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Regulation of rat hepatic phosphatidylcholine biosynthesis Pelech, Steven 1982

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REGULATION OF RAT HEPATIC PHOSPHATIDYLCHOLINE BIOSYNTHESIS by STEVEN PELECH B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Biochemistry) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1982 © S t e v e n Pelech, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Biochemistry  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date December 20, 1982. DE-6 (3/81) • • II ABSTRACT Several model systems were i n v e s t i g a t e d to e l u c i d a t e the mechanisms by which r a t l i v e r p h o s p h a t i d y l c h o l i n e s y n t h e s i s i s c o n t r o l l e d . CTP: p h o s p h o c h o l i n e c y t i d y l y l t r a n s f e r a s e was c l e a r l y the key r e g u l a t o r y enzyme f o r p h o s p h a t i d y l c h o l i n e f o r m a t i o n from c h o l i n e . This a m b i q u i t o u s enzyme was d e t e c t e d i n b o t h the c y t o s o l i c and m i c r o s o m a l f r a c t i o n s o f r a t l i v e r , a l t h o u g h 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 occurred i n the s o l u b l e f r a c t i o n . The d i s t r i b u t i o n of c y t i d y l y l t r a n s f e r a s e between these f r a c t i o n s was a l t e r e d when the r a t e o f p h o s p h a t i d y l c h o l i n e s y n t h e s i s was perturbed. T r a n s l o c a t i o n o f c y t i d y l y l t r a n s f e r a s e was observed i n r a t l i v e r during e a r l y development, w i t h s t a r v a t i o n and during a d i u r n a l r h y t h m . A r e d i s t r i b u t i o n o f c y t i d y l y l t r a n s f e r a s e was a l s o d e t e c t e d i n i s o l a t e d hepatocytes which were t r e a t e d w i t h g l u c a g o n , cAMP analogues or f a t t y a c i d s bound to albumin. The r a t e of phosphatidylcholine s y n t h e s i s was found to r e f l e c t the amount o f microsomal 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 i n h i b i t i o n of p h o s p h a t i d y l c h o l i n e s y n t h e s i s by glucagon or cAMP analogues was l i k e l y due t o p h o s p h o r y l a t i o n and i n h i b i 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 . Several l i n e s o f e v i d e n c e i n d i c a t e d 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 i n f r e s h r a t l i v e r c y t o s o l was p r o b a b l y phosphorylated and a c t i v a t e d upon d e p h o s p h o r y l a t i o n by endogenous p h o s p h o p r o t e i n phosphatases or a l k a l i n e phosphatase from hog i n t e s t i n e . A l t h o u g h the phosphorylation of c y t i d y l y l -t r a n s f e r a s e was apparently k i n e t i c a l l y " s i l e n t " , dephosphorylation r e s u l t e d i n an in c r e a s e d a f f i n i t y of the enzyme f o r membranes. F a t t y a c i d s s t i m u l a t e d de novo p h o s p h a t i d y l c h o l i n e s y n t h e s i s by a c c e l e r 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 - c a t a l y z e d r e a c t i o n . F a t t y a c i d s and t h e i r CoA d e r i v a t i v e s were shown t o s t i m u l a t e the 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 a c t i v i t y . However, these compounds f a i l e d to a c t i v a t e p a r t i a l l y 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 i i i appreciably. Apparently, f a t t y a c i d s , l i k e d e p h o s p h o r y l a t i o n , enhanced the t e n a c i t y of c y t i d y l y l t r a n s f e r a s e f o r membranes. Upon b i n d i n g to membranes, 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 c o u l d be elevated up to 45-fold, and the a f f i n i t y of the enzyme f o r t h e s u b s t r a t e , CTP, was i n c r e a s e d 20-fold. The i n f l u e n c e of glucagon, cAMP analogues and f a t t y a c i d s on the s y n t h e s i s of p h o s p h a t i d y l c h o l i n e by s u c c e s s i v e N-methylation was a l s o examined i n i s o l a t e d rat hepatocytes. Glucagon and cAMP analogues i n h i b i t e d the m e t h y l a t i o n pathway i n these c e l l s , but the a c t i v i t y of microsomal phosphatidylethanolamine m e t h y l t r a n s f e r a s e was e l e v a t e d . Fatty acids also reduced the formation of p h o s p h a t i d y l c h o l i n e from phosphatidylethanolamine. Fatty acids and t h e i r CoA d e r i v a t i v e s d i r e c t l y i n h i b i t e d the phosphatidyl-ethanolamine methyltransferase i n rat l i v e r microsomes. The coordinate c o n t r o l of hepatic phosphatidylcholine synthesis by cAMP and f a t t y acids may be important d u r i n g s t a r v a t i o n when the i n t r a c e l l u l a r l e v e l s of these compounds are increased. i v TABLE OF CONTENTS INTRODUCTION 1 (1.1.1.1) P h o s p h a t i d y l c h o l i n e - S t r u c t u r e and D i s t r i b u t i o n 1 THE ENZYMES AND PATHWAYS OF PHOSPHATIDYLCHOLINE BIOSYNTHESIS ELUCIDATION OF DE NOVO PHOSPHATIDYLCHOLINE BIOSYNTHESIS (1.2.1.1) H i s t o r i c a l P e r s p e c t i v e 2 (1.2.1.2) Choline Kinase 3 (1.2.1 .3) CTP: Phosphocholine C y t i d y l y l t r a n s f e r a s e 7 (1.2.1.4) CDP-Choline: 1 , 2 - D i a c y l - s n - G l y c e r o l Cholinephospho-t r a n s f e r a s e 12 SYNTHESIS OF PHOSPHATIDYLCHOLINE FROM PHOSPHATIDYLETHANOLAMTNE (1.2.2.1) Ph o s p h a t i d y l e t h a n o l a m i n e N - M e t h y l a t i o n 18 FORMATION OF PHOSPHATIDYLCHOLINE BY GROUP EXCHANGE (1.2 .3.1) Base Exchange 24 (1.2 .3.2) F a t t y Acyl Exchange 25 THE FUNCTIONS OF PHOSPHATIDYLCHOLINE AND ITS ROUTE OF SYNTHESIS IN VARIOUS MAMMALIAN TISSUES (1 .3-1.1) B i o l o g i c a l Membranes 26 (1.3.1.2) L i v e r 27 (1 .3.1 -3) B i l e 28 (1 .3.1.4) L i p o p r o t e i n s 29 (1 .3-1.5) Prostaglandins 29 (1 .3.1.6) Lung and Su r f a c t a n t 30 (1 .3.1-7) Brain and Sphingomyelin 33 (1 .3.1.8) I n t e s t i n e . 36 V (1.3.1.9) Heart 36 (1.3.1.10) Blood - C h o l e s t e r o l E s t e r i f i c a t i o n 37 (1.3.1.11) Blood - PE Methylation 37 POSSIBLE REGULATORY FACTORS IN PHOSPHATIDYLCHOLINE BIOSYNTHESIS REGULATION OF PHOSPHATIDYLCHOLINE SYNTHESIS FROM CHOLINE (1.1.1.1) Mechanisms For Regulation 43 (1.4.1.2) Choline Uptake 43 (1.4.1.3) I n t r a c e l l u l a r Supply of Choline f o r Ph o s p h a t i d y l c h o l i n e B i o s y n t h e s i s 47 (1.4.1.4) Choline Oxidation 47 (1.4.1.5) Choline Kinase 50 (1.4.1.6) Phosphocholine Phosphatase 52 (1.4.1.7) C y t i d y l y l t r a n s f e r a s e Catalyzes the Rate-Li m i t i n g Step of de novo PC B i o s y n t h e s i s 53 (1.4.1.8) S u b c e l l u l a r D i s t r i b u t i o n of C y t i d y l y l t r a n s f e r a s e 55 (1.4.1.9) Supply of Substrates f o r the C y t i d y l y l t r a n s f e r a s e Reaction.. 56 (1.4.1.10) Supply of D i g l y c e r i d e 58 (1.4.1.11) Cholinephosphotransferase 59 REGULATION OF PHOSPHATIDYLETHANOLAMINE N-METHYLATION (1.4.2.1) Supply of P h o s p h a t i d y l e t h a n o l a m i n e f o r N-Methylation 59 (1.4.2.2) Supply of Ado-Met 62 (1.4.2.3) Phosphatidylethanolamine Methyltransferase 62 (1.4.2.4) I n h i b i t i o n o f P h o s p h a t i d y l e t h a n o l a m i n e N-Methylation by S-Adenosylhomocysteine 63 PHOSPHATIDYLCHOLINE SYNTHESIS IN VARIOUS MODEL SYSTEMS v i DEVELOPMENTAL STUDIES (1.5.1.1) Rat 66 (1.5.1.2) L i v e r 66 (1.5.1.3) Lung 67 (1.5.1.4) Brain 71 VIRAL STUDIES (1.5-2.1) Viruses and Phospholipid Metabolism 73 TUMOUR PROMOTER STUDIES (1.5.3.1) Ph o s p h a t i d y l c h o l i n e B i o s y n t h e s i s i n Transformed C e l l s 78 SUPPLEMENTATION STUDIES (1.5.4.1) F a t t y Acid Supplementation 81 (1.5.4.2) C h o l e s t e r o l and Cholate Supplementation 82 (1.5.4.3) Monomethylethanolamine and Dimethylethanolamine Supplementation 84 (1.5.4.4) Deazaadenosine Supplementation 84 DEPRIVATION STUDIES (1.5.5.1) E s s e n t i a l F a t t y Acid D e p r i v a t i o n 86 (1.5.5.2) Choline D e p r i v a t i o n 87 (1.5.5.3) . Choline-Methionine D e p r i v a t i o n 90 (1.5.5.4) S t a r v a t i o n . 90 HORMONAL STUDIES (1.5.6.1) Estrogens 93 (1.5.6.2) G l u c o c o r t i c o i d s 95 (1.5.6.3) Thyroxine 96 (1.5.6.4) I n s u l i n 97 a m V I I (1.5.6.5) Glucagon 97 (1.5.6.6) cAMP and P r o t e i n Phosphorylation 98 (1.6.1.1) The Thesis I n v e s t i g a t i o n s 101 v i i i EXPERIMENTAL PROCEDURES 103 MATERIALS (2.1.1.1) Animals 103 (2.1.1.2) Enzymes, Chemicals and Radioisotopes 103 GENERAL PREPARATIVE PROCEDURES (2.2.1.1) I s o l a t i o n and Culture of Adult Rat Hepatocytes 103 (2.2.1.2) S u b c e l l u l a r F r a c t i o n a t i o n of Whole Rat L i v e r or I s o l a t e d Hepatocytes 109 (2.2.1.3) P a r t i a l P u r i f i c a t i o n of L-form 109 3 (2.2.1.4) Enzymatic S y n t h e s i s of [Me- H ] P h o s p h o c h o l i n e 110 (2.2.1.5) Folch L i p i d E x t r a c t i o n 111 (2.2.1.6) B l i g h and Dyer L i p i d E x t r a c t i o n 111 (2.2.1.7) Preparation of Total Rat L i v e r Phospholipid 111 (2.2.1.8) S a p o n i f i c a t i o n of L i p i d s . . . 1 1 3 GENERAL ANALYTICAL PROCEDURES PROTEIN ASSAYS (2.3.1.1) Lowry P r o t e i n Assay 113 (2.3.1.2) Bio-Rad P r o t e i n Assay ... 115 THIN-LAYER CHROMATOGRAPHY (2.3.2.1) Resolution of Aqueous Choline M e t a b o l i t e s . . . . 115 (2.3.2.2) Resolution of Phospholipids 116 (2.3.2.3) Resolution of G l y c e r o l i p i d s 116 METABOLITE POOL SIZE MEASUREMENTS (2.3-3.1) Phosphocholine 117 (2.3-3.2) D i g l y c e r i d e 117 i x (2.3.4.1) L i q u i d S c i n t i l l a t i o n Counting 118 (2.3.5.1) S t a t i s t i c s 118 ENZYME ASSAYS (2.4.1.1) Choline Kinase. 120 (2.4.1.2) CTP: Phosphocholine C y t i d y l y l t r a n s f e r a s e 120 (2.4.1.3) CDP-Choline: D i a c y l g l y c e r o l Cholinephosphotransferase 121 (2.4.1.4) Phosphatidylethanolamine Methyltransferase 122 X RESULTS 123 DISTRIBUTION OF CYTIDYLYLTRANSFERASE IN RAT (3.1.1.1) Rat L i v e r Contains the Largest Amount of C y t i d y l y l t r a n s f e r a s e . 123 (3.1.1.2) Most of the C y t i d y l y l t r a n s f e r a s e i s Located i n Cytosol 125 CHARACTERIZATION OF RAT LIVER CYTIDYLYLTRANSFERASE (3.2.1.1) Rat L i v e r C y t o s o l i c C y t i d y l y l t r a n s f e r a s e A c t i v a t i o n and Aggregation are Time- and Temperature-Dependent Processes 126 (3.2.'.1.2) L i p i d - A s s o c i a t e d C y t i d y l y l t r a n s f e r a s e has a Higher pH Optimum than the Non-Lipid-Associated Enzyme 126 (3.2.1.3) Incubation of Cytosol Increases the A f f i n i t y of C y t i d y l y l t r a n s f e r a s e for CTP But Not f o r Phosphocholine 128 (3.2.1.4) The Microsomal C y t i d y l y l t r a n s f e r a s e Reaction Goes Non-Linear With Time and Protein Sooner than the C y t o s o l i c Reaction 131 PURIFICATION OF CYTIDYLYLTRANSFERASE FROM RAT LIVER (3.3.1.1) Revisions of the O r i g i n a l P u r i f i c a t i o n P r o t o c o l 134 (3.3.1.2) H-form can be Dis s o c i a t e d by HDL or Octy l - G l u c o s i d e 135 (3.3.1.3) C y t i d y l y l t r a n s f e r a s e Binds to DEAE Ion-Exchange Resins 137 (3.3.1.4) C y t i d y l y l t r a n s f e r a s e Binds to Dymatrex Dye-Ligand Resins 140 (3.3.1.5) P a r t i a l l y 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 i s S t a b i l i z e d by Lysophosphatidylethanolamine 143 DEVELOPMENTAL STUDIES WITH RAT LIVER (3.4.1.1) A c t i v i t i e s of the Rat Li v e r P h o s p h a t i d y l c h o l i n e B i o s y n t h e t i c Enzymes During Pre- and Postnatal Development 144 DIURNAL RHYTHM STUDIES WITH RAT LIVER (3.5.1.1) Choline Kinase and Microsomal 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 i e s are Both Reduced Late at Night 148 x i FASTING STUDIES WITH RAT LIVER (3.6.1.1) Choline Kinase and Microsomal 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 i e s are Both Reduced by 24 h f a s t i n g of the Rat 150 (3.6.1.2) C y t i d y l y l t r a n s f e r a s e Seems to be Translocated During F a s t i n g o f the Rat...... 150 (3.6.1.3) F a s t i n g Increases the Level of a 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 A c t i v a t o r 152 GLUCAGON AND cAMP REGULATION OF PHOSPHATIDYLCHOLINE SYNTHESIS IN MONOLAYER CULTURES OF RAT HEPATOCYTES INFLUENCE OF cAMP ANALOGUES AND PHOSPHODIESTERASE INHIBITORS ON THE CDP-CHOLINE PATHWAY (3.7.1.1) cAMP Analogues and Phosphodiesterase I n h i b i t o r s I n i t i a l l y Cause an I n h i b i t i o n and L a t e r a S t i m u l a t i o n of [Me- 3H]-Choline I n c o r p o r a t i o n i n t o Phosphatidylcholine 157 (3.7.1.2) Chlorophenylthio-cAMP Reduces the Pool Size of Phosphocholine. 159 (3.7.1.3) Chlorophenylthio-cAMP I n h i b i t s Choline Uptake 162 (3.7.1.4) Chlorophenylthio-cAMP I n h i b i t i o n of P h o s p h a t i d y l c h o l i n e Synthesis i s Independent of the Concentration of Choline i n the Medium 164 (3.7.1.5) Chlorophenylthio-cAMP and Other cAMP Analogues I n h i b i t P h o s p h a t i d y l c h o l i n e Synthesis 165 (3.7.1.6) Prolongued Exposure of Hepatocytes to Chlorophenylthio-cAMP Stimulates P h o s p h a t i d y l c h o l i n e Synthesis 168 (3-7.1.7) Chlorophenylthio-cAMP does Not A f f e c t the Release of Betaine from Hepatocytes 168 (3.7-1.8) 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 s Reduced i n Microsomes and C y t o s o l s From Hepatocytes Treated with Chlorophenylthio-cAMP.. 170 INFLUENCE OF cAMP ANALOGUES ON PHOSPHATIDYLETHANOLAMINE N-METHYLATION 3 3 (3-7.2.1) cAMP Analogues Reduce [ H ] M e t h i o n i n e and [ H]Ethanolamine I n c o r p o r a t i o n i n t o Phosphatidylcholine 173 (3.7.2.2) Phosphatidylethanolamine Methyltransferase A c t i v i t y i s Stimulated i n Microsomes from Chlorophenylthio-cAMP-Treated Hepatocytes 177 x i i INFLUENCE OF GLUCAGON ON PHOSPHATIDYLCHOLINE BIOSYNTHESIS (3-7.3. D Glucagon Does Not I n h i b i t [Me- H ] C h o i i n e Uptake by Rat Hepatocytes 179 (3.7.3.2) Glucagon I n h i b i t s Phosphatidylcholine Synthesis From Choline and S t i m u l a t e s Betaine Secretion From Rat Hepatocytes 179 (3.7.3-3) Glucagon I n h i b i t s N - M e t h y l a t i o n o f Phosphatidylethanolamine i n Rat Hepatocytes 182 (3.7.3.4) Glucagon I n h i b i t s 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 Stimulates Phosphatidylethanolamine Methyltransferase A c t i v i t y i n Microsomes from Rat Hepatocytes 184 REGULATION OF RAT LIVER CYTIDYLYLTRANSFERASE BY REVERSIBLE PROTEIN PHOSPHORYLATION IN VITRO EVIDENCE FOR INHIBITION OF CYTIDYLYLTRANSFERASE BY PHOSPHORYLATION AND ACTIVATION BY DEPHOSPHORYLATION (3.8.1.1) Mg-ATP Prevents the Time-Dependent A c t i v a t i o n of 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 186 (3.8.1.2) NaF Prevents the Time-Dependent A c t i v a t i o n of 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 189 (3.8.1.3) Mg'ATP Reduces the pH Optimum of the C y t i d y l y l t r a n s f e r a s e Catalyzed Reaction and 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 189 (3.8.1.4) P r o t e i n Kinase I n h i b i t o r s Rapidly A c t i v a t e 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 193 (3.8.1.5) A l k a l i n e Phosphatase Rapidly A c t i v a t e s 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 But Not L-form 195 PHOSPHORYLATION REGULATES THE BINDING OF CYTIDYLYLTRANSFERASE TO MEMBRANES (3.8.2.1) Dephosphorylation Promotes the Aggregation of 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 200 (3.8.2.2) Dephosphorylation Promotes T r a n s l o c a t i o n of C y t i d y l y l t r a n s f e r a s e to Microsomes 203 IS CYTIDYLYLTRANSFERASE KINASE A CALCIUM-DEPENDENT PROTEIN KINASE? m • • XIII (3.8.3.1) Calcium Enhances Mg-ATP I n h i b i t i o n of C y t i d y l y l t r a n s f e r a s e . . . 205 (3-8.3.2) Pro t e i n Kinase I n h i b i t o r Reverses Calcium I n h i b i t i o n of C y t i d y l y l t r a n s f e r a s e 205 (3.8.3.3) Anaesthetics I n h i b i t 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 205 REGULATION OF HEPATIC PHOSPHATIDYLCHOLINE SYNTHESIS BY FATTY ACIDS EXPOSURE OF ISOLATED RAT HEPATOCYTES TO FATTY ACIDS ALTERS THE RATE OF PHOSPHATIDYLCHOLINE SYNTHESIS FROM CHOLINE (3.9-1.1) Long Chain F a t t y A c i d s A c c e l e r a t e the Rate of de novo Phos p h a t i d y l c h o l i n e Synthesis 208 (3.9.1.2) Fatty Acids Do Not Perturb Betaine Secretion From Hepatocytes 210 (3.9.1.3) F a t t y Acid Treatment of Hepatocytes Increases Microsomal 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 212 FREE AND ACTIVATED FATTY ACIDS DIRECTLY EFFECT LIVER CYTOSOLIC CYTIDYLYLTRANSFERASE (3.9.2.1) Fatty Acids A c t i v a t e 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 But Not L-form 214 (3.9.2.2) Fatty Acids Promote T r a n s l o c a t i o n of C y t i d y l y l t r a n s f e r a s e i n I n t a c t Hepatocytes and Aggregation of 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 216 (3.9.2.3) Oleoyl-Coenzyme A Stimula t e s the Conversion of L- to H-form i n L i v e r Cytosol 218 FATTY ACIDS ALTER THE RATE OF PHOSPHATIDYLETHANOLAMINE N-METHYLATION IN CULTURED RAT HEPATOCYTES (3.9 .3.1) Fatty Acids I n h i b i t the Methylation of Phos p h a t i d y l -ethanolamine 219 (3.9.3.2) Fatty Acid Treatment of Hepatocytes Reduces Microsomal Phosphatidylethanolamine Methyltransferase A c t i v i t y 222 COORDINATE REGULATION OF LIPID ANABOLISM BY cAMP, GLYCEROL AND FATTY ACIDS x i v CONTROL OF FATTY ACID SYNTHESIS IN ISOLATED RAT HEPATOCYTES (3.10.1.1) I n s u l i n Stimulates F a t t y Acid Synthesis 223 (3.10.1.2) Glucagon and Chlorophenylthio-cAMP I n h i b i t Fatty Acid Synthesis 223 CONTROL OF GLYCEROLIPID SYNTHESIS (3.10.2.1) cAMP Analogues I n h i b i t T r i g l y c e r i d e Synthesis 225 (3-10.2.2) G l y c e r o l S t i m u l a t e s d_e novo P h o s p h a t i d y l c h o l i n e Synthesis But Not Met h y l a t i o n of Phosphatidylethanolamine.... 229 (3.10.2.3) F a t t y Acids Reverse cAMP Analogue I n h i b i t i o n of T r i g l y c e r i d e Synthesis 232 (3.10.2.4) F a t t y A c i d s Reverse cAMP Analogue I n h i b i t i o n of de novo Ph o s p h a t i d y l c h o l i n e Synthesis 232 X V DISCUSSION 235 MICROSOMAL CYTIDYLYLTRANSFERASE CONTROLS HEPATIC PHOSPHATIDYLCHOLINE  SYNTHESIS VIA THE CDP-CHOLINE PATHWAY (4.1.1.1) C y t i d y l y l t r a n s f e r a s e i s the Key Regulatory Enzyme f o r Hepatic Phosphatidylcholine Synthesis ... 2 3 5 (4.1.1.2) Microsomal- and H-form Probably Represent the Same Species of C y t i d y l y l t r a n s f e r a s e 2 3 6 (4.1.1.3) T r a n s l o c a t i o n of C y t i d y l y l t r a n s f e r a s e i s Commonly Associated with A l t e r a t i o n s i n the Rate of Phosphatidylcholine Synthesis 2 3 7 FACTORS WHICH CONTROL TRANSLOCATION OF CYTIDYLYLTRANSFERASE (4.2.1.1) Phosphorylation Reduces C y t i d y l y l t r a n s f e r a s e Binding to Membranes 238 (4.2.1.2) F a t t y Acids and F a t t y Acyl-CoA Promote C y t i d y l y l t r a n s f e r a s e Binding to Membranes 238 (4.2.1.3) Other Factors which Influence C y t i d y l y l t r a n s f e r a s e Binding to Membranes 2 3 9 (4.2.1.4) P o s s i b l e Importance of P r o t e i n T r a n s l o c a t i o n f o r Regulation of Other Enzymes 240 PHOSPHORYLATION OF CYTIDYLYLTRANSFERASE (4.3.1.1) 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 s Regulated by Protein Phosphorylation 242 (4.3.1.2) Is C y t i d y l y l t r a n s f e r a s e a Phosphorylatable Protein? 242 (4.3.1-3) I d e n t i t y of C y t i d y l y l t r a n s f e r a s e Kinase(s) 243 PHOSPHORYLATION OF PHOSPHATIDYLETHANOLAMINE METHYLTRANSFERASE (4.4.1.1) Phosphatidylethanolamine M e t h y l t r a n s f e r a s e A c t i v i t y i s Stimulated by cAMP-Dependent P r o t e i n Kinase 244 COORDINATE REGULATION OF L I P I D SYNTHESIS BY FATTY ACIDS (4.5.1.1) F a t t y Acids and Acyl-CoA's Modulate L i p i d Synthesis at x v i M u l t i p l e S i t e s 246 (4.5.1.2) F a t t y A c i d s S t i m u l a t e C y t i d y l y l t r a n s f e r a s e and de novo Phosphatidylcholine Synthesis 247 (4.5.1.3) F a t t y Acids I n h i b i t Phosphatidylethanolamine Methyltransferase and Transmethylation of Phosphatidylethanolamine 247 REGULATION OF HEPATIC PHOSPHATIDYLCHOLINE SYNTHESIS DURING FASTING (4.6.1.1) C y t i d y l y l t r a n s f e r a s e i s Translocated During Fasting 249 (4.6.1.2) I n f l u x of F a t t y Acids to the L i v e r During S t a r v a t i o n May Reactivate C y t i d y l y l t r a n s f e r a s e and Phosphatidylcholine Synthesis 250 (4.6.1.3) S t i m u l a t i o n of Phos p h a t i d y l c h o l i n e Synthesis A f t e r Prolonged Exposure of Hepatocytes t o cAMP Analogues 250 (4.7.1.1) Unanswered Questions and Future D i r e c t i o n s 252 References 254 x v i i LIST OF TABLES Table Page 1. K i n e t i c P r o p e r t i e s of Choline Kinase 6 2. A c t i v a t i o n of C y t i d y l y l t r a n s f e r a s e by Phospholipids 9 3. K i n e t i c P r o p e r t i e s of C y t i d y l y l t r a n s f e r a s e 11 4. K i n e t i c P r o p e r t i e s of Cholinephosphotransferase 13 5. Compounds Which Influence Cholinephosphotransferase A c t i v i t y 14 6. S p e c i f i c i t i e s of Rat Cholinephosphotransferase and Ethanolamine-phosphotransferase f o r Various Species of D i a c y l g l y c e r o l 17 7. K i n e t i c P r o p e r t i e s of P h o s p h a t i d y l e t h a n o l a m i n e N-Methyltransferase 21 8. Model Systems Which have been Linked with Methylation of PE 38 9. K i n e t i c s and I n h i b i t o r s of Choline Uptake 46 10. M e t a b o l i t e Pool Sizes 48 11. Changes 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 i n Various Model Systems.. 54 12. Changes i n Cholinephosphotransferase A c t i v i t y i n Various Model Systems. 60 13. Changes i n PE Methyltransferase A c t i v i t y i n Various Model Systems.. 64 14. Influence of Ado-Hcy on PE M e t h y l a t i o n i n V a r i o u s Model Systems.... 65 15. Influence of Viruses on P h o s p h a t i d y l c h o l i n e Synthesis 74 16. Commercial Sources of Research M a t e r i a l s 104 17. D i s t r i b u t i o n of C y t i d y l y l t r a n s f e r a s e i n the Cytosols of Various Rat Organs 124 18. S u b c e l l u l a r D i s t r i b u t i o n of C y t i d y l y l t r a n s f e r a s e i n Rat L i v e r 125 19. P u r i f i c a t i o n of CTP:Phosphocholine C y t i d y l y l t r a n s f e r a s e from Rat L i v e r . . . , 134 20. P a r t i a l P u r i f i c a t i o n of a C y t o s o l i c A c t i v a t o r of C y t i d y l y l -t r a n s f e r a s e 156 21. I n h i b i t i o n of Phosphatidylcholine Synthesis by Various cAMP Analogues and Phosphodiesterase I n h i b i t o r s 1 6 9 22. A c t i v i t i e s of Phosphatidylcholine B i o s y n t h e t i c Enzymes from Rat Hepatocytes Treated with 0.5 mM Chlorophenylthio-cAMP f o r x v i i i 1.5, 6 and 12 h 172 23. E f f e c t of cAMP Analogues and Aminophylline on the Incorporation of [Me- H]Methionine by C u l t u r e d Rat Hepatocytes i n t o P h o s p h o l i p i d s 174 24. E f f e c t of cAMP Analogues and Aminophylline on the Conversion of Phosphatidylethanolamine to Pho s p h a t i d y l c h o l i n e by Cultured Rat Hepatocytes 177 25. A c t i v i t i e s of Phosp h a t i d y l c h o l i n e B i o s y n t h e t i c Enzymes from Rat Hepatocytes Treated with 100 nM Glucagon f o r 1.5 h 185 26. A c t i v i t i e s o f the de novo P h o s p h a t i d y l c h o l i n e B i o s y n t h e t i c Enzymes from Rat Hepatocytes Treated with 2 mM Oleate f o r 1 h 213 27. Phosphatidylethanolamine Methyltransferase A c t i v i t y i n Microsomes from Rat Hepatocytes Treated with 2 mM Oleate f o r 1 h 222 3 28. E f f e c t of Glucagon and Chl o r o p h e n y l t h i o - c A M P on [ HjAcetate I n c o r p o r a t i o n i n t o S a p o n i f i a b l e and Nonsaponifiable L i p i d s 224 29. E f f e c t of Oleate and Chlorophenylthio-cAMP on the D i g l y c e r i d e Pool S i z e i n I s o l a t e d Rat Hepatocytes 228 xix LIST OF FIGURES Figure Page 1. S t r u c t u r e of phosphatidylcholine 1 2. Pathway f o r phosphatidylcholine b i o s y n t h e s i s from c h o l i n e 4 3. P h o s p h a t i d y l c h o l i n e b i o s y n t h e s i s by s u c c e s s i v e N-methylation of phosphatidylethanolamine 19 4. Summary of c h o l i n e and phosphatidylcholine metabolism 42 5. Postulated events during TPA s t i m u l a t i o n of PC b i o s y n t h e s i s . . . . . 80 6. E s t a b l i s h e d and suspected s i t e s of r e g u l a t i o n by p r o t e i n phosphorylation 100 7. P r o t e i n standard curves 114 8. A c t i v a t i o n and aggregation of 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 at 20° C 127 9. Concentration dependence of various b u f f e r s on 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 a c t i v i t y 129 10. pH optima of the various forms of 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 129 11. E f f e c t of i n c u b a t i o n at 20° C and phospholipid on the apparent Km's of the 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 f o r CTP and phosphocholine 130 12. Apparent Km's of the microsomal c y t i d y l y l t r a n s f e r a s e f o r CTP and phosphocholine 132 13. Requirement of the c y t i d y l y l t r a n s f e r a s e f o r magnesium 132 14. L i n e a r i t y of the 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 c a t a l y z e d r e a c t i o n w i t h time and p r o t e i n 133 15- L i n e a r i t y of the microsomal c y t i d y l y l t r a n s f e r a s e c a t a l y z e d r e a c t i o n w i t h time and p r o t e i n 133 16. D i s s o c i a t i o n of H-form by various agents 136 17. Anion-exchange chromatography of 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 . 139 18. Dye-ligand chromatography of 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 141 19. A c t i v i t y of r a t l i v e r c h oline kinase during development 145 20. A c t i v i t y of r a t l i v e r cholinephosphotransferase during development...... 145 X X 21. A c t i v i t i e s of r a t l i v e r c y t o s o l i c and microsomal c y t i d y l y l -t r a n s f e r a s e during development 147 22. A c t i v i t y o f r a t l i v e r PE methyltransferase during development... 147 23. D i u r n a l rhythm of r a t l i v e r c h o l i n e kinase and c y t i d y l y l -t r a n s f e r a s e 149 24. A c t i v i t i e s o f the d_e novo p h o s p h a t i d y l c h o l i n e b i o s y n t h e t i c enzymes of r a t l i v e r during s t a r v a t i o n 151 25. S u b c e l l u l a r d i s t r i b u t i o n of 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 during s t a r v a t i o n 153 26. A c t i v a t i o n of l i v e r c y t o s o l i c c y t i d y l y l t r a n s f e r a s e from fed and f a s t e d r a t s by phospholipid 154 27. Gel f i l t r a t i o n chromatography of l i v e r c y t o s o l i c c y t i d y l y l -t r a n s f e r a s e from fed and fa s t e d r a t s 154 28. P a r t i a l p u r i f i c a t i o n of a l i v e r c y t o s o l i c a c t i v a t o r of c y t i d y l y l t r a n s f e r a s e from fed and 24 h fasted r a t s 155 29. E f f e c t of cAMP analogues and phosphodiesterase i n h i b i t o r s on [Me_ 3 H]choline uptake by c u l t u r e d r a t hepatocytes 158 30. E f f e c t of cAMP analogues and phosphodiesterase i n h i b i t o r s on the r e l a t i v e i n c o r p o r a t i o n o f [Me- 3 H ] c h o l i n e i n t o p h o s p h a t i d y l c h o l i n e 158 3 31. E f f e c t o f c h l o r o p h e n y l t h i o - c A M P on [Me- HJcholine i n c o r p o r a t i o n i n t o phosphocholine and betaine 160 32. E f f e c t of chlorophenylthio-cAMP on the c e l l u l a r phosphocholine pool s i z e 160 33. Concentration dependence of chlorophenylthio-cAMP i n h i b i t i o n of [Me- 3 H ] c h o l i n e c e l l u l a r uptake 163 34. E f f e c t of chlorophenylthio-cAMP on c h o l i n e uptake by c u l t u r e d r a t hepatocytes 163 35. Concentration dependence of chlorophenylthio-cAMP (CPT-cAMP) i n h i b i t i o n of [Me- 3 H ] c h o l i n e i n c o r p o r a t i o n i n t o p h o s p h a t i d y l c h o l i n e 166 36. E f f e c t of chlorophenylthio-cAMP on the i n c o r p o r a t i o n of [Me- ^ H ] c h o l i n e i n t o v a r i o u s c h o l i n e m e t a b o l i t e s 167 37. E f f e c t of c h l o r o p h e n y l t h i o - c A M P on the f a t e of [Me- 3 H]-c h o l i n e i n p r e l a b e l e d r a t hepatocytes 167 x x i 3 38. E f f e c t o f cAMPS on the d i s a p p e a r a n c e of [Me- H] c h o l i n e from c e l l u l a r phosphocholine and accumulation i n t o p h o s p h a t i d y l c h o l i n e 169 39. Latent e f f e c t of chlorophenylthio-cAMP on the i n c o r p o r a t i o n of [Me- 3H]choline i n t o v a r i o u s c h o l i n e m e t a b o l i t e s •••• 171 3 40. E f f e c t of c h l o r o p h e n y l t h i o - c A M P on the i n c o r p o r a t i o n of [ H]-ethanolamine i n t o phospholipids 174 3 41. E f f e c t of c h l o r o p h e n y l t h i o - c A M P on [ H]ethanolamine i n c o r p o r a -t i o n i n t o phosphatidylethanolamine and p h o s p h a t i d y l c h o l i n e by hepatocytes 175 42. E f f e c t of chlorophenylthio-cAMP on the rate of conversion of phosphatidylethanolamine to phosphatidylcholine by c u l t u r e d hepatocytes 178 43. E f f e c t of chlorophenylthio-cAMP on the a c t i v i t y of microsomal phosphatidylethanolamine N - m e t h y l t r a n s f e r a s e i n c u l t u r e d hepatocyes 178 3 44. E f f e c t o f g l u c a g o n on the i n c o r p o r a t i o n o f [Me- H]-c h o l i n e i n t o r a t hepatocytes 180 3 45. E f f e c t o f 100 nM g l u c a g o n on the d i s a p p e a r a n c e of [Me- H]-c h o l i n e from c e l l u l a r phosphocholine and accumulation i n t o p h o s p h a t i d y l c h o l i n e 181 46. E f f e c t of 100 nM glucagon on betaine release from r a t hepatocytes 181 3 47. C o n c e n t r a t i o n dependence o f glucagon i n h i b i t i o n of [Me- H]-phosphocholine i n c o r p o r a t i o n i n t o p h o s p h a t i d y l c h o l i n e 183 3 48. E f f e c t of glucagon on [ H]phosphatidylethanolamine i n c o r p o r a t i o n i n t o p h o s p h a t i d y l c h o l i n e 183 49. E f f e c t of Mg-ATP on time-dependent a c t i v a t i o n of 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 187 50. Concentration dependence of Mg-ATP i n h i b i t i o n of 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 188 51. E f f e c t of NaF on the time-dependent a c t i v a t i o n of 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 at 20 0 C 188 52. E f f e c t of Mg-ATP on the pH optimum of the 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 a c t i v i t y 190 53. E f f e c t of Mg-ATP on the apparent Km's of the 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 f o r CTP and phosphocholine 190 54. E f f e c t of t o t a l r a t l i v e r phospholipid on the s u b s t r a t e i n h i b i t i o n of the 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 by CTP 192 XXII Reversal of Mg-ATP i n h i b i t i o n of 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 by p r o t e i n kinase i n h i b i t o r s 194 E f f e c t of p r o t e i n kinase i n h i b i t o r a c t i v a t i o n of 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 on the s t i m u l a t i o n produced by phospho-l i p i d liposomes 196 E f f e c t of t o t a l r a t l i v e r phospholipid on the i n h i b i t i o n of c y t i d y l y l t r a n s f e r a s e by Mg-ATP 196 E f f e c t of p r o t e i n kinase i n h i b i t o r a c t i v a t i o n on the apparent Km of 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 f o r CTP 197 E f f e c t of t o t a l r a t l i v e r p h o s p h o l i p i d on the apparent Km of the Mg-ATP i n h i b i t e d c y t i d y l y l t r a n s f e r a s e f o r CTP 197 E f f e c t of a l k a l i n e phosphatase 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 . 199 E f f e c t of Mg-ATP and p r o t e i n kinase i n h i b i t o r on the aggregation of 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 201 E f f e c t of NaF on the aggregation of 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 202 E f f e c t of Mg-ATP and p r o t e i n kinase i n h i b i t o r on the d i s t r i b u t i o n of c y t i d y l y l t r a n s f e r a s e i n the pos t - m i t o c h o n d r i a l supernatant... 204 E f f e c t of 2 mM calcium on r a t l i v e r c y t o s o l i c and microsomal 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 206 Reversal of calcium i n h i b i t i o n of r a t l i v e r c y t o s o l i c c y t i d y l y l t r a n s f e r a s e by p r o t e i n kinase i n h i b i t o r 206 E f f e c t o f anaesthetics on 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 a c t i v i t y 207 3 E f f e c t o f 2 mM o l e a t e on the i n c o r p o r a t i o n o f [Me- Hjc h o l i n e i n t o p h o s p h a t i d y l c h o l i n e . . 209 3 E f f e c t of v a r i o u s f a t t y a c i d s on the i n c o r p o r a t i o n of [Me- H]-phosphocholine i n t o p hosphatidylcholine 211 A c t i v a t i o n of c y t i d y l y l t r a n s f e r a s e i n l i v e r c y t o s o l by f a t t y a c i d s 215 E f f e c t o f 100 o l e a t e on the apparent Km values of the 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 for CTP and phosphocholine 215 E f f e c t of oleate on L-form a c t i v i t y 217 S u b c e l l u l a r d i s t r i b u t i o n of c y t i d y l y l t r a n s f e r a s e i n o l e a t e -t r e a t e d hepatocytes 217 E f f e c t of olea t e and oleoyl-CoA on the aggregation of r a t l i v e r x x i i i 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 217 74. E f f e c t of o l e a t e on p h o s p h a t i d y l e t h a n o l a m i n e N-methylation i n r a t hepatocytes 220 75. Concentration dependence of f a t t y a c i d i n h i b i t i o n of phospha-t i d y l e t h a n o l a m i n e N - m e t h y l a t i o n i n r a t hepatocytes 221 3 76. Influence of i n s u l i n on the i n c o r p o r a t i o n of [ H]acetate i n t o s a p o n i f i a b l e and no n - s a p o n i f i a b l e l i p i d s 221 3 77. ' E f f e c t of cAMP analogues on [ 1 ( 3 ) - H ] g l y c e r o l i n c o r p o r a t i o n i n t o hepatocyte g l y c e r o l i p i d s 226 14 78. E f f e c t of cAMP analogues on [ 1 - C] p a l m i t a t e i n c o p o r a t i o n i n t o the g l y c e r o l i p i d s of hepatocytes.. 227 3 79. E f f e c t o f g l y c e r o l on [Me- H ] c h o l i n e metabolism i n r a t hepatocytes 230 3 80. E f f e c t of g l y c e r o l on [ H]ethanolamine i n c o r p o r a t i o n i n t o p h o s p h a t i d y l c h o l i n e i n r a t hepatocytes 231 14 81. E f f e c t of g l y c e r o l on [ 1 - C ] p a l m i t a t e i n c o r p o r a t i o n i n t o hepatocyte g l y c e r o l i p i d s 231 3 82. E f f e c t of p a l m i t a t e on [ 1 ( 3 ) - H ] g l y c e r o l i n c o r p o r a t i o n i n t o r a t hepatocytes t r e a t e d w i t h chlorophenylthio-cAMP 233 83. Reversal of cAMPS i n h i b i t i o n of pho s p h a t i d y l c h o l i n e s y n t h e s i s by o l e a t e 234 xxiv LIST OF ABBREVIATIONS ACS aqueous counting s c i n t i l l a n t Ado-Hcy S-adenosyl-homocysteine Ado-Met S-a d e n o s y l - m e t h i o n i n e ADP adenosine diphosphate ATP adenosine triphosphate BHK baby hamster kidney BSA bovine serum albumin cAMP adenosine 3':5'-monophosphate cAMPS adenosine 3':5'-phosphothioate CHO Chinese hamster ovary Ci c u r i e CK c h o l i n e kinase CM carboxy-methyl CMP c y t i d i n e monophosphate CoA coenzyme A ConA concanavalin A cpm counts per minute CPT cholinephosphotransferase CPT-cAMP 8-(4-chlorophenylthio)-adenosine 3'5'-monophosphate CT phosphocholine c y t i d y l y l t r a n s f e r a s e CTP c y t i d i n e triphosphate DEAE d i e t h y l a m i n o e t h y l DES d i e t h y l s t i l b o e s t r o l DG d i g l y c e r i d e DME dimethylethanolamine DMPE dimethylphosphatidylethanolamine dpm d i s i n t e g r a t i o n s per minute DZA deazaadenosine EDTA ethyle n e d i a m i n e t e t r a a c e t a t e EK ethanolamine kinase EPT ethanolaminephosphotransferase E.R. endoplasmic r e t i c u l u m ET phosphoethanolamine c y t i d y l y l t r a n s f e r a s e F i g . f i g u r e FFA free f a t t y acids g gram g g r a v i t y h hour HDL high d e n s i t y l i p o p r o t e i n IBMX isobutylmethylxanthine Km M i c h a e l i s - M e n t e n constant 1 l i t e r LCAT l e c i t h i n c h o l e s t e r o l a c y l t r a n s f e r a s e LDL low d e n s i t y l i p o p r o t e i n LPC l y s o p h o s p h a t i d y l c h o l i n e LPE lysophosphatidylethanolamine m meter M molar MEM modified Eagle's medium min minute MME monomethylethanolamine MMPE monomethylphosphatidylethanolamine X X V n .s. not s i g n i f i c a n t P s t a t i s t i c a l p r o b a b i l i t y PAF p l a t e l e t a c t i v a t i n g f a c t o r PBS phosphate buffered s a l i n e PC ph o s p h a t i d y l c h o l i n e PE phosphatidylethanolamine PEMT phosphatidylethanolamine methyltransferase PG p h o s p h a t i d y l g l y c e r o l PHA phytohemagglutinin PI p h o s p h a t i d y l i n o s i t o l PS ph o s p h a t i d y l s e r i n e RBL ra t b a s o p h i l i c leukemia r e f . reference Rf r a t i o of di s t a n c e moved by a s o l u t e r e l a t i v e t o the sol v e n t f r o n t S.D. standard d e v i a t i o n S.D.S. sodium l a u r y l s u l p h a t e . , S.E. standard e r r o r Sec. s e c t i o n SV simian v i r u s TG t r i g l y c e r i d e TLC t h i n - l a y e r chromatography TPA 1 2 - 0 - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e T r i s t r i s (hydroxymethyl) aminomethane UV u l t r a v o l e t V volume VLDL very low d e n s i t y l i p o p r o t e i n Vmax maximum v e l o c i t y ( o f an enzyme r e a c t i o n ) N O T E S _2 Standard p r e f i x e s a r e : c ( c e n t i ) - 10 ; m ( m i l l i ) -6 -9 -12 10 ; n (nano) - 10 ; p ( p i c o ) - 10 - 1 0 ; u (micro) -x x v i ACKNOWLEDGEMENTS I am most g r a t e f u l t o my s u p e r v i s o r , Dr. Dennis E. Vance f o r h i s c o n t i n u e d encouragement and g u i d a n c e . A l t h o u g h some o f our n o t i o n s o f how p h o s p h a t i d y l c h o l i n e s y n t h e s i s i s r e g u l a t e d sometimes c o n f l i c t e d , he allowed me complete freedom t o pursue my w i l d ideas. I am a l s o indebted to Dr. P a t r i c k Choy f o r c u l t i v a t i n g my enth u s i a s m f o r r e s e a r c h and h i s a i d e s p e c i a l l y i n the e a r l y s t a g e s o f t h i s s t u d y . I thank Dr. Haydn P r i t c h a r d f o r h i s p r e p a r a t i o n s o f monolayer c u l t u r e s of r a t h e p a t o c y t e s and our c o l l a b o r a t i o n . I a l s o appreciate the t e c h n i c a l a i d that I have r e c e i v e d from Mr. Harry Paddon, Dr. Ronhilda de B r i t o , Dr. Princeton Lim, Dr. Ron Angus, Ms. Amelia Wong, Mr. Anthony P e r c i v a l - S m i t h , Ms. Jane Harvey, Ms. E l l e n Powers and Ms. Fateem Jetha. F i n a l l y I wish t o thank my w i f e , J u d i a , f o r her p a t i e n c e . x x v i i DEDICATION Dedicated to those who c o n t r i b u t e d the most to t h i s work, approximately 250 Wistar r a t s . 1 INTRODUCTION P h o s p h a t i d y l c h o l i n e - S t r u c t u r e and D i s t r i b u t i o n (1.1.1.1). P h o s p h a t i d y l c h o l i n e ( l e c i t h i n ) r e s e a r c h p r o b a b l y began w i t h t h e pio n e e r i n g work of J.L.W. Thudichum, who i s o l a t e d and analyzed the l i p i d s from many animal t i s s u e s , p a r t i c u l a r l y c e r e b r a l , and published h i s f i n d i n g s i n a h i s t o r i c document "A T r e a t i s e on The Chemical C o n s t i t u t i o n o f The B r a i n (1884)." P h o s p h a t i d y l c h o l i n e has s i n c e proven to be ub i q u i t o u s i n animals and higher p l a n t s , where i t i s the most abundant of the phosphorus c o n t a i n i n g l i p i d s . The occurrence of p h o s p h a t i d y l c h o l i n e i n pro-kar y o t e s , however, i s r a r e . P h o s p h a t i d y l c h o l i n e s are d i s t i n g u i s h e d from the other p h o s p h o l i p i d s by t h e i r c h o l i n e h e a d g r o u p , a l t h o u g h a v a r i e t y o f f a t t y a c i d s can be e s t e r i f i e d t o the g l y c e r o l backbone ( F i g . 1 ) . S a t u r a t e d f a t t y a c i d s are u s u a l l y e s t e r i f i e d ,at the C1 p o s i t i o n , w h i l e u n s a t u r a t e d f a t t y a c i d s are mainly found at the C2 p o s i t i o n . ( T h i s i s by no means a u n i v e r s a l r u l e as some b a c t e r i a l and tumour p h o s p h o l i p i d s p e c i e s have q u i t e t h e o p p o s i t e p a t t e r n s ( 1 ) ) . The d i v e r s i t y o f phosphatidylcholines i s e x e m p l i f i e d by milk f a t which contains about 95 d i f f e r e n t species of t h i s l i p i d . Figure 1. S t r u c t u r e of p h o s p h a t i d y l c h o l i n e . 2 P h o s p h a t i d y l c h o l i n e (PC) and the o t h e r p h o s p h o l i p i d s were o r i g i n a l l y c o n c e i v e d as m e t a b o l i c a l l y i n e r t compounds o f p u r e l y s t r u c t u r a l s i g n i f i c a n c e . B u t, w i t h t h e a d v e n t o f r a d i o - l a b e l l e d compounds t h i s s i m p l i s t i c notion was c h a l l e n g e d . In 1935, George Hevesy (2) demonstrated 32 t h a t [ P ] o r t h o p h o s p h a t e was r a p i d l y i n c o r p o r a t e d i n t o t i s s u e p h o s p h o l i p i d s , d i s p e l l i n g t h e myth t h a t p h o s p h o l i p i d s had extremely long turnover times. Instead, p h o s p h o l i p i d synthesis was recognized f o r the f i r s t time as an a c t i v e process even w i t h i n mature t i s s u e s . The Enzymes and Pathways of Phosphatidylcholine Biosynthesis ELUCIDATION OF DE NOVO PHOSPHATIDYLCHOLINE BIOSYNTHESIS H i s t o r i c a l P e r s p e c t i v e ( 1 . 2 . 1 . 1 ) . The e x i s t e n c e o f a c y t o s o l i c enzyme i n y e a s t which p h o s p h o r y l a t e d c h o l i n e with ATP was f i r s t r e p o r t e d by Wittenberg and Kornberg (3) i n 1953. The a b s o l u t e r e q u i r e m e n t o f t h i s c h o l i n e k i n a s e f o r magnesium and i t s occurrence i n l i v e r , b r a i n , k i d n e y and the i n t e s t i n a l mucosa of va r i o u s animals were a l s o d e s c r i b e d ( 3 ) . A year e a r l i e r , Kornberg and P r i c e r (4) had 32 14 d e m o n s t r a t e d t h a t d u a l l a b e l l e d ( P and C) p h o s p h o c h o l i n e was i n c o r p o r a t e d by l i v e r p r e p a r a t i o n s i n t a c t i n t o p h o s p h o l i p i d . These i n v e s t i g a t o r s a l s o r e p o r t e d t h e s y n t h e s i s of p h o s p h a t i d a t e from a c t i v a t e d f a t t y a c i d s and g l y c e r o l - 3 - p h o s p h a t e by c e l l - f r e e l i v e r p r e p a r a t i o n s . Phosphatidate serves as t h e p a r e n t m o l e c u l e f o r the e n t i r e g l y c e r o l i p i d f a m i l y . In the mid 1950's, Kennedy and h i s co-workers (5) i n i t i a l l y noted that l a r g e amounts o f ATP were r e q u i r e d t o s u p p o r t t h e f o r m a t i o n o f PC from 3 phosphocholine. Subsequently, Kennedy proved that the CTP, which was. present as a small contaminant i n the ATP p r e p a r a t i o n s , was the e s s e n t i a l c o f a c t o r i n v o l v e d i n the i n c o r p o r a t i o n o f p h o s p h o c h o l i n e i n t o l i p i d . The enzymatic s y n t h e s i s o f C D P - c h o l i n e from p h o s p h o c h o l i n e and CTP was then described ( 6 ) . T h i s r e a c t i o n was c a t a l y z e d b y C T P : p h o s p h o c h o l i n e c y t i d y l y l t r a n s f e r a s e , which was l o c a t e d i n both the c y t o s o l i c and microsomal f r a c t i o n s of guinea p i g l i v e r ( 6 ) . The f i n a l r e a c t i o n f o r the f o r m a t i o n of PC was shown by Kennedy and Weiss (7) to i n v o l v e the c o n d e n s a t i o n o f d i g l y c e r i d e with CDP-choline with the r e l e a s e o f CMP. The c h o l i n e p h o s p h o t r a n s f e r a s e was found i n the 18,000 X g p e l l e t of a r a t l i v e r homogenate, and the r e a c t i o n c o u l d be s t i m u l a t e d by exogenous d i g l y c e r i d e . The o v e r a l l scheme o f what i s sometimes r e f e r r e d to as the Kennedy Pathway ( a l s o c a l l e d cte novo or CD P - c h o l i n e Pathway) i s shown i n Figure 2. Phosphatidylethanolamine i s synthesized from e t h a n o l -amine by an analogous pathway. C h o l i n e K i n a s e ( 1 . 2 . 1 . 2 ) . C h o l i n e k i n a s e (EC 2 . 7 . 1 . 3 2 ) c a t a l y z e s t h e A T P - d e p e n d e n t phosphorylation of c h o l i n e . In the r a t l i v e r t h i s enzyme has a molecular weight of 120,000 (288c) and i s a p p a r e n t l y e x c l u s i v e l y cytoplasmic (8-10) d e s p i t e an a b e r r a n t r e p o r t o f micros o m a l a c t i v i t y ( 1 1 ) . However, c h o l i n e k i n a se i s attached to the c e l l envelope o f the anaerobic protozoon E n t o d i n i u m c a u d a t u m ( 1 2 ) . I n f a n t e and K i n s e l l a (13) have d e t e r m i n e d t h a t a t low s u b s t r a t e c o n c e n t r a t i o n s , the c h o l i n e k i n a s e r e a c t i o n f o l l o w s a s e q u e n t i a l order mechanism w i t h the r e a c t a n t s i n the o r d e r : c h o l i n e , Mg.ATP +^ and Mg+^. Magnesium was shown t o a c t i v a t e the enzyme d i r e c t l y . Some of the k i n e t i c p r o p e r t i e s of the r a t l i v e r and other c h o l i n e kinases are presented i n Table 4 H.C \ . H H H,C—N—C—C—OH / H H H,C CHOLINE CHOLINE KINASE PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE NH, OH OH DIGLYCERIDE CHOLINEPHOSPHOTRANSFERASE O H,C—O—C—R •II I R—C—C—CH O CH, I II H H « , / H,C—O—P—0-C—C—N—CH. I H H \ 0 o CH 3 PHOSPHATIDYLCHOLINE Figure 2. Pathway f o r p h o s p h a t i d y l c h o l i n e b i o s y n t h e s i s from c h o l i n e . •5 1. Choline kinase was o r i g i n a l l y b e l i e v e d t o possess ethanolamine kinase a c t i v i t y , and f o r two decades t h e r e was no e v i d e n c e t o the c o n t r a r y . Weinhold and Rethy (8) p a r t i a l l y p u r i f i e d (31-fold) the major ethanolamine kinase from r a t l i v e r , e t h a n o l a m i n e k i n a s e I I , and t h i s enzyme c o p u r i f i e d w i t h c h o l i n e k i n a s e . The r a t i o o f ethanolamine and c h o l i n e kinase a c t i v i t i e s from r a t l i v e r remained u n a l t e r e d a f t e r chromatography on DEAE-cellulose and Sephadex G200, and bot h a c t i v i t i e s had s i m i l a r s t a b i l i t y p r o p e r t i e s . L i k e w i s e , Brophy et a l . (10) found t h a t even a f t e r 5 5 0 - f o l d p u r i f i c a t i o n o f r a t l i v e r c h o l i n e k i n a s e b y c h o l i n e - S e p h a r o s e 6B a f f i n i t y chromatography, the a c t i v i t y r a t i o between c h o l i n e and ethanolamine kinase was unchanged. C h o l i n e k i n a s e p u r i f i e d 2 0 0 - f o l d from r a b b i t b r a i n a l s o r e t a i n e d ethanolamine kinase a c t i v i t y (22) . A l l of these s t u d i e s pointed to a s i n g l e enzyme which c a t a l y z e d both a c t i v i t i e s . The f i r s t c l e a r i n d i c a t i o n of d i s t i n c t kinases f o r c h o l i n e and e t h a n o l -amine stemmed f r o m s t u d i e s w i t h E n t o d i n i u m caudatum. Broad and Dawson (26) managed to separate p a r t i a l l y the two kinases by g e l f i l t r a t i o n . Then i n 1976, evidence f o r d i f f e r e n t k i n a s e s f o r c h o l i n e and ethanolamine was obtained from r a t l i v e r ( 1 1 ) , l a c t a t i n g b o v i n e mammary gland (9) and spinach l e a v e s ( 2 7 ) . L a t e r , Brophy e t a l . (10) used d i s c g e l e l e c t r o -p h o r e s i s to r e s o l v e t h e r a t l i v e r k i n a s e s and d i s c o v e r e d a t l e a s t three c h o l i n e kinase isoenzymes which d i d not phosphorylate ethanolamine. Choline kinase has r e c e n t l y been p u r i f i e d 700-900-fold from r o o s t e r l i v e r by choline-Sepharose 6B a f f i n i t y chromatography ( 1 4 ) , and isoenzymes f o r c h o l i n e k i n a s e were a l s o d e t e c t e d (H. Paddon and D. Vance, unpublished o b s e r v a t i o n s ) . The nature and f u n c t i o n of the various isoenzymes f o r c h o l i n e kinase remains an enigma. 6 A l t h o u g h the r a t l i v e r c h o l i n e k i n a s e has yet t o be p u r i f i e d t o homogeneity, such p u r i f i c a t i o n ( 1 0 0 0 - f o l d ) has been reported from A f r i c a n g r e e n monkey l u n g ( 1 7 ) . The p u r i f i e d enzyme f r o m p r i m a t e l u n g a l s o phosphorylated ethanolamine, but the y i e l d o f the p u r i f i c a t i o n was only 10%. 0) •P CO c L. o 4J •H n • U ,—. JZ\ •rH 3 JO e to •rH sz o- c f- M < o JC C H I 11 H E I. •* ---ro a. a. E 01 4J C »— C -H Z 01 rH E t. O w ra £ a. o a. ? I. 00 .rH £ Z -P E a •-' o 3 Q. 4J a. o 0) t-m o CM CM A m o d ^ t— A o d in ,— a- o cn m • •— m • in • r- • CM • CM o o o o in in o o o O • + • • + • • • + • a + • o o o C- CM o in m o o o o • o o o o o I in I CM m o 7 CO m o m t— CO o „_ CM cn z r m CM in CM CM CM CM CM C\J ( TJ rH rH o o •o CM n 01 | rv o i> at o •o •u 3 •rH o 01 •a >. 