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The biosynthesis of sphingomyelin and the role of phosphatidylcholine as its precursor in baby hamster… Ruff, Blair A. 1981

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THE BIOSYNTHESIS OF SPHINGOMYELIN AND THE ROLE OF PHOSPHATIDYLCHOLINE AS ITS PRECURSOR IN BABY HAMSTER KIDNEY-21F CELLS by BLAIR A. RUFF B . S c , U n i v e r s i t y o f L e t h b r i d g e , 1978 \ THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE 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 a c c e p t t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF A p r i l ® B l a i r A. BRITISH COLUMBIA 1981 R u f f , 1981 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of B i o c h e m i s t r y The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e A p r i l 27, 1981 DE-6 (2/79) A b s t r a c t The l a s t step i n sphingomyelin's de novo b i o s y n -t h e t i c pathway was i n v e s t i g a t e d i n Baby Hamster Kidney (BHK-21F) c e l l s i n t i s s u e c u l t u r e . Three types o f p u l s e -chase experiments were done to t r y to i d e n t i f y the p r e c u r s o r f o r sphingomyelin's phosphocholine moiety. F i r s t , [Me- H]-c h o l i n e was used to monitor the movement of the c h o l i n e moiety i n a l l the p o s s i b l e phosphocholine donors: i e . phos-phocholine, CDP-choline, p h o s p h a t i d y l c h o l i n e , lysophospha-t i d y l c h o l i n e , and g l y c e r o p h o s p h o c h o l i n e . R a d i o a c t i v i t y was observed i n phosphocholine b e f o r e appearing i n p h o s p h a t i d y l -c h o l i n e , g l y c e r o p h o s p h o c h o l i n e , and sphingomyelin. S p e c i f i c r a d i o a c t i v i t i e s of p h o s p h a t i d y l c h o l i n e and sphingomyelin r e v e a l e d a p e c u l i a r p a t t e r n , i f r e p r e s e n t a t i v e of a p r e c u r s o r -product r e l a t i o n s h i p between these two p h o s p h o l i p i d s . T h e i r s p e c i f i c r a d i o a c t i v i t i e s became equal a t 22 hours of ;chase and remained q u i t e s i m i l a r f o r the next 24 hours. The ot h e r two types of pulse-chase experiments both u t i l i z e d p r e l a b e l e d BHK phosphatidyl[Me- H ] c h o l i n e i n p h o s p h o l i p i d v e s i c l e s as t h e i r 'pulse' source. P h o s p h o l i p i d Exchange P r o t e i n ( P L E P ) -mediated exchange and p o l y e t h y l e n e g l y c o l / p h y t o h e m a g g l u t i n i n (PEG/PHA)-mediated f u s i o n between p h o s p h o l i p i d v e s i c l e s and BHK c e l l s were used to i n t r o d u c e the l a b e l e d p h o s p h a t i d y l -c h o l i n e . In a d d i t i o n , i n the ' f u s i o n ' experiments, o t h e r l a b e l e d compounds (glycerophosphocholine and sphingomyelin) were s u b s t i t u t e d f o r l a b e l e d p h o s p h a t i d y l c h o l i n e . PLEP-- i i i -mediated exchange of l a b e l e d p h o s p h a t i d y l c h o l i n e d i d not r e s u l t i n enough t r a n s f e r of r a d i o a c t i v i t y i n t o the c e l l to adequately monitor i n d i v i d u a l c e l l p h o s p h o l i p i d s or any t r a n s f e r of l a b e l - i e . from p h o s p h a t i d y l c h o l i n e to sphingo-m y e l i n . However, PEG/PHA-mediated f u s i o n of v e s i c l e s and c e l l s d i d r e s u l t i n enough r a d i o a c t i v i t y showing up i n the c e l l s . When l a b e l e d p h o s p h a t i d y l c h o l i n e was used, the r a d i o -a c t i v i t y r a t i o between i t and sphingomyelin averaged around 16, depending on the l e n g t h of the chase (2-52 h o u r s ) m The use of e i t h e r l a b e l e d glycerophosphocholine or sphingomyelin r e s u l t e d i n a r a t i o of about 1.8. One ' c o l d - t r a p 1 experiment was done by i n c l u d i n g a l a r g e amount of u n l a b e l e d g l y c e r o p h o s p h o c h o l i n e with l a b e l e d p h o s p h a t i d y l c h o l i n e i n the v e s i c l e p r e p a r a t i o n . The r e s u l t a n t r a d i o a c t i v i t y i n sphingomyelin was 50% l e s s than p r e v i o u s l y , but p h o s p h a t i d y l c h o l i n e ' s had remained the same. The evidence seems to i n d i c a t e a r e v e r s i b l e p r e c u r s o r -product r e l a t i o n s h i p between p h o s p h a t i d y l c h o l i n e and sphingo-myelin, but does not c l e a r l y show whether or not any o t h e r i n t e r m e d i a t e (such as g l y c e r o p h o s p h o c h o l i n e ) i s a l s o i n v o l v e d . - i v -LIST OF ABBREVIATIONS AdoHCy S-adenosylhomocysteine AdoMet S-adenosylmethionine ADP adenosine diphosphate ATP adenosine t r i p h o s p h a t e BHK c e i l s Baby Hamster Kidney - 21F c e l l s CDP-choline c y t i d i n e diphosphocholine CDP c y t i d i n e diphosphate C i c u r i e CMP c y t i d i n e monophosphate CoA coenzyme A cone . . c o n c e n t r a t i o n CTP c y t i d i n e t r i p h o s p h a t e dpm d i s i n t e g r a t i o n s per minute EDTA e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d F i g F i g u r e g gram G-P-choline.....glycerophosphocholine h . . . hour (s) 1 l i t e r LPC l y s o p h o s p h a t i d y l c h o l i n e LPE 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 molar MEMA - 5% CS . . . Dulbecco" s M o d i f i e d Eagle':s Medium, and 5% c a l f serum (growth medium) mg m i l l i g r a m M199 - 2% CS . . . E a r l e ' s Unmodified S a l t s S o l u t i o n (maintenance medium when the 2% c a l f serum i s included) -v-Mg magnesium i o n min • • • • .minute ml m i l l i l i t e r mm m i l l i m e t e r NAD n i c o t i n a m i d e adenine d i n u c l e o t i d e NADP n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate nmol nanomole PEG p o l y e t h y l e n e g l y c o l PHA phytohemagglutinin PBS .phosphate b u f f e r e d s a l i n e , pH 7.4 (Dulbecco's) PC p h o s p h a t i d y l c h o l i n e ( l e c i t h i n ) P - c h o l i n e phosphocholine PE phosphatidylethanolamine PI p h o s p h a t i d y l i n o s i t o l PS p h o s p h a t i d y l s e r i n e P i . i n o r g a n i c phosphate PPi i n o r g a n i c pyrophosphate PLEP P h o s p h o l i p i d Exchange P r o t e i n PL, p h o s p h o l i p i d ! R any long c h a i n f a t t y a c i d R^ r a t i o of the d i s t a n c e t r a v e l l e d by a compound to t h a t t r a v e l l e d by the s o l v e n t f r o n t SM sphingomyelin s.d standard d e v i a t i o n TLC . t h i n - l a y e r chromatography T r i s t r i s (hydroxymethyl) aminomethane UV u l t r a v i o l e t umol micromole - v i -TABLE OF CONTENTS Page ABSTRACT • 1 1 ACKNOWLEDGEMENTS x i i LIST OF ABBREVIATIONS IV LIST OF TABLES i X LIST OF FIGURES X INTRODUCTION I. Statement o f the Problem..... 1 I I . Sphingomyelin 1 I I I . S t u d i e s on Sphingomyelin's Phosphocholine.... 2 1. C y t i d i n e Diphosphocholine 2. Phosphocholine 3. P h o s p h a t i d y l c h o l i n e 4. C y t i d i n e Diphosphocholine v i a P h o s p h a t i d y l c h o l i n e 5. Others IV. My Method of I n v e s t i g a t i o n 14 MATERIALS AND METHODS I. Chemicals and R a d i o a c t i v e Compounds 18 I I . General Methods 1. T h i n Layer Chromatography 18 ( i ) S o l v e n t s f o r Water-Soluble Compounds A) S i n g l e Dimensional Systems B) Two Dimensional Systems C) P r e p a r a t i o n of Acid-Washed Charcoal ( i i ) S o l v e n t s For L i p i d s A) S i n g l e Dimensional System - v i i -Page 2. L i q u i d S c i n t i l l a t i o n Counting 21 3. Measurement of Wet Weight and P r o t e i n of C e l l s 22 4. Measurement of P h o s p h o l i p i d -Phosphorus 23 I I I . C e l l C u l t u r e 23 3 IV. I n c o r p o r a t i o n of [Me- H ] c h o l i n e i n t o BHK C e l l s 1. Pulse-Chase Experiments 24 2. Long-Term L a b e l i n g of BHK P h o s p h o l i p i d s 28 V. Pool S i z e Measurements o f P h o s p h o l i p i d s . . . 29 VI. S p e c i f i c . R a d i o a c t i v i t y Determination of P h o s p h o l i p i d s 29 V I I . P r e p a r a t i v e Procedures 1. I s o l a t i o n of PLEP Supernatant F r a c t i o n s From Rat L i v e r 30 2. P r e p a r a t i o n of P h o s p h o l i p i d V e s i c l e s 31 V I I I . Procedures f o r I n t r o d u c i n g [Me-^H]-c h o l i n e Labeled P h o s p h o l i p i d s i n t o BHK C e l l s 1. PLEP-Mediated Exchange of P h o s p h o l i p i d s Between P h o s p h o l i p i d V e s i c l e s and BHK-21F C e l l s 33 2. PEG/PHA-Mediated Fusion Between P h o s p h o l i p i d V e s i c l e s and BHK-21F C e l l s 35 RESULTS AND DISCUSSION I. Growth Parameters of BHK-21F C e l l s During the Chase P e r i o d 38 1. We t We i g h t 2. P r o t e i n C o n c e n t r a t i o n s 3. Phospholipid-Phosphorus Concen-t r a t i o n s - v i i i -Page I I . S e p a r a t i o n and I d e n t i f i c a t i o n o f C h o i i n e - C o n t a i n i n g Compounds 43 1 . Water-Soluble Compounds 2. P h o s p h o l i p i d s 3 I I I . I n c o r p o r a t i o n of [Me- H] c h o l i n e i n t o BHK C e l l s 1 . Pulse-Chase S t u d i e s 5 1 2. Long-Term L a b e l i n g S t u d i e s 6 1 IV. How P h o s p h o l i p i d S p e c i f i c R a d i o a c t i v i t i e s R e l a t e to t h e i r P r e c u r s o r - P r o d u c t R e l a t i o n s h i p s 6 2 V. Mo n i t o r i n g R e l a t i v e P h o s p h o l i p i d Pool S i z e s During the Chase P e r i o d 6 5 3 VI. Can Ph o s p h a t i d y l [Me- H] c h o l i n e be Incorporated i n t o BHK C e l l s ' Plasma Membrane by a PLEP-Mediated Exchange Reaction? 6 9 V I I . I n c o r p o r a t i o n of R a d i o a c t i v e l y Labeled Compounds by PEG/PHA-Mediated Fusion Techniques 7 8 3 1. Ph o s p h a t i d y l [Me- H ] c h o l i n e 2. [Me - l 4 c]glycerophosphocholine 3. [Me-^H]sphingomyelin CONCLUSION I. What Can Pulse-Chase S t u d i e s Show? 9 0 I I . I n t e r p r e t a t i o n o f my R e s u l t s from the Pulse-Chase Stud i e s Presented Here 9 0 I I I . R e s u l t s From Pr e v i o u s I n v e s t i g a t i o n s i n View of t h i s Study 9 5 IV. What Could be Done to Confirm the Conclusions Derived from my I n v e s t i g a t i o n 9 7 REFERENCES 1 0 0 - i x -LIST OF TABLES T a b l e Page 1 Comparison of C e l l Growth Parameters Du r i n g the Chase P e r i o d 40 2 Mole P e r c e n t C o m p o s i t i o n o f P h o s p h o l i p i d s i n BHK C e l l s 68 3 3 I n c o r p o r a t i o n o f [Me- H] c h o l i n e L a b e l e d P h o s p h a t i d y l c h o l i n e i n t o BHK C e l l s 71 4 PLEP-Mediated Exchange o f L a b e l e d P h o s p h a t i d y l [Me-^H] c h o l i n e between P C - v e s i c l e s and BHK C e l l s 75 3 5 I n c o r p o r a t i o n o f P h o s p h a t i d y l [Me- H]-c h o l i n e L a b e l e d PC V e s i c l e s i n t o the Plasma Membrane of BHK C e l l s by PEG-Mediated Fas i o n 79 6 I n c o r p o r a t i o n o f PC/PS V e s i c l e s L a b e l e d w i t h P h o s p h a t i d y l [Me_- H] c h o l i n e i n t o the Plasma Membrane of BHK C e l l s by PEG-Mediated F u s i o n 82 7 I n c o r p o r a t i o n of PC/PS V e s i c l e s C o n t a i n i n g R a d i o a c t i v e l y L a b e l e d Compounds i n t o the Plasma Membrane of BHK C e l l s by PEG-Mediated F u s i o n . 85 8 I n c o r p o r a t i o n o f PC/PS V e s i c l e s C o n t a i n i n g W a t e r - S o l u b l e Compounds and P h o s p h o l i p i d s R a d i o a c t i v e l y L a b e l e d i n t o the P l a s m a Membrane of BHK C e l l s by PEG-Mediated F u s i o n 86 -X-LIST OF FIGURES F i g u r e Page 1 Pathways f o r the B i o s y n t h e s i s of P h o s p h a t i d y l c h o l i n e 2 2 Proposed S t r u c t u r e f o r S p h i n g o s i n e i n 1916 4 3 S t r u c t u r e f o r S p h i n g o s i n e Proposed i n 1933. 5 4 E s t e r i f i c a t i o n o f S p h i n g o m y e l i n Proposed i n 1940 5 5 C u r r e n t C o n c e p t i o n o f S p h i n g o s i n e 1 s S t r u c t u r e 6 6 C u r r e n t C o n c e p t i o n o f S p h i n g o m y e l i n ' s S t r u c t u r e 6 7 D e p i c t i o n o f S p h i n g o s i n e 1 s S t e r e o -c h e m i c a l S t r u c t u r e 7 8 Pathway o f the de novo B i o s y n t h e s i s o f Ceramide 9 9,10 Photographs of BHK-21F C e l l s D e p i c t i n g V a r i o u s Stages o f P l a t e C o n f l u e n c y . . 25,26 11 Parameters o f BHK C e l l Growth During the Chase P e r i o d 39 12 E f f e c t s o f Growth-Medium Changes on the Wet Weight o f C e l l s as a F u n c t i o n o f Time 42 13 E f f e c t s o f C a r r i e r - S t a n d a r d s on the M o b i l i t y o f Sample R a d i o a c t i v i t y on T h i n - L a y e r Chromatograms .. 44 14 E f f e c t o f an A c i d i c S o l v e n t on the M o b i l i t y o f G l y c e r o p h o s p h o c h o l i n e and Sample R a d i o a c t i v i t y on T h i n - L a y e r Chroma tograms 46 - x i -Page 15 E f f e c t s o f C a r r i e r - S t a n d a r d s on the M o b i l i t y of Sample R a d i o a c t i v i t y on T h i n - L a y e r Chromatograms 48 3 16 I n c o r p o r a t i o n o f [Me- H ] c h o l i n e i n t o V a r i o u s C h o l i n e - C o n t a i n i n g Compounds i n BHk C e l l s 52 3 17 E f f e c t s o f Doubled [Me- H ] c h o l i n e C o n c e n t r a t i o n on i t s I n c o r p o r a t i o n i n t o V a r i o u s Compounds i n BHK C e l l s 53 18 E f f e c t s o f I n c r e a s e d C e l l P o p u l a t i o n on the I n c o r p o r a t i o n of [Me-~*H] -c h o l i n e i n t o V a r i o u s Compounds i n BHK C e l l s 54/55 19 S e m i l o g P l o t o f the R a d i o a c t i v i t y i n P h o s p h o c h o l i n e D u r i n g the Chase P e r i o d 58 3 20 C e l l - I n c o r p o r a t e d [Me- H ] c h o l i n e R a d i o a c t i v i t y S u b s e q u e n t l y D e t e c t e d i n the Chase-Medium 60 21 S p e c i f i c R a d i o a c t i v i t y o f [Me- 3H]-c h o l i n e L a b e l e d P h o s p h o l i p i d s i n BHK C e l l s 63 22 P h o s p h o l i p i d P o o l S i z e s i n BHK C e l l s 67 23 PLEP A c t i v i t y Time Course Experiments 74 24 Proposed R e a c t i o n s L e a d i n g t o the Form a t i o n o f S p h i n g o m y e l i n 98 - x i i -Acknowledgements I would l i k e to thank Dr. Dennis E. Vance f o r h i s continued support and guidance. I am a l s o g r a t e f u l t o : Mr. Steve P e l e c h f o r h i s h e l p f u l d i s c u s s i o n s and h i s con-s t r u c t i v e comments on t h i s t h e s i s , Dr. P. Haydn P r i t c h a r d f o r h i s many h e l p f u l s uggestions i n the l a b , Mr. Harry Paddon f o r h i s i d e a of u s i n g c h a r c o a l to h e l p separate glycerophosphocholine from CDP-choline and f o r h i s and Mr. P i e r r e T a l b o t ' s a s s i s t a n c e i n the t i s s u e c u l t u r e of BHK-21F c e l l s , and Ms. Sandi Anderson f o r her p a t i e n c e i n r e c e i v i n g and t y p i n g t h i s t h e s i s . I a l s o wish to express my s p e c i a l thanks to my wif e L a u r i e , f o r her constant love and encour-agement. -1-I n t r o d u c t i o n I. Statement of the Problem There has been a l o t of r e s e a r c h d u r i n g the past 25 years on the r e a c t i o n i n which phosphocholine i s i n c o r -porated i n t o sphingomyelin (SM) during i t s b i o s y n t h e s i s (References 1-42). T h i s r e a c t i o n remains u n c e r t a i n p r i m a r i l y because there are s e v e r a l p o s s i b l e c a n didates f o r the phos-phocholine donor - ( i . e . phosphocholine, CDP-choline, c h o l i n e , and p h o s p h a t i d y c h o l i n e ) . A l l these 'candidates' are i n v o l v e d i n the de novo b i o s y n t h e t i c pathv/ay of another major phos-p h o l i p i d , p h o s p h a t i d y l c h o l i n e (PC) ( F i g u r e 1). T h e r e f o r e , the i n v e s t i g a t i o n of the b i o s y n t h e t i c r e l a -t i o n s h i p s between these two p h o s p h o l i p i d s (PC and SM) and the i d e n t i f i c a t i o n of the donor of the phosphocholine moiety found i n sphingomyelin (SM) have been the major o b j e c t i v e s of t h i s t h e s i s . I I . Sph ingomyelin S t u d i e s on sphingomyelin have progressed i n an o r d e r l y way, beginning with i t s chemical c h a r a c t e r i z a t i o n based on the d i f f e r e n t i s o l a t i o n procedures employed. T h i s was q u i c k l y f o l l o wed by crude s t r u c t u r a l s t u d i e s . The chemical s y n t h e s i s and b i o s y n t h e t i c pathway of sphingomyelin took longer to determine as d i d i t s s t e r e o c h e m i c a l c o n f i g u r a t i o n . In f a c t -2-C h o l i n e Plasma Membrane C h o l i n e ATP-Mg ++ ADP-Mg <• (1) B e t a i n e - a l d e h y d e P h o s p h o c h o l i n e ++ CTP-Mg PPi + Mg + +< (2) V CDP-choline 1 , 2 - d i a c y l g l y c e r o l ++ Mg CMP v P h o s p h a t i d y l -e t h a n o l amine (3) 3 AdoMet 3 AdoHCy Acyl-CoA G-P-choline so-PC + F a t t y a c i d Lyso-PC F i g u r e 1. Pathways f o r the B i o s y n t h e s i s of P h o s p h a t i d y l c h o l i n e . The enzyme names are: (1) C h o l i n e Kinase(EC 2.7.1.32); (2) Phosphocholine C y t i d y l y l t r a n s f e r a s e (EC 2.7.7.15);(3).Choline Phosphotransferase (EC 2.7.8.2); (4) C h o l i n e Dehydrogenase (EC 1.1.99.1); (5) Betaine-aldehyde Dehydrogenase (EC 1.2.1.8). -3-s p h i n g o m y e l i n 1 s b i o s y n t h e s i s remains an area of a c t i v e r e s e a r c h today. The i n v e s t i g a t i o n i n t o s p h i n g o l i p i d s began i n 1874 (by Thudichum) although the term ' s p h i n g o l i p i d 1 wasn't proposed u n t i l 1947, by C a r t e r (43). T h i s f a m i l y of complex l i p i d s i s d e r i v e d from the l o n g - c h a i n aminoalcohol, sphingo-s i n e (Refer to F i g . 6 and 7) or r e l a t e d homologous bases, as w e l l as the hydroxy and s a t u r a t e d d e r i v a t i v e s of these compounds. In animals, sphingosine i s the major base mole-c u l e . T h i s i s d i f f e r e n t i n p l a n t s , where phytosphingosine (hydroxylated d i h y d r o s p h i n g o s i n e ) i s the major l o n g - c h a i n base. The a c t u a l ' d i s c o v e r y ' of sphingomyelin i s a t t r i b u -ted to Thudichum (44) i n 1884. The substance Thudichum worked on, c a l l e d "protagon", was a c t u a l l y comprised of a mixture of b r a i n p h o s p h o l i p i d s . T h i s was demonstrated by subsequent f u r t h e r p u r i f i c a t i o n by Rosenheim and Tebb (45-48). Because of the i m p u r i t i e s i n Thudichum's protagon, the products of h i s h y d r o l y s i s experiments were questioned by Levene (49-51). These products had been i d e n t i f i e d as; s p h i n g o s i n e , neurine, s p h i n g o s t e a r i c a c i d , phosphoric a c i d , and s p h i n g o l . Although Thudichum d i d e s t a b l i s h the p r i n c i p a l ' d i s t i n c t i o n ' of t h i s phosphatide from those of the l e c i t h i n group, most of h i s o t h e r d a t a was judged as not accurate (51). T h i s ' d i s t i n c t i o n ' was the absence of g l y c e r o l i n -4-the sphingomyelin molecule. One other major d i f f e r e n c e t h a t should be noted i s the 2:1 ( r a t i o ) r a t h e r than 1:1 r a t i o of n i t r o g e n atoms to phosphorus atoms per molecule of sphingomyelin. L a t e r work by Levene (49, 50) y i e l d e d a sphingomyelin p r e p a r a t i o n with "a very i n s i g n i f i c a n t p r o p o r t i o n of g a l a c -t o s i d e " . I t was composed of; phosphoric a c i d , l i g n o c e r i c a c i d , c e r e b r o n i c a c i d , c h o l i n e , s p h i n g o s i n e , and another base ( c i 7 H 3 5 N ° ) l a t e r i d e n t i f i e d as a secondary product of sphingosine h y d r o l y s i s (51). The c e r e b r o n i c a c i d was no longer d e t e c t e d a f t e r the p u r i t y of the sphingomyelin p r e p a r a t i o n was, increased and c e r e b r i n was e l i m i n a t e d . The s t r u c t u r e f i n a l l y proposed by Levene (F i g u r e 2) was also based on work with kidney sphingomyelin (51). O-C, -,H_,0 (OH)NH. C0C o oH._ (or another / 1 7 3 2 2 3 4 7 r a d i c l e ) F i g u r e 2) 0 =P-OH . O.C..H.. oN(0H) In 1933, Fr a n k e l and co-workers (52) prepared sphingo-myelin which contained e q u i v a l e n t amounts of s p h i n g o s i n e , c h o l i n e , phosphoric a c i d and f a t t y a c i d s . The f a t t y a c i d s were i d e n t i f i e d as p a l m i t i c (C-, 6), s t e a r i c ( C 1 8 ) and l i g n o -c e r i c (^24) a c i d s . The s t r u c t u r e by F r a n k e l and h i s co-workers i s shown below. -5-OH OH Fi g u r e 3) I I CH., - (CH.,) ,,CH=CH-CH-CH-CH -O-P-O-C H. 3 2 12 | z || | NH ° NE(CH 7). ?