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Characterization of calmodulin effects on calcium transport in cardiac microsomes enriched in sarcoplasmic… Lopaschuk, Gary David 1980

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CHARACTERIZATION OF CALMODULIN EFFECTS ON CALCIUM TRANSPORT IN CARDIAC MICROSOMES ENRICHED IN SARCOPLASMIC RETICULUM ( ^ ^ ) G A R Y DAVID LOPASCHUK B . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES i n THE FACULTY OF PHARMACEUTICAL SCIENCES D I V I S I O N OF PHARMACOLOGY AND TOXICOLOGY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA September 19 80 • 0 Gary D a v i d L o p a s c h u k , 1980 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 t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 s t u d y . I f u r t h e r a g r e e 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r 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 n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Pharmacology. F a c u l t y of Pharmaceutical Sciences The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e September 18, 1980 i i ABSTRACT 2+ Calmodulin prepared from red c e l l hemolysate stimulates Ca -transport i n cardiac microsomal preparations enriched i n sarcoplasmic reticulum (S.R.) i n a concentration-dependent manner (Katz and. Remtulla, 1978). The present study was performed to characterize t h i s calmodulin 2+ + + regulation of Ca -transport. K and Na have also been found to enhance 2+ Ca -transport i n microsomal preparations enriched i n S.R. (Jones e t a l , 1978). I t was observed t h a t i n the presence of K + ( l l O mM) and N a + ( l l O mM) 2+ stimulation of Ca -transport a c t i v i t y by calmodulin was g r e a t l y reduced. 2+ This r e s u l t was obtained at a l l free Ca concentrations studied. That t h i s was not an i o n i c e f f e c t was i n d i c a t e d by the decreased antagonism of calmodulin stimulation by L i + (110 mM). K i n e t i c c h a r a c t e r i z a t i o n determined that calmodulin s i g n i f i c a n t l y 2+ enhanced the maximal Ca a c t i v a t i o n without a f f e c t i n g the apparent 2 + 2 + + Ca a f f i n i t y of the Ca -transport process i n cardiac S.R.. K was 2+ found t o enhance the V 2+, as w e l l as lowering the apparent Ca 2+ a f f i n i t y . Examination of the i n i t i a l rate of Ca -transport i n cardiac S.R. confirmed that calmodulin stimulation i s due mainly to an increase i n the V 2+. CyclicAMP-dependent p r o t e i n kinase , on the other hand, has been shown t o increase the V 2+, as w e l l as decrease the apparent 2+ K m f o r Ca (Hicks et a l , 1979), suggesting a d i f f e r e n t mechanism of a c t i o n . Experiments were performed to investigate whether calmodulin was indigenous t o the preparations used. Attempts were therefore made to i s o l a t e calmodulin from dog cardiac microsomal preparations enriched i n S.R. by methodology used to i s o l a t e calmodulin from other sources (Jung, 1978; Depaoli-Roach et a l , 1979)• These extracts were then 2+ tested to determine t h e i r a b i l i t y t o stimulate Ca -transport i n t o S.R.. I t was observed that neither b o i l i n g nor treatment with 0.6mM EGTA could extract calmodulin from these preparations. This r e s u l t i ndicates that the microsomal preparations u t i l i z e d do not contain indigenous calmodulin. Since calmodulin dose not appear to be a.component of the S.R>, i t was postulated that binding t o s i t e s on the membrane must occurr i n 2+ order f o r calmodulin to augment Ca -transport. I t was also suggested i i i + + 2+ that K and may decrease calmodulin stimulation of Ca -transport 125 t>y a l t e r i n g t h i s binding. Studies were therefore performed using I-l a b e l l e d calmodulin to determine the degree of binding to microsomal preparations i n the presence and absence of K + (llOmM), Na + (llOmM) , • + 125 an d::.Li(llOmM.):... "It was found that I-calmodulin binds t c microsomal 2+ 2+ cardiac S.R. i n a Ca concentration-dependent manner. At Ca - 7 concentrations above 10 M, t h i s binding was s i g n i f i c a n t l y decreased (p<0.05, students " t " t e s t ) i n the presence of K + or Na +. L i + , 2+ previously shown not t o a l t e r Ca . -transport augmentation byucalmodulin, + + did not a l t e r calmodulin binding to a s i g n i f i c a n t extent. K and Na 2+ therefore may i n h i b i t calmodulin stimulation of Ca -transport i n these preparations by decreasing calmodulin binding t o the S.R.. The lack of i n h i b i t i o n by L i + (llOmM) indicates t h a t t h i s r e s u l t i s not due t o a non-specific i o n i c e f f e c t . Our studies therefore have shown that calmodulin i s not an indigenous p r o t e i n of the sarcoplasmic reticulum preparations used. Calmodulin, 2+ though, has been found to bind, i n a Ca -dependent manner, t o these preparations. This binding i s altered by monovalent cations p r e v i o u s l y 2+ shown to i n h i b i t calmodulin stimulation of Ca -transport. Dr. S. Katz (supervisor) Associate Professor D i v i s i o n of Pharmacology and Toxicology Faculty of Pharmaceutical Sciences i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i LIST OF ABBREVIATIONS i x INTRODUCTION 1 I. Calmodulin- . 1 a) P r o p e r t i e s of Calmodulin 2 I I . Calcium Transport i n Sarcoplasmic Reticulum h a) P r o p e r t i e s of Sarcoplasmic Reticulum 6 n \ 2+ „ 2+ B m T, b) Mg -Ca -ATPase 7 c) Regulation of Cardiac Sarcoplasmic Reticulum 9 Calcium Transport d) Regulation by Monovalent Cations 10 e) Regulation by a C y c l i c AMP-dependent P r o t e i n 11 Kinase 2+ f ) Regulation of Ca -transport by Calmodulin 15 I I I . Objectives of the Present Study 1 6 MATERIALS AND METHODS 17 A. M a t e r i a l s 17 1) Animals 17 2) Chemicals 17 i ) Radioisotopes 17 i i ) Chromatography Columns 17 i i i ) SDS-polyacrylamide g e l e l e c t r o p h o r e s i s 17 i v ) F i l t r a t i o n equipment IT v) Assay chemicals IT B. Methods 1) P r e p a r a t i o n of Calmodulin from Outdated 18 Human Erythrocytes 2) P r e p a r a t i o n of Cardiac Microsomes Enriched 19 w i t h Sarcoplasmic Reticulum 3) Measurement of Calcium Uptake by Cardiac Microsomes 20 Enriched i n Sarcoplasmic Reticulum V e s i c l e s k) C a l c u l a t i o n of Calcium Uptake A c t i v i t y by Cardiac 21 Microsomes Enriched i n Sarcoplasmic Reticulum V Page 5) Sodium Dodecyl Sulphate-Polyacrilamide Gel 21 Electrophoresis i ) Preparation of 10%. polyacrilamide gels 21 i i ) Preparation of Calmodulin Protein Samples 22 i i i ) E l e c t r o p h o r e s i s , S t a i n i n g , and Destaining of 22 Samples 6) Iodination of Calmodulin 22 125 7) Measurement of I-calmodulin Binding to 23 Sarcoplasmic Reticulum Enriched Microsomes 8) Miscellaneous Methods 2h RESULTS 26 1) I s o l a t i o n of Calmodulin: 26 2) Characterization of Cardiac Microsomes Enriched 30 i n S.R. 2+ 3) Monovalent Cation Stimulation of Ca -transport 3-? i n Cardiac S.R.: k) Effect of Calmodulin on the K i n e t i c Parameters of !l3 Ca -transport i n Cardiac Microsomal Preparations Enriched i n S.R.: 5) E f f e c t of Microsomal S.R. Extracts On Calcium Uptake 52 A c t i v i t y i n Cardiac Microsomal Preparations Enriched i n Sarcoplasmic Reticulum: 6) Binding of I - l a b e l l e d Calmodulin to Cardiac 58 Microsomes Enriched i n Sarcoplasmic Reticulum DISCUSSION 1) - A c t i v i t y and P u r i t y of the. Red C e l l Calmodulin Used 65 2) E f f e c t of Storage on Calmodulin A c t i v i t y 66 2+ 3) Calmodulin Stimulation of Ca -transport i n 67 Cardiac Microsomes Enriched i n Sarcoplasmic Reticulum E f f e c t of Moi Cardiac S.R. 2+ k) E f f e c t of Monovalent Cations on Ca -transport i n 6? 5) K i n e t ^ Properties of Calmodulin and K Regulation 69 of Ca -transport i n Cardiac S.R. 6) E f f e c t of Calmodulin and gAMP-dependent Protein Kinase 71 on the I n i t i a l Rate of Ca -transport i n Cardiac S.R. 7) I s o l a t i o n of Calmodulin from Microsomal Cardiac S.R. 72. 8) Calmodulin Binding t o Microsomal Cardiac S.R. 73 2+ 9) Calmodulin and i t s Role i n Ca -transport i n Cardiac 75 S.R. SUMMARY AND CONCLUSIONS 78' BIBLIOGRAPHY 79 LIST OF TABLES Table 1) Regulatory Functions of Calmodulin 2+ 2+ 2) Mg -Ca -ATPase Reaction Sequence as Proposed by I n e s i e t a l (1979) 2+ 3) E f f e c t of Various Calmgdulig +Preparations on Mg ATPase A c t i v i t y and Mg -Ca -ATPase A c t i v i t y of EDTA-washed Human Erythroc y t e Membranes 2+ h) Ca -uptake by Various Cardiac Muscle Preparations i n the Presence and Absence of cAMP-dependent P r o t e i n Kinase 5) E f f e c t of Microsomal E x t r a c t s on Calcium Uptake A c t i v i t y i n Cardiac Microsomal Preparations Enriched i n Sarcoplasmic Reticulum 125 6) E f f e c t of Monovalent Cations on I-calmodulin Binding to Cardiac Microsomes Enriched i n Sarco-plasmic Reticulum v i i LIST OF FIGURES Figure Page 1) Amino Acid Sequence of Calmodulin from Bovine Brain 3 as Determined by Vanamari et a l (1977) 2) Possible Mechanism by Which Phospholambam Modulates 12 the A c t i v i t y of the Calcium Pump of the Cardiac Sarcoplasmic Reticulum 3) Proposed Integration of the E f f e c t s of Catecholamines on the Cardiac Contractile Proteins and Sarcoplasmic Reticulum i n Terms of t h e i r P h y s i o l o g i c a l Response h) Determination of Fractions Containing Calmodulin Using 27 the ATPase Assay of EDTA-washed Red C e l l Membranes. 2+ 5) E f f e c t of Red C e l l Calmodulin on Ca -uptake i n Micro- 31 somal Preparations Enriched i n Sarcoplasmic Reticulum 6) Densitometer Tracings of Calmodulin Preparations 33 Subjected to SDS-polyacrylamide Gel Electrophoresis 7) E f f e c t of Varying Concentrations of the Microsomal 35 Preparation Enriched i n Sarcoplasmic Reticulum on Ca -uptake by the S.R. Preparation 2+ 8) E f f e c t of Monovalent Cations on Ca -uptake i n the 39 Presence , and Absence of Calmodulin + 2+ 9) E f f e c t of K on Ca -uptake i n the Presence and Absence i+1 of Sigma Calmodulin + 2+ 10) E f f e c t of K on Ca -uptake i n the Presence of Red • kk C e l l Calmodulin + 2+ 11) E f f e c t of Na on Ca -uptake i n the Presence and U6 Absence of Red C e l l Calmodulin 2+ 12) Effect 2o_f Calmodulin on the K. : f o r Ca -uptake i n Microsomal ^ Preparations Enriche i n Sarcoplasmic Reticulum + 2+ 2+ 13) E f f e c t of K on the K for Ca and the V c 2+ f o r Ca - 50 uptake i n Microsomal Preparations Enriched i n Sarco-plasmic Reticulum .lh) E f f e c t o f Calmodulin on the. I n i t i a l Rate .of-Calcium' 53 "Uptake In Microsomal Preparations Enriched i n .. Sarcoplasmic Reticulum , . . 15) V e l o c i t y of Calcium Uptake i n Cardiac :Micros-omal " 55 . Preparations Enriched i n Sarcoplasmic Reticulum i n the Presence and Absence of Calmodulin f o r Ca and the V n 2+ U8 Ca Figures 125 l6. ) Separation of F r a c t i o n ^ C o n t a i n i n g ' I - l a b e l l e d Calmodulin from Free I Using G-25 Sephadex Column Chromatography 125 17 ) E f f e c t of Monovalent Cations on I-calmodulin Binding to Cardiac Microsomes Enriched i n Sarcoplasmic Reticulum 18.) Possib^| Mechanism by Which Calmodulin Regulates the Ca -pump of Cardiac Sarcoplasmic Reticulum i x L I S T OF ABBREVIATIONS ADP ATP ATPase °C ( C a 2 + - M g 2 + ) - A T P a s e cAMP C i cpm E EDTA EGTA E? e t a l g Xg IO K d i s s K m m n M mg min ml A l mM A M MW NAD nmo1es •P. pmoles RBC SDS-PAGE a d e n o s i n e 5 ' - d i p h o s p h a t e a d e n o s i n e 5 ' - t r i p h o s p h a t e a d e n o s i n e t r i p h o s p h a t a s e d e g r e e c e n t r i g r a d e 2+ Mg -d e p e n d e n t c a l c i u m s t i m u l a t e d ATPase c y c l i c a d e n o s i n e 5 1-monophosphate C u r i e c o u n t s p e r m i n u t e enzyme e t h y l e n e d i a m i n e t e t r a a c e t a t e , d i s o d i u m s a l t e t h y l e n e g l y c o l - b i s - (Ji- amino e t h y l e t h e r ) N , N ' - t e t r a - a c e t i c a c i d p h o s p h o r y l a t e d enzyme i n t e r m e d i a t e and o t h e r s gram a c c e l e r a t i o n of g r a v i t y i n s i d e - o u t d i s s o c i a t i o n c o n s t a n t Miehaelis-Ment-en constant m i l l i m i c r o mo l a r m i l l i g r a m m i n u t e m i l l i l i t e r m i c r o l i t e r m i l l i m o l a r concentration m i c r o m o l a r concentration m o l e c u l a r w e i g h t n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e nanomoles i n o r g a n i c p h o s p h a t e p i c o m o l e s r e d b l o o d c e l l s o d i u m d o d e c y l 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 s t a n d a r d e r r o r o f t h e mean s a r c o p l a s m i c r e t i c u l u m t r i c h l o r o a c e t i c a c i d t r i s ( h y d r o x y m e t h y 1 aminomethane) 2+ maximum v e l o c i t y of Ca - t r a n s p o r t maximum v e l o c i t y of enzyme r e a c t i o n p e r c e n t p e r xi ACKNOWLEDGEMENTS I am deeply g r a t e f u l t o my s u p e r v i s o r , Dr. S. Katz f o r h i s guidance and support throughout t h i s study. I -would l i k e t o thank my committee members ( Drs. J . H . M c N e i l l , B.D.Roufogalis , and J.Diamond) f o r t h e i r c o n s t r u c t i v e c r i t i c i s m and suggestions during t h i s study. I am indebted t o Kurt Henze o f the Department o f Physiology, F a c u l t y o f Medicine, U.B.C, f o r supplying the dog hearts used i n the., study. I -would also l i k e t o g r a t e f u l l y acknowledge the f i n a n c i a l support extended by the F a c u l t y of Pharmaceutical S c i e n c e s , U.B.C. , and the Canadian Heart Foundation throughout t h i s study. I would l i k e to thank B e t t y R i c h t e r and the r e s t o f my collegues i n 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 also wish t o 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 o f Pharmaceutical Sciences, U.B.C. f o r making t h i s Masters program enjoyable. 1 INTRODUCTION Cardiac sarcoplasmic r e t i c u l u m p l a y s an i n t e g r a l p a r t i n heart f u n c t i o n . Calcium r e l e a s e d from t h i s i n t r a c e l l u l a r o r g a n e l l e i s i n -volved i n muscle f i b r i l c o n t r a c t i o n , while reuptake of calcium by the sarcoplasmic r e t i c u l u m i s b e l i e v e d t o mediate the subsequent r e l a x a t i o n of the muscle (Ebashi et a l , 1969). Reaccumulation o f calcium w i t h i n 2+ 2+ the sarcoplasmic r e t i c u l u m i s an a c t i v e process i n v o l v i n g a Mg -Ca -adenosine triphosphatase (ATPase) enzyme, which i s embedded i n the sarcoplasmic r e t i c u l u m membrane (Hasselbach and Makinose, 1961). In recent years various r e g u l a t o r y mechanisms c o n t r o l l i n g the uptake process have been i d e n t i f i e d . A cAMP-dependent p r o t e i n kinase appears t o modulate calcium transport by phosphorylation o f a low MW sarco-plasmic r e t i c u l u m membrane p r o t e i n , phospholambam (Kirchberger et a l , 1978). Monovalent c a t i o n s such as K + and Na + a l s o e f f e c t the t r a n s p o r t mechanism, probably by i n c r e a s i n g the r a t e of dephosphorylation of the 2+ 2+ Mg -Ca -ATPase enzyme (Shigekawa and P e a r l , 1976; Jones et a l , 1978). Recently a calcium-dependent r e g u l a t o r y p r o t e i n , termed cal m o d u l i n , has been shown t o modulate the a c t i v i t y of a number of enzymes i n c l u d i n g 2+ 2+ e r y t h r o c y t e membrane Mg -Ca -ATPase a c t i v i t y (Gopinath and V i n c e n z i , 1977)• Subsequently i t was shown that calmodulin enhanced Ca^ +-uptake i n t o i n s i d e out v e s i c l e s prepared from red c e l l membranes (Sarkadi et a l , 1979). In view of t h i s i t was decided t o i n v e s t i g a t e the r o l e of calmodulin i n the r e g u l a t i o n of calcium transport i n cardiac sarco-plasmic r e t i c u l u m (S.R.). The i n t e r a c t i o n of calmodulin w i t h various mediators o f calcium uptake was a l s o i n v e s t i g a t e d . Calcium uptake was measured i n a microsomal p r e p a r a t i o n enriched i n sarcoplasmic r e t i c u l u m obtained from canine c a r d i a c v e n t r i c l e muscle. BACKGROUND I . Calmodulin I t i s now becoming apparent that a calcium b i n d i n g p r o t e i n , termed calmodulin (Cheung et a l , 1978), acts as a mediator i n many c e l l u l a r 2+ Ca - r e g u l a t e d events. The presence of t h i s a c t i v a t o r p r o t e i n i n animal t i s s u e was f i r s t demonstrated by Cheung i n 19^7• I t was shown that a heat s t a b l e , nondialyzable f a c t o r from bovine b r a i n homogenate was necessary t o maintain c y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t y during 2 p u r i f i c a t i o n of the enzyme from crude e x t r a c t . Using