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Synthesis of affinity derivatives of ouabain and their interaction with the sodium pump MacKenzie, Donald J. 1980

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SYNTHESIS OF A F F I N I T Y DERIVATIVES OF OUABAIN AND THEIR INTERACTION WITH THE SODIUM PUMP by D o n a l d J . M a c K e n z i e B . S c . 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 , 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the FACULTY OF GRADUATE STUDIES D e p a r t m e n t o f B i o 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 a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d f o r t h e d e g r e e o f MASTER OF SCIENCE THE UNIVERSITY OF B R I T I S H COLUMBIA MAY 19 8 0 ©Donald J . MacKenzie, 1980 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department nf Biochemistry The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date March 31, 1980. i i ABSTRACT: A f f i n i t y d e r i v a t i v e s o f t h e c a r d i a c g l y c o s i d e , o u a b a i n , w e r e s y n -t h e s i z e d and c h a r a c t e r i z e d w i t h r e s p e c t t o t h e i r i n t e r a c t i o n s w i t h N a , K A T P a s e . T h e r h a m n o s e s u g a r m o i e t y o f o u a b a i n was s u b j e c t e d t o p e r i o d a t e o x i d a t i o n and t h e r e s u l t i n g a l d e h y d e g r o u p s d e r i v a t i z e d t h r o u g h S c h i f f b a s e f o r m a t i o n w i t h n u c l e o p h i l i c amino r e a g e n t s . S u b s e q u e n t r e d u c t i o n w i t h b o r o h y d r i d e y i e l d e d c h e m i c a l l y s t a b l e c o n j u g a t e s w h i c h c o u l d be i s o l a t e d i n p u r e f o r m . O u a b a i n was c o v a l e n t l y l i n k e d t o c o p o l y m e r m e t h a c r y l a t e m i c r o s p h e r e s v i a a h e p t a n e - 1 , 7 - d i a m i n e s p a c e r a r m . The o u a b a i n - m i c r o s p h e r e c o n j u g a t e s w e r e i n h i b i t o r s o f b e e f b r a i n N a , K A T P a s e a c t i v i t y w i t h an a p p a r e n t K . o f 6 ° 3 . 0 X10~ M ( f o r 200 A d i a m e t e r m i c r o s p h e r e s ) , w i t h r e s p e c t t o t h e i m m o b i -l i z e d g l y c o s i d e c o n c e n t r a t i o n . F l u o r e s c e n t l y t a g g e d o u a b a i n - m i c r o s p h e r e c o n j u g a t e s c o u l d n o t be d e m o n s t r a t e d t o b i n d t o H e l a c e l l s , e i t h e r by f l u o r e s c e n c e m i c r o s c o p y o r r a d i o i m m u n o a s s a y . O u a b a i n was a l s o d e r i v a t i z e d w i t h t h e f l u o r o c h r o m e D A N S - l y s i n e and t h e r e s u l t i n g c o n j u g a t e d i s p l a y e d a K. f o r t h e i n h i b i t i o n o f b e e f b r a i n N a , K A T P a s e o f 7 . 5 X 10~ ^ M. N a t i v e o u a b a i n was shown t o h a v e a K i o f 6 . 5 X Q 10 M f o r t h e i n h i b i t i o n o f b e e f b r a i n N a , K A T P a s e . H a l f - m a x i m a l p r o t e c -q t i o n a g a i n s t t h e b i n d i n g o f ( H ) - o u a b a i n t o b e e f b r a i n m i c r o s o m a l membranes c o u l d be p r o v i d e d by e i t h e r 3 . 0 X 1 0 " 7 M D A N S - l y s - o u a b a i n o r 3 . 0 x 1 0 " 8 M u n t r e a t e d o u a b a i n . S t u d i e s on t h e b i n d i n g o f ( H ) - o u a b a i n t o H e l a c e l l s i n d i c a t e d t h a t 5 t h e s e c e l l s p o s s e s s a b o u t 2 . 5 x 10 o u a b a i n b i n d i n g s i t e s w i t h a s i n g l e P d i s s o c i a t i o n c o n s t a n t o f 5 . 7 x 10 M. T h e b i n d i n g o f D A N S - l y s - o u a b a i n t o i i i H e l a c e l l s c o u l d n o t be d i r e c t l y d e m o n s t r a t e d u n d e r t h e f l u o r e s c e n c e m i c r o s c o p e and a r a d i o i m m u n o a s s a y u t i l i z i n g ( I ) - a n t i - D A N S IgG a l s o g a v e n e g a t i v e r e s u l t s i n t h i s r e g a r d . A h a p t e n s a n d w i c h l a b e l l i n g t e c h n i q u e u s i n g a n t i - D A N S a n t i b o d i e s and DANS d e r i v a t i z e d i r o n m i c r o s p h e r e s was d e v e l o p e d t o d e m o n s t r a t e t h e d i s t r i b u -t i o n o f DANS d e r i v a t i z e d c e l l s u r f a c e p r o b e s a t t h e l e v e l o f t h e s c a n n i n g e l e c t r o n m i c r o s c o p e , and t h e u s e o f X - r a y m i c r o p r o b e a n a l y s i s w i t h r e s p e c t t o c e l l s u r f a c e l a b e l l i n g was i n v e s t i g a t e d . As an e x a m p l e o f t h e h a p t e n s a n d w i c h l a b e l l i n g t e c h n i q u e , mouse f i b r o b l a s t c e l l s w e r e i n i t i a l l y l a b e l l e d w i t h D A N S - r i c i n . C e l l s w e r e t h e n l a b e l l e d s e q u e n t i a l l y w i t h a n t i - D A N S IgG f o l l o w e d by DANS d e r i v a t i z e d i r o n m i c r o s p h e r e s . L a b e l l e d c e l l s w e r e t h e n v i s u a l i z e d i n t h e SEM u s i n g t h e s e c o n d a r y e l e c t r o n mode o r by a n a l y s i s o f e n e r g y d i s p e r s e d X - r a y s . i v ACKNOWLEDGEMENT I would l i k e to acknowledge the e x c e l l e n t support and s u p e r v i s i o n provided by Dr. R. S. Molday. I would a l s o l i k e to extend my thanks to the other members of our l a b f o r t h e i r worthwhile advice on many p r a c t i c a l matters. V L I S T OF ABBREVIATIONS ADP ATP BSA Con A DANS EDC EDTA FITC HEPES N a , K A T P a s e NEM PBS pNPP RBC SEM SDS TLC T R I S a d e n o s i n e d i p h o s p h a t e a d e n o s i n e t r i p h o s p h a t e b o v i n e s e r u m a l b u m i n c o n c a n a v a l i n A 5 - d i m e t h y l a m i n o - 1 - n a p h t h a l e n e s u l f o n y l 1 - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) c a r b o d i i m i d e e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d f l u o r e s c e i n i s o t h i o c y a n a t e N - 2 - h y d r o x y t h y l - N ' - 2 - e t h a n e s u l f o n i c a c i d s o d i u m and p o t a s s i u m 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 N - e t h y l m a l e i m i d e p h o s p h a t e b u f f e r e d s a l i n e p - n i t r o p h e n y l p h o s p h a t e r e d b l o o d c e l l s c a n n i n g e l e c t r o n m i c r o s c o p e s o d i u m d o d e c y l s u l f a t e t h i n l a y e r c h r o m a t o g r a p h y t r i s - ( h y d r o x y m e t h y l ) amino m e t h a n e v i TABLE OF CONTENTS PAGE T I T L E PAGE i ABSTRACT i i ACKNOWLEDGMENT i v L I S T OF ABBREVIATIONS v TABLE OF CONTENTS v i L I S T OF TABLES * L I S T OF FIGURES x i PART I : CHEMICAL DERIVATIVES OF OUABAIN INTRODUCTION 1 ( I ) T h e S o d i u m Pump 1 A . I o n T r a n s p o r t Modes 1 1 . N o r m a l Mode 2 2 . R e v e r s e d Mode 2 3 . N a - N a E x c h a n g e 3 4 . K - K e x c h a n g e 3 5 . U n c o u p l e d Na E f f l u x 4 B . The R o l e o f P h o s p h o r y l a t e d I n t e r m i d i a t e s i n t h e N o r m a l W o r k i n g o f t h e Pump 5 C . I n t e r a c t i o n o f C a r d i a c G l y c o s i d e s w i t h t h e S o d i u m Pump 6 D. P h y s i c a l P r o p e r t i e s o f t h e S o d i u m Pump 8 E. A n t i g e n i c i t y o f t h e S o d i u m Pump 14 ( I I ) P r e v i o u s M e t h o d s f o r t h e C y t o c h e m i c a l D e m o n s t r a t i o n o f N a , K A T P a s e 15 v i i TABLE OF CONTENTS ( c o n t ' d ) PAGE ( I I I ) R o l e o f C h e m i c a l D e r i v a t i v e s o f O u a b a i n i n t h e D e m o n s t r a t i o n o f t h e S o d i u m Pump 17 A . C h e m i c a l D e r i v a t i v e s o f O u a b a i n 17 B . T h e s i s 19 EXPERIMENTAL PROCEDURES 20 ( 1 ) M a t e r i a l s 20 ( 2 ) T i s s u e C u l t u r e 2 0 ( 3 ) A n a l y t i c a l P r o c e d u r e s 21 ( 4 ) O x i d a t i o n o f O u a b a i n 22 ( 5 ) P r e p a r a t i o n o f DANS L y s i n e O u a b a i n 22 ( 6 ) I m m o b i l i z a t i o n o f O u a b a i n on C o p o l y m e r M i s c r o s p h e r e s 23 ( 7 ) Enzyme P r e p a r a t i o n 24 ( 8 ) A T P a s e A c t i v i t y D e t e r m i n a t i o n 25 ( 9 ) B i n d i n g S t u d i e s 26 RESULTS 28 ( 1 ) P r e p a r a t i o n o f O u a b a i n D e r i v a t i v e s 28 A . P e r i o d a t e O x i d a t i o n 28 B . O u a b a i n M i c r o s p h e r e c o n j u g a g e s 28 C . D a n s - L y s - O u a b a i n D e r i v a t e s 32 v i i i TABLE OF CONTENTS ( c o n t ' d ) PAGE ( 2 ) B i n d i n g S t u d i e s 37 A . ( 3 H ) - 0 u a b a i n B i n d i n g D a t a 37 B . O u a b a i n - M i c r o s p h e r e C o n j u g a t e s 41 C . D a n s - L y s - O u b a i n 43 D. C e l l L a b e l l i n g S t u d i e s 46 DISCUSSION 47 (A) O u a b a i n - M i c r o s p h e r e C o n j u g a t e s 47 ( B ) D a n s - L y s - O u a b a i n 47 ( C ) O t h e r C o n s i d e r a t i o n s 4 8 ix TABLE OF CONTENTS ( c o n t ' d ) PAGE PART I I : HAPTEN SANDWICH LABELLING METHODOLOGY INTRODUCTION 50 A . The L a b e l 5 0 B . T h e M a r k e r 51 C . The H a p t e n S a n d w i c h T e c h n i q u e 52 EXPERIMENTAL PROCEDURES 54 ( 1 ) H a p t e n - M o d i f i e d L e c t i n s 54 ( 2 ) P r e p a r a t i o n o f D A N S - L y s i n e - S e p h a r o s e 54 ( 3 ) P r e p a r a t i o n and P u r i f i c a t i o n o f R a b b i t a n t i - D A N S Immune Serum 55 ( 4 ) R a d i o i m m u n o a s s a y 56 ( 5 ) D i r e c t V i s u a l i z a t i o n o f Bound A n t i - H a p t e n A n t i b o d y 57 ( 6 ) P r e p a r a t i o n o f t h e H a p t e n M o d i f i e d M a r k e r 58 ( 7 ) H a p t e n S a n d w i c h L a b e l l i n g 59 RESULTS AND DISCUSSION 60 ( 1 ) R a d i o i m m u n o a s s a y 60 ( 2 ) B i n d i n g o f FITC C o n j u g a t e d A n t i - D A N S IgG 60 ( 3 ) H a p t e n S a n d w i c h M e t h o d o l o g y 62 DISCUSSION 71 BIBLIOGRAPHY 72 X LIST OF TABLES PAGE TABLE I Comparison o f the d i s s o c i a t i o n c o n s t a n t s o f o u a b a i n - r e c e p t o r complexes and o u a b a i n c o n c e n t r a t i o n s c a u s i n g h a l f maximal i n h i b i t i o n o f Na,K ATPase TABLE I I S t r u c t u r e o f some c a r d i a c m o n o g l y c o s i d e s TABLE I I I S t r u c t u r e o f t h e a g l y c o n e s w h i c h form s t e r o i d m o i e t i e s o f g l y c o s i d e s l i s t e d i n T a b l e I 9 10 12 x i LIST OF FIGURES PAGE FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: FIGURE: 1 S t r u c t u r e s o f sugars l i s t e d i n T a b l e I I 2 G e n e r a l i z e d r e a c t i o n schemes f o r t h e c h e m i c a l m o d i f i c a t i o n o f m e t h a c r y l a t e m i c r o s p h e r e s 3 F r a c t i o n a t i o n o f ( % ) - o u a b a i n - m i c r o -sphere c o n j u g a t e s on Sepharose-6B 4 S y n t h e i t i c r o u t e f o l l o w e d i n t h e p r e p -a r a t i o n o f DANS-lys-ouabain 3 5 Chromatography o f ( H)-ouabain on DOWEX (AG50X12)-H i o n exchange r e s i n 6 P u r i f i c a t i o n o f DANS-lys-ouabain on B i o g e l - P 2 3 7 Chromatography o f p u r i f i e d DANS-lys-( H)-o u a b a i n on B i o g e l - P 2 3 8 B i n d i n g o f ( H)-ouabain t o beef b r a i n m i c r o s o m a l membranes 3 9 B i n d i n g o f ( H)-ouabain t o H e l a c e l l s 3 10 S c a t c h a r d a n a l y s i s o f ( H ) - o u a b a i n b i n d i n g 11 I n h i b i t i o n o f Na,K ATPase a c t i v i t y by o u a b a i n m i c r o s p h e r e c o n j u g a t e s 12 I n h i b i t i o n o f Na,K ATPase a c t i v i t y by DANS-lys-ouabain 13 3 C o m p e t i t i v e i n h i b i t i o n o f ( H ) - o u a b a i n b i n d i n g by DANS-lys-ouabain 14 B i n d i n g o f anti-DANS IgG t o human e r y t h r o -c y t e s p r e l a b e l l e d w i t h D A N S - r i c i n 15 Schematic r e p r e s e n t a t i o n o f t h e s t e p s i n v o l v e d i n t h e hapten sandwich l a b e l l i n g t e c h n i q u e 16 Mouse neur o b l a s t o m a c e l l s i n d i r e c t l y l a b e l -l e d w i t h FITC-anti-DANS IgG 11 29 30 33 34 35 36 38 39 40 42 44 45 61 63 65 LIST OF FIGURES (cont'd) Hapten sandwich l a b e l l i n g of Mouse 3T3 f i b r o b l a s t c e l l s Energy d i s p e r s i v e X-ray microprobe a n a l y s of f i b r o b l a s t c e l l l a b e l l e d w i t h DANS-Fe microspheres - 1 -I N T R O D U C T I O N ( I ) THE SODIUM PUMP: T h e a d e n o s i n e t r i p h o s p h a t a s e , a c t i v a t e d by Na and K ( N a , K A T P a s e ) , i s c o n s i d e r e d t o be t h e i n t e g r a l membrane enzyme r e s p o n s i b l e f o r t h e a c t i v e t r a n s p o r t o f s o d i u m and p o t a s s i u m i o n s a c r o s s t h e e u k a r y o t i c p l a s m a m e m b r a n e . The N a , K A T P a s e c o m p l e x h a s b e e n shown t o c o n t a i n b o t h a l a r g e p o l y p e p t i d e o f m o l e c u l a r w e i g h t 9 5 , 0 0 0 and a s m a l l e r g l y c o p r o t e i n o f m o l e c u l a r w e i g h t 4 5 , 0 0 0 ( 7 ) . Under p h y s i o l o g i c a l c o n d i t i o n s t h e enzyme c a t a l y z e s t h e t r a n s p o r t o f 3 Na i o n s o u t o f t h e c e l l , 2 K i o n s i n t o t h e c e l l and t h e h y d r o l y s i s o f one ATP m o l e c u l e i n a s i n g l e c y c l e ( 8 , 9 ) . The i o n movements a r e t h o u g h t t o be o b l i g a t o r i l y c o u p l e d t o ATP h y d r o l y s i s i n a s e r i e s o f i n t e r m e d i a t e s t e p s w h i c h i n v o l v e t h e f o r m a t i o n o f a p h o s p h o e n z y m e c o m p l e x , c h a n g e s i n c o n f o r m a t i o n and c l e a v a g e o f t h e p h o s p h o e n z y m e ( 1 0 , 1 1 ) . A . I o n T r a n s p o r t M o d e s : T h e s o d i u m pump h a s been o b s e r v e d t o o p e r a t e i n a n y o f f i v e d i f f e r e n t i o n t r a n s p o r t m o d e s , d e p e n d i n g on t h e c o m p o s i t i o n o f i n t r a c e l l u l a r and e x t r a -c e l l u l a r c a t i o n s as w e l l as t h e r a t i o o f ATP t o A D P . T h e pump o p e r a t e s i n ( 1 ) n o r m a l N a - K e x c h a n g e as w e l l a s ( 2 ) a r e v e r s e d m o d e , ( 3 ) N a - N a e x c h a n g e , ( 4 ) K - K e x c h a n g e and ( 5 ) Na e f f l u x w h i c h i s n o t c o u p l e d t o t h e u p t a k e o f Na o r K ( 4 7 ) . - 2 -1 . N o r m a l M o d e : The Na-K e x c h a n g e i s d r i v e n by t h e h y d r o l y s i s o f ATP o n t h e i n n e r s u r f a c e o f t h e p l a s m a m e m b r a n e . The h y d r o l y s i s o f a s i n g l e m o l e c u l e o f ATP l e a d s t o t h e a c t i v e t r a n s p o r t o f t h r e e Na i o n s o u t o f t h e c e l l and t h e t r a n s p o r t o f two K i o n s i n t o t h e c e l l . T h i s s t o i c h i o m e t r y h a s b e e n o b s e r v e d f o r human r e d c e l l s ( 4 8 ) as w e l l as n e r v e , m u s c l e and a v a r i e t y o f o t h e r t i s s u e s ( 4 9 ) . 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 t h e s o d i u m pump may be s a i d t o be e l e c t r o g e n i c , i n t h a t i t s a c t i o n l e a d s d i r e c t l y t o a n e t t r a n s f e r o f c h a r g e a c r o s s t h e m e m b r a n e . A o u a b a i n - s e n s i t i v e h y p e r p o l a r i z a t i o n h a s b e e n d e m o n s t r a t e d i n a number o f e x c i t a b l e t i s s u e s when t h e i n t r a c e l l u l a r Na c o n c e n t r a t i o n was r a i s e d by r e p e a t e d s t i m u l a t i o n , by i n j e c t i o n o f N a , o r by s t o r a g e i n K f r e e s o l u t i o n s r i c h i n Na ( 5 0 , 5 1 , 5 2 , 5 3 ) . 2 . R e v e r s e d M o d e : The r e d c e l l Na,K A T P a s e c a n be i n d u c e d t o s y n t h e s i z e ATP i f one l o w e r s t h e i n t r a c e l l u l a r r a t i o o f ( A T P ) / ( A D P ) ( P i ) ( 5 4 ) . T h i s r e v e r s e d mode i s d r i v e n by t h e 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 i o n s a c r o s s t h e m e m b r a n e . The r a t e o f ATP s y n t h e s i s i s r o u g h l y p r o p o r t i o n a l t o t h e e x t e r n a l Na c o n c e n -t r a t i o n ( 5 4 ) and i s i n h i b i t e d by e x t e r n a l K. T h e h a l f m a x i m a l i n h i b i t i o n o f t h e b a c k w a r d r u n n i n g pump by e x t r a c e l l u l a r K i o n s (KQ g 1 . 3 mM a t 150 mM N a C l ) i s s i m i l a r t o t h e KQ ^ f o r a c t i v a t i o n o f t h e f o r w a r d r u n n i n g pump by e x t e r n a l K when n o r m a l c e l l s a r e i n c u b a t e d i n m e d i u m c o n t a i n i n g 150 mM N a C l ( 5 5 ) . - 3 -3 . N a - N a E x c h a n g e A N a - N a e x c h a n g e c a n be o b s e r v e d when c e l l s a r e i n c u b a t e d i n a medium f r e e o f K b u t r i c h i n N a . T h i s o u a b a i n - s e n s i t i v e e x c h a n g e o f i n t e r n a l and e x t e r n a l Na i o n s h a s been d e m o n s t r a t e d w i t h r e d c e l l s ( 5 6 ) as w e l l as f r o g m u s c l e and p a r t i a l l y p o i s o n e d g i a n t a x o n s ( 5 7 , 5 8 ) . The N a - N a e x c h a n g e o b s e r v e d i n e r y t h r o c y t e s i s o n e - f o r - o n e . H o w e v e r t h e r e i s a d i s t i n c t d i f -f e r e n c e i n t h e a f f i n i t y f o r Na on t h e two s i d e s o f t h e m e m b r a n e . The i n t e r n a l b i n d i n g s i t e i s s a t u r a t e d a t 10-15 mM N a , b u t e x t e r n a l l y s a t u r a t i o n o c c u r s w e l l a b o v e 150 mM ( 5 9 ) . E x t e r n a l K i n h i b i t s N a - N a e x c h a n g e and f a c i l i t a t e s N a - K e x c h a n g e ( 5 9 ) . I n t e r n a l K h a s been f o u n d t o e n h a n c e N a - N a e x c h a n g e and a l t h o u g h t h e e x p l a n a t i o n f o r t h i s i s n o t k n o w n , i t d o e s i m p l i c a t e some r o l e f o r K on t h e i n n e r f a c e o f t h e pump. N a - N a e x c h a n g e i s n o t c o u p l e d t o ATP h y d r o l y s i s ( 9 ) , b u t t h e e x c h a n g e i s l i n e a r l y d e p e n d e n t on t h e i n t e r n a l ADP c o n c e n t r a t i o n up t o 0 . 3 mM ( t h e h i g h e s t c o n c e n t r a t i o n t e s t e d ) ( 6 0 ) . T h i s d e p e n d e n c e o f N a - N a e x c h a n g e on t h e i n t r a c e l l u l a r l e v e l s o f ADP i n d i c a t e s t h a t t h e i n f l u x o f Na i s l i n k e d d i r e c t l y or i n d i r e c t l y w i t h t h e t r a n s f e r o f p h o s p h a t e t o ADP f r o m t h e p h o s p h o r y l a t e d pump p r o t e i n . 