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Isolation of rat liver CTP: phosphocholine cytidylyltransferase and regulation of hepatic phosphatidylcholine… Yao, Zemin 1985

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ISOLATION OF RAT LIVER CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE AND REGULATION OF HEPATIC PHOSPHATIDYLCHOLINE BIOSYNTHESIS by ZEMIN YAO B.Sc. East China Normal U n i v e r s i t y , 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of B i o c h e m i s t r y ) We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1985 © Zemin Yao, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date >E-6 (3/81) ABSTRACT Two k i n d s of a f f i n i t y chromatography, C D P - c h o l i n e - and CTP-Sepharose 4B, were i n v e s t i g a t e d f o r p u r i f i c a t i o n of the c y t o s o l i c C T P : p h o s p h o c h o l i n e c y t i d y l y l t r a n s f e r a s e f r o m r a t l i v e r . The enzyme d i d not show s t r o n g a f f i n i t y f o r the CDP-choline Sepharose r e s i n , b u t bound t o t h e C T P - S e p h a r o s e c o l u m n i n t h e p r e s e n c e o f 14 mM magnesium a c e t a t e . The c o m b i n a t i o n of CTP a f f i n i t y chroma-t o g r a p h y w i t h i o n - e x c h a n g e t e c h n i q u e s p r o v i d e d a b o u t 7 0 - f o l d p u r i f i c a t i o n of the c y t o s o l i c enzyme w i t h a s p e c i f i c a c t i v i t y o f about 90 u n i t s per m i l l i g r a m p r o t e i n . The i n f l u e n c e of d i p h e n y l s u l f o n e compounds on the s y n t h e s i s o f p h o s p h a t i d y l c h o l i n e by the C D P - c h o l i n e pathway was examined i n i s o l a t e d r a t h e p a t o c y t e s and HeLa c e l l s . The a d m i n i s t r a t i o n o f the s u l f o n e s (100 ug/ml), except dapsone, to HeLa c e l l s i n h i b i t e d 3 t h e t o t a l [ m e t h y l - H ] c h o l i n e i n c o r p o r a t i o n i n t o t h e c e l l s , b u t d i d not change the r a t e of c o n v e r s i o n of c h o l i n e t o p h o s p h a t i d y l -c h o l i n e . The a d d i t i o n of the s u l f o n e s (100 ug/ml) to r a t hepato-c y t e s d i d not i n h i b i t the b i o s y n t h e s i s o f p h o s p h a t i d y l c h o l i n e and c h o l i n e m e t a b o l i s m . The e f f e c t of v a s o p r e s s i n on the d i s t r i b u 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 between c y t o s o l and microsomes i n r a t h e p a t o c y t e s was a l s o i n v e s t i g a t e d . The d i g i t o n i n - m e d i a t e d r e l e a s e o f c y t o s o l i c c y t i d y l y l t r a n s f e r a s e was r e d u c e d f r o m t h e c e l l s t r e a t e d w i t h v a s o p r e s s i n (5-20 nM) , w h i l e the enhanced r a t e of i n c o r p o r a t i o n o f [ m e t h y l - H ] c h o l i n e i n t o p h o s p h a t i d y l c h o l i n e was not observed. TABLE OF CONTENTS Chapter I. I n t r o d u c t i o n 1 1. S t r u c t u r e of p h o s p h a t i d y l c h o l i n e 1 2. B i o l o g i c a l f u n c t i o n s of p h o s p h a t i d y l c h o l i n e 1 3. Pathways 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 n mammals 4 4. Enzymes 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 v i a Kennedy pathway 4 5. Re 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 v i a Kennedy pathway 7 6. The t h e s i s i n v e s t i g a t i o n s 8 Ch a p t e r I I . P u r i f i c a t i o n of CTP:phosphocho1i ne c y t i d y l -y l t ransf erase from r at l i v e r c y t o s o l by a f f i n i t y chromatography 10 M a t e r i a l s and methods 13 R e s u l t s 18 D i s c u s s i o n 28 Chapter I I I . E f f e c t s of d i p h e n y l s u l f o n e compounds on the metabolism of [methyl- H]choline i n r a t hepatocytes and HeLa c e l l s 32 M a t e r i a l s and methods 36 Re s u l t s 39 D i s c u s s i o n 48 Chapter IV. E f f e c t s of v a s o p r e s s i n on CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e i n rat hepatocytes 52 M a t e r i a l s and Methods 57 Res u l t s 59 D i s c u s s i o n 67 References 72 III LIST OF TABLES Table Page 1. P u r i f i c a t i o n of CTPtPhosphocholine C y t i d y l y l t r a n s f e r a s e f r o m R a t L i v e r C y t o s o l 27 •3 2. E f f e c t s of Dapsone and AUS on the [ M e t h y l - H ] C h o l i n e U p t a k e by HeLa C e l l s 40 3. E f f e c t s of D i p h e n y l s u l f o n e d e r i v a t i v e s ( I I I and IV) on The [Me t h y l- 3 H ] C h o l i n e Uptake by HeLa C e l l s 41 4. E f f e c t of V a s o p r e s s i n on The I n c o r p o r a t i o n of [ M e t h y l -3H] C h o l i n e i n t o PC 66 IV LIST OF FIGURES Figure Page 1. S p a c e - f i l l i n g Model of P h o s p h a t i d y l c h o l i n e 2 2. Kennedy Pathway for 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 from Choline 5 3. 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 by Successive N - M e t h y l a t i o n of P h o s p h a t i d y l e t h a n o l a m i n e 6 4. Scheme of The Synthesis of CDP-Choline Sepharose 4B 19 5. DEAE-Cellulose chromatography of Rat L i v e r C y t o s o l i c C y t i d y l y l t ransf erase 22 6. CDP-Choline Sepharose 4B Chromatography of Rat L i v e r C y t o s o l i c C y t i d y l y l t r a n s f e r a s e 23 7. Phospho-Cellulose Chromatography of Rat L i v e r C y t o s o l i c C y t i d y l y l t r a n s f e r a s e 25 8. CTP Sepharose 4B Chromatography of Rat L i v e r C y t o s o l i c Cyt i d y l y l t r a n s f erase 26 9. S t r u c t u r e of Dapsone ( I ) , AUS (II) and Analogs (III and IV) 35 10. Influence of AUS and IV on The Metabolism of [Methyl- H] C h o l i n e i n HeLa C e l l s 44 11. Influence, of Dapsone and I I I on the Metabolism of [Methyl- H] C h o l i n e i n Rat Hepatocytes 46 12. Influence of AUS and IV on The Metabolism of [Methyl- H] C h o l i n e i n Rat Hepatocytes 47 13. Digitonin-mediated Release of CT from Rat Hepatocytes... 60 14. E f f e c t of D i f f e r e n t C o n c e n t r a t i o n s of Vasopressin on The Digitonin-mediated Release of CT from Rat Hepatocytes 61 15. E f f e c t of Vasopressin on The I n c o r p o r a t i o n of [Methyl- H] Choline i n t o P h o s p h o l i p i d s 63 16. Influence of V a s o p r e s s i n on The I n c o r p o r a t i o n of [Methyl- H]Choline into PC 64 V LIST OF ABBREVIATIONS A absorbance ACS aqueous c o u n t i n g s c i n t i l l a n t ATP adenosine 5 ' - t r i p h o s p h a t e AUS l - [ 4 - ( 4 - s u l f a n i l y l ) p h e n y l ] u r e a BHK baby hamster k i d n e y cAMP adenosine 3 5 1 - m o n o p h o s p h a t e CHO C h i n e s e hamster o v a r y CoA coenzyme A CPT c h o l i n e p h o s p h o t r a n s f e r a s e CT p h o s p h o c h o l i n e c y t i d y l y l t r a n s f e r a s e CTP c y t i d i n e 5 1 - t r i p h o s p h a t e DEAE d i e t h y l a m i n o e t h y l DES d i e t h e y l s t i l b o e s t r o l DG d i a c y l g l y c e r o l DMSO d i m e t h y l s u l f o x i d e dpm d i s i n t e g r a t i o n s per minute DTT d i t h i o t h r e i t o l E.R. endoplasmic r e t i c u l u m FCS f e t a l c a l f serum F i g . f i g u r e g g r a v i t y g(m) gram hr hour HDL h i g h d e n s i t y l i p o p r o t e i n Hepes 4 - ( 2 - h y d r o x y e t h y l ) - 1 - p i p e r a z i n e - e t h a n e s u l p h o n i c a c i d HMG-CoA be t a - h y d r o x y - b e t a - m e t h y l g l u t a r y l - C o A Ig immunoglobulin Km M i c h a e l i s - M e n t e n c o n s t a n t LCAT l e c i t h i n c h o l e s t e r o l a c y l t r a n s f e r a s e LDL low d e n s i t y l i p o p r o t e i n l y s o - P C l y s o p h o s p h a t i d y l c h o l i n e l y s o - P E l y s o p h o s p h a t i d y l e t h a n o l a m i n e m meter M mo 1 a r MEM m o d i f i e d E a g l e ' s medium min minute PBS phosphate b u f f e r e d s a l i n e PC p h o s p h a t i d y l c h o l i n e PE p h o s p h a t i d y l e t h a n o l a m i n e PG p h o s p h a t i d y l g l y c e r o l PGs p r o s t a g l a n d i n s PI p h o s p h a t i d y l i n o s i t o l PMSF p h e n y l m e t h y l s u l p h o n y l f l u o r i d e PS p h o s p h a t i d y l s e r i n e P t d l n s 1 - ( 3 - s n - p h o s p h a t i d y l ) - L - m y o - i n o s i t o l PtdIns4P 1 - ( 3 - s n - p h o s p h a t i d y l ) - L - m y o - i n o s i t o l 4-phosphate P t d l n s ( 4 , 5 ) P 2 1 - ( 3 - s n - p h o s p h a t i d y l ) - L - m y o - i n o s i t o l 4,5-biphos-phate VI r a t i o o f d i s t a n c e moved by a s o l u t e r e l a t i v e t o the s o l v e n t f r o n t rpm r e v o l u t i o n s per minute S.D. s t a n d a r d d e v i a t i o n TLC t h i n - l a y e r chromatography TTP t h y m i d i n e 5 ' - t r i p h o s p h a t e T r i s t r i s (hydroxymethyl) aminomethane UTP u r i d i n e 5 ' - t r i p h o s p h a t e UV u l t r a v i o l e t VLDL v e r y low d e n s i t y l i p o p r o t e i n N o t e s : S t a n d a r d p r e f i x e s a r e : m ( m i l l i ) - 10 ; u ( m i c r o ) -10~ 6; n (nano) - 10" 9 VII ACKNOWLEDGEMENTS I w i s h t o thank Dr. Dennis E. Vance, my s u p e r v i s o r , f o r h i s c o n t i n u e d encouragement and h e l p f u l comments throughout my p o s t -g r a d u a t e work. I a l s o a p p r e c i a t e t h e t e c h n i c a l g u i d a n c e t h a t I r e c e i v e d from Dr. Rosemary B. C o r n e l l , Dr. Howard W. M u e l l e r and Mr. Trang Nguyen. I thank a l l my t e a c h e r s and grad u a t e s t u d e n t f r i e n d s i n the B i o c h e m i s t r y Department of UBC f o r t h e i r c o n s t r u c -t i v e s u g g e s t i o n s on my e x p e r i m e n t s . F i n a l l y , I w i s h t o thank my d e a r m o t h e r l a n d , t h e P e o p l e ' s R e p u b l i c o f C h i n a , and t h e g r e a t c o u n t r y , Canada, f o r t h e i r o f f e r i n g s u c h an o p p o r t u n i t y t h a t e n a b l e s me to pursue my p o s t - g r a d u a t e s t u d i e s . VIII CHAPTER I . INTRODUCTION 1. STRUCTURE OF PHOSPHATIDYLYLCHOLINE P h o s p h a t i d y l c h o l i n e (PC) i s the major p h o s p h o l i p i d p r e s e n t i n e u k a r y o t e s but r a r e l y o c c u r s i n p r o k a r y o t e s . The s t r u c t u r e of PC i s d i s t i n g u i s h e d from the o t h e r p h o s p h o l i p i d s by i t s c h o l i n e headgroup, a l t h o u g h a v a r i e t y of f a t t y a c i d s can be e s t e r i f i e d to t h e g l y c e r o l b a c k b o n e . U s u a l l y , s a t u r a t e d f a t t y a c i d s a r e e s t e r i f i e d a t the CI p o s i t i o n of g l y c e r o l w h i l e u n s a t u r a t e d f a t t y a c i d s are a t C2 p o s i t i o n ( F i g u r e 1). 2. BIOLOGICAL FUNCTIONS OF PHOSPHATIDYLYLCHOLINE The p r i m a r y f u n c t i o n o f PC i s s t r u c t u r a l , w h i c h a r i s e s from i t s a m p h i p a t h i c nat u r e the c h o l i n e headgroup i s h y d r o p h i -l i c w h e reas t h e f a t t y a c i d a c y l c h a i n s a r e h y d r o p h o b i c . The a b i l i t y of PC and o t h e r p h o s p h o l i p i d s t o assume s p o n t a n e o u s l y a b i l a y e r o r g a n i z a t i o n i n t h e p r e s e n c e o f e x c e s s w a t e r and a t c o n c e n t r a t i o n s above the " c r i t i c a l m i c e l l a r c o n c e n t r a t i o n " (CMC) might be the dominant reason f o r Nature's c h o i c e of p h o s p h o l i p i d s as a u n i v e r s a l c o n s t i t u e n t of c e l l membranes. The most abundant p h o s p h o l i p i d i n e u k a r y o t e s i s PC, which a c c o u n t s f o r n e a r l y 50% o f t h e t o t a l membrane p h o s p h o l i p i d ( 1 ) . PC has a l s o been d e s c r i b e d as a r e q u i r e m e n t f o r the a c t i v i t y of s e v e r a l membrane-bound enzymes (2). A p p a r e n t l y , PC o f f e r s a s u i t a b l e environment f o r m a i n t a i n i n g the f u n c t i o n a l c o n f o r m a t i o n of those enzymes. In 1 F i g . l . S p a c e - f i l l i n g model of p h o s p h a t i d y l -c h o l i n e . 2 a d d i t i o n to the membrane component, PC a l s o f u n c t i o n s as a c o n s t i t u e n t of b i l e , plasma l i p o p r o t e i n s and lung s u r f a c t a n t . In b i l e , PC accounts f o r 90% of the t o t a l p h o s p h o l i p i d component i n the r a t (3) and 68% i n humans (4). The b i l e PC t o g e t h e r w i t h conjugated b i l e a c i d s f u n c t i o n s i n s o l u b i l i z a t i o n of c h o l e s t e r o l i n b i l e . The f u n c t i o n of PC i n blood plasma l i p o p r o t e i n i s not c l e a r . I t i s thought PC c o a t s the s u r f a c e of l i p o p r o t e i n to render the core of t r i a c y l g l y c e r o l and c h o l e s t e r o l e s t e r s o l u b l e i n p l a s m a , and a l s o , PC i t s e l f i s t r a n s p o r t e d from l i v e r to t a r g e t t i s s u e s v i a l i p o p r o t e i n s . PC accounts f o r approximately 70% of lu n g s u r f a c t a n t by weig h t and l i n e s the a l v e o l i of l u n g , which lowers the s u r f a c e t e n s i o n and prevents the c o l l a p s e of the a l v e o l a r t i s s u e d u r i n g e x p i r a t i o n (5). F a i l u r e to m a i n t a i n an adequate s u p p l y of t h i s m a t e r i a l i n l u n g a l v e o l i a f t e r b i r t h i s thought to be r e s p o n s i b l e f o r R e s p i r a t o r y D i s t r e s s Syndrome, the major cause of m o r t a l i t y and m o r b i d i t y i n premature i n f a n t s (6). As w e l l as being a s t r u c t u r a l component, PC a l s o f u n c t i o n s as an important metabolic pr e c u r s o r i n v o l v e d i n many b i o c h e m i c a l path-ways. PC s e r v e s as a donor of the f a t t y a c y l m o i e t y f o r the s y n t h e s i s o f c h o l e s t e r o l e s t e r i n a r e a c t i o n c a t a l y z e d by l e c i t h i n - c h o l e s t e r o l a c y l t r a n s f e r a s e (LCAT). A r a c h i d o n i c a c i d from PC i s u t i l i z e d f o r the s y n t h e s i s of p r o s t a g l a n d i n s , throm-boxanes and l e u k o t r i e n e s . R e c e n t l y , a p a r t i c u l a r m o l e c u l a r s p e c i e s of PC ( l - a l k y l - 2 - a c e t y l - P C ) has been d e s c r i b e d as a p l a t e l e t a c t i v a t i n g f a c t o r which causes p l a t e l e t aggregation (7). 3 3. PATHWAYS OF PHOSPHATIDYLYLCHOLINE BIOSYNTHESIS IN MAMMALS There are f i v e s e p a r a t e pathways which l e a d t o the f o r m a t i o n o f PC ( 1 ) . They a r e : 1) t h e m a j o r de novo p a t h w a y ( a l s o known as C D P - c h o l i n e p athway) d e s c r i b e d by Kennedy i n t h e 1950's ( 8 ) , which i n v o l v e s the c o n v e r s i o n of c h o l i n e to p h o s p h o c h o l i n e , CDP-c h o l i n e and PC ( F i g u r e 2); 2) the s t e p w i s e m e t h y l a t i o n of phos-p h a t i d y l e t h a n o l a m i n e (PE) which i s . c a t a l y z e d by PE m e t h y l t r a n s -f e r a s e (9) ( F i g u r e 3 ) ; 3) t h e base e x c h a n g e r e a c t i o n i n w h i c h f r e e c h o l i n e can d i s p l a c e e t h a n o l a m i n e or s e r i n e from a p p r o p r i a t e d i a c y l g l y c e r o p h o s p h o l i p i d (10,11); 4) the a c y l a t i o n of l y s o p h o s -p h a t i d y l c h o l i n e (lyso-PC) by acyl-CoA (12,13); and 5) t r a n s a c y l a -t i o n between two m o l e c u l e s of l y s o - P C (14). In r a t l i v e r , about 70% of t h e t o t a l PC i s s y n t h e s i z e d by t h e C D P - c h o l i n e p a t h w a y and t h e r e m a i n i n g 30% by t h e m e t h y l a t i o n p a t h w a y ( 1 5 ) , w h e r e a s t h e o t h e r t h r e e m e chanisms a r e n o t i m p o r t a n t f o r de novo b i o -s y n t h e s i s . 4. ENZYMES OF PHOSPHATIDYLYLCHOLINE BIOSYNTHESIS VIA KENNEDY PATHWAY S t u d i e s on PC m e t a b o l i s m are c o m p l i c a t e d s i n c e the m a j o r i t y o f enzymes i n v o l v e d i n p h o s p h o l i p i d b i o s y n t h e s i s a r e membrane bound or r e q u i r e l i p i d f o r t h e i r a c t i v i t y . The o n l y e x c e p t i o n i s c h o l i n e k i n a s e (EC 2.7.1.32) which c a t a l y z e s the p h o s p h o r y l a t i o n o f c h o l i n e by ATP i n t h e Kennedy path w a y . C h o l i n e k i n a s e i s n e i t h e r a s s o c i a t e d w i t h membranes n o r - r e q u i r e s p h o s p h o l i p i d s f o r a c t i v i t y , and has r e c e n t l y been p u r i f i e d to homogeneity from r a t k i d n e y i n a d i m e r i c form w i t h the m o l e c u l a r w e i g h t of 80,000(16). 4 \ « H H H,C—N—C—C—OH / H H HjC CHOLINE ATP-ADP-H,c CHOLINE KINASE o \ o H H « H 3 C—N—C—C—O—P—0 s / H H I H,C O e PHOSPHOCHOLINE CTP PPi CYTIDYLYLTRANSFERASE N H 2 H3C 0 C \ F F L H H n 11 o H 3 C — N — C — C — O — P — O — P — O — C H , / H H I I I n H 3 C O E O E ' ' U CDP-CHOLINE N OH OH DIGLYCERIDE CHOLINEPHOSPHOTRANSFERASE H 2 C—O—C—R R— C — O — C H C H 3 H H « . / H.C—O—P—O—C-C—N—CHj I H H \ 0 o CH, PHOSPHATIDYLCHOLINE Fig.2 . Kennedy pathway for phosphatidylcho-line biosynthesis from choline. 