a c y c l i c n u c l e o t i d e phosphodiesterase p r e p a r a t i o n free o f a c t i v a t o r K a k i u c h i et a l (l'9T0) showed t h a t the enzymes c a t a l y t i c a c t i v i t y had an absolute requirement 2+ f o r both Ca and the a c t i v a t o r p r o t e i n f r a c t i o n . Further p u r i f i c a t i o n of the a c t i v a t o r f r a c t i o n showed th a t an a c i d i c p r o t e i n (MW approximately 2+ 18,000) which forms a complex w i t h Ca was the t r u e a c t i v a t o r o f phos-phodiesterase ( L i u et a l , 197k;. Teo-.and Wang., 1973). Independently, Bond and Clough (1973) demonstrated that hemolysate from human e r y t h r o c y t e s , i f added to ery t h r o c y t e membrane preparations 2 + 2 + could s t i m u l a t e Mg -Ca -ATPase a c t i v i t y . The s t i m u l a t o r y a c t i v i t y was found t o be due t o an a c t i v a t o r p r o t e i n found predominantly i n the c y t o s o l of e r y t h r o c y t e s . The a c t i v a t o r was p u r i f e d t o homogeneity, and shown to be a calcium b i n d i n g p r o t e i n of MW approximately 18,000 ( J a r r e t t and Penniston, 1977; Jung, 1978: L u t h r a et a l , 1976). A comp-ar i s o n was made of the a b i l i t y of the erythrocyte a c t i v a t o r and the 2+ 2+ phosphodiesterase a c t i v a t o r , t o st i m u l a t e erythrocyte Mg -Ca -ATPase (Gopinath and V i n c e n z i , 1977; J a r r e t t and Penniston, 1977). I t was found t h a t the p r o p e r t i e s o f the two a c t i v a t o r s were s i m i l a r , suggesting that the two were the same p r o t e i n . I t has subsequently been shown that calmodulin i s ubiquitous i n the animal '. kingdom, and r e c e n t l y has been i s o l a t e d from p l a n t s ( J a r r e t t et a l , 1980). a) P r o p e r t i e s of Calmodulin: Estimates of the molecular weight of calmodulin have v a r i e d between 15,000 and 19,000 depending on the methodology and cond i t i o n s employed (Watterson et a l , 1976; L u l et a l , 197^; Vanaman et a l , 1977; Dedman et a l , 1977). One reason f o r the v a r i a t i o n i s t h a t SDS-polyacrylamide g e l e l e c t r o p h o r e s i s and sedimentation e q u i l i b r i u m MW determinations can 2+ 2+ be a l t e r e d by changing EGTA or Ca concentrations. As the Ca conc-e n t r a t i o n increases the d e n s i t y of the calmodulin increases due t o 2+ conformational changes i n the molecule due t o Ca -binding ( L u i and Cheung, 1976; Wolff et a l , 1977). R e c e n t l y , Vanaman et a l (1977) have worked out the amino a c i d sequence of bovine b r a i n calmodulin, a r r i v i n g at a MW of l6 , 7 2 3 . The sequence as shown i n f i g u r e 1, contains ihQ residues w i t h 27 glutamate and 23 aspartate residues accounting f o r 3 Figure 1: Amino A c i d Sequence o f Calmodulin from Bovine B r a i n , as Determined by Vanaman et a l (1977). A c ( a l a , asx) GLN LEU THR GLU GLU GLN ILE ALA GLU PHE LYS g l u ALA PHE SER LEU PHE ASP l y s ASP GLY THR ILE THR THR LYS GLU LEU GLY THR VAL MET ARG ser LEU GLY GLN ASN PRO THR g l u a l a GLU LEU GLU ASX MET ILE ASN GLU VAL ASP a l a ASP GLY ASX GLY THR ILE ASP PHE pro GLU PHE LEU t h r MET MET ALA ARG l y s MET LYS ASP t h r asp SER GLU GLU GLU ILE arg GLU a l a PHE ARG VAL PHW ARG VAL PHE ASP LYS ASP GLY ASN GLY TYR ILE SER ALA a l a GLU LEU ARG h i s VAL MET t h r asx l e u GLY GLU tml LEU THR ASP GLU GLU VAL ASP GLU MET ILE ARG GLU ALA ASN i l e ASP g l y ASP GLY g l x VAL ASX TYR GLX GLX PHE VAL GLN MET MET t h r ALA l y s COOH the a c i d i c nature of calmodulin. The molecule can be d i v i d e d i n t o four 2+ symmetrical homologous domains, each containg a Ca -binding s i t e (Vanaman e t a l , 1977; K r e t s i n g e r , 1979). Each s i t e contains a hydro-phobic core, w i t h s i x oxygen c o n t a i n i n g amino a c i d s i d e chains a c t i n g as the ca l c i u m b i n d i n g l i g a n d s . The b i n d i n g s i t e s appear i d e n t i c a l 2+ t o the Ca -binding s i t e s o f the calcium b i n d i n g p r o t e i n s t r o p o n i n C and parvalbumin (Vanaman, 1980) and a common e v o l u t i o n a r y o r i g i n has been suggested ( K r e t s i n g e r , I98O; Barker e t a l , 1977; Wang et a l , 1975). The r e g u l a t i o n of various enzymatic fun c t i o n s by calmodulin r e q u i r e s 2+ the presence of Ca and has been described by the. f o l l o w i n g non-stoichic-metric sequence. 2+ 2+ Ca + calmodulin • Ca .calmodulin 2+ 2+ Ca .calmodulin + enzyme,. .. —* » Ca .calmodulin-enzyme/ . x J ( i n a c t i v e ) ( a c t i v e ) 2+ When Ca binds t o calmodulin the h e l i c a l content of the molecule i n -creases .thereby producing a conformational change (Wolff and Brostrom, 1979). I t i s thought that t h i s change allows calmodulin to b i n d to the 2+ enzyme, r e s u l t i n g i n a c t i v a t i o n . The Ca -dependent b i n d i n g of calmodulin k to t h e. enzyme 'is a r e v e r s i b l e process dependent of the concen-t r a t i o n o f Caf i n the medium. In some enzyme systems, such as phosphor-yl'ase kinase (Cohen , 1978) and c y c l i c n u c l e o t i d e phosphodiesterase (Cheung, 1970; Wang, 1980) calmodulin i s a c t u a l l y a subunit of the 2+ enzyme. In t h i s i n s t a n c e , Ca b i n d i n g t o calmodulin present i n the en-zyme complex would r e s u l t i n a c t i v a t i o n . Recently, Klee and Haiech (1980) have suggested that b i n d i n g of 2+ 2+ Ca to calmodulin i s dependent on p r e v i o u s l y bound Ca , and have sug-gested the f o l l o w i n g s t o i c h i o m e t r i c equation: 2+ 2Ca + calmodulin > calmodulin » 2+ 2Ca calmodulin + enzyme i calmodulin-enzyme/. ,.' \ l p+ 1 p+ ' ( i n a c t i v e ) 2Ca 2Ca 2+ calmodulin-enzyme/. ,. s + 2Ca calmodulin-enzyme ( a c t i v e ) I p + ( i n a c t i v e ) > , 2Ca kC& 2+ Since Cheung f i r s t demonstrated t h a t calmodulin mediates Ca -regu l a t e d phosphodiesterase a c t i v a t i o n many researchers have looked at 2+ i t s involvement i n other Ca -mediated events. I t i s now known th a t calmodulin has widespread a c t i o n s which u s u a l l y i n v o l v e a c t i v a t i o n o f an enzyme system c o n t r o l l i n g some c e l l u l a r f u n c t i o n . L i s t e d i n t a b l e 1 are most of the c e l l u l a r f u n c t i o n s and enzymes which have been shown to date t o i n v o l v e calmodulin r e g u l a t i o n . I I . Calcium Transport i n Sarcoplasmic Reticulum 2+ The c e n t r a l r o l e of Ca as an inducer of the c o n t r a c t i l e response i n s k e l e t a l muscle was f i r s t suggested by Heilbrunn and W i e r c i n s k i (19U7). This was l a t e r confirmed by other r e s e a r c h e r s , who demonstrated the a b i l i t y of C a 2 + t o c o n t r o l m y o f i b r i l c o n t r a c t i l i t y (Weber and H e r z % 196l; B o z l e r , 195U) and i d e n t i f i e d a calcium b i n d i n g p r o t e i n , t r o p o n i n , i n the t h i n f i l a m e n t s of the m y o f i b r i l s (Ebashi and Endo, 1968). In 1951 i t was shown by Marsh that an aqueous e x t r a c t of s k e l e t a l muscle could a l s o a l t e r c o n t r a c t i l i t y of m y o f i b r i l l a r bundles. This e x t r a c t was subsequently shown to c o n s i s t of microsomal v e s i c l e s , 5 Table 1: Regulatory Functions of Calmodulin i ) S p e c i f i c Enzymes References Phosphodiesterase Adenylate Cyclase 2+ 2+ Ca -Mg -ATPase Phosphorylase Kinase Phospholipase A^ NAD +Kinase Myosin Light Chain Kinase a) S k e l e t a l Muscle b) Smooth Muscle c) P l a t e l e t Tryptophan 5'-monooxygenase i i ) C e l l u l a r Functions Cheung (1970) Brostrom e t a l :i975) Gopinath and V i n c e n z i (1977) J a r r e t t and Penniston (1977) Cohen (1978) Wong and Cheung (1979) Anderson and Cormier (1978) Yagi et a l (1978) Dabrowski et a l (1975) A d e l s t e i n et a l (1975) Yamauchi and Fujisawa (1979) Microtubule assembly and disassembly Welsh et a l (1978) Ca - t r a n s p o r t a) Sarcoplasmic Reticulum b) E r y t h r o c y t e Membrane c) Synaptic Membrane Neurotransmitter Release I n s u l i n Release P r o t e i n Phosphorylation P r o s t a g l a n d i n Metabolism Axoplasmic Transport Katz and Remtulla (1978) Hinds e t a l (1978) Kuo e t a l (1979) Grab e t a l (1979) Sudgen et a l (1979) Schulman and Greengard (1978) Wong and Cheung (1980) I q b a l and Ochs (1980) Brady et a l (1980) 6 presumably composed of sarcoplasmic reticulum membranes (Ebashi and Lipmann, 2+ 1962). The microsomal v e s i c l e s contain a Mg -dependent ATPase a c t i v i t y 2+ and are capable of removing Ca from medium, i n the presence of ATP and 2+ Mg (Hasselbach and Makinose, 1962; Ebashi and Lipmann, 1962). I t was thus suggested that the sarcoplasmic reticulum a l t e r s m y o f i b r i l contract-2+ l l i t y by changing cytoplasmic Ca l e v e l s , e i t h e r by a c t i v e l y accumulating 2+ 2+ cytoplasmic Ca , or by r e l e a s i n g accumulated Ca i n t o the cytoplasm. Comparitive studies performed by Fawcett (1961) i n d i c a t e d that cardiac muscle also contained sarcoplasmic reticulum, which i s q u a l i t a t i v e l y s i m i l a r to that of s k e l e t a l muscle. Harigaya and Schwartz (1969), as 2+ w e l l as Repke and Katz (1972) have since confirmed th a t , although Ca -transport i n cardiac S.R. i s slower than s k e l e t a l muscle, both transport 2+ 2+ systems are b a s i c a l l y s i m i l a r . Mg -Ca -ATPase a c t i v i t y i n cardiac S.R. 2+ has a V f o r Ca 3-6 times lower than s k e l e t a l muscle S.R. (Shigekawa max ' et a l , 1976). As w e l l phosphoprotein l e v e l s that can be obtained i n cardiac preparations are h times lower than fast s k e l e t a l S.R.. This indicates that cardiac S.R. has k times fewer active s i t e s than s k e l e t a l 2+ 2+ muscle. Since the turnover rate of Mg -Ca -ATPase does not d i f f e r 2+ at saturating Ca concentrations i n s k e l e t a l or cardiac S.R. the 2+ slower Ca -transport rate i n cardiac S.R. must be due t o a lower 2+ density of Ca -pumping s i t e s . Therefore i t can be concluded that cardiac and s k e l e t a l muscle S.R. i s q u a l i t a t i v e l y , but not q u a n t i t a t i v e l y , s i m i l a r . a) Properties of Sarcoplasmic Reticulum: Sarcoplasmic reticulum i n s k e l e t a l and cardiac muscle i s an i n t r a -c e l l u l a r membranous compartment i n close association with the m y o f i b r i l s (Porter and Palade, 1957). This membrane system performs two important 2+ functions; l ) transport of Ca from the m y o f i b r i l l a r space t o the i n t e r i o r of the organelle, therby allowing muscle r e l a x a t i o n , and 2) 2+ release of accumulated Ca , which r e s u l t s i n muscle a c t i v a t i o n . A portion of the S.R. membrane, termed the j u n c t i o n a l S.R., i s located i n close proximity t o the transverse tubules (T-tubules), of the sarcolemma membrane. It has been postulated that t h i s segment of the S.R. acts as a sensor, which detects T-tubule membrane depolarization (Franzini -7 2+ Armstrong, 1980). The mechanism by which Ca i s r e l e a s e d from t h e S.R. i n response to membrane d e p o l a r i z a t i o n has y e t t o be e l u c i d a t e d . Schneider and Chandler (1973) suggest t h a t the charge movements o c c u r r i n g " 'within the T-tubule membrane have a d i r e c t e f f e c t of producing an 2+ increase i n the S.R. p e r m e a b i l i t y t o Ca . Fabiato and Fabiato (1975) 2+ 2+ hypothesized the " t r i g g e r " Ca mechanism, whereby low l e v e l s of Ca which enter the c e l l during d e p o l a r i z a t i o n t r i g g e r a massive r e l e a s e of 2+ Ca from the S.R. The S.R. membrane i t s e l f i s a l i p i d b i l a y e r c o n s i s t i n g p r i m a r i l y of p h o s p h o l i p i d . I t contains f o u r main p r o t e i n s , the 100,000 MW ATPase 2+ p r o t e i n , a high a f f i n i t y Ca b i n d i n g p r o t e i n , c a l s e q u e s t r i n , and low molecular weight p r o t e o l i p i d s (MacLennan et a l , 197*0. The ATPase p r o t e i n , comprising 90% of S.R. p r o t e i n , i s uniforml y d i s t r i b u t e d along the surface of the membrane which i s i n c l o s e s t contact w i t h the myofib-r i l s (Bray e t a l , 1978)• I t i s embedded w i t h i n the membrane (Deamer and Baskin , I969) and appears aggregated i n t o tetramers w i t h other ATPase p r o t e i n s (Hidalgo...and Ikemoto, 1977). The low molecular weight proteo-l i p i d s are a l s o . , i n t r i n s i c p r o t e i n s and are randomly d i s t r i b u t e d through-2+ out the S.R. membrane. C a l s e q u e s t r i n and 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 are e x t r i n s i c p r o t e i n s , which are l o o s e l y a s s o c i a t e d w i t h the inner, surface of the membrane (Ikemoto et a l , 1971). As of y e t no disc e r n a b l e f u n c t i o n has been i d e n t i f i e d f o r the p r o t e o l i p i d s , 2+ c a l s e q u e s t r i n , o r 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 . I t has been suggested that c a l s e q u e s t r i n may act as a calcium storage s i t e , but Repke e t a l (1976) present data which does not support t h i s . I n cardiac S.R. a 22,000 MW p r o t e i n , r e c e n t l y termed phospholambam, which bands on SDS-PAGE i n the region MacLennan i d e n t i f i e d as p r o t e o l i p i d s , plays a 2+- , \ r o l e i n the r e g u l a t i o n of Ca -uptake (Tada e t a l , 1975;• This w i l l be discussed i n a l a t e r s e c t i o n . b) Mg 2 +-Ca 2 +-ATPase: 2+ Sarcoplasmic r e t i c u l u m has been shown to con t a i n a Ca -dependent 2+ ATPase, as w e l l as a Ca -independent ATPase ( i n e s i e t a l , 1976). This 2+ d i s c u s s i o n w i l l concern only the Ca -dependent ATPase a c t i v i t y . 2+ I t has been f a i r l y w e l l e s t a b l i s h e d t h a t the Ca -ATPase p r o t e i n 2+ p l a y s a c e n t r a l r o l e i n Ca - t r a n s p o r t i n S.R.. Meissner and F l e i s h e r 8 (1973), as w e l l as Repke .et a l (1976) have shown t h a t p u r i f i e d S.R. ATPase which has been r e c o n s t i t u t e d i n t o membrane v e s i c l e s w i l l a c t i v e l y 2+ t r a n s p o r t calcium. The tra n s p o r t process i s dependent on ATP and Mg (Hasselbach, 1962) and i t appears that a Mg.ATP complex acts as the sub-2+ s t r a t e f o r the Ca -ATPase a c t i v i t y (Yamamoto and Tonomura, 1967). Another absolute requirement f o r a c t i v i t y i s calcium....binding t o s p e c i f i c s i t e s on the S.R. ATPase. Ikemoto (197*0 demonstrated the presence of 2+ both high and low a f f i n i t y Ca -binding s i t e s , although only the h i g h a f f i n i t y s i t e s appear t o be s p e c i f i c a l l y i n v o l v e d i n ATPase a c t i v a t i o n . (Meissner et a l , 1973). Recently, I n e s i et a l (1979) have demonstrated 2+ a co-operative mechanism of Ca -bi n d i n g . They suggest t h a t b i n d i n g of 2+ Ca to a f i r s t s i t e causes a conformational change i n the ATPase, r e -s u l t i n g i n the development of higher a f f i n i t y at a second b i n d i n g s i t e . 2+ This co-operative mechanism of Ca -bind i n g and t r a n s p o r t , shown m Table 2, i s a m o d i f i c a t i o n of the r e a c t i o n sequence f i r s t proposed by Kanazawa et a l (1971)• Table 2 2+ 2+ Mg -Ca -ATPase r e a c t i o n sequence as proposed by I n e s i e t a l (1979) ?+ 1 ATP ? "3 E + Ca . ' ATP.E.Ca s  D ATP.E'.Ca O u t  7 ^ 7 7 ATP.E'.Ca + C a 2 + , _^_!L, ATP . E " . C a 2 + * L_ ADP.E' Lr P.Ca 0 out 7 2 ADP.E'ly-P.Ca 0 ^ — A D P . E 1 ' '-P.Ca_ * 1—- ADP .E " * -P. Ca + C a 2 + 2 2 7 m ADP.E*".Ca * — r ADP. E'' ' -P + C a 2 + ^ 9 E'"-P 10 11 12 P E'"-P / . E-P / , E-P. * _«<£_±__ E Me 2+ I n e s i e t a l suggest t h a t a f i r s t cytoplasmic Ca i o n r a p i d l y binds to the enzyme, f o l l o w e d by high a f f i n i t y ATP b i n d i n g ( l , 2 ) . This r e s u l t s 2+ i n a slower p r o t e i n t r a n s i t i o n , which forms a second high a f f i n i t y Ca -2+ b i n d i n g s i t e (3). Upon b i n d i n g of a second Ca i o n (h) the bound ATP hydrolyzes forming a high energy phosphorylated.;,,intermediate ( 5 ) (Hasselbach, 196U). Kanazawa et " a l (1970) provide evidence suggesting 9 t h a t the formation of the phosphorylated intermediate i s coupled t o the 2+ t r a n s l o c a t i o n of Ca across the membrane. Once t h i s occurs a r e d u c t i o n • - 2+ m the b i n d i n g a f f i n i t y r e s u l t s , and Ca d i s s o c i a t e s from the enzyme (7,8) (Yamada and Tonomura ,197-2).. Yamada and Ikemoto (1979) suggest 2+ that d i s s o c i a t i o n of the second Ca i o n i s the r a t e l i m i t i n g step of the e n t i r e r e a c t i o n sequence. The f i n a l step i n v o l v i n g decomposition of the phosphorylated intermediate (12) is- g r e a t l y a c c e l e r a t e d i n the 2+ 2+ presence of Mg . Therefore Mg has two r o l e s i n ATPase a c t i v a t i o n , . i ) t o a c c e l e r a t e the decomposition r a t e of the phosphorylated enzyme, and i i ) to a c t as the true s u b s t r a t e f o r the enzyme. From the r e a c t i o n scheme and e a r l i e r s t u d i e s by Hasselbach '. 