UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on the proteolysis of the human erythrocyte calcium-pumping ATPase by endogenous calpain Wang, Kevin Ka-Wang 1989

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1989_A1 W37.pdf [ 17.77MB ]
Metadata
JSON: 831-1.0098268.json
JSON-LD: 831-1.0098268-ld.json
RDF/XML (Pretty): 831-1.0098268-rdf.xml
RDF/JSON: 831-1.0098268-rdf.json
Turtle: 831-1.0098268-turtle.txt
N-Triples: 831-1.0098268-rdf-ntriples.txt
Original Record: 831-1.0098268-source.json
Full Text
831-1.0098268-fulltext.txt
Citation
831-1.0098268.ris

Full Text

STUDIES ON THE PROTEOLYSIS OF THE HUMAN ERYTHROCYTE CALCIUM-PUMPING ATPase BY ENDOGENOUS CALPAIN I by KEVIN KA-WANG WANG B . S c , Un ive rs i t y of Guelph, 1984 A t h e s i s submitted in p a r t i a l f u l f i l l m e n t of the requirement for the degree of Doctor of Philosophy in The Facul ty of Graduate Studies D i v i s i o n of Pharmaceutical Chemistry of the Facul ty of Pharmaceutical Sciences We accept t h i s thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1989 © Kevin Ka-Wang Wang, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT It i s well known that the ery throcyte plasma membrane Ca -pumping ATPase can be ac t iva ted by ca lmodul in . Limited p r o t e o l y s i s of the C a ^ + -ATPase with exogenous proteases can a lso produce a ca lmodul in -1 ike a c t i v a t i o n . Because a calcium-dependent cys te ine protease (ca lpa in I) i s present in the erythrocyte c y t o s o l , i t was proposed that t h i s enzyme could p r o t e o l y t i c a l l y ac t iva te the Ca^ + -ATPase dur ing a sustained e leva t ion of the c y t o s o l i c f ree C a ^ + l e v e l . Upon incubat ion of red blood c e l l membranes with p u r i f i e d ca lpa in I and C a ^ + , the membrane-bound Ca^ + -ATPase a c t i v i t y was increased and i t s s e n s i t i v i t y to calmodulin was l o s t . Calmodulin protected the Ca^ + -ATPase against p r o t e o l y t i c a c t i v a t i o n by c a l p a i n . Both the membrane-bound and the p u r i f i e d Ca^ + -ATPase (136 kDa) Were transformed by c a l p a i n in to two phosphoenzyme intermediate- forming fragments of 125 kDa and 124 kDa, fol lowed by the formation of two phosphoenzyme intermediate- forming fragments of 82 kDa and 80 kDa. The 125 kDa and the 82 kDa fragments bound to and were st imulated by ca lmodul in , whereas, the 124 kDa and the 80 kDa fragments d id not bind and were not st imulated by calmodulin and t h e i r formation corresponded to the observed p r o t e o l y t i c a c t i v a t i o n . In the presence of ca lmodul in , however, the nat ive enzyme was s e q u e n t i a l l y transformed into two phosphoenzyme intermediate- forming fragments of 127 kDa and 85 kDa. Both of these fragments bound to and were st imulated by ca lmodul in . Apparent ly , calmodulin protects the Ca^ + -ATPase from c a l p a i n -i i mediated a c t i v a t i o n by preventing the formation of the ca lmodul in-i n s e n s i t i v e 124 kDa and 80 kDa fragments. Smaller i n a c t i v e fragments were a lso produced with fur ther p r o t e o l y s i s . Fol lowing l i m i t e d ca lpa in treatment of the phosphat idy lcho l ine 1 iposome-reconst i tuted Ca^ + -ATPase, both the i n i t i a l ra tes of C a ^ + uptake and ATP h y d r o l y s i s were increased to near maximal l e v e l s , s i m i l a r to those obtained upon addi t ion of ca lmodul in . The r e c o n s t i t u t e d C a ^ + -ATPase was transformed mainly into 124 kDa and 127 kDa ac t ive fragments, in the absence and the presence of ca lmodul in , r e s p e c t i v e l y . Based on the deduced amino acid sequence of a plasma membrane C a ^ + -ATPase from a human teratoma, the ca lmodul in -b ind ing domain was i d e n t i f i e d to be about 3.5 kDa long and located about 9-10 kDa from the C-terminal end of the enzyme while the acylphosphate s i t e was loca ted about 50 kDa from the N-terminal end. By combining t h i s knowledge with the estimated molecular masses and ca lmodul in-b inding a b i l i t y o f the var ious fragments, i t was postulated that ( i ) in the absence of ca lmodul in , c a l p a i n I i n i t i a l l y c leaves o f f about 11 kDa from the C-terminal end and about 2 kDa of the ca lmodul in-b inding domain, producing the ca lmodul in -s e n s i t i v e 125 kDa fragment; ( i i ) in the absence of ca lmodul in , a second cleavage by c a l p a i n I fu r ther removes most (1.5 kDa) of the remaining ca lmodul in -b ind ing domain and produces the c a l m o d u l i n - i n s e n s i t i v e 124 kDa fragment; ( i i i ) in the presence of ca lmodul in , c a l p a i n c leaves o f f about 9 kDa o f the C-terminal end and produces a c a l m o d u l i n - s e n s i t i v e 127 kDa fragment which reta ined most of the ca lmodul in -b inding domain. A slower i i i cleavage at a s i t e about 42-44 kDa from the N-terminal end was a lso proposed to generate the 85 kDa, 82 kDa and 80 kDa a c t i v e fragments from the 127 kDa, 125 kDa and 124 kDa fragments, r e s p e c t i v e l y . Several other ca lmodul in -b ind ing prote ins were a lso found to be proteolyzed by c a l p a i n I, i nc lud ing adducin from human ery throcy te membrane as well as neuromodulin and c a l c i n e u r i n from bovine b r a i n . A l i t e r a t u r e search fu r ther revealed that s u s c e p t i b i l i t y to c a l p a i n was in f a c t a c h a r a c t e r i s t i c shared by at l e a s t 16 ca lmodu l in -b ind ing p r o t e i n s . Primary s t ruc ture a n a l y s i s showed that the major i ty o f ca lmodul in -b ind ing pro te ins sequenced to date have one or more regions enr iched in p r o l i n e (P) , glutamate (E ) , aspartate (D), ser ine (S) and threonine (T) (PEST sequences) . It was proposed that PEST sequences in ca lmodul in -b ind ing prote ins may serve as recogn i t ion s i t e s f o r c a l p a i n . Signature o f t h e s i s c o - s u p e r v i s o r Signature of t h e s i s c o - s u p e r v i s o r iv ACKNOWLEDGEMENTS I am wholeheartedly gra te fu l to my s u p e r v i s o r s , Dr. B a s i l D. Roufogal is and Dr. Antonio V i l l a ! o b o fo r t h e i r guidance, t i r e l e s s support , i n s p i r a t i o n and f r i e n d s h i p . I am indebted to my Ph.D. Thesis Committee members: Dr. Marc Levine (Chairman), Dr. Frank Abbott , Dr. David Godin and Dr. Sidney Katz fo r t h e i r a s s i s t a n c e . I would l i k e to acknowledge the con t r ibu t ion of Mr. James G i l c h r i s t who suppl ied the junc t iona l sarcoplasmic re t icu lum preparat ions in t h i s work. I would a lso l i k e to thank Dr. Y. L iu and Dr. D. R. Storm f o r prov id ing neuromodul i n , Dr. B. Mutus for prov id ing oncomodulin and Dr. M. Rechsteiner fo r provid ing the PEST-FIND program. I would l i k e to express my apprec ia t ion to my c o l l e a g u e s , past and present , in p a r t i c u l a r , Dr. C h r i s t i n e N i c h o l , Dr. Jon Church, Dr. Shobba Gosh, Dr. Dorothy J e f f e r y , Mr. James H a r r i s , Ms. Cheryl Machan and Mr. Bruce Wilson fo r t h e i r technica l ass is tance and moral support . Specia l thanks are given to Dr. John McNeil l and Dr. Sidney Katz f o r t h e i r strong support when i t was needed most. I a lso l i k e to thank the members of Dr. K a t z ' s l abora tory , e s p e c i a l l y Mr. Bruce A l l e n and Mr. James G i l c h r i s t , f o r sharing experimental experience and i n s p i r a t i o n . I would l i k e to acknowledge the f i n a n c i a l support extended by the Canadian Heart Foundation, the B r i t i s h Columbia Heart Foundation and the U n i v e r s i t y v of B r i t i s h Columbia. I a lso acknowledge the supply of f resh human blood from the Red Cross of Canada (Vancouver branch) . F i n a l l y , I wish to thank a l l members of the f a c u l t y , support ing s t a f f and graduate students in the Facul ty of Pharmaceutical S c i e n c e s , U n i v e r s i t y of B r i t i s h Columbia fo r making my Ph.D. program most enjoyable and memorable. vi DEDICATION To my wife A l i c e and to my parent TABLE OF CONTENTS CONTENT page ABSTRACT i i ACKNOWLEDGEMENTS v DEDICATION v i i TABLE OF CONTENTS v i i i LIST OF FIGURES x i i i LIST OF TABLES x v i i LIST OF ABBREVIATIONS x v i i i INTRODUCTION 1 I. Calcium and c e l l funct ions 1 II. Control of i n t r a c e l l u l a r calcium concentra t ion 7 1. Mitochondria 7 2. Endoplasmic and sarcoplasmic re t icu lum 8 3. Plasma membrane 9 III. The plasma membrane Ca -pumping ATPase 11 1. Overview 11 2. P u r i f i c a t i o n and r e c o n s t i t u t i o n 17 3. Regulations 19 ( i ) Phosphol ipid 19 ( i i ) ATP 20 ( i i i ) Divalent cat ions 20 ( iv ) C a 2 + / c a " l m ° d u l in 21 (v) Monovalent cat ions 26 (v i ) Phosphorylat ion 30 ( v i i ) Other regulators 31 IV. Calmodulin and calmodul in-regulated systems 33 V. Calpain 39 vi i i 1. Overview 39 2. Regulators 47 3. Substrate s p e c i f i c i t y 50 4. Substrates and funct ions of ca lpa in 53 VI . Object ives of the study 58 MATERIALS AND METHODS 60 I. Mater ia ls 60 II. Methods 65 1. Prote in concentrat ion determination 65 2. Determination of inorganic phosphate 65 3. C a l c u l a t i o n s of the concentrat ions of f ree Ca and M g z + 66 4. Polyacrylamide gradient gel e l e c t r o p h o r e s i s and autoradiography 67 5. Preparat ion of membrane-free hemblysate and ca lmodul in-depleted membranes 68 6. S o l u b i l i z a t i o n and p u r i f i c a t i o n of the C a 2 + - A T P a s e 69 7. I s o l a t i o n and p u r i f i c a t i o n of ca lpa in from membrane-free hemolysate 70 8. Determination of the membrane-bound C a z + - A T P a s e 71 9. Determination of the a c t i v i t y of the p u r i f i e d C a z + - A T P a s e 72 10. Determination of the phosphorylated intermediate of the C a z + - A T P a s e 72 11. Reconst i tu t ion of the p u r i f i e d C a 2 + - A T P a s e 73 12. Determination of the C a z + - A T P a s e and C a 2 + t ranspor t a c t i v i t i e s of the reconst i tu ted calc ium pump 73 13. Determination of ca lpa in a c t i v i t y ( c a s e i n o l y s i s ) 74 14. Treatment of calmodul in-depleted membranes with var ious proteases 75 ix 15. Treatment of the p u r i f i e d Ca -ATPase with c a l p a i n 16. Treatment of the reconst i tu ted Ca^ + -ATPase with p u r i f i e d ca lpa in 17. Formation of the phospborylated intermediate of the recons t i tu ted Ca -ATPase 18. PEST sequence i d e n t i f i c a t i o n and PEST score c a l c u l a t i o n 19. Data ana lys is RESULTS I. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of ca lpa in 1. P u r i f i c a t i o n of ca lpa in 2. Charac te r i za t ion of c a l p a i n II. E f f e c t of ca lpa in on membrane-bound the Ca^ -ATPase 1. E f f e c t of t r y p s i n , papain and ca lpa in on the a c t i v i t y of the membrane-bound Ca -ATPase 2. Protect ion by calmodulin of the membrane-bound Ca -ATPase against p r o t e o l y t i c a c t i v a t i o n by ca lpa in 3. Further c h a r a c t e r i z a t i o n of the ca lpa in d i g e s t i o n of the membrane-bound Ca -ATPase in the absence and the presence of calmodulin III. P r o t e o l y s i s of the p u r i f i e d Ca^ + -ATPase by c a l p a i n p • 1. Calpain d i g e s t i o n of the p u r i f i e d Ca^ -ATPase 2. P u r i f i c a t i o n of the ca lmodul in -b inding fragments of the C a z + - A T P a s e 3. T ryps in fragmentation of the Ca^ + -ATPase IV. Charac te r i za t ion of the untreated and c a l p a i n - t r e a t e d 1iposome-reconsituted Ca -ATPase (Ca pump) 1. E f f e c t of A23187, a lamethic in and T r i t o n X-100 on the ATP h y d r o l y t i c a c t i v i t y of the recons t i tu ted C a 2 + pump p • 2. E f f e c t of c a l p a i n on the Ca*- - t ranspor t and ATP h y d r o l y t i c a c t i v i t y of the Ca^ pump 136 3. P ro tec t ive e f f e c t of calmodulin against p r o t e o l y t i c a c t i v a t i o n of the reconst i tu ted C a z + pump by c a l p a i n 149 4. K i n e t i c proper t ies of the in tac t and c a l p a i n - t r e a t e d C a 2 + pump in phosphat idy lchol ine v e s i c l e s 153 V. S u s c e p t i b i l i t y of other ca lmodul in-b inding pro te ins and t roponin C superfamily prote ins to c a l p a i n 156 1. P r o t e o l y s i s of adducin and neuromodulin by c a l p a i n 161 2. E f f e c t of ca lpa in on the junc t iona l SR pro te ins 165 3. Resistance of t roponin C superfamily p ro te ins to ca lpa in 169 DISCUSSION 172 I. D iscuss ion of the experimental r e s u l t s 172 1. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of c a l p a i n 172 2. Ca lpa in ac t iva tes the ATP-hydro ly t i c a c t i v i t y of the C a z + - A T P a s e 173 3. Fragmentation of the C a z + - A T P a s e by c a l p a i n : comparison with t r y p s i n 174 4. Ca lpa in a lso ac t iva tes the C a z + - t r a n s l o c a t i n g a c t i v i t y of the C a z + - A T P a s e 193 5. Comparison of experimental r e s u l t s from t h i s laboratory and from other l a b o r a t o r i e s 197 II. S i g n i f i c a n c e of t h i s work 203 1. P h y s i o l o g i c a l and pa tho-phys io log ica l s i g n i f i c a n c e 203 2. Using c a l p a i n as a tool 207 3. Using the p r o t e o l y t i c pattern of the C a z + - A T P a s e as a means of i d e n t i f i c a t i o n of the enzyme 208 III. Calmodul in-b inding prote ins as ca lpa in substrates 208 1. Calmodulin-dependent enzymes 212 2. C y t o s k e l e t a l / s t r u c t u r a l and other ca lmodul in -b ind ing xi prote ins 215 3. Substrate s p e c i f i c i t y of ca lpa in 216 4. PEST sequences in ca lmodul in-b inding pro te ins 218 5. Recognit ion of ca lmodul in-b inding prote ins by c a l p a i n 224 6. PEST sequences in other ca lpa in substrates 230 7. Resistance of the t roponin-C superfamily to c a l p a i n 240 8. Future d i r e c t i o n s 241 CONCLUSIONS 243 BIBLIOGRAPHY 246 APPENDIX 284 xi i LIST OF FIGURES FIGURE Page 1 Three-dimensional s t ruc tu ra l model of calmodulin 5 2 Reaction' c y c l e of the plasma membrane Ca^ + -ATPase 14 3 Amino ac id sequence of the plasma membrane Ca -ATPase from a human teratoma 23 4 Schematic fragmentation of the Ca^ + -ATPase produced by t r y p s i n 27 5 Diagrammatic representat ion of the f l i p - f l o p mechanism f o r caldesmon/calmodul in-caldesmon/act in and t a u / c a l m o d u l i n - t a u / t u b u l i n 37 i n t e r a c t i o n 6 Schematic of the domain s t ructure of ca lpa in 41 7 Mechanism of hydro lys is of the peptide bond of substrates by ca lpa in 44 8 Schematic of the s t ruc ture of c a l p a s t a t i n 48 9 Schematic presentat ion of the i n t e r a c t i o n between the cleavage s i t e of substrate and the ac t ive s i t e of c a l p a i n 51 10 DEAE-Sephacel chromatography of ca lpa in I from human erythrocyte hemolysate 80 11 Phenyl-Sepharose chromatography of DEAE-Sephacel - i s o l a t e d ca lpa in I 82 12 Omega-hexylamine agarose chromatography of phenyl -Sepharose-pur i f i ed c a l p a i n I 84 13 Sephacryl S-200 chromatography of omega-hexylamine a g a r o s e - p u r i f i e d ca lpa in I 86 14 P u r i f i c a t i o n of ca lpa in from human erythrocytes 88 15 A u t o l y s i s of p u r i f i e d ca lpa in (A) and e f f e c t of assay temperature on ca lpa in a c t i v i t y (B) 93 p. 16 A c t i v a t i o n of ca lpa in by Ca and e f f e c t of var ious i n h i b i t o r s on ca lpa in a c t i v i t y 96 xi i i Time-course of the e f f e c t of proteases on the membrane-bound C a - A T P a s e a c t i v i t y E f f e c t of protease concentrat ion on the membrane-bound Ca -ATPase a c t i v i t y E f f e c t of calmodulin on the act ion of proteases on the membrane-bound Ca -ATPase E f f e c t of calmodulin on ca lpa in act ion on the membrane-bound Ca -ATPase E f f e c t of calmodulin on the p r o t e o l y t i c a c t i v a t i o n of the Ca -ATPase a c t i v i t y by ca lpa in (A) and the C a z + - A T P a s e a c t i v i t y (B) A c t i v i t y of the membrane-bound C a z + - A T P a s e t reated with ca lpa in in the presence and absence of calmodulin Formation of the phosphorylated intermediate of the nat ive membrane-bound Ca -ATPase and the fragments produced by ca lpa in treatment E f f e c t of ca lpa in on the p u r i f i e d Ca -ATPase a c t i v i t y Fragmentation of the s o l u b l i z e d and p u r i f i e d Ca -ATPase by ca lpa in and formation of the phosphorylated intermediate Separat ion of the C a z + - A T P a s e fragments with a calmodul in-agarose column P u r i f i c a t i o n of the ca lmodul in -b inding fragments of the C a - A T P a s e from c a l p a i n - t r e a t e d erythrocyte membranes Comparison of the fragmentation patterns of the p u r i f i e d C a - A T P a s e obtained by ca lpa in and t r y p s i n d i g e s t i o n E f f e c t of A23187, a lamethic in and T r i t o n X-100 on the reconst i tu ted C a - A T P a s e a c t i v i t y E f f e c t of ca lpa in on the i n i t i a l rate of both calc ium uptake and ATP-hydro lys is of the reconst i tu ted C a z + pump E f f e c t of increas ing ca lpa in concentrat ion on the i n i t i a l rate of C a z uptake and ATP-hydro ly t i c a c t i v i t y of the recons t i tu ted C a z + pump Time-course of the e f fec t of ca lpa in d i g e s t i o n on the i n i t i a l rate of Ca uptake and ATP h y d r o l y t i c a c t i v i t y Time-course of ca lpa in - induced p r o t e o l y s i s of the reconst i tu ted C a z + pump E f f e c t of calmodulin on the p r o t e o l y s i s of the recons t i tu ted C a z + pump Calcium dependence of the i n i t i a l rate of Ca uptake of the reconst i tu ted C a 2 + pump Calcium dependence of the Ca^ + -ATPase a c t i v i t y in proteoliposomes E f f e c t of ATP concentrat ion on the i n i t i a l ra te of C a z + uptake of the reconst i tu ted C a 2 + pump P r o t e o l y s i s of human erythrocyte adducin and bovine bra in neuromodulin by ca lpa in P r o t e o l y s i s of junct iona l sarcoplasmic re t icu lum prote ins by ca lpa in Resistance of the troponin C superfamily of Ca -b ind ing prote ins to ca lpa in Proposed model fo r the control of the human er throcyte membrane Ca -ATPase by calmodulin and ca lpa in Flow-chart fo r the fragmentation of the ery throcyte Ca -ATPase by ca lpa in in the presence and absence of calmodulin Schematic of the p r o t e o l y s i s of the C a z + - A T P a s e by t r y p s i n Schematic of the p r o t e o l y s i s of the C a 2 + - A T P a s e by c a l p a i n I Schematic of the formation of ac t i ve fragments of the plasma membrane pump produced by c a l p a i n in the absence (-) and the presence (+) of calmodulin (CaM) Proposed model fo r the dual control of the plasma membrane C a 2 + pump by calmodulin and c a l p a i n in a l i v i n g c e l l xv L o c a l i z a t i o n of PEST reg ions , ca lmodul in-b ind domain and ca lpa in cleavage s i t e wi th in human bra in a - f o d r i n Model of calpain-mediated p r o t e o l y s i s of ca lmodul in -b inding prote ins xvi LIST OF TABLES TABLE page 1 Calpain substrates 54 2 P u r i f i c a t i o n of ca lpa in from human erythrocytes 91 3 E f f e c t of ca lpa in treatment in the absence and presence of calmodulin on Ca -ATPase a c t i v i t y before and a f t e r p u r i f i c a t i o n 129 4 E f f e c t of ca lpa in treatment on the degree of coupl ing and C a z * / A T P r a t i o of the l iposome-reconst i tu ted C a z + pump 140 5 Pro tec t ive e f f e c t of calmodulin against p r o t e o l y t i c a c t i v a t i o n of the recons t i tu ted Ca -ATPase by ca lpa in 152 6 Matching of ca lmodul in -b inding prote ins and c a l p a i n substrates 210 7 E f f e c t of ca lpa in on calmodulin-dependent enzymes 213 8 PEST sequences of ca lmodul in -b inding prote ins 220 9 PEST sequences of PEST-containing c a l p a i n substrates 231 10 Non-PEST-containing ca lpa in substrates 237 xvi i LIST OF ABBREVIATIONS % percent phosphorus-32 A ampere a . a . amino acid ADP adenosine 5 ' -d iphosphate Ah aromatic hydrocarbon compound AMP adenosine 5'-monophosphate ATP adenosine 5 ' - t r iphosphate 9 I Ca f ree calcium ion C a 2 + - ca lc ium-s t imu la ted , magnesium-dependent ATPase ATPase CaM calmodulin CaM-PK II calmodulin-dependent prote in kinase II cAMP adenosine 3 ' : 5 ' - c y c l i c monophosphate cAMP-PK c y c l i c AMP-dependent prote in kinase CANP ca lc ium-ac t iva ted neutral protease CBP calcium binding prote in (ca lsequest r in ) Ci Cur ie cpm counts per minute CRC calcium re lease channel DAG D i a c y l g l y c e r o l DEAE d ie thy l aminoethyl DMD Duchenne muscular dystrophy DTT d i t h i o t h r e i t o l xvi i i E enzyme EDTA e thy lenediaminete t raacet ic ac id EGF epidermal growth f a c t o r EGTA e thy leneglyco l b is(^-aminoethylether) N , N , N ' , N ' -t e t r a a c e t i c ac id EP phosphoenzyme intermediate ER endoplasmic ret iculum FPLC f a s t p ro te in l i q u i d chromatography g gram Hepes 4-(2-hydroxy e t h y l ) - l - p i p e r a z i n e e t h a n e - s u l f o n i c ac id HMG-CoA 3 -hydroxy l -3 -methy l -g lu ta ry l coenzyme A IOV i n s i d e out v e s i c l e K m Michael is-Menten constant kDa k i l o d a l t o n L l i t r e m m i l l i M molar M.W. molecular weight MAP-2 microtubule associated prote in-2 MBP myelin bas ic prote in min minute MLCK-G chicken g izzard myosin l i g h t chain kinase MLCK-S s k e l e t a l muscle myosin l i g h t chain kinase mol mole MOPS morphol inopropanesulfonic ac id M r r e l a t i v e molecular mass PAGE polyacrylamide gel e lec t rophores is xix PDE phosphodiesterase P-j inorgan ic phosphate PI phosphatidyl i n o s i t o l PS phosphatidyl ser ine PK-C pro te in kinase C PM plasma membrane PMSF phenyl methylsul fonyl f l u o r i d e RII regu la tory subunit of type II cAMP-PK s second S . E . M . standard e r ro r of the mean SDS