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Studies on sodium and potassium ion activated adenosine trihosphatase Pang, Yew Choi 1975

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STUDIES ON SODIUM AND POTASSIUM ION ACTIVATED ADENOSINE TRIPHOSPHATASE . BY  YEW  CHOI  '  n  PANG  B.Sc. McGILL UNIVERSITY  1972  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  i n the Department  of Chemistry  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e requi red standard  THE UNIVERSITY OF BRITISH COLUMBIA May,  1975  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y I  further  for  fulfilment  of  the  requirements  the U n i v e r s i t y of B r i t i s h Columbia, I agree  s h a l l make it  freely  available  for  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f  this  representatives. thesis for  It  financial  of  gain s h a l l  Qj^N^T^  The U n i v e r s i t y of B r i t i s h Columbia  2075 Wesbrook Place Vancouver, Canada V6T 1W5  that  this  thesis or  i s understood that c o p y i n g or p u b l i c a t i o n  written permission.  Department  for  r e f e r e n c e and study.  s c h o l a r l y purposes may be granted by the Head of my Department  by h i s of  in p a r t i a l  not  be allowed without my  ABSTRACT  The Na K - a c t i v a t e d ATPase was o b t a i n e d from the r e c t a l , g l a n d s o f dog f i s h . Optimum c o n d i t i o n s f o r e x t r a c t i o n o f the membrane bound enzyme i n t o s o l u t i o n and s t a b i l i z a t i o n by g l y c e r o l was  investigated,  f o l l o w e d by a t t e m p t s t o p u r i f y the enzyme by a f f i n i t y  chromatography  u s i n g i n s o l u b i l i z e d C o n c a n a v a l i n A, wheat germ a g g l u t i n i n and a m o d i f i e d ATP m o l e c u l e as a f f i n i t y l i g a n d s . The s t r u c t u r e and s i z e o f the enzyme as a m a c r o m o l e c u l e  was c h a r a c t e r i z e d by a c o m b i n a t i o n o f  the r e s u l t s from v e l o c i t y s e d i m e n t a t i o n i n an i s o k i n e t i c s u c r o s e g r a d i e n t and g e l f i l t r a t i o n w i t h S e p h a r o s e 6B. The s e d i m e n t a t i o n c o e f f i c i e n t was f o u n d t o be 5.0 S  by v e l o c i t y s e d i m e n t a t i o n , and the  S t o k e s ' r a d i u s was found Lo be .114 A  by g e l f i l t r a t i o n . U s i n g t h e s e  d a t a , t h e m o l e c u l a r w e i g h t o f the i n t a c t enzyme was e s t i m a t e d to be 240,000. T h i s i s i n good agreement w i t h the m o l e c u l a r w e i g h t  derived  from sodium d o d e c y l s u l f a t e g e l e l e c t r o p h o r e s i s o b t a i n e d by o t h e r workers.  CONTENTS  ABSTRACT o » e « 4 » & & e a 9 » » o o e o o « e « a « a o f t o o o o f t e c f t o o > e « 9 « o o & O A C > c « o « « * f t * ' * e e - o o * « * * * B O  ACKNOWLEDGEMENTS  ..  ABREVIATIONS....  "....'.rl  ' . f j i  INTRODUCTION PART I  V.  ; .1  C h a r a c t e r i s t i c s o f N a K - a c t i v a t e d ATPase... +  ....2  +  (1) Mechanism o f c a t a l y s i s a n d c a t i o n t r a n s p o r t (2) I n t e r a c t i o n w i t h c a r d i o a c t i v e s t e r o i d s (3) P u r i f i c a t i o n . .  (5) L i p i d r e q u i r e m e n t  .....6  11  ....18 .-  (2) L i g a n d (3) C o v a l e n t c o u p l i n g t o t h e m a t r i x (4) B i n d i n g o f p r o t e i n t o a f f i n i t y column (5) E l u t i o n o f t h e bound p r o t e i n PART I I I T h e o r y o f V e l o c i t y S e d i m e n t a t i o n & G e l F i l t r a t i o n (1) V e l o c i t y s e d i m e n t a t i o n  ..4  .......15  PART I I A f f i n i t y C h r o m a t o g r a p h y (1) S t a t i o n a r y m a t r i x  .  .9  (4) S u b u n i t s t r u c t u r e a n d s i z e  (2) G e l f i l t r a t i o n .  XX  19 .19 21 .22 .....22 .....24  24 ...27  iv  METHODS  .  32  (1) S y n t h e s i s of t r i methyl d o d e c y l ammonium bromide (2) C o l l e c t i o n of r e c t a l g l a n d s of dog f i s h  32  .'.rrr;:;..  32  (3) L y o p h i l i z a t i o n of t i s s u e s  33  (4) E x t r a c t i o n of N a K - a c t i v a t e d ATPase  33  (5) P r o t e i n d e t e r m i n a t i o n  34  +  +  (6) Na K - a c t i v a t e d ATPase a s s a y  ....  35  (7) P r e p a r a t i o n of a f f i n i t y r e s i n s . . . . .  ,.  36  (a) Cyanogen b r o m i d e a c t i v a t i o n of S e p h a r o s e 4B...............36 (b) C o u p l i n g o f C o n c a n a v a l i n A t o S e p h a r o s e 4B  37  ( c ) C o u p l i n g of wheat germ a g g l u t i n i n to S e p h a r o s e 4B  37  (d) S y n t h e s i s o f h e x a n e d i o i c a c i d d i h y d r a z i d e  37  (e) C o u p l i n g of h e x a n e d i o i c a c i d d i h y d r a z i d e to S e p h a r o s e 4B..38 ( f ) C o u p l i n g o f p e r i o d a t e e x i d i z e d ATP t o S e p h a r o s e hexanedioic acid dihydrazide... (8) A f f i n i t y c h r o m a t o g r a p h y  39 ,  40  (9) V e l o c i t y s e d i m e n t a t i o n  40  (a).Calculation of sucrose concentration gradient.  40  , (b) G e n e r a t i o n o f s u c r o s e c o n c e n t r a t i o n g r a d i e n t  44  (c) C a l i b r a t i o n w i t h s t a n d a r d p r o t e i n s and d e t e r m i n a t i o n of sedimentation c o e f f i c i e n t  ....46 + +  (10) Gel f i l t r a t i o n o f d e t e r g e n t s o l u b i l i z e d Na K - a c t i v a t e d ATPase and S t o d e s  1  radius calibration  ...48  RESULTS  ......49  (1) O p t i m i z a t i o n o f c o n d i t i o n s f o r enzyme e x t r a c t i o n . . . . .  .....49  (a) T e s t o f e f f i c i e n c y o f e x t r a c t i o n by d i f f e r e n t d e t e r g e n t s . . 4 9 (b) E x t r a c t i o n as a f u n c t i o n o f L u b r o l WX c o n c e n t r a t i o n . . . . . . . 4 9 ( c ) S t a b i l i t y o f L u b r o l WX e x t r a c t e d N a K - a c t i v a t e d A T P a s e . . . 5 0 +  +  (2) A f f i n i t y c h r o m a t o g r a p h y  50  (3) V e l o c i t y s e d i m e n t a t i o n  50  (4) G e l f i l t r a t i o n o f N a K - a c t i v a t e d ATPase and S t o k e s ' r a d i u s +  +  51  calibration  (5) , E s t i m a t i o n o f t h e m o l e c u l a r w e i g h t o f N a K - a c t i v a t e d A T P a s e . .52 +  +  DISCUSSION  ...66  (1) E x t r a c t i o n o f Na K - a c t i v a t e d A T P a s e  66  (2) A f f i n i t y c h r o m a t o g r a p h y  67  .  (3) E s t i m a t i o n o f m o l e c u l a r w e i g h t and s u b u n i t c o m p o s i t i o n REFERENCES  ,.  69 71  vi  ACKNOWLEDGEMENTS /  I w o u l d l i k e t o e x p r e s s my most s i n c e r e t h a n k s t o D r . D. G. C l a r k , whose a d v i c e a n d s u g g e s t i o n s have made t h i s work p o s s i b l e . I w o u l d a l s o l i k e t o thank Kathy Chance f o r a sample o f p u r i f i e d c h i c k e n e g g w h i t e l y s o z y m e , Guy B e a u r e g a r d f o r many h e l p f u l d i s c u s s i o n s , P e t e r Morrod f o r h i s c o l l a b o r a t i o n i n t h e development o f t h e c o n s t a n t velocitv prariient  Ladysmith,  and  Mr. Martin  Handel o f  Spa Fresh P a c i f i c Products.  B. C. f o r a f r e e s u p p l y o f r e c t a l g l a n d s o f dog f i s h .  vii  ABBREVIATIONS  ATP  Adenosine triphosphate  ADP  Adenosine diphosphate  AMP  A d e n o s i n e monophosphate  NAD  Nicotinamide adenine d i n u c l e o t i d e  N a K - a c t i v a t e d ATPase  Sodium and p o t a s s i u m i o n a c t i v a t e d adenosine triphosphatase  P  Inorganic orthophosphate  E-P  P h o s p h o r y l a t e d sodium and p o t a s s i u m ion a c t i v a t e d adenosine triphosphatase  +  +  1  INTRODUCTION  2  INTRODUCTION  + + C h a r a c t e r i s t i c s o f Na K - a c t i v a t e d ATPase  PART I  Under normal p h y s i o l o g i c a l c o n d i t i o n s , c o n c e n t r a t i o n o f Na  gradients  and K e x i s t a c r o s s t h e c e l l membrane. [Na ] = 140 mM and [K ] -  5 mM i n human b l o o d p l a s m a , compared t o [ N a ] ' = 10 mM and [ K ] - 150 mM +  in the cytoplasm,  +  d e p e n d i n g on t h e t y p e o f t h e c e l l . T h e s e c a t i o n  g r a d i e n t s a r e r e q u i r e d f o r a v a r i e t y o f f u n c t i o n s o f t h e c e l l . The high K  +  concentration i n s i d e the c e l l i s required f o r protein synthesis  by r i b o s o m e s and a l s o f o r maximal a c t i v i t y o f p y r u v a t e k i n a s e . The  t r a n s p o r t o f amino a c i d s and s u g a r s i n t o t h e c e l l . M o r e o v e r t h e combined c a t i o n g r a d i e n t s g e n e r a t e an e l e c t r o c h e m i c a l p o t e n t i a l d i f f e r e n c e w h i c h may be as h i g h as 100 mV i n muscle and n e r v e c e l l s . The p o t e n t i a l d i f f e r e n c e , w h i c h can a c t u a l l y be measured i n g i a n t s q u i d a x o n s , p l a y s an i m p o r t a n t p a r t , i n t h e t r a n s m i s s i o n o f n e r v e impules ( 1 ) . The e x i s t e n c e o f .such c a t i o n g r a d i e n t s i m p l i e s t h a t t h e r e i s a ' c a t i o n pump', p r e s u m a b l e s u i t u a t e d i n t h e c e l l membrane, w h i c h f u n c t i o n s t o t r a n s p o r t Na  o u t o f and K  i n t o the c e l l . Experiments with resealed  r e d b l o o d c e l l g h o s t s p r o v i d e e v i d e n c e t h a t ATP i s t h e e n e r g y s o u r c e ( 2 ) . In 1957 an i m p o r t a n t b r e a k t h r o u g h understanding  opened t h e molecular b a s i s f o r the  o f t h e c a t i o n pump t o b i o c h e m i c a l  experimentation.  Skou ( 3 ) d i s c o v e r e d t h a t a f r a c t i o n o f h o m o g e n i z e d c r a b n e r v e known  3  t o c o n t a i n c e l l membrane f r a g m e n t s e x h i b i t e d M g - d e p e n d e n t ATPase a c t i v i t y +  t h a t was g r e a t l y s t i m u l a t e d when b o t h K  +  and N a  +  were added. Skou  suggested  t h a t t h i s c e l l membrane f r a c t i o n c o n t a i n e d an enzyme w h i c h i s r e p o n s i b l e for maintaining  the N a  is strongly supported  +  and K  +  concentration g r a d i e n t s . His  suggestion  by the f a c t t h a t b o t h c a t i o n t r a n s p o r t and  the  enzyme a c t i v i t y r e q u i r e ATP, and i n h i b i t i o n o f c a t i o n t r a n s p o r t a l s o l e d t o i n a c t i v a t i o n o f enzyme a c t i v i t y ( 4 , 5 ) . T h i s enzyme, r e f e r r e d t o as Na K - a c t i v a t e d A T P a s e , i s u b i q u i t o u s . I t has been f o u n d i n many t i s s u e s o f mammals, such as b r a i n , m u s c l e and r e d b l o o d c e l l s i n d i f f e r e n t amounts ( 6 ) . However, t h e r i c h e s t s o u r c e s of Na K -activated +  +  ATPase are s p e c i a l i z e d o r g a n s , e i t h e r f o r  generation  o f e l e c t r i c a l p o t e n t i a l , s u c h as the e l e c t r o p l a x o f e l e c t r i c e e l s , o r for  e x t r u s i o n of excess  the  Na  , s u c h as tne  Kianey  cortex  OT  mammals  {/),  the s a l t g l a n d o f s e a g u l l s ( 8 ) , and the r e c t a l g l a n d o f dog f i s h ( 9 ) , w h i c h i s the s o u r c e used i n t h i s s t u d y . The r e c t a l g l a n d o f dog f i s h has  been  shown t o s e c r e t e a f l u i d i n t o the f i s h gut w i t h a s o d i u m  concentration  as h i g h as 540 mM,  100 mM h i g h e r than t h a t o f s e a w a t e r ( 1 0 ) .  I t s f u n c t i o n i s to e x c r e t e e x c e s s s o d i u m c h l o r i d e a c c u m u l a t e d t h r o u g h ingestion. To d a t e , the r e s e a r c h on N a K - a c t i v a t e d +  +  c e n t r a t e d i n the f o l l o w i n g f i v e main a r e a s : (1) Mechanism o f c a t a l y s i s and c a t i o n t r a n s p o r t (Z) I n t e r a c t i o n w i t h c a r d i o a c t i v e s t e r o i d s (3) P u r i f i c a t i o n (4) S u b u n i t s t r u c t u r e . a n d (5) L i p i d r e q u i r e m e n t  size  ATPase has been con-  4  (1) Mechanism o f c a t a l y s i s and c a t i o n t r a n s p o r t  It i s generally agreed that N a K - a c t i v a t e d +  ATP i n a s t e p w i s e  El  + ATP El  _  hydrolyses  f a s h i o n , each s t e p r e q u i r i n g d i f f e r e n t c a t i o n ( s ) .  ^ — ^ ^  P  E i - P + ADP  1  E -P  2  2  E -P + H 0 2  ATPase  +  2  E  2  v  E  vs."  Ei  2  + P  3 4  More e l a b o r a t e and s o p h i s t i c a t e d schemes have been p r o p o s e d ( 1 9 ) , b u t they are e s s e n t i a l l y m o d i f i c a t i o n and r e f i n e m e n t r e p r e s e n t s the p h o s p h o r y l a t e d higher energy conformation  enzyme. t  x  and t  2  o f t h e a b o v e . E-P  r e p r e s e n t a lower and.a  o f the enzyme m o l e c u l e ,  r e s p e c t i v e l y (20,81).  T h e s e p a r t i a l r e a c t i o n s were e s t a b l i s h e d by b i n d i n g s t u d i e s and  substantiated  32  by k i n e t i c d a t a ( 2 0 , 2 1 ) . I t was  found t h a t  P was i n c o r p o r a t e d  from  [y P]ATP into Na K -activated  ATPase p r e p a r a t i o n i n the p r e s e n c e o f  Na  t o p a r t i a l r e a c t i o n 1 and 2. I f K  32  +  and Mg  +  , t h i s corresponds  added t o the enzyme s y s t e m a f t e r N a , +  t h i s corresponds  the  3 2  P  i n the enzyme was  was discharged,  t o p a r t i a l r e a c t i o n 3 ( 2 2 , 2 3 ) . The e v e n t u a l g o a l o f  t h e s e s t u d i e s i s t o e l u c i d a t e the mechanism o f c a t i o n t r a n s p o r t b a s e d on the p a r t i a l r e a c t i o n s . A t the moment, the mechanism o f c a t i o n t r a n s p o r t is poorly understood. Recently, magnetic resonance  t e c h n i q u e s were a p p l i e d to d e a l  w i t h t h i s p r o b l e m . Noggle e t . a l f o u n d t h a t the s p i n l a t t i c e r e l a x a t i o n  5  time T-| o f  23  Na i n a s o l u t i o n c o n t a i n i n g t h e enzyme d e c r e a s e d upon t h e  a d d i t i o n o f ATP, w h i c h i n d i c a t e s t h e b i n d i n g o f Na . When K was added, T-j i n c r e a s e d , w h i c h i n d i c a t e s t h e d i s p l a c e m e n t  o f Na by K ( 2 5 ) .  