2+ ( 196*+) and other workers i t has been suggested t h a t 2 moles of Ca are transported per mole of ATP hydrolyzed. Studies by Suko (1973) suggest a r a t i o of 1:1. A f i x e d r a t i o has been questioned.by Tate et a l (198O) who provide evidence suggesting a f l e x i b l e s t o i c h i o m e t r y . De-pending on f a c t o r s , such as pH and temperature, the s t o i c h i o m e t r i c 2+ r e l a t i o n s h i p between Ca - t r a n s p o r t and energy t r a n s d u c t i o n may. be a dynamic e n t i t y . c) Regulation of Cardiac Sarcoplasmic Reticulum Calcium Transport: Calcium t r a n s p o r t by cardiac S.R. i s r e g u l a t e d by a number of 2+ 2+ systems. These appear to act mainly on the Mg -Ca -ATPase enzyme, not only through i t s r e a c t i o n mechanism, but a l s o through a r e g u l a t o r y system a s s o c i a t e d w i t h the enzyme. 2+ One obvious r e g u l a t o r of Ca -tr a n s p o r t i s the t r u e substrate of 2+ 2+ the Mg -Ca -ATPase enzyme, Mg.ATP. Kanazawa e t a l (l97l) showed there i s an increased formation of the phosphorylated intermediate w i t h i n c r e a s i n g ATP concentration. They suggest t h a t t h i s i s not due t o an increased r a t e of ATP b i n d i n g , but ra t h e r to an ac c e l e r a t e d t r a n s i t i o n of the enzyme-ATP complex to a second complex, which can r a p i d l y convert to the phosphorylated intermediate. The presence of 2+ 2+ Mg i s r e q u i r e d f o r t h i s phosphorylation t o occurr. As w e l l , Mg 2+ plays a re g u l a t o r y r o l e i n Ca -transport by a c c e l e r a t i n g the r a t e o: decomposition of the phosphorylated intermediate ( i n e s i et a l , 197*0 10 This increases the turnover r a t e of the enzyme, thereby i n c r e a s i n g the r a t e of calcium uptake. 2+ 2+ As would be expected, Ca i t s e l f can a l t e r S.R. Ca - t r a n s p o r t . 2+ Increases i n -cytoplasmic Ca w i l l immediately increase the r a t e of ATP 2+ h y d r o l y s i s and Ca - t r a n s p o r t (Hasselbach, '1964). I t i s a l s o p o s s i b l e 2+ 2+ t h a t Ca a t the i n n e r surface of the membrane may i n h i b i t Ca - t r a n s p o r t . 2+ Yamada and Tonomura (1972) have found that Ca can c o m p e t i t i v e l y i n h i b i t 2+ Mg -dependent decomposition of the phosphorylated intermediate. This decomposition of the enzyme phosphate complex i s thought to be as s o c i a t e d w i t h the i n t e r i o r of the membrane (Sumida and Tonomura, 1974).. Two p h y s i o l o g i c a l parameters, pH and temperature, may also c o n t r o l 2+ the Ca - t r a n s p o r t mechanism. A l t e r a t i o n of e i t h e r parameter r e s u l t s 2+ 2+ i n an a l t e r a t i o n of the coupling r a t i o between Ca -uptake and Ca -ATPase a c t i v i t y (Tate et a l , 1980). The s e n s i t i v i t y of the transport mechanism to pH and temperature may be due t o an a l t e r a t i o n of the l i p i d -p r o t e i n i n t e r a c t i o n i n the S.R. membrane. d) Regulation by Monovalent Cations: I t i s now becoming apparent t h a t monovalent c a t i o n s , such as K + + 2+ and Na , p l a y an important r o l e i n the r e g u l a t i o n of S.R. Ca - t r a n s p o r t . E a r l i e r s t u d i e s (DeMeis , 1969; Katz and Repke, 1967) suggested that the 2+ a l k a l i metals i n h i b i t e d Ca -uptake i n t o S.R. v e s i c l e s . They were 2+ 2+ -unable, however, t o show a s i m i l a r decrease i n Mg -Ca -ATPase a c t i v i t y . With the development of b e t t e r experimental techniques i t i s now w e l l e s t a b l i s h e d that these ca t i o n s have a marked s t i m u l a t o r y e f f e c t on the 2+ Ca - t r a n s p o r t process (Shigekawa and P e a r l , 1976; Jones e t a l , 1977; 2+ 2+ Duggan, 1977; Lopaschuk et a l , 1980). Mg -Ca -ATPase a c t i v i t y and ATP + h y d r o l y s i s i n c a r d i a c S.R. v e s i c l e s i s enhanced to such a degree by K + 2+ t h a t Jones et a l (1977) r e f e r to the enzyme as a K -Ca -ATPase. The a l k a l i metals appear to exert t h e i r e f f e c t by i n c r e a s i n g the turnover 2+ 2+ r a t e of the Mg -Ca -ATPase pump (Shigekawa and P e a r l , 1976). K i n e t i c 2+ s t u d i e s suggest t h a t they can r e g u l a t e Ca -tra n s p o r t by two mechanisms. The phosphorylated ATPase can be i n two forms, an ADP-sensitive ( E " ' - P i n t a b l e 2) and A D P - i n s e n s i t i v e form (E-P i n t a b l e 2) (Shigekawa et a l , 1978). In the absence of K + an appreciable p o r t i o n of the phosphorylated 11 enzyme i s i n s e n s i t i v e t o ADP (E-P). I f K + i s added the E-P can convert t o the the ADP-sensitive form, which can r e a c t w i t h ADP t o g i v e the enzyme pl u s ATP. As w e l l , the presence of a l k a l i metals r e s u l t i n an inc r e a s e d decomposition r a t e of the A D P - i n s e n s i t i v e complex (Shigekawa and Akowitz, 1979). These steps r e s u l t i n an increase turnover of the phosphorylated i n t e r m e d i a t e , which increases the r e a c t i o n r a t e o f the enzyme. This work was r e c e n t l y confirmed by Yamada and Ikemoto (1980) who suggest t h a t + 2+ m u l t i p l e forms of the E-P complex e x i s t s depending on K and Mg c o n -c e n t r a t i o n s . They al s o demonstrate that K + w i l l s h i f t the e q u i l i b r i u m of the s e q u e n t i a l ADP-sensitive t o A D P - i n s e n s i t i v e E-P r e a c t i o n back toward the ADP-sensitive form. Although i t has been w e l l e s t a b l i s h e d that the a l k a l i metals can 2+ a l t e r Ca -tra n s p o r t m S.R. i t s p h y s i o l o g i c a l r e l e v a n c e , i f any, i s not 2+ known. Ca -uptake and enzyme phosphorylation experiments t o date have been performed on fragmented S.R. p r e p a r a t i o n s , where the e x t e r n a l i o n concentration can be e a s i l y r e g u l a t e d . In these experiments the a c t i v -a t i o n occurrs i n the range of 0-50mM K + or Na +. In v i v o the S.R. i s bathed i n high K +, i n the range of 100-175mM ( S r e t e r , 1963). I t i s not known whether K + (or Na +) l e v e l s i n v i v o can change to such a degree as 2+ to be p h y s i o l o g i c a l l y s i g n i f i c a n t as a r e g u l a t o r of Ca - t r a n s p o r t , e) Regulation by a C y c l i c AMP-dependent P r o t e i n Kinase: Probably the most unique fea t u r e concerning the r e g u l a t i o n of cardiac S.R. i s the a b i l i t y of the cAMP-dependent p r o t e i n kinase t o s t i m u l a t e 2+ Ca - t r a n s p o r t (Kirchberger e t a l , 1972). C y c l i c AMP-dependent p r o t e i n k i n a s e , which i s present i n c a r d i a c c e l l s (Walsh et a l , 1968; Wray e t a l , 1973), w i l l phosphorylate a p r o t e i n contained i n the c a r d i a c S.R. membrane (LaRaia and Morkin, 197^; Wray et a l , 1973). The phosphorylation of t h i s 22,000 MW p r o t e i n , termed phospholambam (Katz e t a l , 1975),results i n s t i m u l a t i o n of the c a l c i u m pump (Kirchberger et a l , 197^0. Figure 2 (Hicks e t a l , 1979) i s a schematic r e p r e s e n t a t i o n of the proposed. mechanism of a c t i o n of cAMP-dependent p r o t e i n k i n a s e . According t o t h i s scheme phosphorylation of phospholambam, which i s a s s o c i a t e d w i t h the 2+ 2+ Mg -Ca -ATPase enzyme, r e s u l t s i n a conformational change i n the p r o t e i n . 2+ . • This i n c r e a s e s the Ca - s e n s i t i v i t y of the ATPase enzyme by decreasing 12 Insid* Phoiphoprotain PhoipHotote Cyclic AMP-D«p«nd«mt P'0»«in Kinos* Figure 2: P o s s i b l e mechanism- by which phospholambam modulates the a c t i v i t y o f the calcium pump of the c a r d i a c sarcoplasmic r e t i c u l u m . Upper: Dephosphorylation °f phosphlambam i n t e r a c t s w i t h t h e calcium pump ATPase i n the membrane 2+ (shaded), c o n f e r r i n g p o s i t i v e c o - o p e r a t i v i t y on the two Ca b i n d i n g s i t e s 2+ and lowering the Ca - s e n s i t i v i t y of the l a t t e r . Lower: Phosphorylation of phospholambam reduces i t s i n t e r a c t i o n w i t h the calcium pump ATPase, 2+ 2+ i n c r e a s i n g the Ca s e n s i t i v i t y of calcium uptake and a l l o w i n g t h e Ca 2+ b i n d i n g s i t e s of the calcium pump t o i n t e r a c t independently w i t h Ca . 2+ These e f f e c t s increase c a l c i u m t r a n p o r t r a t e a t low Ca con c e n t r a t i o n s . Reproduced from Hicks et a l (1979). 13 2+ the p o s i t i v e c o - o p e r a t i v i t y between the two Ca bi n d i n g s i t e s . The 2+ increase i n the Ca - s e n s i t i v i t y of the ATPase enzyme r e s u l t s i n s t i m u l -2+ a t i o n o f Ca - t r a n s p o r t by the pump. In other words, cAMP-dependent p r o t e i n kinase acts on the ATPase r e a c t i o n sequence i t s e l f . This i s supported by the work of Tada et a l (1979) who demonstrated t h a t phos-p h o r y l a t i o n of phospholambam incr e a s e s the V of the calcium pump, max 2 + 2 + but not the l e v e l of the phosphorylated intermediate of Mg -Ca -ATPase. C y c l i c AMP-dependent p r o t e i n kinase r e g u l a t i o n of cardiac S.R. 2+ Ca - t r a n s p o r t has generated (considerable i n t e r e s t due to the p o s s i b i l i t y that i t may mediate catecholamine a c t i o n on the heart. Current b i o -chemical evidence , as summarized by Katz (1979) ( f i g u r e 3), suggests t h a t catecholamines act through cAMP-dependent p r o t e i n kinase t o l ) 2+ phosphorylate t r o p o n i n I , which decreases the Ca - s e n s i t i v i t y of t r o p o n i n C, and 2) t o phosphorylate an S.R. p r o t e i n , phospholambam, 2+ which r e s u l t s i n s t i m u l a t i o n of Ca -uptake. Together the two processes can r e s u l t i n augmentation of the r a t e and degree of ten s i o n develop-ment i n c a r d i a c muscle. Although cAMP-dependent p r o t e i n kinase had been shown t o be i n v o l v e d i n the two processes mentioned above, as of yet no experimental evidence c l e a r l y demonstrates the involvement of c a t e c h o l -amines. In v i t r o experiments us i n g fragmented S.R. prep a r a t i o n s have 2+ f a i l e d t o show an augmentation of Ca - t r a n s p o r t using adrenergic agonists (Yu and T r i e s t e r , 1969)- Messineo and Katz (1979) have shown that 2+ ^ - a d r e n e r g i c b l o c k i n g agents, such as p r o p r a n a l o l , w i l l i n h i b t Ca -uptake i n t o S.R., but i n concentrations which f a r exceed c l i n i c a l l y observed serum l e v e l s . The p o s s i b l e r o l e of phospholambam i n cardiac S.R. calcium transport i s now becoming wi d e l y accepted. Whether cAMP-dependent p r o t e i n kinase 2+ e g u l a t i o n of the S.R. Ca pump i s unique t o cardiac S.R. i s s t i l l con-t r o v e r s i a l . K i r c h b e r g e r e t a l (1972) f a i l e d to show any s i g n i f i c a n t 2+ s t i m u l a t i o n of Ca -tra n s p o r t by f a s t s k e l e t a l muscle S.R. i n the presence of the kinase enzyme. Schwartz e t a l (1976) were able.to show s l i g h t 2+ s t i m u l a t i o n of Ca - t r a n s p o r t , but not a concomittant phosphorylation of any S.R. v e s i c l e p r o t e i n . E f f o r t s to i s o l a t e phospholambam from f a s t • Possible Role of Phosphorylation of Cardiac Contractile Proteins and Sarcoplasmic Reticulum in Mediating the Mechanical Response of the Heart to Catecholamines 1. Catecholamine Binding to Sarcolemmal P - Receptor I 2. Activation of Adenylyl Cyclase 3 . Increased Intracellular Cyclic AMP 4. Activation of Cyclic AMP-Dependent Protein Kinase 5A: Phosphorylation of Troponin I " ° ' 6A: Decreased Ca 2 *-Sensit ivity of the Troponin Complex / \ 7A: Increased Calcium.Requirement 7B: Facilitation of Calcium for Contraction Removal during Relaxation ^ >i (Decreased Tension) 5B: Phosphorylation of Phospholamban 6B: Increased Ca 2 + -Sens i t iv i ty of the Calcium Pump-Channel Mechanism I \ 7C: Increased Calcium Transport 7D: Increased Calcium Rate Release Rate i i A C C E L E R A T E D R E L A X A T I O N 8. Increased Calcium Store* in AVA.ci.cn Sarcoplasmic Reticulum A C C E L E R A T E D C O N T R A C T I O N INCREASED TENSION < . (Increased Calcium Influx via slow channel) Figure 3: Proposed i n t e g r a t i o n of the e f f e c t s of catecholamines on the ca r d i a c c o n t r a c t i l e p r o t e i n s and sarcoplasmic r e t i c u l u m i n terms of t h e i r p h y s i o l o g i c a l reponse. Reproduced from Katz (1979)-15 s k e l e t a l muscle S.R. (Kirchberger and Tada, 1976) were u n s u c c e s s f u l , and only s m a l l amounts could be obtained from slow s k e l e t a l muscle. The more pronounced r o l e of cAMP-dependent p r o t e i n kinase i n cardiac S.R. conforms w i t h the p h y s i o l o g i c a l r o l e of catecholamines i n muscle. In f a s t s k e l e t a l muscle catecholamines w i l l only s l i g h t l y increase the t e n s i o n developed, u n l i k e the marked changes seen i n cardiac muscle ( G o f f e r t and R i t c h i e , 1952). I f indeed cAMP-dependent p r o t e i n kinase does mediate catecholamine a c t i o n , one would expect t h i s more pronounced a c t i o n i n cardiac S.R. pre p a r a t i o n s . 2+ f ) Regulation of Ca -Transport by Calmodulin: One of the many apparent f u n c t i o n s of calmodulin i s the r e g u l a t i o n 2+ 2+ of v a r i o u s Ca -t r a n s p o r t events. Calmodulin w i l l s t i m u l a t e Ca -uptake i n t o synaptosomes (Ochs and I g b a l , 1978) as w e l l as i n s i d e - o u t v e s i c l e s d erived from e r y t h r o c y t e membranes (Larsen and V i n c e n z i , 1978). Previous work i n t h i s l a b o r a t o r y demonstrated that calmodulin obtained from bovine 2+ b r a i n w i l l s t i m u l a t e Ca -uptake i n t o c a r d i a c S.R. v e s i c l e s (Katz and Remtulla, 1978). The maximum a c t i v a t i o n seen by calmodulin was a d d i t i v e 2+ t o the maximum a c t i v a t i o n of Ca -uptake seen w i t h cAMP-dependent p r o t e i n 2+ kinase. This suggests t h a t the calmodulin 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 a r e s u l t of inc r e a s e d cAMP l e v e l s due t o a c t i v a t i o n of adenylate c y c l a s e . LePeuch e t a l (1979) found t h a t calmodulin• will.,phosphorylate . phospholambam through a p r o t e i n k i n a s e T h i s kinase enzyme d i f f e r s from 2+ cAMP-dependent p r o t e i n kinase i n that i t i s Ca -dependent, and the c a t a l y t i c subunit appears t o be membrane bound. The p r o t e i n i n h i b i t o r 2+ of cAMP-dependent p r o t e i n kinase w i l l not reverse the Ca -calmodulin phosphorylation of phospholambam. This l e d LePeuch e t a l t o b e l i e v e that r e g u l a t i o n of cardiac S.R. calcium t r a n s p o r t occurred through two sep-erate p r o t e i n kinases , each phosphorylating a d i s t i n c t s i t e on phospho-lambam. Phosphorylation experiments performed by Kranias et a l (1980) 2+ support t h i s hypothesis. They found that calmodulin-Ca -dependent phosphorylation and cAMP-dependent phosphorylation both occurred on the 2+ same p r o t e i n . As w e l l , phosphorylation of ca r d i a c S.R. by Ca and calmodulin, i n the presence of cAMP-dependent p r o t e i n kinase,was enhanced above the l e v e l obtained when cAMP-dependent p r o t e i n kinase was present. alone. 