° OH R ( f a t t y acid) A f t e r the i n i t i a l composition was determined, the main s t r u c t u r a l problem to be e l u c i d a t e d i n v o l v e d the o r d e r o f the s i n g l e amino and two hydroxyl groups. These were l o c a t e d at one end of the sphingosine carbon backbone. Klenk and Diebold (53) hel d s i m i l a r views to F r a n k e l e t a l . (52) on the order of these groups i n sph i n g o s i n e . The former group (53) were the f i r s t to propose the 1,2-dihydroxy-3-amino octadecene-4 s t r u c t u r e as seen i n F i g -ure 3. Thannhauser and R e i c h e l (54,55) claimed t h a t sphingo-myelin was present i n animal t i s s u e as a mixture of f r e e and e s t e r i f i e d sphingomyelin. P a l m i t i c a c i d was thought to be j o i n e d i n an e s t e r l i n k a g e (C-3) and l i g n o c e r i c a c i d i n an amide l i n k a g e ( F i g u r e 4 ) . 0 F i g u r e 4) ^ H O - C ( C H 2 ) 1 4 ~ C H 3 ( P a l m i t i c acid) ^ HO 0 I II CH.,(CH„),_-CH=CH-CH-CH-CH„-NH-C-R ( f a t t y acid) 3 2 12 I 2 0 I HO-P=0 I 0-CH 2CH 2-N(CH 3) 3"0H L a t e r work by Thannhauser and Schmidt (56) proved t h a t the e s t e r i f i c a t i o n data came from contamination by a s a t u r a t e d -6-l e c i t h i n . To date, t h e r e i s no evidence that sphingomyelin e x i s t s i n t h i s e s t e r i f i e d form ( F i g u r e 4 ) . The sequence of the amino and two hydroxy groups i n F i g u r e 4 are not new. Lapworth (57) and another group, Levene and West (58-60), had a l s o proposed t h i s arrangement, but i n a C^^ sphingosine molecule: 1-amino-2,3-d ihydroxyheptadecene-4. F i n a l l y , i n 1942, the proposed s t r u c t u r e f o r sphingo-s i n e was again m o d i f i e d (see F i g u r e 5 ) , t h i s time by C a r t e r (61). The s t r u c t u r a l formula i s 1,3-dihydroxy-2-aminooctadiene C o n c l u s i v e evidence using p e r i o d a t e - o x i d a t i o n methods was report e d i n s e v e r a l p u b l i c a t i o n s from t h i s group (43,61-63). Using s i m i l a r methods, a d d i t i o n a l s t u d i e s (64,65) determined the p o s i t i o n of the phosphocholine bond i n sphingomyelin to be in agreement with the sphingosine s t r u c t u r e i n F i g u r e 5. T h i s arrangement i s i n accordance with our pr e s e n t c o n c e p t i o n of sphingomyelin ' s s t r u c t u r e as i l l u s t r a t e d i n F i g u r e 6. F i g u r e 5) CH 3(CH 2) 1 2CH=CH-CH-CH -CH 2 OH NH 2 OH H H H 0 ... .CH. F i g u r e 6) l I I // (+) / 3 CH_— (CH_ ) , C='C -C -C -CH o-0-P-0-CH -CH -N —CH., 3 2 1 2 l | | 2 \ 2 2 \ J H HO NH O (-) CH., I J C=0 I ( C j : H2 ) 22 CH 3 -7-A l l t h a t remained to be determined i n the a b s o l u t e s t r u c t u r e of sphingomyelin was i t s s t e r e o c h e m i s t r y . There are two f e a t u r e s t h a t p l a y an important r o l e i n sphingo-myelin's s t e r e o c h e m i c a l c o n f i g u r a t i o n : 1) the presence of a double bond, and 2) asymmetric carbon atoms. Both of these f e a t u r e s occur i n the sphingosine component o f sphingo-myelin. The f i r s t p o i n t to be r e s o l v e d was the s t e r e o c h e m i s t r y of the double bond between carbons 4 and 5 of sphingosine. Evidence f o r i t s trans-geometry i s now abundant (66-71). The second p o i n t proved doubly c o n f u s i n g as there are two asymmetric carbon atoms i n sphingosine (C-2 and C-3). T h e r e f o r e the base (trans-1,3-dihydroxy-2-amino-4-octadecene) can e x i s t i n four o p t i c a l l y a c t i v e s t e r e o i s o m e r s , i . e . D-and L - e r y t h r o , and D~and L-thr e o forms. N a t u r a l o c c u r i n g sphingosine i s i n the D-erythro form (62,63,72-84) as p r e -sented i n F i g u r e 7. F i g u r e 7) n H / \ C H 2 - ( C H 2 ) I I - C H 3 The chemical s y n t h e s i s of sphingomyelin s t a r t e d with i t s base p r e c u r s o r s , d i h y d r o s p h i n g o s i n e and sphingosine (75,85-89). The f i r s t chemical s y n t h e s i s o f sphingomy-e l i n was by S h a p i r o and h i s co-workers (90). An easy method v/as developed i n 1962 by the same group (83). Dihydrosphingo--8-myelin was al s o s y n t h e s i z e d i n a s i m i l a r manner a few years e a r l i e r (91). St u d i e s on the in_ v i t r o enzymatic s y n t h e s i s of sphingo-s i n e began around 1958 (92,93) and continued throughout the 1960's (6,94). Isotope l a b e l i n g experiments _in v i v o were done e a r l i e r to determine the o r i g i n a l source of the carbon atoms i n sphingosine, d i h y d r o s p h i n g o s i n e , and phyto-sphingosine (95-101). During the l a t e 1960's and e a r l y 1970's most of the enzymatic pathway f o r sphingomyelin b i o -s y n t h e s i s was e s t a b l i s h e d (71,102-10 8 ) . Some of the h i g h -l i g h t s of these i n v e s t i g a t i o n s f o l l o w . In 1953, Zabin and Mead (96,97) demonstrated t h a t i n the r a t , sphingosine ( F i g u r e 5) i s sy n t h e s i z e d p r i m a r i l y from a c e t a t e . When c a r b o x y l - l a b e l e d a c e t a t e was adminis-ter e d to weanling r a t s , the sphingosine i s o l a t e d from the b r a i n and c a r c a s s had the same s p e c i f i c r a d i o a c t i v i t y as the f a t t y a c i d s i n the sphingosine f r a c t i o n . Since carbons 14 1 and 2 contained no C, they b e l i e v e d these r e s u l t s r u l e d out a b i o s y n t h e s i s of sphingosine by condensation o f a C-16 a c i d with ethanolamine. I t was more l i k e l y t h a t s e r i n e was u t i l i z e d i n t h i s b i o s y n t h e s i s (6,92-97). The f i n a l enzymatic pathway f o r the formation of sphingosine i n v o l v e s three steps ( F i g u r e 8) as deduced by s e v e r a l i n v e s t i g a t o r s (71,102-108). The experimental evidence p u b l i s h e d by Braun and S n e l l (102) d e s c r i b e d pyridoxalphosphate as an e s s e n t i a l c o f a c t o r -9-CH 3(CH 2) 1 3CH 2C-S-CoA 0 (palmitoy1-CoA) CoASH HOOC-CH-CH„-OH NH 2 (serine) + H-C=0 0 (-) " 0 W—P-0-H„C-I 2 0 -OH ( p y r i d o x a l phosphate) NADPH + H NADP CH-(CH„) n .-C-CH-CH„-OH -5 2 l f l || j 2 0 NH 2 (3-keto-sphinganine) CH 0 (CH 2) 1 4-CH—CH-CH^-OH 1 1 1 l l OH NH. CH_.(CH„),„-CH=CH-CH CH-CH--OH ' j 2 12 j I 2 OH NH„ (sphinganine) (trans-4-sphingenine) (4) R-C-X H 0 C H 0 ( C H 2 ) 1 4 ~ C H CH-CHo-0H I OH NH I R-C=0 (N-acy1-sphinganine) v — CH_(CH„),„-CH=CH-CH CH-CH„-OH j 2 12 I I 2 OH NH I R-OO (N-acyl-trans-4-sphingenine) (ceramide) F i g u r e 8. Pathway of the de novo B i o s y n t h e s i s of Ceramide. The enzyme r e a c t i o n s are: (1) Synthase and (2) Reductase (both bound to the endoplasmic r e t i c u l u m membrane) ;(3) Desa-tur a s e ;(4) Sphingosine A c y l t r a n s f e r a s e (EC 2.3.1.24). The (X) may r e p r e s e n t a hydrogen atom o r CoAS. - l O -i n the proposed condensation step (103) t h a t l e d to the f i r s t long chain base i n t e r m e d i a t e , 3-dehydrosphinganine ( F i g u r e 8). T h i s synthase r e a c t i o n and the subsequent NADPH dependent r e d u c t i o n o f the c a r b o n y l group was performed e n z y m a t i c a l l y using a microsomal f r a c t i o n (100,000 x g p e l l e t ) from the yeast, Hansenula c i f e r r i , as the enzyme source (104). The reductase enzyme was c h a r a c t e r i z e d as a l i p o p r o t e i n (104). S i m i l a r r e a c t i o n s as those d e s c r i b e d above were a l s o demonstrated with mouse b r a i n microsomal f r a c t i o n s (105). In 1971, F u j i n o and Nakano (106) proposed t h a t the desaturase enzyme used 3-dehydrosphinganine as the s u b s t r a t e to i n t r o d u c e the trans double bond ( i n p o s i t i o n 4 o f sphingosine) b e f o r e r a t h e r than a f t e r (92, 93) i t s r e d u c t i o n ( F i g u r e 8 ) . S e v e r a l o t h e r i n v e s t i g a t o r s (107-109) have suggested t h a t sphinganine ( d i h y d r o s p h i n g o s i n e ) i s a c y l a t e d to form N-acylsphinganine (dihydroceramide) which i s f o l l o w e d by the i n t r o d u c t i o n of the double bond t o form ceramide. Although the exact sequence of r e a c t i o n s has not been f u l l y r e s o l v e d , the o r i g i n a l i n c l u s i o n o f a f l a v i n r e c e p t o r no longer seems t o be favored (107-113). The enzyme, sphingosine a c y l t r a n s f e r a s e , t h a t c a t a l y z e d the l a s t step i n the formation of ceramide ( F i g u r e 8) had been d e s c r i b e d p r e v i o u s l y (10), using both t h r e o - and e r y t h r o -sphingosine s u b s t r a t e s . There are s e v e r a l good reviews by S t o f f e l ( 1 0 9 , 1 1 0 , n i ) and others (112, 113), t h a t d e a l e x t e n s i v e l y w i t h the s y n t h e s i s (chemical and enzymatic) and degradation o f sphingomyelin and i t s p r e c u r s o r s . -11-I I I . S t u d i e s on Sphingomyelin's Phosphocholine Moiety The next major i n t e r m e d i a t e a f t e r sphingosine, i s the formation of i t s N-acyl d e r i v a t i v e , ceramide. T h i s i s achieved i n one simple a c y l t r a n s f e r step ( F i g u r e 8) , which i s supposedly f o l l o w e d by the a d d i t i o n of phosphate and c h o l i n e to the h y d r o x y l group i n the C - l p o s i t i o n of c e r a -mide ( F i g u r e 6). T h i s has been d i s p u t e d from time to time by those who thought t h a t these two compounds co u l d be added t o sphingosine f i r s t (7,13,14,16). However, the m a j o r i t y of the r e s e a r c h has e i t h e r supported (9,17,21,27,40) o r accepted ceramide as the immediate p r e c u r s o r to sphingomyelin (2,3, 4,10-12,15,16,22-24, 30-38, 40, and 41). In the e a r l y 1950*s, i t was s t i l l not known how the phosphate and c h o l i n e groups were added t o ceramide. In 1956, a paper on PC b i o s y n t h e s i s by Kennedy and Weiss (1) suggested t h a t phosphate and c h o l i n e might be t r a n s f e r r e d t o g e t h e r from CDP-choline t o the d i g l y c e r i d e molecule to form p h o s p h a t i d y l c h o l i n e . I t was known t h a t p h o s p h a t i -d y l c h o l i n e and sphingomyelin have s i m i l a r phosphocholine m o i e t i e s i n a monophosphate d i e s t e r l i n k a g e . T h e r e f o r e a r e a c t i o n analogous to the one i n 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 might a l s o apply to sphingomyelin b i o s y n t h e s i s , as proposed by Kennedy's l a b (1,2,3,4) i n the f o l l o w i n g r e a c t i o n : (1) N-acyl sphingosine + CDP-choline ^ r r i SM + CMP Indeed, evidence f o r such a scheme, u s u a l l y i n -v o l v i n g ceramide i n the t h r e o c o n f i g u r a t i o n (3-6,8,9,16,17,21, 26,27,38,40,41), had been p r o v i d e d as e a r l y as 1957 by -12-S r i b n e y and Kennedy (3) and as r e c e n t l y as 1980 by S t o f f e l and Melzner (40). I t was Sribney and Kennedy (3,4) who were the f i r s t to d e s c r i b e the enzyme (CDP-choline: N - a c y l s p h i n g o s i n e 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 ) t h a t they thought was r e s p o n s i b l e f o r c a t a l y s i n g the above r e a c t i o n . A s i m i l a r enzyme (CDP-ethanolamine: ceramide ethanolamine-phosphotransferase) has l i k e w i s e been d e s c r i b e d f o r the s y n t h e s i s o f threo ceramide phosphorylethanolamine i n c h i c k e n and r a t l i v e r (26). Another r e a c t i o n with ceramide, t h i s time i n v o l v i n g phosphocholine, was a l s o c o n s i d e r e d , but no c o n c l u s i o n s r e s u l t e d from those s t u d i e s (9,25). In a d d i t i o n , other i n v e s t i g a t o r s proposed a r e a c t i o n t h a t i n v o l v e d the a c y l a t i o n (by acyl-CoA) o f the compound s p h i n g o s y l p h o s p h o r y l c h o l i n e (7,13,14,16) by an enzyme F u j i n o (16) c a l l e d ' a c y l - C o A - s p h i n g o s y l p h o s p h o r y l c h o l i n e a c y l t r a n s f -e r a s e ' . The enzyme (CDPcholine: sphingosine cholinephospho-t r a n s f e r a s e ) t h a t c a t a l y s e d the formation o f s p h i n g o s y l -p h o s p h o r y l c h o l i n e (Reaction 2) was d e s c r i b e d by F u j i n o e t a l . (14). 2) CDP-choline + sphingosine ^tCMP + sphingosylphos-p h o r y l c h o l i n e Most of the work i n the 1970's has focused on phos-p h a t i d y c h o l i n e (PC) as a donor o f the phosphocholine group -13-(23,24,30,31,34,35,37). T h i s was f i r s t suggested i n two papers by D i r i n g e r et a_l. (23,24) i n 1972 (Reaction 3 ) . I t r e c e i v e d a d d i t i o n a l support from evidence showing t h a t PE can a l s o a c t as a donor t o ceramide to y i e l d ceramide phosphorylethanolamine (18,29). In 1974 Ullman and Radin (35) c h a r a c t e r i z e d an enzyme ( p h o s p h a t i d y l c h o l i n e : ceramide t r a n s f e r a s e ) which c a t a l y z e d the formation o f sphingomyelin i n the f o l l o w i n g r e a c t i o n : 3) P h o s p h a t i d y l c h o l i n e + ceramide ^ ^ s p h i n g o m y e l i n + d i g l y c e r i d e The most recent work, done by S t o f f e l 's l a b (40,41) a l s o s t u d i e d p h o s p h a t i d y l c h o l i n e as a p o s s i b l e donor but c o u l d not f i n d any evidence t o support t h i s p o s s i b i l i t y . Instead they confirmed t h e i r e a r l i e r work us i n g CDP-choline as the donor of phosphocholine to ceramide (see Rea c t i o n 1 ) . In o p p o s i t i o n t o a l l these schemes, a few papers have suggested t h a t t h e r e i s no common p r e c u r s o r i n the b i o s y n t h e t i c pathways of PC and SM. Instead a separate source or p o o l of c h o l i n e f o r each was proposed (19,28,39). T h i s has been c r i t i c a l l y examined by D i r i n g e r (30,31) who c o u l d f i n d no evidence to support t h i s n o t i o n . An endogenous source (phospha-t i d y l c h o l i n e - d e r i v e d ) o f CDP-choline has been i n v e s t i g a t e d as an a l t e r n a t e to the d i r e c t t r a n s f e r (from p h o s p h a t i d y l -c h o l i n e ) o f the phosphocholine moiety needed t o make sphingo-myelin (11,12,19,20,22-24). T h i s has r e c e i v e d support -14-from s t u d i e s on the back r e a c t i o n o f the CDP-choline (-ethanolamine): 1-2- d i g l y c e r i d e c h o l i n e (ethanolamine) -phosphotransferase r e a c t i o n i n r a t l i v e r microsomes (32,33).' F u r t h e r support f o r p h o s p h a t i d y l c h o l i n e *s involvement as p r e c u r s o r to sphingomyelin came from a seemingly unex-pected area o f r e s e a r c h , g e n e t i c s . Esko and Raetz (42) screened f o r g e n e t i c mutations i n Chinese hamster ovary (CHO) c e l l c o l o n i e s unable to generate c h o l i n e - l i n k e d phospho -l i p i d s _in v i v o . They have rep o r t e d a s t r a i n d e f i c i e n t i n CDP-choline and p h o s p h a t i d y l c h o l i n e s y n t h e s i s a t the nonper-m i s s i v e temperature (40°). But a l l other p h o s p h o l i p i d s , i n c l u d i n g sphingomyelin, c o n t i n u e d t o be s y n t h e s i z e d f o r about 20 h. One i n t e r p r e t a t i o n would be t h a t p h o s p h a t i d y l -c h o l i n e 's l a r g e p o o l s i z e was s u f f i c i e n t to supply sphingo-myelin with i t s phosphocholine moiety, p o s s i b l y j u s t u n t i l t h e i r p o o l s i z e s had been e q u a l i z e d - _ie. a f t e r 20 h at 40°, PC was 21% and SM was 13% of the t o t a l p h o s p h o l i p i d s , compared to t h e i r p r e v i o u s values of 41% and 11.1%, r e s p e c -t i v e l y . IV My Method o f I n v e s t i g a t i o n An i n v i v o study o f the b i o s y n t h e s i s o f sphingo-myelin has a t l e a s t one advantage over i n v i t r o s t u d i e s i n t h a t the use of l i v i n g systems preserves more of the "n a t u r a l environment. T h i s reduces l a b o r a t o r y a r t i f a c t s and allows d i r e c t a p p l i c a t i o n o f r e s u l t s to other l i v i n g systems. To minimize the spread of l a b e l i n i s o t o p e experiments, -15-[Me-^Hjcholine and pulse-chase techniques were used. T h i s r e q u i r e d t h a t most or a l l of the p r e c u r s o r s to sphingomyelin and p h o s p h a t i d y l c h o l i n e have r a p i d t u r n - o v e r times. T h i s i s t r u e i n BHK-2IF c e l l s f o r c h o l i n e , phosphocholine, and CDP-c h o l i n e (114-119). A l s o due to the l a r g e p o o l s i z e f o r p h o s p h a t i d y l c h o l i n e , other types of experiments w i t h i s o t o p e s would not be as good. BHK c e l l s are a l s o widely used i n s t u d i e s of the c e l l b i o l o g y o f n e o p l a s t i c t r a n s f o r m a t i o n s and v i r u s -s p e c i f i c membrane f u n c t i o n s (116,120-123). T h i s system i s t h e r e f o r e widely a p p l i c a b l e to s t u d i e s o f the r e g u l a -t i o n of membrane b i o s y n t h e s i s . In a d d i t i o n , t h e r e i s l i t t l e or no m e t h y l a t i o n f o r the c o n v e r s i o n of phosphatidylethanolamine to phos-p h a t i d y l c h o l i n e (123). C h o l i n e o x i d a t i o n l e v e l s are a l s o very low (123,124). T h i s means t h a t the o n l y source o f newly added ( l a b e l e d ) c h o l i n e f o r p h o s p h a t i d y l c h o l i n e and sphingomyelin b i o s y n t h e s i s i s through the CDP-choline pathway. The f i r s t p a r t o f t h i s study c o n s t i t u t e s pulse-chase experiments u s i n g [Me-^H]choline as the l a b e l to take f u l l advantage of the f e a t u r e s mentioned p r e v i o u s l y . BHK-21F c e l l s were exposed ("pulsed") t o l a b e l e d c h o l i n e i n c l u d e d i n maintenance medium, f o r 30 min. When time had elapsed, the c e l l s were washed f r e e o f excess l a b e l e d c h o l i n e and incubated f u r t h e r ("chased") i n growth medium u n t i l h a r v e s t e d . 