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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 ' n YEW CHOI PANG B.Sc. McGILL UNIVERSITY 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department of C h e m i s t r y 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 the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA May, 1975 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f r ee ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permission. Department of Qj^N^T^ The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 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 i n v e s t i g a t e d , 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 t o 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 d e r i v e d f r o m 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 w o r k e r s . 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 X X ACKNOWLEDGEMENTS .. "....'.rl ABREVIATIONS.... ' . f j i INTRODUCTION V. ; .1 PART I 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 A TPase... ....2 (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 . ..4 (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 .....6 (3) P u r i f i c a t i o n . .9 (4) S u b u n i t s t r u c t u r e and s i z e . 11 (5) L i p i d r e q u i r e m e n t .......15 PART II A f f i n i t y C hromatography ....18 (1) S t a t i o n a r y m a t r i x .- 19 (2) L i g a n d .19 (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 21 (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 .22 (5) E l u t i o n o f the bound p r o t e i n .....22 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 & Gel F i l t r a t i o n .....24 (1) V e l o c i t y s e d i m e n t a t i o n 24 (2) G e l f i l t r a t i o n . ...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 32 (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 . ' . r r r ; : ; . . 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 bromide 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 t o 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 t o 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 -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 . . . 39 (8) A f f i n i t y c hromatography , 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 ) . 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 g r a d i e n t . 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 s e d i m e n t a t i o n 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 r a d i u s c a l i b r a t i o n ...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 ATPase.. . 5 0 (2) A f f i n i t y c h romatography 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) Gel 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 c a l i b r a t i o n 51 (5) , 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 o f N a + K + - a c t i v a t e d ATPase. .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 ATPase 66 (2) A f f i n i t y c h romatography . 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 69 REFERENCES ,. 71 vi ACKNOWLEDGEMENTS / I would l i k e t o e x p r e s s my most s i n c e r e t h anks t o Dr. D. G. C l a r k , whose a d v i c e and 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 uld a l s o l i k e t o thank Kathy Chance for a sample o f p u r i f i e d c h i c k e n egg 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 the c o n s t a n t velocitv prariient and Mr. Martin Handel o f Spa Fresh Paci f ic Products. L a d y s m i t h , 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 . v i i ABBREVIATIONS ATP A d e n o s i n e t r i p h o s p h a t e ADP A d e n o s i n e d i p h o s p h a t e AMP A d e n o s i n e monophosphate NAD N i c o t i n a m i d e a d e n i n e 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 a d e n o s i n e t r i p h o s p h a t a s e P E-P I n o r g a n i c o r t h o p h o s p h a t e 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 i o n a c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e 1 INTRODUCTION 2 INTRODUCTION + + PART I 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 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 g r a d i e n t s o f Na 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 lasma, compared t o [ N a + ] ' = 10 mM and [ K + ] - 150 mM i n the c y t o p l a s m , d e p e n d i n g on the t y p e o f the c e l l . These 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 h i g h K + c o n c e n t r a t i o n i n s i d e t h e c e l l i s r e q u i r e d f o r p r o t e i n s y n t h e s i s by ribosomes 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 reover t he 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 hich 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 hich can a c t u a l l y be measured i n g i a n t s q u i d axons, p l a y s an i m p o r t a n t p a r t , i n the t r a n s m i s s i o n o f n e r v e i m p u l e s ( 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', presumable s u i t u a t e d i n the 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 t h e c e l l . E x p e r i m e n t s w i t h r e s e a l e d 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 the 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 opened t h e m o l e c u l a r b a s i s f o r the u n d e r s t a n d i n g 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 e x p e r i m e n t a t i o n . 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 homogenized 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 fra g m e n t s e x h i b i t e d Mg +-dependent 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 s u g g e s t e d 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 which i s r e p o n s i b l e f o r m a i n t a i n i n g the N a + and K + c o n c e n t r a t i o n g r a d i e n t s . H i s s u g g e s t i o n i s s t r o n g l y s u p p o r t e d 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 t h e 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 ATPase, 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 uscle 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 o f N a + K + - a c t i v a t e d ATPase a r e 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 g e n e r a t i o n 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 f o r the e x t r u s i o n o f e x c e s s Na , s u c h as t n e Kianey c o r t e x 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 ich 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 g u t w i t h a sodium c o n c e n t r a t i o n 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 t o 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 i n g e s t i o n . 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 ATPase has been con-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 s i z e (5) L i p i d r e q u i r e m e n t 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 I t i s g e n e r a l l y a g r e e d t h a t N a + K + - a c t i v a t e d ATPase h y d r o l y s e s ATP i n a s t e p w i s e 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 ) . E l + ATP ^ — ^ ^ E i - P + ADP 1 E l_ P E 2 - P 2 s. E 2-P + H 20 v E 2 + P 3 E 2 v " Ei 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 o f t h e above. E-P 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 enzyme. t x and t 2 r e p r e s e n t a lower and.a h i g h e r e n e r g y c o n f o r m a t i o n o f the enzyme m o l e c u l e , r e s p e c t i v e l y ( 2 0 , 8 1 ) . These 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 s u b s t a n t i a t e d 32 by k i n e t i c d a t a ( 2 0 , 2 1 ) . I t was f o u n d t h a t P was i n c o r p o r a t e d f r o m [ y 3 2 P ] A T P i n t o N a + K + - a c t i v a t e d ATPase p r e p a r a t i o n i n t h e p r e s e n c e o f Na and Mg , t h i s c o r r e s p o n d s t o p a r t i a l r e a c t i o n 1 and 2. I f K was added t o the enzyme syst e m a f t e r N a + , t h