a phos-phatase. However, phosphocholine phosphatase a c t i v i t y does not p a r a l l e l the i n c o r p o r a t i o n of Q 3 H - J c h o l i n e i n t o p h o s p h o l i p i d s of two s t r a i n s of HeLa c e l l s (89). CT a c t i v i t y i n a c e t o n e - b u t a n o l - e x t r a c t e d post-microsomal supernatant of r a t l i v e r i s s t i m u l a t e d f i v e - f o l d by lysophos-p h a t i d y l c h o l i n e (LPC). PC d i d not s t i m u l a t e the enzyme a c t i v i t y (90). However, Choy and Vance found that LPC i n h i b i t s the pur-i f i e d forms of r a t l i v e r CT, whereas lysophosphatidylethanolamine (LPE) a c t i v a t e s . At lmg/ml.,.. p i g l i v e r LPE s t i m u l a t e s the low molecular weight form e i g h t e e n - f o l d , whereas p i g l i v e r LPC causes more than 99% i n h i b i t i o n ( 9 l ) . - 19 -Opposite e f f e c t s occur i n CT from r a t i n t e s t i n a l mucosa. The enzyme i s s t i m u l a t e d g r e a t l y by LPC, whereas LPE has no e f -f e c t (92). The p h y s i o l o g i c a l reason f o r LPC a c t i v a t i o n seems c l e a r by the f o l l o w i n g experiments. I f the i n t e s t i n a l mucosa i s d e p r i v e d of b i l e by a f i s t u l a , PC and p r o t e i n l a b e l l i n g from r a d i o a c t i v e p r e c u r s o r s are d i m i n i s h e d , while PE and t r i g l y c e r i d e l a b e l l i n g are u n a f f e c t e d (93). C h o l i n e or LPC added to i n t e s -t i n a l c e l l s r e s t o r e s p r o t e i n l a b e l l i n g and g r e a t l y s t i m u l a t e s PC l a b e l l i n g (93). Chylomicron r e l e a s e by mucosal c e l l s , which was i n h i b i t e d by the f i s t u l a , i s a l s o p a r t l y r e s t o r e d (93). F e t a l lung CT i s a l s o s t i m u l a t e d by p h o s p h o l i p i d , phospha-t i d y l g l y c e r o l ; . 7 being the most a c t i v e (36). Of the three d_e novo enzymes i n the protozoan, Ehtodinium  caudatum, onl y CT i s r e s t r i c t e d to the 144,000 x g_ supernatant f r a c t i o n . I f v a r i o u s amounts of supernatant are added to i n -c u b a t i o n mixtures t h a t c o n t a i n s u b s t r a t e s and c e l l membranes, a roughly l i n e a r s t i m u l a t i o n of 1 ** C c h o l i n e i n c o r p o r a t i o n i n t o PC occurs (31). T h i s r e s u l t suggests that CT i s l i m i t i n g . (iv) 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 F a t t y a c i d s are known to have a v a r i e t y of e f f e c t s on mic-rosomal CPT. O l e i c a c i d , f o r example, s t i m u l a t e s chicken l i v e r (94) or r a t l i v e r (43) CPT, i n the presence of exogenous d i g l y c e r -i d e s . However, the endogenous forward r e a c t i o n (no added d i g l y -c e r i d e ) i n r a t l i v e r i s i n h i b i t e d or u n a f f e c t e d by o l e a t e (43, 54, 95) . - 20 -ATP and CoA added to r a t l i v e r microsomes caused i n c r e a s e d c o n v e r s i o n of l a b e l l e d d i g l y c e r i d e to t r i g l y c e r i d e and reduced formation of PC, but had no e f f e c t on PE formation. P a l m i t o y l CoA had s i m i l a r e f f e c t s , suggesting that a c y l CoA's might regu-l a t e the s y n t h e s i s of d i f f e r e n t g l y c e r o l i p i d s (96). Sribney ejt a l found that ATP p l u s CoA or ATP p l u s pantetheine i n h i b i t r a t l i v e r CPT,but not i r r e v e r s i b l y . An a c y l CoA i s not l i k e l y the i n h i b i t i n g agent, p a r t i c u l a r l y s i n c e adenosine 5 1 - ( aB-methylene) t r i p h o s p h a t e w i l l l a r g e l y r e p l a c e ATP. I t was suggested t h a t CPT may be r e v e r s i b l y i n a c t i v a t e d by enzyme p h o s p h o r y l a t i o n (97). The Mn + +-dependant EPT i s s i m i l a r l y i n h i b i t e d , while Mg + +-depen-dant EPT i s i n h i b i t e d by ATP p l u s pantetheine, but not by ATP p l u s CoA (98). " (e) R e g u l a t i o n of P h o s p h a t i d y l c h o l i n e B i o s y n t h e s i s (i) L i v e r 1. A v a i l a b i l i t y of S u b s t r a t e s PC b i o s y n t h e s i s must be r e g u l a t e d by e i t h e r the a v a i l a b i l i t y of s u b s t r a t e s and/or the l e a s t a c t i v e , l i m i t i n g enzyme. The f i r s t type of r e g u l a t i o n e f f e c t i v e l y p l a c e s c o n t r o l at some p o i n t o u t s i d e the given pathway. T r i g l y c e r i d e and p h o s p h o l i p i d s y n t h e s i s are under independent c o n t r o l i n l i v e r . L i v e r t r i g l y c e r i d e l a b e l l i n g i s decreased and i n c r e a s e d r e s p e c t i v e l y , by f a s t i n g and r e f e e d i n g of r a t s (99). F a t t y a c i d s s t i m u l a t e the l a b e l l i n g of n e u t r a l l i p i d s i n r a t l i v e r s l i c e s (100). F a t t y a c i d s s t i m u l a t e t r i g l y c e r i d e l a b e l l i n g i n human l i v e r s l i c e s (101), and r a t hepatocytes (102). In a l l - 21 -cases, p h o s p h o l i p i d s y n t h e s i s i s much l e s s a f f e c t e d (99-102). As C 1''C I] p a l m i t a t e i s added to the medium of r a t l i v e r c e l l s , p h o s p h o l i p i d s are l a b e l l e d i n a h y p e r b o l i c f a s h i o n , whereas t r i -g l y c e r i d e l a b e l l i n g d i d not i n c r e a s e u n t i l higher c o n c e n t r a t i o n s o f p a l m i t a t e were reached. T r i g l y c e r i d e l a b e l l i n g i n c r e a s e d s i g -m o i d a l l y (103). Thus, as Akesson and Sundler have s t a t e d (49): "The data so f a r i n d i c a t e that the need f o r d i a c y l g l y c e r o l i n p h o s p h o l i p i d s y n t h e s i s w i l l f i r s t be met, then a d d i t i o n a l d i a c y l -g l y c e r o l w i l l be converted i n t o t r i a c y l g l y c e r o l . " Another f a c t o r i n s u b s t r a t e a v a i l a b i l i t y concerns c h o l i n e . Not a l l of the c h o l i n e pool i s c a l c u l a t e d as a v a i l a b l e f o r PC s y n t h e s i s . In r a t l i v e r t h i s p o o l r e p r e s e n t s 15% of the t o t a l (33). P o s s i b l y a separate pool of c h o l i n e might e x i s t i n mito-c h o n d r i a , s i n c e c h o l i n e i s o x i d i z e d i n r a t l i v e r mitochondria (104). S p e c i f i c r a d i o a c t i v i t y r e s u l t s suggest that separate cho-l i n e pools i n r a t h e a r t are used f o r PC and sphingomyelin syn-t h e s i s (105) . The s u b s t r a t e s , c h o l i n e and ethanolamine s t i m u l a t e the l a b e l l i n g of PC and PE, r e s p e c t i v e l y , from [] 3H^] g l y c e r o l , mea-sured i n i s o l a t e d r a t hepatocytes. Maximum s t i m u l a t i o n by ethan-olamine occurs a t 40 yM c o n c e n t r a t i o n , whereas s t i m u l a t i o n by c h o l i n e i s maximal a t 1 mM (72). These s t i m u l a t i o n s c o u l d be p h y s i o l o g i c a l l y r e l e v a n t based on the serum c o n c e n t r a t i o n of ethanolamine (17 yM) (72), and the c o n c e n t r a t i o n , o f c h o l i n e i n l i v e r (approx 0.24 mM) (33). However, the c o n c e n t r a t i o n of ethanolamine i n l i v e r i s approximately 0.1 mM (49, 106), which - 22 -would appear 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 the f r e e base. These l i v e r c o n c e n t r a t i o n s are only e s t i m a t e s , assuming t h a t there i s no compartmentation, and that the d e n s i t y of l i v e r i s 1 g/cc. 2. R a t e - L i m i t i n g Step The CK step i n r a t l i v e r i s c a l c u l a t e d , a c c o r d i n g to Infante (107) , to be f u r t h e r from e q u i l i b r i u m than the CT step. The d i s e q u i l i b r i u m r a t i o , p (mass a c t i o n r a t i o -f the e q u i l i b r i u m constant) i s 49 times smaller over the CK step than over the CT step, i t i s true however, that o n l y a f r a c t i o n of the c h o l i n e pool i s a v a i l a b l e f o r PC s y n t h e s i s , then would become gr e a t e r f o r CK, making the CK r e a c t i o n c l o s e r to e q u i l i b r i u m . M g + + might c o n t r o l the kinase a c t i v i t y s i n c e : 1. Mg + + forms the a c t i v e s u b s t r a t e with ATP and 2. Mg + + i n c r e a s e s the enzyme a f f i n i t y f o r [[ Mg-ATP H * (108). I t would be expected that changes i n f l u x through a r a t e -l i m i t i n g step would cause changes i n the mass a c t i o n r a t i o , and v i c e v e r s a . D i e t a r y c h o l i n e d e f i c i e n c y probably causes an e f -f e c t on the kinase mass a c t i o n r a t i o (107). However, c h o l i n e d e f i c i e n c y i s u n l i k e l y to have a s i n g l e e f f e c t (see below). Over any enzyme step, the f u r t h e r a r e a c t i o n i s from e q u i l -ibrium, the smaller w i l l be the f l u x response to a p r o p o r t i o n -ate change i n p.;, as caused by a change i n the mass a c t i o n r a t i o (109). CK and CT should thus be r e l a t i v e l y i n s e n s i t i v e to sub-s t r a t e and product c o n c e n t r a t i o n s . CPT i s probably c l o s e r to e q u i l i b r i u m _in v i v o (107) , and should be more s e n s i t i v e to meta-b o l i t e c o n c e n t r a t i o n s . - 23 -The maximal v e l o c i t i e s of CK and c y t o s o l i c CT i n r a t l i v e r are 3.1 y moles/min/10 g,_, l i v e r and 1.3 y moles/min/10 g, l i v e r ( c a l c u l a t e d from r e f . 110). The net s y n t h e s i s of PC i n r a t l i v e r i s estimated at 0.2 y mole /min/10 g, l i v e r (50). (This r a t e r e p r e s e n t s c o n v e r s i o n of phosphocholine i n t o CDP-choline p l u s PC, thus e x c l u d i n g the i n f l u e n c e due to the CPT back r e a c -t i o n . ) Hence, the f i r s t two steps of PC b i o s y n t h e s i s cannot be s a t u r a t e d or s u b s t r a t e independent enzymes (109) . Since the f l u x i s estimated to be much l e s s than V m a x f o r both s t e p s , the f l u x would be rather i n s e n s i t i v e to changes i n enzyme a c t i -v i t y . Thus, i t remains u n c e r t a i n as to whether enzyme a c t i v i t y or s u b s t r a t e a v a i l a b i l i t y c o n t r o l the f l u x through de novo PC b i o s y n t h e s i s . By comparison, ET can c l e a r l y be r a t e - l i m i t i n g f o r hepato-cyt e PE s y n t h e s i s s i n c e , a t s a t u r a t i n g c o n c e n t r a t i o n s f o r l a b e l -l i n g of PE, ethanolamine causes an accumulation of ethanolamine phosphate, and not CDP-ethanolamine (72). On the other hand, s t i m u l a t i o n of PE l a b e l l i n g by o l e i c a c i d , and i n h i b i t i o n by l a u r i c a c i d , appear to be d i r e c t e d at EPT. O l e i c and l a u r i c a c i d s i n c r e a s e , and decrease, r e s p e c t i v e l y , the amount of hepato-cyt e d i g l y c e r i d e d u r i n g 15-30 minutes i n c u b a t i o n . The f a t t y a c i d s do not a p p r e c i a b l y a f f e c t phosphoethanolamine or CDP-ethanolamine l e v e l s . (72). 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 Roberts and Bygrave found that r a t l i v e r PE s y n t h e s i s i n  v i t r o was i n h i b i t e d by C a + + , which c o u l d be reversed by Mg + +. - 24 -Mit o c h o n d r i a , which take up Ca , s t i m u l a t e d PE s y n t h e s i s . The authors proposed that p h o s p h o l i p i d s y n t h e s i s is' c o n t r o l l e d by the M g + + / C a + + r a t i o (111). M i t o c h o n d r i a s t i m u l a t e r a t l i v e r PE s y n t h e s i s f i v e times more than PC s y n t h e s i s . T h i s 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 not due to ATP s y n t h e s i s . The C a + + c h e l a t o r , EGTA, mimics such s t i m u l a t i o n (112). A p o s s i b l e e x p l a n a t i o n f o r t h i s e f f e c t i s the higher s e n s i t i v i t y of r a t l i v e r EPT to C a + + i n h i b i t i o n than CPT, which i s known to/.be true f o r the back r e a c t i o n (51) . I t i s i n t e r e s t i n g i n t h i s regard that M g + + and Mn + + form a metal i on complex with the phosphotransferase s u b s t r a t e , CDP-choline, but C a + + does not (113). A l t e r n a t i v e l y , i f CPT i s not a l i m i t i n g enzyme, the e f f e c t s of C a + + might be on CK or CT, except a t ex-treme i n h i b i t i o n of CPT. C a + + uptake i n r a t l i v e r microsomes i s i n v e r s e l y c o r r e l a t e d with the l a b e l l i n g of 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 1 1 +C~ J glycerol-3-phosphate (114). S i m i l a r l y , a d d i t i o n of the C a + + i o n -phore, A23187, reduces the l a b e l l i n g of t r i g l y c e r i d e and phospho-l i p i d from C 3 H ^ ] g l y c e r o l , when t e s t e d i n lymphocytes (115). However, i n t h i s case, d i g l y c e r i d e l a b e l l i n g was i n c r e a s e d , pos-s i b l y through the a c t i o n of a C a + + - s e n s i t i v e t r i g l y c e r i d e l i p a s e . I f the l e v e l of c h o l i n e d i c t a t e d the ra t e of PC s y n t h e s i s a u s e f u l model might be to l i m i t d i e t a r y c h o l i n e . The l e v e l of fr e e c h o l i n e i n r a t l i v e r i s reduced 40-70% when c a l c u l a t e d per gram body weight, by about 1 week of c h o l i n e d e f i c i e n c y (116). However, l i p i d - b o u n d l e v e l s are not changed (116). - 25 -C h o l i n e d e f i c i e n c y a l s o a l t e r s the a c t i v i t i e s of enzymes of g l y c e r o l i p i d s y n t h e s i s . Rat l i v e r d i g l y c e r i d e a c y l t r a n s f e r -ase i s i n c r e a s e d i n a c t i v i t y , whereas 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 is' unchanged, d u r i n g c h o l i n e d e f i c i e n c y (117). Skurdal and Cornatzer r e p o r t e d a decrease of h e p a t i c CPT i n c h o l i n e d e f i -c i e n t r a t s (118, 119), which was not s u b s t a n t i a t e d by Schneider and Vance (110). CK i s u n a f f e c t e d , whereas c y t o s o l i c CT decreases s i g n i f i c a n t l y (110). The amount of CT, measured by antibody t i -t r a t i o n , i s not a l t e r e d (120). Thus, a higher p r o p o r t i o n of CT must be i n a c t i v e i n the c h o l i n e - d e f i c i e n t r a t • l i v e r . L i v e r PEMT i s s t i m u l a t e d by c h o l i n e d e f i c i e n c y i n rats;. (110) . Corresponding to t h i s r e s u l t , c h o l i n e d e f i c i e n c y causes an _in v i v o s t i m u l a t i o n of Q M e - 1 k C m e t h i o n i n e i n c o r p o r a t i o n i n t o l i v e r l i p i d - b o u n d c h o l i n e (116). Increased m e t h y l a t i o n might compen-sate f o r a l a c k of f r e e c h o l i n e . The f a t t y l i v e r and decreased blood l i p i d observed i n cho-l i n e d e f i c i e n c y c o u l d be e x p l a i n e d i f d e f i c i e n c y impairs forma-t i o n of a s p e c i e s of PC necessary f o r s y n t h e s i s or t r a n s p o r t of plasma l i p o p r o t e i n s , which i n turn are necessary f o r h e p a t i c l i p i d r e l e a s e (121). A l t e r n a t i v e l y , a l a c k of phosphocholine or CDP-choline c o u l d be r e s p o n s i b l e f o r d e f e c t i v e l i p o p r o t e i n s y n t h e s i s . Phosphocholine (but not c h o l i n e or CDP-choline) doubles p r o t e i n s y n t h e s i s i n a r a t l i v e r c e l l - f r e e system (122). The pool of phosphocholine i n r a t l i v e r i s l a r g e , comprising 9% of the w a t e r - s o l u b l e phosphate (123). At two days of c h o l i n e d e f i c i e n c y , phosphocholine l e v e l s (u m o l e s / l i ver/gram body weight) - 26 -are 70% lower than i n choline-supplemented r a t s (124). A l s o , i t has been noted t h a t CDP-choline s t i m u l a t e s the g l y c o s y l a t i o n of r a t l i v e r 3 - l i p o p r o t e i n (125) . 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 of p h e n o b a r b i t a l cause an i n -crease i n the r a t i o of r a t l i v e r microsomal p h o s p h o l i p i d to mic-rosomal p r o t e i n (126, 127). An i n c r e a s e i n l i v e r p h o s p h o l i p i d s p e c i f i c r a d i o a c t i v i t y has been observed, s u r p r i s i n g l y , i n male but not female r a t s , a f t e r p h e n o b a r b i t a l treatment (128). The p h o s p h o l i p i d i n c r e a s e i s mainly accounted f o r by a l a r g e r p o o l of PC (127, 129). At 12 hours a f t e r p h e n o b a r b i t a l i n j e c t i o n , the l a r g e r PC pool appears to a r i s e by PE N-methylation (127, 130). Feeding ethanol to r a t s , l i k e p h e n o b a r b i t a l i n j e c t i o n , causes p r o l i f e r a t i o n of h e p a t i c smooth endoplasmic r e t i c u l u m . Ethanol i n g e s t i o n has been shown to produce s i g n i f i c a n t i n c r e a s e s in l i v e r CPT and PEMT, which might account f o r i n c r e a s e d mem-brane s y n t h e s i s (131). 4. Phosphatidate PC s y n t h e s i s may be r e g u l a t e d i n the de novo pathways ( F i g . 3) or i n the m e t h y l a t i o n of PE (Fig.. 2). A t h i r d pos-s i b i l i t y would be r e g u l a t i o n by the r e a c t i v i t y of phosphatidate. Phosphatidate may be converted to C D P - d i g l y c e r i d e by a r e a c t i o n with CTP, c a t a l y z e d by CTP phosphatidate c y t i d y l y l t r a n s f e r a s e ; or to d i g l y c e r i d e by phosphatidate phosphohydrolase. CDP-d i g l y c e r i d e i s the p r e c u r s o r of the a c i d i c p h o s p h o l i p i d s , p h o s p h a t i d y l g l y c e r o l , p h o s p h a t i d y l s e r i n e , and p h o s p h a t i d y l i -n o s i t o l . D i g l y c e r i d e i s a p r e c u r s o r of t r i g l y c e r i d e , PC and PE ( F i g . 2). - 27 -The r a t i o of a c t i v i t i e s of the c y t i d y l y l t r a n s f e r a s e and phosphatase i s constant i n r a t l i v e r microsomes over a wide range of phospholipase-generated membrane-bound phosphatidate (132), i n d i c a t i n g that phosphatidate does not c o n t r o l t h i s r a t i o . The phosphatase may be a r e g u l a t o r y enzyme, s i n c e i t s ac-t i v i t y c o r r e l a t e s with t r i g l y c e r i d e b i o s y n t h e s i s i n l i v e r (133). M g + + and a m p h i p h i l i c c a t i o n i c drugs cause r e l a t i v e l y more con-v e r s i o n of phosphatidate to CDP d i g l y c e r i d e , and l e s s to d i g l y -c e r i d e , probably through p h y s i c a l i n t e r a c t i o n s of the c a t i o n s with phosphatidate (134-136). However, the r e g u l a t o r y r o l e of phosphatidate phophatase i s q u e s t i o n a b l e s i n c e the amount of i t s s u b s t r a t e , phosphatidate, i s o n l y one-quarter of that of d i g l y c e r i d e i n r a t l i v e r micro-somes (137). Often, s u b s t r a t e b u i l d s up at a r e g u l a t o r y step, ( i i ) . . Lung The r e g u l a t i o n of lung PC s y n t h e s i s has been e x t e n s i v e l y s t u d i e d and deserves some comment here. Mammalian lung c o n t a i n s a s u r f a c e - a c t i v e m a t e r i a l c a l l e d s u r f a c t a n t . T h i s m a t e r i a l ( i n dog lung) i s composed of 85% l i p i d , of which 75% i s PC. Sur-f a c t a n t d e f i c i e n c y i s thought to cause i n f a n t r e s p i r a t o r y d i s -t r e s s syndrome, which i s c h a r a c t e r i z e d by a t e l e c t a s i s ( f a i l u r e of lung expansion). D i p a l m i t o y l PC i s an e s s e n t i a l component of s u r f a c t a n t i i i i t s f u n c t i o n of d e c r e a s i n g s u r f a c e t e n s i o n (138). The d i p a l m i t o y l s p e c i e s i s predominant among PC's of r a t , rab-b i t , p i g and sheep lung, but not cow lung (139) . - 28 -S u r f a c t a n t i s s y n t h e s i z e d by Type 2 e p i t h e l i a l c e l l s of lung (Type 1 e p i t h e l i a l c e l l s are the s t r u c t u r a l c e l l s of the a l v e o l a r wall) (140). PE N-methylation i s minor i n lung com-pared to s y n t h e s i s of PC v i a CDP-choline (141). However, the CPT s e l e c t i v i t y would appear to r u l e out an a c t i o n of t h i s en-zyme i n d i p a l m i t o y l PC s y n t h e s i s . Endogenous d i p a l m i t o y l PC and phospholipase-generated d i p a l m i t o y l g l y c e r o l are poor sub-s t r a t e s f o r the back and forward r e a c t i o n s i n mouse lung micro-somes (141). S i m i l a r l y , the enzyme from r a t lung i s p o o r l y ac-t i v e with exogenous d i p a l m i t o y l g l y c e r o l (142). The l a r g e i n c r e a s e of s a t u r a t e d PC observed i n r a t lung p r i -or to b i r t h i s p a r a l l e l e d by an i n c r e a s e i n i n c o r p o r a t i o n of ; [ ] l - l l t C ~ J p a l m i t o y l LPC i n t o s a t u r a t e d PC. In c o n t r a s t , Q 3 H ~J g l y c e r o l i n c o r p o r a t i o n i n t o s a t u r a t e d PC was constant d u r i n g de-velopment. R a d i o a c t i v i t y i n the d i s a t u r a t e d PC's was l o c a t e d about e q u a l l y at C - l and C-2 of g l y c e r o l . T h i s f i n d i n g i n d i -c a t e s that a c y l a t i o n of LPC occurs by LPC:LPC a c y l t r a n s f e r a s e , not d i r e c t a c y l a t i o n by a c y l CoA:LPC a c y l t r a n s f e r a s e (143). Si m i - . l a r l y , i n mouse lung, LPC;LPC a c y l t r a n s f e r a s e i n c r e a s e s drama-t i c a l l y before b i r t h , but a c y l CoA:LPC a c y l t r a n s f e r a s e does not change (144). These two a c y l t r a n s f e r a s e s (and l y s o p h o s p h a t i d i c a c i d a c y l t r a n s f e r a s e ) c o r r e l a t e " w i t h i n c r e a s i n g PC content of r a b b i t lung d u r i n g f e t a l development, while the three de novo enzymes ( F i g . 3) remained constant or d e c l i n e d (145, 146). On the other hand, the o n l y known primary route f o r the r a p i d s y n t h e s i s of lung PC around the time of b i r t h i s by de novo - 29 -s y n t h e s i s . The i s s u e i s s t i l l open s i n c e : 1. For r a t lung, c o n f l i c t i n g f i n d i n g s have shown both i n -creases and lack of i n c r e a s e s of a l l three de novo enzymes dur-ing f e t a l development (145, 147). 2. Perhaps none of the de novo enzymes i s l i m i t i n g f o r PC s y n t h e s i s . Although CPT has the lowest s p e c i f i c a c t i v i t y of the de novo enzymes i n f e t a l monkey lung (147) and i n f e t a l r a t lung (148), t h i s r e s u l t i s i n c o n c l u s i v e s i n c e ijn v i t r o r e a c t i o n r a t e s are under o p t i m a l c o n d i t i o n s which are not l i k e l y met _in v i v o . Thus, enzyme s p e c i f i c a c t i v i t i e s are not proof of a l i m i t i n g step. 3. The a v a i l a b i l i t y of d i g l y c e r i d e may determine PC syn-t h e s i s . P h o s p h a t i d i c a c i d phosphatase, which produces d i g l y -c e r i d e , i n c r e a s e s i n s p e c i f i c a c t i v i t y i n r a b b i t lung d u r i n g l a t e g e s t a t i o n (149). The CT from r a t lung i s s t i m u l a t e d 700% by lung l i p i d four days before b i r t h , but only 67% one day a f t e r b i r t h (148), sug-g e s t i n g a r e g u l a t i o n of enzyme a c t i v i t y . Without a c t i v a t i n g l i p i d , the enzyme a c t i v i t y i s s t i l l low one day before b i r t h , i n c r e a s i n g d r a m a t i c a l l y a f t e r b i r t h (148). C o r r e l a t i n g with t h i s r e s u l t , Q 11*C ]^ c h o l i n e i n c o r p o r a t i o n i n t o l i p i d i n c r e a s e s to a d u l t l e v e l s i n r a t lung s l i c e s j u s t before b i r t h (150). P h o s p h a t i d y l g l y c e r o l i s i m p l i c a t e d i n lung CT a c t i v i t y and p o s s i b l y PC s y n t h e s i s at b i r t h s i n c e : 1. P h o s p h a t i d y l g l y c e r o l i n c r e a s e s i n amount i n r a b b i t ' s u r f a c t a n t by f i v e - f o l d w i t h i n one day a f t e r b i r t h (151) . 2. P h o s p h a t i d y l g l y c e r o l accounts s o l e l y f o r the s t i m u l a t i o n - 30 -of f e t a l CT from r a t lung by a d u l t r a t lung l i p i d ( 3 6 ) . 