UBC Theses and Dissertations

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UBC Theses and Dissertations

Isolation and partial characterization of vesicles derived from the plasma membrane of the chicken gizzard… Azad, Aristotle 1979

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ISOLATION AND PARTIAL CHARACTERIZATION OF VESICLES DERIVED FROM THE PLASMA MEMBRANE OF THE CHICKEN GIZZARD MUSCLE  by ARISTOTLE AZAD B. Sc., The U n i v e r s i t y of B r i t i s h Columbia, 1975  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Anatomy  We a c c e p t t h i s t h e s i s as conforming to t h e r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1979 ©  A r i s t o t l e Azad, 1979  In p r e s e n t i n g t h i s  thesis in partial  an a d v a n c e d d e g r e e a t the L i b r a r y I further for  shall  the U n i v e r s i t y  make i t  agree that  this  thesis for  of ,  V  E  extensive  ANATOMY  i s understood  .—  Columbia  the requirements I agree  r e f e r e n c e and copying of  this  that  not  copying or  for  that  study. thesis  by t h e Head o f my D e p a r t m e n t  f i n a n c i a l gain shall  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  1  It  of  B r i t i s h Columbia,  available for  permission.  Department  75-51  of  s c h o l a r l y p u r p o s e s may be g r a n t e d  written  BP  freely  permission for  by h i s r e p r e s e n t a t i v e s . of  fulfilment  or  publication  be a l l o w e d w i t h o u t  my  ABSTRACT  Plasma membrane v e s i c l e s , i s o l a t e d from the c h i c k e n g i z z a r d u s i n g d i f f e r e n t i a l c e n t r i f u g a t i o n and s u c r o s e c e n t r i f u g a t i o n , were b i o c h e m i c a l l y c h a r a c t e r i z e d . obtained  gradient  Two f r a c t i o n s  from t h e s u c r o s e g r a d i e n t , F r a c t i o n s 4 and 5 ( 3 2 % and  34% s u c r o s e  r e s p e c t i v e l y ) , were judged t o be t h e most pure i n  plasma membranes a s based on 5' n u c l e o t i d a s e and i o d i n a t i o n studies.  Both f r a c t i o n s had t h e same coomassie b l u e and PAS  s t a i n i n g p r o f i l e when e l e c t r o p h o r e s e d  and under e l e c t r o n m i c r o -  scopy b o t h f r a c t i o n s c o n s i s t e d o f membrane v e s i c l e s o f v a r y i n g size. A Mg^  +  s t i m u l a t e d ATPase a c t i v i t y was found t o be p r e s e n t  and h i g h e s t i n F r a c t i o n 5 w h i l e F r a c t i o n 4 e x h i b i t e d activity.  little  T h i s enzyme was i n h i b i t e d i n the p r e s e n c e of h i g h  c o n c e n t r a t i o n s of ATP and Mg.  A s i m i l a r e c t o Mg2+ s t i m u l a t e d  ATPase was observed i n i s o l a t e d smooth m u s c l e c e l l s . Phosphorylation  u s i n g [Y-^P] ATP was o b s e r v e d a t 205,000,  165,000 and 145,000 d a l t o n s i n F r a c t i o n 5 o n l y . dephosphorylation phosphorylation  Mg promoted  o f t h e 205,000 d a l t o n band w h i l e Ca promoted of t h e 165,000 d a l t o n band.  All  peaks were s e n s i t i v e t o h y d r o x y l a m i n e t r e a t m e n t .  phosphorylated These r e s u l t s  would seem t o i n d i c a t e t h a t t h e r e i s a d i f f e r e n c e i n membrane o r i e n t a t i o n between F r a c t i o n 4 and F r a c t i o n 5. The membrane o r i e n t a t i o n i n F r a c t i o n s 4 and 5 was then examined u s i n g a c e t y l c h o l i n e s t e r a s e and s i a l i c a c i d was e x t e r n a l plasma membrane m a r k e r s .  F r a c t i o n 4 was found t o c o n t a i n  mainly  i n s i d e - o u t v e s i c l e s i n c o n t r a s t t o F r a c t i o n 5, w h i c h was thought  iii  to c o n s i s t m a i n l y of r i g h t - s i d e - o u t plasma membrane v e s i c l e s . The o r i e n t a t i o n d i f f e r e n c e s were f u r t h e r examined 125 lactoperoxidase catalyzed i o d i n a t i o n using I.  using  I o d i n a t i o n of F r a c t i o n 4 r e s u l t e d i n the appearance of 125 I i n a band m i g r a t i n g  i n SDS e l e c t r o p h o r e t i g r a m s  w i t h an  apparent m o l e c u l a r w e i g h t of 100,000 d a l t o n s , and minor l a b e l l i n g was seen a t 205,000 and 55,000 d a l t o n s .  0.05%  T r i t o n X-100 s i g n i f i c a n t l y enhanced l a b e l l i n g of a l l bands.  three-  I o d i n a t i o n of F r a c t i o n 5 r e s u l t e d i n l a b e l l i n g of a l l  t h r e e bands, but t r e a t m e n t of the membranes w i t h T r i t o n enhanced l a b e l l i n g o n l y a t 100,000 d a l t o n s .  X-100  I o d i n a t i o n of  i n t a c t s i n g l e c e l l s r e s u l t e d i n an i o d i n a t i o n p a t t e r n  similar  to t h a t of F r a c t i o n 5 i n the absence of T r i t o n X-100. Attempts were made t o f u r t h e r p u r i f y the membranes u s i n g c o n c a n a v a l i n A - Sepharose a f f i n i t y chromatography. A f t e r F r a c t i o n 4 was a p p l i e d t o t h e column, f o u r peaks of p r o t e i n c o u l d be e l u t e d .  The f i r s t two peaks, e l u t e d i n the  absence of a methyl-D-mannoside were thought t o c o n s i s t of i n s i d e - o u t v e s i c l e s as judged by i o d i n a t i o n and sidedness s t u d i e s .  acetylcholinesterase  The o t h e r two peaks, e l u t e d i n t h e  presence o f a methyl-D-mannoside were thought t o c o n t a i n u n s e a l e d plasma membrane v e s i c l e s .  Over 90% o f the o r i g i n a l l y  a p p l i e d p r o t e i n was e l u t e d , 20% b e i n g c o n t a i n e d  i n the two  peaks e l u t e d i n the p r e s e n c e of a methyl-D-mannoside. F r a c t i o n 5 behaved q u i t e d i f f e r e n t l y on the a f f i n i t y columns.  A p p r o x i m a t e l y 90% of the o r i g i n a l l y a p p l i e d  protein  c o u l d n o t be e l u t e d even i n the p r e s e n c e of a methyl-D-mannoside.  iv:-  iv.  Of  t h e two peaks e l u t e d , one peak o b t a i n e d i n the absence o f  a methyl-D-mannoside, was thought t o c o n s i s t of i n s i d e - o u t plasma membrane v e s i c l e s .  The second peak, e l u t e d i n t h e  p r e s e n c e of amethyl-D-mannoside was thought t o c o n t a i n u n s e a l e d membrane v e s i c l e s as i n d i c a t e d by s i d e d n e s s  studies.  I t was c o n c l u d e d t h a t F r a c t i o n s 4 and 5 r e p r e s e n t plasma membrane p r e p a r a t i o n s  of d i f f e r i n g o r i e n t a t i o n ,  F r a c t i o n 5 b e i n g p r e d o m i n a n t l y r i g h t - s i d e - o u t and F r a c t i o n 4 a f t e r a f f i n i t y chromatography m a i n l y i n s i g h t - o u t .  These two  f r a c t i o n s may have some a p p l i c a b i l i t y i n i n v e s t i g a t i n g t h e asymmetry o f v a r i o u s membrane t r a n s p o r t  systems.  V  •TABLE OF CONTENTS  Page Abstract T a b l e of Contents L i s t of T a b l e s L i s t of Figures Acknowledgements  i i v v i v i i x  Introduction A. I s o l a t i o n of Plasma Membranes B. The O r i e n t a t i o n Problem C. Smooth M u s c l e P r e p a r a t i o n s D. R a t i o n a l e  1 3 10 16 21  Materials  27  Methods A. Enzyme Assays • B. Plasma Membrane I s o l a t i o n C. C e l l Sheet and S i n g l e C e l l P r e p a r a t i o n D. G e l E l e c t r o p h o r e s i s E. I o d i n a t i o n Experiments F. Membrane E x t r a c t i o n P r o c e d u r e G. A f f i n i t y Chromatography H. P h o s p h o r y l a t i o n S t u d i e s I. E l e c t r o n Microscopy  27 27 37 39 41 46 51 53 54 57  Results A. G e n e r a l B. Membrane Marker S t u d i e s C. Plasma Membrane Mg2+ ATPase A c t i v i t i e s D. O r i e n t a t i o n S t u d i e s U s i n g A c e t y l c h o l i n e s t e r a s e and S i a l i c A c i d E. I o d i n a t i o n S t u d i e s F. E x t r a c t i o n S t u d i e s G. A f f i n i t y Chromatography H. Summary  58 58 58 66 83 83 108 131 140  Discussion  141  Literature Cited  150  vi  LIST OF TABLES  Table  Page  I  Summary of smooth muscle p r e p a r a t i o n s .  23  II  S u b s t r a t e s o l u t i o n s to t e s t Mg2+ ATPase s e n s i t i v i t y to pH, Na, L i , K, and o u a b a i n .  33  III  G e l l f o r m u l a t i o n s used f o r e l e c t r o p h o r e s i s .  42  IV  E x t r a c t i o n media used i n membrane e x t r a c t i o n .  52  V  Components used i n p h o s p h o r y l a t i o n  55  VI  T o t a l and s p e c i f i c a c t i v i t i e s of s e l e c t e d marker enzymes a t v a r i o u s s t a g e s of the f r a c t i o n a t i o n procedure.  60  Vila  S p e c i f i c a c t i v i t i e s of marker enzymes. o b t a i n e d from s u c r o s e g r a d i e n t s .  Fractions  60  Vllb  S p e c i f i c a c t i v i t i e s of marker enzymes. o b t a i n e d from s u c r o s e g r a d i e n t s .  Fractions  61  VIII  A c c e s s i b i l i t y of markers i n s u c r o s e g r a d i e n t fractions.  84  IX  M o l e c u l a r w e i g h t assignments of bands and peaks d e p i c t e d i n F i g u r e s 15 t o 41.  85  Xa  A c c e s s i b i l i t y of F r a c t i o n s 4 and 5 to lactoperoxidase catalyzed i o d i n a t i o n .  107  Xb  S e l f l a b e l l i n g of l a c t o p e r o x i d a s e presence of T r i t o n X-100.  107  XI  P r o t e i n c o n t a i n e d i n e x t r a c t i o n media and p e l l e t f o l l o w i n g e x t r a c t i o n procedure.  114  XII  C h a r a c t e r i z a t i o n of column f r a c t i o n s e l u t e d from Con A - Sepharose a f f i n i t y columns.  136  studies.  i n the  vii  LIST OF FIGURES Figure  Page  1  Scheme f o r i s o l a t i o n o f smooth muscle plasma membranes from t h e c h i c k e n g i z z a r d .  40  2  E l e c t r o n m i c r o g r a p h o f c h i c k e n g i z z a r d smooth muscle.  59  3  E l e c t r o n m i c r o g r a p h of c h i c k e n g i z z a r d , smooth muscle c e l l .  59  4  E l e c t r o n m i c r o g r a p h o f F r a c t i o n 4 plasma membranes i s o l a t e d from t h e c h i c k e n g i z z a r d smooth muscle.  65  5  E l e c t r o n m i c r o g r a p h of F r a c t i o n 5 plasma membranes i s o l a t e d from t h e c h i c k e n g i z z a r d smooth m u s c l e .  65  6.  Optimization F r a c t i o n 5.  67  7.  E f f e c t s o f pH, Na, L i , K and o u a b a i n on t h e Mg2+ ATPase a c t i v i t y o f F r a c t i o n 4 and F r a c t i o n 5.  69  8.  P l o t o f r a t e v e r s u s l o g a r i t h m o f the s u b s t r a t e [MgATP]-2 f o r t h e Mg2+ ATPase observed i r i F r a c t i o n 5.  71  9.  Phase c o n t r a s t m i c r o g r a p h o f a s u s p e n s i o n o f s i n g l e smooth muscle c e l l s ( 8 0 x ) .  72  10  Phase c o n t r a s t m i c r o g r a p h o f a s i n g l e i s o l a t e d smooth m u s c l e c e l l .  72  11  Phosphorylation p a t t e r n s o f F r a c t i o n 5 a t v a r i o u s i n c u b a t i o n times u s i n g [y- 32p] ATP.  75  12  Phosphorylation patterns F r a c t i o n 5.  o f F r a c t i o n 4 and  76  13  Phosphorylation patterns F r a c t i o n 5.  o f F r a c t i o n 4 and  78  14  Phosphorylation patterns  o f F r a c t i o n 5.  80  15  Coomassie b l u e s t a i n i n g p a t t e r n (top) and PAS p r o f i l e (bottom) o f F r a c t i o n 4.  86  16  Coomassie b l u e s t a i n i n g p a t t e r n (top) and PAS p r o f i l e (bottom) o f F r a c t i o n 5. 125 L a b e l l i n g of muscle cubes w i t h I. 125 L a b e l l i n g of c e l l sheets w i t h I.  87  o f Mg2+ ATPase a c t i v i t y i n  r  17 18  89 90  viii  Figure  Page  19  L a b e l l i n g of a s u s p e n s i o n of i s o l ^ f r e d s i n g l e smooth muscle c e l l s w i t h I.  91  20  I o d i n a t i o n p a t t e r n s of F r a c t i o n 4 u s i n g p r e l a b e l l e d muscle cubes.  92  21  I o d i n a t i o n p a t t e r n s of F r a c t i o n 5 u s i n g p r e l a b e l l e d muscle cubes.  93  22  I o d i n a t i o n p a t t e r n s of F r a c t i o n 4 u s i n g p r e l a b e l l e d c e l l sheets.  94  23  I o d i n a t i o n p a t t e r n s of F r a c t i o n 5 u s i n g p r e l a b e l l e d c e l l sheets.  95  24  I o d i n a t i o n of F r a c t i o n 4.  97  25  I o d i n a t i o n of F r a c t i o n 5.  98  26  I o d i n a t i o n of s u c r o s e f r e e F r a c t i o n 4.  99  27  I o d i n a t i o n of s u c r o s e f r e e F r a c t i o n 5.  100  28  S e l f i o d i n a t i o n of l a c t o p e r o x i d a s e .  102  29  A c c e s s i b i l i t y of F r a c t i o n 4 t o i o d i n a t i o n u s i n g 1251.  103  30  A c c e s s i b i l i t y of F r a c t i o n 5 t o i o d i n a t i o n u s i n g 1251.  105  31  E x t r a c t i o n of F r a c t i o n 4 u s i n g H2O, E t h y l e n e d i a m i n e t e t r a a c e t a t e and D i g i t o n i n .  115  32  E x t r a c t i o n o f F r a c t i o n 5 u s i n g H20, E t h y l e n e d i a m i n e t e t r a a c e t a t e and D i g i t o n i n .  117  33  E x t r a c t i o n of F r a c t i o n 4 u s i n g D i m e t h y l m a l e i c a n h y d r i d e (DMMA).  119  34  E x t r a c t i o n of F r a c t i o n 5 using Dimethyl maleic a n h y d r i d e (DMMA).  121  35  E x t r a c t i o n of F r a c t i o n 4 u s i n g p - C h l o r o m e r c u r i benzene s u l p h o n i c a c i d (pCMBS).  123  36  E x t r a c t i o n of F r a c t i o n 5 u s i n g p-Chloromercuri'benzene s u l p h o n i c a c i d (pCMBS).  125  37  E x t r a c t i o n of F r a c t i o n 4 u s i n g (TX-100).  T r i t o n X-100 '"  .127  ix  Figure  Page  38  E x t r a c t i o n of F r a c t i o n 5 u s i n g T r i t o n X-100 (TX-100).  129  39  Con A - Sepharose a f f i n i t y chromatography of F r a c t i o n 4- (bottom) and F r a c t i o n 5 ( t o p ) .  134  40a  A n a l y s i s o f peak f r a c t i o n s o b t a i n e d by Con A Sepharose a f f i n i t y chromatography of F r a c t i o n 4.  137  40b  A n a l y s i s of peak f r a c t i o n s o b t a i n e d by Con A Sepharose a f f i n i t y chromatography of F r a c t i o n 4.  138  41  A n a l y s i s of peak f r a c t i o n s o b t a i n e d by Con A Sepharose a f f i n i t y chromatography of F r a c t i o n 5.  139  X  ACKNOWLEDGEMENTS  To P r o f e s s o r V. P a l a t y , my humble and most s i n c e r e s u p e r v i s o r , I w h i c h t o extend my s i n c e r e s t t h a n k s , e s p e c i a l l y f o r h i s t o l e r a n c e and g u i d a n c e .  As w e l l , I would l i k e t o  thank Ms. M a r y e t t e Mar f o r h e r e x p e r t and  f r i e n d l y advice.  The t e c h n i c a l e x p e r t i s e o f Mrs.  L a i and Ms. Susan Shinn was a l s o The  technical assistance Virginia  appreciated.  encouragement and immense t o l e r a n c e d i s p l a y e d by  the f a c u l t y and s t a f f o f t h e Department o f Anatomy were prime f a c t o r s i n the completion  of t h i s t h e s i s .  F i n a l l y I w i s h t o thank M i s s Judy Myar f o r t y p i n g t h e t h e s i s d e s p i t e a l l odds and I w i s h t o thank t h e n i g h t p e o p l e , L y l e , F r e d , Lea, P e t e r and Don f o r p r o v i d i n g proof s k i l l s and tremendous encouragement.  reading  To M i k e S i l v e r s t e i n f o r  h i s a l l n i g h t d i s c u s s i o n s on membranes, my s i n c e r e s t t h a n k s .  - 1 -  INTRODUCTION  Although  the s t u d i e s w i t h i s o l a t e d p r e p a r a t i o n s r i c h i n  smooth muscle have p r o v i d e d v a l u a b l e i n f o r m a t i o n on many f e a t u r e s of membrane phenomena i n t h i s type o f c e l l s , p e r u s a l o f t h e l i t e r a t u r e r e v e a l s t h a t some b a s i c q u e s t i o n s have n o t been answered s a t i s f a c t o r i l y as y e t .  F o r example, i t i s r e c o g n i z e d  t h a t the mechanism o f Ca e x t r u s i o n from t h e c e l l p l a y s a key r o l e i n t h e c o n t r o l o f t e n s i o n , b u t t h e p r e s e n t knowledge o f the mechanism i s r a t h e r i n c o m p l e t e .  Indeed, i t has n o t even  been e s t a b l i s h e d whether t h e energy r e q u i r e d f o r e x t r u s i o n o f Ca a g a i n s t a steep g r a d i e n t of the n e g a t i v e c h e m i c a l p o t e n t i a l of t h i s i o n i s p r o v i d e d by h y d r o l y s i s o f ATP, o r spontaneous i n f l u x of Na (BLAUSTEIN, 1977). I t must be a p p r e c i a t e d , however, t h a t study o f these and o t h e r problems i s c o m p l i c a t e d muscle preparations;.  by f a c t o r s l a r g e l y unique t o smooth  The dimensions o f smooth muscle c e l l s  make i t r a t h e r u n l i k e l y t h a t a number o f t h e methods a l r e a d y a p p l i e d s u c c e s s f u l l y t o s t u d i e s on o t h e r t y p e s o f m u s c l e c e l l s , such as t h e m o n i t o r i n g 2+ Ca  of the i n t r a c e l l u l a r c o n c e n t r a t i o n of  by measurement of t h e l u m i n i s c e n c e o f i n j e c t e d a e q u o r i n  (ALLEN & BLINKS, 1978) c a n be used f o r smooth muscle s t u d i e s . Secondly, the l a r g e e x t r a c e l l u l a r space w i t h i t s h i g h conc e n t r a t i o n o f f i x e d charged groups makes i t d i f f i c u l t ,  i f not  i m p o s s i b l e , t o o b t a i n r e l i a b l e i n f o r m a t i o n on t h e transmembrane ion. f l u x e s from e v a l u a t i o n of i s o t o p e f l u x d a t a .  Last, but not  l e a s t , c e l l s other than smooth muscle a r e f r e q u e n t l y  present  i n i s o l a t e d p r e p a r a t i o n s , and u n l e s s t h i s f a c t i s taken  into  a c c o u n t , some o f t h e o b s e r v a t i o n s may be i n t e r p r e t e d i n c o r r e c t l y .  2  - 2 -  To e l i m i n a t e the l a s t two  factors, isolated  c e l l s have been i n t r o d u c e d by the group of FAY FAY,  1977;  (FAY,  individual 1973;  SCHEID e t a l . , 1979), but the s t u d i e s have been  r e s t r i c t e d almost e x c l u s i v e l y to c e l l s i s o l a t e d from the stomach m u s c u l a r i s of Bufo m a r i n u s by enzymic d i g e s t i o n .  It  remains to be shown t h a t t h i s approach would a p p l y e q u a l l y w e l l t o smooth muscle c e l l s from mammaliam t i s s u e s . An a t t r a c t i v e a l t e r n a t i v e i s t o study membrane phenomena u s i n g the i s o l a t e d plasma membrane. approach o f f e r s a r e o b v i o u s .  The advantages t h a t t h i s  Unfortunately, r e l a t i v e l y  little  a t t e n t i o n has been p a i d t o the f a c t t h a t , i f m e a n i n g f u l r e s u l t s a r e to be o b t a i n e d , the p r e p a r a t i o n must meet c e r t a i n c r i t e r i a . I f the p r e p a r a t i o n were to be used, e.g.,  f o r a study of.  Ca transport., i t s h o u l d be a t l e a s t e s s e n t i a l l y f r e e of  contamination  by membranes of s a r c o p l a s m i c r e t i c u l u m .  I n o t h e r t y p e s of m u s c l e 2+ t h i s c e l l u l a r component i s known t o e x h i b i t a Ca - stimulated ATPase a c t i v i t y , w h i c h , i n c o n t r a s t t o the m i t o c h o n d r i a l 2+ Ca  - s t i m u l a t e d ATPase, cannot be i n h i b i t e d  (CARAFOLI &. CROMPTON, 1978).  selectively  C l e a r l y , an i d e a l p r e p a r a t i o n  s h o u l d be f r e e of c o n t a m i n a t i o n  by membranes d e r i v e d from  i n t r a c e l l u l a r organelles. There a r e , however, o t h e r c r i t e r i a w h i c h an p r e p a r a t i o n s h o u l d meet.  I n order to remove the  membranes and components o f t h e c y t o p l a s m ,  ideal  contaminating  a multi-step  f r a c t i o n a t i o n procedure i s u s u a l l y r e q u i r e d .  The problem h e r e  3  - 3 -  i s t h a t , i n the course o f f r a c t i o n a t i o n , some i m p o r t a n t , b u t l o o s e l y bound components o f t h e plasma membrane may become l o s t , o r , c o n v e r s e l y , some components t h a t a r e n o t a s s o c i a t e d w i t h t h e membrane i n s i t u may become f i r m l y a t t a c h e d preparation.  I d e a l l y the process  to the f i n a l  of f r a c t i o n a t i o n s h o u l d produce  a p r e p a r a t i o n whose c o m p o i s i t i o n , c o n f o r m a t i o n  and o t h e r  features  a r e i d e n t i c a l t o those e x h i b i t e d by the plasma membrane i n s i t u . T h i s problem i s much more c o m p l i c a t e d  than i t may seem t o  be a t f i r s t sight., because t h e membrane i n s i t u i s t y p i c a l l y under the i n f l u e n c e of an e l e c t r i c a l f i e l d o f a p p r e c i a b l e s t r e n g t h and the l a t t e r may have a pronounced e f f e c t on t h e c o n f o r m a t i o n and d i s p o s i t i o n i n t h e membrane o f any component p o s s e s s i n g a t l e a s t a d i p o l e o r a charged, group.  The s i g n i f i c a n c e o f t h e f a c t t h a t  the two s u r f a c e s o f the membrane a r e t y p i c a l l y exposed t o s o l u t i o n s of m a r k e d l y d i f f e r e n t c o m p o s i t i o n  should n o t be  underestimated.  F i n a l l y , i f t h e membrane i s o l a t i o n procedure i s t o be of any v a l u e , i t s y i e l d s h o u l d be r e a s o n a b l y  high, p a r t i c u l a r l y  because the q u a n t i t y of t i s s u e s r i c h i n smooth muscle t h a t can be o b t a i n e d from t y p i c a l l a b o r a t o r y a n i m a l s  i s usually quite  limited.  A.  I s o l a t i o n of Plasma Membranes I n g e n e r a l , the i n i t i a l step i n t h e p r e p a r a t i o n of  i s o l a t e d plasma membranes i s h o m o g e n i z a t i o n , w h i c h i s u s u a l l y a c h i e v e d by a p p l i c a t i o n o f shear f o r c e s (BIRNIE, 1972). f o l l o w e d by d i f f e r e n t i a l c e n t r i f u g a t i o n t o s e p a r a t e  This i s  t h e plasma  . . .  0  4  - 4 -  membranes from t h e c e l l d e b r i s , n u c l e i and m i t o c h o n d r i a (GRAHAM, 1972; GRAHAM, 1975; NEVILLE, 1975; SCHAPIRA, 1975; SCHIMMEL & KENT, 1977; WALLACH & SCHMIDT-ULLRICH, 1977). r e s u l t i n g crude " m i c r o s o m a l " p r e p a r a t i o n c a n be f u r t h e r by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n .  The fractionated  T y p i c a l l y , plasma membranes  and endoplasmic r e t i c u l u m a r e found a t lower d e n s i t i e s (1.14 -3 1.15 g x cm ) w h i l e m i t o c h o n d r i a a r e a t h i g h e r d e n s i t i e s -3 (1.16 - 1.18 g x cm ) (PRICE, 1974; TOLBERT, 1974) .  I f warranted,  f u r t h e r p u r i f i c a t i o n o f t h e p r e p a r a t i o n c a n be attempted u s i n g t e c h n i q u e s such as a f f i n i t y chromatography  (CUATRECASES, 1973;  PHARMACIA, 1974; SHARON & L I S , 1975; HYNES, 1976; WALSH e t a l . , 1976; BRUNNER e t . a l . ,  1977).  The h o m o g e n i z a t i o n t e c h n i q u e i s of c r i t i c a l importance i n t h a t i t a l s o d e t e r m i n e s t h e f i n a l y i e l d o f plasma membranes.  There  a r e t h r e e b a s i c types o f h o m o g e n i z a t i o n m e c h a n i c a l , l i q u i d and gaseous (BIRNIE, 1972; GRAHAM, 1975; WALLACH & SCHMIDT-ULLRICH, 1977).  M e c h a n i c a l p r e p a r a t i o n u s i n g m e c h a n i c a l shear employs two  basic techniques.  The f i r s t , t h e f r e e z e thaw t e c h n i q u e , used l e s s  f r e q u e n t l y , i n v o l v e s u s i n g c y c l e s o f f r e e z i n g and thawing w h i c h r e s u l t i n t h e d i s r u p t i o n o f c e l l s by i n t r a c e l l u l a r i c e c r y s t a l formation.  I n t h e second t y p e o f m e c h a n i c a l s h e a r , employed  by commercial u n i t s l i k e t h e P o l y t r o n and MSE homogenizers, t h e sample i s drawn i n t o a w o r k i n g head where i t i s mixed by r o t a t i n g b l a d e s and sheared d u r i n g e x p u l s i o n from t h e w o r k i n g head.  Membranes a r e u s u a l l y o b t a i n e d i n t h e form o f v e s i c l e s  r a t h e r than c e l l s h e e t s .  U n t i l r e c e n t l y , i t was f e l t t h a t t h e  drawbacks o f m e c h a n i c a l shear f a r outweighed  the advantages.  . .  5  - 5 -  Drawbacks to t h i s method i n c l u d e the p o s s i b l e damage to the plasma and i n t r a c e l l u l a r membranes by the h i g h l o c a l generated during homogenization  temperatures  and d i s r u p t i o n of most o r g a n e l l e s .  On the o t h e r hand, m e c h a n i c a l shear of t h i s type i f q u i t e even f o r h o m o g e n i z a t i o n  of t i s s u e s r i c h i n c o l l a g e n and  I n l i q u i d shear h o m o g e n i z a t i o n ,  effective  elastin.  the d i s r u p t i v e f o r c e s a r e  c o n s i d e r a b l y I : weaker than those of m e c h a n i c a l s h e a r .  Tissues  a r e d i s r u p t e d by b e i n g f o r c e d t h r o u g h a narrow space between a moving p e s t l e and t h e w a l l of the c o n t a i n i n g v e s s e l .  Using  t h i s method, s o f t t i s s u e s a r e r e a d i l y homogenized w i t h o u t c o n c o m i t a n t d i s r u p t i o n .of c e l l u l a r o r g a n e l l e s .  I n t h e homogenate,  the plasma membranes a r e u s u a l l y p r e s e n t i n the form of s h e e t s r a t h e r than, v e s i c l e s , but spontaneous w i t h time.  v e s i c l e formation occurs  C o m m e r c i a l l y a v a i l a b l e homogenizers of t h i s  i n c l u d e t h e P o t t e r - E l v e h j e m and the Dounce The t h i r d method of h o m o g e n i z a t i o n  type  homogenizers. i s gaseous s h e a r .  N i t r o g e n c a v i t a t i o n u s i n g a P a r r bomb i n v o l v e s e q u i l i b r a t i n g a s t i r r e d c e l l suspension w i t h oxygen-free n i t r o g e n at pressures 2 between 500 and 800 l b . / i n . COMMERFORD, 1961).  f o r p e r i o d s of 15-20 m i n u t e s  (HUNTER &  C e l l d i s r u p t i o n o c c u r s upon sudden r e l e a s e of  the p r e s s u r e d u e t o the gas expanding w i t h i n the c e l l o r by 1  the  s h e a r i n g f o r c e s of the r a p i d l y f o r m i n g b u b b l e s of gas i n the l i q u i d phase.  Gaseous s h e a r , however, r e q u i r e s t h a t the t i s s u e  be i n the form o f i s o l a t e d s i n g l e c e l l s .  The p r e p a r a t i o n of s i n g l e  c e l l s i t s e l f i s a s s o c i a t e d w i t h drawbacks i n c l u d i n g a l t e r a t i o n of the plasma membranes by p r o t e a s e s found i n commercial c o l l a g e n a s e p r e p a r a t i o n s (R0DBELL, 1964; BAGBY e t a l . ,  1971; FAY  & DELISE,  1973;  6  - 6 -  RODBELL & KRISHNA, 1974;  SMALL, 197.7).  The  plasma membranes, (PMs) ,  endoplasmic r e t i c u l u m and n u c l e a r membranes form v e r y v e s i c l e s , w h i c h makes f u r t h e r d e n s i t y g r a d i e n t necessary.  small  separations  Other c e l l o r g a n e l l e s a r e g e n e r a l l y m a i n t a i n e d  Osmotic l y s i s  (BARBER & JAMIESON, 1973)  intact.  used i n f r e q u e n t l y ,  has been a p p l i e d t o the i s o l a t i o n of membranes from s k e l e t a l m u s c l e (McCOLLESTER, 1962).  I n t h i s method,, segments are i n c u b a t e d  h i g h temperatures and  then excess d i s t i l l e d water i s added.  at  T h i s l e a d s to an a b r u p t d i s s o l u t i o n of i n t r a c e l l u l a r components, l e a v i n g o n l y m u s c l e plasma membranes.  