16 I I I ) O b j ectives of the present study 2+ Although we know that calmodulin regulates c a r d i a c S.R. Ca -t r a n s p o r t , l i t t l e i s known concerning i t s mechanism of a c t i o n . I t has been shown t h a t calmodulin i s i n v o l v e d i n phosphorylation o f phospho-2+ lambam, but what e f f e c t t h i s has on the Ca -pump mechanism remains to be e l u c i d a t e d . As w e l l , the monovalent c a t i o n s p l a y an important r o l e 2+ i n the t r a n s l o c a t i o n of Ca . The i n t e r a c t i o n between calmodulin 2+ r e g u l a t i o n and monovalent c a t i o n r e g u l a t i o n of Ca - t r a n s p o r t has not been p r e v i o u s l y s t u d i e d . In t h i s study we used cardiac microsomes, enriched i n S.R., t o 2+ c h a r a c t e r i z e calmodulin s t i m u l a t i o n of Ca - t r a n s p o r t . These v e s i c l e s 2+ are b e l i e v e d to have an i n t a c t Ca - t r a n s p o r t mechanism, which i s 2+ c h a r a c t e r i s t i c o f Ca -transport i n v i v o . Using t h i s system we s t u d i e d the i n t e r a c t i o n of calmodulin and monovalent c a t i o n s on the t r a n s p o r t system. K i n e t i c a n a l y s i s and i n i t i a l r a t e s t u d i e s were performed t o + 2+ determine what e f f e c t calmodulin and K had on the Ca - s e n s i t i v i t y of the pump. These r e s u l t s can be compared t o r e s u l t s p r e v i o u s l y obtained by Hicks et a l (1979) studying cAMP-dependent p r o t e i n kinase s t i m u l a t i o n 2+ of cardiac S.R. Ca - t r a n s p o r t . From t h i s i t can be determined whether 2+ calmodulin-dependent phosphorylation of S.R. has an e f f e c t on Ca -t r a n s p o r t s i m i l a r to cAMP-dependent p r o t e i n kinase phosphorylation. 2+ Since calmodulin appears t o r e g u l a t e Ca -transport i t i s im-p o r t a n t to determine whether i t i s a c y t o s o l i c p r o t e i n , or an i n t e g r a l p r o t e i n of the S.R.. Therefore experiments were performed to determine i f calmodulin was endogenous to the S.R. membrane. This was done using a number o f techniques which have been used t o i s o l a t e calmodulin from other sources. I f calmodulin i s a c y t o s o l i c p r o t e i n then i t must e i t h e r i n t e r a c t d i r e c t l y w i t h the S.R., or through an intermediate which i n -125 t e r a c t s w i t h the S.R.. Therefore I - l a b e l l e d calmodulin was pre-2+ pared i n order t o determine i f calmodulin w i l l b i n d t o S.R. i n a Ca -dependent manner.- The e f f e c t of monovalent c a t i o n s on calmodulin b i n d i n g t o S.R. was a l s o determined. Although much work must be done t o r e v e a l the mechanism of calmodulin 2+ r e g u l a t i o n of Ca - t r a n s p o r t i n cardiac S.R. the above o b j e c t i v e s should p a r t i a l l y c l a r i f y t h i s problem. •17 MATERIALS AND METHODS A) M a t e r i a l s : 1) Animals: Dogs of e i t h e r sex between s i x months and four years of age were used throughout the study. Hearts were removed from p e n t o b a r b i t o l aneastheti.zed dogs and placed i n c o l d normal s a l i n e s o l u t i o n (k°C). The v e n t r i c l e s were cut i n t o 2-4g p i e c e s , quick frozen i n methyl butane on dry i c e , and sto r e d at -80°C u n t i l use. 2) Chemicals: i ) Radioisotopes - ^ C a i n the form of ( i | 5 C a ) C l ? (-10 Ci/mmole),Y- 3 2P-ATP .( X6.h:_\^ 125 125 Ci/mmole), and J\ i n form of Na( J l ) i n NaOH pH 7-11 (l5mCi^(g) were purchased from Amersham c o r p o r a t i o n (Toronto, O n t a r i o ) . i i ) Chromatography columns -Sephadex G-15, g-25, g-75, g-200, DEAE Sephadex A50:, and Blue Dextran 2000 were purchased from Pharmacia Fine Chemicals^. -AG 50 WX8 (200-400 mesh) a n a l y t i c a l grade c a t i o n exchange r e s i n was purchased from Bio-Rad l a b o r a t o r i e s , i i i ) SDS-polyacrylamide g e l e l e c t r o p h o r e s i s -sodium dodeeyl sulphate and -.mercaptoethanol were, purchased from Siguier chemicals. -Sodium phosphate (monobasic and d i b a s i c ) was purchased from F i s h e r ^ S c i e n t i f i c .Co.® - a l l other reagents were obtained from Bio-Rad . i v ) F i l t r a t i o n equipment -Immersible-CX separator f i l t e r s and c i r c u l a r vacuum f i l t e r s (0 . 4 5 / ^ , 0 .8 /^ , 2.0/\ , and 5.0/\) were obtained from M i l l i p o r e ^ Co. Cr) -PM-10 f i l t e r s (10,000 MW c u t o f f ) were obtained from AmicorX. v) Assay Chemicals -CaClp^ di h y d r a t e , L-ascorbate, 'Trizma o x a l a t e , sodium carbonate, and sodium bicarbonate were purchased from Analar BDlf^.'Chemicals. - L i t h i u m c h l o r i d e , lanthanum c h l o r i d e , and a c t i v i a t e d c h a r c o a l were purchased from F i s h e r S c i e n t i f i c Co. -2-methyl butane was purchased from MCB manufacturing chemists. -Aquasol s c i n t i l l a t i o n f l u i d was purchased from New England Nuclear^. -Sodium hydroxide (10N) was obtained from Baker^ Chemicals. 18 (r) -The folio-win chemicals -were purchased from Sigmar chemical: Trizma Adenosine Triphosphatase (equine muscle) Phosphodiesterase 3 ' , 5 ' - c y c l i c n u c l e o t i d e a c t i v a t o r from bovine h e a r t Adenosine 3 ' , 5 ' - c y c l i c n u c l e o t i d e monophosphate ( T r i s s a l t ) cAMP-dependent p r o t e i n kinase from beef heart (Type I I ) L - H i s t i d i n e f r e e base Bovine serum albumin EGTA Sucrose EDTA Tr'iama base Chloramine T Trizma HCl Imidazole Trizma phosphate Magnesium c h l o r i d e T r i t o n X-100 Potassium c h l o r i d e D i t h i o t h r e o t o l Sodium c h l o r i d e Potassium i o d i d e Sodium azide Urea Sodium potassium t a r t r a t e Copper s u l f a t e B) Methods. l ) P r e p a r a t i o n of Calmodulin from Outdated Human E r y t h r o c y t e s : Calmodulin was prepared by a m o d i f i c a t i o n of the method of V i n c e n z i and co-workers (Jung, 1978). One hundred ml of outdated human blood, obtained from the Canadian Red Cross, was d i v i d e d i n t o four 50ml p l a s t i c c e n t r i f u g e tubes. The tubes were c e n t r i f u g e d at 3,000xg f o r 5 minutes at h°C i n a Beckman®Model J2-21 c e n t r i f u g e . The supernatant was discarded and the packed c e l l s washed w i t h normal s a l i n e (0.9% NaCl). The tubes were r e c e n t r i f u g e d (3,00Q>(g f o r 5 minutes), the supernatant d i s c a r d e d , and the normal s a l i n e wash subsequently repeated three times. Washed packed c e l l s were then l y s e d w i t h 500ml 15.7mM Imidazole pH 7.^ and the r e s u l t a n t hemolysate c e n t r i f u g e d at 30 5000yg f o r 30 minutes. The supernatant was f i l t e r e d through 2^ m i f i I t e r s using a M i l l i -pore® pump, and NaCl added t o b r i n g the hemolysate to 0.3M NaCl. The hemolysate was then passed through a DEAE-Sephadex^ A50 column ( 2 . 6 X 35 cm), p r e - e q u i l i b r a t e d w i t h 0.3 M NaCl and 20mM Imidazole (pH 6 .8). The hemolysate was A e l u t e d from the column using the e q u i l i b r a t i n g b u f f e r . Calmodulin was e l u t e d from the column w i t h a l i n e a r NaCl 19 gradient (0.3-0.8 M NaCl) and 8ml f r a c t i o n s c o l l e c t e d u s i n g a Bromma^ LKB 7000. U l t r a r a c f r a c t i o n c o l l e c t o r . These f r a c t i o n s were assayed f o r 2+ 2+ t h e i r a b i l i t y t o s t i m u l a t e (Mg -Ca )-ATPase a c t i v i t y of human EDTA-washed red c e l l membranes. A c t i v e f r a c t i o n s were pooled and concentrated k times using M i l l i p o r e ^ Immersible CX membranes (10,000 MW'iCUTOFF). A Sephadex G-15 column (2X20cm) was used t o desalt the calmodulin s o l -u t i o n . P u r i t y of the s o l u t i o n was determined by sodium dodecyl-poly-acrylamide g e l e l e c t r o p h o r e s i s (SDS-PAGE) and a Gelman ACD l6 d e n s i t o -meter used t o determine the percent of calmodulin p r o t e i n present i n the p o l y a c r lamide g e l s . Absence of s a l t i n the calmodulin s o l u t i o n was v e r i f i e d u s i n g atomic absorption spectrophotometry. The calmodulin s o l u t i o n was s t o r e d i n a l i q u o t s at -80°C. 2) P r e p a r a t i o n of Cardiac Microsomes Enriched w i t h Sarcoplasmic Reticulum V e s i c l e s : Sarcoplasmic r e t i c u l u m enriched v e s i c l e s were prepared using the methodology of Harigaya and Schwartz (1969) w i t h 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 performed at h®C. Frozen dog v e n t r i c l e (Ug) was minced and put i n t o 30ml of b u f f e r 1 (lOmM NaHCO^ 5mM NaN^, and 0.2 mM ascorbate pH 6.8) contained i n a g l a s s homogenizer tube. This was homogenized using a t e f l o n p e s t l e at 1500 r.p.m. f o r 15 seconds. The homogenized s o l u t i o n was t r a n s f e r r e d t o a 50ml Beckman p l a s t i c c e n t r i f u g e tube and, along w i t h three other s i m i l a r homogenate (r) s o l u t i o n s , was c e n t r i f u g e d at 10,000Xg f o r 20 minutes m a Beckman^ Model J2-21 c e n t r i f u g e . The supernatant from the f o u r tubes was then d i v i d e d i n t o 8 Corex® tubes and c e n t r i f u g e d at ^0,000Xg f o r 60 minutes. The r e s u l t a n t supernatant was discarded and the p e l l e t resuspended by gentle homogenization i n 2ml of B u f f e r 2 (6.6 M KC1, 2.0 mM t r i s CI pH 7.3, 1.0 mM MgClg). The suspensions were pooled, d i v i d e d i n t o 2 co rex0tubes and b u f f e r 2 added t o b r i n g the volumes up to 10ml. These tubes were r e c e n t i f u g e d at U-0,00CXg f o r 60 minutes, the super-natant was discarded, and the two p e l l e t s pooled and suspended i n 10 ml B u f f e r 3 ( lOmM t r i s CI pH 7.3)'. A f t e r re c e n t r i f u g a t i o n at U0,000Xg f o r 60 minutes, the supernatant was d i s c a r d e d , and the two p e l l e t s pooled and suspended i n 3 ml of B u f f e r h (h0% sucrose 10 mM t r i s CI pH 7.U, 5 mM d i t h i o t h r e i t o l ) . The sucrose s o l u t i o n had p r e v i o u s l y 20 been p u r i f i e d by passage through a Bio-Rad® AG 5Cv-X8 cation exchange r e s i n column i n the Na form. The concentrated microsomal suspension (1.2-1.5 mg/ml) was then divided i n t o k t e s t tubes. These were e i t h e r used immediately, or quick frozen, by immersing the aliquot i n methyl butane on dry i c e , and stored at -80°C. When used the concentrated aliquots were d i l u t e d with b u f f e r k such that the f i n a l p r otein content ranged from O.U-0.6mg/ml. A l l microsomal suspensions were used within two weeks of preparation. The a c t i v i t y of the suspension, as measured 2+ by the a b i l i t y t o transport Ca int o the S.R. v e s i c l e s , was s i m i l a r i n fresh and previously frozen ali q u o t s . Measurement of Calcium Uptake by Cardiac Microsomes Enriched i n Sarcoplasmic Reticulum V e s i c l e s : Oxalate f a c i l i t a t e d ATP-dependent calcium uptake by S.R. was measured by the method of Tada et a l (197M with a few modifications. An incubation medium was prepared such that when 0.3 ml was d i l u t e d to the f i n a l incubation volume of 0.5 ml a sol u t i o n of ho mM h i s t i d i n e - H C l pH 6.8, 5mM MgClg, 5 mM t r i s -ATP and 2.5 mM t r i s - o x a l a t e was obtained. Depending on the experiment, calmodulin or monovalent cations such as 110 mM K + were then added to the incubation medium. When monovalent cation strength was a l t e r e d the osmolarity of the incubation medium was maintained by sucrose a d d i t i o n . The microsomes enriched i n S.R. v e s i c l e s (30-50/A.g) were then pre-incubated i n the incubation medium f o r 11 minutes at 30°C. Calcium uptake by the v e s i c l e s was i n i t i a t e d by the addition of CaClg containing ( ^ C a ^ l ^ (10 Ci/mmole). The desired free calcium concentration was maintained by the addition-of ethylene-2+ b i s - ( ^ - a m i n o e t h y l ether) -N,N'-tetraacetate (EGTA) and the free Ca concentration present determined by the equations of Katz e t a l (1970) 2+ taking i n t o consideration temperature, pH, Mg concentration, and ATP concentrations. Calcium uptake a c t i v i t y was terminated, usually a f t e r 5 minutes, by f i l t e r i n g a O.kl ml ali q u o t of the incubation medium through Millipore® f i l t e r s (type HA 0.45/tpore size) with'the a i d of a Millipore® pump. The f i l t e r s were then washed twice with 10 ml of 1+OmM t r i s - C l pH 6.8, dried f o r 5 minutes at 60°C, and placed i n 10 ml of Aquasol® s c i n t i l l a t i o n f l u i d . Samples were counted for 10 minutes i n an IsocapQ 21 s c i n t i l l a t i o n counter. k) C a l c u l a t i o n of Calcium Uptake A c t i v i t y by Cardiac Microsomes Enriched i n Sarcoplasmic Reticulum: 2+ The r a t e o f Ca -uptake by the microsomal p r e p a r a t i o n i s expressed 2+ as nmoles Ca taken up per mg p r o t e i n per minute.. This i s determined by the f o l l o w i n g formula: Sample Counts - Blank Counts 2 + .—— "y.dilution X T o t a l (Ca ) /mg y i n c u b a t i o n T o t a l Counts - Blank Counts f a c t o r / p r o t e i n time where 1+5 2+ Sample Counts = Ca counts obtained per i n d i v i d u a l samples. 1+5 2+ T o t a l Counts= t o t a l Ca counts present i n the i n c u b a t i o n medium 1+5 2+ Blank Counts= Ca counts obtained i n the absence of microsomal p r o t e i n 2+ T o t a l (Ca )= t o t a l c alcium concentration present i n t h e i n c u b a t i o n medium D i l u t i o n factor= c o r r e c t i o n f o r i n c u b a t i o n volume = 0.5ml = 1.21 a l i q u o t volume 0.1+1 ml mg protein= weight o f microsomal p r o t e i n present i n the i n c u b a t i o n medium. Incubation time= l e n g t h of time the microsomal p r o t e i n was incubated i n the presence of CaCl^. 5) Sodium Dodecyl Sulphate-Polyacrylamide G el E l e c t r o p h o r e s i s (SDS-PAGE): i ) P r e p a r a t i o n o f 10% polyacrylamide g e l s : SDS-PAGE was performed according to the p r o t o c o l of Weber and Osborne (1968). A 10% acrylamide stock s o l u t i o n was prepared by d i s -s o l v i n g 22 .2g acrylamide and 0 .6g b i s - a c r y l a m i d e i n 100 ml d i s t i l l e d HgO , and f i l t e r e d through a Whatman #1 f i l t e r . The acrylamide stock s o l u t i o n (13 . 5 ml) was g e n t l y mixed w i t h 15 ml Gel b u f f e r ( 0 . 2 M Na phosphate b u f f e r , pH 7.0, 0.2% SDS). To t h i s , 1.5ml of 1.5% ammonium p e r s u l f a t e and 0.01+5 ml TEMED (N,N,N' ,N'-tetramethylene-diamine) Neat reagent were added and g e n t l y mixed. The mixture was then p i p e t t e d i n t o g l a s s g e l tubes (2 .5ml/tube) sealed at one end w i t h P a r a f i l m . Before the g e l s o l u t i o n s e t , one drop of d i s t i l l e d 22 H^.Q was. added to the top of each g e l . i ) P r e p a r a t i o n : o f Calmodulin P r o t e i n Samples: P r e v i o u s l y frozen calmodulin samples (U0-80/<g/ml) were l y o -p h i l i z e d using a V i r t i s l y o p h i l i z e r , such t h a t approximately 20/^g of s o l i d p r o t e i n was obtained per sample. This was resuspended i n 70/^ 1 of d i s t i l l e d H^O. To t h i s was added 30/t.l of a p r e v i o u s l y prepared s o l u t i o n c o n t a i n i n g 5X b u f f e r (0.5 M Na phosphate b u f f e r , pH 7.0, 5% SDS, k0% g l y c e r o l ) : 0.03% bromophenol b l u e : and 2-mercaptoethanol Neat reagent i n a r a t i o of 20:5:5- The samples were then capped, g e n t l y b o i l e d f o r 5 minutes, cooled t o 20°C, and p i p e t t e d onto t h e top of each g e l . E l e c t r o p h o r e s i s b u f f e r (Gel b u f f e r d i l u t e d 1:1) was then care-f u l l y added t o f i l l each g e l tube. i i ) E l e c t r o p h o r e s i s , S t a i n i n g , and Destaining of Samples: The g e l tubes were then pla c e d i n a Pharmaci EPS 500/1+00 E l e c t r o p h o r e s i s Gel Column Apparatus. A current of 8 mA/tube was app l i e d t o the apparatus f o r 8 hours, or u n t i l the t r a c k i n g dye had migrated t o the bottom of the g e l tubes. The polyacrylamide g e l s were then removed from the tubes by f o r c i n g g e l b u f f e r between the g e l and the tube w i t h a syri n g e . The gels were placed i n i n d i v i d u a l t e s t tubes c o n t a i n i n g s t a i n 1 ( 0.0125% Coomassie b r i l l i a n t b l u e , 10% g l a c i a l a c e t i c a c i d , and 25% i s o p r o p y l a l c o h o l ) . A f t e r l6 hours s t a i n 1 was replaced w i t h s t a i n 2 (0.0125% Coomassie b r i l l i a n t b l u e , 10% g l a c i a l a c e t i c a c i d , and 10% i s o p r o p y l a l c o h o l ) , and the gels l e f t f o r an ad-d i t i o n a l 2k hours. The gels were destained and stored i n a 10% a c e t i c a c i d s o l u t i o n . Molecular weight of the p r o t e i n bands was determined by comparing the Rf value (distance t r a v e l l e d by the band/ m i g r a t i o n distance of the dye) to a standard curve of the r f vs. l o g molecular weight of the standards. 6) I o d i n a t i o n of Calmodulin: R a d i o i o d i n a t i o n of calmodulin was achieved by the method of Hunter and Greenwood (1962). The e n t i r e procedure, i n c l u d i n g e l u t i o n of the 1 2 ^ I - c a l m o d u l i n through a Sephadex® G-25 column, was performed at 1+°C. The column was prepared by soaking 5 g of Sephadex G-25 i n 0.05 M NaH 2P0^.2H 20)pH T.k + 2 g / l B.S.A., and packing i t i n t o a 1>C30 cm jac k e t e d column maintained at U°C. The i o d i n a t i o n r e a c t i o n was performed 23 i n a v i a l c o n t a i n i n g 2 mCi Iodine-125 (Amersham R a l ( 1 2 ^ l ) i n NaOH pH 7-11, 13-17 mCi/Ag). To t h i s was added lOjul (lS^g) pure calmodulin, and l O / i l of 5 mg/ml chloramine T i n 0.05. M HaH2P0^.2H 0 pH '7.U. W i t h i n 15 seconds of the chloramine T a d d i t i o n , 10/CL of 2h mg/ml Na m e t a b i s u l f i t e 0.05. M RaH 2P0^.2H 20 pH 7.