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Rat hepatic phosphatidylethanolamine N-methyltransferase : enzyme purification and characterization Ridgway, Neale David 1988

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RAT HEPATIC PHOSPHATIDYLETHANOLAMLNE W-METHYLTRANSFERASE: ENZYME PURIFICATION AND CHARACTERIZATION  by  Neale David Ridgway B.Sc.  (1982), M.Sc.(1985), Dalhousie University  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Biochemistry)  We accept this thesis as conforming to the required  standards  THE UNIVERSITY OF BRITISH COLUMBIA May,  1988  © Neale Ridgway, 1988  In  presenting  degree  this  at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  publication of  thesis for by  his  or  her  an advanced  Library shall make it  It  is  granted  by the  understood  that  head  of  copying  my or  this thesis for financial gain shall not be allowed without my written  Department of The University of British Columbia Vancouver, Canada  DE-6 (2788)  representatives.  for  agree that permission for extensive  scholarly purposes may be  permission.  Date  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  ^UlAAJL  Z/ffgZ  ii  Thesis Phosphatidylethanolamine  Abstract  (PE)  N -methyltransferase  catalyzes  stepwise transfer of methyl groups from S-adenosyl-L-methionine the amino headgroup of P E . the  two  intermediates,  Successive methylation  phosphatidylcholine  (PC).  protein  localized  primarily  fraction)  of  PE-,  PE  the  PMME-  purified  fold,  respectively.  and  from  methyltransferase indicated  Triton  X-100  The purified  (in the  methyltransferase of  Triton  rat  liver  is  (PMME)  the  an  final  integral  reticulum  enzyme  and  product membrane  (microsomal  N-methyltransferase  enzyme  was composed of  activities  1542-  a single  and  832-  18.3  kDal  Molecular mass analysis of purified P E micelles)  existed  as  a  by  gel  24.7  filtration  kDal  N-  on Sephacryl S-  monomer.  PE  N -  catalyzed the complete conversion of P E to P C and had a p H  10 for  PE  PE  solubilized microsomes 429-,  X-100  all three steps.  developed to assay P E - , P M M E microsomal  and  endoplasmic  PDME-dependent  protein as determined by S D S - P A G E .  optimum  to  liver.  were  300  (PDME),  AT-methyltransferase in  (AdoMet)  results in the formation of  phosphatidyl-N-monomethylethanolamine  p h o s p h a t i d y 1-AT, Af-dimethylethanolamine  the  Triton X-100  mixed micelle assay was  and PDME-dependent activities of both pure and  N-methyltransferase.  P E N-methyltransferase  A  The and  amino  AT-terminal  the  recently  acid  cloned  sequence of  23.1  kDal  S.  cerevisiae P E M 2 were found to be 35% homologous. Double reciprocal plots for P E N-methyltransferase concentrations  and  increasing P E , P M M E  Similar cooperative effects X-100  increased.  or  PDME  at  were  fixed  Triton  highly  were noted when phospholipid was fixed  The cooperativity could be partially  X-100  cooperative. and Triton  abolished if a fixed mol%  of nonsubstrate phospholipid such as P C was included in the assay.  This would  iii indicate that P E /V-methyltransferase  has specific  binding  requirements  for a  site(s) in contact with the micellar substrate.  The occupation of this boundary  layer  expression  by phospholipid  Kinetic  analysis  ordered  Bi-Bi  PE  is essential  revealed  that  mechanism.  to a common site  for full  PMME  and P D M E  The overall  and successive  o f enzyme  methylation  mechanism involves methylation  followed  initial  steps involving  involved  (which  are rapidly  in the catalytic  Reverse-phase  the binding  HPLC  experiments  in the absence  of reduced  Cysteine thiols) are  mechanism.  of P E /V-methyltransferase specificity  oxidized  an  binding of  and release of AdoMet and S-adenosyl-L-homocysteine, respectively. residue(s)  activity.  was used to fractionate into  individual  the phospholipid products  molecular  on P E N - m e t h y l t r a n s f e r a s e  species.  Substrate  in vitro and in vivo  revealed no selectivity for any molecular species of diacyl P E , P M M E or P D M E . The PE-derived P C , which is rich in 16:0-22:6, is rapidly remodeled to conform to the molecular species compositon of total hepatocyte P C The  18.3 kDal P E N-methyltransferase  cAMP-dependent phosphorus/mol  protein of  P E Af-methyltransferase  observed effect  on activity.  choline-deficient  rat liver  levels  of cellular P E .  hepatocyte no  membranes  alteration  indicated that activity  Immunoblotting with  a anti-PE  in enzyme mass.  by substrate  However, was  only  incorporated,  Studies on P E N-methyltransferase  While  are that hepatic P E N-methyltransferase primarily  was found to be a substrate for  in vitro.  kinase  in vivo .  and product  N-methyltransferase  is  levels.  with  regulation  no in  changes were due to elevated  of choline-deficient  more  0.25 m o l  liver microsomes or antibody  revealed  work is needed, initial indications a  constitutive  enzyme  regulated  iv  Table of Contents Page  ii  Abstract List of Tables  viii  List of Figures  ix  List of Abbreviations  xii  Acknowledgements  xiv  Introduction 1.1.  The Centrality  1.2.  Phosphatidylcholine Structure  2  1.3.  Phosphatidylcholine Synthesis  2  1.3.1. 1.3.2. 1.3.3.  4 5 7  1.4.  of Phospholipids to Cell Function  CDP-Choline Pathway Base Exchange Reacylation of Lysophosphatidylcholine  Synthesis of Phosphatidylcholine by Phosphatidylethanolamine Methylation 1.4.1. Structure of Phosphatidyethanolamine  1  8 8  1.5.  Origins of Phosphatidylethanolamine for Methylation 1.5.1. The CDP-Ethanolamine Pathway 1.5.2. Phosphatidylserine Decarboxylation 1.5.3. Acylation of Lysophosphatidylethanolamine  11 11 12 12  1.6.  Characterization of Phosphatidylethanolamine N - M e t h y l t r a n s f e r a s e . . . 1.6.1. Elucidation of the Phosphatidylethanolamine Methylation Pathway 1.6.2. Tissue- and Subcellular-Localization 1.6.3. Topology in Membranes 1.6.4. Molecular Structure and Kinetics 1.6.5. Purification 1.6.6. Phosphatidylethanolamine Molecular Species Specificity 1.6.7. Substrate Specificity  13 13 14 16 17 19 21 22  1.7.  Functions of Hepatic Phosphatidylethanolamine AT-Methyltransferase.23 1.7.1. Contribution to Net P C Synthesis 23 1.7.2. A s a Source of Phosphatidylcholine in Lipoproteins 23 1.7.3. Does Phosphatidylethanolamine N-Methyltransferase Supply the Hepatocyte With Polyunsaturated Phosphatidylcholine? 24  1.8.  Regulation of P E N-Methyltransferase in Liver 1.8.1. Regulation by Substrate Levels 1.8.2. Hormonal Effects in Liver 1.8.3. Hormonal Regulation in the Adipocyte 1.8.4. Other Effectors  25 25 27 29 30  V 1.8.5. 1.8.6. 1.9.  1.10.  1.11.  Developmental Regulation Coordinate Regulation with the  Phosphatidylethanolamine N-Methyltransferase in Eukaryotic Microorganisms 1.9.1. Phosphatidylethanolamine Methylation in Yeast 1.9.2. Phosphatidylethanolamine Methylation in Bacteria 1.9.3. Other Organisms  31 31 33 35  Phosphatidylethanolamine Signaling  36  and  Transmembrane  39  Procedures  2.1.  Materials  2.2.  Phosphatidylethanolamine  40 AT-Methyltransferase  Purification  40  2.2.1. 2.2.2. 2.2.3. 2.2.4. 2.2.5. 2.2.6. 2.2.7.  Isolation of Microsomes Preparation of Microsomal Membranes Solubilization of Microsomal Membranes Chromatogaphy on Whatman DE-52 Cellulose Chromatography on Whatman P - l l Phosphocellulose Chromatography on Octyl Sepharose C L - 4 B Chromatography on P B E 94  40 41 41 41 42 42 43  Assay  of  45  2.3.1.  Assay using [ m e f / t y / - H ] S - A d e n o s y l - L - M e t h i o n i n e  45  2.3.2. 2.3.3.  Assay using [^HJPhosphatidylethanolamine Analysis of Products by Thin-Layer Chromatography  46 46  2.3.4.  Preparation of [l-3H]Ethanolamine Labeled ethanolamine Repurification of 5 - A d e n o s y l - L - M e t h i o n i n e  48 48  2.3.5. 2.4.  Methylation  Rationale and Objectives of the Present Study  Experimental  2.3.  30 30  CDP-Choline Pathway  Phosphatidylethanolamine  Af-Methyltransferase  3  Phosphatidyl-  High Performance Liquid Chromatographic Analysis of Phospholipids 2.4.1. Preparation of Radiolabeled Phosphatidylethanolamine Methyltransferase Products 2.4.2. High Performance Liquid Chromatographic Separation Phospholipid Molecular Species  2.5.  SDS and Two-Dimensional Gel Electrophoresis  2.6.  Purification of Microsomal Phosphatidylcholine  Phosphatidylethanolamine  2.7.  Protein and Phosphorus Assays  2.8.  Animal Maintenance and Hepatocyte Culturing 2.8.1. Animals and Diet 2.8.2. Preparation and Culturing of Primary Monolayers Hepatocytes  48 N48 of 49 50  and 51 51 52 52 of  Rat 52  vi  2.9.  Immunochemistry 2.9.1. Immunoblotting 2.9.2. Immunodetection of Phosphatidylethanolamine / / - M e t h y l transferase on Nitrocellulose Membranes 2.9.3. Production of Anti-Phosphatidylethanolamine N-Methyltransferase IgG 2.9.4. Purification of Rabbit Plasma IgG Fraction  54 54 54 55 56  2.10.  Phosphorylation of Phosphatidylethanolamine N-Methyltransferase...56 2.10.1. Enzyme Phosphorylation In Vitro 56 2.10.2. Analysis of Phosphoamino Acids by Thin-Layer Electrophoresis 57 2.10.3. Effect of Phosphorylation on Enzyme Activity In Vitro 58  2.11.  Photolabeling of in Microsomes  2.12.  2.13.  Phosphatidylethanolamine  N-Methyltransferase 58  TV-Terminal Amino Acid Sequence Analysis of Phosphatidylethanolamine ^-Methyltransferase  59  Data Analysis  59  Results 3.1.  Phosphatidylethanolamine N-Methyltransferase Purification and Molecular Properties 3.1.1. Purification 3.1.2. Analysis of Methylated Products 3.1.3. pH Optima for Methylation 3.1.4. Molecular Mass Determination by SDS-PAGE 3.1.5. Molecular Mass Determination by Gel Filtration 3.1.6. Two-Dimensional Gel Analysis 3.1.7. Immunoblotting of Microsomes and Purified Enzyme  60 60 62 62 66 66 70 70  3.2.  Kinetics of Phosphatidylethanolamine N-Methyltransferase 3.2.1. Analysis of Micellar Substrates 3.2.2. Methylation Adheres to the 'Surface Dilution' Model 3.2.3. Effects of Mixed Micelle Composition and Concentration 3.2.4. Effect of Phosphatidylcholine on Initial Velocities  70 70 73 75 76  3.3.  Kinetic Mechanism of Purified Phosphatidylethanolamine Methyltransferase 3.3.1. Evaluation of the Kinetic Pathway 3.3.2. Kinetic Constants 3.3.3. Free Sulfhydryls are Required for Methylation  81 81 93 94  3.4.  3.5.  N-  Photoaffinity Labeling of Microsomal Phosphatidylethanolamine Methyltransferase 3.4.1. Photolabeling of Microsomes 3.4.2. Photolabeling of a 19 kDal Methyltransferase Specificity  of Phosphatidylethanolamine  ^-Methyltransferase  N97 97 97 101  vii 3.5.1. High Performance Liquid Chromatographic Separation of Phospholipids 3.5.2. In Vitro Molecular Species Specificity 3.5.3. Methylation Rates with Synthetic Phosphatidylethanolamines 3.5.4. Molecular Species Specificity In Vivo 3.6.  In Vitro Phosphorylation  3.7.  Phosphatidylethanolamine N-Methy ltransferase in Choline- and Methionine-Deficient Rat Liver 3.7.1. Effect of Choline Deficiency on Enzyme Activity and Mass 3.7.2. Activity and Mass in Choline- and Methionine-Deficient Rat Hepatocytes  3.8.  of Phosphatidylethanolamine NMethyltransferase by cAMP-Dependent Protein Kinase 3.6.1. Phosphorylation and Phosphoamino Acid Analysis 3.6.2. Stoichiometry of Phosphorylation and Effect on Activity  101 104 104 107 Ill Ill 113 117 117 119  N-Terminal Sequence Analysis of Phosphatidylethanolamine NMethy ltransferase  124  Discussion  4.1.  Purification and Molecular Properties  4.2.  Kinetic Properties of Phosphatidylethanolamine N-  125  Methy ltransferase  131  4.3.  Molecular Species Specificity In Vitro and In Vivo  135  4.4.  In Vitro Phosphorylation  4.5.  Methy ltransferase Phosphatidylethanolamine Methionine-Deficiency  Af-Methyltransferase in Choline- and  Conclusions  Considerations  References  and Future  of Phosphatidylethanolamine  N-  139 142 146 148  viii  Legend  to  Tables  Tables 1. Cell and Tissue Distribution of P E N-Methyltransferse 2.  Page 15  Compilation of Reports For and Against P E in Cell Signaling Events  Methylation  3.  Purification of P E N-Methyltransferase  Rat  4.  Distribution of Methylated transferase Purification  37  From  Phospholipids During  Liver  Microsomes  61  P E AT-Methyl63  5.  Types of Co-Substrate and Product Inhibition Patterns Observed for P E , P M M E and P D M E Methylation by Purified P E //-Methyltransferase 92  6.  Kinetic Constants for P E TV-Methyltransferase  7.  Inhibition of P E AT-Methyltransferase Reagents  8.  by  94 Sulfhydryl-Modifying 95  M o l % Distribution of Microsomal P C , Microsomal P E and Egg P C Molecular Species  103  9.  % Distribution of Labeled P C and P D M E Molecular Species Synthesized by P E N-Methyltransferase In Vitro 105  10.  Distribution of [mefAy/- H]Methionine-Labeled During a 24 Hour Chase  PC  11.  Specific Activity  Molecular  12.  Activity of P E N-Methyltransferase in Endoplasmic Reticulum (Microsomes) from Choline-Deficient (CD) and Choline-Supplemented (CS) Rat Livers 118  13.  Phospholipid Levels in Choline-Deficient (CD) Supplemented (CS) Rat Liver Homogenates  3  of Hepatocyte  [m e thy I- H]PC 3  Molecular  Species 108  Species  Ill  and Choline118  ix Legends  to  Figures  Figure  Page  1.  P C Biosynthetic Pathways  3  2.  Synthesis of P C by P E Methylation  9  3.  P E Biosynthetic Pathways  4.  Octyl-Sepharose C L - 4 B  10  Chromatography  of P E A T - M e t h y l -  transferase  44  5.  Time Course for Methylation of P E , P M M E and P D M E  47  6.  Analysis of Products Formed During the Time Course of P E , P M M E and P D M E Methylation p H Curves for the Methylation of P E , P M M E and P D M E by Purified P E Af-Methyltransferase  64  7. 8.  9.  Electrophoresis of Partially transferase  Purified  and Purified  65  PE Af-Methyl67  Sephacryl S-300 Chromatography  of Purified  P E N-  Methyltransferase  68 and 69  10.  Two-Dimensional  Gel Electrophoresis of P E N-Methyltransferase  11.  Immunoblot  12.  Analysis of Micellar  13.  Surface Dilution of P E , P M M E and P D M E Methylation  14.  Lack of Influence of Micelle  15.  Cooperative Methylation Concentration  of P E N-Methyltransferase  in  Microsomes  71 72  Substrates by Gel Filtration  74 Activities  Concentration on P D M E Methylation  74 77  at Fixed P M M E and Increasing Triton X-100 77  16.  Cooperativity  of PE-Dependent  17.  Cooperativity  of PMME-Dependent  18.  Cooperativity  of PDME-Dependent  19.  Influence of Egg P C on P E , P M M E and P D M E Initial Velocity Curves  82  20.  Influence of Egg P C on InversePDME  83  21.  Methylation Methylation Methylation  78 79 80  and Hill-Plots for P E , P M M E and  Double Reciprocal Plots of Initial Methylation Rates Versus AdoMet at Several Fixed Concentrations of P M M E and P D M E  85  X 22.  23.  24.  25.  Double Reciprocal Plots of Initial Methylation Rates Versus P M M E and P D M E at Various Fixed Concentration of AdoMet  86  Double Reciprocal Plots of Initial P E Methylation Various AdoMet Concentrations  86  Rates Versus  Inhibition of P E , P M M E and P D M E Methylation by AdoHcy at Variable AdoMet  87  Inhibition of P E , P M M E and P D M E Methylation by AdoHcy at Fixed and Saturating AdoMet  89  26.  Dixon Plots Showing Co-Inhibition of P M M E and P D M E Methylation  90  27.  Inhibition of P E Methylation by P M M E and P D M E  91  28.  A  Concerted Kinetic Mechanism for P E Methylation  93  29.  Activation of P E , P M M E and P D M E Methylation by D T T  96  30.  Photolabeling of Microsomal Proteins with [ m e f / i y / - H ] A d o M e t Identification by S D S - P A G E 3  and 98  31.  Time Course of 19 kDal Protein Photolabeling  99  32.  Inhibition of  99  33.  Distribution of [mef/iy/- H]AdoMet Tissues  34.  35.  36.  37.  38.  39.  19 kDal Protein Photolabeling by AdoMet and AdoHcy 3  Labeled Proteins  in  Extrahepatic 100  Separation of Microsomal P E , Microsomal P C and Egg P C by ReversePhase H P L C  102  Rates of Methylation for Various PEs Using Purified P E NMethyltransferase  106  H P L C Profiles of [mef/iy/- H]Methionine-Labeled Cellular Molecular Species During a 24 Hour Chase Period 3  Phosphorylation of Purified and Partially Purified transferase by cAMP-Dependent Protein Kinase Phosphoamino A c i d Analysis of Methyltransferase  32  P-Labeled PE  PE  PC 110  ^-Methyl112  N114  Two-Dimensional Gel Analysis of Phosphorylated P E Methy Itransferase  N-  Phosphorylation  115  40.  Time Course of P E ^-Methyltransferase  41.  Immunoblot of P E N-Methyltransferase in Choline-Deficient Choline-Supplemented Rat Liver Microsomes  In  Vitro  116  and 120  xi 42.  43.  44.  45.  Effect of Choline Supplementation on P E /V-Methyltransferase Activity in Choline- and Methionine-Deficient Hepatocytes  121  Effect of Methionine Supplementation on P E N - M e t h y l t r a n s f e r a s e Activity in Choline- and Methionine-Deficient Hepatocytes  122  Immunoblot Analysis of P E /V-Methyltransferase in Deficient and Methionine-Supplemented Hepatocyte  123  MethionineHomogenates  TV-Terminal Amino Acids of Purified Rat Liver P E NMethyltransferase  124  46.  Hypothetical Catalytic Mechanism for P E Methylation  127  47.  Intramolecular Transalkylation of Nitro-Methylsulfoniumphenylethane  127  l-(2-Methoxycyclopentyl)-2-/? -  xii List AdoEt AdoHcy AdoMet A ATP BSA cAMP CDP CHO Ci CHAPS CMC CoA cpm CTP kDal DEAE DME dpm DTNB DTT DZA EDTA EGTA Fig. g g h Hjj HEPES HPLC IAA K 1 lysoPC lysoPE m M MEM min MME NEPHGE Nonidet P40 m  PAGE PC PDME Ref.  of Abbreviations  S-adenosyl-L-ethionine S-adenosyl-L-homocysteine S-adenosyl-L-methionine ampere adenosine triphosphate bovine serum albumin adenosine 3', 5'-monophosphate cytidine diphosphate Chinese hamster ovary curie 3-[(3-cholamidopropyl)dimethylammonio]-lpropanesulfate critical micellar concentration coenzyme A counts per minute cytidine triphosphate kilodalton diethylaminoethyl N.N-dimethylethanolamine disintegrations per minute 5,5'-dithiobis-(2-nitrobenzoic acid) dithiothreitol deazaadenosine ethylenediaminetetraacetate ethyleneglycol bis-(B-aminoethyl ether)-N,N,N',N'tetraacetic acid figure gram gravity hour hexagonal phase structure of phospholipid N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate high performance liquid chromatography iodoacetic acid Michealis constant of an enzyme reaction litre lysophosphatidylcholine lysophosph at idyl ethanol amine meter molar modified Eagle's medium minute /V-monomethylethanolamine non-equilibrium pH gradient gel electrophoresis same compound as Triton X-100-contains average of 9 moles of ethylene oxide per molecule polyacrylamide gel electrophoresis phosphatidylcholine phosphatidyl-A ,A -dimethylethanolamine reference 7  f  xiii PI PMME rpm PS SDS S.E. SM TCA TEMED THF TLC TLE TP-egg PE T  m  Tris Triton X-100 UV  V V  Vmax w  phosphatidyl inositol phosphati dy 1 -N-m on ome thy lethanol amine revolutions per minute phosphatidyl serine sodium dodecyl sulfate standard error sphingomyelin trichloroacetic acid N,N,N',N'-tetramethylethylenediamine tetrahydrofolate thin-layer chromatography thin-layer electrophoresis PE prepared by transphosphatidylation of egg PC in the presence of ethanolamine gel to liquid-crystaline phase transition temperature of phospholipids tris (hydroxymethyl) aminomethane octyl phenoxy polyethoxyethanol ultraviolet volume volt maximum velocity of an enzyme reaction weight  xiv  Acknowledgements I  would  encouragement  like  gels  had  innumerable,  thank  purification  thousands  early  Special  go  to  in the  proof  manuscript.  subcellular  fractionation  technical  assistance  Marilyn  Matlock  Carpenter  supervisor  Dr.  Dennis  course of my  project.  be  bands.  successful,  His  even  attitude  in  helped  Vance Dennis those  me  for  seemed  bleak  cope  his  days  with  the  studies  of  setbacks.  thanks  this  would  of  methyltransferase read  my  and guidance during the  to have faith that when  to  in  Yao  choline deficient  of  Suzanne rat  enzyme  prepared  performed  Zemin  the  for rat.  his  Zemin  Lingrell  livers.  was  helped  Sandra  purification  collaboration  and  hepatocytes  for  N-terminal  amino  assay.  some  also kind enough to  in  Ungarian  on  lipid  analysis  provided Penney  acid  excellent  Carlson  experiments.  and  and  Michael  sequencing  of  the  methyltransferase. I  would  financial support.  like  to  thank  the  Canadian  Heart  Foundation  for  personal  This work was supported by a M R C grant to Dr. Vance.  1 INTRODUCTION The Centrality  1.1  The ionic,  of Phospholipids  phospholipid bilayer  pH and physical  controled  environment  phenotype  are  organelles,  components  stresses the  which  bilayer are  is  of  the  not,  are constantly  this regard, many of the membrane  structure  cellular membranes  activities  however, stored,  that  bilayers  an  but  is  inert more  synthetic  functions  are in or on the  are a variety of enzymatic  occur.  'bag'  which  a  ubiquitous  membranes  An example  protein  kinase  functions  (4).  pathway  by  sphinganine) responses  increasingly  in  as a  that  embedded  are the specialized  In of in  receptors  ligand with  (2).  phospholipids  C, which  of  hormonal  are  signals  not  just  mimicking been  across  cell  of the products of PI hydrolysis, activates  in turn has  an important  role  in  TPA, a potent tumor promoter, usurps the  (5).  cellular  with no bearing on  binding of an external  apparent  transduction  Diglyceride, one  have  The  Receptor mediated hydrolysis of PI, an acid phospholipid, event  (3).  cellular  described  Also  functions  intracellular event or internalize essential molecules  is  cell  Components of the  itself.  an  structural elements.  in  (1).  the phospholipid bilayer which couple  becoming  house  reactions  in  is  also  the  involved in the maintenance  membrane  the  Within this  describe  accurately  and proteins  from  being degraded and replenished by synthesis.  membrane synthesis or degradation.  It  a barrier  environment.  biochemical  "fluid-mosaic" of various phospholipids bilayer itself  cells  external  Phospholipid specific  conveniently  eukaryotic  multivalent  orchestrated.  within  phospholipid  affords  to Cell Function  diglyceride  (4).  Other  reported  inhibit  protein  to  lipids kinase  regulating  cell  protein kinase C (sphingosine  and  C-mediated  cell  2 Structural amphiphilic which  proteins,  character,  they  are  receptors  much  the  associated.  structural  properties  refractory  to  same This  on the  biochemical  and enzymes as  the  infers  proteins,  of  the  lipid  phospholipid  unique  bilayer have  molecules  solubility,  which in turn makes  with  kinetic  them  and  extremely  analysis.  Pathways for the synthesis of PC, the major phospholipid of eukaryotic cell  membranes  result,  the  are localized  elucidation  biosynthetic enzyme  (6),  of  almost  molecular  and  pathways has been sluggish.  purification  techniques,  exclusively  in membranes.  regulatory  1.2  more  of  the PC  In recent years, the development of  membrane  solubilizing  kinetic models for enzymes using lipophilic substrates biosynthesis  properties  As a  surfactants  and  has made study of PC  amenable.  Phosphatidylcholine  Structure  PC, or lecithin (l,2-diacyl-.yrt-glycero-3-phosphocholine),  the  structure  of which is shown in Fig. 1, was first isolated from egg yolk and brain in 184647 by Gobley (7) and chemically synthesized by Baer and Kates (8) in 1950. amphiphilic nature of PC is conferred by a negatively and positively  charged choline  moiety  In solution, PC will  energetically  structure  favourable  bilayer  environment  charged phosphodiester  (hydrophilic), and two  (hydrophobic) in ester linkage.  from the  aqueous  1.3  Phosphatidylcholine  Synthesis  There are 4 major routes  for the  with  the  The  n-alkyl  spontanously n-alkyl  chains  assume an  chains  shielded  (9).  synthesis of PC.  The majority of  research has been devoted to the liver and lung systems which possess great synthetic references  capacity  owing  to  export  will be made to other tissues.  requirements.  Where  applicable,  3  H(XH OT N(CH ) 2  2  3  3  choline ATP ADP PO 0 ^ 0 1 2 ^ 0 1 3 ) 3 4  phosphocholine  CTP  CYTIDINE- P 0 O l C H N ( C H 3 ) 3 2  7  2  2  CDP-choline 2  diglyceride CMP  f a  " ^  lysophosphatidylcholine "y acid  4  0  fatty acyl C o A  11  CH2-OC—Ri O R2—OO-OI II O  3 AdoHc  3 AdoMet  0^-0-p-o— O-  + 0^0^(013)3  phosphatidyl choline headgroup  phosphatidylethanolamine  Ca2+  choline  Ca2+  phospholipid  Figure 1. P C biosynthetic pathways. The numbers i n bold indicate the enzymes i n v o l v e d ; 1, choline kinase; 2, C T P : p h o s p h o c h o l i n e c y t i d y l y l transferase; 3, C D P - c h o l i n e : 1,2-diglyceride phosphocholinetransferase; 4, phospholipase A2 or A ^ 5, a c y l - C o A : l y s o p h o s p h a t i d y l c h o l i n e acyltransferase; 6, base-exchange enzyme; 7, phosphatidylethanolamine /V-methyltransferase.  4 1.3.1  CDF'-Choline Pathway.  Synthesis most the  important work  via route  of  who  for  PC  Wittenburg  phosphotransferase (11)  CDP-choline and diglyceride synthesis.  and  (choline  Kornberg  kinase,  EC  phosphotransferase, Choline y-phosphate  both  kinase  to  activity  choline  EC  was  who  2.7.1.32),  Unlike  is  A  cytosolic.  and ethanolamine Ishidate  ethanolamine/choline estimated  identity  at  between  activity  in  isoforms  have  the that  formation  other  the rat  liver  by  partially  from  rat  the  of  enzyme (14).  multiple  kinase  the  Kennedy (EC  through  and Weiss  2.7.7.15)  and  (choline  complete  the  pathway,  monkey  lung  purification  Native  a  molecular  experiments  aromatic  isoproteins  (16,17)  it  42  weight  established kinase kinase  lung and intestine  isoforms are unknown,  polycyclic  (12).  of  ethanolamine/choline  kidney,  ATP  choline  ethanolamine/choline  Multiple liver,  of  that phosphorylates  from  kidney.  and  the  enzyme  purified the  transfer  steps in  Immunoprecipation  kidney  certain  the 42 kDal kinase (15). ethanolamine-kinase  the  discovered A T P : c h o l i n e  and  (native)  reported  kDal.  tissues  latter  kDal  was  kinase  the  been demonstrated in rat  function of  in  80  et al. (13)  75-80  CCI4 and  elucidated  cholinephosphotransferase  choline (10).  kDal  note  (10),  synthesizes phosphocholine by  recently,  While  pathway  is quantitatively  2.7.8.2).  More  was  1)  described CTP:phosphocholine cytidylyltransferase  CDP-choline: 1,2-diacylglycerol  kinase  The  (Fig.  (15).  is interesting  hydrocarbons immunologially  will  to  induce  distinct  from  In light of these reports on the duality of choline- and  activities  it  seems that  reports  of  separate  enzymes  (18)  should be re-examined. In limiting step  in  some  transformed  step in P C formation a  biosynthetic  cell  lines,  (19,20).  pathway  is  choline  phosphorylation  is  the  However, the general rule that the rate-limiting  does  not  hold  true  for  ratefirst PC  5 synthesis in the majority of cases. choline  It is now generally recognized that CDP-  synthesis by the cytidylyltransferase  and a variety of cell lines (21). isolated  in soluble  ambiquitous absence  of exogenous  cytidylyltransferase, compartments,  that  Only  forms:  the membrane  phospholipid  (23).  thus  lung  it is termed an  bound form is active in the  It is the  and the translocation governs  in liver,  Cytidylyltransferase is unique in that it can be  and membrane-bound  enzyme (22).  is rate-limiting  of enzyme  CDP-choline  dual  localization of  between  these two  (and PC) production (23).  Translocation is stimulated by long-chain fatty acids, fatty alcohols and monoand  in vivo (24-26).  diglycerides  It should be cautioned that the effects of  oleate are produced at non-physiological concentrations (1 mM medium  or 10 m o l % in PC vesicles, Ref. 27).  provided  evidence  that  Pelech and Vance (28) have  phosphorylation-dephosphorylation  regulation  of cytidylyltransferase.  phosphate  into  the enzyme  To date  has been  no direct  recorded.  recently purified from rat liver cytosol (29).  in cell culture  is important in incorporation of  Cytidylyltransferase was  The enzyme consists of a single  45 kDal protein by SDS-PAGE, and a tetramer when analyzed by gel filtration in the presence of Triton X-100 (29). The diglyceride.  final step in PC synthesis is the condensation  No complete purification has yet been reported.  In vitro, cholinephosphotransferase  oleoyl  with  This reaction is catalyzed by an integral membrane protein of the  endoplasmic reticulum (30).  linoleoyl-  of CDP-choline  shows  a preference  for  and l-palmitoyl-2-docosahexaenoyl-diglyceride (31).  and l-palmitoyl-2-linoleoyl  substrates in vivo (32).  7J.2 Base-Exchange.  diglyceride  seem  l-palmitoyl-2l-Palmitoyl-2-  to be the  preferred  6 The exchange of choline into P C was first microsomal activity mM,  Ref.  34)  ethanolamine  had a K and  (35).  reported  was  competitively  [ H]Choline 3  that  of  that  the  labeling  rate  of  choline  phosphocholine incorporation  Since  there  exist  ethanolamine,  it  still  involved  the  Kanfer  and (39)  exchange  is  inhibited in  rats  enzyme  used  exclusively  with  a  33%  mutagenized  an  to  activity P E or  cells  The  activities  lipid  as  that  activity  for to  from  brain  was  a  number  al.  four  times  serine  and  serine-  enzyme,  Concomitant  and  The  have  with  of  Recently,  microsomes.  serine-exchange  (40,41).  base-exchange  choline,  the  acceptor.  homogeneity  rat  indicated  and  PC.  question  for  (2-3  Contrary to this, Treble et  apparent  in  (33).  both L-serine  and  ethanolaminekDal  PS  been  deletion  enzymes  Suzuki  100  asolectin as a phospholipid acceptor.  