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Lipid-protein interactions: A., Semliki forest virus : B., CTP:phosphocholine cytidylyltransferase from… Farren, S. Blake 1980

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LIPID-PROTEIN INTERACTIONS SEMLIKI FOREST VIRUS CTP:PHOSPHOCHOLIN£ CYTIDY LY LT RAN S FE RASE FROM RAT LIVER by S. BLAKE FARREN B.Sc., Hon., U n i v e r s i t y of New Brunswick, 197^ A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department o f B iochemis t ry ) We accept t h i s t he s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1980 Q S. Blake Far ren, 1980 In present ing t h i s t he 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 at the U n i v e r s i t y of B r i t i s h Columbia, I agree tha 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 re ference and study. I f u r t h e r agree tha t permiss ion f o r ex tens i ve copying of t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t he s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n permi s s ion . Department The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P lace Vancouver, Canada V6T 1W5 D E - 6 B P 75-51 I E ABSTRACT A. Seml ik i Forest v i r u s : L i p i d Headgroup-Protein I n t e r a c t i on s Seml ik i Forest v i r u s i s an a lphav i ru s enveloped by a l i p i d b i l a y e r that conta ins approx imately 200 copies each of three g l y c o p r o t e i n s : E-j, mo lecu la r weight 49,000, E^, molecu lar weight of 52,000, and E^, mo lecu la r weight o f 10,000. Proton magnetic resonance measurements were done on i n t a c t Seml ik i Forest v i r u s and v i r u s that had been d iges ted w i th the rmo ly s i n . The magnetic resonance l i n e de r i ved from a p o r t i o n o f the N-methyl groups o f the cho l i ne con ta i n i n g phospho l ip id s narrowed cons ide rab ly a f t e r p r o t e o l y t i c d i g e s t i o n . This reduct ion i n the N-methyl resonance i s not i n c o n s i s t e n t w i t h increased motion o f the headgroups of the phospho l ip ids as a r e s u l t o f thermolys in removal o f the v i r a l g l y cop r o t e i n sp i ke s . To f u the r i n v e s t i g a t e t h i s phenomenon, experiments were planned us ing deuterium NMR. T r i - t r i d e u t e r o m e t h y l c h o l i n e was chemica l l y s ynthes i zed and added to the medium o f BHK-21 c e l l s w i th the hope o f l a b e l l i n g a la rge m a j o r i t y of the cho l i ne con ta i n i n g 1 ip ids.v However, no i n c o r p o r a t i o n o f t h i s l a b e l l e d spec ies cou ld be observed. Two o the r deu te r -ated c h o l i n e s , mono- t r ideuteromethy lcho l ine and d i - t r i d e u t e r o m e t h y l c h l o l i n e , were synthes i zed and e a s i l y i nco rpo ra ted i n t o the BHK-21 c e l l c ho l i ne con ta in i ng l i p i d s . Due to the low y i e l d s o f Seml ik i Fores t v i r u s , deuterium NMR could not be performed, however, the lack of i n c o r p o r a t i o n of t r i - t r i -deuteromethyl chol i ne was of i n t e r e s t . From l a b e l l i n g s tud ie s i t was concluded tha t t h i s deuterated cho l i ne was not t ranspor ted across the c e l l plasma membrane. i i B. CTP:phosphocholine C y t i d y l y l t r a n s f e r a s e : L i p i d - P r o t e i n I n t e r a c t i on s Ea r l y s tud ie s on the r a t l i v e r CTP:phosphocholine c y t i d y l y l t r a n s -ferase (E.C. 2.7.7.15) (CT) reported tha t the enzyme i s o l a t e d from the c y t o s o l i c f r a c t i o n o f " 0 . 9% NaCl s o l u t i o n homqgena-tes inc reased i n a c t i v t y 4- to 5 - f o l d upon aging severa l days a t 0°C or i n cuba t i on a t 37°C f o r 3 hours. I t was subsequently shown tha t the a c t i v a t i n g agent was lysophospha-t i d y l e thano l am ine (LPE). We have demonstrated tha t the p u r i f i e d r a t l i v e r CT i s dependent upon l i p i d f o r a c i t v i t y and i s a c t i v a t e d by o l eoy l - LPE and i n h i b i t e d by o l e o y l - l y s o p h o s p h a t i d y l c h o l i n e (LPC). The p l o t of CT a c t i v i t y a t var ious concent ra t ions o f LPE y i e l d s a h ype rbo l i c curve w i th a K o f 0.3 mM. The a c t i v a t i o n of CT by LPE r e s u l t s i n a decrease o f the K a tn f o r CTP from2 mM at 0.1 mM LPE, to 0.5 mM at 0.4 mM LPE. LPE had no e f f e c t on the Km f o r phosphochol ine. Hence, the a c t i v a t i o n o f CT by LPE i s due to an i n f l u e n c e on the Km f o r CTP. When CT i s assayed i n the presence of LPE, LPC i n h i b i t s the a c t i v i t y w i th a concent ra t i on f o r hal f -maximal i n h i b i t i o n of 0.16 mM. I n h i b i t i o n by LPC was not compet i t i ve w i th phosphochol ine. i i i TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES i x LIST OF FIGURES x ACKNOWLEDGMENTS xv i INTRODUCTION 1 A. The B i o l o g i c a l Membrane 1 B. Probes o f Membrane S t r uc tu re 3 a) S c a t t e r i n g Methods 5 ( i ) X-ray d i f f r a c t i o n 5 ( i i ) Neutron d i f f r a c t i o n 6 ( i i i ) Raman s c a t t e r i n g . 7 ( i v ) F luorescence probes 8 (v) Magnetic resonance techniques 9 (1) Nuclear Magnetic Resonance 9 (1.1) Headgroup s tud ie s 1 0 (1.2) Order parameter s tud ie s 10 (1.3) 1 3 C NMR s tud ie s 1 2 (1.4) Recons t i t u ted systems ^ 3 (1.5) I n tac t Systems ^ (2) E l e c t r on sp in resonance 20 i v Page ( v i ) Ca lo r imet ry 22 ( v i i ) Freeze f r a c t u r e e l e c t r o n microscopy 23 C Stud ies o f V i r a l Membranes . 23 D. L ip id-dependent p ro te in s 26 (a) L - l a c t a t e dehydrogenase 28 (b) Pyruvate ox idase . . . 29 (c) Mal ate ox idase 31 (d) D-3-hydroxybutyrate dehydrogenase 31 (e) CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e 33 E. Conc lus ions 35 F. The S t r uc tu re o f Seml i k i Forest V i ru s 37 G. The- Thes is I n ve s t i g a t i on s 40 (a) Seml ik i f o r e s t v i r u s : l i p i d headgroup-prote in i n t e r a c t i o n s . 40 (b) CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e : 1 i p i d -p r o t e i n i n t e r a c t i o n s 42 MATERIALS AND METHODS 44 A. Chemicals and Isotopes 44 B. General Methods. 46 ( i ) P r o te i n dete rminat ion 46 ( i i ) Thin l a y e r chromatography 47 ( i i i ) Gas chromatography 47 ( i v ) Phospho l ip id phosphorous ana l y s i s 48 (v) L i q u i d s c i n t i l l a t i o n count ing 49 ( v i ) SDS-polyacrylamide gel e l e c t r o p h o r e s i s 49 v Page ( v i i ) Non-denaturing po lyacry lamide gel e l e c t r o p h o r e s i s 51 ( v i i i ) P repa ra t i on o f [ H]-phosphochol ine 52 C. C e l l Cu l tu re • 53 D. Propagat ion o f Semi ik i Fores t V i ru s 53 E. P repa ra t i on o f Large Amounts o f Semi ik i Forest V i ru s 54 F. Thermolys in D i ge s t i on o f Semi i k i Fores t V i ru s 55 G. P repa ra t i on o f Semi ik i Forest V i ru s L i p i d Ex t rac t s 55 ( i ) P repa ra t i on o f a mock v i r u s l i p i d sample 55 ( i i ) P repara t ion o f au then t i c v i r u s l i p i d l iposomes. . . 56 ( i i i ) P repa ra t i on o f l i p i d v e s i c l e s 56 (a) Mock v i r u s l i p i d sample. . 56 (b) Egg PC l i p i d sample 5 6 H. P repa ra t i on o f Deuterated Chol ines 57 ( i ) Synthes i s o f t r i r t r i d e u t e r o m e t h y l c h o l i n e 57 ( i i ) Synthes i s o f d i - t r i d e u t e r o m e t h y l c ho l i n e 57 ( - i i i ) Synthes i s o f mono- t r ideuteromethy lcho l ine 58 ( i v ) I nco rpo ra t i on o f 1abel1ed cho l i n e i n t o BHK-21 b c e l l s 58 3 (v) P repa ra t i on o f t r i - t r i d e u t e r o m e t h y l - [ l , 2 - H ] -c h o l i n e 59 ( v i ) Cho l ine t r an spo r t 60 I. Enzyme Assays. 60 ( i ) Choi ine k inase 60 ( i i ) CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e 61 J . P u r i f i c a t i o n o f the CTP:phosphocholine c y t i d y l y l -t r an s f e r a se 62 v i Page K. Assay o f the C y t i d y l y l t r a n s f e r a s e from Non-denaturing Po lyacry lamide gels 64 RESULTS 66 A. L i p i d - p r o t e i n I n t e r a c t i o n s i n the Po l a r Headgroup Region o f Seml ik i Forest V i ru s 66 (a) P repa ra t i on o f Seml i k i Fores t v i r u s 67 (b) P r o t e o l y t i c d i g e s t i o n o f Seml ik i Fores t v i r u s 67 (c) High r e s o l u t i o n proton NMR o f i n t a c t and thermo ly s in t r e a t e d Seml i k i Fo res t v i r u s 71 B. I n c o r p o r a t i o n o f Deuterated Species of Cho l ine i n t o BHK-21 C e l l L i p i d s 84 (a) C h a r a c t e r i z a t i o n o f the deuterated cho l i ne s 84 (b) I ncorpora t ion of the deuterated cho l i ne s i n t o BHK-21 c e l l pho spha t i dy l cho l i ne 88 C. Studies on the CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e . . . . 96 (a) P repa ra t i on o f the c y t i d y l y l t r an s f e r a se 96 (b) P u r i t y o f the enzyme 98 (c) E f f e c t s o f l y s o l i p i d s on c y t i d y l y l t r a n s f e r a s e a c t i v i t y 100 (d) E f f e c t o f o l eoy l - LPE on the a c t i v i t y o f the p u r i f i e d enzyme 100 (e) E f f e c t o f o l eoy l - LPC on the a c t i v i t y o f the p u r i f i e d enzyme 103 ( f ) E f f e c t o f o l eoy l - LPE on the k i n e t i c parameters o f the c y t i d y l y l t r an s f e r a se 103 (g) E f f e c t o f o leoy l - LPC on the k i n e t i c parameters o f the c y t i d y l y l t r an s f e r a se 107 (h) Aggregat ion o f the CTP:phosphocholine c y t i d y l y l -t r an s f e r a se I l l -v i i Page DISCUSSION 1-16-A. L i p i d - p r o t e i n i n t e r a c t i o n s w i t h i n the po la r head-group reg ion o f Semi i k i Forest v i r u s 116.. B. I ncorpora t ion of deuterated cho l i ne s i n t o BHK-21 c e l l p h o s p h a t i d y l c h o l i n e 121 ' C. CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e : l i p i d -p r o t e i n i n t e r a c t i o n s 123-(a) Aggregat ion o f the c y t i d y l y l t r an s f e r a se 125; (b) Contro l o f the c y t i d y l y l t r a n s f e r a s e by LPE and LPC. . . . 127, D. Suggest ions f o r Future Work 129 (a) Semi i k i Forest v i r u s 129 (b) CTP:phosphochol ine c y t i d y l y l t r a n s f e r a s e 131. BIBLIOGRAPHY 132 v i i i LIST OF TABLES Table Page 1 Carbohydrate Content of Semi ik i Fores t V i ru s Membrane P ro te in s 39 2 L i p i d Class Composit ion o f Semi ik i Fores t V i r u s 39 3 Number o f D i f f e r e n t Molecules i n Semi i k i Fores t V i ru s 40 4 L i p i d Class Composit ion o f Semi ik i Fores t V i ru s 56 5 Integrated Areas o f the Peaks from Proton NMR Spectra o f Cho l i ne , Mono-t r ide.uteromethy lcho l ine, D i - t r i d e u t e r o -methy l cho l i ne , and T r i - t r i d e u t e r o m e t h y l cho l i ne 86 6 Incorporat ion o f Deuterated Chol ines i n t o BHK-21 C e l l Pho spha t i dy l cho l i ne 90-7 CK A c t i v i t y us ing Cho l i ne , Mono - t r i deu te romethy l cho l i ne , and T r i - t r i d e u t e r o m e t h y l chol ine as Subst rate 91 8 Time Course of I nco rpo ra t i on o f R a d i o a c t i v i t y i n t o the Organic and Aqueous So lub le F rac t i on s o f BHK-21 C e l l s 92 i x LIST OF FIGURES Figure Page 1 I l l u s t r a t i o n o f var ious phases l i p i d s may assume along w i th t h e i r corresponding 31 p NMR spec t r a 17 2 The s t r u c t u r e o f Semi i k i Fores t v i r u s 37 3 CT a c t i v i t y vs. time and p r o t e i n concen t ra t i on 63 4 Schematic drawing of the non-denatur ing po lyacry lamide gel o f the CT. 65 5 P r o f i l e o f a 15-50% l i n e a r sucrose g rad ien t c on ta i n i n g Semi ik i Fores t v i r u s 68 6 SDS-polyacrylamide, gel e l e c t r o p h o r e s i s o f p u r i f i e d Semi ik i Forest v i r u s 69 7 Negative s t a i n i n g t r an smi s s i on e l e c t r o n micrograph o f p u r i f i e d Semi i k i Forest v i r u s 70 8 SDS-polyacrylamide s l ab gel e l e c t r o p h o r e s i s o f t r y p s i n -t r e a t ed Semi ik i Forest v i r u s 72 9 Negative s t a i n i n g t ransmi s s i on e l e c t r o n microscopy o f t r y p s i n - t r e a t e d Semi i k i Forest v i r u s 73 10 SDS-polyacrylamide s l ab gel e l e c t r o p h o r e s i s o f t he rmo l y s i n -t r ea ted Semi ik i Forest v i r u s 74 11 High r e s o l u t i o n proton NMR spectrum of i n t a c t Semi ik i Forest v i r u s 75 12 High r e s o l u t i o n proton NMR spectrum of t he rmo l y s i n -t r e a t ed Semi ik i Forest v i r u s 78 13 High r e s o l u t i o n proton NMR spectrum of i n t a c t Semi i k i Forest v i r u s 79 14 High r e s o l u t i o n proton NMR spectrum of t he rmo l y s i n -t r ea ted Semi ik i Forest v i r u s 80 x Figure Page 15 High r e s o l u t i o n proton NMR spectrum o f Seml i k i Forest v i r u s l i p i d l iposomes. -81 16 High r e s o l u t i o n proton NMR spec t r a o f mock Seml i k i Forest v i r u s l i p i d v e s i c l e s and egg pho spha t i dy l cho l i ne v e s i c l e s 83 17 S t r uc tu re o f deuterated cho l i ne s 85 18 Proton NMR spectrum of c h o l i n e , mono-tr ideuteromethyl c h o l i n e , d i - t r i d e u t e r o m e t h y l c h o l i n e , and t r i - t r i d e u t e r o -methyl c h o l i n e 87 19 Decrease o f r a d i o a c t i v i t y from the c e l l medium 93 20 Uptake o f r a d i o a c t i v i t y i n t o BHK-21 c e l l pho spha t i dy l cho l i ne . 94 21 Chromatography o f CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e from r a t l i v e r c y to so l on Sepharose 6B 97 22 Chromatography o f SDS -d i s soc ia ted CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e from r a t l i v e r cy to so l on Sepharose 6B. . 97 23 Non-denaturing po lyacry lamide gel e l e c t r o p h o r e s i s o f p u r i f i e d CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e 99 24 A c t i v a t i o n o f CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e i n r a t l i v e r cy to so l a t 4°C 101 25 A c t i v a t i o n o f CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e i n r a t l i v e r cy to so l a t 37°C 101 26 TLC and GC of o l eoy l - LPE and o leoy l - LPC 102 27 Lysophosphat idylethanolamine a c t i v a t i o n o f p u r i f i e d CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e from r a t l i v e r . . . . 104 28 Double i nve r se p l o t o f i n i t i a l v e o l o c i t y o f CDP-cho l i n e synthes i s at s a t u r a t i n g concent ra t i ons o f phosphochol ine and CTP and i n c r e a s i n g LPE- concen t ra t i on . . . 105 29 Ly sophosphat idy l cho l i ne i n h i b i t i o n o f p u r i f i e d CTP: phosphocholine c y t i d y l y l t r a n s f e r a s e from r a t l i v e r . . . . . . 106 x i Figure 30 Double r e c i p r o c a l p l o t of i n i t i a l v e l o c i t y o f CDP-cho l i n e synthes i s at s a t u r a t i n g phosphocholine concen-t r a t i o n , va ry ing CTP concen t r a t i on s , and i n c r e a s i n g amount of LPE 108 31 Double r e c i p r o c a l p l o t of i n i t i a l v e l o c i t y of CDP-cho l i ne synthes i s a t s a t u r a t i n g CTP c oncen t r a t i o n , va ry ing phosphocholine concen t r a t i on s , and f i x e d amount o f LPE 109 32 Double r e c i p r o c a l p l o t of i n i t i a l v e l o c i t y of CDP-cho l i ne synthes i s at s a t u r a t i n g phosphocholine con-c e n t r a t i o n , varyiing CTP concen t r a t i on s , and i n c r ea s i n g amounts of LPE 110 33 E f f e c t of phosphat idy l g l y c e r o l on the aggregat ion of CTP: phosphocholine c y t i d y l y l t r a n s f e r a s e i n r a t l i v e r cy to so l . . . 1 1 3 34 E f f e c t of a p h o s p h a t i d y l g l y c e r o l - s p e c i f i c phosphol ipase A on the aggregat ion o f the CTP:phosphocholine c y t i d y l y l -t r an s f e r a se 114 x i i LIST OF ABBREVIATIONS A Angstrom u n i t - 10" cm ABS absorbance ACS aqueous count ing s c i n t i l l a n t ATP adenosine t r iphosphate BHK-21 Baby Hamster Kidney-21 c e l l s Bu f f e r A 20 m M T r i s - H C l , 100 mM NaCl, (pH 7.0) Ci Cu r ie CK cho l i n e k inase CDP-chol ine c y t i d i n e d iphosphochol ine CMP c y t i d i n e monophosphate CHO carbohydrate cpm counts per minute CPT c h o l i n e phosphotransferase CT CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e CTP c y t i d i n e t r i phosphate 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 c e t i c a c i d FAD f l a v i n adenine d i n u c l e o t i d e FCS f e t a l c a l f serum F i g . f i g u r e g gram x i i i g rav i t y hour he r t z a s s o c i a t i o n constant o f an enzyme-act ivator complex Michae l i s -Menten constant 1 i t e r l y sophosphat idy l chol i ne lysophosphat i dy lethano l ami ne meter mol ar minute normal nanomole nuc lear magnetic resonance n i cot inamide adenine d i n u c l e o t i d e phosphate buf fe red s a l i n e , pH 7.4 (0.137M NaCl, 2.68 mM KC1, 1.47 mM KH 2 P0 4 , and 4.29 mM Na 2 HP0 4 ) pho spha t i dy l cho l i ne phosphat idy lethanolamine plaque forming u n i t phosphat idy l g l y c e r o l phosphat idy l i n o s i t o l 1,4 B i s ( 2 - ( 5 - pheny l o xazo l y l ) ) Benzene 2 ,5 -d ipheny loxazo le phosphat idy l s e r i ne i no r gan i c phosphate parts per m i l l i o n x i v r e f . R f RNA SDS SFV t TEMED TLC T r i s TNE T l TPP UV V V 0 V max re ference r a t i o o f d i s t ance moved by a s o l u t e to t ha t moved by the s o l ven t f r o n t r i b o n u c l e i c a c i d sodium dodecy l su lphate Semi i k i Forest v i r u s time N ,N ,N ' ,N ' - t e t ramethy l e thy lened iamine t h i n l a y e r chromatography t r i s (hydroxymethyl) aminoethane 20 mM-. T r i s - H C l , 100 mM- NaCl , 2 mM EDTA, pH 7.4, b u f f e r s p i n - l a t t i c e r e l a x a t i o n time thiamine pyrophosphate u l t r a v i o l e t volume vo id volume maximal v e l o c i t y (o f an enzyme r eac t i on ) NOTES -2 -3 Standard p r e f i x e s a re : c ( cent . ) - 10 ; m ( m i l l i ) - 10 ; \i (micro) 1 0 " 6 ; n (nano) - 1 0 ' 9 xv ACKNOWLEDGEMENTS I wish to thank Dr. Dennis E. Vance f o r h i s s upe r v i s i o n and constant encouragement throughout t h i s work. I a l so wish to thank Dr. P a t r i c k C. Choy, Dr. A lex Mackay, and Mr. Harry B. Paddon f o r t h e i r he lp i n var ious aspects o f the re sea rch . xv i DEDICATION To My Parents The Mountains of British Columbia x v i i 1. INTRODUCTION A. The B i o l o g i c a l Membrane During the l a s t few years membrane b i o l ogy has become one o f the most e x c i t i n g and r a p i d l y advancing f i e l d s o f b i ochemi s t r y . Many books ( 1 - 7 ) , reviews (8 -11) , and papers have been pub l i shed i n . th i s area dea l i n g w i th such t op i c s as membrane p ro te in s (12-17), membrane l i p i d s (18-27), b a c t e r i a l membranes (28,29), membrane t r an spo r t (30 ) , membrane and c e l l m o t i l i t y ( 31 ) , e t c . For some time now, the membranesof euka ryo t i c c e l l s have been known to be f u n c t i o n a l l y s i g n i f i c a n t . The membranes o f the var ious c e l l u l a r o rgane l l e s act as b a r r i e r s , s epa ra t i ng the v i t a l f unc t ions o f the c e l l . Compartmentation o f these i n d i v i d u a l c e l l u l a r f unc t i on s i s necessary f o r the growth of the i n d i v i d u a l c e l l and the organism as a whole. However these b a r r i e r s are not t o t a l l y impermeable. This would defeat the purpose of the complex compartmentation requ i red f o r the c e l l to f u n c t i o n . A number of p r o t e i n s , i n p a r t i c u l a r recepto r and t r an spo r t p ro te in s have evolved over the ages to a i d the var ious o rgane l l e s and indeed whole c e l l s to communicate and nour i sh each o the r . As we l l as having p ro te in s i nvo l ved i n t r an spo r t and communication there a re other v i t a l operat ions r equ i red 2. f o r c e l l f u n c t i o n , f o r example, the e l e c t r o n - t r a n s p o r t cha in o f the m i t o -chondr ia and c h l o r o p l a s t s . The p ro te in s of the endoplasmic r e t i c u l um are re spons i b l e f o r a mu l t i t ude o f a c t i v i t i e s i n c l u d i n g the s yn thes i s o f pho spho l i p i d s , as s t r u c t u r a l components o f the c e l l as we l l as s t e r i o d b i o s y n t h e s i s , p r o t e i n s y n t h e s i s , e t c . Membranes p lay a v i t a l r o l e i n the ex i s tence of the l i v i n g c e l l , be i t from a complex m u l t i c e l l u l a r organism to the s imple y e t complex u n i c e l l u l a r organism. For the l a s t 50 years the evidence has suggested tha t the membrane con s i s t s o f l i p i d which i s o r i e n t a t e d i n a b imo lecu l a r l e a f s t r u c t u r e w i th the hydrophobic t a i l s of the phospho l ip ids forming a nonpolar reg ion and the h y d r o p h i l i c head groups open to the aqueous environment (32). In the e a r l y 1930's t h i s idea was updated to i n c l ude a p r o t e i n l a y e r on the su r face o f the b i l a y e r . S ince tha t time the ba s i c model o f the b i o l o g i c a l membrane has not changed, but the d e t a i l s o f the i n t e r a c t i o n s between the l i p i d s and p ro te in s have. The s t r u c t u r e o f the b i o l o g i c a l membrane i s i n a constant s t a t e o f f l u x . Under p h y s i o l o g i c a l cond i t i on s most membrane l i p i d s e x h i b i t r a the r f r ee l a t e r a l d i f f u s i o n (33-42) w i th t h e i r hydro-carbon chains i n a d i so rdered s t a t e (14,36,43). A number o f membrane pro -t e i n s a l so d i f f u s e r a the r f r e e l y i n the l a t e r a l plane o f the membrane (16,17,44,45), a f i n d i n g which l ed to the S inge r -N i cho l son " f l u i d mosaic model" of the membrane i n 1972 (8-11) . A con s ide rab le amount of suppor t ing evidence has come from work on the r e d i s t r i b u t i o n o f membrane receptors on lymphocyte su r faces (45-52), and o ther c e l l s (53-55) , the i n f l u e n c e of l i p i d f l u i d i t y and phase changes on membrane t r an spo r t (56-61) , the e f f e c t of l i p i d on enzyme a c t i v i t i e s (62-64) , and work on the i n s e r t i o n o f i n t e g r a l p ro te in s i n t o membranes (65,66). 3. Although membrane f l u i d i t y and l a t e r a l d i f f u s i o n o f membrane components appear to be the general r u l e , there i s c l e a r evidence that t h i s motion i s g r e a t l y r e s t r i c t e d i n c e r t a i n membranes or a t l e a s t i n c e r t a i n reg ions of membranes under p a r t i c u l a r c o n d i t i o n s . In the membranes of synapses (67) and gap j unc t i on s (68,69) there are reg ions of s p e c i a l i z e d and ordered s t r u c t u r e . In the p r o ka r yo t i c c e l l membrane o f halebaeterium, s i m i l a r l a r ge regions o f ordered s t r u c t u r e , known as p laques, are observed in an otherwise random membrane mat r i x (70). Recent evidence suggests that these regions c o n s i s t o f a s i n g l e p r o t e i n o r a few p r o t e i n spec ies which form a r egu l a r two-dimensional a r ray by v i r t u e o f s p e c i f i c p r o t e i n -p ro te i n i n t e r a c t i o n s . A much c l e a r a r p i c t u r e o f the b i o l o g i c a l membrane i s beg inn ing to emerge, a very t r i d i m e n s i o n a l s t r u c t u r e being i n t r i c a t e l y i n vo l ved w i t h the ex i s tence o f the c e l l . Work i n t h i s area i s by no means complete. Many areas s t i l l remain a mystery, i n c l u d i n g the exact i n t e r a c t i o n s o f l i p i d s and p ro te in s and indeed i n t e r a c t i o n s w i t h i n the var ious l i p i d c la s ses themselves. Questions such as , why are there so many d i f f e r e n t phospho l i p id headgroups, some being neut ra l w h i l e others are ba s i c or a c i d i c ? ? what i s - t h e r o l e - o f c h o l e s t e r o l ' i n the b i o l o g i c a l membrane?-remain unanswered, as do many o the r s . - . B. Probes o f Membrane S t r uc tu re In the previous s e c t i o n a membrane model i s de sc r ibed which i s compat ib le w i th cu r ren t knowledge. To t h i s po in t however, the p i c t u r e has been more q u a l i t a t i v e than q u a n t i t a t i v e . This model has not been assembled 4. from a few s imple experiments o r on ly a few techn iques . Rather t h i s p i c t u r e has been s yn thes i zed from a myriad of experiments and from a great number o f techn iques , w i th more s o p h i s t i c a t e d methods being developed. For many of the techniques employed today the i n t a c t b i o l o g i c a l membrane s t i l l appears to be too complex to e x t r a c t i n f o r m a t i o n , hence model membranes have been employed which range from s imple l i p i d b i l a y e r s , c o n s i s t i n g o f pure i n d i v i d u a l l i p i d s or complex mixtures i s o l a t e d from b i o l o g i c a l membranes, to the r e c o n s t i t u t e d systems i n v o l v i n g the l i p i d b i l a y e r w i th p ro te in s being r e i n s e r t e d i n t o the b i l a y e r . The development o f such model membrane systems, which are s t r u c t u r a l l y r e l a t e d to the i n t a c t membrane, has g r e a t l y a ided the search f o r i n fo rmat i on on the i n t e r a c t i o n between l i p i d s themselves as we l l as l i p i d s and p r o t e i n s . What types o f phy s i ca l techniques have been usefu l i n the e l u c i -da t ion o f membrane o r gan i za t i on ? The most succes s fu l methods i n c l ude the s c a t t e r i n g techniques ( i . e . X - ray , neut ron, and raman s c a t t e r i n g ) , f l uo re scence measurements, magnetic resonance techn iques , c a l o r i m e t r y , and f r e e z e - f r a c t u r e e l e c t r o n microscopy. As was mentioned be fo re , i t has not been a s i n g l e technique o r experiment t ha t has y i e l d e d a working model o f the b i o l o g i c a l membrane but r a the r a number o f experiments and techn iques , each having i t s advantages and l i m i t a t i o n s . Each prov ides an incomplete p i c t u r e , a par t o f the j i g - s aw puz z l e , however when assembled together they prov ide a d e t a i l e d view o f the b i o l o g i c a l membrane. Before we deal w i th the var ious techniques i n v o l v e d , i t i s of i n t e r e s t to desc r ibe the d i f f e r e n t types o f model systems tha t have been used. B a s i c a l l y three types of model membrane p repa ra t i on s , w i th the l i p i d i n vo l ved i n a b i l a y e r c o n f i g u r a t i o n , have been employed. 5. (1) m u l t i l a m e l l a r d i s p e r s i o n s , i n w h i c h t h e l i p i d i s r e s u s p e n d e d i n an aqueous medium by s h a k i n g r a p i d l y . The s t r u c t u r e s formed a r e r e f e r r e d t o as o n i o n s s k i n s and c o n s i s t o f c l o s e d c o n c e n t r i c l i p i d b i l a y e r s s e p a r a t e d by an aqueous r e g i o n ( 7 1 ) , (2) v e s i c l e s , w h i c h a r e p r e p a r e d by p r o l o n g e d s o n i c a -t i o n o f t h e m u l t i l a m e l l a r d i s p e r s i o n s . These c o n s i s t o f s p h e r i c a l s i n g l e w a l l e d l i p i d b i l a y e r s ( 7 2 ) , and (3) o r i e n t a t e d m u l t i l a y e r s , i n w h i c h t h e b i l a y e r s a r e f l a t and p a r a l l e l t o one a n o t h e r r a t h e r t h a n c u r v e d . These a r e p r e p a r e d i n a number o f ways as d e s c r i b e d i n t h e f o l l o w i n g p a p e r s (73,74-). These p repara t ions can be made w i th a v a r i e t y o f l i p i d m ix tu re s . As w e l l as p repar ing these systems w i th pure l i p i d s o r l i p i d mixtures i t i s a l so po s s i b l e to i nco rpo ra te o the r b i o l o g i c a l l y important molecu les , i n c l u d i n g . p r o t e i n s o r o the r l i p i d s which have been l a b e l l e d i n some way (75) . a) S c a t t e r i n g Methods ( i ) X-ray D i f f r a c t i o n X-ray d i f f r a c t i o n has been a technique long i n vo l ved i n s t r u c -t u r a l determinat ion of small molecules and l a t e l y i n the determinat ion o f the three dimensional s t r u c t u r e o f r a the r l a r ge molecules such as p r o t e i n s . The high r e s o l u t i o n s t r u c t u r a l determinat ions o f these molecules i s due to the a b i l i t y to form a uni form c r y s t a l l i n e ar ray o f the molecule under i n v e s t i g a t i o n . A membrane on the o ther hand i s i ncapab le o f being c r y -s t a l l i z e d or formed i n t o a s t r i c t l y uniform repeat ing u n i t . However s t r u c -t u r a l i n fo rmat ion can s t i l l be obta ined although i t w i l l not be near l y as d e t a i l e d . 