4 . K-K E x c h a n g e K-K e x c h a n g e h a s been s t u d i e d i n human r e d c e l l s and i n r e s e a l e d g h o s t s c o n t a i n i n g l i t t l e Na ( 6 1 ) . I t i s s i m i l a r t o N a - N a e x c h a n g e i n t h a t i t i s o n e - f o r - o n e and i s n o t c o u p l e d t o t h e h y d r o l y s i s o f A T P . T h e r e i s a d i s t i n c t d i f f e r e n c e i n t h e a f f i n i t y f o r K on t h e t w o s i d e s o f t h e c e l l m e m b r a n e , h o w e v e r t h e a s y m m e t r y i s i n t h e r e v e r s e d i r e c t i o n o f t h a t o b s e r v e d f o r N a - N a e x c h a n g e , s u c h t h a t t h e i n t e r n a l f a c e o f t h e pump i s s a t u r a t e d a t a much h i g h e r K c o n c e n t r a t i o n t h a n t h e e x t e r n a l f a c e . T h i s r e v e r s a l o f a s y m m e t r i e s i n g o i n g f r o m N a - N a e x c h a n g e t o K - K e x c h a n g e may i n d i c a t e t h a t 4 -t h e same c a t i o n b i n d i n g s i t e s a r e i n v o l v e d ( 5 5 ) . K - K e x c h a n g e i s n o t d e p e n d e n t on i n t e r n a l ADP ( 6 1 ) b u t i s d e p e n d e n t on t h e p r e s e n c e o f i n o r g a n i c p h o s p h a t e w i t h i n t h e c e l l ( 5 5 ) . Hence i t i s l i k e l y t h a t K - K e x c h a n g e i s d e p e n d e n t on t h e r e v e r s i b l e t r a n s f e r o f p h o s p h a t e b e t w e e n t h e enzyme and w a t e r , r a t h e r t h a n b e t w e e n t h e enzyme and A D P . 5 . U n c o u p l e d Na E f f l u x : I n c u b a t i o n o f r e d c e l l s o r r e s e a l e d e r y t h r o c y t e g h o s t s i n medium c o n t a i n i n g c h o l i n e o r Mg r e s u l t s i n a t r a n s f e r o f i n t r a c e l l u l a r Na i n t o t h e e x t e r n a l medium n o t c o u p l e d t o t h e e n t r y o f Na o r K ( 5 6 , 6 2 ) . T h i s u n c o u p l e d Na e f f l u x i s o u a b a i n s e n s i t i v e and i s l i n k e d t o ATP h y d r o l y s i s s u c h t h a t f o r e a c h m o l e c u l e o f ATP h y d r o l y z e d a b o u t t h r e e Na i o n s a r e t r a n s p o r t e d o u t o f t h e c e l l . U n c o u p l e d e f f l u x i s i n h i b i t e d by i n t e r n a l K a s w e l l a s by l o w c o n c e n t r a t i o n s ( 5 mM) o f e x t e r n a l N a . I n summary i t c a n be s a i d t h a t t h e movements o f Na a c r o s s t h e c e l l m e m b r a n e , b o t h i n w a r d and o u t w a r d , a r e l i n k e d t o t h e r e v e r s i b l e t r a n s f e r o f p h o s p h a t e b e t w e e n t h e enzyme and A T P . T h e o u t w a r d movement o f Na b e i n g c o u p l e d w i t h t h e t r a n s f e r o f p h o s p h a t e f r o m ATP t o t h e e n z y m e , w h i l e t h e i n w a r d movement o f Na i s l i n k e d t o t h e t r a n s f e r o f p h o s p h a t e f r o m t h e p h o s p h o e n z y m e t o A D P . I n w a r d and o u t w a r d movements o f K a r e , on t h e o t h e r h a n d , a s s o c i a t e d w i t h t h e t r a n s f e r o f p h o s p h a t e f r o m t h e p h o s p h o e n z y m e t o w a t e r and f r o m i n o r g a n i c p h o s p h a t e t o t h e e n z y m e . I t i s c l e a r t h e n t h a t t h e c a t i o n c o m p o s i t i o n o f t h e medium w i l l d i c t a t e w h e t h e r t h e p h o s p h e n z y m e w i l l t r a n s f e r i t s p h o s p h a t e t o ADP o r t o w a t e r . In t h e p r e s e n c e o f e x t e r n a l K t h e p h o s p h a t e i s t r a n s f e r r e d t o w a t e r and K e n t e r s t h e c e l l , b u t i n t h e p r e s e n c e o f e x t e r n a l Na t h e p h o s p h a t e i s t r a n s f e r r e d t o ADP and Na i s t r a n s -p o r t e d i n t o t h e c e l l . I n t h e a b s e n c e o f e i t h e r Na o f K i n t h e e x t e r n a l - 5 -m e d i u m , t h e p h o s p h o e n z y m e s l o w l y t r a n s f e r s i t s p h o s p h a t e t o w a t e r and t h e r e i s a s l o w h y d r o l y s i s o f ATP w h i c h r e s u l t s i n an u n c o u p l e d movement o f Na o u t o f t h e c e l l . B . The R o l e o f P h o s p h o r y l a t e d I n t e r m e d i a t e s i n t h e N o r m a l W o r k i n g o f t h e  Pump: 32 3? E x p e r i m e n t s w i t h X - P-ATP h a v e d e m o n s t r a t e d t h a t P was i n c o r -p o r a t e d i n t o t h e membrane l i p o p r o t e i n and t h a t i t c o u l d be r e l e a s e d a s i n o r g a n i c p h o s p h a t e i n t h e p r e s e n c e o f K i o n s . S u c h s t u d i e s h a v e l e d i n v e s t i g a t o r s t o b e l i e v e t h a t t h e h y d r o l y s i s o f ATP by t h e s o d i u m pump may i n v o l v e t h e t r a n s f e r o f p h o s p h a t e f r o m ATP t o a s i t e on t h e enzyme p r i o r t o i t s u l t i m a t e t r a n s f e r t o w a t e r . T h e r e i s g o o d e v i d e n c e t h a t t h e l i p o p r o t e i n bound p h o s p h a t e o c c u r s as a / 3 - a s p a r t y l p h o s p h a t e w i t h a s e r i n e o r t h r e o n i n e on one s i d e o f i t and a l y s i n e on t h e o t h e r ( 6 3 ) . I t i s known t h a t a c y l p h o s p h a t e s h a v e a h i g h f r e e e n e r g y y i e l d on h y d r o l y s i s and i t w o u l d n o t be u n r e a s o n a b l e t o e x p e c t t h e ATP d e p e n d e n t p h o s p h o r y l a t i o n o f t h e enzyme t o be r e v e r s i b l e s u c h t h a t t h e p h o s p h o e n z y m e c o u l d , i n t u r n , p h o s p h o r y l a t e A D P . E x p e r i m e n t s t o t h i s e f f e c t h a v e d e m o n s t r a t e d a Na d e p e n d e n t A T P - A D P e x c h a n g e o n l y i n t h e p r e s e n c e o f v e r y low Mg c o n c e n t r a t i o n s , o r i f t h e enzyme was p r e t r e a t e d w i t h N - e t h y l m a l e i m i d e (NEM) ( 6 4 , 6 5 ) . I n o r d e r t o e x p l a i n t h e e f f e c t o f NEM and o t h e r s u l f h y d r y l r e a g e n t s , i t h a s been s u g g e s t e d t h a t t h e p h o s p h o r y l a t e d compound f i r s t f o r m e d , E ^ P , was c o n v e r t e d t o a l o w e r e n e r g y f o r m , E ^ P , by a r e a c t i o n r e q u i r i n g Mg i o n s and i n h i b i t e d b y NEM ( 6 5 , 1 2 ) . I n t h i s s i t u a t i o n , t h e o v e r a l l h y d r o l y s i s o f ATP may be r e p r e s e n t e d b y : L l (MglSa) * E 1 P b l o c k e d ^ NEM * E 2 P ^ * E 2 * E l - 6 -H e n c e , t h e E^P f o r m o f t h e p h o s p h o e n z y m e i s s e n s i t i v e t o ADP b u t n o t t o K, w h e r e a s , t h e E 2 P f o r m i s s e n s i t i v e t o K b u t n o t t o A D P . T h e f r e e e n e r g y o f h y d r o l y s i s o f E-^ P h a s been e s t i m a t e d f r o m m e a s u r e m e n t s o f t h e f r a c t i o n o f t h e enzyme p h o s p h o r y l a t e d a f t e r i n c u b a t i o n w i t h d i f f e r e n t ATP/ADP m i x t u r e s i n t h e p r e s e n c e o f o l i g o m y c i n , w h i c h i n h i b i t s t h e E^P-* rE^P t r a n s f o r m a t i o n ( 6 6 ) , and i t i s a b o u t 1 k c a l / m o l e l e s s t h a n t h e f r e e e n e r g y o f h y d r o l y s i s o f A T P . A p h o s p h o e n z y m e c o m p l e x s i m i l a r t o t h a t f o r m e d f r o m ATP c a n a l s o be f o r m e d f r o m o t h e r n u c l e o s i d e t r i p h o s p h a t e s and f r o m p - n i t r o p h e n y l p h o s p h a t e ( p N P P ) and a c e t y l p h o s p h a t e ( 6 7 , 6 8 ) . Mg i s a l w a y s e s s e n t i a l ; Na i s e s s e n t i a l f o r p h o s p h o r y l a t i o n by n u c l e o s i d e t r i p h o s p h a t e s and h a s b e e n f o u n d t o i n c r e a s e p h o s p h o r y l a t i o n by a c e t y l p h o s p h a t e u n l e s s K was a l s o p r e s e n t ( 6 7 ) . C . I n t e r a c t i o n o f C a r d i a c G l y c o s i d e s w i t h t h e S o d i u m Pump: The c a r d i a c g l y c o s i d e s h a v e been o f immense c l i n i c a l v a l u e i n t h e t r e a t m e n t o f c h r o n i c c o n j e s t i v e h e a r t f a i l u r e as w e l l as o t h e r c a r d i o v a s c u l a r d i s o r d e r s ( 6 ) . A p a r t f r o m i t s c l i n i c a l v a l u e , t h i s f a m i l y o f m o l e c u l e s h a s a t t r a c t e d t h e i n t e r e s t o f b i o c h e m i s t s and p h y s i o l o g i s t s b e c a u s e o f i t s u n i q u e e f f e c t o n membrane f u n c t i o n w i t h r e s p e c t t o t h e t r a n s p o r t o f s o d i u m and p o t a s s i u m . T h e i n h i b i t o r y e f f e c t o f t h e c a r d i a c g l y c o s i d e s o n t h e s o d i u m pump was f i r s t r e p o r t e d by S c h a t z m a n n i n 1953 ( 1 3 ) . E a r l y w o r k e s t a b l i s h i n g t h e m o l e c u l a r f e a t u r e s n e c e s s a r y f o r i n h i b i t i o n , t h e e x t e r n a l s i t e o f a c t i o n , t h e p a r a l l e l i n h i b i t i o n o f t r a n s p o r t and A T P a s e a c t i v i t y and t h e p r o t e c t i v e e f f e c t o f e x t e r n a l K i s d e s c r i b e d i n d e t a i l b y G l y n n , 1964 ( 1 4 ) . I t i s now g e n e r a l l y b e l i e v e d t h a t t h e c a r d i a c g l y c o s i d e s a c t b y i n h i b i t i n g d e p h o s -p h o r y l a t i o n o f t h e p h o s p h o e n z y m e a s w e l l a s p h o s p h o r y l a t i o n o f t h e d e p h o s p h o e n z y m e ( 1 5 ) . T h e e n z y m e - o u a b a i n i n t e r a c t i o n c a n be d e s c r i b e d by a s i n g l e r e v e r s i b l e e q u i l i b r i u m : E + Ou-* 7 E - 0 u ( 1 6 ) . The a s s o c i a t i o n r a t e h a s b e e n shown t o be f i r s t o r d e r w i t h r e s p e c t t o b o t h enzyme and o u a b a i n c o n c e n t r a t i o n ( 1 7 ) , t h e d i s s o c i a t i o n f o l l o w s f i r s t o r d e r e x p o n e n t i a l k i n e t i c s ( 1 7 , 1 8 ) and t h e m e a s u r e d e q u i l i b r i u m c o n s t a n t i s s i m i l a r t o t h e q u o t i e n t o f d i s s o c i a t i o n and a s s o c i a t i o n r a t e c o n s t a n t s . B i n d i n g c o n s t a n t s o f o u a b a i n t o d i f f e r e n t A T P a s e p r e p a r a t i o n s v a r y , r e f l e c t i n g m a i n l y d i f f e r e n c e s i n t h e r a t e s o f d i s s o c i a t i o n o f t h e e n z y m e - o u a b a i n c o m p l e x ( 1 9 ) . T h e r a t e o f o u a b a i n b i n d i n g h a s been o b s e r v e d t o d e p e n d o n t h e p r e s e n c e o f v a r i o u s p h y s i o l o g i c a l l i g a n d s . L i g a n d s , o r c o m b i n a t i o n s o f l i g a n d s , t h a t s t a b i l i z e t h e o r E^P c o n f o r m a t i o n o f t h e enzyme ( M g ; M g , P ^ ; M g , N a , n u c l e o s i d e t r i p h o s p h a t e s ) a l l i n c r e a s e t h e r a t e o f b i n d i n g and l o w e r t h e d i s s o c i a t i o n c o n s t a n t ( 2 0 ) . L i g a n d s w h i c h t e n d t o s t a b i l i z e t h e o r E-^ P c o n f o r m a t i o n s ( N a ; n o n - p h o s p h o r y l a t i n g ATP a n a l o g u e s ) d e c r e a s e t h e r a t e o f o u a b a i n b i n d i n g ( 2 1 ) . P o t a s s i u m r e d u c e s i n h i b i t i o n by c a r d i a c g l y c o s i d e s b y a f f e c t i n g t h e i r b i n d i n g . T h e r e i s c l e a r e v i d e n c e t h a t t h e r a t e o f o u a b a i n b i n d i n g i n t h e p r e s e n c e o f M g ; o r M g , P ^ ; o r M g , N a , A T P ; i s r e d u c e d b y K. W h i l e K d e c r e a s e s b i n d i n g a f f i n i t i e s i t does not a f f e c t b i n d i n g c a p a c i t y ( 2 2 ) . A v a i l a b l e e x p e r i m e n t a l d a t a on t h e f o r m a t i o n and d i s s o c i a t i o n o f t h e o u a b a i n - e n z y m e c o m p l e x d i f f e r w i d e l y , r e f l e c t i n g , i n p a r t , t h e d i f f e r e n t enzyme p r e p a r a t i o n s s t u d i e d . T h e k i n e t i c p a r a m e t e r s o f t h e o u a b a i n - e n z y m e i n t e r a c t i o n f o r a number o f d i f f e r e n t enzyme s o u r c e s i s g i v e n i n TABLE I . - 8 -W h i l e t h e p r i m a r y d e t e r m i n a n t o f a c t i v i t y o f t h e c a r d i a c g l y c o s i d e s r e s i d e s w i t h i n t h e a g l y c o n e r e g i o n o f t h e m o l e c u l e , t h e s u g a r p o r t i o n i s a l s o i n t i m a t e l y i n v o l v e d i n a s s o c i a t i o n w i t h t h e e n z y m e . I t h a s b e e n f o u n d t h a t t h e 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 t h e g l y c o s i d e - e n z y m e c o m p l e x i s n o t d e p e n d e n t on t h e s t r u c t u r e o f t h e a g l y c o n e p o r t i o n , b u t o n l y on t h a t o f t h e s u g a r p o r t i o n ( 9 0 ) . A c o m p a r i s o n o f t h e 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 f o r a number o f m o n o g l y c o s i d e s r e v e a l e d t h a t t h e 31-/S - h y d r o x y l a n d 5 ' - a - m e t h y l g r o u p s w e r e i m p o r t a n t i n b i n d i n g w i t h t h e e n z y m e . M o d i f i c a t i o n o f t h e 3 ' - / 3 - h y d r o x y l g r o u p b y a c e t y l a t i o n , r e s u l t e d i n a l a r g e r e d u c t i o n i n t h e s t a b i l i t y o f t h e g l y c o s i d e - e n z y m e c o m p l e x . D. P h y s i c a l P r o p e r t i e s o f t h e S o d i u m Pump: O n l y two m a j o r p r o t e i n c o m p o n e n t s a r e o b s e r v e d on SDS 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 o f p u r i f i e d p r e p a r a t i o n s o f N a , K A T P a s e ( 6 9 ) . The m o l e c u l a r w e i g h t o f t h e l a r g e p o l y p e p t i d e i s c l o s e t o 1 0 0 , 0 0 0 , w h e r e a s , t h e s m a l l e r , c a r b o y d r a t e c o n t a i n i n g p o l y p e p t i d e has a m o l e c u l a r w e i g h t o f a b o u t 4 5 , 0 0 0 . T h e l a r g e p o l y p e p t i d e a c c o u n t s f o r 57-72% o f t h e t o t a l p r o t e i n i n p u r i f i e d p r e p a r a t i o n s o f N a , K A T P a s e . T h i s p r o t e i n c a r r i e s t h e p h o s p h a t e i n c o r p o r a t e d f r o m ATP i n t h e p r e s e n c e o f Na and i t p o s s e s s e s s u l f h y d r a ! g r o u p s w i t h s e n s i t i v i t y t o A T P , N a , K and o u a b a i n . T h e s e o b s e r v a t i o n s s e r v e t o i d e n t i f y t h e p r o t e i n as t h e m a j o r c a t a l y t i c s u b u n i t o f t h e e n z y m e . - 9 -TABLE I . COMPARISON OF THE DISSOCIATION CONSTANTS OF OUABAIN-RECEPTOR COMPLEX AND OUABAIN CONCENTRATIONS CAUSING HALF MAXIMAL INHIBITION OF N A , K ATPASE Enzyme P r e p a r a t i o n H a l f M a x i m a l A p p a r e n t I n h i b i t i o n o f D i s s o c i a t i o n N a , K A T P a s e (M) C o n s t a n t , K r f (M) B e e f k i d n e y ( 1 7 ) 5 X I O " 7 0 . 5 X 1 0 " 8 B e e f b r a i n ( 1 7 ) 6 X I O " 7 0 . 3 X 1 0 " 8 B e e f h e a r t ( 1 7 ) 2 X I O " 7 0 . 3 X 1 0 " 8 Dog h e a r t ( 1 7 ) 1 . 6 X I O " 7 0 . 4 X 1 0 " 8 G u i n e a p i g k i d n e y ( 1 7 ) 2 . 0 X 1 0 " 6 1 . 5 X I O " 7 Dog b r a i n ( 1 8 ) 7 . 6 X I O " 7 6 . 4 X 1 0 ~ 8 * * enzyme p r e - i n c u b a t e d w i t h o u a b a i n p l u s A T P , N a f o r 5 m i n . a t 37 C p r i o r t o a d d i t i o n o f K. - 10 -TABLE I I STRUCTURE OF SOME CARDIAC MONOGLYCOSIDES COMPOUND AGLYCONE SUGAR GLYCOSIDE BOND OUABAIN CONVALLATOXIN HELVETICOSIDE D I X I T O X I G E N I N MONODIGITOXIDE DEGLUCOCHEIRO TOXIN DIGIPROSIDE D I G I T O X I G E N I N -6-DEOXY GLUCOSIDE CYMARIN ODOROSIDE H OUABAGENIN STROPHANTHIDIN STROPHANTHIDIN DIGITOXIGENIN STROPHANTHIDIN DIGITOXIGENIN DIGITOXIGENIN STROPHANTHIDIN DIGITOXIGENIN L-RHAMNOSE L-RHAMNOSE D-DIGITOXOSE D-DIGITOXOSE D-6-DE0XYGUL0SE D-FUCOSE D-6-DE0XYGLUC0SE D-CYMAROSE ( 3 ' - M E T H 0 X Y -D-DIGITOXOSE) D-DIGITALOSE ( 3 1 - M E T H -OXY-D-FUCOSE) a a P P P P P P - 11 -1 S T R U C T U R E S OF S U G A R S L I S T E D I N T A B L E I I R = H R = CH 3 B-D-FUCOSE B-D-DIGITALOSE - 12 -T A B L E I I I , S T R U C T U R E S OF THE A G L Y C O N E S W H I C H FORM S T E R O I D M O I E T I E S OF G L Y C O S I D E S L I S T E D I N T A B L E I COMPOUND S U B S T I T U T I O N S D I G I T O X I G N E N I N 1 9 - C H 3 » D I G O X I G E N I N 1 9 - C H 3 , 1 2 - B - O H S T R O P H A N T H I D I N 1 9 - C H O , 5 - B - O H O U A B A G E N I N 1 9 - C H 2 O H , 1 - B - O H , 5 - B - O H , 1 1 - B -- 13 -T h e l a r g e p o l y p e p t i d e h a s been i s o l a t e d by p r e p a r a t i v e 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 i n SDS and t h e r e a c t i v i t y o f i t s s u l f y d r a l g r o u p s e x a m i n e d ( 7 0 ) . T r e a t m e n t o f t h e l a r g e p o l y p e p t i d e w i t h NEM d e m o n s t r a t e d t h a t two S H - g r o u p s c o u l d be a l k y l a t e d p e r ATP b i n d i n g s i t e and 10% o f t h e s e g r o u p s c o u l d be p r o t e c t e d f r o m a l k y l a t i o n by ATP p l u s N a . P h o s p h o r y l a t i o n o f t h e p o l y p e p t i d e b y ATP r e s u l t s i n t h e e x p o s u r e o f a d d i t i o n a l S H - g r o u p s w h i c h a r e s u s c e p t i b l e t o a l k y l a t i o n . Such b e h a v i o r i n d i c a t e s t h a t p h o s p h o -r y l a t i o n i s a c c o m p a n i e d b y a c o n f o r m a t i o n a l c h a n g e i n t h e p r o t e i n . Amino a c i d a n a l y s i s o f t h e l a r g e c h a i n has shown t h a t 5 3 % o f t h e amino a c i d r e s i d u e s a r e h y d r o p h i l i c ( 7 1 ) . T h i s c o n t e n t o f p o l a r r e s i d u e s i s n o t as l o w as t h a t r e p o r t e d f o r o t h e r i n t r i n s i c membrane p r o t e i n s ( 7 2 ) and may i m p l y an a s y m m e t r i c d i s t r i b u t i o n o f p o l a r g r o u p s o r t h a t s m a l l p r o t e o l i p i d s a r e i n v o l v e d i n t h e i n t e r a c t i o n b e t w e e n t h e p r o t e i n and p h o s p h o l i p i d s ( 7 3 ) . The s m a l l e r p o l y p e p t i d e f o u n d i n p u r i f i e d p r e p a r a t i o n s o f N a , K A T P a s e h a s n o t been i d e n t i f i e d w i t h any c a t a l y t i c f u n c t i o n and i t r e m a i n s an open q u e s t i o n as t o w h e t h e r i t i s a s u b u n i t o f t h e e n z y m e . The mass r a t i o o f l a r g e t o s m a l l p o l y p e p t i d e v a r i e s f r o m 1 . 