reduction CHO  into  open  by  base-exchange  exchange  requirements  purified  1959  for choline of 8 m M , a requirement for Ca 2+  m  was a negligible route for P C synthesis (36,37). (38)  described in  of  protein  auxotrophs,  isolated the  from  structural  gene or promoter for the serine exchange enzyme was a loss of 99%  and 50%  choline-  Comparison  of  and  remaining  activities exchange  to  ethanolamine-exchange  activities,  activities  deleted  a  with  choline-,  enzyme  I)  those  serine-  and  (serine exchange enzyme II,  a  and  acceptor, while exchange enzyme  II  as  fulfill the an  which  acceptor. again  cell's requirement These  and  of  function  exchange (serine-  ethanolamine-exchange  uses P E (42).  activity  Lack of decarboxylation  that serine exchange enzyme II  for P S , owing to  auxotrophs  assignment  Serine-exchange enzyme I uses P C as an  PS to P E , due to PS insufficiency, meant not  prompted  (41).  ethanolamine-exchange  serine-  Ref. 42).  respectively  of  were deficient  its own in  suggests P C synthesis by exchange is of  requirement  cellular minimal  PS but  of  could for P E  not P C ,  importance.  It  7  is also interesting to note that choline-exchange in CHO cells requires PC as a lipid acceptor, indicating that exchange is a futile process (42). 1.3.3  Reacylation of Lysophosphatidylcholine  An elevated fatty acid/glycerol [ C] labeling ratio in the PC (relative to 14  TG) of lung slices suggested to Lands (43) that fatty acids in PC were turning over in preference to complete degradation. rat  liver  microsomal  Lands (44,45) latter identified a  acyl-CoA: l-acyl-sw-gl y cerol-3-pho sphocholine  acyltransferase (lysoPC acyltransferase, EC 2.3.1.23) as well as an enzyme that esterifies a fatty acid at the sn-2 position (EC 2.3.1.62). The two activities that acylate the sn-l and sn-2 positions of lysoPC are specific for stearoyl- and linoleoyl-CoA, respectively (46).  Thus, the acyl composition of the lysoPC  acceptor was of little importance compared to the position of the free hydroxyl to be acylated (47,48).  Evidence suggests that two separate microsomal  enzymes exist for the esterification of arachidonoyl-CoA and oleoyl-CoA to 1acyl lysoPC (49).  The arachidonoyl-CoA-specific acyltransferase was recently  purified from rat brain (50). It is apparent that the reacylation pathway is more important in the remodeling of PC molecular species through the concerted action of a phospholipase A2 or A^. However, it should be noted that rat liver is capable of taking up and esterifing  lysoPC, predominantly with arachidonate (51).  LysoPC was also utilized as a precursor for PC in CHO cells cultured in cholinedeficient medium (52). In addition to the acylation pathways just described, there also exists a microsomal activity that catalyzes the transfer of fatty acid from the sn-2 position of intact PC or PI to the sn-2 postion of lysoPC (53) in a CoA-dependent fashion  (independent  of  phospholipase  A2 activity).  A microsomal  8 transacylase  activity  that  forms  PC  and  glycerophosphocholine  from  two  lysoPC molecules has also been reported (54).  1.4  Synthesis of Phosphatidylcholine amine Methylation The  main focus  regulatory  properties  of this of  the  work was  by  Phosphatidylethanol-  investigation  PE methylation  illustrated in Fig. 2, PE N -methyltransferase  of the  pathway  catalyzes  in  the  molecular and  rat  liver.  stepwise  As  transfer  of  methyl groups from AdoMet to the amino headgroup of PE, thus generating the two  partially methylated  intermediates,  PMME  and PDME,  starting a detailed discussion of PE /V-methyltransferase,  and PC. the  Before  properties  and  synthesis of it's lipid substrate will be examined.  1.4.1  Structure of Phosphatidylethanolamine.  PE (Fig. 3) was first isolated from brain by Thudichum (55) in 1884. was  not  until  component aqueous  of  1930 this  that  ethanolamine  phospholipid (56).  environments  was  unequivocally  PE, similar to  such that contact  identified  of the hydrocarbon chains  as  inverted  association bilayer  cylinders  P E , which  with the ethanolamine  and  reversible  of  H J J structure  (59).  was  contain  studies, water  headgroup (57,58). shown  The temperature  to  be  dependence  is  with one  in  In this regard PE H  T T  structure exists  their  interior  m  of the PE in question (59).  biological membranes  membranes does exist.  (6),  so  the  in  The transition between  temperature-dependent related  to  the  fatty  composition of the PE and the transition to H J J structure usually occurs the T  a  Unlike PC, PE exists  in both bilayer and a unique hexagonal ( H J J ) structure (9). Based on 31p_NMR  as  PC, self-associates in  another is maximized and exposure to water is minimized.  exhibits 'lipid polymorphism'.  It  and acid above  PE makes up 15-35% of phospholipid in potential  for nonbilayer structure  in these  9  S-Adenosyl-L-Homocysteine  OH O H  S-Adenosyl-L-Methionine  Figure 2.  Synthesis  of  PC by  PE  methylation.  10 H(XH <^NH ethanolamine ATP 2  2  ADP  1  P0 CH ai,NH 2  4  C  2  phosphoethanolamine CTP P  P  i  CYTIDINE— P 0 CH CH NH 2  7  2  2  2  CDP-ethanolamine ^  O  K^f  triglyceride CMP phosphatidylserine  O ll  CH -OC—R, 2  i R2— C O - C H II  ethanolamine 4 Ca / 5  1  2+  V  •  II  C H - O - P - O— C H C H N H 2  2  2  2  O-  phosphatidylethanol amine lysophosphatidylethanolamine fatty acyl CoA  Figure 3. PE biosynthetic pathways. The bold numbers indicate the enzymes involved in the pathway; 1, ethanolamine kinase; 2, CTP:phosphoethanoIamine cytidyly ltransferase; 3, CDP-choline:l,2 digfyceride phosphoethanolaminetransferase; 4, base-exchange enzyme; 5, phosphatidylserine decarboxylase; 6, phospholipase A or A ; 7, acyl-CoA:lysophosphatidylethanolamine acyltransferase. 2  x  11 1.5  Origins of Phosphatidylethanolamine  Utilized  for Methylation  Biosynthesis of P E occurs by four distinct routes (Fig.3). decarboxylation, knowledge  of  7.5.7  the  pathways  regulation  are  similar  and enzymology  to  is  those  quite  for  Except for PS  PC  synthesis,  but  rudimentary.  The CDP-Ethanolamine Pathway.  This  route  for  PE  synthesis is  from the CDP-choline pathway  enzymologically  for P C synthesis.  similar,  though  distinct  As discussed in Section 1.3.1,  there appears to be good evidence that in rat tissues the same cytosolic enzyme catalyzes  both  choline  In  1956  (11)  manner  analogous  to  that  enzymes,  reversible  (11).  and  dependent 8.0  saline  upon  (61).  kDal  by  cytosolic  of  gel  of  intermediate.  synthesis  pyrophosphate  filtration.  and M g  was  and (EC  2 +  The  It  by  2.7.7.14)  was  separate  ubiquitous  for  enzyme  activity,  in  rat  has since  following  translocation  liver  activity  is  totally  and has a p H optimum of  cytidylyltransferase  seems  to  be  homogenization  between  cytosol  localized  in  and  isotonic  membrane  occurs is unknown.  Ethanolaminephosphotransferase protein  -choline  cytidylyltransferase  fraction  Whether  compartments  CDP-ethanolamine  and reported to have a molecular mass of 49-50 kDal by SDS-  Phosphoethanolamine the  phosphorylation.  and  addition  on reducing agents  in  a  CDP-ethanolamine  100-120  (60).  primarily  P C ; through  Phosphoethanolamine  been purified (60) PAGE  ethanolamine  Kennedy and Weiss discovered that P E was synthesized in a  recognized  tissues  and  the  endoplasmic reticulum  pathway.  Apparently,  this  choline  condensation  enzyme  with  (EC (62),  is  2.7.8.1)  is  an  and catalyzes  separate  diglyceride  from  the  integral  the one  (62,63).  step in  catalyzing  Unlike  phosphotransferase,  which  shows  little  synthesizes  species  of  ethanolaminephosphotransferase  saturated  PC,  specificity  final  membrane  in  the  CDP-  choline-  vitro (31)  and  shows  a  12 preference  for  l-palmitoyl-2-docosahexaenoyl  diglycerides  vivo (31,64,67).  The enzyme is also known to utilize  acyl  (63,66,67).  diglycerides Analogous  to  P C synthesis, the rate-limiting  1-alkyl-  step  occurs at the production of CDP-ethanolamine (68).  in vitro and in and l-alkenyl-2-  in synthesis  of P E  P E synthesis is stimulated  in an unknown fashion by oleic acid (68).  7.5.2 PE  Phosphatidylserine Decarboxylation can also  be  synthesized  in  decarboxylase ( E C 4.1.1.65, Ref. 69,70). synthesized transported or  in  endoplasmic  endoplasmic  with  reticulum  serine  by  phosphatidylserine  Due to the location of this enzyme, PS by  base-exchange  to the mitochondria for decarboxylation.  base-exchange  purified  mitochondria  then  necessitates  reticulum.  The mammalian  and characterized  extensively.  (34,35)  must  be  Methylation  of P E to P C  movement  back  PS decarboxylase  to  has yet  the  to be  The major route for P E synthesis is not as well defined as that for P C . appears are  the  pathway  that  both  major  the CDP-ethanolamine  routes  is in question.  decarboxylated  synthesis,  oversimplified  however,  and decarboxylation  the  net  contribution  while  Wise  and Elwyn  then  ethanolamine  those  one-compartment  from  for growth  model  for  PS  (72)  metabolism  PS (74).  However,  cells  each  found 100%  (73).  do not require  and seem to be able to synthesize P E almost  Acylation of Lysophosphatidylethanolamine.  The  are 3- to 4-times  in culture  from PS (75,76).  1.5.3  of  These values are believed to be overestimates owing to  reported rates of P E synthesis in rat liver from ethanolamine greater  of PS  Yeung and Kuksis (71) reported that 50% of PS was  to P E in rat liver,  conversion of PS to P E . an  of  pathway  It  entirely  13 Merkl lysoPE  in  and  rat  Lands  liver  (77)  have  microsomes.  reported  the  This pathway  acylation  of  is probably  tailoring the fatty acid composition of P E molecules (78).  1-  only  and  important  to rescue ethanolamine  in PS base-exchange activity  1.6  Characterization transferase  in  However, lysoPE can  be used as a precursor for P E (without catabolism to ethanolamine) by its ability  2-acyl  requiring C H O cell mutants  as indicated  with a defect  (75).  of  Phosphatidylethanolamine  ^-Methyl-  1.6.1 Elucidation of the Phosphatidylethanolamine Methylation Pathway As  early  methionine  was  demonstrated labeled  'choline  methyl  precursors lipid  of  fraction  for  methylated  the  methylethanolamine majority  of  to  feeding result  choline  (81)  be  the  to  resulted  in  interpreted  to  to  pathway.  [ H-mef/iy/]methionine-treated 3  the  and  not  of  Stetten  of  indicate into  primary the  (80)  production  incorporation that  group  PC.  In  acceptor  of  water  Acid  hydrolysis  rats,  and  that  of  soluble the  separation  liver  of  the  was in choline and the remainder in mono- and di-  (81,81a).  These finding  methylation  immediate  methyl  rats  phosphatidylethanolamine  CDP-ethanolamine  the  choline.  was  prior  that  of  demonstrated  activity  was  fraction of rat liver homogenate (82,83). to  recognized  synthesis  The  Greenberg  was  of  was  the  phosphatide'.  labeled bases, showed >95%  the  it  15  and  groups  (79)  [ N]ethanolamine  was  Bremer  1941  utilized  that  ethanolamine 1959,  as  donor of  methyl  were later associated  In  expanded to with  the  100,000  addition, AdoMet was  groups to  P E (82,83).  show that  It  x  g  demonstrated was  further  demonstrated, on the basis of product distribution, that the  addition of the  first  methyl  and  (84)  group  to  PE  was  rate-limiting  (82).  Rehbinder  Greenberg  found that methylation could be stimulated 2- to 3-fold by the addition of P M M E and P D M E to deoxycholate solubilized microsomes. methylation  (82,84,85).  In  retrospect,  this  was  Oddly, P E did not due  to  improper  stimulate  delivery  of  14 substrate  to  the  enzyme  since  it  has  been  reported  solubilized enzyme is stimulated 8.9-fold by P E The requires  enzyme  is  detergents  tightly  for  its  associated  release  when subjected to  100,000 x g  sulfhydryl(s)  activity  for  methyltransferase optimum  could  headgroup occurs  of  is be  PE  its a  predominately  pH  optimum  pH  7.5  has  occurs  at  a  x 1 h).  (84,85). alkaline  reflection  (pKa  in  pH of  to  the  highest  activity.  and Greenberg (85)  fractions Brain,  this  compared  requirements such low explanation  have to  specific  all  spleen  original  methyltransferase  in  (ie.  does not  interesting  optimum  (10-10.5,  ionization This  form  feature Ref.  and  sediment  of  infers  that  of P E .  the  NThis  methyl  but 10  PE  ethanolamine  Whether  rate of that at p H (85)  of  81a,84).  state  a physiological significance is unknown,  methyltransferase  microsomal  low  form  membranes  transfer  this  alkaline  methylation  (84,87,88).  and does not require M g  at The  2 +  for  Tissue- and Subcellular-Localization  Bremer  is  X-100  (89).  1.6.2  Since  microsomal  Another  microsomal enzyme is insensitive to E D T A activity  with  9.5).  the  Triton  Enzyme activity is dependent on a free  deprotonated  about one-tenth  the  (86).  soluble  the  approximately  that  and  The  contained  In  all  gene expression.  these tissues so tissue-specific inhibitors  no  that  heart, low  on  liver  lung  had  testis  of  liver),  activity  extrahepatic  instances the  PE  specific  activity  and  summarized in Table  reports exist  cannot be  (85). N-  activity co-factor  1.  tissues is unknown, but  No  the  and  (2-6%  measurable  specific  are  is expressed in extrahepatic  is differential  show  but  studies  localization, activities  to  Kidney,  numerous  been published. liver.  first  measurable,  intestine  observation,  the  activity.  contained  of these extrahepatic  activity  were  Why a good  on enzyme mass  ruled out.  It  is clear,  15  Table 1. Cell and Tissue Distribution of P E N-Methyltransferase  Tissue or Cell  Subcellular fraction  Specific pH AdoMet Activity (pM)  Rat liver  microsomes  9.2 9.2 9.2 8.8 8.8 8.8 8.8 8.0 9.0 10 10 10 6 7 10 8.0  Mouse liver  Rat liver Rat heart Rat heart Hamster heart  450 580 550 microsomes 209 226 673 463 microsomes 31 homogenate 4.3-9.6 sarcolemma 4.3 microsomes 4.6 mitochondria 4.4 sarcolemma 0.01 sarcolemma 0.05 sarcolemma 3.60 plasma membranes 3.66  Rat colonic epithelial cells Rat adipocytes plasma membranes 65.0 Rat adipocytes plasma membranes 4.8 Rat erythrocytes plasma membranes 0.49 Human neutrophils microsomes 0.6 Human lymphocytes microsomes 0.84 Rat aorta microsomes 0.42 Mouse thymus microsomes 0.01 0.02 0.06 Bovine adrenal medulla microsomes 3.87 Whole rat brain microsomes 0.91 synaptsomal plasma 0.93 membrane Rat pituitary homogenate 0.21 homogenate 0.06 0.11 Dog lung microsomes 0.78  2  2  1  Additional cofactors  Ref.  200 200 0.28 mg/ml PMME 200 0.85 mg/ml PDME 24 24 0.6 mM PE 24 0.6 mM PMME 24 0.6 mM PDME 200 200 10 mM MgCl 150 150 150 0.055 1 mM MgCl 10 1 mM MgCl 150 1 mM MgCl 200 —  —  —  2  — — —  2 2 2  —  0.4 mM PMME 0.1 mM PMME 5 mM MgCl  7.4 8.5 8.0  20 10 200  8.0  50  8.0 8.2 7.4 7.4 7.4  50 100 20 20 20  5 mM MgCl 0.4 mg/ml PE 1.0 mg/ml PMME 1.0 mg/ml PDME  239 240 113 113 113  7.0 8 8  4.0 200 200  5 mM MgCl 1 mM MgCl 1 mM MgCl  99 103 103  9.5 6.5 9.5 8.2  200 200 200 200  2  173 171 98 239  —  — 2  2 2 2  10 mM MgCl 241 10 mM MgCl 105 10 mM MgCl 105 6.6 mM MgCl 243 0.3 mM dog lung PE 2  2 2  2t  ^mol/min/mg indicate the pH and AdoMet concentration at which the assay was performed  2  89 89 89 86 86 86 86 96 242 236 236 236 237 237 237 238  16 however, these  that  P E methylation  extrahepatic  microsomes  a  significant  route  or  tissues, P E /V-methyltransferase  plasma  membrane  P E /V-methyltransferase results.  for  fractions  activity  in  rat  (Table  liver  is  PC  synthesis  localized  1).  in  has  majority  of  PDME  methylation  Unfortunately,  methylation.  Contrary  no  to  endoplasmic systematic results  reticulum  evaluation  varied  specific  often  cellular  on the  It  was  membranes from  methyltransferase  and  Golgi  from  activity had  trans-Golgi,  0  estimated  PE  subcellular  to  that  (92).  endoplasmic  in  for  techniques  0.8  Golgi In  organelles  A  revealed Thus,  methyl 2.3%  of  report, above  that all  groups the  total  mitochondria that  Localization  by  8.5).  to  methyltransferase  nmol  same  reticulum.  pH  PE  twice  compared  used (92). but  /V-methyltransferase  found  Golgi  contained  this  endoplasmic  (91)  at  in  conflicting  endogenous  Golgi  (assayed  activity  to  on  Fielding  of  reported that  confined  'cis-enriched'  fractionation  enriched  ranged  methyltransferase  contamination  in  (90)  in  analysis  produced  provided  Higgins  of P E /V-methyltransferase  activities  plasma  was  was  'trans-enriched'  procedures  transferred/min/mg.  report,  activity  or  depending  fractionation  information  this  the P E /V-methyltransferase  activity  primarily  Subcellular  Similar to earlier findings (84,85), Van Golde et al.  reticulum.  and  not  tissues. In  the  is  due  of  PE  immunocytochemistry  to N -  would  be the method of choice in such studies.  1.6.3 The  Topology in Membranes. enzymes  of  glycerol  lipid  synthesis  are  to the cytosolic surface of the microsomal membrane PDME-dependent  PE  trypsin  (95).  (an  treatment  enzyme  of  the  /V-methyltransferase Latency  microsomal  of  lumen)  asymmetrically (93,94).  activities  were  mannose-6-phosphate was  maintained  above  distributed  PE-, PMMEall  degraded  and by  phosphohydrolase 90%  during  the  17 course of digestion. PMME  Contrary to this, it was reported that P E was converted to  on the microsomal lumen and subsequently converted to P C by a second  methyltransferase  on the  cytosolic surface (96).  This conclusion relied  on the  use of phospholipase C as a probe for localizing P C , P M M E and P D M E , but the authors  gave  no  phospholipase  C  evidence (96).  The  asymmetry  has  differential  hydroysis due to  fact  drawbacks  that P M M E  when  the  that use  is  problems  asymmetric Similar  observations  membranes (98)  are  disrupted,  of  were  and  evidence  cells  (104).  The  synthesis  of  of  for  of  phospholipase C  (97).  and The  (96),  intermediates  methylated.  the  membrane  products  of membranes  these  by  even remain  Because of the  phospholipases, conclusions on  for  in  liver  is  methylation  unfounded.  in  erythrocyte  for  model PMME  all  of enzymes involved. PE  methylation  methylation were  enzyme  (98), rat  rat brain  PE  that  events  liver  (84,85).  enzymes.  model  in  bovine  microsomes  synaptosomes (103) a  unique  (methyltransferase  Those researchers  inferred  formulated,  concept of two  proposed from  occurs via a three step process immediately  models  a two  (102,103),  Kinetics  liver  kinetic  membranes  leukemia  use  made  number  characterized  developed in support of the  erythrocyte  hydrolyzed  effects  methyltransferases  Structure and  responsible for  examined  provided  that  completely  the  been  question as to the  was  suggests  until  two  have  Molecular  originally  enzyme  perturbing  'sequestered' from  The fact that P E methylation  that  were  and will be discussed in Section 1.6.4.  1.6.4  raises the  PDME  phospholipases as probes  include  associated with  distribution  or  the heterogenous nature  bound to P E N-methyltransferase technical  of  which  and P D M E  microsome  PMME  a  that  single  As more tissues body  of  literature  Axelrod and coworkers adrenal (100), and  and that  medulla rat  (99),  basophilic  reticulocyte  methyltransferase I)  a  ghosts  catalyzes  the  a second enzyme  18 added  the  majority  last  two  methyl  to  Methyltransferase  showed a p H optimum of 10-10.5, a K requirement data  at  with  both  (98-100,103,105-107).  increasing a  was  requirements  (99)  reported  extracts  and  I  (methyltransferase  by  Prasad  methyltransferase  that  II  intermediates  on  of  the  in the pathway time  measurements their  Edwards  methyltransferase sonication  studies  pH  of  that I.  rat  a steady-state  on  (103)  typically  biphasic  process.  The  tissue,  of  to  rat  this,  only of  the  amount  radioactivity levels.  of microsomal P E .  methylations catalyzed  all  +  It  pituitary Crews  synaptosomes  et  released  methyltransferases PDME,  both  and Vance  of  (88)  This implies that  based  which  are  pointed  out  intermediates  ( P M M E and P D M E ) rapidly reach constant levels in 1 min, after  steady-state  were  2  absolute  were reported.  of  the  end  in P M M E  product  (PC)  and P D M E are  Based on this  increases.  only  concept, formulae  K  m  reported was three  10.5  Thus,  an assessment of were derived that  estimate the actual amounts of P M M E , P D M E and P C formed during  PDME  Mg  fraction.  two  Audubert  II  velocity  the  Contrary  and  6.5-8,  initial  sonication  supernatant  PMME  of  were  brain  of  and no cation  (100,103,105,106).  indicating of  8)  in activity  (105)  in P E to P C conversion. is  (at  The  Methyltransferase  plots  depending  stimulation  accumulation  that P E methylation  which  and  and,  into a 100,000 x g  majority  conclusions  15%  solubilized  reported  The  variable  or only  Lineweaver-Burke  component  m  .  2 +  optimum  for AdoMet of 67-110 u M  m  II).  data, p H optima and divalent  had a reported p H  concentrations  low-K  often  partially  (103)  AdoMet  high-  requirement  al.  PC  for AdoMet of 0.6-4 u M and a requirement for M g  m  was  form  of the evidence was derived from kinetic  cation requirements. a K  groups  methylation  values for AdoMet for the methylation of P E , P M M E and to  be  (88).  50-100 This  methylations.  A  p,M, was  and the taken  similar  to  pH  optimum  indicate  conclusion was  that  for one  reached  all  three  enzyme in  calf  19 brain the  (108).  Reconcilation of these conflicting reports  enzyme(s)  for  Kinetic more  PE  enzymes, is very will determine  will  require  of  methylation.  analysis of  (88)  awaited purification  a  multistep  complex.  the K  a different  While  for  m  pathway, the  whether  formulae  catalyzed  of Audubert  AdoMet, elucidation of the  approach.  The best  by  one  or  and Vance  kinetic mechanism  approach would  involve assaying  the individual steps with P M M E and P D M E , since in both cases only one methyl addition occurs to yield P D M E and P C , respectively (88,89,108,109). the methylation available.  of P E to P M M E poses a problem, for no discrete assay is yet  Since this  step is rate-limiting  production of P C should reflect endogenous membrane attempts  Assaying  substrates  fractions  and  the  rate of P E methylation.  various  necessitates kinetic  at purification  and no intermediates  inhibitors  and  analysis of  the  are discussed in the  accumulate, the The presence of  activators pure  in  crude  enzyme.  Earlier  following section.  7.6.5 Purification P E N-methyltransferase  is tightly  associated with  due to one or more hydrophobic membrane integral  nature,  solubilize and  purification  P E AT-methyltransferase  other  proteins.  conventional  requires  that  detergent  and  A  to  The  techniques.  been extensively  detergent  requires  reviewed a  balance  maintenance  the  from  The  use  methyltransferase.  of  Any  be  struck  of  biological  detergents Most  of  amphiphiles in  then  detergents  achieve this end is largely  variety  use  could  of  (110-112).  spanning domains.  membranes  enzyme  membranes,  a  be in  between  detergents free  purified  optimal  activity.  or  form  of  doubt  Because of its  protein  purification  no  of  by  lipids various  purification  a bioactive  of  the  has  protein  solubilization  Selection  to  with proper  empirical.  have  been  used  purifications  have  utilized  to  solubilize liver  PE  microsomes  N as  20 starting  material  for  methyltransferase sonication  of  the  logical  specific  activity.  liver  microsomes  rat  reason Schneider in  the  released 44%  of methyltransferase  activity.  enzyme  partially  (28-fold  89).  were  A  similar  microsomes,  successful  using 0.2%  solubilized  Triton  with  methyltransferase reported (113). (PE-dependent liver  Triton  from  X-100,  Vance  presence  of  Attempts  at  purification  was  (89)  that X-100  the  soluble  homogenate,  from the  X-100.  Partial  Ref.  mouse  same  thymocytes  purification  using  Triton  liver  time  had  a  specific activity  of  X-100  The activity in thymocytes is so low that the enzyme  Triton  purifying  at  highest  reported  0.2%  from  reported  the  (86).  PE has  1500-fold  similar  to  Nbeen  purified  that  in  rat  microsomes.  solubilized  rat  liver  labeled  showed  the  paper  reported  microsomal  had  photoaffinity that  (114)  et al.  methyltransferase  a  enzyme  was published by  PE  molecular  with  the  experiments 25  kDal  publications  they  protein and activity) methyltransferase. (114,115)  N -methyltransferase. 25  kDal,  and  3  also methylated the  little  fatty  same group (115)  described in  (114,115)  purification  [/wef/j>>/- H]8-azido-AdoMet.  By  concluded that the  protein  200-fold  mass of  protein was P E N-methyltransferase.  step.  and  contains  also showed that the calf brain enzyme could  mouse  activity)  Pajares  the  it  solubilization of P E //-methyltransferase  Percy, Moore and Waechter (108) be  that  direct  50  the  kDal  was that C H A P S  was  protein  alleged  Oddly,  the  Shortly  to  after,  claimed a 50  and confusing a dimer  In  of  evidence  (such  as  a  kDal  actually  (114).  be  authors  was  report  disturbing  The shown  elaborate  earlier  is provided that either Another  a series of  CHAPS  was  acids (114). which now  of  both  of  these  co-chromatography  of  a 25 or 50 kDal protein is P E  N-  feature  removed after the  of first  these DEAE  purifications chromatography  Thus, no effort was made to remove endogenous lipids (or proteins) and  21 the  result  was purification of a large  fragment'.  lipid-protein aggregate or 'membrane  Although all the aforementioned  purifications  were  unsuccessful,  they were unanimous on one point: one enzyme seemed to be responsible for all  three  methylation events.  1.6.6 Phosphatidylethanolamine Molecular Species Specificity of PE NMe thy ltransferase Specificity the  primary  composition),  can imply both selectivity  substrates  or for compounds  but with a primary-, specificity  PE, PMME  secondary-  with  for different molecular species of  and P D M E widely  (varying  divergent  in  fatty  acid  structural properties,  or tertiary-amino group.  Molecular species  will be discussed first.  All reports to date have utilized argentation T L C (116) as a method for separating molecular species of phospholipids. degree of unsaturation, and thus  Separation is on the basis of  'fractions' with the same number of double  bonds, and not individual species, are resolved. have  involved  AdoMet,  in vivo or in vitro labeling  respectively,  argentation T L C .  [mef/iy/- H]-methionine  of the labeled  In vivo, di- and tetra-enoic  (118-120) fractions liver.  and analysis  with  Most reports on specificity 3  or -  PC molecular species by  (117) or tetra- and hexa-enoic  of PC were the chief products of P E methylation  Arvidson (64) reported that [l,2- C]ethanolamine 14  rich PE was the primary substrate for methylation.  labeled  in rat  hexaenate-  This conclusion has since  been shown to be incorrect, and the general consensus is that methylation of any particular PE species is dependent on its concentration (117,120) and not fatty  acid composition.  fraction activity  of PC synthesized  same report (117) showed  by methylation  that the hexaenoic  had the highest  initial  specific  and appeared to turnover rapidly.  Rat specificty  This  brain,  the only  other organ in which PE /V-methyltransferase  was examined, showed polyunsaturated-rich species of PC to be the  22 major  methylation products.  In vitro labeling of synaptosomal P C (121)  vivo labeling of whole rat brain P C (122) species  of  studies,  PC  there  concomitant recent  to  be  was  the  a  pronounced  increase in  development  phospholipid  primary  %  of  label  HPLC  molecular  species  of P E N-methyltransferase  methylation.  turnover  hexaenoic  more  of  saturated  techniques  for  the  (123,124) made  PC  PC  analysis of  Substrate Specificity  There  is  the  unavailability  of  information  indicating  pure  enzyme,  thus  cannot be ascribed to P E N -methyltransferase been  reported  that  rat  liver  A  microsomes  to  report,  hepatic  methylation  in  dimethylethanolamine. by  liver  suggesting  more  of  this  work  that is  These two  required  injected  lyso is  (preferably  in  a  The  products  then P E ,  problem  crude  that  exists  with  rat  brain no  ceramide-phospho-7V,7V-  compete  catalyzed  has  reported  but not lysoPE, are lipids  It  methylates  in  (126)  al.  has  membranes  certainty.  activity  et  other  The  activity  LeKim  and - P D M E ,  methylation  an  similar  intravenously  Lyso-PMME  microsomes (127).  analogues Clearly,  rats  and  individual  methylated  complete  microsomes possess (125).  Contrary  liver  (122).  of  substrates  activities  with  ceramide-phosphoethanolamine (125).  to  species  fractions  P M M E and P D M E are methylated by P E N-methyltransferase. been  and hexa-enoic Similar  separation  in  amenable.  7.6.7  of  products of  in  more  a paucity  showed tetra-, penta-  and  by  with their  the  purified  methylated  same  enzyme)  diacyl  enzyme. to  assign  these activities to P E N-methyltransferase. Modification of the base moiety of P E has given some insight into P E methyltransferase  specificity.  Incubation  monoethylethanolamine,  2-aminopropanol  their  phosphatides  methyl  incorporation group  into  (128).  and  and the  of  hepatocytes  2-aminobutanol resultant  Phosphatidyl-diethanolamine,  addition  with  resulted of  a  -diethylethanolamine  N-  in  single and  23 -dimethylaminopropan-2-ol reported  that  were  (129).  ethanolamine The well  PC, 14  features early  of  study  AdoMet  (85).  [ethyl- C]ethionine 1.5%  compared  injection  was  of  groups  to  (130).  