A t . t h e same t i m e , however,  p a r t i a l s o l u b i l i z a t i o n of p e r i p h e r a l PM p r o t e i n s o c c u r s accompanied by an i n c r e a s e i n membrane p e r m e a b i l i t y . W i t h few provides  exceptions,  none of the shear t e c h n i q u e s  p e r f e c t h o m o g e n i z a t i o n of the s t a r t i n g m a t e r i a l .  I t i s o f t e n n e c e s s a r y , t h e r e f o r e , to remove the unhomogenized fragments by f i l t r a t i o n .  The  f i l t r a t e contains  i n a d d i t i o n to  plasma membranes, i n t a c t and d i s r u p t e d o r g a n e l l e s as w e l l as soluble and.insoluble  components of t h e c y t o p l a s m .  e a r l i e r , the f i l t r a t e . i s  then f r a c t i o n a t e d by  As mentioned  differential  c e n t r i f u g a t i o n , a t e c h n i q u e based on d i f f e r e n c e s i n buoyant d e n s i t i e s of the v a r i o u s c e l l components.  For example, n u c l e i  sediment r a p i d l y i n a g r a v i t a t i o n a l f i e l d of 2000 g. c e l l d e b r i s , n u c l e a r membranes, i n t a c t m i t o c h o n d r i a  After and  the  other  o r g a n e l l e s have been removed by c e n t r i f u g a t i o n s a t l o w e r g f o r c e s (2000 g - 17,000 g ) , the m i c r o s o m a l f r a c t i o n c o n t a i n i n g PM  i s u s u a l l y p e l l e t e d by c e n t r i f u g a t i o n a t 100,000 g (GRAHAM,  1975;  7  - 7 -  SCHIMMEL & KENT, 1977; WALLACH & SCHMIDT-ULLRICH, 1977). Many i n v e s t i g a t o r s c o n s i d e r t h e 100,000 g p e l l e t  sufficiently  e n r i c h e d i n plasma membranes t o w a r r a n t i t s use i n v a r i o u s s t u d i e s . However, i t has c l e a r l y been demonstrated t h a t t h i s e n r i c h e d 100,000 g plasma membrane f r a c t i o n s t i l l c o n t a i n s c o n t a m i n a t i o n from fragmented o r g a n e l l e s . of v e s i c l e s , may  These f r a g m e n t s , o f t e n i n the form  s e r i o u s l y b i a s any r e s u l t s observed i n t h i s  membrane f r a c t i o n .  plasma  The demonstrated presence o f c o n t a m i n a t i o n  w a r r a n t s a t h i r d s t a g e of membrane i s o l a t i o n such as t h e use of density gradient centrifugation.  T h i s i n v o l v e s c e n t r i f u g a t i o n of  the m i c r o s o m a l f r a c t i o n on a s u c r o s e d e n s i t y g r a d i e n t a t h i g h g f o r c e s (120,000 g) f o r l o n g p e r i o d s of t i m e (2-24 h o u r s ) w h i c h a l l o w s the i n d i v i d u a l components of the m i c r o s o m a l f r a c t i o n t o r e a c h t h e i r own buoyant d e n s i t i e s .  The g r a d i e n t used must span  the f u l l range of d e n s i t i e s e x h i b i t e d by the v a r i o u s membrane fragments p r e s e n t .  The g r a d i e n t m a t e r i a l must be water  soluble,  w h i c h r e q u i r e m e n t i s n o t met by e i t h e r d e x t r a n o r F i c o l l . S e p a r a t i o n s t h a t do-use the l a t t e r r e l y on d i f f e r e n c e s i n q u a n t i t a t i v e r i s e s i n buoyant d e n s i t y when f i x e d charges a r e 2+ n e u t r a l i z e d by Mg  (STECK, 1974a; GRAHAM,, 1975) but i n g e n e r a l ,  s u c r o s e g r a d i e n t s a r e more commonly used.  The plasma membranes  f r a c t i o n d e r i v e d from the s u c r o s e g r a d i e n t i s q u i t e o f t e n subs t a n t i a l l y f r e e from c o n t a m i n a t i o n by i n t r a c e l l u l a r membranes, though n o t n e c e s s a r i l y 100% pure. important questions.  T h i s r a i s e s a number of  Can o r s h o u l d . t h e membranes be f u r t h e r  p u r i f i e d and how does one a s s e s s the i n c r e a s e s i n plasma membranes n o t o n l y i n a f o u r t h s t a g e o f membrane i s o l a t i o n but i n each stage?  8  - 8 -  F u r t h e r p u r i f i c a t i o n o f t h e plasma membranes has i n v o l v e d one b a s i c approach. of  That i s , by s e l e c t i v e l y  altering  the density  t h e PM i n s t a g e 1 o r s t a g e 3, b e t t e r s e p a r a t i o n can be e f f e c t e d  based on l a r g e r d i f f e r e n c e s i n the buoyant d e n s i t e s o f plasma membranes as compared  t o i n t r a c e l l u l a r membranes.  T h i s has been  a c h i e v e d by a t t a c h i n g l e c t i n s t o plasma membrane c a r b o h y d r a t e m o i e t i e s (CUATRECASES, 1973; NICHOLSON, 1974; SHARON & L I S , 1975; HYNES, 1976; BARCHI e t a l . , 1977; WALLACH & SCHMIDT-ULLRICH, 1977). the  The l a b e l l i n g of t h e PM w i t h p l a s t i c m i c r o s p h e r e s a f f o r d s  same advantage (LIM e t a l . , 1975).  the m i c r o s o m a l p e l l e t p r i o r  D i g i t o n i n , incubated w i t h  t o g r a d i e n t c e n t r i f u g a t i o n , has been  found t o i n c r e a s e t h e d e n s i t y o f PM over o t h e r  intracellular  membranes (LEWIS e t a l . , 1975; MAGARGAL e t a l . , 1978).  Not o n l y  i s i t p o s s i b l e t o a l t e r t h e d e n s i t y o f t h e plasma membrane, b u t a l s o t h a t o f t h e c o n t a m i n a t i n g components.  A good example o f  t h i s i s seen i n t h e s e p a r a t i o n o f plasma membranes from s a r c o p l a s m i c r e t i c u l u m i n h e a r t muscle  (LEVITSKY e t a l . , 1976).  The SR was a l l o w e d t o accumulate Ca i n t h e p r e s e n c e o f ATP and o x a l a t e and t h e SR then removed by g r a d i e n t c e n t r i f u g a t i o n . The v a l i d i t y o f t h e whole p r o c e s s o f membrane i s o l a t i o n r e s t s on t h e a b i l i t y t o a s s e s s t h e r e s u l t s degree o f plasma membrane p u r i f i c a t i o n ways.  o f each s t e p used.  The  c a n be m o n i t o r e d i n two  Removal of t h e c o n t a m i n a n t s can be a s s e s s e d by  selectively  measuring a f e a t u r e such as an e n z y m a t i c a c t i v i t y o f t h e r e s p e c t i v e o r g a n e l l e i n t h e s u p e r n a t a n t and p e l l e t . e n z y m a t i c markers a r e :  A few examples o f  m i t o c h o n d r i a (BONNER, 1955; DONALDSON  et a l . , 1972; TOLBERT, 1974):  s u c c i n a t e dehydrogenase, cytochrome  c^ o x i d a s e , fumarase and NADH c y t ^ r e d u c t a s e (rotenone s e n s i t i v e ) ;  9  - 9 -  GOLGI APPARATUS (FLEISCHER  & KERVINA, 1974):  galactosyltransferase;  LYSOZOMES (HUBSCHER & NEST, 1965; HODGES & LEONARD, 1974):  acid  phosphatase; ENDOPLASMIC RETICULUM (NORDLIE & ARION, 1966; TOLBERT, 1974):  g l u c o s e - 6 - p h o s p h a t a s e , NADPH c y t £ r e d u c t a s e and  protein synthesis.  Some o f t h e above markers a r e found w i t h a  n o n - s p e c i f i c d i s t r i b u t i o n i n o t h e r o r g a n e l l e s , and t h e r e f o r e have l i m i t e d v a l u e as markers.  The above assessment may be supplemented  by s e m i - q u a n t i t a t i v e e x a m i n a t i o n o f t h e f r a c t i o n under t h e e l e c t r o n microscope. The g r a d u a l enrichment o f plasma membranes can be f o l l o w e d by markers s p e c i f i c f o r t h e plasma membrane (WALLACH & WINZLER, 1974).  Such markers i n c l u d e endogenous c h e m i c a l markers  ( c h o l e s t e r o l / p h o s p h o l i p i d r a t i o s ) , enzyme markers ( 5 ' n u c l e o t i d a s e , a d e n y l a t e c y c l a s e , ATPases)  (RODBELL & KRISHNA, 1974; WIDNELL,  1974), v i r u s r e c e p t o r s , c o v a l e n t l a b e l s and i m m u n o l o g i c a l markers. sites.  C l o s e l y r e l a t e d t o t h e l a s t marker a r e l e c t i n  binding  L e c t i n s a r e thought t o b i n d t o c e l l plasma membrane  s u r f a c e s r e a c t i n g w i t h t e r m i n a l non r e d u c i n g sugars i n g l y c o p r o t e i n s and/or l i p i d s .  I n h e r e n t i n t h i s work i s t h e  a s s u m p t i o n t h a t t h e l a b e l does n o t permeate the plasma membrane (SCHIMMEL & KENT, 1977; WALLACH & SCHMIDT-ULLRICH, 1977). Once i t has been e s t a b l i s h e d t h a t t h e plasma membrane p r e p a r a t i o n i s o f a c c e p t a b l e p u r i t y , one must c o n s i d e r t h e s t a t e o f t h e plasma membranes t h e m s e l v e s .  I t i s known t h a t t h e  plasma membranes can be i n t h e form o f membrane s h e e t s o r vesicles.  A l s o , t h e v e s i c l e s , may be r i g h t - s i d e - o u t (RO) o r  10  - 10 -  i n s i d e - o u t (10).  Therefore, before using the preparation to  i n v e s t i g a t e plasma membrane p r o p e r t i e s we must know t h e o r i e n t a t i o n o f t h e membrane.  As w e l l , i t may be p o s s i b l e t o  i n v o k e a f i f t h stage o f membrane i s o l a t i o n t o s e p a r a t e out t h e u n s e a l e d v e s i c l e s , RO v e s i c l e s and 10 v e s i c l e s . one  T h i s would a l l o w  t o examine s p e c i f i c phenomena a s s o c i a t e d w i t h t h e c y t o p l a s m i c  membrane s u r f a c e and/or t h e e x t e r n a l membrane s u r f a c e . B.  The O r i e n t a t i o n Problem Each b i o l o g i c a l membrane o p e r a t e s  two compartments i t s e p a r a t e s .  d i f f e r e n t l y on t h e  Being a n i s o t r o p h i c i n i t s f u n c t i o n ,  the plasma membranes has been shown t o be a s s y m e t r i c w i t h r e s p e c t to  the composition  o f t h e two s u r f a c e s (STECK, 1974b).  I d e n t i f i c a t i o n of t h e components a t each s u r f a c e would do much t o d e f i n e t h e s t r u c t u r e . I n v e s t i g a t o r s have approached t h e problem o f o r i e n t a t i o n from two d i r e c t i o n s . (STECK, 1974a; STECK & KANT, 1974). one  i n v o l v e s t h e assessment o f membrane s i d e d n e s s  The i n i t i a l  u s i n g marker  enzymes s p e c i f i c f o r e i t h e r t h e e x t e r n a l o r c y t o p l a s m i c  surface.  D e t e r g e n t s can be used t o make b o t h membrane s u r f a c e s e q u a l l y a c c e s s i b l e t o t h e enzyme s u b s t r a t e s used. e x t r a c t i o n also occurs during detergent SIMONS, 1975; TANFORD & REYNOLDS, 1976).  Selective protein  treatment  (HELENIUS &  T h i s must be a p p r e c i a t e d .  R e s u l t s observed u s i n g p r e p a r a t i o n s c o n t a i n i n g a m i x t u r e o f r i g h t - s i d e - o u t , i n s i d e - o u t and u n s e a l e d  plasma, membrane v e s i c l e s  a r e thought t o r e f l e c t t h e g e n e r a l o r i e n t a t i o n o f t h e membranes. The  second approach t o e l u c i d a t i o n o f o r i e n t a t i o n i n v o l v e s  assessment as above, combined w i t h a f i f t h stage o f membrane  11  - 11 -  f r a c t i o n a t i o n i n w h i c h a t t e m p t s a r e made.to s e p a r a t e  the p u r i f i e d  plasma membrane p r e p a r a t i o n f u r t h e r on t h e b a s i s of membrane orientation.  I n general there are three techniques  a f i f t h stage of i s o l a t i o n .  available for  These a r e the use h i g h polymer  g r a d i e n t s (STECK, 1974a) combined w i t h aqueous p a r t i t i o n / c o u n t e r c u r r e n t d i s t r i b u t i o n (AP./CCD) (DODGE e t a l . , 1963; ALBERTSSON, 1970;  WALTER & KROB, 1976; WALTER, 1978), f r e e f l o w e l e c t r o p h o r e s i s  (FFE)  (HANNIG & HEIDRICH, 1974; HANNIG, 1975a; HANNIG, 1975b)  and a f f i n i t y chromatography (MURTHY & HERCZ, 1973; PHARMACIA, 1974;  HYNES, 1976; WALSH e t a l . ,  1976; BRUNNER e t a l . ,  1977).  E'ach has been a p p l i e d w i t h v a r y i n g degrees o f s u c c e s s . H i g h d e n s i t y F i c o l l polymer g r a d i e n t s combined w i t h AP/CCD have p r o v e n i d e a l f o r s e p a r a t i n g membranes o f d i f f e r i n g o r i e n t a t i o n from c e r t a i n t i s s u e s (STECK, 1974a). are f i r s t separated gradients.  from u n s e a l e d  Sealed v e s i c l e s  ones u s i n g F i c o l l d e n s i t y  The s e p a r a t i o n depends on t h e f a c t t h a t s e a l e d  v e s i c l e s a r e n o t c o l l a p s e d as on s u c r o s e g r a d i e n t s , b u t i n s t e a d they expand.  They t h e r e f o r e have buoyant d e n s i t i e s d i f f e r e n t  from u n s e a l e d v e s i c l e s .  I t i s t h e low s o l u b i l i t y and p e r m e a b i l i t y  of F i c o l l w h i c h p e r m i t s  this.  G l y c e r o l , f o r example, i s n o t  a c c e p t a b l e s i n c e i t r e a d i l y permeates s e a l e d v e s i c l e s . s e a l e d membranes have been s e p a r a t e d AP i s used t o s e p a r a t e  Once t h e  from t h e s e a l e d v e s i c l e s ,  t h e two p o p u l a t i o n s o f v e s i c l e s r e m a i n i n g .  Aqueous p a r t i t i o n s e p a r a t i o n s r e l y on t h e e x p l o i t a t i o n o f subtile., physicochemical surfaces.  d i f f e r e n c e s between the two d i f f e r e n t membrane  These s u r f a c e p r o p e r t i e s a r e h i g h l y dependent on  i o n i c c o n d i t i o n s and pH. by m o d i f y i n g  One c a n f u r t h e r e f f e c t  t h e polymers used i n t h e p a r t i t i o n .  separations U t i l i z a t i o n of  12  - 12 -  polymer l i g a n d s , such as d e r i v a t i v e s o f d e x t r a n s  and l e c t i n s ,  s p e c i f i c f o r membrane r e c e p t o r s shows g r e a t promise. The most w e l l known a p p l i c a t i o n o f t h e above i s t h a t of Steck (rbcg).  technique  (STECK, 1974a) u s i n g r e d b l o o d c e l l g h o s t s  I n t h e i n i t i a l i n v e s t i g a t i o n he l o c a l i z e d marker enzymes  on b o t h membrane s u r f a c e s .  L o c a l i z e d on t h e  cytoplasmic  s u r f a c e s o f r b c were Na+/K+ ATPase, g y l c e r a l d e h y d e - 3 - p h o s p h a t e dehydrogenase, a d e n y l a t e cyt  <: r e d u c t a s e .  c y c l a s e , p r o t e i n k i n a s e and NADH  E x t e r n a l l y l o c a l i z e d were a c e t y l c h o l i n e s t e r a s e ,  s i a l i c a c i d r e s i d u e s and the o u a b a i n b i n d i n g s i t e o f Na+/K+ ATPase.  These markers were used t o check on t h e i s o l a t i o n  o f v e s i c l e s w i t h d i f f e r e n t o r i e n t a t i o n s . Unsealed g h o s t s were removed from RO and 10 v e s i c l e s by F i c o l l  gradients.  A d e x t r a n T110 and g l y c o l 6000 p a r t i t i o n was then used t o s e p a r a t e the RO v e s i c l e s from t h e 1 0 v e s i c l e s ; w i t h s e p a r a t i o n o c c u r r i n g o n l y under c e r t a i n i o n i c c o n d i t i o n s and t e m p e r a t u r e s .  Interestingly  i t was observed t h a t h i g h i o n i c s t r e n g t h l e a d t o a c c u m u l a t i o n of a l l the v e s i c l e s a t t h e i n t e r f a c e .  H a l i d e i o n drove t h e  v e s i c l e s i n t o t h e lower phase w h i l e phosphate r e v e r s e d  this  trend. Under i n v e s t i g a t i o n as a t e c h n i q u e of R O . v e s i c l e s HEIDRICH, 1974;  for the separation  i s f r e e f l o w e l e c t r o p h o r e s i s (FFE) HANNIG, 1975a; HANNIG, 1975b).  (HANNIG &  T h i s method  e x p l o i t s d i f f e r e n c e s i n s u r f a c e charge, d e n s i t y and s i z e between R0 and 10 v e s i c l e s .  The m i x t u r e  o f plasma membrane  v e s i c l e s i s i n j e c t e d i n t o a c o n t i n u o u s l y f l o w i n g b u f f e r w i t h an a p p l i e d e l e c t r i c f i e l d a t r i g h t angle to the f l o w d i r e c t i o n .  13  - 13 -  The v e s i c l e s s e p a r a t e a c c o r d i n g t o e l e c t r o p h o r e t i c m o b i l i t y . d u r i n g flow.  Problems a r i s e s i n c e t h e r m a l l y u n d i s t u r b e d  f l o w of the  l i q u i d c u r t a i n can o n l y be o b t a i n e d a t h i g h f l o w v e l o c i t i e s w h i c h may r e s u l t i n t u r b u l e n c e .  S u f f i c i e n t d e f l e c t i o n only  o c c u r s w i t h l o n g e r and s m a l l e r chambers and a t h i g h strengths.  field  The l a t t e r i s r u l e d o u t as t h e heat g e n e r a t e d  i n c r e a s e s w i t h t h e f i e l d s t r e n g t h squared b u t by employing v e r y low i o n i c s t r e n g t h media f o r the s e p a r a t i o n b u f f e r t h e h e a t problem can be a v o i d e d .  As w e l l , i t s h o u l d be noted t h a t h i g h e r  l i q u i d c u r t a i n v e l o c i t i e s f u r t h e r reduce t h e h e a t problem, t h e p r o b a b i l i t y s t i l l e x i s t s t h a t t u r b u l e n c e may The  occur.  l i m i t i n g f a c t o r a t low i o n i c s t r e n g t h s appears t o  be t h e i n s t a b i l i t y o f t h e v e s i c l e s (STECK, 1974a). e f f e c t s can o c c u r w i t h c o n t a m i n a t i o n  Further  from DNA, RNA and c e l l  n u c l e i w h i c h b i n d t o the membrane s u r f a c e s s c r e e n i n g  charge.  To d a t e , RO arid 10 r b c v e s i c l e s , v i r u s e s , b a c t e r i a , p r o t e i n s and n u c l e i a c i d s have been  separated.  The l a s t major method of c u r r e n t p r a c t i c a l u s e i n t h e s e p a r a t i o n of p r e f e r e n t i a l l y o r i e n t e d v e s i c l e s i s a f f i n i t y chromatography and r e l a t e d t e c h n i q u e s .  L e c t i n s , such a s , WGA,  Con A, RGA, a r e c o v a l e n t l y l i n k e d t o s o l i d s u p p o r t s agarose, sepharose o r n y l o n f i b r e s .  like  dextran,  RO v e s i c l e s and u n s e a l e d  v e s i c l e s a r e thought t o b i n d t o t h e c o v a l e n t l y l i n k e d l e c t i n by s p e c i f i c s u g a r m o i t i e s l o c a t e d on t h e e x t e r n a l plasma membrane s u r f a c e w h i l e 10 v e s i c l e s a r e n o t absorbed and pass t h r o u g h the column.  The bound membranes a r e then e l u t e d by adding a sugar  w h i c h competes w i t h t h e membranes f o r t h e l e c t i n b i n d i n g  sites.  14  - 14 -  As mentioned e a r l i e r a v a r i a t i o n on t h i s theme i n c l u d e s adding l e c t i n to a m i x t u r e of RO and .10 v e s i c l e s f o l l o w e d by  density  g r a d i e n t c e n t r i f u g a t i o n to s e p a r a t e the h i g h e r d e n s i t y  RO  v e s i c l e s from the 10 ones (CUATRESAS, 1973). There have been two m a j o r s t u d i e s u s i n g Con A a f f i n i t y chromatography t o s e p a r a t e p o p u l a t i o n s but w i t h c o n f l i c t i n g r e s u l t s . Walsh (WALSH e t a l . . , 1976)  of 10 and RO v e s i c l e s ,  U s i n g p o r c i n e lymphocyte homogenates  i s o l a t e d what appeared to be  10  lymphocyte plasma membranes as judged by marker s t u d i e s , immunoprecipitation,  and f e r r i t i n l i n k e d Con A e x p e r i m e n t s .  40% of the p r o t e i n a p p l i e d to the column was However, 50% c o u l d not be e l u t e d under any to be due The  recovered.  c o n d i t i o n s and  to h i g h a f f i n i t y n o n - s p e c i f i c m u l t i v a l e n t  appeared  binding.  s t u d y a l s o r e v e a l e d a number of drawbacks t o t h i s type of  membrane p u r i f i c a t i o n .  L e c t i n s i n h i b i t c e r t a i n enzymes and  a l s o cause capping of s u r f a c e markers. b u f f e r s may  The  low o s m o l a r i t y of  the  l e a d t o an i n c r e a s e i n membrane p e r m e a b i l i t y .  I n the second study (BRUNNER e t a l . , 1977), anywhere from 60 t o . 7 0 % of the a p p l i e d membranes remained bound t o l e c t i n l i n k e d sepharose beads.  the  The membranes, however, c o u l d  be e l u t e d i n the p r e s e n c e of a methyl-D-mannoside accompanied by m e c h a n i c a l s t i r r i n g of the Con A l i n k e d sepharose beads. Whether the e l u t e d p r o t e i n was  Con A or membranes i s s u b j e c t  c o n j e c t u r e as no e l e c t r o p h o r e t i c g e l s were r u n and marker a s s a y s were done. beads may  no enzyme  As w e l l , m e c h a n i c a l s t i r r i n g of  have been a s s o c i a t e d w i t h  to  the  fragmentation.  15  - 15 -  The above t e c h n i q u e has t h e advantages o f b e i n g i n e x p e n s i v e and easy t o c a r r y out.  relatively  However, t h e f a c t t h a t 50%  of the p r o t e i n i s o f t e n n o t r e c o v e r a b l e does n o t auger w e l l f o r the i s o l a t i o n o f a RO s e t o f membranes. All  t h e p r e v i o u s methods o u t l i n e d i n v o l v e t h e u s e o f a  membrane p r e p a r a t i o n t h a t has a v a r i a b l e r a t i o o f RO:10:unsealed membrane v e s i c l e s w h i c h may be dependent upon t h e homogenzation method used.  The s e p a r a t i o n p r o c e d u r e s c o u l d be e l i m i n a t e d i f i t  became p o s s i b l e t o c o n t r o l the membrane o r i e n t a t i o n d u r i n g p r e p a r a t i o n and p r e d i c t membrane o r i e n t a t i o n based on t h e method o f p r e p a r a t i o n . unanswered.  To date b o t h q u e s t i o n s remain b a s i c a l l y  Only S t e c k (STECK, 1974a; STECK & KANT, 1974) has  s u c c e s s f u l l y prepared  10 and RO v e s i c l e s from r b c g h o s t s , by  varying the i o n i c m i l i e u .  W h i l e i t i s s t i l l n o t c l e a r how t h e  i o n i c m i l i e u determines v e s i c l e s i d e d n e s s , one m i g h t s p e c u l a t e t h a t the l a b e l l i n g o f plasma membranes w i t h l e c t i n s p r i o r t o h o m o g e n i z a t i o n may a l t e r t h e R0:I0 membrane v e s i c l e  ratio.  D e s p i t e t h e many methods a v a i l a b l e and t h e many t h e o r i e s on how t o i n c r e a s e y i e l d s o f p r e f e r e n t i a l l y o r i e n t e d v e s i c l e s , t h e attainment  of t h i s g o a l i s s t i l l q u i t e f a r away.  The 10 and RO  plasma membranes i s o l a t e d from t h e r b c membrane were the r e s u l t of f o r t u n a t e o b s e r v a t i o n and r i g o r o u s c h a r a c t e r i z a t i o n . I t i s o n l y through the l a t t e r t h a t we can b e g i n t o u n d e r s t a n d w h i c h f a c t o r s a r e r e s p o n s i b l e f o r the o r i e n t a t i o n s seen i n plasma membrane p r e p a r a t i o n s .  16  - 16 -  There a r e many plasma membrane p r e p a r a t i o n s from v a r i o u s t i s s u e s w h i c h s u f f e r from problems o f ' c o n t a m i n a t i o n and membrane o r i e n t a t i o n .  indefinite  These p r e p a r a t i o n s o f t e n have f a i l e d t o  use more than 2 s t a g e s i n the membrane i s o l a t i o n w h i l e o t h e r s . h a v e f a i l e d t o c a r r y out p r o p e r c h a r a c t e r i z a t i o n of the membrane preparation.  These d e f i c i e n c i e s a r e no more a p p a r e n t than i n the  c a s e of smooth muscle though more r e c e n t l y some good  studies  have appeared.  C.  Smooth M u s c l e P r e p a r a t i o n s As shown i n T a b l e I , plasma membrane f r a c t i o n s d e r i v e d  from smooth muscle have been p r e p a r e d by l i q u i d and m e c h a n i c a l shear t e c h n i q u e s . of  Many of the i n v e s t i g a t i o n s l a c k p r o p e r e v a l u a t i o n  the p u r i t y of t h e i r plasma membrane p r e p a r a t i o n s .  Most  studies  i n t h e p a s t s i m p l y used d i f f e r e n t i a l c e n t r i f u g a t i o n to o b t a i n a 100,000 g m i c r o s o m a l plasma membrane.preparation (PREISS & BANASCHAK, 1975; ZELCH e t a l . , 1975; GODFRAIND e t a l . , 1976; RANGACHARI e t a l . , 1976; WEBB & BHALLA, 1976; NISHIKORI e t a l . , 1977; BHALLA e t a l . , 1978a, 1978b).  Some have r e p l a c e d the  100,000 g p e l l e t by a 40,000 g p e l l e t (SHIBATA & HOLLANDER, 1974; FITZPATRICK & SVENTIVANYI, 1977).  More r e c e n t s t u d i e s  combine  d i f f e r e n t i a l c e n t r i f u g a t i o n w i t h simple four step gradients (WEI e t a l . , 1976a, 1976b, 1976c; JANIS e t a l . , . 1977; AKERMAN & WIKSTROM, 1978; VALLIERES e t a l . , 1978).  The smooth muscle used  was o b t a i n e d from a r t e r i e s , the u t e r u s and t h e i l e u m . The s i m p l e s t of a l l plasma membrane p r e p a r a t i o n s would be to use the crude homogenate. T h i s k i n d of approach s e r v e s no  real  17  - 17  -  purpose under t h e s e c o n d i t i o n s because of the c o n t a m i n a t i o n o r g a n e l l e s and problems w i t h v e s i c l e o r i e n t a t i o n . of membrane ATPase a c t i v i t i e s i s c o n t r a d i c t e d due of actinomyosin  ATPase.  by  Measurement t o the p r e s e n c e  Furthermore the l y s o z o m a l  enzymes w i l l  e v e n t u a l l y degrade a l l membrane systems. M i c r o s o m a l p r e p a r a t i o n s of smooth muscle have i n v o l v e d the use of a 4,000 g or 100,000 g p e l l e t . were done u s i n g v a s c u l a r smooth muscle. 2+ these s t u d i e s was  Ca  Many of these s t u d i e s The main emphasis of  t r a n s p o r t and measurement of ATPase  a c t i v i t i e s a s s o c i a t e d w i t h the membranes.  The  characterization  of the membranes'varied from the c o m b i n a t i o n ' o f e l e c t r o n m i c r o s c o p y w i t h one marker such as s u c c i n a t e dehydrogenase (ZELCK e t a l . , 1975; 1977;  CYLMAN e t a l . ,  1976;  NISHIKORI e t a l . ,  KRALL e t a l . ,  1978)  to more e x h a u s t i v e marker s t u d i e s  (CHATURVEDI e t a l . ,  1978;  KUTSKY & GOODMAN, 1978;  HAUESLER, 1978; The  MATLIB et a l . , 1979;  THORENS &  THORENS, 1979)  - See T a b l e 1.  l i m i t e d use of markers i n a m i c r o s o m a l p r e p a r a t i o n i s  unacceptable.  To measure Ca^uptake or r e l e a s e i n a membrane  p r e p a r a t i o n w i t h o u t knowing t h e v a r i o u s p o s s i b l e types of membranes p r e s e n t  i s unadvisable.  A good example of t h i s i s  i n 100,000 g m i c r o s o m a l p r e p a r a t i o n s c o n s i d e r e d sarcoplasmic 1976;  by many to be  r e t i c u l u m (SHIBATA & HOLLANDER, 1974;  FITZPATRICK & SZENTIVANYI, 1977;  This hypothesis  WEBB & BHALLA,  BHALLA e t a l . ,  1978a, 1978b).  has been advanced on t h e b a s i s of the marker  enzyme NADH c y t c^ r e d u c t a s e .  While mitochondria  are excluded  by  e l e c t r o n m i c r o s c o p y s t u d i e s the p r o b a b i l i t y t h a t plasma membranes are present  i s acknowledged but r a r e l y checked by u s i n g  standard  marker enzymes and no a t t e m p t s have been made t o determine the  18  - 18 -  o r i e n t a t i o n and/or p e r m e a b i l i t y of the membrane, t h i s  latter  p o i n t b e i n g c r u c i a l i n the o b s e r v a t i o n of e f f l u x or i n f l u x of various ions.  The  use of NADH c y t c_ r e d u c t a s e as a  r e t i c u l u m marker i s now shown to be p r e s e n t  suspect as t h i s a c t i v i t y has a l s o been  i n the plasma membranes and o t h e r  o r g a n e l l e s (SOTTOCASA, 1967; 1979).  sarcoplasmic  SOTTOCASA, 1971;  E l e c t r o n microscopy i s at best only  cell  KELBERG & CHRISTENSEN, semi-quantitative.  A l s o , t h e p r e s e n c e of plasma membranes i n the 100,000 g sarcoplasmic  r e t i c u l u m p e l l e t has been u n e q u i v o c a l l y  (HURWITZ e t a l . , 1973; WUYTACK e t a l . , 1978;  MOORE e t a l . , 1975; MATLIB et a l . , 1979;  VALLIERES e t a l . , THORENS, 1979).  