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Isolation of rat liver CTP: phosphocholine cytidylyltransferase and regulation of hepatic phosphatidylcholine… Yao, Zemin 1985

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ISOLATION OF RAT LIVER CTP:PHOSPHOCHOLINE AND REGULATION  CYTIDYLYLTRANSFERASE  OF HEPATIC PHOSPHATIDYLCHOLINE  BIOSYNTHESIS  by ZEMIN B.Sc.  East China  A THESIS SUBMITTED  YAO  Normal U n i v e r s i t y ,  1982  I N PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES (Department o f B i o c h e m i s t r y ) We a c c e p t  this  thesis  to the required  as  conforming  standard  THE UNIVERSITY OF B R I T I S H COLUMBIA April, ©  1985  Zemin Y a o , 1985  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may  be granted by the head o f  department or by h i s o r her r e p r e s e n t a t i v e s .  my  It is  understood t h a t copying o r p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department of The  U n i v e r s i t y o f B r i t i s h Columbia  1956  Main Mall  V6T  1Y3  Vancouver, Canada  Date  >E-6  (3/81)  written  ABSTRACT  Two  kinds of a f f i n i t y chromatography,  Sepharose  C D P - c h o l i n e - and  4B, w e r e i n v e s t i g a t e d f o r p u r i f i c a t i o n  CTP:phosphocholine  c y t i d y l y l t r a n s f e r a s e from  enzyme d i d n o t show s t r o n g a f f i n i t y  magnesium a c e t a t e .  tography  with  purification about  The  ion-exchange  cytosolic  rat liver.  f o r the CDP-choline  r e s i n , but bound t o the C T P - S e p h a r o s e c o l u m n 14 mM  of the  i n the presence of  techniques provided enzyme w i t h  The  Sepharose  c o m b i n a t i o n o f CTP a f f i n i t y  of the c y t o s o l i c  CTP-  about  a specific  chroma70-fold  activity  of  90 u n i t s p e r m i l l i g r a m p r o t e i n . The  i n f l u e n c e of d i p h e n y l s u l f o n e  compounds on t h e  o f p h o s p h a t i d y l c h o l i n e by t h e C D P - c h o l i n e p a t h w a y was isolated  r a t h e p a t o c y t e s and  the s u l f o n e s  (100 u g / m l ) ,  H e L a c e l l s . The  except dapsone,  synthesis  examined  in  a d m i n i s t r a t i o n of  t o HeLa c e l l s  inhibited  3  the t o t a l  [methyl-  H]choline  i n c o r p o r a t i o n i n t o the c e l l s ,  d i d n o t change t h e r a t e o f c o n v e r s i o n choline.  The  a d d i t i o n of the s u l f o n e s  c y t e s d i d not i n h i b i t choline The  of c h o l i n e to  but  phosphatidyl-  (100 ug/ml) t o r a t h e p a t o -  the b i o s y n t h e s i s of p h o s p h a t i d y l c h o l i n e  and  metabolism. effect  of vasopressin  on t h e d i s t r i b u t i o n  t r a n s f e r a s e b e t w e e n c y t o s o l and m i c r o s o m e s  vasopressin  (5-20  of  H]choline  [methyl-  was  reduced  nM) , w h i l e  the  from  cytidylyl-  i n rat hepatocytes  a l s o i n v e s t i g a t e d . The d i g i t o n i n - m e d i a t e d cytidylyltransferase  of  r e l e a s e of  the c e l l s  enhanced  rate  into phosphatidylcholine  of was  was  cytosolic  treated  with  incorporation not observed.  TABLE  Chapter  I.  OF CONTENTS  Introduction  1  1.  Structure  2.  Biological  3.  Pathways o f p h o s p h a t i d y l c h o l i n e mammals  4.  1  functions of phosphatidylcholine  Enzymes o f p h o s p h a t i d y l c h o l i n e Kennedy pathway  5.  The t h e s i s  4 biosynthesis v i a 4 biosynthesis  investigations  7 8  I I . P u r i f i c a t i o n o f C T P : p h o s p h o c h o 1 i ne c y t i d y l y l t r a n s f e r a s e f r o m r a t l i v e r c y t o s o l by a f f i n i t y chromatography  Materials  1  biosynthesis in  Regulation of phosphatidylcholine v i a Kennedy pathway  6. Chapter  of phosphatidylcholine  and methods  10 13  Results  18  Discussion  28  Chapter  III.  E f f e c t s o f d i p h e n y l s u l f o n e compounds on t h e metabolism of [methyl- H]choline in rat h e p a t o c y t e s and HeLa c e l l s  Materials  and methods  32 36  Results  39  Discussion  48  Chapter  IV.  Effects  of vasopressin  cytidylyltransferase Materials  and Methods  on C T P : p h o s p h o c h o l i n e  i n r a t hepatocytes  52 57  Results  59  Discussion  67  References  72 III  L I S T OF TABLES Table  Page  1.  P u r i f i c a t i o n of CTPtPhosphocholine from Rat L i v e r C y t o s o l  Cytidylyltransferase  2.  E f f e c t s o f Dapsone and AUS on t h e [ M e t h y l U p t a k e by HeLa C e l l s  27  •3  3.  H]Choline 40  E f f e c t s o f D i p h e n y l s u l f o n e d e r i v a t i v e s ( I I I and IV) on The [ M e t h y l - H ] C h o l i n e U p t a k e by HeLa C e l l s  41  E f f e c t o f V a s o p r e s s i n on The I n c o r p o r a t i o n o f H] C h o l i n e i n t o PC  66  3  4.  3  IV  [Methyl-  LIST OF  FIGURES  Figure  Page  1.  S p a c e - f i l l i n g Model o f  2.  Kennedy Pathway f o r P h o s p h a t i d y l c h o l i n e B i o s y n t h e s i s from C h o l i n e  5  P h o s p h a t i d y l c h o l i n e B i o s y n t h e s i s by S u c c e s s i v e N - M e t h y l a t i o n of P h o s p h a t i d y l e t h a n o l a m i n e  6  4.  Scheme o f The  19  5.  D E A E - C e l l u l o s e c h r o m a t o g r a p h y o f Rat C y t i d y l y l t ransf erase  3.  6. 7. 8. 9. 10. 11. 12.  Phosphatidylcholine  2  S y n t h e s i s o f C D P - C h o l i n e S e p h a r o s e 4B Liver  Cytosolic 22  C D P - C h o l i n e S e p h a r o s e 4B C h r o m a t o g r a p h y o f Rat Cytosolic Cytidylyltransferase P h o s p h o - C e l l u l o s e Chromatography of Cytosolic Cytidylyltransferase  Rat  25 Liver  S t r u c t u r e o f Dapsone and IV)  Analogs  ( I I ) and  23  Liver  CTP S e p h a r o s e 4B C h r o m a t o g r a p h y o f Rat Cyt i d y l y l t r a n s f erase ( I ) , AUS  Liver  Cytosolic 26 (III 35  I n f l u e n c e o f AUS and IV on The M e t a b o l i s m [ M e t h y l - H] C h o l i n e i n HeLa C e l l s  of 44  I n f l u e n c e , o f Dapsone and I I I on t h e M e t a b o l i s m [ M e t h y l - H] C h o l i n e i n Rat H e p a t o c y t e s  of 46  I n f l u e n c e o f AUS and IV on The M e t a b o l i s m [ M e t h y l - H] C h o l i n e i n Rat H e p a t o c y t e s  of  13.  Digitonin-mediated  Hepatocytes...  14.  E f f e c t of D i f f e r e n t C o n c e n t r a t i o n s of V a s o p r e s s i n on The D i g i t o n i n - m e d i a t e d R e l e a s e o f CT from Rat Hepatocytes  61  E f f e c t o f V a s o p r e s s i n on The I n c o r p o r a t i o n o f [ M e t h y l - H] C h o l i n e i n t o P h o s p h o l i p i d s  63  I n f l u e n c e o f V a s o p r e s s i n on [ M e t h y l - H ] C h o l i n e i n t o PC  64  15. 16.  Release  o f CT  V  The  from  Rat  47 60  I n c o r p o r a t i o n of  L I S T OF A ACS ATP AUS BHK cAMP CHO CoA CPT CT CTP DEAE DES DG DMSO dpm DTT E.R. FCS Fig. g g(m) hr HDL Hepes HMG-CoA Ig Km LCAT LDL lyso-PC lyso-PE m M MEM min PBS PC PE PG PGs PI PMSF PS Ptdlns PtdIns4P Ptdlns(4,5)P  ABBREVIATIONS  absorbance aqueous c o u n t i n g s c i n t i l l a n t adenosine 5'-triphosphate l-[4-(4-sulfanilyl)phenyl]urea baby h a m s t e r k i d n e y adenosine 35 -monophosphate C h i n e s e hamster ovary coenzyme A cholinephosphotransferase phosphocholine cytidylyltransferase cytidine 5 -triphosphate diethylaminoethyl dietheylstilboestrol diacylglycerol dimethylsulfoxide d i s i n t e g r a t i o n s per minute dithiothreitol endoplasmic r e t i c u l u m f e t a l c a l f serum figure gravity gram hour high density l i p o p r o t e i n 4-(2-hydroxyethyl)-1-piperazine-ethane sulphonic acid beta-hydroxy-beta-methyl glutaryl-CoA immunoglobulin Michaelis-Menten constant lecithin cholesterol acyltransferase low density lipoprotein lysophosphatidylcholine lysophosphatidylethanolamine meter mo 1 a r m o d i f i e d E a g l e ' s medium minute phosphate b u f f e r e d s a l i n e phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol prostaglandins phosphatidylinositol phenylmethylsulphonyl fluoride phosphatidylserine 1-(3-sn-phosphatidyl)-L-myo-inositol 1 - ( 3 - s n - p h o s p h a t i d y l ) - L - m y o - i n o s i t o l 4-phosphate 1 - ( 3 - s n - p h o s p h a t i d y l ) - L - m y o - i n o s i t o l 4,5-biphosphate 1  1  2  VI  r a t i o o f d i s t a n c e moved by a s o l u t e the solvent f r o n t r e v o l u t i o n s per minute standard deviation t h i n - l a y e r chromatography thymidine 5'-triphosphate t r i s ( h y d r o x y m e t h y l ) aminomethane uridine 5'-triphosphate ultraviolet very low d e n s i t y l i p o p r o t e i n  rpm S.D. TLC TTP Tris UTP UV VLDL Notes:  Standard 10~ ; 6  prefixes are:m (milli)  n (nano) -  10"  9  VII  - 10  relative to  ; u (micro) -  ACKNOWLEDGEMENTS  I w i s h t o thank continued  Dr. Dennis  E. V a n c e , my s u p e r v i s o r ,  e n c o u r a g e m e n t and h e l p f u l  comments t h r o u g h o u t  g r a d u a t e work. I a l s o a p p r e c i a t e t h e t e c h n i c a l g u i d a n c e received Mr.  Trang  from  for h i s my p o s t that I  D r . R o s e m a r y B. C o r n e l l , D r . Howard W. M u e l l e r and  Nguyen.  I thank  a l l my t e a c h e r s and g r a d u a t e  student  f r i e n d s i n t h e B i o c h e m i s t r y D e p a r t m e n t o f UBC f o r t h e i r c o n s t r u c tive  s u g g e s t i o n s on my e x p e r i m e n t s .  dear  motherland,  country,  Canada,  Finally,  I w i s h t o thank  my  t h e People's R e p u b l i c o f C h i n a , and t h e g r e a t f o rtheir  e n a b l e s me t o p u r s u e  offering  my p o s t - g r a d u a t e  VIII  such  an o p p o r t u n i t y t h a t  studies.  CHAPTER I .  1.  INTRODUCTION  STRUCTURE  OF PHOSPHATIDYLYLCHOLINE  Phosphatidylcholine in PC  (PC) i s t h e m a j o r  eukaryotes but r a r e l y occurs i s distinguished  headgroup, although the  glycerol  from  phospholipid  i n prokaryotes.  the other  The s t r u c t u r e o f  phospholipids  by i t s c h o l i n e  a v a r i e t y o f f a t t y a c i d s c a n be e s t e r i f i e d t o  backbone.  Usually,  saturated  fatty  e s t e r i f i e d a t the CI p o s i t i o n of g l y c e r o l w h i l e acids  present  acids  unsaturated  are fatty  a r e a t C2 p o s i t i o n ( F i g u r e 1 ) .  2. BIOLOGICAL FUNCTIONS OF PHOSPHATIDYLYLCHOLINE The from lic  primary  f u n c t i o n o f PC i s s t r u c t u r a l ,  i t s amphipathic whereas  ability  nature  the f a t t y  bilayer  organization  concentrations  acyl  chains  phospholipids  a u n i v e r s a l c o n s t i t u e n t of c e l l  phospholipid of  i n eukaryotes  the t o t a l  described  membrane  of excess  as a r e q u i r e m e n t  maintaining  membranes.  i s PC, w h i c h  of  (1).  the f u n c t i o n a l conformation  (CMC)  The most a b u n d a n t  accounts  PC o f f e r s  1  and a t  phospholipids  PC  f o r nearly has  also  f o r the a c t i v i t y of several  bound enzymes ( 2 ) . A p p a r e n t l y , for  water  micellar concentration"  phospholipid  The  t o assume s p o n t a n e o u s l y a  i n the presence  above t h e " c r i t i c a l  are hydrophobic.  m i g h t be t h e d o m i n a n t r e a s o n f o r N a t u r e ' s c h o i c e as  arises  the c h o l i n e headgroup i s h y d r o p h i -  acid  o f PC and o t h e r  which  a suitable  50% been  membrane-  environment  o f t h o s e enzymes. I n  Fig.l.  Space-filling  choline.  2  model o f  phosphatidyl-  addition  t o t h e membrane  constituent bile, the  of b i l e ,  PC a c c o u n t s  plasma  r a t (3) and 68% i n h u m a n s bile  in  The f u n c t i o n  bile.  clear.  the core  plasma,  target  of lung  PC c o a t s  itself  tissue  during  tension  expiration  major As  t o be r e s p o n s i b l e  cause  well  as b e i n g  important ways.  of m o r t a l i t y  PC s e r v e s  synthesis  of  i n lung  and m o r b i d i t y  as a donor  cholesterol  PC i s u t i l i z e d  boxanes  and  species  o f PC  platelet  activating  involved  ester  Recently,  (l-alkyl-2-acetyl-PC) which  3  ester from  soluble liver  to  f o r approximately of lung,  the collapse  o f the  after birth is  Distress  Syndrome, t h e  i n premature PC a l s o  i n many  in a  f o r the synthesis  factor  of l i p o p r o t e i n to  alveoli  of the f a t t y  acyltransferase  leukotrienes.  of c h o l e s t e r o l  F a i l u r e t o m a i n t a i n an  f o r Respiratory  precursor  lecithin-cholesterol from  (5).  with  l i p o p r o t e i n i s not  accounts  a s t r u c t u r a l component,  metabolic  component i n  and l i n e s t h e a l v e o l i  adequate supply of t h i s m a t e r i a l thought  plasma  and p r e v e n t s  In  PC t o g e t h e r  and c h o l e s t e r o l  PC  as a  surfactant.  i s transported  by w e i g h t  which lowers the s u r f a c e alveolar  The b i l e  the surface  v i a lipoproteins.  surfactant  functions  phospholipid  o f PC i n b l o o d  PC  also  in solubilization  of t r i a c y l g l y c e r o l  and a l s o ,  tissues  (4).  functions  I t i s thought  render  70%  acids  PC  l i p o p r o t e i n s and l u n g  f o r 90% o f t h e t o t a l  conjugated  in  component,  functions  a s an  biochemical  path-  acyl  moiety  reaction (LCAT).  i n f a n t s (6).  f o r the  catalyzed  Arachidonic  of prostaglandins, a  particular  has been  by acid  throm-  molecular  described  as a  c a u s e s p l a t e l e t a g g r e g a t i o n (7).  3. PATHWAYS OF  PHOSPHATIDYLYLCHOLINE BIOSYNTHESIS IN MAMMALS  There are f i v e separate pathways which l e a d to the o f PC  (1).  They  as C D P - c h o l i n e which  a r e : 1) t h e m a j o r de n o v o p a t h w a y  choline  and  PC  conversion  (Figure 2);  phatidylethanolamine ferase  2) t h e s t e p w i s e  (PE)  (9) ( F i g u r e 3 ) ;  which  (lyso-PC)  b e t w e e n two of the  total  and  the remaining  the  other  three  PC  methylation  i s . c a t a l y z e d by  (10,11); by  molecules  70%  1950's ( 8 ) ,  PE  of  CDPphos-  methyltrans-  3) t h e b a s e e x c h a n g e r e a c t i o n i n w h i c h  diacylglycerophospholipid phatidylcholine  known  of c h o l i n e to phosphocholine,  f r e e c h o l i n e can d i s p l a c e e t h a n o l a m i n e  tion  (also  p a t h w a y ) d e s c r i b e d by K e n n e d y i n t h e  i n v o l v e s the  formation  4)  acyl-CoA  or s e r i n e from the  acylation  (12,13);  of lyso-PC  (14).  i s s y n t h e s i z e d by  and  of  lysophos-  5) t r a n s a c y l a -  In r a t l i v e r ,  the CDP-choline  30% b y t h e m e t h y l a t i o n p a t h w a y  mechanisms are not  appropriate  important  about  pathway  (15), whereas  f o r de  novo  bio-  synthesis. 4.  ENZYMES OF  PHOSPHATIDYLYLCHOLINE BIOSYNTHESIS VIA  KENNEDY  PATHWAY S t u d i e s on  PC  metabolism  are c o m p l i c a t e d  s i n c e the  of enzymes i n v o l v e d i n p h o s p h o l i p i d b i o s y n t h e s i s are bound o r r e q u i r e l i p i d choline of  kinase  choline  by  for their  activity.  (EC  2.7.1.32) w h i c h  ATP  i n the  Kennedy  The  membrane  only exception i s  c a t a l y z e s the pathway.  majority  phosphorylation  Choline  kinase  is  n e i t h e r a s s o c i a t e d w i t h membranes n o r - r e q u i r e s p h o s p h o l i p i d s f o r activity, kidney  and  has  r e c e n t l y been p u r i f i e d  to homogeneity from  i n a d i m e r i c form w i t h the molecular  4  weight  of  rat  80,000(16).  \ « H H H,C—N—C—C—OH / H H HjC  CHOLINE ATP-  CHOLINE  KINASE  ADP-  o  H,c  \ o H H « H C—N—C—C—O—P—0 / H H I H,C O  s  3  e  PHOSPHOCHOLINE CTP  CYTIDYLYLTRANSFERASE NH  PPi  2  N  0  H3C  C  \ H H n 11 o H C—N—C—C—O—P—O—P—O—CH, / H H I I In H C O O ' ' F F L  3  U  3  E  E  CDP-CHOLINE  OH OH  DIGLYCERIDE  CHOLINEPHOSPHOTRANSFERASE  H C—O—C—R 2  CH H H «./ H.C—O—P—O—C-C—N—CHj I H H \ 0o CH,  R— C — O — C H  3  PHOSPHATIDYLCHOLINE  F i g . 2 . Kennedy pathway for phosphatidylchol i n e biosynthesis from choline.  5  o O  o  CH,-0-C-R,  A d 0 W e  R -C-O-C-H O I II CH -0-P-OCH CH NH 2  2  2  '  X  A d  °  O  H c y  CH -0-C-R, 2  • Rj-C-O—C-H O I II CH -0-P—OCH CH -NH  y  3  2  2  O-  2  O-  Phosphatldylethanolamlne  CH  2  3  N-methylphosphalldylethanolamlne  AdoMel  AdoHcy  o O  o  CH -0-C-R,  A d  2  R,-C-O-i-H  O  . ^  CH -0-P-OCH CH N(CH ) I O" 2  °  H c y  2  2  3  A d  .° '  O  M e  <^  CH -0-C-R, 2  R _C-0-C-H 2  O  H  CH -0--P-OCH CH N-CH CH °  3  2  2  2  3  3  Phosphatidylcholine  N,N-<Jlmethylpho*phatidylethanolamine  Fig.3. Phosphatidylcholine biosynthesis by s u c c e s s i v e N - m e t h y l a t i o n o f p h o s p h a t i d y l ethanolamine.  6  Once the  choline only  has  f a t e of  been this  product  CTPtphosphocholine catalyzes line  and  lism.  the  formation  CTP,  the  Attempts  dominant  energy form  utilized  discussed  in Chapter  choline,  of  catalyzed  phot ransf erase recovered  CPT  was  fication The  of  enzyme  this  5.  REGULATION  properties  of  CT  in rat l i v e r  final  CMP  step  from  i n the  Kennedy  d i a c y l g l y c e r o l (DG)  2.7.8.2). The  the  activity  and  report  only  will  be  pathway and  CDP-  cholinephos-  o f CPT  mitochondria  from c h i c k  plasma  been  in  is mostly mammalians  macrophages  showed  (18).  Partial  puri-  membrane b o u n d e n z y m e h a s  been  reported  .(19).  for  OF  far  metabo-  membrane  requires  phospholipids  so  while a on  phosphocho-  in l i p i d have  enzyme  2.7.7.15)  CT  (microsomes)  located  from  CDP-choline:l,2-diacylglycerol (EC  E.R.  PPi  (EC  of  The  and  by  t i s s u e s ( 1 0 ,17 ) , that  II. PC  (CPT)  from  The  The  (CT)  and  purification  phosphocholine  formation.  CDP-choline  at  formation  PC  form  of  successful.  the  i s for  to  cytidylyltransferase  partially  is  phosphorylated  magnesium  as  a  cofactor  i t s maximum a c t i v i t y  and  microsomal  (19).  P H O S P H A T I D Y L C H O L I N E B I O S Y N T H E S I S VIA  KENNEDY  PATHWAY Towards provided  the  evidence  Kennedy pathway It  has  E.R.,  arises  of  that  1970's, the  is usually  been p o s t u l a t e d  translocation the  ends  o f CT  where  from the  it  is  the  the  PC by  the  activity  (20).  t h a t : 1) CT  7  of  approaches  biosynthesis  of  c y t o s o l , where  activated  observations  number  of  determined  that  from  rate  a  The  activity CT  of  have  via CT  the (20).  is regulated  by  i t i s i n a c t i v e , to conclusion  is ambiquitous  largely in that  it  i s recovered  2) t h e  i n both  c y t o s o l i c CT  activity  while  phospholipid  the  the  is  c y t o s o l i c and  inactive  and  m i c r o s o m a l CT  a c t i v a t i o n ; 3)there  i s a c t i v e and  are  i n s e n s i t i v e to  changes between  conditions.  been  obtained  from  studies  i n a v a r i e t y of mammalians o r g a n s , l i v e r  (21,22),  heart  (23),  by  lines  (26). The  microsomal  Supportive  and  evidence  i n t e s t i n e (25),  yet  proof  of  this  hypothesis  c o m p l e t e p u r i f i c a t i o n of the On  the other  (27)  as  well may  and  is still  in different  has  t r a n s l o c a t i o n p r o c e s s o f CT  r e v e r s i b l e enzyme p h o s p h o r y l a t i o n  (28),  CT  PC  physiological  (24)  of  parallel  for  and  lung  activity  needs p h o s p h o l i p i d  biosynthesis  cell  the  microsomal f r a c t i o n s ;  as be  by  waiting  enzyme f o r  6.  THE  PC  choline  biosynthesis  for  the  and  theoretical evi-  kinase  m i g h t be  a  synthesis  rate-limiting  (29,30).  THESIS INVESTIGATIONS Liver  i s a major organ f o r p h o s p h o l i p i d  phospholipids  synthesized  membrane f o r m a t i o n bile  or  plasma  laboratory regulatory ship  by  acids  enzyme.  h a n d , some e x p e r i m e n t a l  catalyzed  some  regulated  fatty  d e n c e s u g g e s t s t h a t t h e f i r s t r e a c t i o n i n t h e d_e n o v o pathway  in  in  PC  liver  p a r t i c i p a t e not  w i t h i n the organ but  the  only  in  a l s o are t r a n s f e r r e d  the  last  decade the  b i o s y n t h e s i s - and  were PC  focused  revealing the  enzymes i n v o l v e d  i n PC  biosynthesis  liver.  t h e s i s , I have i n v e s t i g a t e d t h e  into this the  relation-  l i p o p r o t e i n metabolism,  the  8  on  biosynthesis,  p u r i f i c a t i o n of In t h i s  and  l i p o p r o t e i n s ( 3 1 ) . H e n c e , many e f f o r t s o f  mechanisms behind  between  in  biosynthesis  and  from  regulatory  on rat  mecha-  nism  of  PC  biosynthesis  diphenylsulfone IV),  and  compounds  also attempted  techniques  in cultured  including  (Chapter  I I I ) or  t o p u r i f y CT  affinity  rat hepatocytes  from  vasopressin rat l i v e r  chromatography  9  by  by  using  (Chapter different  (Chapter I I ) .  CHAPTER I I . PURIFICATION  OF CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFER-  ASE FROM RAT LIVER CYTOSOL BY A F F I N I T Y CHROMATOGRAPHY  CTP:phosphochol ine catalyzes phate  the r e v e r s i b l e  formation of CDP-choline  and  f r o m p h o s p h o c h o l i n e a n d CTP, a n d may c o n t r o l  phosphatidylcholine can a l s o but  c y t i d y l y l t r a n s f e r a s e (CT) (E.C.2.7.7.15)  (PC) b i o s y n t h e s i s  s e r v e as a s u b s t r a t e  pyrophos-  the rate of  (15,32,33,34).  instead  the other nucleoside triphosphate  Deoxy-CTP  o f CTP i n t h i s (ATP, GTP,  UTP,  reaction, o r TTP) do  not (35). CT h a s b e e n associated tissue organs  a s an a m b i q u i t o u s  with both the s o l u b l e  homogenates  and n e e d s  i t i s the dominant  microsomal activated  and m i c r o s o m a l  ( 3 4 ) . The c y t o s o l i c  i s inactive  although  minor  described  by exogenous p h o s p h o l i p i d  enzyme f o r m w i t h i n c e l l s  t h e enzyme b e t w e e n c y t o s o l  lic  o v e r 70% of t h e t o t a l fraction,  associates  with  been observed days  while  for i t s activity, in cells.  The  and i s n o t f u r t h e r  although i t i s always the  and m i c r o s o m e s v a r i e s  i s redistributed  10  most  (21). A s i m i l a r  i n l u n g (36). In f e t a l  liver  of  i s detected i n the c y t o s o -  on t h e d a y o f b i r t h ,  so t h a t t h e m i c r o s o m a l a c t i v i t y  in different  animals. In prenatal  CT p r o t e i n  the microsomes  p r e m a t u r e l y , CT  form  different  (20). The r e l a t i v e d i s t r i b u t i o n o f  developmental stages of i n d i v i d u a l rat,  from  o f t h e enzyme  CT, h o w e v e r , i s t h e a c t i v e  which i s  f r a c t i o n s of the  CT i s o l a t e d  phospholipid  form  enzyme  o f t h e enzyme phenomenon has  rats delivered from  i s increased  cytosol  one o r two  t o microsome  by 6 0 % , w h i l e t h e r e  is  no c h a n g e i n t h e t o t a l  activity  o f CT ( 3 6 ) .  I n a d d i t i o n , t h e r e l a t i v e d i s t r i b u t i o n o f CT b e t w e e n and  microsomes  (37).  If  i s dependent  adult  on c o n d i t i o n s  rat liver  used  i s homogenized  cytosol  i n homogenization  in isotonic  (0.145 M N a C l ) , more t h e n 90% o f t h e enzyme a c t i v i t y  saline  i s recovered  f r o m t h e 1 7 0 , 000 x g , 1 h r s u p e r n a t a n t ( c y t o s o l ) , w h e r e a s homogenization activity  performed  in  being asssociated  distilled with  water  results  the p e l l e t  in  fraction  85% o f t h e (microsomes)  (37) . The s t r a t e g i e s taken  advantage  (38,39,40,41).  i n CT p u r i f i c a t i o n  of the soluble  i n terms  u n i t s per organ ing  material  so f a r h a v e a l l  property of the c y t o s o l i c  The o c c u r r e n c e o f CT i n t h e c y t o s o l  organ to organ, of which l i v e r activity  published  of e i t h e r  (42).  varies  from  e x h i b i t s the greatest c y t o s o l i c units  Thus, l i v e r  f o r preparative  enzyme  p e r gram o f t i s s u e  or  total  a p p e a r s t o be an i d e a l  purposes.  However,the  CT  start-  purification  o f t h e c y t o s o l i c CT i s c o m p l i c a t e d s i n c e t h e s o l u b l e enzyme h a s a strong which  tendency  to aggregate  i n the presence  of  phospholipid  i s u n a v o i d a b l y p r e s e n t i n c y t o s o l due t o t h e b r e a k a g e o f  membranes mers w i t h  during  homogenization.  The a g g r e g a t e d CT f o r m s  poly-  d i v e r s e m o l e c u l a r w e i g h t s r a n g i n g b e t w e e n 5 x 10^ and  7 1.3 x 10 , a n d b e h a v e s sitive form  to phospholipid  l i k e t h e m i c r o s o m a l enzyme w h i c h i s i n s e n activation,  i s e s t i m a t e d t o have a  while  material isolated  enzyme  m o l e c u l a r weight of 2 x 1 0 (28). 5  The a g g r e g a t i o n o f CT w i t h p h o s p h o l i p i d attempts to displace  the unaggregated  seems i r r e v e r s i b l e  t h e enzyme a c t i v i t y  from the p a r t i c u l a t e  from water homogenates o f r a t l i v e r 11  since  were unsuc-  cessful  (21,37).  stabilize to the  On  the  the p a r t i a l l y  other  hand,  purified  enzyme p r e p a r a t i o n  CT.  phospholipid  The  at l a t e r  a d d i t i o n of  purification  appears  phospholipid  stages  can  main-  t a i n t h e enzyme a c t i v i t y w h i c h i s l o s t d r a m a t i c a l l y i n t h e ce  of  cytosol  and  s t u d i e s on  showed  that  the  e q u i l i b r i u m constant phosphocholine  CDP-choline mM)  of  CDP-choline substrate  partially  of  0.80  ( 4 3 ) . The  i n the  from  of the  CT  had  formation  of  CTP  Michaelis constants CT  are  the  be  an  ideal  and  and  same (Km  CPT,  candidate  CPT  f o r CTP  phospholipid  f o r o n l y CT  liver  by  =  (43).  while  f o r s e v e r a l enzymes i n v o l v e d i n l i p i d should  rat  0.21 Since  CTP  is a  biosynthesis,  for a f f i n i t y  column  i s a membrane-associated  enzyme  i s absent i n c y t o s o l . In  graphy,  the  present  studies,  C D P - c h o l i n e and  purification  results  of  the  showed c y t o s o l i c  CDP-choline column i n the affinity  about  70-fold  units  per  the  i n favour  presence of  p u r i f i c a t i o n o f c y t o s o l i c CT.  CTP  CT  reversible reaction catalyzed  i s a substrate  CDP-choline  for  purified  the p a r t i a l l y p u r i f i e d  when a s s a y e d  which  absen-  phospholipid. In v i t r o  an  to  Sepharose  two  kinds  of  affinity  chromato-  C T P - S e p h a r o s e 4B,  h a v e been i n v e s t i g a t e d  cytosolic  rat l i v e r .  CT  CT  d i d not  resin  but  from  have s t r o n g  did  bind  to  p r e s e n c e of magnesium a c e t a t e .  affinity  the The  Preliminary  CTP  with  milligram protein.  later purification  stages  a specific  The was  12  problem still  activity of  losing  unsolved.  of  the  affinity  combination  chromatography w i t h ion-exchange techniques purification  to  of  provided about  90  activity  at  MATERIALS AND METHODS  Chemicalsducts  DE-52 and P - l l i o n i c  exchange r e s i n s were the p r o -  o f Whatman. ATP, CTP, C D P - c h o l i n e ,  and  c h o l i n e k i n a s e were purchased  and  Aqueous C o u n t i n g  Radiochemical uci/umol)  from  Scintillant  Center, Amersham.  was  adipic  acid  dihydrazide  Sigma.  [methyl- H]Choline  (ACS) w e r e  o b t a i n e d from the  3  [ m e t h y l - H] P h o s p h o c h o l i n e  s y n t h e s i z e d e n z y m a t i c a l l y from  (10  [methyl- H]choline 3  and ATP w i t h c h o l i n e k i n a s e b a s e d on t h e m e t h o d o f V a n c e e_t a l . ( 4 3 ) . 2*, 7'-d i c h l o r o - f l u o r e s c e i n was a p r o d u c t o f E a s t m a n K o d a k Co. P r e p a r a t i o n of Rat L i v e r C y t o s o l -  Rat l i v e r  cytosol  was  prepared  b a s e d on t h e m e t h o d d e s c r i b e d b y C h o y e_t a _ l . ( 4 0 ) . W i s t a r  rats  (about  Unit  200 g) f r o m  were,decapitated, homogenate  the U n i v e r s i t y of B r i t i s h  Columbia Animal  and t h e l i v e r s w e r e removed i m m e d i a t e l y .  of l i v e r  was  prepared  (0.145>M N a C l ) c o n t a i n i n g 0.5 mM  in ice-cold  isotonic  phenylmethylsulfonyl  saline fluoride  (PMSF) b y 5 s t r o k e s i n a P o t t e r - E l v e h j e m h o m o g e n i z e r , and fuged  a t 1 7 0 , 000 x g_ f o r 60 m i n a t 4°C.  filtered ty  through  after  loss  cytosolic  layer  which  floated  ultracentrifugation.  s t a b l e , as f r e e z i n g table  centri-  supernatant  was  two l a y e r s o f c h e e s e c l o t h t o remove t h e m a j o r i -  of the l i p i d - r i c h  interface  The  A 25%  CT  a t -70°C and t h a w i n g  o f enzyme a c t i v i t y . fraction.  13  The  at the  in this  air-solution  sample i s very  a t 37°C c a u s e s  s a m p l e was  no  detec-  designated  as  D E A E - C e l l u l o s e C h r o m a t o g r a p h y o f C y t o s o l i c CT- A l l t h e p r o c e d u r e s described at  below  were p e r f o r m e d  a t 4°C.  The l i v e r  -70°C w a s t h a w e d a t 37°C a n d q u i c k l y c o o l e d  longed  incubation  i n 37°C w i l l  cytosol  stored  i n i c e , as p r o -  cause a g g r e g a t i o n o f t h e c y t o s o l i c  CT. The s a m p l e was b r o u g h t t o 30% ammonium s u l f a t e s a t u r a t i o n i n the  presence  discarded  after  supernatant the  X-100,  centrifugation  was b r o u g h t  and t h e p r e c i p i t a t e  6.5) b u f f e r  t o 40% ammonium  The p e l l e t was s u s p e n d e d containing  dithiothreitol  sulfate  i n 20 mM  saturation  and  u n d e r t h e same  Tris-succinate  0.025% T r i t o n X - 1 0 0 , 0.5 mM  (DTT) t o t h e same v o l u m e a s t h a t  nal c y t o s o l , thus ensuring a low c o n d u c t i v i t y . the  was  a t 10,000 x £ f o r 10 m i n . The  precipitate collected after centrifugation  conditions.  