3 . P h o s p h a t i d y l g l y c e r o l i s absent i n lung e f f l u e n t from newborn i n f a n t s with r e s p i r a t o r y d i s t r e s s syndrome and prese n t i n newborn c o n t r o l i n f a n t s ( 1 5 2 ) . C o r t i s o l s t i m u l a t e s the p r o d u c t i o n of PC i n f e t a l lung lavage i n r a b b i t s , but does not s t i m u l a t e sphingomyelin pro-d u c t i o n ( 1 5 3 ) . At present, the enzyme (or enzymes) of de novo s y n t h e s i s which responds, to 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 s i n c e c o n f l i c t i n g r e s u l t s have been repo r t e d ( 1 5 4 ) . ( i i i ) B r a i n I t has been proposed t h a t c h o l i n e i s t r a n s p o r t e d i n the blood to the b r a i n i n a l i p i d - b o u n d form ( 1 5 5 ) . T h i s idea i s tenable s i n c e , 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 of [~ 1''C 3 ethanolamine w i l l l a b e l plasma PC and LPC, but u n e s t e r i f i e d cho-l i n e i s not l a b e l l e d (156) ( Q 1 4C ~ Jethanolamine would l a b e l h e p a t i c PC by N-methylation of PE, which c o u l d then be exported from the l i v e r ) . The o r i g i n of c h o l i n e i n r a t b r a i n remains u n c e r t a i n . B r a i n cannot s i g n i f i c a n t l y c o n vert ethanolamine l i p i d s to cho-l i n e l i p i d s by m e t h y l a t i o n (157) . A f t e r i n t r a p e r i t o n e a l i n j e c -t i o n of r a d i o a c t i v e ethanolamine, A n s e l l and Spanner d i d not d e t e c t l a b e l l e d f r e e c h o l i n e i n the b r a i n ( 1 5 5 ) . By c o n t r a s t , Kewitz and P l e u l found that intravenous i n j e c t i o n s of r a d i o a c -t i v e ethanolamine caused s i g n i f i c a n t l a b e l l i n g of b r a i n c h o l i n e . In f a c t , b r a i n c h o l i n e had a higher s p e c i f i c r a d i o a c t i v i t y than PC. Kewitz and P l e u l proposed t h a t f r e e ethanolamine i s methyl-ated i n r a t b r a i n , forming c h o l i n e ( 1 5 8 ) . - 31 -In neuroblastoma c e l l s , PE and PC appear to be s y n t h e s i z e d by separate pathways. Monomethylethanolamine ente r s the PE pathway and dimethylethanolamine ent e r s the PC pathway (159). Mammalian b r a i n CT was proposed as r a t e - l i m i t i n g i n PC s y n t h e s i s (160). T h i s p r o p o s a l was however based on in v i t r o enzyme a c t i v i t i e s . Phosphocholine i s g e n e r a l l y l a b e l l e d from exogenous c h o l i n e to a higher extent than c h o l i n e or CDP-choline i n embryonic r a t c e r e b r a l c e l l s (15), and i n r a b b i t neuronal and g l i a l c e l l s (161). T h i s f i n d i n g a l s o t e n t a t i v e l y p o i n t s to the CT as l i m i t i n g i n PC s y n t h e s i s . In summary, although s u b s t r a t e a v a i l a b i l i t y cannot be r u l e d out, the main c o n t r o l p o i n t f o r de novo PC s y n t h e s i s may be at the CT step. The l a r g e pool of phosphocholine i n r a t l i v e r sug-gests that CT i s l i m i t i n g . As mentioned, CT a c t i v i t y c o r r e l a t e s with the expected r a t e of PC s y n t h e s i s , f a l l i n g i n the l i v e r d uring c h o l i n e d e f i c i e n c y , and r i s i n g i n lung t i s s u e about the time of b i r t h . (f) E f f e c t s of 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 The use of c e l l c u l t u r e to study l i p i d s y n t h e s i s p r o v i d e s s e v e r a l advantages over whole animal or organ s t u d i e s : 1. Homogenous p o p u l a t i o n s of c e l l s are e a s i l y o b t a i n e d . 2. S m a l l - s c a l e experiments can be e f f i c i e n t l y performed. 3. L a b e l l e d p r e c u r s o r s may be added d i r e c t l y to the c e l l medium. G a l l a h e r and Blough have repo r t e d that PC i n BHK-21 c e l l s has a h a l f - l i f e of l e s s than four hours (162). T h i s r e s u l t was - 3 2 -based on pulse-chase and e q u i l i b r i u m l a b e l l i n g with r a d i o a c t i v e g l y c e r o l or c h o l i n e . The same authors made the i n t e r e s t i n g f i n d i n g t h at s y n t h e s i s of PC i s halved while PE s y n t h e s i s doub-l e s d u r i n g density-dependant i n h i b i t i o n of BHK c e l l s growth. Sy n t h e s i s was estimated by l a b e l l i n g with L7 2- 3H~J g l y c e r o l (163). Serum causes an i n c r e a s e i n the s p e c i f i c r a d i o a c t i v i t y of 3 2 P - l a b e l l e d PC of E h r l i c h a s c i t e s c e l l s . S p e c i f i c r a d i o a c -t i v i t y of the pr e c u r s o r ATP i s s t i m u l a t e d much l e s s . T h i s e f -f e c t on PC i s l i k e l y on turnover, s i n c e the PC po o l s i z e i s not app a r e n t l y i n c r e a s e d (164). The i n c o r p o r a t i o n o f Q 1^C J ace-t a t e i n t o p h o s p h o l i p i d s of e a r l y passage WI-38 c e l l s i s stimu-l a t e d f i v e - f o l d by changing the medium i n the absence of serum. Older c u l t u r e s , however, r e q u i r e d serum p r o t e i n to d i s p l a y t h i s e f f e c t (165) . Many v i r u s i n f e c t i o n s of animal c e l l s are known to p e r t u r b c e l l u l a r PC and PE s y n t h e s i s (Table I ) . No v i r u s i n f e c t i o n has been shown c o n c l u s i v e l y to have a d i f f e r e n t e f f e c t on PC s y n t h e s i s compared to PE syn t h e s i s , a l -though i n c o r p o r a t i o n data i n c e l l s i n f e c t e d with Newcastle d i s -ease v i r u s suggests such an e f f e c t (175). The i n h i b i t i o n of Q 1 hC ~J c h o l i n e i n c o r p o r a t i o n by S i n d b i s v i r u s (Table 1) (172) was examined using v i r u s temperature-sen-s i t i v e mutants. Mutants with d e f e c t i v e n u c l e o c a p s i d or membrane p r o t e i n s i n h i b i t e d c h o l i n e i n c o r p o r a t i o n . However, mutants un-able to s y n t h e s i z e v i r a l RNA d i d not cause i n h i b i t i o n . The authors proposed t h a t i n h i b i t i o n of host RNA and/or p r o t e i n Table 1 Effect of virus infections on animal PC and PE synthesis: Virus Family Virus Picornaviridae Polio virus (166). Picornaviridae Mengovirus (167) Adenoviridae Adenovirus type 5 (168) Herpetoviridae Pseudorabies virus (169) Retroviridae Togaviridae Togaviridae Togaviridae Friend virus (170) Japanese encephalitis virus (171) Sindbis virus (172) Semliki Forest virus (173, 174) Cell Line Time of (or Animal) Infection HeLa L HEK RK BHK-21 BHK-21 9 h 8 h 9 h BALB/c mice 14 days 24 h Chick embryo 10 h fibroblasts 7 h 1 '-•' represents 'incorporation into 1 2 (sa) specific activity 3 represents 'increase' 4 'i' represents 'decrease' 5 PC plus sphingomyelin 6 specific activity = % lipid cpm in PC or PE ; pg protein The numbers in brackets refer to references. Measurement 6 h L~32P]pi *1PC (sa) r-3 2p- pi + pE ( s a ) r_l"C]choline -» PC (sa) jj*c] acetate •> PC ( s a ) 5 , 6 f_"*C]acetate •<• PE (sa) 6 r_3H[] choline •* lipid [^ "•C^  choline •* PC (sa) ["C] ethanolamine •* lipid minus PC (sa) [^'"C^oleate + PE [/"C^oleate •* PC PC PE L~32P>i £?H] choline •» PC [J'^C] ethanolamine ->• PE Effect on: PC PE Synthesis Synthes +370% + 60% + 60% t 20% + 90% +470% + 60% + 40% +120% t 60% + 60% + 70% + 40% + 40% - 34 -s y n t h e s i s was r e s p o n s i b l e f o r the i n c o r p o r a t i o n e f f e c t . In sup-p o r t of t h i s i d e a , actinomycin D (which i n h i b i t s DNA-dependant RNA sy n t h e s i s ) and cycloheximide (which i n h i b i t s p r o t e i n syn-t h e s i s ) i n h i b i t c h o l i n e i n c o r p o r a t i o n i n c h i c k embryo c e l l s . However, i n BHK c e l l s actinomycin D has no e f f e c t and cyc l o h e x -imide has o n l y a slow i n h i b i t o r y e f f e c t on c h o l i n e i n c o r p o r a -t i o n (172). S i n d b i s v i r u s i n f e c t i o n does, on the other hand, cause a smal l r e p r o d u c i b l e decrease i n c h o l i n e i n c o r p o r a t i o n by BHK c e l l s (172). The cause of t h i s decrease i s consequently obscure. (g) E f f e c t of 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 B i o s y n t h e s i s S e m l i k i F o r e s t v i r u s ( l i k e S i n d b i s v i r u s ) i s a member of the genus A l p h a v i r u s , i n the T o g a v i r i d a e f a m i l y (176, 177). Sem-l i k i F o r e s t v i r u s c o n s i s t s of an inner n u c l e o c a p s i d and an outer l i p i d envelope. The l i p i d envelope a p p a r e n t l y o r i g i n a t e s from the host plasma membrane, s i n c e t h e i r l i p i d compositions are s i m i l a r (178). The envelope c o n t a i n s three g l y c o s y l a t e d pro-t e i n s , E i , E 2 and E 3 . The n u c l e o c a p s i d core i s composed of a s i n g l e p r o t e i n s p e c i e s and s i n g l e - s t r a n d e d RNA of molecular weight 4 .5 x 10 6 (177) . Richardson and Vance have pr o v i d e d e v i -dence that envelope p r o t e i n s move from endoplasmic r e t i c u l u m to the plasma membrane of i n f e c t e d BHK c e l l s (179). The v i r u s e x i t s the c e l l by budding, a process which may e x e r t some e f f e c t on PC s y n t h e s i s . S e m l i k i F o r e s t v i r u s i n f e c t i o n caused 50% and 40% i n h i b i -t i o n o f the t o t a l and s p e c i f i c a c t i v i t i e s , r e s p e c t i v e l y , of BHK - 35 -c e l l CPT, 7 hours a f t e r i n o c u l a t i o n (173). Actinomycin D and cycloheximide do not mimic t h i s e f f e c t , so that i n h i b i t i o n of host RNA or p r o t e i n s y n t h e s i s are not l i k e l y causes (173) . I t i s a l s o known that S e m l i k i F o r e s t v i r u s i n f e c t i o n of BHK or c h i c k embryo c e l l s reduces the pool s i z e of ATP, ADP, CTP, and CDP-choline (Table I I ) . One of these pools c o u l d c o n t r o l PC s y n t h e s i s . A l s o , C a + + uptake i n t o c h i c k embryo f i b r o b l a s t mito-cho n d r i a i s e l e v a t e d immediately and 2.5 hours a f t e r SF v i r u s - i n -f e c t i o n (182). C a + + may r e g u l a t e PC s y n t h e s i s although mito-c h o n d r i a l r e l e a s e , not uptake, would be expected to i n h i b i t s y n t h e s i s of the l i p i d . Table W% E f f e c t s of S e m l i k i F o r e s t V i r u s I n f e c t i o n on BHK C e l l Pool S i z e s C e l l Type PFU/cell Time of infection Pool Infected/ Mock-infected Pool Size Ratio Reference CEF CEF lv>> 1 BHK/CEF BHK/CEF1 5 5 10-50 10-50 5h 5h 4h 4h ATP ADP CTP CDP-choline 0.68 1.25 0.15 0.30 (180) (180) (181) (181) 1. C e l l type was not c l e a r l y s t a t e d i n the o r i g i n a l r e f e r e n c e The aim of the t h e s i s p r o j e c t o u t l i n e d here was to e l u c i -date the p r e c i s e mechanism of S e m l i k i F o r e s t v i r u s i n h i b i t i o n of c h o l i n e i n c o r p o r a t i o n i n t o PC. The four steps of de novo syn-t h e s i s o u t l i n e d i n t h i s i n t r o d u c t i o n were examined i n mock-infected - 36 -and v i r u s - i n f e c t e d c e l l s . However, s i n c e i t was r e a l i z e d that i n vierQcenzyme a c t i v i t i e s do not n e c e s s a r i l y correspond with the ' i n v i v o a c t i v i t i e s , p ool s i z e measurements were r e q u i r e d . Sen-s i t i v e enzyme assays of c h o l i n e , phosphocholine, CDP-choline, CTP, and d i g l y c e r i d e were performed. Much of the c u r r e n t metho-dology f o r these assays was i n s u f f i c i e n t , so t h a t m o d i f i c a t i o n s or new methods were necessary. A l s o , the PC pool was measured i n both mock- and v i r u s - i n f e c t e d c e l l s . Since the pool s i z e r e s u l t s d i d not unambiguously d e l i n e a t e the p o i n t of i n h i b i t i o n i t was decided to perform pulse-chase experiments with Q3H^] cho-l i n e . As a r e s u l t , i t was shown that the disappearance of Q3H^j phosphocholine i n i n f e c t e d c e l l s was i n h i b i t e d i n a f a s h i o n p a r a l -l e l to the i n h i b i t i o n of i n c o r p o r a t i o n .into PC. However, because the pool of phosphocholine was enlarged i n i n f e c t e d c e l l s , the turnover r a t e of phosphocholine was not reduced by v i r u s i n f e c -t i o n . The reason f o r an enlarged phosphocholine pool remains un-c e r t a i n , but the l i k e l y cause was a t r a n s i e n t r e d u c t i o n i n CT ac-t i v i t y , as a r e s u l t o f the diminished pool s i z e of CTP. - 37 -' MATERIALS AND METHODS (a) Chemicals and Isotopes C h o l i n e c h l o r i d e , c h o l i n e i o d i d e , phosphocholine, egg yolk PC (Type V-E and 1T-E) , t a u r o c h o l i c a c i d , and T r i s were purchased from Sigma. N u c l e o t i d e s were obtained from e i t h e r Sigma or P-L B i o c h e m i c a l s . L i p i d s (pig l i v e r PC and PE, d i p a l m i t o y l PC, p a l m i t o l e y l LPC, o l e y l LPE, d i p a l m i t i n , and d i o l e i n ) were sup-p l i e d by Serdary Research L a b o r a t o r i e s . N o r i t - A c h a r c o a l , Phenol Reagent S o l u t i o n 2N (for p r o t e i n d e t e r m i n a t i o n ) , and sodium deoyxcholate were products of F i s h e r S c i e n t i f i c . T r i -c h l o r o a c e t i c a c i d was from Amachem, while p e r c h l o r i c ; . a c i d was from M a l l i n c k r o d t . 3-Heptanone was produced by Matheson, C o l e -man & B e l l . Other chemicals were of reagent grade. F e t a l c a l f serum was s u p p l i e d by Flow L a b o r a t o r i e s . Dul-becco's M o d i f i e d Eagle Medium, Medium 199, and E a r l e ' s Balanced S a l t S o l u t i o n were products of Grand I s l a n d B i o l o g i c a l Company. Enzymes ( a l k a l i n e phosphatase, phosphodiesterase, c h o l i n e kinase, g l y c e r o k i n a s e , and i n o r g a n i c pyrophosphatase) were bought from Sigma. The anion exchanger, AG 1-X10 and the c a t i o n exchanger, AG 50W-X8 were purchased from Bio-Rad L a b o r a t o r i e s . r~5-3H~] C y t i d i n e 5 ' - t r iphosphate , [[Methyl- 3H~ J S-adenosyl-L-methionine , Q 3 H ~ J toluene, r~_l, 2- 1 ' • C J J c h o l i n e c h l o r i d e , (jYlethyl- 1 1*C - J c y t i d i n e diphosphocholine , [^Methyl- 1 ^ C j J phospho-c h o l i n e , and [ [ ^ C - ] toluene were obtained from New England Nu-c l e a r . []3H3] Hexadecane was from The Radiochemical Centre, Amer-sham. [_y-32P~2 Adenosine 5 '-triphosphate was from Amersham/ - 38 -S e a r l e . Qyiethyl- 3H^] C h o l i n e c h l o r i d e was s u p p l i e d by New England Nuclear or Amer sham/Sear l e . C 1 ^ ^ ] Glycerophosphocho-l i n e was a product of ICN Pharmaceuticals. [jyiethyl- 3H^] Phos-phocholine was sy n t h e s i z e d by the technique of Paddon and Vance (89). (b) General Methods (i) Thin-Layer Chromatography TLC was performed on 20 cm x 20 cm l a y e r s of s i l i c a g e l , 0.25 mm i n t h i c k n e s s , supported by g l a s s p l a t e s . E i t h e r S i l G-25 (Machery-Nagel & Co., s u p p l i e d by Brinkmann Instruments) or Chromar 7GF ( M a l l i n c k r o d t ) p l a t e s were used. R o u t i n e l y used s o l v e n t systems were: A. CH3OH - 0.6% NaCl - NH.4OH (50/50/5;v/v/v) (72). T h i s system i s u s e f u l f o r s e p a r a t i n g c h o l i n e (R^ 0-0.13), phos-phocholine (R f 0.25-0.38), CDP-choline (R f 0.53-0.63) and be-ta i n e (R:f 0.56-0.66). To see these compounds, the TLC p l a t e was sprayed with D r a g e n d o r f f 1 s reagent (183) and a molybdenum reagent (184). l*C^ ] Glycerophosphocholine, d e t e c t e d by auto-radiography, has an R^ value of 0.50. B. Benzene - CHC1 3 - CH3OH (80/15/5;v/v/v). T h i s system i s u s e f u l i n i s o l a t i n g d i g l y c e r i d e . 1,2- and 1,3-d i a c y l g l y c e r o l s are separated by t h i s system (185). C. C H C I 3 - CH 3OH - H 20 (70/30/4;v/v/v) . Thi s system separates p h o s p h o l i p i d s . (See s e c t i o n (I) ( i i ) . ) - 39 -( i i ) S c i n t i l l a t i o n Counting L i p i d samples were counted i n toluene with 2,5-diphenylox-azole (PPO, 4g/jl) and 1, 4 - b i s - [J2-(5-phenyloxazolyl) ~J -benzene (POPOP, 50mg/jl) . Aqueous samples were counted i n ACS (Aqueous Counting S c i n t i l l a n t , Amersham) or i n T r i t o s o l (186). S c i n -t i l l a t i o n c ounting was done i n an ISOCAP/300 s c i n t i l l a t i o n coun-ter (Nuclear-Chicago) except f o r samples c o n t a i n i n g 3 2 P and 1!*C. 'These samples were counted i n a T r i - C a r b s c i n t i l l a t i o n counter (Packard), which has a window to count 3 2 P while ex-c l u d i n g l l fC. Counting e f f i c i e n c y was determined by the e x t e r -n a l standards r a t i o of chloroform-quenched standards which con-t a i n e d Q 3*Q- or ' 4C~J toluene, or Q 3 H j hexadecane. Stan-dards 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 f l u i d were counted with each s e t of samples. ( i i i ) P r o t e i n P r o t e i n was determined by the method of Lowry e t a l . (187) using bovine serum albumin as a standard. (iv) S t a t i s t i c s T e sts of s i g n i f i c a n c e were those d e s c r i b e d by Woolf (188). (c) C e l l C u l t u r e BHK-21 clone 13 c e l l s were obtained from Flow L a b o r a t o r i e s . C e l l s were grown at 37° as monolayers on l a r g e (150 mm x 15 mm, LUX S c i e n t i f i c ) or on medium (100 mm x 15 mm, Falcon) p l a s t i c d i s h e s . C e l l s were grown i n Dulbecco's M o d i f i e d Eagle Medium (and 5% f e t a l c a l f serum). By v i s u a l i n s p e c t i o n , c e l l s were c o n f l u e n t when used, unless noted otherwise. - 40 -(d) Propagation of S e m l i k i F o r e s t V i r u s The v i r u s o r i g i n a t e d as p r e v i o u s l y d e s c r i b e d (173). For propagation, SF v i r u s was added to r o l l e r b o t t l e s at a m u l t i -p l i c i t y of about 0.2 P F U / c e l l , a l s o adding 10 ml of Medium 199 (and 2% f e t a l c a l f serum). A f t e r 1 hour at 37°, 40 ml of the same medium were added. A f t e r 24 hours at 37° the medium was removed and used as a source of v i r u s . I n f e c t i v i t y was moni-tored by plaque assay. V i r u s p r e p a r a t i o n s were s t o r e d at -70° u n t i l use. (e) I n f e c t i o n of C e l l s with S e m l i k i F o r e s t V i r u s Each l a r g e d i s h of c e l l s was washed twice with 3 ml Medium 199 (and 2% f e t a l c a l f serum). V i r u s was r a p i d l y thawed and added at a m u l t i p l i c i t y of 40 P F U / c e l l , a l s o adding enough of the medium d e s c r i b e d above to make a t o t a l volume of 3 ml. A f t e r 30 minutes i n c u b a t i o n at 37°, the dishes were tip p e d back and f o r t h . A f t e r 1 hour at 37°, the medium was removed. Ten ml of Medium 199 (and 2% f e t a l c a l f serum) were added. Mock-i n f e c t e d c e l l s were t r e a t e d i n the same way, except that v i r u s was not added. C e l l s were f u r t h e r incubated as d e s i r e d . (f) P r e p a r a t i o n of S u b c e l l u l a r F r a c t i o n s The f o l l o w i n g procedure a p p l i e s to 4 l a r g e d i s h e s of c e l l s . C o n f l u e n t c e l l s were washed twice with about 3 ml per d i s h of PBS (189). The c e l l monolayer was removed twice with 3 ml of PBS each time. C e l l s were packed by c e n t r i f u g a t i o n at 900 x £ f o r 10-15 minutes at 4°. The p e l l e t was kept on i c e and - 41 -resuspended in 2.5 ml of 10 mM T r i s - H C l (pH 7.4), with 10 mM KC1 and 15 mM MgCl2• C e l l s were homogenized by 20 s t r o k e s i n a t i g h t - f i t t i n g Dounce homogenizer. To the homogenate was added 2.5 ml of 50% sucrose i n the p r e v i o u s l y mentioned b u f f e r . The suspension of c e l l o r g a n e l l e s was c e n t r i f u g e d at 9,000 x £ f o r 10 minutes at 4°. The supernatant was r e t a i n e d and c e n t r i f u g e d at 100,000 x g f o r 1 hour at 4°. The supernatant ( c e l l c y t o s o l ) and the p e l l e t (microsomes) so obtained were used as sources of CK, CT, and CPT a c t i v i t i e s . The p e l l e t s were resuspended using a l o o s e - f i t t i n g Dounce homogenizer, i n 10 mM T r i s - H C l (pH 7.4) with 1 mM EDTA (unless noted o t h e r w i s e ) . (g) Enzyme Assays (i) C h o l i n e Kinase Enzyme a c t i v i t y was measured e s s e n t i a l l y by the method of Weinhold and Rethy (24). For o p t i m a l a c t i v i t y , the procedure was as f o l l o w s . C y t o s o l from BHK c e l l s was added to a r e a c t i o n mixture c o n t a i n i n g 100 ymoles of T r i s - H C l (pH 8.0), 7.5 ymoles of both ATP and MgCl 2 , and 0.25 pinole . of [~_3HTJ c h o l i n e c h l o r i d e ( e i t h e r 1 or 5 Ci/mole) i n a f i n a l volume of 1 ml. A f t e r i n c u -b a t i o n a t 37° f o r 20 minutes, the r e a c t i o n was stopped by im-mersing the r e a c t i o n tubes i n b o i l i n g water for 2 minutes. Each mixture was d r a i n e d i n t o a 0.5 x 4 cm column of AG 1.-X10 ion exchange r e s i n (OH form), which had p r e v i o u s l y been washed with 1 ml of 0.1 M T r i s - H C l (pH 8.0). Assay tubes were r i n s e d with 1 ml of water and t h i s r i n s e was a l s o d r a i n e d i n t o the columns. Water (about 15 ml) was f o r c e d through the columns to - 42 -e l u t e Q 3 H [ ] c h o l i n e . Q 3 f Q Phosphocholine was e l u t e d with 0.5 ml of IN NaOH followed by 1.5 ml of 0.IN NaOH. G l a c i a l a c e t i c a c i d (about 70 uft) was,added to the e l u a t e to reduce chemilumi-nescence before c o u n t i n g . The above ion exchange method i s q u i t e s e n s i t i v e to ions i n the r e a c t i o n mixture (see R e s u l t s ) . A second method v e r i f i e d the r e s u l t s obtained by ion exchange. In the second method, c y t o s o l was added to a mixture c o n t a i n i n g 10 ymoles of T r i s - H C l (pH 8.0), 0.75 ymole of ATP, 1 ymole of MgCl 2, and 0.025 ymole'." of Q 3 H J c h o l i n e (50 Ci/mole) i n a f i n a l volume of 100 y£. Af-ter i n c u b a t i o n at 37°, the assay was terminated as d e s c r i b e d above. An a l i q u o t of the mixture (50 yjl) , with 40 yg of cho-l i n e and 200 yg of phosphocholine as c a r r i e r s , were spotted onto a s i l i c a g e l TLC p l a t e . The p l a t e was developed i n s o l -vent system A. Ch o l i n e was dete c t e d by s p r a y i n g the p l a t e with Dragendorff's reagent (183), and phosphocholine, by sp r a y i n g with a molybdenum reagent (184). Phosphocholine was e l u t e d from the s i l i c a and counted. The enzyme a c t i v i t y obtained was l i n e a r to at l e a s t 40 minutes. ( i i ) C y t i d y l y l t r a n s f e r a s e C y t i d y l y l t r a n s f e r a s e was assayed by a procedure s i m i l a r to that of A n s e l l and Chojnacki (190), except t h a t TLC, not char-c o a l a d s o r p t i o n , was used to separate phosphocholine from CDP-c h o l i n e . For optimal a c t i v i t y , BHK c e l l c y t o s o l or microsomes were added to a mixture c o n t a i n i n g 2 ymoles of T r i s - s u c c i n a t e (pH 6.0), 1.2 ymoles of Mg a c e t a t e , 0.30 ymole;') of CTP (for the - 43 -c y t o s o l i c enzyme) or 0.20 ymole; ; of CTP (for the microsomal enzyme), and 0.16 umoleQ of ["Methyl-3H~J - or [jMethyl- 1 l*C~J phosphocholine (except f o r mock-infected microsomes, to which 0.20 ymole^/ were added) i n a f i n a l volume of 100 yiU The spe-c i f i c r a d i o a c t i v i t y of phosphocholine was 12.8 or 7.5 Ci/mole. A f t e r 20 minutes i n c u b a t i o n at 37°, the r e a c t i o n was stopped as f o r c h o l i n e k i n a s e . An a l i q u o t of the mixture was spotted on a s i l i c a g e l TLC p l a t e with about 0.6 mg of CDP-choline c a r -r i e r . The p l a t e was developed i n s o l v e n t system A. UV l i g h t was used to v i s u a l i z e CDP-choline. CDP-choline was e l u t e d from the s i l i c a with 0.5 ml of IN NaOH, followed by 1.5 ml of 0.IN NaOH. G l a c i a l a c e t i c a c i d (about 70 y£) was added to the e l u a t e before c o u n t i n g . ( i i i ) 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 CPT was assayed by the p r o t o c o l d e s c r i b e d by Vance and Burke (173), except that the s p e c i f i c r a d i o a c t i v i t y of 1*C-J CDP-choline was 0.3 Ci/mole, the assay i n c u b a t i o n time was 15 minutes, and the e x t r a c t e d Q 1 1 * ^ ] PC was blown dry with a i r . The d i g l y c e r i d e s u b s t r a t e was prepared by a phospholipase C d i g e s t i o n of egg yolk PC (191). D i g l y c e r i d e was separated from unhydrolyzed PC by s i l i c i c a c i d chromatography. A s i l i c i c a c i d column (18 g of s i l i c a ) was washed with 100 ml of petroleum e t h e r , the hydrolyzed sample was loaded onto the column, and d i g l y c e r i d e was e l u t e d with 200 ml of 25% e t h y l ether i n pet-roleum e t h e r . The s o l v e n t was removed with a stream of n i t r o -gen. The i d e n t i t y of d i g l y c e r i d e was v e r i f i e d by TLC i n - 44 -comparison with 1 , 2 - d i o l e i n . (See (£)(iii) below;) D i g l y c e r -ide was prepared f o r the CPT assay as f o l l o w s . A d i g l y c e r i d e s o l u t i o n (50 y& a t 40 mg/ml i n chloroform) was p i p e t t e d i n t o a 5 or 10 ml beaker. The chloroform was evaporated under n i t r o -gen. Water (2.8 ml) and 001% Tween 20 (v/v) (1.2 ml) were then added. An emulsion of d i g l y c e r i d e was formed by two 3 minute p e r i o d s of s o n i c a t i o n with a Sonic Dismembrator (Quigley-Roches-t e r , Inc.) at s e t t i n g 80. The emulsion was cooled on i c e a f t e r the f i r s t s o n i c a t i o n . (iv) Phosphatidylethanolamine M e t h y l t r a n s f e r a s e PEMT was measured e s s e n t i a l l y as d e s c r i b e d by Rehbinder and Greenberg (192). Microsomes prepared without EDTA were added to a mixture c o n t a i n i n g 110 or 148 ymoles of T r i s - H C l (pH 922), 75 yg of phosphatidylmonomethylethanolamine, 400 yg of deoxycho-l a t e , and 0.15 ymole: of Q 3 l Q S-adenosylmethionine (25 Ci/mole) in a f i n a l volume of 0.5 ml. The l i p i d s u b s t r a t e (1.12 mg) had been e m u l s i f i e d i n 1.5 ml o f 0.4% deoxycholate by s o n i c a t i o n fo r 2 minutes with a Sonic Dismembrator at s e t t i n g 60. A f t e r 30 minutes i n c u b a t i o n at 37°, the r e a c t i o n was stopped with 0.2 ml cone. HC1. A s o l u t i o n of 0.9% NaCl (2.5 ml) and n-buta-n o l (2.5 ml) were added. A f t e r mixing and cen t r i f u g i n g , an.: a l i q u o t of the top phase was removed f o r cou n t i n g . (v) C h o l i n e Oxidase To prepare mitochondria, each l a r g e d i s h o f BHK c e l l s was washed three times with 3 ml of i c e - c o l d PBS. C e l l s were i s o -l a t e d as usual ( s e c t i o n ( f )) and homogenized i n 50 mM T r i s - H C l - 45 -b u f f e r (pH 7 .0 ) with 0 . 