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Regulation of the calcium transport atpase of rat heart sarcoplasmic reticulum Mahey, Rajesh 1986

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REGULATION OF THE CALCIUM TRANSPORT ATPASE OF RAT HEART SARCOPLASMIC RETICULUM By RAJESH MAHEY B . S c , The Sunderland P o l y t e c h n i c , England, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Pharmacology and T o x i c o l o g y The F a c u l t y of Pharmaceutical Sciences We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October 1986 © Rajesh Mahey, 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f Pharmacology and Toxicology (Far.. P h a r m a r p n t i r a i Sciences) The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date December 9, 1986 ' ' ' ABSTRACT 2 + The sarcoplasmic r e t i c u l u m Ca -pumping ATPase i s the primary system r e s p o n s i b l e f o r the removal of c a l c i u m from the sarcoplasm d u r i n g r e l a x a t i o n of s k e l e t a l and c a r d i a c muscles. Since the r a t heart SR i s used f r e q u e n t l y i n our l a b o r a t o r y to 2+ 2+ study the Ca - t r a n s p o r t d e f e c t s i n d i s e a s e s t a t e s , the Ca ATPase a c t i v i t y of t h i s system was c h a r a c t e r i z e d . Calmodulin (CaM) and cAMP-dependent p r o t e i n kinase (cAMP-PK) are known to 2+ r e g u l a t e the dog c a r d i a c SR Ca -pump. The e f f e c t s of these 2+ r e g u l a t o r s on the r a t heart SR Ca -pump were s t u d i e d . S t u d i e s were a l s o c a r r i e d out to i n v e s t i g a t e the e f f e c t s of T r i t o n X-100 on SR i by CaM. 2+ . . on SR Ca -ATPase a c t i v i t y and the r e g u l a t i o n of t h i s a c t i v i t y 2+ The r a t heart SR Ca -ATPase was s t i m u l a t e d i n a 2+ 2 + concentration-dependent manner by both Ca and Mg i n the complete absence of the other c a t i o n . Magnesium produced a concentration-dependent i n c r e a s e i n the b a s a l ATPase a c t i v i t y without a f f e c t i n g the maximal ATPase a c t i v i t y . T h i s appeared to 2+ r e s u l t i n a gradual disappearance of the Ca dependency of the ATPase a c t i v i t y . A d d i t i o n of 100uM CDTA (tra n s - 1 , 2 - d i a m i n o c y c l o -hexane-N,N,N',N'-tetraacetic a c i d ) , i n the absence of added 2 + magnesium, produced no e f f e c t on Ca s t i m u l a t i o n of ATPase a c t i v i t y . The r e s u l t s appear to i n d i c a t e the presence of a low a f f i n i t y n o n - s p e c i f i c d i v a l e n t c a t i o n - s t i m u l a t e d ATPase. 2+ At a constant Mg:ATP r a t i o , ATP s t i m u l a t e d the SR Ca ATPase a c t i v i t y i n a concentration-dependent manner. Double-I l r e c i p r o c a l p l o t s of the data suggest that the t r u e s u b s t r a t e f o r 2+ r a t heart SR Ca -ATPase may be ATP and not Mg.ATP. 2+ In the crude SR, CaM d i d not s t i m u l a t e t o t a l or Ca -2+ 2+ s t i m u l a t e d ATPase a c t i v i t y over a range of Ca and Mg c o n c e n t r a t i o n s . CaM a l s o f a i l e d to s t i m u l a t e membrane phosphory-2 + l a t i o n over a range of Mg c o n c e n t r a t i o n s . Furthermore, CaM d i d not produce a s i g n i f i c a n t e f f e c t on c a l c i u m t r a n s p o r t i n t o SR v e s i c l e s . The c a t a l y t i c subunit of cAMP-dependent p r o t e i n kinase was a l s o i n e f f e c t i v e i n s t i m u l a t i n g membrane phosphoryla-2+ . . t i o n and Ca -ATPase a c t i v i t y . Two CaM a n t a g o n i s t s , t n f l u p e r a -z i n e and compound 48/80, d i d not a f f e c t the r a t heart SR ATPase a c t i v i t y . The ATPase a c t i v i t y i n Triton-washed SR membranes appeared to be i n c r e a s e d at low T r i t o n c o n c e n t r a t i o n s . T h i s e f f e c t was probably due to the removal of n o n - i n t r i n s i c p r o t e i n s , leaky v e s i c l e s or a l t e r e d membrane f l u i d i t y . At higher T r i t o n X-100 c o n c e n t r a t i o n s , the ATPase a c t i v i t y was l o s t , probably due to l o s s of the p h o s p h o l i p i d environment. When SR membranes phosphorylated under c o n d i t i o n s s i m i l a r to those used f o r the ATPase assay were analysed by SDS-PAGE (sodium dodecyl s u l p h a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ) f o l l o w e d by autoradiography, a s i n g l e phosphorylated p r o t e i n of 7,500-9,000 d a l t o n was observed. T h i s p r o t e i n may represent the monomeric form of phospholamban. CaM, however, appeared to have no e f f e c t on the p h o s p h o r y l a t i o n of t h i s 7,500-9,000 d a l t o n p r o t e i n i n e i t h e r u n t r e a t e d or Triton-washed SR membranes. I t i s i i i s p e c u l a t e d that the r a t heart SR c o n t a i n s t i g h t l y bound CaM which cannot be removed by treatment with T r i t o n X-100. TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES x ACKNOWLEDGEMENTS x i i LIST OF ABREVIATIONS x i i i DEDICATION XV INTRODUCTION 1 1. Role of Calcium i n B i o l o g i c a l Systems 1 a) H i s t o r i c a l 1 2+ b) Ca as a Second Messenger 2 2 + 2. R e g u l a t i o n of I n t r a c e l l u l a r Ca 3 3. Sarcoplasmic Reticulum and R e g u l a t i o n of 4 I n t r a c e l l u l a r Calcium a) Morphology of SR 4 b) E x c i t a t i o n - C o n t r a c t i o n C oupling i n Cardiac and 5 S k e l e t a l Muscle c) SR as the R e l a x i n g F a c t o r 7 2 + d) The SR Ca -Transport ATPase 8 2 + i ) S t r u c t u r e of SR Ca -Transport ATPase 9 2 + i i ) The Ca Transport C y c l e 11 2 + e) Phospholamban and the R e g u l a t i o n of SR Ca - 13 T r a n s p o r t ATPase OBJECTIVES OF THE PRESENT STUDY 19 MATERIALS AND METHODS 21 A. MATERIALS 21 a) Radiochemicals 21 b) Reagents 21 B. METHODS 24 1. P r e p a r a t i o n of Cardiac Microsomes E n r i c h e d i n 24 Sarcoplasmic Reticulum Method 1 . 24 Method 2. 24 2. P r e p a r a t i o n of "Triton-washed" SR Membranes 25 3. Measurement of Calcium Uptake i n t o SR V e s i c l e s 26 2+ 2 + 4. Assay of (Ca +Mg )-ATPase A c t i v i t y i n SR 27 V e s i c l e s 5. P h o s p h o r y l a t i o n of Rat Cardiac SR V e s i c l e s 29 6. Sodium Dodecyl Sulphate Polyacrylamide Gel 30 E l e c t r o p h o r e s i s (SDS-PAGE) and Autoradiography of Phosphorylated Cardiac SR P r o t e i n s Gel P r e p a r a t i o n 30 Sample P r e p a r a t i o n E l e c t r o p h o r e s i s 31 S t a i n i n g , D e s t a i n i n g and Autoradiography 32 7. P r o t e i n Assay 32 8. Determination of Free Calcium C o n c e n t r a t i o n s 33 9. S t a t i s t i c a l A n a l y s i s 34 RESULTS 35 1. C h a r a c t e r i z a t i o n of Rat Heart Sarcoplasmic 35 Reticulum Calcium Transport ATPase a) E f f e c t of SR P r o t e i n on ATPase A c t i v i t y 35 b) Time-Course f o r ATPase A c t i v i t y 35 c) E f f e c t of Ouabain, Vanadate and Sodium Azide 38 on ATPase A c t i c i t y d) Study of Calcium Transport 38 2. E f f e c t of Magnesium on the ATPase A c t i v i t y i n r a t 38 Heart SR 2 + a) E f f e c t of Magnesium on Ca - A c t i v a t i o n of 38 ATPase A c t i v i t y b) Role of Magnesium as a Co-Substrate 45 3. E f f e c t of Regulators on Rat Heart SR ATPase 45 A c t i v i t y a) E f f e c t of Calmodulin and C-subunit on ATPase 45 A c t i v i t y i n SR V e s i c l e s b) E f f e c t of Calmodulin on Calcium Uptake 48 d) E f f e c t of Calmodulin I n h i b i t o r s on ATPase 48 A c t i v i t y 4. E f f e c t of T r i t o n Treatment on ATPase A c t i v i t y and 53 the R e g u l a t i o n of t h i s A c t i v i t y a) E f f e c t of T r i t o n X-100 on SR ATPase A c t i v i t y 53 b) E f f e c t of Calmodulin on ATPase A c t i v i t y i n 55 Triton-washed Membranes 5. S t u d i e s on P h o s p h o r y l a t i o n of SR Membranes with 59 n"l »- T 4- /^ \ Y"» * - A -» +• m A T~» • or without T r i t o n Treatment a) CaM and C-subunit-Dependent P h o s p h o r y l a t i o n of SR Membranes 59 b) E f f e c t of Magnesium on P h o s p h o r y l a t i o n of 61 SR Membranes c) P h o s p h o r y l a t i o n of Triton-washed SR 61 Membranes Q u a n t i t a t i o n 61 Autoradiography 64 DISCUSSION 66 2+ 2 + 1. The Requirement of Mg f o r SR Ca -Transport 67 A c t i v i t y 2+ 2 + a) The Role of Mg i n Ca Transport 67 b) The N o n - S p e c i f i c C a 2 + (or Mg 2 +) ATPase 71 v i i 2+ 2 + 2. R e g u l a t i o n of SR Ca -ATPase and Ca Transport 73 by CaM 3. E f f e c t of Detergent Treatment on ATPase A c t i v i t y 76 and i t s R e g u l a t i o n SUMMARY AND CONCLUSIONS 80 BIBLIOGRAPHY 82 v i i i LIST OF TABLES TABLE Page 1. E f f e c t of sarcolemmal and m i t o c h o n d r i a l i n h i b i t o r s on t o t a l ATPase a c t i v i t y 39 2. The recovery of ATPase a c t i v i t y i n the I05,000xg f r a c t i o n s of T r i t o n X-100 t r e a t e d SR membranes 57 LIST OF FIGURES FIGURE Page 1. E f f e c t of changing p r o t e i n c o n c e n t r a t i o n s on (Ca 2 ++Mg 2 +)-ATPase a c t i v i t y 36 2+ 2 + 2. Time-course of (Ca +Mg )-ATPase a c t i v i t y i n r a t heart SR membranes 37 3. Time-course of c a l c i u m uptake a c t i v i t y i n r a t heart SR v e s i c l e s 40 E f f e c t of v a r i o u s p r o t e i n c a l c i u m uptake a c t i v i t y . 41 4. p r c o n c e n t r a t i o n s on 2+ 2 + 5a. E f f e c t of magnesium on (Ca +Mg )-ATPase a c t i v i t y at d i f f e r e n t c a l c i u m c o n c e n t r a t i o n s 43 2+ 5b. E f f e c t of magnesium on Ca -dependent ATPase a c t i v i t y 43 6. E f f e c t of magnesium on (Ca +Mg )-ATPase a c t i v i t y at d i f f e r e n t c a l c i u m c o n c e n t r a t i o n s i n the presence of CDTA 44 7a. E f f e c t of changing ATP c o n c e n t r a t i o n s on (Ca 2 ++Mg 2 +)-ATPase a c t i v i t y at f i x e d Mg:ATP r a t i o 46 7b. D o u b l e - r e c i p r o c a l p l o t of ATP h y d r o l y s i s versus ATP c o n c e n t a t i o n 46 7c. D o u b l e - r e c i p r o c a l p l o t of ATP h y d r o l y s i s versus Mg.ATP c o n c e n t r a t i o n 46 8. (a) - (d) E f f e c t of calmodulin on (Ca +Mg )-ATPase a c t i v i t y i n r a t heart SR 47 9. E f f e c t of the C-subunit on (Ca +Mg )-ATPase a c t i v i t y 49 10. E f f e c t of ca l m o d u l i n on c a l c i u m uptake by r a t heart SR v e s i c l e s 50 11. E f f e c t of t r i f l u o p e r a z i n e on t o t a l and b a s a l ATPase a c t i v i t i e s 51 12. E f f e c t of compound 48/80 on t o t a l and b a s a l ATPase a c t i v i t i e s 52 2+ 13. E f f e c t of magnesium on t o t a l (a) and Ca dependent ATPase (b) a c t i v i t y i n Triton-washed SR membranes 54 2 + 14. The l e v e l s of T o t a l (a) and Ca -dependent (b) ATPase a c t i v i t i e s i n I05 r000xg f r a c t i o n s of T r i t o n - t r e a t e d SR membranes 56 2+ 15. E f f e c t of calmodulin on T o t a l (a) and Ca dependent (b) ATPase a c t i v i t y i n Triton-washed SR membranes 58 16. E f f e c t of calmodulin and C-subunit on membrane ph o s p h o r y l a t i o n i n r a t heart SR i n the presence (b) and absence (a) of hydroxylamine 60 17. E f f e c t of magnesium on membrane p h o s p h o r y l a t i o n in r a t heart SR i n the presence and absence of calmodulin and hydroxylamine 62 18. E f f e c t of calmodulin on p h o s p h o r y l a t i o n of Triton-washed SR membranes i n absence (a) and presence (b) of hydroxylamine 63 3 2 19. P-Autoradiogram of SDS-PAGE of phosphorylated crude and Triton-washed SR 65 ACKNOWLEDGEMENTS I am deeply g r a t e f u l to my s u p e r v i s o r , Dr. Sidney Katz f o r a l l the guidance and support throughout t h i s study. I would l i k e to thank my committee members, Drs. B.D. R o u f o g a l i s , J . Diamond, K. MacLeod. I would a l s o l i k e to thank Dr. D. Godin f o r s u b s t i t u t i n g f o r Dr. B.D. R o u f o g a l i s i n h i s absence. I wish to thank Dr. M. Br i d g e s , Dr. D. J e f f e r y and other members of Dr. Katz's l a b o r a t o r y f o r t h e i r t e c h n i c a l a s s i s t a n c e and moral support. F i n a l l y , I wish to thank a l l members of the f a c u l t y , s t a f f , and graduate student body i n The F a c u l t y of Pharmaceutical S c i e n c e s , U.B.C. f o r making t h i s Masters program an enjoyable exper ience. x i i LIST OF ABREVIATIONS ADP adenosine 5'-diphosphate ATP adenosine 5'-triphosphate ATPase adenosine t r i p h o s p h a t a s e °C degrees C e l s i u s C 1 2 E g polyoxyethylene 9 - l a u r y l ether 2+ Ca f r e e i o n i z e d c a l c i u m 2+ 2+ (Ca +Mg )-ATPase c a l c i u m - s t i m u l a t e d , magnesium dependent ATPase CaM calmodulin cAMP adenosine 3 ' , 5 ' - c y c l i c monophosphate CDTA trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic ac i d C i c u r i e cprn counts per minute dpm d i s i n t e g r a t i o n s per minute E 1 the high a f f i n i t y s t a t e of the enzyme E 2 the low a f f i n i t y s t a t e of the enzyme E-C e x c i t a t i o n - c o n t r a c t i o n EGTA e t h y l e n e g l y c o l b i s - ( b e t a - a m i n o e t h y l ether)-N,N,N',N'-t e t r a a c e t i c a c i d 2+ 2+ EP phosphorylated i n t e r m e d i a t e of (Ca +Mg )-ATPase et a l and others g gram xg g r a v i t a t i o n a l u n i t KQ g c o n c e n t r a t i o n at half-maximal response x i i i 1 l i t r e m m i l l i M molar micro mA mi l l i a m p e r e s M.W. molecular weight min minutes n nano P p i c o PAGE pol y a c r y l a m i d e g e l e l e c t r o p h o r e s i s P. 1 i n o r g a n i c phosphate PLB phospholamban PK p r o t e i n kinase rpm r e v o l u t i o n s per minute S.A s p e c i f i c a c t i v i t y SDS sodium dodecyl sulphate S.E.M. standard e r r o r of mean SL sarcolemma SR sarcoplasmic r e t i c u l u m TCA t r i c h l o r o a c e t i c a c i d TEMED N, N, N 1, N'-tetramethylethylenediamine t r i s tris(hydroxy)aminomethane w/w weight per u n i t weight DEDICATION To my mom and dad, f o r t h e i r love and p a t i e n c e . INTRODUCTION 1. Role of Calcium i n B i o l o g i c a l Systems a ) . H i s t o r i c a l 2 + Calcium (Ca ) p l a y s a key r o l e i n many b i o l o g i c a l systems. I t began with the s e r e n d i p i d o u s d i s c o v e r y of Sydney Ringer (1883) t h a t a s o l u t i o n of sodium c h l o r i d e and tap water was more e f f i c a c i o u s i n m a i n t a i n i n g c a r d i a c c o n t r a c t i l i t y than sodium c h l o r i d e and d i s t i l l e d water; Ringer concluded that the minute amounts of c a l c i u m present i n tap water antagonized the " i n j u r i o u s " e f f e c t s of sodium. An important f i r s t s tep i n the e l u c i d a t i o n of the r o l e of ca l c i u m i n b i o l o g i c a l systems was p r o v i d e d by Mines (1911) who found t h a t c a l c i u m , s t r o n t i u m , or barium c o u l d r e v e r s e the gradual d i m i n u t i o n i n the response of f r o g gastrocnemius muscle p e r f u s e d with 0.7% NaCl to e l e c t r i c s t i m u l a t i o n . He concluded that c a l c i u m , s t r o n t i u m and barium had some s p e c i a l chemical p r o p e r t y t h a t enabled them t o i n t e r a c t with some unknown c o n s t i -tuent of the c e l l . In a d d i t i o n to i t s importance i n p r o v i d i n g r i g i d i t y by being a major c o n s t i t u e n t of bone, c a l c i u m i s i n v o l v e d i n a d i v e r s i t y of f u n c t i o n s i n b i o l o g i c a l systems. These i n c l u d e c o n t r a c t i o n of a l l forms of muscle, the metabolic processes of g l y c o g e n o l y s i s and gluconeogenesis, the m o b i l i z a t i o n and s e c r e t i o n of e x o c r i n e , endocrine and n e u r o c r i n e p r o d u c t s , the t r a n s p o r t and s e c r e t i o n of f l u i d s and e l e c t r o l y t e s and the growth of c e l l s (Rasmussen, 1986). 