sodium dodecy lsu l fa te SR sarcoplasmic ret iculum T 3 3 , 3 ' , 5 ' - t r i i o d o t h y r o n i n e T 4 thyroxine TCA t r i c h l o r o a c e t i c ac id TEMED N,N,N' ,N ' - te t ramethylethy lenediamine TLCK N - a - p - t o s y l - L - l y s i n e chloromethyl ketone T r i s t r i s(hydroxymethyl)ami nomethane V m a x maximum v e l o c i t y H micro xx INTRODUCTION I. Calcium and c e l l funct ions Calcium i s one of the e s s e n t i a l elements of eukaryot ic organisms. In ve r tebra tes , inc lud ing man, over 99% of body calc ium i s immobilized in the bones and teeth by complexing with phosphate to form hydroxy-apat i te . The remaining calc ium i s d i s t r i b u t e d between the e x t r a c e l l u l a r f l u i d and the i n t r a c e l l u l a r space. E x t r a c e l l u l a r calc ium concent ra t ion , inc lud ing that of the blood plasma, i s maintained at about 3 mM. This calc ium leve l i s c o n t r o l l e d mainly by the mob i l i za t ion o f calc ium in and out of bone depos i ts and the intake of d ie ta ry ca lc ium. In a d d i t i o n , about 50% of the e x t r a c e l l u l a r calc ium e x i s t s in an ion ized form ( C a 2 + ) (see C a r a f o l i , 1987). On the other hand, the to ta l i n t r a c e l l u l a r calc ium concentrat ion v a r i e s . Erythrocytes conta in only 20 /iM (Long and Mouat, 1972), bra in c e l l s conta in 1.5 mM and heart c e l l s have 4 mM (see C a r a f o l i , 1987). In cont ras t to the e x t r a c e l l u l a r p o o l , only a f r a c t i o n of the to ta l i n t r a c e l l u l a r calc ium i s ion ized C a 2 + (Hodgkin and Keynes, 1957). T y p i c a l l y , c y t o s o l i c f ree C a 2 + concentrat ion i s between 10" 5 and 10" 8 M, which i s at l e a s t 2-orders of magnitude lower than the e x t r a c e l l u l a r l e v e l . Th is r e s u l t s in a strong e lectrochemical gradient of Ca across the plasma membrane of c e l l s . Such an arrangement enables C a 2 + to func t ion as an i n t r a c e l l u l a r messenger. A c e l l can be considered as an e n t i t y in i t s e l f , using the 1 plasma membrane as i t s b a r r i e r to the outside environment. Yet , i t i s important to develop communication between the outs ide and the ins ide of the c e l l . Such communication i s achieved by a so c a l l e d "receptor -e f f e c t o r coupl ing" mechanism. Genera l ly , s t i m u l i , such as hormones and growth f a c t o r s , can be regarded as l igands which i n t e r a c t s e l e c t i v e l y with the e x t r a c e l l u l a r binding s i t e of t h e i r respect ive receptor p r o t e i n s . Upon l igand b ind ing , the receptor molecule i s "ac t iva ted" and exerts c e r t a i n e f f e c t ( s ) on i t s e f f e c t o r ( s ) . Two d i f f e r e n t types of r e c e p t o r - e f f e c t o r coupl ing systems transduce t h e i r respect ive external s igna ls d i f f e r e n t l y : ( i ) i n s u l i n and epidermal growth f a c t o r receptors are a lso t y r o s i n e - k i n a s e s whose a c t i v i t y i s enhanced by the binding of t h e i r respect ive l igands (see White and Kahn, 1986; Carpenter , 1987). There fore , t h i s c l a s s of prote ins funct ions both as a receptor and an e f f e c t o r and coupl ing i s achieved i n t r a m o l e c u l a r l y . ( i i ) another group of receptors i s known to be coupled to s i g n a l - t r a n s d u c i n g G-prote ins (see Gilman, 1987, f o r a review) . These G - p r o t e i n s , in t u r n , i n t e r a c t with the e f f e c t o r s . To date , several e f f e c t o r s have been i d e n t i f i e d as being coupled to G- 'proteins: adenylate cyc lase ( L e v i t z k i , 1987), cGMP-phosphodiesterase (S t ryer , 1986), phosphatidyl i n o s i t o l ( P l ) s p e c i f i c phospholipase C (S ib ley et a l . , 1987), phospholipase A2 (Jelsema and A x e l r o d , 1987) and var ious ion channels , inc lud ing a voltage-dependent C a ^ + channel (A l lende , 1988). Adenylate c y c l a s e , once a c t i v a t e d , w i l l convert ATP to c y c l i c AMP (cAMP), and cAMP in turn ac t i va tes the cAMP-dependent prote in k inases . These kinases can phosphorylate a large number of target p r o t e i n s . One such target prote in i s a voltage-dependent Ca^ channel (Hofmann et a l . . , 1987). cAMP i s therefore regarded as an 2 i n t r a c e l l u l a r messenger. P l - s p e c i f i c phospholipase C mediates the breakdown of plasma membrane phosphatidyl i n o s i t o l 4 ,5 -b isphosphate ( P I P 2 ) to generate i n o s i t o l - t r i p h o s p h a t e ( I P 3 ) and d i a c y l g l y c e r o l (DAG). DAG in turn enhances the C a 2 + s e n s i t i v i t y of prote in kinase C to a c t i v a t e i t even at submicromolar concentrat ions of C a 2 + (N ish izuka , 1984). This kinase a lso regulates c e l l funct ion by phosphorylat ion of target p r o t e i n s . On the other hand, IP3 can mobi l ize C a 2 + from i n t r a c e l l u l a r calc ium stores (such as endoplasmic ret iculum (ER) (Berr idge, 1987). The elevated c y t o s o l i c C a 2 + l eve l w i l l in turn ac t iva te var ious Ca 2 + -dependent systems (see below)' Therefore , both DAG and I P 3 are a l s o regarded as i n t r a c e l l u l a r messengers. The voltage-dependent C a 2 + channel i s a lso demonstrated to be c o n t r o l l e d by G-prote in-coupled receptors (Rosenthal et a l . . . 1988). Various non-voltage dependent C a 2 + channels which appear to be independent of G-protein a lso e x i s t (Spedding, 1987). Conceivably , c e r t a i n types of hormone s t imula t ion w i l l cause b r i e f opening of C a 2 + channels and al low C a 2 + to enter the c e l l p a s s i v e l y and e levate c y t o s o l i c C a 2 + concent ra t ion . It i s thought that the t r a n s i e n t l y e levated c y t o s o l i c f ree C a 2 + concentra t ion ( C a 2 + t rans ient ) funct ions to "ac t i va te" the s o - c a l l e d i n t r a c e l l u l a r C a 2 + r eceptors , which are a group of C a 2 + - b i n d i n g prote ins (Kre ts inger , 1976). For example, upon binding of C a 2 + , t roponin-C undergoes conformational changes (see Leavis and Gergely , 1984) and i n d i r e c t l y d i s s o c i a t e s the adjacent t roponin- I from a c t i n . The end r e s u l t i s a f a c i l i t a t e d act in-myosin i n t e r a c t i o n , which i s involved in the molecular mechanism of muscle cont rac t ion (Squi re , 1983). Calcium ion 3 a lso binds to an ubiquitous prote in c a l l e d calmodulin (Klee and Vanaman, 1982). In f a c t , calmodulin and t roponin C belong to the same superfamily of Ca -b inding p r o t e i n s . These prote ins are h igh ly a c i d i c and contain 2 to 4 ca lc ium-binding domains, termed E-F hands, which i s the name given to the C a 2 + - b i n d i n g loop in parvalbumin (Krets inger and Nockolds, 1973; Kre ts inger , 1976). Genera l ly , the E-F hand s t ruc ture i s formed by the two h e l i c e s connected by a peptide loop (EF loop) that che la tes the C a 2 + ( F i g . 1) (nomenclature a f te r Kre ts inger and Nockolds, 1973). Unl ike the t roponin C s p e c i f i c f u n c t i o n , calmodulin appears to be a mul t i func t iona l Ca^ receptor . Calmodulin i s well known to regulate var ious c e l l u l a r funct ions v i a i t s i n t e r a c t i o n s with var ious ca lmodul in -b ind ing prote ins (Cheung, 1980; Manalan and K lee , 1984). A d e t a i l e d account of calmodulin and i t s binding prote ins w i l l be presented l a t e r . p i Another Ca*- -b ind ing prote in that recen t ly has drawn increas ing a t tent ion i s the ca lc ium-ac t iva ted neutral protease c a l p a i n . Upon C a 2 + -b ind ing , ca lpa in i s ac t iva ted and proteolyzes se lec ted target prote ins (Suzuki et a l . , 1987a). A d e t a i l e d account o f c a l p a i n w i l l be given below. A fami ly of C a 2 + - and phospho l ip id -b ind ing prote ins c a l l e d l i p o c o r t i n or c a l p a c t i n has been descr ibed recen t ly ( fo r review see K l e e , 1988). However, the funct ions of these prote ins are s t i l l unc lear . Recent ly , i t has become c l e a r that not only the magnitude of the C a 2 + t r ans ien t but a lso i t s frequency are important (Berr idge and Ga l ione , 1988). In other words, C a 2 + t r ans ien ts can be in the form of a bundle of short spikes of C a 2 + t r ans ien ts ( C a 2 + o s c i l l a t i o n s ) . Such C a 2 + 4 F i g . 1 Three-dimensional s t ru tu ra l model of ca lmodul in . The main polypept ide chain of calmodulin i s i l l u s t r a t e d by the r ibbon and each segment represents an amino ac id r e s i d u e . Each of the four calcium ions i s represented by a white sphere. Note that calmodulin has two lobes connected by an e i g h t - t u r n cr -hel ix . Each lobe contains two E-F hand C a 2 + -b inding s t r u c t u r e s . Taken from Babu et a l . (1988). 5 gure 1 o s c i l l a t i o n s have already been demonstrated in many c e l l types (see Berr idge and Ga l ione , 1988). By now, i t should be qui te obvious that C a 2 + i s an important s i g n a l -t ransducing messenger and the c y t o s o l i c C a 2 + concentrat ion needs to be c a r e f u l l y c o n t r o l l e d . In f a c t , uncontro l led e leva t ion of c y t o s o l i c C a 2 + l eve l eventua l ly leads to c e l l death (Chien et a l . , 1978; Farber , 1981; Nicotera et a l . , 1988). II. Control o f i n t r a c e l l u l a r ca lc ium concentra t ion As important as c rea t ing t rans ien t c y t o s o l i c C a 2 + e l eva t ion i s the maintenance o f a submicromolar r e s t i n g l e v e l s of c y t o s o l i c C a 2 + concent ra t ion . Three s u b c e l l u l a r compartments that can p o t e n t i a l l y be involved in C a 2 + handling are d iscussed here: the mi tochondr ia , the endoplasmic re t icu lum (E .R. ) ( inc lud ing sarcoplasmic re t icu lum (SR) of muscle c e l l s ) and the plasma membrane. 1. Mi tochondria Ear ly s tudies demonstrated that mitochondria could take up large amounts of C a 2 + (Vasington and Murphy, 1962; DeLuca and Engstrom, 1961). Th is led to the specu la t ion that mitochondria were important in regu la t ing c y t o s o l i c f ree C a 2 + . The uptake of C a 2 + by mitochondria was achieved by an e l e c t r o p h o r e t i c un ipor ter (Vasington and Murphy, 1961), wh i ls t a 7 N a y C a ^ exchanger prote in was demonstrated to re lease Ca^ from heart mitochondria (Cara fo l i et a l . , 1974). However, more recent s tudies showed that the a f f i n i t y of mitochondria f o r C a 2 + was r e l a t i v e l y low (K m about 10 /iM) and there fore mitochondria were u n l i k e l y to regulate c y t o s o l i c C a 2 + at submicromolar concentrat ions (Crompton et a l . , 1976). A l s o , i t became evident that the rate of C a 2 + uptake by mitochondria i s one order of magnitude below that of sarcoplasmic re t icu lum (Crompton, 1985). At present , the mitochondrial calcium t ranspor t systems are be l ieved to be important not in regu la t ing c y t o s o l i c C a 2 + concent ra t ion , but rather in c o n t r o l l i n g the f ree Ca^ concentrat ion in the mitochondrial matrix s ince several dehydrogenases in the matrix are calcium-dependent (Denton et a l . , 1980). 2. Endoplasmic and sarcoplasmic re t icu lum Sarcoplasmic re t icu lum (SR) of both ske le ta l muscle and card iac muscle c e l l s has been the focus of research on C a 2 + t r anspor t . One of the reasons was the abundant amount of C a 2 + - t r a n s l o c a t i n g ATPase (about 90% of t o t a l p ro te in in ske le ta l SR and 40-50% in card iac SR). This C a 2 + pump i s a p ro te in of about 105 kDa. Several isoforms in ske le ta l muscle have recen t ly been sequenced (MacLennan et a l . , 1985; Brandl et a l . , 1986). The calc ium pump, e i t h e r in r i g h t - s i d e - o u t SR v e s i c l e s or in a r t i f i c i a l phosphol ip id v e s i c l e s , was shown to t rans loca te C a 2 + inwardly at the expense of hydrolyz ing ATP (see MacLennan and Reithmeier , 1985 fo r a rev iew) . The Ca*- -ATPase i s apparently regulated by a small a c i d i c p r o t e i n , phospholamban (Tada et a l . , 1975). I n t e r e s t i n g l y , 8 phosphorylat ion of phospholamban by cAMP-dependent pro te in kinase and a calmodulin-dependent pro te in kinase (Kirchberger et a l . , 1974; Katz and Remtul la , 1978; Le Peuch et al_. , 1979) increased the rate of ATP h y d r o l y s i s and C a 2 + t r a n s l o c a t i o n . Another C a 2 + - b i n d i n g pro te in c a l s e q u e s t r i n (45 kDa) (MacLennan and Wong, 1971), e x i s t s mostly in the terminal c i s t e r n a e of SR and has a high capaci ty f o r complexing C a 2 + (up to 40-60 moles C a 2 + / m o l of c a l s e q u e s t r i n ) . The c i s t e r n a e of SR are anchored to the t ransverse tubule system by f e e t l i k e p r o j e c t i o n s ( t r i a d i c j u n c t i o n s ) . Th is region of SR i s re fe r red to as junc t iona l SR. It turns out that the f e e t l i k e s t ructures are formed by hexameric pro te ins which are a lso known as the calc ium re lease channel (Inui et a l . , 1987a; Imagawa et a l . , 1987; Hymel et a l . , 1988). Each i d e n t i c a l subunit i s a pro te in (360-450 kDa) that binds calmodulin and a lso i s the receptor f o r the p lant a l k a l o i d ryanodine (Inui et a l . , 1987b). The ca lc ium re lease channel i s be l ieved to be respons ib le f o r calc ium-induced ca lc ium re lease from SR (Lai et a l 1 9 8 8 ) . Endoplasmic re t icu lum (ER) of nonmuscle c e l l s a lso conta ins a s i m i l a r Ca~ 2 +-ATPase which t ranspor ts C a 2 + in to the lumen (Moore e t _ a l _ . , 1978), much s i m i l a r to that o f SR. However, the C a 2 + - A T P a s e i s only a minor 9+ component of t o t a l ER p r o t e i n s . Recent ly , the re lease of Ca from endoplasmic re t icu lum has been demonstrated to be induced by the second messenger IP 3 (Prentki et a l . . 1984; Muallem et a l . . 1985). It i s now be l ieved that the IP 3 - induced C a 2 + re lease from ER plays an important r o l e in s igna l t r a n s d u c t i o n . 3. Plasma Membrane 9 The t h i r d s u b c e l l u l a r membrane fo r handling Ca^ i s the plasma membrane. An e lectrochemical gradient of C a z + of 2-3 orders in magnitude l i e s across t h i s l i p i d b i l a y e r . To f a c i l i t a t e passive C a 2 + entry in to the c e l l , at l e a s t three c l a s s e s of C a 2 + channels e x i s t (Spedding, 1987). The L-type channel , which i s vol tage-operated and blocked by agents c a l l e d Ca antagonists (eg. d i h y d r o p y r i d i n e s ) , i s the most studied (Hofmann et a l . , 1987). The other two are the T- type and N-type channels , which are both i n s e n s i t i v e to d i h y d r o p y r i d i n e s . The plasma membrane a lso contains two systems f o r extruding C a 2 + ( C a r a f o l i , 1987): ( i ) N a + / C a z + exchanger, which has been studied mostly in heart and nervous t i s s u e prepara t ions . The exchanger i s a lso present in other c e l l types or t i s s u e s , with the exception of mature erythrocytes (B lauste in and Nelson, 1982, f o r a rev iew) . The pro te in exchanges three moles of Na + f o r one mole C a 2 + and i s e l e c t r o g e n i c . It has a low C a 2 + a f f i n i t y (K m 0.2-10 /iff) but a high maximal v e l o c i t y (>15 nmol/mg p r o t e i n / s ) in hear t . Recent attempts to p u r i f y the exchanger revealed that i t could be a pro te in of 70-82 kDa (Hale et a l . . 1984; B a r z i l a i et a l . , 1984) whi le C a r a f o l i and co-workers (So ldat i et a l . , 1985) proposed that i t s bas ic subunit may be a 33 kDa p r o t e i n . ( i i ) The C a 2 + pumping ATPase of plasma membrane, which i s d i s t i n c t from the SR C a 2 + - A T P a s e , i s present in almost a l l t i s s u e s and c e l l types . A d e t a i l e d account of t h i s prote in w i l l be given below. This pro te in has 10 a higher a f f i n i t y f o r C a 2 + ( K M about 1 JJM or l e s s ) , but lower capaci ty (0.5 nmol of Ca*- /mg membrane p r o t e i n / s ) in heart (Caroni and C a r a f o l i , 1981). In e x c i t a b l e c e l l s ( e . g . heart c e l l s ) such a low capaci ty i s l i k e l y to be complemented by the high capac i ty N a + / C a 2 + exchanger. However, in e ry th rocy tes , where the N a + / C a 2 + exchanger i s absent, the 7+ Ca*- pump alone appears s u f f i c i e n t to maintain the submicromolar r e s t i n g c y t o s o l i c C a 2 + concent ra t ion . Due to the lack of C a 2 + s t o r i n g organel les and N a + / C a 2 + exchanger, the plasma membrane of the ery throcyte becomes an inva luab le system to study the funct ion of the C a 2 + - A T P a s e . III. The plasma membrane Ca 2 + -pumpinq ATPase  1. Overview It was almost three decades ago when Dunham and Glynn (1961) f i r s t reported a (Mg 2 + +Ca 2 + ) -ATPase (Ca 2 + -ATPase) in the red c e l l membrane, which has a c t i v i t y s e v e r a l - f o l d higher than the (Na + +K + ) -ATPase. Subsequently, i t was demonstrated that the red c e l l membrane contains a p. Ca^ pumping mechanism which u t i l i z e s ATP as an energy source to extrude ca lc ium against a chemical and e lectrochemical gradient (Schatzmann, 1966; Schatzmann and V i n c e n z i , 1969). Later on, the a s s o c i a t i o n between the C a 2 + pumping a c t i v i t y and the C a 2 + - A T P a s e a c t i v i t y became c l e a r , based on the f i n d i n g tha t : ( i ) both C a 2 + t ransport and Ca 2 + -dependent ATPase a c t i v i t i e s requi re M g 2 + (Lee and Sh in , 1969; Dunham and Glynn, 1961); ( i i ) both the (Mg 2 + +Ca 2 + ) -ATPase (Watson et a l . . , 1971) and ac t ive C a 2 + 11 t ranspor t (Cha et a l . , 1971) s p e c i f i c a l l y use ATP over other nucleot ide t r iphosphates ; ( i i i ) Strontium ion ( S r 2 + ) can subs t i tu te f o r C a 2 + in the (Mg + +Ca 2 + ) -ATPase a c t i v i t y (Wins and S c h o f f e n i e l s , 1966) and S r 2 + i s t ransported from resealed red c e l l ghosts by a mechanism which i s Mg 2 + - and ATP-dependent (Olson and Cazor t , 1969). Other accounts of the a s s o c i a t i o n of C a 2 + t ranspor t and the C a 2 + - A T P a s e have been reviewed in d e t a i l (Schatzmann, 1969; Roufoga l i s , 1979). Since the d iscovery of the C a 2 + pumping ATPase from plasma membrane of red c e l l , a great deal of work was done on the c h a r a c t e r i z a t i o n of t h i s enzyme in d i f f e r e n t types of membrane prepara t ions , inc lud ing permeable membrane fragments and i n s i d e - o u t resealed v e s i c l e s (see Roufoga l i s , 1979). Considerable d iscrepanc ies between ea r ly repor ts on the k i n e t i c proper t ies of the Ca^ -ATPase ( e s p e c i a l l y with respect to i t s maximum v e l o c i t y and a f f i n i t y f o r C a 2 + ) can now be explained by the fac t that the C a 2 + - A T P a s e i s regulated by many i n t e r a c t i n g f a c t o r s , inc lud ing ATP, M g 2 + , C a 2 + and a prote in a c t i v a t o r , calmodulin (see A l - J o b o r e et a l . , 1984). TWs regu la t ion w i l l be d iscussed l a t e r . A d i f f e r e n t approach to the study of the C a 2 + - A T P a s e was developed by Knauf et a l . , (1974) and Katz and B l o s t e i n (1975) to v i s u a l i z e the plasma membrane-bound C a 2 + - A T P a s e . They demonstrated that micromolar concentrat ions of calc ium induced the formation of a 3 2 P - l a b e l l e d hydroxy lamine-sens i t ive acyl -phosphoprote in (EP) intermediate using [ 7 - 3 2 P ] A T P as the subs t ra te . Upon SDS-polyacrylamide gel e lec t rophores is the EP migrated at a molecular weight of about 150 kDa. The EP reaches a steady s ta te leve l in seconds at 4 -6 °C and i s r a p i d l y turned over (Enyedi et a l . , 1980; A l l e n et a l . , 1987). 12 The formation of EP can be understood by examining the p a r t i a l react ion steps of the enzyme. F i g . 2 i s a scheme f o r the reac t ion c y c l e , compiled from the work of a number of i n v e s t i g a t i n g teams (Rega and Garrahan 1975; Graf and Penniston, 1981; Kosk-Kosicka e t _ a J L , 1986; A l l e n et a l . . 1987; Adamo et a l . , 1988): In the model, the c y c l e normally operates clockwise ( s t a r t i n g from p a r t i a l react ion 1 ) C a 2 + . j n represents c y t o s o l i c f ree C a 2 + pi p I and Ca Q U t i s Ca^ extruded to the e x t r a c e l l u l a r medium. P^  i s inorganic phosphate. Ej and ^ represent two conformational s ta tes of the C a 2 + -ATPase. The t ranspor t c y c l e i s i n i t i a t e d upon the binding of the c y t o s o l i c C a 2 + to a high a f f i n i t y binding s i t e on the cytoplasmic region of the enzyme which i s fol lowed by the binding o f ATP (a lso to the cytoplasmic surface) ( react ion 1) , the ATP i s r a p i d l y s p l i t in to ADP and P^  with the inorganic phosphate now l inked to the enzyme (EjP) v i a the carboxyl group of an aspartate residue at the c a t a l y t i c s i t e ( react ion 2) . Apparent ly , formation of CaEjP does not requi re M g 2 + (Rega and Garrahan, 1975). However, M g 2 + appears to acce lera te EP formation (Rega and Garrahan, 1975; Garrahan and Rega, 1978) probably by a f f e c t i n g the subsequent p a r t i a l reac t ions of the c y c l e . The CaEjP complex then undergoes a conformational change to become CaE2P. This p a r t i a l reac t ion i s thought p. to be concomitant with the t r a n s l o c a t i o n o f Ca^ towards the e x t r a c e l l u l a r s ide of the enzyme (Sarkadi , 1980). At the same t ime, the a f f i n i t y fo r C a 2 + i s reduced by several orders of magnitude (Sarkad i , 1980). Excessive concentrat ions of calcium were found to r e s u l t in a la rge b u i l d up of phosphoprotein (L ichtner and Wolf, 1980; A l l e n et a l . , 1987). This was 13 F i g . 