5 o o O CH,-0-C-R, A d 0 W e ' A d ° H c y O C H 2 - 0 - C - R , R -C-O-C-H O X y • Rj-C-O—C-H O I II I II C H 2 - 0 - P - O C H 2 C H 2 N H 3 C H 2 - 0 - P — O C H 2 C H 2 - N H 2 O- O - C H 3 Phosphatldylethanolamlne N-methylphosphalldylethanolamlne AdoMel AdoHcy o o O C H 2 - 0 - C - R , A d ° H c y A d . ° M e ' O C H 2 - 0 - C - R , R , - C - O - i - H O . ^ <^ R 2 _ C - 0 - C - H O H C H 2 - 0 - P - O C H 2 C H 2 N ( C H 3 ) 3 C H 2 - 0 - - P - O C H 2 C H 2 N - C H 3 I C H 3 O" ° Phosphatidylcholine N,N-<Jlmethylpho*phatidylethanolamine F i g . 3 . 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 by s u c c e s s i v e N - m e t h y l a t i o n o f p h o s p h a t i d y l -e t h a n o l a m i n e . 6 Once c h o l i n e has been p h o s p h o r y l a t e d to form p h o s p h o c h o l i n e th e o n l y f a t e of t h i s p r o d u c t i s f o r PC f o r m a t i o n . The enzyme C T P t p h o s p h o c h o l i n e c y t i d y l y l t r a n s f e r a s e (CT) (EC 2.7.7.15) c a t a l y z e s the f o r m a t i o n of CDP-choline and PPi from phosphocho-l i n e and CTP, the dominant energy form u t i l i z e d i n l i p i d metabo-l i s m . A t t e m p t s at p u r i f i c a t i o n of CT so f a r have been o n l y p a r t i a l l y s u c c e s s f u l . The p r o p e r t i e s of CT i n r a t l i v e r w i l l be d i s c u s s e d i n C h a p t e r I I . The f i n a l s t e p i n the Kennedy pathway i s the f o r m a t i o n of PC and CMP from d i a c y l g l y c e r o l (DG) and CDP-c h o l i n e , c a t a l y z e d by C D P - c h o l i n e : l , 2 - d i a c y l g l y c e r o l c h o l i n e p h o s -phot r a n s f e r a s e (CPT) (EC 2.7.8.2). The a c t i v i t y of CPT i s m o s t l y recovered from E.R. (microsomes) and mitochondria i n mammalians t i s s u e s ( 1 0 ,17 ) , w h i l e a r e p o r t from c h i c k macrophages showed th a t CPT was l o c a t e d on the plasma membrane (18). P a r t i a l p u r i -f i c a t i o n of t h i s membrane bound enzyme has been r e p o r t e d .(19). The enzyme r e q u i r e s magnesium as a c o f a c t o r and m i c r o s o m a l p h o s p h o l i p i d s f o r i t s maximum a c t i v i t y (19). 5. REGULATION OF PHOSPHATIDYLCHOLINE BIOSYNTHESIS VIA KENNEDY PATHWAY Towards the ends of 1970's, a number of a p p r o a c h e s have p r o v i d e d e v i d e n c e t h a t the r a t e of PC b i o s y n t h e s i s v i a the Kennedy pathway i s u s u a l l y determined by the a c t i v i t y of CT (20). I t has been p o s t u l a t e d t h a t the a c t i v i t y of CT i s r e g u l a t e d by t r a n s l o c a t i o n of CT from the c y t o s o l , where i t i s i n a c t i v e , to t h e E.R., where i t i s a c t i v a t e d (20). The c o n c l u s i o n l a r g e l y a r i s e s from the o b s e r v a t i o n s t h a t : 1) CT i s a m b i q u i t o u s i n t h a t 7 i t i s r e c o v e r e d i n both the c y t o s o l i c and m i c r o s o m a l f r a c t i o n s ; 2) t h e c y t o s o l i c CT i s i n a c t i v e and needs p h o s p h o l i p i d f o r a c t i v i t y w h i l e t h e m i c r o s o m a l CT i s a c t i v e and i n s e n s i t i v e t o p h o s p h o l i p i d a c t i v a t i o n ; 3 ) t h e r e are p a r a l l e l changes between PC b i o s y n t h e s i s and t h e a c t i v i t y o f m i c r o s o m a l CT i n d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s . S u p p o r t i v e e v i d e n c e has been o b t a i n e d f r o m s t u d i e s i n a v a r i e t y o f mammalians o r g a n s , l i v e r ( 2 1 , 2 2 ) , h e a r t ( 2 3 ) , l u n g (24) and i n t e s t i n e ( 2 5 ) , as w e l l as i n some c e l l l i n e s (26). The t r a n s l o c a t i o n p r o c e s s of CT may be r e g u l a t e d by r e v e r s i b l e enzyme p h o s p h o r y l a t i o n (27) and by f a t t y a c i d s ( 2 8 ) , y e t p r o o f o f t h i s h y p o t h e s i s i s s t i l l w a i t i n g f o r t h e c o m p l e t e p u r i f i c a t i o n of the enzyme. On t h e o t h e r hand, some e x p e r i m e n t a l and t h e o r e t i c a l e v i -d e n c e s u g g e s t s t h a t t h e f i r s t r e a c t i o n i n t h e d_e novo s y n t h e s i s p a t h w a y c a t a l y z e d by c h o l i n e k i n a s e m i g h t be a r a t e - l i m i t i n g enzyme f o r PC b i o s y n t h e s i s (29,30). 6. THE THESIS INVESTIGATIONS L i v e r i s a major organ f o r p h o s p h o l i p i d b i o s y n t h e s i s and the p h o s p h o l i p i d s s y n t h e s i z e d i n l i v e r p a r t i c i p a t e n o t o n l y i n membrane f o r m a t i o n w i t h i n the organ but a l s o are t r a n s f e r r e d i n t o b i l e o r p l a s m a l i p o p r o t e i n s (31). Hence, many e f f o r t s o f t h i s l a b o r a t o r y i n t h e l a s t decade were f o c u s e d on r e v e a l i n g t h e r e g u l a t o r y mechanisms behind the PC b i o s y n t h e s i s , the r e l a t i o n -s h i p between PC b i o s y n t h e s i s - and l i p o p r o t e i n m e t a b o l i s m , and on p u r i f i c a t i o n of the enzymes i n v o l v e d i n PC b i o s y n t h e s i s from r a t l i v e r . In t h i s t h e s i s , I have i n v e s t i g a t e d the r e g u l a t o r y mecha-8 n i s m o f PC b i o s y n t h e s i s i n c u l t u r e d r a t h e p a t o c y t e s by u s i n g d i p h e n y l s u l f o n e compounds (Chapter I I I ) or v a s o p r e s s i n (Chapter I V ) , and a l s o a t t e m p t e d to p u r i f y CT from r a t l i v e r by d i f f e r e n t t e c h n i q u e s i n c l u d i n g a f f i n i t y chromatography (Chapter I I ) . 9 CHAPTER I I . PURIFICATION OF CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFER-ASE FROM RAT LIVER CYTOSOL BY AFFINITY CHROMATOGRAPHY C T P : p h o s p h o c h o l i n e c y t i d y l y l t r a n s f e r a s e (CT) (E.C.2.7.7.15) c a t a l y z e s the r e v e r s i b l e f o r m a t i o n of CDP-c h o l i n e and pyrophos-p h a t e f r o m p h o s p h o c h o l i n e and CTP, and may c o n t r o l t h e r a t e o f p h o s p h a t i d y l c h o l i n e (PC) b i o s y n t h e s i s (15,32,33,34). Deoxy-CTP c a n a l s o s e r v e as a s u b s t r a t e i n s t e a d o f CTP i n t h i s r e a c t i o n , but the o t h e r n u c l e o s i d e t r i p h o s p h a t e (ATP, GTP, UTP, or TTP) do n o t (35). CT has been d e s c r i b e d as an a m b i q u i t o u s enzyme w h i c h i s a s s o c i a t e d w i t h both the s o l u b l e and m i c r o s o m a l f r a c t i o n s of the t i s s u e homogenates (34). The c y t o s o l i c CT i s o l a t e d from d i f f e r e n t o r g a n s i s i n a c t i v e and needs p h o s p h o l i p i d f o r i t s a c t i v i t y , a l t h o u g h i t i s t h e d o m i n a n t f o r m o f t h e enzyme i n c e l l s . The m i c r o s o m a l CT, h o w e v e r , i s t h e a c t i v e f o r m and i s n o t f u r t h e r a c t i v a t e d by exogenous p h o s p h o l i p i d a l t h o u g h i t i s a l w a y s t h e minor enzyme form w i t h i n c e l l s (20). The r e l a t i v e d i s t r i b u t i o n of t h e enzyme b e t w e e n c y t o s o l and m i c r o s o m e s v a r i e s i n d i f f e r e n t d e v e l o p m e n t a l s t a g e s of i n d i v i d u a l a n i m a l s . In p r e n a t a l l i v e r of r a t , over 70% of the t o t a l CT p r o t e i n i s d e t e c t e d i n the c y t o s o -l i c f r a c t i o n , w h i l e on t h e day o f b i r t h , most o f t h e enzyme a s s o c i a t e s w i t h t h e m i c r o s o m e s (21). A s i m i l a r phenomenon has been o b s e r v e d i n l u n g (36). I n f e t a l r a t s d e l i v e r e d one o r two days p r e m a t u r e l y , CT i s r e d i s t r i b u t e d from c y t o s o l t o microsome so t h a t the m i c r o s o m a l a c t i v i t y i s i n c r e a s e d by 60%, w h i l e t h e r e 10 i s no change i n the t o t a l a c t i v i t y of CT (36). In a d d i t i o n , the r e l a t i v e d i s t r i b u t i o n o f CT between c y t o s o l and microsomes i s dependent on c o n d i t i o n s used i n h o m o g e n i z a t i o n (37 ) . I f a d u l t r a t l i v e r i s h o m o g e n i z e d i n i s o t o n i c s a l i n e (0.145 M N a C l ) , more then 90% of the enzyme a c t i v i t y i s r e c o v e r e d f r o m t h e 170, 000 x g, 1 h r s u p e r n a t a n t ( c y t o s o l ) , w h e r e a s homo-g e n i z a t i o n performed i n d i s t i l l e d water r e s u l t s i n 85% of the a c t i v i t y b e i n g a s s s o c i a t e d w i t h the p e l l e t f r a c t i o n (microsomes) (37) . The s t r a t e g i e s i n CT p u r i f i c a t i o n p u b l i s h e d so f a r have a l l t a k e n advantage of the s o l u b l e p r o p e r t y of the c y t o s o l i c enzyme (38,39,40,41). The o c c u r r e n c e o f CT i n t h e c y t o s o l v a r i e s f r o m organ to organ, o f which l i v e r e x h i b i t s the g r e a t e s t c y t o s o l i c CT a c t i v i t y i n t e r m s o f e i t h e r u n i t s p e r gram o f t i s s u e o r t o t a l u n i t s per organ (42). Thus, l i v e r appears t o be an i d e a l s t a r t -i n g m a t e r i a l f o r p r e p a r a t i v e purposes. However,the p u r i f i c a t i o n o f the c y t o s o l i c CT i s c o m p l i c a t e d s i n c e the s o l u b l e enzyme has a s t r o n g t e n d e n c y t o a g g r e g a t e i n t h e p r e s e n c e o f p h o s p h o l i p i d w h i c h i s u n a v o i d a b l y p r e s e n t i n c y t o s o l due t o t h e b r e a k a g e o f membranes d u r i n g h o m o g e n i z a t i o n . The aggregated CT forms p o l y -mers w i t h d i v e r s e m o l e c u l a r w e i g h t s r a n g i n g between 5 x 10^ and 7 1.3 x 10 , and b e h a v e s l i k e t h e m i c r o s o m a l enzyme w h i c h i s i n s e n -s i t i v e t o p h o s p h o l i p i d a c t i v a t i o n , w h i l e the unaggregated enzyme f o r m i s e s t i m a t e d t o have a m o l e c u l a r w e i g h t o f 2 x 1 0 5 (28). The a g g r e g a t i o n of CT w i t h p h o s p h o l i p i d seems i r r e v e r s i b l e s i n c e a t t e m p t s t o d i s p l a c e t h e enzyme a c t i v i t y f r o m t h e p a r t i c u l a t e m a t e r i a l i s o l a t e d from water homogenates of r a t l i v e r were unsuc-11 c e s s f u l (21,37). On t h e o t h e r hand, p h o s p h o l i p i d a p p e a r s t o s t a b i l i z e the p a r t i a l l y p u r i f i e d CT. The a d d i t i o n o f p h o s p h o l i p i d t o t h e enzyme p r e p a r a t i o n at l a t e r p u r i f i c a t i o n s t a g e s can main-t a i n the enzyme a c t i v i t y which i s l o s t d r a m a t i c a l l y i n the absen-ce of p h o s p h o l i p i d . I n v i t r o s t u d i e s on p a r t i a l l y p u r i f i e d CT f r o m r a t l i v e r c y t o s o l showed t h a t the r e v e r s i b l e r e a c t i o n c a t a l y z e d by CT had an e q u i l i b r i u m c o n s t a n t of 0.80 i n f a v o u r of the f o r m a t i o n of CTP and p h o s p h o c h o l i n e (43). The M i c h a e l i s c o n s t a n t s f o r CTP and C D P - c h o l i n e of the p a r t i a l l y p u r i f i e d CT are the same (Km = 0.21 mM) when a s s a y e d i n t h e p r e s e n c e o f p h o s p h o l i p i d ( 4 3 ) . S i n c e C D P - c h o l i n e i s a s u b s t r a t e f o r o n l y CT and CPT, w h i l e CTP i s a s u b s t r a t e f o r s e v e r a l enzymes i n v o l v e d i n l i p i d b i o s y n t h e s i s , C D P - c h o l i n e s h o u l d be an i d e a l c a n d i d a t e f o r a f f i n i t y c o l u m n p u r i f i c a t i o n of c y t o s o l i c CT. CPT i s a membrane-associated enzyme which i s absent i n c y t o s o l . In t h e p r e s e n t s t u d i e s , two k i n d s o f a f f i n i t y c h r o m a t o -graphy, CDP - c h o l i n e and CTP-Sepharose 4B, have been i n v e s t i g a t e d f o r p u r i f i c a t i o n of the c y t o s o l i c CT from r a t l i v e r . P r e l i m i n a r y r e s u l t s showed c y t o s o l i c CT d i d not have s t r o n g a f f i n i t y t o the C D P - c h o l i n e S e p h a r o s e r e s i n b u t d i d b i n d t o t h e CTP a f f i n i t y column i n the presence of magnesium a c e t a t e . The c o m b i n a t i o n of CTP a f f i n i t y chromatography w i t h ion-exchange t e c h n i q u e s p r o v i d e d about 7 0 - f o l d p u r i f i c a t i o n w i t h a s p e c i f i c a c t i v i t y of about 90 u n i t s per m i l l i g r a m p r o t e i n . The problem of l o s i n g a c t i v i t y a t t h e l a t e r p u r i f i c a t i o n s t a g e s was s t i l l u n s o l v e d . 12 MATERIALS AND METHODS C h e m i c a l s - DE-52 and P - l l i o n i c e xchange r e s i n s were t h e p r o -d u c t s o f Whatman. ATP, CTP, C D P - c h o l i n e , a d i p i c a c i d d i h y d r a z i d e and c h o l i n e k i n a s e were purchased from Sigma. [ m e t h y l - 3 H ] C h o l i n e and Aqueous C o u n t i n g S c i n t i l l a n t (ACS) were o b t a i n e d f r o m t h e R a d i o c h e m i c a l C e n t e r , Amersham. [ m e t h y l - H] P h o s p h o c h o l i n e (10 uci / u m o l ) was s y n t h e s i z e d e n z y m a t i c a l l y from [ m e t h y l - 3 H ] c h o l i n e and ATP w i t h c h o l i n e k i n a s e b a s e d on t h e method of Vance e_t a l . (4 3 ) . 2*, 7'-d i c h l o r o - f l u o r e s c e i n was a p r o d u c t o f E a s t m a n Kodak Co. P r e p a r a t i o n of Rat L i v e r C y t o s o l - Rat l i v e r c y t o s o l was p r e p a r e d b a s e d on t h e method d e s c r i b e d by Choy e_t a_l. (40). W i s t a r r a t s (about 200 g) from the U n i v e r s i t y of B r i t i s h Columbia A n i m a l U n i t w e r e , d e c a p i t a t e d , and the l i v e r s were removed i m m e d i a t e l y . A 25% homogenate o f l i v e r was p r e p a r e d i n i c e - c o l d i s o t o n i c s a l i n e (0.145>M N a C l ) c o n t a i n i n g 0.5 mM p h e n y l m e t h y l s u l f o n y l f l u o r i d e (PMSF) by 5 s t r o k e s i n a P o t t e r - E l v e h j e m homogenizer, and c e n t r i -f u g e d a t 170, 000 x g_ f o r 60 min a t 4°C. The s u p e r n a t a n t was f i l t e r e d t h r o u g h two l a y e r s of cheese c l o t h t o remove the m a j o r i -t y o f t h e l i p i d - r i c h l a y e r w h i c h f l o a t e d a t t h e a i r - s o l u t i o n i n t e r f a c e a f t e r u l t r a c e n t r i f u g a t i o n . CT i n t h i s sample i s v e r y s t a b l e , as f r e e z i n g a t -70°C and thawing at 37°C causes no d e t e c -t a b l e l o s s o f enzyme a c t i v i t y . The s a m p l e was d e s i g n a t e d as c y t o s o l i c f r a c t i o n . 13 D E A E - C e l l u l o s e Chromatography of C y t o s o l i c CT- A l l the p r o c e d u r e s d e s c r i b e d below were performed at 4°C. The l i v e r c y t o s o l s t o r e d a t -70°C was thawed a t 37°C and q u i c k l y c o o l e d i n i c e , as p r o -l o n g e d i n c u b a t i o n i n 37°C w i l l cause a g g r e g a t i o n of the c y t o s o l i c CT. The sample was brought to 30% ammonium s u l f a t e s a t u r a t i o n i n t h e p r e s e n c e of 0.05% T r i t o n X-100, and t h e p r e c i p i t a t e was d i s c a r d e d a f t e r c e n t r i f u g a t i o n a t 10,000 x £ f o r 10 min. The s u p e r n a t a n t was brought to 40% ammonium s u l f a t e s a t u r a t i o n and t h e p r e c i p i t a t e c o l l e c t e d a f t e r c e n t r i f u g a t i o n u nder t h e same c o n d i t i o n s . The p e l l e t was suspended i n 20 mM T r i s - s u c c i n a t e (pH 6.5) b u f f e r c o n t a i n i n g 0.025% T r i t o n X-100, 0.5 mM PMSF and 0.5 mM d i t h i o t h r e i t o l (DTT) to the same volume as t h a t of the o r i g i -n a l c y t o s o l , t h u s e n s u r i n g a l o w c o n d u c t i v i t y . T h i s was c a l l e d t h e 40% f r a c t i o n . The 40% s a m p l e ( a p p r o x . 