7 (71) t o 1 . 9 ( 7 4 ) , 2 . 3 ( 7 ) and 2 . 8 ( 7 5 ) . A s s u m i n g a m o l e c u l a r w e i g h t o f 4 0 , 0 0 0 , t h e r a t i o o f 1 . 7 c o r r e s p o n d s t o two s m a l l c h a i n s p e r l a r g e c h a i n and t h e r a t i o 2 . 8 t o o n e s m a l l c h a i n p e r l a r g e c h a i n . T h e s m a l l c h a i n i s a s i a l o g l y c o p r o t e i n ( 7 1 ) and i t h a s b e e n p r o p o s e d t h a t i t s e r v e s as a f i x e d e x t e r n a l r e f e r e n c e f o r t h e l a r g e c h a i n i n t h e m e m b r a n e . The a m i n o a c i d c o m p o s i t i o n o f t h e s m a l l c h a i n ( 7 1 ) i s s i m i l a r t o t h a t o f t h e l a r g e c h a i n and t h e p o s s i b i l i t y t h a t p a r t o r a l l o f t h e s m a l l e r p o l y p e p t i d e c o u l d have a r i s e n by p r o t e o l y s i s o f t h e l a r g e c h a i n , c a n n o t be d i s c o u n t e d . - 14 -E. A n t i g e n i c i t y o f t h e S o d i u m Pump: I m m u n o g l o b u l i n s r a i s e d a g a i n s t N a , K A T P a s e a r e c h a r a c t e r i z e d by t h e i r a b i l i t y t o i n h i b i t t h e N a , K A T P a s e a c t i v i t y i n v i t r o and t o b l o c k t h e a c t i v e t r a n s p o r t o f Na and K, p r o v i d e d t h e y h a v e a c c e s s t o t h e i n n e r s u r f a c e o f t h e c e l l m e m b r a n e . R a b b i t a n t i - s e r u m a g a i n s t a h i g h l y a c t i v e p r e p a r a t i o n o f N a , K A T P a s e f r o m p o r c i n e r e n a l m e d u l l a i n h i b i t e d t h e enzyme a c t i v i t y i n p r e p a r a t i o n s f r o m v a r i o u s t i s s u e s , as w e l l a s , t h e a c t i v e Na t r a n s p o r t i n human e r y t h r o c y t e s ( 8 1 ) . T h i s s u g g e s t s t h a t t h e a n t i g e n i c d e t e r m i n a n t s o f t h e s o d i u m pump a r e t h e same i n d i f f e r e n t s p e c i e s and o r g a n s . T h e a n t i b o d i e s w e r e a c t i v e i n i n h i b i t i n g t h e o u a b a i n s e n s i t i v e N a - K e x c h a n g e , t h e N a - N a e x c h a n g e , t h e K-K e x c h a n g e and t h e u n c o u p l e d Na e f f l u x , when t h e y w e r e i n c o r p o r a t e d i n t o r e s e a l e d human r e d c e l l g h o s t s . T h e r e was no e f f e c t on t h e o u a b a i n i n s e n s i t i v e f l u x e s o f Na and K and t h e a n t i b o d y was i n a c t i v e f r o m t h e o u t e r s u r f a c e o f t h e p l a s m a m e m b r a n e . The d e g r e e o f i n h i b i t i o n o f t r a n s p o r t was f o u n d t o be d e p e n d e n t on t h e l e v e l o f a n t i b o d y and c o r r e l a t e d w i t h t h e d e c r e a s e i n h y d r o l y t i c a c t i v i t y o f t h e r e d c e l l N a , K A T P a s e . The a n t i - s e r u m was s p e c i f i c , i n t h a t t h e r e was no c r o s s r e a c t i o n w i t h t h e Mg o r Mg p l u s Ca a c t i v a t e d A T P a s e s ( 8 2 ) . T h e i m m u n o g l o b u l i n s do n o t i n t e r f e r e w i t h b i n d i n g o f ATP o r o u a b a i n t o t h e enzyme and have l i t t l e o r no e f f e c t on t h e K d e p e n d e n t p N P P a s e a c t i v i t y ( 8 3 ) . I t has been s u g g e s t e d t h a t a n t i b o d i e s d i r e c t e d a g a i n s t N a , K A T P a s e do not p r e v e n t t h e a c c e s s o f l i g a n d s t o t h e e n z y m e , b u t may i n t e r f e r e w i t h c h a n g e s i n c o n f o r m a t i o n o f t h e p r o t e i n w h i c h a r e n e c e s s a r y f o r p r e c i s e o r i e n t a t i o n o f t h e c a t a l y t i c g r o u p s t h a t p e r m i t t h e t r a n s l o c a t i o n o f c a t i o n s . - 15 -( I I ) PREVIOUS METHODS FOR THE CYTOCHEMICAL DEMONSTRATION OF N A , K ATPASE M o s t o f t h e t e c h n i q u e s f o r t h e c y t o c h e m i c a l d e m o n s t r a t i o n o f a d e n o s i n e t r i p h o s p h a t a s e s ( A T P a s e ) h a v e d e p e n d e d upon t h e d e p o s i t i o n o f a h e a v y m e t a l p r e c i p i t a t e o f e n z y m a t i c a l l y r e l e a s e d i n o r g a n i c p h o s p h a t e , w i t h e i t h e r ATP o r pNPP as t h e s u b s t r a t e ( 7 6 , 7 7 , 7 8 ) . The c o n v e n t i o n a l h i s t o c h e m i c a l a p p r o a c h r e q u i r e s t h e i n c u b a t i o n o f t i s s u e s e c t i o n s w i t h ATP as s u b s t r a t e and a s u i t a b l e c a p t u r e r e a g e n t s u c h as Pb o r C a ; t h e s e c a t i o n s f o r m an i n s o l u b l e p r e c i p i t a t e w i t h t h e i n o r g a n i c o r t h o p h o s p h a t e ( P . . ) t h a t i s l i b e r a t e d by h y d r o l y s i s o f A T P . T h i s a p p r o a c h h a s b e e n s u c c e s s f u l i n t h e d e m o n s t r a t i o n o f a c t o m y s i n A T P a s e i n m u s c l e ( 7 9 ) and u n s p e c i f i e d A T P a s e a c t i v i t i e s i n o t h e r c e l l s , b u t h a s f o u n d l i m i t e d u s e i n t h e l o c a l i z a t i o n o f N a , K A T P a s e b e c a u s e t h i s enzyme i s s t r o n g l y i n h i b i t e d by d i v a l e n t c a t i o n s s u c h as Pb and Ca ( 8 0 ) . P r o c e d u r e s u t i l i z i n g p - n i t r o p h e n y l p h o s p h a t e as t h e s u b s t r a t e and S r C l ^ as t h e c a p t u r e r e a g e n t ( 7 8 ) h a v e been m o d e r a t e l y s u c c e s s f u l i n d e m o n s t r a t i n g N a , K A T P a s e , s i n c e , d e s p i t e enzyme i n h i b i t i o n b y S r , t h e r e was s u f f i c i e n t p r e c i p i t a t e t o be s e e n u n d e r t h e e l e c t r o n m i c r o s c o p e . A n o t h e r a p p r o a c h d e p e n d s on t h e e l e c t r o n d e n s i t y o f c e s i u m , w h i c h may be bound t o t h e enzyme i n t h e p l a c e o f p o t a s s i u m i n t h e p r e s e n c e o f ATP and c a r d i a c g l y c o s i d e . T h i s m e t h o d has n o t b e e n w i d e l y u s e d b e c a u s e d e t e c t i o n o f t h e enzyme i s d e p e n d e n t upon c o m p l i c a t e d m i c r o - d e n s i t o m e t r i c m e a s u r e m e n t s o f m i c r o g r a p h s ( 8 4 ) . M o r e r e c e n t l y , t h e N a , K A T P a s e c o m p l e x h a s b e e n l o c a l i z e d b y i m m u n o -c y t o c h e m i c a l t e c h n i q u e s . A n t i b o d i e s r a i s e d a g a i n s t w e l l d e f i n e d p r e p a r a t i o n s o f t h e h o l o e n z y m e h a v e been shown t o r e a c t w i t h N a , K A T P a s e on b o t h s i d e s o f - 16 -t h e p l a s m a membrane ( 8 5 ) . T h e l a r g e and s m a l l p o l y p e p t i d e s o f t h e s o d i u m pump c a n b e s e p a r a t e d f r o m one a n o t h e r by g e l f i l t r a t i o n i n s o d i u m d o d e c y l s u l f a t e , and t h e d e t e r g e n t r e m o v e d ( 7 1 ) . T h e p r o t e i n s a r e t h e n c o m p l e t e l y w a t e r s o l u b l e . P u r i f i e d p r e p a r a t i o n s o f e i t h e r w a t e r s o l u b l e l a r g e c h a i n o r s m a l l c h a i n w e r e e x a m i n e d w i t h r e s p e c t t o t h e i r a b i l i t y t o a b s o r b o u t p o r t i o n s o f a n t i h o l o e n z y m e a n t i - s e r u m ( 8 6 ) , t h e amount o f a b s o r b e d a n t i b o d y b e i n g d e t e r m i n e d by c o m p l e m e n t f i x a t i o n ( 8 7 ) . T h e r e s u l t s o f t h e s e e x p e r i -m e n t s i n d i c a t e d t h a t a n t i - h o l o e n z y m e a n t i - s e r u m c o n t a i n e d X - g l o b u l i n s d i r e c t e d a g a i n s t a n t i g e n i c s i t e s o n b o t h t h e s m a l l and l a r g e c h a i n o f t h e e n z y m e . T h e s e r e s u l t s c o r r e l a t e w i t h t h e a b i l i t y o f t h e a n t i b o d y t o b i n d t o b o t h s i d e s o f t h e membrane s i n c e t h e s m a l l c h a i n i s a s i a l o g l y c o p r o t e i n , p r e s u m a b l y e x p o s e d a t t h e e x t e r n a l s u r f a c e o f . t h e c e l l , w h e r e a s , t h e l a r g e c h a i n c o n t a i n s an a n t i g e n i c s i t e w h i c h i s on t h e c y t o p l a s m i c s i d e o f t h e p l a s m a m e m b r a n e . A n t i b o d i e s h a v e a l s o been r a i s e d a g a i n s t p r e p a r a t i o n s o f t h e w a t e r s o l u b l e ( S D S - d e n a t u r e d ) l a r g e c h a i n ( 8 5 ) . T h e s e a n t i b o d i e s do n o t b i n d t o t h e e x t e r n a l s u r f a c e o f t h e p l a s m a m e m b r a n e , b u t do r e a c t w i t h a n t i g e n i c s i t e s e x p o s e d o n t h e i n t e r n a l s u r f a c e . P r e p a r a t i o n s o f r a b b i t a n t i - s e r a s p e c i f i c f o r t h e s o d i u m pump h a v e been u s e d a s p r i m a r y l i g a n d s i n t r a n s m i s s i o n e l e c t r o n m i c r o s c o p i c s t u d i e s on t h e d i s t r i b u t i o n o f N a , K A T P a s e o v e r t h e s u r f a c e o f t h e p l a s m a membrane o f c a n i n e d i s t a l ( 8 6 ) and p r o x i m a l ( 8 8 ) c o n v o l u t e d t u b u l e c e l l s . F e r r i t i n - c o n j u g a t e d g o a t a n t i - r a b b i t y - g l o b u l i n was u s e d t o l o c a t e t h e s i t e s a t w h i c h t h e p r i m a r y a n t i b o d y was b o u n d ( 8 6 , 8 8 ) . Bound a n t i b o d y has a l s o b e e n v i s u a l i z e d w i t h p e r o x i d a s e l a b e l l e d g o a t a n t i - r a b b i t IgG and s u b s e q u e n t r e a c t i o n w i t h 3 , 3 ' - d i a m i n o b e n z i d i n e p l u s h y d r o g e n p e r o x i d e ( 8 9 ) . - 17 -( I I I ) ROLE OF CHEMICAL DERIVATIVES OF OUABAIN IN THE DEMONSTRATION OF THE  SODIUM PUMP: A number o f c h a l l e n g i n g q u e s t i o n s c o n c e r n i n g o u a b a i n - e n z y m e i n t e r -a c t i o n s w o u l d be a n s w e r e d i f s p e c i f i c o u a b a i n b i n d i n g s i t e s c o u l d be c y t o c h e m i c a l l y v i s u a l i z e d a t t h e u l t r a s t r u c t u r a l l e v e l : 1 . I s t h e N a , K A T P a s e enzyme t h e o n l y o u a b a i n r e c e p t o r , o r p e r h a p s m o r e i m p o r t a n t , i s i t t h e r e c e p t o r o f p h a r m a c o l o g i c a l i n t e r e s t ? 2 . Can t h e f u n c t i o n a l r o l e o f t h e s o d i u m p u m p , i n c e r t a i n t i s s u e s , be e x p l a i n e d b y a d i f f e r e n t i a l d i s t r i b u -t i o n o f t h e enzyme on t h e c e l l ' s s u r f a c e ? 3 . To what e x t e n t i s o u a b a i n i n t e r n a l i z e d by t h e c e l l , and how m i g h t t h i s a c t i v i t y be r e l a t e d t o t h e o b s e r v e d p h y s i o l o g i c e f f e c t ? A . C h e m i c a l D e r i v a t i v e s o f O u a b a i n In o r d e r t o g a i n f u r t h e r i n s i g h t i n t o t h e n a t u r e o f t h e o u a b a i n - e n z y m e i n t e r a c t i o n , i n v e s t i g a t o r s h a v e , i n r e c e n t y e a r s , p r e p a r e d f l u o r e s c e n t o r p h o t o r e a c t a b l e d e r i v a t i v e s o f t h e c a r d i a c g l y c o s i d e ( 2 3 , 2 4 , 2 5 , 9 2 ) . T h e u n d e r -l y i n g s y n t h e t i c r a t i o n a l e f o r t h e s e m o d i f i c a t i o n s i s b a s e d on t h e f a c t t h a t t h e r h a m n o s e m o i e t y o f o u a b a i n c a n u n d e r g o o x i d a t i o n b y p e r i o d a t e t o p r o d u c e a l d e h y d e g r o u p s w h i c h a r e s u s c e p t i b l e t o r e d u c t i v e a m i n a t i o n by s e l e c t e d r e a g e n t s . I n s t u d y i n g t h e n a t u r e o f t h e o u a b a i n r e c e p t o r , p h o t o a f f i n i t y d e r i v -a t i v e s o f o u a b a i n h a v e b e e n s y n t h e s i z e d i n w h i c h t h e 2 - n i t r o - 5 - a z i d o b e n z o y l (NAB) g r o u p was c o u p l e d t o t h e r h a m n o s e s u g a r g r o u p v i a an e t h y l e n e d i a m i n e s p a c e r a r m . T h e s e p h o t o a f f i n i t y a n a l o g u e s have b e e n d e m o n s t r a t e d t o be - 18 -p o t e n t i n h i b i t o r s o f N a , K A T P a s e a c t i v i t y w i t h I^g v a l u e s a r o u n d 0 . 1 ^ . M ( 2 4 ) . P h o t o l a b e l l i n g o f N a , K A T P a s e u s i n g N A B - o u a b a i n , f o l l o w e d by f r a c t i o n -a t i o n v i a SDS 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 , has shown t h a t t h e p h o t o l a b e l i s d i s t r i b u t e d b e t w e e n t h e l a r g e c h a i n o f t h e enzyme and a s m a l l e r m o l e c u l a r w e i g h t p r o t e o l i p i d ( 1 2 0 0 0 D ) . T h e s u g g e s t i o n t h a t p r o t e o l i p i d s may be a s s o c i a t e d w i t h t h e s o d i u m pump i s s i g n i f i c a n t i n t h a t t h e y h a v e b e e n shown t o be c o m p o n e n t s o f t h e p r o t o n t r a n s l o c a t i n g A T P a s e o f m i t o c h o n d r i a , as w e l l a s t h e Ca a c t i v a t e d A T P a s e o f s a r c o p l a s m i c r e t i c u l u m . T h e m i c r o e n v i r o n m e n t o f t h e c a r d i a c g l y c o s i d e b i n d i n g s i t e h a s been i n v e s t i g a t e d t h r o u g h t h e u s e o f a n t h r o y l o u a b a i n , w h i c h i s a s p e c i f i c f l u o r e s -c e n t p r o b e f o r t h e o u a b a i n r e c e p t o r ( 2 3 ) . A n t r o y l o u a b a i n was s y n t h e s i z e d by r e a c t i o n o f a n t h r a c e n e - 9 - c a r b o x y l i c c h l o r i d e w i t h o u a b a i n . The a n t h r a c e n e m o i e t y was shown b y n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o s c o p y t o be e s t e r i f i e d t o t h e r h a m n o s e s u g a r g r o u p o f t h e g l y c o s i d e . T h i s f l u o r e s c e n t a n a l o g u e o f o u a b a i n was a s p e c i f i c i n h i b i t o r o f N a , K A T P a s e and bound t i g h t l y t o t h e e n -zyme w i t h a d i s s o c i a t i o n c o n s t a n t o f a b o u t 2 . 0 x 1 0 ~ 7 M. A n t h r o y l o u a b a i n was i n c u b a t e d i n t h e p r e s e n c e o f p u r i f i e d r a b b i t k i d n e y N a , K A T P a s e and t h e e x c i t a t i o n and e m i s s i o n s p e c t r a , o f t h e a n t h r o y l g r o u p , w e r e m e a s u r e d . C o m p a r i s o n o f t h e a n t h r o y l o u a b a i n f l u o r e s c e n c e p a r a m e t e r s i n t h e enzyme w i t h t h o s e o f m o d e l s y s t e m s s u g g e s t e d t h a t t h e b i n d i n g s i t e was h y d r o p h o b i c a n d / o r v i s c o u s and s h i e l d e d f r o m w a t e r . - 19 -B . T h e s i s : T h e a i m o f t h i s p r e s e n t p r o j e c t i s t o d e v e l o p a d e r i v a t i v e o f o u a b a i n w h i c h c a n be e m p l o y e d as a c e l l s u r f a c e p r o b e f o r t h e l o c a l i z a t i o n o f t h e N a , K A T P a s e e n z y m e . T h e p r i n c i p a l o u a b a i n d e r i v a t i v e s w h i c h w i l l be e x a m i n e d a r e e i t h e r l a t e x m i c r o s p h e r e c o n j u g a t e s o r s m a l l m o l e c u l a r w e i g h t f l u o r e s c e n t d e r i v a t i v e s , u t i l i z i n g t h e 5 - d i m e t h y l a m i n o - l - n a p h t h a l e n e s u l f o n y l (DANS) f l u o r o c h r o m e . I m m u n o l a t e x m i c r o s p h e r e s h a v e , i n r e c e n t y e a r s , been u s e d t o map t h e r e c e p t o r d i s t r i b u t i o n f o r p l a n t l e c t i n s o r c e l l s u r f a c e s p e c i f i c a n t i b o d i e s on a number o f d i f f e r e n t c e l l t y p e s ( 2 , 3 8 , 4 4 ) . C o p o l y m e r l a t i c e s c a n be p r e p a r e d t h r o u g h t h e e m u l s i o n p o l y m e r i z a t i o n o f a g r o u p o f m e t h a c r y l a t e e s t e r comonomers ( 2 6 ) . A t y p i c a l comonomer c o m p o s i t i o n c o n s i s t s o f m e t h y l m e t h a -c r y l a t e ( 5 3 % ) , 2 - h y d r o x y e t h y l m e t h a c r y l a t e ( 3 0 % ) , m e t h a c r y l i c a c i d ( 1 0 % ) , and e t h y l e n g l y c o l d i m e t h a c r y l a t e ( 7 % ) . T h e p o l y m e r i z a t i o n i s c a r r i e d o u t i n t h e p r e s e n c e o f an e m u l s i f i e r a g e n t (SDS) and an ammonium p e r s u l f a t e f r e e r a d i c a l i n i t i a t o r , u n d e r c o n d i t i o n s w h e r e t h e a v e r a g e p a r t i c l e d i a m e t e r i s a l i n e a r f u n c t i o n o f t h e t o t a l monomer c o n c e n t r a t i o n . The r e s u l t i n g l a t e x c o n t a i n s a h i g h p r o p o r t i o n o f h y d r o x y l and c a r b o x y l g r o u p s w h i c h c a n be c o u p l e d t o n u c l e o p h i l i c r e a g e n t s e i t h e r v i a c y a n o g e n b r o m i d e a c t i v a t i o n o r c a r b o d i m i d e c o n d e n s a t i o n . S u c h i m m u n o l a t e x s p h e r e s h a v e p r o v e n t o be h i g h l y s u c c e s s f u l m a r k e r s f o r t h e v i s u a l i z a t i o n o f c e l l s u r f a c e r e c e p t o r s by s c a n n i n g e l e c t r o n m i c r o s c o p y a s w e l l as f l u o r e s c e n c e - l i g h t m o c r o s c o p y . - 20 -E X P E R I M E N T A L P R O C E D U R E S ( 1 ) M A T E R I A L S : T h e f o l l o w i n g c h e m i c a l s were o b t a i n e d f r o m S i g m a C h e m i c a l C o . : o u a b a i n , e q u i n e ATP ( d i s o d i u m s a l t ) , b o v i n e s e r u m a l b u m i n , 1 - e t h y l - 3 - ( 3 -d i m e t h y l a m i n o p r o p y l ) c a r b o d i i m i d e ( E D C ) , C o n c a n a v a l i n A , and t h e f l u o r o -c h r o m e s 5 - d i m e t h y l a m i n o - 1 - n a p h t h a l e n e s u l f o n y l c h l o r i d e ( D A N S - C 1 ) and e - N -D A N S - l y s i n e . S o d i u m d o d e c y l s u l f a t e ( S D S ) was p u r c h a s e d f r o m BDH C h e m i c a l s L t d . ; c y a n o g e n b r o m i d e f r o m MC&B M a n u f a c t u r i n g C h e m i s t s ; a n d 25% a q u e o u s g l u t a r a l d e h y d e was o b t a i n e d f r o m P o l y s c i e n c e s I n c . , W a r r i n g t o n , P a . A l l o t h e r c h e m i c a l s w e r e o f r e a g e n t g r a d e . ( 3 H ) - 0 u a b a i n ( 1 4 . 