2+ b). Ca as a Second Messenger Calcium has been proposed as the u n i v e r s a l i n t r a c e l l u l a r messenger, l i n k i n g events at the c e l l s u r f a c e to f u n c t i o n s i n the c e l l i n t e r i o r . However, c a l c i u m a f f e c t s d i f f e r e n t systems i n 2 + d i f f e r e n t ways. For example, the manner by which Ca couples e x c i t a t i o n to c o n t r a c t i o n i n s k e l e t a l muscle i s very d i f f e r e n t from that i n smooth muscle (Rasmussen, 1986). In b r i e f c e l l u l a r responses such as n e u r o s e c r e t i o n and s k e l e t a l muscle c o n t r a c -t i o n , the magnitude and d u r a t i o n of response i s a f u n c t i o n of 2 + the magnitude and d u r a t i o n of the Ca message. However, i n s u s t a i n e d responses such as i n s u l i n s e c r e t i o n , smooth muscle c o n t r a c t i o n or a l d o s t e r o n e s e c r e t i o n , a more s u b t l e and complex 2 + r e l a t i o n s h i p e x i s t s between the i n t r a c e l l u l a r f r e e Ca and the c e l l u l a r response. 2 + Two branch s of the Ca messenger system, with d i s t i n c t temporal r o l e s , have been proposed: a calmodulin (CaM) branch (Wang and Waisman, 1979) r e s p o n s i b l e f o r e i t h e r b r i e f responses or the i n i t i a l phase of s u s t a i n e d responses and a C-kinase branch ( N i s h i z u k a , 1986) r e s p o n s i b l e f o r the s u s t a i n e d phase of 2+ c e l l u l a r responses. The Ca messenger system i s i n t i m a t e l y r e l a t e d to and m o d i f i e d by the c y c l i c AMP messenger system and the a r a c h i d o n i c a c i d cascade. 2. R e g u l a t i o n of I n t r a c e l l u l a r Ca The f u n c t i o n a l s p e c i e s of c a l c i u m i s the small pool of f r e e 2+ 2+ c y t o s o l i c Ca . The r e s t i n g i n t r a c e l l u l a r f r e e Ca c o n c e n t r a t i o n i s very low (0.1-0.3>JM) i n v i r t u a l l y a l l animal 2 + c e l l s ( B l i n k s et a l , 1982). In c o n t r a s t , the e x t r a c e l l u l a r Ca c o n c e n t r a t i o n i s approximately 1mM (Rasmussen, 1986). Thus, there i s a 4,000-10,000-fold g r a d i e n t a c r o s s the membrane. T h i s g r a d i e n t i s maintained by i ) a low p e r m e a b i l i t y of the plasma 2 + membrane to Ca ( B o r l e , 1981), i i ) a number of mechanisms to 2+ 2 + extrude Ca from the c e l l , and i i i ) i n t r a c e l l u l a r Ca se q u e s t e r i n g systems. Two types of a c t i v e t r a n s p o r t mechanisms 2+ are i n v o l v e d i n e x t r u s i o n of Ca from the c e l l , namely the 2 + + 2 + Ca -ATPase pump (Penniston, 1983) and the Na -Ca exchange system (Reeves, 1985). The r e l a t i v e importance of the two 2 + mechanisms in v a r i o u s c e l l s i s u n c e r t a i n . C y t o s o l i c f r e e Ca i s a l s o removed by two s u b c e l l u l a r o r g a n e l l e s , mitochondria and smooth endoplasmic r e t i c u l u m (Becker et a l , 1980). These 2 + o r g a n e l l e s a l s o serve as a pool of r e l e a s a b l e Ca . A l i m i t e d b u f f e r i n g c a p a c i t y i s pro v i d e d by the c y t o s o l i c b i n d i n g p r o t e i n s such as parvalbumin i n s k e l e t a l muscle (Wnuk et a l , 1982) and vi t a m i n D-induced c a l c i u m b i n d i n g p r o t e i n i n the i n t e s t i n a l columnar e p i t h e l i a l c e l l s (Wasserman and Fullmer, 1982). 3. Sarcoplasmic Reticulum and Regulation of I n t r a c e l l u l a r Calcium a ) . Morphology of SR 2 + In s k e l e t a l and c a r d i a c muscle, the SR Ca -pump i s the 2+ primary system i n v o l v e d i n the r a p i d s e q u e s t r a t i o n of Ca from the sarcoplasm d u r i n g r e l a x a t i o n (Shamoo and Ambudkar, 1984). SR can be r e l a t e d to the endoplasmic r e t i c u l u m of the other c e l l t ypes. As P o r t e r and P a l l a d e d e s c r i b e d , i t " c o n s i s t s of membrane-limited v e s i c l e s , tubules and c i s t e r n a e a s s o c i a t e d i n a continuous r e t i c u l u m s t r u c t u r e which forms f a c e - l i k e s l e e v e s around the m y o f i b r i l s . I t shows a d e f i n a b l e o r g a n i z a t i o n which repeats with each sarcomere of the f i b r e so that the e n t i r e system i s segmented i n phase with the s t r i a t i o n s of the a s s o c i a t e d m y o f i b r i l s " ( P o r t e r and P a l l a d e , 1957). I t i s d i f f e r e n t i a t e d i n t o s p e c i a l i z e d regions c o n s i s t i n g of the j u n c t i o n a l SR, mainly the t e r m i n a l c i s t e r n a e which form c o u p l i n g with the t r a n s v e r s e tubules and the l o n g i t u d i n a l ( f r e e ) SR. The t r a n s v e r s e t u b u l e s run p e r p e n d i c u l a r to the l o n g i t u d i n a l SR and form " t r i a d s " with the t e r m i n a l c i s t e r n a e . The volume of the t u b u l a r network has been estimated as 11-13% and 6-9% of the t o t a l f i b r e volume i n the f a s t s k e l e t a l muscle and c a r d i a c muscle, r e s p e c t i v e l y (Peachey, 1965; N a y l e r , 1977). Terminal c i s t e r n a e account f o r 5% of the t o t a l f i b r e volume i n the f a s t s k e l e t a l muscle (Peachey, 1965). Assuming the i n t r a v e s i c u l a r 2+ volume of 10 pl/mg SR p r o t e i n , the SR Ca c o n c e n t r a t i o n has been estimated to be approximately 1OmM (Nayler and D r e s e l , 1984). The SR i s composed of a l i p i d b i l a y e r c o n t a i n i n g phospha-t i d y l c h o l i n e (60-75%), phosphatidylethanolamine (10-20%), p h o s p h a t i d y l s e r i n e (5-10%), p h o s p h a t i d y l i n o s i t o l (10%), and other p h o s p h o l i p i d s i n smaller c o n c e n t r a t i o n s (MacLennan et a l , 1971; Meissner and F l e i s h e r , 1971). I t a l s o c o n t a i n s a number of p r o t e i n s . The ATPase p r o t e i n of M.W. 100,000 d a l t o n (Meissner et a l , 1973) accounts f o r 60-80% of t o t a l s k e l e t a l SR p r o t e i n (deMeis and I n e s i , 1982; MacLennan et a l , 1978) and 35-40% of c a r d i a c SR p r o t e i n (Suko and Hasselbach, 1976). C a l s e q u e s t r i n , an a c i d i c p r o t e i n with M.W. of 55,000 d a l t o n (Cala and Jones, 1983; Campbell et a l , 1983), two high a f f i n i t y c a l c i u m b i n d i n g p r o t e i n s (M.W. 53,000 and 160,000 d a l t o n ) , a p r o t e o l i p i d of M.W. 12,000 d a l t o n and a g l y c o p r o t e i n of 53,000 d a l t o n (Campbell et a l , 1983) are the other major p r o t e i n c o n s t i t u e n t s . b) E x c i t a t i o n - C o n t r a c t i o n Coupling i n Cardiac and S k e l e t a l Musele The d e p o l a r i z a t i o n of sarcolemma (SL) i s the i n i t i a l s tep in e x c i t a t i o n - c o n t r a c t i o n (E-C) c o u p l i n g . T h i s d e p o l a r i z a t i o n 2 + r e s u l t s i n the opening of the Ca channels i n the SL (Katz, 1985; Shamoo and Ambudkar, 1984) which allow the p a s s i v e i n f l u x 2+ of Ca down a c o n c e n t r a t i o n g r a d i e n t . However, the amount of 2 + Ca e n t e r i n g i n t o the sarcoplasm d u r i n g t h i s process i s not s u f f i c i e n t to account f o r the a c t i v a t i o n of c o n t r a c t i o n on a beat-to-beat b a s i s (Tada and I n u i , 1983). T h e r e f o r e , i t i s 2+ important t h a t an i n t r a c e l l u l a r pool of Ca be m o b i l i z e d . The 2+ SR i s b e l i e v e d to p r o v i d e most of the r e q u i r e d Ca f o r c o n t r a c t i o n . The mechanism by which e x c i t a t i o n r e s u l t s i n the r e l e a s e of 2 + Ca from the SR remains c o n t r o v e r s i a l . There i s evidence f o r 2+ 2+ two d i f f e r e n t mechanisms: a) Ca -induced Ca r e l e a s e and b) 2+ d e p o l a r i z a t i o n - i n d u c e d Ca r e l e a s e . Ford and Podolsky (1972) have shown that d u r i n g d e p o l a r i z a t i o n a s m a l l amount of the 2 + e x t r a c e l l u l a r Ca l e a k s i n t o the myofilament space causing 2+ 2+ r e l e a s e of enough Ca from the SR to r a i s e the cytoplasmic Ca l e v e l s to those r e q u i r e d f o r a c t i v a t i o n of the myofilaments. Furt h e r evidence fo r t h i s has been pr o v i d e d by F a b i a t o and F a b i a t o (1975). A c c o r d i n g to the l a t t e r theory, the d e p o l a r i z a t i o n i s passed down the t r a n s v e r s e t u b u l e s modifying 2 + the SR membrane which r e s u l t s in the r e l e a s e of Ca from SR. T h i s i s shown by a d i r e c t a l t e r a t i o n of the membrane p o t e n t i a l 2 + of SR to cause Ca r e l e a s e (Nakajima and Endo, 1973). F a b i a t o 2 + and F a b i a t o concluded that while d e p o l a r i z a t i o n - i n d u c e d Ca 2+ . r e l e a s e i s important i n s k e l e t a l muscle, the Ca -induced c a l c i u m r e l e a s e i s important in c a r d i a c muscle (Fa b i a t o and F a b i a t o , 1977). 2 + There i s evidence to show that the s i t e of Ca r e l e a s e i s the t e r m i n a l c i s t e r n a e (Huxley and T a y l o r , 1958; Winegrad, 1968). Huxley and T a y l o r showed that the l o c a l e x c i t a t i o n of a s i n g l e t r a n s v e r s e t u b u l e leads to a c o n t r a c t i o n which i s c o n f i n e d to the h a l f sarcomere. A u t o r a d i o g r a p h i c s t u d i e s by 2+ Winegrad showed that Ca i s accumulated by the l o n g i t u d i n a l SR and t r a n s p o r t e d slowly to the t e r m i n a l c i s t e r n a e and r e l e a s e d 2+ from that s i t e upon e x c i t a t i o n . Most of the SR Ca has been shown to be l o c a t e d i n the t e r m i n a l c i s t e r n a e (Winegrad, 1968). More r e c e n t l y , using a French p r e s s to d i s s o c i a t e t r a n s v e r s e t u b u l e s from SR, Ikemoto and c o l l e a g u e s (1984) concluded that the t r a n s v e r s e tubules p l a y an important r o l e i n d e p o l a r i z a t i o n -2 + induced Ca r e l e a s e . c ) . SR as the Relaxing F a c t o r 2 + Muscle r e l a x a t i o n r e s u l t s when the c y t o s o l i c Ca i s taken 2 + .up by the SR, thus lowering the c o n c e n t r a t i o n of f r e e Ca to the r e s t i n g l e v e l s of 10~ 8-10~ 7M ( B l i n k s et a l , 1982). The 2 + a b i l i t y of the SR Ca - t r a n s p o r t to c o n t r o l i n t r a c e l l u l a r f r e e 2+ 2 + Ca i s r e f l e c t e d by i t s 5-10 times gre a t e r a f f i n i t y f o r Ca 2 + showed that the b i n d i n g of Ca by a number of c h e l a t o r s c o r r e -than that of the c o n t r a c t i l e p r o t e i n s . E a r l y s t u d i e s by Ebashi b i n d i n g l a t e d w e l l with t h e i r r e l a x i n g e f f e c t s (Ebashi, 1960) i n d i c a t i n g 2 + that the r e l a x a t i o n i s caused by removal of Ca from the m y o f i b r i l environment. He l a t e r suggested that the ATP-dependent 2 + Ca b i n d i n g c a p a c i t y of " v e s i c u l a r r e l a x i n g f a c t o r " i s compara-ble to the r e l a x a t i o n produced by c h e l a t i n g agents (Ebashi, 2 + 1961). I t was l a t e r shown that i n h i b i t i o n of Ca accumulation in SR r e s u l t e d i n i n h i b i t i o n of r e l a x a t i o n (Ebashi and Lipman, 1962; Hasselbach and Makinose, 1962) and that agents that 2 + augment Ca uptake a l s o p o t e n t i a t e the r e l a x i n g e f f e c t of the SR. 2 + I n s i d e the SR, Ca i s bound to c a l s e q u e s t r i n (MacLennan and Wong, 1971; Campbell et a l , 1983). Cardiac c a l s e q u e s t r i n 2 + has a c a p a c i t y to bind 650 nmoles of Ca /mg p r o t e i n (Cala and Jones, 1983), although the s k e l e t a l c a l s e q u e s t r i n has been 2+ re p o r t e d to bind up to 970 nmoles Ca /mg p r o t e i n (Cala and Jones, 1983; Campbell et a l , 1983). The d i s t r i b u t i o n of c a l -2 + s e q u e s t r i n c o r r e l a t e s w e l l with the major Ca s t o r e s of SR, i . e . the t e r m i n a l c i s t e r n a e (Jorgensen et a l , 1979). 2 + d ) . The SR Ca -Transport ATPase 2 + How i s Ca t r a n s p o r t e d i n t o the SR lumen? I t was noted i n 2 + e a r l i e r s t u d i e s that Ca t r a n s p o r t i n t o SR v e s i c l e s r e q u i r e d ATP (Ebashi and Lipman, 1962; Hasselbach and Makinose, 1962). Hasselbach and Makinose were probably the f i r s t people to demon-2 + s t r a t e that ATP h y d o l y s i s and Ca t r a n s p o r t were l i n k e d (Hasselbach and Makinose, 1962; Hasselbach, 1964). The enzyme 2+ 2 + system r e s p o n s i b l e f o r Ca t r a n s p o r t was named Ca -dependent 2 + adenosine t r i p h o s p h a t a s e (Ca -ATPase). T h i s enzyme i s capable 2 + of t r a n s p o r t i n g two molecules of Ca f o r each molecule of ATP hydrolyzed (Hasselbach, 1978; Tada et a l , 1978; Weber, 1966). 2 + T h i s 2:1 r a t i o has been supported by Ca b i n d i n g s t u d i e s i n s o l u b l e ATPase systems (Kosk-Kosicka et a l , 1983) which showed 2+ that there were 8-10 nmoles hig h a f f i n i t y Ca b i n d i n g s i t e s per mg p r o t e i n and only 4-5 nmoles of phosphoenzyme int e r m e d i a t e per mg p r o t e i n . Other s t u d i e s have shown that the enzyme r e q u i r e s magnesium (Mg) f o r i t s a c t i v i t y and t h e r e f o r e , i t i s normally 2 + 2+ 2+ r e f e r r e d to as the (Ca +Mg )-ATPase. The Mg dependent 2 + a c t i v i t y ( i n absence of Ca ) i s r e f e r r e d to as the ' b a s a l ' 2+ a c t i v i t y while the Ca - s t i m u l a t e d a c t i v i t y i s c a l l e d the 2+ 'e x t r a ' ATPase a c t i v i t y (Hasselbach, 1964, 1978). Ca can p o t e n t i a t e the b a s a l a c t i v i t y by 7-8 f o l d (Hasselbach and 2 + Makinose, 1962). The a c t u a l process of Ca t r a n s p o r t i s coupled to the e x t r a ATPase a c t i v i t y (Hasselbach, 1978). The ATPase molecules are asymmetrically embedded i n the SR membrane (deMeis, 1981). S t a t i c d i f f r a c t i o n s t u d i e s show the ATPase molecule to be a tapered r e c t a n g l e (Herbette and B l a s i e , 1980). The wider p a r t of the r e c t a n g l e , a c c o u n t i n g f o r 62% of the mass, has been shown to be p o l a r and e x t r u d i n g to the sarcoplasmic s u r f a c e . The other p a r t , accounting f o r 38%, i s i n s i d e the l i p i d b i l a y e r (deMeis, 1981; Herbette and B l a s i e , 1980). There are e s s e n t i a l l y no extensions to the lumen of the SR. 2 + i ) . S t r u c t u r e of SR Ca -Transport ATPase R a d i a t i o n - i n a c t i v a t i o n techniques i n d i c a t e the molecular s i z e of the Ca 2 +-pump p r o t e i n to be 213,000-229,000 d a l t o n (Chamberlain et a l , 1983). T h i s molecular weight i s c o n s i s t e n t with a dimer. However, each monomer has been shown to be a b l e to 2 + perform the e n t i r e c a t a l y t i c c y c l e of ATP h y d r o l y s i s and Ca t r a n s p o r t (Moller et a l , 1982). Although Martonosi had r e p o r t e d 2 + p a r t i a l p u r i f i c a t i o n of the ATPase with Ca t r a n s p o r t and ATPase a c t i v i t y (Martonosi, 1968), the f i r s t s u c c e s s f u l p u r i f i c a t i o n of the enzyme was made from s k e l e t a l muscle by MacLennan (1970). The enzyme has been f r a c t i o n a t e d i n t o d i f f e r e n t p r o t e i n fragments. A m i l d d i g e s t i o n with t r y p s i n r e s u l t e d i n two p r o t e i n fragments: the NF^ t e r m i n a l fragment of 55,000 d a l t o n (fragment A) and the COOH t e r m i n a l fragment (fragment B) of 45,000 d a l t o n (Migala et a l , 1973; Stewart and MacLennan, 1974; Thorley-Lawson and Green, 1973). Fur t h e r d i g e s t i o n of ATPase r e s u l t e d i n a fragmentation of the 55,000 d a l t o n p e p t i d e (60,000 d a l t o n fragment of Thorley-Lawson and Green) i n t o 30,000 (fragment A ^ and 20,000 d a l t o n (fragment A 2) p r o t e i n s (Stewart et a l , 1976). A l l three fragments were t i g h t l y a s s o c i a t e d with one another and with the membrane (MacLennan e_t a l , 1976; Thorley-Lawson and Green, 1975). S a i t o et a l (1984) have r e c e n t l y r e p o r t e d a f u r t h e r t r y p s i n cleavage of the A 1 fragment i n t o A l g and A ^ and the appearance of another fragment C (M.W. 27,000-28,000 d a l t o n ) . Based on the o b s e r v a t i o n s i n the f l u o r e s c e i n i s o t h i o c y a n a t e b i n d i n g s t u d i e s , the 45,000 d a l t o n peptide has been i d e n t i f i e d to be the n u c l e o t i d e b i n d i n g p r o t e i n ( M i t c h i n s o n , 1982). D i c y c l o h e x y l c a r b o d i i m i d e b i n d i n g s t u d i e s 2+ have shown the 25,000 d a l t o n fragment to c o n t a i n the Ca bi n d i n g s i t e (Pick and Racker, 1979). The 30,000 d a l t o n fragment has been shown to c o n t a i n the p h o s p h o r y l a t i o n s i t e ( T h o r l e y -Lawson and Green, 1973). The f i r s t cleavage of ATPase i n t o A and 2+ B fragments produced no change i n the ATP h y d r o l y s i s or Ca uptake a c t i v i t i e s (Stewart and MacLennan, 1974). However, the 2+ second cleavage caused a l o s s of the Ca uptake a c t i v i t y without a f f e c t i n g ATP h y d r o l y s i s ( S c o t t and Shamoo, 1982). One major concern i s the l o s s of a c t i v i t y i n the s o l u b i l i z e d enzyme. I t was noted that the detergent removed the p h o s p h o l i p i d environment of the enzyme which was very important for the a c t i v i t y (MacLennan, 1970). The r e f o r e , attempts were made to r e c o n s t i t u t e the s o l u b i l i z e d enzyme by d i a l y s i s to remove the excess detergent and by adding exogenous l i p i d s (MacLennan et a l , 1971; Racker, 1972; Warren et a l , 1974). T h i s treatment r e s u l t e d i n the r e t u r n of a c t i v i t y to o r i g i n a l or higher l e v e l s . I t has r e c e n t l y been r e p o r t e d that a minimum of 23 p h o s p h o l i p i d s per enzyme were r e q u i r e d f o r enzyme a c t i v i t y (Hidalgo et a l , 1986). However, Dean and Tanford (1978) have repor t e d that the enzyme can be d e l i p i d a t e d down to one mole of p h o s p h o l i p i d per mole of ATPase without l o s s of a c t i v i t y . 