130 ml) was a p p l i e d t o a DE-52 c o l u m n (3 x 35 cm) w h i c h had been e q u i l i b r a t e d w i t h t h e s a m p l e b u f f e r . S u b s e q u e n t l y , the column was e l u t e d w i t h 50 ml o f b u f f e r and t h e n w i t h a 400 ml N a C l g r a d i e n t r a n g i n g b e t w e e n 0-0.5 M i n t h e same b u f f e r . F r a c t i o n s (9 ml) were c o l l e c t e d and enzyme a c t i v i t y was assayed. The enzyme a c t i v i t y e l u t e d i n the conduc-t i v i t y r e g i o n around 3 mMHO. T h i s p r o t o c o l i s r o u t i n e l y used i n t h i s l a b o r a t o r y and g i v e s r e p r o d u c i b l e r e s u l t s o f a p p o x i m a t e l y 1 5 - f o l d p u r i f i c a t i o n w i t h 20-40% y i e l d r e l a t i v e t o the c y t o s o l i c f r a c t i o n . The enzyme c o u l d be s t o r e d a t -70°C f o r a t l e a s t 2-3 months w i t h n e g l i g i b l e l o s s of enzyme a c t i v i t y . P h o s p h o - C e l l u l o s e Chromatography o f CT- The procedure o f CT p u r i -f i c a t i o n by p h o s p h o - c e l l u l o s e was developed i n t h i s l a b o r a t o r y 14 (Sanghera, J . , u n p u b l i s h e d r e s u l t ) . The enzyme pool e d from DE-52 c h r o m a t o g r a p h y was a p p l i e d t o a P - l l c o l u m n (2.5 x 8 cm) w h i c h had been e q u i l i b r a t e d w i t h t h e s a m p l e b u f f e r as used i n DE-52 column. The b u l k o f p r o t e i n was e l u t e d w i t h 80 ml of 0.2 M NaCl and t h e enzyme was removed w i t h a 100 ml g r a d i e n t o f 0-0.6 M N a C l i n t o 4.5 ml f r a c t i o n s . The enzyme peak was l o c a t e d i n t h e c o n -d u c t i v i t y r a n g e b e t w e e n 7 and 8.5 mMHO. CT i n t h i s s a m p l e i s e x t r e m e l y u n s t a b l e and s h o u l d be s u b j e c t e d t o t h e s u c c e s s i v e p u r i f i c a t i o n s t e p as q u i c k l y as p o s s i b l e . Enzyme A s s a y - The measurement o f CT a c t i v i t y was e s s e n t i a l l y based on the methods d e s c r i b e d by Vance et. al.. (43). The r e a c t i o n m i x t u r e c o n t a i n e d i n a f i n a l v o l u m e of 100 u l : 0.2 mg o f t o t a l r a t l i v e r p h o s p h o l i p i d ; 10 nmol of o l e a t e ; 7.5 umol o f T r i s -s u c c i n a t e , pH 6.5; 0.75 umol o f magnesium a c e t a t e ; 0.2 umol of CTP; 0.15 umol of [ m e t h y l - H] phosphochol i n e (10 u c i / u m o l ) and an a p p r o p r i a t e amount of enzyme p r o t e i n (2-40 ug). The r e a c t i o n was performed a t 37°C f o r 15-30 min, and stopped by i m m e r s i o n of the t u b e s i n b o i l i n g w a t e r f o r 2 min. The p r o t e i n p r e c i p i t a t e was p e l l e t e d by c e n t r i f u g a t i o n a t 2,500 rpm ( W e s t e r n H-103N c e n -t r i f u g e ) f o r 5 m i n , and 40 u l o f t h e s u p e r n a t a n t was a p p l i e d t o S i l i c a G-60 p l a t e s (Merck) w i t h 3 x 10 cm per l a n e . C D P - c h o l i n e (0.1 mg) and p h o s p h o c h o l i n e (0.6 mg) c a r r i e r s were a p p l i e d f o r each l a n e . The TLC p l a t e s were developed i n CH3OH /0.6% NaCl/NH 3 (10:10:1; v:v:v) f o r 50 min. C D P - c h o l i n e was v i s i b l e under short-wave UV l i g h t a f t e r s p r a y i n g w i t h 2 ' , 7 ' - d i c h l o r o f l u o r e s c e i n (0.01% i n CH^OH), and s c r a p e d f r o m t h e p l a t e s i n t o a p l a s t i c s c i n t i l l a t i o n v i a l w i t h 0.5 ml o f w a t e r and 4.5 ml o f ACS f l u i d . 15 The samples were counted f o r r a d i o a c t i v i t y w i t h 20-30% c o u n t i n g e f f i c i e n c y . The r e g i o n o f p h o s p h o c h o l i n e on t h e p l a t e s was v i s u a l i z e d i n an i o d i n e vapour i n o r d e r to c o n f i r m the s e p a r a t i o n o f C D P - c h o l i n e from p h o s p h o c h o l i n e . One u n i t of enzyme a c t i v i t y i s d e f i n e d as one umole of CDP-choline formed per min. P r o t e i n A s s a y - The Bio-Rad assay based on the method o f B r a d f o r d (44) was used f o r the e s t i m a t i o n of p r o t e i n c o n c e n t r a t i o n . B i o -Rad s t o c k reagent was d i l u t e d i n d i s t i l l e d water (4:13; v : v ) , and 2.5 ml o f t h e d i l u t e d r e a g e n t was added t o 0.5 ml o f p r o t e i n sample w i t h IgG (Bio-Rad Standard) as the p r o t e i n s t a n d a r d . The a s s a y was l i n e a r i n t h e range b e t w e e n 5-80 ug o f p r o t e i n . F o r some a s s a y s , t h e m i c r o a s s a y p r o c e d u r e was a d o p t e d t o d e t e r m i n e p r o t e i n c o n c e n t r a t i o n s l e s s then 10 ug/ml. In t h i s c a s e , 0.2 ml of the c o n c e n t r a t e d Bio-Rad s t o c k reagent was added to 0.8 ml of p r o t e i n s a m p l e . The a b s o r b a n c e was r e a d a t 595 nm a f t e r 15 min but b e f o r e 60 min. P r e p a r a t i o n of T o t a l Rat L i v e r P h o s p h o l i p i d - T o t a l r a t l i v e r p h o s p h o l i p i d was e x t r a c t e d from the 170,000 x g x 1 hr p e l l . e t of r a t l i v e r homogenate by t h e B l i g h and Dyer method ( 4 5 ) . The p e l l e t was re-homogenized i n 80 ml of water, and mixed w i t h 100 ml o f c h l o r o f o r m and 200 ml o f m e t h a n o l by s t i r r i n g f o r 1 h r a t room t e m p e r a t u r e . The m i x t u r e was c e n t r i f u g e d at 8,000 rpm ( i n a S o r v a l l Type GSA r o t o r ) f o r 20 min, and the s u p e r n a t a n t was f i l -t e r e d t h r o u g h g l a s s w o o l . The f i l t r a t e was brought t o the f i n a l r a t i o of CHC1 3/CH 30H/H 20 to 1:1:0.8 (v:v:v) by the a d d i t i o n of 80 ml o f w a t e r and 100 ml o f c h l o r o f o r m . The f i l t r a t e was c e n t r i -16 f u g e d u n d e r t h e same c o n d i t i o n , and t h e c h l o r o f o r m p h a s e was t r a n s f e r r e d t o a p r e w e i g h e d r o u n d - b o t t o m f l a s k t o remove t h e c h l o r o f o r m by r o t a r y e v a p o r a t i o n . The f l a s k was c o o l e d a t -20 °C f o r 20 min and r i n s e d w i t h i c e - c o l d a c e t o n e t o e x t r a c t t h e n e u t r a l l i p i d s . The r e m a i n i n g acetone was evaporated under n i t r o -gen. The p h o s p h o l i p i d was d i s s o l v e d i n c h l o r o f o r m a t a f i n a l c o n c e n t r a t i o n of 20 mg/ml and s t o r e d at -20°C. 17 RESULTS P r e p a r a t i o n o f C D P - C h o l i n e S e p h a r o s e 4B A m o d i f i c a t i o n o f a method o r i g i n a l l y used f o r t h e c o u p l i n g o f C D P - d i g l y c e r i d e t o S e p h a r o s e 4B (46,47) was used t o l i n k c o v a l e n t l y an o x i d i z e d d e r i v a t i v e of C D P - c h o l i n e to the g e l . The scheme of the r e a c t i o n s a r e summarized i n Fig.4. 1) P r e p a r a t i o n o f Cyanogen Bromide A c t i v a t e d Sepharose 4B. Cyanogen b r o m i d e a c t i v a t e d S e p h a r o s e 4B was p r e p a r e d by t h e method d e s c r i b e d by C u a t r e c a s a s (48). S e p h a r o s e 4B (50 ml) was p r e w a s h e d w i t h 1 M N a C l and H 20 and f i n a l l y s u s p e n d e d i n 50 ml o f H2O. Ground CNBr powder (10 g) was p o u r e d i n t o t h e r e s i n s u s p e n s i o n and mixed q u i c k l y . The r e a c t i o n was m a i n t a i n e d a t pH 11 w i t h 6 N NaOH, and t h e t e m p e r a t u r e was c o n t r o l l e d b e l o w 20°C by adding i c e . The r e a c t i o n t i m e was about 15 min and stopped by t h e a d d i t i o n o f i c e t o t h e m i x t u r e . The a c t i v a t e d r e s i n was washed q u i c k l y w i t h i c e - c o l d 0.1 M N a 2 C 0 3 (pH 9.5) and f i n a l l y resuspended i n the same s o l u t i o n . 2) P r e p a r a t i o n o f S e p h a r o s e 4B A d i p i c A c i d D i h y d r a z i d e . A d i p i c a c i d d i h y d r a z i d e (4.5 g) was mixed i n t o the CNBr a c t i v a t e d r e s i n , and the r e a c t i o n was performed f o r 17 hr a t 4°C by g e n t l y s t i r r i n g the s u s p e n s i o n . The r e s i n was washed e x t e n s i v e l y w i t h 1M NaCl°and t h e n 0.1 M s o d i u m a c e t a t e (pH 5.0), and t e s t e d f o r t h e p r e s e n c e o f c o v a l e n t l y b o u n d h y d r a z i d e as d e s c r i b e d by C u a t r e c a s a s (4 8 ) . The r e s i n w i t h l i g a n d t u r n e d a d a r k r e d i n s a t u r a t e d s o d i u m b o r a t e c o n t a i n i n g s e v e r a l d r o p s of 3% 2,4,6-18 NH, N 0 0 J. (CH_) o-N-CH.-CH o-.0-P-0-p'-0-CH o  3 3 OH OH 1 OH OH NalO, -CH„ BASE HC CH 0 0 BrCN ACTIVATED SEPHAROSE 4B H H 0 0 H H HN-N-C-(CH 2) 4-C-N-NH H O 0 H H H0-N-N-C'-(CH0) ,-C-N-N-C-O-SEPHAROSE 4B NH l2'4 0 0 CHOLINE-P-O-P-O-CH OH OH CH BASE HC C C HO N OH NH CO (9 H 2>4 9 ° NH NH C=NH i 0 SEPHAROSE 4B F i g 4. Scheme of The Sy n t h e s i s of CDP-Choline Sepharose 4B„ 19 t r i n i t r o b e n z e n e s u l f a t e , w h i l e u n s u b s t i t u t e d r e s i n was p a l e y e l l o w . 3) O x i d a t i o n o f C D P - C h o l i n e . C D P - c h o l i n e (0.25 mmol; i.e.,5 umol p e r ml o f r e s i n ) was d i s s o l v e d i n 25 ml o f H 20, and the r i b o s y l h y d r o x y l s were o x i d i z e d to aldehydes by the a d d i t i o n o f N a l O ^ (0.25 mmol). The m i x t u r e was a d j u s t e d t o pH 5.55 w i t h a c e t i c a c i d . The r e a c t i o n was c a r r i e d out i n the dark f o r 2 hr a t room t e m p e r a t u r e and t h e n o v e r n i g h t a t 4°C. F o r m a t i o n o f t h e d i a l d e h y d e was d e t e r m i n e d by TLC on S i l i c a g e l w i t h the s o l v e n t CH 3OH/ 0.6% N a C l / N H 3 (10/10/1; v/v/v) . The o x i d i z e d p r o d u c t had an i n c r e a s e d R f (0.73) r e l a t i v e t o C D P - c h o l i n e (0.59). [ m e t h y 1 -J H ] C D P - c h o l i n e was used t o e s t i m a t e the o x i d a t i o n e f f i c i e n c y . 4) C o u p l i n g of O x i d i z e d CDP-Choline t o Sepharose 4B A d i p i c  A c i d D i h y d r a z i d e . The o x i d i z e d C D P - c h o l i n e s o l u t i o n and t h e S e p h a r o s e 4B a d i p i c a c i d d i h y d r a z i d e were c o m b i n e d i n 0.1 M sodium a c e t a t e (pH 5.0) t o g i v e a f i n a l volume of 90 ml and mixed o v e r n i g h t a t 4°C. The r e s i n was washed w i t h 0.1 M sodium a c e t a t e (pH 5.0) c o n t a i n i n g 0.5 M KC1 f o l l o w e d by H 20 and f i n a l l y w i t h t h e b u f f e r t o be used f o r c h r o m a t o g r a p h y . A n a l y s i s o f t h e CDP-c h o l i n e c o n t e n t ( d e t e r m i n a t i o n of the r a d i o a c t i v i t y of [ m e t h y l -3H] C D P - c h o l i n e on t h e r e s i n ) i n d i c a t e d a b o u t 1.7 umol o f CDP-c h o l i n e per ml of g e l . P r e p a r a t i o n o f C T P - S e p h a r o s e 4B. CTP-Sepharose 4B was p r e p a r e d s i m i l a r l y t o t h a t o f C D P - c h o l i n e S e p h a r o s e 4B w i t h s i m i l a r c o u p l i n g e f f i c i e n c y (2.0 umol of CTP per ml of g e l ) . 20 S e p a r 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 by CDP-Choline Sepharose 4B  Chromatography. The f o l l o w i n g p r o c edures were c a r r i e d out at 4°C. C y t o s o l i c CT c o l l e c t e d by s t e p w i s e ammonium s u l f a t e f r a c t i o n a t i o n (30-40%) was p u r i f i e d by D E A E - c e l l u l o s e c h r o m a t o g r a p h y ( F i g . 5 ) and the s p e c i f i c a c t i v i t y of the enzyme a f t e r the chromatography was u s u a l l y 10-20 u n i t s / m g . A f r a c t i o n o f t h e enzyme (12-15ml) a f t e r D E A E - c e l l u l o s e chromatography was brought to a f i n a l c on-c e n t r a t i o n of 14 mM magnesium a c e t a t e and then a p p l i e d to a CDP-c h o l i n e Sepharose 4B column (1.4 x 15 cm) which had been e q u i l i -b r a t e d w i t h 20 mM T r i s - s u c c i n a t e (pH 6.5) b u f f e r c o n t a i n i n g 0.025% T r i t o n X-100, 0.5 mM PMSF, 0.5 mM DTT and 14 mM magnesium a c e t a t e . The f l o w o f t h e c o l u m n was s t o p p e d f o r 30 min t o a c h i e v e e q u i l i b r i u m between enzyme and l i g a n d of the r e s i n b e f o r e s t a r t -i n g t o wash t h e c o l u m n . The c o l u m n was e l u t e d w i t h t h e b u f f e r u n t i l the f i r s t p r o t e i n peak d i m i n i s h e d (monitored by r e c o r d i n g t h e a b s o r b a n c e a t 280 nm) , and t h e n w i t h 0.5 M N a C l i n t h e same b u f f e r . The chromatogram i n F i g u r e 6 shows t h a t l e s s then 15% of th e t o t a l enzyme a c t i v i t y loaded was r e t a i n e d by the column. The a c t i v i t y peak e l u t e d w i t h 0.5 M N a C l had a s p e c i f i c a c t i v i t y o f 33 u n i t s / m g and showed 3 - f o l d p u r i f i c a t i o n (The s p e c i f i c a c t i v i t y of o r i g i n a l enzyme sample b e f o r e CDP-choline Sepharose 4B chroma-t o g r a p h y was 10 u n i t s / m g i n t h i s p a r t i c u l a r e x p e r i m e n t ) . However, s i n c e t h e b u l k o f enzyme d i d n o t b i n d t o t h e C D P - c h o l i n e r e s i n , the t o t a l a c t i v i t y r e c o v e r y was o n l y 3.5%. P r o l o n g e d i n c u b a t i o n o f t h e s a m p l e w i t h i n t h e c o l u m n f o r up t o 1 h r d i d n o t i m p r o v e enzyme b i n d i n g t o t h e c o l u m n . I n an a t t e m p t t o i m p r o v e t h e a f f i n i t y o f enzyme t o t h e C D P - c h o l i n e l i g a n d , t h e s a m p l e was 21 FRACTION NUMBER Fig.5. D E A E - c e l l u l o s e Chromatography of Rat L i v e r C y t o s o l i c  C y t i d y l y l t r a n s f e r a s e . The 40% ammonium s u l f a t e p r e c i p i t a t e of ra t l i v e r c y t o s o l was suspended i n 20 mM T r i s - s u c c i n a t e (pH 6.5) c o n t a i n i n g 0.025% T r i t o n X-100, 0.5 mM PMSF and 0.5 mM DTT. Approx. 130 ml of the sample was a p p l i e d to a DE-52 column (3 x 35 cm) which had been e q u i l i b r a t e d w i t h the sample b u f f e r . The sample-loaded column was washed with 50 ml of the sample and then e l u t e d with a 0-0.5 N NaCl g r a d i e n t i n the same b u f f e r i n t o 9 ml f r a c t i o n s . 22 5 10 15 20 25 30 35 FRACTION NUMBER Fig.6. CDP-Choline Sepharose 4B Chromatography of Rat L i v e r  C y t o s o l i c C y t i d y l y l t r a n s f e r a s e . The enzyme f r a c t i o n s (12-15 ml) pooled from DE-52 column were brought to f i n a l c o n c e n t r t i o n of 14 mM magnesium a c e t a t e and then a p p l i e d to a CDP-choline column (1.4 x 13 cm) which had been e q u i l i b r a t e d with 20 mM T r i s - s u c c i -nate(pH 6.5) c o n t a i n i n g 0.025% T r i t o n X-100, 0.5 mM PMSF, 0.5 mM DTT and 14 mM magnesium acetate. The column was washed with the same b u f f e r u n t i l A^gg d i m i n i s h e d and then e l u t e d w i t h 0.5 N NaCl. F r a c t i o n s i n "the volume of 3.5 ml were c o l l e c t e d . The column was regenerated by washing with 1 N NaCl. 23 brought t o a f i n a l c o n c e n t r a t i o n of 0.2 mg/ml t o t a l r a t l i v e r p h o s p h o l i p i d b e f o r e b e i n g a p p l i e d to the column. However, the p r o t e i n and a c t i v i t y p r o f i l e of the chromatogram was s i m i l a r to those observed i n the absence of p h o s p h o l i p i d (data not shown). Separation of C y t i d y l y l t r a n s f e r a s e by CTP-Sepharose 4B Chromato- graphy. In c o n t r a s t to CDP-choline Sepharose 4B column, the CTP-Sepharose 4B r e s i n showed a f a i r l y strong a f f i n t y f o r the enzyme. In order to improve on the p u r i f i c a t i o n , the enzyme p r e p a r a t i o n was f r a c t i o n a t e d by a p h o s p h o - c e l 1 u l o s e column (pH 6.5) w hich f o l l o w e d ammonium s u l f a t e p r e c i p i t a t i o n and DEA E - c e l l u l o s e chro-matography. F i g u r e 7 r e p r e s e n t s a t y p i c a l chromatogram of CT p u r i f i c a t i o n by ph o s p h o - c e l l u l o s e column. The enzyme pooled from p h o s p h o - c e l l u l o s e column was d i a l y z e d a g a i n s t 20 mM T r i s -s u c c i n a t e b u f f e r (pH 6.5) c o n t a i n i n g 0.025% T r i t o n X-100, 0.5 mM PMSF and 0.5 mM DTT to l o w e r the sample c o n d u c t i v i t y t o 3 mMHO. This procedure caused a severe l o s s of enzyme a c t i v i t y , thus only one t h i r d of the a c t i v i t y was recovered a f t e r d i a l y s i s (see Table 1). The d i a l y z e d sample (30 ml) was brought to a f i n a l c o n c e n -t r a t i o n of 14mM magnesium a c e t a t e , and a p p l i e d to a CTP-Sepharose 4B column (1.5 x 15 cm) which had been e q u i l i b r a t e d w i t h the sample b u f f e r . A f t e r the bulk of the p r o t e i n was e l u t e d with 0.04 M NaCl, the enzyme was removed by a 200 ml g r a d i e n t of 0.04-0.4 M NaCl i n the same b u f f e r (Figure 8). The chromatogram shows that t h e r e was no d e t e c t a b l e enzyme a c t i v i t y i n the f i r s t p r o t e i n e l u a n t , whereas the a c t i v i t y was e l u t e d at a low c o n d u c t i v i t y range (2-3 mMHO) f o l l o w e d by a l o n g t a i l . The r e s u l t s o f a 24 F i g . 7 . P h o s p h o - C e l l u l o s e Chromatography of Rat L i v e r Cyto- s o l i c C y t i d y l y l t r a n s f e r a s e . The enzyme sample (40 ml) pooled from DE-52 column was a p p l i e d to a P - l l column (2.5 x 8 cm) which had been e q u i l i b r a t e d with 20 mM T r i s - s u c c i n a t e (pH 6.5) c o n t a i n i n g 0.025% T r i t o n X-100, 0.5 mM PMSF and 0.5 mM DTT. The column was washed w i t h 80 ml of 0.2 M NaCl and then e l u t e d w i t h 100 ml of 0-0.6 M NaCl g r a d i e n t i n the same b u f f e r i n t o 4.5 ml f r a c t i o n s . 25 F R A C T I O N Fig.8. CTP-Sepharose 4B Chromatography of Rat L i v e r Cytoso- l i c C y t i d y l y l t r a n s f e r a s e . The enzyme f r a c t i o n s (30 ml) from p h o s p h o - c e l l u l o s e column were d i a l y z e d a g a i n s t 20 mM T r i s -s u c c i n a t e (pH 6.5) c o n t a i n i n g 0.025 % T r i t o n X - 1 0 0 , 0.5 mM PMSF and 0.5 mM DTT f o r 1 hr and brought to the f i n a l c o n c e n t r a t i o n of 14 mM magnesium acetate before a p p l i c a t i o n to a CTP-Sepharose 4B column (1.5 x 15 cm) which had been e q u i l i b r a t e d with the d i a l y -s i s b u f f e r c o n t a i n i n g 14 mM magnesium acetate. The sample-loaded column was washed w i t h 0.04 M NaCl i n the sample b u f f e r and then e l u t e d w i t h 200 ml of 0.04-0.4 M NaCl g r a d i e n t i n t o 4 ml f r a c -t i o n s . 