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Anion regulation of Ca2+ transport ATPase of the human erythrocyte membrane Minocherhomjee, A. M. 1982

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ANION REGULATION OF C a 2 + TRANSPORT ATPase OF THE HUMAN ERYTHROCYTE MEMBRANE by A. M. MINOCHERHOMJEE M . S c , The U n i v e r s i t y of T o r o n t o , 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES ( F a c u l t y o f Pharmaceut ical S c i e n c e s ) We accept t h i s t h e s i s as conforming to the r e q u i r e d standards THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1982 (c) A . M . Minocherhomjee, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Pharmaceutical Sciences The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 27, 1982. DE-6 (3/81) ABSTRACT The mechanism o f r e g u l a t i o n o f the Ca pump ATPase o f the human e r y t h r o c y t e membrane by c a l m o d u l i n , c y c l i c AMP and the anion channel was s t u d i e d u s i n g membrane f ragments, r e s e a l e d "ghosts", i n s i d e - o u t v e s i c l e s and a T r i t o n X-100 s o l u b i l i z e d enzyme p r e p a r a t i o n . 2+ 2+ The (Ca + Mg )-ATPase a c t i v i t y i n e r y t h r o c y t e membranes or a T r i t o n X-100 s o l u b i l i z e d enzyme p r e p a r a t i o n showed b i p h a s i c (h igh and low 2+ a f f i n i t y ) Ca a c t i v a t i o n k i n e t i c s . The a n i o n i c c a l c i u m b i n d i n g p r o t e i n , c a l m o d u l i n , i n c r e a s e d both the c a l c i u m s e n s i t i v i t y ( 1 ^ 2 + ) and the maximum v e l o c i t y ( V m a x ) of the enzyme. C e r t a i n polyaniom'c agents ( p o l y - L - a s p a r t i c a c i d , p o l y - L - g l u t a m i c a c i d ) , a l i c y c l i c s u l f o n i c a c i d s ( H E P E S , N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c a c i d , M E S , 2 - N -( m o r p h o l i n o e t h a n e s u l f o n i c a c i d ) ) , and aromatic c a r b o x y l i c a c i d s (benzoic r 2+ but not the V m = v Ca max. 2+ and s a l i c y l i c a c i d s ) i n c r e a s e d the K r 2+ but not the V o f (Ca + 2+ Mg )-ATPase in e r y t h r o c y t e membranes and T r i t o n X-100 s o l u b i l i z e d enzyme p r e p a r a t i o n s . T r i f l u o p e r a z i n e (30 yM) antagonized a c t i v a t i o n of the enzyme by c a l m o d u l i n and p o l y - L - a s p a r t i c a c i d , but not by sodium-HEPES 2+ 2+ or sodium-MES. L i m i t e d t r y p s i n p r o t e o l y s i s o f (Ca + Mg )-ATPase in the e r y t h r o c y t e membrane a b o l i s h e d a c t i v a t i o n by c a l m o d u l i n , p o l y - L -a s p a r t i c a c i d and sodium-HEPES. These r e s u l t s suggest t h a t the modula-2+ 2+ 2+ t i o n o f the Ca s e n s i t i v i t y of (Ca + Mg )-ATPase by c a l m o d u l i n may be a s s o c i a t e d w i t h the a n i o n i c p r o p e r t i e s o f t h i s p r o t e i n , and t h a t t h i s property can be mimicked by some other a n i o n s , p r o b a b l y by i n t e r a c t i n g a t an a n i o n - r e g u l a t o r y s i t e on the enzyme. C y c l i c AMP (5 yM) was found t o i n h i b i t the ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y (approx. 20%) i n e r y t h r o c y t e membranes, probably v i a endogenous c y c l i c AMP p r o t e i n k i n a s e , s i n c e t h i s e f f e c t c o u l d be blocked by c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r C.PKI) from t h e r a b b i t s k e l e t a l m u s c l e , 2+ 2+ By c o n t r a s t , bovine heart PKI s t i m u l a t e d (Ca + Tig )-ATPase a c t i v i t y (approx. 100%) by i n c r e a s i n g the K r 2+ but not the V „ o f the enzyme ua max. i n membrane o r T r i t o n X-100 s o l u b i l i z e d p r e p a r a t i o n s . At a low c a l c i u m c o n c e n t r a t i o n the s t i m u l a t i o n by bovine heart PKI and s a t u r a t i n g l e v e l s o f c a l m o d u l i n was a d d i t i v e , s u g g e s t i n g t h a t the two e f f e c t o r s acted by 2+ 2+ d i s t i n c t mechanisms. The s t i m u l a t i o n o f (Ca + Mg )-ATPase a c t i v i t y by bovine heart PKI was not s o l e l y due to i t s antagonism o f the p r o t e i n k i n a s e because a) m o d i f i c a t i o n o f a r g i n i n e r e s i d u e s o f bovine heart PKI a b o l i s h e d i t s i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e , but had no e f f e c t 2+ 2+ on the s t i m u l a t i o n of (Ca + Mg )-ATPase; b) t r i f l u o p e r a z i n e (20 yM) 2+ 2+ a n t a g o n i z e d the s t i m u l a t i o n o f (Ca + Mg )-ATPase by P K I , s i m i l a r l y to i t s antagonism o f c a l m o d u l i n s t i m u l a t i o n , but i t d i d not a f f e c t the i n h i b i t i o n of p r o t e i n k i n a s e by PKI . I t i s suggested t h a t d i f f e r e n t mechanisms a r e i n v o l v e d i n the i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e 2+ 2+ and the s t i m u l a t i o n o f (Ca + Mg )-ATPase by bovine heart c y c l i c AMP PKI. 2+ 2+ Next , the r o l e o f anion channel b l o c k e r s on the (Ca + Mg ) -ATPase was s t u d i e d . The p h o t o l a b e l i n g reagent N - ( 4 - a z i d o - 2 - m " t r o p h e n y l ) -2+ 2 a m i n o e t h y l s u l f o n a t e ( N A P - t a u r i n e ) was found to i n h i b i t the (Ca + 2+ Mg )-ATPase o f fragmented red c e l l membranes. H a l f maximal i n h i b i t i o n 2+ o c c u r r e d between 25 yM and 50 yM. At t h e s e c o n c e n t r a t i o n s Mg -ATPase and ( N a + + K + ) -ATPase a c t i v i t i e s i n the membranes were not a f f e c t e d . 2+ 2+ The r e v e r s i b l e i n h i b i t i o n o f (Ca + Mg )-ATPase produced by NAP-t a u r i n e i n the dark became i r r e v e r s i b l e a f t e r p h o t o l y s i s i n the presence C i v ) ^ ' 2 + 2 + o f t h i s r e a g e n t . I n c u b a t i o n o f the membranes w i t h Ca , Mg , ATP or c a l m o d u l i n , p r i o r to p h o t o l y s i s 1n the presence o f N A P - t a u r i n e , d i d not p r o t e c t the enzyme from I n h i b i t i o n . L i m i t e d t r y p s i n p r o t e o l y s i s o f 2+ 2+ (Ca + Mg )-ATPase i n fragmented membranes, which a b o l i s h e d a c t i v a t i o n by c a l m o d u l i n , d i d not a f f e c t the i n h i b i t i o n by N A P - t a u r i n e . 2+ 2+ N A P - t a u r i n e was found to I n h i b i t the (Ca + Mg )-ATPase a c t i v i t y from t h e c y t o p l a s m i c s i d e of the membrane, as determined from t h e f o l l o w -ing e x p e r i m e n t s . A d d i t i o n o f N A P - t a u r i n e (50 yM) to r e s e a l e d e r y t h r o c y t e 2+ 2+ ghosts i n h i b i t e d l e s s than b% of the (Ca + Mg )-ATPase a c t i v i t y , compared to 50-60°' I n h i b i t i o n 1n ghosts r e s e a l e d i n the presence o f 50 yM 2+ N A P - t a u r i n e . Furthermore, N A P - t a u r i n e i n h i b i t e d ATP-dependent Ca -t r a n s p o r t i n t o i n s i d e - o u t v e s i c l e s a t a s i m i l a r c o n c e n t r a t i o n (.50 yM). 2+ 2+ The i n h i b i t i o n o f the (Ca + Mg )-ATPase a c t i v i t y o f membranes by NAP-t a u r i n e appeared to be a d i r e c t a c t i o n on the enzyme, r a t h e r than through 2+ 2+ i n h i b i t i o n of the anion c h a n n e l , as (Ca + Mg )-ATPase a c t i v i t y was not i n h i b i t e d i n membranes made from red blood c e l l s r e a c t e d i r r e v e r s i b l y w i t h 50 yM N A P - t a u r i n e or the anion channel b l o c k e r 4 , 4 ' - d i i s o t h i o c y a n o -2 , 2 ' s t i l b e n e d i s u l f o n a t e (DIDS) (5yM) or i n membranes assayed i n the presence o f another anion channel b l o c k e r , probenecid (125 yM). Th is i s 2+ the f i r s t r e p o r t e d s e l e c t i v e a n t a g o n i s t o f the Ca pump, and i t 1s s u g -2+ gested t h a t N A P - t a u r i n e c o u l d be a u s e f u l t o o l f o r s t u d y i n g the Ca -t r a n s p o r t ATPase i n a v a r i e t y of c e l l s . S i g n a t u r e of t h e s i s s u p e r v i s o r TABLE QF CONTENTS page ABSTRACT < . . , ( i 1 ) LIST OF TABLES ( v i 1 1 ) LIST OF FIGURES O x ) LIST OF ABBREVIATIONS ( x i ) INTRODUCTION 1 1. R e g u l a t i o n o f c e l l u l a r c a l c i u m . . . . . . . . . 3 2 . Calcium t r a n s p o r t and ( C a 2 + + M g 2 + ) - A T P a s e 6 a c t i v i t y - B a s i c C h a r a c t e r i s t i c s 3 . R e g u l a t i o n o f t h e c a l c i u m pump ATPase i n human ^ e r y t h r o c y t e s . . . A) Role o f c a l m o d u l i n 15 B) Role of p r o t e i n k i n a s e s 27 C) Role o f anion channel (Band 3) . . . . . . 30 4. Aims o f the present study 31 MATERIALS AND METHODS 33 I. M a t e r i a l s 33 I I . Methods 36 1. P r e p a r a t i o n o f e r y t h r o c y t e ghosts 36 by v a r i o u s procedures . . . . . . . . . 2 . P r e p a r a t i o n of c a l m o d u l i n d e f i c i e n t 37 (EDTA t r e a t e d ) membranes 3 . P r e p a r a t i o n o f r e s e a l e d ghosts . . . . . . . 38 4. P r e p a r a t i o n o f i n s i d e - o u t v e s i c l e s . 39 5. I r r a d i a t i o n o f red c e l l s and membranes 40 w i t h N - ( 4 - A z i d o - 2 - n i t r o p h e n y l ) - 2 -a m i n o e t h y l s u l f o n a t e ( N A P - t a u r i n e ) . . . . . 6. S o l u b i l i z a t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e from 41 e r y t h r o c y t e membranes . . . . ( v i ) page 7. L i m i t e d p r o t e o l y s i s C t r y p s i m ' z a t i o n ) o f membranes 41 8. P u r i f i c a t i o n o f c y c l i c AMP dependent p r o t e i n k i n a s e I n h i b i t o r 42 9. Chemical m o d i f i c a t i o n o f a r g i n y l s i d e c h a i n s . . 43 10. Assay o f ATPases 43 1 1 . P h o s p h o d i e s t e r a s e assay 44 12. P r o t e i n k i n a s e assay . . . . . . . . . . 45 13. SDS p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . . . . 45 a) P r e p a r a t i o n o f p r o t e i n s o l u t i o n s . . . . 46 b) P r e p a r a t i o n o f g e l s 46 c) S t a i n i n g and d e s t a i n i n g 47 RESULTS AND DISCUSSION 48 S e c t i o n I M e c h a n i s t i c study o f c a l m o d u l i n as an a c t i v a t o r o f c a l c i u m s e n s i t i v i t y and maximum v e l o c i t y of (Ca2+ + Mg 2 +)-ATPase 48 2+ 2+ 2+ 1. K i n e t i c study of Ca a c t i v a t i o n o f (Ca + Mg ) -ATPase and i t s m o d u l a t i o n by c a l m o d u l i n . . 48 2 . E f f e c t o f p o l y - L - c a r b o x y l i c a c i d s and o t h e r anions on the Ca2+ a c t i v a t i o n o f (Ca2+ + Mg2+)-ATPase 51 S e c t i o n I I . E f f e c t o f c y c l i c AMP and c y c l i c AMP dependent p r o t e i n k i n a s e i n h i b i t o r s on (Ca2+ + Mg2+) -ATPase a c t i v i t y . . . . . . . . . . . 66 Section IE. Role o f anion channel (band 3) i n h i b i t o r s i n the r e g u l a t i o n o f (Ca2+ + Mg 2 +)-ATPase a c t i v i t y . . 83 1. E f f e c t o f N A P - t a u r i n e on ( C a 2 + + Mg 2 + ) - A T P a s e a c t i v i t y 84 2 . The s idedness o f N A P - t a u r i n e i n h i b i t i o n of (Ca2+ + Mg2+)-ATPase 92 ( v i i ) Page 3 . Does NAP-ta.urlne I n h i b i t the ( C a 2 + + M g 2 + ) -ATPase and C a 2 + t r a n s p o r t by b l o c k i n g a n i o n c h a n n e l ? 92 CONCLUSIONS , , , , , , 104 BIBLIOGRAPHY , 107 APPENDIX 123 TABLE LIST OF TABLES ( v i i i ) page I. E f f e c t o f a n i o n s on the Ca s e n s i t i v i t y o f T r i t o n -X-100 s o l u b i l i z e d (Ca2+ + Mg2+)_ I I . D e t e r m i n a t i o n o f c a l m o d u l i n i n p r o t e i n p r e p a r a t i o n s by phosphodiesterase assay and c a l m o d u l i n r a d i o -immunoassay . . . . . . . . . . . . . . 57 I I I . E f f e c t o f a r g i n y l group m o d i f i c a t i o n o f bovine heart c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r and c a l m o d u l i n on m o d u l a t i o n o f ( C a 2 + + T1g2+)-ATPase a c t i v i t y o r c y c l i c AMP p r o t e i n k i n a s e ( C - s u b u n i t ) a c t i v i t y 78 IV. E f f e c t o f t r i f l u o p e r a z i n e on the a b i l i t y of c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r to s t i m u l a t e (Ca2+ + Mg2+)-ATPase i n 'Dodge ghost' membranes . . . . 79 V. E f f e c t o f t r i f l u o p e r a z i n e on the i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e c a t a l y t i c s u b u n i t (C) c a t a l y s e d p h o s p h o r y l a t i o n o f h i s t o n e by p r o t e i n k i n a s e i n h i b i t o r from bovine heart 80 2+ 2+ V I . E f f e c t o f Mg , Ca , ATP and c a l m o d u l i n on the . i n h i b i t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y o f membranes a f t e r p h o t o l y s i s i n the presence o f N A P - t a u r i n e (25 yM) 89 2+ 2+ V I I . (Ca + Mg )-ATPase a c t i v i t y o f 'ghosts' r e s e a l e d i n the presence and absence o f N A P - t a u r i n e . . . 93 2+ 2+ V I I I . (Ca + Mg )-ATPase a c t i v i t y o f membranes made from c e l l s t r e a t e d w i t h N A P - t a u r i n e or D1DS. . . 95 2+ 2+ IX. E f f e c t of N A P - t a u r i n e on p u r i f i e d (Ca + Mg ) -ATPase a c t i v i t y 98 O x ) LIST OF FIGURES page FIGURE 2+ 2+ 1. Calmodul in a c t i v a t i o n o f (.Ca + Mg )-ATPase a c t i v i t y 1n human e r y t h r o c y t e membranes (Dodge Ghosts) 49 2 . Calmodul in a c t i v a t i o n o f T r i t o n X-100 s o l u b i l i z e d ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y 50 2+ 3 . E f f e c t o f p o l y - L - c a r b o x y l i c a c i d s on Ca a c t i v a t i o n o f human e r y t h r o c y t e membrane ( C a 2 + + M g 2 + ) -ATPase a c t i v i t y 52 2+ 4 . E f f e c t o f p o l y - L - c a r b o x y l i c a c i d s on Ca a c t i v a t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e i n T r i t o n X-100 s o l u b i l i z e d enzyme from human e r y t h r o c y t e membranes . . . . 53 2+ 5. E f f e c t o f s u l f o n i c a c i d anions on the Ca a c t i v a t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e i n human e r y t h r o c y t e membranes 54 6A. E f f e c t o f s u l f o n i c a c i d anions i n the presence and absence o f c a l m o d u l i n on C a 2 + a c t i v a t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e i n T r i t o n X-100 s o l u b i l i z e d ( C a 2 + + M g 2 + ) - A T P a s e from human e r y t h r o c y t e membranes 55 6B. The curves i n F igure 6A were f i t t e d by a n o n - l i n e a r c u r v e - f i t t i n g computer program (see t e x t and Table I) 56 7. E f f e c t of i n o r g a n i c a n i o n s and a r o m a t i c c a r b o x y l i c a c i d s on C a 2 + a c t i v a t i o n o f T r i t o n X-100 s o l u b i l i z e d ( C a 2 + + M g 2 + ) - A T P a s e from human e r y t h r o c y t e s . . . 61 8 . E f f e c t of p o l y - L - c a r b o x y l i c a c i d s and s u l f o n i c a c i d s on t r y p s i n t r e a t e d membranes . 62 9. E f f e c t o f t r i f l u o p e r a z i n e on human e r y t h r o c y t e membrane ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y i n the presence o f p o l y - L - c a r b o x y l i c a c i d s or sodium-HEPES b u f f e r . . 64 1 0 . SDS-PAGE o f p r o t e i n k i n a s e i n h i b i t o r and c a l m o d u l i n . 69 FIGURE , page 1 1 . E f f e c t o f c y c l i c AMP and PKI on ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y i n human e r y t h r o c y t e membranes a t v a r i o u s c o n c e n t r a t i o n s o f f r e e c a l c i u m 71 2+ 12. E f f e c t o f c a l m o d u l i n and bovine heart PKI on (Ca + Mg2+)-ATPase a c t i v i t y i n e r y t h r o c y t e membranes and T r i t o n X-100 s o l u b i l i z e d enzyme at 0.58 yM and 55 yM f r e e c a l c i u m 72 2+ 2+ 1 3 . A c t i v a t i o n o f (Ca + Mg )-ATPase i n human e r y t h r o c y t e membranes by c a l m o d u l i n and bovine heart PKI . . 74 14. E f f e c t o f c a l m o d u l i n and PKI on c a l c i u m a c t i v a t i o n o f (Ca^+ + Mg2 + )-ATPase i n human e r y t h r o c y t e membranes 75 15. E a d i e - H o f s t e e p l o t o f data i n f i g u r e 14 76 16. E f f e c t o f N A P - t a u r i n e on ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y o f membranes 85 1 7 . C o n c e n t r a t i o n dependence o f N A P - t a u r i n e i n h i b i t i o n o f (Ca|+ + Mg2+)-ATPase , ( N a + + K + ) -ATPase and M g z + - A T P a s e a c t i v i t i e s by NAP-t a u r i n e 86 2+ 2+ 18. E f f e c t o f N A P - t a u r i n e on (Ca + Mg )-ATPase i n membranes w i t h and w i t h o u t p h o t o l y s i s 87 2+ 2+ 19. D o u b l e - r e c i p r o c o l p l o t o f (Ca + Mg )-ATPase i n h i b i t i o n by N A P - t a u r i n e i n the presence o f c a l m o d u l i n . . . . . . . • 90 2+ 2+ 2 0 . E f f e c t o f N A P - t a u r i n e on (Ca + Mg )-ATPase i n t r y p s i n t r e a t e d membranes 91 2+ 2 1 . E f f e c t o f N A P - t a u r i n e on Ca - t r a n s p o r t i n i n s i d e - o u t ( 1 . 0 . ) v e s i c l e s 94 2 2 . Calcium uptake i n i n s i d e - o u t v e s i c l e s made from red blood c e l l s t r e a t e d w i t h DIDS (5 yM) at 36°C f o r 30 min 99 LIST OF ABBREVIATIONS ADP ATP UTP ITP GTP CTP DPG P i c y c l i c AMP c y c l i c GMP E EP ATPase ( C a 2 + + M g 2 + ) - A T P a s e PKI DEAE-EDTA EGTA HEPES MES TES DTT adenosine 5"-diphosphate adenosine 5 ' - t r i p h o s p h a t e u r i d i n e t r i p h o s p h a t e I n o s i n e t r i p h o s p h a t e guanosine t r i p h o s p h a t e c y t o s i n e t r i p h o s p h a t e 2 , 3 - d i p h o s p h o g l y c e r a t e i n o r g a n i c phosphate c y c l i c adenosine 5'-monophosphate c y c l i c guanosine 5'-monophosphate enzyme phosphory lated enzyme i n t e r m e d i a t e adenosine t r i p h o s p h a t a s e 2+ Mg dependent c a l c i u m s t i m u l a t e d ATPase p r o t e i n k i n a s e i n h i b i t o r d i e t h y l a m i n o e t h y l -e t h y l e n e d i a m i n e t e t r a a c e t a t e , disodium s a l t e t h y l e n e g l y c o l - b i s - ( 6 - a m i n o e t h y l e ther ) N , N 1 - t e t r a a c e t i c a c i d N - 2 - h y d r o x y e t h y l p i p e r a z i n e -N ' - 2 - e t h a n e s u l f o n i c a c i d 2 - N - ( m o r p h o l i n o e t h a n e s u l f o n i c a c i d ) T r i s ( h y d r o x y - m e t h y l ) - m e t h y l 2 - a m i n o e t h a n e s u l f o n i c a c i d d i t h i o t h r e i t o l PMSF para-methyl s u l f o n y l f l u o r i d e TCA t r i c h l o r o a c e t i c a c i d SDS sodium dodecyl s u l f a t e PAGE p o l y a c r y l amide gel e l e c t r o p h o r l s i s TFP t r i f l u o p e r a z i n e N A P - t a u r i n e N - ( 4 - a z t d o - 2 n l t r o p h e n y l ) 2-aminoethyl s u l f o n a t e DIDS 4 , 4 ' - d 1 i s o t h i o c y a n o - 2 - 2 , - s t i l b e n e d i s u l f o n a t e SITS 4 - a c e t a m i d o - 4 ' - i s o t h i o c y a n o s t i l b e n e - 2 1 - d i s u l f o n a t e 2+ K^a2+ a s s o c i a t i o n c o n s t a n t o f the enzyme f o r Ca g gram IOV i n s i d e - o u t v e s i c l e s K.. d i s s o c i a t i o n c o n s t a n t diss, M i c h a e l i s - M e n t e n c o n s t a n t m m i 1 1 i y m i c r o M molar M m o l e c u l a r weight mg m i l l i g r a m sec second min . minute ml m i l l i l i t e r y l m i c r o l i t e r mM m i l l i m o l a r c o n c e n t r a t i o n yM micromolar c o n c e n t r a t i o n nmoles nanomoles ACKNOWLEDGEMENTS U i i i ) I would l i k e t o thank my t h e s i s s u p e r v i s o r , Dr. B,D. R o u f o g a l i s , f o r h i s a d v i c e , encouragement and help throughout t h i s work. I am g r a t e f u l to Drs. D. B r o o k s , D. G o d i n , S. Katz and J , M c N e i l l , f o r being on my a d v i s o r y committee. I g i v e s p e c i a l thanks t o Dr. A. A l - J o b o r e and Mr.. D. 'Mauldin f o r many h e l p f u l d i s c u s s i o n s and s u g g e s t i o n s . I am g r a t e f u l to the Canadian Red Cross f o r the supply o f blood and to the B.C. Heart Foundation f o r f i n a n c i a l a s s i s t a n c e . F i n a l l y , I wish to thank a l l the members o f the f a c u l t y f o r making my program a t U.B.C. v e r y e n j o y a b l e . D E D I C A T I O N Dedicated to my parents. INTRODUCTION The r o l e o f c a l c i u m as a key m e d i a t o r i n the r e g u l a t i o n o f a v a r i e t y o f c e l l u l a r f u n c t i o n s i s w e l l r e c o g n i z e d . For i n s t a n c e , a nerve a c t i o n p o t e n t i a l t r i g g e r e d by a s t i m u l u s i s modulated by c a l c i u m i n f l u x through the axon membrane (Baker 1972) and muscle c o n t r a c t i o n i s evoked 2+ by c a l c i u m i n f l u x a c r o s s the plasma membrane and Ca r e l e a s e from s a r c o p l a s m i c r e t i c u l u m i n s k e l e t a l muscle o r from membrane bound pools i n smooth muscle (Ebashi 1 9 7 6 ) . I n t r a c e l l u l a r c a l c i u m i s a l s o i n v o l v e d i n maintenance o f red blood c e l l shape (Weed and C h a i l l e y 1973) and c o n t r o l of membrane t r a n s p o r t (Porzi'g 1 9 7 2 ) . In c e l l u l a r responses to d i f f e r e n t s t i m u l i , c a l c i u m may f u n c t i o n e i t h e r d i r e c t l y as an i n t r a -c e l l u l a r "second messenger" o r i t may a f f e c t the p r o d u c t i o n o f another "second messenger" ( c y c l i c AMP) by a l t e r i n g the a c t i v i t i e s o f a d e n y l a t e c y c l a s e and/or p h o s p h o d i e s t e r a s e (Rasmussen and Goodman 1 9 7 7 ) . The r e g u l a t o r y f u n c t i o n s o f c a l c i u m depend upon i t s asymmetric d i s t r i b u t i o n i n t i s s u e s and o r g a n e l l e s . The f r e e c o n c e n t r a t i o n o f - 5 -7 c a l c i u m i n the cytoplasm i s t y p i c a l l y between 10 and 10 M, i n c o n t r a s t - 3 to much h i g h e r l e v e l s o f c a l c i u m ( a p p r o x i m a t e l y 10 M) i n the e x t r a -c e l l u l a r f l u i d s and i n i n t r a c e l l u l a r o r g a n e l l e s such as m i t o c h o n d r i a , endoplasmic r e t i c u l u m or s a r c o p l a s m i c r e t i c u l u m i n m u s c l e . Animal c e l l s m a i n t a i n t h i s low l e v e l o f i n t r a c e l l u l a r c a l c i u m by e x t r u d i n g c a l c i u m by + 2+ the Na - Ca exchange mechanism a n d / o r by the ATP d r i v e n c a l c i u m pump o f the plasma membrane.Human red c e l l s serve as a very u s e f u l model system f o r s t u d y i n g the ATP d r i v e n c a l c i u m pump o f the plasma membrane + 2+ f o r s e v e r a l r e a s o n s . These c e l l s l a c k the Na - Ca exchange system (Schatzmannl975; S a r k a d i and Tosteson 1979) and c a l c i u m a c c u m u l a t i n g i n t r a c e l l u l a r o r g a n e l l e s . The e r y t h r o c y t e membrane i s a l s o o f c o n s i d e r -a b l e i n t e r e s t because i t s s t r u c t u r e and/or f u n c t i o n i s a l t e r e d i n c e r t a i n d i s e a s e s t a t e s ( e . g . d i a b e t e s m e l l i t u s , h e r e d i t a r y s p h e r o c y t o s i s ) (Jacob 1974; M c M i l l a n et_aT 1 9 7 8 ) . The biconcave shape o f the red c e l l g ives i t a high s u r f a c e a r e a / volume r a t i o which favours r a p i d d i f f u s i o n e q u i l i b r a t i o n o f r e s p i r a t o r y g a s s e s . The d e f o r m a b i 1 i t y o r p l a s t i c i t y o f the red c e l l s promotes t h e i r passage through the m i c r o v a s c u l a t u r e where i t s f r e i g h t o f oxygen must be d e l i v e r e d (La C e l l e 1 9 6 9 ) ; the s m a l l e s t v e s s e l s are l e s s than 2JJ i n d i a -meter , whereas the red c e l l has a diameter o f about 7JJ. Both o f these c h a r a c t e r i s t i c s o f the red c e l l depend on the mechanical p r o p e r t i e s o f the red c e l l membrane, the maintenance o f which r e q u i r e s a low l e v e l o f i n t r a c e l l u l a r c a l c i u m . Small i n c r e a s e s i n i n t r a c e l l u l a r c a l c i u m can cause c r e n a t i o n , d i s c o c y t e - e c h i n o c y t e shape t r a n s f o r m a t i o n (Weed et a l 1969) and i n c r e a s e d r i g i d i t y o f red c e l l s (Dunn 1974; Weed e t _aj_ 1969; S a r k a d i e t a i 197.6). Calcium s e l e c t i v e l y i n c r e a s e s potassium p e r m e a b i l i t y o f the red c e l l by s t i m u l a t i o n o f the Ca a c t i v a t e d Ktchannel (Gardos 1956; Simons 1 9 7 4 ) . Calc ium c o n c e n t r a t i o n s above 0.1 mM s t r o n g l y i n h i b i t the o u a b a i n - s e n s i t i v e ( N a + + K + ) -ATPase a c t i v i t y i n i s o l a t e d red c e l l membranes, r e s e a l e d - g h o s t s and i n t a c t red c e l l s (Dunham and Glynn 1 9 6 1 ; Davis and V i n c e n z i 1971; S a r k a d i et_al_ 1 9 7 7 ) . I t i s e v i d e n t from the f o r e g o i n g d i s c u s s i o n t h a t a c c u m u l a t i o n o f i n t r a c e l l u l a r c a l c i u m l e a d s to s e r i o u s d e l e t e r i o u s e f f e c t s on the c e l l s . A l though the p h y s i c o -chemical mechanisms by which i n t r a c e l l u l a r c a l c i u m mediates these changes are not c o m p l e t e l y u n d e r s t o o d , i t i s n e v e r t h e l e s s i m p o r t a n t as a f i r s t s t e p to understand the fundamental mechanisms by which red c e l l s r e g u l a t e the l e v e l s o f i n t r a c e l l u l a r c a l c i u m . R e g u l a t i o n o f C e l l u l a r Calcium The normal c o n c e n t r a t i o n o f c a l c i u m i n human red c e l l s i s between 10 and 20 ymoles/L packed c e l l s ( H a r r i s o n and Long 1968; Schatzmann and V i n c e n z i 1969; Dunn 1 9 7 4 ) , and most o f i t i s bound to 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 b i n d i n g s i t e s on the membrane. Al though the p r e c i s e c o n -c e n t r a t i o n o f f r e e i n t r a c e l l u l a r c a l c i u m i s not known because o f i t s b i n d i n g to the c e l l membrane (Long and Mouat 1971) and c e l l c o n s t i t u e n t s such as h e m o g l o b i n , ATP and 2 , 3 - d i p h o s p h o g l y c e r i c a c i d , i t i s e s t i m a t e d to be i n the low mic romolar range (10~^ - 10"^ M) ( H a r r i s o n and Long 1968; Schatzmann 1973; F e r r e i r a and Lew 1 9 7 6 ) . The c o n c e n t r a t i o n o f c y t o p l a s -mic c a l c i u m i s about t h r e e to f o u r o r d e r s o f magnitude s m a l l e r than t h a t present i n the blood plasma ( a p p r o x . 