2 Reaction c y c l e of the plasma membrane Ca -ATPase. During a normal c y c l e , the reac t ions proceed c lockwise . Ca represents the calc ium i o n . C a ^ n and Ca o u ^ . represent Ca^ in the cy toso l and the e x t r a c e l l u l a r space, r e s p e c t i v e l y . Ej and E2 represent the two d i f f e r e n t conformational forms of the enzyme. Requirements of other l igands in p a r t i a l reac t ions are ind icated in brackets . See text f o r d e t a i l s of i n d i v i d u a l p a r t i a l r e a c t i o n s . 14 C a 2 * in ATP • 1 ^ A (1a) C a E t (1b) ADP C a ^ A T P ^ C a E i P (2) ( M g 2 + ) ? ( M g 2 + ) / (5) E o P (4) (3) V (ATP) C a E o P Pi Ca 2+ out a t t r i b u t e d to i n h i b i t i o n of the conversion from CaEjP to CaE2P. In the same C a z + concentra t ion range i n h i b i t i o n of C a 2 + - A T P a s e was a lso observed (A l len et a l . , 1987). The conversion between the CaE 2 P state to the E 2 s ta te ( react ion 4 and 5) involves at l e a s t two s teps: ( i ) the re lease of C a 2 + from the low a f f i n i t y s i t e to the e x t r a c e l l u l a r medium fol lowed by ( i i ) the rapid react ion o f E 2 P with water, forming E 2 and inorganic phosphate (P^). The rate of step ( i i ) apparently i s st imulated by mM concentra t ions of ATP (Rega and Garrahan, 1978). A p p l i c a t i o n of lanthanum ion ( L a 3 + ) to the e x t r a c e l l u l a r s ide of erythrocytes i n h i b i t s the C a z + - A T P a s e and causes EP accumulation immediately (Szasz et a l . , 1978). Th is suggests that L a 3 + might bind to E 2 P ( react ion 5) r igh t a f te r C a 2 + i s r e l e a s e d , thereby s t a b i l i z i n g the enzyme in the E 2 P s t a t e . The f i n a l p a r t i a l reac t ion i s the conversion of the E 2 s tate (low C a 2 + a f f i n i t y ) back to the Ej s tate (high a f f i n i t y f o r C a 2 + ) . Schatzmann (1985) proposed that M g 2 + acce lera tes the rate of E 2 to Ej conversion s i m i l a r to the e f f e c t of M g 2 + on the CaEjP to CaE 2 P convers ion . Calmodulin apparently enhances the rate and the C a 2 + a f f i n i t y of C a 2 + t ranspor t and ATP hydro lys is (Al -Jobore et a l . . , 1984 f o r a rev iew) . However, l i t t l e i s known regarding the regulatory s i t e ( s ) f o r calmodulin in the r e a c t i o n c y c l e (Enyedi et a l . , 1980; Rega and Garrahan, 1980). Adamo et a l . (1988) recent ly suggested that calmodulin might s t a b i l i z e E j , which may exp la in the enhancement of a f f i n i t y fo r C a 2 + . 16 2. P u r i f i c a t i o n and r e c o n s t i t u t i o n In order to fur ther understand the C a - A T P a s e , there was a need to s o l u b i l i z e and p u r i f y the enzyme. S o l u b i l i z a t i o n of the a c t i v e enzyme from human erythrocyte membranes was f i n a l l y achieved by Wolf and Gietzen (1974). However, subject ing the s o l u b i l i z e d C a 2 + - A T P a s e to fu r ther p u r i f i c a t i o n steps resu l ted in i n a c t i v a t i o n . Th is problem was solved by the add i t ion o f sonicated phosphol ip ids (Wolf et a l . , 1977). However, the u n a v a i l a b i l i t y of a s p e c i f i c means to separate the enzyme from the remaining 99.7% or so of other membrane prote ins made the p u r i f i c a t i o n progress slow. The d iscovery that calmodulin r e v e r s i b l y ac t i va ted both the membrane-bound form and the s o l u b i l i z e d form of the C a 2 + - A T P a s e (Lynch and Cheung, 1978; N igg l i et a l . , 1979a) suggested the use o f ca lmodul in -a f f i n i t y chromatography to pur i fy the C a 2 + - A T P a s e . N i g g l i et a l . (1979b) and Gietzen et a l . (1980) reported almost ly s imultaneously the successfu l p u r i f i c a t i o n of the C a 2 + - A T P a s e using t h i s method. Subsequently, by using phosphat idy lchol ine instead of phosphat idyl ser ine during p u r i f i c a t i o n , the C a 2 + - A T P a s e thus p u r i f i e d was found to e x i s t in a low C a 2 + - a f f i n i t y form and could be converted to the h i g h - C a 2 + a f f i n i t y form by the add i t ion of calmodulin (Niggl i et a l . , 1981a). Th is confirmed the e a r l i e r reports that the ghost membrane-bound C a 2 + - A T P a s e ex is ted in both a high a f f i n i t y and a low a f f i n i t y s tate (Schatzmann and R o s s i , 1971; Quist and Roufoga l i s , 1975). Since Ca^ t ransport i s the actual p h y s i o l o g i c a l func t ion of the p • C a - A T P a s e , t h i s funct ion of the enzyme has long been the focus of 17 e f f o r t s in t h i s f i e l d . E a r l i e r work studying the C a ^ + t ransport funct ions was achieved using resealed erythrocyte plasma membrane i n s i d e -out v e s i c l e s (IOV). Waisman et a l . (1981a, b, c) and Gimble et a l . (1982) have studied the mechanism of C a 2 + - t r a n s p o r t by human ery throcyte IOV in great d e t a i l . A f t e r the s o l u b i l i z a t i o n of C a 2 + - A T P a s e was achieved, attempts were made to recons t i tu te i t into proteoliposomes (Haaker and Racker, 1979; Gietzen et a l . . 1980). Soon a f t e r the a v a i l a b i l i t y of h igh ly p u r i f i e d C a 2 + - A T P a s e fol lowed the r e c o n s t i t u t i o n of t h i s enzyme into proteol iposomes and i t s c h a r a c t e r i z a t i o n (N igg l i et al_. , 1981b; N i g g l i et a l . , 1982b; V i l l a l o b o and Roufoga l is , 1986). Another approach to inves t iga te the C a 2 + pump i s to study the C a 2 + -ATPase a c t i v i t y as well as C a 2 + e f f l u x from i n t a c t e r y t h r o c y t e s . E a r l i e r s tudies appeared to y i e l d c o n f l i c t i n g r e s u l t s : some reported that the C a 2 + pump funct ioned with low C a 2 + a f f i n i t y (Romero and Whittam, 1971; Sarkadi et a l . , 1977; Burgin and Schatzmann, 1979), while others demonstrated that the C a 2 + pump was in a high C a 2 + a f f i n i t y mode, suggesting that the C a 2 + pump was assoc ia ted with calmodulin (Muallem and K a r l i s h , 1982; F e r r e i r a and Lew, 1976; Schar f f et a l . . 1983). Recent ly , T i f f e r t et a l . (1984) demonstrated that coba l t ion can ar res t passive calc ium t ranspor t by the i n t a c t e ry throcytes without a f f e c t i n g calc ium e f f l u x by the C a 2 + pump. Taking advantage of t h i s technique (cobalt -exposed calc ium e f f l u x ) , the p roper t i es of the C a 2 + pump in in tac t red c e l l s were re-examined in d e t a i l by Xu and Roufogal is (1988a, b) and by Dagher and Lew (1988). Xu and Roufogal is (1988b) demonstrated that both C a 2 + e f f l u x and C a 2 + - A T P a s e have absolute dependence on mM concentrat ion of M g 2 + . The C a 2 + - A T P a s e a c t i v i t y 18 showed a K n 5 fo r ATP of 0 . 5 mM and K Q 5 fo r C a ^ + of 0 . 2 - 0 . 4 /iM when the ATP concentrat ion was between 0 .2 to 1.3 mM (Xu and Roufoga l i s , 1989a, 1989b). However, the K 0 - 5 ( C a ) f o r C a 2 + e f f l u x (30-40 /imol/L c e l l s ) was s i g n i f i c a n t l y higher than K N . 5 ( C a ) f o r C a 2 + - A T P a s e a c t i v i t y ( 0 . 2 - 0 . 4 /JM) in the in tac t c e l l s . The authors suggested a number of p o s s i b l e reasons f o r the discrepancy inc lud ing the d i f f i c u l t y in accura te ly est imat ing the f ree C a 2 + concentrat ions ins ide the c e l l s dur ing the C a 2 + e f f l u x experiments. Concurrent ly , Dagher and Lew (1988) showed that C a 2 + e f f l u x and calcium-dependent ATP h y d r o l y s i s by the cobal t -exposed in tac t c e l l s have a sto ichiometry of 1:1. 3. Regulations ( i ) Phosphol ip id Phosphol ip id i s required during p u r i f i c a t i o n to s t a b i l i z e the Ca^ -ATPase (d iscussed above). In a d d i t i o n , phosphol ip id i s a lso required fo r C a 2 + - A T P a s e a c t i v i t y (Ronner et al_. , 1977; Roelofsen and Schatzmann, 1977). Recent ly , Kosk-Kosicka and Inesi (1986) demonstrated tha t , using a combination of the detergent C ^ E g ( instead of T r i t o n X-100) and g l y c e r o l , an ac t ive C a 2 + - A T P a s e can be p u r i f i e d in the absence of p h o s p h o l i p i d . I n t e r e s t i n g l y , t h i s preparat ion of the s o l u b i l i z e d and p u r i f i e d C a 2 + - A T P a s e shows an enzyme concentrat ion-dependent a c t i v a t i o n that mimics that seen in the presence of calmodulin (Kosk-Kosicka and Bzdega, 1988). These authors suggested that the e f f e c t was due to s e l f -a s s o c i a t i o n of the enzyme. 19 ( i i ) ATP Calcium t ranspor t by the C a 2 + pump i s coupled to the energy-producing h y d r o l y s i s of ATP. The enzyme has a s t r i c t s p e c i f i c i t y f o r ATP over other nucleoside t r iphosphates (GTP, CTP, ITP, UTP) (Sarkadi et a l . , 1979; Gra f f et a l . , 1982). ATP st imulates both calc ium t ranspor t and the C a 2 + - A T P a s e (Muallem and K a r l i s h , 1979) in a b iphas ic manner ( K ( A j p j j = 1-7 x 10" 6 M and K ( A T p j 2 = 1-3 x 10" 4 M) (Muallem and K a r l i s h , 1979; V i l l a l o b o et a l . . , 1986). The high a f f i n i t y b inding s i t e f o r ATP i s considered to be the c a t a l y t i c s i t e , while the lower a f f i n i t y s i t e could al low mM concentrat ions of ATP to modulate the a c t i v i t y of the pump (Muallem and K a r l i s h , 1979). Various forms of ATP, inc lud ing MgATP, CaATP and f ree ATP have been suggested to be the actual substrate of the enzyme (Wolf et a l . , 1977; Enyedi et a l . , 1982; Sarkadi et a l . , 1978; Graf and Penniston, 1981). More r e c e n t l y , Muallem and K a r l i s h (1981) suggested that MgATP, CaATP and f ree ATP are equa l ly e f f e c t i v e as subst ra te . To date , no c l e a r - c u t consensus e x i s t s on what form of ATP i s the actual p h y s i o l o g i c a l substrate of the enzyme. ( i i i ) D i v a l e n t ca t ions M g 2 + has several regulatory funct ions on the C a 2 + - A T P a s e . As descr ibed before , M g 2 + s t imulates the dephosphorylat ion step of the enzyme (Sarkad i , 1980). M g z + may a lso be required to complex with ATP s ince Mg-ATP might be the pre fer red p h y s i o l o g i c a l substrate f o r the pump (Enyedi p , et a l . , 1982). M i l l i m o l a r concentrat ions of Mg decreases both the V m a x 20 and the a f f i n i t y fo r C a 2 + of the ery throcyte ghost membrane-bound form (K l inger et a l . , 1980; A l -Jobore and Roufoga l i s , 1981), the s o l u b i l i z e d form (Al -Jobore and Roufoga l is , 1981), the i n s i d e - o u t membrane v e s i c l e -bound form (Akyempon and Roufoga l is , 1982) and the p u r i f i e d form of the enzyme ( V i l l a l o b o et a l . , 1986). I n i t i a l l y , the enzyme was thought to have absolute dependence on M g 2 + f o r a c t i v i t y (Schatzmann, 1975). Later s tud ies showed that a small por t ion of C a 2 + - s t i m u l a t e d ATPase a c t i v i t y e x i s t s in the absence of magnesium (Richards et a l . , 1978). These authors a lso found that M g 2 + enhanced the s t imulatory e f f e c t of C a 2 + without a l t e r i n g the a f f i n i t y fo r C a 2 + or ATP. During the reac t ion c y c l e , M g 2 + acce le ra tes the formation of the phosphorylat ion and dephosphorylat ion of the enzyme (Garrahan and Rega, 1978), although M g 2 + i s not absolute ly required f o r the EP formation (A l len et a l . , 1987). ( i v ) C a 2 + / c a l m o d u l i n The funct ion of the C a 2 + pump i s to t r a n s l o c a t e C a 2 + from the cytosol to the e x t r a c e l l u l a r space. During the r e a c t i o n c y c l e t h i s t r a n s l o c a t i o n o f - C a 2 + and the hydro lys is of ATP are c l o s e l y coupled . Because of t h i s , the phosphoprotein intermediate formation has an absolute dependence on C a 2 + ( C a r a f o l i and Z u r i n i , 1982). The existence of high and low C a 2 + a f f i n i t y C a 2 + - A T P a s e a c t i v i t i e s has been suggested in e a r l y s tudies (Schatzmann and R o s s i , 1971; Schar f f , 1976). Quist and Roufogal is (1975) demonstrated that ex t rac t ion of erythrocyte membranes with EDTA converted the (Ca 2 + +Mg 2 + ) -ATPase from a high C a 2 + a f f i n i t y s ta te to a low C a 2 + a f f i n i t y s ta te and that the readdi t ion of the EDTA-extract of the membrane p I 9 . res tored the high Ca*- a f f i n i t y of the Cac -ATPase a c t i v i t y . This 21 suggested the presence of an a c t i v a t i n g f a c t o r that i s respons ib le fo r t h i s convers ion . Subsequently, J a r r e t t and Penniston (1977a) and Luthra et a l . (1977) both p a r t i a l l y p u r i f i e d the a c t i v a t i n g f a c t o r . Independently, the d iscovery of calmodulin as an a c t i v a t o r of c y c l i c nuc leot ide phosphodiesterase surfaced (Cheung, 1970; Kakiuchi and Yamazaki, 1970). Shor t ly af terward, the l a b o r a t o r i e s of Penniston and Vincenzi simultaneously r e a l i z e d that the a c t i v a t o r p ro te in of the C a z + -ATPase was calmodulin (Gopinath and V i n c e n z i , 1977; J a r r e t t and Penniston, 1977b). The binding of calmodulin to the membrane-bound C a 2 + - A T P a s e r e s u l t s in a 3-4 f o l d increase in maximum v e l o c i t y and up to a 30 - fo ld increase in C a 2 + a f f i n i t y (Roufogal is and Mauldin , 1980; Foder and S c h a r f f , 1981; Schar f f and Foder, 1982). Subsequently, a s i m i l a r s t imulatory e f f e c t of calmodulin on the s o l u b i l i z e d and p u r i f i e d C a 2 + -ATPase was demonstrated (Al -Jobore and Roufoga l i s , 1981; N i g g l i et a l . , 1981a; V i l l a l o b o et a l . , 1986). The studies of Sarkadi and coworkers s t rong ly suggested that the ca lmodul in -b inding domain is near the C-terminal of the C a 2 + - A T P a s e (Sarkadi et a l . , 1986). The l o c a t i o n of the ca lmodul in -b ind ing domain at the C-terminal has recent ly been confirmed from the amino ac id sequence of the enzyme (Shull and Greeb, 1988; Verma et a l . , 1988) ( F i g . 3 ) . A l s o , a puta t ive i n h i b i t o r y domain located next to and upstream from the ca lmodul in -b ind ing domain has been suggested (Cara fo l i et a l . , 1987). It was be l ieved that in the absence of ca lmodul in , the i n h i b i t o r y domain imposes i n h i b i t i o n on the ATP-hydro ly t i c and C a 2 + t ranspor t a c t i v i t i e s of the enzyme. Upon binding of the C a 2 + - c a l m o d u l i n complex, the ca lmodul in-22 F i g . 3 Amino ac id sequence of the plasma membrane C a - A T P a s e from a human  teratoma. The sequence is continuous from the N-terminal to the C-t e r m i n a l , using the o n e - l e t t e r codes (see appendix) f o r i t s amino ac id r e s i d u e s . The number at the begining of each l i n e represents the residue number o f the f i r s t amino acid of that l i n e . The l e t t e r P i d e n t i f i e s the l o c a t i o n o f acylphosphate s i t e . The ca lmodul in -b ind ing domain i s i d e n t i f i e d as i n d i c a t e d . Amino ac id sequence s t a r t s from the N- terminal . Modi f ied from Verma et al . (1988) . 23 Figure 3 1 MGDMANNSVAYSGVKNSLKEANHDGDFGITLAELRALMELRSTDALRKIQ 51 ESYGDVYGICTKLKTSPNEGLSGNPADLERREAVFGKNFIPPKKPKTFLQ 101 LVWEALQDVTLIILEIAAIVSLGLSFYQPPEGDNALCGEVSVGEEEGEGE 151 TGWIEGAAILLSVVCVVLVTAFNDWSKEKQFRGLQSRIEQEQKFTVIRGG 201 QVIQIPVADITVGDIAQVKYGDLLPADGILIQGNDLKIDESSLTGESDHV 251 KKSLDKDPLLLSGTHVREGSGRMVVTAVGVNSQTGIIFTLLGAGGEEEEK 301 KDEKKKEKKNKKQDGAIENRNKAKAQDGAAMEMQPLKSEEGGDGDEKDKK 351 KANLPKKEKSVLQGKLTKLAVQIGKAGLLMSAITVIILVLYFVIDTFWVQ 401 KRPWLAECTPIYIQYFVKFFIIGVTVLVVAVPEGLPLAVTISLAYSVKKM P 451 MKDNNLVRHLDACETMGNATAICSDKTGTLTMNRMTVVQAYINEKHYKKV 501 PEPEAIPPNILSYLVTGISVNCAYTSKILPPEKEGGLPRHVGNKTECALL 551 GLLLDLKRDYQDVRNEIPEEALYKVYTFNSVRKSMSTVLKNSDGSYRIFS 601 KGASEIILKKCFKILSANGEAKVFRPRDRDDIVKTVIEPMASEGLRTICL 651 AFRDFPAGEPEPEWDNENDIVTGLTCIAVVGIEDPVRPEVPDAIKKCQRA 701 GITVRMVTGDNINTARAIATKCGILHPGEDFLCLEGKDFNRRIRNEKGEI 751 EQERIDKIWPKLRVLARSSPTDKHTLVKGIIDSTVSDQRQVVAVTGDGTN 801 DGPALKKADVGFAMGIAGTDVAKEASDIILTDDNFTSIVKAVMWGRNVYD 851 SISKFLQFQLTVNVVAVIVAFTGACITQDSPLKAVQMLWVNLIMDTLASL 901 ALATEPPTESLLLRKPYGRNKPLISRTMMKNILGHAFYQLVVVFTLLFAG 951 EKFFDIDSGRNAPLHAPPSEHYTIVFNTFVLMQLFNEINARKIHGERNVF 1001 EGIFNNAIFCTIVLGTFVVQIIIVQFGGKPFSCSELSIEQWLWSIFLGMG L 1051 TLLWGQLISTIPTSRLKFLKEAGHGTQKEEIPEEELAEDVEEIDHAEREL CaM-bindinq domain 1 1101 RRGQILWFRGLNRIQTQIRVVNAFRSSLYEGLEKPESRSSIHNFMTHPEF 1151 RIEDSEPHIPLIDDTDAEDDAPTKRNSSPPPSPNKNNNAVDSGIHLTIEM 1201 NKSATSSSPGSPLHSLETSL 1220 24 binding domain undergoes conformational changes, which in turn induce conformational changes in the i n h i b i t i n g domain. Subsequently, the i n h i b i t i o n of the enzymatic a c t i v i t y i s re leased and the C a 2 + pump now funct ions at f u l l c a p a c i t y . N i g g l i et a l . , (1981b) and A l - J o b o r e and Roufogal is (1981) demonstrated that a c i d i c phosphol ip ids ( e . g . phosphatidyl s e r i n e ) , but not neutral phosphol ip ids ( e . g . p h o s p h a t i d y l c h o l i n e ) , can mimic the a c t i v a t i n g e f f e c t of ca lmodul in . A s i g n i f i c a n t d i f f e r e n c e appears to e x i s t between the ac t ion of the two a c t i v a t o r s , however: while calmodulin s h i f t s the KQ 5(ca) t o 0 .5-0 .6 /zM, a c i d i c phosphol ip ids s h i f t the Ko .5(Ca) t o 0 , 2 5 ^ M (Enyedi et a l . , 1987). A l s o , the Ca -dependence of calmodulin a c t i v a t i o n i s sigmoidal whi le that of a c i d i c phosphol ip ids i s h y p e r b o l i c . Minocherhomjee et a l . (1982) a lso found that synthe t ic polyanions such as p o l y ( L ) - a s p a r t i c ac id increased the C a 2 + a f f i n i t y of the C a 2 + - A T P a s e but not i t s V m a x . I n t e r e s t i n g l y , l i m i t e d p r o t e o l y s i s by t r y p s i n or chymotrypsin a lso ac t iva tes the C a 2 + - A T P a s e a c t i v i t y and C a 2 + t ransport a c t i v i t i e s and rendered these a c t i v i t i e s calmodulin- independent (Taverna and Hanahan, 1980; Sarkadi et a l . . 1980; A l - J o b o r e and Roufoga l i s , 1981). The p r o t e o l y s i s of the C a 2 + - A T P a s e by t r y p s i n has been studied in great d e t a i l (Zur in i et a l . . 1984; Benaim et a l . , 1984; Sarkadi et a l . , 1986; Sarkadi et a l . , 1987; Enyedi et a l . , 1987; Olorunsogo et a l . , 1988). The l a b o r a t o r i e s of C a r a f o l i and Sarkadi found that a 90 kDa fragment can s t i l l bind and be st imulated by calmodulin while an 85 kDa fragment can bind but i s not st imulated by, ca lmodul in . Smaller 81 kDa and 76 kDa fragments could not bind or be st imulated by, ca lmodul in . I n t e r e s t i n g l y , 25 the 81 kDa fragment but not the 76 kDa fragment s t i l l responded to a c t i v a t i o n by a c i d i c phosphol ip id ( i . e . a s h i f t to lower K m f o r C a 2 + ) . Recent ly , a model of p r o t e o l y s i s of the C a 2 + - A T P a s e by t r y p s i n was presented by C a r a f o l i et a l . (1987) ( F i g . 4 ) . In t h e i r model, t r y p s i n c leaves at the N-terminal and produces a 90 kDa ac t ive fragment. A subsequent cleavage at the C-terminal that removes the ca lmodul in -b ind ing domain produces the 85 kDa fragment which binds but cannot be ac t iva ted by calmodulin (Zur in i et a l . , 1984; Benaim et a l . , 1984). A fu r ther cleavage removes the putat ive i n h i b i t o r y domain to produce the ac t iva ted and non-calmodul in-b inding form. The l a s t cleavage formd an a c i d i c phosphol ip id - and calmodulin- independent 76 kDa ac t ive fragment (the smal lest EP forming fragment) ( F i g . 4 ) . ( v ) M o n o v a l e n t c a t i o n s Sodium i o n , potassium ion and other monovalent ca t ions were found to ac t iva te both the membrane-bound and p u r i f i e d C a 2 + - A T P a s e (Schatzmann and R o s s i , 1971; Graf et a l . , 1982). Recent s tudies (Romero and Romero, 1984) using resealed ghosts in an a l l - c h o l i n e medium demonstrated that Na + from ins ide and K + from e i t h e r s ide of the membrane produced a b i f u n c t i o n a l ( i n h i b i t o r y - s t i m u l a t o r y ) e f f e c t on C a 2 + e x t r u s i o n , depending on the K + concent ra t ion . Sodium ion from outside in h i g h - c h o l i n e loaded ghosts and K + from outs ide in h i g h - K + loaded ghosts e l i c i t e d only s t imulatory e f f e c t s (Romero and Romero, 1984). The authors concluded that both ions may a f f e c t the pump by two d i s t i n c t mechanisms: ( i ) the e l e c t r i c coupl ing between C a 2 + e f f l u x and external ca t ion ( K + , N a + ) , ( i i ) an in f luence on molecular react ions of the C a 2 + - A T P a s e (see below). The 26 F i g . 4 Schematic fragmentation o f the Ca -ATPase produced by t r y p s i n . The r e l a t i v e molecular weight of each polypept ide i s ind ica ted by a number. The dotted area represents the ca lmodul in -b ind ing domain while the shaded area at the end of the molecule represents a domain required fo r c a l m o d u l i n - a c t i v a t i o n . The ac t ive s i t e capable of forming the acylphosphate i s ind icated by the shaded area in the middle of the molecule. Taken from C a r a f o l i et a l . (1987). 27 Figure 4 38 Short trypsin exposure, 0 1 /7 trypsin-enzyme rotfo s h o r t t r y p s i n e x p o s u r e in C a 2 ' - c a l m o d u l i n . 37° . 1 / 2 5 t r y p s i n - e n z y m e r a t i o 8 5 s h o r t t r y p s i n e x p o s u r e i n • Y0«"-n<TJ\ 3 7 ° , 1 / 2 5 -JL t r y p s i n - e n z y m e r a t i o l o n g t r y p s i n e x p o s u r e i n E G T A . 