26 p u r i f i c a t i o n scheme are summarized i n Table 1. The CTP a f f i n i t y chromatography e x h i b i t e d a t h r e e - f o l d p u r i f i c a t i o n and 10% y i e l d r e l a t i v e to the enzyme sample a p p l i e d to the column. However, due to the l o s s o f a c t i v i t y i n the d i a l y s i s s t e p , the o v e r a l l p u r i f i c a t i o n was not improved. Table 1. P u r i f i c a t i o n of CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e from r a t l i v e r c y t o s o l T o t a l S p e c i f i c P u r i f i -Volume P r o t e i n a c t i v i t y a c t i v i t y Recovery c a t i o n F r a c t i o n (ml) (mg) (units) (units/mg) (%) ( f o l d ) C y t o s o l 128 3942 5558 1.4 100 1 ( N H 4 ) 2 S 0 4 40% 130 621 3916 6.3 70.5 4.5 DE-52 45 54 1165 21.6 21.0 15. 4 P-11 31 5 471 94.2 8.5 67. 3 P-11(after d i a l y s i s ) 31 5 151 30.2 2.7 21.6 CTP-Sepha-rose 4B 16 0.14 13 92.9 0.2 66. 4 27 DISCUSSION The major p u r p o s e o f t h i s work was to f i n d a method f o r p u r i f i c a t i o n of CT i n the s o l u b l e , u n a g g r e g a t e d , low m o l e c u l a r w e i g h t form. S i n c e t h e r e i s alw a y s some p h o s p h o l i p i d i n the c y t o s o l f r a c t i o n of 100,000 x g x 1 hr rat l i v e r homogenate, pa r t of the CT has a s s o c i a t e d w i t h l i p i d s p o n t a n e o u s l y and t h e r e are two forms of CT p r e s e n t i n the c y t o s o l (40). These two forms o f c y t o s o l i c CT can be d i s t i n g u i s h e d by t h e i r d i f f e r e n t s e n s i t i v i t y to p h o s p h o l i p i d a c t i v a t i o n , and can a l s o be separated by stepwise ammonium s u l f a t e p r e c i p i t a t i o n (40). Most of the a g g r e g a t e d CT was p e l l e t e d with 30% ammonium s u l f a t e , whereas the unaggregated CT i s p r e c i p i t a t e d by 40% ammonium s u l f a t e . In o r d e r to o b t a i n the unaggregated form, previous s t u d i e s done i n t h i s l a b o r a t o r y found t h a t the a d d i t i o n of 0.5% T r i t o n X-100 i n the c y t o s o l d u r i n g ammonium s u l f a t e p r e c i p i t a t i o n s i g n i f i c a n t l y improved the r e c o v e r y (70-85%) of the unaggregated form from c y t o s o l , and caused no l o s s of a c t i v i t y . The presence of 0.025% T r i t o n X-100 i n the b u f f e r s used i n the r e s t of the p u r i f i c a t i o n p r o c e d u r e seemed to be h e l p f u l i n p r e v e n t i n g enzyme from a g g r e g a t i n g and s t a b l i z i n g the enzyme at e a r l y p u r i f i c a t i o n stages. The two ion-exchange chromatography techniques (DEAE-cellu-l o s e and p h o s p h o - c e l l u l o s e ) used i n t h i s l a b o r a t o r y have been s u c c e s s f u l i n the p a r t i a l p u r i f i c a t i o n of una g g r e g a t e d CT from r a t l i v e r c y t o s o l . F i g u r e 5 and F i g u r e 7 r e p r e s e n t the t y p i c a l chromatograms of these two procedures r e s p e c t i v e l y . The advan-28 tage of using D E A E - c e l l u l o s e chromatography as the f i r s t p u r i f i -c a t i o n step i s the high sample c a p a c i t y of the column, and a l s o , the enzyme p r e p a r a t i o n i s q u i t e s t a b l e i n the presence of 0.025* T r i t o n X-100 and no s i g n i f i c a n t aggregation occurs. The phospho-c e l l u l o s e chromatography step provided a high f o l d p u r i f i c a t i o n and o f f e r e d a h i g h e r r e c o v e r y of a c t i v i t y (Table 1). But the enzyme e l u t e d from the column c o n t a i n e d h i g h c o n c e n t r a t i o n of s a l t hence c o u l d not be subjected d i r e c t l y to the a f f i n t y chroma-tography without d i a l y s i s which, however, caused a severe l o s s of enzyme a c t i v i t y . The l o s s of a c t i v i t y a f t e r p h o s p h o - c e l l u l o s e chromatography i s p o s s i b l y due to the almost complete removal of p h o s p h o l i p i d s from the enzyme t h r o u g h the two s u c c e s i v e i o n -exchange columns. The a d d i t i o n of t o t a l r at l i v e r p h o s p h o l i p i d to the sample can m a i n t a i n the enzyme a c t i v i t y , although the r e l a -t i o n s h i p between p h o s p h o l i p i d and the enzyme i s u n c l e a r . S i n c e p h o s p h o l i p i d c a u s e s the a g g e g a t i o n o f CT and t h e r e f o r e a f f e c t s the f u r t h e r p u r i f i c a t i o n of the unaggregated form of the enzyme, so we c o u l d not use p h o s p h o l i p i d as a s t a b i l i z e r . C h o o s i n g a detergent to o f f e r an a p p r o p r i a t e environment f o r m a i n t a i n i n g CT f u n c t i o n a l s t r u c t u r e and a l s o prevent CT from aggregating remains an u n s o l v e d p r o b l e m . So f a r , T r i t o n X-100 appears the b e s t candidate f o r t h i s purpose. An i n i t i a l i n v e s t i g a t i o n on CT p u r i f i c a t i o n by a f f i n i t y c h r o m a t o g r a p h y was r e p o r t e d from t h i s l a b o r a t o r y 10 y e a r s ago (41) when glyc e r o p h o s p h o c h o l i n e was c o v a l e n t l y l i n k e d to epoxy-a c t i v a t e d Sepharose 6B. The technique was not e a s i l y reproduced s i n c e the r e a c t i o n of glycerophosphocholine c o u p l i n g to the r e s i n 29 was d i f f i c u l t to m a n i p u l a t e . The a f f i n i t y columns p r e p a r e d f o r t h i s t h e s i s used the n a t u r a l s u b s t r a t e s of CT as l i g a n d s . Hence we adopted a method to l i n k c o v a l e n t l y CTP or C D P - c h o l i n e to Sepharose v i a a r i b o s y l group i n o r d e r to expose both c y t i d i n e and phosphate (or p h o s p h o c h o l i n e ) m o i e t i e s to the enzyme. The p u t a t i v e s t r u c t u r e of the a f f i n i t y r e s i n ( F i g u r e 4) might be the b e s t r e p r e s e n t a t i o n based on the s t u d i e s done by Hanske e t a 1. on the s t r u c t u r e of AMP and c a r b o x y l i c a c i d hydrazides (49). The d i f f e r e n c e i n b i n d i n g a f f i n i t y f o r c y t o s o l i c CT to CTP-and CDP-choline-Sepharose r e s i n i s not explained. Obviously, the o x i d i z e d n u c l e o t i d e d e r i v a t i v e s have a l t e r e d t h e i r o r i g i n a l r i b o s y l s t r u c t u r e i n the c o u p l i n g r e a c t i o n . These s t r u c t u r a l changes might a c c o u n t f o r the f a i l u r e of CT to b i n d to the CDP-c h o l i n e r e s i n , but not f o r the binding to the CTP r e s i n . Choy et a l . ( 4 0 ) s p e c u l a t e d t h a t the a b i l i t y of the polymer form and u n a g g r e g a t e d form of the enzyme to b i n d to an a f f i n i t y column with g l y cerophosphocholine as l i g a n d was d i f f e r e n t w h i le the u n a g g r e g a t e d form was r e t a i n e d by the a f f i n i t y m a t e r i a l , the polymer form was not. The o b s e r v a t i o n with the CDP-choline column experiment that a d d i t i o n of p h o s p h o l i p i d to the c y t o s o l i c enzyme sample d i d not improve the enzyme b i n d i n g to the a f f i n i t y m a t e r i a l might be due to the a g g r e g a t i o n e f f e c t on the enzyme caused by p h o s p h o l i p i d . The M i c h a e l i s c o n s t a n t of p a r t i a l l y p u r i f i e d CT f o r C D P - c h o l i n e (Km = 0.21 mM) was o b t a i n e d i n the presence of p h o s p h o l i p i d (40) because of no d e t e c t a b l e a c t i v i t y of the enzyme i n the absence of p h o s p h o l i p i d . Thus, the Km value r e f l e c t e d the a f f i n i t y o f the a g g r e g a t e d form of the enzyme to CDP-choline. L i t t l e i s known about the a f f i n i t y of unaggregated 30 enzyme f o r CDP-choline. However, the r e s u l t from the CDP-choline Sepharose 4B chromatography shows that both the unaggregated and aggregated enzyme forms have no strong a f f i n i t y to CDP-choline. P r e l i m i n a r y s t u d i e s showed that the CTP-Sepharose prepared as d e s c r i b e d p r e v i o u s l y (46,47) was of b e n e f i t to the c y t o s o l i c CT p u r i f i c a t i o n , w h i l e the commercially a v a i l a b l e CTP a f f i n i t y r e s i n (Sigma) d i d not r e t a i n the enzyme. Magnesium i o n s a r e re q u i r e d f o r a c t i v i t y (35), and are a l s o necessary f o r promoting the enzyme a f f i n i t y to the r e s i n . The mechanism of the i n t e r a c -t i o n between the enzyme and CTP i s unclear. One of the p r o b a b i l i -t i e s i s that there i s a hydrophobic i n t e r a c t i o n i n v o l v e d , because the enzyme can be e l u t e d from the a f f i n t y r e s i n by the b u f f e r with a lower i o n i c s t r e n g t h (2-3 mMHO) than that of the o r i g i n a l sample (3 mMHO) (Figure 8). As e n c o u n t e r e d by o t h e r groups (38,39), the s e v e r e l o s s o f a c t i v i t y i n l a t e r p u r i f i c a t i o n s t a g e s i s s t i l l a prob l e m . The p a r t i a l l y p u r i f i e d enzyme w i t h s p e c i f i c a c t i v i t y of about 100 u n i t s per mg p r o t e i n was extremely unstable and more than 50% of the a c t i v i t y was l o s t w i t h i n 3 hr a t 4°C d u r i n g the d i a l y s i s step. Further s t u d i e s are needed to d i s c o v e r a m a t e r i a l ( s ) other than p h o s p h o l i p i d which not on l y maintains the enzyme a c t i v i t y , but a l s o prevents aggregation of the enzyme. In c o n c l u s i o n , the CTP-sepharose 4B a f f i n i t y chromatography may be an encouraging technique f o r c y t o s o l i c CT p u r i f i c a t i o n i n combination with other ion-exchange chromatographies. 31 CHAPTER I I I . EFFECTS OF DIPHENYLSULFONE COMPOUNDS ON THE META-BOLISM OF [METHYL- 3H]CHOLINE IN RAT HEPATOCYTES AND HELA CELLS The b i o s y n t h e s i s of PC has been e x t e n s i v e l y s t u d i e d i n hepatocytes, because the PC s y n t h e s i z e d i n l i v e r i s not o n l y the major membrane component w i t h i n c e l l s but a l s o s e c r e t e d as a c o n s t i t u e n t of b i l e or plasma l i p o p r o t e i n s . Rat l i v e r has been reported to be a major organ f o r plasma l i p o p r o t e i n s y n t h e s i s as 80% of the l i p o p r o t e i n p o o l i n r a t plasma o r i g i n a t e s from the l i v e r (50) and the r e m a i n d e r i s from i n t e s t i n e . The abundant forms of l i p o p r o t e i n s e c r e t e d by l i v e r are e i t h e r VLDL or HDL (51,52,53), i n which p h o s p h o l i p i d accounts f o r around 20% of the m a t e r i a l by w e i g h t (54). The p h y s i o l o g i c a l f u n c t i o n of the p r o -t e i n components i n serum l i p o p r o t e i n s are somewhat understood. The a b n o r m a l i t y of some l i p o p r o t e i n a p o p r o t e i n s (55) or the d e f i c i e n c y of l i p o p r o t e i n r e c e p t o r s on membranes of the t a r g e t c e l l s causes c i r c u l a t o r y d i s e a s e s . The f u n c t i o n of p h o s p h o l i p i d s i n the l i p o p r o t e i n i s p r o b a b l y m a i n l y s t r u c t u r a l . However, the b i o l o g i c a l r o l e of p h o s p h o l i p i d s i n the p r o c e s s of l i p o p r o t e i n s e c r e t i o n i s of i n t r e s t s i n c e the s e c r e t i o n of p r o d u c t s from c e l l s must i n v o l v e s processes mediated by membrane components. S t u d i e s on the s y n t h e s i s and a s s e m b l y of plasma l i p o p r o t e i n s showed t h a t l i p i d and apoproteins were assembled and g l y c o s y l a t e d i n G o l g i a p p a r a t u s b e f o r e b e i n g s e c r e t e d i n t o plasma (57). S e c r e t i o n of newly s y n t h e s i z e d l i p o p r o t e i n s from l i v e r i n t o p lasma was s t o p p e d v e r y q u i c k l y a f t e r the a d m i n i s t r a t i o n of 32 p u r o m y c i n , a s p e c i f i c i n h i b i t o r of p r o t e i n s y n t h e s i s (54), but the h e p a t i c s e c r e t i o n of l i p o p r o t e i n s was not dependent on p r i o r p r o t e i n g l y c o s y l a t i o n (58). However, l i t t l e i s known about the b i o l o g i c a l r o l e of p h o s p h o l i p i d s i n the l i p o p r o t e i n s e c r e t i o n p r o c e s s . One a p p r o a c h f o r s t u d y i n g the c o n t r o l of the l i p o p r o -t e i n s e c r e t i o n i s to c o r r e l a t e changes i n b i o s y n t h e s i s of PC, the major p h o s p h o l i p i d component of l i p o p r o t e i n s , with l i p o p r o t e i n s e c r e t o r y a c t i v i t y . T h i s could be accomplished by using s p e c i f i c , nontoxic i n h i b i t o r s of PC b i o s y n t h e s i s . A s e r i e s of d i p h e n y l s u l f o n e compounds, dapsone and i t s d e r i -v a t i v e s , were reported to 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 t o PC. The s t u d i e s done by S h i g e u r a e_t a_l (18) i n c h i c k macrophages by using one of these compounds (AUS) showed t h i s m a t e r i a l markedly i n h i b i t e d the s y n t h e s i s of PC but e x h i b i t e d no e f f e c t s on the b i o s y n t h e s i s of DNA, RNA or p r o t e i n . Bonney et al. (59) a l s o showed that the r e l e a s e of inflammatory mediators, p r o s t a g l a n d i n s (PGs), l e u k o t r i e n e s (LT) and l y s o s o m a l a c i d h y d r o l a s e s (LAH) by mouse p e r i t o n e a l macrophages, which i s s t i m u l a t e d by endocytic s t i m u l i such as zymosan, could be i n h i b i t e d by the a d m i n i s t r a t i o n of d i p h e n y l s u l f o n e compounds i n a nontoxic manner. Corresponding-l y , the i n h i b i t i o n of PC b i o s y n t h e s i s was a l s o observed i n mouse macrophages. The reason f o r the i n h i b i t i o n of the product r e l e a s e by macrophages t r e a t e d with the s u l f o n e s has been speculated to be due to the s p e c i f i c i n h i b i t i o n of PC b i o s y n t h e s i s v i a the CDP-c h o l i n e pathway, s i n c e the r e l e a s e of p r o d u c t s from c e l l s c e r t a i n l y i n v o l v e s events mediated by plasma membrane components (59,60). C o n s i d e r i n g the s i m i l a r i t y of the s i t u a t i o n s between the r e l e a s e of the products from macrophages and the s e c r e t i o n of 33 l i p o p r o t e i n s from hepatocytes, we hoped the d i p h e n y l s u l f o n e com-pounds would help us to e s t a b l i s h a model system f o r studying the r e l a t i o n s h i p between an i n h i b i t i o n of PC b i o s y n t h e s i s and l i p o -p r o t e i n s e c r e t i o n i n r a t hepatocytes. The i s o l a t i o n of h e p a t o c y t e s and t h e i r m a intenance i n primary monolayer c u l t u r e has enabled the s t u d i e s of h e p a t i c PC b i o s y n t h e s i s and l i p o p r o t e i n s e c r e t i o n under c o n d i t i o n s which r e f l e c t l i p i d metabolism ir\ v i v o yet permit s u b t l e m o d i f i c a t i o n s i n t h e i r n u t r i t i o n a l , hormonal (61), and pharmacological s t a t e s (62). R a d i o a c t i v e l y l a b e l l e d ( m e t h y l - 1 4 C or 3H) c h o l i n e i s widely used as the p r e c u r s o r of PC i n the C D P - c h o l i n e b i o s y n t h e s i s pathway, and the d i s t r i b u t i o n of r a d i o a c t i v e i n the c e l l u l a r c h o l i n e c o n t a i n i n g m a t e r i a l s r e f l e c t s the s y n t h e t i c r a t e . But i n r a t h e p a t o c y t e s , c h o l i n e w i l l a l s o be o x i d i z e d to b e t a i n e . The conversion of c h o l i n e to betaine i s a two-step r e a c t i o n c a t a l y z e d by c h o l i n e dehydrogenase and betaine aldehyde dehydrogenase (63), and t h e p r o d u c t i s r a p i d l y r e l e a s e d f r o m r a t h e p a t o c y t e s (28,32,64) i n t o medium. Resul t s reported here are from experiments i n which we have a t t e m p t e d to use the d i p h e n y l s u l f o n e compounds to p e r t u r b PC b i o s y n t h e s i s v i a the CDP-choline pathway i n rat hepatocytes, as w e l l as i n HeLa c e l l s . The p r e l i m i n a r y s t u d i e s have shown t h a t the a d m i n i s t r a t i o n of t h e s e d i p h e n y l s u l f o n e compounds, e x c e p t dapsone, to HeLa c e l l s i n h i b i t e d the t o t a l [ m e t h y l - H ] c h o l i n e i n c o r p o r a t i o n i n t o the c e l l s , but d i d not change the r a t e of c o n v e r s i o n of c h o l i n e to PC. A l s o , the e f f e c t s of these compounds on PC b i o s y n t h e s i s i n r a t hepatocytes were i n v e s t i g a t e d , but no 34 i n h i b i t i o n was observed. F i g . 9. S t r u c t u r e s of dapsone ( I ) , AUS (II) and analogs (III and IV). 