e 3 2 P i n t h e enzyme was d i s c h a r g e d , t h i s c o r r e s p o n d s 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 i s p o o r l y u n d e r s t o o d . R e c e n t l y , m a g n e t i c r e s o n a n c e t e c h n i q u e s were a p p l i e d t o 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 23 time T-| o f Na i n a s o l u t i o n c o n t a i n i n g the enzyme d e c r e a s e d upon the a d d i t i o n o f ATP, which i n d i c a t e s the 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 , which 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 the 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 , the 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 the c o o r d i n a t i o n s p h e r e 2+ o f Mn by the b i n d i n g o f p h o s p h a t e t o t h e a c t i v e s i t e . F u r t h e r m o r e , 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 s u g g e s t e d t h e b i n d i n g o f p h o s p h a t e i n the 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 s u g g e s t e d t h e b i n d i n g o f p h o s p h a t e i n the p r e s e n c e 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 and K b i n d t o t h e same s i t e , i.e. t h e a c y l p h o s p h a t e o f the 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 the 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 the phosphate monoanion, then K + i s t r a n s p o r t e d i n t o the 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 the phosphate d i a n i o n ( 2 4 ) . C o o r d i n a t i o n o f the metal i o n by pho s p h a t e o c c u r s i n b o t h c a s e s , 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 the 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 which t he T 1 o f 2 ^ T 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 the p r e s e n c e o f the 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 t r e a t m e n t 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 the 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 because o f t h e s e pharma-c 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 V c i y i nl(Ju i uui i C i n "die i jcc i i 'C i i i Oi" tid K - a v » C i v 'aCeu h i r a s e a C C i v i i y . 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 the 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 Pase, 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 he 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 the p h a r m a c o l o g i c a l r e c e p t o r f o r t h e s e 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 m o i e t y i s c l a s s i f i e d i n t o f o u r t y p e s : d i g i t o x i g e n i n , d i g o x i g e n i n , 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 the 33 hy d r o x y l group o f the 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 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 i s a p p r o x i m a t e l y 10 M b u t 7 d i n 4 oH D - g l u c o s i de F i g . 1 S t r u c t u r e o f c a r d i o a c t i v e g l y c o s i d e s 8 + + t h e 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 c a s e s S o u r c e O u t e r m e d u l l a o f dog k i d n e y c o r t e x (27) C a l f h e a r t (28) 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 (26) Cat b r a i n (26) TABLE I I n h i b i t o r N - ( 4 ' - a m i n o - n - b u t y l ) - 3 - a m i n o a c e t y l - s t r o p h a n t h i d i n 4_ d i g o x i n 2_ o u a b a i n 3^  o u a b a i n 3 A p p a r e n t D i s s o c i a t i o n C o n s t a n t 1.92x10~ 9M S t o i chiometr I n h i b i t o r : enzyme 0.8-1,2 0.9 0.5 1.0 ! 1 k . , 4 -Na and d e c r e a s e d by K . A l t h o u g h K r e t a r d s t h e b i n d i n g , i t c a n n o t , n e v e r t h e l e s s , r e v e r s e i t (26,27,28). The mechanism o f b i n d i n g i s s t i l l n o t u n d e r s t o o d . 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 t h e 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 t h e 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 phosphate ( 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 < ouabagenin g l y c o s i d e . I f the 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 the 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- d e o x y g l u c o s i d e = f u c o s i d e < rham n o s i d e . I t was c o n c l u d e d t h a t the 2'a and 3'a o r B-hydroxyl groups o f the s u g a r m o i e t y s t a b i l i z e the b i n d i n g through hydrogen 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 the enzyme t o d i f f e r e n t e x t e n t s f r o m 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 example 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 complete p u r i f i c a t i o n t a k e s 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 p r o c e d u r e s 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 peed w i l l s e d i m e n t membrane fra 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 fra 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 the enzyme, and t h i s a l l o w s t h o s e membrane fra 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 the removal o f l o o s e l y bound membrane p r o t e i n s from the membrane, l e a v i n g the 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 ATPase. 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 Sou r c e O u t e r m e d u l l a o f dog k i d n e y c o r t e x (7) O u t e r m e d u l l a ur uog k i d n e y c o r t e x (13) R e c t a l g l a n d o f dog f i s h (11) 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 (16) P r o c e d u r e D i f f e r e n t i a l c e n t r i f u g a t i o n 'microsomes' c o l l e c t e d a t 50,000 x g Microsomes 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 s t r e n g t h KI t r e a t m e n t o f microsome Microsomes washed w i t h .06% d e o x y c h o l a t e a t 160 mM KC1 Microsomes 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 ) ( i i ) ( i i i ) ( i v ) ( v ) F i n a l y i e l d mg o f p r o t e i n / g o f wet t i s s u e 0.46/150 S p e c i f i c A c t i v i t y ymoles P/ mg p r o t e i n / h 800 ( i ) ( i i ( i i i ( i v ) ( v ) ( v i ) 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 m i c r o s o m e s 1 c o l l e c t e d a t 30,000 x g N a l t r e a t m e n t o f microsomes S o l u t i l i z a t i o n w i t h .35% d e o x y c h o l a t e G l y c e r o l p r e c i p i t a t i o n 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 -c h o l a t e Ammonium s u l f a t e p r e c i p i t a t i o n ( i ) 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 '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 ) Z o n a l c e n t r i g u g a t i o n w i t h 2 t o 22% l i n e a r s u c r o s e g r a d i e n t ( 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 us e d i n s t e a d o f 1.6% 17/100 1,552 19.3/15 1,510 24.5/405 1,260 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 t he 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 p h o s p h a t e 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 o r d i t h i o e r y t h r i t o l f o l l o w e d by d e n a t u r a t i o n by c o a t i n g the p r o t e i n w i t h the 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 the 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 he + + 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 ATPase, 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 the 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 the 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 M o l e c u l a r Weights S u b u n i t S o u r c e Large s u b u n i t ( a ) Small s u b u n i t ( B ) M o l a r r a t i o S t r u c t u r e O u t e r m e d u l l a o f :84,000 57,000. . 