10 M). The red c e l l thus needs a powerful defence mechanism(s) a g a i n s t the i n w a r d l y d i r e c t e d c a l c i u m g r a d i e n t . The two main mechanisms seem to be the low p a s s i v e p e r m e a b i l i -ty o f the red c e l l f o r c a l c i u m and the ATP-dependent, a c t i v e c a l c i u m e x t r u s i o n from the c e l l i n t e r i o r . The p a s s i v e p e r m e a b i l i t y o f f r e s h normal human red c e l l s i s almost u n d e t e c t a b l e when they are i n c u b a t e d i n i s o - o s m o t i c s a l i n e c o n t a i n i n g 2-10 mM c a l c i u m . This low p e r m e a b i l i t y o f the membrane f o r c a l c i u m was demonstrated by b l o c k i n g a c t i v e c a l c i u m e x t r u s i o n by energy d e p l e t i o n o r i n c u b a t i n g the c e l l s i n the c o l d , when c a l c i u m uptake of 1 - 10 ytnoles/L c e l l s per hr was observed (Schatzmann and V i n c e n z i 1969; Lew and F e r r e i r a 1978) . E x h a u s t i v e d e p l e t i o n o f ATP and 2 , 3 - d i p h o s p h o g l y c e r i c a c i d causes i n c r e a s e i n c a l c i u m uptake to l e v e l s a p p r o x i m a t e l y 100 pmoles/L c e l l s per h r . , i n d i c a t i n g a probable r o l e o f ATP i n m a i n t a i n i n g the low p a s s i v e p e r m e a b i l i t y o f the membrane f o r c a l c i u m (Szasz and Gardos 1 9 7 4 ) , o r an e f f e c t o f the d e p l e t i n g agents on the membrane. However, the p e r m e a b i l i t y o f the membrane to c a l c i u m can be i n c r e a s e d i n c e r t a i n d i s e a s e s t a t e s (Eaton e t a\_ 1973; W i l e y and G i l l 1976) or by b i o c h e m i c a l / p h a r m a c o l o g i c a l m a n i p u l a t i o n s , i n c l u d i n g i n c r e a s i n g pH (Romero and Whittam 1971) and osmotic c o n c e n t r a t i o n i n the medium ( P l i s h k e r and Gitelman 1 9 7 6 ) , and by exposure of c e l l s to membrane a c t i v e drugs such as p r o p r a n o l o l (Szasz e t a_l_ 1 9 7 7 ) , p - c h l o r o m e r c u r y -b e n z e n e s u l f o n i c a c i d (Garrahan and Rega 1976) o r by the c a l c i u m ionophore A 23187 (Reed 1973; 1 9 7 6 ) . I t i s o f p a r t i c u l a r i n t e r e s t t h a t the p e r m e a b i l i t y o f red c e l l s to c a l c i u m under s t a t i c c o n d i t i o n s ( i . e . suspending the c e l l s i n i s o t o n i c s a l i n e w i t h c a l c i u m ) may not r e p r e s e n t p h y s i o l o g i c a l c o n d i t i o n s o f shear s t r e s s which the c e l l s are s u b j e c t e d to upon t h e i r passage through the m i c r o c a p i l l a r i e s (which are o f t e n l e s s than h a l f the diameter of the red c e l l s ) . I t was r e c e n t l y shown by Larsen et a]_ (1981) t h a t m e t a b o l i c a l l y d e p l e t e d red c e l l s s u b j e c t e d to shear r a t e (1200 sec"^) take up 10 f o l d more c a l c i u m than under s t a t i c c o n d i t i o n s . Whatever the cause o f i n c r e a s e d c a l c i u m i n f l u x , i f the i n -f l u x i s not compensated f o r by a c t i v e c a l c i u m e x t r u s i o n , the i n c r e a s e d i n t r a c e l l u l a r c a l c i u m w i l l e v e n t u a l l y l e a d to p a t h o l o g i c a l consequences. The p r e c i s e r e l a t i o n s h i p between i n c r e a s e d c a l c i u m and red c e l l patho-p h y s i o l o g y i s not c l e a r , but a r o l e f o r " c o n t r a c t i l e p r o t e i n s " o f the s p e c t r i n - a c t i n network a t t a c h e d to the c y t o p l a s m i c s u r f a c e o f the membranes has been i m p l i c a t e d . The presence o f c a l c i u m i n micromolar c o n c e n t r a t i o n s may a l t e r the c o n f i g u r a t i o n o f t h i s network, thereby c a u s i n g changes i n c e l l shape (Palek et_ a]_ 1 9 7 5 , K i r k p a t r i c k 1974) o r i t may cause c r o s s - l i n k i n g o f s p e c t r i n w i t h a c t i n and other p r o t e i n s , producing i r r e v e r s i b l e s t i f f e n i n g and s p h e r o e c h i n o c y t e f o r m a t i o n (Lorand e_t aj_ 1976) . The i r r e v e r s i b l e c r o s s - l i n k i n g o f p r o t e i n s by c a l c i u m i s 2+ caused by a c t i v a t i o n o f a c y t o p l a s m i c t r a n s g l u t a m i n a s e ( a t Ca > 0 . 5 mM). A l l a n and M i c h e l l ( 1 9 7 5 ; 1977) have suggested a r o l e f o r c a l c i u m - d e p e n -dent changes i n the l i p i d c o m p o s i t i o n o f red c e l l membranes, r e s u l t i n g i n a c c u m u l a t i o n o f 1 , 2 - d i a c y l g l y c e r o l i n the plasma membrane, which may l e a d to e c h i n o c y t e t r a n s f o r m a t i o n and m i c r o v e s i c u l a t i o n o f e r y t h r o c y t e s . Thus, c a l c i u m may r e g u l a t e the r o l e o f both p r o t e i n s and l i p i d s i n the r e g u l a t i o n o f c e l l shape and f u n c t i o n . I t i s i m p e r a t i v e , t h e r e f o r e , t h a t the red c e l l s have a mechanism f o r r a p i d e x t r u s i o n o f c a l c i u m to ensure t h e i r v i a b i l i t y , p a r t i c u l a r l y under adverse p a t h o l o g i c a l c o n d i t i o n s . Calcium Transport and (Ca + Mg ) ATPase a c t i v i t y -B a s i c C h a r a c t e r i s t i c s The m o l e c u l a r b a s i s o f a c t i v e c a l c i u m t r a n s p o r t ( e x t r u s i o n ) i n 2+ 2+ human red blood c e l l s i s a Ca s t i m u l a t e d , Mg dependent ATPase 2+ 2+ ((Ca + Mg )-ATPase) which uses energy from the h y d r o l y s i s o f ATP to a c t i v e l y extrude c a l c i u m a g a i n s t a chemical and e l e c t r o c h e m i c a l g r a d i e n t (Schatzmann 1966; Schatzmann and V i n c e n z i 1 9 6 9 ) . Dunham and Glynn (1961) 2+ 2+ f i r s t demonstrated the presence o f a (Ca + Mg )-ATPase a c t i v i t y which was s e v e r a l f o l d h i g h e r than, the ouabain s e n s i t i v e ( N a + + K + ) -ATPase 2+ 2+ a c t i v i t y . S e v e r a l l i n e s o f ev idence i n d i c a t e t h a t (Ca + Mg )-ATPase 2+ 2+ i s the enzymatic e x p r e s s i o n o f the "Ca -pump", e x t r u d i n g Ca from the red c e l l c y t o p l a s m . 2+ 2+ 2+ 1) Calcium s t i m u l a t e s (Ca + Mg )-ATPase a c t i v i t y and Ca t r a n s -p o r t o n l y a t the i n t r a c e l l u l a r membrane s u r f a c e (Schatzmann and V i n c e n z i 1969) . A d d i t i o n o f e x t e r n a l c a l c i u m (up to 2 - 5 mM) does not a f f e c t the r a t e o f c a l c i u m e f f u x e i t h e r i n r e s e a l e d ghosts o r i n t a c t red c e l l s , i n d i c a t i n g that the u n i d i r e c t i o n a l c h a r a c t e r o f c a l c i u m 2+ e x t r u s i o n i s preserved even a g a i n s t o p p o s i t e l y d i r e c t e d Ca c o n c e n t r a -t i o n r a t i o s o f s e v e r a l thousand (Schatzmann and V i n c e n z i 1969; Lee and S h i n 1969; Schatzmann 1 9 7 3 ) . 2+ 2+ 2) Both a c t i v e c a l c i u m - t r a n s p o r t and (Ca + Mg )-ATPase a c t i v i t y 2+ r e q u i r e Mg and ATP w i t h i n the c e l l (Lee and S h i n 1 9 6 9 ) . The red c e l l c a l c i u m pump accepts v a r i o u s m e t a l - l i g a n d e d forms o f ATP o r f r e e ATP as s u b s t r a t e s , depending on the e x p e r i m e n t a l c o n d i t i o n s ( d e s c r i b e d i n d e t a i l e l s e w h e r e ) . . B a s e d on k i n e t i c c a l c u l a t i o n s S a r k a d i et aj_ (1981) proposed t h a t the t r u e phys io logical s u b s t r a t e i n s i t u i s most p r o b a b l y 2+ 4 - 2+ Mg ATP . In addi t ion to its r o l e as a MgATP s u b s t r a t e , f r e e Mg i s 2+ r e q u i r e d f o r a c t i v e c a l c i u m t r a n s p o r t . Mg i n t h i s r o l e can be s u b s t i -2+ t u t e d by m e t a l s which have an i o n i c r a d i u s s i m i l a r t o Mg but s m a l l e r 2+ 2+ than Ca . Thus, Mg ( i o n i c r a d i u s = 0.065 nm) can be s u b s t i t u t e d by M n 2 + ( 0 . 0 8 nm), C o 2 + ( 0 . 0 7 8 nm), F e 2 + ( 0 . 0 7 6 nm), N i 2 + ( 0 . 0 7 8 nm). C a 2 + ( 0 . 0 9 9 nm), S r 2 + (0.112 nm), B a 2 + ( 0 . 1 3 4 nm) are not accepted a t i t h i s s i t e ( S a r k a d i et a j _ 1 9 8 1 ) . I n o r g a n i c phosphate i s r e l e a s e d i n the c e l l i n t e r i o r . I t i s + 2+ important t o note at t h i s p o i n t t h a t no c o - t r a n s p o r t of H , Mg , or m e t a b o l i t e s was d e t e c t e d t o accompany c a l c i u m e f f l u x (Olson and Cazort 1974) . GTP, ITP, CTP or OTP can s u b s t i t u t e f o r ATP when i n c o r p o r a t e d i n t o r e s e a l e d ghosts (Lee and Shin 1969; Olson and Cazort 1969) a l t h o u g h a higher s p e c i f i c i t y was found f o r ATP i n membrane fragments (Cha et a l 1971) and i n i n s i d e - o u t v e s i c l e s (Sarkadi et a]_ 1 9 7 9 ) . ADP, AMP, pyrophosphate and a c e t y l phosphate cannot support a c t i v e c a l c i u m t r a n s -port (Schatzmann and V i n c e n z i 1969; Olson and Cazort 1969; S a r k a d i et a l 1979b) . 2+ 2+ 3) S t r o n t i u m can s u b s t i t u t e f o r c a l c i u m f o r a c t i v a t i n g (Ca + Mg ) -ATPase i n red c e l l membranes (Wins and S c h o f f e n i e l s 1966; P f l e g e r and Wolf 1 9 7 5 ) . S t r o n t i u m i s t r a n s p o r t e d from r e s e a l e d ghosts (Olson and Cazort 1969, Olson 1979) and i n t o i n s i d e - o u t red c e l l v e s i c l e s and c a l -modul in s t i m u l a t e s t h i s uptake ( S a r k a d i et al_ 1980b)by a mechanism 2+ 2+ 2+ r e q u i r i n g Mg (or Mn ) and ATP on the c y t o p l a s m i c s i d e . Al though Sr 2+ 2+ 2+ and Ca have s i m i l a r a f f i n i t i e s f o r (Ca + Mg )-ATPase (Wins and 2+ 2+ 2+ S c h o f f e n i e l s 1 9 6 6 ) , o n l y Ca i s t r a n s p o r t e d when both Ca and S r are present s i m u l t a n e o u s l y i n e r y t h r o c y t e ghosts (Schatzmann and V i n c e n z i 1969) . Calcium was shown to i n h i b i t a c t i v e s t r o n t i u m t r a n s p o r t non-c o m p e t i t i v e l y by occupying a s i t e o t h e r than the t r a n s p o r t s i t e (Olson 1979). Moreover, t h i s i n h i b i t i o n c o u l d be p a r t i a l l y r e v e r s e d by i n -2+ c r e a s i n g the c o n c e n t r a t i o n of Mg from 1 mM t o 4 mM e i t h e r d u r i n g l y s i s 2+ or a t the t ime o f i n c u b a t i o n . Mn , however, was i n e f f e c t i v e i n r e v e r s -2+ 2+ ing the Ca induced i n h i b i t i o n o f Sr t r a n s p o r t (Olson 1 9 7 9 ) . B a r i u m , magnesium, manganese, c o p p e r , c o b a l t , and i r o n are not t r a n s p o r t e d by the c a l c i u m pump, but these metal i o n s seem t o i n t e r a c t w i t h the c a l c i u m - s i t e and i n h i b i t enzyme a c t i v i t y ( S a r k a d i ejt al_ 1 977, 1980, 1981; Schatzmann and V i n c e n z i 1969; Schatzmann 1975). 4) A c t i v e c a l c i u m e x t r u s i o n from human red c e l l s does not depend upon a c t i v e sodium and potassium t r a n s p o r t , nor upon the transmembrane g r a d i e n t o f N a + or K + ( S a r k a d i and Tosteson 1979) . However, N a + and + + + 2+ 2+ K , as compared t o L i , c h o l i n e or Cs , s t i m u l a t e (Ca + Mg )ATPase a c t i v i t y ( S a r k a d i 1980a,b).Both ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y and C a 2 + e f f l u x from r e s e a l e d ghosts was maximum when K + (=25 mM) was at the i n s i d e and N a + (=130 mM) at the o u t s i d e o f the r e s e a l e d ghost membrane ( W i e r i c h s and Bader 1980). 5) Calcium t r a n s p o r t has a high Q-j0 v a l u e (=3.5) (Schatzmann and V i n c e n z i 1969, Schatzmann 1973, Lee and Shin 1 9 6 9 ) , as expected f o r t r a n s p o r t coupled t o a chemical r e a c t i o n . The e n e r g i e s o f a c t i v a t i o n 2+ f o r Ca - t r a n s p o r t ( 1 3 . 5 - 2 5 . 0 K c a l / ( m o l ) (Schatzmann and V i n c e n z i 1969; Lee and Shin 1969) and ( C a 2 + + M g 2 + ) - A T P a s e ( 1 4 . 4 - 2 7 . 6 K c a l / mol) ( Q u i s t and R o u f o g a l i s 1975a,b) are s i m i l a r . R e a c t i o n sequence of (Ca + Mg )-ATPase proposed by Muallem and K a r l i s h (1980, 1981) A new approach to the study o f the m o l e c u l a r b a s i s o f the r e d -c e l l c a l c i u m pump was used by Katz and B l o s t e i n (1975) and Knauf et a l (1974) who demonstrated c a l c i u m dependent membrane p h o s p h o r y l a t i o n by 32 [y- P]ATP. I t was shown t h a t micromolar c o n c e n t r a t i o n s o f c a l c i u m induced the f o r m a t i o n o f a phosphoprotein [EP] (a l a b i l e a c y l - p h o s p h a t e which i s s e n s i t i v e to hydroxylamine and b a s i c c o n d i t i o n s ) o f m o l e c u l a r weight =150,000 [the sodium s t i m u l a t e d phosphoprotein was around molecu-l a r weight 1 0 0 , 0 0 0 ] . The phosphoprotein i s produced i n seconds a t 0°C [Tj =40-60 sec] (Enyedi e t a l 1980) and shows a r a p i d t u r n o v e r upon a d d i t i o n of excess n o n - r a d i o a c t i v e ATP. I t s f o r m a t i o n c o r r e l a t e s w i t h 2+ 2+ the (Ca + Mg )-ATPase a c t i v i t y i n the membrane (Katz and B l o s t e i n 1975; Rega and Garrahan 1975). This new approach prov ides v a l u a b l e i n f o r m a t i o n on the p a r t i a l r e a c t i o n sequence o f the red c e l l c a l c i u m pump. I t was f i r s t shown by Rega and Garrahan (1975) and l a t e r by Schatzmann and Bu'rgin (1978) and Szasz e_t aj_ (1978) t h a t f o r m a t i o n o f 2+ the phosphory lated i n t e r m e d i a t e d i d not r e q u i r e the presence o f Mg and t h a t the b a s i c r o l e o f magnesium i s i n the d e p h o s p h o r y l a t i o n o f t h e c a l c i u m - t r a n s p o r t ATPase. However, c a l c i u m dependent f o r m a t i o n o f the 2+ phosphoenzyme i s a c c e l e r a t e d by Mg (Tt < 5 sec) (Schatzmann and B u r g i n 1978; Rega and Garrahan 1978; Enyedi et a l 1980) p o s s i b l y by t h e a c t i o n 2+ of Mg a t a l a t e r s tage i n the r e a c t i o n sequence of the pump. Garrahan and Rega (1978) p o s t u l a t e d a magnesium dependent c o n f o r m a t i o n a l change o f the i n i t i a l phosphoenzyme (E^P ) i n t o a form ^ P ) which can r a p i d l y r e a c t w i t h water to r e l e a s e P^. Monovalent c a t i o n s have no e f f e c t on the f o r m a t i o n of phosphoenzyme but the r a t e o f d e p h o s p h o r y l a t i o n of the c a l c i u m pump i n t e r m e d i a t e i s i n c r e a s e d by N a + and K + i n the presence of 2+ Mg (Enyedi ejt a]_ 1980) . Whereas the s u b s t r a t e f o r the c a l c i u m pump was i n i t i a l l y proposed t o be f r e e ATP (Rega and Garrahan 1975; Schatzmann 1 9 7 7 ) , Graf and Penniston (1981) proposed t h a t at low ATP c o n c e n t r a t i o n s , CaATP was the s u b s t r a t e . R e c e n t l y , Muallem and K a r l i s h (.19.81) have made a s y s t e m a t i c study of the e f f e c t s o f nucleotide s p e c i e s and proposed t h a t a t low c o n c e n t r a t i o n s o f the n u c l e o t i d e (1 - 10 y M ) , MgATP, 2+ 2+ CaATP or f r e e ATP s u s t a i n (Ca + Mg )-ATPase a c t i v i t y e f f e c t i v e l y , whereas a t h igher c o n c e n t r a t i o n s o f the n u c l e o t i d e (100 - 2000y M) MgATP a c c e l e r a t e d ATP h y d r o l y s i s (K 360 yM) and CaATP a c t e d as (MgATP) an i n h i b i t o r (K. = 165 yM) by competing w i t h MgATP f o r the (Ca-ATP) r e g u l a t o r y s i t e . MgATP was found to enhance (Ca + Mg )-ATPase by enhancing h y d r o l y s i s o f E £ P , by i n t e r a c t i n g a t the n o n - c a t a l y t i c s i t e ( r e g u l a t o r y s i t e ) . ATP i s not hydrolysed a t the r e g u l a t o r y s i t e and 2+ 2+ 2+ h y d r o l y s i s o f E2-P by ATP does not r e q u i r e Ca or Mg . However, Mg i s needed i n d i r e c t l y f o r the f o r m a t i o n o f E 2 - P , which r a p i d l y r e a c t s w i t h water i n the presence o f mM c o n c e n t r a t i o n s o f ATP (Rega and Garrahan 1978; R i c h a r d s et al_ 1978). I t has a l s o been suggested t h a t high concen-t r a t i o n s o f MgATP (K = 0.143 mM) s t i m u l a t e the maximum v e l o c i t y o f d 2+ 2+ 2+ the (Ca + Mg )-ATPase i n a Mg -dependent f a s h i o n by promoting t h e c o n v e r s i o n o f E£ to E^  i n the f i n a l r e a c t i o n sequence ( R i c h a r d s et a l 1978). 2+ 2+) 2+ Al though the a s s o c i a t i o n between (Ca + Mg -ATPase and Ca -2+ 2+ t r a n s p o r t has been c l e a r l y demonstrated and the r o l e o f Ca , Mg , and 2+ 2+ ATP i n the r e g u l a t i o n o f (Ca + Mg )-ATPase uncovered, c o n s i d e r a b l e d i s c r e p a n c i e s e x i s t i n the l i t e r a t u r e on the k i n e t i c p r o p e r t i e s o f 2+ 2+ (Ca + Mg )-ATPase i n human red c e l l membranes, p a r t i c u l a r l y w i t h 2+ 2+ regard t o the c a l c i u m s e n s i t i v i t y o f the pump/(Ca + Mg )-ATPase a c t i v i t i e s and the s t o i c h i o m e t r y of the c a l c i u m pump. Whereas Romero and Whittam (1971) found t h a t the ATP dependent c a l c i u m e f f l u x from r e s e a l e d ghosts was h a l f - m a x i m a l l y a c t i v a t e d a t a c a l c i u m c o n t e n t o f 0 . 9 mmol/L packed eel 1s,Schatzmann (1973) r e p o r t e d t h a t h a l f maximal 2+ 2+ s t i m u l a t i o n o f Ca e f f l u x o c c u r r e d a t l e s s than 4 yM Ca i n ghosts loaded w i t h calcium-EGTA b u f f e r s . Schatzmann (1973) a l s o showed t h a t 2+ 2+ h a l f maximal a c t i v a t i o n o f (Ca + Mg )-ATPase a c t i v i t y appeared a t 2+ 1000 f o l d lower Ca c o n c e n t r a t i o n than t h a t i n ghosts loaded w i t h 2+ c a l c i u m i n the absence o f EGTA. The f a c t o r s a f f e c t i n g t h e Ca - a f f i n i t y 2+ 2+ 2+ o f (Ca + Mg )-ATPase and Ca - t r a n s p o r t have been reviewed r a t h e r e x t e n s i v e l y by R o u f o g a l i s (1979) and w i l l be summarized b r i e f l y below. The d i s c r e p a n c y between the k i n e t i c s o f c a l c i u m a c t i v a t i o n o f 2+ 2+ (Ca + Mg )-ATPase a l s o e x i s t s i n e r y t h r o c y t e ghost membranes. Horton 2+ 2+ et al_ (1970) showed the presence of two (Ca + Mg )-ATPase a c t i v i t i e s i n e r y t h r o c y t e ghost membranes, f r e q u e n t l y r e f e r r e d to as the high and low C a 2 + a f f i n i t y ( C a 2 + M g 2 + ) - ATPase a c t i v i t i e s to r e f l e c t t h e i r d i f f e r e n t s e n s i t i -v i t i e s f o r c a l c i u m . The c a l c i u m d i s s o c i a t i o n c o n s t a n t s f o r the high and low 2+ 2+ C a 2 + a f f i n i t y (Ca + Mg )-ATPase a c t i v i t i e s were found t o be a p p r o x i m a t e l y 4 yM and 100 y M , r e s p e c t i v e l y (Schatzmann and Rossi 1971; Wolf 1972). S c h a r f f (1972) and Schatzmann ( 1 9 7 3 ) , however, concluded t h a t the low 2+ 2+ a f f i n i t y (Ca + Mg )-ATPase a c t i v i t y was an a r t i f a c t due t o c o n v e r s i o n 2+ of the high Ca a f f i n i t y form o f the enzyme to a form w i t h low a f f i n i t y f o r c a l c i u m , d e r i v e d from p r e p a r a t i o n o f e r y t h r o c y t e ghost membranes i n the presence o f c h e l a t i n g agents such as EGTA or EDTA. However, t h i s c o n c l u s i o n d i d not concur w i t h the r e s u l t s o f Horton ( 1 9 7 0 ) , who used membranes prepared in the absence o f EGTA/EDTA and demonstrated the 2+ 2+ presence o f h igh and low a f f i n i t y (Ca + Mg )-ATPase a c t i v i t i e s . Furthermore, the pump seems to demonstrate a low a f f i n i t y f o r c a l c i u m when the enzyme a c t i v i t y was s t u d i e d i n whole c e l l s ( S a r k a d i e t al_ 1 979) . Quist and R o u f o g a l i s ( 1 9 7 5 ) , u s i n g a v a r i e t y of membrane p r e p a r a t i o n s , a l s o showed b i p h a s i c ( h i g h and low) c a l c i u m a c t i v a t i o n k i n e t i c s and suggested t h a t the " l o w - c a l c i u m a f f i n i t y " enzyme played a fundamental r o l e i n a c t i v e c a l c i u m t r a n s p o r t . As knowledge of the r e g u l a t i o n o f 2+ 2+ 2+ c a l c i u m a f f i n i t y o f the Ca -pump/(Ca + Mg )-ATPase i s germane t o understanding i t s p h y s i o l o g i c a l r o l e i n human e r y t h r o c y t e s , t h i s t o p i c w i l l form an important p a r t o f t h i s t h e s i s . 2+ E f f o r t s t o c h a r a c t e r i z e the s t o i c h i o m e t r y of the Ca pump have a l s o y i e l d e d c o n f l i c t i n g r e s u l t s . Schatzmann and V i n c e n z i (1969) and Schatzmann (1973) a r r i v e d a t a s t o i c h i o m e t r i c r a t i o o f 1:1 f o r the human red c e l l c a l c i u m pump. These authors compared the r a t i o of i n o r g a n i c phosphate l i b e r a t e d from ATP to the r a t i o o f c a l c i u m e x t r u s i o n i n c a l c i u m loaded r e s e a l e d g h o s t s . S i m i l a r r e s u l t s were o b t a i n e d by Larsen et a l 2+ (1978). In c o n t r a s t , when lanthanum was used t o b l o c k Ca t r a n s p o r t i n 2+ c a l c i u m - l o a d e d r e s e a l e d g h o s t s , the r a t i o o f lanthanum s e n s i t i v e Ca -e f f l u x t o lanthanum s e n s i t i v e ATP s p l i t t i n g was 2:1 ( Q u i s t and R o u f o g a l i s 1 975b). S i m i l a r l y , i n i n t a c t c e l l s the molar r a t i o o f lanthanum s e n s i -t i v e c a l c i u m t r a n s p o r t t o lanthanum s e n s i t i v e ATP s p l i t t i n g was 2:1 ( S a r k a d i e_t aj_ 1977) . These l a t t e r authors used ionophore A23187 to l o a d 2+ the c e l l s w i t h Ca and ouabain t o i n h i b i t the sodium pump. Q u i s t and R o u f o g a l i s (1975b) used f r e s h red c e l l ghosts and found lanthanum t o c o m p l e t e l y i n h i b i t the c a l c i u m t r a n s p o r t but o n l y 50% o f the ATPase a c t i -2+ 2+ v i t y measured i n the presence of Ca + Mg . Larsen et al_ (1978) used outdated blood i n a c i d / c i t r a t e / d e x t r o s e to make ghosts and d i d not f i n d 2+ 2+ a s i g n i f i c a n t lanthanum i n s e n s i t i v e (Ca + Mg )-ATPase a c t i v i t y ; hence t h e i r e s t i m a t e o f the s t o i c h i o m e t r y u s i n g the lanthanum method was 1 : 1 . The reason f o r t h i s d i f f e r e n c e i s not c l e a r , but i t has been suggested t h a t the p a s s i v e p e r m e a b i l i t y o f the membrane to both c a l c i u m and lanthanum i n c r e a s e s c o n s i d e r a b l y i n ghosts made from outdated c e l l s (Szasz ejt aj_ 1978 a , b, c ) . Thus, e x t e r n a l l y added lanthanum, which n o r m a l l y does not p e n e t r a t e the membrane g h o s t s , may have pene-t r a t e d the ghosts from outdated blood and i n h i b i t e d the c a l c i u m - s t i m u l a -ted-ATPase a c t i v i t i e s not a s s o c i a t e d w i t h c a l c i u m pumping ( S a r k a d i et al_ 1 9 7 9 ) , R e c e n t l y , i n s i d e - o u t red c e l l membrane v e s i c l e s have been used 2+ to study the c a l c i u m pump s t o i c h i o m e t r y . In such v e s i c l e s the (Ca + 2+ Mg )-ATPase shows an " u p h i l l " c a l c i u m t r a n s p o r t . I t was shown by S a r k a d i (1980) t h a t the s t o i c h i o m e t r y o f the c a l c i u m pump depended on the c o n c e n t r a t i o n o f c a l c i u m and ranged from 0 . 3 : 1 t o 2:1 a t i n c r e a s i n g c a l c i u m c o n c e n t r a t i o n s . R e c e n t l y , Larsen et al_ (1981) and Akyempong and R o u f o g a l i s (1981) showed t h a t the s t o i c h i o m e t r y of the pump can vary depending on the c o n c e n t r a t i o n o f the e f f e c t o r s used. T h i s may be 2+ 2+ due i n p a r t to the complex k i n e t i c s o f (Ca + Mg )-ATPase observed a t 2+ 2+ v a r i o u s c o n c e n t r a t i o n s o f Ca , Mg and ATP used 1n the assay medium 2+ (Katz et al_ 1 979) . The v a r i a b i l i t y i n the apparent a f f i n i t y of (Ca + 2+ 2+ Mg )-ATPase f o r Ca has been suggested to be a f f e c t e d by t h e method o f membrane p r e p a r a t i o n , which i n turn may govern whether c a l m o d u l i n i s 2+ a s s o c i a t e d or d i s s o c i a t e d from t h e Ca pump due to the presence or absence o f c a l c i u m i n the hemolysing b u f f e r . A d d i t i o n a l f a c t o r s 2+ 2+ a f f e c t i n g the k i n e t i c s o f the (Ca + Mg )-ATPase and v a r i a t i o n i n the exper imental f i n d i n g s may i n c l u d e the f o l l o w i n g . 1) The use o f c a l c i u m - c h e l a t i n g s u b s t a n c e s , such as EDTA or EGTA ( S a r k a d i et aj_ 1979; A l - J o b o r e e t aj[ 1981) or the C a 2 + ionophore A23187 ( S c h a r f f and Foder 1978; Haaker and Racker 1979) d u r i n g the ( C a 2 + + M g 2 + ) - A T P a s e 2+ 2+ or Ca - t r a n s p o r t a s s a y . I t was observed t h a t the k i n e t i c s o f (Ca + 2+ 2+ Mg )-ATPase or Ca - t r a n s p o r t depended on the t o t a l c o n c e n t r a t i o n o f 2+ 2+ Ca i n the Ca-EGTA b u f f e r r a t h e r than the c o n c e n t r a t i o n o f f r e e Ca 2+ 2+ 4 - / a l o n e . Thus, an i n t e r a c t i o n of Ca i n the Ca -EGTA form ( S a r k a d i et a i 1979) or EGTA i n the Ca-EGTA 2 ' form ( A l - J o b o r e and R o u f o g a l i s 1981) was p o s t u l a t e d . 2} Human e r y t h r o c y t e membranes c o n t a i n s i g n i f i c a n t amounts o f p r o t e i n k i n a s e and phosphatase a c t i v i t i e s CAvruch and Fai rbanks 1974; G r e e n q u i s t and Shohet 1975; Hosey and Tao 1977) which can be s t i m u l a t e d by m i c r o -2+ molar c o n c e n t r a t i o n s of Ca ( F a i r b a n k s and Avruch 1974; Q u i s t 1 9 8 0 ) . 2+ 2+ Thus, when measuring (Ca + Mg )-dependent i n o r g a n i c phosphate (.P.) l i b e r a t i o n from ATP, the c o n t r i b u t i o n o f P. from k i n a s e o r phosphatase a c t i v i t i e s cannot be d i s t i n g u i s h e d . This problem may be s o l v e d a t l e a s t 2 + 2 + i n p a r t by u s i n g p u r i f i e d p r e p a r a t i o n s o f (Ca +Mg )-ATPase r e c o n s t i t u -ted i n l i p o s o m a l systems o r w i t h the use o f a s p e c i f i c i n h i b i t o r o f the 2+ 2+ Ca -pump ATPase to c h a r a c t e r i z e ATP s p l i t t i n g not a s s o c i a t e d w i t h Ca 2+ t r a n s p o r t . S i n c e a s p e c i f i c i n h i b i t o r o f the Ca -pump has not been r e p o r t e d so f a r , c o n s i d e r a b l e e f f o r t has been expended i n the study o f the mechanism o f r e g u l a t i o n o f the c a l c i u m pump. R e g u l a t i o n of Calcium Pump ATPase i n human e r y t h r o c y t e s A) Role of Calmodul in I t was f i r s t demonstrated by Gopinath and V i n c e n z i (1977) and J a r r e t t and P e n n i s t o n (1977) t h a t a p u r i f i e d b r a i n phosphodiesterase o r a d e n y l a t e c y c l a s e a c t i v a t o r (termed " c a l m o d u l i n " by Cheung et a l i n 2+ 2+ 1978) s t i m u l a t e s (Ca + Mg )-ATPase a c t i v i t y i n i s o l a t e d red c e l l membranes. Hinds et a l ( 1 9 7 8 ) , Larsen and V i n c e n z i (1979) and M a c l n t y r e and Green (1978) l a t e r demonstrated t h a t c a l m o d u l i n a l s o s t i m u l a t e s c a l c i u m uptake i n i n s i d e - o u t red c e l l v e s i c l e s . I t i s known now t h a t 2+ c a l m o d u l i n i s i n v o l v e d i n many processes o f Ca t r a n s p o r t i n v a r i o u s membranes and t h a t i t may p l a y a d i f f e r e n t r o l e i n d i f f e r e n t t r a n s p o r t systems ( C a r a f o l i 1 9 8 1 ) . The area o f c a l m o d u l i n b i o c h e m i s t r y and p h a r -macology i s one o f the f a s t e s t growing i n b a s i c s c i e n c e s . Some o f i t s p r o p e r t i e s and c h a r a c t e r i s t i c s a r e d i s c u s s e d i n the ensueing s e c t i o n . Calmodul in i s a low m o l e c u l a r weight ( M r = 1 6 , 7 0 0 ) , a c i d i c ( i s o -2+ e l e c t r i c p o i n t = 4 . 2 ) , h e a t - s t a b l e , Ca b i n d i n g p r o t e i n ( S c h a r f f , 1 9 8 1 ) . Calmodul in i s u b i q u i t o u s l y d i s t r i b u t e d i n most e u c a r y o t i c c e l l s and shows l a c k of t i s s u e or s p e c i e s s p e c i f i c i t y (Stevens e t a l 1 9 7 6 ) . In c o n t r a s t , a number o f o t h e r c a l c i u m b i n d i n g p r o t e i n s such as t r o p o n i n C, p a r a l b u -m i n , c a l s e q u e s t r i n , v i t a m i n D - i n d u c i b l e p r o t e i n , show l i m i t e d t i s s u e d i s t r i b u t i o n and/or appear to be concerned w i t h s p e c i f i c t i s s u e processes (Brostrom and W o l f f 1 9 8 1 ) . Mammalian b r a i n and t e s t i s (Watterson et a l 1976; Dedman et aj_ 1977) and the e l e c t r o p l a x o f E l e c t r o p h o r u s e l e c t r i c u s ( C h i l d e r s and S i e g e ! 