2 5 M sucrose (14 ml/g c e l l s ) by 10 strokes' of a t i g h t - f i t t i n g Dounce homogenizer. Mi t o c h o n d r i a were i s o -l a t e d i n the same b u f f e r by the method of Myers and S l a t e r ( 1 9 3 ) . C h o l i n e oxidase a c t i v i t y was measured i n mitochondria by the procedure of Schneider and Vance ( 1 1 0 ) . (h) L i p i d A c t i v a t i o n of C y t i d y l y l t r a n s f e r a s e • In t h i s experiment, c y t o s o l i c CT was assayed w i t h i n 5 hours of p r e p a r a t i o n of the c y t o s o l . From 0 to 250 yg of acetone-e x t r a c t e d r a t l i v e r l i p i d s were added to assay tubes. (The s o l -vent was removed with a stream of n i t r o g e n . ) Before a s s a y i n g , the assay components minus enzyme were v i g o r o u s l y vortexed to d i s p e r s e the l i v e r l i p i d s . • (i) Taurocholate A c t i v a t i o n of Cholinephos.photraBsferase ...i., T a u r o c h o l i c a c i d was d i s s o l v e d i n 50 mM T r i s - H C l (pH 7 .4 ) w i t h i n two days of i t s use. The s o l u t i o n of t a u r o c h o l a t e was added to a mixture on i c e , which contained microsomes and the assay components except [_l hC^] CDP-choline. Each tube i n c l u d e d a t o t a l of 50 ymoles of T r i s - H C l (pH 7 . 4 ) . The mixture was i n -cubated f o r 30 minutes on i c e . At the end of t h i s time, Q 1 4C^] CDP-choline (20 y £ at 20 mM, 0 . 3 Ci/mole) was added and the tubes were incubated at 37° f o r 20 minutes. The f i n a l con-c e n t r a t i o n of t a u r o c h o l a t e was from 0 to 2%. ^CJ PC was e x t r a c t e d as usual (Section ( g ) ( i i i ) ) . (j) C h o l i n e T r a n s p o r t Medium-size dishes of c e l l s were used f o r t r a n s p o r t e x p e r i -ments. I n f e c t i o n was a t 40 P F U / c e l l as u s u a l . At 6h hours - 46 -p o s t - i n f e c t i o n , the c e l l s were washed two times with 1.5 ml of 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) n e u t r a l i z e d with 5% NaHC0 3. E 3 l G c h o l i n e (6.3 uCi) with or without c o l d c h o l i n e i o d i d e , was added to the c e l l s i n 2 ml of the same medium. Dishes were kept at 37° by p l a c i n g them i n a water bath at t h i s temperature, with a l i t t l e l e s s water than that which would cause the d i s h e s to f l o a t . At 1, 10, 20, and 30 minutes a f t e r the a d d i t i o n of medium (i n c u b a t i o n 1), a 5% a l i q u o t was removed f o r c o u n t i n g . The amount of c h o l i n e taken up by the c e l l s i n 30 minutes c o u l d be c a l c u l a t e d s i n c e the s p e c i f i c r a d i o a c t i v i t y of c h o l i n e i n the medium was known. A f t e r 30 minutes i n c u b a t i o n , the c e l l s were washed three times, each time with 1.5 ml c o l d (4°) PBS, and scraped o f f three times, each time with 2 ml of 10% t r i c h l o r o -a c e t i c a c i d . The a c i d - i n s o l u b l e p e l l e t s were obtained by cen-t r i f u g a t i o n and homogenized in 5 ml 0.9% NaCl i n a t i g h t - f i t t i n g Dounce homogenizer. A l i q u o t s of the homogenate were taken for p r o t e i n d e t e r m i n a t i o n . In separate c o n t r o l experiments, the extent of Q 3H^] cho-l i n e b i n d i n g to the e x t e r n a l s u r f a c e of the c e l l s was measured. A f t e r 30 minutes of uptake, as d e s c r i b e d above ( i n c u b a t i o n 1), the c e l l s . w e r e washed twice, each time with 1.5 ml of the s a l t s o l u t i o n d e s c r i b e d above, followed by a d d i t i o n of 2 ml of the same s o l u t i o n c o n t a i n i n g 5 mM c h o l i n e i o d i d e . A l i q u o t s of 5% of the medium were removed a f t e r 1 and 30 minutes of i n c u b a t i o n at.37° ( i n c u b a t i o n 2). The percent r e l e a s e was c a l c u l a t e d as: - 47 -cpm, 30 minutes; i n c u b a t i o n 2 x 100 cpm, 0 minutes; i n c u b a t i o n 1 - cpm, 30 minutes; i n c u b a t i o n 1 Cpm at 0 minutes were obtained by e x t r a p o l a t i o n . (k) L a b e l l i n g of C e l l s with Q 3H~J C h o l i n e C e l l s were l a b e l l e d at 6% hours a f t e r a d d i t i o n of v i r u s (6% hours p . i . ) . C e l l s were washed twice with s e v e r a l ml of n e u t r a l i z e d 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). Next, the l a b e l l i n g medium (3 ml of the same me-dium which contained 10 yCi of Q 3 t Q c h o l i n e ) was added. A f t e r 30 minutes i n c u b a t i o n at 37°, the c e l l s were c h i l l e d and pro-cessed as d e s c r i b e d i n s e c t i o n (£)(i). (£) Pool S i z e Measurements (i) C h o l i n e - C o n t a i n i n g Compounds For each p r e p a r a t i o n , 5 l a r g e d i s h e s of c o n f l u e n t c e l l s were used. Each d i s h of c e l l s was washed three times with 3 ml of i c e - c o l d PBS. Water was added (about 2 ml) to each d i s h and the c e l l s from 5 d i s h e s were scraped i n t o a p l a s t i c c e n t r i f u g e tube (or tubes) c o n t a i n i n g 20 ml of C H C I 3 - C H 3 O H (1/1; v / v ) . Re s i d u a l c e l l s were removed i n another 2 ml of water added to each d i s h . Removal of c e l l s with a rubber policeman was done in a c o l d room at 4°. Standard l tC[]-choline, phosphocholine, and CDP-choline were added to the c e n t r i f u g e tubes of s e l e c t e d samples f o r c a l c u l a t i o n of r e c o v e r i e s . A f t e r mixing, the phases were separated by c e n t r i f u g a t i o n at 4°. Keeping the tubes at 4° or c o l d e r , the lower phase was e x t r a c t e d three times f u r t h e r with 20 ml of CH 3OH-H 20 (1/1; v / v ) . Lower phases were r e t a i n e d . fo r counting, TLC, and p h o s p h o l i p i d phosphorus.; a n a l y s i s . - 48 -The upper phases were combined, placed on i c e , and drip p e d by g r a v i t y i n t o a 30 cm x 1 cm column of AG 1-X10 r e s i n (OH form). T h i s step took s e v e r a l hours. The r e s i n was mixed with 20 volumes of CH 3OH-H 20 (1/1; v/v) before packing the column. H y d r o l y s i s of phosphocholine occurs i f the upper phases are f l a s h evaporated before ion exchange chromatography. The c o l -umn was at room temperature, while e l u a t e s were c o l l e c t e d on i c e . During t h i s procedure bubbles developed i n the r e s i n but di d not a f f e c t the column's performance. The c o n t a i n e r at the top of the column was r i n s e d s i x times with C H 3 O H - H 2 O (1/1; v/v) and once with water. These r i n s e s were f o r c e d through the column with a p e r i s t a l t i c pump. The column e l u a t e obtained so f a r , c ontained c h o l i n e . Next, 0.2 M NH 4HCO 3(about 500 ml) was pumped through the column. The second e l u a t e thus o b t a i n e d , contained phosphocholine and CDP-choline. The f i r s t and second e l u a t e s were evaporated at 37°-40° under reduced pressure un-t i l dry. The second e l u a t e was d i s s o l v e d i n 10 ml of water and ap-p l i e d to a c h a r c o a l column (1.3 cm x 1.6 cm). The c h a r c o a l was packed i n a M i l l i p o r e f i l t r a t i o n apparatus over a g l a s s f i b e r f i l t e r . The f l a s k c o n t a i n i n g the second e l u a t e was r i n s e d s i x times with 10 ml of water and three times with 10 ml of 2% e t h a n o l . These r i n s e s were passed through the c h a r c o a l column. The e l u a t e which r e s u l t e d , contained phosphocholine, and was evaporated as d e s c r i b e d above. CDP-choline was obtained by e l u t i o n of the c h a r c o a l with 60 ml 40% ethanol/1% NH 40H. To avoid v i o l e n t b o i l i n g of e t h a n o l , - 49 -the.CDP-choline f r a c t i o n was i n i t i a l l y evaporated at room tem-perature and subsequently warmed to 37°-40°. Phosphocholine and CDP-choline were hydrolyzed to c h o l i n e by enzyme d i g e s t i o n . The phosphocholine f r a c t i o n was d i s s o l v e d i n 7 ml of water to which was added 3 ml of IN NaOH. The CDP-c h o l i n e f r a c t i o n was d i s s o l v e d i n 5 ml of water to which was added 60 y£ of IN NaOH. (In one case, IN NaOH (100 y£) and IN HC1 (40 y£) were added.) The f i n a l pH of each sample was about 10. A l k a l i n e phosphatase (Sigma 111-S, from E. c o l i ) and phosphodiesterase (Sigma, from C r o t a l u s adamanteus venom) were prepared by d i a l y z i n g three times f o r 2 hours each a g a i n s t 200 volumes of 0.1 M borate b u f f e r (pH 9.0). Phosphatase (about 3 u n i t s , i n 150 y£) was added to each phosphocholine sample. Phosphatase (about 2 u n i t s ) and phosphodiesterase (about 0.06 unitQ) i n a t o t a l volume of 200 y£ were added to each CDP-cho-l i n e sample. An enzyme u n i t i n t h i s case i s denoted by the Sigma Chemical Company as h y d r o l y s i s of 1 ymole of s u b s t r a t e per minute. Incubations were at 37° o v e r n i g h t . Since h y d r o l y s i s was not completed by t h i s treatment, as shown by the r e s u l t s of TLC i n s o l v e n t system A, f u r t h e r en-zyme d i g e s t i o n was necessary. Phosphodiesterase and a l k a l i n e phosphatase were d i a l y z e d a g a i n s t 0.1 M N H 4 H C O 3 b u f f e r , pH 8.9 (buffer A) and 1 M N H 4 H C O 3 b u f f e r , pH 10.0 (buff e r B), respec-t i v e l y . D i a l y s i s was done three times a g a i n s t approximately 200 volumes of b u f f e r f o r at l e a s t 2 hours. For the phospho-c h o l i n e samples, a p o r t i o n (1 ml) of the pre v i o u s mixture was - 50 -mixed with 75-300 uJ, of a l k a l i n e phosphatase (about 15-60 u n i t s ) and 0.7-0.925 ml of b u f f e r B, to make 2 ml. For the CDP-choline samples, an a l i q u o t (1.5 ml) of the previous mixture was mixed with 150. \iZ of phosphodiesterase (about 0.12 u n i t ) , 50 u£ of phosphatase (about 10 u n i t s ) , 0.3 ml of water, and 1 ml of buf-f e r A. Incubation was at 37° f o r at l e a s t 4 hours. A f t e r h y d r o l y s i s , the c h o l i n e - c o n t a i n i n g compounds were separated by TLC i n s o l v e n t system A, and e l u t e d from the s i l i -ca two times, each time with 2 ml IN HC1. The e l u a n t s were counted f o r r a d i o a c t i v i t y . The percent of cpm i n the Rf 0-0.2 reg i o n ( c h o l i n e region) compared to the t o t a l cpm from R^ 0-0.7, averaged 92% f o r c h o l i n e , 85% f o r hydrolyzed phosphocholine, and 94% f o r hydrolyzed CDP-choline. The hydrolyzed samples were concentrated by l y o p h i l i z a t i o n . C h o l i n e was i n i t i a l l y measured by the c o l o r i m e t r i c method of Hayashi e_t a l . (194). However, the r e s u l t s by t h i s method were v a r i a b l e (see Re s u l t s s e c t i o n ) . The method e v e n t u a l l y chosen was that of Goldberg and McCaman (195). T h i s method i s based on the i o n - p a i r e x t r a c t i o n of c h o l i n e with t e t r a p h e n y l -boron i n heptanone, r e - e x t r a c t i o n i n t o a s o l u t i o n of HC1, and ph o s p h o r y l a t i o n of c h o l i n e by Q Y - 3 2 P ^ ] ATP with c h o l i n e kinase. E x t r a c t i o n s were performed on the f o l l o w i n g : f i r s t l y , on f r e e c h o l i n e i s o l a t e d from BHK c e l l s , and secondly, on c h o l i n e d e r i v e d from hydrolyzed phosphocholine and CDP-choline pools o f ' BHK c e l l s . In the f i r s t case, samples of i s o l a t e d c h o l i n e were d i s s o l v e d i n 5 ml of water, and e x t r a c t e d twice with 5 ml of - 51 -tetraphenylboron i n heptanone (10 mg/ml). The heptanone l a y e r s were r e e x t r a c t e d with 10 ml of 0.4 N HC1, and then with 4 ml IN HC1. The combined HC1 phases were e x t r a c t e d with 10 ml of heptanone. C h o l i n e d e r i v e d from phosphocholine and CDP-choline was p u r i f i e d i n a s l i g h t l y d i f f e r e n t manner. C h o l i n e was e x t r a c t e d from the l y o p h i l i z e d h y d r o l y s a t e s of phosphocholine and CDP-c h o l i n e with 70% e t h a n o l (33). Three e x t r a c t i o n s of 1 ml each were done. The p r e c i p i t a t e was removed by c e n t r i f u g a t i o n i n a desk-top c e n t r i f u g e . The 70% e t h a n o l was removed by warming the combined supernatants to about 37° under a stream of n i t r o g e n , f o l lowed by l y o p h i l i z a t i o n . The phosphocholine h y d r o l y s a t e s were then d i s s o l v e d i n 2 ml of water and e x t r a c t e d three times with 2 ml of tetraphenylboron i n heptanone (10 mg/ml). The combined heptanone l a y e r s were r e e x t r a c t e d with an equal volume of 0.4 N HC1. F i n a l l y , the HC1 phase was e x t r a c t e d with an equal volume of heptanone. The HC1 phase was l y o p h i l i z e d . CDP-c h o l i n e h y d r o l y s a t e s were t r e a t e d i d e n t i c a l l y , except that o n l y two e x t r a c t i o n s with tetraphenylboron i n heptanone were done. The e x t r a c t i o n c o n d i t i o n s given here were chosen to maximize the y i e l d of []3H~] c h o l i n e . The l y o p h i l i z e d samples were r e -d i s s o l v e d i n water before a s s a y i n g . The assay of c h o l i n e was done i n a t o t a l volume of 50 y£ . T h i s volume i s more convenient than 10 y&, which i s the volume suggested (195), although the s e n s i t i v i t y of the assay i s r e -duced by an i n c r e a s e i n volume. The p u b l i s h e d procedure (195) - 52 -was fo l l o w e d , except i n the f o l l o w i n g d e t a i l s . An a l i q u o t (25 u&) of b u f f e r - s u b s t r a t e (which c o n t a i n s 10 mM MgCl 2> 2 mM ATP, and 100 mM sodium phosphate b u f f e r , pH 8.0) was mixed with 5 y£ of c h o l i n e kinase. T h i s amount of c h o l i n e kinase (Sigma, from yeast) re p r e s e n t s 6 yg of p r o t e i n (0.8 ymole min mg pro-t e i n "*") . P r e i n c u b a t i o n of t h i s mixture was f o r 15 minutes at 25°, not 37°. (Pr e i n c u b a t i o n converts any c h o l i n e contaminant i n the enzyme p r e p a r a t i o n to phosphocholine.) C 3 2 p J A T P (about 0.5 y C l , i n 5 y£) was then added, fo l l o w e d by 15 y£, of sample. Further i n c u b a t i o n was f o r 15 minutes at 25°. The amount of 0.3 M barium acetate used to p r e c i p i t a t e ATP was i n c r e a s e d to 75 y£. Samples were then c e n t r i f u g e d f o r 1 minute at 4°. P a r t of the supernatant (75 yfi,) was a p p l i e d to AG 1-X10 (not 1-X8) columns i n the formate form. The standard curve ( F i g . 4) was l i n e a r to 2.5 nmoles of c h o l i n e i o d i d e . C h o l i n e i o d i d e was used s i n c e i t i s not hydroscopic l i k e c h o l i n e c h l o r i d e (196). Lack of enzyme i n h i b i t i o n by the samples was t e s t e d by as-say of a mixture of h a l f of a given sample with a known amount of c h o l i n e i o d i d e . One sample of c h o l i n e i s o l a t e d from BHK c e l l s d i d c o n t a i n •. an i n h i b i t o r which depressed the assay cpm. However, by d i l u t i n g the sample, t h i s e f f e c t was e l i m i n a t e d , and a l l f r e e c h o l i n e samples assayed d i d not i n h i b i t the enzyme as shown by t h i s method. ( i i j PC L i p i d samples were prepared as d e s c r i b e d i n s e c t i o n (£) (i) , except f o r one sample (of seven) which was obtained by the 0 0 0.4 0.8 1.2 Chol ine, nmoles Figure 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 the procedure of Goldberg and McCaman (195), m o d i f i e d as de 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 apply to the o r d i n a t e and a b s c i s s a of the l a r g e f i g u r e and the i n s e t graph. - 54 -f o l l o w i n g procedure. C e l l s were washed twice with PBS, and r e -moved i n 8-10 ml of i c e - c o l d PBS. L i p i d s were e x t r a c t e d i n t o an equal volume of CHCI3-CH3OH (2/1; v/v) i n a t i g h t - f i t t i n g Dounce homogenizer (20 s t r o k e s ) . The l i p i d phase was e x t r a c t e d twice with 8-10 ml of CH3OH-0..9% NaCl-CHCl 3 (48/47/3 ; v/v/v) (197). P r o t e i n and other s o l i d s were remo.ved by f i l t r a t i o n of the l i p i d samples over a g l a s s f i b e r f i l t e r . Samples were concen-t r a t e d by f l a s h e v a p o r a t i o n at 35° or lower and r e d i s s o l v e d i n CHC13-CH3OH (2/1; v / v ) . A l i q u o t s were then d r i e d and counted. The f i b e r f i l t e r and r i n s e s (about 1 ml, CHCI3-CH3OH (2/1; v/v)) of the f i l t r a t i o n f u n n e l , and the tubes which had contained the l i p i d e x t r a c t s , were measured f o r r a d i o a c t i v i t y (cpm). These combined cpm were an average of 0.3% of the t o t a l cpm recovered. A l i q u o t s of the f i l t e r e d samples were a p p l i e d to s i l i c a g e l TLC p l a t e s which were developed i n s o l v e n t system C. T h i s s y s -tem separated PC from PE, LPC, and LPE. PC was e l u t e d from the s i l i c a with 7 ml of CHCI3-CH3OH (2/1; v / v ) . (Of the cpm e l u t e d i n 14 ml, 85% were recovered i n the f i r s t 7 ml.) Since the t o t a l l i p i d dpm were known, and s i n c e the r a d i o a c t i v i t y appeared to be e x c l u s i v e l y i n PC (see R e s u l t s ) , pool s i z e s were c a l c u l a t e d from a measurement of the s p e c i f i c r a d i o a c t i v i t y of PC. Phos-p h o l i p i d phosphorus: i n i s o l a t e d PC was measured by the proce-dure of Raheja e t a_L (198). T h i s method gave a l i n e a r standard curve to 10 yg of phosphorus: i n d i p a l m i t o y l PC ( F i g . 5). Spe-c i f i c r a d i o a c t i v i t i e s i n dpm/yg phosphorus were thus determined. 16 P h o s p h o r u s , u g D i g l y c e r i d e , n m o i e s Figure 5. Standard curves f o r l i p i d phosphorus ' ( l e f t ) and d i g l y c e r i d e i r i g h t ) d e t e r m i n a t i o n s . L i p i d phosphorus was measured by the method of Raheja et a l . (198). Formation of the coloured complex was determined by i t s absor-bance at 710 nm. D i g l y c e r i d e was measured by Schneider's ,procedure (199), modified 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 ) . I s o l a t e d g l y c e r o l - [ [ 3 2P 2] phosphate was counted f o r r a d i o a c t i v i t y . - 56 -To c a l c u l a t e s p e c i f i c r a d i o a c t i v i t i e s i n dpm/ymole, an average molecular weight of 831.1 f o r PC was estimated. T h i s estimate i s based on the f a t t y a c i d composition of PC repo r t e d f o r BHK-21 c e l l s grown i n Eagle's Minimal E s s e n t i a l Medium with 2.5% f e t a l c a l f serum ("Day 3", Table V, r e f . 163.),. ( i i i ) D i g l y c e r i d e A l i q u o t s of the l i p i d samples from mock- or v i r u s - i n f e c t e d c e l l s were chromatographed on TLC i n s o l v e n t system B. D i o l e i n and d i g l y c e r i d e prepared f o r CPT assay were used as TLC s t a n -dards f o r BHK c e l l d i g l y c e r i d e . The prepared d i g l y c e r i d e had an Rf range of 0.64 to 0.76 and d i o l e i n was between R^ 0.66 and 0.73 (with a contaminant between R:^  0.75 and 0.78). D i g l y c e r -ide from BHK c e l l s was v i s u a l i z e d by i o d i n e vapour and e l u t e d from the s i l i c a with 4 ml of 2-propanol-hexane-water (75/25/1; v/v/v) as d e s c r i b e d by Schneider (199). D i s t i l l a t i o n of the 2-propanol before use was e s s e n t i a l to o b t a i n low blanks i n the d i g l y c e r i d e assay. In a c o n t r o l experiment, the y i e l d of d i -p a l m i t i n a f t e r TLC and e l u t i o n from the s i l i c a was shown to be q u a n t i t a t i v e . H y d r o l y s i s of d i g l y c e r i d e to g l y c e r o l and p h o s p h o r y l a t i o n of g l y c e r o l by g l y c e r o k i n a s e were by Schneider's method (199). A f t e r the enzyme r e a c t i o n , 0.8 ml of water was added to each assay tube. The contents of each tube were then a p p l i e d over a 2 cm column of c h a r c o a l - c e l i t e (1/1; by weight) i n a pasteur p i p e t plugged with g l a s s wool. Two f u r t h e r r i n s e s with 0.8 ml water were passed through the c h a r c o a l . A i r pressure was used - 57 -to d r a i n the columns. The e l u t e s were counted. The standard curve was l i n e a r from 0 to 10 nmoles of d i p a l m i t i n ( F i g . 5). Lack of enzyme i n h i b i t i o n by the samples was checked by mixing one-half of one sample with 2 nmoles of d i p a l m i t i n , which when assayed gave 88% of the expected cpm. (iv) Phosphocholine and CTP Phosphocholine and CTP were assayed independently by an o r i g i n a l method which has been d e s c r i b e d (200). At 6h hours p . i . , each of 5 l a r g e d i s h e s of c e l l s was washed three times with 3 ml of i c e - c o l d PBS. The c e l l s were removed with 6 ml of 10% p e r c h l o r i c a c i d . The c e l l u l a r m a t e r i a l i n p e r c h l o r i c a c i d was homogenized with 5 s t r o k e s of a t i g h t - f i t t i n g Dounce homogenizer. The above steps were done i n a c o l d room at 4°. The e x t r a c t s were c e n t r i f u g e d at 30,000 x £ f o r 10 minutes at 4°, n e u t r a l i z e d with KOH (to pH 7-8), he l d on i c e f o r at l e a s t 1 hour, and r e c e n t r i f u g e d at 6,000 x £ f o r 10 minutes (201). The supernatant obtained was l y o p h i l i z e d and r e d i s s o l v e d i n 10 ml of water. T h i s s o l u t i o n was again c e n t r i f u g e d a t 6,000 x £ fo r 10 minutes at 4° to remove the s a l t p r e c i p i t a t e . CTP i n 8 ml of supernatant was absorbed twice i n a t o t a l of 0.30 g of c h a r c o a l . Phosphocholine remained i n the supernatant. The c h a r c o a l was e x t r a c t e d fb.ur times with 1.5 ml of ethanol-water-ammonium hydroxide (50/49/1; v / v / v ) . A f t e r the f i r s t e x t r a c -t i o n , the c h a r c o a l and e l u a n t were heated to 60° f o r 2 minutes. A f t e r each e x t r a c t i o n , the c h a r c o a l was c o l l e c t e d by c e n t r i f u -g a t i o n at 6,000 x £ f o r 5-10 minutes. The combined e l u a n t s were - 5 8 -f i l t e r e d through a M i l l i p o r e f i l t e r (0.22 ym) to remove f i n e p a r t i c l e s of c h a r c o a l . The s o l u t i o n was f l a s h evaporated a t 37°. The residue c o n t a i n i n g CTP was d i s s o l v e d i n 2 ml of water. T h i s f r a c t i o n and the supernatant which contained phosphocho-l i n e were concentrated by l y o p h i l i z a t i o n . To measure phosphocholine, a sample or a s o l u t i o n of s t a n -dard phosphocholine (20 y£) was mixed with 80 y& of a s o l u t i o n c o n t a i n i n g 1 ymole of T r i s - s u c c i n a t e (pH 7.0), 1.2 ymoles of magnesium a c e t a t e , 0.05 ymole:;, of C5-3H~J CTP (5 Ci/mole ) , 50 y£ (about 0.5 mg p r o t e i n ) o f p a r t i a l l y p u r i f i e d r a t l i v e r CT (200), and 0.008 unit/? (ymole min ^) of i n o r g a n i c pyrophospha-tase (Sigma, from y e a s t ) . (Pyrophosphatase causes the CT re a c -t i o n to favour CDP-choline formation.) The r e a c t i o n proceeded at 37° f o r 45 minutes, and was stopped by p l a c i n g the r e a c t i o n tube i n b o i l i n g water f o r 2 minutes. The mixture was c e n t r i -fuged at 3,000 x £ f o r 10 minutes and p a r t of the supernatant (50 y£) was incubated with 50 y£ of a s o l u t i o n c o n t a i n i n g 0.1 ymolef) of CDP-choline, 20 ymoles of T r i s - g l y c i n e b u f f e r (pH - 1 10.5), and 0.30 units.;.