1 : 1 a& dog k i dney c o r t e x (15) O u t e r m e d u l l a o f 89,000 56,000 1 : 1 ag dog k i d n e y c o r t e x (13) R e c t a l g l a n d o f 97,000 55,000 2 : 1 a 2 g dog f i s h (11,48) -E l e c t r o p l a x o f 93,500 47,000 2 : 1 . a 2 $ e l e c t r i c e e l (16,48) Ox b r a i n 94,000 53,000 (12) 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 c a r d i o -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 t h e a c t i v e s i t e f o r p h o s p h o r y l a t i o n 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 1,13,15). 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 a l s o 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 g e l 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 t a k e n 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 . T h e r e i s a c l e a r d i s c r e p a n c y i n t h i s m o l a r r a t i o ( l a r g e s u b u n i t t o . 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 . The d i s c r e p a n c y 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 g l y c o p r o t e i n , 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 because 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 g e l s i s a c c o m p l i s h e d 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_ . Na03S ( | * ) s ° 3 -V N(C 2H S) 2 In s l i g h t l y a c i d i c media, t h e 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 t o t h e c h a r g e d c a t i o n i c groups o f t h e p r o t e i n . The p r o t e i n - d y e complex has an a b s o r p t i o n maximum a t 549 nm. B ackground s t a i n i n g o f the b l a n k 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 a r e 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 a r e a s under the peaks then y i e l d s t h e 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 f r o m 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 above 0.8 . 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 Beer'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 . F o r e q u a l w e i g h t s 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, t h e 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 d u r i n g d e s t a i n i n g even under 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 t h e 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 , t h e 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 t h e 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 , b u t 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 s u b s t a n t i a t e t h i s . I f t he 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 t h e 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 2 g enzyme. 15 The p o s s i b i l i t y o f the 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 hich c a u s e d an i n c r e a s e i n the c o n d u c t a n c e o f a b l a c k l i p i d membrane ( 5 2 , 5 3 ) , and c o n s e q u e n t l y c e n t r e s o u r a t t e n t i o n on the mechanism o f c a t i o n t r a n s l o c a t i o n . In the e x p e r i m e n t s e i t h e r a t r y p t i c d i g e s t 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 the 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 c o n d u c t a n c e o f t h e b l a c k l i p i d membrane doped w i t h the p r o t e i n b e i n g h i g h e s t when the 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 to 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 he 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 the 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 hich maximized the c o n d u c t a n c e o f t h e b l a c k membrane, i e . a g 2 ( 5 4 ) . However, i t s h o u l d be f iu tcu ciiat- One S u b u i i i l b UScG iiave been u t i i d l u f c u uy buuiuui u u u e c y l s u l f a t e and 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 ) . L i p i d r e q u i r e m e n t L i p i d s were f o u n d 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 the 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 a l s o l e a d s 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 phospha-t i d y l i n o s i t o l , p h o s p h a t i d y l c h o l i n e , p h o s p h a t i d y l e t h a n o l a m i n e 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 the enzyme. I t i s b e l i e v e d t h a t 16 p h o s p h a t i d y l s e r i n e i s t h e 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 ( 3 1 , 3 2 , 3 3 ) . 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) C H 3 ( C H 2 ) - C - ( C H 2 ) n C 0 0 C H 3 0 N-0 7 n = 3,4,5,6 o r 10 \ I „ m+n = 14 o r 15 c o r r e l a t e d the 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 the membrane phospho-l 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 20°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 t e m p e r a t u r e , the 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 freedom f o r the enzyme t o a c h i e v e t h e n e c e s s a r y c o n f o r m a t i o n 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 which the 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 he 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 the 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 ( f r o m d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y ) i n c r e a s e w i t h i n c r e a -s i n g c h a i n l e n g t h o f the 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 the c r i t i c a l t e m p e r a t u r e and the 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 l o w e r t h a n 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 appear s t h a t the 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 he 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 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 the 'a v e r a g e ' phase t r a n s i t i o n t e m p e r a t u r e o f the b u l k s ample, w h i l e the 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 A f f i n i t y 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 p u r i f i e d . 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 : (1) A s u i t a b l e s t a t i o n a r y phase o r m a t r i x , (2) a l i g a n d m o l e c u l e which can s p e c i f i c a l l y b i n d t h e p r o t e i n , (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 m a t r i x , (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 (5) a method t o e l u t e the bound p r o t e i n . (1) S t a t i o n a r y m a t r i x 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 hich 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 form a l o o s e , porous 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 s t r u c t u r e 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 . A g a r o s e g e l , p o l y a c r y l a m i d e beads and g l a s s beads have been used f o r t h i s purpose ( 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 c o m m e r c i a l l y a v a i l a b l e g e l c o n t a i n i n g 2-6 % a g a r o s e . (2) L i g a n d 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 a n a l o g , 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 ( 6 4 , 7 0 ) . T a b l e IV summarizes some examples. 20 P r o t e i n t o be p u r i f i e d N i c o t i n i c a c e t y l c h o l i n e r e c e p t o r (59) A c e t y l c h o l i n e r e c e p t o r (60) A c e t y l c h o l i n e e s t e r a s e (63) N a + K + - a c t i v a t e d ATPase (62) Myosin (84) G l y c e r o l k i n a s e and (68,85^86) G l y c e r a l d e h y d e 3-phosphate d e h y d r o g e n a s e ( 8 7 ) Rhodopsin (61) TABLE IV L i g a n d C o b r a t o x i n Method o f e l u t i o n hexamethoniurn c h l o r i d e - N H(CH 2) 5-C0NH(CH 2 ) 3 N(CH 3); 1 - m e t h y l - 9 - [ N a - ( E - a m i n o -c a p r o y l ) - y - a m i n o p r o p y ! -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_ 6 - ( p u r i n e 5 ' r i b o s y l -t r i p h o s p h a t e ) - 4 - ( l ,3 d i n i t r o p h e n y l ) t h i o e s t e r (ATP a n a l o g ) 8 s e b a c i c a c i d h y d r a z i d e -ATP j o 6 N - ( 6 - a m i n o h e x y l ) -5 1 - A M P n NAD C o n c a n a v a l i n A NaCl g r a d i e n t decamethomi urn ATP KC1 g r a d i e n t KC1 g r a d i e n t NAD D - g l u c o s e 0 0 0 xfTl f "OP'OPOQH... ur^<^ 0 NH'CCH3 (CH2)2CHCNH(CH2)3NH(CH2)3NH2 0 H O P O K U K U ^ 0 0 0 l /CKJ H H H ^ 0 0 0 / HOPOPVPOCH;,^  UiLo H H H < 10 CO(CH2)eC0NHNH2 OH OH HN(CH2)3N0C(CH2)5i!iH3Br~ 9 0 HO^OC, OH N(CH2)6NH2 HO HO F i g . 