37 ° . l / 2 5 t r y p s i n -e n z y m e r a t i o Q "re; V ) 28 second mechanism i s supported by that fac t that high in terna l K enhances C a 2 + a f f i n i t y in h i g h - c h o l i n e ghosts (Romero and Romero, 1984) and that K + increased both the l eve l and turnover of the E-P intermediate (La Rocca et a l . , 1981). I n i t i a l l y the Ca pump was thought not to counter - t ranspor t monovalent or d i v a l e n t ca t ions and therefore would be e lec t rogen ic (due to the net p o s i t i v e charge c a r r i e d by C a 2 + e x t r u s i o n ) . It was thought that e l e c t r o n e u t r a l i t y of the c e l l was restored by net CI" e f f l u x , v i a the anion channel (band 3) of the plasma membrane (see Penniston, 1983). The evidence f o r t h i s came from reports that 4-acetamido-4-i s o t h i o c y a n o s t i l b e n e - 2 - d i s u l f o n a t e (SITS) and N- (4 -az ido-2 -n i t ropheny l )-2-aminoethylsulfonate (NAP-taurine) blocked both C a 2 + t ranspor t and anion t ranspor t with s i m i l a r potency (Waisman et a l . , 1981c). The authors in terpre ted these r e s u l t s as i n d i c a t i n g that the two t ranspor t processes were c l o s e l y coupled . However, Minocherhomjee and Roufogal is (1982) and N i g g l i et a l . (1982a) subsequently showed that NAP-taurine and 4 , 4 ' -d f i s o t h i o c y a n o - 2 - 2 ' - s t i b e n e d i s u l f o n a t e (DIDS), another anion i n h i b i t o r re la ted to SITS, a lso d i r e c t l y i n h i b i t the C a 2 + - A T P a s e . These l a t t e r s tudies cast doubt on the i n t e r p r e t a t i o n o r i g i n a l l y proposed by Waismann et a l . (1981c). On the other hand, Smallwood et a l . (1983) demonstrated that DIDS treatment of whole erythrocytes i n h i b i t e d ATP-dependent C a 2 + t r a n s p o r t . S i n c e , under t h e i r c o n d i t i o n s , DIDS treatment d id not s i g n i f i c a n t l y a l t e r i t s Ca^ -ATPase a c t i v i t y , i t was suggested that a c l o s e - c o u p l i n g e x i s t s between band 3 and the C a 2 + - A T P a s e and that the C a 2 + - A T P a s e ca ta lyzes a C a 2 + : H + exchange. Using 1 iposome-reconst i tuted 29 Ca -ATPase, N i g g l i et a l . (1982b) demonstrated that two extra protons (H +) were found in the medium per ATP hydrolyzed. Based on the assumption that one Ca^ i s pumped into the liposomes per ATP hydrolyzed (N igg l i et a l . , 1981a, N i g g l i et a l . , 1981b) they in terpre ted the r e s u l t s as evidence that the C a 2 + pump i s a C a 2 + - 2 H + an t ipor t that is e lec t roneut ra l ( N i g g l i , 1982b). Using ionophoret ic compounds, V i l l a l o b o and Roufogal is (1986) a lso showed that the C a z + pump counter - t ranspor ts protons. Recent ly , the C a t T pump from both the red c e l l i n s i d e - o u t membrane v e s i c l e s and card iac sarcolemma have been considered to be modulated by membrane potent ia l (Gassner et a l . , 1988; Kuwayama, 1988; Romero and O r t i z , 1988). The r e s u l t s of these s tudies suggested that the C a 2 + pump i s e l e c t r o g e n i c by v i r t u e of a s to ich imetry of 1 C a 2 + : 1H + . (v i ) Phosphorylat ion The laboratory of C a r a f o l i demonstrated that the C a 2 + - A T P a s e from heart sarcolemma and from erythrocyte membrane are both phosphorylated by cAMP-dependent pro te in kinase (Caroni and C a r a f o l i , 1981; Neyses et a l . , 1985). cAMP-dependent phosphorylat ion of the C a 2 + pump from both the heart and the red c e l l appeared to enhance the C a 2 + a f f i n i t y but does not appear to a f f e c t V m a x (Lamers et al_. , 1981; Neyses et al_. , 1985). Subsequently, the plasma membrane C a 2 + pump p u r i f i e d from vascu la r smooth muscle (bovine aorta) was shown to be st imulated by cGMP-dependent prote in kinase (Furukawa and Nakamura, 1987; Furukawa et al_. , 1988). The 7+ phosphorylat ion was p a r a l l e l e d by an increase in both Ca^ a f f i n i t y and p, p i maximum Ca^ uptake by the reconst i tu ted Ca pump. However, recent studies suggested that the s t imula t ion of the C a 2 + - A T P a s e induced by cGMP-30 dependent phosphorylat ion could be i n d i r e c t (Baltensperger et a l . , 1988; V r o l i x et a l . , 1988). Most r e c e n t l y , both the membrane-bound and the p u r i f i e d forms of the human erythrocyte C a 2 + - A T P a s e were found to be st imulated by prote in kinase C (Smallwood et a l . , 1988). However, a d i r e c t demonstration of phosphorylat ion of the C a 2 + - A T P a s e prote in by p ro te in kinase C was not repor ted . ( v i i ) Other regu la tors Besides ca lmodul in , another prote in a c t i v a t o r of the C a 2 + - A T P a s e was found in human erythrocyte membranes (Mauldin and Roufoga l i s , 1980). This pro te in was charac te r i zed as a 56 kDa band on SDS-gel e l e c t r o p h o r e s i s and to e lu te as a 107-178 kDa prote in on gel f i l t r a t i o n (Roufogal is et a l . , 1984). The a c t i v a t o r prote in a lso contained ca lmodul in . It was suggested that the a c t i v a t o r prote in was a ca lmodul in -b ind ing pro te in with calmodulin bound to i t in a Ca 2 + - independent manner. There fore , i t i s qu i te p o s s i b l e that the s t imulatory e f f e c t of t h i s a c t i v a t o r on the C a 2 + -ATPase was produced by the calmodulin molecules bound to t h i s p r o t e i n . Several endogenous pro te in i n h i b i t o r s of the erythrocyte C a 2 + - A T P a s e have a lso been reported (Au, 1978; Pedemonte and Balegro , 1981; Wuthrich, 1982). These prote ins appeared to be of small molecular weight (6 kDa to 20 kDa), but t h e i r modes of i n h i b i t i o n of the C a z + - A T P a s e d i f f e r . Only the i n h i b i t o r reported by Au was fu r ther charac te r i zed (Lee and Au, 1983; Au et a l . . , 1985). Since t h i s i n h i b i t o r a lso appeared to i n h i b i t ca lmodu l in -ac t iva ted adenylate cyc lase and cAMP-phosphodiesterase (Au, 1978), the exact funct ion and s p e c i f i c i t y of t h i s i n h i b i t o r i s unc lear . The erythrocyte membrane C a 2 + - A T P a s e a c t i v i t y was a lso st imulated by 31 thyro id hormones ( T 3 and T 4 ) (Davis , 1981) while r e t i n o i c ac id can i n h i b i t the basal and T^-st imulated a c t i v i t y of t h i s enzyme (Smith et a l . , 1989). The heart sarcolemma! C a 2 + pump was a lso found to be st imulated by N-methylat ion of phosphatidylethanolamine (Panagia et a l . , 1986) i n h i b i t e d by cho les te ro l (Ortega and Mas-O l iva , 1986) and by guanine nucleot ide and i n o s i t o l t r isphosphate (Kuo and Tsang, 1988). The erythrocyte C a 2 + - A T P a s e was p a r t i a l l y i n h i b i t e d by ox ida t ive agents and ac t iva ted oxygen (Lec lerc et a l . , 1987; Hebbel et a l . , 1986). A l s o , i n s u l i n and concanaval in A a lso i n h i b i t e d the adipocyte plasma membrane Ca -ATPase (Pershadsingh and McDonald, 1981). 7+ The most con t rovers ia l plasma membrane Ca^ -ATPase enzymes appeared 7+ to be those reported in the hepatocytes. F i r s t , a high a f f i n i t y (Ca -Mg 2 + ) -ATPase was found to be calmodulin- independent (Lotersz ta jn et a l . , 1981). Lotersz ta jn et a l . (1984) a lso reported on the c h a r a c t e r i z a t i o n of an ATP-dependent C a 2 + pump of l i v e r plasma membrane, which formed a phosphoprotein intermediate of 110 kDa. An i n h i b i t o r y prote in (30 kDa) that mediated the i n h i b i t i o n of the l i v e r ( C a 2 + - M g 2 + ) - A T P a s e by glucagon arfd M g 2 + was p u r i f i e d (Loterszta jn et a l . , 1985) and was suggested to be a G - p r o t e i n , s i m i l a r to a type c a l l e d G s . Th is i s based on the f a c t that cho lera t o x i n , which normally permanently ac t i va tes the G s -mediated s ignal 7+ t r a n s d u c t i o n , blocked the glucagon-mediated i n h i b i t i o n of the Ca^ -ATPase (Lotersz ta ju et al_., 1987). L in and Fain (1984) a lso i s o l a t e d a high a f f i n i t y ( C a 2 + - M g 2 + ) - A T P a s e . Subsequently, L in (1985a, b) claimed that t h i s ( C a 2 + - M g 2 + ) - A T P a s e (70 kDa) was not a C a 2 + pump, based on k i n e t i c d i f f e r e n c e s between ATP h y d r o l y s i s and C a 2 + t r a n s p o r t . L in a lso reported a vanadate - inh ib i tab le phosphoprotein of 118 kDa, which was suggested to 32 be the authent ic plasma membrane ATP-dependent Ca^ pump of hepatocytes ( L i n , 1985a). She a lso suggested that the ATP-hydrolyz ing a c t i v i t y of the non-Ca - t r a n s p o r t i n g ATPase, an ecto-ATPase, is about 10 times that of the Ca 2 + -pumping ATPase (Lin 1985a, b ) . Recent ly , L in (1988) reported that the l i v e r plasma membrane ( C a 2 + - M g 2 + ) - A T P a s e was in f a c t a plasma membrane ecto-ATPase which possessed an e x t r a c e l l u l a r l y located ac t ive s i t e f o r ATP h y d r o l y s i s . Birch-Machin and Dawson (1988) reported that a l i v e r plasma membrane C a 2 + - A T P a s e that they observed had d i f f e r e n t k i n e t i c proper t ies from those involved in ATP-dependent C a 2 + t r a n s p o r t . On the other hand, using l i v e r plasma membrane v e s i c l e s , the laboratory of Lo tersz ta jn demonstrated that glucagon and a glucagon fragment i n h i b i t e d both C a 2 + t ranspor t and the ( C a 2 + - M g 2 + ) - A T P a s e a c t i v i t y (Lotersz ta jn et a l . , 1984; Mall at et a l . . 1987). This would suggest that the ( C a 2 + - M g 2 + ) -9+ ATPase and the Cac pump that they charac ter i zed are the same p r o t e i n . There fore , i t i s p o s s i b l e that the ( C a 2 + - M g 2 + ) - A T P a s e reported by the group of Lo te rsz ta jn and that reported by Lin are two d i f f e r e n t p r o t e i n s . The i d e n t i t y of the C a 2 + - A T P a s e reported by Birch-Machin and Dawson (T988) i s a lso unc lear . IV. Calmodulin and ca lmodul in- regulated systems Since i t s d iscovery (Cheung, 1970; Kakiuchi and Yamazaki, 1970) the p ivo ta l r o l e of calmodulin in Ca 2 + -med ia ted c e l l u l a r regu la t ion has become apparent (Cheung, 1980; Klee and Vanaman 1982; Manalan and K lee , 33 1984; Wang et a l . . , 1985). Calmodulin is present in v i r t u a l l y a l l eukaryot ic organisms and in a l l c e l l types (Klee and Vanaman, 1982). It i s a s m a l l , a c i d i c pro te in (16.5 kDa). The amino ac id sequence from many species has been determined and an excep t iona l l y high degree of conservat ion was demonstrated (see Manalan and K lee , 1984). Calmodulin belongs to the t roponin C superfamily of C a 2 + - b i n d i n g prote ins conta in ing the E-F hand s t ruc ture (d iscussed e a r l i e r ) . Several p o s t - t r a n s l a t i o n a l mod i f i ca t ions o f calmodul in that can p o t e n t i a l l y regulate i t s b i o l o g i c a l a c t i v i t y have been i d e n t i f i e d : enzymatic removal of the carboxyl terminal l y s i n e (Murtaugh et a l . . 1983), N-methylation of l y s i n e 115 (Sitaramayya e t a]. . , 1980), carboxyl methyl a t i on (Gagnon et a l . , 1981) and phosphorylat ion at t y r o s i n e , threonine and ser ine res idues have been demonstrated (Plancke and Lazar ides , 1983; Fukami et a l . , 1986; Colca et a l . , 1987; Meggio et a l . , 1987; Church et a l . . . 1988; Ghosh et a l . . 1988; Kubo and S t r o t t , 1988). Evidence in favour of a regula tory r o l e f o r carboxylmethylat ion has been demonstrated (Gagnon et a l . , 1981). Many c e l l u l a r funct ions appear to be regulated by ca lmodul in , inc lud ing c e l l m o t i l i t y , m i t o s i s , cAMP metabolism, e x o c y t o s i s , prote in phosphory la t ion/dephosphory la t ion and C a 2 + t ranspor t (Means et a l . , 1982; Manalan and K l e e , 1984). The e f f e c t of calmodulin on these c e l l u l a r funct ions are exerted v i a var ious ca lmodul in -b inding p r o t e i n s . Vertebrate ca lmodul in -b ind ing prote ins can be categor ized in to three groups: a) enzymes (b) cy toske le ton ( s t r u c t u r a l ) prote ins and (c) m isce l laneous . In the enzymes category are two metabolic enzymes (phosphorylase kinase and phosphofructose kinase) (Buschmeier et a l . , 1987) and two key enzymes in 34 the contro l of cAMP l e v e l : adenylate cyc lase i s involved in the formation of cAMP and c y c l i c nucleot ide phosphodiesterase (PDE) i s involved in i t s breakdown (Yeager et a l . , 1985; Cheung, 1971). At l e a s t two d i s t i n c t isozymes of calmodulin-dependent PDE has been reported (see Sharma and Wang, 1988). Two ATPases, the plasma membrane Ca 2 + -pumping ATPase and the f l a g e l l a dynein-ATPase, are a lso c a l m o d u l i n - a c t i v a t e d . Six of the calmodulin-dependent enzymes are involved in prote in phosphory la t ion /dephosphory la t ion , i n c l u d i n g : (1) phosphorylase kinase (Chan and Graves, 1985); (2) myosin l i g h t chain kinase (Klee , 1977); calmodulin-dependent prote in kinase I (Nairn et a l . , 1985a); (3) calmodulin-dependent prote in kinase II (which phosphorylates many prote ins inc lud ing tubu l in and phospholamban) (Kennedy et a l . , 1987); (4) calmodulin-dependent kinase III (Nairn et a l . , 1985b); (5) caldesmon which undergoes autophosphorylat ion in the presence of calmodulin (Scott-Woo and Walsh, 1988), and (6) calmodulin-dependent phosphatase ( c a l c i n e u r i n ) (Ta l lan t and Cheung, 1986). Recent ly , i n o s i t o l 1,4,5 t r isphosphate k inase , a key enzyme in inos i to l -phosphate s ignal t ransduct ion was a lso demonstrated to be ca lmodul in -s t imula ted (Johanson et a l . , 1988). I n t e r e s t i n g l y , a l l of the enzymes that are ca lmodu l in -ac t iva ted are involved in the forming or breaking of a bond between a phosphorus and an oxygen. In the c y t o s k e l e t a l / s t r u c t u r a l p ro te in category , e ry th ro id s p e c t r i n binds calmodulin with low a f f i n i t y (Sobue et a l . , 1981a) while f o d r i n binds with high a f f i n i t y ( C a r l i n et a l . , 1983). Tubul in (a and &) a lso binds calmodulin with low a f f i n i t y (Kumagai et a l - , 1982). The microtubule associated prote in 2 (MAP-2) and Tau f a c t o r a lso appear to bind calmodulin (Lee and Wolf f , 1984; Sobue et a l . , 1981b) . Caldesmon 35 appears to bind calmodulin and a c t i n in a f l i p - f l o p mechanism ( i . e . the binding of calmodulin to caldesmon weakens the binding between ac t in and caldesmon) ( F i g . 5 ) . Tau f a c t o r a lso binds calmodulin and t u b u l i n in the same fashion ( F i g . 5) . Adducin, a newly i d e n t i f i e d ca lmodul in -b inding pro te in in the plasma membrane, promotes a s s o c i a t i o n of s p e c t r i n and a c t i n (Bennett et a l . , 1988). Th is a c t i v i t y of adducin i s i n h i b i t e d upon i t s binding to calmodulin (Bennett et a l . . , 1988). Other calmodul i n -binding prote ins include the calc ium re lease channel from junc t iona l SR ( S e i l e r et a l . , 1984), calspermin (Ono et a l . , 1984) and chicken lens f i b r e gap junc t ion prote in (Welsh et a l . , 1982). Phospholamban (Molla et a l . , 1983), myelin basic prote in (Grand and Perry , 1980) and histone 2B (Grand and Perry , 1980) do not conta in an authent ic ca lmodul in -b inding domain. They probably bind calmodulin l e s s s p e c i f i c a l l y v i a c l u s t e r s of bas ic amino ac id r e s i d u e s . Several prote ins has a lso been suggested to be calmodulin regu la ted , i n c l u d i n g : ( i ) the G a 2 + - a c t i v a t e d K + channel of luminal membranes from renal medulla (Klaerke et a l . , 1987), and ( i i ) a phospholipase ° f human p l a t e l e t membranes (Wong and Cheung, 1979). Neuromodulin i s a ca lmodul in -b inding pro te in (a lso c a l l e d GAP-43) that may be involved in normal c e l l growth (Welsh et a l . , 1982). I n t e r e s t i n g l y , neuromodulin appears to bind calmodulin more t i g h t l y in the absence of C a 2 + than in i t s presence (Adreasen et a l . , 1983). F i n a l l y , sphingosine has recent ly been reported to i n h i b i t several calmodulin-dependent enzymes (Je f ferson and Schulman, 1988). 36 F i g . 5 Diagrammatic presentat ion of the f l i p - f l o p mechanism f o r  ca ldesmon/calmodul in-caldesmon/act in and tau f a c t o r / c a l m o d u l i n - t a u  f a c t o r / t u b u l i n i n t e r a c t i o n . Calmodul in, caldesmon, a c t i n , t u b u l i n are represented by CaM, CaD, A and T, r e s p e c t i v e l y . S o l i d l i n e ind ica tes strong i n t e r a c t i o n whereas dotted l i n e ind ica tes weak i n t e r a c t i o n . Taken from Sobue et a l . (1981c). 37 Figure 5 38 V. Ca lpa in 1. Overview A protease (peptide hydrolase) i s an enzyme that c leaves peptide bonds. Proteases are c l a s s i f i e d in to exopeptidases and endopeptidases. Exopeptidases are r e s t r i c t e d to e i t h e r the C- or N-terminal peptide bond whi le endopeptidases are capable of a t tack ing cent ra l peptide bonds as w e l l . Endopeptidases (a lso c a l l e d prote inases) are fu r ther c l a s s i f i e d in to four c lasses mainly based on the e s s e n t i a l c a t a l y t i c residue at these ac t ive s i t e s (Bar re t t , 1986): ser ine (EC 3 .4 .21 ) , cys te ine (EC 3 .4 .22 ) , aspar t i c ac id (EC 3 .4 .23 ) , and meta l lo -protease (EC 3 .4 .5 .24 ) . In recent y e a r s , i t has become apparent that c e l l u l a r p r o t e o l y s i s i s a complex and h ighly c o n t r o l l e d process (Bond and B u t l e r , 1987). In cont ras t to e x t r a c e l l u l a r proteases , which a f t e r t h e i r synthes is are exported to e x t r a c e l l u l a r spaces, i n t r a c e l l u l a r proteases exert t h e i r ac t ion on c e l l u l a r or fore ign peptides or pro te ins i n s i d e the c e l l . For many y e a r s , lysosomes were thought to be the only l o c a t i o n in which c e l l u l a r p r o t e o l y s i s occurred . More r e c e n t l y , i t has become obvious that ( i ) lysosomal p r o t e o l y s i s i s re la ted to general p ro te in degradation and ( i i ) p r o t e o l y s i s a lso occurs in other organe l les as well as in the cytosol (Pontremoli and M e l l o n i , 1986a). One example i s s ignal pept idases of endoplasmic re t icu lum and mitochondr ia . The funct ion of these proteases i s to cleave o f f s ignal peptides from prote ins a f t e r t h e i r d e l i v e r y in to these organel les (a s ignal peptide i s a segment of . a newly synthesized prote in that i s required f o r proper d e l i v e r y of the prote in 39 in to these membrane organel les) (Docherty and S t e i n e r , 1982). Processing proteases are responsib le fo r p o s t - t r a n s l a t i o n a l mod i f i ca t ion of var ious prote ins (Pontremoli and M e l l o n i , 1986a). ATP/ubiqui t in-dependent prote inases (500-700 kDa) have been i d e n t i f i e d in r e t i c u l o c y t e s and some other t i s s u e s (Rechsteiner , 1987a). Upon an ATP-dependent enzymic conjugat ion with u b i q u i t i n , target prote ins become suscept ib le to the ATP-dependent protease. Another c y t o s o l i c protease i s macropain which i s a lso a high molecular weight protease (500-700 kDa) and i s d i s t i n c t from the ATP-dependent protease (R ive t t , 1989). Th is macromolecule appears to conta in a mixture of proteases as subuni ts , as evident from the p a r t i a l i n h i b i t i o n by var ious types of protease i n h i b i t o r s . L a s t l y , growing evidence has suggested that there i s a c y t o s o l i c calcium-dependent neutral protease system present in a l l mammalian and avian t i s s u e s (Suzuki , 1987). The f i r s t report of a Ca 2 + -dependent protease ( in ra t brain) was by Guroff (1964). Meyer et a l . (1964) a lso descr ibed a phosphorylase k inase-a c t i v a t i n g f a c t o r in ske le ta l muscle, which l a t e r turned out to be a Ca -dependent protease (Huston and Krebs, 1968). Th is protease i s a cyste ine protease homologous to papain, and i t i s a lso c a l l e d c a l p a i n (Murachi et a l . , 1979). Calpain has a pH optimum between 7-8. Two isozymes d i f f e r i n g in t h e i r Cac a f f i n i t y e x i s t (micromolar and m i l l i m o l a r concentrat ion of C a 2 + fo r c a l p a i n I and c a l p a i n II, r e s p e c t i v e l y ) . Calpa in i s a heterodimer, having an 80 kDa c a t a l y t i c subunit and a 29 kDa regula tory subuni t . The large subunit was f i r s t cloned and sequenced by Ohno et a l . (1984) from ch icken . This 80 kDa prote in has four domains with domain II being the c a t a l y t i c domain and domain IV a ca lmodul in-1 ike p, p • Ca -b ind ing domain ( F i g . 6 ) . It i s be l ieved that the E-F hand Ca -40 F i g . 6 Schematic of the domain s t ructure of c a l p a i n . For d e t a i l e d explanat ion see t e x t . Modif ied from Suzuki (1987). 41 Figure 6 Catalytic 80 kDa subunit calmodulin-like cysteine protease domain Ca-binding domain 1 100 300 500 700 • i i i i i i i_ 1 1 . 1 . i . .i i. 1 = 11 = 1 III » • • • • 1 » / ' • • • • • 1 V » • ' • • : • : ' • •'• ' : • ; • Cys Wmm His ffi Regulatory 30 kDa subunit I f f l l i l i l l l 100 200 » . • . • . • « • • • « Gly-rich hydrophobic domain calmodulin-like Ca-binding domain 42 binding domain imposes the s t r i c t Ca -dependence of c a t a l y t i c a c t i v i t y . At the c a t a l y t i c domain, Cys-108 i s in c lose proximity to H is -265 . There i s a requirement fo r a reducing environment ( e . g . 10 mM d i t h i o t h r e i t o l ) to keep the su l fhydry l group of Cys-108 reduced. It i s be l ieved tha t , as in papain , the His-265 imidazole r ing ni trogens a s s i s t in the abs t rac t ion of the s u l f h y d r y l proton, thereby f a c i l i t a t i n g the at tack on the substrate amide carbonyl group, as i l l u s t r a t e d in F i g . 