-' (ymole min ) of a l k a l i n e phosphatase (Sigma, from E. c o l i ) . A f t e r 30 minutes at 37°, the r e a c t i o n was stopped as d e s c r i b e d above. [] 3 H L] CDP-choline was sepa-rated from Q3H~J c y t i d i n e by paper chromatography with e t h a n o l -1 M ammonium a c e t a t e , pH 7.1 (7/3; v / v ) . CTP was measured by an assay i n which 20 \il of a sample or standard CTP s o l u t i o n was mixed with 80 \il of a s o l u t i o n con-t a i n i n g 10 ymoles of T r i s - s u c c i n a t e (pH 7.0), 1.2 ymoles of - 59 -magnesium a c e t a t e , 0.1 ymole< of ATP, 0.1 ymole-: of (^Methyl- 3 H~J phosphocholine (7.5 or 15 C i / m o l e ) , 50 y l of CT p r e p a r a t i o n , and 0.008 u n i V ) of pyrophosphatase. ATP was added to p r o t e c t a g a i n s t h y d r o l y s i s of CTP by the p a r t i a l l y p u r i f i e d CT p r e p a r a -t i o n . The assay tubes were incubated, heated, and c e n t r i f u g e d as d e s c r i b e d f o r phosphocholine measurement. [ ~ 3 H C D P - c h o l i n e formation was measured as p r e v i o u s l y d e s c r i b e d ( s e c t i o n ( g ) ( i i ) ) . The i n c o r p o r a t i o n of 10 nmoles of CTP i n t o CDP-choline un-der the assay c o n d i t i o n s d e s c r i b e d i s p l o t t e d a g a i n s t time i n F i g u r e 6. A l i n e a r standard curve was obtained from 1 to 10 nmoles of CTP ( F i g . 7 ) . Formation of CDP-choline was a l s o a l i n e a r f u n c t i o n of added phosphocholine (1 to 10 nmoles), i n the presence of 50 nmoles of Q 3 H J CTP. CTP values were c o r r e c t e d f o r h y d r o l y s i s by 10% p e r c h l o r i c a c i d (6%/hour at 4°) and f o r the o v e r a l l i s o l a t i o n y i e l d (61%) (200). (v) N u c l e o t i d e s High-pressure l i q u i d chromatography was used to separate BHK c e l l : n u c l e o t i d e s . Chromatography on P a r t i s i l - 1 0 SAX (Reeve-Angel) was performed, as suggested by a recent p u b l i c a t i o n of E l i o n e t a l . (202) . Each l a r g e d i s h of c e l l s was washed three times with 3-5 ml of i c e - c o l d PBS before e x t r a c t i o n . N u c l e o t i d e s were measured by two s i m i l a r methods. In the f i r s t , n u c l e o t i d e s were e x t r a c t e d i n 10% p e r c h l o r i c a c i d and e x t r a c t s were homogenized, n e u t r a - .. l i z e d , c e n t r i f u g e d , and l y o p h i l i z e d as d e s c r i b e d i n s e c t i o n - 60 -I o T I M E (MIN) F i g u r e 6. Time course f o r the conver s i o n of CTP i n t o CDP-choline. Ten nmoles of CTP was assayed i n the presence of 100 nmoles of [~J3H~J phosphocholine. CDP-choline forma-t 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 (£,) (iv) at v a r i o u s times of i n c u b a t i o n . - 61 -0 2 4 6 8 1 0 1 2 C T P ( N M O L ) F i g u r e 7. Standard Curve f o r CTP d e t e r m i n a t i o n . CTP (0-10 nmoles) was assayed 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 of CDP-choline formation was c a l c u l a t e d from the dpm i n the product, which has the same s p e c i f i c / r a d i o a c t i v i t y as the s u b s t r a t e , Q 3 H ~ J CTP. - 62 -(1) ( i v ) . N u c l e o t i d e s were separated on a 40 cm x 2.1 mm column equipped with a dual wavelength UV d e t e c t o r (Model 152, A l t e x S c i e n t i f i c Inc.) o p e r a t i n g a t 280 nm and 254 nm and a dual chan-n e l recorder (Linear Instruments Corp.). A l i n e a r g r a d i e n t of 0.01 M KHzPC^ (pH 3.5) and 0.01 M KC1 to 0.5 M KH 2P0 4(pH 3.5) and 0.5 M KC1 ( t o t a l volume: 100 ml) e l u t e d the n u c l e o t i d e t r i p h o s p h a t e s without o v e r l a p . Peak areas 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 by peak widths a t h a l f h e i g h t . The c o n c e n t r a t i o n s of n u c l e o t i d e s were determined i n each sample by d i v i d i n g peak areas by the peak area/nmole values f o r standard n u c l e o t i d e t r i p h o s p h a t e s ( c o r r e c t e d f o r diphosphate i m p u r i t i e s ) . Chromatographic sepa-r a t i o n s and c a l c u l a t i o n s were made by Dr. Everard T r i p i n Dr. Michael Smith's l a b o r a t o r y . In the second method, known amounts of ITP were added to e v e r y . d i s h of c e l l s to serve as an i n t e r n a l standard (202). C e l l s were removed twice with 3 ml of CH 3OH-H 20 (1/0.8; v/v) to which 6 ml of c h l o r o f o r m was added. Phases were separated by c e n t r i f u g a t i o n . The upper phases were evaporated under reduced p r e s s u r e . The r e s i d u e was then d i s s o l v e d i n 1 ml of water, c e n t r i f u g e d to remove a p r e c i p i t a t e , and again concentrated by h e a t i n g to about 40° under a stream of n i t r o g e n . N u c l e o t i d e s were separated on a 45 cm x 2.1 mm column equipped with a s i n g l e wavelength UV d e t e c t o r (Model 153, A l t e x S c i e n t i f i c Inc.) oper^. a t i n g at 280 nm and a recorder (Linear Instruments C o r p . ) . Chromatographic e l u t i o n was with a l i n e a r g r a d i e n t of 0.1 M - 63 -KH2P04 (pH 3.8) to 1 M KH 2P0 4 (pH 3.8) ( t o t a l volume: 80 ml). Peak areas were determined by p l a n i m e t r y . The r e s u l t s from both methods agreed f a i r l y w e l l (see R e s u l t s ) . (m) F a t t y A c i d A n a l y s i s C e l l s were prepared as u s u a l ( s e c t i o n ( f )) at 7 hours p . i . . C e l l p e l l e t s were homogenized in•CHC1 3-CH 3OH (2/1; v/v) and e x t r a c t e d by the procedure d e s c r i b e d by F o l c h e t aL (197). D i -g l y c e r i d e was i s o l a t e d by TLC i n s o l v e n t system B. PC and PE were i s o l a t e d by TLC i n s o l v e n t system C. L i p i d s were v i s u a l -i z e d by s p r a y i n g the p l a t e s with 0.2% d i c h l o r o f l u o r e s c e i n i n methanol. L i p i d s were e l u t e d from the s i l i c a with 10 ml CHC1 3-CH3OH (2/1; v / v ) . The s o l v e n t was evaporated by h e a t i n g to 52-55° and blowing a stream of n i t r o g e n over the samples.. Methanolysis of l i p i d s was done by the method of Vance and Sweeley (203), without the a d d i t i o n of mannitol and the removal of HC1. The r e a c t i o n proceeded at 80° f o r at l e a s t 12 hours. The methyl e s t e r s e x t r a c t e d i n hexane were separated by gas-l i q u i d chromatography on a column of 12% H1-EFF-2BP 80/100 mesh (Applied Science L a b o r a t o r i e s ) . A 7610A High E f f i c i e n c y Gas Chromatograph (Hewlett Packard) was used, with the oven s e t at 180°. Standards i n c l u d e d mixtures which contained methyl e s t e r s of the f a t t y a c i d s : 14:0, 16:0, 18:0, 20:0, 16:1, 18:1, 18:2, 18:3; and the f a t t y a c i d methyl e s t e r s of r a t and t r o u t l i v e r l i p i d s . Peak areas were measured by p l a n i m e t r y . - 64 -(n) \2 3H~J C h o l i n e Pulse-Chase At 6h hours p . i . , the Medium 199 (and 2% f e t a l c a l f serum) was removed from each l a r g e d i s h . Immediately, the pulse me.-dium (7 ml of the same medium which contained 20 y C i of [] 3H~J c h o l i n e ) was added to each d i s h . At 6h hours p . i . , the c e l l s were washed twice with 5 ml of the same medium. To s t a r t the chase, 10ml of the same medium were immediately added. At 20 minute i n t e r v a l s , methanol-water-soluble e x t r a c t s were taken 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 ) . The chloroform l a y e r was e x t r a c t e d with a second 6 ml of CH 3OH-H 20 (1/0.8; v / v ) . The upper phases were concentrated by f l a s h eva-p o r a t i o n and di s s o l v e d , i n 1 ml of water. A l i q u o t s were then taken f o r counting and f o r TLC i n s o l v e n t system A. C a r r i e r c h o l i n e , phosphocholine, and CDP-choline (0.9 mg each) were ad-ded to each 2.25 cm TLC la n e . The c a r r i e r compounds were d e t e c -ted by i o d i n e vapour. Areas of s i l i c a g e l corresponding to each compound were scraped o f f the TLC p l a t e d i r e c t l y i n t o s c i n t i l l a -t i o n v i a l s . To each v i a l was added 2 ml 0.IN NaOH arid 100y£ : g l a c i a l a c e t i c a c i d . The lower c h l o r o f o r m phases were e x t r a c t e d twice more with 6 ml CH 3OH-H 20 (1/0.8; v / v ) , t r a n s f e r r e d to s c i n t i l l a t i o n v i a l s , 3 d r i e d by a i r , and counted. - 6 5 -RESULTS'" (a) C e l l Weight and P r o t e i n C o n c e n t r a t i o n The wet weight of BHK c e l l s from f i v e l a r g e d i s h e s i s shown in Table 3. The weight was about the same at 6% or 7 hours p . i . (Table 3). The average weight (Table 3:); was not changed by SF v i r u s i n f e c t i o n . T h i s average was used to convert r e s u l t s per d i s h of c e l l s to r e s u l t s per gram (wet weight) of c e l l s . Table 3 Wet Weight of BHK C e l l s I n f e c t e d with SF V i r u s 1 Wet weight, g 6h hours 7 hours p . i . p . i . Average Mock-infected 0.70, 1.04 0.97 0.90±0.18 SF v i r u s - i n f e c t e d 0.72, 1.12 0.85 0.90±0.20 1 F i v e dishes of c e l l s were harvested as usual ( 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 weighed i n a tared c e n t r i f u g e tube. 2 The v a r i a t i o n noted 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 enzyme a c t i v i t i e s of BHK c e l l s were measured i n r e l a -t i o n to p r o t e i n c o n c e n t r a t i o n s , these c o n c e n t r a t i o n s were mea-sured i n mock-infected and v i r u s - i n f e c t e d c e l l s . Table 4 shows 1 A l l methods were those that are d e s c r i b e d i n M a t e r i a l s and Methods, except where i t i s otherwise noted. - 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 with SF V i r u s P r o t e i n c o n c e n t r a t i o n (mg/g c e l l s ) S i g n i f i c a n c e of 4 Mock-infected , SF v i r u s - i n f e c t e d d i f f e r e n c e T o t a l , • _ . protein " 1 21.7 ±1.8 (5) 18.0 ±1.8 ( 5 ) J p<0.025 C y t o s o l 2 7.12±0.66(4) 5.50 ±0.52(4) p<0.01 Microsomes 3 2.37±0.56(5) 2.14 ±0.07(5) NS, p>0.25 Determinations were made on BHK c e l l p r o t e i n which 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 at 7 hours p . i . and then homogenized i n 0.9% NaCl ( M a t e r i a l s and Methods ( j ) ) . ^ C y t o s o l and microsomes were prepared a t 7 hours p . i . . The numbers i n brackets i n t h i s and s u c c e s s i v e t a b l e s r e f e r to the number of experiments. 4 S i g n i f i c a n c e of d i f f e r e n c e was measured by a group com-pa r i s o n t t e s t . - 67 -the t o t a l p r o t e i n i n BHK c e l l s i n f e c t e d with SF v i r u s . V i r u s i n f e c t i o n caused a s i g n i f i c a n t r e d u c t i o n i n the t o t a l c e l l pro-t e i n (Table 4). The amount of p r o t e i n i n the c y t o s o l of BHK c e l l s was a l s o reduced by v i r u s i n f e c t i o n , while the amount of microsomal p r o t e i n was not reduced (Table 4). At 6% hours p . i . , the amount of microsomal p r o t e i n (2.89 mg/g of mock- or v i r u s -i n f e c t e d c e l l s ) was s i m i l a r to that at 7 hours p . i . (Table 4). (b) C h o l i n e T r a n s p o r t The purpose of t h i s t h e s i s p r o j e c t was to e s t a b l i s h the mechanism of i n h i b i t i o n of []3H~J c h o l i n e i n c o r p o r a t i o n i n t o PC of BHK c e l l s , an i n h i b i t i o n which was caused by SF v i r u s i n f e c -t i o n . Since such i n h i b i t i o n could be a r e s u l t of i n h i b i t i o n of c h o l i n e t r a n s p o r t , the t r a n s p o r t process was examined in BHK c e l l s . The r e s u l t s of []]3H~J c h o l i n e t r a n s p o r t experiments are shown in F i g u r e 8. From 6% to 7 hours p . i . . . l e s s uptake occ u r r e d i n SF v i r u s - i n f e c t e d c e l l s compared to c o n t r o l c e l l s . I n h i b i -t i o n of uptake by i n f e c t i o n became more marked as the c o n c e n t r a -t i o n of c h o l i n e i n the medium was i n c r e a s e d ( F i g . 8). The up-take in mock-infected c e l l s f o l l o wed Michaelis-Menton k i n e t i c s . The K m value was 17 yM. The V m a x value was 381 pmoles minute 1 mg of t o t a l c e l l p r o t e i n In SF v i r u s - i n f e c t e d c e l l s however, the p l o t of 1/V vs. 1/S was not l i n e a r , so that K and V m „ ^ ' ' m max values were not o b t a i n e d . Since b i n d i n g of Q 3 H D c h o l i n e to the c e l l s u r f a c e would i n c r e a s e the apparent amount of t r a n s p o r t , such b i n d i n g was measured by competition with u n l a b e l l e d c h o l i n e . C e l l s were - 68 -iu i -o ce a. o to U J _ J o Q. < 0. 3 U J o X o 80H 1 60H 40 H 2 0 H 0 M0CK-INFECTED > ^ 0 x VIRUS-^ — INFECTED X 1 5 — F CHOLINE CONCENTRATION (pM) F i g u r e 8. Uptake of c h o l i n e by BHK c e l l s i n f e c t e d with SF v i r u s . Uptake rep r e s e n t s the disappearance of Q3H~J c h o l i n e from the medium of BHK c e l l s over a 30 minute p e r i o d , s t a r t i n g at 6h h j u r s p . i . . R e s u l t s are expressed as pmoles of c h o l i n e minute mg of t o t a l c e l l p r o t e i n - 69 -incubated f i r s t l y with fJ3H~J c h o l i n e and secondly with u n l a b e l l e d c h o l i n e , f o r 30 minutes each time. The percent r e l e a s e of [~J3H^ ] c h o l i n e was measured ( M a t e r i a l s and Methods s e c t i o n ( j ) ) . In c e l l s which were incubated with 0.61 yM fJ3H[] c h o l i n e , the per-cent r e l e a s e was 3.3% f o r mock-infected c e l l s , and 5.2% f o r SF v i r u s - i n f e c t e d c e l l s . (The average of three r e l e a s e r e s u l t s was 6.3% ± 6.2% f o r mock-infected c e l l s , and 5.2% ± 0.7% f o r SF v i r u s - i n f e c t e d c e l l s . ) In three experiments, c h o l i n e - c o n t a i n i n g pools were s e p a - i rated from c e l l s which were l a b e l l e d with 0.32-0.80 yM rj 3rfj c h o l i n e . At these c o n c e n t r a t i o n s , l e s s than 19% i n h i b i t i o n of c h o l i n e uptake by v i r u s i n f e c t i o n would be expected ( F i g . 8 ) . However, i n these experiments the r a t i o of t o t a l dpm i n a l l c h o l i n e - c o n t a i n i n g pools of SF v i r u s - i n f e c t e d c e l l s compared to mock-infected c e l l s was 0.44. Thus, 56% i n h i b i t i o n of uptake o c c u r r e d . (See D i s c u s s i o n . ) (c) Enzyme A c t i v i t i e s The enzymes of de novo PC s y n t h e s i s were examined in order to e x p l a i n the p o s s i b l e i n h i b i t i o n of de novo s y n t h e s i s which was caused by SF v i r u s i n f e c t i o n . The a c t i v i t i e s of c h o l i n e kinase (CK), phosphocholine c y t i d y l y l t r a n s f e r a s e (CT), and cho-l i n e p h o s p h o t r a n s f e r a s e (CPT) were measured i n s u b c e l l u l a r f r a c -t i o n s which were prepared from BHK c e l l s at 7 hours p . i . , unless i t i s otherwise noted. I t was thought that perhaps o n l y the r a t e - l i m i t i n g enzyme would be i n h i b i t e d by i n f e c t i o n . - 70 -The k i n e t i c s of CK were examined f i r s t . CK a c t i v i t y i n BHK c e l l c y t o s o l was l i n e a r l y r e l a t e d to p r o t e i n (to 250 yg) and time of assay (to 35 minutes) ( F i g . 9). CK a c t i v i t y was a p p a r e n t l y s a t u r a t e d with c h o l i n e at a c o n c e n t r a t i o n of 0.25 mM ( F i g . 10). CK a c t i v i t y was a l s o measured as a f u n c t i o n of ATP c o n c e n t r a t i o n ( F i g . 11). The s p e c i f i c a c t i v i t y of the c y t o s o l i c enzyme was not changed by v i r u s i n f e c t i o n ( F i g s . 9-11). In the c a l c u l a t i o n of CK a c t i v i t i e s at v a r i o u s ATP concen-t r a t i o n s , i t was found necessary to c o r r e c t f o r the extent of s e p a r a t i o n of c h o l i n e and phosphocholine. S e p a r a t i o n was de-pendant on the ions i n the assay mixture. In a c o n t r o l e x p e r i -ment, b o i l e d c y t o s o l (50 60-180 yg of p r o t e i n ) and []1'*C~j phosphocholine (10 nmoles) were mixed with the other assay com-ponents. At 0 mM or 5 mM r~/ATP-Mg~J ( i n the assay m i x t u r e ) , a l l the phosphocholine cpm were recovered i n the sodium hydroxide e l u a n t from assay columns. However, from 5 mM-25mM [~ATP-Mg~J , phosphocholine recovery decreased l i n e a r l y from 100% to 34%. Thus, a t each c o n c e n t r a t i o n of [~_ATP-Mg~J , a c o r r e c t i o n was made for l o s s of phosphocholine. F u r t h e r , i t was found u s e f u l to add 100' y£ of 0.1 M MgCl 2 to each assay tube a f t e r t e r m i n a t i o n of the r e a c t i o n but before ion exchange s e p a r a t i o n . T h i s proce-dure reduced the [ ] 3 H ~ J c h o l i n e cpm which were e l u t e d from the ion exchange r e s i n by the sodium hydroxide wash. The r e d u c t i o n of Q3H~J c h o l i n e cpm became more pronounced as the c o n c e n t r a t i o n of r~ATP-Mg~J was reduced. Such a d d i t i o n of MgCl 2 d i d not a f -f e c t the recovery of 4C~J phosphocholine from assay mixtures which contained 0, 5, or 25 mM QATP - M g J J . - 71 -Prote in, mg 12-^ Time, min F i g u r e 9. CK a c t i v i t y vs. p r o t e i n and time. 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 assayed. A f t e r the assay, the product was separated by ion exchange. A c t i v i t y was measured at 10 mM ATP, 10 mM Mg* +, and 0.25 mM. fJ3H^] c h o l i n e (1 C i/mole). The upper graph shows the e f f e c t of 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 lower graph shows the e f f e c t of time on CK a c t i v i t y (in 87-126 yg of c y t o s o l p r o t e i n ) . A blank value was s u b t r a c t e d 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 f i g u r e s and t a b l e s . Blanks contained b o i l e d p r o t e i n or no p r o t e i n . 4 Chol ine, m M F i g u r e 10. CK a c t i v i t y vs. c h o l i n e . CK was assayed 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 assay, the product was separated by ion exchange. Choline c o n c e n t r a t i o n was v a r i e d by the a d d i t i o n of Q3H~J c h o l i n e (5 Ci/mole). ATP'and Mg + + c o n c e n t r a t i o n s were 10 mM. Separate blanks, were assayed at each concen-t r a t i o n o f rj3H~J c h o l i n e . The blank cpm increased with higher fJ3H]] c h o l i n e c oncentra-t i o n s . (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 at 0.75 mM and 1 mM c h o l i n e were not used to p l o t the curve s i n c e they were averages of very 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 -A T P , m M F i g u r e 1 1 . CK a c t i v i t y vs. 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 assayed. A f t e r the assay, the product was separated by the ion exchange method. ATP and Mg +* were added to each assay i n equal amounts. Ac-t 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). - 74 -CK a c t i v i t y was v e r i f i e d by a second assay system, i n which c h o l i n e and phosphocholine were separated by TLC. C y t o s o l from mock- or SF v i r u s - i n f e c t e d c e l l s was incubated with 7.5 mM ATP (and 10 mM Mg + +) . The formation of fJ3H~J phosphocholine was l i n e a r to 30 minutes. The a c t i v i t y of CK by t h i s assay was 0.30 nmoleV min ^ mg p r o t e i n ^ i n c y t o s o l from both i n f e c t e d and con-t r o l c e l l s . T h i s a c t i v i t y i s s i m i l a r to the r e s u l t from the ion exchange assay ( F i g . 11, Table 5). The k i n e t i c s of CT were examined next. C y t o s o l i c CT ac-t i v i t y from BHK c e l l s was p r o p o r t i o n a l to the amount of p r o t e i n in the assay (up to 72 yg) and to the time of assay (up to 40 minutes) ( F i g . 12). CT was s a t u r a t e d by phosphocholine at a c o n c e n t r a t i o n of 1.25 mM ( F i g . 13). C y t o s o l i c CT a c t i v i t y was not a f f e c t e d by SF v i r u s i n f e c t i o n ( F i g s . 12, 13), except t h a t c y t o s o l i c a c t i v i t y a t suboptimal c o n c e n t r a t i o n s of phosphocho-l i n e appeared to be i n c r e a s e d by SF v i r u s i n f e c t i o n ( F i g . 13). The optimal c o n c e n t r a t i o n of CTP f o r c y t o s o l i c CT was 3 mM f o r the enzyme from both mock-infected and SF v i r u s - i n f e c t e d c e l l s ( F i g . 14). The k i n e t i c s of CT i n microsomes were a l s o examined. The CT a c t i v i t y i n BHK c e l l microsomes was p r o p o r t i o n a l to assay p r o t e i n (up to 68 yg) and time (up to 40 minutes). Microsomal CT from SF v i r u s - i n f e c t e d c e l l s was s a t u r a t e d by phosphocholine at 1.25 mM c o n c e n t r a t i o n , whereas CT from mock-infected c e l l s was s a t u r a t e d by phosphocholine at a c o n c e n t r a t i o n of 2 mM or - 75 -o Protein, mg T ime, 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 vs. p r o t e i n and time. CT was assayed at 1.25 mM I*C~J phosphocholine, 3 mM CTP and 12 mM Mg + +. 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 to the p r o t e i n i n the assay. The lower 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 of time of assay. C y t o s o l from mock-infected 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 , ( • ) . - 7 6 -E n z y m e A c t i v i t y , n m o l e s m i n m g p r o t e i n 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 . phosphocholine. CT was assayed at 3 mM CTP, 12 mM Mg + +, and 0.31-1.6 mM 4C~J phosphocholine. Background (blank) cpm were determined a t each c o n c e n t r a t i o n of 4C~J phosphocholine. C y t o s o l from mock-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 , (•). - 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 vs. CTP. CT was assayed at 1.25 mM [_l 4C~J phosphocholine, 12 mM Mg + +, and 0 mM - 16 mM CTP. A blank was used which contained no CTP. T h i s blank had about the same value as a b o i l e d enzyme blank. C y t o s o l from mock-infected 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 - 78 -h i g h e r ^ ( F i g . 15). CT a c t i v i t y was g r e a t e s t at a CTP concen-t r a t i o n of 2 mM i n both mock- and SF v i r u s - i n f e c t e d microsomes. A p o s s i b l e e x p l a n a t i o n f o r the i n h i b i t i o n of fJ3H^] c h o l i n e i n c o r p o r a t i o n i n t o PC a f t e r v i r u s i n f e c t i o n i s t h a t the l e v e l of l i p i d a c t i v a t i o n of c y t o s o l i c CT was lower i n i n f e c t e d c e l l s than i n mock-infected c e l l s . I f t h i s were t r u e , f u r t h e r l i p i d a c t i v a t i o n would s t i m u l a t e CT more i n i n f e c t e d c e l l s . However, r a t l i v e r l i p i d (at 2.5 mg/ml) s t i m u l a t e d c y t o s o l i c CT 2.4-f o l d i n c o n t r o l microsomes and about e q u a l l y , 2 . 8 - f o l d i n mic-rosomes from the v i r u s - i n f e c t e d c e l l s ( F i g . 16). The a c t i v i t i e s of the enzymes of de novo s y n t h e s i s of PC i n BHK c e l l s i n f e c t e d with SF v i r u s are summarized i n Table 5. Not one as expected, but a l l three enzymes of de novo s y n t h e s i s were reduced i n t o t a l a c t i v i t y by SF v i r u s i n f e c t i o n . M i c r o -somal CT and the microsomal enzyme, CPT, were reduced i n a c t i -v i t y because of s i g n i f i c a n t i n h i b i t i o n of the s p e c i f i c a c t i v i -t i e s (Table 6). Greater i n h i b i t i o n of microsomal CT would be expected i f the a c t i v i t y had been measured at 2 mM phosphocho-l i n e (the s a t u r a t i o n c o n c e n t r a t i o n f o r the enzyme from micro-somes of mock-infected c e l l s ) . The c y t o s o l i c CT and the c y t o -solic enzyme, CK, were reduced i n t o t a l a c t i v i t y because of the decrease i n s o l u b l e p r o t e i n i n i n f e c t e d c e l l s (Table 4). 