6. The technique has found success i n determin ing such s t r u c t u r a l features as b i l a y e r t h i c k n e s s , d i s tances between the headgroup regions of the membrane, as we l l as hydrocarbon chain pack ing. More d e t a i l e d i n f o r -mation about the technique i t s e l f and i n fo rmat ion der i ved by i t may be found i n the f o l l o w i n g reviews and papers (76-78). ( i i ) Neutron D i f f r a c t i o n Another s c a t t e r i n g technique which has been r a the r s i l e n t u n t i l r e c e n t l y i s neutron d i f f r a c t i o n . The bas i s o f t h i s technique i s very s i m i l a r to t ha t o f X-ray s c a t t e r i n g , however i n s tead o f e l e c t r on s s c a t t e r i n g an i n c i d e n t beam of X - ray s , n u c l e i s c a t t e r a monochromatic beam of neutrons. An e legant experiment o f Zaccai et al. (79) i n vo l ved the study o f d i p a l m i t o y l -pho spha t i dy l cho l i ne b i l a y e r s d i sper sed i n r e l a t i v e l y small q u a n t i t i e s of normal and; then . deuterated water. Ana l y s i s of the two sets o f data y i e l d e d a p r o f i l e o f the neutron s c a t t e r i n g dens i ty as a f u n c t i o n o f p o s i t i o n across the b i l a y e r . From the p r o f i l e the polarheadgroup l aye r s are ev ident as w e l l as the p o s i t i o n o f the hydrocarbon cha in s . The l e a s t den s i t y was found at the j u n c t i o n o f the two l e a f s o f the b i l a y e r . Sub-t r a c t i n g the two sets o f data leaves a p r o f i l e o f the l o c a t i o n o f the water w i t h i n the b i l a y e r . Other e legant experiments i n vo l ve the use o f s e l e c t i v e l y deuterated molecu les . In one study (80,81), s e l e c t i v e l y deuterated cho-l e s t e r o l (hydrocarbon chain deuterated) was i nco rpo ra ted i n t o d i p a l m i t o y l -pho spha t i dy l cho l i ne b i l a y e r s . By us ing protonated c h o l e s t e r o l i n a s i m i l a r experiment and comparing the two set s o f da ta , i t was po s s i b l e to determine 7. the p o s i t i o n o f the t a i l of the hydrocarbon cha in i n the b i l a y e r . Schoenborn (80) has r e c e n t l y reviewed the use o f neutron s c a t t e r i n g i n i n v e s t i g a t i n g membrane s t r u c t u r e . ( i i i ) Raman S c a t t e r i n g Raman s c a t t e r i n g has not been one o f - t h e more- important techniques i n vo l ved wi th the determinat ion o f membrane s t r u c t u r e . This i s mainly due to the many d i f f e r e n t phy s i ca l e f f e c t s w i t h i n the membrane tha t can a l t e r the measurable featu res o f the techn ique. The assignment of the var ious absorpt ion l i n e s i s normal ly s t r a i g h t forward s i nce they are u s ua l l y very s i m i l a r i n a l l molecu les . The frequency o f p a r t i c u l a r types of v i b r a t i o n s such as C-C and C-H s t r e t c h i n g v i b r a t i o n s do not vary s i g -n i f i c a n t l y from molecule to molecu le . However the i n t e n s i t i e s and shapes o f the bands are i n f l uenced by a myriad o f f a c t o r s i n c l u d i n g , mo lecu la r conformat ion, molecule s i z e and shape, i n t e r m o l e c u l a r and i n t r a m o l e c u l a r i n t e r a c t i o n s . The Raman spectrum conta ins a vast amount o f knowledge as to the molecu lar o r g a n i z a t i o n of the membrane but a major ob s tac l e to be overcome i s to determine which are the important s t r u c t u r a l p r ope r t i e s on a mo lecu la r l e v e l which i n f l u e n c e the l i n e i n t e n s i t i e s and shapes. As a consequence o f such d i f f i c u l t i e s , the conc lus ions drawn from t h i s t e c h -nique are u s ua l l y regarded as support ing ev idence. The f o l l o w i n g papers deal w i th more d e t a i l e d aspects o f Raman spectroscopy and membrane s t r u c t u r e (82-86). 8. ( i v ) F luorescence Probes The use of f l uo re scence s c a t t e r i n g experiments i n membrane research has markedly i nc reased i n the past few yea r s . A common property o f a l l f l uo re scence probes i s the ex i s t ence o f one or more aromatic hydro-carbon r ings and/or conjugated double bonds. Some o f the probes have no resemblence to b i o l o g i c a l l y r e l e van t molecu les , such as 1 - a n i l i n o n a p t h a l i n e -8-su lphonate, but o t he r s , such as dansy l -phosphat idy lethano lamine and p a r i n a r o y l - p h o s p h a t i d y l c h o l i n e have f l u o r e s c e n t f u n c t i o n a l groups at tached to b i o l o g i c a l l y important molecules (87,88). The l a t t e r probe i s o f i n t e r e s t s i n ce i t almost complete ly resembles a normal phospho l i p id w i th the except ion tha t i t has a n o n - b i o l o g i c a l f a t t y a c i d - p a r i n a r i c a c i d (9 ,11 ,13 ,15 -octadecatet raeno ic a c i d ) . As w i th most membrane techn iques , f l uo rescence probe experiments have t h e i r advantages and disadvantages. A se r ious drawback of t h i s t e c h -nique i s the determinat ion o f the l o c a t i o n o f the probe i n the membrane. Many o f these probes are s imply mixed w i th the sample and w i th most o f these probes being hydrophobic i n na tu re , i t i s assumed t h a t they enter the hydrophobic environment o f the membrane. S ince the concent ra t i on of the probe i n the membrane must be kept to a minimum so as not to d i s t u r b the ac tua l membrane s t r u c t u r e , i t i s d i f f i c u l t to be completely sure tha t the environment the probe i s expe r i enc ing i s t y p i c a l o f the whole membrane. F i n a l l y , the measured p rope r t i e s o f the probe are u s ua l l y a f f e c t e d by a v a r i e t y of phy s i ca l i n t e r a c t i o n s as i n the Raman experiment. Therefore the data obta ined must be t r ea ted c a r e f u l l y . The f o l l o w i n g p u b l i c a t i o n s w i l l g ive more d e t a i l e d i n fo rmat i on on t h i s technique (89-91). 9. (v) Magnetic Resonance Techniques Some of the most usefu l techniques i n membrane research a r e the magnetic resonance methods i n c l u d i n g nuc lea r magnetic resonance and e l e c t r o n sp in resonance. (1) Nuclear Magnetic Resonance Most o f the methods p rev i ou s l y mentioned have mainly g iven a t ime-independent view o f the b i o l o g i c a l membrane. The magnetic resonance techniques however, answer quest ions concerned w i th the dynamic p rope r t i e s of the membrane. For some time people have thought t h a t the membrane was not a s t a b l e s p a t i a l l y o r i e n t a t e d e n t i t y . In the 1930's l i p i d s were being desc r ibed as possess ing p rope r t i e s between s o l i d and l i q u i d , thus the term " l i q u i d - c r y s t a l l i n e " s t a t e . In the years o f 1933-39 Rinne, Zermal, and Schmitt were d i scus s ing the ' f l u i d ' p r ope r t i e s of the b i o l o g i c a l membrane. At t ha t po in t a l l observat ions were q u a l i t a t i v e . Today we are s t i l l t a l k i n g about the l i q u i d - c r y s t a l l i n e s t a t e o f membrane l i p i d s but i t i s now time to t rans form these q u a l i t a t i v e fo rmu la t ions i n t o q u a n t i t a t i v e ones. The magnetic resonance methods are i d e a l l y s u i t e d f o r such a task . Due to the complex i ty o f the i n t a c t b i o l o g i c a l membrane, the ma jo r i t y of usefu l i n fo rmat ion us ing the magnetic resonance techniques has been obta ined from the study of model systems. The amount o f research done on these systems i s overwhelming and i t would be near to imposs ib le to cover t h i s area i n g reat d e t a i l . However the re have been s i g n i f i c a n t c o n t r i b u t i o n s made to membrane research us ing these techniques that should be mentioned. 10. The .NMR technique can monitor the l i p i d component o f the membrane, even when p r o t e i n i s present i n the b i l a y e r . B y s tudy ing what happens to the l i p i d upon the a d d i t i o n o f membrane a s soc i a ted po lypept ides i t i s po s s i b l e to deduce the types o f i n t e r a c t i o n s t ha t take p lace between the l i p i d and p r o t e i n . (1.1) Headgroup Stud ies Recent ly th ree groups have been i n v e s t i g a t i n g the headgroup con-31 2 format ion o f phospho l ip ids i n membranes. Using P and H NMR, S e e l i g and Ga i l y (92) s t ud i ed b i l a y e r s of d i pa lm i toy lpho spha t i dy l e thano l am ine , above and below the phase t r a n s i t i o n . They s e l e c t i v e l y deuterated the ethano-1 amine carbon hydrogens f o r the deuter iun NMR study. The data suggested a model i n which the headgroup ro ta te s f l a t on the su r face of the b i l a y e r and makes r ap i d t r a n s i t i o n s between two conformat ions. Stud ies on d i p a l -mi toy ! phosphat idy l c ho l i ne headgroup o r i e n t a t i o n by S e e l i g , G a i l y , and Wohlgemuth (93) suggest a model i n which the cho l i ne headgroup i s a l i g ned 31 p a r a l l e l to the b i l a y e r p lane. Koh ler and K l e i n (94) have measured P NMR spec t r a o f d i pa lm i toy lpho spha t i dy l e thano lam ine , d i p a lm i t o y l pho s -p h a t i d y l c h o l i n e , egg pho spha t i d y l cho l i ne , and b r a i n pho spha t i d y l c ho l i ne . 31 C u l l i s et al. s tudy ing pho spha t i dy l cho l i ne l iposomes, us ing P NMR, d i scuss f a c t o r s a f f e c t i n g the motion of the p o l a r headgroup- .(237). .The spec t ra o f unsaturated and s a tu ra ted phosphat idy l cho l i ne s i n the l i q u i d - c r y s t a l l i n e s t a t e are very s i m i l a r i n d i c a t i n g that the motion of the po l a r headgroup i s not s e n s i t i v e to f a t t y a c i d compos i t ion. They a l s o noted t h a t there was a reduct ion i n the motion o f the phosphate when the phospho l ip ids 11. were taken below t h e i r hydrocarbon phase t r a n s i t i o n temperature. The a d d i t i o n o f equimolar concent ra t ions o f c h o l e s t e r o l e l i m i n a t e d t h i s e f f e c t . 31 A recent review by S e e l i g (213) deals w i t h the use o f P NMR and head-group s t r u c t u r e of phospho l ip ids i n membranes. (1.2) Order Parameter Stud ies Deuterium NMR has severa l advantages over the other n u c l e i used i n membrane re sea rch . S ince the deuterium resonance i s s e n s i t i v e to r e s t r i c t e d mot ion, t h i s a t t r i b u t e can be used t o . y i e l d i n fo rmat i on on the. l o c a l o rder exper ienced by the nuc leus . The parameter tha t can be e a s i l y measured i s known as the order parameter. S e e l i g and S e e l i g us ing deuterated DPL (95) and deuterated 1 - p a l m i t o y l - 2 - o l e o y l PC (96) s t ud i ed the order parameter as a f unc t i on of p o s i t i o n along the cha i n . In a s i m i l a r experiment Stockton et al. s t ud i ed egg PC and egg PC - cho l e s te ro l b i l a y e r s con ta i n i n g deuterated s t e a r i c a c i d (97). This group a l so b i o s y n t h e t i c a l l y i ncorporated deuterated f a t t y a c i d s , deuterated at s e l e c t i v e po s i t i on s along the c ha i n , to study the m o b i l i t y g rad ient along the hydrocarbon c h a i n s , i n A. laidlawii (134,135). The r e s u l t s from each of these experiments are q u a l i t a t i v e l y the same. They noted t h a t the order parameters are near l y constant at the top and middle o f the f a t t y a c i d cha in but then decrease toward the methyl ends. The values o f the o rder parameters are s ub jec t to temperature, degree of un s a tu r a t i on , and mole f r a c t i o n o f c h o l e s t e r o l . Even though the measurement o f such order parameters are r e l a t i v e l y easy to o b t a i n , the actua l i n t e r p r e t a t i o n has not been s imple (95, 99). Most of these 12. analyses i n vo l ve many assumptions and o v e r s i m p l i f i c a t i o n s , making i t d i f f i c u l t sometimes to b e l i e v e the conc lu s i on s . Peterson and Chan (98) have d e a l t w i th t h i s t o p i c i n d i c a t i n g seve ra l o ther po s s i b l e exp lanat ions to account f o r the change i n order parameters, i n c l u d i n g the importance of r e o r i e n t a t i o n of the chains as we l l as r o t a t i o n a l i s o m e r i z a t i o n . Many membrane s tud ie s have been performed us ing son i ca ted v e s i c l e s . The use o f such systems f o r membrane research has been ques-t i o n a b l e f o r some t ime. I t i s not c l e a r that the l o c a l o r i e n t a t i o n a l o rder o f the l i p i d chains i s the same i n the son i ca ted systems and i n the m u l t i l a m e l l a r system. I f the order were cons ide rab ly d i f f e r e n t , then the son i ca ted v e s i c l e system would not be a-" good model f o r " t h e -s t r u c t u r e o f the b i o l o g i c a l membrane. Severa l report s by L i chtenburg (100). and F i ne r (101) have attempted to answer t h i s que s t i on . Recent ly Bloomet al. (129) us ing proton NMR and Stockton et al. (97) have concluded t ha t the l i p i d packing i n v e s i c l e s i s not s u b s t a n t i a l l y more d i so rdered than i n the m u l t i l a m e l l a r d i s p e r s i o n s . (1.3) 1 3 C NMR Stud ies 13 The use o f C i n membrane s tud ie s has been hampered by s t rong 13 p ro ton - C d i p o l a r broadening. Urbina and Waugh (104) have app l i ed a double resonance technique to study DPL d i s p e r s i o n s . This technique not only e l im ina te s d i p o l a r c oup l i n g , but a l so y i e l d s g r ea te r s e n s i t i v i t y . O p e l l a , Yes inowsk i , and Waugh (105) app l i ed the technique to the study of c h o l e s t e r o l i n DPL d i sper sons , us ing c h o l e s t e r o l s p e c i f i c a l l y enr iched w i th carbon-13 i n two p o s i t i o n s . 13. Several other groups have used s h i f t reagents in conjunction with 13 C in model systems, to d i s t inguish signals from the outside and ins ide l ea f l e t s of the b i layer (106,107). (1.4) Reconstituted Systems When proteins were f i n a l l y added to the l i p i d dispersions, many questions began to ar i se as to the nature of the interact ions between the polypeptide and the l i p i d . Using reconstituted systems i t was possible to control the number of components of a system under study. One of the most popular systems was the cytochrome oxidase system i n i t i a l l y studied by Jost et al. (108) using electron spin resonance, and then by Longmuir 19 et al. (109) using both deuterium and F NMR. Early reports indicated the presence of several d i f fe rent classes or types of l i p i d in the sample: (1) n o n e x c h a n g i n g l i p i d , w h i c h was always p r e s e n t even on t h e p u r i f i e d cytochrome o x i d a s e complex and c o u l d n o t be removed by a v a r i e t y o f t e c h n i q u e s ; (2) boundary l i p i d ( a n n u l a r l i p i d ) , w h i c h i s t h a t segment o f t h e l i p i d p o p u l a t i o n t h a t i s i n d i r e c t c o n t a c t w i t h t h e p r o t e i n s u r f a c e ; (3) m o t i o n a l l y p e r t u r b e d l i p i d , w h i c h i s a s s o c i a t e d w i t h t h e ' s o l v a t i o n ' l a y e r s o f l i p i d e x t e n d i n g s e v e r a l l a y e r s away from t h e boundary l i p i d . I t i s t h i s c l a s s a l o n g w i t h t h e boundary l i p i d w h i c h g i v e s r i s e t o a c l a s s o f s l o w l y e x c h a n g i n g , r e s t r i c t e d l i p i d , as o b s e r v e d by d e u t e r i u m NMR; (4) f i n a l l y t h e r e i s t h e f r e e l i p i d , t h o s e r e g i o n s o f l i p i d many ' s o l v a t i o n ' l a y e r s away (109,110). It appears however the Dahlquist et al. have been the only group to observe this 'boundary l i p i d ' phenomenon ..us'ing NMR. OTdfield 'et.at. (1.11;) have studied a 14. number o f deuterated l i p i d s and var ious p ro te i n s i n c l u d i n g g r am ic i d i n A, cytochrome ox ida se , cytochrome b5, myle in p r o t e o l i p i d apopro te in , and bacter iophage f l coat p r o t e i n . Studying the above l i s t e d p r o t e i n s , above and below the phase t r a n s i t i o n us ing DMPC deuterated i n the te rmina l methyl groups and a long w i th r e s u l t s on the i n t e r a c t i o n o f cytochrome ox idase w i t h DPL deuterated i n the termina l methyl groups o f cha in number 1, no examples o f such 'boundary l i p i d 1 cou ld be observed above the t r a n s i t i o n temperature. Instead they conclude t ha t p ro te in s and po lypept ides d i s o rde r the phospho l i p id hydrocarbon chains as judged from the deuterium NMR quadropole s p l i t t i n g s . Below the t r a n s i t i o n s temperature they prevent c r y s t a l l i z a t i o n and as a r e s u l t cause b i l a y e r d i s o r d e r (111). Work r e c e n t l y completed by Devaux et al. (112) s tudy ing rhodopsin boundary l i p i d s i n s p i n - l a b e l l e d r h o d o p s i n - l e c i t h i n complexes po i n t t o avery i n t e r e s t i n g c r i t i c i s m o f work done on r e c o n s t i t u t e d systems. I t was t h e f r exper ience t ha t i f the 1 i p i d / p r o t e i n r a t i o o f the complex being s tud ied was v e r y l o w ( 1 i p i d / p r o t e i n - 10/1) then a two com-ponent system i s observed. Such a r a t i o i s however very much lower than the p h y s i o l o g i c a l r a t i o o f about 80/1. The system they were s tudy ing had rhodospin sp in l a b e l l e d w i t h sp in l a b e l I. SPIN LABEL I 15. With a l i p i d / p r o t e i n r a t i o i n the p h y s i o l o g i c a l range no t r a ce of two com-ponents was observed. I t i s t h e i r op in ion that a low 1 i p i d - p r o t e i n r a t i o would correspond to a sma l l e r d i s tance between rhodops in-molecules which may r e s u l t i n an inc rease i n the boundary e f f e c t s imposed by the p r o t e i n . However an a l t e r n a t i v e exp lana t i on may be po s t u l a t ed (112,113). Immobi l i -z a t i o n o f the probe (spin l a b e l I ) a t low l i p i d / p r o t e i n r a t i o s may r e f l e c t p r o t e i n aggregat ion which i s q u i t e po s s i b l e when the r a t i o i s decreased. Whatever the bas i s o f t h i s immob i l i z a t i o n the problem remains that at p h y s i o l o g i c a l temperature no immob i l i z a t i o n appears to take p l ace . This f a c t must be r e c o n c i l e d w i th the previous experiments on the c y t o -chrome oxidase system (108,114-16), the C a + + ATPase (117) and rhodopsin systems (118). (1.5) I n t a c t Systems Although most membrane research us ing NMR tends to i n vo l ve model systems there are a few report s o f s tud ie s done on i n t a c t systems. The data obta ined from such systems tends to be r a the r complex, but by us ing s p e c i f i c l a b e l l i n g techniques and the knowledge obta ined from model systems i t i s now po s s i b l e to e x t r a c t i n fo rmat i on on the i n t a c t b i o l o g i c a l membrane. Adholeplasma laidlawii has been a system o f con s ide rab le i n t e r e s t 31 f o r the l a s t few yea r s . D e K r u i j f f et al. (119) us ing P NMR s t ud i ed A. laidlawii c e l l membranes and de r i ved l iposomes. I t was noted t ha t the phosphorous spectrum o f the i n t a c t membrane i s very s i m i l a r to the spectrum of the l iposomes. When the membranes were t r e a t ed w i t h pronase, a non-s p e c i f i c p rotease, the spectrum appeared to be i n s e n s i t i v e w i th 40-60% 16. of the membrane p r o t e i n being removed. This i n d i c a t e d t ha t e i t h e r no l ong -l i v e d l i p i d headgroup-protein i n t e r a c t i o n s accur or t ha t the l i p i d - p r o t e i n complexes i n the membrane have a f a s t r o t a t i o n time (t < 10~^s) along an ax i s pe rpend icu la r to the plane of the membrane. D e K r u i j f f et al. (120) have shown evidence f o r i s o t r o p i c motion 31 of phospho l ip ids i n l i v e r microsomal membranes us ing P NMR. This i s o t r o p i c motion was not due to r a p i d tumbl ing of the microsomal v e s i c l e s nor to r ap i d l a t e r a l d i f f u s i o n of the phospho l ip id s . They d i scuss the p o s s i b l e format ion of a t r a n s i t o r y non -b i l a ye r l i p i d c o n f i g u r a t i o n w i t h the bulk l i p i d i n r a p i d exchange. The ex i s t ence o f anyth ing but a b i l a y e r phase i n b i o l o g i c a l membranes has not been cons idered u n t i l r e c e n t l y . However, there i s a s t rong p o s s i b i l i t y t ha t other l i p i d phases can e x i s t i n membranes ( F i g . 1) and p lay f u n c t i o n a l r o l e s . Membrane f u s i o n , f o r example, must r equ i re tha t some of the l i p i d s adopt, a t l e a s t on the shor t term, a non-b i l a y e r c o n f i g u r a t i o n dur ing the in te rmed ia te s teps . S tud ies by C u l l i s 31 and Hope, us ing P NMR, have suggested the involvement of the hexagonal H-JI phase i n the f u s i on event (121). The a d d i t i o n of o l e i c a c i d or g l y c e r o l mono-oleate a t concent ra t ions found to induce f u s i on o f e r y th rocy te s in vitro are found to produce a t r a n s i t i o n of a v a r i a b l e p o r t i o n o f the membrane phospho l ip ids from the c l a s s i c a l b i l a y e r c o n f i g u r a t i o n to the hexagonal H-^ phase. They propose a model f o r o l e i c a c i d induced f u s i on of the e r y t h r o c y te membrane and a l s o suggest t h i s to be the mechanism o f / f u s i on events in vivo. To face page 17. F igure 1. I l l u s t r a t i o n o f the var ious phases l i p i d s may assume along w i th t h e i r corresponding 3 1 P NMR spec t ra (reproduced by permiss ion o f P.R. Cu l l i s ) . 17. Corresponding 5 1 P N M R spectra Hexagonal ( H j)) Bilayer Phosphol ipid phases Phases where isotropic mot ion occurs a, C u b i c b, R h o m b i c c, M i c e l l a r , inverted micellar C u l l i s and V e r k l e i j have s t u d i e d a pho spha t i d y l s e r i ne/pho spha t i d y l -ethanolamine mixture w i th regards to the e f f e c t s of C a ' T and the l o c a l anae s the t i c d ibuca ine (122) on membrane phase behaviour. I t was repor ted that C a + + can induce the b i l a y e r to hexagonal phase t r a n s i t i o n but the a d d i t i o n o f d ibuca ine can reverse t h i s e f f e c t . Th is r e s u l t i s d i scussed i n terms o f a model f o r membrane f u s i o n and the mechanics of anaes thes i a . An a r t i c l e by C u l l i s and McLaughl in (123) d i scusses the recent progress 31 o f P NMR as a probe o f membrane s t r u c t u r e and motion o f phospho l ip ids i n membrane systems. Dratz et al. have cent red t h e i r a t t e n t i o n on the use o f proton NMR i n s tudy ing rod outer segment d i sk membranes (124). The use o f proton NMR presents c e r t a i n problems i n i n t e r p r e t i n g the data due to the c l o s e l y ove r l app ing reasonances. However some general conc lu s ions about mo lecu la r motion can be made. The study o f the l i n e w i d t h s suggest t h a t the rhodopsin does not g r e a t l y a f f e c t r e l a t i v e l y low frequency motions o f the phospho-l i p i d s such as l a t e r a l d i f f u s i o n . However the study o f the s p i n - l a t t i c e d. Vesicles F igure 1 ++ 18. r e l a x a t i o n rates i n d i c a t e that the membrane reasonances can be decomposed i n t o two components, one corresponding to phospho l ip ids i n t e r a c t i n g w i th the rhodopsin and the o ther corresponding to the bulk pho spho l i p i d . The theory f o r the i n t e r p r e t a t i o n of proton NMR l ineshapes has been i n v e s t i g a t e d by severa l groups. Chan and coworkers have pub l i shed severa l a r t i c l e s i n t h i s area (98,125,126) however there appears to be c o n f l i c t i n g views on the sub jec t as seen i n p u b l i c a t i o n s by Ulrnius et al. (127) and Bloom et al. (128,129). Two o f the most popular nuc l e i f o r use i n s tudy ing i n t a c t mem-13 brane systems appear to be deuterium and C. Due to the low natu ra l 13 2 abundance o f these nuc l e i ( C = 1.1%; H = 0.015%) they are usefu l l a b e l s f o r membrane study s i n ce v i r t u a l l y no background s i g na l i s obta ined i n s pec t r a o f i s o t o p i c a l l y enr i ched membranes. London and coworkers (130) repor ted a loc F ou r i e r t rans form (FT) NMR study on the f r a c t i o n a t e d membranes o f Candida utilis. By growing 13 the organism on a medium enr iched w i t h 20 atom % C acetate (doubly l a b e l l e d ) , they were ab le to n o n s p e c i f i c a l l y l a b e l the yea s t . From sp in l a t t i c e measurements they found evidence to suggest a m o b i l i t y g rad ient along the hydrocarbon chain w i th i nc reased m o b i l i t y from the g l y c e r o l backbone towards the termina l methyl g roup ' as we l l as towards the c ho l i n e methyls. Metca l fe et al. (131) repor ted 1 3 c NMR .spectra o f Aohole-13 plasma laidlawii membranes con ta in i ng C l a b e l l e d phospho l i p i d s . The 13 organism was grown on a medium enr i ched w i th [1- C ] - p a l m i t i c a c i d and by doing so the spectrum of the membrane was reduced to a s i n g l e we l l d e f i n e d . resonance. A major disadvantage o f t h i s l a b e l l i n g procedure i s t ha t i t 13 i s expens ive, e s p e c i a l l y f o r p l a c i n g C l a b e l s along the cha in . Smith 19. et al. (132) l a b e l l e d another organism, Aureobasidium pullulans, us ing 13 1- and 2- [ C ] -ace ta te as p r e v i ou s l y desc r ibed by London et al. (130). 13 13 The use o f l - [ C ] - ace ta te and 2- [ C ] - ace ta te a l lows the study o f the odd and even carbon atoms, r e s p e c t i v e l y , o f the f a t t y acy l cha in s . T ana l y s i s of the data obta ined i n d i c a t e d q u a l i t a t i v e l y a t l e a s t , an i nc rea se i n m o b i l i t y from the g l y c e r o l backbone towards the termina l methyl group. S i m i l a r work i s a l so underway on two o ther organisms, Micrococcus freudenreichii and Ealobacteriwn cutirubrum. A. Laidlawii was the f i r s t organism to be s t ud i ed by deuterium NMR (133). O l d f i e l d et al. employed the use o f perdeuterated f a t t y a c i d i n the growth medium to s p e c i f i c a l l y l abe l the phospho l i p i d s . However, due to a l ack o f r e s o l u t i o n o f the i n d i v i d u a l resonances no conc lus ions cou ld be made. Smith et al. (134,135) have r e c e n t l y completed work on the A. Laidlawii system i n which they i nco rpo ra ted i n d i v i d u a l s p e c i f i c a l l y deuterated f a t t y a c i d s . Th is work i s the r e s u l t o f an i n c r e d i b l e amount o f t ime and expense i n p repar ing the i n d i v i d u a l l y l a b e l l e d f a t t y a c i d s . The p r o f i l e s of the order parameters and quadropole s p l i t t i n g s versus p o s i t i o n along the acy l chain are very s i m i l a r to those obta ined w i th the s y n t h e t i c systems f o r egg RC (136,137) and DPL (138). These r e s u l t s are very encouraging because they conf i rm the re levance o f model system s t u d i e s . S t o f f e l et al. (139,140) have used 1 3 C NMR to study the l i p i d o r g a n i z a t i o n o f the enveloped v i r i o n , V e s i c u l a r Stomat itus v i r u s . Moore et al.3 us ing phosphorous-31 NMR, have a l so been s tudy ing t h i s v i r u s (141). These papers w i l l be d i scussed i n a separate s e c t i o n . 