mM  o f 0.05% T r i t o n  (pH  PMSF a n d 0.5 of the o r i g i -  T h i s was c a l l e d  40% f r a c t i o n . The  40% sample  column  130 m l ) was  applied  (3 x 35 cm) w h i c h h a d b e e n e q u i l i b r a t e d w i t h  buffer.  Subsequently,  and  then with  the  same b u f f e r .  activity tivity  (approx.  a 400 ml N a C l g r a d i e n t  around  this laboratory 15-fold  Fractions  was a s s a y e d .  region  t h e c o l u m n was e l u t e d  The enzyme a c t i v i t y  and g i v e s  p u r i f i c a t i o n with  fraction.  20-40% y i e l d  months w i t h  be s t o r e d  the sample  50 ml o f b u f f e r  collected eluted  This protocol  reproducible  The enzyme c o u l d  DE-52  r a n g i n g b e t w e e n 0-0.5 M i n  (9 m l ) w e r e  3 mMHO.  with  to a  and enzyme  i n t h e conduc-  i s r o u t i n e l y used i n  results of appoximately r e l a t i v e to the c y t o s o l i c a t -70°C f o r a t l e a s t  2-3  n e g l i g i b l e l o s s o f enzyme a c t i v i t y .  P h o s p h o - C e l l u l o s e C h r o m a t o g r a p h y o f CT- The p r o c e d u r e o f CT p u r i fication  by  phospho-cellulose  was d e v e l o p e d 14  in this  laboratory  (Sanghera, J., unpublished r e s u l t ) . chromatography had  was  The  bulk  ductivity  range  extremely  unstable  Enzyme A s s a y based  on  should  The  0.15  pH  in a final  6.5;  0.75  umol o f  appropriate performed  each  lane.  (10:10:1; short-wave (0.01%  UV  CT  NaCl  M NaCl  i n the  con-  in this  sample  to the  successive  is  was e s s e n t i a l l y  v o l u m e o f 100 nmol  u l : 0.2  of o l e a t e ;  7.5  reaction  mg  of  total  umol  of  Tris-  0.2  min,  (2-40 ug).  and  umol  light  vial  reaction  s t o p p e d by i m m e r s i o n  of  ( Western  with  3 x 10 cm  50  min.  scraped  with  with  from  cen-  applied  to  CDP-choline  i n CH3OH /0.6%  the  was  NaCl/NH  visible  3  under  2',7'-dichlorofluorescein plates  into  a  0.5 ml o f w a t e r a n d 4.5 m l o f ACS 15  was  c a r r i e r s were a p p l i e d f o r  CDP-choline  a f t e r spraying  of the  H-103N  per lane.  an was  The p r o t e i n p r e c i p i t a t e  p l a t e s were d e v e l o p e d  for  The  40 u l o f t h e s u p e r n a t a n t was  (Merck)  i n CH^OH), a n d  scintillation  M  by Vance et. a l . . (43). The  water f o r 2 min.  TLC  v:v:v)  i n DE-52  ml o f 0.2  located  CT a c t i v i t y  p h o s p h o c h o l i n e (0.6 mg) The  which  [ m e t h y l - H] p h o s p h o c h o l i n e (10 u c i / u m o l ) and  plates  and  80  be s u b j e c t e d  b y c e n t r i f u g a t i o n a t 2,500 rpm  G-60  as used  umol of magnesium a c e t a t e ;  t r i f u g e ) f o r 5 m i n , and  (0.1 mg)  with  mMHO.  amount o f enzyme p r o t e i n  tubes i n b o i l i n g  Silica  10  a t 37°C f o r 15-30  pelleted  8.5  measurement of  phospholipid;  succinate, CTP;  and  7 and  the methods d e s c r i b e d  liver  (2.5 x 8 cm)  buffer  e n z y m e p e a k was  DE-52  as q u i c k l y as p o s s i b l e .  mixture contained rat  The  between  step  eluted  from  r e m o v e d w i t h a 100 ml g r a d i e n t o f 0-0.6  i n t o 4.5 m l f r a c t i o n s .  purification  the sample  o f p r o t e i n was  and t h e e n z y m e was  enzyme p o o l e d  a p p l i e d to a P - l l column  been e q u i l i b r a t e d w i t h  column.  The  plastic fluid.  The  samples  were counted  efficiency. visualized of  The  region  for radioactivity  with  of phosphocholine  i n an i o d i n e v a p o u r  on  20-30% c o u n t i n g the plates  was  i n order to confirm the separation  CDP-choline from phosphocholine.  One u n i t o f enzyme a c t i v i t y  i s d e f i n e d a s one umole o f C D P - c h o l i n e f o r m e d p e r m i n . Protein Assay-  The B i o - R a d a s s a y b a s e d on t h e method o f B r a d f o r d  (44) was u s e d Rad  f o r the estimation  s t o c k r e a g e n t was d i l u t e d  2.5 m l o f t h e sample  with  diluted  of protein  in distilled  r e a g e n t was  concentration.  water  added  Bio-  (4:13; v : v ) , and  t o 0.5 m l o f p r o t e i n  IgG ( B i o - R a d S t a n d a r d ) a s t h e p r o t e i n s t a n d a r d .  a s s a y was l i n e a r  The  i n t h e r a n g e b e t w e e n 5-80 ug o f p r o t e i n .  For  some a s s a y s , t h e m i c r o a s s a y p r o c e d u r e was a d o p t e d t o d e t e r m i n e protein of  concentrations  less  the c o n c e n t r a t e d Bio-Rad  p r o t e i n sample.  t h e n 10 u g / m l .  In t h i s  case,  0.2 ml  s t o c k r e a g e n t was added t o 0.8 ml o f  The a b s o r b a n c e  was r e a d a t 595 nm a f t e r  15 m i n  b u t b e f o r e 60 m i n . P r e p a r a t i o n of T o t a l Rat L i v e r P h o s p h o l i p i d phospholipid rat  liver  pellet  was e x t r a c t e d  from  and Dyer  Sorvall  method  by s t i r r i n g  The m i x t u r e was c e n t r i f u g e d  with  a t 8,000 rpm ( i n a  tered  through glasswool.  ratio  o f C H C 1 / C H 0 H / H 0 t o 1:1:0.8 (v:v:v) by t h e a d d i t i o n 3  The f i l t r a t e  was b r o u g h t  fil-  to the f i n a l  2  m l o f w a t e r a n d 100 m l o f c h l o r o f o r m . T h e f i l t r a t e was  16  100  f o r1 hr at  Type GSA r o t o r ) f o r 20 m i n , and t h e s u p e r n a t a n t was  3  liver  ( 4 5 ) . The  i n 80 ml o f w a t e r , and m i x e d  m l o f c h l o r o f o r m a n d 200 m l o f m e t h a n o l room t e m p e r a t u r e .  rat  t h e 170,000 x g x 1 h r p e l l . e t o f  homogenate by t h e B l i g h  was r e - h o m o g e n i z e d  Total  o f 80  centri-  fuged  under  transferred chloroform for  20  neutral gen.  t h e same  c o n d i t i o n , and t h e c h l o r o f o r m  to a preweighed  round-bottom  b y r o t a r y e v a p o r a t i o n . The f l a s k  m i n and  rinsed  with  l i p i d s . The r e m a i n i n g  The p h o s p h o l i p i d  ice-cold  flask  was d i s s o l v e d i n c h l o r o f o r m  17  a t -20  °C  to e x t r a c t the  a c e t o n e was e v a p o r a t e d  c o n c e n t r a t i o n o f 20 mg/ml and s t o r e d a t -20°C.  was  t o remove t h e  was c o o l e d  acetone  phase  under at a  nitrofinal  RESULTS  Preparation  of  CDP-Choline  method o r i g i n a l l y Sepharose  4B  Sepharose  used f o r the  ( 4 6 , 4 7 ) was  summarized  in  used  to  bromide  method d e s c r i b e d prewashed with of  H2O.  suspension 11 w i t h by the  and  of  activated by  CNBr mixed  i c e . The  addition  of  2)  Sepharose  i n the  H0 (10  quickly.  ice  to  the  of  was  Cuatrecasas  reaction  saturated  prepared (50  was  poured  reaction  was  4B.  by  the  ml)  was  the  ml  resin  maintained  at  pH  c o n t r o l l e d b e l o w 20°C  about  15  The  M Na C0 2  into  min  and  stopped  activated (pH  3  9.5)  resin and  by was  finally  was  4B  mixed  The  Acid  i n t o the  Dihydrazide. CNBr  r e s i n was  washed e x t e n s i v e l y  bound  resin  (pH  5.0),  and  hydrazide  as  with  sodium borate containing 18  17 h r a t  ligand  turned  several  4°C  activated  for  M sodium acetate  covalently  Adipic  performed  s u s p e n s i o n . The  (48).  The  Sepharose  and  of  was  Sepharose  same s o l u t i o n .  resin,  presence  reactions  f i n a l l y s u s p e n d e d i n 50  g)  i c e - c o l d 0.1  (4.5 g)  NaCl°and t h e n 0.1  scheme o f t h e  4B  mixture.  acid dihydrazide  the  to  oxidized  t h e t e m p e r a t u r e was  Adipic  stirring  and  2  powder  Preparation  the  a  an  ( 4 8 ) . S e p h a r o s e 4B  r e a c t i o n t i m e was  washed q u i c k l y w i t h resuspended  covalently  g e l . The  Cuatrecasas  6 N NaOH, and  adding  of C D P - d i g l y c e r i d e  Cyanogen Bromide A c t i v a t e d  1 M N a C l and  Ground  link  of  Fig.4.  1) P r e p a r a t i o n Cyanogen  A modification  coupling  d e r i v a t i v e of C D P - c h o l i n e to the are  4B  by  tested  gently with  1M  for  the  described  by  a dark  in  d r o p s of  3%  red  2,4,6-  NH,  0 0 (CH_) -N-CH.-CH -.0-P-0-p'-0-CH OH OH o  3  o  3  N J. o  1  OH  OH  BrCN  NalO,  -CH„  HC 0  ACTIVATED  SEPHAROSE 4B  H H 0 0 H H HN-N-C-(CH ) -C-N-NH  BASE  2  CH 0  4  HO 0 H H H -N-N-C'-(CH ) ,-C-N-N-C-O-SEPHAROSE 2'4 NH 0  l  0 0 CHOLINE-P-O-P-O-CH OH OH  0  4B  BASE  CH HC C C HO N OH NH CO (9  H  9°  2  >4  NH NH C=NH i 0 SEPHAROSE 4B  Fig  4. Scheme o f The S y n t h e s i s  19  o f CDP-Choline  S e p h a r o s e 4B„  trinitrobenzene  sulfate,  while  unsubstituted  resin  was  pale  yellow. 3)  Oxidation  of CDP-Choline.  i . e . , 5 u m o l p e r m l o f r e s i n ) was the  ribosyl  of NalO^ acetic room  hydroxyls  dialdehyde  was  C H O H / 0.6% an J  increased  Acid  sodium  (pH 5.0)  H]  on  used t o e s t i m a t e  a t 4°C. Silica  The  oxidized  acid  a t 4°C.  The  gel with  containing  content  0.5  used  dihydrazide  was  M KC1  Preparation similarly coupling  solvent had  [methy1-  efficiency. 4B A d i p i c  0.1 M s o d i u m 2  and  i n 0.1  acetate  A n a l y s i s of the  1.7  of  M  mixed  finally  radioactivity  the  with CDP-  [methyl-  umol of  CDP-  of g e l .  of CTP-Sepharose  to  (0.59).  combined  C D P - c h o l i n e on t h e r e s i n ) i n d i c a t e d a b o u t  c h o l i n e p e r ml  the  s o l u t i o n and  f o l l o w e d by H 0  the  of  v o l u m e o f 90 ml and  washed w i t h  of  the  to Sepharose  were  f o r chromatography.  (determination  with  oxidized product  CDP-choline  to give a f i n a l resin  5.55  Formation  the o x i d a t i o n  of O x i d i z e d CDP-Choline  (pH 5.0)  b u f f e r t o be  choline  by TLC  t o pH  out i n the dark f o r 2 hr a t  ( 1 0 / 1 0 / 1 ; v / v / v ) . The  3  adipic  acetate  overnight  the  was  Dihydrazide. 4B  carried  adjusted  (0.73) r e l a t i v e t o C D P - c h o l i n e  f  Coupling  Sepharose  3  R  and  2  then overnight  determined  H]CDP-choline 4)  and  NaCl/NH  3  mmol;  d i s s o l v e d i n 25 m l o f H 0 ,  m i x t u r e was  r e a c t i o n was  temperature  (0.25  were o x i d i z e d t o a l d e h y d e s by t h e a d d i t i o n  (0.25 m m o l ) . The  a c i d . The  CDP-choline  that  efficiency  of  4B.  CDP-choline  (2.0 umol o f CTP  20  C T P - S e p h a r o s e 4B was Sepharose  4B  with  p e r ml o f g e l ) .  prepared similar  Separation  of C y t i d y l y l t r a n s f e r a s e  Chromatography.  The  following  by  CDP-Choline Sepharose  p r o c e d u r e s were c a r r i e d  C y t o s o l i c CT c o l l e c t e d by s t e p w i s e ammonium s u l f a t e ( 3 0 - 4 0 % ) was  purified  and  the s p e c i f i c  was  usually  after  c e n t r a t i o n o f 14 mM Sepharose  brated  with  20  0.025% T r i t o n  the  chromatography  was  (12-15ml)  brought to a f i n a l  m a g n e s i u m a c e t a t e and t h e n a p p l i e d  4B mM  column  (1.4 x 15 cm)  Tris-succinate  X-100,  0.5  mM  (Fig.5)  chromatography  u n i t s / m g . A f r a c t i o n of t h e enzyme  DEAE-cellulose  choline  o f t h e enzyme a f t e r  (pH  PMSF, 0.5 mM  DTT  buffer and  con-  to a  w h i c h had been 6.5)  4°C.  fractionation  by D E A E - c e l l u l o s e c h r o m a t o g r a p h y  activity  10-20  out at  4B  CDP-  equili-  containing  14 mM  magnesium  a c e t a t e . The f l o w o f t h e c o l u m n was s t o p p e d f o r 30 m i n t o a c h i e v e equilibrium ing  between  enzyme and  t o w a s h t h e c o l u m n . The  until  the  first  protein  peak  ligand  column  of the r e s i n b e f o r e  was  eluted  diminished  The  the t o t a l  chromatogram  i n Figure  enzyme a c t i v i t y  a c t i v i t y peak  eluted  with  33 u n i t s / m g and showed of o r i g i n a l  the  ( m o n i t o r e d by  t h e a b s o r b a n c e a t 280 nm) , and t h e n w i t h buffer.  with  buffer  recording  0.5 M N a C l i n t h e s a m e  6 shows t h a t l e s s t h e n 15% o f  l o a d e d was  r e t a i n e d by the column.  0.5 M N a C l h a d a s p e c i f i c a c t i v i t y  3-fold p u r i f i c a t i o n  (The s p e c i f i c  enzyme s a m p l e b e f o r e C D P - c h o l i n e S e p h a r o s e 4B  t o g r a p h y was  start-  The of  activity chroma-  10 u n i t s / m g i n t h i s p a r t i c u l a r e x p e r i m e n t ) . H o w e v e r ,  s i n c e t h e b u l k o f enzyme d i d not b i n d t o t h e C D P - c h o l i n e r e s i n , the t o t a l  activity  of the sample enzyme b i n d i n g affinity  r e c o v e r y was  within  only  the column  to the column.  of enzyme  3.5%.  Prolonged incubation  f o r up t o 1 h r d i d n o t I n an  attempt  to the CDP-choline l i g a n d ,  21  to  improve  improve  the  the sample  was  FRACTION NUMBER  F i g . 5 . D E A E - c e l l u l o s e C h r o m a t o g r a p h y o f Rat L i v e r C y t o s o l i c Cytidylyltransferase. The 40% ammonium s u l f a t e p r e c i p i t a t e o f r a t l i v e r c y t o s o l was s u s p e n d e d i n 20 mM T r i s - s u c c i n a t e (pH 6.5) c o n t a i n i n g 0.025% T r i t o n X - 1 0 0 , 0.5 mM PMSF and 0.5 mM DTT. A p p r o x . 130 ml o f t h e s a m p l e was a p p l i e d t o a DE-52 c o l u m n (3 x 35 cm) w h i c h h a d b e e n e q u i l i b r a t e d w i t h t h e s a m p l e b u f f e r . T h e s a m p l e - l o a d e d c o l u m n was washed w i t h 50 ml of t h e s a m p l e and t h e n e l u t e d w i t h a 0-0.5 N N a C l g r a d i e n t i n t h e same b u f f e r i n t o 9 ml fractions.  22  5  10  15  20 FRACTION  NUMBER  25  30  35  F i g . 6 . C D P - C h o l i n e S e p h a r o s e 4B C h r o m a t o g r a p h y o f Rat L i v e r C y t o s o l i c C y t i d y l y l t r a n s f e r a s e . The e n z y m e f r a c t i o n s ( 1 2 - 1 5 ml) pooled f r o m DE-52 c o l u m n were b r o u g h t t o f i n a l c o n c e n t r t i o n of 14 mM magnesium a c e t a t e and t h e n a p p l i e d t o a C D P - c h o l i n e c o l u m n (1.4 x 13 cm) w h i c h had been e q u i l i b r a t e d w i t h 20 mM T r i s - s u c c i n a t e ( p H 6.5) c o n t a i n i n g 0.025% T r i t o n X-100, 0.5 mM PMSF, 0.5 mM DTT and 14 mM magnesium a c e t a t e . The c o l u m n was washed w i t h t h e same b u f f e r u n t i l A ^ g g d i m i n i s h e d and t h e n e l u t e d w i t h 0.5 N NaCl. F r a c t i o n s i n "the v o l u m e o f 3.5 ml w e r e c o l l e c t e d . The c o l u m n was r e g e n e r a t e d by w a s h i n g w i t h 1 N N a C l .  23  brought  to a final  phospholipid protein those  concentration  before  and a c t i v i t y  observed  Separation  was  o f t h e chromatogram  to  4B r e s i n  showed a f a i r l y  ammonium  on t h e  strong  matography.  Figure  purification  by p h o s p h o - c e l l u l o s e  succinate  buffer  PMSF and  7 represents  one  third  1).  The  tration  column (pH 6.5)  caused  o f the  activity  a typical  sample  4B C h r o m a t o -  ml)  bulk  enzyme.  