2 + i i ) . The Ca Transport C y c l e 2 + The mechanism of Ca t r a n s p o r t by the ATPase i s now becoming c l e a r . In a recent review, Haynes (1983) has proposed a 2 + Ca t r a n s p o r t c y c l e with the f o l l o w i n g 8 s t e p s : a) b i n d i n g of 2 2+ Ca and Mg-ATP to e x t e r n a l s i t e s of the enzyme with high a f f i n i t y and random order, b) enzyme p h o s p h o r y l a t i o n , c) inward 2+ 2+ t r a n s l o c a t i o n of the Ca -laden s i t e s , d) Ca r e l e a s e to the SR lumen and ADP to the e x t e r n a l medium (random o r d e r ) , e) b i n d i n g 2+ + + of Mg or a c h a r g e - s t o i c h i o m e t r i c amount of K p l u s H to the t r a n s l o c a t o r , f ) de p h o s p h o r y l a t i o n , g) the r e t u r n of K + and H + from the t r a n s l o c a t o r to the o u t s i d e , h) d i s s o c i a t i o n of K + and H + from the t r a n s l o c a t o r and completion of the c y c l e with step 2+ a ) . A t i m e - r e s o l v e d d i f f r a c t i o n study has shown that Ca t r a n s p o r t r e s u l t s i n a s h i f t of e l e c t r o n d e n s i t y from the e x t r a -v e s i c u l a r s u r f a c e of the SR membrane to the i n t e r i o r of the 2 + membrane (Herbette and B l a s i e , 1980). The high a f f i n i t y Ca b i n d i n g p r o t e i n s (step a) have been i d e n t i f i e d to be the 20,000 d a l t o n t r y p t i c fragments of ATPase (Pick and Racker, 1979). The 2+ b i n d i n g of the f i r s t Ca leads to c o n f o r m a t i o n a l changes which 2+ r e s u l t i n an i n c r e a s e d a f f i n i t y f o r b i n d i n g of the second Ca ( I n e s i et a l , 1980). A f t e r p h o s p h o r y l a t i o n , the high a f f i n i t y . . 2+ s i t e s are converted to low a f f i n i t y s i t e s and r e l e a s e the Ca i n t o the lumen of the SR (Ikemoto, 1974). The phosphorylated i n t e r m e d i a t e (EP) i n step b) has been shown to be an a c y l phosphate (Shigekawa et a l , 1976) which can be h y d r l y s e d by hydroxylamine (Makinose, 1969). The formatio2+ 2 + of the EP i s Ca dependent and Mg causes dephosphorylation (Shigekawa et a l , 1976). 2 + A scheme proposed by Tada and Katz (1982) f o r the Ca t r a n s p o r t c y c l e i n c o r p o r a t e s these o b s e r v a t i o n s i n t o the f o l l o w i n g sequence of s t e p s : E 1 ' C a o u t Ca 2 + A Mg 2+ E 2.Mg ATP out E 1.ATP.Ca a o u t E 2.Mg fP i * D P o u t E1 P ' C a i n k v Ca Mg 2+ 2+ E 2 P.Mg H 20 in the above diagram r e p r e s e n t s the high a f f i n i t y Ca 2 + b i n d i n g s t a t e of the enzyme while E 2 r e p r e s e n t s the low a f f i n i t y s t a t e . The " i n " and "out" r e f e r to the i n s i d e and the o u t s i d e of the SR v e s i c l e s , r e s p e c t i v e l y . 2+ e ) . Phospholamban and the Re g u l a t i o n of SR Ca -Transport ATPase In the e a r l y s e v e n t i e s , cAMP-dependent p r o t e i n kinase (cAMP-PK) and cAMP were observed to produce a s t i m u l a t i o n of 2 + Ca t r a n s p o r t and ATP h y d r o l y s i s i n c a r d i a c SR (K i r c h b e r g e r et a l , 1972). T h i s s t i m u l a t i o n was shown to be due to the ph o s p h o r y l a t i o n of an SR p r o t e i n ( K i r c h b e r g e r et a l , 1974; Tada et a l , 1974), l a t e r termed phospholamban (PLB) meaning 'phosphate r e c e p t o r ' (Tada et a l , 1975). A recent study with a P L B - s p e c i f i c antibody p r o v i d e s d i r e c t evidence f o r the i n v o l v e -2+ ment of PLB as a r e g u l a t o r y p r o t e i n of the SR Ca pump (Suzuki and Wang, 1986). I t has a l s o been r e p o r t e d that calmodulin (CaM), a c a l c i u m b i n d i n g p r o t e i n present i n most c e l l s (Cheung, 1980), s t i m u l a t e d 2 + Ca uptake (Katz and Remtulla, 1978; Lopaschuk et a l , 1980) and ATPase a c t i v i t y (Lopaschuk et a l , 1980) i n the SR. U n l i k e the 2 + cAMP-PK s t i m u l a t i o n , Ca appears to be e s s e n t i a l f o r the CaM-dependent s t i m u l a t i o n . CaM s t i m u l a t i o n i s a l s o thought to be v i a the p h o s p h o r y l a t i o n of PLB (Kir c h b e r g e r and Antonetz, 1982a; 2+ LePeuch et a l , 1979). An endogenous kinase, c a l l e d Ca -CaM-dependent p r o t e i n kinase (CaM-PK) i s b e l i e v e d to be r e s p o n s i b l e for CaM-dependent p h o s p h o r y l a t i o n of PLB (Davis et a l , 1983; Ki r c h b e r g e r and Antonetz, 1982a; LePeuch et a l , 1979). CaM-PK has, very r e c e n t l y , been p a r t i a l l y p u r i f i e d and c h a r a c t e r i z e d (Molla and Demaille, 1986). I t i s i d e n t i f i e d as a 56,000 d a l t o n p r o t e i n with a broad s u b s t r a t e s p e c i f i c i t y . E a r l y o b s e r v a t i o n s i n d i c a t e d that the s t i m u l a t o r y e f f e c t s 2 + of cAMP-PK and CaM on the SR Ca t r a n s p o r t system were a d d i t i v e (Katz, 1980; Lopaschuk et a l , 1980), suggesting that d i f f e r e n t mechanisms were i n v o l v e d . T h e r e f o r e , i t was suggested that the PLB p h o s p h o r y l a t i o n occurs at separate and d i s t i n c t s i t e s ( B i l e z i k j i a n et a l , 1981; Kranias et a l , 1980). A recent r e p o r t suggests the involvement of three s i t e s , one phosphorylated by cAMP-PK and the other two by CaM-PK (imaqawa et a l , 1986). One of the CaM-PK phosphorylated s i t e s may be the same as the cAMP-PK phosphorylated s i t e . C h i e s i et a l (1983) have r e p o r t e d evidence f o r the p o s s i b i l i t y of two f u n c t i o n a l l y d i s t i n c t p r o t e o l i p i d s , one of which i s s e l e c t i v e f o r CaM and the other for cAMP-PK. Tada and Inui (1983) r e p o r t e d that the cAMP-PK-mediated p h o s p h o r y l a t i o n of PLB can be observed i n the absence of any CaM-dependent p h o s p h o r y l a t i o n . However, some workers have suggested that the CaM-PK system i s r e q u i r e d f o r the cAMP-PK-dependent p h o s p h o r y l a t i o n (LePeuch et a l 1979, Wray and Gray, 1977) and that cAMP can only a m p l i f y the CaM-dependent phospho-r y l a t i o n . Lindemann and Watanabe (1985), on the other hand, have 2+ 2+ suggested that both cAMP and Ca are r e q u i r e d f o r Ca -CaM dependent p h o s p h o r y l a t i o n . 2+ I t has r e c e n t l y been r e p o r t e d t h a t p r o t e i n kinase C (Ca phospholipid-dependent p r o t e i n kinase) a l s o s t i m u l a t e s the 2 + p h o s p h o r y l a t i o n of PLB r e s u l t i n g i n a 2 - f o l d s t i m u l a t i o n of Ca t r a n s p o r t (Movsesian et a l , 1984). The s i t e phosphorylated by t h i s kinase appears to be d i f f e r e n t from the two s i t e s phospho-r y l a t e d by cAMP-PK and CaM-PK. Movsesian et a l a l s o r e p o r t e d a common s i t e which c o u l d be phosphorylated by a l l three k i n a s e s . The PLB content has been suggested to be 3% (Caponi et a l , 1983), 4% (Tada et a l , 1979) or 6% (LePeuch et a l , 1979) of t o t a l SR p r o t e i n . It appears to be an a c i d i c p r o t e i n (Tada and I n u i , 1983) which can be e x t r a c t e d i n t o an a c i d i f i e d c h l o r o f o r m -methanol mixture ( B i d l a c k and Shamoo, 1980). However, r e c e n t l y , Jones and c o l l e a g u e s have r e p o r t e d that dephosphorylated PLB i s s t r o n g l y b a s i c and r i c h i n c y s t e i n e (Jones et a l , 1985). T h i s i s in d i r e c t c o n t r a s t to the p r e v i o u s r e p o r t s which have shown l i t t l e or no c y s t e i n e i n PLB (see Tada and I n u i , 1983). PLB 2 + appears to be s t r o n g l y bound to the Ca -ATPase molecule (Tada et a l , 1975; LePeuch et a l , 1980) with the p h o s p h o r y l a t i o n s i t e s exposed to the c y t o s o l . The s t o i c h i o m e t r y of a s s o c i a t i o n between the two p r o t e i n s i s b e l i e v e d to be 1:1 (LePeuch et a l , 1979). However, Wegener et a l (1986) have r e c e n t l y questioned t h i s 1:1 s t o i c h i o m e t r i c r a t i o and suggested a much smaller c o n c e n t r a t i o n of PLB. K r a n i a s et a l (1983) have r e p o r t e d the i s o l a t i o n of 2+ Ca -ATPase e s s e n t i a l l y f r e e of PLB, i n d i c a t i n g only a loose a s s o c i a t i o n between the two p r o t e i n s . The phosphorylated PLB d i s p l a y s c h a r a c t e r i s t i c s of a phosphoester as shown by i t s s t a b i l i t y i n hot a l k a l i and i n hydroxylamine. The phosphoryla-t i o n i n v o l v e s i n c o r p o r a t i o n of the t e r m i n a l phosphate group of ATP i n t o a s e r i n e r e s i d u e of PLB ( K i r c h b e r g e r et a l , 1974). Phospholamban has been r e p o r t e d to be a p r o t e o l i p i d with an apparent molecular weight of 20,000-25,000 d a l t o n ( B i d l a c k et a l , 1982; Jones et a l , 1985; K i r c h b e r g e r and Antonetz, 1982b; Lamers and S t i n i s , 1980; Tada et a l , 1975; Wegener and Jones, 1984; Wray and Gray, 1977). Reports have suggested PLB to be a dimer of 11,000 d a l t o n (LePeuch et a l , 1979), a t r i m e r of 11,000, 8,000 and 4,000 d a l t o n ( L o u i s et a l , 1982), a tetramer of 5,500 (Kirchberger and Antonetz, 1982b), a pentamer of 9,000 da l t o n (Wegener and Jones, 1984) or more r e c e n t l y , a pentamer of 5,000-6,000 d a l t o n ( F u j i et a l , 1986; Jones et a l , 1985). A p a r t i a l amino a c i d sequence of PLB has r e c e n t l y been r e p o r t e d ( F u j i et a l , 1 9 8 6 ) . The mechanism by which p h o s p h o r y l a t i o n of PLB leads to 2 + s t i m u l a t i o n of Ca t r a n s p o r t i s not known. P o s s i b i l i t i e s i n c l u d e , a) a c c e l e r a t i o n of dephosphorylation of the phosphory-2 + l a t e d i n t e r m e d i a t e of the Ca -ATPase (Katz et a l , 1985; Tada et a l , 1982), and b) c o n f o r m a t i o n a l changes i n the a c t i v e s i t e of 2+ . 2 + the Ca -ATPase which r e s u l t i n the t r a n s l o c a t i o n of the Ca b i n d i n g subunit from the o u t s i d e to the i n s i d e (Tada et a l , 2 + 1982). The s t o i c h i o m e t r i c r e l a t i o n s h i p of 2:1 between the Ca t r a n s p o r t e d and the ATP h y d r o l y s e d i s not a l t e r e d d u r i n g PLB-2 + mediated s t i m u l a t i o n of Ca t r a n s p o r t (Tada et a l , 1974). In a d d i t i o n to the PLB pathway, CaM has been p o s t u l a t e d to 2+ act d i r e c t l y on Ca -ATPase molecules without the mediation of PLB (Katz, 1980). T h i s c o n c l u s i o n was reached from the o b s e r v a t i o n that CaM s t i m u l a t e d the dephosphorylation of the calcium-dependent phosphoprotein intermediate of the ATPase (EP) and thus the turnover r a t e of c a l c i u m t r a n s p o r t (Katz, 1980). More r e c e n t l y , our l a b o r a t o r y has r e p o r t e d that CaM s t i m u l a t e d 2+ . . Ca -ATPase a c t i v i t y under c o n d i t i o n s where no augmentation of PLB phosphoprotein formation was seen (Katz et a l , 1985). L o u i s 2 + and M a f f i t t (1982) have a l s o observed that at Ca c o n c e n t r a -2 + t i o n s where CaM-PK i s i n a c t i v e , CaM s t i m u l a t i o n of Ca -ATPase a c t i v i t y i s present. 1 25 In I-CaM b i n d i n g experiments, L o u i s and J a r v i s observed 1 2 5 that the major product c o n t a i n i n g the I l a b e l was a 40,000 d a l t o n p r o t e i n with a minor component of 120,000 d a l t o n (Louis and J a r v i s , 1982). They suggested that the 40,000 d a l t o n 1 25 component may represent a 1:1 complex of PLB and I-CaM. I t was a l s o p o s t u l a t e d that the 120,000 d a l t o n component was due to a 1 2 + a 1:1 c r o s s - l i n k between the 100,000 d a l t o n Ca -ATPase and I-CaM. T h i s would i n d i c a t e a d i r e c t i n t e r a c t i o n of CaM with the ATPase. A d i r e c t i n t e r a c t i o n between the two p r o t e i n s i s a l s o i n d i c a t e d by the o b s e r v a t i o n s of Mas O l i v a et a l (1983) who r e p o r t e d that f o r the optimum e f f e c t of CaM, approximately 2+ e q u i v a l e n t molar c o n c e n t r a t i o n s of CaM and Ca -ATPase were r e q u i r e d . Although the evidence presented above appears c o n v i n c i n g , some r e p o r t s i n the l i t e r a t u r e c o n t r a d i c t the hy p o t h e s i s of a 2 + d i r e c t e f f e c t of CaM on SR Ca -ATPase. Using the l a b e l l e d CaM g e l o v e r l a y techniques, M o l l a et a l (1985) d e t e c t e d at l e a s t 7 CaM-binding p r o t e i n s i n SR v e s i c l e s . However, none of these was 2+ b e l i e v e d to be the Ca -ATPase. In a d d i t i o n , Caroni and C a r a f o l i 2 + (1981) have found t h a t , u n l i k e sarcolemmal Ca -ATPase, the SR 2 + Ca -ATPase f a i l e d to bind to CaM a f f i n i t y chromatography columns. OBJECTIVES OF THE PRESENT STUDY Most of the work on c a r d i a c SR Ca -ATPase has been done using dogs as the experimental model. However, s i n c e our l a b o r a t o r y i s i n t e r e s t e d i n a number of d i s e a s e models of r a t he a r t , i n c l u d i n g d i a b e t e s and t h y r o i d hormone a l t e r a t i o n s , we decided to conduct our s t u d i e s on r a t c a r d i a c SR p r e p a r a t i o n s . T h e r e f o r e , the knowledge gained i n t h i s study may be p e r t i n e n t to the d i s e a s e models. 2+ In i n i t i a l experiments, we assayed the Ca -ATPase a c t i v i t y in r a t heart SR under i d e n t i c a l c o n d i t i o n s to those used f o r dog he a r t . A high " b a s a l " ATPase a c t i v i t y was observed i n these 2+ experiments with l i t t l e or no r e p r o d u c i b l e Ca -dependent ATPase a c t i v i t y . On reviewing the l i t e r a t u r e , i t was d i s c o v e r e d that Penpargkul (1979) had r e p o r t e d t h i s u n u s u a l l y high b a s a l ATPase a c t i v i t y i n r a t heart SR. Recently, Velema et a l (1985) r e p o r t e d 2 + that i n the absence of c h e l a t o r s , Mg (0.45mM and 5.0mM) caused 2 + an apparent l o s s of Ca -dependency of the r a t heart sarcolemmal 2+ 2 + Ca -ATPase. They concluded that Mg was not e s s e n t i a l f o r SL 2+ . • Ca -ATPase a c t i v i t y . T h e r e f o r e , i n t h i s study, we i n v e s t i g a t e d 2 + the e f f e c t s of Mg on SR ATPase a c t i v i t y . 