M i l d v a n e t . a l , u s i n g e l e c t r o n p a r a m a g n e t i c r e s o n a n c e and w a t e r p r o t o n 2+ 2+ r e l a x a t i o n s p e c t r a , showed t h a t Mn can s u b s t i t u t e f o r Mg. a t one t i g h t b i n d i n g s i t e on t h e enzyme. When i n o r g a n i c p h o s p h a t e was added i n t h e p r e s e n c e o f Na , t h e r e l a x a t i o n time T^ f o r w a t e r p r o t o n was i n c r e a s e d . I t was c o n c l u d e d t h a t t h i s i n c r e a s e was due t o t h e d i s p l a c e ment o f a r a p i d l y e x c h a n g i n g  w a t e r p r o t o n from t h e c o o r d i n a t i o n  sphere  2+ o f Mn  by the b i n d i n g o f phosphate t o t h e a c t i v e s i t e .  Furthermore,  t h i s i n c r e a s e i n T-j was a l m o s t a b o l i s h e d by c h a n g i n g t h e pH from 6.1 t h r o u g h t h e s e c o n d i o n i z a t i o n o f i n o r g a n i c p h o s p h a t e t o 7.5. T h i s suggested  t h e b i n d i n g o f p h o s p h a t e i n t h e p r e s e n c e of Na  i s much more  f a v o r e d when i t i s a monoanion. The o p p o s i t e e f f e c t was o b s e r v e d f o r K , which suggested  the b i n d i n g o f phosphate i n the presence o f K  i s more f a v o r e d when i t i s a d i a n i o n . Based on t h e s e d a t a , a t r a n s p o r t mechanism was p o s t u l a t e d i n w h i c h Na i.e. t h e a c y l p h o s p h a t e  and K b i n d t o t h e same s i t e ,  o f t h e enzyme. The b i n d i n g o f one e x c l u d e s t h e  b i n d i n g o f t h e o t h e r . N a i s t r a n s p o r t e d o u t o f t h e c e l l as a p r o t e i n +  complex, s t a b i l i z e d by t h e p h o s p h a t e monoanion, then K i s t r a n s p o r t e d +  i n t o t h e c e l l as a p r o t e i n complex, s t a b i l i z e d by t h e p h o s p h a t e d i a n i o n ( 2 4 ) . C o o r d i n a t i o n o f t h e metal i o n by p h o s p h a t e o c c u r s i n b o t h  cases,  b u t v i a d i f f e r e n t modes. T h i s p o s t u l a t e r e q u i r e s t h a t t h e c a t i o n b i n d i n g s i t e be v e r y c l o s e t o t h e s i t e f o r ATP h y d r o l y s i s . T h i s was s u b s t a n t i a t e d by f u r t h e r e x p e r i m e n t s i n w h i c h t h e T  of ^T1, 2  1  which, as T l , s u b s t i t u t e s +  6  f o r K , was d e c r e a s e d by t h e p a r a m a g n e t i c r e l a x a t i o n o f Mn* , b u t o n l y +  +  i n t h e p r e s e n c e o f t h e enzyme ( 4 6 ) .  (2) I n t e r a c t i o n w i t h c a r d i o a c t i v e s t e r o i d s  The c a r d i o a c t i v e s t e r o i d s a r e a c l a s s o f compound u s e f u l f o r treatment  o f h e a r t f a i l u r e . T h e i r s t r u c t u r e s a r e shown i n F i g . 1. Much  r e s e a r c h has been d i r e c t e d t o s t u d y i n g t h e b i n d i n g and i n h i b i t i o n o f Na K - a c t i v a t e d ATPase by c a r d i o a c t i v e s t e r o i d s b e c a u s e o f these pharmac e u t i c a l i m p l i c a t i o n s . The i n h i b i t i o n i s h i g h l y s p e c i f i c , and no o t h e r enzyme systems have been f o u n d t o be s e n s i t i v e t o t h e s e s t e r o i d s . T h i s +..+ ' iS  Vc i y  i nl(Ju i uui i C  in  "die  ijccii'Cii  i Oi" tid  K  . .  ,...  .  ....  -av»C i v'aCeu h i r a s e  .  aCCiviiy.  I f t h e enzyme a c t i v i t y i s i n h i b i t e d by a c a r d i o a c t i v e s t e r o i d , then t h e a c t i v i t y i s due t o Na K - a c t i v a t e d A T P a s e , o t h e r w i s e  i t i s due t o o t h e r  ATPase a c t i v i t y , such as C a - a c t i v a t e d ATPase. T h i s s p e c i f i c i t y l e d +  to. t h e s p e c u l a t i o n t h a t N a K - a c t i v a t e d . A T P a s e i s t h e +  +  pharmacological  r e c e p t o r f o r these d r u g s and t h a t t h e i r e f f e c t on t h e enzyme a r e r e s p o n s i b l e f o r t h e i r t h e r a p e u t i c and t o x i c e f f e c t s ( 7 4 ) . The s t e r o i d moiety i s c l a s s i f i e d i n t o four types: d i g i t o x i g e n i n , digoxigenin, o u a b a g e n i n and s t r o p h a n t h i d i n as shown i n F i g . 1. A c a r d i o a c t i v e g l y c o s i d e i s formed when one o r more s u g a r r e s i d u e i s l i n k e d t o t h e 33 hydroxyl  group o f t h e s t e r o i d . ATPase i n h i b i t i o n i s e s s e n t i a l l y i r r e v e r -  s i b l e a t p h y s i o l o g i c a l pH ( 2 6 ) . As shown i n T a b l e I , t h e d i s s o c i a t i o n constant o f the enzyme-inhibitor  complex i s a p p r o x i m a t e l y  10  M but  7  din 4  D - g l u c o s i de  Fig. 1  oH  Structure of cardioactive glycosides  8  +  +  the a c t u a l v a l u e depends on the c o n c e n t r a t i o n s o f Na , K , and  ATP.  A s t o i c h i o m e t r y o f c l o s e t o 1 : 1 b i n d i n g was r e p o r t e d i n most cases TABLE I Inhibitor  Source Outer medulla of dog k i d n e y c o r t e x  N-(4'-amino-n-butyl)-3-amino a c e t y l - s t r o p h a n t h i d i n 4_ (27)  Apparent Dissociation Constant 1.92x10~ M  S t o i chiometr Inhibitor : enzyme  9  0.8-1,2  di g o x i n  2_  0.9  Electroplax of e l e c t r i c e e l s (26)  ouabain  3^  0.5  Cat b r a i n (26)  ouabain  3  1.0  Calf heart  (28)  !1  Na  k  .  and d e c r e a s e d by K . A l t h o u g h K  n e v e r t h e l e s s , r e v e r s e i t (26,27,28). not understood.  ,4-  r e t a r d s the b i n d i n g , i t c a n n o t , The mechanism o f b i n d i n g i s s t i l l  I t has a l s o been shown t h a t o u a b a i n i n h i b i t s t r a n s p o r t  (and by i n f e r e n c e the N a K - a c t i v a t e d +  +  ATPase) o n l y by b i n d i n g ATPase  e x p o s e d on the e x t e r n a l s u r f a c e o f the c e l l membrance ( 2 9 ) . The d i s s o c i a t i o n r a t e c o n s t a n t o f the e n z y m e - i n h i b i t o r  complex  depends on the c o n d i t i o n s f o r b i n d i n g . I t was f o u n d t h a t the complex + 2+ formed i n the p r e s e n c e o f Na , Mg and ATP ( t y p e I) d i s s o c i a t e s f a s t e r 2+ than the c o r r e s p o n d i n g complex formed i n the p r e s e n c e o f Mg  and  p h o s p h a t e ( t y p e I I ) . The d i s s o c i a t i o n r a t e c o n s t a n t s o f v a r i o u s type I complexes w i t h d i f f e r e n t s t e r o i d m o i e t y i n d i c a t e s t h a t t h e s t a b i l i t y o f t y p e I complex i n c r e a s e s i n the o r d e r : d i g i t o x i g e n i n g l y c o s i d e <  9  s t r o p h a n t h i d i n g l y c o s i d e < d i g o x i g e n i n g l y c o s i d e and s t r o p h a n t h i d i n g l y c o s i d e < o u a b a g e n i n g l y c o s i d e . I f t h e s t e r o i d m o i e t y i s t h e same, the s t a b i l i t y o f t h e g l y c o s i d e i n c r e a s e s i n t h e o r d e r d i g i t o x i d e 6-deoxyglucoside  = f u c o s i d e < r h a m n o s i d e . I t was c o n c l u d e d t h a t t h e  2'a a n d 3'a o r B-hydroxyl  groups o f t h e s u g a r m o i e t y s t a b i l i z e t h e  b i n d i n g through h y d r o g e n b o n d i n g ( 4 7 ) .  (3) P u r i f i c a t i o n  S e v e r a l w o r k e r s have p u r i f i e d t h e enzyme t o d i f f e r e n t e x t e n t s from a v a r i e t y o f s o u r c e s . T a b l e II summarizes some e x a m p l e s . A l l o f them u t i l i z e c l a s s i c a l p r o t e i n D u r i f i c a t i o n techniques. The c o m p l e t e p u r i f i c a t i o n takes from f o u r days t o one week, d e p e n d i n g on t h e method. Most  procedures  s t a r t w i t h 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 . Thus c e n t r i f u g a t i o n o f a s u s p e n s i o n o f homogenized t i s s u e a t i n c r e a s i n g s p e e d w i l l s e d i m e n t membrane f r a g m e n t s o f d e c r e a s i n g d e n s i t y . Membrane f r a g m e n t s o f d i f f e r e n t d e n s i t y may c o n t a i n d i f f e r e n t amounts o f t h e enzyme, and t h i s a l l o w s t h o s e membrane f r a g m e n t s r i c h e s t i n Na K - a c t i v a t e d ATPase a c t i v i t y be s e l e c t e d . Sodium o r p o t a s s i u m i o d i d e t r e a t m e n t i s a l s o commonly u s e d . Such t r e a t m e n t i s c a l l e d c h a o t r o p i c e x t r a c t i o n and r e s u l t s i n t h e removal o f l o o s e l y bound membrane p r o t e i n s from t h e membrane, l e a v i n g t h e membrane r e l a t i v e l y r i c h e r i n the more s t r o n g l y bound Na K - a c t i v a t e d A T P a s e . The N a K - a c t i v a t e d +  +  ATPase i s v e r y t i g h t l y bound t o t h e membrane.  S i n c e most p u r i f i c a t i o n t e c h n i q u e s a r e d e s i g n e d t o be c a r r i e d o u t i n  10  TABLE I I  Source Outer medulla o f dog kidney cortex (7)  Outer medulla ur  uog  kidney cortex (13)  Rectal gland o f dog f i s h (11)  Electroplax o f electric eels (16)  Procedure (i) Differential centrifugation 'microsomes' c o l l e c t e d a t 50,000 x g ( i i ) M i c r o s o m e s washed w i t h .06% d e o x y c h o l a t e a t low i o n i c strength ( i i i ) KI t r e a t m e n t o f microsome ( i v ) Microsomes washed w i t h .06% d e o x y c h o l a t e a t 160 mM KC1 (v) M i c r o s o m e s e x t r a c t e d w i t h .6% d e o x y c h o l a t e a t 267 mM KC1 ( v i ) S e p h a r o s e 2B g e l f i l t r a t i o n enzyme c o l l e c t e d i n v o i d volume (i) Differential centrifugation microsomes c o l l e c t e d a t 30,000 x g ( i i Nal treatment o f microsomes ( i i i S o l u t i l i z a t i o n w i t h .35% deoxycholate (iv) Glycerol precipitation (v) S o l u b u l i z a t i o n w i t h .35% m i x t u r e o f 3:1 d e o x y c h o l a t e cholate ( v i ) Ammonium s u l f a t e p r e c i p i t a t i o n  Specific A ctivity Final y i e l d ymoles P/ mg o f p r o t e i n / mg p r o t e i n /h g o f wet t i s s u e 0.46/150  800  17/100  1,552  19.3/15  1,510  24.5/405  1,260  1  (i) Differential centrifugation 'membranes' c o l l e c t e d a t 23,500 x g ( i i ) Membranes e x t r a c t e d w i t h 1.6% L u b r o l WX ( i i i ) Zonal c e n t r i g u g a t i o n with 2 t o 22% l i n e a r s u c r o s e gradient ( i v ) Ammonium s u l f a t e p r e c i p i t a t i o n E s s e n t i a l l y t h e same as above 1% L u b r o l was u s e d i n s t e a d o f 1.6%  11  aqueous phase, an i m p o r t a n t s t e p i n any p u r i f i c a t i o n i s the e x t r a c t i o n o f the enzyme from the membrane i n t o aqueous s o l u t i o n by a d e t e r g e n t . Two d e t e r g e n t s f r e q u e n t l y employed a r e sodium d e o x y c h o l a t e 5_ and L u b r o l  WX.  Sodium d e o x y c h o l a t e i s a s t e r o i d s e c r e t e d from the b i l e f o r e m u l s i f i c a t i o n o f i n g e s t e d l i p i d s to f a c i l i t a t e a b s o r p t i o n . L u b r o l WX, a  commercial  d e t e r g e n t , i s a p o l y o x y e t h y l e n e e t h e r o f the g e n e r a l f o r m u l a FUOCHgCHg) OH where n a v e r a g e s 6. Once d e t e r g e n t e x t r a c t e d , t h e enzyme i s v e r y u n s t a b l e and q u i c k l y l o s e s i t s a c t i v i t y . P r e c i p i t a t i o n by ammonium s u l f a t e as the f i n a l s t e p n o t o n l y improves  the p u r i f i c a t i o n b u t a l s o a l l o w s the  enzyme to be s t o r e d i n a p h y s i c a l s t a t e i n which i t i s more s t a b l e . One c r i t e r i o n f o r a s s e s s i n g the p u r i t y o f a p r e p a r a t i o n o f t h e enzyme i s the s p e c i f i c a c t i v i t y , e x p r e s s e d as t h e number o f moles o f  phosphate  p r o d u c e d by the h y d r o l y s i s o f ATP p e r m i l l i g r a m s o f t o t a l p r o t e i n p e r h o u r . As shown i n t h e l a s t column o f T a b l e I I , t h e s p e c i f i c a c t i v i t y o f the h i g h l y p u r i f i e d enzyme, i r r e s p e c t i v e o f the s o u r c e o r method o f p u r i f i c a t i o n , i s a p p r o x i m a t e l y 1,500  ymoles/mg/hr.  (4) S u b u n i t s t r u c t u r e and s i z e  The s u b u n i t s t r u c t u r e o f p u r i f i e d N a K - a c t i v a t e d ATPase has +  +  been i n v e s t i g a t e d by many w o r k e r s , most o f them r e l y i n g on t h e r e s u l t s  12  from sodium d o d e c y l s u l f a t e g e l e l e c t r o p h o r e s i s . In t h i s method, p r o t e i n s a r e s e p a r a t e d i n t o t h e i r s u b u n i t s by r e d u c t i o n o f any i n t r a m o l e c u l a r d i s u l f i d e bonds u s i n g B - m e r c a p t o e t h a n o l  or dithioerythritol followed  by d e n a t u r a t i o n by c o a t i n g t h e p r o t e i n w i t h t h e a n i o n i c d e t e r g e n t  sodium  d o d e c y l s u l f a t e . The p r o t e i n s a r e t h e n e l e c t r o p h o r e s e d i n a p o l y a c r y l a m i d e g e l . The  v a l u e o f t h e s u b u n i t has a l i n e a r r e l a t i o n s h i p w i t h t h e + +  m o l e c u l a r w e i g h t ( 1 4 ) . With t h e h i g h l y p u r i f i e d Na K - a c t i v a t e d A T P a s e , two bands a r e always f o u n d , r e g a r d l e s s o f t h e s o u r c e . T h i s i n d i c a t e s t h a t t h e enzyme c o n s i s t s o f two d i f f e r e n t s u b u n i t s w i t h d i f f e r e n t m o l e c u l a r w e i g h t s . The a c t u a l v a l u e s o f t h e s u b u n i t m o l e c u l a r w e i g h t depend on t h e s o u r c e , b u t a r e c o n s i s t e n t l y f o u n d t o be a p p r o x i m a t e l y 100,000 and 50,000, as can be seen i n T a b l e I I I TABLE I I I Source  M o l e c u l a r Weights Large s u b u n i t ( a ) Small s u b u n i t ( B )  Outer medulla o f dog k i dney c o r t e x (15)  Molar r a t i o  a&  84,000  57,000.  