3 The p a r t i c u l a r l a b e l used, [Me- H] c h o l i n e , was chosen f o r the low r a t e of exchange of i t s methyl groups. Maintenance -16-medium was used f o r the p u l s e p e r i o d t o reduce changes i n c e l l growth d u r i n g the l a b e l s i n i t i a l i n c o r p o r a t i o n and because i t s low unlabeled c h o l i n e content would not d i l u t e the l a b e l e d p o o l o f c h o l i n e as much. The r e s u l t s o b t a i n e d from these experiments were not c o n c l u s i v e . P r e v i o u s r e s e a r c h on the f i n a l step o f sphingomyelin b i o s y n t h e s i s t h a t was s i m i l a r to my own d i d not r e s o l v e i t s a t i s f a c t o r i l y e i t h e r . T h e r e f o r e , the l a t t e r p a r t o f my study i n v o l v e d a s l i g h t l y d i f f e r e n t approach to the pulse-methodology. I s o l a t e d p h o s p h a t i d y l [Me-3H]choline from p r e v i o u s l y l a b e l e d BHK c e l l s was used as the i n i t i a l source i n a second pulse-chase experiment. However, as i n one other p r e v i o u s study using t h i s approach (34), I too found i t d i f f i c u l t t o get enough of the l a b e l i n c o r p o r a t e d i n t o the c e l l to adequately monitor the l a b e l throughout the chase phase of the experiment. Consequently, I t r i e d t o adopt two d i f f e r e n t and somewhat novel approaches to i n t r o d u c e l a b e l e d phos-p h a t i d y l c h o l i n e i n t o growing BHK-21F c e l l s . The f i r s t r e q u i r e d the use of a p h o s p h o l i p i d exchange p r o t e i n (PLEP) t o h e l p f a c i l i t a t e the uptake of p h o s p h a t i d y l -c h o l i n e from P C - v e s i c l e s added t o the c e l l medium (125-134). The second approach i n v o l v e d f u s i n g the P C - v e s i c l e s w i t h the plasma membrane o f the BHK-21F c e l l s using p o l y -ethylene g l y c o l (PEG) i n the absence and presence o f -17-phytohemagglutanin (135-144). The l a t t e r compound was thought to i n c r e a s e the percentage and v i a b i l i t y o f the fused c e l l - v e s i c l e e n t i t y (135,139). By i n t r o d u c i n g l a b e l e d p h o s p h a t i d y l c h o l i n e , any p r e c u r s o r - p r o d u c t r e l a t i o n s h i p i t might have w i t h sphingo 3 myelin should become apparent from any t r a n s f e r o f [Me- H c h o l i n e from p h o s p h a t i d y l c h o l i n e t o other compounds such as; phosphocholine, CDP-choline, Glycerophosphocholine, LPC, and sphingomyelin. -18-MATERIALS AND METHODS I. Chemicals and R a d i o a c t i v e Compounds Grand I s l a n d B i o l o g i c a l Co. s u p p l i e d the f o l l o w i n g ; Dulbecco's m o d i f i e d Eagle's medium (MEMA), medium 199, p e n i c i l l i n , s treptomycin, fungizone, trypsin-EDTA, and E a r l ' s balanced s a l t s o l u t i o n (E.B.S.S.). P l a s t i c p e t r i d i s h e s were obtained from two sources; the 10 cm s i z e from Fal c o n and the 15 cm s i z e from M i c r o b i o l o g i c a l A s s o c i a t e s . C a l f serum and f e t a l c a l f serum were from Grand I s l a n d B i o -l o g i c a l Co. i n i t i a l l y , but were l a t e r o b t a i n e d from Canadian W i l d l i f e serums. Both c h o l i n e i o d i d e and phosphocholine c h l o r i d e were purchased from Sigma Chemical Co. The l i p i d standards and CDP-choline used i n t h i n - l a y e r chromatography were a l l a c q u i r e d from Serdary Research L a b o r a t o r i e s . Am-3 ersham C o r p o r a t i o n was the source of ACS and [Me- H]-r 14 i c h o l i n e c h l o r i d e . The other l a b e l e d compounds; LMe- CJ -phosphocholine, - CDP-choline, and glycerophosphocholine were purchased•from New England N u c l e a r . The BHK-21F c e l l s used were obtained from Flow L a b o r a t o r i e s o r s u p p l i e d by Dr. C h r i s Richardson of the R o c k e f e l l e r U n i v e r s i t y , New York. I I . G eneral Methods 1. Th i n - L a y e r Chromatography TLC was performed on 20 x 20 cm l a y e r s of s i l i c a g e l , 0.25mm i n t h i c k n e s s , supported by g l a s s o r p l a s t i c p l a t e s . -19-S i l i c a Gel 60 (BDH chemicals A s s o c i a t e of E. Merck), S i l G-25 (Machery-Nagel & Co., s u p p l i e d by Brinkman I n s t r u -ments, I n c . ) , or S i l i c AR-7GF ( M a l l i n c k r o d t Chemical Works) p l a t e s were used. S o l v e n t systems used were: ( i ) S o l v e n t s f o r Water-Soluble Compounds A) S i n g l e Dimensional Systems Methanol/0.6% NaCl/conc. NH^OH (50/50/5;v/v/y). T h i s system was used to separate aqueous compounds con-t a i n i n g c h o l i n e , i . e . c h o l i n e , phosphocholine, g l y c e r o p h o s -phocholine, and CDP-choline - i n ascending order (145). The l a s t two compounds u s u a l l y remained too c l o s e together f o r p o s i t i v e s e p a r a t i o n . They were f u r t h e r separated by f i r s t s c r a p i n g the s i l i c a g e l i n t o c e n t r i f u g e tubes, ad-d i t i o n of 6 ml d i s t i l l e d H 20, shaking f o r 30 min, c e n t r i -f u g a t i o n at 1000 rpm f o r 5-15 min, counting an a l i q u o t (1 ml), adding 1 ml acid-washed c h a r c o a l ( r e f e r to S e c t i o n C ) . A f t e r shaking and c e n t r i f u g i n g as b e f o r e , another 1 ml a l i -quot was counted. Any r a d i o a c t i v e CDP-choline present i n the f i r s t 1 ml supernatant a l i q u o t w i l l have been adsorbed onto the c h a r c o a l and sedimented during the second c e n t r i -f u g a t i o n step. A l l remaining r a d i o a c t i v i t y i n the l a s t supernatant f r a c t i o n would mostly ( 99%) have been r a d i o -a c t i v e g l y c e r o p h o s p h o c h o l i n e . A l l these compounds, while on TLC p l a t e s , were v i s u a l i z e d by I.-, vapors or spray. In -20-a d d i t i o n , CDP-choline on p l a t e s w i t h or without phosphorescent i n d i c a t o r c o u l d a l s o be v i s u a l i z e d under s h o r t wavelength U.V. Methanol/0.6% NaCl (50/50; v / v ) . An a l t e r n a t e method f o r s e p a r a t i n g w a t e r - s o l u b l e c h o l i n e con-t a i n i n g compounds to v e r i f y the i d e n t i t y of compounds v i s u -a l i z e d and t h e i r r a d i o a c t i v i t y c ontent. B) Two Dimensional Systems  Methanol/0.6% NaCl/conc. NH^OH (50/50/5; v/v/v) - f i r s t dimension; Acetone/Methanol/conc. HC1 (10/90/4;  v/v/v) - second dimension. An a l t e r n a t e method used to in c r e a s e the s e p a r a t i o n of gly c e r o p h o s p h o c h o l i n e from phos-phocholine and CDP-choline. Isopropanol/20% TCA/16M NH^OH (75/25/0.3;  v/v/v) - f i r s t dimension; methanol/0.6% NaCl/cOnc. NH^OH  (50/50/5; v/v/v) - second dimension. T h i s system was used to increase the s e p a r a t i o n of phosphocholine, g l y c e r o -phosphocholine, and CDP-choline. C) P r e p a r a t i o n of Acid-Washed Ch a r c o a l Charcoal ( N o r i t A powder) was prepared by the modified method (146) of T h r e l f a l l (147). The f i n a l p r e p a r a t i o n (5g/50ml d i s t i l l e d water) was mixed thoroughly j u s t before being used with the f i r s t s o l v e n t system (meth-anol/0 .6% NaCl/conc. NH.OH) d e s c r i b e d . -21-( i i ) S o l v e n t s For L i p i d s A) S i n g l e Dimensional System  Chloroform/methanol/acetic acid/water  (100/60/16/6; v / v / v / v ) . T h i s system was a m o d i f i c a t i o n of one p r e v i o u s l y p u b l i s h e d by Wagner e_t al^. (148); and was used to separate p h o s p h o l i p i d s . In ascending o r d e r , the s e p a r a t i o n sequence was; LPC, SM, PC, PI/PS, and PE. These compounds were a l l v i s u a l i z e d very e a s i l y with 1^ spray or vapors. 2. L i q u i d S c i n t i l l a t i o n Counting I n i t i a l l y , dry l i p i d and s i l i c a g e l - l i p i d samples were counted i n 5 ml of toluene with 2,5-diphenyloxazole (PPO, 4g/l) and 1,4-bis-(2-(5-phenyloxazoly1)) - benzene (POPOP, 100mg/l). L a t e r , these samples were counted i n 10 ml ACS (Aqueous Counting S c i n t i l l a n t , Amersham) p l u s 1.0 ml d i s t i l l e d water. To avoid quenching, i t was import-ant that a l l of the c h l o r o f o r m was f i r s t evaporated before adding water. Aqueous samples with or without s i l i c a g e l present were i n i t i a l l y counted i n ACS w i t h 2.0 ml of 0.1N NaOH and 100 u l g l a c i a l a c e t i c a c i d . T h i s was l a t e r changed to 10 ml of ACS w i t h 1.0 ml of d i s t i l l e d water. S c i n t i l l a t i o n counting was done i n an ISOCAP/300 s c i n t i l l a t i o n counter ( N u c l e a r - C h i c a g o ) . Counting e f f i -c i e n c y was determined by the c h a n n e l s - r a t i o or e x t e r n a l -- 2 2 -standard c h a n n e l s - r a t i o of chloroform-quenched [~"H]-hexa-r 14 n decane standards o r a s e t of Nuclear Chicago L C ] toluene standards. Standards i n the a p p r o p r i a t e s c i n t i l l a t i o n f l u i d were counted with each set of samples. Samples were e l u t e d from TLC s i l i c a g e l by adding e i t h e r 2.0 ml 0.1 N NaOH and then lOOul a c e t i c a c i d , or 1.0 ml of d i s t i l l e d water f o r a t l e a s t 30 min, before the a d d i t i o n of 10 ml of ACS. 3. Measurement of Wet Weight and P r o t e i n of C e l l s During one of the pulse-chase experiments, I a l s o harvested d u p l i c a t e p l a t e s ( 1 5 0 mm) f o r wet weight and p r o t e i n measurements. The c e l l s were t r e a t e d i d e n t i c a l l y to those t h a t were 3 pulsed with [ Me- H ] - c h o l i n e (see s e c t i o n I V - 1 ) . A f t e r wet weight measurements, the c e l l p e l l e t was stored a t - 2 0 ° u n t i l the p r o t e i n content was to be determined. P r o t e i n was determined by the method of Lov/ry e t a l . ( 1 4 9 ) using bovine serum albumin as a p r o t e i n s t andard. To prepare the samples, they were f i r s t thawed and vortexed i n 1.0 ml of 0.66 N NaOH, then incubated at 3 7 ° o v e r n i g h t . A l i q u o t s o f the stock s o l u t i o n s were used t o determine t h e i r p r o t e i n content. The o v e r n i g h t i n c u b a t i o n i n NaOH d i g e s t s the l i p i d s present which otherwise would i n t e r f e r e w i t h the p r o t e i n assay o r prevent the s o l u b i l i z a t i o n o f the c e l l ' s p r o t e i n ( 1 4 9 ) . -23-4. Measurement o f Phospholipid-Phosphorus P h o s p h o l i p i d s (LPC, SM, PC, Pl/PS, and PE) separated by TLC were e l u t e d from the s i l i c a g e l u s i n g CHCl^/ MeOH/H20 (1:1:0.9) as d e s c r i b e d by Raheja e t a l . (150). T h i s e x t r a c t i o n method, i n p r i n c i p a l , i s the same as f i r s t proposed by B l i g h and Dyer (151) f o r t i s s u e samples with no or very l i t t l e water content. A l i q u o t s of the lower (CHCl^) phase were removed by Hamilton s y r i n g e or g l a s s p i p e t s f o r phospholipid-phosphorus measurement by the procedure of Raheja et. a_l. (150). The same procedure was a p p l i e d t o whole l i p i d samples i s o l a t e d from c e l l s by the method o f F o l c h et. a_l. (152). T h i s method gave a l i n e a r standard curve from zero to 12 mg or more of d i p a l m i t o y l p h o s p h a t i d y c h o l i n e phos-phorus o r BHK l i p i d phosphorus. However, p u b l i s h e d r e s u l t s r e p o r t l i n e a r i t y o n l y from 1-10 ug of p h o s p h o l i p i d phos-phorus using PC, PE, and SM (150). I I I . C e l l C u l t u r e C e l l s were grown a t 37° as monolayers on l a r g e (150 mm x 15 mm, M i c r o b i o l o g i c a l A s s o c i a t e s ) or on medium (100 mm x 15 mm, Falcon) p l a s t i c p e t r i p l a t e s . C u l t u r e s were grown and maintained i n Dulbecco's M o d i f i e d Eagle's Medium (MEMA) p l u s 5% c a l f serum i n a 5% C0 2/95% a i r i n -cubator - eg. a f t e r thawing f r o z e n (-70°) c e l l s t o c k s , they were s u b c u l t u r e d 10-12 times b e f o r e the l i p i d - p h o s p h o r u s d e t e r m i n a t i o n s were performed. By v i s u a l i n s p e c t i o n ( F i g . -24-9 a-lOb) , c e l l s were approx. 50% c o n f l u e n t when used and con-f l u e n t when s u b c u l t u r e d to maintain the c u l t u r e , unless otherwise noted (153). S u b c u l t u r e s were u s u a l l y d i l u t e d by o n e - f o u r t h , a f t e r which the c e l l d e n s i t y was approx. 2.22 x 10 6 c e l l s per medium s i z e p l a t e . T h i s was c a l c u l a t e d from d u p l i c a t e hemocytometer counts performed on the d i l u t e d c e l l suspension. Confluency was u s u a l l y reached w i t h i n 48-72 h a f t e r p l a t i n g the c e l l s . IV. I n c o r p o r a t i o n of [Me- H ] - c h o l i n e i n t o BHK-21F C e l l s 1. Pulse-Chase Experiments The c e l l s were s u b c u l t u r e d ( d i l u t e d one to four) when c o n f l u e n t . A f t e r 18-24 hours, the growth medium (MEMA + 5% C.S.) was removed and the c e l l s were washed once with M199. In 8 ml of maintenance medium (M199 + 2% C.S.), 27-29 uCi 3 of [Me- H^-choline was added to each 150 mm p l a t e . T h i s w i l l be r e f e r r e d to as the p u l s e medium. The same or h a l f t h i s c o n c e n t r a t i o n of l a b e l had been used i n i t i a l l y with 100 mm s i z e p l a t e s . A f t e r 30 min the pulse medium was removed and the c e l l s were prepared f o r h a r v e s t i n g , o r chas-ing w i t h excess c o l d c h o l i n e i n growth medium. Each p l a t e of c e l l s to be chased with c o l d c h o l i n e was f i r s t washed twice with warm (22-37°) PBS and then once with warm growth medium. Then another 10 or 25 ml was added to each 100 or 150 mm s i z e p l a t e . These p l a t e s were kept a t 37° i n a 5% CO„/95% a i r i n c u b a t o r u n t i l h a rvested. F i g u r e 9. BHK C e l l D e n s i t y i n T i s s u e C u l t u r e . C e l l s take about 15 - 30 min t o s e t t l e and a t t a c h f i r m l y t o the bottom of the t i s s u e c u l t u r e p l a t e a f t e r b e i n g s u b c u l t u r e d - i e . as i n ( a ) , were c e l l s were d i l u t e d one t o f o u r , ( b ) , ( c ) , C e l l s t h a t are about 50% c o n f l u e n t o r approx. 24 h a f t e r b e i n g s u b c u l t u r e d , as were used i n the p u l s e - c h a s e e x p e r i m e n t s ( s e c t i o n IV o f M a t e r i a l s and Methods). M a g n i f i c a t i o n s a r e ; ( a ) , 370x, (b) , 370x,and ( c ) , 74Qx. - 2 6 -F i g u r e 10. BHK C e l l C o n f l u e n c y i n T i s s u e C u l t u r e . C e l l s t h a t are round and s l i g h t l y r a i s e d ( b r i g h t e r c o n t r a s t ) a r e u n d e r g o i n g d i v i s i o n , ( a ) , C e l l s t h a t have c o m p l e t e l y c o v e r e d the bottom of the t i s s u e c u l t u r e p l a t e i n a monolayer - 100% c o n f l u e n t , ( b ) , Over c o n f l u e n t c e l l s t h a t have begun t o e l o n g a t e and form s t r i a t e d p a t t e r n s . M a g n i f i c a t i o n i n b o t h photographs was 530x. -27-Growth medium (MEMA + 5% CS) used to 'chase' the c e l l s was c a l l e d 'chase medium'. J u s t before h a r v e s t i n g each plate" of c e l l s , some (1/5).of t h i s chase medium was removed and stored a t + 4 ° . The r e s t was d i s c a r d e d and the r o u t i n e h a r v e s t i n g procedure f o l l o w e d . To h a r v e s t , each p l a t e of c e l l s was washed 3 times with i c e c o l d PBS (153). An i c e c o l d s o l u t i o n o f methanol/ water (1:1) was added to l y s e the c e l l s b e f o r e s c r a p i n g them from the p l a t e . Each p l a t e was r i n s e d , and that s o l u -t i o n of methanol/water was added to the f i r s t . The c e l l suspension was vortexed thoroughly a f t e r the a d d i t i o n of c h l o r o f o r m , and c e n t r i f u g e d a t 200 x g f o r 5 min to speed up phase s e p a r a t i o n (152). The upper or top phase con-tai n e d the w a t e r - s o l u b l e c h o l i n e compounds i n a methanol/ water s o l u t i o n (1:1) while the lower or bottom phase con-t a i n e d the l i p i d compounds i n the c h l o r o f o r m s o l u t i o n . T h i s upper phase was removed and the c h l o r o f o r m phase was washed 1-2 times with 0.69% NaCl/CH 3OH/CHCl (47/48/3; v / v / v ) . Combined, these washings were found to c o n t a i n o n l y 3% or l e s s of the t o t a l r a d i o a c t i v i t y p r e s e n t i n the f i r s t upper phase volume. These washings were d i s c a r d e d i n subsequent experiments, although p e r i o d i c checks ensured that t h e i r dpm l e v e l s had remained i n the 3% range. T h i s kept the upper phase volume and s a l t content to a minimum. Both the TLC work-up time and the s a l t i n t e r f e r -ence were reduced compared to when the f i r s t samples were -28-separated on t h i n - l a y e r chiromatograms (my own o b s e r v a t i o n s ) . In v a r i o u s experiments, the d u p l i c a t e p l a t e s were worked up e i t h e r s e p a r a t e l y o r a f t e r b e i n g scraped and combined. The l a t t e r procedure was used t o o b t a i n enough sphingomyelin i n the samples t o determine i t s s p e c i f i c r a d i o a c t i v i t y . T h i s a l s o h e l p e d reduce e r r o r due to v a r i a t i o n i n the c e l l s ' c o n d i t i o n from p l a t e to p l a t e , and reduced the 'phase' volumes, as p l a t e r i n s e s were doubled up. A f t e r being washed with f r e s h upper phase s o l u t i o n , the lower phase was f i l t e r e d through f i b e r g l a s s wool t o r e -move the p r e c i p i t a t e . The f i b e r g l a s s - p r e c i p i t a t e was r i n s e d through with f r e s h CHC1 3 or CHCl 3/MeOH (1:1). The r e s u l t a n t p r e c i p i t a t e - f i b e r g l a s s wool mixture was checked, o c c a s i o n a l l y , f o r r a d i o a c t i v i t y content. The r i n s e s o l u t i o n s were added t o the ( p r e v i o u s l y ) f i l t e r e d lower phases. N 2 gas was used t o evaporate a l l lower phase samples while immersed i n a warm water bath. They were taken up i n a CHCl 3/MeOH mixture e i t h e r t o be a p p l i e d t o TLC p l a t e s or were counted as d e s c r i b e d p r e v i o u s l y ( s e c -t i o n I1-2). 2. Long-term L a b e l i n g o f BHK P h o s p h o l i p i d s Two methods were used. The f i r s t was s i m i l a r to the pulse-chase procedure on l y m o d i f i e d by extending the 'pulse' stage ( t o 18h) u n t i l the c e l l s were h a r v e s t e d . In the second, s i m p l e r method, an a l i q u o t of the stock s o l u t i o n -29-of [Me- H ] - c h o l i n e was added d i r e c t l y to the p l a t e of c e l l s c o n t a i n i n g growth medium. In e i t h e r method the c e l l s were 40-60% c o n f l u e n t , or about 24-48h before reaching c o n f l u e n c y . The h a r v e s t i n g and i s o l a t i o n of the p h o s p h o l i p i d s and w a t e r - s o l u b l e compounds was as d e s c r i b e d i n the pre-ceeding s e c t i o n . V. Pool S i z e Measurements of P h o s p h o l i p i d s Phospholipid-phosphorus measurements (150) were per-formed to determine the p h o s p h o l i p i d p o o l s i z e s in BHK c e l l s (see s e c t i o n I I - 4 ) . The ug amounts of phosphorus det e c t e d were d i r e c t l y converted i n t o umol of phosphorus u s i n g 30.9738 as i t s atomic weight (154). T h i s was assumed to be d i r e c t l y p r o p o r t i o n a t e to the number of umol of p h o s p h o l i p i d on a 1 to 1 b a s i s (1 phosphorus atom per molecule of phos-p h o l i p i d ) . VI. S p e c i f i c R a d i o a c t i v i t y Determination of P h o s p h o l i p i d s P h o s p h o l i p i d s were f i r s t e l u t e d from c e l l s (152) and from s i l i c a g e l (150,151) a f t e r they had been separated by TLC. Next, one or both of the f o l l o w i n g procedures were used (see a l s o s e c t i o n I I - 4 ) . A l i q u o t s taken from the e l u t e d p h o s p h o l i p i d s o l u t i o n (CHC1 3) were assayed f o r p h o s p h o l i p i d phosphorus or r a d i o a c t i v i t y , s e p a r a t e l y . However, I found t h a t a l i q u o t s used i n the p h o s p h o l i p i d phosphorus assay c o u l d f u r t h e r be used to measure the r a d i o a c t i v i t y content , as f o l l o w s . -30-The assay mixture (99%) was t r a n s f e r r e d to s c i n t i l l a -t i o n v i a l s . The assay tube was r i n s e d twice with 1-2 ml of a CHCl 3/MeOH s o l u t i o n (1:1). The presence of MeOH was necessary to d i s s o l v e a l l of the chromogenic s o l u t i o n (150) p r e s e n t (my own o b s e r v a t i o n s ) . The mixture i n the s c i n t i -l l a t i o n v i a l was then d r i e d under a stream of a i r , a s s i s t e d sometimes with a warm water bath to speed up e v a p o r a t i o n . The d r i e d sample was then ready f o r s c i n t i l l a t i o n counting i n 1 ml of water and 10 ml of ACS. Some c o l o r quenching was e v i d e n t , but w e l l w i t h i n range of the c h a n n e l s ~ r a t i o standard curve. S p e c i f i c r a d i o a c t i v i t y was f i r s t expressed as dpm per ug P i which converted to dpm per umol P i (using 30.9738 a t . wt. P i , 154). T h i s l a t t e r c a l c u l a t i o n i s e q u i v a l e n t to dpm per umol PL. See a l s o s e c t i o n s II-4 and V. V I I . P r e p a r a t i v e Procedures 1. I s o l a t i o n of PLEP Supernant F r a c t i o n From Rat  L i v e r S e v e r a l procedures (125,126,128) were combined or modified to decrease the p r e p a r a t i o n time. The l i v e r s from 3 male r a t s were used to prepare PLEP found i n the c y t o s t o l i c f r a c t i o n . T h e i r l i v e r s were removed, weighed (30g) and placed i n a 10% s o l u t i o n of B u f f e r A (1 mM EDTA, 0.25M sucrose, and 1.0 mM T r i s HC1, pH 7.4). The l i v e r s were homogenized and c e n t r i f u g e d at 600 x g f o r 15 min. a t +4°. The super--31-natant was c a r e f u l l y decanted and c e n t r i f u g e d at 15,000 x g f o r 15 min. a t + 4 ° . Again the supernatant was decanted, and c e n t r i f u g e d a t 100,000 x g f o r 1 hour at +4°. T h i s super-natant (some stored a t -20°) had i t s pH a d j u s t e d to 5.1 with 0.1N HC1 and was l e f t to s t i r s l o w l y f o r 15 min p r i o r to c e n t r i f u g a t i o n a t 15,000 x g f o r 15 min a t +4°. The super-natant was removed, t h i s time to a d j u s t the pH back to 7.4 with IN NaOH. T h i s f i n a l s o l u t i o n was s t o r e d at -20°, and termed the 1 pH 5.1 supernatant 1 f r a c t i o n . PLEP was used from t h i s l a s t 'PH 5«1 supernatant' f r a c -t i o n or from crude supernatant f r a c t i o n s taken a f t e r the 100,000 x g c e n t r i f u g a t i o n . I t was thawed j u s t b e f o r e use. 2. P r e p a r a t i o n of P h o s p h o l i p i d V e s i c l e s Unlabeled p h o s p h o l i p i d s were purchased (from Serdary) or i s o l a t e d from BHK-21F c e l l s . A l l l a b e l e d p h o s p h o l i p i d s were obtained from BHK-21F c e l l s . Refer to s e c t i o n s I I - l , I I - 4 , IV-1, IV-2, and V. P h o s p h o l i p i d s v e s i c l e s c o n s i s t i n g s o l e l y of p h o s p h a t i -dylcholine were used i n both types of ' v e s i c l e ' experiments and PEG-mediated f u s i o n experiments. The i n i t i a l v e s i c l e p r e p a r a -t i v e procedure (by B u t l e r and Thompson, 132) was m o d i f i e d sev-e r a l times to t r y to increase the percentage of l a b e l e d PC ex-changed. A l s o , to o b t a i n the p r e f e r r e d s m a l l b i l a y e r v e s i c l e s r a t h e r than the l a r g e m u l t i l a y e r v e s i c l e s ( f i r s t formed i n aqueous s o l u t i o n s ) longer p e r i o d s of s o n i c a t i o n were re q u i r e d -32-(135-137). S u i t a b l e p r e c a u t i o n s a g a i n s t p h o s p h o l i p i d d e n a t u r a -t i o n from the i n c r e a s e d heat g e n e r a t e d were developed f o r the o v e r a l l p r o c e d u r e , and f o r when s t e r i l i t y was r e q u i r e d . For the PLEP-mediated exchange e x p e r i m e n t s , a l l p h o s p h a t i d y c h o l i n e v e s i c l e p r e p a r a t i o n s were made from a 3 f Me- H ] c h o l i n e p r e p a r a t i o n o f BHK c e l l p h o s p h a t i d y c h o l i n e (approx. 