2 A f f i n i t y L i g a n d s 11 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 . F o r 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 r e c e p t o r -c o b r a t o x i n complex i s 1.13 x 10~ 9M ( 5 9 ) . L i k e w i s e t h a t - o f R h o d o p s i n -C o n c a n a v a l i n A complex i s 2 x 1 0 ~ 7 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 t h e 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 may 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 m a t r i x backbone, and p e r h a p s by t h e 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 t h e s o l v e n t ( 9 1 ) . \ ) oC V a i2ii K v-o i i n y pin- nig i A 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 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 an a g a r o s e g e l m a t r i x . I t makes use o f the t e r m i n a l 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 t h e h y d r o x y l 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 groups a r e a c t i v a t e d by cyanogen bromide 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 . LOCONH, I ^ i -O-CHN ioH gel fOH matrix BrCN. OH H ?0 +-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 t h e t e r m i n a l NH 2 o f the l i g a n d ( 6 5,66,67). ^•ONH H?N-1igand 0 :-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 n e c e s s a r y 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 c o n d i t i o n s such 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 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 . F o r example, t h e 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 ( 8 4 ) . The c o n c e n t r a t i o n o f 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 . R a f t e r y and S c hmidt (60) 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 t h e b i n d i n g 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 , b i n d i n g 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 t h e l i g a n d . In some c a s e s 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 , the 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 (60,68,84). In most c a s e s 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 ) , o r v i c e v e r s a ( 6 1 , 6 9 ) . A f f i n i t y c hromatography i s n o t 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 the 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 hydros-p 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 he optimum d i m e n s i o n to 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 s e p a r a t e a m i x t u r e o f g l y c o g e n p h o s p h o r y l a s e and g l y c o g e n 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 2) NH 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 hich 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 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 & Gel 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 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 under the 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 which has a s m a l l mass does n o t s e d i m e n t because the 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 i a r g e r o u t 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 can 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 a p p r o x i -m a t e l y 200,000 x g, s e d i m e n t a t i o n o f p r o t e i n m o l e c u l e s becomes o b s e r v a b l e 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 e x p e r i e n c e s 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 the 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 the motion o f any mac 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) : ZFx d t . RT 1 dx = M ( l - v P ) D 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 m a c r o m o l e c u l e f r o m c e n t e r o f r o t a t i o n t = time M = m o l e c u l a r w e i g h t 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 m a c r o m o l e c u l e p. = d e n s i t y o f s o l u t i o n D = d i f f u s i o n c o e f f i c i e n t R = gas c o n s t a n t T = a b s o l u t e t e m p e r a t u r e 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 f o r a p a r t i c u l a r e x p e r i m e n t , 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 s i s d e f i n e d as f o l l o w s 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 s e d i m e n t 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 t o 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 words, 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 m a c r o m o l e c u l e s 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 . 1 dx [2] 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 the d i s t a n c e f r o m 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 the 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 X 2 O J . T h i s means t h a t t he 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 the c e n t r i f u g e t u b e . I f t h e ma c r o m o l e c u l e were i n a homo-geneous 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 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 a t a l l t i m e s d u r i n g 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 a i o n g t n e length o f tne centr iTuge t u b e . Thus, t h e m a c r o m o l e c u l e , 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 c o n c e n t r a t i o n 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 f r i c t i o n and an i n c r e a s i n g buoyancy which combine t o compensate f o r the 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 mac r o m o l e c u l e w i l l s e d i m e n t w i t h a c o n s t a n t v e l o c i t y p r o p o r t i o n a l t o 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 . T h i s i s t h e u n d e r l y i n g 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 . 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 c o n c e n t r a t i o n 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 the 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 c o n c e n t r a t i o n and t h u s v i s c o s i t y , and the d i s t a n c e from the 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 g r a d i e n t . 27 (2) Gel 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 , o r 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 t y p e o f p a r t i t i o n c h r omatography 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 . D u r i n g c h r o m a t o g r a p h y , t h e s o l u t i o n c o n t a i n i n g the m o l e c u l e s 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 the 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 the p o r e s o f the 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 the 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 the 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 the p o r e s , i t w i l l be e l u t e d o u t o f the column a f t e r a volume o f s o l v e n t e q u a l t o the i n t e r s t i t u a l s pace 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 the 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 the 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 F C i s t h e volumn o f t h e g e l b e d . 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 c o e f f i c i e n t 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 - 1 1 a v 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 f l v 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 . T h i s 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 s y m m e t r i c , o r g l o b u l a r p r o t e i n s . L a r g e 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 a r e o f t e n a s y m m e t r i c . 