4J -—. CM 0) o •rH c o •rH T3 •rH 4 J r— <- CO *J HI L. co *— lH rH rH 10 T4 3 c L. CO rH 0) c <M a. <u 3 0) •U •o o •rH so c a. >> O CO 01 o 1_ •a o 3 a> t- c •rH •a 01 3 rH rH E rH • U T ) CO 0) 01 CM 01 t- ,—. a o o o rH co rH E rH CO TH •M V rH c« m .c >» o c o E O o t- CM o •o I o 0) 10 rH 0> CO CO E 3 •H E CO rH o •u 00 o o to 1 E 3 o O. i- 3 o o rH rH o o >> c c rH -*-> CO o o E •C 3 4 J CO o o CM CO o 3 o o >> o E o 00 >, a. en >. o to w 1 -— rH E CO a H-> o CM. c a <0 rH •a o 4-9 o o o 00 o >> .c *—• -*H iH 01 rH T3 3 >> H-> J-> o c >« C c o 4-> c 4J CO rH CO T3 o >. >> in 3 0) 0) >> •rH c c CO o H^ •»H o CJ C o o — > rH 01 CJ CO c •rH iH * J >> o 4J CM a rH •»H C L. L. •«H CO CO a 01 1. CO t. 1 E rH L. t- rH •U o CJ c X I «J L. L. CO *> x> < CO in 3 m 01 01 CD E •rH 01 L. X l •Q rH CO E 1 a. CM •rH 01 CJ > > > C 1- c a> CO t- X I C 01 x: i J * ' c c •rH •rH 10 m •iH L. o 4J 4 J 01 11 o •rH CO i— •—1 rH rH rH 3 o CM £1 CM 01 •H •rH c 00 ^ iH 4-3 4-> a t- CM o CO 10 •r-i •rH m XI fH O CJ rH CO M o X I 1 •U HJ •rH 4J 01 1. L. H-> 4-1 3 •O JD > E -H 1. CO CO E CO ro JZ ai •C 3 CO CO O CO CO o o x: x: 01 01 c 01 <- o CM cc •a: a M E u t . X I •C o u >. >. E m 7 CTP: Phosphocholine C y t i d y l y l t r a n s f e r a s e (1.2.1.3). CTP: phosphocholine c y t i d y l y l t r a n s f e r a s e (EC 2.7.7.15) c a t a l y z e s the s y n t h e s i s of CDP-choline and pyrophosphate from CTP and phosphocholine. This enzyme i s ambiquitous (28) i n t h a t i t i s recovered i n both the c y t o s o l i c and microsomal f r a c t i o n s of r a t l i v e r homogenate ( 2 9 ) . The d i s t r i b u t i o n of enzyme a c t i v i t y between these f r a c t i o n s i s dependent on the homogenization medium and method of c e n t r i f u g a t i o n . Schneider (29) observed that 85% of the a c t i v i t y was r e c o v e r e d i n the p a r t i c u l a t e f r a c t i o n i f the r a t l i v e r was homogenized i n d i s t i l l e d water. However, when the t i s s u e was homogenized i n i s o t o n i c s a l i n e 99% of the a c t i v i t y r e m a i n e d i n the 68,000 X g X 1 h supernatant. I f r a t l i v e r s a l i n e homogenate i s c e n t r i f u g e d at 100,000 X g X 1 h, then a p p r o x i m a t e l y 15% o f the t o t a l enzyme a c t i v i t y i s p e l l e t e d . Vance et a l . (30) have shown t h a t the microsomal c y t i d y l y l t r a n s f e r a s e i s exposed on the c y t o p l a s m i c f a c e o f the s e a l e d fragments o f endoplasmic r e t i c u l u m (E.R.) that form during homogenization. C y t i d y l y l t r a n s f e r a s e i n f r e s h r a t l i v e r c y t o s o l has a molecular weight of 200,000 and has been d e s i g n a t e d the L-form (low molecular weight) (31). o o When c y t o s o l i s i n c u b a t e d at 4 f o r 5 days ( 3 D or 20 f o r 8 h (32), 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 can be e l e v a t e d 7 - f o l d . In a d d i t i o n , the enzyme e l u t e s from a Sepharose 2B column over a wide range of molecular weights (0.5-13 X 1 0 ^ ) . The median m o l e c u l a r weight of t h i s H-form (high m o l e c u l a r w e i g h t ) was e s t i m a t e d t o be 1.2 X 10^ ( 3 1 ) . A l a r g e r H-form (5-50 X 10^ daltons) has been shown t o predominate i n f r e s h a d u l t r a t lung c y t o s o l ( 3 3 ) , a l t h o u g h l u n g l a v a g e t r e a t m e n t w i l l reduce the amount of H-form by 72% (34). In c o n t r a s t , the f e t a l r a t lung c y t o s o l has only L-form (190,000 daltons) (33). C y t i d y l y l t r a n s f e r a s e from f r e s h a d u l t r a t b r a i n and kidney c y t o s o l s a l s o e l u t e s from g e l f i l t r a t i o n columns as mostly L-form 8 (34). The L-form i s markedly s t i m u l a t e d by liposomes prepared from t o t a l r a t l i v e r p h o s p h o l i p i d , whereas t h e m i c r o s o m a l and H-forms are much l e s s s e n s i t i v e ( 3 5 ) . F i s c u s and S c h n e i d e r (36) i d e n t i f i e d 3 -sn-lysophospha-t i d y l c h o l i n e (LPC) as a potent a c t i v a t o r o f c y t i d y l y l t r a n s f e r a s e i n acetone-butanol e x t r a c t s of rat l i v e r c y t o s o l . S i m i l a r l y , palmitoyl-LPC s t i m u l a t e d c y t i d y l y l t r a n s f e r a s e 3 3 - f o l d i n a c e t o n e - b u t a n o l e x t r a c t s of r a t i n t e s t i n a l mucosa c y t o s o l ( 3 7 ) . Choy and Vance ( 3 5 ) , h o w e v e r , found LPC t o be i n h i b i t o r y w i t h r a t l i v e r L-form and c o n c l u d e d that f i n d i n g s w i t h acetone-butanol preparations were of d u b i o u s p h y s i o l o g i c a l relevance. As shown i n Table 2, a number o f a c i d i c p h o s p h o l i p i d s can a c t i v a t e L-form from r a t l i v e r , l u n g , kidney and b r a i n . In a d d i t i o n , l y s o p h o s p h a t i d y l e t h a n o l a m i n e (LPE) has been reported to be t h e b e s t a c t i v a t o r of the l i v e r enzyme (35). The conversion of L-form t o H-form i n r a t l i v e r c y t o s o l i s not i n f l u e n c e d by LPE ( 3 5 ) . R a t h e r , t h e d i g l y c e r i d e p r e s e n t i n r a t l i v e r c y t o s o l was i d e n t i f i e d as the p r i n c i p a l agent r e s p o n s i b l e f o r aggregation (38). In lung c y t o s o l , p h o s p h a t i d y l g l y c e r o l s e r v e s a d u a l r o l e as an a c t i v a t o r and aggregator of the c y t i d y l y l t r a n s f e r a s e (39). R e c e n t l y , Feldman e_t a l . ( 4 0 ) have i d e n t i f i e d endogenous f r e e f a t t y a c i d s i n r a t l u n g c y t o s o l as the major a c t i v a t i n g s p e c i e s f o r the c y t i d y l y l t r a n s f e r a s e . I s o p r o p y l e t h e r e x t r a c t i o n o f the l u n g c y t o s o l f a c i l i t a t e d the removal o f f r e e f a t t y a c i d s w i t h a corresponding r e d u c t i o n 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 . When the i s o p r o p y l ether e x t r a c t or long c h a i n u n s a t u r a t e d f a t t y a c i d s were r e c o n s t i t u t e d w i t h the c y t o s o l , the enzyme a c t i v i t y was r e s t o r e d . O l e a t e , l i n o l e a t e and l i n o l e n a t e (200 ^ jM) were found to a c t i v a t e r a t lung 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 2.5-, 2.6-and 2 . 8 - f o l d , r e s p e c t i v e l y ( 4 0 ) . A l t h o u g h the e f f e c t of f a t t y acyl-CoA was Table 2. Ac t i v a t i o n of C y t i d y l y l t r a n s f e r a s e by Phospholipids. DIOLEOYL- LPC PALMITOYL- OLEOYL- PE PG PI PS PA LPC LPE ra t l i v e r cytosol (fresh) (35) r a t l i v e r cytosol (acetone-butanol extract) (35) r a t l i v e r L-form (35) no change (1.1) mM) 2.1-fold (1.1 mM) (3D r a t l i v e r H-form (35) r a t lung f e t a l L-form (3D r a t kidney adult L-form (34) rat brain adult L-form (34) 19% i n h i b i t e d (1.1 mM) 1.8-fold (0.1 mM) 21* i n h i b i t e d (1.1 mM) 7.5-fold (0.6 mM) 75$ i n h i b i t e d (2 mM) (pig l i v e r ) 6-fold (2 mM) (pig l i v e r ) 100$ i n h i b i t e d (2 mM) (pig l i v e r ) 1 . 3-fold (2 mM) 9.3-fold (2 mM) 9.3-fold (2.2 mM) 57.9-fold (2.2 mM) 60$ i n h i b i t e d 17.7-fold (2 mM) (2.2 mM) 4.9-fold (0.6 mM) 5-fold (0.5 mM) 27$ i n h i b i t e d (2 mM) (pig l i v e r ) 3.5-fold (1 mM) (rat lung) 2.9-fold (1.2 mM) (rat lung) 6-fold (1.5 mM) (rat lung) no change (2 mM) 2.5-fold (2.2 mM) 10.5-fold (1 mM) 1.0-fold (1.2 mM) 9.3-fold (1.3 mM) no change (1.3 mM) (pig l i v e r ) 10.7-fold (1.3 mM) (pig l i v e r ) 75$ i n h i b i t e d (1.3 mM) (pig l i v e r ) no change (1.3 mM) (pig l i v e r ) 6.5-fold (0.1 mM) (egg) 11-fold (0.5 mM) (egg) 3.9-fold (0.5 mM) 10-fold (0.4 mM) 4.6-fold (1.2 mM) (pig l i v e r ) 46-fold (1.2 mM) (pig l i v e r ) 11.5-fold (1.2 mM) (pig l i v e r ) 1.5-fold (1.2 mM) (pig l i v e r ) 3.7- f o l d (1.3 mM) (pig brain) 12.7-fold (1.3 mM) (pig brain) 7.8- f o l d (1.3 mM) (pig brain) 1.2-fold (1.3 mM) (pig brain) 2.3-fold (0.2 mM) (bovine brain) r a t i n t e s t i n a l mucosa cytosol (acetone-butanol extract) (37) 33-fold (1.7 mM) 10 not t e s t e d i n the l u n g , Lim (41) has noted t h a t 50 p a l m i t o y l - C o A or stearoyl-CoA a c t i v a t e d r a t l i v e r c y t o s o l i c c y t i d y l y l t r a n s f e r a s e by 14% and 35%, r e s p e c t i v e l y . These f i n d i n g s t end t o i n d i c a t e t h a t the a l k y l p o r t i o n of these compounds i s recognized by the c y t i d y l y l t r a n s f e r a s e . This i s f u r t h e r supported by the s t i m u l a t o r y e f f e c t s of n - a l k a n e 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 i n r a t l i v e r homogenates ( 4 2 ) . At 10% c o n c e n t r a t i o n s , n-hexane, n-octane and n - t e t r a d e c a n e s t i m u l a t e d 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 3.8-, 2.7- and 1.6-fold, r e s p e c t i v e l y . The f u n c t i o n a l r e l a t i o n s h i p between the L-, H- and microsomal forms of the c y t i d y l y l t r a n s f e r a s e i s o b s c u r e . Choy e t a l . (43) have demonstrated t h a t a l l three forms of the r a t l i v e r enzyme are immunologically i d e n t i c a l , and there i s some support f o r the i d e a t h a t the H- and microsomal forms represent the same sp e c i e s . H-form may be p e l l e t e d under c o n d i t i o n s s i m i l a r t o those which w i l l sediment microsomes ( 3 1 ) . As shown i n Table 3, the k i n e t i c p r o p e r t i e s of the H-form are s i m i l a r t o t h o s e r e c o r d e d f o r the m i c r o s o m a l form. F u r t h e r m o r e , b o t h forms are not u s u a l l y s e n s i t i v e t o p h o s p h o l i p i d a c t i v a t i o n , p r e s u m a b l y b e c a u s e t h e s e forms are a l r e a d y p h o s p h o l i p i d - a s s o c i a t e d (35). Sundler (44) has c l e a r l y shown th a t phosphocholine c y t i d y l y l t r a n s f e r a s e i s a very d i f f e r e n t enzyme from phosphoethanolamine c y t i d y l y l t r a n s f e r a s e . Table 3. K i n e t i c P r o p e r t i e s of C y t i d y l y l t r a n s f e r a s e . Ref. PH optimum Mg + 2 optimum (mM) Apparent Km phosphocholine (mM) Apparent Km CTP (mM) CTP Substrate I n h i b i t i o n (mM) - r a t l i v e r homogenate (45) 6.0-6.5 - r a t l i v e r homogenate (46) 6.0-broad 0.3 - r a t l i v e r cytosol (46) 1.0 - r a t l i v e r cytosol (fresh)(Sec . 3 .2 .1 ) 5.6-5.8 5 0.2-0.7 1.3-5 >6 - r a t l i v e r cytosol (fresh) + r a t l i v e r phospholipid (Sec.3.2.1) 6.4-6.6 0.45-0.7 0.5-0.7 >4 - r a t l i v e r cytosol (aged) (Sec.3.2.1) 6.2-6.6 0.7-0.8 0.7 >4 - r a t l i v e r H-form (3D 7.0-broad 0.18 0.3 - r a t l i v e r (100-fold p u r i f i e d ) + r a t l i v e r phospholipid (3D 7.0-broad 0.17 0.2 - r a t l i v e r microsomes (Sec.3.2.1) 6.4-broad 6 0.35 0.65 - r a t lung cytosol (33) 6.0-7.0 12 .1.0 4.0 - chick embryonic muscle c e l l homogenates (23) 0.14 - Entodinium caudatum ( r e c o n s t i -tuted membranes and cytosol (12) 0.4 >1 - BHK-21 c e l l cytosol (25) >3 12 CDP-Choline: 1 , 2 - d i a c y l - s n - g l y c e r o l C h o l i n e p h o s p h o t r a n s f e r a s e (1.2.1.4). The f i n a l s t e p i n the d_e novo pathway i s the f o r m a t i o n of PC and CMP from d i g l y c e r i d e and CDP-choline, c a t a l y z e d by CDP-choline: 1 , 2 - d i a c y l -s n - g l y c e r o l c h o l i n e p h o s p h o t r a n s f e r a s e (EC 2.7.8.2). The contention that t h i s enzyme may a l s o be r e s p o n s i b l e f o r the s y n t h e s i s o f the c h o l i n e plasmalogen precursor, 1 - a l k y l - 2 - a c y l - s n - g l y c e r o p h o s p h o c h o l i n e from CDP-c h o l i n e and 1 - a l k y l - 2 - a c y l - s n - g l y c e r o l (47) has been strengthened by recent s t u d i e s (47b). C h o l i n e p h o s p h o t r a n s f e r a s e i s l a r g e l y m i c r o s o m a l i n d i s t r i b u t i o n , although McCaman and Cook (48) have d e s c r i b e d s i g n i f i c a n t m i t o c h o n d r i a l a c t i v i t y i n r a t b r a i n w h i c h c o u l d n o t be a t t r i b u t e d t o m i c r o s o m a l contamination. Furthermore, Baker and Chang (48b) have detected high cholinephosphotransferase a c t i v i t y , i n the nuclear membranes of neuronal n u c l e i i s o l a t e d from immature r a b b i t c e r e b r a l c o r t e x . The v u l n e r a b i l i t y o f the microsomal cholinephosphotransferase to proteases has i n d i c a t e d that the enzyme i s exposed on the c y t o p l a s m i c s i d e of the E.R. (30,49). This 14 was c o n f i r m e d i_n v i v o by the s e l e c t i v e i n c o r p o r a t i o n of [ C j c h o l i n e i n t o p h o s p h a t i d y l c h o l i n e of the outer l e a f l e t of the r a t l i v e r microsomal membranes (50). The l i g a n d s of the c h o l i n e p h o s p h o t r a n s f e r a s e r e a c t i o n have d i f f e r e n t s o l u b i l i t i e s : d i g l y c e r i d e and PC a r e l i p i d s o l u b l e , whereas CDP-choline and CMP are water s o l u b l e . The a p p l i c a t i o n o f s i m p l e Michaelis-Menten k i n e t i c s t o a system i n v o l v i n g two i m m i s c i b l e phases i s questionable. However, with t h i s r e s e r v a t i o n i n mind, some o f the k i n e t i c p r o p e r t i e s of the c h o l i n e -phosphotransferase from r a t l i v e r and o t h e r t i s s u e s are summarized i n Table 4. Compounds which have been shown t o i n f l u e n c e the Vn v i t r o a c t i v i t y of t h i s enzyme are presented i n Table 5. 13 Table 4. K i n e t i c P r o p e r t i e s o f Cholinephosphotransferase. Ref. PH optimum Mg + 2 optimum (mM) Apparent Km CDP-choline (mM) Apparent Km exogenous DG (mM) - r a t l i v e r homogenate (45) 8.0 - r a t l i v e r microsomes (51) 0.13 - r a t l i v e r microsomes (52) <0.5 - r a t l i v e r (4-5-fold p u r i f i e d ) (53) 8.0-8.5 0.036 0.081 - r a t brain homogenate • (45) 8.0-8.6 25-50 0.22 - r a t b r ain synaptosomes (54) 0.35 1.2 - r a b b i t cerebrum microsomes (55) 8.0 20 0.05 0.026 - r a t adipocyte microsomes (56) 8.5-9-3 0.24 0.05 - chick embryonic muscle c e l l s microsomes (23) 0.033 0.87 - Entodinium caudatum (57) 0.019 - Tetrahymena pyriformis (58) 0.012 - Saccharomyces cerevisiae (59) >20 0.06 14 Table 5. Compounds Which I n f l u e n c e Cholinephosphotransferase A c t i v i t y . A) ACTIVATORS Compound Cone. A c t i v a t i o n System Ref. - d i a c y l g l y c e r o l (2 mM) 6.2-fold rat l i v e r microsomes (60) - d i a c y l g l y c e r o l (15 mM) 25-fold rat brain homogenate (48) - d i a c y l g l y c e r o l (2 mM) 3.2-fold r a t brain microsomes (60) - d i a c y l g l y c e r o l (15 mM) 30-fold rat lung homogenate (18) - d i a c y l g l y c e r o l (15 mM) 21-fold rat l i v e r homogenate (48) - d i p a l m i t o y l g l y c e r o l (1 mM) 11-fold rat lung microsomes (61) - d i o l e i n (2.D mM) 5-fold rat l i v e r homogenate (45) - phosphatidylglycerol (0.8 mM) 8.7-fold rat lung microsomes (61) - d i p a l m i t o y l g l y c e r o l + Tween 20 + phosphatidylglycerol (1 mM) (0.1 mg/ml) (1 mM) 268-fold rat lung microsomes (61) - stearate (1 mM) 1.9-fold rat l i v e r microsomes (60) B) INHIBITORS Compound Cone. % I n h i b i t i o n System Ref. - calcium (1 mM) 100 ra t brain homogenates (48) - calcium (1 mM) 100 E. caudatum homogenates (12) - ATP (20 mM) 70 rat l i v e r microsomes (62) - ATP + Coenzyme A (2 mM) (0.1 mM) 80 ra t l i v e r microsomes (63) - ATP + pantotheine (2 mM) (1 mM) 90 rat l i v e r microsomes (62) - oleoyl-CoA (10 pM) 15 Baker's yeast p a r t i c l e s (59) - oleate + Coenzyme A + ATP (0.8 mM) (1 mM) (15 mM) 79 Baker's yeast p a r t i c l e s (59) - centrophenoxine (30 mM) 99 rat l i v e r microsomes (63) - dimethylethanolamine (30 mM) 69 rat l i v e r microsomes (63) - neophenoxine (5 mM) 50 ra t l i v e r microsomes (64) - dimethylaminoethyl-p. chlorophenoxyacetate (30 mM) 77 ra t l i v e r microsomes (63) 15 Microsomal C D P - c h o l i n e : 1 , 2 - d i a c y l - s n - g l y c e r o l ethanolaminephospho-t r a n s f e r a s e (EPT) (EC 2.7.8.1) s y n t h e s i z e s PE by a r e a c t i o n analogous to t h a t c a t a l y z e d by c h o l i n e p h o s p h o t r a n s f e r a s e . Whether the ethanolamine- and cholinephosphotransferase a c t i v i t i e s r e s i d e i n one or more enzymes has been a c o n t r o v e r s i a l matter (60,65,66). S i n c e CDP-choline and CDP-ethanolamine are competitive i n h i b i t o r s o f the complementary phosphotransferases from c a s t o r bean endosperm (67) and s p i n a c h ( 6 8 ) , a common enzyme may c a t a l y z e s both r e a c t i o n i n higher p l a n t s . However, lack of such competition s u g g e s t s t h a t s e p a r a t e enzymes o c c u r i n t h e membranes of E n t o d i n i u m  caudatum (57). In the case of r a t a d i p o c y t e s (56) and b r a i n (69,70), the s i t u a t i o n i s more c o m p l i c a t e d . CDP-ethanolamine c o m p e t i t i v e l y i n h i b i t e d c h olinephosphotransferase, but i n h i b i t i o n of EPT by CDP-choline was non-c o m p e t i t i v e . The adipocyte t r a n s f e r a s e s could be d i f f e r e n t i a l l y i n h i b i t e d by h e a t i n g , t r y p s i n d i g e s t i o n , M n C l 2 and p a l m i t o y l - C o A and a r e l i k e l y s e parate enzymes ( 5 6 ) . P o l o k o f f e t a l . (71) have a l s o i s o l a t e d Chinese hamster o v a r y (CHO) c e l l mutants w i t h normal c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y , but 6-10-fold lower EPT a c t i v i t y . The most convincing e v i d e n c e f o r d i s t i n c t c h o l i n e - and ethanolamine-phosphotransferases has been obtained from s t u d i e s with r a t l i v e r . Kanoh and Ohno (53,72) c o s o l u b i l i z e d both a c t i v i t i e s from microsomes and p a r t i a l l y r e s o l v e d the phosphotransferases by s u c r o s e d e n s i t y gradient c e n t r i f u g a t i o n a f t e r T r i t o n X-100 treatment. C h o l i n e p h o s p h o t r a n s f e r a s e was separated i n t o magnesium- and manganese- (or magnesium-) r e q u i r i n g components. EPT a c t i v i t y was o n l y a s s o c i a t e d with the manganese-dependent cholinephosphotransferase. EPT has a l s o been s o l u b i l i z e d from r a t l i v e r microsomes with o c t y l - g l u c o s i d e and the e x t r a c t contained v i r t u a l l y no c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y ( 7 3 ). 16 A d d i t i o n a l p r o o f f o r s e p a r a t e p h o s p h o t r a n s f e r a s e s i n r a t b r a i n and l i v e r has been provided by the d i f f e r e n t s p e c i f i c i t i e s of these enzymes f o r the d i g l y c e r i d e substrate (Table 6 ) . The preference of the phosphotransfer-ases with respect to p a r t i c u l a r f a t t y a c i d s i d e chains has been e l u c i d a t e d w i t h exogenous r a d i o - l a b e l l e d d i g l y c e r i d e e m u l s i o n s , t h r o u g h the back r e a c t i o n s t a r t i n g with r a d i o - l a b e l l e d f a t t y a c i d s e s t e r i f i e d to PC, and by e x a m i n a t i o n o f t h e PC s p e c i e s f o r m e d ijn v i v o w i t h r a d i o - l a b e l l e d c h o l i n e . In summary, cholinephosphotransferase favours palmitate r a t h e r than s t e a r a t e at p o s i t i o n 1 o f d i g l y c e r i d e , w h i l e the o p p o s i t e holds true f o r EPT. EPT s e l e c t s f o r e i c o s a h e x a e n o a t e a t p o s i t i o n 2 of d i g l y c e r i d e , while cholinephosphotransferase shows a s l i g h t p r e f e r e n c e f o r l i n o l e a t e at t h i s p o s i t i o n . Weiss et a l . (51) o r i g i n a l l y d e m o n s t r a t e d the r e v e r s i b i l i t y of the c h o l i n e p h o s p h o t r a n s f e r a s e r e a c t i o n Vn v i t r o w i t h microsomes and CMP. . T h i s back r e a c t i o n a l s o o p e r a t e s in v i v o ( 7 8 - 8 0 ) , and Akesson and Sundler (81) have c a l c u l a t e d the r a t i o o f the f o r w a r d to back v e l o c i t y as between 1.2 and 2.3 f o r r a t l i v e r . Such v e l o c i t y e s t i m a t e s , however, are complicated by the f i n d i n g that v a r i o u s s p e c i e s and s u b c e l l u l a r pools of PC are synthesized at d i f f e r e n t rates (76). The p h y s i o l o g i c a l r o l e o f t h e b a c k r e a c t i o n has not been f i r m l y e s t a b l i s h e d , but the degradation o f PC by c h o l i n e p h o s p h o t r a n s f e r a s e may be important i n b r a i n , p a r t i c u l a r l y d u r i n g i s c h e m i a (82), where an a c t i v e microsomal d i g l y c e r i d e l i p a s e g e n e r a t e s f r e e f a t t y a c i d s and g l y c e r o l from the r e l e a s e d d i g l y c e r i d e (83). The back r e a c t i o n has a l s o been i m p l i c a t e d i n the r e c y c l i n g of p o l y -u n s a t u r a t e d d i g l y c e r i d e s from PC t o PE. In i s o l a t e d r a t h e p a t o c y t e s , o n e - h a l f and o n e - t h i r d , r e s p e c t i v e l y , o f t e t r a e n o i c and h e x a e n o i c 17 d i g l y c e r i d e s used f o r PE s y n t h e s i s do not d i r e c t l y o r i g i n a t e from phospha-t i d a t e , but can a r i s e by the c h o l i n e p h o s p h o t r a n s f e r a s e back r e a c t i o n (84). Once formed, the t e t r a e n o i c and hexaenoic PE's are p r e f e r e n t i a l l y methylated to form PC (see S e c t i o n . 1.2.2.1 and F i g . 2 ) . Such r e c y c l i n g o f polyenoic d i g l y c e r i d e u n i t s between PE and PC was i n i t i a l l y proposed by Tinoco et a l . ( 8 5 ) . Table 6. S p e c i f i c i t i e s of Rat Cholinephosphotransferase and Ethanolamine- phosphotransferase f o r V a r i o u s S p e c i e s of D i a c y l g l y c e r o l . Cholinephospho- Ethanolaminephospho- Method System Ref. transf e r a s e transferase A) PREFERRED FATTY ACIDS ESTERIFIED TO CARBON 1 C12:0 -C17:0 >C18:0 C17:0, C18:0. C18:1> exogenous DG l i v e r (71) C16:0 >C12:0 - C15:0 C16:0 >C18:0 C18:0 >C16:0 ( i f C21:1 or exogenous DG l i v e r (75) C18:1 at C2) C18:0 >C16:0 backward rxn. l i v e r (53) C16:0 >C18:0 i n v i v o l i v e r (76) l a b e l i n g of PC C18:1 >C18:0 exogenous DG adipose (56) B) PREFERRED FATTY ACIDS ESTERIFIED TO CARBON 2 no preference bet- marked preference for exogenous DG l i v e r (75) ween unsaturated FA C20:6 s l i g h t preference marked preference for backward rxn. l i v e r (53) for C18:2 C20:6 s l i g h t preference backward rxn. l i v e r (66) for C18:2 s l i g h t preference strong preference for backward rxn brain (77) C18:1 and C18:2 C21:1 18 SYNTHESIS OF PHOSPHATIDYLCHOLINE FROM PHOSPHATIDYLETHANOLAMINE Phosphatidylethanolamine N - M e t h y l a t i o n (1.2.2.1). The existence of an a l t e r n a t e r o u t e f o r PC s y n t h e s i s was evident as e a r l y as 1946 when Horowitz (86) r e p o r t e d t h a t while normal s t r a i n s of the mold Neurospora c r a s s a c o u l d c o n v e r t PE t o PC, t h i s c o n v e r s i o n was blocked i n genetic v a r i a n t s . Much l a t e r i n 1959, Bremer and Greenberg (87) 1 4 found t h a t r a t s g i v e n an i n t r a p e r i t o n e a l i n j e c t i o n o f [M£- C ] -methionine incorporated the r a d i o - l a b e l i n t o l i v e r l i p i d s c o n t a i n i n g mono-and dimethylethanolamine, and c h o l i n e . An enzyme system was subsequently d e t e c t e d i n r a t l i v e r microsomes w h i c h c a r r i e d out the s u c c e s s i v e N-methylation of PE to produce PC ( 8 8 ) . S>-Adenosyl-methionine (Ado-Met) was t h e m e t h y l d o n o r i n e ach o f t h e t h r e e r e a c t i o n s c a t a l y z e d by PE m e t h y l t r a n s f e r a s e (EC 2.1.1.17) ( F i g . 3 ) . A l t h o u g h l i v e r c o n t a i n s the h i g h e s t l e v e l of t r a n s m e t h y l a t i o n a c t i v i t y , t h i s enzyme system i s widely d i s t r i b u t e d i n animal t i s s u e s (89-94), s l i m e mold (95,96) and some b a c t e r i a (97). PE m e t h y l t r a n s f e r a s e (PEMT) a c t i v i t y has been detected i n the E.R., plasma, mitochondrial and nuclear membranes of mammalian c e l l s (98). Studies with microorganisms p r o v i d e d some o f the f i r s t s igns that PE methylation i n v o l v e s at l e a s t two d i s t i n c t m e t h y l t r a n s f e r a s e s . C l o s t r i d i u m  butyricum can produce p h o s p h a t i d y l - N - m e t h y l e t h a n o l a m i n e (MMPE), but not PC (99). A g r o b a c t e r i u m t u m e f a c i e n s " a p p e a r s " t o g e n e r a t e PC e x c l u s i v e l y by t r a n s m e t h y l a t i o n and a s o l u b l e m e t h y l t r a n s f e r a s e p u r i f i e d 4 0 - f o l d from t h i s b a c t e r i a c o u l d o n l y s u p p o r t m e t h y l a t i o n of PE, whereas homogenates c a t a l y z e d a l l t h r e e m e t h y l a t i o n r e a c t i o n s ( 9 7 ) . When Neurospora c r a s s a i s grown on minimal medium, m e t h y l a t i o n o f PE i s the major metabolic source o f P C ( 1 0 0 ) . One mutant s t r a i n o f N_^_ c r a s s a ( 47904) was shown t o 19 R 2 — C - O - C - H O II C H 2 — 0 - C - - R . I 2 1 O I 11 C H , - 0 - P -AdoMet AdoHcy V J O C H 2 C H 2 N H 3 Phosphatidylethanolamine R 2 - C O C H 2 - 0 - C - R , ' O — C - H O I fl C H 2 - 0 - P — O C H X H j - N H , I I O - C H 3 N-rnethylphosphatidylethanolamlne AdoMet AdoHcy O C H . - - 0 — C — R , R , - C - 0 — C - H O AdoHcy AdoMet O C H , — O - C - R , V J "I I « N. y R . - C - O - C - H O H C H 2 — 0 - P - O C H 2 C H 2 N ( C H 3 ) 3 0 " Phosphatldylcholine C H 2 — 0 - - P - O C H 2 C H 2 N - C H 3 N,N-dlmethylpho»phatldylethanolamlne Figure 3. P h o s p h a t i d y l c h o l i n e b i o s y n t h e s i s by successive N-methylation of p h o s p h a t i d y l e t h a n o l a m i n e . 20 accumulate MMPE, while another mutant s t r a i n (34486) was found to methylate MMPE and p h o s p h a t i d y l - N , N d i m e t h y l e t h a n o l a m i n e (DMPE), but not PE(101). Scarborough and Nyc (96) were a b l e t o o b t a i n from c r a s s a microsomes (by s o n i c o s c i l l a t i o n ) a " s o l u b l e " p r e p a r a t i o n t h a t c a t a l y z e d only the two step methylation of MMPE to PC, and pre s e n t e d evidence that one enzyme was r e s p o n s i b l e f o r both m e t h y l a t i o n r e a c t i o n s . The f i r s t m ethyltransferase a c t i v i t y was n o t r e c o v e r e d a f t e r d i s r u p t i o n by s o n i c o c i l l a t i o n . C o l l e c t i v e l y , these s t u d i e s i n d i c a t e one m e t h y l t r a n s f e r a s e (PEMT 1) c a t a l y z e s the formation o f MMPE from PE, w h i l e a second methyltransferase (PEMT 2) converts MMPE to PC by two succ e s s i v e methylations. The existence of PEMT 1 and PEMT 2 i n mammalian t i s s u e s was supported by r e p o r t s o f d i f f e r e n t k i n e t i c s p r o p e r t i e s of these methyltransferases (Table 7 ) . These values f o r the appar e n t K o f PEMT 1 f o r Ado-Met are i n m most cases underestimates s i n c e many i n v e s t i g a t o r s have f a i l e d t o account f o r the MMPE which i s immediately converted to DMPE and PC. Recent s t u d i e s i n our l a b o r a t o r y (F. Audubert and D. Vance, unpublished data) i n d i c a t e t h a t a l l three methylations may have s i m i l a r K 's f o r Ado-Met and no r e q u i r e -m ment f o r Mg There have been a number of r e p o r t s o f p a r t i a l or complete s o l u b i l i z a -t i o n of the phospholipid methyltransferases from mammalian t i s s u e s . Rehbin-der and Greenberg (113) found t h a t a combination o f deoxycholate and sonic o s c i l l a t i o n s o l u b i l i z e d PEMT 2 f r o m r a t l i v e r microsomes, but PEMT 1 a c t i v i t y was destroyed by t h i s p r o c e d u r e . Morgan (94) used the sequence of u l t r a c e n t r i f u g a t i o n , ammonium s u l f a t e p r e c i p i t a t i o n and D E A E - c e l l u l o s e chromatography to achieve a 3 0 0 - f o l d p u r i f i c a t i o n o f pho s p h o l i p i d methyl-t r a n s f e r a s e from dog lung homogenates. U n f o r t u n a t e l y , the recovery was l e s s than \% and the enzymes were s t i l l a s s o c i a t e d w i t h membrane fragments. 21 Table 7. K i n e t i c Properties of Phosphatidylethanolamine N-Methyltransferase. Ref. pH Mg Apparent Km optimum optimum Ado-Met (mM) (pM) ra t hepatocyte homogenates r a t l i v e r plasma membranes r a t l i v e r r a t l i v e r r a t l i v e r microsomes microsomes microsomes r a t l i v e r ( s o l u b i l i z e d from microsomes) guinea pig l i v e r microsomes r a t b r a i n synaptosomes r a t b r a i n cortex synaptosomes r a t b r a i n cortex microsomes ra t b r a i n cortex homogenates ra t p i t u i t a r y homogenates r a t erythrocyte ghosts bovine adrenal medulla microsomes dog lung ( s o l u b i l i z e d from microsomes) Neurospora crassa ( s o l u b i l i z e d from microsomes) Agrobacterium tumefaciens (soluble) PEMT I PEMT I I PEMT I PEMT I I PEMT I PEMT I PEMT I I PEMT I PEMT I I PEMT I PEMT I I PEMT I PEMT I I PEMT I PEMT I I PEMT I PEMT I I PEMT I PEMT I I PEMT I PEMT I I PEMT I I PEMT I (102) (103) 9.2 no (101) 8.0 yes 10 (105) 9.8 . 9-4 (106) (107) (105) ( 98) 7.5 1 10.5 no (108) (109) (110) 8 ( 90) 6.5 + 9-5 no 6.5 + 9.5 no (111) 10.5 2 10.5 (112) 6.5 10 ( 94) 8.0-9.0 ( 96) 8.0 ( 97) 8.4 + 9-6 2 no 0.81 67 18 295 + 87 100 68 22 + 16 302 50 + 1.8 4 110 44 + 29 1 1000 41 ' 6.7 1.4 100 13-18 200 22 C o s o l u b i l i z a t i o n of PEMT 1 and 2 has been accomplished from mouse l i v e r by su c c e s s i v e treatments w i t h deoxycholate and T r i t o n X-100 (114), and from r a t l i v e r by s o n i c a t i o n of microsomes and e x t r a c t i o n with T r i t o n X-100 (107). In both i n s t a n c e s , the m e t h y l t r a n s f e r a s e s were p u r i f i e d approximately 4-5-fold from microsomes, and the o v e r a l l r e c o v e r y was 10% or l e s s . From the data of S c h n e i d e r and Vance ( 1 0 7 ) . i t may be c a l c u l a t e d t h a t the T r i t o n X-100 e x t r a c t i o n f a c i l i t a t e d s o l u b i l i z a t i o n o f 21% of the PEMT 1 a c t i v i t y and 45% of the PEMT 2 a c t i v i t y . Recently, Axelrod's group (104) have reported that repeated e x t r a c t i o n s of r a t l i v e r microsomes w i t h a hypotonic medium which contained EDTA r e s u l t e d i n an enric h m e n t of PEMT 1 a c t i v i t y r e l a t i v e to PEMT 2 a c t i v i t y , but the methyltransferase a c t i v i t i e s were measured at sub-opt i m a l pH. These i n v e s t i g a t o r s a l s o claimed t o have s o l u b i l i z e d PEMT 2 when the enzyme a c t i v i t y was d e t e c t e d i n the 105,000 X g supernatant of a p r e p a r a t i o n o f microsomes t h a t was p r e v i o u s l y subjected to repeated m i l d s o n i c a t i o n . S i m i l a r l y , A x e l r o d ' s group (98) have " s o l u b i l i z e d " the PEMT 2 from r a t b r a i n synaptosomes by s o n i c a t i o n . 23 Mammalian PEMT 1 a p p a r e n t l y behaves as an i n t e g r a l membrane p r o t e i n s i n c e i t i s r e l a t i v e l y r e s i s t a n t t o e x t r a c t i o n from the b i l a y e r by var i o u s treatments, and i t does not accept exogenous PE as a s u b s t r a t e . In c o n t r a s t , PEMT 2 which i s more r e a d i l y s o l u b i l i z e d and can accept exogenous s u b s t r a t e s , seems t o resemble an e x t r i n s i c membrane p r o t e i n . Since s o l u b i l i z e d PEMT 1 always has PEMT 2 a c t i v i t y , a c l o s e a s s o c i a t i o n between the two methyltrans-f e r a s e s may e x i s t . The low c o n c e n t r a t i o n o f MMPE and DMPE intermediates i n t h e membrane a l s o t e n d s t o s u p p o r t t h e view t h a t t h e s e enzymes are i n j u x t a p o s i t i o n . During homogenization of r a t l i v e r , the E.R. fragments to form v e s i c l e s having the same o r i e n t a t i o n with the luminal side of the membrane inward and the cytop l a s m i c s i d e outward (115). These v e s i c l e s are the major c o n s t i t u e n t of the microsomal f r a c t i o n . Since i n c u b a t i o n of microsomes with t r y p s i n f o r 1 min decreased methylation o f PE by 95% ( 3 0 ) , PEMT 1 and 2 are thought to r e s i d e on the cytoplasmic f a c e o f the E.R. Recent s t u d i e s by Higgins (116) using phospholipase C as a probe to monitor the production of r a d i o - l a b e l l e d MMPE, DMPE and PC,suggest t h a t some PEMT 2 a c t i v i t y i s l o c a t e d on the lu m i n a l s i d e of the E.R. A l t h o u g h t r y p s i n t r e a t m e n t o f i n t a c t microsomes 3 i n h i b i t e d 95% of the i n c o r p o r a t i o n o f l a b e l from [Me- H]Ado-Met i n t o the outer l e a f l e t of the b i l a y e r , t h e r e was no r e d u c t i o n of the i n c o r p o r a t i o n i n t o a pool of MMPE, DMPE and PC which was sequestered from phospholipase C i n open and closed v e s i c l e s . F u r t h e r m o r e , i n t r a p e r i t o n e a l i n j e c t i o n of L-14 [Me- C j m e t h i o n i n e r e s u l t e d i n u n i f o r m l a b e l l i n g o f the PC i n each l e a f l e t o f t h e r a t l i v e r m i c r o s o m e s ( 1 1 6 ) . T h u s ^ n v i v o , t h e t r a n s l o c a t i o n of PE v i a m e t h y l a t i o n t o the lu m i n a l s i d e as PC was even more p r o n o u n c e d t h a n in. v i t r o . Crews e t a l . (117) have used p r o t e o l y t i c d i g e s t i o n by t r y p s i n o f 24 i n t a c t and lys e d r a t b r a i n synaptosomes t o show t h a t PEMT 1 i s l o c a l i z e d on the cytoplasmic side of the plasma membrane, while PEMT 2 faces the e x t e r n a l s u r f a c e . These i n v e s t i g a t o r s a l s o found t h a t p h o s p h o l i p a s e C d i g e s t i o n of i n t a c t synaptosomes produced h y d r o l y s i s o f a c o n s i d e r a b l e f r a c t i o n of DMPE and PC, but not MMPE. Hence, MMPE was thought t o be b u r i e d w i t h i n the membrane or l o c a t e d on the cytoplasmic s i d e . H i r a t a and A x e l r o d ( 1 1 1 ) have a l s o shown a s i m i l a r asymmetric arrangement of PEMT 1 and 2 a c r o s s the r a t e r y t h r o c y t e membrane. Trypsin treatment of i n s i d e - o u t ghosts ( c y t o p l a s m i c f a c e exposed) abolished PEMT 1 a c t i v i t y , whereas PEMT 2 a c t i v i t y was u n a f f e c t e d . The converse was t r u e f o r r i g h t - s i d e - o u t ghosts ( e x t r a c e l l u l a r f a c e exposed). The methylated phospho-l i p i d s o f r i g h t - s i d e - o u t ghosts were s e n s i t i v e to d i g e s t i o n by phospholipase C and p h o s p h o l i p a s e A^» but not those o f i n s i d e - o u t g h o s t s . When ghosts were i n c u b a t e d under c o n d i t i o n s i n which MMPE a c c u m u l a t e d , m e t h y l a t e d phos p h o l i p i d s were r e s i s t a n t to d i g e s t i o n by phospholipase C from e i t h e r s i d e of the membrane, i n d i c a t i n g that MMPE i s deeply embedded i n the membrane. FORMATION OF PHOSPHATIDYLCHOLINE BY GROUP EXCHANGE Base Exchange ( 1 . 2 . 3 . 1 ) . The exchange o f f r e e c h o l i n e , e t h a n o l a m i n e and s e r i n e w i t h t h e headgroups o f p r e e x i s t i n g p h o s p h o l i p i d s t o p r o d u c e PC, PE and PS, r e s p e c t i v e l y , has been shown to occur i n microsomes (118,119). These enzyma-t i c processes are c a l c i u m - r e q u i r i n g and energy-independent (48,120-122). The exchange a c t i v i t i e s have been s o l u b i l i z e d from r a t b r a i n microsomes and f r a c t i o n a t i o n s t r o n g l y suggests t h a t s e p a r a t e enzymes c a t a l y z e each of the 25 r e a c t i o n s ( 1 2 3 ) . In r a t b r a i n , c h o l i n e exchange a c t i v i t y was t r y p s i n -s e n s i t i v e and presumably l o c a t e d on the c y t o p l a s m i c side of the E.R. (121). In c o n t r a s t , s e r i n e and ethanolamine exchange were t r y p s i n - i n s e n s i t i v e and assumed t o r e s i d e on the l u m i n a l s i d e o f the E.R. ( 1 2 1 ) . At present, the exchange of L - s e r i n e w i t h the e t h a n o l a m i n e m o i e t y of PE i s b e l i e v e d to represent the p r i n c i p a l route of PS s y n t h e s i s i n animal t i s s u e s (118,125). F a t t y A c y l Exchange (1.2.3.2). There are two ways the f a t t y a c y l m o i e t y at the C2 p o s i t i o n of PC can be r e p l a c e d and the i n i t i a l removal of t h i s chain by the a c t i o n of phospho-l i p a s e A^ i s common to b o t h mechanisms. The r e s u l t i n g 1-saturated-2-lyso-PC i s then e i t h e r r e a c y l a t e d w i t h a c y l - C o A or t r a n s a c y l a t e d w i t h a second molecule of LPC. The a c y l a t i o n of LPC w i t h a c y l - C o A by a c y l Coenzyme-A: 1-acy l g l y c e r o -3-phosphocholine O - a c y l t r a n s f e r a s e (EC 2 .3.1.23) was f i r s t described by Lands (126), and subsequently s t u d i e d i n lung by Webster (127). This enzyme has been p u r i f i e d 4 - 5 - f o l d from microsomes and shows a p r e f e r e n c e f o r a r a c h i d o n y l - C o A , e s p e c i a l l y when the c o n c e n t r a t i o n of LPC i s low (128). There i s e v i d e n c e f o r i s o e n z y m e s o f LPC a c y l t r a n s f e r a s e w i t h v a r y i n g s p e c i f i c i t i e s f o r acyl-CoA species (129). The t r a n s a c y l a t i o n between 2 m o l e c u l e s of LPC as c a t a l y z e d by LPC: LPC t r a n s a c y l a s e (EC 2 . 3 .1.?) was f i r s t demonstrated i n r a t l i v e r by M a r i n e t t i et a l . (130). The t r a n s a c y l a s e (MW 58-60,000) i s p r e s e n t i n the 100,000 X g supernatant of lung homogenates (131) and has been p a r t i a l l y p u r i f i e d from r a t lung (132) and r a b b i t heart (133). The lung enzyme was shown to be b i f u n c t i o n a l , promoting both the d e a c y l a t i o n of LPC and the t r a n f e r of a c y l groups t o an accepting LPC (134,135,135b). Since the a c y l group or g i n a t e s from the C1 p o s i t i o n of LPC, t h i s pathway can generate d i s a t u r a t e d species of PC. 26 The Functions of Phosphatidylcholine and i t s Routes of Synthesis i n Various Mammalian Tissues B i o l o g i c a l Membranes (1.3.1.1). The numerous f u n c t i o n s of PC a r i s e from i t s amphipathic nature - the c h o l i n e h e a d g r o u p i s h y d r o p h i l i c , w h i l e t h e f a t t y a c y l c h a i n s a r e hydrophobic. The a b i l i t y of PC and the o t h e r p h o s p h o l i p i d s to s e l f - i n t e r a c t and spontaneously form a b i l a y e r membrane i s probably the most important f e a t u r e of these g l y c e r o l i p i d s . T h i s s p a t i a l arrangement permits the maximal i n t e r a c t i o n s between the h y d r o p h i l i c groups and ambient water, and between the h y d r o p h o b i c f a t t y a c y l g r oups i n the i n t e r i o r o f the b i l a y e r . Hence the p h o s p h o l i p i d s s e r v e as a s t r u c t u r a l m a t r i x f o r the p r o t e i n component o f a l l b i o l o g i c a l membranes as d e t a i l e d i n t h e f l u i d m osaic model of S i n g e r and N i c h o l s o n ( 1 3 6 ) . In a d d i t i o n , the membrane b i l a y e r serves as anchorage f o r g l y c o p r o t e i n s , and i s a non-aqueous medium fo r r e a c t i o n s i n v o l v i n g l i p i d s o l u b l e m e t a b o l i t e s . Substrates of membrane enzyme c a t a l y z e d r e a c t i o n s tend t o be c o n c e n t r a t e d i n the two-dimensional network of the membrane. The sheer d i v e r s i t y of l i p i d s i n biomembranes a l s o i m p l i e s t h a t l i p i d s p l a y more t h a n a p a s s i v e s t r u c t u r a l r o l e . The p h o s p h o l i p i d s have been i m p l i c a t e d i n the c o n t r o l of such dynamic processes as membrane f u s i o n (137), e n d o c y t o s i s ( 1 3 8 ) , m e t a b o l i t e t r a n s p o r t (139-141) and the c a t a l y t i c a c t i v i t y of membrane-bound enzymes (142). For example, D-beta-hydroxybutyrate dehydrogenase a c t i v i t y has an absolute requirement f o r PC (143). In eukaryotes, PC a c c o u n t s f o r n e a r l y h a l f o f the membrane phospho-l i p i d , and 70-75% of t h i s l i p i d has been reported t o be i n the cytoplasmic 27 l e a f l e t of the- E.R. (144). PE and PS were enriched on the lumi n a l side of of the E.R. (144). Some of the asymmetry o f the phospholipid b i l a y e r of the E.R. may stem from the asymmetrical d i s t r i b u t i o n of the l i p i d b i o s y n t h e t i c enzymes on the c y t o p l a s m i c s i d e , and the l i p i d d egradative enzymes on the l u m i n a l s i d e of the E.R. Other f a c t o r s undoubtably c o n t r i b u t e to the l i p i d asymmetry as w e l l . Higgins (50,116) has obtained evidence w i t h r a t l i v e r microsomes t o suggest t h a t PC s y n t h e s i z e d by the d_e novo pathway i s pre-d o m i n a n t l y l o c a t e d on the c y t o p l a s m i c s i d e , whereas PC produced by met h y l a t i o n o f PE i s d i s t r i b u t e d on both s i d e s . Numerous s t u d i e s have i n d i c a t e d t h a t t h e e x t r a c e l l u l a r l e a f l e t o f the plasma membrane b i l a y e r c o r r e s p o n d s t o the l u m i n a l l e a f l e t o f the E.R. b i l a y e r (145). In plasma membranes, PC, sphingomyelin and c h o l e s t e r o l occur predominantly on the e x t r a c e l l u l a r s i d e , and PE, PS and p h o s p h a t i d y l i n o s i t o l ( P I ) on the i n t r a c e l l u l a r s i d e ( 1 4 6 - 1 4 9 ) . I t i s hard t o r e c o n c i l e the discrepancy i n the o r i e n t a t i o n of the l i p i d asymmetry i n the plasma membrane and E.R. F u r t h e r m o r e , N i l s s o n and D a l l n e r (150) have d e t e r m i n e d w i t h phospholipase t h a t 55? o f the PC and a l l o f the PE and PS are i n the cytoplasmic l e a f l e t o f the E.R. S u n d l e r et. a l . (151) a l s o w i t h phospho-l i p a s e A^ found no evidence f o r p h o s p h o l i p i d asymmetry i n r a t l i v e r micro-somes. For these reasons, the a c t u a l d i s t r i b u t i o n o f phospholipids i n the E.R. remains obscure. L i v e r ( 1 . 3 . 1 . 2 ) . The l i v e r i s the l a r g e s t o r g a n i n humans and i s composed of 65% hepatocytes (parenchymal c e l l s ) and 35% non-parenchymal c e l l s (132). The hepatocytes occupy 72% of the t o t a l l i v e r volume. The non-parenchymal c e l l s have been r e f e r r e d to as "Kupffer c e l l s " although t h i s population of l i v e r c e l l s a l s o i n c l u d e s e n d o t h e l i a l c e l l s , f a t - s t o r i n g c e l l s and p i t c e l l s . Non-28 parenchymal c e l l s f u n c t i o n i n c l e a r i n g f o r e i g n m a t e r i a l s and some of the l i p o p r o t e i n s from the blood, and i n the production of a n t i b o d i e s . The hepa-t o c y t e s c a r r y out the wide spectrum o f b i o s y n t h e t i c and degradative processes that maintain homeostasis i n the whole animal. In the post-absorptive s t a t e , the l i v e r removes plasma chylomicrons remnants and f r e e f a t t y acids (FFA) from the c i r c u l a t i o n and i s the main s i t e f o r e s t e r i f i c a t i o n o f these FFA i n t o g l y c e r o l i p i d s . The major s y n t h e s i s of glycogen and FA from carbohydrate and from p r o t e i n , a l s o occurs i n l i v e r . The l i v e r c o n s t i t u t e s an important source of plasma t r i g l y c e r i d e (TG) and i s the s i t e o f very low density l i p o p r o t e i n (VLDL) p r o d u c t i o n . In the f a s t i n g s t a t e , the l i v e r provides energy by d e g r a d a t i o n of glycogen r e s e r v e s , o x i -d a t i o n o f m o b i l i z e d FA and ketone body formation. The hepatic synthesis of PC i s of p a r t i c u l a r i n t e r e s t since the l i v e r makes PC not o n l y f o r membrane b i o g e n e s i s , b u t a l s o f o r e x p o r t to other t i s s u e s . B i l e ( 1 . 3 . 1 . 3 ) . The l i v e r i s the p r i m a r y source o f b i l e which i s e s s e n t i a l f o r the e f f i c i e n t s o l u b i l i z a t i o n , d i g e s t i o n and a b s o r p t i o n of t r i g l y c e r i d e i n the upper s m a l l i n t e s t i n e . In a d d i t i o n , b i l e f a c i l i t a t e s the e x c r e t i o n o f b i l i r u b i n and o t h e r waste p r o d u c t s . PC i s a component of b i l e and i t f u n c t i o n s t o g e t h e r w i t h c o n j u g a t e d b i l e a c i d s f o r the s o l u b i l i z a t i o n of c h o l e s t e r o l i n b i l e ( 1 5 3 , 1 5 4 ) . B i l e PC i s composed o f m a i n l y the 1 - p a l m i t o y l , 2 - l i n o l e o y l s p e c i e s ( 1 5 5 , 1 5 6 ) and t h i s s p e c i e s i s c h a r a c t e r i s t i c a l l y a p r o d u c t o f t h e d_e novo pathway ( 5 3 , 7 5 ) . The b i l i a r y s e c r e t i o n o f c h o l e s t e r o l , b i l e s a l t s and PC are c l o s e l y coordinated (157,158), and regulated by d i e t and by b i l e a c i d i n f l u x (157). 29 L i p o p r o t e i n s ( 1 - 3 - 1 - 4 ) . In a d d i t i o n to b i l e , PC i s an i m p o r t a n t c o n s t i t u e n t of l i p o p r o t e i n s comprising between 7% (chylomicrons) and 35% (high d e n s i t y l i p o p r o t e i n s ) of the t o t a l w e i g h t . The PC found i n LDL may be s y n t h e s i z e d by the de  novo pathway ra t h e r than by PE N - m e t h y l a t i o n . This p o s s i b i l i t y i s based on the o b s e r v a t i o n t h a t during c h o l i n e - d e f i c i e n c y (Section 1.5.5.2) there i s a r e d u c t i o n i n the h e p a t i c s e c r e t i o n of LDL d e s p i t e an enhancement of the m e t h y l a t i o n pathway (159)- The h e p a t i c s e c r e t i o n of LDL i s of minor impor-tance, however, i n comparison to the r e l e a s e of VLDL. The plasma l i p o p r o t e i n s f a c i l i t a t e the t r a n s p o r t o f c h o l e s t e r o l , c h o l e s t e r o l - e s t e r s and TG between t h e i n t e s t i n e s , l i v e r and a d i p o s e . A t h e r o s c l e r o s i s , the l e a d i n g cause o f death of a d u l t s i n North America, has been l i n k e d with a b n o r m a l i t i e s i n l i p o p r o t e i n metabolism (160). The p r e c i s e f u n c t i o n o f PC i n l i p o p r o t e i n s i s u n c l e a r , b u t t o g e t h e r w i t h the a p o p r o t e i n s , PC i s thought to coat the surface of l i p o p r o t e i n s to render the core of TG and c h o l e s t e r o l - e s t e r s water s o l u b l e . P r o s t a g l a n d i n s ( 1 . 3 . 