35 MATERIALS AND METHODS Chemicals- Female W i s t a r r a t s were from the U n i v e r s i t y of B r i t i s h Columbia Animal Unit. Dulbecco's modified Eagle's medium (MEM) and f e t a l c a l f serum were bought from the Grand I s l a n d B i o l o g i c a l Co., Grand I s l a n d , NY. [ M e t h y l - 3 H ] C h o i i n e was obtained from Amersham. The d i p h e n y l s u l f o n e s , p , p ' - d i a m i n o d i -p h e n y l s u l f o n e (dapsone), [ 1 - [ 4 - ( 4 - s u l f a n i l y l ) p h e n y l ] u r e a (AUS), 4 - M e t h o x y a c e t a m i d o - 4 ' - u r e i d o d i p h e n y l s u l f o n e ( I I I ) and 4-(N-M e t h y l f o r m a m i d o ) - 4 ' - u r e i d o d i p h e n y l s u l f o n e (IV) ,were o b t a i n e d from Merck Sharp & Dohme Research Lab., D i v i s i o n of Merck & Co., Inc. Rahway, NJ. The s t r u c t u r e s of the four compounds are shown i n F i g u r e 9. Pr e p a r a t i o n of Rat Hepatocytes- Hepatocytes were i s o l a t e d from female Wistar r a t s (180 g) by a co l l a g e n a s e p e r f u s i o n technique as d e s c r i b e d by Davis e_t a_l (65), and c u l t u r e d i n p l a s t i c c u l t u r e d i s h e s (Lux Contur, 60 mm, 2-3 x 10 6 c e l l s / d i s h ) i n a r g i n i n e - f r e e MEM w i t h 28 uM c h o l i n e c h l o r i d e , 100 nm i n s u l i n , 0.4 mM o r n i -t h i n e , 100 ug/ml of s t r e p t o m y c i n s u l f a t e , 100 u n i t s / m l of p e n i c i l -l i n G, 10 mM Hepes (pH 7.4) and 5% FCS at 37° C under an atmos-phere of 95% a i r / 5 % C0 2. The c e l l s were maintained i n monolayer c u l t u r e f o r approximate 24 hr p r i o r to a l l experiments. Pulse-Chase S t u d i e s - Monolayer c u l t u r e s of rat hepatocytes i n 60 mm dishes (3 x 10 c e l l s / d i s h ) were washed twice with serum-free MEM and pulsed with 10 uCi [methyl- 3H] c h o l i n e (0.12 Ci/mmol) f o r 36 1 hr. The c e l l s were subsequently chased with MEM that contained 28 uM c h o l i n e i n the absence or p r e s e n c e of 100 ug/ml of d i -p h e n y l s u l f o n e compounds. At v a r i o u s times up to 4 hr, the c e l l s were harvested and the r a d i o a c t i v i t y i n c o r p o r a t e d i n t o c e l l u l a r PC and the water s o l u b l e m e t a b o l i t e s ( c h o l i n e , phosphocholine and C D P - c h o l i n e ) was d e t e r m i n e d . The r a d i o a c t i v i t y i n the water s o l u b l e c h o l i n e m e t a b o l i t e s was separated by TLC on S i l i c a g e l G-60 with CH3OH/0.6% NaCl/NH 3 (50:50:5; v:v:v) as s o l v e n t . P h o s p h a t i d y l c h o l i n e E x t r a c t i o n - The c e l l u l a r PC was e x t r a c t e d by B l i g h and Dyer's method (45). The c u l t u r e medium was removed, and 0.7 ml of c o l d CH 3OH/H 20 (10:4; v:v) was added to each d i s h . The c e l l s were harvested and the d i s h was washed with another 0.7 ml of the same s o l u t i o n . The sample was s o n i c a t e d i n a water b a t h s o n i c a t o r f o r 10 min and an a l i q u o t (50 ul) of sample was removed fo r p r o t e i n assay. The remainder was mixed with 0.5 ml CHC1 3, 0.5 ml CHC1 3, and 0.5 ml H 20 s e q u e n t i a l l y , and then c e n t r i f u g e d at 2,500 rpm (Western H-103N c e n t r i f u g e ) f o r 5 min. The m e t h a n o l -water (upper) phase was removed, and the c h l o r o f o r m (lower) phase was washed w i t h 0.75 ml of t h e o r e t i c a l upper phase (CH 30H/ CHC1 3/H 20; 48/3/47; v/v/v) by c e n t r i f u g a t i o n . The upper phase, which co n t a i n s water s o l u b l e c h o l i n e m e t a b o l i t e s , was combined, and an a l i q u o t was a p p l i e d to TLC p l a t e s . An a l i q u o t of the lower phase c o n t a i n i n g c e l l u l a r PC was d r i e d under n i t r o g e n . The sam-p l e s were counted f o r r a d i o a c t i v i t y as d e s c r i b e d p r e v i o u s l y (Cha-pter I I , M a t e r i a l s and Methods s e c t i o n ) . Growth of HeLa c e l l s - HeLa c e l l s were obtained from Flow Labora-37 t o r i e s ; t h e y o r i g i n a l l y came from American Type C e l l C u l t u r e C o l l e c t i o n HeLa CCL-2. The r o u t i n e growth c o n d i t i o n s were as de s c r i b e d by Pelech ^ t a_l (66). HeLa c e l l s were grown on p l a s t i c c u l t u r e d i s h e s (Lux C o n t u r , 60 mm) i n MEM ( c o n t a i n s 28 uM c h o l i n e c h l o r i d e ) with 0.4 mM o r n i t h i n e , 100 ug/ml streptomycin s u l f a t e , 80 ug/ml a r g i n i n e , 100 u n i t s / m l p e n i c i l l i n G, 10 mM Hepes (pH 7.4) and 5% FCS at 37°C under an atmosphere of 95% a i r / 5 % C0 2» C o n f l u e n t monolayer c e l l c u l t u r e s were used f o r [ m e t h y l - H] c h o l i n e i n c o r p o r a t i o n or p u l s e - c h a s e e x p e r i m e n t s as d e s c r i b e d above, except 80 ug/ml a r g i n i n e was always present i n a l l c u l t u r e media. 38 RESULTS Infl u e n c e of D i p h e n y l s u l f o n e Compounds on [Methyl- H]Choline Up- take by Hela C e l l s : Pulse L a b e l i n g S t u d i e s - Bonney e_t a_l. (60) have shown that d i p h e n y l s u l f o n e s dapsone and AUS i n h i b i t e d i n c o r -p o r a t i o n of [ 1, 2 - 1 4 C ] c h o l i ne i n t o PC, but not the f o r m a t i o n of PE from [ 1,2,-*^C] ethanolamine i n both mouse macrophages and HeLa c e l l s . S i m i l a r r e s u l t s were o b t a i n e d i n the s t u d i e s on c h i c k macrophages by using AUS (18). I have s t u d i e d the e f f e c t of these drugs, dapsone and AUS, as w e l l as the r e l a t e d compounds II I and IV, on the PC s y n t h e s i s i n HeLa c e l l s . The e x p e r i m e n t a l data o b s e r v e d a r e summarized i n T a b l e 2 and T a b l e 3. A f t e r a 1 hr p u l s e , 90% of the r a d i o a c t i v i t y i n the c h l o r o f o r m phase was i n PC and l i k e w i s e , 90% of r a d i o a c t i v i t y i n the m e t h a n o l - w a t e r phase was i n p h o s p h o c h o l i n e . The r a d i o a c t i v e l a b e l i n g of CDP-c h o l i n e was v e r y s m a l l . T h i s i m p l i e s t h a t the c o n v e r s i o n of CDP-choline to PC i s f a s t i n t h i s b i o s y n t h e t i c pathway. The data showed t h a t dapsone (100 ug/ml) had no i n h i b i t o r y e f f e c t on the i n c o r p o r a t i o n of l a b e l e d c h o l i n e , as measured by the t o t a l r a d i o -a c t i v i t y of the c e l l u l a r m a t e r i a l s i n both chloroform phase and methanol-water phase (Table 2). The other three compounds, AUS, I I I and IV, i n h i b i t e d l a b e l e d c h o l i n e i n c o r p o r a t i o n to v a r y i n g d e g r e e (20- 50%) f o l l o w i n g a p r e t r e a t m e n t p e r i o d of 0 or 4 hr (Table 3). Among t h e s e compounds, IV e x h i b i t e d the h i g h e s t i n -h i b i t o r y e f f e c t on [ m e t h y l - H ] c h o l i n e i n c o r p o r a t i o n , s i n c e no p r e t r e a t m e n t of the c e l l s w i t h the compound was n e c e s s a r y to 39 Table 2. E f f e c t s of Dapsone and AUS on the [Methyl- H]Choline Uptake by HeLa C e l l s * Lower phase (dpm x 10 - 5/mg) Upper phase (dpm x 10 /mg) T o t a l (dpm x 10 ~5/mg) A d d i t i o n 0 hr 4 hr 0 hr 4 hr Ohr 4hr C o n t r o l 0. 97+0.09 1.05+0.12 35 .6+4.8 29.1+1. 5 36.7 30.2 Dapsone 1. 08+0.09 0.97+0.05 39 .9+0.3 31.1+1. 8 41.0 32.1 (-) (-) (-) (-) (-) (-) AUS 0. 79+0.11 0.68+0.06 28 .2+3.9 20.5+2. 4 29.0 21.2 (19) (35) (21) (30) (21) (30) * HeLa c e l l s were c u l t u r e d as d e s c r i b e d i n M a t e r i a l and Methods. C e l l s were preincubated i n serum-free MEM c o n t a i n i n g 100 ug/ml of dapsone or AUS d i s s o l v e d i n DMSO f o r 0 or 4 hr and then l a b e l e d w i t h 11 uCi of [ m e t h y l - H] c h o l i n e (0.13 Ci/mmol) per d i s h f o r an a d d i t i o n a l 1 hr i n the pre s e n c e of dapsone or AUS p r i o r to h a r v e s t i n g . A l l c u l t u r e s c o n t a i n e d a f i n a l c o n c e n t r a t i o n of 0.1% DMSO. The [ m e t h y l - H ] c h o l i n e uptake was e s t i m a t e d by q u a n t i t a t i o n of the c e l l u l a r r a d i o a c t i v i t y i n both w a t e r -methanol (upper) phase and chloroform (lower) phase e x t r a c t e d by t h e B l i g h and Dyer's method. P r o t e i n was e s t i m a t e d by Bio-Rad a s s a y . The r e s u l t s are the averages + S.D.; N = 3. Per cent i n h i b i t i o n by AUS w i t h r e s p e c t to c o n t r o l i s given i n parentheses. Table 3. E f f e c t s of Diphenylsulfone D e r i v a t i v e s (III and IV) on the [Methyl- 3H]Choline Uptake by HeLa C e l l s * A d d i t i o n Lower (dpm x pha 10 _ i ) ise /mg) Upper (dpm x phase 10~5/mg) T o t a l (dpm x 10 5/mg) 0 hr 4 hr 0 hr 4 hr 0 hr 4 hr Co n t r o l 4. 02+0.51 1. 93+0. 38 39.7+1.8 32.2+3. 5 43.7 34.1 I I I 3. 61+0.29 1. 08+0. 20 27.4+1.0 19.0+2. 3 31. 0 20.0 (10) (44) (3D (41) (29) (41) IV 2. 21+0.12 1. 17+0. 06 20.4+0.4 16.6+0. 6 22. 6 17.8 (45) (39) (49) (48) (48) (48) * The expermental c o n d i t i o n s were the same as d e s c r i b e d i n the legend of T a b l e 2, except the compounds I I I and IV were used i n s t e a d of dapsone and AUS. The r e s u l t s are the ave r a g e s +S.D.; N = 3. Per c e n t i n h i b i t i o n by III and IV with respect to c o n t r o l i s given i n parentheses. g i v e the g r e a t e s t i n h i b i t i o n ( T a b l e 3). The t o t a l [ m e t h y l - H] c h o l i n e i n c o r p o r a t i o n was reduced i n the c e l l s t r e a t e d with AUS, I I I or IV, but the p r o p o t i o n of r a d i o a c t i v i t y i n the c h l o r o f o r m and the methanol-water phases were s i m i l a r f o r a l l three drugs, which s u g g e s t s t h a t the c o n v e r s i o n of c h o l i n e i n t o PC was not a f f e c t e d . The s t u d i e s done w i t h mouse (60) or c h i c k (18) macro-phages suggested that these compounds i n h i b i t e d the PC b i o s y n t h e -s i s v i a C D P - c h o l i n e pathway at s t e p s f o l l o w i n g p h o s p h o c h o l i n e , i . e . at e i t h e r CT or CPT. However,the d a t a p r e s e n t e d h e r e i m p l y that the reduced i n c o r p o r a t i o n of r a d i o a c t i v i t y i nto PC was due to the r e d u c t i o n of t o t a l [methyl- H ] c h o l i n e i n c o r p o r a t i o n i n t o the c e l l s t r e a t e d with the compounds, rather than the i n h i b i t i o n of the conversion of c h o l i n e i n t o PC. In order to determine the i n f l u e n c e of d i p h e n y l s u l f o n e com-pound on the [ m e t h y l - H ] c h o l i n e metabolism i n HeLa c e l l s , p u l s e -chase s t u d i e s were done i n the presence of AUS or IV. The reason f o r c h o o s i n g t h e s e two compounds i n t h i s e x p e r i m e n t was t h e y e x h i b i t e d h i g h e r i n h i b i t i o n of c h o l i n e uptake. S i n c e t h e r e i s n e i t h e r s i g n i f i c a n t o x i d a t i o n of c h o l i n e to form b e t a i n e nor b i o s y n t h e s i s or s e c r e t i o n of l i p o p r o t e i n i n HeLa c e l l s , the r a d i o a c t i v i t y i n methanol-water and c h l o r o f o r m phases could r e -p r e s e n t the major c h o l i n e m e t a b o l i t e s , p h o s p h o c h o l i n e and PC, r e s p e c t i v e l y , w h i l e the c o u n t s i n medium compounds might be n e g l i g i b l e . A f t e r 1 hr p u l s e of [ m e t h y l - 3 H ] c h o l i n e i n the p r e -sence of AUS and IV, the 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 the c e l l s was observed. The r a d i o a c t i v i t y l a b e l i n g i n both c h l o -r o f o r m (Fig.10 A) and m e t h a n o l - w a t e r phases (Fig.10 B) at the 42 zero t i m e of the chase were l o w e r i n t r e a t e d c e l l s than i n the c o n t r o l s . The rate of PC b i o s y n t h e s i s i n the c e l l s could s t i l l be estimated by comparing the r a t e of [ m e t h y l - 3 H ] c h o l i n e i n c o r p o r a -t i o n i n t o the c h l o r o f o r m phase between t r e a t e d and c o n t r o l c e l l s . There were l i t t l e or no d i f f e r e n c e s i n the decrease of r a d i o a c t i -v i t y from p h o s p h o c h o l i n e ( F i g . l O , B) and the i n c r e a s e of r a d i o -a c t i v i t y i n PC ( F i g . l O , A) by c o m p a r i n g the t r e a t e d and c o n t r o l c e l l s . The d a t a c o n f i r m e d t h a t the d i p h e n y l s u l f o n e compounds i n h i b i t e d o n l y the t o t a l 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 HeLa c e l l s but had no e f f e c t on the c o n v e r s i o n of c h o l i n e into PC. However, as AUS and IV i n h i b i t e d the i n c o r p o r a t i o n of c h o l i n e , hence a l t e r i n g the s p e c i f i c a c t i v i t y of c e l l u l a r c h o l i n e , the slope of the i n c o r p o r a t i o n of [methyl- H ] c h o l i n e i n t o PC might not r e f l e c t the a c t u a l r a t e of PC b i o s y n t h e s i s . Influence of D i p h e n y l s u l f o n e Compounds on [Methyl- H]Choline Me- t a b o l i s m i n Rat Hepatocytes : Pulse-Chase S t u d i e s - S i n c e the ma-j o r organ f o r l i p o p r o t e i n s y n t h e s i s and s e c r e t i o n i n r a t i s l i v e r , c u l t u r e d r a t hepatocytes were t r e a t e d with d i p h e n y l s u l f o n e compounds (100 ug/ml) to show whether these drugs could i n h i b i t s p e c i f i c a l l y PC b i o s y n t h e s i s v i a the C D P - c h o l i n e pathway, and d e t e r m i n e the e f f e c t on PC b i o s y n t h e s i s and l i p o p r o t e i n s e c r e -t i o n . The c e l l s were p r e l a b e l e d with [methyl- H ] c h o l i n e f o r 1 hr i n the absence of any k i n d of d i p h e n y l s u l f o n e compound to av e r t the p o t e n t i a l problem of apparent changes i n the l a b e l i n g of PC due to a l t e r a t i o n s i n c h o l i n e i n c o r p o r a t i o n , as seen i n HeLa c e l l s (Fig.lO). [ M e t h y l - H] C h o l i n e i s taken up by the c e l l s and r a p i d l y p h o s p h o r y l a t e d to p h o s p h o c h o l i n e or o x i d i z e d to 43 c h a s e t i m e < h r > Fig.10. I n f l u e n c e of AUS and IV on the metabolism of [methyl- H ] c h o l i n e i n HeLa c e l l s . HeLa c e l l s c u l t u r e d i n 60 mm d i s h e s were washed with serum-free MEM and pulsed with 10 uCi [methyl-J H ] c h o l i n e (0.12 Ci/mmol) per d i s h i n the absence ( A ) or presen-ce of 100 ug/ml AUS ( A ) or IV (•) d i s s o l v e d i n DMSO f o r 1 hr. The c e l l s were s u b s e q u e n t l y c h ased w i t h 28 uM c h o l i n e i n the unlabeled medium f o r up to 6 hr p r i o r to h a r v e s t i n g . A l l c u l t u r e media contained a f i n a l c o n c e n t r a t i o n of 0.1% DMSO. R a d i o a c t i v i -t i e s i n the c h l o r o f o r m phase (A) and meth a n o l - w a t e r phase (B) e x t r a c t e d by B l i g h and Dyer's method c o r r e s p o n d to PC and phosphocholine, r e s p e c t i v e l y . Each p o i n t represents the mean of three dishes. 44 b e t a i n e (Fig.11 and F i g . 1 2 ) . A f t e r 1 hr p u l s e , a p p r o x i m a t e l y 50% of the r a d i o a c t i v i t y was converted to betaine, whereas phospho-c h o l i n e , PC, CDP-choline and c h o l i n e accounted f o r the remaining 50%, i n which about 70% was p h o s p h o c h o l i n e and 20% was PC. The c e l l s s e c r e t e d b e t a i n e i n t o the medium, so a f t e r 1 hr of c h a s e , more than h a l f of the c e l l u l a r b e t a i n e d i s a p p e a r e d from the c e l l s . The r a d i o a c t i v i t y i n medium compounds i n c r e a s e d r a p i d l y w i t h i n the f i r s t hours of the chase p e r i o d , which represented the r e l e a s e of not o n l y c e l l u l a r b e t a i n e , but a l s o phosphocholine and l i p o p r o t e i n PC. A l l of t h e s e p r o c e s s e s are not i n f l u e n c e d by dapsone, AUS, I I I or IV, s i n c e n e i t h e r the r a t e of d e c r e a s e of r a d i a o c t i v i t y i n c e l l u l a r b e t a i n e nor the rate i n the i n c r e a s e of r a d i o a c t i v i t y i n the medium compounds were s i g n i f i c a n t l y a l t e r e d . The decrease i n l a b e l e d phosphocholine and increase i n PC r e p r e -sented the enzymatic a c t i v i t y i n v o l v e d i n the s u c c e s s i v e conver-s i o n s of phosphocholine to CDP-choline and PC. S t i l l , no a l t e r e d r a t e of these conversions was observed i n e i t h e r d i p h e n y l s u l f o n e compound t r e a t e d c e l l s compared to the c o n t r o l . The s l o w e r i n c r e a s e i n c e l l u l a r PC r a d i o a c t i v i t y , compared with the r a t e of d e c r e a s e i n c e l l u l a r p h o s p h o c h o l i n e , might be e x p l a i n e d as the s e c r e t i o n of l i p o p r o t e i n PC to the medium. These r e s u l t s suggest-ed that the a c t i v i t y of PC b i o s y n t h e s i s and l i p o p r o t e i n s e c r e t i o n i n r a t h e p a t o c y t e s were not a f f e c t e d by t h e s e d i p h e n y l s u l f o n e compounds i n these experimental c o n d i t i o n s . 45 B ETA IN E PHOSPHOCHOLINE c/i X £ a -o r-< cc o a. cc O O z U J o X a . i ' i r o 0 1 2 3 4 MEDIUM COMPOUND 0 1 2 3 4 0 1 2 3 4 c h a s e t i m e <hr > Fig.11. I n f l u e n c e of Dapsone or II on The Metabolism of [me- t h y l - H] Ch o l i n e i n Rat Hepatocytes. Monolayer c u l t u r e s of r a t hepatocytes i n 60 mm dishes (3 x 10 C c e l l s / d i s h ) were washed with serum-free MEM tw i c e , and then pulsed with 10 uCi [me_thyl- H] cho-l i n e (0.12 Ci/mmol) i n the same medium f o r 1 hr. The c e l l s were s u b s e q u e n t l y chased w i t h 28 uM c h o l i n e i n the absence ( A ) or pr e s e n c e of 100 ug/ml dapsone ( A ) or I I I (•) d i s s o l v e d i n 0.1% DMSO f o r up to 4 hr. A l l chase media contained 0.1% DMSO. Radio-a c t i v i t y i n c o r p o r a t e d i n t o the c e l l u l a r c h o l i n e m e t a b o l i t e s and medium compounds was determined as d e s c r i b e d under M a t e r i a l s and Methods. Each p o i n t r e p r e s e n t s the mean of two d i s h e s . The experiment was repeated, and s i m i l a r r e s u l t s were obtained. 