preparation  (pH 6.5)  against  which chroo f CT  20 mM X-100,  l o s s o f enzyme a c t i v i t y ,  x 15 cm) w h i c h  b u f f e r . A f t e r the  f o r the  chromatogram  0.025% T r i t o n  was r e c o v e r e d (30  the CTP-  from Tris-  0.5 mM  s a m p l e c o n d u c t i v i t y t o 3 mMHO.  after  was b r o u g h t  o f 14mM magnesium a c e t a t e , (1.5  shown).  c o l u m n . The enzyme p o o l e d  containing  a severe  d i a l y z e d sample  column  not  and D E A E - c e l l u l o s e  was d i a l y z e d  0.5 mM DTT t o l o w e r t h e  procedure  However, t h e  t h e enzyme column  liver  was s i m i l a r t o  (data  affinty  purification,  sulfate precipitation  phospho-cellulose  rat  C D P - c h o l i n e S e p h a r o s e 4B c o l u m n ,  f r a c t i o n a t e d by a phospho-cel1ulose  This  column.  absence o f p h o s p h o l i p i d  t o improve  followed  4B  t o the  total  o f C y t i d y l y l t r a n s f e r a s e by C T P - S e p h a r o s e  Sepharose order  applied  profile  i n the  graphy. In c o n t r a s t  In  being  o f 0.2 mg/ml  thus  dialysis  (see  toa final  only Table  concen-  and a p p l i e d t o a C T P - S e p h a r o s e  had been  o f the  e q u i l i b r a t e d with the  p r o t e i n was e l u t e d  w i t h 0.04  M N a C l , t h e e n z y m e was r e m o v e d b y a 200 m l g r a d i e n t o f 0.04-0.4 M NaCl there  i n the  was no d e t e c t a b l e  eluant, range  same b u f f e r  whereas (2-3  the  mMHO)  (Figure enzyme  activity  followed  8).  The c h r o m a t o g r a m  activity  was e l u t e d  by a long  24  shows  i n thef i r s t  that  protein  at a lowc o n d u c t i v i t y  tail.  The r e s u l t s  of a  F i g . 7 . P h o s p h o - C e l l u l o s e C h r o m a t o g r a p h y o f Rat L i v e r C y t o s o l i c C y t i d y l y l t r a n s f e r a s e . The enzyme sample (40 ml) p o o l e d f r o m DE-52 c o l u m n was a p p l i e d t o a P - l l c o l u m n (2.5 x 8 cm) w h i c h had been e q u i l i b r a t e d w i t h 20 mM Tris-succinate (pH 6.5) c o n t a i n i n g 0.025% T r i t o n X-100, 0.5 mM PMSF and 0.5 mM DTT. The c o l u m n was w a s h e d w i t h 80 ml o f 0.2 M N a C l a n d t h e n e l u t e d w i t h 100 ml o f 00.6 M N a C l g r a d i e n t i n t h e same b u f f e r i n t o 4.5 ml f r a c t i o n s .  25  FRACTION  Fig.8. C T P - S e p h a r o s e 4B C h r o m a t o g r a p h y o f Rat L i v e r C y t o s o l i c C y t i d y l y l t r a n s f e r a s e . The e n z y m e f r a c t i o n s (30 ml) f r o m phospho-cellulose column w e r e d i a l y z e d a g a i n s t 20 mM Triss u c c i n a t e (pH 6.5) c o n t a i n i n g 0.025 % T r i t o n X - 1 0 0 , 0.5 mM PMSF and 0.5 mM DTT f o r 1 h r and b r o u g h t t o t h e f i n a l c o n c e n t r a t i o n o f 14 mM magnesium a c e t a t e b e f o r e a p p l i c a t i o n t o a C T P - S e p h a r o s e 4B c o l u m n (1.5 x 15 cm) w h i c h had been e q u i l i b r a t e d w i t h t h e d i a l y s i s b u f f e r c o n t a i n i n g 14 mM magnesium a c e t a t e . The s a m p l e - l o a d e d c o l u m n was w a s h e d w i t h 0.04 M N a C l i n t h e s a m p l e b u f f e r and t h e n e l u t e d w i t h 200 ml o f 0.04-0.4 M N a C l g r a d i e n t i n t o 4 ml f r a c tions.  26  purification  scheme a r e s u m m a r i z e d  chromatography relative to  the  exhibited  of  purification  was  Table  a three-fold  t o t h e enzyme s a m p l e loss  1.  activity not  Purification  i n Table  applied  i n the  1.  The CTP  purification  and  to the column.  dialysis  step,  affinity 10%  yield  However, due the  overall  improved.  of CTP:phosphocholine from  rat liver  cytidylyltransferase  cytosol  Volume (ml)  Protein (mg)  Total activity (units)  128  3942  5558  1.4  130  621  3916  6.3  70.5  4.5  DE-52  45  54  1165  21.6  21.0  15. 4  P-11  31  5  471  94.2  8.5  67. 3  P-11(after dialysis)  31  5  151  30.2  2.7  21.6  CTP-Sephar o s e 4B  16  0.14  13  92.9  0.2  66. 4  Fraction  Cytosol (NH ) S0 40% 4  2  4  27  Specific activity Recovery (units/mg) (%)  100  Purification (fold)  1  DISCUSSION  The  major  purification  purpose  of this  form.  cytosol  fraction  Since  there  cytosolic  with  unaggregated  found  that  during  ammonium  recovery  in  precipitation 30% ammonium  i sprecipitated  caused  form,  the b u f f e r s  used  rat  liver  i n the p a r t i a l cytosol.  chromatograms  sulfate. done  In o r d e r to o b t a i n in this  X-100  significantly form  from  cytosol  improved the cytosol,  o f 0.025% T r i t o n  and  X-100  procedure  enzyme f r o m a g g r e g a t i n g and  purification  chromatography  stages.  techniques (DEAE-cellu-  in this  purification  two p r o c e d u r e s  28  laboratory  i n the  laboratory  have  of unaggregated  F i g u r e 5 and F i g u r e 7 r e p r e s e n t  of these  sensitivity  of the p u r i f i c a t i o n  i n preventing  and p h o s p h o - c e l l u l o s e ) used  successful  two f o r m s o f  whereas t h e u n a g g r e g a t e d  The p r e s e n c e  i n the rest  two i o n - e x c h a n g e  part  be s e p a r a t e d by s t e p w i s e  sulfate,  precipitation  t h e enzyme a t e a r l y  homogenate,  different  o f 0.5% T r i t o n  of a c t i v i t y .  i n the  ( 4 0 ) . M o s t o f t h e a g g r e g a t e d CT  of the unaggregated  s e e m e d t o be h e l p f u l  lose  phospholipid  (40). These  previous studies  sulfate  (70-85%)  stablizing  a method f o r  s p o n t a n e o u s l y and t h e r e a r e  by t h e i r  b y 40% ammonium  the a d d i t i o n  no l o s s  The  with l i p i d  a c t i v a t i o n , and c a n a l s o  sulfate  was p e l l e t e d  the  some  o f CT p r e s e n t i n t h e c y t o s o l  to p h o s p h o l i p i d  CT  i s always  CT c a n be d i s t i n g u i s h e d  ammonium  to find  o f 100,000 x g x 1 h r r a t l i v e r  o f t h e CT h a s a s s o c i a t e d forms  was  o f CT i n t h e s o l u b l e , u n a g g r e g a t e d , l o w m o l e c u l a r  weight  two  work  respectively.  been  CT f r o m  the t y p i c a l The  advan-  tage  of using  cation the  step  and  the high  and no  salt  chromatography  eluted  from  hence c o u l d  tography  i s quite  without  enzyme  stable  significant  offered a higher  enzyme  chromatography  step  provided  recovery  a high  contained  phospholipids  from  through  exchange c o l u m n s . The a d d i t i o n o f t o t a l sample  tionship  can m a i n t a i n  between p h o s p h o l i p i d  phospholipid causes the so  could  detergent functional an  candidate An  problem.  for this initial  when  activated since  removal of  rat liver  p h o s p h o l i p i d to  although  the  i s unclear.  form  environment CT f r o m  relaSince  affects  o f t h e enzyme, Choosing  for maintaining aggregating  X-100  appears  a CT  remains the  best  purpose.  reported  on  from  glycerophosphocholine Sepharose  phospho-cellulose  as a s t a b i l i z e r .  f a r ,Triton  investigation  c h r o m a t o g r a p h y was (41)  So  loss  succesive ion-  of the unaggregated  an a p p r o p r i a t e  chroma-  a severe  o f CT a n d t h e r e f o r e  s t r u c t u r e and a l s o p r e v e n t  unsolved  c o n c e n t r a t i o n of  t h e two  and t h e e n z y m e  not use p h o s p h o l i p i d  to o f f e r  1). But the  complete  activity,  the aggegation  further purification we  t h e enzyme  phospho-  (Table  after  i s p o s s i b l y due t o t h e a l m o s t t h e enzyme  The  purification  however, caused  of a c t i v i t y  o f 0.025*  to the a f f i n t y  chromatography  the  purifi-  fold  high  directly  which,  The l o s s  occurs.  of a c t i v i t y  n o t be s u b j e c t e d dialysis  i n the presence  aggregation  the column  activity.  as t h e f i r s t  s a m p l e c a p a c i t y o f t h e c o l u m n , and a l s o ,  preparation  X-100  cellulose  of  is  enzyme  Triton  DEAE-cellulose  6B. The  CT  purification  this  was  technique  laboratory  was  not e a s i l y coupling  affinity  10 y e a r s  covalently linked  the r e a c t i o n o f glycerophosphocholine  29  by  ago  to epoxyreproduced  to the r e s i n  was  difficult  this we  t h e s i s used  adopted  Sepharose and  a  best  to  group  based  o f AMP  difference  oxidized  resin  ( Figure  acid  affinity  resin  have  account  f o r the f a i l u r e  o f CT t o  choline  resin,  but not  f o r the b i n d i n g  t o t h e CTP  with  the  polymer  experiment sample  caused  that  did  material by  was  not  might  was  addition  the  The  CT  for CDP-choline  presence  of  phospholipid  t h e enzyme  reflected  i n the  CDP-choline.  Little  (40)  absence  the a f f i n i t y  was  the  observation  purified  of  by  the  resin.  different  = 0.21  because  of  no  of the aggregated  column  material,  the  to  the on  the  affinity  column enzyme  affinity  the  enzyme  partially  o b t a i n e d i n the  detectable  form  and  the  c o n s t a n t of was  et  while  o f p h o s p h o l i p i d . Thus,  30  form  to the c y t o s o l i c  mM)  CDP-  Choy  affinity  affinity  Michaelis  i s known about  to the  polymer  binding  CTP-  original  w i t h the CDP-choline  enzyme  (Km  to  structural  bind  to the a g g r e g a t i o n e f f e c t  phospholipid.  the  O b v i o u s l y , the  These  t o an  of p h o s p h o l i p i d  improve due  ligand  retained  n o t . The  be  of  o f t h e enzyme t o b i n d  form  form  reaction.  ability  g l y c e r o p h o s p h o c h o l i n e as  unaggregated  The  be  CT  their  might  form  coupling  altered  changes  unaggregated  cytidine  4) m i g h t  explained.  structure  (40)speculated that  to  h y d r a z i d e s (49).  ribosyl  al.  both  for cytosolic  i s not  derivatives  i n the  CDP-choline  m o i e t i e s to the enzyme.  carboxylic  in binding  nucleotide  or  Hence  on t h e s t u d i e s d o n e by H a n s k e e t a 1.  and  CDP-choline-Sepharose  CTP  as l i g a n d s .  i n o r d e r to expose  of the a f f i n i t y  representation  The  covalently  (or p h o s p h o c h o l i n e )  structure  columns prepared f o r  s u b s t r a t e s o f CT  link  via a ribosyl  the s t r u c t u r e  and  the n a t u r a l  method  phosphate  putative  on  t o m a n i p u l a t e . The a f f i n i t y  activity  the Km  value  o f t h e enzyme t o of  unaggregated  enzyme  f o r CDP-choline.  Sepharose  4B c h r o m a t o g r a p h y  aggregated  enzyme f o r m s  Preliminary as d e s c r i b e d CT  for activity  enzyme  that  from  both  the CDP-choline  t h e u n a g g r e g a t e d and  no s t r o n g a f f i n i t y t o C D P - c h o l i n e .  showed  that  (46,47) was  t h e CTP-Sepharose of benefit  the commercially  (Sigma) d i d n o t r e t a i n  required the  while  shows  have  studies  previously  purification,  resin  However, t h e r e s u l t  ( 3 5 ) , and a r e a l s o  a f f i n i t y to the resin.  to the  available  t h e enzyme.  Magnesium i o n s a r e  necessary  f o r promoting  The mechanism o f t h e i n t e r a c -  b e t w e e n t h e enzyme and CTP i s u n c l e a r . One o f t h e  ties  i s that  with  a lower  sample  in later  partially  than  involved,  resin  (2-3 mMHO) t h a n  by o t h e r groups  enzyme  p e r mg p r o t e i n  that  because  by the b u f f e r of the o r i g i n a l  was l o s t  stages i s s t i l l  with  within  specific  a problem.  activity  to discover  not only maintains  The  o f a b o u t 100  3 hr a t 4°C d u r i n g the  a r e needed  p h o s p h o l i p i d which  In  (38,39), t h e s e v e r e l o s s o f  was e x t r e m e l y u n s t a b l e and more t h a n  Further studies  but a l s o  may  the a f f i n t y  purification  purified  activity  step.  strength  encountered  activity  units  ionic  from  interaction  probabili-  (3 mMHO) ( F i g u r e 8 ) .  As  the  there i s a hydrophobic  e n z y m e c a n be e l u t e d  cytosolic  CTP a f f i n i t y  tion  the  prepared  dialysis  a material(s) t h e enzyme  50% o f  other  activity,  p r e v e n t s a g g r e g a t i o n o f t h e enzyme.  conclusion,  t h e CTP-sepharose  be an e n c o u r a g i n g  combination  technique  for cytosolic  w i t h o t h e r ion-exchange  31  4B a f f i n i t y  chromatography  CT p u r i f i c a t i o n i n  chromatographies.  CHAPTER  III.  BOLISM OF  EFFECTS  hepatocytes,  because  membrane  constituent  80%  of  be  the (50)  forms  of  and  the  in  by  weight  causes  of  is by  must  Studies  on  the  showed t h a t Golgi  Secretion was  and  apparatus of  newly  stopped  either  for  some  lipoprotein  apoproteins  processes  on  somewhat  function  i n the  the  the  20%  HDL  of  the pro-  understood. or  the  target  phospholipids  secretion  of  products  by  membrane  assembly  of  plasma  synthesized quickly  32  secreted  the  from  components.  lipoproteins  and  into  lipoproteins after  the  p r o c e s s of l i p o p r o t e i n  mediated  being  as  abundant  (55)  of  a  been  from  membranes o f t h e  a p o p r o t e i n s were a s s e m b l e d  very  has  m a i n l y s t r u c t u r a l . However,  since  before  as  p h y s i o l o g i c a l f u n c t i o n of the are  and  the  VLDL o r  around  lipoproteins  synthesis  lipid  originates  are  accounts  phospholipids  involves  liver  i n t e s t i n e . The  liver  The  in  only  secreted  serum  intrest  studied  i s not  also  META-  lipoprotein synthesis  from  c i r c u l a t o r y diseases.  i s of  but  plasma  l i p o p r o t e i n receptors  r o l e of  secretion  plasma  in rat  l i p o p r o t e i n is probably  biological  in  ( 5 4 ) . The  cells  f o r plasma  phospholipid  in  abnormality  cells  organ  in l i v e r  THE  HELA CELLS  extensively  l i p o p r o t e i n s . Rat  remainder  which  components  the  within  or plasma  major  been  synthesized  component  a  d e f i c i e n c y of  in  PC  has  lipoprotein secreted  material  cells  PC  l i p o p r o t e i n pool  (51,52,53),  The  of  the  of b i l e  to  liver  tein  HEPATOCYTES AND  3  biosynthesis  reported  DIPHENYLSULFONE COMPOUNDS ON  [METHYL- H]CHOLINE IN RAT  The  major  OF  glycosylated plasma  from  (57).  liver  administration  into of  puromycin,  a specific  the  s e c r e t i o n of  hepatic  inhibitor  l i p o p r o t e i n s was  p r o t e i n g l y c o s y l a t i o n (58). biological process. tein  r o l e of One  secretory nontoxic  activity. inhibitors  The  were  one  of  inhibited  This  be  accomplished  PC  reported  showed t h a t (PGs), mouse  of ly,  of  the  such  as  macrophages  be  due  to  choline certainly (59,60).  