has been shown t h a t t h i s p e l l e t c o n t a i n s s u f f i c i e n t 2+ and plasma membranes t o e f f e c t Ca  demonstrated 1978; It  mitochondrial  a c c u m u l a t i o n observed i n the  100,000 g p e l l e t s . Another drawback to s t u d i e s u s i n g 100,000 g p e l l e t s sarcoplasmic  r e t i c u l u m or plasma membranes p r e p a r a t i o n s 2+  has  i s seen  i n s t u d i e s comparing Ca  uptake i n microsomes p r e p a r e d from  the a o r t a s of h y p e r t e n s i v e  and n o r m o t e n s i v e r a t e s (BHALLA e t a l . ,  I t i s thought t h a t c e r t a i n u l t r a s t r u c t u r a l changes occur i n smooth muscle c e l l s of a o r t a s i n h y p e r t e n s i v e  animals.  To t r e a t b o t h  a o r t a s under i d e n t i c a l p r e p a r a t i o n c o n d i t i o n s i s  questionable  because i t i s by no means c e r t a i n t h a t b o t h t i s s u e t y p e s w i l l behave i n the same manner.  F u r t h e r m o r e , i t i s d i f f i c u l t to a s s i g n 2+  much s i g n i f i c a n c e t o s t u d i e s of Ca o r i g i n , o r i e n t a t i o n and c o n t a m i n a t i o n  uptake by v e s i c l e s whose by o t h e r membranes i s not  known. Many i n v e s t i g a t o r s r e a l i z i n g the drawbacks of  the  m i c r o s o m a l p r e p a r a t i o n s have f u r t h e r p u r i f i e d the plasma membranes gradients 1978;  (KIDWAI, 1974;  MAGARGAL e t a l . , 1978;  MATLIB e t a l . , 1979;  THORENS, 1979).  WUYTACK e t a l . ,  Most s t u d i e s have 19  1978b).  - 19 -  utilized  v i s c e r a l smooth muscle.  S e v e r a l i n v e s t i g a t o r s have  c a r r i e d out e x t e n s i v e c h a r a c t e r i z a t i o n of the f i n a l  preparations.  A t y p i c a l example i s seen i n the p r e p a r a t i o n s of m y o m e t r i a l plasma membranes.  The s a r c o p l a s m i c  reticulum, p r i o r to  3  homogenization, was  loaded w i t h  to check the v i a b i l i t y  H-leucine  and t h i s was used  of NADH c y t _c r e d u c t a s e  r e t i c u l u m marker (MATLIB et a l . , 1979).  as a  sarcoplasmic  E l e c t r o n microscopy  can be made s e m i - q u a n t i t a t i v e by a t t a c h i n g WGA  L e c t i n to the  plasma membranes p r i o r to homogenization (VALLIERES e t a l . , 1978). I t was f u l l y a p p r e c i a t e d  t h a t t h i s may  change membrane d e n s i t i e s .  I n v e s i t g a t i o n s by v a r i o u s a u t h o r s for  to f i n d s u i t a b l e markers  smooth muscle c e l l components have met w i t h some  success  (MAGARGAL et a l . , 1978; VALLIERES et a l . , 1978; MATLIB et a l . , 1979).  By changing the d e n s i t y of plasma membranes on the  gradient using  d i g i t o n i n , i t has been shown t h a t o l e y l  CoA:  l y s o l e c i t h i n a c e t y l t r a n s f e r a s e i s a s p e c i f i c marker f o r t h e SR (MAGARGAL et a l . , 1978). I n t e r e s t i n g r e s u l t s have been obtained, u s i n g g r a d i e n t 2+ centrifugation. The f i r s t of these i s a Mg - stimulated ATPase thought to be l o c a t e d i n the plasma membrane f r a c t i o n s 2+ which may  be the Mg  100,000 g s a r c o p l a s m i c 1975; WEI  - s t i m u l a t e d ATPase s e e n . i n the so c a l l e d reticulum preparations  (MOORE e t a l . ,  et. a l . , 1976; JANIS et a l . , 1977; VALLEIRES et a l . , 2+  1978; MATLIB. e t a l . , 1979).  T h i s Mg  obscures any Na+/K+ ATPase a c t i v i t y  - s t i m u l a t e d ATPase  t h a t might be p r e s e n t  i n the  membrane p r e p a r a t i o n .  20  - 20 -  The second i n t e r e s t i n g point- i s t h a t t h e l o c a t i o n o f t h e plasma membranes on t h e g r a d i e n t s appears t o be v a r i a b l e .  In  some p r e p a r a t i o n s u s i n g the r a t a o r t a , t h e plasma membranes a r e found a t h i g h e r d e n s i t i e s i n t h e g r a d i e n t than a r e t h e s a r c o p l a s m i c r e t i c u l u m membranes.  This i s i n contrast to the  r e s u l t s o f o t h e r s t u d i e s i n w h i c h t h e plasma membranes a r e found a t lower d e n s i t i e s r e l a t i v e t o t h e s a r c o p l a s m i c r e t i c u l u m (HURWITZ e t a l . , 1973; MOORE e t a l . , 1975; WEI e t a l . , 1976b; THORENS & HAEUSLER, 1978).  1976a,  I t may be t h a t t h e plasma  membranes a r e b i n d i n g t o denser fragments o f o t h e r membranes. S t u d i e s u s i n g g r a d i e n t c e n t r i f u g a t i o n s t o compare plasma membranes from a r t e r i s of h y p e r t e n s i v e a n i m a l s non-hypertensive  t o those o f  a n i m a l s r a i s e s i m i l a r q u e s t i o n s to those mentioned  e a r l i e r i n t h e d i s c u s s i o n o f m i c r o s o m a l p r e p a r a t i o n s (MOORE e t a l . , 1975; WEI e t a l . , 1976c).  What i s n e c e s s a r y  i n these s t u d i e s  a r e marker and s i d e d n e s s assays o f each f r a c t i o n o b t a i n e d each s t e p i n the p r o c e d u r e . done (VALLIERES e t a l . ,  after  F o r t u n a t e l y , t h i s i s now b e i n g  1978; MATLIB e t a l . ,  1979).  I t would be v e r y improper t o assume t h a t the drawbacks mentioned above a p p l y u n i v e r s a l l y t o a l l plasma membrane preparations.  T h i s i s o b v i o u s l y i n c o r r e c t , s i n c e t h e r e have  been some n i c e s t u d i e s on membrane c h a r a c t e r i z a t i o n o f s k e l e t a l , c a r d i a c and even smooth m u s c l e .  I t i s apparent,  however, t h a t ,  u n t i l r e c e n t l y , t h e m a j o r i t y o f smooth muscle p r e p a r a t i o n s have been i n a d e q u a t e l y c h a r a c t e r i z e d .  21  - 21 -  D.  Rationale The b a s i c aim of the p r e s e n t study was t o p r e p a r e and  c h a r a c t e r i z e a w e l l d e f i n e d p r e p a r a t i o n o f plasma membranes from smooth m u s c l e .  Such a p r e p a r a t i o n r e q u i r e s a t i s s u e r i c h  i n smooth muscle c e l l s and r e l a t i v e l y f r e e from f a t as w e l l as c o n n e c t i v e t i s s u e .  The smooth muscle s h o u l d be r e a d i l y  a v a i l a b l e i n l a r g e q u a n t i t i e s as w e l l .  I d e a l l y s u i t e d f o r these  purposes i s t h e c h i c k e n stomach m u s c u l a r i s .  T h i s m u s c l e , based  on t h e r e s u l t s of v a r i o u s s t u d i e s (CALHOUN, 1954; McCLEOD e t a l . , 1964;  KING & McCLELLAND, 1975; SOBIESZEK.& SMALL, 1976) i s thought  to c o n s i s t p r i n c i p a l l y of v i s c e r a l smooth m u s c l e .  A certain  degree of c a u t i o n i s r e q u i r e d as s k e l e t a l muscle i s p r e s e n t i n neighbouring  r e g i o n s of t h e d i g e s t i v e t r a c t .  As much as  10 t o 15 grams of smooth muscle can be o b t a i n e d from one domestic c h i c k e n . The n e x t stage i n t h e p r e p a r a t i o n of t h e plasma membranes i s h o m o g e n i z a t i o n .  L i q u i d shear would r e q u i r e t h a t t h e  t i s s u e be d i s p e r s e d i n t o s i n g l e c e l l s p r i o r t o h o m o g e n i z a t i o n , a process  t h a t may e f f e c t t h e plasma membranes of t h e c e l l s .  The  b e s t approach a t f i r s t i s t o use m e c h a n i c a l shear of m u s c l e cubes by a P o l y t r o n homogenizer.  I t was borne i n mind t h a t  the c o n d i t i o n s used c o u l d be o p t i m i z e d t o g i v e maximal plasma membrane y i e l d s a t the v a r i o u s stages o f t h e p r o c e d u r e . The crude homogenate c o n t a i n s a mix of c e l l components, many o f w h i c h a r e fragmented.  Differential centrifugation  combined w i t h marker s t u d i e s , a l l o w s r e m o v a l o f much o f t h e contamination.  However, d i f f e r e n t i a l c e n t r i f u g a t i o n i n i t s e l f  22  - 22 -  would n o t y i e l d a s u f f i c i e n t l y p u r i f i e d plasma membrane p r e p a r a t i o n r e q u i r i n g a f o u r t h stage of i s o l a t i o n .  Sucrose  g r a d i e n t s a r e t h e e a s i e s t t o u s e and a l l o w f o r f i n e  separations  of c o n t a m i n a t i n g  organelles.  The g r a d i e n t s used would have t o  be s u f f i c i e n t l y l o n g and l a r g e t o a l l o w f o r t h i s s e p a r a t i o n . The  f i n a l plasma membrane p r e p a r a t i o n o b t a i n e d from t h e g r a d i e n t ,  once c h a r a c t e r i z e d f o r p u r i t y and s i d e d n e s s , was expected t o be b a s i c a l l y f r e e o f contaminants.  I t i s u n l i k e l y , however,  t h a t the v e s i c l e s would have u n i f o r m o r i e n t a t i o n o f t h e membrane. I f t h e membranes a r e i l l d e f i n e d w i t h r e g a r d s  to t h e i r o r i e n t a t i o n  i t would become n e c e s s a r y  t o f u r t h e r p u r i f y the p r e p a r a t i o n .  There wre t h r e e c h o i c e s .  Free flow e l e c t r o p h o r e s i s i s very  expensive,  the r e s o l u t i o n i s poor and t h e e x p e r i m e n t a l  are q u i t e v a r i a l b e .  conditions  AP/CCD i s dependent on many v a r i a b l e s and  t h e r e a r e d i f f i c u l t i e s encountered i n removing p a r t i t i o n i n g compounds from t h e membranes.  The e a s i e s t method t o u s e seemed  to be a f f i n i t y chromatography, and i f a s u i t a b l e l i g a n d c a n be found, the most e f f i c i e n t .  I t s o n l y drawback i s t h e danger o f  h i g h a f f i n i t y non s p e c i f i c b i n d i n g o f the plasma membranes. As a p o s s i b l e method o f f i r s t c h o i c e i t s h o u l d be used.  Failure  of t h i s t e c h n i q u e would mean r e s o r t i n g t o FFE o r AP/CCD. i s extremely  important  What  i s t h a t the membranes be c h a r a c t e r i z e d a t  each s t e p i n t h e p r o c e d u r e w i t h r e s p e c t t o p u r i t y and i f a t a l l p o s s i b l e w i t h respect to sidedness,  the l a t t e r being very  i n t h e l a t t e r stages o f t h e i s o l a t i o n p r o c e d u r e .  critical  I t was hoped  t h a t t h i s approach would make i t p o s s i b l e t o o b t a i n a s u i t a b l e plasma membrane p r e p a r a t i o n .  23  Table I Summary o f smooth muscle p r e p a r a t i o n s . 1. Source o f smooth muscle - a o r t a AUTHORS  METHODS USED  PREPARATION  CHARACTERIZATION  PETERS e t a l . , (1972)  Dounce homogenization Differential centrifugation Sucrose gradients  Single c e l l s Lysozomes  Cytochrome o x i d a s e 5' N u c l e o t i d a s e DNA Phosphatases Glycosidases N - acetyl glucosaminidase Napthylamidase C a t h e p s i n A-D Monamine o x i d a s e  HURWITZ e t a l . (1973)  Homogenization not i n d i c a t e d Differential centrifugation Sucrose gradients  Plasma membranes  Na /K+ ATPase Ca^+ uptake 5" N u c l e o t i d a s e NADH o x i d a s e  HESS & FORD (1974)  Homog e n i za t i o n no t . i n d i c a t ed Differential centrifugation  Microsomal  r.  Polytron homogenization Differential centrifugation Sucrose gradients  Plasma membranes Mitochondria  Suecinate?.^ dehydrogenase 5' N u c l e o t i d a s e NADH o x i d a s e  BHALLA e t a l . , P o l y t r o n (1978a, 1978b) homogenization FITZPATRICK & Differential SZENTTIVANYI centrifugation (1977) MOORE e t a l . , (1975) SHIBATA & HOLLANDER (1974) WEBB & BHALLA (1976)  Microsomes Plasma membranes Sarcoplasmic reticulum  Ca ATPase (N,~ i n s e n s i t i v e ) cAMP Cytochrome o x i d a s e Succinate dehydrogenase Phosphodiesterase I  KIDWAI (1974)  +  2  +  Ca uptake Cytochrome o x i d a s e Na /K+ ATPase 5' N u c l e o t i d a s e Mg 2+ ATPase +  2 +  - .24 Table I (cont'd) 1. a o r t a ( c o n t ' d ) . . . AUTHORS  METHODS USED  PREPARATION  CHARACTERIZATION  WEI e t a l . , (1976a)  Polytron homogenization Differential centrifugation Sucrose gradients  Plasma membranes Mitochondria Endoplasmic reticulum  Phosphodiesterase I 5* N u c l e o t i d a s e Cytochrome c r e d u c t a s e K+ phosphatase (ouabain s e n s i t i v e ) ATPases Ca^+ a c c u m u l a t i o n E l e c t r o n microscopy  CHATURVEDI e t a l . , (1978) THORENS & HAEUSLERQ978)  P o t t e r Elvehjem homogenization Differential centrifugation Sucrose gradients  Microsomes Plasma membranes  Na+/K+ ATPase 5' N u c l e o t i d a s e Glucose-6-phosphatase NADH o x i d a s e Cytochrome o x i d a s e Ca2+ uptake  KUTSKY & GOODMAN (1978)  Polytron homogenization Differential centrifugation  Microsomes  5'Nucleotidase Mg ATPase Succinate-cytochrome c_ r e d u c t a s e NADPH c y t c r e d u c t a s e Ca2+ uptake  MAGARGAL e t a l ., V o r t e x , Dounce (1978) homogenization Differential centrifugation Sucrose gradients Digitonin treatment  WEI e t a l . , (1976b,1976c) THORENS (1979) Mesenteric a r t e r y used.  CLYMAN e t a l . , (1976) Umbilical a r t e r y used.  2 +  Plasma membranes  NADH f e r r i c y a n i d e reductase NADH c y t c r e d u c t a s e (rotenone i n s e n s i t i v e ) NADPH c y t c_ r e d u c t a s e Cytochrome o x i d a s e 5' N u c l e o t i d a s e Monamine o x i d a s e A l k a l i n e Phosphodiesterase I Acyl transferase  Polytron, Plasma membranes P o t t e r Elvehjem M i t o c h o n d r i a homogenization Endoplasmic Differential reticulum centrifugation Sucrose gradients  5' N u c l e o t i d a s e N a / K+ ATPase A l k a l i n e phosphatase Phosphodiesterase I Cytochrome c_ o x i d a s e Ca2+ uptake  P o t t e r Elvehjem homogenization Differential centrifugation  None  Microsomes  +  - 25 Table _I (cont'd) 1. a o r t a  (cont'd)...  AUTHORS  METHODS USED  PREISS & BANASHEK(1975)  Homogenizer Microsomal not i n d i c a t e d Sucrose g r a d i e n t  Adenylate cyclase 5' N u c l e o t i d a s e  P o t t e r Elvehjem homogenization Differential centrifugation Sucrose gradients  C 2+ uptake  Carotid a r t e r y used. WUYTACK e t a l . , (1978) Coronary a r t e r y used.  PREPARATION  Plasma membranes  CHARACTERIZATION  a  NADH cytochrome c r e d u c t a s e (rotenone insensitive) Choline phosphotransf erase Cytochrome o x i d a s e  2. Smooth muscle s o u r c e - i l e u m AUTHORS  METHODS USED  PREPARATION  CHARACTERIZATION  GODFRAIND e t a l . , P o t t e r Elvehjem Microsomes (1973,1976,1977) homogenization M i t o c h o n d r i a Differential centrifugation  Ca uptake ATPases 5' N u c l e o t i d a s e Cytochrome o x i d a s e  ZELCK e t a l . , (1975)  Homogenizer not i n d i c a t e d Differential centrifugation  Nuclei Mitochondria  E l e c t r o n microscopy S u c c i n a t e dehydrogenase  NILSSON e t a l . , (1977)  Homogenizer not i n d i c a t e d Differential centrifugation Sucrose gradients  Plasma membranes Mitochondria Nuclei  5' N u c l e o t i d a s e Phosphodiesterase I ^ H - l e u c i n e uptake Cholesterol/phospholipid Cytochrome c_ o x i d a s e %-ouabain binding  RAEYMAEKERS e t al.,(1977)  P o t t e r Elvehjem homogenization Differential centrifugation  Mitochondria  0 consumption Cytochrome £ o x i d a s e Ca uptake  i T  2  +  - 26 T a b l e I (cont'd) 3. Source o f smooth muscle - myometrium AUTHORS  METHODS USED  PREPARATION  JANIS e t a l . (1976)  Polytron homogenization Differential centrifugation Sucrose gradients  Plasma membranes Mitochondria Smooth endoplasmic reticulum Rough endoplasmic reticulum  RANGACHARI e t a l . , (1976)  P o t t e r Elvehjem homogenization Sucrose gradients  Microsomes  Adenyl c y c l a s e 5' N u c l e o t i d a s e  MATLIB e t a l . (1979)  Polytron homogenization Differential centrifugation Sucrose gradients  Plasma membranes  S u c c i n a t e cytochrome c^ reductase NADH c y t £ r e d u c t a s e (rotenone s e n s i t i v e and insensitive) Monamine o x i d a s e H-WGA l a b e l l i n g o f PM 3H-oxytocin binding s i t e s 2+ p 5' N u c l e o t i d a s e NADPH r e d u c t a s e  CHARACTERIZATION 5 Nucleotidase glucose-6-phosphatase Mg2+ ATPase Ca uptake Ouabain s e n s i t i v e phosphatase Cytochrome c o x i d a s e 1  2 +  3  M g  A  T  a  s  e  NISHIKORI e t a l . , (1977)  Polytron homogenization Differential centrifugation  Microsomal  Succinate  KRALL e t a l . (1978)  Polytron homogenization Differential centrifugation  Microsomes  P r o t e i n Kinase  VALLIERES e t a l . , (1978)  P a r r bomb Differential centrifugation Sucrose gradients  Plasma membranes  dehydrogenase  Adenyl cyclase 5' _ N u c l e o t i d a s e  2 Mg*" ATPase Phosphodiesterase I Cytochrome c^ o x i d a s e 5l-WGA l a b e l l i n g o f plasma membranes (PM) E l e c t r o n microscopy 12  - 27 -  MATERIALS C o n c a n a v a l i n A - l i n k e d t o Agarose (Con A-Agarose)-, amethyl-D-mannoside,  T r i t o n X-100, l a c t o p e r o x i d a s e , a c r y l a m i d e ,  ammonium p e r s u l p h a t e , T r i s (hydroxymethyl) aminomethane ( T R I S ) , 2 (N-Morpholino) ethane s u l p h o n i c a c i d (MES) and a l l enzyme s u b s t r a t e s were o b t a i n e d from Sigma C h e m i c a l Company, vanadium-free ATP from B o e h r i n g e r Manheim, c o n c a n a v a l i n A - l i n k e d t o Sepharose (Con A-Sepharose) and Sepharose 4B from Pharmacia F i n e C h e m i c a l s , c o l l a g e n a s e (Grade A) from C a l Biochem, Coomassie B l u e R-250, sodium d o d e c y l s u l p h a s e (SDS), N,N,N',N' - T e t r a m e t h y l e t h y l e n e d i a m i n e (TEMED) from B i o r a d L a b o r a t o r i e s . 125 Radioactive c a r r i e r free [ i ] N a l (100 mCi/ml) was o b t a i n e d from Amersham and New England N u c l e a r . Adenosine 32 5 ' - t r i p h o s p h a t e , t e t r a (triethylammonium) s a l t , [ y4 was purchased from New  p] (20-40 Ci/mmol)  England N u c l e a r as w e l l as ,0mnifluor.  U n l e s s o t h e r w i s e s t a t e d , s c i n t i l l a t i o n grade t o l u e n e , MIRACLOTH and a l l common c h e m i c a l s were r e c e i v e d from N o r t h American Chemical Supply L i m i t e d . Domestic c h i c k e n s (Rhode I s l a n d Reds, 18-24 months o l d ) were o b t a i n e d from the U.B.C. P o u l t r y Farm U n i t . k i l l e d by b r e a k i n g t h e i r n e c k s .  Animals were  G i z z a r d s were i m m e d i a t e l y e x c i s e d  and used.  METHODS A.  Enzyme Assays  5'-nucleotidase The method used was LOWENSTEIN (1975). 5'-AMP i s measured.  t h a t of WIDNELL (1974) and BURGER &  The r e l e a s e of i n o r g a n i c phsophate  from  To 0.9 m l of s u b s t r a t e s o l u t i o n (11.11  mM  5'-AMP, sodium s a l t ; 1.11 mM m a g n e s i u m : c h l o r i d e ; 0.1 M TRIS-HCL, pH 8.5 a t 22°C) a t 37°C, 50 i s added.  1 of membrane s u s p e n s i o n (10-20 yg p r o t e i n )  A f t e r i n c u b a t i o n f o r 20 m i n u t e s a t 37°C, 50 V1 of 50%  28  - 28 -  trichloroacetic acid  (TCA) and 0.5 ml of Ames reagent (1 p a r t  10% a s c o r b i c a c i d and 6 p a r t s 0.42% ammonium molybdate i n 0.5 M s u l p h u r i c a c i d ) (AMES, 1966) a r e added w i t h s t i r r i n g . are  then i n c u b a t e d a t 37°C f o r a f u r t h e r one hour.  The samples  Colour  development i s then stopped by a d d i t i o n of 0.5 ml i c e c o l d tilled  water.  dis-  The samples a r e then c e n t r i f u g e d a t 2000 g,  0°C, 3 minutes i n a S o r v a l l RC-2B c e n t r i f u g e and the absorbance of  t h e supernatant read a t 660 nm.  Blanks w i t h o u t e i t h e r  5'-AMP o r membranes a r e r u n w i t h each sample.  Nanomoles o f  phosphate r e l e a s e d a r e c a l c u l a t e d based on a standard c u r v e o b t a i n e d u s i n g i n o r g a n i c phosphate and t h e procedure above l e a v i n g out membranes.  R e s u l t s a r e expressed as  mole phosphate  r e l e a s e d p e r hour per mg p r o t e i n .  NADPH/NADH a n t i m y c i n A - i n s e n s i t i v e cytochrome c_ r e d u c t a s e The methods used were those o f TOLBERT (1974), HATEFI & RIESKE (1967) and HODGES & LEONARD (1974). substrate solution  To 2.80 ml of  (0.375 mg/ml cytochrome c; 45 mM sodium  phosphate, .pH 7.0 at 20°C; 1.8 mM sodium c y a n i d e ) i s added 20 y l of  a n t i m y c i n A s o l u t i o n i n e t h a n o l (2 mg/|ml) .  This i s followed  by the a d d i t i o n of 100 to 200 y l o f membrane p r e p a r a t i o n  (40-80 yg  p r o t e i n ) a f t e r which the r a t e of change i n absorbance a t 550 nm i s r e c o r d e d f o r 5 minutes.  Once t h e i n i t i a l r a t e has been  e s t a b l i s h e d , 100 y l of NADH or NADPH (3 mg/ml) i s added. new  The  r a t e o f change i n absorbance a t 550 nm i s r e c o r d e d and  s u b s t r a c t e d from the i n i t i a l  r a t e to y i e l d  the a c t u a l  rate.  Micromoles of c y t c_ consumed per u n i t time a r e c a l c u l a t e d assuming the e x t i n c t i o n c o e f f i c i e n t of c y t c^ a t 550 nm to be  29  - 29 -  18.5 mM  -cm . ,  hour per mg  S p e c i f i c a c t i v i t i e s are expressed i n  moles per  protein.  NADH a n t i m y c i n A - s e n s i t i v e cytochrome £ r e d u c t a s e . T h i s a s s a y i s c a r r i e d o u t as t h e assay f o r NADPH/NADH a n t i m y c i n A - i n s e n s i t i v e cytochrome c^ r e d u c t a s e except t h a t 45  mM  g l y c i n e - s o d i u m h y d r o x i d e b u f f e r , pH 9.0 i s s u b s t i t u t e d f o r sodium phsophate b u f f e r and a n t i m y c i n A o m i t t e d . acid  phosphatase  The method used was a m o d i f i c a t i o n of t h e methods of HODGES & LEONARD (1974) and HUBSCHER & WEST (1965).  To 0.9 ml  s u b s t r a t e s o l u t i o n (3.33 mM p - n i t r o p h e n o l phosphate; 1.67  mM  magnesium s u l p h a t e ; 36.7 mM TRIS-MES, pH 5.5 a t 25°C) i s added 100  1 of membrane s u s p e n s i o n (20-30 yg p r o t e i n ) .  For d e t e r m i n a t i o n  of K+ s t i m u l a t e d a c i d phosphatase the s u b s t r a t e s o l u t i o n i s made to c o n t a i n 55.5 mM p o t a s s i u m c h l o r i d e .  T h i s s o l u t i o n i s then  i n c u b a t e d a t 37°C f o r 20 m i n u t e s a f t e r w h i c h 50 y l o f 50% TCA and 0.5 m l of Ames r e a g e n t (1 p a r t 10% a s c o r b i c and 6 p a r t s 0.42% ammonium molybdate i n 0.5 M s u l p h u r i c a c i d ) (AMES, 1966). The samples a r e then i n c u b a t e d a t 37°C f o r one hour a f t e r w h i c h 0.5 m l of i c e c o l d d i s t i l l e d water i s added.  Samples a r e n e x t  c e n t r i f u g e d a t 2000 g, 3 min., 0°C i n a S a r v a l l RC-2B c e n t r i f u g e . The s u p e r n a t a n t s a r e withdrawn and the absorbance of the s u p e r n a t a n t a t 660 nm determined. are  Nanomoles of phosphate r e l e a s e d  r e a d from a 0-10 nanomole c a l i b r a t i o n c u r v e u s i n g t h e above  procedure.  B l a n k s w i t h o u t e i t h e r p - n i t r o p h e n o l phsophate or  membranes a r e r u n f o r each sample. expressed as  S p e c i f i c a c t i v i t i e s are  moles phosphate r e l e a s e d per hour per mg  protein.  30  - 30 -  succinate  dehydrogenase  The method used was based on t h o s e o f BONNER (1955) and TOLBERT (1974).  To a 4.0 ml q u a r t z c u v e t t e a r e added 0.3 m l  of 0.1 M p o t a s s i u m c y a n i d e ; 0.3 m l of 0.01 M potassium f e r r i c y a n i d e ; 0.2 ml o f 0.2 M sodium s u c c i n a t e and 2.0 m l o f 55 mM phosphate b u f f e r , pH 7.2.  sodium  A t t i m e z e r o , 0.20 m l o f membrane  s u s p e n s i o n (40-80 yg p r o t e i n ) i s added and t h e change i n absorbance. a t 400 nm r e c o r d e d .  C o n t r o l samples w i t h o u t e i t h e r  the membrane p r e p a r a t i o n o r s u c c i n a t e were p r o c e s s e d i n t h e same manner.  S p e c i f i c a c t i v i t i e s a r e c a l c u l a t e d u s i n g an  e x t i n c t i o n c o e f f i c i e n t a t 400 nm, a t 20°C, o f 0.892 x 1 0 M c m 3  f o r potassium f e r r i c y a n i d e .  _ 1  - 1  R e s u l t s a r e e x p r e s s e d i n moles  s u c c i n a t e u t i l i z e d per hour p e r mg p r o t e i n .  glucose-6-phosphatase The method used was t h a t o f NORDLIE & ARION (1966). To 0.90 m l o f s u b s t r a t e s o l u t i o n (3.33 mM  glucose-6-phsophate;  111 mM sodium c a c o d y l a t e b u f f e r , pH6.5) a t 37°C i s added 50 y l of membrane s u s p e n s i o n (10-20  g protein).  This mixture i s  then i n c u b a t e d a t 37°C f o r 20 minutes a f t e r w h i c h 50 y l o f 50% TCA and 0.5 m l o f Ames Reagent (.1 p a r t 10% a s c o r b i c a c i d t o 6 p a r t s 0.42% ammonium molybdate (AMES, 1966) a r e added.  i n 0.5 M s u l p h u r i c a c i d )  The samples a r e then i n c u b a t e d f o r  1 hour a t 37°C a f t e r which 0.5 m l of i c e c o l d d i s t i l l e d i s added.  water  Samples a r e then c e n t r i f u g e d a t 2000 g, 3 m i n . 0°C  i n a S o r v a l l RC-2B c e n t r i f u g e , f o l l o w i n g w h i c h the s u p e r n a t a n t s are withdrawn and t h e i r absorbance a t 660 nm measured. B l a n k s w i t h o u t e i t h e r glucose-6-phosphate o r membrane s u s p e n s i o n were  31  - 31 -  a l s o r u n w i t h each sample.  The absorbance d a t a a r e c o n v e r t e d t o  micromoles of phosphate by u s e of a s t a n d a r d c u r v e o b t a i n e d by the above procedure u s i n g i n o r g a n i c phosphate i n p l a c e of glucose-6-phosphate and membrane s u s p e n s i o n . expressed i n  Results are  moles phosphate r e l e a s e d per hour per mg  protein.  Lowry P r o t e i n Assay The method i s based on the o r i g i n a l procedure of LOWRY et a l . (1951).  The f o l l o w i n g s t o c k s o l u t i o n s a r e p r e p a r e d f r e s h  and s t o r e d a t room temperature p r i o r t o u s e . i n the f o l l o w i n g order:  Reagent A i s p r e p a r e d  2% sodium c a r b o n a t e i n 0.1 M  sodium  h y d r o x i d e , 49 m l ; 2% p o t a s s i u m t a r t r a t e , 0.5 ml; 1% copper s u l p h a t e , 0.5 m l .  Reagent B i s t h e F o l i n - C i o c a l t e a u 2 N d i l u t e d  1:1 w i t h d i s t i l l e d w a t e r . The sample t o be assayed i s made up to 1.0 ml w i t h d i s t i l l e d water.  At time z e r o , 5.0 ml reagent A i s added and t h e  mixsture s t i r r e d .  0.5 m l of reagent B i s added a f t e r 10 m i n u t e s  w i t h s t i r r i n g and t h e absorbance a t 750 nm read 20 m i n u t e s later. BSA  A s t a n d a r d c u r v e i s o b t a i n e d i n t h e same manner u s i n g  (0-250 y g / m l ) .  I f TRIS b u f f e r or s u c r o s e i s p r e s e n t i n the  samples to be assayed a p p r o p r i a t e c o n t r o l samples must be included  (ROBSON e t a l . , 1968; GERHARDT & BEEVERS, 1968). 2+  C h a r a c t e r i z a t i o n of Mg (a)  Stimulated,ATPase  D e t e r m i n a t i o n of Optimum C o n d i t i o n s TRIS-ATP (vanadium f r e e , see HUDGINS & BOND, 1977; BEAUGE  & GLYNN, 1977; JOSEPHSON & CANTLY, 1977) c o n c e n t r a t i o n s of 0,  0.05,  0.10,  The  0.20,  0.40,  0.80,  1.20,  2.00,  3.00,  4.00 mM were used.  s u b s t r a t e s o l u t i o n s were b u f f e r e d w i t h 50 mM  TRIS-HC1, pH7.2.  32  - 32 -  To 0.9 ml o f each s u b s t r a t e s o l u t i o n a t 37°C, 50 y l o f membrane s u s p e n s i o n (10-20 yg p r o t e i n ) a r e added.  The samples  are then i n c u b a t e d f o r 10 minutes a t 37°C f o l l o w i n g which the r e a c t i o n s a r e t e r m i n a t e d u s i n g 50 y l o f 50% TCA and 0.5 ml o f Ames Reagent  (.1 p a r t  10% a s c o r b i c a c i d t o 6 p a r t s 0.42% ammonium  molybdate i n 0.5 M s u l p h u r i c a c i d ) (AMES, 1966).  C o l o u r develop-  ment i s a l l o w e d to proceed f o r 20 minutes a t 37°C a f t e r which 0.5 ml o f i c e c o l d water a r e added and t h e absorbance o f each sample read a t 660 nm.  