20. (2) E l e c t r o n Spin Resonance ESR has been a very popular and usefu l magnetic resonance t e c h -nique i n membrane study. I t has the advantage of being ab le to use small samples and y e t obta in spec t ra i n a very sho r t t ime. The most f r equen t l y used ' s p i n l a b e l s ' are n i t r o x i d e s which conta in a N + 0 group. Th is group can be i nco rpo ra ted i n t o a number of molecules which are s o l ub l e i n both aqueous and non-aqueous phases. An example o f t h i s type o f probe i s 2 , 2 , 6 , 6 , - t e t r amethy l p i pe radone -1 - oxy l . (TEMPO). This type o f probe i s be l i e ved to p a r t i t i o n i t s e l f between the aqueous and hydrophobic reg ions o f the membrane. Other types o f probes i nc lude d e r i v a t i v e s o f f a t t y ac ids w i th the n i t r o x i d e being at tached at var ious po s i t i o n s along the chain (doxyl d e r i v a t i v e s ) . Phospho l ip ids can a l s o be l a b e l l e d , e i t h e r along the f a t t y a c i d c ha i n , or i n the headgroup reg ion . F i n a l l y , s t e r o l d e r i v a t i v e s have been prepared i n c l u d i n g 3-doxyl d e r i v a t i v e s of cholestane-3-one and androstane-3-one- l7-01 (142). An important d i f f e r e n c e between ESR and NMR i s i n the time s c a l e s . -8 ESR i s s e n s i t i v e to motions on the order o f 10 s wh i l e NMR i s s e n s i t i v e to motions on the orders o f 1 0 ' 5 - 1 0 _ 6 s . There fo re , ESR i s s e n s i t i v e , . to motions that take p lace on a much s h o r t e r t ime s c a l e compared to NMR. Theor ies f o r l i n e shape ana l y s i s are d i scussed elsewhere (142). A common method o f ana l y s i ng a spectrum invo lves the e x t r a c t i o n o f the ESR ' o r de r pa rameter ' , which i s conceptua l l y s i m i l a r to the proton and deuterium order parameters. The ESR order parameter i s a number between 0' and +1 and has a w e l l de f ined meaning i n terms of the average a b i l i t y -8 o f the n i t rogen p o r b i t a l to r o t a t e dur ing times on the order o f 10~ s. -8 I f the order parameter i s near u n i t y , then i n a time o f 10" s the p o r b i t a l 21. con ta i n i ng the unpaired e l e c t r o n does not apprec iab l y change d i r e c t i o n . However a value s i g n i f i c a n t l y l e s s than 1 i s c on s i s t en t w i th a wide v a r i e t y o f po s s i b l e motions. ESR experiments have measured a number of membrane p r o p e r t i e s . The p a r t i t i o n i n g of TEMPO between the aqueous and non-aqueous membrane regions has been used to i n v e s t i g a t e . l i p i d phase t r a n s i t i o n s and l a t e r a l d i f f u s i o n (238). TEMPO i s found to be more s o l u b l e i n the membrane i n t e r i o r above the phase t r a n s i t i o n temperature. Using t h i s property o f the probe the e f f e c t o f membrane p ro te in s may be s t u d i e d , l ook i ng f o r changes i n the phase t r a n s i -t i o n temperature o f model membranes induced by the i n t r o d u c t i o n o f the p r o t e i n (117). In t h i s same way i t i s po s s i b l e to est imate the f r a c t i o n o f l i p i d s i n a b i o l o g i c a l membrane which are i n a f l u i d s t a t e (143). The p a r t i t i o n -ing of phospho l i p id sp in l a b e l s between the f l u i d and s o l i d phase i s one method o f s tudy ing the ca lc ium- induced l a t e r a l phase separat ions i n phospho-l i p i d b i l a y e r s (144). By a f f i x i n g doxyl sp in l a b e l s at var ious p o s i t i o n s along the hydrocarbon chains of phospho l ip ids i t i s po s s i b l e to ob ta in i n fo rmat i on about the m o b i l i t y o f the chain as a f u n c t i o n o f d i s tance from the g l y c e r o l back-bone. S tud ies us ing t h i s system have found that the e l e c t r o n sp i n resonance order parameter decreases as the sp in l a b e l i s moved away from the g l y c e r o l backbone. This v a r i a t i o n , r e f e r r e d to as the ' f l e x i b i l i t y g r ad i en t ' o r ' f l u i d i t y g r a d i e n t ' has been observed i n a v a r i e t y of systems us ing deuterium 13 and . C NMR as p rev i ou s l y mentioned (43,145). B u t t e r f i e l d et al. (146) have used sp i n l a b e l s to compare order parameters de r i ved from e ry th rocy te s obta ined from normal sub jec t s and from pa t i en t s w i th myotonic muscular dystrophy, Duchenne muscular dystrophy, 22. and congent ia l myotonia. Increased membrane f l u i d i t y was demonstrated i n both mytonic muscular dystrophy and congent ia l myotonic e th rocy te membranes by means o f ESR. The eythrocyte membranes from pa t i en t s having Duchenne muscular dystrophy e x h i b i t e d normal membrane f l u i d i t y . Although the s p e c i f i c mechanism re spons i b l e f o r these phenomena i s not known, the sp i n l a b e l measurements suggest a c o r r e l a t i o n o f i nc reased e r y th rocy te membrane f l u i d i t y w i t h the presence of myotonia. A number o f papers have been pub l i shed dea l i n g w i th sp in l a b e l l i n g of i n t a c t v i r a l membranes (147-152). These w i l l be d i scussed i n a separate s e c t i o n . ( v i ) Ca lo r imet ry D i f f e r e n t i a l scanning c a l o r ime t r y (DSC) i s a usefu l technique f o r determin ing phase t r a n s i t i o n temperatures. In the case o f l i p i d s , i t • detect s the l a t e n t heat a s s oc i a ted w i t h ' t h e t r a n s i t i o n from the c r y s t a l l i n e ; to the l i q u i d - c r y s t a l l i n e s t a t e . Most o f the work us ing DSC has been on model systems; The t r a n s i t i o n - t e m p e r a t u r e and en tha l p i e s f o r var ious one component systems have been repor ted (153-156). The phase t r a n s i t i o n temperature can be a f f e c t e d by changing the ex te rna l environment o f the l i p i d s such as changing the pH or the concen-++ ++ t r a t i o n o f d i v a l e n t cat ions such as Ca o r Mg (90). B ina ry mixtures of phosphat idy l cho l i ne s have been s t ud i ed as we l l as the e f f e c t o f c h o l e s t e r o l on the phase t r a n s i t i o n temperature of these l i p i d s (156,157). Th i s t e c h -niques has a l so been used to study the phase behaviour o f the membranes o f A. laidlawii (158) and ff. ooli (159). 23. ( v i i ) Freeze F racture E l e c t r on Microscopy For years e l e c t r o n microscopy has been a very powerful t oo l i n membrane re search . Some o f the f i r s t ev idence f o r the b i l a y e r s t r u c t u r e o f the membrane was c on t r i bu ted by the negat ive s t a i n i n g e l e c t r o n microscopy procedure (160). A more popular technique today i s f r eeze f r a c t u r e e l e c t r o n microscopy which invo lves ' photograph ing ' the i n t e r i o r su r face o f the membrane (161). From e l e c t r o n micrographs of one or two component l i p i d m ix tu re s , d i f f e r e n t tex tu res can sometimes be seen, presumably due to d i f f e r e n t phases i n the b i l a y e r . When two or more such tex tu re s appear on the same b i l a y e r , i t i s c l e a r t ha t there i s a l a t e r a l phase sepa ra t i on o f the phospho l i p id s . F r e e z e . f r a c t u r e e l e c t r o n microscopy s tud ie s o f b i o l o g i c a l membranes u sua l l y show p a r t i c l e s on r e l a t i v e l y smooth sur faces which may be homogeneously d i s t r i b u t e d o r concentrated i n patches. These p a r t i c l e s are i n t e r p r e t e d as r e f l e c t i n g intra-membrane p ro te i n s d i sper sed i n smooth l i p i d s u r f a ce s . Van D i j c k et al. (162) and Papahadjopoulos et al. (163,164) have used t h i s technique i n con junc t i on w i th DSC to study the e f f e c t o f C a + + and M g + + on the s t r u c t u r e o f b i l a y e r s composed o f d i m y r i s t o y l phos-pha t i dy l g l y c e r o l . Chapman et al. (165) s t ud i ed the e f f e c t of monovalent ions on the phase t r a n s i t i o n o f pho spha t i dy l cho l i ne b i l a y e r s wh i l e V e r k l e i j et al. (166) s t ud i ed the outer membrane of E. ooli mutants. C. Membrane Stud ies o f V i ruses Stud ies on b i o l o g i c a l membranes have been hampered by t h e i r complex nature. As seen i n the previous s e c t i o n , most work has centred on 24. model and r e c o n s t i t u t e d membrane systems. L i p i d enveloped v i ru se s on the o ther hand o f f e r an oppor tun i t y to study a b i o l o g i c a l membrane which i n most cases i s more s i m p l i f i e d compared to whole c e l l membranes. These v i ru ses o f f e r a number o f a t t r a c t i v e features i n c l u d i n g : ( i ) t h e y can be o b t a i n e d w i t h a h i g h degree o f p u r i t y ; ^ ( i i ) t h e y have a l i m i t e d number o f p o l y p e t i d e s a s s o c i a t e d w i t h t h e membrane,.which s i m p l i f i e s t h e number o f i n t e r a c t i o n s w i t h i n t h e b i l a y e r ; ( i i i ) by g r o w i n g t h e v i r u s i n d i f f e r e n t h o s t c e l l s i t i s p o s s i b l e t o a l t e r t h e l i p i d c o m p o s i t i o n w h i l e k e e p i n g t h e p r o t e i n c o m p o s i t i o n t h e same, and ( i v ) t h e r e v e r s e case i s a l s o p o s s i b l e - by g r o w i n g d i f f e r e n t v i r u s e s , w h i c h v a r y i n p r o t e i n compo-s i t i o n i n t h e same h o s t c e l l , t h e l i p i d c o m p osi-t i o n w i l l be t h e same w h i l e t h e p r o t e i n c o n t e n t w i l l be d i f f e r e n t . A number of v i ru se s i n c l u d i n g i n f l u e n z a , p a r a i n f l u e n z a , SV-5 (147,149,167), Rauscher Murine Leukemia v i r u s (148), S indb i s v i r u s (150), Venezualan Equine Encepha lomye l i t i s v i r u s (151), and V e s i c u l a r S t o m a t i t i s 1 3 v i r u s (152,139-141) have been s t ud i ed by such techniques as ESR, C 3.1. NMR, P NMR, and f l uo re scence s c a t t e r i n g . Landsberger et al. (147-149, 152) have been very a c t i v e s tudy ing i n f l u e n z a , Rauscher leukemia, pa r a i n f l uenza SV5, and V e s i c u l a r S t o m a t i t i s v i ru ses us ing ESR probes. The i r s tud ie s have i n d i c a t e d t h a t a l l of these v i ru ses have the c l a s s i c b i l a y e r s t r u c t u r e , and t ha t the host c e l l membrane i s cons ide rab l y more f l u i d than the v i r a l l i p i d . Protease d i g e s t i o n o f these v i ru ses leads to the removal of the su r face g l y cop ro te i n " s p i k e s " u s ua l l y observed by e l e c t r o n microscopy s tud ie s (149). Removal o f these su r face " s p i k e s " from i n f l u e n z a v i ru s (147) does not appear to a f f e c t the o r g a n i z a t i o n o f 25. the l i p i d b i l a y e r , however i n a study on V e s i c u l a r S t o m a t i t i s V i rus the removal o f the " s p i k e s " increases the f l u i d i t y o f the l i p i d b i l a y e r (152). By growing i n f l u e n z a and pa ra i n f l uenza SV5 v i r u s on two d i f f e r e n t c e l l l i n e s , BHK21-F and MBDK c e l l s (149), Landsberger et al. us ing ESR were ab le to show tha t the r i g i d i t y o f the v i r a l membrane depends l a r g e l y on the l i p i d composit ion and i s not a f f e c t e d by the d i f f e r e n t p r o t e i n com-p o s i t i o n o f the two v i r u s e s . Sefton and Gaffney (150) s t ud i ed the f l u i d i t y o f the l i p i d s in the membrane o f S indb i s V i rus us ing ESR. Again the v i r a l membrane was found to be more r i g i d than the host c e l l membrane, however t h e i r data suggests that the d i f f e r e n c e i n f l u i d i t y i s not due s imply to d i f f e r ence s i n l i p i d composit ion but r a the r the r e s u l t o f the i n t e r a c t i o n o f the v i r a l p ro te in s w i th the membrane l i p i d s . They found tha t the v i r a l l i p i d s were more f l u i d a f t e r chloroform/methanol e x t r a c t i o n than i n the i n t a c t v i r u s and the v i r a l membrane i s made more f l u i d by p r o t e o l y t i c d i g e s t i o n o f the v i r a l g l y c o p r o t e i n s . Using 1 3 C NMR S t o f f e l et al. (1.39,140) s t ud i ed the l i p i d . 1 3 o r g an i z a t i on of V e s i c u l a r S t o m a t i t i s v i r u s . From C T^ r e l a x a t i o n data i t was concluded tha t the motions o f the phospho l ip id c ho l i n e headgroups o f V e s i c u l a r S t o m a t i t i s v i r u s are more r e s t r i c t e d by the d i g e s t i o n o f the v i r a l g l y c o p r o t e i n w i th t r y p s i n . They were a l s o ab le to study the m o b i l i t y o f the cen t r a l par t o f the f a t t y a c i d chains by growing the v i r u s on host c e l l s p r e l a b e l l e d w i th [ 1 3 C ] - o l e i c a c i d . Using [ 3 - 1 3 C ] - and [ T l - 1 3 C ] - 0 1 e i c a c i d 13 and [16- C } pa lm i t i c a c i d as l a b e l s , the data suggested a high r i g i d i t y o l i m i t e d to a depth o f about 15 A which i s p r i m a r i l y due to the high c h o l e s t e r o l content of the membrane as w e l l as i n t e r a c t i o n s o f the membrane a s soc i a ted G- and po s s i b l y M-prote in w i th the l i p i d s . The i nne r core o f the b i l a y e r 26. was found to be more f l u i d . T r y p t i c d i g e s t i o n o f the g l y c o p r o t e i n " s p i k e s " o f the G -p ro te in caused a reduc t i on i n the f l u i d i t y of the i nne r core o f the v i r a l membrane, po s s i b l y due to the f u r t h e r ent ry o f the remaining hydrophobic pept ide o f the G-p ro te in i n t o the b i l a y e r . 31 Moore et al. us ing P T-j r e l a x a t i o n measurements to study head-group motion i n the same v i r u s , V e s i c u l a r S t o m a t i t i s v i r u s , concluded t ha t p r o t e o l y t i c d i g e s t i o n o f the G-prote in leads to an i nc rea se i n the motion o f the phosphate, and t he r e f o r e presumably an inc reased m o b i l i t y o f the phospho l ip id headgroups (141) which i s opposed to the conc lu s i on o f S t o f f e l et al. (139,140). The r e s u l t s obta ined us ing T-| r e l a x a t i o n data i s sometimes d i f f i c u l t to i n t e r p r e t e when s tudy ing such complex heterogenous systems. D. L ip id-dependent P ro te i n s In the previous s e c t i o n s the t o p i c o f i n t e r e s t has been the bulk i n t e r a c t i o n s o f l i p i d s and p r o t e i n s . Using such techniques as NMR, ESR, DSC, f l uo re s cence , X- ray, and neutron d i f f r a c t i o n , i t i s the average i n t e r a c t i o n s between l i p i d s and p ro te in s t ha t are being s t u d i e d . For some time now i t has been known t h a t c e r t a i n p r o t e i n s , be they membrane bound or s o l u b l e , are dependent on c e r t a i n c l a s se s o f l i p i d s o r l i p i d mixtures f o r a c t i v i t y . I n ve s t i g a t i on s i n t h i s area have proven to be r a the r d i f f i c u l t due to the low monomer concent ra t ions of l i p i d s i n aqueous s o l u t i o n . Most s tud ie s r epo r t p r o t e i n - 1 i p i d i n t e r a c t i o n s on s e l f - a s s o c i a t e d l i p i d s ( pho spho l i p i d l iposomes) i n contact w i th p ro te i n s o l u t i o n s . A wide range o f p r o t e i n c la s ses have been s t ud i ed i n c l u d i n g h y d r o p h i l i c p r o t e i n s , serum a p o l i p o -p r o t e i n s , s o l ub l e pe r i phe ra l membrane p r o t e i n s , i n s o l u b l e p ro te in s and 27. small pept ides . Of i n t e r e s t are the group o f enzymes which have been p u r i f i e d and found to be dependent on l i p i d f o r a c t i v i t y . Ma l a te - v i t am in K reductase i s o l a t e d from M. phlev ( p u r i f i e d to near homogeneity) i s found to be dependent upon added phospho l i p id f o r a c t i v i t y (171). The enzyme a c t i v i t y i s found to be dependent upon the degree o f aggregat ion o f the p r o t e i n . In high s a l t s o l u t i o n the p r o t e i n e x i s t s as a monomer and i s most a c t i v e , wh i l e i n low s a l t i t aggregates and i s l e s s a c t i v e . P r o t e i n i n l i p i d complexes have been i s o l a t e d and i t i s suggested t h a t a phospho l i p id b ind ing s i t e ( s ) appears to be i n vo l ved w i th the agg regat ion -d i sagg regat ion process. Whi le s tudy ing the a c t i v a t i o n process by phospho l ip ids Imai et al. found a s o l e c t i n (major components are PC and PE) to be most e f f e c t i v e . I nd i v i dua l l i p i d s alone o r c a r d i o l i p i n e x h i b i t e d l e s s a b i l i t y to a c t i v a t e . S ince the i s o l a t e d p r o t e i n - 1 i p i d com-plexes can be d i s s o c i a t e d us ing high s a l t , i t would seem that e l e c t r o s t a t i c i n t e r a c t i o n s may be important. D i f f e r e n t i a l c e n t r i f u g a t i o n of s on i ca ted c e l l extracts were used to l o c a l i z e the enzyme. The p r o t e i n a c t i v i t y was p r e d o m i n a n t l y found i n the supernatant f r a c t i o n w i th some r e s i dua l a c t i v i t y i n the p a r t i c u l a t e f r a c t i o n . However, ghost p reparat ions obta ined by the treatment o f c e l l s w i th lysozyme i n the presence o f g l y c i n e , conta ined >85% o f the enzymatic a c t i v i t y which can then be re leased by s on i c o s c i l l a t i o n (172). Th i s suggests t ha t the enzyme i s l o o s e l y a s s oc i a ted w i th the c e l l membrane and cou ld be c l a s s i f i e d as a " p e r i p h e r a l " p r o t e i n us ing the S inger and N icho l son nomen-c l a t u r e ( 8 ) . A phospho l i p id r e q u i r i n g enzyme, NAD-dependent malate dehydro-genase, i s o l a t e d from M. sp . s t r a i n Takeo (173), i s found to be d i f f e r e n t 28. from the enzyme i s o l a t e d from M.phlei although they have s i m i l a r mo lecu la r we ights . Enzyme a c t i v i t y i s dependent upon the presence o f c a r d i o l i p i n , w i thout which the enzymatic a c t i v i t y i s on ly about 3% o f maximum. (a) L - l a c t a t e Dehydrogenase L - l a c t a t e dehydrogenase from E. coli i s i s o l a t e d by detergent treatment o f b a c t e r i a l membranes. The enzyme i s a c t i v a t e d about 3 - f o l d w i th the a d d i t i o n o f p h o s p h a t i d y l g l y c e r o l , c a r d i o l i p i n , o r a l i p i d mixture (174). When a c t i v a t e d by phospho l ip ids the enzyme e x h i b i t s a s i m i l a r Km value f o r L - l a c t a t e to t ha t o f the membrane bound enzyme. Severa l po ints of i n fo rmat i on i n d i c a t e d t h a t the enzyme has a d i f f e r e n t conformation i n the detergent f r e e system. The a - h e l i c a l content o f the p r o t e i n i s i nc reased 1 . 7 - f o l d dur ing p re incubat i on w i th the l i p i d s and the a - h e l i x becomes more s t a b l e dur ing heat t reatment. Th i s suggests that the enzyme i s showing monomolelcular d i s pe r s i o n i n the l i p i d b i l a y e r . In the detergent f r ee system the p r o t e i n i s an aggregate o r o l i g o m e r i c complex o f 10 o r more molecules i n aqueous s o l u t i o n . In t h i s form the enzyme i s on ly p a r t i a l l y s e n s i t i v e to a s p e c i f i c ant ibody suggest ing tha t not a l l o f the a n t i g e n i c s i t e s i n the o l igomer are a c c e s s i b l e . However upon i n cuba t i on o f the aggregate w i t h pho spha t i dy l g l y ce ro l o r c a r d i o l i p i n , the enzyme became completely s e n s i t i v e to the ant ibody presumably due to a l l the p r o t e i n molecules becoming d i sper sed i n the monomolecular form i n the l i p i d b i l a y e r and thus exposing t h e i r a n t i g e n i c s i t e s . K i n e t i c evidence a l s o supports a d i f f e r e n t conformation o f the enzyme i n the de te r gen t - f r ee system. I t was found t h a t both the Km and V increase f o r L - l a c t a t e when the enzyme i s incubated w i th phospho l i p i d s , max 29. From p re l im i na r y s tud ie s i t appears t ha t the s p e c i f i c i t y o f phospho l ip id f a t t y a c i d chains i s r a t he r broad s i n ce c a r d i o l i p i n from E. aoli or bovine heart have s i m i l a r e f f e c t s even though they d i f f e r con s i de r -ably i n f a t t y a c i d compos i t ion. Phosphat idy lethanolamine o r pho spha t i d y l -c ho l i n e along w i th cho la te have a s i m i l a r e f f e c t as phosphat idy l g l y c e r o l suggest ing t ha t pho soph i l i p i d s i n s u i t a b l e l iposomes w i th an a c i d i c com-ponent have s i m i l a r e f f e c t s regard les s o f f a t t y a c i d compos i t ion. (b) Pyruvate Oxidase Th i s enzyme from E. Coli, which i s a s o l u b l e t e t r ame r i c f l a v o -p r o t e i n , has been p u r i f i e d to homogeneity and c r y s t a l l i z e d (175-177). I t binds both th iamine pyrophosphate and FAD and i s a c t i v a t e d 15- to 100 - fo ld by phospho l ip ids and long chain f a t t y a c i d s . Maximal a c t i v a t i o n requ i res p re incubat i on o f the enzyme f o r at l e a s t s i x min wi th the l i p i d a c t i v a t o r i n the presence o f subs t ra tes and co fac to r s (pyruvate, TPP, and MgC^ ) . Minimal a c t i v a t i o n occurs i f any o f these components are mi s s ing o r i f the enzyme i s pre incubated w i th l i p i d before the subs t ra tes are added. I n i t i a l l y l y sophosphat idy lethano lamine was found to be the on ly a c t i v a t i n g l i p i d , however water s o l u b l e m i c e l l a r p reparat ions o f o ther phosphol ip ids were found to e x h i b i t h i gher s t i m u l a t i o n o f a c t i v i t y . I t appears t h a t the nature o f the phosphoryl base i s not o f primary importance i n determin ing the capac i t y o f the phospho l i p id to a c t i v a t e pyruvate ox idase , s i n ce pho spha t i dy l cho l i ne and the hydrophobic mo iet ie s o f l e c i t h i n f u l l y a c t i v a t e the enzyme, wh i l e 1 -a -g lycerophosphate and 1 -a -g lycerphosphochol ine have no e f f e c t on enzyme a c t i v i t y . I t was a l so noted that severa l f a t t y 30. ac ids can a c t i v a t e the enzyme w i th a s p e c i f i c a c t i v i t y on ly s l i g h t l y lower than that given by the phospho l i p id s . P a l m i t o l e i c and o l e i c ac ids are p a r t i c u l a r l y e f f e c t i v e i n enzyme a c t i v a t i o n . This a c t i v a t i o n by f a t t y ac ids cou ld serve as an important con t r o l mechanism i n E.ooli, metabol ism. Phosphatides d r a m a t i c a l l y a f f e c t the k i n e t i c parameters of pyruvate ox idase. The K f o r pyruvate and TPP are lowered 13- and 3- to 4 - f o l d r e s p e c t i v e l y i n the presence o f phospho l i p id s . In a d d i t i o n , the phosphatides bestow c o - o p e r a t i v i t y to TPP b ind ing to the enzyme. In the absence of pho spho l i p i d , TPP binds w i t h the usual M ichae l i s -Menten type s a t u r a t i o n k i n e t i c s , however i n the presence of pho spho l i p i d s , TPP i s found to b ind c o - o p e r a t i v e l y and s h i f t s the 1^ . f o r TPP to a lower va lue . Using s topped-f low k i n e t i c s i t i s c l e a r l y ev ident tha t i n measuring the r a te of FAD r e d u c t i o n , the presence o f phospho l i p id a f f e c t s the r a t e - c o n t r o l l i n g s tep lead ing to the format ion of the enzyme-FADH^ complex. The ra te o f reduc-t i o n of enzyme bound FAD i s increased some 100 - fo ld i n the presence o f l i p i d . I t would seem tha t there i s a good case f o r a r e g u l a t i o n mechanism i n v o l v i n g phospho l ip ids and long cha in f a t t y ac ids a c t i n g as a l l o s t e r i c modulators o f pyruvate ox idase a c t i v i t y . By s tudy ing the pathways of E. ooli f o r convers ion o f pyruvate to the aceta te l e v e l , a small b u i l d up o f f r e e f a t t y a c i d cou ld serve to sw i tch on the pyruvate ox idase to produce acetate and carbon d i o x i d e , which would be a low energy pathway f o r pyruvate u t i l i z a t i o n i n s tead o f having pyruvate dehydrogenase producing a c e t y l -CoA and carbon d i o x i d e . 31. (c) Malate Oxidase In mutant s t r a i n s o f E. coli, l a c k i n g NAD-dependent malate dehy-drogenase a c t i v i t y , malate oxidase i s present which u t i l i z e s L-malate and FAD as subs t ra tes (178). The enzyme i s l o c a l i z e d on the i nne r face o f the cytop lasmic membrane to which i t i s l o o s e l y bound and e a s i l y re leased by s o n i c a t i o n or membrane d i s r u p t i o n . As w i th pruvate ox idase , malate ox idase , considered, a pe r i phe ra l enzyme, can be obta ined in a s o l u b l e s t a t e f r ee o f l i p i d s and detergent . Th is makes i t an i d e a l model system f o r the study o f l i p i d - p r o t e i n i n t e r a c t i o n s . Pho spha t i d y l g l y ce ro l and c a r d i o l i p i n are the two major, a c i d i c phos-pho l i p i d s produced by E. coli c e l l s (179) and both are potent a c t i v a t o r s of malate ox idase. The enzyme i s a l so a c t i v a t e d by non ion ic detergents such as TX-100 and some other l i p i d s , such as a s o l e c t i n , o l e o y l - a c e t a t e , and p a l m i t o l e o y l a c e t a t e . The a l l o s t e r i c s ub s t r a te f o r malate ox idase i s FAD and not malate. The a d d i t i o n of, phospho l i p id to the enzyme does not a f f e c t the of malate o r f e r r i c y a n i d e ( a r t i f i c a l subs t ra te ) but has a l a r ge e f f e c t on the Km f o r FAD (9 uM i n the absence o f phospho l i p id to 0.2 uM i n i t s presence). (d) D-g^hydroxybutyrate Dehydrogenase D-3- hydroxybutyrate dehydrogenase i s t i g h t l y bound to the m i to -chondr i a l membrane and can be re lea sed from the. membrane by d i g e s t i o n w i th phosphol ipase A o r by detergents such as c h o l i c a c i d (180). As w i th most of these systems the l i p i d - f r e e p r o t e i n e x h i b i t s no a c t i v i t y , however upon r e c o n s t i t u t i o n w i t h m i tochondr ia l l i p i d s o f l e c i t h i n s from a v a r i e t y of 32. sources, an a c t i v e 1 i p i d - p r o t e i n complex i s formed (181-183). The enzyme s p e c i f i c a l l y requ i re s unsaturated phosphat idy l cho l i ne s f o r maximum a c t i v a t i o n (184). Most o ther common l i p i d s have been shown to be i n e f f e c t i v e i n the format ion o f an a c t i v e 1 i p i d - p r o t e i n complex and o f t en i n h i b i t the enzyme (184,185). From s tud ie s on the a c t i v a t i o n o f the enzyme by pho spha t i d y l -cho l ines , the minimal requirement f o r a c t i v a t i o n was a hydrophobic cha in and a phosphory lcho l ine group such as s t e a r o y l phosphory l cho l i ne . I t i s not enough to have the two charges and a hydrophobic chain p resent , as i n a mixture o f SDS and N- t r imethy l -n -dodecy lamine. This suggests that there i s a s p a t i a l requirement as w e l l . These r e s u l t s i n d i c a t e t h a t the enzyme has a s p e c i f i c a c t i v a t i n g s i t e ( s ) which has a s p e c i f i c requirement f o r a hydro-phobic chain l i n k e d to phosphory l cho l i ne . However, a membrane-like l iposome s t r u c t u r e appears necessary f o r the s t a b i l i z a t i o n o f an enzyme-l. ipid complex. Gazzo t t i et al. (186,187) s t ud i ed the i n t e r a c t i o n o f phospho l ip ids and 3-hydroxybutyrate dehydrogenase i n terms o f r e s t o r a t i o n o f enzymatic a c t i v i t y and the a b i l i t y o f the phospho l ip ids to complex w i th the enzyme. Optimal r e a c t i v a t i o n takes p lace when var ious l e c i t h i n s are m ic rod i sper sed w i th phosphat idy lethanolamine. S ince water s o l u b l e pho spha t i dy l cho l i ne s (PC6:0 and PC8:0) were ab le to r e a c t i v a t e the enzyme i t suggests t ha t a b i l a y e r s t r u c t u r e i s not necessary f o r a c t i v a t i o n however, as shown before (180) a b i l a y e r s t r u c t u r e i s necessary f o r enzyme s t a b i l i z a t i o n . S tud ies on the format ion of pho spho l i p i d - p r b t e i n complexes i n d i c a t e s tha t those phospho l ip ids r e a c t i v a t i n g the enzyme a l so form complexes and the amount o f r e a c t i v a t i o n i s p r opo r t i ona l to the phospho l i p id b ind ing a f f i n i t y . 