the  PC or  PC  reason treated  specific  which be  the  products  33  al. (59)  has  the  by  the  (LAH)  by  endocytic  the  been  v i a the from  to CDP-  cells  components  situations the  release  speculated  membrane  m a c r o p h a g e s and  i n mouse  product  products  plasma of  also  administration  biosynthesis of  the  prostaglandins  a l s o observed  sulfones  by  et  by  manner. C o r r e s p o n d i n g -  of  similarity from  by  inhibition  mediated  e f f e c t s on  stimulated  was  release  no  PC.  markedly  acid hydrolases  is  PC  macrophages  mediators,  in a nontoxic  of  into  material  Bonney  inhibited  inhibition  events  r e l e a s e o f the  exhibited  lysosomal  the  the  in chick  protein.  and  with  involves  lipoprotein  incorporation  showed t h i s  but  f o r the  since  Considering  choline  biosynthesis  pathway,  the  its deri-  inflammatory  compounds of  PC,  compounds, dapsone and  (AUS)  zymosan, c o u l d  inhibition  by  RNA  (LT)  by  of  specific,  e_t a_l (18)  macrophages,  diphenylsulfone the  of  r e l e a s e of  peritoneal  m a c r o p h a g e s . The  the  DNA,  with  lipopro-  using  inhibit  Shigeura  leukotrienes  stimuli  to  synthesis  biosynthesis  the  biosynthesis.  t h e s e compounds  the  c o n t r o l of the  lipoproteins,  of  prior  lipoprotein secretion  of  could  on  but  i s known a b o u t  component  s t u d i e s d o n e by  using  the  (54),  dependent  c o r r e l a t e changes i n b i o s y n t h e s i s  A s e r i e s of d i p h e n y l s u l f o n e vatives,  i n the  approach for studying  phospholipid  not  However, l i t t l e  phospholipids  s e c r e t i o n i s to  major  of p r o t e i n s y n t h e s i s  between  secretion  of  lipoproteins  from  hepatocytes,  pounds would h e l p relationship protein  us  The primary  an  in  inhibition  rat  isolation monolayer  of  reflect  metabolism  their  nutritional,  as  the  pathway, choline  the  choline  dehydrogenase  product  (28,32,64)  into  Results attempted  the  as  conversion PC  of  PC  in  and  of  will  and  the  of  subtle  or  3  H)  the  a l s o be  oxidized  aldehyde  released  widely  biosynthesis cellular  r a t e . But  to b e t a i n e .  i s a two-step r e a c t i o n  betaine  which  states  is  i n the  synthetic  rat  in The  catalyzed  dehydrogenase  from  PC  modifications  choline  radioactive  in  hepatic  pharmacological  CDP-choline  reflects  rapidly  via  here  the the  administration to  lipo-  (63),  hepatocytes  medium.  use  HeLa  incorporation  on  is  The  of  experiments  in  pathway  preliminary  inhibited  to  cells, PC.  rat  the  but  Also,  in  hepatocytes  34  we  have  rat hepatocytes,  s t u d i e s have shown  total  did  the  i n which  compounds to p e r t u r b  these diphenylsulfone  the  choline  biosynthesis  from  CDP-choline  cells  into  are  diphenylsulfone  i n HeLa c e l l s .  dapsone,  and  the  maintenance  studies  permit  1 4  to betaine  reported  to  biosynthesis well  choline  yet  (methyl- C  materials  by  the  their  the  (61),  distribution  of  and  vivo  of  conversion choline  ir\  labelled  containing  rat hepatocytes,  for studying  biosynthesis  and  enabled  hormonal  precursor  and  PC  com-  l i p o p r o t e i n s e c r e t i o n under c o n d i t i o n s  (62). R a d i o a c t i v e l y used  diphenylsulfone  hepatocytes.  c u l t u r e has  and  in  the  of  hepatocytes  biosynthesis lipid  hoped  t o e s t a b l i s h a model s y s t e m  between  secretion  we  not  compounds, [methylchange  e f f e c t s of were  as  that  except  H]choline  the  these  PC  rate  of  compounds  investigated,  but  no  inhibition  was  (II)  observed.  F i g . 9. S t r u c t u r e s o f dapsone and a n a l o g s ( I I I and I V ) .  35  ( I ) , AUS  MATERIALS  ChemicalsBritish (MEM)  Female  Columbia  Animal  and f e t a l  Biological obtained  calf  Co.,  from  Wistar  serum  were  from  Dulbecco's were  NY.  medium  the Grand  Island  3  [1-[4-(4-sulfanilyl)phenyl]urea  Methylformamido)-4'-ureidodiphenyl  I n c . Rahway, NJ.  of  was  The d i p h e n y l s u l f o n e s , p , p ' - d i a m i n o d i -  (dapsone),  Merck S h a r p  from  Eagle's  [Methyl- H]Choiine  4-Methoxyacetamido-4'-ureidodiphenyl  from  the University  modified  bought  Island,  Amersham.  phenylsulfone  rats  Unit.  Grand  AND METHODS  & Dohme R e s e a r c h The s t r u c t u r e s  sulfone  (AUS),  ( I I I ) and 4-(N-  s u l f o n e (IV) ,were o b t a i n e d  Lab., D i v i s i o n  of the four  o f Merck  compounds  & Co.,  a r e shown  i n F i g u r e 9.  Preparation female as  Wistar  MEM  (Lux C o n t u r ,  with  thine,  e_t a_l (65), and c u l t u r e d  60 mm,  2-3 x 1 0  Hepes  o f 95% a i r / 5 %  dishes  C0 . 2  Studies(3 x 10  and p u l s e d  with  isolated  chloride,  100 nm  from  technique  in plastic  culture  i n arginine-free  insulin,  0.4 mM  orni-  100 u n i t s / m l o f p e n i c i l -  (pH 7.4) a n d 5% FCS a t 3 7 ° C u n d e r an a t m o s -  f o r approximate  Pulse-Chase  were  cells/dish)  6  100 ug/ml o f s t r e p t o m y c i n s u l f a t e ,  culture  MEM  Hepatocytes  (180 g) by a c o l l a g e n a s e p e r f u s i o n  28 uM c h o l i n e  G, 10 mM  phere  mm  rats  d e s c r i b e d by D a v i s  dishes  lin  o f Rat Hepatocytes-  The c e l l s  were  24 h r p r i o r  Monolayer  cells/dish)  maintained  i n monolayer  to a l l experiments.  c u l t u r e s of rat hepatocytes were washed  twice  10 u C i [ m e t h y l - H ] c h o l i n e 3  36  with  i n 60  serum-free  (0.12 Ci/mmol) f o r  1 hr.  The  28  choline  uM  cells  phenylsulfone were PC  in  harvested  and  the  soluble  the  and  the  was  0.7  metabolites  The  cells  of  cold  3  2,500 rpm water was  0.5  and  and  ml  (Western  washed  with  and  contains  an  phase  a l i q u o t was containing  pter  II, Materials  Growth o f  was  removed, and ml  of  soluble  cellular  were c o u n t e d  dish  for and  HeLa c e l l s -  ug/ml  4 hr,  TLC  of  the  into  on  as  to PC  dicells  cellular  the  medium  gel  G-  extracted  by  was  added  removed,  to each  with  sonicated  and  with  another  p l a t e s . An  was  dried  r a d i o a c t i v i t y as  ml  removed 0.5  then c e n t r i f u g e d  at  The  upper  methanol-  phase  The  was  3  phase,  combined, the  nitrogen.  described  phase  (CH 0H/  upper  a l i q u o t of  under  3  (lower)  metabolites,  TLC  0.7  CHC1 ,  centrifugation. choline  0.5  chloroform  theoretical  dish.  in a water bath  f o r 5 min.  the  water  Silica  was  washed  mixed  and  solvent.  PC  was  was  H-103N c e n t r i f u g e )  applied  ples  by  culture  sequentially,  2  water  contained  an a l i q u o t (50 u l ) o f s a m p l e was  H 0  0.75  to  cellular  s a m p l e was  2  which  The  the  C H C 1 / H 0 ; 4 8 / 3 / 4 7 ; v / v / v ) by 3  The  remainder  (upper) p h a s e was  100  up  v:v:v)  (10:4; v:v)  2  a s s a y . The  C H C 1 , and  of  that  radioactivity in  separated  (45).  3  were h a r v e s t e d  protein  MEM  (choline, phosphocholine  (50:50:5;  3  o f t h e same s o l u t i o n . The  for  times  The  was  CH OH/H 0  s o n i c a t o r f o r 10 m i n  ml  various  Extraction-  Dyer's method  ml  presence  metabolites  3  and  with  r a d i o a c t i v i t y incorporated  CH OH/0.6% N a C l / N H  and  ml  At  or  determined.  Phosphatidylcholine Bligh  absence  water s o l u b l e  choline  with  s u b s e q u e n t l y chased  compounds.  CDP-choline)  60  were  lower  The  sam-  previously  (Cha-  Methods s e c t i o n ) .  HeLa  cells  37  were o b t a i n e d  from  Flow  Labora-  tories;  they  Collection described  originally  HeLa C C L - 2 .  by P e l e c h  came The  from  routine  ^ t a_l (66).  (Lux C o n t u r , 60 mm)  chloride)  0.4  80 u g / m l  arginine,  7.4) and  5% FCS  Confluent choline  mM  100 u n i t s / m l  monolayer  incorporation  above, e x c e p t  ornithine,  a t 37°C  under  cell  growth  HeLa c e l l s  culture dishes with  American  i n MEM  80 ug/ml a r g i n i n e  G,  10 mM  used  38  choline sulfate,  Hepes  (pH C0 »  f o r [methyl-  e x p e r i m e n t s as  as  plastic  o f 95% a i r / 5 %  was a l w a y s p r e s e n t  media.  28 uM  streptomycin  an a t m o s p h e r e  or p u l s e - c h a s e  were  were grown on  penicillin  were  Culture  conditions  (contains  100 ug/ml  cultures  Type C e l l  2  H]  described  in a l l culture  RESULTS  Influence  of  Diphenylsulfone  t a k e by H e l a C e l l s : P u l s e have shown t h a t poration PE f r o m cells.  IV,  Similar  results  t h e PC  were  AUS  dapsone and AUS  obtained  as w e l l  synthesis  are summarized  inhibited incorthe f o r m a t i o n  i n the s t u d i e s  likewise,  p h a s e was choline  cells.  i n Table  CDP-choline  very  small.  to PC  i s fast  that  dapsone  incorporation activity  of  The  2 and  of  The  This  3. A f t e r  (Table  ( 2 0 - 50%)  following  (Table  3 ) . Among  these  pretreatment  of the  that  biosynthetic  materials  degree  e f f e c t on  data  a  1 hr  p h a s e was i n  r a d i o a c t i v e l a b e l i n g of  implies  in this  2).  I I I a n d IV, i n h i b i t e d l a b e l e d  hibitory  these  I I I and  CDP-  the c o n v e r s i o n pathway.  The  c h o l i n e , as measured by t h e t o t a l  the c e l l u l a r phase  chick  of data  (100 u g / m l ) had no i n h i b i t o r y e f f e c t on t h e  of l a b e l e d  methanol-water  HeLa  experimental  Table  of  r a d i o a c t i v i t y i n the methanol-water  in phosphocholine.  was  showed  90%  on  the e f f e c t of  90% o f t h e r a d i o a c t i v i t y i n t h e c h l o r o f o r m  and  and  as t h e r e l a t e d compounds  i n HeLa  Up-  e_t a_l. (60)  mouse macrophages  (18). I have s t u d i e d  and AUS,  H]Choline  Bonney  C ] c h o l i ne i n t o PC, b u t n o t  by u s i n g  dapsone  observed  PC  1 4  [Methyl-  Studies-  [ 1,2,-*^C] e t h a n o l a m i n e i n b o t h  on  pulse,  Labeling  diphenylsulfones  o f [ 1, 2 -  macrophages drugs,  Compounds on  The  cells  other  choline  39  three  phase  compounds,  period  IV e x h i b i t e d  H]choline with  chloroform  incorporation  a pretreatment  compounds,  [methyl-  i n both  radio-  to  and AUS,  varying  of 0 or 4 hr  the highest i n -  incorporation,  t h e c o m p o u n d was  since  no  n e c e s s a r y to  Table  2. E f f e c t s  o f Dapsone and AUS on t h e [ M e t h y l Uptake by HeLa  Addition  Cells*  Lower phase (dpm x 10 /mg)  Upper phase (dpm x 10 /mg)  0 hr  0 hr  4 hr  -5  4 hr  H]Choline  (dpm  Total x 10 ~ /mg) 5  Ohr  4hr  Control  0. 97+0.09  1.05+0.12  35 .6+4.8  29.1+1. 5  36.7  30.2  Dapsone  1. 08+0.09  0.97+0.05  39 .9+0.3  31.1+1. 8  41.0  32.1  (-)  (-)  (-)  0.68+0.06  28 .2+3.9  20.5+2. 4  (-) AUS  0. 79+0.11 (19)  (35)  (21)  (30)  (-)  (-)  29.0  21.2  (21)  (30)  * HeLa c e l l s w e r e c u l t u r e d a s d e s c r i b e d i n M a t e r i a l a n d M e t h o d s . C e l l s were p r e i n c u b a t e d i n s e r u m - f r e e MEM c o n t a i n i n g 100 ug/ml o f dapsone o r AUS d i s s o l v e d i n DMSO f o r 0 o r 4 h r a n d t h e n l a b e l e d w i t h 11 u C i o f [ m e t h y l - H] c h o l i n e (0.13 C i / m m o l ) p e r d i s h f o r an a d d i t i o n a l 1 h r i n t h e p r e s e n c e o f d a p s o n e o r AUS p r i o r t o h a r v e s t i n g . A l l c u l t u r e s c o n t a i n e d a f i n a l c o n c e n t r a t i o n o f 0.1% DMSO. The [ m e t h y l - H ] c h o l i n e u p t a k e was e s t i m a t e d by q u a n t i t a t i o n o f t h e c e l l u l a r r a d i o a c t i v i t y i n b o t h w a t e r m e t h a n o l (upper) phase and c h l o r o f o r m (lower) phase e x t r a c t e d by t h e B l i g h and D y e r ' s m e t h o d . P r o t e i n was e s t i m a t e d by B i o - R a d a s s a y . The r e s u l t s a r e t h e a v e r a g e s + S.D.; N = 3. P e r c e n t inhibition b y AUS w i t h r e s p e c t to c o n t r o l i s g i v e n i n p a r e n t h e s e s .  Table  3. E f f e c t s  o f D i p h e n y l s u l f o n e D e r i v a t i v e s ( I I I and IV)  on t h e [ M e t h y l - H ] C h o l i n e 3  Lower pha ise (dpm x 1 0 /mg) _i)  Addition  0 hr  4 hr  Uptake by HeLa  Cells*  Upper phase (dpm x 10~ /mg) 5  0 hr  (dpm  Total x 10 /mg) 5  4 hr  0 hr  4 hr  Control  4. 02+0.51  1. 93+0. 38  39.7+1.8  32.2+3. 5  43.7  34.1  III  3. 61+0.29  1. 08+0. 20  27.4+1.0  19.0+2. 3  31. 0  20.0  (44)  (3D  (41)  (29)  (41)  20.4+0.4  16.6+0. 6  22. 6  17.8  (49)  (48)  (48)  (48)  (10) IV  2. 21+0.12 (45)  1. 17+0. 06 (39)  * The e x p e r m e n t a l c o n d i t i o n s were t h e same as d e s c r i b e d i n t h e l e g e n d o f T a b l e 2, e x c e p t t h e c o m p o u n d s I I I and IV w e r e u s e d i n s t e a d o f d a p s o n e and AUS. The r e s u l t s a r e t h e a v e r a g e s +S.D.; N = 3. P e r c e n t i n h i b i t i o n by I I I and IV w i t h r e s p e c t t o c o n t r o l i s g i v e n i n p a r e n t h e s e s .  give  the  choline  greatest inhibition incorporation  was  ( T a b l e 3).  reduced  i n the  The  total  cells  III  o r IV, b u t t h e p r o p o t i o n o f r a d i o a c t i v i t y  and  the  which  methanol-water  suggests  a f f e c t e d . The  that  that  s i s v i a CDP-choline i.e. at e i t h e r  to the of  the  the  order  chase for  with  exhibited neither  the  compounds,  choline into the  higher  two  significant  secretion  radioactivity  in  methanol-water  present  the  major  respectively,  After  sence  and  o f AUS  cells  roform  was  PC  the  the  observed.  and  counts  was  and  due  incorporation the  into  inhibition  i n HeLa c e l l s ,  o f AUS  in this  or  IV.  c h o l i n e to in  chloroform  HeLa  they  there  nor  cells,  the  phases  compounds  could  PC,  might  be  pre-  of c h o l i n e i n c o r p o r a t i o n  methanol-water  phases  i n both  (Fig.10  re-  and  3  labeling  is  betaine  [ m e t h y l - H ] c h o l i n e i n the  radioactivity  42  reason  was  Since  form  com-  pulse-  The  experiment  i n medium  inhibition The  imply  of d i p h e n y l s u l f o n e  lipoprotein  1 hr p u l s e of  IV,  ( F i g . 1 0 A)  here  choline metabolites, phosphocholine  while  negligible.  of  macro-  biosynthe-  into  c h o l i n e uptake.  o x i d a t i o n of  or  not  phosphocholine,  r a t h e r than  influence  of  was  PC.  compounds  inhibition  AUS,  drugs,  (18)  t h e PC  radioactivity  [methyl- H]choline  biosynthesis  the  of  s t u d i e s were done i n t h e p r e s e n c e these  PC  (60) o r c h i c k  [ m e t h y l - H ] c h o l i n e metabolism  choosing  with  for a l l three  inhibited  H]  chloroform  However,the data presented  to determine  pound on t h e  mouse  compounds  total  c o n v e r s i o n of  In  i n the  pathway at s t e p s f o l l o w i n g  r e d u c t i o n of  the  these  incorporation  treated  similar  treated  c o n v e r s i o n of c h o l i n e i n t o  CT o r CPT.  reduced  cells  the  were  s t u d i e s done w i t h  phages s u g g e s t e d  that  phases  [methyl-  B)  into chlo-  at  the  zero  time  of  the  chase  controls.  