B l a n k s w i t h o u t enzyme were r u n w i t h each  sample, c o n t r o l s w i t h o u t ATP a t f r e q u e n t i n t e r v a l s .  Micromoles  phosphate r e l e a s e d a r e c a l c u l a t e d based on a phosphate curve u s i n g i n o r g a n i c phosphate.  standard  Specific a c t i v i t i e s are  expressed i n umoles. phosphate l i b e r a t e d per hour p e r mg p r o t e i n . 2+ Optimum Mg  t o t a l and ATP t o t a l c o n c e n t r a t i o n s were then used  f o r the next s e r i e s of experiments. (b)  pH Optima and N a , Li" ", K +  1  +  and Ouabain  Sensitivity  For d e t e r m i n a t i o n of the pH optimum, the f o l l o w i n g pH's at 37°C were used:  6.00, 6.50, 7.00, 7.20, 7.40, 7.60, 7.80,  8.00, 8.50 and 9.00.  F o r each pH v a l u e , s u b s t r a t e  solutions  were p r e p a r e d as i n T a b l e I I to t e s t f o r s t i m u l a t i o n by i o n s and i n h i b i t i o n by ouabain.  33  -  ^-33  Table I I S u b s t r a t e S o l u t i o n s t o T e s t Mg + 2  + L i ,K S e n s i t i v i t y t o pH, Na', +  [ATP]  2 [ATP]  max  30 mM B u f  1  max  3 [ATP]  - Stimulated +  and Ouabain  4 [ATP]  max  50 mM Buf  50 mM Buf  max 50 mM Buf  [Mg2+] max  [ 2+] ° max 120 mM N a  [Mg +] max 120 mM L i  a  5 [ATP]  2  Mg  max  +  6 [ATP]  max  50 mM Buf  50 mM Buf  [Mg +] max  [Mg2+] max  120 mM Na+  120 mM N a  20 mM K+  20 mM K +  2  a  +  ATPase  +  2 mM Ouabain  o p t i m a l c o n c e n t r a t i o n s based on the r e s u l t s o f p a r t ( a ) . :,pH a t 37°C - 6.0, 6.5 C a c o d y l a t e B u f f e r , - 7.0-8.5 TRIS-HC1, - 9.0 Glycine-NaOH B u f f e r  b  To 0.9 m l o f each s u b s t r a t e s o l u t i o n a t 37°C a r e added 50 y l of membrane s u s p e n s i o n (10-20 jS-g p r o t e i n ) . incubated  The samples a r e then  a t 37°C f o r 10 m i n u t e s a f t e r w h i c h 50 y l o f 50% TCA  s o l u t i o n and 0.50 m l o f Ames reagent (1 p a r t  ascorbic a c i d to 6  p a r t s 0.42% ammonium molybdate i n 0.5 M s u l p h u r i c a c i d ) (AMES, 1966) are added w i t h s t i r r i n g .  F o l l o w i n g f u r t h e r i n c u b a t i o n a t 37°C  f o r 20 m i n u t e s , 0.50 m l o f i c e c o l d d i s t i l l e d water a r e added. The absorbance a t 660 nm of each sample i s read and r e c o r d e d . B l a n k s were r u n f o r each sample d e l e t i n g t h e 50 y l o f membrane suspension.  C o n t r o l s w i t h o u t ATP were a l s o r u n on a random spot  .34  - 34 -  check b a s i s .  M i c r o m o l e s phosphate r e l e a s e d were c a l c u l a t e d  based on a s t a n d a r d curve of 0 t o 50 umoles I n o r g a n i c phosphate. S p e c i f i c a c t i v i t i e s were expressed  i n m i c r o m o l e s phosphate  r e l e a s e d per hour per mg p r o t e i n . (c)  Velocity Profile Twenty m l of s u b s t r a t e s o l u t i o n were prepared 2+  the o p t i m a l Mg  t o t a l and ATP t o t a l c o n c e n t r a t i o n s  from p a r t ( a ) , u s i n g 50 mM TRIS-HG1, pH7.2.  derived  F o r t h e time  0.9 m l of s u b s t r a t e were used w i t h 50 y'JL membrane (10-20 u g p r o t e i n ) f o r each time.  using  course  suspension  The r e a c t i o n times used  were 0, 3, 5, 10, 15 and 30 m i n u t e s a t 37°C, a f t e r which the r e a c t i o n s were t e r m i n a t e d by t h e a d d i t i o n o f 50 y 1 o f 50% TCA and 0.50 m l o f Ames Reagent (see p a r t s (a) and ( b ) ) . Phosphate r e l e a s e d was c a l c u l a t e d based on s t a n d a r d  curves.  acetylcholinesterase The a s s a y was based on t h e methods o f STECK & KANT (1974) . The a s s a y was done i n d u p l i c a t e w i t h 100 y 1 o f t h e membrane suspension  p e r sample.  The 100 y l a l i q u o t s o f membrane  suspension  (50-60 y g p r o t e i n ) were p i p e t t e d i n t o t h e bottom of a 1 cm semi-micro q u a r t z c u v e t t e and mixed w i t h an e q u a l volume of e i t h e r 5 mM sodium phosphate, pH 8.0 o r (0.40% T r i t o n X-100 (v/v) i n 5 mM sodium phosphate, pH 8.0).  The c o n c e n t r a t i o n o f  d e t e r g e n t was chosen so t h a t a l l l a t e n t enzymatic a c t i v i t y would be r e l e a s e d w i t h l i t t l e or no a c t i v a t i o n .  The volume  was made t o 0.70 m l w i t h 100 mM sodium phosphate, pH 7.5, then 50 y l o f 5, 5' - d i t h i o b i s ( 2 - n i t r o b e n z o i c a c i d ) (DTNB) s t o c k  35  - 35  s o l u t i o n (10 mM bicarbonate:  DTNB; 100 mM  DTNB, 3:8;  -  sodium, phosphate, pH  s t o r e d a t -5°C  7.5;  sodium  u n t i l used) i s added.  F i n a l l y , 50 p i of a c e t y l c h o l i n e c h l o r i d e or bromide (12.5 i n H^O),stored f r o z e n ) i s added. then mixed by a t 412  nm  i n v e r s i o n and  recorded.  c o r r e s p o n d s t o 1 um 4-1-1 of 13.6  x 10  M  per mg  An i n c r e a s e  contents  cm  a t 412  nm  i n absorbance of  17.0  f o r DTNB.  To e s t a b l i s h  the  r e c o r d e d a l s o p r i o r to a d d i t i o n of  F i n a l r e s u l t s a r e e x p r e s s e d i n nmoles per  hour  protein. acid  The method used was WARREN (1955; 1959)  and  Clostridium Perfringens C h e m i c a l Co.)  The  p r o p e r , the s t o c k  A neuraminidase  p r e p a r e d by d i s s o l v i n g 1 mg  aqueous s o l u t i o n of b o v i n e  s o l u t i o n was  s t o r e d a t 4°C.  For  + 0.40%  enzyme i s a c t i v e w i t h r e s p e c t  T r i t o n X-100.  to 0.05  the  and  the  by the enzyme  by a 1 hour exposure of  the  M s u l p h u r i c a c i d a t 80°C.  P r i o r to use membrane p r e p a r a t i o n Amicon ML-2  M TRIS-  to the s i a l o g l y c o p r o t e i n s  neuramic a c i d (NANA) r e l e a s e d  compared to t h a t r e l e a s e d  assay  To e n s u r e t h a t  s i a l o g l y c o l i p i d s p r e s e n t i n t h e membrane p r e p a r a t i o n ,  preparation  the  s b l t u i o n i s d i l u t e d 10 f o l d w i t h 0.1  b u f f e r , . pH 5.7  amount of N - a c e t y l  of  n e u r a m i n i d a s e (type G from Sigma  i n 1 ml of 0.03%  serum a l b u m i n .  a m o d i f i c a t i o n of the methods of  STECK & KANT (1974).  ( s i a l i d a s e ) s o l u t i o n was  was  are  of p r o d u c t based on an e x t i c t i o n c o e f f i c i e n t  measurement of s i a l i c  acetate  cuvette  the r a t e of change i n absorbance  b a s e l i n e , the absorbance was the s u b s t r a t e .  The  mM  a r e d i a l y z e d i n an  d i a f i l t r a t i o n u n i t to remove i n t e r f e r i n g chromophores  36  - 36 -  as 2 - d e o x y r i b o s e .  D u p l i c a t e 100 y 1 a l i q u o t s o f membrane s u s p e n s i o n  (60-80 y g p r o t e i n ) a r e mixed w i t h o r w i t h o u t t h e d e t e r g e n t . The sample i s i n c u b a t e d f o r 30 m i n u t e s a t room temperature i n a t e s t tube w i t h a T e f l o n l i n e d screw cap. R e l e a s e d NANA i s then determined as f o l l o w s . A f t e r t h e 30 minute i n c u b a t i o n .100 y 1 o f sodium m e t a p e r i o d a t e (0.2 M i n 9 M p h o s p h o r i c a c i d ) was t h o r o u g h l y mixed i n t o each sample and a l l o w e d t o r e a c t f o r a t l e a s t 20 minutes.  F o l l o w i n g t h i s , 1.5 m l o f sodium a r s e n i t e  solution  (10% w/v i n 0.5 M sodium phosphate) i s mixed i n t o each sample and a l l o w e d t o r e a c t f o r a t l e a s t 20 m i n u t e s . . F o l l o w i n g  this,  1.5 m l o f sodium a r s e n i t e s o l u t i o n (10% w/v i n 0.5 M sodium phosphate) i s mixed i n v i g o r o u s l y w i t h t h e samples.  After 2  minutes t h e m i x i n g i s r e p e a t e d , and i s f o l l o w e d by t h e a d d i t i o n of 3.0 m l o f t h i o b a r b i t u a r i c a c i d s o l u t i o n (0.6% o f w/v i n 0.5 M sodium s u l p h a t e ) . Next t h e t i g h t l y capped tubes a r e p l a c e d i n a b o i l i n g water b a t h f o r e x a c t l y 15 m i n u t e s and t h e r e a f t e r c o l l e d tap water t o room temperature.  under  Two m l from each sample i s  withdrawn and e x t r a c t e d w i t h 2 m l c y c l o h e x a n o n e . by v i g o r o u s s h a k i n g f o r 15 seconds.  T h i s was done  The two phases were  s e p a r a t e d by c e n t r i f u g a t i o n o f the samples a t 2000 rpm/20°C/5 m i n u t e s i n an I n t e r n a t i o n a l SBV type c e n t r i f u g e .  The r o s y p i n k  cyclohexanone (upper phase) l a y e r i s t r a n s f e r r e d t o a 1 cm path l e n g t h c u v e t t e and t h e absorbances a t 549, 562, and 532 nm read.  M i c r o m o l e s NANA r e l e a s e d were c a l c u l a t e d i n two ways.  37  - 37 -  The f i r s t : for  moles NANA = v o l (ml) x A549 nm doesn't c o r r e c t  2-deoxyribose contamination which i n t e r f e r e s .  If  2 - d e o x y r i b o s e c o n t a m i n a t i o n i s s u s p e c t e d , the NANA absorbance maxima (A562 nm) and t h e 2 - d e o x y r i b q s e maxima (A532 nm) a r e used t o c a l c u l a t e t h e micromoles o f NANA r e l e a s e d , w h i c h =  vol  , imi;  133 A562 nm (32.6)  (133)-C26)(8)  -  8 A532 nm (32.6) (133)-(26) (8)  Other e q u a t i o n s a r e a v a i l a b l e u s i n g the absorbance a t 549 and 532 nm f o r o t h e r i n t e r f e r i n g chromophores. as nmoles s i a l i c B.  Results are expressed  a c i d r e l e a s e d p e r mg o f p r o t e i n .  Plasma Membrane I s o l a t i o n The method used f o r i s o l a t i o n o f smooth muscle plasma  membrane; (PM) was m o d i f i e d from t h e p r o c e d u r e o f KIDWAI (1974). The g i z z a r d s were e x c i s e d from f r e s h l y s a c r i f i c e d  chicken,  c l e a n e d o f f a t and p l a c e d i n i c e c o l d b u f f e r A (0.25 M s u c r o s e ; 3.0mMMg  ; 1 mM TRIS-MES, pH 7.4, a t 22°C).  o p e r a t i o n s were c a r r i e d o u t a t 4°C.  A l l subsequent  The stomach m u s c u l a r i s was  removed and 2. grams of v i s c e r a l smooth muscle ( v i s i b l y f r e e o f c o n n e c t i v e t i s s u e ) were minced w i t h s c i s s o r s and p l a c e d i n 30 m l b u f f e r A. homogenized  The muscle (chunks 2 mm x 2 mm  3 mm) was then  f o r 30 seconds a t a s e t t i n g o f 6.0 u s i n g a Brinkmann  P o l y t r o n PT-20.  A f t e r one m i n u t e the muscle was rehomogenized  for  Homogenization t i m e s were s e l e c t e d on o p t i m a l  40 seconds.  y i e l d s o f 5' - n u c l e o t i d a s e s p e c i f i c a c t i v i t y i n t h e f i r s t 5 g r a d i e n t f r a c t i o n s (see d i s c u s s i o n page 141 ) .  38  - 38 -  The homogenate was then f i l t e r e d through 2 c r o s s e d l a y e r s of M i r a c l o t h .  The f i l t r a t e was made up t o 38 m l and  c e n t r i f u g e d a t 2000 g, 10 m i n u t e s , 0°C i n a SS-34 a n g l e r o t o r i n a S o r v a l l RC-2B c e n t r i f u g e . removed and saved.  The s u p e r n a t a n t was c a r e f u l l y  The p e l l e t was n o r m a l l y d i s c a r d e d , u n l e s s  marker a s s a y s were t o be done.  The s u p e r n a t a n t was then  c e n t r i f u g e d as above except a t 15,000 g , 15 m i n u t e s , 0°C. av The p e l l e t was d i s c a r d e d , and t h e s u p e r n a t a n t c e n t r i f u g e d a t 100,000 g , 75 m i n u t e s , 0°C, i n a SW-27 r o t o r i n a av L5-65 Beckman U l t r a c e n t r i f u g e .  The r e s u l t i n g p e l l e t was  saved and t h e s u p e r n a t a n t d i s c a r d e d u n l e s s marker a s s a y s were to be done.  The p e l l e t was then resuspended  i n a 2.5 m l o f  0.25 M s u c r o s e and l a y e r e d on a d i s c o n t i n u o u s s u c r o s e g r a d i e n t c o n s i s t i n g of 2.5 m l each o f 27.0, 30.0, 32.0, 34.0, 35.0, 36.5, 40.0, 43.0, 45.0 and 66.0% s u c r o s e .  The l a y e r e d g r a d i e n t s  were then c e n t r i f u g e d a t 12,000 g , 1 5 0 m i n u t e s , 0°C i n a av Beckman L5-65 u l t r a c e n t r i f u g e u s i n g a SW-27 s w i n g i n g bucket rotor.  F o l l o w i n g c e n t r i f u g a t i o n each l a y e r was c a r e f u l l y  removed by a hypodermic s y r i n g e and e i t h e r used i m m e d i a t e l y or s t o r e d f r o z e n a t -20°C.  Membrane enzymatic a c t i v i t y was  s t a b l e f o r 2 weeks. To remove t h e b u l k o f s u c r o s e from t h e f r a c t i o n s , w h i c h was n e c e s s a r y i n those s i t u a t i o n s when s u c r o s e i n t e r f e r e s w i t h t h e enzyme a s s a y , the. f r a c t i o n s were d i l u t e d 1:1 w i t h d i s t i l l e d water and c e n t r i f u g e d a t 140,000 g > 150 m i n u t e s , a v  0°C u s i n g a Type 65 Beckman a n g l e r o t o r in. a L5-65 Beckman ultracentrifuge.  Recovery o f membranes was g r e a t e r than 85%  using t h i s procedure.  A d d i t i o n o f 5.0 mM C a C ^ t o t h e d i l u t i n g  s o l u t i o n p r i o r t o c e n t r i f u g a t i o n i n c r e a s e d y i e l d s o n l y 3%.  39  - 39 -  Membranes can be s t o r e d f o r 3-4  weeks i n the p e l l e t form.  I t s h o u l d be added t h a t an Amicon u l t r a f i l t r a t i o n u n i t was  a l s o used to remove s u c r o s e .  Using t h i s procedure  40% of the membranes were l o s t by a b s o r p t i o n to the  filter.  Sucrose was most e f f e c t i v e l y removed by u s i n g d i a l y s i s and d i a l y z i n g a g a i n s t 20 volumes of 20 mM 1 mM magnesium c h l o r i d e , 1 mM  EDTA and  Ml-2  tubing  TRIS-HC1, pH  1 mM  CaC^  7.0,  f o r 2 days  w i t h r e g u l a r changes of d i a l y z i n g b u f f e r . C.  C e l l Sheet and  Single C e l l Preparation  C e l l sheets  (LEWIS et a l . ,  1975)  0.25  mm  t h i c k were  o b t a i n e d from c h i c k e n g i z z a r d smooth muscle u s i n g a S o r v a l l Tissue S l i c e r .  The c e l l sheets were immediately  TRIS-MES b u f f e r (1 mM  TRIS-MES, pH 7.2;  1 mM magnesium c h l o r i d e ) t w i c e .  0.25  M  washed w i t h sucrose;  I f the c e l l s h e e t s were to  be used f o r i s o l a t i o n of plasma membranes, they were suspended i n 35 ml o f B u f f e r A (see e a r l i e r ) and  t r e a t e d as d e s c r i b e d i n  the plasma membrane i s o l a t i o n p r o c e d u r e . was  t h a t the P o l y t r o n h o m o g e n i z a t i o n was  The o n l y d i f f e r e n c e done i n two -  10  second b u r s t s ; one m i n u t e a p a r t ( a g a i n o p t i m i z e d on the b a s i s of 5' n u c l e o t i d a s e a c t i v i t y of the f i n a l  product).  I f the c e l l s h e e t s were to be used to p r e p a r e i n d i v i d u a l c e l l s (BAGBY e t a l . , 1971; 1964;  FAY  & DELISE, 1973;  LEWIS e t a l . , FAY  1975;  & SINGER, 1977;  they were suspended i n 9.0 ml of B u f f e r Bphosphate, pH 7.4;  0.25  M sucrose)  RODBELL e t a l . , SMALL,  (100 mM  c o n t a i n i n g 0.03%  1977)  sodium grade  40  - 40 -  Freshly excised gizzard - 2.0 grams minced i n 30 m i s 0.25 M s u c r o s e + 1.0 mM TRIS-MES pH 7.4 + 3fl mM MgCl . ~ - homogenized w i t h a PT 20 P o l y t r o n probe f o r 30 + 45 seconds a t 0°C. - homogenate f i l t e r e d t h r o u g h 2x l a y e r s of M i r a c l o t h  R e s i d u e (D) ( c e l l debris)  F i l t r a t e (S) j volume made up to 38 mis  •• c e n t r i f u g e d a t 2000 g, 10 min.  P e l l e t (D) (mito, er)  Supernatant  0°C  (S)  j c e n t r i f u g e d a t 15,000 g, 15 min. 0 C  P e l l e t (D) (mito, e r )  Supernatant  (S)  7 c e n t r i: f u g e d at-100,000 75 min. .n. . 0°C  P e l l e t (S) (pm, m i t o )  Supernatant (D) ( l y s o , m i t o , pm)  j resuspended i n 2.5 mis 0.25 M sucrose - applied to a discontinuous sucrose g r a d i e n t 27% - 45% - c e n t r i f u g e d a t 120,000 g, 150 min. 0 C C o l l e c t and assay f r a c t i o n s Figure 1  Scheme f o r i s o l a t i o n o f smooth muscle plasma membranes from t h e c h i c k e n g i z z a r d . A b b r e v i a t i o n s a r e as f o l l o w s : pm=plasma membranes;. l y s o = l v s o z o m e s ; m i t o = m i t o c h o n d r i a ; er=endoplasmic r e t i c u l u m ; S=saved; D=discarded f r a c t i o n .  41  g,  - 41 -  A collagenase  (CALBIOCHEM) f o r 30 m i n u t e s a t 37°C w i t h  a e r a t i o n by O^^CO^ (95:5).  The r e s u l t i n g c e l l s h e e t s and  c e l l s were then p e l l e t e d by c e n t r i f u g a t i o n a t 1000 g  , 5 minutes, clV  0 C i n a S o r v a l l RC-2B c e n t r i f u g e . c a r e f u l l y removed and d i s c a r d e d .  The s u p e r n a t a n t was The p e l l e t was g e n t l y  resuspended i n 9.0 m l B u f f e r B, w i t h 0.15% grade A c o l l a genase and a e r a t e d The  a t 37°C by 0^:CO^ (95:5) f o r 20 m i n u t e s .  c e l l s u s p e n s i o n was then, r e c e n t r i f u g e d as above and t h e  formed p e l l e t resuspended i n B u f f e r A o n l y . were i m m e d i a t e l y c e n t r i f u g e d as above.  The s i n g l e c e l l s  T h i s p r o c e d u r e was  r e p e a t e d f i v e times t a k i n g g r e a t c a r e t o n o t d i s c a r d t h e p e l l e t each time. A and used.  The f i n a l p e l l e t was resuspended i n B u f f e r  C e l l s were examined by phase c o n t r a s t m i c r o s c o p y  (See F i g u r e s 9 and 10) and were t r e a t e d w i t h t r y p a n b l u e t o test c e l l D.  Gel Electrophoresis The  gel  viability.  o v e r a l l approach used f o r SDS (sodium d o d e c y l s u p h a t e )  e l e c t r o p h o r e s i s was t h a t o f BARTON (1978) .  The  m o d i f i c a t i o n s used by LAMELLI e t a l . (1973) and FAIRBANKS e t a l . (1971a) were a l s o i n c o r p o r a t e d . are g i v e n below i n T a b l e I I I .  Reservoir  Gel formulations b u f f e r s were 0.1%  SDS i n 0.1 M sodium phosphate.  42  - 42 -  Table I I I Gel Formulations  Used f o r E l e c t r o p h o r e s i s  COMPONENT  5% s e p a r a t i n g g e l STOCK M l Used  3% s t a c k i n g g e l STOCK M l Used  Acrylamide  22.2%  30.0%  Methylene-bisacrylamide  0.60%  Ammonium p e r s u l p h a t e  15mg/ml  TEMED  9.0 m l  2.5 m l  1.50%  -  2.0 m l  0.75  10 mg/ml  30. y l  10 y l  H 0  distilled  9.0 m l  distilled  19.25  Buffer  0.2 M sodium phosphate, pH 7.2; + 0.2% SDS  20.0 m l  1.25 M TRISHG1, pH6.8; +1.0% SDS  2.50  2  ml ml ml  A f t e r p r e p a r a t i o n o f t h e s e p a r a t i n g g e l s o l u t i o n , 9 cm l o n g g e l s were formed i n 0.4 cm x 12 cm a c i d washed f l i n t g l a s s tubes.  Each column was o v e r l a y e r e d w i t h 1.0 cm o f water o r  isobutanol.  F o l l o w i n g p o l y m e r i z a t i o n , t h e o v e r l a y was  removed and 1 cm s t a c k i n g g e l s o l u t i o n aided atop gel  the'separating  and o v e r l a y e r e d w i t h water o r i s o b u t a n o l u n t i l p o l y m e r i z a t i o n .  The o v e r l a y was removed a f t e r p o l y m e r i z a t i o n and r e p l a c e d by reservoir buffer.  The g e l s were then p l a c e d i n a Pharmacia  Ge-4 e l e c t r o p h o r e s i s apparatus and p r e - r u n a t 8 ma/gel and 8-13 v o l t s / c m f o r 4 h o u r s . As t h e g e l s were p r e - r u n n i n g , membrane s u s p e n s i o n i n 15.0%  s u c r o s e was prepared  membrane s u s p e n s i o n  for electrophoresis.  To 150 y l o f  wae added 125 y l o f sample b u f f e r  43  - 43 -  (0.01 M sodium phosphate, pH 7.2; 1% SDS; 0.14 M 10% v / v g l y c e r o l ; 0.002 M bromophenol b l u e ) .  mercaptoethanol,  The samples were  then capped and p l a c e d i n a b o i l i n g water b a t h f o r 5-10 m i n u t e s t o reduce d i s u l p h i d e bonds and t o promote by SDS.  solubilization  M o l e c u l a r w e i g h t markers were i n c u b a t e d as above.  Those used were BSA (MW=70,000), A l d o l a s e (MW=161,000) egg albumin (MW=43,000), c y t c (MW=12,398), E l a s t a s e (MW=25,900), a c t i n (MW=43,000) and myosin (MW=210,000). A f t e r sample p r e p a r a t i o n was complete, each sample was t a k e n up i n a d i s p o s a b l e m i c r o p i p e t t e and d i s c h a r g e d g e n t l y onto the top o f t h e s t a c k i n g g e l .  E l e c t r o p h o r e s i s was performed  w i t h the v o l t a g e a t 4-5v/cm and c u r r e n t a t 4-5mA/gel. r u n n i n g time was about 10 h o u r s .  The  Variations i n absolute  m i g r a t i o n d i s t a n c e s were m i n i m i z e d by removing tubes i n d i v i d u a l l y from t h e e l e c t r o p h o r e s i s a p p a r a t u s one by one as the bromphenol b l u e t r a c k i n g dye m i g r a t e d 9.0 cm from t h e origin.  T r a c k i n g dye p o s i t i o n s were marked by s l o t s i n t h e  gels. The g e l s were n o r m a l l y s t a i n e d f o r p r o t e i n w i t h B l u e R-250 ( B i o r a d ) .  G e l s were s t a i n e d as f o l l o w s .  Coomassie  After  r i n s i n g each g e l t w i c e w i t h d i s t i l l e d water they were p l a c e d i n 16 x 150 mm capped c u l t u r e tubes.  To each tube 30 m i s of  f i x i n g , s t a i n i n g and d i s t a i n i n g s o l u t i o n s were added as g i v e n below.  The tubes were g e n t l y a g i t a t e d a t room t e m p e r a t u r e i n  a Dubnoff s h a k e r .  44  - 44 -  (.1) 25% i s o p r o p a n o l ;  10% a c e t i c a c i d ; 0.05%  Coomassie B l u e - 12 h o u r s . (2)  10% i s o p r o p a n o l ;  10% a c e t i c a c i d ; 0.002%  Coomassie B l u e - 10 h o u r s . (3)  10% a c e t i c a c i d ; 0.001% Coomassie B l u e - 8 hours.  (4) The  10% a c e t i c a c i d - 3 x 10 hours each. t h i r d and f o u r t h s t e p s a r e a b s o l u t e l y  necessary i f  the background s t a i n i n g i s t o be reduced t o minimum. methanol was s u b s t i t u t e d d e s t a i n as w e l l .  When  f o r isopropanol the gels did not  G e l s s t a i n e d w i t h Coomassie B l u e from  Sigma c o u l d n o t be d e s t a i n e d e i t h e r by d i f f u s i o n o r electrophoretic The  methods.  Periodic-Acid  S c h i f f ' s (PAS)  p r o c e d u r e was used t o  s t a i n t h e g e l s f o r c a r b o h y d r a t e (NEVILLE & GLOSSMAN, 1974). Because h i g h c o n c e n t r a t i o n s o f SDS produce an i n t e n s e  background,  i t was n e c e s s a r y t o remove t h e d e t e r g e n t by c a r r y i n g o u t t h e steps 1 - 4  d e s c r i b e d above, b u t w i t h o u t t h e p r e s e n c e of t h e  protein stain.  The f i x e d g e l s were then p l a c e d i n d i v i d u a l l y  i n t o s l o t t e d p l e x i g l a s s tubes and suspended f o r t h e s p e c i f i e d period  t o t h e f o l l o w i n g sequence o f s o l u t i o n s .  required  Each g e l  100 m l .  45  - 45 -  (1)  0.5% p e r i o d i c a c i d - 2 hours  (.2) 0.5% sodium a r s e n i t e ; 5% a c e t i c a c i d - 1 hour (3)  0.1% sodium a r s e n i t e ; 5% a c e t i c a c i d - 2 x 20 m i n u t e s  (4)  5% a c e t i c a c i d - 10-20 m i n u t e s  Each s o l u t i o n was s t i r r e d v i g o r o u s l y The  g e l s were t h e n t r a n s f e r r e d  a t room t e m p e r a t u r e .  t o tubes c o n t a i n i n g  10 m l of  S c h i f f ' s r e a g e n t (ACHARIUS & ZELL, 1969; NEVILLE & GLOSSMAN, 1974)  and l e f t o v e r n i g h t .  F i n a l l y , they were r e t u r n e d t o t h e  s l o t t e d tubes and i n c u b a t e d i n 0.1% sodium b i s u l p h i t e i n 0.01  M HC1 f o r s e v e r a l hours ( s o l u t i o n changed once e v e r y  hour) u n t i l t h e r i n s e s o l u t i o n f a i l e d t o t u r n p i n k upon a d d i t i o n o f formaldehyde.  Rose p i n k bands appeared a f t e r 2  hours i n t h e S c h i f f ' s r e a g e n t .  No v a r i a b l e background  a b s o r p t i o n was seen. The  g e l s were scanned u s i n g a G i l f o r d s p e c t r o p h o t o m e t e r  equipped w i t h a Model 2420 L i n e a r  Transport Accessory.  The  Coomassie B l u e s t a i n e d g e l s were scanned a t 530 nm, those s t a i n e d by t h e PAS p r o c e d u r e a t 560 nm.  The s l i t w i d t h was  0.1 mm i n e i t h e r case. When samples c o n t a i n i n g  radioactively  l a b e l l e d components were r u n , g e l s were n o t s t a i n e d b u t r i n s e d i n water t w i c e , f i x e d w i t h 8% TCA (15 m i n u t e s ) and 32 then s l i c e d i n t o 2 mm s e c t i o n s ATP  l a b e l l i n g experiments).  f o r counting (see [ y  Duplicate  P ]  gels f o r s t a i n i n g only  were a l s o r u n .  46  - 46  E.  -  I o d i n a t i o n Experiments  (a)  I o d i n a t i o n of M u s c l e Chunks, C e l l Sheets and F r e e C e l l s with I . 1  2  5  A p p r o x i m a t e l y 2 grams of c e l l s h e e t s (or 2 x 2 x 2 muscle chunks) were washed w i t h 200 ml of i c e c o l d 0.25 sucrose  i n 50 mM  sodium phosphate, pH 7.2  c o n t a i n i n g 0.43 C and al.,  1  Morrison,  & SCHONBAUM, 1976;  1970,  20 m i n u t e s .  2  5  I ] Nal  1974;  c e n t r i f u g e tube  (.1.2 m C i ) (ROMBAUTS e t  MORRISON & BAYSE, 1970;  Hynes, 1976).  hydrogen p e r i o x i d e was for  The  ml of 10 nM l a c t o p e r o x i d a s e , 9.025 ml of B u f f e r  12 y l of c a r r i e r f r e e [ 1967;  M  ( B u f f e r C).  sheets were then p l a c e d i n t o a p o l y p r o p y l e n e  mm  MORRISON  A t o t a l of 40 y l of 1.6  mM  added i n 1 y l p o r t i o n s every 30 seconds  F o l l o w i n g the l a s t a d d i t i o n , the c e l l  were washed on a Buchner f u n n e l f i l t r a t i o n u n i t w i t h l i t r e s of B u f f e r C c o n t a i n i n g 5 mM  sodium i o d i d e .  sheets 1.5  The  cell  s h e e t s were then homogenized as d e s c r i b e d under the membrane i s o l a t i o n procedures. 4 and  F o l l o w i n g membrane i s o l a t i o n ,  Fractions  5 of the s u c r o s e g r a d i e n t were i m m e d i a t e l y c o l l e c t e d and  samples w i t h d r a w n f o r SDS  gel electrophoresis.  Two  g e l s were  r u n f o r each sample, one f o r Coomassie B l u e s t a i n i n g and 125 other f o r subjected  I counting.  The  remaining  F4 and F5 were then  t o the i o d i n a t i o n procedure to be d e s c r i b e d  S e c t i o n B.  Controls without  p e r i o x i d e were a l s o r u n .  the  in  l a c t o p e r o x i d a s e and/or hydrogen  Samples of homogenized c e l l  sheets 125  or muscle chunks were removed f o r g e l e l e c t r o p h o r e s i s . was  detected  using a Nuclear  c) (FAIRBANKS e t a l . , 1967;  Chicago gamma c o u n t e r  I  (see  B0GDAN0VE & STRASH, 1975;  Section  LEINEN &  WITCLIFFE, 1978).  .  47  - 47  -  When f r e e c e l l s i s o l a t e d from 1.0 grams of m u s c l e 125 were t o be l a b e l l e d w i t h I , the p r o c e d u r e was s i m i l a r e x c e p t t h a t the volume was reduced to 3.0 ml because of the s m a l l e r q u a n t i t y of the c e l l s but 125  the f i n a l c o n c e n t r a t i o n s  lactoperoxidase,  hydrogen p e r o x i d e remained  unaltered. ml of 0.1% The  The  I and  r e a c t i o n was  0.75  sodium i o d i d e s o l u t i o n .  c e l l s were then i m m e d i a t e l y p e l l e t e d by c e n t r i f u g a t i o n p e l l e t was  as above. was  stopped by the a d d i t i o n of  sodium a z i d e i n a 25 mM  a t lOOOg, 5 m i n u t e s , 0°C The  of  i n a S o r v a l l RC-2B c e n t r i f u g e .  suspended i n 3.0  T h i s p r o c e d u r e was 125  v i r t u a l l y f r e e of  I.  i n 3.0 ml B u f f e r C and  ml of B u f f e r C and  recentrifuged  r e p e a t e d 5x u n t i l the  supernatant  The  p e l l e t was  then resuspended  used i m m e d i a t e l y f o r e l e c t r o p h o r e s i s .  