2 + As d i s c u s s e d in the INTRODUCTION, dog c a r d i a c SR Ca ATPase i s r e g u l a t e d by CaM, cAMP-PK and p r o t e i n kinase C. Although i t s p h y s i o l o g i c a l r o l e i s not known, CaM has been suggested to be important i n the beat to beat r e g u l a t i o n of 2 + c a r d i a c Ca - t r a n s p o r t ( K i r c h b e r g e r and Antonetz, 1982a) or i n c e r t a i n p a t h o l o g i c a l c o n d i t i o n s (Tada et a l , 1983). The r o l e of CaM and cAMP-PK i n the r e g u l a t i o n of r a t c a r d i a c SR Ca t r a n s p o r t has not been e l u c i d a t e d . The e f f e c t s of these 2+ p o t e n t i a l r e g u l a t o r s on SR Ca -ATPase a c t i v i t y and SR membrane ph o s p h o r y l a t i o n were t h e r e f o r e i n v e s t i g a t e d i n the c u r r e n t study. Detergent treatment of SR membranes has been shown to s o l u b i l i z e r e g u l a t o r y p r o t e i n s and a l t e r ATPase a c t i v i t y . In t h i s present i n v e s t i g a t i o n , Triton-washed membranes were s t u d i e d 2+ 2+ fo r p o s s i b l e a l t e r a t i o n s i n the Mg and Ca components of ATPase a c t i v i t y and membrane p h o s p h o r y l a t i o n . MATERIALS AND METHODS A. MATERIALS a ) . Radiochemicals: Y 3 2 P-ATP (10-40 Ci/mmole) was purchased from Amersham and 4 5 C a C ^ (10-40 mCi/mg calcium) was purchased from Amersham or New England N u c l e a r . b ) . Reagents: The f o l l o w i n g chemicals were purchased from Sigma Chemical Company: adenosine t r i p h o s p h a t e (disodium), adenosine t r i p h o s p h a t e ( t r i s ) , bovine serum albumin, Bromophenol Blue, c a t a l y t i c subunit of c y c l i c AMP dependent p r o t e i n kinase, CDTA, compound 48/80, EGTA, g l y c e r o l , g l y c i n e , L - h i s t i d i n e , hydroxylamine, magnesium c h l o r i d e , 2-mercaptoethanol, sodium a z i d e , sucrose, tetraethyl-methylenediamine (TEMED), t r i c h l o r o a c e t i c a c i d ( c r y s t a l l i n e ) t r i c h l o r o a c e t i c a c i d (100% s o l u t i o n ) , t r i f l u o p e r a z i n e , T r i s - b a s e , T r i s - h y d r o c h l o r i d e , T r i s - m a l e a t e , T r i s - o x a l a t e , T r i t o n X-100. The f o l l o w i n g chemicals were purchased from BDH Bio c h e m i c a l s : calcium c h l o r i d e , sodium b i c a r b o n a t e , sodium dodecyl sulphate, sodium hydroxide and potassium c h l o r i d e . The f o l l o w i n g e l e c t r o p h o r e s i s chemicals were obtained from Bio-Rad: acrylamide, N,N'-methylene-bis acrylamide, ammonium pe r s u l p h a t e , Coomassie B r i l l i a n t Blue R-250 and high and low molecular weight standards. The dye reagent f o r the p r o t e i n assay was a l s o purchased from Bio-Rad. Gamma-globulin was obtained e i t h e r from Calbiochem or Sigma Chemical Co. R TM C e l l o - s e a l and S c i n t i - V e r s e II were obtained from F i s h e r S c i e n t i f i c Co. Aquasol was from New England N u c l e a r . Potassium phosphate (monobasic) was purchased from Amechem and sodium c h l o r i d e was from Chemonics S c i e n t i f i c . G l a c i a l a c e t i c a c i d was from M a l l i n c k r o d t Chemical Works L t d , while a c t i v a t e d c h a r c o a l was purchased e i t h e r from BDH Biochemicals or F i s h e r S c i e n t i f i c Co. B. METHODS 1. P r e p a r a t i o n of Ca r d i a c Microsomes E n r i c h e d i n Sarcoplasmic  Reticulum Method 1. In i n i t i a l s t u d i e s , r a t c a r d i a c microsomes e n r i c h e d i n SR were prepared by a m o d i f i c a t i o n of the method of Sumida et a l (1978). The e n t i r e procedure was c a r r i e d out at 4°C. Wistar r a t s of both sexes (250-300 grammes) were k i l l e d by stunning and d i s l o c a t i o n of the neck. The h e a r t s were immediate-l y d i s s e c t e d out and p l a c e d i n i c e c o l d 0.9% NaCl. The v e n t r i c l e s were trimmed of a t r i a , b l o o d v e s s e l s , f a t and connec-t i v e t i s s u e , cut i n t o s m a l l p i e c e s and homogenized i n 25 ml lOmM T r i s - m a l e a t e , pH 6.8 with a t e f l o n p e s t l e by 5 passes at 1500 rpm. The homogenate was c e n t r i f u g e d i n 50 ml Beckman c e n t r i f u g e tubes a t 4,000xg f o r 10 minutes. The supernatant was passed through four l a y e r s of cheese c l o t h and c e n t r i f u g e d at I5,000xg f o r 20 minutes. The second supernatant was again passed through cheese c l o t h and the f i l t r a t e c e n t r i f u g e d at 40,000xg f o r 90 minutes. The r e s u l t i n g p e l l e t was resuspended i n 10 ml lOmM T r i s - m a l e a t e , pH 6.8 c o n t a i n i n g 0.6M KCl and c e n t r i f u g e d i n 15 ml Corex tubes at 40,000xg f o r 110 minutes. The f i n a l p e l l e t , e n r i c h e d i n SR, was resuspended i n a medium c o n t a i n i n g lOmM T r i s - m a l e a t e and 40% sucrose, quick f r o z e n i n l i q u i d n i t r o g e n and s t o r e d a t -80°C. Method 2. In the l a t e r s t u d i e s , the SR v e s i c l e s were pre-pared by the method of Jones et a l (1979) with s l i g h t m o d i f i c a -t i o n s . The e n t i r e procedure was c a r r i e d out at 4°C. The h e a r t s were i s o l a t e d and trimmed as before and homogenized i n Beckman 50 ml c e n t r i f u g e tubes i n 15 ml 1OmM NaHCOg, pH 7.4 f o r 3x15 seconds using a P o l y t r o n homogenizer (Kinematica PT 10-35) at a s e t t i n g of 4.0. The homogenate was d i l u t e d to 25 ml and c e n t r i -fuged at 500xg f o r 5 minutes. The supernatant was f i l t e r e d through four l a y e r s of cheese c l o t h and c e n t r i f u g e d at 7,000xg fo r 15 minutes. The p e l l e t was d i s c a r d e d and the supernatant was c e n t r i f u g e d at 31,000xg f o r 30 minutes. The p e l l e t was resus -pended i n 10 ml 30mM h i s t i d i n e - C l , pH 7.0, c o n t a i n i n g 0.6M KC1 and c e n t r i f u g e d i n 15 ml Corex tubes at 31,000xg f o r 30 minutes. The r e s u l t i n g p e l l e t , e n r i c h e d i n SR, was resuspended i n a medium c o n t a i n i n g 0.25M sucrose, 0.3M KC1 and 0.1M T r i s base, pH 7.2. These resuspended SR v e s i c l e s were quick fro z e n i n l i q u i d n i t r o g e n and s t o r e d at -80°C, p r i o r to use, normally w i t h i n one week. 2. P r e p a r a t i o n of "Triton-washed" SR Membranes The SR v e s i c l e s were d i l u t e d i n the f i n a l resuspending medium (0.25M sucrose, 0.3M KC1 and 0.1M T r i s base, pH 7.2) c o n t a i n i n g d i f f e r e n t T r i t o n X-100 c o n c e n t r a t i o n s to give the r e q u i r e d d e t e r g e n t / p r o t e i n r a t i o and incubated at 4°C f o r 60 minutes. T h i s mixture was c e n t r i f u g e d at l05,000xg f o r 60 minutes to separate the s o l u b i l i z e d p r o t e i n s from the p e l l e t . T h i s !05,000xg p e l l e t was termed the "Triton-washed" membranes. 3. Measurement of c a l c i u m uptake i n t o SR v e s i c l e s ATP-dependent c a l c i u m t r a n s p o r t i n t o c a r d i a c SR v e s i c l e s was measured by a m o d i f i c a t i o n of the method of Tada et a l (1974). The r e a c t i o n medium c o n t a i n e d 40mM h i s t i d i n e - C l , pH 6.8, 110mM KC1, 5mM MgCl 2, 2.5mM T r i s - o x a l a t e , 5mM NaN^ and 5mM ATP. The v e s i c l e s (approx. 40 ug) were pr e i n c u b a t e d with the medium f o r 6.5 minutes at 30°C and the r e a c t i o n was s t a r t e d by the a d d i t i o n of the d e s i r e d C a C l 2 s o l u t i o n s ( c o n t a i n i n g 4 5 C a C l 2 ; 2+ 200,000 dpm/sample). The d e s i r e d f r e e Ca c o n c e n t r a t i o n s were maintained by a d d i t i o n of EGTA (ethylene g l y c o l bis(/S-aminoethyl ether)-N,N' t e t r a a c e t i c a c i d ) and determined by a F o r t r a n prog-ram (see below). The r e a c t i o n was terminated a f t e r 5 minutes by f i l t e r i n g a 0.41 ml a l i q u o t of the r e a c t i o n mixture through a 0.45 urn M i l l i p o r e f i l t e r (HA 45, M i l l i p o r e R Co.). The f i l t e r was washed twice with 20 ml of 40mM T r i s - H C l , pH 7.2, d r i e d and R R counted i n Aquasol u s i n g a TRI-CARB 4530 S c i n t i l l a t i o n counter (Packard). 2 + The r a t e of Ca uptake by SR v e s i c l e s , expressed i n nanomoles c a l c i u m t r a n s p o r t e d per mg p r o t e i n per minute, was c a l c u l a t e d u s i n g the f o l l o w i n g formula: (sample counts - blank counts) X d i l u t i o n f a c t o r X t o t a l c a l c i u m ( t o t a l counts - blank counts) X i n c u b a t i o n time X mg p r o t e i n where: 45 sample counts = Ca counts (dpm) obtained per sample 45 t o t a l counts = t o t a l Ca counts (dpm) added to each tube blank counts = Ca counts (dpm) obtained i n the absence of microsomal p r o t e i n d i l u t i o n f a c t o r = c o r r e c t i o n f o r in c u b a t i o n volume sampled (=1.21) i n c u b a t i o n time = len g t h of time microsomal p r o t e i n s were incubated i n the presence of C a C l 2 (5min) t o t a l c a l c i u m = t o t a l amount of cal c i u m present i n the i n c u -b a t i o n medium (=62.5 nmoles) mg p r o t e i n = weight of microsomal p r o t e i n present i n the inc u b a t i o n medium 2+ 2 + 4. Assay of (Ca +Mg )-ATPase A c t i v i t y i n SR V e s i c l e s 2+ 2 + (Ca +Mg )-ATPase a c t i v i t y was measured by a m o d i f i c a t i o n of the method of Katz and B l o s t e i n (1975). SR v e s i c l e s (25 pq/ml) were prei n c u b a t e d fo r 5 minutes at 30°C i n a medium c o n t a i n i n g 40mM h i s t i d i n e - C l , pH 6.8, lOOmM KC1, 5mM NaN^, lOOpM 2 + EGTA, 0-2mM MgCl 2 and C a C l 2 to giv e the d e s i r e d f r e e Ca as determined by a F o r t r a n program (see below). Whenever CaM, c a t a l y t i c subunit of cAMP dependent p r o t e i n kinase, t r i f l u o -p e r a z i n e (TFP) or compound 48/80 were used, they were i n c l u d e d i n the medium. The r e a c t i o n was s t a r t e d by the a d d i t i o n of 500pM 3 2 ATP ( c o n t a i n i n g P-ATP; 200,000 dpm/sample). The r e a c t i o n was terminated a f t e r 2.5 min (except i n the time-course experiments) with i c e c o l d "TCA stop s o l u t i o n " c o n t a i n i n g 5% t r i c h l o r o a c e t i c a c i d (TCA), 5mM Na 2ATP and 2mM KH 2P0 4. A suspension of a c t i v a t e d c h a r c o a l (0.15 g per ml) was added to the samples to adsorb unhydrolyzed ATP. The samples were shaken i n an Eppendorf R Shaker 5432 (Brinkman Instruments) f o r 5 minutes followed by c e n t r i f u g a t i o n at 1500xg f o r 5 minutes. An a l i q u o t of the super-• • 32 R natant, c o n t a i n i n g l i b e r a t e d P i was counted i n Aquasol or Scinti-Verse™ I I . 2+ Ca -dependent ATPase a c t i v i t y was c a l c u l a t e d by s u b t r a c -t i n g the " b a s a l " a c t i v i t y ( i n absence of C a 2 + ) from the " t o t a l " 2+ a c t i v i t y ( i n presence of Ca ). The ATPase a c t i v i t y , expressed i n nmoles ATP h y d r o l y s e d per mg p r o t e i n per minute, was c a l c u l a t e d by the f o l l o w i n g formula: ATPase A c t i v i t y = (sample counts - background) X d i l u t i o n f a c t o r S.A. X mg p r o t e i n per sample X r e a c t i o n time i n minutes where: d i l u t i o n f a c t o r = r e a c t i o n volume + volume sto p s o l u t i o n + volume c h a r c o a l volume counted S.A. = s p e c i f i c a c t i v i t y = t o t a l cpm added to each sample nmoles ATP i n each sample 32 sample counts = P i counts (cpm) obtained per sample background = counts (cpm) obtained from s c i n t i l l a t i o n f l u i d alone 5. P h o s p h o r y l a t i o n of Rat Cardiac SR V e s i c l e s The c o n d i t i o n s f o r p h o s p h o r y l a t i o n were s i m i l a r to those used f o r the ATPase assay. SR membranes (50 pg) were p r e i n c u -bated f o r 10 minutes at 30°C i n a medium c o n t a i n i n g 40mM h i s t i d i n e - C l , pH 6.8, lOOmM KCl, 200 or 500;uM MgCl 2, 5 mM NaN^, 100;JM EGTA and 40 pM C a C l 2 (0.5JJM f r e e C a 2 + ) . CaM (3 ug/ml) or c a t a l y t i c subunit of cAMP-dependent p r o t e i n kinase (125 U n i t s per tube) were added to the d e s i r e d r e a c t i o n tubes. The r e a c t i o n 32 was i n i t i a t e d by the a d d i t i o n of 200JJM ATP ( c o n t a i n i n g P-ATP; 2x10^ dpm/sample) and was terminated a f t e r 1 minute with TCA stop s o l u t i o n (5% TCA, 5mM Na 2ATP and 2mM K H 2 P 0 4 ) . Bovine serum albumin (0.125% f i n a l c o n c e n t r a t i o n ) was added to the tubes. The tubes were mixed by v o r t e x i n g and c e n t r i f u g e d at 850xg f o r 5 min at 4°C. The supernatant was d i s c a r d e d . The p e l l e t was resuspen-ded in 0.5 ml of e i t h e r 0.6M hydroxylamine/0.8M sodium a c e t a t e , pH 5.2 or 0.6M NaCl/0.8M sodium a c e t a t e , pH 5.2 ( c o n t r o l ) . A f t e r a 10 minute i n c u b a t i o n at room temperature, 1 ml i c e c o l d 15% TCA was added to the samples and the membranes p e l l e t e d at 850xg fo r 5 min at 4°C. The supernatant was d i s c a r d e d and the p e l l e t was resuspended i n 5% TCA, a p p l i e d to g l a s s f i b r e f i l t e r s (Whatman GF/A) and washed with 30 ml 5% TCA s o l u t i o n . The 32 R f i l t e r s were d r i e d and counted f o r P i i n Aquasol or S c i n t i -TM Verse I I . 32 32 The amount of P bound, expressed i n pmoles P bound per mg p r o t e i n , was c a l c u l a t e d as f o l l o w s : 3 2 P bound = (sample counts - background) s p e c i f i c a c t i v i t y X mg p r o t e i n per sample where: . . . 32 s p e c i f i c a c t i v i t y = P counts per picomole of ATP t o t a l counts t o t a l ATP i n picomoles 32 sample counts = P counts (cpm) obtained per sample 32 t o t a l counts = t o t a l P counts added t o each tube 6. Sodium Dodecyl Sulphate P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s  (SDS-PAGE) and Autoradiography of Phosphorylated C a r d i a c SR  P r o t e i n s Gel P r e p a r a t i o n : P o l y a c r y l a m i d e s l a b g e l s (12%) were c a s t a c c o r d i n g t o the method of Laemmli and Favre (1973), using a 5% s t a c k i n g g e l . The " s e p a r a t i n g " g e l con t a i n e d 12% a c r y l a m i d e - b i s acrylamide mixture (30:0.8 w/w), 375mM T r i s - H C l b u f f e r , pH 8.8, 0.1% sodium dodecyl sulphate (SDS), 3.45% g l y c e r o l , 0.15 mg/ml ammonium pe r s u l p h a t e and 0.03% tetraethyl-methylenediamine (TEMED). A f t e r mixing, the g e l s o l u t i o n was immediately added to the g e l chamber. About 1.0 ml of d i s t i l l e d water was c a r e f u l l y l a y e r e d on top to keep the s u r f a c e of the g e l from d r y i n g and the g e l was allowed to polymerize f o r 2 hours or more at room temperature. The " s t a c k i n g " g e l c o n s i s t e d of 5% a c r y l a m i d e - b i s a c r y l -amide mixture (30:0.8 w/w), 3l5mM T r i s - C l b u f f e r , pH 6.8, 0.1% SDS, 0.4 mg/ml ammonium persulp h a t e and 0.136% TEMED. The d i s t i l l e d water was removed from the s e p a r a t i n g g e l . The s t a c k i n g g e l s o l u t i o n was mixed and very c a r e f u l l y added to the top of the s t a c k i n g g e l . A t e f l o n comb was i n s e r t e d i n t o the s t a c k i n g g e l and the g e l was allowed to polymerize at room temperature f o r at l e a s t one hour. Sample P r e p a r a t i o n and E l e c t r o p h o r e s i s : When phosphorylated SR membranes were to be analysed on SDS-PAGE fo l l o w e d by auto-radiography, the i n c u b a t i o n c o n d i t i o n s were i d e n t i c a l to those above except higher p r o t e i n c o n c e n t r a t i o n s (40-100 jug) were used 32 6 and the s p e c i f i c a c t i v i t y of P-ATP was i n c r e a s e d (4x10 cpm/sample). The r e a c t i o n was stopped with 1.0 ml i c e - c o l d 15% TCA s o l u t i o n . The samples were then c e n t r i f u g e d at I500xg f o r 10 min and the supernatant d i s c a r d e d . The p e l l e t was resuspended i n 25 u l of sample b u f f e r c o n t a i n i n g 43mM SDS, 1.25M urea, 0.l25mM d i t h i o t h r e i t o l , 12.5mM T r i s - H C l , pH 6.8 and a small amount of Bromophenol Blue. Samples were vortexed, n e u t r a l i z e d to pH 7.0 with 2M T r i s base and 60 ; j l of each sample was a p p l i e d to separate w e l l s i n the g e l . The running b u f f e r contained 0.25M T r i s b u f f e r , pH 8.3, 1.92M g l y c i n e and 1% SDS. The g e l s were run overnight at room temperature under a constant c u r r e n t of 10mA (PS 1200 DC Power Supply, Hoefer S c i e n t i f i c Instruments) per s l a b . The p r o t e i n standards used f o r e s t i m a t i o n of molecular weight ( i n d a l t o n s ) were: myosin (200,000), fl-galactosidase (116,250), phosphorylase b (92,500), bovine serum albumin (66,200), ovalbumin (45,000), c a r b o n i c anhydrase (31,000), soyabean t r y p s i n i n h i b i t o r (21,500) and lysozyme (14,400). S t a i n i n g , D e s t a i n i n g and Autoradiography: The g e l s were s t a i n e d i n a mixture c o n t a i n i n g methanol, a c e t i c a c i d , water (5:1:5) and 0.25% Coomassie B r i l l i a n t Blue R-250 f o r 30 minutes at room temperature. The g e l s were then d e s t a i n e d i n a mixture of methanol, a c e t i c a c i d and water (5:1:5) f o r one hour (3 changes) and then i n methanol:acetic acid:water (4:1:15) u n t i l the background became c l e a r . The g e l s were f i x e d i n a c e t i c a c i d : g l y c e r o l : w a t e r (10:3:87) f o r 30 minutes and then i n 70% methanol f o r 20 minutes. The g e l s were immediately d r i e d under vacuum at 80°C f o r 90 minutes and exposed to X-ray f i l m (Kodak Min-R) using an i n t e n s i f i e r screen (Cronex L i g h t n i n g P l u s , Du3 pont) f o r 7-14 days at -80°C. The f i l m s were developed to v i s u a l i z e the phosphorylated bands. 