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 p r o t e i n s h o u l d e l u t e e a r l i e r ( s m a l l e r 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 , m o l e c u l a r w e i g h t i s n o t n e c e s s a r y 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 o f S t o k e s ' r a d i u s R g . The S t o k e s ' r a d i u s o f . a n asymmetric m a c r o m o l e c u l e 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 S t o k e s - E i n s t e i n E q u a t i o n . 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 o f t h e i r S t o k e s 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 c an be u s e d t o f i n d t he 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 bromide, 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 Y o r k . 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 (sodium 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 (sodium 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 - a c e t y l g l u c o s a m i n e , a-methyl-D-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, M i c r o c o c u s  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. F l u o r e s c a m i n e ( 4 - p h e n y l s p i r o [ f u r a n - 2 ( 3 H ) , l ' - p h t h a l a n ] - 3 - 3 ' d i o n e ) 31 was o b t a i n e d from Hoffman-La 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 Koch-L i g h t Lab. L t d . , E n g l a n d . - • .. Ammonium mo 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 -phosphate 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 . , Rehovoth, I s r a e l . 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 Co 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 B r -E x p e c t e d 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 the 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 min., f r o z e n i n a d r y i c e - a c e t o n e m i x t u r e , 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 the 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 f o r N a ' V . . ' - a c t i v a t e d ATPase. 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 ( a p p r o x i m a t e l y 3 g) was homogenized i n 45 ml. 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 he p r o p o r t i o n a l i n c r e a s e o r d e c r e a s e 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 i m i d a z o l e , 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 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 . 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 ( I v a n S o r v a l l I n c . , Norwalk, C o n n e c t i c u t ) a t 14,000 r.p.m. (max. 23,600 x g) f o r 30 min. The s u p e r n a t a n t was d i s c a r d e d . The p e l l e t was homogenized i n 45 ml o f 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 was d i s c a r d e d . T h e - p e l l e t - w a s homogenized i n 12.5 ml o f the same b u f f e r 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 I n c . , P a l o A l t o , C a l i f . ) a t 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 d i s c a r d e d . 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 t he "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, Rosenbrough, 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 al b u m i n as t h e s t a n d a r d . A b s o r b a n c e was measured a t 650 nm on a C a r l Z e i s s PMQII s p e c t r o p h o t o m e t e r . 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 uld 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 u s e d . 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 -s p i r o [ f u r a n - 2 ( 3 H ) , 1 ' - p h t h a l a n ] - 3 - 3 ' d i o n e ]Z_, known as f l u o r e s c a m i n e , 35 w i t h p r i m a r y 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 ( 3 5 , 3 6 ) . 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 and 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 a s s a y 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 C u t i vi sou . uin b i c a r b o n a t e , pn u.o mi o r u.uib& (w/v) 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 N a + K + - a c t i v a t e d 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 a t 480 nm and e x c i t a t i o n w a v e l e n g t h a t 390 nm. B o v i n e serum a l b u m i n was used as the s t a n d a r d 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 mM p o t a s s i u m c h l o r i d e , 7.mM magnesium c h l o r i d e , 4 mM ATP ( s o d i u m 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 ml. A c o n t r o l , c o n t a i n i n g a l l the above p l u s TO" 4-M o u a b a i n , was i n c u b a t e d 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 and 30 min., 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 by a d d i n g the enzyme, and t e r m i n a t e d by the 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 the 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 Se p h a r o s e 4B 10 ml o f Seph a r o s e 4B was washed t h o r o u g h l y w i t h w a t e r , s u s p e n d e d 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 the r e s t o f the 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 to t h e Sepha r o s e w i t h s t i r r i n g w h i l e the 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 sodi u m 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 S e p h a r o s e 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 t o 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 added 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 s o d i u m 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 S e p h a r o s e 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 S e p h a r o s e 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 oles) 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 f r o m 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 p r o d u c t . % by wei ght E l e m e n t C H N E x p e c t e d 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 4B S e p h a r o s e 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-t a i n i n g 0.01 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 m i x t u r e 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 S e p h a r o s e . ( 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  d i h y d r a z i d e (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 taken 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 s t i r r e d 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 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 Sepha-r o s e , 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 -dures were c a r r i e d out 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 sodium 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 as d e s c r i b 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 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 ed volumes o f b u f f e r had p a s s e d t h r o u g h , a n o t h e r b u f f e r , c o n t a i n i n g a c o m p e t i t o r f o r the a f f i n i t y l i g a n d 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 i d e n t i c a l to t h e 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 hyl-mannoside, o r 5 inq/mi N-acetylgiucosamine, o r \c mg/mi sodium A i r t o r t h e a f f i n i t y l i g a n d s 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 , o r p e r i o d a t e o x i d i z e d 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 bed 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 s e d i m e n t a t i o n (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 g r a d i e n t 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 the Beckman SW 41 r o t o r w hich was used f o r t h i s s t u d y . The method o f Hans N o l l (43) was used 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 V x ) = m x [ p P " p m ( x ) ] . C 6 ] n t P [ 7 ] where m = — 7 — r x t ( P p - p t ) v i s c o s i t y a t the 