3000 dpm/nmol) o b t a i n e d from c e l l s grown i n t h i s l a b . T h i s m i x t u r e was s t o r e d as a c h l o r o f o r m s o l u t i o n under gas a t -20° b e f o r e b e i n g used i n p h o s p h o l i p i d v e s i c l e p r e p a r a t i o n s . I n BHK c e l l / p h o s p h o l i p i d v e s i c l e f u s i o n e x p e r i m e n t s , a mixed p h o s p h o l i p i d v e s i c l e p r e p a r a t i o n was used most o f the t i m e , i n s t e a d of the pure p h o s p h a t i d y l c h o l i n e v e s i c l e s . The mixed v e s i c l e s were composed of a p h o s p h a t i d y l c h o l i n e (750 o r 900 dpm/nmol): p h o s p h a t i d y l s e r i n e m i x t u r e , w i t h a molar r a t i o o f 80:20, r e s p e c t i v e l y . Rat l i v e r p h o s p h o l i p i d s ( p h o s p h a t i d y l c h o l i n e - 6 0 % and p h o s p h a t i d y l s e r i n e - 2 0 % ) had been added to the p r e v i o u s l y l a b e l e d (approx. 3000 dpm/nmol) BHK c e l l p h o s p h a t i d y l c h o l i n e ( 2 0 % ) . Any w a t e r - s o l u b l e compound t o be i n c l u d e d was added b e f o r e v o r t e x i n g . The v o r t e x i n g d i s p e r s e s the p h o s p h o l i p i d s i n t o s o l u t i o n i n the form of m u l t i l a m e l l a r v e s i c l e s . Any a q u e o u s - s o l u b l e compounds p r e s e n t w i l l a l s o be d i s p e r s e d , i n s i d e and o u t s i d e o f these l a r g e v e s i c l e s ( 1 3 7 ) . The s o n i -c a t e d v e s i c l e p r e p a r a t i o n s ( 132,135-137) were k e p t under N <y gas and a t 0-4° u n t i l u sed, u s u a l l y w i t h i n 1 hour. -33-Any v e s i c l e p r e p a r a t i o n not used was stored a t -20 . I f used again, i t was thawed and r e s o n i c a t e d . Sometimes the aqueous volume had to be in c r e a s e d during s o n i c a t i o n , i n which case d i s t i l l e d water was added. When I expected prolonged exposure of the BHK c e l l s to the v e s i c l e p r e p a r a t i o n o r a l e n g t h l y chase p e r i o d , s t e r -i l e c o n d i t i o n s were maintained as much as p o s s i b l e . T h i s e n t a i l e d c a r r y i n g out the e n t i r e precedure i n s i d e a u n i f l o w t i s s u e c u l t u r e hood, and using s h o r t e r s o n i c a t i o n p e r i o d s as sometimes a water bath was not used. Normally, phospho-l i p i d s were taken up i n d i s t i l l e d water, but to preserve s t e r i l e c o n d i t i o n s , s e v e r a l s t e r i l i z e d s o l u t i o n s were used; HBSS (Hank's Balanced S a l t S o l u t i o n ) , M199, and MEMA. 3 V I I I . Procedures For I n t r o d u c i n g f Me-. H~l - e holine Labeled  P h o s p h o l i p i d s I n t o BHK C e l l s 1. PLEP-mediated Exchange of P h o s p h o l i p i d s Between  P h o s p h o l i p i d V e s i c l e s and BHK-21F C e l l s BHK c e l l s were i n i t i a l l y used at 50% confluency, but t h i s was changed to c e l l s of about 90% co n f l u e n c y . A t i s s u e c u l t u r e hood was used throughout (Laminar Flow Cabinet by Labgard) . The c e l l s ' maintenance medium (10 or 25 ml of MEMA + 5% C.S., f o r 100 mm or 150 mm s i z e p l a t e s ) was f i r s t r e -moved, followed by one washing with prewarmed (37°) M199. The c e l l s were then exposed to a "pulse" s o l u t i o n (4 or 8 ml -34-of MEMA with p h o s p h a t i d y l ["Me- H] c h o l i n e v e s i c l e s ) , with or without ( c o n t r o l ) a r a t l i v e r PLEP f r a c t i o n . Rat l i v e r c y t o s o l c o n t a i n i n g PLEP and f r o z e n at -70° was s u p p l i e d by Steve Pelech from t h i s l a b . Once thawed, the c y t o s o l was c e n t r i f u g e d at 170,000 x g f o r 60 min i n a Beckman LB-70 ul t r a c e n t r i f uge. The p e l l e t was d i s c a r d e d . The supernatant, c a l l e d the 'crude PLEP supernatant' f r a c -t i o n , was used the same day or f r o z e n at -20°. If s t o r e d , i t was used w i t h i n 2 weeks. Further p u r i f i c a t i o n c o u l d be achieved by f o l l o w i n g the pH 5.1 adjustment procedure o u t l i n e d i n s e c t i o n V I I I - 1 . T h i s p r e p a r a t i o n , because o f the low p r o t e i n c o n c e n t r a t i o n , was not as s t a b l e . When f r e s h pH 5.1 supernatant PLEP was prepared ( s e c -t i o n V I I I - 1 ) , the pulse s o l u t i o n contained B u f f e r A (1 mM EDTA, 0.25 M sucrose, and 1.0 mM T r i s HC1» pH 7.4) in s t e a d of the MEMA component. The p u l s e s o l u t i o n f o r each p l a t e of c e l l s was made up j u s t before use, under s t e r i l e c o n d i t i o n s and kept at 37°. A l l i n c u b a t i o n s and 'chase' p e r i o d s were c a r r i e d out i n a 5% CO,, in c u b a t o r , a t 37°. At the end of the p u l s e p e r i o d (20 min-48 h), 1.0 ml a l i q u o t s of the pu l s e s o l u t i o n were (sometimes) removed. The r e s t of the pulse s o l u t i o n was a s p i r a t e d and d i s c a r d e d . The p l a t e s of c e l l s were e i t h e r harvested or r e - i n c u b a t e d w i t h 'chase' (growth) medium. In both s i t u a t i o n s , the pro-cedures o u t l i n e d i n s e c t i o n IV-1 were f o l l o w e d . -35-Pulse-medium r a d i o a c t i v i t i e s were u s u a l l y counted i n whole or e x t r a c t e d (152) samples (water-soluble and l i p i d f r a c t i o n s ) . C e l l u l a r r a d i o a c t i v i t i e s were counted i n the e x t r a c t e d (152) aqueous and l i p i d f r a c t i o n s . As w e l l , the l i p i d f r a c t i o n was f u r t h e r separated (by TLC) i n t o i n d i v i d u a l p h o s p h o l i p i d f r a c t i o n s which were assayed f o r dpm's. and sometimes l i p i d - p h o s p h o r u s (see s e c t i o n s I I - l , II-2, I I-4, and IV-1). 2. PEG/PHA-Mediated Fusion Between P h o s p h o l i p i d  V e s i c l e s and BHK-2IF C e l l s The procedures of Oi and Herzenberg (141), and Poste and N i c o l s o n (143)-were followed except f o r t h e i r techniques i n resuspending the c e l l s throughout the experiment (see R e s u l t s and D i s c u s s i o n s e c t i o n V I I ) . To h a r v e s t the c e l l s , they had to be loosened from the p l a t e ' s s u r f a c e by in c u b a t i n g them at 37°, i n a t r y s p i n s o l u t i o n (M199 + tryspin-EDTA) f o r 5-10 min (153). The c e l l s were t r i t u r a t e d o f f the bottom with MEMA. Once i n suspen-s i o n , the c e l l s were t r a n s f e r r e d to the c e n t r i f u g e tubes ( s t e r i l i z e d with ethanol beforehand) and c e n t r i f u g e d at s e t t i n g '1800' on a bench top c e n t r i f u g e ( P h i l l i p s L-708) f o r 10 min. I i n i t i a l l y t r i e d one c o n f l u e n t 150 mm p l a t e of c e l l s per tube. However, due to c e l l l o s s during the experiment and l o s s of c e l l v i a b i l i t y during the chase p e r i o d , I found i t necessary to use two p l a t e s ( c o n f l u e n t ) of c e l l s -36-per tube. The c e l l p e l l e t was g e n t l y s w i r l e d while the ph o s p h o l i p i d v e s i c l e p r e p a r a t i o n (100 ul - 300 ul) was added, and the whole mixture was again s w i r l e d g e n t l y by hand. The c e l l p e l l e t - s u s p e n s i o n was then ready f o r the PEG mixture (50%, wt./wt. i n MEMA c o n t a i n i n g PHA, 50.ug/ ml) which was added l a s t (143). T h i s was p r e v i o u s l y prepared under s t e r i l e c o n d i t i o n s (141), and could be kept at room temper-ature ( i n d e f i n i t e l y ) . I t was heated t o 37° j u s t before being used. The PEG/PHA s o l u t i o n was added very slowly (137) using a p l a s t i c t i p p e d p i p e t . T h i s worked b e t t e r than g l a s s p i p e t s s i n c e very l i t t l e of the v i s c o u s PEG/PHA mix-tu r e adhered to the s i d e s of the p l a s t i c t i p s . The u s u a l i n c u b a t i o n and washing procedures then followed (143). Some of the f i n a l i n c u b a t i o n supernatant was removed to be checked f o r r a d i o a c t i v i t y l e v e l s , the r e s t was d i s -carded . The l a s t w a s h i n g / c e n t r i f u g a t i o n using warm MEMA preceded the f i n a l resuspension i n 5 ml 'chase' medium. T h i s chase medium (MEMA and 5% C.S.) was p i p e t t e d out i n t o 150 mm pla t e s , , one tube of c e l l s per two p l a t e s with 10 ml f i n a l chase medium volume per p l a t e . The c e l l s were incubated f o r the d e s i r e d l e n g t h of time i n the CO„ in c u b a t o r , a t 37° (141). -37-Th e h a r v e s t procedure was that o u t l i n e d i n s e c t i o n IV-1. R a d i o a c t i v i t y was counted i n whole aqueous and l i p i d c e l l e x t r a c t s as w e l l as i n t h e i r i n d i v i d u a l c h o l i n e -c o n t a i n i n g compounds ( s e c t i o n s I I - l , and 11-2). -38-RESULTS AND DISCUSSION I. Growth Parameters of BHK-21F C e l l s During the  Chase P e r i o d The course of the pulse-chase experiments was c a r -r i e d out from 22 to 68 h a f t e r the c e l l s had been passed. I t was f e l t that during the l a s t 24h p e r i o d , t h e r e might be some density-dependant i n h i b i t i o n o f c e l l growth or p h o s p h o l i p i d s y n t h e s i s (119). These c e l l s reached 'con-f l u e n c e * between 72-96 h when subcultured a t a r a t i o of 1:4. T h e r e f o r e , c e l l wet weight and p r o t e i n c o n c e n t r a t i o n ( F i g . 11) were two of the parameters by which t h i s would be s t u d i e d . Other, more s u b j e c t i v e parameters used, i n v o l v e d p e r i o d i c v i s u a l checks on medium c o l o r (pH dependent), and c e l l shape and d e n s i t y . The l a t t e r i n v o l v e d the use of a pha s e - c o n t r a s t microscope (10/0.40 or 20/0.60 phase). Refer to the photographs i n Fi g u r e s 9 and 10. The wet weight of BHK c e l l s i s shown i n F i g u r e 11, along with the p r o t e i n content of these wet weight samples. A l i n e a r growth p a t t e r n was observed on the b a s i s o f these two parameters ( F i g u r e 11). The c e l l volume d i d seem to decrease d u r i n g the second 24 h of the case p e r i o d , i n d i -cated by an increased p r o t e i n to wet weight r a t i o . (Table 1 ) . V i s u a l l y the c e l l s began l o o k i n g t h i n n e r and formed s t r i a t e d p a t t e r n s as c e l l - c e l l c o n t a c t increased d u r i n g p r o l i f e r a t i o n ( F i g u r e 10). However v i s u a l o b s e r v a t i o n s -39-4-> x: 01 +J 0) T3 c (T3 OJ O 800 h-600 U 400 r~ 200 \-I o O E 03 P i-l o ,G O x: Cu I •H Cu •f-i H O a o ,c ft 10 20 30 40 Chase Time (h) F i g u r e 11. Parameters of BHK C e l l Growth During the Chase P e r i o d . mm sxze phospho were pe vested, been ex method t i o n s I Wet weight measurement p l a t e s of c e l l s ( M a t e r i a l l i p i d - p h o s p h o r u s (•) determ rformed on 5, 150 mm p l a t e so the wet weight(•) and pressed t h a t way a l s o . Mea of Lowry e t a l . ( 1 4 9 ) . (Ref 1-3 and II-4) . s were performed on s i n g l e , 150 s and Methods s e c t i o n I I - 3 ) . A l l i n a t i o n s (Raheja e t al.,150) s of c e l l s combined when har-p r o t e i n content(•) v a l u e s have surement of p r o t e i n was by the er to M a t e r i a l s and Methods sec--40-Table 1 Comparison of C e l l Growth Parameters During the Chase P e r i o d S i n g l e , l a r g e p l a t e s of BHK c e l l s were used to measure the wet  weight and p r o t e i n v a l u e s used to c a l c u l a t e the r a t i o s below. The p h o s p h o l i p i d v a l u e s used were o b t a i n e d from c e l l s com-bined from 4 or 5 l a r g e p l a t e s , and r e p r e s e n t a l l d e t e c t a b l e p h o s p h o l i p i d s r ecovered from t h i n - l a y e r chromatograms. See a l s o s e c t i o n s I I - l , I I - 3 , I I I , IV, and V of M a t e r i a l s and Methods f o r f u r t h e r d e t a i l s . Hours i n Chase Medium :— Average :( ± s. d.) 0.0 3.0 22.0 32.0 45.5 mg p r o t e i n 3 2 . 9 + 4 , 4 1 34.8±2*8 35.016.5 39.113.4 39.5+5.7 36.3110.7 g c e l l s nmol PL 8.22+1.5 9.7411.4 9.5511.2 9.34+0.7 10.610.4 9.49+2.5 mg c e l l s nmol PL mg p r o t e i n 250148 281+47 273148 239115 268139 262193 1 Expressed as the standard d e v i a t i o n (s.d.) of the mean value -41-o f the c e l l s was l i m i t e d t o two dimensions o n l y . The h e i g h t o f the c e l l s c o u l d not be determined, so t h i s s t r i a t e d appearance may have been the r e s u l t of squeezing . I t had been observed t h a t even a f t e r ( f l a t t e n e d ) c e l l s covered the e n t i r e p l a t e bottom, c e l l v i a b i l i t y con-t i n u e d f o r another 24 h r . I t was du r i n g t h i s time t h a t the c e l l s had begun t o appear s t r i a t e d ( F i g . 10), but t h e i r v i a b i l i t y would not d e c l i n e r a p i d l y u n t i l t h i s s t r i a t e d f o rmation dominated. T h i s i s r e f l e c t e d i n the unchanged phospholipid-wet weight r a t i o s (Table 1, legend) d u r i n g the chase p e r i o d . I t seems more l i k e l y t h a t there was no c e l l volume change but only an o v e r a l l i n c r e a s e i n the c e l l l s p r o t e i n content, r e l a t i v e to i t s wet weight and p h o s p h o l i p i d c o n c e n t r a t i o n ( F i g . 11 and Table 1). C e l l growth was a l s o monitored on the b a s i s of i n -creased p h o s p h o l i p i d p o o l s i z e s per 5 l a r g e p l a t e s o f c e l l s ( r e f e r t o s e c t i o n V). These r e s u l t s as w e l l as those i n F i g u r e 11 and Table 1 had not i n d i c a t e d any decrease i n c e l l growth due to c e l l d e n s i t y i n h i b i t i o n d u r i n g the l a t t e r 24 h o f the chase p e r i o d , as p r e d i c t e d by G a l l a h e r and Blough (119). The medium change t h a t o c c u r r e d because o f the i n -t e r v e n i n g p u l s e p e r i o d had the e f f e c t o f p r o l o n g i n g the h e a l t h y appearance of the medium (pH/color a f f e c t e d l e s s ) . C e l l growth as based on wet weight measurements ( F i g . 12) -42-F i g u r e 12. E f f e c t of Growth-Medium Changes on the Increase of BHK C e l l Wet Weight as a F u n c t i o n of Time. Measure-ments f o r wet weight d e t e r m i n a t i o n were made u s i n g s i n g l e , l a r g e p l a t e s of c e l l s . The c i r c l e s (•,0) r e p r e s e n t one s e t of v a l u e s o b t a i n e d from the same s u b c u l t u r e d c e l l s used i n one of the pulse-chase experiments (see F i g . 11). S o l i d c i r c l e s (•), r e p r e s e n t c e l l s w i t h a growth-medium change a t 22 h and are the average of two d e t e r m i n a t i o n s . The open symbols (0,D), are o n l y s i n g l e d e t e r m i n a t i o n s . The squares (•), r e p r e s e n t v a l u e s o b t a i n e d from c e l l s t h a t have been f u r t h e r s u b c u l t u r e d once ( l a s t two) or twice ( f i r s t two). The l a s t two p o i n t s (squares) r e p r e s e n t the weights of c e l l s t h a t have had a medium change at 64 h. The f i r s t two squares and the open c i r c l e s r e p r e s e n t the weights of c e l l s t h a t have not had any medium change s i n c e the l a s t time they were s u b c u l t u r e d . The symbol, #], denotes o v e r l a p p i n g s o l i d c i r c l e and open square v a l u e s . (Refer a l s o to M a t e r i a l s and Methods s e c t i o n I I - 3 ) . -43-and c e l l appearance was unchanged or p r o l o n g e d p a s t the average time p e r i o d (48-72 h ) . U s u a l l y the e f f e c t s o f c o n f l u e n c y ( i . e . c e l l - c e l l c o n t a c t i n h i b i t i o n ) m a n i f e s t t h e m s e l v e s by t h e n , so i f the c e l l s a re l e f t i n the same medium p a s t t h i s p o i n t , c e l l v i a b i l i t y d e c r e a s e s v e r y q u i c k l y i n the next 24-48 h. The e f f e c t o f removing c e l l u l a r m e t a b o l -i t e s a l o n g w i t h the ' o l d ' medium seems to have some e f f e c t ( b e n e f i c i a l ) on BHK c e l l s . T h e r e f o r e , t h i s c o u l d be a f a c t o r i n f l u e n c i n g the b i o s y n t h e t i c p a t t e r n o f the c e l l ' s phospho-l i p i d s as r a p i d growth may be a v a l u a b l e parameter to main-t a i n when s t u d y i n g t u r n o v e r and s y n t h e s i s o f p h o s p h o l i p i d s ( 1 1 8 ) . I I . S e p a r a t i o n and I d e n t i f i c a t i o n o f C h o l i n e - C o n t a i n i n g  Compounds R a d i o a c t i v i t y measurements i n c h o l i n e , p h o s p h o c h o l i n e and C D P - c h o l i n e d i d not r e q u i r e v e r y much of these com-pounds. T h e r e f o r e f o l l o w i n g t h e i r F o l c h e x t r a c t i o n (152) from BHK c e l l s , , a TLC approach was used ( 1 4 5 ) , though q u a n t i t a t i v e f o r r a d i o a c t i v i t y o n l y ( F i g . 1 3 ) . C D P - c h o l i n e and G - P - c h o l i n e m i g r a t e d t o o c l o s e t o g e t h e r (R^=0.60-0.77) t o i d e n t i f y s e p a r a t e l y by t h i s i n i t i a l TLC system ( F i g . 13a, 13b). F o r t u n a t e l y l i t t l e r a d i o a c t i v i t y was e x p e c t e d i n C D P - c h o l i n e , due t o i t s s m a l l p o o l s i z e (114,123). The Rf v a l u e s f o r c h o l i n e and P - c h o l i n e were 0.18-0.40 and 0.49-59 ( F i g . 1 3 ) , r e s p e c t i v e l y . To m o n i t o r r a d i o a c t i v e l e v e l s ro I O rH X OJ C •rH rH o Xi o ro I si C a r r i e r ( s ) Added With Sample  |l5 'CDP-choline (1.0 and 1.0 mg) C h o l i n e 5 (1.0 and 0.8 mg) CDP-choline (1.0 and 1.0 mg) G-P-choline (2.5 and 3.0 mg) (L5 G-P-choline (2.5 and 3.0 mg) 5 P - c h o l i n e (1.5 and 0.75 mg) - f 2 0 p - c h o l i n e (1.5 and 1.8 mg) -£0 O r i g i n 2.6 7.7 10 Origin 3.8 Distance Migrated (cm) F i g u r e 13. E f f e c t s of C a r r i e r - S t a n d a r d s on the M o b i l i t y of Sample R a d i o a c t i v i t y on T h i n - Layer Chromatograms. Water-soluble compounds c o n t a i n i n g a c h o l i n e moiety were separated by TLC u s i n g the s o l v e n t system of Sundler and Akesson ( 1 4 5 ) - methanol/0.6% NaC cone. NH4OH ( 5 0 / 5 0 / 5 ) . The samples used were obtained from the e x t r a c t e d ( 1 5 2 ) BHK c e l l s a f t e r a 30 min p u l s e w i t h [Me-^H]choline. (a) No chase p e r i o d ( 0 h ) , and (b) with a chase p e r i o d of 22 h. D i f f e r e n t c a r r i e r - s t a n d a r d s were added to each lane with the same amount of sample i n (a) and (b), r e s p e c t i v e l y . ( M a t e r i a l s and Methods s e c t i o n s I I - l and I V - 1 ) . -45-i n CDP-choline, s e v e r a l methods (covered l a t e r ) were de-veloped. Since only small amounts o f the i s o l a t e d l a b e l e d compounds ( c h o l i n e , P - c h o l i n e , G-P-choline, and CDP-choline) were needed on the t h i n - l a y e r chromatograms, the e x t r a c t e d (152) aqueous phase o f BHK c e l l s d i d not r e q u i r e any a d d i t i o n a l c o n c e n t r a t i n g procedures. T h i s d i d neces-s i t a t e the use o f c a r r i e r - s t a n d a r d s on the t h i n - l a y e r chrom-atograms which p e r m i t t e d v i s u a l i z a t i o n with 1^ vapors and prevented t r a i l i n g of r a d i o a c t i v i t y ( F i g . 