1975) are very r i c h sources o f c a l m o d u l i n . Sub-c e l l u l a r and f r a c t i o n a t i o n s t u d i e s have l o c a l i z e d c a l m o d u l i n predominant-l y i n the c y t o p l a s m i c o r s o l u b l e f r a c t i o n s , a l t h o u g h a s i g n i f i c a n t amount i s a s s o c i a t e d w i t h p a r t i c u l a t e f r a c t i o n s as w e l l . Calmodul in b i n d i n g 2+ to p a r t i c u l a t e f r a c t i o n s r e q u i r e s Ca and occurs a t s p e c i f i c s i t e s (Vandermeers et al_ 1978) . B i n d i n g i s a l s o s a t u r a t a b l e , r e v e r s i b l e , temperature and t r y p s i n s e n s i t i v e (Brostrom and W o l f f 1 9 8 1 ) . Calmodul in has been shown to p l a y a r o l e i n r e g u l a t i o n o f a v a r i e t y o f c e l l u l a r f u n c t i o n s . I t i s i n v o l v e d i n s y n a p t i c t r a n s m i s s i o n (De Lorenzo et al_ 1 9 7 9 ) , c a l c i u m induced i n s u l i n r e l e a s e and i n t e s t i n a l s e c r e t i o n (Richman and Klee 1 9 7 9 ) . Calmodul in was a l s o shown to be i n v o l v e d i n the r e g u l a t i o n o f c a l c i u m t r a n s p o r t i n s a r c o p l a s m i c r e t i c u l u m (Katz and R e m t u l l a 1978) and i n synaptosomes (Sobue et^  aj_ 1 9 7 9 ) , i n r e g u l a t i o n o f c e l l m u l t i p l i c a t i o n ( W h i t f i e l d e t al^ 1979) and f e r t i l i z a -t i o n o f oocytes by spermatozoa (Jones et al_ 1978) . The enzymes r e g u l a t e d by c a l m o d u l i n i n c l u d e the c a l c i u m t r a n s p o r t ATPases, p h o s p h o d i e s t e r a s e , a d e n y l a t e c y c l a s e , myosin l i g h t c h a i n k i n a s e i n muscle/non-muscle c e l l s , phosphorylase B k i n a s e and s e v e r a l o t h e r p r o t e i n k i n a s e s . ( T h e reader i s r e f e r r e d to a r e c e n t rev iew by Brostrom and W o l f f (1981) f o r the r o l e o f c a l m o d u l i n i n r e g u l a t i n g t h e s e enzyme systems) . Calmodul in thus seems t o possess t h e i n h e r e n t a b i l i t y to modulate a l a r g e number o f c e l l u l a r f u n c t i o n s and e s p e c i a l l y those r e g u l a t e d by c a l c i u m . Calmodul in from animal sources has o n l y one h i s t i d i n e , no t r y p t o -phan or c y s t e i n e , but one t r i m e t h y l l y s i n e r e s i d u e and a predominance o f a c i d i c over b a s i c amino a c i d r e s i d u e s . R e c e n t l y , chemical m o d i f i c a t i o n s t u d i e s have been used to prov ide an overview of the amino a c i d s which 2+ may be i n v o l v e d i n the b i n d i n g o f Ca - c a l m o d u l i n complex to the enzymes (Walsh and Stevens 1977). I t was shown by T h i r y et aj_ (1980) t h a t m o d i f i c a -t i o n of the s i n g l e h i s t i d i n e r e s i d u e or both t y r o s i n e r e s i d u e s had no e f f e c t on the a c t i v a t i o n p r o p e r t i e s o f c a l m o d u l i n u s i n g a d e n y l a t e c y c l a s e and p h o s p h o d i e s t e r a s e . The c a r b o x y m e t h y l a t i o n of two m e t h i o n i n e r e s i d u e s or the amidat ion o f s e v e r a l carboxy l groups reduced the a c t i -v a t i o n p r o p e r t i e s o f c a l m o d u l i n on a d e n y l a t e c y c l a s e and p h o s p h o d i e s t e r -a s e . M o d i f i c a t i o n o f a r g i n y l groups (4 o f the 6) r e s u l t s i n a d e r i v a t i v e t h a t shows 1.3 f o l d higher f o r a d e n y l a t e c y c l a s e . However, t h i s i n c r e a s e d Km f o r c a l m o d u l i n i s a l s o accompanied by a 50% i n c r e a s e i n the V m , v o f a d e n y l a t e c y c l a s e . In c o n t r a s t , m o d i f i c a t i o n o f a r g i n y l r e s i d u e s ma x of c a l m o d u l i n had no e f f e c t on i t s a b i l i t y t o a c t i v a t e p h o s p h o d i e s t e r a s e (Thi ry et a l 1980) . R e c e n t l y , Gagnon e_t aj_ (1981) showed t h a t c a r b o x y -methylated c a l m o d u l i n was l e s s e f f e c t i v e i n s t i m u l a t i n g p h o s p h o d i e s t e r a s e , In our l a b o r a t o r y we found t h a t carboxymethylated c a l m o d u l i n was a l s o 2+ 2+ i n e f f e c t i v e i n s t i m u l a t i n g (Ca + Mg )-ATPase a c t i v i t y o f the e r y t h r o -c y t e membranes ( A . A l - J o b o r e and B.D. R o u f o g a l i s - unpubl ished r e s u l t s ) . These s t u d i e s prompted f u r t h e r c h a r a c t e r i z a t i o n of the r o l e o f f r e e carboxy l and a r g i n y l groups i n the a c t i v a t i o n p r o p e r t i e s o f c a l m o d u l i n 2+ 2+ on (Ca + Mg )-ATPase o f red c e l l membranes. Our s t u d i e s i n d i c a t e d t h a t c e r t a i n p o l y a n i o n i c compounds ( v i z . p o l y - L - a s p a r t i c a c i d M f = 20,000 and p o l y - L - g l u t a r a i c - a c i d M r = 14,000 and 26,000) i n c r e a s e the Ca s e n s i t i v i -ty o f the ( C a 2 + + M g 2 + ) - A T P a s e wihout a f f e c t i n g the V ( A l - J o b o r e e t a l max 1980) . In c o n t r a s t , b l o c k i n g a r g i n y l groups o f c a l m o d u l i n had no e f f e c t 2+ 2+ on i t s a c t i v a t i o n o f (Ca + Mg )-ATPase (Minocherhom.jee and R o u f o g a l i s , 1981) . In a d d i t i o n t o i t s r a t h e r unique a m i n o - a c i d c o m p o s i t i o n , calmo-d u l i n a l s o e x h i b i t s f o u r i n t e r n a l l y homologous sequences o r domains, each o f which c o n t a i n s the a p p r o p r i a t e amino a c i d s forming the h e l i x - l o o p -h e l i x , c a l c i u m b i n d i n g s t r u c t u r e P a t t e r s o n e t a]_ 1980) . This l a t t e r p r o p e r t y i s a l s o common to o t h e r c a l c i u m b i n d i n g p r o t e i n s such as p a r v a l -2+ bumin and t r o p o n i n C ( R e i d and Hodges 1 9 8 0 ) . Calmodul in binds f o u r Ca 2+ ions and the f o u r Ca b i n d i n g s i t e s appear to cor respond to the f o u r domains. S t a r t i n g a t the amino terminus c a l m o d u l i n shows the h i g h e s t degree o f homology between the f i r s t and t h i r d and between the second and f o u r t h domains. Despi te the general agreement i n the l i t e r a t u r e 2+ t h a t c a l m o d u l i n b inds 4 Ca i o n s , there appears to be o n l y poor agree-ment r e g a r d i n g the a f f i n i t i e s o f these s i t e s f o r c a l c i u m (Brostrom and W o l f f 1 9 8 1 ) . P a r t o f the d i s c r e p a n c i e s has been a t t r i b u t e d to the c o n d i t i o n s and procedures employed i n d e t e r m i n i n g the b i n d i n g d a t a , e s p e c i a l l y the use of EGTA to c o n t r o l the l e v e l o f f r e e c a l c i u m . W o l f f et aj_ (1977) w i t h reagents f r e e d o f c o n t a m i n a t i n g d i v a l e n t c a t i o n s , used e q u i l i b r i u m d i a l y s i s and determined the c o n c e n t r a t i o n o f bound and t o t a l c a l c i u m by atomic a b s o r p t i o n s p e c t r o p h o t o m e t r y . T h i s t e c h n i q u e 2+ e l i m i n a t e s the need f o r EGTA to c o n t r o l t h e l e v e l o f f r e e Ca . I t was 2+ shown t h a t o f t h e f o u r Ca b i n d i n g s i t e s , three s i t e s e x h i b i t e d high a f f i n i t y f o r c a l c i u m (K d - 0,2 yM) but low a f f i n i t y f o r M g 2 + (K d = 140 nM). The f o u r t h s i t e has s i m i l a r a f f i n i t i e s f o r both c a l c i u m and magnesium ( K d j C g 2 + = 1 P M and K r f M g 2 + = 20 yM) (Wol f f e t aj_ 1 9 7 7 ) . I t was t h e r e -f o r e suggested t h a t the f u n c t i o n a l i n t r a c e l l u l a r form o f c a l m o d u l i n i s 2+ 2+ p r o b a b l y a 3 Ca : 1 Mg m o l e c u l e . A v a r i e t y o f data a l s o supports 2+ ?+ the s u g g e s t i o n t h a t Mg and o t h e r d i v a l e n t metal ions compete w i t h the Ca^ 2+ 2+ b i n d i n g s i t e on c a l m o d u l i n , a) Calmodul in binds 4 moles o f Mg o r Mn 2+ per mole o f p r o t e i n and these ions can be d i s p l a c e d by Ca (Wolf f e t a l 2+ 1977). b) An i n c r e a s e i n the apparent Km f o r Ca of v a r i o u s c a l m o d u l i n dependent enzymes ( e . g . ATPase, a d e n y l a t e c y c l a s e , phosphodiesterase) 2+ occurs w i t h i n c r e a s i n g Mg c o n c e n t r a t i o n s (Katz ejt al_ 1979; Brostrom and W o l f f 1976; Brostrom et a]_ 1 9 7 7 ) . Thus b i n d i n g o f c a l m o d u l i n to v a r i o u s enzymes i n the presence o f d i f f e r e n t d i v a l e n t c a t i o n s may i n v o l v e complex k i n e t i c s . R e g u l a t i o n o f enzyme (E) by c a l m o d u l i n (C) i s g e n e r a l l y b e l i e v e d to proceed by 2 s e q u e n t i a l , f u l l y r e v e r s i b l e mass a c t i o n equat ions n C a 2 + + C ^ = = i ( C a 2 + ) n C (1) x ( C a 2 + ) C + E. 4 . . ' ^ : ^ [ ( C a 2 + ) C] E «. (2) 'n i n a c t i v e L ^ 'n J x a c t i v e v ' 2+ where n = number o f b i n d i n g s i t e s which must be occupied by Ca to c o n v e r t c a l m o d u l i n to an a c t i v a t i n g form f o r the enzyme. For the 2+ 2+ (Ca + Mg )-ATPase i t was suggested t h a t n = 3 > 4 > 2 > l , a n d t h a t c a l m o d u l i n d i s s o c i a t e s from the c a l m o d u l i n s a t u r a t e d enzyme i n the range o f 10"^ - 10" 6 M even a t c a l m o d u l i n c o n c e n t r a t i o n s o f 5^.M (Foder and S c h a r f f 1 9 8 1 ) , w h i l e n = 4 f o r p h o s p h o d i e s t e r a s e (Huang et al_ 1981) and myosin l i g h t c h a i n k i n a s e (Blumenthal and S t u l l 1 9 8 0 ) . However, i t i s 2+ not known i f Ca binds s e q u e n t i a l l y or randomly t o the v a r i o u s domains (Brostrom and W o l f f 1 9 8 1 ) ; 2+ x = unknown number of (Ca ) n ~ c a l m o d u l i n complexes. S a t u r a t i o n o f c a l -2+ modulin w i t h Ca r e s u l t s i n a c o n f o r m a t i o n a l change i n v o l v i n g an i n c r e a -se i n the a - h e l i c a l c o n t e n t from 28% t o 42%, and a c o n f o r m a t i o n a l change to a more compact form. The i n c r e a s e i n a - h e l i c a l c o n t e n t i s r e f l e c t e d by an i n c r e a s e i n e l l i p t i c i t y , an enhanced t y r o s i n e f l u o r e s c e n c e and a n e g a t i v e t y r o s i n e d i f f e r e n c e spectrum (Wolf f e_t al_ 1977) . Tanaka and Hidaka (1980) have shown t h a t the c o n f o r m a t i o n a l change produced i n 2+ c a l m o d u l i n by b i n d i n g o f Ca t o the high a f f i n i t y s i t e s exposes hydro-phobic groups which i n t u r n appear t o be i n v o l v e d i n a c t i v a t i n g calmo-d u l i n - d e p e n d e n t enzymes. Thus, both the a c i d i c nature o f c a l m o d u l i n and 2+ i t s Ca - i n d u c i b l e hydrophobic p r o p e r t i e s seem to be important i n r e g u l a t i n g c a l m o d u l i n dependent enzymes. I t i s important t o note from e q u a t i o n (1) t h a t the s e n s i t i v i t y of the enzyme f o r c a l c i u m w i l l vary as a f u n c t i o n o f c a l m o d u l i n c o n c e n t r a t -i o n , a c c o r d i n g t o the law o f mass a c t i o n . S e c o n d l y , c a t i o n s which com-2+ pete f o r Ca b i n d i n g s i t e s may a l s o a f f e c t the s e n s i t i v i t y o f the enzyme 2+ f o r Ca . Equat ion (2) i n d i c a t e s t h a t the c a l c i u m s e n s i t i v i t y a l s o 2+ depends on the a f f i n i t y o f the i n a c t i v e enzyme f o r Ca - c a l m o d u l i n -2+ complex. Thus, enzymes w i t h r e l a t i v e l y h igh a f f i n i t y f o r the Ca -c a l m o d u l i n - c o m p l e x may be a c t i v a t e d p r e f e r e n t i a l l y over those w i t h lower a f f i n i t i e s i n c e l l s exposed t o s t i m u l i . Indeed, a range o f a f f i n i t i e s f o r c a l m o d u l i n e x i s t among v a r i o u s p r o t e i n s (Brostrom and W o l f f 1 9 8 1 ) . Based on k i n e t i c d a t a , Graf and Penniston (1981) and Foder and S c h a r f f 2+ (1S81) showed t h a t c a l m o d u l i n i n t e r a c t s w i t h p u r i f i e d s o l u b i l i z e d Ca -ATPase from human e r y t h r o c y t e i n a molar r a t i o o f 1 : 1 . Furthermore, the 2+ 2+ apparent a f f i n i t y o f c a l m o d u l i n f o r (Ca + Mg )-ATPase i s s t r o n g l y dependent on the c o n c e n t r a t i o n of c a l c i u m and decreased a p p r o x i m a t e l y 1000 t imes when the c o n c e n t r a t i o n o f c a l c i u m was reduced from 112 to 0.5 yM ( F o d e r and S c h a r f f 1981). The c o n c e n t r a t i o n o f c a l m o d u l i n i n the e r y t h r o c y t e c y t o s o l i s a p p r o x i m a t e l y 5 yM, c o r r e s p o n d i n g to 50-80 2+ 2+ t imes the c e l l u l a r c o n c e n t r a t i o n of (Ca + Mg ) -ATPase(approx imately 10 ymol/g membrane p r o t e i n ) . S i n c e the c o n c e n t r a t i o n o f i n t r a c e l l u l a r c a l c i u m i s n o r m a l l y l e s s than 10~^M, i t was proposed by Foder and S c h a r f f 2+ (1981) t h a t i n v i v o most o f the c a l m o d u l i n i s d i s s o c i a t e d from the Ca -t r a n s p o r t - A T P a s e and t h a t c a l m o d u l i n b i n d i n g and i t s subsequent a c t i v a t -2+ 2+ 2+ ion o f (Ca + Mg )-ATPase r e q u i r e s t h a t the Ca c o n c e n t r a t i o n r i s e s to 1 0 " 6 - 1 0 " 5 M . I t i s g e n e r a l l y accepted t h a t i n human red c e l l s , c a l m o d u l i n 2+ 2+ a f f e c t s (Ca + Mg )-ATPase a c t i v i t y and a c t i v e c a l c i u m t r a n s p o r t through a c a l c i u m dependent b i n d i n g to the enzyme (Hanahan et a l 1978; 2+ N i g g l i et aj_ 1979) . T h i s p r o p e r t y has been u t i l y s e d to p u r i f y t h e (Ca + M g 2 + ) - A T P a s e ( G i e t z e n e t a]_ 1980; N i g g l i et a l 1981). 2+ 2+ The r o l e o f c a l m o d u l i n i n the r e a c t i o n sequence o f (Ca + Mg ) -ATPase i s summarized below: 2+ 2+ 1) Calmodul in b i n d i n g to the (Ca + Mg )-ATPase i n c r e a s e s the apparent 2+ c a l c i u m a f f i n i t y o f the system and i n c r e a s e s the V o f the Ca -max t r a n s p o r t - A T P a s e ( M a c l n t y r e and Green 1976; Hinds e t a l 1981; S a r k a d i et a l 1980) . 2+ 2) Calmodul in has no apparent e f f e c t on the a f f i n i t y of ATP or Mg f o r 2+ the Ca - t r a n s p o r t - A T P a s e ( S a r k a d i et a]_ 1978) al though i t may a l t e r the r e g u l a t o r y r o l e o f high ATP c o n c e n t r a t i o n s (Muallem and K a r l i s h 1980). 3) In the absence o f Mg , but i n the presence o f Ca , ca lmodul in i n c r e a s e s the r a t e and the steady s t a t e l e v e l o f the phosphoenzyme (EP complex) . (Muallem and K a r l i s h 1980, 1981; Rega and Garrahan 1980) . 2+ 2+ 4) In the presence o f Mg + Ca , c a l m o d u l i n does not a f f e c t the steady s t a t e l e v e l o f EP a t low c o n c e n t r a t i o n s but decreases the l e v e l a t high c o n c e n t r a t i o n s ( J e f f e r y et aj_ 1 9 8 0 ) , and a c c e l e r a t e s the r a t e o f EP f o r m a t i o n (Muallem and K a r l i s h 1980) and decomposi t ion (Muallem and K a r l i s h 1981) . 5) Calmodul in has a l s o been suggested t o s t i m u l a t e EP d e p h o s p h o r y l a t i o n 2+ when measured i n the absence of Mg (Rega and Garrahan 1980) . 2+ 6) J e f f e r y e t al_ (1980) have shown t h a t both Mg and c a l m o d u l i n s t i m u -l a t e the decomposi t ion o f EP complex and t h a t the e f f e c t s o f these two e f f e c t o r s was a n t a g o n i s t i c , s u g g e s t i n g a s i m i l a r mechanism of EP d e c o m p o s i t i o n . 7) Muallem and K a r l i s h (1980) proposed t h a t the £2 s t a t e of the enzyme i s converted t o E-| by c a l m o d u l i n i . e . c o n v e r s i o n o f the enzyme from a 2+ s t a t e o f low a f f i n i t y to a s t a t e of high a f f i n i t y f o r Ca Although r e l a t i v e l y much i s known about the r o l e o f c a l m o d u l i n i n d i f f e r e n t enzyme/protein systems, l i t t l e i s known about the f a c t o r s which c o n t r o l the l e v e l s or f u n c t i o n s o f c a l m o d u l i n . In 1977, Wang and Desai i s o l a t e d and c h a r a c t e r i z e d a c a l m o d u l i n - b i n d i n g p r o t e i n (CaM-BP) from the bovine b r a i n which e x h i b i t e d c a l c i u m dependent b i n d i n g t o c a l m o d u l i n . Larsen et a]_ (1978) showed t h a t t h i s p r o t e i n (CaM-BP) 2+ 2+ 2+ antagonized the a c t i v a t i o n o f (Ca + Mg )-ATPase and Ca - t r a n s p o r t o f red blood c e l l membranes. S i n c e t h e n , s e v e r a l types o f CaM-BP's have been i d e n t i f i e d . Klee e t al_ (1 979) demonstrated the presence o f a heat l a b i l e CaM-BP i n nerve t i s s u e , which they termed c a l c i n e u r i n . Sharma et a]_ (1978) r e p o r t e d the i s o l a t i o n o f heat s t a b l e CaM-BP from bovine b r a i n . Au (1978) and S a r k a d i et aj_ (1980) showed t h a t a c o n c e n t r a t e d heat l a b i l e p r o t e i n f r a c t i o n o f red c e l l cytoplasm prevents c a l m o d u l i n 2+ 2+ 2+ a c t i v a t i o n o f the red c e l l membrane (Ca + Mg )-ATPase and Ca -t r a n s p o r t . L i t t l e i s known about the f a c t o r s governing the l e v e l s o r f u n c t i o n s o f c a l m o d u l i n b i n d i n g p r o t e i n s i n the c e l l s . Furthermore, i n the o r i g i n a l i n t r a c e l l u l a r envi ronment, these p r o t e i n s may n u l l i f y the e f f e c t o f c a l m o d u l i n ( S a r k a d i et a ! J 9 8 0 ; P e n n i s t o n 1980; Wang 1981) and the a c t u a l c o n t r o l o f enzyme a c t i v i t y may be a complex f u n c t i o n o f 2+ 2+ Ca , Mg , ATP and f r e e c a l m o d u l i n c o n c e n t r a t i o n s . S a r k a d i (1980) suggest t h a t the c a l m o d u l i n e f f e c t s i n the i n t a c t red c e l l s may be 2+ l a r g e l y suppressed because the Ca pump i n i n t a c t c e l l s demonstrates 2+ both a low a f f i n i t y f o r c a l c i u m and low Ca t r a n s p o r t r a t e s as compared to ghosts r e s e a l e d i n the presence o f p u r i f i e d c a l m o d u l i n or i n i n s i d e -out v e s i c l e s i n the presence o f c a l m o d u l i n i n the i n c u b a t i o n medium. The f u n c t i o n o f c a l m o d u l i n as an a c t i v a t o r o f s e v e r a l enzymes was shown to be blocked not o n l y by CaM-BP's but a l s o by p h e n o t h i a z i n e s . Levin and Weiss (1977) f i r s t r e p o r t e d t h a t c e r t a i n a n t i p s y c h o t i c d r u g s , such as t r i f l u o p e r a z i n e and r e l a t e d p h e n o t h i a z i n e s , b l o c k e d c a l m o d u l i n a c t i v a t i o n o f p h o s p h o d i e s t e r a s e . These authors a l s o demonstrated t h a t the b i n d i n g o f p h e n o t h i a z i n e s t o c a l m o d u l i n was c a l c i u m dependent and t h a t there appeared to be a c o r r e l a t i o n between n e u r o l e p t i c potency and a n t i c a l m o d u l i n a c t i v i t y . I t was t h e r e f o r e i m p l i e d t h a t antagonism o f c a l m o d u l i n formed the b a s i s o f n e u r o l e p t i c a c t i v i t y . R o u f o g a l i s (1981) Raess and V i n c e n z i (1980) and G i e t z e n et al_ (19.80) demonstrated t h a t 2+ 2+ p h e n o t h i a z i n e s a l s o antagonized c a l m o d u l i n a c t i v a t i o n o f (Ca + Mg ) -2+ ATPase and Ca - t r a n s p o r t i n human e r y t h r o c y t e membranes. However, l i t t l e o r no c o r r e l a t i o n was found between c a l m o d u l i n antagonism and a n t i p s y c h o t i c a c t i v i t y e . g . h a l o p e r i d o l , a member o f the butyrophenone c l a s s o f c l i n i c a l a n t i p s y c h o t i c s i s 50 f o l d more potent than c h l o r p r o -mazine c l i n i c a l l y , y e t i t b inds to c a l m o d u l i n w i t h lower a f f i n i t y than c h l o r p r o m a z i n e ( L e v i n and Weiss 1979) and has a s i m i l a r o r s l i g h t l y lower potency i n a n t a g o n i z i n g c a l m o d u l i n - a c t i v a t e d p h o s p h o d i e s t e r a s e ( L e v i n and Weiss 1979) or i s i n e f f e c t i v e a g a i n s t c a l m o d u l i n a c t i v a t i o n 2+ 2+ o f membrane bound e r y t h r o c y t e (Ca + Mg )-ATPase compared to c h l o r r promazine (Raess and V i n c e n z i 1 9 8 0 ) . Another problem i n r e l a t i n g c a l m o d u l i n i n h i b i t i o n w i t h c l i n i c a l a n t i p s y c h o t i c potency i s the l a c k o f s t e r e o s p e c i f i c i t y o f c a l m o d u l i n i n h i b i t i o n i n c o n t r a s t to a n t i p s y c h o -t i c a c t i v i t y . R o u f o g a l i s (1981) r e p o r t e d t h a t a l l o f a s e r i e s o f c h l o r -promazine a n a l o g s , i n which the c h l o r i n e s u b s t i t u t i o n was v a r i e d from p o s i t i o n 1 to 4 on the A r i n g o f t h e t r i c y c l i c nucleus, i n h i b i t e d calmodu-2+ 2+ Tin a c t i v a t i o n o f e r y t h r o c y t e (Ca + Mg ) -ATPase, y e t o n l y the compound w i t h c h l o r i n e s u b s t i t u t e d on p o s i t i o n 2 shows t r a n q u i l i z e r a c t i v i t y i n v i v o . S i m i l a r l y , Raess and V i n c e n z i (1980) showed t h a t both (+)- and ( - ) - i s o m e r s o f butaclamol (a n o n - p h e n o t h i a z i n e n e u r o l e p t i c ) were 2+ 2+ i n e f f e c t i v e i n b l o c k i n g c a l m o d u l i n - s e n s i t i v e (Ca + Mg ) -ATPase, even though (+)-butaclamol has t r a n q u i l i z e r a c t i v i t y . I t was thus concluded t h a t the potency o r s t e r e o c h e m i s t r y o f c a l m o d u l i n a n t a g o n i s t s e i t h e r w i t h i n a d i v e r s e s e r i e s o f a n t i p s y c h o t i c agents o r w i t h i n a subgroup o f p h e n o t h i a z i n e analogs does not c o r r e l a t e w e l l w i t h the c l i n i c a l or exper imental e f f i c a c y o f compounds as a n t i - p s y c h o t i c agents ( R o u f o g a l i s 1 9 8 1 ) . I t was i n d i c a t e d i n the above s e c t i o n t h a t p h e n o t h i a z i n e s b i n d to c a l m o d u l i n i n a c a l c i u m dependent manner. I t was shown t h a t the t r i f l u o -2+ p e r a z i n e s i t e s are d i s t i n c t from the Ca b i n d i n g s i t e s C K l e v i t e t a l 2+ 1981) and t h a t Ca dependent exposure o f hydrophobic s i t e s i s important f o r b i n d i n g o f t r i f l u o p e r a z i n e t o c a l m o d u l i n . I t may be noted t h a t 2+ al though Ca induces t h e exposure o f hydrophobic s i t e s i n two o t h e r c a l c i u m b i n d i n g p r o t e i n s v i z . t r o p o n i n C and S - 1 0 0 , o n l y t r o p o n i n C b inds t r i f l u o p e r a z i n e and i n a c a l c i u m dependent manner ( L e v i n and Weiss 1 9 7 8 ; Kobayashi et al_ 1 9 7 9 ) . However, r e c e n t l y S-100 was found to i n t e r a c t 2+ w i t h i m m o b i l i z e d p h e n o t h i a z i n e i n a Ca -dependent manner. P h e n o t h i a z i -nes are not the o n l y compounds c a p a b l e o f i n h i b i t i n g calmodul in-dependent a c t i v a t i o n o f the enzyme s y s t e m s , as a v a r i e t y o f compounds are a b l e to a n t a g o n i z e the a c t i o n o f c a l m o d u l i n . These agents i n c l u d e a n t i p s y c h o t i c agents, a n t i d e p r e s s a n t s , l o c a l a n e s t h e t i c s , v i n c r i s t i n e , r a u w a l f i a a l k a -l o i d s , a n t i - h y p e r t e n s i v e s , N - ( 6 - a m i n o h e x y l ) - 5 - c h l o r o - 1 - n a p h t h a ! e n e -sul fonamide ( W - 7 ) , l - [ b i s ( p - c h l o r o p h e n y l ) m e t h y l ] - 3 - [ 2 , 4 - d i c h l o r o - e - ( 2 , 4 -d i c h l o r o b e n z y l o x y ) p h e n e t h y l ] i m i d a z o l i n i u m c h l o r i d e ( R 2 4 5 7 1 ) , and T r i t o n X-100 ( R o u f o g a l i s 1 9 8 2 ) . A f e a t u r e common to these agents i s t h a t they are hydrophobic or a m p h i p a t h i c and g e n e r a l l y c a t i o n i c a t p h y s i o l o g i c a l pH. These compounds have otherwise l i t t l e s i m i l a r i t y i n t h e i r s t r u c t u r e s and p h a r m a c o l o g i c a l a c t i o n s ( V i n c e n z i 1 9 8 1 ; R o u f o g a l i s 1 9 8 2 ) . A l though the p h e n o t h i a z i n e s are u s e f u l t o o l s to s tudy the c a l m o d u l i n a c t i v a t i o n o f v a r i o u s enzyme systems i n v i t r o , i t i s i m p o r t a n t to note t h a t these agents do not b i n d to c a l m o d u l i n a l o n e . Indeed, p h e n o t h i a z i n e s have been shown to b i n d some " c a l m o d u l i n - l i k e " p r o t e i n s i s o l a t e d from Chlamydompnas f l a g e l l a (Van E l d i k et aj_ 1980) and r a t heart (MacManus, 1 9 8 1 ) , P h e n o t h i -a z i n e s a l s o a n t a g o n i z e ( N a + + K + ) -ATPase and the a c t i v a t i o n o f a number o f enzymes fay l i p i d s , and they a l s o e x h i b i t r e c e p t o r b l o c k i n g a c t i o n s (a - a d r e r t e r g i c , m u s c a r i n i c , dopamine and s e r o t o n i n ) . For a c r i t i c a l d i s -c u s s i o n on the s p e c i f i t y and s e l e c t i v i t y o f p h e n o t h i a z i n e s the r e a d e r i s r e f e r r e d t o a r e c e n t review by R o u f o g a l i s ( 1 9 8 2 ) . I t i s , however, c l e a r t h a t p h e n o t h i a z i n e s are f a i r l y n o n - s p e c i f i c i n t h e i r a c t i o n , a t l e a s t when used i n v i v o . We have shown t h a t t r i f l u o p e r a z i n e i n h i b i t s a c t i v a t -2+ 2+ i o n o f e r y t h r o c y t e (Ca + Mg ) -ATPase, both membrane bound and T r i t o n X - 1 0 0 s o l u b i l i z e d , by two a c i d i c p r o t e i n s ( v i z . p o l y - L - a s p a r t i c a c i d ( M r = 20,000) and cAMP p r o t e i n k i n a s e i n h i b i t o r from bovine heart) a p p a r e n t l y u n r e l a t e d to c a l m o d u l i n . The a c t i v a t i o n was i n h i b i t e d a t a c o n c e n t r a t i o n o f t r i f l u o p e r a z i n e (30 yM) s i m i l a r to t h a t r e q u i r e d f o r i n h i b i t i o n o f c a l m o d u l i n a c t i v a t i o n . The mechanism o f i n h i b i t i o n w i l l be d i s c u s s e d l a t e r under r e s u l t s and d i s c u s s i o n . 2+ 2+ An i n t e r e s t i n g o b s e r v a t i o n was made t h a t (Ca + Mg )-ATPase 2+ a c t i v i t y and Ca - t r a n s p o r t c o u l d be s t i m u l a t e d not o n l y by c a l m o d u l i n but a l s o by a number of a c i d i c p h o s p h o l i p i d s ( N i g g l i e t al_ 1980; A l - J o b o r e and R o u f o g a l i s 1981),by long c h a i n u n s a t u r a t e d f a t t y - a c i d s ( V i n c e n z i 1 9 8 1 ; A l - J o b o r e and R o u f o g a l i s 1 9 8 1 ) , c e r t a i n poly anions (Minocherhomjee and R o u f o g a l i s 1981) and by l i m i t e d p r o t e o l y s i s (Taverna and Hannahan 1980; N i g g l i e t aj_ 1 9 8 1 ) . I t was a l s o shown t h a t t r i f l u o -p e r a z i n e (approx. 