1.7.1  recovered  but  in  AdoEt  ethyl  PC  in  methylation  from  [ H]ethanolamine-labeled  occupy  occurs  of  of PE  assessed. liver  [methy l-  the  only  AdoMet slowly.  A -Methyltransferase 7  the  total  liver  (68).  3  methylation  Earlier  reports  P E estimated  that  Since  methylation  of  PE  contributes  significantly  to  pool, it is feasible that this P C has some preordained function. are  actively  these  lipoproteins (133),  secreted is  PE  of P C was  (131).  As A Source of Phosphatidylcholine in Lipoproteins  HDL  contributes  monitored  that 3-10%  7.7.2  SM  not  quoted value is that P C synthesis via methylation  that  and  inhibitor  was  not  analogue  following  can  the  are  was recovered in  transfer  Phosphatidylethanolamine  of  derived by  competitive  rats, label  in  transfer  sulfonium  PE  single  headgroup.  methyl  into  a  alterations  amino  ethyl  a  7  Contribution to Phosphatidylcholine Synthesis  The widely 20-40%  of  into  Apparently  7  Functions in Liver  ethyl  that  active site on P E A -methyltransferase,  1.7  for  was  phosphatidyl-A -  to  of the  essential  It  undergoes  sensitive  (AdoEt),  was injected  14  C]methionine  is  indicated that the  Incorporation  to  subsequently  and to AT-alkyl substitutions  An  only  and  vivo  S-adenosyl-L-ethionine  but  converted  7  structural  methylation When  in  microsomes (128).  by  is  P E A -methyltransferase  moiety  defined.  AdoMet,  methylated  N-isopropylethanolamine  isopropylethanolamine methylation  not  by  hepatocytes  composed primarily  which  function  to  (132).  and H D L  total  surface  monolayer  the  choline-containing  solubilize  the  apolar  core  cellular  Both V L D L  of  cholesterol ester) components of these particles. and secretion of V L D L  The  the  lipids,  (triglyceride  and of PC and  The P C required for assembly  from hepatocytes  in monolayer culture seems  24 to be synthesized by both the CDP-choline- and methylation-pathway made  via methylation  However, than  cellular  of ethanolamine-derived  specific activity  hepatocyte  culture  lipoprotein  P C by  hepatocytes  with D Z A .  and inhibits  methylation  of this  medium  This  The relative  was further  adenosine analogue  lack  of  demonstrated  lipoprotein  methylation the  in  lipoproteins.  rapid  is  sequestered  secretion  decarboxylation reticulum  occur  is in  to mitochondria  interpretation  even separate  in the methyl While  because the labeled  from  organelles  it  appears  that  it is worth noting  used  to resume V L D L  to  indicate  that  and transit  in  manner.  methylation from  This  in these  some  and  endoplasmic  is not the only  studies  (134,135)  will  A large proportion of the label in P C would be  P E methylation that V L D L  hepatocytes  since  at 6 h (75).  serine  and rapidly (30  seem  inhibitor  peculiar  group and not the ethanolamine  methionine-deficient sufficient  more  would  P E into P C  by a AdoHcy-insensitive enzyme or  away  is estimated  label the AdoMet pool via T H F .  secretion,  results  of PS-derived P E is performed  methyltransferase  This  These  treating  secretion (135).  (134,136), and in fact this serine-derived P C was preferentially secreted  into  AdoHcy pools  Oddly, D Z A did not inhibit the conversion of [3-3H]serine-labeled  min)  greater  input by  raises cellular  of P E , but does not affect  PC  in lipoproteins.  P C was found to be 2-fold  (134).  P E methylation  P E is secreted  (134).  moiety (71). is not required  secretion is impaired  and supplementation  for  lipoprotein  in choline- and  of methionine  alone  is  secretion (137).  1.7.3 Does Phosphatidylethanolamine N-Methyltransferase Supply the Hepatocyte With Polyunsaturated Phosphatidylcholine? Saturates,  monoenes, dienes and trienes  of P C formed by the CDP-choline pathway Section 1.6.6, P C formed via methylation species.  It is tempting  are the major  (74,138).  molecular species  However,  as discussed in  of P E is rich in tetra- and hexa-enoic  to speculate that P E N -methyltransferase  supplies  the  25 cell with P C enriched in unsaturated  species by virtue of enzyme specificity or  due to the highly unsatuated nature of P E fatty acids (138).  There is still little  direct  data  proof  for  this  methyltransferase  and  reacylation  pathway  incorporation  of  1.8  apart  from  specificity  cholinephosphotransferase.  in  microsomes  may  long-chain polyunsaturated  be  fatty  the  of  hepatocytes  increase in methylation  TV-methyltransferase  the  in  blocking  reagent  PE  route  P C (Section  for  1.3.3).  TV-Methyltrans-  of ethanolamine  assayed  pathway)  on enzyme of  cultured  was  in vitro  concentration  rates (128).  ethanolamine  produces  a  75%  and an increase in cellular P E from 20  in  striking,  been  MME  substrates  the  caused  of P C (the  has not  to  on P E  but  some  showed that reduction  with  microsomes  cells with  synthesis of the P E TV-methyltransferase  less  treatment  The effect  activity  supplementation  Experiments  (by  methylacetimidate)  Supplementation  142).  mM  was observed (139).  reduction in methylation methylation  0.5  The effect  activity  activity  endogenous  with  of P E (139)  of total phospholipid.  stimulation  N-  deacylation-  primary  acids into  PE  Regulation by Substrate Levels.  Incubation  30%  on  The  Regulation of Phosphatidylethanolamine ferase in Liver 1.8.1  of  hypothesis  amino a  group-  simultaneous  end product of the  reported.  and  PMME  DME and  resulted PDME  in  the  (68,140-  In L M cells cultured with M M E or D M E up to 60% of cellular lipid was  P M M E or P D M E (140).  Because L M cells are deficient in P E methylation, little of  the  is converted to  PMME  and P D M E  supplementation phospholipid PDME little  raised the  and  are trace  stimulated  In  hepatocytes, M M E  phosphatides of these bases to P E methylation  components in hepatic  physiological  PC.  relevance.  by  40%  (68).  10-20% of  and the  Since P M M E  phospholipids (143), these  DME total and  studies have  26 In the  two)  addition to P E , AdoMet and AdoHcy (or more importantly modulate  methionine  P E methylation.  stimulated  concentrations  had  PE  no  to  further  Incubation  PC  of  conversion  affect.  hepatocytes  by  Normal  2-fold  perfused  the ratio of  with  0.1  (68). rat  mM  Higher  liver  has  an  AdoMet concentration of 32 nmol/g of tissue (144).  This level is elevated to  120  and  methionine,  are  300  nmol/g tissue when  included  in  the  perfusate  0.05  and 2.25  (144).  This  accompanied by increased AdoHcy. homocysteine nmol/g  and 4.0  tissue  and  AdoMet/AdoHcy  mM  increase  from  in  respectively,  AdoMet  levels  is  not  However, perfusion of livers with 3.4  adenosine elevates  AdoMet  ratios  mM  to  1250  5.6  to  nmol/g  0.3  mM  A d o H c y levels  from  8 to  tissue  This  change  results in  (144).  a complete  abolition  4000 in  of P E  methylation. Further  insights on the  influence of  have been made using a variety intracellular AdoHcy levels. compound  that  The most widely used of these compounds is D Z A , a  potently  reaction of  inhibits  AdoHcy  hydrolase  nmol/g  AdoHcy hydrolase (147).  respectively).  Administration  [mer/iy/- H]methionine 3  mM)  Intraperitoneal injection to  1.6  and  of  labeling of  DZA  to  P C (146).  of  DZA  in 4 h (146).  3-  compared to  125  levels were found to be similar to AdoHcy (151  liver,  decrease in  (I50 =0.008  As well, D Z A is a good substrate for the  into rats decreased the AdoMet/AdoHcy ratio from 4.5 DZA-AdoHcy  methylation  of A d o H c y analogues or compounds that raise  elevates cellular A d o H c y (145,146). reverse  AdoMet/AdoHcy on P E  rats  caused  Thus,  a  90%  inhibition  of  P E methylation  is due to appearance of two  competitive inhibitors (AdoHcy and  DZA-AdoHcy)  of  (146,148).  methyltransferase analogues pentanoic  PE  /V-methyltransferase  Activity  in isolated microsomes was not affected (148).  5-7-deazaadenosyl-homocysteine acid)adenosine  (Sinefungin)  also  and  inhibit  of  PE  N-  The AdoHcy  5'-deoxy-5'(l ,4-diaminoPE  methylation  (the  latter  27 only  in  Ref.  vitro,  arabinofuranosyladenine,  149).  Other  adenosine  analogues,  5'-deoxy-5'-isobutylthioadenosine  (SIBA)  9-P -D -  and A[(-)-  9-[trans-2,trans-3-dihydroxy-4-(hydroxymethyl)cyclopent-4-enyl]adenine (Neplanocin  A ) , are potent  inhibitors  of P E methylation  in vivo (135,149) and  appear to raise cellular AdoHcy levels in a manner analogous to D Z A (149,150).  1.8.2 The hepatocytes effects  Hormonal Effects in Liver reported vary  effects  greatly.  of various  The majority  hormones  on P E methylation  of the studies have  centered  on  in the  of glucagon, insulin, vasopressin and angiotensin on microsomal P E N-  methyltransferase  activity  [mef/ty/-3H]methionine messenger  cAMP,  a Ca -mediated  labeling.  while  conversion of P E to P C measured by  Glucagon  appears  to act v i a the second  vasopressin and angiotensin  pathway  2+  Geelen  and in vivo  exert  their  influence by  (151).  et al. (152) were  the first  to demonstrate  that  pretreatment of  hepatocytes with glucagon for 3 h caused a small (20%) increase in P E to P C conversion. methylation  Since  glucagon  may have  Experiments performed PC  conversion after  Incubation  been  also  enhanced  P E synthesis,  due to an expanded  in a similar manner  reported  2.5 hours in the presence of  of hepatocytes  with  the c A M P  the effect  substrate  from  cAMP  analogues  choline.  100 n M glucagon  analogue  methylation  by  methyltransferase  glucagon activity  that  showed  (153) and c A M P in  respectively.  a  microsomal  stimulated  2-fold,  Castano  stimulation  o f P E TV-methyltransferase  P E into P C P C production  in vivo inhibition analogues  fraction  (154),  in  of P E P E N-  was unchanged or  et al. (156) reported  activity  (153).  chlorophenylthio-cAMP  (155) and glucagon (153) also inhibit  In the two reports  (152).  a 33% reduction in P E to  reduced by 50% the incorporation of [l-3H]ethanolamine-labeled (154).  pool  on  a  2-fold  homogenates  from  28 hepatocytes treated with glucagon.  In  accord with a role  for cAMP-dependent  phosphorylation, treatment of rat  liver microsomes with c A M P  a  activity  2-fold  Pelech  stimulation et al.  protein  in  (158)  enzyme  reported  phosphatase  1  or  no  (157).  effect  2A,  of  casein  In  opposition to  cAMP-dependent  kinase  protein kinase on P E TV-methyltransferase  and A T P caused  II  and  activity  in  this  protein  result, kinase,  calmodulin-dependent  microsomes.  However,  A T P or G T P and cytosol caused a 50% increase in activity (158). Results that  from  various  cAMP-dependent  laboratories,  phosphorylation  Various experiments  were initiated  A -methyltransferase  (Section  7  though  was  quite  activating  to demonstrate  1.6.5)  was  protein kinase in response to glucagon.  contradictory,  PE  AT-methyltransferase.  that the putative  phosphorylated A  suggested  by  50 kDal P E  cAMP-dependent  50 kDal protein was phosphorylated  on a serine residue by cAMP-dependent kinase with a resultant  4-fold  in  P E methylation  phosphorylation  or  inhibit  the  50  hepatocytes  (162).  (159).  dephosphorylation kDal  activated  activity  protein  was  and shown to  PE  of  AdoMet  partially  pure  have  incorporated and  7  evidence reported  activate  methyltransferase  immunoprecipitated  A -methyltransferase  However,  seemed to  3 2  P  from  (161).  (160).  glucagon  Also, treated  Protein kinase C also  phosphorylated  in this thesis will  increase  the  50  kDal  protein  show that the  50  kDal  protein is not P E /V-methyltransferase. Vasopressin,  angiotensin  and  the  Ca  2+  ionophore  A23187  inhibited  choline labeling of P C (163), but activated by 2-fold P E /V-methyltransferase hepatocyte  homogenates  (164).  methyltransferase  activity  calmodulin (165).  Apparently, the  also  stimulates  PE  Ca  2-fold,  2 +  and  ATP  presumably p-adrenergic  /V-methyltransferase  also  stimulated  through receptor  activity  in  the agonist  in  microsomal  mediation  of  isoprenaline  hepatocytes  from  29 adrenalectomized rats and, to a lesser extent, control animals (166,167).  Again,  this activation appeared to be mediated by c A M P . Insulin methylation  has no of  PE  effect  on  (168).  glucagon-dependent  PC  However,  stimulation  mechanisms  Accordingly,  in  a  manner  adrenocorticotropin  adipocytes.  cAMP  reported  to  or via  inhibit  activity  seems to be activated  similar  (169),  to  that  epinephrine  the and  of  PE  of  by  cAMP-  hepatocytes.  (170),  isoproterenol  methylation  activity  Administration of oxytocin, a hormone that does not act  (172), to adipocytes resulted in a time-  activation  (153)  in hepatocytes (168).  and forskolin (170,171) were all shown to stimulate  in intact via  reported  methyltransferase  of adipocytes  7  (170)  was  choline  Hormonal Regulation in the Adipocyte  P E A -methyltransferase dependent  from  insulin  of  phosphorylation of a 50 kDal protein  1.8.3  synthesis  methylation  to stimulate  (170).  methylation  Phorbol  activity  2-fold  and dose-dependent  1.5-fold  12-myristate-12-acetate  was  in rat adipocytes, presumably  via protein kinase C (171). Insulin through  cAMP-dependent  methylation reported  kDal 1.8.2).  in  that  adipocyte activity,  was reported  intact insulin  plasma  stimulated  membranes  methyltransferase  membranes  by  and  (169,170)  by 40%  (179).  (174)  in  a manner  analogous  phospho-oligosaccharide  phosphorylation  presumed to be cleaved  from  C  itself,  as  to  well of  as  inhibit group  isolated  stimulating  hepatocytes from  digestion)  both  its lipid  in  acting  the putative  (prepared  (174).  to  the same  activity  phosphorylation  phospholipase  hormones  Strangely,  Isoproterenol,  enhance  Pi-specific  and, by  7  to  a  (169).  of various  P E A -methyltransferase  shown  isoproterenol-dependent oligosaccharide,  mechanisms  adipoctyes  was recently  Insulin  to abolish the effects  The moiety  50  (Section rat  liver  inhibited phospho-  by an insulin-  30 responsive insulin  phospholipase  action (175).  methyltransferase, and  C, is proposed to serve Considering that the  one  50  as a second  kDal  messenger of  protein is  not PE N-  should be wary of reports on enzyme phosphorylation  effects on activity.  1.8.4 Other Effectors There  appear  methyltransferase low  molecular  (158,176). from  to  be cytosolic  factors  activity (158), but they weight  inhibitor  has  that  influence  remain unidentified.  been  identified  in  A  rat  PE  N-  heat-stable,  liver  cytosol  This inhibitor may be similar to a 25 amino acid peptide isolated  rabbit  liver  methyltransferases  cytosol  that  is  proposed  to  inhibit  various  (177).  Unsaturated fatty acids are potent inhibitors of PE methylation in intact hepatocytes effective  and microsomes (178).  Long-chain fatty acyl-CoA esters were also  inhibitors and inhibition was reversible upon addition of BSA.  1.8.5  Developmental Regulation  Two reports on PE TV-methyltransferase in pre- and post-natal  rat liver  demonstrated a steady rise in activity from -5 to +15 or +20 days and a slow decline to adult values thereafter (179,180).  Activities in prenatal rabbit livers  were about 33% lower than values at birth and reached a maximum at +14 days (181).  Similarly, rat brain PE TV-methyltransferase activity  to be the highest between day 5 and 30 (182). correlated with activity, it is feasible  was  demonstrated  Since enzyme mass was not  that activity changes  noted after birth  could be due to altered PE or AdoMet/AdoHcy levels.  1.8.6 Several for  Coordinate Regulation with the CDF'-Choline Pathway examples  PC synthesis  potently  have  inhibiting  PE  of coordinate regulation of the been  demonstrated.  methylation  (178),  two major pathways  Unsaturated fatty were  found  to  acids, stimulate  while PC  31 production  from  synthesis  choline  in cultured  rat hepatocytes  via the CDP-choline pathway  phosphocholine The  cytidylyltransferase  elevated  cellular  P E methylation  synthesis  v i a the CDP-choline pathway activity  Maintenance metabolic  of  levels,  but caused  rats  on  a  change  hepatic  reduction  in microsomal  recent  report  by  cytidylyltransferase deficient  redistribution  1.9  increase  increase  activity  of activity  7  evidence  to P C .  about mutants mutant  P C (184,185).  (186,187).  choline-  and  However,  no a  that  microsomal  and  methionine-  to the cytosol occurred upon  methionine.  T h e mechanism  of  this  is as yet unknown.  in  Phosphatidylethanolamine Methylation in Yeast.  P E A -methyltransferase  synthesized  circulating  activity  (188) showed in  various  in P E N - m e t h y l t r a n s f e r a s e  activity  was elevated  of  decreased  7  genetic  in P C  in microsomal  caused  cytidylyltransferase  and Vance  choline  diet  Phosphatidylethanolamine A -Methyltransferase Eukaryotic Microorganisms 1.9.1  PE  liver,  elevation  cytidylyltransferase  activity  in enzyme  by D Z A treatment  P E and decreased hepatic  in cytosolic  Y a o , Jamil  with  translocation of  a 2- to 3-fold  fatty  hepatocytes, and that a shift  supplementation  enhanced  to microsomes.  caused  choline-deficient  including  increased  a 2-fold  with  cytosol  and a 3-fold  Accompanying these changes is a 2-fold activity,  This  (148).  perturbations  lipoprotein  from  of A d o H c y  inhibited  cytidylyltransferase  (146),  was correlated  activity  levels  (183).  points  In yeast,  has yet to be purified  to the involvement unlike  by P E methylation  4-times  more  effectively  in P E methylation (191) appeared  have  mammalian (189,190).  of two enzymes cells,  Methionine  to be defective  isolated  yeast,  from  than 5.  in methylation  however,  in conversion of  the majority  for P C synthesis been  from  seemed choline  of P C can  be  to be utilized (190).  cerevisiae. of P M M E  Two  The opi 3 to P C  and  32 accumulated P M M E and, to a lesser extent, P D M E . 3  H]methionine  is defective in the conversion of P E to P M M E (192).  this strain on M M E restores P C levels to normal these two mutations  two  are the result  with several other  and Yamashita  two S. cerevisiae double  mutant  choline auxotrophs of S.  (195) have  carrying  in an intact  of genetic  predicted  amino  recently, genes for A  and was defective  but P C levels  with pern 2  were  about one-  Thus, one enzyme converts P E to P C (PEM 2 )  of normal P C levels. acid  More  complementation.  accumulated P M M E  pathway,  whether  also indicated that  Transfection of the double mutant  P E methylation  The  or structural genes.  cerevisiae  and the other catalyzes P M M E synthesis only (PEM for the maintenance  It is unclear  of P C (193,194).  by means  the pern 1 gene  half that of wild type values (195).  the  [methyl-  Growth of  successfully cloned the structural  methyltransferases  in synthesis of P D M E and P C . resulted  (192).  of lesions in regulatory  enzymes were required for the formation  Kodaki  of  into P C was about 10-20% of that in wild type strains (191).  cho 2 mutant  Work  Incorporation  sequence  1), but both are necessary  Internal homology was observed in  of the PEM  1 gene product,  as well as  to be inhibited  by choline  between PEM 1 and PEM 2 (195). Yeast P E /V-methyltransferases in  the growth  reversibly the  (196).  P E methylation  presence  showed  medium  that  of choline choline  inositol alone  alone  does  for full  reported  M y o-inositol  in S. cerevisiae (194).  is required  were  A  was also  (193),  detailed  an effect  survey  not repress  by inositol  restoration  of  cycloheximide  and choline  enzyme (193).  activities  is due to reduced removal  of  activity. (192).  enzyme  inositol)  inhibit  expressed only in conditions  P E TV-methyltransferase,  expression of enzyme  (by  to  o f all growth  choline together led to 6- to 10-fold repression of activity activity  observed  and  Inositol and Repression of  synthesis is  since  abolished by  The 5. cerevisiae sterol and fatty acid auxotroph G L - 7 , which is deficient in squalene epoxide cyclase (197), displays unusual sterol synergism. of  limiting  quantities  of cholesterol or ergosterol did not support growth, but  addition of both sterols produced a marked increase in growth PE  methylation,  incorporation one  of  (199).  measured by [mef/ty/- H]methionine  in vivo and into  Since the total  stimulated amount  plus ergosterol  of sterol  was the same  cytidylyltransferase methylation selective  (supplemented activities  was the major  inhibition  4-fold source  of methylation  synthesis from choline in both mutant Two mutant phospholipid PE  with lower  wild  of P C in these by ethionine  indicated  a specific  It was also observed that  ergosterol) then  supplementation  in cholesterol versus  supplemented cells, the results (199).  (198,199).  was found to be  by ergosterol/cholesterol  7  G L - 7 mutant  rates  [methyl-^H]AdoMet  microsomal P C , respectively,  effect of ergosterol on P E A -methyltransferase the  and  3  the processes  cholesterol  Addition  had phosphocholine type,  cells  resulted  and as such P E (190).  Evidently,  in enhanced P C  and wild type cells (190).  strains of the mold Neurospora crassa were shown to have  compositions consistent  to P C conversion (200,201).  appeared to be defective  with  a requirement  for two enzymes in  Microsomes from one of these mutant  in P E methylation  to P M M E , while  strains  the second strain  accumulated P M M E , and thus may have a defect in the conversion of P M M E to P C (202).  Partial purification of the P M M E - and PDME-dependent activities was  also consistent with the notion of two enzymes for P E to P C conversion in N. crassa.  (203).  It  is possible that, like  5.  cerevisiae, N.  crassa has a P E  methyltransferase that converts P E to P C and not just P M M E to P C .  7.9.2  Phosphatidylethanolamine Methylation in Bacteria  As a general rule, bacteria do not contain P C or the partially intermediates  of the P E methylation  pathway  (204).  There  methylated  are some  notable  exceptions found  to contain  bacteria as also  to this  (family  rule.  Twelve  of hydrogen-oxidizing  23 to 47% P C (205), Chromatiaceae)  P C (205) and several contain  strains  P C as well  6 strains  contained  strains  10-17%  o f methane-  as P M M E  bacteria  of the phototrophic of their  (207,208).  bacteria  Phospholipids of  thiobacilli were shown to be composed of P C , P M M E and P D M E (209).  strains  of Clostridium  a  beijerinckii,  butyric  contained P M M E but no other methylated the methyl  direct methyl donor (209,209a). among  these  membrane function  bacteria  systems  is correlated  are tenuous  and P D M E  combination  were  that  found  would  Characterization  the presence  since  rich  membrane  the presence  within  Such  bacterial  Goldfine (210) noted that fatty  acids; a  stability.  system  soluble  enzyme  structure-  a particular  in unsaturated  bilayer  of a  and a particulate  from endogenous P E (211).  The source of  o f intracytoplasmic  (205).  others do not.  of the P E methylating  PMME  bacteria,  and AdoMet appears to be the  pigments  in bacteria  enhance  tumefaciens revealed synthesis of only  with  at best,  genus some species contain P C while PC  Eight  It has been proposed that the occurrence of P C  or photosynthetic  relationships  acid-producing  phospholipids (209b).  groups in bacterial P C is methionine  purple  total phospholipid  and methanol-utilizing  and P D M E  were  of  Agrobacterium  enzyme  catalyzing the  that produced primarily P C  The P E /V-methyltransferase  of  Rhodopseudomonas  sphaeroides (212) was found to be exclusively (>90%) associated with a soluble cell  fraction.  A single  enzyme  catalyzing  all three  methylations  purified from Triton X-100 solubilized membranes of Zymomonas  to P C  was  mobilis (213).  The putative enzyme is composed of a single subunit of 42 kDal (213). As  yet, no choline  synthesis of bacterial P C .  nucleotide  pathway  has been  identified  for  the  This would indicate that of the two pathways for P C  synthesis, P E methylation was the first to evolve.  The prevalence of P M M E and  35 PDME  (often in the absence of P C ) in some bacterial  pathway  evolved in a stepwise manner  strains suggests that the  (205,210).  1.9.3 Other Organisms The ciliate 20%  protozoan  2-aminoethylphosphonolipid  aminoethylphosphonate  is peculiar owing to the presence of  Tetrahymena  as the base)  (an  analogue  of  PE  in its phospholipid (214).  with  2-  Tetrahymena  contains about 29% P C , of which 60% is synthesized by P E methylation Interestingly,  2-aminoethylphosphonolipid  dimethylaminoethylphosphonolipid  are  (AEP-lipid)  not  substrates  (215,216) and AT.N.AMrimethylaminoethylphosphonolipid a natural  component  of Tetrahymena  lipids (214).  medium containing T M A E P incorporate this base into represents 20% of the total phospholipid (217). lipid,  cell  growth  is seriously  makes it imperative  diglyceride, bond  is yet unknown.  A E P injected derivative  slime  methylation  (TMAEP-lipid)  is not  grown on  Tetrahymena  T M A E P - l i p i d such that it  The toxicity  a phosphoester  is necessary for methylation The  (217).  requires  but the trimethyl  for  N,N-  At this proportion of the total  that A E P - l i p i d is not methylated.  P E N -methyltransferase and phosphate  inhibited  and  bond into  of T M A E P - l i p i d  Whether  the mammalian  between  the headgroup  rats  was found  was not observed  indicating  in A E P the  conclusion  mold Dictyostelium  discoideum  has been  found  to possess P E N-  of D. discoideum appears to be composed of two enzymes, a  based on the biphasic nature  velocity data (220).  development),  of reciprocal plots  of AdoMet  The major product of P E A -methyltransferase 7  to be P M M E (219,220). mold  O-P  (218).  enzyme systems for the methylation of both neutral lipids and P E (219). methyltransferase  (214).  initial  was reported  It has been suggested that cell aggregation (a stage in  mediated  by cellular  cGMP  levels  that  are elevated  in  response to occupation of c A M P receptors on the cell surface, is accompanied  36 by  a parallel  activity  in  cGMP  cell  and  correlate  stimulation  homogenates  calmodulin  with  in P E methylation was  enhanced  (220,221).  developmental  as  sugars  and  modification Genetic  amino  manipulation  case,  this  cells,  but  protein such  with  pituitary,  parotid  aforementioned observed  in  between  PE  formulated  techniques  for  coincided  may  series in  of  to the  with  also play  that  role  in  and  signal in  by  methyl  of  PE  In  from  methylation  labeled lipids preceeded the  prostaglandins. inhibitors  of  Also, PE  DZA,  suggested response  of  methionine  on  the  that  increased  in  motile  or  was  reports  observation  and to  cells  Protein from  AdoMet  was  association  membranes  was  correlated  with  based that  the  effector second-  synthesis  arachidonate, C a  2 +  homocysteine-thiolactone, attenuate  the  course of these  across  appearance of c A M P ,  appeared  eukaryotic  agonist or chemotactic  these  the  in  and the  P E methylation  3-deaza-SIBA  methylation,  tested  P E methylation,  summary,  A s is often  was during the  summarized  2.  was  (222).  methylation  hormones  transmission  which  protein  such  covalent  movements  secretion It  label  of  and  (223).  are  function  not  seemed  effectors  spermatids  response of a tissue or cell to a particular  messenger  did  instead  binding  response.  chemotactic and  a  role  Evidence  the  methyl  Instances  Table  with  Transmembrane  flagellar  response  the  in  activity  (chemotactic  chemotactic  results.  phospholipids, presumably  (99,224).  treatment  and  and  receptor  the  chemotactic  neutrophils  methylation  cAMP,  specific  and adrenal glands (223). studies  by  stimuli  supported  spectacular  leukocytes,  carboxymethylation  a  culminate  model  less  methylation as  by  being central  bacterial  upon  methylation  Methylation  environmental  that  2-fold  PE  TV-methyltransferase  synthesis (219).  acids)  episodes  and demethylation  to  about  However,  1.10 Phosphatidylethanolamine Signaling respond  PE  changes mediated  to be involved in membrane  Bacteria  (221).  the  of or all  response  37  Table 2.  Compilation of Reports For and Against P E Methylation i n Cell Signaling Events  Tissue and Effect  In Disagreement  Rat basophilic leukemia cell, mast cell and thymocyte histamine and Ca release  Moore et al. (229) Boam et al. (254)  Axelrod et al. (246) Axelrod et al. (247) Axelrod et al. (225) Kannagi et al. (248) Pike et al. (249)  Lymphocyte mitogenesis Leukocyte chemotaxis Reticulocyte cAMP levels Platelet activition Macrophage chemotaxis  Moore et al. (255)  Axelrod et al. (250)  cAMP and Ca levels in fibroblasts Na transport in A6 epithelia Stimulation of SR Ca -ATPase Differentation of lens fiber epithelia Myogenic cell cAMP Paroid gland cAMP Hepatocyte cAMP  In Favour Axelrod Axelrod Axelrod Axelrod  et al. et al. et al. et al.  (101) (102) (244) (245)  Wiesman et al. (251) Ganguly et al. (252) Zelenka et al. (253)  2+  Randon et al. (256) Sung and Silverstein (257) Aksamit et al. (258)  2+  +  2+  Koch et a/.(259) Padel et al. (260) Colard, et al. (261) Schanche et a/(262)  38 of cells to various agonists (224). was  It was suggested that P E  in close association with the  occurred  in  increased with  response  membrane  adenylate  changes  to  fluidity,  cyclase  were  ligand  binding.  the  and  observed  B-adrenergic  result  upon  receptor  and  Generation of  production  /V-methyltransferase  which  of  incubation  cAMP  of  of  was  PE  methylation  PMME  coupling  (225).  (and of  receptor  Membrane  erythrocyte  ghosts  PC)  fluidity  with  AdoMet  (226).  