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Phosphatidylcholine biosynthesis in hypercholesterolemic rats Lim, Princeton H. 1981

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PHOSPHATIDYLCHOLINE BIOSYNTHESIS IN HYPERCHOLESTEROLEMIC RATS by PRINCETON H. LIM •Sc., Hon., University of British Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Biochemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1980 0 Princeton H. Lim, 1980 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C olumbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and stud y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r - p u b l i c a t ion o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date CkfsUUOM,/ /J)/fcf/ i. ABSTRACT The regulation -of the rate of phosphatidylcholine biosynthesis was investigated i n the present- study. Rats fed a high cholesterol/cholate d i e t had elevated l e v e l s of plasma c h o l e s t e r o l and phospholipids and t h i s suggested that the rate of phosphatidylcholine biosynthesis might be a l t e r e d during \ hypercholesterolemia. The a c t i v i t i e s of the enzymes of phosphatidylcholine biosynthesis were measured and the a c t i v i t i e s of phosphocholine c y t i d y l y l t r a n s f e r a s e i n cytosol and microsomes were increased 2- to 3-fold i n the l i v e r s from hypercholesterolemia rats when compared to c o n t r o l s . The a c t i v i t i e s of choline kinase, phosphocholine-transferase and phosphatidylethanolamine methyltransferases, however, were unchanged. The stimulation of the c y t i d y l y l t r a n s f e r a s e was not due to an adaptive increase in the quantity of enzyme and r e s u l t s suggested that a phospholipid modulator was responsible for the a c t i v a t i o n of the enzyme. The rate of phosphatidylcholine biosynthesis i n the l i v e r was measured a f t e r i n t r a p o r t a l i n j e c t i o n of l a b e l l e d choline. The estimated rate of synthesis was 2- to 3-fold higher i n the hypercholesterolemia r a t s and t h i s correlated with the 2- to 3-fold stimulation of phospho-choline 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 r e s u l t s c l e a r l y showed that 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 the l i v e r was r a t e - l i m i t i n g i n the de novo synthesis of phosphatidylcholine during hypercholesterolemia and strongly indicated that the increase i n plasma phospholipids during hypercholesterolemia was, at l e a s t i n part, the r e s u l t of increased synthesis of phosphatidylcholine i n the l i v e r . ii. TABLE OF CONTENTS Page ABSTRACT 2..- i. LIST OF TABLES vii. LIST OF FIGURES DE-LIST OF ABBREVIATIONS xii. ACKNOWLEDGEMENTS XV. DEDICATION xvi. INTRODUCTION , ' 1. I. Biological Roles of Phosphatidylcholine 1. II. Pathways for the Synthesis of Phosphatidylcholine 3• 1. The De Novo Pathway 4. (a) Choline Kinase 7. (b) Phosphocholine Cytidylyltransferase 8. (c) Phosphocholinetransferase 15. 2>. Phosphatidylethanolamine N-Methylation Pathway 23. 3. Other Pathways for Phosphatidylcholine Biosynthesis... 33. I l l . Regulation of the Rate of Phosphatidylcholine Biosynthesis 34. 1. The De Novo Pathway 34. 2. Phosphatidylethanolamine N-Methylation Pathway 41. IV. Mechanisms of Regulation of the Rate of Phosphatidylcholine Biosynthesis 42 . V. Hepatic Phosphatidylcholine Biosynthesis and Hypercholesterolemia 43. MATERIALS AND METHODS 46. I. Animals, Enzymes, Chemicals and Radioactive Compounds 46. 1. Animals 46. 2. Enzymes, Chemicals and Radioactive Compounds 46. iii. Page II. Preparative Procedures 48. 1. Isolation of Total Rat Liver Phospholipid 48. 2. Preparation of Partially Purified Phospho-choline Cytidylyltransferase 48. 3. Delipidation of Phosphocholine Cytidylyltransferase by Extraction with Acetone and Butanol 49. 4. Enzymatic Synthesis of [Me-3H]Phosphocholine 49. 5. Preparation of [1,2-ethanolamine-l^C]-Phosphatidylethanolamine and Radio-Labelled Phosphatidylethanolamine-Phospholipid Vesicles 50. 6. Preparation of Buffers 51. I l l . General Procedures 51. 1. Measurement of Protein 51. 2. Measurement of Phosphorus in Phospholipids 52. 3. Liquid Scintillation Counting 52. 4. Thin Layer Chromatography 53. (a) Solvents for Water-Soluble Compounds 53. i) Single Dimensional Systems 53. i i ) Two Dimensional Systems 54. (b) Solvents for Lipids 54. i) Single Dimensional Systems 54. i i ) Two Dimensional Systems 54. 5. Column Chromatography 55. IV. Enzyme Assays 55. 1. Choline Kinase 55. 2. Phosphocholine Cytidylyltransferase 57. (a) Cytosolic Activity 59. (b) Microsomal Activity 59. iv. Page 3. Phosphocholinetransf erase 59. 4. Phosphatidylethanolamine methyltransferase 62. V. Methods Used in In Vitro Studies 65. 1. Care and Feeding of Experimental Animals 65. 2. Preparation of Experimental Diets 65. 3. Measurement of Total Plasma Cholesterol and Phospholipids 66. 4. Blood Sampling, Preparation of Cytosol and Microsomes and Perfusion of Rat Liver 66. 5. Measurement of Diacylglycerol in Cytosol 67. 6. Ul t r a f i l t r a t i o n and Phospholipase C Treatment of Cytosols 68. 7. Purification of Phosphocholine Cytidylyl-transferase and Assessment of Purity 69. ) 8. Preparation of Antibodies Against the Purified cytidylyltransferase and Immunotitration of the Enzyme from Cytosol 70. 9. Treatment of Cytosols with Trypsin and Column Chromatography of Trypsin-inactivated Cytosols 72. 10. Procedure for the Large Scale Isolation of Cytosolic Lipids and the Fractionation of These Lipids by Thin Layer Chromatography 73. 11. Analysis of Lysophosphatidylethanolamine by Mass Spectrometry 73. 12. Quantitation of Lysophosphatidyl-ethanolamine by Ninhydrin 74. 13• Analysis of Fatty Acids on Lysophosphatidyl-ethanolamine by Gas-Liquid Chromatography 75. VI. Methods Used in In Vivo Studies 75. 1. Intraportal Injection of [Methyl-3H-]-Choline and Freeze Clamping of Livers 75. 2. Bligh and Dyer Extraction 76. V. Page 3. Separation of Choline, Phosphocholine, CDP-Choline and Betaine 77. 4. Measurement of Phosphocholine Pool 77. 5. Calculation of the Rate of Phosphatidylcholine Biosynthesis 79. VII. St a t i s t i c a l Methods 80. RESULTS 82. I. Characterization of Phosphocholine Cytidylyl- transferase: The Effect of pH, Phospholipids,  Acyl-CoAs, Various Adenosine-Containing Coenzymes,  and Nucleotides on Enzyme Activity. 82. II . Effects of Various Diets on the Levels of Plasma Lipids and Phosphocholine Cytidylyl- transferase Activity 88. I l l . Cholesterol/Cholate Feeding: In Vitro Studies 92. 1. The Effect of the Cholesterol/Cholate-rich Diet on Body Weight, Liver Weight and Plasma Lipid Levels 92. 2. Effect of Diet on Enzymes of Phosphatidylcholine Biosynthesis 92. 3. The Effect of the Cholesterol/Cholate-rich Diet on the Aggregation of Phosphocholine Cytidylyltransferase and Diacylglycerol Concentration in Cytosol 94. 4. Studies on Phosphocholine Cytidylyltransferase in Cytosols from Normal and Cholesterol/ Cholate-fed Rats 94. 5. Immunotitration of Phosphocholine Cytidylyltransferase 101. 6. Characterization of the Modulator of Phosphocholine Cytidylyltransferase 105. 7. Analysis of Palmitoyl-lysophosphatidyl-ethanolamine by Mass Spectrometry 117. 8. The Fatty Acid Composition and the Amount of Lysophosphatidylethanolamine in Cytosol 119. vi. Page 9. Lysophosphatidylethanolamine i n Cytosol was not Generated During the Preparation of Subcellular Fractions 1 2 3 . IV. Cholesterol/Cholate Feeding: In Vivo Studies 123. 1. The Amount of Radioactivity i n Choline, Phosphocholine, CDP-Choline, Betaine and Phos-phatidylcholine at Several Time Periods After I n t r a p o r t a l Injection of Labelled Choline 124. 2. An Estimate of the Rate of Phosphatidylyl choline Biosynthesis i n Livers from Control and Cholesterol/Cholate-fed Rats 126. DISCUSSION 133-I. Phosphatidylcholine Biosynthesis i n Rats Fed a Normal and Cholesterol/Cholate-Rich Diet 133. I I . Measurement of the Rate of Phosphatidylcholine Biosynthesis 134. I l l . Enzymes of Phosphatidylcholine Biosynthesis i n Normal and Hypercholesterolemia Rats. 136. IV. The Regulation of the De Novo Pathway for Phosphatidylcholine Biosynthesis 140. V. Suggestions for Further Studies. 144. BIBLIOGRAPHY 145. vii. LIST OF TABLES Table Page 1 Concentrations of Substrates and Intermediates of the Pathways for Phosphatidylcholine Biosynthesis i n Rat L i v e r 5. 2. Properties of 31-Fold P u r i f i e d Ethanolamine and Choline Kinase from Rat Liver 9. 3. Physical and K i n e t i c Properties of Phosphocholine C y t i d y l y l -transferase from Rat Lung and Liver 12. 4. Modulators of the A c t i v i t y of Phosphocholine C y t i d y l y l -transferase from Lung, L i v e r , Intestine and Brain 13. 5. S p e c i f i c i t i e s of Phosphocholinetransferase and Phospho-ethanolaminetransferase from Rat L i v e r for Various Species of D i a c y l g l y c e r o l s 18. 6. Properties of Phosphocholinetransferase and Phospho-ethanolaminetransferase from Rat Adipocyte Microsomes 20. 7. Properties of P a r t i a l l y P u r i f i e d Phosphocholinetrans-ferase and Phosphoethanolaminetransferase from Rat Liver Microsomes 21. 8. Modulators of Phosphocholinetransferase and Phospho-ethanolaminetransf erase from Rat Liver Microsomes 22. 9. Properties of Phosphatidylethanolamine Methyltransferase from Crassa Microsomes 27. 10. Properties of Phosphatidylethanolamine Methyltransferases from Agrobacterium Tumefaciens 28. 11. Properties of Phosphatidylethanolamine Methyltransferases from Bovine Adrenal Medulla 29. 12. Properties of P a r t i a l l y P u r i f i e d Phosphatidylethanolamine Methyltransferases from Dog Lung 30. 13. Properties of Phosphatidylethanolamine Methyltransferases from Rat Liver 31-14. Properties of S o l u b i l i z e d Phosphatidylethanolamine Methyltransferases from Rat Liver 32. viii. Table Page 15. The E f f e c t of Development, Diet, Hormones and Other Compounds on the Enzymes and the Incorporation of Labelled Choline into Phosphatidylcholine i n Rat L i v e r and Lung 38. 16. M o b i l i t i e s of Phospholipids on Thin Layer Plates Developed i n Various Solvent Systems 56. 17. E f f e c t of Various Adenosine-Containing Coenzymes and Nucleotides on the A c t i v i t y of Phosphocholine C y t i d y l y l t r a n s f e r a s e 85. 18. Plasma L i p i d s 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 Liver Cytosols from Rats Fed a Control High Fat Diet and a Basal Atherogenic Diet (BAD) 90. 19. E f f e c t of Diets Containing 2% Cholate, 5% Cholesterol or 2% Cholate and 5% Cholesterol on the Level of Plasma L i p i d s and the C y t o s o l i c A c t i v i t y of Hepatic Phosphocholine C y t i d y l y l t r a n s f e r a s e 91 • 20. S p e c i f i c A c t i v i t i e s of Enzymes of Phosphatidylcholine Biosynthesis from Rat Liver 93 • 21. Degradation of l^C-Labelled CDP-Choline by Cytosol and Microsomes from Normal and Cholesterol/Cholate-Fed Rats 97. 22. Stimulation of Delipidated C y t i d y l y l t r a n s f e r a s e by the Water-Soluble and Lipid-Soluble Components of Column Fractions that Contain the Activator 110. 23. Composition of Phospholipids i n Cytosols from Normal and Hypercholesterolemia Rats 112. 2k. Stimulation of P a r t i a l l y P u r i f i e d L-form of phospho-choline C y t i d y l y l t r a n s f e r a s e by C y t o s o l i c Phospho-l i p i d s from Normal and Hypercholesterolemia Rats 113. 25. Recovery of the A b i l i t y of C y t o s o l i c L i p i d s to Stimulate 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 After Thin Layer Chromato graphy on S i l i c i c Acid 115. 26. Analysis of Eluted and Stock Palmitoyl-lysophosphatidyl-ethanolamine by Thin Layer Chromatography 118. 27. Structure and Predicted Fragments of the S i l y l a t e d Derivative of Palmitoyl-lysophosphatidylethanolamine 121. ix. Table Page 28. The Incorporation of [ 1 ^ - l ^ C j C h o l i n e into Various Compounds i n the Livers from Control and Cholesterol/ Cholate-Fed Rats 127. 29. Incorporation of Radioactivity into Various Choline-Containing Metabolites After I n t r a p o r t a l Injection of [Me-3H]Choline 129. 30. Incorporation of Radioactivity i n t o Betaine and the percent of Injected Dose Incorporated After Intraportal I n j e c t i o n of [Me-3H]Choline 130. ft 31. E f f e c t of Cholesterol/Cholate Feeding on the Rate of Phosphatidylcholine Biosynthesis 132. X. LIST OF FIGURES Figure Page 1 Structure of Phosphatidylcholine 2. 2. Pathways for the Biosynthesis of Phosphatidylcholine 6. 3. Choline Kinase A c t i v i t y as a Function of Time and Protein.... 58. 4. A c t i v i t y of C y t o s o l i c Phosphocholine C y t i d y l y l t r a n s f e r a s e with Time and Various Amounts of Protein 60. 5. A c t i v i t y of Microsomal Phosphocholine C y t i d y l y l t r a n s f e r a s e with Time and Various Amounts of Protein 61. 6. A c t i v i t y of Phosphocholinetransferase with Time and Various Amounts of Protein 63 • 7. A c t i v i t y of Phosphocholinetransferase i n the Presence of Various Amounts of D i a c y l g l y c e r o l s 64. 8. A c t i v i t y of the High and Low Molecular Weight Forms of Phosphocholine C y t i d y l y l t r a n s f e r a s e at Various Values of pH 83. 9. Double Reciprocal Plots of the I n i t i a l V e l o c i t y of Phosphocholine C y t i d y l y l t r a n s f e r a s e Versus Various Concentrations of CTP or Phosphocholine i n the Presence of ATP 86. 10. Double Reciprocal Plots of the I n i t i a l V elocity of Phosphocholine C y t i d y l y l t r a n s f e r a s e Versus Various Concentrations of CTP or Phosphocholine i n the Presence of NAD 87. 11. Chromatography of Cytosols from Normal and Cholesterol-fed Rats 95. 12. A c t i v a t i o n of Phosphocholine C y t i d y l y l t r a n s f e r a s e i n Control and Experimental Cytosols by Incubation at 4 ° C . . . 99. 13- A c t i v a t i o n of Phosphocholine C y t i d y l y l t r a n s f e r a s e i n Cytosol from Control and Cholesterol/Cholate-fed Rats 100. 14. Stimulation by Rat Liver Phosphoplipids of Phosphocholine C y t i d y l y l t r a n s f e r a s e which Remained i n the Supernatant After Immunoprecipitation 102. 15. P r e c i p i t a t i o n of Phosphocholine C y t i d y l y l t r a n s f e r a s e by Control and A n t i - c y t i d y l y l t r a n s f e r a s e Antibodies at Various pH 103. xi. Figure v Page 16. Immunotitration of Phosphocholine C y t i d y l y l t r a n s f e r a s e i n Cytosols from Control and Cholesterol/Cholate-fed Rats with Control and A n t i - c y t i d y l y l t r a n s f e r a s e Antibody 104. 17. Immunochemical Quantitation of Cytosolic Phosphocholine C y t i d y l y l t r a n s f e r a s e from Normal and Cholesterol/ Cholate-fed Rats 106. 18. E f f e c t of Phospholipase C ( c l . Welchii) Digestion on the A c t i v i t y of Phosphocholine C y t i d y l y l t r a n s f e r a s e i n Cytosols from Normal and Cholesterol/Cholate-fed Rats 107. 19. Chromatography of Trypsin-Treated Cytosol from Control and Cholesterol/Cholate-fed Rats 109. 20. Stimulation of 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 by L i p i d Extracts from Control and Cholesterol/Cholate-fed Rat Liver Cytosol 111. 21. Stimulation of 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 by Standard Lysophosphatidylethanolamine and by the Same L i p i d which had been Applied on and Eluted from S i l i c a 116. 22. Mass Spectra of the S i l y l a t e d Derivative of Palmitoyl-lysophosphatidylethanolamine 120 . 23. Incorporation of [Me-3H]Choline i n t o Hepatic Pools of Choline, Phosphocholine, CDP-choline and Betaine 125. 24. Regulation of the Rate of Phosphatidylcholine Biosynthesis 142. LIST OF ABBREVIATIONS AdoHcy S-adenosylhomocysteine AdoMet S-adenosylmethionine ATP adenosine triphosphate Ci curie CDP.-choline cytidine-diphosphocholine CDP c y t i d i n e diphosphate CL C a r d i o l i p i n CMP c y t i d i n e monophosphate cpm counts per minute CTP c y t i d i n e triphosphate DG d i a c y l g l y c e r o l dpm d i s i n t e g r a t i o n s per minute EDTA ethylenediaminetetracetic acid ev electron v o l t s g grams GMP guanosine 5'-monophosphate GTP guanosine 5'-triphosphate hr hour IgG immunoglobulin G Ki i n h i b i t i o n constant Km Michaelis-Menten constant 1 l i t e r LPC lysophosphatidylcholine LPE lysophosphatidylethanolamine m meter xi-Li. M molar Mg + + magnesium ion min minutes Mn + + manganese ion moi mole N normal sol u t i o n NAD nicotinamide-adenine dinucleotide NADP nicotinamide-adenine dinucleotide phosphate PC phosphatidylcholine PE phosphatidylethanolamine PG phosphatidylglycerol PI phosphatidylinositol POPOP 1,4 Bis(2-(5-phenyloxazolyl))Benzene PPO 2,5-diphenyloxazole PS phosphatidylserine P i inorganic phosphate PPi inorganic pyrophosphate Rf r a t i o of the distance t r a v e l l e d by a compound to that t r a v e l l e d by the solvent front sec second SDS sodium dodecylsulphate SM sphingomyelin TLC t h i n layer chromatography T r i s tris(hydroxymethyl)amino ethane UMP uridine 51-monophosphate UTP uridine 5'-triphosphate UV u l t r a v i o l e t V V max w volume maximum v e l o c i t y weight Standard Prefixes K K i l o - 10 3 C c e n t i - 10"' m m i l l i - 10" \i micro - 10" n nano - 10" XV. ACKNOWLEDGEMENTS I would like to thank Dr. Dennis E. Vance for his support and guid-ance. I would also like to thank the following people: Dr. Patrick C. Choy for his .invaluable help in the lab, Dr. P. Haydn Pritchard for his collaboration in the in vivo studies, Ms. Sandra Fenn for her many patient hours in typing this thesis, and Mr. Steve Pelech for his many construc-tive comments. Lastly, I wish to express my special thanks to Faith Lynn for her constant love and encouragement. DEDICATION To My Parents, Benjamin and Josephine the beginning God created..." Genesis 1:1 1. INTRODUCTION I. B i o l o g i c a l Roles of Phosphatidylcholine Phospholipids are found i n a l l l i v i n g organisms and they are major components of c e l l u l a r membranes and blood l i p o p r o t e i n s i n animals. In b i o l o g i c a l membranes, phospholipids are arranged i n a b i l a y e r where the polar head groups face the aqueous environment on the outside of the b i l a y e r while the f a t t y a c y l chains face inward to form a hydrophobic matrix. The membrane i s not merely a s t a t i c structure of the c e l l but performs many v i t a l functions such as ion transport and endo- and exocytosis. Although proteins found i n membranes are responsible f o r many membrane functions, evidence suggests that phospholipids may play a ro l e i n some c e l l u l a r processes such as membrane fusion ( l ) . The importance of phospholipids i n the structure and function of lip o p r o t e i n s i s unclear at the present, but i t i s very possible that phospholipids and apoproteins may coat the surface of the lip o p r o t e i n s and render the core of t r i g l y c e r i d e s and cho l e s t e r o l esters water soluble. Phosphatidylcholine i s the major class of phospholipids found i n animal t i s s u e s . A g l y c e r o l molecule forms the backbone of t h i s l i p i d and f a t t y acids are e s t e r i f i e d to carbons 1 and 2 while phosphocholine i s e s t e r i f i e d to carbon 3 ( F i g . ! ) • Phosphatidylcholine performs several s p e c i a l i z e d 0 II CH2-0-C-R1 Ro-C-0-CH 0 CH2-0-P-0-CH2CH2N(CH3)3 0-. 1 . Structure of Phosphatidylcholine. In most species, saturated and R£ is unsaturated. 3. functions i n the body. In lung, the major component of surfactant i s dipalmitoylphosphatidylcholine which f a c i l i t a t e s the expansion and contraction of the lungs by lowering the surface tension i n the a l v e o l i (2) . Phosphatidylcholine i s also an important component of b i l e which aids i n the s o l u b i l i z a t i o n , digestion and absorption of dietary t r i g l y c e r i d e s . In the blood, PC plays an important r o l e i n the metabolism of l i p o p r o t e i n c h o l e s t e r o l . L e c i t h i n c h o l e s t e r o l acyltransferase (LCAT) e s t e r i f i e s c h o l e s t e r o l by t r a n s f e r r i n g a f a t t y acid chain from PC. This reaction i s e s s e n t i a l for clearance of free cholesterol from c e l l membranes (3) . The regulation of PC biosynthesis i s not well understood, although the two major biosynthetic pathways were elucidated i n the 1950's and early 60's. However, i n the past 20 years, the soluble enzymes i n the pathways were i s o l a t e d and characterized and the membrane bound enzymes were suc c e s s f u l l y s o l u b i l i z e d . The following sections discuss some of the properties of the biosynthetic enzymes and the regulation of PC biosynthesis i n lung and l i v e r . I I . Pathways for the Synthesis of Phosphatidylcholine The major pathway for PC biosynthesis was discovered by Kennedy and Weiss i n the 1950's (4). Their work extended from the e a r l i e r studies of 32 14 Kornberg (5) who reported that P- and C-labelled phosphocholine was converted by l i v e r enzymes to a l i p i d , presumably PC and proposed the following scheme for PC synthesis: phosphatidic acid + phosphorylcholine l e c i t h i n + P i 32 The reaction as shown should be r e v e r s i b l e and P should be r e a d i l y incorporated into phospholipids i n v i t r o . Work i n Kennedy's laboratory, however, f a i l e d to demonstrate such a reaction and i n the course of further 4. investigations a' new type of c y t i d i n e containing coenzyme was discovered (4). The cytidine-diphosphocholine (CDP-choline) and cytidine-diphosphoethanolamine (CDP-ethanolamine) coenzymes were found to play an e s s e n t i a l r o l e i n the de novo biosynthesis of PC and PE (4). A second pathway was discovered around I960 by Greenberg and co-workers (6) who were mainly interested i n choline biosynthesis. They found that 14 when C-ethanolamine was given to r a t s , the l a b e l was incorporated into PC but not into phosphocholine. Extensive experiments f a i l e d to demonstrate choline formation from free ethanolamine and when 14 LMe- C]methionine was used, r a d i o a c t i v i t y was found i n mono- and dimethylPE as well as in PC. The turnover rates of the methylated PEs were very much fas t e r than PC and t h i s suggested that the mono- and dimethylated derivatives were intermediates i n the synthesis of PC. These and other observations supported the conclusion that PE i s methylated to form PC, with S-adenosylmethionine serving as the donor of a l l the methyl groups. The de novo pathway i s the major route for PC biosynthesis i n the l i v e r , lung, and other tissues, whereas the N-methylation pathway, which i s responsible for about 20% of hepatic PC synthesis i n rats (7) and humans (8), i s of s i g n i f i c a n c e only i n l i v e r (9). The enzymes i n both of these pathways have been studied and the known physical and enzymological properties are given below. The concentrations of substrates and intermediates of both pathways are given i n Table 1. 1. The De Novo Pathway The _de novo pathway converts choline to PC by three separate reactions with phosphocholine and CDP-choline as intermediates ( F i g . 2). Three enzymes catalyzed these reactions and choline which i s water-soluble i s eventually converted into a l i p i d - s o l u b l e molecule. Table 1 Concentrations of Substrates and Intermediates of the Pathways f o r Phosphatidylcholine Biosynthesis i n Rat Li v e r De Novo Pathway Infante (70) Substrate ATP Choline ADP Phosphocholine M g++ CTP CDP-Choline PPi Concentration' (yimol'g wet l i v e r wt.~i) 1.95 + 0.06 0.23 ± 0.10 1.4 ± 0.20 1.1 ± 0.03 0.7 0.083 + 0.005 0.051 + 0.003 0.0062 ± 0.0003 Phosphatidylethanolamine N-Methylation Pathway-Katyal and Lombardi (96) Substrate Phosphatidylethanolamine Phosphatidyl N-monomethyl-ethanolamine Phosphatidyl N-dimethyl-ethanolamine Phosphatidylcholine Concentration* (nmol'g l i v e r wt.~l) 11 x 103 12 18 18 x 103 * Concentration was calculated with the assumption that the l i v e r of a 100 g rat weighs 5 g. 6. 3AdoMet Phosphatidyl-ethanolamine Choline ATP-Mg ++ ADP-Mg ++ Choiine Kinase (Cytosol) Phosphocholine Phosphocholine Itransi _. and Microsomes) ^vtidylvitra ferase CDP-Choline Diglyceride Mg++) CMP 3AdoHCy 4 Phosphocholinetransferase (Microsomes) .Phosphatidylcholine Phosphatidylethanolamine-P Methyltransferases (Microsomes) Fig. 2. Pathways for the Biosynthesis of Phosphatidylcholine 7. (a) Choline Kinase The f i r s t enzyme of the pathway i s choline kinase. This c y t o s o l i c enzyme was f i r s t described i n yeast by Wittenberg and Romberg (10) who established the stoichiometry of the reaction and demonstrated an absolute requirement of the enzyme for magnesium ions. Romberg's early studies also showed that the kinase was most active between pH 8.0 and 9.0, and that i t was able to catalyze the phosphorylation of monoethyl- and diethylethanolamine, monomethyl- and dimethylethanolamine and ethanolamine. However, substrate binding data indicated that the a f f i n i t y of the enzyme for choline was several orders of magnitude higher than for ethanolamine. Phosphorylation of choline was demonstrated i n l i v e r , brain, kidney and i n t e s t i n a l mucosa of various animals (10). The f i r s t evidence which indicated that the ethanolamine and choline phosphorylations might be catalyzed by separate kinases stemmed from the work of Sung and Johnstone (11). They found that E h r l i c h a s c i t e s c e l l extracts converted choline and ethanolamine i n t o t h e i r phosphorylated derivatives but the choline phosphorylating a c t i v i t y was much more stable during storage at -10°C. Cysteine and calcium ions i n h i b i t e d ethanolamine phosphorylation but did not a f f e c t choline phosphorylation. Furthermore, various a c t i v a t o r s of choline phosphorylation i n h i b i t e d ethanolamine phosphorylation. The r e s u l t s suggested that separate enzymes were involved but physical separation of the two was not achieved. The choline and ethanolamine phosphorylating a c t i v i t i e s were p a r t i a l l y separated by Weinhold and Rethy (12). By using DEAE-cellulose chromatography, they were able to pur i f y the enzymes 31-fold and dissoc i a t e d ethanolamine kinase into two d i s t i n c t forms. Ethanolamine kinase I was much smaller than ethanolamine kinase II which had choline kinase a c t i v i t y . The 8. properties of the kinases are summarized i n Table 2. Ethanolamine kinase I and kinase II have d i s t i n c t c h a r a c t e r i s t i c s and aside from the d i f f e r e n t Km's for ATP and ethanolamine, ethanolamine kinase II was strongly i n h i b i t e d by choline (Ki = 0 . 