13a and 13b). The w a t e r - s o l u b l e r a d i o a c t i v i t y from the c e l l s seemed to migrate f u r t h e r even when s p e c i f i c c a r r i e r - s t a n d a r d s had been excluded ( F i g . 13). T h i s i n d i c a t e d t h a t the non-s p e c i f i c a d s o r p t i o n by the s i l i c a g e l ( s t a t i o n a r y phase) had been reduced or e l i m i n a t e d p r e f e r e n t i a l l y by c e r t a i n c a r r i e r - s t a n d a r d s . T h i s c o u l d be used as a crude q u a n t i t a -t i v e method f o r i d e n t i f y i n g the r a d i o a c t i v i t y content i n CDP-choline and G-P-choline as they had co-migrated i n the system ( F i g . 13). To c o n f i r m these estimates other TLC procedures were employed. The f i r s t a l t e r n a t e method used to separate CDP-c h o l i n e from G-P-choline was a two-dimensional TLC system ( F i g . 14). T h i s two-solvent system (see M a t e r i a l s and Methods s e c t i o n I I - l ) would only 'work' i n one s o l v e n t sequence ( d i r e c t i o n ) . If the a c i d i c s o l v e n t system was used f i r s t ( F i g . 14a) degradation of G-P-choline o c c u r r e d , -46-(ii) /j\ Origin ° t i > < - c l ) F i g u r e 14. E f f e c t of an A c i d i c S o l v e n t on the M o b i l i t y of Gly- cerophosphocholine and Sample R a d i o a c t i v i t y on T h i n - Layer Chromatograms. The s o l v e n t systems used were;(i) A c i d i c s o l v e n t - acetone/methanol/conc. HC1 (10/90/4), a n d ( i i ) B a s i c s o l -vent- methanol/0.6% NaCl/conc. NH4OH (50/50/5). An equal amount of sample from BHK c e l l s ( p u l s e d f o r 30 min with [Me-^H]choline and chased f o r 4 h) was s p o t t e d on chromatograms i n ( a ) , (b) , and ( c ) . Chromatogram (d) had o n l y c a r r i e r G-P-choline on i t . Refer to M a t e r i a l s and Methods s e c t i o n I I - l . -47-as i l l u s t r a t e d by the two I 2 s t a i n e d areas f o r G-P-choline on l y ( F i g . 14d). T h i s a c i d - d e g r a d a t i o n of G-P-choline may have occured i n the forward d i r e c t i o n a l s o , s i n c e the deg r a d a t i v e products would only be separated by the f i r s t or b a s i c s o l v e n t ( F i g . 14b versus F i g . 14c). T h e r e f o r e , when the two s o l v e n t s were used i n the reverse sequence, both d e g r a d a t i v e products would have remained together ( F i g . 14c). A l s o , when j u s t c a r r i e r G-P-choline was used, o n l y one I 2 s t a i n e d spot was observed .(results not included) . A second two-dimensional system was t r i e d (not shown) i n which an i s o p r o p a n o l - s o l v e n t was used i n combination with the ' b a s i c 1 s o l v e n t system i n F i g u r e 13. Using the l a t t e r s o l v e n t f i r s t was a l i t t l e b e t t e r , but e i t h e r o r d e r o f use r e s u l t e d i n only marginal s e p a r a t i o n o f CDP-choline and G-P-choline. In ascending o r d e r , the c a r r i e r - s t a n d a r d s mi-grated i n the isopropanol/20% TCA/16M NH^OH (75/25/0.3; v/v/v) system (only) as f o l l o w s ; CDP-choline remained a t the o r i g i n , P - c h o l i n e and G-P-choline stayed together (R =0.07-0.15), and c h o l i n e moved f u r t h e s t (R =0.25-0.38). The r e s u l t s from these two-dimensional t h i n - l a y e r chromato-grams d i d support the pulse-chase data on the 22 h chase-time sample (see R e s u l t s and D i s c u s s i o n s e c t i o n I I I ) - i e . about 2/3 of the sample's r a d i o a c t i v i t y was i n the G-P-choline/ CDP-choline area on the t h i n - l a y e r chromatogram. The t h i r d a l t e r n a t e method t r i e d was a simple one-dimension TLC system ( F i g . 15). The s o l v e n t used, MeOH/0.69% -48-I T3 0) o u i— i I 0)1 si 25 15 25 15 5 k P-choline G--P-choline Choline P-choline CDP-choline SI E Choline S i n g l e C a r r i e r Added A l l C a r r i e r s Added Origin 3.2 4.8 10.2 13.7 Distance Migrated (cm) C a r r i e r Added With Sample C h o l i n e (1.0 mg) P - c h o l i n e (1.5 mg) CDP-choline (1.0 mg) G-P-choline (2.5 mg) F i g u r e 15. E f f e c t s of C a r r i e r - S t a n d a r d s on the M o b i l i t y of Sample R a d i o a c t i v i t y on Thin-Layer Chromatograms. Water-soluble compounds wit h a c h o l i n e moiety were separated by TLC u s i n g the s o l v e n t system- methanol/0.6% NaCl (50/50; v / v ) . The sample used was o b t a i n e d from the extracted(152) BHK c e l l s a f t e r a 30 min p u l s e with [Me-3H]choline. A chase p e r i o d of 22 h f o l l o w e d b e f o r e the c e l l s were harvested. D i f -f e r e n t c a r r e r i e r - s t a n d a r d s were added to each lane on the t h i n - l a y e r chromatogram along w i t h equal amounts of sample. Refer to M a t e r i a l s and Methods s e c t i o n s I I - l and IV-1. -49-NaCl (50/50; v / v ) , was a m o d i f i c a t i o n of the s o l v e n t used i n F i g u r e 13. In t h i s system, the c a r r i e r standards d i d not migrate cons i s t a n t l y . T h e i r order remained the same -P - c h o l i n e , CDP-choline, G-P-choline, and c h o l i n e ( i n ascending order) - but t h e i r 1 s changed ( F i g . 15). T h i s depended on whether the c a r r i e r s were used i n separate lanes or together i n one l a n e . The main problem was poor separ-a t i o n of G-P-choline and CDP-choline, although t r a i l i n g without c a r r i e r - s t a n d a r d s was g r e a t l y reduced ( F i g . 15) as compared to the o r i g i n a l s o l v e n t system ( F i g . 13). Because the i n i t i a l ' b a s i c ' s o l v e n t was more c o n s i s t e n t and p r e v i o u s l y documented (145), I went back to t h a t TLC procedure. S e p a r a t i o n of CDP-choline and G-P-choline was achieved using c h a r c o a l ' s p r o p e r t y o f b i n d i n g c y t i d i n e nuc-l e o t i d e s (146,147). A f t e r samples had been separated on t h i n - l a y e r chromatograms, the area i n q u e s t i o n was removed. The r a d i o a c t i v i t y was c h a r a c t e r i z e d as to which compound i t a s s o c i a t e d with, as d e s c r i b e d i n M a t e r i a l s and Methods s e c t i o n s 11-1,2. T h i s was t e s t e d with p u r i f i e d [ "^C ] CDP-r 14 n c h o l i n e and L CJ G-P-choline. In the former, 98% of the r a d i o a c t i v i t y was i n the i o d i n e - s t a i n e d spot. When t r e a t e d with c h a r c o a l , over 99% of the r a d i o a c t i v i t y was removed from s o l u t i o n and p e l l e t e d with the c h a r c o a l . As f o r the 14 [ C] G-P-choline, 86% of the r a d i o a c t i v i t y was recovered from the i o d i n e - s t a i n e d area. Of t h i s , over 94% remained i n s o l u t i o n a f t e r treatment with the c h a r c o a l . T h i s pro--50-cedure was used to o b t a i n a l l the valu e s ( f o r aqueous cho-1 i n e - c o n t a i n i n g compounds) i n the R e s u l t s and D i s c u s s i o n s e c t i o n I I I . P h o s p h o l i p i d s were separated by a m o d i f i c a t i o n o f Wagner's (148) s o l v e n t system. The R f v a l u e s obtained were; LPC, 0.05-0.11; SM, 0.11-0.21; PC, 0.28-0.43; PI/PS, 0.46-0.66; and PE, 0.79-0.89. The R f v a l u e s of c a r r i e r -standards and the corresponding BHK p h o s p h o l i p i d s were the same, except that alone, BHK LPC could not be v i s u a l i z e d with I^ vapors. Sphingomyelin from BHK c e l l s , although w i t h i n the R^ range mentioned, was v i s u a l i z e d as a d i s t i n c t d o u b l e t . T h i s i s probably due to the two predominant f a t t y a c i d chain l e n g t h s (16:0; 24:1 and 24:0) i n the N-acyl p o s i t i o n of sphingomyelin (35,167,175). Otherwise, t h i s system was ex-c e l l e n t , e s p e c i a l l y f o r s e p a r a t i o n o f PC from SM. When aqueous counts from BHK c e l l s were run i n t h i s system, 95-98% of the r a d i o a c t i v i t y remained a t the o r i g i n , between R f v a l u e s of 0.0-0.05. The r e s t , 1-4% was found between an R f of 0.05 to 0.11. Conversely, when p h o s p h o l i p i d c a r r i e r - s t a n d a r d s were run i n the ' b a s i c ' s o l v e n t system used to separate aqueous compounds ( F i g . 13), no movement at a l l was det e c t e d (by i o d i n e s t a i n ) . -SI-II I. I n c o r p o r a t i o n of [Me- H~l-choline i n t o BHK C e l l s 1. Pulse-Chase S t u d i e s Pulse-chase experiments were done to show how the i n c o r p o r a t i o n of [Me- H ] - c h o l i n e i n t o sphingomyelin i s r e l a t e d to the disappearance of [Me- H ] - c h o l i n e from p h o s p h a t i d y l c h o l i n e and i t s w a t e r - s o l u b l e p r e c u r s o r s . In each o f four separate pulse-chase experiments using [Me- H]-c h o l i n e , the r a d i o a c t i v i t y i n phosphocholine decreased ( e x p o n e n t i a l l y ) while r a d i o a c t i v i t y i n p h o s p h a t i d y l c h o l i n e , sphingomyelin, and g l y c e r o p h o s p h o c h o l i n e increased ( F i g . 16-18). Such an i n v e r s e r e l a t i o n s h i p i n d i c a t e s t h a t phosphocholine i s a p r e c u r s o r f o r these l a t e r compounds (155-160). I t was unclear whether the r a d i o a c t i v i t y i n sphingomyelin and g l y c e r o p h o s p h o c h o l i n e came from phospho-c h o l i n e d i r e c t l y , v i a p h o s p h a t i d y l c h o l i n e , or both. In each experiment, the r a d i o a c t i v i t y i n c e l l - e x t r a c t e d c h o l i n e ( a f t e r 0.5h of chase) and CDP-choline was n e g l i g i b l e , through-out the chase p e r i o d - i e . l e s s than 5% and 2% r e s p e c t i v e l y . T h i s was expected s i n c e exogenous c h o l i n e taken up by the c e l l i s phosphorylated extremely q u i c k l y (124,161). As f o r CDP-choline, i t s pool s i z e i s r e l a t i v e l y s m a l l and i t i s turned over q u i c k l y i n the de novo b i o s y n t h e t i c pathway o f p h o s p h a t i d y l c h o l i n e . As t h i s i m p l i e s , the r a t e - l i m i t i n g s t e p o f that pathway i s the c o n v e r s i o n of phosphocholine t o CDP-choline (114,163,164). I t f o l l o w s that the l a r g e s t -52-(a) (b) Chase Time (h) 3 F i g u r e 16. I n c o r p o r a t i o n of [Me- H|choline i n t o V a r i o u s C h o l i n e -C o n t a i n i n g Compounds i n BHK C e l l s . In 4 ml of main-tenance medium, 100 mm p l a t e s of c e l l s were pul s e d f o r 30 min with 5.7 uCi of JMe-^ifj c h o l i n e . The c e l l s were r i n s e d and i n c u -bated (chased) i n growth medium at 37 i n a CC^ i n c u b a t o r . D u p l i c a t e p l a t e s of c e l l s were combined when har v e s t e d at each of the time p o i n t s ( M a t e r i a l s and Methods s e c t i o n IV-1). (a), Water-soluble compounds; (•) , c h o l i n e ; (O) ,phosphocholine; and (•), g l y c e r o p h o s p h o c h o l i n e . (b), P h o s p h o l i p i d s ; (•), p h o s p h a t i d y l -c h o l i n e ; (•), sphingomyelin. - 5 3 -F i g u r e 17. E f f e c t o f Doubled [Me- JH]choline C o n c e n t r a t i o n s on I t s I n c o r p o r a t i o n i n t o V a r i o u s Compounds i n BHK  C e l l s . C o n d i t i o n s were the same as f o r F i g u r e 16, except 12 uCi of [Me- 3H]choline was used per(100 mm) p l a t e o f c e l l s . The pulse-chase procedure used here i s o u t l i n e d i n s e c t i o n IV-1 of M a t e r i a l s and Methods, ( a ) , Water-soluble compounds; (T ) , c h o l i n e ; ( O ) , p h o s p h o c h o l i n e ; ( • ) , g l y c e r o p h o s p h o c h o l i n e . (b), P h o s p h o l i p i d s ; (•), p h o s p h a t i d y l c h o l i n e ; and(B), sphingomyelin. -54-F i g u r e 18. E f f e c t o f L a r g e r C e l l P o p u l a t i o n s on the I n c o r p o r t i o n of |_Me-3HJcholine i n t o BHK C e l l s . C o n d i t i o n s were sim-i l a r t o those f o r F i g u r e 17. But l a r g e ( 1 5 0 mm) p l a t e s o f c e l l s were used ( i n s t e a d o f medium(100 mm) s i z e d ) , w i t h 4 o r 5 b e i n g combined when the c e l l s were h a r v e s t e d a t each time p o i n t ( M a t e r -i a l s and Methods s e c t i o n I V - 1 ) . The open and s o l i d symbols r e p -r e s e n t time p o i n t v a l u e s from two independent p u l s e - c h a s e e x p e r -i m e n t s , (a) , W a t e r - s o l u b l e compounds; (T, v) , c h o l i n e ; (•,(")), phos-p h o c h o l i n e ; (•,•), g l y c e r o p h o s p h o c h o l i n e . Jb_)_, P h o s p h o l i p i d s ; (•,0) , p h o s p h a t i d y l c h o l i n e ; (•, D),sph i n g o m y e l i n . S t a n d a r d d e v i a t i o n s shown are tho s e l a r g e r than the symbol i t s e l f , e x c e p t t h a t f o r s p h i n g o m y e l i n (open s q u a r e ) v a l u e s o n l y one d e t e r m i n a t i o n was made. -55-•(a) (b) 10 20 30 40 50 Chase Time (h) -56-p r o p o r t i o n o f r a d i o a c t i v e l a b e l i n p h o s p h a t i d y l c h o l i n e ' s p r e c u r s o r s should be i n phosphocholine ( F i g . 1 ) . In the p h o s p h o l i p i d f r a c t i o n , I found no s i g n i f i c a n t amount of r a d i o a c t i v i t y i n the PE and PI/PS spots on the TLC p l a t e s used to separate the p h o s p h o l i p i d s . There was only a s l i g h t amount i n LPC 1 s area, which might only be the r e s u l t of t r a i l i n g . These f i n d i n g s held true f o r the d u r a t i o n of the chase p e r i o d . The g r e a t e s t p e r i o d o f f l u x i n r a d i o a c t i v i t y l e v e l s o ccurred i n the f i r s t 4 to 8 h of chase ( F i g . 16-18). The ' t r a n s i e n t ' r a d i o a c t i v i t y f i r s t seen i n phosphocholine q u i c k l y showed up i n p h o s p h a t i d y l c h o l i n e , a t a l e s s e r r a t e i n g l y c e r o -phosphocholine and e v e n t u a l l y a l s o i n sphingomyelin ( F i g . 16 -18 ) . The r a d i o a c t i v i t y i n phosphocholine decreased to n e g l i -g i b l e l e v e l s a f t e r 3 2 h . In p h o s p h a t i d y l c h o l i n e and sphingo-myelin, there never seems to be a r e d u c t i o n i n t h e i r dpm l e v e l s . T h i s was a l s o true f o r gl y c e r o p h o s p h o c h o l i n e although there were more o v e r a l l f l u c t u a t i o n s ( F i g . 16-18). I t seems t h a t there was v e r y l i t t l e n e t - l o s s of l a b e l e d c h o l i n e from c e l l u l a r p h o s p h o l i p i d s . T h i s was i n c o n t r a d i c t i o n to p r e v i o u s s t u d i e s (110-114,156-15 9) which u s u a l l y showed sphingomyelin as the o n l y c h o l i n e - l a b e l e d p h o s p h o l i p i d to r e t a i n i t s r a d i o a c t i v i t y i n BHK c e l l s , presumably because i t had a long h a l f - l i f e ; 18h (113). Glycerophosphocholine has not been reported i n very many j o u r n a l s , e s p e c i a l l y i n conjunc--57-t i o n with p h o s p h o l i p i d s . There had been some i n v e s t i g a t i o n s e a r l i e r i n 1949 by Ducet (169) and by Dawson during the middle 1950's (170,171). However, t h i s work was d i r e c t e d toward g l y c e r o 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 r o l e g l y c e r o -phosphocholine and -ethanolamine might p l a y as i n t e r m e d i a t e s i n p h o s p h a t i d y l c h o l i n e and -ethanolamine s y n t h e s i s , r e s p e c -t i v e l y . T h e r e f o r e , t h e r e are no comparable s t u d i e s a v a i l a b l e w i t h which to compare data on g l y c e r o p h o s p h o c h o l i n e . The e x p o n e n t i a l l o s s of l a b e l e d c h o l i n e from phospho-c h o l i n e seemed to represent two d i f f e r e n t r a t e s . A semi-log p l o t of i t s r a d i o a c t i v i t y (dpm) a g a i n s t chase time (h) i n F i g u r e 19, r e v e a l e d a d i s t i n c t p a t t e r n change sometime a f t e r 8 h of chase with c o l d c h o l i n e ( i n growth medium). I t was d i f f i c u l t , with so few time p o i n t s and the f l u c t u a t i o n s be-tween experiments, to determine whether the change in slope a f t e r the 4 8.h r e g i o n represented a p l a t e a u or a very slow r a t e of r a d i o a c t i v i t y being l o s t from phosphocholine ( F i g . 19). A s i m i l a r p l a t e a u , but beginning as e a r l y as 2h a f t e r the p u l s e , was a l s o observed f o r p h o s p h a t i d y l c h o l i n e ( F i g . 16-18). Even a f t e r doubling the c o n c e n t r a t i o n o f l a b e l e d c h o l i n e ( F i g . 16 compared to F i g . 17) and averaging more (2 to 5) and l a r g e r (100mm to 150mm) p l a t e s as from F i g u r e 17 to F i g u r e 18, the r a d i o a c t i v i t y p a t t e r n s had remained very much the same. The l a r g e s t d i f f e r e n c e occured near the end of the chase p e r i o d , i n p h o s p h a t i d y l c h o l i n e s ' dpm l e v e l s ( F i g . 16b,19b,20b). In two of the four experiments 44 \~ i o CD c o -C u i—i K I 0)1 SI 24 U 12 10 20 30 Chase Time (h) 50 F i g u r e 19. Semilog P l o t of the R a d i o a c t i v i t y A s s o c i a t e d with Phosphochline During the Chase  P e r i o d . The r a d i o a c t i v i t y l e v e l s from the P - c h o l i n e r e g i o n s on t h i n - l a y e r chrom-atograms i s shown above. The v a l u e s from four independent experiments have been no r m a l i z e d f o r comparison h e r e - i e . the v a l u e s from F i g u r e s 16 and 18 were converted to the same s c a l e as those i n F i g u r e 17 by; (1), adjustment due to d i f f e r e n t p l a t e s i z e s ( 1 0 0 mm versus 150 mm) (2), the number of p l a t e s used(2, 100 mm versus 4-5, 150mm), and (3), the c o n c e n t r a t i o n of [ M e - 3 H ] c h o l i n e used i n the p u l s e medium(1.5 u C i / m l / p l a t e versus 3.0 u C i / m l / p l a t e ) . (•), v a l u e s from F i g u r e 16; ( O ),values from F i g u r e 17; and ( A ),values from F i g u r e 18. -59-shown, one f o r each p l a t e s i z e used, the amount of r a d i o -a c t i v i t y i n p h o s p h a t i d y l c h o l i n e had i n c r e a s e d ( F i g . 16b and 18b). The o n l y d i f f e r e n c e t h a t c o u l d have been a c o n t r i b u t i n g f a c t o r was i n both these experiments - the BHK c e l l d e n s i t y during the pulse with l a b e l e d c h o l i n e was 10-15% more than i n the o t h e r two experiments. The i n i t i a l uptake of r a d i o -a c t i v i t y d u r i n g the 30 min pulse was about the same i n a l l four experiments; approx. 9% of the t o t a l amount of l a b e l i n the p u l s e medium. I t does not seem l i k e l y t h a t t h i s would have had any d i r e c t e f f e c t on t h e i r r e l a t i v e p h o s p h a t i d y l c h o l i n e dpm l e v e l s 30-40 h afterwards. However, the c e l l s had 'leaked' r a d i o a c t i v i t y i n t o the medium ( F i g . 20). The amount of r a d i o a c t i v i t y i n the aqueous f r a c t i o n of the medium decreased between 24 and 48. h w h i l e the o p p o s i t e occurred f o r the l i p i d a s s o c i a t e d dpm l e v e l s ( F i g . 20). Due to high amounts of o t h e r o r g a n i c and i n o r g a n i c m a t e r i a l a l r e a d y present i n the chase medium, i t was not p o s s i b l e to i d e n t i f y with which c h o l i n e - c o n t a i n -ing compounds the l a b e l was a s s o c i a t e d . The l o s s of r a d i o -a c t i v e l y l a b e l e d compounds from the c e l l i n t o the chase medium has been s t u d i e d p r e v i o u s l y (118,172-17 4). The r e - i n c o r p o r a t i o n o f these compounds may have caused the i n c r e a s e in p h o s p h o l i p i d r a d i o a c t i v i t y l e v e l s so l a t e i n the chase p e r i o d ( F i g . 