1 - 5 ) . In i s o l a t e d r a t hepatocytes, S u n d l e r and Akesson (161) have shown th a t w h i l e t h e de novo pathway i s t h e m a j o r r o u t e f o r PC s y n t h e s i s , under s p e c i a l c o n d i t i o n s , methylation can account f o r 30% of newly formed PC. The a c t i v i t y of the methylation pathway i s higher i n l i v e r than any other t i s s u e (162), although the reason fo'r t h i s i s e l u s i v e . The methylation pathway may be l a r g e l y responsible f o r the formation of the 2-arachidonyl PC (163). The a c t i o n of phospholipase on t h i s species of PC may be the f i r s t step i n the s y n t h e s i s of p r o s t a g l a n d i n s . Prostaglandins a r e l i p i d hormones which have been shown t o c o n t r o l a wide v a r i e t y o f p h y s i o l o g i c a l p r o c e s s e s . Some o f the e f f e c t s o f p r o s t a g l a n d i n s i n c l u d e 30 s t i m u l a t i o n of inflammation, r e g u l a t i o n o f blood flow to p a r t i c u l a r organs, c o n t r o l o f i o n t r a n s p o r t a c r o s s membranes, s t i m u l a t i o n o f p a r t u r a t i o n , c o n t r o l of p l a t e l e t aggregation and modulation of s y n a p t i c t r a n s m i s s i o n . In r a t l i v e r , c h o l i n e base exchange i s a r e l a t i v e l y minor pathway. Less than 5% of the r a d i o - l a b e l l e d c h o l i n e taken up by r a t l i v e r (163,165) or r a t hepatocytes (165) i s d i r e c t l y i ncorporated i n t o PC v i a base exchange. Lung and S u r f a c t a n t (1.3-1-6). The l u n g i s a h e t e r o g e n o u s o r g a n composed o f a p p r o x i m a t e l y 40 d i f f e r e n t c e l l types. While a l l the c e l l t y p e s s y n t h e s i z e PC f o r membrane b i o g e n e s i s , only the type I I a l v e o l a r e p i t h e l i a l c e l l s are r e s p o n s i b l e f o r s u r f a c t a n t s y n t h e s i s and s e c r e t i o n (166-168). S u r f a c t a n t i s the s u r f a c e a c t i v e m a t e r i a l which l i n e s the a l v e o l i of the lungs to lower the s u r f a c e t e n s i o n a t the a i r - a l v e o l a r i n t e r f a c e and thereby prevent a l v e o l a r c o l l a p s e d u r i n g e x p i r a t i o n . S u r f a c t a n t d e f i c i e n c y i s thought to be r e s p o n s i b l e f o r I n f a n t R e s p i r a t o r y D i s t r e s s Syndrome (169) which i s the major cause of death o f the newborn. S u r f a c t a n t , i t s e l f , i s almost 80% phospholipid by weight, of which PC accounts f o r 80-90% (170). In r a t , r a b b i t , p i g and s heep l u n g s u r f a c t a n t , d i p a l m i t o y l PC i s the predominant species and can be 70% of the t o t a l PC (171,172). The s y n t h e s i s of d i p a l m i t o y l PC was p r e v i o u s l y a s c r i b e d to remodelling mechanisms, d e s p i t e the f i n d i n g t h a t t h e de_ novo pathway i s t h e major route of PC s y n t h e s i s i n l u n g ( 1 73,174). Abe and A k i n o (175,176) found t r a n s a c y l a t i o n a c t i v i t y was h i g h e r i n the l u n g than i n any of the other organs. Furthermore, during p r e n a t a l development, the a c t i v i t y of LPC: LPC t r a n s a c y l a s e was elevated s e v e r a l f o l d i n the lungs of mouse (177) and r a t (178), and was a maximum v a l u e about one day b e f o r e b i r t h . The r e l a t i v e importance of t r a n s a c y l a t i o n f o r b i o s y n t h e s i s of s u r f a c t a n t PC i n mature 31 lung i s c o n t r o v e r s i a l (179). S t u d i e s with mouse pulmonary adenoma type I I c e l l s i n d i c a t e that the r e a c y l a t i o n of LPC w i t h p a l m i t o y l - C o A i s q u a n t i t a t i v e l y more s i g n i f i c a n t than t r a n s a c y l a t i o n ( 1 8 0 ) . Van Heusden et a l . (181) found that r a t lung PC synthesized from i n t r a v e n o u s l y i n j e c t e d LPC stereoisomers c o n s i s t e d e n t i r e l y o f sn-3-PC. A c y l - C o A : LPC a c y l t r a n s f e r a s e o n l y a c c e p t s the n a t u r a l sn-3-LPC i s o m e r , w h i l e the LPC: LPC t r a n s a c y l a s e e x p r e s s e s no s t e r e o s p e c i f i c i t y between s j i - 1 and s n - 3 - L P C . T h e r e f o r e , the i n v i v o f i n d i n g s were i n t e r p r e t e d as proof that the LPC:LPC t r a n s a c y l a t i o n r e a c t i o n does not c o n t r i b u t e towards the s y n t h e s i s of d i p a l m i t o y l PC, at l e a s t not from plasma LPC. The l a c k of PC s y n t h e s i s from model-membrane embedded LPC i n v i t r o with s o l u b l e LPC: LPC t r a n s a c y l a s e c o r r o b o r a t e d t h i s view (181). The c o n t r i b u t i o n o f t h e d_e n o v o p a t h w a y f o r d i p a l m i t o y l PC formation was o r i g i n a l l y down p l a y e d because a t t e m p t s t o demonstrate the i n v i t r o s y n t h e s i s , of d i p a l m i t o y l PC f r o m d i p a l m i t o y l g l y c e r o l were u n i f o r m l y unsuccessful (177,182-184). Furthermore, r a d i o - t r a c e r experiments i n v i v o (185,186), w i t h p e r f u s e d l u n g (187-189), w i t h l u n g s l i c e s (190) and w i t h i s o l a t e d type I I c e l l s (191) i n d i c a t e d t h a t l a b e l l e d p a l mitate was p r e f e r e n t i a l l y i n c o r p o r a t e d i n t o the sn-2 p o s i t i o n of d i p a l m i t o y l PC. D i s a t u r a t e d PC i s a c t i v e l y produced i n the lung (192,193), however, and very recent s t u d i e s have demonstrated t h a t cholinephosphotransferase can accept d i p a l m i t o y l g l y c e r o l as a s u b s t r a t e iin v i t r o (61,172,194). M i l l e r and Weinhold (61) have di s c o v e r e d t h a t the presence of p h o s p h a t i d y l g l y c e r o l , the minor p h o s p h o l i p i d c o n s t i t u e n t of s u r f a c t a n t , i n c r e a s e s the c h o l i n e -phosphotransferase a c t i v i t y 4-5-fold with d i o l e o y l - g l y c e r o l and 16-fold with d i p a l m i t o y l - g l y c e r o l . F i n a l l y , Buechler and Rhoades (195) have r e c e n t l y 32 shown t h a t i n i s o l a t e d perfused r a t lung over k0% of the p a l m i t a t e e s t e r i f i e d i n t o d i s a t u r a t e d PC was d e r i v e d from de novo s y n t h e s i s . 33 B r a i n and S p h i n g o m y e l i n (1.3.1.7). PC i s a c o n s t i t u e n t o f b r a i n , b u t s p h i n g o l i p i d s a c c o u nt f o r the m a j o r i t y o f the b r a i n l i p i d c o n t e n t . A l a r g e p o r t i o n o f the membranous ph o s p h o l i p i d i s found i n the m y e l i n s h e a t h which wraps around neurons and permits f a s t e r r a t e s of impulse c o n d u c t i o n . O l i g o d e n d r o g l i a l c e l l s produce myelin i n the white matter o f the b r a i n , w h i l e Schwann c e l l s c a r r y out the s y n t h e s i s i n the p e r i p h e r a l nervous system. S p h i n g o m y e l i n i s the major p h o s p h o l i p i d i n myelin and a c c o u n t s f o r 30% o f the t o t a l dry weight of the human myelin sheath (196). S p h i n g o m y e l i n (SM) a l s o a c c o m p a n i e s PC i n the plasma membrane o f mammalian c e l l s and i n the s u r f a c e monolayer o f plasma l i p o p r o t e i n s . In e r y t h r o c y t e s , SM c o m p r i s e s from 23% (human) t o 61% (bovine ) o f the t o t a l membrane p h o s p h o l i p i d s (197). SM was i n i t i a l l y thought t o be produced from CDP-choline and ceramide (198), but very recent s t u d i e s (199-202) c l e a r l y i d e n t i f y PC as the source of the phosphocholine moiety that i s t r a n s f e r r e d t o ceramide. Esko and Raetz (203) have found t h a t temperature s e n s i t i v e mutants of Chinese hamster ovary c e l l s produced' n o r m a l l e v e l s o f SM u n d e r c o n d i t i o n s o f CDP - c h o l i n e d e p l e t i o n . The cholinephosphotransferase which c a t a l y z e s the formation of SM has been detected i n the plasma membranes f r a c t i o n s o f mouse f i b r o b l a s t s ( 1 9 9 , 2 0 1 ) , r a t l i v e r ( 2 0 2 ) , BHK c e l l s (202) and i n mouse l i v e r microsomes (200). PC: ceramide c h o l i n e p h o s p h o t r a n s f e r a s e has a l s o been measured i n G o l g i f r a c t i o n s from mouse f i b r o b l a s t s , where i t could be st i m u l a t e d 6-fold by exogenous ceramide (201). In b r a i n , c h o l i n e i s a l s o r e q u i r e d f o r the s y n t h e s i s of the neurotrans-m i t t e r , a c e t y l c h o l i n e . B l u s z t a j n and Wurtman (204) have shown l a t e l y that r a t b r a i n synaptosomes can m e t a b o l i z e the PC generated by N-methylation of 34 PE t o l i b e r a t e f r e e c h o l i n e . A l t h o u g h t h e s e a u t h o r s d i d not determine the mechanism of PC degradation t o r e l e a s e c h o l i n e , they speculated t h a t the c h o linephosphotransferase r e a c t i o n may be i n v o l v e d . The r e v e r s i b i l i t y o f the c h o l i n e p h o s p h o t r a n s f e r a s e r e a c t i o n i n b r a i n l e d G o r a c c i £t a l . (83) t o p r o p o s e t h a t t h i s i s the f i r s t s t e p i n a major pathway f o r PC d e g r a d a t i o n . An a l k a l i n e d i g l y c e r i d e l i p a s e i n r a t b r a i n microsomes (83,205) was p o s t u l a t e d t o f u r t h e r degrade d i g l y c e r i d e to r e l e a s e g l y c e r o l and FFA. P o r c e l l a t i e t a l . (206) have described l a r g e i n c r e a s e s i n both FFA and CDP-choline i n g e r b i l b r a i n s during the f i r s t 3 min of ischemia with no a l t e r a t i o n i n the l e v e l of d i g l y c e r i d e s . In a d d i t i o n , Marion and Wolfe (82) have r e p o r t e d t h a t PC i s the source of 63% of the arachidonate released i n r a t f o r e b r a i n d u r i n g 30 min of p o s t - d e c a p i t a t i o n i s c h e m i a , although an a c t i v a t i o n of p h o s p h o l i p a s e A^ might e x p l a i n t h i s r e s u l t . A d e f e c t i n b r a i n PC m e t a bolism has been i m p l i c a t e d i n Parkinson's disease (207). CDP-choline has been used as an a n t i p a r k i n s o n i a n drug, but' the biochemical b a s i s of the t h e r a p e u t i c e f f e c t of exogenous CDP-choline i s e l u s i v e . However, C D P - c h o l i n e i n c r e a s e s dopamine l e v e l s and d e c r e a s e s s e r o t o n i n l e v e l s i n whole mouse b r a i n (207) and i n the b r a i n stem of r a t ( 2 0 8 ) . M a r t i n e t £t al^ ( 2 0 9 ) h a v e a l s o n o t e d t h a t C D P - c h o l i n e a d m i n i s t r a t i o n produced a 20% s t i m u l a t i o n o f t y r o s i n e hydroxylase i n r a t b r a i n , although C D P - c h o l i n e d i d not s t i m u l a t e t h i s enzyme in_ v i t r o . When r a t s are administered r a d i o - l a b e l l e d c h o l i n e , accumulation of the l a b e l i n t o b r a i n l i p i d i s b i p h a s i c (210) and c h o l i n e i s p r e f e r e n t i a l l y i n c o r p o r a t e d i n t o PC r a t h e r than p h o s p h o c h o l i n e f o r the f i r s t few minutes a f t e r i n j e c t i o n (211). Base exchange can e x p l a i n the e a r l y i n c o r p o r a t i o n of l a b e l i n t o PC and perhaps the enhanced i n c o r p o r a t i o n observed a f t e r i n t r a -35 c e r e b r a l i n j e c t i o n of l a b e l l e d c h o l i n e w i t h hemicholinium-3 (212). Hemi-cholinium-3 caused reduced c h o l i n e p h o s p h o r y l a t i o n i n the cerebellum, yet c h o l i n e i n c o r p o r a t i o n i n t o PC was i n c r e a s e d . The pool of PC i n r a t b r a i n a v a i l a b l e f o r exchange has been estimated by p r e l a b e l l i n g PC w i t h c h o l i n e , f o l l o w e d by c o m p e t i t i v e removal of the base w i t h c h o l i n e , ethanolamine or s e r i n e l a b e l l e d w i t h a second i s o t o p e (213)• This pool represented only 3-6% of the t o t a l b r a i n microsomal PC. • 36 I n t e s t i n e ( 1 . 3 - 1 - 8 ) . De novo s y n t h e s i s v i a C D P - c h o l i n e and a c y l a t i o n o f LPC are two pathways f o r PC s y n t h e s i s i n the i n t e s t i n e . PE m e t h y l t r a n s f e r a s e a c t i v i t y i s not d e t e c t a b l e i n t h i s t i s s u e ( 1 0 6 ) . R e a c y l a t i o n o f LPC i s an a t t r a c t i v e route f o r PC s y n t h e s i s i n the i n t e s t i n e since LPC i s r e a d i l y a v a i l a b l e a f t e r d i g e s t i o n and a b s o r p t i o n . Only two h i g h energy phosphate bonds are required t o synthesize PC from LPC, whereas seven ar e r e q u i r e d i f PC i s generated from s n - g l y c e r o l - 3 - p h o s p h a t e , or f i v e i f PC i s s y n t h e s i z e d from sn-mono-a c y l g l y c e r o l . Mansbach and P a r t h a s a r a t h y (214) have examined the i n f l u e n c e of PC i n f u s i o n i n v i v o on PC s y n t h e s i s i n r a t i n t e s t i n e . The PC content of the microsomes was doubled a f t e r PC was i n f u s e d i n t o a c t i v e l y l i p i d absorbing, b i l e f i s t u l a t e d r a t s . The r a t s w i t h PC i n c l u d e d i n t h e i r i n f u s a t e had a 32 reduced i n c o r p o r a t i o n o f [ P]phosphate i n t o C D P - c h o l i n e and PC, but not i n t o phosphocholine. These i n v e s t i g a t o r s concluded t h a t the f l u x of LPC was increased i n t o t h e i n t e s t i n a l mucosal c e l l s when PC was i n c l u d e d i n the i n f u s a t e , and t h a t the 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 f e r a s e (35) and phosphocholinetransferase (215) catalyzed r e a c t i o n s . H e a r t ( 1 . 3 - 1 - 9 ) . Choy and a s s o c i a t e s have shown t h a t more than 90% o f the PC i n the hamster h e a r t i s formed cie novo f r o m c h o l i n e ( 2 1 6 ) . Only 2.5% of the t o t a l PC i n hamster heart was generated by p r o g r e s s i v e N-methylation of PE. Furthermore, the c o n t r i b u t i o n of base-exchange t o t o t a l PC s y n t h e s i s was minor i n t h i s organ. Savard and Choy (217) a l s o examined the the formation o f PC by r e a c y l a t i o n and t r a n s a c y l a t i o n o f exogenous LPC s i n c e LPC i s t r a n s p o r t e d i n t o the h e a r t . P e r f u s i o n o f the i s o l a t e d hamster heart with 14 3 1-[ C ] p a l m i t o y l g y c e r o p h o s p h o [ M e - H ] c h o l i n e i n d i c a t e d t h a t l a b e l l e d PC 37 was e x c l u s i v e l y formed by r e a c y l a t i o n o f LPC w i t h a c y l - C o A , and not by t r a n s a c y l a t i o n w i t h another molecule of LPC. B l o o d - C h o l e s t e r o l E s t e r i f i c a t i o n (1.3•1•10). In t h e b l o o d , PC p l a y s an i m p o r t a n t r o l e i n t h e m e t a b o l i s m o f l i p o p r o t e i n c h o l e s t e r o l . The f a t t y a c y l moiety at C2 of PC i s t r a n s f e r r e d to the 3-hydroxyl group o f c h o l e s t e r o l i n a r e a c t i o n c a t a l y z e d by l e c i t h i n : c h o l e s t e r o l a c y l t r a n s f e r a s e (LCAT) (EC 2 . 3.1.43). This r e a c t i o n i s c r u c i a l f o r t h e c l e a r a n c e o f f r e e c h o l e s t e r o l from c e l l membranes and f o r the c a t a b o l i s m o f p l a s m a t r i g l y c e r i d e s ( 2 1 8 ) . LCAT d e f i c i e n c y may be a pr e d i s p o s i n g f a c t o r f o r development o f a t h e r o s c l e r o s i s (219). Blood-PE M e t h y l a t i o n ( 1 . 3 . 1 . 1 1 ) . H i r a t a and Axelrod (220) have a t t r i b u t e d l a r g e changes i n the f l u i d i t y of r e d b l o o d c e l l membranes t o a l t e r a t i o n s i n PE m e t h y l a t i o n . These i n v e s t i g a t o r s reported that the m i c r o v i s c o s i t y o f r a t ery t h r o c y t e ghosts was reduced 33% i n the pre s e n c e o f 100 uM Ado-Met. T h i s h y p o t h e s i s has since been challenged by Vance and De K r u i j f f (162) who have c a l c u l a t e d that l e s s than 0.00033% o f t h e t o t a l membranous PE was m e t h y l a t e d i n t h e s e e x p e r i m e n t s . The d r a m a t i c changes i n m i c r o v i s c o s i t y were d i f f i c u l t to r e c o n c i l e w i t h the minute changes i n p h o s p h o l i p i d methylation. Vance and De K r u i j f f " s c o n c l u s i o n s are f u r t h e r s u p p o r t e d by the s t u d i e s of Schroeder et a l . ( 2 2 1 ) . There was no change i n membrane f l u i d i t y (as m o n i t o r e d with s e v e r a l d i f f e r e n t probes) when the p o l a r head group o f the pho s p h o l i p i d i n the plasma or s u b c e l l u l a r membranes was a l t e r e d by more than 50%. There i s b e t t e r evidence t h a t a l t e r a t i o n s i n phospholipid methylation may be a common step i n the t r a n s d u c t i o n ' o f a v a r i e t y of s i g n a l s a c t i n g on the c e l l s u r f a c e ( T a b l e 8 ) . M c G i v n e y e_t a l . (92) o b t a i n e d data which i n d i c a t e d m e t h y l a t i o n of PE may be i m p o r t a n t f o r the IgE-mediated 38 Table 8. Model Systems Which have been Linked with Methylation of PE. Ref. Model System Effector PE Methylation C e l l u l a r Process (222) murine T lymphocytes mitogenic l e c t i n s stimulated from BALB/c mice e.g. ConA, PHA (223) r a b b i t peritoneal fMet-Leu-Phe leukocytes -decreased due to degrad-ation of PC (221) human natural k i l l e r 3-deazaadenosine decreased c e l l s (225) mouse mammary gland Ado-Hcy increased mitogenesis and increased DNA synthesis PL A degradation of PC generated v i a methylation with C20:4 release impaired c y t o t o x i -c i t y increased binding of human growth hormone (226) r a t mast c e l l s anti-IgE receptor increased antibodies increased cAMP + calcium i n f l u x + histamine release (227) mouse macrophage c e l l l i n e RAW 261 (228) r a t r e t i c u l o c y t e ghosts (229) human erythrocyte ghosts (223) rabbit neutrophils 3-deazaadenosine L - i s o p r o t e r e n o l Ado-Hcy fMet-Leu-Phe decreased increased increased decreased i n h i b i t i o n of chemotaxis st i m u l a t i o n of adenylate cyclase s t i m u l a t i o n of calcium dep-ATPase st i m u l a t i o n of chemotaxis (230) C6 rat glioma c e l l s paramyxovirus e.g. SSPE, CDV decreased (23D human polymorpho-nuclear leukocytes ( 92) r a t basophilic leukemia c e l l s zymosan p a r t i c l e s decreased coated with comple-ment increased reduction i n num-ber of beta-adre-nergic receptors stimulated release of PAF and beta-glucuronidase histamine release (232) D. discoideum (233) r a t p i t u i t a r y homogenates cAMP increased lysine-vasopressin increased s t i m u l a t i o n of chemotaxis 39 r e l e a s e of h i s t a m i n e from r a t b a s o p h i l i c leukemia (RBL) c e l l s . RBL c e l l l i n e s d e f e c t i v e i n PEMT enzymes were unable t o evoke an i n f l u x of Ca +^ or re l e a s e histamine when challenged with IgE. In human polymorphonuclear l e u k o c y t e s , PEMT a c t i v i t y was reduced when phagocytosis of zymosan p a r t i c l e s coated with complement t r i g g e r e d the s e c r e t i o n of p l a t e l e t - a c t i v a t i n g f a c t o r (PAF) ( 2 3 1 ) . I n c o r p o r a t i o n of [Me-3 H]methionine i n t o PC was a l s o reduced by the zymosan p a r t i c l e s . Homo-c y s t e i n e l a c t o n e and 3-deaza-adenosine, i n h i b i t o r s of PEMT, a l s o stimulated the r e l e a s e of PAF from the leukoc y t e s . 40 Phospholipid m e t h y l a t i o n has a l s o been i m p l i c a t e d i n beta-receptor l i n k e d i n c r e a s e s i n cAMP l e v e l s i n r a t r e t i c u l o c y t e s (228,234), chemotaxis of n e u t r o p h i l s (235) and mitogenesis i n lymphocytes (222). In these systems, i n h i b i t i o n o f methylation blocked the a b i l i t y of the c e l l s t o respond t o an e x t e r n a l s i g n a l . Other processes a s s o c i a t e d w i t h t h i s s i g n a l t r a n s d u c t i o n , such as calcium i n f l u x and p h o s p h o l i p a s e A^ i n d u c e d a r a c h i d o n a t e r e l e a s e , were a l s o purportedly prevented by i n h i b i t i o n o f ph o s p h o l i p i d m e t h y l a t i o n . H o t c h k i s s e t a l . (236) have f o u n d t h a t t h r o m b i n s t i m u l a t i o n o f human p l a t e l e t s f a i l e d t o produce the changes i n the methylation of PE t h a t have been a s s o c i a t e d with r e c e p t o r a c t i v a t i o n o f other c e l l t ypes. This i s somewhat s u r p r i s i n g s i n c e t h e 2 - a r a c h i d o n y l s p e c i e s o f PC which i s p r e f e r e n t i a l l y h y d r o l y z e d d u r i n g p l a t e l e t a c t i v a t i o n by phospholipase A^ may l a r g e l y o r i g i n a t e from m e t h y l a t i o n o f PE (93). Randon et a l . (237) have a l s o noted t h a t collagen-induced p l a t e l e t aggregation was not accom-panied by an increase i n PE m e t h y l a t i o n . F u r t h e r m o r e , drugs which r a p i d l y blocked methylation (e.g. 3-deaza-adenosine) d i d not a l t e r p l a t e l e t aggre-g a t i o n . Recently, Moore e t a l . (238) c o n c l u d e d t h a t an e a r l y increase i n PE me t h y l a t i o n i s not an o b l i g a t o r y component o f the mitogenic s t i m u l a t i o n of lymphocytes by concanavalin A. Furthermore, Pike and Snyderman (238b) have shown t h a t reduced methylation of PE i s a poor e x p l a n a t i o n f o r the i n h i b i -b i t i o n of chemotaxis of macrophages by methylation i n h i b i t o r s . Rather t r e a t -ment of the macrophages with methylation i n h i b i t o r s decreased the a f f i n i t y of the N-formylated chemoattractant receptor present on these c e l l s by a f a c t o r of 4.5- I t seems that t h i s receptor on macrophages can e x i s t i n more than one a f f i n i t y s t a t e and t h a t an ongoing methylation r e a c t i o n r e a c t i o n i s r e q u i r e d f o r the maintenance of the receptor i n i t s high a f f i n i t y form. Pike 41 and Snyderman speculated t h a t i n h i b i t i o n of methylation lowered the a f f i n i t y of the receptor and rendered i t non-function or "uncoupled" i n i t s a b i l i t y to produce chemotaxis, superoxide and the release of a r a c h i d o n i c a c i d from membrane ph o s p h o l i p i d s . Hepatic microsomes c o n t a i n the h i g h e s t l e v e l s o f PEMT a c t i v i t y , yet methylation of PE i s a secondary pathway i n the l i v e r . Therefore, i n r e t r o -s p e c t , i t i s hard to see how such s i g n i f i c a n t p h y s i o l o g i c a l changes can be a t t r i b u t e d t o methylation i n o t h e r c e l l s and t i s s u e s (111,112,239) where the i n v i t r o a c t i v i t y o f PEMT i s 1 0 0 0 - f o l d lower ( 1 6 2 ) . However, i f PEMT i n plasma membrane i s an e a r l y component o f a cascade, then the a c t i v i t y of t h i s enzyme s h o u l d be low so t h a t i t i s s e n s i t i v e t o s t i m u l a t i o n by an e f f e c t o r at the top of the cascade. In r a t r e t i c u l o c y t e s , the s t i m u l a t i o n of b e t a - a d r e n e r g i c r e c e p t o r s i n c r e a s e s p h o s p h o l i p i d m e t h y l a t i o n which i n tur n i s thought to enhance the co u p l i n g o f the rece p t o r w i t h adenylate c y c l a s e (228). Since PEMT a c t i v i t y i s already high i n r a t l i v e r , a r e l a t i o n -ship between adenylate c y c l a s e and ph o s p h o l i p i d methylation would be unexpected. The l a c k of such a r e l a t i o n s h i p i n r a t l i v e r plasma membrane has been w e l l documented (103 ,239b) . * * * * « x * * x x In summary, PC i s a major component o f e u k a r y o t i c membranes, and i s required f o r the a c t i v i t y of s e v e r a l membrane-bound enzymes. In the l i v e r , PC i s synthesized f o r s e c r e t i o n o f b i l e and l i p o p r o t e i n s . PC i s the predomi-nant c o n s t i t u e n t of lung s u r f a c t a n t , and i s a pre c u r s o r of sphingomyelin. This p h o s p h o l i p i d i s a l s o a r e s e r v o i r f o r f a t t y a c i d s used t o e s t e r i f y c h o l e s t e r o l and fo r s y n t h e s i s o f p r o s t a g l a n d i n s . Evidence i s accumulating from s t u d i e s w i t h b l o o d - a s s o c i a t e d c e l l s that m e t h y l a t i o n o f PE may a l s o be 42 coupled to r e c e p t o r — l i n k e d responses. Since PC i s made a v a r i e t y ways and f o r s e v e r a l needs, the pathways f o r PC s y n t h e s i s are p r o b a b l y c o o r d i n a t e l y c o n t r o l l e d . Furthermore, the r e g u l a t i o n of these pathways i s l i k e l y complex si n c e the c e l l must be able t o respond to a multitude of s i t u a t i o n s i n which PC metabolism i s a f f e c t e d . Figure 4 o u t l i n e s the routes of PC b i o s y n t h e s i s and secondary pathways path-ways which i n f l u e n c e PC metabolism. Figure 4. Summary of c h o l i n e and p h o s p h a t i d y l c h o l i n e metabolism.  A b b r e v i a t i o n s : Ac-cho, a c e t y l c h o l i n e ; ado-met, S - a d e n o s y l - m e t h i o n i n e ; ado-hcy, S - a d e n o s y l - h o m o c y s t e i n e ; c h o , c h o l i n e ; CDP-cho, C D P - c h o l i n e ; CDP-EA, CDP-ethanolamine; DG, d i a c y l g l y c e r o l ; DMgly, d i m e t h y l g l y c i n e ; EA, ethanolamine; g l y , g l y c i n e ; G3P, g l y c e r o l - 3 - p h o s p h a t e ; GPC, g l y c e r o - 3 -phosphocholine; hey, h o m o c y s t e i n e ; LPA, l y s o p h o s p h a t i d a t e ; LPC, lysophos-p h a t i d y l c h o l i n e ; met, m e t h i o n i n e ; N^MTHF, fj5 m e t h y l t e t r a h y d r o f o l a t e ; PA, phosphatidate; PC, p h o s p h a t i d y l c h o l i n e ; PE, phosphatidylethanolamine; PS, p h o s p h a t i d y l s e r i n e ; pho-cho, p h o s p h o c h o l i n e ; pho-EA, phosphoethanolamine; and TG, t r i a c y l g l y c e r o l . LPA S bet a ine 43 Possible Regulatory Factors i n Phosphatidylcholine Biosynthesis REGULATION OF PHOSPHATIDYLCHOLINE SYNTHESIS FROM CHOLINE Mechanisms•for R e g u l a t i o n (1.1.1.1). There are numerous ways i n which PC anabolism might be c o n t r o l l e d . The supply of s u b s t r a t e s and c o f a c t o r s f o r PC b i o s y n t h e t i c r e a c t i o n s could be r e s t r i c t e d at the l e v e l o f c h o l i n e u p t a k e , by c o m p a r t m e n t a l i z a t i o n or by competing r e a c t i o n s . A l t e r n a t i v e l y , the PC b i o s y n t h e t i c enzymes, themselves, might be r e g u l a t e d by modulators, covalent m o d i f i c a t i o n or by changes i n the l e v e l of enzyme p r o t e i n . The remainder o f t h i s t h e s i s w i l l focus on the r e g u l a t i o n o f the two major p a t h w a y s o f PC f o r m a t i o n , de novo and PE m e t h y l a t i o n . C h o l i n e Uptake ( 1 . 4 . 2 . 1 ) . Choline i s a d i e t a r y requirement f o r most mammals. Choline absorbed i n the small i n t e s t i n e i s d e l i v e r e d t o the l i v e r where i t i s r a p i d l y c l e a r e d from the p o r t a l c i r c u l a t i o n . Z e i s e l e_t a l . ( 2 4 0 ) have s t u d i e d the k i n e t i c s o f c h o l i n e uptake by i s o l a t e d perfused l i v e r and found c h o l i n e was t r a n s p o r t e d by b o t h a s a t u r a b l e and a n o n - s a t u r a b l e mechanism. The non-saturable mechanism was a t t r i b u t e d to simple d i f f u s i o n o f c h o l i n e across the plasma membrane, while the s a t u r a b l e mechanism was reported to have an a pparent K of 170 uM f o r c h o l i n e ( 2 4 0 ) . S i n c e the r a t plasma c h o l i n e m ' c o n c e n t r a t i o n normally ranges between 10 to 20 uM (241,242), the f a c i l i t a t e d t r a n s p o r t o f c h o l i n e was not expected t o be s a t u r a t e d i j i v i v o . S t u d i e s with N o v i k o f f hepatoma c e l l s (243) c o n f i r m the e x i s t e n c e of s a t u r a b l e and non-saturable components f o r c h o l i n e uptake, but the s a t u r a b l e component had an a p p a r e n t K of o n l y 4-7 uM f o r c h o l i n e . A l t h o u g h the k i n e t i c s o f m ' 44 c h o l i n e uptake were not d i r e c t l y p r e s e n t e d , an apparent K value of 10 uM m ' f o r c h o l i n e can be estimated f o r i s o l a t e d r a t hepatocytes (244). Therefore, i t i s l i k e l y that the f a c i l i t a t e d t r a n s p o r t of c h o l i n e can be saturated at p h y s i o l o g i c a l plasma . c h o l i n e concentrations, although there i s no r e s t r i c t i o n of c h o l i n e entry by passive d i f f u s i o n . S i n c e c h o l i n e i s a l s o a p r e c u r s o r f o r t h e n e u r o t r a n s m i t t e r , a c e t y l c h o l i n e , b r a i n c h o l i n e t r a n s p o r t has r e c e i v e d the most a t t e n t i o n . Crude synaptosomes from r a t c e r e b r a l c o r t e x showed two c h o l i n e uptake systems: one had a high a f f i n i t y f o r c h o l i n e and was sodium-dependent, while the other had a l ower a f f i n i t y f o r c h o l i n e and was r a t h e r i n s e n s i t i v e to sodium (245-247). The high a f f i n i t y c h o l i n e uptake by guinea p i g neocortex syaptosomes was a l s o shown to be sodium-dependent (248). In PC 12, a c l o n a l line of r a t pheochromocytoma c e l l s t h a t r a p i d l y convert c h o l i n e i n t o a c e t y l -c h o l i n e , uptake of c h o l i n e had an apparent K of 12 uM and was sodium and m energy independent (249). Other b r a i n c e l l s which have a h i g h a f f i n i t y c h o l i n e uptake besides c h o l i n e r g i c neurons i n c l u d e g l i a l c e l l s (250), neuroblastoma c e l l s (251) and embryonic cortex c e l l s ( 2 5 2 ) . R a b b i t ( 2 5 3 ) , r a t ( 2 5 4 ) , c h i c k e n (255) and t u r t l e (256) r e t i n a l p h o t o r e c e p t o r c e l l s a c c u m u l a t e c h o l i n e from the e x t r a c e l l u l a r environment by h i g h a f f i n i t y c h o l i n e uptake f o r subsequent metabolism to PC r a t h e r than a c e t y l c h o l i n e . S i m i l a r l y , the high a f f i n i t y uptake of c h o l i n e by d i s s o c i a t e d r a t embryo b r a i n c e l l s was a s s o c i a t e d with a higher p r o p o r t i o n of p h o s p h o c h o l i n e f o r m a t i o n ( 2 5 2 ) . However, at higher c o n c e n t r a t i o n s of c h o l i n e i n the medium, the added c h o l i n e was i n c r e a s i n g l y recovered as a c e t y l c h o l i n e and f r e e c h o l i n e , w i t h a s m a l l e r p r o p o r t i o n of phosphocholine. The reported a p p a r e n t K 's of c h o l i n e uptake by s e v e r a l t i s s u e s and 45 c e l l t y p e s a r e l i s t e d i n Table 9- A samp l i n g o f v a r i o u s i n h i b i t o r s o f c h o l i n e t r a n s p o r t i s a l s o presented. Normally, the r a t e o f PC s y n t h e s i s i s not i n f l u e n c e d by the r a t e of c h o l i n e t r a n s p o r t . A s m a l l i n c r e a s e ( 4 0 % ) i n the r a t e of s y n t h e s i s of PC i n i s o l a t e d r a t h e p a t o c y t e s has been r e p o r t e d when t h e medium c h o l i n e c o n c e n t r a t i o n was v a r i e d from 5 - 4 0 yH ( 2 4 4 ) . Some e v i d e n c e f o r c h o l i n e t r a n s p o r t s e r v i n g as the r a t e - l i m i t i n g s t e p i n PC f o r m a t i o n has been obtained from N o v i k o f f hepatoma c e l l s ( 2 4 3 ) At con c e n t r a t i o n s below 2 0 ^ J M , c h o l i n e i n c o r p o r a t i o n i n t o PC was l i m i t e d by the r a t e o f f o r m a t i o n o f phosphocholine which i n t u r n was l i m i t e d by the r a t e o f c h o l i n e entry i n t o the c e l l s . However, at c o n c e n t r a t i o n s exceeding 2 0 ^ J M, the u l t i m a t e r a t e of c h o l i n e i n c o r p o r a t i o n i n t o PC was i n d e p e n d e n t o f the medium c h o l i n e c o n c e n t r a t i o n and the i n t r a c e l l u l a r l e v e l of phosphocholine ( 2 4 3 ) . 46 Table 9. k i n e t i c s and I n h i b i t o r s of Choline Uptake. Ref. Nonsaturable Component Apparent Km of Saturable Component ( p M ) - i s o l a t e d perfused l i v e r (210) yes 170 - Novikoff hepatoma c e l l s ( 2 1 3 ) yes 1-7 - Novikoff hepatoma c e l l s (257) 20 - human erythrocytes (215) 600 _ r a t c e r ebral cortex (216.217) 1.2-1.8; synaptosomes 50-90 - rat embryo brain c e l l s (252) 16; 950 _ i s o l a t e d perfused hamster heart (216) 100 - E h r l i c h - L e t t r e Ascites (258) yes 59; 220 - r a t blood brain b a r r i e r (259) 110 - BHK c e l l s (260) 17 - Entodinium caudatum ( 12) 20 INHIBITORS OF CHOLINE UPTAKE Simple D i f f u s i o n Carrier-mediated 2,1 din i t r o p h e n o l (258) 2,1 dinitrophenol (258) p-chloromercuribenzoate (258) deoxyglucose (258) chlorocholine (258) benzoylcholine (258) phenethylalcohol (257) 2-(1-anisidino)-1,6-bis(2-(diethylmethylammoni-um)ethylamino)-1,3.5-triazine d i i o d i d e (261) 1-(1-(1-sulfanilyl)phenyl)urea (262) hemicholinium-3 (12,218) theophylline (218) 47 I n t r a c e l l u l a r Supply of C h o l i n e f o r P h o s p h a t i d y l c h o l i n e B i o s y n t h e s i s (1.4.1.3).. Estimates of the pool s i z e of c h o l i n e i n r a t l i v e r and other c e l l s are shown i n Table 10. Not a l l o f t h e i n t r a c e l l u l a r c h o l i n e , however, i s committed f o r PC s y n t h e s i s . In r a t l i v e r , i t has been c a l c u l a t e d t h a t only 15% of the c h o l i n e i s a v a i l a b l e to c h o l i n e kinase (165). Likewise i n BHK-21 c e l l s , t h e r e i s ample e v i d e n c e f o r a s e p a r a t e p o o l o f c h o l i n e which i s a c t i v e i n PC s y n t h e s i s (260). 3 P u l s e l a b e l i n g s t u d i e s w i t h BHK-21 c e l l s s uggest t h a t [Me- H ] -c h o l i n e does not e q u i l i b r a t e w i t h t h e l a r g e i n t r a c e l l u l a r c h o l i n e p o o l , but i s r a p i d l y phosphorylated i n s t e a d (260). This might be f e a s i b l e i f c h o l i n e kinase phosphorylates c h o l i n e j u s t a f t e r t r a n s p o r t across the plasma membrane as has been p r o p o s e d f o r E n t o d i n i u m c a u d a t u m ( 1 2 ) . B u t, c h o l i n e k i n a s e i n e u k a r y o t e s i s a s o l u b l e enzyme. Another p o s s i b l e e x p l a n a t i o n i s th a t the bulk of the i n t r a c e l l u l a r c h o l i n e i s i n a c c e s s i b l e to c h o l i n e k i n a s e , p o s s i b l y i n s i d e the mitochondria. A l t h ough c h o l i n e i s l a r g e l y o b t a i n e d from the d i e t , the l i v e r can synthesize c h o l i n e from the m e t h y l a t i o n o f PE. Perhaps base-exchange of ethanolamine f o r the c h o l i n e head group o f PC p e r m i t s the release of free c h o l i n e and the r e g e n e r a t i o n of PE f o r subsequent m e t h y l a t i o n . Wise and Elwyn (278) have estimated that t h e m e t h y l a t i o n pathway can provide 13 pmol of c h o l i n e per day per g o f l i v e r , or e q u i v a l e n t t o the normal d i e t a r y i n t a k e of c h o l i n e f o r the r a t . C h o l i n e O x i d a t i o n ( 1 . 4 . 1 . 4 ) . In r a t l i v e r m i t o c h o n d r i a , c h o l i n e can be o x i d i z e d to betaine i n two s t e p s . The f i r s t r e a c t i o n i s c a t a l y z e d by c h o l i n e dehydrogenase (EC 1.1.99.1) which converts c h o l i n e to betaine aldehyde. The second r e a c t i o n i s Table 10. M e t a b o l i t e Pool S i z e s . ( m M ) Metabolite Rat Liver DHK C e l l s He La C e l l s (261) F e t a l 3 Rabbit Lung (265) Adult Rabbit Lung (216) Hamster Heart (216) Rat I n t e s t i n a l Mucosa (211) Chicken L i v e r (266) Rat S k e l e t a l Muscle (267) Rat Brain (268) Choline 0.23 (263) 0.15 (25) 0.13 0.21 0.36 0.28 ATP 1.95 (263) 1.8 (25) 2.3 Mg 0.7 (263) ADP 1.1 (263) Phosphocholine 1.7 (268) 1.3 (21"0 0.031 (25) 0.07 (269) 1.8 0.6 0.05 0.21 0.5 0.16 0.39 CTP Pyrophosphate CDP-choline Diglyceride Phosphatidyl-choline Phosphatidyl-ethanolamine Ado-Met MMPE DMPE 1.2 (269) 1.1 (263) 0.08 (263) 0.06 (269) 0.06 (268) 0.006 (263) 0.1 (7) 0.05 (263) 0.05 (270) 0.03 (27D 0.009 (165) 1.9 (272) 1.9 (273) 0.2-0.1 (27t) 16 (273) 15 (275) 11 (272) 9 (275) 7 (273) 6.3 (272) 0.3-0.6 (276) 0.07 (277) 0.01 (275) 0.015 (275) 0.12 (25) 0.16 (269) 0.006 (25) 0.05 (25) 3.0 (25) 0.1 0.03 0.23 5.1 0.021 1.3 0.016 0.09 17 0.05 0.031 0.35 OO 0.007 assumed 1 g wet t i s s u e = 1 ml 26 days gestation 49 c a t a l y z e d by betaine aldehyde dehydrogenase (EC 1.2.1.8) which o x i d i z e s the aldehyde t o b e t a i n e . Choline dehydrogenase has r e c e n t l y been p u r i f i e d from r a t l i v e r (279) and shown to be a monomer of 70,000 daltons (280). Haubrich and Gerber (281) have measured appreciable c h o l i n e dehydrogenase a c t i v i t y i n the kidneys and l i v e r s o f a wide range of mammals. The s p e c i f i c enzyme a c t i v i t y o f c h o l i n e dehydrogenase was 1 0 0 - f o l d lower i n r a t b r a i n and l u n g , 10,000-fold lower i n blood, spleen and h e a r t , and not detectable i n muscle or f a t . In the l i v e r , c h o l i n e k i n a s e and c h o l i n e dehydrogenase compete f o r c h o l i n e (164,165,240,282,283). The apparent K of the dehydrogenase f o r m c h o l i n e i s 0.7 mM (284), while the apparent K of the kinase f o r c h o l i n e i s m 0.03 mM (Table 1). Therefore, c h o l i n e k i n a s e has a greater a f f i n i t y f o r cho-l i n e than the dehydrogenase. In i s o l a t e d r a t hepatocytes (161,244), c h o l i n e i s p r e f e r e n t i a l l y i n c o r p o r a t e d i n t o p h o s p h o c h o l i n e at the expense of b e t a i n e s y n t h e s i s when the s u p p l y of c h o l i n e i s r e s t r i c t e d . However, at c o n c e n t r a t i o n s of c h o l i n e i n the medium i n excess of 5 JJM, c h o l i n e oxidation' i s favoured. With 50 c h o l i n e i n the medium, 75% of the c h o l i n e taken up by hepatocytes i s transformed i n t o b e t a i n e . Hence, the l i v e r has a greater c a p a c i t y f o r o x i d a t i o n of c h o l i n e than phosphorylation, c The betaine produced by o x i d a t i o n o f c h o l i n e i s r a p i d l y r e l e a s e d back i n t o the c i r c u l a t i o n by t h e l i v e r . The b e t a i n e must be s u b s e q u e n t l y metabolized s i n c e b e t a i n e does not n o r m a l l y appear i n u r i n e . Betaine can donate a methyl group to homocysteine f o r the s y n t h e s i s of methionine i n a r e a c t i o n c a t a l y z e d by betaine-homocysteine m e t h y l t r a n s f e r a s e . This methionine can, i n t u r n , be converted t o Ado-Met and used to methylate PE f o r the s y n t h e s i s of PC. Recently, the hepatic a c t i v i t y of betaine-homocysteine m e t h y l t r a n s f e r a s e was shown to be increased when r a t s were maintained on 50 e i t h e r methionine-free or excessive methionine (1.0%) r a t i o n s (284b). C h o l i n e K i n a s e ( 1 . 4 . 1 . 5 ) . In r a t l i v e r , c h o l i n e k i n a s e c a t a l y z e s the f i r s t committed step of PC s y n t h e s i s s i n c e phosphocholine and CDP-choline are o b l i g a t e precursors f o r PC. Hence t h i s r e a c t i o n would be an a t t r a c t i v e s i t e f o r r e g u l a t i o n of the r a t e of PC f o r m a t i o n . O f t e n i n m e t a b o l i c pathways r a t e - l i m i t i n g steps are c a t a l y z e d by r e g u l a t o r y enzymes. I n f a n t e (263) has advanced t h e o r e t i c a l arguments th a t while c h o l i n e k i n a s e and c y t i d y l y l t r a n s f e r a s e r e a c t i o n s are b o t h p o t e n t i a l l y r a t e - l i m i t i n g , t h e k i n a s e s t e p i s 4 9 - t i m e s more r a t e - l i m i t i n g than the c y t i d y l y l t r a n s f e r a s e c a t a l y z e d s t e p . However, Inf a n t e ' s c o n c l u s i o n s were based on the premise t h a t the e n t i r e pool of c h o l i n e was a v a i l a b l e f o r PC s y n t h e s i s . S t u d i e s w i t h r a t l i v e r (165) and BHK-21 c e l l s (260) challenge t h i s assumption. Perhaps i n E h r l i c h a s c i t e s , the c h o l i n e k i n a s e r e a c t i o n l i m i t s the r a t e of PC s y n t h e s i s ( 2 5 6 ) . P u l s e - c h a s e s t u d i e s w i t h t h e s e tumour c e l l s suggest t h a t once r a d i o - l a b e l l e d c h o l i n e i s incorporated i n t o the c e l l s , i t accumulates as c h o l i n e r a t h e r than p h o s p h o c h o l i n e . At low c h o l i n e con-c e n t r a t i o n s i n the medium, c h o l i n e k i n a s e a c t i v i t y a l s o can be r a t e - l i m i t i n g f o r c h o l i n e uptake and i n c o r p o r a t i o n i n t o PC i n E n t o d i n i u m caudatum (12). The s u p p l y o f p h o s p h o c h o l i n e f r o m t h e c h o l i n e k i n a s e r e a c t i o n r e s t r i c t s the r a t e of PC p r o d u c t i o n i n r o o s t e r l i v e r (14). When r o o s t e r s were t r e a t e d w i t h d i e t h y l s t i b e s t r o l (DES) f o r one day, c h o l i n e k i n a s e a c t i v i t y was e l e v a t e d 1 . 7 - f o l d (285) and PC s y n t h e s i s was s t i m u l a t e d 2-f o l d (266). The elevated c h o l i n e k i n a s e a c t i v i t y by DES treatment was shown to be due to an i n c r e a s e i n the amount of enzyme p r o t e i n (286). 5 1 Although the c h o l i n e k i n a s e r e a c t i o n does not u s u a l l y appear to be r a t e - l i m i t i n g i n r a t l i v e r , under c e r t a i n circumstances i t may. Infante and K i n s e l l a (287) have r e p o r t e d t h a t the r a t e of h e p a t i c PC s y n t h e s i s was s t i m u l a t e d 3.8-fold i n r a t s f e d a d i e t d e p r i v e d of e s s e n t i a l f a t t y a c i d s , and t h i s c o r r e l a t e d w i t h a 3 . 5 - f o l d i n c r e a s e i n c h o l i n e kinase a c t i v i t y . A f t e r a 48 h f a s t , the content of PC i n r a t l i v e r was reduced 39% (272), and t h i s was accompanied by a 50% r e d u c t i o n i n choline kinase a c t i v i t y (273). The a d m i n i s t r a t i o n of 3 - m e t h y l - c h o l a n t h r e n e , 3, 4-benzo-(a) pyrene and b e t a - n a p h t h o f l a v o n e t o r a t s produced a 60% r i s e i n the h e p a t i c c h o l i n e kinase a c t i v i t y (288,288b). The l e v e l of phosphocholine i n l i v e r c y t o s o l was doubled by 3-methyl-cholanthrene a d m i n i s t r a t i o n (288b) and f u r t h e r i n d i c a t e d a s t i m u l a t i o n of c h o l i n e kinase a c t i v i t y . The i n c l u s i o n of e i t h e r cyclohex-amide or actinomycin D c o m p l e t e l y d e p r e s s e d the e l e v a t i o n of CK a c t i v i t y induced by these p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s . Therefore, the elevated a c t i v i t y caused by these c a r c i n o g e n i c c h e m i c a l s could be explained by changes i n the enzyme l e v e l . I s h i d a t e e t a l . (288c) f u r t h e r showed that 3-methylcholanthrene treatmentof the r a t d i d not a l t e r the molecular weight of c h o l i n e kinase (120,000), the surface i o n i c charge, nor the a f f i n i t y f o r ATP, but the apparent K v a l u e f o r c h o l i n e was increased from 0.11 to 0.2 m mM. Despite the i n d u c t i o n of c h o l i n e kinase by 3-methylcholanthrene, hepatic PC s y n t h e s i s i s apparently i n h i b i t e d i n t r e a t e d r a t s (288b). This i n h i b i t i o n may be due to the reduced cholinephosphotransferase a c t i v i t y noted i n micro-somes from the p o l y c y c l i c aromatic hydrocarbon-administrated r a t s (2886). A d m i n i s t r a t i o n of p o l y c h l o r i n a t e d b i p h e n y l s to r a t s has a l s o been shown t o increase h e p a t i c c h o l i n e kinase a c t i v i t y 1 . 3 - f o l d , but again PC s y n t h e s i s was apparently i n h i b i t e d . (289). 0 Fukuya and Yamashita (290) have demonstrated tha^fe 2 mM spermine can 52 S t i m u l a t e r a t l i v e r c h o l i n e k i n a s e 9 - f o l d tn v i t r o . Spermine, and the other polyamine, spermidine increased the a f f i n i t y of the enzyme f o r Mg.ATP. Since polyamines accumulate d u r i n g r a p i d t i s s u e growth, perhaps polyamines can a l s o stimulate PC s y n t h e s i s . Polyamines have been found to i n c r e a s e 4.5-14 f o l d the i n c o r p o r a t i o n o f [ C ] c h o l i n e i n t o PC i n a polyamine auxotroph of Saccharomyces c e r e v i s i a e ( 2 9 1 ) . However, i n t h i s c a s e , p o l y a m i n e s induced the synthesis of the c h o l i n e t r a n s p o r t system without s i g n i f i c a n t l y a l t e r i n g the a c t i v i t i e s of any of the PC b i o s y n t h e t i c enzymes. The h e p a t i c c o n c e n t r a t i o n o f ATP (1.95 mM (263)) i s l o w e r than the apparent K (3-7 mM) of the c y t o s o l i c c h o l i n e kinase ( 8 ) . Therefore, the m energy charge o f the c e l l might p o t e n t i a l l y r e g u l a t e PC s y n t h e s i s under c o n d i t i o n s where the ATP pool becomes depleted. Magnesium might a l s o c o n t r o l c h o l i n e kinase a c t i v i t y s i n c e i t forms the a c t i v e s u b s t r a t e w i t h ATP and a l s o i n c r e a s e s the enzyme's a f f i n i t y f o r Mg.ATP (13). Phosphocholine Phosphatase (1.4.1.6). Phosphocholine phosphatase might p o s s i b l y degrade phosphocholine before i t could be metabolized to C D P - c h o l i n e . The a c t i v i t y of t h i s enzyme could account f o r the reduced p o o l s i z e s o f p h o s p h o c h o l i n e i n e x t r a - h e p a t i c t i s s u e s . The l i v e r , however, does not seem to hydrolyze phosphocholine back to c h o l i n e . Phosphocholine phosphatase a c t i v i t y has been detected i n HeLa c e l l s (292) and hamster heart (216). In HeLa c e l l s , p h o s p h o c h o l i n e p h o s p h a t a s e was found t o resemble a l k a l i n e phosphatase (EC 3.1 .3. 1 ) ( 2 9 2 ) . However, i t was deduced that the bulk o f the phosphatase a c t i v i t y r e s i d e d on the o u t s i d e o f the HeLa plasma c e l l membrane, and t h a t the i n t r a c e l l u l a r p o o l o f p h o s p h o c h o l i n e was r e l a t i v e l y i n a c c e s s i b l e to the phosphatase. C y t i d y l y l t r a n s f e r a s e C a t a l y z e s the R a t e - L i m i t i n g Step of de novo PC 53 B i o s y n t h e s i s ( 1 . 4 . 1 . 7 ) . In most t i s s u e s , the r a t e - l i m i t i n g s t e p o f PC s y n t h e s i s seems t o be c a t a l y z e d by c y t i d y l y l t r a n s f e r a s e . T h i s i s p r o b a b l y most c l e a r f o r r a t l i v e r . In t h i s o r g a n , t h e p o o l s i z e o f p h o s p h o c h o l i n e i s 6-times t h a t of c h o l i n e and 4 3 - t i m e s l a r g e r t h a n t h e C D P - c h o l i n e p o o l ( T a b l e 10). S i m i l a r l y , i n HeLa c e l l s t h e p h o s p h o c h o l i n e p o o l s i z e i s 6 0 - f o l d l a r g e r than the CDP - c h o l i n e p o o l ( 2 6 4 ) . P u l s e - c h a s e s t u d i e s i n i s o l a t e d r a t hepatocytes (244), r a t lung a l v e o l a r type I I c e l l s (244b), HeLa c e l l s (264), BHK-21 c e l l s ( 2 5 ) , i s o l a t e d hamster heart (216) and r a t i n t e s t i n a l v i l l u s c e l l s (293) a l s o suggest 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 - c a t a l y z e d r e a c t i o n i s r a t e - l i m i t i n g . F i n a l l y , there i s a high degree o f p a r a l l e l i s m between t he a c t i v i t y o f c y t i d y l y l t r a n s f e r a s e and the r a t e of r a d i o - l a b e l l e d c h o l i n e i n c o r p o r a t i o n i n t o PC i n a v a r i e t y of model systems (Table 11). Of t h e t h r e e d_£ novo enzymes i n E n t o d i n i u m c a u d a t u m , o n l y the c y t i d y l y l t r a n s f e r a s e i s f o u n d i n t h e 144 , 000 X g X 1 h s u p e r n a t a n t f r a c t i o n (12). I f v a r i o u s amounts o f s u p e r n a t a n t are added to i n c u b a t i o n mixtures that contain the membrane f r a c t i o n and s u b s t r a t e s , a roughly l i n e a r 14 s t i m u l a t i o n o f [ C J c h o l i n e i n c o r p o r a t i o n i n t o PC i s o b s e r v e d . Such a r e s u l t i s expected provided t h a t c y t i d y l y l t r a n s f e r a s e c a t a l y z e s the r a t e -l i m i t i n g step. Table 11. Changes 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 In Various Model Systems. Model System Ref. Effect on PC Synthesis Enzyme A c t i v i t y Relative to Control Cytosol Cytosol+PL Microsomes Rat f e t a l lung - premature b i r t h ( 15) Stimulated 3 15* + - 1.9-fold• Rat f e t a l l i v e r - development (+day1/-day1) (29D Stimulated 3 2-fold • - 4.3-foldt Rabbit f e t a l lung - 17B e s t r a d i o l treatment (295) Stimulated 1.6-foldt - -Rooster l i v e r - d i e t h y l s t i l b e s t r o l treatment (285) Inhibited 50** 10*+ n.s. -Rabbit f e t a l lung - 17B e s t r a d i o l treatment (265) Stimulated 1.7-foldt no change CHO c e l l s - temp, s e n s i t i v e mutant (203) I n h i b i t e d 3 (homogenate - 97?+ ) Embryonic chick muscle c e l l s - PLC treatment ( 23) Stimulated Rat l i v e r - 48 h starvation (273) I n h i b i t e d 3 18%+ n.s. - -Rat l i v e r - 3-deazaadeno3ine treatment (268) Stimulated 1.6-foldt 23** 1.9-foldt Mouse l i v e r - quaking ( 20) I n h i b i t e d ? 3 1.4-foldt - 1.1-foldt HeLa c e l l s - p o l i o virus i n f e c t i o n (264) Stimulated 38*+ n.s. - 47* + Rat l i v e r - hypercholesterolemia ( 41) Stimulated 3.1-foldt 12* + 1.9-foldt BHK-21 c e l l s - Semliki f o r e s t virus i n f e c t i o n (260) Inhibited? no change 1.2-foldt 47*+ n.s. HeLa c e l l s - TPA treatment (296) Stimulated no change - -Rat l i v e r - choline deficiency (297) I n h i b i t e d 3 60** - -Rat l i v e r - 3-methylcholanthrene treatment ( 52) I n h i b i t e d 3 13* + - 8* + Rat l i v e r - polychlorinated biphenyl treatment ( 52) I n h i b i t e d 3 11* + - 42* + Rat l i v e r - phenobarbital treatment ( 52) I n h i b i t e d ? 3 8* + - 41* + aAn increase i n r a d i o - l a b e l e d p r e c u r s o r incorporation i n t o PC was noted, but the actual rates of synthesis were not determined. 