As noted in Table 2, the second messenger role of P E methylation  largely  advocated by  Axelrod and coworkers.  Results from  refute most of the conclusions reached by these workers are  noted  in  transduction lipid  Table  was  2).  based  synthesis or  The either  a lack  to various stimuli (Table The  low  changes  in  mouse  influence that  the  kinase  C  to  PI  observed  of  PE  examples  methylation  stimulation  methyltransferase  of  to  in  methyl  inhibitors  signal labeled  on response  2). activity  in most extrahepatic  membranes This  cells, Ref.  proportion  of  A n interesting of  rat  with  PE  hydrolysis  mediate  to  not  observation  tissues led Vance  account  was  and  liposomes  PMME  and  PDME  report  by  Moore  cells,  of histamine  by  inositol  Ca  2 +  supported by (228),  in  which  membranes  mast  the and  in  showed did  not  demonstrated  cells  initial  and  mouse signal,  response  diglyceride,  (3,229) or  large  work  2+  Instead,  of  the  a Ca -dependent  polyphosphates  release  for  et al. (229)  leukemia  methylation.  intracellular  (230), respectively.  could  140)  basophilic  and subsequent release  was  against  (specific  labs tend  to speculate that the 0.00033% of total P C derived via P E  (LM  coincident  known  effect  erythrocyte  fluidity.  not  no  microviscosity (226).  stimulation  agonists are  in  of  thymocytes, was  (227)  fibroblasts  alteration  on  P E methylation  and de Kruijff methylation  of  evidence  other  was  activate  to  which protein  39 Rationale and Objectives of the Present Study  1.11  A multitude of studies on P E N-methyltransferase 27  years  since  this  enzyme  toward  characterization  lagged,  primarily  utilizes  lipid  incorrect  last  which  40 is  substantiate  methylation  because  the  enzyme  is  As  a  result  of  regulation  pages of  devoted this  kinetic  that  characterization  efforts  researchers  reports  had  contaminating  were  of  of  quite  the  the  only  protein  of  isolated in  reported  purification a  their  (using the  following  liver  Work  has  often  protein  of  have  the  and  information,  been  ascribed  strong in light  remainder  of  this  methyltransferase,  of  text,  should  results have  from  rat  and  in  the  were  partially  pure  liver  microsomes  was  subsequent molecular  and  revealed  literature.  properties  very  Results suggested  erronous,  and  enzyme  and  in  fact  these  characterized  a  preparation.  choline-deficient  indicated a serious re-evaluation of the  the  enzyme  Regulation of P E TV-methyltransferase levels  paucity  seem rather  fruitful  purified  those previously  previous  the  However  level  membrane  functions  may  of P E TV-methyltransferase  These  from  of  in  microsomes.  molecular  integral  questionable  review.  liver  the  an  This statement  characterization  dissimilar  and  literature  to  direct  at  in  appeared  view.  Purification undertaken.  identified  PE  to P E TV-methyltransferase. the  first  of  substrates.  modes  was  have  rat  by  phosphorylation  as a model)  was  of published reports  appeared  in publication  and  investigated  was required. (231-235).  substrate and A  again portion  40  E X P E R I M E N T A L PROCEDURES 2.1  Materials PMME,  P D M E , dipalmitoyl-PE,  2-oleoyl-PE, (prepared  dioleoyl-PE,  by  AdoMet 3  3  dilinoleoyl-PE,  phospholipase  ethanolamine)  were  distearoyl-PE, dimyristoyl-PE,  purchased  D  action  from  dilinolenoyl-PE  on  egg  Avanti  PC  Polar  [mef/iy/- H]methionine,  H]ethanolamine  were  phosphoserine, methionine,  y  3  from  Amersham  X-100,  catalytic  P-ATP,  1 2 5  of  Birmingham,  I-protein  DTT,  AB.  A  and  choline  were mm)  CL-4B  purchased silica gel  plates were  were from  and P B E 94 from  60,  from  NY.  Oxnard,  2.2  salt  Primaria  CA.  Laboratory,  analytical  chloride,  protein  kinase  Molecular mass standards  mm)  HPLC  grade  culture  Gelatin  and  Richmond, C A .  in  the  resin  gel  (arginine-,  dishes  were  nitrocellulose  chromatofocusing  cellulose  chloroform  from  and  and  (2.0  thin-layer acetonitrile  methionine-free)  Gibco Laboratory,  Becton  were  DEAE  Preparative  and  choline-,  from  for  Inc.  60  methanol,  A l l other materials  Dickinson  purchased  from  were of reagent  A -Methyltransferase 7  Grand  and  Co.,  Biorad  grade.  Purification  Isolation of Microsomes  Microsomes were isolated g)  silica  solutions were obtained  Phosphatidylethanolamine 2.2.1  exchange  Biotechnology  (0.2  Dulbecco's M E M  and Hanks' balanced Island,  Pharmacia-LKB  Merck.  Fisher.  polybuffer  [1-  insulin,  for gel filtration and S D S - P A G E , octyl Sepharose C L - 4 B , Sephacryl S-300, Sepharose  of  [me thy l-  collagenase,  cAMP-dependent  and B S A were from Sigma Chemical Co., St. Louis, M O .  presence  Canada,  phosphotyrosine, subunit  TP-egg-PE  the  Lipids,  Corp., U K .  phosphothreonine,  Triton  3 2  and  in  and A T P was purchased from Boehringer Mannheim,  H]AdoMet,  1-palmitoyl-  following  the livers immediately  manner.  from Rats  the  livers of female Wistar  were sacrificed by  cervical  rats (175-225 dislocation  and  removed and placed in ice cold 10 m M Tris HC1 (pH  7.2)  41 buffer  containing  150  mM  fluoride and 1 m M D T T . concentration of 25% motor  driven  12,000  x  NaCI,  for  10  mM  (w/v)  7.9)  apparatus.  min.  The  dounce.  The  treated  prepared  from  specific  activity.  2.2.2  an  were  identical  perfused  was  was  centrifuged  at  subjected  to  then  The cytosol was immediately decanted and m M potassium phosphate buffer  (pH  1 m M D T T and 250 m M sucrose using a hand held  Microsomes  in  and homogenized using a  fraction  also  isolated  perfused with 150 m M NaCI and 0.5 m M E G T A . were  phenylmethylsulfonyl  homogenate  supernatant  resuspended in a 20  containing 1 m M E D T A ,  glass  1 mM  in the Tris-saline buffer  centrifugation at 120,000 x g for 1 h. the microsomal pellet  EDTA,  The liver was cut into small pieces, suspended at a final  Potter-Elvehjem  g  1  livers  manner had  as  that  a higher  from  livers  that  had  been  Following perfusion the livers described  initial  PE  above.  Microsomes  /V-methyltransferase  Preparation of Microsomal Membranes  Microsomes  (20-30 mg/ml  protein)  were suspended in  100  mM  Na2CC«3  and 5 m M D T T , at a final protein concentration of 4 mg/ml, and stirred at 4 ° C for  30  min.  membrane  The  pellet  suspension was centrifuged at  collected and resuspended (using  potassium phosphate buffer DTT.  This buffer  was  referred to as buffer A .  2.2.3  1 h.  7.9)  used in  containing  all  for 1 h and the  a glass dounce)  10%  (v/v)  20  mM  glycerol and 5  mM  subsequent purification  in  steps and will  be  A l l purification steps were performed at 4 ° C .  Solubilization of Microsomal Membranes  Microsomal (w/v)  (pH  120,000 x g  membranes  were  suspended in  buffer  A  (containing  0.7%  Triton X-100) to a final protein concentration of 4 mg/ml and stirred for The mixture  was centrifuged at  120,000 x g  for  1 h and the supernatant  collected and used as a source of soluble enzyme.  2.2.4  Chromatography on Whatman DE-52 Cellulose  42 Soluble P E TV-methyltransferase cellulose (30 100,  at  in  ml/min.  unbound  column  fractions  1.0  in buffer  a column of  A plus 0.7%  P E /V-methyltransferase  DE-52  Triton  activity  Xwas  fractions.  Chromatography on Whatman P-ll Phosphocellulose  equilibrated  0.25  rate of  the  2.2.5 A  passed through  x 2.5 cm), previously equilibrated  a flow  recovered  was  in  from  of  Whatman  buffer  complete  Triton  1.6  the  succession with 100 ml of buffer A containing 0.7% ml containing 0.25%  (w/v)  Triton X-100.  X-100.  cm)  was  rate of  was  (w/v)  x  the previous step were applied to the column at a flow loading  0.7%  (16  pooled  When  containing  phosphocellulose  The  ml/min.  A  P-ll  column (w/v)  was  flushed  Triton X-100  P E TV-methyltransferase  and  100  activity  was  eluted from the column with a 350 ml linear gradient of NaCl from 0 to 0.8 M 0.25%  (w/v)  Triton X-100.  peak from 0.2 to 0.6 M . had  been  NaOH  used  for  according to  recoveries.  P E TV -methyltransferase  We found it necessary to use P - l l  several the  activity  purifications  manufacturers  (and  not  specifications)  eluted  a  in  broad  phosphocellulose that  regenerated in order  The phosphocellulose could also be pretreated  in  in  with to  HCI  achieve  with 0.2%  and good  (w/v)  BSA  in Buffer A containing 0.7% Triton X-100 followed by elution with 2.0 M NaCl in the  same  resin  buffer.  that  change  its  would  otherwise  Active diluted with (w/v).  X-100.  recovery  characteristics  block of  on  high  affinity  sites  on  P E N-methyltransferase  octyl  the and  Sepharose.  Chromatography on Octyl Sepharose CL-4B fractions  from  the  phosphocellulose  column  were  Buffer A (no Triton X-100) to a final Triton X-100 The enzyme solution was pumped (0.5  octyl Sepharose (17 Triton  seemed to  reduce  chromatographic  2.2.6  0.05%  This procedure  The  x  1.6  cm)  equilibrated  column was  flushed  in buffer with  100  pooled  and  concentration of  ml/min) onto a column of A ml  containing 0.05% of buffer  A  (w/v)  containing  43 0.05%  Triton X-100.  column  be  eluted  separated from results  were  It  so that  the  major  obtained  containing 0.15%  is critical to the the  protein  by  (w/v)  main  2.2.7 The  of the enzyme that this  that precedes it  the  Triton X-100,  Triton X-100 from 0.15 to 0.5%  purity  P E TV-methyltransferase  peak  eluting  final  (w/v)  column  with  followed by  peak  (Figure 100  a 250  is  4).  ml  well  The best  of  Buffer  A  ml linear gradient  of  in the same buffer.  Chromatography on PBE 94 final  step  in  the  purification  nature of P E TV-methyltransferase.  The  takes  advantage  pooled  fractions  of  the  from  very  the  basic  previous  step, adjusted to p H 9.4 with 250 m M ethanolamine, were applied to a column of P B E 94 (15 x 1.6 cm) equilibrated in 25 m M ethanolamine (pH 9.4), 10% (v/v)  glycerol and 0.1%  (w/v)  Triton X-100.  loaded at a flow rate of 0.2 ml/min. methyltransferase Concentration the LKB  enzyme  of  activity the  was  dilute,  solution to  Biotechnology Inc.).  buffer A containing 0.1%  not  bound  to  enzyme  this  can  S column (cation  Briefly,  (w/v)  P E //-methyltransferase  was  Similar to the DE-52 cellulose step, P E N-  purified  a Mono  5 mM DTT,  the  Mono  Triton X-100.  be  anion  exchange  achieved  exchange  S column  by  resin.  reapplying  resin, Pharmacia-  was  equilibrated  in  Purified P E / V - m e t h y l t r a n s f e r a s e  was adjusted to p H 7.9 with 0.5 M KH2PO4 and applied to the column at a flow rate of  0.2  ml/min.  Enzyme was  subsequently eluted  at  a flow  ml/min with a linear gradient of NaCl from 0 to 1 M in buffer A . of the gradient  was  15 ml.  rate of  Total volume  Concentration by Mono S chromatography did not  alter the specific activity of P E /V-methyltransferase using P E , P M M E or as substrates and recoveries averaged 75-80%. salt eluate at 4 ° C in 0.1% Triton X-100. least two  months.  0.6  PDME  Enzyme was stored in the high  There was no loss of activity after at  44  Fraction number (4.8 ml/fraction) 4. Octyl Sepharose CL-4B chromatography of P E N methyltransferase. Pooled fractions from the P - l l phosphocellulose step  Figure  were applied to an octyl Sepharose column as described in Section 2.2.6. Protein concentrations ( • ) in the column fractions were measured by the silver binding method (267). PE //-methyltransferase activity was determined in the presence of 0.25 mM PMME and 0.5 mM Triton X-100 (X\ Triton X-100 concentrations (—) were determined by relating the absorbance of unknown samples to those of standards at 275 nm.  45 2.3  Assay of Phosphatidylethanolamine A -Methyltransferase 7  2.3.1  Assay using  The fractions  presence (step  concentrations to  achieve  complete  1 of  [methyl-^H]S-Adenosyl-L-Methionine  of  to  microsomal  4,  refer  Triton  maximal  X-100  removal  of  Table  of  PE  endogenous  3)  3)  in  necessitated  partially the  use  A -methyltransferase  D T T , with a final  of  higher  phospholipids  after  order  activity.  7  The  phosphocellulose  allowed the use of lower Triton X-100  lipid substrate concentrations.  purified  and exogenous phospholipid substrates in  expression  chromatography (Table  5 mM  to  phospholipids  and exogenous  A l l assays were in 125 m M Tris HC1 (pH 9.2)  assay volume of  150  ul.  and  Samples from steps 1 to 4  contained 1.0 m M Triton X-100 and either no addition, 2.0 m M P E , 0.4 m M P M M E or 0.4 m M P D M E . steps.  No more than 25 ug of protein were assayed in these first 4  Purification steps 5 to 7 (Table  3)  were assayed in the presence of  m M Triton X-100 and 2.0 m M P E , 0.25 m M P M M E or 0.45 m M P D M E . the enzyme source to 0.5 m M Triton X-100  were  added to the  Lipids  were  dried  vacuum (10 mM  for  NY)  identical manner assay  9.2),  1 min  Ultrasonic  Farmingdale,  assay as vesicles prepared a  stream  of  nitrogen  microns of Hg) for 30 min.  Tris HC1 (pH  vortexing IM-1  under  0.01%  (w/v)  and immediately  Processor at  a  100  EDTA  with  watt  setting.  was  in the  following  further  dried  manner.  under  high  and 0.02%  (w/v)  Triton  X-100  by  sonicated at 3 7 ° C for 3 min using a Son-  equiped  mixture  and  P E , P M M E and  The dry lipids were resuspended in 20  a  and sonicated above their  components, the  Dilution of  was the major factor in deciding the  volume of enzyme to be assayed in the final 3 purification steps. PDME  0.5  Microtip  (Heat Systems-Ultrasonics,  Synthetic  PEs were  T  m  was added to a final concentration of 200  in  an  Following the addition of these  placed on ice and the  added and allowed to equilibrate for 10 min.  prepared  enzyme  [mef/ty/- H]AdoMet 3  u,M and the mixture  (21  source was LiCi/umol)  was incubated at  46 3 7 ° C for 10 min to assay P M M E - and PDME-dependent activity or 30 min to assay PE-dependent The (2:1,  unless  assay was  v/v).  (w/v)  activity,  stopped by  Methylated  N a C l , vortexed  (2:1,  Counter methyl PDME  (Beckman groups  and  2  ml  of  by  chloroformrmethanol  addition  were  dried  of 2 ml  0.5%  to separate organic  under  redissolved  radioactivity  was  evaporated  from  the  CA)  and  phospholipid/min/mg  3801  expressed  was  in  of  a  0.1  prior  v/v)  to  soluble  Scintillation  as  amount  PE, linear  of  ml  sample  Liquid  protein.  by purified P E /V-methyltransferase  0.2  (2:1,  0.5%  stream  determined  chloroform :methanol  Fullerton,  a  in  was  using a Beckman L S  Instruments, to  and  the  Total  directly  transferred  methylation  min (Fig.  v/v)  counting.  counts were determined  v/v/v)  residues  Chloroform:methanol  scintillation  of  (2000 rpm for 5 min)  (50:50:4,  dried  chloroform/methanol  liquid  addition  The organic phase was washed 3 times with 2 ml of  The  aliquot.  specified.  phospholipids were extracted  NaCl:methanol:chloroform  ml  the  and centrifuged  and aqueous phases.  nitrogen.  otherwise  of  PMME  for  30  and  to  40  5).  2.3.2  Assay using [3 H ] P  Methylation  activity  hosphatidylethanolamine  assayed  with  [3H]PE  was  determined  manner as described in Section 2.3.2, but in the presence of 200 AdoMet and 2 m M  [^HJPE (2500 cpm/nmol).  in pM  a  similar  unlabeled  Following incubation for 30  P C , P M M E and P D M E were separated by T L C and radioactivity  min,  was determined as  described in Section 2.3.2.  2.3.3  Analysis  of Products  by Thin-Layer  Products of P E /V-methyltransferase system PE,  of  PC,  chloroform:methanol:acetic PMME  and  PDME  were  were  Chromatography separated  acid:water added  to  the  by  (50:30:5:2, extracts  TLC  in  a solvent  v/v/v/v).  Carrier  prior  to  separation.  47  0  0  10  10  20  30  20  40  30  minutes  Figure 5. Time course for methylation of PE, PMME and PDME. 57, 60 and 24 ng of purified PE N-methyltransferase was assayed in the presence of 0.5 mM Triton X-100 and 2.0 mM PE, 0.25 mM PMME and 0.5 mM PDME, respectively, for the indicated periods of time.  48 Phospholipids scintillation Scintillant  were vials  visualized  by  containing  (Amersham  exposure  250  ul  of  to  iodine  water,  Canada Ltd., Oakville,  5  Ont.)  vapours,  ml  was  of  scraped  into  Aqueous Counting  added and  radioactivity  was measured after 24 h. 2.3.4  Preparation of ethanolamine  [ H]PE  Phosphatidyl-  3  was prepared by a 3 h continuous pulse of freshly prepared  3  hepatocyte  [l- H]Ethanolamine-Labeled  monolayers  with  15  u C i of [l-^HJethanolamine.  rat  At the end of this  time, medium was aspirated, cells were scraped from dishes and P E was purified from  the  extracted  phospholipids by the  method of  Arvidson (64)  as described  in Section 2.4.1. 2.3.5  Repurification  AdoMet Cellex-P,  and  (molar  experiments  was  were  3  were  absorption  S-Adenosyl-L-Methionine  [methyl- H]AdoMet  respectively,  concentrations  of  as  previously  determined  coefficient not  spectrophotometrically  15,000  repurified,  at  257  nm  described  absorbance  by  =  repurified  M  and (molar  measurements  concentration  absorption  1-X8  at  was  coefficient  =  and  AdoMet 257  AdoHcy used in  - 1  its  Dowex  (269,270).  cm ).  _ 1  on  nm  kinetic  determined 15,000  M  _ 1  cm" ). 1  2.4  High Performance Liquid Phospholipids 2.4.1  Methyl previously  Preparation of Radiolabeled Methyltransferase Products labeled for  methyltransferase mM  PDME,  Chromatographic Analysis of  and  methyltransferase  PDME  the  assay  activities 2.0  and P C  mM  of  were prepared PE-,  (Section 2.3.1). TP-egg  Phosphatidylethanolamine  PE  or  under  PMME-,  N-  conditions described  and  PDME-dependent  Methylation  of 0.25  mM  microsomal  PE  purified  was in 0.5 m M Triton X-100,  by  PMME,  125 m M T r i s - H C l (pH 9.2)  PE  0.4 N-  and 5  49 mM  DTT.  The  components for 3H]AdoMet 37°C  enzyme  10 min.  (20  was  preincubated  on  Incubations  2.3.1.  and  The  nitrogen,  P E in whole microsomes was methylated  the  methylated  redissolved  v/v/v/v). ethanol  were stopped by the  in  a solvent  addition of 2 ml  extracts  chloroform  and  (w/v)  2.4.2  (2:1,  v/v)  of  PC,  PE  and  150  mm  using  a  Pelosphere C18 Perkin-Elmer  PDME  4  stream  were  acid:water  light.  of  separated (50:30:5:2, 95%  Bands were scraped Phospholipids were  Analysis species  HPLC.  of was  performed  Separations were performed on a  column (Perkin-Elmer  solvent  Section  dichlorofluorescein in  molecular  (123).  cartridge  Series  a  at - 2 0 ° C prior to separation by  essentially as described by Patton et al. x  classes  High Performance Liquid Chromatographic Phospholipid Molecular Species  Separation  chloroform:methanol  under  according to the of method of Arvidson (64).  stored in chloroform:methanol  CT)  phospholipid  were sprayed with 0.2%  and incubated at  as described in  evaporated  and phospholipids were visualized under U V  and extracted  4.6  were  [methyl-  in a similar fashion  of  system of chloroform:methanol:acetic  The plates  aforementioned  were absent.  phospholipids extracted  chloroform:methanol  by T L C in  the  p C i / p m o l ) to a final concentration of 200 p M  for 20 min.  v/v)  with  The reaction was initiated by the addition of  except exogenous lipid substrates and Triton X-100  (2:1,  ice  delivery  Corp.,  system.  Norwalk,  Elution  was  monitored at 205 nm using a Perkin-Elmer Bio L C 90 spectrophotometer.  PC, PE  or  absolute  PDME  ethanol. a 20  applied  to  the  reverse-phase column  in  10-25  pl  of  Phospholipid molecular species were eluted in an isocratic mode using  solvent mM  were  system  of  methanol:acetonitrile:water  choline chloride, at a flow  individual  molecular  a Pharmacia Frac  species were  100  fraction  (90.5:2.5:7,  rate of 2 ml/min.  collected using the  collector.  v/v/v),  containing  Peaks corresponding to peak  cutting  program  on  50 Individual dissolved  in  extraction  peaks  2  ml  with  were  of  2  pooled,  dried  chloroform:methanol  ml  of  NaCl:methanol:chloroform  under  a  stream  (2:1,  v/v).  This  and  0.5%  (w/v)  NaCl  (50:50:4,  v/v/v).  The  and dissolved in 2 ml of chloroform:methanol species were identified in  absolute  methyl  5%  methanol  esters  column of  were  (Pierce analyzed  Chemical by  of individual  Company,  gas-liquid  succinate,  using  fractions 100.  were  PE  Molecular 14  IL). on  (w/v)  evaporated  peaks with  chromatography  a  %  BF3  Fatty  acid  6'  x  1/8"  Bellefonte, P A ) , coated with  temperature  precipitated  Precipitates  and  N-methyltransferase with  10%  programming.  partially  trichloroacetic  were set on ice for  10,000 rpm for 5 min  30  min  purified  acid  in a  2%  buffer.  glycerol and 0.25  Electrophoresis was done in 10%  SDS as described by Laemmli (263), or in 5-15% gradient  gels were  solution (w/v)  prepared  in  the  following  was prepared by combining 2.8  acrylamide-0.8% bis-acrylamide solution with 4.23 and 9.8 ml of distilled water. of 60%  of Tris HCI  glycerol (v/v)-30%  M  and 4.14 ml of distilled water.  SDS (w/v) in a final volume of 17 ml.  at  1:1  buffer  with this  acrylamide gels containing acrylamide gradient gels.  manner.  ml of a 20%  A  5%  acrylamide  glycerol (v/v)-30%  ml of 1.5 M Tris HCI (pH  8.8)  contained 8.5  bis-acrylamide solution, 4.23  Both solutions were brought to  T E M E D (6 |j.l)  X-  Precipitates  Tris HCI  The 15 % acrylamide solution (w/v) acrylamide-0.8%  Triton  air dried and dissolved  Samples that did not require concentration were diluted  same SDS 0.1%  0.15%  in a bench top Eppendorf microcentrifuge.  S D S , 5% 2-mercaptoethanol, 20%  (pH 6.8).  in  microsomal  and subsequently centrifuged  were washed twice with one volume of acetone at -20 ° C ,  ml  was  by  SDS and Two-Dimensional Gel Electrophoresis Purified  The  0.5%  -20°C.  Rockford,  and  followed  of  phase  and stored at  nitrogen  was  ml  organic  100-120 mesh Supelcoport (Supelco Inc.,  diethyleneglycol.  2.5  by transmethylation  2  of  ml  0.1%  was added and the solutions  51  were degassed.  Ammonium persulfate was added to a final concentration of  0.02% (w/v) and the solutions were placed in an acrylic gradient former. A linear gradient was poured over a 5 min period.  The stacking gel solution  consisted of 3% acrylamide (w/v), 0.125 M Tris HC1 (pH 6.6) and 0.1% SDS (w/v), with ammonium persulfate and TEMED concentrations the same as the running gel. All SDS-PAGE was run at 25 mA constant current. Purified PE /V-methyltransferase was resolved in a two-dimensional electrophoresis system described by O'Farrell et. al. (264). Briefly, NEPHGE gels (first dimension) contained 9.2 M urea, 2% (w/v) Triton X-100 and 5% (v/v) pH 3-10 Ampholytes.  PE /V-methyltransferase was loaded at the anode and  electrophoresed for 2000 V/h. The first dimensional gel was extruded, soaked in 60 mM Tris HC1 (pH 6.8), 2-mercaptoethanol, 2% SDS and 10% glycerol for 10 minutes, applied to a 1% agarose cushion on a 5-15% gradient gel and electrophoresised at 25 mA.  Gels were fixed in water:ethanol:acetic acid  (50:40:10, v/v/v) and silver stained (279). 2.6  Purification of Microsomal Phosphatidylcholine  Phosphatidylethanolamine  and  Microsomes (400 mg of microsomal protein, 25 mg/ml) were mixed with 8 volumes of chloroform :methanol (1:1, v/v) and stirred at 20° C for 30 min. The mixture was filtered through a plug of glass wool, total lipids extracted by the method of Folch (265) and the organic phase flash evaporated.  The lipids  were dissolved in a small volume of chloroform and applied to preparative thin-layer plates (silica gel 60, 2.0 mm).  Plates were developed in a solvent  system of chloroform:methanol:water:acetic acid (50:30:2:5, v/v/v/v) and the PE and PC zones identified and eluted according to the method of Arvidson (64). Purified microsomal PE was stored in chloroform:methanol (2:1, v/v) under nitrogen at -20°C. 2.7  Protein and Phosphorus  Assays  52 Protein was determined by the method of Lowry et. al. (266), modified to contain deoxycholate  at a final concentration of 0.04% (w/v), or by a sensitive  silver binding assay (267).  Both protein assays used BSA as a standard.  very dilute protein concentrations in the PE /V-methyltransferase  The  purification  scheme (particularly the final 3 steps) required the use of a sensitive protein assay  to  avoid using  determinations. concentrations  In  most  of  regions  could  be  the of  pure sample the  determined  to  purification  accurately  by  obtain  accurate protein  scheme both  where  methods,  protein  the  silver  binding assay gave values that were 10-20% lower than Lowry determinations. Lipid phosphorus was determined by the method of Rouser et al. (268). Sterile solutions of 1 mM KH2PO4 were used to generate standard curves.  2.8  Animal Maintenance and Hepatocyte Culturing 2.8.1  Animals and Diets  Male or female  Wistar rats (150-175 gm) were maintained on standard  Purina rat chow and water ad  libitum.  In the choline deficiency studies, male  Sprague-Dawley rats (45-50 gm) were fed a diet consisting of 10% vitamin-free casein, 10% a protein, 56% sucrose, a 4% salt mixture and vitamins, but with no choline (w/w)  (ICN Biochemicals, choline  Canada).  included in this  Choline-supplemented  ICN diet.  In the  rats  had  molecular species  0.4%  studies,  hepatocytes were isolated from the livers of female Wistar rats (150-175 gm). New Zealand white rabbits were maintained on Purina rabbit chow (and an occasional  lettuce leaf).  2.5.2  Preparation and Culturing of Primary Monolayers of Rat Hepatocytes  Rats  were  intraperitoneal Hepatocytes  lightly  injection  were  modification (137).  isolated  anesthetized of  5  mg  as  previously  with  diethylether  pentobarbitone/100 described  g  (271,272),  followed body but  by  weight.  with some  Briefly, livers were perfused through the portal vein with  53 40-50 ml  of Hanks' solution (Ca^+  and Mg2+  free) supplemented with 20  glucose, 25 m M Hepes, 0.5 m M E G T A and 10 pg/ml insulin. 50-60 ml  of collagenase (6  excised and further at 3 7 ° C .  mg/ml)  digested in  dissolved in  resuspended in Dulbecco's M E M containing plated at a density of 3 x 10*> 17% fetal bovine serum. in a 5% CO2 For  in  which  PC  with  were sampled for  medium  was  and  mol%  extracted and  with  our  fractionation  of and  methyltransferase  Cells were  of Dulbecco's M E M  choline-deficient  containing  MEM  supplemented  with  In  by  our  in  were  pM  probed,  200 24  by  the  30  min  addition of  1 h incubation, the pM h.  methionine,  of  v/v),  molecular  15  medium  was  and cells  and  Phospholipid from (2:1,  cells and  separated species in  by  TLC  PC  was  HPLC.  collaborative  immunoblots  (containing 200  were  rat model was developed by Zemin Yao, a graduate  following  rats,  species  were initated  distribution  choline-deficient  activity  was probed in the  containing  label  laboratory.  assays  Dulbecco's  agitation  for 2 min) and  fetal bovine serum.  chloroform:methanol  methyl  following  maintenance  Enzyme  x g  was  medium was exchanged for Dulbecco's M E M  a period up to  The choline-deficient  for  17%  Following a  Dulbecco's M E M  medium  in  centrifuged (50  molecular  Experiments  p C i [mefAy/-3H]methionine/dish.  student  liver  atmosphere prior to the start of experiments.  experiments  determined  The  Cells were allowed to plate for at least 5 hours at 3 7 ° C  without serum or methionine.  the  Hanks' buffer.  cells/dish in 3 ml  prior to the start of the experiment  replaced  This was followed by  10 ml of collagenase solution by gentle  The cell suspension was filtered,  mM  were  cholinemanner.  plated 17%  rats  for  and  and  Zemin  by  h  or  100  of  responsible hepaotcytes.  myself.  methionine-deficient  Hepatocytes, isolated 6  was  culturing  performed  delipidated  methionine  effort,  in  choline-  bovine pM  from and  fetal  choline.  N -  hepatocytes the  livers  of  methionine-free  serum) At  PE  and  then  various times,  54  cells were rinsed with PBS (pH 7.4) and homogenized in 20 mM potassium phosphate (pH 7.9), 1 mM EDTA, 1 mM DTT and 250 mM sucrose by 40 strokes of a motor driven Potter-Elvehjem apparatus.  A membrane fraction was prepared  by centrifugation at 120,000 x g for 1 h and PE N-methyltransferase  activity  was assayed as in Section 2.3.1. 2.9  Immunochemistry 2.9.1  Immunoblotting  Proteins separated by SDS-PAGE were transferred to nitrocellulose according to the method of Tobin et al. (273), with several modifications. Briefly, 5-15% acrylamide gradient gels were soaked in transfer buffer (25 mM Tris HCI, 192 mM glycine, 20% methanol (v/v) and 0.03% SDS at pH 8.3) for 15 min.  The transfer cassette was placed horizontally and a Scotch-Brite pad and  filter paper (Whatman 4Chr), both soaked in transfer buffer, were placed on top.  The SDS-gel was placed on the filter paper and any bubbles removed.  Meantime, a nitrocellulose sheet was soaked in transfer buffer for 15 min. The surface of the SDS-gel was wetted with 2 ml of transfer buffer and the nitrocellulose placed on top. surface with a finger.  Contact was assured by gently smoothing the  Finally, filter paper and a Scotch-Brite pad were placed  on the nitrocellulose, the cassette was closed, placed in a transfer chamber (Biorad Transblot Cell, Biorad, Richmond, CA) with the nitrocellulose facing the anode, immersed in transfer buffer and subjected to a field strength of 150 V (0.55 A) for 4 h. 2.9.2  Immunodetection of Phosphatidylethanolamine transferase on Nitrocellulose Membranes  N-Methyl-  Nitrocellulose blots were incubated in 25-50 ml of buffer B (150 mM NaCl, 5 mM EDTA, 50 mM Tris HCI, 0.05% (v/v) Nonidet P40 and 0.25% (w/v) gelatin at pH 7.4) containing 150 ug/ml of rabbit anti-PE N-methyltransferase IgG for 4 h at 20°C. The antibody solution was replaced with 100 ml of buffer B  55  and incubated for 1 h at 20° C. buffer B containing 0.1 uCi/ml  This wash solution was replaced with 20 ml I-protein A and incubated for 2 h at 37°C.  125  The nitrocellulose was then washed with buffer C (1 M NaCl, 5 mM EDTA, 50 mM Tris HCI, 0.25% (w/v) gelatin and 0.4% (w/v) sodium lauryl sarcosine at pH 7.4) for 2 h at 37°C.  