7 (Bar re t t , 1986). I n t e r e s t i n g l y , ca lpa in has to f i r s t undergo a u t o l y s i s to be f u l l y a c t i v a t e d . For example, the c a t a l y t i c subunit of e ry throcyte c a l p a i n I (80 kDa) was found to autolyze sequent ia l l y to 78 kDa and 76 kDa (Pontremoli and M e l l o n i , 1986b). The autolyzed c a l p a i n I and II a lso appeared to have a much lower requirement f o r ca lc ium (Pontremoli and M e l l o n i , 1986; Murachi , 1983). Recent ly , i t was demonstrated that a u t o l y s i s i s both intramolecular and in termolecular (Inomata et a]_., 1988). Further au toca ta lys is produced i n a c t i v a t i o n of ac t i va ted c a l p a i n (Pontremoli and M e l l o n i , 1985). Calpain a c t i v i t y can be i n h i b i t e d by t y p i c a l cys te ine protease i n h i b i t o r s : t h i o l - r e a c t i v e agents ( e . g . iodoace ta te ) , epoxysuccinyl peptides ( e . g . E64) and microb ia l peptide aldehydes ( leupept in and ant ipain) (Suzuki et a l . , 1981). E64 appeared to a l k y l a t e the sulphydryl group while leupept in mimics the geometry of the t r a n s i t i o n s ta te fo r an enzyme-substrate complex and thus binds t i g h t l y to the c a l p a i n molecule, as in the case of papain ( B a r r e t t , 1986). I n t e r e s t i n g l y , i t has been demonstrated that the binding of both leupept in and E64 to c a l p a i n i s calcium-dependent and the i n h i b i t i o n by leupept in i s r e v e r s i b l e by d i a l y s i s in the presence of EGTA but not that of E64 (Suzuki et al_. , 1981). Calpain a c t i v i t y i s a lso dependent on temperature. At 43 F i g . 7 Mechanism of hydro lys is of peptide bond of substrates by c a l p a i n . React ion proceeds from (1) to (5) . The conserved cys te ine (Cys) and h i s t i d i n e (His) residues of the c a t a l y t i c domain of la rge subunit of c a l p a i n (see F i g . 6) are i l l u s t r a t e d . Modi f ied from Barre t t (1986). 44 Figure 7 9 o C ^ - S H (Cys -S / H- lm J 1 His , 0 R-*. - 0 ' (Cys___Jm-His^ (3) It R-C-OH -—> ^Cys H*lm-His^ (3) JSfHs .HN' HgNR- x C f ^ R-C-NHjR' ' S (CyS* Im-His ^ 9 R-C-OH SH Im HiSp 45 3 7 ° C , the enzyme i s inact iva ted in about 30-40 min while the a c t i v i t y remains s tab le f o r at l eas t 50 min at 21°C (Mellgren et a l . 1982). Like the la rge c a t a l y t i c subuni t , the small 30 kDa subunit a lso binds C a 2 + . In f a c t , an E-F hand Ca -b inding domain at the C-terminal end of the small subunit was i d e n t i f i e d (Aoki et a l . . 1986; Emori et a l . , 1986b; Suzuk i , 1987) ( F i g . 6 ) . It has been observed that ery throcyte c a l p a i n binds to 7+ the plasma membrane in a Ca^ -dependent manner and in the bound form the 80 kDa subunit can undergo a u t o l y t i c a c t i v a t i o n to a 78 kDa form (Pontremoli et al_., 1985a). I n t e r e s t i n g l y , a s i m i l a r b inding of neutrophi l c a l p a i n to i t s plasma membrane i s observed except that in t h i s case plasma membrane-bound ca lpa in was a c t i v e without undergoing a u t o l y s i s (Pontremoli et a l . , 1985c). A lso phosphat idyl i n o s i t o l reduced the Ca 2 + - r equ i rement of ca lpa in fo r a u t o l y s i s (Cool ican and Hathaway, 1984). Subsequently, i t was i l l u s t r a t e d that the g l y c i n e - r i c h domain near the N-terminal of the small subunit of c a l p a i n was e s s e n t i a l f o r such a c t i v a t i o n by phosphatidyl i n o s i t o l (Imajoh et a l . , 1986). Suzuki (1987) proposed that the hydrophobic region of the small subunit may be d i r e c t l y respons ib le f o r ca lpa in binding to the plasma membrane v i a phosphatidyl i n o s i t o l . A u t o l y t i c cleavage of the small subunit of c a l p a i n II a lso appears to r e s u l t in a c t i v a t i o n of the protease (DeMartino et a l . , 1986). 2. Regulators The most important regula t ion of c a l p a i n a c t i v i t y i s perhaps the co-ex is tence of an endogenous ca lpa in i n h i b i t o r p ro te in ( c a l p a s t a t i n ) in most 46 t i s s u e s studied (Murachi, 1983a). It i s now c l e a r that there are two types ( l i v e r and erythrocyte types) of c a l p a s t a t i n . The l i v e r type i s about 107 to 110 kDa on SDS-PAGE while the erythrocyte type i s about 68-70 kDa. Each molecule of the former i n h i b i t s 4-10 molecules of c a l p a i n while that of the l a t t e r only i n h i b i t s 3-5 molecules of c a l p a i n (Emori et a l . , 1987; Suzuki et a l . . . 1987b). From the m-RNA of these p r o t e i n s , i t was found that ( i ) the l i v e r i n h i b i t o r has a c a l c u l a t e d molecular weight of 68 kDa (although i t appears as 110 kDa on SDS-PAGE) and conta ins four repeated domains ( F i g . 8 ) ; ( i i ) the erythrocyte i n h i b i t o r has a c a l c u l a t e d molecular weight of 46 kDa ( i t appears as 68 kDa on SDS-PAGE) and has three repeated uni ts ( F i g . 8) (Suzuki et al_. , 1987b). The erythrocyte c a l p a s t a t i n appears to form an oligomer of 230 kDa in the presence of EGTA and d i s s o c i a t e s into monomers in the presence of C a 2 + (Melloni et a l . , 1982a). The l i v e r type c a l p a s t a t i n appears to undergo very s i m i l a r changes (see Pontremoli et a l . . 1985a). There fore , i t i s l i k e l y that the monomeric form of capastat in i n t e r a c t s with c a l p a i n . The binding of c a l p a s t a t i n to ca lpa in i s Ca 2 + -dependent and i s reversed upon removal of C a 2 + (Mel loni et a l . , 1982a). Upon binding to c a l p a s t a t i n , c a l p a i n i s incapable of a u t o l y s i s or p r o t e o l y s i s of substra te p r o t e i n s . I f the amount of c a l p a i n i s in excess, however, the i n h i b i t o r i s proteolyzed (Imajoh and Suzuki , 1985). Most r e c e n t l y , i t has been demonstrated that a l l four i n t e r n a l l y r e p e t i t i v e domains of c a l p a s t a t i n possess i n h i b i t i n g a c t i v i t i e s against ca lpa in (Maki et a l . , 1987). Although c a l p a i n and c a l p a s t a t i n are found mostly in the c y t o s o l i c f r a c t i o n s , there are several reports showing that both c a l p a i n and 47 F i g . 8 Schematic of the s t ructure of c a l p a s t a t i n . I-IV in te rna l repeats of about 14 kDa are shown. Each repeat unit corresponds to a func t iona l uni t of i n h i b i t i o n . Note that the erythrocyte i n h i b i t o r l acks in te rna l repeat I. Taken from Suzuki et a l . (1987b). 48 Figure 8 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 I I I 1 I I L _ P r i m a r y t r w u U t t o n p r o d u c t L M « T inhibitor E r y t h r o c y i o inhib i tor 7 1 8 H 2 N 1 II III IV • COOH 9 0 7 1 8 H3N- 1 II III IV -COOM 2 9 0 7 1 8 X-HN- II III IV -COOH 49 c a l p a s t a t i n are found in var ious s u b c e l l u l a r o r g a n e l l e s , inc lud ing plasma membrane/sarcolemma and sarcoplasmic re t icu lum (Gopalakrishna and Barsky, 1986; Mellgren et a l . , 1987a, b ) . Other prote in regula tors of ca lpa in a lso e x i s t , i nc lud ing a human erythrocyte protease i n h i b i t o r prote in (240 kDa) that i n h i b i t s both the high molecular weight protease and ca lpa in (Murakami and E t l i n g e r , 1986). An endogenous prote in a c t i v a t o r which increases the neutrophi l c a l p a i n 9 . a f f i n i t y fo r C a c (Pontremoli et a l . , 1988) and an endogenous a c t i v a t o r 9 . that increases the V m a x but not the Ca a f f i n i t y of bra in c a l p a i n have been reported (DeMartino and Blumenthal, 1982; Takeyama et_al_-> 1986). Hincke and Tolnai (1986) reported that prote in kinase C can phosphorylate c a l p a i n although others have f a i l e d to demonstrate such phosphorylat ion in v ivo (Adachi et a l . , 1986). 3. Substrate S p e c i f i c i t y Calpain I and II appear to hydrolyze only a s e l e c t i v e group of prote in substrates (Zimmerman and Sch laepfer , 1984). Small pept ides are usua l ly very poor substrates or not -hydrolyzed at a l l (Murachi, 1983b). Some degree of cleavage s i t e preference appears to apply: a Lys , T y r , Arg or Met res idue in the Pj p o s i t i o n preceded by e i t h e r a Leu or Val in the ?2 p o s i t i o n would favor cleavage at the carboxyl s ide of the res idue in the Pj p o s i t i o n (Sasaki et a l . , 1984) ( F i g . 9 ) . Th is aspect w i l l be fu r ther analyzed in d e t a i l under the DISCUSSION s e c t i o n . 50 F i g . 9 Schematic presentat ion of the i n t e r a c t i o n between the cleavage  s i t e of substrate and the ac t ive s i t e of c a l p a i n . Amino ac id res idues P3 to ?2 at the cleavage s i t e of substrate are shown while corresponding subs i tes S3 to S 2 ' on the ca lpa in molecules are shown. Based on the s t ruc ture of papain (Schechter and Berger, 1967). 51 Figure 9 52 4 . Substrates and funct ions of ca lpa in The p h y s i o l o g i c a l ro les of ca lpa in are s t i l l unc lear . Recent ly , Mel lgren (1987) and Suzuki et al_. (1987a) reviewed some of the p h y s i o l o g i c a l events that are be l ieved to be in f luenced by c a l p a i n . Ca lpa in substrates can be categor ized in to the fo l lowing general groups according to t h e i r funct ion and s u b c e l l u l a r l o c a t i o n : (a) membrane-bound p r o t e i n s ; (b) cy toske le ta l p r o t e i n s ; (c) receptor p r o t e i n s ; (d) m y o f i b r i l l a r p r o t e i n s ; (e) enzymes; (f) blood c l o t t i n g f a c t o r s ; (g) miscel laneous prote ins and (h) sma l l , natural peptides (Table 1) . M y o f i b r i l l a r p r o t e i n s , as a group, probably were the e a r l i e s t i d e n t i f i e d substrates fo r ca lpa in (Dayton et al_. , 1976). P r o t e o l y t i c degradation of membrane prote ins such as ery throcyte band 3 pro te in and s p e c t r i n i s l inked to f a c i l i t a t i o n of membrane fus ion ( G i l a s a and Kosower, 1986), while c a l p a i n - h y d r o l y s i s of f o d r i n in p o s t - s y n a p t i c membranes i s suggested to expose the o r i g i n a l l y l a ten t glutamate r e c e p t o r s , which may cont r ibu te to the observed long-term p o t e n t i a t i o n (Lynch and Baudry, 1984). The s u s c e p t i b i l i t y of microtubule pro te ins ( tubu l ins and MAP-2), intermediate f i lament prote ins (v iment in, desmin, f i l a m i n , kera t in and nebul in) and neurofi laments suggested that c a l p a i n can d i s r u p t the assembly of these three types of networks (Table 1) . The s t e r o i d r e c e p t o r s : g l u c o c o r t i c o i d , estrogen and progesterone receptors appeared to have a l te red proper t ies a f te r ca lpa in - f ragmenta t ion (see Table 1). Ca lpa in a lso hydrolyzes the s o l u b l e , s t e r o i d r e c e p t o r - l i k e Ah receptor , 53 Table 1 Substrates of c a l p a i n Substrate E f f e c t of ca lpa in Reference (a) Membrane prote ins Fodr in (a , /J) S p e c t r i n (a , 0) Ankyrin (band 2.1) Band 3 Band 4.1 (b) Cvtoske le ta l prote ins Tubul in (a , fl) MAP-1, MAP-2 Nebulin T a l i n Vimentin Desmin Kera t in F i lamin Neurofi lament (H,M & L) Adducin (a , /S) Crys ta l 1 in (a , /}) Lamin (nuclear) (c) Receptor prote ins a j - a d r e n e r g i c receptor EGF receptor Estrogen receptor Progesterone receptor Ah receptor G l u c o c o r t i c o i d receptor 1 fragmented fragmented fragmented fragmented fragmented fragmented degraded 2 degraded fragmented degraded degraded degraded fragmented degraded degraded degraded degraged becomes GTP/GDP-independent increased auto-phosphorylat ion can enter nucleus l o s t DNA-binding a b i l i t y fragmented fragmented Simon et a l . , 1984 Pant et a l . , 1983 Hal l & Bennet, 1987 Au et a l . , 1988 Pant et a l . , 1983 B i l l g e r et a l . , 1988 B i l l g e r et a l . , 1988 Gol l et a l . , 1983 Beckerle et a l . , 1986; O 'Ha l loran et a l . , 1985 Nelson & Traub, 1986 Nelson & Traub, 1986 Ando et a l . , 1988a T r u g l i a & S t racher , 1981 Mel ik et a l . , 1983 Wang et a l . , 1989b David & Shearer , 1986 Traub et a l . , 1988 Lynch et a l . , 1986 Gates & K ing , 1983 Puca et a l . , 1977 Vedeckis et a l . , 1980 Poland & G lover , 1988; Naray, 1981 54 Table 1 (cont . ) Substrate E f f e c t of ca lpa in Reference (d) M y o f i b r i l l a r pro te ins Myosin heavy chain degraded Myosin l i g h t chains degraded C-pro te in degraded a - a c t i n i n degraded Troponin T degraded Troponin I degraded Tropomyosin degraded Dayton et a l . , 1975 Ish iura et a l . , 1979 Dayton et a l . , 1975 Ish iura et a l . , 1979 Dayton et a l . , 1975 Dayton et a l . , 1975 Dayton et a l . , 1975 (e) Enzymes Phosphorylase kinase Phosphorylase phosphatase Glycogen synthase (D-form) Tryptophan hydroxylase HMG-CoA reductase Pyruvate kinase Myosin l i g h t chain kinase C y c l i c n u c l e o t i d e -phosphodiesterase C a l c i n e u r i n C a 2 + - A T P a s e Prote in kinase C Calpa in Adenylate c y c l a s e Transglutaminase P l -phosphol ipase C I n h i b i t o r - 1 -phosphatase Regulatory subunit (Rj) of cAMP-PK cAMP-independent p ro te in kinases ac t iva ted ac t iva ted ac t iva ted ac t iva ted re leased a a c t i v e fragment from ER act iva ted inac t iva ted ac t iva ted & CaM-independent ac t iva ted & CaM-independent ac t iva ted & CaM-independent independent o f C a z + , PS & DAG f i r s t ac t iva ted then i n a c t i v a t e d ac t iva ted ac t iva ted fragmented act iva ted d i s s o c i a t e d from c a t a l y t i c subunit ac t iva ted 55 Huston & Krebs, 1968 Mel lgren et a l , 1979 Hiraga & T s u i k i , 1986 Hamon & Bourgoin , 1979 Li scum et a l . , 1983 Ekman & E r i k s s o n , 1980 Ito et a l . , 1987 Ito et a l . , 1987 T a l l ant et a l . , 1988 Wang et a l , 1988a, b; Au, 1987 Kishimoto et a l . , 1980 Suzuki et a l , 1987b Tremblay & Hamet, 1984 Ando et a l . , 1987 Low et a l . , 1984 Waelkens et a l . , 1985 Beer et a l . , 1984 Tahara & Traugh, 1982 Table 1 (cont . ) Substrate E f f e c t of c a l p a i n Reference (f) Blood c l o t t i n g f a c t o r s Factor XIII ( t rans-glutaminase Factor V F ibr inogen (a,/}) Kininogen Von Wi l l brand f a c t o r Glycoprote in lb (a) (h) Natural pept ides Insu l in Protamine Glucogen Dynorphin ac t iva ted degraded ac t iva ted degraded fragmented fragmented fragmented fragmented fragmented fragmented Ando et a l . , 1987 McGowan et a l . , 1983 Kunicki et a l . , 1984 Schmaier et a l . , 1986 Kunicki et a l . , 1985 Yamamoto et a l . , 1986 Suzuki et a l . , 1987b Murachi , 1983b Mel loni et a l . , 1984 Sakai et a l . , 1987 B e l l e s et a l . , 1988 S e i l e r et a l . , 1984 Wang et a l . , 1989b Haraguchi et a l . , 1987 Ish iura et a l . , 1979 Hayashi et a l . , 1985 Sasaki et a l . , 1984 Sasaki et a l . , 1984 (g) Miscel laneous prote ins C a l p a s t a t i n Casein ( a i s , &p s & p) Hemoglobin (a , ft) Histone (IIA, IIB & III) Calcium channel (L- type) Calcium re lease channel Neuromodulin Thyroglobul in fragmented degraded fragmented degraded degraded fragmented fragmented fragmented "fragmented" i n d i c a t e s that the o r i g i n a l p ro te in undergoes only l i m i t e d p r o t e o l y s i s "degraded" i n d i c a t e s that the o r i g i n a l p ro te in undergoes m u l t i p l e and extensive p r o t e o l y s i s 56 which, upon binding to compounds l i k e 2 , 3 , 7 , 8 , - t e t r a c h l o r o d i b e n z o - p -d i o x i n , induces the synthesis of cytochrome -450 and other drug metabol iz ing enzymes (Poland and Glover , 1988). The a-adrenergic r e c e p t o r s , upon p r o t e o l y s i s , become agonist- independent (Lynch et a l . . , 1986). Ca lpa in a lso appeared to be involved in m y o f i b r i l l a r prote in turnover (Dayton et a l . , 1975). Blood c l o t t i n g f a c t o r s : f a c t o r V, f ib r inogen (a , 0 ) , kininogen and von Wil lebrand f a c t o r s , are considered poten t ia l substrates i f enough c a l p a i n can be re leased upon p l a t e l e t a c t i v a t i o n (Table 1). The most i n t e r e s t i n g group of ca lpa in substrates i s the "enzymes". It appears that upon p r o t e o l y s i s by c a l p a i n most of the enzyme substra tes are a c t i v a t e d , although the mode of a c t i v a t i o n can be very d i f f e r e n t (Table 1). For example, several calmodulin-dependent enzymes (phosphorylase k inase , phosphodiesterase and c a l c i n e u r i n ) are ac t iva ted and made independent of ca lmodul in . Prote in kinase C could be proteolyzed by ca lpa in to produce a s o - c a l l e d M-form of the kinase which i s independent of C a 2 + and phosphol ip id (Kishimoto et a l . , 1983). In n e u t r o p h i l s , such p r o t e o l y t i c a c t i v a t i o n of p ro te in kinase C appears to occur in response to st imulus (Melloni et a l . , 1986; Pontremoli et a l . , 1986a; 1988). I n t e r e s t i n g l y , phosphorylat ion of some substrates increases t h e i r s u s c e p t i b i l i t y to c a l p a i n (Toyo-Oka, 1982; Parker et a l . , 1986; Pontremoli et a_l., 1987) while phosphorylat ion renders other substrates l e s s s u s c e p t i b l e (Zhang et a l . , 1988; Pant, 1988). The c a l p a i n - c a l p a s t a t i n a c t i v i t y balance appears to be a l te red in 57 some disease s t a t e s . In Duchenne muscular dystrophy (DMD) p a t i e n t s , c a l p a i n a c t i v i t y was increased in muscle as well as in p l a t e l e t s (Rabbani et a l . , 1984). Both the erythrocytes and kidneys of Mi lan hypertensive ra ts had decreased l e v e l s of c a l p a s t a t i n , while the c a l p a i n leve l was the same as that in normal Milan ra ts (Pontremoli et a l . , 1986b, 1987a). As was d iscussed e a r l i e r , the erythrocyte Ca 2 + -pumping ATPase can be ac t iva ted by ca lmodul in . A l t e r n a t i v e l y , l i m i t e d p r o t e o l y s i s with exogenous protease a lso ac t iva tes the C a 2 + - A T P a s e . On the other hand, the erythrocyte a lso has a large quant i ty of c a l p a i n in the c y t o s o l . There fore , i t i s proposed that the C a 2 + - A T P a s e may be a substrate fo r c a l p a i n and that such p r o t e o l y s i s may serve to a c t i v a t e the C a 2 + - A T P a s e i r r e v e r s i b l y . Since c a l p a i n may be involved in c e r t a i n d iseases or d isorders ( e . g . DMD and e s s e n t i a l hyper tens ion) , i t i s important to e l u c i d a t e i t s r o l e in the contro l of the C a 2 + pump a c t i v i t y . V f . Object ives o f the study (1) to p u r i f y ca lpa in I from human e ry th rocy tes . (2) to charac te r i ze the p u r i f i e d c a l p a i n . (3) to inves t iga te whether the C a 2 + - A T P a s e i s an in v i t r o substrate of c a l p a i n . (4) to examine and to charac te r i ze changes in A T P - h y d r o l y t i c a c t i v i t y , C a 2 + t ranspor t funct ion and k i n e t i c p roper t ies of the C a 2 + - A T P a s e upon ca lpa in - t rea tment . 58 (5 ) to examine the s t ruc tu ra l changes of treatment. ( 6 ) to c o r r e l a t e the s t ruc tura l changes a c t i v i t y / k i n e t i c property changes mechanism of act ion of ca lpa in in the C a - A T P a s e upon c a l p a i n -of the C a 2 + - A T P a s e with the of the enzyme and to provide a i t s e f f e c t s on the C a 2 + - A T P a s e . 59 MATERIALS AND METHODS I. Mater ia ls The chemicals and/or prote ins were purchased from the fo l lowing sources: 1. Sigma Chemical Co. 2-mercaptoethanol 5 ' - n u c l e o t i d a s e (Crotalus adamamteus venom, grade III) Ac t iva ted charcoal ADP ( M g 2 + s a l t ) A lamethic in Arsenazo III Ascorb ic ac id Bovine serum albumin Bromophenol blue Calmodulin-agarose cAMP Casein C i t r i c ac id Dansylated calmodulin EDTA EGTA Hepes Histone ( c a l f thymus, type 11-A) 60 Hydroxylamine monohydrochloride L - l a c t i c dehydrogenase ( rabbi t muscle, type II) Lanthanum c h l o r i d e Leupeptin Male ic ac id Manganese c h l o r i d e MOPS NADH (yeast , grade III) Nickel (II) c h l o r i d e o -Phospho-L- tyros ine Ouabain Papain (papaya l a t e x , type III) Phosphoenolpyruvate PMSF pNPP Pyruvate kinase ( rabbi t muscle, type II) S-100 A prote in (bovine brain) S-100 B prote in (bovine brain) Sodium azide Sodium cholate Sodium deoxycholate Sodium-ortho-vanadate TEMED TLCK T r i c h l o r o a c e t i c ac id T r i s base 61 T r i s - H C l Tr i ton-X lOO Troponin ( rabbi t ske le ta l muscle) T ryps in (bovine pancreas, type III) T ryps in i n h i b i t o r (soybean, type IV) Omega-aminohexyl-agarose 2. BDH Chemicals Calcium c h l o r i d e Chloroform Dimethylformamide G l a c i a l a c e t i c ac id Glutaraldehyde Glycero l G lyc ine Hydrochlor ic ac id Magnesium c h l o r i d e Methanol Phosphoric ac id Potassium c h l o r i d e Potassium hydrogen phosphate Potassium hydroxide S i l v e r n i t r a t e Sodium carbonate Sodium c h l o r i d e Sodium hydroxide 62 3. Amersham [gamma-^P]-ATP ( s p e c i f i c a c t i v i t y 20-40 Ci/mmol) 4. Calbiochem A23187 Calmodulin (bovine brain) Myel in bas ic pro te in (porcine spinal cord) Parvalbumin (rat muscle) 5. Bio-Rad Laborator ies Acrylamide Ammonium persu l fa te Coomassie b r i l l a n t blue R-250 N,N' -methylene-bi sacrylamide SDS SDS-PAGE high molecular weight standards SDS-PAGE low molecular weight standards 6. Pharmacia DEAE-Sephacel Gel f i l t r a t i o n molecular weight standards Phenyl-Sepharose CL-4B Sephacryl S-200 Superose-12 63 7. Boehrinqer Mannheim D i t h i o t h r e i t o l ATP ( M g 2 + and Na + s a l t s ) 8 . L i p i d Products Co. Egg l e c i t h i n (phosphat idy lcho l ine , grade I) 9 . MCB Manufacture Chemists. Inc. A s o l e c t i n 1 0 . New England Nuclear Aquasol ( l i q u i d s c i n t i l l a t i o n f l u i d ) 64 11. Methods 1. Prote in concentrat ion determination Prote in concentrat ions of var ious samples were determined by the method of Lowry et a l . (1951), using bovine serum albumin as a standard. B r i e f l y , up to 100 /il of sample was mixed with 4 mL of copper reagent, which was f r e s h l y made by f i r s t combining 800 /xL of 2 % (w/v) NaK-tar t rate and 800 /zL of 1 % (w/v) of CuS0 4 and then 80 mL of 2 % (w/v) N a 2 C 0 3 in 0.1 M NaOH). A f t e r 10 min, 400 /zL of 50 % phenol reagent . Th is reagent conta ins phosphomolybdictungstic complex, L i 2 S 0 4 and bromine water) . When reduced by phenol groups of t y ros ine and tryptophan res idues in a p r o t e i n , the phosphomolybdictungstic complex g ives a blue c o l o r at a l k a l i n e pH. The add i t ion of L i 2 S 0 4 prevented t u r b i d i t y while bromine water maintained the ox id i zed state of the reagent dur ing s torage. A l k a l i n e copper apparently increases the c o l o r developed markedly (Lowry et a l . , 1951). The mixture o f Na 2C03 and NaOH maintained the pH around 10 and they helped the denaturing (unfolding) of p r o t e i n s . A f t e r a l lowing c o l o r development fo r 60 min at room temperature or 20 min at 3 7 ° C , absorbance at 660 nm was read . If prote in concentrat ions of samples were low, then pro te ins were f i r s t p r e c i p i t a t e d with 10 % (w/v) TCA in the presence of 0.05 % (w/v) of sodium deoxycholate and are resuspended to 100 /zL with water before pro te in determinat ion. 2. Determination of inorganic phosphate 65 The production of inorganic phosphate as a r e s u l t of ATP breakdown was measured by the method descr ibed by Raess and Vincenzi (1980). B r i e f l y , at the end of the r e a c t i o n , 200 of 10 % (w/v) SDS was added to the reac t ion mixture (400 /xL) to stop the r e a c t i o n , 200 [xl of 9 % (w/v) of ascorb ic ac id was added and fol lowed by 200 /zL of 1.25 % (w/v) ammonium molybdate in 6.5 % (v/v) H 2 S 0 4 . The p r i n c i p l e of t h i s method l i e s in the complex formation between molybdate and f ree inorganic phosphate. The complex gives a blue c o l o r in a reducing environment (provided by ascorb ic a c i d ) . Absorbance at 660 nm i s read a f te r exact ly 30 min of c o l o r development at room temperature. K2HPO4 was used as a standard. Since molybdate a lso ca ta lyzes the breakdown of unhydrolyzed ATP, blanks conta in ing ATP were run as c o n t r o l s . 