7 C i / m m o l e ) and c a r r i e r f r e e 1 2 5 i o d i n e ( 1 0 0 m C i / m l ) w e r e o b t a i n e d f r o m A m e r s h a m . P h o s p h a t e - b u f f e r e d s a l i n e ( P B S ) i s c o m p o s e d o f 8 . 0 g N a C l , 0 . 2 K C 1 , 0 . 2 g K H 2 P 0 4 and 1 . 1 5 g N a 2 H P 0 4 p e r l i t e r o f d i s t i l l e d H 2 0 and a d j u s t e d t o pH 7 . 4 . D u l b e c c o ' s m o d i f i e d P B S , c o n t a i n i n g C a C l 2 , was p r e p a r e d a s p r e v i o u s l y d e s c r i b e d ( 2 8 ) . ( 2 ) T I S S U E CULTURE: C e l l c u l t u r e l i n e s o f H e l a ( S 3 ) , mouse n e u r o b l a s t o m a c l o n e ( 2 A ) and 3T3 f i b r o b l a s t s were g r o w n i n E a g l e ' s miniumum e s s e n t i a l m e d i u m , c o n t a i n i n g 10% f o e t a l c a l f s e r u m ( G i b c o ) , p e n i c i l l i n ( 1 0 0 u n i t s / m l ) , s t r e p t o m y c i n ( 1 0 0 / x g / m l ) as w e l l a s f u n g i z o n e ( 1 . 2 5 / z g / m l ) . C e l l s w e r e grown a t 3 7 ° C i n a h u m i d i f i e d i n c u b a t o r c o n t a i n i n g 5% C 0 9 . - 21 -C e l l s t o be u s e d f o r l a b e l l i n g s t u d i e s w e r e p l a t e d o u t o n t o a l c o h o l s t e r i l i z e d g l a s s c o v e r s l i p s 24 h o u r s p r i o r t o u s e . ( 3 ) ANALYTICAL PROCEDURES: C a r d i a c s t e r i o d was a s s a y e d by t h e m e t h o d o f K e d d e ( 1 9 4 7 ) ( 1 ) . To 0 . 0 5 - 0 . 2 m l o f s a m p l e was a d d e d 1 . 0 m l o f 1 .2% d i n i t r o b e n z o i c a c i d i n m e t h a -n o l f o l l o w e d by 0 . 5 ml o f 1 M NaOH. T h e o p t i c a l d e n s i t y a t 5 4 0 nm was d e t e r -m i n e d w i t h i n 5 m i n u t e s . T h i s a s s a y i s q u i t e s p e c i f i c f o r t h e c a r d e n o l i d e s and i s d e p e n d e n t on an i n t a c t l a c t o n e r i n g . A s t a n d a r d c u r v e was c o n s t r u c t e d u t i l i z i n g an a c c u r a t e l y w e i g h e d s t o c k s o l u t i o n o f o u a b a i n o c t a h y d r a t e . The a s s a y was f o u n d t o be l i n e a r o v e r t h e r a n g e 5 /J. g t o 100 fj.q o u a b a i n . P r i m a r y a m i n e was a s s a y e d a s p r e v i o u s l y d e s c r i b e d ( 2 ) . To a 0 . 0 5 - 0 . 2 ml a l i q u o t o f s a m p l e was added 0 . 5 ml o f 0 . 3 % 2 , 4 , 6 - t r i n i t r o b e n z e n e s u l f o n i c a c i d f o l l o w e d by 1 . 0 ml o f s a t u r a t e d N a 2 B 4 0 y . T h e o p t i c a l d e n s i t y o f 4 2 0 nm was d e t e r m i n e d a f t e r 5 m i n u t e s . T h i s a s s a y was u s e d f o r q u a l i t a t i v e p u r p o s e s o n l y . P r o t e i n was d e t e r m i n e d a c c o r d i n g t o t h e m e t h o d o f L o w r y , R o s e b r o u g h F a r r and R a n d a l l ( 1 9 5 1 ) ( 3 ) , u s i n g c r y s t a l l i n e BSA a s t h e s t a n d a r d . I n o r g a n i c p h o s p h a t e was a s s a y e d as p e r t h e p r o c e d u r e o f F i s k and S u b b a r o w ( 4 ) . To an a l i q u o t o f s a m p l e c o n t a i n i n g 5% t r i c h l o r o a c e t i c a c i d was a d d e d an e q u a l v o l u m e o f 4% f e r r o u s s u l p h a t e i n 1 M ^ S O ^ c o n t a i n i n g 1% ammonium m o l y b d a t e (made up f r e s h ) . A s t a n d a r d c u r v e was c o n s t r u c t e d u s i n g an a c c u r a t e l y w e i g h e d s o l u t i o n o f NaH^PO^. The c o n c e n t r a t i o n o f t h e 5 - d i m e t h y l a m i n o - 1 - n a p h t h a l e n e s u l f o n y l (DANS) g r o u p was d e t e r m i n e d f r o m i t s a b s o r p t i o n a t 330 nm u s i n g an e x t i n c t i o n c o - e f f i c i e n t o f 4 5 7 0 l i t r e m o l e ^ c m ' ^ (5). - 22 -( 4 ) OXIDATION OF OUABAIN: The f o l l o w i n g s t a n d a r d p r o c e d u r e was u s e d i n t h e p r o d u c t i o n o f p e r i o d a t e o x i d i z e d o u a b a i n . O u a b a i n ( 1 0 0 mg) was d i s s o l v e d i n 1 0 . 0 m l o f h o t d i s t i l l e d w a t e r and a l l o w e d t o c o o l t o room t e m p e r a t u r e w i t h s t i r r i n g . To t h i s was added 100 mg o f N a l O ^ and t h e m i x t u r e was i n c u b a t e d f o r 45 m i n u t e s a t 2 5 ° C i n t h e d a r k . F o l l o w i n g o x i d a t i o n , e x c e s s o x i d i z i n g a g e n t was a b s o r b e d o u t on an a n i o n e x c h a n g e c o l u m n o f Dowex A G ! - X 1 0 ( C l ~ f o r m ) . The c o l u m n was e l u n t e d w i t h w a t e r and t h e f r a c t i o n s c o n t a i n i n g t h e o x i d i z e d o u a b a i n w e r e p o o l e d and c o n c e n t r a t e d on a r o t a r y e v a p o r a t o r . T h e s o l u t i o n o f o x i d i z e d o u a b a i n was s t o r e d a t 4 ° C u n t i l u s e d . ( 5 ) PREPARATION OF DANSYL L Y S I N E OUABAIN: D a n s y l l y s i n e ( 5 0 0 mg) was d i s s o l v e d i n 5 . 0 ml o f H,,0 and a d j u s t e d t o pH 9 . 5 - 1 0 . 0 . To t h i s was a d d e d 500 mg o f o x i d i z e d o u a b a i n ( i n 10 m l o f w a t e r ) and t h e r e a c t a n t s w e r e i n c u b a t e d f o r 1 h o u r a t 2 5 ° C w i t h s t i r r i n g . A t t h e end o f t h i s t i m e 5 0 0 mg o f NaBH^ w e r e a d d e d and t h e r e a c t i o n m i x t u r e was m a i n t a i n e d a t pH 9 . 0 f o r a f u r t h e r 30 m i n u t e s . The r e a c t i o n m i x t u r e was t h e n c o o l e d t o 4 ° C and t h e u n r e a c t e d b o r o h y d r i d e was d e -c o m p o s e d by t h e a d d i t i o n o f 1 N HC1 t o pH 4 . 5 f o l l o w e d by r e t i t r a t i o n t o pH 6 . 5 w i t h NaOH. U n r e a c t e d o u a b a i n was s e p a r a t e d f r o m t h e D A N S - l y s - o u a b a i n a d d u c t b y c h r o m a t o g r a p h y o n Dowex A G 5 0 - X 1 2 ( H + f o r m ) c a t i o n e x c h a n g e r e s i n . T h e e l u a n t and w a t e r w a s h , c o n t a i n i n g f r e e o u a b a i n , w e r e d i s c a r d e d ; t h e s p e c i f i c a l l y a d s o r b e d m a t e r i a l was e l u t e d w i t h 1 . 0 M t r i e t h y l a m i n e i n 20% - 2 3 -a q u e o u s e t h a n o l . T h e e l u t e d m a t e r i a l was c o n c e n t r a t e d o n a r o t a r y e v a p o r a t o r and c h r o m a t o g r a p h e d on a B i o G e l P - 2 g e l f i l t r a t i o n c o l u m n , i n o r d e r t o s e p a r a t e u n r e a c t e d D A N S - l y s i n e f r o m t h e f i n a l p r o d u c t , D A N S - l y s - o u a b a i n . T h e c o l u m n was e l u t e d w i t h 5 mM NaHCG"3 and f r a c t i o n s w h i c h g a v e a p o s i t i v e t e s t f o r o u a b a i n and a b s o r b e d s t r o n g l y a t 330 nm w e r e p o o l e d and c o n c e n t r a t e d . T h e f i n a l p r o d u c t e l u t e d as a s i n g l e peak when r e - c h r o m a t o g r a p h e d on t h e same c o l u m n . ( 6 ) IMMOBILIZATION OF OUABAIN ON COPOLYMER MICROSPHERES: The c a r d i a c g l y c o s i d e was c o v a l e n t l y a t t a c h e d t o m e t h y l m e t h a c r y l a t e o o c o p o l y m e r l a t i c e s o f two d i f f e r e n t p a r t i c l e s i z e s , e i t h e r 200 A o r 1000 A . The c o p o l y m e r m i c r o s p h e r e s u s e d i n t h i s s t u d y w e r e p r e p a r e d by e m u l s i o n p o l y m e r i z a t i o n t e c h n i q u e s and w e r e a g e n e r o u s g i f t o f S . P . S . Yen f r o m t h e J e t P r o p u l s i o n L a b o r a t o r y , P a s a d e n a C a l i f o r n i a . The s y n t h e t i c p r o c e d u r e s d e s c r i b e d b e l o w a r e g e n e r a l f o r b o t h l a t e x p r e p a r a t i o n s and c o n s i s t o f f i r s t t a g g i n g t h e m i c r o s p h e r e s w i t h t h e f l u o r o c h r o m e F I T C ; s u b s e q u e n t t o t h i s t h e s p h e r e s a r e f u r t h e r d e r i v a t i z e d w i t h d i a m i n o h e p t a n e p r i o r t o c o u p l i n g t o p e r i o d a t e o x i d i z e d o u a b a i n . H y d r o x y l g r o u p s on t h e l a t e x were a c t i v a t e d w i t h c y a n o g e n b r o m i d e (5 m g / m l ) a t pH 9 . 5 , f o l l o w i n g t h e m e t h o d o f C u a t r e c a s s u s ( 1 9 7 0 ) ( 3 3 ) . The m i x t u r e was t h e n c h i l l e d on i c e and e t h y l e n e d i a m i n e (pH 9 . 5 ) was added t o a f i n a l c o n c e n t r a t i o n o f 50 mM and a l l o w e d t o r e a c t f o r 12 h o u r s a t 4 ° C . E x c e s s d i a m i n o r e a g e n t was r e m o v e d b y d i a l y s i s a g a i n s t 4 mM s o d i u m c a r b o n a t e (pH 9 . 5 ) a f t e r w h i c h FITC was added t o 0 . 5 mg/ml and r e a c t e d a t 2 5 ° C f o r 3 h o u r s . The m i c r o s p h e r e s w e r e t h e n d i a l y z e d a g a i n s t 10 mM HEPES pH 7 . 2 p r i o r - 24 -t o b e i n g r e - d e r i v a t i z e d w i t h h e p t a n e d i a m i n e ( 5 0 mM) u s i n g t h e a q u e o u s c a r b o -d i i m i d e , EDC, (5 m g / m l ) f o r 2 h o u r s a t 4 ° C . E x e s s r e a g e n t s w e r e r e m o v e d b y e x t e n s i v e d i a l y s i s a g a i n s 0 . 1 M . N a C l . The d e r i v a t i z e d l a t e x was t h e n r e a c t e d w i t h p e r i o d a t e o x i d i z e d o u a b a i n (2 mM) a t pH 9 . 5 f o r 2 h o u r s a t room t e m p e r a t u r e , f o l l o w e d by s o d i u m b o r o h y d r i d e r e d u c t i o n as p r e v i o u s l y d e s c r i b e d . T h e l a t e x c o n j u g a t e was t h e n p u r i f i e d by s t e p - w i s e s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n u s i n g a t w o s t e p g r a d i e n t o f 15% (w/w) and 60% (w/w) s u c r o s e . T h e s a m p l e was o v e r l a y e d o n t o t h e 15% s u c r o s e and c e n t r i f u g e d f o r 3 h o u r s a t 1 0 0 , 0 0 0 x g i n t h e SW 27 r o t o r , u s i n g a Beckman L 5 - 6 5 u l t r a c e n t r i f u g e . T h e p u r i f i e d m i c r o s p h e r e c o n j u g a t e was c o l l e c t e d a t t h e i n t e r f a c e o f t h e 15% and 60% s u c r o s e l a y e r s and d i a l y z e d e x t e n s i v e l y a g a i n s t 0 . 0 5 M T r i s - C l pH 7 . 4 and s t o r e d a t 4 ° C u n t i l u s e d . ( 7 ) ENZYME PREPARATION: A m i c r o s o m a l f r a c t i o n o f N a , K A T P a s e was p r e p a r e d f r o m b e e f b r a i n , f o l l o w i n g t h e m e t h o d o f S c h o n e r e t a l . ( 1 9 6 7 ) ( 2 9 ) . A l l o p e r a t i o n s w e r e p e r f o r m e d a t 0 - 4 ° C . B e e f b r a i n was o b t a i n e d i m m e d i a t e l y a f t e r s l a u g h t e r and p a c k e d on i c e . A p p r o x i m a t e l y 150 g o f b e e f b r a i n was h o m o g e n i z e d ( 3 m i n u t e s ) i n a W a r i n g b l e n d e r w i t h 5 0 0 ml o f i c e c o l d 0 . 2 5 M s u c r o s e c o n t a i n i n g 1 mM EDTA. The h o m o g e n a t e was c e n t r i f u g e d a t 9 , 5 0 0 rpm f o r 4 0 m i n u t e s i n a S o r v a l l RC-2B c e n t r i f u g e . T h e r e s u l t i n g p e l l e t was r e - h o m o g e n i z e d i n 500 m l 0 . 2 5 M s u c r o s e (1 mM EDTA) f o r 2 - 3 m i n u t e s . S o d i m d e o x y c h o l a t e (20%) was t h e n added t o a f i n a l c o n c e n t r a t i o n o f 0 . 2 % ( w / v ) . T h e s u s p e n s i o n was t h e n s t o r e d a t 0 - 4 ° C f o r 5 d a y s . - 25 -F o l l o w i n g t h i s t r e a t m e n t t h e s u s p e n s i o n was m i x e d w i t h an e q u a l v o l u m e o f 0 . 2 5 M s u c r o s e c o n t a i n i n g 10 mM t r i s - a c e t a t e pH 7 . 4 and 1 . 0 mM EDTA, and c e n t r i f u g e d a t 1 3 , 0 0 0 rpm f o r 4 0 m i n u t e s i n a S o r v a l ! S S 3 4 r o t o r . T h e r e s u l t i n g s u p e r n a t a n t s were p o o l e d and r e - c e n t r i f u g e d a t 1 0 0 , 0 0 0 x g f o r 1 h o u r i n t h e 6 0 T i r o t o o f a Beckman L 5 - 6 5 u l t r a c e n t r i f u g e . E a c h p e l l e t ( f r o m 45 ml o f c r u d e h o m o g e n a t e ) was r e s u s p e n d e d i n 2 . 0 ml o f 0 . 2 5 M s u c r o s e , w h i c h was b u f f e r e d w i t h T r i s - a c e t a t e t o pH 7 . 4 . T h i s c r u d e m i c r o s o m a l p r e p a r a t i o n had a s p e c i f i c N a , K A T P a s e a c t i v i t y o f 450 n m o l e s o f ATP h y d r o l y z e d m i n ~ ^ m g ~ ' o f p r o t e i n a t 3 7 ° C . ( 8 ) A T P a s e A C T I V I T Y DETERMINATION: T h e c r u d e m i c r o s o m a l A T P a s e p r e p a r a t i o n s ( u s u a l l y 35 fjug o f p r o t e i n ) w e r e p r e - i n c u b a t e d f o r 15 m i n u t e s a t 3 7 ° C , i n medium c o n t a i n i n g 50 mM T r i s - C l pH 7 . 4 , 100 mM N a C l , 3 mM M g C l 2 , 3 mM A T P - T r i s s a l t , i n t h e p r e s e n c e o r a b s e n c e o f o u a b a i n o r i t s d e r i v a t i v e s . F o l l o w i n g t h i s p r e -i n c u b a t i o n , KC1 was added t o a f i n a l c o n c e n t r a t i o n o f 20 mM. T h e enzyme was i n c u b a t e d f o r a f u r t h e r 30 m i n u t e s a t 3 7 ° C and t h e r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f an e q u a l v o l u m e o f i c e c o l d 10% t r i c h l o r o a c t i c a c i d . The p r e c i p i t a t e d p r o t e i n was r e m o v e d by c e n t r i f u g a t i o n i n an E p p e n d o r f m i c r o -c e n t r i f u g e ( 1 2 , 0 0 0 x g f o r 5 m i n u t e s ) and t h e s u p e r n a t a n t a s s a y e d f o r t h e p r e s e n c e o f i n o r g a n i c p h o s p h a t e b y t h e method p r e v i o u s l y d e s c r i b e d . O u a b a i n s e n s i t i v i t y and n o n e n z y m a t i c ATP h y d r o l y s i s w e r e d e t e r m i n e d i n t u b e s c o n t a i n i n g 1 mM o u a b a i n o r no e n z y m e , r e s p e c t i v e l y . ATP h y d r o l y s i s was l i n e a r w i t h t i m e u n d e r t h e a b o v e c o n d i t i o n s . - 26 -( 9 ) BINDING S T U D I E S : 3 9 T h e b i n d i n g o f ( H ) - o u a b a i n ( 1 . 4 x 10 d p m / ^ t m o l e ) t o t h e b e e f b r a i n m i c r o s o m a l membrane p r e p a r a t i o n was d e t e r m i n e d by f i l t r a t i o n t e c h n i q u e s . A l i q u o t s c o n t a i n i n g 0 . 4 mg o f p r o t e i n w e r e i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f t h e l i g a n d i n 1 . 0 m l o f i n c u b a t i o n b u f f e r f o r 40 m i n u t e s a t 3 7 ° C . T h e i n c u b a t i o n b u f f e r c o n t a i n e d 50 mM T r i s - C l pH 7 . 4 , 100 mM N a C l , 3 mM M g C l 2 , and 3 mM ATP ( T r i s s a l t ) . F o l l o w i n g i n c u b a t i o n s , t h e s a m p l e s w e r e m i x e d r a p i d l y w i t h an e q u a l v o l u m e o f i c e c o l d 0 . 0 5 M M g C l 2 and c o l l e c t e d on Mi 1 1 i p o r e f i l t e r s ( 0 . 4 5 HAWP). T h e f i l t e r s w e r e o r i n s e d w i t h 1 0 . 0 ml o f i c e c o l d w a t e r , d r i e d i n a i r a t a b o u t 8 0 C and d i s s o l v e d i n 1 0 . 0 m l o f ACS a q u e o u s s c i n t i l l a t i o n f l u i d . I n a l l c a s e s t h e " s p e c i f i c b i n d i n g " was d e t e r m i n e d f r o m t h e d i f f e r e n c e b e t w e e n t h i s v a l u e and t h e amount o f ( H ) - o u a b a i n bound i n t h e p r e s e n c e o f 1 mM n a t i v e o u a b a i n . C o m p e t i t i v e b i n d i n g e x p e r i m e n t s i n w h i c h t h e a f f i n i t y d e r i v a t i v e s o f o u a b a i n were u s e d t o c o m p e t e o f f ( H ) - o u a b a i n b i n d i n g s i t e s w e r e c o n d u c t e d i n t h e f o l l o w i n g m a n n e r . M i c r o s o m a l membrane a l i q u o t s ( 0 . 2 5 mg p r o t e i n ) w e r e p r e - i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f t h e n o n - r a d i o a c t i v e l i g a n d o , f o r 4 0 m i n u t e s a t 37 C . ( H ) - o u a b a i n was added t o a f i n a l c o n c e n t r a t i o n o f 0.2/J.M and t h e s a m p l e s w e r e i n c u b a t e d f o r a f u r t h e r 15 m i n u t e s . A t t h e end o f t h i s t i m e t h e s a m p l e s w e r e q u e n c h e d o n i c e w i t h 0 . 0 5 M M g C l2 and p r o c e s s e d by f i l t r a t i o n as p r e v i o u s l y d e s c r i b e d . T h e b i n d i n g o f ( 3 H ) - o u a b a i n ( 1 . 4 x 1 0 9 d p r n / ^ m o l e ) t o H e l a ( S 3 ) c e l l s was c o n d u c t e d i n t h e f o l l o w i n g m a n n e r . H e l a c e l l s w e r e grown t o c o n f l u e n c e i n E a g l e s minimum e s s e n t i a l medium ( c o n t a i n i n g 10% f o e t a l c a l f s e r u m ) and h a r v e s t d w i t h 1 mM EDTA i n P B S . D i s s o c i a t e d c e l l s w e r e w a s h e d - 27 -t w i c e i n t h e i n c u b a t i o n b u f f e r ( 0 . 0 5 M s o d i u m p h o s p h a t e pH 7 . 4 , 0 . 1 4 5 M N a C L , 3 mM MgCL^) b y c e n t r i f u g a t i o n and r e s u s p e n d e d t o a c o n c e n t r a t i o n o f 7 fi 5 . 0 x 10 c e l l s / m l . I n c u b a t i o n s w e r e c a r r i e d o u t u s i n g 9 . 0 x 10 c e l l s and v a r i o u s c o n c e n t r a t i o n s o f ( H ) - o u a b a i n , i n a t o t a l v o l u m e o f 0 . 2 m l , o f o r 4 0 m i n u t e s a t 37 C . F o l l o w i n g e q u i l i b r i u m t h e s a m p l e s w e r e l a y e r e d o n t o 0 . 2 ml o f 5% BSA i n 0 . 4 ml p o l y p r o p y l e n e m i c r o c e n t r i f u g e t u b e s and p e l l e t e d a t 1 2 , 0 0 0 x g f o r 1 m i n u t e i n an E p p e n d o r f m i c r o f u g e . T h e c e l l p e l l e t s w e r e t h e n s o l u b i z e d i n c o n c e n t r a t e d s u l f u r i c a c i d and c o u n t e d i n ACS a q u e o u s s c i n t i l l a t i o n f l u i d . - 28 -R E S U L T S ( 1 ) PREPARATION OF OUABAIN DERIVATIVES A . PERIODATE OXIDATION: The o x i d a t i o n o f t h e c a r d i a c g l y c o s i d e by N a I 0 4 was e s s e n t i a l l y q u a n t i t a t i v e as d e t e r m i n e d by TLC on S i l i c a g e l G p l a t e s . N a t i v e o u a b a i n had an R^ o f 0 . 3 5 as c o m p a r e d t o an R^ o f 0 . 6 3 f o r p e r i o d a t e o x i d i z e d o u a b a i n u s i n g c h o l o r o f o r m - m e t h a n o l ( 2 : 1 ) as t h e d e v e l o p i n g s o l v e n t . The p r e s e n c e o f u n t r e a t e d o r p e r i o d a t e o x i d i z e d g l y c o s i d e was d e t e c t e d by a s p r a y r e a g e n t c o n t a i n i n g 1.2% 3 , 5 - d i n i t r o b e n z o i c a c i d / 0 . 