Finally, the blot was rinsed in distilled water, dried and exposed  to Kodak XAR-5 film at -70°C.  Film cassettes were equiped with intensifying  screens. 2.93  Production of Anti-Phosphatidylethanolamine transferase IgG.  N-Methyl-  PE TV -methyltransferase antibodies were raised in female New Zealand white rabbits (1.5 kg) using TCA-precipitated enzyme or native enzyme. The TCA-precipitated enzyme was prepared by bringing unconcentrated purified enzyme solutions to 10% (w/v) TCA, stirring at 4°C for 1 h and centrifuging at 12,000 x g for 10 min. Supernatant was decanted and the pellet washed twice with 20 ml of -20°C acetone and each time centrifuged at 12,000 x g for 10 min. Residues were dried under nitrogen and dissolved in 1 ml of 20 mM Tris HCI (pH 7.0), 150 mM NaCl and 0.5% SDS. Native antigen was prepared by concentrating enzyme against solid polyetheneglycol 8000 to a volume of 1-2 ml and dialyzing versus 20 mM potassium phosphate (pH 7.4), 150 mM NaCl, and 0.1 mM DTT for 24 h. Both antigens (75-100 ug) were emulsified in equal volumes of Freund's complete adjuvent and injected subcutaneously at 5-6 sites on the rabbit's neck and back. After 4 weeks, a booster of 25 pg PE N-methyltransferase in Fruend's incomplete adjuvent was given in a manner identical to primary injections. After 2 weeks, a blood sample was drawn from an ear vein and the presence of PE N -methyltransferase antibody was tested by immunoblotting.  When titre  was detected, the rabbit was bled by cardiac puncture, blood clotted at room temperature for 2 h and serum separated by centrifugation for 10 min at  56 12,000 x g. to  Antibodies against the native and denatured enzyme could be used  detect  neither kDal  enzyme  of the  protein  antibodies under  nitrocellulose  IgG  Walker  immunoprecipitate  of detergent  against  the  and buffer  Unfortunately,  activity  or the  conditions.  enzyme  18.3  Anti-PE  was  used  in  Nall  herein.  fraction of rabbit  (274).  Briefly,  serum was purified by the method of  serum  was  4  1.75  M  phosphate,  (NH4) S0 . 2  1 mM  The  4  EDTA,  1 mM  buffer (2 litres) for 24-34 h. column  enzyme  denatured  ( N H ) 2 S C > 4 , centrifuged at 10,000 x g with  membranes.  Purification of Rabbit Plasma IgG Fraction.  The and  would  raised  described  2.9.4  on  a variety  methyltransferase experiments  protein  of  DEAE  phosphate buffer,  pellet NaN  3  adjusted  to  50%  Mayer  saturation  with  for 10 min and the pellet washed twice was (pH  redissolved in 8.0)  10  mM  potassium  and dialyzed against the same  The dialyzed sample was applied to a 1.6 x 20 cm  Sepharose  CL-4B,  equilibrated  in  the  aforementioned  and unbound fractions were collected, pooled and stored at -  70°C.  2.10  Phosphorylation transferase. 2.10.1  subunit  Phosphorylation potassium  Phosphatidylethanolamine  was  of of  pure  7  bovine  done in  phosphate (pH  P E A -methyltransferase  7.0),  1.5 10  heart ml  mM  3 2  P-ATP  MgCl  2  and 10 m M  500 ng) were added. were  at  30° C  for  pg  of  times  using  in  the  mM  buffer system.  cAMP-dependent kinase,  and pure P E /V-methyltransferase  indicated  the  kinase.  using a 50  DTT  The final volume of the cocktail was 150 the  tested protein  plastic Eppendorf tubes  mM  (200-500 cpm/pmol)  was  cAMP-dependent  Following the addition of these components, 1.5 0.1  ^-Methyl-  Enzyme Phosphorylation In Vitro  Phosphorylation catalytic  of  pertinent  (100-  pl.  Incubations  Figures  and Tables.  Reactions were terminated by the addition of 150 p l of 20% (w/v) T C A , set on ice  57 for  20 min and centrifuged  10 min). acetone enzyme  Supernatant and dried  was then  in an Eppendorf microcentrifuge  was removed, the pellet  at  room  temperature  exposed to Kodak X A R - 5 film at - 7 0 ° C .  20  The  phosphorylated  If the amount of phosphate incorporated band  7  gel and subjected to liquid  min.  stained with Coomassie Blue R and  was to be determined, the P E A -methyltransferase dried  rpm for  was washed once with ice cold  for  subjected to S D S - P A G E ,  (14,000  scintillation  was excised  from  the  counting.  2.10.2 Analysis of Phosphoamino Acids by Thin-Layer Electrophoresis. Phosphorylated  amino  acids of P E N-methyltransferse  by acid hydrolysis and T L E (275). methyltransferase  (Section  were  identified  Phosphorylated and TCA-precipitated P E N-  2.10.1) was washed  successively with  20% (w/v) T C A , 5% T C A and ethenacetone (1:1, v/v).  1 ml ice cold  The sample was dissolved  in 0.2 ml 6 N HC1, transferred to a glass screw cap vial with a teflon cap liner, flushed with nitrogen  and heated  at 1 1 0 ° C for 2 h.  HC1 was removed under a stream of nitrogen 10 |xl of T L E electrode formic  acid:distilled  phosphothreonine  buffer,  Following hydrolysis, the  and the residues were dissolved in  the composition of which  water  (78:25:897,  and phosphotyrosine  v/v/v).  standards  (16  with  electrode  chamber (Shandon 600 X both  ends  ensure  and a clean  good  subjected  to  electrophoresis  contact  buffer,  placed  horizontally  The plate was lightly in  oven.  an  between  electrophoresis was complete,  the T L E plate toward  the  and wicks.  anode  for 3  the plate was air dried  h  electrophoresis wicks placed at  on top to prevent  evaporation and  The samples at  0.5  at room  30 min, sprayed with 0.2% (w/v) ninhydrin in 95% ethanol 160°C  Phosphoserine,  100 model, Cheshire, U K ) , Cambrelle glass plate placed  acid:88%  pg) were added to the  sample and it was spotted on a cellulose thin-layer plate. sprayed  was acetic  kV.  were When  temperature  for  and developed in a  The plate was exposed to Kodak X A R - 5 film at - 7 0 ° C and the  58 mobility  of the  standards.  3 2  P-labeled  amino  Phosphorylated  phosphoserine  acids  and  compared  hydrolyzed  to  histone  the ninhydrin was  also  stained  run  as a  standard.  2.10.3 Effect of Phosphorylation on Enzyme Activity In Vitro. Phosphorylation ATP  was used.  removed  from  dependent  was performed  A t various  2.10.1,  cocktail  of the purified  and assayed  the enzyme  Azido-derivatives Fortunately,  of  are  was achieved,  by photoaffinity  expensive  iV-Methyl-  or  methyltransferases  labeling  difficult  (277,278).  AdoMet  in microsomes  membranes  (100-200  Spectronics Corp.,  to  in the following  manner.  Microsomes or  Westbury,  4%  SDS disruption buffer  the  samples were  (hand-held  DuPont, Boston, M A ) . - 7 0 ° C for 7-14 days.  of 1 cm.  ENF-24  Irradiation  Following irradiation, 50 p i of  (refer to Section 2.5) was added to the cocktail and  acid (40:50:10, v/v/v),  10% (v/v)  and irradiated  Spectroline model  N Y ) at a pathlength  subjected to S D S - P A G E  ethanol:water:acetic  photolabel  ug) were suspended in 0.15 ml of 50 m M Tris-  was performed in 0.5 ml Falcon microtitre wells.  in  synthesize.  utilized  for 20 min at 4 ° C with 254 nm U V light  R  made.  was  3  Blue  were  to  H C l (pH 9.5), 5 m M D T T , 4 u C i (0.35 nmol) of [methyl- H]AdoMet  lamp,  attempts at  AdoMet is photosensitive and has been used to photolabel cytosolic  methyltransferases  microsomal  in microsomes AdoMet  PMME-  enzyme as described in Section 2.3.1.  Before purification of P E AT-methyltransferase identifying  unlabeled  for P E - or  Photolabeling of Phosphatidylethanolamine transferase in Microsomes  2.11  except  time points during phosphorylation a sample was  the phosphorylation  activities  as in Section  acetic  acid  (samples stained  were not boiled),  fixed in  with 0.04% (w/v) Coomassie  and impregnated  with  fluor  (En Hance, 3  After drying, the gel was exposed to Kodak X A R - 5 film at  59  2.12 A -Terminal Amino Acid Sequence Analysis of PE ^-Methyltransferase 7  A-terminal amino acid sequencing was performed by the Northern 7  Alberta Peptide Institute, University of Alberta, for a cost covering the use of reagents.  Purified enzyme was precipitated with TCA as described in Section  2.10.1 and subsequently dissolved in 0.01% (w/v) DTT and 0.2% (w/v) SDS. This material was used directly for amino acid sequence analysis. 2.13 Data Analysis Assays of PE A-methyltransferase activity were performed in duplicate 7  and expressed as an average of the two values.  Linear regression analysis  (performed on a Macintosh Plus computer using the Cricket graph program) was used to determine the best fit of lines through data points on all Hill and double reciprocal plots as well as for replots of kinetic data.  In cases where  double reciprocal plots were nonlinear, curves were drawn by hand. Values in tables were expressed as the average of the indicated number of determinations with either the range or standard deviation given.  Where  appropriate, significance of difference between the means of two sets of data was determined by the students T-test  60  RESULTS 3.1  P E N-Methyltransferase Properties 3.1.1  Purification  and  Molecular  Purification  Table 3 shows a typical purification of PE //-methyltransferase crude microsomal fraction of rat liver.  Activities for methylation of PE, PMME  and PDME copurified, but not to the same degree. methylation of PMME and PDME phosphocellulose  from a  The activities for the  purified to the same degree to the  step, after which the PMME-dependent activity showed a  substantially higher fold purification (1542- versus 832-fold). dependent methylation activity was found to purify 429-fold.  Microsomal PEThese results can  be rationalized by considering that the individual lipid substrates are also providing the enzyme with an unique phospholipid environment in each case. Differences  in phospholipid head group and fatty acid composition would  affect the properties of the mixed micelle substrate, thereby influencing PE Nmethyltransferase activity.  Unlike previous purification schemes that relied  on endogenous PE in order to assay PE AT-methyltransferase (114), it was found that PE N-methyltransferase activity was completely dependent on exogenous substrate following the phosphocellulose step.  This is consistent with the  observation that all measurable lipid phosphorus was removed following this step (Table 3). The hydrophobic character of PE //-methyltransferase  is illustrated by  the high Triton X-100 concentration necessary to release it from microsomal membranes and by its high affinity for octyl Sepharose CL-4B (Fig. 4).  This  step offered the most substantial purification (100-fold) and was critical with regard to final enzyme purity. As illustrated in Fig. 4, PE A^-methyltransferase elutes  in  a  broad  peak  following  single  step  elution  with  TABLE 3. PURIFICATION OF PHOSPHATIDYLETHANOLAMINE /V-METHYLTRANSFERASE  Specific activities  1  Fraction  Volume (ml)  Protein Lipid (mg) phosphorus (pmol)  NA^  PE  PMME  PDME  Total % Recovery^ Fold Activity2»3 Purification  Microsomes  35  1227.8  742  1.05  1.47  5.57  4.51  6838  100  —  Microsomal Membranes Soluble Membranes DE52 Cellulose  41  743.7  623  1.49  1.73  8.96  5.29  6663  97  1.6  180  478.8  577  0.25  0.44  6.18  7.55  2959  43  1.1  206  350.2  585  0.40  0.64  12.8  10.8  4486  65  2.3  P-ll Cellulose  183  40.3  N.D.  5  N.D.  5.96  34.25  44.5  1380  20  6.1  Octyl Sepharose CL-4B PBE 94  140  0.25  N.D.  N.D.  156  3560  1575  871  12  639  138  0.041  N.D.  N.D.  631  8590  3750  352  5  1540  Specific activities expressed as nanomoles of methyl groups transferred/minute/mg of protein. 2 xpressed as nmol/minute. ^Total activity, recovery and fold purification were calculated for PMME-dependent methylation. ^No addition of exogenous lipid substrates. Not detected 1  E  5  62 0.15%  Triton  X-100.  pooled fractions to  3.1.2  (88),  was necessary to  keep  the  volume  of  the  a minimum.  distribution  all three  PMME  of  methylated  products  and  PC,  Triton  X-100  respectively.  mixed  radioactivity  found  the  at  each step  lipid substrates is shown in Table 4.  and P D M E methylation  PDME  throughout  gradient  Analysis of Methylated Products  The and for  The  micelle in  entire  resulted  in  Methylation  of  assay showed the  these  same  two  purification.  the  P C (the  two  purification  of  reported  predominately  substrates  same trend  major  the  As previously  formation  these  products.  of  using  with 95-99%  This  trend  was  product of P E  of  the the  observed  methylation)  showed a 10-15% increase in content, with a concomitant decrease in P M M E and P D M E , as the enzyme was purified. Methylation of P E , P M M E and P D M E was found to be linear for up to 30 min  (Fig.  5).  methylation formed  As  is P C .  was  proportional  PDME  previously  reported  (88),  When P M M E methylation (Fig.  6).  However,  the  of  PDME  was examined, the major  product  with  major  increasing  product  time  there  increase in P C counts and decrease in P D M E counts.  was  This  a  result  would indicate that as the proportion of P D M E in the mixed micelle increases it can  compete  effectively  for  methylation  with  PMME.  Methylation  resulted in the linear accumulation of P C for 40 min (Fig. 6). reached steady state levels in 2 to 5 min. 6) produced P C linearly is  a  good  between  micelles  3.1.3 As enzyme  indication  that  is not  the  rate  P M M E and P D M E  As expected, P D M E methylation  The linearity of  exchange  of the methylation of  PE  (Fig.  reactions  phospholipid  substrate  rate-limiting.  pH Optima for Methylation.  illustrated  have  for 30 min.  of  a  pH  in F i g . 7, optimum  all three at  10.  methylation  The  alkaline  activities pH  optima  of the of  purified  the  three  63  Table 4.  DISTRIBUTION OF METHYLATED PHOSPHOLIPIDS DURING PE /V-METHYLTRANSFERASE PURIFICATION  Distribution of the individial phospholipids are expressed as the percentage of the total counts in PC, PMME and PDME. The recovery of applied radioactivity was 60-70% with > 95% of the recovered counts in the three methylated products. Results are expressed as the average ±_ S.D for 3 determinations unless indicated by numbers in parentheses. Fraction  Substrate  PC  %  Product distribution PDME PMME  Microsomes  NA PE PMME PDME  77.1±17.7 76.7±14.8 3.2±1.8 96.7±1.6  10.5+3.4 11.7+3.9 95.7±2.3 2.0±0.9  12.5+14.9 11.5+12.3 1.1+0.5 1.4+0.6  Membranes  NA PE PMME PDME  75.1+25.2 81.3±14.8 6.8±2.4 95.5±1.6  12.3+8.3 10.3±3.8 92.8+2.6 3.5±0.8  12.5+17.2 8.4+11.1 0.5+0.2 1.0±0.8  Soluble Membranes  NA PE PMME PDME  46.6(2) 82.0±9.7 1.9±1.2 98.1±1.0  32.4(2) 9.1+4.1 96.9+1.6 1.6±0.6  21.1(2) 8.8+6.7 0.5+0.6 0.3±0  DE 52 Cellulose  NA PE PMME PDME  39.6(2) 71.8+15.9 3.0±1.7 97.5+1.1  32.4(2) 20.6+16.3 96.5+2.0 2.3±0.9  21.1(2) 7.6+4.8 0.2+0.3 1.2+1.3  P-ll Cellulose  PE PMME PDME  78.1±4.7 2.9±1.6 98.8+0.8  16.5±2.5 97.7±2.1 1.1+0.9  5.4+3.4 0.5+0.3 0.2±0.1  Octyl Sepharose CL-4B  PE PMME PDME  81.2+12.9 3.9±4.1 99.0±0.4  14.7+8.3 96.1+4.4 0.6±0.4  3.8+4.8 0.6+0.4 0.4+0.2  PBE 94  PE PMME PDME  92.3±6.7 2.5±1.1 95.8±4.3  6.6+5.9 96.5+1.2 2.5+1.7  1.1+0.3 1.0±0.8 1.7+2.0  no addition  1  64 60  0  10  20 minutes  30  Figure 6. Analysis of products formed during the time course of PE, PMME and PDME methylation. Following methylation, radioactive PC (•), PMME ( X ) and PDME (•) were separated by TLC, counted and expressed as total dpm recovered. 65-70% of the applied counts were recovered for all time points. Purified PE //-methyltransferase was used in all experiments.  65 300  E c E o E  E  o E  Figure 7. pH curves for the methylation of PE, PMME and PDME by purified PE N-methyltransferase. PE TV-methyltransferase was assayed as described in Section 2.3.1. Assays contained 125 mM potassium phosphate ( X ) , Tris HCI ( • ) or glycine KOH (•).  66 methylation group.  It  activities  probably  reflects  the  p K a of  the  substrate's  amino  is feasible that protonation of the substrate's amino group results in  poor binding to the active site of P E N-methyltransferase.  These p H optima are  similar to that reported for microsomes (84,87,88).  3.1.4  Molecular Mass Determination by SDS-PAGE  Analysis of the purified enzyme by S D S - P A G E  in 10% acrylamide gels  indicated that P E AT-methyltransferase was composed of a single 1 8 . 3 ± 0 . 7 kDal subunit (Fig. 8, lane 1). N -methyltransferase a  18.3 kDal  from  protein.  methyltransferase  (n=3)  It was apparent that during the purification of P E  microsomes there  The 50 kDal  (114,115,159,162)  was an increase in the amount of  protein  steadily  previously thought  decreased in content  to be P E N(lane 7 to 2)  and was absent from the pure enzyme.  3.7.5  Molecular Mass  Molecular  Determination by Gel Filtration.  mass analysis of the native  enzyme  in Triton  showed that P E - , P M M E -  X-100,  by gel  filtration  on Sephacryl S-300,  and PDME-dependent  activities  co-chromatographed with a Stokes radius of 55.2 A (n=2, F i g . 9A).  Pure Triton X-100 micelles were found to have a Stokes radius of 53.1 A when chromatographed difference  on  between  micelle.  same  column.  P E N-methyltransferase  to be 24.7 kDal. Triton  the  The  apparent  and pure  These results indicated that there Indeed,  dimethylsuberimidate  were  attempts negative.  to  cross  link  micelles  of  concentrated  methyltransferase methylation  protein  activities.  column (insert)  was determined  the enzyme  Analysis of the elution  fractions  mass  was a single subunit per  18.3 kDal protein on Sephacryl S-300 is shown in Fig. 9B. staining  molecular  subunits profile  SDS-PAGE  revealed  co-chromatographed  that  of the  and silver  the  with  with  all  putative three  This is strong evidence to suggest that the 18.3 kDal  67  1 2  3  4  5  6  7  Figure 8. Electrophoresis of partially purified and purified PE Nmethyltransferase. 1 ug of purified PE N-methyltransferase (lane 1) was subjected to SDS-PAGE in a 10% acrylamide gel and silver stained. Lanes 2-7 are protein profiles of the various steps from the purification scheme. Lane 2, octyl Sepharose (1 ug); lane 3, P-ll cellulose (1 ug); lane 4, DE 52 cellulose (25 ug); lane 5, soluble microsomal membranes (25 ug); lane 6, microsomal membranes (25 ug); lane 7, microsomes (50 ug).  68  400  fraction number (1.25 ml/fraction)  Figure 9A. Sephacryl S-300 chromatography of purified PE N • m e t h y l t r a n s f e r a s e . A 96 x 1.6 cm column of Sephacryl S-300 was equilibrated in buffer A containing 0.1% Triton X-100. Panel A, 3.68 ng of PE Nmethyltransferase (1.5 ml) was chromatographed at a flow rate of 16 ml/h. Fractions were assayed for P E - ( ^ ) , P M M E - ( X ) and P D M E - ( H ) dependent activities as described in Section 2.3.1, except that incubation times were 20 min. Triton X-100 micelle size was determined by equilibrating the column in 0.35 mM Triton X-100 and chromatographing 1 ml of 8 mM Triton X-100 at a flow rate of 16 ml/h. The elution position of the Triton X-100 micelles was determined by absorbance at 275 nm. The column was calibrated using aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa) and thyroglobulin (669 kDa). The void volume was determined using Dextran Blue 2000. A plot of Stokes radii versus V-log Kav is shown in the insert. Arrows 1 and 2 show the positions of PE A -methyltransferase and pure Triton X-100 micelles, respectively. 7  69  t  -45.0  -21.0  -14.4  47  48  49  50  51  52  S3  54  55  56  57  58  2 Ul  40  60 Fraction number (1.75 mllraclion)  80  Figure 9B. 3.68 ng of enzyme was chromatographed at a flow rate of 16 ml/h and fractions were assayed for PE-(B), PMME-(D) and PDME-(4) dependent methyltransferase activity. Fractions around the activity peak were precipitated with 10% TCA and analyzed by SDS-PAGE in a 10% acrylamide gel (insert).  70 protein is indeed PE N-methyltransferase and that all  a single  enzyme performs  three methylation reactions.  3.1.6  Two-Dimensional Gel Analysis  During  purification it became  possessed an extremely  basic pi.  This observation was corroborated by the  finding that PE //-methyltransferase focusing gels (pH 3-10). NEPHGE  (the  method  apparent that PE //-methyltransferase  would  not  enter  conventional  isoelectric  A two dimensional electrophoresis system, employing of  choice  for resolving basic  proteins)  in the  first  dimension and SDS-PAGE in the second, revealed that PE N-methyltransferase is composed of two (or possibly three) isoproteins (Fig. 10). microheterogeneity  3.1.7  is  The reason for the  unknown.  Immunoblotting of Microsomes and Purified Enzyme  A rabbit polyclonal antibody was raised against the purified enzyme and used to analyze microsomes by immunoblotting. purified  enzyme  enzyme.  was  a proteolysis  The concern was whether the  product and not  the  native microsomal  This concern proved to be unfounded since, as shown in Fig. 11, both  the purified and microsomal enzyme were of identical molecular mass.  3.2.  Kinetics 3.2.1 We  determine  of Phosphatidylethanolamine N-Methyltransferase.  Analysis of Micellar Substrates have  developed  individually  a simple PE-,  methyltransferase activities.  Triton  PMME-  X-100  and  mixed  micelle  PDME-dependent  assay PE  to N-  The purified enzyme was found to be maximally  active when assayed in the presence of 0.5 mM Triton X-100 and 2.0 mM (88 mol%) PE or 0.25 mM (49 mol%) PMME and 0.4 mM (61 mol%) PDME in 1.0 mM Triton X-100. methylation  The mol% of Triton X-100 at which maximum PMME and PDME  occurred is  micelles exists (279).  in  the  region  where  a homogenous  population of  The 88 mol% of PE required for maximal activity is well  71  4NEPHGE  C/3  GO  5  Figure 10. Two-dimensional gel electrophoresis of PE TV methyltransferase. A, two pg of purified enzyme was resolved in a NEPHGE system containing pH 3-10 ampholytes, followed by SDS-PAGE in the second dimension. The gel was fixed in 30% (v/v) methanol/10% (w/v) trichloroacetic acid/3.5% (w/v) sulfosalicyclic acid for 2 h prior to silver staining. B, a schematic drawing of the gel in A with background removed for clarity. The 3 isoforms are shown.  72  Figure 11. Immunoblot of PE /V-methyltransferase in microsomes. 200 ug of microsomes and 150 ng purified enzyme were separated by 5-15% SDS-PAGE, transferred to nitrocellulose and probed with an anti-PE Nmethyltransferase antibody. The autoradiogram shown was exposed for 7 h.  73 above 68  mol% (Triton  X-100  mole fraction of 0.32)  sphingomyelin, which is  the effective limit for a monodisperse population of mixed micelles (279).  Also,  freeze  large  fracture  multilamellar A  analysis  vesicles  of  by  gel  mol%  PE  revealed  the  presence  of  (280).  study was made  substrates  91  of the  filtration  on  structure  a  1.6  x  of P E , P M M E 96  cm  and P D M E micellar  column  of  Sephacryl  S-300  equilibrated in 125 m M Tris HCI (pH 9.2), 5 m M D T T , 0.3 m M Triton X-100 and 3.3 mM  potassium phosphate.  enzyme  assay, but  filtration  column.  room temperature. the  0.75  Phospholipid substrates were prepared ml  x 10  6  the  mixture  was  Separations were performed It  at  instead a flow  loaded  on  rate of  20  for the  an gel  ml/hr  at  is evident from the elution profiles shown in Fig. 12 that  88 mol% P E mixture  (>1.5  of  as if  eluted at the void volume and thus consists of large  dal) multilamellar  structures.  On the other hand, 49 mol% P M M E  and 61 mol% P D M E mixed micelles are retained on the column and elute with Stokes radii of 52  and 63  Stokes radius of 55 A . radii  to  micelles  methyltransferase Methylation of  which  of  of  pure  Triton  substrates  PE  are  is maximal  if  in  X-100  indicated  indeed large  seen  for  non-micellar  these and  two  PE  N-  monodisperse.  aggregates,  the  structure  Methylation Adheres to the 'Surface Dilution' Model in the mixed micelle assay was investigated to  P E /V-methyltransferase  activities  enzymes  methylation  that  homogenous  had  kinetic  described for other membrane bound enzymes. methylation  micelles had a  undefined.  The role of Triton X-100 determine  Pure Triton X-100  The similarity of P M M E and P D M E mixed micelle Stokes  is presently  5.2.2  A , respectively.  are  that  activities  subject  act  to  properties  similar  micelle  showed a definite  peak  those  F i g . 13 illustrates that all  'surface dilution' inhibition,  on mixed  to  a result  substrates (281,282). of  activity  between  three often  All 0.5  and  three 2.5  74 c  500  Fraction number (1.3 ml)  Figure 12. Analysis of micellar substrates by gel filtration. PE (88 mol%), P M M E (49 mol%) and P D M E (61 mol%) were separated by gel filtration as described in Section 3.2.1. Fractions were analyzed for lipid phosphorous following extraction by the method of Folch et al. (265).  mM Triton X-100 Figure 13. Surface dilution of PE, PMME and PDME methylation activities. Assay conditions were as described in Section 2.3.1, except 2 m M P E (•), 0.5 m M P M M E ( • ) and 0.5 m M P D M E ( X ) were assayed at various Triton X100 concentrations. The insert shows P E - , P M M E - and PDME-dependent activities assayed at 88, 49 and 61 mol%, respectively, over a range of Triton X 100 concentrations.  75 mM  Triton  inhibited The  X-100.  as  much  inhibition  increasing  However, as  of  PE  PMME  methylation  PE, P M M E  micelle  and P D M E  and P D M E  concentration  methylation  by  low  surfactant  methylation  (insert).  was  Optimal  essentially linear over a range of Triton X-100  activities  were  not  concentrations.  not  the  enzyme  result  activity  of was  concentrations and at fixed P E ,  P M M E and P D M E mol%s of 88, 49 and 61, respectively.  These results are taken  to  activity  suggest  that  phospholipid  substrate  3.2.3  deviations  catalysis  in  catalyze  reactions  the  order to  fixed  the  reversibly  mixed  inhibits  catalysis classical  in  mixed  at  the  Michaelis-Menten  interface  these in  This can be  of  systems the  the  micelle  a  fixed  large  must  substrate,  and Triton  act on mixed  be  varied  ways:  (i)  was  concentration was fixed and the bulk  Triton  X-100.  In  of  micellar substrates  micellar  micelle  concentration  some  Instead  particle.  substrate  independently  varied,  Triton X-100  in F i g . 13 that enzyme activity  X-100  has  detergent-amphiphile  was  mM  diluting  substrates  kinetics.  concentration of  achieved in three  phospholipid  micelle  concentration  0.5  by  micellar phase.  aqueous phase, enzymes that  analyze  mol%  of  from  concentration  another.  in  enzymology  notable  lipid  X-100  Effects of Mixed Micelle Composition and Concentration  The  In  Triton  of  and one  concentration, at  (ii)  bulk  was  varied,  lipid varied.  It  substrate and  (iii)  was shown  was independent of micelle concentration up to  Fig.  14,  this  observation was extended  concentrations of Triton X-100 close to its C M C of 0.24 m M (110)  to include  and at various  fixed mol% of P M M E (similar results were obtained for P E and P D M E ) .  Here it is  evident that no minimum activity is displayed even at the C M C of Triton X-100, and  thus  constructed  no in  saturation  curves  the  of  range  related  surfactant  This is contrary to the kinetic model for  to  micelle  concentrations  concentration where  can  micelles  be  exist.  N. naja naja venom phospholipase A2  76 (283,284) saturable  E. coli phosphatidyserine  or  binding to micelles occurs.  decarboxylase  (285)  for which  F i g . 15 shows a substrate velocity  true curve  and double reciprocal plot (insert) for fixed P M M E and increasing Triton X-100 concentration.  In  this  case  the micelle  concentration  is constant  but the  surface concentration of P D M E  is varied.  A sigmoidal substrate velocity  and  plot  indicate  resultant  parabolic  inverse  again  curve  apparent cooperativity for  lipid substrate methylation.  Identical results were obtained for P E and P M M E .  For  reciprocal  situation  substrate and  double  concentration  highly  velocity  (iii),  (at  cooperative.  plots  fixed  Triton  Thus,  for  responses were obtained.  of  X-100)  initial were  situations  Hill for  number  and (iii)  similar  inital  at 0.5 m M  Similar behaviour was noted  X-100 were  (Fig. 17).  Hill  numbers  3.1, 4.7 and 6.4, respectively.  numbers of 2.5, 2.8, 6.1 and 12.8 were determined  for P D M E  methylation at 0.5, 1.0, 2.0 and 3.0 m M Triton X-100, respectively (Fig. 18). type  of  cooperative  kinetic  behaviour  phospholipase  A  mixed  as the concentration  micelle  increases.  2  Another  and been attributed  plausible  P E N -methyltransferase) achieve full  catalytic  result of partial  3.2.4  have  activity.  theory  has been  described  (283,284)  This using  to changes in the size and shape of the o f phospholipid is that  boundary  layers  integral  the mixed  membrane  of lipid  The nonlinear inital  in  that  velocity  must  micelle  proteins  (like  be filled to  curves may be the  filling of this domain.  Effect of Phosphatidylcholine on Initial Velocities  Curve fitting of data from Figs. 15-18 is complex (286). and  lipid  to be non-linear  Plots of PE-dependent activity  with higher concentrations of detergent  Hill  versus  In the case of P M M E , there was a marked increase in the  0.5, 1.0 and 2.0 m M Triton  Similarly,  found  (ii)  Triton X-100 (Fig. 16) gave a Hill number of 3.7. for P M M E and P D M E .  velocity  coefficients  were  formulated  under  the  The Hill equation  assumption  of  infinite  77  3  0.0  0.2  0.4  0.6  mM PDME  Figure 14.  of influence of micelle concentration on PDME Methyltransferase activity was assayed at increasing micelle concentrations and at fixed PDME/Triton X-100 ratios of 1:1 ( X ) , 1:2 (•), 1:3 (•) and 1:5 ( A ) . Lack  methylation.  