0 3 mM) which competes with ATP, whereas kinase I was not af f e c t e d . The authors suggested that the l e v e l s of ATP and choline could possibly regulate the rate of PE biosynthesis. The choline kinase a c t i v i t y was i n h i b i t e d by high concentrations of ethanolamine which competes with choline (Ki = 4.5 mM) and ATP (Ki = 9.8 mM) for the enzyme. The controversy of whether choline kinase and ethanolamine kinase II were one or two separate enzymes remained unresolved for some time. Brophy and Vance (13) reported i n 1976 that the kinases from rat l i v e r were c o - p u r i f i e d 500-fold by a f f i n i t y chromatography and the r e s u l t s supported the single enzyme hypothesis. However, i n a l a t e r study (14), ethanolamine kinase and choline kinase were separated by g e l electrophoresis at pH 8.7 from a highly p u r i f i e d preparation and ethanolamine kinase was resolved i n t o two components which were d i s t i n c t from choline kinase. Furthermore, when fresh cytosol was subjected to d i s c g e l electrophoresis, four isozymes of choline kinase and ethanolamine kinase were found (14) and the function of these multiple forms of the enzymes remains unclear at present. (b) Phosphocholine C y t i d y l y l t r a n s f e r a s e Phosphocholine c y t i d y l y l t r a n s f e r a s e catalyzes the formation of CDP-choline from phosphocholine and CTP and i s the second enzyme of the de novo pathway. In e a r l y studies (15), Kennedy and h i s co-workers found that a large amount of ATP was required to support the synthesis of PC from phosphocholine. In l a t e r experiments, however, t h i s reaction was shown to s p e c i f i c a l l y require CTP which was a contaminant i n the ATP preparation 9. Table 2 Properties of 31-Fold P u r i f i e d Ethanolamine and Choline Kinase from Rat Liver EKI EKII CK Weinhold and Rethy (12) Molecular Weight 36,000 166,000 (EKII and CK co-purified) pH Optimum 7.5-8.5 8.0-9.5 8.0 Substrates i ) ATP i i ) Ethanolamine i i i ) Choline 14.3 0.4 Km for Substrate (mM) 0.5 1.7 3-7 0.03 Competitive I n h i b i t o r s  (Substrate) ( i ) Ethanolamine (choline) i i ) Ethanolamine (ATP) K i , I n h i b i t i o n Constant (mM) 4.5 9.8 i i i ) Choline (ATP) 0.03 EK - ethanolamine kinase CK - choline kinase 10. used i n e a r l i e r studies. Shortly a f t e r Kennedy elucidated the r o l e of CDP-choline i n PC synthesis, he and co-workers described the enzymatic synthesis of CDP-choline i n guinea pig l i v e r (16). Their early work showed that t h i s enzyme required magnesium or manganese ions for a c t i v i t y and u t i l i z e d CTP. The pyrophosphorolysis of CDP-choline was also r e a d i l y catalyzed by t h i s enzyme. The c y t i d y l y l t r a n s f e r a s e could also u t i l i z e dCTP i n the synthesis of dCDP-choline as was f i r s t shown by Kennedy, Borkenhagen, and Smith (.17)• This was confirmed by Schneider and Behki (18) and subsequent studies by Schneider (19) revealed some other i n t e r e s t i n g r e s u l t s . He found that 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 p r e f e r e n t i a l l y p a r t i t i o n e d into the cytosol when the l i v e r was homogenized i n s a l i n e , whereas most of the a c t i v i t y was found i n the p a r t i c u l a t e f r a c t i o n when d i s t i l l e d water was used. The enzyme a c t i v i t y p a r t i t i o n e d almost equally between the soluble and p a r t i c u l a t e f r a c t i o n s when the l i v e r was homogenized i n sucrose. He also found that storage of the 100,000 x g supernatant obtained from 20% l i v e r homogenate at 0°C r e s u l t e d i n a 5-fold stimulation 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 . These r e s u l t s l a t e r f a c i l i t a t e d the development of a p u r i f i c a t i o n procedure for the c y t i d y l y l t r a n s f e r a s e (22). Further ground work was established by Fiscus and Schneider (20) when they reported that the a c t i v 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 was stimulated by phospholipids. The enzyme was delipidated by extraction with cold acetone and butanol, and t h i s resulted i n l o s s of almost a l l a c t i v i t y . Addition of phospholipids resulted i n a 25-fold stimulation of enzyme a c t i v i t y and with t h i s delipidated form of the enzyme, they found that LPC and LPE were potent a c t i v a t o r s , whereas p u r i f i e d egg PC showed l i t t l e stimulative a c t i v i t y . The authors suggested a p o s i t i v e feedback mechanism i n the regulation of PC synthesis. 11. Early attempts to p u r i f y t h i s enzyme were unsuccessful because severe l o s s of enzyme a c t i v i t y occurred during ion exchange chromatography (20) and Sephadex G-200 chromatography (21). The enzyme was f i n a l l y p u r i f i e d to homogeneity by Choy et a l . (22) by Sepharose 6B chromatography. The authors also reported the presence of a high molecular weight (H-form) and low molecular weight (L-form) form of the 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 c y t o s o l . The existence of the two forms i n lung had been reported e a r l i e r by Stern, Kovac and Weinhold (23) and subsequent studies from the same laboratory showed that PG, CL and PI were potent a c t i v a t o r s of the enzyme (24) . However, the e f f e c t of PG on the enzyme was unique since t h i s phospholipid also aggregated the enzyme to the high molecular weight form. The physical and k i n e t i c properties of the enzyme i n lung and l i v e r , as well as the phospholipid modulators of the lung, l i v e r and 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 , are summarized i n Tables 3 and 4. The e f f e c t s of various phospholipids on the a c t i v 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 rat l i v e r were investigated by Choy and Vance (25) . They showed that the delipidated form of the c y t i d y l y l t r a n s f e r a s e , f i r s t used by Fiscus and Schneider (20), was stimulated by a l l classes of phospholipids. In contrast, the L-form, H-form and enzyme i n cytosol were i n h i b i t e d by c e r t a i n phospholipids. The delipidated form of the enzyme may have been desensitized during acetone-butanol extraction and was probably not representative of native c y t i d y l y l t r a n s f e r a s e . Of the d i f f e r e n t classes of phospholipids tested, LPE was the most potent a c t i v a t o r of the enzyme but unlike PG t h i s l i p i d did not aggregate the enzyme. LPC, however, was a potent i n h i b i t o r of the enzyme. Phospholipids have detergent properties and the requirement of the 12. Table 3 Physical and Ki n e t i c Properties of Phosphocholine  Cy t i d y l y l t r a n s f e r a s e from Rat Lung and Liver LUNG Stern et a l . (23) LIVER Choy et a l . (22) Molecular Weight Low Molecular Weight Form ( l i p i d - r e q u i r i n g form) Molecular Weight Range High Molecular Weight Form (n o n - l i p i d r e q u i r i n g form) pH Optimum 1.9 x 105 5-50 x 10 6 6.0 - 7-0 2.0 x 10° 0.5-13 x 10 6 7-0 Reactants i ) phosphocholine i i ) CTP i i i ) Mg++ iv) CDP-choline v) Inorganic pyrophosphate Km for Substrates (mM)  Lung Cytosol L-Form 1.0 4.0 12.0 0.167 0.208 0.210 0.004 H-Form 0.180 0.296 0.640 0.007 13. Table 4 Modulators of the Activity of Phosphocholine Cytidylyltransferase  from Rat Lung, Liver, Intestine and Brain LUNG LIVER INTESTINE CHICKEN BRAIN Feldman Choy et.al. et.al.(24) (25,26) O'Doherty et.al(27) Chojnacki et.al(28) Aggregators PG PG.DG Activators PG, PS, PG, LPE, PI, CL PI, PS 1-sn and 3-sn LPC (C^5 and CIQ more effective than species with shorter chains) 1-stearoyl LPC Inhibitors Neutral Lipids, LPC Ik. e y t i d y l y l t r a n s f e r s e was studied by using a v a r i e t y of detergents. Almost a l l of the detergents tested i n h i b i t e d the a c t i v i t y of the enzyme from lung (23) and l i v e r (25) while none stimulated the enzyme. The i n h i b i t i o n i n lung was shown to be r e l i e v e d , at l e a s t i n part, by the addition of phospholipids, suggesting that the detergents i n h i b i t e d a c t i v i t y by removing phospholipids from the enzyme. The f a c t that the detergents themselves do not stimulate the enzyme suggested that the a c t i v a t i o n by PG and LPE involve s p e c i f i c i nteractions between the l i p i d s and enzyme. The aggregation of the 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 was further studied. Choy e_t a l . (26) showed that the concentration of PG i n r a t l i v e r cytosol was i n s u f f i c i e n t to a f f e c t the c y t i d y l y l t r a n s f e r a s e and a survey of various classes of l i p i d s showed that d i a c y l g l y c e r o l was the only l i p i d from rat l i v e r that aggregated the enzyme. Cy t i d y l y l t r a n s f e r a s e from crypt and v i l l u s c e l l s was studied by O'Doherty, Smith, and Kuksis (27). They found that acetone and butanol extracted enzyme from i n t e s t i n e was stimulated by 3-sn-LPC and showed s p e c i f i c i t y for the f a t t y a c i d side chain. LPC with and f a t t y acids were several times more e f f e c t i v e i n stimulating enzyme a c t i v i t y than the l y s o - l i p i d s with shorter f a t t y acids. However, LPE as well as the serine and i n o s i t o l phosphoglycerides were not stimulatory i n contrast to the findings i n l i v e r and lung. The authors suggested that the stimulation by LPC may be rel a t e d to i t s detergent c h a r a c t e r i s t i c s since 1-sn-LPC was as e f f e c t i v e as the n a t u r a l l y occurring 3-sn analog. This idea, however, was not supported by studies i n lung and l i v e r which showed that detergents do not stimulate the c y t i d y l y l t r a n s f e r a s e . 15. (c) Phosphocholinetransferase Phosphocholinetransferase i s the l a s t enzyme of the'de novo pathway and i t i s associated with the endoplasmic reticulum (29). The c h a r a c t e r i z a t i o n of t h i s enzyme had been hindered by two main f a c t o r s . The transferase i s t i g h t l y bound to membrane and attempts to p u r i f y the enzyme were unsuccessful (30). A further complication stems from the physical properties of the substrate and products: DG and PC are both l i p i d soluble, whereas CDP-choline and CMP are water soluble. The enzyme k i n e t i c s for a reaction which involves two immiscible phases i s not yet understood. However, the s p e c i f i c i t y of the enzyme for the f a t t y acid groups on the DG has been extensively investigated by various workers. Kennedy and Weiss (4) were the f i r s t to describe phosphocholine-transferase i n r a t l i v e r microsomes. They found that the p a r t i c u l a t e f r a c t i o n converted CDP-choline to PC and t h i s a c t i v i t y was stimulated 10-to 20-fold by the addition of Tween 20 and DG. The transferase s p e c i f i c a l l y u t i l i z e d CDP-choline and magnesium or manganese ions were required for a c t i v i t y whereas calcium ions i n h i b i t e d phosphocholinetransferase even i n the presence of magnesium ions. In a l a t e r study, Kennedy, Borkenhagen and Smith (17) found that dCDP-choline was as active as CDP-choline i n the synthesis of PC but t h i s f i n d i n g was l a t e r refuted by Schneider and Behki (18) who found that CDP-choline had a 10-fold higher binding constant than dCDP-choline for the formation of PC. The s e l e c t i v i t y of phosphocholinetransferase for DG i n mouse lung was studied by Sarzala and Van Golde (31). They found that the enzyme showed a d i s t i n c t preference for 1-unsaturated DG over disaturated species. The membrane-bound DG substrates were prepared by treatment of lung microsomes with phospholipase C. In the presence of CMP, phosphocholinetransferase 16. catalyzed the formation of CDP-choline and i n t h i s reaction PC containing an unsaturated f a t t y a c i d was also p r e f e r e n t i a l l y u t i l i z e d while dipalmitoyl-PC was not used p r e f e r e n t i a l l y . The r e s u l t s suggested that the de novo pathway p r i m a r i l y functions i n the production of unsaturated PCs and the remodelling pathways (discussed l a t e r ) may be mainly responsible for the production of disaturated species i n lung. The s u b s t r a t e - s e l e c t i v i t y of phosphocholine transferase i n r a t l i v e r microsomes was also studied with endogenous substrates (32). The enzyme appeared to u t i l i z e 1-myristoyl-PC most r a p i d l y followed by the 1- palmitoyl- and 1-stearoyl-PC. The f a t t y acids on the C-2 p o s i t i o n were not characterized, and i n adipocytes Coleman and B e l l (33) showed that the optimal a c y l chain length of DG for phosphoethanolaminetransferase was longer than that f o r phosphocholinetransferase. The s p e c i f i c i t y of phosphocholinetransferase and phosphoethanolaminetransferase i n r a t l i v e r were subsequently studied more rigo r o u s l y by Holub (34). With DG's with known C- l and C-2 f a t t y acids as substrates, he found that the phosphocholinetransferase showed a marked preference f o r the 1-palmitoyl over the 1-stearoyl DG and t h i s was observed whether the f a t t y a c i d on carbon 2 was a mono- ( o l e o y l ) , d i - ( l i n o l e o y l ) , t e t r a - (arachidonoyl), or hexaene (docohexaenoyl) species. But the enzyme also showed s e l e c t i v i t y f or the f a t t y acids on C-2, although to a l e s s e r degree. The l-palmitoyl-2-arachidonoyl species was only s l i g h t l y preferred 1.5 f o l d over the 1-stearoyl species, while the l - p a l m i t o y l - 2 - o l e o y l , 2- l i n o l e o y l and 2-docohexaenoyl DG's were preferred 3-fold over t h e i r 1-stearoyl counterparts. Thus, the phosphocholine transferase appeared to be mainly s e l e c t i v e for f a t t y a c i d at the C-l p o s i t i o n . I n t e r e s t i n g l y , phosphocholinetransferase u t i l i z e d 1-oleoyl 2-stearoyl and 1-oleoyl 17. 2-palmitoyl g l y c e r o l s equally well ( 3 5 ) . This suggested that the influence of saturated f a t t y acids on the s e l e c t i v i t y of the enzyme depended on the lo c a t i o n of the f a t t y acid and that the C - l p o s i t i o n appeared to be most important for s p e c i f i c i t y . On the other hand, ethanolaminephosphotransferase showed a d i s t i n c t preference for 1-saturated 2-docohexaenoyl species over the mono-, d i - and tetraene DGs, but the enzyme also showed s p e c i f i c i t y for the C-l f a t t y a c i d . In d i r e c t contrast to the phosphocholinetransferase, phosphoethanolaminetransferase showed a preference for the 1-stearoyl over the 1-palmitoyl species when arachidonic acid was at the C-2 p o s i t i o n ( 3 4 ) . The s p e c i f i c i t y of the transferases i n a s o l u b i l i z e d preparation were studied by Morimoto and Kanoh ( 3 5 ) . By using chemically synthesized 1-acyl 2-oleoylglycerols having 12 to 18 carbons, they found that phosphocholinetransferase have s i m i l a r Vmax when f a t t y acids with 12 to 17 carbons were used. However, a marked difference was observed with the 1- stearoyl species which had a Vmax that was only 60% of the 1-palmitoyl, 2- o l e o y l g l y c e r o l . The phosphoethanolaminetransferase, i n contrast, was most active with d i o l e o y l , 1-heptadecanoyl and 1-stearoyl 2- o l e o y l g l y c e r o l s . The 1-palmitoyl species was 40% as active as the 1-stearoyl DG and 1-acyl 2-oleoylglycerol species shorter than were only 10% as a c t i v e . These r e s u l t s agree very well with Holub's study and both r e s u l t s are summarized i n Table 5. Analysis of f a t t y acids from r a t l i v e r phospholipids showed that the C - l p o s i t i o n of PC was more enriched for palmitate than stearate when compared to PE and suggested that the s p e c i f i c i t i e s of phosphocholinetransferase and phosphoethanolaminetransferase may indeed be operating i n vivo ( 3 6 ) . 18. Table 5 S p e c i f i c i t i e s of Phosphocholinetransferase and Phosphoethanolamine-transferase from Rat Li v e r for Various Species of Di a c y l g l y c e r o l s Phosphocholinetransferase Phosphoethanolamine-transferase Morimoto and Kanoh (35) Carbon 1 vmax c12:0 t o c17:0 greater than V m a x c l 8 : 0 vmax c17:0» c l 8 : 0 and C i 8 : l greater than V m a x Ci6:0 which i n turn i s greater than V m a x  c12:0 to C i 5 : 0 Holub (34) Carbon 1 ^16:0 preferred over c l 8 : 0 Cl8:0 preferred over Cl6:0 only i n 2-arachidonoyl species Carbon 2 No preference between Marked preference f o r unsaturated f a t t y 2-docosahexaenoyl species over a l l others tested. Although the k i n e t i c s for a biphasic system are not understood, attempts were made to characterize the k i n e t i c properties of the phosphocholine- and phosphoethanolaminetransferases from rat adipocytes and r a t l i v e r . The Km's of the transferases for the various substrates are shown i n Tables 6 and 1. Coleman and B e l l (37) found that both transferases from f a t c e l l s had s i m i l a r dependencies on MgCl 2 and pH, and both a c t i v i t i e s were i n h i b i t e d by C a C l 2 > detergents and d i t h i o t h r e i t o l . Furthermore, CDP-ethanolamine and CDP competitively i n h i b i t e d phosphocholinetransferase. CDP-choline appeared to be a non-competitive i n h i b i t o r of phosphoethanolamine transferase while the i n h i b i t i o n by CDP appeared to be of a mixed type (Table 6). The transferases were d i f f e r e n t i a l l y i n h i b i t e d by heating, t r y p s i n digestion, MnCl 2 and palmitoyl CoA, and the authors suggested that phosphocholinetransferase and phosphoethanolaminetransferase are separate enzymes. The transferases were s o l u b i l i z e d from r a t l i v e r microsomes by Kanoh and Ohno (32). The s o l u b i l i z e d preparation which was 4- to 5-fold p u r i f i e d contained both transferase a c t i v i t i e s and t h e i r properties were studied (Tables 7 and 8). I n t e r e s t i n g l y , phosphocholinetransferase required phospholipids for a c t i v i t y while phosphoethanolamine a c t i v i t y was i n h i b i t e d by phospholipids. The p a r t i a l l y p u r i f i e d preparation was treated with T r i t o n X-100 and then applied to a sucrose density gradient and centrifuged. The phosphocholinetransferase was separated into a magnesium and a manganese-requiring component. Phosphoethanolaminetransferase a c t i v i t y on the other hand, formed a single band which co-migrated with the manganese re q u i r i n g component of the phosphocholinetransferase. These r e s u l t s , along with the k i n e t i c properties of the transferases, strongly indicate that phosphocholinetransferase and phosphoethanolaminetransferase are two d i s t i n c t enzymes. 2 0 . Table 6 Properties of Phosphocholinetransferase and Phosphoethanolamine-transferase from Rat Adipocyte Microsomes Phosphocholine-transferase Phosphoethanolamine-transferase Coleman and B e l l ( 3 7 ) pH Optimum Substrate CDP-choline CDP-ethanolamine 1 , 2 - d i o l e o y l - s n -g l y c e r o l 8 . 5 to 9 - 3 8 . 5 to 9 . 3 Apparent Km (mM) 0 . 2 3 9 0 . 0 1 8 0.051 0 . 0 1 2 I n h i b i t o r s CDP-choline CDP-ethanolamine CDP K i , I n h i b i t i o n Constants (mM)  (Type of In h i b i t i o n ) 1 . 6 2 (non-competitive) 0 . 2 3 (competitive) O.36O (competitive) (mixed type) Other I n h i b i t o r s C a C l 2 Organic Solvents T r i t o n X - 1 0 0 Tween X - 2 0 D i t h i o t h r e i t o l Palmitoyl CoA 21. Table 7 Properties of P a r t i a l l y P u r i f i e d Phosphocholinetransferase and Phospho-ethanolaminetransferase from Rat Liver Microsomes Kanoh and Ohno (32) Phosphocholine-transferase Phosphoethanolamine-transferase pH Optimum 8.0-8.5 8.0-8.5 Substrate Apparent Km (mM) i ) D i a c y l g l y c e r o l 0.081 0.063 i i ) CDP-choline 0.036 -i i i ) CDP-ethanolamine - 0.022 Other Properties 1. Requires phospho-l i p i d s for a c t i v i t y Inhibited by phospho-l i p i d s 2. Consists of two ph y s i c a l l y d i s t i n c t components: a mg + + r e q u i r i n g and a mn + + r e q u i r i n g a c t i v i t i e s One a c t i v i t y dependent on Mn + + 22. Table 8 Modulators of Phosphocholinetransferase and Phosphoethanolamine-transferase from Rat Liver Microsomes Phosphocholine- Phosphoethanolamine-transf erase transferase Kanoh and Ohno ( 3 2 ) Competitive Inhibitor (Substrate) Ki, Inhibition Constant (mM) i) CDP-choline - 0 . 3 5 (CDP-ethanolamine) i i ) CDP-ethanolamine 0 . 3 5 (CDP-choline) Parthasarathy e_t a l . ( 3 8 ) Drug Inhibitors ( 3 0 mM) Centrophenoxine Dimethylethanolamine Percent Inhibition of Enzyme Activity 99 69 Dimethylaminoethyl p-chlorophenoxyacetate 77 23. 2. Phosphatidylethanolamine N-Methylation Pathway The enzymes in this pathway catalyze the N-methylation of PE to form PC with S-adenosylmethionine serving as donor of methyl groups. The d i f f i c u l t i e s in characterizing the methyltransferases were similar to those encountered with the phosphocholinetransferase. The methyltransferases are membrane bound and secondly, the reactants PE and i t s N-methylated derivatives, as well as the product PC, are water insoluble while S-adenosylmethionine and S-adenosylhomocysteine, are water-soluble. But in spite of these obvious d i f f i c u l t i e s , successful attempts have been made in characterizing the enzymes. The i n i t i a l characterization of the methyltransferases from rat liver was carried out by Bremer and Greenberg (39). They found that enzyme activ i t i e s were optimal at very alkaline conditions (pH'~10); The methyltransferases were inhibited by sulfhydryl reagents while S-adenosylethionine was a competitive inhibitor. Studies with N_;_ crassa, Agrobacterium tumefaciens, rat liver and bovine adrenal medulla indicated that two distinct methyltransferases were involved in the pathway. This suggestion was made as early as 1946 by Horowitz (40) who studied two mutants of N. crasea which were defective in PC synthesis. N. crassa does not have the enzymes for-de novo synthesis of PC and these mutants each carry a mutation on separate genes (41). Strain 34,486 was unable to synthesize phosphatidylmonomethylethanolamine but was able to u t i l i z e this intermediate when supplied, while strain 47,904 had a partial block between phosphatidylmonomethylethanolamine and PC. The result of this mutation was an accumulation of the monomethyl intermediate in the cells and medium. Furthermore, this strain was also unable to u t i l i z e dimethylaminoethanol as readily as strain 34,846 when grown on agar 24. and, Horowitz suggested that the same enzyme catalyzed the methylation of the mono- and dimethyl intermediates. Subsequent studies of Scarborough and Nyc (42) showed that strain 34,486 had subnormal PE methyltransferase activity and strain 47,904 was unable to convert either phosphatidylmonomethyl- or phosphatidyldimethylethanolamine to PC. These in vitro studies also support the hypothesis that the last two methylations in the formation of PC are catalyzed by one enzyme. The properties of the methyltransf erases' from .N.. ..crassa are summarized in Table 9. Phosphatidylcholine is the most abundant phospholipid in many plants and animals but is rarely found in bacteria. Agrobacterium, however, produces PC and other methylated derivatives of PE using methionine as the donor of methyl groups (43), while Clostridium butyricum only produce's phosphatidylmonomethylethanolamine and no PC (44). The methyltransferase system in Agrobacterium tumefaciens was studied by Kaneshiro and Law (45). They found a soluble methyltransferase which only catalyzed the methylation of PE with S-adenosylmethionine as donor. This enzyme was purified 40-fold and did not show any cofactor requirement in contrast to the rat liver enzymes (46). Furthermore, the soluble methyltransferase was not inhibited by sulfhydryl reagents but was strongly inhibited by S-adenosylhomocysteine and exogenously supplied PE was utilized by the enzyme (Table 10). However, as in rat li v e r , the particulate fraction from this bacteria catalyzed a l l three methylation reactions. The methyltransferases from rat liver microsomes were further studied by Rehbinder and Greenberg (46). The enzyme was solubilized from microsomes by deoxycholate or by sonication and the removal of phospholipid substrate made the enzyme completely dependent upon added substrates. Like the enzyme in i t s native state, the solubilized preparation could not 25. methylate exogenously supplied PE. Kinetic studies showed that the amounts of phosphatidyldimethylethanolamine and PC produced _in vitro were dependent upon the ratios of the concentration of phosphatidylmonomethylethanolamine, S-adenosylmethionine and the enzyme. When the ratio of phosphatidylmonomethylethanolamine to S-adenosylmethionine was 10, the product was predominantly the dimethyl intermediate while PC was the major product when the ratio was 1.0. The results suggested to the authors that o the last two methylation steps were catalyzed by the same active site, i.e. the same enzyme. Recently, two methyltransferases from bovine adrenal medulla were studied by Hirata et a l . (47). Both enzymes were localized in the microsomal fraction. The enzyme that catalyzes the f i r s t methylation could only be solubilized by detergents while washing the microsomes with EDTA removed an enzyme that catalyzes the conversion of phosphatidylmonomethylethanolamine to PC. The results from various studies strongly support the involvement of two enzymes in the pathway. The methyltransferases have been successfully solubilized and partially purified. Morgan (48) reported the solubilization of the enzyme from dog lung microsomes and partial purification was achieved by ammonium sulfate precipitation and DEAE-cellulose chromatography. In rat liver, Schneider and Vance (49) solubilized the methyltransferases by sonic disruption of the microsomes in the presence of 0.2% Triton X-100 and the enzyme was purified by a combination of octyl-Sepharose and Sepharose 6B chromatography. The partially purified enzymes were characterized and the results are summarized in Table 14, along with the properties of the methyltransferases from N_. crassa (Table 9), Agrobacterium tumefaciens (Table 10), bovine adrenal medulla (Table 11), dog lung (Table 12) and rat 26. l i v e r (Tables 13 and 14). More recently, Tanaka et a l . (50) reported the s o l u b i l i z a t i o n of the methyltransferases from mouse l i v e r by treatment of microsomes with deoxycholate and T r i t o n X-100. I n t e r e s t i n g l y , the s o l u b i l i z e d enzyme was strongly stimulated by PE and i t s methylated derivatives as well as PC. The authors suggested that PC, and to a l e s s e r degree, PE, activated the enzymes by maintaining i t i n an active conformation. The methylation of PE i n brain has been investigated and e a r l i e r experiments i n vivo (51) indicated that t h i s pathway was absent. Subsequent studies, however, showed that the N-methylation reactions occur i n brain (52 , 5 3 ) . Recently, Mozzi and P o r c e l l a t i (54) have investigated N-methylation i n r a t brain homogenate and found that the a c t i v i t i e s of the methyltransferases were very much lower i n brain than i n l i v e r . The methyltransferases i n l i v e r have a preference f o r unsaturated PE's ( 3 4 ) , while i n lung, Morgan suggested that the enzymes preferred more saturated species ( 5 5 ) . However, i n contrast, experiments with r a t lung (130) and rabbit lung (131) showed that the methylation of PE resulted primarily i n the formation of unsaturated PC. The methyltransferase from Agrobacterium was quite d i f f e r e n t from the r a t l i v e r enzyme. The b a c t e r i a l system possesses a soluble PE methyltransferase, which i s absent i n r a t l i v e r , as well as the three p a r t i c l e bound methyltransferase a c t i v i t i e s . The b a c t e r i a l system required exogenous substrate while the l i v e r enzymes can only u t i l i z e endogenous PE. Furthermore, the soluble methyltransferase, unlike the rat l i v e r 27. Table 9 Properties of Phosphatidylethanolamine Methyltransferase from N. crassa Microsomes Scarborough and Nyc (56) pH Optimum  Substrate S-adenosylmethionine Phosphatidylmonomethyl-ethanolamine Phosphatidylmonomethylethanolamine Methyltransferase 8.0 Apparent Km (mM) 0.013 Results Variable 0 28. Table 10 P r o p e r t i e s of Phosphatidylethanolamine Methyltransferase from Agrobacterium tumefaciens Kaneshiro and Law (45) pH optima  Substrates S-adenosylmethionine Phosphatidylethanolamine Other P r o p e r t i e s Phosphatidylethanolamine Methyltransferase ( 4 0 - f o l d p u r i f i e d preparation) 8.4 and 9.6 Apparent Km (mM) 0.02 k i n e t i c p l o t s n on-linear i ) No requirement f o r d i v a l e n t c a t i o n i i ) U t i l i z e d exogenous phosphatidyl-ethanolamine i i i ) Enzyme found i n s o l u b l e f r a c t i o n i v ) I n s e n s i t i v e to s u l f h y d r y l i n h i b i t o r s v) S-adenosylhomocysteine i s a potent i n h i b i t o r v i ) Unable to.methylate s u l f h y d r y l groups Table 11 Properties of Phosphatidylethanolamine Methyltransferase from Bovine Adrenal Medulla Hirata et a l . (47) pH Optimum  Reactants S-Adenosylmethionine Mg++ Competitive I n h i b i t o r i ) S-Adenosylhomo-cysteine Other Properties i ) Mg + + i i ) Substrate Phosphatidylethanolamine Methyltranferase Phosphatidylmonomethyl-ethanolamine Methyl-transferase 6.5 10 Apparent Km (mM) 0.0014 0.1 0.4 K i , I n h i b i t i o n Constant (mM) 0.0016 required u t i l i z e d exogenous phospha-tidylethanolamine not required 30. Table 12 Properties of P a r t i a l l y P u r i f i e d Phosphatidylethanolamine  Methyltransferases from Dog Lung Morgan (48) pH Optimum Substrate S p e c i f i c i t y  S u l f h y d r y l I n h i b i t o r s Other Properties Methyltransf erases 8.0-9-0 Saturated phosphatidylethanolamine preferred over unsaturated species. HgCl 2 P-Chloromercuribenzoate N-ethylmaleimide i ) Requires Mg + + for maximum A c t i v i t y i i ) Requires cysteine for s t a b i l i t y 31. Table 13 Properties of Phosphatidylethanolamine  Methyltransferases from Rat Liver Bremer and Greenberg (39,57) N-Methyltransferases pH Optimum 10 Competitive Inhibitor of S-Adenosylmethionine  Sulfhydryl Inhibitors (10 mM) HgCl 2 p-Chlorornercuribenzoate p-Chloromercuriphenylsulphonate Iodoacetic Acid Other Properties S-adenosylethionine Percent Enzyme Activity Remaining  44 14 33 90 i) Methylates sulfhydryl compounds i i ) Does not u t i l i z e exogenous phosphatidylethanolamine i i i ) Slightly stimulated by cholate iv) Does not require Mg + + Table 14 Properties of S o l u b i l i z e d Phosphatidylethanolamine Methyltransferases from Rat Liver Schneider and Vance (49) Substrate Phosphatidylmonomethyl-ethanolamine Phosphatidyldimethyl-ethanolamine S-adenosylmethionine Competitive I n h i b i t o r s (Substrate (mM)  S-adenosylhomocysteine (S-adenosylmethionine) Phosphatidylcholine at 0.36 \iM Other Properties i ) pH optimum at 9.5 i i ) Molecular weight of 2.0 x 10^ i i i ) U t i l i z e d exogenous phosphatidylethanolamine iv) Does not require Mg + + v) Required s u l f h y d r y l reagents for a c t i v i t y v i ) A c t i v i t i e s i n h i b i t e d by T r i t o n X-100 Methyltransferase A c t i v i t i e s (5-fold p u r i f i e d ) PME+PDE PDE+PC Apparent Km (mM) 0.08 0.45 0.022 0.016 K i I n h i b i t i o n Constants (mM) 0.0049 0.0067 Percent Enzyme A c t i v i t y 56 38 A l l Methyltransferases PME - phosphatidylmonomethylethanolamine PDE - phosphatidyldimethylethanolamine 33. enzyme, was i n s e n s i t i v e to s u l f h y d r y l reagents and was not capable of methylating foreign s u l f h y d r y l compounds. The evidence indicates that the soluble b a c t e r i a l enzyme i s not the same as the enzyme found i n rat l i v e r . 