5 M NaOH. B . OUABAIN-MICROSPHERE CONJUGATES: P r e l i m i n a r y e x p e r i m e n t s , i n w h i c h t h e c a r d i a c g l y c o s i d e was c o v a l e n t l y a t t a c h e d t o m e t h a c r y l a t e c o p o l y m e r m i c r o s p h e r e s , w e r e q u i t e p r o m i s i n g . H y d r o x y l g r o u p s on t h e l a t e x were a c t i v a t e d w i t h c y a n o g e n b r o m i d e and s u b s e q u e n t l y d e r i v a t i z e d w i t h t h e f l u o r o c h r o m e F I T C . C a r b o x y l g r o u p s on t h e s p h e r e s w e r e t h e n r e a c t e d w i t h h e p t a n e - 1 , 7 - d i a m i n e i n t h e p r e s e n c e o f an a q u e o u s c a r b o d i i m i d e , and t h e s p h e r e s w e r e s u b s e q u e n t l y r e a c t e d w i t h p e r i o d a t e o x i d i z e d o u a b a i n . T h e S c h i f f b a s e was r e d u c e d w i t h N a B H 4 a t pH 8 . 0 and t h e r e s u l t i n g c o n j u g a t e was p u r i f i e d on a d i s c o n t i n u o u s g r a d i e n t o f 15% (w/w) and 6 0 % (w/w) s u c r o s e . A r e a c t i o n scheme o u t l i n i n g t h e g e n e r a l s t e p s f o r c h e m i c a l m o d i f i c a t i o n o f m e t h a c r y a t e s p h e r e s i s g i v e n i n F I G : 2 . 29 HO-^C OOH CN Br ethylene diamine FITC H00C-^^NH-(CH 2) 2- NH E DC FLUORESCEIN heptane diamine OUABAIN (oxidized) 0 HO-OcNH-(CH„)-NH 2 7 OUABAIN FIG 2 C H E M I C A L MODIFICATIONS OF C O P O L Y M E R M I C R O S P H E R E S - 30 -r o ' O 6 Q. O > h -O < o Q < cr F I G : 244-20 + I64> 12 + 8 + 44-( H ) - 0 U A B A I N I v o i d v o l u m e 8 1 * ( J O O O (D O 24 32 40 FRACTION NUMBER 48 F r a c t i o n a t i o n o f a ( J H ) - o u a b a i n - 1 a t e x c o n j u g a t e ( 2 0 0 A d i a m e t e r ) on S e p h a r o s e ( C L ) - 6 B ( 1 . 0 x 20 c m ) . T h e c o l u m n was e l u t e d w i t h 0 . 0 5 M t r i s - a c e t a t e pH 7 . 4 a t a f l o w r a t e o f 8 m l / h o u r a n d 30 d r o p f r a c t i o n s w e r e c o l l e c t e d a n d a s s a y e d f o r r a d i o a c t i v i t y . S a m p l e s o f l a t e x c o n j u g a t e w e r e c h r o m a t o g r a p h e d p r i o r t o s u c r o s e d e n s i t y g r a d i e n t p u r i f i c a t i o n ( • • ) a n d a g a i n f o l l o w i n g p u r i f i c a t i o n (O O) . T h e v o i d v o l u m e o f t h e c o l u m n was d e t e r m i n e d w i t h b l u e d e x t r a n a n d t h e p o s i t i o n o f e l u t i o n o f o u a b a i n was d e t e r m i n e d by an i n d e p e n d e n t f r a c t i o n a t i o n o f a s a m p l e o f ( 3 H ) - o u a b a i n . - 31 -I n some c o u p l i n g p r o c e d u r e s t h e s e l e c t i v e r e d u c i n g a g e n t NaCNBH^ was e m p l o y e d a s a s u b s t i t u t e f o r N a B H ^ . T h e c y a n o b o r o h y d r i d e a n i o n h a s b e e n r e p o r t e d t o be s e l e c t i v e f o r t h e i m m i n i u m m o i e t y s u c h t h a t r e d u c t i o n c a n be c a r r i e d o u t u n d e r m i l d e r c o n d i t i o n s w i t h f e w e r p o s s i b l e s i d e r e a c t i o n s ( 3 0 ) . T y p i c a l l y , r e d u c t i o n w i t h c y a n o b o r o h y d r i d e was p e r f o r m e d a t pH 6 . 5 i n s o d i u m a c e t a t e f o r 72 h o u r s a t room t e m p e r a t u r e . O u a b a i n - l a t e x c o n j u g a t e s w h i c h w e r e p r e p a r e d e i t h e r v i a c y a n o b o r o h y d r i d e o r b o r o h y d r i d e r e d u c t i o n d i s p l a y e d c o m p a r a b l e d e g r e e s o f s u b s t i t u t i o n and w e r e e q u a l l y a c t i v e as N a , K A T P a s e i n h i b i t o r s . I n c e r t a i n e x p e r i m e n t s , t h e l a t e x s p h e r e s w e r e d e r i v a t i z e d w i t h ( H ) - o u a b a i n . P r e v i o u s l y i t has been r e p o r t e d t h a t m e t h y l - m e t h a c r y l a t e c o p o l y m e r l a t i c e s h a v e a d e n s i t y o f 1 . 2 3 2 g cm ( 9 1 ) . T h i s f a c t a l o n g w i t h t h e l a t e x d i a m e t e r was u t i l i z e d i n c a l c u l a t i n g t h e a p p r o x i m a t e e x t e n t o f d e r i v a t i z a t i o n . I t was c a l c u l a t e d t h a t a p p r o x i m a t e l y 50 o u a b a i n m o l e c u l e s c o u l d be i m m o b i l i z e d p e r m i c r o s p h e r e u n d e r t h e r e a c t i o n c o n d i t i o n s u s e d . I n o r d e r t o d e m o n s t r a t e t h a t t h e o u a b a i n - m i c r o s p h e r e c o n j u g a t e s w e r e f r e e o f a n y u n d e r i v a t i z e d c a r d i a c g l y c o s i d e , a s a m p l e o f ( H ) - o u a b a i n d e r i v a t i z e d l a t e x was c h r o m a t o g r a p h e d on a S e p h a r o s e ( 6 B ) c o l u m n b e f o r e and a f t e r p u r i f i c a t i o n on a s u c r o s e d e n s i t y g r a d i e n t . ( S e e F I G : 3 ) . I n t h e c r u d e s a m p l e , t h e m a j o r i t y o f t h e t r i t i u m c o u n t s w e r e e l u t e d a t t h e s a l t v o l u m e o f t h e c o l u m n . W i t h t h e p u r i f i e d c o n j u g a t e , a l l o f t h e r a d i o a c t i v i t y o e l u t e d a t t h e v o i d v o l u m e , i n d i c a t i n g t h a t t h e r e was no f r e e ( H ) - o u a b a i n p r e s e n t . - 32 -C . DANS-LYS-OUABAIN D E R I V A T I V E S : S i n c e i t was a p p a r e n t , f r o m t h e i n i t i a l e x p e r i m e n t s on t h e o u a b a i n - l a t e x c o n j u g a t e s , t h a t s t e r i c a c c e s s i b i l i t y c o u l d be a v e r y r e a l p r o b l e m ( d i s c u s s e d i n a l a t e r s e c t i o n ) , i t was d e c i d e d t o c o n c e n t r a t e on a s m a l l m o l e c u l a r w e i g h t d e r i v a t i v e o f o u a b a i n . The c a r d i a c g l y c o s i d e was c o v a l e n t l y c o u p l e d t o D A N S - l y s i n e w i t h t h e a i m t h a t t h i s d e r i v a t i v e c o u l d be l o c a l i z e d on t h e c e l l s u r f a c e e i t h e r d i r e c t l y by f l u o r e s c e n c e m i c r o s c o p y o r i n d i r e c t l y u s i n g t h e m o d i f i e d h a p t e n s a n d w i c h t e c h n i q u e , d e s c r i b e d i n P a r t I I . ( S e e F I G : 4 f o r t h e r e a c t i o n s t e p s i n v o l v e d i n t h e p r e p a r a t i o n o f D A N S - l y s - o u a b a i n ) . T h e r e a c t i o n p r o d u c t b e t w e e n o x i d i z e d o u a b a i n and D A N S - l y s i n e was p u r i f i e d by i o n e x c h a n g e and g e l f i l t r a t i o n c h r o m a t o g r a p h y . I n o r d e r t o d e m o n s t r a t e t h a t o u a b a i n w o u l d n o t be a d s o r b e d o n t o DOWEX i o n e x c h a n g e r e s i n , an a l i q u o t o f p e r i o d a t e o x i d i z e d ( H ) - o u a b a i n was c h r o m a t o g r a p h e d on t h e i o n e x c h a n g e c o l u m n . ( F I G : 5 ) . A l l o f t h e r a d i o a c t i v i t y c o u l d be r e c o v e r e d i n t h e c o l u m n w a s h , i n d i c a t i n g t h a t t h e r e was no i n t e r a c t i o n b e t w e e n t h e c a r d i a c g l y c o s i d e and t h e DOWEX r e s i n . U n r e a c t e d o u a b a i n was s e p a r a t e d f r o m t h e DANS d e r i v a t i v e on a DOWEX-H c o l u m n . M a t e r i a l w h i c h was b o u n d t o t h e DOWEX-H c o l u m n was e l u t e d w i t h 1 . 0 M t r i e t h y l a m i n e i n 20% e t h a n o l . T h i s s p e c i f i c a l l y e l u t e d m a t e r i a l was c o m -p r i s e d o f D A N S - l y s - o u a b a i n and u n r e a c t e d D A N S - l y s i n e . As shown i n F i g u r e 6 D A N S - l y s - o u a b a i n c o u l d be s e p a r a t e d f r o m D A N S - l y s i n e by g e l f i l t r a t i o n on B i o G e l P - 2 . I n o r d e r t o d e m o n s t r a t e i t s s t a b i l i t y , an a l i q u o t o f p u r i f i e d o D A N S - l y s - o u a b a i n was s t o r e d a t - 2 0 C f o r a p e r i o d o f 4 m o n t h s and t h e n c h r o m a t o g r a p h e d on B i o G e l P - 2 ( s e e F I G : 7 ) . - 33 -D A N S - l y s i n e - OUABAIN FIG: 4 S y n t h e t i c r o u t e f o l l o w e d i n t h e p r e p a r a t i o n o f D A N S - l y s - o u a b a i n . The v i c i n a l h y d r o x y l groups o f t h e rhamnose sugar m o i e t y o f o u b a i n a r e s u s c e p t i b l e t o p e r i o d a t e o x i d a t i o n , g i v i n g r i s e t o f o r m i c a c i d and t h e d i a l d e h y d e i n t e r m e d i a t e . The o x i d i z e d o u a b a i n i s h i g h l y r e a c t i v e towards p r i m a r y amines and upon r e d u c t i v e a m i n a t i o n w i t h sodium b o r o h y d r i d e w i l l form the m o r p h o l i n o d e r i v a t i v e i n which t h e amino n i t r o g e n i s i n c o r p o r a t e d i n t o a six-membered r i n g . - 3 4 -20 + r o i _ E Q . O < g < or 4 8 12 FRACTION 16 20 24 NUMBER F I G : 5 C H R O M A T O G R A P H Y OF H - O U A B A I N ON DOWEX ( A G 5 0 X 1 2 ) - H  I O N E X C H A N G E R E S I N A D O W E X - H + c o l u m n o f a b o u t 3 . 0 m l b e d v o l u m e w a s l o a d e d w i t h o . l m l o f ( 3 H ) - o u a b a i n ( 9 . 9 8 x 1 0 $ c p m t o t a l ) a n d t h e c o l u m n w a s e l u t e d w i t h d i s t i l l e d w a t e r . F r a c t i o n s ( 3 0 d r o p : 0 . 5 3 m l ) w e r e c o l l e c t e d a n d 0 . 0 5 m l a l i q u o t s o f e a c h f r a c t i o n , w e r e a s s a y e d f o r ( H ) - o u a b a i n b y s c i n t i l l a t i o n t e c h n i q u e s . I t w a s c a l c u l a t e d t h a t - 9 8 . 5 % o f t h e a p p l i e d r a d i o a c t i v i t y w a s c o l l e c t e d i n t h e w a t e r w a s h . - 35 -FRACTION NUMBER F I G : 6 B i o G e l P - 2 f r a c t i o n a t i o n o f t h e m i x t u r e o f a m i n e s , c o n t a i n i n g D A N S - l y s - o u a b a i n a n d D A N S - l y s i n e , w h i c h had p r e v i o u s l y b e e n e l u t e d f r o m a D o w e x - H + e x c h a n g e c o l u m n . The B i o G e l c o l u m n (1 . 2 x 50 cm) was d e v e l o p e d w i t h 0 . 0 2 % Na, CCXj and 35 d r o p f r a c t i o n s w e r e c o l l e c t e d and a s s a y e d f o r a b s o r b a n c e a t 330 nm ( • #) a s w e l l a s f o r t h e p r e s e n c e o f c a r d i a c s t e r o i d (O O) • F r a c t i o n s c o n t a i n i n g D A N S - L Y S - o u a b a i n w e r e t h e n p o o l e d , c o n c e n t r a t e d and r e c h r o m a t o g r a p h e d t o a s i n g l e p e a k . - 36 -FRACTION NUMBER F I G : 7 3 A s a m p l e o f p u r i f i e d D A N S - l y s - ( H ) - o u a b a i n , w h i c h had b e e n s t o r e d a t - 2 0 ° C f o r s e v e r a l m o n t h s was r e c h r o m a t o g r a p h e d o n a B i o G e l P - 2 g e l f i l t r a t i o n c o l u m n (1 . 2 x 50 c m ) . T h e c o l u m n was e l u t e d w i t h 0 . 0 2 % N a 2 C 0 3 a n d 35 d r o p f r a c t i o n s w e r e c o l l e c t e d a n d a s s a y e d f o r a b s o r b a n c e a t 330 nm 3 (O—O) a s w e l l as r a d i o a c t i v i t y ( • - — • ) . The p o s i t i o n o f e l u t i o n o f ( H) - o u a b a i n i s i n d i c a t e d (a ©) f r o m an i n d e p e n d e n t f r a c t i o n a t i o n u n d e r i d e n t i c a l c o n d i t i o n s . T h e a b s e n c e o f a n y d e t e c t a b l e f r e e o u a b a i n i n t h e s a m p l e o f D A N S - l y s - o u a b a i n , e v e n a f t e r l o n g t e r m s t o r a g e , i s e v i d e n c e f o r t h e s t a b i l i t y o f t h e c o v a l e n t l i k a g e b e t w e e n t h e DANS m o i e t y a n d t h e c a r d i a c g l y c o s i d e . - 37 -( 2 ) BINDING STUDIES A . ( 3 H ) - O U A B A I N BINDING DATA: T h e i n h i b i t o r y a c t i v i t y o f n a t i v e o u a b a i n was d e t e r m i n e d by i n c u b a t i n g a l i q u o t s o f c r u d e b e e f b r a i n m i c r o s o m a l N a , K A T P a s e i n t h e p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s o f o u a b a i n . T h e h a l f m a x i m a l c o n c e n t r a t i o n n e c e s s a r y f o r i n h i b i t i o n o f enzyme a c t i v i t y by t h e c a r d i a c g l y c o s i d e was c a l c u l a t e d t o p be 6 . 5 x 1 0 " M when t h e enzyme was p r e i n c u b a t e d w i t h o u a b a i n p l u s ATP and Na p r i o r t o t h e a d d i t i o n o f K . . T h e e q u i l i b r i u m d i s s o c i a t i o n c o n s t a n t o f n a t i v e o u a b a i n was d e t e r m i n e d f r o m d i r e c t b i n d i n g s t u d i e s u s i n g t h e b e e f b r a i n m i c r o s o m a l N a , K A T P a s e . The b i n d i n g c u r v e f o r ( 3 H ) - o u a b a i n (1.4 x 1 0 9 c p m / / x m o l e ) a t 3 7 ° C i n K f r e e , medium i s shown i n F i g u r e 8 . S c a t c h a r d a n a l y s i s o f t h e d a t a was a K d o f 2 . 8 x 1 0 " 8 M f o r t h e b i n d i n g o f ( 3 H ) - o u a b a i n . ( F I G : 1 0 ) . T h e b i n d i n g o f ( H ) - o u a b a i n t o m i c r o s o m a l N a , K A T P a s e was a l s o s t u d i e d u s i n g an i n d i r e c t a s s a y t e c h n i q u e . In t h i s p r o c e d u r e a l i q u o t s o f membrane enzyme w e r e i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f u n l a b e l l e d n a t i v e o u a b a i n u n t i l e q u i l i b r i u m had b e e n a c h i e v e d . The enzyme was f u r t h e r i n c u b a t e d w i t h a s a t u r a t i n g c o n c e n t r a t i o n o f ( H ) - o u a b a i n and t h e amount o f b o u n d r a d i o a c t i v i t y was d e t e r m i n e d as a p e r c e n t a g e o f t h e t o t a l amount bound i n t h e a b s e n c e o f a n y p r e i n c u b a t i o n . I t was c a l c u l a t e d t h a t h a l f -m a x i m a l p r o t e c t i o n a g a i n s t ( H ) - o u a b a i n b i n d i n g was a c h i e v e d a t a c o n c e n t r a t i o n o f 3 . 0 x I O - 8 M u n l a b e l l e d o u a b a i n . ( S e e F I G : 1 3 ) . T h i s v a l u e a g r e e s c l o s e l y w i t h t h e d i s s o c i a t i o n c o n s t a n t f o r o u a b a i n d e t e r m i n e d f r o m d i r e c t b i n d i n g a s s a y s . - 38 -ro i g 0.2 0.4 0.6 0.8 3 ( H)-0UABAIN CONCENTRATION (/i.M) F I G : 8 3 B i n d i n g o f ( H ) - o u a b a i n t o b e e f b r a i n m i c r o s o m a l membrane p r e p a r a t i o n . A l i q u o t s o f membrane (0.4 mg p r o t e i n ) w e r e i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f ( ^ H ) - o u a b a i n a t 3 7 ° C . T h e enzyme was t h e n 3 c o l l e c t e d on M i l l i p o r e HAWP f i l t e r s a n d a s s a y e d f o r bound ( H ) - o u a b a i n ( O O ) . - 39 -F I G : 9 3 B i n d i n g o f ( H ) - o u a b a i n t o H e l a c e l l s grown i n t i s s u e c u l t u r e . H e l a c e l l s ( 9 . 4 4 x 1 0 6 / i n c u b a t i o n ) w e r e i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s 3 - 4 o f ( H ) - o u a b a i n i n t h e p r e s e n c e a n d a b s e n c e (• O ) o f 10 M c o l d o u a b a i n . C e l l s w e r e c e n t r i f u g e d t h r o u g h 5% BSA and t h e p e l l e t s o l u b i l i z e d a n d c o u n t e d by s c i n t i l l a t i o n t e c h n i q u e s . - 4 0 -f 3 H)"OUABAIN BOUND (p moles) F I G : 10 3 S c a t c h a r d a n a l y s i s f o r t h e b i n d i n g o f ( H ) - o u a b a i n t o e i t h e r b e e f b r a i n m i c r o s o m a l N A , K A T P a s e ( • — - # ) , o r t o i n t a c t H e l a c e l l s (O O) • 3 - 8 ( H ) - o u a b a i n b i n d s t o t h e m e m b r a n a l s y s t e m w i t h an a p p a r e n t K n o f 2 . 8 x 10 M , 3 8 w h e r e a s t h e K Q f o r ( H ) - o u a b a i n b i n d i n g t o H e l a c e l l s was 5 . 7 x 1 0 " M . H e l a c e l l s p o s s e s s 2 . 5 x 10^ o u a b a i n r e c e p t o r s p e r c e l l a n d t h e l i n e a r i t y o f t h e S c a t c h a r d p l o t s u g g e s t t h a t t h e s e s i t e s a r e o f a s i n g l e c l a s s . - 41 -Studies were also carried out to quantitate the extent of binding of ( H)-ouabain to Hela cells grown in tissue culture. The binding curve is shown in Figure 9. Scatchard analysis of the data revealed that Hela cells 5 possess about 2.5 x 10 ouabain binding sites per cell with a dissociation constant of 5.7 x 10~8M. (FIG: 10). B. OUABAIN-MICROSPHERE CONJUGATES: The microsphere conjugates were found to be potent inhibitors of the o o Na,K ATPase from beef brain. The apparent Ki values for the 200 A and 1000 A ouabain-microsphere adducts were 3.0'x 10~ 6" M and 2.0 x 10~ 5 M (See ;.FIG 11) . I t should be noted that the above Ki values are expressed in terms of the concentration of derivatized cardiac glycoside present during the enzyme reaction. Fluorescently tagged microspheres which had not been derivatized with ouabain were found to have no effect on ATPase activity. Previous experiments on the binding of ( H)-ouabain to Hela cells had indicated that these cells possess on the order of 10 ouabain binding sites, however, attempts at measuring specific binding of the ouabain-latex conjugates to these cells gave negative results. The fluorescein moiety of the glycoside-microsphere conjugate was iodinated with carrier free ( X " I ) using the chloramine T method (31). The radiolabelled product was purified by sucrose density gradient centrifugation to a specific activity of 8.7 x 10~7 cpm/microsphere. Hela cells were incubated with the radioactive ligand in the presence or absence of native ouabain, following which, the cells were pelleted through 5% (w/w) BSA and assayed for bound ( I ) in a gamma counter. The degree of binding in both the test and control samples - 42 -F I G : 11 , Comparison of the inhibitory activities of two different ouabain latex conjugates and native ouabain. A membraneous preparation of Na,K ATPase was incubated with different concentrations of the inhibitors, as described in Methods and the ouabain sensitive ATPase activitiy was determined. The log-dos curves displayed above are for native ouabain — • ) and ouabain-microsphere conjugates of either 200 A (O O) or 1 ,000 ^  (•----•) in diameter. - 43 -was f o u n d t o be l i n e a r and n o n - s a t u r a b l e and a t a l e v e l o n l y 5 - 1 0 % o f t h a t m e a s u r e d w i t h ( H ) - o u a b a i n . S i n c e a g r e a t d e a l o f c a r e was t a k e n t o e n s u r e t h a t t h e o u a b a i n - m i c r o s p h e r e c o n j u g a t e s w e r e f r e e o f a n y u n r e a c t e d o u a b a i n , t h i s i n a b i l i t y t o d e t e c t s p e c i f i c b i n d i n g o f t h e c o n j u g a t e t o H e l a c e l l s was a t t r i b u t e d t o a p r o b l e m o f s t e r i c a c c e s s i b i l i t y . C . D A N S - L Y S - O U A B A I N : T h e o u a b a i n a d d u c t w i t h D A N S - l y s i n e was p r e p a r e d i n a p p r o x i m a t e l y 10-15% y i e l d and was a s p e c i f i c i n h i b i t o r o f N a , K A T P a s e a c t i v i t y . I n e x p e r i m e n t s c o n d u c t e d w i t h b e e f b r a i n m i c r o s o m a l N a , K A T P a s e , D A N S - l y s -o u a b a i n had an a p p a r e n t K i v a l u e o f 7 . 