55 S u b c e l l u l a r D i s t r i b u t i o n o f C y t i d y l y l t r a n s f e r a s e (1.4.1.8) Previous studies have t r i e d to c o r r e l a t e the 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 a c t i v i t y w i t h a l t e r a t i o n s i n the r a t e o f PC s y n t h e s i s . T h i s was probably because s a l i n e l i v e r c y t o s o l contains over 75% of the c y t i d y l y l -t r a n s f e r a s e p r o t e i n , and o n l y t he s o l u b l e form o f the enzyme i s s e n s i t i v e t o l i p i d modulators. However, from the r e s u l t s s e c t i o n o f t h i s t h e s i s , the concept w i l l emerge that the microsomal c y t i d y l y l t r a n s f e r a s e a c t u a l l y r e p r e -sents the a c t i v e form of t h i s enzyme. The c y t o s o l probably serves as a r e s e r v o i r of i n a c t i v e c y t i d y l y l t r a n s f e r a s e . T h e r e f o r e , a c t i v a 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 may be a s s o c i a t e d w i t h a t r a n s l o c a t i o n o f the enzyme from the c y t o p l a s m to the E.R. The s o l u b l e c y t i d y l y l t r a n s f e r a s e has u s u a l l y been measured i n the 100,000 X g X 1 h s u p e r n a t a n t o f c e l l homogenates. U n f o r t u n a t e l y , these c y t o s o l preparations s t i l l c o n t a i n c o n t a m i n a t i n g membrane fragments which c a t a l y z e microsomal a c t i v i t y . The s i t u a t i o n i s f u r t h e r complicated by the f a c t that more than 10% o f the microsomes i s l o s t i n the m i t o c h o n d r i a l f r a c t i o n (268). Since the 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 a c t i v i t y i s low when measured i n the absence o f exogenous phospholipid while the microsomal enzyme i s f u l l y a c t i v a t e d , t r a n s l o c a t i o n events may have been l e s s obvious i n the past. 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 can be a c t i v a t e d 7 - f o l d by exogenous p h o s p h o l i p i d . Furthermore, g e l - f i l t r a t i o n s t u d i e s , w i t h 100,000 X g X 1 h supernatants have i n d i c a t e d t h a t r o u g h l y 5% o f the c y t i d y l y l t r a n s f e r a s e p r o t e i n i s m e m b r a n e - b o u n d ( 4 1 ) . What w o u l d h a p p e n t o t h e 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 f a t r a n s l o c a t i o n of the enzyme occurred such t h a t the amount of enzyme p r o t e i n i n the E.R. f r a c t i o n d oubled? In the absence o f exogenous p h o s p h o l i p i d i n t h e enzyme a s s a y , t he c y t o s o l i c 56 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 w o u l d a p p a r e n t l y i n c r e a s e by 1 . 1 - f o l d , while the microsomal enzyme a c t i v i t y would i n c r e a s e only 1.7-fold. Again, t h i s problem stems from the s l i g h t c o n t a m i n a t i o n o f the 100,000 X g X 1 h s u p e r n a t a n t w i t h microsomes, and t h e l o s s o f some microsomes w i t h the m i t o c h o n d r i a l p e l l e t . As there i s t y p i c a l l y 6-times more 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 compared t o microsomes, t r a n s l o c a t i o n c o u l d p o t e n t i a l l y s t i m u l a t e the enzyme a c t i v i t y i n the c e l l an a d d i t i o n a l 6 - f o l d . T r a n s l o c a 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 to the E.R. has been i m p l i c a t e d i n the s t i m u l a t i o n of de novo PC s y n t h e s i s o b s e r v e d i n t h e l i v e r s o f r a t s a d m i n i s t e r e d 3-deaza-adenosine (268), i n f e t a l l u n g w i t h premature d e l i v e r y (15), and i n c h i c k embryonic muscle c e l l s t r e a t e d w i t h p h o s p h o l i p a s e C ( 2 3 ) . In a d d i t i o n , i t i s p o s s i b l e ' t h a t a change i n the i n t r a c e l l u l a r d i s t r i b u 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 can e x p l a i n changes i n the r a t e s of PC s y n t h e s i s i n h ypercholesterolemia r a t s (41) and S e m l i k i - f o r e s t v i r u s i n f e c t e d BHK-21 c e l l s (260). Supply of Substrates For The C y t i d y l y l t r a n s f e r a s e Reaction (1.4.1.9). For r a t l i v e r , the pool s i z e o f phosphocholine (Table 10) i s t w i c e the apparent K of c y t i d y l y l t r a n s f e r a s e f o r t h i s s u b s t r a t e ( T a b l e 3 ) . Hence m the i n t r a c e l l u l a r c o n c e n t r a t i o n of phosphocholine probably does not r e s t r i c t the r a t e of PC s y n t h e s i s i n r a t l i v e r . However, i n r o o s t e r l i v e r , the pool s i z e of phosphocholine i s below the apparent K and may l i m i t the m r a t e of PC f o r m a t i o n ( 2 8 5 ) . I t i s p o s s i b l e t h a t p h o s p h o c h o l i n e may a l s o i n f l u e n c e the r a t e of PC s y n t h e s i s i n BHK-21 c e l l s , since the pool s i z e was a l m o s t 1 0 - f o l d lower t h a t the a p p a r e n t K of the c y t i d y l y l t r a n s f e r a s e m f o r t h i s phosphocholine (Table 10). R e g u l a t i o n of PC s y n t h e s i s by t h e s u p p l y of CTP i s a f a s c i n a t i n g 57 prospect since CTP i s r e q u i r e d i n the a n a b o l i s m of a l l the p h o s p h o l i p i d s . Estimates of the apparent K (0.2-5 mM) of the c y t i d y l y l t r a n s f e r a s e f o r m CTP from r a t l i v e r vary over 2 5 - f o l d (Table 3 ) . For the c y t o s o l i c c y t i d y l y l -t r a n s f e r a s e , the apparent K i s 2 0 - 7 5 - f o l d h i g h e r than the estimated pool m s i z e of CTP i n r a t l i v e r (Table 10). However, f o r microsomal c y t i d y l y l t r a n s -f e r a s e , the apparent K i s o n l y about 9 - f o l d h i g h e r than the i n t r a c e l l u l a r m l e v e l of CTP. A t r a n s l o c a 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 to the E.R. could t h e r e f o r e p o t e n t i a l l y s t i m u l a t e the c y t i d y l y l t r a n s f e r a s e - c a t a l y z e d r e a c t i o n 2-8-fold i f the amount of CTP i s r a t e - l i m i t i n g . Studies with p o l i o - v i r u s i n f e c t e d HeLa c e l l s have c o r r e l a t e d a 2 - f o l d s t i m u l a 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 step and PC b i o s y n t h e s i s w i t h a 2 - 3 -f o l d i n c r e a s e i n the c o n c e n t r a t i o n of CTP i n the c y t o p l a s m i c compartment (298). In S e m l i k i - f o r e s t v i r u s i n f e c t e d BHK-21 c e l l s , the CTP p o o l was reduced an average of 77? ( 2 5 , 2 9 9 ) , and both the c y t i d y l y l t r a n s f e r a s e c a t a l y z e d r e a c t i o n and PC b i o s y n t h e s i s a r e probably i n h i b i t e d . In 3-deaza-adenosine-treated r a t s , enhancement of PC formation was accompanied by a 26% i n c r e a s e i n the hepatic c o n c e n t r a t i o n of CTP (268). F i n a l l y , i n myopathic hamsters, the c a r d i a c CTP l e v e l was 34% lower than found i n c o n t r o l s (299b) . The r a t e of PC s y n t h e s i s i n these hearts was comparable to c o n t r o l s because of a compensating increase i n the microsomal 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 . ,58 Supply o f D i g l y c e r i d e (1.1.1.10). D i g l y c e r i d e i s produced from p h o s p h a t i d a t e i n a r e a c t i o n c a t a l y z e d by phosphatidate phosphohydrolase (EC 3.1.3.1). D i g l y c e r i d e i s a s u b s t r a t e f o r cholinephosphotransferase, ethanolaminephosphotransferase, d i g l y c e r i d e a c y l -t r a n s f e r a s e and d i g l y c e r i d e l i p a s e . A l l o f t h e s e r e a c t i o n s compete f o r d i g l y c e r i d e , but during s t a r v a t i o n d i g l y c e r i d e i s p r e f e r e n t i a l l y channelled i n t o r a t hepatocyte p h o s p h o l i p i d s a t the expense of t r i g l y c e r i d e s y n t h e s i s ( 2 7 2 ) . S i m i l a r l y , exposure o f r a t h e p a t o c y t e s t o g l u c a g o n r e s u l t e d i n decreased d i g l y c e r i d e i n c o r p o r a t i o n i n t o t r i g l y c e r i d e , with l i t t l e e f f e c t on p h o s p h o l i p i d f o r m a t i o n ( 3 0 0 ) . When PE s y n t h e s i s was s t i m u l a t e d i n r a t h e p a t o c y t e s supplemented w i t h e t h a n o l a m i n e , t r i g l y c e r i d e s y n t h e s i s was reduced while PC synth e s i s was s c a r c e l y a l t e r e d (301). Groener and van Golde (273) have discounted the l e v e l o f d i g l y c e r i d e as a rate-determining f a c t o r i n the s y n t h e s i s of r a t l i v e r PC. This c o n c l u s i o n was based on the r e l a t i v e i n s e n s i t i v i t y o f the PC pool s i z e to a l t e r a t i o n s a f t e r a 18 h f a s t and 21 h r e f e e d i n g on a h i g h s u c r o s e , f a t f r e e d i e t , d e s p i t e a 10-fold increase i n the l e v e l of d i g l y c e r i d e . S t i l l , t here i s some evidence that the a v a i l a b i l i t y of d i g l y c e r i d e can regula t e PC s y n t h e s i s . F a t t y a c i d s s t i m u l a t e <d£ H£.vo PC s y n t h e s i s i n c u l t u r e d r a t hepatocytes (302) and the i n t r a c e l l u l a r l e v e l o f d i g l y c e r i d e i s more than doubled (161). Furthermore, i n h y p e r c h o l e s t e r o l e m i a r a t s , the hepa t i c l e v e l of d i g l y c e r i d e was 2 . 8 - f o l d h i g h e r than c o n t r o l r a t s , and PC sy n t h e s i s i n c r e a s e d 2 - 3 - f o l d ( 1 1 ) . S i n c e d i g l y c e r i d e has been shown to a c c e l e r a t e the a g g r e g a t i o n o f c y t i d y l y l t r a n s f e r a s e i n v i t r o ( 3 0 7 ) , perhaps i n these model systems d i g l y c e r i d e promoted the t r a n s l o 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 t o the E.R. where i t was a c t i v a t e d . Phospholipase £ treatment of mouse embryonic muscle c^J.ls produced an apparent t r a n s l o c a t i o n 59 of the c y t i d y l y l t r a n s f e r a s e to the membrane f r a c t i o n and a s t i m u l a t i o n of PC s y n t h e s i s (23). Phospholipase C d i g e s t i o n of membranous ph o s p h o l i p i d s w i l l l i b e r a t e d i g l y c e r i d e . Such a m e c h a n i s m i n which one s u b s t r a t e o f the cholinephosphotransferase r e a c t i o n i n c r e a s e s the a v a i l a b i l i t y of the second s u b s t r a t e i s rather novel. C h o l i n e p h o s p h o t r a n s f e r a s e (1.1.1.11). Since the c h o l i n e p h o s p h o t r a n s f e r a s e r e a c t i o n operates near e q u i l i b r i u m i n v i v o (51,84,263 . 3 0 3 ) , t h i s s t e p s h o u l d be s e n s i t i v e t o m e t a b o l i t e c o n c e n t r a t i o n s . A l t h o u g h t h e PC p o o l i s 4 0 - 6 0 - t i m e s l a r g e r t han the d i g l y c e r i d e pool i n r a t l i v e r and BHK-21 c e l l s (Table 10), the a v a i l a b i l i t y of CMP and CDP-choline seem to i n f l u e n c e the net d i r e c t i o n of the r e a c t i o n . Despite the overwhelming e v i d e n c e t h a t the c h o l i n e p h o s p h o t r a n s f e r a s e c a t a l y z e d r e a c t i o n i s n o t r a t e - l i m i t i n g , t h i s enzyme may s t i l l be r e g u l a t o r y . The Vn v i t r o a c t i v i t y o f t h e c h o l i n e p h o s p h o t r a n s f e r a s e c o r r e l a t e s with a l t e r a t i o n s i n the s y n t h e s i s of PC i n many,model systems (Table 12). However, i n most systems such a c o r r e l a t i o n i s not found. S r i b n e y £t a l . (62) have r e p o r t e d t h a t c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y i n r a t l i v e r microsomes can be i n h i b i t e d more than 80% by the c o m b i n a t i o n of ATP and CoA, o r ATP and p a n t e t h e i n e . These compounds st i m u l a t e d sphingomyelin s y n t h e s i s a p p r o x i m a t e l y 2 0 - f o l d . The p h y s i o l o g i c a l importance of c h o l i n e p h o s p h o t r a n s f e r a s e i n h i b i t i o n by these compounds was obscure as was the mechanism of the i n h i b i t i o n . REGULATION OF PHOSPHATIDYLETHANOLAMINE N-METHYLATION Supply of P h o s p h a t i d y l e t h a n o l a m i n e For N-Methylation (1.4.2.1). Exogenous PE i n c u b a t e d w i t h r a t l i v e r microsomes i s not methylated, 60 Table 12.Changes i n C h o l i n e p h o s p h o t r a n s f e r a s e A c t i v i t y In V a r i o u s Model Systems A) POSITIVE CORRELATION WITH PHOSPHATIDYLCHOLINE SYNTHESIS. Model System Ref. E f f e c t on PC Enzyme A c t i v i t y R e l a t i v e t o C o n t r o l S y n t h e s i s - d i g y l c e r i d e + d i g l y c e r i d e R a b b i t cerebrum - development ( 55) S t i m u l a t e d - 1 - f o l d * R a b b i t c e r e b r a l c o r t e x - development ( 18) S t i m u l a t e d 2 - f o l d t -Rat l i v e r - methyl d e f i c i e n c y (277) I n h i b i t e d - 861* Rat i n t e s t i n e - p h o s p h a t i d y l c h o l i n e p e r f u s i o n (211) I n h i b i t e d -BHK c e l l s - S e m l i k i f o r e s t v i r u s i n f e c t i o n - (301) I n h i b i t e d - l o t * BHK-21 c e l l s - Semlike f o r e s t v i r u s i n f e c t i o n (260) I n h i b i t e d - 571* BHK-21 c e l l s - S i n d b i s v i r u s i n f e c t i o n (305) I n h i b i t e d - 131* Rat l i v e r - 3-rciethylcholanthrene treatment (289) I n h i b i t e d - 35%* Rat L i v e r - p o l y c h l o r i n a t e d b i p h e n y l t r e a t m e n t (289) I n h i b i t e d - 50** B) NEGATIVE OR NO CORRELATION WITH PHOSPHATIDYLCHOLINE SYNTHESIS Hat l i v e r - development (near b i r t h ) (29D S t i m u l a t e d no change no change 3T3-L1 p r e a d i p o c t e s - d i f f e r e n t i a t i o n (306) Unchanged 2 . 3 - f o l d • -Rat h e p a t o c y t e s - glucagon treatment (307) I n h i b i t e d no change -Chick embryonic m u s c l e c e l l s - PLC tre a t m e n t ( 23) S t i m u l a t e d no change -Rat l i v e r - 5* e t h a n o l f e e d i n g (308) Unchanged -HeLa c e l l s - p o l i o - v i r u s i n f e c t i o n (261) S t i m u l a t e d - 6S* n.s. Rat l i v e r - c a s t r a t e d male - e s t r a d i o l t r e a t m e n t (309) 7 - 21** Rat l i v e r - c h o l i n e d e f i c i e n c y (297) I n h i b i t e d - 15*+ n.s. Chick l i v e r - p r o p y l t h i o u r a c i l - i n d u c e d hypo-t h y r o i d i s m HeLa c e l l s - TPA treatment (310) (296) 7 S t i m u l a t e d 90*4 no change Rat l i v e r - h y p e r c h o l e s t e r o l e m i a ( 11) S t i m u l a t e d - 11** n.s. Rat l i v e r - deazaadenosine treatment (268) S t i m u l a t e d - no change BHK c e l l s - Dengue type 2 v i r u s i n f e c t i o n (311) 7 - 51** F e t a l r a t l u n g e x p l a n t s - a n i n o p h y l l i n e t r e a t -ment Rooster l i v e r - d i e t h y l s t i l b e s t r o l t r e a t m e n t (312) (285) S t i m u l a t e d S t i m u l a t e d no change 15** n.s. no change Rat l i v e r - t r i o d o t h y r o n i n e treatment ( 52) S t i m u l a t e d ? - 1.1-fold • Rat l i v e r - k e t o t i c a l l o x a n - i n d u c e d d i a b e t e s ( 52) S t i m u l a t e d ? - 2 . 3 - f o l d • F e t a l r a b b i t l u n g - 17f - e s t r a d i o l treatment (265) S t i m u l a t e d - no change Rat l i v e r - 18 h s t a r v a t i o n (273) I n h i b i t e d - no change Rat h e p a t o c y t e s - cAMP analogue treament (313) I n h i b i t e d - 1 . 6 - f o l d * Mouse b r a i n - qu a k i n g ( 20) I n h i b i t e d ? _ 1 . 2 - f o l d * 61 although MMPE and DMPE are a c c e p t a b l e s u b s t r a t e s (106,113). This could be i n t e r p r e t e d t o mean t h a t t h e s u p p l y o f PE does not l i m i t PE methylation even though the f i r s t methylation i s r a t e - l i m i t i n g . Akesson (314) has shown tha t the amount of membranous PE can be increased by supplementing c u l t u r e d r a t hepatocytes with ethanolamine . When the hepatocytes were incubated with 14 1 mM e t h a n o l a m i n e , the amount o f [ C j m e t h i o n i n e i n c o r p o r a t e d i n t o PC doubled. I t seems l i k e l y , t h e n , t h a t the r a t e of PE N-methylation can be i n f l u e n c e d by the synth e s i s of PE. Although PE synthesis may be a l t e r e d under a v a r i e t y of circumstances, i t i s no t e w o r t h y t h a t e x c e s s c h o l i n e w i l l reduce PE s y n t h e s i s ( 3 1 5 ) . S p e c i f i c a l l y , c h o l i n e i n h i b i t s the phosphorylation of ethanolamine by ethan-olamine kinase (315,316,316b). When r a t s are fed a c h o l i n e - d e f i c i e n t d i e t , t h e i n t r a c e l l u l a r p o o l s i z e s o f e t h a n o l a m i n e , phosphoethanolamine and CDP-ethanolamine are at l e a s t 3 - f o l d l a r g e r , and the amount of PE about 15% g r e a t e r than i n t h e l i v e r s o f r a t s f e d a c h o l i n e - s u p p l e m e n t e d d i e t ( 3 1 5 , 3 1 7 ) . The r a t e - l i m i t i n g s t e p o f PE s y n t h e s i s i n i s o l a t e d r a t hepatocytes i s catalyzed by phosphoethanolamine c y t i d y l y l t r a n s f e r a s e (161). P l a n t a v i d e t a l . (318) have shown t h a t 1 mM Ado-Met w i l l reduce t h e r a t e o f t h i s r e a c t i o n by 5 0 % i n r a t l i v e r c y t o s o l . A d o - H c y , S - a d e n o s y 1 e t h i o n i n e n o r A T P h a d a n y s i g n i f i c a n t a c t i o n on phosphoethanolamine c y t i d y l y l t r a n s f e r a s e , nor i n f l u e n c e d t h e i n h i b i t o r y a c t i o n of Ado-Met on t h i s c y t i d y l y l t r a n s f e r a s e . S i n c e the i n h i b i t i o n by Ado-Met was time-dependent, t h e s e a u t h o r s s p e c u l a t e d t h a t the enzyme was i n h i b i t e d by non-enzymatic m e t h y l a t i o n . 62 Supply o f Ado-Met (1.4.2.2). Transmethylation of PE may be l i m i t e d by the supply of methionine f o r the s y n t h e s i s of Ado-Met ( 2 7 6 ) . S u n d l e r and Akesson (161) have shown t h a t the a d d i t i o n of m e t h i o n i n e t o i s o l a t e d r a t h e p a t o c y t e s i n c r e a s e s the m e t h y l a t i o n of PE 2 - f o l d . Maximum s t i m u l a t i o n was achieved with 100 u^M m e t h i o n i n e i n the medium. S i n c e the c o n c e n t r a t i o n o f m e t h i o n i n e i n r a t plasma i s 55-90 yM (319.320), me t h y l a t i o n i s probably not u s u a l l y r e s t r i c t e d by the a v a i l a b i l i t y of methionine. Phosphatidylethanolamine M e t h y l t r a n s f e r a s e (1.4.2.3). The r a t e - l i m t i n g s t e p i n t h e f o r m a t i o n o f PC by s u c c e s s i v e N-methylation of PE i s the f i r s t step (106). As shown i n Table 8, b e t a - a d r e n e r g i c a g o n i s t s , m i t o g e n i c l e c t i n s , i m m u n o g l o b u l i n s and o t h e r compounds can a f f e c t PE m e t h y l a t i o n r n v i v o which can be c o r r e l a t e d w i t h a l t e r a t i o n s i n various c e l l u l a r processes. The p r e c i s e mechanism by which PE m e t h y l t r a n s f e r a s e a c t i v i t y i s a l t e r e d , however, i s not known. Castano e t a l . (102) have r e p o r t e d t h a t glucagon and cAMP w i l l a c t i v a t e PE m e t h y l t r a n s f e r a s e i n homogenates from i s o l a t e d r a t hepatocytes. I t i s p o s s i b l e t h e n , t h a t PE m e t h y l t r a n s f e r a s e i s an enzyme which might be a c t i v a t e d by p h o s p h o r y l a t i o n . There i s e v i d e n c e from the s l i m e mold D i c t o s t e l i u m d i s c o i d e u m (95) and very r e c e n t l y f o r r a t l i v e r (320b) that the PE m e t h y l t r a n s f e r a s e r e a c t i o n can be a c t i v a t e d by c a l m o d u l i n . The a d d i t i o n of bovine b r a i n calmodulin enhanced l i p i d t r a n s m e t h y l a t i o n w h i l e antiserum a g a i n s t r a t b r a i n calmodulin i n h i b i t e d the c o n v e r s i o n . EGTA and c h l o r p r o m a z i n e , i n h i b i t o r s o f c a l m o d u l i n , a l s o reduced PE m e t h y l a t i o n . Hence, PE m e t h y l t r a n s f e r a s e might p o t e n t i a l l y be c o n t r o l l e d by the i n t r a c e l l u l a r l e v e l s of c a l c i u m . PE m e t h y l t r a n s f e r a s e a c t i v i t y has been measured i j i v i t r o from l i v e r 63 and i s o l a t e d hepatocytes where p h o s p h o l i p i d methylation has been perturbed i n v i v o ( T a b l e 13). While sometimes a p o s i t i v e c o r r e l a t i o n was f o u n d , more times than n o t , the i j i v i t r o enzyme a c t i v i t y d i d not correspond to the a l t e r a t i o n of PE m e t h y l a t i o n i n the i n t a c t system. I t i s p o s i b l e that d u r i n g the p r e p a r a t i o n of the microsomes, c y t o s o l i c f a c t o r s are removed which might i n f l u e n c e the enzyme a c t i v i t y . I n h i b i t i o n o f P h o s p h a t i d y l e t h a n o l a m i n e N - M e t h y l a t i o n by S-Adenosylhomo- c y s t e i n e ( 1 . 4 . 2 . 4 ) . S-Adenosylhomocysteine (Ado-Hcy) i s a p r o d u c t of the t r a n s m e t h y l a t i o n r e a c t i o n s i n which Ado-Met s e r v e s as the methyl donor. Since Ado-Hcy i s a potent i n h i b i t o r of the PE m e t h y l t r a n s f e r a s e (324-326) and other m e t h y l t r a n s -f e r a s e s (327-330), Ado-Hcy may f u n c t i o n as a b i o r e g u l a t o r y compound. Ado-Hcy tends to have a greater a f f i n i t y f o r the methyltransferase than Ado-Met (Table 14). The a c t i v i t y o f the PE m e t h y l t r a n s f e r a s e might t h e r e f o r e be s e n s i t i v e to the i n t r a c e l l u l a r Ado-Met:Ado-Hcy r a t i o . Hoffman and Cornatzer (324,331,332) have surveyed the Ado-Met:Ado-Hcy r a t i o i n the brains and l i v e r s of v a r i o u s animals (Table 14). Measurement of the Ado-Hcy concentration was d i f f i c u l t s i n c e the Ado-Hcy l e v e l s immediately r i s e f o l l o w i n g death ( 3 3 2 ) . A d i r e c t c o r r e l a t i o n e x i s t s between the Ado-Met:Ado-Hcy r a t i o and m i c r o s o m a l PE m e t h y l t r a n s f e r a s e a c t i v i t y i n r a t s s u b j e c t e d t o e i t h e r 72 h s t a r v a t i o n or d i e t a r y methyl group d e f i c i e n c y (324). As shown i n Table 14, the Ado-Met :Ado-Hcy r a t i o i s 15-fold higher i n the r a t cerebellum than i n r a t l i v e r . Y e t , the methylation pathway i s more a c t i v e i n the l i v e r than i n any o t h e r organ ( 7 8 ) . The guinea p i g l i v e r , which has an Ado-Met: Ado-Hcy r a t i o t w i c e t h a t of r a t l i v e r , has 10-fold lower PE m e t h y l t r a n s f e r a s e a c t i v i t y . .Hence, under normal n u t r i t i o n a l c o n d i t i o n s , i t seems d o u b t f u l t h a t Ado-Hcy a c t s as a major r e g u l a t o r of p h o s p h o l i p i d methylation. 64 Table 13. Changes i n PE M e t h y l t r a n s f e r a s e A c t i v i t y i n Various Model Systems A) POSITIVE CORRELATION WITH PHOSPHATIDYLCHOLINE SYNTHESIS Model System Ref. Effect on PE Methylation Enzyme A c t i v i t y Relative to Control Rat l i v e r - castrated male -e s t r a d i o l treated (309. 321) Stimulated 1.H-fold* Rat l i v e r - choline d e f i c i e n c y (322. 323) Stimulated • Rat l i v e r - choline d e f i c i e n c y (297) Stimulated 1.6-fold• B) NEGATIVE OR NO CORRELATION WITH PHOSPHATIDYLCHOLINE SYNTHESIS Rat l i v e r - 3-deazaadenosine treatment (268) Inhibited no change Rat l i v e r - methyl d e f i c i e n c y (277) Inhibited 3.2-fold • Rooster l i v e r - d i e t h y l s t i l b e s t r o l treatment (285) ? 2.0-fold • Rat hepatocytes - cAMP analogue treatment (313) Inhibited 2.0-foldt Rat hepatocytes - glucagon treatment (102) Inhibited 2.0-fold• 65 Table 14. Influence of Ado-Hcy on PE M e t h y l a t i o n i n Various Model Systems. Animal Tissue Age Apparent Km f o r Ado-Met Apparent K i f o r Ado-Hcy Ado-Met/Ado-Hcy r a t i o Ref. Agrobacterium tumefaciens 200 pM 4 pM - (326) Rat l i v e r newborn 12/1 (324) Guinea Pig l i v e r 15 weeks old 302 uM 68 pM 10/1 (324) Rat l i v e r 100 days old 18 MM 3.8 MM 5/1 (324) Mouse l i v e r 50 days old 5/1 (324) Rabbit l i v e r 50 days old 3/1 (324) Rat l i v e r 35-40 days old (fed) 2.7/1 (333) Rat l i v e r 35-40 days old (24h fasted) 2.4/1 (333) Rat cerebrum 100 days o l d 42/1 (332) Rat cerebrum newborn 110/1 (332) Rat cerebellum 100 days old 75/1 (332) Rat cerebellum newborn 130/1 (332) 66 Phosphatidylcholine Synthesis i n Various Model Systems DEVELOPMENTAL STUDIES  Rat ( 1 . 5 . 1 . 1 ) . More i s known about the pre- and p o s t n a t a l development o f the r a t than any other mammal. Rats are born between the 21st and 22nd day of g e s t a t i o n and b e g i n t h e w e a n i n g p r o c e s s n e a r t h e 16th p o s t n a t a l day ( f o r a comprehensive review see 334). L i v e r ( 1 . 5 . 1 . 2 ) . During p r e n a t a l and e a r l y p o s t n a t a l development there i s a s i g n i f i c a n t d a i l y i n c r e a s e i n t h e l i v e r w e i g h t o f t h e r a t ( 3 3 5 , 3 3 6 ) . However, immediately a f t e r b i r t h the amount of PC per g of l i v e r r a p i d l y accumulates so t h a t i t i s almost doubled by the 4th day of neonate (335,336). A f t e r the 6th day, the amount of PC per g o f l i v e r has almost a t t a i n e d a d u l t values and p l a t e a u s . The enhanced a b i l i t y o f the l i v e r t o produce PC a f t e r b i r t h 32 i s a l s o i n d i c a t e d by the reduced i n c o r p o r a t i o n of [ P]phosphate (336) and 14 [Me- C ] c h o l i n e (294) i n t o PC by f e t a l l i v e r s l i c e s i n co m p a r i s o n t o s l i c e s from neonatal l i v e r . Weinhold e_t a l . (294) have m e a s u r e d t h e l e v e l s o f the r a t l i v e r de novo b i o s y n t h e t i c enzymes d u r i n g development. C h o l i n e kinase a c t i v i t y peaked about two days p r i o r t o t e r m , w h i l e c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y s t e a d i l y increased d u r i n g development u n t i l day 5 where i t matched a d u l t v a l u e s . On day 1, however, cholinephosphotransferase a c t i v i t y was only h a l f of mature values. Only 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 i n both the microsomes and c y t o s o l c o r r e l a t e d w i t h the i n c r e a s e d r a t e o f h e p a t i c PC s y n t h e s i s a f t e r b i r t h . 67 The p o s t n a t a l developmental changes i n r a t l i v e r PE methyltransferase have been reported by Hoffman et_ a l . ( 3 3 2 ) . Both PEMT 1 and DMPE methyl-t r a n s f e r a s e a c t i v i t i e s s t e a d i l y i n c r e a s e d from b i r t h u n t i l day 20 and slow l y dropped to a d u l t v a l u e s by day 50. The a c t i v i t i e s a t day 20 were almost 2 - f o l d higher than a t b i r t h or day 50. The Ado-Met: Ado-Hcy r a t i o i n r a t l i v e r dropped markedly from 12:1 a t b i r t h t o 5:1 at day 30 ( 3 2 4 ) . The r e d u c t i o n i n t h i s r a t i o p r o b a b l y r e f l e c t e d i n c r e a s e d production of Ado-Hcy from enhanced phospholipid m e t h y l a t i o n s i n c e the Ado-Met c o n c e n t r a t i o n was constant throughout the r a t ' s l i f e span. PE methyltransferase a c t i v i t y has r e c e n t l y been examined i n p r e n a t a l r a b b i t l i v e r (337). T r a n s m e t h y l a t i o n was unchanged at -12 and -9 days, but inc r e a s e d 5 - f o l d at b i r t h . The dramatic i n c r e a s e i n PE m e t h y l a t i o n i n the neonate might account f o r the changes i n the m o l e c u l a r s p e c i e s o f r a t l i v e r PC with development (335). During the pr e n a t a l p e r i o d , the major species are d i p a l m i t o y l - P C and monoenoic PC. A f t e r b i r t h , the l i v e r content of long polyunsaturated species i n c r e a s e s r a p i d l y , while the p r o p o r t i o n o f saturated and monoenoic PC species subsides. Lung ( 1 . 5 . 1 . 3 ) . PC s y n t h e s i s i s c r u c i a l f o r s u r f a c t a n t p r o d u c t i o n i n the newborn. I n s u f f i c i e n t s u r f a c t a n t a t b i r t h i s b e l i e v e d t o be r e s p o n s i b l e f o r R e s p i r a t o r y D i s t r e s s Syndrome, w h i c h i s a major cause o f m o r t a l i t y i n premature i n f a n t s . F o r t h i s and o t h e r r e a s o n s , PC s y n t h e s i s d u r i n g pulmonary development has re c e i v e d c o n s i d e r a b l e a t t e n t i o n . The r a b b i t has been a u s e f u l model t o s t u d y developmental changes i n lung PC metabolism as the l a r g e r lungs o f f e t a l r a b b i t s are e a s i e r t o mani-p u l a t e than those of f e t a l r a t s . Rooney e t a l . (338) have r e p o r t e d that 68 the r a t e of c h o l i n e i n c o r p o r a t i o n i n t o PC i n f e t a l r a b b i t lung s l i c e s i n -creased s e v e r a l - f o l d between 26-29 days g e s t a t i o n (term, 31 days). The t i s -sue content and s e c r e t i o n of PC i n t o the a l v e o l i was a l s o increased during t h i s p e r i o d ( 3 3 9 ) . S i m i l a r l y , Tokmakjian e_t a l . (340) noted t h a t the 14 a b i l i t y of a d u l t l u n g s l i c e s t o c o n v e r t [Me- C ] c h o l i n e i n t o PC was 30% lower than s l i c e s from f e t a l l u n g s at 25 days g e s t a t i o n , even though the uptake of r a d i o - l a b e l l e d c h o l i n e i n t o the s l i c e s was the same. In the same study, the pool s i z e s of the c h o l i n e m e t a b o l i t e s i n lung were i n v e s t i g a t e d . Between 25 and 30 days g e s t a t i o n the p o o l s i z e s of c h o l i n e and PC increased 1.5- and 2 - f o l d , r e s p e c t i v e l y . The p o o l s i z e s o f p h o s p h o c h o l i n e and C D P - c h o l i n e were d e c r e a s e d 85% and 21%, r e s p e c t i v e l y , d u r i n g the same p e r i o d . Together, the p u l s e - l a b e l l i n g s t u d i e s and p o o l s i z e measurements s u g g e s t e d t h a t t h e f l u x t h r o u g h t h e c y t i d y l y l t r a n s f e r a s e s t e p was a c c e l e r a t e d d u r i n g pulmonary development. The s l i g h t r e d u c t i o n i n the C D P - c h o l i n e l e v e l between 27 and 30 days g e s t a t i o n i n d i c a t e d t h a t the m e t a b o l i c f l u x t h r o u g h the c h o l i n e p h o s p h o t r a n s f e r a s e r e a c t i o n was a l s o enhanced. In the r a t , the PC content i n -1 day o l d f e t a l lung i s h a l f of newborn and a d u l t v a l u e s ( 3 3 6 ) . W e i n h o l d (1 7 3 ) s u b s e q u e n t l y showed t h a t the i n c o r p o r a t i o n of r a d i o - l a b e l l e d c h o l i n e i n t o PC was low i n lung s l i c e s from 19 day o l d f e t u s e s , but increased t o a d u l t values i n 22 day o l d f e t u s e s . On the other hand, i n c o r p o r a t i o n o f the l a b e l i n t o phosphocholine was high i n 19 day o l d f e t u s e s , but d e c r e a s e d i n o l d e r f e t u s e s . S i n c e the s p e c i f i c r a d i o a c t i v i t y o f phosphocholine was u n a l t e r e d , the increased i n c o r p o r a t i o n of l a b e l i n t o PC was a t t r i b u t e d t o a s t i m u l a t i o n o f the enzyme r e a c t i o n s between phosphocholine and PC. 6? In rhesus monkey (341), the i n c o r p o r a t i o n o f r a d i o a c t i v e c h o l i n e i n t o PC by s l i c e s of f e t a l lung a l s o i n c r e a s e d during the l a s t 10% of g e s t a t i o n . As observed f o r the r a b b i t and r a t (342) , the s t i m u l a t i o n c o r r e l a t e d w i t h i n c r e a s e d amounts o f s u r f a c t a n t i n l u n g l a v a g e or a m n i o t i c f l u i d o f the si m i a n . The a c t i v i t i e s o f the l u n g PC b i o s y n t h e t i c enzymes from r a b b i t , r a t mouse and guinea p i g have been measured d u r i n g development. In most cases (342-345), there was l i t t l e change i n t h e p r e n a t a l a c t i v i t i e s o f c h o l i n e k i n a s e and c h o l i n e p h o s p h o t r a n s f e r a s e . However , i n a few s t u d i e s the pulmonary a c t i v i t i e s of cho l i n e kinase i n r a t (346,347). and cholinephospho-t r a n s f e r a s e i n r a t (348) and mouse (343) were r e p o r t e d t o peak 1-3 days p r i o r to term. Despite the c o n f u s i n g and sometimes c o n t r a d i c t o r y d a t a , the incr e a s e d production of PC c o r r e l a t e s b e s t with a s t i m u l a t i o n of c y t i d y l y l -t r a n s f e r a s e a c t i v i t y p r i o r t o or immediately a f t e r p a r t i t i o n (33,339,342, 343.346,347.349). Weinhold et. a l . (350) have r e c e n t l y examined l u n g PC s y n t h e s i s i n f e t a l r a t s which were d e l i v e r e d one and two days premature. Although the 14 33 r a t e s o f i n c o r p o r a t i o n o f [Me- C J c h o l i n e and [ P]phosphate i n t o PC were low immediately a f t e r d e l i v e r y , 3 h l a t e r the i n c o r p o r a t i o n s were e l e v a -ted 60%. The increased choline i n c o r p o r a t i o n i n t o PC was p r i m a r i l y due t o an incre a s e d s p e c i f i c r a d i o a c t i v i t y o f p h o s p h o c h o l i n e which r e s u l t e d from the r a d i o - l a b e l l e d p h o s p h o c h o l i n e b e i n g d i l u t e d i n t o a s m a l l e r p o o l o f phosphocholine (15). The reduction i n the l e v e l o f phosphocholine f o l l o w i n g premature d e l i v e r y was a s s o c i a t e d w i t h i n c r e a s e d 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 ( 15 ) . There was no change i n the t o t a l a c t i v i t i e s o f c h o l i n e k i n a s e , c y t i d y l y l t r a n s f e r a s e , p h o s p h a t i d a t e p h o s p h o h y d r o l a s e nor cholinephosphotransferase, but the c y t i d y l y l t r a n s f e r a s e was r e d i s t r i b u t e d so 70 t h a t t h e m i c r o s o m a l a c t i v i t y i n c r e a s e d by 60%. In a d d i t i o n , t h e 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 n the 100,000 X g X 1 h supernatant of the f e t a l r a t lung changed from p r e d o m i n a n t l y L-form t o more than 60% H-form, a p a t t e r n s i m i l a r to that found i n a d u l t lung (33). I f the H-form and m i c r o s o m a l c y t i d y l y l t r a n s f e r a s e both r e p r e s e n t membrane-bound enzyme, how i s the t r a n s l o c a t i o n of the lung c y t i d y l y l t r a n s -f e r a s e from the c y t o p l a s m t o t h e E.R. a c h i e v e d d u r i n g development? P h o s p h a t i d y l g l y c e r o l (PG) i s the n e x t major p h o s p h o l i p i d component of s u r f a c t a n t a f t e r PC. T h e r e f o r e , perhaps the s y n t h e s i s of PG can t r i g g e r t h e f o r m a t i o n of PC. Feldman e_t a l . ( 3 9 ) have shown t h a t the f e t a l (L-form) c y t i d y l y l t r a n s f e r a s e can be a c t i v a t e d by p h o s p h a t i d y l g l y c e r o l (PG). Furthermore, PG promoted the a g g r e g a t i o n of the enzyme. Hence i t may be s i g n i f i c a n t t h a t the a c t i v i t y o f c y t i d y l y l t r a n s f e r a s e i n r a b b i t lung i n c r e a s e s i m m e d i a t e l y a f t e r b i r t h when t h e r e i s a l s o r e p o r t e d to be an i n c r e a s e i n the amount of PG i n l a m e l l a r bodies (351). I t i s noteworthy t h a t g l u c o c o r t i c o s t e r o i d s and e s t r o g e n s which s t i m u l a t e 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 a l s o a c c e l e r a t e the s y n t h e s i s o f PG (352-354). Furthermore, lung 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 a c t i v i t y f r o m r a b b i t f e t u s e s i s not s t i m u l a t e d by 1 7 - b e t a - e s t r a d i o l i f PG i s i n c l u d e d i n the enzyme a s s a y (265,295). C o l l e c t i v e l y , these f i n d i n g s i m p l i c a t e PG as a r e g u l a t o r y f a c t o r f o r s u r f a c t a n t s y n t h e s i s . I t i s e q u a l l y f e a s i b l e t h a t d i g l y c e r i d e c o u l d serve a r e g u l a t o r y r o l e i n l u n g PC s y n t h e s i s . The c o n t e n t o f d i g l y c e r i d e i n f e t a l r a t l u n g i s approximately 36% o f a d u l t v a l u e s , and r a p i d l y i n c r e a s e s to a d u l t l e v e l s w i t h i n a day a f t e r b i r t h ( 1 9 3 ) . In a d d i t i o n t o b e i n g a s u b s t r a t e f o r PC s y n t h e s i s , d i g l y c e r i d e has been shown t o aggregate the 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 (38) and w i l l l i k e l y do the same fo r the lung enzyme. 71 B r a i n ( 1 . 5 . 1 . 4 ) . The a c t i v i t i e s o f t h e d_e novo PC b i o s y n t h e t i c enzymes d u r i n g p r e n a t a l development have not been w e l l c h a r a c t e r i z e d i n b r a i n . However, Fimbres et a l . (55) have measured c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y i n the developing r a b b i t cerebrum. The a c t i v i t y o f t h i s enzyme peaked 5 days p r i o r to b i r t h , d e c l i n e d t o 50% a t b i r t h , and peaked a g a i n near the 15th p o s t n a t a l day. The age-related changes i n cholinephosphotransferase a c t i v i t y appeared to c o r r e l a t e with both t h e p r e n a t a l m u l t i p l i c a t i o n of neurons and g l i a l c e l l s , and w i t h t h e p o s t n a t a l e l a b o r a t i o n o f plasma membrane a s s o c i a t e d with d e n d r i t i c a r b o r i z a t i o n and m y e l i n a t i o 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 was not determined. McCaman and Cook (48) examined c h o l i n e k i n a s e and ph o s p h o c h o l i n e -t r a n s f e r a s e a c t i v i t i e s i n t h e r a b b i t c o r t e x and m e d u l l a d u r i n g e a r l y p o s t n a t a l d e v e l o p m e n t . I n t h e c o r t e x , b o t h c h o l i n e k i n a s e and phosphocholinetransferase a c t i v i t i e s peaked between day 16 and day 20. The a c t i v i t i e s of these enzymes i n t h e m e d u l l a d i d not f l u c t u a t e i n the f i r s t few week a f t e r b i r t h . The p h o s p h o l i p i d m e t h y l t r a n s f e r a s e a c t i v i t i e s d u r i n g p o s t n a t a l development have been studied i n r a t cerebrum and cerebellum by Hoffman et a l . (332). Both PEMT 1 and DMPE m e t h y l t r a n s f e r a s e a c t i v i t i e s were s l o w l y reduced i n the cerebrum d u r i n g development so t h a t the values at 50 days were 40? lower than a t b i r t h . In c o n t r a s t , the p h o s p h o l i p i d methyltrans-f e r a s e a c t i v i t i e s of the cerebellum were unchanged throughout the p o s t n a t a l p e r i o d . Hitzemann (108) u s i n g c o r t i c a l synaptosomes, found that PE, MMPE and DMPE methyltransferase a c t i v i t i e s were h i g h e r i n 14 day ol d than 7 day o l d or a d u l t r a t b r a i n s . The a f f i n i t y o f the phospholipid methyltransferases f o r 72 o Ado-Met at 20 C a l s o i n c r e a s e d w i t h development. The t r a n s i e n t r i s e i n p h o s p h o l i p i d methylation a c t i v i t y on day 14 c o r r e l a t e d with a t r a n s i e n t i n c r e a s e i n the concentrations of the a r a c h i d o n y l species of PC i n c o r t i c a l s y n a p t i c membranes. 73 VIRAL STUDIES V i r u s e s and P h o s p h o l i p i d M e t a b o l i s m (1.5.2.1). V i r u s e s have been shown t o p e r t u r b t h e b i o s y n t h e s i s of PC i n va r i o u s e s t a b l i s h e d c e l l l i n e s (Table 1 5 ) . T h e r e f o r e , v i r u s e s can be u s e f u l probes to d e l i n a t e the r e g u l a t i o n of PC a n a b o l i s m . Unfortunately, only two systems have been c r i t i c a l l y e v a l u a t e d , and i n t h e case of one of them the a c t u a l e f f e c t o f the v i r u s on PC synthe s i s i s c o n t r o v e r s i a l . When c h i c k embryo f i b r o b l a s t s were i n f e c t e d w i t h S i n d b i s v i r u s , 32 d e c r e a s e d i n c o r p o r a t i o n o f [ P ] p h o s p h a t e i n t o s e v e r a l c l a s s e s o f ph o s p h o l i p i d s r e s u l t e d (355, 356). S i m i l a r l y , when Vance and Lam (305) 3 i n f e c t e d BHK c e l l s w i t h t h i s v i r u s a reduced r a t e o f [Me- H ] c h o l i n e i n c o r p o r a t i o n i n t o PC was observed . I n f e c t i o n o f BHK c e l l s with a r e l a t e d toga v i r u s , S e m l i k i F o r e s t v i r u s , a l s o produced the same r e s u l t (304). In bo t h c a s e s , the v i r u s e s reduced c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y by approximately 45% (304,305). Whitehead (260) f u r t h e r examined the e f f e c t s of S e m l i k i Forest v i r u s on PC s y n t h e s i s i n BHK-21 c e l l s , and a l s o detected a 47% i n h i b i t i o n o f the m i c r o s o m a l 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 . When c y t i d y l y l t r a n s f e r a s e was assayed i n the presence of a c t i v a t i n g p h o s p h o l i p i d , the c y t o s o l s from v i r u s - i n f e c t e d c e l l s had approximately 20% higher a c t i v i t y than c o n t r o l c y t o s o l s . Hence, i t i s p o s s i b l e 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 r e a c t i o n may have been i n h i b i t e d by a t r a n s l o c a t i o n o f the enzyme t o the cytoplasm. The 3.5-fold increased pool s i z e o f phosphocholine i n the S e m l i k i Forest v i r u s - i n f e c t e d c e l l s tends to support t h i s contention (260). The 2.6-f o l d l a r g e r pool s i z e of CDP-choline i n the v i r u s - i n f e c t e d c e l l s a l s o con-firmed t h a t the rate of the c h o l i n e p h o s p h o t r a n s f e r a s e r e a c t i o n might be i n -h i b i t e d i n v i v o . F u r t h e r m o r e , t h i s v i r u s reduced the CTP pool s i z e by Table 15. Influence of V i r u s e s on Phosphatidylcholine Synthesis V i r U 3 Host Time of Ef f e c t on Change i n S p e c i f i c Infection PC Synthesis Enzyme A c t i v i t i e s (357) adenoviridae adenovirus type 5 HEK c e l l s 8 h 1 . 6 - f o l d ta f (358) adenoviridae simian viru3 40 EHSVi ( v i r u s transformed c e l l l i n e ) 2 . 5 - f o l d t c 6 .5 r-foldt PLMTe (359) herpetoviridae psuedorabis v i r u s RK c e l l s 9 h 1 . 2 - f o l d ta f (264) picornaviridae p o l i o virus HeLa c e l l s 3.5 h 2-fold t f 2 - f o l d t CT (360) picornaviridae mengovirus L c e l l s 9 h 1 . 6 - f o l d t a f (358) PY vi r u s PY-BHK-21 (virus transformed c e l l l i n e ) 1 . 4 - f o l d tc 3 . 8 - f o l d t PLMTe (361) r e t r o v i r i d a e Friend virus BALB/c mice 14 days 1 . 9 - f o l d *a f (305) togoviridae sindbis virus BHK-21 c e l l s 6 h 7 3 * *a f 43* CPT + (355) togaviridae sindbis v i r u s chick embryo f i b r o b l a s t s 10 h 60*+ f (260) togaviridae Semliki forest v i r u s BHK-21 c e l l s 7 h ? * a f 47*+ CT1; 57*+ CPT (362) togaviridae Japanese encepha-l i t i s v irus BHK-21 c e l l s 21 h 7 0 * * ' (311) togaviridae dengue virus type 2 BHK-21 c e l l s 45 min ? 50*+ CPT a - represents the rate of incorporation of a radio-labeled precursor b - microsomal c - methylation of PE d - compared to uninfected c e l l l i n e e - phospholipid methyltransferase (PLMT) f - de novo pathway 75 almost 70% (260,299). Since the c o n c e n t r a t i o n of CTP i s w e l l below the appar-ent K o f the c y t i d y l y l t r a n s f e r a s e , t h i s r e d u c t i o n would be e x p e c t e d m t o i n h i b i t s e v e r e l y the m e t a b o l i c f l u x t h r o u g h the c y t i d y l y l t r a n s f e r a s e c a t a l y z e d s t e p . I t i s t h e r e f o r e hard to accept the conclusion t h a t S e m l i k i F o r e s t v i r u s does not i n h i b i t PC b i o s y n t h e s i s i n BHK c e l l s (25). This pro-nouncement was based on the r e s u l t s of a pulse-chase experiment. However, i t 3 s h o u l d be noted t h a t at the end o f t h e p u l s e w i t h [Me- H ] c h o l i n e , the amount o f l a b e l a s s o c i a t e d w i t h p h o s p h o c h o l i n e and PC was the same f o r v i r u s - and mock-infected c e l l s . This c o n t r a d i c t s the e a r l i e r f i n d i n g 3 t h a t [Me- H ] c h o l i n e uptake was r e d uced by r o u g h l y 40% i n the v i r u s -i n f e c t e d c e l l s (25). This model system should be re-examined to c l e a r up t h i s discrepancy. The s i t u a t i o n w i t h t h e p i c o r n a v i r u s t h a t c a u s e s p o l i o i s l e s s ambiguous. In 1965, i t was f i r s t r e p o r t e d t h a t p o l i o v i r u s s t i m u l a t e d the 3 i n c o r p o r a t i o n of [Me- H ] c h o l i n e i n t o the t r i c h l o r o a c e t i c a c i d - p r e c i p i t -able f r a c t i o n of HeLa c e l l s ( 3 6 3 ) . T h i s f i n d i n g was confirmed by Vance et a l . (264) who proceeded to i n v e s t i g a t e the mechanism f o r the p o l i o v i r u s e f f e c t on PC b i o s y n t h e s i s . In summary, 3.5 h p o s t - i n f e c t i o n , p o l i o v i r u s 3 a c c e l e r a t e d the c o n v e r s i o n of H from p h o s p h o c h o l i n e i n t o PC more than 2-f o l d , and t h i s was c o r r e l a t e d w i t h an i n c r e a s e d t u r n o v e r r a t e f o r the c y t i d y l y l t r a n s f e r a s e c a t a l y z e d r e a c t i o n . The s t i m u l a 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 r e a c t i o n was a t t r i b u t e d t o a 3 - f o l d i n c r e a s e i n the cytoplasmic pool of CTP 4 h p o s t - i n f e c t i o n ( 2 9 8 ) . There are two p o i n t s , however, which warrant comment. F i r s t l y , i f the c y t i d y l y l t r a n s f e r a s e r e a c t i o n were a c c e l e r a t e d , a s l i g h t r e d u c t i o n i n the p o o l s i z e of phosphocholine might have been noted. This was not the case ( 2 6 4 ) . Secondly, i f the microsomal c y t i d y l y l t r a n s f e r a s e was reduced 47% by the v i r u s as r e p o r t e d , t h i s i s 76 r a t h e r at odds with the concept t h a t the microsomal enzyme governs the r a t e of PC s y n t h e s i s . Although t h e e f f e c t on PC s y n t h e s i s was not evaluated,. Malewicz et a l . (311) have reported t h a t i n f e c t i o n o f BHK-21 c e l l s with dengue type 2 v i r u s was accompanied by a r a p i d , but t r a n s i e n t s t i m u l a t i o n of phospholipase A^ a c t i v i t y . F u r t h e r m o r e , m i c r o s o m a l c h o l i n e p h o s p h o t r a n s f e r a s e was i n h i b i t e d i n phase w i t h the p h o s p h o l i p a s e k^ a c t i v a t i o n . The a u t h o r s suggested t h a t enhanced l y s o - P C p r o d u c t i o n might be r e s p o n s i b l e f o r the r e d u c e d c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y s i n c e l y s o - P C can have an i n h i b i t o r y a c t i o n on t h i s enzyme in_ v i t r o (215). A n d e r t o n ejb a 1 . ( 3 6 4 ) h a v e m e a s u r e d t h e p o o l s i z e s o f t h e p h o s p h o l i p i d s i n A f r i c a n G r e e n Monkey c e l l s which were u n i n f e c t e d , p e r s i s t e n t l y i n f e c t e d or l y t i c a l l y i n f e c t e d with measles v i r u s . An i n c r e a s e i n the lyso-PC c o n c e n t r a t i o n i n p e r s i s t e n t l y i n f e c t e d c e l l s suggested t h a t phospholipase A^ might be a c t i v a t e d i n t h e s e c e l l s . However,, i n l y t i c a l l y i n f e c t e d c e l l s , the l y s o - P C c o n c e n t r a t i o n was 73% lower than i n mock-i n f e c t e d c e l l s . Consequently, measles v i r u s probably i n h i b i t s phospholipase A^ a c t i o n d u r i n g l y t i c i n f e c t i o n s . The i n c r e a s e p o o l o f PC i n the l y t i c a l l y - i n f e c t e d c e l l s m i g h t a l s o be due t o a s t i m u l a t i o n o f PE m e t h y l a t i o n s i n c e the PE pool s i z e was reduced by 15% i n these, c e l l s . The b e s t e v i d e n c e t h a t p h o s p h o l i p i d m e t h y l a t i o n may be a l t e r e d i n v i r u s - i n f e c t e d c e l l l i n e s has been o b t a i n e d by M a z i e r e e_t a l . (358). PY 14 or SV 40-transformed hamster c e l l s i n c o r p o r a t e d up t o 50% more [Me- C ] -methionine i n t o p h o s p h o l i p i d than u n i n f e c t e d hamster c e l l s . Furthermore, p h o s p h o l i p i d m e t h y l t r a n s f e r a s e a c t i v i t i e s from SV40- and PY-transformed c e l l s were 6.5- and 3-5-fold h i g h e r , r e s p e c t i v e l y , than measured from normal hamster f i b r o b l a s t s or BHK c e l l s . The a u t h o r s speculated that m o d i f i c a t i o n s 77 i n p h o s p h o l i p i d m e t h y l a t i o n might be i n v o l v e d i n s e v e r a l p e r t u r b a t i o n s of c e l l membrane s t r u c t u r e and f u n c t i o n seen a f t e r oncogenic t r a n s f o r m a t i o n . Monzel and K o s c h e l (230) have r e c e n t l y r e p o r t e d t h a t the paramyxo-v i r u s e s , subacute s c l e r o s i n g p a n e n c e p h a l i t i s v i r u s and c a n i n e distemper v i r u s both cause reduced m e t h y l a t i o n o f PE i n p e r s i s t e n t l y - i n f e c t e d C6 r a t glioma c e l l s . This was c o r r e l a t e d w i t h an impairment o f the catecholamine-induced beta-adrenergic r e c e p t o r - d e p e n d e n t cAMP generation i n these c e l l s . 