2 Two experiments were combined to 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 , microsomal CT ac-t i v i t i e s were determined at up to 1.56 mM phosphocholine. In the second experiment, the f i r s t p o i n t (at 1.6 mM phosphocholine) was f i t t e d onto the curve from the f i r s t experiment by l i n e a r r e g r e s s i o n of a p l o t of 1/V vs. 1/S. Further values of a c t i v i -t i e s a t higher phosphocholine c o n c e n t r a t i o n s were not f i t t e d onto the curve, but were c a l c u l a t e d as a p r o p o r t i o n of the f i r s t p o i n t at 1.6 mM phosphocholine. "> u < ® E > N C Phosphochol ine, m M F i g u r e 15. Microsomal CT a c t i v i t y vs. phosphocholine. CT was assayed at 2 mM CTP, and 0-2.8 mM rjlltC~J- or £_3ti~\ phosphocholine (7.4-12.8 Ci/mole) i n microsomes from mock-infected (0,0) or 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 at high phospho-c 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 separate experiment. 12 mM Mg + +, - 80 -0 > 1 1 1 1 " i — 0 0.5 1.0 1.5 2.0 2.5 RAT LIVER LIPID IN ASSAY (MG ML~') F i g u r e 16. 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 l i v e r l i p i d . CT was assayed i n c y t o s o l which was prepared from 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 hours p . i . . Enzyme a c t i v i t y was measured at 1.6 mM []3H~] phosphocholine, .3 mM CTP, and 12 mM Mg + +. Of the 100 u£ of assay mixture, an a l i q u o t (40 y&) was spotted onto a TLC p l a t e f o r s e p a r a t i o n of phosphocholine and CDP-choline. Acetone-extracted r a t l i v e r l i p i d was added to the f i n a l c o n c e n t r a t i o n s which are shown above. C y t o s o l from mock-infected 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). - 8 1 -Table 5 Enzymes of De Novo S y n t h e s i s of PC i n BHK C e l l s I n f e c t e d with SF V i r u s A c t i v i t i e s Mock- i n f e c t e d SF v i r u s - i n f e c t e d Enzyme 1 S p e c i f i c 2 a c t i v i t y T o t a l 3 a c t i v i t y S p e c i f i c 2 a c t i v i t y T o t a l 3 a c t i v i t y CK 0.432 7.3 0.435 5.2 CT ( c y t o s o l i c ) 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 assayed i n c y t o s o l or microsomes from a s i n g l e p r e p a r a t i o n of 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 - 1 mg p r o t e i n - ^ -nmoles m i n - 1 g c e l l s -Table 6 I n h i b i t i o n of Microsomal CT and CPT by SF V i r u s I n f e c t i o n of BHK C e l l s Enzyme A c t i v i t i e s 1 Mock-infected SF v i r u s - i n f e c t e d S i g n i f i c a n c e of d i f f e r e n c e CT (micro- 3 somal) ^  1.61±0.54(5) 1.19±0. 56(5) p<0.10 CPT 3.32±0.74(5) 2.37±0. 23(5) p<0.025 2 3 nmoles min 1 mg p r o t e i n 1 CT a c t i v i t y was measured at 1.25 mM phosphocholine. Since the a c t i v i t i e s were not 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 group comparison, and s i n c e the 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 comparison t t e s t was made. 4 S i g n i f i c a n c e was determined by a group comparison t t e s t - 82 -The assay c o n d i t i o n s f o r the t h i r d enzyme of de novo syn-t h e s i s o f PC, namely CPT, have been e s t a b l i s h e d (173). A c t i v a -t i o n of CPT by the detergent, t a u r o c h o l a t e , i t was reasoned, might r e l i e v e the v i r u s - i n d u c e d i n h i b i t i o n . CPT i n microsomes from BHK c e l l s at 6h hours p . i . was a c t i v a t e d by t a u r o c h o l a t e (0.4%; w/v). T h i s treatment d i d not reverse the i n h i b i t i o n of CPT caused by v i r u s i n f e c t i o n , s i n c e the enzyme from mock-in-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 was a c t i v a t e d about e q u a l l y ( i . e . , 3 . 8 - f o l d and 3 . 7 - f o l d , r e s p e c t i v e l y ) . CPT which was ac-t i v a t e d by 0.6% t a u r o c h o l a t e was not s o l u b i l i z e d , s i n c e the ac-t i v i t y c o u l d be sedimented by c e n t r i f u g a t i o n at 100,000 x £ f o r 1 hour. Two other enzymes, p e r i p h e r a l to de novo s y n t h e s i s of PC, were examined. Phosphatidylethanolamine m e t h y l t r a n s f e r a s e ac-t i v i t y was not detected i n mock-infected or v i r u s - i n f e c t e d BHK c e l l microsomes a t 7 hours p . i . . The l i m i t o f d e t e c t i o n was 0.19 nmole'x min mg p r o t e i n . By comparison, the a c t i v i t y i n r a t l i v e r microsomes was 2.5 nmoles min ^  mg p r o t e i n ^. Sim-i l a r l y , c h o l i n e oxidase a c t i v i t i e s were below the d e t e c t i o n l i m i t s of 0.39 nmole": min mg p r o t e i n i n mitochondria from mock-infected c e l l s and 0.60 nmole;:'\min mg p r o t e i n i n mito-chondria from v i r u s - i n f e c t e d c e l l s , both at 6h hours p . i . . Rat l i v e r m itochondria (which were s u p p l i e d by Miss Linda Grieve) had an a c t i v i t y of 0.66 nmoleC> min 1 mg p r o t e i n 1 . Low cho-l i n e oxidase a c t i v i t y was expected since most of the w a t e r - s o l -uble r a d i o a c t i v i t y i n fJ3H[] c h o l i n e - l a b e l l e d c e l l s was i d e n t i f i e d - 83 by TLC as c h o l i n e or an e s t e r of c h o l i n e ( M a t e r i a l s and Methods s e c t i o n (£) ( i ) ) . (d) Separation and I d e n t i f i c a t i o n of C h o l i n e - C o n t a i n i n g Com-pounds In the measurement of pool s i z e s o f c h o l i n e , phosphocholine, and CDP-choline, the f i r s t necessary step was to separate these compounds on a p r e p a r a t i v e s c a l e . Reportedly, the three com-pounds should be separated by column chromatography on AG 1 r e s i n (formate form) e l u t e d with a l i n e a r g r a d i e n t of 0-0.02N formic a c i d (33). However, i n t h i s l a b o r a t o r y , Q 1 " * ^ ] phospho-c h o l i n e was e l u t e d from AG 1 (formate form) by water alone. Moreover, the s e p a r a t i o n of phosphocholine and CDP-choline was not complete. Because a workable p u b l i s h e d procedure f o r s e p a r a t i n g cho-l i n e , phosphocholine, and CDP-choline was not found, a new method was developed f o r t h i s purpose. In t h i s method, c h o l i n e was separated from phosphocholine and CDP-choline by chroma-tography on AG 1 ion exchange r e s i n . Phosphocholine and CDP-c h o l i n e were separated by chromatography on c h a r c o a l . In order to monitor t h e i r s e p a r a t i o n and y i e l d , e i t h e r 4C]] ^ c h o l i n e , phosphocholine, or CDP-choline was added to the p r e l i m i n a r y ex-t r a c t i o n mixture of BHK c e l l s (water-methanol-chloroform, 1/0.5/0.5; v / v / v ) . The r e s u l t of the s e p a r a t i o n of c h o l i n e , phosphocholine, and CDP-choline i s shown i n Table 7. For each compound, 1>*C r a d i o a c t i v i t y (cpm) was recovered q u a n t i t a t i v e l y in the a p p r o p r i a t e f r a c t i o n s from columns of AG 1 r e s i n (cho-l i n e ) or colums of c h a r c o a l (phosphocholine and CDP-choline). - -84 -Table 7 Separa t i o n of Q ^ C f ] C h o l i n e - C o n t a i n i n g Compounds Added Percent of cpm i n each f r a c t i o n Number of compound Cho l i n e Phospho-c h o l i n e CDP-c h o l i n e exper iments ^CfJ C h o l i n e 96.0% 3.5% 0.4% (1) rjlltC"J Phospho-c h o l i n e 0.6% 97.2% 2 * 2 *6 (2) fJ l l fC~J CDP-c h o l i n e 1.5% 6. 6% 91.9% (3) The method i s d e s c r i b e d i n M a t e r i a l s and Methods sec-t i o n (I) (i) . A f t e r enzyme h y d r o l y s i s of phosphocholine and CDP-choline, the product o f h y d r o l y s i s was v e r i f i e d as c h o l i n e by TLC (Ma-t e r i a l s and Methods s e c t i o n (£,) ( i ) ) . A l s o , the h y d r o l y s i s product of r a d i o a c t i v e l y l a b e l l e d phosphocholine was i d e n t i f i e d by ion exchange chromatography. In t h i s experiment, the hydro-l y s i s to c h o l i n e was found to be 80% complete when determined by TLC. Most of the r a d i o a c t i v i t y (from.the h y d r o l y s a t e ) which was e l u t e d from AG 50W-X8 r e s i n , was i n a s i n g l e peak which e l u t e d a t the same p o i n t as a u t h e n t i c . (~J3H~J c h o l i n e ( F i g . 17). A second peak, which e l u t e d before c h o l i n e , was assumed to be - 85 -C h o l i n e F r a c t i o n N u m b e r F i g u r e 17. 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. H^ J - L a b e l l e d phosphocholine from mock-in-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 (the e l u a n t was evaporated and then d i s s o l v e d i n 5 ml of water) and on a c o l -umn of c h a r c o a l , as u s u a l . Phosphocholine was hydrolyzed with about 3 u n i t s of a l k a l i n e phosphatase i n a t o t a l volume of 8.2 ml at pH 9.2. P a r t of the h y d r o l y s a t e was a p p l i e d to a column (10 cm x 1.5 cm) of AG 50W-X8 (ammonium form). The column was e l u t e d with a l i n e a r g r a d i e n t of ammonium formate (0.1M-0.5M; t o t a l volume, 400 ml). A l i q u o t s of 5 ml f r a c t i o n s of e l u a n t were counted. 3H cpm were not c o r r e c t e d f o r 1 4 C . ( T 1 4C~| phosphocholine was added to the c e l l e x t r a c t . ) r j 3 H j C h o l i n e e l u t e d at the p o s i t i o n of the arrow. - 86 -phosphocholine, s i n c e phosphocholine binds l e s s t i g h t l y than c h o l i n e to t h i s type of r e s i n (155). Q 3H[] C h o l i n e , which was i s o l a t e d by i o n - p a i r e x t r a c t i o n , was again t e s t e d f o r p u r i t y by TLC i n s o l v e n t system A. T e s t s were made on c h o l i n e from one pool each of f r e e c h o l i n e , phos-phocholine, and CDP-choline pools of mock- and SF v i r u s - i n -f e c t e d c e l l s . . The cpm i n the c h o l i n e r e g i o n , expressed as the percent of the combined cpm i n c h o l i n e , phosphocholine, and CDP-c h o l i n e r e g i o n s , averaged 96.6%. (e) I n c o r p o r a t i o n of Q 3 H Q C h o l i n e I f the f l u x over a s i n g l e enzyme step was reduced by v i r u s i n f e c t i o n , then the s u b s t r a t e of that enzyme would accumulate. Thus, i t was u s e f u l to measure the c o n c e n t r a t i o n s of the sub-s t r a t e s of PC s y n t h e s i s i n i n f e c t e d BHK c e l l s . I n i t i a l l y , the i n c o r p o r a t i o n of Q 3H^] c h o l i n e was measured s i n c e the l a b e l c o u l d a l s o accumulate i n the s u b s t r a t e of an enzyme with reduced f l u x . The i n c o r p o r a t i o n of [] 3H]] c h o l i n e i n t o c h o l i n e , phospho-c h o l i n e , CDP-choline, and PC i s shown i n Table 8. In these ex-periments, c e l l s were l a b e l l e d with Q 3 l Q c h o l i n e (10 uCi i n 3 ml of medium f o r each l a r g e dish) f o r 30 minutes, s t a r t i n g a t 6^-7 hours p . i . . The i n c o r p o r a t i o n i n t o c e l l u l a r c h o l i n e was doubled by v i r u s i n f e c t i o n . I n c o r p o r a t i o n i n t o phosphocholine was a l s o i n c r e a s e d (but not s i g n i f i c a n t l y ) , whereas the i n c o r -p o r a t i o n i n t o CDP-choline remained u n a l t e r e d a f t e r v i r u s i n f e c -t i o n . These i n c o r p o r a t i o n r e s u l t s supported the idea that CK was reduced i n a c t i v i t y i n BHK c e l l s which were i n f e c t e d with Table 8 I n c o r p o r a t i o n of []3H^] Choline i n t o PC and i t s Precursors i n BHK C e l l s I nfected with SF V i r u s " -6 I n c o r p o r a t i o n (dpm x 10 /g c e l l s ) Mock-infected SF v i r u s - i n f e c t e d S i g n i f i c a n c e of d i f f e r e n c e co C h o i i n e Phosphocholine CDP-choline P h o s p h a t i d y l c h o l i n e T o t a l 1.10± 0.18 (3) 4.1 ± 2.5 (3) 0.78± 0.21 (3) 26.4 ±13.1(7) 32.4 2.28±0.48 (3) 9.5 ±5.4(3) 0.84±0.33 (3) 4.8 ±4.4(7) 17.4 p<0.05 NS, p>0.1 NS, p>0.5 p<0.005 C e l l s were l a b e l l e d for 30 minutes, s t a r t i n g at 6%-7 hours p . i . . C h o l i n e -c o n t a i n i n g compounds were separated 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). 2 S i g n i f i c a n c e was determined by a group comparison t t e s t . - 88 -SF v i r u s , s i n c e the l a b e l l i n g of i n t r a c e l l u l a r c h o l i n e was i n -creased. However, i n c o r p o r a t i o n r e s u l t s such as these do not take isotope d i l u t i o n i n t o account. (Compare R e s u l t s s e c t i o n Over seven experiments, the i n c o r p o r a t i o n of Q 3 H ^ ] c h o l i n e i n t o PC was i n h i b i t e d by an average of 77% ± 23% (s.d.) as a r e -s u l t of v i r u s i n f e c t i o n . However, the t o t a l i n c o r p o r a t i o n -of Q 3H^] c h o l i n e (Table 8) was o n l y 54% as g r e a t i n i n f e c t e d c e l l s as i n mock-infected c e l l s . T h i s r e s u l t i m p l i e d an i n h i b i t i o n of 46% of E 3H[] c h o l i n e uptake by SF v i r u s i n f e c t i o n , which ac-counted f o r a l a r g e p a r t of the i n h i b i t i o n o f i n c o r p o r a t i o n i n t o PC. In a methanol-water e x t r a c t of BHK c e l l s which were l a b e l l e d with Q 3H[] c h o l i n e , the r a d i o a c t i v i t y seemed to be e x c l u s i v e l y in c h o l i n e , phosphocholine, or CDP-choline ( M a t e r i a l s and Meth-ods s e c t i o n (£) ( i ) ) . The l i p i d which was l a b e l l e d by Q 3H^] c h o l i n e was i d e n t i f i e d as PC by TLC ( F i g . 18). L i t t l e or no 3H was i n LPC, LPE, or PE ( F i g . 18). I t i s not l i k e l y t h a t s p h i n -gomyelin was l a b e l l e d , s i n c e i n t h i s s o l v e n t system, i t migrates to a p o s i t i o n with an Rf value l e s s than t h a t of PC (but g r e a t -er than that of LPC) (204). (f) P o o l / S i z e Measurements - 1. PC and PC P r e c u r s o r s i S ince i n c o r p o r a t i o n r e s u l t s may be biased by a change i n pool s i z e of the l a b e l l e d compound, s p e c i f i c r a d i o a c t i v i t i e s and pool s i z e s of PC and i t s p r e c u r s o r s were measured. B 1 5 co • O E a o I 1 0 H 0 .5 1.0 K f « f F i g u r e 18. TLC of Q 3 H j c h o l i n e - l a b e l l e d 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 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,) ( i ) . P a r t of the l i p i d was a p p l i e d to a s i l i c a g e l TLC p l a t e , which was developed i n s o l v e n t system C. PC (R^ 0.13-0.18) was separated from LPC (R f 0.03-0.06), LPE (R f 0.19-0.24) , and PE (R f 0.46-0.52). T r i - and d i g l y c e r i d e s moved to the s o l v e n t f r o n t . S e c t i o n s of 10% of the s i l i c a g e l lane were scraped o f f and e l u t e d with 7 ml of chloroform-methanol (2/1; v / v ) . The e l u a n t was d r i e d and then counted for 3H. L i p i d was from mock- (A) or SF v i r u s - i n f e c t e d ( B X l e e l l s , - 90 -The c o l o r i m e t r i c d e t e r m i n a t i o n of c h o l i n e of Hayashi e t a_L (194), was i n i t i a l l y used to measure c h o l i n e . However, the r e -s u l t s were v a r i a b l e . C3H~J C h o l i n e , which was separated., as • usual, from fJ3H^] c h o l i n e - l a b e l l e d BHK c e l l s and then measured by the c o l o r i m e t r i c t e s t , had a s p e c i f i c r a d i o a c t i v i t y of 1360 dpm/nmole. Further p u r i f i c a t i o n of c h o l i n e by TLC (in s o l v e n t system A) or by column chromatography on AG 50W, ammonium form (which was e l u t e d with a g r a d i e n t of 0.IM to 0.5M ammonium f o r -mate) decreased the s p e c i f i c r a d i o a c t i v i t y to 584 and 345 dpm/ nmole, r e s p e c t i v e l y . In the case of c h o l i n e p u r i f i e d by column chromatography, the decrease appeared to be caused by contamina-t i o n of the c h o l i n e with ammonium formate. When p a r t of the pur-i f i e d c h o l i n e sample was passed through a column of AG 1 i n the hydroxyl form (which should remove formate) and evaporated (which should remove ammonia), the s p e c i f i c r a d i o a c t i v i t y became 672 dpm/nmole. A l s o , ammonium formate caused an in c r e a s e d ab-sorbance i n the c o l o r i m e t r i c d e t e r m i n a t i o n of standard c h o l i n e . The s p e c i f i c r a d i o a c t i v i t y of the c h o l i n e - c o n t a i n i n g com-pounds was measured (Table 9). The compounds were i s o l a t e d from BHK c e l l s which had been l a b e l l e d with fJ3H^] c h o l i n e f o r 30 min-utes. Free c h o l i n e and c h o l i n e which was d e r i v e d from phospho-c h o l i n e and CDP-choline were measured e s s e n t i a l l y by the proce-dure of Goldberg and McCaman (195). Phosphoruss i n PC was meas-ured by the method of Raheja et a_L (198). The s p e c i f i c r a d i o -a c t i v i t y of c h o l i n e i n c r e a s e d , while that of phosphocholine and CDP-choline decreased, as a r e s u l t of v i r u s i n f e c t i o n . (Phos-phocholine s p e c i f i c r a d i o a c t i v i t y would be f u r t h e r decreased, - 91 -Table 9 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 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 1 S p e c i f i c r a d i o a c t i v i t y S i g n i f y Mock-infected SF v i r u s - i n f e c t e d icance C h o l i n e Phosphocholine CDP-choline P h o s p h a t i d y l c h o l i n e (dpm/praole) 9.5+ 3.4(3) 38.9+ 7.5(3) p<0.01 128 +48 (3) 83 +28 (3) NS,p>0.10 149 +59 (3) 59.2+ 9.2(3) p<0.10 (dpm/nmole) 7400+1700(7) "1700+1500(7) p<0.005 Pool s i z e S i g n i f y Mock-infected SF v i r u s - i n f e c t e d icance (nmoles/g c e l l s ) o Ch o l i n e 146 + 71 (3) 68 + 27 (3) NS,p>0.1 Phosphocholine 34 + 12 (3) 120 + 40 (3) p<0. 025 CDP-choline 6. ,1 + 1 • 0(3) 15. 7+ 5. • 6 (3) p<0 . 05 (ymoles/g c e l l s ) P h o s p h a t i d y l c h o l i n e 3. ,4 + 1 • 4 (7) 3. 0+ 1. • 4 (7) NS,p>0.5 C e l l s were l a b e l l e d with £ 3 H ^ | c h o l i n e f o r 30 minutes. L a b e l l i n g began a t 6^-7.hours p . i . . 2 S i g n i f i c a n c e of d i f f e r e n c e was determined by a group comparison t t e s t . 3 By a p a i r e d comparison t t e s t , p was l e s s than 0.1. - 92 -except that there was a n o n s i g n i f i c a n t i n c r e a s e i n i n c o r p o r a t i o n i n t o phosphocholine (Table 8).) C u r i o u s l y , the s p e c i f i c r a d i o -a c t i v i t y o f c h o l i n e was l e s s than that of phosphocholine. (See Di s c u s s i o n . ) Values of pool s i z e s were c a l c u l a t e d by d i v i d i n g (a), the t o t a l 3H dpm i n c h o l i n e , phosphocholine,or CDP-choline ( a f t e r s e p a r a t i o n by ion exchange or c h a r c o a l columns), or PC ( i n the l i p i d e x t r a c t ) by (b), the s p e c i f i c r a d i o a c t i v i t y of each com-pound. Pool s i z e s are shown in Table 9 and F i g u r e 19. The r e -s u l t s i n Table 9 and F i g u r e 19 were from the same experiments as those of Table 8. Of the three w a t e r - s o l u b l e compounds, cho-l i n e predominated i n mock-infected c e l l s while phosphocholine was the most abundant i n v i r u s - i n f e c t e d c e l l s . The pool of cho-l i n e was made s m a l l e r , the pools of phosphocholine and CDP-cho-l i n e were made g r e a t e r , and the pool of PC was u n a f f e c t e d by SF v i r u s i n f e c t i o n (Table 9, F i g . 19). Accumulation of phospho-c h o l i n e (and CDP-choline) l i k e l y r e s u l t e d from r e d u c e d - a c t i v i t y over the CT (and CPT).steps. (See D i s c u s s i o n . ) The t o t a l amount of a l l three w a t e r - s o l u b l e pools was very s i m i l a r i n mock-in-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 ( i . e . , 186 and 204 nmoles/g c e l l s , r e s p e c t i v e l y ) . D i g l y c e r i d e was measured in some of the same l i p i d e x t r a c t s which were used f o r measurement of PC. At 7 to 7% hours p . i . , the pool s i z e of d i g l y c e r i d e i n mock-infected BHK c e l l s was 47± 16 (s.d.) nmoles/g c e l l s . At the same time, i n SF v i r u s - i n f e c t e d - 93 -o 7 0 3 I t o 7 0 3 9 u 7 0 (A •o o 0 I n 7 0 3 ID 7 0 at 0 7 = V 3 2 9 a Nanomoles / 5 15 cm. dishes of cells o o 00 o -J ] I D = 5 5 I si ID 1 ? 1 Micromoles / 5 15 cm. dishes of cells Ul to © io in u a 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 . C e l l s were l a b e l l e d and compounds were separated as d e s c r i b e d i n Table 8. The e r r o r bars r e p r e s e n t s t a n -dard e r r o r s of the mean. R e s u l t s are expressed per 5 l a r g e dishes of BHK c e l l s . C h o l i n e phosphate i s synonymous with phosphocho-l i n e . 94 -c e l l s , the pool s i z e was 43±17 (s.d.) nmoles/g c e l l s . These two values were each an average of 5 d e t e r m i n a t i o n s . The two values were not s i g n i f i c a n t l y d i f f e r e n t (p>0.5, group comparison t t e s t ) . 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 could not have caused any virus-mediated p e r t u r b a t i o n s of PC s y n t h e s i s . ' Phosphocholine and CTP were each measured by two indepen-dent methods. The r e s u l t s of these methods were i n f a i r agree-ment (Table 10). The d i f f e r e n c e s i n phosphocholine determina-t i o n s which were observed (Table 10) were probably caused by the d i f f e r e n t i n c u b a t i o n s of c e l l s before e x t r a c t i o n of phosphocho-l i n e . (See D i s c u s s i o n . ) The pool of CTP i n BHK c e l l s was s i g -n i f i c a n t l y reduced i n s i z e by SF v i r u s i n f e c t i o n (Table 10). However, a sm a l l e r n u c l e o t i d e pool s i z e was not l i m i t e d to CTP alone (Results s e c t i o n ( g ) ) . (g) Pool S i z e Measurements - 2. N u c l e o t i d e s N u c l e o t i d e c o n c e n t r a t i o n s i n p e r c h l o r i c a c i d e x t r a c t s of mock-infected and SF v i r u s - i n f e c t e d BHK c e l l s were measured by the f i r s t method 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 (£) (v). C e l l s were taken from the incubator at 6h hours p . i . f o r e x t r a c t i o n of n u c l e o t i d e s . The r e s u l t s of one experiment are shown in F i g u r e s 20 and 21. In t h i s experiment, ITP was added to each d i s h of c e l l s (23.5 nmoles/large dish) a f t e r the PBS wash, but before removal of the c e l l s i n 10% p e r c h l o r i c a c i d . The c o n c e n t r a t i o n of ITP i n the sample which was i n j e c t e d onto the high pressure column was compared with the expected concen-t r a t i o n . The recovery was 77% f o r e x t r a c t s from both mock-- 95 -Table 10 Pool S i z e s of C y t i d y l y l t r a n s f e r a s e S u b s 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 Pool s i z e Mock-infected SF v i r u s - i n f e c t e d S i g n i f i c a n c e - ^ f (nmoles/g c e l l s ) d i f f e r e n c e Phosphor c h o l i n e ^ 34±12(3) 120±40(3) p<0.025 Phospho-choline- 3 64± 7(3) 147±37(3) p<0.02 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 determined by a group comparison t t e s t . 2 Pool s i z e s were determined by the method mo d i f i e d from Goldberg and McCaman (195). C e l l s were taken from the incubator (for e x t r a c t i o n ) at 7-7% hours p . i . . 3 Pool s i z e s were determined by the method of Choy e t a l (200). C e l l s were taken at 6-1/2 - 6-5/6 hours p . i . . ^ P o o l s i z e s were determined by high p r e s s u r e l i q u i d chroma-tography. C e l l s were taken at 6h hours 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 -] fected BHK c e l l s . See tex t f o r • ' d e t a i l s . 