3. C a l c u l a t i o n s of the concentrat ions of f ree C a ^ and M q 2 + Free Ca*- and Mg*- concentrat ions were c a l c u l a t e d by computer using a FORTRAN program (Cations BC), descr ibed by Goldste in et a l - (1979). Binding constants f o r C a 2 + , M g 2 + and H + to ATP and EGTA were taken from Marte l l and Smith (1974-1982) except in the case of monoprotonated l igands which were c a l c u l a t e d as descr ibed by B l inks et a l . (1982). P r i o r to a p p l i c a t i o n of the program, constants were corrected f o r temperature using a BASIC program (LOGTEMP), based on the formula given by Tinoco et a l . (1978) and using enthalpy values tabulated by Marte l l and Smith (1974-1982). The constants were then adjusted fo r i o n i c strength (Martel l and Smith, 1974-1982; B l inks et a l . , 1982). 66 4. Polvacrylamide gradient gel e lec t rophores is and autoradiography ( i ) Sample p repara t ion . Regular prote in samples (20-40 /xL) were mixed with 25 /iL d i g e s t i o n buf fer conta in ing 5 % (w/v) SDS, 10 % (v/v) g l y c e r o l , 1 mM 2-mercaptoethanol, 0.02 % (w/v) bromophenol blue and 100 mM T r i s - H C l (pH 6 .8 ) . I f sample volume was l a r g e , prote in was f i r s t p r e c i p i t a t e d with 20 % (w/v) TCA. The p e l l e t was resuspended with 25 /xL of d i g e s t i o n buf fer and neu t ra l i zed with T r i s - b a s e . When a sample contained proteol iposomes, the p r o t e i n / 1 i p i d mixture was f i r s t p r e c i p i t a t e d with 20 % (w/v) TCA. The p e l l e t was then t reated with 500 /xL chloroform to ext ract the excess amount of phospho l ip id . The mixture was cent r i fuged ( 5 min at 10,000 x g m a x ) and the chloroform phase was removed before the add i t ion o f d i g e s t i o n b u f f e r . I f samples were to be analyzed f o r EP formation on an ac id gel (see below), the d i g e s t i o n buf fer contained 100 mM Tr is -phosphate (pH 6.8) instead of T r i s - H C l . ( i i ) SDS-polyacrylamide s lab ge ls of 1.5 mm th ickness were cast according to the method of Laemmli (1971). The separat ing gel conta ins a l i n e a r gradient (5-20 % (w/v)) of to ta l acrylamide (ac ry lamide /N ,N ' -methylene-bisacrylamide (30:0 .8 ) ) , 375 mM T r i s - H C l (pH 8 .8 ) , 0.1 % (w/v) SDS, 1.35-5.75 % (v/v) gradient of g l y c e r o l , whi le the s tack ing gel contained 3.5 % (w/v) t o t a l acrylamide (see above), 330 mM T r i s - H C l (pH 6 .8 ) , 0.1 % (w/v) SDS. The gel was run overnight (16-18 h) at 18 mA/slab in 25 mM T r i s - b a s e , 192 mM g l y c i n e and 0.1 % (w/v) SDS (pH 8.3) at 4 ° C . For the measurement of the formation of EP, " a c i d " gel was cast as 67 descr ibed above, except that the pH values of the separat ing and stacking ge ls were 7.0 and 6 .0 , r e s p e c t i v e l y . The acid gel was run overnight at 30 mA/slab in 25 mM MOPS (pH 6 .8 ) , 0.1 % (w/v) SDS, 192 mM g l y c i n e at 4 ° C . Gels were sta ined with 0.25 % (w/v) Coomassie B r i l l i a n t blue R-250 in s o l u t i o n A (methanol /water /acet ic ac id (5:4:1)) f o r 60 min and destained with s o l u t i o n A u n t i l the background became t ransparent . The ge ls were then stored in 7.5 % (v/v) a c e t i c a c i d . For autoradiography, the ge ls were d r ied on Whatmam No. 1 paper and a Kodak X-0MAT AR f i l m was then exposed to i t f o r 36-64 h at - 7 0 ° C . 5. Preparat ion of membrane-free hemolyzate and ca lmodul in -deple ted  ery throcyte membranes A l l procedures were c a r r i e d out at 4 ° C . One uni t (approximately 250 g) of packed human red c e l l s (4-5 days o l d ) , suspended in c i t r a t e -phosphate-dextrose medium conta in ing adenine, was obtained from the Canadian Red Cross and washed three times in 1 L of an i s o t o n i c s o l u t i o n conta in ing 150 mM KC1 and 20 mM T r i s - H C l (pH 7.4) (buf fer A-150) . The c e l l s were c o l l e c t e d by c e n t r i f u g a t i o n at 3900 x g m a x f o r 5-10 min. The washed red c e l l s were hemolyzed in 10 volumes of a hypotonic buf fe r conta in ing 20 mM T r i s - H C l , 5 mM EGTA and 1 mM d i t h i o t h r e i t o l , 500 /iM PMSF, 10 /iM TLCK and 20 mg.L" 1 soybean t r y p s i n i n h i b i t o r (pH 7.4) (buf fer H) f o r 15 to 20 min. The hemolyzate was then passed through the M i l l i p o r e Pe l l icon Casset te system (equipped with f i l t e r s of 0.5 fim diameter pore s i ze ) in the concentrat ion mode un t i l the volume was reduced to approximately 1 L. The f i r s t 5 l i t r e s of f i l t r a t e was c o l l e c t e d and used as membrane-free hemolyzate fo r the p u r i f i c a t i o n of c a l p a i n (see below). 68 The concentrated membranes were then washed with 10 to 12 L of 20 mM potassium-Hepes (pH 7 .4) , 1 mM EDTA, 500 /xM PMSF and 10 mM a s c o r b i c ac id with the casset te system operat ing in the constant volume mode, in order to remove ca lmodul in , hemoglobin and other so luble p r o t e i n s . F i n a l l y , the white membranes were washed with 1 to 2 L of 130 mM KC1, 2 mM d i t h i o t h r e i t o l , 1 mM EDTA and 20 mM potassium-Hepes (pH 7.4) and 500 /xM PMSF (buf fer IV -E) . The membranes were then concentrated by c e n t r i f u g a t i o n at 20,000 x g m a x fo r 20 min. The f i n a l concentrated suspension o f membranes was qu ick - f rozen in l i q u i d n i t rogen ( - 1 9 6 ° C ) in a l i q u o t s of 2 to 4 mL and stored at - 7 0 T u n t i l used. 6. S o l u b i l i z a t i o n and p u r i f i c a t i o n of the C a 2 + - A T P a s e The s o l u b i l i z a t i o n and p u r i f i c a t i o n of the C a 2 + - A T P a s e , performed at 4 ° C , was c a r r i e d out e s s e n t i a l l y as descr ibed by V i l l a l o b o et a l . (1986): 150-200 mL of white calmodul in-depleted erythrocyte membrane suspension (600 to 1000 mg prote in) were concentrated by c e n t r i f u g a t i o n at 125,000 x 9max f ° r min. The p e l l e t s were s o l u b i l i z e d by resuspension in a determined amount of buf fer conta in ing 0.5 % (w/v) T r i t o n X-100, 300 mM KC1, 10 mM potassium-Hepes (pH 7 .4) , 1 mM M g C l 2 , 100 /xM C a C l 2 , 2 mM DTT, 500 tiM PMSF and 100 /xM leupept in to achieve a d e t e r g e n t / p r o t e i n r a t i o of 1/1 (or o c c a s i o n a l l y 1.5/1) (about 150-200 mL). The mixture was incubated f o r 15 min at 4°C with rapid s t i r r i n g . The s o l u b i l i s a t e was cen t r i fuged at 125,000 x g m a x f o r 30 min and the supernatant was c a r e f u l l y c o l l e c t e d . To the supernatant was added a pre-determined volume of presonicated s o l u t i o n conta in ing 4 % (w/v) a s o l e c t i n , 2 % (w/v) T r i t o n X-100 and 80 mM DTT (TAD 69 so lu t ion ) to achieve 0.1 % (w/v) a s o l e c t i n , 0.05 % (w/v) T r i t o n X-100 and 2 mM DTT in the f i n a l so lu t ion mixture. The s o l u t i o n was then loaded onto a calmodul in-agarose column (15 x 1.5 cm) conta in ing 20-30 mg cova len t ly bound calmodulin e q u i l i b r a t e d in buf fer EQ (0.5 % (w/v) T r i t o n X-100, 0.1 % (w/v) sonicated a s o l e c t i n , 200 mM KC1, 10 mM potassium-Hepes (pH 7.4), 1 mM M g C ^ , 100 /xM C a C ^ , 2 mM DTT. The column was then washed overnight (15-17 h) with 700-800 mL of buf fer W, which i s i d e n t i c a l to buf fer EQ except that the concentrat ion of T r i t o n X-100 was only 0.05 % (w/v) . The enzyme was e luted from the column with buf fer E, which i s the same as buf fe r W except that 2 mM EDTA replaced the 100 /xM C a C l 2 . Two mL- f rac t ions were c o l l e c t e d and the peak f r a c t i o n s of C a 2 + - A T P a s e a c t i v i t y were pooled and d iv ided in to small a l iquots which were f rozen in l i q u i d n i t rogen . F i n a l l y , a l i q u o t s were stored in a f reezer ( - 7 0 ° C ) p r i o r to use. The enzyme a c t i v i t y appeared to be s tab le f o r at l e a s t a few months. A f te r each use, the calmodul in-agarose was t reated s e q u e n t i a l l y with ( i ) 100 mL of 20 mM potassium-Hepes and 2 mM EDTA (pH 7.4), ( i i ) 100 mL of 2 M NaCl and 3 % (v/v) a c e t i c a c i d , ( i i i ) 100 mL of 6 M urea and ( iv ) 200 mL of 200 mM KC1, 50 mM potassium-Hepes, 50 jxM C a C ^ , 0.02 % (w/v) sodium azide (pH 7.4) and stored in t h i s buf fer at 4°C u n t i l used aga in . 7. I s o l a t i o n and p u r i f i c a t i o n of c a l p a i n from membrane-free hemolyzate Ca lpa in was prepared from the membrane-free hemolyzate according to Mel loni et a l . (1982b) and with the fo l lowing m o d i f i c a t i o n s (Waxman, 1981; Gopalakrishna and Head, 1985): the pH of the hemolysate was adjusted to 6.5 with a c e t i c ac id before batch-b inding to 150 mL DEAE-Sephacel for 90 min. The s l u r r y of r es in was washed with 20 mM T r i s - H C l (pH 6 .6 ) , 1 mM 70 DTT and 5 mM EGTA packed into a g lass column (diameter 2.5 cm) and fu r ther washed with 20 mM T r i s - H C l (pH 7.4), 1 mM DTT and 1 mM EGTA (buf fer A ) . The prote ins were e luted with a l i n e a r gradient (0-350 mM) of NaCl in buf fer A. Ac t ive f r a c t i o n s were pooled and appl ied onto a column of phenyl-Sepharose CL-4B (1.5 x 8.5 cm) p rev ious ly e q u i l i b r a t e d with 20 mM T r i s - H C l , 200 mM NaCl , 1 mM EGTA and 1 mM DTT at pH 7.4 (buf fer A-200) . The column was washed with buf fer A-200 and the pro te ins were e luted with buf fe r A (pH 7.4). The ac t ive f r a c t i o n s were pooled and appl ied to a omega-hexylamine-agarose column (1.5 x 5.5 cm). The prote ins were e luted with a l i n e a r gradient (0-350 mM) of NaCl in buf fer A (pH 7.4). The pooled ac t ive f r a c t i o n s were concentrated to 5-10 mL with a PM-30 membrane (30 kDa cu to f f ) in an u l t r a - f i l t r a t i o n uni t (Amicon) and loaded onto a gel f i l t r a t i o n column (Sephacryl S-200, 1.5 cm x 60 cm). The column was e luted with buf fe r F (50 mM T r i s - H C l (pH 7.4), 1 mM DTT and 1 mM EGTA). These preparat ions o f ca lpa in (5-20 unit.mg p r o t . " 1 ) conta in both the la rge and small subunits of ca lpa in (80 kDa and 29 kDa) which c o n s t i t u t e more than 90-95% of the to ta l p ro te in using e l e c t r o p h o r e t i c c r i t e r i a , and do not have Ca 2 + - independent p r o t e o l y t i c a c t i v i t y . Preparat ions of ca lpa in conta in ing 50-200 iig.mL"* (3-10 uni t .mL"*) are stored in bu f fe r F at 4°C u n t i l used (one uni t of ca lpa in a c t i v i t y i s def ined in a l a t e r s e c t i o n ) . 7+ 8. Determination of the membrane-bound Ca^ -ATPase a c t i v i t y Calmodul in-depleted membranes (50-100 /ig prote in .mL"* ) were incubated at 37°C f o r 30 min in a to ta l volume of 0.4 mL conta in ing 55 mM T r i s -maleate (pH 7 .2) , 66 mM KC1, 0.1 mM ouabain, 6.5 mM M g C l 2 , 120 nM calmodulin (when added), 2 mM ATP, 0.1 mM EGTA and var ious concentrat ions 71 of C a C ^ to obtain the f ree Ca"1 concentrat ion ind ica ted in the legends of the f i g u r e s . The Mg 2 + -ATPase a c t i v i t y (assayed in the absence of added C a C ^ and in the presence of 2.5 mM EGTA) was subtracted from the to ta l a c t i v i t y assayed in the presence of ca lc ium. The inorganic phosphate l i b e r a t e d was determined c o l o r i m e t r i c a l l y as descr ibed above. 9. Determination of the a c t i v i t y of the p u r i f i e d C a 2 + - A T P a s e P u r i f i e d C a 2 + - A T P a s e (1 to 5 /ig prote in) was incubated at 37°C f o r 30 min in 0.4 mL of 66 mM KC1, 55 mM Tr is -malea te (pH 7 .2 ) , 6.5 mM M g C l 2 , 120 nM calmodulin (when added), 2 mM ATP, 2 mM DTT, 0.05% (w/v) T r i t o n X-100, 0.1% (w/v) sonicated a s o l e c t i n , 100 /iM EGTA and var ious concentra t ions of EDTA and C a C ^ to a t t a in the des i red f ree C a 2 + concen t ra t ion . 10. Determination of the phosphorylated intermediate of the C a 2 + - A T P a s e Plasma membranes (50-100 /ig prote in) were incubated at 4°C f o r 15 s in 150 /iL of 50 mM KC1, 5 mM potassium-Hepes, 40 mM T r i s - m a l e a t e , 0.1 mM ouabain, 0.1 mM EGTA, 0.13 mM EDTA, 2 /iM MgCl 2 (not e s s e n t i a l ) , 2.5 mM C a C l 2 , 100 /iM L a C l 3 and 2 /iM ATP conta in ing 6 mCi.mmol" 1 [ -y- 3 2 P]ATP (pH 7 .2 ) . A l t e r n a t i v e l y , the p u r i f i e d C a 2 + - A T P a s e (5-20 /tg prote in ) was incubated at 4°C f o r 15 s in 300 /iL of 50 mM KC1, 40 mM T r i s - m a l e a t e , 0.1 mM EGTA, 0.5 mM EDTA, 50 /iM M g C l 2 , 2.6 mM C a C l 2 , 100 /iM L a C l 3 , 0.05 % (w/v) T r i t o n X-100, 0.1 % (w/v) sonicated a s o l e c t i n and 4 /iM ATP conta in ing 6 mCi. mmol"* [ 7 - 3 2 P ] A T P (pH 7 .2 ) . The reac t ion was i n i t i a t e d by the add i t ion of [ 7 - 3 2 P ] A T P and terminated by the add i t ion of i c e - c o l d 10% (w/v) t r i c h l o r o a c e t i c a c i d . The p r e c i p i t a t e d pro te in was processed fo r ac id gel e l e c t r o p h o r e s i s at pH 6.6 as descr ibed above. 72 11. Reconst i tu t ion of the p u r i f i e d Ca -ATPase A c h o l a t e - d i a l y s i s method (Kagawa and Racker, 1971), as modif ied by V i l l a l o b o and Roufogal is (1986), was used fo r the r e c o n s t i t u t i o n of the p u r i f i e d enzyme. P u r i f i e d C a 2 + - A T P a s e (0 .1-0.3 mg prote in) was added to a 7-15 mL presonicated phosphat idylchol ine-sodium cholate mixture. The f i n a l mixture contained 1.5 % (w/v) phosphat idy lcho l ine , 1 % (w/v) sodium c h o l a t e , 100 mM KCL, 20 mM potassium-Hepes, 2 mM DTT, 1 mM MgCl 2 and 50 /zM C a C l 2 (pH 7.4) . The mixture was put ins ide two to four Spectropore d i a l y s i s bags (50 kDa cu to f f ) and d ia lyzed at 4°C fo r 24 to 36 h against 7-11 L (5-7 changes of 1-2 L each) of the buf fer descr ibed above, except that i t d id not contain phosphat idy lchol ine or c h o l a t e . 12. Determination of the C a 2 + - A T P a s e and C a 2 + t ranspor t a c t i v i t i e s of the  recons t i tu ted Ca*- pump The ATP h y d r o l y t i c a c t i v i t y of the recons t i tu ted enzyme was determined e i t h e r by measuring the formation of inorganic phosphate by a coTor imetr ic method (Raess and V i n c e n z i , 1980) or by monitor ing the rate of NADH ox idat ion at 340 nm-400 nm in a dual wavelength spectrophotometer using an ATP regenerat ing system c o n s i s t i n g of pyruvate kinase and l a c t a t e dehydrogenase. The f i r s t method (Method A) was chosen when determinat ion of the i n i t i a l rate of ATP hydro lys is was not an e s s e n t i a l requirement. Proteoliposome suspensions (40 ill conta in ing 0.6-1.2 /zg pro te in plus 0.65 mg phosphol ip ids) were incubated at 37°C for 30 min in 0.4 mL of a medium conta in ing a f i n a l concentrat ion of 55 mM Tr is -ma lea te (pH 7 .2 ) , 66 mM KC1, 6.5 mM M g C l 2 , 120 nM calmodulin (when added), 2 mM ATP and var ious 73 concentrat ions of C a C l 2 (around 70 /xM) and EGTA (around 110 xxM) to a t t a in 0.4 /xM f ree calc ium ion (unless stated otherwise) . A second method (Method B) was employed when determination of the i n i t i a l rate of ATP h y d r o l y s i s was r e q u i r e d . Proteoliposomes (6-12 /xg pro te in p lus 7 mg phosphol ip ids) were incubated at 37°C in 1.5 mL of a medium conta in ing a f i n a l concent ra t ion of 73 mM KC1, 17 mM potassium-Hepes (pH 7 .4 ) , 5 mM DTT, 730 /xM M g C l 2 , 130 txM NADH, 5 mM phosphoenolpyruvate, 1 /xM calmodulin (when added), 20 un i ts pyruvate k inase, 90 uni ts l a c t a t e dehydrogenase, and var ious concentrat ions of C a C l 2 (240-280 /xM) and EGTA (around 300-370 /xM) to obta in 0.4 /xM f ree calcium i o n . The reac t ion was i n i t i a t e d by the add i t ion of 27 jM ATP. C a 2 + uptake by the proteoliposomes was monitored at 650 nm-720 nm with a dual wavelength spectrophotometer using the cond i t ions descr ibed above in the presence of the metallochromic dye Arsenazo III (10 /xM). To determine ra tes of ATP hydro lys is and C a 2 + t ranspor t in a 1.5-mL to ta l volume, a 3-mL cuvette was used under magnetic s t i r r i n g and the upper part of the l i g h t path o f the spectrophotometer was blocked with black tape to avoid i n t e r f e r e n c e by the turbulence produced in the l i q u i d / a i r i n t e r f a c e . 13. Determination of ca lpa in a c t i v i t y ( c a s e i n o l y s i s ) Ca lpa in samples were assayed using casein as substra te in a t o t a l volume of 0.5 mL conta in ing 2 mg.mL"^ c a s e i n , 50 mM T r i s - H C l , 100 /xM EGTA, 1 mM d i t h i o t h r e i t o l and 400 /xM C a C l 2 (pH 7 .4 ) , unless otherwise s t a t e d . The r e a c t i o n was c a r r i e d out at 37°C f o r 30 min and terminated by the add i t ion o f 0.5 mL of 5% (w/v) t r i c h l o r o a c e t i c a c i d . A f t e r 10 min on i c e , 74 the samples were cent r i fuged at 4000 x g m a x fo r 5 min and the absorbance of the supernatant was measured spectrophotometr ica l ly at 278 nm. Blanks were run under the same condi t ions except that CaCl2 was not added. The absorbance of the blanks was subtracted from the t o t a l absorbance in the presence of ca lc ium. A uni t of ca lpa in a c t i v i t y w i l l produce an increase of one absorbance uni t at 278 nm under the standard assay c o n d i t i o n s . 14. Treatment of ca lmodul in-depleted plasma membranes with var ious  proteases Calmodul in-depleted plasma membranes were t reated with the proteases ind ica ted with one of the fo l lowing methods: Method a) plasma membranes (1-2 mg prote in) suspended in buf fer IV-E were d i l u t e d (1:1) to obta in the fo l lowing f i n a l concent ra t ions : 65 mM KC1, 19 mM potassium-Hepes, 250 /iM M g C l 2 , 10 mM d i t h i o t h r e i t o l , the ind icated protease and a combination o f EGTA and C a C l 2 to obtain a concentrat ion of f ree C a 2 + of 200 /iM (pH 7 .4 ) . Method b) plasma membranes (50-75 ng prote in) suspended in buf fe r IV were incubated with the protease ind icated in an incubat ion medium conta in ing 55 mM T r i s - m a l e a t e , 66 mM NaCl , 0.1 mM ouabain, 150 /xM EGTA, 6.5 mM M g C l 2 , 1 mM d i t h i o t h r e i t o l and 350 /iM C a C l 2 (pH 7 .4 ) . The reac t ion was s ta r ted by adding the protease and incubat ing at 25°C f o r 60 min, unless stated otherwise. The p r o t e o l y s i s was stopped by the add i t ion of at l e a s t a 10 f o l d excess of soybean t r y p s i n i n h i b i t o r in the case of t r y p s i n , or leupept in in the case of papain or c a l p a i n . When r e q u i r e d , the membranes were cooled on ice f o r 10 min and cent r i fuged at 4000 x g m a x f o r 5 min. The p e l l e t was washed 4 times with 1 mM EGTA, 20 mM T r i s - H C l (pH 7.4) and 3 times with buf fe r IV-E . The f i n a l ca lmodu l in - f ree p e l l e t was 75 resuspended in buf fer IV-E to the s t a r t i n g volume before assaying fo r ATPase a c t i v i t y or the 3 2 P - p h o s p h o r y l a t e d intermediate . 15. Treatment of the p u r i f i e d C a 2 + - A T P a s e with c a l p a i n P u r i f i e d C a 2 + - A T P a s e (40-60 /ig prote in) was incubated with 0.02 uni t .mL"* c a l p a i n at 25°C in an appropriate volume of 55 mM T r i s - m a l e a t e , 5 mM potassium-Hepes, 6.5 mM M g C l 2 , 0.5 mM EDTA, 10 mM d i t h i o t h r e i t o l , 300 nM calmodul in (when added), 65 mM K C 1 , and a combination o f EGTA and C a C l 2 to obta in a f ree C a 2 + concentrat ion of 200 /zM (pH 7 .4 ) . At the des i red t ime, a l i q u o t s were taken fo r Ca 2 + -ATPase assay and determinat ion of the phosphorylated intermediate in the presence of 200 /iM leupept in and subjected to gel e l e c t o p h o r e s i s . 