33. Th i s was the f i r s t enzyme i n which the r o l e o f a s p e c i f i c l i p i d i n a p a r t i c u l a r s tep o f the r e a c t i o n mechanism was demonstrated (187). The enzyme when complexed w i th pho spha t i dy l cho l i ne o r a l i p i d mixture con-t a i n i n g pho spha t i dy l cho l i ne binds NADH w i th a = 6-16 uM w h i l e , w i t h no pho spha t i dy l cho l i ne present , no NADH i s bound. The b i nd ing o f NADH i s dependent on the fo rmat ion o f a p r o t e i n - 1 i p i d complex. (e) CTP:phosphocholi ne C y t i d y l y l t r a n s f e r a s e The synthes i s o f CDP-chol ine from CTP and phosphocholine by CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e (E.C. 2.7.7.15) was f i r s t de sc r ibed by Kennedy and Weiss (188). I t was a l so shown tha t CDP-chol ine was an i n t e r -mediate i n the b i o s yn the s i s o f pho spha t i d y l cho l i ne . Schneider (189) repor ted that the c y t i d y l y l t r a n s f e r a s e , i s o l a t e d from the s o l u b l e f r a c t i o n o f r a t l i v e r 0.9% NaCl s o l u t i o n homogenates, inc reased i n a c t i v i t y about 4-5 f o l d upon storage at 0°C f o r severa l days o r i n cuba t i on a t 38°C f o r 3 h. Sub-sequent ly F i scus and Schneider (190) found t ha t the enzyme cou ld be s t imu -l a t e d by the a d d i t i o n o f b o i l e d , p r e v i ou s l y aged p a r t i c u l a t e f r a c t i o n s o f r a t l i v e r . This s t i m u l a t i o n was a t t r i b u t e d to the phospho l ip ids present i n the p a r t i c u l a t e f r a c t i o n s . I t was i n t e r e s t i n g to f i n d t h a t l i p i d e x t r a c t s from f re sh r a t l i v e r cy to so l had- l i t t l e a c t i v a t i n g p o t e n t i a l unless they were o x i d i z e d by being exposed to a stream of a i r f o r 15 h (190). L i p i d ana l y s i s i n d i c a t e d a h igher p ropo r t i on o f l y s o l i p i d s i n the o x i d i z e d sample. This f i n d i n g r a i s e d the p o s s i b i l i t y o f some type o f c on t r o l mechanism v i a degraded pho spho l i p i d s . To t h i s po i n t a l l of the work had been done us ing crude homogenates or at best s e m i - p u r i f i e d enzyme. Choy et al. desc r ibed the f i r s t succes s fu l 34. p u r i f i c a t i o n o f the c y t i d y l y l t r a n s f e r a s e (191). Two forms o f the enzyme were i d e n t i f i e d i n the cy to so l as judged by gel f i l t r a t i o n chromatography, a low molecu la r weight form (L-form) and a high molecu la r weight aggregate (H-form). Fresh r a t l i v e r cy to so l conta ins predominately the L-form of the c y t i d y l y l t r a n s f e r a s e , which i s found to r equ i re l i p i d f o r a c t i v i t y . Upon aging the cy to so l a t 4°C f o r severa l days the L-form i s converted to the H-form, which has no dependence on l i p i d f o r enzyme a c t i v i t y . A study o f a l l the l i p i d s i n r a t l i v e r cy to so l revea led t ha t l y sophosphat idy lethano lamine (LPE) was the most potent a c t i v a t o r o f the enzyme although pho spha t i d y l -s e r i ne and phosphat idy l i n o s i t o l cou ld a c t i v a t e but to a l e s s e r ex tent (212). Some spec ies o f l y s opho spha t i d y l c ho l i n e (LPC) i n h i b i t e d the c y t i d y l y l t r a n s -fe rase by 80%. A l l of these e f f e c t s cou ld be demonstrated on the p u r i f i e d enzyme. The i nc rease i n enzyme a c t i v i t y by s t o r i n g the cy to so l a t 4°C f o r severa l days was c o r r e l a t e d to an i nc rease i n LPE concen t ra t i on as w e l l as a decrease i n phosphat idy lethanolamine concen t r a t i on . Th i s r e s u l t i m p l i c a t e d LPE as a po s s i b l e p h y s i o l o g i c a l a c t i v a t o r o f the r a t l i v e r c y t i d y l y l t rans ferase/! In a subsequent study Choy et al. i n v e s t i g a t e d the aggregat ion process t ha t accompanies the a c t i v a t i o n of the enzyme when s t o red at 4°C (192). An exhaust ive ana l y s i s o f the l i p i d s o f r a t l i v e r cy to so l revea led t ha t d i a c y l g l y c e r o l was the a c t i v e aggregat ing f a c t o r . The study o f the c y t i d y l y l t r a n s f e r a s e from r a t lung has some i n t e r e s t i n g d i f f e r e n c e s compared to the r a t l i v e r enzyme. As w i th the r a t l i v e r enzyme, the adu l t lung enzyme i s l o ca ted i n both the microsomal and c y t o s o l i c f r a c t i o n s when the t i s s u e i s homogenized i n i s o t o n i c s a l i n e . However, when f e t a l r a t s were s t u d i e d , the enzyme a c t i v i t y was predominately 35. l o ca ted i n the c y t o s o l i c f r a c t i o n . Fur ther i n v e s t i g a t i o n revea led t ha t two forms o f the enzyme were present i n the adu l t l ung , a s i t u a t i o n s i m i l a r to the r a t l i v e r system. The f e t a l form has a low molecu lar weight and requ i re s l i p i d f o r a c t i v i t y , w h i l e the adu l t form i s an aggregate o f the f e t a l form and has no l i p i d dependence. A s i g n i f i c a n t d i f f e r e n c e between these two systems however i s t ha t phosphat idy l g l y c e r o l i s both the a c t i v a t i n g and aggregat ing f a c t o r i n the r a t lung. I t was a l s o noted t ha t the convers ion o f the f e t a l form o f the c y t i d y l y l t r a n s f e r a s e to the a d u l t form p a r a l l e l e d an i nc rease i n phosphat idy l g l y c e r o l concen t ra t i on i n the l ung , i n d i c a t i n g the p o s s i b i l i t y t ha t pho spha t i dy l cho l i ne b i o s yn the s i s may be regu la ted by the concen t ra t i on o f phosphat idy l g l y c e r o l , E. Conclus ions The ma jo r i t y o f enzymes which are a f f e c t e d by the presence o f l i p i d are a s s oc i a ted i n some way w i t h membraneous o rgane l l e s such as m i t o -chondr ia (193), microsomes (194), o r b a c t e r i a l membranes (195). The a s s o c i a -t i o n o f these p ro te i n s w i t h membranes might l ead to the conc lu s i on tha t r e a c t i v a t i o n by the a d d i t i o n o f membrane l i p i d merely r e f l e c t s the need o f the enzyme to be i n a hydrophobic environment. T h i s . p o s s i b i l i t y i s f u r t h e r s ub s t an t i a t ed i n that most o f these p ro te in s respond i n a n o n - s p e c i f i c manner to the added l i p i d . However the quest ion t h a t remains i s , can phospho l ip ids perform a regu la to ry f u n c t i o n as we l l as forming the suppor t -ing s t r u c t u r e s o f the c e l l . From the s tud ie s on l a c t a t e dehydrogenase, pyruvate ox idase , malate ox idase , B-hydroxybutyrate dehydrogenase, and the CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e i t would appear they can and do 36. ac t as regu la to r s of enzyme a c t i v i t y although the s p e c i f i c i t y normal ly a s soc i a ted w i th enzyme r e g u l a t i o n appears to be much broader. The i n t e r -ac t i on s between l i p i d s and p ro te in s are very complex and w i t h f u r t h e r study o f more p u r i f i e d and c o n t r o l l e d systems and the improvement of e x i s t i n g techn iques , more knowledge about these i n t e r a c t i o n s w i l l be d i s cove red . 37. F. The S t r uc tu re o f Semi i k i Fores t V i rus The s t r u c t u r e s o f a l l the Group A Togaviruses are almost i f not e n t i r e l y i d e n t i c a l (224,225). These v i r u s e s , o f which Seml i k i Forest v i r u s and S indb i s v i r u s are the most s t u d i e d , c o n s i s t o f an i co sahedra l nuc leocaps id surrounded by a s phe r i c a l l i p i d envelope. Three g l ycop ro te in s denoted as E-|, and are s i t u a t e d i n the envelope and l i e i n c l o se p rox im i ty to the v i r u s nuc leocaps id (226). The s t r u c t u r e of Seml i k i Forest v i r u s i s shown d iag rammat i ca l l y i n F i g . 2. The v i r u s genome con s i s t s o f a s i n g l e s t r and o f 42S RNA. £ .| M W 52,000 C h o l e s t e r o l PL E_ M W 49,000 Figure 2. SEMLIKI FOREST VIRUS The s t r u c t u r e o f Seml i k i Fo res t v i r u s . 38. The s t r u c t u r a l p ro te in s of Seml i k i Fores t v i r u s have been re so l ved w i t h SDS po lyacry lamide gel e l e c t r o p h o r e s i s . O r i g i n a l l y s t ud i e s were performed us ing the gel system of Weber and Osborne (227). E-j and E 2 were not r e so l ved i n the s tud ie s o f Hay, Skehel and Burke (228), Kaa r i a inen et al. (229), and Acheson and Tamm (230). Nuc leocaps id p r o t e i n was c l e a r l y ev ident wh i l e no t r ace o f E^ was found on the gels and i t s ex i s t ence remained unknown. More r e c e n t l y the d i scont inuous b u f f e r SDS gel e l e c t r o p h o r e s i s systems o f N e v i l l e (231) and Laemmli (232) were a p p l i e d to p u r i f i e d v i r u s p reparat ions by Simons (233) and P f e f f e r k o r n (225). These r e s u l t s show the molecu la r weights o f E^, E^ and nuc leocaps id to be 52,000, 49,000 and 34,000 r e s p e c t i v e l y . The ex i s tence o f E^ i n Seml i k i Forest v i r u s was not ev ident u n t i l very r e c e n t l y (234). As y e t , t h i s p r o t e i n has not been demonstrated to be present i n S indb i s v i r u s . Garo f f and Simons showed t h a t although E^ cannot be detected on 7.5% and 10% SDS acry lamide gels by c l a s s i c a l 35 s t a i n i n g techn iques , the small p r o t e i n could be detected when [ S]Met l a b e l l e d SFV was app l i ed to 10% SDS gels - the gels were then s l i c e d and assayed f o r r a d i o a c t i v i t y . More conc l u s i ve evidence f o r the ex i s t ence o f E^ was presented when d e l i pi dated membrane p r o t e i n was e l u t e d from an SDS h y d r o x y l a p a t i t e column (234). E^, E^, E^, and nuc leocaps id p ro te in s appear i n equimolar amounts i n the mature v i r i o n and c o n s t i t u t e 35.7%, 35.7%, 4.9%, and 23.7% o f the t o t a l p r o t e i n r e s p e c t i v e l y . A l l three SFV membrane p ro te in s are g l y c o s y l a t e d . Residues o f N-acety lg lucosamine, mannose, ga l a c to se , fucose , and s i a l i c a c i d appear i n a l l th ree p ro te in s (234). The carbohydrate sequences f o r some o f the g l y cop ro te in s o f Seml ik i Forest v i r u s have r e c e n t l y been proposed (235). 39. Table 1 (12) Moles CHO Residue per Mole P r o t e i n P r o t e i n N -acety l glucosamine Mannose Galactose Fucose Si a l i c A c i d Tota l CHO. % by weight E l 7 5 3 1 2 7.5% E 2 8 12 3 1 4 11.5% E 3 9 4 4 2 3 45.1% The l i p i d s o f the v i r a l membrane c o n s i s t o f 32% neut ra l l i p i d s , 61% phospho l ip ids and 7% g l y c o l i p i d s (205). The neut ra l l i p i d f r a c t i o n of SFV con s i s t s almost e x c l u s i v e l y o f f r e e c h o l e s t e r o l w h i l e the main components o f the phospho l ip ids are sph ingomyel in , pho spha t i dy le thano la -mine, phosphat idy l cho l i ne and pho spha t i dy l s e r i ne . The g l y c o l i p i d f r a c t i o n conta ins almost e x c l u s i v e l y s i a l i c - l a c t o s y l ceramides. The d i s -t r i b u t i o n of the var ious l i p i d types i s shown as mole r a t i o s i n Table 2 (205). Table 2 L i p i d Class Composit ion o f SFV shown as Mole Rat io R e l a t i v e to Phospho l ip ids L i p i d C lass Mole Rat io Cho le s te ro l 0.99 G l y c o l i p i d s 0.08 Phospho l ip ids 1.00 PE 0.23 PC 0.33 PS 0.13 PI 0.02 Spingomyelin 0.20 40. I t i s b e l i e v e d tha t the l i p i d c l a s s compos i t ion resembles t h a t o f the host plasma membrane. Such a r e l a t i o n s h i p i s a l s o r e f l e c t e d i n the f a t t y a c i d composit ion of the phosphol ip ids i n the v i r u s and plasma membranes o f i n f e c t e d BHK-21 c e l l s (205). Recent s t ud ie s -by Richardson and Vance (236) have shown tha t Seml i k i Forest v i r u s obta ins i t s l i p i d envelope by "budding " from the plasma membrane o f the host c e l l . In summary, a s i n g l e p a r t i c l e o f Seml ik i Forest v i r u s conta ins the mo lecu la r composit ion l i s t e d i n Table 3. Table 3 (234,235) Number of D i f f e r e n t Molecules i n SFV Cons t i tuent Number o f Molecules Per V i r i o n RNA Nuc leocaps id Membrane p ro te in s Cho l e s te ro l P o l a r l i p i d s 15,000 16,000 200 550 Sphingomyelins Gang! ios ides PE PC PS PI 3,500 6,400 2,000 200 2,400 1,000 G. The ;Thesis* I n ve s t i g a t i on s a) Seml ik i Forest V i r u s : L i p i d Headgroup-Protein I n t e r a c t i o n s As has been mentioned seve ra l times i n the i n t r o d u c t i o n , up to the present time most s tud ie s of l i p i d - p r o t e i n i n t e r a c t i o n s have been done on model and r e c o n s t i t u t e d systems. Although these systems have been 41. extremely h e l p f u l i n e l u c i d a t i n g the var ious i n t e r a c t i o n s between l i p i d s and p r o t e i n s , they do have t h e i r l i m i t a t i o n s . To overcome the problems a s soc ia ted w i th the p repara t i on o f such r e c o n s t i t u t e d systems but more impo r t an t l y , to be con f i den t that the p ro te in s w i t h i n the membrane are exper ienc ing na tu ra l 1 i p i d - p r o t e i n i n t e r a c t i o n s , the only s o l u t i o n i s to study an i n t a c t b i o l o g i c a l membrane. But as has been mentioned, these i n t a c t systems tend to be extremely complex and as a r e s u l t , not much usefu l i n fo rmat ion has been obta ined. L i p i d enveloped v i r u s e s , on the o the r hand, are an a t t r a c t i v e a l t e r n a t i v e to the complex membranes o f most euka ryo t i c and p r o k a r y o t i c c e l l s . Some of the advantages of these v i ru ses have been o u t l i n e d p rev i ou s l y ( I n t r o d u c t i o n , sec. C). We chose to study the 1 i p i d - p r o t e i n i n t e r a c t i o n s w i t h i n the polarheadgroup o f Seml ik i Forest v i r u s us ing high r e s o l u t i o n proton NMR. The use o f proton NMR to study membrane systems has gene ra l l y not been very f r u i t f u l , due to the complex i ty o f the spectrum and the c l o se over -l app ing resonances. However i n s tudy ing changes i n the motion o f the polarheadgroups o f the cho l i ne con ta i n i ng l i p i d s , the ana l y s i s o f the spectrum i s s i m p l i f i e d , s i n ce the N ( C H 3 ) 3 resonance i s s u b s t a n t i a l l y separated from the remaining p o r t i o n o f the spectrum. I n i t i a l l y experiments were planned to i n v e s t i g a t e the e f f e c t of protease d i g e s t i o n o f the v i r a l g l y c o p r o t e i n ' s p i ke s ' on the motion o f the polarheadgroup reg ion of the membrane. I f there are i n t e r a c t i o n s between the g l y cop ro te i n ' sp i kes 1 ' and the l i p i d polarheadgroups, then protease d i g e s t i o n should remove the ' s p i ke s ' and t he r e f o r e the i n t e r -a c t i o n s . A decrease i n any i n t e r a c t i o n s should be r e f l e c t e d by changes i n the l i n e w i d t h o f the c ho l i n e resonance. Fur ther experiments were 42. planned i n v o l v i n g the s e l e c t i v e deu te ra t i on o f the cho l i ne methyl groups. By growing the host c e l l on medium supplemented w i th deuterated c h o l i n e we should be ab le to en r i ch the deuterium content o f the c ho l i n e con ta i n i n g l i p i d s . S ince the v i r u s obta ins i t s envelope by ' budd ing ' from the host c e l l plasma membrane (236) the v i r u s would a l s o become enr i ched w i th deuterium l a b e l e d cho l i ne con ta in i ng l i p i d s . By us ing deuterium NMR we would then be able to q u a n t i t a t i v e l y eva luate changes i n the motion o f the l i p i d polarheadgroups. However, due to the l ack o f i n c o r p o r a t i o n o f t r i - t r i d e u t e r o m e t h y l c ho l i ne i n t o the BHK-21 c e l l l i p i d s and the o v e r a l l low y i e l d s of Seml i k i Forest v i r u s , t h i s aspect of the p r o j e c t was d i s ca rded . Ins tead, the lack of -uptake o f t r i - t r i d e u t e r o m e t h y l c ho l i n e was s t ud i ed . b) CTP:phdsphocho1 ine C y t i d y l y l t r a n s f e r a s e : L i p i d - P r o t e i n I n t e r a c t i o n s The second po r t i on o f t h i s t h e s i s deals w i th the i n t e r a c t i o n s between the two l y s o l i p i d s , o l eoy l - LPE and o l e o y l - L P C , and the CTP:phospho-cho l i ne c y t i d y l y l t r a n s f e r a s e . Schneider (189) i n i t i a l l y repor ted that t h i s enzyme was a c t i v a t e d by s torage a t 4°C f o r severa l days and subse-quent ly F i scus and Schneider (190) showed t h a t t h i s a c t i v a t i o n was due to pho spho l i p i d . Choy et al. (212) found t ha t LPE was the a c t i v a t i n g l i p i d i n r a t l i v e r cy to so l s t o red at 4°C f o r severa l days. They a l so noted tha t pho spha t i dy l se r i ne and phosphat idy l i n o s i t o l cou ld a l s o a c t i v a t e the enzyme but not to the same ex ten t . Lysophqsphat idy l chol ine was found to s t r ong l y i n h i b i t the c y t i d y l y l t r a n s f e r a s e . The f i n d i n g tha t LPE a c t i v a t e d and LPC i n h i b i t e d the enzyme was i n t e r e s t i n g wi th regards to a system f o r s tudy ing l i p i d - p r o t e i n i n t e r a c t i o n s and p o s s i b l y as a mechanism f o r phospho l i p id r e g u l a t i o n . 43. I n i t i a l l y experiments were planned us ing phy s i ca l techniques such as e l e c t r o n s p i n resonance and e q u i l i b r i u m b ind ing s t u d i e s , however the amounts o f p r o t e i n needed f o r such s tud ie s cou ld not be ob ta ined . Therefore a k i n e t i c approach was adopted by s tudy ing the e f f e c t of these l y s o l i p i d s on the k i n e t i c parameters o f the c y t i d y l y l t r a n s f e r a s e . The l a s t pa r t o f the s tud ie s on the c y t i d y l y l t r a n s f e r a s e deal w i th the mechanism of the aggregat ion process o f t h i s enzyme when s t o red at 4°C f o r severa l days. 44. MATERIALS AND METHODS A. Chemicals and Isotopes Chemicals and i sotopes were obta ined from;the f o l l o w i n g s u p p l i e r s . Sigma Chemical Company, P.O. Box 14508, S a i n t L o u i s , M i s s o u r i , 63178 U.S.A. Cho l ine c h l o r i d e , c ho l i n e i o d i d e , phosphochol ine, T r i s , yea s t cho l i ne k inase, t he rmo l y s i n , t r y p s i n , chymotryps in, bovine serum albumin, and p h o s p h a t i d y l - D L - g l y c e r o l . Serdary Research L a b o r a t o r i e s , 1643 Kathyrn D r i ve , London, O n t a r i o , N6G 2R7. O leoy l - l y sophosphat idy le thano lamine and o l e o y l -l y s o p h o s p h a t i d y l c h o l i n e . F i s he r Chemical Company, 196 West Th i r d Avenue, Vancouver, Canada, V5Y 1E9 Phenol reagent ( F o l i n s , 2N), Reneicke s a l t , and P0P0P P-L B i ochemica l s , 1037 West McKinley Avenue, Milwaukee, Wicons in , 53205, U.S.A. C y t i d i n e t r i phosphate and c y t i d i n e d iphosphocho l ine. Swartz-Mann, 2646 South Sher idan Way, M i s s i s sauga , O n t a r i o , L5J 2M8 Enzyme grade sucrose. 45. J .T. Baker Chemical Company, c/o Canadian Laboratory Supp l i e s , 237-7080 R i ve r Road, Richmond, B.C., V6X 1X5 Potassium t a r t r a t e . Matheson, Coleman, and B e l l , c/o North American S c i e n t i f i c Chemical  L t d . , 268 East Second.AVenue. Vancouver, B.C., V5T 1B7 Aery1 amide. Bio-Rad L a b o r a t o r i e s , 2580 Wharton Glen Avenue., M i s s i s sauga , On ta r i o , L4X 2A9 M-N'-Methylene b i s - a c r y l a m i d e , c a t i o n exchange r e s i n AG50W-X8, and anion exchange r e s i n AG1-X10. B r i t i s h Drug House Chemicals , 15 West 6th Avenue, Vancouver, B.C., V5Y 1K2 Sodium dodecy l su lphate . Grand I s l and B i o l o g i c a l Company, 4534 M a n i l l a Road S.E., Ca l ga ry , A l b e r t a . Dulbecco ' s Mod i f i ed Eagles Medium and Medium 199. Flow L abo r a t o r i e s , 1625 Sismet Road, Un i t 10, M i s s i s sauga , On ta r i o , L4W 1V6 Feta l c a l f serum and BHK-21 c lone 13 c e l l s . Pharmacia, 2044 S t . Regis B l v d . , Do r va l , Q u e b e c , H9P 1H6 Sepharose 6B. New England Nuc lear , 2453 46th Avenue, Lach ine , Quebec, H8T 3C9 3 3 [Methy l - H ] - c ho l i n e and [ H ] - to luene . Amersham/Searle, 505 I roquois Road, O a k v i l l e , O n t a r i o , L6H 2R3 [ M e t h y l - 3 H ] - c h o l i n e , n - [ l , 2 - 3 H] -hexadecane , ACS, and 3 [ 1 - H]-ethanolamine. 46. Brinkman Instruments, 50 Galaxy B l v d . , Rexdale, On ta r i o , M9W 4Y5 Prespread s i l i c a gel (G-25) t h i n l a y e r chromatography p l a t e s , 20cm x 20cm x 0.25mm. M a l l i n c k r o d t Company, c/o North American S c i e n t i f i c Company (see above). Prespread s i l i c a gel (Chromar 7GF) t h i n l a y e r chromato-graphy p l a t e s , 20cm x 20cm x 0.25mm, bulk s i l i c a gel (chromar 7GF), and PP0. Merck, Sharpe, and Dohme, Mont rea l , Canada. Tr ideuteromethy l i o d i d e , Deuterium ox ide . Eastman Organic Chemicals , c/o North American S c i e n t i f i c Company (see above). Ethanolamine, monomethylethanolamine, and di methylethanolami ne. A l l o ther chemicals were o f reagent grade. B. General Methods ( i ) P r o t e i n Determinat ion P r o t e i n was determined by the method of Lowry et al. (196), w i t h bovine serum'albumin as a s tandard . -The assay o f p r o t e i n i n membrane f r a c t i o n s r equ i red a minor m o d i f i c a t i o n o f the general procedure. In t h i s i n s tance the samples, i n 0.66N NaOH, were immersed i n a b o i l i n g water bath f o r 5 min, coo led , and assayed i n the normal way. Reagent A: 100. ul 4% NaK T a r t r a t e 100 u l 2% CuS0 4 70 ml 2% K 2 C 0 3 Reagent B: 300 y l IN Fo l i n s reagent 47. General procedure: ( i ) Samples are made up to 200 u l w i t h water ( i i ) Three hundred u l IN NaOH i s added and the samples are immersed i n a b o i l i n g water bath f o r 5 min and then , coo led. ( i i i ) Add 3 ml Reagent A. ( i v ) Wait 15 min. (v) Add 300 u l Reagent B. ( v i ) Wait 45 min and read absorbance at 550 nm. A standard curve was prepared s imu l taneous ly and shown to be l i n e a r from 0-100 yg bovine serum albumin. ( i i ) Th in -Layer Chromatography TLC was performed on 20cm x 20cm x 0.25mm prespread (or homemade) s i l i c a gel p l a t e s . Rout ine ly used s o l v en t systems were: A. CHC1 3 - CH30H - H 20 (70/30/4 ; v/v/v) Th i s system i s used f o r the s epa ra t i on o f phospho l ip id s . B. CH30H - 0.6% NaCl - NH^OH (50/50/5 ; v/v/v) (197). This system i s u se fu l f o r s epa ra t i ng cho l i ne ( R f 0 -0 .13) , phosphochol ine (R. 0.25-0.38), and CDP-chol ine (R - 0.53-0.63). ( i i i ) Gas Chromatography Fat ty a c i d ana l y s i s o f pho spho l i p i d s . Phospho l i p id samples (l-5mg) i n chlioroform were d r i ed down under 48. a stream of N,,. Three ml of IN HC1 i n anhydrous methanol was added to each sample and N^ bubbled through the s o l u t i o n before t i g h t l y capping and i n cuba t i on a t 80°C ove rn i gh t . The samples were then d r i e d under a stream of N 2 and r e d i s s o l v e d i n 200 ul of hexane f o r d i r e c t a na l y s i s (198). The analyses were performed on a Hewlett -Packard high e f f i c i e n c y gas chromatograph (Model 7610A) operated a t 170°C w i t h a c a r r i e r gas (N 2 ) f low ra te of 50 ml/min. The column (180 cm) conta ined 12% (w/v) po l y -ethy lene g l y co l s u c c i na te supported on Gaschrom P (80-100 mesh) (App l i ed Sc ience L a b o r a t o r i e s ) . Fat ty a c i d standards were prepared from pure f a t t y a c i d spec ies and were e s t e r i f i e d as desc r ibed above. ( i v ) Pho spho l i p i d Phosphorous Ana l y s i s Tota l o rgan ic phospho l i p id phosphorous was measured by the method o f Raheja et al. (199). The l i p i d sample (1-10 ug l i p i d P) i n ch loroform was added to a g lass tube and d r i e d under a stream o f H^. Chloroform (0.4 ml) and chromogenic s o l u t i o n (0.1 ml) were added and the tubes p laced i n a b o i l i n g water bath f o r 90 s. A f t e r c oo l i n g to room temperature, an a d d i t i o n a l 5 ml o f ch loroform was added and the tubes gent ly shaken. The absorbance at 710 nrn was then measured. A standard curve (0-10 ug l i p i d P) was prepared us ing d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e as s tandard. The assay was found to be l i n e a r over the range of 1-10 ug l i p i d P (about 25-250 ug phospho l ip id ) . 49. (v) L i q u i d S c i n t i l l a t i o n Counting Rad ioac t i ve l i p i d samples were counted i n a to luene based s c i n -t i l l a n t con ta i n i ng PPO (4 g/1) and POPOP (50 mg/1). Rad ioac t i ve aqueous samples were counted i n ACS. L i q u i d s c i n t i l l a t i o n count ing was done i n an IS0CAP/300 counter (Nuclear Ch icago). Counting e f f i c i e n c y was determined by the ex te rna l 3 standards r a t i o o f chloroform-quenched standards con ta i n i ng e i t h e r [ H ] -3 to luene or [ H]-hexadecane. Standards i n the appropr ia te s c i n t i l l a t i o n f l u i d were counted w i t h each se t o f samples. ( v i ) SDS-Polyacrylamide Gel E l e c t r opho re s i s One dimensional SDS-polyacry lamide s l ab gel e l e c t r o p h o r e s i s was performed by the method o f Laemmli and Favre (200). The sepa ra t i on gel was 8.5cm h igh , 14cm wide, and 1.5mm t h i c k . A 1cm high s t a ck i n g gel which conta ined ten 8mm sample s l o t s was cas t on top o f the sepa ra t i on g e l . The sepa ra t i on gel c on s i s t ed o f a s p e c i f i e d percentage o f ac ry l amide , 0.375M T r i s -HC l (pH 8.8 ) , and 0.1% SDS. The s t a c k i n g gel c on s i s t ed of 4% (w/v) ac ry lamide, 0.08% (w/v) N-N 1 -methylene b i s - a c r y l a m i d e , 0.125M T r i s -HC l (pH 6 .8 ) , and 0.1% SDS. Buf fer s and s o l u t i o n s : 1. Lower gel b u f f e r (prepared 4X f i n a l concent ra t ion ) 1.5M T r i s - H C l pH 8.8 (36.4 g) 0.4% SDS (0.8 g) Make up to 200 ml w i th water. 2. Upper gel b u f f e r (prepared 4X f i n a l concent ra t i on ) 0.5M T r i s - H C l pH 6.8 (6.06 g) 0.4% SDS (0.4 g) Make up to 100 ml w i th water . 3. SDS sample b u f f e r 10% g l y c e r o l 2% 3-mercaptoethanol 1% SDS 0.0625M T r i s - H C l pH 6.8 4. Running b u f f e r (prepare 5X f i n a l concent ra t ion ) 0.125M T r i s (15.15 g) 0.96M g l y c i n e (72.0 g) 0.5% SDS (5.0 g) Make up to l £ w i t h water NOTE: pH 8.3 DO NOT ADJUST. 5. Gel s tock s o l u t i o n 30.0 g aery1 amide 0.8 g N,N'-methylene b i s - a c r y l am ide Make up to 100 ml w i th water . P repa ra t i on o f gels Lower gel ( sepa ra t i ng ge l ) (1) Lower gel b u f f e r 7.5 ml (2) water 22.5 ml - X ml (3) gel s tock X ml 51. (4) TEMED 30 y l (5) Ammonium persu lphate 150 u l (10%; w/v) (FRESHLY PREPARED) NOTE: X corresponds to the percentage gel d e s i r e d . Upper gel ( s t a c k i n g gel) 0 ) Upper gel b u f f e r 2.5 ml (2) water 6.5 ml (3) gel stock 1.0 ml (4) TEMED 20 y l (5) Ammonium persu lphate 30 y l (10%; w/v) The sepa ra t i ng gel was a l lowed to polymerize f o r 1-2 h wh i l e the s t a c k i n g gel r equ i red 0.5 h to po lymer ize . The r e s e r v o i r b u f f e r c on s i s t ed o f 0.25M T r i s , 0.192M g l y c i n e and 0.1% SDS (pH 8.3). The gel was e l e c t r o -phores i sed at a constant cu r rent o f 30 mA f o r 2.5 h. The gels were s t a i ned f o r p r o t e i n us ing a s o l u t i o n o f 0.1% (w/v) Coomassie Blue and 50% (w/v) t r i c h l o r o a c e t i c a c i d f o r a pe r i od of 1 h and desta ined overn ight w i th 7.5% (v/v) a c e t i c a c i d . (v i i ) Non-denaturing • Po lyacry lamide G e l , E lect-rophores is Non-denaturing po lyacry lamide gel e l e c t r o p h o r e s i s was performed as desc r ibed by Nelson et al. (201). P repa ra t i on o f the po lyacry lamide gels i n vo l ved mixing 11.1 ml- of 0.1M T r i s - g l y c i n e (pH 8.7) , 5 ml o f water ( f o r 5% po lyacry lamide g e l s ) , 5 ml o f s tock acry lamide s o l u t i o n (22.2 g aery1 amide 52. and 0.6 g N-N 1 -methylene b i s - a c r y l am ide i n a f i n a l volume o f 100 ml of w a t e r ) , 1.1 ml o f f r e s h l y prepared ammonium persu lphate (15 mg/ml) and 30 u l of TEMED. The mixture was poured i n t o g lass tubes (0.5 cm x 7 cm), covered w i t h a few m i l l i m e t e r s of water and a l lowed to polymer ize f o r 2 h. The upper and lower r e s e r v o i r b u f f e r con s i s t ed of 50 mM T r i s - g l y c i n e (pH 8.7). The gels were e lec t rophoresed a t a constant cu r rent o f 2 mA/gel. The samples, up to 75 u l , were added i n 10% (w/v) g l y c e r o l plus 0.001% bromophenol b lue as a dye marker. P r o t e i n was detected as desc r ibed above. 