3• Other Pathways for Phosphatidylcholine Biosynthesis Phosphatidylcholine i n lung and l i v e r i s synthesized mainly by the de  novo and the N-methylation pathways. However, these two pathways which p r e f e r e n t i a l l y synthesize l-saturated-2-unsaturated PCs (see previous sections) do not f u l f i l l the requirements of the lung for disaturated PC's. Two "remodelling" pathways i n lung have been demonstrated which convert 1-saturated, 2-unsaturated PC's into disaturated species. Another pathway for PC biosynthesis involved the Ca + +-dependent exchange of phospholipid headgroups with free nitrogen bases. The remodelling pathways and base exchange reactions are b r i e f l y discussed below. The substrates for the remodelling reactions are 1-acyl-LPC which are formed by the action of phospholipases. One pathway, f i r s t described i n rat l i v e r by M a r i n e t t i (58), required two l y s o l e c i t h i n s but not f a t t y acids or CoA. A saturated f a t t y acid from LPC i s transacylated to the second po s i t i o n of another molecule to form a disaturated PC. This reaction was studied by Abe and Akino (59,60) and they found higher a c t i v i t i e s i n lung than i n other organs. However, the r e l a t i v e importance of t h i s pathway i s i n dispute since others (61) have found i t to be of minor importance. The a c y l a t i o n of LPC by acyl-CoA was f i r s t demonstrated by Lands (62) and was subsequently studied i n lung by Webster (63). Saturated f a t t y acids from acyl-CoAs are used to acylate 1-saturated PC to form disaturated species. 34. In mammalian brain, choline and ethanolamine can be incorporated into phospholipids d i r e c t l y by base exchange„(64,65)• This reaction requires C a + + but does not u t i l i z e energy and does not r e s u l t i n net synthesis of phospholipids. Recent studies suggest that base exchange i s a very f a s t reaction and may be important i n vivo (66). In l i v e r , base exchange appears to be unimportant for the synthesis of PC. Experiments have shown that the rate of ale novo biosynthesis of PC i s at l e a s t 20 times faster than base exchange (67). I l l . Regulation of the Rate of Phosphatidylcholine Biosynthesis The control of the rate of the de_ novo and the N-methylation pathways i n l i v e r and to a l e s s e r extent i n lung, i s b r i e f l y discussed below. The r a t e - l i m i t i n g enzyme of each pathway as well as the e f f e c t s of various hormones and chemicals on the rate of PC biosythesis are also discussed. 1. De Novo Pathway The regulation of PC biosynthesis i n r a t l i v e r during development was studied by Weinhold and co-workers (68). They found that the concentration of phosholipids i n f e t a l l i v e r increased a f t e r b i r t h and attained adult values 22 days l a t e r . Studies with l i v e r s l i c e s showed that the incorporation of J P i n t o f e t a l l i v e r was low but reached adult values 5 days a f t e r b i r t h . In a subsequent study (69), the a c t i v i t i e s of the enzymes i n the de novo pathway were measured and the developmental pattern 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 the only one found to c l o s e l y p a r a l l e l the pattern of l a b e l l e d choline incorporation i n t o tissue s l i c e s , the developmental studies suggested that the c y t i d y l y l t r a n s f e r a s e may play a rate l i m i t i n g and regulatory r o l e i n the pathway. This idea was further reinforced by r e s u l t s from studies with rat hepatocytes. Sundler and Akesson (7) found that by increasing the concentration of choline i n the 35. culture medium, they were able to saturate the capacity of the de_ novo pathway. The synthesis of PC was stimulated several f o l d at low concentrations of choline. At higher concentrations, however, choline and phosphocholine accumulated i n the c e l l s while CDP-choline concentration as well as PC synthesis remained unaltered. The b e l i e f that the c y t i d y l y l t r a n s f e r a s e was the only r a t e - l i m i t i n g enzyme of the pathway i s not shared by a l l workers. By a mathematical analysis of the pathway based on "the l o g i c derived from t h e o r e t i c a l p r i n c i p l e s of metabolic regulation", Infante (70) showed t h e o r e t i c a l l y that both choline kinase and phosphocholine c y t i d y l y l t r a n s f e r a s e were p o t e n t i a l l y r a t e - l i m i t i n g . Furthermore, a quantitative determination of the d i s e q u i l i b r i u m r a t i o for the reactions catalyzed by both enzymes showed that the choline.kinase step was forty times "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 step. Evidence that the a c t i v i t y of choline kinase can change the fl u x of molecules through the pathway i n vivo was subsequently presented by Infante and K i n s e l l a (71). In rats deprived of e s s e n t i a l f a t t y acids, the pool s i z e of the C 1g. 2 and C20-4 PC were decreased while the l e v e l of C l 6 > 1 and C l 8 > 1 PC's were elevated (72). The authors calculated the rate of PC biosynthesis, by a method which was not c l e a r l y explained, and found i t to be 3.8 times higher than normal. The a c t i v i t i e s of choline kinase and the c y t i d y l y l t r a n s f e r a s e were measured i n normal and d e f i c i e n t r a t s . The a c t i v i t y of choline kinase was increased 3.5-fold while 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 unchanged i n the d e f i c i e n t animals. The authors concluded that choline kinase must regulate the rate of de novo PC biosynthesis at l e a s t during e s s e n t i a l f a t t y a c i d d e f i c i e n c y . 3 6 . Studies by Schneider and Vance (73) showed that the activities of choline kinase and phosphocholine transferase were unchanged during choline deficiency but the cytidylyltransferase activity was reduced about half. Although the rate of PC biosynthesis was not measured in choline deficient rats, the results further indicate that the cytidylyltransferase may play a regulatory role in the pathway. Further work by Choy et a l . (74) showed that the amount of immunotitratable enzyme in normal and choline deficient rats was the same. The results suggested that the decrease in cytidylyltransferase activity was not due to an adaptive decline in enzyme quantity. The various results presented above indicate that the phosphocholine cytidylyltransferase, and perhaps choline kinase, regulate the rate of de novo PC synthesis. The results presented in this thesis show that the activity of the cytidylyltransferase regulates the rate of PC biosynthesis in the livers from hypercholesterolemia rats. The effects of hormones, n-alkanes, ethanol and phenobarbital on the rate of biosynthesis have been investigated. In propylthiouracil-induced hypothyroid chicks, the specific activity of phosphocholinetransferase in the liver declined to about 10% of controls after 15 days of treatment and the transferase activity was increased to normal values 2 days after administration of L-thyroxine (75). Other hormones, however, inhibited phosphocholinetransferase activity. Administration of estradiol to castrated and normal rats decreased the specific activity of the transferase in both groups but stimulated PE methyltransferase activity (76). Prostaglandins also affect PC synthesis in rat l i v e r . Prostaglandin E^, administered intraperitoneally for 4 weeks stimulated the incorporation of 3 2P into liver PC (77). 37. Non-physiological agents also a f f e c t PC synthesis. In an i n v i t r o system c o n s i s t i n g of r a t l i v e r cytosol and/or microsomes, several n-alkanes, such as n-hexane and n-octane, stimulated the incorporation of l a b e l l e d choline into PC (78) and the added organic solvents were found to stimulate the reaction catalyzed by the c y t i d y l y l t r a n s f e r a s e . Feeding of r a t s with 5% ethanol for two weeks stimulated phosphocholinetransferase a c t i v i t y (79). The l i t e r a t u r e on the e f f e c t of phenobarbital on PC biosynthesis i s somewhat confusing. E a r l i e r studies (80,81) showed that chronic administration of the anaesthetic i n r a t s stimulated the incorporation of 3 2 P into hepatic PC's and t h i s was associated with p r o l i f e r a t i o n of endoplasmic reticulum. Results from a recent study (82), however, indicated that the increase i n PC was due to a decrease i n breakdown rather than an increase i n synthesis. The amount of glyeerophosphorylcholine which i s one of the breakdown products of PC was decreased by 50% i n the treated animals. The e f f e c t s of development, various d i e t s , hormones and other substances on l i v e r PC biosynthesis are summarized i n Table 15. The i n t e r e s t i n the production of surfactant has stimulated research i n t o the regulation of PC biosynthesis i n lung. Developmental studies of Weinhold (68) showed that the amount of PC in 20-day old r a t fetuses were 50% of adult values while newborns had as much lung phospholipids as adults. A subsequent study (83) showed that the incorporation of l a b e l l e d choline into lung s l i c e s from 19-day old fetuses was low but increased to adult values i n 22-day old fetuses. A d i f f e r e n t pattern, however, was observed for the incorporation of r a d i o a c t i v i t y into water-soluble precursors. Incorporation into phosphocholine was high i n 19-day old fetuses but decreased i n older fetuses. The s p e c i f i c r a d i o a c t i v i t y of 38. T a b l e 15 The E f f e c t o f Development, D i e t , Hormones and Other Compounds on the  B i o s y n t h e t i c Enzymes and the I n c o r p o r a t i o n o f L a b e l l e d 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 i n Rat L i v e r and Lung E f f e c t on the I n c o r -p o r a t i o n o f 32p o r l a b e l l e d c h o l i n e E f f e c t on t h e i n t o p h o s p h a t i d y l - A c t i v i t i e s o f the S t i m u l i c h o l i n e B i o s y n t h e t i c Enzymes Development o f Rat Lung Weinhold e t a l . (69) p e r i n a t a l period- I n c r e a s e d a f t e r b i r t h Only the a c t i v i t y o f p h o s p h o c h o l i n e c y t i -d y l y l t r a n s f e r a s e f o l l o w e d the changes observed w i t h i n c o r -p o r a t i o n o f l a b e l l e d c h o l i n e L i v e r E s s e n t i a l F a t t y A c i d D e f i c i e n c y K i n s e l l a and I n f a n t e (71) C a l c u l a t e d 3 . 8 - f o l d i n c r e a s e A c t i v i t y o f c h o l i n e k i n a s e was i n c r e a s e d 3 . 5-fold C h o l i n e D e f i c i e n c y A c t i v i t y o f phospho-S c h n e i d e r and Vance (73) 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 h a l v e d E s t r a d i o l Treatment o f  Male Rats Young (76) A d m i n i s t r a t i o n o f  P r o s t a g l a n d i n E H o r i u c h i (77) A d d i t i o n o f n-Alkanes  t o an In V i t r o System T a k a g i (78) E t h a n o l F e e d i n g Uthus et a l . (79) I n c r e a s e d 2 to 3 - f o l d i n c r e a s e P h 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 y was d e c r e a s e d F o r m a t i o n o f CDP-c h o l i n e was s t i m u l a -t e d A c t i v i t y o f Phospho-c h o l i n e t r a n s f e r a s e was i n c r e a s e d 3 9 . phosphocholine, however, were the same i n f e t a l and adult lungs. The r e s u l t s suggested that the increase i n l a b e l l e d choline incorporation into lung PC between day 19 and 22 of gestation was due to a stimulation of the enzymatic reactions between phosphocholine and PC. Thus, the c y t i d y l y l t r a n s f e r a s e during development was.investigated ( 2 3 ) . 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 e t a l lung was about a quarter of the adult a c t i v i t y when assayed under normal condition. However, addition of phospholipids stimulated the f e t a l enzyme to values comparable to that found i n adult lung. Two forms of the c y t i d y l y l t r a n s f e r a s e were found i n c y t o s o l : a high molecular weight form which does not require addition of phospholipid for maximum a c t i v i t y and a low molecular weight form which required addition of phospholipids. The f e t a l enzyme existed predominantly i n the low molecular weight form while the high molecular weight form was predominant i n adult lung. The developmental pattern of choline incorporation into PC i n lung s l i c e s correlated with the pattern of c y t i d y l y l t r a n s f e r a s e e a c t i v i t y . However, the r i s e i n enzyme a c t i v i t y could not t o t a l l y account for the more rapid incorporation: the rate of choline incorporation i n 1-day old fetuses increased to adult values while 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 remained at 40$ of adult values. The synthesis of PC i n lung during the early post-natal period was also studied by Chida et a l . (84). The incorporation of l a b e l l e d choline into PC as well as the a c t i v i t i e s of the enzyme of the'de_ novo pathway were measured i n minced lung from rats at various stages of development. They found that the developmental pattern 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 d i f f e r e n t from the other two enzymes. The a c t i v i t i e s of choline kinase and phosphocholinetransferase were high during the prenatal and newborn period while the a c t i v 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 the same period was low. 40. But, more s i g n i f i c a n t l y , these workers found that the developmental pattern of choline incorporation was s i m i l a r to patterns 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 both reached maximum values f i v e days a f t e r b i r t h . These r e s u l t s further support the idea that the c y t i d y l y l t r a n s f e r a s e catalyzed the r a t e - l i m i t i n g step of the pathway. The conversion of the c y t i d y l y l t r a n s f e r a s e from the low molecular weight form i n f e t a l lung to the high molecular weight form i n adult lung was investigated (24). The stimulation of the enzyme by various phospholipids were tested and compared to the stimulation by t o t a l lung l i p i d and only PG was as active i n stimulating the enzyme. In contrast, PS and PI were only h a l f as e f f e c t i v e as t o t a l lung l i p i d i n a c t i v a t i n g the enzyme. The e f f e c t of PG on the c y t i d y l y l t r a n s f e r a s e i s also unique because only t h i s l i p i d aggregated the enzyme. The workers speculated that the a c t i v 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 , and therefore the rate of PC synthesis, may be coupled with the l e v e l of PG i n lung. I n t e r e s t i n g l y , the l e v e l of g l y c e r o l phosphatide increased c o i n c i d e n t a l l y during the l a t e r stages of lung development. The production of lung surfactant was stimulated by various hormones (85). For studies with f e t a l lung from various animals, g l u c o c o r t i c o i d and estrogen administration increased the amount of PC i n lung by at l e a s t 2-fold and incorporation of l a b e l l e d choline into PC i n lung s l i c e s was also increased to the same extent. 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 measured in_ v i t r o was also increased when experimental animals were treated with hormones but the increase was not as extensive as the elevation i n lung PC and the incorporation of l a b e l l e d choline. 41. As mentioned e a r l i e r , the a c t i v i t y o f 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 s t i m u l a t e d by PG and, s i g n i f i c a n t l y , the s y n t h e s i s o f t h i s l i p i d was a l s o s t i m u l a t e d by g l u c o c o r t i c o i d s and e s t r o g e n . The a c t i v i t i e s o f the c y t i d y l y l t r a n s f e r a s e from c o n t r o l and hormone t r e a t e d a n i m a l s were s i m i l a r when assayed i n the presence o f PG s u g g e s t i n g t h a t the e l e v a t e d l e v e l s o f t h i s l i p i d may be r e s p o n s i b l e f o r the s t i m u l a t i o n o f the c y t i d y l y l t r a n s f e r a s e . The e v i d e n c e i n d i c a t e s t h a t PG may p l a y a r e g u l a t o r y r o l e i n the c o n t r o l o f PC s y n t h e s i s i n the l u n g . R e s u l t s from s t u d i e s i n l u n g and l i v e r s t r o n g l y s u p p o r t the h y p o t h e s i s 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 s r e g u l a t o r y and r a t e l i m i t i n g i n the de  novo s y n t h e s i s o f PC. The c o n c e n t r a t i o n s o f i n t r a c e l l u l a r p h o s p h o c h o l i n e and CDP-choline a r e c o n s i s t e n t w i t h t h i s h y p o t h e s i s . The c o n c e n t r a t i o n o f C DP-choline i s twenty times lower than the c o n c e n t r a t i o n o f p h o s p h o c h o l i n e ( T a b l e 1) i n r a t l i v e r and the a v a i l a b i l i t y o f CDP-choline appears t o be the r a t e l i m i t i n g f a c t o r i n PC s y n t h e s i s 2. 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 Pathway The r e g u l a t i o n o f the r a t e o f c o n v e r s i o n o f PE t o PC has not been i n v e s t i g a t e d as e x t e n s i v e l y as the r e g u l a t i o n o f de novo PC b i o s y n t h e s i s . However, s t u d i e s i n r a t s and y e a s t have y i e l d e d some important i n s i g h t s . E a r l y s t u d i e s o f Greenberg (39) suggested t h a t two enzymes a r e i n v o l v e d i n the pathway and t h a t PE m e t h y l t r a n s f e r a s e , the f i r s t enzyme o f the pathway, appear t o be r a t e l i m i t i n g . These o b s e r v a t i o n s have now been c o n f i r m e d by o t h e r workers. The a c t i v i t i e s o f the m e t h y l t r a n s f e r a s e s were a f f e c t e d by hormones and d i e t . 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 to normal and c a s t r a t e d r a t s i n c r e a s e d 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 (76). In d i e t a r y s t u d i e s , i n c o r p o r a t i o n o f 42. methyl l a b e l l e d S-adenosylmethionine i n vivo into phospholipids was stimulated i n r a t s fed a choline d e f i c i e n t d i e t ( 8 6 ) and the a c t i v i t y of PE methyltransferase was increased i n the microsomes ( 7 3 , 8 7 ) . Regulation of N-methyltransferases has been studied i n Saccharomyces  cerevisiae ( 8 8 ) . The presence of N-methylethanolamine, N,N-dimethylethanolamine or choline i n the growth medium resulted i n reduced a c t i v i t i e s of a l l the methyltransferase a c t i v i t i e s involved i n the formation of that p a r t i c u l a r phosphatidyl ester. The a c t i v i t y of the l a s t methyltransferase for instance, was 9 0 ? lower i n c e l l s grown with s u f f i c i e n t choline than i n c e l l s grown i n choline d e f i c i e n t medium. C e l l s grown i n medium supplemented with any of the three methylated aminoethanols also resulted i n increased c e l l u l a r l e v e l s of the corresponding phosphatidyl esters and diminished amounts of precursor phosphatidyl esters. The mechanisms by which the methyltransferase a c t i v i t i e s are regulated i n yeast and rats remain to be investigated. IV. Mechanisms of Regulation of the Rate of Phosphatidylcholine Biosynthesis Studies in r a t lung, l i v e r and i n various other systems strongly indicated that phosphocholine c y t i d y l y l t r a n s f e r a s e i s rate l i m i t i n g as well as regulatory i n the control of de_ novo PC biosynthesis. Two mechanisms by which the a c t i v 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 may be regulated have emerged from these studies. As described i n a previous section, PG appear to be important i n regulating PC biosynthesis during lung development by modulating the a c t i v 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 . A second mechanism was elucidated by Vance e_t a l . i n studies with HeLa c e l l s . I nfection of the c e l l s with P o l i o v i r u s stimulated the incorporation of l a b e l l e d choline i n t o PC and the reaction catalyzed by phosphocholine c y t i d y l y l t r a n s f e r a s e , which i s rate l i m i t i n g i n HeLa c e l l s , was found to be stimulated ( 8 9 ) . A 4 3 . subsequent study by Choy et_ al_. ( 9 0 ) showed that an increase i n c y t o s o l i c CTP concentration i n P o l i o v i r u s - i n f e c t e d c e l l s was responsible for the stimulation of i n vivo a c t i v 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 which i n turn re s u l t e d i n a higher rate of PC biosynthesis. The concentration of CTP i n cytosol under normal circumstances appear to be below the Km of the enzyme for CTP and a 2 - to 3 - f o l d increase i n c y t o s o l i c CTP would r e s u l t i n a proportional increase i n enzyme a c t i v i t y . The control of PC biosynthesis by changes i n the l e v e l s of the biosynthetic enzymes has not been demonstrated. The amount of c y t i d y l y l t r a n s f e r a s e has been shown to be unchanged during choline deficiency, where enzyme a c t i v i t y i s decreased. The amount of immunoprecipitable enzyme i n l i v e r cytosols from choline d e f i c i e n t r a t s were unchanged even though the a c t i v 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 was h a l f the control value ( 7 3 ) . Thus, the presence of a l i p i d modulator or a l t e r a t i o n of i n vivo l e v e l s of substrate appear to be two mechanisms by which the rate of PC biosynthesis may be regulated. V. Hepatic Phosphatidylcholine Biosynthesis and Hypercholesterolemia A r e l a t i o n s h i p between the l e v e l of plasma ch o l e s t e r o l and phospholipids has been observed i n humans and i n various animals. In normal persons, the r a t i o of serum c h o l e s t e r o l to serum phopspholipids i s e s s e n t i a l l y constant. Peters and Man (91) found that l i p i d phosphorus increased i n a l i n e a r fashion with c h o l e s t e r o l i n serum when serum ch o l e s t e r o l concentration exceeded 100 mg/100 ml. A s i m i l a r r e l a t i o n s h i p e x i s t s i n swine and r a t s . Cholesterol feeding of miniature swine ( 9 2 ) resulted i n a 4 - f o l d increase i n plasma c h o l e s t e r o l and an almost 3 - f o l d increase i n plasma phospholipids. The hypercholesterolemia was also associated with hyperlipoproteinemia and the experimental animals subsequently developed a t h e r o s c l e r o s i s . In rats fed a d i e t with high 4 4 . l e v e l s of c h o l e s t e r o l , cholate and p r o p y l t h i o u r a c i l , plasma c h o l e s t e r o l was very dramatically increased while plasma phospholipid was also elevated ( 9 3 ) . The r e l a t i o n s h i p between plasma cholate, phospholipids, and ch o l e s t e r o l i n rats was studied by Friedman and Byers ( 9 4 ) . They found that a f t e r b i l i a r y obstruction, the l e v e l of plasma cholate was increased and t h i s was followed by a r i s e i n plasma phospholipid which i n turn was followed by an increase i n plasma c h o l e s t e r o l . Intravenous i n f u s i o n of cholate resulted i n increased l e v e l s of plasma phospholipids and cholesterol while i n j e c t i o n of phospholipids led to an elevation of plasma ch o l e s t e r o l only. The r e s u l t s suggested that d u r i n g / b i l i a r y obstruction, elevated l e v e l s of cholate, rather than c h o l e s t e r o l , was responsible for the increase i n plasma phospholipids and t h i s may also be the case during diet-induced hypercholesterolemia. The increase i n plasma phospholipids during c h o l e s t e r o l feeding could be explained i n a number of ways. F i r s t , there could be decreased degradation of plasma phospholipids or, secondly, phospholipid synthesis could be stimulated i n the l i v e r which i s a major s i t e of l i p o p r o t e i n synthesis. Is the rate of PC synthesis stimulated i n the l i v e r s from hypercholesterolemia animals? And i f i t i s , which enzymes are involved? What are the regulatory mechanisms? The hepatic synthesis of PC i n normal and hypercholesterolemia rats was investigated and the r e s u l t s are reported here. The l e v e l of plasma ch o l e s t e r o l i n adult rats was d i f f i c u l t to manipulate. Thus, young rats whose plasma cholesterol l e v e l s could be more r e a d i l y a l t e r e d ( 9 5 ) , were used to study the e f f e c t s of cholesterol/cholate feeding on hepatic PC biosynthesis. The r e s u l t s presented i n t h i s thesis consist of two major components. The f i r s t segment deals with the in v i t r o studies of the enzymes of PC biosynthesis, with p a r t i c u l a r emphasis on the c y t i d y l y l t r a n s f e r a s e . The second part deals with the _in vivo rate of PC biosynthesis i n l i v e r s from normal and cholesterol/cholate fed r a t s . 