20). I had kept the c a l f serum ( l i p o p r o t e i n ) content to a minimum i n the p u l s e medium to reduce l i p o p r o t e i n - c e l l exchange of p h o s p h o l i p i d s (172). -60-I O rH OJ C •H rH O U I 25 H 20 15 \-10 5 \-10 2 0 3 0 Ghase Time (h) 40 50 F i g u r e 20. C e l l - I n c o r p o r a t e d [ Me-^Hlcholine R a d i o a c t i v i t y  Subsequently Detected i n the Chase Medium. The chase medium from each of 5, 150 mm p l a t e s of c e l l s was removed a f t e r 24 or 48 hours of chase, j u s t p r i o r to har-v e s t i n g the c e l l s . The r a d i o a c t i v i t y l e v e l s had not v a r i e d s i g n i f i c a n t l y between the p l a t e s of c e l l s a t each time p o i n t , so o n l y two samples from each time were s e l e c t e d f o r both e x t r a c t i o n ( 1 5 2 ) and t h i n - l a y e r chromatography. However the s e p a r a t i o n on the chromatograms was not s u f -f i c i e n t enough to r e s o l v e the compounds a s s o c i a t e d w i t h the r a d i o a c t i v i t y i n the chase medium's p h o s p h o l i p i d ( y ) and aqueous(v) f r a c t i o n s . The c i r c l e s ( # , 0 ) r e p r e s e n t the r a d i o a c t i v i t y v a l u e s f o r the e x t r a c t e d (152) BHK c e l l p h o s p h o l i p i d ( • ) and aqueous(O) f r a c t i o n s . -61-T h i s was e s p e c i a l l y important i n o r d e r to minimize c a l f serum as a source of new sphingomyelin f o r the c e l l . But o n l y by i n c l u d i n g the r a d i o a c t i v i t y from the chase mediums of the 24h and 48 h c e l l samples could a l l 7 the i n c o r p o r a t e d l a b e l be taken i n t o account(0 h, 2.8x10 7 7 dpm; 24 h, 2.6x10 dpm; and 48 h,2.7x10 dpm). T h i s was e s p e c i a l l y s i g n i f i c a n t as the r a d i o a c t i v i t y i n the aqueous f r a c t i o n of the e x t r a c t e d (152) chase medium was l e s s a t 48 h than a t 24 h ( F i g . 20). The above t o t a l s seemed to v e r i f y the i n i t i a l o b s e r v a t i o n i n F i g u r e 20 t h a t the aqueous-soluble r a d i o a c t i v i t y was r e - i n c o r p o r a t e d i n t o the c e l l s during the l a t t e r 24 h chase p e r i o d . 2. Long-term L a b e l i n g S t u d i e s T h i s was done f o r three reasons. The f i r s t was to i n c r e a s e the amount o f r a d i o a c t i v i t y i n sphingomyelin and p o s s i b l y l y s o p h o s p h a t i d y l c h o l i n e a l s o . The second reason was to make l a b e l e d BHK p h o s p h a t i d y l c h o l i n e of high s p e c i -f i c r a d i o a c t i v i t y to use i n the making of p h o s p h o l i p i d v e s i c l e s . The l a s t reason was to observe any e f f e c t s l o n g -term l a b e l i n g would have on the r e l a t i v e r a d i o a c t i v i t y l e v e l s of p h o s p h a t i d y l c h o l i n e and sphingomyelin and t h e i r s p e c i f i c r a d i o a c t i v i t i e s . By using the pulse-chase method or j u s t by adding l a b e l e d c h o l i n e to the growth medium, the amount of l a b e l taken up i n 17-18 hours was 10-15 times more than during a one-half hour p u l s e . However, the PC/SM r a d i o a c t i v i t y -62-r a t i o (14/1) was approximately the same as those i n the 22.hand 24h chase time samples ( F i g . 16-18). The s p e c i f i c r a d i o a c t i v i t i e s of PC and SM were c a l c u l a t e d i n d i r e c t l y as f o l l o w s . The p r o t e i n content of a s i m i l a r p l a t e o f c e l l s h arvested a t the same time, was 0.86+ 0.018 mg. From Table 1, nmol PL/mg p r o t e i n r a t i o s (239-281) were used i n conjunc-t i o n w i t h the (TLC) p h o s p h o l i p i d Mol% v a l u e s f o r PC (60/.92%) and SM (6.3/.92%) found i n Table 2. These f i g u r e s gave v a l u e s of 13 2-15 7 nmol and 14-16 nmol f o r PC and SM per 150mm p l a t e o f c e l l s . The t o t a l r a d i o a c t i v i t y found i n PC was 1 6 x l 0 6 dpm and i n SM i t was 1.19xl0 6 dpm. T h e i r s p e c i f i c r a d i o a c t i v i t i e s were c a l c u l a t e d as 10 2,000-122,000 dpm/nmol and 62,500-74,200 dpm/nmol, r e s p e c t i v e l y . T h i s value f o r PC was 10-15 times more than the h i g h e s t value recorded ( f o r PC) i n the pulse-chase experiments ( r e f e r to F i g . 21; 3 h time p o i n t ) . Again t h e i r s p e c i f i c r a d i o a c t i v i t i e s were very c l o s e (as i n 20-24.h of chase) though they were not expected to become equal f o r another 24 h (118). The amount of r a d i o a c t i v i t y found i n LPC's area on the t h i n - l a y e r chromatogram was not enhanced a t a l l , r e -maining l e s s than 100,000 dpm. IV How P h o s p h o l i p i d S p e c i f i c R a d i o a c t i v i t i e s R e l a t e to  T h e i r P r e c u r s o r - P r o d u c t R e l a t i o n s h i p s Experiments to determine the s p e c i f i c r a d i o a c t i v i t y o f p h o s p h a t i d y l c h o l i n e and sphingomyelin ( F i g . 21) r e --63-I o 10 20 30 40 50 Chase Time (h) F i g u r e 21. S p e c i f i c R a d i o a c t i v i t y of [Me-^Hlcholine L a b e l e d Phos- p h o l i p i d s i n BHK C e l l s . The open and s o l i d symbols r e p r e s e n t the r e s u l t s from two independent experiments. The r e -s u l t s from the f i r s t experiment(0,d) were obtained from determin-a t i o n s on s i n g l e samples(5, 150 mm p l a t e s of c e l l s combined) a t each time p o i n t . The second s e t o f experimental v a l u e s (•,•) were obt a i n e d from d u p l i c a t e ( f i r s t t h ree time p o i n t s ) or s i n g l e ( l a s t two time p o i n t s ) samples of c e l l s combined from 4 or 5, 150 mm s i z e p l a t e s . The average standard d e v i a t i o n of the mean values f o r phosphatidylcholine(0,#) was + 9.1%, and f o r sphingomyelin (•,•) , i t was ± 14.6%. -64-q u i r e d the h a r v e s t i n g of l a r g e r amounts of BHK c e l l s per time p o i n t . T h i s was necessary to o b t a i n enough sphingo-myelin ( v i a TLC s e p a r a t i o n ) to assay i t a c c u r a t e l y f o r l i p i d phosphorus (150). The a c t u a l amount decided upon, 4 or 5 l a r g e p l a t e s , was a crude estimate c a l c u l a t e d from work o f o t h e r s on the p h o s p h o l i p i d content o f BHK c e l l s (114,115-119,12 3,16 2,16 6-168). If p h o s p h a t i d y l c h o l i n e i s the immediate p r e c u r s o r of sphingomyelin, then the s p e c i f i c r a d i o a c t i v i t y o f both l i p i d s should obey the c r i t e r i a f i r s t proposed by Z i l v e r -smit ejt a_l. (155). Such c r i t e r i a p r e d i c t e d f o r a p r e -c u r s o r - p r o d u c t r e l a t i o n s h i p were as f o l l o w s ; ( i ) I n i t i a l l y , the s p e c i f i c r a d i o a c t i v i t y o f the p r e c u r s o r w i l l be g r e a t e r than t h a t of the product, subsequently l e s s than t h a t of the product; ( i i ) The p o i n t a t which the s p e c i f i c r a d i o -a c t i v i t y - t i m e curves of p r e c u r s o r and product cross w i l l be the same moment f o r which the s p e c i f i c r a d i o a c t i v i t y of the product i s a maxim. Z i l v e r s m i t ' s c r i t e r i a (155-157, 160) are a c c o r d i n g to Reiner (158,159), not r e s t r i c t e d t o steady s t a t e systems. However, the p a r t i c i p a t i o n of more than one immediate pr e -c u r s o r curve would not f i t these c r i t e r i a . I f the p r e c u r s o r curve c r o s s e s the product curve b e f o r e the product's s p e c i f i c r a d i o a c t i v i t y a t t a i n s i t s maximum, then the pre-c u r s o r would not be an immediate one to the product (158,159). -65-My experiments y i e l d e d s p e c i f i c r a d i o a c t i v i t y - t i m e curves (Fig.21) t h a t d i d not e x a c t l y f i t e i t h e r of the two curves p r e v i o u s l y d e s c r i b e d . The s p e c i f i c r a d i o a c t i v -i t y - t i m e curves f o r p h o s p h a t i d y l c h o l i n e and sphingomyelin i n i t i a l l y f i t the c r i t e r i a ' s p a t t e r n f o r a p r e c u r s o r - p r o d u c t r e l a t i o n s h i p ( F i g . 21). However, t h e i r l i n e s never seemed t o c r o s s , but reach a k i n d of ' e q u i l i b r i u m ' p l a t e a u a t 22 h i n t o the chase p e r i o d ( F i g . 21). Except f o r s m a l l f l u c t u a -t i o n , t h i s p l a t e a u remains u n t i l the end of the chase p e r i o d (48 h l o n g ) . The r e s u l t s from two separate e x p e r i -ments, (open and s o l i d symbols i n F i g u r e 21) seemed to o v e r l a p or match, to support t h i s p a t t e r n . These data suggest t h a t p h o s p h a t i d y l c h o l i n e i s a p r e c u r s o r f o r sphingomyelin, but not the immediate donor o f sphingomyelins' phosphocholine moiety. F u r t h e r s t u d i e s u s i n g p h o s p h a t i d y l c h o l i n e as the p u l s e source (and t h e r e -f o r e o n l y source) of l a b e l e d c h o l i n e were hoped to r e s o l v e t h i s ambiguity. V. M o n i t o r i n g R e l a t i v e P h o s p h o l i p i d Pool S i z e s During  the Chase P e r i o d Values f o r l a b e l e d - l i p i d p o o l s i z e s were c a l c u l a t e d from a l i q u o t s used i n phospholipid-phosphorus assays ( M a t e r i a l s and Methods s e c t i o n II-4) and c o r r e c t i n g f o r recovery from TLC by using the sample la n e s ' p e r c e n t value f o r t o t a l r a d i o a c t i v i t y r e c o v e r e d . Pool s i z e s are shown -66-i n F i g u r e 22. For p h o s p h o l i p i d s (PE and PI/PS) t h a t con-t a i n e d very l i t t l e or no r a d i o a c t i v i t y , p o o l s i z e s were ap-proximations o n l y . T h e i r percent r e c o v e r y from TLC s i l i c a g e l v/as c a l c u l a t e d as above, but the r a d i o a c t i v i t y recov-ered was mostly (98%) a s s o c i a t e d with PC and SM, and t h e r e -f o r e r e a l l y only p r o p o r t i o n a l t o PC and SM nmol re c o v e r y . The 'mol %' of the t o t a l p h o s p h o l i p i d content r e -covered from the s i l i c a g e l (as c a l c u l a t e d above) was shown i n t a b l e 2. These val u e s should have been around 95%, w i t h the o t h e r 5% a t t r i b u t e d to p h o s p h a t i d i c a c i d and minor p h o s p h o l i p i d s (LPC, LPE, e t c . ) not d e t e c t e d on the TLC p l a t e . The i n d i v i d u a l mol % v a l u e s were s i m i l a r t o those o f other r e s e a r c h e r s f o r BHK c e l l s (116-119, 162). The l a r g e s t d ivergences seemed to be i n a 10% h i g h e r v a l u e f o r phos-p h a t i d y l c h o l i n e (60%) and a corresponding lower value f o r phosphatidylethanolamine (17%). But the l e a s t % v a r i a n c e was a l s o i n the p h o s p h a t i d y l c h o l i n e v a l u e s (Table 2 ) . T h i s was probably due to the l a r g e amounts of p h o s p h a t i d y l c h o l i n e i n the samples and the c o r r e c t i o n method based on dpm r e -covery from t h i n - l a y e r chromatograms. T h i s l a t t e r f a c t o r was more a r e f l e c t i o n of p h o s p h a t i d y l c h o l i n e r e c o v e r y than of any o t h e r p h o s p h o l i p i d . More than 90% of the r a d i o a c t i v i t y i n the l i p i d f r a c t i o n was a s s o c i a t e d w i t h the p h o s p h a t i d y l -c h o l i n e area on t h i n - l a y e r chromatograms (see R e s u l t s and D i s c u s s i o n s e c t i o n I I I ) . -67-10 20 30 40 50 Chase Time (h) F i g u r e 22. P h o s p h o l i p i d Pool S i z e s i n BHK C e l l s During the Chase P e r i o d . Pool s i z e s are expressed as nmol per 5 l a r g e (150 mm) p l a t e s of c e l l s . The open symbols (0,V,D) r e p r e s e n t the average value of 1-2 phospholipid-phosphorus determinations(150), while the s o l i d symbols ( • j Y .B ) r e p r e s e n t the average ± s.d. of 3-6 d e t e r m i n a t i o n s . The l i n e , ( -) , r e p r e s e n t s an o v e r l a p p i n the two curves f o r sphingomyelin (•,•) . P h o s p h a t i d y l c h o l i n e (0/#), and phosphatidylethanolamine(V/V)• Refer to M a t e r i a l s and Meth-ods s e c t i o n s II-4', IV-1, V, and VI. To convert to 'time a f t e r the c e l l s were s u b c u l t u r e d , ' add approximately 20 h to the times i n the above F i g u r e . -68-Table 2 Mole Percent Composition of P h o s p h o l i p i d s i n BHK C e l l s Molar percentages o f i n d i v i d u a l p h o s p h o l i p i d s were determined from the r a t i o of the L i p i d - P i f o r a g i v e n p h o s p h o l i p i d to the L i p i d - P i from whole l i p i d f r a c t i o n s e x t r a c t e d from c e l l samples. Refer to ' M a t e r i a l s and Methods' s e c t i o n s I I - l , I I - 4 , IV, V, and 'Results and D i s c u s s i o n ' s e c t i o n I. Hours i n Chase Medium P h o s p h o l i p i d Mean Value (Mol% ± s.d.) 0.0 3.0 22.0 32.0 45.5 (0.0) (24.0) (48.0) SM T o t a l P h o s p h o l i p i d (Mol%) 4.9 3.9 6.6 8.6 6.2 6 . 3 ± 1 . 5 (6.3) (5.9) (7.8) PI/PS 8.6 12.6 6.7 10.6 9.3 9.6 1 2.2 PE 10.4 10.8 12.6 22.6 19.5 17.2 1 5.1 (21.6) (18.7) (21.2) PC 64 51 73 67 50 59.9 1 7.9 (59) (56) (59) TLC T o t o a l 1 88 79 98 109 85 91.8 1 12 (87). (81) (89) (85.7 1 4.2) 1 Includes a l l p h o s p h o l i p i d s d e t e c t e d and recovered from each t h i n - l a y e r chrmatogram. The v a l u e s i n p a r e n t h e s i s were from a separate experiment and do not i n c l u d e PI/PS v a l u e s . -69-VI. Can Phosphatidyl!" Me-3H ] c h o l i n e be Incorporated i n t o BHK C e l l s Plasma Membrane by a PLEP-Mediated Exchange  Reaction? The f i r s t o b j e c t i v e here was to f i n d out i f phosphati-d y l c h o l i n e p r e v i o u s l y l a b e l e d with [Me- 3H] c h o l i n e (Mater-i a l s and Methods s e c t i o n IV-2) could be i n s e r t e d i n t o the plasma, membrane of BHK c e l l s by the a c t i o n of a pho s p h o l i p i d exchange p r o t e i n (PLEP). T h i s exchange would have to be r a p i d enough to get a large amount of l a b e l i n t o the c e l l (membrane). Upon achieving t h i s , the second o b j e c t i v e would be to incubate ('chase') the c e l l s long enough to observe ( i n the c e l l s ) a lo s s of l a b e l ( i e . [ Me-3H ] c h o l i n e ) from p h o s p h a t i d y l c h o l i n e and i d e n t i f y , any compound with which i t i s subsequently a s s o c i a t e d . These two o b j e c t i v e s should allow some kind of con-c l u s i o n s about the precursor-product r e l a t i o n s h i p (between p h o s p h a t i d y l c h o l i n e and sphingomyelin) whose e x i s t e n c e had been i n d i c a t e d i n previous s e c t i o n s . The experimental procedure proved very d i f f i c u l t to develop. O v e r a l l , the presence of a PLEP f r a c t i o n u s u a l l y produced a two-fold enhancement i n the exchange r e a c t i o n . Unlabeled p h o s p h a t i d y l c h o l i n e i n BHK c e l l plasma membranes was presumably r e p l a c e d by l a b e l e d p h o s p h a t i d y l c h o l i n e (mon-i t o r e d as dpm uptake) from p h o s p h o l i p i d v e s i c l e s i n an aque-ous s o l u t i o n . Without PLEP, each large (150mm) p l a t e of c e l l s (ap-prox. 50% confluent) incorporated 0.46% of the r a d i o a c t i v i t y i n t o c e l l u l a r p hospholipids a f t e r almost 5 h of incuba t i o n (Table 3). A f t e r 50 h of incuba t i o n t h i s was increased to 3.7-4.9% for the whole c e l l - l i p i d f r a c t i o n (Table 3). T h i s appeared to be a s t r i c t l y l i n e a r phenomenon, probably based on. random contact and d i f f u s i o n . Higher c e l l d e n s i t y (from 50% to over 90% confluent) and s p e c i f i c r a d i o a c t i v i t y of lab e l e d phosphatidyl [Me-^H]-c h o l i n e (from 3100 to 3600 dpm/nmol PC) i n PC v e s i c l e s ( M a t e r i a l s and Methods se c t i o n s VII-2 and VIII - 1 ) , produced an increased percent t r a n s f e r of r a d i o a c t i v i t y . T h i s reached 3.8-4.2% f o r c o n t r o l (no PLEP) c e l l s ' l i p i d - f r a c t i o n (1380-1640 dpm) and 6.1-7.4% for the l i p i d - f r a c t i o n (2400-2900 dpm) of c e l l s with a crude PLEP supernatant f r a c -t i o n present. The incuba t i o n time was only 80 min i n each case. The water-soluble r a d i o a c t i v i t y was never more than 350 dpm and u s u a l l y was about 2 50 dpm for c e l l s with or with-out PLEP i n the pulse medium (MEMA + v e s i c l e s o l u t i o n ) . The increased percent t r a n s f e r mentioned above could probably be a t t r i b u t e d to the higher r a t i o ( s p e c i f i c r a d i o -a c t i v i t y ) of a v a i l a b l e l a b e l e d PC. Also, an increased 'mem-brane' pool s i z e ( c e l l density) would increase the proximi t y of v e s i c l e s and c e l l s as w e l l as decrease the p r o b a b i l i t y that l a b e l e d PC i n c e l l membranes would be exchanged back i n t o the ph o s p h o l i p i d v e s i c l e s . The composition of the pulse medium was changed, using the PBS s o l u t i o n i n s t e a d of the MEMA mixture. The PBS s o l u t i o n had fewer a d d i t i v e s than MEMA, which might have T a b l e 3 I n c o r p o r a t i o n o f [Me- H ] c h o l i n e L a b e l e d P h o s p h a t i d y l c h o l i n e i n t o BHK C e l l s Each 150 mm p l a t e o f c e l l s (approx. 50% c o n f l u e n t ) was 'pulsed' (37°) w i t h 8 m l o f growth medium i n c l u d i n g PC v e s i c l e s (3100 dpm/nmol PC). T h i s was i n c r e a s e d t o 2 5 ml o f growth medium e i t h e r by r e p l a c i n g the p u l s e medium c o m p l e t e l y (chase) o r by ad d i n g 17 ml o f f r e s h growth medium (to m a i n t a i n the p u l s e s i t u a t i o n ) . The w a t e r - s o l u b l e r a d i o a c t i v i t y r e p r e s e n t s t h e combined P - c h o l i n e and G - P - c h o l i n e a r e a s from a t h i n l a y e r chromatogam. See a l s o s e c t i o n V I I I - 1 o f M a t e r i a l s and Methods. R a d i o a c t i v i t y (dpm) P u l s e Chase Time Time (h) (h) PC- Water- PC SM LPC R a t i o v e s i c l e s s o l u b l e (PC/SM) 4.7 44.7 74000 720 570 60 0 9 4.7 44.7 74000 765 ( 2 2 2 0 ) 1 4.7 44.7 148000 880 1050 45 0 23 4.7 44.7 148000 670 630 60 0 10.5 49.3 0 74000 1400 (3650) 49.3 0 148000 2450 6820 520 170 13 49.3 0 148000 2800 5540 400 160 14 1 R e p r e s e n t s T o t a l F o l c h - e x t r a c t e d (152) l i p i d r a d i o a c t i v i t y i n the c e l l s . ' reduced p o s s i b l e i n t e r f e r e n c e , s i n c e even the c o n t r o l c e l l l i p i d - f r a c t i o n was now 20% of the i n i t i a l PC v e s i c l e r a d i o -a c t i v i t y a f t e r a 60 min p u l s e - i n c u b a t i o n . However, when c e l l s were ha r v e s t e d and e x t r a c t e d (152) a f t e r a 9 h chase p e r i o d , only 10-13.4% of the i n i t i a l r a d i o a c t i v i t y was found a s s o c i a t e d w i t h the c o n t r o l c e l l s ' l i p i d - f r a c t i o n . In c e l l s t h a t had a 'pH 5.1 supernatant' PLEP f r a c t i o n i n c l u d e d , the i n i t i a l 12-15% t r a n s f e r dropped t o 8.7-10.4% a f t e r a 9 h chase p e r i o d a l s o . During the pH adjustment ( r e f e r to M a t e r i a l s and Methods s e c t i o n VII-1 and VIII-1) of the crude PLEP supernatant f r a c t i o n used p r e v i o u s l y , o c c a s i o n a l pH f l u c t u a t i o n s up to 11.4 were observed. What e f f e c t ( i f any) t h i s had on the p h o s p h o l i p i d exchange p r o t e i n (PLEP) i t s e l f i s not known. But t h i s may be the reason why the c o n t r o l c e l l s had a h i g h e r p e r c e n t t r a n s f e r than c e l l s exposed to PLEP. The b e n e f i t s of the pH adjustment step were f u r t h e r p r o t e i n p u r i f i c a t i o n and reduced endogenous p h o s p h o l i p i d con-tent (128). The l a t t e r f a c t o r may have had a d i l u t i o n e f f e c t on the s p e c i f i c r a d i o a c t i v i t y of the prepared phos-p h a t i d y l [ Me-3H ] c h o l i n e v e s i c l e s ( M a t e r i a l s and Methods sections VII-2 and V I I I - 1 ) . The endogenous p h o s p h o l i p i d content i n r a t l i v e r c y t o s o l i s about 2 ug PL-Pi per 10 mg p r o t e i n (128). I u s u a l l y used 10-15 mg p r o t e i n from the crude supernatant f r a c t i o n ( c y t o s o l ) per p l a t e of c e l l s incubated. The p h o s p h a t i d y l c h o l i n e v e s i c l e p r e p a r a t i o n (used per p l a t e of c e l l s ) u s u a l l y contained 0.32-1.24 ug PL-Pi. Therefore 2 ug more could have c o n c e i v a b l y decreased p h o s p h a t i d y l c h o l i n e ' s s p e c i f i c r a d i o a c t i v i t y by 62-86%. -73-I c a r r i e d out a time course experiment ( F i g u r e 23) w i t h and w i t h o u t the a d d i t i o n of a pH 5.1 f r a c t i o n of PLEP. I a l s o changed t o 100mm s i z e p l a t e s t o reduce the volume of the i n c u b a t i o n m i x t u r e . T h i s a l s o meant a r e d u c t i o n i n the number of c e l l s , s i n c e the p l a t e s u r f a c e was reduced 2 . 