46 B E T A I N E P H O S P H O C H O L I N E o X E o. •o a U J < cc O Q . CC O a UJ 2 _ i O X o I n 0' 1 2 3 4 M E D I U M C O M P O U N D c h a s e t i m e <hr > F i g . 12. I n f l u e n c e of AUS and IV on The Metabolism of [roethyl- J H ] C h o l i n e i n Rat H e p a t o c y t e s . The e x p e r i m e n t a l p r o c e d u r e was s i m i l a r to t h a t d e s c r i b e d i n the l e g e n d of Fig.11. AUS (•) and IV (•) i n the f i n a l c o n c e n t r a t i o n of 100 ug/ml were used h e r e . Each p o i n t r e p r e s e n t s the average of two dishes. The experiment was repeated, and s i m i l a r r e s u l t s were obtained. 47 DISCUSSION There i s no known i n h e r i t e d d e f i c i e n c y i n PC b i o s y n t h e s i s , so the s t u d i e s on the b i o l o g i c a l f u n c t i o n of PC i n l i p o p r o t e i n s e c r e t i o n have been p e r f o r m e d o n l y i n " e x p e r i m e n t a l " d i s e a s e s , such as c h o l i n e d e f i c i e n c y i n the d i e t (67). The s i t u a t i o n has become more complicated s i n c e PC b i o s y n t h e s i s v i a the CDP-choline pathway seems to be h i g h l y r e s i s t a n t to the environmental v a r i a -t i o n s and the q u a n t i t a t i v e amount of the r a t e - l i m i t i n g enzyme CT i s a l m o s t unchanged i n any p h y s i o l o g i c a l c o n d i t i o n . The r e p o r t from Bonney e t a^ (60) t h a t d i p h e n y l s u l f o n e compounds c o u l d s p e c i f i c a l l y i n h i b i t PC b i o s y n t h e s i s i n macrophages i n a non-t o x i c manner encouraged us to study PC b i o s y n t h e s i s and l i p o p r o -c t e i n s e c r e t i o n i n h e p a t o c y t e s by u s i n g t h e s e compounds. U n f o r -t u r n a t e l y , our r e s u l t s showed t h a t t h e r e were no e f f e c t s on 3 [methyl- H]choline metabolism i n hepatocytes administered these compounds (100 ug/ml). The s i m p l e s t e x p l a n a t i o n of t h i s phenome-non might be the d e t o x i f i c a t i o n a b i l i t y of l i v e r c e l l s . However, the d a t a o b t a i n e d from the s t u d i e s on c u l t u r e d macrophages by using [ 1 4C]dapsone or [ 1 4C]AUS showed that the compounds were not accumulated by the c e l l s (60), which i m p l i e d that the d i p h e n y l -s u l f o n e s d i d n o t p e n e t r a t e t h e c e l l membrane. Thus i t i s u n c o n v i n c i n g to e x p l a i n the l a c k o f e f f e c t s i n h e p a t o c y t e s t r e a t e d w i t h the compounds by a c e l l u l a r d e t o x i f i c a t i o n mecha-nism. On the o t h e r hand, i t i s s t i l l p o s s i b l e t h a t we have not 48 found an e f f e c t i v e dosage of these diphenysulfones on rat hepato-c y t e s , s i n c e we only d i d experiments i n which the compound con-c e n t r a t i o n was 100 ug per ml. The p a r e n t compound of t h i s s e r i e s d i p h e n y l s u l f o n e s , dapsone, i s an e s t a b l i s h e d a n t i m a l a r i a l and a n t i l e p r o t i c agent (68), and l e p r o s y i s a d i s e a s e which i s c h a r a t e r i z e d by polymor-phonuclear i n f i l t r a t i o n . Bonney et_ a_l (59) found that dapsone and i t s d e r i v a t i v e s i n h i b i t e d the r e l e a s e of the i n f l a m m a t o r y m e d i a t o r s by macrophages which i s s t i m u l a t e d by zymosan. The mechanism of the i n h i b i t i o n of PC b i o s y n t h e s i s by the d i p h e n y l -s u l f o n e s i n mouse and c h i c k macrophage has been p o s t u l a t e d by Bonney e_t al. (60) and S h i g e u r a ejt al. (18) to be because s u l f o n e s acted at the s i t e ( s ) of c o n v e r s i o n of phosphocholine to PC. They found l i t t l e or no i n h i b i t i o n of the c h o l i n e t r a n s p o r t , or of c h o l i n e p h o s p h o r y l a t i o n . The r e s u l t s from the experiments done on HeLa c e l l s p r e s e n t e d here does not agree w i t h t h i s p r e v i o u s c o n c l u s i o n . We found t h a t a l l the d i p h e n y l s u l f o n e s , e x c e p t dapsone, i n h i b i t e d o n l y the c h o l i n e i n c o r p o r a t i o n i n t o the c e l l s , but not the c o n v e r s i o n of l a b e l from c h o l i n e to PC. The r a d i o a c t i v i t y d i s t r i b u t i o n i n c e l l u l a r i n t e r m e d i a t e s of PC syn-t h e s i s i n HeLa c e l l s from both pulse l a b e l i n g (Table 2 and Table 3) and p u l s e - c h a s e e x p e r i m e n t (Fig.10) s u p p o r t t h i s p o i n t of view. 3 The reason f o r reduced t o t a l [methyl- H]choline i n c o r p o r a -t i o n i n t o HeLa c e l l s was not examined here as the major purpose of the e x p e r i m e n t was to v e r i f y the e f f e c t of the compounds on the c o n v e r s i o n of c h o l i n e to PC. I t i s p o s s i b l e that the reduced 49 c h o l i n e i n c o r p o r a t i o n i s due to i n h i b i t e d c h o l i n e t r a n s p o r t and t h e r e are s e v e r a l p r o b a b i l i t i e s which might a c c o u n t f o r the r e s u l t . I t has been r e p o r t e d t h a t t h e r e were two mechanisms i n v o l v e d i n c h o l i n e t r a n s p o r t i n r a t l i v e r , s a t u r a b l e and un-s a t u r a b l e uptake (69), and the a p p a r e n t Km f o r c h o l i n e f o r the s a t u r a b l e mechanism was 10 uM i n r a t h e p a t o c y t e s (70). S i n c e the c o n c e n t r a t i o n of c h o l i n e i n r a t plasma n o r m a l l y ranges be-tween 10 to 20 uM (71), the f a c i l i t a t e d t r a n s p o r t of c h o l i n e by l i v e r p r o b a b l y a pproaches s a t u r a t i o n , a l t h o u g h t h e r e seems no l i m i t on c h o l i n e entry by p a s s i v e d i f f u s i o n . The reduced c h o l i n e i n c o r p o r a t i o n i n t o the HeLa c e l l s t r e a t e d with d i p h e n y l s u l f o n e s might be due to the i n h i b i t i o n of the membrane c a r r i e r s which are s p e c i f i c f o r c h o l i n e t r a n s p o r t . On the o t h e r hand, s t u d i e s on c h o l i n e t r a n s p o r t with N o v i k o f f hepatoma c e l l s argued t h a t the c h o l i n e t r a n s p o r t served as the r a t e - l i m i t i n g step i n PC b i o s y n -t h e s i s when the c o n c e n t r a t i o n of c h o l i n e was below 20 uM, i n which the 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 was l i m i t e d by the r a t e of f o r m a t i o n of phosphocholine c a t a l y z e d by c h o l i n e kinase (72), while at c o n c e n t r a t i o n s above 20 uM, the r a t e 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 was i n d e p e n d e n t of medium c h o l i n e c o n c e n t r a t i o n (72). Reduced c h o l i n e p h o s p h o r y l a t i o n i n the t r e a t e d HeLa c e l l s might a l s o be the cause of the o b s e r v a t i o n s of reduced c h o l i n e i n c o r p o r a t i o n . That i s , the i n h i b i t i o n of c h o l i n e kinase r e s u l t s i n l e s s c o n v e r s i o n of c h o l i n e to p h o s p h o c h o l i n e , t h e r e f o r e the 3 medium [methyl- H]choline e n t e r i n g v i a p a s s i v e d i f f u s i o n i n t o the d i p h e n y l s u l f o n e t r e a t e d c e l l s i s reduced due to an i n c r e a s e i n c e l l u l a r c h o l i n e c o n c e n t r a t i o n . We do not know whether or not 50 the d i p h e n o l s u l f o n e 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 t o r at hepa-t o c y t e s . P r i t c h a r d and Vance (32) have s t u d i e d the r o l e of c h o l i n e t r a n s p o r t i n PC b i o s y n t h e s i s i n r a t h e p a t o c y t e s , and found the rate of hepatic PC b i o s y n t h e s i s was almost not i n f l u -enced by the rate of c h o l i n e t r a n s p o r t when 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 c u l t u r e medium ranged between 5-40 uM. The r e s u l t s from HeLa c e l l s presented here confirmed t h i s statement. In c o n c l u t i o n , the d i p h e n y l s u l f o n e compounds ap p e a r s not s u i t a b l e f o r the s t u d i e s on the i n h i b i t i o n of PC b i o s y n t h e s i s v i a CDP-choline pathway i n rat hepatocytes or HeLa c e l l s . T h e r e f o r e , i t i s s t i l l of i n t e r e s t to f i n d a s p e c i f i c i n h i b i t o r of PC b i o -s y n t h e s i s v i a C D P - c h o l i n e pathway i n l i v e r s to s t u d y the r e l a -t i o n s h i p between PC b i o s y n t h e s i s and l i p o p r o t e i n s e c r e t i o n . 51 CHAPTER IV. EFFECTS OF VASOPRESSIN ON CTP:PHOSPHOCHOLINE CYTIDYL-YLTRANSFERASE IN RAT HEPATOCYTES The r e g u l a t i o n of PC b i o s y n t h e s i s has been s t u d i e d under a v a r i e t y of developmental and p h y s i o l o g i c a l c o n d i t i o n s (24,34,73, 74,75,76,76a) and by g e n e t i c m a n i p u l a t i o n s (77,78). Based on analyses of the a c t i v i t i e s of enzymes i n PC s y n t h e s i s as w e l l as l e v e l s of metabolic i n t e r m e d i a t e s , i t was concluded t h a t the CT was r e g u l a t o r y i n the CDP-choline pathway under the experimental c o n d i t i o n s . The s t u d i e s on the r e g u l a t o r y mechanisms of CT a c t i -v i t y and the b i o s y n t h e s i s of PC have f o c u s e d on the s h o r t term c o n t r o l . The e f f e c t of s u b s t r a t e c o n c e n t r a t i o n on PC s y n t h e s i s v a r i e s i n d i f f e r e n t c e l l s . In p o l i o v i r u s - i n f e c t e d HeLa c e l l s (79,80), the c o n c e n t r a t i o n of c y t o s o l i c CTP seems to be c r i t i c a l f o r r e g u l a t i o n of the CT c a t a l y z e d r e a c t i o n and PC a n a b o l i s m , whereas i n the c o c k e r e l t r e a t e d with d i e t h y l s t i l b o e s t r o l (DES), i t was shown t h a t the c o n c e n t r a t i o n o f c e l l u l a r p h o s p h o c h o l i n e r e s t r i c t e d t h e r a t e o f PC s y n t h e s i s i n t h e l i v e r ( 81,82). However, i n BHK c e l l s , both CTP and p h o s p h o c h o l i n e appeared to l i m i t the rate of PC b i o s y n t h e s i s (83). S i n c e CT has a b i m o d a l d i s t r i b u t i o n w i t h i n c e l l s and i t s a c t i v i t y i s phospholipid-dependent, t h i s suggests that the enzyme i s r egulated v i a a t r a n s l o c a t i o n mechanism. The bulk of CT l o c a t -ed i n c y t o s o l i s i n a c t i v e and f u n c t i o n s as an enzyme r e s e r v o i r . But once the enzyme becomes a s s o c i a t e d with membrane, i t w i l l be a c t i v a t e d by p h o s p h o l i p i d s . S e v e r a l d i f f e r e n t mechanisms have 52 been proposed to e x p l a i n how c e l l s c o n t r o l the r e l a t i v e d i s t r i b u -t i o n of CT. S t u d i e s done by S l e i g h t and Kent on CHO c e l l s (75) and e m b r y onic c h i c k muscle c e l l s (76) d e m o n s t r a t e d t h a t the CT might be r e g u l a t e d by a membrane r e p a i r i n g mechanism. Exposure of the i n a c t i v e c y t o s o l i c enzyme to membranes depleted of PC, which could be formed by phospholipase C treatment, r e s u l t s i n both the b i n d i n g and a c t i v a t i o n of the enzyme. One q u e s t i o n a s s o c i a t e d with t h i s proposol i s whether the i n c r e a s e d membrane-associated CT i s caused d i r e c t l y by the d e p l e t i o n of PC i n the membrane or by higher c o n c e n t r a t i o n of d i a c y l g l c e r o l (DG) which i s a product of phospholipase. DG has been demonstrated to promote aggregation of CT ir\ v i t r o , and t h i s e f f e c t was m i m i c k e d by an exogenous phospholipase C from B a c i l l u s cereus (84). There are two other models of r e g u l a t i o n . The f i r s t i s the r e v e r s i b l e p h o s p h o r y l a t i o n hypothesis. Covalent m o d i f i c a t i o n of CT v i a a p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n c y c l e c o r r e l a t e d with changes i n the d i s t r i b u t i o n of CT a c t i v i t y between c y t o s o l and microsome. Regul a t i o n of PC b i o s y n t h e s i s by t h i s mechanism has been proposed i n rat hepatocytes (61,74) and i n _i_n v i t r o s t u d i e s w i t h l i v e r c y t o s o l (27). In the p r e s e n c e of cAMP a n a l o g u e s and cAMP phosphodiesterase i n h i b i t o r s , there i s more s o l u b l e CT and l e s s membrane-associated enzyme i n hepaocytes (61,74). S i m i l a r l y , i n c u b a t i o n of r a t l i v e r c y t o s o l j_n v i t r o with Mg-ATP or phospho-p r o t e i n p h o s p h a t a s e i n h i b i t o r , NaF, p r e v e n t s the c y t o s o l i c CT from a s s o c i a t i n g w i t h membranes upon i n c u b a t i o n a t 37° C (27). T h i s e f f e c t c o u l d be a b o l i s h e d by the a d d i t i o n of cAMP p r o t e i n k i n a s e i n h i b i t o r s i n t o the i n c u b a t i o n system and r e s u l t s i n 53 increased p r o p o r t i o n of CT a c t i v i t y i n c y t o s o l (27). The dephos-phorylated CT has a tendency to a s s o c i a t e with membrane (ge n e r a l -l y E.R.) and to be a c t i v a t e d by p h o s p h o l i p i d s , w h i l e the phos-phory l a t e d CT i s i n a c t i v e and l o c a t e d i n the c y t o s o l . The r e v e r -s i b l e p h o s p h o r y l a t i o n mechanism i n r e g u l a t i o n of PC b i o s y n t h e s i s i s assumed to be s i m i l a r to the mechanisms i n v o l v e d i n f a t t y a c i d and c h o l e s t r o l b i o s y n t h e s i s (85). P h o s p h o r y l a t i o n of acetyl-CoA c a r b o x y l a s e (86) and HMG-CoA r e d u c t a s e (87) a l s o r e n d e r t h e s e r e g u l a t o r y enzymes i n a c t i v e i n t h e i r r e s p e c t i v e pathways. Pelech and Vance (88) speculated that the p h o s p h o r y l a t i o n of CT might be hormonally c o n t r o l l e d by glucacon, s i n c e cAMP i s the e s t a b l i s h -ed second messenger of t h i s hormone. However, the d i r e c t i n c o r -3 2 p o r a t i o n of [ P]phosphate i n t o the enzyme remains to be demons-t r a t e d . A second mechanism d e r i v e d from the s t u d i e s done on r a t hepatocytes (28) and HeLa c e l l s (66), suggested that the t r a n s l o -c a t i o n of CT from c y t o s o l to microsomes and the s t i m u l a t i o n of PC s y n t h e s i s i s r e g u l a t e d by f a t t y a c i d s and f a t t y a c y l - C o A (28). I t was shown i n r a t h e p a t o c y t e s t h a t t h e e f f e c t of u n s a t u r a t e d l o n g c h a i n f a t t y a c i d s (1 mM p a l m i t a t e or 3 mM o l e a t e ) on CT t r a n s l o c a t i o n i s so strong that i t w i l l reverse the phosphoryla-tion-mediated decrease i n CT a c t i v i t y a s s o c i a t e d with microsomes (61). However, the mechanism of f a t t y acid-mediated t r a n s l o c a t i o n i s p o o r l y understood. I t has been s p e c u l a t e d that DG may account f o r some of the t r a n s l o c a t i o n of CT, s i n c e the pool s i z e of DG i n r a t h e p a t o c y t e s s u p p l e m e n t e d w i t h 1 mM o l e a t e was i n c r e a s e d (28,15). S t i l l , the q u e s t i o n r e m a i n i n g i s whether or not DG can 54 enhance the b i n d i n g of the enzyme to microsomes j_n v i v o . V a s o p r e s s i n , an a l p h a - a d r e n e r g i c a g o n i s t , has been shown to cause a t r a n s i e n t ( w i t h i n 4 min) i n c r e a s e of 50% i n the t o t a l c o n c e n t r a t i o n of DG i n r a t h e p a t o c y t e s (89). The DG formed i n the presence of v a s o p r e s s i n i s considered to be d e r i v e d from the h y d r o l y s i s of Ptdlns(4,5) P 2, PtdIns4P and Ptdlns and/or perhaps the other p h o s p h o l i p i d s (89). T h i s r e a c t i o n has been shown to be c a t a l y z e d by p h o s p h o l i p a s e C which a p p e a r s to be a c t i v a t e d by v a s o p r e s s i n i n c u l t u r e s (90). The q u e s t i o n r e l a t i n g to the e f f e c t s of v a s o p r e s s i n on the PC b i o s y n t h e s i s i s whether or not the t r a n s i e n t i n c r e a s e of DG w i l l promote the t r a n s l o c a t i o n of CT from c y t o s o l to microsomes and hence a c t i v a t e the C D P - c h o l i n e formation. To answer t h i s q u e s t i o n , hepatocytes were t r e a t e d with v a s o p r e s s i n and the e f f e c t s of the hormone on CT d i s t r i b u t i o n and PC s y n t h e s i s were determined i n t h i s paper. A p r o t o c o l f o r e s t i m a t i o n of the s u b c e l l u l a r l o c a t i o n of c e l l u l a r enzymes has been developed by MacKall e_t a_l (91). C e l l s a r e d i s r u p t e d by t r e a t m e n t w i t h d i g i t o n i n , and the r a t e of r e -l e a s e of s o l u b l e enzyme d e t e r m i n e d . The r e l e a s e of c y t o s o l i c enzymes from c u l t u r e d c e l l s i s more r a p i d than membrane-a s s o c i a t e d enzymes. Thus, i f the i n c r e a s e of DG, caused by the a d m i n i s t r a t i o n of v a s o p r e s s i n to the c e l l s , r e s u l t s i n the t r a n s l o c a t i o n of CT from c y t o s o l to micrsomes, a re d u c t i o n i n the r a t e of r e l e a s e of c y t o s o l i c CT from the c e l l s i n t o the medium should be observed. The r e s u l t s presented here shows that the a d d i t i o n of vaso-p r e s s i n (5-20 nM) to r a t h e p a t o c y t e s r e s u l t e d i n a reduced r a t e 55 of r e l e a s e of c y t o s o l i c CT i n t o the c u l t u r e medium. However, the 3 increased rate of [methyl- H ] c h o l i n e i n c o r p o r a t i o n into PC was not observed i n e i t h e r p u l s e - l a b e l i n g or i n pulse-chase e x p e r i -ments . 