3 ( v i i i ) P repa ra t i on o f [ H]-phosphochol ine 3 [ H]-phosphochol ine was prepared accord ing to the method of Paddon and Vance (202). yea s t cho l i ne k inase [ 3 H ] - c h o l i n e + ATP > [ 3 H]-phosphochol ine + ADP Mg 3 B r i e f l y , 1 mCi o f [ H ] - c ho l i n e was incubated w i th 20 y l o f 100 mM ATP, 20 y l 1M Tr i s -HCL (pH 7 ) , 20 y l 0.1M MgC l 2 , and 150 y l d i a l y s a d yeas t c ho l i n e k inase (0.2 un i t s ) . The r e a c t i o n was terminated a f t e r 2 h a t 37°C by immersing the tube i n a b o i l i n g water bath f o r 2 min. The whole r e a c t i o n mixture was spot ted on a s i l i c a gel (G-25) TLC p l a t e over a length of 2 cm. The chromatogram was developed i n s o l ven t B. A marker l ane , con ta i n i ng a smal l amount o f the r e a c t i o n m ix tu re , was a l so spot ted to enable l o c a t i o n of the r a d i o a c t i v e product. Th i s lane was scraped at 1 cm i n t e r v a l s and the s i l i c a gel p laced i n t o s c i n t i l l a t i o n v i a l s con ta i n i ng 2 ml 0.1N NaOH plus 10 ml ACS. Before l i q u i d s c i n t i l l a t i o n 53. count ing 100 u l o f g l a c i a l a c e t i c a c i d was added. Upon l o c a t i n g the r a d i o -a c t i v e phosphocholine i n the marker l ane , the corresponding area i n the major lane was scraped and the s i l i c a gel washed w i th 8 ml of water to e l u t e the 3 [ H]-phosphochol ine. Th is sample was evaporated in vacuo and the res idue no rma l l y ' d i s s o l v ed i n 5 mM phosphocholine a t a r a d i o a c t i v e concent ra t i on of 100-150 uC i/ml . C. C e l l Cu l tu re The c e l l s used throughout t h i s work were Baby Hamster kidney-21 c e l l s , c lone 13 (BHK-21). The c e l l s were grown as monolayer cu l t u r e s a t 37°C i n a c o n t r o l l e d atmosphere of 5% C02/95% a i r and 100% humid i ty . They were grown on l a r ge (150mm x 15mm, Lux S c i e n t i f i c ) and medium (100mm x 15mm, 2 Falcon) p e t r i d ishes or r o l l e r b o t t l e s (725 cm ). The c e l l s were mainta ined on Dulbecco ' s Mod i f i ed Eagles Medium w i t h 5% f e t a l c a l f serum. By v i s u a l i n s p e c t i o n , c e l l s were used when they were near l y con f l uen t unless otherwise s t a t e d . D. Propagat ion o f Seml ik i Forest V i ru s Seml i k i Forest v i r u s o r i g i n a t e d as desc r ibed by Vance and Burke (203). For p ropagat ion , the v i ru s was added to nea r l y con f luent r o l l e r b o t t l e cu l t u r e s of c e l l s a t a m u l t i p l i c i t y of i n f e c t i o n o f about 0.05 plague forming u n i t s / c e l l ( p . f . u . ) i n 10 ml o f Medium 199 plus 2% f e t a l c a l f serum. A f t e r adsorpt ion f o r 1 h, an a d d i t i o n a l 40 ml o f the same medium was added. The cu l t u r e s were incubated at 37°C f o r 18-24 h ( r o l l i n g ra te about 1-2 r.p.m.) 54. at which time the medium was removed and s t o red i n 3 ml a l i q u o t s at -70°C u n t i l needed. I n f e c t i v i t y o f each p repa ra t i on was determined by the mono-l a y e r plaque assay (204). E. P repa ra t i on o f Large Amounts o f Pure Seml ik i Forest V i ru s The p repara t ion o f l a r ge amounts o f Seml i k i Fores t v i r u s r equ i red up to 200 r o l l e r b o t t l e s o f BHK-21 c e l l c u l t u r e s . The r egu l a r growth medium was removed and the c e l l s i n f e c t e d w i th v i r u s a t a m u l t i p l i c i t y of i n f e c t i o n o f 0.01-0.05 p . f . u . / c e l l i n 10 ml o f Medium 199 plus .2% f e t a l c a l f serum. A f t e r 1 h of adsorpt ion at 37°C an a d d i t i o n a l 30 ml of Medium 199 plus 2% f e t a l c a l f serum was added and the cu l t u r e s incubated overn ight (18-24 h ) . The medium was removed and cen t r i f u ged at 10,000 x £ f o r 10 min a t 4°C to remove dead c e l l s and deb r i s . S o l i d ammonium su lphate was added to the super-natant f r a c t i o n over a pe r i od o f 20 min to y i e l d 65% s a t u r a t i o n at 0°C (430.g ammonium sulphate/1 o f supernatant f r a c t i o n ) . The s o l u t i o n was s t i r r e d dur ing the a d d i t i o n and the pH mainta ined at 7 by dropwise a d d i t i o n of IN NaOH. The s o l u t i o n was a l lowed to s t i r f o r an a d d i t i o n a l 1 h a t 0°C and was then c e n t r i f u g e d at 10,000 x g f o r 20 min. The p r e c i p i t a t e was resuspended i n i c e - c o l d PBS and c e n t r i f u g e d at 10,000 x g f o r 10 min to remove any i n s o l u b l e d e b r i s . The supernatant f r a c t i o n was then l aye red onto e i t h e r 15-50% l i n e a r sucrose g rad ients or 15-50% l i n e a r potassium t a r t r a t e g rad ients and cen t r i f u ged at 65,000 x g (25,000 r.p.m. i n a SW27 r o to r ) f o r 3 h. The v i r u s band was removed e i t h e r by d r i pp i n g the tube from the bottom or using- a p i p e t t e to s e l e c t i v e l y remove the band from the g r ad i en t . To prepare the v i r u s f o r proton NMR i n v e s t i g a t i o n s the sample was cen t r i f u ged at 100,000 x g f o r 1 h to sediment the v i r u s . The p e l l e t was 55. resuspended i n 8 ml D 20/0.9% NaCl and r e c e n t r i f u g e d at 100,000 x g f o r 1 h The p e l l e t was again resuspended i n 8 ml D 20/0.9% NaCl and recen-t r i f u g e d before f i n a l l y resuspended i n 0.25 ml D 20/0.9% NaCl f o r the ac tua l NMR experiment. A l l NMR measurements were done at 30° C. F. Thermolys in D i ge s t i on o f Seml i k i Forest V i rus The v i ru s sample (45 mg p ro te in ) was resuspended i n 250 y l 0.1M T r i s - H C l (pH 7.5) , 250 y l water , and 500 yl the rmo ly s in (2.4 mg i n 0.05M T r i s - H C l , pH 7.5) and incubated a t 37°C f o r 1 h. At the end o f the i n c u -bat ion the t r e a t ed v i r u s was coo led to 4°C and r e p u r i f i e d on a 15-50% l i n e a r potassium t a r t r a t e g rad ien t . The sample was then prepared f o r the NMR experiment as desc r ibed above. In another experiment the t o t a l v i r u s sample (28 mg p ro te in ) was resuspended i n 6 ml PBS. One-ha l f was t r e a t e d w i t h 2.4 mg thermolys in plus 35 ymol C a C l 2 f o r 1 h at 37°C. The o ther h a l f o f the sample was t r e a t e d s i m i l a r l y except w i thout the a d d i t i o n o f the t he rmo l y s i n . At the end o f the i n cuba t i on both tubes were coo led i n an i c e bath and subsequently c e n t r i f u g e d at 100,000 x g f o r 1 h. The samples were not r e p u r i f i e d on potassium t a r t r a t e g rad ient s due to the low y i e l d o f v i r u s . The samples were prepared f o r the NMR experiment as desc r ibed above. Th is experiment r equ i red about 200 r o l l e r b o t t l e c u l t u r e s to ob ta in 28 mg of v i r a l p r o t e i n . G. P repa ra t i on o f a Mock Seml i k i Forest V i rus L i p i d Sample ( i ) P repa ra t i on o f the Sample A l i p i d sample c o n s i s t i n g o f the same l i p i d c l a s s compos i t ion as Seml i k i Fo res t v i r u s was prepared accord ing to the p ropor t ions i n d i c a t e d i n Table 4 (205). 56. Table 4 L i p i d C lass Composit ion o f Seml ik i Forest V i ru s L i p i d Class Mole Rat io Cho le s te ro l 0.99 G l y c o l i p i d s 0.08 Phospho l ip ids 1.00 PE 0.23 PC 0.33 PS 0.13 PI 0.02 Sphingomyelin 0.20 A l l l i p i d s except PI were shown to be ch romatograph ica l l y pure. The appropr i a te amount o f each l i p i d (except PI and g l y c o l i p i d ) , i n ch l o ro fo rm, was placed i n a f l a s k and the so l ven t was removed i n vacuo. ( i i ) P repa ra t i on o f Liposomes A sample o f Seml ik i Fores t v i r u s (28 mg p ro te i n ) was e x t r a c t ed by the procedure o f Folch et al. (207). The l i p i d e x t r a c t was t r a n s f e r r e d to a 5mm NMR tube and the so l ven t was removed under a stream o f N,,. Deuterium ox ide/ 0.9% NaCl (0.25 ml) was added and the sample was v i go rou s l y a g i t a t e d . ( i i i ) P repa ra t i on o f V e s i c l e s (a) Mock V i ru s L i p i d Sample A l i p i d mixture (30 mg) composed o f the same l i p i d c l a s s compos i t ion as Seml ik i Forest v i r u s (Table 4) except w i thout PI and g l y c o l i p i d was s o n i -cated 3 x 30s ( F i s he r SONIC Dismembrator, s e t t i n g 8) i n 2 ml lOOmM NaCl, 10'mM T r i s - a c e t a t e , pH 7.4 i n 100% D,,0. The sample was immediately t r a n s f e r r e d to a 5 mm NMR tube f o r the experiment. (b) Egg Pho spha t i dy l cho l i ne L i p i d Sample V e s i c l e s o f egg pho spha t i dy l cho l i ne (50 mg) were prepared as desc r ibed above. 57. H. P repa ra t i on of Deuterated Chol ines Three d i f f e r e n t l y l a b e l l e d cho l i nes were prepared by deutero-ni methy la t i on o f ethanolamine o r methylated d e r i v a t i v e s o f ethanolamine w i t h t r i deute romethy l i o d i d e . ( i ) Synthes i s o f T r i - t r i d e u t e r o m e t h y l c h o l i n e Ethanolamine (18 mmol) was d i s s o l v e d i n 5 ml i c e - c o l d e thano l . CD^I (55 rnmol) was added s l ow ly to the r ea c t i on f l a s k w i th constant s t i r r i n g . The f l a s k was kept c o l d i n an i c e - e thano l bath and i n the dark by cover ing w i th t i n f o i l . The r e a c t i o n was a l lowed to proceed over 3 h wh i l e s l ow l y warming up to room temperature. Some product was found to p r e c i p i t a t e at t h i s t ime. The r e a c t i o n mixture was then kept at 4°C f o r seve ra l days. Afterwards the s o l v e n t was evaporated in vacuo to y i e l d a ye l l ow o i l . Some product was ab le to be c r y s t a l l i z e d a t t h i s po in t (725 mg). The remaining po r t i on o f the mixture was d i s s o l v ed i n water and passed through a Dowex AG50-X8 c a t i on exchange r e s i n (3cm x 25cm column) and e l u t ed w i th a 800 ml 0-4N HC1 g r ad i en t . The cho l i n e con ta i n i n g f r a c -t i on s were detected by assay ing w i th rene icke s a l t (Ammonium t e t r a t h i o -cyanodiammonchromate), a reagent s p e c i f i c f o r de tec t i n g t e r t i a r y amino compounds. The f r a c t i o n s were pooled and the so l ven t removed in vacuo. Tota l y i e l d o f t r i - t r i d e u t e r o m e t h y l c h o l i n e c h l o r i d e was 1.5 g (10.1 mmol). ( i i ) Synthes i s o f D i - t r i d e u t e r o m e t h y l c h o l i n e Monomethylethanolamine (33 mmol) was d i s s o l v ed i n 5 ml i c e - c o l d e thano l . Tr ideuteromethy l i od i de (69 mmol) was added s l ow ly to the r ea c t i on 58. f l a s k with_ constant, s t i r r i n g . - Tiie vessel was kept, i n an i c e - e thano l bath and i n the dark. The r e a c t i o n was a l lowed to warm up to room temperature over 3 h and was then cooled to -20°C f o r ove rn i gh t . The product c r y -s t a l l i z e d from s o l u t i o n and was f i l t e r e d . To ta l y i e l d o f d i - t r i d e u t e r o -methy l cho l i ne i o d i d e was 2.6 g (11 mmol). ( i i i ) Synthes i s o f Mono- t r ideuteromethy lcho l ine Dimethylethanolamine (63 mmol) was d i s s o l v e d i n 5 ml o f i c e -co l d e t hano l . Tr ideuteromethy l i od i de (69 mmol) was added s l ow l y to the f l a s k w i th constant s t i r r i n g . The r e a c t i o n was again kept c o l d and i n the dark. The product p r e c i p i t a t e d almost immediately. The f l a s k was kept a t -20°C overn ight at which time the product was f i l t e r e d . To ta l y i e l d o f mono-tr/ideuteromethyl chol i ne i o d i d e was 13.6 g (58 mmol). ( i v ) I n co rpo ra t i on o f L a b e l l e d Chol ines i n t o BHK-21 C e l l s Baby Hamster Kidney-21 c e l l s have been shown to i n co rpo ra te exogenous cho l i ne which has been added to the c u l t u r e medium (203). There-fore by supplementing the c u l t u r e medium w i th these l a b e l l e d (deuterated) spec ies o f c h o l i n e , one would expect to observe i n c o r p o r a t i o n i n t o the cho l i ne con ta in i ng l i p i d s . The a b i l i t y of each o f the deuterated cho l i nes to be i nco rpo ra ted i n t o the c e l l u l a r c h o l i n e con ta i n i n g l i p i d s was d e t e r -mined by a long term l a b e l l i n g experiment. The c e l l s were grown on Dulbecco ' s Mod i f i ed Eagles Medium supplemented wi th the deuterated cho l i ne s plus [ H ] - c h o l i n e . By knowing 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 c ho l i n e i n the medium and by determin ing 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 c h o l i n e 59. c on t a i n i n g l i p i d s i t should be p o s s i b l e to determine the a b i l i t y o f the deuterated cho l i ne s to be i n co rpo ra ted . In d e t a i l , th ree l a r ge d i shes o f BHK-21 c e l l s were grown on Du lbecco ' s Mod i f i ed Eagles Medium supplemented w i th 80 ug/ml o f t r i - t r i - , 3 deuteromethy lcho l ine c h l o r i d e plus 0.167 uCi/ml [ H ] - c h o l i n e . Another three p la te s were grown on medium supplemented w i th 130 ug/ml o f protonated 3 c h o l i n e i od ide plus 0.167 uCi/ml [ H ] - c h o l i n e . F i n a l l y three dishes o f 3 c e l l s were grown on medium supplemented w i th on ly 0.167 uCi/ml [ H ] - c h o l i n e . (Note: Du lbecco ' s Mod i f i ed Eagles Medium has 4 ug/ml o f protonated c h o l i n e c h l o r i d e ) . A f t e r the c e l l s reached conf luence the c e l l s were harvested and the l i p i d s were ex t r ac ted (207) and p u r i f i e d by t h i n l a y e r chromatography us ing so l vent A. The var ious l i p i d c l a s se s were v i s u a l i z e d us ing Ir> vapours. The pho spha t i dy l cho l i ne band was i d e n t i f i e d and scraped from the p l a t e . The l i p i d was e l u ted w i th 10 ml CHC1 3 - CH30H - NH^OH (1/1/0.1 ; v/v/v). Total l i p i d phosphorous was determined by the method o f Rajeha et-al. as desc r ibed e a r l i e r , and the r a d i o a c t i v e content measured by l i q u i d s c i n t i l l a t i o n count ing . 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 pho spha t i d y l -c h o l i n e was then c a l c u l a t e d . The same experiment was performed when s tudy ing the i n co rpo ra^ t i o n o f mono- t r ideuteromethy lcho l ine and d i ^ t r i d e u t e r o m e t h y l c h o l i n e . 3 (v) P repara t ion o f t r i - t r i deute romethyTrp l ,2- H l -choTine fo'dide 3 [ 1 , 2 - H ] - e than - l - o l - am ine (2 mCi) was d i s s o l v ed i n 0.5 ml ethanol at 0°C. Tr ideuteromethy l i od ide (6.9 mmol) was added to the r e a c t i o n 60. vessel w i th constant a g i t a t i o n . The r e a c t i o n mixture was kept at room temperature f o r 3 h at which time the so l ven t was removed by evaporat ion under a stream of U^. The mixture was d i s s o l v e d i n a smal l volume o f ethanol and spot ted on a s i l i c a gel G-25 t h i n l a y e r chromatography p l a t e and developed us ing s o l ven t B. A marker lane was a l so spot ted (2 y l o f the r ea c t i on mixture) to l o ca te the product of the r e a c t i o n . The t o t a l y i e l d 3 of t r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - H ] -eho l i ne i od i de was 10 y C i . (.vi) Chol ine T r a n s p o r t Large dishes o f c e l l s were used f o r the t r an spo r t s t u d i e s . The c e l l s 3 were washed 3 times w i th Medium 199. T r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - H ] - cho l i ne (2.86 yC i ) was added i n 6 ml Medium 199. The c e l l s were incubated a t 37°C and at 37, 60, and 126 min, 2 x 50 y l a l i q u o t s o f medium were removed and the r a d i o a c t i v i t y determined by l i q u i d s c i n t i l l a t i o n count ing . At these same time p o i n t s , c e l l s were harvested to determine the amount o f t r an spo r t i n t o BHK-21 c e l l s . The medium was removed and the c e l l s were washed w i th 5 x 10 ml i c e - c o l d PBS. The c e l l s were scraped o f f the d i sh w i th a rubber policeman and e x t r a c t e d accord ing to the procedure o f Fo lch et al. (207). The t o t a l aqueous and o rgan ic s o l u b l e r a d i o a c t i v i t y was determined by l i q u i d s c i n t i l l a t i o n count ing . 3 The same experiment was repeated us ing [ H ] - c ho l i n e as a c o n t r o l . I. Enzyme Assays ( i ) Chol ine Kinase Cho l ine k inase was assayed e s s e n t i a l l y by the method o f Weinhold and Rethy (208). Cytosol was prepared from BHK-21 c e l l s and added to a 61. r e a c t i o n mixture con ta in i ng 100 umol T r i s - H C l (pH 8 ) , 7.5 umol MgCl^, 7.5 umol [ y - 3 2 P ] - A T P (0.03 C i /mo l ) , and 0.25 umol c h o l i n e , us ing e i t h e r protonated cho l i n e o r the deuterated spec ies ( t r i - t r i d e u t e r o m e t h y l c h o l i n e or mono- t r ideuteromethy lcho l ine ) i n a f i n a l volume o f 1 m l . A f t e r i n cuba t i on at 37°C f o r 20 min, the r e a c t i o n was terminated by immersing the tubes i n b o i l i n g water f o r 2 min. Each r e a c t i o n mixture was d ra ined i n t o a 0.5cm x 2cm column o f a c t i v a t e d c h a r c o a l / c e l i t e (1/1). The assay tubes were r i n sed w i t h 2 x 1 ml water and t h i s r i n s e was a l s o d ra ined i n t o the column. Water (5 ml) was fo rced through the column to e l u t e the product, o f the r e a c t i o n . R a d i o a c t i v i t y i n the product was determined by l i q u i d s c i n t i l -l a t i o n spectrometry. ( i i ) CTP:phosphocholine C y t i d y l y l t r a n s f e r a s e The c y t i d y l y l t r a n s f e r a s e was assayed.by a procedure s i m i l a r to tha t o f An se l ! and Chojnacki (209) except t h a t t h i n l a y e r chromatography on s i l i c a gel was used to separate CDP-chol ine from phosphochol ine, i n s t ead o f charcoal ad so rp t i on . Rat l i v e r cy to so l o r p u r i f i e d c y t i d y l y l t r a n s f e r a s e was added to a r e a c t i o n mixture con ta i n i ng 10 y l 1M T r i s - s u c c i n a t e (pH 7) and 0.1M MgC l 2 , 20 y l lOmM CTP, and 20 y l 5mM [ 3 H]-phosphocho l ine (100-150 yCi/ml) i n a f i n a l volume o f 100 y l . The mixture was incubated at 37°C f o r 15 min and the r e a c t i o n stopped by immersing the tubes i n t o a b o i l i n g water bath f o r 2 min. U sua l l y 60 y l o f the r e a c t i o n mixture was spot ted onto a s i l i c a gel t h i n l a y e r p l a t e along w i th 0.5 mg CDP-chol ine as c a r r i e r . The TLC p l a t e was developed i n s o l ven t B. The product o f the r e a c t i o n was v i s u a l i z e d under UV l i g h t and the band scraped from the p l a t e i n t o a s c i n t i l l a t i o n v i a l con ta i n i ng 2 ml 0.1N NaOH and 10 ml ACS. G l a c i a l 62. a c e t i c a c i d (100 y l ) was added to reduce chemiluminescence before l i q u i d s c i n t i l l a t i o n count ing . The assay was"found to be l i n e a r up to 15 min a t 37°C and up ' t o 3.5,mg cy- toso l i c p r o t e i n ( F i g . 3 ) . J . P u r i f i c a t i o n of the CTP:phosphocholine C y t i d y l y l t r a n s f e r a s e The p u r i f i c a t i o n o f the c y t i d y l y l t r a n s f e r a s e i s desc r ibed by Choy et al. (191). B r i e f l y , f re sh r a t l i v e r obta ined from 150-200 g male o r female Wi s ta r r a t s , was homogenized i n i s o t o n i c s a l i n e (20% w/v) and c e n t r i f u g e d a t 100,000 x g f o r 1 h. The supernatant f r a c t i o n was aged at 4°C f o r 5 days and was then c e n t r i f u g e d at 10,000 x g to remove any d e b r i s . The supernatant f r a c t i o n was adjus ted to 25% s a t u r a t i o n w i th ammonium su lphate (pH 7) and, l e f t a t 4°C f o r 1 h. The s o l u t i o n was c e n t r i f u g e d a t 10,000 x g f o r 10 min and the p e l l e t was resuspended i n B u f f e r A (20mM T r i s - H C l , pH 7 and 0.1M NaCl) a t 1/10 the i n i t i a l volume. A 4 ml a l i q u o t was a p p l i e d to a Sepharose 6B column (2.5cm x 80cm) i ,equ i l ib rated w i th B u f f e r A. The m a j o r i t y o f the c y t i d y l y l t r a n s f e r a s e a c t i v i t y was l o ca ted i n the vo id volume of the column ( F i g . 21 ) . F rac t i on s con ta i n i n g c y t i d y l y l t r a n s f e r a s e a c t i v i t y were pooled and SDS was added to a f i n a l concen t ra t i on of 0.05%. The sample was kept a t 5°C f o r 1 h and then concentrated by u l t r a f i l t r a t i o n , us ing an XM100A Amicon f i l t e r , to a volume o f 4 m l . Th i s sample was then a p p l i e d to a second Sepharose 6B column (2.5cm x 80cm) e q u i l i b r a t e d w i th B u f f e r A plus 0.001% SDS. Upon assay ing the column f r a c t i o n s f o r c y t i d y l y l t r a n s f e r a s e a c t i v i t y , the enzyme a c t i v i t y was found to s p l i t i n t o two peaks, some remaining i n the vo id volume o f the column and a new f r a c t i o n l o ca ted i n the i nc luded volume o f the column ( F i g . 22 ) . Th is second peak o f a c t i v i t y , which To face page 63. F igure 3. CT a c t i v i t y vs. t ime and p r o t e i n . The enzyme was assayed i n 10 y l 1M T r i s - s u c c i n a t e (pH 7) and 0.1M MgC l 2 , 20 y l 10 mM CTP, and 20 y l 5mM [ 3 H ] -phosphocholine (100-150 yCi/ml) i n a f i n a l volume o f 100 y l . The upper graph shows the e f f e c t o f i n cuba t i on time at 37°C on enzyme a c t i v i t y . The assay was l i n e a r to approx imately 15 min. The lower graph shows the e f f e c t o f c y t o s o l i c p r o t e i n concen t r a t i on on CT a c t i v i t y . The assays were performed a t 37°C f o r 15 min w i th vary ing amounts o f p r o t e i n . The assay was l i n e a r up to ~ 3.5 mg c y t o s o l i c p r o t e i n . 63. Protein (pg • 10~3) Figure 3. 64. requ i res l i p i d f o r a c t i v i t y , was pooled and concentrated by u l t r a f i l t r a -t i o n to a volume o f about 1 ml f o r use i n subsequent exper iments. K. Assay o f the C y t i d y l y l t r a n s f e r a s e from Non-Denaturing Po lyacry lamide Gels Non-denaturing 5% po lyacry lamide gels were prepared as desc r ibed i n s e c t i o n B ( v i i ) . The c y t i d y l y l t r a n s f e r a s e was i n i t i a l l y too l a r ge to en te r the 5% po lyacry lamide g e l . Therefore SDS ( f i n a l concen t ra t i on 0.05%) was added and the sample was e l e c t r o p h o r e s i s e d f o r 2.5 h a t 2 mA/gel. Dup l i ca te gels were e l e c t r o p h o r e s i s e d w i th one being s t a i ned f o r p r o t e i n , and the. other being aassayed- f o r c y t i d y l y l t r a n s f e r a s e a c t i v i t y . Only one band was observed i n the gel although some mate r i a l s t a i n e d f o r p r o t e i n a t the top o f the g e l . Three sec t i on s were cut from the gel corresponding to the areas i n d i c a t e d i n F i g . 4 . Each s e c t i o n was crushed and assayed f o r c y t i d y l y l t r a n s f e r a s e a c t i v i t y . The assay conta ined the f o l l o w i n g components: ( i ) 20 y l T r i s - s u c c i n a t e (pH 7) ( i i ) 40 u l lOmM CTP ( i i i ) 40 u l 5mM [ 3 H]-phosphocho l ine (150 uCi/ml) and ( i v ) 100 y l water The r e a c t i o n mixture was incubated a t 37°C f o r 1 h a t which time the r e a c t i o n was terminated by immersing the tubes i n a b o i l i n g water bath f o r 2 min. A s e r i e s o f a l i q u o t s (5 x 20 ul) were spot ted on a s i l i c a gel G-25 TLC p l a t e along w i t h 0.5 mg CDP-chol ine as c a r r i e r . The p l a t e was developed i n s o l v en t B and the r a d i o a c t i v i t y i n CDP-chol ine determined as desc r ibed above. 65. 0 TOP MID BAND Figure 4. Schematic drawing o f a non-denatur ing po lyacry lamide gel o f the CT. 66. RESULTS A. L i p i d - P r o t e i n I n t e r a c t i on s i n the P o l a r Headgroup Region o f Seml i k i  Forest V i rus The study of 1 i p i d - p r o t e i n i n t e r a c t i o n s has mainly used model membrane p repa ra t i on s . However severa l groups have attempted to i n v e s t i g a t e the e f f e c t of p r o t e i n on the motion o f the phospho l i p id polarheadgroup i n i n t a c t b i o l o g i c a l membrane systems (119,139-141). Severa l s tud ie s on V e s i c u l a r S t o m a t i t i s v i r u s by two groups are of p a r t i c u l a r i n t e r e s t s i n ce they are s tudy ing a r e l a t i v e l y s imple and uniform membrane p repara t i on (139,140,141). As mentioned p rev i ou s l y i n the i n t r o d u c t i o n , l i p i d enveloped v i ru ses o f f e r a number o f a t t r a c t i v e featu res over whole c e l l membranes. S t o f f e l et al. (139,140) and Moore et al. (141) i n s tudy ing the e f f e c t of t r y p s i n d i g e s t i o n o f the v i r u s on the motion of the polarheadgroups of the l i p i d s o f V e s i c u l a r S t o m a t i t i s v i r u s , a r r i v e d a t opposing conc lus ions as to the e f f e c t o f protease d i g e s t i o n . This may be r e l a t e d to problems a s soc i a ted w i t h the i n t e r p r e t a t i o n of s p i n - l a t t i c e r e l a x a t i o n da ta . We have chosen to examine the e f f e c t of thermo ly s in removal of the v i r a l g l y cop ro te i n s on the headgroup motion of the membrane l i p i d s o f Seml i k i Forest v i r u s us ing high r e s o l u t i o n proton magnetic resonance. The in fo rma-t i o n obta ined about the motion of the headgroups of c ho l i n e con ta i n i n g l i p i d s w i th t h i s technique i s l e s s ambiguous than t h a t obta ined from s p i n -l a t t i c e r e l a x a t i o n s t u d i e s . 67. (a) Preparation of Semliki Forest Virus Semliki Forest virus was prepared by infecting monolayer cultures of BHK-21 cells and harvested as described previously (Materials and Methods, see E). Briefly, the virus was precipitated from Medium 199 plus 2% fetal calf serum by addition of ammonium sulphate and subsequently purified on either 15-50% linear potassium tartrate gradients or 15-50% linear sucrose gradients (Fig. 5). The purity of the preparation was determined by SDS-polyacrylamide slab gel electrophoresis in 9% polyacryla-mide (Fig. 6). In all cases the virus was shown to be pure with the gel pattern exhibiting only two bands, a higher molecular weight band corre-sponding to the E-jE2 combination, and a lower molecular weight band corre-sponding to the nucleocapsid protein (NC). As mentioned before, the third membrane protein of Semliki Forest virus, E^. is not detected in this system due to its low molecular weight and high carbohydrate composition. The integrity of the virus particules was determined by negative staining transmission electron microscopy using phosphotungstic acid. The virus preparations consisted of intact, uniformly shaped particles o of about 650 A diameter (Fig. 7). (b) Proteolytic Digestion of Semliki Forest Virus Initially trypsin and chymotrypsin were chosen to remove the glycoprotein "spikes" of Semliki Forest virus. It was felt that since these enzymes were readily available in pure form, the use of other proteases such as pronase and thermolysin would be avoided since they are relatively impure and non-specific. However the use of trypsin or chymotrypsin To face page 68. F igure 5. P r o f i l e of a 15-50% l i n e a r sucrose g rad ient c on ta i n i n g Seml i k i Fores t v i r u s . Seml ik i Fores t v i r u s , grown i n BHK-21 c e l l s , was p r e c i p i t a t e d from Medium 199 plus 2% f e t a l c a l f serum using ammonium su lphate (65% s a t u r a t i o n a t 4°C) as desc r ibed i n Ma te r i a l s and Methods, sec. E. The resuspended p r e c i p i t a t e , i n PBS, was l aye red onto a 15-50% l i n e a r sucrose g rad ient and c e n t r i f u g e d at 65,000 x £ f o r 3 h. The g rad ient was dr ipped from the bottom of the tube and 0.5 ml f r a c t i o n s were c o l l e c t e d . The p u r i f i e d v i r u s was detected by i t s absorbance at 260 nm. 3h 0 4 8 12 16 20 24 ' 74 78 F R A C T I O N N O . Figure 5. CP) CO To face page 69. Figure 6. SDS-polyacrylamide gel electrophoresis of pu r i f i ed Semliki Forest v i rus. Semliki Forest virus was pu r i f i ed as outl ined in Materials and Methods and so l ub i l i zed in sample buffer which contained 0.5% 3-mercaptoethanol and 0.5% SDS, f i n a l concentration. The virus proteins (20 ug protein) were separated on a 9% polyacrylamide slab ge l . Proteins were stained with Coomassie Blue. Envelope proteins are designated E - ^ since they resolve poorly in th i s system, and nucleocapsid protein is denoted NC. E^ was not detected on these gels. F igure 6. To face page 70. F igure 7. Negat ive s t a i n i n g t ransmi s s i on e l e c t r o n micrograph o f p u r i f i e d Seml i k i Forest v i r u s . (89,367 x magn i f i c a t i on ) 70. Figure 7. 71. r e s u l t e d i n no d i g e s t i o n o f the v i r a l g l y c o p r o t e i n ' s p i k e s ' as judged by SDS-polyacrylamide s l ab gel e l e c t r o p h o r e s i s ( F i g . 8) or negat ive s t a i n i n g t ran smi s s i on e l e c t r o n microscopy ( F i g . 9 ) . Th is r e s u l t nece s s i t a t ed the use of thermolys in which has a l ready been shown to d i ge s t the v i r a l g l y c o -p ro te in s o f Seml i k i Forest v i r u s (210, 211). A f t e r i n cuba t i on o f the v i r u s w i th thermolys in and p a r t i c l e r e p u r i f i c a t i o n (Ma te r i a l s and Methods, see F ) , a small a l i q u o t (50 ug protein)was removed and sub jec ted to SDS-polyacrylamide s l ab gel e l e c t r o -pho re s i s . From the gel pa t te rn i t was demonstrated t ha t the p r o t e i n band corresponding to the E - ^ combination had been complete ly removed ( F i g . 10). (c) High Reso lu t i on Proton NMR o f I n t ac t and Thermolys in Treated Seml i k i Forest V i rus The e l e c t r o n micrographs o f Seml i k i Forest v i r u s show p ro t rud ing ma te r i a l from the envelope encapsu la t ing the core o f the v i r u s . We were i n t e r e s t e d to see what e f f e c t , i f any, the removal o f these ' s p i k e s ' would have on the motion o f the headgroup of the cho l i ne con ta i n i ng l i p i d s . Although the proton NMR spectrum o f the v i r u s i s r a t he r complex, the resonance o f the N- tCH^g group o f the cho l i ne con ta i n i n g phospho l ip ids e x h i b i t s a d i s t i n c t l i n e . I f there were an a l t e r a t i o n o f the membrane so that i n t e r a c t i o n s between membrane p r o t e i n and l i p i d were d i s t u rbed ( i f any i n t e r a c t i o n s e x i s t a t a l l ) , we might detect these changes by observ ing v a r i a t i o n s i n the l i n e w i d t h o f the c ho l i n e methyl resonance. The proton NMR spectrum of i n t a c t Seml i k i Forest v i r u s i s shown i n F i g . 