C o n t r o l s were r u n as d e s c r i b e d p r e v i o u s l y and a c o n t r o l u s i n g 125 125 homogenized I l a b e l l e d c e l l s was a l s o run. I was d e t e r m i n e d as i n p a r t c and as d e s c r i b e d under g e l e l e c t r o p h o r e s i s . :  (b)  I o d i n a t i o n of Sucrose G r a d i e n t For r e a s o n s t o be d i s c u s s e d  Fractions l a t e r three versions  of  the i o d i n a t i o n p r o c e d u r e were employed to l a b e l i s o l a t e d membrane f r a c t i o n s . of 32%  s u c r o s e (.34%  phosphate, pH 7.2, 125 in t  I ] Nal  of 1.6 mM 30 V1)  f o r F r a c t i o n 5) made 50 mM 0.8 pM  i n lactoperoxidase  (carrier free).  i n sodium  and  0.15  mCi/ml  At room t e m p e r a t u r e , 1 p i a l i q u o t s  hydrogen p e r o x i d e were added ( t o t a l volume added  a t 30 second i n t e r v a l s f o r 15 m i n u t e s .  terminated 28 mM  (1) Membranes were i o d i n a t e d i n 4 0 0 u 1  w i t h t h e a d d i t i o n of 35 P 1 of 0.1%  sodium i o d i d e or by a d d i n g 100 U1  sample b u f f e r .  of  R e a c t i o n s were sodium a z i d e i n  electrophoresis  The membranes i n b o t h c a s e s were used  48  - 48 -  immediately  f o r SDS  gel electrophoresis.  I n the e x p e r i m e n t ,  l a c t o p e r o x i d e and/or hydrogen p e r o x i d e wre (2)  400  y l of membranes i n s u c r o s e  w i t h d i s t i l l e d water.  The  o m i t t e d as c o n t r o l s .  (32-34) were d i s t i l l e d  i o d i n a t i o n was  c a r r i e d out 125  the f i n a l c o n c e n t r a t i o n s of l a c t o p e r o x i d a s e , [ hydrogen p e r o x i d e and  100  Following  y l of  sodium a z i d e i n 25 mM.sodium i o d i d e s o l u t i o n was the sample c e n t r i f u g e d a t 140,000g  using  I] Nal,  soium phosphate as i n ( 1 ) .  the l a s t a d d i t i o n to hydrogen p e r o x i d e ,  1:1  0.1% added  and  , 150 m i n u t e s , 0°C  in a  clV  L5-65 Beckmann U l t r a c e n t r i f u g e u s i n g a Type 65 a n g l e r o t o r . The  r e s u l t i n g p e l l e t was  sucrose-50 mM  resuspended i n 0.25  sodium phosphate b u f f e r , pH 7.2  electrophoresis. (3)  immediately  and used f o r  C o n t r o l s were run as i n ( 1 ) .  Membranes i n 0.8  ml were p e l l e t e d or d i a l y z e d as  under membrane p r e p a r a t i o n to remove s u c r o s e . resuspended i n .400 y l of 100 mM a syringe.  The  M  described  The p e l l e t  sodium phosphate, pH 7.2  was using 125  f i n a l c o n c e n t r a t i o n s of l a c t o p e r o x i d a s e ,  and hydrogen p e r o x i d e were as i n ( 1 ) . and samples prepared  as i n ( 1 ) .  f r a c t i o n s i n detergent  R e a c t i o n s were  For i o d i n a t i o n of  I  terminated  sucrose  the o n l y changes from (1) a r e t h a t  membranes were made 0.05%  and 0.40%  i n T r i t o n X-100  the  20 m i n u t e s  p r i o r to the a d d i t i o n of hydrogen p e r o x i d e a l i q u o t s to s t a r t the r e a c t i o n . (c)  I o d i n a t i o n of l a c t o p e r o x i d a s e - " i o d o l a c t o p e r o x i d a s e " F i v e y l a l i q u o t s of a 64 yM l a c t o p e r o x i d a s e s t o c k  s o l u t i o n ( f r e s h l y prepared  since self i o d i n a t i o n increases  w i t h a g i n g of the enzyme) were added t o 10 d i f f e r e n t 400 p o r t i o n s of sodium i o d i d e s o l u t i o n s , c o n t a i n g 0.00, 0.30,  0.40,  0.50,  1.00,  iodide respectively.  1.50,  2.00,  and 5.00  Each s o l u t i o n was  mM  yl  0.10,  0.20,  sodium  a l d o made t o be  .  .  49  0.15  mCi/ml i n  pH 7.2.  f  I ] N a l and  50 mM  i n sodium phosphate,  To s t a r t the r e a c t i o n 1 u l a l i q u o t s of a 1.6  hydrogen p e r o x i d e  s o l u t i o n were added every 30 seconds f o r  15 m i n u t e s , a f t e r w h i c h 600 suspension  mM  u l of 12.5%  TCA was  added.  The  were then c e n t r i f u g e d a t 5000g, 10 m i n u t e s ,  0°C  i n a S o r v a l l RC-2B c e n t r i f u g e u s i n g a SS-34 r o t o r l e a d . The  supernatants  were poured o f f and saved.  were then counted i n a N u c l e a r counter.  The  pellets  Chicago Model 1020 Gamma  S u p e r n a t a n t s were counted as a check to ensure  a l l samples c o n t a i n e d the same i n i t i a l r a d i o a c t i v e c a r r i e r 125 free [ I ] N a l . C o n t r o l s were run o m i t t i n g l a c t o p e r o x i d a s e and/or hydrogen p e r o x i d e . The s u p e r n a t a n t s and p e l l e t s were a l s o s u b j e c t e d t o e l e c t r o p h o r e s i s , s t a i n e d and counted f o r 125 I t o ensure t h a t o n l y l a c t o p e r o x i d a s e was  labelled  and  t h a t e q u a l amounts of the enzyme were used. A f t e r d e t e r m i n a t i o n of the o p t i m a l sodium i o d i d e c o n centration required f o r optimal I  -  i n c o r p o r a t i o n by  l a c t o p e r o x i d a s e , the above experiment was constant  i o d i d e c o n c e n t r a t i o n of 0.3 mM  hydrogen p e r o x i d e c o n c e n t r a t i o n used.  the  repeated  using a  but v a r y i n g After  the  determination  of the optimum hydrogen p e r o x i d e c o n c e n t r a t i o n ( l u l / 3 0 seconds f o r 15 m i n u t e s ) , was  done.  0.5 mM  the f o l l o w i n g c o l d l a b e l l i n g of  lactoperoxidase  F i v e -5 y l of l a c t o p e r o x i d a s e were l a b e l l e d w i t h  sodium i o d i d e ( c o l d ) and  c o n c e n t r a t i o n above.  the optimum hydrogen  F o l l o w i n g the l a s t hydrogen  peroxide  a d d i t i o n , t h e l o t s were p o o l e d , d i a f i l t e r e d a t 0°C and  the c o n c e n t r a t e  column a t 0°C.  The  peroxide  a p p l i e d t o a 1 x 40 cm Sephadex  to 250  V1  G-100  l a c t o p e r o x i d a s e came o f f the column s h o r t l y  - 5b -  a f t e r the v o i d volume as i n d i c a t e d by UV a b s o r p t i o n a t 280 nm 125 p r e v i o u s runs u s i n g  I l a b e l l e d l a c t o p e r o x i d a s e on the column.  The f r a c t i o n s w i t h the l a c t o p e r o x i d a s e were p o o l e d  and  'the  non-radioactive iodine iodinated lactoperoxidase  concentrated  by d i a f i l t r a t i o n t o 200 y l .  assayed f o r  T h i s was  immediately  s p e c i f i c a c t i v i t y p r o t e i n and e l e c t r o p h o r e s e d . a c t i v i t y found was assessed  The  specific  i d e n t i c a l to the u n l a b e l l e d enzyme.  S e l f - i o d i n a t i n g a b i l i t y of the c o l d l a b e l l e d was  and  lactoperoxidase  by comparison w i t h n o n - i o d i n a t e d  The c o n d i t i o n s used were those above.  I t was  lactoperoxidase. found t h a t  the  non r a d i o a c t i v e l y l a b e l l e d i o d o l a c t o p e r o x i d a s e s e l f  labelling  with I  non-iodinated  was  l e s s than. 10% o f t h a t observed w i t h the  variety. (d)  E f f e c t of T r i t o n X-100  on L a c t o p e r o x i d a s e  S i x 400 y l a l i q u o t s of a 0.25 phosphate, pH 7.2. 0.10,  0.20  made 0.8 Nal.  Self Labelling  M s u c r o s e -50 mM  b u f f e r s o l u t i o n c o n t a i n i n g 0.00,  and 0.40%  sodium 0.05,  of TX-100 (v/v) r e s p e c t i v e l y were a l l 125  M i n l a c t o p e r o x i d a s e and 0.15  mCi/ml i n  [  I ]  To each tube f o r 15 m i n u t e s a t 30 second i n t e r v a l s , 1 y l  a l i q u o t s of 1.6 mM  hydrogen p e r o x i d e were added  subsequently.  R e a c t i o n s were t e r m i n a t e d by the a d d i t i o n of 50 y l of sodium a z i d e i n 25 mM  sodium i o d i d e s o l u t i o n or by  0.1%  the  a d d i t i o n of 100 y l g e l e l e c t r o p h o r e s i s sample b u f f e r proceeded by h e a t i n g a t 100°C f o r 10 m i n u t e s and a l l the samples s u b j e c t e d to g e l e l e c t r o p h o r e s i s .  G e l s were prepared  e i t h e r s t a i n i n g w i t h Coomassie B l u e or  as d e s c r i b e d f o r  counting.  5<1  -  (e)  51  -  S t u d i e s o f Membrane I o d i n a t i o n Times and E f f e c t o f N a l on S p e c i f i c A c t i v i t y  [  I]  Nine s e t s of membranes were i o d i n a t e d and t h e samples prepared  as d e s c r i b e d i n s e c t i o n b ( l ) e x c e p t f o r t h e f o l l o w i n g  changes:  a) F o r t h e f i r s t 6 s e t s of membranes, t h e 1.6 mM  hydrogen p e r o x i d e a d d i t i o n s were 1 y l a l i q u o t s / 3 0 seconds f o r 0, 5, 10, 15, 25 and 30 m i n u t e s r e s p e c t i v e l y ; b) F o r t h e remaining  3 s e t s , hydrogen p e r o x i d e was added as i n b ( l ) b u t 125  using three d i f f e r e n t s p e c i f i c a c t i v i t i e s of 0.075 mCi/ml, 0.15 mCi/ml, and 0.30 mCi/ml.  [  I] N a l o f  C o n t r o l s were  run as d e s c r i b e d i n b ( l ) . F.  Membrane E x t r a c t i o n Procedure, \. To make the e x t r a c t i o n e x p e r i m e n t s f e a s i b l e ,  et a l . , 1976;  (FAIRBANKS  1971; STECK & YU, 1973; STECK, 1974b; COLEMAN e t a l . ,  KAHLENBURG, 1976) t h e membranes had t o be a v a i l a b l e  i n s u f f i c i e n t l y l a r g e q u a n t i t y so t h a t the p r o t e i n s e x t r a c t e d d u r i n g t h e e x t r - c t i o n p r o c e d u r e s c o u l d be d e t e c t e d on SDS g e l electrophoresis.  To a c c o m p l i s h  t h i s , s i x t e e n 0.8 m l f r a c t i o n s  of membranes o b t a i n e d d i r e c t l y from s u c r o s e g r a d i e n t s F4 and F5 were d i l u t e d 1:1 w i t h d i s t i l l e d water and c e n t r i f u g e d as d e s c r i b e d i n t h e Membrane P r e p a r a t i o n s e c t i o n .  The p e l l e t s  were p o o l e d and suspended i n 15 mM sodium phosphate pH 7.5. The  suspension  contained  1.0 mg o f p r o t e i n / m l .  The  supernatants  and p e l l e t s u s p e n s i o n were s u b j e c t e d t o g e l e l e c t r o p h o r e s i s as d e s c r i b e d e a r l i e r t o see I f changes i n t h e p r o t e i n comp o s i t i o n of the membranes o c c u r r e d due t o t h e water d i l u t i o n p r i o r to sedimentation.  5.2  - 52 -  For  t h e e x t r a c t i o n e x p e r i m e n t s , 50 u l o f membrane  s u s p e n s i o n was used and e x t r a c t e d  w i t h 250 u l e x t r a c t i n g  media l i s t e d i n Table I V . F o l l o w i n g were c e n t r i f u g e d  a t 150,000g  e x t r a c t i o n , samples  , 120 m i n u t e s , 0°C i n a  Type 65 r o t o r u s i n g a Beckman L5-65 u l t r a c e n t r i f u g e . Table IV E x t r a c t i o n Media Used i n Membrane E x t r a c t i o n Media  Concentrations  Conditions  H 0  20 m i n . , 0 C  sodium phosphate, pH 7.5 5 mM  20 m i n . 0 C  2  pCMBS i n 5 mM sodium phosphate, pH 7.5  0.01,  0.20, 2.00 mM  TX-100 i n 5 mM sodium phosphate pH 7.5  0.01,  0.05, 0.50% ( y / v )  b DMMA  E D T A i n 5 mM sodium phosphate, pH 7.5  25 m i n . , 25 C  0.05 mg/ml, 2.0 mg/ml 5.0 mg/ml n e u t r a l i z e d to pH 7.5 w i t h sodium hydroxide  C  Digitonin  0.50  mM  0.36 mg/ml i n 5 mM sodium phosphate, pH 7.5  pCMBS r e f e r s t o p - C h l o r o m e r c u r i b e n z e n e s u l p h o n i c  25 m i n . , 0 C  25 m i n . , 0 C acid  DMMA r e f e r s t o D i m e t h y l m a l e i c a n h h y d r i d e  :>  "EDTA r e f e r s t o E t h y l e n e d i a m i n e  tetraacetate  5-3  - 53 -  F o l l o w i n g c e n t r i f u g a t i o n t h e p e l l e t s were suspended i n 150 y l b u f f e r (sodium phospaate, pH 8.0 and s u p e r n a t a n t 300 y l ) were a n a l y z e d f o r p r o t e i n and then i m m e d i a t e l y e l e c t r o p h o r e s e d as d e s c r i b e d under t h e g e l e l e c t r o p h o r e s i s s e c t i o n ( u s i n g 150 y l s u p e r n a t a n t and 75 y l p e l l e t s u s p e n s i o n ) . s t a i n i n g was done and a l l g e l s scanned.  Coomassie B l u e Peaks were a s s i g n e d  v a l u e s and compared.  G.  Column Chromatography on Con A-Agarose o r Con A-Sepharose  (a)  One m l o f membrane s u s p e n s i o n (1-2 mg p r o t e i n ) i n 50 mM  sodium phosphate, pH 6.8, was l o a d e d a t room t e m p e r a t u r e onto a Con A-Agarose column ( 1 x 5 cm) (CUATRECASES, 1973; MURTHY & HENEZ, 1973; SHARON & L I S , 1975; WALSH e t a l . , 1976; BRUNNER e t a l . , 1977).  The column had p r e v i o u s l y been p r e p a r e d by washing w i t h  (.1) 20 m l o f a c e t a t e b u f f e r (0.2 M sodium c h l o r i d e ; 6.0 mM magnesium c h l o r i d e ; 5 mM sodium a c e t a t e , pH 6.5), (2) 60 ml o f 0.20 M sodium c h l o r i d e i n 5 mM sodium a c e t a t e , pH 6.5, and finally,  (3) 20 m l o f 50 mM sodium phosphate, pH 6.5  The  a p p l i e d membranes were e l u t e d i n i t i a l l y w i t h 50 mM sodium phosphate, pH 6.5, a t a f l o w r a t e o f 12 m l / h r . f o r 4 h o u r s , a f t e r w h i c h t h e e l u t i n g b u f f e r was supplemented w i t h 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 mM.  amethyl-D-mannoside  The column was a l l o w e d t o  run  f o r a n a d d i t i o n a l 4 hours a t t h e above r a t e , a f t e r w h i c h  the  e l u t i n g b u f f e r , was r e p l a c e d by (50 mM_ sodium b o r a t e , pH 7.5;  150 mM  a-methyl-D-mannoside).  The e l u t i o n r a t e i n c r e a s e d t o  24 m l / h r . and t h e column r a n f o r 2 h o u r s .  54  - 54^-  The  1 m l f r a c t i o n s c o l l e c t e d were i m m e d i a t e l y assayed  f o r p r o t e i n using  t h e Lowry a s s a y and t h e B i u r e t method.  Samples o f peak f r a c t i o n s were then s u b j e c t e d e l e c t r o p h o r e s i s as d e s c r i b e d  earlier.  to g e l  Peak f r a c t i o n s  were a l s o used f o r enzyme a s s a y s and i o d i n a t i o n . (b) was  U s i n g Con A-Sepharose t h e above mentioned p r o c e d u r e repeated y i e l d i n g s i m i l a r r e s u l t s .  using (c)  C o n t r o l s were r u n  Sepharose 4B and Agarose columns. R a d i o a c t i v e l y l a b e l l e d membranes from F4 o r F5 were  p r e p a r e d as d e s c r i b e d column.  e a r l i e r and a l s o a p p l i e d t o t h e a f f i n i t y  Samples were t r e a t e d as i n (a) except t h a t no  i o d i n a t i o n of t h e e f f l u e n t was c a r r i e d o u t , o n l y Coomassie B l u e s t a i n i n g and r a d i o a c t i v e c o u n t i n g H. (a)  Phosphorylation  of the gels.  Studies  General Membrane F r a c t i o n s 4 and 5 C25 - 50 u g o f p r o t e i n i n t h e  r e s p e c t i v e s u c r o s e s o l u t i o n ) were made up t o t h e f i n a l volume o f I . 05 m l by a d d i t i o n o f components l i s t e d i n T a b l e V and mixed. T h i s and a l l t h e subsequent s t e p s were c a r r i e d o u t a t 0°C (NAGANO e t a l . , 1965; AVRUCH & FAIRBANKS, 1972; KNAUF e t a l . , 1975; CHA & SOOLEE, 1976; LANE, 1976).  55-  - 5f, -  Table V  Components Used i n P h o s p h o r y l a t i o n Sample 1 2 0.1mMMgCl„  0.1mMMgCl„ 0.5 mM C a C l 2  5 6 0.1 mM MgCl 0.1 mM M g C l 100 mM C h o l i n e 100 mM NaCl 25 mM KC1  2  Studies  3  4  0.1 mM MgCl 100 mM N a C l  0.1 mM MgCl 100 mM L i C l  7 0.1 mM M g C l 100 mM NaCl 0.5 mM CaCl„  8 Control  ?  9 Hydroxylamine  a - Used 0.4 M h y d r o x y l a m i n e h y d r o c h l o r i d e f r e s h l y p r e p a r e d . E x t r a c t i o n done as by Knauf e t a l . (.1974) e x c e p t t h a t t h e sample was c e n t r i f u g e d a t 150,000g ; 47,000 rpm, 2.5 h r . , i n a Beckman L5-65 u l t r a c e n t r i f u g e u s i n g a Type 65 r o t o r . 32 [  P ] - ATP was added t o g i v e the f i n a l ATP c o n c e n t r a t i o n o f  3.74  M and t h e f i n a l s p e c i f i c a c t i v i t y of 28 C i / m o l .  After  30 seconds, 100 y l of 50% TCA was added and the samples spun down a t 1 5 0 , 0 0 0 g , 60 m i n u t e s , 0°C i n a Beckman 25-65 u l t r a av  c e n t r i f u g e u s i n g a Type 65 r o t o r .  The TCA p r e c i p i t a t e d p e l l e t s  were then washed once w i t h and resuspended i n 200 u l 10% s u c r o s e - 50 mM TRIS-HC1, pH 7.2.  Each sample was  i n 3% SDS sample b u f f e r a t 37^C f o r 25 m i n u t e s .  solubilized  The t o t a l  volume of the s o l u b i l i z e d samples, 350 u l now, was d i v i d e d i n t o two 175 u l a l i q u o t s t h a t were e l e c t r o p h o r e s e d .  Following  e l e c t r o p h o r e s i s , one sample was used f o r c o u n t i n g and t h e o t h e r  56  a  - 56-  f o r Coomassie B l u e s t a i n i n g .  The c o u n t i n g of  radiation  was done by s l i c i n g t h e g e l i n t o 2 mm s l i c e s and p l a c i n g them i n t o s c i n t i L l a t i o n v i a l s w i t h 1 m l o f 0.05% SDS.  The  v i a l s were then i n c u b a t e d f o r 24 hours a t 37°C.  N e x t , 20 "1  of T r i t o n toluene s c i n t i l l a t i o n f l u i d  (1 l i t r e TX-100; 2 l i t r e s  t o l u e n e ; 16.9. grams o f OMNIFLUOR) were added t o each v i a l .  Samples  were c o o l e d t o 0°C and then counted u s i n g a Mark I I N u c l e a r 32 Chicago S c i n t i l l a t i o n c o u n t e r . P c o u n t i n g e f f i c i e n c y was 32 a p p r o x i m a t e l y 85% as determined  by adding a known  to t h e v a r i o u s v i a l s . V a r i o u s c o n t r o l s were r u n throughout  P  dpm  t h e experiments.  To see whether p h o s p h o r y l a t i o n was a f f e c t e d by t r a c e i m p u r i t i e s 32 on t h e l a b e l l e d [y - P ] ATP, p h o s p h o r y l a t i o n was measured 32 using  [y. -  was found  P ] ATP a t 25 f o l d lower s p e c i f i c a c t i v i t i e s .  t h a t t h e r e s u l t s wre u n a f f e c t e d by v a r i a t i o n s i n t h e  specific activity.  R e a c t i o n s were t e r m i n a t e d u s i n g 10% TCA w i t h  0.2 mM ATP and 1.0 mM orthophosphate  o r 10% TCA w i t h 0.2 mM ATP  or 10% TCA w i t h 1.0 mM orthophosphate (b)  It  r a t h e r than TCA a l o n e .  Time Course I n o r d e r t o determine  optimum p h o s p h o r y l a t i o n , membranes  (100 y l p r o t e i n ) i n 1000 y 1 of 30% s u c r o s e w i t h 20 mM TRIS H C l , ;  pH 7.8 a t 0°C, were p h o s p h o r y l a t e d  f o r 0, 15, 30, and 45 seconds 32  u s i n g 3.7 yM ATP made 28 mCi/mmol w i t h  [y-  P ]ATP.  The  r e a c t i o n s were t e r m i n a t e d as d e s c r i b e d e a r l i e r , and t h e samples p r o c e s s e d i n a s i m i l a r manner. (c)  Time Course  t e h Mg  2+  I n o r d e r to determine more p r e c i s e l y t h e n a t u r e o f 2+' d e p h o s p h o r y l a t i o n and Ca " p h o s p h o r y l a t i o n t h e  membranes were p h o s p h o r y l a t e d as above f o r 15 seconds a f t e r  5 7  - 57 -  w h i c h t h e sample was made 0.1 mM i n Mg f o r a further.15 seconds.  o r 0.5 mM i n Ca  The r e a c t i o n s were t h e n  terminated  as d e s c r i b e d i n p a r t ( a ) . C o n t r o l s were r u n c h e c k i n g 2+ p h o s p h o r y l a t i o n w i t h Mg and ATP added a t t h e same time f o r 1 5 2+ 30 seconds.  The same a p p l i e s t o Ca  .  Samples were p r o c e s s e d  as  usual. I.  E l e c t r o n Microscopy F4 and F5 were o b t a i n e d  d i r e c t l y from t h e s u c r o s e  g r a d i e n t and d e p o s i t e d on a M i l l i p o r e F i l t e r means o f a Sweeney S y r i n g e .  ( 0 . 2 2 u pore) by  The d e p o s i t e d membranes o r 0.5  mm  cubes o f m u s c l e were p o s t f i x e d f o r 1 hour w i t h K a r n o v s k y ' s f i x a t i v e (KARNOVSKY, 1969) i n 1% o s m i u m r t e t r o x i d e ,  stained f o r  1 hour i n s a t u r a t e d aqueous u r a n y l a c e t a t e , d e h y d r a t e d t h r o u g h a graded a l c h o l s e r i e s and embedded u s i n g s t a n d a r d i n Epon A r a l d l t e r e s i n .  Gold  procedure  s e c t i o n s c u t on a R e i c h a r t  OMU  3 ultramicrotome,  were s t a i n e d w i t h Reynolds l e a d  citrate,  and  examined under P h i l l i p s 300 t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e .  58-  - 58 -  RESULTS  A.  General The muscle i n t h e c h i c k e n stomach m u s c u l a r i s has been  shown t o c o n s i s t p r i n c i p a l l y of v i s c e r a l smooth muscle ( F i g u r e s 2 and 3) (CALHOUN, 1954).  As seen i n t h e e l e c t r o n m i c r o g r a p h s t h e  c e l l s a r e of v a r y i n g s i z e and each c e l l c o n t a i n s a c e n t r a l n u c l e u s . A l s o , p r e s e n t a r e m i t o c h o n d r i a , however, t h e r e was l i t t l e of s a r c o p l a s m i c r e t i c u l u m b e i n g p r e s e n t .  evidence  This l a t t e r p o i n t  being  e s p e c i a l l y i n t e r e s t i n g i n v i e w of t h e enzyme marker a s s a y s done f o r t h e s a r c o p l a s m i c r e t i c u l u m (SR).  Using P o l y t r o n  homogenization  of t h e smooth m u s c l e , d i f f e r e n t i a l c e n t r i f u g a t i o n , and  gradient  c e n t r i f u g a t i o n we o b t a i n e d membrane f r a c t i o n s e n r i c h e d i n plasma membranes ( F i g u r e 1, see page 40 ) .  The homogenization  t i m e s and  c e n t r i f u g a t i o n r a t e s were o p t i m i z e d t o y i e l d maximal s p e c i f i c and t o t a l a c t i v i t i e s o f 5' n u c l e o t i d a s e i n t h e f i r s t 5 l a y e r s of t h e s u c r o s e g r a d i e n t s . ( 4 5 seconds: F4 - 0.50 um/mg/hr, 0.005 um/mg/hr; F5 - 0.61 um/mg/hr, 0.006 um/hr: 60 seconds: F4 - 0.61 um/mg/hr, 0.007 .um/mg/hr; F5 - 0.90 um/mg/hr, 0.009 .um/hr . : : 75 s e c o n d s : F4 - 0.58 um/mg/hr, 0.022 um/hr; F5 - 0.99 um/mg/hr, 0.025 um/hr: 90 seconds: F4 - 0.47 um/mg/hr, 0.007 um/hr; F5 - 0.73 um/mg/hr, 0.006 um/hr). B.  Membrane Marker S t u d i e s The i s o l a t i o n o f t h e plasma membranes (PMs) was m o n i t o r e d  by measuring t h e s p e c i f i c a c t i v i t i e s of t h e enzymes l i s t e d T a b l e V I . Based on 5' n u c l e o t i d a s e a c t i v i t i e s , t h e PMs  in  were  59  -  F i g u r e 2.  59  -  E l e c t r o n m i c r o g r a p h o f c h i c k e n g i z z a r d smooth m u s c l e .  f i e l d shows a number of c e l l s .  The  M a g n i f i c a t i o n a t 12,000x.  PM  N / NM F i g u r e 3.  E l e c t r o n m i c r o g r a p h of a c h i c k e n g i z z a r d smooth muscle c e l l .  The f i e l d shows v a r i o u s o r g a n e l l e s (M - m i t o c h o n d r i a , n u c l e a r membrane, PM - plasma membrane).  N - n u c l e u s , NM -  M a g n i f i c a t i o n a t 21,000x.  Table V I T o t a l and s p e c i f i c a c t i v i t i e s o f s e l e c t e d marker enzymes a t v a r i o u s  s t a g e s of t h e f r a c t i o n a t i o n p r o c e d u r e . The  s p e c i f i c a c t i v i t i e s a r e i n umole / mg p r o t e i n / hour, t h e t o t a l a c t i v i t i e s a r e i n ymole / hour.  Marker Enzyme  Residue  Filtrate  a c i d phosphatase specific activity total activity % yield  0.033 0.65 11.4  0.044 5.01 89.0  0.046 0.91  0.026 2.96  2,000 x g pellet 0.026 0.24 4.0  2,000 x g supernatant  15,000 x g pellet  15,000 x g supernatant  0.056 4.36 77.0  0.036 0.15 2.6  0.056 4.62 81.0  0.057 0.29 5.0  0.032 3.99 70.0  0.068 5.30  0.051 0.18  0.017 1.40  0.063 0.33  0.026 3.24  100,000 x g pellet  100,000 x g supernatant  4-  K - stimulated a c i d phosphatase specific activity total activity  0.10 0.94  succinate dehydrogenase specific activity total activity % yield  0.75 14.80 8.0  1.45 165.00 92.0  8.59 80.70 45.0  1.24 97.00 54.0  2.88 14.40 8.0  0.96 79.04 41.6  2.87 14.80 8.2  0.53 66.50 37.0  NADPH c y t c_ reductase specific activity total activity % yield  0.21 4.17 14.0  0.23 26.11 86.0  0.37 3.43 11.0  0.16 12.50 41.5  0.72 3.60 12.0  0.07 10.20 34.0  0.17 0.86 2.8  0.05 6.25 21.0  0.031 0.59 15.0  0.030 3.42 85.0  0.072 0.68 17.0  0.040 3.12 78.0  0.310 1.60 40.0  0.005 0.62 16.0  ;  5  nucleotidase specific activity total activity % yield 1  0.077 0.27 6.7'  0.028 2.26 56.0  Table  Vila  S p e c i f i c a c t i v i t i e s o f marker enzymes. F r a c t i o n s o b t a i n e d from sucrose g r a d i e n t s . S p e c i f i c a c t i v i t i e s i n ymole/mg p r o t e i n / h o u r . See Table V l l b a l s o . Fraction Number  5 ' nucleotidase  Mg  2 +  ATPase  3  I c.p.m./yg p r o t e i n 1  2  5  succinate dehydrogenase  NADH c y t c reductase  15  Protein (yg)  % sucro:  1  0.05  0.33  420  1.20  1.09  563  2  0.86  8.06  703  0.34  0.63  775  27.0  3  0.93  17.25  714  n.d.  n.d.  500  30.0  4  0.57  8.97  743  2.29  0.11  387  32.0  5  1.11  23.30  760  0.37  0.01  263  34.0  8.0  6  1.10  22.72  690  0.37  0.01  225  35.0  7  0.27  19.86  420  10.39  0.62  263  36.5  8  0.17  14.57  310  9.90  1.70  200  40.0  9  0.12  14.43  200  0.11  0.65  113  42.0  10  0.05  4.20  n.d.  4 .58  1.13  250  43.0  11  0.07  n.d.  n.d.  0.09  0.28  463  44.0  12  0.04  n.d.  n.d.  n.d.  n.d.  375  45.0  13  0.01  n.d.  n.d.  n.d.  n.d.  100  66.0  a  S p e c i f i c a c t i v i t i e s i n ymole/:mg p r o t e i n / m i n u t e • b „ ^ A n t i m y c i n s e n s i t i v e NADH cyt* c r e d u c t a s e . Refers to  I ON  T a b l e .VIlb S p e c i f i c a c t i v i t i e s o f marker enzymes. F r a c t i o n s o b t a i n e d from s u c r o s e g r a d i e n t s . NADH c y t c r e d u c t a s e a c t i v i t y measured i n t h e p r e s e n c e o f A n t i m y c i n A. S p e c i f i c a c t i v i t i e s i n umol/ mg p r o t e i n / h o u r . The r e s u l t s o f t h i s t a b l e c a n be compared d i r e c t l y t o those o f Table V i l a . Fraction Number  NADH c y t c_ reductase  NADPH c y t c reductase  glucose-6phosphatase  a c i d phosphatase -K rfc"" +  1  Protein (yg)  % sucrose  1  3.24  0.23  0.11  0.19  0.25  563  2  7.08  0.63  0.25  0.21  0.31  775  27.0  3  7.50  0.34  0.11  0.28  0.39  500  30.0  4  4.70  0.44  0.09  0.10  0.11  387  32.0  5  4.60  0.28  0.09  0.05  0.05  263  34.0  6  4.80  0.23  0.04  n.d.  n.d.  225  35.0  7  6.67  0.37  0.07  n.d.  n.d.  263  36.5  8  4.38  0.32  0.03  0.09  0.10  200  40.0  9  0.24  0.25  0.03  0.11  0.13  113  42.0  10  0.81  0.09  0.10  n.d.  n.d.  250  43.0  11  2.82  0.18  0.12  n.d.  n.d.  463  44.0  12  n.d.  n.d.  0.01  n.d.  n.d.  375  45.0  13  n.d.  n.d.  n.d.  n.d.  n.d.  100  66.0  8.0  63 -  p u r i f i e d t e n f o l d d u r i n g the f i r s t f i v e s t e p s of the procedure ( F i g u r e 1 ) . the PMs values  isolation  I n the p r o c e s s of the g r a d i e n t c e n t r i f u g a t i o n ,  were p u r i f i e d another 2 to 4 f o l d based on 5' (Tables V i l a and V l l b ) , the  specific  5' n u c l e o t i d a s e b e i n g h i g h e s t i n F r a c t i o n 5. u s i n g 5' n u c l e o t i d a s e as a PM marker was  nucleotidase  a c t i v i t y of The v a l i d i t y o f  checked by  using  l a c t o p e r o x i d a s e c a t a l y z e d i o d i n a t i o n of the PM p r i o r to h o m o g e n i z a t i o n . T a b l e V i l a shows t h a t F r a c t i o n 4 (F4) and F r a c t i o n 5 (F5) c o n t a i n e d 125 the h i g h e s t s p e c i f i c a c t i v i t y of I . The d i f f e r e n c e between 125 the  I l a b e l l i n g r e s u l t s and  the 5' n u c l e o t i d a s e r e s u l t s gave us  the f i r s t i n d i c a t i o n s t h a t t h e r e may  be a d i f f e r e n c e between the  o r i e n t a t i o n of the membranes i n F4 compared to F5. The c o n t a m i n a t i o n  of the v a r i o u s PM g r a d i e n t f r a c t i o n s was  f o l l o w e d by s t a n d a r d markers f o r the SR, m i t o c h o n d r i a , and Mitochondria, assessed  lysozomes.  