7. P r o t e i n Assay The p r o t e i n contents of the c a r d i a c SR-enriched microsomal p r e p a r a t i o n s were determined by the method of B r a d f o r d (1976) as s i m p l i f i e d by Bio-Rad (Bio-Rad P r o t e i n Assay, I n s t r u c t i o n Manual). Bovine gamma-globulin was used as the standard to y i e l d p r o t e i n ranges of 4-20 pq (micro assay) or 20-100 pq (macro a s s a y ) . A f u l l p r o t e i n c o n c e n t r a t i o n curve was produced f o r each assay. In the i n i t i a l macro assays the gamma-globulin or sample p r o t e i n was d i l u t e d to 100 u l i n d i s t i l l e d water. The d i l u t e d dye reagent (5 ml of 5 - f o l d d i l u t e d ) was added, and the tubes were read at 595 nm i n a spectrophotometer. The p r o t e i n concen-t r a t i o n s were determined by e x t r a p o l a t i o n from the standard curves. In the l a t e r experiments, the procedure was m o d i f i e d so that the e n t i r e assay c o u l d be performed i n d i s p o s a b l e c u v e t t e s . For t h i s , 1 volume of dye reagent concentrate was d i l u t e d with 3 volumes of d i s t i l l e d water i n s t e a d of a 1:4 d i l u t i o n and 3 ml of t h i s d i l u t e d dye reagent was used, i n s t e a d of 5 ml, to develop c o l o u r . The r e s t of the procedure was the same as b e f o r e . The micro assay was c a r r i e d out i n the exact manner as d e s c r i b e d i n the Bio-Rad Manual. 8. Determination of Free Calcium C o n c e n t r a t i o n s Free c a l c i u m c o n c e n t r a t i o n s were determined using the F o r t r a n program "CATIONS" w r i t t e n by G o l d s t e i n (1979). A s s o c i a -t i o n c o n s t a n t s f o r c a t i o n s and l i g a n d s were obtained from M a r t e l l and Smith (1979, 1982) and were c o r r e c t e d f o r i o n i c s t r e n g t h , pH and temperature a c c o r d i n g to the methods d e s c r i b e d by these a u t h o r s . The c o r r e c t e d l o g a s s o c i a t i o n constants of c h e l a t i n g l i g a n d s ( i n order of f i r s t to f o u r t h proton a s s o c i a -t i o n ) used were: 9.440, 8.820, 2.770 and 2.110 f o r EGTA; 12.345, 6.210, 3.640 and 2.530 f o r CDTA; 6.635, 4.125, 0.0 and 0.0 f o r ATP. A s s o c i a t i o n c o n s t a n t s f o r monoprotonated s p e c i e s were c a l c u l a t e d a c c o r d i n g to the procedure of B l i n k s et a l . (1982). Log a s s o c i a t i o n c o n s t a n t s f o r unprotonated and monoprotonated c a t i o n - l i g a n d complexes ( i n order) were: 5.341 and 3.631 f o r Mg-EGTA; 11.114 and 0.0 f o r Mg-CDTA; 4.114 and 2.128 f o r Mg-ATP; 10.762 and 5.222 f o r Ca-EGTA; 13.109 and 0.0 f o r Ca-CDTA; 3.758 and 1.923 f o r Ca-ATP. 9. S t a t i s t i c a l A n a l y s i s Whenever two means were compared, the s t a t i s t i c a l a n a l y s i s was performed using Student's unpaired t - t e s t . The n u l l -h y pothesis was r e j e c t e d only i f the two means were s i g n i f i c a n t l y d i f f e r e n t at a 0.05 or 0.01 s i g n i f i c a n c e l e v e l . RESULTS 1. C h a r a c t e r i z a t i o n of Rat Heart Sarcoplasmic Reticulum Calcium  T r a n s p o r t ATPase 2+ The r a t heart SR Ca - t r a n s p o r t ATPase has not been w e l l c h a r a c t e r i z e d . T h e r e f o r e , i n our i n i t i a l experiments we determined the optimum assay c o n d i t i o n s . a ) . E f f e c t of SR P r o t e i n C o n c e n t r a t i o n on ATPase A c t i v i t y To determine the optimum p r o t e i n c o n c e n t r a t i o n f o r the assay, the e f f e c t of a range of p r o t e i n c o n c e n t r a t i o n s (5-100 mg/ml) was i n v e s t i g a t e d . The ATPase a c t i v i t y i n c r e a s e d with p r o t e i n c o n c e n t r a t i o n i n an almost l i n e a r manner up to a concen-t r a t i o n of 35 jjg/ml and then s t a r t e d t o p l a t e a u ( f i g u r e 1). A p r o t e i n c o n c e n t r a t i o n of 25 jug/ml was s e l e c t e d f o r use i n f u t u r e experiments. T h i s c h o i c e was made s i n c e t h i s c o n c e n t r a t i o n produced a c t i v i t y i n the l i n e a r p a r t of the curve. b ) . Time-Course f o r ATPase A c t i v i t y The time-course of ATP h y d r o l y s i s was s t u d i e d i n crude SR p r e p a r a t i o n s . The r e s u l t s u s i n g SR prepared by method I are shown i n f i g u r e 2. The i n c r e a s e i n ATPase a c t i v i t y w i t h time showed l i n e a r i t y at l e a s t up t o 4 minutes. A r e a c t i o n time of 2.5 minutes was chosen f o r f u r t h e r e x perimentation. At t h i s r e a c t i o n time and SR p r o t e i n c o n c e n t r a t i o n , the ATP h y d r o l y t i c 2+ 2 + E f f e c t of changing p r o t e i n c o n c e n t r a t i o n s on (Ca +Mg )-ATPase a c t i v i t y . The ATPase a c t i v i t y , at 200^M added MgCl 2 and 7.0>iM 2+ f r e e Ca , was determined as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from two d i f f e r e n t SR p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. 2+ 2 + Time-course of (Ca +Mg )-ATPase a c t i v i t y i n the r a t heart SR p r e p a r a t i o n . The ATPase a c t i v i t y , at 200>JM added MgCl 2 and 7.0;JM 2+ f r e e Ca , was determined as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from two d i f f e r e n t SR p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. 1800-1 Time, minutes a c t i v i t y was l e s s than 15% and thus below the P i accumulation that might produce product i n h i b i t i o n and i n c r e a s e d l e v e l s of non-enzymatic h y d r o l y s i s . c) . E f f e c t of Ouabain, Vanadate and Sodium Azide on ATPase A c t i v i t y As shown i n t a b l e I, ouabain was found to have no e f f e c t on ATPase a c t i v i t y under our experimental c o n d i t i o n s . Vanadate (2uM) produced an 8.5% i n h i b i t i o n of the ATPase a c t i v i t y , while 2+ 5mM sodium a z i d e , a m i t o c h o n d r i a l Ca - t r a n s p o r t i n h i b i t o r , produced a 19% i n h i b i t i o n of ATPase a c t i v i t y . In view of these r e s u l t s showing some p o s s i b l e m i t o c h o n d r i a l contamination, sodium a z i d e was r o u t i n e l y added to the r e a c t i o n medium. d) . Study of Calcium Transport The SR p r e p a r a t i o n used was able to support o x a l a t e -2 + f a c i l i t a t e d Ca t r a n s p o r t i n d i c a t i n g that the p r e p a r a t i o n i s v e s i c u l a t e d to some degree. T h i s was shown i n time-course s t u d i e s ( f i g u r e 3) and a p r o t e i n c o n c e n t r a t i o n curve ( f i g u r e 4) 2+ f o r Ca uptake. Calcium was accumulated i n a l i n e a r manner f o r at l e a s t 15 minutes i n c u b a t i o n time at 30°C ( f i g u r e 3 ). T h i s accumulation was l i n e a r up to 80 jjg/ml p r o t e i n . 2. E f f e c t of Magnesium on the ATPase A c t i v i t y i n Rat Heart SR 2 + a ) . E f f e c t of Magnesium on Ca - A c t i v a t i o n of ATPase A c t i v i t y Table I The e f f e c t of sarcolemmal and m i t o c h o n d r i a l i n h i b i t o r s on t o t a l ATPase a c t i v i t y . The r e s u l t s represent mean of o b s e r v a t i o n s from two d i f f e r e n t experiments. I n h i b i t o r ATPase a c t i v i t y (nmoles/mg/min) Percent I n h i b i t i o n None 1mM Ouabain 5mM Sodium azid e 2uM Sodium vanadate 327.18 342.77 264.56 299.40 19.1 8.5 Time-course of c a l c i u m uptake a c t i v i t y i n r a t heart SR v e s i c l e s . 2+ • Calcium uptake, at 2pM f r e e Ca , was measured as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from two i n d i v i d u a l SR p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. 140-i E f f e c t of changing p r o t e i n c o n c e n t r a t i o n s on c a l c i u m uptake . . . . . 2+ a c t i v i t y . Calcium uptake a c t i v i t y , at 2pM f r e e Ca , was d e t e r -mined as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean + S.E.M. of o b s e r v a t i o n s from at l e a s t three d i f f e r e n t SR p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. 350H 1 1 ! 1 1 1 1 0 20 40 60 80 100 Protein Concentration, /xg/ml In e a r l y experiments we observed a high b a s a l ATPase a c t i v i t y i n our p r e p a r a t i o n s and were unable to d e t e c t a repro-2 + d u c i b l e Ca -dependent ATPase a c t i v i t y (data not shown). We, t h e r e f o r e , decided to determine the e f f e c t of magnesium on the 2 + Ca -dependent component of ATPase a c t i v i t y . F i g u r e 5a shows the 2+ . . Ca a c t i v a t i o n of ATPase a c t i v i t y at d i f f e r e n t magnesium c o n c e n t r a t i o n s (0-2mM). The ATPase was found to be a c t i v a t e d by 2+ Ca i n the complete absence of added magnesium. Co n c e n t r a t i o n s of Mg ( t o t a l added magnesium) up to 1 0 uM had l i t t l e or no e f f e c t on t h i s a c t i v a t i o n . However, higher Mg produced a c o n c e n t r a t i o n -dependent i n c r e a s e i n the b a s a l ATPase a c t i v i t y and the t o t a l 2 + ATPase a c t i v i t y at the lower Ca c o n c e n t r a t i o n s without any s i g n i f i c a n t e f f e c t on the maximal ATPase a c t i v i t y at 300uM f r e e 2 + Ca . For example, 80,uM Mg i n c r e a s e d the b a s a l ATPase a c t i v i t y 1 1 - f old as compared to zero Mg, while the t o t a l ATPase a c t i v i t y 2 + at 300uM f r e e Ca was not i n c r e a s e d . These o b s e r v a t i o n s can be 2 + i n t e r p r e t e d as a concentration-dependent i n h i b i t i o n of the Ca dependent ATPase a c t i v i t y by Mg. T h i s i s i l l u s t r a t e d i n f i g u r e 2+ 2 + 5b which shows the e f f e c t of Ca on Ca -dependent ATPase 2 + a c t i v i t y at d i f f e r e n t Mg c o n c e n t r a t i o n s . The Ca c o n c e n t r a t i o n curves s h i f t e d to the r i g h t and downward with i n c r e a s i n g Mg 2+ c o n c e n t r a t i o n . At 350JJM Mg, the Ca dependency appeared to be almost completely l o s t . 2+ To determine whether the Ca a c t i v a t i o n at zero Mg was due to endogenous f r e e Mg, 100UM CDTA was i n c l u d e d i n the r e a c t i o n medium. As seen i n f i g u r e 6, CDTA d i d not i n h i b i t t h i s a c t i v a -a ) . E f f e c t of magnesium on Ca a c t i v a t i o n of (Ca +Mg )-ATPase a c t i v i t y . The ATPase a c t i v i t y was assayed i n the absence ( A ) , or presence of 1.0uM 0 , 10uM ( X ) , 20pM ( V ) , 80pM (•), 350pM ( O ) , 1mM (•) or 2mM (•) M g C l 2 . 2+ b ) . E f f e c t of magnesium on Ca -dependent ATPase a c t i v i t y . The ATPase a c t i v i t y was assayed i n the absence ( A ) , or presence of 1.0uM (O), 20pM (V), 80>iM O , or 350JJM (O) M g C l j . The C a 2 + -ATPase a c t i v i t y was c a l c u l a t e d by s u b t r a c t i n g the b a s a l a c t i v i t y 2+ 2 + ( i n presence of Mg but no Ca ) from the t o t a l ATPase a c t i v i t y 2+ 2 + ( i n presence of Mg and Ca ). The ATPas.e a c t i v i t y was determined as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from three i n d i v i d u a l SR p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. Ca -ATPase Activity, nmoles/mg/min 2+ 2 + E f f e c t of magnesium on (Ca +Mg )-ATPase a c t i v i t y i n the prese-nce of 100JJM CDTA. The ATPase a c t i v i t y was assayed i n the absence ( A ) , or presence of 10juM ( X ) , 20;uM ( V ) , 80juM (•), 350juM (O), 1mM (•) or 2mM (•) MgCl 2. The ATPase a c t i v i t y was d e t e r -mined as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from two i n d i v i d u a l SR p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. 1800 - i t i o n of ATPase a c t i v i t y . Under these c o n d i t i o n s , 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 r e s u l t e d i n a s i m i l a r p a t t e r n of ATPase a c t i v i t y as seen i n the absence of CDTA ( f i g u r e 5a). b ) . Role of Magnesium as a Co-Substrate 2+ At zero f r e e Ca , when the Mg to ATP r a t i o was kept cons t a n t , the ATPase a c t i v i t y i n c r e a s e d i n a h y p e r b o l i c f a s h i o n 2 + ( f i g u r e 7a). The a d d i t i o n of 0.8JJM f r e e Ca r e s u l t e d i n stimu-l a t i o n of t h i s a c t i v i t y as shown by a l e f t w a r d movement of the . • . 2 + curve. An i n c r e a s e i n f r e e Ca to 7.0juM had l i t t l e f u r t h e r e f f e c t on the ATPase a c t i v i t y . A d o u b l e - r e c i p r o c a l p l o t of ATPase a c t i v i t y vs. ATP c o n c e n t r a t i o n produced s t r a i g h t l i n e s at 2 + a l l three Ca c o n c e n t r a t i o n s ( f i g u r e 7b). However, when the r e c i p r o c a l of ATPase a c t i v i t y was p l o t t e d a g a i n s t the r e c i p r o c a l of the c o n c e n t r a t i o n of the Mg.ATP complex, a n o n - l i n e a r graph was obtained ( f i g u r e 7 c ) . 3. E f f e c t of Regulators on Rat Heart SR ATPase A c t i v i t y a ) . E f f e c t of Calmodulin and C-subunit on ATPase A c t i v i t y i n SR V e s i c l e s The e f f e c t of CaM on ATPase a c t i v i t y was i n v e s t i g a t e d at 2 + v a r i o u s Ca and Mg c o n c e n t r a t i o n s . We were unable t o show a s t i m u l a t o r y e f f e c t of CaM at zero ( f i g u r e 8a), IOJUM ( f i g u r e 8b), 50JJM ( f i g u r e 8c) and 500>iM ( f i g u r e 8d) Mg and with a l l f r e e Ca 2+ 2 + a ) . E f f e c t of changing ATP c o n c e n t r a t i o n s on (Ca +Mg )-ATPase a c t i v i t y . The ATPase a c t i v i t y was assayed i n the absence ( A ) , or 2+ presence of 0.8JLJM (X) or IjM (•) f r e e Ca . The c o n c e n t r a t i o n of MgCl 2 was a d j u s t e d to give a 1:1 r a t i o of Mg:ATP. The ATPase a c t i v i t y was determined as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from two i n d i v i d u a l SR p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. b ) . D o u b l e - r e c i p r o c a l p l o t of ATP h y d r o l y s i s versus ATP concen-t r a t i o n . c ) . D o u b l e - r e c i p r o c a l p l o t of ATP h y d r o l y s i s versus Mg.ATP concentrat i o n . 