Outer medulla o f dog k i d n e y c o r t e x (13)  89,000  56,000  1:1  ag  Rectal gland o f dog f i s h (11,48)  97,000  55,000  2:1  a g  Electroplax of electric eel (16,48)  93,500  47,000  2:1  . a $  Ox b r a i n (12)  94,000  53,000  :  . 1 : 1  Subunit Structure  2  2  13  The l a r g e r s u b u n i t was f o u n d t o be the one t h a t b i n d s a c t i v e s t e r o i d (17) and a l s o c o n t a i n s the a c t i v e s i t e f o r  cardio-  phosphorylation  by ATP ( 1 2 , 1 8 ) , The s m a l l e r s u b u n i t was f o u n d t o be a g l y c o p r o t e i n ( 1 1 , 1 3 , 1 5 ) . The m o l a r r a t i o f o r the l a r g e and s m a l l s u b u n i t was  also  e s t i m a t e d . The r a t i o o f the i n t e n s i t y o f the two s t a i n e d bands i n t h e c  gel e l e c t r o p h o r e s i s e x p e r i m e n t was taken as t h e mass r a t i o , t h i s combined w i t h the m o l e c u l a r w e i g h t s , gave t h e m o l a r r a t i o as shown i n T a b l e I I I . There i s a c l e a r discrepancy i n t h i s molar r a t i o ( l a r g e subunit to . s m a l l s u b u n i t ) between r e s u l t s o b t a i n e d f r o m d i f f e r e n t s o u r c e s . discrepancy  i s open t o s e v e r a l i n t e r p r e t a t i o n s : ( i ) The  The  glycoprotein,  o r the s m a l l s u b u n i t , may n o t be an i n t e g r a l p a r t o f the enzyme. T h i s i s u n l i k e l y b e c a u s e none o f the d i f f e r e n t p u r i f i c a t i o n methods a p p l i e d cn Ua Is - a c t i v a t e d A T T s s e from d i f f e r e n t s o u r c e s c o u l d e l i m i n a t e i t . ( i i ) The e s t i m a t i o n o f the r e l a t i v e i n t e n s i t y o f the two s t a i n e d bands may n o t be q u a n t i t a t i v e enough. S t a i n i n g a f t e r e l e c t r o p h o r e s i s o f the gels i s accomplished  Na0 S 3  w i t h a dye known as c o o m a s s i e b r i l l i a n t b l u e 6_ .  V (|*)  s  N(C H ) 2  S  °3  -  2  In s l i g h t l y a c i d i c media, the d y e - a n i o n i s e l e c t r o s t a t i c a l l y a t t r a c t e d to t h e c h a r g e d c a t i o n i c groups o f the p r o t e i n . The p r o t e i n - d y e has an a b s o r p t i o n maximum a t 549 nm. B a c k g r o u n d s t a i n i n g o f the  complex blank  gel i s h i g h , and has t o be removed w i t h d e s t a i n i n g s o l u t i o n s . Then the g e l s are s c a n n e d f o r the a b s o r b a n c e o f the dye. P r o t e i n bands a p p e a r as peaks i n a p l o t o f a b s o r b a n c e v e r s e s d i s t a n c e a l o n g the g e l . I n t e g r a t i o n  14  o f the areas under the peaks then y i e l d s the mass r a t i o . Fazekas e t . a l have c a r e f u l l y a n a l y s e d the q u a n t i t a t i v e a s p e c t o f the s t a i n i n g p r o c e s s ( 4 9 ) . D e v i a t i o n from B e e r ' s Law was o b s e r v e d when a b s o r b a n c e was 0.8  above  . S i n c e the amount d f s t a i n i n g depends on the number o f e x p o s e d  c a t i o n i c g r o u p s , d i f f e r e n t p e p t i d e s c o n t a i n i n g d i f f e r e n t amounts o f c a t i o n i c r e s i d u e s w i l l be s t a i n e d t o d i f f e r e n t e x t e n d s even when B e e r ' s Law i s o b e y e d . T h i s was i n d e e d o b s e r v e d .  For equal weights o f f i v e d i f f e r e n t  p r o t e i n s , the a b s o r b a n c e s d e v i a t e d as much as ±21 %  o f t h e mean. F u r t h e r -  more, the coomassi'e b l u e - p r o t e i n complex d i s s o c i a t e d a p p r e c i a b l e  during  d e s t a i n i n g even u n d e r c a r e f u l l y c o n t r o l l e d c o n d i t i o n s . T h i s l e a d s t o an u n c e r t a i n s y s t e m i c e r r o r o v e r and above the random d e v i a t i o n due to the d i f f e r e n c e i n the d i s t r i b u t i o n o f c a t i o n i c r e s i d u e s . A l t h o u g h t h i s a n a l y s i s w?s made on c e l l u l o s e a c e t a t e e l e c t r o p h o r e t i c s t r i p s , s i m i l a r d e v i a t i o n has been r e p o r t e d f o r p o l y a c r y l a m i d e  g e l e l e c t r o p h o r e s i s ( 5 0 ) . I t has  a l s o been o b s e r v e d t h a t the p r e s e n c e o f f a t t y a c i d s i n a p r e p a r a t i o n o f membrane p r o t e i n can i n f l u e n c e the i n t e n s i t y o f s t a i n i n g ( 5 1 ) . Base on t h e s e c o n s i d e r a t i o n s , the r a t i o o f the i n t e n s i t y o f the c o o m a s s i e b l u e s t a i n o f the l a r g e s u b u n i t to s m a l l s u b u n i t i s a t b e s t a measure o f the r a t i o o f c a t i o n i c amino a c i d s o f the two s u b u n i t s , r a t h e r than t h e i r mass r a t i o , ( i i i ) I t i s a l s o p o s s i b l e t h a t the d i s c r e p a n c y i s r e a l , due t o s p e c i e s d i f f e r e n c e , but t h e r e i s n o t enough d a t a on o t h e r s p e c i e s t o substantiate this. I f the s m a l l s u b u n i t i s an i n t e g r a l p a r t o f the enzyme, then  the  minimum m o l e c u l a r w e i g h t w o u l d be 150,000 f o r an ag enzyme, and 250,000 f o r an a g enzyme. 2  15  The p o s s i b i l i t y o f t h e l a r g e and s m a l l s u b u n i t s a c t i n g as an i o n o p h o r e has a l s o been i n v e s t i g a t e d . An i o n o p h o r e was d e f i n e d as t h e m a t e r i a l w h i c h c a u s e d an i n c r e a s e i n t h e conductance membrane ( 5 2 , 5 3 ) , and c o n s e q u e n t l y  o f a black l i p i d  c e n t r e s o u r a t t e n t i o n on t h e mechanism  o f c a t i o n t r a n s l o c a t i o n . In t h e e x p e r i m e n t s  either a tryptic digest  o f t h e s m a l l s u b u n i t o r a m i x t u r e o f t h e l a r g e and s m a l l s u b u n i t were a b l e t o a c t as a N a s p e c i f i c i o n o p h o r e . The conductance +  o f the black  l i p i d membrane doped w i t h t h e p r o t e i n b e i n g h i g h e s t when t h e m o l a r r a t i o o f t h e l a r g e s u b u n i t t o t h e s m a l l s u b u n i t was 1 : 2. The r e s u l t s were i n t e r p r e t e d t o mean t h a t t h e N a s p e c i f i c s i t e i s i n t h e s m a l l s u b u n i t +  and t h e r a t i o o f t h e s u b u n i t s i n t h e i n t a c t enzyme i s t h a t w h i c h m a x i m i z e d the c o n d u c t a n c e be  fiutcu  ciiat-  o f t h e b l a c k membrane, i e . a g One S u b u i i i l b  UScG i i a v e  been  ( 5 4 ) . However, i t s h o u l d  2  utiidlufcu  uy b u u i u u i  uuuecyl  s u l f a t e a n d were no l o n g e r a c t i v e e n z y m a t i c a l l y .  (5).Lipid  requirement  L i p i d s were found t o be r e q u i r e d f o r N a K - a c t i v a t e d +  +  ATPase  a c t i v i t y . Removal o f l i p i d s w i t h o r g a n i c s o l v e n t l e a d s t o i n a c t i v a t i o n , b u t a c t i v i t y can be r e s t o r e d by a d d i n g back t h e e x t r a c t e d l i p i d s , o r new l i p i d s w i t h c h o l e s t e r o l ( 3 0 ) . T r e a t m e n t w i t h p h o s p h o l i p a s e s  also  leads t o i n a c t i v a t i o n . A v a r i e t y o f p h o s p h o l i p i d s , i n c l u d i n g phosphatidyl i n o s i t o l , phosphatidylcholine, phosphatidylethanolamine  and  p h o s p h a t i d y l s e r i n e can r e a c t i v e t h e enzyme. I t i s b e l i e v e d t h a t  16  phosphatidyl s e r i n e i s the p h o s p h o l i p i d r e q u i r e d f o r a c t i v i t y under p h y s i o l o g i c a l c o n d i t i o n (31,32,33). U s i n g a n i t r o x y l s p i n l a b e l l e d f a t t y a c i d e s t e r 7, B a r n e t t (55)  CH (CH ) -C-(CH ) C00CH 3  2  0  \I  2  n  N-0  3  7  „  n = 3,4,5,6 o r 10 m+n = 14 o r 15  c o r r e l a t e d t h e enzyme a c t i v i t y w i t h t h e m o b i l i t y o f t h e membrane phosphol i p i d . A r r h e n i u s p l o t s o f b o t h m o b i l i t y and enzyme a c t i v i t y changed s l o p e a t t h e same " c r i t i c a l " t e m p e r a t u r e o f 2 0 ° C . T h i s was i n t e r p r e t e d to i n d i c a t e t h a t enzyme a c t i v i t y i n c r e a s e s w i t h i n c r e a s i n g l i p i d m o b i l i t y . A t the c r i t i c a l temperature,  t h e l i p i d undergoes a phase  f o r enzyme c a t a l y s i s , p r o b a b l y by a l l o w i n g more f r e e d o m f o r t h e enzyme to achieve the necessary conformation  change. P a p a h a d j o p o u l o s (56)  o b s e r v e d t h a t a c t i v a t i o n by p h o s p h a t i d y l g l y c e r o l depends on t h e f a t t y a c i d s i d e c h a i n s t o w h i c h t h e p h o s p h a t i d y l g l y c e r o l was e s t e r i f i e d . Both t h e c r i t i c a l t e m p e r a t u r e o b t a i n e d from t h e A r r h e n i u s p l o t o f enzyme a c t i v i t y , and the phase t r a n s i t i o n t e m p e r a t u r e o f p h o s p h a t i d y l g l y c e r o l s (from d i f f e r e n t i a l scanning c a l o r i m e t r y ) i n c r e a s e with i n c r e a s i n g c h a i n l e n g t h o f t h e p h o s p h a t i d y l g l y c e r o l , as e x p e c t e d . However, a l t h o u g h t h e c r i t i c a l t e m p e r a t u r e and t h e t r a n s i t i o n t e m p e r a t u r e a r e s i m i l a r , t h e y a r e n o t e x a c t l y e q u a l , and one can be h i g h e r o r lower than t h e o t h e r , d e p e n d i n g on t h e p a r t i c u l a r p h o s p h a t i d y l g l y c e r o l , I t a p p e a r s t h a t t h e r e l a t i o n s h i p between c r i t i c a l t e m p e r a t u r e as d e t e c t e d  17  by t h e A r r h e n i u s p l o t o f enzyme a c t i v i t y and t h e phase t r a n s i t i o n temperature  d e t e c t e d by c a l o r i m e t r y i s n o t s t r a i g h t f o r w a r d . D u r i n g  phase t r a n s i t i o n , l i p i d s may be s e p a r a t e d i n t o d i f f e r e n t domains w i t h d i f f e r e n t e x t e n t s o f f l u i d i t y . C a l o r i m e t r y measures t h e 'average' phase t r a n s i t i o n t e m p e r a t u r e o f t h e b u l k s a m p l e , w h i l e t h e enzyme may be c o n c e n t r a t e d i n a domain w i t h a h i g h e r o r l o w e r degree o f d i s o r d e r .  18  PART II  Affinity  Chromatography  The p u r i f i c a t i o n o f N a K - a c t i v a t e d ATPase by c o n v e n t i o n a l methods +  +  has been d e s c r i b e d i n P a r t I o f t h i s i n t r o d u c t i o n . These p r o c e d u r e s a r e l e n g t h l y and o f t e n i n v o l v e t h e use o f t e c h n i q u e s r e q u i r i n g e x p e n s i v e i n s t r u m e n t a t i o n such as z o n a l c e n t r i f u g a t i o n . T h i s drawback i s n o t u n i q u e , i t i s a common p r o b l e m i n many p u r i f i c a t i o n methods. The r e a s o n f o r t h i s i s the l a c k o f s p e c i f i c i t y o f t h e t e c h n i q u e f o r the p r o t e i n t o be purified. A f f i n i t y chromatography  i s a more modern p u r i f i c a t i o n t e c h n i q u e  which i s d e s i g n e d t o be h i g h l y s p e c i f i c f o r the p r o t e i n t o be p u r i f i e d . I t makes use o f the s p e c i f i c i n t e r a c t i o n o f t h e p r o t e i n w i t h some o t h e r m o l e c u l e . These s p e c i f i c i n t e r a c t i o n s i n c l u d e such examples as t h e a f f i n i t y between enzyme and s u b s t r a t e , enzyme and i n h i b i t o r , r e c e p t o r and t o x i n , and p r o t e i n and c a r b o h y d r a t e ( 8 2 , 8 3 ) . The p r o t e i n to be p u r i f i e d i s p a s s e d t h r o u g h a column c o n t a i n i n g a c r o s s l i n k e d p o l y m e r o r g e l to which a l i g a n d m o l e c u l e s p e c i f i c f o r the p r o t e i n has been c o v a l e n t l y a t t a c h e d . A l l p r o t e i n s , w i t h o u t s u b s t a n t i a l a f f i n i t y f o r the bound m o l e c u l e w i l l pass d i r e c t l y t h r o u g h t h e column, whereas one t h a t r e c o g n i z e s t h e bound m o l e c u l e w i l l be r e t a r d e d i n p r o p o r t i o n t o i t s a f f i n i t y c o n s t a n t . E l u t i o n o f the bound p r o t e i n i s a c h i e v e d by c h a n g i n g the s a l t c o n c e n t r a t i o n o r pH, o r by e l u t i o n w i t h a n o t h e r b i n d a b l e m o l e c u l e t o  19  d i s s o c i a t e the p r o t e i n f r o m the l i g a n d . The s u c c e s s f u l a p p l i c a t i o n o f t h i s method depends on a number o f f a c t o r s : phase o r m a t r i x , the p r o t e i n ,  (1)  A suitable stationary  (2) a l i g a n d m o l e c u l e w h i c h can s p e c i f i c a l l y b i n d  (3) a method t o c o v a l e n t l y l i n k the l i g a n d t o the  (4) c o n d i t i o n o f b i n d i n g o f p r o t e i n t o a f f i n i t y column and  matrix,  (5) a method  t o e l u t e the bound p r o t e i n .  (1)  Stationary  matrix  T h i s s h o u l d be an i n e r t m a t e r i a l w h i c h shows minimum i n t e r a c t i o n w i t h p r o t e i n s i n g e n e r a l ; i t must f o r m a l o o s e , p o r o u s network t h a t p e r m i t s e a s y e n t r v and e x i t o f m a c r o m o l e c u l e s . I t s c h e m i c a l  structure  must p e r m i t e x t e n s i v e c o v a l e n t a t t a c h m e n t o f the s p e c i f i c l i g a n d . Agarose g e l , polyacrylamide  beads and g l a s s beads have been used f o r  t h i s p u r p o s e ( 5 7 , 5 8 ) . An a g a r o s e g e l i n common use i s S e p h a r o s e , a commercially  (2)  a v a i l a b l e gel c o n t a i n i n g 2-6 %  agarose.  Ligand The l i g a n d m o l e c u l e may be an i n h i b i t o r , a s u b s t r a t e  analog,  a c o f a c t o r f o r an enzyme, an a g o n i s t o r a n t a g o n i s t f o r a r e c e p t o r p r o t e i n o r i t can be a h a e m a g g l u t i n a t i n g  agent f o r a g l y c o p r o t e i n  T a b l e IV summarizes some e x a m p l e s .  (64,70).  20  TABLE IV P r o t e i n t o be p u r i f i e d  Ligand  Method o f e l u t i o n  Nicotinic acetylcholine r e c e p t o r (59)  Cobratoxin  hexamethoniurn c h l o r i d e  Acetylcholine receptor (60)  -NH(CH ) -C0NH(CH )3N(CH );  NaCl g r a d i e n t  Acetylcholine esterase (63)  1-methyl-9-[N -(E-aminocaproyl)-y-aminopropy! a m i n o ] a c r i d i n urn bromide h y d r o b r o m i d e 9_  decamethomi urn  N a K - a c t i v a t e d ATPase (62)  6-(purine 5 ' r i b o s y l t r i p h o s p h a t e ) - 4 - ( l ,3 dinitrophenyl)thioester (ATP a n a l o g ) 8  ATP  Myosin ( 8 4 )  sebacic acid hydrazideATP j o  KC1 g r a d i e n t  G l y c e r o l k i n a s e and  N  +  2  3  a  +  6  KC1 g r a d i e n t  -(6-aminohexyl)-  5 -AMP  n  1  (68,85^86)  NAD  NAD  G l y c e r a l d e h y d e 3-phosphate dehydrogenase(87) Rhodopsin  2  5  D-glucose  Concanavalin A  (61) 0 NH'CCH3  (CH 2 ) 2 CHCNH(CH 2 ) 3 NH(CH 2 ) 3 NH 2  0 0 0 xfTl f  HOPOKUKU^  "OP'OPOQH... 0 0 0 l/CKJ H HH ^  0  00 0  /  HOPOPVPOCH;,^  UiLo H HH <  ur^<^  CO(CH )eC0NHNH2 2  10  OH OH  N(CH 2 ) 6 NH 2 HN(CH ) N0C(CH ) i!iH Br~ 2  3  2  5  3  0 HO^OC,  9  OH  11 HO HO  F i g . 2 A f f i n i t y Ligands  The l i g a n d s h o u l d have a h i g h and s p e c i f i c a f f i n i t y f o r the p r o t e i n . For example, the d i s s o c i a t i o n c o n s t a n t o f n i c o t i n i c c o b r a t o x i n complex i s 1.13 x 10~ M  (59). Likewise  9  C o n c a n a v a l i n A complex i s 2 x 1 0 ~  7  receptor-  that-of Rhodopsin-  M ( 6 1 ) . O f t e n a h y d r o c a r b o n backbone  i n s e r t e d between the l i g a n d and the g e l m a t r i x was  f o u n d t o be n e c e s s a r y  i n c a s e s where the b i n d i n g between the l i g a n d and the p r o t e i n i s n o t v e r y s t r o n g (Kg > 10~  M) ( 5 7 ) . The e f f e c t o f i n c r e a s i n g d i s t a n c e  be p a r t l y e x p l a i n e d by r e l i e f o f s t e r i c h i n d r a n c e imposed by the  may matrix  b a c k b o n e , and p e r h a p s by the i n c r e a s e d f l e x i b i l i t y and m o b i l i t y o f the l i g a n d as i t p r o t u d e f u r t h e r i n t o the s o l v e n t  \ ) oC Va i2ii K v-o  i i ny  pin- nig  (91).  iA  An e f f i c i e n t t e c h n i q u e has been d e v e l o p e d to c o v a l e n t l y the l i g a n d t o an a g a r o s e g e l m a t r i x . I t makes use o f the  terminal  p r i m a r y amino group o f p r o t e i n s o r s y n t h e t i c l i g a n d s , and the groups o f a g a r o s e . The v i c i n a l h y d r o x y l  link  hydroxyl  groups are a c t i v a t e d by cyanogen  b r o m i d e t o y i e l d e i t h e r an i n e r t carbamate o r a c t i v e i m i d o c a r b a m a t e . gel  fOH  matrix  OH  BrCN.  -O-CHN  I  ^ H?0  ioH  LOCONH,  i+-0H  , N>NH  The a c t i v e i m i d o c a r b a m a t e then r e a c t s w i t h the t e r m i n a l N H o f 2  the l i g a n d ( 6 5 , 6 6 , 6 7 ) . ^•ONH  0 H?N-1igand  :  -0-C-NH-ligand  -OH  22  (4) B i n d i n g o f p r o t e i n t o a f f i n i t y column  S t r o n g b i n d i n g between the p r o t e i n and l i g a n d i s . o b v i o u s l y necessary  f o r a good p u r i f i c a t i o n , hence e x p e r i m e n t a l  as t e m p e r a t u r e ( 8 8 ) , pH ( 8 9 ) , m e t a l i o n c o n c e n t r a t i o n s  c o n d i t i o n s such e t c . have t o  be o p t i m i z e d to f a v o r b i n d i n g . For example, the b i n d i n g o f myosin t o ATP r e q u i r e d the p r e s e n c e o f Ca  o r Mg  l i g a n d i n the g e l m a t r i x i s a l s o i m p o r t a n t .  ( 8 4 ) . The c o n c e n t r a t i o n R a f t e r y and S c h m i d t  f o u n d t h a t i n c r e a s i n g the l i g a n d c o n c e n t r a t i o n i m p r o v e d the  of (60)  binding  c a p a c i t y , b u t i t a l s o c a u s e d the l i g a n d t o a c t as a n o n s p e c i f i c i o n e x c h a n g e r and r e d u c e d the s e l e c t i v i t y . G e n e r a l l y s p e a k i n g ,  binding  s h o u l d be a l l o w e d t o t a k e p l a c e u n d e r p h y s i o l o g i c a l c o n d i t i o n s when the p r o t e i n i s most a c t i v e .  (5) E l u t i o n o f the bound p r o t e i n  A f t e r the bound p r o t e i n i s washed f r e e o f o t h e r p r o t e i n s , i t has t o be d i s s o c i a t e d from the l i g a n d . In some cases where the a f f i n i t y o f t h e p r o t e i n f o r the l i g a n d i s a f f e c t e d by s a l t c o n c e n t r a t i o n , e l u t i o n can be a c h i e v e d by c h a n g i n g the s a l t c o n c e n t r a t i o n  the  (60,68,84).  In most cases the p r o t e i n i s d i s s o c i a t e d and e l u t e d w i t h a s o l u t i o n c o n t a i n i n g a m o l e c u l e to compete w i t h the l i g a n d f o r the enzyme ( 5 9 , 6 2 ) , or vice versa (61,69). A f f i n i t y c h r o m a t o g r a p h y i s not l i m i t e d t o t h e use o f ' p h y s i o l o g i c a l '  23  l i g a n d s such as i n h i b i t o r o r s u b s t r a t e a n a l o g . H y d r o p h o b i c i n t e r a c t i o n has a l s o been used t o d e s i g n an a f f i n i t y l i g a n d . In aqeous s o l u t i o n , h y d r o p h o b i c bonds may be t h e most i m p o r t a n t i n t e r a c t i o n - b e t w e e n a hydrosp h o b i c s i t e o r " p o c k e t " o f a p r o t e i n and a s m a l l m o l e c u l e h a v i n g t h e optimum d i m e n s i o n t o f i t t h e p o c k e t ( 7 1 ) . T h i s c o n c e p t was a p p l i e d t o separate a mixture o f glycogen phosphorylase and glycogen s y n t h t a s e . Both enzymes have h y d r o p h o b i c p o c k e t s w h i c h w i l l b i n d t h e l i g a n d -NH(CH ) NH 2  2  b u t s i n c e g l y c o g e n p h o s p h o r y l a s e has a l a r g e r h y d r o p h o b i c  p o c k e t , i t w i l l b i n d o n l y when n > 4 , whereas g l y c o g e n s y n t h e t a s e w h i c h has a s m a l l e r h y d r o p h o b i c p o c k e t w i l l b i n d the l i g a n d when n < 4 . U s i n g a l i g a n d o f a p p r o p i a t e l e n g t h , t h e two enzymes c o u l d be s e p a r a t e d and p u r i f i e d ( 7 2 ) .  24  PART I I I  Theory o f V e l o c i t y Sedimentation & Gel F i l t r a t i o n  (1) V e l o c i t y  sedimentation  A l l p a r t i c l e s i n s o l u t i o n have a t e n d e n c y t o s e d i m e n t u n d e r t h e i n f l u e n c e o f g r a v i t y and a t e n d e n c y t o d i s p e r s e randomly i n s o l u t i o n v i a d i f f u s i o n . A s m a l l m o l e c u l e w h i c h has a s m a l l mass does n o t s e d i m e n t b e c a u s e t h e g r a v i t a t i o n a l f o r c e i s t o o s m a l l t o overcome d i f f u s i o n , r o r m a c r o m o l e c u l e s . t n e g r a v i tar.iona'1 f o r c e i s c o n s i d e r a b l e s t i l l n o t enough. The f o r c e n e c e s s a r y f o r s e d i m e n t a t i o n  c a n be i n c r e a s e d  by c e n t r i f u g a t i o n . When t h e c e n t r i f u g a l f o r c e i s l a r g e r than m a t e l y 200,000 x g, s e d i m e n t a t i o n  iarger out  approxi-  o f p r o t e i n m o l e c u l e s becomes  observable  on a t i m e s c a l e o f tens o f m i n u t e s . A p a r t i c l e s u b j e c t e d t o c e n t r i f u g a t i o n experiences  two o p p o s i n g f o r c e s , t h e c e n t r i f u g a l f o r c e and the f r i c t i o n  between t h e p a r t i c l e and t h e s o l v e n t . T a k i n g t h e s e two f o r c e s i n t o c o n s i d e r a t i o n , S v e d b e r g d e r i v e d an e q u a t i o n  g o v e r n i n g t h e m o t i o n o f any  m a c r o m o l e c u l e i n any medium i n a c e n t r i f u g a l f o r c e f i e l d (42) :  1 ZFx  dx dt  =  M(l-v )D . RT P  (  Cl]  25  oo = a n g u l a r v e l o c i t y x = d i s t a n c e o f macromolecule  from c e n t e r o f r o t a t i o n  t = time M=  molecular weight  v = p a r t i a l s p e c i f i c volume o r i n v e r s e d e n s i t y o f  macromolecule  p. = d e n s i t y o f s o l u t i o n D = diffusion coefficient R = gas c o n s t a n t T = absolute  temperature  I t can be seen t h a t the r i g h t s i d e o f e q u a t i o n [1] i s a c o n s t a n t for a p a r t i c u l a r experiment,  and the l e f t s i d e c o n t a i n s t h e v a r i a b l e s  t o be measured. The s e d i m e n t a t i o n c o e f f i c i e n t  1  s  i s d e f i n e d as f o l l o w s  dx  [2]  U s i n g t h i s d e f i n i t i o n and r e a r r a n g i n g e q u a t i o n [1]  When more than one type o f m a c r o m o l e c u l e  :  i s p r e s e n t , each w i l l  sediment p a s t a g i v e n p o i n t a t an i n s t a n t a n e o u s v e l o c i t y p r o p o r t i o n a l to i t s s e d i m e n t a t i o n c o e f f i c i e n t . In o t h e r w o r d s , a f t e r a g i v e n l e n g t h o f time o f c e n t r i f u g a t i o n , t h e macromolecules  w i l l be s e p a r a t e d , h a v i n g  t r a v e l l e d d i f f e r e n t d i s t a n c e s a l o n g the l e n g t h o f the c e n t r i f u g e t u b e .  26  Those w i t h a l a r g e r s e d i m e n t a t i o n c o e f f i c i e n t w i l l be f u r t h e r away f r o m the a x i s o f r o t a t i o n . Thus t h e d i s t a n c e from t h e c e n t e r o f r o t a t i o n can be c a l i b r a t e d w i t h m a c r o m o l e c u l e s  o f known s e d i m e n t a t i o n c o e f f i c i e n t ,  and t h e s e d i m e n t a t i o n c o e f f i c i e n t o f t h e m a c r o m o l e c u l e  o f i n t e r e s t can  be f o u n d by i n t e r p o l a t i o n . The c e n t r i f u g a l f o r c e i s p r o p o r t i o n a l t o  X2OJ.  T h i s means t h a t  the m a c r o m o l e c u l e w i l l e x p e r i e n c e a l a r g e r c e n t r i f u g a l f o r c e when i t i s f u r t h e r down t h e c e n t r i f u g e t u b e . I f t h e m a c r o m o l e c u l e were i n a homogeneous medium, i t w i l l a c c e l e r a t e due t o t h e i n c r e a s i n g c e n t r i f u g a l f o r c e . C o n s e q u e n t l y , t h e v e l o c i t y o f s e d i m e n t a t i o n w i l l n o t be d i r e c t l y p r o p o r t i o n a l t o the sedimentation c o e f f i c i e n t at a l l times during the e x p e r i m e n t . T h i s d i f f i c u l t y can be overcome by h a v i n g an i n c r e a s i n g v i s c o s i t y and d e n s i t y  yracnent  Thus, t h e m a c r o m o l e c u l e , concentration  aiong tne  length  o f tne  tube.  centriTuge  s e d i m e n t i n g t h r o u g h , s a y , an i n c r e a s i n g  o f s u c r o s e s o l u t i o n , w i l l e x p e r i e n c e an i n c r e a s i n g  friction  and an i n c r e a s i n g buoyancy which combine t o compensate f o r t h e i n c r e a s i n g c e n t r i f u g a l f o r c e . Under such c o n d i t i o n , t h e m a c r o m o l e c u l e  will  sediment  with a constant v e l o c i t y proportional t o i t s sedimentation c o e f f i c i e n t . This i s the underlying p r i n c i p l e o f constant v e l o c i t y sedimentation. The t h e o r y o f v e l o c i t y s e d i m e n t a t i o n i n a s u c r o s e  concentration  g r a d i e n t was d e v e l o p e d by Hans N o l l (43). The d e s i r e d g r a d i e n t w i l l depend on t h e t e m p e r a t u r e and on t h e d i m e n s i o n o f t h e r o t o r b e i n g u s e d . I f t h e d i m e n s i o n o f t h e r o t o r i s known, a f u n c t i o n r e l a t i n g t h e s u c r o s e concentration  and t h u s v i s c o s i t y , and t h e d i s t a n c e from t h e a x i s o f  r o t a t i o n can be c a l c u l a t e d . T h i s i s known as an i s o k i n e t i c s u c r o s e  gradient.  27  (2) G e l f i l t r a t i o n  Gel f i l t r a t i o n , a l s o known as m o l e c u l a r s i e v e c h r o m a t o g r a p h y , or g e l p e r m e a t i o n c h r o m a t o g r a p h y ,  i s a type o f p a r t i t i o n  chromatography  which s e p a r a t e s m o l e c u l e s a c c o r d i n g t o t h e i r s i z e s . The g e l i s made o f porous m a t e r i a l and a c t s as t h e s t a t i o n a r y phase. A v a r i e t y o f g e l s a r e a v a i l a b l e f o r f r a c t i o n a t i o n o f m o l e c u l e s o f d i f f e r e n t s i z e (M.W.) r a n g e s . The one used i n t h i s s t u d y i s S e p h a r o s e 6B ( P h a r m a c i a ) , which c o n s i s t s o f 6% a g a r o s e . During chromatography, the s o l u t i o n c o n t a i n i n g the molecules to be f r a c t i o n a t e d a r e p a s s e d t h r o u g h t h e g e l column. The s o l u t e m o l e c u l e s p a r t i t i o n t h e m s e l v e s between t h e s t a t i o n a r y phase ( t h e g e l ) , and t h e m o b i l e phase (th*3 s o l v e n t ) . M o l e c u l e s with.a. s m a l l s i z e w i l l be a b l e t o e n t e r most o f t h e p o r e s o f t h e g e l and spend a c o m p a r a t i v e l y l o n g e r time i n s i d e the g e l b e f o r e e l u t e d o u t o f t h e column, whereas m o l e c u l e s w i t h a l a r g e s i z e w i l l be e x c l u d e d from e n t e r i n g most o f t h e p o r e s and hence e l u t e d f a s t e r . In t h e extreme c a s e when a m o l e c u l e i s l a r g e enough t o be e x c l u d e d from a l l t h e p o r e s , i t w i l l be e l u t e d o u t o f t h e column  after  a volume o f s o l v e n t e q u a l t o t h e i n t e r s t i t u a l space between t h e g e l beads has p a s s e d t h r o u g h . T h i s i s c a l l e d t h e v o i d volume, VQ '.' In t h e o t h e r extreme when a m o l e c u l e i s s m a l l enough t o e n t e r a l l t h e p o r e s , i t w i l l be e l u t e d o u t o f t h e column a f t e r a volume o f s o l v e n t e q u a l t o  - VQ  has p a s s e d t h r o u g h t h e column, where V i s t h e volumn o f t h e g e l b e d . FC  An i m p o r t a n t p a r a m e n t e r i n g e l f i l t r a t i o n i s t h e p a r t i t i o n coefficient K  a w  . The p a r t i t i o n c o e f f i c i e n t f o r a m o l e c u l e i s d e f i n e d as  where V  G  i s the volume o f s o l v e n t w h i c h has p a s s e d t h r o u g h when t h a t  m o l e c u l e i s e l u t e d o u t o f t h e column. The p a r t i t i o n c o e f f i c i e n t i s a measure o f how f a s t a m o l e c u l e t r a v e l s t h r o u g h t h e column compared t o t h e s o l v e n t , o r the r e l a t i v e amount o f t i m e t h a t the m o l e c u l e spends i n t h e g e l p h a s e . The p a r t i t i o n c o e f f i c i e n t i s i n d e p e n d e n t o f the compactness o f the g e l and the s o l v e n t used w i t h i n e x p e r i m e n t a l e r r o r ( 7 3 ) . A m o l e c u l e which i s n e i t h e r t o t a l l y e x c l u d e d n o r a b l e to e n t e r a l l t h e p o r e s o f the g e l phase has a p a r t i t i o n c o e f f i c i e n t between 0 and 1. From t h e d e f i n i t i o n o f VQ, a t o t a l l y ~_i..  j_ J  u»-  -  i/  .