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 the 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 f r o m the a x i s o f r o t a t i o n d e n s i t y o f s u c r o s e s o l u t i o n a t the 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 e n s i t y o f the m a c r o m o l e c u l e d i s t a n c e f r o m the a x i s o f r o t a t i o n v i s c o s i t y o f the s u c r o s e s o l u t i o n a t x d e n s i t y o f s u c r o s e s o l u t i o n a t 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 the b e g i n n i n g o f the 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 he 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 the 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 the 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 3 and 2.073 c e n t i -p o i s e ( 4 4 ) , r e s p e c t i v e l y , 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 i n t o e q u a t i o n [ 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 nm(x) p m ( x ) 42 the v i s c o s i t y and 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 ( x ) a n d 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 T a b l e V. TABLE V S u c r o s e Cone. pm(x) n m ( x ) , » % (w/v) (g/cm 3) ( c e n t i p o i s e ) p p " p m ( x ) x (cm) x ~ V c m ; Volume (ml) 10 1.041 2.073 0.302 7.00 0.00 0.000 11 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 i n i n o n o n n i n o / ^ o - I I / ^ T /I C " » "7 * n 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 There i s a d i r e c t r e l a t i o n s h i p between the d i s t a n c e x and the 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 the m a n u f a c t u r e r i s 1 .605 cm2-, so t h a t Volume = 1.605 (x - x t ) ml o r V = 1.605 (x - 7.0) ml. ' [ 8 ] . T h i s c o r r e s p o n d i n g 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. The s o l i d l i n e o f F i g . 3 shows the shape o f the 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 ) s i m u l a t i o n (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 needed. 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 convex 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 g r a d i e n t 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. F i g . 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 = c o n c e n t r a t i o n i n r e s e r v o i r S u c r o s e s o l u t i o n i s pumped from t he m i x i n g chamber and d e l i v e r e d t o the 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 t h e m i x i n g 45 chamber, then the 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 to 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 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 the 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 ) . volume V has been pump out o f the m i x i n g chamber. The p a r a m e t e r s C 2 , C-j and V-| d e t e r m i n e t h e a c t u a l shape o f the g e n e r a t e d c o n c e n t r a t i o n g r a d i e n t . These a r e t o be a d j u s t e d so t h a t e q u a t i o n [ 9 ] s i m u l a t e s the 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 the 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 2 and V-| were f o u n d by s u b s t i t u t i n g the f i f t h and e i g t h row o f d a t a from T a b l e V i n t o e q u a t i o n [ 9 ] . T h i s g i v e s 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 = C 2 - ( C 2 - C,) exp ( V / V 1 ) [ 9 ] 17 = C 2 - ( C 2 -10) exp (-5.26/V-,) [ 1 0 ] 23 = C 2 - ( C 2 -10) exp (-13.10/V-j) [1 1 ] 46 S o l v i n g e q u a t i o n [ 1 0 ] and [11] 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 = 29.3 V, = 11.7 S u b s t i t u t i n g t h e s e v a l u e s i n t o e q u a t i o n [ 9 ] C = 29.3 - 19.3 exp(-V/11.7) [ 1 2 ] E q u a t i o n [ 1 2 ] d e f i n e s t he 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 the r e s e r v o i r . 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 a p p r o x i m a t i o n . (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  c o e f f i c i e n t 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 to 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 ATPase, as o u t l i n e d i n page 33 to 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 each 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-c a l c u l a t e d from e q u a t i o n [ 1 2 ] . I t can 47 d r o g e n a s e , c a t a l a s e and lysozyme 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 the 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 the 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 and 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 min. 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 d e h y d r o g e n a s e , c a t a l a s e , lysozyme and Na K - a c t i v a t e d ATPase a c t i v i t y . A l c o h o l d ehydrogenase a s s a y (79) : Samples o f 25 to 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 and 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 (80) : 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 pho 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 ( P h a r m a c i a ) 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 d e t e r m i n e d 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 al b u m i n 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 f r o m 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 ATPase, 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 . 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 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 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) D e t e r g e n t 0.1%(w/v) 1.0%(w/v) T r i methyldodecylammoni urn bromi de 0.03 0.00 T r i t o n X-100 0.09 0.01 Sodium d e o x y c h o l a t e 0.15 0.00 L u b r o l WX 0.34 0.96 (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 the 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 t h e 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 a r e 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 t h e 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 i m m e d i a t e l y a f t e r e x t r a c t i o n . (2) A f f i n i t y chromatography 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 hicn the S e p h a r o s e 46 was n o t l i n k e d t o 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 ml. (3) V e l o c i t y s e d i m e n t a t i o n 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 . 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 s u c r o s e g r a d i e n t . 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 s o f the t h r e e s t a n d a r d p r o t e i n s (44) : c a t a l a s e , a l c o h o l d e hydrogenase and lysozyme 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 d i s t a n c e o f m i g r a t i o n , t a k i n g the 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 the 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 the s t a n d a r d i s i n d i c a t e d by t h e arrow. 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 u c i i' i i i rd i i On Oi iia k - a t i l V a c e u M I r a s e aim i uUNtiS ' rriu'l US c a l i b r a t i o n 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' s e c t i o n . 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 ATPase, and the t h r e e s t a n d a r d p r o t e i n s : 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 al 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 i n d i c a t e s 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 to 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 l o g a r i t h m (base 10) o f t h e S t o k e s ' r a d i u s ( R g ) o f t h e t h r e e s t a n d a r d p r o t e i n s (44) i n Angstroms was p l o t t e d a g a i n s t t h e i r p a r t i t i o n c o e f f i -c i e n t s . They a re 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 the arrow. 