100 uM) was a b l e to a n t a g o n i z e the a c t i v a t i o n produced by a c i d i c p h o s p h o l i p i d s , f a t t y - a c i d s , poly a n i o n s , and a l t h o u g h o n l y p a r t i a l l y , by l i m i t e d p r o t e o l y s i s (see C a r a f o l i 1 9 8 1 ) . I t was proposed 2+ by S a r k a d i (1980) t h a t a subunit o f the Ca t r a n s p o r t enzyme i s e a s i l y d i g e s t e d a t the i n t e r n a l membrane s u r f a c e and p o s s i b l y the f u n c t i o n i n g 2+ o f t h i s c a l m o d u l i n - b i n d i n g r e g u l a t o r y s u b u n i t determines the Ca -a f f i n i t y o f the pump. B) Role o f P r o t e i n Kinases I t was shown i n the p r e y i o u s s e c t i o n t h a t many membrane f u n c t i o n s are r e g u l a t e d by the c a l c i u m - c a l m o d u l i n complex. However, i n a d d i t i o n t o c a l c i u m , membrane p h o s p h o r y l a t i o n may a l s o r e g u l a t e c e r t a i n c e l l u l a r f u n c t i o n s . Membrane p h o s p h o r y l a t i o n can be c a t a l y s e d hy p r o t e i n k i n a s e enzymes a s s o c i a t e d w i t h the membrane or by c y t o p l a s m i c p r o t e i n k i n a s e s . S e v e r a l p r o t e i n k i n a s e a c t i v i t i e s a r e p r e s e n t i n the red c e l l ( B o i v i n e t a l 1 9 8 1 ) . C y c l i c n u c l e o t i d e dependent hi stone k inases and c y c l i c n u c l e o -t i d e independent c a s e i n k inases have been i d e n t i f i e d i n the human e r y t h r o c y t e (Rubin et aj_ 1982; F a i r b a n k s and Avruch 1974; Avruch and Fai rbanks 1 9 7 4 ) . Both c y c l i c AMP dependent and independent p r o t e i n k i n a s e s were shown to be compartmental ized between the membrane and c y t o s o l i n human e r y t h r o c y t e s (Drey fuss et aj_ 1978; B o i v i n and Galand 1 9 8 0 ) . Although the r o l e o f these k inases i n membrane f u n c t i o n s remains to be e s t a b l i s h e d , i t has been known f o r q u i t e some time t h a t red c e l l membrane c o n t a i n s s e v e r a l p r o t e i n s which a r e p h o s p h o r y l a t e d by these k i n a s e s (Rubi n et aj_ 1972; Avruch and F a i r b a n k s 1 9 7 4 ) . In membranes from v a r i o u s mammalian t i s s u e s and c e l l s , p h o s p h o r y l a t i o n o f membrane p r o t e i n s was shown to c o r r e l a t e w i t h changes i n p e r m e a b i l i t y to ions 2+ (Greengard 1 9 7 6 ) , drugs ( C a r l s e n et al_ 1 9 7 7 ) , Ca ( W e l l e r and Morgan 1 9 7 7 ) , g l u c o s e t r a n s p o r t (Chang et al_ 1 9 7 4 ) , arrangement o f membrane p r o t e i n s ( G a z i t t e t aj_ 1 9 7 6 ) , and membrane f l u i d i t y (Kury and McConnel 1 9 7 5 ) . In the case o f human e r y t h r o c y t e membranes, i t has been suggested t h a t the degree o f p h o s p h o r y l a t i o n c o u l d r e g u l a t e the i n t e r a c t i o n between c e r t a i n p r o t e i n s which may be c r u c i a l i n m a i n t a i n i n g e r y t h r o c y t e shape and deformabi 1 i t y (Yawata e t al_ 1976; Kury and .McConnel 1 1 9 7 5 ) . In p a r t i c u l a r , i n the p a s t few y e a r s i n c r e a s i n g i n t e r e s t has been d i r e c t e d toward the s tudy o f the p h o s p h o r y l a t i o n o f s p e c t r i n , the roost abundant membrane p r o t e i n l o c a t e d on the c y t o s o l i c s u r f a c e , s i n c e i t s p h o s p h o r y l a t i o n s t a t e may modulate i t s i n t e r a c t i o n w i t h a c t i n and o t h e r membrane components, which i n t u r n may c o n t r o l the shape, d e f o r m a b i l i t y and f u n c t i o n a l p r o p e r t i e s o f the membrane. Al though c o n t r o v e r s i a l , i t i s b e l i e v e d t h a t s p e c t r i n (band 2) and band 3 ( t h e most abundant i n t e g r a l membrane p r o t e i n ) a r e poor s u b s t r a t e s f o r c y c l i c AMP dependent hi stone k i n a s e (Type I) (Suzuki et al_ 1981; C l a r i e t al_ 1 9 8 1 ) , whereas both these p r o t e i n s are good s u b s t r a t e s f o r c y c l i c AMP independent p r o t e i n k i n a s e ( c a s e i n - k i n a s e ) (Suzuki et a l 1981; C l a r i e t a l 1981; V i c k e r s et aj_ 1979; Simkowski and Tao 1980) . C y c l i c AMP dependent h i s t o n e k i n a s e , however, has been shown to p h o s p h o r y l a t e membrane p r o t e i n s , e s p e c i a l l y a n k y r i n (band 2 . 1 ) , bands 4 . 5 and 4 . 8 ( B o i v i n et a l 1 9 8 1 ) . A n k y r i n ( M r = 215,000) i s l o c a l i z e d on the c y t o p l a s m i c s u r f a c e o f the membrane and may be r e s p o n s i b l e f o r the attachment o f s p e c t r i n to the membrane through i t s high a f f i n i t y b i n d i n g s i t e s f o r s p e c t r i n (Bennett and Stenbuck 1 9 7 9 ) . A n k y r i n a l s o i n t e r a c t s d i r e c t l y w i t h band 3 (Bennett and Stenbuck 1 9 8 0 ) . A s s o c i a t i o n o f s p e c t r i n w i t h a n k y r i n has been demonstrated to r e s t r i c t the l a t e r a l m o b i l i t y o f band 3 and o t h e r i n t e g r a l membrane p r o t e i n s i n the e r y t h r o c y t e ghost (Fowler and Bennett 1 9 7 8 ) . Thus, a l though s p e c t r i n may not be a very good s u b s t r a t e f o r c y c l i c nucleotide dependent h i s t o n e k i n a s e , i t i s p o s s i b l e t h a t i t s s t a t e can be a l t e r e d i n d i r e c t l y through c y c l i c AMP-dependent p h o s p h o r y l a t i o n o f a n k y r i n . I t i s r a t h e r i n t r i g u i n g t h a t the human red c e l l has l i t t l e o r no a d e n y l a t e c y c l a s e or p h o s p h o d i e s t e r a s e a c t i v i t y but a very high p o t e n t i a l c y c l i c AMP dependent h i s t o n e k i n a s e a c t i v i t y ( B o i v i n and Galand 1 9 7 8 ) . Recent r e p o r t s , however, i n d i c a t e t h a t d e s p i t e the absence o f a c t i v e c y c l i c AMP metabol ism i n the human e r y t h r o c y t e . c y c l i c AMP can e n t e r the e r y t h r o c y t e by passive uptake tn s u f f i c i e n t amounts t o a l t e r t h e a c t i v i t y o f c y c l i c AMP dependent p r o t e i n k i n a s e s o r t o a l t e r the r a t e o f t u r n o v e r o f c e r t a i n phosphory lated membrane p e p t i d e s (Thomas et aj_ 1979; Tsukamato et a]_ 1 9 8 0 ) . The l e v e l s o f c y c l i c AMP thus appear to be r e g u l a t e d by d i f f u s i o n a c r o s s the red c e l l membrane. A recent r e p o r t by Varghese and Cunningham (1980) i n d i c a t e d t h a t 2+ 2+ micromolar c o n c e n t r a t i o n s o f c y c l i c AMP I n h i b i t (Ca + Mg )-ATPase i n red c e l l membrane f ragments, p r o b a b l y v i a membrane a s s o c i a t e d c y c l i c AMP p r o t e i n k i n a s e . S i n c e c y c l i c AMP dependent p r o t e i n k i n a s e mediated p h o s p h o r y l a t i o n can be s p e c i f i c a l l y blocked by c y c l i c AMP dependent p r o t e i n k i n a s e i n h i b i t o r s (PKI) which have been i s o l a t e d from v a r i o u s t i s s u e s (Whitehouse et a l 1 9 8 0 ) , the e f f e c t ( s ) o f PKI's on the k i n e t i c s of 2+ 2+ Ca a c t i v a t i o n o f Ca - t r a n s p o r t ATPase has been i n v e s t i g a t e d i n t h i s t h e s i s . I t i s important to note t h a t i n c o n t r a s t to c a l m o d u l i n , which shows l a c k o f s p e c i e s o r t i s s u e s p e c i f i c i t y , the PKI's i s o l a t e d from d i f f e r e n t animal t i s s u e s such as the r a b b i t s k e l e t a l m u s c l e , bovine heart or r a t t e s t i s a r e not i d e n t i c a l . However, a l l the PKI's a r e a c i d i c , heat s t a b l e p r o t e i n s (Walsh et aj_ 1971; Weber and Rosen 1977; Beal e t al_ 1977; D e m a i l l e e t a l 1978; F u r s t e n t h a l 1980) . S i n c e both PKI's and c a l m o d u l i n are h e a t - s t a b l e a c i d i c p r o t e i n s , the e f f e c t o f PKI on 2+ 2 + e r y t h r o c y t e (Ca + Mg )-ATPase was i n v e s t i g a t e d i n f u r t h e r d e t a i l and compared to the a c t i v a t i o n o f t h i s enzyme by c a l m o d u l i n . C) Role o f Anion Channel (Band H I ) In the model f o r c a l c i u m e x t r u s i o n from human red c e l l s , i t was shown t h a t a c t i v e c a l c i u m t r a n s p o r t i s not coupled to c o - o r c o u n t e r -t r a n s p o r t o f monovalent or d i v a l e n t c a t i o n s . E l e c t r o - n e u t r a l i t y d u r i n g 2+ a c t i v e Ca e x t r u s i o n i s probably m a i n t a i n e d by net CI e f f l u x , s i n c e the CI" conductance o f the human red c e l l membrane i s h igh enough to 2+ permit Ca movement (Gunn 1978). Waisman e_t a l _ ( l 9 8 1 ) , u s i n g i n s i d e - o u t v e s i c l e s , r e p o r t e d t h a t band 3 (anion channel) i n h i b i t o r s such as 4 - a c e t a m i d o - 4 ' - i s o t h i o cyano s t i l b e n e - 2 ' - d i s u l f o n a t e (SITS) and N - ( 4 - a z i d o - 2 - n i t r o p h e n y l ) - 2 - a m i n o e t h y l s u l f o n a t e ( N A P - t a u r i n e ) blocked 2+ c a l m o d u l i n s t i m u l a t e d Ca t r a n s p o r t and anion t r a n s p o r t w i t h s i m i l a r potency. T h i s o b s e r v a t i o n l e d t h e s e authors t o suggest t h a t these two t r a n s p o r t processes were c l o s e l y c o u p l e d . Furthermore, they suggested t h a t a t low c o n c e n t r a t i o n s (.K. = 45 - 80 y M ) , N A P - t a u r i n e blocked c a l m o d u l i n dependent c a l c i u m t r a n s p o r t a c t i v i t y v i a blockade of a n i o r i -t r a n s p o r t through the anion c h a n n e l . The band 3 p r o t e i n , which i s the major transmembrane p r o t e i n , n o r m a l l y f a c i l i t a t e s the e l e c t r o n e u t r a l exchange o f anions a c r o s s the e r y t h r o c y t e membrane (Cabantchik and R o t h s t e i n 1974; Ho and G u i d o t t i , 1975). Band 3 p r o t e i n i s a l s o known t o f u n c t i o n as an anion u n i p o r t i n response to an e l e c t r o c h e m i c a l g r a d i e n t a c r o s s the membrane (Knauf et a i l 9 7 1 ; Wolosin et a l 1 9 7 7 ) . G r i n s t e i n et a l (1979) showed t h a t 4 , 4 ' - d i i s o t h i o c y a n o - 2 , 2 ' - s t i l b e n e d i s u l f o n a t e (DIDS) i s a t r a n s p o r t s i t e s p e c i f i c , n o n - p e n e t r a t i n g i r r e v e r s i b l e i n h i b i t o r o f band 3 and t h a t i t i s e f f e c t i v e as an i n h i b i t o r o f band 3 o n l y when a p p l i e d e x t r a c e l l u l a r l y . Knauf e t aj_ (1978) showed t h a t N A P - t a u r i n e a c t s as a r e v e r s i b l e i n h i b i -t o r o f band 3 ( i n the dark) when i t i s present e i t h e r w i t h i n the c e l l o r i n the e x t e r n a l medium. However, t h e r e i s a s u b s t a n t i a l d i f f e r e n c e i n the c o n c e n t r a t i o n o f N A P - t a u r i n e r e q u i r e d to b l o c k the anion channel from the two s i d e s o f the membrane. On the c y t o p l a s m i c s i t e , N A P - t a u r i n e i s a r e l a t i v e l y low a f f i n i t y i n h i b i t o r o f c h l o r i d e exchange (K. = 370 yM) and competes w i t h CI" f o r the s u b s t r a t e s i t e o f the anion exchange system. On the o t h e r hand, e x t e r n a l N A P - t a u r i n e i s a f a r tnore potent i n h i b i t o r o f anion-exchange (K. - 20 yM) and causes n o n - c o m p e t i t i v e i n h i b i t i o n o f the anion-channel by b i n d i n g t o a "modi f ier" s i t e . I t was suggested by t h e s e authors t h a t the m o d i f i e r s i t e i s a c c e s s i b l e t o NAP-t a u r i n e o n l y from the o u t s i d e , whereas the t r a n s p o r t s i t e may be a c c e s s i b l e from e i t h e r s i d e . S t a r o s and R i c h a r d s (.1974) showed t h a t i n e r y t h r o c y t e membrane p r e p a r a t i o n s N A P - t a u r i n e , upon p h o t o a c t i v a t i o n , l a b e l s s e v e r a l p r o t e i n bands i n a d d i t i o n to band 3 . These o b s e r v a t i o n s prompted f u r t h e r s t u d i e s i n the present t h e s i s on (a) the s idedness o f a c t i o n of NAP-2+ t a u r i n e as an i n h i b i t o r o f Ca - t r a n s p o r t ATPase, (b) the mechanism and 2+ s p e c i f i c i t y of N A P - t a u r i n e i n i n h i b i t i o n o f Ca - t r a n s p o r t , and (c) a 2+ comparison between DIDS and N A P - t a u r i n e as i n h i b i t o r s o f Ca - t r a n s p o r t i n human e r y t h r o c y t e i n s i d e out v e s i c l e s . O b j e c t i v e s o f the Present Study 1) To study the i n f l u e n c e o f c a l m o d u l i n on the K c a ^ + and V of ( C a 2 + + M g 2 + ) - A T P a s e . max. 3 2) To determine the r o l e o f cAMP mediated p r o t e i n p h o s p h o r y l a t i o n on the 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y , u s i n g cAMP dependent p r o t e i n k i n a s e i n h i b i t o r s . 3) To determine the r o l e o f heat s t a b l e a c i d i c p r o t e i n s , o t h e r than 2 t 2+ c a l m o d u l i n , and o t h e r anions, i n the r e g u l a t i o n .Qf (Ca. t Mg )-ATP-ase a c t i v i t y , 4) To study the r o l e o f the anion channel (Band 3) i n the r e g u l a t i o n o f 2+ Ca - t r a n s p o r t i n the red c e l l . 5) To s e p a r a t e the e f f e c t s o f N A P - t a u r i n e as an i n h i b i t o r o f the a n i o n -channel and c a l c i u m - t r a n s p o r t ; and i t s comparison w i t h the a c t i o n o f the anion channel b l o c k e r , DIDS. MATERIALS AND flETHQPS [ I ] M a t e r i a l s : The c h e m i c a l s and/or p r o t e i n s were purchased from the f o l l o w i n g s o u r c e s : 1) Sigma Chemical Company: EDTA EGTA Trizma base Imidazole (grade I) HEPES MES M a l e i c a c i d PMSF T r i s - A T P T r i t o n X-100 P o l y - L - c a r b o x y l i c a c i d s H i s t o n e ( t y p e 11-A) C y c l i c AMP dependent p r o t e i n k i n a s e (bovine heart ) P r o t e i n k i n a s e c a t a l y t i c s u b u n i t (bovine heart ) C y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r (bovine heart and r a b b i t s k e l e t a l muscle) Calmodul in d e p l e t e d p h o s p h o d i e s t e r a s e C y c l i c AMP Bovine serum albumin ( f a t t y - a c i d f r e e ) Sodium t u n g s t a t e B o r i c a c i d Taur ine DIDS T r y p s i n (Type I) Soya bean t r y p s i n i n h i b i t o r (Type 1-S) Laury l s u l f a t e Coomassie blue 2) J . T . Baker Chemical Company: 1 , 2 - c y c l o h e x a n e d i o n e TCA Sodium hydroxide Sodium c h l o r i d e Monosodium dihydrogen phosphate (NaHgPQ^) Disodium monohydrogen phosphate (J^HPO^) 3) F i s h e r S c i e n t i f i c Company: Calcium c h l o r i d e ( C a C ^ ) Carbowax (PEG 20,000) Lanthanum c h l o r i d e 4) P i e r c e N A P - t a u r i n e SITS DIDS 5) Amersham: [ Y _ 3 2 P ] _ A T P ( s p e c i f i c a c t i v i t y = 5 - 1 0 x 1 0 6 DPM/nmole) C a C l 2 ( s p e c i f i c a c t i v i t y = l , 5 j * 1 0 6 CPM/^mole), 6) Whatman: DE-52 anion exchange r e s i n , 7) A l l i e d C h e m i c a l s : H y d r o c h l o r i c a c i d (HC1) G l a c i a l A c e t i c A c i d (CHg COOH) S u l f u r i c A c i d ( H 2 S 0 4 ) 8) B i o r a d L a b o r a t o r i e s : SDS 9) Merck, Sharp and Dohme Probenecid (a g i f t ) 10) S m i t h , K l i n e and French Co. ( M o n t r e a l ) : T r i f l u o p e r a z i n e h y d r o c h l o r i d e (a g i f t ) . 11) C a l b i o c h e m ; C a l m o d u l i n . 12) Eastman: Acrylamide(enzyme grade) N , N ' - m e t h y l e n e - b i s a c r y l a m i d e N , N , N ' , N " - t e t r a m e t h y l - e t h y l e n e - d i a m i n e . [ I I ] Methods 1) P r e p a r a t i o n o f E r y t h r o c y t e Ghosts Human blood preserved i n a c i d - c i t r a t e - d e x t r o s e s o l u t i o n was o b t a i n e d from the l o c a l Red Cross Blood Bank and used w i t h i n 5 days o f c o l l e c t i o n f o r p r e p a r a t i o n o f e r y t h r o c y t e ghosts (membranes). Membranes were prepared by the methods o u t l i n e d by a) Dodge et a l ( 1 9 6 3 ) , b) S c h r i e r (1967) and c ) C a r a f o l i et a l (1980). a) Membrane p r e p a r a t i o n by the procedure o f Dodge et a l : E r y t h r o c y t e s c o l l e c t e d by c e n t r i f u g a t i o n a t 2,500 x ^ were washed three t imes w i t h 310 i d e a l mosm-sodium phosphate b u f f e r , pH 7.4 (P 310 b u f f e r ) . The washed c e l l s were suspended i n an equal volume o f P 310 b u f f e r and 10 ml o f t h i s suspension was r a p i d l y p i p e t t e d i n t o 140 mis o f 20 i d e a l mosm-sodium phosphate b u f f e r , pH. 7.4 (P 20 b u f f e r ) , f o l l o w e d by s w i r l i n g . The hemolysed c e l l s were c e n t r i f u g e d a t 22,000 x £ f o r 40 m i n . and the hemolysate was c a r e f u l l y decanted. The c e l l s were washed t w i c e more i n 140 ml p o r t i o n s o f P 20 b u f f e r . The 'ghosts' from the i n d i v i d u a l c e n t r i -fuge b o t t l e s were pooled and washed once more by c e n t r i f u g a t i o n a t 40,000 x £ f o r 20 mins i n an equal volume o f P 20 b u f f e r . One m i l l i l i t e r of ghosts was found to c o n t a i n between 3 . 8 - 4 . 2 mg o f p r o t e i n (determined by an automated Lowry procedure (1951) w i t h a Technicon a u t o a n a l y s e r ) . These ghosts were whi te and s t o r e d at - 2 0 ° C f o r ! 6 h r before use and used w i t h i n 4 days o f p r e p a r a t i o n . b) Membrane p r e p a r a t i o n by s tepwise hemolysis procedure as o u t l i n e d  by S c h r i e r : The red c e l l s were washed t h r i c e i n i s o t o n i c s a l i n e s o l u t i o n a t 2500 x g_. The washed c e l l s were c o n s e c u t i v e l y hemolysed 1n 10 volumes o f 0.08M, 0.06M and 0.04M NaCl and c e n t r i f u g e d a t 8 , 0 0 0 , 13,000 and 15,000 x £ f o r 10 m i n . at 4 ° C , r e s p e c t i v e l y . The c e l l s were f i n a l l y hemolysed i n 0.015M NaCl and 0.005M t r i s - m a l e a t e (pH 7.1) a t 2 - 4°C and washed twice i n t h i s s o l u t i o n . These ghosts were s l i g h t l y pink i n c o l o r and were used immediately f o r the p r e p a r a t i o n o f r e s e a l e d ghosts or i n s i d e - o u t v e s i c l e s . c) Membrane p r e p a r a t i o n by the procedure d e s c r i b e d by C a r a f o i l ' et a l : The red c e l l s were washed t h r i c e i n 5 volumes of 130mM KC1, 20mM T r i s - C l , pH 7 . 4 . The c e l l s were hemolysed i n 5 volumes o f ImM Na-EDTA, lOmM T r i s - C l , pH 7 . 4 , and c e n t r i f u g e d at 18,000 x £ f o r 10 min. The ghosts were then washed f i v e times i n the hemolysis b u f f e r , and f o u r more t imes i n lOmM HEPES, pH 7 . 4 , w i t h o u t EDTA. The ghosts were whi te and were f i n a l l y resuspended i n 130mM KC1, 20mM HEPES, pH 7 . 4 , 500yM M g C l 2 , 50yM C a C l 2 , 2mM d i t h i o t h r e i t o l and s t o r e d a t - 8 0 ° C and used w i t h i n 3-4 weeks. 2) P r e p a r a t i o n o f Calmodul in d e f i c i e n t (EDTA-treated) membranes: One volume o f Dodge 'ghosts' was suspended i n f i v e volumes of O.lmM EDTA and 1.OmM T r i s - m a l e a t e (pH 8 . 0 ) and incubated a t 37°C f o r 15 min. The suspension was then c e n t r i f u g e d at 2 0 , 0 0 0 x £ a t 4°C f o r 15 m i n . The supernatent was c a r e f u l l y d i s c a r d e d and the p e l l e t was resuspended i n 15mM NaCl and 5mM T r i s - m a l e a t e (pH 7.1) t o the o r i g i n a l volume o f ghosts used. The EDTA-treated membranes were 2+ 2+ used immediately f o r assay Of (Ca t Mg )-ATPase a c t i y i t y , as these membranes a r e r e l a t i v e l y l a b i l e under these c o n d i t i o n s . 3) P r e p a r a t i o n o f r e s e a l e d ghosts Resealed ghosts were prepared e s s e n t i a l l y as d e s c r i b e d by Q u i s t and R o u f o g a l i s ( 1 9 7 5 ) . The main steps i n the procedure were as f o l l o w s . Red c e l l "ghosts" were prepared from human b l o o d , s t o r e d i n a c i d - c i t r a t e - d e x t r o s e (Canadian Red Cross) f o r no more than 4 days a t 4 ° C , by a s t e p w i s e hemolysis procedure as o u t l i n e d by S c h r i e r ( 1 9 6 7 ) . These ghosts were used immediately f o r r e s e a l i n g . Ghosts (.1 ml) were suspended i n the "loading-medium" (3 m l ) , which c o n t a i n e d ( i n f i n a l c o m p o s i t i o n ) 4mM M g C I 2 , 3mM C a C l 2 , 4mM T r i s - A T P , O.lmM o u a b a i n , lOmM T r i s - m a l e a t e , pH 7.1 at 4°C ± N A P - t a u r i n e . The suspension was e q u i l i b r a t e d f o r 10 min a t 2 - 4 ° C . R e s e a l i n g was achieved by r e s t o r a t i o n o f i s o t o n i c i t y w i t h N a C l , f o l l o w e d by i n c u -b a t i o n i n a water bath at 25°C f o r 10 m i n . The samples were r e t u r n e d to the i c e - b a t h and washed t w i c e w i t h 5 ml o f 2mM M g C ^ , 125mM N a C l , O.lmM ouabain,' 20mM T r i s - m a l e a t e , pH 7.5 a t 5,000 x g_ f o r 10 min a t 4 ° C . The r e s e a l e d ghosts were then suspended i n the same s o l u t i o n which c o n t a i n e d i n a d d i t i o n ImM C a C ^ . When NAP-t a u r i n e (50 yM) was added t o r e s e a l e d ghosts the sample was a l lowed to i n c u b a t e i n the absence o f l i g h t a t 4°C f o r 10 min p r i o r to i n i t i a t i o n o f the a s s a y . The ATP h y d r o l y s i s was i n i t i a t e d by p l a c i n g the tubes i n a bath a t 3 7 ° C . The ATPase a c t i v i t y was stopped by adding SDS t o a f i n a l c o n c e n t r a t i o n o f 2%. The ATPase a c t i v i t y was measured by d e t e r m i n i n g the r e l e a s e of i n o r g a n i c phosphate (P..) 2+ from ATP by the method o f F i s k e and SubbaRow (1925). Mg -ATPase a c t i v i t y was determined i n the absence o f C a C l 2 . b y d i r e c t i n c u b a t i o n o f ghosts ( p r i o r to re sea. l i n g w i t h NaCl) w i t h ATP a t 3 7 ° C , The c o n t r i b u t i o n of " l e a k y - g h o s t s " to t o t a l ATPase a c t i v i t y was e s t i m a t e d by r e s e a l i n g ghosts i n the absence o f ATP. The f o r m a t i o n o f P.. a f t e r a d d i t i o n of ATP to the e x t e r n a l medium a t 37°C g i v e s ATPase a c t i v i t y due to "leaky ghosts". Using t h i s method o f a n a l y -s i s , a p p r o x i m a t e l y 60-65% o f the ghosts were found to be r e s e a l e d . 2+ The Mg -ATPase a c t i v i t y (17%) was s u b t r a c t e d from t o t a l ATPase a c t i v i t y t o o b t a i n the c a l c i u m s t i m u l a t a b l e a c t i v i t y . 4) P r e p a r a t i o n of i n s i d e - o u t v e s i c l e s I n s i d e - o u t v e s i c l e s were prepared from ghosts as p r e v i o u s l y d e s c r i b e d ( Q u i s t and R o u f o g a l i s 1977). The main steps i n the p r o c e -dure were as f o l l o w s . On the same day o f p r e p a r a t i o n , the ghosts (prepared a c c o r d i n g to the method o f S c h r i e r ) were suspended to the o r i g i n a l volume o f the packed c e l l s i n 15mM NaCl and 5mM T r i s - m a l e a t e , pH 7 . 1 , d i l u t e d w i t h 5 volumes (25 ml) o f a s o l u t i o n of O.lmM EGTA and ImM T r i s - m a l e a t e , pH 8 and incubated a t 37°C f o r 30 min w i t h c o n s t a n t s h a k i n g . A f t e r c e n t r i f u g a t i o n a t 20,000 x £ a t 4°C f o r 10 m i n , the p e l l e t s were resuspended to 1 volume (5 -ml) i n 15mM NaCl and 5mM T r i s - m a l e a t e , pH 7 . 1 . The v e s i c l e p r e p a r a t i o n was suspended i n 3 volumes o f lOmM T r i s - m a l e a t e , 4mM M g C l 2 and 0.5mM CaClg a t pH 7.1 and incubated f o r 5 min a t 0 - 4 ° C . I s o t o n i c i t y was r e s t o r e d by t h e a d d i t i o n o f 1 ml o f 2.9M NaCl and the v e s i c l e s i n c u b a t e d a t 30°C f o r 10 m i n . The v e s i c l e s were p e l l e t e d a t 20,000 x £ f o r 10 min and washed t w i c e in 66mM NaCl and 55mM T r i s - m a l e a t e at pH 7 . 1 . The f i n a l p e l l e t s were combined and suspended to 4 volumes i n the same washing medium, Sidedness p f the y e s i c l e s was determined by the a s s a y o f a c e t y l c h o l i n e s t e r a s e i n the absence and presence o f T r i t o n X-100 (0.2%). A p p r o x i m a t e l y 50-55% o f the v e s i c l e s were i n s i d e - o u t . 5) I r r a d i a t i o n o f c e l l s and membranes w i t h N A P - t a u r i n e Whole c e l l s , washed w i t h 140mM KC1 and 20mM T r i s - H C l , pH 7 . 4 , 2+ 2+ were suspended i n the b u f f e r used f o r assay o f (Ca + fig )-ATPase a t 10% h e m a t o c r i t i n the absence and presence o f N A P - t a u r i n e . The sample was i r r a d i a t e d f o r 40 min a t 4°C w i t h a S y l v a n i a 3M lamp (500 W) focussed on the sample. A f t e r i r r a d i a t i o n the c e l l s were c e n t r i f u g e d at 3,000 x £ and washed t w i c e w i t h t h e above b u f f e r c o n t a i n i n g 0.5% bovine serum albumin and one more t ime w i t h o u t a l b u m i n . Membranes were then prepared from these c e l l s as d e s c r i b e d above. Membranes o b t a i n e d from DIDS t r e a t e d c e l l s were processed s i m i l a r l y . The c e l l s were a l l o w e d to r e a c t w i t h DIDS (5 yM) a t 37°C f o r 30 min p r i o r to h e m o l y s i s . For i r r a d i a t i o n o f membranes i n the presence o f N A P - t a u r i n e , the membranes (approx. 4 mg protein/ml f i n a l c o n c e n t r a t i o n ) were 2+ 2+ i n c u b a t e d i n the (Ca + Mg )-ATPase assay b u f f e r (wi thout ATP) i n the absence and presence o f N A P - t a u r i n e and p h o t o l y s e d f o r 40 min a t 4 ° C , as above. 50 y l o f the p h o t o l y s e d membrane p r e p a r a t i o n was then 2+ 2+ assayed f o r (Ca + Mg )-ATPase a c t i v i t y i n 0.6 ml o f the s t a n d a r d assay medium. As the enzyme p r e p a r a t i o n was d i l u t e d 1 2 - f o l d , the c o n c e n t r a t i o n o f unreacted N A P - t a u r i n e was reduced to a c o n c e n t r a t i o n (approx. 2 yM) which was shown i n c o n t r o l experiments not t o i n h i b i t the ( C a 2 + + Mg 2 + )ATPase a c t i v i t y . In experiments t o p r o t e c t a g a i n s t N A P - taurine i n h i b i t i o n , membranes were photolysed w i t h N A P - t a u r i n e (25 jiM) as d e s c r i b e d 2+ 2+ above, but i n the presence of v a r i o u s e f f e c t o r s o f (Ca + Mg ) -2+ 2+ ATPase v i z . Ca , Mg , ATP and c a l m o d u l i n . C o n t r o l a c t i v i t i e s were e s t a b l i s h e d i n the same c o n d i t i o n s w i t h i r r a d i a t i o n i n t h e absence of N A P - t a u r i n e . 2+ 2+ 6) S o l u b i l i z a t i o n o f (Ca + Mg )-ATPase from human e r y t h r o c y t e mem- branes 2+ 2+ (Ca + Mg )-ATPase was s o l u b i l i z e d from e r y t h r o c y t e membra-nes as d e s c r i b e d by C a r a f o l i e t al_ ( 1 9 8 0 ) . E s s e n t i a l l y , the membrane (4 - 4 . 5 mg p r o t e i n / m l ) was t r e a t e d w i t h T r i t o n X-100 (.1 -mg T r i t o n / mg membrane p r o t e i n ) a t 4°C f o r 10 m i n , f o l l o w e d by c e n t r i f u g a t i o n a t 100,000 x £ f o r 30 m i n . The supernatent was c a r e f u l l y removed and used as a source o f enzyme. A p p r o x i m a t e l y , 10-12% o f membrane p r o t e i n was s o l u b i l i z e d by t h i s procedure. 