The  r a t e of  estimated  by  comparing  tion  i n t o the  There  i n PC  cells.  The  inhibited  as  had  data  IV  the  the  actual  tabolism  i n Rat for  determine t i o n . The  PC  potential  of  due  to  rapidly  increase  choline  choline  cellular  of  and  compounds  PC.  cells  However,  choline,  choline,  i n t o PC  control  i n t o HeLa  into of  radio-  the  hence  slope  m i g h t not  of  reflect  H]Choline  : Pulse-Chase  Studies-  Since  synthesis  and  show  secretion  PC  any  problem  [Methyl-  phosphorylated  the  in  ma-  rat  via  CDP-choline pathway,  the  with  of of  and  drugs  H] C h o l i n e  43  inhibit and  lipoprotein secre-  diphenylsulfone  to  could  [methyl- H]choline  apparent  is  diphenylsulfone  these  biosynthesis  kind  with  Me-  whether  alterations in choline  (Fig.lO).  be  cells.  [Methyl-  were p r e l a b e l e d  the  control  Compounds on  biosynthesis  absence of  cells  of  still  incorpora-  diphenylsulfone of  the  biosynthesis.  e f f e c t on  cells  and  treated  r a t h e p a t o c y t e s were t r e a t e d  avert  and  PC  the the  [methyl- H]choline  ug/ml) t o  the  of  could  in  decrease of r a d i o a c t i -  incorporation  activity  lipoprotein  (100  specifically  PC  the  Hepatocytes  cultured  compounds  the  of  and  the  conversion  of D i p h e n y l s u l f o n e  organ  liver,  rate  i n the  incorporation  the  of  treated  comparing  that  cells  than  3  ( F i g . l O , B) by  cells  [methyl- H]choline  differences  total  specific  of  i n the  phase b e t w e e n  inhibited  incorporation  Influence  rate  confirmed  the  the  HeLa  no  e f f e c t on  and  altering  in  the  ( F i g . l O , A)  only  no  AUS  jor  or  in treated  biosynthesis  from phosphocholine  activity  but  PC  chloroform  were l i t t l e  vity  were l o w e r  changes  for  1  compound in  the  incorporation,  hr to  labeling  as  seen  i s taken  up  by  the  phosphocholine  or  oxidized  in  cells to  c h a s e  t i m e  <hr>  F i g . 1 0 . I n f l u e n c e o f AUS and IV on t h e m e t a b o l i s m o f [ m e t h y l H ] c h o l i n e i n HeLa c e l l s . H e L a c e l l s c u l t u r e d i n 60 mm d i s h e s were washed w i t h s e r u m - f r e e MEM and p u l s e d w i t h 10 u C i [ m e t h y l H ] c h o l i n e (0.12 Ci/mmol) p e r d i s h i n t h e a b s e n c e ( A ) o r p r e s e n ce o f 100 u g / m l AUS ( A ) o r I V (•) d i s s o l v e d i n DMSO f o r 1 h r . The c e l l s w e r e s u b s e q u e n t l y c h a s e d w i t h 28 uM c h o l i n e i n t h e u n l a b e l e d medium f o r up t o 6 h r p r i o r t o h a r v e s t i n g . A l l c u l t u r e media c o n t a i n e d a f i n a l c o n c e n t r a t i o n o f 0.1% DMSO. R a d i o a c t i v i t i e s i n t h e c h l o r o f o r m p h a s e (A) a n d m e t h a n o l - w a t e r p h a s e (B) e x t r a c t e d by B l i g h and Dyer's method c o r r e s p o n d t o PC a n d phosphocholine, r e s p e c t i v e l y . Each p o i n t r e p r e s e n t s t h e mean o f three dishes. J  44  betaine of  the  ( F i g . 1 1 and radioactivity  choline, 50%,  Fig.12). A f t e r  PC,  was  cells  converted  CDP-choline  i n which about  and  70%  than  half  c e l l s . The within  of  was  o f not  lipoprotein  hours  d a p s o n e , AUS,  the  accounted  f o r the  and  20%  m e d i u m , so a f t e r betaine  of the chase  A l l of  I I I or  whereas  was  disappeared  period,  b e t a i n e , but  these  IV,  also  processes  since  neither  sions rate  the  enzymatic  are the  these  compound  cells  increase  in cellular  decrease  in cellular  secretion  of l i p o p r o t e i n  ed  that  the a c t i v i t y  in  rat hepatocytes  compounds  PC  involved  to CDP-choline  c o n v e r s i o n s was  treated  the  phosphocholine  activity  of phosphocholine of  b e t a i n e nor  which  the  r e p r e s e n t e d the  phosphocholine not  rate  observed  compared  to  radioactivity,  phosphocholine, PC  o f PC  to the  rate  and  by  of decrease  of  and  i n these experimental  altered. repre-  no  altered  in either diphenylsulfone the  control.  compared  with  The the  slower rate  m i g h t be e x p l a i n e d a s  by  results  lipoprotein  of the  suggestsecretion  these d i p h e n y l s u l f o n e  conditions.  45  Still,  medium. These  affected  i n c r e a s e of  successive conver-  PC.  b i o s y n t h e s i s and  were not  i n the  i n c r e a s e i n PC  i n the  and  influenced  i n t h e medium compounds were s i g n i f i c a n t l y  sented  The  chase,  from  radioactivity  in labeled  PC.  1 hr of  in cellular  decrease  phosphoremaining  radiaoctivity  The  50%  i n medium compounds i n c r e a s e d r a p i d l y  only c e l l u l a r  PC.  betaine,  phosphocholine  cellular  radioactivity  the f i r s t  release  the  to  choline  secreted betaine into  more  1 hr p u l s e , a p p r o x i m a t e l y  PHOSPHOCHOLINE  B ETA IN E  c/i  X  £ a -o r<  cc  o  a. cc O O z  0  1 2  3  4  MEDIUM COMPOUND  UJ  o X  a  .i 'i ro  0  1  2  3  4  0  chase  1  2  3  4  t i m e <hr >  F i g . 1 1 . I n f l u e n c e o f Dapsone o r I I on The M e t a b o l i s m o f [met h y l - H] C h o l i n e i n R a t H e p a t o c y t e s . Monolayer c u l t u r e s of r a t h e p a t o c y t e s i n 60 mm d i s h e s (3 x 1 0 c e l l s / d i s h ) were washed w i t h s e r u m - f r e e MEM t w i c e , and t h e n p u l s e d w i t h 10 u C i [me_thyl- H] c h o l i n e (0.12 Ci/mmol) i n t h e same medium f o r 1 h r . The c e l l s were s u b s e q u e n t l y c h a s e d w i t h 28 uM c h o l i n e i n t h e a b s e n c e ( A ) o r p r e s e n c e o f 100 u g / m l d a p s o n e ( A ) o r I I I (•) d i s s o l v e d i n 0.1% DMSO f o r up t o 4 h r . A l l c h a s e media c o n t a i n e d 0.1% DMSO. R a d i o activity i n c o r p o r a t e d i n t o t h e c e l l u l a r c h o l i n e m e t a b o l i t e s and medium compounds was d e t e r m i n e d as d e s c r i b e d under M a t e r i a l s and M e t h o d s . E a c h p o i n t r e p r e s e n t s t h e mean o f two d i s h e s . The e x p e r i m e n t was r e p e a t e d , and s i m i l a r r e s u l t s were o b t a i n e d . C  46  PHOSPHOCHOLINE  B ETA I N E  o X  E  o. •o  a UJ  < cc O  0'  1  MEDIUM  2  3  4  COMPOUND  Q. CC  O  a UJ  2 _i  O X  o I  n  chase  time  <hr >  F i g . 12. I n f l u e n c e o f AUS and IV on The M e t a b o l i s m o f [ r o e t h y l H ] C h o l i n e i n Rat H e p a t o c y t e s . The e x p e r i m e n t a l p r o c e d u r e was s i m i l a r t o t h a t d e s c r i b e d i n t h e l e g e n d o f F i g . 1 1 . AUS (•) and IV (•) i n t h e f i n a l c o n c e n t r a t i o n o f 100 u g / m l w e r e u s e d h e r e . Each p o i n t r e p r e s e n t s t h e a v e r a g e o f two d i s h e s . The e x p e r i m e n t was r e p e a t e d , and s i m i l a r r e s u l t s were o b t a i n e d . J  47  DISCUSSION  There so  i s no  the studies  known  inherited  on t h e b i o l o g i c a l  s e c r e t i o n have been p e r f o r m e d such  as c h o l i n e  deficiency  become more c o m p l i c a t e d pathway tions is  seems  and t h e q u a n t i t a t i v e  almost  from  unchanged  Bonney  et a^  only  i n PC  biosynthesis,  o f PC i n l i p o p r o t e i n  i n "experimental"  diseases,  ( 6 7 ) . The s i t u a t i o n h a s  PC b i o s y n t h e s i s  v i a the CDP-choline  r e s i s t a n t to the environmental amount  (60) t h a t  inhibit  toxic  encouraged  varia-  o f t h e r a t e - l i m i t i n g enzyme  i n a n y p h y s i o l o g i c a l c o n d i t i o n . The  specifically manner  function  i n the d i e t  since  t o be h i g h l y  deficiency  diphenylsulfone  PC b i o s y n t h e s i s us t o s t u d y  compounds  CT  report could  i n macrophages i n a non-  PC b i o s y n t h e s i s  and l i p o p r o -  c  tein  secretion  turnately,  i n h e p a t o c y t e s by u s i n g  our  results  showed  that  t h e s e compounds. U n f o r -  there  were  no  effects  on  3  [methyl- H]choline  metabolism  i n hepatocytes  compounds  (100 u g / m l ) .  non  might  be t h e d e t o x i f i c a t i o n a b i l i t y  the  data  using  obtained  The s i m p l e s t  from  explanation  the s t u d i e s  of l i v e r  on c u l t u r e d  [ C ] d a p s o n e o r [ C ] A U S showed t h a t 1 4  1 4  accumulated sulfones  by t h e c e l l s  d i d not  unconvincing treated  to  (60), w h i c h  penetrate  explain  the  the  cell  lack  of  hand, i t i s s t i l l 48  these  of t h i s  phenome-  cells.  However,  m a c r o p h a g e s by  t h e compounds were n o t  implied  that  the  membrane. effects  w i t h t h e c o m p o u n d s by a c e l l u l a r  n i s m . On t h e o t h e r  administered  in  diphenyl-  Thus  i t is  hepatocytes  d e t o x i f i c a t i o n mecha-  possible  that  we  have not  found  an  cytes,  effective  since  centration  we  was  The  t h e s e d i p h e n y s u l f o n e s on  experiments  per  of  established  this  derivatives  inhibited  the  mechanism  the  inhibition  i n mouse and  B o n n e y e_t al. (60) acted  at  found  site(s)  little  choline HeLa  the  or  cells  We  inhibited  cells,  not  radioactivity  the  al. (18)  distribution  i n HeLa c e l l s  3)  pulse-chase  from  not  in  the  The  postulated  to  by  sulfones PC.  They or  of  e x p e r i m e n t s done  on  transport,  with  from  this  previous except into  choline  intermediates  labeling  (Fig.10)  and  diphenyl-  incorporation  label  pulse  experiment  the  diphenylsulfones,  cellular  both  dapsone  t o be b e c a u s e  agree  choline of  been  polymor-  zymosan.  by  choline  from  a l l the the  by  agent  inflammatory  phosphocholine  the  does  that  only  of  of  conversion  thesis and  ejt  by  that  the  biosynthesis  results  here  found  dapsone, but  The  of  m a c r o p h a g e has  inhibition  presented  conclusion.  PC  Shigeura  phosphorylation.  antileprotic  found  release  of c o n v e r s i o n  no  con-  diphenylsulfones,  is stimulated  of  chick  and  hepato-  compound  is charaterized  Bonney et_ a_l (59)  macrophages which  sulfones  the  series  which  m e d i a t o r s by of  which  a n t i m a l a r i a l and  i s a disease  infiltration.  in  rat  ml.  compound  leprosy  phonuclear  did  ug  parent  and  its  only 100  d a p s o n e , i s an (68),  dosage o f  (Table  support  to of  PC. PC  2 and  this  the The synTable  point  of  view. 3 The tion of the  reason  into  the  for  HeLa c e l l s  experiment  conversion  of  reduced  total  was  examined  was  not  to v e r i f y  choline  to  PC. 49  [methyl- H]choline  the It  here effect  as of  is possible  incorpora-  the  major  the  compounds  that  the  purpose on  reduced  choline there  incorporation are  result.  several  It  involved  has  been  in choline  saturable  mechanism  probably  limit  on  was  (71),  10  entry  into  by  the  to the  are  for choline  on  specific choline  choline thesis which of  transport  transport when  the  the  formation  while  at  ration  of  PC  passive  a l s o be  of  the  incorporation. in  less  choline PC  cause of  the  i s , the of  the  other  ranges  be-  choline  by  seems  hand, argued  step  was  which  studies that  i n PC  below  20  uM,  by  choline  kinase  r a t e of c h o l i n e  of  rate (72),  incorpo-  HeLa  reduced  choline  the  in  concentration  treated of  the  biosyn-  by  observations  no  choline  limited  i n the  inhibition  choline  of  Since  was  catalyzed uM,  the  diphenylsulfones  i n d e p e n d e n t of medium c h o l i n e  That  conversion  the  rate-limiting  above 20  for  membrane c a r r i e r s On  into  un-  reduced  hepatoma c e l l s  of  and  (70).  there  with  the  mechanisms  for choline  The  and  for  saturable  although  the  transport.  phosphocholine  the  two  transport  treated  (72). R e d u c e d c h o l i n e p h o s p h o r y l a t i o n might  Km  diffusion.  cells  incorporation  was  were  liver,  saturation,  as  account  in rat plasma normally  with Novikoff  concentrations  into  rat  transport  in rat hepatocytes  concentration  choline  there  apparent  inhibition  served  might  facilitated  HeLa  m i g h t be due  in  uM  the  choline  which  that  the  approaches  choline  incorporation  and  of c h o l i n e  uM  liver  reported  (69),  concentration t o 20  inhibited  transport  uptake  t w e e n 10  to  probabilities  saturable  the  i s due  cells  choline  kinase  results  to p h o s p h o c h o l i n e , t h e r e f o r e  the  3 medium the in  [methyl- H]choline  diphenylsulfone cellular  choline  treated  entering  via  passive  cells  is  reduced  concentration.  We  do  50  not  due  diffusion to  an  into  increase  know w h e t h e r or  not  the  diphenolsulfones  tocytes. choline  Pritchard transport  found  the r a t e  enced  by t h e r a t e  choline from  suitable  and  i n PC  of hepatic  choline  Vance  PC  of c h o l i n e  presented  conclution,  transport  here  of i n t e r e s t to f i n d  PC b i o s y n t h e s i s  51  the  role  almost  not  this  uM.  of and  influ-  when t h e c o n c e n t r a t i o n  of  The r e s u l t s  statement.  compounds  appears not  o f PC b i o s y n t h e s i s v i a  o r HeLa  a specific  v i a CDP-choline pathway between  was  on t h e i n h i b i t i o n  it  r a t hepa-  in rat hepatocytes,  confirmed  i n rat hepatocytes  into  studied  r a n g e d b e t w e e n 5-40  pathway  tionship  have  biosynthesis  CDP-choline  synthesis  (32)  the d i p h e n y l s u l f o n e  f o r the s t u d i e s  is still  incorporation  biosynthesis  i n t h e c u l t u r e medium  HeLa c e l l s In  inhibit  cells.  Therefore,  inhibitor  o f PC b i o -  in livers  to study the  and l i p o p r o t e i n s e c r e t i o n .  rela-  CHAPTER IV. EFFECTS OF VASOPRESSIN YLTRANSFERASE  The  IN RAT  of developmental  74,75,76,76a) analyses  was  of metabolic  varies  The e f f e c t  p a t h w a y under  on t h e r e g u l a t o r y  of s u b s t r a t e cells.  i n the c o c k e r e l  restricted However, limit  i n BHK  the rate Since  activity  rate cells,  o f c y t o s o l i c CTP  ed  in cytosol  t h e CT  experimental  on PC  with  synthesis  cells  critical  anabolism,  diethylstilboestrol of c e l l u l a r  term  synthesis  t o be  and PC  acti-  (DES),  phosphocholine  i n the  liver  (81,82).  