11. The cho l i ne methyl resonance i s found to be d i s t i n c t from To face page 72. F igure 8. SDS-polyacrylamide s l ab gel e l e c t r o p h o r e s i s o f t r y p s i n -t r e a t e d Seml i k i Forest V i r u s . Seml i k i Forest v i r u s (70 yg p ro te in ) was incubated w i th 5 yg t r y p s i n i n 55 y l TNE b u f f e r a t 37°C f o r 20 min. T r yp s i n i n h i b i t o r (20 yg) was added to terminate the r e a c t i o n . The sample was prepared f o r SDS-polyacrylamide gel e l e c t r o -phores is i n a 7.5% po lyacry lamide s l ab gel as desc r ibed i n the M a t e r i a l s and Methods. E 1 E 2 NC dye To face page 73. F igure 9. Negat ive s t a i n i n g t ran smi s s i on e l e c t r o n microscopy of t r y p s i n -t r ea ted Seml ik i Forest v i r u s . (89,367 x m a g n i f i c a t i o n ) . The v i ru s was t r e a t ed as noted i n F i g . 8l Figure 9. To face page 74. F igure 10. SDS-polyacrylamide s l ab gel e l e c t r o p h o r e s i s o f t he rmo l y s i n -t r e a t e d Seml ik i Forest v i r u s . Seml i k i Forest v i r u s (45 mg p ro te i n ) was resuspended i n 250 y l water , 250 y l 0.1M Tr i s -HCL (pH 7.5) , and 500 y l thermolys in (2.4 mg i n 0.05M T r i s - H C l , pH 7.5) and incubated a t 37°C f o r 1 h. A t the end o f the i n cuba t i on the sample was coo led to 0°C and l aye red onto a 15-50% l i n e a r potassium t a r t r a t e g rad ien t . The g rad ient was c e n t r i f u g e d at 65,000 x £ f o r 3 h a t 4°C and the t he rmo l y s i n - t r e a t ed v i r u s band i s o l a t e d . An a l i q u o t was removed (50 yg p ro te i n ) and s o l u b i l i z e d i n sample b u f f e r con ta i n i ng 0.5% 3-mercaptoethanol and 0.5% SDS, f i n a l c oncen t r a t i on . The sample was then a p p l i e d to the top o f a 9% SDS-polyacrylamide s l ab gel and e l e c t r opho re s i s ed a t 30 mA f o r 2.5 h. The p ro te in s were detected by s t a i n i n g w i t h Coomassie B lue and de s t a i n i n g overn ight w i th 7.5% a c e t i c a c i d . NC dye To face page 75. F igure 11. High r e s o l u t i o n proton NMR spectrum of i n t a c t Seml i k i Forest v i rus . Seml ik i Fo res t v i r u s (45 mg) was prepared f o r the NMR e x p e r i -ment as desc r ibed i n Ma te r i a l s and Methods, sec. E. The v i r u s was resuspended i n 0.25 ml D 20/0.9% NaCl . The spectrum was recorded on a N i c o l e t TT.23 100 MHz NMR spectrometer us ing a 2 KHz s p e c t r a l width and a 2 s a c q u i s i t i o n t ime. The spectrum requ i red 1000 scans. 75: 76. the rather broad resonances o f the f a t ty acyl terminal methyl groups and the methylene protons. A f t e r thermolysin d i g e s t i o n of the v i rus and r e p u r i f i -ca t ion of the p a r t i c l e s , the NMR spectrum exh ib i ted a narrow resonance superimposed on a broader resonance, both corresponding to the absorpt ion frequency of the chol ine N-^Hg)^ group ( F i g . 12). The l inewidth of a p a r t i c u l a r resonance i s d i r e c t l y re la ted to the motion experienced by the nucleus as descr ibed in equation 1. where t i s the ro ta t iona l c o r r e l a t i o n time which can be obtained as a s o l u t i o n of the ro ta t iona l d i f f u s i o n equation (239) as where a is the radius of the v e s i c l e and D d l - f f i s the d i f f u s i o n ra te . D d l - f f can be d iv ided in to two components, ro ta t iona l d i f f u s i o n due to Brownian tumbling (D ) which i s given by the S t o k e s - E i n s t e i n r e l a t i o n I O C lc '2 (1) (2) as D r= kT/8Traii (where n i s the v i s c o s i t y of the medium) and l a t e r a l d i f f u s i o n D. (240). Therefore equation 2 becomes, (3) It can be seen that the ro ta t iona l d i f f u s i o n component (D r ) va r ies as the cube of the r a d i u s , whereas the l a t e r a l d i f f u s i o n component (D.) va r ies 77. as the square of the rad iu s . The l i n e w i d t h of a resonance from a nucleus which i s unable to r e o r i e n t r a p i d l y w i l l be much broader than t h a t from a nucleus which i s exper ienc ing r ap i d motion. S ince the thermolys in on ly d iges t s p r o t e i n on the out s ide o f the membrane (210,211), the narrow resonance (4 Hz, F i g . 12) superimposed on the broad resonance i s ass igned to the cho l i ne con ta i n i ng l i p i d s on the i n s i d e o f the membrane, which are now exper ienc ing more r ap i d motion. Consequently, the broad under l y ing resonance i s ass igned to the cho l i ne con ta i n i ng l i p i d s on the i n s i d e of the membrane. A second experiment ( F i g s . 13 and 14) was performed, however, t h i s time two> samples of v i r u s were prepared and both t r ea ted i d e n t i c a l l y except one po r t i on o f v i r u s was not incubated i n the presence of the rmo ly s i n . The thermolys in t r ea ted v i r u s ( F i g . 14) again i n d i c a t e d a narrowing of the cho l i ne resonance (6 Hz), but not to the same extent as shown in F i g . 12. There are severa l po s s i b l e mechanisms f o r producing an N-methyl resonance as narrow as i l l u s t r a t e d i n F i g s . 12 and 14. During the d i g e s t i o n w i th t he rmo l y s i n , i t may have been po s s i b l e to have degraded the t o t a l s t r u c t u r e , of a small p o r t i on o f the v i r u s , causing the format ion of smal l v e s i c l e s which may have a narrower l i n e width than the i n t a c t v i r u s . However, s i nce the t r e a t ed v i r u s ( i n experiment #1) was r e p u r i f i e d on a potassium t a r t r a t e g r ad i en t , such small s t r u c t u r e s would have been removed, r u l i n g out such a mechanism. Another mechanism would i n vo l ve the use o f thermolys in i t s e l f . S ince the enzyme p repara t i on i s not pure, a po s s i b l e phosphol ipase A contam-i n a t i o n could lead to the product ion of l y s o p h o l i p i d s , i n p a r t i c u l a r l y s o -phosphat idy l chol i ne . The format ion of such a product could lead to d i s r u p t i o n of the v i r u s membrane w i th the format ion of m i c e l l e s which would e x h i b i t a very narrow N-methyl resonance. Although t h i s mechanism cannot be complete ly r u l ed out , the f a c t t ha t the p u r i f i e d t r e a t ed v i r u s p a r t i c l e - had the same To face page 78. F igure 12. High r e s o l u t i o n . proton NMR spectrum of t he rmo l y s i n - t r e a t ed Seml i k i Forest v i r u s . Seml i k i Fores t v i r u s was t r e a t e d w i th thermo ly s in as desc r ibed i n Fig.10.- A f t e r treatment and r e p u r i f i c a t i o n the sample was prepared f o r the NMR experiment as desc r ibed i n the Ma te r i a l s and Methods, sec. E. The spectrum was recorded under the same cond i t i on s as i n F i g . 11. .78. To face page 79,. F igure 13. High r e s o l u t i o n proton NMR.spectrum of i n t a c t Seml i k i Forest v i r u s . The v i r u s sample (14 mg p ro te i n ) was incubated w i th 35 umol C a C l j i n 3 ml PBS f o r 1 h a t 37°C and subsequently c e n t r i f u g e d 100,000 x £ f o r 1 h. The v i r u s was then prepared f o r the NMR experiment as desc r ibed i n M a t e r i a l s and Methods, sec . E. The spectrum was recorded on a Var ian XL-100 100 MHz s p e c t r o -meter us ing a 2 KHz s p e c t r a l w id th and a 2 s a c q u i s i t i o n t ime. The spectrum requ i red 30,000 scans. To face page '80. F igure 14. High r e s o l u t i o n proton NMR spectrum o f thermo ly s in t r e a t e d Seml i k i Forest v i r u s . The v i r u s sample (14 mg p r o t e i n ) , i n 3 ml PBS, was t r e a t e d w i th 2.4 mg thermolys in plus 35 umol C a C l 2 f o r 1 h a t 37°C. At the end of the i n cuba t i on the sample was cooled and cen-t r i f u g e d at 100,000 x £ f o r 1 h to p e l l e t the p a r t i c l e s . The sample was prepared f o r the NMR experiment as desc r ibed p re -v i o u s l y (Ma te r i a l s and Methods, sec. E) . The s p e c t r a l con-d i t i o n s were the same as desc r ibed i n F i g . 13. 80.. To face page 81. F igure 15. High r e s o l u t i o n proton NMR spectrum of Seml i k i Fo res t v i r u s l i p i d l iposomes. A sample o f Seml i k i Fo res t v i r u s (28 mg v i r a l p ro te i n ) was e x t r a c t ed by the procedure of Folch et dl. (207). The l i p i d phase was i s o l a t e d and t r a n s f e r r e d to a 5 mm NMR tube and the cho lor form evaporated under a stream of N^. D^O/O^ NaCl (0.25 ml) was added and the sample r a p i d l y a g i t a t e d . The spectrum was recorded on a Var ian XL-100 100 MHz s p e c t r o -meter us ing a 2 KHz s p e c t r a l width and a 2 s a c q u i s i t i o n .. t ime. The spectrum requ i red 1000 scans. 81.. 82. dens i t y as non- t reated v i r u s leads one to be l i e ve t ha t the t r e a t ed p a r t i c l e s were i n t a c t . During the r e p u r i f i c a t i o n the p a r t i c l e s are c e n t r i f u g e d seve ra l times a t 100,000 x £ and resuspended i n 0.9% NaCl/D 20 before c a r r y i n g out the NMR measurements. Such treatment may cause d e s t r u c t i o n of the v i r a l membrane l ead ing to the format ion of sma l l e r l i p i d s t r u c t u r e s . However, t h i s same procedure was employed when prepar ing the i n t a c t v i r u s p a r t i c l e s f o r the NMR experiment and c l e a r l y no small s t r u c t u r e s were formed. To see i f a v e s i c l e sample would e x h i b i t a cho l i ne resonance l i n e -width o f comparable s i z e to tha t obta ined i n F i g . 12, a l i p i d sample was prepared tha t was comprised o f the l i p i d c l a s s compos i t ion o f Seml i k i Fo res t v i r u s l e s s p h o s p h a t i d y l i n o s i t o l and g l y c o l i p i d (Table 2) (these l i p i d s represent les s than 10% of the t o t a l l i p i d ) (205). This sample was son i ca ted (Ma te r i a l s and Methods,sec. G ( i i i ) ) and the proton NMR spectrum taken. From F i g . 16 the c ho l i n e resonance was measured as 16 Hz, s u b s t a n c i a l l y broader than the resonance i n F i g . 12. This might suggest the the l i n e w i d t h o f the cho l i ne resonance observed i n F i g . 12 i s anomalously narrow. However, the r e s u l t o f t h i s experiment may not mean much s i n ce the sample does not conta in the exact l i p i d compos i t ion o f the i n t a c t v i r u s . A v e s i c l e p repa ra t i on o f egg pho spha t i dy l cho l i ne was a l s o s tud ied ( F i g .16 ) . From the spectrum a c ho l i n e resonance l i n e w i d t h o f 7 Hz was observed, comparable to tha t pub l i shed i n the l i t e r a t u r e (242). A l iposomal p repa ra t i on of the e x t r a c t ed v i r a l l i p i d was prepared. From F i g . 15 the l i n e w i d t h of the cho l i ne resonance was 10 Hz. Due to the l a rge s t r u c t u r e s i n a l iposomal l i p i d sample some broadening of the resonaces w i l l occur. Cons ider ing the cho l i ne l i new id th s obta ined from the v i r a l l iposomal sample and the egg pho spha t i dy l cho l i ne v e s i c l e s , i t i s not unreasonable to observe such a narrow resonance a f t e r thermolys in d i g e s t i o n o f the v i r u s . To face page 83. F igure 16. High r e s o l u t i o n proton NMR spec t r a o f mock Seml i k i Fores t v i r u s l i p i d v e s i c l e s and egg pho spha t i dy l cho l i ne v e s i c l e s . A. A l i p i d mixture (30 mg) composed of the same l i p i d c l a s s compos i t ion as Seml i k i Forest v i r u s (205) was prepared as descr ibed i n Ma te r i a l s and Methods, sec. G ( i ) . The sample was s on i ca ted 3 x 30 s ( F i s h e r Sonic Dismembrator, s e t t i n g 8) i n 2 ml 100 mM NaCl , lOOmM T r i s - a c e t a t e , pH 7.4 i n 100% D 2 0. B. Ve s i c l e s o f egg pho spha t i dy l cho l i ne (50 mg) were prepared i n a s i m i l a r manner as desc r ibed above. The spec t r a were recorded on a Bruker WP200 200 MHz spectrometer us ing a 5 KHz s p e c t r a l w i d th . The spectrum o f the mock v i r u s l i p i d sample requ i red 32 scans w h i l e the egg pho spha t i d y l -cho l i ne v e s i c l e spectrum requ i red 4 scans. 83, Figure 16. 84. B. Incorporat ion of Deuterated Species of Cho l ine i n t o BHK-21 C e l l L i p i d s The magnetic resonance techniques are u se fu l f o r s tudy ing the dynamic aspects o f the b i o l o g i c a l membrane. The use of proton NMR i s however, somewhat l i m i t e d i n i t s a b i l i t y to quan t i f y changes i n the motion o f a p a r t i c u l a r nuc leus. Therefore i t would be advantageous to f i n d a nucleus which i s s e n s i t i v e to r e s t r i c t i v e motion and at the same time y i e l d a q u a n t i t a t i v e e va l ua t i on of changes i n motion. Deuterium i s such a nuc leus , however, i t s u f f e r s from the problem o f low s e n s i t i v i t y , r e q u i r i n g the product ion of l a r ge samples. We wanted to ob ta i n a more q u a n t i t a t i v e e va l ua t i on o f the inc reased motion o f the headgroups o f the c h o l i n e con ta i n i n g l i p i d s as a r e s u l t o f protease d i g e s t i o n of the v i r u s . A s e r i e s of i s o t o p i c a l l y enr i ched cho l i ne s were prepared f o r i n c o r p o r a t i o n i n t o the cho l i n e con ta i n i n g l i p i d s of BHK-21 c e l l s . (a) C h a r a c t e r i z a t i o n of the Deuterated Chol ines A s e r i e s o f three i s o t o p i c a l l y l a b e l l e d cho l i ne s were prepared (Ma te r i a l s and Methods, sec. H ( i , i i , i i i ) ) w i th e i t h e r 1,2, or 3 deuterated N-methyl groups ( F i g . 17). The synthes i s of a l l three deuterated cho l i ne s f o l l owed the same general scheme, tha t i s , the methy la t i on o f an amine w i t h t r i deu te romethy l i o d i d e . A l l three l a b e l l e d products were p u r i f i e d by c r y s t a l l i z a t i o n and shown to r eac t s t r ong l y w i t h re inecke s a l t (ammonium t e t r a t h i o c y a n o -diammonochromate) forming a red i n s o l u b l e complex i n aqueous s o l u t i o n . 85. i | 3 S I 3 H 3 C - N - C H 3 H 3 C - N - C D 3 C H , C H , C H 2 C H , 1 2 l 2 OH O H Chol ine mono-tr ideuteromethyl Cho l ine ^C D a C D 3 © l 3 © I 3 H 3 C - N - C D 3 D 3 C - N - C D 3 C H 2 C H , 1 2 l 2 C H 2 C H , 1 1 2 O H OH d i - t r i d e u t e r o m e t h y l Cho l ine t r i - t r i d e u t e r o m e t h y l Cho l ine F igure 17. 86. IMHgCHgCHgOH + 3 CD 3I > Ethanolamine t r i deute romethy l Iodide e CD. D Q C-N-CH-CH-OH + 2HI o I 2 2 QQ Hydrogen Iodide 3 t r i - t r i d e u t e r o m e t h y l Cho l ine Iodide This reagent i s s p e c i f i c f o r d e t e c t i n g t e r t i a r y ^amino -:' compounds.. NMR—-spec t ra were taken- o f each o f the three l a b e l l e d species•«(Figs. 18 A , B, .0, D), The i n t e g r a t e d areas of the peaks are g iven i n * Table. 5; - * Table 5 Integrated Areas o f the Peaks from Spect ra Obtained from Cho l i ne , Mono-t r i deu te romethy l Cho l i ne , D i - t r i deu te romethy l Cho l i ne , and T r i - t r i d e u t e r o m e t h y l Cho l ine Ra t i o of areas under the peaks HO - CH 2 - CH 2 - N + ( C H 3 ) 3 r egu l a r c h o l i n e mono-tr ideuteromethyl c ho l i n e d i - t r i d e u t e r o m e t h y l c ho l i n e t r i - t r i d e u t e r o m e t h y l c h o l i n e 2 2 2 2 2 2 2 2 9 6 3 0 To face page 87. F igure 18. Proton NMR spectrum of c h o l i n e , mono-tr ideuteromethyl c h o l i n e , d i - t r i d e u t e r o m e t h y l c h o l i n e , and t r i - t r i d e u t e r o m e t h y l c h o l i n e . Cho l ine and the three deuterated spec ies o f c ho l i ne (100 mg each) were d i s s o l v ed s epa ra te l y i n 0.3 ml D 2 O . The spec t r a o f the deuterated cho l i ne s were recorded on a Bruker WP200 200 MHz NMR spectrometer us ing a 2 KHz s p e c t r a l w i d t h . The spec t ra r equ i red 5 scans. The spectrum of c ho l i n e was . recorded on a Var ian T-60 CW NMR spectrometer us ing a 500 Hz s p e c t r a l w i d t h . A. c h o l i n e , B. mono-tr ideuteromethyl c h o l i n e , C. d i - t r i - d e u t e r o m e t h y l c h o l i n e , and D. t r i - t r i d e u t e r o m e t h y l c h o l i n e . F igure 18. 88. The succes s i ve deu te ra t i on o f the cho l i ne methyl groups w i l l r e s u l t i n the los s of 3 protons per deuterated methyl group. (b) I nco rpo ra t i on o f the Deuterated Chol ines i n BHK-21 C e l l Cho l ine  Conta in ing L i p i d s 3 Regular cho l i ne and the three deuterated spec ies p lus [ H ] -cho l i ne were added to the medium above the BHK-21 c e l l s as desc r ibed p re -v i o u s l y (Ma te r i a l s and Methods, sec . H ( i v ) ) . The c e l l s were grown to conf luence, the c e l l l i p i d s were i s o l a t e d and the pho spha t i d y l cho l i ne p u r i f i e d . By c a l c u l a t i n g 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 added c h o l i n e and measuring 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 pho spha t i d y l c ho l i n e , i t was po s s i b l e to determine the a b i l i t y of each spec ies o f c h o l i n e to be i nco rpo ra ted . T h e o r e t i c 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 pho spha t i d y l -cho l i ne should be the same as 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 added c h o l i n e , t ha t i s i f there are no problems i n i n c o r p o r a t i n g the var ious cho l i ne s pec i e s . From Table 6, the s p e c i f i c r a d i o a c t i v i t y of the pho spha t i d y l -cho l i ne l a b e l l e d w i th r e gu l a r c h o l i n e , mono-tr ideutermethyl c h o l i n e , and d i - t r i d e u t e r o m e t h y l c h o l i n e i s very s i m i l a r to 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 added cho l i n e suggest ing t h a t the deuterated and t r i t i a t e d spec ies of c ho l i n e are f i n d i n g no d i f f i c u l t y i n being i nco rpo ra ted i n t o the pho spha t i d y l cho l i ne . However, 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 pho spha t i d y l -c ho l i n e i s o l a t e d from c e l l s grown on medium supplemented w i th t r i - t r i d e u -te romethy l cho l i ne i s much h igher than the pho spha t i dy l cho l i ne i s o l a t e d from c e l l s grown on r egu l a r c h o l i n e . The s p e c i f i c r a d i o a c t i v i t y o f the 89. phospha t i dy l cho l i ne from c e l l s incubated w i th t r i - t r i d e u t e r o m e t h y l c h o l i n e 3 plus [ H ] - cho l i ne i s very s i m i l a r to 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 3 pho spha t i dy l cho l i ne from c e l l s incubated w i th on ly [ H ] - cho l i ne ( s p e c i f i c r a d i o a c t i v i t y o f pho spha t i dy l cho l i ne - 4.66, 3.43, and 5.0 uC i/umol . in three d i f f e r e n t exper iments ) . This suggests t ha t t h i s deuterated spec ies of c ho l i ne i s not being i nco rpo ra ted i n t o the l i p i d . Several po s s i b l e exp lanat ions f o r t h i s lack o f i n c o r p o r a t i o n i n t o pho spha t i dy l cho l i ne may be: (1) The i n a b i l i t y o f the c e l l to t r an spo r t t h i s h e a v i l y deuterated spec ies of c ho l i ne across the plasma membrane o f the c e l l , or (2) the enzymes i n vo l ved i n the b i o s yn the s i s of pho spha t i d y l -cho l i ne cannot u t i l i z e such a h e a v i l y deuterated molecule. S ince c h o l i n e i s the sub s t r a te f o r the f i r s t enzyme i n the b i o s yn the s i s o f pho spha t i d y l -c h o l i n e , we were i n t e r e s t e d to see i f these deuterated cho l i ne s would be e f f e c t i v e as a s ub s t r a te f o r the enzyme c h o l i n e k inase . Cho l ine k inase from BHK-21 c e l l c y to so l was found to be e f f e c t i v e i n phosphory la t ing both mono-tr ideuteromethyl c ho l i ne and t r i - t r i d e u t e r o m e t h y l c ho l i ne (Table 7) . Unless the o the r two enzymes i n the b i o s y n t h e t i c pathway, the CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e or the cho l i ne phosphotrans-f e r a s e , are unable to u t i l i z e the deuterated phosphochol ine, the on ly a l t e r n a t i v e f o r the l ack o f i n c o r p o r a t i o n o f t r i - t r i d e u t e r o m e t h y l cho l i ne i n t o pho spha t i dy l cho l i ne i s due to problems i n t r an spo r t . 3 To i n v e s t i g a t e t h i s p o s s i b i l i t y , t r i - t r i d e u t e r o m e t h y l - [ l , 2 - H ] -3 cho l i n e was prepared by deuteromethy lat ion o f [ 1 , 2 - H]-ethanolamine w i th CD^I (Ma te r i a l s and Methods, Sec. H ( v ) ) , f o r use i n t r an spo r t s t u d i e s . I f the l ack o f i n c o r p o r a t i o n o f t r i - t r i d e u t e r o m e t h y l c ho l i n e were due to f a i l u r e to t r an spo r t the molecule across the plasma membrane o f the c e l l , . 9 0 . Table 6 I nco rpora t ion o f Deuterated Chol ines i n t o BHK-21 Pho spha t i dy l cho l i ne S p e c i f i c Rad i o a c t i v i t y o f cho l i ne i n the medium (uCi/umol) S p e c i f i c r a d i o a c t i v i t y o f phospha-t i d y l c h o l i n e i n c e l l s grown on the f o l l o w i n g i s o t o p i c a l l y l a b e l l e d cho l i ne s (uCi/umol) c ho l i n e mono^ d i 2 t r i 3 1.4 1.44 _ — — — 1.4 1.44 --0.86 0.42 0.47 — 0.87 0.46 -- 0.6 0.8 -- -- 9.0 0.8 0.62 -- 5.0 1. mono = mono- t r ideuteromethy lcho l ine 2. d i = d i - t r i d e u t e r o m e t h y l c h o l i n e 3. t r i = t r i - t r i d e u t e r o m e t h y l c h o l i n e BHK-21 c e l l s (3 l a rge d i shes ) were grown on Dulbecco ' s Mod i f i ed Eagles Medium supplemented w i th 80 ug/ml o f t r i - t r i d e u t e r o m e t h y l c h o l i n e c h l o r i d e plus 0.167 uCi/ml [ ^H j - cho l i ne . Another three d ishes were grown on medium supplemented wi th 130 ug/ml o f c ho l i n e i od i de plus 0.167 uCi/ml [^H]-c h o l i n e . F i n a l l y three dishes o f c e l l s were grown on medium supplemented w i th on ly 0.167 yCi/ml [ 3 H ] - c h o l i n e . (Note: Du lbecco ' s Mod i f i ed Eagles Medium has 4 ug/ml c ho l i n e c h l o r i d e : ) A f t e r the c e l l s reached conf luence they were harvested and the l i p i d s e x t r a c t ed (207) and p u r i f i e d by TLC ( so l ven t A ) . The pho spha t i dy l cho l i ne was i s o l a t e d and e x t r a c t ed from the s i l i c a gel w i th 10 ml C H C l 3 / C H 3 0 H / N r U 0 H (1/1/0.1 : v/v/v). Tota l l i p i d phosphorous was determined by the method of Rajeha et dl. (199), and the r a d i o a c t i v e content measured by l i q u i d s c i n t i l l a t i o n count ing , 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 pho spha t i dy l cho l i ne was then c a l c u l a t e d . The same experiment was performed when s tudy ing the i n c o r p o r a t i o n o f mono- t r ideute rmethy l cho l i ne and d i - t r i d e u t e r o m e t h y l c h o l i n e . 91, Table 7 Cho l ine Kinase A c t i v i t y Using Cho l i ne , Mono-tr ideuteromethyl Cho l i n e , Cho l ine k inase from BHK-21 c e l l c y to so l was assayed using c h o l i n e , mono-t r i deuteromethyl c h o l i n e , and t r i - t r i d e u t e r o m e t h y l c ho l i ne as s ub s t r a t e . The assay was performed as desc r ibed i n M a t e r i a l s and Methods us ing 32 [y- P]-ATP as a l a b e l l e d s ub s t r a t e . Enzyme a c t i v i t i e s are expressed as counts above background. 3 then no uptake of the t r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - H ] - c ho l i n e should take p l ace . Un fo r tunate l y there was some contaminat ion o f the t r i - t r i d e u t e r o -3 3 m e t h y l - [ l , 2 - H ] - cho l i ne w i th [ 1 ,2 - H]-ethanolamine, t h e r e f o r e , some uptake o f r a d i o a c t i v i t y i n t o the c e l l was expected. However a l l o f the r a d i o -a c t i v i t y absorbed by the c e l l should be a s soc i a ted w i th pho spha t i d y l -ethanolamine and i t s p recu r so r s . As i n d i c a t e d i n F i g . 19, the re was an i n i t i a l l o s s o f r a d i o a c t i v i t y from the medium above the c e l l s which were 3 incubated w i th t r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - H ] - c h o l i n e . However a f t e r 37 min the uptake o f r a d i o a c t i v i t y p lateaued i n d i c a t i n g tha t no f u r t h e r t r an spo r t was t ak i ng p l a ce . Ana l y s i s o f the o rgan ic s o l u b l e r a d i o a c t i v i t y i n these c e l l s showed t ha t a l l o f the r a d i o a c t i v i t y was a s s oc i a ted w i t h phosphat idy lethanolamine (Table 8 ) . There was no i n copo r a t i on i n t o BHK-21 c e l l pho spha t i dy l cho l i ne ( F i g . 20). 3 In comparison, c e l l s incubated w i th [methy l - H ] - cho l i ne showed no p la teau i n uptake ( F i g . 19), w i th the l abe l being r a p i d l y i n co rpo ra ted and T r i - t r i d e u t e r o m e t h y l Cho l ine as Subst rate Background Cho l ine Mono-tr i deuteromethyl r ehol ine T r i - t r i d e u t e r o m e t h y l c h o l i n e . 95 cpm 643 cpm 755 cpm 870 cpm Table 8 Time Course o f I ncorpora t ion o f R a d i o a c t i v i t y i n t o the Organic and Aqueous So lub le F rac t i on s o f BHK-21 C e l l s C e l l s incubated w i th t r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - 3 H ] - c h o l i ne C e l l s incubated w i th [me thy l - 3 H] - cho l i ne Time (min) Aqueous (dpm) Organic (dpm) Aqueous (dpm) Organic (dpm) 0 0 0 0 0 37 0.26 x 10 6 2.7 x 10 6 0.42 x 10 6 2.8 x 10 6 60 0.12 x 10 6 3.1 x 10 6 0.25 x 10 6 3.8 x 10 6 126 0.28 x 10 6 2.9 x 10 6 0.23 x 10 6 4.3 x 10 6 BHK-21 c e l l s ( l a r ge d i shes ) were incubated a t 37°C w i th 6 ml Medium 199 con ta in i ng e i t h e r t r i -t r i d e u t e r o m e t h y l - [ l , 2 - 3 H ] - c h o l i n e (2.86 y C i - d i s h ) or [ 3 H ] - c h o l i n e (3 uC i /d i s h ) . At 37, 60, and 126 min the c e l l s were harvested to determine the amount of t r an spo r t o f r a d i o a c t i v i t y i n t o the c e l l s . The medium was removed and the c e l l s were washed wi th 5 x 10 ml i c e - c o l d PBS. The c e l l s were scraped o f f the d ishes w i th a rubber policeman and ex t r ac ted accord ing to the procedure o f Folch et al. (207). The t o t a l aqueous and o rgan i c s o l u b l e r a d i o a c t i v i t y was determined by l i q u i d s c i n t i l l a t i o n count ing , (a ) . A l l o rgan ic s o l u b l e r a d i o a c t i v i t y a s soc ia ted w i th c e l l s incubated wi th the t r i - t r i d e u t e r o -m e t h y l - [ 1 , 2 - 3 H ] - c h o l i n e was a s soc i a ted w i th phosphat idy lethanol amine. To face page 9 3 - . F igure 1 9 . Decrease of r a d i o a c t i v i t y from the c e l l medium. B H K - 2 1 c e l l s were incubated over a 1 . 5 h pe r i od w i t h 6 ml Medium 1 9 9 c on ta i n i n g e i t h e r t r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - 3 H ] -cho l i ne ( 2 . 8 6 uC i/d i sh ) (•) or [ m e t h y l- 3H ] - c h o l i n e ( 3 uC i/ d ish (A) as c o n t r o l . At 3 7 , 6 0 , and 1 2 6 min, 2 x 5 0 y l a l i q u o t s o f medium were removed and the r a d i o a c t i v i t y determined by l i q u i d s c i n t i l l a t i o n count ing . [ 3 H , 2 H ] -cho l i n e = t r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - 3 H ] - c h o l i n e . Figure 19. <X3 To face page 94'.,. F igure 20. Uptake o f r a d i o a c t i v i t y i n t o BHK-21 c e l l p ho spha t i d y l cho l i ne . BHK-21 c e l l s were t r ea ted as desc r ibed i n F i g . 19. At 37, 60, and 126,min, c e l l s were harvested and the t o t a l l i p i d s e x t r a c t ed (207). The pho spha t i dy l cho l i ne was p u r i f i e d by TLC ( s o l ven t A) and the r a d i o a c t i v i t y determined by l i q u i d s c i n t i l l a t i o n count ing . [ 3 H , 2 H ] - c h o l i n e = t r i - t r i -d e u t e r o m e t h y l - [ l , 2 - 3 H ] - c h o l i n e . 9.4;.: (9_<H X ujdp) u o u e j o d j o o u i 9 A ! i o e o ! p e y 9.5. i n t o pho spha t i dy l cho l i ne ( F i g . 20). S ince the uptake o f r a d i o a c t i v i t y i n 3 c e l l s incubated w i th the t r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - H ] - cho l i ne (contami -3 nated w i th [1 ,2 - H]-ethanolamine) p lateaued at about 37 min, t h i s suggests tha t there was a r a p i d uptake o f most o f the contaminat ing ethanolamine 3 l eav i ng the t r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - H ] - cho l i ne i n the medium. The data suggests tha t t r i - t r i d e u t e r o m e t h y l c h o l i n e i s unable to be i nco rpo ra ted i n t o BHK-21 c e l l c ho l i n e con ta i n i ng l i p i d s because i t i s unable to be t r an spo r ted across the plasma membrane of the c e l l . 