3  20  30  40  50  60  70  80  m o l % PMME  Figure 15.  at fixed PMME and increasing Methyltransferase activity was determined at a fixed PMME concentration of 0.75 mM and titrated with 0.5 to 2.2 mM Triton X-100. The corresponding inverse plot is shown in the insert. Triton  Cooperative  methylation  X-100 concentration.  78  30  20 oi E c  J  o E  >  -  10 -  0.011  0.012  0.013  0.014  0.015  0.016  0.017  1/mol% PE  Figure 16. Cooperativity of PE-dependent methylation. P E Nmethyltransferase activity was assayed in the presence of various P E concentrations at a fixed Triton X-100 concentration of 0.5 m M . The data is expressed in the form of a double reciprocal plot. The insert shows a H i l l plot of the P E initial velocity data.  79  80  0.02  0.04  0.06  0.08  0.10  1/mol% PMME  Figure 17 Cooperativity of PMME-dependent methylation. P E Nmethyltransferase activity was determined in the presence of various concentrations of P M M E at 0.5 m M ( X ) , 1.0 m M ( • ) and 2.0 m M ( • ) Triton X-100 and expressed in the form of a double reciprocal plot. The insert shows the corresponding Hill plot for the three Triton X-100 concentrations.  80  E  o E  >  0.02  0.06  0.10  0.14  1/mol% PDME  Figure 18. Cooperativity  of  PDME-dependent methylation.  PE N-  methyltransferase activity was determined in the presence of various concentrations of PDME at 0.5 mM ( X ) , 1.0 mM ( • ) , 2.0 mM (•) and 3.0 mM (+) Triton X-100. The insert shows the corresponding Hill plot for the four Triton X-100 concentrations.  81 cooperativity, In  a situation that would not apply  addition, the apparent  cooperativity  to steady-state  observed  for activation  proteins  Still,  coefficients are useful empirical measures of cooperativity.  The  cooperativity  apparent  of P E , P M M E ,  on E.coli  of phospholipid (287).  or P D M E  (289), both of which require nonsubstrate lipids for activation. F i g .  increasing  bulk  velocity curves of P E - , P M M E -  activities  substrate.  at  fixed  However,  Triton  this  reaches a maximum  population of mixed micelles exist. small peak of PE-dependent activity substrate  velocity  The  +  X - 1 0 0 concentration and  sigmoidicity  is largely  obviated  A s noted in Section  out of the range  where  a uniform  However, it should be noted that in Fig. 19 a occurred in the mixed micelle range of the  curve.  return  to Michaelis-Menten  illustrated in F i g . 20. data in F i g . 19.  (Na -  and PDME-dependent  when a fixed mol% of egg P C is included in the micelle. 3.2.1, P E methylation  (288) and  is  K )ATPase  P E /V-methyltransferase  kinase  methylation  of previous  19 shows sigmoidal initial  diglyceride  integral  reminiscent +  work  binding  of  membrane Hill  is due to non-cooperative  enzyme kinetics.  This figure  kinetics  afforded  by egg P C is further  shows inverse- and Hill-plots (insert) for the  Hill coefficients for P E , P M M E and P D M E in the absence of egg  P C were 3.6, 2.5 and 4.7 (average of 2 experiments), respectively.  However, with  the addition of an increasing fixed mol% of egg P C (refer to Fig. 19 for details) the coefficients were reduced to 1.2, 1.1 and 0.9 (average  of 2 experiments) for  P E , P M M E and P D M E , respectively.  3.3  Kinetic Mechanism Methyltransferase 3.3.1 The  evaluation Cleland  of  Purified  Phosphatidlyethanolamine  N-  Evaluation of the Kinetic Pathway kinetic  mechanism  of initial  (290).  It  velocitywould  of P E /V-methyltransferase and product  be virtually  inhibition-patterns  impossible  was studied  by  as described by  to determine  the kinetic  82 1.0  0.0  0.3  0.6 0.9 mM PDME  1.2  Figure 19. Influence of egg PC on PE, PMME, and PDME initial velocity curves. Increasing PE, PMME and PDME were methylated at fixed Triton X-100 concentrations of 0.5, 1.0, and 1.0 mM, respectively (X). PMME and PDME were methylated in the presence of 30 mol% egg PC and PE methylation was with 40 mol% egg PC (•).  83  50  0.007  0.00  0.10 1/mol%PMME  0.20  0.018 1/mol% PE  0.02  0.027  0.06  0.10  0.14  l/mol%PDME  Figure 20. Influence of egg P C on inverse and Hill plots for P E , P M M E and P D M E . A , varied mol% of P E was methylated at a fixed Triton X-100 concentration of 0.5 m M and fixed egg P C of 0 ( • ) , 20 ( • ) and 40 ( • ) mol%. B, varied mol% of P M M E , 1.0 m M Triton X-100 and 0 ( • ) , 10 ( A ) , 20 ( • ) , and 30 ( • ) fixed mol% egg P C was methylated. C , a varied mol% of P D M E was methylated in the presence of 1.0 m M Triton X-100 and 0 ( • ) , 10 ( A ) , 20 ( • ) , and 30 ( • ) fixed mol% of egg P C . In all instances, the corresponding Hill plot is shown as an insert. Refer to the text for Hill coefficients.  84  mechanism on the in  basis of P E methylation  alone  since steady  1 min, after which no accumulation of P M M E  shown  in  product  analysis  (Section  3.1.2),  the  state  is reached  or P D M E occurs (88). two  intermediate  As  methylation  steps could be assayed individually with P M M E and P D M E ; in both instances 95100 % of the product was P D M E and P C , respectively. PMME  has yet  nature  of  the  conversion  to  be  first  step  provided  Inverse  assayed as a discrete allows  no  plots  it  to  intermediates  for  variable  be  intersecting  lines  addition  substrates.  of  were  However,  assayed on the  are  present  AdoMet  P M M E (Fig. 21 A) or P D M E (Fig. 2 IB)  step.  The conversion of P E to  at  or  rate-limiting  basis of  P E to  PC  accumulate.  several  are shown.  the  fixed  concentrations  of  In both instances two sets of  obtained,  which  is  partial  evidence  Varying  PMME  or  PDME  (Fig.  22)  for at  a  sequential  several  fixed  concentrations of AdoMet, but in the presence of 30 mol% egg P C , also yielded intersecting and  lines.  PDME  Evidence for  methylation  by  previously been reported (89). PC)  at several fixed  Slope or intercept PE,  PMME  consistent  inverse  plots  partially  with lipid  fixed the  addition  of  substrates for  PMME  P E yV-methyltransferase  has  Inverse plots of variable P E (plus 40 mol% egg  replots of initial  and  pure  concentrations of AdoMet  and P D M E  nature,  a sequential  velocity  were also intersecting (Fig.  patterns,  without egg P C and with  and varied, were highly previous  binding  irregular  discussion detailing  requirements  of  23).  PE  the  or parabolic  in  nonlinearity  of  //-methyltransferase.  Replots of variable P M M E (with 30 mol% PC) versus fixed AdoMet (Fig. 22) were used to determine Product variable lines  kinetic constants and will be discussed in a latter section.  inhibition  AdoMet  intersecting  patterns  and several on the  fixed  horizontal  for  PE, PMME  and  PDME  methylation,  concentrations of A d o H c y , revealed axis  (Fig.  24).  Noncompetitive  at  a set of inhibition  85  Figure  21. D o u b l e  versus  AdoMet  at  reciprocal plots of initial various fixed concentations of  methylation PMME and  rates PDME.  The indicated fixed mol% of PMME (A) and PDME (B) was methylated at varied AdoMet concentrations.  86  -1  0 1 2 3 4 5 6 1/PMME (mM)  -1  0 1 2 3 4 5 6 1/ PDME (mM)  Figure 22. Double reciprocal plots of initial methylation rates versus P M M E and P D M E at various fixed concentrations of AdoMet. Methylation rates at 10 ( X ) , 20 ( • ) , 30 ( • ) , and 40 ( • ) um AdoMet were determined in the presence of 30 mol% egg PC as described in Section 2.3.1. PDME methylation was not analyzed at 30 u M AdoMet.  -0.02  0.02 0.06 0.10 1/AdoMet (uM)  Figure 23. Double reciprocal plots of initial P E methylation rates versus various AdoMet concentrations. Methylation rates at 0.3 (•), 0.4 ( • ) , 0.6 (•), 0.8 (A), and 1.0 ( X ) mM PE were determined in the presence of 40 mol% egg PC and variable AdoMet concentrations as described in Section 2.3.1  87  24. Inhibition of PE, PMME and PDME methylation by AdoHcy at variable AdoMet. Methylation of 88 mol% F E (A), 49 mol%  Figure  P M M E (B), and 61 mol% P D M E (C) was determined at various fixed concentrations of 0 ( X ) , 100 ( • ) and 400 ( • ) Lim and variable AdoMet.  AdoHcy  88  indicates bind  that  the  the  same  two  ligands  enzyme  form  are  separated  (290).  by  a reversible  Product  inhibition  step, but  patterns  do  for  not  several  fixed mol% of P E , P M M E and P D M E at variable AdoHcy were uncompetitive (Fig. 25),  and  therefore  separated  in  the  made so by the presence of 200 inhibition  was  the  reversion  uM  to  remaining  piece  scheme  by  an  irreversible  AdoMet.  Consistant  with  noncompetitive  kinetics  when  indicate  an  lowered to nonsaturating levels (25 One  kinetic  step  uncompetitive AdoMet  was  uM).  of  evidence  to  ordered  Bi-Bi  mechanism is direct competition between P M M E and P D M E for the free enzyme form.  Data in support of this is shown in Fig. 26A for the inhibition of P M M E  methylation slope  by P D M E and is presented in the form of a Dixon plot (291).  replot  (insert)  phospholipid PMME  intersect  methylation  competitively inhibition  was  found  the  by  PDME.  and  full  in  mixed  activity.  exert  conversion nonspecific  and the  However, of  [^HJPE  high  surface dilution  of  PE  was  high  mol%  inhibition  observed  methylation  This  of  to  were  of  inhibit  fraught  inhibition at  and P D M E was  not  No  achieve  inhibit  the  result  concentrations of  inhibition of P C formation.  with  toward  P E necessary to  PMME  since P C lacked effect  and P D M E that produced 50%  at  of P E Af-methyltransferase  in F i g . 27,  3  points  competitive  PMME  concentration  [ H]PC.  but  Experiments to show what type of  over  activity  as shown to  indicative  (Fig. 26B).  PDME  micelles  parabolic,  Similarly,  technical problems due to the low PE  be  origin,  P D M E methylation  PMME  to  A  the of  PMME  accumulation of  labeled P M M E or P D M E was observed at high concentration of cold P M M E or PDME, addition.  indicative The  of  results  inhibition of  are summarized in Table 5.  these  of  PE  initial  methylation velocity  at  the  and product  first  methyl  inhibition  group patterns  89  0  r-  o' 0  o.o  0  100  200  1  1  100  200  •  100  1  200  liM A d o H c y  300  400  1  300  1  300  400  1  400  Figure 25. Inhibition of PE, PMME, and PDME methylation by A d o H c y at fixed and saturating A d o M e t . P E (A), P M M E (B) and P D M E (C) methylation rates, at the indicated fixed m o l % , were determined at variable A d o H c y concentrations and constant AdoMet of 200 u M . Data is presented in the form of a Dixon plot.  90 0.4  0  10  20  1/mol% P M M E  Figure 26. Dixon Plots showing co-inhibition of P D M E and P M M E Methylation. 20.8 ( X ) , 28.3 (•), 34.5 ( A ) and 49.9 (•) mol% PMME (panel A) was methylated at various mol% of PDME. Radioactive PDME was isolated by T L C and radioactivity determined as described in Section 2.3.3. A replot of slope versus l/mol% PMME is shown in the insert. 20.8 ( X ) , 44.0 ( • ) and 50.0 ( • ) mol% PDME (panel B) was methylated in the presence 5 to 20 mol% PMME and the radioactivity in PC determined. A replot is shown in the insert.  91  140  0.00  0.10  0.20  0.30  Phospholipid Concentration (mM)  Figure 27. Inhibition of PE methylation by PMME and PDME. [ H]PE (2 mM) was methylated in the presence of 0.5 mM Triton X-100, 200 uM AdoMet, and various concentrations of PMME (•), PDME (•) and PC ( X ) (Section 2.3.2). Products were isolated by TLC and counted as described in Section 2.3.3. The radioactivity recovered in PC is shown. 3  Table 5. TYPES OF COSUBSTRATE AND PRODUCT INHIBITION PATTERNS OBSERVED FOR PE, P M M E A N D PDME METHYLATION B Y PURIFIED PE /V-METHYLTRANSFERASE  PHOSPHOLIPID SUBSTRATE  COSUBSTRATE EFFECT PRODUCT EFFECT OF OF ADOMET ADOHCY ON Vf( ADOMET)  PRODUCT EFFECT OF ADOHCY ON Vf(PMME ,PDME)  PRODUCT EFFECT OF PMME,PDME on Vf(PDME,PMME)  PE  INTERSECTING  NONCOMPETITIVE  UNCOMPETITIVE  PMME  INTERSECTING  NONCOMPETITIVE  UNCOMPETITIVE  COMPETITIVE  PDME  INTERSECTING  NONCOMPETITIVE  UNCOMPETITIVE  COMPETITIVE  Vf,  forward  reaction  velocity  93 The used  to  various  generate  combinations the  and  inverse-  and  permutations  of  Dixon-plots  substrates  herein  are  and  inhibitors  indicative  of  an  ordered B i - B i mechanism for the methylation of P M M E to P D M E and P D M E to P C (290).  Evidence for a single lipid substrate  patterns  for P E methylation  active  site and product  were used to formulate  a complete  inhibition  model (Fig.  28)  for the methylation of P E to P C based on the nomenclature of Cleland (292).  PE  AdoMet  AdoHcy AdoMet  ,11  AdoHcy AdoMet  t1 tI (E-PMME-AdoMet)  (E-PDME-AdoMet)  (E-PMME-AdoHcy)  (E-PDME-AdoHcy)  (E-PC-AdoHcy)  Briefly, site  dissociates.  the model predicts P E initally followed The  linked together. effect a  by  AdoMet.  next two  The only deficiency  of P C on substrate binding.  classic  methylation.  product  inhibitor  kinetic  binds to a common lipid  PMME  steps are  PC  tt  (E-PE-AdoMet)  Figure 28. A - concerted mechanism for PE methylation.  binding  "AdoHcy  is  essentially  formed two  and  the  ordered  first  Bi-Bi  in this model is the inability  substrate AdoHcy  mechanisms to show the  As shown previously, instead of P C acting as  it  actually  activates  PE,  PMME  and  PDME  By a process of elimination, P C has tentatively been placed as the  last product to depart.  3.3.2  Kinetic Constants  Table  6 is a compilation  and P D M E methylation. for microsomes (88). replots  of  Fig.  24)  The K  of kinetic m  to  for P E , P M M E  values for AdoMet are similar to those reported  AdoHcy Kj appear  constants determined  values  decrease  (determined as  the  PE  from  slope and  molecule  is  intercept  progressively  94  methylated. about Kj  The Kj value of 277 u M for the inhibition of P E methylation is  100-fold  uM).  In addition, the  values for inhibition of P M M E and P D M E methylation by AdoHcy are 10- to  20-fold (89).  greater  than  those  previously  PMME  reported  for the partially  pure  enzyme  Vmax values for P M M E and P D M E methylation,  The 5- and 10-fold greater  respectively, to  greater than that found for microsomes (3  compared to that for P E methylation, conversion is rate-limiting.  support the notion that P E  Furthermore,  was greater than values for P M M E or P D M E .  the apparent  K  m  for P E  Vmax values determined in the  presence of egg P C followed the pattern P D M E > P M M E > P E , but were increased 2- to 10-fold over values determined in the absence of egg P C . data from Figs. found  when  PMME  Slope replots of  21 and 25 were nonlinear, consistent with the sigmoidal curves  lipid  substrate  was variable.  Only  slope and intercept  replots of  data (Fig. 22) in the presence of 30 m o l % egg P C were linear.  AdoMet ° f 11-9 calculated.  A K _ m  uM , Vmax of 32.3 umol/min/mg and K _ p j Y [ M E of 0.4 m M were m  These data are similar to values presented in Table 6.  Table 6. KINETIC CONSTANTS FOR PE A^METHYLTRANSFERASE  1  Lipid Substrate PE PMME PDME 1  K -AdoMet (pM) m  36.6±14.7 39.7(14.9) 13.6(3.5)  K  m-PL (mM)  5.00(1.02) 0.74+0.30 2.12(0.07)  Values are the average 3 determinations  2 Values determined from replots and variable and AdoMet variable. 49 and 61 m o l % , respectively. 3  Constants determined  K  i-AdoHcy (uM) 277.5±175.9 138.3(23.5) 73.9(9.7)  (±S.D)  V  max max (^mol/min/mg) 2  v  0.95±0.20 9.70(0.95) 4.25(1.10)  3  3.38(0.78) 20.36±6.35 40.95(5.11)  or 2 determinations  (range).  of Lineweaver-Burk data with A d o H c y fixed P E , P M M E and P D M E were held fixed at 88,  from Lineweaver-Burk plots at 200  u M AdoMet,  variable  concentrations of P E , P M M E or P D M E and a fixed mol% of egg P C .  3.3.3 The evaluated  Free Sulfhydryls are Required for Methylation role  of sulfhydryls in P E N-methyltransferase  using the purified  enzyme.  A s reported  catalysis  previously  was  (84,85),  a  refree  95 sulfhydryl(s) to  have  2.2).  is  required  a  reducing  DTT  was  for  methylation  agent,  such  removed  from  as  of microsomal P E  DTT,  present  purified  PE  concentration on a Mono S column exactly buffer PE-,  A  minus D T T ) .  PMME-  However,  of all activities. 29).  is essential  purification  (Section  A -methyltransferase  during  7  as described in Section 2.2.7  The enzyme was completely  and PDME-dependent  when D T T  during  and it  activities  in the  inactive  when  assayed for  absence of D T T  was titrated into the assay there  (using  (Fig.  29).  was a complete recovery  Maximum reactivation was achieved at about 10 m M D T T (Fig.  It is possible that removal of D T T results in rapid oxidation of two or more  cysteine  residues  Formation  of  and  formation  intermolecular  disulfide  of  intramolecular  linkages  cannot be  disulfide ruled  out,  bridges. however.  As expected, methylation was inhibited 70-90% by a 2.5 m M excess of I A A and greater then 95% by 2.5 m M D T N B (Table 7). DTNB it  inhibition, but not inhibition by I A A .  seems that cysteine(s)  Table 7.  are required for  Based on these preliminary  catalytic  activity.  Inhibition of PE //-Methyltransferase by Sulfhydryl Modifying Reagents Assay  Reagent  %  D T T (a 2-fold excess) alleviated  1  IAA IAA+DTT Reactivation  DTNB DTNB+DTT % Reactivation  PE 33.4 46.9 13.5%  (3.3) (7.5) (10.8)  2.2 (2.0) 81.9 (2.9) 80.1% (0.9)  Substrate  (%Activity)  PMME  PDME  7.7 (4.8) 13.9 (7.8) 6.2% (3.0)  29.3 (8.3) 40.9 (1.8) 11.7% (6.5)  1.5 (1.7) 64.0 (12.0) 62.5% (10.3)  1.2 90.4 (3.4) 89.2% (2.8)  P u r i f i e d enzyme was incubated with 5 m M I A A or D T N B . A 10 min reactivation with 10 m M D T T was initiated after a 10 min incubation on ice with inhibitor. Results are the average of two experiments with the range in brackets.  results  96  0  10  20  mM DTT  Activation of P E , P M M E and P D M E methylation by DTT. D T T - f r e e methyltransferase  Figure 29.  was incubated with the indicated concentrations of D T T for 10 min on ice and assayed as described in Section 2.3.1. The distribution of products using P E (X), PMME (•) and P D M E ( • ) as substrates was similar to that described in Table 4.  97  3.4 Photoaffinity Labeling of Microsomal ethanolamine /V-Methyltransferase 3.4.1  Photolabeling of Microsomes  Photolabeling investigated cellular 3  of  liver  as a means of  fractions.  H]AdoMet,  and  fluorography,  microsomal  identifying  Irradiation  of  identification  revealed  the  as the  result  microsomes  of  the  of  suggesting  incorporation  in  labeled  photoactivation  AdoMet in  presence  proteins  by  was  tissues of  [me thy l-  SDS-PAGE  bands (Fig.  and  30).  and  The most  The proteins of 19 and 30 kDal were since  both  it  appears  as  a  were  The  19  result kDal  in molecular mass to the P E A -methyltransferase 7  was not  using  absent  in incubations  The band at 25 kDal was labeled in the absence of  (carboxymethylation).  (Section 3.1.4).  the  presence of four major  which were not irradiated. irradiation,  proteins  P E Af-methyltransferase  heavily labeled was a protein of 19 kDal. labeled  Phosphatidyl-  of  direct  protein  was  purified  methyl almost  from  group identical  microsomes  A band was observed slightly above the 19 kDal protein, but this  a consistent observation.  3.4.2 The  Photolabeling of the Microsomal 19 kDal Methyltransferase time  min (Fig. 31).  course of  photolabeling was  essentially  linear  for  least  30  The efficiency of labeling was estimated to be 0.01-0.005% (for a  20 min irradiation) and labeling was independent of p H between illustrated  at  6 and 10.  in F i g . 32, photolabeling was inhibited completely by the  As  addition of  a 300-fold excess of cold AdoMet or AdoHcy, but not by adenosine or adenine. Distribution  of  the  19  kDal  protein  was  examined  in  various  (Fig.  33).  organs  and  correlated with the activity  of P E A -methyltransferase  activities  in liver microsomes, and addition of P M M E resulted  in  a  were the greatest  3.6-fold  methylation liver,  in  however,  stimulation other the  7  of  organs. former  activity. The  19  No  such  and 30  was completely  stimulation  kDal  proteins  absent from  was were  Methylation  observed for observed in  kidney, lung and heart.  98  Figure 30. Photolabeling of microsomal proteins with [methylH]AdoMet and identification by SDS-PAGE. Microsomes were photolabeled for 20 min and subjected to SDS-PAGE. The gel was dried and exposed to film for 6 days. Non-irradiated microsomes were kept in the dark at 4°C for 20 min. 3  99  Figure 31. Time course of 19 kDal protein photolabeling. Microsomes (100 ug) were labeled with [methylH]AdoMet for the indicated times (in min) and identified as described in Section 2.11. The fluorogram shown in the Figure was the result of a 7 day exposure. 3  10  20  30  Figure 32. Inhibition of 19 kDal protein photolabeling by AdoMet and AdoHcy. Microsomes (100 ug) were photolabeled with [mef/ty/-H]AdoMet for 20 min in the presence of 100 um unlabeled AdoMet, AdoHcy, adenosine or adenine (these concentrations are in 300-fold excess compared to labeled AdoMet). Following electrophoresis and treatment with fluor, the gel was exposed to film for 8 days. NA, no addition. 3  100  LIVER  PMME  +  RELATIVE ACTIVITY  3.6  o.6mm  -  KIDNEY  _  +  1.0  0.1  0.1  LUNG  +  0.2  HEART  -  0.2  +  -  0.1 0.1  Figure 33. Distribution of [methy/- H]AdoMet labeled proteins in extrahepatic tissues. 100,000 x g fractions of tissue post-mitochondrial supernatants (100 pg) were photolabeled for 20 min, separated by S D S - P A G E , soaked in fluor and exposed to film for 11 days. The corresponding fluorogram is shown. P E N-methyltransferase activity was assayed with and without P M M E as described in Section 2.3.1. Triton X-100 was not included in the photolabeling cocktails. 3  101 Traces  of the 30 kDal  band were observed in kidney  PMME  in the irrradiation  cocktail  inhibited  labeling  and lung.  of the 19 kDal  liver and the 30 kDal band to a lesser extent (Fig. 33). 30  kDal  proteins  likely  that  the  19  Attempts  [mef/ty/- H]AdoMet  have  3  into  AdoMet-labeled  phospholipid  3.5  3.5.1  34.  is  achieved  of  elution  is  indeed  it seems PE  this  profiles  of  of the enzyme  inhibition  alleviated  will  be  19 kDal  and  protein.  N - M e t h y l transferase.  Chromatographic  of microsomal  with  Once reconstitution  of the microsomal  Liquid  N-  to the 'quenching' effect  Phosphatidylethanolamine  High Performance Phospholipids  Typical molecular  vesicles  process.  should establish the identity  Specificity  protein  been unsuccessful owing  in  The identity of the 25 and  to label the purified P E N-methyltransferase  X-100 on the photolabeling  photolabeling  Fig.  kDal  of  protein  is unknown, but based on molecular mass similarities  methyltransferase.  Triton  Inclusion  P C , microsomal  Separation  of  P E and egg P C  species from a 4.5 x 150 mm C18 reverse-phase column are shown in The resolution of molecular  species is similar  to previous reports on  fractionation of whole liver P E and P C (123) and egg P C (124).  The shorter C18  column allows separation times to be reduced from 2 h (123) to 1 h, but lessened resolution interfere  of peaks 9-10 and 15-16 for microsomal P E and P C . in the interpretation  Peak  identification  shown in Table 8. palmitoyl-  and  their  stearate the  mass  and  results.  m o l % distribution  l-stearoyl-2-docosahexaenoyl  in  molecular  species  are  two  species  relative  to  that  reported  Microsomal P E and P C contain 85% and 91%, respectively, groups  in the one position  two position.  of  Microsomal P E and P C were both found to be enriched in 1-  for whole liver (123). of  of  This did not  of  molecular  and linoleate,  species;  arachidonate  those  with  palmitate  or  or docosahexaenoate  in  Interestingly, microsomal P E contains only traces of saturated  Figure 34. Separation of microsomal PE, microsomal PC and egg PC by reverse-phase HPLC. Eighty pg microsomal PE, 120 pg microsomal PC, and 220 pg of egg PC were dissolved in absolute ethanol and separated by reverse-phase HPLC as described in Section 2.4.2. Peak identification is given in Table 8.  103  Table 8.  M o l % Distribution of Microsomal PC, Microsomal PE and Egg PC Molecular Species  Peak Number  Molecular Species  Microsomal PC (n=3)  Microsomal PE (n=7)  Egg PC or TP-Egg PE (n=3)  1,2 3 4 5 6 7 8 9,10 11 12 13 14 15,16 17 18  18:2-18:3,14:0-22:6 16:1-18:2 18:2-22:6 18:2-20:4 18:2-18:2 16:0-22:6 16:0-20:4 16:0-18:2,18:1-18:2 18:0-20:5,16:0-22:5 16:0-18:1 18:0-22:6 18:0-20:4 18:0-18:2,18:0-22:5 18:0-20:3,18:0-22:5 18:0-18:1  1.5+1.4 0.7±0.4  ND 0.5+0.3  1.5+1.0 3.4+0.2  1.5+0.4  ND ND ND 2.6+1.1  4.9+0.5 9.7+1.5 12.0+1.5 4.6+0.7 ND 11.7+2.8 30.5+3.3 15.9+0.9 2.5+1.1 ND  19.1±2.2 10.5+1.6 8.5+1.5 5.1±0.9 ND 14.2±4.5 32.0+4.8 6.2+1.3 1.7+0.3 ND  1  3.4±1.0 4.4+1.4 20.5+2.7 4.1+1.4 41.6+4.6  L  6.6+1.4 5.4+4.6 3.3+1.9 8.9+0.7  Values are expressed as the average of the indicated number of determinations + S.D. Peak distribution was determined by pooling individual phospholipid species and quantitating lipid phosphorus as described under "Experimental Procedures". ^ND, 2  i.,  not detected indicates  species mol% distribution was pooled with preceding value.  104 or monoenoic molecular species (293).  TP-egg PE contains 60 mol% of 1-  palmitoyl with oleate or linoleate in the 2 position. 3.5.2  In Vitro Molecular Species Specificity  Because  of  their molecular species  composition,  microsomal PE are excellent PE //-methyltransferase enzyme specificity.  TP-egg PE and  substrates  for  testing  Table 9 shows the results of such experiments.  When  microsomes were incubated with [methyl-^H]AdoMet, and the PC fractionated by HPLC, it was found that the % distribution of radioactivity did not parallel PC mol% distribution but was almost identical to that of microsomal PE (Table 8). A similar pattern was obtained when pure PE N-methyltransferase with microsomal PE and the PC molecular species analyzed.  was incubated  Methylation of TP-  egg PE, PMME and PDME resulted in a pattern of labeling that was similar to the mol% composition of molecular species in egg PC. The one exception noted was an  apparent preferential labeling of  l-palmitoyl-2-linoleoyl and l-oleoyl-2-  linoleoyl species of PC and PDME.  These results indicated that PE N-  methyltranferase shows little substrate specificity,  and methylates molecular  species of PE, PMME and PDME on the basis of their mol% within a mixture. 3.5.3  Methylation Rates with Synthetic Phosphatidylethanolamines  Specificity studies were extended to examine the rates of methylation of various synthetic PEs at saturating concentrations of 2 mM. First, it should be noted that while there was no selectivity for any given species of PE between microsomal- and TP-egg-PE, the former was methylated at about 3 times the rate (Fig. 35B).  In addition, l-palmitoyl-2-oleoyl PE (which makes up 41% of TP-egg  PE) and TP-egg PE were methylated at approximately the same rate. Enhanced methylation  of  dilinolenoyl-, dilinoleoyl- and  dioleoyl-PE compared to  105  Table 9.  % Distribution of Labeled P C and P D M E Molecular Species Synthesized by P E N-Methyltransferase In Vitro  Substrate Methylated/Product Analyzed Peak Microsomes/PC Number (n=3)  2  1  Microsomal PE/PC (n=3)  TP-Egg PE/PC  PDME/PC  (n=3)  (n=3)  (n=4)  3  3  PMME/PDM]  3  1.2 3 4 5,6 7 8 9,10 11 12 13 14 15,16 17 18  0.4+0.1 0.3+0.1 0.4±0.1 2.6±0.6 23.9+1.8 12.5±1.5 8.9±0.8 3.6±0.2 ND 18.0+1.2 22.3+2.1 5.9+0.1 1.0+0.2 ND  0.3+0.1 0.3+0.2 0.5±0.1 1.9±0.6 23.0±4.1 11.9±1.6 8.9±0.6 2.9+1.6 ND 17.6+1.8 25.2±2.9 5.5+J0.7 1.3+0.7 ND  ND ND ND 1.0+0.4 2.8+0.7 4.3±1.3 32.8±0.7 3.2+0.2 36.4+3.3  ND ND ND 0.8+0.1 1.8+0.1 1.9±0.4 26.5±1.0 1.6+0.2 49.6+1.0  ND ND ND 0.8+0.5 1.4+0.3 1.8+0.3 28.1+2.6 2.2+0.2 45.7+3.2  5.2+1.2 8.7+0.5 1.2+0.5 4.4+TJ.6  2.9+0.3 7.9+0.5 0.8+0.1 6.2+0.7  5.3+1.8 9.6+1.0 0.7+0.2 4.2±1.2  % Recovery  76.6±10.4  81.9+18.2  90.2+0.2  72.9±7.2  88.9+13.5  4  Values are expressed as the average of the indicated number of determinations +S.D. 1  Refer to Table I for molecular species identification  Rat liver microsomes were incubated with [m«r/iy/- H]AdoMet and the labeled PC analyzed by HPLC as described in Section 2.3.1.  2  3  Purified PE N-methyltransferase was incubated with purified microsomal PE, TP-egg PE, PMME, or PDME and the indicated product analyzed by HPLC as described in Section 2.4. 3  4  5  ND, not detected. i , indicates % distribution of label in species was included in preceding value.  106  0  10  20  30  T i m e (min) Figure 35. Rates of methylation for various PEs using purified PE /V-methyltransferase A, PE //-methyltransferase (0.12 mg) was assayed with 2 mM microsomal-^ ), diIauroyl-(A ), dimyristoyl-(Q) dipalmitoyl-(* ), distearoyl-(B ), l-palmitoyI-2-oleoyl-(B ), dioleoyl-(X ), dilinoleoyl-(D), dilinolenoyl-(A) and TP-egg-PE (+) for up to 30 min. Product analysis revealed 90-95% of the label in PC in all cases. B, methylation rates were determined as described above except 40 mol% microsomal PC was included in the assay.  107 distearoyl-PE was observed, indicating that the introduction of double bonds in the PE molecule markedly enhanced methylation rates. difference  (average of 2 exp.)  in methylation rates between the species  containing 6 double bonds (dilinolenoyl) (dioleoyl).  There was only a 16%  and the species containing two  All the disaturated PEs tested had minimal activity, with the  exception of dilauroyl PE.  Dipalmitoyl, distearoyl and dimyristoyl were the only  PEs tested that had phase transition temperatures (60-66°C, 71°C and 49.5-47.5°C, respectively) preference  above  the  assay temperature  by PE N -methyltransferase  (293).  for  This may indicate a  substrate  and  bulk  phase  phospholipids in the liquid-crystalline phase. Results presented in Section 3.2.4 indicated a role for boundary lipids in the modulation of PE AT-methyltransferase  activity.  