56 MATERIALS AND METHODS Chemicals- W i s t a r r a t s (180 g) were s u p p l i e d by the U n i v e r s i t y of B r i t i s h C o l u m b i a A n i m a l U n i t . D i g i t o n i n (80%) and a r g i n i n e v a s o p r e s s i n (Grade VIII) were o b t a i n e d from Sigma. V a s o p r e s s i n was d i s s o l v e d i n 0.9% NaCl as a s t o c k s o l u t i o n of 5 ug/ml and kept at -20° C. Dulbecco's phosphate b u f f e r e d s a l i n e (PBS) c o n t a i n s 2.7 mM KC1, 1.5 mM KH 2P0 4, 137 mM NaCl and 8.1 mM NaHP0 4, pH 7.4. Incubation of Hepatocytes with V a s o p r e s s i n - Rat hepatocytes were i s o l a t e d from n o r m a l l y f e d W i s t a r r a t s and m a i n t a i n e d i n 60 mm c u l t u r e d i s h e s (2 x 10 c e l l s / d i s h ) as p r e v i o u s l y d e s c r i b e d ( i n Chapter I I I , M a t e r i a l s and Methods). For the study on d i g i t o n i n -m e d i a t e d r e l e a s e of c y t o s o l i c CT, the c e l l s were washed w i t h serum-free MEM twice, and incubated i n the same medium c o n t a i n i n g 5-20 nM v a s o p r e s s i n f o r 0-8 min. For the [ m e t h y l - 3 H ] c h o l i n e i n c o r p o r a t i o n study, each d i s h of hepatocytes was l a b e l e d with 10 3 uCi of [ m e t h y l - H ] c h o l i n e (0.12 Ci/mmol) i n the same medium c o n t a i n i n g 10 nm v a s o p r e s s i n f o r 0-60 min. For a p u l s e - c h a s e study, the c e l l s were washed with serum-free MEM twice and then p u l s e d w i t h 10 uCi of [ m e t h y l - 3 H ] c h o l i n e (0.12 Ci/mmol) i n the same medium f o r 30 min. The c e l l s were washed with serum-free MEM c o n t a i n i n g 28 uM c h o l i n e and chased i n the same medium i n the absence or p r e s e n c e of 10 nM v a s o p r e s s i n f o r up to 10 min. In order to avoid any e f f e c t s of serum i n the medium on the e x p e r i -ment, i n some s t u d i e s the medium was rep l a c e d by serum-free MEM 57 12 hr before the pulse-chase p e r i o d . Release of C y t o s o l i c CT from Rat Hepatocytes- D i g i t o n i n - m e d i a t -ed r e l e a s e of c y t o s o l i c CT from c u l t u r e d r a t h e p a t o c y t e s was performed e s s e n t i a l l y as d e s c r i b e d by M a c k a l l e_t a_l. (91). A f t e r removal of c u l t u r e medium, the c e l l monolayer was washed c a r e f u l -l y w i t h 2.5 ml of i c e - c o l d PBS. C o l d d i g i t o n i n - r e l ease b u f f e r (1.0 ml per 60 mm c l u t u r e d i s h ) c o n t a i n i n g 10 mM T r i s - H C l (pH 7.4), 250 mM s u c r o s e , 0.5 mM PMSF and 0.5 mg/ml d i g i t o n i n was c a r e f u l l y p i p e t t e d i n t o each c u l t u r e d i s h to i n i t i a t e enzyme r e l e a s e . The d i s h e s were p l a c e d on an i c e - c o l d t r a y and the c e l l s were incubated with d i g i t o n i n up to 8 min. The dishes were o c c a s i o n a l l y g e n t l y s w i r l e d d u r i n g the i n c u b a t i o n p e r i o d . At the times i n d i c a t e d the d i g i t o n i n - r e l e a s e b u f f e r was removed, and an a l i q u o t of the sample was used f o r CT assay. Other Methods- The a s s a y of CT a c t i v i t y and the s e p a r a t i o n of c h o l i n e m e t a b o l i t e s by TLC are d e s c r i b e d under the p r e v i o u s M a t e r i a l s and Methods s e c t i o n . C D P - c h o l i n e was s e p a r a t e d from b e t a i n e by TLC w i t h a c e t o n e / m e t h a n o l / H C l (10:90:4; v:v:v) as d e s c r i b e d p r e v i o u s l y by Lim et a l (92). 58 RESULTS E f f e c t of Va s o p r e s s i n on The D i g i t o n i n - m e d i a t e d Release of CT  from Rat Hepatocytes Exposure of hepatocytes to 10 nM vasopres-s i n l e d to a reduced r a t e of r e l e a s e o f c y t o s o l i c CT i n t o the c u l t u r e medium (Fig.13). The maximal e f f e c t was observed when the c e l l was incubated with v a s o p r e s s i n f o r 3 min. A f t e r 3 min i n c u -b a t i o n , l e s s than 50% (1 min) and 65% (2 min) of the c e l l u l a r CT, compared w i t h t h o s e of the c o n t r o l s , was r e l e a s e d i n t o the medium. The reduced r e l e a s e of CT was a l s o o b s e r v e d i n c e l l s t r e a t e d with v a s o p r e s s i n f o r only 1 min. Prolonged i n c u b a t i o n (5 and 10 min) of the c e l l s with v a s o p r e s s i n at the same conc e n t r a -t i o n r e s u l t e d i n a r e t u r n t o normal r a t e of r e l e a s e of CT. T h i s suggested that the e f f e c t of v a s o p r e s s i n on the t r a n s l o c a t i o n of CT was t r a n s i e n t . The reduced r e l e a s e of CT from the c e l l s t r e a t e d with v a s o p r e s s i n was maintained up to 8 min. E f f e c t of D i f f e r e n t C o n c e n t r a t i o n s of V a s o p r e s s i n on The D i g i t o n i n  Mediated Release of CT from Rat Hepatocytes In order to d e t e r -mine the maximally e f f e c t i v e c o n c e n t r a t i o n of v a s o p r e s s i n on the d i g i t o n i n - m e d i a t e d r e l e a s e of CT i n r a t h e p a t o c y t e s , d i f f e r e n t c o n c e n t r a t i o n s (5-20 nM) of v a s o p r e s s i n were i n v e s t i g a t e d ( F ig. 14). P r e l i m i n a r y s t u d i e s showed t h a t the maximal e f f e c t of v a s o p r e s s i n was obtained with a c o n c e n t r a t i o n of 5 nM. The c e l l s t r e a t e d w i t h 20 nM o f v a s o p r e s s i n d i d n o t show a f u r t h e r i n h i b i t i o n i n the rate of CT r e l e a s e compared with those t r e a t e d 59 CM ' ' ' • 1 1 1 L . 1 2 3 4 5 6 7 8 T i m e <min> F i g 13. D i g i t o n i n - m e d i a t e d Release of CT from Rat Hepa- t o c y t e s . Rat hepatocytes i n 60 mm dishes (2 x 10 c e l l s ) were i n c u b a t e d f o r 0 min (O) , 1 min (A), 3 min ( • ) , 5 min (£) or 10 min (•) i n s e r u m - f r e e medium c o n t a i n i n g 10 nM v a s o p r e s s i n . The c e l l medium was r e p l a c e d w i t h 1 ml of i c e - c o l d d i g i t o n i n (0.5 mg/ml) a f t e r washing the c e l l s w i t h 2.5 ml of i c e - c o l d PBS, and the i n c u b a t i o n c o n t i n u e d on an i c e - c o l d m etal t r a y f o r up to 8 min. CT a c t i v i t y i n the d i g i t o n i n e x t r a c t was subsequently de-t e r m i n e d i n the p r e s e n c e of 0.2 mg of t o t a l r a t l i v e r p h o s p h o l i p i d and 10 nmol of o l e a t e . Each p o i n t r e p r e -s e n t s the a v e r a g e o f two d i s h e s . The e x p e r i m e n t was repeated, and the same r e s u l t s were obtained. 60 o i .—•—• . -1 2 3 4 T i m e (min> Fig.14. E f f e c t of D i f f e r e n t C o n c e n t r a t i o n s of Vaso- p r e s s i n on The D i g i t o n i n - m e d i a t e d Release of CT  from Rat H e p a t o c y t e s . Rat h e p a t o c y t e s i n 60 mm d i s h e s (2 x 10 c e l l s ) were i n c u b a t e d f o r 3 min i n s e r u m - f r e e medium i n the absence (O) o r p r e s e n c e of 5 nM ( • ) , 10 nM (•) or 20 nM (#) of v a s o p r e s -s i n . The c e l l s were washed w i t h 25 ml of i c e - c o l d PBS and i n c u b a t e d i n 1 ml of i c e - c o l d d i g i t o n i n (0.5 mg/ml) f o r up to 4 min. CT a c t i v i t y i n the d i g i t o n i n e x t r a c t was measured as d e s c r i b e d under the l e g e n d of Fig.15. Each p o i n t r e p r e s e n t s the mean of two dishes. 61 with 10 nM of hormone. The r e l e a s e of CT returned to the c o n t r o l l e v e l by 4 min (Fig. 14). E f f e c t of Va s o p r e s s i n on The I n c o r p o r a t i o n of [ m e t h y l - 3 H ] c h o l i n e  i n t o P h o s p h o l i p i d s ; Pulse L a b e l i n g S t u d i e s - The reduced release of CT from the hepatocytes exposed to v a s o p r e s s i n i n d i c a t e d that p a r t of the c y t o s o l i c CT became membrane-associated. Therefore, i t was expected that PC b i o s y n t h e s i s i n the c e l l s incubated with v a s o p r e s s i n would be s t i m u l a t e d . However, h e p a t o c y t e s l a b e l e d with [methyl- H ] c h o l i n e i n the presence of 10 nM v a s o p r e s s i n d i d not e x h i b i t an i n c r e a s e i n the r a t e o f 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 (Fig.15 A) d u r i n g 60 min. The r a d i o a c t i v e p o o l s i z e of CDP-choline i s too s m a l l to be examined (data not shown). The rate of t o t a l [methyl- H ] c h o l i n e i n c o r p o r a t i o n i n t o the c e l l s i n the presence of 10 nM v a s o p r e s s i n was not a l t e r e d r e l a t i v e to the c o n t r o l i n 60 min p e r i o d (Fig.15 B). 3 I n f l u e n c e of V a s o p r e s s i n on [ M e t h y l — H ] C h o l i n e Metabolism i n Rat  Hepatocytes : pulse-Chase S t u d i e s - Since the e f f e c t of vasopres-s i n on CT t r a n s l o c a t i o n was t r a n s i e n t ( F i g . 13) and the amount of [methyl- H]choline i n c o r p o r a t i o n i n t o the c e l l s was s m a l l i n the f i r s t 5 min ( F i g . 15A), the p u l s e l a b e l i n g e x p e r i m e n t might not be a b l e to show the e f f e c t of v a s o p r e s s i n on s t i m u l a t i n g CT a c t i v i t y . In t h i s e x p e r i m e n t , c e l l s were p r e l a b e l e d w i t h [me-t h y l - H]choline f o r 30 min i n the absence of v a s o p r e s s i n and then c h a s e d i n the medium c o n t a i n i n g 10 nM v a s o p r e s s i n f o r up to 10 min. The i n c o r p o r a t i o n of l a b e l e d c h o l i n e i n t o PC was d e t e r -mined. The r e s u l t s from two s e p e r a t e e x p e r i m e n t s were shown i n 62 T ime <min> T ime <min> Fig.15. E f f e c t of Va s o p r e s s i n on The I n c o r p o r a t i o n of [methyl- H]Choline i n t o P h o s p h o l i p i d s ^ At zero time, r a t hepatocytes In 60 mm d i s h e s (2 x 10 c e l l s ) were l a b e l e d w i t h 10 uCi of [ m e t h y l -H ] c h o l i n e (0.12 Ci/mmol) per d i s h i n serum-free medium f o r up to 60 min i n the absence (O/A) or p r e s e n c e of 10 nM v a s o p r e s s i n ( # , • ) . R a d i o a c t i v i t y i n c o r p o r a t e d i n t o the c e l l u l a r PC (A) and the t o t a l c e l l u l a r c h o l i n e m e t a b o l i t e s (B) were d e t e r m i n e d as d e s c r i b e d under M a t e r i a l s and Methods. Each p o i n t represents the mean of three d i s h e s . 63 s: 0 2 4 6 8 10 0 2 4 6 8 10 C h a s e T i m e <rnin> C h a s e T ime <min> F i g . 16. I n f l u e n c e of V a s o p r e s s i n on The I n c o r p o r a t i o n  of [Methyl- H]Choline i n t o PC i n Rat Hepatocytes. M o n o l a y e r c u l t u r e s of r a t h e p a t o c y t e s i n 60 mm d i s h e s (2 x 10° c e l l s / dish) were washed with serum-free MEM tw i c e , and then pulsed w i t h 10 uCi [ m e t h y l - 3 H ] c h o l i n e (0.12 Ci/mmol) i n the same medium f o r 30 min. The c e l l s were subsequently chased with 28 uM c h o l i n e i n the absence (O) or p r e s e n c e of 10 nM v a s o -p r e s s i n ( 0 ) f o r up to 10 min. R a d i o a c t i v i t y i n c o r p o r a t e d i n t o c e l l u l a r PC was d e t e r m i n e d as d e s c r i b e d u n d e r M a t e r i a l s and Methods. The p o i n t s i n p a n e l A r e p r e s e n t the mean of two dishes while i n panel B represent the average of three dishes. 64 F i g . 16 A and B, r e s p e c t i v e l y . The data from the f i r s t experiment showed that the c e l l s chased i n the presence of 10 nM v a s o p r e s s i n e x h i b i t e d an in c r e a s e by about 50% i n l a b e l e d PC r e l a t i v e to the c o n t r o l s at 4 min chase time (Fig. 16A). However, the same r e s u l t could not be obtained when the same experiment was repeated ( Fig. 16B) . In o r d e r to s u b s t a n t i a t e the e f f e c t of v a s o p r e s s i n on PC b i o s y n t h e s i s , the hepatocytes were preincubated i n serum-free MEM fo r 12 hr before s t a r t i n g the pulse-chase experiment to avoid any t r a c e amount of v a s o p r e s s i n from serum i n the c u l t u r e medium which might i n t e r f e r e the c e l l r e s p o n s e to the a d d i t i o n o f v a s o p r e s s i n . The r e s u l t s summarized i n Table 4 showed t h a t w i t h i n 4-min chase p e r i o d t h e r e were no s t a t i s t i c l l y s i g n i f i c a n t a l t e r n a t i o n s i n r a d i a c t i v i t y i n c o r p o r a t i o n i n t o PC between the c e l l s t r e a t e d w i t h v a s o p r e s s i n and t h o s e of c o n t r o l s . A l s o , the d i s a p p e a r a n c e of r a d i a c t i v i t y from the m e t h a n o l - w a t e r phase w i t h i n 4-min chase p e r i o d showed the same p a t t e r n i n both hormone t r e a t e d c e l l s and c o n t r o l s (Table 4). The d e c r e a s e d c e l l u l a r r a d i o a c t i v i t y r e c o v e r y was due to the s e c r e t i o n of b e t a i n e , a product of c h o l i n e o x i d a t i o n , i n t o c u l t u r e medium by hepatocytes (see Chapter I I I ) . 65 T a b l e 4. E f f e c t of V a s o p r e s s i n on The I n c o r p o r a t i o n o f [Methyl- 3H]Choline into PC* Chloroform Phase Methanol-water Phase A d d i t i o n (dpm x 10" 5/dish) (dpm x 10~ 5/dish) 0 min 4 min 0 min 4 min C o n t r o l 0.43+0.02 0.45+0.08 17.0+0.9 12.1+0.7 Vasopres- 0.49+0.04 0.43+0.07 17.5+0.7 12.1+0.8 s i n * Rat h e p a t o c y t e s were c u l t u r 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. A f t e r monolayer of c e l l s were formed i n the c u l t u r e d i s h e s , the c e l l s were i n c u b a t e d i n s e r u m - f r e e medium f o r 12 hr b e f o r e p u l s e l a b e l i n g . Each d i s h of c e l l s were l a b e l e d with 10 uCi of [methyl-3 H ] c h o l i n e (0.12 Ci/mmol) f o r 30 min i n the absence of v a s o p r e s s i n and then chased f o r 4 min i n the medium c o n t a i n i n g 10 nM v a s o p r e s s i n . The [ m e t h y l - H ] 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 and the other c h o l i n e m e t a b o l i t e s was d e t e r m i n e d i n both c h l o r o f o r m phase and m e t h a n o l -water phase e x t r a c t e d by the B l i g h and Dyer's method. The r e s u l t s are the average+S.D.; N=5. 