u s i n g s u c c i n a t e dehydrogenase and NADH -.v..  a n t i m y c i n A s e n s i t i v e c y t c^ r e d u c t a s e a c t i v i t i e s , were removed mainly  i n the 2,000g c e n t r i f u g a t i o n s t e p .  T h i s r e s u l t was  quite  s u r p r i s i n g s i n c e h i g h e r g f o r c e s a r e u s u a l l y r e q u i r e d to sediment out m i t o c h o n d r i a .  D e s p i t e t h i s r e s u l t , t h e r e was  s t i l l a 2 to 4  f o l d i n c r e a s e i n the s p e c i f i c a c t i v i t y of s u c c i n a t e dehydrogenase i n the m i c r o s o m a l p e l l e t , f o l l o w e d by a f u r t h e r 4 to 5 f o l d increase after gradient centrigugation. s p e c i f i c a c t i v i t y was  This increase i n  found i n F r a c t i o n s 7 and 8 o f the  gradient.  K  mainly  lysozomes, showed l i t t l e i n c r e a s e i n s p e c i f i c  in  +  sucrose  s t i m u l a t e d a c i d phosphatase, thought to be found  a t any stage of the PM i s o l a t i o n p r o c e d u r e .  activity  Maximal s p e c i f i c  a c t i v i t i e s were found i n the f i r s t 3 l a y e r s of the g r a d i e n t .  64  - 64 -  While dependable markers f o r the SR a r e r a t h e r c o n t r o v e r s i a l , i t has been f e l t  t h a t NADH a n t i m y c i n A i n s e n s i t i v e c y t c_ r e d u c t a s e  was s p e c i f i c f o r the SR.  R e c e n t l y , some doubt has been c a s t on  t h i s h y p o t h e s i s as PMs and other o r g a n e l l e s a r e thought the above mentioned enzyme as w e l l (SOTTOCASA e t a l . , SOTTOCASA,  1971; KELBERG & CHRISTENSEN, 1979).  glucose-6-phosphatase the SR. used  to contain 1967;  Similarily  i s no l o n g e r regarded as b e i n g s p e c i f i c f o r  However, NADPH c y t c^ r e d u c t a s e , one o f the more commonly  SR markers, i s thought  to be s p e c i f i c f o r these membranes.  Based on s t u d i e s w i t h t h i s enzyme, the SR was l o c a l i z e d to f r a c t i o n s 1 to 3, 7, 10 and 11.  There was d e f i n i t e  contamination  of  F r a c t i o n s 4 and 5 but t h i s r e p r e s e n t e d l e s s than a 2 f o l d i n c r e a s e  in  s p e c i f i c a c t i v i t y over the f i l t r a t e .  glucose-6-phosphatase  The d i s t r i b u t i o n s o f  and NADH a n t i m y c i n A i n s e n s i t i v e c y t c,  r e d u c t a s e were n o t e x a c t l y the same as t h a t of NADPH c y t c^ reductase. section  The exact meaning of t h i s i s t r e a t e d i n the D i s c u s s i o n  (see a l s o T a b l e s V i l a and V l l b ) . Based on the above r e s u l t s , F4 and F5 were judged  p u r e s t PM f r a c t i o n s o b t a i n e d from to and  the g r a d i e n t .  to be the  They were shown  c o n s i s t o f v e s i c l e s under the e l e c t r o n microscope  (Figures 4  5) but no q u a n t i t a t i v e r e s u l t s were d e r i v e d from the e l e c t r o n  micrographs  other than v e s i c l e s i z e  (which appeared  quite variable).  F u r t h e r c h a r a c t e r i z a t i o n o f the membranes u s i n g g e l e l e c t r o p h o r e s i s , showed the PAS and coomassie b l u e s t a i n i n g p r o f i l e s of F4 and F5 to be the same ( F i g u r e s 15 and 16, T a b l e I X ) . i n d i c a t e t h a t any d i f f e r e n c e s observed appear t o a r i s e from  These r e s u l t s would :'.  i n membrane p r o p e r t i e s do n o t  s t r u c t u r a l heterogeneity.  65  -65-  Figure  4. E l e c t r o n micrograph of F r a c t i o n 4 plasma membranes i s o l a t e d from  the c h i c k e n  Figure  g i z z a r d smooth muscle. M a g n i f i c a t i o n  a t 47,000x.  5. E l e c t r o n micrograph o f F r a c t i o n 5 plasma membranes i s o l a t e d from  the c h i c k e n  g i z z a r d smooth muscle. M a g n i f i c a t i o n  a t 60,000x.  - 66 -  C.  Plasma Membrane Mg  2+  Stimulated  ATPase A c t i v i t i e s  (a) O p t i m i z a t i o n of Mg2+ s t i m u l a t e d ATPase a c t i v i t y When the g r a d i e n t f r a c t i o n s were being to contamination, activity.  characterized with  respect  attempts were made to observe some N a / K 2+ +  +  As shown i n T a b l e V i l a , o n l y a v e r y a c t i v e Mg  ATPase was observed.  ATPase stimulated  Maximal s p e c i f i c a c t i v i t y was observed i n F5,  t h i s i n c o n t r a s t , to the r a t h e r low s p e c i f i c a c t i v i t y observed i n F4.  These r e s u l t s when combined w i t h  r e s u l t s , provided  f u r t h e r evidence  the aforementioned PM marker  f o r F4 having  the o p p o s i t e  o r i e n t a t i o n to F5.  I t was f e l t a t t h i s time t h a t a thorough a n a l y s i s 2+ should be done of the Mg s t i m u l a t e d ATPase a c t i v i t y observed i n F5 2+ s i n c e there have been many r e p o r t s of s i m i l a r Mg ATPases i n other smooth muscle p r e p a r a t i o n s . Some i nsvteismtuil ga at e t do r s 2+ now now c co on ns si id de er r Mg Mg sstimu. t i m u l a t e d ATPase as a s p e c i f i c PM marker ( VALLIERES  e t a l . , 1978)  Using  F5 , the s p e c i f i c a c t i v i t y of the Mg 2+  ATPase was o p t i m i z e d of o p t i m a l a c t i v i t y  f o r Mg  and ATP  the enzyme was  2+  stimulated  ( F i g u r e 6) and under c o n d i t i o n s  t e s t e d f o r N a , L i and  K  +  (Figure 7). K  +  +  s e n s i t i v i t y , ouabain i n h i b i t i o n and pH e f f e c t s  s t i m u l a t i o n was o n l y observed a t v e r y h i g h pH v a l u e s ,  +  while  ouabain and the v a r i o u s o t h e r c a t i o n s t e s t e d had l i t t l e  effect.  The pH optima of the enzyme was 7.6 a t 37°C.  stimulation 2+  was t r i e d no e f f e c t was noted.  When C a ^  +  The enzyme appeared to be a Mg  s t i m u l a t e d ATPase. Assumingthat [MgATP] of v e l o c i t y v e r s u s  was  the enzyme s u b s t r a t e , a p l o t  s u b s t r a t e c o n c e n t r a t i o n was done ( F i g u r e 8 ) .  67  - 67 -  F i g u r e 6. O p t i m i z a t i o n o f Mg P l o t of s p e c i f i c various  a c t i v i t y o f Mg  ATPase a c t i v i t y i n F r a c t i o n 5. 2+  [ ATP ] = 0.00 mM;  [ ATP ] = 0.10 mM;  O,  , [ ATP ] = 4.00 mM.  mean o f 5 v a l u e s .  •,  [ ATP ] = 0.05 mM;  [ ATP ] = 0.20 mM;  , [ ATP ] =0.80 mM;^,  mM; A  C O Ucl-L  at  [ ATP ] . ATPase assay was as d e s c r i b e d under " M a t e r i a l s  and Methods " . A ,  O  2-f [ Mg ]  ATPase v e r s u s  [ ATP ] = 0.40 mM;  [ ATP ] = 1.20 mM; • Specific  activities  •,  , [ ATP ] = 2.00 represent  the  SPECIFIC ACTIVITY in jjmoles Pj/minmg protein 01  o  cn  8  S  8  - 69 -  F i g u r e 7. E f f e c t s o f pH, Na, K, L i and o u a b a i n on t h e Mg"'  ATPase  a c t i v i t y o f F r a c t i o n 4 (bottom) and F r a c t i o n 5 ( t o p ) . F r a c t i o n 4: A , 0.40 mM  ATP; # , 0.40 mM  ATP + 0.20 mil M g ; O ,  mM  + 120 mM Na*; A ,  0.40 mM  Mg  20 mM  2 +  K ; • , 0.40 mM +  ATP + 0.20 mM M g (all ATP I  2 +  2 +  ATP + 0.20 mM M g * + 120 mM N a 2  ATP 4 0.20 mM  + 120 mM N a  +  0.40 mM ATP + 0.20  Mg  4 20 mM K  4 120 mM L i ; • , 0.40 mM  2 +  +  +  4 1 mM o u a b a i n . F r a c t i o n 5  c o n c e n t r a t i o n s as f o r F r a c t i o n 4 ) : A , ATP; • , ATP + M g ; 2 +  + Mg  2 +  + N a ; A , ATP + M g  , ATP 4 M g  +  2 +  4 Na  mean o f 5 v a l u e s .  +  4 K  +  2 +  + Na  +  +  +  4 K "; 1  4 ouabain. S p e c i f i c  • , ATP * M g  2 +  O,  + L i ;  a c t i v i t i e s are  +  the  SPECIFIC ACTIVITY  in  iimoles Pj/minmg protein oi  o  SPECIFIC ACTIVITY in jumoles Pj/minmg protein oi  _ L  o  _ L  cn  ro o  ro en  01  -71-  30h  c  «_i  -6  i  i  i  i  t  -5  -4  -3  -2  -1  LOG F i g u r e 8. P l o t of r a t e v e r s u s  [SUBSTRATE] l o g a r i t h m of the  substrate  [ MgATP ]  for  +2 the Mg  ATPase observed i n F r a c t i o n 5. The  high substrate  concentrations.  enzyme appears to be  inhibited  by  -  F i g u r e 10. muscle c e l l  72  -  Phase c o n t r a s t m i c r o g r a p h of an i s o l a t e d  ( see arrows ).  Magnification  160x.  smooth  -  The  73 -  r e s u l t s a r e r e p r e s e n t a t i v e of an enzyme t h a t i s i n h i b i t e d  by h i g h s u b s r t a t e c o n c e n t r a t i o n s . Based on t h e maxima, we might -5 -3 c a l c u l a t e a Km of 5 x 10 M, K s ' o f 2 x 10 M and a Vmax of 2.5 x 10^ M/sec.  As we s h a l l see l a t e r  the assumption t h a t  [MgATP]  2  i s the s u b s t r a t e , has to be somewhat m o d i f i e d . The presence of the 2+ Mg s t i m u l a t e d ATPase on t h e e x t e r n a l s u r f a c e o f F5's PM 2+ c o r r e l a t e d w e l l w i t h the ecto Mg s t i m u l a t e d ATPase a c t i v i t y noted i n suspensions of s i n g l e smooth muscle c e l l s d e r i v e d from t h e 2+ c h i c k e n g i z z a r d ( F i g u r e s 9 and 10). The ecto Mg stimulated 2+ ATPase on t h e c e l l s had s i m i l a r c h a r a c t e r i s t i c s to the Mg s t i m u l a t e d ATPase found i n F5. (b) P h o s p h o r y l a t i o n All  o f the plasma membranes i n F4 and F5 -  the r e s u l t s i n t h i s s e c t i o n a r e d i r e c t l y comparable.  The  amounts of p r o t e i n , as judged by g e l e l e c t r o p h o r e s i s , were i d e n t i c a l i n a l l runs.  The ATPase was f u r t h e r c h a r a c t e r i z e d by p h o s p h o r y l a t i o n 32 32 of F4 and F5 PMs w i t h [ Y - P] ATP. F o r F5 i n the presence o f [ p] 32 ATP o n l y , the P i n c o r p o r a t e d was maximized w i t h r e s p e c t to 32 i n c u b a t i o n times used  ( F i g u r e 11).  I n the presence of [  p] ATP,  F5 e x h i b i t e d t h r e e major peaks; Peak A - 205,000 d a l t o n s ; Peak B 165,000 d a l t o n s ; Peak C - 145,000 d a l t o n s . wi th [  p] ATP, o n l y a s m a l l amount of  When F4 was l a b e l l e d  P was i n c o r p o r a t e d when  compared w i t h F5 ( F i g u r e s 12a, 12b and 12c) . toward an ATP b i n d i n g s i t e being surface only, while  These r e s u l t s p o i n t  a c c e s s i b l e from the e x t e r n a l PM  the f a c t t h a t some F4 was l a b e l l e d may have  been i n d i c a t i v e o f some of the v e s i c l e s i n t h i s f r a c t i o n  being 32 e i t h e r l e a k y , unsealed o f o f mixed RO/IO o r i e n t a t i o n . [ p] ATP 2+ 32 added i n the presence of Mg caused a decrease i n the P  74  -  74 -  i n c o r p o r a t i o n of Peak A i n F5 ( F i g u r e 12b) .  A decrease o f 35%  was seen i n Peak B o f F5, t h i s b e i n g h a l f o f t h a t noted f o r Peak A ( F 5 ) , whereas i n F4 no d e t e c t a b l e changes were noted. When 2+ 2+ Ca was s u b s t i t u t e d f o r Mg i n F5 a s l i g h t decrease was noted i n the p h o s p h o r y l a t i o n of Peak A, t h i s compared to a 50% i n c r e a s e i n 32 the  P i n c o r p o r a t i o n of Peak B ( F i g u r e 12c).  A g a i n , no r e a l  e f f e c t was noted i n F4. 2+ To show t h a t Mg was promoting d e p h o s p h o r y l a t i o n o f the 32 2+ bound P i n F5, a time d e l a y study was done i n which Mg was 32 added 15 seconds a f t e r t h e a d d i t i o n of [ P ] ATP. The r e a c t i o n was t e r m i n a t e d a f t e r an a d d i t i o n a l 15 seconds. The r e s u l t s 2+ ( F i g u r e s 14a and 14b) show t h a t Mg promotes d e p h o s p h o r y l a t i o n 2+ 2+ of Peak A. When Ca r e p l a c e d Mg i n the time d e l a y experiment 32 ( F i g u r e 14a), a 3 f o l d i n c r e a s e was noted i n the P incorporation of Peak B.  Peak A showed l i t t l e  or no i n c r e a s e .  i n F i g u r e 14a, a d d i t i o n o f hydroxlamine  As i l l u s t r a t e d 32 reduced the P activity 32  i n c o r p o r a t e d i n t o F5 membranes ( l a b e l l e d wi t h [  p]  ATP) which  i n d i c a t e s t h a t the l a b e l l e d p h o s p h o r y l groups i n Peaks A, B and C were bound to a c y l m o i e t i e s . A and B appear  Both t h e enzymes i n v o l v e d i n Peak  to have s i t e s l o c a l i z e d e x t e r n a l l y on the F5  membranes b u t these s i t e s a r e i n a c c e s s i b l e i n F4 due to an apparent difference i n orientation. 32 and C) a r e b i n d i n g Mg^  +  A l l p h o s p h o r y l a t e d enzymes  P by a c y l m o i e t i e s .  (Peaks A, B  The c o r r e l a t i o n of the  s t i m u l a t e d ATPase a c t i v i t y a t 37°C w i t h t h e l a b e l l i n g by ^ P  a t 205,000 d a l t o n s i s extremely tempting.  2  Based on these r e s u l t s  t h i s may be p a r t i a l l y j u s t i f i a b l e , but i t i s by no means c e r t a i n .  7-5  -  -75  •  •  •  •  •  i  5  10  15  20  25  GEL SLICE F i g u r e 11. P h o s p h o r y l a t i o n  i  30  i  35  i  i  40  i  45  NUMBER  p a t t e r n s of F r a c t i o n 5 at v a r i o u s  incubation  32 times u s i n g  [ y-  P ] ATP.  Procedure i s as d e s c r i b e d under " M a t e r i a l s  Methods " . P e a k assignments are as f o l l o w s r A - 205,000 MW; C - 14 5,000 MW.  Peaks i n F i g u r e s 12 to 14 a r e a s s i g n e d  B - 165,000  similarly.  and MW;  F i g u r e 12. P h o s p h o r y l a t i o n  p a t t e r n s of  F r a c t i o n 4 (•—•)  and F r a c t i o n 5 ( Q — O ) . C o n d i t i o n s a r e  as d e s c r i b e d under " M a t e r i a l s and Methods ". ( a ) : 3 .74 uM ATP ( [ y 3.74  uM ATP  ] ATP,  32  nmol ) + 0.50 mM Ca  Mg ; 2+  ( [ y- V  2+  .  40 Ci/nmol  ) + 0.10  mM  3 2  (c) 3.74 uM  P  ] ATP, 40 Ci/nmol ) ; (b) : ATP ( [  y -  3  2  ?  ] ATP, 40 C i /  [ P] DPM INCORPORATED 32  [ P] DPM INCORPORATED 32  F i g u r e 13. P h o s p h o r y l a t i o n contained Li  +  (.#—•) and F r a c t i o n 5 (O—O) • A l l media  3.74 uM ATP ( [ Y ~ P ] ATP, 40 Ci/nmol ) . ( a ) : ATP + 0 1 0 mM Mg"*" + 120 mM N a o r 3 2  or c h o l i n e ; ( b ) : ATP + 0.10 mM  2+ Ca  p a t t e r n s of F r a c t i o n 4  + +• + 120 mM Na or L i , These  2  +  t  Mg  2 +  results  +0.50 mM  Ca  2 +  ;  ( c ) : ATP +• 0.10 mM M g  2 +  + 0.50  a r e d i r e c t l y comparable to F i g u r e s 11 t o 14.  mM  [ P] D P M INCORPORATED  [ P] D P M INCORPORATED  32  _L  o o  ho o o  co o o  32  ^. o o  N>  1  T  CO  I  [ P] D P M INCORPORATED 32  -i  -u-  ro  CO  Js,  F i g u r e 14. P h o s p h o r y l a t i o n p a t t e r n s o f F r a c t i o n 5. A l l media c o n t a i n e d 3.74 uM ATP ( [ jATP,  40  Ci/nmol ) . ( a ) : (O—O), ATP + 0.10 mM  M g ; ( • — # ) , ATP + 0.50 mM C a 2 +  2 +  (the  P ] latter 2+  being  added  15 seconds  after  the a d d i t i o n  of  the ATP), (b) : ( O — O ) ,  l a t t e r b e i n g added 15 seconds a f t e r the a d d i t i o n o f the membranes p h o s p h o r y l a t e d w i t h ATP f o r 15 seconds.  ATP + 0.10 mM Mg  (the  ATP); ( # — • ) , hydroxylamine treatment o f  [ P] DPM INCORPORATED 32  hO O O  - L  •  O O  1  1  CO O O I  ^. o O 1  [ P] DPM INCORPORATED 32  -i  -IS-  N>  CO  ^.  -  8-2 -  F i n a l l y F5, i n the p r e s e n c e of Mg ( F i g u r e 13b), showed a major decrease Peak B showed l i t t l e o r no change. effect.  2+  , Ca  2+  i n Peak A  and [  32 P ]  ATP  phosphorylation.  F4 as b e f o r e showed no r e a l  These r e s u l t s f o r F5 may be due to c o m p e t i t i o n  of the  two c a t i o n s f o r a s i n g l e b i n d i n g s i t e i n Peak B of they a r e due to the e f f e c t s of more than one b i n d i n g s i t e i . e . independent s i t e s 2+ 2+ 2+ f o r Mg and Ca . Mg i s promoting d e p h o s p h o r y l a t i o n of t h i s 2+ Peak B, w h i l e Ca i s promoting p h o s p h o r y l a t i o n . When the 32 2~f" membranes were p h o s p h o r y l a t e d i n the presence of [ P ] ATP, Mg and 120 mM N a or L i or c h o l i n e , no i n c r e a s e i n p h o s p h o r y l a t i o n +  +  of any of the t h r e e peaks was noted i n F4 o r F5 ( F i g u r e 13a). 2+ However when Ca was added to the above i n c u b a t i n g media, non ->cic s p e c i f i c i n c r e a s e s i n Peaks A and B p h o s p h o r y l a t i o n were seen ( F i g u r e 13c). We can e x p l a i n the i n c r e a s e in,Peak B s i n c e we 2+ know Ca increases i t s phosphorylation. However, the i n c r e a s e i n 2+ Peak A i s much more conformational  puzzling.  Whether the Ca  i s causing  large  changes i n the membrane enzymes i s u n c e r t a i n .  For each of the above experiments e x h a u s t i v e c o n t r o l 32 s t u d i e s were done. To ensure t h a t the P bound was n o t adsorbed r 32 32 3 [ P ] ATP o r PO^ , r e a c t i o n s were stopped by means o t h e r than the a d d i t i o n of 5% TCA (see Methods s e c t i o n ) . To see whether 32 p h o s p hp oh ro ys lp ah to ir oy nl a twas f f e cmeasured t e d by t r ua sc ien gi m 20-30 p u r i t i feosl di nlower the [y s-p e c iPf i]c ATP, i o n ewas 32 a c t i v i t i e s of [y - P ] ATP. The same r e s u l t s wei were o b t a i n e d 32 r e g a r d l e s s of the s p e c i f i c a c t i v i t y of t h e [ y - P ] ATP used. T  Our o r i g i n a l h y p o t h e s i s t h a t [ MgATP ] i s the s u b s t r a t e 2+ f o r the Mg s t i m u l a t e d a c t i v i t y observed has to be m o d i f i e d somewhat. I t appears t h a t ATP i s b i n d i n g p r i o r to the b i n d i n g of 24* 2— Mg . The i n t e r m e d i a t e may s t i l l be t h e [ MgATP] but t h i s i s purely  conjecture.  .  .83  - 83 -  D.  O r i e n t a t i o n S t u d i e s Using I t was f e l t  of d i f f e r i n g out  A c e t y l c h o l i n e s t e r a s e and S i a l i c  a t t h i s stage  orientation.  Acid  t h a t F4 and F5 were c e r t a i n l y  F4 appeared t o be e n r i c h e d  in  inside-  (10) v e s i c l e s w h i l e F5 was thought to c o n t a i n predominantly  right-side-out  (RO) v e s i c l e s .  This hypothesis  c o n c l u s i v e a t t h i s stage so i t was d e c i d e d  was s t i l l  f a r from  t o c h a r a c t e r i z e F4 and  F5 w i t h r e s p e c t to t h e i r o r i e n t a t i o n u s i n g a c e t y l c h o l i n e s t e r a s e (AchE) and s i a l i c localized  a c i d sidedness  on the c e l l PM.  a s s a y s , b o t h being e x t e r n a l l y  A 1.5 f o l d  i n c r e a s e i n AchE  specific  a c t i v i t y was observed i n F4 when both membrane s u r f a c e s were made e q u a l l y a c c e s s i b l e t o the s u b s t r a t e by the a d d i t i o n o f 0.05% TX-100 prior  t o the assay (Table V I I I ) .  sialic  S i m i l a r i l y , a f t e r ; c l e a v a g e of  a c i d by neuraminadase t h e r e was a 3 f o l d  accessible s i a l i c  a c i d content  increase i n  of F4 w i t h the a d d i t i o n o f TX-100.  F5 membranes showed l i t t l e or no i n c r e a s e i n AchE s p e c i f i c a c t i v i t y and s i a l i c If  a c i d content  i n the presence of 0.05% TX-100.  one examines the a c t u a l i n c r e a s e s i n the s p e c i f i c a c t i v i t y o f  AchE and s i a l i c we f i n d  a c i d content  f o r F4 w i t h  the a d d i t i o n of TX-100,  t h a t the r e l a t i v e i n c r e a s e s of the two markers d i f f e r s .  T h i s appears to i n d i c a t e t h a t t h e r e i s n o t a homogeneous d i s t r i b u t i o n of these markers on the membrane s u r f a c e .  I t should  a l s o be p o i n t e d out t h a t the AchE s p e c i f i c a c t i v i t y was h i g h e s t i n F r a c t i o n s 4 to 7.  S i a l i c a c i d was found to be h i g h e s t i n  F r a c t i o n 8, the m i t o c h o n d r i a l  E.  Iodination  enriched  fraction.  Studies  I o d i n a t i o n was the next step i n the i n v e s t i g a t i o n of membrane o r i e n t a t i o n .  The i n v e s t i g a t i o n began w i t h the l a b e l l i n g  . . .  84  Table  VIII  A c c e s s i b i l i t y of markers i n sucrose g r a d i e n t f r a c t i o n s . S u l p h u r i c a c i d heading r e f e r s t o t a l nanomoles of N - a c e t y l n e u r a m i c a c i d / mg The  c o n c e n t r a t i o n of d e t e r g e n t used was  Sucrose g r a d i e n t f r a c t i o n number  0.05%  acetylcholinesterase - T r i t o n X-100 + T r i t o n X-100  protein  p r e s e n t i n each  T r i t o n X-100  to  assayed : f r a c t i o n .  v/v.  sialic acid^ +• T r i t o n X-100 + Sulphuric  3  - T r i t o n X-100  1  1.21  3.36  3.82  3.80  3.90  2  11.22  9.13  3.22  2.90  3.15  3  9.05  10.80  9.99  10.16  10.01  4  8.00  13.60  3.01  10.38  10.50  5  9.17  9.69  10.19  8.90  9.86  6  10.08  11.24  9.01  9.32  9.30  7  8.05  12.06  5.51  7.50  8.00  8  0.84  3.68  10.19  26.16  9  3.70  5.51  2.43  3.76  3.63  10  4.12  6.28  10.50  16.34  17.17  11  1.06  1.07  n.d.  n.d.  1.03  12  n.d.  n.d.  n.d.  n.d.  n.d.  13  n.d.  n.d.  n.d.  n.d.  n.d.  Expressed as nanomoles of product per m i l l i g r a m  protein  Expressed as nanomoles of N - a c e t y l n e u r a m i c a c i d per  per  minute.  milligram  protein.  28.67  Acid  - 85 -  Table IX M o l e c u l a r weight assignments o f bands and peaks d e p i c t e d in' F i g u r e s 15 to 41. Assignments a r e based on standard m o l e c u l a r weight markers as d e s c r i b e d under " M a t e r i a l s and Methods ".  Band 1 2  Peak A, r  Molecular  Weight  207,000  -  205,000 - 210,000  4  -  191,000  5  B  165,000  6  C  136,000 - 145,000  7  -  130,000  8  s  100,000  9  -  82,000  10  t  55,000  11  -  45,000  12  -  13  -  14  -  31,000  15  -  93,000  114'  r'  207,000  115*  s'  100,000  : 16'  t*  55,000  3  T r a c k i n g Dye _  -86-  1  2  3  4  5  6  7  8  9  GEL LENGTH (cm)  F i g u r e 15. of F r a c t i o n used f o r PAS  Coomassie b l u e s t a i n i n g p a t t e r n  4.  Protein  staining  (top)  and  used f o r coomassie b l u e s t a i n i n g (30  yg).  PAS (15  profile yg)  was  (bottom) half  that  -87-  1  2  3  4  5  6  7  8  9  GEL LENGTH (cm)  F i g u r e 16.  Coomassie b l u e s t a i n i n g p a t t e r n  of F r a c t i o n  5. P r o t e i n  used f o r PAS  staining  (top)  and  used f o r coomassie b l u e s t a i n i n g (30  yg).  PAS (15  profile yg)  was  (bottom) half  that  - 88  -  of the PM e x t e r n a l l y u s i n g 2 mm cubes o f m u s c l e , c e l l and  s i n g l e c e l l s u s p e n s i o n s ( F i g u r e s 17,18, and 19). 125  cubes ( F i g u r e 17) were l a b e l l e d w i t h Band 16' (55,000 MW, 14' (205,000 MW,  sheets, When muscle  I , l a b e l l i n g was seen a t  peak t ' ) . C e l l s h e e t s were l a b e l l e d a t Band  peak r ' ) and Band 16'.  l a b e l l i n g i n the 100,000 MW r e g i o n .  There was no prominent  The l a b e l l i n g of s i n g l e c e l l  s u s p e n s i o n s ( F i g u r e 19) y i e l d e d s i m i l a r r e s u l t s , however, t h e r e was a n o t a b l e i n c r e a s e i n t h e l a b e l l i n g of the 100,000 MW (Band 15', peak s ' ) .  region  I t should be p o i n t e d out t h a t i f c e r t a i n 125  r e g i o n s of t h e p r o t e i n p r o f i l e have a low  I specific  activity,  i t does n o t i m p l y t h a t the PM p r o t e i n s have been l a b e l l e d w i t h a low s p e c i f i c a c t i v i t y .  These g e l s ( F i g u r e s 17 t o 19) i n c l u d e t h e  PM p r o t e i n s and a l l o t h e r c e l l u l a r p r o t e i n s .  Based on t h e above  d a t a we can c o n c l u d e t h a t a t l e a s t two r e g i o n s of the PM a r e a c c e s s i b l e to the l a b e l l i n g species e x t e r n a l l y . Next, the l a b e l l i n g of the m u s c l e cubes and c e l l was r e p e a t e d .  However, t h i s time the m u s c l e t i s s u e was homogenized  and p r o c e s s e d t o y i e l d F4 and F5. 125 then examined f o r a t 205,000 MW 55,000 MW  The c o l l e c t e d F4 and F5 were  I i n c o r p o r a t i o n , F4 showing l a b e l  (Band 1, peak r ) , 100,000 MW  Upon r e - l a b e l l i n g  ( F i g u r e s 21 and 2 3 ) .  MW.  E x a m i n a t i o n o f F5 f o r  i n i t i a l i n c o r p o r a t i o n showed the l a b e l a t 205,000 MW, loading i n these regions.  and  I , l a b e l l i n g was o n l y noted a t 100,000  F5 showed a r a t h e r d i f f e r e n t b e h a v i o r . 55,000 MW  incorporated  (Band 8, peak s)  (Band 10, peak t ) ( F i g u r e s 20 and 2 2 ) . 125  of t h i s f r a c t i o n w i t h  and  sheets  100,000  MW  R e - l a b e l l i n g increased the  Not o n l y do these p r e l i m i n a r y r e s u l t s  p r o v i d e f u r t h e r e v i d e n c e f o r the p r e f e r r e d o r i e n t a t i o n s o f F4 and F5 b u t they a l s o p r o v i d e us w i t h i n f o r m a t i o n c o n c e r n i n g  the  d i s p o s i t i o n o f c e r t a i n p r o t e i n s i n the membrane. 89  -89-  0.4  1,000  r  CL Q CO ID  0.2  H500  <  LO CM  0"-  1  2 3 4 5 6 7 8 9  GEL LENGTH (cm)  F i g u r e 17. L a b e l l i n g o f muscle cubes w i t h  125  d e s c r i b e d under " M a t e r i a l s and Methods ". (•  I . The procedure  used i s  ), coomassie b l u e s t a i n i n g 125  p a t t e r n o f i o d i n a t e d muscle; (•-#), i o d i n a t i o n muscle cubes.  p a t t e r n of  I  labelled  -90-  F i g u r e 18. L a b e l l i n g of c e l l sheets w i t h d e s c r i b e d under " M a t e r i a l s and Methods ".  ±  (  i  J  I.  The  procedure  used  is  ), coomassie b l u e s t a i n i n g 125  pattern  of i o d i n a t e d  labelled c e l l  sheets.  cell  sheets;  ()•—•) , iodination  p a t t e r n of  I  -91  -  0.4  £ c O CO  n  1,000  CL Q 0.2  H500  <  i __i  m CM i  1 -  i  0  1  2 3 4 5 6 7 8 9  GEL LENGTH  F i g u r e 19. c e l l s with Methods ". cells; cells.  125 (  (•—•),  (cm)  L a b e l l i n g of a s u s p e n s i o n of i s o l a t e d I. The  procedure used i s as d e s c r i b e d  ), coomassie  blue staining  iodination pattern  of  125.  I  pattern  s i n g l e smooth muscle under '' M a t e r i a l s of  iodinated  and  single  l a b e l l e d s i n g l e smooth muscle  -92-  -.1,000  E c  Q  O  500  CO  m  to  <  CM  1  2 3 4 5 6 7 8 9  GEL LENGTH (cm)  Figure  20. I o d i n a t i o n p a t t e r n s  cubes. (-—•—), coomassie  of F r a c t i o n 4  blue s t a i n i n g  pattern  using of  p r e l a b e l l e d muscle F r a c t i o n 4; (O-O),  i o d i n a t i o n p a t t e r n o f F r a c t i o n 4 o b t a i n e d u s i n g muscle cubes l a b e l e d w i t h  125 I p r i o r to homogenization: (•-#), observed  upon r e l a b e l l i n g  iodination  of F r a c t i o n 4  pattern  of F r a c t i o n 4  prepared from i o d i n a t e d muscle  cubes. F o r the exact procedure see under " M a t e r i a l s and Methods ".  - 9 3 -  1  2 3 4 5 6 7 8 9  GEL LENGTH (cm)  Figure cubes. (  21. I o d i n a t i o n p a t t e r n s ), coomassie  blue  of F r a c t i o n 5 staining pattern  u s i n g p r e l a b e l l e d muscle of  F r a c t i o n 5; (O—O),  i o d i n a t i o n p a t t e r n of F r a c t i o n 5 o b t a i n e d u s i n g muscle cubes l a b e l l e d w i t h 125  _ I  p r i o r to  homogenization; ( • - • ) , i o d i n a t i o n  observed upon r e l a b e l l i n g  of  Fraction 5  pattern of  prepared from  cubes. For the exact procedure see under " M a t e r i a l s  Fraction 5  i o d i n a t e d muscle  and Methods ".  0.4  -,1,000  r  Q_ Q CO ID  H500  0.2  I D  <  CM  o  0  J  1  2 3  4 5 6  7 8 9  GEL LENGTH  Figure ( pattern  )  s  22. I o d i n a t i o n p a t t e r n s coomassie b l u e  of F r a c t i o n 4  homogenization;  r-=i  of F r a c t i o n 4 u s i n g  staining pattern  obtained using  (•—•),  (cm)  iodination  cell  of F r a c t i o n  4;  pattern  of F r a c t i o n  and Methods ".  