1, ADP; 2, GDP; 3, CTP j 4, UTP; 5, ATP; 6, ITP ( i n t e r n a l standard); 7, GTP. SF VIRUS :l=rh - 98 -i 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 . In other experiments, ITP was not added to the d i s h e s , and the r e s u l t s were c o r r e c t e d f o r 77% recovery. The pool s i z e s of n u c l e o t i d e s i n BHK c e l l s which were i n f e c t e d with SF v i r u s are shown i n Table 11 . The amounts of the p y r i m i d i n e n u c l e o t i d e t r i p h o s p h a t e s , CTP and UTP, were reduced by v i r u s i n f e c t i o n to about o n e - t h i r d of the c o n t r o l v a l u e . S i m i l a r l y , both purine n u c l e o t i d e t r i p h o s p h a t e s , ATP and GTP, were reduced e q u a l l y , to about t h r e e - f i f t h s of the con-t r o l v a l u e . In c o n t r a s t , both purine n u c l e o t i d e diphosphates were i n c r e a s e d , again e q u a l l y so (Table 11) > Use of n u c l e o t i d e t r i p h o s p h a t e s i n v i r a l RNA s y n t h e s i s may be the reason f o r the smaller pools which were the r e s u l t of v i r u s i n f e c t i o n . Since l a c t a t e dehydrogenase has been shown to leak out of c e l l s (chick embryo c e l l s ) which were i n f e c t e d with SF v i r u s (205) , i t was thought that n u c l e o t i d e s a l s o might leak out. The absorbance of the medium of i n f e c t e d c e l l s was monitored (Table 1 2 ) . A leakage of 10 nmoles of ATP/dish (5% of the pool i n v i r u s - i n f e c t e d c e l l s ) would have i n c r e a s e d the absorbance a t 260 nm of 3 ml of medium by 0 . 051 . The i n c r e a s e i n absorbance at 260 nm (or 280 nm) which was caused by v i r u s i n f e c t i o n , was a b o l i s h e d by treatment with t r i c h l o r o a c e t i c a c i d (Table 1 2 ) . T h i s treatment presumably removed v i r u s (or p r o t e i n s ) i n the medium. Thus, very l i t t l e of the i n t r a c e l l u l a r pools of n u c l e -o t i d e s leaked out of the i n f e c t e d c e l l s . - 99 -Table 11 Pool S i z e s of 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 Pool size"*" Mock- SF v i r u s - R a t i o of S i g n i f i c a n c e i n f e c t e d i n f e c t e d pool s i z e s of 2 (nmoles/g c e l l s ) (virus/mock) d i f f e r e n c e CTP 116±135 36+. 13 0.30 p<0.05 UTP 213± 77 76± 34 0.35 p<0 . 05 ATP 1800±520 1080±320 0.60 p<0 .05 GTP 213± 67 122± 42 0.57 p<0 . 05 ADP 405± 99 654±100 1.65 p<0.02 GDP 61± 21 97± 21 1.69 p<0.10 1 R e s u l t s were an average of three d e t e r m i n a t i o n s . C e l l s were taken f o r e x t r a c t i o n a t 6h hours p . i . . 2 Since measurements were c o v a r i a n t , s i g n i f i c a n c e was determined by a p a i r e d comparison t t e s t . - 100 -Table 12 Absorbance of the Medium of BHK C e l l s I n f e c t e d with SF V i r u s Absorbance 280nm 260nm BSS-2% DCS 1.689 1.129 BSS-2% DCS from mock-infected c e l l s 1 1.688 1.212 BSS-2% DCS from SF v i r u s - i n f e c t e d c e l l s 1 1.701 1.243 2 A f t e r t r i c h l o r o a c e t i c a c i d treatment: Supernatant from mock-infected c e l l medium 0.210 1.198 Supernatant from SF v i r u s - i n f e c t e d c e l l medium 0.194 1.199 Large d i s h e s of c e l l s were i n f e c t e d as u s u a l . At 6-3/4 hours p . i . , the c e l l s were washed twice with 3 ml of E a r l e ' s Balanced 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). Each d i s h of c e l l s was incubated with 3 ml of the same medium for 30 minutes at 37 . The medium was then removed and c e n t r i -fuged (6,ooo x c[, 10 minutes). The absorbance of the supernatant was measured. A f t e r c e n t r i f u g a t i o n of the medium, an a l i q u o t (0.75 ml) was mixed with 40% t r i c h l o r o a c e t i c a c i d (0.25 ml). The mixture was c e n t r i f u g e d (6,000 x £, 10 minutes). The absorbance of the supernatant was measured. - 101 -(h) Time Sequence of N u c l e o t i d e Pool S i z e s During SF V i r u s I n f e c t i o n Since both CTP and ATP pools were decreased i n s i z e by SF v i r u s i n f e c t i o n , i t was decided to i n v e s t i g a t e the time sequence of n u c l e o t i d e pool s i z e s d u r i n g i n f e c t i o n . E i t h e r CTP or ATP c o n c e n t r a t i o n s could c o n t r o l PC s y n t h e s i s through t h e i r e f f e c t s on CT or CK. The time sequence of Q 3 H 2 ] c h o l i n e i n c o r p o r a t i o n i n t o PC has been examined p r e v i o u s l y (173). N u c l e o t i d e concen-t r a t i o n s were measured by the second method of M a t e r i a l s and Methods s e c t i o n (£) ( v). The r e s u l t s are presented i n F i g u r e s 22 and 23. The pools of CTP and UTP in c r e a s e d i n s i z e from 2h to lh hours p . i . i n mock-infected c e l l s , but remained s t a t i c i n v i r u s -i n f e c t e d c e l l s ( F i g . 22). GTP pools i n c r e a s e d i n s i z e over t h i s time i n both mock-infected and v i r u s - i n f e c t e d c e l l s ( F i g . 22). The pool s i z e of ATP was about the same at 2h or 7% hours p . i . i n both mock-infected and v i r u s - i n f e c t e d c e l l s ( F i g . 23). In a l l four n u c l e o t i d e p o o l s , some r e d u c t i o n i n pool s i z e had a l -ready been caused by v i r u s i n f e c t i o n , at the e a r l i e s t time p o i n t of 2\ hours p . i . . The changes i n any of the n u c l e o t i d e pool s i z e s at v a r i o u s times p . i . ( F i g s . 22, 23), do not match the changes i n Q3H^] c h o l i n e i n c o r p o r a t i o n i n t o l i p i d (173), f o r both mock-infected and v i r u s - i n f e c t e d c e l l s . However, t h i s i s of minor importance s i n c e l a b e l l i n g does not r e f l e c t the true ra t e o f s y n t h e s i s (Results s e c t i o n ( j ) ) . - 102 -0 -I 1 1 1 -r- 1 r 1 2 3 4 5 6 7 T ime, hours pos t - in fect ion F i g u r e 2 2 . E f f e c t of d u r a t i o n of i n f e c t i o n on CTP, UTP, and GTP pool s i z e s . BHK c e l l s were i n f e c t e d with SF v i r u s as u s u a l . N u c l e o t i d e s were measured by the second method of 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 are expressed in nmoles per l a r g e d i s h of c e l l s . Pools from mock-infected c e l l s are: CTP, (o); UTP, (•); GTP, (A ) . The s o l i d symbols represent the e q u i v a l e n t pools 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 o o E c XL < 200] 100 2 3 4 5 6 Time, hours post-infection F i g u r e 23. E f f e c t of d u r a t i o n of i n f e c t i o n on ATP pool s i z e . The experiment was performed and r e s u l t s are expressed as noted i n F i g u r e 21. ATP from mock-infected 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 , (•). - 104 -The c e l l s which were used i n the time sequence experiment were not q u i t e c o n f l u e n t . However, i f i t was assumed that these c e l l s weighed the same as c o n f l u e n t c e l l s (0.18 g/la r g e d i s h ) , then the r e s u l t s by the two methods of n u c l e o t i d e a n a l y s i s were in f a i r agreement (Table 13). The two methods d i f f e r e d i n the procedure 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 , and i n the type of standard which was used to c a l c u l a t e amounts of 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 s t a n d a r d s ) . They a l s o d i f f e r e d i n the g r a d i e n t which was used to e l u t e the chromatography column, and in the method of measurement of peak areas. Table 13 Comparison of N u c l e o t i d e R e s u l t s by Two Procedures Percent r e s u l t 2 3 Mock-infected 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 Procedures are d e s c r i b e d i n M a t e r i a l s and Methods sec-t i o n (&) (v) . 2 Percentages are: r e s u l t s by second method r e s u l t s by f i r s t method x 100. 3 C e l l s were taken f o r e x t r a c t i o n at 6h hours p . i . . - 105 -(i) F a t t y A c i d A n a l y s i s of L i p i d s from BHK C e l l s I n f e c t e d with SF V i r u s The i n h i b i t i o n of i n c o r p o r a t i o n of fJ3H]] c h o l i n e i n t o PC a f t e r v i r u s i n f e c t i o n could have been a s s o c i a t e d with a change in the r a t e of s y n t h e s i s of some p a r t i c u l a r s p e c i e s of PC from i t s p r e c u r s o r , d i g l y c e r i d e . Consequently, the f a t t y a c i d com-p o s i t i o n s of PC and d i g l y c e r i d e were measured. A l s o , PE was analyzed. ( S i m i l a r to Q 3H]] c h o l i n e i n c o r p o r a t i o n , **C]] e t h -anolamine i n c o r p o r a t i o n i n t o PE i s 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 (174).) For f a t t y a c i d a n a l y s i s , BHK c e l l s were i n f e c t e d u n t i l 7 hours p . i . , at which time the c e l l s were removed from the i n -cubator and processed (as d e s c r i b e d in M a t e r i a l s and Methods s e c t i o n (m)). The f a t t y a c i d composition o f l i p i d s from v i r u s -i n f e c t e d c e l l s i n comparison with mock-infected c e l l s was very s i m i l a r f o r PC, PE, or d i g l y c e r i d e (Tables 14, 15). The compo-s i t i o n of PC and PE from BHK c e l l s was s i m i l a r to t h a t of a p r evious r e p o r t (163). The p r o p o r t i o n of 18:2 was a p p a r e n t l y decreased i n both PC and PE a f t e r v i r u s i n f e c t i o n . (However, no s i m i l a r change occurred i n d i g l y c e r i d e . ) Lack of a change i n f a t t y a c i d composition of PC i n d i c a t e d that the r e l a t i v e s y n t h e s i s of v a r i o u s s p e c i e s of PC from d i g l y c e r i d e (or synthe-s i s by r e a c y l a t i o n ) was probably not g r e a t l y a f f e c t e d by v i r u s i n f e c t i o n . - 106 -Table 14 Percent D i s t r i b u t i o n of 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 F a t t y a c i d PC Mock- SF v i r u s i n f e c t e d i n f e c t e d (% of t o t a l ) 14:0 0.8 0.8 n d 1 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 (Other>18:3) (1.4) (nd) (9.1) (8.8) 1 2 longer not d e t e c t a b l e T h i s l i n e r e p r e s e n t s than t h a t of 18:3. f a t t y a c i d s with r e t e n t i o n times PE • Mock- SF v i r u s -i n f e c t e d i n f e c t e d (% of t o t a l ) - 107 -Table 15 Percent D i s t r i b u t i o n of 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 F a t t y A c i d Mock-infected SF v i r u s - i n f e c t e d (% of 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 n d 1 Other 10.4 11.7 (Other>18 : 3 ) 2 (8.2) (10.0) „ not d e t e c t a b l e T h i s l i n e r epresents f a t t y a c i d s with r e t e n t i o n times longer than t h a t of 18:3. - 108 -(j) Pulse-Chase of \2'$lQ C h o l i n e i n BHK C e l l s I n f e c t e d with SF V i r u s Pulse-chase experiments were done to show more p r e c i s e l y how the i n c o r p o r a t i o n of []3Hy] c h o l i n e i n t o PC r e l a t e d to the d i s a p -pearance of Q 3 H ~ J c h o l i n e from the wa t e r - s o l u b l e p r e c u r s o r s of PC. In each of three separate []3H^] c h o l i n e pulse-chase experiments, the r a d i o a c t i v i t y i n phosphocholine decreased d u r i n g the chase while the r a d i o a c t i v i t y i n PC in c r e a s e d i n both mock-infected and v i r u s - i n f e c t e d c e l l s . Such a converse r e l a t i o n s h i p i n d i c a t e d t h a t phosphocholine i s a precursors:of PC. In each experiment, the amount of r a d i o a c t i v i t y i n c h o l i n e and CDP-choline was much s m a l l -er than that of phosphocholine. The amount of r a d i o a c t i v i t y i n c h o l i n e and CDP-choline -, u n l i k e phosphocholine, did. not vary much over 60 minutes of chase. The r e s u l t s of one of these experiments are shown i n F i g . 24. Over the chase p e r i o d , the disappearance of 3H from phospho-c h o l i n e accounted f o r the appearance of 3H i n PC. In mock-infected c e l l s , the disappearance of 3H from phosphocholine was 106% of the appearance i n PC; i n v i r u s - i n f e c t e d c e l l s i t was 148% ( F i g . 24). I n c o r p o r a t i o n of r~_3H^ ] c h o l i n e i n t o l i p i d (PC) was 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 . C o n c u r r e n t l y , the r a t e of d i s a p -pearance of Q3H~J phosphocholine was reduced by v i r u s i n f e c t i o n ( F i g . 24). I n h i b i t i o n of fJ3H~J c h o l i n e uptake d i d not occur i n t h i s experiment, s i n c e the t o t a l w a t e r - s o l u b l e r a d i o a c t i v i t y - 109 -Time of Chase, min F i g u r e 24. Pulse chase of Q3H^[ c h o l i n e i n BHK c e l l s i n -f e c t e d with SF V i r u s . BHK c e l l s which were not q u i t e c o n f l u e n t were i n f e c t e d with 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 with Q 3H]] c h o l i n e i n a 15 minute pulse which began at 6h hours p . i . . Both pulse and chase media were Medium 199 (and 2% f e t a l c a l f serum). The r e s u l t s are expressed i n dpm per two l a r g e d i s h e s of c e l l s . R a d i o a c t i v i t y ( 3H) was measured i n : c h o l i n e (A ) , phosphocholine (o), CDP-choline (V), and PC (•) from mock-infected c e l l s . The s o l i d symbols 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 -f e c t e d c e l l s . - 110 -(c h o l i n e p l u s phosphocholine p l u s CDP-choline) was about the same, a t the beginning of the chase, i n mock-infected or v i r u s - i n f e c t e d c e l l s ( F i g . 24). The r a t e of disappearance of phosphocholine was e x p o n e n t i a l between 20 and 60 minutes of chase ( F i g . 24). Were i t assumed that t h e ! p o o l : s i z e of phosphocholine d i d not vary i n t h i s exper-iment between 20 and 60 minutes of chase (6 hours 50 minutes -7 hours 30 minutes p . i . ) , then the f r a c t i o n a l turnover rate (k) and the h a l f - l i f e of phosphocholine could be c a l c u l a t e d . (The f r a c t i o n a l turnover r a t e denotes the f r a c t i o n of a p o o l which i s turned over per u n i t of time (206) .) The value of k was c a l c u -- k t l a t e d by the equation: R^/RQ = e (206). R represented r a d i o -a c t i v i t y ( 3 H dpm). RQ was the i n i t i a l r a d i o a c t i v i t y (at 20 min-utes of chase) and R^ was the subsequent r e a d i o a c t i v i t y (at 40 or 60 minutes of chase). The time i n t e r v a l was represented by t . The phosphocholine h a l f - r i i f e e q u a l l e d 0.693/k (206). Thus, f o r two measurements, the average h a l f - l i f e of phosphocholine i n min-utes was 42.4+4.2 (s.d.) i n mock-infected c e l l s and 67.4±3.0 (s.d.) i n c e l l s i n f e c t e d . w i t h SF v i r u s . The turnover rate (or f l u x ) , q, e q u a l l e d kB, where B was the pool s i z e (206) . I f the pool s i z e s of phosphocholine at 6-1/2-6-5/6 hours p . i . (Table 10) were used as values of B, then the turnover r a t e of phosphocholine was 1.05 nmoles min ^ g c e l l s ^ i n mock-infected c e l l s and 1.51 nmoles min g c e l l s 1 i n v i r u s -i n f e c t e d c e l l s . I f phosphocholine was o n l y converted j to PC, which - I l l -was assumed to be the case, then this rate also represented the rate of synthesis of PC from phosphocholine in 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 did not mean that synthesis of PC was in h i b i t e d . It should be noted that the flux represents synthesis of PC and not turnover of PC, since the c e l l s may have s t i l l been grow-ing (although the c e l l s were incubated in a maintenance medium rather than a growth medium, for galsercHase experiments) . In c e l l s which are growing, the rate of synthesis of PC would ex-ceed the rate, of degradation,. The values of the f r a c t i o n a l turnover rate (k) for choline, phosphocholine, and CDP-choline are shown in Table 16. Values of k for phosphocholine" were calculated as described previously in this section. Since the flux (q) over each step of de novo syn-thesis of PC was considered^constant, k was e a s i l y calculated for choline and CDP-choline from the equation, k = q B. The pool size (B) values for choline and CDP-choline were from Table 9 (at 7-7% hours p . i . ) . As can be seen in Table 16, for phospho-choline .and CDP-choline, the value of k i s reduced by virus in^-fection. (as. a consequence of the larger pool sizes in infected c e l l s ) . For choline, the value of k i s apparently increased by i n f e c t i o n . However, not a l l of the choline pool may be active in PC synthesis. (See Discussion.) A hypothetical "active" cho-li n e pool was estimated as the pool size of choline 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 - 112 -p o o l , the value of k was a l s o reduced by v i r u s i n f e c t i o n (since t h i s pool was l a r g e r i n i n f e c t e d c e l l s ) . Table 16 F r a c t i o n a l Turnover Rates of 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 1 k, min Mock-infected SF v i r u s - i n f e c t e d C h o l i n e 0.0072 0.022 " A c t i v e " c h o l i n e 2 0.097 0.047 Phosphocholine 0.016 0.010 CDP-choline 0.17 0.096 Values of k were based on the ra t e of decay of Q3H^] phosphocholine ( F i g . 24) and on pool s i z e s (Tables 9, 10) which were both measured between 6h and lh hours p . i . . 2 The pool s i z e of " a c t i v e " c h o l i n e was the amount of cho-l i n e which would be necessary to d i l u t e the Q 3H]] c h o l i n e i n the c e l l to 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 (Table 9). T h i s p o o l of c h o l i n e i s 11 (and 32) nmoles/g c e l l s i n mock-i 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 ) . - 113 -DISCUSSION (a) Does SF V i r u s I n f e c t i o n A f f e c t the Rate of PC S y n t h e s i s in BHK C e l l s ? Since SF v i r u s i n f e c t i o n causes an i n h i b i t i o n of i n c o r p o r a -t i o n of fJ3H~J c h o l i n e i n t o PC of BHK c e l l s (173), our i n i t i a l h y pothesis was that PC s y n t h e s i s was i n h i b i t e d . The r a t e of PC s y n t h e s i s i s c o n s i d e r e d equal to the turnover r a t e o f fJ3H~J phos-phocholine. T h i s turnover r a t e or f l u x , q, equals kB. Although k i s lower a f t e r v i r u s i n f e c t i o n , B i s much high e r , so that the turnover r a t e : i s a c t u a l l y higher i n i n f e c t e d c e l l s than mock-in-f e c t e d c e l l s (Results s e c t i o n ( j ) ) . Consequently, the r a t e of s y n t h e s i s of PC i s not 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 but may indeed be s t i m u l a t e d . (b) Why Is the I n c o r p o r a t i o n of rj3H~J 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 of BHK C e l l s ? T h i s q u e s t i o n was the b a s i s f o r the p r e s e n t t h e s i s p r o j e c t . []3H^] C h o l i n e i n c o r p o r a t i o n i n t o PC i s i n h i b i t e d by an average of 77% (Results s e c t i o n (e)) by SF v i r u s i n f e c t i o n . Such i n h i b i -t i o n exceeds the i n h i b i t i o n of uptake of rj3H~J c h o l i n e . (See D i s c u s s i o n s e c t i o n (c) ( i ) . ) I n h i b i t i o n of i n c o r p o r a t i o n can be simply e x p l a i n e d . The i n h i b i t i o n p a r a l l e l s an i n h i b i t i o n of d i s -appearance of fJ3H~J phosphocholine ( F i g . 24). In other words, the f r a c t i o n of rj3H]] phosphocholine which turned over per u n i t - 114 -of time i s l e s s i n i n f e c t e d c e l l s . However, the pool s i z e of phosphocholine i s 2.3 times l a r g e r i n v i r u s - i n f e c t e d c e l l s com-pared to the c o n t r o l c e l l s a t 6-1/21- 6-5/6 hours p . i . (Table 10). (See D i s c u s s i o n s e c t i o n (f) ( i i ) . ) Thus, Q 3 H^J phospho-c h o l i n e which i s formed i n the c e l l , i s d i l u t e d by the l a r g e r pool of phosphocholine of i n f e c t e d c e l l s . The d i l u t i o n of the i s o t o p e , rather than i n h i b i t i o n of PC s y n t h e s i s , i s the cause of i n h i b i t i o n of i n c o r p o r a t i o n . (c) What Causes the E f f e c t s of SF V i r u s I n f e c t i o n on Each Step of PC S y n t h e s i s ? (i) C h o l i n e T r a n s p o r t The mechanism of i n h i b i t i o n of c h o l i n e t r a n s p o r t i s un-known. However, c h o l i n e uptake i n BHK c e l l s i s a s a t u r a b l e process ( F i g . 8), which suggests t h a t i t i s c a r r i e r - m e d i a t e d . The uptake of c h o l i n e has a K^ of 17 yM,. compared to a K^ f o r c h o l i n e i n the c h o l i n e kinase r e a c t i o n , of 110 yMr, which sug-gests that these two a c t i v i t i e s are not l i n k e d . Perhaps i n -h i b i t i o n of host p r o t e i n s y n t h e s i s by SF v i r u s r e s u l t s ^ i n a smaller amount of a c h o l i n e - s p e c i f i c t r a n s p o r t p r o t e i n . A l t e r -n a t i v e l y , s i n c e SF v i r u s p a r t i c l e s are extruded from the plasma membranes of i n f e c t e d c e l l s (178, 179, 207), d i s r u p t i o n of the plasma membrane o r g a n i z a t i o n may cause an i n h i b i t i o n of c h o l i n e uptake. Furthermore, s i n c e the v i r i o n l i p i d envelope c o n t a i n s no host p r o t e i n s (208), a mechanism f o r e x c l u d i n g such p r o t e i n s 3 The K value was obtained by l i n e a r r e g r e s s i o n of 1/V as a f u n c t i o n of 1/S. The data used were from F i g . 10 (from mock-i 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 to 0.5 mM and 1.25 mM. - 115 -i s necessary. Whatever the mechanism, i t too c o u l d d i s r u p t the p r o t e i n and l i p i d s t r u c t u r e of the plasma membrane. A f i n a l p o s s i b i l i t y i s that i n h i b i t i o n of c h o l i n e t r a n s p o r t could be caused by a r e d u c t i o n i n s u r f a c e area of i n f e c t e d c e l l s . At 7 hours p . i . , BHK c e l l s which were i n f e c t e d with SF v i r u s u s u a l l y were more rounded i n shape than mock-infected c e l l s . Such a shape change could r e f l e c t a decreased s u r f a c e area as a r e s u l t of v i r u s i n f e c t i o n . T r a n s p o r t i n h i b i t i o n averages 56% i n three i n c o r p o r a t i o n experiments (Results s e c t i o n ( b ) ) , although an i n h i b i t i o n of l e s s than 19% might be expected from t r a n s p o r t experiments ( F i g . 8). The higher l e v e l of i n h i b i t i o n which i s observed i n these i n c o r p o r a t i o n experiments may be a t t r i b u t a b l e to a d d i t i o n a l cho-l i n e i n the medium, e i t h e r from incompletely d i a l y z e d c a l f serum or because of incomplete washing of the c e l l s . A d d i t i o n a l cho-l i n e i n the medium w i l l i n c r e a s e i n h i b i t i o n of c h o l i n e t r a n s p o r t in SF v i r u s - i n f e c t e d c e l l s ( F i g . 8 ) . Perhaps a g r e a t e r i n h i -b i t i o n of c h o l i n e t r a n s p o r t i n the present work e x p l a i n s the higher i n h i b i t i o n of c h o l i n e i n c o r p o r a t i o n i n t o PC here, com-pared to p r e v i o u s experiments (173). Oddly, i n h i b i t i o n of r3H^j c h o l i n e uptake (Results s e c t i o n (b)) does not occur i n SF v i r u s - i n f e c t e d c e l l s of the pulse-chase experiment (Results s e c t i o n ( j ) ) . One d i f f e r e n c e between the the two experiments i s that Medium 199 with un-d i a l y z e d serum was the medium f o r the pulse-chase experiment, whereas E a r l e ' s Balanced S a l t S o l u t i o n with d i a l y z e d serum was - 116 -used i n the t r a n s p o r t experiments. Some f a c t o r present i n one of the media may e x p l a i n why t r a n s p o r t i n h i b i t i o n i s not repro-d u c i b l e . ( i i ) C h o l i n e Kinase Because the amount of c y t o s o l i c p r o t e i n i n BHK c e l l s (mg/g c e l l s ) i s diminished by SF v i r u s i n f e c t i o n (Table 4 ) , the t o t a l CK a c t i v i t y i s reduced (Table 5). C y t o s o l i c p r o t e i n l e -v e l s are probably reduced because o f v i r a l i n h i b i t i o n o f host p r o t e i n s y n t h e s i s . ( i i i ) C y t i d y l y l t r a n s f e r a s e C y t o s o l i c CT i s a f f e c t e d by v i r u s i n f e c t i o n i n the same manner as CK (Table 5). The other form of the enzyme, found i n microsomes, i s reduced i n s p e c i f i c a c t i v i t y (Table 6 ) . I n h i -b i t i o n i s l i k e l y because of turnover of the enzyme p r o t e i n a f t e r v i r a l i n h i b i t i o n of host p r o t e i n s y n t h e s i s . The accumulation of v i r a l envelope and n u c l e o c a p s i d p r o t e i n s , which are a s s o c i a t e d with microsomes (179), c o u l d mask l o s s e s of c e l l u l a r microsomal p r o t e i n s . Such an hypothesis e x p l a i n s the s t a t i c amount of microsomal p r o t e i n a f t e r v i r u s i n f e c t i o n of BHK c e l l s (Table 4 ) . The apparent d i f f e r e n c e s i n a c t i v a t i o n by phosphocholine between CT from i n f e c t e d and mock-infected c e l l s ( F i g s . 