78 TUMOUR PROMOTER STUDIES P h o s p h a t i d y l c h o l i n e B i o s y n t h e s i s i n Transformed C e l l s (1.5.3.1). Tumour promoters c o n s t i t u t e a f a m i l y of compounds which although not c a r c i n o g e n i c or mutagenic by themselves, d r a m a t i c a l l y i n c r e a s e the incidence of tumours when a p p l i e d r e p e a t e d l y t o a n i m a l s which have rece i v e d a sub-t h r e s h o l d dose o f a c a r c i n o g e n . D i h y d r o t e l e o c i d i n B from the mycelia of Streptomyces (365) and 1 2 - 0 - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e (TPA) from croton o i l (366) a r e , r e s p e c t i v e l y , t he f i r s t and second most potent tumour promoters known. The a c t i o n o f TPA has been i n t e n s i v e l y i n v e s t i g a t e d and t h i s tumour promoter evokes a v a r i e t y o f b i o l o g i c a l and biochemical changes when added to c u l t u r e c e l l s (367-370). Enhancement of PC s y n t h e s i s i s one o f the e a r l i e s t m e t a b o l i c events that occurs d u r i n g the i n i t i a l s t i m u l a t i o n of c e l l growth and p r o l i f e r a t i o n by TPA and o t h e r p h o r b o l e s t e r s . K r e i b i c h et_ a l . (371) and others (372, 373) have r e p o r t e d t h a t TPA a d m i n i s t r a t e d t o mouse s k i n i n c r e a s e d 3 32 [Me- H ] c h o l i n e and [ P ] p h o s p h a t e l a b e l l i n g o f PC. S i m i l a r l y , p u l s e l a b e l l i n g experiments have demonstrated t h a t p h o r b o l e s t e r s s t i m u l a t e PC s y n t h e s i s i n HeLa c e l l s (296,374-376), bovine lymphocytes (377), c h i c k em-bryo myoblasts (378,379), r a t myoblasts (380), HL-60 promyelocytic leukemia c e l l s (381) and human n e u t r o p h i l s ( 3 8 1 b ) . Sakamoto et^ a l . (382) have shown th a t the i n d o l e a l k a l o i d s , d i h y d r o t e l e o c i d i n B and lyngbyatoxin A, although s t r u c t u r a l l y d i s t i n c t from p h o r b o l ester tumour promoters, a l s o increased PC s y n t h e s i s i n HeLa c e l l s . The r e l a t i v e l y weak, non-phorbol 3 e s t e r promoter, m e z e r e i n a l s o s t i m u l a t e d [Me- H ] c h o l i n e i n c o r p o r a t i o n i n t o PC (374.375). 79 TPA and the o t h e r p h o r b o l e s t e r s e x e r t t h e i r e f f e c t on PC anabol ism w i t h i n 15 min (374,378,381) and when human polymorphonuclear n e u t r o p h i l s were exposed to phorbol my r i s t a te a c e t a t e , the l e v e l o f PC i n c r e a s e d w i t h i n 15 sec (381b) . Hence i t i s u n l i k e l y tha t the s t i m u l a t i o n o f PC s y n t h e s i s i s due to i n c r e a s e d p r o t e i n s y n t h e s i s o f any o f the de_ novo b i o s y n t h e t i c enzymes. Th is c o n c l u s i o n i s con f i rmed by the f i n d i n g tha t the response to TPA i s i n s e n s i t i v e to a c t i n o m y c i n D and cyc l ohex im ide (374,377, 379) . In a d d i t i o n , when the p h o r b o l - e s t e r s are removed from HeLa c e l l s (383) and HL-60 c e l l s ( 3 8 1 ) , i n c o r p o r a t i o n o f r a d i o a c t i v e c h o l i n e i n t o PC r a p i d l y r e t u r n s to norma l . T h e r e f o r e , c o n t i n u e d r e c e p t o r occupa t i on on the plasma membrane of the c e l l seems t o be r e q u i r e d t o ma in ta in the promoter induced a l t e r a t i o n s i n phospho l i p i d metabol ism. Paddon and Vance (296) have shown t h a t TPA a c c e l e r a t e s PC b i o s y n t h e s i s i n HeLa c e l l s by s t i m u l a t i o n o f t h e r e a c t i o n c a t a l y z e d by c y t i d y l y l t r a n s f e r a s e . When the a c t i v i t i e s o f c h o l i n e k i n a s e , 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 or c h o l i n e p h o s p h o t r a n s f e r a s e were examined from TPA-t r e a t e d HeLa c e l l s , there were no apparent changes. However, H i l l and Sanwal (380) have repor ted tha t t r e a t m e n t o f r a t myob las t s w i th TPA i n c r e a s e d the s p e c i f i c a c t i v i t y o f c y t i d y l y l t r a n s f e r a s e 1 . 5 - 2 - f o l d . R e c e n t l y , TPA has been shown to no t o n l y s t i m u l a t e PC s y n t h e s i s , but i t a l s o a c t i v a t e s PC c a t a b o l i s m . Grove and Schimmel (378) found tha t TPA produced a doub l i ng o f the d i g l y c e r i d e concen t ra t i on i n the plasma membranes o f c h i c k embryo d i f f e r e n t i a t e d m y o b l a s t s . The d i g l y c e r i d e was subsequent ly shown to be de r i ved from PC by the a c t i o n o f phospho l ipase C (379 ) . Guy and Murray (375) have s i m i l a r l y demonstrated tha t TPA s t i m u l a t e s a phospho l ipase 3 C i n HeLa c e l l s , and c o u l d d u p l i c a t e the enhancement o f []4e- H ] cho l i ne i n c o r p o r a t i o n i n t o PC w i t h e x o g e n o u s l y added p h o s p h o l i p a s e C . T h i s i s 80 p a r t i c u l a r l y i n t e r e s t i n g s i n c e S l e i g h t and Kent (23) have reported t h a t treatment of c h i c k embryonic muscle c e l l s w i t h exogenous phospholipase C promoted the t r a n s l o c a t i o n of c y t i d y l y l t r a n s f e r a s e from the cytoplasm t o the E.R. and i n c r e a s e d PC s y n t h e s i s . Choy et a l . (38) have noted th a t d i g l y c e r i d e w i l l a g g r e g a t e r a t l i v e r c y t o s o l i c c y t i d y l y l t r a n s f e r a s e . Therefore i t i s f e a s i b l e t h a t t h e d i g l y c e r i d e g e n e r a t e d i n TPA-treated c e l l s causes the c y t i d y l y l t r a n s f e r a s e t o b i n d to the E.R. where the enzyme i s a c t i v a t e d , and t h i s r e s u l t s i n i n c r e a s e d rates of PC s y n t h e s i s . A scheme which o u t l i n e s t h i s model f o r a c c e l e r a t e d PC turnover i n TPA-treated c e l l s i s presented as Figure 5 . T P A PHOSPHOCHOLINE X PHOSPHATIDYLCHOLINE DIGLYCERIDE \ inactive y X CYTOPLASM DP-CHOLINE CMP active E.R» 11 iiiiiiMiiMfiirtiiia* Figure 5 . P o s t u l a t e d e v e n t s d u r i n g TPA s t i m u l a t i o n o f PC b i o s y n t h e s i s .  A b b r e v i a t i o n s : E.R., e n d o p l a s m i c r e t i c u l u m ; CPT, c h o l i n e p h o s p h o t r a n s f e r -ase; CT, c y t i d y l y l t r a n s f e r a s e ; P.M., plasma membrane; PL C, phospholipase C; and TPA, 1 2 - 0 - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e . 81 SUPPLEMENTATION STUDIES F a t t y A c i d S u p p l e m e n t a t i o n (1.5.4.1). F a t t y a c i d s (FA) d e r i v e d f r o m e i t h e r t h e p l a s m a or d_e novo s y n t h e s i s can be u t i l i z e d f o r h e p a t i c g l y c e r o l i p i d s y n t h e s i s . Although FA's tend to be channelled i n t o t r i g l y c e r i d e (TG) s y n t h e s i s i n the fed animal, t h i s happens o n l y when the p h o s p h o l i p i d r e q u i r e m e n t s o f the a n i m a l are f i r s t s a t i s f i e d . When the c y t o p l a s m i c p o o l of a c t i v a t e d f a t t y a c i d s i s d i m i n i s h e d , FA are p r e f e r e n t i a l l y e s t e r i f i e d i n t o p h o s p h o l i p i d a t the expense of TG formation (384). S t u d i e s w i t h r a t l i v e r s l i c e s (385,386) and i s o l a t e d hepatocytes (161, 302) showed th a t 1 mM c o n c e n t r a t i o n s o f albumin-bound . f a t t y a c i d 3 s t i m u l a t e d [ H ] g l y c e r o l i n c o r p o r a t i o n i n t o TG over 1 5 - f o l d , whereas e n t r y i n t o PC was enhanced only 2 - f o l d . S i m i l a r l y , oleate (1 mM) has a l s o produced 32 a 3 - f o l d s t i m u l a t i o n o f [ P]phosphate i n c o r p o r a t i o n i n t o t h e PC of r a t hepatocytes (161). The increased l a b e l l i n g of PC probably r e f l e c t e d enhanced p h o s p h o l i p i d s y n t h e s i s , but the p o s s i b i l i t y t h a t these r e s u l t s arose from is o t o p e d i l u t i o n due to changes i n precursor pool s i z e s was not e l i m i n a t e d . Recently, Urade and K i t o (368b) have reported t h a t a d d i t i o n of p a l m i -t a t e to Chinese Hamster V79-R c e l l s caused severe i n h i b i t i o n o f c e l l growth. In the presence of p a l m i t a t e , the t o t a l phospholipid and t r i g l y c e r i d e per c e l l i n c reased to 2- and 13 - f o l d , r e s p e c t i v e l y . These c e l l s could be pro-t e c t e d from the i n h i b i t i o n by pa l m i t a t e by the simultaneous a d d i t i o n of o l e a t e . Apparently p a l m i t a t e caused an increase i n the p r o p o r t i o n o f p a l m i -t a t e with concomitant decrease of o l e a t e i n PC, PE and t r i g l y c e r i d e . Sundler and Akesson (161) have demonstrated t h a t i n c u b a t i o n o f r a t h e p a t o c y t e s w i t h FA and g l y c e r o l p r o d u c e s more than a d o u b l i n g o f the 82 i n t r a c e l l u l a r l e v e l of d i g l y c e r i d e . B ut, i t i s u n l i k e l y that the supply of d i g l y c e r i d e n o r m a l l y l i m i t s t h e r a t e o f PC s y n t h e s i s . S t i l l , i t i s f a c i n a t i n g that free FA have been found t o stimulate l i v e r cholinephospho-t r a n s f e r a s e a c t i v i t y ijn v i t r o p r o v i d e d t h a t exogenous d i g l y c e r i d e was in c l u d e d i n the enzyme assay ( 6 0 , 3 8 7 ) . S r i b n e y and Lyman (387) speculated t h a t f r e e FA may a l t e r the conformation of cholinephosphotransferase so tha t i t i s more able t o accept the DG s u b s t r a t e . The f i n d i n g that f r e e FA's a c c e l e r a t e the c y t i d y l y l t r a n s f e r a s e r e a c t i o n would be a more s a t i s f y i n g e x p l a n a t i o n f o r the s t i m u l a t i o n of PC s y n t h e s i s s i n c e the amount of C D P - c h o l i n e seems t o be r a t e - l i m i t i n g i n r a t l i v e r . Feldman e_t a l . (40) have r e c e n t l y r e p o r t e d t h a t v a r i o u s u n s a t u r a t e d f r e e FA's a c t i v a t e the l u n g 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 almost 3 - f o l d . Furthermore, endogenous f r e e FA's were found to be the predominant species 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 a t o r s i n l u n g c y t o s o l . Perhaps the elevated supply of d i g l y c e r i d e from FA s u p p l e m e n t a t i o n ensures that the c y t i d y l y l -t r a n s f e r a s e c a t a l y z e d r e a c t i o n c o n t i n u e s t o be r a t e - l i m i t i n g d e s p i t e the increased production of CDP-choline. C h o l e s t e r o l and C h o l a t e S u p p l e m e n t a t i o n (1 .5 .4 .2) . Hypercholesterolemia has been a s s o c i a t e d with h y p e r l i p o p r o t e i n e m i a and the development of a t h e r o s c l e r o s i s . In humans (388), m i n i a t u r e swine (389) and r a t s (390) a h i g h d i e t a r y i n t a k e o f c h o l e s t e r o l w i l l d r a m a t i c a l l y e l e v a t e serum c h o l e s t e r o l l e v e l s and t h i s i s accompanied by increased plasma p h o s p h o l i p i d c o n c e n t r a t i o n s . F r i e d m a n and Byers (391) have shown t h a t b i l i a r y o b s t r u c t i o n i n r a t s r e s u l t s i n e l e v a t e d serum l e v e l s of c h o l a t e , c h o l e s t e r o l and p h o s p h o l i p i d . I n t r a v e n o u s i n f u s i o n o f c h o l a t e produced increased l e v e l s of plasma p h o s p h o l i p i d s and c h o l e s t e r o l , while i n j e c t i o n o f p h o s p h o l i p i d s l e d t o an e l e v a t i o n o f plasma c h o l e s t e r o l o n l y . Hence, 83 d u r i n g b i l i a r y o b s t r u c t i o n , i n c r e a s e d serum c h o l a t e was p r o b a b l y r e s p o n s i b l e f o r the r i s e i n plasma phospholipids. Lim ('41) i n v e s t i g a t e d the e f f e c t o f a 2% c h o l a t e and 5% c h o l e s t e r o l d i e t on the hepatic s y n t h e s i s o f PC i n 60 g female Wistar r a t s . This d i e t produced 8- and 2 - f o l d i n c r e a s e s i n the serum c h o l e s t e r o l and p h o s p h o l i p i d c o n c e n t r a t i o n s , r e s p e c t i v e l y . When the hypercholesterolemia r a t s were given 3 p o r t a l i n j e c t i o n s o f [Me- H ] c h o l i n e , i n c o r p o r a t i o n i n t o PC was 3 - f o l d higher than seen f o r c o n t r o l r a t s . The a c t u a l r a t e o f PC s y n t h e s i s was estimated to be 3.2-fold higher i n the hypercholesterolemia r a t s . Lim a l s o examined the a c t i v i t i e s o f the PC b i o s y n t h e t i c enzymes i n the hypercholesterolemia r a t s . Choline k i n a s e , cholinephosphotransferase and PE m e t h y l t r a n s f e r a s e a c t i v i t i e s were unchanged by t h e 2% c h o l a t e / 5% c h o l e s t e r o l d i e t . On the other hand, the c y t o s o l i c and microsomal 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 i e s were e l e v a t e d 3.1- and 1 . 9 - f o l d , r e s p e c t i v e l y . A p p r o x i m a t e l y 30% of t h e 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 n the l i v e r c y t o s o l s o f h y p e r c h o l e s t e r o l e m i a r a t s o c c u r r e d as H-form (compared t o 10% H-form i n c o n t r o l c y t o s o l s ) . Although immunochemical t i t r a t i o n s t u d i e s were performed and i n d i c a t e d the amount o f c y t i d y l y l t r a n s f e r a s e was unchanged i n the c y t o s o l s from h y p e r c h o l e s t e r o l e m i a r a t s , the increased amount of H-form which may r e p r e s e n t m i c r o s o m a l c o n t a m i n a t i o n i n v a l i d a t e s t h e s e r e s u l t s . C y t o sols from hypercholesterolemia r a t s had h a l f as much L-form as c y t o s o l s from purina r a t chow fed r a t s . A t r a n s l o c a 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 to the E.R. might t h e r e f o r e e x p l a i n the i n c r e a s e d r a t e o f PC s y n t h e s i s d u r i n g h y p e r c h o l e s t e r o l e m i a . The c y t o s o l i c d i g l y c e r i d e l e v e l s were el e v a t e d 2 . 8 - f o l d by the c h o l a t e - c h o l e s t e r o l r i c h d i e t . Perhaps the i n c r e a s e d d i g l y c e r i d e c o n c e n t r a t i o n c o u l d have promoted the t r a n s l o 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 (38). 84 Monomethylethanolamine and D i m e t h y l e t h a n o l a m i n e Supplementation ( 1 . 5 . 4 . 3 ) . The u t i l i z a t i o n of monomethylethanolamine (MME) and d i m e t h y l e t h a n o l -amine (DME) f o r phospholipid s y n t h e s i s has been demonstrated i n Neurospora  c r a s s a ( 3 9 2 ) , MOPC-31 C c e l l s ( 3 9 3 ) , LM f i b r o b l a s t s ( 3 9 4 - 3 9 6 ) and r a t hepatocytes ( 1 6 1 ) . When LM c e l l s a r e supplemented w i t h e i t h e r MME or DME, the' corresponding phospholipid can account f o r 3 0 - 5 0 % of the t o t a l phospho-l i p i d ( 3 9 4 , 3 9 5 ) . A l t e r a t i o n s o f the membrane c o m p o s i t i o n o f LM c e l l s i n t h i s way r e s u l t e d i n a reduced c a p a c i t y f o r p h a g o c y t o s i s or p i n o c y t o s i s ( 3 9 5 ) . Since phosphoethanolamine c y t i d y l y l t r a n s f e r a s e w i l l not form the CDP-e s t e r o f DME ( 1 6 1 ) , DME i s not i n c o r p o r a t e d i n t o DMPE v i a t h e de novo pathway f o r P E s y n t h e s i s . K a n f e r ( 3 9 7 ) has r e c e n t l y proposed t h a t MME and DME are incorporated i n t o MMPE and DMPE, r e s p e c t i v e l y , by base exchange. A calcium-dependent r e a c t i o n was found to enhance the i n c o r p o r a t i o n o f MME and DME i n t o the phospholipid of r a t b r a i n microsomes. 1 4 Maeda ej; j s l ^ ( 3 9 6 ) h a v e s h o w n t h a t [ M e - C ] m e t h i o n i n e i n c o r p o r a t i o n i n t o DMPE and PC can be enhanced 8 0 - f o l d when LM c e l l s are incubated with MME and DME. S i m i l a r , but l e s s s t r i k i n g r e s u l t s have been obtained f o r M0PC-31C c e l l s ( 3 9 3 ) . This enhanced methylation can be a s c r i b e d t o t h e PEMT I I c a t a l y z e d r e a c t i o n s . P h o s p h o l i p i d m e t h y l a t i o n was d r a m a t i c a l l y s t i m u l a t e d , presumably because the r a t e - l i m i t i n g step o f the pathway was bypassed with these methylated bases. Deazaadenosine S u p p l e m e n t a t i o n ( 1 . 5 . 4 . 4 ) . A d m i n i s t r a t i o n of 3-deazaadenosine (DZA), a s t r u c t u r a l analogue of adenosine, has been found t o r a i s e t h e i n t r a c e l l u l a r l e v e l s of Ado-Hcy and 3-deaza-Ado-Hcy, both o f which r e s t r i c t numerous m e t h y l a t i o n r e a c t i o n s ( 3 9 8 ) . In p a r t i c u l a r , DZA a d m i n i s t r a t i o n has been shown toreduce drama-85 3 t i c a l l y [Me- H ] m e t h i o n i n e i n c o r p o r a t i o n i n t o the PC of r a t l i v e r (399) and i s o l a t e d r a t hepatocytes (268) . A l t h o u g h PE methy l t r a n s f e r a s e a c t i v i t y i n v i t r o i s not a l t e r e d by DZA t r e a t m e n t , 3-deaza-Ado-Hcy i s a p o t e n t c o m p e t i t i v e i n h i b i t o r with Ado-Met (268). While DZA s e v e r e l y r e s t r i c t s PE N - m e t h y l a t i o n , t h i s drug produces a compensating i n c r e a s e i n the d_e novo s y n t h e s i s o f PC. Chiang and Cantoni (399) o r i g i n a l l y demonstrated t h a t DZA a d m i n i s t r a t i o n to r a t s or hamsters 14 enhanced the i n c o r p o r a t i o n of [ 1 , 2 - C ^ c h o l i n e i n t o PC. S u b s e q u e n t l y , P r i t c h a r d e t a l . (268) showed t h a t DZA s t i m u l a t e d 3 . 1 - f o l d the r a t e of de novo PC s y n t h e s i s i n i s o l a t e d r a t h e p a t o c y t e s . A l t h o u g h t he t o t a l h e p a t i c a c t i v i t i e s o f c h o l i n e k i n a s e , c y t i d y l y l t r a n s f e r a s e and c h o l i n e -p h o s p h o t r a n s f e r a s e were u n a f f e c t e d i n D Z A - t r e a t e d r a t s , t h e r e was a r e d i s t r i b u 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 a c t i v i t y . The 1 . 9 - f o l d i n c r e a s e i n the amount of c y t i d y l y l t r a n s f e r a s e i n the microsomes could be accounted f o r by a c o r r e s p o n d i n g r e d u c t i o n i n the amount of enzyme p r o t e i n i n the c y t o p l a s m . The mechanism by w h i c h DZA e l i c i t e s a s t i m u l a t i o n and r e d i s t r i b u 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 remains a mystery. 86 DEPRIVATION STUDIES E s s e n t i a l F a t t y A c i d D e p r i v a t i o n (1.5.5.1). E s s e n t i a l FA d e f i c i e n t d i e t s have been used to probe the r e g u l a t i o n o f PC s y n t h e s i s i n r a t l i v e r . T rewhella and C o l l i n s (400) found that l i n o l e a t e d e p r i v a t i o n i n c r e a s e d the b i o s y n t h e t i c f l u x o f the s p e c i e s o f PC t h a t c o n t a i n p a l m i t a t e and o l e a t e . The p o o l s i z e s o f the l i n o l e o y l - and a r a c h i -donyl-PC species were reduced, a l t h o u g h the content of arachidonate i n the t o t a l pool of phospholipids was s t r o n g l y conserved throughout the period of e s s e n t i a l FA d e f i c i e n c y ( 4 0 0 ) . I n f a n t e and K i n s e l l a (287) estimated t h a t the r a t e of PC s y n t h e s i s was e n h a n c e d 3 . 8 - f o l d d u r i n g e s s e n t i a l FA d e p r i v a t i o n , and t h i s was c o r r e l a t e d w i t h a 3 . 5 - f o l d e l e v a t i o n o f c h o l i n e kinase a c t i v i t y . A s m a l l e r s t i m u l a t i o n ( 1 . 5 - f o l d ) o f r a t l i v e r c h o l i n e kinase a c t i v i t y has been found by o t h e r s (401). Infante and K i n s e l l a s t a t e d t h a t the 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 a c t i v i t y was unchanged by the d i e t , but the m i c r o s o m a l enzyme a c t i v i t y was not i n v e s t i g a t e d . E s s e n t i a l FA d e f i c i e n c y has been shown to e l e v a t e 1 . 6 - f o l d the pool s i z e of CDP-choline i n r a t s k e l e t a l muscle (267) and t h i s i n c r e a s e could be due to an a c c e l e r a -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 c a t a l y z e d r e a c t i o n or i n h i b i t i o n of c h o l i n e -phosphotransferase . Nakamura et a l . (401) have shown t h a t a l i n o l e a t e d e f i c e n t d i e t p r oduces a s i g n i f i c a n t d e c r e a s e i n d i p a l m i t o y l PC l e v e l i n r a t l u n g . A p p a r e n t l y none o f t h e d_e novo PC b i o s y n t h e t i c enzymes' ji_n v i t r o a c t i v i t i e s were a f f e c t e d by the d e f i c i e n t s t a t e . The e f f e c t of e s s e n t i a l FA d e p r i v a t i o n on PE N-methylation has not been p u r s u e d . However, t r a n s m e t h y l a t i o n may be enhanced s i n c e d e l e t i o n o f e s s e n t i a l FA from the d i e t r e s u l t s i n a 1.5-fold increase i n r a t hepatic PE 87 s y n t h e s i s (400). The s t i m u l a t i o n o f PE s y n t h e s i s c o r r e l a t e d with a 1.5-fold e l e v a t i o n of ethanolamine k i n a s e and phosphoethanolamine c y t i d y l y l t r a n s -f erase a c t i v i t i e s . C h o l i ne D e p r i v a t i o n (1.5.5.2). C h o l i n e d e f i c i e n c y i n the r a t s h o u l d be a u s e f u l model system f o r s t u d y i n g the c o o r d i n a t e c o n t r o l o f PC s y n t h e s i s . A p r i o r i , under these c i r c u m s t a n c e s an i n h i b i t i o n o f d^ e novo s y n t h e s i s and a c o m p e n s a t i n g i n c r e a s e i n PE methylation might be expected. This turns out to be so. The removal of c h o l i n e from the d i e t depletes the l e v e l of f r e e c h o l i n e i n r a t l i v e r by up to 70% a f t e r one week (159). However, a f t e r o n ly two days of c h o l i n e d e f i c i e n c y , the phosphocholine l e v e l s are 70% lower than found i n choline-supplemented r a t s and the h e p a t i c concentrations o f betaine are reduced by 98% (402). E v e n t u a l l y the l i v e r s of c h o l i n e - d e f i c i e n t r a t s assume an enlarged, yellow appearance. The amount of PC i s reduced i n these f a t t y l i v e r s , but the c o n c e n t r a t i o n s of PE and e s p e c i a l l y TG are e l e v a t e d (403-406). The impairment of hepatic l i p i d r e l e a s e i s b e l i e v e d to a r i s e from a r e d u c t i o n i n those species of PC which are r e q u i r e d for the assembly and t r a n s p o r t of plasma l i p o p r o t e i n s (159). The a c t i v i t i e s o f t h e d_e novo b i o s y n t h e t i c enzymes have been measured by Schneider and Vance (297) i n r a t s which were maintained on a c h o l i n e f r e e d i e t f o r up t o 18 d a y s . C h o l i n e k i n a s e and cholinephospho-t r a n s f e r a s e a c t i v i t i e s were unchanged t h r o u g h o u t the s t u d y . Skurdal and Cornatzer (407,408) have r e p o r t e d an e l e v a t i o n of hepatic cholinephospho-t r a n s f e r a s e a c t i v i t y w i t h c h o l i n e d e p r i v a t i o n , but the r e s u l t of Schneider and Vance has been confirmed by o t h e r s (331,409). The a c t i v i t y of c y t o s o l i c c y t i d y l y l t r a n s f e r a s e was found t o be reduced a f t e r one day o f c h o l i n e d e f i c i e n c y and decreased to 63% of c o n t r o l v a l u e s by the second day (297). 88 Choy e_t a l . (43) d e m o n s t r a t e d by i m m u n o c h e m i c a l t i t r a t i o n t h a t t h e amount o f c y t i d y l y l t r a n s f e r a s e i n l i v e r c y t o s o l s from c o n t r o l and c h o l i n e -d e f i c i e n t r a t s was the same. S t i l l , i f the reduced 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 n c y t o s o l was due t o a r e d u c t i o n i n c o n t a m i n a t i n g microsomal a c t i v i t y , t h i s t e c h n i q u e would not be s e n s i t i v e enough t o detect i t . For example, i f the microsomal a c t i v i t y were reduced by 40% a f t e r t r a n s l o c a t i o n of the enzyme, then the c y t o s o l i c a c t i v i t y measured i n the presence of phos-p h o l i p i d liposomes would increase by only 7%. Thomas et^ a l . (402) d i d not f i n d any changes i n the a c t i v i t i e s of c h o l i n e dehydrogenase and b e t a i n e a l d e h y d e dehydrogenase with short-term c h o l i n e d e f i c i e n c y . However, Schneider and Vance (297) were able to detect a 42% r e d u c t i o n i n the m i t o c h o n d r i a l o x i d a t i o n o f c h o l i n e to bet a i n e a f t e r four days of d i e t a r y omission of c h o l i n e . H a ines and Rose (403) have shown t h a t c h o l i n e d e f i c i e n c y i n r a t s i n c r e a s e s the h e p a t i c l e v e i s o f e t h a n o l a m i n e , phosphoethanolamine, CDP-ethanolamine and PE. A subsequent s t u d y (315) confirmed and extended these o b s e r v a t i o n s . When r a t s w e r e a d m i n i s t e r e d p o r t a l i n j e c t i o n s o f 1 4 [ 1 , 2 - c ^ l ^ h a n o l a m i n e , the l a b e l was much h i g h e r i n e t h a n o l a m i n e and phosphoethanolamine, but much l o w e r i n CDP-ethanolamine and PE i n the c h o l i n e - d e f i c i e n t r a t s . The reduced l a b e l l i n g of CDP-ethanolamine and PE was due to d i l u t i o n of the r a d i o a c t i v e i s o t o p e i n t o the l a r g e r ethanolamine and p h o s p h o e t h a n o l a m i n e p o o l s ( 3 1 5 ) . When t h e d_e novo enzymes f o r PE b i o s y n t h e s i s were a s s a y e d , the i j i v i t r o a c t i v i t i e s were u n a l t e r e d by c h o l i n e d e f i c i e n c y ( 2 9 7 ) . The increased h e p a t i c p r o d u c t i o n o f PE i n the c h o l i n e d e f i c i e n t r a t p r o v i d e s more s u b s t r a t e f o r PE m e t h y l a t i o n . W e l l s and Remy (159) have 14 r e p o r t e d t h a t [Me- C] m e t h i b n i n e i n c o r p o r a t i o n i n t o l i v e r p h o s p h o l i p i d 89 i s enhanced by c h o l i n e d e p r i v a t i o n . Moreover, increased PE m e t h y l t r a n s f e r -ase a c t i v i t y w i t h c h o l i n e d e f i c i e n c y has been noted by s e v e r a l i n v e s t i g a t o r s 1 4 ( 2 9 7 , 4 0 7 ) . However, i n o t h e r s t u d i e s , t h e i n c o r p o r a t i o n of [Me- C ] -14 32 methionine (405,410), [ C ] e t h a n o l a m i n e (411) and [ P]phosphate (410) imply t h a t PE m e t h y l a t i o n i s reduced a f t e r removal o f c h o l i n e from the d i e t . Since the s p e c i f i c r a d i o a c t i v i t i e s o f the i n t e r m e d i a t e p r e c u r s o r s were not d e t e r m i n e d i n t h e s e s t u d i e s , t h e s e f i n d i n g s are d i f f i c u l t t o i n t e r p r e t . Recently, Pascale et a l . (411b) examined l i p o p r o t e i n s e c r e t i o n by hepatocytes i s o l a t e d from choline-supplemented and c h o l i n e - d e f i c i e n t r a t s . The s e c r e t i o n of t r i g l y c e r i d e i n l i p o p r o t e i n s was impaired i n the hepato-cy t e s from c h o l i n e - d e f i c i e n t r a t s . However, a d d i t i o n of Ado-Met to the cho-l i n e d e f i c i e n t c e l l s r e stored l i p o p r o t e i n s e c r e t i o n . The supplementation of 3 c h o l i n e - d e f i c i e n t r a t s with Ado-Met enhanced the appearance of [ ] e t h a n o l -amine i n t o membrane PC by 7.4-fold and i n t o l i p o p r o t e i n PC by 3 3-fold. Although c y t i d y l y l t r a n s f e r a s e and PE m e t h y l t r a n s f e r a s e appear to be t a r g e t enzymes f o r c o o r d i n a t e r e g u l a t i o n of PC s y n t h e s i s d u r i n g c h o l i n e d e f i c i e n c y , the a c t u a l mechanisms by which t h e s e enzymes are c o n t r o l l e d remain e l u s i v e . I I 90 C h o l i n e - M e t h i o n i n e D e p r i v a t i o n (1-5.5.3). Hoffman e t a l . (277,331 ) have f u r t h e r s t r a i n e d the c a p a c i t y o f r a t s to synthesize PC by f e e d i n g them a s y n t h e t i c L-amino ac i d d i e t , f r e e of c h o l i n e , methionine, v i t a m i n B and f o l i c a c i d , and supplemented with the methyl group a c c e p t o r g u a n i d o a c e t i c a c i d . The v i t a m i n B and f o l i c a c i d were excluded to p r e v e n t c o n v e r s i o n o f c e l l u l a r homocysteine back to methionine. The severe c h o l i n e - m e t h i o n i n e d e f i c i e n t d i e t , even a f t e r o n l y one day, produced a 38% i n h i b i t i o n o f c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y (331)• A f t e r two week on t h i s d i e t , the cholinephosphotransferase a c t i v i t y was reduced by 86%, w h i l e the PE m e t h y l t r a n s f e r a s e a c t i v i t y was e l e v a t e d 2.2-fold (277). U n f o r t u n a t e l y , 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 not examined i n these s t u d i e s . In o r d e r t o a s s e s s the r a t e o f PE ^ - m e t h y l a t i o n i r i v i v o , 14 day choline-methionine deprived r a t s were g i v e n an i n t r a p e r i t o n e a l i n j e c t i o n of 1 4 [ 1 , 2 - C^lethanolamine 3 h p r i o r t o s a c r i f i c e ( 2 7 7 ) . A l t h o u g h the s p e c i f i c r a d i o a c t i v i t y of the h e p a t i c PE pools were the same i n the c o n t r o l and m e t h y l - d e f i c i e n t r a t s , the i n c o r p o r a t i o n of l a b e l i n t o PC was almost 40% lower i n the d i e t a r y - d e f i c i e n t r a t s . Hoffman ejt a l . (277) a l s o examined the Ado-Met :Ado-Hcy r a t i o s i n the same s t u d y and found the r a t i o was 67% l o w e r i n t h e l i v e r s o f c h o l i n e - m e t h i o n i n e d e f i c i e n t r a t s . T h ese i n v e s t i g a t o r s concluded that the apparent s t i m u l a t i o n of the PE m e t h y l t r a n s -f e r a s e a c t i v i t y i j i v i t r o m i g h t be due t o t h e i n c r e a s e d amount o f PE (1.7-fold) i n the microsomes from the d i e t a r y d e p r i v e d animals. However, i n v i v o PE m e t h y l a t i o n was i n h i b i t e d by the d e p l e t i o n of Ado-Met (46% decrease) and accumulation of Ado-Hcy ( 1 . 7 - f o l d i n c r e a s e ) . S t a r v a t i o n ( 1 . 5 . 5 . 4 ) . The i n f l u e n c e of complete food d e p r i v a t i o n on r a t hepatic PC s y n t h e s i s 91 has l a r g e l y been studi e d by Groener and van Golde (272,273). A 24 and 48 h f a s t were found t o reduce the h e p a t i c p o o l s i z e of PC by 15 and 39%, r e s p e c t i v e l y (272). The f a t t y acid composition of PC i n r a t s fasted f or 48 h was not d r a s t i c a l l y a l t e r e d , a l t h o u g h the p e r c e n t a g e of l i n o l e a t e species was decreased by 26% (273). Evidence t h a t the anabolism of PC was i n f l u e n c e d by s t a r v a t i o n has been provided by p u l s e - l a b e l l i n g s t u d i e s ijn v i v o and i n i s o l a t e d r a t hepato-3 c y t e s . F o l l o w i n g i n t r a v e n o u s i n j e c t i o n o f [ 2 - H ] g l y c e r o l i n t o 24 and 48 h fas t e d r a t s , the i n c o r p o r a t i o n of l a b e l i n t o l i v e r PC was reduced by 38 and 47%, r e s p e c t i v e l y (272). S i m i l a r l y , the i n c o r p o r a t i o n of t h i s same r a d i o -l a b e l l e d compound i n t o PC was reduced by 54% by hepatocytes i s o l a t e d from 48 h f a s t e d r a t s compared t o h e p a t o c y t e s o b t a i n e d from fed a n i m a l s . These s t u d i e s w i t h g l y c e r o l as the r a d i o l a b e l l e d precursor, however, are s l i g h t l y suspect s i n c e h e p a t o c y t e s i s o l a t e d from 24 h f a s t e d r a t s have 62% l e s s g l y c e r o l - 3 - p h o s p h a t e than c e l l s from f e d a n i m a l s ( 4 1 2 ) . T h e r e f o r e , the s p e c i f i c r a d i o a c t i v i t y of g l y c e r o l - 3 - p h o s p h a t e may be higher i n hepatocytes 3 from f a s t e d r a t s . C o n sequently, the ap p a r e n t i n h i b i t i o n of [2- H ] g l y c e r o l i n c o r p o r a t i o n i n t o PC with s t a r v a t i o n may underestimate the a c t u a l degree of reduced PC s y n t h e s i s . G r o e n e r e_t a l . ( 2 7 3 ) have m e a s u r e d t h e a c t i v i t i e s o f t h e de  novo PC b i o s y n t h e t i c enzymes i n l i v e r s from 48 h f a s t e d r a t s . C h o l i n e kinase a c t i v i t y was reduced 50% and 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 a c t i v i t y -was decreased by 18% (not s t a t i s t i c a l l y s i g n i f i c a n t ) with s t a r v a t i o n . The s p e c i f i c a c t i v i t y of c h o l i n e p h o s p h o t r a n s f e r a s e was unaltered by s t a r v a t i o n , but the a c t i v i t y expressed as nmol/min/liver was reduced 50%. Although the hepatic pool s i z e o f d i g l y c e r i d e was reduced by 73% a f t e r 48 h s t a r v a t i o n , i t i s u n l i k e l y t h a t PC s y n t h e s i s was s o l e l y r e s t r i c t e d by 92 •the a v a i l a b i l i t y o f d i g l y c e r i d e ( 2 7 3 ) . There was c e r t a i n l y s u f f i c e n t d i g l y c e r i d e f or PE synthesis s i n c e the PE pool s i z e was only reduced by 15% a f t e r 48 h s t a r v a t i o n . Hepatic PC s y n t h e s i s i s probably f a r more s e n s i t i v e to the CTP pool s i z e s i n c e the a v a i l a b i l i t y o f t h i s s u b s t r a t e can l i m i t the c y t i d y l y l t r a n s f e r a s e r e a c t i o n . L e e l a v a t h i and Guynn (413) have reported t h a t the c o n c e n t r a t i o n of CTP i n l i v e r i s decreased by 50% a f t e r 24 h s t a r v a t i o n . Hoffman et a l . (413b) have examined the e f f e c t o f s t a r v a t i o n on the a c t i v i t y of r a t l i v e r PE methy l t r a n s f e r a s e . Assay of the f i r s t and l a s t m e t h y l a t i o n r e a c t i o n s revealed depressed a c t i v i t y (30% decrease) 20-27 h a f t e r the food was removed. Methyltransferase a c t i v i t y p a r t i a l l y recovered by 68-72 h. P a r a l l e l changes i n the hepatic Ado-Met/Ado-Hcy r a t i o s were detected throughout the 72 h s t a r v a t i o n p e r i o d . Hence, during f a s t i n g the meth y l a t i o n of PE may be in f l u e n c e d by the r e l a t i v e concentrations of Ado-Met and Ado-Hcy i n r a t l i v e r . 93 HORMONAL STUDIES E s t r o g e n s ( 1 . 5 . 6 . 1 ) . The l i v e r i s a major extra g e n i t a l t a r g e t organ f o r endogenous sex s t e r o i d s , and many of i t s e s s e n t i a l f u n c t i o n s are a f f e c t e d by these substan-ces. The i n f l u e n c e o f e s t r o g e n s on PC m e t a b o l i s m has been i n v e s t i g a t e d almost e x c l u s i v e l y i n male an i m a l s s i n c e the female counterparts normally produce l a r g e q u a n t i t i e s o f e s t r o g e n s . N a t o r i ( 4 1 4 ) , Bjornstad and Bremer ( 4 1 5 ) . and Lyman < ; a ( 4 1 6 ) h a v e n o t e d an i n c r e a s e d i_n v i v o 3 i n c o r p o r a t i o n o f r a d i o - l a b e l from [Me- H ] m e t h i o n i n e i n t o the hepatic PC of female as opposed to male r a t s . Lyman e t a l . ( 3 2 1 ) r e p o r t e d a s i m i l a r 3 i n c r e a s e i n [^ 1e_- H ] m e t h i o n i n e i n c o r p o r a t i o n i n t o l i v e r PC a f t e r a d m i n i s t r a t i o n of e s t r a d i o l t o c a s t r a t e d male r a t s . Young (309) found t h a t h e p a t i c PE m e t h y l t r a n s f e r a s e a c t i v i t y was e l e v a t e d 41 and 19% a f t e r a d m i n i s t r a t i o n o f e s t r a d i o l t o c a s t r a t e d and n o r m a l male r a t s , r e s p e c t i v e l y . In the same study, e s t r a d i o l treatment reduced cholinephospho-t r a n s f e r a s e a c t i v i t y 15% i n c a s t r a t e d and 24% i n normal male r a t s . Hence i n female and e s t r a d i o l - t r e a t e d male r a t s , an i n c r e a s e d p r o p o r t i o n o f the hepatic PC may be synthesized by t r a n s m e t h y l a t i o n of PE. This c o n c l u s i o n i s f u r t h e r s u b s t a n t i a t e d by the observation that a f t e r e s t r a d i o l a d m i n i s t r a t i o n there i s a red u c t i o n i n the l i n o l e i c s p e c i e s of PC and an increase i n long chain polyunsaturated f a t t y acid species of PC (321). In the e a r l y 1940's, s e v e r a l i n v e s t i g a t o r s (417, 418) reported t h a t i n j e c t i o n o f s e x u a l l y immature c h i c k e n s w i t h e s t r o g e n s or s y n t h e t i c estrogens r e s u l t e d i n markedly i n c r e a s e d c o n c e n t r a t i o n s of plasma phospho-32 l i p i d s and e n h a n c e d i n c o r p o r a t i o n o f [ P ] p h o s p h a t e i n t o t h e s e p h o s p h o l i p i d s . These f i n d i n g s were c o n f i r m e d by Vigo and Vance (266) who 94 found t h a t d i e t h y l s t i l b o e s t r o l (DES) a d m i n i s t r a t i o n to 7 day o l d r o o s t e r s 3 s t i m u l a t e d [Me- H ] c h o l i n e i n c o r p o r a t i o n i n t o h e p a t i c PC by up t o 3.4-f o l d . The apparent s t i m u l a t i o n of PC s y n t h e s i s i n cockerels t r e a t e d with DES f o r 2 days could be c o r r e l a t e d w i t h a 2 . 4 - f o l d s t i m u l a t i o n of l i v e r c h o l i n e k i n a s e and a 2 - f o l d i n c r e a s e i n the i n t r a c e l l u l a r phosphocholine c o n c e n t r a t i o n (266). Apparently i n c h i c k e n l i v e r , the pool s i z e of phospho-c h o l i n e r e s t r i c t s the r a t e of PC s y n t h e s i s . The i n c r e a s e d c h o l i n e kinase a c t i v i t y i n the D E S - t r e a t e d r o o s t e r l i v e r s was due t o a d o u b l i n g of the amount of enzyme p r o t e i n (14). 3 A f t e r 3 days t r e a t m e n t w i t h DES, t h e i n c o r p o r a t i o n of [Me- H ] -c h o l i n e i n t o hepatic PC was reduced by 61% (266). Hence prolonged exposure t o DES p r o b a b l y i n h i b i t e d c[e novo PC s y n t h e s i s i n r o o s t e r l i v e r . The i n h i b i t i o n was l i k e l y due to the 50% r e d u c t i o n i n 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 a c t i v i t y . Cholinephosphotransferase a c t i v i t y was unaltered by the DES treatment, while PE m e t h y l t r a n s f e r a s e a c t i v i t y was elevated 2 - f o l d a f t e r 3 days treatment with DES. The i n f l u e n c e of DES on r a d i o a c t i v e ethanolamine or methionine i n c o r p o r a t i o n i n t o PC was not examined. While' DES i n h i b i t s c h i c k e n l i v e r c y t o s o l i c c y t i d y l y l t r a n s f e r a s e , 1 7 - b e t a - e s t r a d i o l has been shown t o s t i m u l a t e f e t a l r a b b i t lung 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 by a p p r o x i m a t e l y 64% ( 2 6 5 , 2 9 5 ) . T h i s e s t r a d i o l d e r i v a t i v e had no a p p a r e n t e f f e c t on r a b b i 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 a c t i v i t y (295). A d m i n i s t r a t i o n of 1 7 - b e t a - e s t r a d i o l to pregnant r a b b i t s at 25 days g e s t a t i o n produced a 1 . 6 - 2 - f o l d i n c r e a s e i n the rate of r a d i o a c t i v e c h o l i n e i n c o r p o r a t i o n i n t o the PC o f f e t a l l u n g s l i c e s (265,295). Maternal treatment with 1 7 - b e t a - e s t r a d i o l a l s o reduced the phosphocholine pool s i z e by 50% while the CDP-choline pool s i z e was increased by 46% i n r a b b i t f e t a l lungs (26 days g e s t a t i o n ) ( 2 6 5 ) . Thus the c y t i d y l y l t r a n s f e r a s e c a t a l y z e d 95 r e a c t i o n was a c c e l e r a t e d i n the 1 7 - b e t a - e s t r a d i o l - t r e a t e d f e t u s e s , and de  novo PC synth e s i s was a l s o s t i m u l a t e d s i n c e t h e pulmonary pool s i z e of PC was increased by 20?. G l u c o c o r t i c o i d s (1.5.6.2). The i n f l u e n c e o f g l u c o c o r t i c o i d s on PC s y n t h e s i s has been p r i m a r i l y examined i n the d e v e l o p i n g l u n g . Maternal treatment with c o r t i c o s t e r i o d s has been reported to promote s u r f a c t a n t p r o d u c t i o n and pulmonary maturation i n the f e t u s (346, 419-422). The r a t e o f r a d i o a c t i v e c h o l i n e i n c o r p o r a t i o n i n t o the PC o f f e t a l r a b b i t l u n g s l i c e s (353) and r a t l u n g a l v e o l a r t y p e I I c e l l s (354) was a c c e l e r a t e d by a d d i t i o n of C o r t i s o l , and a 22% i n c r e a s e i n f e t a l r a b b i t l u 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 c o u l d be d e t e c t e d a f t e r t r e a t m e n t w i t h C o r t i s o l ( 4 2 3 ) . A d m i n i s t r a t i o n o f betamethasone t o the p r e g n a n t r a b b i t does a l s o s t i m u l a t e d r a d i o a c t i v e c h o l i n e i n c o r p o r a t i o n i n t o the PC o f f e t a l l u n g s l i c e s ( 3 38,424), and produced a 50% increase 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 , although c h o l i n e kinase a c t i v i t y was s l i g h t l y d e p r e s s e d ( 3 3 8 ) . Dexamethasone treatment of f e t a l r a t l u n g i n organ c u l t u r e s i m i l a r l y e l e v a t e d 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 (425) and increased r a d i o a c t i v e c h o l i n e and palmitate i n c o r p o r a t i o n i n t o PC (426,427). C h o l i n e p h o s p h o t r a n s f e r a s e has been r e p o r t e d t o be st i m u l a t e d by g l u c o c o r t i c o i d s i n some s t u d i e s (343. 346,419), but not i n others (338,353,423,424). The mechanism by which g l u c o c o r t i c o i d s s t i m u l a t e the c y t i d y l y l t r a n s -ferase c a t a l y z e d r e a c t i o n and pulmonary PC s y n t h e s i s i s unclear. However, g l u c o c o r t i c o i d s have been shown t o e l e v a t e glycerophosphate p h o s p h a t i d y l -t r a n s f e r a s e a c t i v i t y i n r a b b i t f e t a l l u n g (353.428-430) and increased production of PG could e x p l a i n the a c t i v a t i o n of c y t i d y l y l t r a n s f e r a s e (34). Melby et a l . (430b) have noted d e c r e a s e d PC l e v e l s (25%) i n l i v e r 96 microsomes from adrenalectomized c o r t i s o l - t r e a t e d r a t s . The f a t t y a c i d com-p o s i t i o n of PC was a l t e r e d . P a l m i t a t e , o l e a t e and l i n o l e a t e were in c r e a s e d , whereas s t e a r a t e and arachidonate were decreased i n the adrenalectomized, c o r t i s o l - t r e a t e d r a t s . T h y r o x i n e ( 1 . 5 . 6 . 3 ) . The production o f s u r f a c t a n t by the f e t a l l u n g can be stim u l a t e d by th y r o x i n e (346,131,432) and t h i s hormone has been used c l i n i c a l l y f o r the preventi o n of R e s p i r a t o r y D i s t r e s s Syndrome (433) . The mechanism through which t h y r o x i n e a c c e l e r a t e s the s y n t h e s i s o f s u r f a c t a n t has not yet been e l u c i d a t e d . A l v e o l a r type I I c e l l s p o s s e s s t h y r o i d hormone receptors (434), but exposure of i s o l a t e d r a t l u n g a l v e o l a r t y p e I I c e l l s to thyroxine d i d not a f f e c t the f o r m a t i o n o f t o t a l o r d i s a t u r a t e d PC as judged w i t h r a d i o - l a b e l l e d precursors (354). 97 I n s u l i n (1 .5.6.4). I n s u l i n c l e a r l y p r o m o t e s g l y c o g e n e s i s , g l y c o l y s i s , f a t t y a c i d s y n t h e s i s , f a t t y a c i d e s t e r i f i c a t i o n and c h o l e s t e r o g e n e s i s i n the l i v e r , although the molecular mechanisms by w h ich i n s u l i n e x e r t s i t s a c t i o n s are p o o r l y d e f i n e d . I n s u l i n ' s i n f l u e n c e on t r i g l y c e r i d e , PC and PE formation i n i s o l a t e d r a t h e p a t o c y t e s has been b r i e f l y examined by Geelen e_t a l . (300). I n s u l i n d i d not s i g n i f i c a n t l y a f f e c t the synthesis of g l y c e r o l i p i d s 14 from r a d i o a c t i v e g l u c o s e or g l y c e r o l , but the i n c o r p o r a t i o n of [ 1 - C ] -acetate i n t o PC was i n c r e a s e d 1 . 4 - f o l d . The e f f e c t of i n s u l i n on e i t h e r r a d i o - l a b e l l e d c h o l i n e or ethanolamine i n c o r p o r a t i o n i n t o hepatic PC has not been i n v e s t i g a t e d . However, Sharp e_t a l . (434b) have reported t h a t 14 i n s u l i n s t i m u l a t e d [ C j c h o l i n e i n c o r p o r a t i o n i n t o the PC of primary e p i -t h e l i a l c e l l c u l t u r e s . Exposure of r a t 3A2 l i v e r c e l l s t o i n s u l i n f o r 18 h has been shown to double c h o l i n e kinase a c t i v i t y (434c). The i n c r e a s e i n s p e c i f i c a c t i v i t y was due to the s y n t h e s i s of new enzyme r a t h e r than a c t i v a t i o n of p r e e x i s t i n g c h o l i n e k i n a s e . Glucagon ( 1 . 5 . 6 . 5 ) . Under c o n d i t i o n s of food d e p r i v a t i o n , g l u c a g o n i s r e l e a s e d from the a l p h a - c e l l s of the pancreas and the l i v e r i s the p r i m a r y t a r g e t organ of t h i s hormone. Glucagon s t i m u l a t e s g l y c o g e n o l y s i s , gluconeogenesis and f a t t y a c i d o x i d a t i o n i n the l i v e r and a c t s i n o p p o s i t i o n t o the a c t i o n s o f i n s u l i n . The i n f l u e n c e of glucagon on the i n c o r p o r a t i o n o f r a d i o a c t i v e p r e c u r s o r s i n t o the p h o s p h o l i p i d s o f i s o l a t e d r a t h e p a t o c y t e s has been i n v e s t i g a t e d by G e e l e n e_t a l . ( 3 0 0 , 4 3 5 ) . G l u c a g o n s i g n i f i c a n t l y 14 14 decreased [U- C ] g l u c o s e and [ 1 - C ] a c e t a t e i n c o r p o r a t i o n , whereas the 14 entry of [2- C ] g l y c e r o l i n t o PC was a c t u a l l y s t i m u l a t e d 1.7-fold (300). 98 32 In the subsequent s t u d y ( 4 3 5 ) , g l u c a g o n was shown t o reduce [ P ] -14 phosphate and [M£- C ] c h o l i n e i n c o r p o r a t i o n i n t o PC, a l t h o u g h t he i n h i b i t i o n o f r a d i o a c t i v e c h o l i n e e n t r y i n t o PC was not s t a t i s t i c a l l y s i g n i f i c a n t . Even though the p o o l s i z e s o f the c h o l i n e metabolites and the de novo p a t h w a y e nzyme a c t i v i t i e s w e r e n o t d e t e r m i n e d , t h e s e i n v e s t i g a t o r s concluded t h a t d i g l y c e r i d e i s p r e f e r e n t i a l l y channelled i n t o p h o s p h o l i p i d s y n t h e s i s a t the expense of t r i g l y c e r i d e f o r m a t i o n under a glucagon l o a d . G e e l e n e_t £_l_i. (**35) f o u n d t h a t g l u c a g o n s t i m u l a t e d t h e 14 i n c o r p o r a t i o n of [1,2- C ] e t h a n o l a m i n e i n t o PC by 1.3-fold. However, a s i m i l a r i n c r e a s e i n the entry o f t h i s r a d i o a c t i v e compound i n t o PE was a l s o d e t e c t e d . Hence the ap p a r e n t r i s e i n PE m e t h y l a t i o n when the hepatocytes were exposed to glucagon c o u l d be e x p l a i n e d by an increase i n the s p e c i f i c r a d i o a c t i v i t y o f PE. G l u c a g o n has a l s o been r e p o r t e d t o i n c r e a s e 3 [Me- H ] m e t h i o n i n e i n c o r p o r a t i o n i n t o t h e p h o s p h o l i p i d s of f r e s h l y i s o l a t e d mouse h e p a t o c y t e s ( 4 3 6 ) . Castano e t a l . (102) have observed a doubling of PE m e t h y l t r a n s f e r a s e a c t i v i t y i n homogenates prepared from r a t h e p a t o c t y e s exposed t o p h y s i o l o g i c a l c o n c e n t r a t i o n s o f g l u c a g o n . T h i s a c t i v a t i o n was due to an i n c r e a s e i n the V of the PE methyltransferase max c a t a l y z e d r e a c t i o n w i t h o u t a f f e c t i n g t h e a f f i n i t y o f the enzyme f o r Ado-Met. C o l l e c t i v e l y , t h e s e s t u d i e s p o i n t t o a s t i m u l a t i o n of hepatic PE t r a n s m e t h y l a t i o n i n response to glucagon. cAMP and P r o t e i n P h o s p h o r y l a t i o n (1.5.6.6). Although cAMP i s the secondary messenger f o r glucagon i n the l i v e r , i t a l s o serves as an i n t r a c e l l u l a r messenger f o r numerous hormones throughout the body. Since cAMP does not r e a d i l y c r o s s the plasma membrane, s y n t h e t i c a n a l o g u e s o f cAMP have been used t o i n v e s t i g a t e the i n f l u e n c e of t h i s 99 compound on c e l l u l a r metabolism. A l t e r n a t i v e l y , drugs such as aminophylline which s p e c i f i c a l l y i n h i b i t cAMP p h o s p h o d i e s t e r a s e a c t i v i t y have been used to boost i n t r a c e l l u l a r cAMP l e v e l s (437). To date, the brunt of the i n f o r m a t i o n a v a i l a b l e about cAMP r e g u l a t i o n of PC me t a b o l i s m has been r e s t r i c t e d t o f e t a l l u n g development. Gluco-c o r t i c o i d s and t h y r o x i n e (see s e c t i o n s 1.5.6.2 & 1.5.6.3) have been shown to enhance s u r f a c t a n t production. In o t h e r t i s s u e s , g l u c o c o r t i c o i d s e l e v a t e cAMP c o n c e n t r a t i o n s by i n h i b i t i o n o f cAMP p h o s p h o d i e s t e r a s e (438 - 441), w h i l e t h y r o x i n e does so by s t i m u l a t i o n o f a d e n y l a t e c y c l a s e ( 4 4 2 ) . Therefore, i t i s not unexpected t h a t a d m i n i s t r a t i o n o f a m i n o p h y l l i n e to preg n a n t r a b b i t s a c c e l e r a t e s f e t a l l u n g m a t u r a t i o n ( 4 2 0 , 4 4 3 ) . The p h o s p h o l i p i d c o n t e n t o f l u n g t i s s u e h o m o g e n a t e was e l e v a t e d by approximately 14% from a m i n o p h y l l i n e - t r e a t e d f e t u s e s (420,443). F u r t h e r -14 more, [ C]choline i n c o r p o r a t i o n i n t o f e t a l l u n g PC was sti m u l a t e d by 26% a f t e r maternal a d m i n i s t r a t i o n of aminophylline (420). Gross and Rooney (312) found t h a t a m i n o p h y l l i n e and d i b u t y r y l - c A M P 3 s e p a r a t e l y s t i m u l a t e d a b o u t 1 . 