^ was added, f o l l o w e d by 0.2ml of lOmg/ml KI i n 0.05 M NaH 2P0^.2H 20 pH 7-^. The contents of the v i a l were then p i p e t t e d onto the s.urface of the Sephadex G-25 column. The i o d i n a t i o n v i a l was r i n s e d w i t h a f u r t h e r 0.2 ml KI s o l u t i o n , which was a l s o 125 added to the column. The I-calmodulin was. e l u t e d w i t h 0.05 M Na 2P0^. 2H2Q pH J.k + 2 g/1 B.S.A. and 6 drop f r a c t i o n s c o l l e c t e d i n p o l y -styrene tubes (Amersham/Searle®). These tubes were counted i n a Nuclear-Chicago II85 Gamma counter f o r 1 second, and the f r a c t i o n from the f i r s t peak w i t h the highest a c t i v i t y were pooled and st o r e d a t -70°C. A 15/^1 sample of the i o d i n a t e d calmodulin was suspended i n 1 ml of 10$ t r i c h l o r o a c e t i c a c i d (TCA) and counted i n the -Gamma counter. This, was then c e n t r i f u g e d and 0.5 ml of the supernatant counted s e p a r a t e l y . I t 125 was determined that approximately 50$ of the I i n the sample was bound t o calmodulin. 125 7) Measurement of I-calmodulin B i n d i n g to Sarcoplasmic Reticulum Enriched Microsomes: Bindi n g s t u d i e s were performed us i n g i n c u b a t i o n procedures s i m i l a r 2+ to those u t i l i z e d m the Ca -uptake experiments. Microsomal S.R. (approximately 150^g/0.5 ml i n c u b a t i o n volume) was pre-incubated f o r 7 minutes at 30°C i n Corex tubes c o n t a i n i n g kO mM h i s t i d i n e - C l pH 6.8, 5 mM MgClg, 110 mM KC1, 5 mM ATP, and 2.5 mM t r i s . o x a l a t e . The i o d i n a t e d calmodulin (approximately 80,000 cpm) was added t o the i n c u b a t i o n medium • " " • • • 2+ wi t h the b u f f e r e d - c a l c i u m -solution'. - The amount.of • Ca necessary t o maintain, a desired-free-*G"a c o n c e n t r a t i o n I n the presence: of "0. kmM -.EGTA was determined by the equations of Katz et a l (1970). A f t e r 5 minutes 'of i n c u b a t i o n the medium was c e n t r i f u g e d at kO ,000*g f o r ho minutes and the supernatant discarded. The p e l l e t was c a r e f u l l y washed 2+ w i t h h0 mM t r i s - C I pH 7-2, c o n t a i n i n g O.lt mM EGTA and the Ca conc-e n t r a t i o n t h a t was present i n the i n c u b a t i o n medium (O.Ol/jm-10 mM). The p e l l e t was then suspended i n 0.75ml of t r i s - C l s o l u t i o n by homo-g e n i z a t i o n w i t h a Tef l o n p e s t l e (200 RPM.X20 seconds). The content of 2h the Gorex tube was pi p e t e d i n t o p o l y s t y r e n e tubes (Amersham/Searle). The Gorex tube was then r i n s e d w i t h a f u r t h e r 0.75- ml of t r i s - C l s o l u t i o n , which was als o p i p e t e d i n t o the polystyrene tube. These tubes were counted f o r 10 minutes i n a Gamma spectrophotometer (Nuclear-0 125 Chicago ll85) which measured the photopeak f o r I . 125 Binding of I-calmodulin t o the microsomal S.R..was expressed as counts per minute/ mg sarcoplasmic r e t i c u l u m p r o t e i n . 8) Miscellaneous Methods: EDTA-washed human er y t h r o c y t e membranes were prepared by the method of B l o s t e i n (1968). 2+ 2+ Measurement of Mg -Ca -ATPase a c t i v i t y was performed by the technique of Katz and B l o s t e i n (1975). Determination of Na + concentrations i n calmodulin samples was c a r r i e d out by Atomic Absorption Spectrophotometry, according to the operation manual procedure of the V a r i a n Techtron A.A.-5 Spectrophoto-meter. The process was performed on samples containing calmodulin i n s o l u t i o n , or calmodulin p r e c i p i t a t e d w i t h 6% TCA. Procedures used f o r i s o l a t i n g calmodulin from cardiac S.R. membranes are described i n d e t a i l i n the r e s u l t s s e c t i o n . P r o t e i n assays were performed by the method of Lowry et a l (l95l) u s i n g Bovine Serum Albumin as a standard. . S t a t i s t i c a l a n a l y s i s was by the student's " t " t e s t f o r ungrouped data. In a niamber of f i g u r e s appearing i n the r e s u l t s ( f i g u r e 5,8,9, 10,11,14) experimental data was presented as t y p i c a l experiments, r a t h e r than the average of a number of experiments. This i s due t o the v a r i a b i l i t y from experiment to.experiment i n the c o n t r o l r a t e o f Ca -uptake i n c a r d i a c microsomes enriched i n sarcoplasmic r e t i c u l u m . The r a t e of t r i s - o x a l a t e f a c i l i t a t e d Ca^ +-uptake i n the presence of 2+ 1 y*l M fr e e Ca and absence of KC1 ranged from 5-0 t o 30 nmoles/mg/ min depending on the microsomal p r e p a r a t i o n used. In the presence of 2+ 110 mM KC1 the ra t e of t r i s - o x a l a t e f a c i l i t a t e d Ca -uptake i n t h e 2+ presence of 1/\ M f r e e Ca ranged from 10 t o 50 nmoles/mg/min. When the 25 e f f e c t of calmodulin on Ca -uptake by the cardiac microsomal S.R. was determined, maximal s t i m u l a t o r y concentrations of calmodulin were used. Depending on the calmodulin p r e p a r a t i o n used, t h i s ranged from 2.25 to 6.5 g/0.5ml f i n a l i n c u b a t i o n volume. 26 RESULTS l ) I s o l a t i o n of Calmodulin: In the m a j o r i t y of experiments performed i n t h i s study calmodulin was i s o l a t e d from hemolysates of human erythrocytes using a m o d i f i c a t i o n of the method of Jung•- (1978). F r a c t i o n s c o l l e c t e d from the DEAE Sephadex column were t e s t e d f o r t h e i r a b i l i t y t o s t i m u l a t e Mg -Ca -ATPase a c t i v i t y i n EDTA-washed red c e l l membranes. Figure k shows a 2+ 2+ t y p i c a l p r e p a r a t i o n i n which Mg -Ca -ATPase a c t i v i t y i s enhanced by 2+ a narrow range of f r a c t i o n s , w h i l e Mg -ATPase a c t i v i t y remains unal t e r e d . 2+ 2+ This suggests that the f r a c t i o n s are s p e c i f i c a l l y s t i m u l a t i n g Mg -Ca -ATPase. Once the a c t i v e f r a c t i o n s were concentrated 4 - f o l d i t was necessary t o d e s a l t the samples. I n i t i a l samples were des a l t e d by d i a l y s i s against low - concentrations of EDTA. In most instances t h i s r e s u l t e d i n 2+ 2+ an unexplained l o s s i n the a b i l i t y of calmodulin t o s t i m u l a t e Mg -Ca -ATPase i n EDTA-washed red c e l l membranes. I t was then decided t o O d e s a l t the calmodulin samples by passage through a G-15 Sephadex column. In order t o i n s u r e t h a t the sample was r e l a t i v e l y f r e e of s a l t i t was subjected t o atomic absorption spectrophotometry. This was performed e i t h e r w i t h the calmodulin free i n s o l u t i o n , or p r e c i p i t a t e d out w i t h 6% TCA. In a l l calmodulin samples i t was determined t h a t t h e r e was 0.6 mM or l e s s Na + present. I f the samples were d e s a l t e d through a G-25 Sephadex column the calmodulin was r e t a i n e d c l o s e to the v o i d volume of the column. Once the v o i d volume e l u t e d • Na + w i l l begin to be e l u t e d from the column. In the only sample des a l t e d w i t h the G-25 Sephadex*^ column i t was determined that 9 mM Na + was s t i l l present i n s o l u t i o n . To ensure that calmodulin a c t i v i t y was maintained during the concentrating and d e s a l t i n g procedure the sample was again t e s t e d 2+ 2+ f o r i t s a b i l i t y t o s t i m u l a t e Mg -Ca -ATPase i n EDTA-washed red c e l l membranes. Table 3 i n d i c a t e s t h a t these calmodulin preparations 2+ 2+ markedly s t i m u l a t e red c e l l Mg -Ca -ATPase a c t i v i t y without a l t e r i n g 2+ Mg -ATPase a c t i v i t y . Depending on the degree of s t i m u l a t i o n , the sample was e i t h e r concentrated f u r t h e r or used as i s . During the course of the study, calmodulin obtained from beef heart became com-m e r c i a l l y a v a i l a b l e from the Sigma Chemical Co.. Table 3 shows t h a t 27 Figure h: Determination of F r a c t i o n s Containing Calmodulin Using the ATPase Assay of EDTA-washed Red C e l l Membranes. ATPase assay, calmodulin p r e p a r a t i o n , and p r e p a r a t i o n of EDTA-washed red c e l l membranes were as described i n methods. The top l i n e {* 2+ 2+ represents Mg -Ca -ATPase a c t i v i t y i n each f r a c t i o n i n the presence 2+ 2+ of 50/^M Ca , w h i l e the bottom l i n e (• •) represents Mg -ATPase 32 a c t i v i t y . ATPase a c t i v i t y i s c a l c u l a t e d as counts/minute of P i ' 15,0 o o L C 3 0 u 10 2 0 30 fraction number 29 Table 3 2+ E f f e c t of Various Calmodulin Preparations on Mg -ATPase and 2+ 2+ Mg -Ca -ATPase a c t i v i t y of EDTA-washed Human Erythroc y t e Membranes Sample. Mg -ATPase A c t i v i t y Mg -Ca -ATPase A c t i v i t y % (pmoles/mg/min) (pmoles/mg/min) increase C o n t r o l 2566 3562 39 Calmodulin C - 5U32 (2.5/fg/ml) Calmodulin D 19^9 5620 188 (3.26/tg/ml) Calmodulin E 2578 8U58 228 (6.2/tg/ml) Sigma calmodulin 2225 5003 125 (20ng/ml) Sigma calmodulin 2H97 6302 152 (UO/g/ml) -calmodulin samples C,D,. and E are three t y p i c a l calmodulin preparations from hemolysate of human eryt h r o c y t e s . The r calmodulin purchased from Sigma -Chemical Co.., i s derived from beef heart. -ATPase a c t i v i t y was measured i n the presence and absence of 2+ 50 f\M Ca as described i n methods. - data was normalized due t o d i f f e r e n c e s i n membrane a c t i v i t y between experiments. 30 2+ 2+ t h i s calmodulin w i l l a l s o s t i m u l a t e Mg -Ca -ATPase, but that much higher concentrations are necessary t o produce a s i m i l a r e f f e c t . The reason f o r t h i s decreased potency w i l l be discussed i n a l a t e r s e c t i o n . E a r l y on i n the study i t was found that calmodulin i n the ab-sence of K + i n the i n c u b a t i o n medium cou l d s u b s t a n t i a l l y s t i m u l a t e Ca - t r a n s p o r t i n c a r d i a c microsomal S.R.. I t was decided t h a t t h i s assay could be used t o determine r e l a t i v e a c t i v i t y of the calmodulin sample prepared . Figure 5 demonstrates the calmodulin c o n c e n t r a t i o n -2+ dependent increase i n Ca -uptake observed w i t h a t y p i c a l sample prep-a r a t i o n under these c o n d i t i o n s . In a d d i t i o n t o the determination of the a c t i v i t y of the calmodulin-p r e p a r a t i o n i t was necessary t o .ensure t h a t the sample was r e l a t i v e l y pure. In order t o do t h i s , samples were subjected to SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . Shown i n f i g u r e 6 are 3 densitometer t r a c i n g s obtained from SDS-PAGE g e l s . The f i r s t t r a c i n g c o n s i s t s of compounds of known MW used as a reference standard. The t r a c i n g of calmodulin p r e p a r a t i o n A and calmodulin p r e p a r a t i o n B are i n d i c a t i v e of what was obtained f o r most of the calmodulin samples. The sample p r o t e i n mig-r a t e d e i t h e r t o a s i n g l e band of 18,000 MW, or t o a major band at 18,000 MW w i t h minor bands at 50-65,000 MW. As determined by d e n s i t o -metry, the 18,000 MW peak i n calmodulin p r e p a r a t i o n A comprised 85$ of the t o t a l p r o t e i n present. The 18,000 MW band i s thought to be calmodulin, and w i l l co-migrate w i t h a red c e l l calmodulin p r e p a r a t i o n i s o l a t e d i n the l a b o r a t o r y of DR. B. Rou f o g a l i s . 2) C h a r a c t e r i z a t i o n of Cardiac Microsomes Enriched i n S.R.: 2+ Ca -uptake was measured a t var y i n g concentrations of S.R. prepared 2+ as described i n the methods. Figure 7 i l l u s t r a t e s that Ca -uptake increased l i n e a r l y as the microsomal S.R. concentration increased. In other words, the r a t e of Car -uptake was not a f f e c t e d by v a r i a t i o n i n the S.R. concentration used. Most of the experiments described were performed a t microsomal S.R. concentrations between 0.05 and 0.1 mg/ml. 2+ The m a j o r i t y of Ca -uptake experiments performed during the course of the study i n v o l v e d i n c u b a t i o n of the S.R. f o r 5 minutes i n the presence 2+ 2+ of Ca . To obtain an accurate measurement of the amounti-of Ca - t r a n s -Figure 5 2+ E f f e c t of Red C e l l Calmodulin on Ca -uptake i n Microsomal Preparations Enriched i n Sarcoplasmic Reticulum 2+ Ca -uptake was determined as described i n methods i n the absence of 2+ KC1, and i n the presence of 1/<.M f r e e C a . The calmodulin used (2.25 A g/50/tl) was a t y p i c a l p r e p a r a t i o n . 2+ This r e s u l t i s t y p i c a l of 9 experiments performed. Ca -uptake a c t i v i t y i n these experiments i n the absence o f added calmodulin ranged from 5-0 t o 30.0 nmoles/mg/min. Figure 6 Densitometer Tracings of Calmodulin Preparations Subjected to SDS-Polyacrylamide Gel Electrophoresis. SDS-PAGE was performed as described i n the methods. Calmodulin preparations A and B represent two calmodulin samples obtained by the method of Jung (1978). The number by each peak represents the apparent molecular weight of the p r o t e i n which comprises that peak. Molecular weight standard proteins include phosphorylase B (94,000 MW) , Bovine Serum Albumin (67,000 MW), Ovalbumin (43,000 MW), Carbonic Anhydrase (30,000 MW), Soybean Trypsin I n h i b i t o r (20,100 MW), and C<-1 act albumin (l4,000 MW). I 3k Figure 7 E f f e c t of Varying Concentrations of the Microsomal P r e p a r a t i o n 2+ Enriched i n Sarcoplasmic Reticulum on Ca -uptake by the S.R. P r e p a r a t i o n . 2+ Ca -uptake was determined as described i n methods, i n the pres 2+ of l / i M f r e e Ca and 110 mM KC1. 36 37 2+ ported/minute i t was necessary t h a t Ca -uptake be l i n e a r over the p e r i o d of i n c u b a t i o n . I n i t i a l r a t e s t u d i e s shown.in f i g u r e 15 (discussed . 2+ l a t e r ; confirm that Ca -uptake was indeed l i n e a r over the i n c u b a t i o n periods u t i l i z e d . At the time t h i s study was being performed other researchers i n the l a b o r a t o r y were attempting t o p u r i f y the crude micro-somal S.R. p r e p a r a t i o n using sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n techniques. Cardiac S.R. preparations at various stages of p u r i f i c a t i o n were t e s t e d t o determine the a b i l i t y of cAMP-dependent p r o t e i n kinase 2+ to s t i m u l a t e Ca -uptake. As i n d i c a t e d i n t a b l e h the crude micro-somal S.R. was q u a l i t a t i v e l y s i m i l a r t o the p u r i f i e d S.R. f r a c t i o n s i n 2+ the a b i l i t y of cAMP-dependent p r o t e i n kinase to st i m u l a t e Ca -uptake. 2+ As w e l l , the crude microsomal S.R. was found to be higher i n Ca -tr a n s p o r t a c t i v i t y than the other S.R. preparations t e s t e d . These r e -2+ s u i t s are f u r t h e r evidence t h a t the Ca -uptake a c t i v i t y observed i n crude microsomal preparations i s due to the.presence of S.R. and not due to contamination from other o r g a n e l l e s . 2+ 3) Monovalent Cation S t i m u l a t i o n of Ca -transport i n Cardiac S.R.: + + 2 + The e f f e c t of K and L i on Ca - t r a n s p o r t i n microsomal S.R., m the presence and absence of calmodulin, i s shown i n f i g u r e 8 . In the 2+ presence of 110 mM K C 1 , Ca -uptake a c t i v i t y i s enhanced compared t o t h a t a c t i v i t y noted i n the presence of 110 mM L i C l , or i n the presence of no monovalent c a t i o n s ( 2 0 0 mM sucrose was used t o maintain osmolarity) at every f r e e calcium c o n c e n t r a t i o n s t u d i e d . In the presence of 110 mM 2+ K C 1 , calmodulin increased Ca -uptake a c t i v i t y by 2 0 - 4 0 % as noted pre-v i o u s l y (Katz and Remtulla, 1 9 7 8 ) . However, i n the presence of 110 mM L i C l or i n the absence of monovalent c a t i o n s , calmodulin s t i m u l a t e d 2+ Ca -uptake to a much grea t e r degree. In f a c t , i n the absence o f _ K C l , 2+ calmodulin r e s t o r e d Ca -uptake to the maximum a c t i v i t y noted m the presence of 110 mM K C 1 alone. S i m i l a r experiments were performed i n the presence and absence of KC1 using commercially obtained Sigma calmodulin i n place of red c e l l <2> calmodulin. As shown i n f i g u r e 9 , the Sigma calmodulin, at conc-e n t r a t i o n s comparable t o the amount of red c e l l calmodulin needed t o 2+ produce maximal a c t i v a t i o n , only s l i g h t l y enhanced Ca -uptake a t a l l 38 Table 4 Ca -uptake by Various Cardiac Muscle Preparations i n the Presence and Absence of cAMP-dependent Pro tein Kinase 2+ Sample Ca -uptake A c t i v i t y (nmoles/mg/min) % increase -cAMP-dependent +cAMP-dependent p r o t e i n kinase p r o t e i n kinase Washed p a r t i c l e s 1 4.26 6.44 51 Crude microsomal 8.96 14.23 59 S.R. P u r i f i e s | S.R. 2.20 3.88 76 l e v e l 1 P u r i f i e d S.R. 2.29 4.12 79 l e v e l 2 1 - washed p a r t i c l e s , p r e p a r e d by Bet t y R i c h t e r , c o n s i s t of dog heart homogenized i n Na phosphate b u f f e r , f o l l o w e d by a K C 1 b u f f e r and t i r s b u f f e r wash. 2- crude microsomal S.R. was prepared as described i n methods. 