16. Treatment of the reconst i tu ted Ca^-ATPase with p u r i f i e d c a l p a i n Proteoliposomes (20-60 /ig pro te in .mL" 1 p lus 11 mg phospho l ip ids .mL" 1 ) were incubated at 25° C f o r 120 min (unless stated otherwise) in a medium conta in ing 67 mM K C 1 , 50 mM potassium-Hepes (pH 7 .4 ) , 0.67 mM M g C l 2 , 10 mM d t t h i o t h r e i t o l , 2 u n i t . m L " 1 ca lpa in I (unless stated o therwise ) , 40-100 /iM EGTA and var ious concentrat ions of C a C l 2 (440-500 /iM) to a t t a i n 400 /iM f ree ca lc ium i o n . P r o t e o l y s i s was arrested by the add i t ion of 200 /iM l e u p e p t i n . 17. Formation of the phosphorylated intermediate of the recons t i tu ted  C a 2 + - A T P a s e Proteol iposomes (60-120 /tg prote in plus 32 mg phosphol ip id ) were incubated at 4°C fo r 15 s in 400 /iL of 50 mM K C 1 , 25 mM potassium-Hepes, 76 pH 7 .4 , 0.1 mM EGTA, 0.1 mM EDTA, 0.5 mM M g C l 2 , 2.5 mM C a C l 2 , 100 /xM L a C l 3 , 300 nM calmodulin and 4 /xM ATP contain ing 12 mCi.mmol" 1 [ 7 - 3 2 P ] A T P . The r e a c t i o n was i n i t i a t e d by the addi t ion of [ 7 - 3 2 P ] A T P and terminated by the add i t ion of i c e - c o l d 10 % (w/v) t r i c h l o r o a c e t i c a c i d . The mixture was spun at 10,000 g m a x fo r 5 min. The p e l l e t was then t reated with 500 /xL chloroform to ext ract the phospho l ip id . The mixture was spun again and the chloroform phase was removed. The remaining pro te in p r e c i p i t a t e was then processed f o r ac id -ge l e lec t rophores is at pH 6.6 as descr ibed above. The d r i ed gel was exposed to X-ray f i l m fo r 3-6 days at - 7 0 ° C . 18. PEST sequence i d e n t i f i c a t i o n and PEST score c a l c u l a t i o n PEST sequences are i d e n t i f i e d using the PEST-FIND computer program developed by Rogers et a l . (1986). PEST sequence was def ined as a s t re tch of amino ac id residues beginning and ending with p o s i t i v e l y charged res idues (H,K or R) with a number of in terna l res idues of at l e a s t 8, conta in ing residue P, E, D, S and T. A PEST score i s c a l c u l a t e d as descr ibed by Rogers et a l . , (1986); in b r i e f , PEST score of the sequence = 0.55 (MPpES j ) " 0-5 (H0)> where MPpr/^j = mole percent of P, E, D, S and T a f t e r subt rac t ing one mole equivalent of P, E/D and S / T , and H 0 = average hydrophobic i ty of the s t re tch (Rogers et a l . , 1986). To q u a l i f y as a PEST sequence, the PEST score value has to be l a r g e r than - 5 . 0 . When the s t r e t c h i s missing e i ther P, E/D or S / T , then i t s PEST score has to be l a r g e r than 0 to q u a l i f y as a PEST sequence (see legend of Table 8 fo r a l i s t o f the o n e - l e t t e r codes of amino acid r e s i d u e s ) . 19. Data ana lys is The fragmentation patterns shown in several f i gures of t h i s thes is 77 were a l l reproduced in separate experiments (n = 2 to 5 ) . The t o t a l number of separate experiments performed i s i n d i v i d u a l l y stated in the legend of these f i g u r e s . Due to the v a r i a b i l i t y in enzyme a c t i v i t y of the C a 2 + -ATPase and c a l p a i n , in c e r t a i n f i g u r e s , i t was decided tha t , ra ther than showing mean values ± S . E . M . , data from t y p i c a l experiments would be shown. However, the trends and patterns of the i l l u s t r a t e d experiments were reproduced in separate experiments (n= 2 to 4 ) . A l s o , mean value ± S . E . M . of untreated (contro l ) enzyme a c t i v i t y from separate experiments i s given in the legend of these f i g u r e s . 78 RESULTS I P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of c a l p a i n 1. P u r i f i c a t i o n of ca lpa in In order to study the e f f e c t of ca lpa in I on the erythrocyte C a 2 + -ATPase, i t was important to p u r i f y the protease, p re fe rab ly from the same source. There fore , ca lpa in I was p u r i f i e d from a human erythrocyte c y t o s o l i c f r a c t i o n . B r i e f l y , the p u r i f i c a t i o n involved (a) i s o l a t i o n of plasma membrane-free cytosol prote in f r a c t i o n using l a r g e - s c a l e u l t r a f i l t r a t i o n ( P e l l i c o n casset te system) and (b) four steps of column chromatography. Each step exp lo i ted a d i f f e r e n t property of the protease: 1) i o n i c i n t e r a c t i o n with the DEAE-Sephacel ( F i g . 10), 2) hydrophobic i n t e r a c t i o n with phenyl-Sepharose ( F i g . 11), 3) mixed ion ic /hydrophobic i n t e r a c t i o n with omega-hexylamine-agarose ( F i g . 12) and 4) separat ion by molecular s i z e on a gel f i l t r a t i o n (Sephacryl S-200) column ( F i g . 13). F i g . 14 shows the prote in p r o f i l e a f te r each step of p u r i f i c a t i o n . Note that s i g n i f i c a n t p u r i f i c a t i o n was achieved a f te r the phenyl-Sepharose step ( F i g . 14, lane 3) . A f t e r the l a s t step of chromatography, apparent homogeneity of ca lpa in I was achieved ( F i g . 14, lane 5) . It was of i n t e r e s t to note that i n i t i a l l y when only 1 mM EGTA was used in the hemolysis b u f f e r , p u r i f i e d c a l p a i n I c o n s i s t e n t l y contained the 80 kDa subunit and i t s ac t iva ted fragments (78 and 75 kDa). When 5 mM EGTA was 79 F i g . 10 DEAE-Sephacel chromatography of ca lpa in I from human erythrocyte  hemolysate. 150 mL of DEAE-Sephacel was subjected to batch-b inding with 4 L of membrane-free hemolysate, packed into a g lass column and subsequently washed with 2 L of buf fe r A . The column was then e luted with a gradient (0-350 mM) of NaCl in buf fer A (dashed l i n e ) . Prote in concentra t ion was monitored from the absorbance at 280 nm ( s o l i d l i n e ) . Calpa in a c t i v i t y was measured as calcium-dependent p r o t e o l y s i s of c a s e i n , as descr ibed in Mater ia ls and Methods ( f i l l e d c i r c l e s ) . Each e l u t i o n f r a c t i o n was 7 mL and the pooled f r a c t i o n s are ind icated by a s o l i d bar. Data presented here are from a t y p i c a l preparat ion of c a l p a i n . 80 18 ( ) NaCI gradient (mM) —» rO OJ O O O O O O O o H 1 1 1 ( • • ) C a l p a i n a c t i v i t y ( A A 2 7 8 ) o p 0 o o M ^ o> o p 0 ^ O CO rO ( ) A b s o r b a n c e A 2 8 O 01 3Jn6Lj F i g . 11 Phenyl-Sepharose chromatography of DEAE-Sephace l - iso la ted ca lpa in JL Pooled c a l p a i n f r a c t i o n s from DEAE-Sephacel were appl ied to a column of phenyl-Sepharose (1.5 x 8.5 cm) ( load ) , the column was then washed ex tens ive ly with buf fe r A conta in ing 200 mM NaCl (wash). The column was f i n a l l y e luted with buf fer A (e lu te ) . Prote in concentra t ion ( s o l i d l i n e ) and c a l p a i n a c t i v i t y ( f i l l e d c i r c l e s ) were measured as in F i g . 10. Each f r a c t i o n was 7 mL and the pooled f r a c t i o n s are ind ica ted with a s o l i d bar . Data presented here from a t y p i c a l ca lpa in p repara t ion . 82 £8 F i g . 12 Omega-hexylamine-agarose chromatography of phenyl-Sepharose- p u r i f i e d ca lpa in I. Pooled ca lpa in f r a c t i o n s from the phenyl-Sepharose column were appl ied d i r e c t l y to a omega-hexylamine agarose column (1.5 x 5.5 cm). The column was washed with 100 mL of buf fe r A before being eluted with a gradient (0-350 mM) of NaCl in buf fer A (dashed l i n e ) . Protein concentrat ion ( s o l i d l i n e ) and ca lpa in a c t i v i t y ( f i l l e d c i r c l e s ) were monitored as in F i g . 10. Each f r a c t i o n was 5 mL and the pooled ca lpa in f r a c t i o n s are ind icated with a s o l i d bar . Data presented here are from a t y p i c a l preparat ion of c a l p a i n . 84 S8 ( ) NaCl gradient (mM) ( ) A b s o r b a n c e A 2 5 0 Zl aan6 L J F i g . 13 Sephacryl S-200 chromatography of omega-hexylamine-agarose- p u r i f i e d ca lpa in I. Pooled ca lpa in f r a c t i o n s from omega-hexylamine-agarose column chromatography were concentrated to 2 mL with an u l t r a f i l t r a t i o n uni t (Amicon) f i t t e d with a PM-10 membrane. The concentrated sample was then appl ied to a Sephacryl S-200 column (1.5 x 60 cm) coupled to an FPLC system. The column was developed with buf fer F and f o r t y 2 mL- f rac t ions were c o l l e c t e d at a flow rate of 10 m L . h " 1 . The void volume (40 mL) as determined using Blue Dextran-2000 i s ind ica ted ( V Q ) . E l u t i o n volume of gel f i l t r a t i o n prote in standards: human 7 -g lobul in (160 kDa) (a ) , bovine serum albumin (67 kDa) (b) , ovalbumin (43 kDa) ( c ) , chymotrypsinogen A (22 kDa) and r ibonuclease A (14 kDa) are 49.5 , 63.2 , 69.5 , 84.2 and 92.8 mL, r e s p e c t i v e l y . The e l u t i o n volume (60 mL) of ca lpa in I i s ind ica ted ( V e ) . Prote in concentrat ion ( s o l i d l i n e ) and ca lpa in a c t i v i t y ( f i l l e d c i r c l e s ) were monitored as in F i g . 10. Pooled f r a c t i o n s are ind ica ted with a s o l i d bar . Data presented here are from a t y p i c a l ca lpa in p repara t ion . 86 F i g . 14 P u r i f i c a t i o n of ca lpa in from human e r y t h r o c y t e s . SDS-PAGE gel showing the peptide pattern at var ious stages of p u r i f i c a t i o n : (1) membrane-free hemolysate, (2) a f te r DEAE-Sephacel, (3) a f t e r phenyl -Sepharose, (4) a f t e r omega-hexylamine agarose and (5) a f t e r Sephacryl S-200. The pro te in patterns presented here were r o u t i n e l y reproduced in separate c a l p a i n prepara t ions . 88 F i g u r e 14 2 3 4 5 KDa 89 used, the 80 kDa subunit remained i n t a c t . This ind ica tes that a por t ion of ca lpa in was ac t iva ted in the hemolysate f r a c t i o n , despi te the f a c t that c a l p a s t a t i n was in excess when compared to ca lpa in in human erythrocytes (see Murachi , 1989). The recovery of ca lpa in a c t i v i t y was a lso fol lowed (Table 2 ) . Twenty-two percent recovery was found while the protease was p u r i f i e d 4011-fold (means f o r three p repara t ions ) . S ta r t ing with 300-400 mL of red c e l l s , about 1 mg of ca lpa in I can be p u r i f i e d . Since human erythrocytes conta in only ca lpa in I but not ca lpa in II, from t h i s point on, f o r convenience, "ca lpa in" r e f e r s to ca lpa in I, unless stated otherwise. 2. Charac te r i za t ion of ca lpa in The autohydro lys is of two preparat ions of c a l p a i n was analysed by SDS-PAGE. It was c l e a r l y i l l u s t r a t e d that in the presence of 10 mM DTT and 200 /zM f ree C a 2 + , the 80 kDa c a t a l y t i c subunit was transformed into a t rans ien t 78 kDa fragment fol lowed by the accumulation of the 75 kDa fragment p rev ious ly i d e n t i f i e d to be the ac t iva ted form of the protease ( F i g . 15A, lane 2, 3, 5 and 6 ) . The small subunit was a lso transformed into a smal ler fragment. The fac t that the 80 kDa subunit can hydrolyze i t s e l f i nd ica tes that t h i s i n t a c t form of c a l p a i n must a lso express some degree of p r o t e o l y t i c a c t i v i t y . This argument s e t t l e s a common misconception that the 80 KDa subunit i s the i n a c t i v e form while the 76 kDa fragment i s the ac t ive form of ca lpa in (see Pontremoli et a l . , 1985a). 90 Table 2 P u r i f i c a t i o n of ca lpa in I from human erythrocytes STEP S p e c i f i c Vol Protein A c t i v i t y P u r i f i c a t i o n Recovery (mL) (mg) (unit/mg p ro t . ) ( fo ld ) Membrane-free 4000 hemolysate DEAE-Sephacel 50 Phenyl-Sepharose 75 Omega-hexylamine- 35 agarose 18810 0.0126 3 78.1 3.03 13.7 23.87 3.6 41.02 V 240 1894 3256 100c 100 138 62 Sephacryl S-200 8 1.03 50.55 4011 22 a assuming f u l l recovery from step 1 to step 2 Values presented are averages of three preparat ions F i g . 15 A u t o l y s i s of p u r i f i e d ca lpa in (A) and e f f e c t of assay temperature  on c a l p a i n a c t i v i t y (B). In (A), two c a l p a i n preparat ions (1.5 /ig prote in) p u r i f i e d to the omega-hexylamine-agarose step ( lane 1, 2 and 3) and to the Sephacryl S-200 step (lane 4, 5 and 6 ) , r e s p e c t i v e l y , were incubated in the presence of 200 /iM f ree C a 2 + , 10 mM DTT, 50 mM T r i s - H C l f o r 0 min (lane 1 and 4) , 30 min (lane 2 and 5) or 60 min (lane 3 and 6) at 2 5 ° C . P r o t e o l y s i s was stopped with 20 % (w/v) TCA and the samples were processed fo r SDS-PAGE. The p r o t e o l y s i s pat terns were reproduced in two other experiments. In (B), p u r i f i e d c a l p a i n (4 /tg) was assayed as descr ibed in Mater ia ls and Methods using casein as a substra te at 25°C (open c i r c l e s ) or 37°C ( f i l l e d c i r c l e s ) . P r o t e o l y s i s was stopped with the add i t ion of 10 % (w/v) TCA at the ind icated t ime. The f i g u r e in panel B presents the mean values for 3 d i f f e r e n t experiments. 92 F i g u r e 15A KDa 1 2 3 4 5 93 Figure 15 B B 94 The e f f e c t of assay temperature on ca lpa in a c t i v i t y was assessed ( F i g . 15B). Calpa in a c t i v i t y p rogress ive ly decreased with time at 37°C a f t e r 30 min. whereas at 25°C ca lpa in a c t i v i t y remains r e l a t i v e l y l i n e a r f o r at l e a s t 60 min, as descr ibed by Mellgren et al_. (1982). For the determinat ion of ca lpa in a c t i v i t y , the re fo re , the protease was assayed r o u t i n e l y at 37°C fo r only 30 min. However, to tes t the e f f e c t of the p u r i f i e d c a l p a i n on the C a 2 + - A T P a s e , incubat ion at 25°C was chosen s ince p r o t e o l y s i s time beyond 30 min was usua l ly requ i red . In the next s e r i e s of experiments, i t was confirmed that the preparat ions of ca lpa in exhib i ted the expected p roper t i es of c a l p a i n I. F i g . 16A shows that the a c t i v i t y of p u r i f i e d c a l p a i n has an absolute requirement f o r ca lc ium. Half-maximal a c t i v a t i o n of the enzyme occurred at 20 iiM f ree C a 2 + . Thus the p u r i f i e d protease belongs to the c l a s s designated c a l p a i n I, which requi res micromolar concent ra t ions of C a 2 + to be f u l l y a c t i v e . Calpain a c t i v i t y was found to increase with the concentra t ion of DTT in the assay medium up to 20 mM. Ca lpa in i s a lso known to be h igh ly suscept ib le to cys te ine -pro tease i n h i b i t o r s (eg. leupept in) and a l k y l a t i n g agents (eg. iodoaceta te ) . F i g . 16B shows that the protease was i n h i b i t e d by these compounds as expected. On the other hand, PMSF was considerably l e s s potent as an i n h i b i t o r , whi le soybean t r y p s i n i n h i b i t o r increased the ca lpa in a c t i v i t y observed ( F i g . 16B). II. E f f e c t of c a l p a i n on the membrane-bound C a 2 + - A T P a s e 95 F i g . 16 A c t i v a t i o n of ca lpa in by C a ^ + and the e f f e c t of var ious  i n h i b i t o r s on ca lpa in a c t i v i t y . (A) The p u r i f i e d enzyme (4 /ig prote in) was assayed with var ious concentrat ions of C a 2 + as ind ica ted in Mater ia ls and Methods. (B) The p u r i f i e d enzyme (4 /ig prote in) was assayed with 100 7+ /iM f ree Ca in the presence of the ind icated concentrat ions of leupept in ( c i r c l e s ) , iodoacetate (squares) , PMSF ( inver ted t r i a n g l e ) or soybean t r y p s i n i n h i b i t o r ( t r i a n g l e ) . The maximum a c t i v i t y ( c a s e i n o l y s i s ) achieved was 0.20 u n i t . h " 1 (see Mater ia ls and Methods fo r uni t d e f i n i t i o n ) and was taken as 100% ca lpa in a c t i v i t y . Data presented here are t y p i c a l of three separate experiments. The untreated c a l p a i n a c t i v i t y (mean ± S . E . M . ) of the three experiments was 0.34 ± 0.15 u n i t . h " 1 . KQ 5 (c a ) obtained from the three experiments (mean ± S . E . M . ) i s 20 ± 8 /iM. 96 Figure 16 120 -i -10 - 8 - 6 -4 - 2 Log [Inhibitor] (M) 97 1. E f f e c t of t r y p s i n , papain and ca lpa in on the a c t i v i t y of the membrane-bound C a ^ - A T P a s e The plasma membrane-bound Ca -ATPase a c t i v i t y was tes ted a f te r progress ive exposure to c a l p a i n , t r yps in or papain ( F i g . 17). Both t r y p s i n and papain ac t iva ted the Ca -ATPase a c t i v i t y maximally w i th in 10 min, when assayed in the absence of ca lmodul in , but the a c t i v a t i o n was fol lowed immediately by a progress ive i n a c t i v a t i o n of the C a 2 + - A T P a s e a c t i v i t y at longer per iods of pre incubat ion with the proteases ( F i g . 17A and 17B). However, in the case of ca lpa in the a c t i v a t i o n of the C a 2 + - A T P a s e a c t i v i t y under these cond i t ions was not fol lowed by i n a c t i v a t i o n at l e a s t fo r 120 min of treatment with the protease ( F i g . 17C). When assayed in the presence of calmodulin the C a z + - A T P a s e a c t i v i t y was not f u r t h e r inc reased . Th is was true f o r the three proteases, suggesting that c a l p a i n may have ac t iva ted the Ca -ATPase by a s i m i l a r mechanism to that of t r y p s i n or papain . There fore , i t i s conceivable that c a l p a i n , l i k e t r y p s i n or papain , might a c t i v a t e the C a 2 + - A T P a s e by c leav ing the ca lmodul in -b ind ing domain of the enzyme (Sarkadi et a l . , 1986). F i g . 17D shows that pre incubat ion of the plasma membrane with ca lpa in in the presence of 2.5 mM EGTA has no e f f e c t on the C a 2 + - A T P a s e a c t i v i t y in the presence or in the absence of ca lmodul in . The e f f e c t of pre incubat ion of the plasma membrane with d i f f e r e n t protease concentrat ions on the C a 2 + - A T P a s e a c t i v i t y was examined ( F i g . 18). When assayed in the absence of ca lmodul in , the C a 2 + - A T P a s e a c t i v i t y was ac t iva ted maximally at t r y p s i n and papain concentra t ions of 10"^ M and 98 F i g . 17 Time-course of the e f fec t of proteases on the membrane-bound C a - ATPase a c t i v i t y . Membranes (60 /ig prote in) were t rea ted with 8.5 x 10~ 7 M t r y p s i n (A) , 9.1 x 10~ 7 M papain (B) or 6.4 x 10~ 7 M c a l p a i n (C and D), according to method (b) (see Mater ia ls and Methods), except that in A, B and C membranes were incubated in the presence of 250 /iM C a C l 2 whereas in D, membranes were incubated in the absence of added C a C l 2 and in the presence of 2.5 mM EGTA. Protease was added to s t a r t the p r o t e o l y s i s . Protease i n h i b i t o r was added a f te r the incubat ion was completed and p r i o r to the add i t ion of proteases to obtain the zero time va lues . The membranes were assayed thereaf ter fo r C a 2 + - A T P a s e a c t i v i t y at 37°C f o r 30 min at 100 /iM f ree Ca , in the absence (open c i r c l e s ) or in the presence (c losed c i r c l e s ) of 120 nM calmodul in . Data presented here are from a t y p i c a l experiment. The trends and patterns presented were reproduced in three separate experiments but the t ime-courses var ied s l i g h t l y . The untreated (zero time) basal and ca lmodul in -s t imulated membrane-bound C a 2 + -ATPase a c t i v i t y at 200 /iM f ree C a 2 + (mean ± S .E .M . ) from the three experiments was 26.7 ± 7.2 and 48.5 ± 10.1 nmol .mg p r o t . _ 1 . m i n " * , r e s p e c t i v e l y . 99 Figure 17 100 F i g . 18 E f f e c t of protease concentrat ion on the membrane-bound Ca - ATPase a c t i v i t y . The ind icated protease concentrat ions were used f o r t r y p s i n (A), papain, (B) , or ca lpa in (C) during pretreatment at 25°C f o r 60 min according to method (b) (see Mater ia ls and Methods). The membranes (60 fig prote in) were then assayed fo r C a 2 + - A T P a s e a c t i v i t y at 37°C f o r 30 min at 20 zzM f ree Ca^ in the absence (open c i r c l e s ) or in the presence (c losed c i r c l e s ) of 120 nM calmodul in . Data presented here are t y p i c a l of two experiments. 101 Figure 18 10"' M, r e s p e c t i v e l y . Fu l l a c t i v a t i o n was associa ted with the loss of s e n s i t i v i t y to exogenously added ca lmodul in . The C a 2 + - A T P a s e a c t i v i t y was d ramat ica l l y decreased at s l i g h t l y higher t r y p s i n or papain concent ra t ions . In con t ras t , the C a 2 + - A T P a s e was maximally ac t iva ted at 10"^ M c a l p a i n and the a c t i v a t i o n was maintained when the ca lpa in concentrat ion was increased by two orders of magnitude (up to 10"^ M). A decrease in Ca^ -ATPase a c t i v i t y was subsequently observed at higher concentrat ions of c a l p a i n . 2. Protect ion by calmodulin of the membrane-bound C a 2 + - A T P a s e against  p r o t e o l y t i c a c t i v a t i o n by ca lpa in The presence of calmodulin (0.3 /iM) dur ing t r y p s i n or papain incubat ion d id not prevent the p r o t e o l y t i c a c t i v a t i o n of the C a 2 + - A T P a s e a c t i v i t y ( F i g . 19). However, the p r o t e o l y t i c a c t i v a t i o n of the C a 2 + -ATPase a c t i v i t y induced by ca lpa in was s i g n i f i c a n t l y reduced by calmodulin in a concentrat ion dependent manner ( F i g . 20) . The maximum percent p ro tec t ion achieved ranges from 67% to 84% depending on the f ree C a 2 + concentrat ion in the assay system (see F i g . 20) . From a p l o t of the percent p ro tec t ion against p r o t e o l y t i c a c t i v a t i o n of the C a z + - A T P a s e versus the concentrat ion of added ca lmodul in , the concentra t ion needed fo r h a l f maximum pro tec t ion (KQ 5 ) was found to be 1.9 ± 0.4 nM ( F i g . 21A), which was comparable to the KQ 5 fo r a c t i v a t i o n of the C a 2 + - A T P a s e by calmodulin (2.2 ± 0.3 nM) ( F i g . 21B). 103 F i g . 