7) L i m i t e d p r o t e o l y s i s ( t r y p s i n i z a t i o n ) o f membranes To 1 ml o f membranes (4 mg/ml) prepared by the procedure of C a r a f o l i et_ al_ ( 1 9 8 0 ) , was added 1 ml o f lOmM sodium-HEPES b u f f e r , pH 7 . 4 , c o n t a i n i n g t r y p s i n (0.2 yg/mg p r o t e i n ) . In the c o n t r o l sample t r y p s i n was o m i t t e d . The samples were incubated a t 37°C f o r 3 m i n . P r o t e o l y s i s was stopped by a d d i t i o n o f 1 ml o f a soyabean t r y p s i n - i n h i b i t o r ( 0 . 4 yg/mg p r o t e i n ) i n the above b u f f e r . The samples were c e n t r i f u g e d a t 20,000 x £ f o r 10 m i n . The membranes were washed one more t ime i n lOmM sodium-HEPES b u f f e r and r e c o n s t i -t u t e d to the o r i g i n a l volume (1 m l ) . T r y p s i n i z a t i o n o f membranes 2+ l e d to a l o s s o f a p p r o x i m a t e l y 22% of membrane p r o t e i n s . (Ca + 2+ Mg )-ATPase a c t i v i t y was measured w i t h i n 30 min a f t e r t r y p s i n i z a t -i o n . 8) P u r i f i c a t i o n o f c y c l i c AMP dependent p r o t e i n k i n a s e i n h i b i t o r The PKI from bovine heart o b t a i n e d from Sigma Chemical Company, a l though r e p o r t e d t o be f r e e o f "heat s t a b l e modulator" ( c a l m o d u l i n ) , was found to c o n t a i n small but s i g n i f i c a n t c o n t a m i n a t i o n by calmodu-l i n , as determined by the p h o s p h o d i e s t e r a s e assay and by r a d i o -immunoassay. The PKI p r e p a r a t i o n was p u r i f i e d on a Whatman DE 52 column. The PKI p r e p a r a t i o n from Sigma (5 mg/5 ml) was d i a l y z e d a g a i n s t 1OmM i m i d a z o l e - H C l , pH 6.8,and a p p l i e d to a DE 52 column (2.1 x 20 cm) p r e v i o u s l y e q u i l i b r a t e d in the same b u f f e r . The column was washed w i t h 25 ml o f the b u f f e r and e l u t e d w i t h 200 ml o f a l i n e a r g r a d i e n t o f 10 to 300mM NaCl i n lOmM i m i d a z o l e - H C l , pH 6 . 8 . F r a c t i o n s of 2-3 ml were c o l l e c t e d . T h i s was f o l l o w e d by washing w i t h 100 ml o f 1M NaCl i n the i m i d a z o l e b u f f e r . The f r a c t i o n s c o n t a i n i n g the c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r (as determined by t h e i r i n h i b i t i o n o f the p h o s p h o r y l a t i o n of hi stone by the c a t a -l y t i c s u b u n i t ) were pooled and c o n c e n t r a t e d by d i a l y s i s (M cut o f f 3,000) a g a i n s t p o l y e t h y l e n e g l y c o l . The p r o t e i n s o l u t i o n , c o n c e n t r a -ted to approx. 5 m l , was then d i a l y z e d a g a i n s t lOmM i m i d a z o l e b u f f e r c o n t a i n i n g 5mM EDTA, f o l l o w e d by e x t e n s i v e d i a l y s i s a g a i n s t i m i d a z o l e b u f f e r to remove EDTA. The 1M NaCl f r a c t i o n was t r e a t e d i n an i d e n t i c a l manner. Both the pooled g r a d i e n t f r a c t i o n s and the 1M NaCl f r a c t i o n were t e s t e d f o r the presence o f c a l m o d u l i n by c a l -modul in radioimmunoassay and by t h e i r a b i l i t y t o s t i m u l a t e c a l m o d u l i n - d e p l e t e d p h o s p h o d i e s t e r a s e . 9) Chemical m o d i f i c a t i o n o f a r g i n y l - s i d e c h a i n ? A r g i n i n e r e s i d u e s were m o d i f i e d by t reatment o f the p r o t e i n s ( p r o t e i n k i n a s e i n h i b i t o r and c a l m o d u l i n ) w i t h 0.05M 1 , 2 - c y c l o h e x a n e -dione i n 0.15M b o r a t e b u f f e r , pH 9 f o r 3hours a t 3 7 ° C , e s s e n t i a l l y by the method o f D e m a i l l e et al_ (1 977) . C o n t r o l samples were t r e a t e d i n the same way i n the absence o f 1 , 2 - d i c y c l o h e x a n e d i o n e . S i n c e T r i s b u f f e r s were shown t o decrease the s t a b i l i t y o f the m o d i f i e d a r g i n i n e product (Patthyand S m i t h , 1 9 7 5 ) , assay o f ATPase a c t i v i t i e s was performed i n sodium maleate b u f f e r , pH 6.9 before and a f t e r a r g i n i n e m o d i f i c a t i o n . 10) Assay o f ATPases 2+ 2+ 32 (Ca + Mg )-ATPase a c t i v i t y was determined from P^  r e l e a s e d 32 from [y- P]ATP (Katz and B l o s t e i n , 1975). The i n c u b a t i o n medium c o n t a i n e d ( u n l e s s i n d i c a t e d o t h e r w i s e ) 66mM NaCl (or the a p p r o p r i a t e a n i o n ) , 6.5 mM MgCl^, O.lmM o u a b a i n , O.lmM EGTA, v a r i o u s c o n c e n t r a -t i o n s of f r e e c a l c i u m and 55mM o f e i t h e r T r i s , HEPES, MES or TES a d j u s t e d to the r e q u i r e d pH (pH 6.9 or 7.2) i n the assay medium a t 37°C by a d d i t i o n of maleate ( T r i s - m a l e a t e ) , HC1 ( T r i s - C l ) or sodium hydroxide (sodium-HEPES, sodium-MES, sodium-TES and s o d i u m - m a l e a t e ) . The e f f e c t s o f p o l y - L - c a r b o x y l i c a c i d s and v a r i o u s anions were 2+ s t u d i e d i n a T r i s - m a l e a t e b u f f e r . Free Ca c o n c e n t r a t i o n s were c a l c u l a t e d by s o l v i n g a s e r i e s o f q u a d r a t i c e q u a t i o n s as d e s c r i b e d p r e v i o u s l y (Akyempon and R o u f o g a l i s 1981) and checked d i r e c t l y by a c a l c i u m s e l e c t i v e e l e c t r o d e a t a p p r o p r i a t e pH ( 6 . 9 or 7.2) as p r e v i o u s l y d e s c r i b e d ( A l - J o b o r e and R o u f o g a l i s 1981). Dodge 'ghosts' ( 0 . 1 8 mg p r o t e i n / 0 . 6 ml a s s a y ) , T r i t o n X-100 s o l u b i l i z e d ( C a 2 + + Mg24) -ATPase (0.02 mg p r o t e i n / 0 . 6 ml assay) o r p u r i f i e d ( C a 2 + + M g 2 + ) - A T P -ase ( 1 . 8 yg p r o t e i n / 0 . 6 ml assray) were assayed w i t h 2mM ATP ( T r i s -s a l t ) f o r 60 min (/ghosts') or 30 min ( . p u r i f i e d ( C a 2 + + M g 2 + ) - A T P a s e ) o r w i t h 20 TJM ATP ('ghosts') were assayed f o r 3 min and w i t h 0.5mM ATP 2+ 2+ ( T r i s - s a l t ) s o l u b i l i z e d CCa + Mg )-ATPase was assayed f o r 10 min a t 3 7 ° C . P u r i f i e d human e r y t h r o c y t e c a l m o d u l i n o r other e f f e c t o r s o f the enzyme were added to the enzyme p r e p a r a t i o n 10 min p r i o r t o s t a r t -i n g the r e a c t i o n w i t h ATP. 2+ Mg -ATPase a c t i v i t y was determined by measuring ATP h y d r o l y z e d i n the above i n c u b a t i o n medium i n the presence o f O.lmM ouabain but 2+ w i t h o u t added Ca and s u b t r a c t e d from the t o t a l ATPase a c t i v i t y t o 2 + + + o b t a i n the Ca s t i m u l a t a b l e a c t i v i t y . Ouabain s e n s i t i v e (Na + K ) -ATPase a c t i v i t y was measured i n a medium c o n t a i n i n g 55mM T r i s - m a l e a t e (pH 7 . 2 ) , 66mM N a C l , 5mM KC1, 2mM M g C l 2 , O.lmM EGTA w i t h o r w i t h o u t O.lmM ouabain and 2mM T r i s - A T P , and i n c u b a t e d f o r 60 min a t 3 7 ° C . 11) P h o s p h o d i e s t e r a s e assay P h o s p h o d i e s t e r a s e a c t i v i t y was determined by the method o f Sharma e t al_ ( 1 9 7 8 ) . The procedure i n v o l v e d c o u p l i n g o f the phospho-d i e s t e r a s e r e a c t i o n to a 5 ' - n u c l e o t i d a s e r e a c t i o n which was f o l l o w e d by a n a l y s i n g the r e s u l t i n g i n o r g a n i c phosphate by an automated F i s k e and Subbarow assay as d e s c r i b e d by Raess and V i n c e n z i ( 1 9 8 0 ) . C a l -modul in s t i m u l a t i o n o f the enzyme a c t i v i t y was measured a t 3 0 ° C , pH 7 . 5 , i n a r e a c t i o n m i x t u r e c o n t a i n i n g 40mM T r i s - H C l , 40mM i m i d a -z o l e , 5mM M g C l 2 » 1.2mM c y c l i c AMP, 0.05 u n i t s o f p h o s p h o d i e s t e r a s e , 2+ 0 . 3 u n i t s o f 5 ' - n u c l e o t i d a s e and e i t h e r O.lmM Ca and p r o t e i n k i n a s e i n h i b i t o r ( o r c a l m o d u l i n ) o r O.lmM EGTA ( n o n - a c t i v a t e d a c t i -v i t y ) . The r e a c t i o n was a l l o w e d t o proceed f o r 15 min and stopped by the a d d i t i o n o f SDS to a f i n a l c o n c e n t r a t i o n o f 2%. I n o r g a n i c phos-phate r e l e a s e d was determined as d e s c r i b e d above. 12) P r o t e i n k i n a s e assay I n h i b i t i o n o f p r o t e i n k i n a s e by p r o t e i n k i n a s e i n h i b i t o r was determined w i t h e i t h e r p r o t e i n k i n a s e and c y c l i c AMP o r w i t h f r e e c a t a l y t i c s u b u n i t . The r e a c t i o n m i x t u r e c o n t a i n e d 24 mg/ml h i s t o n e i n 50mM MES, pH 6 . 9 , 5 yM c y c l i c AMP, 5 yg c y c l i c AMP p r o t e i n k i n a s e ( o r 5 yg p r o t e i n k i n a s e c a t a l y t i c s u b u n i t ) , 6.5mM M g C ^ , O.lmM EGTA, 5mM DTT, and [y- P] ATP (100 dpm/pmol) a t a f i n a l ATP c o n c e n t r a t i o n o f 100 yM i n a t o t a l volume o f 200 y l . The r e a c t i o n m i x t u r e was i n c u b a t e d f o r 10 min i n a s h a k i n g water bath a t 3 0 ° C . The r e a c t i o n was t e r m i n a t e d by the a d d i t i o n o f 4 ml o f i c e c o l d 5% TCA and 0.25% sodium t u n g s t a t e , pH 2 , f o l l o w e d immediately by 0.2 ml o f 0.63% bo-v i n e serum albumin as a c a r r i e r p r o t e i n . The m i x t u r e was a l l o w e d to s t a n d f o r 5 min i n i c e and c e n t r i f u g e d f o r 15 m i n . The s u p e r n a t a n t was c a r e f u l l y removed by a s p i r a t i o n and the p r e c i p i t a t e d i s s o l v e d i n 0.1 ml o f 1 N NaOH, f o l l o w e d by 2 ml o f 5% TCA-0.25% sodium t u n g s t a t e , pH 2 . The p r o t e i n was r e p r e c i p i t a t e d by adding 1.1 N ^ S O ^ . The p r e c i p i t a t e was washed two more t imes as above and f i n a l l y d i s s o l v e d i n 0.1 ml o f 1 N NaOH. The r a d i o a c t i v i t y i n t h i s sample was measured i n a l i q u i d s c i n t i l l a t i o n c o u n t e r . 13) SDS Polyacry!amide Gel E l e c t r o p h o r e s i s P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s was performed i n a gel s l a b e l e c t r o p h o r e s i s system. The procedure used was e s s e n t i a l l y as o u t -l i n e d hy Weber and Osborn Cl969), by p o l y a c r y l a m i d e gel e l e c t r o p h o r e -s i s i n the presence o f the a n i o n i c d e t e r g e n t sodium dodecyl s u l f a t e CSDS). a) P r e p a r a t i o n of p r o t e i n s o l u t i o n s : To 20 y l o f the p r o t e i n sample 0 2 - 1 0 yg ) was added 30 y l o f the sample b u f f e r c o n t a i n i n g ( i n a f i n a l c o n c e n t r a t i o n ) 62.5mM T r i s - C l (pH 6 . 8 ) , 3% SDS, 10% g l y c e r o l , 5% B-mercaptoethanol 0.001% bromo-phenol b l u e . The sample was b o i l e d f o r 2 min and c o o l e d . A s t a n d a r d m o l e c u l a r weight p r o t e i n s o l u t i o n (Pharmacia) was t r e a t e d s i m i l a r l y and used to c a l i b r a t e the g e l s . b) P r e p a r a t i o n o f g e l s : The ' r e s o l v i n g ' or ' running' gel (12.5%) was prepared by m i x i n g 1 0 . 5 ml o f 1.5M T r i s - C l (pH 8 . 8 ) , 0.42 ml o f 10% SDS, 1 7 . 5 ml 30% a c r y l a m i d e , 0.8% m e t h y l e n e - b i s a c r y l a m i d e and 6.4 ml d i s t i l l e d w a t e r . A f t e r degassing f o r 5 m i n , 2 . 9 4 mis o f 0.5% ammonium p e r s u l f a t e and 4 . 2 mis o f 0.5% t e t r a e t h y l m e t h y l e n e - d i a m i n e (TEMED) were added. 42 ml o f t h i s gel was s u f f i c i e n t to produce good r e s o l u t i o n o f the p r o t e i n bands. A f t e r m i x i n g , t h i s s o l u t i o n was immediately poured i n t o the gel chamber c a r e f u l l y w i t h a 10 ml p i p e t t e . The gel was covered very c a r e f u l l y w i t h 2 ml o f 0.375 M T r i s - C l b u f f e r (pH 8 . 8 ) . The gel was a l l o w e d to p o l y m e r i z e f o r 12 hours a t room t e m p e r a t u r e . A f t e r t h i s t ime the ' b u f f e r ' was c a r e f u l l y removed w i t h a Pasteur p i p e t t e . The ' s t a c k i n g g e l ' (.3%) was prepared by m i x i n g 1 ml o f 1.25 M T r i s - C l (pH 6 . 8 ) , 0.1 ml o f 10% SDS, 1 ml o f 30% a c r y l a m i d e - 0.8% methylene h i s - a c r y l a m i d e and 6.2 jnl o f d i s t i l l e d w a t e r . A f t e r degasing f o r 5 jni.n, 0.7 ml o f 0.5% ammonium p e r s u l f a t e and 1 ml o f TEMED were added. A f t e r m i x i n g , s t a c k i n g gel s o l u t i o n was poured w i t h a 5 ml p i p e t t e i n t o the gel chamber over the p o l y m e r i z e d r e s o l v i n g - g e l . This was f o l l o w e d by i n s e r t i n g a t e f l o n "comb" i n t o the s t a c k i n g gel making sure t h a t no a i r bubbles were t rapped between the comb's t e e t h and the s t a c k i n g - g e l s o l u t i o n . The s t a c k i n g - g e l was a l l o w e d to p o l y m e r i z e f o r a t l e a s t 12 h o u r s . Both chambers o f the e l e c t r o p h o r e s i s c e l l were f i l l e d w i t h T r i s - G l y c i n e b u f f e r (25mM Tris-192mM G l y c i n e ) (pH 8.3) c o n t a i n i n g 0.1% SOS and the "comb" was c a r e f u l l y removed. 25 - 50 pi o f the p r o t e i n sample was a p p l i e d to the gel w i t h a 50 pi Hamil ton s y r i n g e and run a t 400 V and 25 mA ( c o n -s t a n t c u r r e n t ) u n t i l the t r a c k i n g dye had moved a p p r o x i m a t e l y 10 cm. c) S t a i n i n g and D e s t a i n i n g : The g e l s were s t a i n e d w i t h 0.2% Coomassie B r i l l i a n t B l u e i n 50% t r i c h l o r o a c e t i c a c i d f o r 15 min a t room t e m p e r a t u r e . The g e l s were then washed w i t h d i s t i l l e d water and d e s t a i n e d w i t h 7.5% a c e t i c a c i d and 5% methanol u n t i l the background was t r a n s p a r e n t . To photograph the g e l , the gel was soaked i n 1% g l y c e r o l - 10% a c e t i c a c i d s o l u t i o n f o r 30 m i n . I t was t r a n s f e r r e d onto a p l a i n g l a s s p l a t e and any excess l i q u i d was removed. RESULTS AND DISCUSSION S e c t i o n I. M e c h a n i s t i c study o f c a l m o d u l i n as an a c t i v a t o r o f C a 2 +  s e n s i t i v i t y and maximum v e l o c i t y o f ( C a 2 * + Mq2"1")- ATPase 1. K i n e t i c study o f C a + a c t i v a t i o n o f ( C a 2 * + M g 2 + ) - A T P a s e and  i t s modulat ion by c a l m o d u l i n . Calc ium a c t i v a t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e i n 'Dodge ghosts' and T r i t o n X-100 s o l u b i l i z e d enzyme p r e p a r a t i o n s was b i p h a s i c when the enzyme a c t i v i t y was measured i n the absence o f c a l m o d u l i n ( F i g s . 1 and 2 ) . The k i n e t i c s o f c a l c i u m a c t i v a t i o n can be d e s c r i b e d by assuming a mixed popu-l a t i o n of high and low c a l c i u m a f f i n i t y s t a t e s o f the enzyme ( A l - J o b o r e and R o u f o g a l i s 1 9 8 1 ) . Calmodul in i n c r e a s e d both the apparent C a 2 + a f f i n i t y and maximum v e l o c i t y of the ( C a 2 + + M g 2 + ) - A T P a s e i n ghosts ( F i g . 1) and T r i t o n X-100 s o l u b i l i z e d ( C a 2 + + M g 2 + ) - A T P a s e ( F i g . 2 ) . In the presence o f c a l m o d u l i n , both enzyme p r e p a r a t i o n s d i s p l a y e d s i n g l e h i g h a f f i n i t y C a 2 + a c t i v a t i o n k i n e t i c s . The a b i l i t y o f c a l m o d u l i n to a c t i v a t e ( C a 2 + + Mg 2 + )ATPase o f e r y t h r o c y t e membranes was l o s t upon c a r -boxymethyl a t i o n o f c a l m o d u l i n ( A l - J o b o r e and R o u f o g a l i s , unpubl ished o b s e r v a t i o n ) . Moreover , carboxymethylated c a l m o d u l i n a l s o showed reduced a c t i v a t i o n p r o p e r t i e s on another c a l m o d u l i n s e n s i t i v e enzyme, phosphodi-e s t e r a s e (Gagnon et a l . 1 9 8 1 ) . These o b s e r v a t i o n s suggested t h a t the a n i o n i c nature o f c a l m o d u l i n due to the presence o f several f r e e carboxy l groups may be important i n the r e g u l a t i o n o f c a l m o d u l i n s e n s i t i v e enzymes. cm t (3 3 -LogCC^reetM) 2+ 2+ F i g u r e 1. Calmodul in a c t i v a t i o n of (Ca + Mg )-ATPase a c t i v i t y i n human e r y t h r o c y t e membranes (Dodge G h o s t s ) . The enzyme a c t i -v i t y was measured i n t h e absence (•) and i n the presence (o) o f c a l m o d u l i n (2 yg/0.6 m l ) . The i n c u b a t i o n medium c o n t a i n e d 55 mM T r i s - m a l e a t e , pH 7 . 2 , 0.1 mM EGTA, 0.1 mM o u a b a i n , 6.5 mM M g C I £ » 66 mM NaCl and v a r i o u s c o n c e n t r a t i o n s o f f r e e c a l c i u m . The enzyme a c t i v i t y was assayed witff 2 mM T r i s - A T P f o r 60 min at 3 7 ° C . TJie r e s u l t s shown are means ± S . E . M , f o r f o u r s e p a r a t e e x p e r i m e n t s . P < 0.01 compared t o c o n t r o l at a l l C a 2 + c o n c e n t r a t i o n s . T -LogCCa?*]^ (M) 2+ F i g u r e 2 . Calmodul in a c t i v a t i o n o f T r i t o n X-100 s o l u b i l i z e d (Ca + 2+ Mg )-ATPase a c t i v i t y . The enzyme a c t i v i t y was measured i n the absence (•) and i n the presence (o) o f c a l m o d u l i n (2 yg/0.6 m l ) . The i n c u b a t i o n medium was i d e n t i c a l t o t h a t d e s c r i b e d i n F i g u r e 1. The enzyme a c t i v i t y was assayed w i t h 0.5 mM ATP f o r 15 min at 3 7 ° C . The r e s u l t s shown are means ± S . E . M . f o r at. l e a s t t h r e e s e p a r a t e e x p e r i m e n t s . P < 0 . 0 1 , compared to c o n t r o l . 2. E f f e c t o f p o l y - L - c a r b o x y l i c a c i d s and o t h e r anions on the C a 2 +  a c t i v a t i o n o f ( C a 2 + + Mg 2 +)-ATPase To study the e f f e c t o f f r e e carboxyl groups and the a n i o n i c nature o f c a l m o d u l i n i n r e g u l a t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e , c e r t a i n p o l y - a n i o n i c and a n i o n i c compounds were s t u d i e d . The ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y i n e r y t h r o c y t e membranes was a c t i v a t e d at low c a l c i u m c o n c e n t r a t i o n s and s h i f t e d to a predominantly h igh a f f i n i t y form by p o l y - L - a s p a r t i c a c i d ( M r = 20,000) and p o l y - L - g l u t a m i c a c i d ( M r = 20,000 and M r = 26,000) ( F i g . 3 ) . In an attempt to d e l i n e a t e the mechanism and k i n e t i c s o f the a c t i v a t i o n by these p o l y a n i o n s on the ( C a 2 + + M g 2 + ) - A T P a s e i n e r y t h r o c y t e membranes, t h e i r e f f e c t ( s ) on T r i t o n X-100 s o l u b i l i z e d ( C a 2 + + M g 2 + ) - A T P a s e was s t u d i e d . It was shown t h a t p o l y - L - a s p a r t i c a c i d ( M r = 20,000) and p o l y - L - g l u t a m i c a c i d s ( M r = 20,000 and 2 6 , 0 0 0 ) i n c r e a s e d the detergent s o l u b i l i z e d ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t i e s as w e l l , by a f f e c t i n g the sen-s i t i v i t y of the pump f o r C a 2 + ( F i g . 4 ) . An i n c r e a s e i n the apparent c a l c i u m s e n s i t i v i t y of ( C a 2 + + M g 2 + ) -ATPase was observed not o n l y i n the presence o f p o l y - L - c a r b o x y l i c a c i d s but a l s o i n the presence of c e r t a i n s u l f o n i c a c i d s , i n c l u d i n g sodium-HEPES (55 mM) or sodium-MES (55 mM) i n e r y t h r o c y t e ghosts ( F i g . 5 ) . In the absence o f s u l f o n i c a c i d s b i p h a s i c C a 2 + a c t i v a t i o n of the enzyme was observed i n 55 mM T r i s - m a l e a t e , 55 mM T r i s - H C l o r 55 mM sodium m a l e a t e , pH 7.2 ( F i g . 5 ) . The s u l f o n i c a c i d s a l s o i n c r e a s e d the C a 2 + s e n s i t i v i t y o f the T r i t o n X - 1 0 0 s o l u b i l i z e d enzyme ( F i g . 6 ) . In T r i s - m a l e a t e b u f f e r the C a 2 + a c t i v a t i o n was best f i t t e d to the equat ion f o r b i p h a s i c C a 2 + a c t i v a t i o n d e s c r i b e d p r e v i o u s l y ( A l - J o b o r e and R o u f o g a l i s , 1981) and i n Table I, w i t h a o f 2 . 2 ± 0 . 7 uM f o r the high C a 2 + a f f i n i t y component and a K,j o f 40 uM or more f o r the low C a 2 + a f f i n i t y component. >» 5 6 5 4 2+ F i g u r e 3 . E f f e c t o f p o l y - L - c a r b o x y l i c a c i d s on Ca a c t i v a t i o n o f human 2+ 2+ e r y t h r o c y t e membrane (Ca + Mg )-ATPase a c t i v i t y . The enzyme a c t i v i t y was measured i n a medium c o n t a i n i n g 55 mM T r i s -m a l e a t e , pH 7 . 2 , 0.1 mM EGTA, 0.1 mM o u a b a i n , 6.5 mM M g C l 2 , 66 mM N a C l , 2 mM ATP and v a r i o u s c o n c e n t r a t i o n s o f f r e e c a l c i u m as i n d i c a t e d . Enzyme a c t i v i t y was measured i n the absence (•) and i n the presence of 81 yM p o l y - L - a s p a r t i c a c i d , M f = 20,000 57 yM p o l y - L - g l u t a m i c a c i d , M = 20,000 (•), and 44 yM p o l y - L - g l u t a m i c a c i d , M = 2 6 , 0 0 0 (•). The r e s u l t s shown are means ± S . E . M . o f at l e a s t 3 s e p a r a t e e x p e r i m e n t s . *P < 0.05 compared t o c o n t r o l . 6 5 4 - L o g C C a 2 + 3 f r e e ( M ) 2+ F i g u r e 4. E f f e c t of p o l y - L - c a r b o x y l i c a c i d s on Ca a c t i v a t i o n o f 2+ 2+ (Ca + Mg )-ATPase i n T r i t o n X-100 s o l u b i l i z e d enzyme from human e r y t h r o c y t e membranes. The enzyme a c t i v i t y was measured i n the absence (•) and i n the presence of 81 yM p o l y - L - a s p a r t i c a c i d , M = 20,000 (*), 57 yM p o l y - L - g l u t a m i c a c i d , M 4 = 20,000 (•), and 44 yM p o l y - L - g l u t a m i c a c i d , M f = 26,000 (•). The assay medium was i d e n t i c a l to t h a t i n F i g u r e 3 except t h a t the c o n c e n t r a t i o n o f ATP was 0 . 5 mM and the assay t ime 10 m i n . Data r e p r e s e n t the means o f three s e p a r a t e e x p e r i m e n t s . re £. CO & I is CO o .c ~ E + CM co o E a. + C M re O - L o g C C a 2 + J f r e e ( M ) 2+ ure 5. E f f e c t o f s u l f o n i c a c i d anions on the Ca a c t i v a t i o n o f 2+ 2+ (Ca + Mg )-ATPase i n human e r y t h r o c y t e membranes. Ghosts were assayed i n 55 mM T r i s - maleate b u f f e r (•), 55 mM T r i s - H C l (•), 55 mM sodium-maleate (•), 55 mM sodium-HEPES (^) and 55 mM sodium-MES ( A ) . The pH o f the assay m i x t u r e s was 6.9 a t 3 7 ° C . The assay m i x t u r e c o n t a i n e d the b u f f e r (as i n d i c a t e d , 0.1 mM EGTA, 0.1 mM o u a b a i n , 66 mM N a C l , 6.5 mM M g C l 2 , and v a r i o u s c o n c e n t r a t i o n s o f f r e e c a l c i u m . Data r e p r e s e n t the means o f three s e p a r a t e e x p e r i m e n t s . T F i g u r e 6A. E f f e c t o f s u l f o n i c a c i d a n i o n s i n the presence and absence o f 2+ 2+ 2+ c a l m o d u l i n on Ca a c t i v a t i o n of (Ca + Mg )-ATPase i n 2+ 2+ T r i t o n X-100 s o l u b i l i z e d (Ca + Mg )-ATPase from human e r y t h r o c y t e membranes. The s o l u b i l i z e d enzyme was assayed i n 55 mM T r i s - m a l e a t e b u f f e r (•), 55 mM sodium-HEPES (*), 55 mM sodium-KES ( A ) and 55 mM sodium-HEPES p l u s 0.6 ug c a l m o d u l i n / 0 . 6 ml ( o ) . The pH o f the a s s a y m i x t u r e was 7.2 at 3 7 ° C . The o t h e r components o f the a s s a y m i x t u r e were i d e n t i c a l t o those shown i n F i g u r e 5 . Each p o i n t r e p r e s e n t s the means o f f o u r e x p e r i m e n t s . An a n a l y s i s o f v a r i a n c e was performed (see Appendix I ) , V/Ca2H(xiO) (n moles Pj. mg-"1 mirr1) LCa 2 +3 OjM) F i g u r e 6B. The curves i n F i g u r e 6A were f i t t e d by a n o n - l i n e a r c u r v e -f i t t i n g computer program (see t e x t and Table I ) . Enzyme a c t i v i t y i n T r i s - m a l e a t e ( o ) , Na-HEPES( A ) and c a l m o d u l i n i n Na-HEPES (•). In sodium-HEPES o r sodium-MES (55 mM i n anion) the C a 2 + a c t i v a t i o n was b e s t f i t t e d by a s i n g l e high C a 2 + a f f i n i t y component (K^ = 0.35 ± 0.08 yM). F i g . 6B shows the f i t o f the data by a computer generated non-l i n e a r curve f i t t i n g program, i n the form o f E a d i e - H o f s t e e p l o t s . As i n g h o s t s , the maximum v e l o c i t y o b t a i n e d was not s i g n i f i c a n t l y d i f f e r e n t i n T r i s or s u l f o n i c a c i d b u f f e r s . The a d d i t i o n of c a l m o d u l i n (0.6 yg/0.6 ml assay medium) i n the presence o f sodium-HEPES b u f f e r i n c r e a s e d the maximum v e l o c i t y of the ( C a 2 + + M g 2 + ) - A T P a s e without f u r t h e r a f f e c t i n g i t s apparent C a 2 + a f f i n i t y (Kd = 0.3 ± 0.04 uM) ( F i g . 6 A , B ) . T h i s i s in c o n t r a s t to the e f f e c t o f c a l m o d u l i n i n T r i s - m a l e a t e b u f f e r , where oalmodulin i n c r e a s e s both the apparent C a 2 + a f f i n i t y and the maximum v e l o c i t y of ( C a 2 + + Mg^"1")-ATPase, both in ghosts and s o l u b i l i z e d ( C a 2 + + Mg2 + )-ATPase p r e p a r a t i o n s ( F i g u r e s 1 and 2 ) . The s h i f t i n C a 2 + a f f i n i t y o f the enzyme i n the presence of sodium-HEPES or sodium-MES cannot be a t t r i b u t e d to a change i n the concen-t r a t i o n of sodium (the counter i o n i n the s u l f o n i c a c i d b u f f e r s ) , because no a c t i v a t i o n was o b t a i n e d i n sodium maleate b u f f e r at a s i m i l a r sodium c o n c e n t r a t i o n (see F i g . 5 ) . In a d d i t i o n , d e l e t i o n o f sodium c h l o r i d e from the assay medium i n the presence o f T r i s - m a l e a t e b u f f e r d i d not a l t e r the b i p h a s i c C a 2 + k i n e t i c p a t t e r n observed in 'ghosts' or s o l u b i l i z e d pre-p a r a t i o n s ( r e s u l t s not shown). It i s a lso u n l i k e l y that the low C a 2 + a f f i n i t y component r e s u l t e d from the presence o f T r i s , as i t was a l s o seen i n sodium maleate b u f f e r . The f a c t t h a t these anions s h i f t e d the C a 2 + a f f i n i t y o f s o l u b i l i z e d enzyme p r e p a r a t i o n s suggests t h a t they act d i r e c t -l y on the C a 2 + - t r a n s p o r t ATPase complex, r a t h e r than by an i n d i r e c t e f f e c t on membrane s t r u c t u r e . In support o f t h i s was the f i n d i n g t h a t t h e anions a l s o a c t i v a t e d a p r e p a r a t i o n o f p u r i f i e d ( C a 2 + + M g 2 + ) - A T P a s e i n a s o l e c t i n , o b t a i n e d from Dr. J . T . P e n n i s t o n , Mayo C l i n i c , Minnesota ( r e s u l t s not shown). Fur thermore, to t e s t the p o s s i b i l i t y t h a t p o l y a n i o n s may have s h i f t e d the a f f i n i t y o f the ( C a 2 + + M g 2 + ) - A T P a s e i n d i r e c t l y by m o b i l i z i n g t i g h t l y bound c a l m o d u l i n ( i . e . c a l m o d u l i n not removed by hypo-t o n i c l y s i s i n the absence o f C a 2 + ) , membranes were preincubated w i t h p o l y a n i o n s and washed t w i c e w i t h 20 mM sodium phosphate, pH 7.4 to remove any l o o s e l y bound c a l m o d u l i n . The washed membranes s t i l l r e t a i n e d t h e i r s e n s i t i v i t y to p o l y a n i o n s t i m u l a t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e , s i m i l a r to the u n t r e a t e d membranes ( r e s u l t s not shown). It was determined t h a t n e i t h e r p o l y a n i o n s nor s u l f o n i c a c i d s a l t e r e d the c a l c i u m s e n s i t i v i t y by a l t e r i n g the s e n s i t i v i t y o f the enzyme to MgCl2 o r ATP ( r e s u l t s not shown). To study the nature of anion a f f e c t s on the modulat ion of the Ca2"1" s e n s i t i v i t y of the c a l c i u m t r a n s p o r t ATPase, v a r i o u s c l a s s e s of anions were used (Table I ) . Of the range o f i n o r g a n i c and o r g a n i c anions t e s t e d , o n l y the aromatic c a r b o x y l i c a c i d s , benzoic a c i d and s a l i c y l i c a c i d , i n c r e a s e d the c a l c i u m s e n s i t i v i t y and s h i f t e d the b i p h a s i c k i n e t i c s t o a predominantly s i n g l e high C a 2 + a f f i n i t y s t a t e (Table I, F i g . 