46. MATERIALS AND METHODS I• Animals, Enzymes, Chemicals and Radioactive Compounds 1. Animals Animals were purchased from the University of B r i t i s h Columbia Animal Unit. Female Wistar r a t s , 18 days old, weighing 30-35 g, were used i n a l l feeding experiments while older female ra t s weighing 150 to 250 g were used for preparing phosphocholine c y t i d y l y l t r a n s f e r a s e and t o t a l r a t l i v e r phospholipids. New Zealand buck rabbits were used to r a i s e antibodies against the c y t i d y l y l t r a n s f e r a s e . 2. Enzymes, Chemicals and Radioactive Compounds The following companies supplied the enzymes, chemicals, radioactive compounds and other materials l i s t e d below. A l l other chemicals used were of reagent grade. Ralston Pur-ina Company Purina Rat Chow and Rabbit Chow ICN Pharmaceuticals, Inc. Special basal atherogenic d i e t , modified and choline d e f i c i e n t d i e t supplemented with 4% choline (used as control diet) Matheson, Coleman, and B e l l Cholesterol and Cholate 47. Sigma Chemical Company B i s ( t r i m e t h y l s i l y l ) - t r i - f l u o r o a c e t a m i d e (BSTFA), choline chloride, choline iodide, phosphocholine, T r i s , ATP, CTP, CDP, CMP, GTP, GDP, GMP, UTP, UDP, UMP, NAD, NADH, NADP, NADPH, acetyl-CoA, stearoyl-CoA, malonyl-CoA, palmitoyl-CoA, standard even numbered f a t t y acids, egg PC, PE, PS, synthetic palmitoyl-LPE, bovine serum albumin, a l k a l i n e phosphatase, phospholipase c from B^ cereus and CI. perfringens, t r y p s i n and tr y p s i n i n h i b i t o r , g l y c e r o l dehydrogenase, choline kinase, pyrophosphatase. Serdary Research Laboratories CDP-choline, synthetic palmitoyl-LPC, pig l i v e r PC, pig brain PE, pig l i v e r PI, pig brain SM, pig l i v e r LPE, synthetic palmitoyl- and oleoyl-LPE. Difco Laboratories Complete and incomplete Freund's adjuvant. Pharmacia Sepharose 6B. Mallincrodt Company Chromar plates, 7 GF, 20 cm x 20 cm precoated glass plates, 0.25 mm layer thickness Macherey-Nagel Precoated TLC plates, S i l G-25, 0.25 mm layer without gypsum. E. Merck Laboratories Inc. Precoated TLC plates, S i l i c a Gel 60 (without fluorescence i n d i c a t o r ) , 0.25 mm layer thickness. Amersham/Searle [Me- 3H]choline chloride New England Nuclear Canada [ 1,2-^CJcholine ch l o r i d e , [Me-^C]cytidine diphosphocholine, 14 3 [1,2- C]ethanolamine hydrochloride, S-[Me- H]adenosyl-L-methionine 48. ICN, Chemical and Radioisotope D i v i s i o n [5-- >H]cytidine 5'-triphosphate, tetrasodium s a l t . I I . Preparative Procedures 1. I s o l a t i o n of Total Rat L i v e r Phospholipid The method of Folch et a l . (97) was used. Approximately 20 g of l i v e r obtained from 2 female r a t s , each weighing about 220 g, were homogenized i n 400 ml of CH^OH i n a Thomas tiss u e grinder, with Teflon pestle. In a 3 1 f l a s k , 800 ml of CHCl^ were added to the homogenate and the mixture was agitated extensively. The extract was then f i l t e r e d and the f i l t r a t e was c o l l e c t e d i n a 1 1 separating funnel. To the organic phase, 250 ml of 0.145 M NaCl were added and the two-phase sol u t i o n was vigorously extracted. The phases were allowed to separate and the lower organic phase was drained into a preweighed 1 1 round bottom f l a s k . The solvent was evaporated under reduced pressure and the dry l i p i d residue was washed 3 times with 50 ml of ic e - c o l d acetone. The acetone wash, which contained mostly neutral l i p i d s , was discarded and the residue was dissolved i n chloroform at a concentration of 20 to 30 mg/ml. This extraction procedure yielded 27 mg of phospholipid per g l i v e r . 2. Preparation of P a r t i a l l y P u r i f i e d Phosphocholine C y t i d y l y l t r a n s f e r a s e The enzyme was enriched from fresh 20% (w/v) rat l i v e r cytosol by passage of 5 ml of cytosol through a 200 ml Sepharose 6B column. The fr a c t i o n s i n the included volume were assayed for enzyme a c t i v i t y i n the presence of 5 mg/ml r a t l i v e r phospholipid. Ten of the fr a c t i o n s that contained the highest enzyme a c t i v i t y were pooled and concentrated to 2 ml by u l t r a f i l t r a t i o n with an Amicon XM-100 membrane f i l t e r . In l a t e r experiments where a much more active enzyme preparation was required the enzyme was f i r s t p r e c i p i t a t e d from cytosol by (NH^^SO^ (25 to 50% f r a c t i o n ; w/v). The p r e c i p i t a t e d enzyme was redissolved i n 5 ml 49. of column buffer and chromatographed as described above. The four f r a c t i o n s that contained the most a c t i v i t y were pooled and used immediately or stored i n small a l i q u o t s at -70°C. 3. D e l i p i d a t i o n of Phosphocholine C y t i d y l y l t r a n s f e r a s e by Extraction with  Acetone and Butanol The extraction procedure was f i r s t described by Fiscus and Schneider (20). The acetone and butanol used i n the procedure were cooled i n an ethanol-dry i c e bath before use. B r i e f l y , 50 ml of f r e s h l y prepared r a t l i v e r c ytosol were f i r s t extracted with 4 volumes of cold acetone and the p r e c i p i t a t e d proteins were c o l l e c t e d by c e n t r i f u g a t i o n . The extraction procedure was repeated but cold butanol was used and the protein p e l l e t from the f i n a l e x traction was purged of butanol i n a l y o p h i l i z e r . By t h i s procedure, 14 mg of p r e c i p i t a t e was obtained per ml cytosol while Fiscus and Schneider obtained 23 mg of p r e c i p i t a t e per ml c y t o s o l . The enzyme was stored as a s o l i d at -20°C and when required 50 mg of the p r e c i p i t a t e was added to 1 ml of d i s t i l l e d water i n a small t e s t tube. The mixture was vigorously vortex mixed and any undissolved p a r t i c l e s were sedimented by c e n t r i f u g a t i o n . The brownish supernatant contained phosphocholine c y t i d y l y l t r a n s f e r a s e which was active only i n the presence of phospholipids. This preparation was used as the source of enzyme i n some experiments. 4. Enzymatic Synthesis of [ M e - P h o s p h o c h o l i n e 3 Commercially a v a i l a b l e [Me- H]choline was converted to 3 [Me- H]phosphocholine by the action of choline kinase. In each 3 preparation, 0.24 umol of [Me- H]choline (8.3 mCi/umol) was treated with 0.2 units of choline kinase (1 u n i t w i l l catalyze the phosphorylation of 1.0 umole of choline per min at pH 8.5 at 25°C) i n the presence of 100 mM T r i s - H C l , pH 8.0, 10 mM MgCl and 10 mM ATP i n a f i n a l volume of 0.25 50. ml. The reaction was incubated at 37°C for 1 h, and then boiled f o r two min. The mixture was spotted on a 2.5 cm lane on a Brinkman S i l G-25 TLC plate. A small amount of the reaction mixture was spotted on an adjacent lane and the plate was developed i n CE^ou/0.6% NaCl/25$ NH^OH (50/50/5; v/v/v). The mobility of the radioactive choline and phosphocholine was determined by measuring the amount of r a d i o a c t i v i t y i n 1 cm sections of the lane that had a small amount of r a d i o a c t i v i t y . The Rf's for choline and phosphocholine are 0.10 and 0.33, re s p e c t i v e l y . The [Me-^Hlphosphocholine was eluted from the s i l i c a with 10 ml of d i s t i l l e d water and the y i e l d of the procedure varied between 70 and 90%. 5• Preparation of [l,2-ethanolamine--^C]Phosphatidylethanolamine and  Labelled Phosphatidylethanolamine-Phospholipid V e s i c l e s . c R a d i o a c t i v e l y - l a b e l l e d PE was made by incubating 75 x 10 fr e s h l y i s o l a t e d hepatocytes (98), maintained i n monolayer culture, with 0.013 mmol of [1,2- I l +C]ethanolamine (3.85 mCi/mmol) for 24 h at 37°C. The c e l l s were subsequently harvested and extracted by the Bligh and Dyer procedure (99). Labelled PE was i s o l a t e d by thin-layer chromatography with CHC13/CH30H/H20 (70/30/4; v/v/v) as solvent. Of the 50 vimCi of r a d i o a c t i v i t y added, 4.9$ of the l a b e l was incorporated into PE and 2.5$ in t o PC. The l a b e l l e d PE had a s p e c i f i c r a d i o a c t i v i t y of 1.75 nCi/mmol. Radioactive PE was suspended in* s o l u t i o n as phosphatidylethanolamine-phospholipid mixed v e s i c l e s which were prepared with 0.5 mg of [1,2-ethanolamine-^Cjphosphatidylethanolamine (1.75 vimCi/mmol) and 0.5 mg of t o t a l rat l i v e r phospholipid i n 10 ml of s a l i n e . The mixture was sonicated at 0°C, two times with a Quigley-Rochester Sonic Dismembrator for 1 min at a se t t i n g of 80. The sonicated v e s i c l e s were used within 2 hr a f t e r preparation. 51. 6. Preparation of Buffers A systematic study of a va r i e t y of buffers has been reported (100) and procedures for preparing buffers have also been published (101). Tris-HCl was used for buff e r i n g solutions between pH 7*0 and 9.0 while T r i s succinate was used between pH 6.0 and 7.0. Buffers were prepared s t a r t i n g with a concentrated s o l u t i o n of T r i s and the solu t i o n was t i t r a t e d with 1 N HCI or 0.2 M succ i n i c a c i d . When the desired pH was obtained, the buffer s o l u t i o n was d i l u t e d to the appropriate concentration with d i s t i l l e d water and the pH of the f i n a l buffer s o l u t i o n was measured and adjusted accordingly i f necessary. I l l . General Procedures 1. Measurement of Protein Protein content was determined by the procedure of Lowry e_t a l . (102). Approximately 60 ug of protein (10 y l of cytosol and 5 u l microsomes) were added to 0.66 N NaOH to make up a f i n a l volume of 1.5 ml and samples that contained l i p i d were f i r s t incubated at 37°C overnight before use. The following stock solutions were prepared: Solution A 100 ml of 13% (w/v) Na 2C0 3 3 ml of k% (w/v) NaKC^H^Og 4H 20 3 ml of 2% (w/v) Cu 2S0 n To the sample i n 0.66 N NaOH, 1.5 ml of solu t i o n A was added and mixed. After 10 min, 0.5 ml of 2 N phenol reagent so l u t i o n was added and the mixture was again mixed. Absorbance of the so l u t i o n at 625 nm was measured 30 to 60 min l a t e r . A standard curve was generated by. using 10 to 100 ug of f a t t y acid free bovine serum albumin and buffer, d i s t i l l e d water or s a l i n e was used as blank. 52. 2. Measurement of Phosphorus i n Phospholipids A rapid c o l o r i m e t r i c method that does not involve acid digestion of phospholipid was used (103). To the dry l i p i d samples contained i n tes t tubes, 0.4 ml of chloroform and 0.1 ml of chromogenic sol u t i o n were added. The chromogenic s o l u t i o n contained molybdate, mercury, s u l f u r i c and hydrochloric acids. The tubes were placed i n b o i l i n g water for 80 sec and then cooled at room temperature for 30 min. The chromogen was d i l u t e d with 5 ml of CHCl^, the mixture agitated, and the absorbance of the CHCl^ at 710 nm was measured immediately. A stock so l u t i o n of 0.237 mg per ml CHCl^ (equivalent to 10 yg l i p i d phosphorus per ml) of dipalmitoyl-PC was used to generate a standard curve which was l i n e a r from 1 to 10 yg of l i p i d phosphorus. 3. Liq u i d S c i n t i l l a t i o n Counting Aqueous radioactive samples were counted i n 10 ml of ACS. Samples i n 0.1 N NaOH were f i r s t a c i d i f i e d with 0.1 ml of g l a c i a l a c e t i c a c i d . With l i p i d samples, the solvent was f i r s t evaporated before s c i n t i l l a t i o n counting i n 10 ml of a toluene based s c i n t i l l a n t containing PPO (4 g/1 and POPOP (50 mg/1). Radioactive samples on TLC plates were scraped with the s i l i c a into s c i n t i l l a t i o n v i a l s and 1 ml of water was added to deactivate the s i l i c a . The samples were counted i n 10 ml of ACS with recovery ranging from 95$ to 100$ for water soluble samples and 78$ to 83$ for l i p i d samples. A l l l i q u i d s c i n t i l l a t i o n counting was done i n an ISOCAP/300 counter and counting e f f i c i e n c i e s were determined by either channels r a t i o or external standard r a t i o . Each set of samples was counted with quenched standard of eith e r 3H-hexadecane i n ACS or ^C-toluene i n toluene, PPO, POPOP 53. 4. Thin-Layer Chromatography (a) Solvents for Water-Soluble Compounds Various solvent systems were used to separate choline, phosphocholine, CDP-choline and betaine (167). Machery-Nagel S i l G-25 or chromar plates were used. When only one solvent system was used, samples and standards were applied as a thi n band, 2.5 cm long and 2 cm above the bottom edge of the plate. In two dimensional chromatography, one sample was applied as a small spot at the bottom r i g h t hand corner of the plate and 2 cm from each edge. The plate was developed i n the f i r s t solvent from bottom to top, and af t e r a i r drying overnight, the plate was developed from r i g h t to l e f t with the second system. In the in vivo studies where the amount of r a d i o a c t i v i t y incorporated into water-soluble pools was measured, 0.20 mg of choline, 0.75 mg of phosphocholine, 0.30 mg of CDP-choline and/or 0.30 mg of betaine were chromatographed with the samples. The c a r r i e r s increased the recovery and re s o l u t i o n and also f a c i l i t a t e d v i s u a l i z a t i o n of each compound. When chromar plates were used, the amount of CDP-choline c a r r i e r was reduced to 0.10 mg. CDP-choline was v i s u a l i z e d under U.V. while the other compounds were v i s u a l i z e d by iodine s t a i n i n g . i ) Single Dimensional Systems CH3OH/0.6% NaCl/27% NH^OH (50/50/5; v/v/v) Choline Rf 0.10, phosphocholine Rf 0.33, betaine and CDP-choline Rf 0.66. This system was mainly used i n the phosphocholine c y t i d y l y l t r a n s f e r a s e assay as a means to separate radioactive CDP-choline from l a b e l l e d phosphocholine. This system was also used to separate l a b e l l e d phosphocholine from choline during the preparation of [Me- 3H]phosphocholine. 54. i i ) Two D i m e n s i o n a l Systems System I : As i n I I I . 4 . a . i System I I : C H ^ O C H y C H ^ H / c o n c e n t r a t e d HCI (10/90/4/; v/v/v) C h o l i n e Rf 0.31, ph o s p h o c h o l i n e Rf 0 .39, CDP-choline Rf 0.21, b e t a i n e Rf 0.49. A l l f o u r compounds were c o m p l e t e l y r e s o l v e d by t h i s two d i m e n s i o n a l system. (b) S o l v e n t s f o r L i p i d s S i n g l e d i m e n s i o n a l TLC was r o u t i n e l y used t o s e p a r a t e the major c l a s s e s o f p h o s p h o l i p i d s . A more complete r e s o l u t i o n o f the d i f f e r e n t c l a s s e s was a c h i e v e d by two d i m e n s i o n a l TLC (104,105). S i l i c a g e l 60 p l a t e s from Merck were heat a c t i v a t e d a t 110°C f o r 1 h b e f o r e use and samples were s p o t t e d on TLC p l a t e s a c c o r d i n g t o the procedure d e s c r i b e d above ( S e c t i o n I I I . 4 . a . ) i ) S i n g l e D i m e n s i o n a l Systems System I ( n e u t r a l ) : CHC1 3/CH 30H/H 20 (70/30/4; v/v/v) System I I ( a c i d ) : CHCiyCH^H/CH^OOH/HgO (100/60/16/8, v / v / v / v ) . The m o b i l i t i e s o f the d i f f e r e n t p h o s p h o l i p i d s a r e g i v e n i n T a b l e 1 6 . i i ) Two D i m e n s i o n a l Systems System I I I : B a s i c CHCl 3/CH 3OH/40$ CH 3NH 2 (13/6/1.5; v/v/v) A c i d CHCl 3/CH 3COCH 3/CH 3OH/CH 3COOH/H 20 (10/4/2/3/1; v/v/v/v) System IV: B a s i c CHCl 3/CH 3OH/25$ NH^OH/H^ (90/54/5.7/5.3; v/v/v/v) A c i d CHCL 3/CH 3OH/CH 3COOH/H 20 (60/30/8/2.85; v/v/v/v) 55. With system I I I , a f t e r d e v e l o p i n g i n the b a s i c s o l v e n t , the p l a t e was a i r - d r i e d and a c i d i f i e d w i t h HCI fumes b e f o r e d e v e l o p i n g the p l a t e i n the a c i d system. The m o b i l i t i e s o f p h o s p h o l i p i d s f o r both systems a r e g i v e n i n T a b l e 1 6 . 5. Column Chromatography A l l p r e p a r a t i o n s o f c y t o s o l were f r a c t i o n a t e d i n a 200 ml Sepharose 6B column which had been e q u i l i b r a t e d w i t h 20 mM T r i s , pH 7.0 b u f f e r c o n t a i n i n g 100 mM N a C l . Enzyme grade s u c r o s e was d i s s o l v e d i n each sample a t a c o n c e n t r a t i o n o f 0.2 g/ml and the dense s o l u t i o n was c a r e f u l l y l a y e r e d on top o f the g e l bed. The column f l o w r a t e was about 50 ml/h and 8 ml f r a c t i o n s were c o l l e c t e d . The column was washed w i t h b u f f e r e q u i v a l e n t t o f i v e times the bed volume o f the column a f t e r each r u n . IV. Enzyme Assays 1. C h o l i n e K i n a s e C h o l i n e k i n a s e was assayed a c c o r d i n g t o the procedure o f Weinhold and Rethy ( 1 2 ) . The a s s a y c o c k t a i l c o n s i s t e d o f the f o l l o w i n g : 150 u l 0.1 M M g C l 2 100 y l 1.0 M T r i s - H C l pH 8.0 20 u l 25 mM [Me- H ] c h o l i n e c h l o r i d e , 1 yCi/umole 150 u l 100 mM ATP 100 y l enzyme 480 y l d i s t i l l e d H 20 56. Table 16 M o b i l i t i e s of Phospholipids on Thin-Layer Plates Developed  i n Various Solvent Systems Solvent Systems I II III IV Neutral Acid Basic Acid Basic Acid Phospholipids M o b i l i t i e s (Rf) PC 0 . 2 2 0 . 2 8 0 . 5 7 0 . 3 1 0 . 5 4 0 . 3 5 PE 0 . 5 3 0 . 7 5 0 . 6 9 0.64 0 . 5 8 0 . 8 3 PS smeared 0 . 2 7 0.56 0 . 3 9 0.50 PI 0 . 4 3 0 . 3 7 LPC 0 . 0 8 0 . 0 9 0 . 3 2 0.08 0 . 2 2 0.07 LPE 0 . 2 6 0 . 4 3 0 . 5 9 0 . 2 2 0 . 3 9 0 . 3 2 SM 0 . 3 9 0.17 0 . 1 2 5 7 . The 1 ml r e a c t i o n was incubatSd a t 37°C f o r 20 min and the tube immersed i n b o i l i n g water f o r 2 min. The l a b e l l e d p h o s p h o c h o l i n e produced was bound to a 3 x 0.5 cm column o f AG1-X10 and l a b e l l e d c h o l i n e was washed from the column w i t h 10 ml o f water. The r a d i o a c t i v e p h o s p h o c h o l i n e was e l u t e d w i t h 1 ml o f 0.1 N NaOH and r a d i o a c t i v i t y was measured by s c i n t i l l a t i o n c o u n t i n g . The r a t e o f the r e a c t i o n was l i n e a r f o r a t l e a s t 25 min w i t h up t o 1.4 mg o f p r o t e i n from both c o n t r o l and c h o l e s t e r o l / c h o l a t e - f e d r a t l i v e r c y t o s o l ( F i g . 3 ) . In r o u t i n e measurements, c h o l i n e k i n a s e was assayed w i t h 1.0 to 1.4 mg o f p r o t e i n f o r 10 min a t 37°C and under these c o n d i t i o n s , 21% o f the s u b s t r a t e was c o n v e r t e d t o p h o s p h o c h o l i n e . 2 . 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 The assay used f o r measuring the 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 s and microsomes was the same (69) and c o n s i s t e d o f the f o l l o w i n g components: 10 u l 10 o r 40 mM CTP 10 u l 10 mM p h o s p h o c h o l i n e , 20 yCi/vimole 10 p i 1.0 M T r i s - s u c c i n a t e , pH 6.0 10-40 y l enzyme The f i n a l volume o f the a s s a y was made up to 0.1 ml w i t h d i s t i l l e d water. When the enzyme was to be a s s a y e d i n the presence o f p h o s p h o l i p i d s , an a p p r o p r i a t e amount was f i r s t added t o the assay tubes and the s o l v e n t was e v a p o r a t e d under a stream o f n i t r o g e n . The enzyme and e v e r y o t h e r component o f the a s s a y e x c e p t f o r the l a b e l l e d p h o s p h o c h o l i n e were added and the p h o s p h o l i p i d was e m u l s i f i e d i n t o s o l u t i o n by a g i t a t i o n on a V o r t e x m i x e r . The m i x t u r e was i n c u b a t e d a t 37°C f o r 3 min b e f o r e s t a r t i n g the r e a c t i o n w i t h p h o s p h o c h o l i n e . 58. 0 10 20 25 Time (min) 90r 0 1 2 Protein (mg) Figure 3. Choline Kinase Activ i ty as a Function of Time and Pro- tein. The enzyme was assayed according to the procedure given in the text. In the upper graph, 1.0 mg of protein was used in the assay and i'n the lower f igure, the reaction was incubated at 37°C for 20 min. The figures show the results obtained with control cytosol. A similar result was obtained when experimental cytosol was used. 59. The reaction was incubated at 37°C for the appropriate time and the tubes immersed in boiling water for 2 min. The protein was pelleted by centrifugation and 60 ul of the supernatant along with CDP-choline carrier was subjected to TLC (see Section III.4.a.i). The amount of labelled CDP-choline produced was determined by liquid s c i n t i l l a t i o n counting (see Section III.3). In routine assays, less than 5% of the substrate was converted. The concentration of CTP in the assay varied depending upon the state of the enzyme. The purified and partially purified enzymes were measured in the presence of 1 mM CTP (23) whereas 1 or 4 mM CTP was used when the cytidylyltransferase was measured in cytosol. (a) Cytosolic Activity The rate of the reaction was linear with up to 260 uS of protein and up to 10 min incubation time for both control and cholesterol/cholate-fed rat liver cytosols (Fig. 4). Thus, the measurement of cytosolic cytidylyltransferase activity was carried out with 150 yg of protein for 10 min. (b) Microsomal Activity The activity of the cytidylyltransferase was linear with up to 240 uS of protein and up to 5 min incubation time (Fig. 5)• Thus, 120 ug of protein and 3 min of incubation were routinely used in measuring enzyme activity in l i v e r microsomes from control and cholesterol/cholate-fed rats. 3. Phosphocholinetransferase Phosphocholinetransferase activity was measured according to the procedure of Vance and Burke (106). The reaction mixture contained the following: 60. Time (min) Protein (mg) Figure 4. Act iv i ty of Cytosolic Phosphocholine Cytidylyltransferase  With Time and Various Amounts of Protein. The cytidylyltransferase was assayed according to the procedure g~iven in the text. In the upper f igure, 100 yg of cytosolic protein was used and in the lower graph, the reaction was incubated for 10 min at 37°C for both control (•) and experimental (•) cytosols. Under the same conditions, con-trol cytosol converted half as much phosphocholine to CDP-choline as the experimental cytosol. 61. Protein (mg) Figure 5. Act iv i ty of Microsomal Phosphocholine Cytidylyltransferase  with Time and Various Amounts of Protein. The conditions used in the assay were as described in the text. In the upper graph, 240 pg of prote'in were used and in the lower f igure, the reactions were incuba-ted for 5 min at 37°C. The data presented were from experiments with microsomes from control (•) and cholesterol/cholate-fed rats (•). 62. 14 20 y l 20 mM [Me- C]CDP choline (0.3 yCi/ymol) 40 y l 1 M T r i s - H C l , pH 8.5 75 y l 0.1 M MgCl 2 20 y l microsomes 200 y l sonicated DG (1 mg/ml) 65 y l H 20 The reaction mixture was preincubated for 5 min at 37°C and the assay was started by addition of l a b e l l e d CDP-choline. Two ml of CHCl^CH OH (1/1) was added to stop the reaction, followed by 1 ml of 0.9 NaCl and 1 ml of CHCl^. The mixture was extracted and the organic phase was recovered and washed twice with 3 ml of t h e o r e t i c a l upper phase. F i n a l l y , the bottom phase was q u a n t i t a t i v e l y transferred into a s c i n t i l l a t i o n v i a l , the solvents were evaporated under a stream of dry a i r and r a d i o a c t i v i t y was measured by s c i n t i l l a t i o n counting. The DG preparation contained 2 mg of DG, 60 y l of 1% Tween 20 and 1.94 ml of H 20. The mixture was sonicated with a Quigley-Rochester Sonic Dismembrator, at a s e t t i n g of 80, for two 3-min periods. The optimal r a t i o of Tween 20 to d i g l y c e r i d e was not determined. The rate of the reaction was l i n e a r up to 80 yg of microsomal protein and up to 12 min of incubation ( F i g . 6). The amount of DG used was optimal for the rate of the reaction ( F i g . 7) and about 5% of the substrate was converted to PC. 4. Phosphatidylethanolamine Methyltransferase This enzyme was assayed by the procedure of Schneider and Vance (73). The reaction contained: 50 y l 100 mM T r i s - H C l , pH 9-2 50 y l microsomes 3 15 y l 0.6 mM S-[Me- H]adenosylmethionine, 100 yCi/ymole 6 3 . o E Protein (mg) Figure 6. Act iv i ty of Phosphocholinetransferase with Time and Var i - ous Amounts of Proteins. The conditions used in the assays were des-cribed in the text. The results were obtained with control micro-somes. In the top f igure, 58 ug of microsomal protein were used and in the bottom graph the reactions were incubated for 10 min at 37°C. Similar results were obtained when microsomes from cholesterol/-cholate-fed rats were used. 64. Diacylglycerol Concentration (mg-ml-1 x 101) Figure 7. Act iv i ty of Phosphocholinetransferase in the Presence of  Various Amounts of Diacylglycerols. The assay conditions are des-cribed in the text. The assay contained 58 ug of control microsomal protein and act iv i ty of the transferase was maximal when 175 ug of diacylglycerol were added to the reaction. Thus, in routine assays 200 ug of diacylglycerol were used. 65. The assay was incubated for 40 min and stopped with 2.1 ml of 0.1 N HCI. The mixture was extracted with 2 ml of butanol and 0.25 ml was used for s c i n t i l l a t i o n counting. The rate of the reaction was l i n e a r up to 30 min. Since a s i g n i f i c a n t proportion of the t o t a l incorporated r a d i o a c t i v i t y was associated with the monomethyl and dimethyl d e r i v a t i v e s of PE, a c t i v i t y of the methyltransferase was therefore expressed as nanomoles of methyl groups transferred rather than nanomoles of PC formed. V. Methods Used i n In V i t r o Studies 1. Care and Feeding of Experimental Animals Female Wistar r a t s (18 day) weighing 30 to 35 g were maintained on Purina r a t chow and tap water ad l i b i t u m i n a l i g h t - and temperature-controlled room. Three days l a t e r , the di e t of h a l f the animals was alt e r e d to one containing 2% cholate, 5% cholesterol and 93% purina rat chow. The ch o l e s t e r o l and cholate content of t h i s d i e t was the same as that found i n other experimental d i e t s (93) and the rats were maintained on t h e i r respective d i e t s for s i x or nine days. This same protocol was used when other experimental diets were tested. New Zealand buck rabbits weighing about 2 kg were maintained on Purina rabbit chow and tap water ad lib i t u m . 