If this  were true,  methylation rates for the saturated and moneonic PEs tested in Fig. 35B should be affected in a positive manner by the inclusion of unsaturated PC. results of this experiment are shown in Fig 35B. distearoyl-PE methylation  was  stimulated  The  Dimyristoyl-, dipalmitoyl- and  by the  inclusion of 40 mol%  microsomal PC. This stimulation was no doubt due to a fluidizing effect of the microsomal PC such that the PE-PC mixture assumes a T temperature of 37°C.  requirement  methyltransferase.  below the assay  Stimulation of dilauryl-, l-palmitoyl-2-oleoyl- and TP-  egg-PE methylation is not due to a shift in T structural  m  for  lipids  in  m  values but is a reflection of some  the  boundary  layer  of  PE N -  The requirement would be for PE or PC molecules with  more than one double bond in their acyl chains.  In agreement with these  observations, methylation rates for the more unsaturated PEs were not altered by inclusion of PC. 3.5.4  Molecular Species Specificity In Vivo  It could be argued that the lack of PE TV-methyltransferase specificity in  Table 10. Distribution of [mef/iy/-3H]Methionine-Labeled During a 24 Hour Chase.  PC Molecular Species  Time (hours) Peak number  0  2  6  12  18  24  mol% cellular PC  1,2,3 4 5,6 7 8 9,10 11 13 14 15 16  1.2+0.8 0.6+0.2 1.6+1.2 21.0+6.2 13.9+0.9 7.5+1.3 2.8+0.4 18.2+2.4 27.7+6.2 6.+1.4 0.8+0.4  0.9+0.5 0.5+0.1 1.2+0.3 16.0+5.7 17.0+3.4 8.3+1.4 2.5+0.3 17.6+1.7 27.9+4.6 6.8+2.6 0.8+0.4  0.8+0.4 0.6+0.2 1.0+0.2 12.3+4.9 15.1+1.3 7.8+2.7 2.7+0.5 17.5+2.8 30.9+5.6 9.8+2.1 1.1±0.4  1.5+0.7 0.6+0.1 1.1+0.5 10.4+4.4 14.7+1.9 9.5+1.1 2.1+0.1 16.6+2.2 33.2+6.6 8.5+2.6 1.2+0.4  1.4+0.6 0.7+0.4 0.9+0.3 9.4+3.1 15.7+1.8 9.2+0.8 2.1+0.4 15.2+2.1 32.4+7.2 11.3+1.3 1.2+0.1  1.1+0.2 0.6+0.2 0.9+0.4 8.6+4.0 17.2+1.9 9.6+1.3 2.2+0.5 14.0+1.4 34.3+8.1 10.0+1.5 1.2+0.3  0.9+1.2 1.4+0.9 1.9+0.5 10.2+4.0 16.3+3.5 10.1+0.9 2.8+0.2 14.1+1.8 29.4+3.8 10.9+1.4 1.6+1.1  %Recovery 78.9+5.0  86.7±9.1  87.2+4.6  76.3+11.0  81.6+3.6  83.6±6.4  79.2+6.4  Results are expressed as the average of 4 separate pulse-chase experiments + S.D.  109  vitro using complex mixtures of PE was due to pH, detergent or buffer effects and does not represent true in vivo conditions.  Hence, PE /V-methyltransferase  specificity in vivo was tested by pulsing monolayers of rat hepatocytes for 1 h with [mer/iy/-^H]methionine.  Reverse-phase HPLC was used to monitor the fate  of PC molecular species produced via methylation during a 24 h chase period. Table 10 shows the % distribution of radioactivity in PC molecular species over the 24 h chase period. At the end of the 1 h pulse, the distribution of PC species was very similar to the mol% distribution in microsomal PE (Table 8) and to the label distribution observed for microsomal PE methylation in vitro (Table 9). During the course of the chase, however, there was a progressive reduction in the % of label in l-palmitoyl-2-docosahexaenoyl PC. Concomitant with this loss were less significant increases in 1-palmitoyl and 1-stearoyl species with oleate and arachidonate in the two position.  By 24 h the % distribution of label in PC  molecular species was almost identical to the mol% distribution in total cell PC (Table 10).  HPLC elution profiles for PC molecular species at 0, 12 and 24 h  reveal in a graphic manner the striking decay of label from l-palmitoyl-2docosahexaenoyl PC (Fig. 36).  The loss of this species was not the result of  preferential secretion, since % distribution of label in medium PC was identical to that in the hepatocyte from 6 to 24 h. The remodeling of PE derived PC to conform to total cell molecular species composition is further demonstrated in Table 11. the 6 major species are shown. pulse  period  For simplicity only  It is evident that immediately following the  l-palmitoyl-2-docosahexaenoyl  PC had the  highest  specific  activity, while species containing 2 and 3 double bonds had the lowest. Similar to  % label  distribution  data,  there  was  a decay  in  l-palmitoyl-2-  docosahexaenoyl PC and increases in the other species specific activity. one exception being l-stearoyl-2-docosahexaenoyl  PC.  The  It should be noted that  110  80  100  120  20  40  60  80  1 00  1 20  20  40  60  80  1 00  1 20  8000 6000 4000 • 2000 -  0  fraction number ( 1 . 0 ml/fraction) Figure 36. HPLC elution profiles of [mef/iy/-3H]methionine-labeled cellular PC molecular species during a 24 hour chase period. Cellular PC (90,000 cpm) was fractionated by HPLC, collected fractions allowed to evaporate to dryness and radioactivity was measured. Recoveries of radioactivity were 85-95%. Identification of the major molecular species is shown in the 0 h frame.  Ill decay in l-palmitoyl-2-docosahexaenoyl specific  activity increased.  The increases  stearoyl-2-arachidonyl-PC specific cell PC specific activity.  PC occurred even though total cell PC in l-palmitoyl-2-arachidonyl- and 1-  activity  followed  closely  the net  increase in  At 24 h the specific activities of the 6 fractions were  not significantly different, indicating that PE-derived PC is being remodeled to conform to the molecular species composition of total cell PC.  Table 11.  Specific Activity of Hepatocyte Molecular Species Time  Molecular  0  [methyl-^K]PC  (hours) 12  24  Specific Activity 1  Species 16:0-22:6 16:0-20:4 16:0-18:2,18:1-18:2 18:0-22:6 18:0-20:4 18:0-18:2,18:0-22:5  1575±294 627+58 501+144 823±330 774±41 331±73  1194+36 753+180 609+296 894+303 932+294 669±205  899+83 912+54 806+59 813+114 931+63 735±128  Specific Activity of Total PC  619+J30  750±47  857±7  cpm/nmole and 40%). 1  PC (counting  efficiency  was  constantly  between  35  Vitro Phosphorylation of Phosphatidylethanolamine NMethyltransferase by cAMP-Dependent Protein Kinase.  3.6  In  3.6.1 As  Phosphorylation and Phosphoamino Acid Analysis  an initial step toward determining if phosphorylation plays a role in  PE N-methyltransferase  regulation  in vivo, its viability as a substrate for the  catalytic subunit of cAMP-dependent protein kinase was tested in vitro. Fig. 37 shows  an  autoradiogram  methyltransferase  of  purification  phosphorylated scheme  fractions  separated  by  from  the  SDS-PAGE.  PE  NBoth  microsomes (A and B) and microsomal membranes (C and D) contain a plethora of  proteins phosphorylated by cAMP-dependent kinase or endogenous  kinases.  112  A  B  C  D  E  F  G  H  I  Figure 37. Phosphorylation of partially purified and purified PE /V-methyltransferase by cAMP-dependent protein kinase. Samples were incubated with cAMP-dependent protein kinase for 60 min, separated by 10% SDS-PAGE, stained with Coomassie Blue and exposed to Kodak XAR-5 film for 18 h. Lane A, microsomes (25 pg); lane B, microsomes (50 pg); lane C, soluble microsomal membranes (25 ug); lane D, soluble microsomal membranes (50 ug); Lane E, octyl Sepharose step (1 pg); lane F, octyl Sepharose step (0.5 ug); lane G, Pure PE //-methyltransferase (0.45 pg); lane H, minus methyltransferase; lane I, minus kianse.  113 One of the most abundant is a 50 kDal protein. The octyl Sepharose fraction (E and F) contains both a 50 and 18.3 kDal phosphoprotein, while the purified enzyme (lane G) is composed of only the 18.3 kDal methyltransferase.  The  minor bands in lane G, with the exception of minor contaminantes at 25 and 30 kDal, are due to proteins in the kinase preparation since a control incubation (lane H) with no PE N-methyltransferase also had these contaminants.  This  figure not only showed that PE A-methyltransferase is a kinase substrate, but 7  also  revealed that the 50 kDal  protein previously thought to be the  methyltransferase (114,115,159,162) is a phosphorylated contaminant. Phosphoamino acid analysis of the P-labeled PE Af-methyltransferase 32  revealed that cAMP-dependent kinase phosphorylates on a serine residue (Fig. 38,  lane A).  Identity  was based on both the mobility of authentic  phosphoserine and of labeled phosphoserine from hydrolyzed histone (lane D).  Hydrolyzed preparations containing only kinase or methyltransferase  showed no phosphoamino acids (lanes B and C). Phosphorylated dimensional  gel  PE A -methyltransferase 7  electrophoresis  was  subjected  to  two-  and the position of the phosphoenzyme  isoproteins compared to the stained gel.  The result of such an experiment is  shown in Fig. 39 for 20 and 60 min incubations with cAMP-dependent protein kinase.  Evidently, phosphorylation shifts both the major isoproteins and a  third minor isoprotein to the anode on the NEPHGE gel, indicative of an increase in negative charge.  Futile attempts were made to shift the mobility  or alter the distribution of isoproteins by treatment with potato acid- or alkaline-phosphatase.  This gives an early indication that the isoproteins are  not the result of phosphorylation prior to or during purification. 3.6.2  Stoichiometry  Phosphate  of Phosphorylation and  incorporation into  Effect on Activity  PE Af-methyltransferase  reached  a  114  # Ser(P)^ Tyr(P)^ Thr(P)^  t  Phosphoamino acid methyltransferase. Purified P E  analysis  of  P-labeled P E N /V-methyltransferase (0.18 p g ) was phosphorylated, hydrolyzed and subjected to T L E as described in Section 2.10.2. The T L E plate was exposed to Kodak X A R - 5 film for 14 h. Lane A , P E Nmethyltransferase plus kinase; lane B , P E N-methyltransferase only; lane C , kinase only; lane D, 20,000 cpm of phosphorylated, hydrolyzed histone. O, point of sample application. Figure  38.  32  115  Figure 39. Two-dimensional gel analysis of phosphorylated P E /V methyltransferase. 0.39 pg of phosphorylated PE /V-methyltransferase was subjected to two-dimensional gel electrophoresis as described in Section 2.5. The dried gel was exposed to Kodak XAR-5 film for 18 h. Triangles indicate the positions of the stained isoproteins.  116  LU 0.  CL O  20  40  Incubation Time (Minutes)  20 Minutes  Figure 40. Time course of PE N-methyltransferase phosphorylation in vitro. Panel A, enzyme was phosphorylated with the catalytic subunit or cAMP-dependent protein kinase as described in Section 2.10.1. Radioactivity in the PE N -methyltransferase band was determined and expressed as mole phosphate/mole enzyme (average of four experiments). The insert shows a representive autoradiogram from an experiment in Panel A. Panel B, PE N -methyltransferase was phosphorylated for 30 min and then assayed for PMME-dependent activity. Identical results were obtained for PEdependent activity.  117 maximum at 30 min with a stoichiometry of only 0.25 mol Pi/mol enzyme (Fig. 40A).  The addition of PE, PMME or PDME had no effect on incorporation nor did  varying  the  Triton  X-100  concentration.  Interestingly,  no  effect  of  phosphorylation was observed on enzyme activity (Fig. 40B insert) in the presence of saturating AdoMet and PMME.  The same lack of effect was  observed for PE-dependent activity. 3.7  Phosphatidylethanolamine Deficient Rat Liver.  N-Methyltransferase  in  Choline-  3.7.1 Effect of Choline Deficiency on Activity and Mass  It has previously been reported that microsomes from choline-deficient rat livers were increased in PE N-methyltransferase (186,187).  activity  about  2-fold  However, the reason for this elevation in activity had not been  elucidated. Using new information on the enzyme assay and an anti-PE Nmethyltransferase antibody, PE methylation in the choline-deficient rat was again addressed. A 2-fold elevation of PE N -methyltransferase activity was observed in choline-deficient microsomal fractions when endogenous PE was a substrate (Table 12).  However, when a saturating quantity of exogenous methyl  acceptor (PMME) and 1.0 mM Triton X-100 was added the observed differences in activity between the choline-deficient and -supplemented liver microsomes were no longer significant.  It is apparent that methylation activity is being  affected by the level of substrate PE in the microsome. further  corroborated by the  reaffirmation  (185)  results in a slight elevation in hepatic PE (Table 13).  This observation was  that  choline-deficiency  PC levels were reduced in  the deficient liver, and more importantly the PC/PE ratio was reduced from 1.88 to 1.22.  A characteristic accumulation of triglyceride (185, 295) was also  noted (23.9±_6.0 compared to 3.7 ±.0.9 pmol/g liver in choline-deficient and -  118  Table  12. Activity  of  Reticulum  P E iV-Methyltransferase  (Microsomes)  Choline-Supplemented  Endoplasmic  Choline-Deficient  (CD)  and  Livers  Exogenous PMME  Endogenous PE CD  from  (CS) Rat  in  CS  CD  CS  ER I  1.45+0.34 0.74+0.19 (0.01<p>0.02)  8i.06+2.85 6.70+1.68 (n.s)  ER II  1.09±0.17 0.61+0.15 (0.001<p<0.01)  6.15±1.05  5.36±0.91 (n.s)  ER I and II were isolated by the method of Croze and Morre (297). Both CD and CS fractions were enriched 10-fold and had identical NADPH-cytochrome c reductase activity. Methyltransferase activity is expressed as nmol/min/mg + S.D. Results are the average of 4 determinations, n.s., not significant.  Table  13.  Phospholipid Levels in Choline-Deficient (CD) and Supplemented (CS) Rat liver Homogentates PE  1  PC  PC/PE  CD  9.36+0.79  11.40+2.13  1.22  CS  8.13±0.82 p<0.1  15.27±1.02 p<0.02  1.88  Choline-  *PE and PC levels are expressed as pmol/g liver+S.D. 55-60 g rats were maintained on deficient or 0.3% (w/w) choline supplemented diet for 3 days prior to sacrifice.  119  supplemented livers, respectively). As further confirmation that enzyme activity was affected by substrate availability, the amount of methyltransferase protein in choline-deficient and -supplemented rat liver microsomes was determined by immunoblot analysis using a rabbit polyclonal anti-PE N-methyltransferase antibody.  Fig. 41 shows  an autoradiogram of a nitrocellulose blot of two different amounts of deficient and supplemented difference deficient  in  rat liver microsomes plus an enzyme  immuno-detectable  mass  or -supplemented microsomes.  methyltransferase (18.3  was  observed  standard.  No  between choline-  The molecular mass of PE N-  kDal) in microsomes was identical to that of the  purified enzyme standard. 3.7.2 Activity and Mass in Choline- and Methionine-Deficient Rat Hepatocytes.  Choline- and methionine-deficient rat hepatocytes were cultured in the presence of choline or methionine, and changes in PE N-methyltranferase mass and activity were examined.  Supplementation of deficient hepatocytes  with 100 pM choline for a 4 h period resulted in a 12% decrease in activity when assayed with endogenous  PE (Fig. 42A).  saturating PMME was unchanged.  Activity measured with  During this time the cellular content of PC  increased from 100 to 130 nmol/mg protein, while PE levels remained constant (Fig. 42B).  With addition of 200 pM methionine, however, there was a 50%  decrease in activity (Fig 43A) accompanied by a similar drop in cellular PE mass (Fig. 43B). constitutive  even  Again, immunoblotting showed enzyme protein to be though  supplementation (Fig. 44).  activity  was  halved  methionine  These results provide strong support for the role of  PE in auto-regulation of PE N-methyltransferase deficiency.  upon  in choline and methionine  120  Q UJ  - oc 2 aa!  MICROSOMES CS  CD  CS CD  18.3 • Kdal  Figure 41. Immunoblot of PE N -methyltransferase in cholinedeficient and choline-supplemented rat liver microsomes. 100 and 300 pg of microsomes were separated on 5-15% gels, transferred to a nitrocellulose membrane and probed with an anti-PE //-methyltransferase antibody. The autoradiogram was exposed for 6 h. A purified enzyme standard is shown. The recovery of microsomes from choline-deficient and cholinesupplemented liver homogenates was 76.5±_9.8% and 80.9+4.7% (n=3), respectively, based on NADPH-cytochrome c reductase activities (298).  121  140  0.2  1 2  3  hours  4  40  1 2  3  4  hours  Figure 42. Effect of choline supplementation on PE N methyltransferase activity in cholineand methionine-deficient hepatocytes. Panel A , at the indicated times hepatocytes were collected, homogenized and a membrane fraction isolated by centrifugation. PE Nmethyltransferase activity was assayed for 20 min under the following conditions; • , choline-deficient plus PMME; • , choline-supplemented plus PMME; • , choline-deficient with endogenous PE; X , choline-supplemented with endogenous PE. Panel B, hepatocyte PE and PC were extracted, separated by T L C and lipid phosphorous determined. A , choline-deficient PE; • , cholinesupplemented PE; • , choline-deficient PC; X , choline-supplemented PC.  122  Figure 43. Effect of methionine supplementation on PE N • methyltransferase activity in cholineand methionine deficient hepatocytes. Panel A , at the indicated times hepatocytes were collected, homogenized and a membrane fraction isolated by centifugation. P E Nmethyltranferase activity was assayed for 20 m i n under the following conditions; A , methionine-deficient plus P M M E ; • , methionine-supplemented plus P M M E ; • , methionine-deficient with endogenous P E ; X , methioninesupplemented with endogenous P E . Panel B , hepatocyte P E and P C were extracted, separated by T L C and lipid phosphorous determined. A , methioninedeficient P E ; • , methionine-supplemented P E ; • , methionine-deficient P C ; X , methionine-supplemented PC.  s  123  METHIONINE TIME(h)  0  + 4  4  + 8  - + 8  12 12  Figure 44. Immunoblot of methionine-supplemented and methhomogenates with an anti-PE N• ionine-deficient hepatocyte 200 pg of methionine-supplemented and methyltransferase antibody. deficient hepatocyte membranes was probed with an anti-methyltransferase antibody. The nitrocellulose blot was exposed to Kodak XAR-5 film for 12 h.  124  3.8  A -Terminal Sequence A - Methyltransferase 7  Analysis  of  Phosphatidylethanolamine  7  The first 30 A -terminal amino acids of purified rat liver ' PE A 7  7  methyltransferase are shown in Fig. 45. separate  determinations  to  residue  The sequence was identical for two  15.  Residues  determination and the chance of a misread is greater. acids  are observed  hydrophobic.  in this  sequence; the  15-30  are from one  Only 2 charged amino  remainder being  neutral or  Significant identity (35%) was noted between this sequence and  the predicted A-terminal amino acid sequence of the 21 kDal S. cerevisiae PEM 7  2 (195). The sequence of rat liver enzyme started at residue 10 of the yeast sequence indicating possible post- or co-translational cleavage of some of the N-terminal amino acids.  Homology (25%) between the rat liver Af-terminal  sequence and that of bovine phenylethanolamine A -methyltransferase 7  (296)  was also noted.  10  QQ SS  20  IHS VD L Q S  LlLGY V D P T E P 5  K  -40, I V C T MFNP IFWNIV 30  QL  V N AlVLTI H F N P L L Y N.V  Yeast PEM Rat Liver PEM  25  15  Figure 45. A -terminal amino acids of rat liver PE A -methyltransferase. Homology with the 5. cerevisiae PEM 2 (195) A -terminal is indicated by the enclosed areas. Conservative substitutions are indicated by inverted triangles. 7  7  7  23 kDa  PE^PC  18.3 kDa  PE*PC  125  DISCUSSION 4.1  Purification and Molecular Properties PE methylation is considered to be a minor route for the synthesis of PC  in liver, but may be more important in situations where PC synthesis via the CDP-choline pathway is compromised (186,187).  Alternatively, it has been  demonstrated that factors that stimulate the CDP-choline pathway inhibit PE methylation (148,178,183,232,233).  As a prelude to understand better the  factors that regulate hepatic PC synthesis, PE //-methyltransferase  was  purified to apparent homogeneity from rat liver microsomes. The 7 step purification scheme illustrates some properties of this enzyme.  Contrary to previous reports from this laboratory (89), we were  successful in using the nonionic detergent Triton X-100 as a solubilizing agent after substituting 20 mM potassium phosphate and 10% glycerol for the Tris HCI  buffers  used  in  other  purification attempts.  Purified  PE N -  methyltransferase showed no perceivable loss in activity for at least 2 months when stored in buffer A plus 0.1% Triton X-100 at 4°C.  This is in sharp contrast  to the 80% loss of activity reported for the partially pure enzyme after 16 h at 2°C (89). A second property that became apparent during our purification efforts was that PE N -methyltransferase  is  a very  basic  protein.  PE TV -  methyltransferase passed unretained through an anion exchange resin (PBE 94, step 7) at pH 9.4 and can only be resolved into two isoproteins when electrophoresised toward the cathode in a NEPHGE system. alkaline pi has to enzyme function is yet unknown. cerevisiae  PEM 2 is also a basic protein (195).  What relation this  Interestingly, the 5.  This enzyme contains 23 basic  residues (13 lysine, 7 arginine and 3 histidine residues) and only 14 acidic amino acids (9 glutamate and 5 aspartate residues).  The pi of the native  126 purified enzyme has not been determined.  Other Af-methytransferases  possess basic pH optima (8.5-9.5) do not have basic pis (299-303). basic residues in PE //-methyltransferase  which  Thus, the  may have a structural function,  perhaps for charge-pairing with the phosphate groups in the surrounding phospholipids. One  apparent  cytosolic  domain  (residue  46-61)  of  methyltransferase contains six basic residues and two cysteines.  the  yeast  This may  represent the catalytic domain since two cysteines have been implicated in methylation in the rat liver enzyme.  The hypothetical role of these two  cysteines in methyl transfer is shown in Fig. 46.  This mechanism involves  enhancement of the nucleophilicity of the PE amino group by general base catalysis.  The two cysteines would abstract both amino group protons during  PDME formation, and then, due to the electron-donating capacity of the two methyl groups on PDME, final methylation would occur without general base catalysis.  General base catalysis has been suggested to be the probable  mechanism in enzymatic  O -methyltransferase  reactions  (304,305).  This  conclusion was based on studies using various compounds that undergo intramolecular transalkylation (305a-305c). shown in Fig. 47.  An example of such a reaction is  The intramolecular reaction rate shows a strong pH  dependence above 9.0, indicative of ionization of the nucleophilic cis-alcohol. Also, the reaction is catalyzed by oxyanion buffers (phosphate), but not by amines, which acted as nucleophiles and abstracted the sulfonium methyl group (305a).  An ionized cysteine residue (in the methyltransferase active  site) could act as a general base catalyst in much the same way as phosphate. Most of the N-methyltransferases examined possess essential cysteine residues, but their exact role in catalysis has not been elucidated (299-303).  127  +AdoMet  +AdoMet CH3  HL Enz-S  t  \„  -AdoHcy  H  >V ~y  CH  c  S—Enz  C.H-2 s  I  —  Enz-SH  CH2  Figure 46. Hypothetical transferase.  ^CH, 03  1  3  -S-Enz  1  CH  -AdoHcy  A  CH  Enz— SH  2  3  V  C H 3  1 CH  SH—Enz 2  I CH  CH?  catalytic  mechanism  for  2  PE N -methyl-  Figure 47. Intramolecular transalkylation of l-(2-methoxycyclopentyl)-2-p-nitro-methylsuIfoniumphenylethane. The Figure was adopted from Ref. 305.  128 It is feasible that the cysteines are not involved directly in the catalytic mechanism but have some regulatory role.  Short term regulation of PE  methylation could be achieved by thiol-disulfide exchange mechanisms in the cell by a variety of biologically active disulfides.  Such a mechanism has been  proposed for regulation of rat liver guanidoacetate //-methyltransferase The extremely alkaline pH optimum of PE //-methyltransferase been a point of contention.  Assay of activity in vitro consistently  (302).  has long revealed a  pH optimum between 9 and 10.5 (84,87-89) for the methylation of PE, but workers still insist on assaying at 'physiological' pH (7.4) or at values between 8.0 and 8.5 (refer to Table 1 for specifics).  The alkaline lability of AdoMet has  often been used as an excuse to assay at suboptimal pH, but this premise seems unfounded  (88).  Activity measurements  at 7.4  would be credible if  measurements were at the same time done at pH 9.5-10.0. Why both the microsomal and purified methyltransferase alkaline pH optima is unknown.  have such  Does the enzyme within the intact hepatocyte  also function at an alkaline pH and could pH act to regulate enzyme activity? These are questions that at this moment remain unanswered.  Interestingly,  the ethanolamine headgroup of PE would have a pKa in the range of the PE Nmethyltransferase pH optimum (pKa of the ethanolamine amino group is 9.5). This indicates that the deprotonated species of PE maybe the active nucleophile involved in methyl group abstraction from AdoMet. beyond pH 9.5,  as would be expected  Activity does increase  if activity were related to the  concentration of unprotonated PE, and then declines sharply (Fig. 7), maybe as the result of protein denaturation.  Alternatively, the pH optimum could  reflect the ionization state of amino acids in the enzyme (cysteines or basic groups) involved in catalysis.  Since the amino group pKa for PE in  129  endoplasmic reticulum membranes and of catalytic residues in the enzyme has not been determined, the dependence of activity on these factors is unknown. In a highly speculative vein, perhaps PE N- methyltransferase  is  localized in a portion of the endoplasmic reticulum where hydroxide ions are concentrated as the result of ion translocation processes.  The bulk cellular pH  is perhaps the sum of that in various microenvironments. The subunit molecular mass (18.3 kDal) of PE AT -methyltransferase unusually  small  considering  the  fairly  complex  series  of  is  methylation  reactions it catalyzes. Analysis of the pure enzyme in Triton X-100 micelles by gel filtration indicated that a single subunit is present per micelle.  No  information is yet available concerning the subunit structure in phospholipid membranes.  Low molecular masses have been reported for several other  phospholipid biosynthetic enzymes: (306)  the 34 kDal phosphatidylinositol synthase  and 23 kDal phosphatidylserine synthase (282)  cerevisiae  purified from S.  and the 13.2 kDal diglyceride kinase (307) and 27 kDal CDP-  diglyceride synthetase (308) from E.coli. Two reports by Pajares et al. (114,115) have claimed purification of PE Nmethyltransferase from rat liver.  These researchers have co-purified PE N-  methyltransferase activity with a 50 kDal protein.  The specific activity of this  preparation (assayed in the presence of a mixture of PE, PMME and PDME) was 0.27^mol/min/mg protein (115).  Based on the data presented herein, several  lines of argument would indicate that the 50 kDal protein bears no relation to PE N-methyltransferase.  First, we have achieved final specific activities with  PE, PMME and PDME as substrates that are 2.3-, 32- and 14-fold higher, respectively,  than that reported by Pajares et al. (115).  Although these  authors assay PE N-methyltransferase activity at 20°C, pH 8.35 and 100 uM AdoMet, all of which are suboptimal assay conditions in our hands, direct  130  comparison would indicate a substantially greater purification in our case. Second, examination of Fig. 8 and Table 3 would indicate that the 50 kDal protein is the major protein in partially purified fractions (steps 2-5), but is less abundant in step 6 relative to the 18.3 kDal protein and completely absent in the pure fraction.  Third, a polyclonal anti-PE  N-methyltransferase  antibody raised against the purified enzyme recognized only the 18.3 kDal methyltranferase in liver microsomes (Figs. 11 and 41) and in hepatocyte membranes (Fig. 44).  No cross-reactivity with proteins of 50 kDal was  Fourth, AT-terminal sequence of the rat liver methyltransferase was  observed.  35% homologous with PEM 2 of S. cerevisiae,  an enzyme of similar molecular  mass which catalyzes an identical reaction. Fifth, experiments indicated that a microsomal methyltransferase of 19 kDal was the major protein photolabeled by [me thy l-^H] AdoMet.  The molecular mass of this photolabeled protein was  identical to the purified microsomal PE Af-methyltransferase and no proteins of 50 or 25 kDal were photolabeled.  These five points taken together indicate  that the 50 kDal protein is only a persistent contaminant and that a microsomal protein of 18.3 kDal is PE N-methyltransferase. The distribution of products of PE, PMME and PDME methylation was observed to remain constant throughout the purification of the microsomal enzyme.  As with the microsomal enzyme, the major product of PE and PMME  methylation by the purified enzyme was PC and PDME, respectively.  Another  interesting observation from Table 4, that is not immediately apparent, was that PMME appeared to inhibit the formation of PC from endogenous PE in microsomes.  Implications for a single active site for phospholipid substrates  and the competition among them for this site is discussed further in Section 4.3.  131  The time course of product formation from PE and PDME (Fig. 6) using the purified enzyme is similar to that reported for microsomes (88).  The time  course  a gradual  for the  formation of P M M E  methylation products revealed  increase in the formation of PC and reduction of PDME concentration of newly competes  efficiently  production. for  formed PDME  with P M M E  approaches  0.04  for methylation,  formation.  mM in the  resulting  As the assay,  it  in enhanced PC  These results would seem to indicate that PMME and PDME compete  methylation by PE /V-methyltransferase.  This  result  was  corroborated by  kinetic analysis (Section 4.2).  It is interesting to note that, like methylation of  microsomal  of  P E , methylation  methyltransferase  results  in  pure  the  microsomal  formation  of  PE by  PC  purified PE  (92%).  N-  Clearly,  PE  methylation is the rate-limiting step in the reaction sequence and PE does not compete 4.2  with and release the two partially methylated  Kinetic Properties transferase  of  intermediates.  