7 ) . A l i p h a t i c c a r b o x y l i c and s u l f o n i c a c i d s (sodium g l u c o n a t e , h i p p u r a t e , a s p a r t a t e , g lutamate and TES) were i n e f f e c t i v e at the same c o n c e n t r a t i o n s (Table I ) . S i m i l a r l y , none o f the i n o r g a n i c anions t e s t e d , which i n c l u d e both water s t r u c t u r e 'toakers" and "breakers", i n c r e a s e d the C a 2 + s e n -s i t i v i t y of the enzyme at 66 mM, w h i l e F" and to a l e s s e r extent NO3" s l i g h t l y i n h i b i t e d the enzyme a c t i v i t y (Table I ) . In an attempt to i n v e s t i g a t e the s i t e and mechanism o f a c t i o n o f t h e s e anions and p o l y a n i o n s i n r e l a t i o n to c a l m o d u l i n , two types of exper-iments were performed. F i r s t l y , the enzyme was t r e a t e d w i t h t r y p s i n . T r y p s i n t reatment o f ghosts i n c r e a s e d both the maximum v e l o c i t y and the c a l c i u m s e n s i t i v i t y of the ( C a 2 + + M g 2 + ) - A T P a s e ( F i g . 8 ) , probably by 2+ E f f e c t o f a n i o n s on the Ca s e n s i t i v i t y o f T r i t o n X-100 s o l u b i l i z e d 2 + 2 + (Ca +Mg )-ATPase. The assay-medium c o n t a i n e d 55 mM T r i s - m a l e a t e (pH 7.2 at 3 7 ° C ) , 0.1 mM EGTA, 6.5 mM MgCl 2mM, 0.1 mM o u a b a i n , v a r i o u s c o n c e n t r a t i o n s o f f r e e c a l c i u m and 66 mM i n a n i o n , t e s t e d as the sodium 2+ s a l t s . The k i n e t i c s o f the Ca a c t i v a t i o n were examined over a range 2+ o f Ca c o n c e n t r a t i o n s i n experiments s i m i l a r t o those shown i n F i g . 2 . 2+ 2+ The Ca a c t i v a t i o n was f i t t e d t o t h e e q u a t i o n f o r b i p h a s i c Ca a c t i -v a t i o n V l t C a 2 + ] 2 + J K ? 1 [Ca ] K1 + r c a ^ + ] 1 2+ where V-| and K-j are the maximum v e l o c i t y and Ca d i s s o c i a t i o n c o n s t a n t 2+ ^ of t h e high Ca a f f i n i t y component and K ? ' = - 2 - ( the c o r r e s p o n d i n g 2+ 2+ maximum v e l o c i t y and Ca d i s s o c i a t i o n c o n s t a n t o f the low Ca a f f i n i t y component). The c r i t e r i o n used f o r d e c i d i n g whether the k i n e t i c s f i t t e d the b i p h a s i c a c t i v a t i o n o r a h y p e r b o l i c f u n c t i o n ( l e f t hand term of e q u a t i o n o n l y ) was the c o r r e l a t i o n c o e f f i c i e n t o f the f i t ( r ) and the magnitude o f t h e standard d e v i a t i o n o f t h e e s t i m a t e s of the c o n s t a n t s . V-| i s i n nmoles Pyminper mg p r o t e i n and K-j i s in yM. K2" i s the r a t i o o f V 2 and K 2 -2+ K i n e t i c s o f Ca A c t i v a t i o n Anion C a 2 K i n e t i c Constants A c t i v a t i o n ^1 ^1 ^2* CI" B i p h a s i c 322 + 27 2 . 2 + 0.7 0.30 + 0,02 B r ' B i p h a s i c 315 + 25 2.1 + 0 ,6 0.32 + 0.022 r B i p h a s i c 320 + 26 2.1 + 0 . 6 5 0,30 + 0.02 F" B i p h a s i c 308 + 18 2 . 5 + 0.72 0.27 + 0.019 2 -SO4 Bi phasic 315 + 26 2 . 3 + 0.66 0,29 + 0.02 C 0 3 2 - B i p h a s i c 318 + 29 2.1 + 0 , 6 5 0.29 + 0.02 HCO3" B i p h a s i c 315 + 25 2.1 + 0.66 0,33 + 0.03 CH 3C00" B i p h a s i c 320 + 26 2.3 + 0.67 0,30 + 0,025 NO3- B i p h a s i c 305 + 20 2 . 6 + 0.78 0.26 + 0,023 Gluconate" B i p h a s i c 325 + 26 2 . 3 + 0 . 6 9 0.29 + 0.03 H i p p u r a t e " Bi phasic 318 + 18 2.2 + 0.71 0,31 + 0,032 S a l i c y l a t e " Hyperbo1ic 340 + 32 0.60 + 0.08 — Benzoate" H y p e r b o l i c 330 + 29 0.62 + 0.08 --A s p a r t a t e " B i p h a s i c 315 + 23 2.2 + 0.08 0,29 + 0.021 Glutamate" B i p h a s i c 320 + 26 2 , 0 + 0.85 0.28 + 0.019 Taur ine* Bi phasic 315 + 25 2 . 2 + 0.68 0.30 + 0.022 TES* Bi p h a s i c 309 + 18 2.1 + 0.71 0,32 + 0.023 •Taurine and *TES were used a t a c o n c e n t r a t i o n o f 55 mM I _ 400 6 5 4 - L o g C C a P ^ t M ) Figure 7. E f f e c t o f i n o r g a n i c anions and a r o m a t i c c a r b o x y l i c a c i d s on C a 2 + a c t i v a t i o n o f T r i t o n X-100 s o l u b i l i z e d ( C a 2 + + M g 2 + ) -2+ 2+ ATPase from human e r y t h r o c y t e s . (Ca + Mg )-ATPase a c t i v i t y was assayed i n a medium c o n t a i n i n g 55 mM T r i s - m a l e a t e , pH 7 . 2 , 0.1 mM EGTA, 0.1 mM o u a b a i n , 6.5 mM M g C l 2 » v a r i o u s c o n -c e n t r a t i o n s o f f r e e c a l c i u m and 66 mM NaCl (•), 66 mM sodium b i c a r b o n a t e (•), 66 mM sodium carbonate (*), 66 mM sodium benzoate (A) or 66 mM sodium s a l i c y l a t e (•). Data r e p r e s e n t the means o f at l e a s t three s e p a r a t e e x p e r i m e n t s . 6 5 - L o g C C a 2 + D f r e e (M) F i g u r e 8 . E f f e c t o f p o l y - L - c a r b o x y l i c a c i d s and s u l f o n i c a c i d s on 2+ 2+ t r y p s i n t r e a t e d membranes, (Ca + Mg )-ATPase a c t i v i t y o f n o n - t r y p s i n t r e a t e d ( c o n t r o l ) membranes was assayed i n T r i s - m a l e a t e b u f f e r (55 mM) (•). T r y p s i n - t r e a t e d membranes were assayed i n T r i s - m a l e a t e (55 mM) (o) o r sodium-HEPES (55 mM) (A ) . In the presence o f 56 uM p o l y - L - a s p a r t i c a c i d ( M = 20 000) ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y was assayed before (•) and a f t e r t r y p s i n t reatment (•) i n T r i s - m a l e a t e (55 mM). The data shown i s t y p i c a l o f three s i m i l a r e x p e r i m e n t s . c l e a v i n g the c a l m o d u l i n b i n d i n g s i t e on the (Ca2"1" + M g 2 + ) - A T P a s e (Sarkadi et a l 1980; Enyedi et a l 1980; N i g g l i et a l 1 9 8 1 ) . A d d i t i o n o f e i t h e r p o l y - L - a s p a r t i c a c i d or sodium-HEPES to the t r y p s i n t r e a t e d membranes f a i l e d to f u r t h e r a f f e c t the C a 2 + s e n s i t i v i t y or a c t i v i t y of the enzyme compared to t h a t i n T r i s - m a l e a t e b u f f e r a l o n e . These r e s u l t s s u g -gest t h a t pretreatment o f the membrane w i t h t r y p s i n l e a d s to a change i n the enzyme s i m i l a r to t h a t produced by b i n d i n g o f the a n i o n s . However, u n l i k e a n i o n s , but s i m i l a r l y to c a l m o d u l i n , t r y p s i n t reatment has the f u r -t h e r e f f e c t o f i n c r e a s i n g the maximum v e l o c i t y o b t a i n e d . While these r e s u l t s show t h a t the anions t e s t e d have s i m i l a r e f f e c t s to c a l m o d u l i n and t r y p s i n t r e a t m e n t , i t cannot be concluded from the k i n e t i c a n a l y s i s t h a t the v a r i o u s anions and c a l m o d u l i n n e c e s s a r i l y bind at the same s i t e . S e c o n d l y , the e f f e c t o f t r i f l u o p e r a z i n e on the a c t i v a t i o n by a n i o n s was examined, s i n c e i t i s known to b l o c k the c a l m o d u l i n a c t i v a t i o n of ( C a 2 + + M g 2 + ) - A T P a s e . Whereas 30 uM t r i f l u o p e r a z i n e blocked ( C a 2 + + M g 2 + ) - A T P a s e a c t i v a t i o n by p o l y - L - a s p a r t i c a c i d , i t d id not antagon-i z e the e f f e c t o f sodium-HEPES on the enzyme a c t i v i t y ( F i g . 9 ) . It i s not known at present i f t r i f l u o p e r a z i n e i n h i b i t s the p o l y a n i o n a c t i v a t i o n of ( C a 2 + + M g 2 + ) - A T P a s e by b i n d i n g to the p o l y a n i o n through i o n i c i n t e r -a c t i o n i n the absence or presence o f c a l c i u m or to the enzyme. U n t i l the mechanism o f the c a l m o d u l i n blockade by t r i f l u o p e r a z i n e i s e s t a b l i s h e d , i t i s not p o s s i b l e to analyse the p r e c i s e mechanism by which p o l y - L - c a r b o x y -1 i c a c i d s and s u l f o n i c a c i d s a l t e r the C a 2 + s e n s i t i v i t y o f the ( C a 2 + + M g 2 + ) - A T P a s e . These s t u d i e s suggest t h a t d i s t i n c t s t r u c t u r a l f e a t u r e s of c a l m o d u l i n may c o n t r i b u t e to the i n c r e a s e i n the C a 2 + s e n s i t i v i t y and maximum v e l o c i t y of ( C a 2 + + M g 2 + ) - A T P a s e . The r e s u l t s o b t a i n e d w i t h p o l y - a n i o n s and some o f the o t h e r anions examined suggest t h a t the a c i d i c nature o f Figure 9. E f f e c t o f t r i f l u o p e r a z i n e on human e r y t h r o c y t e membrane 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y i n the presence o f p o l y - L -c a r b o x y l i c a c i d s or sodium-HEPES b u f f e r . The enzyme a c t i v i t y was measured i n sodium-HEPES b u f f e r e i t h e r i n the absence (n) or i n the presence o f 30 yM t r i f l u o p e r a z i n e (•). The enzyme was assayed i n 55 mM T r i s - m a l e a t e b u f f e r (•) and i n the presence of 56 yM p o l y - L - a s p a r t i c a c i d (M = 20,000) w i t h o u t t r i f l u o p e r a z i n e (A) o r w i t h 30 yM t r i f l u o p e r a z i n e (*). The data shown i s t y p i c a l of t h r e e s i m i l a r e x p e r i m e n t s . c a l m o d u l i n may be i n v o l v e d i n a l t e r i n g the C a 2 + s e n s i t i v i t y ( K - a c t i v a -t i o n ) . The mechanism o f K - a c t i v a t i o n by the anions may be through a l l o -s t e r i c modulat ion o f the enzyme conformation to a high C a 2 + a f f i n i t y s t a t e , through a r e g u l a t o r y s i t e on the enzyme. I t was proposed t h a t c a l -modul in and phosphat idy l s e r i n e bind to o v e r l a p p i n g but d i s t i n c t r e g i o n s on the r e g u l a t o r y s i t e , thereby a l t e r i n g i t s c o n f o r m a t i o n , and removing the c o n s t r a i n t on C a 2 + a c t i v a t i o n o f the ( C a 2 + + M g 2 + ) - A T P a s e ( A l - J o b o r e and R o u f o g a l i s 1 9 8 1 ) . The a b i l i t y o f c a l m o d u l i n t o a l s o i n c r e a s e the maximum v e l o c i t y ( V - a c t i v a t i o n ) may be due to i t s hydrophobic n a t u r e , as demon-s t r a t e d by s i m i l a r (K + V) a c t i v a t i o n by c e r t a i n a c i d i c p h o s p h o l i p i d s ( A l - J o b o r e and R o u f o g a l i s 1 9 8 1 ; 1982) which are both n e g a t i v e l y charged and h y d r o p h o b i c . I t remains to be determined i f endogenous anions i n t h e red c e l l a f f e c t the s t a t e o f the c a l c i u m pump i n v i v o . S e c t i o n I I , E f f e c t o f c y c l i c AMP and c y c l i c AMP dependent p r o t e i n k i n a s e i n h i b i t o r s (PKI's) on ( C a 2 + + t )g 2 + )^ATPase a c t i v i t y The interdependency o f v a r i o u s c y c l i c AMP and c a l c i u m - c a l m o d u l i n dependent c e l l u l a r events i s not c l e a r l y u n d e r s t o o d . Calmodul in has been 2+ shown to s t i m u l a t e b r a i n a d e n y l a t e c y c l a s e , phosphodiesterase and (Ca + 2+ Mg )-ATPase enzymes, among o t h e r s (Cheung 1 9 8 0 ) . The c y c l i c AMP PKI's have been shown t o i n h i b i t p h o s p h o r y l a t i o n by c y c l i c AMP p r o t e i n k i n a s e s by i n t e r a c t i n g w i t h the f r e e c a t a l y t i c s u b u n i t s o f t h e s e enzymes (Ashby and Walsh 1 9 7 3 ) . They a l s o i n h i b i t c a l m o d u l i n - s t i m u l a t e d phos-p h o d i e s t e r a s e i n S e r t o l i c e l l s o f t e s t e s , which c o n t a i n both c a l m o d u l i n and PKI (Beale e_t al_. 1 9 7 7 ) . In the e r y t h r o c y t e plasma membrane, c y c l i c 2+ 2+ AMP has been shown under c e r t a i n c o n d i t i o n s t o i n h i b i t the (Ca + Mg ) -ATPase a c t i v i t y p r o b a b l y y_1_a an endogenous c y c l i c AMP p r o t e i n k i n a s e (Varghese and Cunningham 1 9 8 0 ) . I t was the purpose o f t h i s study t o c h a r a c t e r i z e the r o l e o f c y c l i c AMP dependent p r o t e i n k i n a s e p h o s p h o r y l a t -2+ 2+ ion on (Ca + Mg )-ATPase a c t i v i t y u s i n g the s p e c i f i c p r o t e i n i n h i b i t o r s o f c y c l i c AMP p r o t e i n k i n a s e s . Bovine h e a r t PKI from Sigma, f u r t h e r p u r i f i e d on a Whatman DE 52 column, was shown to be f r e e o f c a l m o d u l i n by a number o f c r i t e r i a (Table I I ) . Pure PKI d i d not s t i m u l a t e c a l m o d u l i n - d e f i c i e n t p h o s p h o d i e s t e r a s e , whereas the PKI p r e p a r a t i o n from Sigma before p u r i f i c a t i o n and pure c a l m o d u l i n s t i m u l a t e d t h i s enzyme. The absence of any s i g n i f i c a n t l e v e l s o f c a l m o d u l i n i n the p u r i f i e d PKI was conf i rmed by c a l m o d u l i n radioimmuno-assay ( 1 . 3 ng o f c a l m o d u l i n which was found per 2 ug o f PKI (Table I I ) , was shown to be w e l l below the t h r e s h o l d (50 ng) r e q u i r e d to s t i m u l a t e ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y ) . Moreover, 0.1% - S D S - 1 2 . 5 % - p o l y a c r y l a m i d e D e t e r m i n a t i o n o f c a l m o d u l i n tn v a r i o u s p r o t e i n p r e p a r a t i o n s , Calmodul in was e s t i m a t e d u s i n g the phosphodiesterase assay and c a l m o d u l i n r a d i o -immunoassay, as d e s c r i b e d 1n the t e x t . The phosphodiesterase a c t i v i t y -1 -1 was 100 nmoles P^  min mg p r o t e i n i n the absence of added p r o t e i n s . The c a l m o d u l i n c o n t e n t o f v a r i o u s p r e p a r a t i o n s i s expressed per 2 yg o f t o t a l p r o t e i n . P r e p a r a t i o n Phosphodiesterase a c t i v a t i o n (%) Calmodul in radioimmunoassay Bovine heart PKI (Sigma) 75% 75 "ng/2 y g Rabbit s k e l e t a l muscle PKI (Sigma) none 0.16 ng/2 yg P u r i f i e d bovine heart PKI none 1.33 ng/2 yg Calmodul in 230% 420 ng (400 ng) Calmodul in immunoassays were k i n d l y performed by Dr. C h a f o l e a s i n the l a b o r a t o r y o f Dr. Means, B a y l o r C o l l e g e o f M e d i c i n e , Texas. gel e l e c t r o p h o r e s i s showed a s i n g l e p r o t e i n band o f M f = 15,500 ( f i g . 1 0 ) . The p u r i f i e d p r o t e i n k i n a s e I n h i b i t o r s from bovine heart and r a b b i t s k e l e -t a l muscle were used i n f u r t h e r s t u d i e s . In agreement w i t h the r e p o r t o f Varghese and Cunningham (.1980), I t was found t h a t c y c l i c AMP (5 yM) produced an i n h i b i t i o n (approx. 20%) o f t h e ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y 1n 'Dodge g h o s t s ' assayed a t 20 yM ATP ( F i g . 1 1 ) . C y c l i c AMP (5 y M ) , however, had no e f f e c t on the enzyme a c t i -v i t y when i t was assayed a t 2 mM ATP ( r e s u l t s not shown). The i n h i b i t i o n 2+ 2+ o f (Ca + Mg )-ATPase a c t i v i t y produced by c y c l i c AMP was p r o b a b l y v i a c y c l i c AMP mediated p r o t e i n k i n a s e p h o s p h o r y l a t i o n , because t h e i n h i b i -t i o n o f ATPase a c t i v i t y produced by c y c l i c AMP c o u l d be a b o l i s h e d by r a b b i t s k e l e t a l muscle PKI ( F i g . 1 1 ) . However, when the e f f e c t o f c y c l i c 2+ 2+ AMP (5 yM) on (Ca + Mg )-ATPase a c t i v i t y was s t u d i e d i n the presence o f bovine heart P K I , i t was found t h a t the enzyme a c t i v i t y was s t i m u l a t e d approx. 100% i n the presence or absence o f c y c l i c AMP ( F i g . 1 1 ) . S i n c e the p r o t e i n k i n a s e i n h i b i t o r s a r e small a c i d i c p r o t e i n s (Ashby and Walsh 1973; Whitehouse and McPherson 1 9 8 0 ) , the e f f e c t ( s ) o f t h e s e p r o t e i n s on 2+ 2+ the k i n e t i c s o f (Ca + Mg )-ATPase was f u r t h e r i n v e s t i g a t e d and compared to t h a t o f c a l m o d u l i n . 2+ 2+ Bovine h e a r t PKI a l s o s t i m u l a t e d (Ca + Mg )-ATPase a c t i v i t y i n T r i t o n X-100 s o l u b i l i z e d p r e p a r a t i o n (assayed a t 0 , 5 mM ATP) ( F i g . 1 2 ) . 2+ 2+ However, whereas c a l m o d u l i n s t i m u l a t e d (Ca + Mg )-ATPase a c t i v i t y a t 2+ both low ( 0 . 5 8 yM) and high (55 yM) f r e e Ca , bovine heart PKI s t i m u l a t e d 2+ o n l y a t the low Ca c o n c e n t r a t i o n , both i n 'ghosts' ( F i g . 12) and i n T r i t o n X-100 s o l u b i l i z e d p r e p a r a t i o n s ( r e s u l t not shown). At 0.58 yM 2+ 2+ 2+ Ca bovine heart PKI s t i m u l a t e d (Ca + Mg )-ATPase i n the presence o f 2+ a s a t u r a t i n g l e v e l . (3 yg/0.6 ml) o f c a l m o d u l i n , w h i l e a t 55 yM Ca , PKI 69 Molecular Weight 94,000 67,000 43,000 30,000 21,000 14,000 F i g u r e 10. SDS-PAGE o f p r o t e i n k i n a s e i n h i b i t o r and c a l m o d u l i n . The p r o t e i n s were separated on a 12.5%-acry lamide-0.1%-SDS s l a b gel as d e s c r i b e d i n the Methods. The f o l l o w i n g p r o t e i n s were loaded on the gel ( s t a r t i n g from l e f t ) ; Lane 1, s tandard m o l e c u l a r weight p r o t e i n s , 3 yg each o f phosphorylase B ( M r = 9 4 , 0 0 0 ) , bovine serum albumin ( M r = 6 7 , 0 0 0 ) , ovalbumin (M 4 3 , 0 0 0 ) , c a r b o n i c anhydrase ( M r = 3 0 , 0 0 0 ) , soyabean t r y p s i n i n h i b i t o r (M = 21,000) and lysozyme (M = 1 4 , 3 0 0 ) ; Lane 2 , 10 yg calmo-d u l i n ; Lane 3 , 2 yg r a b b i t s k e l e t a l muscle PKI c o n t a m i n a n t ; Lane 4 , 10 yg PKI (bovine h e a r t ) ; Lane 5 , 5 ^ g PKI ( b o v i n e h e a r t ) ; Lane 6, 2 p g - c a l m o d u l i n . C o ? * C o n c e n t r a t i o n (M' ure 1 1 . E f f e c t o f c y c l i c AMP and PKI on (Ca + Mg )-ATPase a c t i v i t y i n human e r y t h r o c y t e membranes a t v a r i o u s 2+ 2+ c o n c e n t r a t i o n s o f f r e e c a l c i u m . (Ca + Mg )-ATPase was determined i n 'Dodge ghost' membranes i n the absence (*) and i n the presence o f 5 yM c y c l i c AMP (o), 5 yM c y c l i c AMP plus 2 yg/0.6 ml o f r a b b i t s k e l e t a l muscle PKI ( • ) » 2 yg/0.6 ml o f bovine heart PKI ( a ) , and 5 yM c y c l i c AMP p l u s 2 yg bovine heart PKI (•). ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y was measured a t 20 yM ATP f o r 3 m i n . The r e s t o f t h e e x p e r i m e n t a l procedure was the same as t h a t d e s c r i b e d i n the Methods. Each p o i n t r e p r e s e n t s the means o f d u p l i c a t e o r t r i p l i c a t e d e t e r m i n a t i o n s . The curves shown are t y p i c a l o f t h r e e s i m i l a r e x p e r i m e n t s . F i g u r e 1.2. E f f e c t of c a l m o d u l i n and bovine heart PKI on (Ca + Mg ) -ATPase a c t i v i t y i n e r y t h r o c y t e membranes and T r i t o n X-100 s o l u b i l i z e d enzyme a t 0.58 yM and 55 uM f r e e c a l c i u m . 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y i n 'Dodge ghost' membranes (DG) was measured a t 2 mM ATP f o r 1 h a t 37°C and s o l u b i l i z e d enzyme a c t i v i t y (SE) a t 0 . 5 mM ATP f o r 10 min ( f o r d e t a i l s o f i n c u b a t i o n m i x t u r e see M a t e r i a l s and Methods) .A,PKI (2 yg/0,6 m l ) ; B, Calmodul in (.3 pg/0.6 m l ) ; C, PKI (2 p g / 0 . 5 ml) + c a l m o d u l i n (3 yg/0.6 m l ) . Data shown i s t y p i c a l o f t h r e e s i m i l a r e x p e r i m e n t s . d i d not f u r t h e r s t i m u l a t e a.boye t h a t i n the presence o f c a l m o d u l i n ( F i g . 12) , 2+ 2+ The c o n c e n t r a t i o n - d e p e n d e n c e o f a c t i v a t i o n o f (Ca + Wg )-ATPase 2+ by bovine heart PKI and c a l m o d u l i n was i n v e s t i g a t e d a t 0.58 yM Ca ( F i g . 13) . Whi le the enzyme a c t i v i t y was a c t i v a t e d maximal ly a t a lower c o n c e n -t r a t i o n o f PKI (2 yg/0.6 ml) than o f c a l m o d u l i n (3 yg/0.6 m l ) , the maximum a c t i v a t i o n by c a l m o d u l i n was h i g h e r than t h a t o f PKI. The e f f e c t o f PKI 2+ 2+ 2+ on the k i n e t i c s of Ca a c t i v a t i o n o f (Ca + Mg )-ATPase was i n v e s t i g a -2+ ted ( F i g . 1 4 ) . In the absence o f the a c t i v a t o r s the Ca a c t i v a t i o n was 2+ b i p h a s i c , due to a mixed p o p u l a t i o n o f h igh and low Ca a f f i n i t y s t a t e s 2+ o f the enzyme ( A l - J o b o r e and R o u f o g a l i s 1981) . The K^  o f the high Ca 2+ a f f i n i t y i s approx. 2 yM and the K^  f o r the low Ca a f f i n i t y component i s 40 yM or more. In the presence o f e i t h e r c a l m o d u l i n or P K I , the low 2+ Ca a f f i n i t y component was v i r t u a l l y a b o l i s h e d , and the a c t i v a t i o n was f i t t e d by a s i n g l e high C a 2 + a f f i n i t y (K d = 0.4 yM) ( F i g . 1 4 ) . However, 2+ 2+ u n l i k e c a l m o d u l i n , PKI d i d not i n c r e a s e the v"m a x o f the (Ca + Mg ) -2+ ATPase a c t i v i t y . The s h i f t i n the Ca s e n s i t i v i t y o f the enzyme by PKI and c a l m o d u l i n and the i n c r e a s e i n V _ v by c a l m o d u l i n i s seen more c l e a r l y max i n E a d i e - H o f s t e e p l o t s o f the data ( F i g . 1 5 ) . 2+ The PKI from r a b b i t s k e l e t a l muscle d i d not s t i m u l a t e the (Ca + 2 + Mg )-ATPase ( F i g . 1 1 ) , even though i t s i s o - e l e c t r i c p o i n t and m o l e c u l a r weight are s i m i l a r t o the bovine heart PKI (Whitehouse ejt al_. 1980) . The reason f o r t h i s i s not known, but i t may be due to s p e c i e s d i f f e r e n -ces i n the amino a c i d c o m p o s i t i o n and o t h e r s t r u c t u r a l p r o p e r t i e s o f the p r o t e i n s . Whitehouse «rt aj_. (1980) have r e c e n t l y demonstrated t h r e e charge isomers o f PKI from bovine h e a r t , a l l o f which were c a p a b l e o f i n h i b i t i n g the c y c l i c AMP p r o t e i n k i n a s e . Because a l l the charge isomers Protein (jig/assay) Figure 1 3 . A c t i v a t i o n o f (Ca + Mg )-ATPase i n human e r y t h r o c y t e 2+ membranes by c a l m o d u l i n and bovine heart P K I , (Ca + 2+ 2+ Mg )-ATPase a c t i v i t y was determined a t 0,58 -\ffl Ca , w i t h PKI (A ) and c a l m o d u l i n ( p ) . Each p o i n t r e p r e s e n t s the means o f three e x p e r i m e n t s . F i g u r e 14. E f f e c t o f c a l m o d u l i n and PKI on c a l c i u m a c t i v a t i o n o f 2+ 2+ (Ca + Mg )-ATPase i n human e r y t h r o c y t e membranes. 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y was determined i n the absence (*) and i n the presence o f 3 ug c a l m o d u l i n / 0 . 6 ml (•) and 2 yg bovine heart PKI/0.6 ml (o) . Data r e p r e -sent the means o f t h r e e s e p a r a t e e x p e r i m e n t s . ± JL -L -L -L 0 2 4 6 8 10 12 14 V/ri+Ojmoles Pi/h per ml ghost/pM) F i g u r e 15. E a d i e - H o f s t e e p l o t o f data i n f i g u r e 14. Curves were o b t a i n e d i n the absence (*) and i n the presence of 3 yg c a l m o d u l i n / 0 . 6 ml (•) and 2 yg bovine heart PKI/ 0.6 mis (o). were presumably present i n t h i s s t u d y , i t i s not known i f any s p e c i f i c 2+ 2+ charge Isomer i s more potent than the o t h e r s i n s t i m u l a t i n g (Ca + Mg ) -ATPase. 2+ 2+ Bovine heart PKI a l s o s t i m u l a t e d (Ca + Mg )-ATPase i n 'Dodge g h o s t s ' t r e a t e d w i t h EDTA (EDTA-treated membranes), which a r e d e p l e t e d o f endogenous c a l m o d u l i n ( R o u f o g a l i s and M a u l d i n 1980) ( r e s u l t s not shown). These membranes a l s o l a c k the endogenous c y c l i c AMP p r o t e i n k i n a s e ( B o i v i n 2+ and Galand 1977) which f u r t h e r suggests t h a t PKI d i d not s t i m u l a t e (Ca + 2+ Mg )-ATPase by an a c t i o n on c y c l i c AMP p r o t e i n k i n a s e . D e m a i l l e ejt al_. (1979) showed t h a t m o d i f i c a t i o n o f the a r g i n y l groups o f PKI a b o l i s h e d i t s i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e . M o d i f i c a t i o n o f a r g i n y l groups of bovine heart PKI or c a l m o d u l i n had no 2+ 2 + e f f e c t on t h e i r a b i l i t y t o s t i m u l a t e (Ca + Mg )-ATPase a c t i v i t y (Table I I I ) . A r g i n y l group m o d i f i c a t i o n o f P K I , however, antagonized i t s i n h i b i t i o n o f the p h o s p h o r y l a t i o n of hi stone by c y c l i c AMP p r o t e i n k i n a s e . 2+ TFP has been shown to i n h i b i t c a l m o d u l i n s t i m u l a t i o n of (Ca + M g 2 + ) - A T P a s e ( L e v i n and Weiss 1979). TFP (20 yM) i n h i b i t e d a c t i v a t i o n o f 2+ 2 + (Ca + Mg )-ATPase by PKI i n the presence or absence o f c a l m o d u l i n (Table IV) . By c o n t r a s t , the a b i l i t y o f bovine heart PKI to i n h i b i t c y c l i c AMP p r o t e i n k i n a s e was not a f f e c t e d by TFP (Table V ) , s u g g e s t i n g t h a t d i f f e r e n t s i t e s or conformat ions o f the PKI m o l e c u l e may be i n v o l v e d 2+ 2+ i n s t i m u l a t i o n o f (Ca + Mg )-ATPase and i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e . I t i s a l s o o f i n t e r e s t t h a t i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e by PKI does not r e q u i r e c a l c i u m , as i n h i b i t i o n of h i s t o n e p h o s p h o r y l a t i o n occurs even i n the presence o f EGTA ( r e s u l t not shown). 2+ 2+ Thus PKI s t i m u l a t i o n o f (Ca + Mg )-ATPase resembles t h a t o f 2+ c a l m o d u l i n , as both i n c r e a s e the s e n s i t i v i t y f o r Ca . However, u n l i k e E f f e c t of a r g i n y l group m o d i f i c a t i o n o f bovine heart c y c l i c AMP p r o t e i n k i n a s e I n h i b i t o r 2+ 2+ and c a l m o d u l i n on modulat ion of (Ca + Mg )-ATPase a c t i v i t y or c y c l i c AMP p r o t e i n k i n a s e ( C - s u b u n i t ) a c t i v i t y . M o d i f i c a t i o n o f PKI and c a l m o d u l i n a r g i n y l groups and the assay o f h i s t o n e p h o s p h o r y l a t i o n and ATPase a c t i v i t y are d e s c r i b e d i n Methods, Reagent Residue M o d i f i e d C-subunit c a t a l y z e d t r a n s f e r of 32p t o h i s t o n e (pmoles P-j t r a n s f e r r e d per 10 min) PKI ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y (ymoles P^  per ml 'ghosts') PKI Calmodul in Unmodified 1,2-Cyclohexane-dione A r g i n i n e 38 130 ( 1 4 0 ) a 3 . 5 3 . 5 4 . 5 4 . 3 The v a l u e in p a r e n t h e s i s represents c o n t r o l va lue w i t h o u t p r o t e i n k i n a s e i n h i b i t o r . Data r e p r e s e n t the means o f two ( h i s t o n e p h o s p h o r y l a t i o n ) or t h r e e ( ( C a 2 + + M g 2 + ) - A T P a s e ) s e p a r a t e experiments E f f e c t o f t r i f l u o p e r a z i n e (TFP) on the a b i l i t y o f c y c l i c AMP 2+ 2+ p r o t e i n k i n a s e i n h i b i t o r t o s t i m u l a t e (Ca + Mg )-ATPase i n 'Dodge ghost' membranes (j)G). When u s e d , TFP was added to the r e a c t i o n m i x t u r e i n the dark 10 min b e f o r e the r e a c t i o n 2+ 2+ was s t a r t e d w i t h ATP, (Ca + Mg )-ATPase a c t i v i t y was 2+ assayed at 2 mM ATP and 0,58 yM Ca , as d e s c r i b e d i n Methods, Data r e p r e s e n t the means o f a t l e a s t three s e p a r a t e e x p e r i m e n t s . Enzyme P r e p a r a t i o n TFP (Ca + Mg )-ATPase (20 yM) Dodge ghosts (DG) 1.5 + 1.4 DG + PKI (2 yg) 3 . 