2. Preparation of Experimental Diets Purina r a t chow p e l l e t s were ground into powder form by a C h r i s t i e and Norris eight inch laboratory m i l l . The experimental diets were prepared by slowly adding the appropriate amount of c h o l e s t e r o l , cholate or ch o l e s t e r o l and cholate to the powdered rat chow i n a food mixer set a medium speed. For example, 2 g of c h o l i c acid and 5 g of cho l e s t e r o l were added to 93 g of powdered chow. The experimental chows were mixed for another 15 min u n t i l a homogenous powder was obtained. The powder foods 66. were made s o l i d by compressing i n a food p e l l e t e r or a l t e r n a t i v e l y , the chows were moistened with d i s t i l l e d water and dispensed into small food j a r s . The chows were compressed i n the jars and dried overnight i n a l y o p h i l i z e r . 3• Measurement of T o t a l Plasma Cholesterol and Phospholipid A c o l o r i m e t r i c method for measuring t o t a l plasma ch o l e s t e r o l developed by Zak was used (107). B r i e f l y , 0.10 ml of plasma was added to 10 ml of ethanol. The mixture was centrifuged and 2 ml of the supernatant was reacted with 2.0 ml of colour reagent which contained f e r r i c c hloride, phosphoric acid and s u l f u r i c a c i d . T h i r t y to s i x t y minutes l a t e r , the absorbance of the mixture at 550 nm was measured. Standards were used to generate a curve ranging between 0 and 1,000 mg c h o l e s t e r o l per 100 ml. Plasma phospholipids were extracted from 0.20 ml of Plasma with 3 ml of CHCl^/CH^OH (2/1, v/v) and p r e c i p i t a t e d proteins were removed by f i l t r a t i o n through glass wool. Phospholipids were measured by estimating l i p i d phosphorus (see Section I I I . 2 ) . 4. Blood Sampling, Preparation of Cytosol and Microsomes and Perfusion of  Rat Liver Rats were decapitated and blood c o l l e c t e d i n a heparinized tube. The red and white blood c e l l s were sedimented by centrifugation (2,000 x g_) at 4°C and the plasma l i p i d s were analyzed. The r a t l i v e r s were quickly excised and placed i n i c e - c o l d 0.145 M NaCl. Previous studies have shown that the a c t i v 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 cytosol increases when stored at 4°C (22). Thus as a precautionary measure, a control rat was s a c r i f i c e d f i r s t followed by an experimental rat and t h i s sequence was repeated. Subsequently, 4 l i v e r s from control animals were homogenized i n 4 volumes of i c e - c o l d 0.145 M NaCl i n a glass homogenizer with t e f l o n 67. pestle and the experimental l i v e r s were treated i d e n t i c a l l y . The homogenates were centrifuged at 10,000 x g_ for 30 min, and the supernatants were removed and centrifuged at 100,000 x g_ for 90 min. The high speed supernatant, excluding the upper layer of white l i p i d , was taken as cytosol and the microsomal p e l l e t was resuspended by homogenization i n 0.145 M NaCl (5 mg protein/ml). Both were immediately stored at -70°C. The l i v e r s obtained by the above procedure contain a s i g n i f i c a n t amount of blood. Since plasma l i p i d s and proteins could a f f e c t the a c t i v i t i e s of the enzymes of PC biosynthesis, blood was washed out of the l i v e r by perfusion i n a few control experiments. The rats were k i l l e d by c e r v i c a l d i s l o c a t i o n and the l i v e r s were perfused in_ s i t u through the po r t a l vein with 50 ml of i c e - c o l d 0.145 NaCl. The perfused l i v e r s , which had a l i g h t pink appearance, were subsequently treated as normally i s o l a t e d l i v e r s . 51 Measurement of D i a c y l g l y c e r o l i n Cytosol The concentration of DG i n cytosol was measured according to the procedure of Choy e_t a_l. (26). The measurement b a s i c a l l y involves the i s o l a t i o n and subsequent conversion of di g l y c e r i d e to g l y c e r o l . The amount of g l y c e r o l was then measured enzymatically by g l y c e r o l dehydrogenase. This enzyme converted g l y c e r o l to dihydroxyacetone and at the same time produced NADH from NAD+. The amount of NADH produced was determined by measuring the absorbance at 340 nm. Li p i d s were extracted from 12 ml of cytosol by CHCiyCH^OH (2/1) and the solvents i n the extract were evaporated under reduced pressure. The residue was dissolved i n CHC13 and passed through an alumina column. The neutral l i p i d s were eluted with CHCl^/CH^OH (95/5; v/v) and digly c e r i d e was i s o l a t e d by thin-layer chromatography with C,H,/CHC1 /CH_0H (80/15/5; v/v/v) as solvent. The DG was eluted 68. from the s i l i c a and hydrolyzed i n potassium hydroxide. Free f a t t y acids were absorbed by albumin and i t was removed by u l t r a f i l t r a t i o n . The amount of g l y c e r o l was measured by g l y c e r o l dehydrogenase and the assay contained hydrazine which converted dihydroxyacetone to the hydrazone and f a c i l i t a t e d complete conversion of the substrate. Various amounts of d i a c y l g l y c e r o l s which were hydrolyzed and assayed i n the same manner were used as standards. 6. U l t r a f i l t r a t i o n and Phospholipase C Treatment of Cytosols The stimulation of the 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 1 i n hypercholesterolemia r a t l i v e r c ytosol could have been caused by the presence of a low molecular weight modulator. This p o s s i b i l i t y was examined by u l t r a f i l t r a t i o n . Cytosols were d i l u t e d and then subjected to u l t r a f i l t r a t i o n through an Amicon XM-100A membrane f i l t e r . Only molecules with molecular weight larger than 100,000 were retained and the d i l u t e d cytosols were concentrated back to t h e i r o r i g i n a l volume. Cy 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 subsequently measured i n the u l t r a f i l t r a t e d c y t o s o l s . The c y t i d y l y l t r a n s f e r a s e i s strongly stimulated by phospholipids and the presence of a l i p i d a c t i v a t o r could explain the stimulation of the enzyme i n experimental c y t o s o l . I f t h i s were the case, removal of phospholipids from both cytosols would abolish the difference i n enzyme a c t i v i t y . Cytosols containing 1 mM CaCl^ and Tris-succinate pH 7.0 were treated with 4 units per ml of phospholipase C from CI. Welchii and the reaction was stopped with 3 mM f i n a l concentration of EGTA. Aliquots of the treated cytosol were subsequently assayed for 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 . 69. 7. P u r i f i c a t i o n of Phosphocholine C y t i d y l y l t r a n s f e r a s e and Assessment of  Pur i t y The procedure f o r the p u r i f i 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 was that of Choy e_t a_l. (22). The 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 two d i s t i n c t species i n cyt o s o l : a high molecular weight (H-form) and a low molecular weight (L-form). The p u r i f i c a t i o n scheme took advantage of the fac t that the c y t i d y l y l t r a n s f e r a s e which e x i s t s predominantly as the L-form i n fresh cytosol aggregates i n t o the H-form upon incubation at 4°C for several days. In the f i r s t step of p u r i f i c a t i o n , the H-form was separated from low molecular weight proteins by gel f i l t r a t i o n . Subsequently, the H-form was diss o c i a t e d to the L-form by sodium dodecyl s u l f a t e and p u r i f i e d L-form was i s o l a t e d by a second g e l f i l t r a t i o n step which eliminated any high molecular weight contaminants. In a t y p i c a l p u r i f i c a t i o n , r a t l i v e r homogenate (20% w/v) was prepared i n i s o t o n i c s a l i n e and centrifuged at 100,000 x g for 60 min. One hundred ml of the supernatant (cytosol) was stored at 4°C for 5 days, a f t e r which p r e c i p i t a t e d materials were removed by cent r i f u g a t i o n at 10,000 x g_ for 10 min. The cyt o s o l was adjusted to 20% ammonium s u l f a t e saturation and the p r e c i p i t a t e was removed by cent r i f u g a t i o n (10,000 x g for 10 min). The supernatant was readjusted to 25% ammonium s u l f a t e saturation and the p r e c i p i t a t e formed a f t e r 2 h of storage at 4°C was c o l l e c t e d by cen t r i f u g a t i o n (10,000 x j| for 10 min) and resuspended i n 10 ml of column buffer (see Section I I I . 5 ) . Half of t h i s sample was applied to 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 the same buffer. Enzyme a c t i v i t y was detected i n the f r a c t i o n s eluted with the void volume and a c t i v e f r a c t i o n s were combined. The chromatography step was repeated with the second h a l f of the ammonium s u l f a t e f r a c t i o n . Sodium dodecyl 70. s u l f a t e (.1%) was added to the pooled f r a c t i o n s to a f i n a l concentration of 0.05$ and the sample was stored at 4°C for 2 h. Part of t h i s s o l u t i o n (4 ml) was applied to a Sepharose 6B column (2.5 x 80 cm) eq u i l i b r a t e d with column buffer containing 0.005$ SDS and the cholinephosphate 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 separated i n t o two d i s t i n c t f r a c t i o n s . The second f r a c t i o n of enzyme a c t i v i t y (which required l i p i d for maximum a c t i v i t y ) was c o l l e c t e d and concentrated by u l t r a f i l t r a t i o n over an Amicon PM 30 membrane. A 1,000-fold p u r i f i c a t i o n of the enzyme was achieved with an o v e r a l l y i e l d of 10$. The p u r i t y of the preparation was determined by nondenaturing polyacrylamide g e l (5$) electrophoresis at pH 7.5 (108). A single band was observed when the g e l was stained f o r p r o t e i n . When duplicate gels were electrophoresed and one stained f o r protein and the other s l i c e d and assayed, 17$ 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 was recovered and i t coincided with the protein band. Since each g e l (0.55 x 8 cm)' was loaded with 50 pg of protein and no other protein bands were observed, the enzyme appears to be pure. Choline kinase, ethanolaminephosphate c y t i d y l y l t r a n s f e r a s e , and cholinephosphotransferase a c t i v i t i e s were not detected i n p u r i f i e d preparations. The p u r i f i e d enzyme was found to be unstable and the presence of d i t h i o t h r e i t o l , CTP-Mg 2 +, or bovine serum albumin did not s i g n i f i c a n t l y s t a b i l i z e the enzyme. When the enzyme was frozen and thawed once, more than 90$ of the a c t i v i t y was l o s t . 8. Preparation of Antibodies Against the P u r i f i e d C y t i d y l y l t r a n s f e r a s e  and Immunotitration of the Enzyme from Cytosol The c y t i d y l y l t r a n s f e r a s e was p u r i f i e d according to the above procedure and 12 preparations were pooled, concentrated to 1 mg protein per ml and dialyzed against 100 volumes of 0.145 M NaCl, 10 mM T r i s HCI, pH 7.4. A 2 kg rabbit was immunized subcutaneously at two s i t e s with a t o t a l of 0.5 mg enzyme per kg body wt, emulsified i n Freund's complete adjuvant. This was followed by two i n j e c t i o n s of 0.25 mg enzyme per kg body weight, emulsified i n Freund's incomplete adjuvant at 7 day i n t e r v a l s . Another rabbit was treated i d e n t i c a l l y except that only buffer was used i n the i n j e c t i o n s . A week a f t e r the l a s t i n j e c t i o n , 10 ml of blood was obtained from each rabb i t and immunoglobulin G was prepared. The c l o t t e d blood c e l l s were sedimented at 1,000 x g for 5 min. The sera were allowed to stand overnight at 4°C and the formed c l o t s were' sedimented at 12,000 x g for 10 min. The p e l l e t s were discarded and one volume of i c e - c o l d saturated (NH^SO^) so l u t i o n was added to each serum. After 30 min on i c e , the protein p r e c i p i t a t e s were pel l e t e d by centrifugation at 6,000 x £ for 15 min and the p e l l e t s were washed 4 times with 5 ml of 40% ( N H ^ S C ^ ) . F i n a l l y , the p e l l e t s were dissolved i n 10 ml of 10 mM phosphate buffer, pH 7.0 and dialyzed 2 times against 100 volumes of 10 mM phosphate buffer, pH 7.0 for 6 h at 4°C. The dialyzed immunoglobulin G preparations were d i l u t e d to give a f i n a l concentration of 7 mg of protein per ml. The immunoglobulin G preparations were analyzed by c e l l u l o s e acetate electrophoresis and had one band which corresponded to the immunoglobulin G band of whole serum. The s p e c i f i c i t y of the anti-serum was tested by double d i f f u s i o n analysis i n agar gel against cytosol or L-form and i n both cases a sin g l e sharp p r e c i p i t i n l i n e was observed. Normal rabbit serum analyzed s i m i l a r l y did not show any reaction. The p u r i f i c a t i o n of immunoglobulin G did not a f f e c t the antigen-antibody reaction nor the s p e c i f i c i t y as tested by double d i f f u s i o n a n a l y s i s . 72. Agar plates used f o r double d i f f u s i o n a nalysis were prepared by p i p e t t i n g 5 ml of hot 1% agar containing 20 mM Tri s - H C l , pH 7.0, 0.1 M NaCl and 0.1% sodium azide i n t o 7 cm p e t r i dishes. After the agar had s o l i d i f i e d , 0.4 cm wells were made with d i f f u s i o n distances of 0.8 cm. The center well was f i l l e d with 50 p i of L-form and immune and con t r o l sera or immunoglobulin G were added to the peripheral wells. The immunoprecipitin l i n e s were allowed to develop for 72 h at 4°C. The plates were washed with s a l i n e and water and stained f o r 15 min with 0.04$ ponceau S i n 2% t r i c h l o r o a c e t i c a c i d and 5% a c e t i c a c i d . Destaining was c a r r i e d out i n 5% a c e t i c a c i d f o r 24 h. The i s o l a t e d immunoglobulins were used to t i t 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 from c y t o s o l . In these experiments, 25 p i (about 0.3 mg protein) of normal and experimental cytosols were d i l u t e d with 75 p i of 0.20 M phosphate buffer, pH 8.0. Duplicate sets of incubations were made f o r each cytosol where 0 to 150 p i (1.1 mg) of control antibodies were added to one set of incubations and the same amount of a n t i - c y t i d y l y l t r a n s f e r a s e antibodies were added to another set. The volumes of a l l the incubations were f i n a l l y made up to 250 p i with 10 mM phosphate buffer, pH 8.0. The antigen-antibody complexes were allowed to form for 8 h at 4°C and the p r e c i p i t a t e was sedimented at 4,200 x g_ for 30 min at 4°C. The tubes were handled very gently and 60 p i of the supernatant was used to measure c y t i d y l y l t r a n s f e r a s e a c t i v i t y . The enzyme assay contained 1 mM CTP and i t was incubated f o r 15 min at 37°C. 9. Treatment of Cytosols with Trypsin and Column Chromatography of  Trypsin-inactivated Cytosols. The cytosols were treated with 0.1 mg of t r y p s i n per ml for 15 min at 37°C and p r o t e o l y s i s was stopped with 0.15 mg of t r y p s i n i n h i b i t o r per ml. 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 completely eliminated a f t e r t h i s treatment and 40 p i of each of the inactivated cytosols were reconstituted with 20 y l of delipi d a t e d or p a r t i a l l y p u r i f i e d enzyme. Enzyme a c t i v i t y was measured as previously described (see Section IV.2.a). A t r y p s i n - r e s i s t a n t a c t i v a t o r was found and i t s properties were further studied by subjecting 4 ml of t r y p s i n treated control and experimental cytosol to Sepharose 6B chromatography. The a c t i v a t o r was assayed i n the column f r a c t i o n s by measuring the a c t i v i t y of 20 p i of delipidated or p a r t i a l l y p u r i f i e d enzyme i n the presence of 40 p i of column eluate. 10. Procedure for the Large Scale I s o l a t i o n of Cytos o l i c L i p i d s and the  Fractionation of These l i p i d s by Thin Layer Chromatography A large amount of ac t i v a t o r was required for further studies. Previous experiments showed that the a c t i v a t o r had a high molecular weight and i t was therefore p r e c i p i t a t e d from cytosol by 25% ammonium s u l f a t e . The p r e c i p i t a t e was sedimented by cen t r i f u g a t i o n and the p e l l e t obtained was extracted 3 times with CHCl^/CH^OH (2/1; v/v) under nitrogen. The extracts were pooled and concentrated to 1/10 the volume of the cytosol extracted. The c y t o s o l i c l i p i d s were fractionated by one dimensional and two dimensional th i n - l a y e r chromatography. L i p i d s were i d e n t i f i e d by iodine and eluted from the s i l i c a with either three 4 ml volumes of CHCl3/CH3OH/25 NHj^ OH (50/50/2; v/v/v) or CHC13/CH30H/H20 (100/50/2; v/v/v). 11. Analysis of Lysophosphatidylethanolamine by Mass Spectrometry Lysophosphatidylethanolamine was i s o l a t e d by preparative two dimensional t h i n layer chromatography (solvent system IV, Section I I I . 4 . b . i i ) . The amount of l i p i d extracted from 30 ml of control and 74. experimental cytosol (2.0 to 3.0 mg) was each applied to three 2.5 cm lanes on a thin layer plate. After development in one solvent, the standard LPE was visualized by iodine and the s i l i c a in the corresponding areas on the sample lanes were scraped and eluted with 30 ml of CHClg/CH OH (1/1). The eluted lipids were concentrated and rechromatographed as described above except that the plate was developed in the second solvent system. Isolated LPE was silylated according to published procedures (109). The solvents in the preparations were evaporated under a stream of N 2 and 0.1 ml of dry pyridine and 0.1 ml of bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane were added. The mixtures were agitated and incubated at room temperature for 1 h. After the reaction solvents were evaporated and the derivatized products, either solid or disolved in CHCl^, were analyzed in an AEI-902 Mass Spectrometer. Each sample was applied on a probe which was introduced directly through the vacuum lock. Insertion probe temperature was 150-l80°C and the spectra was obtained at 70 ev. 12. Quantitation of Lysophosphatidylethanolamine by Ninhydrin The amount of l i p i d in 1.5 ml of control and experimental cytosols (.87 and .165 mg, respectively) was chromatographed by TLC with CHC1 /CH OH/CH COOH/HgO (100/60/16/8; v/v/v/v). A suitable 2-dimensional TLC solvent system was not available at the time. The lipids were eluted from the s i l i c a into test tubes and the solvent was evaporated under N2. The amount of LPE was measured by a procedure developed by Lea and Rhodes (110). Briefly, 1 ml of methylcellosolve and 1 ml of Stein and Moore ninhydrin reagent, which contains 0.80 g of SnCl2*2 H^O in 500 ml of 0.2 M citrate buffer, pH 5.0 and 20 g of ninhydrin in 500 ml of methylcellosolve, were added to the samples. After mixing, the tubes, capped with marbles, were heated in boiling water for 20 min and 5 ml of 75. methylcellosolve were added to each tube a f t e r a b r i e f period of c o o l i n g . The absorbance of the solutions at 575 nm were measured. Synthetic palmitoyl-LPE was treated i d e n t i c a l l y as the samples and was used to generate a standard curve ranging up to 50 ug of LPE (4 ug l i p i d phosphorus). 13- Analysis of Fatty Acids on Lysophosphatidylethanolamine by Gas-Liquid  Chromatography Lysophosphatidylethanolamine was i s o l a t e d from c y t o s o l i c l i p i d s by two dimensional t h i n layer chromatography (see Section I I I . 4 . b . i i ) i n two separate experiments using d i f f e r e n t solvent systems. The LPE's from 0.2 mg of c y t o s o l i c l i p i d s were eluted from the s i l i c a and reacted with 1 ml of 0.75 N HCI i n methanol for 16 h at 80°C. The excess methanol HCI was evaporated under a stream of and the f a t t y a c i d methyl esters, dissolved i n 10 y l of hexane, were analyzed with a Hewlett-Packard 76IOA High E f f i c i e n c y Gas Chromatograph. A few m i c r o l i t r e s of the samples were inject e d i n t o a column of 12% HI-EFF-2BF (ethylene g l y c o l succinate) at 170°C. Methyl esters derived from synthetic palmitoyl-LPE and methyl esters of standard C ^ . Q , C ^ . Q ' C I8*0 a n d ^20*0 w e r e a n a l y z e d i d e n t i c a l l y . The f a t t y acids from the samples were i d e n t i f i e d by comparing retention times of t h e i r methyl esters with the retention times of the standard f a t t y a c i d methyl esters. VI. Methods Used i n In Vivo Studies 1. I n t r a p o r t a l I n j e c t i o n of [Me-3H]Choline and Freeze Clamping of Liver s The procedure used was s i m i l a r to that described by Akesson e_t a l . (111). A co n t r o l r a t was anaesthetized with ether and i t s abdomen was cut open and the p o r t a l vein was exposed. A 100 y l s o l u t i o n of s a l i n e •3 containing 60 yM [Me- H]choline (50 yCi/100 y l , 8.3 Ci/mmol) was i n j e c t e d i n t o the p o r t a l vein. After a predetermined time, not longer than 4 min, approximately 1 g of l i v e r from the r i g h t side of the median lobe was excised and immediately frozen between two aluminum blocks which have been cooled i n l i q u i d nitrogen. The frozen tissue was stored i n l i q u i d N 2 and was extracted within a few h. A cholesterol/cholate-fed r a t was treated i d e n t i c a l l y and the sequence was repeated u n t i l a l l ra t s were k i l l e d . Excess l i v e r tissues from a l l animals were excised and weighed immediately a f t e r k i l l i n g . 2. Bligh and Dyer Extraction The frozen tissues were extracted by the procedure developed by Bligh and Dyer (99). The frozen tissue (1 g) was homogenized with 3 ml of CHCl^/CH^OH (1/2, v/v) i n a Sorval Omni-mixer micro chamber. The tissue was homogenized at maximum speed for four 15 s i n t e r v a l s at 0°C and the homogenate was poured into a 30 ml glass centrifuge tube. The micro chamber was washed with 3.8 ml of CHCl^/CH^OH/HgO (1/2/0.8; v/v/v) which was pooled with the homogenate. The one phase extract was made in t o a two phase system by addition of 2 ml of CHCl^ and d i s t i l l e d water. The mixture, which consisted of CHCl^/CH^OH/HgO (1/1/0.9) was vigorously mixed and the phases were separated by centrifugation at 6,000 x g for 20 min at" 4°C. The t o t a l amount of r a d i o a c t i v i t y incorporated was calculated by adding the t o t a l amount of r a d i o a c t i v i t y incorporated into each of the phases. Radioactivity i n each phase was determined by using 100 y l of upper and lower phases for s c i n t i l l a t i o n counting. Previous experiments showed that over 95$ of the r a d i o a c t i v i t y was incorporated into water soluble compounds i n the upper phase. Thus to minimize contamination of r a d i o a c t i v i t y from upper phase, the lower organic phase was washed 2 times with 4 ml of t h e o r e t i c a l upper phase and the washings were discarded. Theoretical upper phase was obtained from the top phase of an eq u i l i b r a t e d mixture of CHC13/CH30H/H20 (1/1/0.9). 77. 3. Separation of Choline, Phosphocholine, CDP-choline and Betaine The water-soluble compounds i n the upper phase were separated by th i n layer chromatography. In e a r l i e r experiments, an aliq u o t containing 200,000 cpm was subjected to two dimensional th i n layer chromatography (see Section I I I . 4 . a . i i ) and a l l four compounds were well resolved. But because the amount of r a d i o a c t i v i t y incorporated into CDP-choline was i n s i g n i f i c a n t (about 0.1 to 0.5 of t o t a l r a d i o a c t i v i t y ) , a single dimensional t h i n layer system was used i n a l l subsequent experiments. This basic solvent system (see Section III.4.a.i) separated choline, phosphocholine and betaine but CDP-choline co-chromatographed with betaine. 4. Measurement of Phosphocholine Pool The amount of phosphocholine i n l i v e r was measured enzymatically (112). An aliq u o t of upper phase containing about 100 nanomoles of / phosphocholine was f i r s t extracted with acid-washed charcoal. Endogenous CTP and CDP-choline which i n t e r f e r e with the measurement were adsorbed to the charcoal and sedimented at 6,000 x £ for 10 min. The supernatant was f i l t e r e d through glass wooi to remove any r e s i d u a l charcoal and the f i l t r a t e was concentrated to a f i n a l phosphocholine concentration of 80 nanomoles per ml. The measurement of phosphocholine involved two enzymatic reactions. The f i r s t reaction converted phosphocholine to CDP-choline by enzyme phosphocholine c y t i d y l y l t r a n s f e r a s e . The second reaction digested a l l the unreacted radioactive cytidine-5'-triphosphate to cyt i d i n e and inorganic phosphate by a l k a l i n e phosphatase. The l a b e l l e d CDP-choline was then separated from l a b e l l e d c y t i d i n e by paper chromatography and the r a d i o a c t i v i t y i n c y t i d i n e diphosphocholine was measured by s c i n t i l l a t i o n counting. 78. The reaction c o c k t a i l s and conditions used for the two reactions are given below: Reaction I: C o c k t a i l 10 y i 1 M Tris-succinate, pH 7.0; 0.12 M Mg(0Ac) 2 10 u l 0.8 umol/min/ml of inorganic pyrophosphatase 5 u l [5- 3H]-CTP, 10 mM, 10 uCi/umol 50 u l p a r t i a l l y p u r i f i e d L-form from rat l i v e r 25 u l sample containing 1-3 nmol of phosphocholine or standards containing 1-4 nmol of phosphocholine The reaction, i n a f i n a l volume of 100 ul> was c a r r i e d out at 37°C for 45 min and stopped by immersion of the tubes i n b o i l i n g water for 2 min. The p r e c i p i t a t e d proteins were sedimented and 50 u l of the supernatant was used i n the second reaction. Reaction I I : C o c k t a i l 20 u l 5 mM CDP-choline; 1 M T r i s - g l y c i n e , pH 10.5 30 u l 10 umol/min/ml of alkaline"phosphatase 50 u l supernatant from reaction I The reaction was allowed to proceed at 37°C for 30 min and stopped by b o i l i n g for 2 min. A 25 u l aliq u o t of reaction II was subjected to descending paper chromatography along with 100 ug of CDP-choline c a r r i e r . The aliquots were applied as spots not larger than 1 cm i n diameter and a mixture of ethanol/1 M ammonium acetate, pH 7.1 (7/3; v/v) was used as solvent. After development, the dried chromatograms were placed under U.V. and the dark CDP-choline spots were marked. The areas of the paper containing the spots were cut into thin s t r i p s and placed i n a s c i n t i l l a t i o n v i a l containing 1 ml of water. Subsequently, r a d i o a c t i v i t y was determined by s c i n t i l l a t i o n counting. A standard s o l u t i o n of phosphocholine was used to generate a l i n e a r curve from 0 to 4 nanomoles. The concentration of phosphocholine i n each sample was determined twice where one measurement contained twice as much sample than the other. 