5 X 1 0 ~ 7 M, ( S e e FIG 1 2 ) . A l t h o u g h a m e a s u r e o f t h e d i r e c t b i n d i n g o f D A N S - l y s - o u a b a i n t o N a , K A T P a s e c o u l d n o t be a c h i e v e d , an i n d i r e c t a s s a y was p e r f o r m e d . T h i s a s s a y was c o n d u c t e d e s s e n t i a l l y as d e s c r i b e d i n t h e p r e v i o u s s e c t i o n , i n t h a t , a l i q u o t s o f membrane enzyme w e r e i n c u b a t e d w i t h v a r y i n g a m o u n t s o f D A N S - l y s -o u a b a i n and t h e n p o s t l a b e l l e d w i t h a s a t u r a t i n g c o n c e n t r a t i o n o f ( H ) -o u a b a i n ( 1 . 4 x 1 0 9 c p m / / x m o l e ) . I n s u c h an a s s a y i t was c a l c u l a t e d t h a t a c o n c e n t r a t i o n o f 3 . 0 x 1 0 " 7 M D A N S r l y s - o u a b a i n was a d e q u a t e t o p r o t e c t 50% o f t h e c a r d i a c g l y c o s i d e b i n d i n g s i t e s . T h i s v a l u e i s c o n s i s t e n t w i t h t h e a p p a r e n t K i f o r D A N S - l y s - o u a b a i n i n t h a t b o t h p a r a m e t e r s a r e a p p r o x i m a t e l y one o r d e r o f m a g n i t u d e g r e a t e r t h a n t h e c o r r e s p o n d i n g v a l u e s d e t e r m i n e d f o r n a t i v e o u a b a i n ( S e e FIG 1 3 ) . - 44 -F I G : 12 A c o m p a r i s o n o f t h e d o s e - r e s p o n s e c u r v e s f o r t h e i n h i b i t i o n o f m i c r o s o m a l N a , K A T P a s e by n a t i v e o u a b a i n ( # - - - - # ) a n d D A N S - l y s - o u a b a i n (O——O). The a p p a r e n t K i v a l u e s f o r t h e c a r d i a c s t e r o i d a n d i t s D A N S - l y s i n e c o n j u g a t e -8 - 7 a r e 6 . 5 x 10 M a n d 7 . 5 x 10 M r e s p e c t i v e l y . - 45 -tool 8 0 0.8 1.6 2.4 3.2 4.0 LIGAND CONCENTRATION (/iM) F I G : 13 A l i q u o t s o f m i c r o s o m a l N a , K A T P a s e ( 0 . 2 5 mg) w e r e p r e - i n c u b a t e d a t 3 7 ° C w i t h v a r y i n g c o n c e n t r a t i o n s o f e i t h e r n a t i v e o u a b a i n ( # — - • ) o r D A N S - l y s - o u a b a i n (O O) . F o l l o w i n g e q u i l i b r i u m t h e s a m p l e s w e r e 3 i n c u b a t e d w i t h 0 . 2 M ( H ) - o u a b a i n , t h e enzyme was c o l l e c t e d on M i l l i p o r e 3 HAWP f i l t e r s a n d t h e a m o u n t o f bound ( H ) - o u a b a i n was d e t e r m i n e d a s a p e r c e n t a g e o f t h e t o t a l amount b o u n d i n t h e a b s e n c e o f a n y p r e - i n c u b a t i o n . 3 - 8 H a l f - m a x i m a l p r o t e c t i o n a g a i n s ( H ) - o u a b a i n b i n d i n g i s g i v e n by 3 x 1 0 " M o u a b a i n o r 3 . 0 x 1 0 " 7 M D A N S - l y s - o u a b a i n . - 46 -D. CELL L A B E L L I N G S T U D I E S : A t t e m p t s w e r e made a t v i s u a l i z i n g c e l l s u r f a c e bound D A N S - l y s - o u a b a i n u n d e r t h e f l u o r e s c e n t - l i g h t m i c r o s c o p e . H e l a c e l l s , w h i c h had been g r o w n upon g l a s s c o v e r s l i p s , w e r e i n c u b a t e d i n t h e p r e s e n c e o f a s a t u r a t i n g c o c o n c e n t r a t i o n o f D A N S - l y s - o u a b a i n (1 x 10 M) f o r 1 h o u r a t 37 C . F o l l o w i n g l a b e l l i n g t h e c o v e r s l i p s w e r e r i n s e d 3x i n i n c u b a t i o n medium c o n t a i n i n g 0 . 0 5 M s o d i u m p h o s p h a t e pH 7 . 4 , 0 . 1 4 5 M N a C l , 3 mM M g C l 2 , 1 mM C a C ^ and e x a m i n e d u n d e r a f l u o r e s c e n t - l i g h t m i c r o s c o p e . C o n t r o l p r o -c e d u r e s i n c l u d e d a p r e - i n c u b a t i o n w i t h 1 x 1Q~% n a t i v e o u a b a i n . B o t h t e s t and c o n t r o l s p e c i m e n s e x h i b i t e d a l o w l e v e l o f f l u o r e s c e n c e , when a s e n s i t i v e o i l i m m e r s i o n l e n s was u s e d . I n o r d e r t o d e t e r m i n e i f t h e o u a b a i n c o n j u g a t e was i n f a c t b i n d i n g t o t h e c e l l s u r f a c e , H e l a c e l l s w h i c h had been p r e - l a b e l l e d w i t h D A N S - l y s -1 /"S o u a b a i n were i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f ( I ) - r a b b i t - a n t i -DANS I g G . The p r e p a r a t i o n and p u r i f i c a t i o n o f s p e c i f i c a n t i - D A N S immune s e r u m i s d e s c r i b e d f u l l y i n P a r t I I . F o l l o w i n g t h e i n c u b a t i o n w i t h r a d i o -l a b e l l e d a n t i - D A N S IgG t h e s a m p l e s w e r e c e n t r i f u g e d t h r o u g h 5% BSA and c e l l p e l l e t s w e r e c o u n t e d i n a gamma c o u n t e r . T h e b i n d i n g o f r a d i o l a b e l e d i m m u n o g l o b u l i n was i d e n t i c a l i n b o t h t e s t and c o n t r o l s i t u a t i o n s , i n t h a t i t was l i n e a r and n o n - s a t u r a t i n g and r e p r e s e n t e d l e s s t h a n 5% o f t h e known c a r d i a c g l y c o s i d e r e c e p t o r s . - 47 -D I S C U S S I O N ( A ) OUABAIN-MICROSPHERE CONJUGATES: C o n j u g a t e s o f t h e c a r d i a c g l y c o s i d e w i t h m e t h a c r y l a t e c o p o l y m e r m i c r o -s p h e r e s w e r e a c t i v e i n i n h i b i t i n g N a , K A T P a s e f r o m b e e f b r a i n b u t d i d n o t e x h i b i t d i r e c t b i n d i n g t o H e l a c e l l s . T h i s r e s u l t i n d i c a t e d t h a t s t e r i c a c c e s s i b i l i t y o f t h e l i g a n d t o t h e enzyme p o s e d a m a j o r p r o b l e m and t h a t t h e r e may be s i g n i f i c a n t d i f f e r e n c e s i n t h e e n v i r o n m e n t o f t h e A T P a s e enzyme b e t w e e n t h e b e e f b r a i n m i c r o s o m a l s y s t e m and t h e p l a s m a membrane o f i n t a c t H e l a c e l l s . I n o r d e r t o c i r c u m v e n t t h e s e d i f f i c u l t i e s i t was d e c i d e d t o c o n c e n t r a t e on a s m a l l m o l e c u l a r w e i g h t d e r i v a t i v e o f o u a b a i n , D A N S - l y s -o u a b a i n . ( B ) D A N S - l y s - O U A B A I N : The b i n d i n g d a t a f o r t h e D A N S - l y s - o u a b a i n d e r i v a t i v e c o m p a r e s f a v o r a b l y w i t h r e s u l t s q u o t e d by o t h e r i n v e s t i g a t o r s s t u d y i n g o u a b a i n d e r i v a t i v e s . C o n j u g a t e s o f t h e c a r d i a c g l y c o s i d e w i t h N - ( 2 - n i t r o - 4 - a z i d o p h e n y l ) e t h y l e n e d i a m i n e w e r e r e p o r t e d t o g i v e h a l f m a x i m a l p r o t e c t i o n a g a i n s t ( H ) - o u a b a i n b i n d i n g a t c o n c e n t r a t i o n s r a n g i n g f r o m 0.22 - 0.50 f tM (24 ,25 ) , w h e r e a s t h e e q u i v a l e n t v a l u e f o r D A N S - l y s - o u a b a i n was c a l c u l a t e d t o be 0.30 ^ . M . The f a i l u r e t o d e m o n s t r a t e a s p e c i f i c i n t e r a c t i o n b e t w e e n D A N S - l y s -o u a b a i n and H e l a c e l l s , e i t h e r d i r e c t l y u s i n g t h e f l u o r e s c e n t - l i g h t I pc m i c r o s c o p e o r i n d i r e c t l y u s i n g ( I ) - r a b b i t - a n t i - D A N S I g G , s u p p l i e d f u r t h e r e v i d e n c e t h a t t h e o u a b a i n b i n d i n g s i t e may be l o c a t e d d e e p w i t h i n t h e p l a s m a membrane o f t h e s e c e l l s . - 48 -S i n c e i t was n o t p o s s i b l e t o r a d i o l a b e l t h e D A N S - l y s - o u a b a i n d e r i v a t i v e a m e a s u r e o f t h e d i r e c t b i n d i n g o f t h i s d e r i v a t i v e t o d i f f e r e n t N a , K A T P a s e s y s t e m s was n o t p o s s i b l e . Such d a t a w o u l d be v e r y i m p o r t a n t i n p r o v i n g c o n -c l u s i v e l y t h a t t h e D A N S - l y s - o u a b a i n d e r i v a t i v e was n o t c o n t i m i n a t e d by a s m a l l p e r c e n t a g e o f f r e e o u a b a i n , a l t h o u g h s u c h c o n t a m i n a t i o n was n o t i n d i c a t e d by c h e m i c a l a n a l y s i s ( i . e . : c a r d e n o l i d e s p e c i f i c c o l o r t e s t ) o r by t h e enzyme i n h i b i t i o n p r o f i l e s . ( C ) OTHER CONSIDERATIONS: F u t u r e e x p e r i m e n t s d i r e c t e d t o w a r d s t h e d e v e l o p m e n t o f a s p e c i f i c m i c r o s c o p i c p r o b e f o r t h e N a , K A T P a s e enzyme c o u l d be m o r e f r u i t f u l i f o t h e r c l a s s e s o f o u a b a i n d e r i v a t i v e s w e r e s t u d i e d . I f s t e r i c a c c e s s i b i l i t y i s , i n f a c t , as much o f a p r o b l e m a s t h e r e s u l t s p r e s e n t e d h e r e a p p e a r t o i n d i c a t e , t h e n i t w o u l d be d e s i r a b l e t o s y n t h e s i z e d e r i v a t e s i n w h i c h a l o n g " s p a c e r a r m " was i n t e r p o s e d b e t w e e e n t h e o u a b a i n m o i e t y and i n d i c a t o r g r o u p ( i . e . : DANS, F I T C , e t c . ) . Two p o s s i b l e c a n d i d a t e s f o r s u c h a " s p a c e r arm" w o u l d be e i t h e r l i n e a r p o l y a c r y l a m i d e s o r s y n t h e t i c p o l y p e p t i d e s , o f d e f i n e d l e n g t h . I n a t t e m p t i n g t o l o c a l i z e t h e N a , K A T P a s e enzyme a t t h e u l t r a s t r u c t u r a l l e v e l , i t m u s t be r e a l i z e d t h a t t h e r e a r e , a t l e a s t , two d i v e r g e n t p a t h w a y s . I n t h i s r e s e a r c h I h a v e c h o s e n t o e x a m i n e c h e m i c a l d e r i v a t i v e s o f a s p e c i f i c i n h i b i t o r o f N a , K A T P a s e . An a l t e r n a t e a p p r o a c h w o u l d be t o u s e a n t i b o d i e s s p e c i f i c f o r N a , K A T P a s e as a c y t o c h e m i c a l r e a g e n t f o r l o c a l i z i n g t h e s o d i u m pump. S u c h a n t i b o d y p r e p a r a t i o n s h a v e a l r e a d y b e e n u s e d i n t r a n s m i s s i o n e l e c t r o n m i c r o s c o p i c s t u d i e s on t h e l o c a l i z a t i o n o f N a , K A T P a s e ( 8 6 , 8 8 ) and i t i s p r o b a b l e t h a t s u c h a m e t h o d o l o g y c o u l d be m o d i f i e d f o r SEM i n v e s t i g a t i o n s as w e l l . - 49 -P A R T I I : H A P T E N S A N D W I C H L A B E L L I N G M E T H O D O L O G Y - 50 -I N T R O D U C T I O N T h e s c a n n i n g e l e c t r o n m i c r o s c o p e h a s p r o v e n t o be an i d e a l t o o l f o r s t u d y i n g t h e s u r f a c e t o p o g r a p h y o f c e l l s , a s w e l l a s , t h e o r g a n i z a t i o n o f c e l l t y p e s w i t h i n a g i v e n t i s s u e . T h e s u r f a c e s o f t h e s e c e l l s d i s p l a y n u m e r o u s m o l e c u l a r u n i t s w h i c h p l a y i m p o r t a n t r o l e s i n c e l l f u n c t i o n as w e l l as c e l l - c e l l i n t e r a c t i o n . I t i s i n t h e i d e n t i f i c a t i o n o f d i f f e r e n c e s i n t h e p l a s m a membrane c o m p o n e n t s o f v a r i o u s c e l l p o p u l a t i o n s t h a t c e l l s u r f a c e l a b e l l i n g t e c h n i q u e s a r e u s e f u l . T h e g o a l o f a n y l a b e l l i n g t e c h n i q u e i s t h e l o c a l i z a t i o n o f s p e c i f i c m o l e c u l e s on t h e c e l l s u r f a c e . The e f f i c a c y o f t h e t e c h n i q u e d e p e n d s on t h e s p e c i f i c i t y o f a t t a c h m e n t o f a m a r k e r t o t h e membrane c o m p o n e n t o f i n t e r e s t and t o no o t h e r . G e n e r a l l y s p e a k i n g , c e l l s u r f a c e l a b e l l i n g i n v o l v e s ( 1 ) a l a b e l t h a t b i n d s t o a s p e c i f i c m o l e c u l e o r m o l e c u l e s a t t h e c e l l s u r f a c e , ( 2 ) a. m a r k e r t h a t c a n be v i s i b l y a p p r e c i a t e d i n t h e m i c r o s c o p e , and ( 3 ) a way o f l i n k i n g t h e l a b e l t o t h e m a r k e r . A . T h e L a b e l : In g e n e r a l t e r m s t h e n a t u r e o f t h e s i t e a t t h e c e l l s u r f a c e t o be l a b e l l e d w i l l d i c t a t e w h a t may be u s e d as . a l a b e l . I t i s d e s i r a b l e t o u s e a l a b e l t h a t h a s as h i g h an a f f i n i t y and s p e c i f i c i t y f o r i t s b i n d i n g s i t e as p o s s i b l e . I n o r d e r t o d e m o n s t r a t e t h e s p e c i f i c i t y o f l a b e l l i n g , a s p e c i f i c i n h i b i t o r o f t h e i n t e r a c t i o n i s u s e f u l . L a b e l l i n g w i t h a n t i b o d i e s i s a w i d e l y u s e d a p p r o a c h and i s one i n w h i c h t h e c h e m i s t r y i s w e l l u n d e r s t o o d and s p e c i f i c i t y c a n be shown u s i n g a - 51 -v a r i e t y o f i m m u n o l o g i c a l t e c h n i q u e s . R a t h e r e x t e n s i v e p u r i f i c a t i o n o f t h e a n t i b o d y i s u s u a l l y n e c e s s a r y i n o r d e r t o r e d u c e b a c k g r o u n d l a b e l l i n g . W h o l e i m m u n o g l o b u l i n c a n be d i g e s t e d t o Fab f r a g m e n t s i f a s m a l l e r o r m o n o v a l e n t l a b e l i s d e s i r e d . A n o t h e r g r o u p o f l a b e l s i s t h e l e c t i n s , w h i c h i n r e c e n t y e a r s h a v e g a i n e d p o p u l a r i t y as c e l l s u r f a c e p r o b e s . L e c t i n s c o m p r i s e a c l a s s o f p r o t e i n s w h i c h d i s p l a y a h i g h d e g r e e o f s p e c i f i c i t y f o r b i n d i n g n u m e r o u s mono and o l i g o s a c c h a r i d e s ( 3 5 , 3 6 ) . P l a n t l e c t i n s have b e e n u s e d as s p e c i f i c p r o b e s t o map t h e d i s t r i b u t i o n o f s a c c h a r i d e s on t h e c e l l s u r f a c e i n t h e hope o f d e f i n i n g t h e i n t e r a c t i o n s b e t w e e n g l y c o p r o t e i n s and g l y c o l i p i d s , w h i c h a r e s u r e t o p l a y an i m p o r t a n t r o l e i n t h e c o n t r o l o f c e l l b e h a v i o u r ( 3 7 , 3 8 , 3 9 ) . B . The M a r k e r : T h e c r i t e r i a f o r s e l e c t i n g m a r k e r s v a r y w i t h t h e m i c r o s c o p i c t e c h n i q u e u s e d . A m a r k e r w i t h a g o o d a b s o r p t i o n s p e c t r u m o r f l u o r e s c e n c e e m i s s i o n i s u t i l i z e d f o r f l u o r e s c e n t / l i g h t m i c r o s c o p y . M a r k e r s u s e d f o r t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y m u s t h a v e h i g h e l e c t r o n d e n s i t y , w h e r e a s , SEM m a r k e r s m u s t be o f s u f f i c i e n t s i z e and h a v e a d i s t i n c t u n i f o r m s h a p e , i n o r d e r t o be c l e a r l y d i s t i n g u i s h e d . A number o f d i f f e r e n t r e a g e n t s h a v e been u s e d by i n v e s t i g a t o r s t o v i s u a l i z e c e l l s u r f a c e c o m p o n e n t s u n d e r t h e SEM. T h e s e m a r k e r s i n c l u d e m a c r o m o l e c u l a r c o m p l e x e s s u c h as h e m o c y a n i n ( 4 0 ) , f e r r i t i n ( 4 1 ) , t o b a c c o m o s s a i c v i r u s ( 4 2 ) , as w e l l as s y n t h e t i c p o l y m e r p a r t i c l e s ( 2 7 , 4 3 ) . - 52 -C . T h e H a p t e n S a n d w i c h T e c h n i q u e : I n t h e n o r m a l o r d i r e c t c e l l l a b e l l i n g t e c h n i q u e , t h e l a b e l i s l i n k e d t o t h e m a r k e r v i a a c o v a l e n t c h e m i c a l bond o r v i a a s p e c i f i c c h e m i c a l i n t e r a c t i o n b e t w e e n t h e l a b e l and t h e m a r k e r . I n t h e h a p t e n s a n d w i c h t e c h n i q u e t h e l a b e l i s l i n k e d t o t h e m a r k e r b y a b i v a l e n t a n t i b o d y m o l e c u l e w h i c h s e r v e s as a b r i d g i n g g r o u p ( S e e FIG 1 5 ) . T h e h a p t e n s a n d w i c h l a b e l l i n g method u s e s a t h r e e s t e p p r o c e d u r e t o l a b e l c e l l s u r f a c e c o m p o n e n t s w i t h m a r k e r s v i s i b l e i n t h e SEM. I n t h i s p r o c e d u r e , s u r f a c e s p e c i f i c a n t i b o d i e s , w h i c h h a v e p r e v i o u s l y been d e r i v a t i z e d w i t h a s m a l l i m m u n o g e n i c m o l e c u l e ( i . e . : a h a p t e n ) a r e a p p l i e d t o c e l l s . L a b e l l e d c e l l s a r e t h e n i n c u b a t e d , i n s e q u e n c e , w i t h a n t i - h a p t e n a n t i b o d y and h a p t e n m o d i f i e d s u r f a c e m a r k e r s . R a t h e r t h a n u t i l i z e an a n t i -c e l l s u r f a c e i m m u n o g l o b u l i n as t h e i n i t i a l l i g a n d we h a v e e m p l o y e d h a p t e n m o d i f i e d l e c t i n s . A h a p t e n i s a c h e m i c a l g r o u p w h i c h r e s u l t s i n t h e p r o d u c t i o n o f s p e c i f i c a n t i - h a p t e n a n t i b o d y when h a p t e n - p r o t e i n c o n j u g a t e s a r e u s e d as i m m u n o g e n s ; t h e h a p t e n a l o n e i s n o t a n t i g e n i c . A w i d e v a r i e t y o f c o m m e r c i a l l y a v a i l a b l e a m i n o o r n i t r o p h e n y l h a p t e n s ( a r s a n i l i c a c i d , s u l f a n i l i c a c i d , p - a m i n o b e n z o i c a c i d , e t c . ) have been e m p l o y e d by p r e v i o u s i n v e s t i g a t o r s . The h a p t e n we h a v e c h o s e n t o u s e i s t h e DANS ( 