2 + 1.6 DG + c a l m o d u l i n 4 . 0 (3 yg) + 1,75 E f f e c t o f t r i f l u o p e r a z i n e (JFP) on the i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e c a t a l y t i c (X) s u b u n i t c a t a l y z e d phospho-r y l a t i o n o f h i s t o n e by p r o t e i n k i n a s e I n h i b i t o r from bovine h e a r t . H i s t o n e p h o s p h o r y l a t i o n was performed as d e s c r i b e d i n Methods. TFP and PKI were p r e i n c u b a t e d w i t h the r e a c t i o n m i x t u r e f o r 10 min i n the dark p r i o r to s t a r t i n g the r e a c t i o n w i t h ATP. Data shown i s t y p i c a l o f t h r e e s e p a r a t e e x p e r i m e n t s . Assay C o n d i t i o n s H i s t o n e P h o s p h o r y l a t i o n (pmoles 32p t r a n s f e r r e d / 1 0 min) C o n t r o l 140 PKI (2 yg) 38 20 yM TFP 140 PKI (2 yg) + TFP (20 yM) 40 c a l m o d u l i n , PKI does not i n c r e a s e the maximum v e l o c i t y , The s t i m u l a t i o n 2+ by PKI a t low Ca c o n c e n t r a t i o n s i s not due s o l e l y to an e f f e c t on ,the c y c l i c AMP p r o t e i n k i n a s e p h o s p h o r y l a t i o n because a) c y c l i c AMP caused a c o m p a r a t i v e l y small I n h i b i t i o n o f the enzyme a c t i v i t y Capprox. 20%) at a l l c a l c i u m c o n c e n t r a t i o n s u s e d , w h i l e the s t i m u l a t i o n by PKI i s about 100% and independent o f added c y c l i c AMP. Furthermore, PKI s t i m u l a t i o n o c c u r r e d a t 2 mM ATP, c o n d i t i o n s under which c y c l i c AMP I n h i b i t i o n o f 2+ 2+ (Ca + Mg )-ATPase was not o b s e r v e d , b) M o d i f i c a t i o n o f a r g i n y l groups o f PKI l e d t o a l o s s i n the i n h i b i t i o n by PKI o f h l s t o n e p h o s p h o r y l a t i o n by c y c l i c AMP p r o t e i n k i n a s e , but had no e f f e c t on the s t i m u l a t i o n o f 2+ 2+ (Ca + Mg )-ATPase. This suggests t h a t the s i t e s o f i n t e r a c t i o n o f PKI 2+ 2 + w i t h (Ca + Mg )-ATPase and p r o t e i n k i n a s e are d i f f e r e n t or t h a t a r g i -nine r e s i d u e s are not r e q u i r e d f o r a c t i v a t i o n o f ATPase, as i s a l s o the case w i t h c a l m o d u l i n (Table I I I ) , c) PKI s t i m u l a t e d EDTA-treated membra-nes which are d e p l e t e d o f c y c l i c AMP p r o t e i n k i n a s e (.Boivin and Galand 1 9 7 7 ) . Khandelwal e t al_ (1980) have r e p o r t e d t h a t under c e r t a i n c o n d i t -ions the "phosphoprotein-phosphatase" a c t i v i t y o f l i v e r i s i n c r e a s e d by c a l m o d u l i n and t r o p o n i n - C and they suggested t h a t t h i s e f f e c t may be due to the n e g a t i v e charge of these compounds, as the e f f e c t showed l a c k o f 2+ c a l c i u m dependency. Al though i t i s p o s s i b l e t h a t the i n c r e a s e i n (Ca + 2+ Mg )-ATPase a c t i v i t y by PKI observed i n t h i s study c o u l d be due to i n c r e a s e d phosphatase a c t i v i t y , as a r e s u l t o f i t s n e g a t i v e charge a l o n e , t h i s i s l e s s l i k e l y because r a b b i t s k e l e t a l muscle P K I , which has a s i m i -l a r c h a r g e , was i n e f f e c t i v e and the e f f e c t i s observed i n EDTA-treated membranes which l a c k the c y c l i c AMP p h o s p h o r y l a t i n g system. The i n h i b i -2+ 2+ t o r y e f f e c t o f TFP on both PKI and c a l m o d u l i n s t i m u l a t i o n o f (Ca + Mg ) -ATPase suggests t h a t caution should be used i n i n v o k i n g a r o l e f o r c a l m o -d u l i n 1n a c e l l u l a r process on the b a s i s o f t h i s ev idence a l o n e . The l a c k o f TFP e f f e c t on c y c l i c AMP p r o t e i n k i n a s e p h o s p h o r y l a t i o n 1n the presence (or absence) o f PKI f u r t h e r supports the c o n c l u s i o n t h a t d i f f e r e n t mecha-nisms or i n t e r a c t i o n s a r e i n v o l v e d 1n the two a c t i o n s o f PKI . In summary, i t was shown t h a t PKI from bovine heart s t i m u l a t e s 2+ 2+ (Ca + Mg )-ATPase at low c a l c i u m c o n c e n t r a t i o n s by i n c r e a s i n g the s e n -2+ s i t i v i t y f o r f r e e Ca , w i t h o u t a f f e c t i n g the maximum v e l o c i t y o f the enzyme. The s t i m u l a t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e at low C a 2 + by PKI i s seen even i n the presence o f s a t u r a t i n g c o n c e n t r a t i o n s o f c a l m o d u l i n . I t 2+ 2+ was found t h a t a number o f anions s t i m u l a t e (Ca + Mg )-ATPase ( A l -Jobore et al_. 1 9 8 1 ) , and i t i s p o s s i b l e t h a t the s t i m u l a t i o n by bovine heart PKI i s r e l a t e d , at l e a s t in p a r t , to i t s a n i o n i c n a t u r e . The 2+ 2+ s p e c i f i c p a r t i a l r e a c t i o n s o f the (Ca + Mg )-ATPase r e a c t i o n sequence a f f e c t e d by t h i s c l a s s o f a c t i v a t o r s remains to be e s t a b l i s h e d . S e c t i o n I I I . R o l e o f anion channel (band 3) i n h i b i t o r s i n t h e r e g u l a t i o n  o f ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y Waisman et a l . (1981) and Gimble et a l . (1981) r e c e n t l y r e p o r t e d t h a t band 3 (anion channel) i n h i b i t o r s , such as 4 - a c e t a m i d o - 4 ' - i s o t h i o c y a n o s t i l b e n e - 2 ' - d i s u l f o n a t e (SITS) and N - ( 4 - a z i d o - 2 - n i t r o p h e n y l ) - 2 - a m i n o e t h y l s u l f o n a t e ( N A P - t a u r i n e ) , b l o c k e d both c a l m o d u l i n s t i m u l a t e d c a l c i u m t r a n s -port and anion t r a n s p o r t i n i n s i d e - o u t v e s i c l e s w i t h s i m i l a r potency. T h i s o b s e r v a t i o n l e d these authors to suggest t h a t these two t r a n s p o r t p r o c e s s e s were c l o s e l y coupled and t h a t the C a 2 + pump i s e l e c t r o g e n i c . Fur thermore, they suggested t h a t at low c o n c e n t r a t i o n s (K^ = 45-80 uM), N A P - t a u r i n e blocked the c a l m o d u l i n dependent c a l c i u m t r a n s p o r t a c t i v i t y v i a blockade o f a n i o n - t r a n s p o r t through the anion c h a n n e l . In t h i s t h e s i s the a c t i o n o f anion channel i n h i b i t o r s was s t u d i e d f u r t h e r using fragmented membranes, r e s e a l e d ghosts and i n s i d e - o u t v e s i -c l e s , to t e s t i f the anion channel b l o c k e r s i n h i b i t e d the c a l c i u m - t r a n s -port ATPase a c t i v i t y p r i m a r i l y by b l o c k i n g the anion channel or by a d i r -e c t a c t i o n on the ( C a 2 + + M g 2 + ) - A T P a s e enzyme. Furthermore, the e f f e c t o f the anion channel b l o c k e r s on Mg 2 + -ATPase and ouabain s e n s i t i v e ( N a + + K + ) -ATPase a c t i v i t i e s was a l s o s t u d i e d . The s t r u c t u r e s o f S I T S , N A P - t a u r i n e and DIDS ( 4 , 4 ' - d i i s o t h i o c y a n o - 2 - 2 ' - s t i l b e n e d i s u l f o n a t e ) are shown below. N A P - t a u r i n e » 3 S D j DIDS SITS 1. E f f e c t o f N A P - t a u r i n e on (Ca** + h V + ) - A T P a s e a c t i v i t y The ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y o f red c e l l membranes was i n h i b i t e d i n the presence o f N A P - t a u r i n e i n the absence o f i r r a d i a t i o n or d i r e c t d a y l i g h t ( F i g . 1 6 ) . The i n h i b i t i o n o f enzyme a c t i v i t y by 50 \tl NAP-taur-i n e was seen at a l l c o n c e n t r a t i o n s o f c a l c i u m examined ( F i g . 1 6 ) . F i g . 17 shows the c o n c e n t r a t i o n - r e s p o n s e curves f o r i n h i b i t i o n o f ( C a 2 + + M g 2 + ) -ATPase, Mg 2 + -ATPase and (Na + + K + ) -ATPase by N A P - t a u r i n e . H a l f maximal i n h i b i t i o n of ( C a 2 + + M g 2 + ) - A T P a s e o c c u r r e d at around 50 yM. Mg 2 + -ATPase and ( N a + + K + ) -ATPase a c t i v i t i e s were not i n h i b i t e d over the same con-c e n t r a t i o n range. At higher c o n c e n t r a t i o n s (>200 yM), N A P - t a u r i n e began t o i n h i b i t the o t h e r ATPases, probably by a n o n - s p e c i f i c a c t i o n on the membranes. The p h o t o l a b e l ing reagent N A P - t a u r i n e o f f e r s the advantage o f being a r e v e r s i b l e i n h i b i t o r o f ( C a 2 + + M g 2 + ) - A T P a s e before i r r a d i a t i o n and an i r r e v e r s i b l e i n h i b i t o r a f t e r l i g h t induced p h o t o a c t i v a t i o n ( F i g . 1 8 ) . The r e v e r s i b l e i n h i b i t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e by NAP-taur ine was shown by i n c u b a t i n g membranes i n the absence and presence o f N A P - t a u r i n e (25 yM) i n the dark ( i n a p r e i n c u b a t i o n medium i d e n t i c a l to t h a t used f o r measuring ATPase a c t i v i t y but from which ATP was excluded) and d i l u t i n g 50 y l o f t h e i n c u b a t i o n mixture to 600 y l o f the assay medium ( F i g . 1 8 ) . The ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y was u n a f f e c t e d by these c o n d i t i o n s , i n d i c a t i n g t h a t the b i n d i n g o f N A P - t a u r i n e t o the enzyme was r e v e r s i b l e ( F i g . 1 8 ) . o 6 5 4 y- -Log|Can f r e e („) 2+ 2+ F igure 16. E f f e c t o f N A P - t a u r i n e on (Ca + Mg )-ATPase a c t i v i t y o f membranes. The enzyme a c t i v i t y was measured i n the dark i n the absence (•) and i n the presence (o) of NAP-2+ t a u r i n e (50 yM) at v a r i o u s c o n c e n t r a t i o n s o f f r e e Ca Data r e p r e s e n t the means o f two s e p a r a t e e x p e r i m e n t s . o 40 80 NAP-taurine (uM) 120 200 F i g u r e 17. C o n c e n t r a t i o n dependence o f N A P - t a u r i n e i n h i b i t i o n of ( C a 2 + + M g 2 + ) - A T P a s e (•), ( N a + + K + ) -ATPase (A) and 2+ ( C a 2 + + Mg*"'-ATPase (o) a c t i v i t i e s by N A P - t a u r i n e . 9 i Mg )-ATPase a c t i v i t y was measured at 0.58 yM f r e e c a l c i u m . Each p o i n t r e p r e s e n t s the means o f t r i p l i c a t e d e t e r m i n a t i o n s , The curves shown are t y p i c a l o f t h r e e s i m i l a r e x p e r i m e n t s . =C l .5 r 'o> E •5 1 0 E a> o o D Q_ < 0.5 D u 0.0 0 6 5 - L o g [ C a 2 + ] f ree (M) 2+ 2+ F i g u r e 18. E f f e c t o f N A P - t a u r i n e on (Ca + Mg )-ATPase i n membranes w i t h and w i t h o u t p h o t o l y s i s . Membranes (4 mg/ml) i n the presence o f N A P - t a u r i n e (25 yM) were e i t h e r kept i n the dark (o) or i r r a d i a t e d f o r 15 min (•) or 30 min ( o ) . Membranes were a l s o i r r a d i a t e d i n the absence o f N A P - t a u r i n e (•). 50 y l o f these membranes were d i l u t e d to 0.6 ml i n 2+ 2+ the standard assay medium to measure (Ca + Mg )-ATPase 2+ 2+ a c t i v i t y . ( A ) Represents (Ca + Mg )-ATPase a c t i v i t y o f c o n t r o l membranes i n the presence o f N A P - t a u r i n e (25 yM) i n the assay medium i n the d a r k . Data shown i s t y p i c a l o f three s i m i l a r e x p e r i m e n t s . However, p h o t o l y s i s o f membranes wi th 25 uM NAP-taur ine f o r 15 min or 30 min r e s u l t e d i n r e t e n t i o n o f the i n h i b i t i o n o f the ( C a 2 + + M g 2 + ) - A T P a s e a f t e r 5 0 - f o l d d i l u t i o n i n the assay medium ( F i g . 1 8 ) . The extent o f i r r e v e r s i b l e i n h i b i t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e seen upon p h o t o l y s i s was s i m i l a r to the r e v e r s i b l e i n h i b i t i o n o f the enzyme assayed i n the presence o f N A P - t a u r i n e i n the dark. These r e s u l t s suggest t h a t N A P - t a u r i n e could be used as a p h o t o l a b e l i n g probe because the i r r e v e r s i b l e i n h i b i t i o n upon p h o t o l y s i s c l o s e l y r e f l e c t s the r e v e r s i b l e b i n d i n g i n the dark. In an attempt to d e f i n e the s i t e o f a c t i o n o f NAP-taur ine on the ( C a 2 + + M g 2 + ) - A T P a s e , membranes were exposed to l i g h t i n the presence of N A P - t a u r i n e (25 yM) i n the absence and presence o f v a r i o u s c o n c e n t r a t i o n s o f C a + , M g 2 + , ATP and c a l m o d u l i n . I t was f i r s t e s t a b l i s h e d t h a t i r r a d i a -t i o n o f c a l m o d u l i n i n the presence o f 25 yM N A P - t a u r i n e d i d not a f f e c t i t s a c t i v i t y , as shown by i t s a b i l i t y to s t i m u l a t e ( C a 2 + + M g 2 + ) - A T P a s e ( r e s u l t s not shown). None o f the agents p r o t e c t e d a g a i n s t i n h i b i t i o n by N A P - t a u r i n e (Table V I ) . These r e s u l t s suggest t h a t N A P - t a u r i n e i n h i b i t e d the ( C a 2 + + M g 2 + ) - A T P a s e n o n - c o m p e t i t i v e l y w i t h respect to C a 2 + , M g 2 + , ATP, and c a l m o d u l i n . The n o n - c o m p e t i t i v e nature o f i n h i b i t i o n by NAP-t a u r i n e wi th r e s p e c t to c a l m o d u l i n i s a l s o shown i n a d o u b l e - r e c i p r o c o l p l o t ( F i g . 1 9 ) . S i n c e t r y p s i n t reatment o f membranes a b o l i s h e s the a c t i -v a t i o n of ( C a 2 + + M g 2 + ) - A T P a s e by c a l m o d u l i n , probably by l o s s of a r e g u l a -t o r y component o f the enzyme ( N i g g l i et a l . 1980; Sarkadi et a l . 1980; Enyedi et a l . 1 9 8 0 ) , the e f f e c t o f N A P - t a u r i n e on t r y p s i n - t r e a t e d mem-branes was i n v e s t i g a t e d . T r y p s i n t reatment s t i m u l a t e d the ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y and a b o l i s h e d i t s s t i m u l a t i o n by c a l m o d u l i n , i n concurrence w i t h p r e v i o u s r e p o r t s ( F i g . 2 0 ) . N A P - t a u r i n e (50 yM) i n h i b i -ted the ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y o f t r y p s i n - t r e a t e d membranes to the E f f e c t o f Mg , Ca , ATP and c a l m o d u l i n on the i n h i b i t i o n o f 2+ 2+ (Ca + tig )-ATPase a c t i v i t y o f membranes a f t e r p h o t o l y s i s i n the presence o f N A P - t a u r i n e (25 yM). The membranes (~4 mg/ml f i n a l c o n c e n t r a t i o n ) were i n c u b a t e d i n a medium c o n t a i n i n g 55 mM T r i s - m a l e a t e (pH 7 . 2 ) , 66 mM N a C l , 0,1 mM o u a b a i n , N A P - t a u r i n e (25 yM) and v a r i o u s e f f e c t o r s o f the enzyme, as I n d i c a t e d . 2+ 2+ 2+ When Mg was v a r i e d , the c o n c e n t r a t i o n o f f r e e Ca was 10 yM. When Ca 2+ was v a r i e d , the c o n c e n t r a t i o n o f Mg was kept a t 6.5 mM, and when ATP o r 2+ 2+ c a l m o d u l i n were v a r i e d , the c o n c e n t r a t i o n s o f Ca and Mg were 10 yM and 6.5 mM, r e s p e c t i v e l y . The membranes were photolysed at 4°C f o r 40 m i n ; 2+ 2+ 50 y l o f t h i s enzyme p r e p a r a t i o n was used f o r measurement o f (Ca + Mg ) -ATPase a c t i v i t y at 55 yM f r e e c a l c i u m ) 2+ 2+ Medium o f P h o t o l y s i s (Ca + Mg )-ATPase a c t i v i t y (ymoles Pi mg"^ hr"^) C o n t r o l (No N A P - t a u r i n e ) 0.75 N A P - t a u r i n e (25 yM) 0.45 N A P - t a u r i n e (25 yM) + M g 2 + ( 1 mM) (10 mM) 0.47 0.46 N A P - t a u r i n e + C a 2 + ( 5 yM) ( 55 yM) 0.46 0.48 N A P - t a u r i n e + ATP ( 0 . 5 mM) ( 2 . 0 mM) 0.43 0.44 N A P - t a u r i n e + c a l m o d u l i n (0.1 y g ) (1.2 y g) • 0.46 Data r e p r e s e n t the means of at 1 e a s t t h r e e s e p a r a t e e x p e r i m e n t s . F i g u r e 19. D o u b l e - r e c i p r o c a l p l o t o f CCa + Mg )-ATPase i n h i b i t i o n by N A P - t a u r i n e i n t h e presence o f c a l m o d u l i n . (•) Represents enzyme a c t i v i t y i n the presence o f c a l m o d u l i n ; (o) Represents enzyme a c t i v i t y i n the presence o f c a l m o d u l i n and N A P - t a u r i n e (50 yM) i n the assay medium. N A P - t a u r i n e was added to membra-nes i n the assay medium ( 0 . 6 ml) and incubated i n the d a r k . E O E CD or> O 1.5 1.0 ^ 0.5 D u 0.0 B D 2+ 2+ F i g u r e 2 0 , E f f e c t o f N A P - t a u r i n e on (Ca + Mg )-ATPase i n t r y p s i n t r e a t e d membranes. 2+ 2+ A: (Ca + Mg )-ATPase a c t i v i t y of n o n - t r y p s i n t r e a t e d mem-2+ branes determined at 55 yM f r e e Ca 2+ 2+ B: Calmodul in (2 yg) a c t i v a t i o n o f (Ca + Mg )-ATPase a c t i v i t y of n o n - t r y p s i n t r e a t e d membranes. ( C a 2 + + M g 2 + ) - A T P a s e a yg t rypsin/mg p r o t e i n . E f f e c t o f c a l m o d u l i n ( a c t i v i t y o f t r y p s i n t r e a t e d membranes, E f f e c t of N A P - t a u r i n e (50 yM) on ( C a 2 H a c t i v i t y o f t r y p s i n t r e a t e d membranes. C: ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y a f t e r t reatment w i t h 0.2 D: E f f e c t o f c a l m o d u l i n (2 yg) on ( C a 2 + + M g 2 + ) - A T P a s e E: E f f e c t of N A P - t a u r i n e (50 yM) on ( C a 2 + + M g 2 + ) - A T P a s e same extent as i n n a t i v e membranes. These r e s u l t s f u r t h e r support the s u g g e s t i o n t h a t N A P - t a u r i n e i n h i b i t s the ( C a 2 + + M g 2 + ) - A T P a s e at a s i t e d i s t i n c t from t h a t o f the c a l m o d u l i n r e g u l a t o r y s i t e . 2 . The s idedness o f N A P - t a u r i n e i n h i b i t i o n o f ( C a 2 + + M q 2 + ) - A T P a s e A number o f experiments were performed to determine from which s i d e o f the membrane N A P - t a u r i n e i n h i b i t e d the ( C a 2 + + M g 2 + ) - A T P a s e . D i r e c t access t o the c y t o p l a s m i c face o f the membrane was achieved by r e s e a l i n g ghosts i n the presence o f N A P - t a u r i n e (Table V I I ) . NAP-taur ine had v i r t u -a l l y no e f f e c t on the ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y when added to ghosts r e s e a l e d i n the absence o f N A P - t a u r i n e . However, when ghosts were r e -s e a l e d i n the presence o f 50 uM N A P - t a u r i n e , 60% i n h i b i t i o n o f the ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y was observed. These r e s u l t s suggested that the s i t e ( s ) at which N A P - t a u r i n e b inds to cause i n h i b i t i o n of ( C a 2 + + M g 2 + ) - A T P a s e i s a c c e s s i b l e from the i n n e r or c y t o p l a s m i c s ide of the membrane. This c o n c l u s i o n i s f u r t h e r supported by the a c t i o n o f NAP-t a u r i n e on C a 2 + - t r a n s p o r t i n i n s i d e - o u t v e s i c l e s ( F i g . 2 1 ) . I t can be seen t h a t 50 yM N A P - t a u r i n e i n h i b i t e d the C a 2 + - t r a n s p o r t i n i n s i d e - o u t v e s i c l e s to a s i m i l a r extent to i t s i n h i b i t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e . F i n a l l y , the ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y i n membranes prepared from red c e l l s photolysed i n the presence o f 50 yM N A P - t a u r i n e was s i m i l a r to t h a t from untreated c e l l s , suggest ing t h a t N A P - t a u r i n e d id not i n h i b i t the enzyme from the o u t e r membrane s u r f a c e (see Table V I I I ) . At t h i s concen-t r a t i o n N A P - t a u r i n e b inds to a m o d i f i e r s i t e on the outer membrane sur face (Knauf et a l . 1978) and i t does not permeate the red c e l l under these con-d i t i o n s ( S t a r o s et a l . 1974; Cabantchik et a l . 1 9 7 8 ) . 3 . Does N A P - t a u r i n e i n h i b i t t h e ( C a 2 + + M g 2 + ) - A T P a s e and  Ca2"1"- t r a n s p o r t by b l o c k i n g the a n i o n channel ? A number o f compounds have been r e p o r t e d to i n h i b i t band 3 (anion CCa + Mg )-ATPase a c t i v i t y o f ghosts r e s e a l e d i n the presence and absence of N A P - t a u r i n e , The r e s e a l i n g medium c o n t a i n e d (.in f i n a l c o n c e n t r a t i o n ) 4 mM M g C l 2 , 3 mM C a C l 2 , 4 mM T r i s - A T P , 0.1 mM o u a b a i n , 10 mM T r i s - m a l e a t e , pH 7.1 ± N A P - t a u r i n e (50 y M ) . Data r e p r e s e n t the means o f t h r e e s e p a r a t e e x p e r i m e n t s . Composit ion o f Loading Medium (Ca + Mg )-ATPase a c t i v i t y -1 -1 (umoles P. mg hr ) C o n t r o l (no N A P - t a u r i n e ) 0.63 C o n t r o l ( N A P - t a u r i n e added to the assay medium) 0.58 N A P - t a u r i n e (50yM) i n r e s e a l i n g medi urn 0.25 Time (min) F i g u r e 2 1 . E f f e c t of N A P - t a u r i n e on Ca - t r a n s p o r t i n i n s i d e - o u t 2 + ( 1 . 0 . ) v e s i c l e s . Ca - t r a n s p o r t was measured i n the absence o f ATP (•), i n the presence o f 2 mM ATP (•) and i n the p r e -sence o f ATP plus N A P - t a u r i n e (.50 yM) i n the dark ( o ) . The r e a c t i o n medium c o n t a i n e d 55 mM T r i s - m a l e a t e , pH 7 . 2 , 66 mM 45 N a C l , 6.4 mM M g C l 2 , 0.1 mM EGTA and 3 C a C l 2 , to g i v e a f r e e c a l c i u m c o n c e n t r a t i o n o f 55 yM. The r e a c t i o n was i n i t i a t e d a f t e r a 5 min p r e i n c u b a t i o n ( 3 7 ° C ) by a d d i t i o n o f ATP (2 mM, f i n a l c o n c e n t r a t i o n ) . The data shown i s t y p i c a l o f t h r e e s i m i l a r e x p e r i m e n t s . 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y o f membranes made from c e l l s t r e a t e d w i t h NAP-taur1ne or DIDS Human e r y t h r o c y t e s (.10% h e m a t o c r i t ) were e i t h e r incubated w i t h N A P - t a u r i n e (25 yM) and photolysed a t 4°C or incubated a t 37°C w i t h DIDS (5 yM) f o r 30 m i n . The c e l l s were washed and then hemolysed f o r the p r e p a r a t i o n o f membranes. The enzyme a c t i v i t y o f membranes was measured a t 55 yM f r e e c a l c i u m . 2+ 2+ Membrane p r e p a r a t i o n (Ca + Mg )-ATPase a c t i v i t y (ymoles P^  mg~^ hr~^ C o n t r o l 0.62 50 yM N A P - t a u r i n e 0.57 5 yM DIDS 0.62 Data r e p r e s e n t the means o f four experiments f o r the c o n t r o l and t h r e e experiments f o r the N A P - t a u r i n e and DIDS t r e a t e d c e l l s . c h a n n e l ) . N A P - t a u r i n e was shown to be a r e v e r s i b l e i n h i b i t o r ( i n the dark) o f the anion channel when i t was present e i t h e r i n s i d e or o u t s i d e the c e l l (Knauf et a l . 1 9 7 8 ) . However, t h e r e i s a s u b s t a n t i a l d i f f e r e n c e ( 1 5 - f o l d ) i n the c o n c e n t r a t i o n r e q u i r e d to i n h i b i t the anion channel from e i t h e r s i d e . On the i n s i d e , N A P - t a u r i n e has a r e l a t i v e l y low a f f i n i t y for band 3 (K-j = 370 uM), whereas e x t e r n a l N A P - t a u r i n e i s a f a r more potent i n h i b i t o r o f band 3 (Ki = 20 uM) (Knauf et a l . 1 9 7 8 ) . Four l i n e s o f e v i d e n c e i n d i c a t e t h a t N A P - t a u r i n e i n h i b i t s the ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y o f membranes d i r e c t l y r a t h e r than i n d i r e c t l y by i n h i b i t i n g t h e anion channel (assuming t h a t the ATPase and the anion channel were indeed i n some way t i g h t l y c o u p l e d ) . a) Whereas 50 uM N A P - t a u r i n e and 5 pM DIDS (Knauf et a l . 1978) i n h i b i t the anion channel from the e x t e r n a l membrane s u r f a c e , ( C a 2 + + M g ^ + ) -ATPase a c t i v i t y i n membranes made e i t h e r from 50 pM N A P - t a u r i n e or 5 pM DIDS t r e a t e d c e l l s was comparable to t h a t from c o n t r o l c e l l s under c o n d i -t i o n s of i r r e v e r s i b l e band 3 i n h i b i t i o n (Table V I I I ) . b) P r o b e n e c i d (125 uM) has a l s o been r e p o r t e d to be an anion channel b l o c k e r (Motais and Cousin 1 9 7 6 ) . However, 125 pM probenecid had no e f f e c t on the ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y i n unsealed membranes ( r e s u l t s not shown). c) P r e - i r r a d i a t e d N A P - t a u r i n e ( i . e . NAP-taur ine photolysed i n b u f f e r a lone) was able to i n h i b i t the C a 2 + - t r a n s p o r t ATPase to the same extent as n o n i r r a d i a t e d N A P - t a u r i n e ( r e s u l t s not shown), whereas n e i t h e r r e v e r s -i b l e nor i r r e v e r s i b l e blockade of the anion channel was observed w i t h NAP-t a u r i n e which was p r e - i r r a d i a t e d (Knauf et a l . 1978; R o t h s t e i n et a l . 1 9 7 7 ) . d) The most c o n v i n c i n g evidence came from experiments i n which N A P - t a u r -i n e at c o n c e n t r a t i o n s between 25 pM and 50 pM i n h i b i t e d p u r i f i e d ( C a 2 + + M g 2 + ) - A T P a s e (Table I X ) . The i n h i b i t i o n by 50 uM NAP-taur ine o f the p u r i f i e d enzyme i s more pronounced than t h a t o f the enzyme i n membranes. The e f f e c t o f pretreatment o f i n t a c t red c e l l s wi th an anion channel b l o c k e r on c a l c i u m t r a n s p o r t i n t o i n s i d e - o u t v e s i c l e s prepared from these c e l l s i s shown i n F i g . 2 2 . Treatment o f red c e l l s wi th 5 \M DIDS l e d to ' an i n h i b i t i o n o f c a l c i u m t r a n s p o r t i n i n s i d e - o u t v e s i c l e s , d e s p i t e the l a c k of i n h i b i t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y i n membranes from DIDS t r e a t e d c e l l s (Table V I I I ) . These r e s u l t s , t h e r e f o r e , concur w i t h the o b s e r v a t i o n o f Waisman et al_. (1981) t h a t the i n h i b i t i o n of c a l c i u m t r a n s p o r t i n i n s i d e - o u t v e s i c l e s a f t e r p r e i n c u b a t i o n o f c e l l s w i t h an anion channel b l o c k e r (these authors used SITS) i s a r e s u l t of i n h i b i t i o n o f anion t r a n s p o r t through band 3 . T h e r e f o r e , w h i l e net uptake o f c a l c i u m can be i n h i b i t e d by b l o c k i n g band 3 (Waisman et a l . 1 9 8 1 ) , the converse s i t u a t i o n , i n which the net uptake o f anions may be reduced due to b l o c k -ade o f net C a 2 + - u p t a k e by the i n h i b i t i o n o f the C a 2 + - t r a n s p o r t ATPase, must a l s o be c o n s i d e r e d . In c o n c l u s i o n , the present r e s u l t s show that N A P - t a u r i n e s e l e c t i v e l y i n h i b i t s the ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y o f red c e l l membranes. The o u a b a i n - s e n s i t i v e ( N a + , K + ) -ATPase and Mg 2 + -ATPase are i n h i b i t e d o n l y at much higher c o n c e n t r a t i o n s . The i n h i b i t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e of membranes by N A P - t a u r i n e appears to be independent o f anion channel i n h i -b i t i o n , as determined by a number of c r i t e r i a . The f a c t t h a t N A P - t a u r i n e at c o n c e n t r a t i o n s between 25-50 uM i n h i b i t e d a pure ( C a 2 + + M g 2 + ) - A T P a s e p r e p a r a t i o n p r o v i d e s f u r t h e r c o m p e l l i n g evidence f o r a d i r e c t i n h i b i t o r y e f f e c t o f N A P - t a u r i n e on the enzyme. P r e v i o u s l y a v a i l a b l e i n h i b i t o r s are e i t h e r n o n s p e c i f i c ( e . g . lanthanum, ruthenium red) or predominantly i n h i -b i t the c a l m o d u l i n a c t i v a t i o n o f the pump ( e . g . R 24571 ( G i e t z e n et a l . 