2 5 - f o l d . T h i s may have a d v e r s e l y a f f e c t e d the net t r a n s -f e r of ( r a d i o a c t i v e ) p h o s p h a t i d y l c h o l i n e , as t h e r e was an observed drop t o about 2.0% net t r a n s f e r i n a l l the p l a t e s , a f t e r 60 min of i n c u b a t i o n i n the presence and absence of PLEP ( F i g u r e 23). D o u b l i n g the v e s i c l e c o n c e n t r a t i o n had not h e l p e d much, nor had d o u b l i n g the c o n c e n t r a t i o n of the pH 5.1 supernatant f r a c t i o n ( F i g u r e 23). The v e s i c l e p r e p a r a t i o n used here was o b t a i n e d from long-term l a b e l i n g o f BHK c e l l s ( M a t e r i a l s and Methods s e c t i o n I V - 2 ) . Consequently, the s p e c i f i c r a d i o a c t i v i t y of p h o s p h a t i d y l c h o l i n e used was now about 28,000 dpm/nmol. However, the p r e v i o u s net t r a n s f e r of 10-20% of the i n i t i a l v e s i c l e r a d i o a c t i v i t y was not i n c r e a s e d or even o b t a i n e d ( F i g u r e 23), as a r e s u l t of t h i s much h i g h e r s p e c i f i c r a d i o -a c t i v i t y . Perhaps the p e r c e n t t r a n s f e r would have been even lower w i t h o u t the s u b s t a n t i a l l y h i g h e r s p e c i f i c r a d i o a c t i -v i t y of the PC v e s i c l e p r e p a r a t i o n s used. F i n a l l y , t o t e s t the v i a b i l i t y of the p r e v i o u s l y f r o z e n PLEP f r a c t i o n s t h a t were used, f r e s h l y prepared r a t l i v e r PLEP was used i n c o n j u n c t i o n w i t h the 28,000 dpm/nmol ve-s i c l e p r e p a r a t i o n (Table 4 ) . A pH 5.1 supernatant f r a c t i o n ( M a t e r i a l s and Methods s e c t i o n VII - 1 ) was p r e p a r e d w i t h o u t -74-m I Cu TJ OJ c •H rH o Xi O i—i w I > 1 TJ •H -P (0 a, o p-1 510 U 480 450 420 I— 20 h 10 15 30 45 Incu b a t i o n Time (min) 60 F i g u r e 23. PLEP A c t i v i t y Time Course Experiments. BHK c e l l s ( 90% c o n f l u e n t ) i n medium s i z e (100 mm) p l a t e s were incubated i n the presence or absence of a pH 5.1 supernatant PLEP f r a c t i o n . The i n i t i a l P C - v e s i c l e r a d i o a c t i v i t i e s were; 500,000 dpm f o r co n t r o l ( O ) and +PLEP (•) c e l l s ; 474, 000 dpm f o r 2x[+PLEP] c e l l s ( • ) ; a n d 955,000 dpm f o r 2 x [ P C - v e s i c l e ] c e l l s (T) . (a), Pulse-medium r a d i o a c t i v i t y . The val u e s f o r the 2x [PC-v e s i c l e ] c e l l s ( T ) have been halved, (b), P h o s p h o l i p i d f r a c t i o n r a d i o a c t i v i t y from the extracted(152) c e l l s . -75-Table 4 PLEP-Mediated Exchange o f Labeled P h o s p h a t i d y l c h o l i n e F r e s h l e y prepared pH 5.1 supernatant f r a c t i o n o f r a t l i v e r PLEP was used w i t h a PC v e s i c l e p r e p a r a t i o n of about 28,000 dpm/nmol. The c e l l s (90% c o n f l u e n t i n 100 mm p l a t e s ) were incubated at 37° i n a C O 2 i n c u b a t o r f o r the d u r a t i o n o f the p u l s e . I n i t i a l l y , 491,000 dpm and 495,000 dpm of p h o s p h a t i d y l [Me- 3H]choline were i n c l u d e d i n the pu l s e medium (0.25 M sucrose, ImM EDTA, 1.0 mM T r i s HCL; pH 7.4) of c o n t r o l and PLEP-exposed c e l l s . (Refer t o M a t e r i a l s and Methods S e c t i o n V I I I - 1 ) . R a d i o a c t i v i t y (dpm) Pulse Time (min) C o n t r o l +PLEP Ra t i o ( l i p i d / a q u e o u s ) Water- L i p i d s s o l u b l e Water- L i p i d s C o n t r o l +PLEP s o l u b l e 20 650 920 1010 5150 7380 6520 4720 8500 18680 15940 7.9 8.0 6.5 4.0 1.9 46 22 2 0 3770 4100 14910 14920 15400 11750 10770 14170 17310 6.7 4.0 3.8 1.2 1.6 65 4 710 4090 6670 13690 16010 12680 8450 3840 19670 22170 2 . 9 3.9 1.9 2.3 5.8. -76-the high pH f l u c t u a t i o n s observed e a r l i e r . The percent of i n i t i a l v e s i c l e r a d i o a c t i v i t y t r a n s f e r r e d was now 3.8-4.1% and 5.3-5.7% f o r c o n t r o l and +PLEP c e l l s r e s p e c t i v e l y , a f t e r 65 min of pulse time (Table 4 ) . To reduce the e f f e c t s of endogenous components i n the pulse medium (from PBS of MEMA), the same b u f f e r s o l u t i o n (0.25M sucrose, ImM EDTH, l.OmM T r i s - H c l ; pH 7.4) that was used to prepare the pH 5.1 PLEP supernatant f r a c t i o n , was a l s o used i n the pulse medium instead of PBS or MEMA. This was a l s o the same b u f f e r s o l u t i o n used by others i n PLEP-me-di a t e d exchange experiments (125,126,128). A f t e r 60 min, both c o n t r o l and +PLEP c e l l s showed increased percent uptake of v e s i c l e r a d i o a c t i v i t y a s s o c i a t e d with t h e i r l i p i d - f r a c t i o n s , from 2% ( i n Figure 23) to 2.6-33% and 4-4.5% r e s p e c t i v e l y (Table 4). But the c e l l - e x t r a c t e d (152) l i p i d r a d i o a c t i v i t y was much lower r e l a t i v e to the c e l l - e x t r a c t e d aqueous r a d i o -a c t i v i t y (Table 4), than p r e v i o u s l y observed. Before, ( f o r p u l s e s under 70 min) t h i s r a d i o a c t i v i t y r a t i o had ranged from 6 to 17 ( r e s u l t s not shown). When c o n t r o l or +PLEP p l a t e s of c e l l s were l e f t exposed to the i s o t o p e f o r about 4 8 h, the percent r a d i o a c t i v i t y t h a t was t r a n s f e r r e d i n c r e a s e d d r a m a t i c a l l y , to 21% and 31% r e s p e c t i v e l y . .These were the h i g h e s t percentages y e t observed f o r any l e n g t h of 'pulse' time. A l s o the c e l l s ' -77-l i p i d / a q u e o u s r a d i o a c t i v i t y r a t i o was now at l e a s t a t the lower end of the range o b t a i n e d p r e v i o u s l y ; i e . 107,000 dpm/ 14,000 dpm (or a r a t i o of 7.6) f o r c o n t r o l c e l l s , and 149,000 dpm/23,000 dpm (or a r a t i o of 6.5) f o r +PLEP c e l l s . The r a d i o a c t i v i t y observed i n c e l l s observed f o r 48 h (to the isotope) f i n a l l y r e p r e s e n t e d dpm l e v e l s high enough to be detectable i n i n d i v i d u a l phospholipds to be recovered from t h i n - l a y e r chromatograms. But the lengthy d u r a t i o n of the ex-p o s u r e / i n c u b a t i o n c o u l d have i n t r o d u c e d unknown f a c t o r s t h a t might a f f e c t the p l a u s i b i l i t y of p h o s p h a t i d y l c h o l i n e being the only ( s i g n i f i c a n t ) source of la b e l e d c h o l i n e . I t was a l s o not c l e a r that the PLEP f r a c t i o n s used were of any great b e n e f i t (Figure 23). Therefore, another method was sought f o r i n t r o d u c i n g s u f f i c i e n t amounts of l a b e l e d p h o s p h a t i d y l c h o l i n e i n t o BHK c e l l s . -78-V I I . I n c o r p o r a t i o n of Radio-actively Labeled Compounds by  PEG/PHA-Mediated Fu s i o n Techniques The o v e r a l l o b j e c t i v e s were the same as those men-ti o n e d i n the preceding s e c t i o n ( V I ) . An added f e a t u r e t h a t r e s u l t e d from i n c o r p o r a t i n g ' i n t a c t ' p h o s p h o l i p i d v e s i c l e s was the p o s s i b i l i t y of i n t r o d u c i n g w a t e r - s o l u b l e compounds (that the c e l l membrane was p r e v i o u s l y impermeable to) w h i l e i n s i d e the v e s i c l e s (137). Two d i f f i c u l t i e s were encountered while d e v e l o p i n g t h i s procedure. The f i r s t , of course, was g e t t i n g enough l a b e l e d p h o s p h a t i d y l c h o l i n e i n t o BHK c e l l s so that the c h o l i n e - l a b e l could be t r a c e d i f and when i t was t r a n s -f e r r e d to any o t h e r compound - _ie_. sphingomyelin. The second problem was to maintain the v i a b i l i t y o f the c e l l s a f t e r exposure to the PEG mixture. C e l l s are able to take up much p h o s p h o l i p i d i n the v e s i c l e s with l i t t l e adverse e f -f e c t (135-137). I t i s the PEG i t s e l f which seems to be harmful to the c e l l . For t h i s reason the compound phyto-hemagglutinin (PHA) was i n c l u d e d . The l e n g t h of c o n t a c t between c e l l s and the 50% PEG s o l u t i o n (and when 1/10 d i l u t e d ) was kept at a minimum a l s o . A mixed v e s i c l e p r e p a r a t i o n was used (except i n the f i r s t t e s t ; Table 5), c o n t a i n i n g 0,8 umol phosphati-d y l c h o l i n e and 0.2 umol p h o s p h a t i d y l s e r i n e , , per tube .of c e l l s (Tables 6-8). The PEG/PHA s o l u t i o n was added l a s t ( r e f e r to M a t e r i a l s and Methods s e c t i o n V I I I - 2 ) . No t r a n s f e r of r a d i o a c t i v i t y from l a b e l G-P-choline to unlabeled p h o s p h a t i d y l c h o l i n e i n the v e s i c l e p r e p a r a t i o n s -79-T a b l e 5 I n c o r p o r a t i o n o f P h o s p h a t i d y l [Me.- H ] c h o l i n e L a b e l e d P C - V e s i c l e s  i n t o the Plasma Membrane o f BHK C e l l s by PEG-Mediated F u s i o n I n i t i a l l y , 254 , 0 0 0 dpm o f l a b e l e d P C - v e s i c l e s j w e r e added t o each o f two tubes o f c e l l s . Each tube c o n t a i n e d c e l l s (90% c o n f l u e n t ) from one l a r g e p l a t e . The c e l l s w i t h o u t PEG were t r e a t e d the same as thos e w i t h PEG, e x c e p t 1 ml o f MEMA i n s t e a d o f PEG, was added. A f t e r t h e PEG i n c u b a t i o n and washing p r o c e d u r e ( s e c t i o n V I I I - 2 o f Methods and M a t e r i a l s ) , 1/10 o f the c e l l s from each tube was h a r v e s t e d , a n o t h e r 8/10 was c u l t u r e d i n two l a r g e p l a t e s each. One p l a t e from each tube was h a r v e s t e d a t each r e m a i n i n g time p o i n t . The v a l u e s below have been c o r r e c t e d f o r t h i s d i l u t i o n e f f e c t . V a l u e s i n p a r e n t h e s i s r e p r e s e n t combined dpm t o t a l s . R a d i o a c t i v i t y (dpm) 1 Chase Time (h) P - c h o l i n e G-P-c h o l i n e SM PC R a t i o (PC/SM) (-) PEG 0.0 (8770) 4660 17870 3.8 10.5 (1280) 810 4400 5.1 2 3.0 (1360) 3110 4300 1.4 (+) PEG 0.0 (3770) 6380 94910 15.0 10.5 9380 4750 2950 31200 10.6 23.0 4750 462 0 3250 25520 7.9 -80-was observed. T h i s was checked by p u t t i n g l a b e l e d G-P-c h o l i n e i n the unlabeled PC/PS v e s i c l e p r e p a r a t i o n onto t h i n - l a y e r , chromatograms, and using the ' p h o s p h o l i p i d ' s o l v e n t system ( M a t e r i a l s and Methods s e c t i o n I l - l ( i i ) ) . Approx. 93-96% of the r a d i o a c t i v i t y remained a t the o r i g i n . Labeled PC i n the PC/PS v e s i c l e p r e p a r a t i o n v/as sub j e c t e d to the same procedure, and 82-87% of the r e -covered r a d i o a c t i v i t y was found t o be a s s o c i a t e d with the p h o s p h a t i d y l c h o l i n e - i o d i n e spot on the chromatogram The low value was probably caused by t r a i l i n g of the l a b -e l e d PC/PS v e s i c l e r a d i o a c t i v i t y on the chromatogram. T h i s c o u l d have been one source f o r the r a d i o a c t i v i t y which was observed i n sphingomyelins' chromatograph area (Tables 5-8). However, when l a b e l e d PC i n PC v e s i c l e s (no PS added) had .been chromatographed, 96% of the r a d i o a c t i v i t y was found i n the PC area. T h i s confirmed the v i s u a l o b s e r v a t i o n that the smeared i o d i n e s t a i n i n the f i r s t i n s t a n c e was due to the excess PS present on the chromatogram. Since most of the PC/PS V e s i c l e p r e p a r a t i o n was washed f r e e o f the c e l l s , excess PS should not have been a problem f o r the c e l l u l a r p h o s p h o l i p i d samples prepared f o r s e p a r a t i o n on t h i n - l a y e r chroma tograms. In the f i r s t group of experiments (Table 5), no phytohemagglutinin (PHA) was used. The v e s i c l e prepara-t i o n contained o n l y BHK p h o s p h a t i d y l c h o l i n e (28,000 dpm/ nmol) prepared e a r l i e r ( M a t e r i a l s and Methods s e c t i o n IV-2). -81-Each tube of c e l l s contained approx. 254,000 dpm of phos-p h a t i d y [ Me- H] c h o l i n e , o r 9 nmol of the p h o s p h o l i p i d . The c e l l s w i t h PEG exposure i n i t i a l l y r e t a i n e d 5 times as much r a d i o a c t i v i t y as those c e l l s not exposed ( c o n t r o l ) . Both groups of c e l l s l o s t r a d i o a c t i v i t y (approx. 75%) during the chase p e r i o d (Table 5), though c o n t r o l c e l l s l o s t a g r e a t e r percentage. T h i s l o s s of r a d i o a c t i v i t y was probably due to the r e l e a s e o f v e s i c l e s t h a t had ad-hered to the c e l l s u r f a c e but were not fused o r adsorbed. E f f l u x of c e l l membrane p h o s p h o l i p i d s ( F i g . 20) (118, 172-174) may a l s o have accounted f o r some of t h i s l o s s , e s p e c i a l l y i f p h o s p h o l i p i d s from fused v e s i c l e s had been p r e f e r e n t i -a l l y exchanged or l o s t to the chase medium. The r a d i o -a c t i v i t y r a t i o s of PC/SM v/ere i n c o n s i s t e n t f o r the c o n t r o l c e l l s , but ranged from 8 to 14 f o r the c e l l s exposed to PEG. Those v a l u e s were a l s o a r e f l e c t i o n o f PC and SM's r e l a t i v e pool s i z e s . T h i s c o u l d be a c o i n c i -dence or the r e s u l t o f an e q u i l i b r i u m between t h e i r s p e c i -f i c r a d i o a c t i v i t i e s . The r e s t of the experiments (Tables 6-8) u t i l i z e d PHA i n the PEG s o l u t i o n and a mixed p h o s p h o l i p i d v e s i c l e p r e p a r a t i o n ( M a t e r i a l s and Methods s e c t i o n s VI1-2 and V I I I - 2 ) . Two p l a t e s of c o n f l u e n t c e l l s were harvested f o r each tube of c e l l s used i n these experiments (Tables 6-8). T h i s had been done to i n c r e a s e the t o t a l c e l l s u r f a c e a v a i l a b l e to fuse with the v e s i c l e s , and to compensate f o r - 8 2 -Table 6 I n c o r p o r a t i o n of PC/PS V e s i c l e s C o n t a i n i n g P h o s p h a t i d y l [Me_-3H]  c h o l i n e i n t o the Plasma Membrane of BHK C e l l s by PEG-Mediated F u s i o n Two l a r g e p l a t e s of c e l l s per tube were used, but t h e i r v i a b i l i t y was much l e s s than thoes c e l l s used f o r Tab l e 5 v a l u e s . One-ten t h of the c e l l s were ha r v e s t e d a t time zero, the ot h e r 9/10 was c u l t u r e d i n two l a r g e p l a t e s which were l a t e r combined .when harvested. See Accompanying t e x t f o r e x c e p t i o n s . Exper-ment No. R a d i o a c t i v i t y (dpm)2 Chase-Time (h) P - c h o l i n e G-P-choline LPC SM PC R a t i o (PC/SM) 0.0 22.5 (3000) (400) 4660 6320 196000 31 9000 1500 17300 12 0.0 (56000) 25.0 1310 1170 0.0 (18000) 25.0 .1200 1760 52.0 2700 10600 (287000) 3100 2350 71800 31 (262000) 6900 2900 62000 21 2000 1700 17500 10-27.0 (7000) 27.0 2700 7200 2.7. 0 (5700) 27.0 3450 7000 (52000) 340 1820 37800 21 (55000) 260 1640 33400 20 1 " I n i t i a l dpm's added;. (2), 610,000 dpm/tube; (3), 588,000 dpm/ tube; and (4), 626,000 dpm/tube. The s p e c i f i c r a d i o a c t i v i t y of p h o s p h a t i d y l [ M e - 3 H ] c h o l i n e was approximately 680 dpm/nmol. 2 R a d i o a c t i v i t y i n each s e t of time p o i n t s r e s u l t e d from one tube of c e l l s . -83-c e l l s l o s t during the washing procedure and because of the PEG s o l u t i o n s t o x i c i t y ( M a t e r i a l s and Methods s e c t i o n s V I I -2 and V I I I - 2 ) . The next three groups' experimental r e s u l t s are shown i n Table 6. They r e f l e c t measures taken to i n c r e a s e the c e l l s ' v i a b i l i t y . In experiment 2 (Table 6), approx. 30% of the i n i t i a l r a d i o a c t i v i t y taken up by the c e l l s was l o s t d uring the washing procedure. T h i s was s i m i l a r to that observed i n the f i r s t experiment. But in an attempt to expose more c e l l s u r f a c e , the c e l l p e l l e t was vortexed to resuspend the c e l l s . T h i s caused the c e l l s ' v i a b i l i t y during the f o l l o w i n g chase p e r i o d to decrease d r a s t i c a l l y . T h i s was e v i d e n t from v i s u a l o b s e r v a t i o n s and the l o s s o f r a d i o a c t i v i t y from the c e l l u l a r p h o s p h o l i p i d f r a c t i o n w i t h 22.5 h of chase time (Table 6). The r e s u l t s f o r the 25 h chase time i n experiment 3, represented ' f l o a t i n g ' c e l l s (from two se t s of chase medium) which could be p e l l e t e d as normal v i a b l e c e l l s would have been. The r e s u l t i n g c e l l u l a r p h o s p h o l i p i d s were e x t r a c t e d by the method of F o l c h et a_l. (152). The 52 h time p o i n t represented the v i a b l e (attached to the p l a t e s ' surface) c e l l s from both ( i n i t i a l ) tubes. The PC/SM r a t i o a t 251 h. was 31 and 21, while i t was only 10 at 52 h of chase. I t was d i f f i c u l t to determine whether t h i s d i f f e r e n c e in PC/SM r a t i o s was a r e f l e c t i o n o f the c e l l s ' v i a b i l i t y o r of a completed trend toward e q u i l i b r i u m of the r a d i o a c t i v i t y between the two p h o s p h o l i p i d s . The l a t t e r p o s s i b i l i t y was -84-supported somewhat by the PC/SM r a t i o s of 31 a t 0 h and 12 a t 22.5 h of chase time (Table 6;Experiment 2 ) . T h i s might have i n d i c a t e d that the e q u i l i b r i u m was h a l t e d or slowed because of the poor c o n d i t i o n o f the c e l l s used f o r the 25h time p o i n t (experiment 3, Table 6 ) . The c e l l s ' c o n d i t i o n was improved d u r i n g experiment 5 (Table 6). Whole a l i q u o t s (2%) of the c e l l - e x t r a c t e d aqueous and l i p i d f r a c t i o n s were counted b e f o r e they were s u b j e c t e d t o t h i n - l a y e r chromatography. I n i t i a l l y , 483,000 dpm had been taken up by the c e l l s but t h i s was reduced to approx. 50,000 dpm due to washing, and chasing the c e l l s f o r 27 h. The PC/SM r a t i o was n e a r l y the same i n each p l a t e o f c e l l s (21 and 20) as were t h e i r r e s p e c t i v e p h o s p h o l i p i d r a d i o -a c t i v i t y counts (Table 6). The l a s t two groups of experiments were performed to determine i f G-P-choline was an int e r m e d i a t e i n the 3 t r a n s f e r of [Me- H ] c h o l i n e from i n c o r p o r a t e d v e s i c l e p h o s p h a t i d y l c h o l i n e to endogenous BHK sphingomyelin (Tables 7 and 8) and whether t h i s t r a n s f e r was r e v e r s i b l e (Table 8 ) . In t h i s experiment (Table 8) very l i t t l e d e r b i s was observed i n the chase medium. Th e r e f o r e i n t a c t c e l l s s t i l l i n suspension t h a t c o u l d be p e l l e t e d the same as v i a b l e c e l l s , were c o n s i d e r e d adequate 'membrane-models'. They were i n c l u d e d i n the e x t r a c t i o n (152) of the r e g u l a r c e l l p e l l e t to ensure more recover y o f c e l l - a s s o c i -ated r a d i o a c t i v i t y . -85-T a b l e 7 I n c o r p o r a t i o n o f PC/PS V e s i c l e s C o n t a i n i n g R a d i o a c t i v e l y L a b e l e d  Compounds i n t o the Plasma Membranes o f BHK C e l l s by PEG-Mediated  F u s i o n The z e r o time p o i n t s were c o r r e c t e d f o r d i l u t i o n s i n c e o n l y 1/10 of t h e c e l l s from one tube were used. B r a c k e t s denote whole c e l l r a d i o a c t i v i t y v a l u e s . The remainder (9/10) o f t h e tube c e l l s u s p e n s i o n was c u l t u r e d f u r t h e r (chased) i n two l a r g e p l a t e s . These p l a t e s of c e l l s were combined when th e c e l l s were h a r v e s t e d . The v a l u e s i n p a r e n t h e s i s r e p r e s e n t r a d i o a c t i v i t y i n each of the two c e l l - e x t r a c t e d (152) phases. 1 2 L a b e l e d Chase Compound Time (h) R a d i o a c t i v i t y (dpm) P - c h o l i n e G - P - c h o l i n e LPC SM PC R a t i o (PC/SM) [ H]PC 0 [156000] 22 (3760) (54700) 22 2100 2100 830 1890 49600 26 22 (2740) (101000) 22 1000 1800 800 3350 64700 19 [ 1 4 C ] G - P - 0 [900] c h o l i n e 22 47 . 5 1 82 91 286 3.1 [ 1 4 C ] G - P - 0 [360] c h o l i n e 22 35 44 7 26 125 4.8 ( c o n t r o l ) 1 I n i t i a l amounts added were; 1,550,000 dpm [ H]PC/tube ;200,000 dpm's o f [ ^ c ] G - P - c h o l i n e / t u b e ( w i t h PC/PS v e s i c l e s ) ; and 120,000 dpm [ i 4 C ] G - P - c h o l i n e / t u b e ( w i t h o u t PC/PS v e s i c l e s . 