96. C. Stud ies on the CTP:phosphocholine C y t i d y l y l t r a n s f e r a s e I t has been known f o r some time t ha t the a c t i v i t y o f the r a t l i v e r CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e i s modulated by l i p i d (190). I n ve s t i g a t i on s by Choy et al. (212) have shown tha t the a c t i v a t i o n of the enzyme, which i s observed when the r a t l i v e r c y to so l i s aged at 4° C f o r severa l days, i s due to an i nc rease i n the concent ra t ions of LPE i n the c y t o s o l . Two other l i p i d s , p h o s p h a t i d y l s e r i n e and p h o s p h a t i d y l i n o s i t o l , were a l s o ab le to a c t i v a t e the enzyme but not to the same l e v e l as LPE. Another l y s o l i p i d , LPC, was found to s t r o n g l y i n h i b i t the c y t i d y l y l t r a n s f e r a s e . The f i n d i n g that two d i f f e r e n t l y s o l i p i d s are capable of r e g u l a t i n g the c y t i d y l y l t r a n s -fe ra se a c t i v i t y l ed to the present i n v e s t i g a t i o n , which was to study the e f f e c t s of these two l y s o l i p i d s on the c y t i d y l y l t r a n s f e r a s e . (a) P repa ra t i on of the CTP:phosphocholine C y t i d y l y l t r a n s f e r a s e The c y t i d y l y l t r a n s f e r a s e was p u r i f i e d accord ing to the method of Choy et al. (191) (Ma te r i a l s and Methods, sec. J ) . The p u r i f i c a t i o n of the enzyme i s dependant on the aggregat ion of the l i g h t form (L-form) of the enzyme to the heavy form (H-form) o f the enyzme. The c y t i d y l y l t r a n s f e r a s e was p r e c i p i t a t e d form aged cy to so l us ing ammonium su lphate (25% at 4°C). This f r a c t i o n con ta i n i n g the heavy ':' form of the enzyme was app l i ed to the f i r s t of two Sepharose 6B columns ( F i g . 21). The m a j o r i t y o f the c y t i d y l y l t r a n s f e r a s e , l o c a t e d : i n the vo id volume of the column, was pooled and t r e a t ed w i th SDS ( f i n a l To face page 97. F igure 21. Chromatography o f CTP:phosphocholine c y t i d y d l y l t r a n s f e r a s e from r a t . i l i v e r c y to so l on Sepharose 6B. Rat l i v e r cy to so l (50 ml o f a 20% homogenate i n i s o t o n i c s a l i n e ) was aged f o r 5 days a t 4°C. The cy to so l was c e n t r i f u g e d at 10,000 x £ f o r 10 min to remove any d e b r i s . The supernatant f r a c t i o n was adjus ted to 25% s a t u r a t i o n w i th ammonium su lphate (pH 7) and l e f t a t 4°C f o r 1 h. The s o l u t i o n was c e n t r i f u g e d at 10,000 x £ f o r 10 min and the p e l l e t resuspended i n Bu f f e r A at 1/10 the i n i t i a l volume. A 4 ml sample was then app l i ed to a Sepharose 6B column (2.5 cm x 80 cm) e q u i l i b r a t e d w i th B u f f e r A. F r ac t i on s (6 ml) were c o l l e c t e d and assayed f o r enzyme a c t i v i t y i n the presence o f exogeneous r a t l i v e r phospho l i p id (1 mg/ assay) . A c t i v i t y i s expressed per ml o f column e l uan t . F igure 22. Chromatography o f SDS -d i s soc ia ted CTP:phosphocholine c y t i d y l y l -t r an s f e r a se from r a t l i v e r cy to so l on Sepharose 6B. The f r a c t i o n s con ta i n i n g c y t i d y l y l t r a n s f e r a s e a c t i v i t y ( F i g . 21) were pooled and SDS added to a f i n a l concen t ra t i on o f 0.06%. The sample was l e f t a t 4°C f o r 1 h and then u l t r a f i l t r a t e d , us ing an Amicon XM100A f i l t e r , to a volume o f about 4 ml . This sample was then app l i ed to a second Sepharose 6B column (2.5 cm x 80 cm) e q u i l i b r a t e d w i th B u f f e r A plus 0.001% SDS. F rac t i on s (6 ml) were c o l l e c t e d and assayed f o r c y t i d y l y l t r a n s -ferase i n the presence o f r a t l i v e r phospho l i p id (1 mg/assay). A c t i v i t y i s expressed per ml o f column e luan t . 97.-0 5 10 15 20 25 Fraction Number 98, concent ra t i on 0.06%) f o r 1 h a t 4°C. Th i s sample was then u l t r a f i l t r a t e d and app l i ed to the second Sepharose 6B column which had been e q u i l i b r a t e d w i th Bu f f e r A plus 0.01% SDS ( F i g . 22). The column p r o f i l e showed t h a t the c y t i d y l y l t r a n s f e r a s e a c t i v i t y s p l i t i n t o two f r a c t i o n s . The a c t i v i t y l o ca ted w i t h i n the i nc l uded volume of the column was pooled and concen-t r a t e d by u l t r a f i l t r a t i o n f o r use i n subsequent exper iments. (b) P u r i t y o f the C y t i d y l y l t r a n s f e r a s e Although SDS was able to d i s s o c i a t e the H-form o f the enzyme to the L- form, which was l o ca ted w i t h i n the i nc luded volume of the second Sepharose 6B column, the s i z e o f the p r o t e i n (o r aggregate) d i d not a l l ow i t to en te r a 5% nondenaturing po lyacry lamide g e l . To overcome t h i s problem, SDS ( f i n a l concen t ra t i on of 0.05%) was added to the sample o f L-form. A f t e r e l e c t r o p h o r e s i s and s t a i n i n g the gel f o r p r o t e i n , on ly one band o f p r o t e i n was observed i n s i d e the gel although some mate r i a l s t i l l r e s i ded on top o f the gel ( F i g . 23). By running d u p l i c a t e g e l s , s t a i n i n g one f o r p r o t e i n and assay ing the other f o r c y t i d y l y l t r a n s f e r a s e a c t i v i t y (Ma te r i a l s and Methods, sec . K) the s i n g l e band i n the gel was found to conta in enzyme a c t i v i t y . The mate r i a l on top o f the gel a l s o conta ined c y t i d y l y l t r a n s -fe ra se a c t i v i t y suggest ing t ha t not a l l o f the enzyme was complete ly d i s -s o c i a t ed to the L- form, o r i t had reaggregated a f t e r the column chromatography. \ To face page 99., F igure 23. Non-denaturing po lyacry lamide gel e l e c t r o p h o r e s i s o f p u r i f i e d CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e . The p u r i f i e d c y t i d y l y l t r a n s f e r a s e was e l e c t r opho re s i s ed on 5% po lyacry lamide tube g e l s , prepared as desc r ibed i n M a t e r i a l s and Methods, sec. B ( v i i ) . The d i s s o c i a t i o n o f the H-form to the L-form was not t o t a l l y complete, t h e r e f o r e , SDS was added to a f i n a l concen t ra t i on o f 0.05%. Dup l i ca te gels were e l e c -t r opho re s i s ed (2 mA/gel f o r 2 h a t 4°C) w i t h one gel being s t a i ned f o r p r o t e i n us ing Coomassie B lue , and the o the r gel being assayed f o r c y t i d y l y l t r a n s f e r a s e a c t i v i t y . Three s ec t i on s were cut out o f the gel corresponding to the three reg i on s , TOP, MID, and BAND. Each s e c t i o n was crushed and assayed f o r c y t i d y l y l t r a n s f e r a s e a c t i v i t y as desc r ibed i n M a t e r i a l s and Methods, Sec. K. The enzyme a c t i v i t y a s s o c i a t ed w i th each reg ion i s expressed as a percentage o f the t o t a l recovered a c t i v i t y . 99. Recovered Enzyme A c t i v i t y e TOP 6 i % MID n % BAND 28% F igure 23. 100. (c) E f f e c t s o f L y s o l i p i d s on CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e  A c t i v i t y I t was the i n i t i a l ob se rva t i on by Schneider (189) t ha t storage o f r a t l i v e r y cy to so l a t 4°C f o r severa l days l e d to an i nc rease i n c y t i d y l y l t r a n s f e r a s e a c t i v i t y ( F i g . 24). The enzyme can a l so be a c t i v a t e d over a s ho r t e r t ime per iod by i n cuba t i on o f the f resh cy to so l a t 37°C f o r a few hours ( F i g . -25). Choy et al. (212) s t ud i ed t h i s a c t i v a t i o n phenomenon i n more d e t a i l and reported t ha t l y sophosphat idy lethano lamine (LPE) was re spons ib le f o r the a c t i v a t i o n o f the c y t i d y l y l t r a n s f e r a s e . I t was a l s o noted that l y s opho spha t i d y l cho l i ne (LPC) s t r ong l y i n h i b i t e d c y t i d y l y l -t r an s f e r a se a c t i v i t y . (NOTE: Throughout the study on ly the o l e o y l -d e r i v a t i v e s o f both . l y s o l i p i d s . were used due to t h e i r s o l u b i l i t y i n aqueous s o l u t i o n . ) The p u r i t y o f o l e o y l - l y s o p h o s p h a t i d y l c h o l i n e and o l e o y l -l y sophosphat idy lethano lamine was determined by s i l i c a gel TLC (So lvent A) and gas chromatography (Ma te r i a l s and Methods, Sec. B ( i i i ) ) . Both l y s o l i p i d s were shown to be 100% pure by l i p i d c l a s s ( F i g . 26A). O l e o y l -LPC conta ined >99% o f the C^g.^-species o f f a t t y a c i d wh i l e o l eoy l - LPE conta ined >90% of the C-jg.-j-species w i t h minor contaminat ion w i th C^g.Q and C.jg.-|-species (3.5% each) ( F i g . 26B). (d) E f f e c t of LPE on the A c t i v i t y the P u r i f i e d CTP-phosphocholine  C y t i d y l y l t r a n s f e r a s e The p u r i f i e d enzyme was assayed at opt imal s ub s t r a te concent ra t i ons To face page 101. F igure 24. A c t i v a t i o n of CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e i n r a t l i v e r cy to so l a t 4°C. Rat l i v e r cy to so l (20% homogenate i n i s o t o n i c s a l i n e ) was s t o red at 4°C. The a c t i v i t y o f the enzyme was determined as p r e v i ou s l y desc r ibed (Ma te r i a l s and Methods, sec . I ( i i ) ) , a f t e r 1, 2, 3, 4, and 5 days. Enzyme a c t i v i t y was d e t e r -mined i n the absence o f exogenous r a t l i v e r l i p i d . F igure 25. A c t i v a t i o n o f CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e i n r a t l i v e r cy to so l a t 37°C. Rat l i v e r cy to so l (20% homogenate i n i s o t o n i c s a l i n e ) was incubated at 37°C f o r 4 h. At hour ly i n t e r v a l s an a l i q u o t (20 u l ) o f cy to so l was removed and assayed f o r enzyme a c t i v i t y i n the absence o f exogenous r a t l i v e r l i p i d (Ma te r i a l s and Methods, sec . I ( i i ) ) . Figure 24. TIME (days) To face page 102. : Figure 26. A. Thin l a y e r chromatography o f o l e o y l - l y s o p h o s p h a t i d y l e -thanolamine and o l e o y l - l y s o p h o s p h a t i d y l c h o l i n e on s i l i c a gel G-25. L i p i d s were v i s u a l i z e d us ing I 9 vapor. B. Gas chromatography o f the f a t t y a c i d methyl e s te r s o f o l e o y l -l y sophosphat idy lethano lamine and o l e o y l - l y s o p h o s p h a t i d y l -c h o l i n e (Ma te r i a l s and Methods, sec. B ( i i i ) ) . LPE 03 LPC P r i g A. Figure 26. 103, i n the presence o f i n c r ea s i n g amounts o f LPE. The c y t i d y l y l t r a n s f e r a s e was found to be s t imu l a ted about 1 0 - f o l d ( F i g . 27 and F i g . 28) w i th a K a f o r LPE of about 0.3 mM. (e) E f f e c t o f LPC on the A c t i v i t y o f P u r i f i e d CTP:phosphocholine  C y t i d y l y l t r a n s f e r a s e In con t r a s t to the a c t i v a t i n g a b i l i t y o f LPE, the o l e o y l -d e r i v a t i v e o f LPC was found to s t r ong l y i n h i b i t the c y t i d y l y l t r a n s f e r a s e ( F i g . 29). The p u r i f i e d enzyme was assayed at opt imal s ub s t r a te concent ra -t i on s i n the presence o f an i n c r e a s i n g LPC/LPE r a t i o . The enzyme a c t i v i t y i s very low i n the presence o f LPC a lone. The re fo re , to ob ta in more r e l i a b l e r e s u l t s , LPE (20 ug/assay, 0.402 mM) was added to r a i s e the basal l e v e l o f a c t i v i t y . The i n i t i a l approach to the study o f the i n t e r a c t i o n s between these two l y s o l i p i d s and the c y t i d y l y l t r a n s f e r a s e was to be o f a phy s i ca l b iochemical na tu re , however due to the low y i e l d s of p u r i f i e d enzyme a k i n e t i c approach was adopted. ( f ) E f f e c t of LPE on the K i n e t i c Parameters o f the CTP:phosphocholine  C y t i d y l y l t r a n s f e r a s e The c y t i d y l y l t r a n s f e r a s e ca ta l y se s the r e a c t i o n between phospho-chol ine and CTP to produce CDP-chol ine and pyrophosphate. From F i g . 27 i t i s obvious t ha t LPE i s having a pronounced e f f e c t on the a c t i v i t y of the enzyme. An i n i t i a l ques t ion to be answered was, what e f f e c t i n t h i s l y s o l i p i d having on the a b i l i t y o f the two subs t ra te s to b i nd (or i n t e r a c t ) To face page 104. F igure 27. Lysophosphat idylethanolamine a c t i v a t i o n o f p u r i f i e d CTP: phosphocholine c y t i d y l y l t r a n s f e r a s e from r a t l i v e r . P u r i f i e d c y t i d y l y l t r a n s f e r a s e was assayed at opt imal s ub s t r a te concent ra t ions w i th i n c r e a s i n g LPE concent ra -t i o n s . Enzyme a c t i v i t i e s are expressed as nmols of CDP-cho l i n e formed per minute x 1 0 " 2 . 104, F igure 27. To face page 105;. F igure 28. Double inver se p l o t o f i n i t i a l v e l o c i t y o f CDP-chol ine s ynthes i s a t s a t u r a t i n g concent rat ions o f phosphocholine and CTP and i n c r e a s i n g LPE concen t r a t i on . To face page 106.: F igure 29. Ly sophosphat idy l cho l i ne i n h i b i t i o n o f p u r i f i e d CTP:phospho-cho l i n e c y t i d y l y l t r a n s f e r a s e from r a t l i v e r . P u r i f i e d c y t i d y l y l t r a n s f e r a s e was assayed a t opt imal sub-s t r a t e concent ra t ions w i t h i n c r e a s i n g LPC concen t r a t i on s . Each assay had 20 yg LPE added to r a i s e the basa l l e v e l o f a c t i v i t y . 'No l i p i d a d d i t i o n ' i n d i c a t e s the a c t i v i t y of the enzyme when assayed w i th no LPE o r LPC present . '0.55mM L P C i n d i c a t e s the enzyme a c t i v i t y i n the presence o f 0.55mM LPC wi th no added LPE. Enzyme a c t i v i t i e s are expressed as nmols o f CDP-chol ine formed per minute x 10~ 2 . no lipid addition 0.55 mM LPC 0 . 1 0 . 2 0 . 3 OA 0 5 0 . 6 LPC (mM) Figure 29. 107. w i th the c y t i d y l y l t r a n s f e r a s e ? To t h i s end, the e f f e c t of LPE on the Km f o r CTP was s t u d i e d . The enzyme was assayed at var ious concent ra t ions o f CTP and constant ( s a tu r a t i n g ) phosphocholine c o n c e n t r a t i o n , as w e l l as at var ious concent ra t ions o f LPE. A double r e c i p r o c a l p l o t o f the data revea led a chan i n the Km f o r CTP i n such a way as to cause an i nc rea se i n the a f f i n i t y o f the c y t i d y l y l t r a n s f e r a s e f o r CTP ( F i g . 30) . The e f f e c t o f LPE on the Km f o r phosphocholine was then s t u d i e d . In performing a s i m i l a r experiment except va ry ing the phosphocholine concen-t r a t i o n at s a t u r a t i n g CTP c oncen t r a t i o n , the i nve r se p l o t i n d i c a t e d l i t t l e e f f e c t on the Km f o r t h i s s ub s t r a t e ( F i g . 31) . From these two r e s u l t s i t would appear t ha t the a c t i v a t i o n o f the c y t i d y l y l t r a n s f e r a s e by LPE i s p r i m a r i l y due to the decrease i n the K f o r CTP. r J m (g) E f f e c t o f LPC on the K i n e t i c Parameters o f the CTP:phosphocholine  C y t i d y l y l t r a n s f e r a s e With the above r e s u l t i t was o f i n t e r e s t to i n v e s t i g a t e what e f f e c t LPC was having on the a f f i n i t y o f the subs t ra tes f o r the enzyme. I n i t i a l l y the e f f e c t o f LPC on the K^ f o r CTP was s t u d i e d . The p u r i f i e d enzyme was assayed a t var ious concent ra t i on s o f CTP and constant ( s a t u r -a t i ng ) phosphocholine c o n c e n t r a t i o n , as w e l l as i n c r ea s i n g the LPC/LPE r a t i o . The i nve r se p l o t o f the data revea led an i n t e r e s t i n g t rend ( F i g . 32) With an i n c r e a s i n g concen t ra t i on of LPC, the K f o r CTP was s h i f t e d to a • m l a r g e r va l ue , i n d i c a t i n g t ha t t h i s l y s o l i p i d was decreas ing the subs t ra tes To face page 108;. F igure 30. Double r e c i p r o c a l p l o t o f i n i t i a l v e l o c i t y of CDP-chol ine synthes i s a t s a t u r a t i n g phosphochol ine c o n c e n t r a t i o n , va ry ing CTP concen t r a t i on s , and i n c r ea s i n g amounts o f LPE. The amounts of LPE were 5 (•), 1 0(A), and 20 (o) yg. To face page 109. F igure 31... Double r e c i p r o c a l p l o t o f i n i t i a l v e l o c i t y o f CDP-chol ine s yn thes i s a t s a t u r a t i n g CTP c o n c e n t r a t i o n , va ry ing phospho-cho l i n e concen t r a t i on s , and f i x e d amounts of LPE. The f i x e d amounts o f LPE were 0 (•), 1 0 ( A ) , and 20 (o ) yg . •601 To face page 1.10>* F igure 32... Double r e c i p r o c a l p l o t o f i n i t i a l v e l o c i t y o f CDP-chol ine synthes i s a t s a t u r a t i n g phosphochol ine c o n c e n t r a t i o n , va ry -ing CTP concen t r a t i on s , and i n c r e a s i n g amounts o f LPC. The f i x e d amounts o f LPC were 0 ;_(©), 5 ( o ) , 10 ( A ) , and 15 ( • ) ug; Each assay a l so conta ined 20 yg (0.4mM) LPE to r a i s e the basal l e v e l of a c t i v i t y . "GLL 111. a f f i n i t y f o r the enzyme. This would account f o r the i n h i b i t i o n of the c y t i d y l y l t r a n s f e r a s e which i s observed. However, the i n t e r p r e t a t i o n o f t h i s data i s compl icated by the f a c t t ha t two e n z y m e : l y s o l i p i d complexes are present. S ince each po in t i s assayed i n the presence of 0.4 mM LPE, as mentioned i n the f i g u r e legend, the assay w i l l conta in a CT:LPE complex, which has a low Km f o r CTP, and a CT:LRC complex which appears to have a high K f o r CTP. In a d d i t i o n the V of each o f these two forms are not the same, m max The presence o f both o f these forms i n the assay leads to an exper imental v e l o c i t y equat ion y i e l d i n g square terms a s soc i a ted w i th the v e l o c i t y and K . The r e s u l t o f t h i s i s one f i nd s curved p l o t s r a the r than c l a s s i c a l M i c h a e l i s -Menten k i n e t i c p l o t s ' ( i . e . s t r a i g h t l i n e s ) . I t i s obvious i n F i g . 32 that as the LPC/LPE r a t i o i s increased there i s d e v i a t i o n from l i n e a r i t y p r ov i d i n g evidence f o r the presence of these two l y so l ip id/enzyme complexes. (h) Aggregat ion of the CTP:phosphocho1ine c y t i d y l y l t r a n s f e r a s e Another i n t e r e s t i n g phenomenon a s soc i a ted w i th t h i s enzyme i s i t s a b i l i t y to aggregate from a low molecu la r weight form to a high molecu la r weight form. As mentioned p r e v i o u s l y , t h i s aggregat ion can be seen to take p lace over seve ra l days i n r a t l i v e r c y to so l s to red at 4°C (191). Reports from Weinhold et aZ.(214) s tudy ing the r a t lung form of the c y t i d y l y l t r a n s -ferase a l s o noted t h i s aggregat ion process and i m p l i c a t e d pho spha t i d y l -g l y c e r o l (PG) as the aggregat ion and a c t i v a t i n g f a c t o r . We were i n t e r e s t e d to see what e f f e c t t h i s l i p i d had on the r a t l i v e r form of the enzyme. 112. Fresh r a t l i v e r cy to so l (3 ml) was incubated w i th 1 ml (0.15 mg) o f a pho spha t i dy l g l y ce ro l suspension ( F i g . 33) f o r 24 h at 4°C. A c on t r o l sample o f r a t l i v e r c y to so l (3 ml) was incubated w i t h 1 ml s a l i n e f o r the same time pe r i od . Although PG i s ab le to aggregate the c y t i d y l y l -t r an s f e r a se in vitro, the r o l e of t h i s l i p i d i n the aggregat ion o f the enzyme i n r a t l i v e r cy to so l s t i l l remained unknown. A major reason f o r b e l i e v i n g t ha t PG i s not i n vo l ved i n the aggregat ion o f the c y t i d y l y l -t r an s f e r a se i n r a t l i v e r i s because PG i s not present i n r a t l i v e r cy to so l (<0.6 uM, P.C. Choy). Th i s i s not the case w i th r a t lung where PG i s a major component o f the l i p i d s . I t i s po s s i b l e however tha t the amount o f PG necessary to cause aggregat ion over a pe r i od o f days i s very small and po s s i b l y undectable by convent iona l i s o l a t i o n procedures. In an attempt to ensure complete removal o f any PG i n r a t l i v e r c y t o s o l , a P G - s p e c i f i c phosphol ipase A was i s o l a t e d from r a t red c e l l s (215). The i s o l a t e d enzyme has a s p e c i f i c a c t i v i t y of 0.3 umol•min"^ «mg p r o t e i n " ^ (P.C. Choy). I t s a c t i v i t y was approximately 100 - fo ld h igher w i t h PG as s ub s t r a t e than w i th e i t h e r pho spha t i dy l cho l i ne o r phosphat idy lethanolamine. The product o f the r e a c t i o n , l y s o p h o s p h a t i d y l g l y c e r o l , was i n e f f e c t i v e i n caus ing aggre-gat ion o f the c y t i d y l y l t r a n s f e r a s e (P.C. Choy). The e f f e c t o f t h i s phosphol ipase A on the aggregat ion o f the c y t i d y l y l t r a n s f e r a s e was s t ud i ed w i th f r e s h l y prepared r a t l i v e r c y t o s o l . Four ml a l i q u o t s o f cy to so l were incubated w i t h e i t h e r 0.5 ml (2 mg) o f the phosphol ipase A or w i th 0.5 ml (2 mg) o f albumin s o l u t i o n as c o n t r o l , f o r 48 h a t 4°C. As dep i c ted i n F ig .34 % s l i g h t l y l e s s H-form was generated i n the pho spho l i pa seA t r ea ted cy to so l compared to c o n t r o l , but the r a t i o To face page 113? F igure 33. E f f e c t o f phosphat idy l g l y c e r o l on the aggregat ion o f CTP: phosphocholine c y t i d y l y l t r a n s f e r a s e i n r a t l i v e r c y t o s o l . Fresh r a t l i v e r cy to so l (3 ml of 20% homogenate i n i s o t o n i c s a l i n e ) was incubated w i th 1 ml (0.15 mg) o f a pho spha t i d y l -g l y c e r o l suspension (•—•) o r w i th 1 ml s a l i n e (o—a) f o r 24 h a t 4°C. The H-form and L-form o f the c y t i d y l y l t r a n s f e r a s e were re so l ved by chromatography on a Sepharose 6B column (2.6 cm x 40 cm) e q u i l i b r a t e d w i t h B u f f e r A. F r a c t i on s (7 ml) were c o l l e c t e d and 40 y l a l i q u o t s from each f r a c t i o n was assayed f o r enzyme a c t i v i t y i n the presence of r a t l i v e r l i p i d . The H-form o f the enzyme was e l u t e d near the vo id volume (VQ) of the column. Figure 33. To face page 114. F igure 34. E f f e c t of a p h o s p h a t i d y l . g l y c e r o l - s p e c i f i c phosphol ipase A on the aggregat ion o f the CTP:phosphocholine c y t i d y l y l t r a n s f f e r a s e . Fresh r a t l i v e r c y to so l (4 ml o f a 20% homogenate i n i s o t o n i c s a l i n e ) was incubated w i t h 0.5 ml (2 mg) of phosphat idy l -g l y c e r o l - s p e c i f i c phosphol ipase A ( •— • ) i s o l a t e d from r a t red b lood c e l l s (215), o r w i th 0.5 ml albumin (2 mg) (• a ) , f o r 48 h a t 4°C. The H-form and L-form o f the enzyme were re so l ved by Sepharose 6B chromatography as desc r ibed i n F i g . 114. Figure .34. 1 1 5 . of the H-form/L-form remained the same. S ince the r a te o f aggregat ion o f the c y t i d y l y l t r a n s f e r a s e was not s i g n i f i c a n t l y a f f e c t e d by such t reatment, i t was concluded t ha t PG i s not important f o r the aggregat ion o f . t h e c y t i d y l y l t r a n s f e r a s e i n r a t l i v e r c y t o s o l . The on ly l i p i d i s o l a t e d from r a t l i v e r which was capable o f aggregat ing the c y t i d y l y l t r a n s f e r a s e was d i a c y l g l y c e r o l (192). 1-1.6. DISCUSSION A. L i p i d - P r o t e i n I n t e r a c t i on s i n the P o l a r Headgroup Region o f Seml i k i  Fores t V i ru s The b i o l o g i c a l membrane has been, u n t i l recent t ime, a r e l a t i v e l y unknown q u a n t i t y . Even though the components o f the membrane have been i d e n t i f i e d , the ac tua l d e t a i l s o f the phy s i ca l i n t e r a c t i o n s between these components s t i l l remain unc lea r . One o f the reasons f o r t h i s i s due to the extreme complex ity of the i n t a c t b i o l o g i c a l membrane, w i th most membranes c o n s i s t i n g o f a wide v a r i e t y o f l i p i d s and numerous p ro te in s i n var ious stages o f immersion i n t o the b i l a y e r . The number o f i n t e r a c t i o n s t a k i n g p lace w i t h i n these systems does not a l l ow the study o f s p e c i f i c l i p i d - p r o t e i n i n t e r a c t i o n s , r a t h e r one i s s tudy ing an average l i p i d - p r o t e i n i n t e r a c t i o n , which i s an o v e r s i m p l i f i e d i n t e r p r e t a t i o n o f the ac tua l i n t e r a c t i o n s tha t take p lace w i t h i n the membrane. The s o l u t i o n to such a problem would seem to be the c r e a t i o n o f a model system c o n s i s t i n g o f only a few components. Most of the usefu l i n fo rmat ion on l i p i d - p r o t e i n i n t e r a c t i o n s has come from the study of these model systems as p r e v i ou s l y de sc r ibed . However, these systems are not w i thout problems. When p ro te in s are added to phospho l i p id mixtures i t i s d i f f i c u l t to know whether they are rega in ing t h e i r na tu ra l conformat ion a n d o i n t e r a c t i o n s w i th the l i p i d s . 117. Therefore i t would be usefu l to f i n d an i n t a c t b i o l o g i c a l membrane system that conta ins only a few components which w i l l reduce the number of i n t e r a c t i o n s w i t h i n the membrane. Seml i k i Forest v i r u s o f f e r s a number o f a t t r a c t i v e features over most i n t a c t b i o l o g i c a l membranes and r e c o n s t i t u t e d model systems. As mentioned i n the i n t r o d u c t i o n , Seml i k i Fo res t v i r u s i s formed by ' budd ing ' from the plasma membrane o f the host c e l l . The l i p i d envelope o f the v i ru s can e f f e c t i v e l y be cons idered a pure plasma membrane o f a c e l l except that on ly v i r a l l y coded membrane p ro te in s are found i n the membrane. This reduct ion i n the d i f f e r e n t types o f p ro te in s w i l l s u b s t a n t i a l l y reduce the number o f l i p i d - p r o t e i n i n t e r a c t i o n s . Another convenient fea tu re o f the v i r u s i s i t s s i z e . The v i r u s p a r t i c l e s are s p h e r i c a l and o f uniform s i z e (65 nm). ( F i g . 7 ) . Th is i s o f s i g n i f i c a n c e when s tudy ing the membrane us ing proton NMR (129). The only drawback i s the great d i f -f i c u l t y i n producing l a rge quant i te s o f v i r u s which are needed f o r NMR experiments. The study o f i n t a c t b i o l o g i c a l membranes by proton NMR has gene ra l l y r e s u l t e d i n the f a i l u r e to determine any usefu l i n fo rmat ion as to the dynamic o r g an i z a t i on o f the membrane. This i s i n pa r t due to the l a rge number o f i n t e r a c t i o n s i n the b i o l o g i c a l membrane but a l s o to the c o n t r i b u t i o n o f many nuc l e i to the proton NMR spectrum which causes problems i n ana l y s i ng the spectrum. Although the spectrum of Seml i k i Fores t v i r u s i s complex, the resonance o f the cho l i ne methyl groups i s q u i t e d i s t i n c t from the remaining po r t i on o f the spectrum ( F i g . 11). This makes i t cons ide rab ly e a s i e r to monitor changes i n the l i n e w i d t h o f the c h o l i n e methyl resonance. 118. What f a c t o r s can i n f l u e n c e the l i n e w i d t h o f the cho l i ne methyl resonance i n a b i o l o g i c a l membrane ? The proton magnetic resonance s i g na l from the methyl groups of the c ho l i n e moiety o f the phospho l ip id s i s broadened by magnetic d i p o l a r i n t e r a c t i o n s from other protons w i t h i n the methyl group, from protons on the neighbour ing two methyl groups, and from protons on neighbour ing molecu les . In the absence of mot ion, the spectrum f o r a non -o r i en ta ted phospho l i p id c ho l i n e would be over ten kHz i n l i n e w i d t h (239), however, when the phospho l i p id i s pa r t of a membrane, the c h o l i n e undergoes con s ide rab le motion which causes ex ten s i ve motional narrowing o f the proton NMR resonances. The d e t a i l s of t h i s headgroup motion i n the membrane of Seml ik i Forest v i r u s may be very complex. [The dynamics of the pho spha t i dy l cho l i ne headgroup i n a model membrane i s d i scussed by S e e l i g et al. ( 93 ) ] . The cho l i ne proton NMR s i gna l i n the v i r u s i s narrowed by a number of motions i n c l u d i n g : (a) r o t a t i o n of each methyl group about i t s symmetry a x i s , ( b ) . r o t a t i o n of the three methyl groups about t h e i r common symmetry a x i s , (c) f l u c t u a t i o n s i n the o r i e n t a t i o n of the headgroup moiety about the plane normal to the b i l a y e r , (d) r o t a t i o n o f the headgroup (or the e n t i r e molecule) about the plane normal to the b i l a y e r , (e) r o t a t i o n a l Brownian tumbl ing of the v i r u s i n the suspending medium,and ( f ) l a t e r a l d i f f u s i o n of the phospho l i p id molecules around the s phe r i c a l v i r a l membrane.'