Pharmaceutical Sciences, Faculty of
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UBCV
Lopaschuk, Gary David
Master of Science - MSc
University of British Columbia
Calmodulin prepared from red cell hemolysate stimulates Ca²⁺ -transport in cardiac microsomal preparations enriched in sarcoplasmic reticulum (S.R.) in a concentration-dependent manner (Katz and. Remtulla, 1978). The present study was performed to characterize this calmodulin regulation of Ca²⁺-transport. K⁺ and Na⁺ have also been found to enhance Ca²⁺-transport in microsomal preparations enriched in S.R. (Jones et al, 1978). It was observed that in the presence of K⁺ (110 mM) and Na⁺ (110 mM) stimulation of Ca²⁺-transport activity by calmodulin was greatly reduced. This result was obtained at all free Ca concentrations studied. That this was not an ionic effect was indicated by the decreased antagonism of calmodulin stimulation by Li⁺ (110 mM). Kinetic characterization determined that calmodulin significantly enhanced the maximal Ca²⁺activation without affecting the apparent Ca²⁺affinity of the Ca²⁺-transport process in cardiac S.R.. K⁺ was found to enhance the V[sub Ca²⁺] as well as lowering the apparent Ca²⁺ affinity. Examination of the initial rate of Ca²⁺-transport in cardiac S.R. confirmed that calmodulin stimulation is due mainly to an increase in the V[sub Ca²⁺]. CyclicAMP-dependent protein kinase, on the other hand, has been shown to increase the V[sub Ca²⁺], as well as decrease the apparent K[sub m] for Ca²⁺ (Hicks et al, 1979), suggesting a different mechanism of action. Experiments were performed to investigate whether calmodulin was indigenous to the preparations used. Attempts were therefore made to isolate calmodulin from dog cardiac microsomal preparations enriched in S.R. by methodology used to isolate calmodulin from other sources (Jung, 1978; Depaoli-Roach et al, 1979). These extracts were then tested to determine their ability to stimulate Ca²⁺-transport into S.R.. It was observed that neither boiling nor treatment with 0.6mM EGTA could extract calmodulin from these preparations. This result indicates that the microsomal preparations utilized do not contain indigenous calmodulin. Since calmodulin dose not appear to be a component of the S.R., it was postulated that binding to sites on the membrane must occur in order for calmodulin to augment Ca²⁺- transport. It was also suggested that K⁺ and NA⁺ and may decrease calmodulin stimulation of Ca²⁺-transport by altering this binding. Studies were therefore performed using ¹²⁵I-labelled calmodulin to determine the degree of binding to microsomal preparations in the presence and absence of K⁺(110mM), Na⁺ (110mM) , and Li⁺(110mM.). It was found that ¹²⁵I-calmodulin binds to microsomal cardiac S.R. in a Ca²⁺ concentration-dependent manner. At Ca²⁺ concentrations above 10⁻⁷M, this binding was significantly decreased (p<0.05, students "t" test) in the presence of K⁺ or Na⁺. Li⁺, previously shown not to alter Ca²⁺-transport augmentation byucalmodulin, did not alter calmodulin binding to a significant extent. K⁺ and Na⁺ therefore may inhibit calmodulin stimulation of Ca²⁺-transport in these preparations by decreasing calmodulin binding to the S.R.. The lack of inhibition by Li⁺ (110mM) indicates that this result is not due to a non-specific ionic effect. Our studies therefore have shown that calmodulin is not an indigenous protein of the sarcoplasmic reticulum preparations used. Calmodulin, though, has been found to bind, in a Ca²⁺-dependent manner, to these preparations. This binding is altered by monovalent cations previously shown to inhibit calmodulin stimulation of Ca²⁺-transport.
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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Thesis/Dissertation
eng
Pharmaceutical Sciences
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Graduate
Characterization of calmodulin effects on calcium transport in cardiac microsomes enriched in sarcoplasmic reticulum
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