b o t h CTP a n d p h o s p h o c h o l i n e a p p e a r e d t o  o f PC b i o s y n t h e s i s (83). distribution  within  suggests  cells that  v i a a t r a n s l o c a t i o n m e c h a n i s m . The b u l k i s i n a c t i v e and f u n c t i o n s  once t h e enzyme by  as  that  the  seems  reaction  i s phospholipid-dependent, t h i s  regulated  activated  PC  CT h a s a b i m o d a l  is  as w e l l  I n p o l i o v i r u s - i n f e c t e d HeLa  treated  of  on  on t h e s h o r t  concentration  that the c o n c e n t r a t i o n the  Based  mechanisms o f CT  o f PC h a v e f o c u s e d  the c o n c e n t r a t i o n  was s h o w n  concluded  under a  (24,34,73,  (77,78).  i n PC s y n t h e s i s  i t was  r e g u l a t i o n o f t h e CT c a t a l y z e d  whereas  But  o f enzymes  i n the CDP-choline  in different  (79,80),  manipulations  intermediates,  The s t u d i e s  has been s t u d i e d  physiological conditions  genetic  and t h e b i o s y n t h e s i s  control.  it  by  and  of the a c t i v i t i e s  conditions.  for  and  regulatory  vity  HEPATOCYTES  r e g u l a t i o n o f PC b i o s y n t h e s i s  variety  levels  ON CTP:PHOSPHOCHOLINE CYTIDYL-  becomes  phospholipids.  associated Several 52  membrane,  different  t h e enzyme  o f CT l o c a t -  a s an enzyme  with  and i t s  reservoir. i t will  mechanisms  be  have  been p r o p o s e d tion and  o f CT.  t o e x p l a i n how  Studies  embryonic  m i g h t be the  cells  done by  Sleight  c h i c k muscle c e l l s  cytosolic  f o r m e d by p h o s p h o l i p a s e  binding  and  this  activation  proposol  CT  i s caused  by  higher  ir\ v i t r o ,  There reversible via a  by  Kent  C  are  DG  has  and  two  e n z y m e . One the  Bacillus  other  was  cereus  models  of  with  liver  cAMP  phosphodiesterase  less  membrane-associated  incubation protein  c y t o s o l (27).  of  a s s o c i a t i n g with  This  effect  c o u l d be  inhibitors  presence there  NaF,  product  aggregation  an  exogenous  the  first  is  the  modification  this  i n _i_n v i t r o  studies  with  and  s o l u b l e CT  (61,74).  and  Similarly,  Mg-ATP or the  and has  o f cAMP a n a l o g u e s i s more  of  with  mechanism  phospho-  cytosolic  i n c u b a t i o n a t 37°  C  CT  (27).  a d d i t i o n o f cAMP p r o t e i n  incubation  53  associated  is a  The  prevents  m e m b r a n e s upon  the  the  between c y t o s o l  i n hepaocytes  a b o l i s h e d by  into  activity  c y t o s o l j_n v i t r o  inhibitor,  from  kinase  enzyme  i n both  cycle correlated  (61,74) and  inhibitors,  rat l i v e r  phosphatase  In t h e  by  Covalent  b i o s y n t h e s i s by  rat hepatocytes  which  regulation.  microsome.  proposed  which  (84).  o f CT  been  PC,  i n t h e membrane o r  mimicked  hypothesis.  PC  of  to p r o m o t e  changes i n the d i s t r i b u t i o n of  CT  membrane-associated  (DG)  phosphorylation-dephosphorylation  Regulation  (75)  t h a t the  question  d e p l e t i o n o f PC  effect  cells  results  increased  diacylglcerol  phosphorylation  in  CHO  distribu-  mechanism. E x p o s u r e of  been d e m o n s t r a t e d  this  from  on  demonstrated  C treatment,  the  the  c o n c e n t r a t i o n of  phospholipase  CT  of  i s whether  directly  of p h o s p h o l i p a s e . CT  (76)  relative  enzyme t o membranes d e p l e t e d  c o u l d be  of  and  the  r e g u l a t e d by a membrane r e p a i r i n g  inactive  with  control  system  and  results  in  increased  proportion  phorylated ly  E.R.)  CT  and  phorylated sible  has to  CT  be  and  and  ed  second  in  (85).  messenger  of  the  PC  phosrever-  biosynthesis in fatty of  also  respective  acid  acetyl-CoA  render  these  pathways.  phosphorylation  hormone.  the  c y t o s o l . The  (87)  since  dephos(general-  while  Phosphorylation  glucacon,  this  i n the  reductase  that  membrane  r e g u l a t i o n of  inactive in their  h o r m o n a l l y c o n t r o l l e d by  with  The  mechanisms i n v o l v e d  HMG-CoA  speculated  (27).  phospholipids,  mechanism  biosynthesis  (86)  (88)  in cytosol  located  s i m i l a r t o the  enzymes  Vance  by  i s i n a c t i v e and  cholestrol  regulatory  activity  activated  phosphorylation  carboxylase  CT  a tendency to a s s o c i a t e  i s assumed t o be and  of  o f CT  Pelech  might  be  cAMP i s the e s t a b l i s h -  However, t h e  direct  incor-  32 poration  of  [  P]phosphate  into  the  enzyme  remains  to be  demons-  trated . A  second  hepatocytes cation  of  It  was  long  (28)  CT  synthesis  mechanism and  HeLa  cells  is regulated  (66),  by  fatty  fatty  tion-mediated  acids  i s so  some o f  the  rat  hepatocytes  (28,15). S t i l l ,  i n CT  mechanism  understood.  for  (1 mM  strong  decrease  (61). However, t h e poorly  the  studies  suggested  acids  shown i n r a t h e p a t o c y t e s  chain  from  f r o m c y t o s o l t o m i c r o s o m e s and  translocation  is  derived  I t has  that  of  fatty  been  CT,  with  3 mM  reverse  the  since  the  pool  1  oleate  (28).  on  CT  phosphoryla-  with  that  PC  unsaturated  oleate)  acid-mediated  mM  rat  translo-  acyl-CoA of  associated  remaining 54  effect  speculated  the  on  s t i m u l a t i o n of  fatty  or  it will  activity  supplemented question  the  palmitate  t r a n s l o c a t i o n of  the  that  that  the  and  done  microsomes  translocation DG  may size  was  account of  DG  in  increased  i s w h e t h e r or not  DG  can  enhance t h e  binding  Vasopressin, cause  presence  hydrolysis the  an  a transient  concentration the  of  other  of  of  of  the  (within DG  vasopressin  e f f e c t s of v a s o p r e s s i n transient increase  formation.  To  vasopressin PC  the  are  has  d i s r u p t e d by  lease  of  from  associated  translocation  should  The pressin  o f CT  is  the  perhaps  been shown to  be  be  by  activated  relating  to  the  i s whether or  not  CT  CT  CDP-choline with  distribution  and  paper. the by  s u b c e l l u l a r l o c a t i o n of MacKall  The  more  the  e_t a_l (91).  and  the  release rapid  increase to  of  DG,  Cells  rate of  of  than  cells,  from c y t o s o l to micrsomes, CT  from  and/or  a c t i v a t e the  digitonin,  vasopressin  r e l e a s e of c y t o s o l i c be  of  enzymes. Thus, i f the  in  t r a n s l o c a t i o n of  hormone on  determined. cells  formed  derived  to  to  total  h e p a t o c y t e s were t r e a t e d  in this  with  DG  question  developed  enzyme  of  appears  hence  the  The  has  promote the  estimation  cultured  administration  r a t e of  will  of  treatment  soluble  enzymes  PC  i n the  Ptdlns  biosynthesis  o f DG  been  and  been shown  50%  be  the  were d e t e r m i n e d  enzymes  to  The  effects  for  (89).  (90).  m i c r o s o m e s and  A protocol cellular  on  C which  has  of  reaction  answer t h i s q u e s t i o n ,  and  synthesis  PtdIns4P  (89). T h i s  cultures  from c y t o s o l to  increase  i s considered  phospholipase in  agonist,  in rat hepatocytes  2  vasopressin  the  4 min)  Ptdlns(4,5) P ,  by  m i c r o s o m e s j_n v i v o .  alpha-adrenergic  phospholipids  catalyzed  enzyme t o  re-  cytosolic membrane-  caused results  a reduction  by  the  in  the  i n the  from the  cells  i n t o the  medium  shows t h a t  the  a d d i t i o n of  observed. results  (5-20  nM)  presented  here  to r a t h e p a t o c y t e s  55  r e s u l t e d i n a reduced  vasorate  of  release  o f c y t o s o l i c CT i n t o t h e c u l t u r e  medium. However, t h e  3  increased not  rate  observed  of  [methyl- H]choline  in either  pulse-labeling  ments .  56  incorporation or  into  i n pulse-chase  PC  was  experi-  MATERIALS  Chemicals-  Wistar  of  Columbia  British  vasopressin was  (Grade  dissolved  kept  at  2.7  N a H P 0 , pH  C.  Animal  V I I I ) were o b t a i n e d  KC1,  1.5  Incubation of Hepatocytes from  normally  culture dishes Chapter  mM  KH P0 , 2  nM  Sigma. V a s o p r e s s i n  137  4  o f 5 ug/ml  buffered  (2 x 10  with Vasopressinfed Wistar  mM  saline  NaCl  and  and  (PBS) 8.1  mM  MEM  Rat h e p a t o c y t e s  r a t s and m a i n t a i n e d  were  i n 60  mm  c e l l s / d i s h ) as p r e v i o u s l y d e s c r i b e d ( i n  r e l e a s e of c y t o s o l i c  serum-free  from  solution  phosphate  I I I , M a t e r i a l s and M e t h o d s ) .  mediated  5-20  as a s t o c k  (80%) and a r g i n i n e  7.4.  4  isolated  Unit. Digitonin  Dulbecco's  mM  METHODS  (180 g) w e r e s u p p l i e d b y t h e U n i v e r s i t y  i n 0.9% N a C l  -20°  contains  rats  AND  CT,  For the study the c e l l s  on  were  digitonin-  washed  with  t w i c e , and i n c u b a t e d i n t h e same medium c o n t a i n i n g  vasopressin  i n c o r p o r a t i o n study,  f o r 0-8  min.  each d i s h  For the  of hepatocytes  [methyl- H]choline 3  was  labeled  with  10  3 uCi  of  [methyl-  containing  10  nm  study,  the c e l l s  pulsed  with  same medium containing absence  H]choline  (0.12 C i / m m o l )  vasopressin were  washed  f o r 0-60  with  i n t h e same  min. F o r a  serum-free  MEM  medium  pulse-chase  twice  and  10 u C i o f [ m e t h y l - H ] c h o l i n e (0.12 C i / m m o l ) 3  f o r 30 min. The c e l l s 28 uM  choline  or presence  o f 10 nM  i n the  were washed w i t h s e r u m - f r e e  and c h a s e d  i n t h e same medium  then  MEM  i n the  v a s o p r e s s i n f o r up t o 10 m i n . I n  order  t o a v o i d any e f f e c t s o f serum  ment,  i n some s t u d i e s t h e medium 57  i n t h e medium  was  on t h e e x p e r i -  r e p l a c e d by s e r u m - f r e e  MEM  12  hr  before  Release of ed  the  pulse-chase  Cytosolic  release  of  performed  CT  period.  f r o m Rat  cytosolic  e s s e n t i a l l y as  CT  from  medium, t h e  ly (1.0  ml  7.4),  2.5  ml  of  ice-cold  per  60  mm  cluture  250  mM  carefully release. cells  sucrose,  pipetted The  were  incubated  occasionally times  indicated  aliquot  Other  gently  of  the  Methods-  choline  betaine described  and  by  TLC  on  during  was  used  assay  of  up  by  Methods with  TLC  section.  8 min.  buffer  Lim  et  and  described CDP-choline  al  58  was  buffer (pH  digitonin  was  tray The  enzyme and  the  dishes  period.  were  At  the  removed, and  an  separation  of  assay.  acetone/methanol/HCl by  After  Tris-HCl  initiate  incubation  activity  are  mM  mg/ml to  was  washed c a r e f u l -  ice-cold to  the  f o r CT  CT  e_t a_l. (91).  10  dish  an  digitonin-release  The  0.5  culture  placed  hepatocytes  d i g i t o n i n - r e l ease  containing  swirled  sample  previously  Cold  digitonin  the  rat  Mackall  PMSF and  each  Digitonin-mediat-  m o n o l a y e r was  with  metabolites  Materials  PBS.  mM  were  by  cell  dish)  0.5  into  dishes  cultured  described  r e m o v a l of c u l t u r e with  Hepatocytes-  (92).  the under was  the  previous  separated  (10:90:4;  from  v:v:v)  as  RESULTS  Effect  of Vasopressin  on The D i g i t o n i n - m e d i a t e d  from Rat Hepatocytes sin  cell  medium  less  compared medium. treated and  than  with The  with  with  of release  50%  those  release  vasopressin  was  o f CT  f o r only with  the e f f e c t  transient.  treated Effect  that  with  The  vasopressin  was  concentrations  observed  in cells  a t t h e same  on t h e t r a n s l o c a t i o n o f  release  o f CT  vasopressin  was o b t a i n e d  with  inhibition  20  from  of Vasopressin  of vasopressin  nM  i n the rate  with  that  o f CT r e l e a s e  59  cells  on The D i g i t o n i n  In o r d e r  to  deter-  of vasopressin  on t h e  were  effect  o f 5 nM.  d i d n o t show  compared  different  investigated (Fig.  the maximal  a concentration  of v a s o p r e s s i n  the  up t o 8 min.  concentration  showed  concentra-  r a t e o f r e l e a s e o f CT. T h i s  was m a i n t a i n e d  studies  the  i n c u b a t i o n (5  r e l e a s e o f CT i n r a t h e p a t o c y t e s ,  (5-20 nM)  Preliminary  treated  also  vasopressin  reduced  CT,  into  Prolonged  of vasopressin  effective  digitonin-mediated  3 min i n c u -  released  R e l e a s e o f CT f r o m R a t H e p a t o c y t e s  mine t h e m a x i m a l l y  when t h e  was  1 min.  of D i f f e r e n t Concentrations  Mediated  was o b s e r v e d  (1 min) and 65% (2 min) o f t h e c e l l u l a r of the c o n t r o l s ,  reduced  vasopres-  CT i n t o t h e  f o r 3 min. A f t e r  r e s u l t e d i n a return to normal  suggested  of c y t o s o l i c  effect  vasopressin  10 min) o f t h e c e l l s  tion  14).  rate  ( F i g . 1 3 ) . The m a x i m a l  was i n c u b a t e d  bation,  CT  E x p o s u r e o f h e p a t o c y t e s t o 10 nM  l e d to a reduced  culture  R e l e a s e o f CT  with  The a  those  of  cells  further treated  CM  '  '  '  •  1  2  3  4  1  1  5  6  1  7  L.  8  T i m e <min> F i g 13. D i g i t o n i n - m e d i a t e d R e l e a s e o f CT f r o m R a t Hepat o c y t e s . R a t h e p a t o c y t e s i n 60 mm d i s h e s (2 x 10 cells) w e r e i n c u b a t e d f o r 0 m i n (O) , 1 m i n (A), 3 m i n ( • ) , 5 min ( £ ) o r 10 m i n (•) i n s e r u m - f r e e m e d i u m c o n t a i n i n g 10 nM v a s o p r e s s i n . T h e c e l l m e d i u m was r e p l a c e d w i t h 1 m l o f i c e - c o l d d i g i t o n i n (0.5 mg/ml) a f t e r w a s h i n g t h e c e l l s w i t h 2.5 ml o f i c e - c o l d PBS, a n d t h e i n c u b a t i o n c o n t i n u e d on a n i c e - c o l d m e t a l t r a y f o r up t o 8 m i n . CT a c t i v i t y i n t h e d i g i t o n i n e x t r a c t was s u b s e q u e n t l y d e termined i n t h e p r e s e n c e o f 0.2 mg o f t o t a l r a t l i v e r p h o s p h o l i p i d a n d 10 n m o l o f o l e a t e . E a c h p o i n t r e p r e s e n t s t h e a v e r a g e o f t w o d i s h e s . T h e e x p e r i m e n t was r e p e a t e d , and t h e same r e s u l t s were o b t a i n e d .  60  o i  .—•—• 1  2  .  -  3  4  T i m e (min> Fig.14. E f f e c t o f D i f f e r e n t C o n c e n t r a t i o n s o f V a s o pressin on The D i g i t o n i n - m e d i a t e d R e l e a s e o f CT from Rat Hepatocytes. Rat h e p a t o c y t e s i n 60 mm d i s h e s (2 x 10 c e l l s ) were i n c u b a t e d f o r 3 min i n s e r u m - f r e e m e d i u m i n t h e a b s e n c e (O) presence o f 5 nM ( • ) , 10 nM (•) o r 20 nM ( # ) o f v a s o p r e s s i n . The c e l l s w e r e w a s h e d w i t h 25 ml o f i c e - c o l d PBS and i n c u b a t e d i n 1 ml o f i c e - c o l d digitonin (0.5 mg/ml) f o r up t o 4 m i n . CT a c t i v i t y i n t h e d i g i t o n i n e x t r a c t was measured as d e s c r i b e d under the l e g e n d of Fig.15. Each p o i n t r e p r e s e n t s the mean o f two d i s h e s . o  61  r  with  10 nM  level  by  Effect into of  4 min  of Vasopressin  CT f r o m  with not  expected  [methyl-  choline size  in  that  would  exhibit  The  ; Pulse  an  into  CT  PC  to the c o n t r o l  increase  i n the  (Fig.15  A) d u r i n g  [methyl-  t h e p r e s e n c e o f 10 nM  The  reduced  i n the c e l l s  release  indicated  rate  o f 10 nM of  the  that  Therefore,  incubated  However, h e p a t o c y t e s  with  labeled  vasopressin did  incorporation  60 m i n . The  radioactive  t o be e x a m i n e d  H]choline  (data  of  pool  n o t shown).  