Phosphatidylethanolamine  A -Methyl7  A common problem encountered in the assay of PE //-methyltransferase has been the efficient exogenous  lipid  preparations would  be  substrates  with  no  limited  microsomes.  delivery of lipid substrates to the enzyme. have  been  detergent.  by  the  rate  added  Under of  to  these  exchange  PE /V-methyltransferase assayed  assay  mixtures  conditions,  of  vesicle  as  enzyme  vesicle activity  phospholipids  in the presence  has been reported to be activated by PMME and PDME (86). also  Previously,  into  of Triton X-100 Tanaka et al. (86)  reported that exogenous PE was methylated by Triton X-100 solubilized  microsomes. developed addition  Here,  that of  a simple  can be used  increasing  to  Triton assay  concentrations  X-100  mixed  all three of  micelle  methylation  surfactant  assay  has  activities.  was  found  to  be  independent  The  inhibited methylation  PE, PMME and PDME by dilution of phospholipid on the micelle surface. inhibition  been  (displaying  apparent  first  of This  order  132 kinetics) of the concentration of mixed micelles when a fixed mol% of phospholipid substrate was used. relation  to  micelle  intramicellar reaction.  The apparent first-order kinetics with  concentration  indicated  that  methylation  is  an  The linearity of PE, PMME and PDME methylation (Fig.  5), and more importantly the absence of an initial lag in rates, indicates that exchange of phospholipid between micelles is not rate-limiting. Optimal PE methylation, unlike PMME or PDME, occurs at a mol% where non-micellar structures no doubt exist. than PE in a micellar form, methyltransferase.  It is conceivable that pure PE, rather  is  more readily methylated  by PE N -  Pure PE in solution is proposed to exist in a hexagonal II  array (309). This is probably not the case in our assay where the pH is >9.0 and 12 mol% of Triton X-100 is present.  Both pH >9.0 (59) and a Triton X-100/PE  ratio of 0.1 or 9 mol% Triton X-100 (280) favour the formation of bilayer structure.  Robinson and Waite (310) reported a lysosomal phospholipase A  2  that preferentially hydrolyzed PE in what appears to be hexagonal II phase. PC, PI and phosphatidylglycerol underwent maximal hydrolysis when in mixed micelles. micellar  The propensity of PE N-methyltransferase for PE bilayers versus structures  is  quite  intriguing and requires  more  study  in a  reconstituted system free of detergent. Catalysis by purified PE N-methyltransferase in PE-, PMME- or PDMETriton X-100 mixed micelles has been systematically studied to determine the kinetic mechanism of PE methylation.  During the construction of initial  velocity curves it became apparent that PE, PMME and PDME methylation, either at fixed Triton X-100 or phospholipid, was sigmoidal and cooperative in nature (Fig. 15-19).  There could be a variety of reasons for this type of  response; a noncatalytic binding site that interacts with the catalytic site, multiple interacting catalytic sites on the same subunit or inhibition by Triton  133 X-100 that is relieved when its mol% in micelles is reduced.  Theoretical and  practical experimentation has lead to the formulation of a model which explains  kinetic  cooperativity  in terms  of  non-cooperative  binding of  boundary- or annular-lipid to integral membrane enzymes (287,289,311). Electron spin resonance studies have confirmed that PC molecules bind to the (Na -K)ATPase at multiple non-interacting sites (312), and binding is sensitive +  to the lipid headgroup charge (313,314).  Sarcoplasmic reticulum Ca -ATPase 2+  activities were reported to be sensitive to the fatty acid chain length of PC in membranes in which it has been reconstituted (315). that integral membrane enzymes  These studies indicated  required phospholipid fatty  and/or polar head groups localized in a boundary lipid layer.  acyl chains  If even 1-5 of  the 20-100 (287,289,316) lipid binding sites of integral membrane proteins are unoccupied,  catalytic efficiency  could be greatly  diminished.  PE N-  methyltransferase would seem to fall into this catagory of enzymes, judging from the sigmoidal velocity curves for PE, PMME, and PDME methylation and Hill kinetics shown in Figs. 16-18 and 20.  The effect of egg PC on PE N-  methyltransferase activity at nonsaturating concentrations of PE, PMME and PDME  indicates  occupation  of  the  phospholipid is sufficient for activation.  boundary  layer  by  non-substrate  Triton X-100 is not an activator of PE  N -methyltransferase, and it exerts inhibition both in terms of classic surface dilution and by occupying phospholipid binding sites in the boundary layer. In this regard, Triton X-100 does not have the structural features necessary for PE N-methyltransferase activation.  This two-fold inhibition could explain why  a high mol% of PE, PMME or PDME is required for full activity. The structural features of phospholipids required for activation of PE Nmethyltransferase have not been systematically evaluated. It was observed that monoenoic PEs were methylated at a lower rate than species containing  134 two or more double bonds (Fig. 35). molecular species specificity.  At the same time the enzyme exhibited no  This was interpreted to indicate that monoenoic-  and saturated-PEs occupied the enzymes boundary layer and there affected methylation rates in a negative fashion by influencing enzyme conformation. This interpretation is supported by the increased PE methylation rates, particularly of the saturated species, upon inclusion of 40 mol% microsomal PC.  The effect of other phospholipids and cholesterol on methylation rates in  mixed micelles has not yet been examined.  It is feasible that the anionic  phospholipids, PI and PS, could charge-pair with basic residues on PE Nmethyltransferase  and provide  a more  suitable  lipid  boundary layer.  Interestingly, it has been reported that reconstituted S. cerevisiae PS synthase is modulated by the PI/PS ratio in PC and PE containing vesicles (317). Whether positive cooperativity prevailed or not (as when egg PC was present), analysis of PE A -methyltransferase 7  initial  velocity  and product  inhibition patterns were reproducible and fit the mechanism proposed in Fig. 28.  The more salient features of this mechanism are: (i) PE and the two  intermediates compete for a common active site,  (ii) separate methylation of  PMME or PDME follows an ordered Bi-Bi mechanism and (iii) PE methylation to PMME is rate-limiting; a conclusion supported by the product distribution data discussed in Section 4.1 and comparison of K , Vmax and inital rate values for m  PE, PMME and PDME.  Now, a more systematic analysis has shown conclusively  that PMME and PDME compete with one another for a common active site. Inhibition of PE methylation by PMME and PDME was demonstrated, but we cannot  say  with complete  certainty  that  it  is  competitive.  Key to  understanding the kinetic mechanism is individual analysis of PMME and PDME methylation, and fit of initial velocity- and product inhibition-patterns to an ordered Bi-Bi mechanism.  During concerted methylation of PE, both  135 mechanisms are linked and neither PMME or PDME diffuse from the active site. As with any concerted pathway it is advantageous for the rate-limiting step to be the first.  This allows for control over the accumulation of intermediates  (which could be potential feed-back inhibitors) and prevents unnecessary expenditure of substrates on a partial process.  There is compelling evidence  that PE methylation is regulated by AdoHcy levels in vivo (144,146,148,149), but it now appears that PC plays no such role, at least for catalysis in mixed micelles.  It is feasible that at high concentrations of PC (>40 mol%) surface  dilution of substrate would occur. In conclusion, evidence has been presented that hepatic PE methylation is catalyzed by a single enzyme and occurs via a concerted mechanism. Apparent  cooperativity related to the binding of substrate phospholipids and  nonsubstrate PC in Triton X-100 mixed micelles, and structural requirements by  the enzyme with regard to activation, could have some relevance to  regulation of PE N-methyltranferase in vivo. 4.3  Molecular Species Specificity In  Vitro  and In  Vivo  This work is the first report on the specificity methyltransferase  of pure PE N -  for molecular species of PE and to extend  observations to cultured rat hepatocytes.  in  vitro  From these studies we can conclude  that the enzyme does not display specificity for molecular species of PE, PMME or PDME.  The molecular species of these lipids are methylated according to the  mol% in the membrane or micelle.  This result is obtained with microsomal  enzyme, pure enzyme or in intact hepatocytes.  Experiments on PE methylation  rates showed that the PE //-methyltransferase is affected by the fluidity of the lipid environment.  The enzyme is inactive in a gel phase lipid environment,  such as dipalmitoyl PE, and its activity in the liquid-crystalline phase is  136 proportional to the degree of unsaturation of the activating and substrate lipids. The estimated 20% of total cellular PC produced by methylation in hepatocytes (68) is small and its function is unknown. regarding methyltransferase  function would be to  rich PC to the cellular pool.  An attractive hypothesis supply polyunsaturated-  This would depend both on the molecular species  composition of microsomal PE and on the specificity of the methyltransferase. Previous reports, which utilized isotopic labeling of hepatic PC and PE in whole rats  and molecular species fractionation  by argentation  TLC, generally  concluded that tetraenoic or hexaenoic PC were the major products formed by methylation specific  (117-119).  The conclusions  presented  in molecular species identification,  herein, though more  are in agreement  with an  enrichment in docosahexaenoate containing species at the expense of more saturated species.  The content in microsomal PE and PC of arachidonate  containing species with palmitate and stearate in the sn-1 position are very similar so methylation would not enrich the PC pool significantly in these species.  In this regard, results herein showed no enrichment in PC tetraenoic  species as was alluded to previously (117,118). Studies on PE N -methyltransferase similar to those for liver.  in rat brain reached conclusions  PDME, an intermediate in the methylation process,  was reported to a have a fatty acid composition similar to its precurser PE (318). In vitro  labeling of synaptosomal PC (121) and in vivo labeling of whole rat  brain PC (122) respectively,  with [methy/-  indicated  3  HJAdoMet  preferential  pentaenoic and hexaenoic species.  and  synthesis  of  [methyl- }!] 3  PC rich  methionine, in  tetraenoic,  PE methylation activity in brain is 100- to  500-fold lower than liver, and the contribution to net PC synthesis is very small.  137 Reverse-phase  HPLC  offers  a distinct  technical  advantage  over  argentation TLC in that individual species, and not pooled fractions, can be identified.  Using this technique to identify the PC molecular species products  formed from two complex mixtures of PE, no difference in % distribution of label relative to the abundance of a particular substrate PE could be identified. Similar results using PMME, PDME and microsomes as substrates lend credence to the proposal that PE A -methyltransferase 7  shows little  regards to acyl chain length and degree of unsaturation.  specificity  with  As shown previously,  choline- (120) and fatty acid-deficiency (117) caused marked alterations in the molecular species composition of hepatic PE and PC.  In both instances, PC  formed by methylation reflected the molecular species in precursor PE. The lack of molecular species specificity using pure enzyme and crude microsomes was also observed  in vivo  using monolayers of rat hepatocytes.  The  % distribution of label in PC molecular species immediately following the [mer/iy/- H]methionine pulse period was 3  distribution of PE in microsomes.  almost  identical to the mol%  An interesting finding in the hepatocyte  studies was the rapid remodeling of PE derived PC such that by 12 h it no longer resembled PE in its molecular species composition, but was very similar to that of cellular PC.  The molecular species that turned over most rapidly was 1-  palmitoyl-2-docosahexaenoyl PC.  Work in whole animals (117,120) has also  shown that a methyl-labeled hexaenoic fraction of PC decayed to a new steadystate by 5-6 h.  In the present study l-stearoyl-2-docosahexaenoyl PC showed  only a 23% decrease in distribution, and no change in specific activity, compared to a 59% decrease in distribution of l-palmitoyl-2-docosahexaenoyl PC, and a 1.8-fold decrease in specific activity. docosahexaenoyl unknown.  The fate of l-palmitoyl-2-  PC and the mechanisms by which it is metabolized are  A possible fate could be hydrolysis by the action of various  138 phospholipases to constitutive components.  If such were the case, 1-palmitoyl-  2-docosahexaenoyl PC could be an immediate source of choline for the cell in case of dietary insufficiency. action of  a phospholipase  Another scenario would involve the concerted A2  and  acyl CoA:lysophosphatidylcholine  acyltransferase (28,29) to generate a new PC molecular species with the choline label intact. The decay from 21.0% to 8.6% (Table III) in l-palmitoyl-2docosahexaenoyl PC more than accounts for the net 5.4% increase in other 1palmitoyl containing species (peaks 8, 9, 10).  The decay in l-stearoyl-2-  docosahexaenoyl PC distribution from 18.2% to 14.0% cannot account for the net 10.5% net increase in the major 1-stearoyl containing species (peaks 14, 15, 16 & 17).  These two points indicate that, besides deacylation-reacylation at the sn-  2 position, more complete degradation of l-palmitoyl-2-docosahexaenoyl PC and recycling of the choline head group is occuring. the sn-l fatty acid may occur.  Remodeling in this fashion would lead to  redistribution of label and specific  activity for PE-derived PC so that it  eventually conforms to cell PC composition. palmitoyl-2-docosahexaenoyl  Alternatively, remodeling at  It is of interest to speculate that 1-  PC could be prone to oxidation of its sn-2  acylchain, and thus be hydrolyzed by a phospholipase A2 specific for oxidized or fragmented, short-chain phospholipids (319,320). Based on pulse-chase studies, PE N-methytransferase  supplies  the  hepatocyte with PC containing more than twice its complement of 1-palmitoyl2-docosahexaenoyl remodeled.  PC, the majority of which seems to be degraded or  Preliminary pulse-chase experiments in hepatocytes have shown  that 14% of label, at the end of a 1 h pulse with 15 pCi of [mer/iy/- H]choline, is 3  in l-palmitoyl-2-docosahexaenoyl PC.  During the chase period this value  decays to the 10% normally found in cell PC. Indeed, in vitro experiments on diglyceride  utilization  by  CDP-choline: 1,2-diacyl-s n -g 1 y c ero 1  139 cholinephosphotransferase  have  shown  l-palmitoyl-2-docosahexaenoyl  synthesis from the corresponding diglyceride (31).  PC  This would seem to indicate  that the CDP-choline pathway can also supply the hepatocyte with this PC species.  Since the CDP-choline pathway appears to be quantitatively more  important for PC synthesis, the role of PE methylation in polyunsaturated PC synthesis is still open to debate. In conclusion, PE A -methyltransferase shows little substrate 7  specificity  in vitro or in vivo, and methylates PE at maximal rates provided the molecular  species contains two or more double bonds.  The variation in methylation rates  for synthetic PEs is further evidence for a role of the lipid environment in modulating activity in vitro. 4.4  In Vitro Phosphorylation Methyltransferase  Controversy system has  surrounding the  also extended  methyltransferase  of  Phosphatidylethanolamine  molecular nature of  to possible  modes of  A 7  the methylation  regulation.  PE N -  is regulated by cellular levels of AdoMet and AdoHcy  (148,149), fatty acids (178) and PE/PC ratios (139).  There is a growing body of  evidence that suggests PE methylation is regulated by phosphorylation (refer to Section 1.8.2).  Most of the studies in question have correlated elevated  cellular cAMP with an increase in phosphorylation state; implicating a cAMPdependent protein kinase.  It should be reiterated that the cAMP mediated  effects are small and often inconsistent.  As well, the direct phosphorylation  studies indicated a 50 kDal protein (presumed to be the methyltransferase) was a cAMP-dependent protein kinase substrate.  The 50 kDal protein is indeed a  kinase substrate (Fig. 37), but this phosphorylated protein is completely absent from purified PE A -methyltransferase. 7  We can now say with certainty that  this 50 kDal protein is not PE A -methyltransferase 7  claiming identity require re-evaluation.  and most  conclusions  140 As  an  initial  step  toward  delineating  the  role  of  reversible  phosphorylation in PE N-methyltransferase regulation, the enzyme was tested as a substrate for cAMP-dependent protein kinase in vitro. The enzyme was found to be a substrate for the kinase, albeit a poor one (only 0.25 mol Pi/mol methyltransferase branched-chain lipase,  was incorporated in 30 min). 2-oxoacid  dehydrogenase,  Enzymes such as the  adipocyte  hormone-sensitive  6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase,  mono-oxygenase  and ornithine decarboxylase  (reviewed  tyrosine-3-  in Ref. 321) all  undergo rapid (<10 min) phosphorylation by their respective kinases in vitro. In  most  instances  the  incorporation of  phosphate  Sluggish phosphorylation of PE //-methyltransferase obvious reasons.  was  stoichiometric.  could occur for two  Although various assay conditions, such as the inclusion of  PE, PMME and PDME and manipulation of the Triton X-100 concentration, did not affect phosphorylation, conditions may not yet be ideal for maximum phosphorylation.  Also,  the  methyltransferase  phosphorylated state following purification.  may  This  be  in  a highly  seems unlikely since  phosphatase  treatment did not enhance phosphorylation or alter isoprotein  distribution.  Clearly more work is needed to solve this problem.  Phosphorylation was observed to have no effect on methylation of PE or PMME when assayed at saturating concentration of substrates. possible  that  phosphorylation  methyltransferase substrates.  may  alter  the  K  for one of the  A good example of this type of effect can be  seen with liver pyruvate kinase.  cAMP-dependent protein kinase was  observed to cause a 3-fold increase in the K  m  for phosphoenolpyruvate (in the  absence of the allosteric effector fructose-1,6-diphosphate) the Vmax (322,323).  m  However, it is  without changing  Perhaps phosphorylation alters the pH optimum of PE N-  141 methyltransferase.  Effects of phosphorylation on kinetic constants and pH  optima are areas of future consideration. Consistent with the specificity of cAMP-dependent kinase (324), PE Nmethyltransferase was phosphorylated on a serine residue. site and number of sites has yet to be delineated.  The nature of the  The consensus sequence of  the cAMP-dependent kinase phosphorylation site was shown to consist of two basic residues (one of which is arginine) adjacent or several residues Nterminal to a serine or (rarely) a threonine residue (324). extremely basic pi of PE A -methyltransferase, 7  arginine  residues  would not  be unexpected.  the  Considering the  presence  of multiple  No potential  sites for  phosphorylation were evident in the enzymes N-terminal region (Fig. 45). Amino acid sequencing of the phosphopeptide from the methyltransferase should identify the cAMP-dependent kinase phosphorylation site and indicate if more than one site is involved. Two-dimensional  gel  methyltransferase indicated that  electrophoresis in vitro  distribution of the two isoproteins. the anode.  of  the  phosphorylated  phosphorylation could not shift the  Instead, both isoproteins were shifted to  This indicated that if the isoproteins  phosphorylation then different sites were being labeled  were the result of in  vitro.  Much work is required to show conclusively that phosphorylation regulates PE A-methyltransferase. 7  Of paramount importance is the generation  of a precipitating antibody that can be used to analyze the enzyme's phosphorylation state in hepatocytes.  Exposure of hepatocytes to hormones  which modulate cAMP levels should also alter PE //-methyltransferase phosphorylation state.  142 4.5  Phosphatidylethanolamine N -Methyltransferase and Methionine-Deficiency  in  Choline-  Choline deficiency produces a variety of gross physiological changes in liver lipid metabolism and in the levels of circulating lipoproteins. of  all  plasma  lipids  (cholesterol  ester,  cholesterol,  The levels  triglyceride and  phospholipids) were depressed when rats were maintained on a cholinedeficient diet (184,318).  The reduction in circulating triglyceride levels is  concurrent with an 5- to (137,185,295,325,326).  10-fold  accumulation of this  lipid  in liver  The accumulation is probably related to a cessation of  VLDL secretion (137,184).  Yao and Vance (137) noted that supplementation of  choline or methionine to deficient hepatocytes resulted in a resumption of VLDL secretion, but did not change cellular triglyceride mass or synthetic rates (monitored by [9,10- H]oleate 3  labeling).  Maintenance of choline-  deficient rats on a choline-supplemented diet for 3 days reduced triglyceride levels from 220 to 142 pmol/g body weight (control value was 35 pmol/g body weight, Ref. 327).  Thus, triglyceride levels and synthetic rates are slow to  normalize upon administration of choline. The mass of hepatic PC was shown to be reduced in choline-deficiency (185,319,327,328,329). for  The primary reason for this effect is lack of substrate  the CDP-choline pathway.  The activities of the CDP-choline pathway  enzymes appear to be largely unchanged (186).  However, Hoffman et al.  (187,328) reported that microsomal cholinephosphotransferase reduced 5-fold by choline, methionine, folate and vitamin B  activity was 1 2  deficiency.  Preliminary results have suggested that phosphocholine cytidylyltransferase activity undergoes redistribution from cytosol to microsomes in the cholinedeficient state (188), but total activity is unchanged. An elevation in the activities of the PE biosynthetic enzymes cannot explain the elevated hepatic levels of PE (185,319,326,328,329) since Schneider  143 and Vance (186) enzymes.  observed no changes in the CDP-ethanolamine pathway  The elevated PE levels are, no doubt, the result of lack of conversion  to PC via methylation. choline-deficient 1 2  While PE A-methyltransferase activity is elevated in 7  microsomes (186,187,329), the in vivo methylation of [1,2-  C]ethanolamine-labeled PE to PC is  impaired (329).  The impaired  methylation is related to the reduced levels of AdoMet and the low AdoMet/AdoHcy ratio in choline-deficient livers (329).  Supplementation of  choline- and methionine-deficient hepatocytes with methionine was found to result in a 2-fold reduction in PE concentration (137), indicating that methyl group availability was limiting the reaction. Armed with new information on the molecular properties of PE A 7  methyltransferase,  the regulation of this enzyme in choline-deficient rat  liver was re-examined.  The auto-regulation of PE A-methyltransferase by PE 7  levels and the mass of enzyme was monitored under conditions where the concentrations of PC and PE changed significantly. Similar to previous reports, choline-deficiency  resulted in a marked  reduction in liver PC concentrations and a less significant elevation in PE. More importantly, the ratio of PC to PE in hepatic membranes was reduced in the deficient state. in  microsomal  choline-deficient  Concomitant with the increase in PE was a 2-fold elevation endogenous  PE-dependent  liver microsomes.  methyltransferase  Differences  activity  in  between deficient and  supplemented microsomes were obviated when an excess of PMME was added to the assay. lipid  The methyltransferase, which possesses a single active site for all  substrates,  is  clearly regulated by PE levels.  Furthermore, the  concentration of PE (or its mol% in membranes) in normal liver microsomes is not saturating and increases, due to choline deficiency, results in elevated in vitro  activities.  This dependency of methylation on PE levels was previously  144 observed  in ethanolamine-supplemented  hepatocytes  occur in the choline-deficient liver (139).  and hypothesized  to  Previously, Hoffman et al. (187)  reported that PDME-dependent methyltransferase activity was reduced 1.2-fold and activity with endogenous PE was stimulated 1.4-fold by choline and methionine  deficiency.  However,  these  researchers  were  under the  impression that two separate enzymes, under different regulatory controls, were involved in PE methylation. We now know that this is not the case. The regulation of PE A -methyltransferase by PE levels in choline 7  deficiency is not related to changes in enzyme mass. effectively by the immunoblot in Fig. 41.  This is illustrated most  Clearly, immuno-detectable enzyme  mass was not altered by maintenance on a choline-deficient diet for three days.  The relationship between the endogenous PE assay versus exogenous  PMME assay can now be understood in light of the constitutive nature of enzyme mass.  The PMME-dependent assay will give enzyme activities that are  proportional to enzyme mass, while the endogenous PE assay is a reflection of PE substrate levels.  The discovery of conditions under which PE N-  methyltransferase mass and substrate is altered (ie. developmental induction) should give further insights into regulation. The relationship between PE levels and methyltransferase activity was further established hepatocytes,  in choline- and methionine-deficient  hepatocytes.  In  supplementation of these phospholipid precursors, and their  effects on lipid levels, could be correlated to changes in enzyme mass and activity.  It was observed that supplementation with methionine alone caused a  55% decrease in cellular PE concentrations (Fig. 43B).  This corresponded to a  47% decrease in endogenous PE-dependent methyltransferase activity and no change in PMME-dependent activity (Fig. 43A). altered by methionine supplementation.  Enzyme protein was not  Under identical conditions, only a  145 12%  decrease  in  PE-dependent  supplementation (Fig. 42A).  activity  was  observed  upon choline  Thus, in a well defined culture system, changes in  enzyme activity are related to altered PE levels and not to changes in enzyme mass. Are there any conditions that may be expected to change expression of enzyme mass?  By analogy to S.cerevisiae PE methylation, choline and  methionine do not repress PE methylation (190,192), but choline does in the presence of inositol.  This does not seem to be the case in choline-deficient  hepatocytes, when addition of choline to inositol containing medium (0.04 mM in MEM) did not affect enzyme activity (Fig. 42).  Multivalent regulation at the  level of substrate availability, phosphorylation, transcription and translation may be involved in regulation of rat liver PE AT-methyltransferase.  While  there is evidence for the first two regulatory mechanisms, the latter two are only hypothetical.  146 Conclusions  and  Future  Considerations  The impetus of this work was to promote research on the molecular properties and regulation of PE N-methyltransferase.  Neither of these goals  can be addressed properly without purification of the enzyme. A -methyltransferase 7  was  To this end, PE  purified from rat liver microsomes  solubilization with Triton X-100.  following  A single 18.3 kDal protein catalyzed the  complete conversion of PE to PC. The kinetic mechanism occurs in a concerted manner with partially methylated  intermediates  remaining in a common  active site until PC is produced. Studies on modes of regulation showed that cAMP-dependent protein kinase phosphorylated the methyltransferase  in vitro.  Phosphorylation had  no effect on enzyme activity when assayed at saturating substrate levels. information is yet available on phosphorylation choline-deficient  rat model  methyltransferase  regulation  reiterated by  in  previous  substrate  vivo.  Work with the  observations  levels.  No  Changes  on PE Nin enzyme  activities were found to be related to PE levels, and not to changes in enzyme mass.  In this regard, enzyme mass appeared to be constitutive, even when  dietary choline and methionine were withheld. With the availability of purified enzyme, knowledge  of molecular  properties and preliminary results on regulation, work can begin in earnest on enzyme structure and regulation.  The following approaches could be quite  fruitful. 1.  The enzyme could be cloned from a rat liver cDNA library using an oligonucleotide probe complimentary to the A-terminal of the enzyme or 7  by the use of polyclonal antibodies. could be sequenced.  If this were unsucessful, the protein  This is not such a daunting task considering the size  of the enzyme (aprox. 160-170 amino acids).  147 While it is apparent that different molecular species of PE influence the rate of methylation, the effect of other phospholipid classes has not been determined.  These experiments could be done in mixed micelles or in a  reconstituted  system.  More indepth analysis of enzyme phosphorylation sites, effects on catalytic properties and possible endogenous kinases and phosphatases could be performed. In vivo phosphorylation and cellular half-live of the methyltransferase could be determined as soon as a precipitating antibody is raised. all  antibodies  that have been raised against  immunoprecipitate activity or protein.  the  enzyme  Oddly, do not  148 REFERENCES  1.  Singer, S. L., and Nicholson, G. L. (1972) Science 175, 720-731.  2.  Sudhof, T. C , Goldstein, J. L., Brown, M. S., and Russell, D. W. (1985) Science 228, 815-822.  3.  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