sheets.  (O—O), i o d i n a t i o n  sheets l a b e l l e d w i t h  r e l a b e l l i n g o f F r a c t i o n 4 prepared from i o d i n a t e d c e l l procedure see under " M a t e r i a l s  prelabelled c e l l  1  2  5  I p r i o r to  4 observed  s h e e t s . For the  upon exact  -95  -  F5 cs  I  I  1  1  I  I  I  I  •  •  •  •  2 3 4 5 6 7 8 9  GEL LENGTH (cm) Figure (  23. I o d i n a t i o n p a t t e r n s  ) , coomassie b l u e  of F r a c t i o n 5 u s i n g  staining pattern  prelabelled c e l l  of F r a c t i o n  5; (O—O),  sheets.  iodination  125 p a t t e r n of F r a c t i o n 5 homogenization;  obtained using  (•—#),  iodination  cell  sheets l a b e l l e d w i t h  pattern  r e l a b e l l i n g of F r a c t i o n 5 prepared from i o d i n a t e d procedure see under " M a t e r i a l s  and Methods ".  of F r a c t i o n cell  5  I p r i o r to observed upon  s h e e t s . For the  exact  - 96 -  S i m i l a r r e s u l t s were o b t a i n e d when F4 and F5 were l a b e l l e d 125  with  I u s i n g the t h r e e d i f f e r e n t methods o u t l i n e d i n the Methods  section  ( t h e r e s u l t s of two of the methods a r e p r e s e n t e d ) .  labelling  The  of F4, d i r e c t l y from the g r a d i e n t , shows t h a t the l a b e l  was i n c o r p o r a t e d a t 100,000 MWwith minor l a b e l l i n g 205,000 MW and 55,000 MW  ( F i g u r e s 24 and 26).  of bands a t  As F4 was thought  to c o n s i s t predominantly of 10 v e s i c l e s t t h e l a b e l l i n g a t 205,000 MW and  55,000 MW  appears due t o e i t h e r l e a k y v e s i c l e s ,  v e s i c l e s or r i g h t - s i d e - o u t v e s i c l e s . noted a t 205,000 MW, and  27).  dye,  100,000 MW  unsealed 125  I n c o r p o r a t i o n of  and 55,000 MW  I was  f o r F5 ( F i g u r e s 25  Some l a b e l was a l s o seen t o m i g r a t e w i t h t h e t r a c k i n g 125 125  representing possibly free  I  or  I-labelled  phospholipids.  Based on the above we can conclude t h a t F4 c o n t a i n s a 100,000 MW p r o t e i n t h a t i s a c c e s s i b l e to i o d i n a t i o n from the e x t e r n a l s u r f a c e of F4.  F5 c o n t a i n s 3 p r o t e i n s t h a t a r e a c c e s s i b l e to the i o d i n a t i n g  species e x t e r n a l l y . 55,000 MW  bands.  These a r e the 205,000 MW,  100,000 MW and  I f the 100,000 MW band i s a s i n g l e p r o t e i n , i t  c o u l d be p o s t u l a t e d t h a t i t spans t h e membrane. be s a i d t h a t the 205,000 MW  I t cannot, however,  and 55,000 MW p r o t e i n s do n o t span the  membrane, because the c y t o p l a s m i c  s i t e s o f l a b e l l i n g may not be  a c c e s s i b l e or may not c o n t a i n any i o d i n a t a b l e r e s i d u e s . Itis worth n o t i n g . t h a t the 205,000 MW band i s thought t o be the s i t e 2+ of the  e c t o Mg  s t i m u l a t e d ATPase d e s c r i b e d e a r l i e r .  Using the  above i n f o r m a t i o n i t should be p o s s i b l e to show i n a c o n t r o l l e d study  t h a t F4 i s indeed  10 and t h a t F5 c o n s i s t s of m a i n l y RO  o r i e n t e d membrane v e s i c l e s .  97  -97  1  -  2 3 4 5 6 7 8 9  GEL LENGTH (cm) i  Figure  24. I o d i n a t i o n  p r o f i l e of iodinated  of F r a c t i o n 4. 0  F r a c t i o n 4;  ), coomassie  staining  ( • — • ) , i o d i n a t i o n p r o f i l e of F r a c t i o n  F r a c t i o n 4 was prepared f o r i o d i n a t i o n as d e s c r i b e d Methods, i o d i n a t i o n s t u d i e s ,  blue  s e c t i o n B2 ".  under " M a t e r i a l s  4. and  -98-  1  2 3 4 5 6 7 8 9  GEL LENGTH (cm)  Figure  25. I o d i n a t i o n  p r o f i l e of iodinated  of F r a c t i o n 5. (  ), coomassie b l u e  staining  F r a c t i o n 5; ( # - # ) , i o d i n a t i o n p r o f i l e of F r a c t i o n 5.  F r a c t i o n 5 was prepared f o r i o d i n a t i o n as d e s c r i b e d Methods, i o d i n a t i o n s t u d i e s ,  s e c t i o n B2 ".  under " M a t e r i a l s  and  0.4  CO LO  r  0.2  <  OL  1  2 3 4 5 6 7 8 9  GEL LENGTH  Figure  26.  Iodination  of s u c r o s e f r e e F r a c t i o n 4.  s t a i n i n g p r o f i l e of sucrose f r e e i o d i n a t e d p r o f i l e of sucrose f r e e F r a c t i o n 4, " Materials  and  (cm)  The  (  F r a c t i o n 4;  procedure  Methods, i o d i n a t i o n s t u d i e s ,  ), coomassie b l u e (•—#),  used i s  s e c t i o n B3  ".  iodination  described  under  -100-  0.4  r  E c  o  co  <  1  2 3 4 5 6 7 8 9  GEL LENGTH  (cm)  Figure 27. Iodination of sucrose free Fraction 5. ( staining  p r o f i l e of sucrose  ), coomassie blue  free iodinated Fraction 5; ( • — • ) , iodination  p r o f i l e of sucrose free Fraction 5. The procedure used i s described " Materials and Methods, iodination studies, section B3  ".  under  - 101 -  I t can be argued t h a t the l a b e l l i n g and  100,000 MW  labelling  i s due t o s e l f l a b e l l i n g o f . l a c t o p e r o x i d a s e and  of a 205,000 MW  preparations.  contaminant i n commercial  To d e a l w i t h  w i l l label itself,  lactoperoxidase  t h i s p o t e n t i a l l y s e r i o u s problem, the  f o l l o w i n g experiments were done.  ( F i g u r e 28).  seen a t 205,000 MW  I t i s w e l l known l a c t o p e r o x i d a s e  e s p e c i a l l y as the age of the enzyme  increases  F r e s h l a c t o p e r o x i d a s e was i o d i n a t e d to s a t u r a t i o n  with non-radioactive  i o d i n e , p u r i f i e d and then examined f o r  s p e c i f i c a c t i v i t y , s e l f l a b e l l i n g and p u r i t y .  Its self  labelling  a b i l i t y was reduced t o l e s s than 10% o f t h a t found f o r the f r e s h enzyme, whereas t h e s p e c i f i c a c t i v i t y o f t h e enzyme was e q u i v i l a n t to t h a t found f o r the f r e s h enzyme. as a s i n g l e band a t 98,000 Using  The enzyme, on SDS g e l s , r a n  MW.  e q u a l amounts of F4 membranes  i o d i n a t i o n c o n d i t i o n s , F4 was l a b e l l e d w i t h  and i d e n t i c a l freshly  l a c t o p e r o x i d a s e and c o l d l a b e l l e d l a c t o p e r o x i d a s e r i g h t and l e f t ) .  The r e s u l t s ,  T a b l e Xa, show t h a t l a b e l l i n g w i t h 100,000 MW was m a i n l y l a b e l l e d .  of the 55,000 MW band. contamination  ( F i g u r e 29,  As w e l l , F4 was l a b e l l e d w i t h f r e s h  i n the presence o f 0.05% TX-100.  The  made  lactoperoxidase  tabulated i n  the two enzymes was  identical.  There was a l s o some l a b e l l i n g  T h i s l a t t e r l a b e l l i n g may be due t o  of F4 by u n s e a l e d ,  RO or l e a k y membrane  vesicles.  In the p r e s e n c e o f 0.05% TX-100, l a r g e i n c r e a s e s i n the l a b e l l i n g of the 100,000 MW were n o t e d .  and 55,000 MW  bands (Bands 8 and 10 r e s p e c t i v e l y )  We can add here t h a t s e l f — l a b e l l i n g ' b y  lactoperoxidase  i n the presence o f 0.05% TX-100 i s 50% o f t h a t found i n the absence  102  o  Q.I O O  o E E  o  1.0  2.0  3.0  4.0  5.0  [I ] in mM -1  F i g u r e 28, S e l f i o d i n a t i o n o f l a c t o p e r o x i d a s e . ( • — • ) , f r e s h l y prepared enzyme;  (•—•),, t h r e e week o l d enzyme s t o r e d f r o z e n ;  (A~A), p r e v i o u s l y l a b e l l e d lactoperoxidase. For  of l a b e l l e d l a c t o p e r o x i d a s e see under " M a t e r i a l s and Methods  preparation  F i g u r e 29. A c c e s s i b i l i t y o f F r a c t i o n 4 to b l u e s t a i n i n g p r o f i l e o f F r a c t i o n 4; ( • — • ) , of 0.05% previously  T r i t o n X-100. LEFT: ( O — O ) , l a b e l l e d with cold iodine;  T r i t o n X-100.  125  iodination  using  iodination  p r o f i l e o f F r a c t i o n 4 i n the  iodination  pattern  I . RIGHT:  of F r a c t i o n  4 using  (  ) , coomassie absence  lactoperoxidase  ( • — • ) , i o d i n a t i o n of F r a c t i o n 4 i n the presence o f 0.05%  40,000  H 20,000  1  GEL LENGTH (cm)  2  3  4  5  6  7  8  9  GEL LENGTH (cm)  Figure  30. A c c e s s i b i t y o f F r a c t i o n 5 to i o d i n a t i o n u s i n g  b l u e s t a i n i n g p r o f i l e o f F r a c t i o n 5; ( • — • of 0.05% T r i t o n X-100. LEFT: previously  (O—O),  l a b e l l e d with cold iodine;  T r i t o n X-100.  iodination  iodination pattern (•—•) ,  X  ^ I. J  RIGHT:  (-  ),  coomassie  p r o f i l e o f F r a c t i o n 5 i n the absence of F r a c t i o n 5 using  lactoperoxidase  i o d i n a t i o n o f F r a c t i o n 5 the presence of 0.05%  1  2  3  4  5  6  7  8  9  GEL LENGTH (cm)  GEL LENGTH (cm)  - 107 -  T a b l e 'Xa Accesslbity  o f F r a c t i o n s , 4 and 5 t o l a c t o p e r o x i d a s e  catalyzed  iodination.^  125 Sucrose g r a d i e n t f r a c t i o n number 4  5  I c.p.m./12 yg p r o t e i n (55% e f f i c i e n c y ) Band 8 - 100,000 d a l t o n s Band 10 -55,000 d a l t o n s  iodolactoperoxidase  83,000  42,000  - T r i t o n X-100  85,000  45,000  + T r i t o n X-100  149,000  145,000  iodolactoperoxidase  61,000  143,000  - T r i t o n X-100  70,000  153,000  + T r i t o n X-100  140,000  155,000  ~* P l e a s e see F i g u r e s 29 and 30 a l s o .  T a b l e Xb S e l f l a b e l l i n g o f l a c t o p e r o x i d a s e i n the presence o f " T r i t o n X-100. L a c t o p e r o x i d a s e used was one day o l d .  [ T r i t o n X-100 ]  125  I c.p.m./25'yg enzyme  0.00%  1.00 x 1 0  5  0.05%  0.50 x 1 0  5  0.10%  0.50 x 1 0  5  0.20%  0.40 x 1 0  5  0.40%  0.30 x 1 0  5  - 108 -  of d e t e r g e n t  (Table Xb)•  On the b a s i s o f the p r e c e e d i n g  f a l s e peaks due to s e l f - l a b e l l i n g p r e p a r a t i o n s , can be r u l e d o u t . revealing..  of commercial l a c t o p e r o x i d a s e The r e s u l t s f o r F5 a r e j u s t as  In the absence o f d e t e r g e n t , u s i n g f r e s h  l a b e l l e d l a c t o p e r o x i d a s e , l a b e l l i n g was observed and  55,000 MW m a i n l y .  were i d e n t i c a l ,  or c o l d  a t 100,000 MW  The r e s u l t s u s i n g the two d i f f e r e n t enzymes  thus a g a i n r u l i n g  l a b e l l i n g g i v i n g f a l s e peaks.  out l a c t o p e r o x i d a s e s e l f -  Labelling  was shown to be i n c r e a s e d o n l y a t  i n the presence  100,000 MW.  change i n the l a b e l l i n g a t 55,000 MW. extremely  results,  o f TX-100  There was no  These r e s u l t s p r o v i d e  s t r o n g arguements i n favour of the h y p o t h e s i s t h a t  F5  PMs a r e m a i n l y RO o r i e n t e d and t h a t F4 c o n t a i n s a predominantly 10  PM v e s i c l e p o p u l a t i o n w i t h some  F.  E x t r a c t i o n Studies  contamination.  At t h i s stage, g i v e n t h e i n d i c a t e d o r i e n t a t i o n s o f F4 and  F5 we had hoped t o c o r r o b o r a t e t h i s d i f f e r e n c e i n o r i e n t a t i o n  using extraction studies.  As w e l l , we had hoped to e x t r a c t  p e r i p h e r a l membrane p r o t e i n s t o s i m p l i f y the coomassie b l u e staining profile.  Examination  o f the r e s u l t s y i e l d s a r a t h e r  c o n f u s i n g p i c t u r e indeed  ( F i g u r e s 31 to 38, T a b l e X I ) .  used were D i m e t h y l m a l e i c  anhydride  tetraacetate  (EDTA), H 0, D i d i t o n i n 2  and p-Chloromercuribenzene was based  (DMMA),  (STECK & YU, 1973) and Kahlenberg pCMBS e i t h e r denature  Ethylenediamine  (DT), T r i t o n X-100 (TX-100),  sulphonic acid  on s t u d i e s by F a i r b a n k s  The agents  (pCMBS).  T h e i r use  (FAIRBANKS e t a l . , (KAHLENBERG, 1976).  or c o v a l e n t l y modify  1971),  Steck  DMMA and  p r o t e i n s , pCMBS by  109  - 109 -  breaking  disulphide linkages.  I n the human r e d b l o o d  cell,  these  agents s e l e c t i v e l y s o l u b i l i z e a c e r t a i n group o f membrane polypeptides, lipid  l e a v i n g the remainder s t i l l  a s s o c i a t e d w i t h a l l the  and cabohydrate i n the membrane r e s i d u e .  of the n o n - i o n i c  detergent  The s p e c i f i c a c t i o n  TX-100, a Type A amphiphile, i s n e a r l y  r e c i p r o c a l t o t h a t seen w i t h p e r t u r b a n t s  such as DMMA.  Polypeptides  anchored i n the memnrane through a p o l a r a s s o c i a t i o n s w i t h  lipids  can be s o l u b i l i z e d by TX-100 and h o p e f u l l y the l i p i d s can be d i s placed  from the hydophobic p r o t e i n s without d e n a t u r a t i o n .  Digitonin,  though c l a s s e d as a Type B amphile, a c t s by a d i f f e r e n t mechanism compared t o TX-100 but the end r e s u l t i s the same. to r e l e a s e membrane f i b r i l l a r  EDTA i s thought  p r o t e i n s by c h e l a t i n g membrane bound  divalent. The r e s u l t s a r e presented  band by band.  I t i s important  to r e a l i x e t h a t F4 may n o t be e n t i r e l y homogeneous w i t h  respect  to 10 o r i e n t a t i o n w h i l e F5 i s thought t o be m a i n l y RO.  The  behavior 2+ of Band 1 (205,000 MW), which may be a s s o c i a t e d w i t h an ecto Mg s t i m u l a t e d ATPase, was q u i t e i n t e r e s t i n g . We a l r e a d y knew t h a t 125 Band 1 was a c c e s s i b l e e x t e r n a l l y based on the  I studies.  When  the membranes were e x t r a c t e d w i t h ^ 0 , a s m a l l amount o f the Band 1 p r o t e i n was e x t r a c t e d  from F5 (The appearance o f Band 1 i n the  supernatant o f F4 e x t r a c t e d w i t h 1^0 i s n o t s e l e c t i v e . seeing v e s i c l e s w i t h a buoyant d e n s i t y s u f f i c i e n t sedimentation  under the c o n d i t i o n s used.).  t o t a l l y i n F4 w h i l e  We a r e  to not a l l o w  DMMA removed Band 1  25% was o n l y removed i n F5.  High  concentrations  of pCMBS removed 50% o f Band 1 i n F5 but the band was not touched  110  - 110 -  i n F4. but,  I n t e r e s t i n g l y Band 1 was t o t a l l y removed by TX-100 i n F4,  i n F5 the band was not d i s t u r b e d .  e f f e c t on Band 1 i n F4 or F5.  D i g i t o n i n and EDTA had no  Based on the above we can summarize  t h a t Band 1 c o n t a i n s more than one p r o t e i n , p a r t of the band i s p e r i p h e r a l l y l o c a t e d and p a r t i s embedded i n the membrane. There i s a l s o d i s u l p h i d e bond c h a r a c t e r  i n the band p r o t e i n s .  A r a t h e r unusual f e a t u r e of our membrane p r e p a r a t i o n s Band 2-3.  As a shoulder  or 200,000 d a l t o n s .  was  on Band 1 i t appeared a t 210,000 d a l t o n s  The p o s i t o n c o u l d not be p r e d i c t e d , f o r example  i n the TX-100 e x t r a c t i o n of F4 ( F i g u r e 37) i t appeared a t 200,000 d a l t o n s , whereas i n the presence of EDTA and K^O m i g r a t e d a t 210,000 MW. and  F5 were s u b j e c t e d  ( F i g u r e 31) i t  Even when a s e r i e s of samples from F4  to g e l e l e c t r o p h o r e s i s under i d e n t i c a l  c o n d i t i o n s , t h i s band appeared randomly a t one p o s i t i o n or the other.  Under treatment w i t h  the v a r i o u s e x t r a c t i n g media  this  band behaved l i k e Band 1. Band 4 was t o s m a l l t o be f o l l o w e d i n these s t u d i e s . 5 was p a r t i a l l y removed by DT i n F4, i n c o n t r a s t Band 5 was by EDTA from F5 o n l y . and  F5.  However,  Using  used i n the e x t r a c t i o n of  Band 5 from F5 was lower than f o r F4. removed a t low pCMBS c o n c e n t r a t i o n s  Band 5 i n F5 was  partially  of 0.01 mM but i t was not  r e a d i l y apparent whether Band 5 was e x t r a c t e d  present.  extracted  DMMA Band 5 was removed from both F4  the c o n c e n t r a t i o n  t h e r e was a l a r g e shoulder  Band  i n F4 by pCMBS, as  on Band 1 i n which Band 5 may have been  T r i t o n X-100 t o t a l l y e x t r a c t e d Band 5 i n F4 whereas  only  a s m a l l amount of Band 5 was removed i n F5 under the same c o n d i t i o n s .  Ill  - I l l-  There might a l s o have been some Band 5 hidden under the  shoulder  of Band 1 i n F5 e x t r a c t e d w i t h 0.05% TX-100. I t should be n o t e d 2+ t h a t a p o r t i o n of t h i s band d i s p l a y e d Ca dependent i n c r e a s e s 2+ i n phosphorylation  and t h a t the b i n d i n g  sites  f o r ATP and Ca  were a c c e s s i b l e from the e x t e r n a l s u r f a c e o n l y .  We can thus  conclude t h a t Band 5 c o n t a i n s d i s u l p h i d e l i n k a g e s , may  c o n s i s t of  more than one p r o t e i n component and i t l i e s p a r t i a l l y embedded i n the hydrophobic r e g i o n of the membrane.  That t h e r e must be  a  p e r i p h e r a l component can be shown by the EDTA e x t r a c t i o n of Band 5. Bands 6.1 and 6.2 were a p p a r e n t l y  p a r t i a l l y removed by  H^O  e x t r a c t i o n of F4 and F5 but i t appeared t h a t t h e r e was no a c t u a l e x t r a c t i o n of 6.1 and 6.2 i n F4.  As p o i n t e d  p r o f i l e of the supernatant o b t a i n e d represents  out e a r l i e r  from the H^O  the g e l  e x t r a c t i o n of F4  s m a l l v e s i c l e s that c o u l d not be sedimented.  In some  cases such as the EDTA e x t r a c t i o n of F4, the Bands 6.1 and 6.2 cou l d not be i d e n t i f i e d . F5 w h i l e  low c o n c e n t r a t i o n s  i n F4 and F5. and  DT removed n e i t h e r 6.1 or 6.2 from F4 or  6.2 but  of DMMA p a r t i a l l y e x t r a c t e d  these bands  Treatment w i t h pCMBS y i e l d e d no e x t r a c t i o n o f 6.1 TX-100 removed the bands p a r t i a l l y i n both F4 and F5.  We can t h e r e f o r e conclude that Bands 6.1 and 6.2 a l s o c o n s i s t of more than one p r o t e i n and are p a r t l y embedded i n the hydrophobic p a r t of the membrane. Band 7 was a v e r y minor band, a t times d i f f i c u l t t o d e t e c t . The o n l y o b s e r v a t i o n selectively  t h a t can be made i s t h a t TX-100 appeared to  remove Band 7 from F5 w h i l e Band 7 was o n l y  removed from F4 by t h i s  treatment.  partially  I t t h e r e f o r e appears t h a t  Band 7 may be found i n the hydrophobic r e g i o n s  of the membrane.  112  - 112 -  Band 8, i f a s i n g l e p r o t e i n was thought to span t h e membrane based on the i o d i n a t i o n s t u d i e s . the band from F4 and F5.  EDTA and DT removed  High c o n c e n t r a t i o n s  of Band 8 i n F4 but l e s s than 50% i n F5. e x t r a c t e d by TX-100 i n both f r a c t i o n s .  o f DMMA removed 100%  The band was completely  Using  pCMBS Band 8 was not  a f f e c t e d i n F4 but appears to have been e x t r a c t e d from F5 ( i n a broad peak on the shoulder  o f Band 9 ) .  We can summarize t h a t  Band 8 appears to span the membrane based on t h e i o d i n a t i o n s t u d i e s . T h i s i s v e r i f i e d by the TX-100 removal o f t h e band i n F4 There may be d i s u l p h i d e c h a r a c t e r  and F 5 .  and more than one p r o t e i n component  i n t h i s band. Of a l l t h e bands, Band 9 d i s p l a y e d the most v a r i a b l e behavior.  EDTA and DT p a r t i a l l y removed Band 9 from F4 and F 5 .  DMMA t o t a l l y removed Band 9 i n F4 but o n l y 50% o f t h i s band was extracted  i n F5 u s i n g h i g h c o n c e n t r a t i o n s  o f DMMA.  The b e h a v i o r  of t h i s band i n t h e presence o f pCMBS was a g a i n q u i t e i n F4 and F5.  different  T h i s treatment t o t a l l y removed t h e band from F5  whereas i t was v i r t u a l l y u n a f f e c t e d  i n F4.  I n t e r e s t i n g l y , the  TX-100 e x t r a c t i o n o f F4 appeared t o r e s u l t i n some k i n d o f m o d i f i c a t i o n o f the Band 9 p r o t e i n as i t became the predominant peak i n the g e l p a t t e r n , p o s s i b l y a t t h e  expense o f Band 10.  Band 9 i n F5 was 75% removed by treatment w i t h TX-100.  We can  t e n t a t i v e l y conclude t h a t Band 9 appears t o be one p r o t e i n w i t h d i s u l p h i d e bond c h a r a c t e r , and i s embedded p a r t l y i n the hydrophobic  r e g i o n s o f the membrane.  I t can a l s o be added t h a t  Bands 9 and 11 were i o d i n a t e d o n l y from the e x t e r n a l membrane surface  ( F i g u r e s 25 t o 28), b u t the e x t r a c t i o n s t u d i e s seem to  i n d i c a t e t h a t Band 9 does have c y t o p l a s m i c  s i t e s which may n o t be  accessible to i o d i n a t i o n .  113  - 113 -  Band 10, which had been shown to have e x t e r n a l l y i o d i n a t e d s i t e s o n l y , c o u l d be e x t r a c t e d by EDTA and DT from F4 and t o a l e s s e r degree i n F5. and  extracted  DMMA appeared to have m o d i f i e d  50% o f the band i n F5.  an i n c r e a s e i n Band 9 c o r r e s p o n d i n g p o s s i b l y implying  the band i n F4  There appeared t o have been  t o a decrease i n Band 10 and  some type o f chemical  modification.  removed p a r t of Band 10 i n both F4 and F5 (50%).  PCMBS  The o n l y anomalous  r e s u l t was noted i n the e x t r a c t i o n o f F4 by 0.05% TX-100.  In t h i s  e x t r a c t i o n the decrease i n Band 10 was a g a i n accompanied by an i n c r e a s e i n Band 9.  Based on i o d i n a t i o n s t u d i e s and these r e s u l t s  Band 10 has d i s u l p h i d e bond c h a r a c t e r , may c o n s i s t o f more than one p r o t e i n and p o s s e s s e s e x t e r n a l s i t e s t h a t can be i o d i n a t e d but i s thought t o extend i n t o the hydrophobic r e g i o n . Band 11 i n both F4 and F5 was n o t a f f e c t e d by DT, but i t was, however, removed from F4 by EDTA. was removed by EDTA.  I n F5, o n l y 50% o f Band 10  DMMA a l s o removed a l l o f Band 10 from E4 b u t  o n l y 50% o f the band i n F5. removed Band 11 i n F4 w h i l e ,  Low c o n c e n t r a t i o n s  o f pCMBS t o t a l l y  i n c o n t r a s t , much h i g h e r  of pCMBS were r e q u i r e d to e l u t e the band i n F5.  concentrations  A g a i n TX-100  s e l e c t i v e l y removed Band 11 from F4, whereas, i n F5 o n l y 50% was removed.  Iodination studies  ( F i g u r e s 25 t o 28) showed Band 11  c o u l d o n l y be i o d i n a t e d e x t e r n a l l y . Band 11 may have and  Our r e s u l t s i n d i c a t e t h a t  d i s u l p h i d e bonds, c o n s i s t o f more than one p r o t e i n  possesses some c y t o p l a s m i c  s i t e s i n a d d i t i o n t o the known  externally accessible sites. There were v a r i o u s anomalies recorded  i n the e x t r a c t i o n  114  - 114 -  Table XI Protein (yg) contained i n extraction media (300 y l ) and p e l l e t (resuspeded i n 150 y l ) following extraction procedure. For g e l electrophoresis 150 y l of supernatant and 75 y l of p e l l e t were were used unless otherwise indicated. Please see Figures 31 to 38 also.  Extracting Agent pCMBS 0.01 mM 0.10 mM 2.00 mM T r i t o n X-100 0.0]% 0.05% 0.50% DMMA 0.1 mg/ml 0.4 mg/ml 1.0 mg/ml  Fraction 4 pellet 32 40 32  24 22 16 6  -4  Digitonin 0.36 mg/ml  24  EDTA 0.50 mM  30  H0  48  2  a  Fraction 5  supernatant 12 6 10  pellet 38 32 20  supernatant 16 14 20  40 30 42  30 40 40  *°a 40  20 20 16  26 26 44  14  34  10  44  32  62  8  38& 60 56 a  a  <  16 12  a  a Refers to 38 y l used f o r g e l electrophoresis.  - 115 -  F i g u r e 31. E x t r a c t i o n of F r a c t i o n 4 u s i n g H^O,  Ethylenediamine  t e t r a a c e t a t e and D i g i t o n i n . F o r yg p r o t e i n i n s u p e r n a t a n t (S) and pellet  (P) see T a b l e X.  (Right), p e l l e t obtained a f t e r e x t r a c t i o n procedure, (Left ), supernatant obtained a f t e r e x t r a c t i o n procedure.  GEL LENGTH (cm)  - 117 -  F i g u r e 32. E x t r a c t i o n o f F r a c t i o n 5 u s i n g H^O, tetraacetate pellet  Ethylenediamine  and D i g i t o n i n . For yg p r o t e i n i n the supernatant  (S) see T a b l e X.  (Right), p e l l e t obtained a f t e r e x t r a c t i o n  procedure.  ( L e f t ), supernatant o b t a i n e d a f t e r e x t r a c t i o n  procedure.  (S)  GEL LENGTH (cm)  - 119 -  F i g u r e 33. E x t r a c t i o n of F r a c t i o n 4 u s i n g Dimethyl m a l e i c anhydride (DMMA). For yg p r o t e i n i n supernatant  (S) and p e l l e t  (Right), p e l l e t obtained a f t e r e x t r a c t i o n  (P) see T a b l e  procedure.  ( L e f t ), supernatant o b t a i n e d a f t e r e x t r a c t i o n  procedure.  X.  -120-  i  01  i  1  •  '  •  •  •  •  2 3 4 5 6 7 8 9  •  •  10  GEL LENGTH (cm)  - 121  -  F i g u r e 34. E x t r a c t i o n of F r a c t i o n 5 u s i n g Dimethyl m a l e i c anhydride (DMMA). For yg p r o t e i n i n supernatant  (S) and p e l l e t  (P) see T a b l e  (Right), p e l l e t obtained a f t e r e x t r a c t i o n procedure. ( L e f t ), s u p e r n a t a n t o b t a i n e d a f t e r e x t r a c t i o n  procedure.  X.  -122~  r 1  0.4mg/ml 10  u 61,6.2  2-3  J  9  I  6.1,6.2  11  I  01  1  1  '  I  1  1  1  9  !  8 5A 7 /  I  I  2 3 4 5 6 7 8 9  •  10  II \  •  10  GEL LENGTH (cm)  14 A  1  2  - 123 -  F i g u r e 35. E x t r a c t i o n  of  Fraction 4  using  p-Chloromercuribenzene  s u l p h o n i c a c i d (pCMBS). For yg p r o t e i n i n s u p e r n a t a n t (S) and p e l l e t (P) see T a b l e X. (Right), p e l l e t obtained a f t e r e x t r a c t i o n  procedure.  ( L e f t ), s u p e r n a t a n t o b t a i n e d a f t e r e x t r a c t i o n  procedure.  -124  -  '  01  I—I  1—I  2 3 4 5 6 7 8 9  1  10  GEL LENGTH (cm)  - 125  Figure sulphonic  36. E x t r a c t i o n acid  of  Fraction 5  -  using  p-Chloromercuribenzene  (pCMBS) . For yg p r o t e i n i n supernatant  (S) and p e l l e t  see T a b l e X. (Right), p e l l e t obtained a f t e r e x t r a c t i o n  procedure.  ( L e f t ), supernatant o b t a i n e d a f t e r e x t r a c t i o n  procedure.  (P)  '126-  F5  pCMBS  S 10  I  0.01 mM  2.00 mM 9 10 1  1 |  11  J  •  1  01  1  A Im  2-3  J  LJ 1  10  1  5  9  1  2 3 4 5 6 7 8 9  10  GEL LENGTH (cm)  A  - 127  -  F i g u r e 37. E x t r a c t i o n o f F r a c t i o n 4 u s i n g T r i t o n X-100 For  yg p r o t e i n i n s u p e r n a t a n t (S)  and p e l l e t (P)  see  ( R i g h t ) , p e l l e t obtained a f t e r e x t r a c t i o n procedure. ( L e f t ), supernatant obtained a f t e r e x t r a c t i o n procedure.  (TX-100). T a b l e X.  -m-  F4  s  TX-100  p  0.01 %  10  10  9 .  9  u  1  11 /  8  A  —<  0.05% 9  6.1 6 . 2 10 11  2,-3  1/ i 5  8  12  0 1  2  3  4  5  6  7  11  8  9  10  GEL LENGTH (cm)  1 2  - 129  F i g u r e 38. E x t r a c t i o n o f F r a c t i o n For  yg  protein  in  supernatant  -  5 u s i n g T r i t o n X-100  (S) a n d  (Right), p e l l e t obtained after extraction  p e l l e t (P)  see  procedure.  (Left ), supernatant obtained a f t e r e x t r a c t i o n  procedure.  (TX-100). Table  X.  -130-  F5  TX-100 0.01%  1  10  6.1,6.2  61;  1°  ?  6.2  i  12  ^ Hi 3  0.05%  V  i  n  12  UL  1  6.1,6.2  9  i  \  !  1 5  :  '  i I  7  a |  10 10  II  6.1,62  i 2-3!  ;  I  9/I I  Ijjj*/  GEL LENGTH (cm)  I  14  \J\  12  - 131  studies;  two  -  of these b e i n g Bands 14 and  appeared both i n the DMMA and  15.  Band 14  TX-100 e x t r a c t i o n s  of F4  Whether t h i s band r e p r e s e n t s c h e m i c a l m o d i f i c a t i o n t h i s , p o i n t but  (93,000 MW).  