-I,.  o.„  n  1..  —1-11  -  av  enough t o e n t e r p o r e s o f a l l s i z e s has a K  flv  e q u a l s t o 1.  Gel f i l t r a t i o n has been used to e s t i m a t e m o l e c u l a r w e i g h t s o f p r o t e i n s by assuming a l i n e a r r e l a t i o n s h i p between the l o g a r i t h m o f m o l e c u l a r w e i g h t and p a r t i t i o n c o e f f i c i e n t ( 7 5 ) . A column can be c a l i b r a t e d by m e a s u r i n g the K  a v  f o r s e v e r a l p r o t e i n s w i t h known m o l e c u l a r  w e i g h t , then the m o l e c u l a r w e i g h t o f the p r o t e i n o f i n t e r e s t e d can be f o u n d by i n t e r p o l a t i o n i n a p l o t o f m o l e c u l a r w e i g h t v e r s e s K  a y  . This  c a l i b r a t i o n i s o n l y v a l i d f o r symmetric, or g l o b u l a r p r o t e i n s . Large p r o t e i n s w i t h m o l e c u l a r w e i g h t l a r g e r than 1 0  5  are o f t e n asymmetric.  F o r t h e s e asymmetric p r o t e i n s , t h e c a l i b r a t i o n i n terms o f m o l e c u l a r w e i g h t i s no l o n g e r v a l i d . F o r example, i t was o b s e r v e d t h a t f i b r i n o g e n which has a m o l e c u l a r w e i g h t o f 330,000 e l u t e d e a r l i e r than u r e a s e w h i c h has a h i g h e r m o l e c u l a r w e i g h t o f 482,700 ; a l s o a l d o l a s e w i t h  29  a m o l e c u l a r w e i g h t o f 149,000 e l u t e d e a r l i e r t h a n a l c o h o l  dehydrogenase  w i t h a m o l e c u l a r w e i g h t o f 160,000 ( 7 6 ) . S i n c e i t i s e x p e c t e d t h a t l a r g e r protein should elute e a r l i e r (smaller K  ), t h e s e r e s u l t s i n d i c a t e d av  t h a t f o r a s y m e t r i c p r o t e i n s , molecular weight i s not necessary p r o p o r t i o n a l t o the " e f f e c t i v e s i z e " . The e f f e c t i v e s i z e i s b e t t e r e x p r e s s e d i n terms of Stokes' radius R  g  . The S t o k e s ' r a d i u s o f . a n asymmetric  macromolecule  i s d e f i n e d as the r a d i u s o f a r i g i d s p h e r e which has t h e same d i f f u s i o n c o e f f i c i e n t as t h a t m a c r o m o l e c u l e .  T h i s d e f i n i t i o n i s e x p r e s s e d by t h e  Stokes-Einstein Equation.  e  OTrnU  S e v e r a l e q u a t i o n s have been p r o p o s e d t o r e l a t e S t o k e s ' r a d i u s and t h e p a r t i t i o n c o e f f i c i e n t (73,77,78,79).  Demassieux and  Lachance  s e l e c t e d n i n e p r o t e i n s w i t h d i f f e r e n t asymmetry and f o u n d t h a t t h e l o g a r i t h m of their Stokes  1  r a d i i have a l i n e a r r e l a t i o n s h i p w i t h t h e p a r t i t i o n  c o e f f i c i e n t s ( 7 8 ) . Thus a g e l f i l t r a t i o n column c a l i b r a t e d by s t a n d a r d p r o t e i n s w i t h known S t o k e s ' r a d i i o r d i f f u s i o n c o e f f i c i e n t s can be u s e d t o f i n d the S t o k e s ' r a d i u s o f t h e p r o t e i n b e i n g s t u d i e d . The d i f f u s i o n c o e f f i c i e n t can then be c a l c u l a t e d from e q u a t i o n [ 5 ] . To summarise, i n d e p e n d e n t e x p e r i m e n t s on v e l o c i t y s e d i m e n t a t i o n and g e l f i l t r a t i o n can be used t o e s t i m a t e s e d i m e n t a t i o n c o e f f i c i e n t ' and d i f f u s i o n c o e f f i c i e n t r e s p e c t i v l e y , and the d a t a can be  combined  i n e q u a t i o n [ 3 ] t o g i v e an e s t i m a t i o n o f the m o l e c u l a r w e i g h t .  30  MATERIALS  R e c t a l g l a n d s o f dog f i s h were k i n d l y s u p p l i e d as a g i f t by Mr. M a r t i n Handel o f Sea F r e s h P a c i f i c P r o d u c t s , L a d y s m i t h , B. C. P u r i f i e d c h i c k e n egg w h i t e lysozyme was k i n d l y s u p p l i e d by Ms. K a t h l e e n Chance. A l l i n o r g a n i c and o r g a n i c c h e m i c a l s used were o f r e a g e n t g r a d e . D o d e c y l a m i n e , methyl b r o m i d e , i m i d a z o l e , cyanogen bromide  and  d i e t h y l a d i p i c a c i d were o b t a i n e d f r o m Eastman Kodak Co., R o c h e s t e r , New  York.  sodium p o t a s s i u m t a r t r a t e and sodium m e t a p e r i o d a t e were o b t a i n e d f r o m F i s c h e r S c i e n t i f i c Co., F a i r Lawn, New J e r s e y . A c e t o n i t r i l e , d i o x a n e , g l y c e r o l and s u c r o s e were o b t a i n e d from M a l l i n c k r o d t C h e m i c a l Co., S t . L o u i s , Mo. L u b r o l WX, b o v i n e serum a l b u m i n , ATP ( s o d i u m s a l t ) , o u a b a i n , T r i t o n X-100, C o n c a n a v a l i n A ( J a c k b e a n ) , c a c o d y l i c a c i d ( s o d i u m s a l t ) . 2 , 4 , 6 - t r i n i t r o b e n z e n e 1 - s u l f o n a t e a c i d , N-acetylglucosamine, a-methylD-mannoside, a l c o h o l d e h y d r o g e n a s e , c a t a l a s e , T r i s m a b a s e , NAD,  Micrococus  l y s o d e i k t i c u s and b o v i n e f i b r i n o g e n were o b t a i n e d f r o m Sigma C h e m i c a l Co. S t . L o u i s , Mo. F o l i n r e a g e n t was o b t a i n e d from H a r l e c o Co. Phi l a . , Pa. Fluorescamine (4-phenylspiro [furan-2(3H),l'-phthalan]-3-3'dione)  31  was o b t a i n e d from H o f f m a n - L a Roche L t d . , Quebec. P h o s p h a t i d y l - L - s e r i n e ( b o v i n e b r a i n ) was o b t a i n e d from KochL i g h t Lab. L t d . , England.  -  • ..  Ammonium m o l y b d a t e t e t r a h y d r a t e and d i s o d i u m hydrogen o r t h o p h o s p h a t e were o b t a i n e d from The B r i t i s h Drug House L t d . , E n g l a n d . S e p h a r o s e 4B, S e p h a r o s e 6B and B l u e D e x t r a n 2000 were o b t a i n e d f r o m P h a r m a c i a F i n e C h e m i c a l s , U p p s a l a , Sweden. H y d r a z i n e h y d r a t e (99-100%) was o b t a i n e d from J . T. B a k e r C h e m i c a l Co., P h i l l i p s b u r g , New J e r s e y . Horse s p l e e n a p o f e r r i t i n , d e o x y c h o l i c a c i d ( s o d i u m s a l t ) were o b t a i n e d from C a l b i o c h e m , San D i e g o , C a l i f . Wheat germ a g g l u t i n i n was o b t a i n e d from M i l e s - Y e d a L t d . , R e h o v o t h , Israel. Ami con CF50A membrane u l t r a f i I t e r s were o b t a i n e d f r o m Ami con C o r p . , L e x i n g t o n , Mass. M i l l i p o r e f i I t e r s w e r e o b t a i n e d from M i l l i p o r e C o r p . , B e d f o r d , ;  Mass.  32  METHODS  (1) S y n t h e s i s o f t r i m e t h y l d o d e c y l a m m o n i u m  bromide  D o d e c y l a m i n e (30.Og, 0.162 m o l e ) , m e t h y l bromide (39.3ml, 0.694 m o l e ) and p o t a s s i u m b i c a r b o n a t e (64.8g, 0.647 mole) were r e f l u x e d i n a c e t o n i t r i l e (70 ml) f o r 12 h r s . P r o d u c t was e x t r a c t e d from the m i x t u r e w i t h h o t e t h a n o l . I t was r e c r y s t a l l i z e d once from b e n z e n e - e t h e r and once from a c e t o n e - e t h e r , d e c o m p o s i t i o n t e m p e r a t u r e : 2 2 8 ° C . M i c r o a n a l y s e s f o r C, H, N and B r c o n f i r m e d t h a t i t was t h e d e s i r e d p r o d u c t .  % by w e i g h t Element  C  H  N  Br -  Expected  58.39  11.12  4.54  25.92  Found  58.66  11.29  4.55  26.10  (2) C o l l e c t i o n o f r e c t a l g l a n d s o f dog f i s h  Dog f i s h were o b t a i n e d i n t h e summer o f 1973. R e c t a l g l a n d s were removed f r o m t h e f i s h on t h e same day t h e y were c a u g h t , wrapped i n aluminum f o i l and s t o r e d i n d r y i c e . They were t r a n s f e r r e d t o a -70°C f r e e z e r f o r s t o r a g e a f t e r b e i n g s h i p p e d back t o t h e l a b o r a t o r y .  33  (3) L y o p h i l i z a t i o n o f t i s s u e s T i s s u e s (42.8 g, 30 g l a n d s ) were homogenized i n a Model 23 V i r t i s h o m o g e n i z e r (The V i r t i s Co., G a r d i n e r , N.Y.)  a t maximum s p e e d  i n 440 ml o f w a t e r a t 0-5°C f o r 2 m i n . , f r o z e n i n a d r y i c e - a c e t o n e mixture,  and then l y o p h i l i z e d . 1 g d r y w e i g h t o f l y o p h i l i z e d t i s s u e  i s e q u i v a l e n t t o 6.7 g o f t h e wet t i s s u e .  (4) E x t r a c t i o n o f N a K - a c t i v a t e d +  +  ATPase  E i t h e r the l y o p h i l i z e d o r t h e f r o z e n t i s s u e can be e x t r a c t e d for Na'V..'-activated  A T P a s e . A l l p r o c e d u r e s w e r e c a r r i e d o u t a t 0-5°C.  In a t y p i c a l e x p e r i m e n t , 0.5 g o f l y o p h i l i z e d t i s s u e was r e s u s p e n d e d i n 50 ml o f b u f f e r , o r 2 g l a n d s  (approximately  3 g) was homogenized  i n 45 m l . The amount o f l y o p h i l i z e d o r f r o z e n t i s s u e can be s c a l e d up o r down t o s u i t a p a r t i c u l a r e x p e r i m e n t , w i t h t h e p r o p o r t i o n a l  increase  or decrease  imidazole,  i n t h e amount o f b u f f e r . The b u f f e r c o n t a i n s 30 mM  100 mM N a C l , 1 mM s o d i u m EDTA, t i t r a t e d t o pH 7.0 w i t h  concentrated  h y d r o c h l o r i c a c i d . T h i s t i s s u e homogenate was c e n t r i f u g e d i n a SS-34 r o t o r (Ivan S o r v a l l I n c . , N o r w a l k , C o n n e c t i c u t ) (max. 23,600 x g) f o r 30 min. The s u p e r n a t a n t was homogenized i n 45 ml o f  a t 14,000 r.p.m. was d i s c a r d e d . The p e l l e t  the same b u f f e r . T h i s p e l l e t homogenate  was a g a i n c e n t r i f u g e d a t 14,000 r.p.m. f o r 30 min. The s u p e r n a t a n t discarded. The-pellet was -  homogenized i n 12.5 ml o f the same b u f f e r  was  34  p l u s 10% ( v / v ) g l y c e r o l . To t h i s p e l l e t homogenate was added 1.3 ml o f a 16% (w/v) L u b r o l WX s o l u t i o n , p e r v i o u s l y t i t r a t e d w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d to pH 7.0 and f i l t e r e d t h r o u g h a 0.45..y M i l l i p o r e f i l t e r . The m i x t u r e was s t i r r e d f o r 15 min., and t h e n c e n t r i f u g e d in a Beckman 42.1 r o t o r (Beckman I n s t r u m e n t s  Inc., Palo A l t o , C a l i f . ) at  42,000 r.p.m. (max. 195,000 x g) f o r 3 h r s . The p e l l e t was  discarded.  The r e s u l t i n g s u p e r n a t a n t was f i l t e r e d t h r o u g h a M i l l i p o r e f i l t e r . T h i s i s c a l l e d the "enzyme e x t r a c t " .  (5) P r o t e i n d e t e r m i n a t i o n T o t a l p r o t e i n i r : a sample was a s s a y e d by e i t h e r one o f two methods. The method o f Lowry, R o s e n b r o u g h , F a r r and R a n d a l l (34) was used i n samples where L u b r o l WX c o n c e n t r a t i o n was l o w e r than 0.2 % (w/v). A c a l i b r a t i o n was p r e p a r e d u s i n g b o v i n e serum albumin as t h e s t a n d a r d . Absorbance was measured a t 650 nm on a C a r l Z e i s s PMQII  spectrophotometer.  When t h e L u b r o l WX c o n c e n t r a t i o n i s h i g h e r than 0.2%, i n t e r f e r e n c e w i l l o c c u r w i t h Lowry's method. A y e l l o w p r e c i p i t a t e w o u l d be  formed  upon a d d i t i o n o f the F o l i n r e a g e n t and t h e s u b s e q u e n t e x t i n c t i o n w o u l d be t o o low. Hence, f o r sample w i t h h i g h L u b r o l WX c o n c e n t r a t i o n , so h i g h t h a t d i l u t i o n t o 0.2% was n o t p r a c t i c a l , a f l u o r e s c e n c e method was  used. The f l u o r e s c e n c e method i s b a s e d on the r e a c t i o n o f 4 - p h e n y l -  spiro[furan-2(3H) ,1'-phthalan]-3-3'dione  ]Z_, known as f l u o r e s c a m i n e ,  35  with primary amines.  o  The p r o d u c t o f the r e a c t i o n f l u o r e s c e s i n t e n s e l y , whereas f l u o r e s c a m i n e i t s e l f i s n o n f l u o r e s c e n t , and e x c e s s r e a g e n t i s h y d r o l y s e d to form n o n f l u o r e s c e n t , w a t e r s o l u b l e p r o d u c t s  (35,36).  T h i s f l u o r e s c a m i n e w i l l l i k e w i s e r e a c t w i t h the N - t e r m i n a l any e x p o s e d l y s i n e r e s i d u e o f a p e p t i d e , p r o v i d i n g a c o n v e n i e n t  and assay  f o r s m a l l q u a n t i t i e s o f p r o t e i n . 100 y l sample c o n t a i n i n g 10 t o 100 yg o f p r o t e i n was mixed w i t h 2 ml o f a b u f f e r c o n t a i n i n g 0.01 NI sodium c a r b u n a t e and  Cut  ii v  sou.  uin  b i c a r b o n a t e , pn  u.o  mi o r  (w/v)  u.uib&  f l u o r e s c a m i n e i n a c e t o n e was added w h i l e m i x i n g . R e l a t i v e f l u o r e s c e n c e was measured a g a i n s t a b l a n k w i t h the b u f f e r i n which  Na K -activated  ATPase was r o u t i n e l y e x t r a c t e d , w i t h e m i s s i o n w a v e l e n g t h e x c i t a t i o n wavelength  +  +  a t 480 nm and  a t 390 nm. B o v i n e serum a l b u m i n was used as the  standard f o r c a l i b r a t i o n .  (6) N a K - a c t i v a t e d ATPase a c t i v i t y a s s a y +  +  The a p p r o p i a t e amount o f enzyme was i n c u b a t e d a t 37°C i n a pH 7 medium c o n t a i n i n g 30 mM i m i d a z o l e - H C l , 120 mM sodium c h l o r i d e , 20 p o t a s s i u m c h l o r i d e , 7.mM  mM  magnesium c h l o r i d e , 4 mM ATP (sodium s a l t ) ,  36  0.006% (w/v) p h o s p h a t i d y l s e r i n e i n a f i n a l volume o f 0.5 o r 1.0 m l . A c o n t r o l , c o n t a i n i n g a l l t h e above p l u s TO" -M o u a b a i n , was i n c u b a t e d 4  s i m u l t a n e o u s l y w i t h e v e r y a s s a y . The d u r a t i o n o f i n c u b a t i o n was between 5 a n d 30 m i n . , d e p e n d i n g on t h e a c t i v i t y o f t h e enzyme p r e p a r a t i o n b e i n g used. R e a c t i o n was s t a r t e d b y a d d i n g t h e enzyme, and t e r m i n a t e d by t h e a d d i t i o n o f 0.5 o r 1.0 ml o f 3% (w/v) p e r c h l o r i c a c i d . The r e s u l t i n g s o l u t i o n was a s s a y e d f o r t h e amount o f i n o r g a n i c phosphate l i b e r a t e d by h y d r o l y s i s o f ATP, u s i n g t h e method o f See and F i t t ( 3 7 ) . Absorbance was measured a t 650 nm. A c a l i b r a t i o n was p r e p a r e d u s i n g + +  d i s o d i u m hydrogen o r t h o p h o s p h a t e as t h e s t a n d a r d . Na K - a c t i v a t e d ATPase a c t i v i t y i s d e f i n e d as t h e d i f f e r e n c e i n a c t i v i t y i n t h e absence and p r e s e n c e o f o u a b a i n .  (7) P r e p a r a t i o n o f a f f i n i t y r e s i n s  (a) Cyanogen bromide a c t i v a t i o n o f S e p h a r o s e 4B 10 ml o f Sepharose 4B was washed t h o r o u g h l y w i t h w a t e r ,  suspended  i n an e q u a l amount o f w a t e r and c o o l e d i n i c e f o r t h e r e s t o f t h e p r o c e d u r e . The a c t i v a t i o n was done i n t h e fumehood. Cyanogen bromide (250 mg) was d i s s o l v e d i n a minimum volume o f d i o x a n e and added t o t h e Sepharose w i t h s t i r r i n g w h i l e t h e pH was m a i n t a i n e d a t 10.5 ± 0.1 w i t h 0.4 N NaOH. When y e r y l i t t l e change o f pH w i t h time was d e t e c t e d ( a f t e r a b o u t 20 m i n . ) , t h e S e p h a r o s e was washed w i t h c o l d w a t e r , and then with, a pH 6.8 sodium a c e t a t e b u f f e r on a s i n t e r e d g l a s s f u n n e l . The method u s e d  i s e s s e n t i a l l y the same as d e s c r i b e d by C u a t r e c a s a s ( 3 8 ) . The a c t i v a t e d Sepharose has t o be c o u p l e d w i t h the d e s i r e d a f f i n i t y l i g a n d i m m e d i a t e l y .  (b) C o u p l i n g o f C o n c a n a v a l i n A to S e p h a r o s e  4B  The a c t i v a t e d S e p h a r o s e 4B as p r e p a r e d from p a r t (a) was t o a s o l u t i o n o f 15 mg o f C o n c a n a v a l i n A i n 7.5 ml o f the pH 6.8  added sodium  a c e t a t e b u f f e r . The m i x t u r e was g e n t l y s t i r r e d f o r 12 h r s a t 4 ° C . The unbound C o n c a n a v a l i n A was removed by w a s h i n g the S e p h a r o s e w i t h a _3  s o l u t i o n c o n t a i n i n g 0.5 M sodium c h l o r i d e , 10  M c a l c i u m c h l o r i d e and  - 3  10  M manganese c h l o r i d e . The a b s o r b a n c e o f the w a s h i n g s o l u t i o n c o n t a i n i n g the unbound  C o n c a n a v a l i n A was measured a t 280 nm. By s u b t r a c t i n g the t o t a l amount o f p r o t e i n i n the w a s h i n g s o l u t i o n f r o m the o r i g i n a l amount o f C o n c a n a v a l i n A used, the c o n c e n t r a t i o n o f C o n c a n a v a l i n A c o v a l e n t l y c o u p l e d was e s t i m a t e d to be 1.8 mg p e r ml o f S e p h a r o s e  4B.  (c) C o u p l i n g o f Wheat germ a g g l u t i n i n t o Sepharose  4B  The method used was e s s e n t i a l l y the same as t h a t f o r C o n c a n a v a l i n A. 1.35 mg o f wheat germ a g g l u t i n i n i n 3 ml o f w a t e r was added t o the a c t i v a t e d S e p h a r o s e 4B. A 0.05 M sodium c a c o d y l a t e b u f f e r , pH 7.2,  was  used t o wash t h e c o u p l e d S e p h a r o s e , i n s t e a d o f sodium a c e t a t e . The c o n c e n t r a t i o n o f c o u p l e d wheat germ a g g l u t i n i n was 0.88 mg p e r ml o f Sepharose  4B.  (d) S y n t h e s i s o f h e x a n e d i o i c a c i d ( a d i p i c a c i d ) d i h y d r a z i d e  38  H e x a n e d i o i c a c i d d i e t h y l e s t e r (100 m l , 0.497 m o l e ) , h y d r a z i n e h y d r a t e (99-100%, 200 ml, 4.04 m o l e s ) and e t h a n o l (100%, 200 ml) were r e f l u x e d f o r 3 h r s i n a one l i t r e f l a s k . The r e s u l t i n g s o l i d s were washed t h o r o u g h l y w i t h h o t e t h a n o l ( 1 0 0 % ) , r e c r y s t a l l i z e d from a w a t e r - e t h a n o l m i x t u r e , and t h e n r e c r y s t a l l i z e d a g a i n from w a t e r . The c o l o r l e s s c r y s t a l s were d r i e d under vaccum w i t h c a l c i u m s u l f a t e f o r 3 h r s . The m.p.  (180-2°C) and  m i c r o a n a l y s e s f o r C, H and N c o n f i r m e d t h a t t h e c r y s t a l s were the d e s i r e d product.  % by wei ght Element  C  H  N  Expected  41.37  8.10  32.17  Found  41.67  8.06  31.86  (e) C o u p l i n g o f h e x a n e d i o i c a c i d d i h y d r a z i d e t o Sepharose Sepharose t a i n i n g 0.01  4B  4B was a c t i v a t e d as i n p a r t ( a ) . A pH 9.4 b u f f e r con-  M sodium c a r b o n a t e and 0.04 M sodium b i c a r b o n a t e was  used  to wash t h e a c t i v a t e d S e p h a r o s e i n s t e a d o f the sodium a c e t a t e b u f f e r . 10 ml o f a 38 g/1 h e x a n e d i o i c a c i d d i h y d r a z i d e was added. The  mixture  was s t i r r e d f o r 12 h r s a t 4°C. I t was t h e n washed w i t h w a t e r and 0.2 M sodium c h l o r i d e . The w a s h i n g s o l u t i o n was m o n i t o r e d f o r unbound h e x a n e d i o i c a c i d d i h y d r a z i d e by t h e 2 , 4 , 6 - t r i n i t r o b e n z e n e 1 - s u l f o n i c a c i d t e s t ( 3 9 ) . To a 0.5 ml sample c o n t a i n i n g 0.05 t o 0.4 ymole o f h e x a n e d i o i c a c i d d i h y d r a z i d e was added 0.5 ml o f 4% (w/v)  sodium  b i c a r b o n a t e and 0.5 ml o f 0.1% (w/v) 2 , 4 , 6 - t r i n i t r o b e n z e n e 1 - s u l f o n i c ' " ' '  39  a c i d . The m i x t u r e was k e p t i n t h e dark a t 40°C f o r 2 h r s . I t was then d i l u t e d w i t h 1 ml o f w a t e r and the a b s o r b a n c e was measured a t 340  nm.  The amount o f h e x a n e d i o i c a c i d d i h y d r a z i d e c o u p l e d was 70 ymole p e r ml o f Sepharose. ( f ) C o u p l i n g o f p e r i o d a t e o x i d i z e d ATP t o S e p h a r o s e - h e x a n e d i o i c a c i d dihydrazide  (40,41)  5 ml o f 0.02 M sodium m e t a p e r i o d a t e was mixed w i t h 5 ml o f 0.02 M sodium ATP p r e v i o u s l y a d j u s t e d to pH 11 w i t h 1 N NaOH. The m i x t u r e was a l l o w e d t o s t a n d a t 0°C i n the dark f o r 1 h r . A ,7 ml a l i q u o t was then t a k e n and added to 18 ml o f 0.1 M sodium a c e t a t e , pH 5 a t 0°C. The m i x t u r e was s t i r r e d and i m m e d i a t e l y added to 10 ml o f S e p h a r o s e h e x a n e d i o i c a c i d d i h y d r a z i d e p r e p a r e d as d e s c r i b e d i n p a r t (e) and e q u i l i b r a t e d i n the same pH 5 b u f f e r a t 4°C. The s u s p e n s i o n was f o r 3 h r s i n the dark a t 4°C. 75 ml o f 2 M sodium c h l o r i d e was  stirred added  and s t i r r i n g c o n t i n u e d f o r 30 min. I t was t h e n washed f r e e o f unbound n u c l e o t i d e by m o n i t o r i n g the a b s o r b a n c e a t 260 nm. The amount o f ATP bound was a l s o e s t i m a t e d by a c i d h y d r o l y s i s o f ATP t o y i e l d i n o r g a n i c p h o s p h a t e . 0.1 ml o f t h e c o u p l e d S e p h a r o s e was added t o 0.9 ml o f 1 N h y d r o c h l o r i c a c i d and h e a t e d i n a b o i l i n g w a t e r b a t h f o r 10 min. The sample was then a s s a y e d f o r i n o r g a n i c p h o s p h a t e by t h e method o f See and F i t t ( 3 7 ) . The amount o f l i g a n d e s t i m a t e d by e x t i n c t i o n a t 260 nm and i n o r g a n i c p h o s p h a t e d e t e r m i n a t i o n was 1.8 and 2.2 ymole p e r ml o f Sepharose, r e s p e c t i v e l y .  40  (8) A f f i n i t y Chromatography In a t y p i c a l e x p e r i m e n t , t h e a f f i n i t y column was p r e p a r e d by p a c k i n g 2 t o 3 ml o f t h e a f f i n i t y r e s i n i n a p a s t e u r p i p e t . A l l p r o c e d u r e s were c a r r i e d o u t a t 0-5°C. The column was e q u i l i b r a t e d and e l u t e d w i t h a b u f f e r c o n t a i n i n g 30 mM i m i d a z o l e - H C l ,  100 mM s o d i u m c h l o r i d e ,  10% g l y c e r o l , pH 7.0 . 0.5 ml o f enzyme e x t r a c t , p r e p a r e d i n page 33 t o 34, was l o a d e d on t h e column. A p p r o x i m a t e l y  as d e s c r i b e d 0.55 ml  f r a c t i o n s were c o l l e c t e d . When f o u r b e d volumes o f b u f f e r had p a s s e d through, another b u f f e r , containing a competitor o r Na K - a c t i v a t e d ATPase b u t o t h e r w i s e  f o r the a f f i n i t y l i g a n d  i d e n t i c a l to the o r i g i n a l b u f f e r ,  was used f o r e l u t i o n . T h i s b u f f e r c o n t a i n e d s a t u r a t e d a - m e t h y l - m a n n o s i d e , o r 5 inq/mi N-acetylgiucosamine, o r \c mg/mi s o d i u m A i r t o r t h e a f f i n i t y ligands Concanavalin  A, wheat germ a g g l u t i n i n , o r p e r i o d a t e  oxidized  ATP r e s p e c t i v e l y . A f u r t h e r f o u r b e d volume o f f r a c t i o n s were c o l l e c t e d . These f r a c t i o n s were a s s a y e d f o r enzyme a c t i v i t y and t o t a l p r o t e i n .  (9) V e l o c i t y  sedimentation  (a) C a l c u l a t i o n o f s u c r o s e c o n c e n t r a t i o n  gradient  A s u c r o s e c o n c e n t r a t i o n g r a d i e n t was c a l c u l a t e d f o r t h e Beckman SW 41 r o t o r w h i c h was used f o r t h i s s t u d y . The method o f Hans N o l l ( 4 3 ) was u s e d w i t h some m o d i f i c a t i o n . The e q u a t i o n 5 he d e r i v e d i s e q u i v a l e n t t o e q u a t i o n [ 6 ] .  41  Vx)  =  where  m x [ p  P " m(x) p  m = —7  n  xt(Pp  t  .  ]  [7]  — r P  -  C 6 ]  p ) t  v i s c o s i t y a t t h e b e g i n n i n g o f .the s u c r o s e g r a d i e n t d i s t a n c e o f t h e b e g i n n i n g o f t h e s u c r o s e g r a d i e n t from the a x i s o f r o t a t i o n density o f sucrose s o l u t i o n a t the beginning o f the sucrose gradient density o f the macromolecule d i s t a n c e from t h e a x i s o f r o t a t i o n n  m(x)  v i s c o s i t y o f the sucrose s o l u t i o n a t x  p  m(x)  density o f sucrose s o l u t i o n at x  x^ i s s u p p l i e d by t h e m a n u f a c t u r e r t o be 7.0 cm. The c o n c e n t r a t i o n o f s u c r o s e a t t h e b e g i n n i n g o f t h e g r a d i e n t was chosen t o be 10% (w/v). A t t h i s c o n c e n t r a t i o n , t h e s u c r o s e s o l u t i o n i s d e n s e r than t h e b u f f e r c o n t a i n i n g t h e p r o t e i n s , so t h a t minimum d i s t u r b a n c e w i l l o c c u r when t h e sample i s l o a d e d on t o p o f t h e s u c r o s e g r a d i e n t . The d e n s i t y and v i s c o s i t y o f a 10% (w/v) s u c r o s e s o l u t i o n a t 5°C a r e 1.041 g/cm and 2.073 c e n t i 3  poise (44), respectively,p  was chosen t o be 1.343. S u b s t i t u t i n g t h e s e  into equation [ 7 ] , m = 0.979 S u b s t i t u t i o n o f m i n t o e q u a t i o n [ 6 ] , and u s i n g c o m p i l e d d a t a o f  42  the v i s c o s i t y a n d d e n s i t y o f s u c r o s e s o l u t i o n as a f u n c t i o n o f i t s c o n c e n t r a t i o n a t 5°C ( 4 4 ) , a r e l a t i o n between c o n c e n t r a t i o n C ( ) m  the d i s t a n c e x can be c a l c u l a t e d . The r e s u l t s a r e s h o w n i i n  a n  x  d  T a b l e V.  TABLE V S u c r o s e Cone. % (w/v)  p  m(x)  n  m(x)  (g/cm )  (centipoise)  10  1.041  11  , » ~V  p" m(x)  x (cm)  2.073  0.302  7.00  0.00  0.000  1.045  2.150  0.298  7.37  0.37  0.594  13  1.053  2.319  0.290  8.17  1.17  1.878  15  1.062  2.513  0.281  9.13  2.13  3.42  17  1.071  2.736  0.272  10.28  3.28  5.26  in  i n  o n n i  no/^o  -II/^T  /I  21  1.089  3.290  0.254  13.10  6.10  9.79  23  1.098  3.636  0.245  15.16  8.16  13.10  3  on  p  p  x  c m ;  Volume ( m l )  C"»  "7  * n  T h e r e i s a d i r e c t r e l a t i o n s h i p between t h e d i s t a n c e x and t h e volume i n t h e c e n t r i f u g e t u b e . The c r o s s - s e c t i o n a l a r e a o f t h e c e n t r i f u g e tube as s u p p l i e d by t h e m a n u f a c t u r e r i s 1 .605 cm -, s o t h a t 2  Volume = 1.605 (x - x ) ml t  or  V = 1.605 (x - 7.0) ml.  This corresponding  volumes a r e l i s t e d on t h e l a s t column o f T a b l e V.  ' [8] .  The s o l i d l i n e o f F i g . 3 shows t h e shape o f t h e s u c r o s e c o n c e n t r a t i o n g r a d i e n t as a f u n c t i o n o f the c e n t r i f u g e tube volume.  •  a  a  c  = 29.3 - l 9 . 3 e x p ( - V / l l . 7 )  simulation  (b) G e n e r a t i o n o f t h e s u c r o s e c o n c e n t r a t i o n g r a d i e n t F o r the e x p e r i m e n t a l a p p l i c a t i o n o f t h e p r i n c i p l e o f c o n s t a n t v e l o c i t y s e d i m e n t a t i o n , a method o f p r e p a r i n g the c a l c u l a t e d g r a d i e n t i s n e e d e d . The c o n s t a n t v e l o c i t y g r a d i e n t as shown i n F i g . 