13, 15) may not be a d i f f e r e n c e i n the p r o p e r t i e s of CT. Instead, some com p e t i t i v e r e a c t i o n (such as a phosphocholine phosphatase) could vary i n a c t i v i t y as a r e s u l t of v i r u s i n f e c t i o n . However, in microsomes the f o r phosphocholine i s almost the same f o r CT a c t i v i t i e s from mock-infected and v i r u s - i n f e c t e d c e l l s • - 117 -(0.34 mM and 0.35 mM, respectively).'* This result makes unlikely the suggestion of a variation in a competitive reaction. On the other hand, in cytosol the for phosphocholine i s 0.32 mM for the enzyme from mock-infected c e l l s and 0.18 mM for that of 4 virus-infected c e l l s . . Thus, the idea of a competitive reaction, higher in a c t i v i t y in cytosol from mock-infected c e l l s , i s more tenable in this case. CT from fresh cytosol of BHK c e l l s i s activated by l i p i d s , as are the enzymes from rat l i v e r (91) , rat intestine (92) , and rat lung (36) . While the function of l i p i d a c tivation of CT remains unclear, i t is certain that SF virus i n f e c t i o n does not markedly a f f e c t this activation (Fig. 16). (iv) Choliriephosphotransferase The a c t i v i t y of CPT i s inhibited by virus infection (Table 6), probably for the same reason that microsomal CT is i n h i b i t e d . A preliminary experiment was performed to test the hypo-thesis of Ca i n h i b i t i o n . Ca has been proposed as a control agent in phospholipid biosynthesis (111). The ion may act p r i -marily by i n h i b i t i n g CPT ;(afid2EPT) . In the experiment, micro-somes were treated with the Ca chelator, EGTA.(20 mM for 5 minutes at 37°) and then collected by centrifugation. Such treatment does not however, lessen the i n h i b i t i o n of CPT from microsomes of virus-infected c e l l s . S i m i l a r l y , while the deter-gent, taurocholate, activates CPT, i t does not relieve the 4 K values were obtained by linear regression of 1/V as a function of 1/S. - 118 -v i r u s - i n d u c e d i n h i b i t i o n . ( R e s u l t s s e c t i o n ( c ) ) . The s i g n i f i c a n c e of CPT a c t i v i t i e s i s q u e s t i o n a b l e f o r at l e a s t two reasons. F i r s t , the enzyme was assayed with d i g l y -c e r i d e which was d e r i v e d from egg yolk PC. T h i s s u b s t r a t e could have a f a t t y a c i d composition q u i t e d i f f e r e n t from that of BHK c e l l d i g l y c e r i d e . Secondly, the s u b s t r a t e was d i s p e r s e d by s o n i c a t i o n i n 0.03% Tween 20. The p h y s i c a l form of t h i s sub-s t r a t e i s probably r a d i c a l l y d i f f e r e n t from the form _in v i v o . Any u n c e r t a i n t y about absolute a c t i v i t i e s does not however, n u l l i f y the r e l a t i v e i n h i b i t i o n of CPT i n i n f e c t e d c e l l s com-pared to mock-infected c o n t r o l c e l l s . '(d) Do ATP and CTP I n h i b i t CK and CT In Vivo? I t i s i n t e r e s t i n g that both CK and CT are i n h i b i t e d by high c o n c e n t r a t i o n s of the n u c l e o t i d e s u b s t r a t e s , ATP and CTP ( F i g s . 11, 14). Such i n h i b i t i o n could have p h y s i o l o g i c a l r e l e -vance. A s i m i l a r e f f e c t has been reported i n a r e c o n s t i t u t e d system from E. caudatum. In that system, QATP-Mg ]] above 2.5 mM1 and Q CTP-Mg ~\ above 1 mM i n h i b i t the i n c o r p o r a t i o n of c h o l i n e i n t o PC (31). A l s o , one ethanolamine kinase from r a t l i v e r i s ;;^^rnh^b±tedM? by [_ ATP-Mg "2 (above 16 mM) (24). However, i n c o n t r a s t , p u r i f i e d CK from r a t l i v e r i s />jv i n h i -b i t e d by [] ATP-Mg [] (24). A l s o , i t seems that i n h i b i t i o n of CT by [_ CTP-Mg has not been p r e v i o u s l y observed. Indeed, i t c o uld be argued t h a t , s i n c e the enzyme a c t i v i t i e s of the present work were measured in crude s u b c e l l u l a r f r a c t i o n s , such i n h i b i t i o n c o u l d merely represent an a c t i o n by n u c l e o t i d e s on - 119 -some other p r o t e i n . To r e a l i s t i c a l l y assess the s i g n i f i c a n c e of such i n h i b i t i o n r e q u i r e s knowledge of the cy t o p l a s m i c con-c e n t r a t i o n of both n u c l e o t i d e s , and the e f f e c t s of the n u c l e -o t i d e s on p u r i f i e d enzymes. (e) Are Pool S i z e Measurements an Accurate I n d i c a t i o n o f In  Vi v o C o n c e n t r a t i o n s ? (i) Is the Pool of 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? A c u r i o u s f e a t u r e of BHK c e l l s i s the f a c t , t h a t the cho-l i n e pool i s l a r g e r than that of phosphocholine. The reverse i s true i n r a t l i v e r (33) and-HeLa c e l l s (209). A p o s s i b l e e x p l a n a t i o n f o r t h i s r e s u l t would be i f ethanolamine was i n a d -v e r t a n t l y measured with c h o l i n e . T h i s i s u n l i k e l y s i n c e l e s s than a t h i r d of the ethanolamine, which has a pK of 9.5 (210), a i n the f r e e c h o l i n e f r a c t i o n which was i s o l a t e d from BHK c e l l s (pH>10), would be i o n i z e d . Thus, l e s s than a t h i r d of the ethanolamine would be a v a i l a b l e to form an ion p a i r with t e t r a -phenylboron. In any case, ethanolamine probably does not i n t e r f e r e with the assay o f c h o l i n e , s i n c e n e i t h e r monomethyl-or dimethylethanolamine i n t e r f e r e g r e a t l y (195). Another pos-s i b l e i e x p l a n a t i o h i s t h a t the c h o l i n e pool i s unevenly d i s t r i -buted i n more than one s u b c e l l u l a r compartment, so t h a t cho-l i n e from on l y one compartment e q u i l i b r a t e s with e x t r a c e l l u l a r c h o l i n e . Evidence f o r a small pool of c h o l i n e which i s a c t i v e i n PC s y n t h e s i s i s as f o l l o w s : 1. The s p e c i f i c r a d i o a c t i v i t y of c h o l i n e (in both - 120 -mock-infected and v i r u s - i n f e c t e d c e l l s ) i s unexpectedly lower than that of phosphocholine (Table 9 ) . A/precursor ..would be expected to have a higher or equal s p e c i f i c r a d i o a c t i v i t y com-pared to the corresponding product. 2. In the pulse-chase experiment, most of the r a d i o -a c t i v i t y i s i n phosphocholine, and very l i t t l e remains i n cho-l i n e a f t e r the pulse ( F i g . 24). Since _. a l l o f the [~ 3 lT] c h o l i n e which ent e r s the c e l l i s expected to go through an i n t r a c e l l u l a r p ool o f c h o l i n e , t h i s r e s u l t i n d i c a t e s t h a t the f r a c t i o n a l turnover r a t e of such an i n t r a c e l l u l a r p ool i s very f a s t . In other words, the value of the f r a c t i o n a l turnover r a t e (k) would be much gr e a t e r f o r c h o l i n e than phosphocholine (Table 16). The f l u x (q) equals kB (Results s e c t i o n ( j ) ) . The f l u x should be constant over each step of t h i s l i n e a r pathway (c h o l i n e to PC). Thus,, i f k i s gr e a t e r f o r c h o l i n e than phos-phocholine, and q remains the same, then the pool s i z e (B) of c h o l i n e should be much smal l e r than t h a t of phosphocholine. Similarly/,-/more than one pool, of c h o l i n e has been i n d i c a t e d f o r r a t l i v e r (33) and rat. h e a r t (105). In c o n c l u s i o n , ^ l a r g e amount of the c e l l u l a r p o o l of cho-l i n e does not mix with incoming Q 3 l T ] c h o l i n e . There are at l e a s t 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 e f f e c t . One i s the p h y s i c a l i s o l a t i o n of a c h o l i n e p o o l , f o r example i n the.mito-chondrion, which i s not a v a i l a b l e f o r s y n t h e s i s o f PC from Q 3H]] c h o l i n e . A second p o s s i b i l i t y i s the p h o s p h o r y l a t i o n of Q 3E~2 c h o l i n e while c h o l i n e i s t r a n s p o r t e d i n t o the c e l l . - 1 2 1 -In t h i s case, the i n a c t i v e pool of c h o l i n e c o u l d be the product of phosphocholine (or PC) d e g r a d a t i o n . However, i n t h i s p o s s i -b i l i t y . o n e would have to imagine t h a t CK i s f o r some reason i n a c c e s s i b l e to t h i s l i b e r a t e d p ool of c h o l i n e , s i n c e the p o o l i s a p p a r e n t l y not a c t i v e i n PC s y n t h e s i s . ( i i ) Why Are Phosphocholine Pool S i z e R e s u l t s V a r i a b l e ? The measurement of phosphocholine pools a t 7 - lh hours p . i . (Table 9) appears to be s m a l l e r than that a t 6-1/2 - 6-5/6 hours p . i . (Table 10). However, f o r the former measurement, the c e l l s were incubated i n 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) f o r 30 minutes before p r o c e s s i n g . Thus, these c e l l s were s t a r v e d of c h o l i n e over t h a t time, but the phosphocholine pool would continue to turn over. The p o o l s i z e of phosphocholine which remains a f t e r the 30 minutes of s t a r v a -t i o n would equal: Pool s i z e without i n c u b a t i o n (at 6-1/2 - . 6-5/6 hours p . i . ) - q(30). For t h i s c a l c u l a t i o n , two assump-t i o n s are made. F i r s t , i t i s assumed t h a t the pool of phospho-c h o l i n e i n BHK c e l l s , e i t h e r mock-infected or v i r u s - i n f e c t e d , which were incubated i n Medium 199 (and 2% f e t a l c a l f serum) does not change from 6% to 7% hours p . i . . Second, i t i s assumed that q does not vary as the pool of phosphocholine i s p a r t l y d e p l e t e d . Thus, p o o l . s i z e s . o f phosphocholine of 32 nmoles per gram of mock-infected c e l l s and 102 nmoles per gram of v i r u s - i n f e c t e d c e l l s are d e r i v e d by the above c a l c u l a t i o n . These values are s i m i l a r to the experimental r e s u l t s (Table 10). - 122 -(f) How Does SF V i r u s I n f e c t i o n Cause Changes i n the Pool S i z e of PC P r e c u r s o r s ? (i) N u c l e o t i d e s The decrease i n pool s i z e s of n u c l e o t i d e t r i p h o s p h a t e s which i s caused by SF v i r u s i n f e c t i o n may be the r e s u l t of i n -h i b i t i o n of n u c l e o t i d e s y n t h e s i s or due to i n c r e a s e d use of the t r i p h o s p h a t e s , probably because of v i r a l RNA s y n t h e s i s . The d i f f e r e n c e between pools of purine and.pyrimidine n u c l e o t i d e t r i p h o s p h a t e s a f t e r v i r u s i n f e c t i o n (Table 11, F i g s . 22, 23) i s not because of p r e f e r e n t i a l use of p y r i m i d i n e s and purines i n v i r a l RNA s y n t h e s i s . Adenyl- and guanyl- r e s i d u e s comprise j u s t over h a l f of the base composition of v i r a l RNA, and 22S, 26S, and 42S i n t r a c e l l u l a r RNAs of SF v i r u s - i n f e c t e d c e l l s (211). The d i f f e r e n c e between purine and p y r i m i d i n e pools could be a r e s u l t of i n c r e a s e d i n h i b i t i o n of p y r i m i d i n e b i o s y n -t h e s i s compared to purine b i o s y n t h e s i s . Another p o s s i b i l i t y i s t h a t GTP and ATP are used l e s s f o r p r o t e i n s y n t h e s i s i n i n -f e c t e d c e l l s , as a r e s u l t of i n h i b i t i o n of host p r o t e i n synthe-s i s . The unused purine n u c l e o t i d e s would then accumulate u n t i l new steady s t a t e c o n c e n t r a t i o n s are reached. :(ii) Phosphocholine I f CT 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 ) ) , then i t becomes easy to e x p l a i n the opposite changes i n pool s i z e s of CTP and phosphocholine a f t e r v i r u s i n f e c t i o n . The p o o l s i z e of CTP becomes sma l l e r i n i n f e c t e d c e l l s (compared to - 123 -mock-infected c e l l s ) e a r l y i n i n f e c t i o n ( F i g . 22). In a pre-l i m i n a r y experiment, the i n h i b i t i o n of i n c o r p o r a t i o n of rj 3 rfJ c h o l i n e i n t o PC, which depends on an i n c r e a s e i n the pool s i z e of phosphocholine, does not become n o t i c e a b l e u n t i l a f t e r 6h hours p . i . . Thus, the change in the CTP pool a p p a r e n t l y occurs f i r s t . T h i s change would l e s s e n the forward v e l o c i t y (V^) of the r e a c t i o n (since equals k [jZTP [^phosphocholine ). In other words, CT would be i n h i b i t e d ; the f l u x through the reac-t i o n would d i m i n i s h (since f l u x equals forward v e l o c i t y minus reverse v e l o c i t y ) . Since the f l u x through the c h o l i n e kinase step continues unabated, phosphocholine must of n e c e s s i t y accu-mulate. However, an i n c r e a s e i n phosphocholine would i n c r e a s e the forward v e l o c i t y of the CT r e a c t i o n and thus, the f l u x through CT c o u l d be r e s t o r e d . ( i i i ) CDP-choline The i n c r e a s e i n the pool s i z e of CDP-choline i s not as easy to e x p l a i n as t h a t of phosphocholine. Contamination of -i:. the CDP-choline with phosphocholine (Table 7) which would amount to 12% of the apparent CDP-choline pool of mock-infected c e l l s and 17% of the pool of v i r u s - i n f e c t e d c e l l s , does not account f o r the 2 . 6 - f o l d i n c r e a s e i n the p o o l s i z e of CDP-cho-l i n e a f t e r i n f e c t i o n (Table 9). However, by reasoning s i m i l a r to that presented above, i f the pool s i z e of the CPT product, CMP, were to i n c r e a s e i n i n f e c t e d c e l l s , then a subsequent i n -crease i n the CDP-choline p o o l . s i z e could r e s u l t . Thus, a constant f l u x would be maintained. S i m i l a r to the case f o r CT, - 124 -the i n i t i a l p o ol s i z e change would cause a t r a n s i e n t i n h i b i t i o n of f l u x over the CPT s t e p . (g) What C o n t r o l s the Rate of 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 of F l u x Two g e n e r a l parameters, i f v a r i e d , may produce a c o o r d i n a t e response i n f l u x over a given enzyme r e a c t i o n . These f a c t o r s are s u b s t r a t e and enzyme c o n c e n t r a t i o n s . Corresponding to these two parameters are two types of enzyme r e a c t i o n s which may occur i n a metabolic pathway. They are n e a r - e q u i l i b r i u m , substrate-dependant r e a c t i o n s ( h e r e a f t e r c a l l e d Type 1) and n o n - e q u i l i b r i u m , s u b s t r a t e - independent r e a c t i o n s (Type 2) (212). In a Type 1 r e a c t i o n , the enzyme i s not f l u x - g e n e r a t i n g . In other words, i t i s not r a t e - l i m i t i n g . The d i s e q u i l i b r i u m r a t i o n , p , of t h i s enzymatic step w i l l be c l o s e to 1. p i s equal to the reverse v e l o c i t y of the enzyme r e a c t i o n (V r) d i -vided by the forward v e l o c i t y (Vf) (109). The c l o s e r V.^/V^ i s to 1, the g r e a t e r must be both Vf and V r, i f a constant f l u x i s to be maintained, s i n c e the f l u x .is equal to V f - V r . . As the r a t i o approaches u n i t y , V^ and V f become much gr e a t e r than the f l u x . I t f o l l o w s that the V_~£ value of a Type 1 r e a c t i o n w i l l Ilia 2\ be much gr e a t e r than the f l u x (212). For t h i s type of enzyme r e -a c t i o n , s m a l l changes i n s u b s t r a t e (or product) c o n c e n t r a t i o n s w i l l have a g r e a t e f f e c t on f l u x (109). Type 2 r e a c t i o n s generate (or c o n t r o l ) the f l u x through a given pathway (212). A c r i t e r i o n f o r a c o n t r o l l i n g enzyme step - 125 -i s to have a V m a x value approximately equal to the f l u x over t h a t step (213). Thus, i n t h i s case the enzyme operates at maximal v e l o c i t y _in v i v o . I f V m a x i s equal to the f l u x , i t i s impli e d that the enzyme i s h i g h l y s a t u r a t e d with s u b s t r a t e ( s ) and the d i s -e q u i l i b r i u m r a t i o i s very s m a l l . However, e i t h e r of these l a t t e r two i n d i c a t o r s , s e p a r a t e l y , are thought not to be c r i t e r i a f o r a c o n t r o l l i n g enzyme step (213). A Type 2 r e a c t i o n i s a l s o termed he r e i n as a r a t e - l i m i t i n g r e a c t i o n . For such a r e a c t i o n , any modulator, which a f f e c t s the V m a x of the enzyme, w i l l i d e n t i c a l l y a f f e c t the f l u x through that enzyme step. To i l l u s t r a t e these two types of r e a c t i o n , c o n s i d e r the CT r e a c t i o n . In a Type 1 r e a c t i o n , the f l u x over the CT step would be c o n t r o l l e d by the mass a c t i o n r a t i o : [_ CDP-choline]] [Pyrophosphate]] .QCTP]] [[Phosphocholinej -In a Type 2 r e a c t i o n , the f l u x would be c o n t r o l l e d by the concen-t r a t i o n of enzyme. The extremes of these:two types of r e a c t i o n s are: an en-zyme r e a c t i o n which i s completely at e q u i l i b r i u m , with no net f l u x ^ o v e r the step (Type 1) and a r e a c t i o n f o r which a change in enzyme c o n c e n t r a t i o n would cause a p r o p o r t i o n a l change i n f l u x (Type 2). Both extremes are improbable, i f not im p o s s i b l e , i n a b i o l o g i c a l system (213). The f l u x through enzyme steps which are midway between the two extremes would be c o n t r o l l e d by both the mass a c t i o n r a t i o and enzyme c o n c e n t r a t i o n . Such enzymes could be c l a s s e d as a t h i r d type. - 126 -( i i ) What Controls.;, the Flux i n BHK C e l l s ? To answer t h i s q u e s t i o n , pulse-chase experiments were per-formed. Perhaps the most conspicuous r e s u l t of the pulse-chase experiment i s the converse r e l a t i o n s h i p between the disappearance of fJ3HJ phosphocholine and the appearance of rj3H~J PC ( F i g . 24). Free c h o l i n e and CDP-choline are l a b e l l e d to a much smaller ex-tent than phosphocholine ( F i g . 24). These r e s u l t s may be ex-p l a i n e d by the pool s i z e s of the p r e c u r s o r s of PC. As d i s c u s s e d i n s e c t i o n (e), the c h o l i n e pool i s l i k e l y much smaller than that of phosphocholine. In a d d i t i o n to c h o l i n e , the pool of CDP-c h o l i n e i s a l s o much smaller than that of phosphocholine (Table 9). I f one c o n s i d e r s the' flow of c h o l i n e molecules to PC, which occurs at a constant f l u x , the s i t u a t i o n i s analogous to a r i v e r which flows at a constant v e l o c i t y . The phosphocholine pool i s l i k e a very deep s e c t i o n of the r i v e r , i n which the flow appears to slow down.(the f r a c t i o n a l turnover r a t e i s smaller) but the water v e l o c i t y does not change (the f l u x i s c o n s t a n t ) . In BHK c e l l s , the l a r g e r e s e r v o i r of phosphocholine thus a c t s as a sink for the 3H l a b e l , which i s o n l y s l o w l y f l u s h e d out. S t i l l , the question remains: What c o n t r o l s the f l u x of c h o l i n e to PC? Three l i n e s o f evidence support the idea that the three: enzymes of de novo PC s y n t h e s i s i n BHK c e l l s are of Type 1. 1. I f the maximal v e l o c i t i e s of CK, CT, and CPT in v i t r o are compared with the f l u x iri v i v o , f o r mock-infected BHK c e l l s , then one can observe that the enzyme a c t i v i t i e s operate i n the c e l l at a v e l o c i t y below the maximum (Table 17). The enzyme - 127 -Table 17 Comparison of Values of V m a x and Flux f o r Mock-Infected BHK C e l l s V e l o c i t y , nmoles min g c e l l s In v i t r o - V ^ 1 CK 7 - 3 CT (Microsomal and C y t o s o l i c ) 31.9 CPT 4 7 - 6 • 2  In v i v o Flux I - 0 5 3= Maximal v e l o c i t i e s are" from Table 5. 2 Flux i s from R e s u l t s s e c t i o n (j). a c t i v i t i e s may be underestimates s i n c e the crude enzyme prepara-t i o n s l i k e l y c o n t a i n other enzymes, such as phosphatases, which compete f o r the s u b s t r a t e s and products. In c o n c l u s i o n , s i n c e the f l u x i n v i v o i s much l e s s than the values of V m •, the f l u x — — nic ix should be rather i n s e n s i t i v e to changes i n enzyme c o n c e n t r a t i o n . Thus, the: r e a c t i o n s cannot be of Type 2. T h i s c o n c l u s i o n a l s o holds true f o r the f l u x i n BHK c e l l s i n f e c t e d with SF v i r u s . (See Table 5, R e s u l t s s e c t i o n ( j ) . ) 2. I f i t i s assumed t h a t pool s i z e s i n nmoles/g c e l l s r e -present yM c o n c e n t r a t i o n s , then the c o n c e n t r a t i o n s of CTP and phosphocholine i n BHK c e l l s , e i t h e r i n f e c t e d or mock-infected, are under 12% of the c o n c e n t r a t i o n f o r maximal v e l o c i t y of CT (Table 10, F i g s . 13-15, R e s u l t s s e c t i o n ( c ) ) . ATP pools would ) - 128 -r e p r e s e n t 24% and 14% of the optimum f o r CK from mock-infected and v i r u s - i n f e c t e d c e l l s , r e s p e c t i v e l y (Table 11, F i g . 11). C h o l i n e p o o l s , on the other hand, would rep r e s e n t 58% (mock-i n f e c t e d c e l l s ) and 27% ( v i r u s - i n f e c t e d •cells) of the c o n c e n t r a t i o n of c h o l i n e (0.25 mM) at which CK is . r:saturated (Table 9, F i g . 10). However, not a l l c e l l u l a r c h o l i n e may be a v a i l a b l e f o r PC synthe-s i s , as p r e v i o u s l y d i s c u s s e d . In a d d i t i o n , t h i s kind of compari-son ignores compartmentation which would cause d i f f e r e n t concen-t r a t i o n s of m e t a b o l i t e s i n differentccompartments. Compartmenta-t i o n a s i d e , t h i s comparison pro v i d e s f u r t h e r evidence t h a t CK and CT are not s a t u r a t e d i n v i v o , and thus these r e a c t i o n s are of Type 1. 3. The t o t a l a c t i v i t i e s of a l l three enzymes are reduced by v i r u s i n f e c t i o n (Table 5), y e t the f l u x (phosphocholine t u r n -over rate) i s not i n h i b i t e d (Results s e c t i o n ( j ) ) . I f any of the enzymes are of Type 2, then a decrease i n a c t i v i t y should c o r -respond to a decrease i n the f l u x of c h o l i n e to PC. ( i i i ) P r e d i c t i o n Since a l l three enzyme r e a c t i o n s of d_e novo PC s y n t h e s i s appear to be n e a r - e q u i l i b r i u m (Type 1), each one should be very s e n s i t i v e to the mass a c t i o n r a t i o ofireach r e a c t i o n . One pre-d i c t i o n of t h i s concept i s as f o l l o w s . I f the c h o l i n e c o n c e n t r a -t i o n i n the medium of BHK c e l l s i s v a r i e d so that c h o l i n e uptake a l s o v a r i e s ( F i g . 8), then the i n t r a c e l l u l a r pool of c h o l i n e would a l s o vary i n the same way, at l e a s t t r a n s i e n t l y . At i n c r e a s e d l e v e l s of uptake, i t i s p r e d i c t e d that the r a t e of s y n t h e s i s of PC would a l s o i n c r e a s e . - 129 -(h) Are Routes of PC Synt h e s i s Other than the De Novo Pathway of Importance i n BHK C e l l s ? De novo s y n t h e s i s of PC may be the only important route of s y n t h e s i s i n BHK c e l l s . In support of t h i s c o n c l u s i o n , while CPT i n BHK microsomes has a s p e c i f i c a c t i v i t y of about o n e - t h i r d that of r a t l i v e r (Table 6 and re f e r e n c e 110), PEMT s p e c i f i c a c t i v i t y i n BHK microsomes i s l e s s than one-tenth t h a t of r a t l i v e r (Results s e c t i o n ( c ) ) . A l s o , the pr e c u r s o r - p r o d u c t r e l a -t i o n s h i p between phosphocholine and PC ( F i g . 