3- p u r i f i e d S.R. , prepared by Bet t y R i c h t e r , i n v o l v e s p l a c i n g t h e crude microsomal S.R. onto a sucrose d e n s i t y gradient and assaying the .resultant l a y e r s . 2+ -Ca -uptake was measured as described i n the methods, i n the 2+ presence of 1 /^M f r e e Ca and absence of K C 1 . „ The experiment was performed e i t h e r i n the absence or presence of cAMP ( l y V t M ) and cAMP-dependent p r o t e i n kinase (type 1 , 25/^g/0.5 ml f i n a l i n c u b a t i o n volume). Figure 8 E f f e c t of Monovalent Cations on Ca -uptake i n the Presence, and Absence of Calmodulin 2+ Ca -uptake was detemined as described i n methods i n the presence and absence of 110 mM KC1, 110 mM L i C l , or 200 mM sucrose (0 KC1, 0 L i C l ) w i t h ( • — • ) and without (*•— -•) calmodulin ( 3 . 0 ^ g/0.5 ml f i n a l i n c u b a t i o n volume). This r e s u l t i s t y p i c a l of the average of h experiments performed. 2+ Ca -uptake a c t i v i t y i n these experiments, i n the absence of added 2+ KC1 and calmodulin at l/( M free Ca ,ranged from 4.25 t o 13.38 nmoles/mg/min. ko uiiu/Buj/sa|oaju :a^D|df| 03 Figure 9 E f f e c t of K on Ca -uptake i n the presence and absence of Sigma Calmodulin 2+ Ca -uptake was determined as described m methods i n the presence and absence of 110 mM KC1 w i t h (•——•) and without (•• •) Sigma* calmodulin (5.0 ^ g/0.5 ml f i n a l i n c u b a t i o n volume). This r e s u l t i s t y p i c a l of 3 experiments performed. 2+ Ca -uptake a c t i v i t y i n these experiments, i n the absence of added 2+ KC1 and calmodulin at 1j\ M f r e e Ca , ranged from 6.6 t o 8.8 nmoles/mg/min. •43 2+ . Ca concentrations s t u d i e d . When the amount of Sigma calmodulin was increased'to'15/1g/0.5 ml i n c u b a t i o n tube i t was p o s s i b l e t o obt a i n a 2+ 3*+. 1%. increase i n Ca -uptake by the microsomal S.R. i n the absence of + 2+ <2> K , at 1/<M f r e e Ca . The cost and a v a i l a b i l i t y of the Sigmsr calmodulin 2+ made i t u n f e a s i b l e to t r y higher concentrations over a range of Ca concentrations. The e f f e c t of i n c r e a s i n g K + concentrations on calmodulin s t i m u l a -2+ t i o n of Ca - t r a n s p o r t i n cardiac microsomal S.R. was i n v e s t i g a t e d ( f i g u r e 10). Increasing the K + concentration increased the degree of 2+ 2+ Ca -uptake to a maximum at 30 mM KC1, f o l l o w i n g which Ca -uptake de-2+ creased s l i g h t l y . Calmodulin, though, s t i m u l a t e d Ca -uptake to a greater degree i n the absence of added KC1. As the KC1 concentration 2+ was i n c r e a s e d the degree of calmodulin s t i m u l a t i o n of Ca -uptake decreased, from over 100% s t i m u l a t i o n at 0 K + t o 15-25% at llOmM KC1. A q u a l i t a t i v e l y s i m i l a r r e s u l t was obtained w i t h i n c r e a s i n g NaCl 2+ concentrations ( f i g u r e l l ) . Maximal Ca -uptake occurred at 100 mM NaCl. Calmodulin had the highest s t i m u l a t o r y e f f e c t at 0 Na +, and no s t i m -u l a t i o n was noted a t NaCl concentrations above 80 mM. 2+ 4) E f f e c t of Calmodulin on the K i n e t i c Parameters of Ca -tranport i n Cardiac Microsomal Preparations Enriched i n S.R.: Calmodulin (2/(g/0.5 ml) had no s i g n i f i c a n t e f f e c t on the apparent 2+ K^ f o r Ca (0.5l6/^M i n t h e absence of calmodulin and 0.676/<^M i n the presence of calmo d u l i n ) , but s i g n i f i c a n t l y i n c reased the 2+ of t h i s p r e p a r a t i o n (20.2 nmoles/mg/min i n the absence of calmodulin and 48.2 nmoles/mg/min i n the presence of calmodulin), ( f i g u r e 12). By c o n t r a s t , 2+ KC1 (110 mM) s i g n i f i c a n t l y a l t e r e d the apparent K^ f o r Ca (0.5l6/*VM i n the absence of KC1 and 2.32/^M i n the presence of 110 mM KCl) ( f i g u r e 13). Figure 10 + 2+ E f f e c t of K on Ca -uptake i n the Presnece of Red C e l l Calmodulin Ca -uptake was determined as described i n methods at l/\ M f r e e C a 2 + i n the presence (•——•#) and absence °) of red c e l l calmodulin (3.0/<g/0.5 ml f i n a l i n c u b a t i o n volume). This r e s u l t i s t y p i c a l of the average of 5' experiments. Calmodulin - 2+ s i g n i f i c a n t l y - s t i m u l a t e d Ca -uptake at a l l KC1 concentrations t e s t e d (p < 0.05 students " t " t e s t , p a i r e d data.) 2+ Ca -uptake a c t i v i t y i n these experiments, i n the absence of added KC1 and calmodulin, ranged from 5.3 t o 18.7 nmoles/mg/min. Ca 4 4 " Uptake : nmoles/mg/min Figure 11 + 2+ E f f e c t of Na on Ca -uptake i n the Presence and Absence of Red C e l l Calmodulin 2+ Ca -uptake was determined as described i n methods at 1 / t M f r e e 2+ Ca i n the presence (•——•) and absence ( ©•—-*) of red c e l l calmodulin (3-0/^g/0.5 ml f i n a l i n c u b a t i o n volume). This r e s u l t i s t y p i c a l of.the average of k experiments. Calmodulin 2+ s i g n i f i c a n t l y s t i m u l a t e d Ca -uptake a t a l l NaCl concentrations t e s t e d below 55 mM (p < 0.05, students " t " t e s t , p a i r e d d a t a ) . 2+ Ca -uptake a c t i v i t y i n these experiments, i n the absence of added NaCl and calmodulin, ranged from 7.7 t o l6.7 nmoles/mg/min. 1+8 Figure 12 E f f e c t of Calmodulin on the K, f o r C a 2 + and the V„ 2+ f o r C a 2 + -d Ca Uptake i n Microsomal Preparations Enriched i n Sarcoplasmic Reticulum 2+ Ca -uptake vas determined as described i n the methods at various free 2+ 2+ Ca concentrations (0.2/^. M-30.0/<M free Ca ) and the data p l o t t e d as r e c i p r o c a l s of r e s u l t s obtained i n the absence (• • ) and presence (O- — - o ) of calmodulin. Data obtained i n assays performed on separate days was normalized to that a c t i v i t y found i n the absence of calmodulin 2+ at 1 free Ca . This r e s u l t i s t y p i c a l of the average of 3 experiments performed. 2+ Ca -uptake a c t i v i t y i n these experiments, i n the absence of added 2+ KC1 and calmodulin at 1 / f M free Ca , ranged from 7.1+ t o 19.3 nmoles/mg/min. 49 / Figure 13 E f f e c t of K + on the K, f o r Ca + and the V 2+ f o r Ca +-uptake i n d ca Microsomal Preparations Enriched i n Sarcoplasmic Reticulum 2+ Ca -uptake was determined as described i n methods at v a r i o u s f r e e ?+ 2+ Ca concentrations ( 0 .2/* M-30.0 /\M f r e e Ca ) and the data p l o t t e d as r e c i p r o c a l s of r e s u l t s obtained i n the absence (* *) and presence ( o — — o ) of K +. Data obtained i n assays performed on separate days was normalized t o that a c t i v i t y found i n the absence of K + at 1 /\U f r e e Ca This r e s u l t i s t y p i c a l o f the average of 3 experiments performed. 2+ Ca -uptake a c t i v i t y i n these experiments, i n the absence of added 2+ KC1 and calmodulin at l/*{ M f r e e Ca , ranged from 27.2 to 5U.6 nmoles/mg/min. 51 52 2+ k) E f f e c t of Calmodulin on the I n i t i a l Ca -uptake V e l o c i t y i n Cardiac Microsomal Preparations Enriched i n Sarcoplasmic Reticulum: 2+ The time course of calcium uptake at various free Ca concentra-tions i n the presence and absence of red c e l l calmodulin (2/^/0.5 ml f i n a l incubation colume) i s shown i n figure 15. These experiments were conducted using tris-phosphate instead of t r i s - o x a l a t e to f a c -2+ l l i t a t e k i n e t i c analysis of the data. Increasing the free Ca conc-2+ centration produced an increase i n the i n t i a l rate of Ca -uptake i n 2+ the microsomal S.R. to a maximum at 10/^M Ca free. Calmodulin 2+ 2+ stimulated the i n i t i a l rate of Ca -uptake at a l l free Ca -concentra-2+ tions tested; maximal stimulation of Ca -uptake by calmodulin.occured 2+ at 5.0/^M'free^.Ca.: ... Figure 16 indicates the calcium uptake v e l o c i t y obtained i n the presence and absence of calmodulin at the various free 2+ Ca -concentrations studied. Reciprocal p l o t s of this data indicate 2+ t h a t calmodulin s i g n i f i c a n t l y increased the ^^2+ of the Ca -transport system (3.07 nmoles/mg/min i n the absence of calmodulin and k.65 nmoles/ mg/min i n the presence of calmodulin) without changing the apparent 2+ for Ca (0.791 /\ M" i n the absence of • calmodulin and 0.6l9/\M i n the presence of calmodulin). 5) E f f e c t of Microsomal S.R. Extracts On Calcium Uptake A c t i v i t y i n Cardiac Microsomal Preparations Enriched i n Sarcoplasmic Reticulum: In order to determine whether calmodulin was indigenous to the S.R. membrane, attempts were made to extract calmodulin from microsomal S.R.. Using methodology developed for i s o l a t i n g calmodulin from other sources (described i n t a b l e 5) extracts of microsomes enriched i n S.R. were prepared. As shown i n table 5 when these extracts were added back to i n t a c t microsomal S.R. , both i n the presence and absence of KC1, 2+ no stimulation of Ca -uptake occurred. This would suggest that calmodulin was not present i n the microsomal preparations r o u t i n e l y used. As a co n t r o l , a calmodulin sample was subjected t o the same b o i l i n g and i s o l a t i o n procedures. The a b i l i t y of t h i s sample to 2+ stimulate Ca -uptake i n microsomal S.R. was unaltered by this procedure, Figure 1^ E f f e c t of Calmodulin on the I n i t i a l Rate of Calcium Uptake i n Microsomal Preparations Enriched i n Sarcoplasmic Reticulum. 2+ Ca -uptake was measured as described i n methods at v a r i o u s times 2+ such t h a t no g r e a t e r than 5% of the t o t a l Ca present was ac-2+ cumulated by the microsomes during the i n c u b a t i o n p e r i o d . Ca -uptake r a t e s are i n d i c a t e d by t h e i r r e s p e c t i v e p l o t s i n the presence (•——•) and absence ( O O ) of calmodulin (2y^g/0.5 ml f i n a l 2+ i n c u b a t i o n volume) f o r each f r e e Ca concentration used. This r e s u l t i s t y p i c a l o f 3 experiments performed. 2+ Ca -uptake a c t i v i t y i n these experiments, i n the absence of 2+ calmodulin at 1 ^ M f r e e Ca , ranged from 3.75 t o k.kf nmoles/mg/min. 5h Figure 15 V e l o c i t y of Calcium Uptake i n Cardiac Microsomal Preparations Enriched i n Sarcoplasmic Reticulum i n the Presence and Absence of Calmodulin 2+ Ca -uptake was measured as described i n methods at various times i n the presence of v a r y i n g f r e e calcium concentrations and 2+ the i n i t i a l r ates of Ca -uptake determined as described i n methods i n the presence (• •) and absence ( o—--o) of calmodulin (2,»<g/C"5 ml f i n a l i n c u b a t i o n volume). The i n s e r t shows a r e c i p r o c a l p l o t of t h i s data. This r e s u l t i s t y p i c a l of 3 experiments performed. 2+ Ca - u p t a k e . a c t i v i t y m these experiments, i n the absence of added 2+ KC1 and calmodulin a t l ^ t j M f r e e Ca , ranged from 3.07 t o 5-93 nmoles/mg/min. 56 Table 5 E f f e c t of Microsomal E x t r a c t s on Calcium Uptake A c t i v i t y i n Cardiac Microsomal Preparations Enriched i n Sarcoplasmic Reticulum. E x t r a c t Volume ( l ) Calcium Uptake as Percent of C o n t r o l Method A ^ Method ' Method C ^ a) Absence of KC1 b) Pres 0 100 100 100 10 95.5 88.1 95-9 20 95.6 88.1+ 93.1 1+0 107.7 80.6 92.0 50 88.9 88.7 92.0 mce of 110 mM KC1 0 100 100 100 10 93.3 97 .9 10U.7 20 95.1 95.8 96.5 30 91.9 92.5 95.5 50 97.0 91.5 94.2 ( l ) concentrated microsomal S.R. (1.1+ mg protein/ml) was b o i l e d g e n t l y f o r 5 min i n the presence of 0.6 mM EGTA. The p r e p a r a t i o n was c e n t r i f u g e d at l+0,000g f o r 30 min and the supernatant d e s a l t e d by s e r i a l d i l u t i o n through an Amicon PM-10 f i l t e r . ((2) same procedure as ( l ) , except the supernatant was d e s a l t e d by passage through a Sephadex G-15 column (2 30 cm). (3) same procedure as ( l ) , except the microsomal sarcoplasmic r e t i c u l u m was b o i l e d i n the presence of 1 mM EGTA. 58 Experiments vere a l s o performed i n an attempt to remove any i n -digenous calmodulin during the p r e p a r a t i o n of the microsomal S.R.. Treatment w i t h e i t h e r 0.02$ T r i t o n X-100, 0.02% EGTA, or 9 M Urea i n 75 mM t r i s - C l before the l a s t t r i s - C l wash r e s u l t e d i n a t o t a l l o s s o f 2+ the a b i l i t y of the microsomal S.R. to t r a n s p o r t Ca 125 6) B i n d i n g of I - l a b e l l e d Calmodulin t o Cardiac Microsomes Enriched i n Sarcoplasmic Reticulum: 125 I - l a b e l l e d calmodulin was s y n t h e s i z e d by the method of Hunter and Greenwood (1962) as described i n the methods. Using a Sephadex 125 125 G-25 column the synthesized I-calmodulin and free I could be seperated due t o d i f f e r e n t i a l e l u t i o n . Figure 17 shows the e l u t i o n p r o f i l e o b t a i n e d , the f i r s t peak r e p r e s e n t i n g the f r a c t i o n s c o n t a i n i n g 125 I - l a b e l l e d calmodulin. The four f r a c t i o n s t h a t comprised t h i s peak were then pooled and d i l u t e d k times. As determined by 6% TCA p r o t e i n 125 p r e c i p i t a b i l i t y , k9.9% of the t o t a l I present i n these f r a c t i o n s was bound t o calmodulin. I t was decided t h a t f u r t h e r removal of f r e e 125 I from the f r a c t i o n s was unnecessary s i n c e i n c o n t r o l s t u d i e s i t was found 125 that there was n e g l i g i b l e b i n d i n g of f r e e I t o cardiac microsomes enriched i n S.R. 125 I n i t i a l l y , measurement of I-calmodulin b i n d i n g t o cardiac 2+ microsomal S.R. was performed i n a s i m i l a r manner t o the Ca -uptake 125 45 assay, except that I-calmodulin was s u b s t i t u t e d f o r Ca i n the i n -cubation medium. However, a high degree of n o n - s p e c i f i c b i n d i n g of " ^ I - c a l m o d u l i n t o the f i l t e r ( M i l l i p o r e ^ ) occurred; the b i n d i n g of 125 I-calmodulin to the f i l t e r could be a l t e r e d by v a r y i n g the conc-e n t r a t i o n of EGTA present i n the i n c u b a t i o n medium. Therefore, as an a l t e r n a t e method, the b i n d i n g technique described i n the methods was 2+ developed. Using t h i s technique a Ca -concentration dependent l n -125 crease i n I-calmodulin b i n d i n g occurred ( f i g u r e 18). This b i n d i n g was decreased i n the presence of monovalent c a t i o n s , w i t h 110 mM KC1 and 110 mM NaCl having the g r e a t e s t e f f e c t . Table '6' shows the e f f e c t of monovalent c a t i o n s on .."^''i-labelled- b i n d i n g a t 10 M f r e e 2 + 2 + . Ca , the Ca concentration used i n the m a j o r i t y of the previous 125 s t u d i e s . B i n d i n g of -I-calmodulin to microsomes enriched i n S.R. 59 was s i g n i f i c a n t l y decreased i n the presence of 110 mM KCL and 110 mM 2+ NaCl (p <- 0.05, students " t " t e s t ) a t a l l f r e e Ca concentrations above 10 M. In the presence of 110 mM L i C l b i n d i n g was decreased, but 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 t h a t degree of b i n d i n g observed -7 2+ i n the c o n t r o l s , except at 10 M f r e e Ca concentrations. In these experiments there was a s u b s t a n t i a l amount of non-125 s p e c i f i c b i n d i n g of I-calmodulin t o the microsomal S.R.. The 125 amount of I-calmodulin which would b i n d t o the microsomal S.R. i n a c t i v a t e d by b o i l i n g was found t o be 6h% of that which would b i n d t o 2+ i n t a c t microsomal S.R. in . the absence of Ca . However, the b i n d i n g 125 2+ of I-calmodulin t o i n a c t i v e S.R. was not Ca -concentration-dependent. Therefore t h i s n o n - s p e c i f i c b i n d i n g was subtracted from the values obtained i n the r e s u l t s shown ( f i g u r e 18, t a b l e 6). Figure 16 Separation of F r a c t i o n s Containing. ? I - l a b e l l e d . Calmodulin From 125 <f> Free I Using G-25 Sephadex^ Column Chromatography I o d i n a t i o n and i s o l a t i o n of calmodulin are as described i n methods. F r a c t i o n s (8 drops) were counted f o r 1 second on a Gamma spectro -125 photometer. The f i r s t peak represents I-calmodulm e l u t e d from 125 the column, while subsequent peaks c o n s i s t of f r e e I e l u t e d from the column. 