2+ 2 + E f f e c t of calmodulin on (Ca +Mg )-ATPase a c t i v i t y i n r a t heart SR. The ATPase a c t i v i t y was measured as d e s c r i b e d i n Methods, i n the absence (a) or presence of 10uM (b), 50juM (c) or 500JJM (d) 2 MgCl,. The a c t i v i t y was assayed at zero ( A ) , O.ljuM ( X ) , I.ChjM (V), 10.UM (•), lOChjM (O) and 300>iM (•) f r e e C a 2 + . 700-i a ) 600-, b ) BOO-i c) 700 ^ 600 CD i o £ 500H • — > • — < 400-| O I D O 300H X 200 A CX o o \ / \ • o > .JO V / \ / » * 6 1 1 1—I—I I I I I -| 1 1 1—I I I I I 1 I 1 1 — M l l | 10 100 Calmodulin Concentration, /xg/ml c o n c e n t r a t i o n s s t u d i e d . The e f f e c t of cAMP-dependent p r o t e i n kinase (cAMP-PK) on . . 2+ the ATPase a c t i v i t y of another r e g u l a t o r of the c a r d i a c SR Ca pump was a l s o i n v e s t i g a t e d . C a t a l y t i c subunit of cAMP-PK (625 2 + units/ml) appeared to i n h i b i t the ATPase a c t i v i t y at high Ca 2+ c o n c e n t r a t i o n s with l i t t l e or no e f f e c t at low Ca c o n c e n t r a -t i o n s ( f i g u r e 9). b) . E f f e c t of Calmodulin on Calcium Uptake 2+ To determine whether CaM s t i m u l a t e d Ca - t r a n s p o r t i n the 45 2+ r a t heart SR, uptake of Ca i n t o SR v e s i c l e s was s t u d i e d i n the absence and presence of 3 ^ig/ml CaM. Under our experimental 2 + c o n d i t i o n s , CaM d i d not produce a s i g n i f i c a n t e f f e c t on Ca uptake ( f i g u r e 10). c) . E f f e c t s of Calmodulin I n h i b i t o r s on ATPase A c t i v i t y To i n v e s t i g a t e whether the l a c k of response to CaM was due to the presence of endogenous CaM, we s t u d i e d the e f f e c t of CaM a n t a g o n i s t s on ATPase a c t i v i t y . TFP had no e f f e c t on b a s a l ATPase a c t i v i t y , but produced a s l i g h t i n h i b i t i o n of t o t a l ATPase a c t i v i t y at c o n c e n t r a t i o n s up to 100JUM ( f i g u r e 11). At higher c o n c e n t r a t i o n s , the t o t a l a c t i v i t y d e c l i n e d towards the b a s a l l e v e l s . Another CaM a n t a g o n i s t , Compound 48/80 (0-100 ;ug/ml) f a i l e d to i n h i b i t the b a s a l and the t o t a l ATPase a c t i v i -t i e s ( f i g u r e 12). E f f e c t of the c a t a l y t i c - s u b u n i t of cAMP-dependent p r o t e i n kinase 2+ 2 + on (Ca +Mg )-ATPase a c t i v i t y . The ATPase a c t i v i t y , i n the absence ( A ) or presence ( X ) of c a t a l y t i c - s u b u n i t (625 u n i t s / m l ) , 2+ was measured, at 10pM added M g C l 2 and 1 OJJM f r e e Ca , as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from two i n d i v i d u a l p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. E f f e c t of calmodulin on c a l c i u m uptake by SR v e s i c l e s . The uptake a c t i v i t y was assayed i n the absence ( A ) and presence ( X ) of 3 ,ug/ml calm o d u l i n , using the procedure d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean + S.E.M. of o b s e r v a t i o n s from three i n d i v i d u a l p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. Calcium uptake, nmoles/mg protein/min O o o o o o oo o _J K>-H S3 CJ1 o o Q + E f f e c t of t r i f l u o p e r a z i n e on the b a s a l ( A ) and t o t a l ( X ) ATPase a c t i v i t y i n the r a t heart SR. The ATPase a c t i v i t y , at 1OjuM added 2 + M g C l 2 and 1 OuM f r e e Ca , was measured as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from two i n d i v i d u a l p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. E f f e c t of compound 48/80 on the b a s a l ( A ) and t o t a l ( X ) ATPase a c t i v i t y i n the r a t heart SR. The ATPase a c t i v i t y , a t 10;JM added 2+ MgCl 2 and IOJJM f r e e Ca , was measured as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from two i n d i v i d u a l p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. 4. E f f e c t of T r i t o n Treatment on ATPase A c t i v i t y and the  Reg u l a t i o n of T h i s A c t i v i t y a) E f f e c t of T r i t o n X-100 on SR ATPase A c t i v i t y The e f f e c t of three d i f f e r e n t Mg c o n c e n t r a t i o n s on ATPase a c t i v i t y was s t u d i e d i n Triton-washed membranes. A l l three Mg c o n c e n t r a t i o n s produced a s i m i l a r p a t t e r n of t o t a l ATPase a c t i v i t y ( f i g u r e 13a); the a c t i v i t y was higher at 500uM Mg compared to zero and 1OuM Mg. There was a s m a l l , but i n s i g n i f i -cant i n c r e a s e i n a c t i v i t y with a d e t e r g e n t / p r o t e i n r a t i o of 0.2 (0.005% T r i t o n at a p r o t e i n c o n c e n t r a t i o n of 0.3125 ug/ml). The a c t i v i t y d e c l i n e d s h a r p l y at d e t e r g e n t / p r o t e i n r a t i o s higher than 0.2. At a d e t e r g e n t / p r o t e i n r a t i o of 0.8, the a c t i v i t y was 10.7% (500uM Mg), 19.0% OOuMMg) and 12.4% (zero Mg) of the ATPase a c t i v i t y i n the c o n t r o l membranes. The d e c l i n e i n ATPase a c t i v i t y with T r i t o n was f a s t e r at 500JJM added Mg than zero and 10UM Mg. 2 + The Ca -dependent ATPase a c t i v i t y was h i g h e s t at zero Mg and lowest at 500uM Mg ( f i g u r e 13b). While the t o t a l ATPase a c t i v i t i e s at zero and 10;uM added Mg were almost i d e n t i c a l 2 + ( f i g u r e 13a), the Ca -dependent ATPase a c t i v i t y at 10;JM Mg was lower than at zero added Mg ( f i g u r e 13b). T h i s decrease was s i g n i f i c a n t i n c o n t r o l membranes and i n Triton-washed membranes 2+ at a T r i t o n / p r o t e i n r a t i o of 0.2. The d e c l i n e of Ca -dependent a c t i v i t y with T r i t o n was f a s t e s t at zero Mg and slowest at SOOjuM Mg. 2 + E f f e c t of magnesium on T o t a l (a) and Ca -dependent (b) ATPase a c t i v i t y i n the Triton-washed SR membranes. The Triton-washed membranes were obtained as d e s c r i b e d i n 2+ Methods. The ATPase a c t i v i t y , at lOpM f r e e Ca , was assayed i n the absence O or presence of 1 OJJM ( A ) or 500pM ( X ) MgCl 2. The r e s u l t s shown represent the mean + S.E.M. of o b s e r v a t i o n s from at l e a s t three i n d i v i d u a l p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. 1800-1 A ) F i g u r e 14a compares the t o t a l ATPase a c t i v i t y of T r i t o n -washed membranes and I05,000xg supernatant f r a c t i o n s . The t o t a l ATPase a c t i v i t y of the s o l u b i l i z e d f r a c t i o n s d e c l i n e d with i n c r e a s i n g T r i t o n c o n c e n t r a t i o n r e a c h i n g 9.3% of c o n t r o l s at a 2 + d e t e r g e n t / p r o t e i n r a t i o of 1.6. On the other hand, Ca dependent ATPase a c t i v i t y of the supernatant f r a c t i o n from membranes t r e a t e d with low T r i t o n c o n c e n t r a t i o n (detergent/pro-t e i n of 0.2) was s t i m u l a t e d 2 . 4 - f o l d ; t h i s a c t i v i t y d e c l i n e d to 23% of c o n t r o l at a d e t e r g e n t / p r o t e i n r a t i o of 1.6 ( f i g u r e 14b). Table II shows the recovery p a t t e r n of the ATPase a c t i v i t y as c a l c u l a t e d by m u l t i p l y i n g the s p e c i f i c ATPase a c t i v i t y with the t o t a l p r o t e i n recovered. The p e l l e t from membranes t r e a t e d with low detergent c o n c e n t r a t i o n s showed the highest recovery of 2 + both to.tal and Ca -dependent ATPase a c t i v i t y . The recovery d e c l i n e d a f t e r a d e t e r g e n t / p r o t e i n r a t i o of 0.4, r e a c h i n g about 10% of c o n t r o l at a d e t e r g e n t / p r o t e i n r a t i o of 1.6. The super-2 + natant f r a c t i o n showed a high recovery of Ca -dependent a c t i v i t y at a d e t e r g e n t / p r o t e i n r a t i o of 0.2 and a hi g h recovery 2 + of both t o t a l and Ca -dependent a c t i v i t i e s at high d e t e r g e n t / p r o t e i n r a t i o s i . e . 0.8. b ) . E f f e c t of Calmodulin on ATPase A c t i v i t y i n Triton-washed  Membranes When CaM was added to the r e a c t i o n medium, i n the presence 2+ . 2+ of 1OuM f r e e Ca i t f a i l e d to a l t e r the t o t a l or Ca -dependent ATPase a c t i v i t y of Triton-washed membranes ( f i g u r e 15a and 15b). 2 + The l e v e l s of T o t a l (a) and Ca -dependent (b) ATPase a c t i v i t y in I05,000xg f r a c t i o n s of T r i t o n - t r e a t e d SR membranes i n the presence of i n c r e a s i n g c o n c e n t r a t i o n s of T r i t o n X-100. The f r a c t i o n s were obtained as d e s c r i b e d i n Methods. The ATPase a c t i v i t y , i n the p e l l e t ( A ) and the supernatant ( X ) f r a c t i o n s , was measured as d e s c r i b e d i n Methods. The MgCl 2 c o n c e n t r a t i o n i n 2+ the assay medium was 200;JM and the f r e e Ca c o n c e n t r a t i o n was kept constant at 10JJM. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from two i n d i v i d u a l p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. 700-1 a ) Table II The recovery of ATPase a c t i v i t y i n the I05,000xg c e n t r i f u g a t i o n f r a c t i o n s of T r i t o n - t r e a t e d SR membranes. The data represent mean of o b s e r v a t i o n s from 2 separate p r e p a r a t i o n s . Supernatant 2+ T o t a l Ca -dep D e t e r g e n t / p r o t e i n r a t i o P e l l e t T o t a l o 2+ , Ca -dep 0 0.2 0.4 0.8 1 .6 142.56 170.20 177.17 67.86 12.74 12.90 37.39 27.46 15.12 1 .20 7.56 8.12 6.76 10.18 6.96 0.82 3.30 0.98 2.52 2.10 The ATPase recovery was c a l c u l a t e d by m u l t i p l y i n g the s p e c i f i c a c t i v i t y with the t o t a l p r o t e i n recovered and i s expressed i n micromoles per min 2 + E f f e c t of calmodulin on T o t a l (a) and Ca -dependent (b) ATPase a c t i v i t y i n Triton-washed SR membranes. The Triton-washed membranes were obtained as d e s c r i b e d i n Methods. The ATPase 2+ a c t i v i t y was assayed, at 1 QpM added M g C l 2 and 10>IM f r e e Ca , i n the absence (O) or presence (A) of 3 jjg/ml c a l m o d u l i n , as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean + S.E.M. of o b s e r v a t i o n s from three i n d i v i d u a l p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. A s t e r i s k i n d i c a t e s s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s (no calmodulin) at p < 0.05 (*) or p < 0.01 (**). 5 0 0 - i a ) CaM produced a s i g n i f i c a n t i n h i b i t i o n of the Ca -dependent ATPase a c t i v i t y i n the c o n t r o l membranes and Triton-washed membranes at the 1.6 T r i t o n / p r o t e i n r a t i o ( f i g u r e 15b). However, at i n t e r m e d i a t e T r i t o n / p r o t e i n r a t i o s , CaM d i d not produce a s i g n i f i c a n t e f f e c t on t h i s ATPase a c t i v i t y . 5. S t u d i e s on P h o s p h o r y l a t i o n of SR Membranes With and Without T r i t o n Treatment a ) . CaM and C-subunit-Dependent P h o s p h o r y l a t i o n of SR Membranes E f f e c t s of CaM and C-subunit on SR membrane p h o s p h o r y l a t i o n were i n v e s t i g a t e d i n the absence and presence of hydroxylamine to determine the e f f e c t s of the two r e g u l a t o r s on t o t a l ( a c y l and s e r i n e p h o s p h o r y l a t i o n ) and s e r i n e p h o s p h o r y l a t i o n , r e s p e c t i v e l y . Both CaM (3 ug/ml) and C-subunit (625 u n i t s per 2 + ml) had s l i g h t e f f e c t s on t o t a l p h o s p h o r y l a t i o n at zero Ca . At 2+ . 32 0.5;JM and 7. ChiM Ca , both r e g u l a t o r s s t i m u l a t e d P i n c o r p o r a -t i o n i n t o SR membrane p r o t e i n s ( f i g u r e 16a). However, only the 2+ C-subunit s t i m u l a t e d p h o s p h o r y l a t i o n at 7.0JJM f r e e Ca at s i g n i f i c a n t l y d i f f e r e n t l e v e l s from the c o n t r o l s . When both CaM and C-subunit were present, the s t i m u l a t i o n was a d d i t i v e at zero 2+ 2 + and 0.5juM f r e e Ca , while at 7.0;uM fr e e Ca , the combination r e s u l t e d i n an intermediate l e v e l of p h o s p h o r y l a t i o n . However, 2+ only a t 0.5pM Ca was t h i s p h o s p h o r y l a t i o n s i g n i f i c a n t l y d i f f e -rent from c o n t r o l . Membrane p h o s p h o r y l a t i o n was s t i m u l a t e d by 2+ 0.5/iM and 7.0uM f r e e Ca both i n the absence and presence of the r e g u l a t o r s . E f f e c t of calmodulin and the c a t a l y t i c subunit of cAMP-dependent p r o t e i n kinase on p h o s p h o r y l a t i o n of r a t heart SR i n the absence (a) or presence (b) of 0.6M hydroxylamine. P h o s p h o r y l a t i o n of SR 2+ membranes was measured, at zero, 0.5;JM or 7. OjuM f r e e Ca i n the absence of a r e g u l a t o r (£Z3) or i n the presence of 3 pg/ml calmodulin (•) , 625 u n i t s / m l C-subunit (SS) or both ( • ) . The 32 c o n c e n t r a t i o n of MgCl 2 was 200;uM. The amount of P-mcorpora-t i o n was determined as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean + S.E.M. of o b s e r v a t i o n s from three i n d i v i d u a l p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. A s t e r i s k i n d i c a t e s s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s (no r e g u l a t o r present) at p < 0.05 (*). In the presence of 0.6M hydroxylamine, 3 jjg/ml CaM produced 32 2+ no e f f e c t on P i n c o r p o r a t i o n at zero and 7.0juM f r e e Ca ( f i g u r e 16b). Although t h i s c o n c e n t r a t i o n of CaM showed some s t i m u l a t i o n of h y d r o x y l a m i n e - i n s e n s i t i v e p h o s p h o r y l a t i o n at 2+ 0.5JJM f r e e Ca , i t was not s t a t i s t i c a l l y s i g n i f i c a n t . C-subunit appeared to i n h i b i t h y d r o x y l a m i n e - i n s e n s i t i v e p h o s p h o r y l a t i o n at 2+ zero and 7.OuM f r e e Ca . However, t h i s i n h i b i t i o n was not s t a t i s t i c a l l y s i g n i f i c a n t . b ) . E f f e c t of Magnesium on Ph o s p h o r y l a t i o n of SR Membranes F i g u r e 17 shows the e f f e c t of changing Mg c o n c e n t r a t i o n on the p h o s p h o r y l a t i o n of SR membranes i n the presence and absence 32 of 0.6M hydroxylamine. P i n c o r p o r a t i o n was s t i m u l a t e d by Mg i n a concentration-dependent manner both i n the absence and presence of hydroxylamine. H y d r o x y l a m i n e - i n s e n s i t i v e phosphory-l a t i o n appeared to be more s e n s i t i v e than t o t a l p h o s p h o r y l a t i o n as shown by s t i m u l a t i o n at lower Mg c o n c e n t r a t i o n s . CaM had.no e f f e c t on e i t h e r t o t a l or h y d r o x y l a m i n e - i n s e n s i t i v e phosphoryla-t i o n . c ) . P h o s p h o r y l a t i o n of Triton-washed SR Membranes Quant i t a t i o n : CaM-dependent p h o s p h o r y l a t i o n of T r i t o n -washed SR was s t u d i e d i n the presence and absence (NaCl c o n t r o l ) of 0.6M hydroxylamine. In the absence of hydroxylamine ( f i g u r e 32 18a), P i n c o r p o r a t i o n i n t o Triton-washed membranes decreased E f f e c t of magnesium on p h o s p h o r y l a t i o n of SR membrane p r o t e i n s i n the absence (A, X) and presence Q O ) of 0.6M hydroxylamine. 32 I n c o r p o r a t i o n of P i n t o SR membrane p r o t e i n s , i n the absence (A, • ) or presence (X, O ) of 3 pg/ml calmodulin was measured at 2 + 0.5;JM f r e e Ca , as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean + S.E.M. of o b s e r v a t i o n s from three i n d i v i d u a l p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. E f f e c t of calmodulin on p h o s p h o r y l a t i o n of Triton-washed SR membranes i n the absence (a) or presence (b) of 0.6M h y d r o x y l -32 amine. The i n c o r p o r a t i o n of P i n t o SR membrane p r o t e i n s , i n the absence (A) or presence (X) of 3 ug/ml calmodulin, was 2 + measured at 500uM MgCl 2 and 0. 