i n c o r p o r a t i o n i n t o the c e l l s  vasopressin  i n 60 min p e r i o d  3  to v a s o p r e s s i n  i n the presence  i s too s m a l l  [methyl- H]choline  membrane-associated.  be s t i m u l a t e d .  of CDP-choline  control  exposed became  of  Studies-  PC b i o s y n t h e s i s  H]choline  rate of t o t a l  the  Labeling  the hepatocytes  vasopressin  o f CT r e t u r n e d  on The I n c o r p o r a t i o n  of the c y t o s o l i c  was  release  ( F i g . 14).  Phospholipids  part it  o f hormone. The  was  not a l t e r e d r e l a t i v e to  (Fig.15 B). 3  Influence  of Vasopressin  Hepatocytes sin  first  H]choline  t o show  activity.  chased min.  the e f f e c t  i n c o r p o r a t i o n i n t o the c e l l s  the e f f e c t  labeling  of  was  vasopres-  on  containing  stimulating  were p r e l a b e l e d  10 nM  vasopressin  i n c o r p o r a t i o n of l a b e l e d c h o l i n e  m i n e d . The r e s u l t s  from  two s e p e r a t e 62  i n the  might not  with  f o r 30 min i n t h e a b s e n c e o f v a s o p r e s s i n  i n t h e medium  i n Rat  small  experiment  of v a s o p r e s s i n  In t h i s e x p e r i m e n t , c e l l s  H]choline  The  Since  Metabolism  t r a n s i e n t ( F i g . 13) and t h e amount o f  5 min ( F i g . 15A), t h e p u l s e  able  thyl-  [Methyl—H]Choline  Studies-  on CT t r a n s l o c a t i o n was  [methyl-  be  : pulse-Chase  on  CT  [me-  and t h e n  f o r up t o 10  i n t o PC was  deter-  e x p e r i m e n t s were shown i n  T i m e <min>  T i m e <min>  Fig.15. E f f e c t o f V a s o p r e s s i n on The I n c o r p o r a t i o n o f [ m e t h y l H]Choline i n t o P h o s p h o l i p i d s ^ At zero time, r a t h e p a t o c y t e s In 60 mm d i s h e s (2 x 10 c e l l s ) w e r e l a b e l e d w i t h 10 u C i o f [ m e t h y l H ] c h o l i n e (0.12 Ci/mmol) p e r d i s h i n s e r u m - f r e e medium f o r up t o 60 m i n i n t h e a b s e n c e ( O / A ) o r p r e s e n c e o f 10 nM v a s o p r e s s i n (#,•). R a d i o a c t i v i t y i n c o r p o r a t e d i n t o t h e c e l l u l a r PC (A) and t h e t o t a l c e l l u l a r c h o l i n e m e t a b o l i t e s (B) w e r e d e t e r m i n e d a s d e s c r i b e d under M a t e r i a l s and Methods. Each p o i n t r e p r e s e n t s t h e mean o f t h r e e d i s h e s .  63  s:  0  2  4  6  8  10  0  C h a s e T i m e <rnin>  2  4  6  8  10  C h a s e T i m e <min>  F i g . 16. I n f l u e n c e o f V a s o p r e s s i n on The I n c o r p o r a t i o n o f [ M e t h y l - H ] C h o l i n e i n t o PC i n R a t H e p a t o c y t e s . Monolayer c u l t u r e s o f r a t h e p a t o c y t e s i n 60 mm d i s h e s (2 x 1 0 ° c e l l s / d i s h ) were washed w i t h s e r u m - f r e e MEM t w i c e , and t h e n p u l s e d w i t h 10 u C i [ m e t h y l - H ] c h o l i n e (0.12 C i / m m o l ) i n t h e same medium f o r 30 min. The c e l l s were s u b s e q u e n t l y c h a s e d w i t h 28 uM c h o l i n e i n t h e a b s e n c e (O) or p r e s e n c e o f 10 nM v a s o p r e s s i n ( 0 ) f o r up t o 10 m i n . R a d i o a c t i v i t y i n c o r p o r a t e d into cellular PC was d e t e r m i n e d as d e s c r i b e d under M a t e r i a l s and M e t h o d s . The p o i n t s i n p a n e l A r e p r e s e n t t h e mean o f two d i s h e s w h i l e i n p a n e l B r e p r e s e n t t h e a v e r a g e o f three dishes. 3  64  Fig.  16 A and B, r e s p e c t i v e l y . The d a t a  showed t h a t exhibited controls could  the c e l l s  an i n c r e a s e  chased  the f i r s t  i n t h e p r e s e n c e o f 10 nM  by about  a t 4 min c h a s e t i m e  n o t be o b t a i n e d  from  50% i n l a b e l e d  PC  ( F i g . 16A). However,  when t h e same e x p e r i m e n t  experiment vasopressin  relative  to the  t h e same  result  was  repeated ( F i g .  16B) . In  order  biosynthesis, for  amount  which  might  vasopressin. 4-min  interfere  treated  disappearance  treated  period  from  the  there  of vasopressin  cells  were  radiactivity  and  recovery  of choline  was  oxidation,  MEM any  medium  that  of  within  significant  i n t o PC b e t w e e n t h e  and t h o s e o f c o n t r o l s . A l s o , t h e from  the methanol-water  showed t h e same p a t t e r n  controls  PC  the a d d i t i o n  statisticlly  incorporation  with vasopressin of  to  i n T a b l e 4 showed no  to avoid  i n the c u l t u r e  response  on  i n serum-free  experiment  serum  cell  in radiactivity  4-min c h a s e p e r i o d  radioactivity product  the pulse-chase  The r e s u l t s s u m m a r i z e d  alternations  within  starting  of v a s o p r e s s i n  chase  cells  the e f f e c t  t h e h e p a t o c y t e s were p r e i n c u b a t e d  12 h r b e f o r e  trace  (see  to substantiate  (Table  4 ) . The  decreased  due t o t h e s e c r e t i o n into  Chapter I I I ) .  65  culture  i n both  medium  phase hormone  cellular  of betaine, by  a  hepatocytes  Table  4. E f f e c t  o f V a s o p r e s s i n on The I n c o r p o r a t i o n o f [Methyl- H]Choline 3  Chloroform Addition  Phase  (dpm x 1 0 " / d i s h ) 5  into  PC*  Methanol-water (dpm x  Phase  10~ /dish) 5  0 min  4 min  0 min  4 min  Control  0.43+0.02  0.45+0.08  17.0+0.9  12.1+0.7  Vasopressin  0.49+0.04  0.43+0.07  17.5+0.7  12.1+0.8  * Rat h e p a t o c y t e s were c u l t u r e d as d e s c r i b e d i n M a t e r i a l s and Methods. A f t e r m o n o l a y e r o f c e l l s were f o r m e d i n t h e c u l t u r e d i s h e s , t h e c e l l s were i n c u b a t e d i n s e r u m - f r e e m e d i u m f o r 12 h r b e f o r e p u l s e l a b e l i n g . Each d i s h o f c e l l s were l a b e l e d w i t h 10 u C i o f [ m e t h y l H ] c h o l i n e (0.12 C i / m m o l ) f o r 30 m i n i n t h e a b s e n c e o f v a s o p r e s s i n and t h e n c h a s e d f o r 4 m i n i n t h e medium c o n t a i n i n g 10 nM v a s o p r e s s i n . The [ m e t h y l - H ] c h o l i n e i n c o r p o r a t i o n i n t o PC and t h e o t h e r c h o l i n e m e t a b o l i t e s was d e t e r m i n e d i n b o t h c h l o r o f o r m p h a s e and m e t h a n o l w a t e r p h a s e e x t r a c t e d b y t h e B l i g h and D y e r ' s m e t h o d . The r e s u l t s a r e t h e average+S.D.; N=5. 3  66  DISCUSSION  The  effects  hydrates lates  and p h o s p h o l i p d s  different  resynthesis (94). to  of vasopressin  membrane  the hormonal  Ptdlns with  breakdown  (90,95).  will  hepatocytes,  diminish  which  within  observed  DG-dependent stimulates  exposed  CT c a u s e d  form  o f CT i n t h e c y t o s o l c o r r e s p o n d i n g  i n the reactions  other  i t i s well  a 2.5-fold  catalyzed  (PC, PE) m i g h t  67  CT f r o m r a t  hormone. The  DG  cytosolic  CT  cytosol  increase  e l e v a t i o n of  the phospholipid  also  that  pretreated  o f aggregated  to a twofold  argue  as the  known t h a t  purified  by p h o s p h o l i p a s e  studies  (10 nM)  CT c a n be t r a n s l o -  to t h i s  done i n r a t l i v e r  (84). A l t h o u g h  t o be P I , some  phospholipids  vasopressin  c a n be e x p l a i n e d  of p a r t i a l l y  C exhibited  treated  by t h e a d m i n i s t r a t i o n o f  experiment  phospholipase  accepted  that  the c y t o s o l i c  with  DG  i n hepatocytes  by e x o g e n o u s p h o s p h o l i p a s e C  rat liver  of  (84). S t u d i e s  t o be due  and  the c e l l s  the aggregation  fluxes  2  that  in this  o f DG  suggested  + +  of PtdIns(4,5)P , PtdIns4P  CT t r a n s l o c a t i o n , s i n c e  concentration  (93), and C a  and  The  from  the  stimu-  the release of i n t r a c e l l u l a r  release of c e l l u l a r  vasopressin  The hormone  C activity.  demonstrated  implies  t o membrane  carbo-  of phospholipase  o f DG f o r m a t i o n  have  of  i n c l u d i n g the degradation  c a n be m i m i c k e d  Our s t u d i e s  treatment  studied.  o f PI has been  i n the c o n c e n t r a t i o n  and t h e i n c r e a s e  reduced  events  stimulation  vasopressin  cated  been  the metabolism  o f P I , t h e s o - c a l l e d PI c y c l e  The enhanced  decrease  have  on  precursor  C i s generally  the h y d r o l y s i s of  account  f o r the i n c r e a s e  in  DG  ther  ( 8 9 ) . I t w o u l d be or  not  there  vasopressin, The  vasopressin with  the  for  3 min.  the  majority  of  release  This  concentration the  returned  o f DG  restored CT  of  fast  within  the  v i v o . The ed  the  result).  on  is compatible  4 min.  t r a n s l o c a t i o n o f CT cells  with  of  CT  with  treated the  of  with  addition  release adding  of  culture  by  between  membrane and  CT,  albumin CT  the  oleate  and  as  to  rapidly  restore  the  oleate  rapid  in  DG  prolonged in a of  reversible in  the  reverse  and  result  been  observ-  unpublished cell  w i t h membrane and of  min  translocation  seems  into  of  cellular  1  ( C o r n e l l , R.B.,  binding  will  the  CT  hormone  increase  t r a n s l o c a t i o n has  oleate of  around  appeared  vasopressin  CT  cells  vasopressin,  t o membranes,  Thus,  p r o m o t e s a s s o c i a t i o n o f c y t o s o l i c CT decreased  at  the  total  by  transient  vasopressin (Fig.13).  treated  i n the  levels  of  with  observation  induced  The  treated  with  the  of  PC.  t r a n s l o c a t i o n of  with  was  whe-  addition  incubation  the  increase  value  by  is  been p r e t r e a t e d  maximal  cells  While  effect  a transient  the  hepatocytes time of  in hepatocytes  release  cells  the  had  same r e v e r s i b i l i t y  i n HeLa  from  on  to  phospholipid  reached  causes a t r a n s i e n t incubation  cells  that  to b a s a l  CT  maximal  result  increase  of  to depend  when t h e  Hughes e_t _al (89)  and  corresponding  hormone. The  observed  i n v e s t i g a t i o n to see  PC-cycle  appeared  was  further  is a  since  reduced  worth  culture  causes  from  this  the  the cell  association  release  of  CT  from  cytosol. It  has  cytosol  to  choline  and  formation  been  proposed  microsomes  of  CTP PC.  will  that  the  stimulate  t o C D P - c h o l i n e and This  i s because  68  CT  translocation  of  CT  the  of  phospho-  PPi is a  conversion  and  from  hence s t i m u l a t e  rate  limiting  enzyme  the in  PC  synthesis  v i a the  vasopressin synthesis in  t r e a t m e n t e x p e r i m e n t , an  was  not  pulse-chase  decreased  observed  ( F i g . 16B,  , caused  conversion ferase Thus  (CPT),  so  the  Ca  CT.  possible  experiments One  PC  vasopressin  of  bio-  on  of  of  of t h i s  the dis-  concentration  of the  cholinephosphotrans-  inhibitor  vasopressin  due  in spite  or  PC  of  this  enzyme.  synthesis  CDP-choline  in  is  the  to the b l o c k a g e of the  not  vaso-  conver-  m i g h t have been o b s e r v e d .  Unfortunate-  r a d i o a c t i v e p o o l s i z e of C D P - c h o l i n e i n the  hepatocytes  small  that on  total  was  the  different and  I  PC,  our  (94), i n h i b i t s  c a t a l y z e d by  accumulation  to  r a t e o f PC  explanation cellular  i s a known  + +  effect  increased  increased  treated hepatocytes,  Pollard of  since  A  Studies using  4)  a d d i t i o n of  of CDP-choline  l y , the is  the  mass  observable. pressin  Table  o f C D P - c h o l i n e to  the  sion  by  that  (88). However, i n  i n e i t h e r p u l s e - l a b e l i n g ( F i g . 15A)  r e l e a s e of c y t o s o l i c  c r e p a n c y m i g h t be Ca  CDP-choline pathway  effect  precusors  Brindley  (97)  phospholipids  rat  hepatocytes  the  other  unable  after  of  to  vasopressin  have  been  to  the  done  f o u n d a 19%  (mainly  PC)  vasopressin  same c e l l s  on  PC  in  rat  possibility. synthesis  [1- C]  oleate  1 4  claimed a  (100  by  hepatocytes.  i n the  treatment  induced  this  increase  from  hand, Alemany et: a_l (96)  vasopressin  examine  nM,  synthesis in 30  Wistarmin).  the  a d d i t i o n of  rapid,  transient  On  100  nM  (within 3  4  min)  inhibition  choline present to  into  PC.  of  the  These  r a t e of aspects  i n c o r p o r a t i o n of were  work. In a d d i t i o n , I have not  show  membrane  whether and  there  hence  i s any  interfere  69  investigated  done the  effect with  not  the  of  [methyl-  control  vasopressin  permeability  in  H] the  experiment on of  plasma cellular  proteins  i n the d i g i t o n i n - m e d i a t e d  precise effect  of v a s o p r e s s i n  release  o f CT e x p e r i m e n t .  on PC s y n t h e s i s , i f a n y ,  The  remains  unknown. Another  possible  translocation  is a  regulatory  promotes binding  phospholipids increased bited  hepatic  carbohydrate  independent  mechanism  cytosolic  methylation  pathway  methyltransferase  controlled  simulate  that  mechanism  + +  to  stimulate  -requiring,  cAMPe_t a ^  the p h o s p h o r y l a t i o n  of  - l i n k e d , cAMP-independent t h e PC b i o s y n t h e s i s  regulated (85) and  by  a  vasopressin  o f PC  stimulated  v i a PE  phosphorylation-  calmodulin-dependent  will  t o an  is inhi-  s t u d i e s done by G a r r i s o n  the b i o s y n t h e s i s  by a v a s o p r e s s i n  specific  cAMP-dependent  a Ca  + +  of the  stimulated  protein  kinase  i n r a t hepatocytes i s  phosphorylation  be  an  interesting  topic  the treatment  of  rat hepatocytes  or de-  for fur-  studies. In  pressin into  a  (27,61,  leads  PC s y n t h e s i s  through  showed  mechanism  whether  phosphorylation  can  was.also  through  in turn  recognized  proteins v i a a Ca  evidence  dephosphorylation  ther  This  i s well  (98). R e c e n t  vasopressin  mechanism. S t i l l ,  So,  metabolism  cycle  where  o f CT b y a p u t a t i v e  (85). V a s o p r e s s i n  shown  10 h e p a t i c  (100) .  t o t h e E.R.  enzyme.  phosphorylation  kinase  have  this  i n t h e CT  that dephosphorylation  o f t h e enzyme  activate  protein  (99)  showed  r a t e o f PC b i o s y n t h e s i s , w h i l e  after  involved  reversible phosphorylation  74,85). I n d i r e c t e v i d e n c e CT  mechanism  conclusion, (5 nM)  caused  a diminished  t h e c u l t u r e medium,  implying  with  vaso-  release of i n t r a c e l l u l a r  CT  t r a n s l o c a t i o n of the c y t o s o l i c 3  enzyme  t o membrane.  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