1 9 8 1 ) ) . The i n h i b i t i o n produced by N A P - t a u r i n e c o u l d not be p r o -E f f e c t o f N A P - t a u r i n e on p u r i f i e d C . C a 2 + + W g 2 + ) - A T P a ? e 2+ 2+ P u r i f i e d (Ca + Mg )-ATPase a c t i v i t y i n the absence o r presence o f N A P - t a u r i n e 1n the assay medium was measured i n t h e d a r k , 1,8 yg o f enzyme p r o t e i n was incubated i n a -medium c o n t a i n i n g 55 mM T r i s - m a l e a t e , 66 mM N a C l , 0.1 mM o u a b a i n , 6.5 mM t l g C l 2 » 10 yM f r e e c a l c i u m , 2 mM ATP, w i t h and w i t h o u t N A P - t a u r i n e , f o r 30 min a t 3 7 ° C . P u r i f i e d enzyme was k i n d l y s u p p l i e d by Dr. J . T . P e n n i s t a n , Mayo C l i n i c , R o c h e s t e r . 2+ 2+ A d d i t i o n s (Ca + Mg )-ATPase a c t i v i t y -1 -1 (ymoles P* mg min ) C o n t r o l + 25 yM N A P - t a u r i n e + 50 yM N A P - t a u r i n e 4.7 2 , 0 1,0 Time (min) Figure 2 2 . Calcium uptake i n i n s i d e - o u t v e s i c l e s made from red blood c e l l s t r e a t e d w i t h DIDS (5 yM) at 37°C f o r 30 m i n . A f t e r t reatment c e l l s were washed and ghosts prepared by h e m o l y s i s . Fresh ghosts were used f o r the p r e p a r a t i o n o f i n s i d e - o u t v e -2+ s i d e s . Ca - t r a n s p o r t was measured 1n v e s i c l e s from u n t r e a t e d c e l l s i n the absence of ATP (•), and i n the presence of 2 mM ATP i n v e s i c l e s from u n t r e a t e d c e l l s (•) and i n v e s i c l e s from c e l l s t r e a t e d w i t h 5 yM DIDS ( o ) . The data shown i s t y p i c a l o f t h r e e s i m i l a r e x p e r i m e n t s . TOO t e c t e d by p r e i n c u b a t i o n o f the enzyme w i t h C a 2 + , M g 2 + , ATP o r c a l m o d u l i n . Indeed, t r y p s i n t r e a t e d membranes which l o s e t h e i r s e n s i t i v i t y to s t i m u l a -t i o n by c a l m o d u l i n , due probably to a l o s s of the c a l m o d u l i n b i n d i n g com-ponent, were i n h i b i t e d by N A P - t a u r i n e at s i m i l a r c o n c e n t r a t i o n s to the n o n - t r y p s i n t r e a t e d p r e p a r a t i o n s . The s i t e o f a c t i o n o f N A P - t a u r i n e on the ( C a 2 + + Mg2 + ) -ATPase i s u n c l e a r at p r e s e n t , but i s probably an a l l o s t e r i c s i t e on the enzyme d i s t i n c t from the Ca2"1", M g 2 + , ATP and c a l m o d u l i n s i t e s . The f i n d i n g t h a t DIDS i n h i b i t e d C a 2 + t r a n s p o r t without i n h i b i t i n g ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y supports the suggest ion of Waisman et a l . (1981) t h a t net movement o f anions may occur d u r i n g C a 2 + t r a n s p o r t i n i n s i d e - o u t v e s i c l e s . However, the present r e s u l t s show t h a t NAP-taur ine (25-50 yM) i n h i b i t s ( C a 2 + + M g 2 + ) - A T P a s e by a d i r e c t a c t i o n on the enzyme, r a t h e r than by the i n h i b i t i o n o f band 3 suggested by these a u t h o r s . Thus, t h e r e appear to be d i s t i n c t d i f f e r e n c e s in the mechanism o f i n h i b i t i o n of net t r a n s p o r t o f C a 2 + and anions by DIDS and N A P - t a u r i n e . P r e l i m i n a r y r e s u l t s i n d i c a t e t h a t N A P - t a u r i n e (25 yM) i n h i b i t s the ( C a 2 + + M g 2 + ) -ATPase a c t i v i t y i n canine heart sarcolemma and s a r c o p l a s m i c r e t i c u l u m mem-b r a n e s , but not the C a 2 + -ATPase a c t i v i t y i n the p a n c r e a t i c a c i n a r c e l l s . T a u r i n e (up to 55 mM) had no e f f e c t on the red c e l l ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y . A study o f the 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 i s c u r r e n t l y under way to o b t a i n i n f o r m a t i o n on the m o l e c u l a r mechanisms o f ( C a 2 + + M g 2 + ) - A T P a s e i n h i b i t i o n by N A P - t a u r i n e . These r e s u l t s suggest that N A P - t a u r i n e may be a general s e l e c t i v e a n t a g o n i s t of the C a 2 + pump i n a v a r i e t y o f t i s s u e s . General D i s c u s s i o n 2+ 2+ The Ca a f f i n i t y o f the Ca t r a n s p o r t ATPase i n i n t a c t e r y t h r o -2+ 2+ c y t e s i n v i v o i s u n c e r t a i n . In i s o l a t e d membranes the (Ca + Mg ) -2+ 2+ ATPase a c t i v i t y can be i s o l a t e d i n a low Ca a f f i n i t y s t a t e , a high Ca a f f i n i t y s t a t e or w i t h both components depending on the procedures u s e d . A number o f f a c t o r s can i n f l u e n c e the r e l a t i v e d i s t r i b u t i o n o f the two and s t a t e s . Calmodul in was shown to i n c r e a s e both the apparent 1^2+ 2+ the Vmax. o f the Ca t r a n s p o r t ATPase. I t was suggested t h a t both the 2+ a n i o n i c nature o f c a l m o d u l i n and Ca induced exposure of hydrophobic r e s i d u e s may be important i n r e g u l a t i o n o f the enzyme by c a l m o d u l i n . The carboxy l groups o f c a l m o d u l i n are i n v o l v e d not o n l y the b i n d i n g o f 2+ 2+ Ca a t the 4 s p e c i f i c Ca b i n d i n g s i t e s but i t i s c o n c e i v a b l e t h a t they 2+ a l s o make the b i n d i n g o f Ca at these s i t e s e a s i e r . Although t h e r e i s no reason a p r i o r i to b e l i e v e t h a t the a n i o n i c nature o f c a l m o d u l i n i s important f o r o t h e r aspects o f c a l m o d u l i n ' s f u n c t i o n s , the s t u d i e s w i t h p o l y - L - c a r b o x y l i c a c i d s suggest t h a t the a n i o n i c nature o f these compounds may be important i n m e d i a t i n g changes i n the K^fl2+ o f the ATPase. Because p o l y - L - c a r b o x y l i c a c i d s , s u l f o n i c a c i d b u f f e r s (Na-HEPES and Na-MES) and a r o m a t i c c a r b o x y l i c a c i d produce s i m i l a r e f f e c t s on the 1(^2+ of the ATPase, i n s p i t e o f t h e i r very d i f f e r e n t s t r u c t u r e s (and hence p h y s i c o - c h e m i c a l p r o p e r t i e s ) , a l t e r n a t i v e e x p l a n a t i o n s f o r t h e i r 2+ e f f e c t s on the Ca t r a n s p o r t ATPase must a l s o be c o n s i d e r e d . For i n -2+ s t a n c e , these agents may i n c r e a s e the l o c a l c o n c e n t r a t i o n o f f r e e Ca a t 2+ the Ca b i n d i n g s i t e on the enzyme or enhance a p o s s i b l e r a t e l i m i t i n g 2+ 2+ d i f f u s i o n o f Ca t o the a c t i v e s i t e (or an a c c e s s o r y Ca b i n d i n g s i t e ) 2+ on the Ca t r a n s p o r t ATPase. Furthermore, at the pH o f a s s a y , the b u f f e r s HEPES and MES c o n t a i n a m i x t u r e o f z w i t t e r i o n s and s i n g l y charged n e g a t i v e ions which vary due to the d i f f e r e n c e s i n t h e i r pK ' s . Thus, the a z w i t t e r i o n i c form o f these compounds may a l s o c o n t r i b u t e t o t h e i r observed e f f e c t s on the ATPase. U n t i l b i n d i n g s t u d i e s o f these agents w i t h the ATPase i s done, i t i s not p o s s i b l e t o d e f i n e the s i t e ( r e g u l a t o r y component) or the p r e c i s e mechanism o f a c t i o n o f these compounds 2+ Although the mechanism o f r e g u l a t i o n o f the Ca pump o f human e r h t h r o c y t e membrane i s b e g i n n i n g t o emerge, i t i s f a r from c l a r i f i e d . 2+ For i n s t a n c e , 1t i s not e x a c t l y c l e a r at present whether the Ca pump i s e l e c t r o g e n i c o r o b l i g a t o r i l y compensated by c o - o r counter t r a n s p o r t 2+ of o t h e r i o n . Waisman et a l (1981) p o s t u l a t e d t h a t the Ca pump i s e l e c t r o g e n i c , r e s u l t i n g i n c r e a t i o n o f a p o s i t i v e membrane p o t e n t i a l i n s i d e the 1.0. v e s i c l e s which serves as the d r i v i n g f o r c e o f anion uptake. S i n c e t h i s process i s i n h i b i t e d by the anion channel b l o c k e r N A P - t a u r i n e or by p r i o r t reatment o f c e l l s w i t h SITS, they suggested 2+ t h a t these compounds blocked Ca t r a n s p o r t by b l o c k i n g the o b l i g a t o r y movement o f anions through the anion c h a n n e l . However, the r e s u l t s shown 2+ i n t h i s t h e s i s i n d i c a t e t h a t N A P - t a u r i n e blocked Ca t r a n s p o r t by a 2+ 2+ d i r e c t a c t i o n on the Ca t r a n s p o r t ATPase. The i n h i b i t i o n o f Ca t r a n s p o r t o b t a i n e d wi th DIDS t r e a t e d c e l l s , however, suggests t h a t t h i s i n h i b i t i o n was probably mediated through blockade o f the anion c h a n n e l . However, an a l t e r n a t i v e e x p l a n a t i o n i s a l s o worth c o n s i d e r i n g . For example, DIDS may have r e a c t e d w i t h some amino groups on the e x t e r n a l 2+ membrane s u r f a c e i n some u n s p e c i f i e d way i n h i b i t i n g Ca t r a n s p o r t w i t h o u t 2+ i n h i b i t i n g Ca t r a n s p o r t ATPase a c t i v i t y . R e c e n t l y , N i g g l i , V . , S i g e l , E. and C a r a f o l i , E. ( J . B i o l . Chem. 257, 2 3 5 0 - 2 3 5 6 , 1982) have shown t h a t 2+ + the c a l c i u m pump c a t a l y s e s an e l e c t r o n e u t r a l exchange o f Ca - 2H . I t i s hoped t h a t f u t u r e work i n t h i s and r e l a t e d areas w i l l c l a r i f y the 2+ fundamental mechanism(s) o f the Ca t r a n s p o r t ATPase o f the human e r y t h r o c y t e membrane. CONCLUSIONS 2 + 2+ 1) The (Ca + Mg )-ATPase a c t i v i t y fn e r y t h r o c y t e membranes or T r i t o n X-100 s o l u b i l i z e d enzyme p r e p a r a t i o n showed b i p h a s i c (.high and 2+ low a f f i n i t y ) Ca a c t i v a t i o n k i n e t i c s , 2) Calmodul in i n c r e a s e d both the c a l c i u m s e n s i t i v i t y and the maximum v e l o c i t y o f ( C a 2 + + M g 2 + ) - A T P a s e . 3) C e r t a i n p o l y - L - c a r b o x y l i c a c i d s ( . p o l y - L - a s p a r t i c a c t d and p o l y - L -g l u t a m i c a c i d ) , a l i c y c l i c s u l f o n i c a c i d s (HEPES, MES) and aromatic c a r b o x y l i c a c i d s (benzoic a c i d and s a l i c y l i c a c t d ) i n c r e a s e d the 2+ 2+ c a l c i u m s e n s i t i v i t y but not the maximum v e l o c i t y o f the (Ca + flg ) -ATPase i n e r y t h r o c y t e membranes and T r i t o n X-100 s o l u b i l i z e d p r e p a -2+ r a t i o n s . I t i s suggested t h a t m o d u l a t i o n o f the Ca s e n s i t i v i t y o f 2+ 2+ (Ca + Mg )-ATPase by c a l m o d u l i n 1s due, a t l e a s t i n p a r t t o i t s a n i o n i c p r o p e r t i e s (low I s o e l e c t r i c pH) and t h a t t h i s p r o p e r t y can be mimicked by some o t h e r a n i o n s , probably by t h e i r i n t e r a c t i o n at an a n i o n - r e g u l a t o r y s i t e on the enzyme. 2+ 2+ 4) C y c l i c AMP (5 yM) i n h i b i t e d (Ca + Mg )-ATPase a c t i v i t y (approx. 20%) i n e r y t h r o c y t e membranes and t h i s e f f e c t c o u l d be blocked by c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r from the r a b b i t s k e l e t a l m u s c l e . 2+ 2+ I t i s suggested t h a t c y c l i c AMP r e g u l a t e s (Ca + Mg )-ATPase a c t i v i t y v i a an endogenous c y c l i c AMP p r o t e i n k i n a s e . 5) Bovine heart c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r * on the o t h e r hand, s t i m u l a t e d ( C a 2 + + T lg 2 + ) -ATPase a c t i v i t y (."100.%) by i n c r e a s i n g the C a 2 + s e n s i t i v i t y o f the enzyme, S t i m u l a t i o n o f ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y by bovine heart PKI (.approx, 100%) o c c u r r e d i n the presence or absence o f c y c l i c AMP and tinder c o n d i t i o n s (2 mM ATP) where c y c l i c AMP had no e f f e c t . The s u g g e s t i o n t h a t the s t i m u l a t i o n was a d i r e c t 2+ e f f e c t on the Ca t r a n s p o r t ATPase was supported by a r g l n y l group m o d i f i c a t i o n s t u d i e s . I t 1s suggested t h a t d i f f e r e n t mechanisms are i n v o l v e d i n the i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e and s t i m u l a -2+ 2+ t i o n o f (Ca + Mg )-ATPase a c t i v i t i e s by bovine h e a r t c y c l i c AMP PKI 6) In agreement w i t h the s u g g e s t i o n o f Waisman et_ al_ (.1981) and Gimble et al_(1981) i t was found t h a t the anion channel b l o c k e r 4 , 4 ' - d i i s o -2+ t h i o c y a n o - 2 , 2 ' s t i l b e n e d i s u l f o n a t e (DIDS) (5 yM) I n h i b i t e d Ca t r a n s p o r t i n i n s i d e - o u t v e s i c l e s . I t i s concluded t h a t the e f f e c t 2+ o f DIDS on the e l e c t r o g e n i c Ca pump o c c u r s v i a i t s i n h i b i t i o n o f 2+ 2+ band 3 , as DIDS (5 yM) had no e f f e c t on (Ca + Mg )-ATPase i n f r a g -mented membranes. 7) N - ( 4 - a z i d o - 2 - n i t r o p h e n y l ) - 2 aminoethyl s u l f o n a t e (a p h o t o l a b e l i n g 2+ r e a g e n t ) , was found to i n h i b i t d i r e c t l y t h e Ca - t r a n s p o r t i n i n s i d e -out v e s i c l e s . D e s p i t e i t s known e f f e c t s as an anion channel b l o c k e r , under the c o n d i t i o n s o f t h i s s t u d y , N A P - t a u r i n e (50 yM) was found to 2+ 2+ 2+ i n h i b i t d i r e c t l y (Ca + Mg )-ATPase a c t i v i t y . Mg -ATPase and ( N a + + K + ) -ATPase a c t i v i t i e s were not i n h i b i t e d a t these c o n c e n t r a -t i o n s of N A P - t a u r i n e . T 06 2+ 2+ N A P - t a u r i n e i n h i b i t s the (Ca t Mg )-ATPa?e a c t i v i t y from t h e c y t o -plasmic s i d e o f the membrane and the i n h i b i t i o n c o u l d not be p r o t e c t e d 2+ 2+ by Ca , Mg , ATP or c a l m o d u l i n . I t i s concluded t h a t a) t h e i n h l b i -2+ t i o n o f Ca t r a n s p o r t 1n 1ns1de-out v e s i c l e s by N A P - t a u r i n e i s not v i a I n h i b i t i o n o f t h e anion c h a n n e l , as proposed by Walsman et a l ( 1 9 8 1 ) ; and b) NAP-taur1ne c o u l d be used as s e l e c t i v e a n t a g o n i s t 2+ o f the Ca -pump ATPase. BIBLIOGRAPHY 107 Akyempon, C. and R o u f o g a l i s , B.D. ( 1 9 8 2 ) , K i n e t i c s o f Ca t r a n s p o r t and ( C a 2 + + M g 2 + ) - A T P a s e a c t i v i t y i n human e r y t h r o c y t e i n s i d e - o u t v e s i c l e s : M o d u l a t i o n by C a 2 + , M g 2 + , ATP and c a l m o d u l i n . C e l l C a l c i u m , i n p r e s s . 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Occurrence o f a C a 2 + and modulator p r o t e i n a c t i v a t a b l e ATPase i n the s y n a p t i c plasma membranes o f b r a i n . FEBS L e t t . 9 9 , 1 9 9 - 2 0 2 . S t a r o s , J . V . and R i c h a r d s , F.M. ( 1 9 7 4 ) . Photochemical l a b e l l i n g o f the s u r f a c e p r o t e i n s o f human e r y t h r o c y t e s . B i o c h e m i s t r y 1 3 , 2720-2726. S t e v e n s , F . C . , W a l s h , M . , Ho, H . , Teo, T . S . and Wang, J . H . ( 1 9 7 6 ) . Comparison o f c a l c i u m b i n d i n g p r o t e i n s : bovine h e a r t and b r a i n p r o t e i n a c t i v a t o r s o f c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e and r a b b i t s k e l e t a l muscle t r o p o n i n C. J . B i o l . Chem. 2 5 1 , 4495-4500. S u z u k i , K., Terao, T. and Osawa, T. ( 1 9 8 0 ) . 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' T h i r y , P . , Vandermeers, A . , V a n d e r m e e r s - P i r e t , M . C . , Rathe J . and C h r i s t o p h e r , J . ( 1 9 8 0 ) . The a c t i v a t i o n o f b r a i n a d e n y l a t e c y c l a s e and b r a i n c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e by seven c a l m o d u l i n d e r i v a -t i v e s . Eur . J . Biochem. 1 0 3 , 4 0 9 - 4 1 4 . Thomas, E . L . , K i n g , L . L . and M o r r i s o n , M. ( 1 9 7 9 ) . The uptake of c y c l i c AMP by human e r y t h r o c y t e s and i t s e f f e c t on membrane p h o s p h o r y l a t i o n . A r c h . Biochem. B i o p h y s . 1 9 6 , 4 5 9 - 4 6 4 . Tsukamoto, T . , Suyama, K. , Germann, P. and Sonenberg, M. ( 1 9 8 0 ) , Adeno-s i n e c y c l i c 3',5 1 -monophosphate uptake and r e g u l a t i o n o f membrane p r o t e i n k i n a s e i n i n t a c t human e r y t h r o c y t e s . B i o c h e m i s t r y 1 9 , 918-924. Vandermeers, A . , Vandermeers, M . 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V i c k e r s , J . D . B r i e r b y , J . and Rathbone, M.P. ( 1 9 7 9 ) . P h o s p h o r y l a t i o n o f c a s e i n by human e r y t h r o c y t e membrane bound p r o t e i n k i n a s e s : c o m p e t i -t i o n o f c a s e i n w i t h endogenous s u b s t r a t e s . J . Membr. B i o l . 4 9 , 1 2 3 - 1 3 8 . V i n c e n z i , F. ( 1 9 8 1 ) . Calmodul in pharmacology. C e l l Calcium 2 , 3 8 7 - 4 0 9 . Waisman, D . M . , G i m b l e , J . M . , Goodman, D.B. and Rasmussen, H. ( 1 9 8 1 ) . S t u d i e s on the C a 2 + t r a n s p o r t mechanism of human e r y t h r o c y t e i n s i d e out plasma membrane v e s i c l e s . J . B i o l . Chem. 256, 4 1 5 - 4 1 9 . Walsh, D.A. , Ashby, D.A., G o n z a l e z , C , C a l k i n s , D., F i s c h e r , E. and K r e b s , E.G. ( 1 9 7 1 ) . P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a p r o t e i n i n h i b i t o r o f adenosine 3',5'-monophosphate-dependent p r o t e i n k i n a s e s . J . B i o l . Chem. 246, 1977-1985. W a l s h , M. and S t e v e n s , F.C. ( 1 9 7 7 ) . Chemical m o d i f i c a t i o n s t u d i e s on the C a 2 + - d e p e n d e n t p r o t e i n modulator o f c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e . B i o c h e m i s t r y 1 6 , 2742-2749. Wang, J . H . and Desai R. (1977) Modulator b i n d i n g p r o t e i n - A bovine b r a i n p r o t e i n e x h i b i t i n g C a 2 + - d e p e n d e n t a s s o c i a t i o n . J . B i o l . Chem. 252, 4175-4184. 2+ Wang, J . H . ( 1 9 8 1 ) . S t r u c t u r e and r e g u l a t i o n o f Ca - a c t i v a t a b l e c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e . Recent advances i n C a 2 + and c e l l f u n c t i o n c a l m o d u l i n and i n t r a c e l l u l a r C a 2 + r e c e p t o r s on I n t e r n a t i o n a l Symposium K y o t o , Japan. W a t t e r s o n , D . M . , H a r r e l s o n , W . G . , K e l l e r , P . M . , S h a r i e f . F. and Vanaman, T .C. ( 1 9 7 6 ) . S t r u c t u r a l s i m i l a r i t i e s between the C a 2 + - d e p e n d e n t r e g u -l a t o r y p r o t e i n s o f 3' : 5 ' c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e and actomyosin ATPase. J . B i o l . Chem. 2 5 1 , 4 5 0 1 - 4 5 1 3 . W a t t e r s o n , D., S h a r i e f , F. , Vanaman, T . C . ( 1 9 8 0 ) . The complete amino a c i d sequence o f the C a 2 + - d e p e n d e n t modulator p r o t e i n ( c a l m o d u l i n ) o f bovine b r a i n . J . B i o l . Chem. 255, 9 6 2 - 9 7 5 . Weber, H. and Rosen, 0. ( 1 9 7 7 ) . P u r i f i c a t i o n o f a p r o t e i n i n h i b i t o r o f adenosine 3':5'-monophosphate-dependent p r o t e i n k i n a s e from bovine myocardium by a n o n - d e n a t u r i n g p r o c e d u r e . J . C y c l i c N u c l e o t i d e Res. 3 , 4 1 5 - 4 2 7 . Weed, R . I . , L a C e l l e , P . L . , M e r r i l l , E .Q. , C r a i b , G . , Gregory , A . , K a r c h , F. and P i c k e r s , E. ( 1 9 6 9 ) . M e t a b o l i c dependence of red c e l l deforma-b i l i t y . J . C l i n . I n v e s t . 4 8 , 7 9 5 - 8 0 9 . Weed, R.F. and C h a i l l e y , B. ( 1 9 7 3 ) . Calcium pH i n t e r a c t i o n s i n the p r o -d u c t i o n o f shape charges i n e r y t h r o c y t e s , i n Red C e l l Shape ( B e s s i s , M . , Weed, R.F. and L e b l o n d s , D . F . , eds. ) p p . 5 5 - 6 7 . S p r i n g e r - V e r l a g , B e r l i n. W e l l e r , M. and Morgan, I . G . ( 1 9 7 7 ) . A p o s s i b l e r o l e o f p h o s p h o r y l a t i o n o f s y n a p t i c membrane p r o t e i n s i n the c o n t r o l of c a l c i u m i o n p e r m e a b i l i -t y . B i o c h i m . B i o p h y s . Acta 4 6 5 , 527-534. Whitehouse, S . , McPherson, J . M . and W a l s h , D.A. ( 1 9 8 0 ) . C h a r a c t e r i z a t i o n o f m u l t i p l e charge isomers o f the i n h i b i t o r p r o t e i n o f c y c l i c AMP-dependent p r o t e i n k i n a s e from bovine h e a r t and r a b b i t s k e l e t a l m u s c l e . A r c h . Biochem. B i o p h y s . 2 0 3 , 7 3 4 - 7 4 3 . W h i t f i e l d , J . F . , B o y n t o n , A . L . , MacManus, J . P . , S i k o r s k a , M. and Tsang, B.K. ( 1 9 7 9 ) . The r e g u l a t i o n o f c e l l p r o l i f e r a t i o n by c a l c i u m and c y c l i c AMP. M o l . C e l l . Biochem. 2 7 , 1 5 5 - 1 7 9 . W i e r i c h s , R. and B a d e r , H. ( 1 9 8 0 ) . I n f l u e n c e o f monovalent c a t i o n s on the a c t i v i t y o f the ( C a 2 + + M g 2 + ) - A T P a s e and c a l c i u m t r a n s p o r t o f human r e d blood c e l l s . B i o c h i m . B i o p h y s . Acta 5 9 6 , 3 2 5 - 3 2 8 . W i l e y , J . S . and G i l l , F.M. ( 1 9 7 6 ) . Red c e l l c a l c i u m l e a k i n c o n g e n i t a l h e m o l y t i c anemia w i t h extreme m i c r o c y t o s i s . Blood 4 7 , 1 9 7 - 2 1 0 . Wins, P. and S c h o f f e n i e l s , E. ( 1 9 6 6 ) . S t u d i e s on the red c e l l ghost ATP-ase systems: P r o p e r t i e s o f a (Mg2+ + C a 2 + ) - d e p e n d e n t ATPase. B i o c h i m . B i o p h y s . Acta 1 2 0 , 3 4 1 - 3 5 0 . W o l f , H.V. ( 1 9 7 2 ) . E f f e c t o f e t h y l e n e d i a m i n e t e t r a a c e t a t e and deoxycholate on k i n e t i c c o n s t a n t s o f c a l c i u m i o n dependent a d e n o s i n e t r i p h o s p h c t a s e o f human e r y t h r o c y t e membrane. Biochem. J . 1 3 0 , 3 1 1 - 3 1 4 . W o l f f , D . J . P o i r i e r , P . G . , B r o s t r o m , C O . and B r o s t r o m , M.A. ( 1 9 7 7 ) . D i v a l e n t c a t i o n b i n d i n g p r o p e r t i e s o f bovine b r a i n C a 2 + - d e p e n d e n t r e g u l a t o r p r o t e i n . J . B i o l . Chem. 2 5 2 , 4108-4177. W o l o s i n , J . M . , G i n s b u r g , H. and C a b a n t c h i c k , Z . I . ( 1 9 7 7 ) . F u n c t i o n a l c h a r a c t e r i z a t i o n o f the anion t r a n s p o r t system i s o l a t e d from human e r y t h r o c y t e membranes. J . B i o l . Chem. 2 5 2 , 2419-2427. Yawata, Y . , J a c o b , H . S . , Matsumoto, N. and W h i t e , J . ( 1 9 7 6 ) . A p o s s i b l e r o l e o f c y c l i c n u c l e o t i d e s i n the r e g u l a t i o n o f e r y t h r o c y t e shape and p e r m e a b i l i t y . J . Lab. C l i n . Med. 8 8 , 5 5 5 - 5 6 2 . APPENDIX A n a l y s i s o f V a r i a n c e on Data i n F i g u r e 6 A t y p i c a l example o f the a n a l y s i s o f v a r i a n c e o f the data i s shown f o r 0.58 yM f r e e C a 2 + . C o n t r o l HEPES Calmodul in 86 280 450 K = 3 95 300 447 n T = 12 105 310 418 I = 278 110 305 435 S 2 = 166 " A " " 4 n B = 4 n c = 4 V 9 9 X B = 298 X, = 437 SV 114 SV 173 S 2 C = 211 where n^, n g , n^ r e p r e s e n t the number of r e p l i c a t i o n s f o r t r e a t m e n t ; i n t h i s case they are each f o u r . The t o t a l sample (12) i s denoted by n-p The mean o f the t h r e e t reatment means i s r e p r e s e n t e d by X; a l s o c a l l e d the grand mean. The v a r i a n c e w i t h i n each t reatment group has been computed by d i v i d i n g the sum of squared d e v i a t i o n s from each t reatment mean by (n^ - 1 ) , ( n g - 1) and n c - 1 ) , r e s p e c t i v e l y . S i n c e , the t reatment n-numbers are a l l the same ( 4 ) , the mean o f the group v a r i a n c e i s c a l c u l a t e d s i m p l y as the mean o f 114, 1 7 2 . 9 and 2 1 1 , which i s 166. This i s r e p r e -sented by S and i s c a l l e d w i t h i n the group or e r r o r v a r i a n c e . The number o f t reatment groups 1s denoted by the symbol i n t h i s case 3 . S i n c e w i t h i n - g r o u p s v a r i a n c e (S ) i s 166, t h i s becomes an e s t i m a t e 2 of o , which i s the random v a r i a t i o n i n the p o p u l a t i o n . Now, the sum o f squared d e v i a t i o n s from the grand mean or "sum o f squares" (SS) SS = (99 - 2 7 8 ) 2 + (299 - 2 7 8 ) 2 + (437 - 2 7 8 ) 2 a n H Q2 _ SS _ SS a n d SI " -RTT - T = 28,861 T h e r e f o r e , 2 ^2 = n ^X = 4 x 28,861 = 1 1 5 , 4 4 4 . 2 2 T h e r e f o r e , S which i s an e s t i m a t e o f a i s o b t a i n e d from the v a r i a b i l i t y among the t reatment means. The p r i n c i p l e o f v a r i a n c e s t a t e s t h a t , any v a r i a b i l i t y among the 2 t r e a t m e n t means c o n s i s t o f an e s t i m a t e of a p lus any e x t r a v a r i a l i t y t h a t i s due t o and o r i g i n a t i n g from d i f f e r e n c e s produced by t reatment e f f e c t s i e . F = a 2 + K a where, K i s the v a r i a b i l i t y i n the t reatment means beyond t h a t which i s 2 expected as an e s t i m a t e o f a a l o n e . 115,444  h ( 2 , 9 ) ~ 166 = 695 ( > 8 . 0 2 at 0.01 l e v e l o f s i g n i f i c a n c e ) The s u b s c r i p t s , 2 and 9 r e p r e s e n t the t reatment and w i t h i n group degrees o f freedom. Thus F i s s i g n i f i c a n t beyond 0.01 l e v e l . The h i g h l y s i g n i f i c a n t F - v a l u e , ( 6 9 5 , P < 0 . 0 0 1 ) , i n d i c a t e s s t a t i s -t i c a l s i g n i f i c a n c e e x i s t s between a t l e a s t one p a i r o f t reatment means and p o s s i b l y b o t h . We can now compare the v a r i o u s mean p a i r s t o see which are s t a t i s t i c a l l y d i f f e r e n t from w h i c h . F i r s t , we can c a l c u l a t e an e s t i m a t e of the standard e r r o r o f the 2 mean as an e s t i m a t e o f a Next we f i n d the e r r o r degrees o f freedom a s s o c i a t e d w i t h the a n a l y s i s . The Student-Neumann-Kuels Test S 2 166 = 6.44 X n 4 q ( 0 . 0 1 l e v e l ) q S Y 4 . 6 5.43 29.62 34.97 The top row c o n t a i n s K va lues r e l e v a n t t o our exper iment. For example, K = 2 i s a s s o c i a t e d w i t h any two means t h a t are a d j a c e n t , K = 3 r e f e r s to comparison o f two means t h a t are a t extremes of t h r e e means. qSy i s the minimum d i f f e r e n c e t h a t must be found between two means a s s o c i a t e d w i t h a s p e c i f i c v a l u e K. Thus, X A X B X C I 11 , l X c - X A = 338 (>34.97)* X B - X A = 199 (>29.62)* X c - X B = 139 (>29.62)* Thus, the r e s u l t s suggest t h a t HEPES and c a l m o d u l i n are somehow 2+ a s s o c i a t e d w i t h an i n c r e a s e i n the enzyme a c t i v i t y a t 0.58 yM f r e e Ca . 2+ F v a l u e s s i g n i f i c a n t at 0.01 l e v e l up t o 25 yM f r e e Ca . Beyond 100 yM f r e e C a 2 + , no s t a t i s t i c a l s i g n i f i c a n c e e x i s t s between c o n t r o l and HEPES. However, s t a t i s t i c a l s i g n i f i c a n c e e x i s t s between c o n t r o l c a l m o d u l i n membrances at a l l v a l u e s o f f r e e c a l c i u m . 

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