5 - d i m e t h y l a m i n o - l - n a p t h a l e n e s u l f o n y l ) m o i e t y . S p e c i f i c a n t i - s e r u m was r a i s e d a g a i n s t a c o n j u g a t e o f DANS-C1 w i t h b o v i n e s e r u m a l b u m i n and s u b s e q u e n t l y p u r i f i e d by a f f i n i t y c h r o m a t o g r a p h y on D A N S - l y s - S e p h a r o s e . T h e s p e c i f i c i t y o f t h e a n t i - h a p t e n a n t i b o d y was d e t e r m i n e d by r a d i o i m m u n o a s s a y , as w e l l a s , by d i r e c t v i s u a l i z a t i o n o f b o u n d FITC c o n j u g a t e d a n t i - D A N S I g G . - 53 -A n t i - h a p t e n a n t i b o d y was v i s u a l i z e d u n d e r t h e SEM t h r o u g h t h e u s e o f i r o n c o n t a i n i n g m i c r o s p h e r e s (44) w h i c h had b e e n d e r i v a t i z e d w i t h D A N S - l y s i n e . S u c h m i c r o s p h e r e s c o u l d be d e t e c t e d i n t h e SEM b y b o t h s e c o n d a r y e l e c t r o n e m i s s i o n as w e l l as X - r a y m i c r o p r o b e a n a l y s i s . When a specimen i n t h e SEM i s bombarded w i t h a h i g h energy-e l e c t r o n beam t h e sample w i l l e m i t X-rays whose e n e r g i e s a r e c h a r a c t e r i s t i c o f t h e at o m i c s p e c i e s p r e s e n t . The in c o m i n g e l e c t r o n knocks a bound e l e c t r o n from an i n n e r o r b i t a l s h e l l and i f t h e e l e c t r o n cannot be d i s p l a c e d t o a h i g h e r s h e l l , because o f t h e P a u l i e x c l u s i o n p r i n c i p l e , i t i s r e l e a s e d from t h e atom. The atom i s now i n an i o n i z e d s t a t e and v e r y q u i c k l y r e t u r n s t o t h e ground s t a t e by c a p t u r i n g an e l e c t r o n from a h i g h e r energy e l e c t r o n s h e l l . I n t h i s p r o c e s s an X-ray photon o f energy e q u a l t o t h e d i f f e r e n c e between t h e i n i t i a l and f i n a l s t a t e s o f t h e c a p t u r e d e l e c t r o n , i s r e l e a s e d . The e m i t t e d X - r a y s a r e a d e t e c t a b l e r a d i a t i v e phenomenon and t h e i r e n e r g i e s can be measured u s i n g an energy d i s p e r s i v e X-ray s p e c t r o m e t e r . C h a r a c t e r i s t i c X-ray s p e c t r o s c o p y c a n , t h e r e f o r e be used as an e f f e c t i v e method f o r m i c r o a n a l y s i s o f c e r t a i n elements p r e s e n t i n t h e sample. - 54 -E X P E R I M E N T A L . P R O C E D U R E S (1) HAPTEN-MODIFIED L E C T I N S : T h e l e c t i n s , C o n - A and r i c i n , w e r e d e r i v a t i z e d w i t h DANS-C1 and s u b -s e q u e n t l y p u r i f i e d by a f f i n i t y c h r o m a t o g r a p h y . R i c i n u s communis l e c t i n ( 5 . 0 m l ; 1 . 5 m g / m l ) , i n 4 mM NaHC0 3 pH 8 . 5 was r e a c t e d w i t h 0 . 1 ml o f DANS-C1 ( 1 0 mg/ml i n d r y a c e t o n e ) f o r 2 h o u r s a t room t e m p e r a t u r e . F o l l o w i n g a l o w s p e e d c e n t r i f u g a t i o n t o r e m o v e any a g g r e g a t e s , t h e r e a c t i o n m i x t u r e was l o a d e d o n t o a S e p h a r o s e 4B ( 2 - 3 ml b e d v o l u m e ) a f f i n i t y c o l u m n a t room t e m p e r a t u r e . T h e c o l u m n was w a s h e d t h o r o u g h l y w i t h PBS u n t i l t h e 0D2RQ was l e s s t h a n 0 . 0 2 and D A N S - r i c i n was s p e c i f i c a l l y e l u t e d w i t h 0 . 1 M D - g a l a c t o s e . E x c e s s s u g a r was r e m o v e d by d i a l y s i s a g a i n s t PBS a t 4 ° C . The d e g r e e o f s u b s t i t u t i o n was c a l c u l a t e d t o be a p p r o x i m a t e l y t h r e e DANS p e r IM l e c t i n on a m o l e : m o l e b a s i s , u s i n g an e x t i n c t i o n c o e f f i c i e n t o f e = 4 5 7 0 3 3 3 0 l i t r e - m o l e - 1 - ™ - 1 f o r D A N S - C 1 . C o n c a n a v a l i n A was d e r i v a t i z e d w i t h DANS-C1 b y a p r o c e d u r e s i m i l a r t o t h a t o u t l i n e d a b o v e , e x c e p t t h a t c a l c i u m and m a n g a n e s e i o n s w e r e p r e s e n t i n t h e r e a c t i o n medium i n o r d e r t o m a i n t a i n a c t i v i t y o f t h e l e c t i n . DANS-Con A was a f f i n i t y p u r i f i e d on S e p h a d e x G100 and s p e c i f i c a l l y e l u t e d w i t h 0 . 1 M m e t h y l m a n n o s i d e . ( 2 ) PREPARATION OF D A N S - L Y S I N E - S E P H A R O S E : S e p h a r o s e 4B ( 1 0 . 0 m l ) was a c t i v a t e d w i t h 3 g o f c y a n o g e n b r o m i d e f o l l o w i n g t h e method o f C u a t r e c a s s u s ( 3 3 ) . T h e a c t i v a t e d g e l was m i x e d w i t h - 55 -an e q u a l v o l u m e o f 2 mM D A N S - l y s i n e , d i s s o l v e d i n 7 . 5 % ( v / v ) a q u e o u s d i o x a n e o pH 9 . 5 . T h e c o u p l i n g was a l l o w e d t o p r o c e e d f o r 12 h o u r s a t 4 C i n a L a b q u a k e s h a k i n g a p p a r a t u s . The d e r i v a t i z e d S e p h a r o s e was w a s h e d i n a B u c h n e r f u n n e l w i t h f i v e v o l u m e s o f H^O; 3 . 0 M NaSCN; r^O: and was o s t o r e d a t 4 C i n t h e p r e s e n c e o f 0 . 1 % s o d i u m a z i d e as a b a c t e r i o s t a t i c a g e n t . ( 3 ) PREPARATION AND PURIFICATION OF RABBIT ANTI-DANS IMMUNE SERUM: The i m m u n i z i n g a n t i g e n D A N S - B S A , u s e d f o r t h e p r o d u c t i o n o f r a b b i t a n t i - D A N S IgG was p r e p a r e d a c c o r d i n g t o H o f f m a n n and Kuo ( 1 9 7 7 ) ( 4 5 ) . BSA ( 0 . 7 g) was d i s s o l v e d i n 2 0 . 0 ml o f 0 . 1 % N a H C 0 3 c o n t a i n i n g 10 mM EDTA, 7 . 0 m l o f d i o x a n e was a d d e d and t h e pH a d j u s t e d t o 8 . 0 . The r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f 1 . 0 ml o f DANS-C1 ( 4 0 mg/ml i n d r y a c e t o n e ) and t h e pH was m a i n t a i n e d a t 8 . 0 w i t h 0 . 5 M NaOH. A f t e r 1 h o u r , a n o t h e r a l i q u o t o f DANS-C1 o was added and t h e r e a c t i o n was a l l o w e d t o p r o c e e d o v e r n i g h t a t 25 C w i t h s t i r r i n g . D e r i v a t i z e d BSA was s e p a r a t e d f r o m o t h e r r e a c t a n t s v i a c h r o m a t o -g r a p h y on S e p h a d e x G - 2 5 t h a t had been e q u i l i b r a t e d w i t h 10 mM T r i s - C l pH 8 . 0 , 10 mM EDTA. F r a c t i o n s w i t h an 0 D £ g Q g r e a t e r t h a n 1 . 0 w e r e p o o l e d and a n a l y z e d s p e c t r o p h o t o m e t r i c a l l y f o r p r o t e i n a t 280 nm and f o r DANS a t 330 nm. P r i m a r y i m m u n i z a t i o n o f a w h i t e New Z e a l a n d r a b b i t was c a r r i e d o u t w i t h 5 . 0 mg o f DANS-BSA i n 0 . 4 m l o f F r e u n d ' s c o m p l e t e a d j u v a n t i n j e c t e d s u b c u t a -n e o u s l y , i n two e q u a l l y d i v i d e d d o s e s . S e c o n d a r y i m m u n i z a t i o n s w e r e p e r f o r m e d 5 weeks a f t e r t h e p r i m a r y i m m u n i z a t i o n and a t 6 week i n t e r v a l s t h e r e a f t e r u s i n g t h e same r e g i m e n . T h e r a b b i t was b l e d o n t h e 1 0 t h and 1 5 t h days a f t e r e a c h s e c o n d a r y i m m u n i z a t i o n . - 56 -Immune s e r u m was s p e c i f i c a l l y p u r i f i e d u s i n g a D A N S - l y s - S e p h a r o s e ( 4 B ) a f f i n i t y c o l u m n . T h e a f f i n i t y c o l u m n was w a s h e d f i r s t w i t h two c o l u m n v o l u m e s o f 3 . 0 M NaSCN and t h e n e q u i l i b r a t e d w i t h P B S . Immune s e r u m ( 1 0 - 1 5 m l ) was l o a d e d o n t o t h e D A N S - l y s - S e p h a r o s e c o l u m n ( 3 - 4 ml bed v o l u m e ) and t h e c o l u m n was w a s h e d e x t e n s i v e l y w i t h PBS u n t i l t h e OD^ Q^ was l e s s t h a n 0 . 0 5 . S p e c i f i c a l l y bound m a t e r i a l was e l u t e d w i t h 3 . 0 M NaSCN i n P B S , and f r a c t i o n s w i t h an ODp^Q g r e a t e r t h a n 0 . 5 w e r e p o o l e d and d i a l i z e d a g a i n s t PBS i n t h e c o l d ( 0 - 4 ° C ) . F o l l o w i n g d i a l y s i s , t h e p u r i f i e d a n t i DANS-a n t i b o d y was s t o r e d a t - 7 0 ° C i n 0 . 1 ml a l i q u o t s u n t i l n e e d e d . ( 4 ) RADIOIMMUNOASSAY: T h e s p e c i f i c i t y o f b i n d i n g o f a n t i - D A N S a n t i b o d i e s was d e t e r m i n e d I p c u s i n g ( I ) - l a b e l l e d a n t i b o d y . B r i e f l y , human e r y t h r o c y t e s w h i c h had b e e n p r e - i n c u b a t e d w i t h s a t u r a t i n g a m o u n t s o f D A N S - r i c i n w e r e l a b e l l e d w i t h v a r y i n g c o n c e n t r a t i o n s o f r a d i o a c t i v e a n t i b o d y . A p p r o x i m a t e l y 2 . 0 mg o f a f f i n i t y p u r i f i e d a n t - D A N S IgG was r e a c t e d 1 w i t h I o d i n e ( 2 5 0 / i . C i ) i n t h e p r e s e n c e o f c h l o r a m i n e T , a t pH 7 . 0 ( 3 1 ) . R a d i o l a b e l e d a n t i b o d y was p u r i f i e d by a f i n i t y c h r o m a t o g r a p h y on D A N S - l y s -S e p h a r o s e , f o l l o w e d by d i a l y s i s a a i n s t P B S . T h e a n t i b o d y had a p r o t e i n c o n c e n t r a t i o n o f 0 . 3 mg/ml w i t h an i n i t i a l s p e c i f i c a c t i v i t y o f 5 . 1 4 x 1 0 4 cpm//i g . F r e s h human e r y t h r o c y t e s were f i x e d w i t h 2% g l u t a r a l d e h y d e , i n P B S , f o r one h o u r a t room t e m p e r a t u r e and t h e n i n c u b a t e d f o r a f u r t h e r 30 m i n u t e s i n t h e p r e s e n c e o f 0 . 1 M g l y c i n e . P r e - f i x i n g w i t h g l u t a r a l d e h y d e was n e c e s s a r y t o p r e v e n t a g g r e g a t i o n o f R B C ' s when t h e y w e r e i n c u b a t e d w i t h D A N S - r i c i n . - 57 -F i x e d e r y t h r o c y t e s w e r e t h e n w a s h e d t h r e e t i m e s i n P B S , by c e n t r i f u g a t i o n , Q and r e s u s p e n d e d t o a c o n c e n t r a t i o n o f 8 . 8 x 10 c e l l s / m l . An a l i q u o t o f c e l l s ( 0 . 2 5 m l ) was i n c u b a t e d w i t h an e q u a l v o l u m e o f D A N S - r i c i n ( 1 . 0 mg/ml o f i n a l c o n c e n t r a t i o n ) f o r 30 m i n u t e s a t 25 C . C o n t r o l c e l l s w e r e l a b e l l e d w i t h D A N S - r i c i n i n t h e p r e s e n c e o f 0 . 1 M D - g a l a c t o s e . F o l l o w i n g t h i s p r e - i n c u b a t i o n , c e l l s w e r e w a s h e d t h r e e t i m e s w i t h PBS and r e s u s p e n d e d t o a t o t a l v o l u m e o f 1 . 0 m l . A l i q u o t s o f t h i s c e l l s u s p e n s i o n ( 0 . 0 5 m l ; 6 . 7 x 10 c e l l s ) were i n c u b a t e d w i t h s e r i a l d i l u t i o n s o f ( I ) - a n t i - D A N S o I g G , i n a t o t a l v o l u m e o f 150 / x l , f o r 15 m i n u t e s a t 25 C . A f t e r w a r d s , 125 / i 1 p o r t i o n s o f e a c h s a m p l e w e r e l a y e r e d o n t o 250 /x 1 ml o f 5% BSA (w/w) i n a 400 / i 1 p o l y p r o p y l e n e m i c r o c e n t r i f u g e t u b e . S a m p l e s w e r e c e n t r i f u g e d a t 1 2 , 0 0 0 x g f o r 1 m i n u t e i n an E p p e n d o r f m i c r o f u g e , a f t e r w h i c h t h e b o t t o m s o f t h e t u b e s , c o n t a i n i n g t h e c e l l p e l l e t , were c u t o f f and a s s a y e d f o r ( I ) i n a gamma c o u n t e r . ( 5 ) DIRECT V I S U A L I Z A T I O N OF BOUND ANTI-HAPTEN ANTIBODY: T h e b i n d i n g o f a n t i - h a p t e n IgG t o n e u r o b l a s t o m a c e l l s was v i s u a l z e d d i r e c t l y u n d e r t h e f l u o r e s c e n c e - 1 i g h t m i c r o s c o p e u s i n g FITC l a b e l l e d a n t i -b o d i e s . S p e c i f i c a l l y p u r i f i e d r a b b i t a -DANS immune s e r u m ( 4 . 0 m l ; 0 . 3 9 m g / m l ) was a d j u s t e d t o pH 8 . 5 - 9 . 0 w i t h s o d i u m c a r b o n a t e . F l u o r e s c e i n i s o t h i o c y a n a t e ( F I T C ) was a d d e d t o a f i n a l c o n c e n t r a t i o n o f 0 . 2 mg/ml and o a l l o w e d t o r e a c t f o r 3 h o u r s a t 25 C . F l u o r e s c e n t l y t a g g e d immuno-g l o b u l i n was p u r i f i e d by a f f i n i t y c h r o m a t o g r a p h y on D A N S - l y s - S e p h a r o s e , f o l l o w e d by d i a l y s i s a g a i n s t PBS t o r e m o v e NaSCN. - 58 -N e u r o b l a s t o m a c e l l s w e r e grown up on g l a s s c o v e r s l i p s i n E a g l e ' s m i n i m u m e s s e n t i a l medium ( c o n t a i n i n g 10% f o e t a l c a l f s e r u m ) and u s e d as s u c h f o r l a b e l l i n g . A l l i n c u b a t i o n s w e r e p e r f o r m e d i n D u l b e c c o ' s m o d i f i e d PBS c o n t a i n i n g c a l c i u m , a t a t o t a l v o l u m e o f 0 . 1 m l . C e l l s w e r e i n t i a l l y o l a b e l l e d w i t h D A N S - r i c i n ( 0 . 1 m g / m l ) , f o r 10 m i n u t e s a t 25 C , b o t h i n t h e p r e s e n c e and a b s e n c e o f 0 . 1 M D - g a l a c t o s e , t h e c o m p e t i t i v e i n h i b i t o r o f r i c i n b i n d i n g . A f t e r r i n s i n g t h r e e t i m e s i n D u l b e c c o ' s P B S , c e l l s w e r e i n c u b a t e d w i t h F I T C - a n t i - D A N S IgG ( 0 . 1 m g / m l ) f o r 10 m i n u t e s a t r o o m t e m p e r a t u r e . Bound a n t i - D A N S a n t i b o d y was v i s u a l i z e d by e x a m i n a t i o n o f t h e N e u r o b l a s t o m a c e l l s u n d e r a f l u o r e s c e n c e - l i g h t m i c r o s c o p e . ( 6 ) PREPARATION OF THE HAPTEN MODIFIED MARKER: o P o l y m e r i c m i c r o s p h e r e s ( 5 0 0 A d i a m e t e r ) c o n t a i n i n g F e . ^ , were o b t a i n e d c o u r t e s y o f S . P . S . Yen and A . Rembaum o f t h e J e t P r o p u l s i o n L a b o r a t o r y i n P a s a d e n a , C a l i f o r n i a . T h e s e i r o n c o n t a i n i n g m i c r o s p h e r e s p o s s e s s a l a r g e number o f e x p o s e d c a r b o x y l and h y d r o x y l g r o u p s w h i c h a r e q u i t e ammendable t o e a s y d e r i v a t i z a t i o n . A 5 . 0 m l a l i q u o t o f F e - m i c r o s p h e r e s ( 3 . 0 m g / m l ) w e r e d e r i v a t i z e d w i t h D A N S - l y s i n e (2 mM) u s i n g t h e a q u e o u s c a r b o d i i m i d e , EDC, (5 m g / m l ) f o r 12 h o u r s a t 4 ° C . T h e h a p t e n d e r i v a t i z e d s p h e r e s w e r e p u r i f i e d b y c h r o m a t o g r a p h y on S e p h a r o s e - 6 B ( 1 . 5 x 20 cm) w h i c h had b e e n e q u i l i b r a t e d w i t h P B S . F r a c t i o n s e l u t i n g i n t h e v o i d v o l u m e , c o n t a i n i n g t h e D a n s - F e - m i c r o s p h e r e s , w e r e p o o l e d and s t o r e d f r o z e n a t - 2 0 ° C i n t h e p r e s e n c e o f 5-10% g l y c e r o l . - 59 -( 7 ) HAPTEN SANDWICH L A B E L L I N G : 3T3 f i b r o b l a s t c e l l s w e r e grown up on g l a s s c o v e r s l i p s and f i x e d w i t h 1 . 2 5 % g l u t a r a l d e h y d e p r i o r t o l a b e l l i n g . C e l l s w e r e i n c u b a t e d w i t h e i t h e r D A N S - r i c i n o r DANS-Con A ( 0 . 1 m g / m l ) f o r 10 m i n u t e s a t room t e m p e r a t u r e . C o n t r o l s p e c i m e n s w e r e t r e a t e d as a b o v e p l u s e i t h e r 0 . 1 M D - g a l a c t o s e o r 0 . 1 M a - m e t h y l m a n n o s i d e . C e l l s w e r e t h e n l a b e l l e d i n s e q u e n c e w i t h a n t i - D A N S IgG ( 0 . 1 m g / m l ) and D A N S - F e - m i c r o s p h e r e s , f o r 10 m i n u t e i n t e r v a l s . A l l i n c u b a t i o n s w e r e p e r f o r m e d i n D u l b e c c o ' s m o d i f i e d P B S , a t a t o t a l v o l u m e o f 0 . 1 m l . F o l l o w i n g t h e f i n a l l a b e l l i n g c y c l e , c e l l s w e r e a g a i n f i x e d w i t h 1 . 2 5 % g l u t a r a l d e h y d e i n PBS f o r 1 h o u r . S a m p l e s w e r e d e h y d r a t e d t h r o u g h a g r a d e d s e r i e s o f e t h a n o l b a t h s f r o m 50%-100% and c r i t i c a l p o i n t d r y e d f r o m l i q u i d CO2. P r i o r t o b e i n g e x a m i n e d u n d e r t h e ETEC A u t o S c a n S E M , t h e 0 s a m p l e s w e r e s p u t t e r e d w i t h a 200 A t h i c k l a y e r o f g o l d : p a l l a d i u m . - 60 -R E S U L T S A N D D I S C U S S I O N : ( 1 ) RADIOIMMUNOASSAY: R a b b i t a n t i - D A N S a n t i b o d y was i s o l a t e d i n p u r e f o r m b y a f f i n i t y c h r o -m a t o g r a p h y o n D A N S - l y s - S e p h a r o s e . The d e g r e e o f s p e c i f i c i t y o f t h e a n t i b o d y was d e t e r m i n e d by r a d i o i m m u n o a s s a y u s i n g R B C ' s w h i c h had been p r e - l a b e l l e d I pc w i t h D A N S - r i c i n . T h e maximum l e v e l o f ( I ) - a n t i - D A N S IgG b i n d i n g was f o u n d t o be o f t h e same o r d e r o f m a g n i t u d e a s t h e e s t i m a t e d number o f r i c i n 1 ? R b i n d i n g s i t e s p e r r e d c e l l . B i n d i n g e x p e r i m e n t s w i t h ( I ) - R C A i n d i c a t e d c t h a t e r y t h r o c y t e s p o s s e s s a b o u t 2 x 10 r i c i n b i n d i n g s i t e s and f r o m t h e r a d i o i m m u n o a s s a y d a t a i t was d e t e r m i n e d t h a t a b o u t 1 . 2 5 x 10 a n t i - D A N S IgG m o l e c u l e s c o u l d be bound p e r e a c h D A N S - r i c i n p r e - l a b e l l e d RBC ( F I G : 1 4 ) . A l t h o u g h t h i s i s n o t a o n e - t o - o n e c o r r e l a t i o n , t h e d a t a d o e s i n d i c a t e t h a t t h e r e i s an o r d e r o f m a g n i t u d e c o r r e s p o n d e n c e b e t w e e n t h e number o f r i c i n r e c e p t o r s and t h e l e v e l o f a n t i - h a p t e n IgG b i n d i n g . T h e d e g r e e o f n o n -s p e c i f i c b i n d i n g o f r a d i o a c t i v e i m m u n o g l o b u l i n was e s t i m a t e d t o be b e t w e e n 1-5% o f t h e t o t a l , i m p l y i n g a h i g h d e g r e e o f s p e c i f i c i t y . ( 2 ) BINDING OF F I T C CONJUGAGED ANTI-DANS I g G : A n o t h e r t e s t f o r t h e s p e c i f i c i t y and a c t i v i t y o f t h e a n t i - D A N S i m m u n o g l o b u l i n was t o d i r e c t l y v i s u a l i z e t h e b i n d i n g o f F I T C c o n j u g a t e d a n t i b o d y t o c e l l s p r e - l a b e l l e d w i t h a h a p t e n m o d i f i e d p r o b e . I n t h i s e x p e r i m e n t , mouse n e u r o b l a s t o m a c e l l s w e r e f i r s t l a b e l l e d w i t h D A N S - r i c i n , a t s a t u r a t i n g c o n c e n t r a t i o n s , and t h e n s t a i n e d w i t h F I T C - a n t i - D A N S I g G . T h e d e g r e e o f s p e c i f i c v e r s u s n o n - s p e c i f i c f l u o r e s c e n c e was t h e n e v a l u a t e d u n d e r - 61 -LIGAND CONCENTRATION (/tg/ml) F I G . 