3 e x h i b i t s a convex c u r v a t u r e . N o l l (43) has d e m o n s t r a t e d t h a t t h i s c o u l d be s i m u l a t e d by a c o n v e x e x p o n e n t i a l g r a d i e n t . An e x p o n e n t i a l  gradient  maker d e s i g n e d f o r g e n e r a t i o n o f such a g r a d i e n t i s shown i n F i g . 4.  Fig. 4  E x p o n e n t i a l g r a d i e n t maker Arrow i n d i c a t e s d i r e c t i o n o f f l o w o f s u c r o s e s o l u t i o n C-| = i n i t i a l c o n c e n t r a t i o n i n m i x i n g chamber V-| = i n i t i a l volume i n m i x i n g chamber = concentration i n reservoir  S u c r o s e s o l u t i o n i s pumped from t h e m i x i n g chamber and d e l i v e r e d to t h e c e n t r i f u g e t u b e , w h i l e a more c o n c e n t r a t e d s u c r o s e s o l u t i o n i s b e i n g pumped i n t o t h e m i x i n g chamber. The s o l u t i o n i n t h e m i x i n g  chambe  i s b e i n g s t i r r e d a l l the t i m e . I f C^, t h e s u c r o s e c o n c e n t r a t i o n i n t h e r e s e r v o i r i s h i g h e r than the i n i t i a l c o n c e n t r a t i o n  i n the mixing  45  chamber, then t h e s u c r o s e s o l u t i o n b e i n g pumped i n t o t h e c e n t r i f u g e tube w i l l have an i n c r e a s i n g c o n c e n t r a t i o n . Low c o n c e n t r a t i o n s u c r o s e s o l u t i o n i n t r o d u c e d f i r s t i s a l l o w e d t o f l o a t t o t h e t o p , as s o l u t i o n o f i n c r e a s i n g c o n c e n t r a t i o n i s f e d i n . The two f l o w r a t e s i n d i c a t e d by t h e arrows i n F i g . 4 were made t o be e q u a l by t h e use o f two i d e n t i c a l c h a n n e l s o f a p e r i s t a l t i c pump. More than one g r a d i e n t s can be made s i m u l t a n e o u s l y u s i n g more c o n n e c t i n g t u b e s , and t h e t o t a l number i s l i m i t e d o n l y by t h e number o f a v a i l a b l e c h a n n e l s i n t h e pump. The g r a d i e n t p r o d u c e d i s d e f i n e d by e q u a t i o n [ 9 ] ( 4 2 , 4 5 ) .  C = C  [9]  - ( C - C,) exp ( V / V )  2  2  1  volume V has been pump o u t o f t h e m i x i n g chamber. The p a r a m e t e r s C C-j a n d V-| d e t e r m i n e t h e a c t u a l shape o f t h e g e n e r a t e d  2  ,  concentration  g r a d i e n t . These a r e t o be a d j u s t e d s o t h a t e q u a t i o n [ 9 ] s i m u l a t e s t h e d e s i r e d g r a d i e n t as shown i n F i g . 3.  w h i c h becomes t h e c o n c e n t r a t i o n  o f t h e b e g i n n i n g o f t h e g r a d i e n t , s h o u l d o b v i o u s l y be 10% ( w / v ) . C and 2  V-| were found by s u b s t i t u t i n g t h e f i f t h and e i g t h row o f d a t a from Table V i n t o equation [9]. This gives  17 = C  2  - ( C -10) exp (-5.26/V-,)  [10]  23 = C  2  - ( C -10) exp (-13.10/V-j)  [11]  2  2  46  S o l v i n g e q u a t i o n [ 1 0 ] and [ 1 1 ] by s u c e s s i v e a p p r o x i m a t i o n :  C  2  V, = 11.7  = 29.3  S u b s t i t u t i n g these values i n t o equation [ 9 ]  C = 29.3 - 19.3 e x p ( - V / 1 1 . 7 )  [12]  E q u a t i o n [ 1 2 ] d e f i n e s t h e g r a d i e n t used i n t h i s work, made by p u t t i n g 11.7 ml o f 10% (w/v) s u c r o s e s o l u t i o n i n t h e m i x i n g chamber p e r g r a d i e n t made a t one t i m e , and 29.3% (w/v) s u c r o s e s o l u t i o n i n t h e reservoir. c a l c u l a t e d from e q u a t i o n [ 1 2 ] . I t can be seen t h a t t h i s g r a d i e n t s i m u l a t e s t h e d e s i r e d g r a d i e n t t o a good approximation. (c) C a l i b r a t i o n w i t h s t a n d a r d p r o t e i n s and d e t e r m i n a t i o n o f s e d i m e n t a t i o n coeffi cient F o u r s u c r o s e g r a d i e n t s were made i n a 30 mM i m i d a z o l e - H C l , pH 7.0 b u f f e r as d e s c r i b e d i n p a r t (b) and c o o l e d t o 5 ° C . One r e c t a l g l a n d was used f o r e x t r a c t i o n o f N a K - a c t i v a t e d A T P a s e , as o u t l i n e d i n page 33 t o +  +  34. The s u p e r n a t a n t o b t a i n e d a f t e r c e n t r i f u g a t i o n a t 42,000 r.p.m. was p a s s e d t h r o u g h a M i l l i p o r e f i l t e r and c o n c e n t r a t e d t o 3 ml on an Ami con CF50A membrane u l t r a f i l t e r . 100 y l o f t h i s c o n c e n t r a t e d enzyme e x t r a c t was l o a d e d on e a c h o f • t w o s u c r o s e g r a d i e n t s . 1 mg each o f a l c o h o l dehy-  47  d r o g e n a s e , c a t a l a s e and l y s o z y m e were d i s s o l v e d i n 0.5 ml o f b u f f e r and f i l t e r e d t h r o u g h a M i l l i p o r e f i l t e r . 100 y l o f t h i s s t a n d a r d p r o t e i n s o l u t i o n was l o a d e d on each o f t h e two r e m a i n i n g s u c r o s e g r a d i e n t s . C e n t r i f u g a t i o n was c a r r i e d o u t i n a Beckman L3-50 u l t r a c e n t r i f u g e a t 41,000 r.p.m. u s i n g a SW-41 r o t o r w i t h t h e t h e r m o s t a t s e t a t 5°C. The run was s t a r t e d as soon as t h e t e m p e r a t u r e was s t a b i l i z e d and t h e vacuum was down t o 50 m i c r o n s . I t was s t o p p e d a f t e r 16.5 h r s . U s i n g a Desaga (W. Germany) p e r i s t a l t i c pump a n d a G i l s o n MF M i n i - E s c a r g o t f r a c t i o n a t o r ( M i d d l e t o n , W i s . ) , t h e c o n t e n t o f each tube was f r a c t i o n a t e d i n t o 28 e q u a l f r a c t i o n s w i t h pump r a t e s e t on 1 and a time c o u n t o f 1.7 m i n . f o r each f r a c t i o n . These f r a c t i o n s were a s s a y e d f o r a l c o h o l dehydrogenase,  c a t a l a s e , lysozyme and Na K - a c t i v a t e d ATPase  activity. A l c o h o l dehydrogenase  a s s a y ( 7 9 ) : Samples o f 25 t o 100 y l were  mixed w i t h 3 ml o f an a s s a y medium w h i c h i s a m i x t u r e  o f 4 ml o f 1 M  T r i s - H C l (pH 7 . 6 ) , 100 y l o f 95% e t h a n o l , 70 mg o f NAD a n d 72 ml o f w a t e r . The a c t i v i t y was f o l l o w e d a t 340 nm. C a t a l a s e was a s s a y e d by m e a s u r i n g d i r e c t l y t h e e x t i n c t i o n a t 405 nm. Lysozyme a s s a y ( 8 0 ) : 50 y l samples were mixed w i t h 2.95 ml o f an a s s a y medium c o n t a i n i n g 0.25 mg o f M i c r o c o c u s l y s o d e i k t i c u s i n a 0.05 M sodium c h l o r i d e and 0.66 M p h o s p h a t e b u f f e r . The d e c r e a s e i n t u r b i d i t y was i m m e d i a t e l y m o n i t o r e d a t 540 nm, w i t h a r e f e r e n c e c o n t a i n i n g o n l y the b u f f e r s o l u t i o n . The a c t i v i t y o f lysozyme was e x p r e s s e d as t h e d e c r e a s e i n a b s o r b a n c e p e r 100 s e c o n d s .  48  (10) Gel f i l t r a t i o n o f d e t e r g e n t s o l u b i l i z e d N a \ - a c t i v a t e d ATPase +  and S t o k e s ' r a d i u s c a l i b r a t i o n  157 ml o f S e p h a r o s e 6B was p a c k e d i n t o a Kl5/90 column  (Pharmacia)  and e q u i l i b r a t e d a t 4°C i n a pH 7.0 b u f f e r c o n t a i n i n g 30 mM i m i d a z o l e HC1, 100 mM NaCl and 10% ( v / v ) g l y c e r o l . The v o i d volume was  determined  b y . l o a d i n g 3 mg o f B l u e D e x t r a n 2000 i n 1.5 ml o f the same b u f f e r . 5 mg each o f b o v i n e f i b r i n o g e n and b o v i n e serum albumin and 3 mg o f h o r s e s p l e e n a p r o f e r r i t i n were d i s s o l v e d i n 2 ml o f the same b u f f e r and c e n t r i f u g e d a t 13,000 r.p.m. i n a S o r v a l SS-34 r o t o r f o r 10 min. 1.5 ml o f the s u p e r n a t a n t was l o a d e d on t h e column. 0.88 ml f r a c t i o n s were c o l l e c t e d . The f r a c t i o n s were m o n i t o r e d f o r e x t i n c t i o n a t 280 nm u n t i l 1.5 l i t r e s o f b u f f e r had p a s s e d t h r o u g h . The column was t h e n r e e q u i V i b r a t e d w i t h the same b u f f e r p l u s 1.5% (w/v) L u b r o l WX, p r e p a r e d from a s t o c k s o l u t i o n o f 10%(w/v) L u b r o l WX f i l t e r e d t h r o u g h M i l l i p o r e . One r e c t a l g l a n d was u s e d f o r e x t r a c t i o n o f N a K - a c t i v a t e d A T P a s e , as o u t l i n e d i n page 33. t o 34. The +  +  s u p e r n a t a n t o b t a i n e d a f t e r c e n t r i f u g a t i o n a t 42,000 r.p.m. was  passed  t h r o u g h a M i l l i p o r e f i l t e r and c o n c e n t r a t e d t o 3 ml on an Ami con CF50A membrane u l t r a f i l t e r . 1.5 ml o f t h i s enzyme e x t r a c t was l o a d e d . 0.80 ml f r a c t i o n s were c o l l e c t e d . u n t i 1 1.5 l i t r e s o f b u f f e r has p a s s e d t h r o u g h . These f r a c t i o n s were a s s a y e d f o r Na K - a c t i v a t e d ATPase a c t i v i t y .  49  RESULTS  (1) O p t i m i z a t i o n o f c o n d i t i o n s f o r enzyme e x t r a c t i o n  (a) T e s t o f e f f i c i e n c y o f e x t r a c t i o n by d i f f e r e n t d e t e r g e n t s  + +  The e f f i c i e n c y o f f o u r d e t e r g e n t s f o r t h e e x t r a c t i o n o f Na K - . a c t i v a t e d ATPase was t e s t e d a t 0.1% (w/v) and 1.0% (w/v) d e t e r g e n t c o n c e n t r a t i o n . The r e s u l t s a r e summaried i n T a b l e V I . TABLE VI N a K - a c t i v a t e d ATPase a c t i v i t y (ymole/mg/hr) 1.0%(w/v) 0.1%(w/v) +  Detergent  +  T r i methyldodecylammoni urn bromi de  0.03  0.00  T r i t o n X-100  0.09  0.01  Sodium  0.15  0.00  0.34  0.96  deoxycholate  L u b r o l WX  (b) E x t r a c t i o n as a f u n c t i o n o f L u b r o l WX c o n c e n t r a t i o n The c o n c e n t r a t i o n o f L u b r o l WX was v a r i e d from. 0.1'to 2.0% (w/v) i n t h e p r e s e n c e o f 0, 5, 10 o r 20% ( v / v ) g l y c e r o l i n t h e e x t r a c t i o n medium. The r e s u l t s a r e shown i n F i g . 5, 6, 7 and 8.  50 + +  ( c ) S t a b i l i t y o f L u b r o l WX e x t r a c t e d Na K - a c t i v a t e d ATPase Enzyme e x t r a c t s i n the p r e s e n c e o f 0, 5, 10 and 20% (v/v) g l y c e r o l were t e s t e d f o r t h e i r s t a b i l i t i e s . The enzyme e x t r a c t s , were s t o r e d a t 4°C and a s s a y e d f o r Na K - a c t i v a t e d ATPase a t a p p r o x i m a t e l y 24 h r i n t e r v a l s . The r e s u l t s are shown i n F i g . 9. The Na K - a c t i v a t e d ATPase a c t i v i t y i s e x p r e s s e d as the p e r c e n t a g e o f the a c t i v i t y o b t a i n e d  immediately  after extraction.  (2) A f f i n i t y c h r o m a t o g r a p h y The p r o c e d u r e has been d e s c r i b e d under the 'Method' s e c t i o n i n d e t a i l . A c o n t r o l , i n w h i c n the S e p h a r o s e 46 was n o t l i n k e d to any a f f i n i t y l i g a n d , i s shown i n F i g . 10. The r e s u l t s o f C o n c a n a v a l i n A, wheat germ a g g l u t i n i n and p e r i o d a t e o x i d i z e d ATP used as a f f i n i t y l i g a n d are shown i n F i g . 11, 12 and 13, r e s p e c t i v e l y . In a l l c a s e s enzyme a c t i v i t y and amount o f p r o t e i n were p l o t t e d a g a i n s t f r a c t i o n number. Each f r a c t i o n was a p p r o x i m a t e l y 1/2 t o 3/4  (3) V e l o c i t y  ml.  sedimentation  The p r o c e d u r e has been d e s c r i b e d u n d e r the 'Method' s e c t i o n . F i g . 14 shows the p r o f i l e o f the d i s t r i b u t i o n o f enzyme a c t i v i t i e s i n the s u c r o s e g r a d i e n t a f t e r s e d i m e n t a t i o n .  S i n c e o n l y the p o s i t i o n s o f  51  maximum enzyme a c t i v i t y were r e q u i r e d  f o r c a l c u l a t i o n , t h e enzyme  a c t i v i t i e s were e x p r e s s e d as a r b i t r a r y a b s o r b a n c e u n i t s . F i g . 15 i s a c a l i b r a t i o n o f the i s o k i n e t i c sucrose gradient. c o e f f i c i e n t s o f the three standard proteins  The s e d i m e n t a t i o n  (44) : catalase,  alcohol  d e h y d r o g e n a s e a n d l y s o z y m e were p l o t t e d a g a i n s t t h e i r r e l a t i v e  distance  o f m i g r a t i o n , t a k i n g t h e t o p o f t h e s u c r o s e g r a d i e n t as o r i g i n . They a r e shown as t h e t h r e e d a t a p o i n t s i n t h e p l o t . The p o s i t i o n o f N a K +  +  a c t i v a t e d ATPase r e l a t i v e t o t h e s t a n d a r d i s i n d i c a t e d b y t h e a r r o w . From t h i s c a l i b r a t i o n , t h e s e d i m e n t a t i o n c o e f f i c i e n t o f Na K - a c t i v a t e d ATPase was e s t i m a t e d t o be 5.0 S.  (ty  uci  i' i i i r d i i On Oi  iia  k  - a t i l Vaceu MI r a s e  a i m i uUNtiS '  rriu'l US  calibration The p r o c e d u r e has been d e s c r i b e d  under t h e 'Method'  section.  F i g . 16 shows how B l u e D e x t r a n 2000, N a K - a c t i v a t e d A T P a s e , a n d t h e +  three standard proteins  +  : f i b r i n o g e n , a p o f e r r i t i n and b o v i n e serum  a l b u m i n were f r a c t i o n a t e d by t h e column. The B l u e D e x t r a n peak  indicates  the v o i d volume. The p a r t i t i o n c o e f f i c i e n t s o f Na K - a c t i v a t e d  ATPase  and o t h e r p r o t e i n s were c a l c u l a t e d a c c o r d i n g t o e q u a t i o n [ 4 j . F i g . 17 i s a c a l i b r a t i o n o f t h e g e l f i l t r a t i o n column. The logarithm proteins  (base 10) o f t h e S t o k e s ' r a d i u s  ( R ) o f the three standard g  ( 4 4 ) i n Angstroms was p l o t t e d a g a i n s t  their partition coeffi-  c i e n t s . They a r e shown as t h e t h r e e d a t a p o i n t s . The p a r t i t i o n c o e f f i -  52  c i e n t o f N a K - a c t i v a t e d ATPase i s i n d i c a t e d by t h e a r r o w . From t h i s c a l i b r a t i o n , t h e S t o k e s ' r a d i u s o f N a V - a c t i v a t e d ATPase was e s t i m a t e d to be 114 A .  (5) E s t i m a t i o n o f t h e m o l e c u l a r w e i g h t o f N a K - a c t i v a t e d ATPase +  +  C o m b i n i n g e q u a t i o n [ 3 ] and [ 5 ] ,  M =  e 1 - 7p  [13]  6 s N l T 1 l R  Siihst.i t.uti nn t.hp  fnllowinn into  s = 5.0 x 1 0 "  sec  1 3  n = 0.0102 p o i s e  ( v i s c o s i t y o f water a t 20°C)  v = 0.73 cm g "  (assumed)  3  1  = 1.00 g cm" R„= e 1.14 x 1 0 " cm 3  P  6  gives  pnuation [1.^]  M = 240,000  umoles o f p h o s p h a t e p e r h o u r  ( D-CW3 )  umoles o f p h o s p h a t e p e r h o u r  (D-D-Q) ro •  o i  1  i  1  r  1  1  1 — T  HQ  co -afD  -o  -so c+  O  -h n  a  m X + r+ -s CU 1 + or+  BJ  —i. O  o c+  o  t> <  <—i.  1T  3  <+— i . jiT CUrtfD c+  &  —^ „—^  1  Cu l/i  ex.  Cu  J=  -h  — •o1 (fD/> Cu o c+  — .  <  r+  X  O 3  o o 3o  1.  O -+)  —1.  r-  <<r+  .  1 ^  1  3 c+ -s  CU  <-!-  a  i.  X  |  1  fD  c cr -so ^  .,—, —  -  cr. :-S O —'  c: '3O  Cu  I  O1 *  r  o ^  o  ofD 3 r+ -s Cu  \  s: <  -  '  •  rt. i . O  3 3 r+ 3"fD •a'  -s fD Ln fD  ro  .  o  V  3O  'v  -  O  1  t  o  cn  1  1  V.  — i  1  \  \ °  n  1  fD  \  i  t  ' OJ  ro  co  >  t  -P»  t  1  1  I I  cn  _  CM  •  pmoles o f p h o s p h a t e p e r mg p r o t e i n p e r h r . (o—o—o)  < << O fD -S  o  o o  «  •  <  » o ro  i  mg o f p r o t e i n  1— o co  (A-A—A )  • — — — —  '  umoles o f p h o s p h a t e p e r h o u r  (U—•O—Q)  99  57  Days Fig. 9  Stability without 5%(v/v) 10%(v/v) 20%(v/v)  o f L u b r o l WX e x t r a c t e d Na K - a c t i v a t e d ATPase glycerol (Q~U—O) g l y c e r o l ( o-O-o ) g l y c e r o l (jHHi) glycerol  58  5  10  15  20  F r a c t i o n number 10 S e p h a r o s e 4B f i l t r a t i o n a s c o n t r o l f o r a f f i n i t y c h r o m a t o g r a p h y NaV-activated ATPase a c t i v i t y ( • — • — • ) P r o t e i n measured by f l u o r e s c e n c e method (o-o.-o)  59  6.0  0.20  5.0 0.15 4.0  0.10  CD  os-  Q.  D-mannoside  2.0  O CD  0.05 1.0  0.0  0.00  1—9-4—«  30 25 20 15 10 F r a c t i o n number Concanavalin A a f f i n i t y chromatography F i g , 11 N a K < - a c t i v a t e d A T P a s e a c t i v i t y (©—« — •') P r o t e i n measured by f l u o r e s c e n c e method (o--o--o) 5  +  +  60  I 9  7.0  h  6.0  >  5.0  I  0.20  S13 O  s-  <D Q<D 4-> <B  J=  Q-  0.15 4.0  10  o  to  3.0  0.10  »•  o s-  CD  o  E 3.  £  +J  2.0  N-acetylglucosamine  I  CL O  0.05 o  1.0  h  A 0.00  0.0  5 F i g . 12  10 F r a c t i o n number  15  20  25  30  Wheat germ a g g l u t i n i n a f f i n i t y c h r o m a t o g r a p h y N a K - a c t i v a t e d ATPase a c t i v i t y ( » — ) P r o t e i n measured by f l u o r e s c e n c e method (O--O--0) +  +  9  61  F r a c t i o n number F i g . 13  P e r i o d a t e o x i d i z e d ATP a f f i n i t y c h r o m a t o g r a p h y N a K - a c t i v a t e d ATPase a c t i v i t y ( e — » — 9 ) . P r o t e i n measured by f l u o r e s c e n c e method (0—0--O) f  +  62  0.15 h  cu  CD  10  <a (/>  0.10  c  I— et  a)  CJ) o  -o  CD  S-  +->  -o  >  s= CD  •I-  o (O  i— o  I  +  -!=  o  +  o  i —  A3  E  E  0.05 r  cu o c  _Q iO (/)  0.00 15  10  20  25  Relative distance F i g . 14  Enzyme a c t i v i t y p r o f i l e i n s u c r o s e g r a d i e n t a f t e r c e n t r i f u g a t i o n Catalase (OHCH3) A l c o h o l dehydrogenase N a K - a c t i v a t e d ATPase_. ({MM?) Lysozyme ( O ° 0 H ? ) +  +  63  64 0.7  0.6 0.5  0.20  0.5  0.18 0.4  0.16  0.4  0.14 ii) E  c  0.3  0.3  4-> rC -E CL co O  LO CM +J ra a> u c ro  o. cu  0.2 °-  0.2  0.10  -J 0.08 -t 0.0b  S_  O </) -Q  0.12  o.i  0.1  .  50 F i g . 16  >.l.  60  r  «-»''•  70  I  I  I  »  80 90 100 Volume (ml)  t  I  110  I  I  120  I  I  130  t  I  i  0.04  140  l  -I  150  S e p h a r o s e 6B g e l f i l t r a t i o n o f Na K - a c t i v a t e d ATPase & standard proteins B l u e D e x t r a n 2000 (&H&—A ) Na+K+-acti v a t e d ATPase (O—O-O) S t a n d a r d p r o t e i n s (©-<&—-9)  CO CNJ +->  rO CD U  c  rO O  IO  0.02 0.0  o  0.00  65  2.5  N a K - a c t i v a t e d ATPase +  +  fibrinogen  2.0 CD  apoferritin  b o v i n e serum a l b u m i n 1.5  JL  1,0  0.0  i  0.1  I  i  I  0.2  0.3 K  F i g . 17  i  av  I  I  . 0.4  1  1  1  0.5  1  0.6  1  1  0.7  -  S t o k e s ' r a d i u s as a f u n c t i o n o f p a r t i t i o n  coefficient  L  0.8  66  DISCUSSION  (1) E x t r a c t i o n o f N a K - a c t i v a t e d +  +  ATPase  From T a b l e VI, i t i s o b v i o u s t h a t o u t o f the f o u r  detergents  t e s t e d , L u b r o l WX i s b e s t s u i t e d f o r the e x t r a c t i o n o f Na K - a c t i v a t e d ATPase from the r e c t a l g l a n d o f dog f i s h . A l t h o u g h  trimethyldodecyl-  ammonium bromide was not u s e f u l f o r t h i s work, i t has been f o u n d t o b e p r o m i s i n g i n the e x t r a c t i o n o f a c e t y l c h o l i n e s t e r a s e from the e l e c t r o pi ax o f E l e c t r i c E e l s and T o r p e d o e s ( 9 2 ) , and c o u l d be a p o t e n t i a l l y -J  i U  I  i  ^Li  1L  I V> t  O  *,i i C . I  i h U H U i  U i k u  uUul>d  pi  U  lllJ •  t h e r e f o r e chosen f o r t h i s work. I t has been o b s e r v e d  u U U i  J  I  ««/%  H U O  that a detergent  which c o u l d be used f o r e x t r a c t i o n a t low c o n c e n t r a t i o n c o u l d become i n h i b i t o r y at higher concentrations  (93). Thus, i t i s d e s i r a b l e t o  i n v e s t i g a t e e x t r a c t i o n as a f u n c t i o n o f L u b r o l WX c o n c e n t r a t i o n . F i g . 5 shows t h a t e x t r a c t i o n o f b o t h Na K - a c t i v a t e d ATPase and t o t a l p r o t e i n i n c r e a s e s f o r i n c r e a s i n g L u b r o l WX c o n c e n t r a t i o n , but the i n c r e a s e i n t o t a l p r o t e i n i s l e s s d r a s t i c when L u b r o l WX c o n c e n t r a t i o n i s h i g h e r than 0.5%  (w/v), such t h a t the s p e c i f i c a c t i v i t y i n c r e a s e s m o d e r a t e l y  w i t h L u b r o l WX c o n c e n t r a t i o n . I t a p p e a r s t h a t h i g h L u b r o l WX c o n c e n t r a t i o n can be used t o a d v a n t a g e , b u t the t h r e s h o l d o f i t s s o l u b i l i t y a t 5°C is approximately extraction. '  2 % (w/v). Hence 1.5%  (w/v) was chosen f o r r o u t i n e  67  The impure enzyme i s h i g h l y u n s t a b l e once d e t e r g e n t - e x t r a c t e d ,  but  g l y c e r o l i s known to s t a b i l i z e i t ( 7 ) . The e f f e c t s o f the p r e s e n c e o f 5, 10,and 20% (v/v) g l y c e r o l d u r i n g e x t r a c t i o n were shown i n F i g . 6,  7,  and 8. The p r e s e n c e o f g l y c e r o l does not s i g n i f i c a n t l y change the e x t r a c t i o n . In o r d e r t o f i n d the optimum c o n c e n t r a t i o n  of glycerol f o r  s t a b i l i z a t i o n , the a c t i v i t y o f the enzyme e x t r a c t e d i n d i f f e r e n t concentrations  o f g l y c e r o l was  f o l l o w e d w i t h t i m e and the r e s u l t s were  shown i n F i g . 9. I t can be s e e n t h a t w i t h o u t g l y c e r o l f o r s t a b i l i z a t i o n , the a c t i v i t y f a l l s to l e s s t h a n h a l f the o r i g i n a l v a l u e upon  storage  a t 4°C f o r two d a y s . The enzyme i s more s t a b l e i n 5% (v/v) g l y c e r o l , and even more so i n 10% (v/v) g l y c e r o l , but t h e r e i s no  detectable  improvement i n s t a b i l i t y f r o m 10% to 20%. Hence 10% (v/v) g l y c e r o l  was  (2) A f f i n i t y c h r o m a t o g r a p h y C o n c a n a v a l i n A and wheat germ a g g l u t i n i n are h a e m a g g l u t i n a t i n g a g e n t s known to have a s t r o n g a f f i n i t y f o r D - g l u c o s i d e  or  D-mannoside  (94) and N - a c e t y l g l u c o s a m i n e (90) r e s p e c t i v e l y . C o n c a n a v a l i n A has been a p p l i e d s u c c e s s f u l l y f o r the p u r i f i c a t i o n o f R h o d o p s i n ( 6 1 ) , and i t i s a l s o known t o b i n d a c e t y l c h o l i n e s t e r a s e  strongly (95). Since Na K -  a c t i v a t e d ATPase i s known to be a g l y c o p r o t e i n  +  ( 7 , 1 1 ) , i t was  +  hoped  t h a t the c a r b o h y d r a t e p o r t i o n m i g h t c o n t a i n e i t h e r D-gl.ucose, D-mannose o r N - a c e t y l g l u c o s a m i n e a t the n o n r e d u c i n g end and be a c c e s s i b l e to  68  C o n c a n a v a l i n A o r wheat germ a g g l u t i n i n . As shown i n F i g . 11 and 12, n e i t h e r o f t h e s e l i g a n d s was a b l e t o b i n d Na K - a c t i v a t e d ATPase s t r o n g l y enough. The a c t i v i t y and p r o t e i n p r o f i l e o f t h e C o n c a n a v a l i n A column i n F i g . 11 a r e e s s e n t i a l l y t h e same as t h a t o f t h e c o n t r o l i n F i g . 10.  No a c t i v i t y c o u l d be d e t e c t e d upon  e l u t i o n w i t h a-D-methylmannoside. The i n i t i a l peak a t f r a c t i o n 5 was p r o b a b l y due to l a r g e a g g r e g a t e s o f p r o t e i n s e l u t i n g i n the v o i d volume. The a c t i v i t y p r o f i l e o f t h e wheat germ a g g l u t i n i n column i n F i g . 12 s u g g e s t s t h a t t h e r e m i g h t have been some weak b i n d i n g . A s m a l l peak o f a c t i v i t y a p p e a r e d i n f r a c t i o n 21 t o 26 a f t e r e l u t i o n o f N - a c e t y l g l u c o s a m i n e s t a r t e d , b u t i t o n l y a c c o u n t s f o r a p p r o x i m a t e l y 4% o f t h e t o t a l i n p u t activity. As shown i n F i g . 13  5  the p e r i o d a t e o x i d i z e d ATP l i g a n d d i d n o t  have a s t r o n g a f f i n i t y f o r Na K - a c t i v a t e d A T P a s e . No s i g n i f i c a n t a c t i v i t y c o u l d be d e t e c t e d upon e l u t i o n w i t h ATP. The f a i l u r e o f b i n d i n g i s n o t due t o h y d r o l y s i s o f t h e l i g a n d . I n o r g a n i c p h o s p h a t e was found to be a b s e n t i n f r a c t i o n 18 t o 31, and a p h o s p h a t e a n a l y s i s o f t h e g e l r e c o v e r e d a f t e r t h e e x p e r i m e n t showed t h a t i t c o n t a i n e d e s s e n t i a l l y t h e same c o n c e n t r a t i o n o f i n t a c t l i g a n d s as b e f o r e . T h e s e r e s u l t s s u g g e s t t h a t an i n t a c t r i b o s e r i n g may be n e c e s s a r y f o r p r o p e r b i n d i n g to o c c u r . The t o t a l amount o f l i g a n d was a p p r o x i m a t e l y 100 f o l d i n e x c e s s o f t h e t o t a l amount o f Na K - a c t i v a t e d ATPase u s e d , on a one t o one b i n d i n g b a s i s f o r C o n c a n a v a l i n A and wheat germ a g g l u t i n i n . F o r p e r i o d a t e o x i d i z e d ATP i t was more than 1,000 f o l d i n e x c e s s . In a l l t h r e e c a s e s the r e c o v e r y o f t o t a l p r o t e i n was q u a n t i t a t i v e (>95%). The r e c o v e r y o f  69  t o t a l a c t i v i t y , i n c l u d i n g t h e c o n t r o l , was p o o r (60% o r l e s s ) . I t a p p e a r s t h a t a p p r e c i a b l e amount o f NaV-activated ATPase was d e n a t u r e d i n t h e column.  (3) E s t i m a t i o n o f m o l e c u l a r w e i g h t and s u b u n i t c o m p o s i t i o n The p l o t o f t h e s e d i m e n t a t i o n c o e f f i c i e n t s o f t h e s t a n d a r d p r o t e i n s v e r s u s t h e i r r e l a t i v e d i s t a n c e s o f m i g r a t i o n i n F i g . 15 i s l i n e a r , and the l i n e p a s s e s t h r o u g h the o r i g i n . These are i n agreement w i t h t h e t h e o r y . F i g . 17 shows t h a t t h e l o g a r i t h m o f t h e S t o k e s ' r a d i u s has a l i n e a r + +  r e l a t i o n s h i p w i t h t h e p a r t i t i o n c o e f f i c i e n t . I t a p p e a r s t h a t Na Ina c t i v a t e d A'lrase i s a r e l a t i v e l y  c o y mine Ir i c p t u l c i n .  Aa coinpui';  J W M ,  6 - g a l a c t o s i d a s e , a p r o t e i n w i t h an u n u s u a l l y h i g h m o l e c u l a r w e i g h t o f 515,300  and an e f f e c t i v e S t o k e s ' r a d i u s o f 71 A  (44),  consistently  e l u t e d a f t e r N a K - a c t i v a t e d ATPase i n Sepharose 6B g e l f i l t r a t i o n ( d a t a +  +  n o t shown). The m o l e c u l a r w e i g h t o f  240,000  f o r Na K - a c t i v a t e d ATPase e s t i m a t e d  from the combined r e s u l t o f v e l o c i t y s e d i m e n t a t i o n and g e l f i l t r a t i o n i s t h a t o f the c a t a l y t i c a l l y a c t i v e s u b u n i t assembly b e c a u s e the enzyme was d e t e c t e d i n t h e s e methods b y i t s own a c t i v i t y . Sodium d o d e c y l s u l f a t e g e l e l e c t r o p h o r e s i s measured the m o l e c u l a r w e i g h t s o f the s e p a r a t e d s u b u n i t s . The m o l e c u l a r w e i g h t o f  250,000  o b t a i n e d f o r Na K - a c t i v a t e d  ATPase f r o m the r e c t a l g l a n d o f dog f i s h and e l e c t r o p l a x o f e l e c t r i c e e l s i s b a s e d o n the a s s u m p t i o n o f an a B z  subunit structure  (11,16,48),  as  70  d e s c r i b e d i n p a r t I o f the i n t r o d u c t i o n . The agreement between the m o l e c u l a r w e i g h t o b t a i n e d f r o m t h i s work and t h o s e mentioned suggests that a B 2  in solution.  above  i s the c o r r e c t s u b u n i t s t r u c t u r e o f t h e i n t a c t enzyme  71  REFERENCES  (1) Duncan C. J . : "The m o l e c u l a r p r o p e r t i e s and e v o l u t i o n o f e x c i t a b l e c e l l s " 1 s t e d . , pp. 1-11 & 50-75, Pergamon P r e s s , 1967. (2) Hoffman J . F., F e d . P r o c . 19, 127 (1960) (3) Skou, J . C , B i o c h i m . B i o p h y s . A c t a 2^3, 394 (1957) (4) Dunham P. B., Hoffman J . F., P r o c . N a t . A c a d . 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