61 Figure 17 E f f e c t of Monovalent Cations on ^ ^ I - c a l m o d u l i n Binding t o Cardiac Microsomes Enriched i n Sarcoplasmic Reticulum. 125 I-calmodulin b i n d i n g , expressed as CPM/mg sarcoplasmic r e t i c u l u m p r o t e i n , was determined i n the absence of monovalent ca t i o n s (• •) , and i n the presence of 110 mM KC1 ( 0 - - 0 ) , 110 mM NaCl or 110 mM L i C l ( A *t). Result shown i s the mean of 3 experiments ' (maximum•• v a r i a b i l i t y - +. l800 count s/mg 125 ~ ' • p r o t e i n ) . N o n - s p e c i f i c b i n d i n g of I-calmodulin t o denatured" microsomal S.R. ,- whieh-is e q u i v a l e n t t o 64%-of.the counts obtained ' - 2+ i n the absence of. added Ca , was subtracted from the t o t a l counts obtained f o r each i n c u b a t i o n sample. Table 6 E f f e c t of Monovalent Cations on I-calmodulin Binding to Cardiac Microsomes Enriched i n Sarcoplasmic Reticulum Incubation Conditions C o n t r o l 110 mM L i C l 110 mM NaCl 110 mM KC1 125 Counts I-calmodulin/mg S.R. P r o t e i n IO" 6 M C a 2 + 7,402 +_ 1795. (5) 6,598 ±. 1595 (3) 4,768 i 1582 ( 3 ) a 4,423 ± 1 6 1 1 ( 3 ) a a - s i g n i f i c a n t l y d i f f e r e n t (p<0.05, student ' t ' t e s t ) than c o n t r o l _ 1 2 5 I - c a l m o d u l i n b i n d i n g as described i n the methods was performed 2+ at 1 /IM f r e e Ca . -bracketed numbers i n d i c a t e the "n" value. 65 DISCUSSION l ) A c t i v i t y and P u r i t y of our Red C e l l Calmodulin: The present study was c a r r i e d out t o c h a r a c t e r i z e the r e g u l a t o r y 2+ r o l e of calmodulin on Ca -transport m cardiac S.R.. In order t o do t h i s i t i s necessary to use an a c t i v e and r e l a t i v e l y pure calmodulin p r e p a r a t i o n . In t h i s study, calmodulin a c t i v i t y was determined by i t s 2+ 2+ a b i l i t y to s t i m u l a t e Mg -Ca -ATPase a c t i v i t y i n EDTA-washed red c e l l 2+ membranes. At 50 /{ M f r e e Ca , maximal concentrations of calmodulin 2+ 2+ r e s u l t e d i n a 236% increase i n Mg -Ca -ATPase a c t i v i t y ( t a b l e h). This i s comparable t o r e s u l t s obtained by a number of other researchers. Katz et a l (1979) showed t h a t maximal calmodulin concentrations would 2+ 2+ s t i m u l a t e Mg -Ca -ATPase 111% i n i d e n t i c a l membranes, at 0.1 /(M f r e e 2+ Ca . N i g g l i et a l (1979) u s i n g EDTA t r e a t e d membranes, which they 2+ 2+ r e f e r to as hypotonic ghosts, obtained a 301% s t i m u l a t i o n of Mg -Ca ATPase a c t i v i t y i n the presence of 2/4g calmodulin/mg ghost p r o t e i n and 2+ 50/vj M Ca . E a r l i e r s t u d i e s by Gopinath and V i n c e n z l (1977) using r e d c e l l membranes prepared i n low o s m o l a r i t y imidazole b u f f e r showed that calmodulin d e r i v e d e i t h e r from r e d c e l l s or b r a i n t i s s u e would 2+ 2+ s t i m u l a t e Mg -Ca -ATPase a c t i v i t y 370%. In a l l of the above s t u d i e s the red c e l l ghosts were prepared such t h a t during i s o l a t i o n some, i f not a l l , of the calmodulin normally associated w i t h the membrane was f r e e d . When determining the p u r i t y of the sample p r e p a r a t i o n using SDS-polyacrilamide g e l e l e c t r o p h o r e s i s , calmodulin migrated t o the apparent MW of 18,000. Vanaman et a l (1977) determined the amino a c i d dequence of calmodulin and a r r i v e d at a MW of 16,723. The discrepancies 2+ i n these two values i s probably due to the a b i l i t y of EGTA or Ca to a l t e r the m o b i l i t y of calmodulin on SDS gels (as discussed i n the i n t r o -d u c t i o n ) . Molecular weight determination of calmodulin by a number of other researchers using SDS-PAGE have produced values that vary -between-. 15,000..and 19,200 MW (Wolff and Brostrom, 1979). The f a c t that our samples co-migrate w i t h calmodulin prepared by other workers (see r e s u l t s ) suggests t h a t the 18,000 MW band i s indeed calmodulin. The small amount of a 50,000 MW t o 65,000 MW contaminant p r o t e i n o f t e n 66 seen i n the sample has not been i d e n t i f i e d . E a r l i e r p r e p a r a t i o n s , which contained l a r g e r q u a n t i t i e s of t h i s p r o t e i n were found to i n h i b i t 2+ Ca - t r a n s p o r t i n cardiac microsomal S.R. once the maximal s t i m u l a t o r y concentration of calmodulin was exceeded. This r e s u l t l e d to the conc l u s i o n that the 51,000. MW or 65 ,000 MW peak i n calmodulin prepara-2+ 2+ t i o n A ( f i g u r e 6) may have been an i n h i b i t o r of calmodulin or Mg -Ca -ATPase. Au (1978) reported that a 56,000 MW and 35,000 MW i n h i b i t o r 2+ 2+ of Mg -Ca -ATPase co u l d be i s o l a t e d from p i g red c e l l hemolysate 2+ 2+ along w i t h the Mg -Ca -ATPase a c t i v a t o r ; s i m i l a r t o our r e s u l t s , i f the preparations were d i l u t e d the e f f e c t of the i n h i b i t o r was masked by the a c t i v a t o r which was present i n higher concentrations. As our study progressed and experimental techniques improved the amount of contaminant p r o t e i n i n the calmodulin p r e p a r a t i o n was decreased or t o t a l l y e l i m i n a t e d ( f i g u r e 6, calmodulin p r e p a r a t i o n B). 2) E f f e c t of Storage on Calmodulin A c t i v i t y During the course of t h i s study a commercial calmodulin-' . 0 p r e p a r a t i o n became a v a i l a b l e from the Sigma Chemical Company. When compared to our calmodulin p r e p a r a t i o n i t d i s p l a y e d a decreased potency 2+ 2+ i n the s t i m u l a t i o n of Mg -Ca -ATPase a c t i v i t y i n EDTA-washed e r y t h -2+ rocyte membranes ( t a b l e 3) and Ca -t r a n s p o r t i n microsomal S.R. ( f i g u r e 9). The decreased a c t i v i t y of Sigma calmodulin was probably due t o the l y o p h i l i z a t i o n and storage of the pr e p a r a t i o n . We have found that l y o p h i l i z a t i o n of a s o l u t i o n c o n t a i n i n g calmodulin and storage at room temperature, or -20°C, r e s u l t s i n a s u b s t a n t i a l l o s s i n a c t i v i t y . A pure l y o p h i l i z e d calmodulin sample obtained from Dr. Penniston which was s t o r e d at room temperature showed a s i m i l a r decrease i n potency. When subjected t o SDS-PAGE the presence of smaller MW breakdown products was detected. K r e t s i n g e r (1980) reported s i m i l a r r e s u l t s using calmodulin prepared i n h i s l a b o r a t o r y ; i n order t o maintain calmodulin a c t i v i t y over time i t was necessary to store calmodulin at -70°C. The 2+ presence and absence of Ca i n the calmodulin p r e p a r a t i o n during storage can a l s o a l t e r a c t i v i t y . Since our calmodulin samples were not l y o p h i l i z e d we d i d not encounter any problems w i t h decreases i n 67 potency over time. 2+ 3) Calmodulin S t i m u l a t i o n of Ca - t r a n s p o r t i n Cardiac Microsomes Enriched i n Sarcoplasmic Reticulum: As mentioned e a r l i e r , previous work i n t h i s l a b o r a t o r y has shown 2+ that calmodulin w i l l s t i m u l a t e Ca -t r a n s p o r t i n c a r d i a c S.R. (Katz and Remtulla, 1978). This has been v e r i f i e d i n t h i s study, as w e l l as by the work of LePeuch et a l (1979), Kranias et a l (1980), and C a r a f o l i et a l (1980). LePeuch e t a l (1979) have shown t h a t calmodulin w i l l c a t a l y z e a membrane bound p r o t e i n kinase phosphorylation of phospho-lambam, the same p r o t e i n phosphorylated by cAMP-dependent p r o t e i n kinase. They a l s o showed that the phosphorylation due t o the two kinases occurred at d i s t i n c t s i t e s on phospholambam. Kranias et a l (1980) have obtained s i m i l a r r e s u l t s . LePeuch et a l t h e r e f o r e p o s t u l a t e that c a l -2+ modulm st i m u l a t e s cardiac S.R. Ca -transport through the s t i m u l a t i o n 2+ of a Ca -dependent, c y c l i c AMP-independent p r o t e i n kinase t h a t phos-phorylates phospholambam. This explanation d i f f e r s from the explanation presented by our l a b o r a t o r y (Lopaschuk et a l , 1980), although we d i d not measure phosphorylation under c o n d i t i o n s t h a t would enable us to v e r i f y t h i s r e s u l t . One of the reasons LePeuch e t a l p o s t u l a t e d t h i s mechanism of calmodulin a c t i o n was tha t they could not show an increase 2 + 2 + i n Mg -Ca -ATPase a c t i v i t y i n the presence of calmodulin. U n l i k e LePeuch et a l we have shown i n our l a b o r a t o r y t h a t calmodulin w i l l 2+ 2+ sti m u l a t e Mg -Ca -ATPase a c t i v i t y i n cardiac S.R.. Kranias et a l have since confirmed t h i s r e s u l t . Therefore, calmodulin may be i n t e r -a c t i n g d i r e c t l y w i t h the ATPase enzyme i t s e l f , r a t h e r than through the phosphorylation of phospholambam. 2+ k) E f f e c t of Monovalent Cations on Ca -transport i n Cardiac S.R. E a r l y on i n the study i t was determined t h a t monovalent c a t i o n s , e s p e c i a l l y K +, had a pronounced e f f e c t on the a b i l i t y of calmodulin 2+ t o s t i m u l a t e Ca - t r a n s p o r t i n cardiac microsomal S.R.. I t was decided to f u r t h e r i n v e s t i g a t e t h i s f i n d i n g since there i s strong evidence + 2+ that K i s also a r e g u l a t o r of S.R. Ca - t r a n s p o r t (Shigekawa and P e a r l , 1976; Jones e t a l , 1978; Shigekawa and Akowitz, 1979; and Yamada and Ikemoto, 1980). In the presence of 110 mM KC1 calmodulin w i l l 68 2+ stimulate (between 10~kO%) Ca -transport, i n microsomal S.R. at 2+ various free Ca concentrations (figure 8). I f KC1 i s replaced with 2+ an equimolar sucrose s o l u t i o n , calmodulin, stimulates Ca -transport 2+ 100-2*40% at the same free Ca -concentrations. It therefore appears + 2+ that K i s decreasing calmodulin stimulation of S.R. Ca -uptake. This 2+ can also be observed i f Ca -transport i n t o microsomal S.R. i s measured at increasing KC1 concentrations i n the presence and absence of calmod-u l i n . (figure 9). The p o s s i b i l i t y e x i s t s that K + maximally stim-2+ ulates Ca -transport i n S.R. and that addition of calmodulin would therefore not result i n a further increase. This seems u n l i k e l y , 2+ however, since cAMP-dependent protein kinase which also stimulates Ca:: -2+ transport i n S.R. w i l l s i g n i f i c a n t l y increase Ca -uptake i n the presence of K +. This suggests that the presence of K + does not maximally 2+ stimulate Ca -transport. 2+ When studying the e f f e c t of KC1 on calmodulin stimulation of Ca uptake by microsomal S.R. the e f f e c t of the Cl concentration must be considered. Kasai and Miyamoto (1976), as well as Campbell and Shamoo, 2+ (1980) have demonstrated that chloride ions w i l l release Ca from i s o l a t e d sarcoplasmic reticulum v e s i c l e s . Therefore, increasing the Cl concentration by increasing the KC1 l e v e l s could r e s u l t i n an i n -2+ creased Ca release from S.R.. I t i s possible that calmodulin stim-2+ u l a t i o n of Ca -uptake although appearing t o be i n h i b i t e d by KC1 could 2+ be due to an increased Ca -release from S.R. by chloride ions. This seems u n l i k e l y since replacement of. KC1 with NaCl or L i C l r e s u l t s i n 2+ a smaller stimulation of Ca -uptake, as we l l as a l e s s pronounced 2+ decrease i n calmodulin stimulation of Ca -uptake, while C l concen-t-rations remain the same. One has to be c a r e f u l i n i n t e r p r e t i n g these 2+ r e s u l t s , however, since Inesi and Malan (1976) reported that Ca can be released from i s o l a t e d S.R. when K + i s exchanged for a less permeable cation. Therefore, the l e s s pronounced effect of Na + on C a 2 + -2+ accumulation may be a res u l t of an increase i n Ca release due t o de-2+ creased permeability of these ions. An increase In Ca release by NaCl or L i C l would not, however, explain why i n the presence of these 2+ ions a less pronounced • decrease- i n the stimulation of Ca -uptake by calmodulin was observed. I t thus seems l i k e l y that a more di r e c t i n t e r -69 a c t i o n e x s i s t s between calmodulin and monovalent c a t i o n r e g u l a t i o n of 2+ S.R. Ca -accumulation. Studies i n t h i s l a b o r a t o r y have shown t h a t calmodulin w i l l i n -2+ crease the turnover r a t e of - the.S .it . Ca -pump (Lopaschuk et a l , 198o). t h i s i s due to an increased decomposition of the phosphoprotein i n t e r -2+ 2+ mediate which i s the r a t e l i m i t i n g step of the Mg -Ca -ATPase r e a c t i o n sequence. Work done by Shigekawa and P e a r l (1976) and Jones et a l (1978) has shown t h a t K + also increases the turnover of the phosphorylated 2+ intermediate of the Ca -pump. The p o s s i b i l i t y e x i s t s t h a t calmodulin + 2+ and K act m a s i m i l a r manner t o s t i m u l a t e Ca -uptake. The decreased 2+ s t i m u l a t i o n of the Ca -pump by calmodulin i n the presence of KC1 may be due t o the s i m i l a r mechanism of a c t i o n of these two p o t e n t i a l reg-u l a t o r s . Since the S.R. under p h y s i o l o g i c a l c o n d i t i o n s i s bathed i n + 2+ h i g h K , calmodulin normally may not s t i m u l a t e Ca - t r a n s p o r t . There-fore the p h y s i o l o g i c a l r o l e of calmodulin may be as a 'backup' t o K + 2+ r e g u l a t i o n of S.R. Ca - t r a n s p o r t . Under c e r t a i n c o n d i t i o n s such as • i. + disease s t a t e s where K l e v e l s are decreased calmodulin may then become 2+ important i n r e g u l a t i n g the Ca -pump. + 2+ 5) K i n e t i c P r o p e r t i e s of Calmodulin and K Regulation of Ca -transport i n Cardiac S.R.: In c a r d i a c microsomes enriched i n sarcoplasmic r e t i c u l u m calmodulin 2+ s t i m u l a t e d Ca - t r a n s p o r t mainly by i n c r e a s i n g the V 2+ and had no 2+ s i g n i f i c a n t e f f e c t on the apparent K^ f o r Ca ( f i g u r e 12). This d i f f e r s from r e s u l t s obtained i n i n s i d e - o u t e r y t h r o c y t e v e s i c l e s where calmodulin not only i n c r e a s e d the V but a l s o caused a s h i f t i n the max K,. f o r Ca from high t o low (Sarkadi et a l , 1978; Maclntyre and d i s s :— 2+ Green , 1978). The e f f e c t of calmodulin oh e r y t h r o c y t e membrane Mg -C a 2 + - A T P a s e p a r a l l e l s i t s e f f e c t on erythrocyte C a 2 + _ t r a n s p o r t . 2+ 2+ Membranes prepared using EDTA, which have a Mg -Ca -ATPase a c t i v i t y of both low and high a f f i n i t y (Schatzman, 1975! ; Quist and R o u f o g a l i s , 2+ 1975), w i l l s h i f t to the high Ca - a f f i n i t y form i n the presence of c a l m o d u l i n ( J a r r e t t and Penniston, 1978; Katz et a l , 1979). In c a r d i a c microsomes enriched i n S.R. an e f f e c t of calmodulin on the K d i s g f o r 70 2+ Ca may depend, .on the .me.thod of pr e p a r a t i o n of the microsomes. However, due to the s e n s i t i v i t y of the microsomes , our attempts to prepare the S.R. v e s i c l e s i n the presence of EDTA or EGTA r e s u l t e d i n 2+ complete l o s s of Ca -uptake a c t i v i t y . The e f f e c t of calmodulin on the 2+ 2+ 2+ V and K f o r Ca of the Mg -Ca -ATPase a c t i v i t y i n S.R. has not been determined. 