5. C a l c u l a t i o n of the Rate of Phosphatidylcholine Biosynthesis The rate of PC biosynthesis was estimated by measuring the conversion of phosphocholine to PC. Evidence to date strongly indicates that the rate l i m i t i n g step of the de novo pathway i s the reaction catalyzed by phosphocholine c y t i d y l y l t r a n s f e r a s e . Since the f i r s t and l a s t reactions of the pathway are believed not to be rate l i m i t i n g , the rate of conversion of phosphocholine to PC should be equivalent to the rate of PC biosynthesis. o Experimentally, [Me- H] choline was injected into the portal vein of control and experimental r a t s and the r a d i o a c t i v i t y incorporated into choline, phosphocholine, CDP-choline, betaine and PC were followed with time. Early experiments showed that the s p e c i f i c r a d i o a c t i v i t y of phosphocholine remained almost unchanged between 1 and 4 minutes a f t e r i n j e c t i o n . Therefore, the rate of biosynthesis was measured between 1 and 2 minutes p o s t - i n j e c t i o n . The amount of r a d i o a c t i v i t y incorporated per min per l i v e r into PC between 1 and 2 min was calculated by subtracting the dpm at 1 min from the dpm at 2 min. S p e c i f i c r a d i o a c t i v i t y of phosphocholine was calculated by d i v i d i n g the average dpm per l i v e r at 1 and 2 min by the average number of nanomoles of phosphocholine per l i v e r at the same time points. F i n a l l y , the rate of PC biosynthesis per l i v e r was calculated by d i v i d i n g the amount of dpm incorporated into PC per min per l i v e r by the s p e c i f i c r a d i o a c t i v i t y of phosphocholine. The estimated rate of PC biosythesis was expressed as nanomoles per min per l i v e r . 80. VIII. S t a t i s t i c a l Methods A standard s t a t i s t i c a l test was used (113). For each set of data, the mean, standard deviation, T value, and degree of freedom were calculated as follows: MEAN n = t n = 1 t where X = mean Xi to Xt = i n d i v i d u a l value t = t o t a l number of values Standard Deviation I n = t 1 (X n - X)2 j t - 1 where S = standard deviation T Value 0 X - Y T = -—= z z = z = r / Z2 + where t x , ty = number of values i n X and Y resp e c t i v e l y z2 _ + >^y 2 i f , and only i f , the number of X values equal Y values, 81. Degree of- Freedom DF = t + t - 2 x y where DF = degree of freedom To determine i f the mean of the X (control) population was equal to that of the Y (experimental) population, value a was obtained from a table by using the T value and DF. The a value i s the p r o b a b i l i t y that the mean of the control and experimental populations are the same. In the analysis of a l l experimental r e s u l t s , the means of the control and experimental populations were considered to be d i f f e r e n t only i f the a values calculated were les s than 0.05. 82. RESULTS I. Characterization of Phosphocholine C y t i d y l y l t r a n s f e r a s e : The E f f e c t of  pH, Phospholipids, Acyl-CoAs, Various Adenosine-containing Coenzymes  and Nucleotides on Enzyme A c t i v i t y The r e s u l t s from e a r l i e r studies suggested that phosphocholine c y t i d y l y l t r a n s f e r a s e may play an important regulatory r o l e i n the control of de novo phosphatidylcholine biosynthesis. Therefore, the e f f e c t of pH and various p h y s i o l o g i c a l compounds on the a c t i v 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 was investigated. Two d i s t i n c t forms of the c y t i d y l y l t r a n s f e r a s e have been described i n rat lung (23) and rat l i v e r (22). The a c t i v i t i e s of the two forms of the enzyme at various pH's were measured ( F i g . 8). A c t i v i t y of the low molecular weight form (L-form) was maximal at pH 6.5, whereas the high molecular weight form .(H-form) was most active at pH 7.5. Furthermore, while the a c t i v i t y of the L-form f a l l s dramatically at high and low pH, the H-form a c t i v i t y remained high under a l k a l i n e conditions but was severely i n h i b i t e d under more a c i d i c conditions. Synthesis of PC requires DG and i t i s conceivable that the DG and f a t t y acid synthesis may be coordinately regulated with the synthesis of PC. Therefore, the e f f e c t 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 of some important 16r PH Figure 8. Act iv i t ies of the High (#) and Low (•) Molecular Weight  Forms oT~ Phosphocholine Cytidylyltransferase at Various Values of  pH. The enzyme assay and the preparation of part ia l ly purified L-form was described in the Materials and Methods section. H-form was prepared by chromatography of aged cytosol (4 ml) on Sepharose 6B and the fractions near the void volume which contained the highest H-form activity were used. The curves have been matched for convenience of comparison. Tris-HCl buffer (50 mM) was used between pH 7.0 and 9.0 while Tris-succinate (50 mM) was used between pH 6.0 ana 7.0. 84. intermediates of f a t t y acid and phosphatidic acid biosynthesis were investigated. The compounds tested did not exert a marked e f f e c t on the enzyme. Acetyl-CoA at 5 uM and malonyl-CoA at 50 uM had no e f f e c t while 50 uM palmitoyl-CoA and 50 uM stearoyl-CoA s l i g h t l y stimulated enzyme a c t i v i t y by 14 and 35% r e s p e c t i v e l y . The synthesis of PC from choline and DG requires the expenditure of 1 ATP and 1 CTP molecule. Phosphatidylcholine biosynthesis must therefore be, i n some manner, coupled to the energy l e v e l of the c e l l . With t h i s i n mind, the e f f e c t s of various compounds involved i n the energy metabolism of the c e l l were examined (Table 17). NAD and NADP i n h i b i t e d enzyme a c t i v i t y to h a l f the control value at 5 mM while NADH, and to a lesser extent NADPH, stimulated the enzyme. In contrast, e f f e c t s of nucleotides did not f a l l into a simple l o g i c a l pattern. Although UMP 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 , GMP had no e f f e c t . Furthermore, a l l the nucleoside triphosphates tested i n h i b i t e d enzyme a c t i v i t y and the most potent was ATP which i n h i b i t e d 80% of enzyme a c t i v i t y at 10 mM. Interestingly, a c t i v 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 cytosol was also i n h i b i t e d by high concentrations of CTP. The a c t i v i t y of the enzyme when assayed with 12 mM CTP was les s than 50% of the enzyme a c t i v i t y when 4 mM CTP was used. A preliminary study of the mode of i n h i b i t i o n by ATP and NAD with respect to CTP and phosphocholine was performed ( F i g . 9 and 10) and r e s u l t s from k i n e t i c experiments suggested that both ATP and NAD were non-competitive with respect to CTP. Further experiments are required before any conclusions can be made. 85. Table 17 E f f e c t of Various Adenosine-Containing Coenzymes and Nucleotides  on the A c t i v i t y of Phosphocholine C y t i d y l y l t r a n s f e r a s e Relative Enzyme A c t i v i t y (o/o control a c t i v i t y ) 1 mM 5mM 10 mM NAD 65 39 -NADH 142 142 -NADP 88 57 -NADPH 128 112 -ATP - - 20 GTP 129 110 69 UTP 109 86 75 GMP 114 - 105 UMP 78 69 56 / The reaction contained 30 y l of 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 , 1 mM CTP and the appropriate concentration of the compound to be tested. The assay was incubated f o r 20 min at 37°C and a l l the other conditions were according to the procedure described i n the Materials and Methods se c t i o n . 86. Phosphocholine -l (mM_i) Figure 9. Double Reciprocal Plots of the Init ial Velocity of Phos- phocholine Cytidylyltransferase Versus Various Concentrations of CTP  or Phosphocholine in the Presence of ATP. Reactions were carried out in the presence (#) and absence (•) of 10 mM ATP, and in a l l experi-ments the concentration of only one substrate was varied. The assay contained 30 u l of part ia l ly purified enzyme and the concentration of the non-varied substrate was saturating. CM | -10 0 10 20 CTP-1 (mM-1) Phosphocholine -l (mM-l) Figure 10. Double Reciprocal Plots of the Init ia l Velocity of Phos- phocholine" Cytidylyltransferase Versus Various Concentrations of CTP  or Phosphocholine in the presence of NAD. The conditions used were similar to F ig. 9 except that reactions were carried out in the pres-ence (•) and absence (•) of 5 mM NAD. In contrast, the modes of i n h i b i t i o n by ATP and NAD with respect to phosphocholine were quite d i f f e r e n t . ATP increased the Km of the enzyme for phosphocholine from a control value of 0.25 mM to 0.50 mM and competes with phosphocholine for the same s i t e on the enzyme. The r e s u l t s indicated that ATP was a competitive i n h i b i t o r of the c y t i d y l y l t r a n s f e r a s e with respect to phosphocholine. Results from experiments with NAD were more d i f f i c u l t to i n t e r p r e t since the v e l o c i t y as well as the a f f i n i t y of the enzyme for phosphocholine appeared to be diminished by the i n h i b i t o r . The p a r a l l e l l i n e s obtained from the double r e c i p r o c a l plot suggested that NAD was an uncompetitive i n h i b i t o r of the enzyme with respect to phosphocholine. I I . E f f e c t s of Various Diets on the Levels of Plasma L i p i d s 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 Phosphocholine c y t i d y l y l t r a n s f e r a s e has been implicated as the regulatory enzyme of the pathway for PC biosynthesis. Further i n s i g h t s into the regulation of PC biosynthesis could be obtained from studies with an animal model whose rate of PC biosynthesis was s i g n i f i c a n t l y altered from normal. As discussed i n the introduction, rats with diet-induced hypercholesterolemia had elevated l e v e l s of plasma phospholipids and t h i s could be the r e s u l t of elevated rates of phospholipid synthesis i n the l i v e r where plasma l i p o p r o t e i n s are synthesized. This idea was tested i n young female rats whose plasma ch o l e s t e r o l l e v e l s could be e a s i l y manipulated (95). The e f f e c t of feeding a commercially a v a i l a b l e Basal Atherogenic Diet was investigated and the r e s u l t s are shown i n Table 18. The plasma ch o l e s t e r o l and phospholipids i n the experimental group were increased over controls by 4- and 2-fold a f t e r 3 days and by 10- and 3-fold, respectively a f t e r 6 days of feeding. The a c t i v i t y of phosphocholine 89. c y t i d y l y l t r a n s f e r a s e was also measured i n both groups (Table 18) and was found to be elevated i n the l i v e r cytosols from rats fed the Basal Atherogenic Diet. 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 a f t e r 3 and 6 days of feeding by 3.5- and 2.5-fold, r e s p e c t i v e l y . The d i e t s used i n these experiments have d i f f e r e n t compositions aside from the 5.3$ c h o l e s t e r o l and 2$ cholate contained i n the atherogenic d i e t . The control d i e t consisted of 20$ l a r d and 56$ sucrose, while the experimental d i e t contained 40$ butterfat and 20.3$ sucrose. I t was possible that the stimulation of the c y t i d y l y l t r a n s f e r a s e could have been caused by a difference i n c a l o r i e uptake rather than by the intake of cholesterol and cholate. In a subsequent study, experimental r a t s were fed a modified control d i e t (Purina Rat Chow) supplemented with either 2$ cholate, 5$ c h o l e s t e r o l , or 2$ cholate and 5$ ch o l e s t e r o l f o r d i f f e r e n t lengths of time (Table 19). The rats fed the di e t containing cholate and cho l e s t e r o l had a 6-fold elevation of plasma c h o l e s t e r o l , a 2-fold increase i n plasma phospholipids and a 2-fold increase i n c y t o s o l i c a c t i v i t y of l i v e r phosphocholine c y t i d y l y l t r a n s f e r a s e when compared to rats fed ordinary purina rat chow. A c t i v 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 was also stimulated, although to a much lesser degree, when rats were fed die t s containing only c h o l e s t e r o l or cholate. Plasma c h o l e s t e r o l was increased while the l e v e l of plasma phospholipids remained unchanged i n the rats fed the diet containing 90. Table 18 Plasma L i p i d s 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  Liver Cytosols from Rats Fed a Control High Fat Diet and  a Basal Atherogenic Diet (BAD) Plasma L i p i d s Phosphocholine (mg per 100 ml) c y t i d y l y l t r a n s -ferase A c t i v i t y Day on Diet Cholesterol Phospholipids (nmol/min/mg) Control (2) 3 164 116 0.78 (3) 6 149 ± 10 157 ± 14 0.54 ± 0.05 BAD (2) 3 633 205 2.7 (3) 6 1514 + 236 427 ± 98 1.5 + 0.06 The conditions and animals used have been described i n the Materials and Methods section. The numbers i n brackets indicate the number of animals used and values are given as mean + S.D. 91. Table 19 E f f e c t of Diets Containing 2% Cholate, 5% Cholesterol or 2% Cholate and  5% Cholesterol on the Level of Plasma L i p i d s and the Cytosolic  A c t i v i t y of Hepatic Phosphocholine C y t i d y l y l t r a n s f e r a s e Diet Days on Diet Plasma L i p i d s (mg per 100 ml) Enzyme A c t i v i t y Total Cholesterol Phospholipids Control 6 100 (2) 91 (2) 0.46 ± O.'l (10) 2% Cholate 3 162 (2) 77 (2) 0.89 (2) 6 146 + 14 (3) 85 ± 26 (3) 0.72 ± 0.33 (3) 5% Cholesterol 6 - - 0.84 (2) 2% 5% Cholate Cholesterol 3 809 ± 144 (3) 191 ± 22 (3) 1.21 ± 0.34 (3) 6 672 + 98 (3) 197 ± 51 (3) 1.1 ± 0.19 (10) 9 664 (2) 1.0 (2) Control rats were fed purina rat chow while the experimental rats were fed a d i e t composed of purina r a t chow supplemented with c h o l e s t e r o l , cholate or c h o l e s t e r o l and cholate. The values are expressed as means or mean + S.D. while the numbers i n brackets ind i c a t e the number of rats used. only cholate. Since the only difference between the control and experimental d i e t s was the content of c h o l e s t e r o l and cholate, the stimulation of the c y t i d y l y l t r a s f e r a s e i n experimental rats must be the r e s u l t of intake of these l i p i d s . The d i e t containing 2% cholate and 5% cholesterol induced the most marked e f f e c t and t h i s d i e t was used i n a l l succeeding experiments. I l l . Cholesterol/Cholate Feeding: In V i t r o Studies 1. The E f f e c t of the Cholesterol/Cholate-rich Diet on Body Weight, L i v e r  Weight and Plasma L i p i d Levels Rats fed the d i e t containing 5% c h o l e s t e r o l and 2% cholate for s i x days gained le s s weight (P < 0.05) than rats fed a normal diet (52 + 6 and 67 + 3 g respectively, mean + S.D., with 24 r a t s per group) although the amount of food consumed per r a t i n each group was the same. However, the average weight of the l i v e r s from cholesterol/cholate-fed rats was only s l i g h t l y l e s s (P < 0.05) than control values, (3.1 + 0.5 and 3.3 + 0.2 g r e s p e c t i v e l y ) . The protein content of cytosol and microsomes, as well as the recoveries of cytosols from homogenates, were s i m i l a r i n both groups. The cholesterol/cholate-fed r a t s had a 6-fold (p < 0.05) elevation of plasma c h o l e s t e r o l (631 +_ 78 compared to 99 + 18 mg/dl) and almost a 2-fold (P < 0.05) increase i n plasma phospholipid (243 ± 31 compared to 132 + 24 mg/dl). The e f f e c t s of t h i s d i e t on the enzymes of PC biosynthesis were subsequently investigated. 2. E f f e c t of Diet on Enzymes of Phosphatidylcholine Biosynthesis The c h o l e s t e r o l / c h o l i c acid d i e t had a marked e f f e c t on the c y t i d y l y l t r a n s f e r a s e from l i v e r (Table 20). A 2 to 3-fold stimulation i n t h i s enzyme a c t i v i t y i n cytosol and microsomes from experimental r a t s was observed whether enzyme a c t i v i t y was expressed per mg protein, per g l i v e r or per l i v e r . The a c t i v i t i e s of choline kinase, phosphocholinetransferase 93. Table 20 S p e c i f i c A c t i v i t i e s of Enzymes of Phosphatidylcholine  Biosynthesis from Rat Liver Cholesterol/Cholate Enzyme Diet Control Diet (nmol. min~l' mg~l) a Choline Kinase 5.6 + 0.3 4.5 ± 0.7 CTP:Phosphocholine c y t i d y l y l t r a n s f e r a s e C y t o s o l i c 1.5 + 0.4* 0.49 ± 0.06 Microsomal 2.7 + 0.3* 1.4 + 0.1 Phosphocholinetransferase 24 ± 1 27 ± 4 Phosphatidylethanolamine methyltransferase 0.12 + 0.01 0.13 + 0.02 a A c t i v i t i e s are ± S.D., n = 3 * P < 0.05 94. and phosphatidylethanolamine methyltransferase from l i v e r , however, were * s i m i l a r to control values. Subsequent studies were directed toward understanding the 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 . We investigated the 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 between the H- and L-forms by chromatography of fresh cytosol on Sepharose 6B ( F i g . 11) and showed that 25-30% of the t o t a l c y t o s o l i c enzyme from hypercholesterolemia rats was i n the H-form, compared to 10% for control r a t s . Thus, the c h o l e s t e r o l / c h o l i c a c i d d i e t resulted i n an increased aggregation as well as a stimulation of the 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 . 3• The E f f e c t of the Cholesterol/Cholate-rich Diet on the aggregation of Phosphocholine C y t i d y l y l t r a n s f e r a s e and D i a c y l g l y c e r o l Concentration i n Cytosol D i a c y l g l y c e r o l promotes aggregation of the c y t i d y l y l t r a n s f e r a s e i n cytosol from r a t l i v e r (26). Thus, the concentration of DG i n the cytosols were determined. The experimental rats had an elevated l e v e l (P < 0.05) of DG (4.25 and 4.23 compared to 1.66 and 1.41 nmoles/1 i n control r a t s as measured i n the 2 sets of pooled l i v e r cytosols from 4 control and 4 o cholesterol/cholate fed r a t s ) . A previous experiment had shown that trypsin-treated cytosol from cholesterol-fed r a t s caused an increase i n aggregation of the p a r t i a l l y p u r i f i e d L-form to the H-form (26). Together, these r e s u l t s suggest that the increased DG i n the cytosol from the experimentally fed rats was responsible for the increased concentration of H-form. 4. Studies on Phosphocholine C y t i d y l y l t r a n s f e r a s e i n Cytosols from Normal  and Cholesterol/Cholate-fed Rats The l i v e r s i s o l a t e d i n the usual way (see Materials and Methods section) contain a s i g n i f i c a n t amount of blood and since plasma phospholipids were increased i n the hypercholesterolemia animals, i t was 95. 2.8 r 0 8 1 6 2 4 Fraction Number Fig. 11. Chromatography of Cytosols from Normal Q and Choles- terol /Choiate-Fed (#) Rats. Four ml of freshly prepared cytosol were applied to a column of Sepharose 6B which had been equilibrated with 100 mM NaCl, 20 mM Tris-HCl, pH 7.0. Fractions (7 ml) were collected and assayed for cytidylyltransferase activity in the presence of 0.5 mg rat l iver phospholipid per 0.1 ml of assay mixture. The data shown were the averaged results obtained from three separate experi-ments'. 96. reasonable to think that plasma l i p i d s from r e s i d u a l blood i n the experi-mental l i v e r s could be responsible f o r stimulation of the c y t i d y l y l t r a n s -ferase. In a few experiments with cytosols prepared from l i v e r s perfused with i c e cold s a l i n e , the a c t i v 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 was s i m i l a r to the a c t i v i t y found i n cytosols prepared from normally i s o l a t e d l i v e r s . Thus plasma l i p i d s do not appear to be involved i n the stimulation of the enzyme. A c t i v 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 was determined i n cytosol which contained a large v a r i e t y of other enzymes. I t was conceivable that the higher a c t i v 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 observed i n c h o l e s t e r o l / cholate-fed r a t l i v e r c ytosol, as measured by the amount of CDP-choline formed i n the reaction mixture, was r e a l l y due to a slower rate of break-down of CDP-choline i n the experimental c y t o s o l . To te s t t h i s p o s s i b i l i t y , the conditions used i n the c y t i d y l y l t r a n s f e r a s e assay were duplicated, ex-cept that none of the usual substrates were used. Instead, 0.65 nmol of C-labelled CDP-choline, the amount normally produced i n the assay, were added to the reaction c o c k t a i l . The l a b e l l e d CDP-choline was not degraded i n the presence of the cytosols but 50% of the l a b e l was degraded when microsomes were used (Table 21). The unrecovered CDP-choline was most l i k e l y used by cholinephosphotransferase f o r PC synthesis. The recovery of r a d i o a c t i v i t y i n PC, phosphocholine and choline were not determined however. There are a number of possible explanations f o r the stimulation of the c y t i d y l y l t r a n s f e r a s e i n experimental cytosol and some of the p o s s i b i l i t i e s involve the presence or absence of an enzyme modulator, covalent modification of the enzyme or an increase i n the amount of enzyme molecules. These three p o s s i b i l i t i e s were explored i n the succeeding experiments. The presence or absence of a soluble low molecular weight modulator was f i r s t tested. Low molecular weight molecules were removed 97. Table 21 14 Degradation of C-labelled CDP-choline by Cytosol and Microsomes from Normal and Cholesterol/Cholate-Fed Rats dpm Remaining i n CDP-Choline Control Samples Experimental Samples Boiled Cytosol 36,808 34,664 Cytosol 36,443 37,565 Boiled Microsomes 38,580 39,417 Microsomes 20,052 21,765 The conditions used i n t h i s experiment are given i n the text. 98. from cytosols and the a c t i v i t i e s of the c y t i d y l y l t r a n s f e r a s e before and a f t e r u l t r a f i l t r a t i o n were measured. I f the s p e c i f i c a c t i v i t i e s of the enzyme i n con t r o l and experimental cytosols a f t e r u l t r a f i l t r a t i o n were sim-i l a r , then the stimulation of enzyme a c t i v i t y could be due to the presence of a small soluble modulator i n eit h e r of the cytosols. S p e c i f i c a c t i v i -t i e s of the enzyme i n both cytosols increased by about 2-fold a f t e r u l t r a -f i l t r a t i o n and t h i s was i n part due to the loss of 30% of t o t a l c y t o s o l i c protein; however, the u l t r a f i l t r a t e d cytosol from experimental animals s t i l l had 2 times more 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 control samples. When cont r o l and experimental cytosols were kept at 4°C, the a c t i v 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 was markedly stimulated. After three days, enzyme a c t i v i t y was maximally stimulated and the difference i n c y t i d y l y l -transferase a c t i v i t i e s i n control and experimental cytosols was e s s e n t i a l l y eliminated ( F i g . 12). The 2-fold d i f f e r e n c e i n enzyme a c t i v i t y observed i n fresh cytosol was reduced to a 10% difference a f t e r 3 days of incubation at 4°C. Previous studies have shown that the stimulation of c y t i d y l y l t r a n s -ferase a c t i v i t y during storage of cytosol at 4°C was probably due to a r i s e i n LPE (25). The increase i n LPE could be accounted for by a small amount of phospholipase A which was present i n c y t o s o l . In the present experi-ment, the stimulation 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 i e s i n control and experimental cytosols was most probably due to an increase i n the concen-t r a t i o n of LPE i n the cy t o s o l s . The differ e n c e i n the a c t i v i t i e s of the c y t i d y l y l t r a n s f e r a s e i n control and experimental cytosols observed i n fresh c y t o s o l was masked by the f i v e f o l d stimulation 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 . Since phosphoplipids are potent a c t i v a t o r s of the c y t i d y l y l t r a n s f e r a s e , cytosols from the two groups of rats were assayed f o r enzyme a c t i v i t y i n the presence of various amounts of phospholipid ( F i g . 13). At saturating 99. Time (Days) Figure 12. Activation of Phosphocholine Cytidylyltransferase Activ-ity in Control (1) and Experimental (#) Cytosols by incubation at 4 UC. 100. Concentration of Phospholipid (mg-ml-1) Figure 13. Activation of Phosphocholine Cytidylyltransferase in  Cytosol from Control (•) and Cholesterol/Choiate-Fed (#) Rats. The enzyme Tn cytosol (10 yl) was assayed in the presence of various amounts of phospholipid from rat l iver . 1 0 1 . l e v e l s of phospholipid, the difference i n c y t o s o l i c enzyme a c t i v i t i e s was eliminated. This r e s u l t implied that the amount of enzyme i n the two cytosols was the same and that an ac t i v a t o r of the c y t i d y l y l t r a n s f e r a s e was present i n the cytosol from c h o l e s t e r o l / c h o l i c acid-fed r a t s . Further studies were directed toward these two p o s s i b i l i t i e s . 5. Immunotitration of Phosphocholine C y t i d y l y l t r a n s f e r a s e The c y t i d y l y l t r a n s f e r a s e i n cytosol was t i t r a t e d with immunoglobulin from normal and immunized r a b b i t s . Some nonspecific i n h i b i t i o n of enzyme a c t i v i t y was observed when control gamma glo b u l i n G was incubated with c y t o s o l . This i n h i b i t i o n was probably due to the binding of phospholipid i n the cytosol by the gamma glo b u l i n since the i n h i b i t i o n was re l i e v e d by the addition of phospholipids to the assay tubes ( F i g . 14). Hence, 1 .6 mg of phospholipids was added to each assay tube for the immunotitration experiments. The c y t i d y l y l t r a n s f e r a s e was incubated with gamma globulin at d i f f e r e n t pH for 8 h at 4°C ( F i g . 15). The antigen-antibody complex was sedimented by centrifugation at 5,000 x g for 30 min. The i n h i b i t i o n of the cy 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 constant between pH 7.5 and 9.0 which indicates that the antibody-antigen complex was not formed by non-specific e l e c t r o s t a t i c i n t e r a c t i o n s . The amount of enzyme present i n control and c h o l e s t e r o l / c h o l i c acid-fed rat l i v e r cytosols was measured by immunotitration. Cyt o s o l i c protein (0.35 mg) was incubated with various amounts of immunoglobulins from normal and immunized r a b b i t s . After centrifugation the supernatant was c a r e f u l l y removed and assayed for c y t i d y l y l t r a n s f e r a s e a c t i v i t y ( F i g . 1 6 ) . The amount of antibodies required to p r e c i p i t a t e h a l f of the enzyme a c t i v i t y was the same for control and c h o l e s t e r o l / c h o l i c acid-fed rat l i v e r cytosol 102. Figure 14. Stimulation by Rat Liver Phospholipids of Phosphocholine  Cytidylyltransferase which Remained in the Supernatant after Immuno- precipitat ion. The incubation contained 0.6 mg of cytosolic protein and 2.2 mg 5? control (•) or anti-cytidylyltransferase (•) ant i -bodies. Other conditions were described in the Materials and Methods section. 103. PH Figure 15. Precipitation of Phosphocholine Cytidylyltransferase by  Control C ) and Anti-Cytidylyltransferase (#) Antibodies at Various  pH. The enzyme in 0.42 mg of cytosolic protein was reacted with 1.0 mg of antibodies and the amount of enzyme remaining in the super-natant was assayed for act iv i ty. All other conditions were described in the Materials and Methods section. 