8 - f o l d t h e i n c o r p o r a t i o n o f [Me- H ] -c h o l i n e i n t o the p h o s p h o l i p i d o f e x p l a n t s o f f e t a l r a t l u n g i n organ c u l t u r e . The a c t i v i t i e s of c h o l i n e k i n a s e and cholinephosphotransferase i n the explants were not s i g n i f i c a n t l y a l t e r e d by exposure to aminophylline (312). N i l e s and Makarski (444) have c o r r e l a t e d the a b i l i t y of a v a r i e t y of 3 cAMP a n a l o g u e s t o s t i m u l a t e [Me_- H ] c h o l i n e e n t r y i n t o t h e PC o f a l v e o l a r type I I c e l l s (A549 c e l l s ) w i t h t h e i r a b i l i t y t o a c t i v a t e d cAMP-dependent p r o t e i n kinase i n these c e l l s . A c t i v a t i o n of p r o t e i n k i n a s e by cAMP i s the p r i n c i p a l means by which glucagon and b e t a - a d r e n e r g i c drugs promote p h o s p h o r y l a t i o n of r e g u l a t o r y enzmes. In g e n e r a l , c A M P - m e d i a t e d p r o t e i n p h o s p h o r y l a t i o n has been 100 a s s o c i a t e d with a s t i m u l a t i o n o f b i o d e g r a d a t i v e pathways and an i n h i b i t i o n of b i o s y n t h e t i c pathways. As d e t a i l e d i n F i g u r e 6, a growing number of enzymes are a p p a r e n t l y r e g u l a t e d by t h i s form o f r e v e r s i b l e c o v a l e n t m o d i f i c a t i o n . I t i s e n t i r e l y r e a s o n a b l e t o exp e c t t h a t p h o s p h o l i p i d metabolism may a l s o be s u b j e c t e d t o t h i s type o f c o n t r o l . A s u b s t a n t i a l p o r t i o n o f t h i s t h e s i s has been d e v o t e d t o d e f i n i n g the i m p o r t a n c e o f p r o t e i n phosphorylation f o r r e g u l a t i o n of hepatic PC s y n t h e s i s . g l u c o s e — * G 6 P G XT GLYCOGEN UDP-glu triose-P- >G3P glycerol cholesterol esters AcCoA*fc- ... k '^carnitine 7 « - O H cholesterol bile salts CHOLESTEROL j citrate PEP ,acyl i I 'd I III l ] br 11 mevalonate citrate H MG-CoA«— AcCo A ^ > ma I-CoA CDP-cho pho-cho Figure 6. E s t a b l i s h e d and s u s p e c t e d s i t e s o f r e g u l a t i o n by p r o t e i n phos- p h o r y l a t i o n . A b b r e v i a t i o n s : AcCoA, acetyl-Coenzyme A; CDP-cho, CDP-c h o l i n e ; DG, d i a c y l g l y c e r o l ; FA, f a t t y a c i d ; FA-CoA, f a t t y acyl-Coenzyme A; F1.6-P2, fructose-1,6-diphosphate; F6P, fructose-6-phosphate; F2,6-P 2, f r u c -tose-2, 6-diphosphate ; G1P, g l u c o s e - 1 - p h o s p h a t e ; G6P, glucose-6-phosphate ; G3P, glycerol-3-phosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-Coenzyme A; LPA, l y s o p h o s p h a t i d a t e ; mal-CoA, malonyl-Coenzyme A; PA, phosphatidate; PC, p h o s p h a t i d y l c h o l i n e ; PE, phosphatidylethanolamine; pho-cho, phospho-c h o l i n e ; PEP, p h o s p h o e n o l p y r u v a t e ; p y r , p y r u v a t e ; TG, t r i a c y l g l y c e r o l ; t r i o s e - P , triose-phosphate and UDP-glu, UDP-glucose. O, a c t i v a t e d by phos-p h o r y l a t i o n . #, i n h i b i t e d by phosphorylation. ©, both a c t i v a t e d and i n h i b i t e d by p h o s p h o r y l a t i o n . © , s i l e n t phosphorylation i n which the k i n e t i c a c t i v i t y o f the enzyme i s unchanged. 101 The Thesis I n v e s t i g a t i o n s (1.6.1.1). We o r i g i n a l l y used s t a r v a t i o n o f r a t s as a means t o p e r t u r b the hepatic s y n t h e s i s of p h o s p h a t i d y l c h o l i n e so t h a t we could e l u c i d a t e how the pathway i s r e g u l a t e d . F a s t i n g produced changes i n the a c t i v i t i e s o f c h o l i n e kinase and c y t i d y l y l t r a n s f e r a s e , and a l s o i n c r e a s e d t h e l i v e r c y t o s o l i c c o n c e n t r a t i o n o f a 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 a t o r . W h i l e t h e s e s t u d i e s p o i n t e d t o the c y t i d y l y l t r a n s f e r a s e s t e p as a major c o n t r o l s i t e f o r p h o s p h a t i d y l c h o l i n e a n a b o l i s m , i t was d i f f i c u l t t o e s t a b l i s h the p r e c i s e mechanisms which are r e s p o n s i b l e f o r the a l t e r a t i o n s i n c y t i d y l y l t r a n s f e r -ase a c t i v i t y . We e x p l o i t e d the use o f monolayer c u l t u r e s o f r a t hepatocytes to r e s o l v e the f a c t o r s which might i n f l u e n c e p h o s p h a t i d y l c h o l i n e s y n t h e s i s d u r i n g s t a r v a t i o n . Glucagon i s r e l e a s e d i n t o the c i r c u l a t i o n during f a s t i n g and i n c r e a s e s the h e p a t i c l e v e l of cAMP. We found t h a t g l u c a g o n , cAMP analogues and cAMP p h o s p h o d i e s t e r a s e i n h i b i t o r s 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 - c a t a l y z e d r e a c t i o n and p h o s p h a t i d y l c h o l i n e s y n t h e s i s i n the i s o l a t e d h e p a t o c y t e s . F a t t y a c i d s a r e m o b i l i z e d from the a d i p o s e and d e l i v e r e d to the l i v e r a f t e r prolonged f a s t i n g . F a t t y a c i d s s t i m u l a t e the c y t i d y l y l t r a n s f e r a s e r e a c t i o n and p h o s p h a t i d y l c h o l i n e s y n t h e s i s i n the hepatocytes. The i n f l u e n c e o f cAMP and f a t t y a c i d s on the transm e t h y l a t i o n of p h o s p h a t i d y l e t h a n o l a m i n e i n h e p a t o c y t e s was a l s o i n v e s t i g a t e d . F a t t y a c i d s and f a t t y acyl-CoA d i r e c t l y a f f e c t 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 r a t l i v e r c y t o s o l . We a l s o o b t a i n e d e v i d e n c e t h a t 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 a c t i v i t y i s r e g u l a t e d by p r o t e i n p h o s p h o r y l a t i o n . F a t t y a c i d s promote the b i n d i n g of c y t i d y l y l t r a n s f e r a s e to membranes, while phosphory-l a t i o n prevents t h i s i n t e r a c t i o n . The membrane-bound form of c y t i d y l y l -t r a n s f e r a s e probably represents the more a c t i v e form, while s o l u b l e form i n c y t o s o l i s l e s s a c t i v e . S i n c e a t r a n s l o c a t i o n o f c y t i d y l y l t r a n s f e r a s e 102 between the m i c r o s o m a l and c y t o s o l i c f r a c t i o n s o f r a t l i v e r was evident during s t a r v a t i o n , f a t t y a c i d s u p p l e m e n t a t i o n and cAMP analogue treatment, we a l s o measured and d e t e c t e d a r e d i s t r i b u t i o n o f c y t i d y l y l t r a n s f e r a s e d u r i n g development and a d i u r n a l rhythm. S i n c e we had o n l y . s u g g e s t i v e evidence that the c y t i d y l y l t r a n s f e r a s e i s phosphorylated by p r o t e i n k i n a s e , we planned t o demonstrate t h a t c y t i d y l y l t r a n s f e r a s e can be l a b e l e d with 32 32 [ P]phosphate from [gamma- P]ATP. U n f o r t u n a t e l y , t h e a v a i l a b i l i t y of 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 i s a p r e r e q u i s i t e f o r t h i s s o r t o f l a b e l i n g e x p e r i m e n t . Many approaches were e x p l o r e d i n an attempt t o p u r i f y the c y t i d y l y l t r a n s f e r a s e and e n c o u r a g i n g r e s u l t s were o b t a i n e d . However, t h i s enzyme s t i l l remains to be p u r i f i e d to homogeneity. 103 EXPERIMENTAL PROCEDURES MATERIALS Anim a l s ( 2 . 1 . 1 . 1 ) . Wistar r a t s were obtained from the U n i v e r s i t y of B r i t i s h Columbia Animal U n i t . For most s t u d i e s , female r a t s weighing 125-150 g were used rou-t i n e l y . For the developmental study (Section 3-4.1.1), 6 day f i r s t - t i m e pregnant r a t s were s u p p l i e d . Enzymes, Chemicals and R a d i o i s o t o p e s (2.1.1.2) The commercial sources of many of the m a t e r i a l s which were used during t h i s i n v e s t i g a t i o n are l i s t e d i n Table 16. A l l other chemicals used were of reagent grade. GENERAL PREPARATIVE PROCEDURES I s o l a t i o n and C u l t u r e o f A d u l t Rat Hepatocytes (2.2.1.1). Hepatocytes can be rel e a s e d from ad u l t r a t l i v e r s by a collagenase per f u s i o n technique (445). The c e l l s are p u r i f i e d by 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 and can be c u l t u r e d f o r periods i n excess of 7 days on collagen-coated p l a s t i c p e t r i - d i s h e s i n a c u l t u r e medium supplemented with f e t a l c a l f serum and i n s u l i n . P r e p a r a t i o n of c o l l a g e n - c o a t e d c u l t u r e d i s h e s . Reagents: 20 ml V i t r o g e n 100 (3 mg/ml) 104 Table 16. Commercial Sources of Research M a t e r i a l s . S-adenosyl-L-tMe- H]methionine - Amersham i n s u l i n - Sigma ACS f l u i d - Amersham a l k a l i n e phosphatase - Sigma aminophylline - Sigma arachidonate - Sigma ATP - Sigma bovine serum albumin - Calbiochem 8-bromo-cAMP - Sigma cAMP- Sigma cAMP-dependent protein kinases - Sigma carboxymethyl-Sepharose - Pharmacia CDP-choline - Serdary CDP-[Me- 3H]choline - New England Nuclear cGMP - Sigma chlorophenylthio-cAMP - Boehringer Mannheim chlorpromazine - Sigma choline - Sigma [Me- 3H]choline - Amersham choline kinase from yeast - Sigma collagenase type IV - Sigma Contur c u l t u r e dishes - Lux S c i e n t i f i c CTP - Sigma DEAE-Sepharose - Pharmacia dibucaine - Sigma dibutyryl-cAMP - Sigma Dulbecco's modified Eagles medium -Grand Island B i o l o g i c a l Dyematrex resins - Amicon t1- 3H]ethanolamine - Amersham f e t a l c a l f serum - W i l d l i f e Serums glucagon - Sigma [ 3 H ] g l y c e r o l - Amersham A l l the phospholipids were purchased from Serdary. immunoglobulin G - Bio-Rad isobutylmethylxanthine - Sigma l i n o l e a t e - Sigma l i n o l e n a t e - Sigma [Me- 3H]methionine - Amersham oleate - Sigma oleoyl-CoA - Sigma palmitate - Sigma [ 1 4 C ] p a l m i t a t e - Amersham phenylmethylsulfonylfluoride -Sigma DL-propanolol - Sigma protein kinase i n h i b i t o r s - Sigma rat chow - Purina Sepharose 2B and 6B - Pharmacia S i l i c a G60 p l a s t i c plates - Merck sodium f l u o r i d e - Sigma soybean t r y p s i n i n h i b i t o r - Sigma tetracaine - Sigma t r i f l u o p e r a z i n e - Smith, K l i n e and French T r i s - Sigma t r y p s i n - Sigma Vitrogen 100 - Flow Lab. 105 160 ml 0.01 M HC1 The Vitrogen 100 i s bovine s k i n c o l l a g e n . Rat t a i l c o l l a g e n i s b e t t e r o but more expensive. The V i t r o g e n 100 i s s t o r e d at 4 C and can denature at higher temperatures. Procedure: 1) D i l u t e the Vitrogen 10-fold with c o l d 0.01 M HC1 and mix w e l l . 2) Dispense 2 ml i n t o each d i s h (LUX Contur; 60 mm). 3) P a r t i a l l y remove the l i d , and all o w the s o l u t i o n to evaporate i n a flow of f i l t e r e d a i r f o r 12 h under UV l i g h t . 4) When the p l a t e s are dry, replace the l i d s , and use the same day. I s o l a t i o n o f l i v e r c e l l s . S o l u t i o n s : o 100 ml f e t a l c a l f serum ( s t o r e d at -20 C) 100 ml collagenase b u f f e r (Normally 500 ml of the s o l u t i o n are prepared at once and fr o z e n f o r convenience i n 100 ml a l i q u o t s . To 50 ml of 10-times concentrated Hanks s a l t s s o l u t i o n (without HCO^-) are added 0.175 g of glucose ( d e x t r o s e ) , 0.175 g of NaHCO^, 2.975 g of HEPES and 450 ml of water. The pH of the s o l u t i o n i s adjusted to 7.4 with NaOH, and the s o l u t i o n i s f i l t e r e d and stored s t e r i l e i n 100 ml a l i q u o t s . P r i o r to the op e r a t i o n , add 100 mg of collagenase type 1 106 (Sigma) ( i . e . not pure c o l l a g e n a s e ) and i n s u l i n to a conce n t r a t i o n of 10 pg/ml ( i n s u l i n stock s o l u t i o n i s 1 mg/ ml i n water which has been adjusted to pH 2.5 with HC1).) 100 ml p e r f u s i o n b u f f e r (Hanks s a l t s s o l u t i o n w i t h o u t C a + ^ , Mg +^ and HCO^-This s o l u t i o n contains 4.5 g/1 of glucose, 0.5 mM EGTA, 25 mM HEPES and 10 ^ig/ml of i n s u l i n (added p r i o r to the o p e r a t i o n ) . EGTA i s added from a 50 mM stock s o l u t i o n i n d i l u t e d a c e t i c a c i d . ) 600 ml c u l t u r e media (The 100 ml of f e t a l c a l f serum i s "added to 500 ml of MEM (modified Eagle's medium) and 6 ml of i n s u l i n (1 mg/ml)). Modified Eagle's medium i s s p e c i a l l y prepared by Dulbecco - Formula #79-5141. One package of the MEM i s d i s s o l v e d i n t o 5 1 of d i s t i l l e d water with 22.5 g of glucose, 525 mg of l e u c i n e , 550 mg of sodium pyruvate, 150 mg of methionine, 11.9 g of HEPES, 40^1 of 500 mg/ml ch o l i n e c h l o r i d e , 335 mg of o r n i t h i n e and f i n a l l y 18.5 g NaHCO^. The p e r f u s i o n b u f f e r i s gassed w i t h 0^, and the c u l t u r e media are gassed with ®2^®2 (95:5) p r i o r t o use. The p e r f u s i o n b u f f e r and collagenase b u f f e r are i n c u b a t e d at 40° C, and the c u l t u r e media kept at 4° C. Procedure: 1) Anesthetize the 100-125 g female Wistar r a t with an i n t r a p e r i t o n e a l i n j e c t i o n of Nembutal (50 mg/ml) (100 pi/ 100 g body weight). 107 2) Place i n flow of s t e r i l e a i r and swab abdomen with e t h a n o l . 3) Turn on heat lamp to maintain body temperature. 4) Make a m i d l i n e i n c i s i o n through the s k i n and cut from thorax to. bladder. 5) Tear back s k i n . 6) Open and cut back abdominal w a l l . 7) Move i n t e s t i n e s to the r i g h t . 8) Place a loose l i g a t u r e around the vena cava above r e n a l v e i n . 9) Place loose l i g a t u r e s around the p o r t a l v e i n . 10) Prime cannula tube and needle w i t h p e r f u s i o n medium. 11) Clamp o f f and cut p o r t a l v e i n . 12) I n s e r t cannula needle with pump running ( f l o w r a t e at 60 s e t t i n g ) . 13) S t a r t c l o c k . 14) Cut vena cava below r e n a l v e i n . 15) Secure p o r t a l v ein cannula t i g h t l y . 16) Turn pump to maximum s e t t i n g f o r 15 sec to f l u s h blood out of the l i v e r . 17) D i s e c t thorax and cut' out r i b cage. 18) Place a loose l i g a t u r e around the vena cava above diaphragm and cut the heart at the r i g h t a t r i a ( l e f t hand s i d e ) . 19) Tie o f f the vena cava above the r e n a l v e i n . 20) Tie o f f the vena cava above the diaphragm. 21) Make 1-2 small cuts at the t i p s of each l i v e r lobe. 22) Perfuse with p e r f u s i o n b u f f e r (continued oxygenation) f o r 4 min. 23) Change p e r f u s i o n to collagenase b u f f e r f o r another 10 min. 24) C a r e f u l l y remove the l i v e r , mince i n a p e t r i d i s h and t r a n s f e r to c e n t r i f u g e tube which a l s o c o n t a i n s 10 ml of collagenase b u f f e r . 108 25) Shake w e l l f o r 5 min a t 37° C i n an 0 2 atmosphere. 26) F i l t e r the c e l l s through a s t e r i l e coarse nylon f i l t e r i n t o c u l t u r e media a t 4° C. 27) Make up i s o l a t e d c e l l suspension to 100 ml with c u l t u r e media. 28) D i v i d e i n t o 2 X 50 ml s t e r i l e c e n t r i f u g e tubes. 29) Spin 1 min a t 90 X £ (min speed). 30) Discard supernatant and wash p e l l e t two more times with c u l t u r e media. 31) F i l t e r c e l l s through a 75 s t e r i l e nylon mesh. 32) Adjust volume of c e l l suspension to 300 ml. 6 33) Count c e l l s i n hemocytometer and a d j u s t to 1 X 10 c e l l s / m l . (The // of c e l l s i n 16 squares X 0.01 = // of m i l l i o n c e l l s / m l ) . 34) Assess c e l l v i a b i l i t y i n presence of 0.04% trypan blue (should exclude the dye a f t e r 5 min). 35) P l a t e out 3 ml per d i s h and place i n incubator f o r 24 h. 109 S u b c e l l u l a r F r a c t i o n a t i o n of Whole Rat L i v e r or I s o l a t e d Hepatocytes (2.2.1.2) Female Wistar r a t s (125-150 g) were d e c a p i t a t e d , and the l i v e r s were immediately removed. The excised l i v e r s were r i n s e d i n i c e - c o l d s a l i n e , cut i n t o pieces and homogenized i n f o u r volumes (ml/g l i v e r ) of i s o t o n i c s a l i n e (0.145 M NaCl) with 7 strokes up and down i n a Potter-Elvehjem homogenizer. The homogenate was c e n t r i f u g e d a t 16,000 X g f o r 10 min, and the post-m i t o c h o n d r i a l supernatant was c e n t r i f u g e d a t 170,000 X g (50,000 rpm i n a Beckman Ti-70 r o t o r ) f o r 60 min. The r e s u l t a n t supernatant was designated the c y t o s o l i c f r a c t i o n . The m i c r o s o m a l p e l l e t was resuspended i n 0.25 M sucrose, 10 mM T r i s - H C l , pH 7.4 with a g l a s s Dounce homogenizer. For p r e p a r a t i o n of microsomal and c y t o s o l i c f r a c t i o n s from i s o l a t e d r a t hepatocytes, the c e l l s from 4 or 5 d i s h e s were pooled i n t o 2 ml of i c e - c o l d 0.145 M N a C l , 10 mM T r i s - H C l , pH 7.4, 20 mM NaF and 2.5 mM EDTA. The suspended c e l l s were broken with 40 . s t r o k e s o f a t i g h t f i t t i n g g l a s s Dounce homogenizer. The homogenate was c e n t r i f u g e d a t 13,000 g f o r 10 min, and the s u p e r n a t a n t was then c e n t r i f u g e d a t 150,000 X g (46,000 rpm i n a Beckman Ti-75 r o t o r ) f o r 60 min. The m i c r o s o m a l p e l l e t was resuspended i n 0.6 ml of 0.25 M sucrose, 10 mM T r i s - H C l , pH 7.4, 20 mM NaF and 2.5 mM EDTA. P a r t i a l P u r i f i c a t i o n o f L-form (2.2.1.3). Fresh l i v e r c y t o s o l from two female r a t s was adjusted to 28% ammonium s u l f a t e s a t u r a t i o n , and the p r e c i p i t a t e was removed by c e n t r i f u g a t i o n (10,000 X g X 10 mi n ) . The s u p e r n a t a n t was a d j u s t e d t o 40% ammonium s u l f a t e , and the p r e c i p i t a t e formed a f t e r 1 h at 4° C was c o l l e c t e d by c e n t r i f u g a t i o n (10,000 X g X 10 min) and resuspended i n 4 ml of Bu f f e r A (100 mM NaCl, 20 mM T r i s - H C l , pH 7 - 0 ) . T h i s sample was a p p l i e d t o a Sepharose 6B column (2.5 X 80 cm) which had been e q u i l i b r a t e d with B u f f e r 110 A and e l u t e d with Buffer A. L-form a c t i v i t y was pooled and concentrated by a d j u s t m e n t o f t h e enzyme s o l u t i o n t o 4 0$ ammonium s u l f a t e . C y t i d y l y l t r a n s f e r a s e when assayed i n the presence of phospholipid liposomes was p u r i f i e d 6-fold from c y t o s o l by t h i s p r o t o c o l . 3 Enzymatic S y n t h e s i s o f [Me-~H]Phosphocholine (2.2.1.4). 3 Choline kinase was used t o g e n e r a t e [Me- H]phosphocholine from 3 [Me- H ] c h o l i n e . The k i n a s e was a p r e p a r a t i o n from baker's y e a s t (Sigma grade II ) . One u n i t o f enzyme was d i s s o l v e d i n 700 pi of d i s t i l l e d water and d i a l y z e d a g a i n s t 20 mM T r i s - H C l , pH 8.0 f o r 3 h at 4° C. The c h o l i n e k i n a s e s o l u t i o n was d i v i d e d i n t o 3 a l i q u o t s which c o u l d be o c o n v e n i e n t l y s t o r e d a t -20 C f o r m o n t h s . Throughout the p r e p a r a t i o n procedure p l a s t i c was used^as much as p o s s i b l e s i n c e phosphocholine had a tendency to bind to g l a s s . Two mCi o f the l a b e l e d c h o l i n e were added to a p l a s t i c t e s t tube and the s o l v e n t e v a p o r a t e d under a stream of n i t r o g e n . T w e n t y - f i v e yl of M g C l 2 (100 mM) , 25 pi of ATP (100 mM) , 10 )Jl o f 1 M T r i s - H C l , pH 8.0, and 190 ^ 1 o f c h o l i n e k i n a s e were added to the tube. The o r e a c t i o n m i x t u r e was i n c u b a t e d at 37 C f o r 1 h, b o i l e d f o r 2 min, and c e n t r i f u g e d at low speed f o r 5 min. The s u p e r n a t a n t was applied as two 4 cm s t r e a k s t o s i l i c a g e l 60 TLC p l a t e s (Brinkman) and developed i n the s o l v e n t s y s t e m CH 'OH/ 0.6% N a C l / NH^ ( 1 0 / 1 0 / 1 ; v / v / v ) . The p h o s p h o c h o l i n e had an Rf of a b o u t 0.23 and m i g r a t e d f u r t h e r than the c h o l i n e . I t was i d e n t i f i e d when 0.5 cm l e n g t h s o f s i l i c a g e l were scraped i n t o d i f f e r e n t t e s t tubes which a l s o c o n t a i n e d 2 ml of water. A l i q u o t s (20 ^jl) were counted f o r r a d i o a c t i v i t y . The s i l i c a t h a t contained r a d i o a c t i v e phosphocholine was e x t r a c t e d w i t h t h r e e X 2 ml o f d i s t i l l e d water and the w a s h i n g s a f t e r c e n t r i f u g a t i o n w e r e p o o l e d . The c o n c e n t r a t i o n o f I l l r a d i o a c t i v i t y was adjusted to 0.3 mCi/ml , and then unlabeled phosphocholine was added so t h a t the f i n a l c o n c e n t r a t i o n was 15 mM. The y i e l d was greater than 80%. Folch L i p i d E x t r a c t i o n (2.2.1.5). I s o l a t e d r a t hepatocytes and whole r a t l i v e r s were e x t r a c t e d by the method o f F o l c h e_t a 1. ( 4 1 6 ) . In t h i s p r o c e d u r e the c e l l s were suspended or the t i s s u e h o m o g e n i z e d i n CH^OH/H^O (1/0.8; v / v ) . The l i p i d s were e x t r a c t e d i n t o the lower phase when CHCl^ was added t o the system. The f i n a l r a t i o s o f CHCl^/CH^OH/h^O were 2/1/0.8 ( v / v / v ) . B l i g h and Dyer L i p i d E x t r a c t i o n (2.2.1.6). For some s t u d i e s , the B l i g h and Dyer (447) procedure was adopted sin c e the i n i t i a l one phase system f a c i l i t a t e d the e x t r a c t i o n of the l y s o l i p i d s . In t h i s method the t i s s u e was homogenized i n d i s t i l l e d water (4 ml/ g of t i s s u e ) and to 0.8 volumes o f the homogenate was added 1 volume of CHC1 and 2 volumes of CH OH. The system was mixed and i n c u b a t e d at 3 3 4° C f o r a p p r o x i m a t e l y 1 h. Then t h e r a t i o s o f CHC1 /CH 0H/H 20 were a d j u s t e d t o 2/1/0.8 ( v / v / v ) . A f t e r m i x i n g , t h e phases were r a p i d l y separated by low speed c e n t r i f u g a t i o n . P r e p a r a t i o n of T o t a l Rat L i v e r P h o s p h o l i p i d (2.2.1.7). Rat l i v e r phospholipids were p a r t i t i o n e d i n t o the lower phase o f the CHC1 / CH OH/ H 0 e x t r a c t i o n system t h a t was d e s c r i b e d i n S e c t i o n 3 3 2 2.2.1.6. The lower phase was t r a n s f e r r e d t o a pre-weighed round bottom f l a s k and d r i e d by r o t a r y f l a s h evaporation'. The f l a s k was flu s h e d with n i t r o g e n , o s t o p p e r e d and s t o r e d a t -20 C f o r 20 min. Most o f the n e u t r a l l i p i d s 112 were e x t r a c t e d when the c o n t e n t s o f the f l a s k were washed with three X 20 ml of i c e - c o l d acetone. The remaining acetone was evaporated under n i t r o g e n , and the temperature o f the f l a s k was brought t o room temperature p r i o r to measurement o f t h e amount o f p h o s p h o l i p i d r e s i d u e i n the f l a s k . The phospholipid was d i s s o l v e d i n c h l o r o f o r m to a f i n a l concentration of 20 o mg/ml and s t o r e d a t -20 C under n i t r o g e n . 113 S a p o n i f i c a t i o n o f L i p i d s (2.2.1.8). 3 The i n c o r p o r a t i o n o f [ H ] a c e t a t e i n t o s a p o n i f i a b l e l i p i d s l a r g e l y r e f l e c t e d the sy n t h e s i s of f a t t y a c i d s i n i s o l a t e d hepatocytes. C e l l s were e x t r a c t e d w i t h 7-6 ml of CHC1 /CH^OH/H^O (2/1/0.8; v / v / v ) . T o t a l r a t l i v e r p h o s p h o l i p i d (0.4 mg) was i n c l u d e d , and the pH of the two phase system was adjusted dropwise to below 2 w i t h 6M HC1 (thymol blue i n d i c a t o r showed p i n k ) . The lower phase was e x t r a c t e d t h r e e t i m e s w i t h pH 2.0 t h e o r e t i c a l upper phase. Two ml of the lower phase were evaporated, and the l i p i d s were r e f l u x e d f o r 1 h at 80° C i n 1 ml of 1 M KOH i n 95% et h a n o l . The s o l u t i o n was c o o l e d p r i o r t o e x t r a c t i o n w i t h t h r e e X 2 ml o f hexane. The hexane washes (upper phase) were pooled and c o n t a i n e d the non-s a p o n i f i a b l e l i p i d s ( l a r g e l y s t e r o l s ) . The pH of the lower phase was adjusted to 3 with 6 M HC1 and e x t r a c t e d w i t h t h r e e X 2 ml o f hexane . The po o l e d hexane washes contained the s a p o n i f i a b l e l i p i d s ( f a t t y a c i d s ) . GENERAL ANALYTICAL PROCEDURES PROTEIN ASSAYS Lowry P r o t e i n Assay (2.3-1.1) For some of the e a r l y s t u d i e s , the p r o t e i n content was estimated by the method o f Lowry ^ t a l . ( 4 4 8 ) . P r o t e i n samples were p r e i n c u b a t e d over-n i g h t a t 37° C (or 15 min at 100° C) i n 1.5 ml of 0.66 M NaOH. To the b a s i c samples, 1.5 ml of s o l u t i o n A (100 ml of 13% (w/v) sodium carbonate; 3 ml o f 4% (w/v) sodium p o t a s s i u m t a r t r a t e ; and 3 ml o f 4% (w/v) copper 114 25 50 75 100 125 A M O U N T O F PROTEIN ( p g ) F i g u r e 7. P r o t e i n s t a n d a r d c u r v e s . P r o t e i n was de t e r m i n e d e i t h e r by A, the pr o c e d u r e o f Lowry ejt a l . ( 4 4 8 ) o r B, the method o f B r a d f o r d (449). The p r o t o c o l f o r t h e s e a s s a y s a r e d e t a i l e d i n se c t i o n s 2.3-1-1 a i d 2.3-1-2. Bovine serum albumin (•) and immunoglobulin G ( A ) . 115 s u l f a t e ) were added and i m m e d i a t e l y mixed. A f t e r 15 min, 0.5 ml of 2 M phenol reagent s o l u t i o n ( F o l i n - C i o c a l t e a u ) were i n c l u d e d and the mixture i v ortexed. Absorbance o f the b l u e s o l u t i o n at 625 nm was measured 3 0 to 60 min l a t e r . F a t t y a c i d d e f i c i e n t bovine serum albumin was used as the p r o t e i n standard, and the assay was l i n e a r f o r up to 125 pg ( F i g . 7A). Bio-Rad P r o t e i n Assay (2.3.1.2) For most s t u d i e s , the Bio-Rad p r o t e i n assay based on the method of Bradford (449) proved t o be the most c o n v e n i e n t and economical. Two and a h a l f ml o f d i l u t e d p r o t e i n r e a g e n t (Bio-Rad Stock Reagent/r^O ( 1 / 3 . 2 5 ; v/v) were added t o 0.5 ml o f sample s o l u t i o n c o n t a i n i n g up t o 125 pg of p r o t e i n . A f t e r 10 min, the absorbance o f the brown to blue s o l u t i o n at 595 nm was determined. IgG was used as the p r o t e i n standard, and the assay was l i n e a r f o r up to 125 pg of p r o t e i n ( F i g . 7B). THIN-LAYER CHROMATOGRAPHY Res o l u t i o n o f Aqueous C h o l i n e M e t a b o l i t e s ( 2 . 3 . 2 . 1 ) . Aqueous metabolites of r a d i o - l a b e l e d c h o l i n e were re s o l v e d by TLC i n a s i n g l e dimension. The samples were s t r e a k as t h i n - b a n d s , 2.5 cm l o n g , and 1.5 cm above the bottom edge o f a S i l i c a G-60 p l a s t i c - b a c k e d TLC p l a t e . Unlabeled c h o l i n e (200 _ug) , p h o s p h o c h o l i n e (750 pg) , CDP-choline (500 pg) and/or betaine (300 ^ig) were r o u t i n e l y chromatographed with the samples to improve recovery and r e s o l u t i o n , and to f a c i l i t a t e the v i s u a l i z a t i o n of each compound. The s p o t t e d TLC p l a t e was d e v e l o p e d i n CH OH/0.6? NaCl/27% NH^OH (10/10/1; v/v/v) f o r a d i s t a n c e o f 10 cm. A f t e r the TLC p l a t e was d r i e d , CDP-choline and phosphocholine were v i s i b l e over a white l i g h t box as f a i n t bands. The other compounds were d e t e c t e d by iod i n e s t a i n i n g . C h o l i n e , 116 Rf = 0 . 1 ; p h o s p h o c h o l i n e , Rf = 0 . 4 5 ; b e t a i n e , Rf = 0.66 and CD P - c h o l i n e , Rf = 0.70. Resolution o f P h o s p h o l i p i d s (2 . 3.2.2). Phospholipids were s e p a r a t e d by TLC i n a s i n g l e dimension on S i l i c a G-60. The p l a t e was d e v e l o p e d , i n CHC1 ^ / C H ^ O H / a c e t o n e / g l a c i a l a c e t i c a c i d / H 2 0 (10/2/4/2/1; v / v / v / v / v ) . A f t e r development, the TLC p l a t e was d r i e d , and the ph o s p h o l i p i d s were v i s u a l i z e d with i o d i n e s t a i n i n g . R e s o l u t i o n o f G l y c e r o l i p i d s (2 . 3.2 . 3 ) . To separate the n e u t r a l and p h o s p h o l i p i d s , t h i n - l a y e r chromatography was performed s e q u e n t i a l l y i n two solvent systems i n the same d i r e c t i o n . The sample was ap p l i e d to the o r i g i n o f a 20X20 cm S i l i c a Gel G-60 p l a t e , and the p l a t e was d e v e l o p e d i n CHC1 ^ /CH^OH/acetone/ g l a c i a l CH^COOH/H^O (10/2/4/2/1; v/v/v/v/v) u n t i l the s o l v e n t f r o n t m i g r a t e d 9 cm from the o r i g i n . The p l a t e was d r i e d and TLC was resumed i n the same d i r e c t i o n with h e x a n e / d i e t h y l e t h e r / g l a c i a l CH^COOH (60/40/1 ; v/v/v) u n t i l the p l a t e was f u l l y developed. The n e u t r a l l i p i d s migrated f u r t h e r than the ph o s p h o l i p i d s , and a l l l i p i d s were v i s u a l i z e d with i o d i n e . 117 METABOLITE POOL SIZE MEASUREMENTS Ph o s p h o c h o l i n e ( 2 . 3 . 3 . 1 ) . The phosphocholine pool s i z e i n r a t h e p a t o c y t e s was determined by the method o f Choy e t a l . ( 2 6 9 ) . In summary, p h o s p h o c h o l i n e i n t h e upper 3 phase of a Folch e x t r a c t i o n o f r a t h e p a t o c y t e s was incubated with [ H]CTP 3 and the L-form of c y t i d y l y l t r a n s f e r a s e . The [ H]CDP-choline formed was 3 r e s o l v e d from [ H]CTP by paper chromatography i n the s o l v e n t system of ethanol/1 M ammonium ac e t a t e , pH 7.1 ( 7 / 3 ; v / v ) . CDP-choline was v i s u a l i z e d under u l t r a v i o l e t l i g h t and counted f o r r a d i o a c t i v i t y . D i g l y c e r i d e ( 2 . 3 . 3 - 2 ) . D i g l y c e r i d e l e v e l s i n h e p a t o c y t e s were measured by the procedure of S c h n e i d e r (450) w i t h a few m o d i f i c a t i o n s . T h i s a s s a y r e l i e s on the h y d r o l y s i s of d i g l y c e r i d e t o g l y c e r o l and p h o s p h o r y l a t i o n of g l y c e r o l with 32 [ P]ATP by g l y c e r o k i n a s e . D i g l y c e r i d e was p u r i f i e d from the lower phase of a Folch e x t r a c t i o n o f two dishes of r a t h e p a t o c y t e s (6 X 10^ c e l l s ) by TLC on S i l i c a g e l G-60 with benzene/CHCl^/methanol (80/15/5; v/v/v) as the developer. The d i g l y -c e r i d e was eluted from the s i l i c a w i t h 2-propanol/hexane/ H^ O (75/25/1; v/v/v) and evaporated under n i t r o g e n . The d i g l y c e r i d e was hydrolyzed when 50 ^ i l of benzene and 100 u l o f 0.1 M KOH i n methanol were added, and the o sample was heated a t 55 C f o r 30 min. The s o l v e n t was evaporated under n i t r o g e n , and the g l y c e r o l which p a r t l y migrated up the w a l l s the t e s t tube was resuspended i n 50 ^ J l of 0. 1 M HC1 . To the t e s t tube was added 50 ^ J l of a g l y c e r o k i n a s e r e a c t i o n c o c k t a i l ( T r i s - H C l , 10 jimol, pH 9.0; EDTA, 200 nmol; MgCl o, 500 nmol; ATP (pH 7.0), 20 nmol; b o v i n e serum albumin, 0.5 H 8 mg; b e t a - m e r c a p t o e t h a n o l , 1 umol and g l y c e r o k i n a s e , 0.16 U n i t s -o 32 p r e i n c u b a t e d f o r 30 min a t 20 C b e f o r e 0.1 yd o f [gamma- P]ATP were added), and the m i x t u r e was i n c u b a t e d a t room temperature f o r 60-90 o min. (The r e a c t i o n s c o u l d be c o n v e n i e n t l y f r o z e n at -20 C a f t e r t h i s s t e p . ) Ten pi o f the r e a c t i o n m i x t u r e w e r e s p o t t e d on a 2 cm l a n e on c e l l u l o s e p l a s t i c backed TLC p l a t e s ( M e r c k ) and developed f o r 3 h i n methanol/ 1 M ammonium a c e t a t e , pH 8.5/0.2 M EDTA (70/20/0.5; v / v / v ) . The 32 P-labeled g l y c e r o l - 3 - p h o s p h a t e m i g r a t e d about 3 cm above the o r i g i n . The r a d i o a c t i v i t y i n g l y c e r o l - 3 - p h o s p h a t e was d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n counting. 32 This assay gave r e p r o d u c i b l e r e s u l t s p r o v i d e d t h a t f r e s h [ P]ATP was used and no g l y c e r o l - 3 - p h o s p h a t e was used as c a r r i e r during the TLC. L i q u i d S c i n t i l l a t i o n C o u n t i n g (2.3.4.1). L i p i d and aqueous samples were c o u n t e d i n ACS (Aqueous C o u n t i n g S c i n t i l l a n t , Amersham). A l l s c i n t i l l a t i o n counting was done i n an ISOCAP/300 s c i n t i l l a t i o n c o u n t e r ( N u c l e a r - C h i c a g o ) , and c o u n t i n g e f f i c i e n c y was 3 determined from the c h a n n e l - r a t i o s of s t a n d a r d s which contained [ H]hexa-14 decane or [ C ] t o l u e n e . S t a t i s t i c s ( 2 . 3 . 5 . 1 ) . The student t.-test was used t o a s s e s s the p r o b a b i l i t y (j?) t h a t the means of two s e t s of data a r e the same. Only p_ v a l u e s below 0.05 were considered as s i g n i f i c a n t . The £ v a l u e s were determined from the T scores and the degrees of freedom (DF) w i t h a t a b l e f o r checking both the upper and lower l i m i t s . 119 DF = n + n - 2 where n and n r e p r e s e n t the number x y x y of "x" and "y" values, r e s p e c t i v e l y T = X - Y V s p 2 (1/n + 1/n ) where X = mean of "x" v a l u e s v x y Y = mean of 11 y" values „ 2 Sp = pooled e s t i m a t e Sp 2 = S D 2 + S D 2 —; x y -where SD and SD r e p r e s e n t x y the standard d e v i a t i o n o f "x" and "y" values, respec-t i v e l y , provided that n = n ^  y ' 120 ENZYME ASSAYS C h o l i n e K i n a s e ( 2 . 4 . 1 . 1 ) . Enzyme a c t i v i t y was measured e s s e n t i a l l y as described by Weinhold and Rethy ( 8 ) . The r e a c t i o n m i x t u r e c o n t a i n e d i n a f i n a l volume of 100 yl: 10 ymol of T r i s - H C l , pH 8.5; 1 pmol o f MgCl ; • 1 pmol o f ATP; 100 nmol o f 3 [Me- H ] c h o l i n e (2 yCi/ymol); and 300 ug o f c y t o s o l i c p r o t e i n . The o r e a c t i o n mixture was incubated f o r 20 min a t 37 C, and the r e a c t i o n was terminated by immersion of the r e a c t i o n t u b e s i n t o b o i l i n g water f o r 2 min. The p r o t e i n p r e c i p i t a t e was removed by c e n t r i f u g a t i o n at 7500 X g f o r 10 min. F i f t y yl of the s u p e r n a t a n t were a p p l i e d t o the o r i g i n o f a lane (2.5 X 10 cm) on S i l i c a G-60 p l a s t i c - b a c k e d p l a t e s (Merck). Phosphocholine c a r r i e r (0.75 mg) was a p p l i e d i n a t h i n - s t r e a k j u s t below the o r i g i n . The TLC p l a t e was developed i n CH^H/0.6% N a C l / NH^ (10/10/1; v/v/v) f o r 50 min. The p l a t e was d r i e d , and the p h o s p h o c h o l i n e (R£ = 0.45) was v i s i b l e over a standard l i g h t box. The region o f the p l a t e where phosphocholine migrated was cut i n t o a s c i n t i l l a t i o n v i a l w i t h 1.5 ml of H^O. A f t e r 5 min o f shaking, 10 ml of ACS f l u i d (Amersham) were added and the sample was counted f o r r a d i o a c t i v i t y . This assay was l i n e a r f or up to 25 min and at l e a s t 0.75 mg of c y t o s o l i c p r o t e i n . CTP: Phosphocholine C y t i d y l y l t r a n s f e r a s e (2.4.1.2). The c o n d i t i o n s f o r the c y t i d y l y l t r a n s f e r a s e assay were s u b s t a n t i a l l y r e v i s e d from the p r o t o c o l o f A n s e l l and Choynacki ( 4 5 1 ) . The r e a c t i o n mixture contained i n a f i n a l volume of 100 u l : 7.5 umol of T r i s - s u c c i n a t e , pH 6.4; 750 nmol o f magnesium a c e t a t e ; 200 nmol o f CTP; 150 nmol of [Me-3 H]phosphocholine (20 yCi/ymol) and a p p r o x i m a t e l y 250 yg of c y t o s o l i c or 121 o microsomal p r o t e i n . The r e a c t i o n m i x t u r e was incubated for 10 min at 37 C and the r e a c t i o n was terminated a f t e r immersion of the r e a c t i o n tubes i n b o i l i n g water f o r 2 min. The p r o t e i n p r e c i p i t a t e was p e l l e t e d by c e n t r i f u g a -t i o n at 7500 X g f o r 10 min and 50 u l of the s u p e r n a t a n t was a p p l i e d to the o r i g i n of a lane (2.5 X 10 cm) on S i l i c a G-60 p l a s t i c backed p l a t e s . CDP-choline c a r r i e r (0.5 mg) was a p p l i e d i n a t h i n - s t r e a k j u s t below the o r i g i n . The TLC p l a t e was de v e l o p e d i n CH OH /0 .6% NaCl/NH^ (10/10/1; v/v/v) f o r 50 min. The p l a t e was d r i e d , and the CDP-choline (Rf = 0.7) was v i s i b l e as a f a i n t band over a s t a n d a r d l i g h t box. The region of the TLC p l a t e where CDP-choline migrated was cut i n t o a s c i n t i l l a t i o n v i a l w i t h 1.5 ml of H^ O- A f t e r 5 min s h a k i n g , 10 ml o f ACS f l u i d were added and the samples were counted f o r r a d i o a c t i v i t y . When 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 determined i n the presence of exogenous phospholipid liposomes, 0.8 mg of t o t a l r a t l i v e r p h o s p h o l i p i d were evaporated i n t o the r e a c t i o n tube under a stream of nitrogen p r i o r to t h e a d d i t i o n o f t h e o t h e r a s s a y i n g r e d i e n t s . The m i c r o s o m a l c y t i d y l y l t r a n s f e r a s e assay was l i n e a r f o r up t o 8 min or with up to 300 yg of microsomal p r o t e i n . The 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 assay was l i n e a r f o r up to 30 min or with up to 1 mg of c y t o s o l i c p r o t e i n . CDP-Choline: D i a c y l g l y c e r o l C h o l i n e p h o s p h o t r a n s f e r a s e (2.4.1.3)-Cholinephosphotransferase was assayed much as described by Vance and Burke (304). The assay c o n t a i n e d i n a f i n a l volume of 250 u l : 12.5 umol of T r i s - H C l , pH 8.5; 5 umol of magnesium c h l o r i d e ; 0.1 mg of Tween 20; 0.5 mg 14 of bovine serum a l b u m i n ; 250 nmol o f EGTA; 250 nmol o f [ C]CDP-choline; and 1-2 mg of microsomal p r o t e i n . The d i g l y c e r i d e emulsion f o r the c h o l i n e -phosphotransferase assay was p r e p a r e d by s o n i c a t i o n of the d i g l y c e r i d e i n 122 the presence o f the Tween 20, T r i s - H C l , magnesium a c e t a t e , bovine serum albumin and EGTA s i x t i m e s f o r 60 sec w i t h a S o n i c Dismembrator (Quigley-o Rochestor, Inc.) at s e t t i n g 80. The r e a c t i o n commenced at 37 C with the 14 a d d i t i o n of the [ C ] C D P - c h o l i n e and was t e r m i n a t e d 15 min l a t e r upon a d d i t i o n o f 1.6 ml o f CHC1 /CH OH ( 1 / 1 ; v / v ) . The r a d i o - l a b e l e d 3 3 p h o s p h a t i d y l c h o l i n e was e x t r a c t e d i n t o the lower phase when 0.8 ml o f CHC1 , 0.35 ml o f w a t e r , 20 u l o f 50 mg/ml CDP-choline and 20 yl of 20 mg/ml t o t a l r a t l i v e r p h o s p h o l i p i d were a l s o added t o the t e s t tube. The upper phase was discarded, and the lo w e r phase was washed two times with 2 ml of t h e o r e t i c a l upper.phase. The lower phase was tr a n s f e r e d to a s c i n t i l l a -t i o n v i a l and evaporated. One ml o f water and 10 ml of ACS were added to the v i a l which was counted f o r r a d i o a c t i v i t y . Phosphatidylethanolamine M e t h y l t r a n s f e r a s e (2.4.1.4). PE N - m e t h y l t r a n s f e r a s e a c t i v i t y was a s s a y e d by the t r a n s f e r o f 3 t r i t i u m f r o m S-adenosyl-L-[M£- H ] m e t h i o n i n e t o p h o s p h o l i p i d . The assay mixture contained i n a f i n a l volume of 100 ^ J l : 12.5 jJmol of T r i s - H C l , 3 pH 9-2; 100 nmol o f L - c y s t e i n e , 15 nmol o f S - a d e n o s y l - L - [ M e - H ] -methionine (150 yCi/ymol) and 0.3 mg o f microsomal p r o t e i n . PE meth y l t r a n s -o ferase was assayed f o r 10 min at 37 C, and the r e a c t i o n was terminated upon a d d i t i o n o f 2 ml of CHCl^/CH^OH ( 2 / 1 ; v / v ) . The o r g a n i c e x t r a c t was t r a n s f e r r e d to a screw-capped tube . An a d d i t i o n a l ml of the chlo r o f o r m -methanol mixture was used to wash the i n c u b a t i o n tube and combined with the 2 ml e x t r a c t . Seven hundred yl of 0.145 M NaCl were added t o the tube, vortexed f o r 30 sec and c e n t r i f u g e d . The o r g a n i c phase was washed 3 times with 2 ml of t h e o r e t i c a l upper phase. F i n a l l y , the organic phase was d r i e d under n i t r o g e n i n a s c i n t i l l a t i o n v i a l , and the r a d i o a c t i v i t y was subse-quently determined. 123 RESULTS DISTRIBUTION OF CYTIDYLYLTRANSFERASE IN RAT Rat L i v e r Contains the L a r g e s t Amount o f C y t i d y l y l t r a n s f e r a s e (3.1.1.1). The occurrence o f 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 i c f r a c t i o n of va r i o u s r a t organs was examined (Table 17). The l i v e r was found to have 8.5-f o l d g r e a t e 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 than l u n g , 2 9 - f o l d more than b r a i n and 257-fold more than i n t e s t i n e . The b r a i n c y t o s o l i c c y t i d y l y l t r a n s -f e r a s e was s e n s i t i v e t o a c t i v a t i o n by exogenous p h o s p h o l i p i d while the corresponding enzyme a c t i v i t y i n i n t e s t i n e was unaffected by ph o s p h o l i p i d . Since the i n t e s t i n a l 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 so low i n comparison w i t h l i v e r , the pH optimum of the i n t e s t i n a l enzyme was checked and found to be 6.5. o When the r a t l u n g and b r a i n c y t o s o l s were i n c u b a t e d at 20 C f o r 8 h, about 42% o f the a c t i v i t y was r e c o v e r a b l e as L-form (28-40% ammonium s u l f a t e f r a c t i o n ) . Less than 10% of the l i v e r c y t o s o l i c enzyme could be recovered as L-form under i d e n t i c a l c o n d i t i o n s . Ammonium s u l f a t e p r e c i p i t a t i o n o f c y t o s o l i c p r o t e i n a l l o w e d r e c o v e r i e s 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 g r e a t e r than 70% from r a t b r a i n or lung p r e p a r a t i o n s , w h i l e r e c o v e r y o f enzyme a c t i v i t y from l i v e r c y t o s o l was c o n s i s t e n t l y l e s s than 45%. 124 Table 17. D i s t r i b u t i o n of C y t i d y l y l t r a n s f e r a s e i n the Cytosols of Various  Rat Organs. Organ Weight (E) Cytosolic Enzyme (nmol/min-g ti s s u e ) A c t i v i t y (nmol/min-organ) % of t o t a l l i v e r a c t i v i t y A c t i v a t i o n by phospholipid L i v e r 8.1 190 1542 100 5-fold Lung 1.U 130 182 12 3-fold Brain 1.7 88 1SO 10 8-fold I n t e s t i n e s 2.3 2.7 6 0.3 no a c t i v a t i o n C y t o s o l i c enzyme a c t i v i t i e s were determined i n the presence of t o t a l rat l i v e r phospholipid. Each value represents the mean of 7 rats (150-175 g weight). 125 Most of 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 i s Located i n Cytosol (3-1-1-2). Homogenization of r a t l i v e r i n i s o t o n i c s a l i n e f a c i l i t a t e d the maximum recovery of 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 i c f r a c t i o n (Table 18). In the past, r e p o r t s have u n d e r e s t i m a t e d t h e amount o f c y t i d y l y l t r a n s f e r a s e p r o t e i n i n the c y t o s o l s i n c e t o t a l r a t l i v e r p hospholipid was not included i n the enzyme a s s a y s . The c y t i d y l y l t r a n s f e r a s e a p p a r e n t l y has increased t e n a c i t y f o r membranes i f the i o n i c s t r e n g t h of the homogenization medium i s reduced. Table 18. Su b c e l l u l a r D i s t r i b u t i o n o f C y t i d y l y l t r a n s f e r a s e i n Rat L i v e r . Rat l i v e r was homogenized i n e i t h e r d i s t i l l e d water, 0.25 M sucrose, or 0.145 M NaCl with 7 strokes up and down i n a Potter-Elvehjem homogenizer. The homogenate was centr i f u g e d at 16,000 X g X 15 min. The post-mitochond-r i a l supernatant was c e n t r i f u g e d at 170,000 X g X 1 h. The 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 a c t i v i t y was determined i n the absence and presence of t o t a l r a t l i v e r phospholipid (PL). Homogenization % Recovered A c t i v i t y J Recovered A c t i v i t y Medium (Without PL Stimulation) (With PL Stimulation) Microsomes Cytosol Microsomes Cytosol D i s t i l l e d water 65 35 54 46 0.25 M sucrose 37 63 29 71 0.1.45 M NaCl 8 92 126 CHARACTERIZATION OF RAT LIVER CYTIDYLYLTRANSFERASE Rat L i v e r C y t o s o l i c C y t i d y l y l t r a n s f e r a s e A c t i v a t i o n and A g g regation Are Time and Temperature-Dependent Processes (3-2.1.1). o Incubation of r a t l i v e r c y t o s o l at 4 C f o r 4 days caused a 5 - f o l d s t i m u l a t i o n of c y t i d y l y l t r a n s f e r a s e a c t i v i t y and produced complete conver-s i o n of the enzyme to the H-form ( d a t a not shown). The r a t e s of a c t i -o v a t i o n and a g g r e g a t i o n c o u l d be a c c e l e r a t e d a t 37 C. P h o s p h o l i p i d -o s t i m u l a t e d 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 d e c l i n e d by 50% a f t e r 4 h at 37 C, but the i n c l u s i o n of the p r o t e a s e i n h i b i t o r p h e n y l m e t h y l s u l f o n y l f l u o r i d e reduced the r a t e of c y t i d y l y l t r a n s f e r a s e d e g r a d a t i o n . 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 i n c y t o s o l was d o u b l e d a f t e r i n c u b a t i o n a t 37° C f o r 15 min, but prolonged i n c u b a t i o n at t h i s t e m p e r a t u r e f a i l e d t o a c t i v a t e t h i s enzyme f u r t h e r ( d a t a not shown). However, 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 could be elevated 5 - f o l d i n 6 h i f the c y t o s o l were incubated at room temp-er a t u r e ( F i g . 8A) w i t h l i t t l e l o s s of enzyme a c t i v i t y due to p r o t e o l y s i s provided that p h e n y l m e t h y l s u l f o n y l f l u o r i d e was included i n the c y t o s o l ( F i g . 8B) . Incubation of c y t o s o l at 20° C a l s o promoted aggregation of c y t i d y l -y l t r a n s f e r a s e ( F i g . 8 C ) . G e l f i l t r a t i o n o f t h e a g g r e g a t e d enzyme on Sepharose 2B demonstrated that the H-form i s a very heterogenous species which eluted w i t h a wide range of molecular weights ( F i g . 8D). L i p i d - A s s o c i a t e d C y t i d y l y l t r a n s f e r a s e has a H i g h e r pH Optimum than the  N o n - L i p i d - A s s o c i a t e d Enzyme (3-2.1.2). The e f f e c t of s e v e r a l b u f f e r 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 i n f r e s h c y t o s o l was i n v e s t i g a t e d ( F i g . 9 ) . T r i s - s u c c i n a t e proved to be the b u f f e r of c h o i c e . Phosphate b u f f e r which i s v e r y e f f e c t i v e at pH 5.8 was i n h i b i t o r y , 127 T — i — i — i — i — i — n I—i— i— i—i—i—i—r U j PREINCUBATION TIME (h) PREINCUBATION TIME (h) F R A C T I O N N U M B E R . F R A C T I O N N U M B E R Figure 8. A c t i v a t i o n and a g g r e g a t i o n o f c y t o s o l i c c y t i d y l y l t r a n s f e r a s e at 2 0 a C. Rat l i v e r c y t o s o l was i n c u b a t e d at 20° C i n the pr e s e n c e o f 0.5 mM p h e n y l m e t h y l s u l p h o n y l f l u o r i d e f o r up t o 14 h. 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 s u b s e q u e n t l y m e a s u r e d : P a n e l A, absence; and Pa n e l B, presence of 750 ug of t o t a l r a t l i v e r p h o s p h o l i p i d . P a n e l C, four m l Q o f f r e s h c y t o s o l ( A ) or c y t o s o l which had been incubated f o r 8 h at 20 C (•) were a p p l i e d to a Sepharose 6B (180 ml bed volume) column and elu t e d w i t h Buffer A (20 mM T r i s - H C l , pH 7-0; 100 mM NaCl) i n t o 250 drop (7-9 ml) f r a c t i o n s . Panel D, the 0-25% ammonium s u l p h a t e f r a c t i o n of c y t o s o l which had been incubated a t 20° C f o r 8 h was lo a d e d on to a Sepharose 2B (180 ml bed volume) column and e l u t e d w i t h B u f f e r A i n t o 200 drop (6.3 ml) f r a c t i o n s . 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 absorbance at 280 nm ( O ) . 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 n the column f r a c t i o n s were determined i n the presence of pho s p h o l i p i d liposomes. 128 presumably because t h i s s p e c i e s resembled the p r o d u c t s o f the c y t i d y l y l -t r a n s f e r a s e c a t a l y z e d r e a c t i o n . C i t r a t e b u f f e r was even more i n h i b i t o r y and t h i s was due to c h e l a t i o n of magnesium ions i n the enzyme assay. These b u f f e r s were used to d e t e r m i n e the pH optima o f the v a r i o u s forms of 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 ( F i g . 