(216). The e f f e c t of thermolys in d i g e s t i o n of Seml ik i Fores t v i r u s i s to remove the su r face g l y cop ro te i n sp ikes wh i l e l e av i ng the hydrophobic reg ions o f the p ro te i n s i n the membrane (210,211). I t i s u n l i k e l y t ha t i n t e r a c t i o n s between the phospho l ip ids and the g l y cop ro te i n s can i n h i b i t motions (a) and (b) app rec i ab l y . We a n t i c i p a t e , t h e r e f o r e , that p r o t e o l y s i s 119.. of the v i r u s would a f f e c t on ly the motional narrowing mechanisms ( c ) , ( d ) , and ( e ) , f o r t he " pho spho l i p i d headgroups on the out s ide o f the membrane and on ly mechanism (e) f o r the c ho l i n e headgroups on the i n s i d e o f the membrane. When the g l y c o p r o t e i n sp ikes of the v i r u s are removed, the e f f e c t i v e rad ius o f the v i r u s decreases from a value of not more t ha t 32 nm to about 25 nm as determined by e l e c t r o n microscopy (210). For s t r u c t u r e s on the o rder o f 50 nm i n d iameter and assuming a l a t e r a l o 9 d i f f u s i o n constant of D^- 2.6 x 10 cm /s (241 )> the c o n t r i b u t i o n of both D^ , ;and D^. (Equation ' . 3). to the r o t a t i o n a l c o r r e l a t i o n t ime , - t , i s s i g n i f i c a n t . The removal of the g l y c o p r o t e i n sp ikes lead, to a 1 . 3 - f o l d r educ t i on i n the v i r u s r ad i u s . This r educ t i on i n p a r t i c l e s i z e w i l l lead to a l a r g e r c o n t r i b u t i o n o f D r o n ' t than D t due to i t s cub ic r e l a t i o n s h i p . * the r e s u l t - o f t h i s r educ t i on i n the r o t a t i o n a l c o r r e l a t i o n time w i l l l ead to a reduct ion i n the l i n e w i d t h o f a l l proton NMR resonances^ A f t e r thermolys in treatment ( F i g s . 12 and 14) the s i n g l e cho l i ne resonance f o r the i n t a c t v i r u s s p l i t s i n t o two peaks - presumably one from the out s ide and one from the i n s i d e of the v i r u s membrane (the narrow component of the c ho l i n e resonance i n F i g . 12 comprises about 40% of the t o t a l c h o l i n e resonance). S i hee the thermo ly s in would d i g e s t p r o t e i n on l y on the out s ide of the v i r a l membrane, the broad peak i s ass igned to the c h o l -ine con ta i n i n g l i p i d s on the. i n s i d e of the membrane and the narrow peak to the cho l i ne con ta i n i n g l i p i d s on the out s ide of the membrane. This dramatic narrowing cannot be a t t r i b u t e d on ly to an i nc rease i n Brownian tumbl ing s i nce a narrowing o f a t most a f a c t o r o f about two can be expected by the decrease i n the v i r u s r ad i u s . 120; We undertook severa l experiments us ing the v i r u s l i p i d e x t r a c t , a mock v i r u s l i p i d sample, and egg pho spha t i d y l cho l i ne . The cho l i n e resonance obta ined w i th the v i r u s l i p i d l iposome sample (10 Hz, F i g . 15) and the egg phospha-t i d y l c h o l i n e v e s i c l e s ( 7 Hz, F i g . 16) would i n d i c a t e tha t the c ho l i n e resonance of the t h e r m o l y s i n - t r e a t e d v i r u s may not be unreasonably narrow. The r e s u l t obta ined us ing the mock v i r u s l i p i d sample ( F i g . 16) i s compl icated by the f a c t t ha t the sample does not have the same f a t t y a c i d composit ion and does not have g l y c o l i p i d and phosphat idy l i n o s i t o l . However, t h i s i n fo rmat ion i s not i n c o n s i s t e n t w i th increased motion of the c ho l i n e head-groups as a r e s u l t of thermolys in d i g e s t i o n of the v i r a l g l y c o p r o t e i n sp i ke s . The on ly other system to be s t ud i ed w i th regard to the e f f e c t o f protease d i g e s t i o n of the g l y c o p r o t e i n sp ikes has been V e s i c u l a r 31 S t o m a t i t i s v i r u s . Two groups have been i n v e s t i g a t i n g t h i s v i r u s us ing P NMR (141) and 1 3 C NMR (139,140). From 1 3 C s p i n - l a t t i c e r e l a x a t i o n (T-,) da ta , S t o f f e l et al. concluded t ha t the motion of the phospho l i p id c ho l i n e headgroups of V e s i c u l a r S t o m a t i t i s v i r u s are more r e s t r i c t e d by the d i g e s t i o n of the v i r a l g l y cop ro te i n s w i th t r y p s i n . On the other hand, Moore et al. 31 us ing P s p i n - l a t t i c e r e l a x a t i o n data concluded tha t t h i s same treatment of the v i r u s leads to an inc rease i n the motion of the phosphate of the head-group and presumably to an inc rease i n the m o b i l i t y o f the headgroup i t s e l f . That the two groups reached opposing conc lu s ions i s a man i f e s t a t i on of the d i f f i c u l t i e s i n i n t e r p r e a t t i o n of nuc lear s p i n - l a t t i c e r e l a x a t i o n data i n such heterogeneous systems. -12:1. To t h i s po in t however the nature of the i n t e r a c t i o n s between the p o l a r -headgroups of the phospho l ip ids and the membrane p r o t e i n remain unknown. B. I nco rpora t ion o f Deuterated Chol ines i n t o BHK-21 C e l l s . Proton magnetic resonance i s h e l p f u l when s tudy ing a resonance which i s r e so l ved from the bulk of the proton resonances. However, i t does not e a s i l y lend i t s e l f to a q u a n t i t a t i v e e va l ua t i on o f the changes i n the motion o f a p a r t i c u l a r nuc leus . Therefore i t would be advantageous to s e l e c t a nucleus which i s s e n s i t i v e to a n i s o t r o p i c motion and gives q u a n t i t a t i v e i n fo rmat ion on the l o c a l o rder exper ienced by the nuc leus. Deuterium i s such a nuc leus . I t a l s o has the advantage of having a low natura l abundance (0.015%), t he r e f o r e by s e l e c t i v e l y deu te ra t i ng a c e r t a i n area o f a molecule one can ob ta in i n fo rmat ion about the l o c a l o rder exper ienced by the nucleus only at t h a t s i t e i n the molecu le. The r e s u l t s obta ined us ing proton NMR y i e l d on ly a q u a l i t a t i v e e va l ua t i on o f changes i n the motion o f the c ho l i n e methyl groups. In o rder to conf i rm the r e s u l t obta ined us ing proton NMR and to a l s o ob ta in a more q u a n t i t a t i v e eva l ua t i on o f the inc reased motion o f the c ho l i n e headgroup we chose to grow the v i r u s host c e l l on medium supplemented w i th t r i - t r i d e u t e r o m e t h y l c h o l i n e i n the hope o f l a b e l i n g a high percentage of the cho l i ne con ta i n i ng l i p i d s . As desc r ibed i n the Resu l t s sec. B(b) (Table 6) t h i s l a b e l e d spec ies was not i nco rpo ra ted i n t o pho spha t i d y l -c h o l i n e . . Two o ther deuterated s pec i e s , mono- t r ideuteromethy lcho l ine and d i - t r i d e u t e r o m e t h y l c h o l i n e , were s yn thes i zed and shown to be e a s i l y i n c o r -porated i n t o the cho l i ne con ta i n i ng l i p i d s . The f a c t t ha t both o f the l a t t e r two deuterated spec ies were i nco rpo ra ted and not the 122.. t r i - t r i d e u t e r o m e t h y l c h o l i n e was thought to be anomalous. Th is spec ies has been shown to be incorporated i n t o r a t l i v e r c ho l i n e con ta i n i n g l i p i d s as we l l as i n t o mouse LM c e l l c h o l i n e c o n t a i n i n g l i p i d s (217). I t was o f i n t e r e s t to determine the s i t e o f blockage o f i n c o r p o r a -t i o n . Several p o s s i b i l i t i e s e x i s t : 1) I t i s po s s i b l e tha t the molecule cannot be t r an spo r ted across the plasma membrane o f the c e l l , o r , 2) the molecule may not be u t i l i z e d by the enzymes o f the pho spha t i dy l cho l i ne b i o s y n t h e t i c pathway. The r e s u l t s o f Table 7 show tha t t r i - t r i d e u t e r o -methy lcho l ine i s an e f f e c t i v e sub s t ra te f o r c h o l i n e k i na se , the f i r s t enzyme i n the pathway l ead i ng to pho spha t i d y l cho l i ne . There fo re , unless the subsequent two enzymes i n t h i s pathway are unable to u t i l i z e the deuterated phosphochol ine, then the on ly a l t e r n a t i v e f o r the l ack o f i n c o r p o r a t i o n would be due to i n h i b i t i o n o f t r an spo r t . 3 To i n v e s t i g a t e t h i s a spect , t r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - H ] -cho l i ne was chem ica l l y synthes i zed f o r use i n t r an spo r t s t u d i e s . Unfor -3 t una te l y t h i s product was contaminated w i th [ 1 ,2 - H]-ethanolamine. I f 3 t r i - t r i d e u t e r o m e t h y l - [ l , 2 - H ] - cho l i ne i s not ab le to be t ranspor ted then there should be no uptake o f the l abe l i n t o the c e l l . Due to the con-taminat ion o f the l a b e l l e d c ho l i n e w i th [1 ,2 - H]-ethanolamine we d i d expect to observe some uptake o f r a d i o a c t i v i t y but i t should a l l be a s soc i a ted w i th phosphat idy lethanolamine and i t s p re su r so r s . As shown i n F i g . 19 there was an i n i t i a l l o s s o f r a d i o a c t i v i t y from the medium but a t 37 min t h i s uptake plateaued l e a v i n g r a d i o a c t i v i t y i n the medium. The uptake 3 o f [ H ] - cho l i ne d i d not show t h i s behaviour. Ana l y s i s o f the o rgan ic 3 s o l ub l e f r a c t i o n from c e l l s incubated w i th t r i - t r i d e u t e r o m e t h y l - [ 1 , 2 - H ] -cho l i ne showed t ha t a l l o f i t was a s soc i a ted w i th phospha t idy le -thanolamine. No r a d i o a c t i v i t y was found to be i nco rpo ra ted i n t o pho spha t i dy l cho l i ne ( F i g . 20). Although the aqueous s o l ub l e f r a c t i o n 123.. was not ana ly sed, s i nce only 10% o f the t o t a l absorbed r a d i o a c t i v i t y was conta ined i n t h i s f r a c t i o n (Table 8 ) , i t i s most l i k e l y t ha t the l a b e l e d ma te r i a l s are r a d i o a c t i v e precursors o f phosphat idy lethanolamine. I t i s concluded t he re fo re t ha t t r i - t r i d e u t e r o m e t h y l c h o l i n e i s unable to be i nco rpo ra ted i n t o c e l l u l a r pho spha t i dy l cho l i ne because i t cannot be-'; t r an spo r ted . C. CTP:phosphocholine C y t i d y l y l t r a n s f e r a s e : L i p i d - P r o t e i n I n t e r a c t i on s The ma jo r i t y o f the s tud ie s on l i p i d - p r o t e i n i n t e r a c t i o n s deal w i t h the e f f e c t s o f the membrane p r o t e i n i n the behaviour o f the bulk l i p i d . S tud ies on systems such as the cytochrome ox idase (108,114-116), the C a + + ATPase (117), and rhodopsin (118) a l l deal w i th the s t r u c t u r e of the l i p i d surrounding the p r o t e i n . However i n a b i o l o g i c a l membrane the s i t u a t i o n i s not one s i ded . Although p ro te in s may be ab le to o rder or d i s o r de r membrane l i p i d s , these l i p i d s are a l so capable o f a f f e c t i n g the performance o f an enzyme which i s a s soc i a ted d i r e c t l y o r p e r i p h e r a l l y w i th the membrane l i p i d . Very l i t t l e study has been done i n t h i s area presumably due to the comp l i ca t i on s a s s oc i a ted w i t h the i n s o l u b i l i t y o f l i p i d s i n aqueous s o l u t i o n . I t i s c l e a r however, t h a t some p ro te in s have s p e c i f i c requirements f o r the presence o f l i p i d f o r enzymatic a c t i v i t y . Often the a c t i v a t i o n o f an enzyme by the a d d i t i o n o f exogenous l i p i d merely r e f l e c t s the p ro te in s need f o r a hydrophobic environment. Even though most o f these p ro te in s can be i s o l a t e d i n a s o - c a l l e d ' s o l u b l e ' s t a t e , the m a j o r i t y o f them are a s s o c i a t e d , a t l e a s t p e r i p h e r a l l y , w i th the membranes o f the c e l l . But some o f these p r o t e i n s , such as pyruvate ox idase (175-177), L - l a c t a t e dehydrogenase (174), 3-hydroxybutyrate T24. dehydrogenase (181-183), malate ox idase (178), and ma la te - v i t amin K reductase (171) respond i n a very s p e c i f i c manner to added l i p i d . Only one o f the above enzymes however responds to a s p e c i f i c l i p i d c l a s s and f a t t y a c i d compos i t ion. 3-hydroxybutyrate dehydrogenase i s shown to r equ i re unsaturated phosphat idy l cho l i ne s i n o rder to b i nd NADH, which i s e s s e n t i a l f o r enzyme a c t i v i t y . The ea r l y work o f F i scus and Schneider (190) on the r a t l i v e r CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e suggested that t h i s enzyme may be dependent on l i p i d f o r a c t i v i t y . Fu r ther i n v e s t i g a t i o n by Choy et al. (212) i m p l i c a t e d LPE as the a c t i v a t i n g f a c t o r i n r a t l i v e r cy to so l s t o r ed at 4°C f o r seve ra l days. They a l s o noted t h a t LPC was a potent i n h i b i t o r of c y t i d y l y l t r a n s f e r a s e a c t i v i t y in vitro. With these r e s u l t s we thought tha t t h i s system might be i d e a l f o r the study of l i p i d - p r o t e i n i n t e r a c t i o n s as we l l as a po s s i b l e model f o r enzyme r e g u l a t i o n by l i p i d s . S ince we were unable to ob t a i n l a rge amounts o f p u r i f i e d p r o t e i n , which would be r equ i r ed f o r phy s i ca l b iochemical s t u d i e s , a c l a s s i c a l k i n e t i c approach was adopted to i n v e s t i g a t e the e f f e c t o f these two l y s o l i p i d s on the behaviour o f the p r o t e i n . Our r e s u l t s i n d i c a t e d t ha t the a c t i v a t i o n o f the c y t i d y l y l -t r an s f e r a se by LPE was due to a reduct ion i n the K f o r CTP ( F i g . 30). This l y s o l i p i d showed l i t t l e e f f e c t on the Km f o r phosphocholine ( F i g . 31). LPC on the o the r hand e x h i b i t e d the exact oppos i te e f f e c t o f LPE, by i n c r e a s i n g the Km f o r CTP ( F i g . 32). The e f f e c t o f LPC i n h i b i t i o n cou ld not be a t t r i b u t e d to compet i t i ve i n h i b i t i o n w i th phosphochol ine. 125. These r e s u l t s would seem to suggest tha t some type of enzyme r e g u l a t i o n could be t a k i n g p lace i n v o l v i n g the two l y s opho spho l i p i d s . However, before such a conc lu s i on can be reached, i t must be determined i f the events being observed in vitro are a c t u a l l y t ak i ng p lace in vivo. Fur ther i n the d i s cu s s i on evidence w i l l be presented which supports a p h y s i o l o g i c a l r o l e f o r LPE i n the r e g u l a t i o n of the CTPrphosphocholine c y t i d y l y l t r a n s f e r a s e . (a) Aggregat ion o f the CTP:phosphocholine C y t i d y l y l t r a n s f e r a s e The aggregat ion o f the c y t i d y l y l t r a n s f e r a s e f o r a low molecu la r weight L-form to a heav ie r molecu la r weight H-form i s an i n t e r e s t i n g phenomenon i n i t s e l f as we l l as being the key to the p u r i f i c a t i o n of the enzyme (191). A s i m i l a r process takes p lace i n the r a t lung (214). The f e t a l r a t lung conta ins predominately the L-form of the enzyme, however, w i th lung maturat ion there i s an inc rease i n the amount of H-form as w e l l a s . an inc rease i n the pho spha t i d y l g l y ce ro l c oncen t r a t i on . In the r a t l ung , pho spha t i d y l g l y ce ro l has been i m p l i c a t e d as being both the a c t i v a t i n g and -. aggregat ing f a c t o r (214). S ince the r a t l i v e r form of the c y t i d y l y l t r a n s f e r a s e i s a c t i v a t e d by LPE (212), i t appeared tha t the l i p i d requirements of the two enzymes are d i f f e r e n t . Although pho spha t i d y l g l y ce ro l w i l l a c c e l e r a t e the in vitro 126. aggregat ion o f the enzyme ( F i g . 3 3 ) , our r e s u l t s i n d i c a t e tha t t h i s l i p i d i s not important i n the aggregat ion o f the c y t i d y l y l t r a n s f e r a s e when s t o r ed a t 4°C f o r seve ra l days. Stud ies w i th the r a t lung and r a t l i v e r enzymes i n d i c a t e tha t the aggregat ion requ i re s between 0.05-0.5mM pho spha t i d y l -g l y c e r o l and s i n ce no pho spha t i d y l g l y ce ro l cou ld be detected i n r a t l i v e r cy to so l ( lower de tec t i on l i m i t < 0.6 uM) i t s r o l e i n c y t i d y l y l t r a n s f e r a s e aggregat ion was u n l i k e l y . This conc lu s i on was f u r t h e r supported by the lack o f e f f e c t on aggregat ion when r a t l i v e r cy to so l was t r e a t e d w i t h a p h o s p h a t i d y l g l y c e r o l - s p e c i f i c phosphol ipase A ( F i g . 3 4 ) . i f t race quan-t i t i e s o f pho spha t i d y l g l y ce ro l were present and re spons i b l e f o r the aggre-gat ion o f the c y t i d y l y l t r a n s f e r a s e i n the c y t o s o l , the phosphol ipase A should have removed i t and prevented aggregat ion. The f a c t t h a t no changes were observed i n the r a te of aggregat ion negated the r o l e o f t h i s l i p i d i n the aggregat ion of the r a t l i v e r form of the enzyme. A r epo r t by Choy et al. has i m p l i c a t e d d i a c y l g l y c e r o l as the only l i p i d capable of aggregat ing the r a t l i v e r c y t i d y l y l t r a n s f e r a s e (192). Several quest ions s t i l l remain to be answered w i th regards to the p h y s i o l o g i c a l s i g n i f i c a n c e o f the a c t i v a t i o n and aggregat ion phenomena. Work r e c e n t l y completed w i t h i n our l abo ra to r y by Lim and.Vance (223) suggests a p h y s i o l o g i c a l r o l e f o r LPE a c t i v a t i o n and po s s i b l y d i a c y l g l y c e r o l aggregat ion o f the c y t i d y l y l t r a n s f e r a s e . Previous s tud ie s w i th ra t s (218) and swine (219) fed on high c h o l e s t e r o l d i e t s e x h i b i t e d markedly a l t e r e d concent ra t ions and d i s t r i b u -t i on s of plasma l i p o p r o t e i n s as w e l l as a severa l f o l d e l e v a t i o n o f plasma c h o l e s t e r o l and pho spho l i p i d . By feed ing female w i s t a r r a t s a high c h o l e s t e r o l / c h o l a t e d i e t , Lim and Vance observed a 2 - f o l d i n c r e a s e - i n 127. c y t i d y l y l t r a n s f e r a s e a c t i v i t y compared to con t ro l which was c o r r e l a t e d w i th a 3 - f o l d inc rease i n c y t o s o l i c LPE. However, to t h i s po in t an i nc rease i n pho spha t i d y l cho l i ne b i o s yn the s i s has y e t to be demonstrated. They a l s o noted t h a t the hypercho le s te ro lemic r a t l i v e r cy to so l had 25-30% o f the t o t a l c y t o s o l i c c y t i d y l y l t r a n s f e r a s e i n the H-form whereas on ly 10% o f the enzyme i n the con t ro l r a t l i v e r c y to so l was i n the H-form. This 2 . 5 - f o l d i nc rease i n the H-form was c o r r e l a t e d w i th an approximately 2 . 5 - f o l d i nc rease i n the d i g l y c e r i d e content o f the hypercho le s te ro lemic r a t l i v e r c y t o s o l . Cons ider ing these f i nd i ng s i t i s p o s s i b l y not unreason-able to cons ider the p h y s i o l o g i c a l re levance o f CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e con t ro l by LPE and d i g l y c e r i d e . (b) Contro l o f the CTP:phosphocholine C y t i d y l y l t r a n s f e r a s e by  LPE and LPC The i n i t i a l s t ud i e s on the r a t l i v e r c y t i d y l y l t r a n s f e r a s e i n crude cy to so l repor ted t h a t l i p i d e x t r a c t s from f resh r a t l i v e r cy to so l had l i t t l e a c t i v a t i n g p o t e n t i a l unless they were, o x i d i z e d under an a i r stream f o r 15 h caus ing the format ion o f a h igher p ropor t i on of l y s o l i p i d s (190). The r e g u l a t i o n o f the c y t i d y l y l t r a n s f e r a s e by l y s o l i p i d s suggests a p o s i t i v e feedback mechanism whereby degraded phospho l ip ids r egu la te the s ynthes i s of pho spha t i d y l cho l i ne . A s i m p l i s t i c model f o r such a r e g u l a t i o n mechanism may be viewed, as f o l l o w s . The growth o f a c e l l r equ i re s t ha t phospho l ip ids be s yn thes i zed to form new membrane s t r u c t u r e s as w e l l as f o r o ther f u n c t i o n s . S ince most phospho l ip ids are i n a con t i nua l s t a t e o f tu rnove r , the r a t i o o f the amount o f a phospho l i p id to i t s degraded component(s) ( i . e . l y s o l i p i d s ) 128. should be r e l a t i v e l y constant . Assuming t h i s to be t r u e , then a bu i l dup o f pho spha t i dy l cho l i ne i n a c e l l w i l l a l so lead to an i nc rea se i n LPC concent ra t i on which w i l l i n t e r a c t w i t h the c y t i d y l y l t r a n s f e r a s e . In t h i s case, the overproduct ion o f pho spha t i dy l cho l i ne w i l l l ead to a reduct ion o f c y t i d y l y l t r a n s f e r a s e a c t i v i t y and a subsequent reduct ion i n pho spha t i d y l -cho l i ne b i o s y n t h e s i s . The r o l e o f LPE i n the r e g u l a t i o n o f t h i s system may be envisaged as he l p i n g to ma inta in a r e l a t i v e l y constant r a t i o o f phosphati dy1chol i ne/phosphati dy lethanolami ne. I f phosphat idy lethanolami ne synthes i s i s s t i m u l a t e d f o r some reason, then the l e v e l s o f LPE w i l l a l s o i nc rease caus ing s t i m u l a t i o n o f the c y t i d y l y l t r a n s f e r a s e . In t h i s way pho spha t i dy l cho l i ne b i o s yn the s i s w i l l be s t imu l a t ed and the pho spha t i d y l -cho l ine/phosphat idy lethano lamine r a t i o w i l l be mainta ined. The r e g u l a t i o n o f the c y t i d y l y l t r a n s f e r a s e i s based on changes i n the Km f o r CTP by the a c t i o n o f these two l y s o l i p i d s . I t must be determined whetherthe in vivo concent ra t i on o f CTP i s i n the proper range to a l l ow changes i n the Km f o r t h i s s ub s t r a te to regu la te the enzyme. Measurement of the CTP l e v e l i n r a t l i v e r (220) i n d i c a t e as average concent ra t i on of about 0.07mM. The t rue M i chae l i s constant f o r CTP, as determined by Choy et al. (191) and An se l l et al. (209) i s approximately 0.3mM. The l e v e l s o f CTP w i t h i n the r a t l i v e r should be low enough to permit changes i n the f o r t h i s s ub s t r a te to regu la te the enzyme. The involvement, i f any, o f the aggregat ion process i n the r e g u l a t i o n o f the c y t i d y l y l t r a n s f e r a s e and t he r e f o r e pho spha t i dy l cho l i ne s y n t h e s i s , i s obscure. Not enough i n fo rmat i on has been determined about the in vivo l e v e l s of the H-form ( i t may po s s i b l y be an a r t i f a c t a s s oc i a ted w i th the homogenization o f the l i v e r ) , which makes i t d i f f i c u l t to 1.29, specu la te on the p h y s i o l o g i c a l s i g n i f i c a n c e o f aggregat ion i n the r e g u l a -t i o n of phospho l i p id s yn the s i s . This model as presented i s very s i m p l i s t i c and conta ins severa l anomalies which cannot be so lved u n t i l more knowledge about the o v e r a l l scheme of phospho l i p id metabolism i n the r a t l i v e r has been obta ined . An i n i t i a l problem i s to determine the p h y s i o l o g i c a l s i g n i f i c a n c e o f LPC, i f any e x i s t s , i n c y t i d y l y l t r a n s f e r a s e r e g u l a t i o n . To t h i s po in t no in vivo r o l e i n c y t i d y l y l t r a n s f e r a s e r e g u l a t i o n has been demonstrated f o r t h i s l y s o l i p i d . I t does however occur w i t h i n the r a t l i v e r c e l l (222) which at l e a s t g ives i t the c a p a b i l i t y o f being i n vo l ved i n such a r egu l a to r y mechanism. A r o l e f o r LPE i n the r e g u l a t i o n o f pho spha t i dy l cho l i ne b i o s yn the s i s would seem to have some p h y s i o l o g i c a l s i g n i f i c a n c e i n l i g h t o f the r e s u l t s of Lim and Vance.(223). Phosphoglycer ide metabolism.and r e g u l a t i o n are very complex sub jec t s i n the r a t l i v e r c e l l . What i s p re -sented i n t h i s t he s i s may or may not be of p h y s i o l o g i c a l s i g n i f i c a n c e to the r e g u l a t i o n o f pho spha t i dy l cho l i ne b i o s y n t h e s i s , but a t best i t i s an extremely smal l p iece o f a vast puzz le which at the present time i s only beginning to be s o l ved . D. Suggestions f o r Future Work (a) Seml i k i Forest V i r u s r L i p i d Headgroup-Protein I n t e r a c t i on s When t h i s p r o j e c t was s t a r t e d , we d i d not r e a l i z e the d i f f i -c u l t i e s a s soc i a ted w i th the p repa ra t i on o f 50-100 mg samples of pure Seml i k i Forest v i r u s which are r equ i red f o r NMR exper iments. This i s one aspect of the p r o j e c t which must be improved before any f u tu re work can 130,. be done. To t h i s po in t i n t ime the b a s i c ob se rva t i on i s t ha t thermolys in d i g e s t i o n o f Seml i k i Fores t v i r u s appears to lead t o i nc reased motion o f the polarheadgroups o f the c ho l i n e con ta i n i n g l i p i d s . However be fo re the d e t a i l s o f these i n t e r a c t i o n s can be exp la i ned there are a number of areas to be i n v e s t i g a t e d . The nature o f the t he rmo l y s i n - t r ea t ed v i r u s p a r t i c l e must be r i g o r o u s l y analysed w i th respect to compos i t ion , s i z e , and f r a g i l i t y . Although the i n t a c t v i r u s appears to be s t a b l e to m u l t i p l e c e n t r i f u g a t i o n s and other man ipu la t i on s , the t he rmo l y s i n - t r ea t ed v i r u s may not be as s t a b l e . I t would a l so be i n f o rma t i ve to study the l i p i d asymmetry o f the v i r a l membrane. To t h i s po i n t i n time there have been no repor t s i n t h i s a rea. S ince we are removing the sugar and p r o t e i n po r t i on o f these ' s p i k e s ' from the membrane su r face i t i s not po s s i b l e to d i s t i n g u i s h whether the i n t e r a c t i o n s between the l i p i d and g l y cop ro te i n are mediated v i a the sugar po r t i on o f the p r o t e i n o r the p ro te i n p o r t i o n . Experiments i n v o l v i n g g l ycos idases may help to d i s t i n g u i s h between the two p o s s i b i l i t i e s . The use of deuterium NMR to i n v e s t i g a t e t h i s system i s very necessary. By s p e c i f i c a l l y l a b e l i n g the cho l i ne headgroups o f the . I ; phospho l i p i d s , a more q u a n t i t a t i v e determinat ion o f changes i n the mo lecu la r motion of the headgroups can be made. However the use o f deuterium NMR requ i res at l e a s t severa l f o l d inc reases i n the amount o f sample compared t o what i s r equ i r ed f o r proton NMR. Therefore u n t i l methods are dev i sed to obta in vast q u a n t i t i e s o f Seml ik i Fores t v i r u s , i t would not be adv i sab le to cont inue s tud ie s w i th t h i s system. 131; (b) CTP:Phosphocholi ne Cyt i d y l y l t r a n s f e r a s e : L i pi d -P ro te i n I n t e r a c t i o n The CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e has the p o t e n t i a l o f being an i n t e r e s t i n g system f o r the study o f l i p i d - p r o t e i n i n t e r a c t i o n s as we l l as po s s i b l y being i n vo l ved i n the r e g u l a t i o n o f phospho l i p id r e g u l a t i o n i n the r a t l i v e r . The s tud ie s presented i n t h i s t he s i s on ly deal w i t h the e f f e c t o f LPE and LPC on the k i n e t i c parameters o f the enzyme. However there are many quest ions tha t can be asked concerned w i t h the number o f b i nd i ng s i t e s f o r each o f the l y s o l i p i d s , and whether o r not they are b ind ing at the same or d i f f e r e n t s i t e s on the enzymes s u r f ace . Our i n i t i a l ob se rva t i on ( F i g . 33) suggests tha t they do not compete w i th each o ther f o r b ind ing however the r e s u l t i s not e n t i r e l y c l e a r . The use o f e q u i l i b r i u m b ind ing s tud ie s us ing r a d i o a c t i v e l y l a b e l e d l i p i d s may help to determine the number o f l i p i d s b i nd ing s i tes/enzyme. The s p e c i f i c i t y o f b ind ing i s an aspect of i n t e r e s t . The on ly d i f f e r e n c e between o l eoy l - LPE and o leoy l - LPC i s i n the headgroup reg ion and y e t each l y s o l i p i d has such an opposing e f f e c t on the enzymes a c t i v i t y . 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