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 Combining e q u a t i o n [ 3 ] and [ 5 ] , M = 6 s N l T 1 l R e [ 1 3 ] 1 - 7p Siihst.i t.uti nn t.hp fnllowinn into pnuation [1.^] s = 5.0 x 1 0 " 1 3 s e c n = 0.0102 p o i s e ( v i s c o s i t y o f w a t e r a t 20°C) v = 0.73 cm 3 g " 1 (assumed) P = 1.00 g cm" 3 R„= 1.14 x 1 0 " 6 cm e g i v e s M = 240,000 umoles o f p h o s p h a t e p e r hour ( D-CW3 ) umoles o f p h o s p h a t e p e r h o u r (D-D-Q) HQ cn < << O fD -S o co -o m -a -s BJ X fD o + r+ O c+ -s + CU -h 1 o a r+ n o — i . c+ O 3 o t> < <+ T CU Cu — i . ji rt- l/i r -< T fD —i. 1 ex. Cu cr. c+ & :-S J = - h O — ^ —1 c: — ' •o '3 „ — ^ Cu O (/> r+ X fD 1 O o Cu 3 o I o 3 1 c+ O o O * 1. -+) fD — . < 3 —1. r - c+ r+ c -s << cr CU -s ^ <-!-o i . .,—, . 1 a — 1 X | o 1 o s: ^ ^ o < fD - ' 3 r+ -s • Cu rt . i . O 3 3 r+ 3" fD •a' -s fD Ln fD 3 O fD O i 1 1 i 1 r 1 1 — T r o • o r o o \ \ V \ . 1 1 n \ ° 'v V. - 1 t 1 1 i ' t > t t 1 I I 1 o OJ _ • — i r o co -P» 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) « • < » i 1— • — — — — ' o o o o r o co mg o f p r o t e i n (A-A—A ) umoles o f phosphate p e r h o u r (U—•O—Q) 99 57 Days F i g . 9 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 w i t h o u t g l y c e r o l (Q ~ U — O ) 5%(v/v) g l y c e r o l ( o-O-o ) 10%(v/v) g l y c e r o l (jHHi) 2 0 % ( v / v ) g l y c e r o l 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 as c o n t r o l f o r a f f i n i t y chromatography 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 5.0 4.0 2.0 1.0 0.0 1—9-4—« F i g , D-mannoside 0.20 15 20 25 30 0.15 0.10 CD o s-Q. O CD 0.05 0.00 5 10 F r a c t i o n number 11 C o n c a n a v a l i n A 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--o) 60 I 9 S-13 O s-<D Q-<D 4-> <B J = Q-10 o to CD o E 3. o 7.0 h 6.0 > 5.0 I 4.0 3.0 »• 2.0 I 1.0 h 0.0 0.20 0.15 0.10 £ N - a c e t y l g l u c o s a m i n e +J o s-C L O 0.05 A 5 10 F r a c t i o n number 15 20 25 30 0.00 F i g . 12 Wheat germ a g g l u t i n i n a f f i n i t y chromatography N a + K + - a c t i v a t e d ATPase a c t i v i t y ( » — 9 ) 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) 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 chromatography N a f 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) 62 cu 10 CD <a (/> I— c e t a) CJ) - o o CD S-+-> - o > s= • I - CD o (O i — I o + -!= o + o A 3 i — E E cu o c _Q i-O (/) 0.15 h 0.10 0.05 r 0.00 10 15 20 25 R e l a t i v e d i s t a n c e 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 C a t a l a s e ( O H C H 3 ) A l c o h o l dehydrogenase N a + K + - a c t i v a t e d ATPase_. ({MM?) Lysozyme (O°0H?) 63 64 E c LO CM +J ra a> u c ro S_ O </) -Q 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.5 0.4 ii) 0.3 o. cu 4-> rC - E CL co O 0.2 °-o.i i . > . l . r « - » ' ' • I I I » t I I I I I t I -I 50 60 70 80 90 100 110 120 130 140 150 Volume (ml) 0.20 0.18 0.16 0.14 0.12 0.10 -J 0.08 -t 0.0b 0.04 0.02 l0.00 o CO CNJ +-> rO CD U c rO O IO F i g . 16 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 & s t a n d a r d p r o t e i n s 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) 65 2.5 2.0 CD 1.5 1,0 JL N a + K + - a c t i v a t e d ATPase f i b r i n o g e n a p o f e r r i t i n b o v i n e serum albumin i I I i i I I 1 1 1 1 1 1 L 0.0 0.1 0.2 0.3 . 0.4 0.5 0.6 K a v -0.7 0.8 F i g . 17 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 c o e f f i c i e n t 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 V I , i t i s o b v i o u s t h a t o u t o f t h e f o u r d e t e r g e n t s 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 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 from t h e 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 t r i m e t h y l d o d e c y l -ammonium bromide was n o t 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 be 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 -p i 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 ^ L i 1 L I V> t O * , i i C . I i h U H U i U i k u u U u l > d p i U l l l J • u U U i J I ««/% H U O 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 t h a t a d e t e r g e n t 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 a t h i g h e r c o n c e n t r a t i o n s ( 9 3 ) . T h us, 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 , b u t 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 appears 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 i s a p p r o x i m a t e l y 2% (w/v). Hence 1.5% (w/v) was chosen f o r r o u t i n e e x t r a c t i o n . ' 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 , b u t 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 no t s i g n i f i c a n t l y change t h e e x t r a c t i o n . In o r d e r t o f i n d t h e optimum c o n c e n t r a t i o n o f 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 o f the enzyme e x t r a c t e d i n d i f f e r e n t c o n c e n t r a t i o n s o f 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 t h e 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 , t h e a c t i v i t y f a l l s t o l e s s t h a n h a l f t h e o r i g i n a l v a l u e upon s t o r a g e 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 , b u t t h e r e i s no d e t e c t a b l e improvement i n s t a b i l i t y f r o m 10% t o 20%. Hence 10% ( v / v ) g l y c e r o l was (2) A f f i n i t y c hromatography 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 a r e h a e m a g g l u t i n a t i n g a g e n t s known t o 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 o r 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 odopsin ( 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 s t r o n g l y ( 9 5 ) . S i n c e N a + K + -a c t i v a t e d ATPase i s known t o 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 t h e c a r b o h y d r a t e p o r t i o n might 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 t h e n o n r e d u c i n g end and be a c c e s s i b l e t o 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 the 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 the 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 the 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 ght 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 a c t i v i t y . As shown i n F i g . 135 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 ATPase. 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 the l i g a n d . I n o r g a n i c p hosphate 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 the g e l r e c o v e r e d a f t e r the 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 . These 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 the 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 th 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 In-a c t i v a t e d A'lrase i s a r e l a t i v e l y coy mine Ir i c p t u l c i n . Aa co inpui ' ; 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 5 1 5 , 3 0 0 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 ( 4 4 ) , c o n s i s t e n t l y 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 S e p h a r o s e 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 2 4 0 , 0 0 0 f o r Na K - a c t i v a t e d ATPase e s t i m a t e d f r o m 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 by 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 2 5 0 , 0 0 0 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 on the a s s u m p t i o n o f an azB s u b u n i t s t r u c t u r e ( 1 1 , 1 6 , 4 8 ) , 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 m e n t i o n e d above s u g g e s t s t h a t a 2B 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 i n s o l u t i o n . 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 . S c i . USA 66, 936 (1970) (5) Dunham P. B., Hoffman J . F., J . Gen. P h y s i o l . 58, 94 (1971) (6) B o n t i n g S. L., Simon K. A., Hawkin N. M., A r c h . Biochem. B i o p h y s . 