104. Amount of Immunoglobulin (mg) x 10* Figure 16. Immunotitration of Phosphocholine Cytidylyltransferase in  Cytosols from Control (p) and Cholesterol/Cholate-Fed (#) Rats with  Control (~—) and Anti-Cytidylyltransferase (—) antibody. Each cytosol was incubated with various amounts 67 immunoglobulins from normal and immunized rabbits. After 8 h incubation at 4°C in 60 mM sodium phosphate buffer, pH 8.0, the mixture was centrifuged at 5,000 x g for 30 min and the supernatant was assayed for cyt idyly l transfer-ase in the presence of 1.6 mg of phospholipid. 105. ( F i g . 17). I t appears that the two cytosols contain the same amount of enzyme. The amount of enzyme found i n l i v e r cytosols from r a t s fed the basal atherogenic d i e t was t i t r a t e d i n the same way with another preparation of antibodies and a s i m i l a r r e s u l t was obtained. 6. Characterization of the Modulator of Phosphocholine C y t i d y l y l t r a n s f e r a s e The immunotitration experiments showed that the stimulation of the c y t i d y l y l t r a n s f e r a s e was not due to an increase i n enzyme protein and the experiments which involved the 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 by phospholipids suggested that a phospholipid modulator was responsible f o r the stimulation of the enzyme. I f t h i s were indeed the case, then elimination of phospholipids from the cytosols followed by r e a c t i v a t i o n with an i d e n t i c a l preparation of phospholipids should abolish the difference i n enzyme a c t i v i t i e s . Experimentally, phospholipids i n the co n t r o l and experimental cytosols were digested with phospholipase C and 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 i e s i n both cytosols were completely l o s t a f t e r 1 h at 37°C ( F i g . 18). The a c t i v 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 experimental cytosol appear to be more s e n s i t i v e to phospholipase than enzyme a c t i v i t y i n control c y t o s o l . The attempts to reactivate the phospholipase treated enzymes, however, were unsuccessful and the i d e n t i t y of the enzyme modulator remained unresolved. I f the modulator were a phospholipid, then p r o t e o l y t i c treatment of the cytosols should not damage the modulator and i t s e f f e c t s should s t i l l be observed upon readdition of p a r t i a l l y p u r i f i e d L-form. Rat l i v e r cytosol was incubated with t r y p s i n which eliminated a l l 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 . After t r y p s i n i n h i b i t o r was added, the same amount of delipidated form or p a r t i a l l y p u r i f i e d L-form of the enzyme was added to the two cytosols and twice as much a c t i v i t y was detected with the 106. Figure 17. Immunochemical Quantitation of Cytosolic Phosphocholine Cytidylyltransferase from Normal Q and Cholesterol/Choiate-Fed Q rats. See legend to Figure 16. 107. Time (min) Figure 18. Effect of Phospholipase C (CI. welchii) Digestion on the  Act iv i ty of Phosphocholine Cytidylyltransferase in Cytosols from  Normal Q and Cholesterol/Choilate-Fed Q Rats. Control and experi-mental cytosols (1 ml) were incubate^ with 10 mM CaCl2 and 2 units (umol/min) of phospholipase C at 37°C. At various times, aliquots of the cytosols were removed and phospholipase C activity was inh ib i -ted by 30 mM EGTA. The aliquots were kept at 4°C until the cy t idy l -yltransferase activity was measured. 108. cytosol from the experimental r a t s . The s p e c i f i c a c t i v i t i e s of the added enzymes though varied from one preparation to another. This experiment indicated the presence of a t r y p s i n - r e s i s t a n t a c t i v a t o r . The inactivated cytosols were chromatographed on Sepharose 6B and the f r a c t i o n s assayed i n the presence of p a r t i a l l y p u r i f i e d L-form. An a c t i v a t o r eluted near the void volume and the cytosol from c h o l e s t e r o l / c h o l i c acid-fed r a t s contained twice as much as the c o n t r o l cytosol ( F i g . 19). When the f r a c t i o n s that contained the a c t i v a t o r were extracted with CHClyci^OH (2/1), a l l the a c t i v a t o r was recovered i n the organic phase (Table 22). The r e s u l t s strongly suggested that the a c t i v a t o r i n cytosol i s a l i p i d . A larger amount of c y t o s o l i c l i p i d was prepared from a 25% (NH^^SO^ extract of t r y p s i n - i n a c t i v a t e d cytosol and greater than 90% of c y t o s o l i c phospholipid was recovered by t h i s procedure. The l i p i d extract from cholesterol/cholate-fed rats had more phospholipid (P < 0.05) than the l i p i d extract from control rats [110 + 53 (S.D.) and 56 + 20 (S.D.) ug/ml of cytosol as measured i n 6 d i f f e r e n t batches of cytosols] and was more e f f e c t i v e i n stimulation of the c y t i d y l y l t r a n s f e r a s e ( F i g . 20). The classes of phospholipids present i n the cytosols were determined i n two experiments and the composition was s i m i l a r i n both groups (Table 23). In order to determine which phospholipid i n cytosol was responsible for the 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 , a portion of each extract was fractionated by t h i n layer chromatography on s i l i c a g e l . L i p i d s were eluted and areas on the plate with no detectable l i p i d were also extracted. The l i p i d s were added to assay, tubes, the solvent evaporated and a c t i v i t y of p a r t i a l l y p u r i f i e d L-form was determined (Table 24). In the experiment using a neutral solvent system ( I ) , l i p i d from only one area 109. 5 . 0 p . o U T L U H 2.5 Fraction Number Figure 19. Chromatography of Trypsin-Treated Cytosol from Control  C) and Chblesterol/Cholate-Fed (» ) Rats. Four ml cytosol (which had been incubated with 0.1 mg trypsin/ml for 15 min at 37°C) from con-trol and cholesterol-fed rats was applied to a column of Sepharose 6B and 7 ml fractions were collected. Part ial ly purified L-form of the cytidylyltransferase was assayed in the presence of 40 yl of each fract ion. Table 22 Stimulation of Delipidated C y t i d y l y l t r a n s f e r a s e by the Water-Soluble  and Lipid-Soluble Components of Column Fractions Which Contained the Activator C y t i d y l y l t r a n s f e r a s e (nmol/min) Column Fractions from Control Cytosol Water-Soluble Fraction 0 Lipid-Soluble Fraction 0.021 Water-Soluble and Lipid-Soluble Fractions 0.023 Column Fractions from Experimental Cytosol Water-Soluble Fraction 0 Lipid-Soluble Fraction 0.074 Water-Soluble and Lipid-Soluble Fractions 0.050 The amount of water- and l i p i d - s o l u b l e materials obtained from column fr a c t i o n s were used to stimulate 20 y l of delipidated enzyme. 111. Amount of Cytosolic Lipid Extract ( u l ) Figure 20. Stimulation of Part ia l ly Purified Cytidylyltransferase by  Lipid Extracts from Control Q and Cholesterol/Choiate-Fed (#) Rat  Liver Cytosol. The l ip id was extracted from cytosol as described in the Materials and Methods section. Lipids from equivalent volumes of the cytosols were added to assay tubes, the organic solvent evaporated and the act iv i ty of the enzyme was determined. The concentrations of phospholipid in the extracts were 0.43 and 0.93 mg/ml for the control and cholesterol/cholate-fed group, respectively. 112. Table 23 Composition of Phospholipids i n Cytosols from  Normal and Hypercholesterolemia Rats Amount of Phospholipid (ug ml c y t o s o l - 1 ) Controls Experimental Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Lysophosphatidylethanolamine 18 (63) 6 (20) 2 (7) 2.8 (10) 73 (74) 13 (13) 6 (6) 6.9 (7) L i p i d s from control and experimental cytosols were fractionated by 2-dimensional t h i n - l a y e r chromatography (solvent system IV). Numbers i n brackets i n d i c a t e the mole percent of the phospholipid with respect to the t o t a l amount 113. Table 24 Stimulation of P a r t i a l l y P u r i f i e d L-form by Cytosolic Phospholipids  from Normal and Hypercholesterolemia Rats Solvent System I Solvent System II CHCl3/CH3OH/H20 CHI3/CH3OH/CH3COOH/H2O (70/30/4) (100/60/16/8) Enzyme A c t i v i t y (nmol/min) X10l Experi- Experi-RF Control mental Rf Control mental No Addition _ 0.33 0.33 _ 0.10 • 0.10 PC 0.27 0.16 0.27 0.32 0.28 0.58 PE 0.59 0.32 0.34 0.77 0.12 0.22 LPC 0.13 0.32 0.34 0.10 - -LPE 0.31 0.72 0.88 0.41 0.20 0.27 SP 0.13 O r i g i n 0-0.05 0.22 0.26 - - -Unknowns 1 0.18 0.30 0.27 0.45 '0.13 0.17 2 0.21 0.11 0.19 0.50 0.13 0.21 3 0.36 0.35 0.38 0.66 0.13 0.13 4 0.81 . 0.12 0.11 - - -Spaces A 0.35-0.51 0.13 0.10 0-0.29 0.07 0.15 B 0.57 0.28 0.28 0.39-0.48 0.11 0.14 C - - - 0.51-0.63 0.08 0.10 D - - - 0.68-0.74 0.12 0.19 E - — - 0.79-1.0' 0.03 0.06 L i p i d s from 2 ml of control and 1 ml of experimental cytosols were chromatographed with solvent system I while the l i p i d s from 1.5 ml of each cytosol were used i n another experiment with solvent system I I . The d e t a i l s were described i n the Materials and Methods section. Unknowns r e f e r to un i d e n t i f i e d bands which were detected by iodine s t a i n i n g while the spaces r e f e r to areas on the plate which did not contain any iod i n e - p o s i t i v e material. 114. on the plate stimulated the p a r t i a l l y p u r i f i e d enzyme and t h i s l i p i d chromatographed with LPE. In t h i s experiment l i p i d s from 2 ml of c o n t r o l and 1 ml of experimental cytosols were used and when t h i s difference was accounted f o r , the l i p i d a c t i v a t o r from experimental cytosol was 2 times more active than co n t r o l i n stimulating the added enzyme (1.43 and 0-72 nmol/min for the experimental and con t r o l l i p i d s , r e s p e c t i v e l y ) . This experiment was repeated with another set of cytosols except that l i p i d s from an equivalent amount of normal and experimental cytosols were used and an a c i d i c solvent system (II) was employed. The l i p i d s that chromatographed with PC and LPE were most act i v e stimulators of the enzyme. Previous studies showed that PC does not activa t e the c y t i d y l y l t r a n s f e r a s e (25) and the stimulation observed i n t h i s experiment could be due to contamination with LPE. Phosphatidylethanolamine and an unknown l i p i d band from experimental c y t o s o l were more active i n stimulating the enzyme than the corresponding l i p i d bands from control cytosols and there were no r e a d i l y obvious explanations for these r e s u l t s . The recovery from s i l i c a of the a b i l i t y of the c y t o s o l i c l i p i d s to stimulate the c y t i d y l y l t r a n s f e r a s e was determined (Table 25) and 70% or more was l o s t whether the neutral or a c i d i c solvent system was used. The low recoveries made i t d i f f i c u l t to i n t e r p r e t the r e s u l t s given i n Table 24. Results from a previous study suggested that LPE might be a p h y s i o l o g i c a l l y important modulator of the c y t i d y l y l t r a n s f e r a s e (25)-Thus, the stimulation of enzyme a c t i v i t y by palmitoyl-LPE before and a f t e r spotting on and e l u t i o n from s i l i c a was tested. Standard palmitoyl-LPE did not s i g n i f i c a n t l y stimulate 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, the same l i p i d which had been eluted from s i l i c a was very much more potent i n stimulating the enzyme ( F i g . 21). This s u r p r i s i n g r e s u l t could be 115. Table 25 Recovery of the A b i l i t y of Cytosolic L i p i d s to Stimulate C y t i d y l y l - transferase A c t i v i t y After Thin-Layer Chromatography on S i l i c i c Acid Stimulation 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 (nmol* min-1) Applied Recovered (percent recovered) Solvent System I Control Experimental Solvent System II Control Experimental 0.17 0.98 0.14 0.40 0.04 (23%) 0.06 (6%) 0.04 (31%) 0.12 (30%) Recovery was calculated by the sum of the a b i l i t y of a l l the l i p i d components detected by iodine s t a i n i n g to activate the c y t i d y l y l t r a n s f e r a s e . The a b i l i t y to stimulate enzyme a c t i v i t y was determined from the difference 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 i e s i n the presence and absence of l i p i d . The other d e t a i l s of the experiments were given i n Table 24. 116. 1 4 r 0 3 0 6 0 1 0 0 Amount of Lysophosphatidylethanolamine (ug) Figure 21. Stimulation of Phosphocholine Cytidylyltransferase Act iv - ity by Standard Palmitoyl-Lysophosphatidylethanolamine and by the  Same Lipid Which Had Been Applied on and Eluted from S i l i c a . Various amounts of palmitoyl-LPE were used to stimulate cytidylyltransferase act iv i ty . The same amounts of this l ip id were applied on a TLC plate ana immediately extracted from s i l i c a with CHCI3/CH3OH (2/1). The eluted l ip id (•) and stock LPE (•) were subsequently used to stimulate enzyme activity and cytidylyltransferase activity was measured as described in the Materials and Methods section. 117. explained i n a number of ways but the most l i k e l y p o s s i b i l i t y appears to be degradation of LPE during the spotting and e l u t i o n procedure. This idea was supported by r e s u l t s from an experiment where eluted and stock palmitoyl-LPE were analyzed by TLC. The stock l i p i d contained a major component (Rf 0.16); presumably LPE, and a minor component (Rf 0.34) (Table 26). In contrast, LPE which had been eluted from s i l i c a resolved i n t o 5 components. Two bands chromatographed i n the same area as LPE and three other components, which had high m o b i l i t i e s , were also detected (Table 26). I t appears that a portion of LPE was degraded during e l u t i o n with CHCl^/CH^OH (2/1) and a degradation product may be responsible for stimulation 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 . The i d e n t i t y of the a c t i v a t o r remains unresolved at the present time. There i s no convincing evidence which in d i c a t e s that LPE i s the a c t i v a t o r and i n f a c t , the d i f f e r e n c e i n the e f f e c t of e l u t i o n from s i l i c a on the stimulation 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 by c y t o s o l i c l i p i d s and LPE suggest that the a c t i v a t o r i s not a l y s o l i p i d . Furthermore, i t i s apparent that s i l i c i c a c i d chromatography i s not s u i t a b l e f or use i n further experiments and various other means of f r a c t i o n a t i o n need to be employed before the i d e n t i t y of the a c t i v a t o r can be c l e a r l y established. 7. Analysis of Palmitoyl-lysophosphatidylethanolamine by Mass Spectrometry In the course of i d e n t i f y i n g the a c t i v a t o r of the c y t i d y l y l t r a n s f e r a s e , mass spectrometry was used to determine whether the a c t i v a t o r and LPE had s i m i l a r chemical structures. Phospholipids had been previously analyzed by f i e l d desorption (114) and electron i o n i z a t i o n (110) mass spectrometry. By f i e l d desorption mass spectrometry, palmitoyl- as well as oleoyl-LPE show fragments which appear to correspond to phosphoethanolamine, M-RC00 and 118. Table 26 Analysis of Eluted and Stock Palmitoyl-lysophosphatidylethanolamine by Thin Layer Chromatography Number of Bands Rf's Eluted LPE 6 0.14, 0.17, 0.32, 0.75, 0.82, 0.90 Stock LPE 2 0.16, 0.34 (minor) •Standard palmitoyllysophosphatidylethanolamine (0.20 mg) was applied on a TLC plate and was immediately eluted from s i l i c a by CHCI3/CH3OH (2/1). The eluted l i p i d was applied to a TLC plate, along with stock l i p i d , and developed i n CHCI3/CH3OH/H2O (65/25/4). The l i p i d s were subsequently v i s u a l i z e d by iodine s t a i n i n g . 119. glycerophosphorylethanolatnine as well as an intense M + H peak (114). F i e l d desorption mass spectrometry was the method of choice but because i t was not r e a d i l y a v a i l a b l e , electron i o n i z a t i o n mass spectrometry was used instead. Lysophosphatidylethanolamine was made v o l a t i l e by s i l y l a t i o n and a t y p i c a l spectrum i s shown i n F i g . 22. The molecular ion was not found but several c h a r a c t e r i s t i c ions formed from the f a t t y acid and ethanolamine moieties were evident. Fragment 174 was i n d i c a t i v e of ethanolamine (110) while fragments 211 and 239 appear to be derived from palmitic acid (Table 27). Fragments 627, 538, 449 and 385 may be useful f o r i d e n t i f i c a t i o n but not fragment 357 which was found i n the spectra of various other s i l y l a t e d derivatives of phospholipids (110). Several attempts were made to analyze LPE i s o l a t e d from cytosol by mass spectrometry and i n every instance, the spectrum obtained was very weak and was not s u i t a b l e f o r a n a l y s i s . And i n l i g h t of the r e s u l t s which showed that LPE was p a r t i a l l y degraded during e l u t i o n from s i l i c a , t h i s experiment was terminated. 8. The Fatty Acid Composition and the Amount of Lysophosphatidyl-ethanolamine i n Cytosol The nature of the f a t t y a c i d of LPE markedly a f f e c t s i t s a b i l i t y to stimulate the c y t i d y l y l t r a n s f e r a s e (25). Hence, the f a t t y acid residues from LPE were analyzed and were found to be s i m i l a r i n both groups of rat s - 70% C l 6 - Q and 30% C-^ .Q f a t t y acids. The t o t a l amount of LPE recovered from c h o l e s t e r o l / c h o l i c acid-fed rat l i v e r cytosol was greater than the amount i s o l a t e d from control cytosol (6.9+ 0.6 and 2.8 ± 0.8 yg/ml cytosol, r e s p e c t i v e l y ) . The s i g n i f i c a n c e of the elevation of LPE i n experimental cytosol remains to be established m/e Figure ??.. Mass Spectra of the Silylated Derivative of Palmitoyl Lysophosphatidylethanol- amine. The sample was prepared by s i ly lat ing 0.25 mg of standard paImitoyl-LPE and analysis was performed in an AEI-902 Mass Spectrometer. Other conditions are described in the Materials and Methods section. 121'. Table 27 Structure and Predicted Fragments of the S i l y l a t e d Derivative of  Palmitoyl-lysophosphatidylethanolamine M/E Predicted Structure 0 II 741 H 2C-0-C-(CH 2)i4-CH 3 TMS-O-CH I o < II H2C-0-P-0-CH2CH2N(TMS)2 0 I TMS 627 M-ll4(CHCH 2NTMSi) 611 M-115(CH 2CH2NTMSi)-15(-CH 3) 555 M-186 538 M-l88(CH 2CH 2N(TMSi) 2)-15(CH 3) 449 M-203-90(HO-TMS) 0 II 385 H 2C-0-C-(CH 2) 1n-CH 3 TMS-O-C-H I CH 2 371 M-356-l4(CH 2) (cont'd) M/E Predicted Structure 341 357-16 0 II 239 CH 3(CH 2)i4C 211 CH 3(CH 2)i4 174 CH 2N(TMS) 2 146 (147) CH2-0 TMS-O-CH CH 2 103 TMS-0-CH2 123. 9. Lysophosphatidylethanolamine In Cytosol was not Generated During the  Preparation of Subcellular Fractions The LPE found i n the cytosol could possibly be generated i n v i t r o during the preparation of the cytosol and two experiments were done to t e s t t h i s p o s s i b i l i t y . I t i s well established that a c t i v i t y of phospholipases i s dependent on the presence of C a + + ions. Thus, control and cholesterol/cholate-fed r a t l i v e r cytosols were prepared i n the presence of 10 mM EDTA and 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 i e s were measured and found to be 0.41 and 0.81 nmol/min/mg, respectively, not d i f f e r e n t from the a c t i v i t i e s found i n cytosols prepared without EDTA (Table 20). 14 The second experiment involved the a d d i t i o n of C-labelled PE, i n the form of PE/rat l i v e r phospholipid (1/1; w/w) sonicated v e s i c l e s , to the tissue and s a l i n e p r i o r to homogenization. Of the 23,000 dpm per ml homogenate added, approximately 12,500 dpm were recovered i n PE per ml of cytosol, and i n both co n t r o l and experimental l i p i d extracts, no r a d i o a c t i v i t y was found to be associated with LPE a f t e r f r a c t i o n a t i o n of the phospholipids by 2-D chromatography. The unrecovered r a d i o a c t i v i t y was most probably associated with v e s i c l e s which were sedimented during the preparation of c y t o s o l . I t i s possible that the added PE v e s i c l e s may be poor substrates for t i s s u e phospholipase A when compared to endogenous PE. Nevertheless, the r e s u l t s strongly indicated that LPE found i n cytosol was not generated i n v i t r o . IV. 'Cholesterol/Cholate Feeding: In Vivo Studies The _in v i t r o studies showed that the a c t i v 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 , when measured under optimal conditions, was stimulated 2-fold i n the l i v e r s of r a t s fed a high c h o l e s t e r o l / c h o l i c acid d i e t and the stimulation of enzyme a c t i v i t y was not due to an increase i n enzyme protein but to an a c t i v a t i o n of the enzyme by a l i p i d . The studies up to t h i s point were l i m i t e d to experiments with s u b c e l l u l a r f r a c t i o n s and the actual status of PC synthesis i n the i n t a c t l i v e r remains unclear. 124. The i i i vivo studies were designed to co r r e l a t e the 2-fold stimulation of the 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 with the rate of PC biosynthesis i n vivo. The rate of biosynthesis i n l i v e r was measured by monitoring the 3 r a t e of incorporation of [Me- H]choline i n t o PC and the following sections describe the r e s u l t s . 1. The Amount of Radioactivity i n Choline, Phosphocholine, CDP-Choline, Betaine and Phosphatidylcholine at Several Time Periods After . I n t r a p o r t a l I n j e c t i o n of Labelled Choline Radioactive choline was introduced i n t o the rat s by the p o r t a l vein as described i n the Materials and Methods sec t i o n . In the f i r s t experiment, 0.5 mM choline was in j e c t e d ( F i g . 23) and as expected, r a d i o a c t i v i t y incorporated i n t o the various pools. The amount of r a d i o a c t i v i t y i n choline reached a peak before 60 sec and t h i s was followed by maximum incorporation of r a d i o a c t i v i t y into phosphocholine and betaine s h o r t l y a f t e r . R a d i o a c t i v i t y i n phosphocholine increased very r a p i d l y and reached a maximum value within 1 min a f t e r i n j e c t i o n . The r a d i o a c t i v i t y i n phosphocholine remained almost unchanged between 1 and H min a f t e r i n j e c t i o n and the rate of PC biosynthesis was measured within t h i s time 3 period i n a l a t e r experiment. [Me- H]choline was incorporated i n t o PC i n a l i n e a r manner and h a l f of the t o t a l i n j e c t e d choline was oxidized to betaine while a n e g l i g i b l e amount (0.1%) was incorporated i n t o the CDP-choline pool. 3 When [Me- H]choline was used i n these experiments, there were two possible ways i n which r a d i o a c t i v i t y could be incorporated into PC. Aside from the d i r e c t incorporation of l a b e l l e d choline by the de novo pathway, r a d i o a c t i v i t y could also be introduced v i a the PE methylation pathway. Methyl l a b e l l e d S-adenosylmethionine could be made i n vivo from l a b e l l e d 125. Time (min) Figure 23. Incorporation of [Me-^HjCholine into Hepatic Pools of  Choline (A), Phosphocholine (M), CDP-Choline (+) Betaine (A) a~nd~  Phosphatidylcholine Q . " A 0T26 ml solution of labelled choline (0.5 mM, 360 yCi/ymol) was injected intraportally into control rats and the amount of radioactivity incorporated into various pools was de-termined as described in the Materials and Methods section. 126. methionine which could be synthesized by methylation of homocysteine with [Me-^H]betaine as methyl donor. Therefore, to eliminate any possible incorporation of r a d i o a c t i v i t y into PC by the PE methylation pathway, 14 C-backbone l a b e l l e d choline was used i n the next experiment. The incorporation of the i n j e c t e d l a b e l into l i v e r s from control and hypercholesterolemia r a t s at the time of sampling varied between 30 and 50%. At 30 sec a f t e r i n j e c t i o n , r a d i o a c t i v i t y i n choline and betaine i n the experimental group was only h a l f that found i n controls, while the incorporation of r a d i o a c t i v i t y into phosphocholine and CDP-choline was markedly increased i n the experimental l i v e r s . In addition, incorporation of r a d i o a c t i v i t y into PC was three times higher i n the experimental group (Table 28). These r e s u l t s suggested that hepatic PC biosynthesis was stimulated i n the experimental animals which appear p r e f e r e n t i a l l y to channel injected choline into phospholipid synthesis. The concentration of choline i n the i n j e c t i o n s o l u t i o n could a f f e c t the uptake of choline into the l i v e r . Under normal circumstances, the concentration of choline i n r a t serum was between 3.6 and 21.4 yM (115). Therefore, i n order to simulate i n vivo conditions as c l o s e l y as possible, choline concentration i n the i n j e c t i o n s o l u t i o n was adjusted to between 50 and 100 yM i n a l l subsequent experiments. Commercially a v a i l a b l e 14 C-labelled choline had very low s p e c i f i c r a d i o a c t i v i t y and was not used i n any further experiments. Instead, [Me- H]choline which had a much higher s p e c i f i c a c t i v i t y was used. 2. An Estimate of the Rate of Phosphatidylcholine Biosynthesis i n L i v e r s  from Control and Cholesterol/Cholate-fed Rats The rate of PC biosynthesis was determined by c a l c u l a t i n g the rate of conversion of phosphocholine to PC. A 100 y l s o l u t i o n of 60 yM 127. Table 28 The Incorporation of [1,2- l l tC]Choline into Various Compounds i n the L i v e r s from Control and Cholesterol/Cholate-Fed Rats. Incorporated Radioactivity (dpm/Liver) x 10-i* Phospho- CDP- Phosphatidyl-Choline choline choline Betaine choline Control 70 90 0.7 168 7.2 Experimental 39 170 8.6 82 21 Each of a group of 3 control and 3 experimental rats was injected with a 0.25 ml s o l u t i o n of 4.5 mM [ 1 ^ - l ^ C j c h o l i n e ( 3 uCi/umol) containing 3 - 3 uCi of r a d i o a c t i v i t y . Both groups incorporated between 30 and 50% of the t o t a l injected dose and the amount of r a d i o a c t i v i t y incorporated into various pools was determined 30 sec post i n j e c t i o n . 