and  0.50%  I t can  Bands  7 and  8 should be  Similarily,  be argued t h a t Band 15's  d e s i g n a t e d 6.2  on Rf v a l u e s however, t h i s d e s i g n a t i o n  formation  assignment  as a r e s u l t the  and  33).  i s unclear at  TX-100 r e s u l t e d i n the  should r e a l l y be d e s i g n a t e d as Band 8 and  MW)  (Figure  s i n c e i t i s p r e s e n t i t must be r e p o r t e d .  treatment of F5 w i t h 0.05% of Band 15  (31,000  designated  7 respectively.  Based  though d e s i r a b l e c o u l d not  be  made. Even though the r e s u l t s of the biased  by u n s e a l e d v e s i c l e s i n F4  these s t u d i e s . differences  The  e x t r a c t i o n s t u d i e s were  some i n f o r m a t i o n  r e s u l t s a t times appeared to be  i n membrane o r i e n t a t i o n of the  two  was  gained  i n d i c a t i v e of  f r a c t i o n s F4 and  F u r t h e r p u r s u i t of the e x t r a c t i o n s t u d i e s , however, was as  G.  the a d d i t i o n a l i n f o r m a t i o n  Affinity  gained was  pure F4 and  orientation. c a r r i e d out  terminated  questionable.  to determine as a c c u r a t e l y  F5 were w i t h r e s p e c t  c o n t a m i n a t i o n o f F4 by v e s i c l e s o f Con using  A - sepharose a f f i n i t y F4 and  F5  as  possible  to t h e i r o r i e n t a t i o n s .  a l s o i n t e n d e d to f u r t h e r p u r i f y the membranes as we the p o s s i b l e  F5.  Chromatography  I t seemed d e s i r a b l e how  to be  by  We  were aware of  differing  chromatography  was  i n the hopes of f u r t h e r p u r i f y i n g  and  c h a r a c t e r i z i n g the f r a c t i o n s . When F4 membranes, l a b e l l e d 125 with I, were a p p l i e d to the a f f i n i t y column, 2 peaks were  132  - 132 -  obtained.  An a d d i t i o n a l  2 peaks b e i n g o b t a i n e d w i t h e l u t i n g  c o n t a i n i n g a methyl-D-mannoside ( F i g u r e 39). 125 peaks were a n a l y z e d f o r  I profiles  l a b e l l i n g was observed e x c l u s i v e l y  When the f i r s t  a t 100,000 d a l t o n s , t h i s  not noted, though t h e r e was an  fraction  17 was i n d i c a t i v e  vesicles  w i t h r e s p e c t to t h i e r s i z e .  interesting  and AchE  results.  fact  an i n c r e a s e i n AchE  i n c r e a s e i n the s p e c i f i c a c t i v i t y (see D i s c u s s i o n ) .  35 f o r i o d i n a t i o n  2  ( F i g u r e s 40a and 40b),  being c o n s i s t e n t o n l y w i t h 10 v e s i c l e s b u t , a c c e s s i b i l t y was s u r p r i s i n g l y  The peak a t  of E4 b e i n g a p o p u l a t i o n of heterogeneous A n a l y s i s of column f r a c t i o n  s p e c i f i c a c t i v i t i e s showed 125  The  I p r o f i l e showed  rather  the same  number of  counts i n Band 8 as i n Band 10, t h i s f a c t b e i n g c o n s i s t e n t unsealed v e s i c l e s  (Table X a ) .  w i t h the above i n t e r p r e t a t i o n with higher similar  the  with  The AchE a c c e s s i b i l t y r e s u l t s (Table X I I ) .  Fraction  to f r a c t i o n 35, a g a i n i n d i c a t i n g  agree  52, e l u t e d  a methyl-D-mannoside c o n c e n t r a t i o n s , y i e l d e d  90% of the F4 p r o t e i n  buffer  results  unsealed v e s i c l e s .  In a l l ,  a p p l i e d to the column c o u l d be e l u t e d  from  column. F5 when a p p l i e d to the a f f i n i t y column y i e l d e d  elutable protein  only  two  peaks and these peaks r e p r e s e n t e d o n l y 5-10% o f the t o t a l o r i g i n a l l y a p p l i e d to the column. A f u l l  90% c o u l d not be  e l u t e d even i n the presence of h i g h c o n c e n t r a t i o n s of e l u t i n g 125 and  borate buffer.  Column f r a c t i o n 6 when examined f o r  p r o f i l e and AchE s p e c i f i c a c t i v i t i e s y i e l d e d  results  o n l y w i t h t h i s f r a c t i o n being m a i n l y 10 v e s i c l e s Fraction  73 s t u d i e s i n d i c a t e  sugar  I  consistent  (Figure 41).  t h a t t h i s f r a c t i o n c o n s i s t e d of  u n s e a l e d membranes.  133  - 133  -  I t should be p o i n t e d out t h a t the pages 134  to 139  f o r the a f f i n i t y  chromatography  r e p r e s e n t the mean v a l u e s f o r t h r e e to f o u r S i m i l a r d a t a was  r e s u l t s presented  on  experiments  experiments.  o b t a i n e d f o r Con A - Agarose  affinity  chromatography. T h i s r e s u l t s a r e not presented here f o r t h a t reason.  E x t e n s i v e c o n t r o l s were run w i t h each column used.  These  a r e d e a l t w i t h a t l e n g t h i n the D i s c u s s i o n s e c t i o n .  134  - 134 -  Figure  39. Con A - Sepharose 4B a f f i n i t y chromatography o f F r a c t i o n 125  4  (top) and  membranes  Fraction 5  e l u t e d from  (bottom). (.#—#),  the column;  eluting  buffer.  r e f e r s to  the  labelled  (O—O), p r o t e i n p r o f i l e o f column  e f f l u e n t u s i n g Lowry p r o t e i n assay, a mannoside. Borate  I d.p.m. o f  MM  refers  to a - m e t h y l - D -  b u f f e r used i n p l a c e o f the  normal  -  - 1 3 5  10  20  30  40  F R A C T I O N  50  60  70  80  N U M B E R  F 5 +100 m M  10  20  30  oc MM  40  F R A C T I O N  50  60  70  N U M B E R  80  - 136 -  Table . XII C h a r a c t e r i z a t i o n o f column f r a c t i o n s e l u t e d from Con A - Sepharose a f f i n i t y columns. A c e t y l c h o l i n e s t e r a s e sp. a c t i v i t y expressed as nm/mg p r o t e i n / m i n .  125  Total  I d.p.m. a p p l i e d  Fraction 4  Fraction 5  3.6 x . 1 0  4.0 x 1 0  7  Recovery  90 - 95%  10  T o t a l p r o t e i n on column  0.735 mg  0.850 mg  Recovery  90 - 95%  8-10%  Fraction Number  Column F r a c t i o n Number  7  - 15%  acetylcholinesterase - T r i t o n X-100 + T r i t o n X-  4  stock 5 17 35 52  2.72 8.61 22.96 30.64 29.01  15.43 26.11 42.13 30.64 30.10  5  stock 6 28 73  14.16 7.00  13.50 22.50  26.70  27.09  -  -  -  -137  F 4 STOCK  s  1 G E L  2  3  4  5  6  L E N G T H  7  8  9 (cm)  Figure 40a. Analysis of peak fractions obtained a f f i n i t y chromatography of Fraction 4. (  by Con A r- Sepharose  ), coomassie blue  staining  p r o f i l e of peak f r a c t i o n s ; ( O — O ) , iodination pattern of protein eluted i n peak f r a c t i o n s . Fraction 4 stock refers to the membranes applied to the column. Greater than 85% of the protein  originally  applied to  the  column was eluted. See also Figure 40b f o r peak fractions 17,37 and 54.  -  - 1 3 8  J  1 G E L  I  I  2  I  3  I  4  1  5  6  I  I  7  8  L E N G T H  L  9 (cm)  Figure 40b. Analysis of peak fractions obtained by Con A - Sepharose a f f i n i t y chromatography of Fraction 4. (  ),  coomassie blue staining  p r o f i l e of peak f r a c t i o n s ; (O—O) , iodination pattern of protein eluted i n peak f r a c t i o n s . Greater than  85%  of the  protein  applied to  the  column was eluted. See also Figure 40a f o r peak f r a c t i o n 5 and Fraction 4 stock membranes o r i g i n a l l y applied to the column.  - 1 3 9 -  1 G E L  Figure  41. A n a l y s i s  2  3  4  5  6  7  L E N G T H  8  9 (cm)  of peak f r a c t i o n s o b t a i n e d by  a f f i n i t y chromatography of F r a c t i o n 5. (  Con A - Sepharose  ) , coomassie  blue s t a i n i n g  p r o f i l e of peak f r a c t i o n s ; ( P — O ) , i o d i n a t i o n p a t t e r n o f p r o t e i n i n peak f r a c t i o n s . F r a c t i o n 5 s t o c k applied  to the  column was  column. Less than  eluted.  eluted  r e f e r s to the membranes o r i g i n a l l y 10% of the  protein applied  to the  - 140- -  H.  Summary In summary we can say t h a t F4 was found t o c o n t a i n 20-25%  unsealed  membrane v e s i c l e s and t h a t these were the contaminating  components i n the p r e p a r a t i o n s used f o r sidedness  studies.  F5  r e p r e s e n t s a membrane p r e p a r a t i o n a l r e a d y 90% r i g h t - s i d e - o u t . attempt to improve on t h i s would be u n l i k e l y t o succeed. blem of e l u t i n g The  the 90% o f p r o t e i n bound  Any  The p r o -  ( i n F5) was not s o l v e d .  b i n d i n g to the a f f i n i t y column appears to be n o n - s p e c i f i c  adsorption.  Perhaps c o n d i t i o n s c o u l d be m o d i f i e d  f o r the e l u t i o n  of these membranes b u t f u r t h e r work would be r e q u i r e d . In c o n c l u s i o n , we have prepared.two s e t s o f membrane v e s i c l e s with enriched o r i e n t a t i o n s . the n a t u r e  At the same time we have demonstrated  o f some of the p r o t e i n s i n the membrane.  Methods  f o r the i d e n t i f i c a t i o n o f RO and 10 membranes were a l s o and  i n d i c a t i o n s were g i v e n o f a p o s s i b l e technique  usable  presented  f o r preparing  PM v e s i c l e s o f pure d e f i n e d o r i e n t a t i o n .  141  "141  "  Discussion The  c h i c k e n g i z z a r d , a l t h o u g h known to be r i c h i n smooth  muscle, has not been used e x t e n s i v e l y i n membrane work. i t was  felt  an i s o l a t e d  t h a t t h i s was  an  ideal  However  source of smooth muscle f o r  pure plasma membrane p r e p a r a t i o n .  A Polytron  used f o r the i n i t i a l homogenization s i n c e the t i s s u e i s to homogenize by o t h e r methods. f o r muscle cubes and specific  cell  a c t i v i t i e s and  Homogenization times chosen  sheets were o p t i m i z e d  No  to y i e l d maximal  t h i s procedure-  The  P o l y t r o n , the most commonly used from v i s c e r a l  smooth muscle,  found to be q u i t e s u c c e s s f u l i n our work. D i f f e r e n t i a l c e n t r i f u g a t i o n was  quite successful i n  enrichming the 100,000 g p e l l e t w i t h plasma membranes, as of the 5' n u c l e o t i d a s e and  a  other marker s t u d i e s were performed  homogenizer i n membrane p r e p a r a t i o n s was  difficult  t o t a l a c t i v i t i e s of 5' n u c l e o t i d a s e ,  plasma membrane marker. during  was  crude f i l t r a t e .  a c t i v i t y was  There was  r e t a i n e d from the  a 10 f o l d p u r i f i c a t i o n  plasma membranes based on the marker.5' n u c l e o t i d a s e . latter result literature  being  similar  (see Table  i n the s p e c i f i c  40%  residue of  the  This  to those noted elsewhere i n the  I, gradient  preparations).  No  increase  a c t i v i t y of NADPH cytochrome c_ r e d u c t a s e  noted, t h i s r e s u l t  being  i n c o n t r a s t to o t h e r  was  s t u d i e s which show  i n c r e a s e s i n the s p e c i f i c a c t i v i t i e s of markers f o r the SR 100,000 g p e l l e t s .  Slight  of a c i d phosphatase and  i n c r e a s e s i n the s p e c i f i c  s u c c i n i c dehydrogenase.  important i s to n o t e t h a t t h e r e was u n i t s of these enzymes.  in  activities  What i s more  a decrease i n the  At the same time i t must be  total pointed  142  - 142 "  out t h a t no o r i e n t a t i o n s t u d i e s were done on the p e l l e t s and supernatants p r i o r to sucrose gradient c e n t r i f u g a t i o n .  In  r e t r o s p e c t t h i s s h o u l d have been done t o check i f t h e v a l u e s f o r t o t a l u n i t s of a c t i v i t y were indeed c o r r e c t .  I t must be  remembered, however, t h a t r e s u l t s a p p l i c a b l e , t o one type o f smooth need n o t n e c e s s a r i l y a p p l y t o smooth m u s c l e o b t a i n e d from another  source.  The f r a c t i o n a t i o n o f the v a r i o u s c e l l membrane types a c h i e v e d by t h e u s e o f t h e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n was s i m i l a r t o t h a t observed  i n v a r i o u s o t h e r plasma membrane  p r e p a r a t i o n s b u t t h e d i s t r i b u t i o n of t h e d i f f e r e n t membranes was  i n disagreement w i t h these o t h e r s t u d i e s .  I n our c a s e ,  m i t o c h o n d r i a were found i n t h e r e g i o n between 36 and 40% s u c r o s e , w h i c h was i n agreement w i t h t h e l i t e r a t u r e . was  d i s t r i b u t e d throughout  The SR  the g r a d i e n t w i t h t h e s p e c i f i c  a c t i v i t y o f marker enzymes f o r t h e SR b e i n g h i g h e s t i n F r a c t i o n 2 (27% s u c r o s e ) . Three peaks o f a c t i v i t y were observed  u s i n g the marker  NADPH c y t c_ r e d u c t a s e , b u t these r e s u l t s d i d n o t agree w i t h t h e d i s t r i b u t i o n o f t h e m a r k e r s glucose-6-phosphatase c_ r e d u c t a s e .  and NADH c y t  The r e s u l t s o f MATLIB (MATLIB e t a l . , 1979)  showed NADH c y t c_ r e d u c t a s e t o be an u n a c c e p t a b l e marker of t h e SR.  specific  Our r e s u l t s seem t o b e a r t h i s o u t .  I t may  be t h a t we a r e s e e i n g t h e e f f e c t s o f d i f f e r i n g o r i e n t a t i o n o r a non s p e c i f i c enzyme d i s t r i b u t i o n o f glucose-6-phosphatase NADH c y t c_ r e d u c t a s e .  or  The plasma membranes were g e n e r a l l y  143  - 143 "  found a t lower d e n s i t i e s , however, SR was a l s o p r e s e n t fractions.  i n certain  The d i s t r i b u t i o n observed was i n agreement w i t h the  r e s u l t s of Moore (MOORE e t a l . , 1975) and Hurwitz (1974).  There  were no i n d i c a t i o n s t h a t the. plasma membranes were b i n d i n g h i g h e r d e n s i t y membrane fragments.. separations  On the other hand, most  on g r a d i e n t s y i e l d r e s u l t s d i s s i m i l a r  above as t h e SR i s found a t h i g h e r membrane i n these  to these  d e n s i t i e s than plasma  studies.  A r a t h e r i n t e r e s t i n g f e a t u r e o f our plasma membrane 2+ f r a c t i o n s was the Mg s t i m u l a t e d ATPase observed on the e x t e r n a l 32 membrane s u r f a c e of F5 and s i n g l e c e l l s . L a b e l l i n g w i t h [ p] ATP seemed to i n d i c a t e t h a t the ATPase was l o c a l i z e d t o an apparent M.W. 2+ of 205,000 d a l t o n s . associated with  Mg  s t i m u l a t e d ATPases have been found  the plasma membrane of a o r t i c smooth muscle,  myometrial and i n t e s t i n a l smooth muscle, b u t t h e i r f u n c t i o n i s 2+ c u r r e n t l y under c o n s i d e r a b l e d i s p u t e .  A Mg  s t i m u l a t e d ATPase  s i m i l a r to t h a t observed here has r e p o r t e d l y been found on the e x t e r n a l s u r f a c e of t h e r e d b l o o d  c e l l plasma membrane.  enzyme e x h i b i t s h i g h s u b s t r a t e i n h i b i t i o n and other  similar  p r o p e r t i e s t o our enzyme (SMOLEN & WEISSMAN, 1978). 2+ authors  s p e c u l a t e t h a t Mg  and superoxide  anion  i t remains t o be seen whether these a r e r e a l l y 2+  i n v e s t i g a t i n g any Mg  The  s t i m u l a t e d ATPases may have some  r o l e i n chemotaxis, p h a g o c y t o s i s but  This  generation  true.  When  s t i m u l a t e d ATPase i n muscle t h e r e i s the  added danger o f myosin ATPase contaminating  the p r e p a r a t i o n , as  myosin may be absorbed to the membrane s u r f a c e d u r i n g homogenization. 2+ However, we f e e l t h a t we can r u l e t h i s o u t as t h e Mg stimulated  144  -  144 -  ATPase a c t i v i t y noted i n F5 was t h e same as that found i n f r e e 2+ cells. Another problem w i t h the h i g h Mg s t i m u l a t e d ATPase a c t i v i t y observed i n our F r a c t i o n 5 was t h a t i t p r o h i b i t e d 2+ the measurement of any Na+/K+ ATPase or Ca I t was o n l y  through [ 32p ] ATP l a b e l l i n g  of t h e p o s s i b i l i t y  ATPase a c t i v i t y .  t h a t we became aware  t h a t more than one ATPase may be p r e s e n t .  I t i s were to be demonstrated c o n v i n c i n g l y t h a t t h e 165,000 MW 2+ peak r e p r e s e n t s a Ca ATPase, s e l e c t i v e e x t r a c t i o n of these 2+ bands or s e l e c t i v e removal of the Mg have t o be achieved  first  stimulated  (JORGENSON, 1974).  added t h a t t h e p h o s p h o r y l a t i o n  ATPase would  I t might be f u r t h e r  c o n d i t i o n s used by other  authors  (see Methods s e c t i o n ) were n o t found to work i n our s t u d i e s whatsoever, and our c o n d i t i o n s were chosen as the r e s u l t of many d i f f e r e n t v a r i a t i o n s . experiments r e s u l t s y i e l d they p r o v i d e d  Not o n l y d i d the p h o s p h o r y l a t i o n information  of p o s s i b l e ATPases b u t  f u r t h e r evidence f o r t h e d i f f e r e n t o r i e n t a t i o n s of  F4 and F5. Perhaps the most i n t e r e s t i n g r e s u l t s were o b t a i n e d the i o d i n a t i o n experiments f o r t h e l a t t e r p r o v i d e d  from  another means  f o r d e t e r m i n i n g o r i e n t a t i o n and degree of p u r i f i c a t i o n of plasma membranes.  The i n h e r e n t  that the l a b e l l i n g & SCHONBAUM, 1976). catalyzed  assumption i n a l l such s t u d i e s i s  s p e c i e s w i l l n o t permeate the membrane (MORRISON Though the exact mechanism of l a c t o p e r o x i d a s e  i o d i n a t i o n i s n o t f u l l y known, i t i s f e l t  iodinating species  s a t i s f i e s this condition.  t h a t the  We might f u r t h e r  p o i n t out t h a t e x t e n s i v e any  inherent  c o n t r o l s must be r u n to account f o r 125 p e r o x i d a s e a c t i v i t y and non s p e c i f i c I binding.  145  -  145 -  A simple washing of the membranes a f t e r i o d i n a t i o n i s q u i t e inadequate, t h e r e f o r e , c o n t r o l s d e l e t i n g H O or l a c t o p e r o x i d a s e 125 _ must be run. Free I can be removed by g e l e l e c t r o p h o r e s i s , if  the l a t t e r i s done p r o p e r l y .  f a c t o r s i n t o account and  Our  r e s u l t s have taken a l l these 125  they r e p r e s e n t  the  I  incorporated  a f t e r background c o n t r o l s have been subtracted'.. of membranes w i t h T r i t o n X-100 the s u b s t r a t e was  reported  treatment  to i n c r e a s e a c c e s s i b i l i t y  to enzymes l o c a t e d on by  The  Steck (1974a) but  the i n n e r membrane  of  surface  u n t i l r e c e n t l y t h i s has  not  been a p p l i e d to the l a b e l l i n g of membranes (HARTIG & RAFTERY, 1977). H a r t i g and was  Raftery  found to i n h i b i t  contrary self  results.  labelling  much to our 0.40% The  used Emulphogene as the d e t e r g e n t s i n c e TX-100  Our  surprise..  I t was  due  not  labelling  of the membranes, above  observed decreased l a b e l l i n g of the membranes.  of s e l f - l a b e l l e d  lactoperoxidase  c o u l d now  be  easily  c o v a l e n t l y l i n k e d to sepharose  T h i s would g r e a t l y f a c i l i t a t e d e a l i n g w i t h c o n t a m i n a t i o n  toself labelling.  unequivocally two  experiments showed  only at concentrations  a v o i d e d by use of l a c t o p e r o x i d a s e beads.  our  i n v e s t i g a t i o n showed t h a t TX-100 i n h i b i t e d  of the enzyme but,  TX-100 t h a t we use  the enzyme, but,  A l l in a l l ,  the i o d i n a t i o n s t u d i e s  demonstrated the p r e f e r e n t i a l o r i e n t a t i o n of  s e t s of membranes.  of f o l l o w i n g the two  They a l s o p r o v i d e d  the  a convenient means  s e t s of membranes i n any  further  purification  procedure. The ways.  We  e x t r a c t i o n r e s u l t s were q u i t e u n s u c c e s s f u l  had  i n many  hoped to s e l e c t i v e l y remove c e r t a i n bands and  them as w e l l as the r e s i d u a l bands but  investigate  there were no c l e a r c u t  r e s u l t s as were seen i n the e x t r a c t i o n s t u d i e s performed on  red  146  - 146  blood c e l l s  (rbc).  "  Our r e s u l t s , however, seemed to r e f l e c t  d i f f e r e n c e s i n membrane o r i e n t a t i o n between F4 and F5. was n o t seen i n s t u d i e s done on r b c by F a i r b a n k s al.,  1971 b) and Steck (STECK & YU, 1973).  This  (FAIRBANKS e t  These s t u d i e s  u s i n g the r e d b l o o d c e l l s have shown: t h a t TX-100 removes g l y c o p r o t e i n s embedded p a r t i a l l y i n t h e hydrophobic r e g i o n o f the membrane and t h a t p r o t e i n p e r t u r b a n t s remove the i n n e r cytoplasmic proteins.  However, whether these agents a c t i n  the same way on membranes other than t h e r b c i s q u e s t i o n a b l e based on our r e s u l t s .  A good example of t h i s i s Band 1.  Band  1 has been shown to be e x t e r n a l l y l o c a l i z e d , but i t was removed t o t a l l y by TX-100 i n F4 and remained unchanged i n F5. on the o t h e r hand, removed Band 1 i n F5 but not i n F4.  pCMBS It  would seem then t h a t a c c e s s i b i l i t y i s the l i m i t i n g f a c t o r i n extraction.  The r e s u l t s a r e f u r t h e r c o m p l i c a t e d by the l a c k  of knowledge of t h e exact mechanism of e x t r a c t i o n of the v a r i o u s agents used.  A d e s i r a b l e a d d i t i o n to these experiments would be  to measure the ATPase a c t i v i t i e s of the e x t r a c t e d membranes i n c o n j u n c t i o n w i t h e l e c t r o n microscopy and marker assay  studies.  T h i s however, was beyond the scope of our work. The f i n a l step i n our membrane i s o l a t i o n procedure was p u r i f i c a t i o n o f the membranes u s i n g a f f i n i t y chromatography. The r e s u l t s , p r e s e n t e d f o r F r a c t i o n s 4 and 5 i n T a b l e X I I and F i g u r e s 39-41, a r e s i m i l a r to those o b t a i n e d by Walsh (WALSH et al.,  1976).  Based on the i o d i n a t i o n s t u d i e s a l o n e , the l a b e l l i n g  p a t t e r n observed f o r the f i r s t  two peaks e l u t e d when F4 was  a p p l i e d to the column i s c o n s i s t e n t o n l y w i t h the membranes  147  - 147  being i n s i d e - o u t .  -  As a c o n t r o l , n o n - i o d i n a t e d membranes  e l u t e d from the column were i o d i n a t e d . was to  The p a t t e r n observed  s i m i l a r to t h a t i n F i g u r e s 40a and 40b. a minor degree was  However, l a b e l l i n g  a l s o seen a t 205,000 and  55,000 d a l t o n s .  These c o n t r o l experiments would seem to i n d i c a t e t h a t membranes, though i n i t i a l l y pure, become l e a k y or due The  latter  based  the 10 v e s i c l e s , may  have  e l u t e d i n the presence of amethyl-D-mannoside,  on the i o d i n a t i o n s t u d i e s appeared  yielded similar  the  to t h e i r u n s t a b l e n a t u r e , R0 or u n s e a l e d .  two peaks,  membrane v e s i c l e s .  (F4)  to c o n s i s t of u n s e a l e d  C o n t r o l s t u d i e s u s i n g n o n - i o d i n a t e d membranes  results.  The above r e s u l t s wre  confirmed u s i n g AchE s i d e d n e s s  assays of the v a r i o u s e l u t e d membrane f r a c t i o n s .  There  was  no i n c r e a s e i n a c c e s s i b i l i t y of column f r a c t i o n s 5 and  17 when  compared to the i n i t i a l l y  In  actuality, was  t h e r e was  noted was  AchE.  a 1-2  T h i s may  a p p l i e d F4 s t o c k membranes.  a decrease i n the a c c e s s i b i l i t y .  What  f o l d i n c r e a s e i n the s p e c i f i c a c t i v i t y of  have been due  to removal of m i t o c h o n d r i a l  c o n t a m i n a t i o n or c o n t a m i n a t i o n from p l a u s i b l e as the s i a l i c  the SR.  T h i s would  be  a c i d d e t e r m i n a t i o n s showed the  presence of s i a l o g l y c o p r o t e i n s i n f r a c t i o n s e n r i c h e d w i t h SR and m i t o c h o n d r i a  (see T a b l e s V i l a , V l l b and V I I I ) .  these c o n t a m i n a t i n g o r g a n e l l e membranes were RO may  have been bound to the column.  t h e r e may to  the  If  oriented  they  It i s also plausible  that  have been some m o d i f i c a t i o n o f the membranes  capping which i s known to occur i n the presence of  The decrease i n AchE a c c e s s i b i l i t y may  due lectins.  be a t t r i b u t e d to e i t h e r  148  - 148 -  l e a k y 10 v e s i c l e s or 10 v e s i c l e s becoming RO and/or The  unsealed.  h i g h o s m o l a r i t i e s o f t h e b u f f e r s used would seem to r u l e  out osmotic e f f e c t s being permeability. assays,  r e s p o n s i b l e f o r the i n c r e a s e i n  What would be n e c e s s a r  i s to do f u r t h e r marker  l e c t i n b i n d i n g s t u d i e s and maybe some c r o s s - l i n k i n g  s t u d i e s to determine the exact  cause of t h e apparent  increase  i n membrane p e r m e a b i l i t y . Based on the AchE assays t h e l a t t e r  two e l u t e d peaks  from t h e F4 column appeared t o c o n t a i n unsealed vesicles.  These l a t t e r r e s u l t s being  iodination studies.  membrane  i n agreement w i t h the  The i n c r e a s e i n t h e s p e c i f i c a c t i v i t y of  the AchE may have been due to ,the reasons d i s c u s s e d  earlier.  On t h e b a s i s o f the i n c r e a s e i n s p e c i f i c a c t i v i t y o f AchE, i t may be s t a t e d t h a t the membranes i n F4 were p u r i f i e d T h i s , when combined w i t h 5'-nucleotidase  the p u r i f i c a t i o n of F4 based on  s t u d i e s , would make an o v e r a l l p u r i f i c a t i o n o f  40-60 f o l d o f t h e plasma membranes. higher  1-2 f o l d .  Though t h i s i s c o n s i d e r a b l y  than most plasma membrane p r e p a r a t i o n s , a c e r t a i n degree  of c a u t i o n i s r e q u i r e d as the f i n a l "10" membrane p r e p a r a t i o n appears to be v e r y u n s t a b l e  o r permeable.  These l a t t e r  issues  may be r e s o l v e d b e f o r e u s i n g these membranes f o r membrane transport i n v e s t i g a t i o n s .  I t s h o u l d a l s o be p o i n t e d o u t  t h a t y i e l d s of membrane p r o t e i n u s i n g the e a r l i e r  described  procedure a r e low and would r e q u i r e a b a t c h p r e p a r a t i o n o f plasma membranes f o r proper i n v e s t i g a t i o n .  The behaviour o f F5 membranes was r a t h e r expected based on the r e s u l t s o f Walsh (WALSH e t a l . , 1976).  Only two peaks  149  - 149  were e l u t e d , protein.  these, r e p r e s e n t i n g  The  i n i t i a l peak was  membrane v e s i c l e s . , w h i l e the  "  8-10%  of the t o t a l  applied  a s s e s s e d to c o n s i s t of 10 plasma  second peak, e l u t e d  i n the  presence  of amethyl-D-mannoside, found to c o n t a i n unsealed membrane vesicles.  These c o n c l u s i o n s  AchE s i d e d n e s s s t u d i e s . found to be S i n c e 90%  As  were based on the i o d i n a t i o n seen u s i n g  F4,  e i t h e r permeable or c o n t a i n  and  the 10 f r a c t i o n  was  u n s t a b l e 10 v e s i c l e s .  of the a p p l i e d p r o t e i n c o u l d not be  eluted  the  a p p l i c a b i l i t y of t h i s method f o r f u r t h e r p u r i f i c a t i o n of a s e t of RO membrane v e s i c l e s must be i t must be  q u e s t i o n e d as  vesicles., already The  two  90%  but  a s e t of RO  Further,  plasma membrane  pure, would r e q u i r e f u r t h e r p u r i f i c a t i o n .  s e t s of membranes i s o l a t e d (F4 and  many d e s i r a b l e f e a t u r e s transport  to why  suspected.  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