95, 416 (1961) </) Kyte J . , J . B i o l . Chem. ^ 4 b , 415/ (19/1) (8) B o n t i n g S. L., C a r a v a g g i o L. L., Candy M. R., Hawkin N. M., A r c h . Biochem. B i o p h y s . J 0 6 , 49 (1964) (9) B o n t i n g S. L., Comp. 'Biochem. P h y s i o l . J_7, 953 (1966) (10) B u r g e r J . W., Hess W. N., S c i e n c e J31_, 670 (1960) ( I T ) H o k i n E. L., Dahl J . L., Deupree J . D., Dixon J . F., Hackney J . F., Perdue J . F., J . B i o l . Chem. 248, 2593 (1973) (12) Uesugi S., Dulak N. C , Dixon J . F., HexumT., Dahl J . L . , Perdue J . T., H o k i n L. E., J . B i o l Chem. 246, 531 (1970) (13) Lane L. K., Copenhaven J . H., Lindenmayer G. E., S c h w a r t z A., J . B i o l . Chem. 248, 7197 (1973) • ~ -(14) Ro.dbard K., Chrambach A., A n a l . Biochem. 40, 95 (1971) (15) Kyte J . , J . B i o l . Chem. 247, 7642 (1972) 72 (16) Dixon J . F., Hokin L. E . , A r c h . Biochem. B i o p h y s . 163, 749 (1974) (17) Ruoho A., Kyte J . , P r o c . N a t . Acad. S c i . USA 71_, 2352 (1974) (18) Kyte J . , Biochem. B i o p h y s . Res. Commun. 43, 1259 (1971). (19) S i e g e l G. J . , Goodwin B., J . B i o l . Chem. 247, 3630 (1972) (20) Ro b i n s o n J . D., Biochem. 6, 3250 (1967) (21) Masiak S. J . , Green J . W., B i o c h i m . B i o p h y s . A c t a 159, 340 (1968) (22) A l b e r s R.W., Fahn S., Kova l G. J . , P r o c . N a t . A c a d . S c i . USA 50, 474 (1963) (23) B l o s t e i n R., Biochem. B i o p h y s . Res. Commun. 24, 598 (1966) (24) Gr i s h a m C. M., M i l d v a n A. S., J . B i o l . Chem. 249, 3187 (1974) (25) O s t r o y F., James T. L., Noggle J . H., S a r r i f A., H o k i n E. H., A r c h . Biochem. B i o p h y s . 162, 421 (1974) (26) A l b e r s R. W., Koval G. J . , S i e g e ! G. J . , MoT. Phami 4, 324 (1968) (27) Kyte J . , J . B i o l . Chem. 247, 7634 (1972) (28) M a t s u i H., S c h w a r t z A., B i o c h i m . B i o p h y s . A c t a 15J_, 655 (1968) (29) P e r r o n e J . R., B l o s t e i n R., B i o c h i m . B i o p h y s . A c t a 291, 680 (1973) (30) J a r n e f e l t J . B i o c h i m . B i o p h y s . A c t a 266, 91 (1972) (31) Tanaka R., S t r i c k l a n d K. P., A r c h . Biochem. B i o p h y s . 1J1, 583 (1965) (32) S t a h l W., A r c h . Biochem. B i o p h y s . 154, 56 (1973) (33) Hokin L. E., Hexum T. D., A r c h . Biochem. B i o p h y s . 151, 4 5 3 (1972) (34) Lowry 0. H., Rosenbrough N. J . , F a r r A. L., R a n d a l l R. J . , J . B i o l . Chem. 193, 265 (1951) (35) W e i g e l e M., De B e r n a r d o S., T e n g i J . , L e i m g r u b e r W., J , Amer. Chem. Soc. 94, 5927 (1972) 73 (36) U n d e n f r i e n d S., S t e i n S., B o h l e n P., Dairman W., Leimgruben W., Wei g e l e M., S c i e n c e 178, 871 (1972) (37) See Y. P., F i t t P. S., A n a l . Biochem. 49, 430 (1972) (38) C u a t r e c a s a s P., J . B i o l . Chem. 245, 3059 (1970) (39) S a t a k e K., Okuyama T., Ohashi M., Tomotaka S., J . Biochem. Japan 47, 654 (1960) (40) Lamed R., L e v i n U., W i l c h e k M., B i o c h i m . B i o p h y s . A c t a 304, 231 (1973) (41) G i l h a m P. T., Methods Enzymol. 21_, 191 (1971) (42) M c C a l l J . S., P o t t e r B. J . : " U l t r a c e n t r i f u g a t i o r t " B a i l l i e r e T i n d a l l , London, 1973. (43) N o l l H., N a t u r e 2j_5, 360 (1967) (44) Sober H. A. : "CRC Handbook o f B i o c h e m i s t r y " 2nd e d . , The C h e m i c a l Rubber Co., C l e v e l a n d , 1970. (45) Bock R. M., L i n g N. S., A n a l . Chem. 26_, 1543 (1954) (46) Grisham C. M., Gupta R. K., B a r n e t t R. E., M i l d v a n A. S., J . B i o l . Chem. 249, 6738 (1974) (47) Yoda A., Yoda S., Mol. Pharm. 10, 494 (1974) (48) Dahl J . , Hokin L. E., Ann. Rev. Biochem. 43, 327 (1974) (49) Fazekas de S t . G r o t h S., Webster R. G., D a t y n e r A., B i o c h i m . B i o p h y s . A c t a 71, 377 (1963) (50) Schnaitman C. A., P r o c . Nat. Acad. S c i . USA 6_3, 412 (1969) (51) Fessenden-Raden J . M., Biochem. B i o p h y s . Res. Commun. 46_, 1347 (1972) (52) Shamoo A. E., Myers M. M., B l u m e n t h a l R., A l b e r s R. W., J . Membrane B i o l . 19, 129 (1974) (53) B l u m e n t h a l R., Shamoo A. E., J . Membrane B i o l . 19_, 141 (1974) 74 (54) Shamoo A. E . , Myers M., J . Membrane B i o l . J_9, 163 (1974) (55) G r i s h a m C M . , B a r n e t t R. E . , Biochem., ]Z, 2635 (1973) (56) K i m e l b e r g H. K., P a p a h a d j o p o u l o s K., J . B i o l . Chem. 249, 1071 (1974) (57) C u a t r e c a s a s P., J . B i o l . Chem. 245, 3059 (1970) (58) w e i b e l M. K., W e e t a l l H. H., B r i g h t H. J . , Biochem. B i o p h y s . Res. Commun. 44, 347 (1971) (59) K l e t t R. P., F u l p i u s B. W., Cooper D. } Smith M,, R e i c h E . , P o s s a n i L. D., J . B i o l . Chem. 248, 6841 (1973) -(60) S c h m i d t J . , R a f t e r y M. A., Biochem. 1_2, 852 (1973) (61) Steinemann A., S t r y e r L., Biochem. 1_2, 1499 (1973) (62) A n d e r s o n B. H., H u l l a F. W., F a s o l d H., White H. A., FEBS L e t t e r s 37, 338 (1973) (63) Dudai Y., S i l m a n I . , S h i n i t z k y M., Blumberg S., P r o c . N a t. A c a d . S c i . USA 69, 2400 (1972) ' (64) A s p b e r g K., P o r a t h J . , A c t a Chem. S c a n d . '24, 1839 (1970) (65) Axen R., P o r a t h J . , E r n b a c k S., N a t u r e 21_4, 1302 (1967) (66) P o r a t h J . , Axen R., E r n b a c k S., N a t u r e 21_5, 1491 (1967) (67) Axen R., E r n b a c k S., E u r . J . Biochem. 1_8, 351 (1971 ) (68) Lowe C. R., Harvey M. J . , Dean P. D. G., E u r . J . Biochem. 41_, 341 (1974) (69) Shaper J . H., B a r k e r R., H i l l R. L., A n a l . Biochem. 53_, 564 (1973) (70) C u a t r e c a s a s P., A n f i n s e n C. B., Ann. Rev. Biochem. 40_, 753 (1971 ) (71) H o f s t e e B. H. J . , A n a l . Biochem. 52,. 430 (1973) (72) S h a l t i e l S., E r - e l Z v i . , P r o c . Nat. A c a d . S c i . USA 70, 778 (1973) (73) T a n f o r d C , N o z a k i Y., Re y n o l d s J , A., Makino S., Biochem. 13. 2369 (1974) 75 (74) A r c h i l b a l d J . T., White T . D., N a t u r e 252, 595 (1974) (75) L o c a s c i o G. A., T i g i e r H. A., B a t t l e A. M. de C , J . Chromatogr. 40, 453 453 (1969) -~ ^ (76) Lehmann G. G., C l i n . Chim. A c t a 28, 335 (1970) (77) S i e g e l L. M., Monty K. J . , B i o c h i m . B i o p h y s . A c t a 226, 346 (1966) (78) Demassieux S., L a c h a n c e J - P . , J . Chromatogr. 89_, 251 (1974) (79) M a s s o u l l i e J . , R i e g e r F., E u r . J . Biochem. JJ_, 441 (1969) (80) Chance K. D., B.Sc. T h e s i s , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1974. (81) H a r t W. M., T i t u s E. 0., J . B i o l . Chem. 248, 4674 (1973) (82) So L. L., G o l d s t e i n I . J . , B i o c h i m . B i o p h y s . A c t a 165, 398 (1968) (83) L l o y d K., A r c h . Biochem. B i o p h y s . 137, 460 (1970) (84) Lamed R., O p l a t k a A., Biochem. 13_, 3137 (1974) (85) Craven D. B., Harvey M. J . , Lowe C. R., Dean P. D. G., E u r . J . Biochem. 4J_, 329 (1974) (86) Harvey M. J . , Lowe C. R., Craven D. B., Dean P. D. G., E u r . J . Biochem. 4]_, 335 (1974) (87) H o c k i n g J . D., H a r r i s J . I . , FEBS L e t t e r s 34, 280 (1973) (88) Harvey M. J . , Lowe C. R., Dean P. D. G., E u r . J . Biochem. 41_, 353 (1974) (89) Lowe C. R., Harvey M. J . , Dean P. D. G., E u r . J . Biochem. 41_, 347 . (1974) (90) B u r g e r M. M., G o l d b e r g A. R., P r o c . Nat. Acad. S c i . USA 57, 359 (1967) (91) C u a t r e c a s a s P., A n f i n s e n C , Methods Enzymol. 22, 345 (1971) (92) H o l u b i t s k y D., Morrod P., p e r s o n a l communications (93) B a n e r j e e S. P., Dwosh L. L., Khanna V. K., Sen A. K., B i o c h i m . B i o p h y s . A c t a 211, 345 (1970) 76 (94) G o l d s t e i n I. J . , Holler-man C. E., Smith E . E., Biochem. 4, 876 (1965) (95) W i e d i n e r T., G e n t i n e t t a R., Brodbeck U., FEBS L e t t e r s 47, 260 (1974) 

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