5uM f r e e Ca , as d e s c r i b e d i n Methods. The r e s u l t s shown represent the mean of o b s e r v a t i o n s from three i n d i v i d u a l p r e p a r a t i o n s prepared and assayed on d i f f e r e n t days. 800-1 a ) 0.5 1 1.5 Triton X-100/Protein Ratio s l i g h t l y at 0.2 T r i t o n / p r o t e i n r a t i o and i n c r e a s e d at higher d e t e r g e n t / p r o t e i n r a t i o s . However, none of these changes were s i g n i f i c a n t when compared with membranes that were not t r e a t e d 32 with T r i t o n . CaM had no s i g n i f i c a n t e f f e c t on the amount of P i n c o r p o r a t e d . The h y d r o x y l a m i n e - i n s e n s i t i v e p h o s p h o r y l a t i o n showed a s i m i l a r p a t t e r n ( f i g u r e 18b). Again, higher T r i t o n c o n c e n t r a -t i o n s appeared to in c r e a s e the h y d r o x y l a m i n e - i n s e n s i t i v e p h o s p h o r y l a t i o n . However, t h i s i n c r e a s e was not s i g n i f i c a n t l y d i f f e r e n t from membranes washed i n c o n t r o l b u f f e r . CaM appeared to produce an i n h i b i t i o n of p h o s p h o r y l a t i o n , but t h i s i n h i b i t i o n was not s i g n i f i c a n t . Autoradiography: Gel e l e c t r o p h o r e s i s f o l l o w e d by a u t o r a d i o -graphy of the phosphorylated membranes showed that a low molecu-l a r weight p r o t e i n (Mr approx. 9,000) was phosphorylated i n crude SR membranes ( f i g u r e 19, lane 1). CaM (3 ug/ml) had no e f f e c t on the amount or the m i g r a t i o n p r o p e r t i e s of t h i s p r o t e i n ( f i g u r e 19, lane 2). The amount of p h o s p h o r y l a t i o n of T r i t o n -washed membranes appeared to i n c r e a s e with T r i t o n c o n c e n t r a t i o n s up to 0.4 mg Triton/mg p r o t e i n ( f i g u r e 19, lanes 3, 5 and 7). No p h o s p h o r y l a t i o n was observed i n the supernatant f r a c t i o n of these low T r i t o n - t r e a t e d membranes ( f i g u r e 19, lanes 4, 6 and 8). However, at 0.8 and 1.6 T r i t o n / p r o t e i n r a t i o s ( f i g u r e 23, lanes 10 and 12, r e s p e c t i v e l y ) , a p r o t e i n of 7,500 d a l t o n was phosphorylated i n the supernatant f r a c t i o n s , while the 9,000 d a l t o n p r o t e i n i n Triton-washed membranes g r a d u a l l y disappeared (lanes 9 and 11). 32 P-Autoradiogram of SDS-polyacrylamide g e l electrophoretogram of SR and Triton-washed membrane p r o t e i n s phosphorylated with 32, 2 + Ca ), e l e c t r o p h o r e s i s and autoradiography were c a r r i e d out as "P-ATP. The p h o s p h o r y l a t i o n (at 500;JM M g C l 2 and 0.5.UM f r e e d e s c r i b e d i n Methods. R e s u l t s shown are t y p i c a l of two d i f f e r e n t experiments. The lanes r e p r e s e n t : 1 - crude SR i n absence of CaM, 2 - crude SR i n presence of 3ug/ml CaM, 3 - membranes washed i n zero T r i t o n , 4 - supernatant from zero T r i t o n - t r e a t e d membranes, 5 - membranes washed i n 0.2 mg Triton/mg p r o t e i n , 6 - supernatant from membranes t r e a t e d with 0.2 mg Triton/mg p r o t e i n , 7 - membranes washed i n 0.4 mg Triton/mg p r o t e i n , 8 - supernatant from membranes t r e a t e d with 0.4 mg Triton/mg p r o t e i n , 9 - membranes washed i n 0.8 mg Triton/mg p r o t e i n , 10 - supernatant from membranes t r e a t e d with 0.8 mg Triton/mg p r o t e i n , 11 - membranes washed i n 1.6 mg Triton/mg p r o t e i n , 12 - supernatant from membranes t r e a t e d with 1.6 mg Triton/mg p r o t e i n , M Wt kDa 200 92.5 45 31 21.5 14.4 4 * Lane # 1 2 3 4 5 6 7 8 9 10 11 12 DISCUSSION I t i s now we l l e s t a b l i s h e d that the c o n t r a c t i l e process i n s k e l e t a l and c a r d i a c muscle i s terminated by the a c t i v e removal of f r e e c a l c i u m from the sarcoplasm v i a the sarcoplasmic r e t i c u -lum c a l c i u m pump. Most of the e a r l y knowledge i n t h i s area came from work with s k e l e t a l muscle. More r e c e n t l y , d i f f e r e n c e s 2 + between the s k e l e t a l and c a r d i a c SR Ca -pump have been shown. For example, the SR from the two sources d i s p l a y a d i f f e r e n t 2 + a f f i n i t y f o r Ca ( K Q 5 of 4.7;jM f o r c a r d i a c and 1 . 3JJM f o r s k e l e t a l SR; Shamoo and Ambudkar, 1984). In a d d i t i o n , the r a t i o 2 + of Ca -pump p r o t e i n to t o t a l p r o t e i n i s d i f f e r e n t (approx. 40% in c a r d i a c -- Suko and Hasselbach, 1976; and 60-80% i n s k e l e t a l SR -- deMeis and I n e s i , 1982). There a l s o appears to be a marked 2 + d i f f e r e n c e i n the r e g u l a t i o n of the Ca -pump and the r o l e of the p r o t e i n , phospholamban. The m a j o r i t y of the pr e v i o u s s t u d i e s have been done on dog c a r d i a c muscle SR. I t i s normally assumed that c a r d i a c SR from a l l s p e c i e s would be s i m i l a r with re s p e c t 2+ to the Ca -pumping ATPase. However, our i n i t i a l experiments with r a t heart SR proved that t h i s i s not the case. The assay 2+ . . of Ca -ATPase a c t i v i t y i n r a t heart SR using c o n d i t i o n s i d e n t i c a l to those used with dog heart SR re v e a l e d a high b a s a l 2 + ATPase a c t i v i t y with l i t t l e or no Ca -dependent a c t i v i t y (data not shown). T h i s prompted us to i n v e s t i g a t e the e f f e c t on t h i s a c t i v i t y of the m o d i f i c a t i o n of our assay parameters; i n 2+ 2+ p a r t i c u l a r , the requirement of SR Ca -ATPase f o r Mg and i t s r e g u l a t i o n by CaM was i n v e s t i g a t e d . 2+ 2 + 1. The Requirement of Mg f o r SR Ca -Transport A c t i v i t y 2+ 2 + a ) . The Role of Mg i n Ca -Transport The r a t heart SR p r e p a r a t i o n used i n our s t u d i e s appears to be very a c t i v e . P r o t e i n c o n c e n t r a t i o n s as low as 25 pq/ml (5 ;jg per tube) produced s i g n i f i c a n t ATP h y d r o l y s i s at short r e a c t i o n times (2.5 min). T h i s i s a l s o s u b s t a n t i a t e d by the f a c t that the 2 + 100,000 d a l t o n p r o t e i n corresponding to the Ca -ATPase molecule appears to be a very small percentage of the t o t a l SR p r o t e i n i n our p r e p a r a t i o n (data not shown). The amount of p r o t e i n used i n our assays i s very small compared to that used i n other s t u d i e s (Kirchberger and Antonetz, 1982a; L e v i t s k y et a l , 1976; Lopaschuk et a l , 1980). 2 + In our p r e p a r a t i o n , the Ca -ATPase c o u l d be f u l l y a c t i -2+ 2+ vated by Ca i n the complete absence of added Mg . T h i s o b s e r v a t i o n c o u l d not be e x p l a i n e d i n l i g h t of the c u r r e n t l y 2 + accepted view that Mg i s i n v o l v e d i n the t r a n s p o r t c y c l e of 2+ 2+ . the SR Ca pump. Mg i s thought to be i n v o l v e d in two ways: 1) the "tru e s u b s t r a t e " for the ATPase enzyme i s thought to be Mg.ATP (Makinose and B o l l , 1979; Vianna, 1975) and 2) f r e e Mg 2 + i s thought to be e s s e n t i a l f o r dephosp h o r y l a t i o n of the enzyme (Kanazawa et a1, 1971; Makinose and B o l l , 1979; Souza and deMeis, 1976). Vianna (1975) repo r t e d t h a t i f Mg.ATP i s the tr u e s u b s t r a t e , the d o u b l e - r e c i p r o c a l p l o t between ATP (when Mg:ATP r a t i o i s 1:1) and ATPase a c t i v i t y would be n o n - l i n e a r . However, when our data, showing the dependence of ATP h y d r o l y s i s on ATP (at constant Mg:ATP r a t i o of 1:1), was p l o t t e d on a double-r e c i p r o c a l p l o t , a l i n e a r graph was obtained. Furthermore, a s i m i l a r p l o t of ATPase a c t i v i t y v s . Mg.ATP was n o n - l i n e a r . These 2 + r e s u l t s i n d i c a t e that i n r a t heart SR Ca -ATPase ATP may be the tru e s u b s t r a t e i n s t e a d of Mg.ATP. The r a t c a r d i a c SR ATPase appeared to be s t i m u l a t e d i n a 2 + concentration-dependent manner to maximal a c t i v i t y by Mg or 2 + Ca , in the complete absence of the other c a t i o n . Thus, when both c a t i o n s were present, any i n c r e a s e i n ATPase a c t i v i t y due 2+ 2+ to i n c r e a s e d Mg r e s u l t e d i n a l o s s of Ca -dependent a c t i v i t y . These r e s u l t s e x p l a i n the high b a s a l a c t i v i t y and the absence of 2 + Ca -dependent a c t i v i t y i n our i n i t i a l experiments where 500 uM * 1- _ — 1 t m n . ^ .! _ ' i 2 + Mg was r o u t i n e l y used. A high b a s a l ATPase a c t i v i t y i n r a t heart SR has p r e v i o u s l y been r e p o r t e d (Nayler et a l , 1975; Penpargkul, 1979). However, an e x p l a n a t i o n f o r t h i s o b s e r v a t i o n was not pr o v i d e d . Nayler et a l (1975) compared the r a t SR ATPase a c t i v i t y with that of guinea-pig and found an almost 3 - f o l d d i f f e r e n c e i n the b a s a l ATPase a c t i v i t i e s of the two s p e c i e s . Rat heart has a l s o been reported to d i f f e r from other s p e c i e s 2+ with respect to i t s Ca accumulating a c t i v i t y and c o n t r a c t i o n and r e l a x a t i o n c h a r a c t e r i s t i c s (Penpargkul, 1979). T h i s appears to be a s p e c i e s d i f f e r e n c e and not a phenomenon of the r a t heart SR, s i n c e Velema et a l (1985) have r e p o r t e d very s i m i l a r r e s u l t s 2+ in r a t heart sarcolemmal p r e p a r a t i o n s . They found that Ca was 2+ ab l e to a c t i v a t e the sarcolemmal Ca -ATPase i n the absence of 2+ 2+ 2+ added Mg ; upon a d d i t i o n of 0.45mM or 5 mM f r e e Mg the Ca dependency was completely l o s t . No c h e l a t o r system was used i n 2+ these experiments; t h e r e f o r e , t r a c e amounts of endogenous Mg 2+ may have been pres e n t . In the presence of a c h e l a t o r , Mg 2 + produced a v a r i a b l e e f f e c t on the Ca a f f i n i t y depending on the 2+ c h e l a t o r and the Ca c o n c e n t r a t i o n s used. The reason f o r not observing the same e f f e c t i n the presence of c h e l a t o r s may be the high c o n c e n t r a t i o n of c h e l a t o r s (2mM) used i n t h e i r study. At such a high c o n c e n t r a t i o n , c h e l a t o r s may have t h e i r own e f f e c t on the ATPase a c t i v i t y . 2+ . 2+ The requirement of Mg i n the Ca t r a n s p o r t c y c l e of the 2 + SR Ca -pump has r e c e n t l y been questioned. The f i n d i n g s of 2 + Velema et a l (1985) as w e l l as ours i n d i c a t e that Mg i s not 2+ r e q u i r e d f o r ATP h y d r o l y s i s by r a t heart SR and SL Ca -ATPases. S i m i l a r c o n c l u s i o n s were drawn by Shigekawa et a l (1983) i n 2 + p u r i f i e d r a b b i t s k e l e t a l muscle SR Ca -ATPase; using Ca.ATP as 2+ a s u b s t r a t e i n s t e a d of Mg.ATP, they showed that Mg was not 2 + r e q u i r e d f o r the turnover of the Ca -pump ATPase. In t h e i r s t u d i e s , ATP h y d r o l y s i s c o u l d be observed with Ca.ATP as sub-2+ . s t r a t e with no Mg p r e s e n t . ATP h y d r o l y s i s and c o n v e r s i o n of E 1 P to E 2 P was slower with Ca.ATP compared to Mg.ATP. The 2 + c o n v e r s i o n of E ^ to E 2 P i s suggested to be one of the Mg 2 + r e q u i r i n g steps i n the Ca t r a n s p o r t c y c l e (Shigekawa and Dougherty, 1978). Ca.ATP and Mg.ATP appear to act as competi-t i v e i n h i b i t o r s of each other (Shigekawa et a l 1983; Vianna, 1975). Yamamoto et a l (1985) have shown that i n C l 2 E g -s o l u b i l i z e d SR ATPase, ATP can be h y d r o l y s e d without going 2 + through the Mg -dependent --> E 2 P s t e p . They suggested that E ^ may be d i r e c t l y h y d r o l y s e d to E and P^. However, Shigekawa et a l (1983) r e p o r t e d that i n the presence of Ca.ATP, the c o n v e r s i o n of E ^ to E 2 P does take p l a c e , but slowly, l e a d i n g to a slow turnover r a t e . These obsevations support our r e s u l t s 2 + which show a decreased Ca dependence of ATPase a c t i v i t y with 2+ 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 (see f i g u r e 3b). If the E ^ --> E 2 P c o n v e r s i o n i s indeed stopped or slowed 2+ down, Ca - t r a n s p o r t would be i n t e r r u p t e d s i n c e t h i s i s an 2 + e s s e n t i a l step f o r d e l i v e r i n g bound Ca to the i n t e r i o r of the SR (Tada and Katz, 1982). A recent report by Costa and Madeira 2 + (1986) suggests that Mg i s r e q u i r e d f o r the c o u p l i n g of ATP 2 + h y d r o l y s i s to Ca t r a n s p o r t , while ATP h y d r o l y s i s i s indepen-2 + dent of Mg . I t should be mentioned, though, that these authors d i d not c l e a r l y e x p l a i n t h e i r methodology and d i d not i n d i c a t e 2+ the source of the SR p r e p a r a t i o n used. In a d d i t i o n , the Mg c o n c e n t r a t i o n s used (0.2-50mM) in t h e i r study appeared to be high. 2+ Mg has been proposed as the c o u n t e r - t r a n s p o r t e d c a t i o n d u r i n g c a l c i u m uptake by SR (Kanazawa et a l , 1971). However, in 2+ t h e i r s t u d i e s , C h i e s i and I n e s i (1980) showed that Mg was not 2 + c o u n t e r - t r a n s p o r t e d . Ueno and Sekine (1978) observed Ca 2+ t r a n s p o r t i n r a b b i t s k e l e t a l muscle SR i n the absence of Mg Salama and Scarpa (1985) provided f u r t h e r evidence f o r t h i s by 2+ . . showing that Mg was not r e l e a s e d from SR v e s i c l e s d u r i n g 2+ a c t i v e Ca t r a n s p o r t . 2+ 2 + b ) . The N o n - S p e c i f i c Ca (or Mg )-ATPase An a l t e r n a t i v e e x p l a n a t i o n f o r the c o m p e t i t i v e nature of 2+ 2+ Mg and Ca s t i m u l a t i o n of r a t heart SR ATPase, can be p r o v i d e d . There i s a strong p o s s i b i l i t y t h a t we have been stud y i n g a low a f f i n i t y n o n - s p e c i f i c d i v a l e n t c a t i o n - s t i m u l a t e d ATPase which has been shown to be present i n plasma membrane p r e p a r a t i o n s of d i f f e r e n t t i s s u e s (Iwasa et a l , 1982; L o t e r s z t a j n et a l , 1981; Pershadsingh and McDonald, 1980; Verma and Penniston, 1981). L o t e r s z t a j n and c o l l e a g u e s showed that the b a s a l ATPase a c t i v i t y i n n a t i v e r a t l i v e r plasma membranes was 2+ 2 + d r a m a t i c a l l y i n c r e a s e d by 5JJM Mg and that 50uM Mg r e s u l t e d 2+ rn a complete l o s s of Ca s t i m u l a t i o n . Verma and Penniston r e p o r t e d that i n t h e i r i n i t i a l experiments with r a t corpus 2+ 2 + luteum ATPase using 6mM Mg , Ca d i d not produce a s t i m u l a t i o n above the b a s a l a c t i v i t y . These r e s u l t s are e x p l a i n e d by the presence of a n o n - s p e c i f i c d i v a l e n t c a t i o n - s t i m u l a t e d ATPase 2 + ( a l s o c a l l e d Mg -ATPase). T h i s ATPase has a l s o been demon-s t r a t e d in r a t kidney cortex (Parkinson and Radde, 1971), r a t adi p o c y t e plasma membranes (Pershadsingh and McDonald, 1980), and r a t osteosarcoma plasma membranes (Murray et a l , 1983). 2 + Furthermore, a high a f f i n i t y Ca -ATPase has been i s o l a t e d from plasma membrane p r e p a r a t i o n s of most of these t i s s u e s (Iwasa et a l , 1982; L o t e r s z t a j n et a l , 1981; Verma and Penniston, 1981). The ' n o n - s p e c i f i c ' ATPase present appears to hinder the a c t i v i t y of the high a f f i n i t y ATPase when s t u d i e d i n the n a t i v e plasma 2 + membranes under high Mg c o n d i t i o n s ( L o t e r s z t a j n et a l , 1981). 