2+ When comparing the k i n e t i c s of Ca - t r a n s l o c a t i o n , i t cannot be 2+ 2+ assumed that changes i n Ca - a c t i v a t i o n and Ca - a f f i n i t y produced by calmodulin i n e r y t h r o c y t e membranes would a l s o r e s u l t i n S.R.. Indeed, calmodulin has been shown to have va r y i n g e f f e c t s on the k i n e t i c para-meters of other enzyme systems. In i n v e s t i g a t i o n s of c y c l i c AMP-dep-endent phosphodiesterase, calmodulin has been reported t o e i t h e r lower the apparent K m f o r cAMP (Brostrom and W o l f f , 1976), e f f e c t s o l e l y the V (Gnegy et a l , 1976) ,or e f f e c t both k i n e t i c parameters (Uzonov et a l , 1976). Calmodulin increases the V of adenylate c y c l a s e without chang-i n g the apparent K m f o r ATP (Lynch and Cheung, 1979). I t has also r e -c e n t l y been reported by Lynch and Cheung (1979) t h a t s t i m u l a t i o n of 2+ 2+ eryt h r o c y t e Mg -Ca -ATPase by. calmodulin i s due to an increase i n V w i t h only a small a l t e r a t i o n of the apparent K f o r ATP. Therefore, max m i t does not seem improbable that calmodulin could increase the V of 2+ 2 + m a X Ca - t r a n s p o r t i n cardiac S.R. without a l t e r i n g the K-, f o r Ca + <2+ Since.K and calmodulin both appear to reg u l a t e Ca -transport i n S.R. the e f f e c t of K + a c t i v a t i o n on the k i n e t i c parameters of the transport process was simultaneously i n v e s t i g a t e d . U n l i k e c a l m o d u l i n , K + not only increased the .V 2+, but a l s o increased the apparent K 2+ 2+ m f o r Ca • '(figure 13). The decrease i n Ca - a f f i n i t y i n the presence of K + i s s i m i l a r to the r e s u l t s obtained by Yamado and Ikemoto (1979), 2+ 2+ as w e l l as Shigekawa and Akowitz (1979), i n s t u d i e s on the Mg -Ca -ATPase a c t i v i t y of s k e l e t a l nuscle S.R.. They demonstrated t h a t K + 2+ 2+ w i l l s h i f t the A D P - i n s e n s i t i v e Mg -Ca -ATPase phosphorylated i n t e r -mediate (E-P) towards the ADP-sensitive phosphorylated intermediate. They a l s o demonstrated that of the two forms of the.E-P the ADP-2+ i n s e n s i t i v e , form ...has the higher a f f i n i t y f o r Ca . Therefore, the 2"t" a d d i t i o n of K w i l l lower the a f f i n i t y of the enzyme system f o r Ca . 2+ 2+ Since Mg -Ca -ATPase plays an . i n t e g r a l r o l e i n c a r d i a c muscle S.R. 71 C a 2 + - t r a n s p o r t , i t would be expected t h a t K + would also lower the C a 2 + -a f f i n i t y of the tra n s p o r t process. Although these researchers d i d not i n v e s t i g a t e cardiac S.R. i t has been shown by Jones et a l (1977) t h a t + 2+ the o v e r a l l e f f e c t s of K on cardiac and s k e l e t a l muscle S.R. Ca -transport are s i m i l a r . 6) E f f e c t of Calmodulin and cAMP-dependent P r o t e i n Kinase on t h e I n i t i a l 2+ Rate of Ca - t r a n s p o r t i n Cardiac S.R.: In t h i s study the e f f e c t of calmodulin on the i n i t i a l r a t e of S.R. 2+ Ca -transport was als o determined. Phosphate was used as the p r e c i p -i t a t i n g anion t o overcome problems associated w i t h the use of oxalate 2+ (Hicks e t a l , 1979). At a l l Ca concentrations t e s t e d i t was shown 2+ t h a t calmodulin i n c r e a s e d the i n i t i a l r a t e of Ca -transport ( f i g u r e 2+ 15). A double r e c i p r o c a l p l o t o f the i n i t i a l r a t e s vs_ Ca -concentra-t i o n ( f i g u r e 16) confirmed t h a t calmodulin a l t e r s the i n i t i a l r a t e of 2+ Ca -t r a n s p o r t by i n c r e a s i n g the V_ 2+ , , , , , T„ Ca r a t h e r t h a n the apparent K 2+ d f o r Ca . Since calmodulin and cAMP-dependent p r o t e i n kinase both increase 2+ phosphorylation of phospholambam the k i n e t i c parameters of Ca -tr a n s p o r t s t i m u l a t i o n i n cardiac-S.R. were compared. Hicks e t a l (1979) determined the e f f e c t of cAMP-dependent p r o t e i n kinase on the i n i t i a l 2+ ra t e of Ca -transport u s i n g the same procedure u t i l i z e d i n our l a b o r -atory. S i m i l a r t o r e s u l t s obtained w i t h calmodulin ,cAMP-dependent 2+ 2+ p r o t e i n kinase s t i m u l a t e d the i n i t i a l r a t e of Ca - t r a n s p o r t at a l l Ca concentrations t e s t e d . However, cAMP-dependent p r o t e i n k i n a s e , u n l i k e calmodulin, not only increased the V 2+ but a l s o decreased the apparent 2+ 2+ K f o r Ca . They suggest that the higher a f f i n i t y f o r Ca i s due t o m 2 + 2 + a decrease i n the p o s i t i v e c o - o p e r a t i v i t y between the two Mg -Ca -2+ ATPase Ca b i n d i n g s i t e s . Tada et a l (1979) demonstrated that phos-p h o r y l a t i o n of phospholambam by cAMP-dependent p r o t e i n kinase d i d 2+ 2+ not a l t e r the amount of Mg -Ca -ATPase phosphoenzyme (E-P), but d i d accel e r a t e the turnover r a t e of the calcium pump. Studies i n our l a b o r a t o r y (Lopaschuk et a l , 1980) have shown that calmodulin a l s o 2+ increased the turnover r a t e of the Ca -pump, but t h i s was accompanied 72 by a s l i g h t decrease i n E-P l e v e l s . Kranias et a l (personal communica-tions) have also found that cAMP-dependent protein kinase increased E-P le v e l s i n cardiac S.R. preparations. Therefore, even though calmodulin 2+ and cAMP-dependent protein kinase can enhance Ca -transport the mech-anism of this regulation may not be the same. 7) I s o l a t i o n of Calmodulin from Microsomal Cardiac S.R.: Calmodulin has previously been i s o l a t e d from-cardiac muscle (Teo et a l , 1973) and has been found to be associated with both p a r t i c u l a t e and supernatant fract i o n s i n various t i s s u e homogenate (Kakiuchi et a l , 1978). In most systems, calmodulin i s lo o s e l y associated with membran-2+ ous or cytoplasmic p r o t e i n s , and requires the presence of Ca f o r bind-2+ ing (Cheung, 1971). Removal of Ca with agents such as EGTA r e s u l t s i n d i s s o c i a t i o n of calmodulin from these proteins. Treatment of mem-branes i n muscle homogenates with high s a l t concentrations, urea, or TCA w i l l also r e s u l t i n t r a n s l o c a t i o n of calmodulin from the p a r t -i c u l a t e to the soluble f r a c t i o n . In microsomal S.R., the presence of calmodulin associated with the p a r t i c u l a t e f r a c t i o n could a l t e r the 2+ k m e t i c parameters of Ca -transport as determined by addition of exogenous calmodulin. Therefore i t was necessary to determine i f the microsomal cardiac S.R. used i n t h i s study contained any indigenous calmodulin. Intact microsomal S.R. was subjected t o procedures used to i s o l a t e calmodulin from other sources (DePaoli-Roach et a l , 1979; Au, 1978, Jung, 1978). B o i l i n g i n the presence of EGTA would r e -sult i n s o l u b i l i z a t i o n of any calmodulin present. Table 5 indicates, that calmodulin could not be i s o l a t e d from our microsomal preparation. This would i n d i c a t e that the characterization of calmodulin stimulation 2+ of Ca -transport i n cardiac S.R. preparations i s not complicated by the presence of indigenous calmodulin. Since the microsomal S.R. i s t reated with 0.6 M KC1 during preparation i t i s l i k e l y that any calmodulin that may have been associated with the S.R. was s o l u b i l i z e d . 2+ 2+ C a r a f o l i et a l (1980) were able to stimulate S.R. Mg -Ca -ATPase with a heat-treated extract from microsomal cardiac S.R. . However, the microsomal preparation had not previously been t r e a t e d with high s a l t concentrations. 73 8) Calmodulin B i n d i n g to Microsomal Cardiac S.R. Since calmodulin does not appear to be an i n t e g r a l component of the S.R. i t was p o s t u l a t e d t h a t b i n d i n g t o a s i t e on the membrane must 2+ occur m order f o r calmodulin to augment Ca - t r a n s p o r t . I t i s p o s s i b l e 2+ that the monovalent ca t i o n s decrease calmodulin s t i m u l a t i o n of Ca -tra n s p o r t by a l t e r i n g t h i s b i n d i n g . Experiments were therefore performed 125 u s i n g I - l a b e l l e d calmodulin t o determine the degree of b i n d i n g to microsomal preparations i n the presence and absence of K + (110 mM), Na + (llOmM), and L i + (110 mM). I n i t i a l experiments, which u t i l i z e d (55 12 5 0.45/| M i l l i p o r e f i l t e r s to separate bound from f r e e I - c a l m o d u l i n , showed that calmodulin e x h i b i t e d a high degree of n o n - s p e c i f i c b i n d i n g . This n o n - s p e c i f i c b i n d i n g was enhanced by i n c r e a s i n g EGTA concentra-t i o n s . A p o s s i b l e explanation i s that an a l t e r a t i o n i n the conformation 2+ of calmodulin due t o decreased Ca l e v e l s (Klee, 1977) may e i t h e r enhance b i n d i n g t o the f i l t e r , or ob s t r u c t the passage of unbound calmodulin through the f i l t e r . When a r e v i s e d technique was developed to measure calmodulin b i n d i n g t o S.R. without u t i l i z a t i o n of f i l t e r s (as described i n methods) there was s t i l l a s u b s t a n t i a l amount of 2+ calmodulin b i n d i n g i n the absence of Ca . This i s probably due t o a high degree of hydrophobic b i n d i n g to membrane p r o t e i n s as suggested by Storm et a l (1980). Therefore measurement of s p e c i f i c calmodulin b i n d i n g t o microsomal S.R. was co r r e c t e d f o r n o n - s p e c i f i c b i n d i n g by su b t r a c t i n g calmodulin bound t o microsomal S.R. i n a c t i v a t e d by b o i l i n g . 125 B i n d i n g of I - l a b e l l e d calmodulin t o i n a c t i v e microsomal S.R. was 2+ Ca -independent, suggesting that i t was indeed n o n - s p e c i f i c m nature. 125 As i n d i c a t e d i n f i g u r e 18 I - l a b e l l e d calmodulin binds t o 2+ microsomal preparations enriched i n S.R. i n a Ca concentration dependent manner. Penniston et a l (1980) has demonstrated that 1 2 ^ i _ l a b e l l e d calmodulin w i l l a l s o b i n d t o p u r i f i e d e r y t h rocyte membrane 2+ 2+ 2+ Mg -Ca -ATPase i n a Ca -concentration-dependent manner. Since 2+ 2+ 2+ calmodulin s t i m u l a t e s Mg -Ca -ATPase and Ca -transport i n both c a r d i a c S.R. and ery t h r o c y t e membranes i t i s p o s s i b l e t h a t calmodulin may be 2+ 2+ b i n d i n g t o the Mg -Ca -ATPase enzyme i n c a r d i a c S.R. as w e l l . At Ca 2 +' concentrations (.10 ^ - 10. ^  M) where monovalent cations 2+ were shown.to decrease calmodulin stimulation of S.R. Ca -transport there i s a s i g n i f i c a n t decrease i n calmodulin binding' i n the presence of K + (110 mM) or Na + (110 mM). In the presence of L i + (110 mM), which has a less pronounced i n h i b i t o r y e f f e c t on calmodulin stimulation 125 of S.R. Ca -transport, there i s less a l t e r a t i o n i n I-calmodulin binding (table 6 ) . This suggests that the monovalent cations decrease 2+ calmodulin augmentation of Ca -transport i n S.R. by decreasing the binding of calmodulin to the S.R. membrane. Since K + and calmodulin 2+ both increase the turnover rate of the Ca -pump i t i s concievable that they both may be competing f o r s i m i l a r binding s i t e s on the S.R. membrane. .Jones et a l (1978) have suggested that K + acts by binding 2+ 2+ d i r e c t l y t o the Mg -Ca -ATPase enzyme i n S.R.. Therefore, calmodulin 2+ may also stimulate cardiac S.R. Ca -transport by binding d i r e c t l y 2+ 2+ to the Mg . -Ca •• -ATPase enzyme. On the other hand, the monovalent cations may be i n h i b i t i n g calmodulin binding le s s s p e c i f i c a l l y . Wolff and Brostrom (1979) have shown that KC1 can cause an i o n i c strength-dependent conformational change i n calmodulin. This non-specific con-formational change may a l t e r calmodulin binding to the S.R. membrane. However, th i s hypothesis does not explain why equal i o n i c strengths of L i C l would not evoke s i m i l a r conformational changes. When analyzing the re s u l t s of these calmodulin binding studies one must be cautious i n i n t e r p r e t i n g the data. Although the s p e c i f i c • a c t i v i t y of the pure calmodulin was determined p r i o r t o iod i n a t i o n the -125 small y i e l d of I - l a b e l l e d calmodulin made i t impossible t o insure that calmodulin retained a c t i v i t y subsequent to i o d i n a t i o n . As w e l l , 125, highlevels of non-specific 1-labelled calmodulin binding make i t d i f f i c u l t to d i s t i n q u i s h p h y s i o l o g i c a l relevant calmodulin binding to non-specific hydrophobic i n t e r a c t i o n . Future experiments should concentrate on developing methodology which would enable one t o determine 125 the s p e c i f i c a c t i v i t y of I - l a b e l l e d calmodulin, as well as decreasing the l e v e l s of non-specific calmodulin binding. 75 2+ 9) Calmodulin and i t s r o l e i n Ca -transport i n Cardiac S.R.: Evidence presented i n t h i s study, and the work, of other researchers (Katz and Remtulla, 1978; LePeuch et a l , 1979; Kranias e t _ _ a l , C a r a f o l i et a l , 1980), s t r o n g l y suggest that calmodulin p l a y s a r e g u l a t o r y r o l e 2+ + m c a r d i a c S.R. Ca - t r a n s p o r t . Along w i t h K and cAMP, calmodulin 2+ 2+ mcreases the turnover r a t e of Mg -Ca -ATPase i n S.R. membrane. Figure 19 i s a schematic r e p r e s e n t a t i o n of the p o s s i b l e mechanism by which- these r e g u l a t o r s act. C y c l i c AMP a c t i v a t e s cAMPr-dependent .protein-"kinase, ' wnich-.phosphorylates -an. S-.R.- membrane p r o t e i n , phospholambam, r e s u l t i n g i n an increased rate of decomposition of the phosphorylated intermediate of Mg -Ca: -ATPase (E-P) . Calmodulin may a c t i v a t e a Ca -dependent p r o t e i n kinase which also phosphorylates phospholambam, r e s u l t i n g i n an increased turnover r a t e of the E-P. As w e l l , calmodulin may a c t 2+ 2+ + d i r e c t l y on the Mg -Ca -ATPase enzyme, s i m i l a r to K , a l s o enhancing the turnover r a t e of the E-P. LePeuch e t a l (1979) have suggested t h a t 2+ a concerted r e g u l a t i o n o f c a r d i a c S.R. Ca -tra n s p o r t by cAMP and c a l -modulin e x i s t s . Indeed, many other c e l l u l a r processes e x h i b i t dual r e g u l a t i o n by cAMP and calmodulin (Wang and Waisman, 1979)• In the 2+ + complicated r e g u l a t i o n of cardiac S.R. Ca - t r a n s p o r t , K may be a t h i r d r e g u l a t o r which i n t e r a c t s w i t h calmodulin to a greater extent than 2+ w i t h cAMP. Regulation of Ca - t r a n s p o r t by cAMP may become more im-portant during sympathetic s t i m u l a t i o n of cardiac muscle, w h i l e K and calmodulin r e g u l a t i o n may be more important i n the absence of t h i s 2+ s t i m u l u s . B e t t e r models, which represent p h y s i o l o g i c a l S.R. Ca -t r a n s p o r t , must be developed before a hypothesis such as t h i s can be tes t e d . 76 Figure 18 2+ P o s s i b l e Mechanisms by Which Calmodulin Regulates the Ca -pump of Cardiac Sarcoplasmic Reticulum. 2+ 2+ EP, phosphorylated intermediate of Mg -Ca -ATPase 2 + 2 + E , non-phosphorylated intermediate of Mg -Ca -ATPase S.R. , cardiac sarcoplasmic r e t i c u l u m C A L M O D U L I N 78 SUMMARY AND CONCLUSIONS + 2+ 1) In the presence of K , calmodulin s t i m u l a t e s Ca -tra n s p o r t i n microsomal preparations enriched i n cardiac S.R. 100 t o 250$ at 2+ p h y s i o l o g i c a l y obtainable Ca concentrations. 2+ 2) In the presence of llOmM KC1, calmodulin s t i m u l a t e s Ca -transport i n microsomal c a r d i a c S.R. 10 t o k0% at p h y s i o l o g i c a l y .obtainable Ca conce n t r a t i o n s . + + 2+ 3) K and Na decrease calmodulin s t i m u l a t i o n o f Ca -tr a n s p o r t to a greater degree than L i + . h) I t t h e r e f o r e appears t h a t monovalent c a t i o n s , such as K and Na , 2+ i n h i b i t calmodulin s t i m u l a t i o n of cardiac S.R. Ca - t r a n s p o r t . 2+ 5) Calmodulin s t i m u l a t e s Ca -transport i n microsomal cardiac S.R. by 2+ i n c r e a s i n g the VQ a2+ without a l t e r i n g the apparent K f o r Ca , whi l e K increases the V 2+, as w e l l as i n c r e a s i n g the apparent 2 Ca fo r Ca d 2+ 6) Calmodulin increases the i n i t i a l r a t e o f Ca - t r a n s p o r t , by 2+ i n c r e a s i n g the V 2+ without a l t e r i n g the apparent K f o r Ca L/ a d Since cAMP-dependent p r o t e i n kinase increases the i n i t i a l r a t e of 2+ Ca - t r a n s p o r t by i n c r e a s i n g the V 2+ as w e l l as decreasing the . 2+ L a apparent f o r Ca i t can be concluded t h a t calmodulin and cAMP-dependent p r o t e i n kinase do not act by s i m i l a r mechanisms. 7) There i s no indigenous calmodulin i n the microsomal c a r d i a c S.R. used i n t h i s study. 125 2Jc 8) I - l a b e l l e d calmodulin binds t o microsomal S.R. i n a Ca dose dependent manner. 9) K + (llOmM) and Na + (llOmM) s i g n i f i c a n t l y decreased (p<0.05, students ' 1 2 5 " t " t e s t ) I - l a b e l l e d calmodulin b i n d i n g t o microsomal cardiac —7 + S..R.., at calcium concentrations above 10' M, wh i l e L i (llOmM) does not a l t e r calmodulin b i n d i n g t o a s i g n i f i c a n t e x t e n t . 10) These st u d i e s t h e r e f o r e i n d i c a t e t h a t calmodulin i s not an indigenous 2+ S.R. p r o t e i n , but binds i n a Ca -concenter at ion-dependent manner t o 2+ s i t e s on the sarcoplasmic r e t i c u l u m t o st i m u l a t e Ca - t r a n s p o r t . 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