10). The L-form i n f r e s h c y t o s o l had a pH optimum of 5.6 or lower ( F i g . 10A) , w h i l e the H-form i n c y t o s o l t h a t had o been preincubated f o r 8 h at 20 C had a broad pH optimum between 6.4 and 7.6 ( F i g . 10B). The i n c l u s i o n o f t o t a l r a t l i v e r p h o s p h o l i p i d i n the enzyme assay was found to s h i f t the pH optimum o f the L-form to that of the H-form ( F i g . 10C). The pH optimum of the microsomal c y t i d y l y l t r a n s f e r a s e a l s o resembled t h a t o f the H-form ( F i g . 10D). Hence, t h e p r e s e n c e of a l i p i d environment was l i k e l y the key f a c t o r which produced a s h i f t i n the pH optimum to p h y s i o l o g i c a l values. Incubation of Cytosol I n c r e a s e s the A f f i n i t y of C y t i d y l y l t r a n s f e r a s e f o r  CTP But Not f o r P h o s p h o c h o l i n e (3.2.1.3). o Neither the i n c u b a t i o n of r a t l i v e r c y t o s o l at 20 C f o r 8 h, nor the i n c l u s i o n o f t o t a l r a t l i v e r p h o s p h o l i p i d i n the enzyme assay a l t e r e d the a p p a r e n t K 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 p h o s p h o c h o l i n e (0.5 mM), m although the V of the r e a c t i o n was i n c r e a s e d ( F i g . 11B). On the other max hand, i n c u b a t i o n a t 20° C reduced t h e apparent K f o r CTP from 1.9 t o m 0.8 mM ( F i g . 11A). P h o s p h o l i p i d l i p o s o m e s f u r t h e r reduced the apparent K m f o r CTP to 0.5 mM ( F i g . 11A). The microsomal c y t i d y l y l t r a n s f e r a s e had an apparent K of 0.35 mM f o r m phosphocholine and 0.65 f o r CTP ( F i g . 12). Since the microsomal-, H- and L-form with p h o s p h o l i p i d had s i m i l a r a f f i n i t i e s f o r CTP, the phospholipid environment was p r o b a b l y r e s p o n s i b l e f o r the reduced K v a l u e s for t h i s m Figure Q. C o n c e n t r a t i o n dependence  of v a r i o u s b u f f e r s on 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 a c t i v i t y . In-form a c t i v i t y i n f r e s h r a t l i v e r cy-t o s o l was determined i n the presence of 2 5 - 1 5 0 mM concentrations of T r i s -s u c c i n a t e , pH 6 . 0 ( A ) ; phosphate, pH 5 . 8 (•) and c i t r a t e , pH 5 -8 ( O ) b u f f e r s . The f i n a l concentration of magnesium i n the enzyme assay was 12 mM. 50 100 150 CONC. BUFFER (mM) Figure 10 . pH optima o f the v a r i o u s forms of 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 . The a c t i v i t y of c y t i d y l y l t r a n s f e r a s e was measured at various pH with 100 mM concentrations o f c i t r a t e ( O . f ) ; phosphate ( • , • ) , T r i s - s u c c i n a t e ( A , A , A ) and T r i s - H C l (<£>) b u f f e r s . Open symbols, f r e s h c y t o s o l ; clo s e d symbols, 8 h at 20 C i n c u b a t e d c y t o s o l ; and h a l f - t o n e symbols, microsomes. S o l i d l i n e s , absence; and dashed l i n e , presence of t o t a l r a t l i v e r phospholipid i n the c y t i d y l y l t r a n s f e r a s e assay. 130 A 1 2 0 IT E 9 0 o E 3 6 0 >° T -i — J — o / o / l l i - 2 . 4 - 1 . 6 - 0 . 8 B 1 2 0 E 9 0 o E 3 6 0 >° 0 0 - 8 1.6 2 . 4 1/[CTP] (mM)"1 ~ o^ 1 1 1 - 2 4 - 1 6 - 0 . 8 0 0 . 8 1.6 2 .4 1/[PHOSPHOCHOLINE] (mM) 1 Figure 11. E f f e c t of i n c u b a t i o n a t 20° C and ph o s p h o l i p i d on the  apparent Km's of the 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 f o r CTP and  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 n f r e s h c y t o s o l ( o , » ) or c y t o s o l incubated a t 20 C f o r 8 h ( A , A ) was measured: A, i n the presence of 1 mM phosphocholine and v a r y i n g c o n c e n t r a t i o n s of CTP; or B, i n the presence of 4 mM CTP and v a r y i n g c o n c e n t r a t i o n s o f phosphocholine. Open symbols, absence; and c l o s e d symbols, presence o f t o t a l r a t l i v e r p h ospholipid i n the enzyme assay. 131 s u b s t r a t e . The 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 had a magnesium optimum near 5 mM, and the microsomal enzyme had a s i m i l a r magnesium requirement ( F i g . 13). The Microsomal C y t i d y l y l t r a n s f e r a s e R e a c t i o n Goes Non-Linear With Time And  P r o t e i n Sooner than the C y t o s o l i c Reaction (3-2.1.4). The c y t o s o l i c c y t i d y l y l t r a n s f e r a s e was l i n e a r w i t h time f o r up to 30 min ( F i g . 14A), and w i t h up t o 1 mg of p r o t e i n i n the enzyme assay ( F i g . 14B) . However, the assay was n o n - l i n e a r w i t h r e s p e c t t o p r o t e i n when the amount of c y t o s o l i c p r o t e i n i n the enzyme assay was below 120 yg ( F i g . 14C). The microsomal c y t i d y l y l t r a n s f e r a s e c a t a l y z e d r e a c t i o n went n o n - l i n e a r when more than 700 pmol of C D P - c h o l i n e was generated i n the enzyme assay ( F i g . 15). The C D P - c h o l i n e was presumably c o n v e r t e d t o p h o s p h a t i d y l c h o l i n e by cholinephosphotransferase which i s a l s o present i n microsomes. A l t e r n a t i v e l y , CDP-choline may have been degraded by a microsomal CDP-choline hydrolase. 132 *7 IT16 -E CTP c -PHOSPHOCHOLINE 1 1 1 - 3 . 2 - 2 . 4 -1 .6 - 0 . 8 0 0 . 8 1.6 2.4 1 /[SUBSTRATE] (mM)'1 Figure 12. Apparent Km's o f the microsomal c y t i d y l y l t r a n s f e r a s e f o r CTP and phosphocholine. Rat l i v e r microsomes were assayed f o r c y t i d y l y l t r a n s -ferase a c t i v i t y i n the presence of 1.5 mM phosphocholine and 0.5-3 mM CTP ( A ) or 2 mM CTP and 0.2-1.8 mM phosphocholine ( A ) . CONC. MgAc (mM) CONC. MgCl2 (mM) Figure 13. Requirement of the c y t i d y l y l t r a n s f e r a s e f o r magnesium. C y t i d y l -y l t r a n s f e r a s e was assayed i n the presence of up to 25 mM magnesium acetate (Panel A) or magnesium c h l o r i d e ( P a n e l B) . F r e s h r a t l i v e r c y t o s o l (•) and microsomes ( • ) . 133 Figure 14. L i n e a r i t y of the 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 c a t a l y z e d  r e a c t i o n w i t h time and p r o t e 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 was measured i n f r e s h r a t l i v e r c y t o s o l (<>.•) and c y t o s o l preincubated at 20° C f o r 8 h ( A , A ) f o r up .to 35 min w i t h 0.25 mg o f c t o s o l i c p r o t e i n (Panel A) or f o r 15 min with up t o 1.25 mg of c y t o s o l i c p r o t e i n (Panel B and Panel £ ) . Open symbols, absence; and c l o s e d symbols, presence o f t o t a l r a t l i v e r p h o s p h o l i p i d i n the enzyme assay. Figure 15. L i n e a r i t y of the m i c r o s o m a l c y t i d y l y l t r a n s f e r a s e c a t a l y z e d  r e a c t i o n w i t h time and p r o t e 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 was measured i n r a t l i v e r microsomes. A, t h e enzyme was assayed f o r up to 21 min with 0.14 mg (•) and 0.28 mg (•) o f microso m a l p r o t e i n . B, the enzyme was assayed with up to 1.4 mg of m i c r o s o m a l p r o t e i n f or 7 min ( V ) and 10 min ( T ) . 134 PURIFICATION OF CYTIDYLYLTRANSFERASE FROM RAT LIVER Revisions of the O r i g i n a l P u r i f i c a t i o n P r o t o c o l (3-3-1-1)-Choy £t a l . ( 3 D d e v e l o p e d a c l e v e r p u r i f i c a t i o n p r o c e d u r e which e x p l o i t e d the tendency o f c y t o s o l i c c y t i d y l y l t r a n s f e r a s e to aggregate. In o t h e i r p u r i f i c a t i o n p r o t o c o l , c y t o s o l was i n c u b a t e d a t 4 C f o r 5 days. The aggregated c y t i d y l y l t r a n s f e r a s e was c o n c e n t r a t e d by ammonium sulphate p r e c i p i t a t i o n and app l i e d to a Sepharose 6B column. The void volume m a t e r i a l o from t h i s column was t r e a t e d w i t h 0.05% S.D.S. a t 4 C f o r 2 h and re a p p l i e d to the g e l f i l t r a t i o n column. 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 which e l u t e d i n the f r a c t i o n a t i o n range o f the column was shown to migrate as a s i n g l e p r o t e i n band on a 5% polyacrylamide g e l at pH 7.5. The p r o t o c o l o f Choy e^ t a l . ( 3 D i s the o n l y r e p o r t e d s u c c e s s f u l p u r i f i c a t i o n procedure to date. However, the o v e r a l l recovery 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 was poor and the enzyme was unstable. In a d d i t i o n , the a g g r e g a t i o n s t e p r e q u i r e d s e v e r a l d a y s . The a g g r e g a t i o n s t e p c o u l d be o ac c e l e r a t e d by incu b a t i o n o f c y t o s o l at 20 C for 8 h ( F i g . 8C). As shown i n Table 19, the con c e n t r a t i o n of H-form by ammonium sulphate p r e c i p i t a t i o n , and d i s s o c i a t i o n of the H-form by 0.05% S.D.S. were res p o n s i b l e f o r major l o s s e s of enzyme a c t i v i t y . Table 1 9- PUKI I ICA TION OF C T P : PHOSPIKK IIOI INK C Y I IDYI.YI I KANSI EHASK I K U M R A T L IVER Fraction Volume (ml) Protein (mg) Total activity (units) Specific activity (units/mg) x |0 3 Recovery (%) Purification (fold) Total activity6 (units) Specific activity* (units/mg) x |0 3 Cytosol 152 2100 0.420 0.20 100 1 3.27 1.56 Aged cytosol 152 2040 3.47 1.70 826 8.5 3.20 1.57 ( N H J . S O , 20-30% 12 281 1.46 5.20 348 26 2.67 9.50 First Sepharose 6B 22.8 57 0.85 14.9 202 74.5 1.36 23.9 Second Sepharose 6B 36 0.67 0.07" 105" 17" 525 0.07 105 " The purified enzyme has no activity unless assayed in the presence of phospholipid. * All fractions were assayed in the presence of phospholipid. 135 Ammonium sul p h a t e p r e c i p i t a t i o n o f H-form r e s u l t e d i n 39 + 6% (mean + S. Dev. of 7. experiments) r e c o v e r y of enzyme a c t i v i t y , but the ammonium o sulphate p r e c i p i t a t e d c y t i d y l y l t r a n s f e r a s e was stab l e at -70 C as a pow-der or frozen i n s o l u t i o n f o r months. Several a l t e r n a t i v e avenues were e x p l o r e d f o r concentration of the H-form. In one approach the aged c y t o s o l was l y o p h i l i z e d t o a powder and resuspended i n d i s t i l l e d w a t e r . The c y t o s o l i c c y t i d y l y l t r a n s f e r a s e was o extremely s t a b l e t o l y o p h i l i z a t i o n and c o u l d be s t o r e d a t -70 C as a powder or r e s u s p e n d e d enzyme f o r months w i t h no l o s s o f a c t i v i t y . A djustment of the pH o f aged c y t o s o l t o 5.2 w i t h s u c c i n i c a c i d a l s o p r e c i p i t a t e d the c y t i d y l y l t r a n s f e r a s e w i t h over 70% r e c o v e r y o f enzyme a c t i v i t y i n t h e resuspended pH 5.2 p e l l e t . U n f o r t u n a t e l y , the pH 5.2 p r e c i p i t a t e d c y t i d y l y l t r a n s f e r a s e was not r e a d i l y s o l u b i l i z e d . H-form can be D i s s o c i a t e d by HDL or O c t y l - G l u c o s i d e (3.3-1.2). The major f a u l t w i t h 0.05% S.D.S. d i s s o c i a t i o n o f H-form was t h a t e i t h e r the enzyme was l a r g e l y i n a c t i v a t e d or o n l y p a r t i a l d i s s o c i a t i o n was achieved ( F i g . 16A). Hence o t h e r means by which the H-form could be d i s -s o c i a t e d were t e s t e d . Although c y t i d y l y l t r a n s f e r a s e was completely s t a b l e i n 2 M NaCl f o r 14 h at 4° C, a f t e r Sepharose 6B chromatography more than 90% of the t o t a l a c t i v i t y e l u t e d as H-form (data not shown). The l i p o p r o t e i n , HDL has been reported to d i s s o c i a t e the l i p a s e complex from r a t adipose (452), so the e f f e c t of HDL from r a t plasma on the c y t i d y l -y l t r a n s f e r a s e was i n v e s t i g a t e d . HDL was f o u n d t o a c t i v a t e L-form approximately 4 - f o l d , presumably because the HDL c o n t a i n e d p h o s p h o l i p i d (data not shown). On the other hand, the same amount of HDL i n h i b i t e d H-form o by 50%. When H-form was t r e a t e d w i t h HDL f o r 90 min at 4 C, t y p i c a l l y 70% 136 Figure 16. D i s s o c i a t i o n of H-form by v a r i o u s agents. A, H-form (6 ml) was t r e a t e d w i t h 0.05% S.D.S. f o r 2 ,h at 1 C p r i o r to Sepharose 6B chromatography. The 180 ml bed volume column was e q u i l i b r a t e d and e l u t e d with 0.005% S.D.S.; 20 mM T r i s - H C l , pH 7-0 and 100 mM NaCl. The f l o w r a t e was 1 drop e v e r y 3 s e c and 250 d r o p (6 ml) f r a c t i o n s were c o l l e c t e d . The t o t a l r e c o v e r y 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 from the column was 50%, and 28% of the recovered a c t i v i t y was e l u t e d as L-form. B, H-form (2 ml) was i n c u b a t e d w i t h 200 p i o f HDL f o r 90 min at 4° C p r i o r t o g e l f i l t r a t i o n on a Sepharose 6B (180 ml bed volume) column. C y t i d y l y l t r a n s f e r a s e was eluted from the column with 20 mM T r i s - H C l , pH 7.0 and 100 mM N a C l , and 250 drop (7.9 ml) f r a c t i o n s were c o l l e c t e d . T o t a l enzyme a c t i v i t y recovery from the column was approximately 85%. C, H-form ( 1 ml) was t r e a t e d w i t h 25 mM o c t y l - b e t a - D - g l u c o s i d e f o r 2 h at 4° C p r i o r to chromatography on a Sepharose 6B (45 ml bed volume) column. The column was e q u i l i b r a t e d and e l u t e d w i t h 8 mM o c t y l - g l u c o s i d e ; 20 mM T r i s - H C l , pH 7.0; and 100 mM N a C l , and 100 drop (1.1 ml) f r a c t i o n s were c o l l e c t e d . T o t a l enzyme a c t i v i t y r e c o v e r e d from the column was approximately 95%. 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 measured i n the presence of t o t a l r a t l i v e r p h o s p h o l i p i d ( • ) ; absorbance at 280 nm ( O ) . 137 of the enzyme a c t i v i t y a p p l i e d t o a Sepharose 6B column el u t e d as L-form ( F i g . 16B). Strangely, no a p p r e c i a b l e d i s s o c i a t i o n was produced by HDL i f the c y t o s o l was p r e v i o u s l y c o n c e n t r a t e d by l y o p h i l i z a t i o n i n s t e a d o f ammonium sulphate p r e c i p i t a t i o n . Hopes o f e x p l o i t i n g HDL d i s s o c i a t i o n f o r p u r i f i c a t i o n of c y t i d y l y l t r a n s f e r a s e were squelched when i t was found that HDL p r o t e i n s c o e l u t e d w i t h L-form from Sepharose 6B and D E A E - c e l l u l o s e columns (data not shown). The most promising agent f o r c o n v e r s i o n o f the H-form back to L-form proved t o be o c t y l - b e t a - D - g l u c o s i d e ( F i g . 16C). T h i s compound produced t y p i c a l l y 60-70% d i s s o c i a t i o n of H-form w i t h 95% t o t a l recovery 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 from Sepharose 6B columns. C y t i d y l y l t r a n s f e r a s e was found to be c o m p l e t e l y s t a b l e i n 10 mM o c t y l - g l u c o s i d e f o r 6 h at 4° C, although c o n c e n t r a t i o n s i n excess of 5 mM s e v e r e l y i n h i b i t e d the c y t i d y l -y l r a n s f e r a s e - c a t a l y z e d r e a c t i o n ( d a t a not shown). Another e x c e l l e n t f e a t u r e of o c t y l - g l u c o s i d e was that t h i s compound could be e a s i l y d i a l y z e d from the L-form a f t e r d i s s o c i a t i o n . 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 u s i n g o c t y l - g l u c o s i d e i n place o f S.D.S. When Sepharose 6B column e l u a t e t h a t contained L-form was examined by S.D.S. denaturing p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , more than 14 bands c o u l d be d i s t i n g u i s h e d . A d d i t i o n a l p u r i f i c a t i o n s t e p s were t h e r e f o r e r e q u i r e d to p u r i f y the c y t i d y l y l t r a n s f e r a s e t o homogeneity. Consequently, the behaviour of c y t i d y l y l t r a n s f e r a s e on va r i o u s column media was examined. C y t i d y l y l t r a n s f e r a s e Binds t o DEAE Ion-Exchange Resins (3.3.1.3). The i n t e r a c t i o n of c y t i d y l y l t r a n s f e r a s e i n d i a l y z e d r a t l i v e r c y t o s o l with c a r b o x y m e t h y l - (CM-) c e l l u l o s e was s t u d i e d . At pH 6.0, most of the enzyme d i d not bind to t h i s c a t i o n - e x c h a n g e r and eluted with the column 138 wash. The small f r a c t i o n (20$) 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 which r e t a i n e d by CM-cellulose was removed with 100 mM NaCl (data not shown). o 139 T 1 1 1 i 1 r O 0 8 16 2 4 3 2 4 0 4 8 5 6 FRACTION NUMBER FRACTION NUMBER Figure 17. Anion-exchange chromatography o f c y t o s o l i c c y t i d y l y l t r a n s f e r a s e . A, Fresh c y t o s o l was d i a l y z e d o v e r n i g h t a g a i n s t 100 mM T r i s - s u c c i n a t e , pH 6.0; 25 mM NaCl, and 4 ml were a p p l i e d t o a DEAE-cellulose (180 ml bed volume) column a f t e r adjustment o f the pH o f the c y t o s o l to 8.5. Bound p r o t e i n was e l u t e d a f t e r the column was washed with'100 mM T r i s - s u c c i n a t e , pH 6.0 and 100 mM c i t r a t e , pH 4.5. Two hundred and f i f t y drop (7.5 ml) f r a c t i o n s were c o l l e c t e d , and t o t a l 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 from the ion-exchange column was 47% when t o t a l r a t l i v e r phospholipid was included i n the enzyme assays. 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 ( • ) ; absorbance at 280 nm ( O ) and pH ( • ). B, Two ml of d i a l y z e d c y t o s o l were a p p l i e d t o a DEAE-Sepharose 6B (100 ml bed volume) column and eluted with 3 column volumes o f a l i n e a r 25 mM - 750 mM NaCl g r a d i e n t which a l s o contained 100 mM T r i s - H C l at pH 8.5. Two hundred and f i f t y drop (8 ml) f r a c t i o n s were c o l l e c t e d and t o t a l c y t i d y l y l t r a n s f e r -ase a c t i v i t y from the ion-exchange column was 63% when phospholipid l i p o -somes were i n c l u d e d i n the enzyme assay. 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 (•); absorbance at 280 nm ( O ) and c o n d u c t i v i t y ( A ) . 140 When the pH of the d i a l y z e d c y t o s o l was a d j u s t e d to 8.5, most of the c y t i d y l y l t r a n s f e r a s e p r o t e i n was r e t a i n e d by D E A E - c e l l u l o s e ( F i g 17A). App r o x i m a t e l y h a l f of 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 l o a d e d on t h i s anion-exchanger column was r e c o v e r e d i n t h e wash a f t e r e l u t i o n w i t h pH 6.0 b u f f e r . Rat l i v e r c y t o s o l i c c y t i d y l y l t r a n s f e r a s e a l s o bound to a DEAE-Sepharose 6B column at pH 8.5 ( F i g . 17B) . When the column was washed with a l i n e a r s a l t g r a d i e n t a t pH 8.5, t h e c y t i d y l y l t r a n s f e r a s e was e l u t e d a t a c o n d u c t i v i t y c o r r e s p o n d i n g t o 150 mM N a C l . The t o t a l r e c o v e r y of enzyme a c t i v i t y from t h i s column was about 63%. S i n c e DEAE-Sepharose 6B combines ion-exchange chromatography w i t h g e l f i l t r a t i o n , t h i s r e s i n may be p o t e n t i a l l y u s e f u l f o r p u r i f i c a t i o n of c y t i d y l y l t r a n s f e r a s e . C y t i d y l y l t r a n s f e r a s e Binds t o Dymatrex Dye-Ligand Resins (3.3.1.4). In the s e a r c h f o r s t e p s which c o u l d f a c i l i t a t e the p u r i f i c a t i o n of c y t i d y l y l t r a n s f e r a s e , the i n t e r a c t i o n o f t h i s enzyme w i t h d y e - l i g a n d s a t t a c h e d c o v a l e n t l y t o a g a r o s e was i n v e s t i g a t e d . .A p r i o r i i t was impossible to p r e d i c t which of the f i v e Dyematrex r e s i n s marketed by Amicon would be most s u i t a b l e , so a l l were t e s t e d . The most s p e c t a c u l a r r e s u l t s were o b t a i n e d w i t h Dyematrex Blue B. C y t o s o l i c c y t i d y l y l t r a n s f e r a s e was alm o s t completely r e t a i n e d by t h i s r e s i n at pH 6.0, whereas the bulk o f the p r o t e i n washed through the column. Over 85% of the a p p l i e d enzyme a c t i v i t y c o u l d be e l u t e d w i t h 150 mM NaCl, and t y p i c a l l y 40-fold or be t t e r p u r i f i c a t i o n from c y t o s o l was accomplished a f t e r t h i s s i n g l e step ( F i g . 18A). Although these r e s u l t s were h i g h l y r e p r o d u c i b l e (5-times) with the f i r s t b a t c h o f Dyematrex B l u e B, batches obtained from Amicon at l a t e r dates f a i l e d t o d u p l i c a t e t h e s e i n i t i a l f i n d i n g s . C y t i d y l -CYTIDYLYLTRANSFERASE ACTIVITY (nmolmin1) X10 0) w w DJ «< M O «< ct C L ct ~s 0) 3 CO "> a> -t 01 01 CD 01 O Ct i-3 Q> 0 O ci- c+ Ql H-h-" < "J d-01 «< Ct " 5 I—1 I H- O < 3 CD -J c t 3" CD •o ar o o o w M T3 C ar 3 o 3 H- £ •D M ct 3 O t—1 c o o 3 C L C o ct < Ci-VJ 3 0 1 01 * M cr „ cn O o " l -J cr 0) ci- 3 3- O CD fD O 01 «< ct c t H - ro C L O O o —j i-ui H* 3 CL CD -S 01 0 -s •o 0Q ^ s —i 0) . CL CD 3 3 I—1 ct ^— O T 01 ui 0 o ct H- 3 o 3 01 o £ fD ui "J o fD I O U l o o I-1 o M fD 3 o 3: ct fD Z Q- 0) • O t—1 •-3 o <-• r r 3 01 -fcr O -s fD 3 O 3 O < H fD I T O l O 01 C L CD -J - O ~i I— CD cf O C fD Q. H- "O ct 3 H- < fD 01 H - s-^ CL > d - - » «< «< • 01 N -cr ct _* fD W ) fD O Q. ~! 3 T3 0) w s:01 fD 00 O - «< ct -J C L Co CD «< 01 M 3" v ; c+ 3" ^ fD £ 0) H* M C O << ct O O d" d- ar H' o ^ ro o fD 1 £ 1 C ON O • «< O d" - • O CO o S ° I—1 c 3 W u fD fl) " o ft H- 3 M fD CO O _ O I— O 1—1 " l 0) ci" 3 ° 01 0) ^ d - d" O 3* fD -3 fl) CL C" S o o w ' £ 3 »2 g 2 01 o ro. ct • «< W O < ct fD fD 3 '"J* d" 3 *< £ 0) C L W "S fD O I—' •o c d-fD ^ C L ^ S -U l fD •a 3 s ^ 01 fD 3" H-O 3" 01 01 T> "1 fD I fD £ 1 •< C 01 *- X) H- X) cr i—1 era -s fD W 3-0) d--J O 3 C L fD C L 0) 3 O Q . 01 CO I o w ' o o o »< H-d" 3 H- 01 CL d -«< fD < U l - t o 2. -s o fD g O rt- O 2 d- o ^ "> o 3 fD 0) «< 3 «< I—'3 I—' TD d - d - 3C -i ^  ~i 0) r> oi cr> 3 u 3 • ca ^ w ui !I oi ro o o 3 3 C O «< M o d -H- fl) 3 C L ct 3 «< h-1 "O ^ ac > 3 o o 3 fD ~i 0) W fD fD -J fl) 0) 01 3 fD C L C ct fD O C L •< fD £ 3 H- 0) Ct Ct ar -s fD 0) x O f t ON w -J . . fl) O 3 01 0) 3 fD C L -J fl) ro oi o fD O s: 3" o 3" 0) ca fD C L "a - i ct ar fD O O c 3 3 £ 0) w C O ui "i Ct fl) 3" fD C L Ct o 0) 3 > 3 o o 3 fD cn oi o s- ar H- fD 3=> r _ 8 o. 3 H-0) o «< fD N g fD 0) C L ct -J fl) fD CK) X 0) 3d 3 fD Cfl C L c t > U) U l —3 f i . 0 1 M O fD O 0) ^ §. fD C L U l O 0) ° 3 3 C L 2 ro 3 2 -i fD O O < fD -1 «< O 0) o O X 3 CO 0) M ct C fD fD CO ac ^ ro ON • 3 o i—1 cr ro fD o C L 3 < o •z. c 0) 3 O fD o fD I OQ 0) 3 C L o ar ~t o 3 0) c t O Ion ~i oi TI ar !*< o o «< ct O Ca O O «< ct C L Ct "S 01 3 ca •-*) fD 1 A) ca fD •a ac o 03 O O M c c 3 3 C L 3 O U l 3 >  PROTEIN ( Mg) Xtcro CYTIDYLYLTRANSFERASE ACTIVITY (nmolmin1) X10 ABSORBANCE at 280 nm X 1 0 O CYTIDYLYLTRANSFERASE ACTIVITY (nmolmin1) X10 ro co ABSORBANCE at 280 nm X 1 0 O 1 4 2 y l t r a n s f e r a s e completely bound to the subsequent batches of Blue B, but only 15-25% of the a p p l i e d enzyme a c t i v i t y c o u l d be recovered a f t e r e l u t i o n with 0.25 M NaCl ( d a t a not s h o w n ) . A d j u s t m e n t o f t h e pH t o 9.0 or h i g h e r c o n c e n t r a t i o n s of KC1 (up t o 1.5 M) d i d not improve the recovery of enzyme a c t i v i t y from t h i s column. I t seems l i k e l y t h a t the poor s t a b i l i t y of the c y t i d y l y l t r a n s f e r a s e i n d i l u t e s o l u t i o n may be r e s p o n s i b l e f o r the poor recovery. Dyematrex Red A ( F i g . 18B) seemed to show some promise f o r p u r i f i c a t i o n of c y t i d y l y l t r a n s f e r a s e . This r e s i n bound c y t i d y l y l t r a n s f e r a s e , while most of the c y t o s o l i c p r o t e i n s washed t h r o u g h the Red A column. T r a g i c a l l y , as l a t e r found f o r Blue B, 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 could not be recovered i n the column e l u a t e , even a f t e r t r e a t m e n t o f the Red A column with 1.5 M NaCl at pH 8.0. A g a i n , i t i s l i k e l y t h a t i n a c t i v e enzyme was e l u t e d at higher i o n i c s t r e n g t h . I f the c y t i d y l y l t r a n s f e r a s e could be s t a b i l i z e d i n d i l u t e s o l u t i o n , c h r o m a t o g r a p h y on Dyematrex Red A or Blue B c o u l d p o t e n t i a l l y permit a high degree o f p u r i f i c a t i o n . The most c o n s i s t e n t success was o b t a i n e d with Dyematrex Green A r e s i n . C y t i d y l y l t r a n s f e r a s e c o m p l e t e l y bound to Green A a t pH 6.0, but could be e l u t e d w i t h 1 M NaCl b u f f e r . The enzyme was p u r i f i e d 4 - f o l d w i t h 70% recovery of enzyme a c t i v i t y under these c o n d i t i o n s . Step wise e l u t i o n o f the Green A column with 0.25, 0.5 and 1 M NaCl a t pH 8.0 caused the re l e a s e of c y t i d y l y l t r a n s f e r a s e a t each i o n i c s t r e n g t h ( d a t a not shown). A higher degree of p u r i f i c a t i o n could be a c h i e v e d w i t h a l i n e a r gradient of 0.05-1.5 M NaCl, but the t o t a l r e c o v e r y 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 from t h i s column was about 45% ( F i g . 18C). T h i s p a r t i c u l a r r e s i n and the CM-cellulose provided column chromatography e v i d e n c e t h a t 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 e x i s t s as i s o m e r s . The peaks which e l u t e d a t 0.85 M and 1 M NaCl both 143 corresponded to the L-form of the enzyme s i n c e the enzyme i n these f r a c t i o n s required exogenous phospholipid f o r d e t e c t a b l e a c t i v i t y . The a f f i n i t y of c y t i d y l y l t r a n s f e r a s e f o r Dyematrex Orange A and Blue A was a l s o examined. The enzyme d i d not bind to Orange A at pH 7.4 (70% of the a p p l i e d 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 r e c o v e r e d i n the wash). In one s i n g l e e x p e r i m e n t , a l t h o u g h 34% 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 a c t i v i t y a p p l i e d to Blue A was not r e t a i n e d by t h i s r e s i n , 61% of the a p p l i e d enzyme a c t i v i t y was bound and e l u t e d w i t h 1 M NaCl at pH 7.4 w i t h q u a n t i t a t i v e recovery. These f i n d i n g s w i t h Blue A a l s o h i n t e d the existence of isozymes f o r c y t i d y l y l t r a n s f e r a s e i n c y t o s o l . P a r t i a l l y 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 i s S t a b i l i z e d by Lysophosphatidyl- e t h a n o l a m i n e ( 3 . 3 . 1 - 5 ) . A major o b s t a c l e i n the p u r i f i c a t i o n o f r a t l i v e r c y t o s o l i c c y t i d y l -y l t r a n s f e r a s e was the i n s t a b i l i t y o f t h i s enzyme i n d i l u t e s o l u t i o n . When L-form was r e a p p l i e d to a Sepharose 6B column, l e s s than 20% of the ap p l i e d enzyme a c t i v i t y was recovered ( d a t a not shown) . However, i f 0.4 mM o l e o y l -lysophosphatidylethanolamine was i n c l u d e d i n the column b u f f e r , over 80% of the a p p l i e d enzyme a c t i v i t y was r e c o v e r e d as L-form. P h o s p h a t i d y l g l y c e r o l and p h o s p h a t i d y l s e r i n e a l s o s t a b i l i z e d 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 , but th e s e p h o s p h o l i p i d s a l s o promoted a g g r e g a t i o n of the enzyme ( d a t a not shown). 1 4 4 DEVELOPMENTAL STUDIES WITH RAT LIVER A c t i v i t i e s of the Rat L i v e r P h o s p h a t i d y l c h o l i n e B i o s y n t h e t i c Enzymes  During Pre- and P o s t n a t a l Development (3.4.1.1). The a c t i v i t i e s o f the enzymes r e s p o n s i b l e f o r p h o s p h a t i d y l c h o l i n e s y n t h e s i s i n l i v e r were examined as a f u n c t i o n of pre- and p o s t n a t a l age to assess which of these enzymes i s r e s p o n s i b l e f o r the surge o f p h o s p h a t i d y l -c h o l i n e s y n t h e s i s at b i r t h . Between -5 and -1 days, t h e s p e c i f i c a c t i v i t y o f c h o l i n e k i n a s e increased 4.7-fold ( F i g . 19). However, the s p e c i f i c a c t i v i t y of c h o l i n e k i n -ase peaked a day p r i o r to b i r t h and s t e a d i l y d e c l i n e d over the next f i v e days. Although the spike of c h o l i n e kinase a c t i v i t y d i d not c o i n c i d e with the increased r a t e o f p h o s p h a t i d y l c h o l i n e s y n t h e s i s a day l a t e r , the elevated a c t i v i t y of t h i s enzyme p r o b a b l y ensured t h a t s u f f i c e n t phosphocholine was a v a i l a b l e f o r when ph o s p h a t i d y l c h o l i n e formation was s t i m u l a t e d . The a c t i v i t y of c h o l i n e p h o s p h o t r a n s f e r a s e , which peaked at b i r t h , was • 90-fol d higher than 5 days p r i o r ( F i g . 2 0 ) . However on the t e n t h p o s t n a t a l day, cholinephosphotransferase a c t i v i t y was even 2 - f o l d higher than detected at b i r t h . Since p h o s p h a t i d y l c h o l i n e s y n t h e s i s d i d not appear to be enhanced at +10 days, the a c t i v i t y of c h o l i n e p h o s p h o t r a n s f e r a s e did not c o n s i s t e n t l y c o r r e l a t e with the rat e of p h o s p h a t i d y l c h o l i n e s y n t h e s i s throughout develop-ment. Nevertheless, the small peak of cholinephosphotransferase a c t i v i t y at b i r t h may have been necessary to accomodate an increased production o f CDP-c h o l i n e . As shown i n Figure 21, the s p e c i f i c a c t i v i t y o f c y t o s o l i c c y t i d y l y l -t r a n s f e r a s e was reduced 67% between -1 and +1 days w h i l e the microsomal c y t i d y l y l t r a n s f e r a s e s p e c i f i c a c t i v i t y was i n c r e a s e d 3-3-fold during t h i s -1 1 3 5 10 AGE (days) 20 A F i g u r e 19. A c t i v i t y o f r a t l i v e r c h o l i n e  k i n a s e d u r i n g development. F e t a l and p o s t -n a t a l r a t s were d e c a p i t a t e d and the excised l i v e r s homogenized i n 0.145 M NaCl; 10 mM T r i s -HCl, pH 7.0; 10 mM NaF and 1 mM EDTA. Cytosol and microsomes were prepared as described i n Section 2.2.2.1. The microsomes were resus-pended i n 0.25 M s u c r o s e ; 10 mM T r i s - H C l , pH 7.0; 10 mM NaF and 1 mM a f t e r 10 s t r o k e s up and down w i t h a g l a s s Dounce homogenizer. Choline k i n a s e a c t i v i t y : nmol-min""1-mg pro-t e i n i c * ) and nmol-min^-g l i v e r - 1 ( A ) . Each p o i n t r e p r e s e n t s the mean of 4-6 animals and the standard e r r o r i s i n d i c a t e d by bars. "A" represents 175-225 g r a t s . Ln - 1 1 3 5 10 AGE (days) 15 2 0 A Figure 20. A c t i v i t y o f r a t l i v e r c h o l i n e - phosphotransferase d u r i n g development. Rat l i v e r microsomes were prepared from f e t a l and po s t n a t a l r a t s as described i n F i g . 19. Cho-linephosphotransferase a c t i v i t y : nmol-min - 1 - mg p r o t e i n - 1 ( A , A ) and nmol-min - 1-g l i v e r " 1 (• , • ). Open symbols, dashed l i n e s , enzyme a c t i v i t y without exogenous d i g l y c e r i d e . Closed symbols, s o l i d l i n e s , enzyme a c t i v i t y with exogenous d i g l y c e r i d e . Each po i n t r e p r e -sents the mean of 4-6 animals and S.E. i s shown by bars. 146 same 24 h p e r i o d . By the t h i r d p o s t n a t a l day, the 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 a c t i v i t y was again e l e v a t e d , while the microsomal enzyme a c t i v i t y was returned to -1 day values. Hence the s t i m u l a t i o n of PC s y n t h e s i s observed at b i r t h ( 3 3 5 , 3 3 6 ) c o r r e l a t e d w e l l w i t h a t r a n s l o c a t i o n of the c y t i d y l y l t r a n s -f erase to the endoplasmic r e t i c u l u m . Most of the c y t i d y l y l t r a n s f e r a s e pro-t e i n returned to the c y t o s o l by the t h i r d p o stnatal day, and a smaller secondary increase i n the microsomal enzyme a c t i v i t y occurred on the f i f t h p o s t - n a t a l day. This second minor peak o f microsomal 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 presumably due to increased p r o t e i n s y n t h e s i s s i n c e both the c y t o s o l i c and microsomal enzyme a c t i v i t i e s were elevated at t h i s time. During the f i r s t few weeks o f p o s t - n a t a l development, the r a t e of PC production seems to o p e r a t e a t a g r e a t e r c a p a c i t y than found i n a d u l t r a t l i v e r . In t h i s r e s p e c t , i t i s i n t e r e s t i n g t h a t the microsomal a c t i v i t y i n the +19 day o l d r a t l i v e r was s t i l l 3 . 3 - f o l d higher than measured f o r a d u l t r a t l i v e r , while the 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 a c t i v i t y (measured i n the presence of phospholipid) was lower ( F i g . 21B). Dramatic changes i n the a c t i v i t y o f p h o s p h a t i d y l e t h a n o l a m i n e methyl-t r a n s f e r a s e were a l s o noted d u r i n g e a r l y development ( F i g . 22). Between - 5 and +10 days, the m e t h y l t r a n s f e r a s e a c t i v i t y was s t e a d i l y e s c a l a t e d 17-f o l d . A l t h o u g h the m e t h y l t r a n s f e r a s e a c t i v i t y d e c l i n e d t h e r e a f t e r , the a c t i v i t i e s i n +19 day o l d and a d u l t r a t l i v e r s were s t i l l 2 - f o l d or higher than pre- and neonatal values. 1 4 7 -5 -11 3 5 10 15 20 A AGE (days) -5 -11 3 5 10 15 20 A AGE (days) Figure 21. A c t i v i t i e s of r a t l i v e r c y t o s o l i c and microsomal c y t i d y l y l - t r a n s f e r a s e during development. Rat l i v e r c y t o s o l and microsomes were obtained from pre- and p o s t n a t a l r a t s as described i n F i g . 19. 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 : P a n e l A, nmol- min - 1-mg p r o t e i n - 1 and Panel B, nmol-min - 1-g l i v e r - 1 . C y t o s o l i c a c t i v i t y ( A ) ; c y t o s o l i c a c t i v i t y i n presence of t o t a l r a t l i v e r phospholipid ( • ); and microsomal a c t i v i t y ( o ) . Each p o i n t represents the mean of 4-6 animals and the standard e r r o r i s i n d i c a t e d by b a r s . "I— —n—i— l 1 1 AS. 1—r -•VsS */ */ ^ -- * a . T -• i . ' i . - i . . . • • • _i—i 12 - 1 1 3 5 10 15 A G E (days) 20 A <] I I 6 = O) § 3 1 Figure 22. A c t i v i t y of r a t l i v e r PE m e t h y l t r a n s f e r a s e during development. Rat l i v e r microsomes were prepared from f e t a l and p o s t n a t a l r a t s as described i n F i g . 19. PE m e t h y l t r a n s f e r a s e a c t i v i t y : nmol-min-1-mg p r o t e i n " ( O ) and nmol-min - 1-g l i v e r - 1 ( A ) . Each point represents the mean of 4-6 animals and S.E. i s shown by bars. 1 4 8 DIURNAL RHYTHM STUDIES WITH RAT LIVER Choline Kinase and Microsomal 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 i e s are Both  Reduced L a t e a t N i g h t (3.5-1.1). The a c t i v i t i e s of many r e g u l a t o r y enzymes f l u c t u a t e d u r i n g the day, so the a c t i v i t i e s of c h o l i n e kinase and c y t i d y l y l t r a n s f e r a s e were determined as a f u n c t i o n of the time of the day ( F i g . 2 3 ) . The 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 a c t i v i t y was h i g h and the m i c r o s o m a l a c t i v i t y low during the n i g h t when these n o c t u r n a l c r e a t u r e s were f e e d i n g . However, between noon and e a r l y evening, there was a r i s e i n the m i c r o s o m a l 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 concomitant drop i n the c y t o s o l i c enzyme a c t i v i t y ( F i g . 23B). During t h i s same p e r i o d , c h o l i n e k i n a s e a c t i v i t y was a l s o e l e v a t e d ( F i g . 23D). These data suggest that hepatic p h o s p h a t i d y l c h o l i n e s y n t h e s i s i s most a c t i v e when the r a t s have c o n c l u d e d d i g e s t i n g and a b s o r b i n g t h e i r food from the sma l l i n t e s t i n e . 1 4 9 ( J 1 5 9 13 17 21 1 1 5 9 13 17 21 1 TIME 24 h clock Figure 23. Di u r n a l rhythm o f r a t l i v e r c h o l i n e kinase and c y t i d y l y l t r a n s - f e r a s e • Six r a t s were s a c r i f i c e d e v e r y 4 h, and the excised l i v e r s were homogenized i n 4 volumes of 0.25 M sucrose; 20 mM T r i s - H C l , pH 7.0 with 10 strok e s up and down i n a Potter-Elvehjem homogenizer. S u b c e l l u l a r f r a c t i o n -a t i o n was performed as des c r i b e d i n Sectio n 2.2.2.1 and the c h o l i n e kinase and 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 i e s were determined. 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 a c t i v i t y was assayed i n the presence of s a t u r a t i n g amounts of t o t a l r a t l i v e r p h o s p h o l i p i d . Panels A & B, c y t i d y l y l t r a n s f e r -ase a c t i v i t y i n the c y t o s o l ( O ) and microsomes ( • ). Panels C & D, c h o l i n e kinase a c t i v i t y ( A ) . Each p o i n t represents the mean of 6 animals and the standard d e v i a t i o n i s i n d i c a t e d by bars. 1 5 0 FASTING STUDIES WITH RAT LIVER Choline Kinase and Microsomal 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 i e s are Both  Reduced by 24 h F a s t i n g o f t h e Rat (3.6.1.1). Female W i s t a r r a t s (100-125 g) were f a s t e d f o r up t o 48 h p r i o r to s a c r i f i c e and t h e a c t i v i t i e s o f t h e d_e novo p h o s p h a t i d y l c h o l i n e b i o s y n t h e t i c enzymes were measured as a f u n c t i o n o f s t a r v a t i o n ( F i g . 24). F a s t i n g f o r two days reduced the mean l i v e r weight by 33% compared to r a t s f e d ac[ l i b i t u m . C h o l i n e k i n a s e a c t i v i t y was reduced 37% (p<0.005) a f t e r 48 h f a s t i n g , a l t h o u g h 13% i n h i b i t i o n (p<0.05) was evident, even a f t e r a 12 h f a s t . The 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 a c t i v i t y was increased 1.5-fold (p<0.005) a f t e r 12 h and remained e l e v a t e d f o r up t o 36 h. On the other hand, microsomal 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 decreased by 19% (p<0.05) a f t e r 12 h and 30% (p<0.005) a f t e r a 24 h f a s t . Both the microsomal and 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 a c t i v i t i e s returned to c o n t r o l values a f t e r 48 h f a s t i n g . The s p e c i f i c a c t i v i t y of c h o l i n e p h o s p h o t r a n s f e r -ase d i d not s i g n i f i c a n t l y f l u c t u a t e d u r i n g f a s t i n g . Groener e t a l . (273) reported that c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y expressed as nmol/min/ g l i v e r was reduced by 50% a f t e r 48 h s t a r v a t i o n . In our hands, however, cho-linephosphotransferase a c t i v i t y e x p r e s s e d i n t h i s manner was 23+3 i n fed r a t s and 27+3 i n f a s t e d r a t s . C y t i d y l y l t r a n s f e r a s e Seems t o be T r a n s l o c a t e d D u r i n g F a s t i n g of the Rat (3.6.1.2). The r e d u c t i o n of m i c r o s o m a l 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 concomitant increase i n the c y t o s o l i c enzyme a c t i v i t y i m p l i e d a t r a n s l o c a -t i o n event. This was p a r t i a l l y s u b s t a n t i a t e d by the f i n d i n g t h a t the t o t a l 1 5 1 0) o Q. E T-C | o E c .24- T T 1 i B 0.14-.05 CYTIDYLYLTRANSFERASE 2.51 i 1.5 1 0.5 CYTIDYLYLTRANSFERASE CHOLINE KINASE ± _L CHOLINEPHOSPHO-TRANSFERASE ± 12 24 36 TIME FASTED (h) 48 1.2 24 36 TIME FASTED (h) 48 A c t i v i t i e s of t h e de novo p h o s p h a t i d y l c h o l i n e b i o s y n t h e t i c Figure 24 enzymes o f r a t l i v e r d u r i n g s t a r v a t i o n .  the food was removed from t h e i r cages up to 48 h p r i o r t o s a c r i f i c e Rats were fed ad l i b i t u m or A l l r a t s had access to tap water and were k i l l e d by carbon d i o x i d e a s p h y x i a t i o n at around 9 a.m. The l i v e r s were^ e x c i s e d , s u b c e l l u l a r f r a c t i o n a t i o n performed, and the enzyme a c t i v i t i e s determined as described i n " E x p e r i -mental Procedures." P r o t e i n was d e t e r m i n e d by the method of Lowry et a l . ( S e c t i o n 2.3-1.1). A, 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 , c y t o s o l i c ( O ) and microsomal ( • ) . B, 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 a c t i v i t y measured i n the presence of ph o s p h o l i p i d l i p o s o m e s ( T ) . C, c h o l i n e kinase a c t i v i t y ( A ) . D, c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y ( • ) . Each po i n t represents the mean of 8-16 animals and the standard d e v i a t i o n i s shown by bars. 1 5 2 microsomal 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 reduced by 40% at 24 h and in c r e a s e d again t o w i t h i n 19% o f c o n t r o l s a t 48 h. s t a r v a t i o n ( F i g . 25). A corresponding increase i n the t o t a l c y t o s o l i c enzyme a c t i v i t y could not be dis c e r n e d since the changes i n the m i c r o s o m a l a c t i v i t i e s were w i t h i n the standard d e v i a t i o n s o f the c y t o s o l i c v a l u e s . However, a f t e r a d d i t i o n of t o t a l r a t l i v e r p h o s p h olipid t o the c y t o s o l i c enzyme assays, more a c t i v i t y was g e n e r a l l y detected i n the sample from faste d animals ( F i g . 24b and 26). This enhanced a c t i v i t y was due t o i n c r e a s e d amounts of L-form i n these c y t o s o l s ( F i g . 2 7 ) . Hence a t r a n s l o c a t i o n e v e n t c o u l d e x p l a i n t h e a l t e r a t i o n s 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 during the f i r s t day and a h a l f of s t a r v a t i o n . The i n c r e a s e d amount o f c y t i d y l y l t r a n s f e r a s e p r o t e i n i n the l i v e r c y t o s o l and microsome samples from 48 h f a s t e d r a t s i n d i c a t e d t h a t more enzyme may have been produced through new p r o t e i n s y n t h e s i s . F a s t i n g Increases the Level o f a 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 A c t i v a t o r (3.6.1.3). S i n c e t h e d i f f e r e n c e between t h e 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 a c t i v i t i e s o f f e d and 24 h f a s t e d r a t s was reduced upon i n c l u s i o n o f ph o s p h o l i p i d i n the enzyme assay, i t was p o s s i b l e that increased amounts of an a c t i v a t o r was present i n the c y t o s o l s o f fasted r a t s . Attempts were made to q u a n t i f y and p a r t i a l l y i s o l a t e t h i s a c t i v a t o r from l i v e r c y t o s o l (Table 20 and F i g . 28). The endogenous c y t i d y l y l t r a n s f e r a s e was digested w i t h t r y p -s i n , and the t r y p s i n was s u b s e q u e n t l y i n a c t i v a t e d w i t h soybean t r y p s i n i n h i b i t o r . When L-form was added t o the t r y p s i n i z e d c y t o s o l s , the c y t o s o l from 24 h fa s t e d r a t s produced 1 . 9 - f o l d more s t i m u l a t i o n of enzyme a c t i v i t y t h a n c y t o s o l f r o m f e d r a t s . T h e a c t i v a t o r was a p p a r e n t l y 1 5 3 t r y p s i n - i n s e n s i t i v e . When t h e t r y p s i n i z e d c y t o s o l s were a p p l i e d t o a Sepharose 6B column, o v e r 67% o f t h e a c t i v a t o r from f a s t e d r a t s was recovered i n the v o i d volume o f the column. The subsequent e x t r a c t i o n of the p o o l e d v o i d volume w i t h CHC1 /CH OH a b o l i s h e d t h e d i f f e r e n c e 3 3 between the samples from c o n t r o l and f a s t e d r a t s . A l l o f the remaining L-form a c t i v a t i n g p o t e n t i a l was c o n f i n e d t o the lower phase of the l i p i d e x t r a c t i o n , which i n d i c a t e d that the a c t i v a t o r might be a l i p i d . The p r e c i s e i d e n t i t y of the a c t i v a t o r which was e l e v a t e d i n l i v e r c y t o s o l s from f a s t e d r a t s remains a mystery. TIME FASTED (h) Figure 25. S u b c e l l u l a r d i s t r i b u t i o n o f 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 d u r i n g  s t a r v a t i o n . Rats were handled as d e s c r i b e d i n F i g u r e 24. S o l i d b ars, microsomal 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 . Half-tone bars, 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 a c t i v i t y measured i n the presence o f t o t a l r a t l i v e r p h o s p h o l i p i d . Height of the bar represents the mean of 4 animals and standard d e v i a t i o n i s i n d i c a t e d . 1 5 4 z v ... -°~ O JL 5 10 15 2 0 [PHOSPHOLIPID] (mg-mr1) Figure 26. A c t i v a t i o n of l i v e r c y t o s o l i c c y t i d y l y l t r a n s f e r a s e from fed and  f a s t e d r a t s by p h o s p h o l i p i d . Rat l i v e r c y t o s o l was 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 i n the presence of 0-20 mg/ml t o t a l r a t l i v e r p h o s p h o l i p i d . Fed ( O ) ; 12 h fa s t e d ( A ) ; 24 h fasted ( v ) and 36 h f a s t e d ( • ) . c § o E ,e >. t-S g J" CO 1 2 0 16 12 1 I T "T I T i i l 9 -/\ \ -ih -\\ 11 • / * ; \\\ - / / • ' i \ i -Vi - jjl Jtl J -i 1 T^ *-8 10 12 14 16 18 2 0 FRACTION NUMBER F i g u r e 27. Gel f i l t r a t i o n chromatography o f l i v e r c y t o s o l i c c y t i d y l y l - t r a n s f e r a s e from fed and f a s t e d r a t s . Rat l i v e r c y t o s o l (5 ml) was a p p l i e d t o a Sepharose 6B (180 ml bed volume) column e q u i l i b r a t e d with b u f f e r (100 mM NaCl; 20 mM T r i s - H C l , pH 7.0). C y t i d y l y l t r a n s f e r a s e was elu t e d i n 250 drop (7.9 ml) f r a c t i o n s and the enzyme a c t i v i t y measured i n the presence of t o t a l r a t l i v e r p h o s p h o l i p i d . Fed ( o ) ; 12 h fasted ( A ) ; 24 h f a s t e d ( V ) and 36 h fasted ( • ) . This experiment was repeated 4 times with d i f f e r e n t c y t o s o l p r e p a r a t i o n s , but with s i m i l a r r e s u l t s . Figure 28. P a r t i a l p u r i f i c a t i o n of a l i v e r  c y t o s o l i c a c t i v a t o r o f c y t i d y l y l t r a n s f e r a s e  from fed and 24 h f a s t e d r a t s . L i v e r c y t o s o l s from fed and 24 h fasted r a t s were t r e a t e d with 0.2 mg/ml t r y p s i n f or 15 min at 37° C.. P r o t e o l y s i s was terminated upon a d d i t i o n of 0.3 mg/ml soybean t r y p s i n i n h i -b i t o r . Endogenous 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 abolished i n the t r y p s i n i z e d c y t o s o l s . The t r y p s i n i z e d c y t o s o l s (10-50 u l ) were incubated with 20 p i of p a r t i a l l y p u r i -f i e d L-form ( P a n e l A) , and 50. u l of the t r y p s i n i z e d c y t o s o l s were incubated with 5-20 u l of L-form ( P a n e l B) . The t r y p s i n i z e d c y t o s o l s (5 ml) were subjected to Sepharose 6B chromatography and the void volumes c o l l e c t e d . The void volumes (10-50 u l ) were incubated with 20 Ml of L-form ( P a n e l C ) , and 50 p i of the void volume were incubated with 5-20 p i of L-form (Panel D) . The v o i d volume s o l u t i o n s were e x t r a c t e d by the method of Folch et  a l . ( S e c t i o n 2.2.5.1) upon a d d i t i o n of chloroform and methanol. The lower phase was evaporated and resuspended i n t o 5 ml of d i s t i l l e d water. F i f t y p i of t h i s l i p i d e x t r a c t were incubated with 5-20 p i of L-form ( P a n e l E ) . Open symbols, s o l i d l i n e s , from l i v e r c y t o s o l p o oled from 2 fed r a t s . Closed  symbols, dashed l i n e s , from l i v e r c y t o s o l pooled from two 24 h fasted r a t s . S i m i l a r r e s u l t s were obtained with preparations of l i v e r c y t o s o l from other fed and 24 h fasted r a t s . •A 12 h -4 e\-H 4 h — ! 1 r B + 50 ul of TRYPSINIZED CYTOSOL 0 15 30 45 60 VOLUME TRYPSINIZED CYTOSOL T 5 10 15 20 VOLUME L-FORM (u l ) r 0 15 30 45 60 VOLUME "VOID VOLUME" (u l ) 1 5 10 15 20 VOLUME L-FORM (u l ) ~ i r E + 50 ul of. CHC1 3 / EXTRACT 5 10 15 20 VOLUME L-FORM (p i ) 1 5 6 Table 20. P a r t i a l P u r i f i c a t i o n of a C y t o s o l i c A c t i v a 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) SPECIFIC ACTIVITY OF ENDOGENOUS CYTOSOLIC CYTIDYLYLTRANSFERASE i ) A c t i v i t y p r i o r to t r y p s i n i z a t i o n . (nmol/ min-mg protein) Fed 24h Fasted Fasted/Fed without phospholipid 0.12 0.81 1.9 with phospholipid 2.6 3.1 1-2 i i ) A c t i v i t y a f t e r t r y p s i n i z a t i o n . (nmol/ min-mg protein) 0.00 0.00 B) PARTIAL PURIFICATION AND QUANTITATION OF CYTOSOLIC ACTIVATOR Volume Units/ml Total Units (ml) i ) Trypsinized c y t o s o l , Fed 5 9.1 17 21 h Fasted 5 17.6 88 i i ) Void Volume of Sepharose 6B Column. Fed 17.0 0.6 10 21 h Fasted 16.9 3.5 59 i i i ) Chloroform/Methanol Extract. Fed 5 5.0 25 21 h Fasted 5 4.9 25 1 u n i t = amount of a c t i v a t o r which stimulated exogenous L-form a c t i v i t y by 10,000 cpm i n a 15 min c y t i d y l y l t r a n s f e r a s e assay. 