128., [Me- H]choline was i n j e c t e d i n t r a p o r t a l l y into control and experimental rats and the amount of r a d i o a c t i v i t y incorporated into phosphocholine, PC and other r e l a t e d compounds at 1, 2 and 3 min p o s t - i n j e c t i o n were measured (Table 29 and 30). L i v e r s from both groups incorporated about h a l f of the inje c t e d dose and the same proportion of the incorporated r a d i o a c t i v i t y was found i n betaine at a l l time points. In contrast, when 4.5 mM choline was used i n the i n j e c t i o n s o l u t i o n as i n a previous experiment (Table 28), the experimental group had only h a l f the amount of r a d i o a c t i v i t y i n betaine when compared to the control group. The data seem to suggest that the proportion of exogenous choline oxidized to betaine i n the l i v e r depends on the rate of hepatic PC synthesis as well as on the abundance of choline. Control and experimental r a t s incorporated r a d i o a c t i v i t y into choline and phosphocholine i n a s i m i l a r manner at 1 and 2 min (Table 29). Incorporation of r a d i o a c t i v i t y into PC was elevated by about 2-fold i n the experimental rats at 1 and 2 min. At 3 min, however, the control data had very large experimental error and i t was d i f f i c u l t to determine i f a r e a l d i f f e r e n c e existed between co n t r o l and experimental values. Therefore, the rate of PC biosynthesis was determined between 1 and 2 min. An estimate of the rate of PC synthesis i n r a t l i v e r was calculated from the s p e c i f i c r a d i o a c t i v i t y of phosphocholine and the net amount of r a d i o a c t i v i t y incorporated i n t o PC between 1 and 2 min p o s t - i n j e c t i o n . The rates calculated i n t h i s way were d i r e c t l y comparable since the percent of the injected dose incorporated into the l i v e r s of control and experimental rats were s i m i l a r . The c a l c u l a t i o n of the rate was based on several assumptions. F i r s t , the rate of PC synthesis was assumed to be equal to the rate of conversion of phosphocholine to PC. Second, the s p e c i f i c r a d i o a c t i v i t y of CDP-choline and PC formed by the de novo pathway i s equal 129. Table 29 Incorporation of Radioactivity into Various Choline-Containing Metabolites  After Intraportal Injection of 60 u.M [Me-%jCholine Amount of Radioactivity Time (DPM x 10"6 L i v e r - 1 ) (min) Phosphatidyl-Choline Phosphocholine choline Control . 1 9.4 + 2.8 (7) 36.0 + 14.6 (7) 0.98 0.29 (7) 2 4.4 + 2.3 (8) 31.4 + 12.5 (8) 3.04 ± 1.93 (8) 3 3.2 + 2.3 (7) 27.0 + 11.0 (7) 7.2 + 7-2 (7) Experimental 1 11.5 + 3.3 (7) 35.4 + 12.1 (7) 1.82 ± 0.82 (7) 2 3.1 ± 1.6 (6) 31.6 + 12.6 (6) 8.53 + 2.47 (6) 3 5.4 + 4.5 (8) 33.0 11.3 (8) 8.4 + 3.4 (8) Brackets indicate the number of animals used. Values are given as mean +_ S.D. 130. Table 30 Incorporation of Radioactivity into Betaine and the Percent of Injected  Dose Incorporated After I n t r a p o r t a l Injection  of 60 yM [Me- 3H]Choline Percent Radioactivity Time of Dose i n Betaine (min) Incorporated (dpm x 10 _6 Liver) Control 1 66 + 15 (7) 25.5 + 7-8 (7) 2 51 + 12 (8) 17.3 + 5.0 (8) 3 53 +_ 21 (8) 18.0 + 10 (8) Experimental 1 65 + 17 (7) 23-4 + 6.3 (7) 2. 54 + 20 (6) 16.4 + 6.6 (6) 3. 59 + 16 (8) 20.0 + 7-7 (8) Percent of dose incorporated was calculated by d i v i d i n g the sum of the t o t a l amount of r a d i o a c t i v i t y incorporated into the upper and lower phases of the Bligh and Dyer extraction by the amount of r a d i o a c t i v i t y i n j e c t e d . The numbers i n brackets indicate the number of animals used. Values are given as mean + S.D. 131. to that of phosphocholine. And l a s t , within t h i s same time period, the incorporation of r a d i o a c t i v i t y into PC by the phospatidylethanolamine methylation pathway was assumed to be n e g l i g i b l e . Estimates of the rates of PC biosynthesis and the parameters used i n the c a l c u l a t i o n s are given i n Table 31• The concentration of phosphocholine as well as the amount of r a d i o a c t i v i t y i n the pool as expressed per l i v e r was s i m i l a r i n both groups. Thus, the calculated s p e c i f i c r a d i o a c t i v i t y of phosphocholine was also s i m i l a r . From the rate of incorporation of r a d i o a c t i v i t y into PC and the s p e c i f i c r a d i o a c t i v i t y of phosphocholine, the rates of synthesis were calc u l a t e d . The estimated rate of PC biosynthesis i n rats fed the high cholesterol/cholate d i e t was three times higher than i n the controls and t h i s correlated well with the 2- to 3-fold stimulation 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 observed i n v i t r o . 132. Table 31 The E f f e c t of Cholesterol/Cholate Feeding on the Rate  of Phosphatidylcholine Biosynthesis Control Treated Phosphocholine concentration (nmoles l i v e r " 1 ) 3,574 + 1,052 (22) 3,522 + 988 (21) Average Radioactivity i n Phospho-choline (dpm x l O " 6 l i v e r " 1 ) 33.7 (15) 33.5 (13) S p e c i f i c Radioactivity (dpm x 10"3 x nmole - 1) 9.4 9.5 Rate of Incorporation of Radioactivity i n t o PC (dpm l i v e r " 1 min" 1 x 10" 6) 2.1 (15) 6.7 ( 13 ) Rate of PC Biosynthesis (nmoles l i v e r " ! min - 1) 223 705 Each r a t was in j e c t e d with 50 uCi of [Me3H]choline and the incorporation of r a d i o a c t i v i t y into phosphocholine and phosphatidylcholine was measured. Control r a t s had s l i g h t l y larger l i v e r s than experimental r a t s (3.2 +0.5 and 2.7+0.4 g per l i v e r r e s p e c t i v e l y ) . The numbers i n brackets i n d i c a t e the number of animals used. 133. DISCUSSION I • Phosphatidylcholine Biosynthesis i n Rats Fed a Normal and  Cholesterol/Cholate-Rich Diet A l i n e a r r e l a t i o n s h i p between plasma c h o l e s t e r o l and phospholipids suggested that the metabolism of cholesterol and phospholipids i n the l i v e r , where the majority of blood l i p o p r o t e i n s are synthesized, may be c l o s e l y r e l a t e d . Induction of hypercholesterolemia by dietary means resul t e d i n elevated l e v e l s of phospholipids i n the plasma while i n f u s i o n of phosphatides to r a i s e the l e v e l of plasma phospholipids resulted i n increased l e v e l s of plasma c h o l e s t e r o l (see Introduction). On the basis of these observations, PC biosynthesis was investigated i n r a t s fed a c h o l e s t e r o l / c h o l a t e - r i c h d i e t . Phosphatidylcholine biosynthesis was perturbed i n hypercholesterolemia r a t s . The observed e f f e c t s were s p e c i f i c a l l y induced by the intake of c h o l e s t e r o l and cholate and not by any other n u t r i t i o n a l factors since the control and experimental d i e t s have very s i m i l a r c a l o r i c content and composition. Results from the present i n v e s t i g a t i o n indicate that young rats have a higher rate of PC synthesis than adults. The estimated rate of PC biosynthesis i n control r a t s (Table 31) was s i m i l a r to rates determined 134. by various other workers (117). However, the rate per g l i v e r obtained i n the present study with young r a t s , was almost 3-times higher than published values for adult r a t s (116). This f i n d i n g , however, was i n agreement with a previous observation that l i v e r s l i c e s from 25-day old r a t s incorporated 50 to 100$ more radioactive choline i n t o phosphatidylcholine than l i v e r s l i c e s from adult r a t s (117). Together, the evidence indicated that the rate of PC biosynthesis per unit weight of l i v e r was indeed markedly influenced by development. Growing ra t s require more phospholipids, not only for assembly of plasma l i p o p r o t e i n s , but also for the synthesis of new membranes i n the l i v e r (118). The l e v e l of plasma phospholipids was increased i n the cholesterol/cholate-fed r a t s . This increase was most probably the r e s u l t of the 2- to 3-fold stimulation i n the rate of hepatic PC biosynthesis. An e a r l i e r study showed that PC's i n the l i v e r and plasma are i n rapid equilibrium (9) and suggested that increased synthesis may indeed by responsible for the hyperphospholipidemia. I I . Measurement of the Rate of Phosphatidylcholine Biosynthesis In experiments where the rate of PC biosynthesis was measured, 2 d i f f e r e n t concentrations of choline were used i n the i n j e c t i o n s o l u t i o n and i n both cases, r a t s fed the high cholesterol/cholate d i e t incorporated 2-to 3-times more r a d i o a c t i v i t y than r a t s fed the control d i e t . The manner i n which choline was u t i l i z e d i n the l i v e r s , however, depended upon the concentration of choline i n the i n j e c t i o n mixture. When 60 yM choline was used, the amount of r a d i o a c t i v i t y incorporated i n t o betaine and phosphocholine was s i m i l a r i n both groups (Tables 29 and 30). In contrast, when the concentration of choline i n the i n j e c t i o n s o l u t i o n was 4.5 mM, the amount of r a d i o a c t i v i t y i n phosphocholine was several f o l d higher and the 135. r a d i o a c t i v i t y i n betaine was 50% lower i n the hypercholesterolemia r a t s when compared to control (Table 28). The data i l l u s t r a t e d an aspect of regulation which may be important i n vivo. When the rate of PC biosynthesis was stimulated, as during hypercholesterolemia, excess choline which was oxidized to betaine i n normal l i v e r s , was converted to phosphocholine and committed for PC biosynthesis. The fate of choline i n l i v e r appeared to be regulated by the requirements of PC biosynthesis and the supply of choline. The p r e f e r e n t i a l channeling of l a b e l l e d choline into phosphocholine and PC has been observed i n d i e t h y l s t i l b e s t r o l - t r e a t e d roosters which have an elevated rate of biosynthesis (119) and also i n hepatocytes when the culture medium contained l e s s than 20 uM choline (120). The mechanism which determines the fate of choline i n l i v e r remains an important subject for future research. Results from experiments with 4.5 mM choline i n the i n j e c t i o n s o l u t i o n suggested that the a c t i v i t i e s of choline kinase and phosphocholine c y t i d y l y l t r a n s f e r a s e were dramatically increased i n the experimental animals. The amount of r a d i o a c t i v i t y i n phosphocholine and CDP-choline was increased by 2- and 20-fold respectively i n the hypercholesterolemia rats (Table 28). The a c t i v i t y of choline kinase i n v i t r o , however, was not increased while 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 only increased 2- to 3-fold i n the experimental cytosol (Table 20). The apparent stimulation of phosphocholine synthesis at high i n t r a c e l l u l a r concentrations of choline remains unclear at present. An increase i n the l e v e l of choline i n the c e l l , would not r e s u l t i n stimulation of choline kinase a c t i v i t y since the normal i n t r a c e l l u l a r concentration of choline (Table 1) i s about seven times the Km of choline kinase for choline (Table 2). 136. The method used for the estimation of the rate of PC biosynthesis was subject to several l i m i t a t i o n s and the assumptions used i n the c a l c u l a t i o n have been discussed previously (see Results). There was considerable v a r i a t i o n i n the amount of r a d i o a c t i v i t y incorporated into betaine and the various choline-containing intermediates (Table 29) i n both groups of rats even though i d e n t i c a l conditions were used throughout the whole study. This v a r i a t i o n could be explained by several f a c t o r s . F i r s t , the concentration of choline i n the portal veins probably d i f f e r e d from one rat to another and consequently, the d i l u t i o n of the injected isotope would vary and second, v a r i a t i o n i n the metabolism of the animals may also be a contributing f a c t o r . I l l • Enzymes of Phosphatidylcholine Biosynthesis i n Normal and  Hypercholesterolemia Rats. Rats fed the high cholesterol/cholate d i e t had a 2- to 3-fold stimulation of the a c t i v i t y of hepatic phosphocholine c y t i d y l y l t r a n s f e r a s e when compared to controls (Table 20). The a c t i v i t i e s of the other enzymes in the de_ novo pathway as well as the a c t i v i t i e s of the PE-N-methyltransferases, however, were unchanged and these r e s u l t s provided evidence that dietary c h o l e s t e r o l and cholate does not cause a general stimulation of enzymes i n cytosol or microsomes but that the d i e t s p e c i f i c a l l y affected the a c t i v 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 . Phosphocholine c y t i d y l y l t r a n s f e r a s e i s the rate l i m i t i n g enzyme i n the de novo synthesis of PC during hypercholesterolemia. The 2- to 3-fold stimulation of PC synthesis i n the experimental group was s p e c i f i c a l l y the r e s u l t of a 2- to 3-fold increase i n 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 . The suggestion by Infante (see Introduction) that choline kinase was also r a t e - l i m i t i n g i n the de_ novo pathway was not consistent with the 1-37. r e s u l t s from the present study where choline kinase a c t i v i t y and the concentration of phosphocholine were not increased i n the l i v e r s from cholesterol/cholate-fed r a t s (Tables 20 and 3 1 ) . And even i f the phosphocholine pool was increased, i t i s d i f f i c u l t to understand how the o v e r a l l rate of the pathway could be stimulated when the concentration of phosphocholine i n normal l i v e r s i s more than f i v e times the Km of the c y t i d y l y l t r a n s f e r a s e for phosphocholine (Tables 1 and 3 ) . The data show that choline kinase i s not r a t e - l i m i t i n g during hypercholesterolemia. The enzyme, however, may be involved i n the regulation of the fate of choline in l i v e r . Fresh r a t l i v e r cytosol from cholesterol/cholate-fed r a t s had 2.5 times more H-form than control cytosol ( F i g . 11) and t h i s was correlated with a 2.5-fold increase i n the concentration of DG i n the experimental c y t o s o l . These r e s u l t s agreed with a previous study which showed that DG was the only l i p i d from r a t l i v e r that aggregated the c y t i d y l y l t r a n s f e r a s e (26). The c y t i d y l y l t r a n s f e r a s e i s found both i n cytosol and microsomes, and the soluble a c t i v i t y was resolved into 2 forms on the basis of molecular s i z e . Studies with antibodies raised against the p u r i f i e d L-form showed that the H-form, L-form as well as the microsome associated enzyme were immunologically i d e n t i c a l (74) and strongly suggested that a l l the c y t i d y l y l t r a n s f e r a s e i n r a t l i v e r was one and the same. In vivo, i s the enzyme i n the cytoplasm and on the endoplasmic reticulum of the c e l l or i s the c y t i d y l y l t r a n s f e r a s e e x c l u s i v e l y membrane bound? There i s no evidence to support either p o s s i b i l i t y at the present but various circumstantial evidence seems to support the l a t t e r hypothesis. When rat l i v e r was homogenized i n s a l i n e , the majority of the c y t i d y l y l t r a n s f e r a s e was found i n the c y t o s o l . In contrast, most of the enzyme a c t i v i t y was found i n the 138. microsomal f r a c t i o n when d i s t i l l e d water was used while an intermediate d i s t r i b u t i o n between microsomes and cytosol was obtained when 0.25 M sucrose was used (19). This suggested that the c y t i d y l y l t r a n s f e r a s e i s loosely bound to the endoplasmic reticulum in_ vivo and homogenization of the t i s s u e i n s a l i n e or sucrose promoted s o l u b i l i z a t i o n of the enzyme. The H-form i s a heterogeneous population of enzyme aggregates which possess a wide range of molecular weights (Table 3) and they appear to be formed by random aggregation of L-form with no apparent stoichiometry. The composition of phospholipids i n cytosols from normal and hypercholesterolemia r a t s (Table 23) was very s i m i l a r to the composition of phospholipids i n microsomes where the mole per cent of PC, PE, PS and p i are 73, 19, 3 and 4 r e s p e c t i v e l y (121). These observations suggested that the s o l u 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 which has a natural a f f i n i t y f o r membranes, probably randomly aggregate on to cytomembrane components which are present i n 100,000 x g supernatant (122). In the present study, the cytosol from cholesterol/cholate-fed r a t s contain 2-fold more l i p i d phosphorus and DG than control cytosol and the l i p i d s are most probably associated with membrane fragments. The increase i n the amount of H-form i n the experimental c y t o s o l , therefore, could very well be the r e s u l t of increased amounts of membrane fragments. The H- and L-form are probably a r t i f a c t s produced during the preparation of s u b c e l l u l a r f r a c t i o n s and the c y t i d y l y l t r a n s f e r a s e most l i k e l y e x i s t s as one species which i s associated with the endoplasmic reticulum in_ v i v o . However, the p o s s i b i l i t y that the enzyme e x i s t s i n a free and membrane bound form in vivo has not been excluded. The stimulation of phosphocholine c y t i d y l y l t r a n s f e r a s e i n experimental cytosol was investigated. The a c t i v 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 was measured by determination of the amount of CDP-choline formed i n the 139. assay. An apparent stimulation 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 would occur i f CDP-choline was more rap i d l y degraded i n control c y t o s o l . The rates of degradation of CDP-choline i n control and experimental cytosols were s i m i l a r (Table 21) and the assay does t r u l y r e f l e c t a stimulation 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 i n experimental c y t o s o l . The stimulation 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 could be explained i n a number of ways. F i r s t , the amount of enzyme protein could be increased i n the experimental c y t o s o l . Second, the amount of enzyme was unchanged but a more active species of enzyme was present i n the experimental cytosol and t h i r d , a modulator of the c y t i d y l y l t r a n s f e r a s e could be present i n or absent from either of the cytosols. The amount of enzyme i n con t r o l and experimental cytosols was s i m i l a r as determined by immunotitration experiments ( F i g . 17) and the regulation of PC biosynthesis does not appear to involve an adaptive change i n the concentration of the 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 s from hypercholesterolemia r a t s . This r e s u l t i s i n l i n e with findings from experiments with c h o l i n e - d e f i c i e n t l i v e r s where the a c t i v 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 was one h a l f of that i n controls and immunotitration studies showed that the quantity of enzyme was unchanged. The p o s s i b i l i t y that an enzyme modulator was responsible for the stimulation of the c y t i d y l y l t r a n s f e r a s e was explored. A t r y p s i n - i n s e n s i t i v e a c t i v a t o r which i s soluble i n CHCl^/CH^OH (2/1; v/v) was found i n the cytosols. The a c t i v a t o r appeared to be a l i p i d and there was twice as much ac t i v a t o r i n the experimental cytosol as i n controls. The presence of a l i p i d a c t i v a t o r i s consistent with r e s u l t s from studies with lung and l i v e r which showed that the c y t i d y l y l t r a n s f e r a s e was strongly stimulated by phospholipids. 140. Several attempts were made to i d e n t i f y the act i v a t o r by TLC (see Results) and t h i s method was found to be unsuitable. When l i p i d extracted from cytosol was fractionated on s i l i c i c acid plates more than 10% of the act i v a t o r was l o s t (Table 25). In d i r e c t contrast, however, palmitoyl lysophosphatidylethanolamine, which was implicated as a potent a c t i v a t o r of the enzyme (25), was activated by spotting and elu t i o n from s i l i c a ( F i g . 21). The r e s u l t s indicated two things: f i r s t , the data suggested that the act i v a t o r of the c y t i d y l y l t r a n s f e r a s e was not LPE i n view of the e f f e c t of s i l i c a and second, another technique needed to be employed i n future studies before the i d e n t i t y of the ac t i v a t o r can be established. IV. The Regulation of the De Novo Pathway for Phosphatidylcholine  Biosynthesis The regulation of de novo synthesis of PC i s complex and every enzyme in the pathway i s involved. The fate of choline as i t enters the l i v e r c e l l appears to be determined, at le a s t i n part, by choline kinase, the f i r s t enzyme of the pathway. The proportion of the t o t a l incoming choline that i s converted to phosphocholine and thus committed for PC biosynthesis, i s dependent upon the rate of PC biosynthesis and the supply of choline. The second enzyme of the pathway, phosphocholine c y t i d y l y l t r a n s f e r a s e , determines the o v e r a l l rate of the pathway as demonstrated i n the present study ( F i g . 24). The f i r s t committed step i n a pathway which involves a dissociated enzyme system i s normally the rate l i m i t i n g reaction whereas i n a multienzyme complex, i t has been proposed that any reaction i n the pathway can be r a t e - l i m i t i n g (123). A multienzyme complex composed of a l l three enzymes of the de novo pathway has not been demonstrated, but the c y t i d y l y l t r a n s f e r a s e and phosphocholinetransferase have been shown to be situated i n the cytoplasmic side of the endoplasmic reticulum (124). 141. Figure 24. Regulation of the Rate of Phosphatidylcholine Biosyn- thesis. The de novo synthesis of PC is i l lustrated as an assembly line process where the cytidylyltransferase is the slowest worker in the chain (Frame 1 ) . An alteration in the activity of the cy t idy l y l -transferase (Frames 2 and 3) would result in a change in the overall rate Df production of the assembly l ine. 142. Although the s p a t i a l r e l a t i o n s h i p of the two enzymes on the membrane has not been investigated, i t i s tempting to speculate that the c y t i d y l y l t r a n s f e r a s e and phosphocholinetransferase are associated i n some manner which f a c i l i t a t e s the u t i l i z a t i o n of CDP-choline by phosphocholinetransferase. This management would prevent wasteful accumulation of intermediates, as i n a multienzyme complex (123), and the concentration of CDP-choline i n vivo (Table 1) seem to support the idea that some physical r e l a t i o n s h i p e x i s t s between the c y t i d y l y l t r a n s f e r a s e and phosphocholinetransferase. The e f f e c t s of various metabolites on the a c t i v 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 suggest that the rate of PC biosynthesis may be s e n s i t i v e to the energy state of the c e l l . 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 i n h i b i t e d by NAD and NADP (Table 17) which are metabolites i n d i c a t i v e of low energy while enzyme a c t i v i t y was stimulated by NADH and NADPH. I t seems l o g i c a l that the c e l l would not synthesize more PC when energy i s scarce and would permit synthesis where energy i s adequate. The e f f e c t s of the nucleotides on the c y t i d y l y l t r a n s f e r a s e (Table 17), however, were more d i f f i c u l t to understand. I n h i b i t i o n of enzyme a c t i v i t y by nucleoside triphosphates was d i f f i c u l t to r a t i o n a l i z e and i t remains to be established whether the e f f e c t s on PC biosynthesis of the various metabolites used i n the present study have any r e a l s i g n i f i c a n c e in_ v i v o . The mechanism by which the rate of PC biosynthesis i s stimulated during cholesterol/cholate feeding of rats remains unclear at the present but r e s u l t s from various studies suggest that a hormonal mechanism i s probably involved. There are two main l i n e s of evidence. F i r s t , d i e t s containing high f a t or high carbohydrate have been shown to markedly a l t e r the l e v e l of glucagon i n the blood (125) and second, the incorporation of l a b e l l e d choline into PC was affected by various hormones (see Introduction). A recent study with f e t a l r at lung i n organ culture (126) showed that the incorporation of l a b e l l e d choline into PC was stimulated 2-fold by aminophylline, a c y c l i c AMP phosphodiesterase i n h i b i t o r , and also by c y c l i c AMP. In another study, the t o t a l amount of PC and disaturated PC as well as the incorporation of l a b e l l e d choline into these f r a c t i o n s were increased i n alv e o l a r type II c e l l s treated with c y c l i c AMP (127). Various analogs of c y c l i c AMP increased phosphatidylcholine l e v e l s and the degree of stimulation by the d i f f e r e n t compounds was correlated to t h e i r a b i l i t y to activate protein kinase (127). The data suggested that a protein phosphorylation mechanism may be involved. The a c t i v i t i e s of choline kinase and phosphocholinetransferase i n f e t a l lung explants were not s i g n i f i c a n t l y affected by aminophylline (126). In contrast, phosphocholine c y t i d y l y l t r a n s f e r a s e was increased i n f e t a l rabbit lung when pregnant rabbits were treated with c o r t i c o s t e r o i d s (128) and some of the e f f e c t s of t h i s hormone have been suggested to be mediated by c y c l i c AMP (129). Together, the evidence suggest that a hormonal mechanism-which involves c y c l i c AMP as second messenger may be involved i n c o n t r o l l i n g the rate of PC biosynthesis and phosphorylation i s one way by which the a c t i v i t y of phosphocholine c y t i d y l y l t r a n s f e r a s e may be regulated. The l a s t enzyme of the de novo pathway i s phosphocholinetransferase and i t determines the degree of unsaturation of PC by s e l e c t i v e l y u t i l i z i n g c e r t a i n DG. The phosphocholinetransferase i s a p o t e n t i a l point for regulation even though the enzyme appears to poorly u t i l i z e disaturated species of DG i n lung where dipalmitoyl PC i s required for surfactant synthesis (see Introduction). Nevertheless, i t i s conceivable that the s p e c i f i c i t y of the enzyme could be regulated so that DG's having c e r t a i n 144. saturated f a t t y acids on C - l or unsaturated f a t t y acids on C-2 may be p r e f e r e n t i a l l y u t i l i z e d . Whether the species of f a t t y acids on PC are alter e d during hypercholesterolemia remains to be determined. V. Suggestions for Further Studies There are several areas i n the present study which warrant further i n v e s t i g a t i o n . F i r s t and foremost, the modulator responsible for the 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 i n cytosol from cholesterol/cholate fed rats should be i d e n t i f i e d . Subsequent investigations should be directed ultimately towards e l u c i d a t i o n of the chain of events which lead to the 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 and stimulation of PC synthesis. These studies could also y i e l d some insi g h t into the re l a t i o n s h i p between the regulation of cho l e s t e r o l and PC biosynthesis and may f a c i l i t a t e i n the understanding of l i p o p r o t e i n synthesis i n the l i v e r . The mechanism which determines the fate of choline i n l i v e r also warrants further study. The various isozymes of choline kinase may very l i k e l y be involved i n t h i s process. The l i v e r synthesizes PC's which are exported as b i l e or blood l i p o p r o t e i n s and how PC synthesis i s regulated to f u l f i l l the various requirements for export and for the needs of the l i v e r remains to be investigated. 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