66 DISCUSSION The e f f e c t s o f v a s o p r e s s i n on the m e t a b o l i s m of c a r b o -hydrates and phospholipds have been s t u d i e d . The hormone s t i m u -l a t e s d i f f e r e n t membrane e v e n t s i n c l u d i n g the d e g r a d a t i o n and r e s y n t h e s i s of PI, the s o - c a l l e d PI c y c l e (93), and C a + + f l u x e s (94). The enhanced breakdown of PI has been suggested to be due to the hormonal s t i m u l a t i o n of p h o s p h o l i p a s e C a c t i v i t y . The d e c r e a s e i n the c o n c e n t r a t i o n of P t d I n s ( 4 , 5 ) P 2 , P t d I n s 4 P and P t d l n s and the i n c r e a s e of DG f o r m a t i o n i n h e p a t o c y t e s t r e a t e d w i t h v a s o p r e s s i n can be mimicked by exogenous p h o s p h o l i p a s e C (90,95). Our s t u d i e s have demonstrated that v a s o p r e s s i n (10 nM) treatment w i l l d i m i n i s h the r e l e a s e of i n t r a c e l l u l a r CT from rat hepatocytes, which i m p l i e s that the c y t o s o l i c CT can be t r a n s l o -cated to membrane w i t h i n the c e l l s exposed to t h i s hormone. The reduced r e l e a s e of c e l l u l a r CT caused by the a d m i n i s t r a t i o n of v a s o p r e s s i n observed i n t h i s experiment can be e x p l a i n e d as the DG-dependent CT t r a n s l o c a t i o n , s i n c e i t i s w e l l known t h a t DG s t i m u l a t e s the a g g r e g a t i o n o f p a r t i a l l y p u r i f i e d c y t o s o l i c CT from rat l i v e r (84). Studies done i n rat l i v e r c y t o s o l p r e t r e a t e d with phospholipase C e x h i b i t e d a 2.5-fold i n c r e a s e of aggregated form of CT i n the c y t o s o l corresponding to a t w o f o l d e l e v a t i o n of the c o n c e n t r a t i o n of DG (84). Although the p h o s p h o l i p i d precursor of DG i n the r e a c t i o n s c a t a l y z e d by phospholipase C i s g e n e r a l l y a c c e p t e d to be PI, some s t u d i e s argue t h a t the h y d r o l y s i s of other p h o s p h o l i p i d s (PC, PE) might a l s o account f o r the inc r e a s e 67 i n DG (89). I t would be worth f u r t h e r i n v e s t i g a t i o n to see whe-the r or not there i s a PC-cycle corresponding to the a d d i t i o n of v a s o p r e s s i n , s i n c e the m a j o r i t y of p h o s p h o l i p i d i s PC. The reduced r e l e a s e of CT from h e p a t o c y t e s t r e a t e d w i t h v a s o p r e s s i n appeared to depend on the time of i n c u b a t i o n of c e l l s w i t h the hormone. The maximal e f f e c t on the t r a n s l o c a t i o n of CT was observed when the c e l l s had been p r e t r e a t e d with the hormone f o r 3 min. T h i s r e s u l t i s c o m p a t i b l e w i t h the o b s e r v a t i o n of Hughes e_t _al (89) that a t r a n s i e n t i n c r e a s e i n the t o t a l c e l l u l a r c o n c e n t r a t i o n of DG i n h e p a t o c y t e s was i n d u c e d by v a s o p r e s s i n , and the i n c r e a s e r e a c h e d maximal l e v e l s at around 1 min and r e t u r n e d to b a s a l v a l u e by 4 min. The t r a n s i e n t i n c r e a s e i n DG causes a t r a n s i e n t t r a n s l o c a t i o n of CT to membranes, as prolonged i n c u b a t i o n of the c e l l s with v a s o p r e s s i n appeared to r e s u l t i n a r e s t o r e d f a s t r e l e a s e of CT (Fig.13). Thus, the t r a n s l o c a t i o n of CT w i t h i n the c e l l s t r e a t e d with v a s o p r e s s i n seems r e v e r s i b l e i n  v i v o . The same r e v e r s i b i l i t y of CT t r a n s l o c a t i o n has been observ-ed i n HeLa c e l l s t r e a t e d with o l e a t e ( C o r n e l l , R.B., unpublished r e s u l t ) . W h i l e the a d d i t i o n of o l e a t e i n t o the c e l l c u l t u r e promotes a s s o c i a t i o n of c y t o s o l i c CT with membrane and causes the d e c r e a s e d r e l e a s e of CT, the b i n d i n g of o l e a t e from the c e l l c u l t u r e by adding albumin w i l l r a p i d l y reverse t h i s a s s o c i a t i o n between membrane and CT and r e s t o r e the r a p i d r e l e a s e of CT from c y t o s o l . I t has been p r o p o s e d t h a t the t r a n s l o c a t i o n of CT from c y t o s o l to microsomes w i l l s t i m u l a t e the c o n v e r s i o n of phospho-c h o l i n e and CTP to C D P - c h o l i n e and PPi and hence s t i m u l a t e the f o r m a t i o n of PC. T h i s i s because CT i s a r a t e l i m i t i n g enzyme in 68 PC s y n t h e s i s v i a the C D P - c h o l i n e pathway (88). However, i n our v a s o p r e s s i n t r e a t m e n t e x p e r i m e n t , an i n c r e a s e d r a t e of PC b i o -s y n t h e s i s was not observed i n e i t h e r p u l s e - l a b e l i n g (Fig. 15A) or i n p u l s e - c h a s e ( F i g . 16B, T a b l e 4) e x p e r i m e n t s i n s p i t e of the d e c r e a s e d r e l e a s e of c y t o s o l i c CT. One e x p l a n a t i o n of t h i s d i s -c r e p a n c y might be t h a t the i n c r e a s e d c e l l u l a r c o n c e n t r a t i o n of Ca , caused by the a d d i t i o n of v a s o p r e s s i n (94), i n h i b i t s the c o n v e r s i o n of CDP-choline to PC c a t a l y z e d by cholinephosphotrans-f e r a s e (CPT), s i n c e C a + + i s a known i n h i b i t o r of t h i s enzyme. Thus the mass e f f e c t of v a s o p r e s s i n on PC s y n t h e s i s i s not observable. A p o s s i b l e accumulation of CDP-choline i n the vaso-p r e s s i n t r e a t e d h e p a t o c y t e s , due to the b l o c k a g e of the c o n v e r -s i o n of CDP-choline to PC, might have been observed. Unfortunate-l y , the r a d i o a c t i v e p o o l s i z e of C D P - c h o l i n e i n the h e p a t o c y t e s i s so s m a l l t h a t I was u n a b l e to examine t h i s p o s s i b i l i t y . S t u d i e s on the e f f e c t of v a s o p r e s s i n on PC s y n t h e s i s by u s i n g d i f f e r e n t p r e c u s o r s have been done i n r a t h e p a t o c y t e s . P o l l a r d and B r i n d l e y (97) found a 19% i n c r e a s e i n the s y n t h e s i s of t o t a l p h o s p h o l i p i d s (mainly PC) from [ 1 - 1 4 C ] o l e a t e i n W i s t a r -r a t hepatocytes a f t e r v a s o p r e s s i n treatment (100 nM, 30 min). On the other hand, Alemany et: a_l (96) claimed the a d d i t i o n of 100 nM v a s o p r e s s i n to the same c e l l s induced a r a p i d , t r a n s i e n t ( w i t h i n 3 4 min) i n h i b i t i o n of the r a t e of i n c o r p o r a t i o n of [ m e t h y l - H] c h o l i n e i n t o PC. These a s p e c t s were not i n v e s t i g a t e d i n the present work. In a d d i t i o n , I have not done the c o n t r o l experiment to show whether t h e r e i s any e f f e c t of v a s o p r e s s i n on plasma membrane and hence i n t e r f e r e with the p e r m e a b i l i t y of c e l l u l a r 69 p r o t e i n s i n the d i g i t o n i n - m e d i a t e d r e l e a s e of CT experiment. The p r e c i s e e f f e c t of v a s o p r e s s i n on PC s y n t h e s i s , i f any, remains unknown. Another p o s s i b l e r e g u l a t o r y mechanism i n v o l v e d i n the CT t r a n s l o c a t i o n i s a r e v e r s i b l e p h o s p h o r y l a t i o n c y c l e (27,61, 74,85). I n d i r e c t e v i d e n c e showed t h a t d e p h o s p h o r y l a t i o n of the CT promotes b i n d i n g of the enzyme to the E.R. where s p e c i f i c p h o s p h o l i p i d s a c t i v a t e t h i s enzyme. T h i s i n t u r n l e a d s to an i n c r e a s e d r a t e of PC b i o s y n t h e s i s , w h i l e PC s y n t h e s i s i s i n h i -b i t e d a f t e r p h o s p h o r y l a t i o n of CT by a p u t a t i v e cAMP-dependent p r o t e i n kinase (85). Vasop r e s s i n i s w e l l recognized to s t i m u l a t e h e p a t i c carbohydrate metabolism through a C a + + - r e q u i r i n g , cAMP-independent mechanism (98). Recent s t u d i e s done by G a r r i s o n e_t a^ (99) have shown v a s o p r e s s i n can s i m u l a t e the p h o s p h o r y l a t i o n of 10 h e p a t i c c y t o s o l i c p r o t e i n s v i a a C a + + - l i n k e d , cAMP-independent mechanism. S t i l l , evidence showed that the PC b i o s y n t h e s i s v i a PE m e t h y l a t i o n pathway w a s . a l s o r e g u l a t e d by a p h o s p h o r y l a t i o n -d e p h o s p h o r y l a t i o n mechanism (85) and v a s o p r e s s i n s t i m u l a t e d m e t h y l t r a n s f e r a s e through a calmodulin-dependent p r o t e i n kinase (100) . So, whether the b i o s y n t h e s i s of PC i n r a t hepatocytes i s c o n t r o l l e d by a v a s o p r e s s i n s t i m u l a t e d p h o s p h o r y l a t i o n or de-p h o s p h o r y l a t i o n mechanism w i l l be an i n t e r e s t i n g t o p i c f o r f u r -t h e r s t u d i e s . In c o n c l u s i o n , the treatment of rat hepatocytes with vaso-p r e s s i n (5 nM) caused a d i m i n i s h e d r e l e a s e of i n t r a c e l l u l a r CT i n t o the c u l t u r e medium, impl y i n g t r a n s l o c a t i o n of the c y t o s o l i c 3 enzyme to membrane. However, no a l t e r e d r a t e of [ m e t h y l - H] 70 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 was observed i n the hormone t r e a t e d c e l l s . 71 REFERENCES 1. A n s e l l , G.B. & Spanner, S. (1982) i n P h o s p h o l i p i d s (Hawthorne, J.N. & Ansell,G.B., eds.) pp. 1-49, E l s e v i e r Biomedical Press, Amsterdam 2. F o u r c a n s , B. & J a i n , M.K. (1974) Adv. L i p i d Res. 12, 147-226, 3. Kawamoto, T., A k i n o , T., Nakamura, M. & M o r i , M. (1980) Biochim. Biophys. Acta 619, 35-47 4. Ro b i n s o n , N. (1961) J . Pharm. P h a r m a c o l . 13, 321-354 5. Rooney, S.A., Canavan, P.M. & Motoyama, E.K. (1974) B i o c h i m.  Biophys. Acta 360, 56-67 6. F a r r e l l , P.M. & Aver y , M.E. (1975) Am. Rev. Resp. P i s . I l l , 657-688 7. B i l l a h , M.M. & L a p e t i n a , E.G. (1983) P r o c . N a t l . Acad. S c i . , USA 80, 965-968 8. Kennedy, E.P. (1962) Harvey Lect. 57, 143-171 9. B e l l , R.M., B a l l a s , L.M. & Coleman, R.A. (1981) J . L i p i d  Res. 22, 391-403 10. Van Gol d e , L.M.G., Raben, J., B a t e n b u r g , J.J., F l e i s c h e r , B., Zambrano, F. & F l e i s c h e r , S. (1974) B i o c h i m . B i o p h y s .  Acta 360, 179-192 11. J e l s e m a , C.L. & Morre, D.J. (1978) J . B i o l . Chem. 253, 7960-7971 12. Lands, W.E.M. (1960) J . B i o l . Chem. 235, 2233-2237 13. Webster, G.R. (1965) Biochim. Biophys. Acta 98, 512-519 14. M a r i n e t t i , G.V., E r b l a n d , J ., W i t t e r , R.F., P e t i x , J . & S t o l t z , E. (1958) Biochim. Biophys. Acta 30, 223 15. S u n d l e r , R. & Akesson, B. (1975) J . B i o l . Chem. 250, 3359-3367 16. I s h i d a t e , K., Nakagomi, K. & Nakazawa, Y. (1984) J . B i o l .  Chem. 259, 14706-14710 17. McCaman, R.E. & Cook, K. (1978) J . B i o l . Chem. 241, 3390-3394 72 18. S h i g e u r a , H.T., Hen, A.C., Burg, R.W., S k e l l y , B.J. & Hoogsteen,K. (1975) Biochem. Pharmac. 24, 687-691 19. Kanoh, H. & Ohno, K. (1976) Eur. J . Biochem. 66, 201-210 20. Vance, D.E. & P e l e c h , S.L. (1984) Trends Biochem. S c i . 9, 17-20, 21. P e l e c h , S.L., Power, E. & Vance, D.E. (1983) Can. J .  Biochem. C e l l B i o l . 61, 1147-1152 22. Vance, D.E. & Choy, P.C. (1979) T r e n d s Biochem. S c i . 4, 145-148 23. A r t h u r , G., Tarn, S.W. & Choy, P.C. (1984) Can. J . Biochem.  C e l l B i o l . 62, 1059-1063 24. W e i n h o l d , P.A., Feldman, D.A., Quade, M.M., M i l l e r , J.C. & Brooks, R.L. (1981) Biochim. Biophys. Acta 665, 134-144 25. O'Doherty, P.J.A. (1980) Can. J . Biochem. 58, 527-533 26. Paddon, H.B. & Vance, D.E. (1980) B i o c h i m . B i o p h y s . A c t a 620, 636-640 27. P e l e c h , S.L. & Vance, D.E. (1982) J . B i o l . Chem. 257, 14198-14202 28. P e l e c h , S.L., P r i t c h a r d , P.H., B r i n d l e y , D.N. & Vance, D.E. (1983) J . B i o l . Chem. 258, 6782-6788 29. N i s h i j i m a , M., Kuge, 0., Maeda, M., Nakano, A. & Akamatsu, Y. (1984) J . B i o l . Chem. 259, 7101-7108 30. I n f a n t e , J.P. & K i n s e l l a , J.E. (1978) Biochem. J . 176, 631-633 31. Coleman, R. (1973) i n Form and Function of Pho s p h o l i p i d s ( A n s e l l , G.B., Hawthorne, J.M. & Dawson, R.M.C., eds.) 2nd edn., pp. 345-375. E l s e v i e r S c i e n t i f i c P u b l i s h i n g Co., Amsterdam 32. P r i t c h a r d , P.H. & Vance, D.E. (1981) Biochem. J . 196, 261-267 33. Vance, D.E., T r i p , E.M. & Paddon, H.B. (1980) J . B i o . Chem. 255, 1064-1069 34. Choy, P.C, Paddon, H.B. & Vance, D.E. (1980) J . B i o l . Chem. 255, 1070-1073 35. A n s e l l , G.B. & C h o j n a c k i , T. (1969) Methods Enzymol. V o l . 14, 121-125 73 36. S t e r n , W., Kovac, CR. & W e i n h o l d , P.A. (1976) B i o c h i m .  Biophys. Acta 441, 280-293 37. S c h n e i d e r , W.C. (1963) J . B i o l . Chem. 238, 3572-3578 38. F i s c u s , W.G. & S c h n e i d e r , W.C. (1966) J ^ B i o l . Chem. 241, 3324-3330 39. Radominska-Pyrek, A., Matysiak, Z. & Chojnacki, T. (1969) Acta Biochim. Polon. 16, 357-363 40. Choy, P.C., Lim, P.H. & Vance, D.E. (1977) J ^ B i o l . Chem. 252, 7673-7677 41. Choy, P.C. & Vance, D.E. (1976) Biochem. B i o p h y s . Res.  Commun. 72, 714-719 42. Pelech, S.L. (1983) T h e s i s , UBC 43. Vance, D.E., P e l e c h , S.L. & Choy, P.C. (1981) Methods  Enzymol. Vol.71, 576-581 44. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254 45. B l i g h , E.G. & Dyer, W.J. (1959) Can. J . Biochem. P h y s i o l . 37, 911-917 46. Dowhan, W. & H i r a b a y a s h i , T. (1981) Methods Enzymol. V o l . 71, 555-561 47. L a r s o n , T.J., H i r a b a y a s h i , T. & Dowhan, W. (1976) B i ochem. lj>, 974-979 48. C u a t r e c a s a s , P. (1970) J . B i o l . Chem. 245, 3059-3065 49. Hansske, F., S p r i n z l , M. & Cramer, F. (1974) B i o o r g . Chem. 3, 367-376 50. Wu, A.L. & W i n d m u e l l e r , H.G. (1979) J . B i o l . Chem. 254, 7316-7322 51. Marsh, J.B. (1976) J . L i p i d Res. 17, 85-90 52. H a m i l t o n , R.L., W i l l i a m s , M.C, F i e l d i n g , C.J. & H a v e l , R.J. (1976) J . C l i n . I n v e s t . 58, 667-680 53. F e l k e r , T.E., F a i n a r u , M., H a m i l t o n , R.L. & H a v e l , R.J. (1977) J . L i p i d Res. 18, 465-473 54. G u r r , M.I. & James, A.T. (1980) i n L i p i d B i o c h e m i s t r y , 3rd edn., pp. 178-215, Chapman & H a l l , London 55. Z a n n i s , V.I., Lees, A.M., Lee s , R.S. & B r e s l o w , J.L. (1982) J. B i o l . Chem. 257, 4978-4986 74 56. Brown, M.S. & G o l d s t e i n , J.L. (1984) S c i . Amer. 251, 58-66 57. S m i t h , L.C., P o w n a l l , H.J. & G o t t o , A.M. (1978) Ann. Rev.  Biochem. 47, 751-777 58. B e l l - Q u i n t , J ., F o r t e , T., & Granam, P. (1981) Biochem. J . 200, 409-414 59. Bonney, R.J., Wightman, P.D., D a h l g r e n , M.E., Sadowski, S.J., D a v i e s , P., J e n s e n , N., Lanza, T. & Humes, J.L. (1983) Biochem. Pharmac. 32, 361-366 60. Bonney, R.J., Wightman, P.D. & D a v i e s , P. (1979) Biochem.  Pharmac. 28, 2471-2478 61. P e l e c h , S.L., P r i t c h a r d , P.H., B r i n d l e y , D.N. & Vance, D.E. (1983) Biochem. J . 216, 129-136 62. P r i t c h a r d , P.H., C h i a n g , P.K., C a n t o n i , G.L. & Vance, D.E. (1982) J . B i o l . Chem. 257, 6362-6367 63. Tsuge, H., Nakano, Y., O n i s h i , H., Futamura, Y. & O h a s h i , K. (1980) Biochim. Biophys. Acta 614, 274-284 64. W e i n h o l d , P.A. & Sand e r s , R. (1973) L i f e . S c i . 13, 621-629 65. D a v i s , R.A., E n g e l h o r n , S.C., Pangburn, S.H., W e i n s t e i n , D.B. & S t e i n b e r g , D. (1979) J . B i o l . Chem. 254, 2010-2016 66. P e l e c h , S.L., Cook, H.W., Paddon, H.B. & Vance, D.E. (1984) Biochim. Biophys. Acta 795, 433-440 67. M o o k e r j e a , S. (1969) Can. J . Biochem. 47, 125-133 68. Goodman, L.S. & Gilman, A. (1975) The Pharmacological B a s i s  of T h e r a p e u t i c s , pp. 1216-1218. Macmi11ian, London 69. Z e i s e l , S.H., S t o r y , D.L., Wurtman, R.J. & Br uneng r abe r , H. (1980) P r o c . Nat. Acad. S c i . , USA. 77, 4417-4419 70. P e l e c h , S.L., P r i t c h a r d , P.H. & Vance, D.E. (1982) B i o c h i m . Biophys. Acta 713, 260-269 71. Cohen, E.L. & Wurtman, R.J. (1976) S c i e n c e 191, 561-562 72. Plagemann, P.G.W. (1971) J . L i p i d Res. 12, 715-724 73. Z e l i n s k i , T.A., S a v a r d , J.D., Man, R.Y.K., & Choy, P.C. (1980) J . B i o l . Chem. 255, 11423-11428 74. P e l e c h , S.L., P r i t c h a r d , P.H. & Vance, D.E. (1981) J . B i o l .  Chem. 256, 8283-8286 75. S l e i g h t , R. & Kent, C. (1983) J . B i o l . Chem. 258, 824-830, 75 831-835 76. S l e i g h t , R. & Kent, C. (1980) J . B i o l . Chem. 255, 10644-10650 76a. C o r n e l l , R.B. & Maclennan, D.H. (1985) B i o c h i m . B i o p h y s .  Acta In press 77. Esko, J.D. & Raetz, C.R.H. (1980) Pr o c . N a t l . Acad. Sci.,USA 77, 5192-5196 78 Esko, J.D. Wermuth, M.M. & Raetz, C.R.H. (1981) J . B i o l .  Chem. 256, 7388-7393 79. Vance, D.E., T r i p , E.M. & Paddon, H.B. (1980) J . B i o l .  Chem. 255, 1064-1069 80. Choy, P.C, Paddon, H.B. & Vance, D.E. (1980) J . B i o l . Chem. 255, 1070-1073 81. V i g o , C , Paddon, H.B., M i l l a r d , F.C, P r i t c h a r d , P.H. & Vance, D.E. (1981) Biochim. Biophys. Acta 665, 546-550 82. V i g o , C. & Vance, D.E. (1981) Biochem. J . 200, 321-326 83. Whitehead, F.W., T r i p , E. & Vance, D.E. (1981) Can. J .  Biochem. 59, 38-47 84. Choy, P.C, F a r r e n , S.B. & Vance, D.E. (1979) Can. J . Biochem. 57, 605-612 85. P e l e c h , S.L. & Vance, D.E. (1984) i n Enzyme R e g u l a t i o n by  R e v e r s i b l e P h o s p h o r y l a t i o n - F u r t h e r Advances (Cohen P. ed.), pp. 63-80, E l s e v i e r Science P u b l i s h e r s B.V. 86. Hardie, D.G. (1980) i n Mo l e c u l a r Aspects of C e l l u l a r  Regula t i on, V o l . _1. (Cohen , P., ed.) , pp.33-62. E l s e v i e r , Amsterdam. 87. I n g i b r i t s e n , T.S. & Gibson, D.M. (1980) i n Mo l e c u l a r Aspects  of C e l l u l a r R e g u l a t i o n , V o l . 1. (Cohen, P., ed) , pp. 135-152, E l s e v i e r , Amsterdam v 88. P e l e c h , S.L. & Vance, D.E. (1984) B i o c h i m . B i o p h y s . A c t a 779, 217-251 89. Hughes, B.P., Rye, K.A., P i c k f o r d , L.B., B a r r i t t , G.J. & Chalmers, A.H. (1984) Biochem. J . 222, 535-540 90. P f e i l s c h i f t e r , J., K u r t z , A. & Bauer, C. (1984) Biochem. J . 223, 855-859 91. M a c K a l l , J., M e r e d i t h , M. & Lane, M.D. (1979) A n a l . Biochem. 95, 270-274 76 92. Lim, P.H., P r i t c h a r d , P.H., Paddon, H.B. & Vance, D.E. (1983) Biochim. Biophys. Acta 753, 74-82 93. Thomas, A.P., Marks, J.S., C o l l , K.E. & W i l l i a m s o n , J.R. (1983) J . B i o l . Chem. 258, 5716-5725 94. B e r r i d g e , M.J. (1981) Mol. C e l l . Endocr. 24, 115-140 95. B e r r i d g e , M.J. (1983) Biochem. J . 212, 849-858 96. Alemany, S., V a r e l a , I. & Mato, J.M. (1982) Biochem. J . 208, 453-457 97. P o l l a r d , A.D. & B r i n d l e y , D.N. (1984) Biochem. J . 217, 461-469 98. Hems, D.A. & Whitton, P.D. (1980) P h y s i o l . Rev. 60, 1-50 99. G a r r i s o n , J.C., Jo h n s e n , D.E. & C a m p a n i l e , C P . (1984) J . B i o l . Chem. 259, 3283-3292 100. Alemany, S., V a r e l a , I. Har p e r , J.F. & Mato, J.M. (1982) J .  B i o l . Chem. 257, 9249-9251 77 

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