2 R a d i o a c t i v i t y i n each s e t o f time p o i n t s r e s u l t e d from one tube o f c e l l s . -86-Table 8 I n c o r p o r a t i o n of PC/PS V e s i c l e s C o n t a i n i n g Water S o l u b l e  Compounds and P h o s p h o l i p i d s R a d i o a c t i v e l y Labeled i n t o the  Plasma Membrane of BHK C e l l s by PEG-Mediated F u s i o n . The v a l u e s i n p a r e n t h e s i s r e p r e s e n t r a d i o a c t i v i t y i n whole c e l l s w ith no chase (other than the washing procedure; s e c t i o n VIII-2 of M a t e r i a l s and Methods). These c e l l s were i n an a l i q u o t of the c e l l suspension to be c u l t u r e d f u r t h e r (chased). Labeled Compound Chase' Time (h) R a d i o a c t i v i t y (dpm) P- c h o l i n e G-P-choline LPC SM PC Rat i o (PC/PS) [ JH]PC 0 19 0 19 (127000) 7840 9470 2460 3000 38800 13 (84000) 620 8470 2020 2630 38400 15 [ 1 4C]G-P-c h o l i n e 0 19 76 (4700) 174 9 510 930 1.8 [ 3H]SM 0 19 1220 (32000) 2390 2010 7720 14000 1.8 [ 3H]PC + G-P-choline 0 20 831 (131000) 6082 1420 1040 43800 42 1 I n i t i a l dpm's added were; 744,000 dpm's of [ H]PC per tube; 180,000 dpm's of [ 1 4 C ] G - P - c h o l i n e per tube; 345,000 dpm's of [ 3H]SM per tube; and 620,000 dpm's of [ 3H]PC (with u n l a b e l e d G-P-choline) per tube. 2 The r a d i o a c t i v i t y i n each s e t o f time p o i n t s r e s u l t e d from one tube of c e l l s . Refer a l s o to Table 7. -87-Th e r a d i o a c t i v i t y r a t i o of PC/SM ranged from 13 to 26 f o r chase times of 2 2 h o r l e s s (Tables 7 and 8) although i n experiment 1 (Table 5), the PC/SM r a t i o was lower f o r a l l time p o i n t s ; Ob, 15; 10.5h, 10.5; and 23h, 7.9. T h i s c o u l d have been due to the h i g h e r i n i t i a l s p e c i f i c r a d i o -a c t i v i t y of the v e s i c l e p h o s p h a t i d y l c h o l i n e i n experiment 1 (approx. 28,000 dpm/nmol) than in experiments 2-4 (approx. 750 dpm/nmol) and experiments 5-6 (approx. 900 dpm/nmol). To i n v e s t i g a t e the p o s s i b l e involvement o f G-P-c h o l i n e i n the t r a n s f e r of l a b e l e d P - c h o l i n e , p h o s p h o l i p i d v e s i c l e s were u t i l i z e d (137) to best i n c o r p o r a t e [ ^ 4 C ] -G-P-choline i n t o BHK c e l l s (as i l l u s t r a t e d i n Table 7 -c o n t r o l c e l l s ) . The r e s u l t s showed r a d i o a c t i v i t y a s s o c i -ated w i t h both p h o s p h a t i d y l c h o l i n e and sphingomyelin, which was 2.3 times (Table 7) and 7.7 times (Table 8) as much as when o n l y f r e e [ ^ C ] G-P-choline was exposed to the c e l l s (Table 7). Unlabeled G-P-choline (6 mg) was a l s o used f o r a c o l d - t r a p experiment by i n c l u d i n g i t i n a p h o s p h a t i d y l c h o l i n e l a b e l e d p r e p a r a t i o n of PC/PS v e s i c l e s (Table 8). The r a d i o -a c t i v i t y r a t i o between p h o s p h a t i d y l c h o l i n e and sphingomy-e l i n remained high (45) even a f t e r 20 h o f chase (Table 8 ) . The presence of so much G-P-choline a l s o had an adverse e f f e c t on the c e l l s . Only approx. 1% of the c e l l s had p l a t e d out. The remainder, although s t i l l i n t a c t , were o n l y suspended i n the chase medium and d i d not appear to be -88-v i a b l e . As mentioned p r e v i o u s l y (page 84 ) these c e l l s were i n c l u d e d i n the p r e p a r a t i o n of the r e g u l a r c e l l p e l l e t I o b t a i n e d . The main d i f f e r e n c e here was that these c e l l s accounted f o r 90% of the whole c e l l s i n s t e a d of the 10-20% observed f o r the o t h e r c e l l s i n Table 8. The r e s u l t s f o r these 'other' c e l l s i n Table 8 had not d i f f e r e d s i g n i f i -c a n t l y from those obtained when only the v i a b l e c e l l s were harvested (Table 7 ) . Perhaps t h i s v/as an i n d i c a t i o n t h a t i n t a c t suspended c e l l s had provided the same 'environment' ( f o r the p h o s p h o l i p i d s incorporated) as had the v i a b l e c e l l s . T h i s was probably not true f o r the d e b r i s c e l l s (used i n experiment 3; Table 6) as v i s u a l o b s e r v a t i o n had i n d i c a t e d i n t e r n a l damage and p a r t i a l rupture of plasma membranes. When BHK sphingomyelin ( p r e v i o u s l y l a b e l e d with [Me- H ] c h o l i n e ) was i n c o r p o r a t e d i n t o c e l l s , almost twice the r a d i o a c t i v i t y found i n sphingomyelin was found i n p h o s p h a t i d y l c h o l i n e . T h i s would seem to c l e a r l y i n d i -c a t e the r e v e r s i b i l i t y of the P - c h o l i n e t r a n s f e r from p h o s p h a t i d y l c h o l i n e t o sphingomyelin. The s p e c i f i c a c t i v -i t y of the i n i t i a l sphingomyelin i n the PC/PS v e s i c l e pre-p a r a t i o n was approx. 2800 dpm/nmol (SM). In a l l the experimental r e s u l t s i n Tables 5-8, meth-anol/water s o l u b l e r a d i o a c t i v i t y from the c e l l s was ob-served. Taken i n l i g h t of the ["^C ]G-P-choline e x p e r i -ments, i t would seem that the source o f t h i s r a d i o a c t i v i t y may be the r e s u l t of l a b e l e d p h o s p h a t i d y l c h o l i n e enzymatic -89-de g r a d a t i o n (181,182). T h i n - l a y e r chromatography of the c e l l - e x t r a c t e d (152) aqueous f r a c t i o n never produced r e l i a b l e s e p a r a t i o n of the c a r r i e r - s t a n d a r d s ( P - c h o l i n e and G-P-choline) to q u a n t i t a t e the r a d i o a c t i v i t y a s s o c i a t e d w i t h each. -90-CONCLUSION I. What Can Pulse-chase S t u d i e s Show? Pulse-chase s t u d i e s can be of v a l u a b l e a s s i s t a n c e i n such areas as; d e t e r m i n a t i o n of a compound's r a t e of s y n t h e s i s , h a l f - l i f e , o r turnover r a t e ; d e t e r m i n a t i o n of a metabolic pathway sequence; and e s p e c i a l l y i n a i d i n g the d e t e r m i n a t i o n of p r e c u r s o r - p r o d u c t r e l a t i o n s h i p s (155-160). T h i s l a s t area was of s p e c i a l i n t e r e s t to t h i s study as i t was the l a s t r e a c t i o n i n sphingomyelin's de novo b i o s y n t h e s i s t h a t was i n v e s t i g a t e d . T h e r e f o r e , any informa-t i o n t h a t could be determined about sphingomyelin's d i r e c t p r e c u r s o r s would be u s e f u l i n determining the donor of sphingomyelin's phosphocholine moiety. I I . I n t e r p r e t a t i o n of My R e s u l t s from the Pulse-Chase  S t u d i e s Presented Here P h o s p h a t i d y l c h o l i n e l o s t very l i t t l e (or none) of i t s l a b e l during the e n t i r e 48h of chase time ( F i g . 16-18). Nor was there any a p p r e c i a b l e i n c r e a s e in the r a d i o a c t i v i t y l e v e l s i n the compounds of the de novo b i o s y n t h e t i c path-way f o r p h o s p h a t i d y l c h o l i n e ( F i g . 1 ) . I t seems most l i k e l y t h a t the [Me- H ] c h o l i n e i n c o r p o r a t e d i n t o p h o s p h a t i d y l -c h o l i n e ( F i g . 16-18) was conserved or reused immediately (exchange r e a c t i o n ? ) r a t h e r than being r e c y c l e d through i t s de novo pathway ( F i g . 1 ) . The most c o n v i n c i n g evidence -91-f o r t h i s was the low l e v e l o f r a d i o a c t i v i t y i n P - c h o l i n e from about 20.0 h through to the end of the chase p e r i o d ( F i g . 16-19). T h i s produced two e f f e c t s on t h i s study. The f i r s t was nega t i v e , i n t h a t very l i t t l e of the r a d i o a c t i v e l a b e l was g e t t i n g p a s t the p h o s p h a t i d y l c h o l i n e p o o l t o be i n c o r p o r -ated elsewhere ( ie_. sphingomyelin) . The second e f f e c t , was a c t u a l l y the same but taken p o s i t i v e l y . Since very l i t t l e o r no [Me- H ] c h o l i n e l a b e l was being r e c y c l e d , 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 sphingomyelin (during the l a t t e r h a l f o f the chase period) was probably coming from the l a b e l e d p h o s p h a t i d y l c h o l i n e p o o l ( F i g . 16-18). That was the reason why o n l y the s p e c i f i c r a d i o a c -t i v i t i e s f o r p h o s p h a t i d y l c h o l i n e and sphingomyelin were of i n t e r e s t . The o n l y o t h e r p o s s i b l e c a n d i d a t e as a source o f 3 [Me- H ] c h o l i n e d u r i n g the l a s t 24 h o f chase time was gl y c e r o p h o s p h o c h o l i n e . However, i t was most l i k e l y t h a t t h i s compound was only a byproduct of one o r both o f the f o l l o w i n g r e a c t i o n s : (1) 2 LPC—>PC + G-P-choline (177-180) (2) LPC + H 20 ->G-P-choline + f a t t y a c i d (181,182) In f i g u r e 21 ( s p e c i f i c r a d i o a c t i v i t y versus chase time), the curves f o r p h o s p h a t i d y l c h o l i n e and sphingomyelin were analyzed a c c o r d i n g t o the p r e c u r s o r - p r o d u c t c r i t e r i a -92-(155) o u t l i n e d p r e v i o u s l y on page 64 . Although more i n -formation was needed f o r the chase p e r i o d from 8 h to 20h (see F i g . 21), i t d i d appear that p h o s p h a t i d y l c h o l i n e was the donor of sphingomyelin's P - c h o l i n e moiety. Phosphati-c y l c h o l i n e ' s s p e c i f i c r a d i o a c t i v i t y would normally be ex-pected to d e c l i n e c o n t i n u a l l y throughout the chase p e r i o d , but t h i s was not observed ( F i g . 21). One e x p l a n a t i o n would be the a d d i t i o n of more l a b e l e d c h o l i n e . But r e c y c l i n g of the l a b e l had a l r e a d y been r u l e d out, so a n a l y s i s of the chase medium was c a r r i e d out ( F i g . 20). I t was d e t e r -mined that r a d i o a c t i v i t y i n aqueous-soluble compounds pre-v i o u s l y 'exported' by the c e l l had l a t e r been taken up ( F i g . 20). T h i s was confirmed when a l l sources o f r a d i o a c t i v i t y ( c e l l - l i p i d , c e l l - a q u e o u s , and chase medium) were needed to account f o r the i n i t i a l amount incorporated a f t e r 30 min i n the pulse medium (j^e. 0 h chase time) . T h i s d i d not e x p l a i n why sphingomyelin's s p e c i f i c r a d i o a c t i v i t y d i d not continue to i n c r e a s e , unless i t had peaked between the 4 hand 22h time p o i n t s shown i n F i g u r e 21. Then, as both p h o s p h o l i p i d p o o l s i z e s i n c r e a s e d , the ad-d i t i o n a l r a d i o a c t i v i t y taken up from the medium would a f -f e c t them i n a s i m i l a r f a s h i o n . T h i s s t i l l posed the q u e s t i o n o f why both s p e c i f i c r a d i o a c t i v i t i e s ( F i g . 21) remained a t the same l e v e l as t h e i r pool s i z e s were o f a 10 f o l d d i f f e r e n c e , p h o s p h a t i d y l -c h o l i n e ' s being l a r g e r ( F i g . 22 and Table 2 ) . Now, suppose -93-sphingomyelin and p h o s p h a t i d y l c h o l i n e had a common ( l a b e l e d ) p r e c u r s o r . Sphingomyelin's s p e c i f i c r a d i o a c t i v i t y should i n c r e a s e more than p h o s p h a t i d y l c h o l i n e ' s when the c e l l had r e - i n c o r p o r a t e d the r a d i o a c t i v i t y from the chase medium. Sphingomyelin's s p e c i f i c r a d i o a c t i v i t y would a l s o have been expected to be (1 0 - f o l d ) more than p h o s p h a t i d y l c h o l i n e ' s at the beginning of the chase p e r i o d . N e i t h e r of these was observed ( F i g . 21). The only remaining p o s s i b i l i t y i s that of a p r e c u r s o r - p r o d u c t r e l a t i o n s h i p between phospha-t i d y l c h o l i n e and sphingomyelin. One that i s capable of a c h i e v i n g an e q u i l i b r i u m between t h e i r r e s p e c t i v e s p e c i f i c r a d i o a c t i v i t i e s . r 3 - i To t e s t t h i s h y p o t h e s i s , experiments using |_ Me- H J -c h o l i n e l a b e l e d p h o s p h a t i d y l c h o l i n e i n a pulse-chase format were c a r r i e d out. PLEP-mediated exchange between l a b e l e d p h o s p h a t i d y l -c h o l i n e v e s i c l e s and BHK c e l l s r e s u l t e d i n very l i t t l e 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 those c e l l s (Tables 3,4 and F i g . 23 ). The r a d i o a c t i v i t y t h a t was monitored appeared to be a s s o c i a t e d p r i m a r i l y with the BHK c e l l p h o s p h o l i p i d f r a c t i o n . Because of the low i n c o r p o r a t i o n , l e n g t h o f i n c u -b a t i o n , and appearance of some r a d i o a c t i v i t y i n aqueous c h o l i n e compounds, t h i s methodology was abandoned. The experiments f u s i n g p h o s p h o l i p i d v e s i c l e s with BHK c e l l s produced b e t t e r r e s u l t s (Tables 5-8). There was always some r a d i o a c t i v i t y a s s o c i a t e d with sphingomyelin on -94-th i n - l a y e r chromatograms. T h i s was observed even f o r 0 h of chase. The average washing procedure f o r the c e l l s took approx. 2 h so t h a t t h i s was the a c t u a l l e n g t h of time s i n c e the c e l l s (at 0 h of chase) had been removed from the 1/10 d i l u t e d v e s i c l e - PEG s o l u t i o n . Increased chase time was to p r o v i d e e q u i l i b r a t i o n (of i n c o rporated r a d i o a c t i v i t y ) between the two phosphol-i p i d s ; p h o s p h a t i d y l c h o l i n e and sphinomyelin. The appearance o f aqueous-soluble r a d i o a c t i v i t y remained an unresolved area. There are a t l e a s t two r e a c t i o n s (177-182) t h a t produce G-P-choline from LPC, which i n turn i s produced from p h o s p h a t i d y l c h o l i n e (181,182). I t i s l o g i c a l to assume that G-P-choline can be f u r t h e r degraded to P-choline and c h o l i n e a l s o . How important a r o l e G-P-c h o l i n e p l a y s i n BHK c e l l s i s not known, e s p e c i a l l y with r e s p e c t to the t r a n s f e r of P - c h o l i n e from p h o s p h a t i d y l c h o l i n e to sphingomyelin. Experiments using two l a b e l e d p r e c u r s o r s other than PC i n the f u s i o n of v e s i c l e s and BHK c e l l s produced i n t e r -e s t i n g r e s u l t s (Tables 7 and 8 ) . These r e s u l t s seemed to i n d i c a t e the r e v e r s i b l e nature of the ' t r a n s f e r ' r e a c t i o n and the r e a c t i o n where two LPC produce PC and G-P-choline (177-180). E i t h e r t h a t or they i m p l i c a t e G-P-choline (or LPC) as an intermediate f o r the t r a n s f e r of P - c h o l i n e be-tween p h o s p h a t i d y l c h o l i n e and sphingomyelin. -95-I I I . R e s u l t s From Previous S t u d i e s i n View of T h i s Study I n i t i a l i n v e s t i g a t i o n s on the i d e n t i t y and mechanism of the t r a n s f e r of P - c h o l i n e to sphingomyelin began i n the middle 1950's. The focus was on the r o l e CDP-choline might p l a y i n t h i s r e a c t i o n s i n c e i t had j u s t then been i m p l i -cated as the immediate source o f p h o s p h a t i d y l c h o l i n e ' s P - c h o l i n e moiety (1,2). When CDP-choline was coupled to ceramide during i n v i t r o r e a c t i o n s , s p h i n g o m y e l i n - l i k e products were pro-duced (3,4). The main o b j e c t i o n s to these s t u d i e s and ot h e r s that followed (9,13-17,21,26,27 and 38) were; the threo c o n f i g u r a t i o n o f ceramide was needed or p r e f e r r e d over the n a t u r a l o c c u r r i n g e r y t h r o c o n f i g u r a t i o n ; the len g t h of the N-acyl c h a i n on ceramide had to be very s h o r t ( a c e t y l ) or was h i g h l y p r e f e r r e d (3,4,17,21,38) over longer chain l e n g t h s , u s u a l l y 16 carbons being the l o n g e s t ; many of the enzyme sources were from m i t o c h o n d r i a l p r e p a r a t i o n s (13, 14,16) even though the major s i t e of p h o s p h o l i p i d s y n t h e s i s i n mammals i s the endophasmic r e t i c u l u m ; and f i n a l l y , when the e r y t h r o c o n f i g u r a t i o n f o r ceramide does work, s p e c i a l c o n d i t i o n s were necessary (3,4), such as s u l f h y d r y l reagents (17,21). Arguments i n defense of these o b j e c t i o n s i n c l u d e ; s t e r e o - i s o m e r i z a t i o n of ceramide j u s t before o r a f t e r the enzymatic r e a c t i o n with CDP-choline (4); there i s no st e r e o c h e m i c a l s p e c i f i c i t y f o r the threo or e r y t h r o c o n f i --96-g u r a t i o n of ceramide (7); and long c h a i n ceramides are i n s o l u b l e so they may be i n a c c e s s i b l e to the enzyme i n  v i t r o (38). L a t e r p u b l i c a t i o n d e a l t p r i m a r i l y with in v i v o and i n v i t r o s t u d i e s i n v o l v i n g p h o s p h a t i d y l c h o l i n e as the d i r e c t p r e c u r s o r (23,24,30,31,34,35,37) or the i n d i r e c t p r e c u r s o r (11,12,20,22) f o r sphingomyelin's P- c h o l i n e moiety. Some of these p u b l i c a t i o n s have suggested t h a t i n v i t r o enzymatic c o n v e r s i o n o f ceramide to sphinomyelin occurs by f i r s t r e a c t i n g with endogenous d i g l y c e r i d e to form p h o s p h a t i d y l c h o l i n e (35). I t has a l s o been argued t h a t the enzyme r e s p o n s i b l e f o r C D P - c h o l i n e 1 s r e a c t i o n with ceramide i s the same enzyme r e s p o n s i b l e f o r p h o s p h a t i d y l c h o -l i n e ' s formation from CDP-choline and d i g l y c e r i d e (35, f o r and 38, a g a i n s t ) . The r e a c t i o n i n v o l v i n g ceramide and p h o s p h a t i d y l c h o -l i n e has a l s o been a t t r i b u t e d to an a l r e a d y known enzyme; ph o s p h o l i p a s e C (37). P r e v i o u s s t u d i e s t h a t best support the r e s u l t s i n t h i s i n v e s t i g a t i o n were p u b l i s h e d by D i r i n g e r (23,31) and by Ullman and Radin (35). The former lab has p u b l i s h e d i n v i v o and ir\ v i t r o s t u d i e s while the l a t t e r group used i n v i t r o experiments o n l y . But D i r i n g e r ' s i n i t i a l p u l s e -chase s t u d i e s (23) are not enough evidence by themselves and the in_ v i t r o evidence by both groups i s not so e a s i l y e x t r a p o l a t e d to l i v i n g systems. T h i s i s why more innova--97-t i v e ix\ v i v o experiments are e s p e c i a l l y h e l p f u l , such as those presented i n t h i s i n v e s t i g a t i o n . A l s o the r e c e n t work by S t o f f e l ' s lab (40,41) s t i l l supports CDP-choline as the immediate donor. An o v e r a l l schematic diagram of the p o s s i b l e r e -a c t i o n s l e a d i n g to sphingomyelin that have been d i s c u s s e d i s shown i n F i g u r e 24. IV. What Could Be Done to Confirm the C o n c l u s i o n s  Derived From My I n v e s t i g a t i o n Beginning with the [Me- H ] c h o l i n e pulse-chase s t u d i e s , many f a c t o r s should be i n v e s t i g a t e d f u r t h e r . Among these are the e f f l u x and r e - 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 during extended chase p e r i o d s , c o n f l u e n c y e f f e c t s on c e l l growth, development of a p r e p a r a t i v e method f o r i s o l a t i n g r a d i o a c t i v e w a t e r - s o l u b l e m a t e r i a l s from the growth (chase) medium, and taking enough time p o i n t s throughout a long chase p e r i o d to more a c c u r a t e l y p l o t a compounds s p e c i f i c r a d i o a c t i v i t y curve. The BHK c e l l / p h o s p h o l i p i d - v e s i c l e f u s i o n experiments worked f a i r l y w e l l . However, the p u r i t y of the l a b e l e d BHK p h o s p h a t i d y l c h o l i n e and sphingomyelin p r e p a r a t i o n s i s more c r i t i c a l here, so more than a s i n g l e t h i n - l a y e r chromato-graph system i s necessary. Pulse-chase s t u d i e s using the g e n e t i c mutant Esko and Raetz (42) had screened and i s o l a t e d (see I n t r o d u c t i o n -98-F i g u r e 24 Proposed Reactions Leading to the Formation of Sphingomyelin cho. .ine \ \ Plasma Membrane P- c h o l i n e sphingosine -^j CDP-choline sphirigosylphosphoryl-c h o l i n e p-CDP-choline < p h o s p h a t i d y l c h o l i n e ^LPC (+ f a t t y a c i d ), <4^r c e r a mi^ e + LPC G-P-choline r.^-ceramide , ^ . SPHINGOMYELIN <• A A A f r e e ethanolamine >> >^ c h o l i n e acyl-CoA -99-s e c t i o n I I I ) would be very i n f o r m a t i v e . 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