24) i s evidence that d i r e c t base exchange does not occur between c h o l i n e and PC. (i) Some Unanswered Questions Measurement of p would provide additional evidence as to the nature of the c o n t r o l of PC s y n t h e s i s i n BHK c e l l s . For Type 1 r e a c t i o n s , -p- i s c l o s e to 1. The measurement r e q u i r e s knowledge of the r a t i o s of s u b s t r a t e and product c o n c e n t r a t i o n s (absolute pool s i z e s are not n e c e s s a r y ) . A l s o , f o r CPT, the e q u i l i b r i u m constant- should be determined with a p u r i f i e d en&yme, so that c o m p e t i t i v e r e a c t i o n s are e l i m i n a t e d . The compartmentation of c e l l u l a r pools of c h o l i n e and C;TP c e r t a i n l y warrants f u r t h e r study. S u b c e l l u l a r pools could be i d e n t i f i e d by monitoring the s p e c i f i c r a d i o a c t i v i t y i n s u b c e l l u l a r f r a c t i o n s from c e l l s which are l a b e l l e d with exogenous c h o l i n e or c y t o s i n e . Changes i n c e l l u l a r CTP content i n i n f e c t e d c e l l s may be caused by changes o c c u r r i n g e x c l u s i v e l y i n the nucleus. However, - 130 -the pool of CTP which i s important for PC synthesis i s presumably that of the cytosol. In HeLa c e l l s infected with polio v i r u s , the cytoplasmic concentration of CTP correlates with the rate of synthesis of PC (214). It would be interesting to see whether the apparent time lag between CTP pool size changes and phospho-choline pool size changes i s indeed, r e a l . To answer this ques-tion, phosphocholine pool sizes should be measured at various times p . i . . Such a time lag could represent the time required for changes in the CTP pool of the nucleus to be reflected in the cytoplasm. The choline transport process also deserves further study. The s p e c i f i c i t y of the process, and the kinds of compounds that would act as competitive and noncompetitive i n h i b i t o r s , would help to characterize the putative choline c a r r i e r protein. It would also be of interest to repeat the measurement of flux to see i f the rate of PC synthesis i s t r u l y increased in virus-infected c e l l s . This re s u l t seems unlikely, since in no experiment was choline uptake stimulated by virus i n f e c t i o n . Other general unanswered questions about the regulation of PC synthesis include: 1. What i s the relationship of choline transport to choline kinase a c t i v i t y ? 2. What are the significance of multiple forms of CK (24, 26, 28), CT (35, 36)", and CPT (39, 43, 45, 46)? 3. What role does l i p i d a ctivation of CT (36, 90-93) play in PC synthesis? - 131 -4. What i s the p h y s i c a l r e l a t i o n s h i p of the s o l u b l e enzymes to the microsomal enzymes of de novo PC s y n t h e s i s ? Is there a loose a s s o c i a t i o n ? 5. What i s the 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 , such as M g + + and C a + + , i n the c o n t r o l of PC s y n t h e s i s ? 6. Are m u l t i p l e pools of c h o l i n e found i n other c e l l types? I f so, what i s the source of the i n a c t i v e pool? What i s i t s f u n c t i o n ? 7. How does an e x t e r n a l agent (such as C o r t i s o l (153)), a c t to s p e c i f i c a l l y s t i m u l a t e PC s y n t h e s i s ? (j) Concluding Remarks' The r e s u l t s of t h i s t h e s i s demonstrate the n e c e s s i t y of i n t e r p r e t i n g i n c o r p o r a t i o n r e s u l t s with c a u t i o n u n t i l s p e c i f i c r a d i o a c t i v i t i e s are known. S i m i l a r l y , enzyme a c t i v i t i e s are more understandable when compared with the in v i v o f l u x . For example, the low a c t i v i t y of CK (Table 5) and the accumulation of Q 3H]] c h o l i n e a f t e r v i r u s i n f e c t i o n (Table 8) c o u l d be taken as e v i -dence that the a c t i v i t y of CK c o n t r o l s PC s y n t h e s i s and that t h i s a c t i v i t y i s i n h i b i t e d a f t e r v i r u s i n f e c t i o n , which causes an ac-cumulation of Q 3H]] c h o l i n e and an i n h i b i t i o n of PC s y n t h e s i s . Such a c o n c l u s i o n i s not j u s t i f i e d . Yet, c o n t r o l l i n g steps of PC s y n t h e s i s have been proposed on the b a s i s of i n v i t r o enzyme a c t i v i t i e s (142, 146, 147, 149, 160), or i n c o r p o r a t i o n r e s u l t s (10, 68, 143) . I t i s c l e a r by comparison of f l u x and maximal v e l o c i t y v a l r - . z . ues, that i n v i t r o enzyme a c t i v i t i e s i n t h i s system, assayed under - 132 -o ptimal c o n d i t i o n s , are not a r e l i a b l e guide to the i n v i v o ac-t i v i t i e s . Since the f l u x i s much l e s s than the maximal v e l o c i -t i e s of the three de novo enzymes from BHK c e l l s , i t i s c l e a r t h a t the three step pathway i s c o n t r o l l e d by the c o n c e n t r a t i o n s of s u b s t r a t e s and products. Information about f l u x e s , mass ac-t i o n r a t i o s , and e q u i l i b r i u m constants w i l l add g r e a t l y to our understanding of t h i s pathway. I t i s t r u l y a c h a l l e n g e ' t o b i o -chemists to f i t the dynamic i n t e r p l a y of events of the microcosm of the l i v i n g c e l l i n t o a broad t h e o r e t i c a l framework; a c h a l l e n g e which w i l l h o p e f u l l y extend the h o r i z o n s of our understanding of the f o r c e s which i n t e r a c t to maintain the l i v i n g system. - 133 -1. White, D.A. (1973) i n The P h o s p h o l i p i d Composition of Mam-malian T i s s u e s i n Form and Fu n c t i o n of P h o s p h o l i p i d s ( A n s e l l , G.B., Hawthorne, J.N., and Dawson, R.M.C., eds) pp. 441-482, E l s e v i e r , Amsterdam 2. Brotherus, J . , and Renkonen, 0. (1977) Biochim. Biophys. Acta 416, 243-253 3. Singer, S.J. (1971) i n S t r u c t u r e and Fu n c t i o n of B i o l o g i c a l Membranes ( R o t h f i e l d , L . I . , ed) pp. 145-222, Academic Pr e s s , New York 4. S i n g e r , S.J., and N i c o l s o n , G.L. (1972) Science 175, 720-731. 5. Rothman, J.E., and Lenard, J . (1977) Science 195, 743-753 6. N i l s s o n , O.S., and D a l l n e r , G. (1977) Biochim. Biophys. Acta 464, 453-458 7. N i l s s o n , O.S., and D a l l n e r , G. (1977) J . C e l l B i o l . 72, 568-583 8. Sundler, R. , S a r c i o n e , S.L,., A l b e r t s , A.W., and Vagelos, P.R. (1977) Proc. N a t l . Acad. S c i . U.S.A. 74/ 3350-3354 9. Denton, R.M., and Pogson, C.I. (1976) i n M e t a b o l i c R e g u l a t i o n pp. 7-20, Chapman and H a l l , London 10. Haeffner, E.W. (1975) Eur. J . Biochem. 51, 219-228. 11. K u c z l e r , F . J . , Nahrwold, D.L., and Rose, R.C. (1977) Biochim. Biophys. Acta 465, 131-137 12. Plagemann, P.G.W., and Richey, D.P. (1974) Biochim. Biophys. Acta 3_4_4 , 263-305 . 13. Simon, J.R., Atweh, S., and Kuhar, M.J. (1976) J . Neurochem. 26, 909-922 14. Kuhar, M.J., and Murr i n , L.C. (1978) J . Neurochem. 3_0, 15-21. 15. Yavin, E. (1976) J . B i o l . Chem. 251, 1392-1397 16. Bader, C.R., Baughman, R.W., and Moore, J.L. (1978) Proc. N a t l . Acad. S c i . U.S.A. 7_5, 2525-2529 17. Diamond, I., and Kennedy, E.P. (1969) J . B i o l . Chem. 244, 3258-3263 18. Co r n f o r d , E.M., Braun, L.D., and Oldendorf, W.H. (1978) J . Neurochem. 3_0, 299-308 - 134 -19. Bremer, J . , and Greenberg, D.M. (1961) Biochim. Biophys. Acta 46, 205-216 20. C h o j n a c k i , T., K o r z y b s k i , T., and A n s e l l , G.B. (1964) Biochem. J . 9_0, 18P-19P 21. M a r s h a l l , E.F., Ch o j n a c k i , T., and A n s e l l , G.B. (1965) Biochem. J . 9_5, 30P-31P 22. Wittenberg, J . , and Kornberg, A. (1953) J . B i o l . Chem. 202, 431-444 23. Kornberg, A., and P r i c e r , W.E. (1952) Fed. Proc. 11, 242 ; 24. Weinhold, P.A., and Rethy, V.B. (1974) B i o c h e m i s t r y 13, 5135-5141 25. U p r e t i , R.K., Sanwal, G.G., and Krishnan, P.S. (1976) Arch. Biochem. Biophys. 174, 658-665 26. Brophy, P.J., Choy, P.C., Toone, J.R. and Vance, D.E. (1977) Eur. J . Biochem. 78, 491-495 27. Spanner, S., and A n s e l l , G.B. (1977) Biochem. Soc. Trans. 5_, 164-165 28. Burt, A.M. (1977) J . Neurochem. 28, 961-966 29. Dykes, C.W., Kay, J . , and Harwood, J.L. (1976) Biochem..."J. 158, 575-581 30. Broad, T.E., and Dawson, R.M.C. (1975) Biochem. J . 146, 317-328 31. Bygrave, F.L., and Dawson, R.M.C. (1976) Biochem. J . 160, 481-490 32. I n f a n t e , J.P., and K i n s e l l a , J.E. (1976) L i p i d s 11, 727-735 33. Sundler, R., Ar v i d s o n , G., and Akesson, B. (1972) Biochim. Biophys. Acta 280, 559-568 34. Kennedy, E.P., and Weiss, S.B. (1956) J . B i o l . Chem. 222, 193-214 35. Choy, P.C., Lim, P.H., and Vance, D.E. (1977) J . B i o l . Chem. 252, 7673-7677 36. Feldman, D.A., Kovac, C.R., D r a n g i n i s , P.L., and Weinhold, P.A. (1978) J . B i o l . Chem. 253, 4980-4986 - 135 -37. Sundler, R. (1975) J . B i o l . Chem. 251' 8585-8590 38. Radominska-Pyrek, A., Matysiak, Z., and C h o j n a c k i , T. (1969) Acta Biochim. P o l . 16, 357-363 39. Kanoh, H., and Ohno, K. (1976) Eur. J . Biochem. 66, 201-210 40. Lord, J.M. (1975) Biochem. J . 151, 451-453 41. Dawson, R.M.C., and Le t c h e r , A. (1977) FEBS L e t t . 7_7, 179-181 42. F r e y s z , L., Horrocks, L.A., and Mandel, P. (1977) Biochim. Biophys. Acta 489, 431-439 43. Radominska-Pyrek, A., S t r o s z n a j d e r , J . , Dabrowiecki, Z., Cho j n a c k i , T., and Horrocks, L.A. (1976) J . L i p i d Res.17, 657-662 44. Coleman, R., and B e l l , R.M. (1977)J. B i o l . Chem. 252, 3050-3056 45. F r e y s z , L., Lastennet, A., and Mandel, P. (1972) J . Neuro-chem 19, 2599-2605 46. F r e y s z , L., and Mandel, P. (1974) FEBS L e t t . 40, 110-113 47. .Bj0rnstad, P., and Bremer, J . (1966) J . L i p i d Res. 1_, 38-45 48. Sal e r n o , D.M., and B e e l e r , D.A. (1973) Bichom, Biophys. Acta 326, 325-338 49. Akesson, B., and Sundler, R. (1977) Biochem. Soc. Trans. 5, 43-48 50. Sundler, R. , and Akesson, B. (1975) Biochem. J . 146, 309-315 51. Kanoh, H., and Ohno, K. (1973) Biochim. Biophys. Acta 306, 203-217 52. Kanoh, H., and Ohno, K. (1973) Biochim. Biophys. Acta 326, 17-25 53. Kanoh, H., and Ohno, K. (1975) Biochim. Biophys. Acta 380, 199-207. 54. Kanoh, H., and Ohno, K. (1976) i n L i p i d s ( P a o l e t t i , R., P o r c e l l a t i , G., and J a c i n i , G., eds) V o l . 1, pp. 39-48, Raven Press, New York 55. Holub, B.J. (1978) J . B i o l . Chem. 253, 691-696 - 136 -56. Morimoto, K., and Kanoh, H. (1978) J . B i o l . Chem. 253, 5056-5060 57. Holub, B.J. (1977) Can. J . Biochem. 5_5, 700-705 58. Sundler, R., Akesson, B. , and N i l s s o n , A. (1974) Biochim. Biophys. Acta 337, 248-254 59. A r v i d s o n , G.A.E. (1968) Eur. J . Biochem 5, 415-421 60. Kanoh, H. (1969) Biochim. Biophys. Acta 176, 756-763 61. MacDonald, \-G. , and Thompson, W. (1975) Biochim. Biophys. Acta 398, 424-432 62. Tinoco, J . , Sheehan, G., Hopkins, S., and Lyman, R.L. (1970) L i p i d s 5, 412-416 63. T r e b l e , D.H., Frumkin, S., B a l i n t , J.A., and B e e l e r , D.A. (1970) Biochim. Biophys. Acta 202, 163-171 64. Moriya, T., and Kanoh, H. (1974) Tohoku J . Exp. Med. 112, 241-256 65. Vance, D.E., Choy, P.C., F a r r e n , S.B., Lim, P.H., and Schneider, W.J. (1977) Nature 270, 268-269 66. Coleman, R.,. and B e l l , R.M.. (1978) J . C e l l B i o l . 76, 245-253 67. Bremer, J . , and Greenberg, D.M. (1959) Biochim. Biophys. Acta 35, 287-288 68. Bremer, J . , and Greenberg, D.M. (1960) Biochim. Biophys. Acta 37, 173-175 69. K a t y a l , S.L., and Lombardi, B. (1976) L i p i d s 11, 513-516 70. H i r a t a , F., V i v e r o s , O.H., D i l i b e r t o , E . J . J r . , and A x e l r o d , J . (1978) Proc. N a t l . Acad. S c i . U.S.A. 7_S, 1718-1721 71. H i r a t a , F., and A x e l r o d , J . (1978) Proc. N a t l . Acad. S c i . U.S.A. 75, 2348-2352 72. Sundler, R., and Akesson, B. (1975) J . B i o l . Chem. 250 3359-3367 73. P l a n t a v i d , M., Maget-Dana, R., and Douste-Blazy, L. (1976) FEBS L e t t . 7_2, 169-172 74. D i l s , R.R., and Hubscher, G. (1961) Biochim. Biophys. Acta 46, 505-513 - 137 -75. Lunt, G.G., and L a p e t i n a , E.G. (1970) B r a i n Res. 18, 451-. 4 5 9 76. Kanfer, J.N. (1972) J . L i p i d Res. 13, 468-476 77. Hubscher, G., D i l s , R.R., and Pover, W.F.R. (1959) Biochim. Biophys. Acta 36, 518-528 78. Borkenhagen, L.F., Kennedy, E.P., and F i e l d i n g , L. (1961) J . B i o l . Chem. 236, PC28-PC30 79. B j e r v e , K.S. (1973) Biochim. Biophys. Acta 296, 549-562 80. Plagemann, P.G.W. (1971) J . L i p i d Res. 12, 715-724 81. G a i t i , A., B r u n e t t i , M., Woelk, H., and P o r c e l l a t i , G. (1976) L i p i d s 11, 823-829 82. A r i e n t i , G., C o r a z z i , L., Woelk, H., and P o r c e l l a t i , G. (1976) J . Neurochem. 21_, 203-210 83. Orlando, P., A r i e n t i , G., C e r r i t o , F., M a s s a r i , P., and P o r c e l l a t i , G. (1977) Neurochem. Res. 2, 191-201 84. A n s e l l , G.B., and Spanner, S. (1975) Biochem. Pharmacol. 24:, 1719-1723 85. G o r a c c i , G., Blomstrand, C , A r i e n t i , G., Hamberger, A., and P o r c e l l a t i , G. (1973) J . Neurochem. 20, 1167-1180 86. Plagemann, P.G.W. (1968) Arch. Biochem. Biophys. 128, 70-87 87. Plagemann, P.G.W., and Roth, M.F. (1969) B i o c h e m i s t r y 8, 4782-4789 88. Fukuyama, H., and Yamashita, S. (1976) FEBS L e t t . 71' 33-36 89. Paddon, H.B., and Vance, D.E. (1977) Biochim. Biophys. Acta 488, 181-189 90. F i s c u s , W.G., and Schneider, W.C. (1966) J . B i o l . Chem. 241, 3324-3330 91. Choy, P . C , and Vance, D.E. (1978) J . B i o l . Chem. 253, 5163-5167 92. O'Doherty, P.J.A., Smith, N.B., and Kuks i s , A. (1977) Arch. Biochem. Biophys. 180, 10-18 93. O'Doherty, P.J.A., Yousef, I.M., Kakis, G., and Kuksis, A. (1975) Arch. Biochem. Biophys. 169, 252-261 - 138 -94. S r i b n e y , M., and Lyman, E.M. (1973) Can. J . Biochem. 51, 1479-1486 95. McMurray, W.C. (1974) Biochim. Biophys. Res. Commun. 58, 467-474 96. De K r u y f f , B., Van Golde, L.M.G., and Van Deeneh, L.L.M. (1970) Biochim. Biophys. Acta 210, 425-435 97. Sribney/ M., Knowles, C.L., and Lyman, E.M. (1976) Biochem. J . 156, 507-514 98. L i t e p l o , R.G., and Sribney, M. (1977) Can. J . Biochem. 55, 1049-1056 99. I r i t a n i , N., Yamashita, S., and Numa, S. (1976) J . Biochem. 80, 217-222 100. Rose, H., Vaughan, M., and S t e i n b e r g , D. (1964) Am. J . P h y s i o l . 206, 345-350 101. N i l s s o n , S., and Schersten, T. (1969) Scand. J . C l i n . Lab. Invest. 24, 237-249 102. Sundler, R., Akesson, B., and N i l s s o n , A. (1974) J . B i o l . Chem. 249, 5102-5107 103. Ontko, J.A. (1972) J . B i o l . Chem. 247, 1788-1800 104. W i l l i a m s , J.N. (1952) J . B i o l . Chem. 19_4, 139-142 105. K i s s , Z. (1977) Biochem. J . 168, 387-391 106. Sundler, R. (1973) Biochim. Biophys. A c t a 306, 218-226 107. Infante, J.P. (1977) Biochem. J . 167, 847-849 108. I n f a n t e , J.P., and K i n s e l l a , J.E. (1976) I n t . J . Biochem. 1_, 483-496 109. Ferdinand, W. (1976) i n The Enzyme Molecule, pp. 225-228, 236-237, John Wiley & Sons, London 110. Schneider, W.J., and Vance, D.E. (1978) Eur. J . Biochem. 8_5, 181-187 111. Roberts, J.B., and Bygrave, F.L. (1973) Biochem. J . 136, 467-475 112. Osanai, A., and Sakagami, T. (1977) J . Biochem. 8_1, 1651-1659 - 139 -113. Cook, G.A., Perry, W.D., and Daron, H.H. (1976) Biochem. Biophys. Res. Commun. 6j3, 411-416 1 114. S o l e r - A r g i l a g a , C , Russe l l , R. L. , and Heimberg, M. (1977) Biochem. Biophys. Res. Commun. 7_8' 1053-1059 115. A l l a n , D., and M i c h e l l , R.H. (1977) Biochem J . 164, 389-397 116. W e l l s , I.C., and Remy, C.N. (1965) Arch. Biochem. Biophys. 112, 201-206 117. Young, D.L. (1967) C l i n . Res. 15, 246 118. SkurdaL D.N., and ..Cornatzer, W.E. (1974) Proc. Soc. Exp. B i o l . Med. 145, 992-995 119. Skurdal.,D.N., and Co r n a t z e r , W.E. (1975) I n t . J . Biochem 6, 579-583 120. Choy, P.C., Schneider, W.J., and Vance, D.E. (1978) Eur. J . Biochem. 8_5, 189-193 121. Kuksis, A., and Mookerjea, S. (1978) Nutr. Rev. 3_6, 201-207 122. Mookerjea, S., and Marai, E. (1971) J . B i o l . Chem. 246, 3008-3017 123. Dawson, R.M.C. (1955) Biochem. J . 60, 325-328 124. Thompson, W., MacDonald, G., and Mookerjea, S. (1969) Biochem. Biophys. Acta 176, 306-315 125. Delahunty, T., and Mookerjea, S. (1974) Can. J . Biochem. 52, 359-365 126. O r r e n i u s , S., E r i c s s o n , J.L.E., and E r n s t e r , L. (1965) J . C e l l B i o l . 25, 627-639 127. Young, D.L., Powell, G., and McMil l a n , W.O. (1971) J . L i p i d Res. 1_2, 1-8 128. Holtzman, J.L., and G i l l e t t e , J.R. (1968) J . B i o l . Chem. 243, 3020-3028 129. O r r e n i u s , S. (1965) J . C e l l B i o l . 26, 725-733 130. Davison, S.C., and W i l l s , E.D. (1974) Biochem. J . 142, 19-26 131. Uthus, E.O., Skur d a l , D.N., and Corn a t z e r , W.E. (1976) L i p i d s 11, 641-644 - 140 -132. Van Heusden, G.P.H., and Van den Bosch, H. (1978) Eur. J . Biochem. 8_4, 405-412 133. F a l l o n , H.J., Lamb, R.G., and Jamdar, S.C. (1977) Biochem. Soc. Trans. 5, 37-40 134. S t u r t o n , R.G., and B r i n d l e y , D.N. (1977) Biochem. J . 162, 25-32 135. B r i n d l e y , D.N., Bowley, M., S t u r t o n , R.G., P r i t c h a r d , P.H., B u r d i t t , S.L., and C o o l i n g , J . (1977) Biochem. Soc. Trans. 5, 40-43 136. Bowley, M., C o o l i n g , J . , B u r d i t t , S.L., and B r i n d l e y , D.N. (1977) Biochem. J . 165, 447-454 ( 137. F a l l o n , H.J., Barwick, J . , Lamb, R.G., and Van den Bosch, H. (1975) J . L i p i d Res. 16, 107-115 138. F a r r e l l , P.M., and Avery, M.E. (1975) Am. Rev. R e s p i r . D i s . I l l , 657-688 139. Montfoort, A., Van Golde, L.M.G., and Van Deenen, L.L.M. (1971) Biochim. Biophys. A c t a 231, 335-342 140. West, J.B. (1977) Pulmonary Pathophysiology - The E s s e n t i a l s p. 94, W i l l i a m s & W i l k i n s , B a l t i m o r e 141. S a r z a l a , M.G., and Van Golde, L.M.G. (1976) Biochim. B i o -phys. Acta 441, 423-432 142. Possmayer, F., Duwe, G., Hahn, M., and Buchnea, D. (1977) Can. J . Biochem. _55, 609-617 143. Okano, G., and Akino, T. (1978) Biochim. Biophys. A c t a 528, 373-384 144. Oldenborg, V., and Van Golde, L.M.G. (1976) Biochim. B i o -phys. Acta 441, 433-442 145. Rooney, S.A. , Wai-Lee, T.S., Gobran,. L., and Motoyama, E.K. (1976) Biochim. Biophys. Acta 431, 447-458 146. Tsao, F.H.C., and Zachman, R.D. (1977) P e d i a t r . Res. 11, 858-861 147. F a r r e l l , P.M., and Morgan, T.E. (1977) i n Development of the Lung (Hodson, W.A., ed) pp. 309-347, Marcel Dekker, New York - 141 -148. Weinhold, P.A., Sanders, R., and St e r n , W. (1973) i n Res-p i r a t o r y D i s t r e s s Syndrome ( V i l l e e , C.A., V i l l e e , D.B., and Zuckerman, J . , eds) pp. 29-45, Academic P r e s s , New York 149. S c h u l t z , F.M., Jimenez, J.M., MacDonald, P.C., and Johnston, J.M. (1974) Gynecol. Invest. 5, 222-229 150. Weinhold, P.A. (1968) J . L i p i d Res. 9, 262-266 151. Hallman, M. , and Gluck, L. (1975) Fed. Proc. 3_4, 274 152. Hallman, M., Feldman, B.H., K i r k p a t r i c k , E., and Gluck, L. (1977) P e d i a t r . Res. 11, 714-720 153. Rooney, S.A., Gobran, L., Gross, I., Wai-Lee, T.S., Nardone, L.L., and Motoyama, E.K. (1976) Biochim. Biophys. Acta 450, 121-130 154. Oldenborg, V., and Van Golde, L.M.G. (1977) Biochim. B i o -phys. Acta 489, 454-465 155. A n s e l l , G.B., and Spanner, S. (1971) Biochem. J . 122, 741-750 156. Spanner, S., H a l l , R.C., and Ansell,; G.B. (1976) Biochem. J . L5_4, 133-140 157. A n s e l l , G.B., and Spanner, S. (1967) J . Neurochem. 14, 873-885 158. Kewitz, H., and P l e u l , 0. (1976) Proc. N a t l . Acad. S c i . U.S.A. 7_3, 2181-2185 159. Yavin, E. (1977) Biochim. Biophys. Acta 489, 278-289 160. A n s e l l , G.B., and Spanner, S. (1972) Biochem. Soc. Symp. 35, 151-159 161. F r a n c e s c a n g e l i , E., G o r a c c i , G. , P i c c i n i n , G.L., Mozzi, R., Woelk, H., and P o r c e l l a t i , G. (1977) J . Neurochem. 28, 171-176 162. G a l l a h e r , W.R., and Blough, H.A. (1975) Arch. Biochem. Biophys. 168, 104-114 163. G a l l a h e r , W.R., and Blough, H.A. (1976) Arch. Biochem. Biophys. 173, 739-746 164. Schneider, P.B. (1977) J . L i p i d Res. 18, 239-245 165. Howard, B.V., Howard, W.J., and K e f a l i d e s , N.A. (1976) J . C e l l P h y s i o l . 8_9, 325-336 - 142 -166. Co r n a t z e r , W.E., Sandstrom, W. , and Fischer., R.G. (1961) Biochim. Biophys. Acta 4j), 414-415 167. Amako, K., and Dales, S. (1967) V i r o l o g y 3_2, 201-215 168. Mcintosh, K., Payne, S., and R u s s e l l , W.C. (1971) J . Gen. V i r o l . 10, 251-265 169. Ben-Porat, T., and Kaplan, A.S. (1971) V i r o l o g y , 45, 252-264 170. S k u r d a l , D.N., R y t t e r , D.J., and Co r n a t z e r , W.E. (1974) Proc. Soc. Exp. B i o l . Med. 146, 844-848 171. Makino, S., and J e n k i n , H.M. (1975) J . V i r o l . 1_5, 515-525 172. Waite, M.R.F., and P f e f f e r k o r n , E.R. (1970) J . V i r o l . 6, 637-643 173. Vance, D.E., and Burke, D.C. (1974) Eur. J . Biochem. 43, 327-336 174. Vance, D.E., and Dahlke, R.M. (1975) Can. J . Biochem. 53, 950-957 175. I s r a e l , A., Audubert, F., and Semmel, M. (1975) Biochim. Biophys. Acta 375, 224-235 176. Fenner, F. (1976) V i r o l o g y 71, 371-378 177. S t r a u s s , J.H., and S t r a u s s , E.G. (1977) i n The Molecular B i o l o g y of Animal V i r u s e s (Nayak,^D.P., ed) V o l . 1, pp. 111-124, Marcel Dekker,New York 178. Renkonen, 0., Kaarainen, L., Simons, K., and Gahmberg, C.G. (1971) V i r o l o g y . 46, 318-326 179. Richardson, CD., and Vance, D.E. (1976) J . B i o l . Chem. 251, 5544-5550 180. C a s s e l l s , A.C., and Burke, D.C. (1973) J . Gen. V i r o l . 18, 135-141 181. Hammer, G., Schwarz, R.T., and S c h o l t i s s e k , C. (1976) V i r o l o g y 70, 238-240 182. Peterhans, E., Browse, E., and Wyler, R. (1977) E x p e r i e n t i a 13, 827 - 143 -183. B r e g o f f , H.M., Roberts, E., and Delwiche, C.C. (1953) J . B i o l . Chem. 205, 565-574 184. Dittmer, J.C., and L e s t e r , R.L. (1964) J . L i p i d Res. 5, 126-127 185. Akesson, B. (1969) Eur. J . Biochem. 9, 463-477 186. F r i c k e , U. (1975) A n a l . Biochem. 63, 555-558 187. Lowry, O.H., Rosebrough, N.J., F a r r , A.L., and R a n d a l l , R.J. (1951) J . B i o l . Chem. 193/ 265-275 188. Woolf, CM. (1968) P r i n c i p l e s of Biometry, D. Van Nostrand, P r i n c e t o n , New J e r s e y 189. Dulbecco, R., and Vogt, M. (1954) J . Exp. Med. 99, 183-199 190. A n s e l l , G.B., and Ch o j n a c k i , T. (1969) i n Methods i n Enzymology (Lowenstein, J.M., ed) V o l . 14, pp. 121-125, Academic P r e s s , New York 191. Hanahan, D.J., and Vercamer, R. (1954) J . Am. Chem. Soc. 76, 1804-1806 192. Rehbinder, D., and Greenberg, D.M. .(1965) Arch. Biochem. Biophys. ip_9, 110-115 193. Myers, D.K., and S l a t e r , E.G. (1957) Biochem. J . 67, 558-572 194. Hayashi, M., Unemoto, T., and M i y a k i , K. (1962) Chem. Pharm. B u l l . 10, 533-535 195. Goldberg, A.M., and McCaman, R.E. (1974) i n C h o l i n e and A c e t y l c h o l i n e : Handbook of Chemical Assay Methods (Hanin, I., ed) pp. 47-61, Raven Press, New York 196. Dittmer, J.C., and We l l s , M.A. (1969) i n Methods i n Enzymology (Lowenstein, J.M., ed) V o l . 14, pp. 482-530, Academic Press, New York 197. F o l c h , J . , Lees, M., and St a n l e y , G.H.S. (1957) J . B i o l . Chem. 2^6, 497-509 198. Raheja, R.K., Kaur, C , Singh, A., and B h a t i a , I.S. (1973) J . L i p i d Res. 14_, 695-697 199. Schneider, P.B. (1977) J . L i p i d Res. 18, 396-399 - 144 -200. Choy, P.C., Whitehead, F.W., and Vance, D.A. (1978) Can. J . Biochem. 56_, 831-835 201. Williamson, D.H., Lund, P., and Krebs, H.A. (1967) Biochem J . 103, 514-527 202. E l i o n , G.B., Furman, P.A., F y f e , J.A., De P i r a n d a , P., Beuachamp, L., and S c h a e f f e r , H.J. (1977) Proc. N a t l . Acad. S c i . U.S.A. 74, 5716-5720 203. Vance, D.E., and Sweeley, C.C. (1967) J . L i p i d Res. 8, 621-630 204. Wagner, H., Horhammer, L. , and Wolff, P./ (1961) Biochem. Z. 334, 175-184 205. C a s s e l l s , A.C. (1973) J . Gen. V i r o l . 18, 203-205 206. Z i l v e r s m i t , . D.B. (1960) Am. J . Med. 29, 832-848 207. Acheson, N.H., and Tamm, I. (1967) V i r o l o g y 3_2, 128-143 208 . Simons, K., K a a r i a i h e s , . L. , Renkonen, 0., Gahmberg, C.G., G a r o f f , H., H e l e n i u s , A.,.Keranen, S., La i n e , R./ Ranki, M., Soderlund, H., and Utermann,G. (1973) i n Membrane - Medi-ated Information (Kent, P.W., ed) V o l . 2, pp. 81-99, Med i c a l and T e c h n i c a l P u b l i s h i n g , Lancaster, England. 209. Vance, D.E., and Paddon, H.B. -(1978) Can. Fed. B i o l . Soc. Proc. 21, 181 210. CRC Handbook of Chemistry and P h y s i c s , 1977-1978 e d i t i o n (Weast, R.C., ed) p. D-147, CRC P r e s s , C l e v e l a n d 211. K a a r i a i h : e f t ^ L . , and Gomatos, P.J. (1969) J . Gen. V i r o l . 5, 251-265 212. Newsholme, E.A., and C r a b t r e e , B. (1973) Symp. Soc. Exp. B i o l . 2 2 , 429-460 213. Kacser, H., and Burns, J.A.. (1973) Symp. Soc.~Exp. B i o l . .27, 65-104 214. Paddon, H.B., Choy, P.C., and Vance, D.E. (1979) I n t . Congr. Biochem. Abs.tr., i n press 

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