14 4 The s p e c i f i c i t y o f r a d i o l a b e l l e d a n t i - D A N S IgG ( 5 . 1 4 x 10 c p m / ^ g ) was d e t e r m i n e d by m e a s u r i n g i t s b i n d i n g t o human e r y t h r o c y t e s w h i c h h a d b e e n p r e - l a b e l l e d w i t h D A N S - r i c i n . The a m o u n t o f s p e c i f i c b i n d i n g (O O) became s a t u r a t i n g a t a p p r o x i m a t e l y 1 . 2 5 x 10^ s i t e s p e r c e l l . The d e g r e e o f n o n - s p e c i f i c b i n d i n g ( • — • ] was d e t e r m i n e d f r o m s a m p l e s w h i c h had b e e n p r e - l a b e l l e d w i t h D A N S - r i c i n i n t h e p r e s e n c e o f 0 . 1 M D - g a l a c t o s e . - 62 -t h e f l u o r e s c e n c e - l i g h t m i c r o s c o p e u s i n g a p p r o p r i a t e f i l t e r s w h i c h c o u l d d i s t i n g u i s h b e t w e e n DANS and F I T C . T e s t c e l l s p e c i m e n s e x h i b i t e d t h e c h a r a c t e r i s t i c " r i m " o f f l u o r e s c e n c e , a r o u n d t h e i r e x t e r i o r , w h e r e a s , c o n t r o l s p e c i m e n s s h o w e d v e r y l i t t l e l a b e l l i n g ( F I G : 1 6 ) . S i n c e l a b e l l i n g was p e r f o r m e d f o r a r e l a t i v e l y s h o r t p e r i o d o f t i m e a t room t e m p e r a t u r e , t h e r e was l i t t l e r e d i s t r i b u t i o n o f r i c i n b i n d i n g s i t e s , a s e x a m i n e d u n d e r t h e f l u o r e s c e n t m i c r o s c o p e . I t h a s p r e v i o u s l y been r e p o r t e d t h a t e x t e n s i v e r e a r r a n g e m e n t and i n t e r n a l i z a t i o n o f l e c t i n r e c e p t o r s o c c u r s when o n e u r o b l a s t o m a c e l l s a r e l a b e l l e d a t 37 C f o r l o n g p e r i o d s o f t i m e ( 4 6 ) . ( 3 ) HAPTEN SANDWICH METHODOLOGY: T h e v i a b i l i t y o f t h e h a p t e n s a n d w i c h l a b e l l i n g t e c h n i q u e i s d e p e n d e n t on t h e c o n d i t i o n t h a t t h e a n t i - h a p t e n a n t i b o d i e s a r e b i v a l e n t and a b l e t o a c t as a b r i d g i n g m o l e c u l e . S i n c e p r o c e s s i n g s a m p l e s f o r SEM i s a t e d i o u s and t i m e c o n s u m i n g p r o c e d u r e , a q u i c k m e t h o d f o r d e t e r m i n i n g t h e e f f i c a c y o f t h e t e c h n i q u e was r e q u i r e d . G l u t a r a l d e h y d e f i x e d R B C ' s w e r e l a b e l l e d by t h e h a p t e n s a n d w i c h m e t h o d u s i n g D A N S - r i c i n as t h e i n i t i a l p r o b e and D A N S - F e -m i c r o s p h e r e s as t h e m a r k e r s . C e l l s w h i c h h a v e been s p e c i f i c a l l y l a b e l l e d i n t h i s m a n n e r s h o u l d now be s u s c e p t i b l e t o movement i n a m a g n e t i c f i e l d , due t o t h e p r e s e n c e o f bound i r o n on t h e i r s u r f a c e s . L a b e l l e d c e l l s w e r e l a y e r e d o n t o a s o l u t i o n o f 10% BSA i n a c l o s e d - t i p P a s t e u r p i p e t and p l a c e d i n t h e i n f l u e n c e o f a s m a l l h o r s e s h o e m a g n e t . S p e c i f i c a l l y l a b e l l e d c e l l s w e r e a t t r a c t e d t o t h e w a l l s o f t h e p i p e t by t h e m a g n e t , w h e r e a s , c o n t r o l c e l l s w e r e n o t a t t r a c t e d and s e d i m e n t e d t o t h e b o t t o m o f t h e p i p e t . T h e - 63 -F I G : 15 A s c h e m a t i c r e p r e s e n t a t i o n o f t h e s t e p s i n v o l v e d i n t h e h a p t e n s a n d w i c h l a b e l l i n g t e c h n i q u e . I n t h i s i l l u s t r a t i o n , c a r b o h y d r a t e c o n t a i n i n g g r o u p s on t h e c e l l s u r f a c e a r e l a b e l l e d w i t h t h e h a p t e n m o d i f i e d p l a n t l e c t i n . The bound l e c t i n c a n be v i s u a l i z e d by l a b e l l i n g i n s e q u e n c e w i t h a n t i - h a p t e n a n t i - b o d i e s f o l l o w e d by t h e h a p t e n m o d i f i e d m a r k e r . - 64 -F I G . 16 Mouse n e u r o b l a s t o m a c e l l s , w h i c h had been p r e - l a b e l l e d w i t h D A N S - r i c i n , w e r e s t a i n e d w i t h FITC c o n j u g a t e d a n t i - D A N S I g G . C o n t r o l c e l l s w e r e p r e - l a b e l l e d w i t h D A N S - r i c i n i n t h e p r e s e n c e o f 0 . 1 M D - g a l a c t o s e and show l i t t l e , i f a n y , b i n d i n g o f t h e f l u o r e s c e n t a n t i - h a p t e n a n t i b o d y . ( m a g n i f i c a t i o n 240 X ) C O N T R O L - 66 -s u c c e s s o f t h i s t e s t was a g o o d i n d i c a t i o n t h a t t h e h a p t e n s a n d w i c h m e t h o d o l o g y was w o r k i n g a s e x p e c t e d . T h e u l t i m a t e t e s t o f t h e m e t h o d was a c t u a l v i s u a l i z a t i o n by SEM. G l u t a r a l d e h y d e f i x e d mouse n e u r o b l a s t o m a l i n e n e u r o 2A c e l l s w e r e l a b e l l e d w i t h e i t h e r D A N S - r i c i n o r DANS-Con A f o l l o w e d by a n t i - D A N S IgG and D A N S - F e -m i c r o s p h e r e s , i n s e q u e n c e . S p e c i f i c a l l y l a b e l l e d c e l l s e x h i b i t e d a h e a v y u n i f o r m d i s t r i b u t i o n o f s p h e r e s o v e r t h e e n t i r e c e l l s u r f a c e . ( S e e F I G : 1 7 ) . I n c o n t r o l e x p e r i m e n t s , i n w h i c h c e l l s were p r e - l a b e l l e d w i t h D A N S - l e c t i n s i n t h e p r e s e n c e o f t h e a p p r o p r i a t e s a c c h a r i d e i n h i b i t o r , t h e r e was no s i g n i f i c a n t l a b e l l i n g . The f a c t t h a t t h e m a r k e r l a t e x p a r t i c l e s c o n t a i n e d an i r o n c o r e o f F e ^ O ^ , was u s e d t o a d v a n t a g e i n v e r i f y i n g t h e p r e s e n c e o f b o u n d D A N S - F e - m i c r o s p h e r e s on t h e c e l l s u r f a c e . R e g i o n s o f b o t h t e s t and c o n t r o l c e l l s e x a m i n e d u n d e r t h e SEM w e r e a n a l y z e d f o r e n e r g y d i s p e r s e d X - r a y s and t h e p r e s e n c e o r a b s e n c e o f a c h a r a c t e r i s t i c i r o n p e a k a t 6 . 4 0 KeV ( d e s i g n a t e d 0 ( - ^KLJJJ ) . T e s t c e l l s g a v e a s t r o n g s i g n a l , w h e r e a s c o n t r o l c e l l s g a v e no a p p r e c i a b l e s i g n a l . ( S e e F I G : 1 8 ) . The X - r a y m i c r o p r o b e a n a l y s i s i s a v e r y u s e f u l t e c h n i q u e i n t h e i d e n t i f i c a t i o n o f s u c h s u r f a c e m a r k e r s , e s p e c i a l l y i n t h e c a s e w h e r e t h e c e l l i n q u e s t i o n p o s s e s s e s a l a r g e number o f m i c r o v i l l i o r o t h e r c o m p l i c a t e d s u r f a c e f e a t u r e s . F I G 17 S c a n n i n g e l e c t r o n m i c r o g r a p h s o f 3T3 f i b r o b l a s t c e l l s w h i c h h a v e b e e n l a b e l l e d b y t h e h a p t e n s a n d w i c h t e c h n i q u e . G l u t a r a l d e h y d e f i x e d c e l l s w e r e p r e - l a b e l l e d w i t h D A N S - r i c i n ( c o n t r o l s i n p r e s e n c e o f 0.1 M D - g a l a c t o s e ) a n d s u b s e q u e n t l y l a b e l l e d i n s e q u e n c e w i t h a n t i - D A N S I g G a n d D A N S - F e - m i c r o -s j p j h e r e s . T e s t c e l l s ( A ) e x h i b i t e d a d e n s e u n i f o r m l a b e l l i n g p a t t e r n , w h e r e a s c o n t r o l c e l l s ( B ) w e r e v i r t u a l l y f r e e o f D A N S - F e - m i c r o s p h e r e s . O u t l i n e d a r e a s o f c e l l s i n d i c a t e r e g i o n o f e n e r g y d i s p e r s e d X - r a y a n a l y s i s . M a g n i f i c a t i o n o f m i c r o -g r a p h s i s 7,37 0 X . - 6 8 -- 69 -F I G . 18 D i r e c t v e r i f i c a t i o n o f t h e p r e s e n c e o f bound D A N S - F e - m i c r o s p h e r e s on 3T3 f i b r o b l a s t c e l l s . C e l l s w e r e l a b e l l e d v i a t h e h a p t e n s a n d w i c h t e c h n i q u e , as p r e v i o u s l y d e s c r i b e d f o r F I G : 1 7 . , u s i n g D A N S - r i c i n as t h e p r i m a r y l i g a n d . E q u i v a l e n t r e g i o n s o f b o t h t h e t e s t (B) and c o n t r o l (A) c e l l s w e r e s u b j e c t e d t o X - r a y m i c r o p r o b e a n a l y s i s and bound D A N S - F e -m i c r o s p h e r e s c o u l d be i d e n t i f i e d by t h e p r e s e n c e o f a s t r o n g i r o n p e a k a t 6 . 4 0 K e V . X-ray microprobe a n a l y s i s was performed u s i n g an ORTEC energy d i s p e r s i v e X-ray spectrometer i n c o n j u n c t i o n w i t h an ETEC Autoscan e l e c t r o n microscope. These f a c i l i t i e s were generously p r o v i d e d by the department of M e t a l l u r g y on the U.B.C. campus. REGION OF INTEREST 0"IO KeV .- . • • : — % t • • •. • %« • • • • • • • • • : : . « . o B. •• • *.* •* • _ • • • • m • ...... No Al Si Au S Cl Pd K K Ti Ti H III!—I—I 1—I 1 h Fe Fe Cu Zn Zn Au — H -- 71 -D I S C U S S I O N The d e v e l o p m e n t o f t h e h a p t e n s a n d w i c h l a b e l l i n g t e c h n i q u e was r e l a t i v e l y s t r a i g h t f o r w a r d and i t s u s e i n v i s u a l i z i n g l e c t i n r e c e p t o r s r e p r e s e n t e d a s i m p l e a p p l i c a t i o n o f t h e m e t h o d o l o g y . T h e t r u e v a l u e o f t h i s t e c h n i q u e may be r e a l i z e d i n i t s a p p l i c a t i o n t o t h e l o c a l i z a t i o n o f s m a l l m o l e c u l a r w e i g h t l i g a n d b i n d i n g s i t e s w h e r e s t e r i c a c c e s s i b i l i t y i s a p o t e n t i a l p r o b l e m o r w h e r e a m p l i f i c a t i o n o f t h e p r i m a r y s u r f a c e s p e c i f i c p r o b e i s n e c e s s a r y . 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S o d i u m - p o t a s s i u m -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 o f E l e c t r o p h o r u s e l e c t r i c o r g a n . I I . E f f e c t s o f N - e t h y l m a l e i m i d e and o t h e r s u l f h y d r a l r e a g e n t s . J . B i o l . Chem. 2 4 1 : 1 8 9 0 P o s t , R . L . , Kume, S . , R o g e r s , F . N . ( 1 9 7 3 ) . A l t e r n a t i n g p a t h s o f p h o s -p h o r y l a t i o n o f t h e s o d i u m a n d p o t a s s i u m i o n pump o f p l a s m a m e m b r a n e s . I n " M e c h a n i s m s i n B i o E n e r g e t i c s " , e d . G . F . A z z o n e , L . E r n s l e r , S . P a p a , E. Q u a g l i a r i e l l o , N . S i l i p r a n d i , 2 0 3 - 2 1 8 . New Y o r k : A c a d e m i c . B o n d , G . H . , B a d e r , H . , P o s t , R . L . ( 1 9 7 1 ) . A c e t y l p h o s p h a t e as a s u b -s t i t u t e f o r ATP i n ( N a + + K + ) d e p e n d e n t A T P a s e . B i o c h i m . B i o p h y s . 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Chem. 2 4 7 : 7 6 4 2 . C a p a l d i , R . A . , V a n d e r k o o i , G . ( 1 9 7 2 ) . T h e l o w p o l a r i t y o f many membrane p r o t e i n s . P r o c . N a t n . A c a d . S c i . U . S . A . 6 9 : 9 3 0 . M a c L e n n a n , D . H . , Y i p , C . C , l i e s , G . H . , S e e m a n , P . ( 1 9 7 3 ) . I s o l a t i o n o f S a r c o p l a s m i c r e t i c u l u m p r o t e i n . C o l d S p r i n g H a r b . Symp. Q u a n t . B i o l . 3 7 : 4 6 9 . L a n e , L . K . , C o p e n h a v e r , J . H . , L i n d e n m a y e r , G . E . , S c h w a r t z , A . ( 1 9 7 3 ) . P u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f and o u a b a i n b i n d i n g t o t h e t r a n s p o r t A T P a s e f r o m o u t e r m e d u l l a o f c a n i n e k i d n e y . J . B i o l . Chem. 2 4 8 : 7 1 9 7 . J o r g e n s e n , P . L . ( 1 9 7 4 ) . E s t i m a t i o n o f t h e p u r i t y o f N a , K A T P a s e and o f t h e m o l e c u l a r w e i g h t and p o l y p e p t i d e c o n t e n t p e r enzyme u n i t i n p r e p a r a t i o n s f r o m t h e o u t e r m e d u l l a o f r a b b i t k i d n e y . B i o c h e m . B i o p h y s . A c t a 3 5 6 : 5 3 . W a c h s t e i n , M . , M e i s e l , E . ( 1 9 5 7 ) . H i s t o c h e m i s t r y o f h e p a t i c p h o s -p h a t a s e s a t a p h y s i o l o g i c p H : w i t h s p e c i a l r e f e r e n c e t o t h e d e m o n s t r a t i o n o f b i l e c n a n a l i c u l i . Am. J . C l i n . P a t h . 2 7 : 1 3 A b e l , J . H . J r . ( 1 9 6 9 ) . E l e c t r o n m i o r o s c o p i c d e m o n s t r a t i o n o f a d e n o s i n e t r i p h o s p h a t e p h o s p h o h y d r o l a s e a c t i v i t y i n h e r r i n g g u l l s a l t g l a n d s . J . H i s t o c h e m . C y t o c h e m . 1 7 : 5 7 0 . E r n s t , S . A . ( 1 9 7 2 ) . T r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e c y t o c h e m i s t r y . I I . C y t o m c h e m i c a l l o c a l i z a t i o n o f o u a b a i n - s e n s i t i v e , p o t a s s i u m d e p e n d e n t p h o s p h a t a s e a c t i v i t y i n t h e s e c r e t o r y e p i t h e l i u m o f t h e a v i a n s a l t g l a n d . J . H i s t o c h e m . C y t o c h e m . 2 0 : 2 3 . G i l l i s , J . M . , P a g e , S . G . ( 1 9 6 7 ) . L o c a l i z a t i o n o f A T P a s e a c t i v i t y i n s t r i a t e d m u s c l e and p r o b a b l e s o u r c e s o f a r t i f a c t . J . C e l l . S c i . 2 : 1 1 3 . M o s e s , H . L . , R o s e n t h a l , A . S . ( 1 9 6 8 ) . P i t f a l l s i n t h e u s e o f l e a d i o n f o r h i s t o c h e m i c a l l o c a l i z a t i o n o f n u c l e o s i d e p h o s p h a t a s e s . J . H i s t o c h e m . C y t o c h e m . 1 6 : 5 3 0 . J o r g e n s e n , P . L . , H a n s e n , 0 . , G l y n n , I . M . , C a v i e r e s , J . D . ( 1 9 7 3 ) . A n t i b o d i e s t o p i g k i d n e y N a , K A T P a s e i n h i b i t t h e Na pump i n human r e d c e l l s p r o v i d e d t h e y h a v e a c c e s s t o t h e i n n e r s u r f a c e o f t h e c e l l m e m b r a n e . B i o c h i m . B i o p h y s . A c t a 2 9 1 : 7 9 5 - 77 -( 8 2 ) G l y n n , I . M . , K a r l i s h , S . J . D . , C a v i e r e s , J . D . , E l l o r y , J . C . , L e w , V . L . , J o r g e n s e n , P . L . ( 1 9 7 4 ) . The e f f e c t s o f a n t i s e r u m t o N a , K A T P a s e on t h e i o n t r a n s p o r t i n g and h y d r o l y t i c a c t i v i t i e s o f t h e e n z y m e . A n n . N . Y . A c a d . S c i . ( 8 3 ) A s c a r i , A . , R a o , S . N . ( 1 9 7 2 ) . N a , K A T P a s e c o m p l e x , e f f e c t o f a n t i -c o m p l e x a n t i b o d y on t h e p a r t i a l r e a c t i o n s c a t a l y z e d by t h e c o m p l e x . B i o c h i m . B i o p h y s . R e s . Commun. 4 9 : 1 3 2 3 . ( 8 4 ) C h a r n o c k , J . S . , T r e b i l c o c k , H . A . , C a s l e y - S m i t h , J . R . ( 1 9 7 2 ) . Demon-s t r a t i o n o f t r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e i n t h e p l a s m a membrane o f e r y t h r o c y t e g h o s t s by q u a n t i t a t i v e e l e c t r o n m i c r o s c o p y . J . H i s t o c h e m . C y t o c h e m . 2 0 : 1 0 6 9 . ( 8 5 ) K y t e , J . ( 1 9 7 4 ) . The r e a c t i o n s o f s o d i u m 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 w i t h s p e c i f i c a n t i b o d i e s . J . B i o l . Chem. 2 4 9 : 3 6 5 2 . ( 8 6 ) K y t e , J . ( 1 9 7 6 ) . I m m u n o f e r r i t i n d e t e r m i n a t i o n o f t h e d i s t r i b u t i o n o f N a , K A T P a s e o v e r t h e p l a s m a membranes o f r e n a l c o n v o l u t e d t u b u l e s : I . D i s t a l s e g m e n t . J . C e l l . B i o l . 6 8 : 2 8 7 . ( 8 7 ) K y t e , J . ( 1 9 7 5 ) . S t r u c t u r a l s t u d i e s o f s o d i u m 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 . J . B i o l . Chem. 2 5 0 : 7 4 4 3 . ( 8 8 ) K y t e , J . ( 1 9 7 6 ) . I m m u n o f e r r i t i n d e t e r m i n a t i o n o f t h e d i s t r i b u t i o n o f N a , K A T P a s e o v e r t h e p l a s m a membranes o f r e n a l c o n v o l u t e d t u b u l e s . I I . P r o x i m a l s e g m e n t . J . C e l l B i o l . 6 8 : 3 0 4 . ( 8 9 ) Wood, J . G . , J e a n , D . H . , W h i t a k e r , J . N . , M c L a u g h l i n , B . J . , A l b e r s , R . W . ( 1 9 7 7 ) . I m m u h o c y t o c h e m i c a l l o c a l i z a t i o n o f t h e s o d i u m , p o t a s s i u m a c t i v a t e d A T P a s e i n k n i f e f i s h b r a i n . J . N e u r o c y t o l . J 5 : 5 7 1 . ( 9 0 ) Y o d a , A . ( 1 9 7 3 ) . S t r u c t u r e a c t i v i t y r e l a t i o n s h i p s o f c a r d i o t o n i c s t e r o i d s f o r t h e i n h i b i t i o n o f N a , K A T P a s e . I . 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 enzyme - c a r d i a c g l y c o s i d e c o m p l e x e s f o r m e d i n t h e p r e s e n c e o f m a g n e s i u m and p h o s p h a t e . M o l . P h a r m a c o l . _ 9 : 5 1 . ( 9 1 ) L i m , R . W . , M o l d a y , R . S . , H u a n g , H . V . , H e n , S . P . S . ( 1 9 7 5 ) . A p p l i c a t i o n o f l a t e x m i c r o s p h e r e s i n t h e i s o l a t i o n o f p l a s m a m e m b r a n e s : A f f i n i t y d e n s i t y p e r t u r b a t i o n o f e r y t h r o c y t e m e m b r a n e s . B i o c h i m i c a e t B i o p h y s i c a . A c t a 3 9 4 : 3 7 7 (92) Ruoho, A., Ky t e J . (1974). P h o t o a f f i n i t y l a b e l i n g o f t h e o u a b a i n - b i n d i n g s i t e on (Na + K) a d e n o s i n e t r i p h o s p h a -t a s e . 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