2+ 2+ The high a f f i n i t y Ca -ATPase i s probably the Ca -pumping enzyme (Verma and Penniston, 1981). The l a t t e r ATPase from a l l three t i s s u e sources showed maximal s t i m u l a t i o n of ATPase • • 2+ a c t i v i t y at 0.5-1.0uM f r e e Ca and no requirement f o r exogenous 2+ 2+ Mg . However, small amounts of Mg were r e q u i r e d f o r t h i s a c t i v i t y . T h i s requirement i s normally s a t i s f i e d by endogenous 2 + Mg as shown by a complete i n h i b i t i o n of the ATPase a c t i v i t y by CDTA (Pershadsingh and McDonald, 1980; Verma and Penniston, 1981). In our s t u d i e s , however, 100JJM CDTA f a i l e d to i n h i b i t the ATPase a c t i v i t y . One e x p l a n a t i o n may be that the c o n c e n t r a t i o n 2+ of CDTA used i n our study was too low to c h e l a t e a l l the Mg present i n the r e a c t i o n medium. The r e f o r e , i t i s p o s s i b l e that i n our study, the a c t i v i t y 2+ of the high a f f i n i t y Ca -pumping ATPase was masked by a low a f f i n i t y n o n - s p e c i f i c ATPase. F u r t h e r i n v e s t i g a t i o n u t i l i z i n g 2+ 2 + lower f r e e Ca c o n c e n t r a t i o n s (0-1.0JJM) and no added Mg would be necessary to determine t h i s p o s s i b i l i t y . From the above d i s c u s s i o n i t appears that the n o n - s p e c i f i c ATPase may be s p e c i e s - s p e c i f i c and not t i s s u e - s p e c i f i c s i n c e a l l the s t u d i e s d i s c u s s e d above used r a t t i s s u e s . A s i m i l a r ATPase has a l s o been r e p o r t e d i n r a t l i v e r endoplasmic r e t i c u l u m (Kraus-Friedman, S a t e l l i t e symposium of the 30th congress of IUPS, S e a t t l e , 2 + 1986). Furthermore, Ca -ATPases from other s p e c i e s , of both plasma membrane and SR o r i g i n , have been shown to be a c t i v e i n 2 + the presence of nigh Mg The ATPase a c t i v i t i e s r e p o r t e d here were completely i n s e n s i t i v e to ouabain, i n d i c a t i n g that the ATP h y d r o l y s i s was not due to Na +/K +-ATPase a c t i v i t y . Vanadate (2pH) produced an 8.5% i n h i b i t i o n of the ATPase a c t i v i t y . T h i s c o n c e n t r a t i o n of vanadate has been shown to almost completely i n h i b i t the s arco-2+ 2+ lemmal Ca -ATPase, while i n h i b i t i n g only 2% of the SR Ca ATPase (Caroni and C a r a f o l i , 1981). T h e r e f o r e , i t appears that the sarcolemmal contamination of our SR p r e p a r a t i o n was minimal. The i n h i b i t o r y e f f e c t of sodium a z i d e i n d i c a t e d that the major contaminant of our SR p r e p a r a t i o n was mitochondria. However, s i n c e 5mM sodium azid e was present i n a l l our assay media, the m i t o c h o n d r i a l ATPase made l i t t l e c o n t r i b u t i o n to the t o t a l a c t i v i t y . A s i m i l a r n o n - s p e c i f i c ATPase has been reported i n r a t p a n c r e a t i c plasma membranes (Ansah et a l , 1984; Forget and H e i s l e y , 1976; Hamlyn and S e n i o r , 1983; Hurley et a l , 1984). However, t h i s ATPase has been i d e n t i f i e d to be a diphosphohydro-l a s e due to i t s a b i l i t y to h y d r o l y s e a l l n u c l e o t i d e d i - and t r i - p h o s p h a t e s . The n o n - s p e c i f i c ATPase d e s c r i b e d above, however, i s more s e l e c t i v e towards n u c l e o t i d e t r i p h o s p h a t e s (Parkinson and Radde, 1971). 2+ 2 + 2. R e g u l a t i o n of SR Ca -ATPase and Ca Transport by CaM One of the major d i f f e r e n c e s between s k e l e t a l muscle and 2 + c a r d i a c SR i s that c a r d i a c SR Ca - t r a n s p o r t i s r e g u l a t e d by cAMP-dependent p r o t e i n kinase, CaM (Tada and I n u i , 1983) and p r o t e i n kinase C (Movsesian et a l , 1984). cAMP-mediated r e g u l a -t i o n i s thought to be r e s p o n s i b l e f o r the i n o t r o p i c e f f e c t s of catecholamines (Tada and Katz, 1982). The p h y s i o l o g i c a l relevance of CaM and C-kinase-mediated r e g u l a t i o n i s not known. 2+ CaM r e g u l a t i o n may be o p e r a t i o n a l i n Ca -o v e r l o a d c o n d i t i o n s 2 + (Tada et a l , 1983) or beat to beat c o n t r o l of Ca t r a n s p o r t ( K i r c h b e r g e r and Antonetz, 1982a). The second p a r t of our study was concerned with an i n v e s t i g a t i o n of the mechanism of r e g u l a -2+ t i o n of Ca - t r a n s p o r t i n rat heart SR. In our s t u d i e s , CaM from two d i f f e r e n t commercial sources f a i l e d to s t i m u l a t e the ATPase a c t i v i t y at low and high Mg 2 + c o n c e n t r a t i o n s (0-500JJM) and d i f f e r e n t Ca c o n c e n t r a t i o n s . Furthermore, CaM d i d not produce a s i g n i f i c a n t e f f e c t on 45 2+ . t r a n s p o r t of Ca i n t o SR v e s i c l e s . T h i s , together with the lack of s t i m u l a t i o n of t o t a l and h y d r o x y l a m i n e - r e s i s t a n t membrane p h o s p h o r y l a t i o n by CaM at a range of Mg c o n c e n t r a t i o n s , 2 + p r o v i d e s c o n v i n c i n g evidence that the r a t heart SR Ca -ATPase cannot be s t i m u l a t e d by exogenous CaM. T h i s c o n c l u s i o n p o i n t s to two p o s s i b i l i t i e s : a) there i s no endogenous CaM-PK and/or PLB present i n r a t heart or b) t h i s p r e p a r a t i o n c o n t a i n s l a r g e q u a n t i t i e s of endogenous CaM. In our s t u d i e s , the C-subunit of cAMP-PK produced no s t i m u l a t i o n of h y d r o x y l a m i n e - i n s e n s i t i v e 2 + membrane p h o s p h o r y l a t i o n or Ca -ATPase a c t i v i t y at low f r e e 2+ 2 + Ca c o n c e n t r a t i o n s and a small i n h i b i t i o n at higher f r e e Ca c o n c e n t r a t i o n s . T h i s , and the l a c k of CaM s t i m u l a t i o n , may i n d i c a t e a complete absence of the PLB r e g u l a t o r y system i n r a t heart SR and supports the f i r s t p o s s i b i l i t y . The l a c k of i n h i b i t i o n by the two CaM a n t a g o n i s t s , TFP and compound 48/80, may i n d i c a t e absence of endogenous CaM. The i n h i b i t o r y e f f e c t of TFP at high c o n c e n t r a t i o n s may be due to i t s n o n - s p e c i f i c e f f e c t s on the membrane. Under the c o n d i t i o n s of our experiments, more than 50% of the p h o s p h o r y l a t i o n monitored was i n s e n s i t i v e to hydroxylamine treatment, i n d i c a t i n g that the phosphoprotein formed was not an a c y l phosphate. Phosphorylation of a 9,000-11,000 d a l t o n p r o t e i n has p r e v i o u s l y been shown to account f o r more than 90% of the h y d r o x y l a m i n e - i n s e n s i t i v e phosphoprotein formed under s i m i l a r assay c o n d i t i o n s (Plank et a l , 1983). The autoradiography r e s u l t s ( f i g u r e 19) i n our s t u d i e s i n d i c a t e an apparent molecu-l a r weight of the phosphorylated p r o t e i n to be 7,500-9,000 d a l t o n . S i m i l a r p r o t e i n s have been suggested to be monomers of PLB (Louis et a l , 1982; Wegener and Jones, 1984). If t h i s h y d r o x y l a m i n e - i n s e n s i t i v e p h o s p h o r y l a t i o n was due to i n c o r p o r a -32 t i o n of P i n t o PLB or a s i m i l a r r e g u l a t o r y p r o t e i n , i t must be f u l l y s t i m u l a t e d by endogenous CaM, s i n c e i t was observed both in the absence and presence of exogenously added CaM. An e x p l a -nation f o r the c o n f l i c t between t h i s f i n d i n g and the lack of i n h i b i t i o n by CaM a n t a g o n i s t s may be that the endogenous CaM was t i g h t l y bound as r e c e n t l y demonstrated i n r a t l i v e r ( G a z z o t t i et a l , 1985) and c a l f heart sarcolemma (Caroni et a l , 1983) and that t h i s membrane-bound CaM was occluded from the CaM a n t a g o n i s t s . 3. E f f e c t of Detergent Treatment on ATPase A c t i v i t y and i t s  Re g u l a t i o n At low c o n c e n t r a t i o n s (0.2 mg Triton/mg p r o t e i n ) , T r i t o n appeared to s t i m u l a t e the ATPase a c t i v i t y i n Triton-washed membranes. These r e s u l t s are c o n s i s t a n t with the f i n d i n g s of Hidalgo et a l (1986) who observed a 10-20% i n c r e a s e i n the ATPase a c t i v i t y i n membranes sedimented a f t e r treatment with 0.1 mg deoxycholate/mg p r o t e i n . T h i s i n c r e a s e was probably due to the removal of n o n - i n t r i n s i c p r o t e i n s and leaky v e s i c l e s . An a l t e r n a t e e x p l a n a t i o n may be that the detergent s t i m u l a t e s the ATPase a c t i v i t y d i r e c t l y by b i n d i n g to i t (Mcintosh and Davidson, 1984) and/or a l t e r i n g membrane f l u i d i t y (LeMaire e_t a l , 1983). In a d d i t i o n , the recovery of ATPase a c t i v i t y a l s o showed an e l e v a t i o n at 0.2 T r i t o n / p r o t e i n r a t i o . T h i s may be due to aggregation of membrane v e s i c l e s (Helenius et a l , 1979) l e a d i n g to p r e c i p i t a t i o n of more p r o t e i n than the c o n t r o l membranes du r i n g c e n t r i f u g a t i o n . The l o s s of ATPase a c t i v i t y at higher T r i t o n c o n c e n t r a t i o n s appears to be due to s o l u b i l i z a t i o n of p r o t e i n s and removal of the p h o s p h o l i p i d environment (Hidalgo et a l , 1986). The i n c r e a s e d recovery of ATPase a c t i v i t y i n the s o l u b i l i z e d f r a c t i o n at high T r i t o n / p r o t e i n r a t i o s i s b a s i c a l l y a r e f l e c t i o n of higher p r o t e i n recovery, s i n c e the s p e c i f i c a c t i v i t i e s i n the I05,000xg supernatant f r a c t i o n s d e c l i n e d with i n c r e a s i n g T r i t o n c o n c e n t r a t i o n s . 2+ . . Again, the Ca -dependent ATPase a c t i v i t y decreased with 2 + 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 i n the Triton-washed membranes. 2 + While the Ca -dependent ATPase a c t i v i t y at 1OpM added Mg was lower than at zero Mg, the t o t a l a c t i v i t i e s at the two Mg c o n c e n t r a t i o n s were almost i d e n t i c a l . T h i s i n d i c a t e s that the e f f e c t of Mg on ATPase a c t i v i t y may be a c o m p e t i t i v e one, i . e . 2+ Mg may be producing i t s e f f e c t by occupying Ca - b i n d i n g s i t e s . Hence, the a d d i t i v e e f f e c t of the two c a t i o n s was the same as 2+ 2 + that of Ca alone, while i n the presence of Mg, the Ca dependent e f f e c t was reduced. T r i t o n appeared to d i m i n i s h t h i s 2 + e f f e c t of Mg on Ca -dependent ATPase a c t i v i t y , s i n c e the a c t i v i t y appeared to merge as the T r i t o n c o n c e n t r a t i o n was i n c r e a s e d . T h i s may be due to the enzyme becoming more s p e c i f i c 2 + f o r Ca , which may r e s u l t from s e l e c t i v e removal or 2+ . . i n a c t i v a t i o n of Mg -ATPase a c t i v i t y . T h i s i s i n p a r a l l e l with . . 2 + the f i n d i n g s that the p u r i f i e d SR Ca .-ATPase c o n t a i n s a lower percent b a s a l ATPase a c t i v i t y as compared to the membrane bound enzyme (Nakamura et a l , 1983). As d i s c u s s e d e a r l i e r , CaM d i d not s t i m u l a t e e i t h e r the 2+ . . Ca -ATPase a c t i v i t y or membrane p h o s p h o r y l a t i o n i n r a t heart SR. We, hence, s t u d i e d the e f f e c t of CaM on Triton-washed membranes to i n v e s t i g a t e whether T r i t o n X-100 treatment removed endogenous CaM and/or exposed CaM-binding s i t e s . CaM (3 pg/ml) was once again found to be without e f f e c t . T h i s may i n d i c a t e that endogenous CaM i s not removed by T r i t o n X-100 suggesting that i f i t i s present, i t i s t i g h t l y bound to the SR membranes. The i n c r e a s e i n p h o s p h o r y l a t i o n seen both i n the a u t o r a d i o -grams and i n the q u a n t i t a t i v e p h o s p h o r y l a t i o n experiments with the 0.4 T r i t o n / p r o t e i n r a t i o , may be due to exposure of the p h o s p h o r y l a t i o n s i t e s by the d e t e r g e n t . The appearance of the phosphorylated p r o t e i n i n the supernatant f r a c t i o n at higher T r i t o n / p r o t e i n r a t i o s i n d i c a t e s o l u b i l i z a t i o n of t h i s p r o t e i n with other SR p r o t e i n s . In summary, the r a t heart SR appears to have a d i f f e r e n t p r o f i l e of ATPase a c t i v i t y to other SR systems that have been 2+ 2 + s t u d i e d , i n c l u d i n g the dog heart SR. Ca and Mg were e q u a l l y 2 + e f f e c t i v e at s t i m u l a t i n g the ATPase a c t i v i t y . S i m i l a r Ca (or 2 + Mg )-ATPases have been demonstrated in other r a t t i s s u e s i n d i c a t i n g that t h i s ATPase i s s p e c i e s s p e c i f i c . T h i s enzyme i s not e x c l u s i v e to plasma membrane s i n c e our s t u d i e s were c a r r i e d out i n SR v e s i c l e s which were r e l a t i v e l y f r e e of sarcolemmal . . 2 + contamination. In a d d i t i o n , the r e g u l a t i o n of r a t heart SR Ca ATPase appears to be d i f f e r e n t from other c a r d i a c systems s i n c e CaM and C-subunit d i d not s t i m u l a t e the ATPase a c t i v i t y , 2 + membrane p h o s p h o r y l a t i o n or Ca uptake. Our r e s u l t s p o i n t to the p o s s i b i l i t y that the r a t heart SR p r e p a r a t i o n s used i n t h i s study c o n t a i n t i g h t l y - b o u n d endogenous CaM. T h i s would, probably be best confirmed by d i r e c t determina-t i o n s of CaM u s i n g radioimmuno assay. A l t e r n a t i v e l y , CaM may be e x t r a c t e d from SR v e s i c l e s by b o i l i n g and the e f f e c t s of the b o i l e d e x t r a c t s s t u d i e d on a CaM-sensitive system such as 2 + e r y t h r o c y t e Ca -ATPase f o l l o w i n g a procedure s i m i l a r to that of E i b s c h u t z et a l (1984) or G a z z o t t i et a l ( 1985). F i n a l l y , p h o s p h o r y l a t i o n of a p r o t e i n with a s i m i l a r apparent molecular weight to monomeric PLB was seen i n r a t heart SR membranes. However, there i s no concre t e evidence that t h i s p r o t e i n i s PLB, s i n c e i t cannot be phosphorylated by exogenous CaM or C-subunit. Recently r e p o r t e d P L B - s p e c i f i c monoclonal a n t i b o d i e s (Suzuki and Wang, 1986) co u l d be used to determine whether the 7,500-9,000 d a l t o n p r o t e i n i s i n f a c t the monomer of PLB. SUMMARY AND CONCLUSIONS 1. Our r e s u l t s show that r a t heart SR i s d i f f e r e n t from s k e l e t a l muscle and dog c a r d i a c SR. I t possesses an ATPase 2+ 2 + a c t i v i t y which can be s t i m u l a t e d by e i t h e r Ca or Mg T h i s ATPase appears to be s i m i l a r to the n o n - s p e c i f i c d i v a l e n t - c a t i o n s t i m u l a t e d ATPase that has been reported i n a number of plasma membrane p r e p a r a t i o n s from d i f f e r e n t r a t 2 + t i s s u e s . T h i s n o n - s p e c i f i c ATPase may not be the Ca t r a n s p o r t i n g ATPase, but may be masking the a c t i v i t y of the l a t t e r system. I f the system i s indeed s i m i l a r to that of ra t plasma membranes, i t may be p o s s i b l e t o study the high 2+ . 2 + a f f i n i t y Ca t r a n s p o r t i n g ATPase at low Ca c o n c e n t r a t i o n s in the absence of magnesium. 2. Experiments with v a r i o u s Mg and ATP c o n c e n t r a t i o n s i n d i c a t e 2+ 2 + that the s u b s t r a t e f o r the r a t heart SR Ca (or Mg )-ATPase may be f r e e ATP and not the Mg.ATP complex. 3. CaM f a i l e d to s t i m u l a t e the ATPase a c t i v i t y and phosphoryla-t i o n of SR p r o t e i n s . CaM a n t a g o n i s t s were i n e f f e c t i v e at i n h i b i t i n g ATPase a c t i v i t y . However, p h o s p h o r y l a t i o n of a 7,500-9,000 d a l t o n p r o t e i n was observed even i n the absence of exogenous CaM. T h e r e f o r e , i t i s s p e c u l a t e d that the r a t heart SR membranes c o n t a i n t i g h t l y - b o u n d endogenous CaM. 2 + 4. The Ca - s p e c i f i c i t y of ATPase appeared to in c r e a s e i n Triton-washed membranes with i n c r e a s i n g T r i t o n c o n c e n t r a -t i o n s . T h i s may i n d i c a t e a s e l e c t i v e removal or i n a c t i v a t i o n 2 + of the Mg -ATPase a c t i v i t y . T r i t o n X-100 had no e f f e c t on the r e g u l a t i o n of r a t heart SR Ca 2 +-ATPase by CaM. BIBLIOGRAPHY 2 + Ansah, T-A., M o l l a , A. and Katz, S. (1984). 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