1 5 7 GLUCAGON AND cAMP REGULATION OF PHOSPHATIDYLCHOLINE BIOSYNTHESIS IN MONOLAYER CULTURES OF RAT HEPATOCYTES INFLUENCE OF cAMP ANALOGUES AND PHOSPHODIESTERASE INHIBITORS ON THE CDP-CHOLINE PATHWAY cAMP Analogues and Phosphodiesterase I n h i b i t o r s I n i t i a l l y Cause an I n h i -3 b i t i o n and Later a S t i m u l a t i o n o f [ M e - — H ] C h o l i n e I n c o r p o r a t i o n i n t o P h o s p h a t i d y l c h o l i n e (3.7.1.1). The time-dependent e f f e c t s of v a r i o u s cAMP analogues and phosphodi-3 esterase i n h i b i t o r s on the uptake of [Me- H ] c h o l i n e are shown i n Figure 29. A f t e r 90 min treatment, uptake was reduced by aminophylline (9%, p<0.05) and c h l o r o p h e n y l t h i o - c A M P (28%, p<0.001). Br-cAMP had no e f f e c t , while 3 [Me- H j c h o l i n e uptake was s t i m u l a t e d by IBMX ( 9 % , p<0.05) and d i b u t y r y l -cAMP (28%, p<0.01). The various e f f e c t s o f these analogues and i n h i b i t o r s on c h o l i n e uptake remained unaltered f o r at l e a s t 15 h. In the same e x p e r i m e n t , the t i m e - d e p e n d e n t e f f e c t s o f t h e s e cAMP 3 analogues and p h o s p h o d i e s t e r a s e i n h i b i t o r s on [Me- H ] c h o l i n e i n c o r p o r a -t i o n i n t o p h o s p h a t i d y l c h o l i n e was i n v e s t i g a t e d . Since the various compounds had d i f f e r e n t e f f e c t s on c h o l i n e u p t a k e , the r e l a t i v e i n c o r p o r a t i o n of 3 [Me- H]choline i n t o p h o s p h a t i d y l c h o l i n e was e s t i m a t e d ( F i g . 30). A l l the cAMP anal o g u e s and p h o s p h o d i e s t e r a s e i n h i b i t o r s i n i t i a l l y reduced the 3 r e l a t i v e i n c o r p o r a t i o n of [Me H ] c h o l i n e i n t o p h o s p h a t i d y l c h o l i n e but d i f f e r e d i n t h e i r degree o f i n h i b i t i o n . At 3 h, a 40% reduction was noted f o r c h l o r o p h e n y l t h i o - c A M P - , Br-cAMP-, d i b u t y r y l - c A M P - and ami n o p h y l l i n e -t r e a t e d c e l l s . The cAMP analogues produced a comparable i n h i b i t i o n i n the Figure 29- E f f e c t o f cAMP analogues and p h o s p h o d i e s t e r a s e i n h i b i t o r s on  [ M e - 3 H ] c h o l i n e uptake by c u l t u r e d r a t h e p a t o c y t e s . C e l l s (3 X 10 y) were preincubated f o r 0-13.5 h w i t h v a r i o u s cAMP analogues and phospho-d i e s t e r a s e i n h i b i t o r s . The medium was replaced and the c e l l s were pulsed with 10 uCi of [ M e - 3 H j c h o l i n e (0.14 Ci/mmol) per d i s h f o r an a d d i t i o n a l 90 min i n the presence o f the analogues and i n h i b i t o r s p r i o r to h a r v e s t i n g . Total [Me- 3 H ] c h o l i n e uptake was estimated by q u a n t i t a t i o n of t o t a l c e l l u l a r r a d i o a c t i v i t y and medium r a d i o a c t i v i t y corresponding to betaine. Incorporation of the l a b e l : untreated ( O ) ; 0.2 mM IBMX (T); 1.0 mM am i n o p h y l l i n e ( A ) ; 0.5 mM c h l o r o p h e n y l t h i o - c A M P ( • ) ; 0.5 mM Br-cAMP ( • ) ; and dibutyryl-cAMP ( • ) . IBMX (0.2 mM) was included with the cAMP analogues to prevent t h e i r h y d r o l y s i s . Each point represents the mean of three d i s h e s , and the standard e r r o r i s i n d i c a t e d by bars. This e x p e r i -ment was performed three t i m e s with s i m i l a r r e s u l t s . F i g u r e 30. E f f e c t o f cAMP anal o g u e s and p h o s p h o d i e s t e r a s e i n h i b i t o r s  on the r e l a t i v e i n c o r p o r a t i o n o f [Me - ^ H J c h o l i n e i n t o p h o s p h a t i d y l c h o l i n e . Hepatocytes were t r e a t e d with v a r i o u s compounds as described i n Figure 29. R e l a t i v e i n c o r p o r a t i o n i n t o p h o s p h a t i d y l c h o l i n e was estimated by d i v i s i o n of the r a d i o a c t i v i t y i n p h o s p h a t i d y l c h o l i n e by the t o t a l amount of r a d i o -a c t i v i t y taken up by the c e l l s . R e l a t i v e i n c o r p o r a t i o n of the l a b e l : untreated ( • ) ; 0.2 mM IBMX ( v ) ; 1 .0 mM aminophylline ( A ) ; 0.5 mM chlorophenylthio-cAMP ( O ) ; 0.5 mM Br-cAMP (•) and 0.5 mM dibutyryl-cAMP ( 0 ) . IBMX (0.2 mM) was i n c l u d e d w i t h the cAMP analogues t o prevent t h e i r h y d r o l y s i s . Each point represents the mean of three d i s h e s , and the standard e r r o r s were a p p r o x i m a t e l y 6% o f the mean. This experiment was r e p e a t e d t h r e e t i m e s w i t h s i m i l a r r e s u l t s . 0 ^ 2 0 s? LU 2 18 _J g : U 12 rx1 • 6 9 12 TIME (h) 10 ? 6 li. o z o o £ 4 o o 5 2 LU > < uJ 0 CE 1 T j p- i — p * * * i i t i i I f f • * t i $ # / — — — i I i 1 $ I 1 I I F / / N O * • i i 6 9 TIME (h) 12 15 1 5 9 absence of 0.2 mM IBMX (data not shown). However, by 6-9 h, c h l o r o p h e n y l -thio-cAMP no longer i n h i b i t e d the r e l a t i v e i n c o r p o r a t i o n i n t o p h o s p h a t i d y l -c h o l i n e , and the r e l a t i v e i n c o r p o r a t i o n was s t i m u l a t e d 6.5-fold by 15 h. Br-cAMP and aminophylline s t i m u l a t e d the r e l a t i v e i n c o r p o r a t i o n i n t o phospha-t i d y l c h o l i n e a t 12 h. By 15 h t r e a t m e n t , IBMX and d i b u t y r y l - c A M P a l s o s l i g h t l y s t i m u l a t e d ( 1 . 4 - f o l d , p<0.05) the r e l a t i v e i n c o r p o r a t i o n . The time-dependent e f f e c t s o f c h l o r o p h e n y l t h i o - c A M P and IBMX on the 3 i n c o r p o r a t i o n of [Me- H ] c h o l i n e i n t o c e l l u l a r p h o s p h o c h o l i n e and betaine 3 are shown i n F i g u r e 31. A f t e r 90 m i n , t h e i n c o r p o r a t i o n o f [Me~ H ] -c h o l i n e i n t o phosphocholine was reduced by 40%. S i m i l a r l y , a 38% red u c t i o n i n the i n c o r p o r a t i o n i n t o betaine was a l s o recorded f o r hepatocytes t r e a t e d 3 with the analogue and IBMX. The amount o f H l a b e l i n the phosphocholine pool and o x i d i z e d to b e t a i n e f o r both u n t r e a t e d and chlorophenylthio-cAMP and IBMX-treated c e l l s remained c o n s t a n t between 1.5 and 15 h. The reduced amount of l a b e l i n p h o s p h o c h o l i n e and b e t a i n e f o r ch l o r o p h e n y l t h i o - c A M P t r e a t e d c e l l s l a r g e l y r e f l e c t e d the r e d u c t i o n i n c h o l i n e uptake. IBMX alone 3 s t i m u l a t e d [Me- H ] c h o l i n e i n c o r p o r a t i o n i n t o b e t a i n e by a p p r o x i m a t e l y 19%, and de c r e a s e d i n c o r p o r a t i o n i n t o p h o s p h o c h o l i n e by 20% r e l a t i v e to untreated c e l l s between 1.5 and 15 h (data not shown). Chloropheriylthio-cAMP Reduces the P o o l S i z e o f Phosphocholine (3.7.1.2). Although i n i t i a l exposure o f h e p a t o c y t e s t o c h l o r o p h e n y l t h i o - c A M P 3 produced an i n h i b i t i o n of [Me- H ] c h o l i n e i n c o r p o r a t i o n i n t o phospha-t i d y l c h o l i n e and prolonged exposure a s t i m u l a t i o n , these changes need not have r e f l e c t e d a c t u a l changes i n the r a t e s o f p h o s p h a t i d y l c h o l i n e s y n t h e s i s . A l t e r n a t i v e l y , isotope d i l u t i o n e f f e c t s might have explained the data. For example, i n i t i a l exposure to c h l o r o p h e n y l t h i o - c A M P might have increased the 3 6 9 12 TIME (h) 6 9 12 15 TIME (h) Figure 31. E f f e c t of c h l o r o p h e n y l t h i o - c A M P on [Me- 3H]choline i n c o r p o r a - t i o n i n t o phosphocholine and b e t a i n e . C u l t u r e d r a t hepatocytes i n 60 mm dishes were preincubated f o r 0-13.5 h with chlorophenylthio-cAMP (0.5 mM) and IBMX (0.2 mM), and then p u l s e - l a b e l e d f o r an a d d i t i o n a l 90 min with 10 uCi of [Me- 3H]choline (0.14 Ci/mmol) per d i s h . The betaine released i n t o the medium was accounted f o r i n the e s t i m a t i o n o f t o t a l betaine pro-duced. Incorporation of the l a b e l - A, c e l l u l a r phosphocholine: untreated ( O ) and ch l o r o p h e n y l t h i o - c A M P - and IBMX-treated ( • ) . B, be t a i n e : un-tr e a t e d (•) and chlorophenylthio-cAMP- and IBMX-treated ( • ) . Each p o i n t represents the mean of three s e p a r a t e experiments and the standard e r r o r s between experiments are shown. ON O 4 8 12 TIME (h) 16 F i g u r e 32. E f f e c t o f c h l o r o p h e n y l t h i o - c A M P on the c e l l u l a r phospho- ch o l i n e pool s i z e . H e patocytes were i n c u b a t e d i n the absence or presence o f 0.5 mM c h l o r o p h e n y l t h i o - c AMP f o r 1.5-15 h p r i o r t o h a r v e s t . Phosphocholine concentrations were measured i n the water-soluble e x t r a c t of the c e l l s as d e s c r i b e d i n S e c t i o n 2 . 3 - 5 . 1 . T h i s a s s a y r e l i e d on the c o n v e r s i o n of p h o s p h o c h o l i n e and [ 3 H ] C T P i n t o [ 3 H ] C D P - c h o l i n e by p a r t i a l l y p u r i f i e d CTP: p h o s p h o c h o l i n e c y t i d y l y l t r a n s f e r a s e . C o n t r o l ( O ) and c h l o r o p h e n y l t h i o - cAMP-treated ( • ) . Each p o i n t represents the mean of three dishes, and the standard e r r o r i s i n d i c a t e d by bars. 1 6 1 pool s i z e of phosphocholine, while prolonged exposure to t h i s analogue might have decreased the s i z e of t h i s p o o l i n t r e a t e d hepatocytes. The pool s i z e s of phosphocholine i n c o n t r o l and 0.5 mM chlorophenylthio-cAMP tr e a t e d c e l l s were measured as a f u n c t i o n o f t i m e ( F i g . 3 2 ) . In c o n t r o l c e l l s the phosphocholine concentration was s t e a d i l y reduced over 12 h by approximately 2 - f o l d , whereas i n t r e a t e d c e l l s the c o n c e n t r a t i o n was reduced almost 3 -3 f o l d . Hence the r e d u c t i o n of [Me- H ] c h o l i n e i n c o r p o r a t i o n i n t o phospha-t i d y l c h o l i n e seen i n analogue t r e a t e d h e p a t o c y t e s c o r r e s p o n d e d w i t h an i n h i b i t i o n of the a c t u a l r a t e of p h o s p h a t i d y l c h o l i n e s y n t h e s i s . Furthermore, the r e d u c t i o n of the p h o s p h o c h o l i n e p o o l s i z e i n c h l o r o p h e n y l t h i o - c A M P t r e a t e d c e l l s r e l a t i v e t o c o n t r o l c e l l s c o u l d account for only a f r a c t i o n 3 of the a c c e l e r a t e d r a t e o f t r a n s f e r o f t h e H l a b e l i n t o p h o s p h a t i d y l -c h o l i n e . The r e d u c t i o n i n the p h o s p h o c h o l i n e l e v e l s i n c o n t r o l and t r e a t e d hepatocytes probably r e f l e c t e d a d e p l e t i o n o f c h o l i n e i n the medium during extended i n c u b a t i o n s s i n c e h e p a t o c y t e s w i l l i n c o r p o r a t e 9% of the medium c h o l i n e i n the f i r s t h. 1 6 2 Chlorophenylthio-cAMP I n h i b i t s C h o l i n e Uptake (3-7.1-3)-Since c h l o r o p h e n y l t h i o - c A M P produced the most marked e f f e c t on [Me-3 H ] c h o l i n e uptake by r a t h e p a t o c y t e s , t h e e f f e c t of t h i s compound on c h o l i n e t r a n s p o r t was examined i n greater d e t a i l . The c o n c e n t r a t i o n dependence f o r the acute i n h i b i t i o n of c h o l i n e uptake by chlorophenylthio-cAMP i s shown i n F i g u r e 33- At analogue con c e n t r a t i o n s below 0.1 mM there was no s i g n i f i c a n t i n h i b i t i o n of c h o l i n e uptake. With 2.5 mM chlorophenylthio-cAMP, c h o l i n e uptake was reduced 2.4-fold compared to c o n t r o l s . The k i n e t i c s o f c h o l i n e u p t a k e by r a t h e p a t o c y t e monolayers was 3 i n v e s t i g a t e d . The t o t a l i n c o r p o r a t i o n o f [Me- H ] c h o l i n e i n t o c e l l u l a r c h o l i n e and i t s m e t a b o l i t e s was l i n e a r f o r up t o 30 min (data not shown). Hepatocytes were a b l e t o p r o l o n g the i n i t i a l r a t e of c h o l i n e t r a n s p o r t , probably because c h o l i n e does not a c c umulate w i t h i n t h e s e c e l l s but i s e i t h e r r a p i d l y phosphorylated or o x i d i z e d to b e t a i n e . The uptake of c h o l i n e i n c l u d e d s a t u r a b l e and n o n - s a t u r a b l e components i n the absence or presence of 0.5 mM c h l o r o p h e n y l t h i o - c A M P ( F i g . 3 4 ) . For c h o l i n e c o n c e n t r a t i o n s i n excess of 40 ^iM, the i n c o r p o r a t i o n of c h o l i n e was a l i n e a r f u n c t i o n of medium c o n c e n t r a t i o n . The s a t u r a b l e component was estimated by s u b t r a c t i o n of the l i n e a r component from the e x p e r i m e n t a l data. The values derived f o r the s a t u r a b l e component of the u p t a k e were r e p l o t t e d by the method o f E i s e n t h a l and Cornish-Bowden (453) f o r the e s t i m a t i o n o f the M i c h a e l i s -Menten c o n s t a n t s . For c o n t r o l c e l l s , K = 1 2 + 3 (S.D.) 11M and V = m — ' max 280 + 16 (S.D.) pmol/min per d i s h . For h e p a t o c y t e s t r e a t e d w i t h c h l o r o -p h e n y l t h i o - c A M P , K . = 9 + 3 (S.D.) uM and V = 7 7 + 7 (S.D.) pmol/ m — ' max " min per d i s h . Therefore, w h i l e t h e r e was l i t t l e or no e f f e c t on the K f o r m Figure 33- Concentration dependence o f chlorophenylthio-cAMP i n h i b i t i o n of  [ M e - 3 H ] c h o l i n e c e l l u l a r u p t a k e . Monolayer c u l t u r e s of r a t he p a t o c y t y e s were preincubated f o r 1 h w i t h 28 uM c h o l i n e and 0.005-2.5 mM chlorophenylthio-cAMP i n the medium, and pulsed f o r an a d d i t i o n a l 30 min i n equivalent medium c o n t a i n i n g 20 uCi of [ M e - 3 H ] c h o l i n e per d i s h . A f t e r h a r v e s t i n g , c e l l s were extracted by the method of Folch (Section 2.2.5.1), and the r a d i o a c t i v i t y quantitated a f t e r t h i n - l a y e r chromatography by l i q u i d s c i n t i l l a t i o n c o u n t i n g . Uptake of [Me- 3 H ] c h o l i n e ( • ). Label r e l e a s e d i n t o the medium as b e t a i n e was accounted f o r when t o t a l uptake was estimated. Each p o i n t ' r e p r e s e n t s the mean of three d i s h e s , and the standard e r r o r i s shown by bars. o ><; E Q & § O Q. o: O O g hi g O I 10h 6r-1 1 1 1 1 . — \ -j r . / — / * * _ w § t t « -1 1 1 I 1 5-10"3 10"3 5-1CTa 10"" 5-10*5 10"5 [ C P T - c A M P ] ( M ) O N C-A4 20 Figure 34. E f f e c t o f c h l o r o p h e n y l t h i o - c A M P on c h o l i n e uptake by c u l t u r e d  r a t h e p a t o c y t e s . C e l l s were i n c u b a t e d w i t h 0.5 mM c h o l i n e f o r 1 h, and incubated f o r an a d d i t i o n a l 30 min i n medium c o n t a i n i n g 5-80 uM c h o l i n e i n the absence or presence of 0.5 mM c h l o r o p h e n y l t h i o - c A M P . C e l l s were pulsed f o r 30 min i n an e q u i v a l e n t medium w i t h 20 pCi of [Me- 3H] ch o l i n e and the c e l l s h arvested. Uptake of c h o l i n e : c o n t r o l , ( 0 , » ) ; chlorophenylthio-cAMP-treated, ( O , ^ ) . C l o s e d symbols, experimental data; open symbols, saturable component of up t a k e . Each point represents the mean of three dishes and the standard e r r o r s were approximately 6% of the mean. o E v S 1 6 o LU CE o Q. o 8 o 2 LU 4 2 _l O I O 0 . . . . .,.„T 1 1 1 -- / / • y r W i i i i 20 40 60 80 C O N C . CHOLINE ( p M ) 1 6 4 c h o l i n e uptake, V was reduced 3 - 6 - f o l d by the cAMP analogue, max Chlorophenylthio-cAMP I n h i b i t i o n o f P h o s p h a t i d y l c h o l i n e Synthesis i s Inde- pendent o f the C o n c e n t r a t i o n o f C h o l i n e i n the Medium (3-7-1-4). The r a t e of p h o s p h a t i d y l c h o l i n e s y n t h e s i s i n c u l t u r e d r a t hepatocytes may be s l i g h t l y s t imulated ( 1 . 4 - f o l d ) when the medium c h o l i n e c o n c e n t r a t i o n i s i n c r e a s e d from 5 t o 40 u^M ( 2 4 4 ) . T h e r e f o r e , t h e i n f l u e n c e o f the co n c e n t r a t i o n of c h o l i n e i n the medium on p h o s p h a t i d y l c h o l i n e b i o s y n t h e s i s i n chlorophenylthio-cAMP-treated c e l l s was i n v e s t i g a t e d . A f t e r 1 h treatment with 0.5 mM chlorophenylthio-cAMP there was a 40% i n h i b i t i o n of the r e l a t i v e 3 i n c o r p o r a t i o n of [Me- H j c h o l i n e i n t o p h o s p h a t i d y l c h o l i n e when the medium c h o l i n e c o n c e n t r a t i o n was v a r i e d from 5 t o 80 _uM (data not shown). In other experiments, the c h o l i n e c o n c e n t r a t i o n i n the medium was adjusted to 28 ^ JM, which i s s i m i l a r to the conc e n t r a t i o n reported f o r r a t serum (241). The e f f e c t of the c h o l i n e c o n c e n t r a t i o n i n the medium on the r e l a t i v e 3 i n c o r p o r a t i o n o f [Me- H ] c h o l i n e i n t o p h o s p h o c h o l i n e and b e t a i n e was examined. When the medium c h o l i n e c o n c e n t r a t i o n was increased from 5 t o 40 3 yM, the r e l a t i v e i n c o r p o r a t i o n o f [He- H ] c h o l i n e i n t o p h o s p h o c h o l i n e decreased from 50 to 22%, whereas that o x i d i z e d to betaine increased from 50 3 to 78% (data not shown). A l t h o u g h the t o t a l c e l l u l a r uptake of [Me- H]-c h o l i n e was r e s t r i c t e d by c h l o r o p h e n y l t h i o - c A M P t r e a t m e n t , the r e l a t i v e i n c o r p o r a t i o n of t r i t i u m l a b e l i n t o p h o s p h o c h o l i n e and bet a i n e resembled c o n t r o l c e l l s (data not shown). Therefore, the cAMP analogue d i d not seem to i n f l u e n c e t h e p r o p o r t i o n o f c e l l u l a r c h o l i n e t h a t was o x i d i z e d or phosphorylated. 1 6 5 Chlorophenylthio-cAMP and Other cAMP Analogues I n h i b i t P h o s p h a t i d y l c h o l i n e  S y n t h e s i s ( 3 . 7 . 1 . 5 ) . Since chlorophenylthio-cAMP produced t h e most s t r i k i n g e f f e c t on [Me-3 H]choline i n c o r p o r a t i o n i n t o p h o s p h a t i d y l c h o l i n e d u r i n g p u l s e - l a b e l i n g s t u d i e s ( F i g . 3 0 ) , the dose r e s p o n s e c u r v e f o r t h i s cAMP analogue was det e r m i n e d ( F i g . 3 5 ) . A l t h o u g h c h o l i n e u p t a k e was not s i g n i f i c a n t l y i n h i b i t e d by 90 min i n c u b a t i o n w i t h 0.1 mM chlorophenylthio-cAMP ( F i g . 3 3 ) , 3 the i n c o r p o r a t i o n o f [Me- H j c h o l i n e i n t o p h o s p h a t i d y l c h o l i n e was a c t u a l l y reduced 2.0-fold ( F i g . 3 5 ) . For most s t u d i e s 0.5 mM c h l o r o p h e n y l -thio-cAMP was used. The e f f e c t of a c u t e exposure t o c h l o r o p h e n y l t h i o - c A M P on the a c t u a l r a t e of p h o s p h a t i d y l c h o l i n e s y n t h e s i s was e v a l u a t e d t h r o u g h pulse-chase s t u d i e s . The presence o f c h l o r o p h e n y l t h i o - c A M P reduced the r a t e o f t r a n s f e r of the t r i t i u m l a b e l from p h o s p h o c h o l i n e i n t o the p h o s p h a t i d y l c h o l i n e pool ( F i g . 36). But, the r e s u l t s o f t h i s p u l s e - c h a s e experiment were somewhat comp l i c a t e d by the f a c t t h a t c h l o r o p h e n y l t h i o - c A M P t r e a t e d h e p a t o c y t e s contained l e s s r a d i o - l a b e l e d phosphocholine than c o n t r o l c e l l s at the end of the p u l s e p e r i o d , as w e l l as a s l i g h t l y reduced p h o s p h o c h o l i n e p o o l . However, from Figure 36A and v a l u e s f o r the phosphocholine pool s i z e at 2 h i n t o the chase ( F i g . 32), the r a t e s o f p h o s p h a t i d y l c h o l i n e s y n t h e s i s during 1 to 3 h i n the chase p e r i o d can be c r u d e l y e s t i m a t e d ( S e c t i o n 2.5.1.2). Chlorophenylthio-cAMP treatment f o r 3.5 h reduced the rate of p h o s p h a t i d y l -c h o l i n e synthesis (nmol p h o s p h a t i d y l c h o l i n e formed/ h per dis h ) by a p p r o x i -mately 31% ( c o n t r o l , 5.1; t r e a t e d , 3.5). 3 Since t h e r e was a d i f f e r e n c e i n the uptake o f [Me- H ] c h o l i n e a f t e r c h l o r o p h e n y l t h i o - c A M P e x p o s u r e , a m o d i f i e d p u l s e - c h a s e e x p e r i m e n t was 3 performed i n which the h e p a t o c y t e s were p r e l a b e l e d f o r 1 h with [Me- H]-166 c h o l i n e ( F i g . 3 7 ) . The analogue was o n l y i n t r o d u c e d t o the c e l l s i n the chase medium. T h e r e f o r e , t h e p o o l s i z e s o f t h e p h o s p h a t i d y l c h o l i n e precursors and the t o t a l amount o f l a b e l i n the c o n t r o l and t r e a t e d c e l l s were i d e n t i c a l at the s t a r t of the chase. At the end of the 1 h puls e , most o f t h e l a b e l was a s s o c i a t e d w i t h t h e p h o s p h o c h o l i n e p o o l . Hence, the re d u c t i o n i n the rate o f disappearance o f the l a b e l from phosphocholine and i t s appearance i n t o p h o s p h a t i d y l c h o l i n e ( F i g 37B) c o u l d not be due to an i n h i b i t i o n of c h o l i n e k i n a s e . O 5 - 1 0 " J 1 0 ~ J 5 -10 1 0 5 - 1 0 3 1 0 X [CPT-cAMP] (M) Q_ F i g u r e 35. C o n c e n t r a t i o n dependence o f chlorophenylthio-cAMP(CPT-cAMP)  i n h i b i t i o n of [Me- aH]choline i n c o r p o r a t i o n i n t o p h o s p h a t i d y l c h o l i n e . Rat hepatocytes were t r e a t e d as described i n F i g . 34 and the r a d i o a c t i v i t y i n p h o s p h a t i d y l c h o l i n e a f t e r a 30 min p u l s e q u a n t i t a t e d . [Me- 3HJCholine incorp o r a t e d i n t o p h o s p h a t i d y l c h o l i n e , ( • ). Each p o i n t represents the mean of three d i s h e s , and the standard e r r o r i s shown by bars. 1 6 7 CHASE TIME (h) Figure 36. E f f e c t of c h l o r o p h e n y l t h i o - c A M P on the i n c o r p o r a t i o n of [Me- 3 H ] c h o l i n e i n t o v a r i o u s c h o l i n e m e t a b o l i t e . Rat h e p a t o c y t e monolayer c u l t u r e s were preincubated f o r 1 h i n the presence of 0.5 mM c h l o r o p h e n y l -thio-cAMP and 0.2 mM IBMX. The c e l l s were then p u l s e d with 20 uCi of [Me-3 H ] c h o l i n e f o r 30 min and subsequently chased with 28 pM c h o l i n e f o r up to 3 h. Chlorophenylthio-cAMP and IBMX were a l s o present i n the pulse and chase mediums. Incorp o r a t i o n of the l a b e l : A, c e l l u l a r phosphocholine, ( O t » ) ; c e l l u l a r p h o s p h a t i d y l c h o l i n e , ( A , A ) ; B, c e l l u l a r b e t a i n e , ( • , • ) ; and medium compounds, ( V , T ) . Open symbols, s o l i d l i n e s , c o n t r o l s ; c l o s e d  symbols, dashed l i n e s , t r e a t e d . Each p o i n t r e p r e s e n t s the mean of three d i s h e s , and the standard e r r o r was approximately U% o f the mean. Figure 37. E f f e c t o f c h l o r o p h e n y l t h i o - c A M P on the f a t e of [Me- 3H]choline  i n p r e l a b e l e d r a t h e p a t o c y t e s . C u l t u r e d r a t h e p a t o c y t e s i n 60 mm dishes were pulse l a b e l e d f o r 1 h w i t h 10 u C i of [ M e - 3 H ] c h o l i n e . The c e l l s were washed and f r e s h medium w i t h or w i t h o u t 0.5 mM chlorophenylthio-cAMP and 0.2 mM IBMX was a d d e d . At v a r i o u s t i m e s , t h e c e l l s and media were c o l l e c t e d , and the r a d i o a c t i v i t y q u a n t i t a t e d i n A, c e l l u l a r phospho-c h o l i n e , ( O t » ) ; c e l l u l a r p h o s p h a t i d y l c h o l i n e , ( A , A ) ; B, c e l l u l a r b e t a i n e , ( • , • ) ; and medium compounds , ( V , T ) . Open symbols, s o l i d  l i n e s , c o n t r o l s ; c l o s e d s y m b o l s , d a s h e d l i n e s , t r e a t e d . Each p o i n t represents the mean o f t h r e e d i s h e s , and the s t a n d a r d e r r o r was a p p r o x i -mately k% of the mean. 1 6 8 3 As shown i n T a b l e 2 1 , t h e i n h i b i t i o n o f [IMe- H ] p h o s p h o c h o l i n e i n c o r p o r a t i o n i n t o p h o s p h a t i d y l c h o l i n e c o u l d be d u p l i c a t e d by o t h e r compounds which e i t h e r e l e v a t e o r m i m i c e l e v a t e d i n t r a c e l l u l a r cAMP c o n c e n t r a t i o n s . I t can be c a l c u l a t e d f rom T a b l e 21 t h a t the r a t e o f i n c o r p o r a t i o n of the t r i t i u m l a b e l i n t o p h o s p h a t i d y l c h o l i n e during the f i r s t 2 h of the chase was reduced 35% by the presence of chlorophenylthio-cAMP. This i s i n rough agreement w i t h the 31% r e d u c t i o n of the r a t e of phospha-t i d y l c h o l i n e b i o s y n t h e s i s estimated from Figure 36. A f t e r these s t u d i e s were p u b l i s h e d , Dr. Eckstein generously provided us w i t h the cAMP analogue, a d e n o s i n e 3', 5 ' - c y c l i c p h o s p h o r o t h i o a t e (cAMPS). cAMPS d i f f e r s from cAMP by o n l y one atom; a s u l p h u r atom r e p l a c e s an oxygen i n the phosphate r i n g . When c u l t u r e d r a t hepatocytes which had been 3 p r e l a b e l e d w i t h [Me- H ] c h o l i n e were exposed to 0.5 mM cAMPS, the r a t e of p h o s p h a t i d y l c h o l i n e s y n t h e s i s was i n h i b i t e d by 40% ( F i g . 38). Prolonged Exposure of H e p a t o c y t e s t o C h l o r o p h e n y l t h i o - c A M P S t i m u l a t e s P h o s p h a t i d y l c h o l i n e S y n t h e s i s (3.7.1.6). Pulse-chase s t u d i e s w i t h h e p a t o c y t e s t h a t had been preincubated f o r over 12 h w i t h c h l o r o p h e n y l t h i o-c AMP r e v e a l e d a 1 . 7 - f o l d i n c r e a s e d 3 i n c o r p o r a t i o n o f [Me- H ] c h o l i n e i n t o p h o s p h a t i d y l c h o l i n e ( F i g . 39A). Since the s p e c i f i c r a d i o a c t i v i t y o f phosphocholine i n t r e a t e d c e l l s was 41% lower than c o n t r o l c e l l s a f t e r 15 h i n c u b a t i o n with chlorophenylthio-cAMP, the r a t e of s y n t h e s i s (nmol p h o s p h a t i d y l c h o l i n e formed/ h per d i s h ) was a c t u a l l y s t i m u l a t e d 2 . 8 - f o l d r e l a t i v e t o c o n t r o l c e l l s ( c o n t r o l , 2.0; t r e a t e d , 5.7)• C h l o r o p h e n y l t h i o - c A M P Does Not A f f e c t the Release o f B e t a i n e from 1 6 9 Table 21. I n h i b i t i o n of P h o s p h a t i d y l c h o l i n e Synthesis by Various cAMP  Analogues and Phosphodiesterase I n h i b i t o r s . Monolayer cu l t u r e s of r a t hepatocytes were labeled f o r 1 h with 10 |iCi of [Me- 3H]choline. cAMP analogues and phosphodiesterase i n h i b i t o r s were introduced at the s t a r t of a 2 h chase, and the incorporation of l a b e l i n t o phosphatidylcholine during the chase period was estimated. Data are expressed as the mean + SD f o r t r i p l i c a t e samples. Additive Phosphatidylcholine - 5 6 dpm X 10 / 10 c e l l s None IBMX (0.2 mM) Aminophylline (1 .0 mM) Chlorophenylthio-cAMP (0 .5 mM)1 Bromo-cAMP (0 .5 mM) b b Dibutyryl-cAMP (0 .5 mM) 1.53 + 0.11 1.51 + 0.13 1.16 + 0.1H 3 1.00 + 0 .16 S 1.06 + 0 . 1 1 ° 1.10 + 0 . i o a p< 0 . 05 IBMX (0.2 mM) was i n c l u d e d w i t h the cAMP analogues. p< 0 . 01 PHOSPHOCHOLINE PHOSPHATIDYLCHOLINE 0 0.5 1 1.5 2 CHASE TIME (h) 0 0.5 1 15 2 CHASE TIME (h) Figure 3 8 . E f f e c t o f cAMPS on the d i s a p p e a r a n c e o f [ M e - 3 H ] c h o l i n e from  c e l l u l a r p h o s p h o c h o l i n e and a c c u m u l a t i o n i n t o p h o s p h a t i d y l c h o l i n e . I s o l a t e d r a t hepatocytes were p u l s e l a b e l e d f o r 30 min with 15 uCi of [Me-3 H ] c h o l i n e . The c e l l s were washed and f r e s h medium with or without 0.5~mM aden o s i n e 3 ' , 5 ' - c y c l i c p h o s p h o r o t h i o a t e (cAMPS) was added. At v a r i o u s times, the c e l l s and media were c o l l e c t e d , and the r a d i o a c t i v i t y determined i n A, p h o s p h o c h o l i n e , c o n t r o l ( v ) and t r e a t e d ( T ) ; and B , phospha-t i d y l c h o l i n e , c o n t r o l ( A ) and t r e a t e d ( A ) . Each po i n t represents the mean of three d i s h e s , and standard e r r o r i s i n d i c a t e d by bars. 1 7 0 H e p a t o c y t e s ( 3 - 7 - 1 - 7 ) . C h l o r o p h e n y l t h i o - c A M P d i d not i n f l u e n c e the p r o p o r t i o n o f c e l l u l a r c h o l i n e t h a t was o x i d i z e d or p h o s p h o r y l a t e d . In a d d i t i o n , p u l s e - c h a s e s t u d i e s a f t e r s h o r t term ( F i g . 3 6 B and 3 7 B ) and l o n g term ( F i g . 3 9 B ) exposure to t h i s analogue demonstrated t h a t t h i s analogue does not a f f e c t the r a t e at which c e l l u l a r betaine leaves the c e l l s . 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 s Reduced i n Microsomes and Cy t o s o l s From  Chlorophenylthio-cAMP-Treated Hepatocytes (3.7.1.8). The a c t i v i t i e s o f t h e d_e novo p h o s p h a t i d y l c h o l i n e b i o s y n t h e t i c enzymes from c o n t r o l and h e p a t o c y t e s exposed t o -chlorophenylthio-cAMP f o r 1.5, 6 and 12 h were measured t o d e t e r m i n e i f therewere a c o r r e l a t i o n between the rat e s o f p h o s p h a t i d y l c h o l i n e s y n t h e s i s and the a c t i v i t y o f any of the enzymes. As shown i n Table 22, both c h o l i n e kinase and chol i n e p h o s -photransferase a c t i v i t i e s were unaffected compared to c o n t r o l s , although the ch o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y o f both c o n t r o l and t r e a t e d c e l l s was elevated during the course o f the i n c u b a t i o n . The increase i n cholinephos-p h o t r a n s f e r a s e a c t i v i t y between 1.5 and 15 h i n c o n t r o l c e l l s d i d not c o r r e l a t e with the reduced r a t e o f ph o s p h a t i d y l c h o l i n e s y n t h e s i s . Changes i n the a c t i v i t i e s of c y t o s o l i c and microsomal c y t i d y l y l t r a n s f e r a s e corresponded with the r a t e of p h o s p h a t i d y l c h o l i n e b i o s y n t h e s i s d u r i n g the 1.5 and 6 h i n c u b a t i o n s . For the 12 h time p o i n t t h e r e was a s t i m u l a t i o n observed f o r 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 i_n v i t r o . However, the magnitude of the increase was much smaller than the 2.8-fold s t i m u l a t i o n o f p h o s p h a t i d y l -c h o l i n e s y n t h e s i s estimated f o r i n t a c t c e l l s t r e a t e d with c h l o r o p h e n y l t h i o -cAMP f o r 12 h. In a l l cases, the d i f f e r e n c e i n 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 -ase a c t i v i t y from c o n t r o l and t r e a t e d c e l l s could be abolished by i n c l u s i o n of s a t u r a t i n g amounts of t o t a l r a t l i v e r phospholipid i n the enzyme assay. 1 7 1 t 1 r C H A S E TIME (h) Figure 39. Latent e f f e c t of c h l o r o p h e n y l t h i o - c A M P on the i n c o r p o r a t i o n of  [ M e - 3 H ] c h o l i n e i n t o v a r i o u s c h o l i n e m e t a b o l i t e s . Rat h e p a t o c y t e s were preincubated f o r 12 h i n the p r e s e n c e o f 0.5 mM chlorophenylthio-cAMP and 0.2 mM IBMX. The c e l l s were then p u l s e d w i t h 10 u C i of [Me- 3H]choline per d i s h f o r 1 h, and s u b s e q u e n t l y chased w i t h 28 pM c h o l i n e f o r up to 3 h. Chlorophenylthio-cAMP and IBMX were a l s o p r e s e n t i n the p u l s e and chase mediums. Incor p o r a t i o n o f the l a b e l : A, c e l l u l a r phosphocholine, ( 0 , » ) ; c e l l u l a r p h o s p h a t i d y l c h o l i n e , ( A , A ) ; B, c e l l u l a r b e t a i n e , ( • , • ) ; and medium compounds (v , T ). Open s y m b o l s , s o l i d l i n e , untreated; close d  symbols, d o t t e d l i n e , t r e a t e d . Each p o i n t r e p r e s e n t s the mean of three d i s h e s , and the standard e r r o r s were approximately 4% of the mean. 1 7 2 of s a t u r a t i n g amounts of t o t a l r a t l i v e r phospholipid i n the enzyme assay. T A B L E 2.2. T H E ACTIVITIES O F PHOSPHATIDYLCHOLINE BIOSYNTHETIC ENZYMES F R O M RAT H E P A T O C Y T E S T R E A T E D WITH 0.5 mM CHLOROPHENYLTHIO-cAMP FOR 1.5. 6 A N D 12 h Subcellular fractionation was performed in the presence of 20 mM NaF and 2.5 mM EDTA. Values are the mean = S.D. The number of preparations are indicated in parentheses. Enzyme Specific activity (nmol/min per mg protein) U h 6h 12 h C O N T R O L T R E A T E D C O N T R O L T R E A T E D C O N T R O L T R E A T E D Choline kinase 1.35=: 0.14 1.50=0.19 1.39 = 0.09 1.25 = 0.11 1.45 = 0.20 1.43 = 0.10 (8) (4) (4) C7) Cytidylyltransferase Cytosol 0.57 = 0.08 0.38 = 0.09* 0.39=0.07 0.37=0.04 0.24 = 0.06 0.34 = 0.05* Cytosol with (8) (8) (4) (4) (7) (7) phospholipid 0.69 = 0.10 0.79 = 0.12 0.81=0.12 0.83 = 0.11 0.82 = 0.06 0.84 = 0.10 (14) (14) (4) (4) (7) (7) microsomes 0.58 = 0.07 0.40=0.06" 0.38 = 0.09 0.43 = 0.02 0.40 = 0.07 0.56 = 0.02 b (8) (8) (4) (4) (4) (4) Phosphocholineiransferase 0.11=0.02 0.11=0.01 0.13 = 0.02 0.18 = 0.03 0.18 = 0.02 0.22 = 0.02 (4) (4) (4) (4) (4) (4) * /><0.05; b P<0.0\. 1 7 3 INFLUENCE OF cAMP ANALOGUES ON PHOSPHATIDYLETHANOLAMINE N-METHYLATION 3 3 cAMP Analogues Reduce [-H]Methionine and [-HjEthanolamine I n c o r p o r a - t i o n i n t o P h o s p h a t i d y l c h o l i n e (3-7.2.1). 3 Cultured r a t h e p a t o c y t e s were p u l s e - l a b e l e d w i t h [Me- H]methionine t o monitor the e f f e c t of cAMP a n a l o g u e s and aminophylline on the J J - m e t h y l a t i o n o f p h o s p h o l i p i d s ( T a b l e 2 3 ) . In the c o n t r o l and t r e a t e d 3 c e l l s , t y p i c a l l y 46% o f t h e [Me- H j m e t h i o n i n e l a b e l w h i c h was i n c o r p o r a t e d i n t o t h e c e l l s was a s s o c i a t e d w i t h p h o s p h o l i p i d . C h l o r o p h e n y l t h i o - c A M P d e c r e a s e d t h e amount o f t r i t i u m r e c o v e r e d i n p h o s p h a t i d y l c h o l i n e by 58%. However, t h i s may have been due i n par t t o a 34% r e d u c t i o n i n t h e amount o f l a b e l e d m e t h i o n i n e t a k e n up by the hepatocytes. Br-cAMP a l s o s i g n i f i c a n t l y reduced t h e t r a n s f e r of l a b e l e d methyl groups t o ph o s p h o l i p i d , but t h i s analogue had no e f f e c t upon the t o t a l uptake of methionine. S i n c e c h l o r o p h e n y l t h i o - c A M P p r o d u c e d the most s t r i k i n g e f f e c t on ph o s p h a t i d y l e t h a n o l a m i n e m e t h y l a t i o n , t h e i n f l u e n c e o f t h i s compound on 3 [ H ] e t h a n o l a m i n e i n c o r p o r a t i o n i n t o h e p a t o c y t e p h o s p h o l i p i d s was o determined. The presence of 0.5 mM c h l o r o p h e n y l t h i o - c A M P i n the c u l t u r e 3 medium decreased by 20% the a c c u m u l a t i o n o f [ H]ethanolamine i n t o l i p i d by t h e c e l l s ( F i g . 4 0 ). T h i s a n a l o g u e reduced i n c o r p o r a t i o n i n t o b o t h phosphatidylethanolamine ( F i g . 41A) and p h o s p h a t i d y l c h o l i n e ( F i g . 41B). The 70% i n h i b i t i o n of t r i t i u m i n c o r p o r a t i o n i n t o p h o s p h a t i d y l c h o l i n e may have r e f l e c t e d changes i n the uptake o f e t h a n o l a m i n e or a l t e r a t i o n s i n the pool s i z e s o f phosphatidylethanolamine and i t s p r e c u r s o r s rather than an a c t u a l decrease i n the rate of p h o s p h a t i d y l c h o l i n e s y n t h e s i s . When the experimental 3 data were expressed i n terms o f r e l a t i v e i n c o r p o r a t i o n of the [ H]ethanol-1 7 4 Table 2 3 THE EFl-ECT OF CYCLIC AMP ANALOGUES AND AMINOPHYLLINE ON THE INCORPORATION OF [methyl-3H[METHIONINE BY CULTURED RAT HEPATOCYTES INTO PHOSPHOLIPIDS Hepatocytes cultured for 24 h (approx. 2.5 • 106 cells/dish) were incubated in the presence of cyclic AMP analogues or aminophylline as indicated. After 1 h, 10 uCi of [mef/ry/-3H]methioninc was added to each dish of cells and the incubations continued for a further hour. Cells were harvested and the radioactivity recovered in the lipid classes was determined. The results are the mean ±S.D. for three dishes of hepatocytes. Significance of difference: a P < 0.05;b/>< 0.001. Total uptake Recovery of [methyl^H]methionine (dpm X lO^/dish) Monomethyl-phosphatidyl-ethanolamine Dimethyl-phosphatidyl-ethanolamine Phosphatidyl-choline Lyso-phosphatidyl choline Sphingomyelin Control 50.7 ±3 .3 0.19 ±0.03 0.41 ±0.04 22.6 ±0.1 0.23 ±0.03 0.05 ± 0.01 1.0 mM Aminophylline 49.2 ± 3.2 0.17 ±0.03 0.37 ±0.03 22.1 ± 1.9 0.15 ±0.07 0.05 ± 0.01 0.5 mM 8-bromo-cyclic AMP 45.5 ±2.5 0.17 ±0.02 0.37 ± 0.05 17.5 ± 2.9 b 0.13 ±0.07 0.03 ±0.001 a 0.5 mM dibutyryl-cyclic AMP 56.8 ±5 .2 0.15 ±0.03 0.37 ±0.05 25.6 ±1.6 a 0.19 ±0.03 0.05 ±0.01 0.5 mM CPT-cyclic AMP 33.6 ±2.1 b 0.16 ±0.01 0.36 ±0.02 9.5 ± 1.1 b 0.08 ±0.01 b 0.02 ±0.003 b T 1 r 15 30 45 60 Time (min) F i g u r e 40. E f f e c t of c h l o r o p h e n y l t h i o - c A M P on the i n c o r p o r a t i o n of [ 3 H ] - ethanolamine i n t o p h o s p h o l i p i d s . H e p a t o c y t e s t h a t had been c u l t u r e d f o r 24 h were incubated i n serum-free medium th a t contained 0.5 mM c h l o r o p h e n y l -thio-cAMP. A f t e r ' 1 h, 10 pCi of [ 3H]ethanolamine were added to each d i s h of c e l l s and the inc u b a t i o n s continued f o r the times i n d i c a t e d . L i p i d s were e x t r a c t e d from the c e l l s and assayed for r a d i o a c t i v i t y . Results are expressed as means + standard d e v i a t i o n . C o n t r o l ( • ) ; c h l o r o p h e n y l t h i o -cAMP-treated ( O ) . 1 7 5 Figure 41. E f f e c t of c h l o r o p h e n y l t h i o - c A M P on [ 3H]ethanolamine i n c o r p o r a - t i o n i n t o phosphatidylethanolamine and p h o s p h a t i d y l c h o l i n e by hepatocytes. Monolayer c u l t u r e s of r a t h e p a t o c y t e s were p u l s e d w i t h 10 uCi of [ 1- 3H]-ethanolamine f o r 15-60 min i n the p r e s e n c e or absence of 0.5 mM c h l o r o -phenylthio-cAMP. Ac t u a l t r i t i u m i n c o r p o r a t e d i n t o phosphatidylethanolamine ( P a n e l A) and p h o s p h a t i d y l c h o l i n e ( P a n e l B) . P e r c e n t of t o t a l l i p i d r a d i o a c t i v i t y i n c o r p o r a t e d i n t o p h o s p h a t i d y l e t h a n o l a m i n e ( P a n e l C) and p h o s p h a t i d y l c h o l i n e ( P a n e l D) . Open s y m b o l s , s o l i d l i n e s , c o n t r o l s . Closed symbols, dashed l i n e s , 0.5 mM c h l o r o p h e n y l t h i o - c A M P - t r e a t e d . Each p o i n t represents the mean of three dishes and standard e r r o r i s i n d i c a t e d by b a r s . 1 7 6 amine, the disappearance of t r i t i u m from phosphatidylethanolamine ( F i g . 41C) and i t s a c c u m u l a t i o n i n t o p h o s p h a t i d y l c h o l i n e ( F i g . 41D) was reduced i n c h l o r o p h e n y l t h i o - c A M P - t r e a t e d c e l l s d u r i n g the p u l s e . T h i s i m p l i e d t h a t t r a n s m e t h y l a t i o n o f phosphatidylethanolamine was i n h i b i t e d i n cAMP analogue-t r e a t e d c e l l s , although changes i n the p o o l s i z e s of p h o s p h a t i d y l e t h a n o l -amine and i t s c y t o s o l i c precursors could not be discounted. To a v e r t the p o t e n t i a l p r o b l e m o f i s o t o p e d i l u t i o n as a f a c t o r r e s p o n s i b l e f o r the apparent i n h i b i t i o n o f p h o s p h o l i p i d m e t h y l a t i o n , a modified pulse-chase s t u d y was d e s i g n e d i n which the p h o s p h a t i d y l e t h a n o l -3 amine pool was p r e l a b e l e d w i t h [ H ] e t h a n o l a m i n e b e f o r e the cAMP analogue was i n t r o d u c e d . At the end o f t h e 2 h p u l s e , 80% o f the t r i t i u m was recovered i n phosphatidylethanolamine and the remaining 20% accumulated i n p h o s p h a t i d y l c h o l i n e ( F i g . 4 2 ) . O n l y v e r y s m a l l q u a n t i t i e s o f t r i t i u m accumulated i n phosphatidylmonomethylethanolamine, p h o s p h a t i d y l d i e t h y l e t h -anolamine, l y s o p h o s p h a t i d y l c h o l i n e or s p h i n g o m y e l i n . Chlorophenylthio-cAMP decreased by 2 - f o l d the rate at which t r i t i u m was removed from the phospha-t i d y l e t h a n o l a m i n e pool d u r i n g the chase i n c u b a t i o n ( F i g . 42A). The r a t e at which t r i t i u m accumulated i n p h o s p h a t i d y l c h o l i n e was decreased to a s i m i l a r extent ( F i g . 42B). The e f f e c t s of aminophylline and o t h e r analogues of cAMP were s t u d i e d when the c e l l s were harvested a t a s i n g l e t i m e point (2 h) a f t e r the s t a r t of the chase i n c u b a t i o n . Table 24 shows t h a t Br-cAMP and probably amino-p h y l l i n e a l s o i n h i b i t e d the c o n v e r s i o n o f phosphatidylethanolamine t o phos-p h a t i d y l c h o l i n e . D i b u t y r y l - c A M P had l i t t l e or no e f f e c t w i t h i n t h e 2 h in c u b a t i o n p e r i o d . 1 7 7 Phosphatidylethanolamine M e t h y l t r a n s f e r a s e A c t i v i t y i s Stimulated i n  Microsomes from C h l o r o p h e n y l t h i o - c A M P - T r e a t e d Hepatocytes (3.7.2.2). Since the r a t e o f p h o s p h o l i p i d m e t h y l a t i o n i s i n h i b i t e d i n c h l o r o -phenylthio-cAMP t r e a t e d h e p a t o c y t e s , the a c t i v i t y o f p h o s p h a t i d y l e t h a n o l -amine methyltransferase i n microsomes from analogue-.treated hepatocytes was measured ( F i g . 43)- S u r p r i s i n g l y , t h e iM v i t r o a c t i v i t y o f t h i s enzyme was a c t u a l l y stimulated 2-3-fold by the presence of chlorophenylthio-cAMP i n the c u l t u r e medium. Table 2'U E F F E C T OF CYCLIC AMP ANALOGUES AND AMINOPHYLLINE ON T H E CONVERSION OF PHOSPHATIDYLETHANOL-AMINE TO PHOSPHATIDYLCHOLINE BY CULTURED RAT HEPATOCYTES The phosphatidylethanolamine pool of cultured rat hepatocytes was labeled with [3HJethanolamine as described for Fig.4/J,The medium was removed and replaced with unlabeled medium that contained various compounds. Cells were harvested after 2 h incubation and the lipids extracted. Radioactivity recovered in phosphatidylcholine and phosphatidylethanolamine was deter-mined after thin4ayer chromatography. Significance of difference; 3 P < 0.05; b P < 0.01 ; c P < 0.001. Time (h) Radioactivity recovered in: Phosphatidylcholine (dpm X 10"*/dish cells) Phosphatidylethanolamine Control 0 2.01 ± 0 . 0 6 8.17 ±0 .06 Control 2 3.95 ± 0.04 6.30 ± 0 . 1 0 1.0 mM Aminophylline 2 3.61 ± 0.09 b 6.53 ± 0.06 a 0.5 mM 8-bromo-cyclic AMP 2 3.19 ± 0 . 2 3 a 7.01 ±0 .23 b 0.5 mM dibutyryl-cyclic AMP 2 3.74 ± 0 . 0 9 b 6.53 ±0 .21 0.5 mM CPT-cyclic AMP 2 2.86 ± 0.05 c 7.32 +0.05 ' Figure 42. E f f e c t o f c h l o r o p h e n y l t h i o - c A M P on t h e r a t e o f conversion of OO phosphatidylethanolamine t o p h o s p h a t i d y l c h o l i n e by c u l t u r e d hepatocytes. Hepatocytes that had been c u l t u r e d f o r 24 h were i n c u b a t e d i n serum-free medium that contained 10 u C i [ 3 H ] e t h a n o l a m i n e . A f t e r 2 h, the medium was removed and the c e l l s washed f r e e o f u n i n c o r p o r a t e d ethanolamine. Some of the c e l l s were harvested at t h i s time w h i l e others were incubated f o r up to 4 h i n the presence ( o ) or absence (•) of 0.5 mM chlorophenylthio-cAMP. L i p i d s were e x t r a c t e d from the c e l l s a f t e r h a r v e s t i n g and were separated i n t o the l i p i d c l a s s e s by t h i n - l a y e r chromatography. A, p h o s p h a t i d y l -e t h a n o l a m i n e ; El, p h o s p h a t i d y l c h o l i n e . R e s u l t s a r e e x p r e s s e d as mean _+ standard d e v i a t i o n . Figure 43. E f f e c t o f c h l o r o p h e n y l t h i o - c A M P on the a c t i v i t y of microsomal  p h o s p h a t i d y l e t h a n o l a m i n e N - m e t h y l t r a n s f e r a s e i n c u l t u r e d h e p a t o c y t e s . I s o l a t e d r a t hepatocytes were incubated i n the presence (O ) or absence ( • ) of 0.5 mM c h l o r o p h e n y l t h i o - c A M P f o r the tim e s i n d i c a t e d . A mi c r o s o m a l f r a c t i o n was i s o l a t e d from the c e l l homogenates and phosphatidylethanolamine N-methyltransferase a c t i v i t y was d e t e r m i n e d . R e s u l t s are mean + standard d e v i a t i o n f or four microsomal preparations at each p o i n t . 1 7 9 INFLUENCE OF GLUCAGON ON PHOSPHATIDYLCHOLINE BIOSYNTHESIS 3 Glucagon Does Not A f f e c t [ M e - - H ] C h o l i n e Uptake by Rat Hepatocytes ( 3 . 7 . 3 - D . The i n f l u e n c e of glucagon on the uptake of c h o l i n e by hepatocytes was examined si n c e the v a r i o u s cAMP a n a l o g u e s had opposing e f f e c t s ( F i g . 2 9 ) . 3 [Me- H ] C h o l i n e i n c o r p o r a t i o n i n t o t h e c e l l s was l i n e a r d u r i n g the i n i t i a l 2 0 min of the p u l s e , and glucagon d i d not perturb the r a t e o f uptake w i t h 2 8 ^ iM c h o l i n e i n the medium ( F i g . 44A) . At t h i s c o n c e n t r a t i o n o f c h o l i n e i n the medium, simple d i f f u s i o n was the major mode of c h o l i n e e n t r y . T h e r e f o r e , the e f f e c t o f g l u c a g o n on the c a r r i e r - m e d i a t e d t r a n s p o r t of c h o l i n e that predominates when the medium c h o l i n e c o n c e n t r a t i o n i s below 10 ^ J M was i n v e s t i g a t e d . The f a c i l i t a t e d u ptake of c h o l i n e was a l s o i n s e n s i t i v e to glucagon a c t i o n ( F i g . 4MB). Glucagon I n h i b i t s P h o s p h a t i d y l c h o l i n e S y n t h e s i s From C h o l i n e and Stimulates Betaine S e c r e t i o n From Rat Hepatocytes ( 3 - 7 . 3 . 2 ) . The e f f e c t of glucagon (100 nM) on p h o s p h a t i d y l c h o l i n e s y n t h e s i s from c h o l i n e was studied i n i s o l a t e d r a t hepatocytes ( F i g . 45). Although glucagon 3 d i d not a f f e c t [Me- H ] c h o l i n e uptake i t was f e a s i b l e t h a t t h i s hormone could reduce the pool s i z e o f p h o s p h o c h o l i n e as has been demonstrated f o r chlor o p h e n y l t h i o - c A M P - t r e a t e d c e l l s ( F i g . 3 2 ) . A decrease i n the l e v e l of phosphocholine would r e s u l t i n a h i g h e r s p e c i f i c r a d i o a c t i v i t y f o r t h i s metabolite and increased l a b e l i n g o f p h o s p h a t i d y l c h o l i n e which could mask any i n h i b i t i o n o f t h i s p a t h w a y by g l u c a g o n . To a v e r t t h i s p o t e n t i a l problem, the h e p a t o c y t e s were p r e l a b e l e d f o r 3 0 min w i t h 15 ^ C i of [Me-3 H ] c h o l i n e . At the end o f the p u l s e p e r i o d , 9 0 % of the l a b e l that had not 180 Figure 44. E f f e c t o f g l u c a g o n on the i n c o r p o r a t i o n o f [Me- 3H]choline i n t o  r a t hepatocytes. C e l l s ~( 3 X 10**) were p r e i n c u b a t e d f o r 1 h i n serum-free medium which contained 28 uM c h o l i n e and f o r an a d d i t i o n a l h i n the absence or presence o f 75 nM g l u c a g o n , k, the medium was r e p l a c e d and the c e l l s were p u l s e d f o r 10-40 min w i t h 20 u C i o f 28 uM [ M e - 3 H ] c h o l i n e i n the absence ( A ) or p r e s e n c e ( A ) o f glucagon p r i o r t o h a r v e s t i n g . B, a f t e r the p r e i n c u b a t i o n , the c e l l s were p u l s e d w i t h 15 uCi of [Me- 3H]choline f o r 10 min i n the absence ( O ) o r p r e s e n c e ( • ) o f gl u c a g o n and 2-120 uM c h o l i n e . For both uptake s t u d i e s , the c e l l s were subsequently scraped i n t o 4 ml of i c e - c o l d PBS and an a l i q u o t o f the suspension was counted f o r r a d i o -a c t i v i t y . Each p o i n t r e p r e s e n t s the mean of f o u r d i s h e s f o r Panel A and three dishes f o r P a n e l B, and the s t a n d a r d e r r o r i s i n d i c a t e d with b a r s , each experiment was repeated twice with s i m i l a r r e s u l t s . CHASE TIME (min) CHASE TIME (min) CHASE TIME (min) CHASE TIME (min) Figure 45. E f f e c t o f 100 nM glucagon on the d i s a p p e a r a n c e of [Me- 3H]cho- l i n e from c e l l u l a r p h o s p h o c h o l i n e and a c c u m u l a t i o n i n t o p h o s p h a t i d y l - c h o l i n e . C u l t u r e d r a t h e p a t o c y t e s were p u l s e l a b e l e d f o r 30 min with 15 u C i of [ M e - 3 H ] c h o l i n e . The c e l l s were washed and f r e s h medium w i t h or without 100 nM glucagon was added. At v a r i o u s t i m e s , the c e l l s and media were c o l l e c t e d , and the r a d i o a c t i v i t y was q u a n t i t a t e d i n A, phospho-c h