19 E f f e c t of calmodulin on the act ion of proteases on the  membrane-bound C a c - A T P a s e . Membranes (1.5 mg prote in) were t reated with protease as f o l l o w s : none (A), 4.3 x 10~ 7 M t r y p s i n (B), 9.1 x 10~ 8 M papain (C) at 25°C fo r 60 min in the absence ( c i r c l e s ) or in the presence (squares) of 300 nM calmodulin according to method (a) (see Mater ia ls and Methods). The membranes were subsequently washed as descr ibed in Mater ia ls and Methods and then assayed (60 /iM prote in) f o r C a 2 + - A T P a s e a c t i v i t y at 37°C f o r 30 min in the absence (open symbols) or in the presence ( f i l l e d symbols) of 120 nM ca lmodul in . Data presented here are t y p i c a l of three separate experiments. The untreated basal and ca lmodul in -st imulated membrane-bound C a 2 + - A T P a s e a c t i v i t y at 4.3 /iM f ree C a 2 + (mean ± S .E .M. ) from the three experiments was 7.2 ± 3.7 and 40.1 + 13.1 nmol.mg p r o t . ~ * . m i n " * , r e s p e c t i v e l y . 104 Figure 19 F i g . 20 E f f e c t of calmodulin on ca lpa in act ion on the membrane-bound  C a - A T P a s e . Membranes (1.5 mg prote in) were untreated ( inver ted f i l l e d t r i a n g l e s ) or t reated with ca lpa in (6.4 x 10" 7 M) in the presence of var ious concentrat ions of calmodul in: none ( f i l l e d squares) , 2.7 x 10 M (open squares) , 2.7 x 1 0 " 1 1 M (X), 2.7 x 1 0 " 1 0 M (+), 2.7 x 10" 9 M _Q _7 ( inver ted open t r i a n g l e s ) , 2.7 x 10 M (open t r i a n g l e s ) , 2.7 x 10 M (open c i r c l e s ) , 2.7 x 10"^ M ( f i l l e d c i r c l e s ) according to method (a) (see Mater ia ls and Methods). The membranes were subsequently washed as descr ibed in Mater ia ls and Methods and then assayed (60 /ig prote in) fo r C a 2 + - A T P a s e in the absence (A) or in the presence (B) of 120 nM ca lmodul in . Data presented here are t y p i c a l of three separate experiments. The untreated basal and calmodul in -s t imu la ted membrane-bound C a 2 + - A T P a s e a c t i v i t y at 4.3 /iM f ree C a 2 + (mean ± S .E .M . ) i s as given in the legend of F i g . 19. 106 F i g . 21 E f f e c t of calmodulin on the p r o t e o l y t i c a c t i v a t i o n of the Ca 2" 1"- ATPase a c t i v i t y by ca lpa in (A) and the C a 2 + - A T P a s e a c t i v i t y (B) . In (A) , membranes (60 /ig prote in) were t reated with ca lpa in (6.7 x 10"^ M) at 25°C f o r 60 min according to method (b) (see Mater ia ls and Methods) in the presence of the ind ica ted concentrat ion of ca lmodul in . Then membranes were subsequently washed as descr ibed in Mater ia ls and Methods and assayed fo r C a 2 + - A T P a s e a c t i v i t y at 37°C f o r 30 min at 4.3 /iM of f ree C a 2 + in the absence of ca lmodul in . The a c t i v a t i o n of the C a 2 + - A T P a s e a c t i v i t y by c a l p a i n in the absence of calmodulin ( A c t i v a t i o n c a - | p a i - n ) or presence of d i f f e r e n t concentrat ions of calmodulin ( A c t i v a t i o n c a - | p a ^ n ( C a M ) ) was c a l c u l a t e d as the d i f f e r e n c e between the C a 2 + - A T P a s e a c t i v i t y ( in the absence o f calmodulin) of the c a l p a i n - t r e a t e d membranes and that of the untreated membranes. The calmodulin pro tec t ion against ca lpa in-mediated p r o t e o l y t i c a c t i v a t i o n of the C a 2 + - A T P a s e a c t i v i t y was then c a l c u l a t e d by the fo l lowing equat ion: Calmodulin Pro tec t ion (%) = A c t i v a t i o n c a l p a i n ( C a M ) A c t i v a t i o n ca lpa in x 100 In (B) , the Ca*- -ATPase was assayed as descr ibed in Mater ia ls and Methods in the absence or presence of the ind icated concentrat ion of ca lmodul in . The calmodul in a c t i v a t i o n was ca lcu la ted as the d i f f e r e n c e of the C a 2 + -ATPase a c t i v i t y in the presence versus the absence of calmodulin at 4.3 /iM p. of f ree Ca . The maximum calmodulin a c t i v a t i o n was 47 nmol.mg p r o t . " * . m i n " 1 and was taken as 100%. Data presented here are t y p i c a l of three separate experiments. From these experiments, the KQ 5 108 F i g . 21 (Cont. ) f o r p ro tec t ion (mean ± S . E . M . ) was found to be 1.9 ± 0.4 nM while the KQ 5 f o r a c t i v a t i o n (mean ± S . E . M . ) was found to be 2.2 ± 0.3 nM. 109 Figure 2 1 1 1 0 The p o s s i b i l i t y that calmodulin has a d i r e c t e f f e c t on c a l p a i n was a lso excluded by showing that calmodulin d id not modify c a l p a i n a c t i v i t y when case in was used as a substrate ( r e s u l t s not shown). 3. Further c h a r a c t e r i z a t i o n of the ca lpa in d i g e s t i o n of the membrane-bound  C a 2 + - A T P a s e in the absence and the presence of calmodulin A t ime-course of the e f f e c t of c a l p a i n on the membrane-bound C a 2 + -ATPase a c t i v i t y i s presented in F i g . 22. In the absence o f calmodulin the protease p r o g r e s s i v e l y ac t iva ted the C a 2 + - A T P a s e and decreased i t s capac i ty to be st imulated by ca lmodul in . However, in the presence of calmodulin the p r o t e o l y t i c a c t i v a t i o n of the C a z + - A T P a s e was s i g n i f i c a n t l y reduced and the calmodulin s t imula t ion was preserved. Binding of calmodulin to the membrane-bound C a 2 + - A T P a s e could prevent i t s p r o t e o l y s i s or induce a d i f f e r e n t pathway of f ragmentat ion. Therefore , the fragmentation of the membrane-bound C a 2 + - A T P a s e was op examined by fo l lowing the P-phosphorylated intermediate of the enzyme. It was found that the fragmentation pattern of the membrane-bound C a 2 + -ATPase was indeed d i f f e r e n t in the absence versus the presence of calmodulin ( F i g . 23). In the absence of ca lmodul in , the nat ive enzyme (136 kDa) was r a p i d l y transformed into a 124 kDa fragment wi th in 1-2 min. This was fol lowed therea f te r by the progress ive formation of a second fragment of lower molecular weight (80 kDa). In the presence of ca lmodul in , however, 111 F i g . 22 A c t i v i t y of the membrane-bound Ca -ATPase t reated with ca lpa in in  the presence and absence of ca lmodul in . Membranes (1.2 mg prote in) were t reated with c a l p a i n in the absence ( c i r c l e s ) or in the presence (squares) of calmodulin as descr ibed in Mater ia ls and Methods. A l iquo ts (56 /ig membrane prote in) were taken at the ind ica ted time and the membranes were washed and therea f te r assayed fo r C a z + - A T P a s e a c t i v i t y with 4.3 /iM of f ree C a 2 + in the absence (open symbols) or in the presence ( f i l l e d symbols) of calmodulin (see Mater ia ls and Methods). Data presented are t y p i c a l of three separate experiments. The untreated (zero time) basal and ca lmodul in -s t imula ted membrane-bound C a z + - A T P a s e a c t i v i t y at 4.3 /iM f ree C a 2 + (mean ± S .E .M . ) from the three experiments was 14.7 ± 6 . 1 and 72.1 ± 17.0 nmol.mg p r o t . _ 1 . m i n " 1 , r e s p e c t i v e l y . 112 Figure 22 113 F i g . 23 Formation of the phosphorylated intermediate by the nat ive  membrane-bound C a 2 + - A T P a s e and the fragments produced by ca lpa in  treatment. Membranes (2.1 mg prote in) were t reated with ca lpa in in the absence (-) or in the presence (+) of calmodulin (CaM), as descr ibed in Mate r ia ls and Methods. At the ind ica ted t imes, a l iquo ts (100 /ig membrane prote in) were taken and t ransfered to 1.5 mL-microcentr i fuge tubes conta in ing 1 mL of buf fe r IV-E and 200 /iM l e u p e p t i n . The membranes were washed and resuspended f o r phosphory la t ion , e l e c t r o p h o r e s i s and autoradiography, as descr ibed in Mater ia ls and Methods. The fragmentation patterns presented here were reproduced in four other separate experiments. 114 F i g u r e 23 115 the r e l a t i v e molecular masses of the ^ P - l a b e l l e d fragments were 127 kDa and 85 kDa, r e s p e c t i v e l y . The phosphorylat ion of the bands was dependent on the presence of C a 2 + and they were s e n s i t i v e to hydroxylamine treatment ( r e s u l t s not shown), demonstrating that they indeed represented fragments of the Ca^ -ATPase capable of forming the acylphosphate intermediate . I I I . P r o t e o l y s i s of the p u r i f i e d C a - A T P a s e by c a l p a i n  1. Ca lpa in d i g e s t i o n of the p u r i f i e d C a 2 + - A T P a s e The p u r i f i e d C a 2 + - A T P a s e was a lso subjected to ca lpa in d i g e s t i o n . F i g . 24A shows a t ime-course of ca lpa in d i g e s t i o n on the a c t i v i t y of the p. p u r i f i e d Ca*- -ATPase. Calpain p r o t e o l y s i s in the absence of calmodulin produced a s l i g h t a c t i v a t i o n of the C a 2 + - A T P a s e in the f i r s t 15 min while the a c t i v i t y in the presence of calmodulin remained approximately the same during t h i s time per iod . The basal and the ca lmodul in -s t imulated a c t i v i t i e s dec l ined progress ive ly t h e r e a f t e r . A f t e r 120 min of p r o t e o l y s i s , the calmodulin s t imulat ion was decreased from the o r i g i n a l 3 . 3 - f o l d to 1 .4 - fo ld ( F i g . 24A). Calpain d i g e s t i o n in the presence of calmodul in a lso produced a progressive decrease of the maximum C a 2 + -ATPase a c t i v i t y ( F i g . 24A). Next, the e f f e c t of d i f f e r e n t ca lpa in concentrat ions on the C a 2 + -ATPase a f t e r a prolonged period of incubat ion (2 hours) was fo l lowed. F i g . 24B shows that in the absence of calmodulin increas ing concentrat ions 116 F i g . 24 E f f e c t of ca lpa in on the p u r i f i e d Ca -ATPase a c t i v i t y . (A) P u r i f i e d Ca^ -ATPase (6.5 zzg prote in) was treated with c a l p a i n in the absence ( c i r c l e s ) or in the presence (squares) of ca lmodul in , as descr ibed in Mater ia ls and Methods. At the ind ica ted t imes, a l i q u o t s were taken and added to the assay medium (a lso conta in ing 200 /zM l e u p e p t i n ) , and the samples were then assayed fo r C a z + - A T P a s e a c t i v i t y at 2 /iM f ree C a 2 + in the absence (open symbols) or in the presence of calmodulin (c losed symbols), as ind ica ted in Mater ia ls and Methods. (B) P u r i f i e d C a 2 + -ATPase (8 /xg protein) was incubated with the ind ica ted a c t i v i t y of ca lpa in in the absence ( c i r c l e s ) or in the presence (squares) of ca lmodul in , as ind icated in Mater ia ls and Methods. A f t e r 2 hours, a l iquo ts were taken and added to the above mentioned assay medium conta in ing 200 /zM leupept in . The samples were then assayed f o r C a 2 + -ATPase a c t i v i t y at 2 /zM free C a 2 + in the absence (open symbols) or in the presence (c losed symbols) of ca lmodul in , as ind icated in Mate r ia ls and Methods. Data presented here are t y p i c a l of four separate experiments. However, the t ime-course of p r o t e o l y t i c e f f e c t var ied s l i g h t l y among experiments. The untreated (zero time) basal and ca lmodul in -s t imulated p u r i f i e d C a 2 + - A T P a s e a c t i v i t y at 2 /zM f ree C a z + (mean ± S .E .M. ) from the four experiments was 1.1 ± 0.3 and 3.6 ± 1.5 /zmol .mg p r o t . ' ^ . m i n " 1 , r e s p e c t i v e l y . 117 Ca 2 + -ATPase Activity (pmol'mg prof."1* min" ) ro ^ O) H i - * - ' i r 4 ^ rr o • • o, • • \ // 1/ / * 0 • CD Ca 2 + -ATPase Activity (fimol-mg prof."1* min"1) of c a l p a i n ac t i va te the basal Ca -ATPase a c t i v i t y (assayed in the absence o f ca lmodul in ) . Furthermore, increas ing the ca lpa in concentrat ion produced a gradual dec l ine of the ca lmodul in -s t imula ted Ca -ATPase a c t i v i t y when the p u r i f i e d enzyme was t reated e i t h e r in the absence or the presence of ca lmodul in . The fragmentation pattern of the p u r i f i e d C a 2 + - A T P a s e produced by c a l p a i n was a lso examined ( F i g . 25B). In the absence of ca lmodul in , the p u r i f i e d C a 2 + - A T P a s e band (136 kDa) was q u i c k l y transformed into a broad 125-124 kDa band. Th is was fol lowed by the formation of another broad band of 82-80 kDa, and smal ler fragments of 55 kDa, 39 kDa, 37 kDa and 32 kDa. In c o n t r a s t , in the presence of ca lmodul in , the most prominent fragments have r e l a t i v e molecular masses o f 127 kDa and 85 kDa, and smal ler fragments of 55 kDa, 39 kDa, 37 kDa and 32 kDa. F i g . 25A shows, as a c o n t r o l , the fragments produced by c a l p a i n a u t o l y s i s (76 kDa, 46 kDa, 36 kDa, 27 kDa and 18 kDa). F i g . 25C shows which fragments of the p u r i f i e d C a 2 + - A T P a s e are capable of forming 3 2 P - l a b e l l e d phosphorylated intermediate . The 125-124 kDa and 82-80 kDa, in the absence of ca lmodul in , and the 127 kDa and 85 kDa, in the presence of ca lmodul in , formed EP in termediates . Therefore , the 3 2 P - l a b e l l e d acylphosphate-forming fragments of the p u r i f i e d C a 2 + - A T P a s e were s i m i l a r to those of the membrane-bound enzyme. It was of i n t e r e s t to e s t a b l i s h the e f f e c t of calmodulin on the ATP h y d r o l y t i c a c t i v i t y of the fragmented form of the C a 2 + - A T P a s e . Therefore , the p u r i f i e d C a 2 + - A T P a s e was t reated with c a l p a i n in the absence of 119 F i g . 25 Fragmentation of the s o l u b l i z e d and p u r i f i e d Ca -ATPase by  c a l p a i n and formation of the phosphorylated in termediate . (A) P u r i f i e d c a l p a i n (10 /zg prote in) (lane 1) was incubated at 25°C f o r 30 min in 50 mM T r i s - H C l (pH 7 .4 ) , 10 mM d i t h i o t h r e i t o l , 0.5 mM EGTA, and 0.7 mM C a C l 2 (pH 7.4) ( lane 2) . The a u t o l y t i c react ion was stopped with 10% (w/v) i c e -co ld t r i c h l o r o a c e t i c ac id and the p r e c i p i t a t e d p ro te in was processed fo r e l e c t r o p h o r e s i s at pH 8 .3 . (B) P u r i f i e d C a 2 + - A T P a s e (47 /zg prote in) was t reated with ca lpa in (0.5 /zg prote in) in the absence (-) or in the presence (+) of calmodulin (CaM) as descr ibed in Mate r ia ls and Methods. At the ind ica ted t ime, a l iquots were taken and the p ro te in p r e c i p i t a t e d with 10% (w/v) i c e - c o l d t r i c h l o r o a c e t i c ac id before being subjected to e l e c t r o p h o r e s i s (pH 8 .3 ) . Calpain a u t o l y t i c fragments are ind ica ted by s o l i d t r i a n g l e s and the band of calmodulin i s ind ica ted by the open 7+ t r i a n g l e . (C) P u r i f i e d Ca^ -ATPase (20 /zg prote in) was e i t h e r untreated (0') or incubated with ca lpa in (0.5 /zg prote in) f o r 60 min (60') in the absence (-) or in the presence (+) of ca lmodul in , as ind ica ted in Mate r ia ls and Methods. P ro teo lys is was stopped with 200 /zM l e u p e p t i n . The samples were then subjected to phosphorylat ion with [-y- 3 2 P]ATP (see Mater ia ls and Methods). The react ion was stopped with 10 % (w/v) i c e - c o l d t r i c h l o r a c e t i c ac id and the p r e c i p i t a t e d prote in was subjected to gel e l e c t r o p h o r e s i s under a c i d i c condi t ions (pH 6 .6 ) , and autoradiography. The p r o t e o l y s i s patterns presented here were reproduced in three other separate experiments. 120 B C CaM - + - + - + - + - + - + caM - + - + 1 3 6 — — _ _ - = ^ 1 2 7 1 3 6 — ^ W ^ 1 2 7 ^ 1 2 5 - 1 2 4 125-124 76 — 8 2 - 8 0 46 — 5 5 18 0 ' 15 ' 3 0 ' 6 0 ' 9 0 ' 120 ' =^85 8 2 - 8 0 0' 60 ' calmodul in and the r e s u l t i n g heterogeneous fragments ( F i g . 26B, lane 1) were passed through a calmodulin-agarose column in the presence of ca lc ium. The column was then washed with a C a 2 + - c o n t a i n i n g buf fer before e l u t i o n with an EGTA-containing b u f f e r . F i g . 26A presents the p r o f i l e of the column f r a c t i o n a t i o n . A f i r s t peak of C a 2 + - A T P a s e a c t i v i t y (not st imulated by calmodulin) was obtained in the f low-through f r a c t i o n s ( F i g . 26A) and e l e c t r o p h o r e t i c ana lys is o f that peak demonstrated that i t conta ins 124 kDa and 80 kDa prote in band ( F i g . 26B, lane 2) . A second C a - A T P a s e peak (st imulated by calmodulin) was obtained a f t e r e l u t i o n of the calmodul in-agarose column with EGTA ( F i g . 26A). E l e c t r o p h o r e t i c a n a l y s i s of t h i s second peak demonstrated that i t contains a prominent 125 kDa component and a f a i n t e r 82 kDa pro te in band ( F i g . 26B, lane 3 ) . There fore , the heterogeneous 125-124 kDa and 82-80 kDa bands prev ious ly i d e n t i f i e d could be fur ther resolved in to calmodulin-dependent and calmodulin- independent fragments. 2. P u r i f i c a t i o n of the calmodul i n - b i n d i n g fragments of the Ca2~*"-ATPase In these experiments, two batches of ca lmodul in-depleted membranes were t reated with c a l p a i n , one in the absence and the other in the presence of sa tura t ing concentrat ions (5 /jg.mg membrane p r o t . " * ) of exogenously added calmodul in . A f t e r treatment, both batches of membranes were washed with an EDTA-containing buf fer to remove calmodulin and/or p. c a l p a i n , s o l u b i l i z e d with T r i ton X-100 and the Ca^ -ATPase fragments p u r i f i e d with two ind iv idua l calmodul in-agarose columns. As a c o n t r o l , a 122 F i g . 26 Separat ion of the C a - A T P a s e fragments with a calmodul in-agarose  column. (A) P u r i f i e d C a 2 + - A T P a s e (85 /tg prote in) was t reated with 1.3 un i t .mL"! c a l p a i n fo r 120 min as descr ibed in Mater ia ls and Methods. P r o t e o l y s i s was stopped by the addi t ion of 200 /iM l e u p e p t i n . The mixture (1.2 mL) was then loaded onto a calmodul in-agarose column (5 cm x 1.5 cm) p r e - e q u i l i b r a t e d with 300 mM KC1, 20 mM potassium-Hepes (pH 7 .4 ) , 50 /iM C a C l 2 , 1 mM M g C l 2 , 2 mM d i t h i o t h r e i t o l , 0.05% (w/v) T r i t o n X-100 and 0.1% (w/v) sonicated a s o l e c t i n . The column was washed with the same buf fe r and f low-through f r a c t i o n s of 1.5 mL were c o l l e c t e d . The column was then e luted with a buf fer conta in ing 300 mM KC1, 20 mM potassium-Hepes (pH 7 .4 ) , 2 mM EGTA, 2 mM d i t h i o t h r e i t o l , 0.05% (w/v) T r i t o n X-100 and 0.1% (w/v) sonicated a s o l e c t i n ( ind icated as "EGTA") and 1.5 mL f r a c t i o n s of the e f f l u e n t were c o l l e c t e d . Both the f low-through and the EGTA e l u t i o n f r a c t i o n s were assayed for C a 2 + - A T P a s e a c t i v i t y at 4 /iM f ree C a 2 + in the absence (open c i r c l e s ) or the presence of 120 nM calmodulin (c lose c i r c l e s ) . (B) A l iquo ts of 100 /iL of the t reated C a 2 + - A T P a s e samples ( lane 1) , and 1 mL of both the unbound (lane 2) and EGTA eluted a c t i v i t y peak f r a c t i o n s (lane 3) were subjected to gel e lec t rophores is (pH 8 .3 ) . The apparent molecular masses (kDa) of the Commassie B lue -s ta ined pro te in bands of the re levant part of the gel are i n d i c a t e d . The r e s u l t s presented here were e s s e n t i a l l y reproduced in another separate experiment. 123 batch of untreated membranes was used and the nat ive C a - A T P a s e p u r i f i e d as above. F i g . 27A i l l u s t r a t e s the formation of the 3 2 P - l a b e l l e d phosphorylated intermediate (EP) of the nat ive membrane-bound C a 2 + - A T P a s e (136 kDa) (lane 1) , the 124 kDa and 80 kDa membrane-bound fragments produced by ca lpa in in the absence of calmodulin (lane 2) , and the 127 kDa and 85 kDa membrane-bound fragments produced by ca lpa in in the presence of calmodulin (lane 3 ) . Panel B shows the prote in pattern of the p u r i f i e d nat ive enzyme (136 kDa) plus a smal ler contaminant of 100 kDa (lane 1) , and the fragments p u r i f i e d with the calmodulin-agarose column from membranes t reated with c a l p a i n in the absence (lane 2) or in the presence (lane 3.) of ca lmodul in . The major fragments p u r i f i e d from the ca lmodul in -dep le ted , c a l p a i n -t reated membranes were the 125 kDa fragment, a small amount of the 82 kDa fragment, and the 39-37 kDa doublet . No fragment of 80 kDa was observed. However, both the 127 kDa and 85 kDa fragments were p u r i f i e d from the ca lmodul in -sa tura ted , ca lpa in t reated membranes. In a d d i t i o n , l a rger amounts of the 39-37 kDa doublet were a lso present . When the above mentioned p u r i f i e d nat ive and fragmented enzymes were incubated with [ T - 3 2 P ] A T P , only the i n t a c t enzyme (136 kDa) ( F i g . 27C, lane 1) and the 127 kDa, 125 kDa, 85 kDa and 82 kDa fragments ( F i g . 27C, o o lanes 2 and 3) formed the J d P - p h o s p h o r y l a t e d intermediate . There fore , i t was reconfirmed that the 124 and 80 kDa fragments formed in the absence 125 F i g . 27 P u r i f i c a t i o n of the ca lmodul in-b inding fragments of the Ca - ATPase from c a l p a i n - t r e a t e d erythrocyte membranes. Calmodul in-depleted membranes were incubated for 60 min with 0.07 u n i t . m L " 1 c a l p a i n in the absence or presence of ca lmodul in , as descr ibed in Mate r ia ls and Methods. Membranes without treatment were used as c o n t r o l s . (A) Autoradi -ography showing 3 2 P - a c y l p h o s p h a t e formation of the membrane-bound C a 2 + -ATPase (18 /xg prote in) e i ther untreated (lane 1) , or t reated with ca lpa in in the absence (lane 2) or in the presence of calmodulin (lane 3 ) . (B) Coomassie Blue s ta in ing of the nat ive or fragmented C a 2 + - A T P a s e p u r i f i e d from 130 mg membrane prote in mentioned in panel A using a ca lmodul in -agarose column. Lanes 1, 2 and 3 correspond to the p u r i f i e d C a 2 + - A T P a s e from the membrane preparat ions shown in lanes 1, 2 and 3 of panel A, r e s p e c t i v e l y . (C) Autoradiogram showing 3 2 P - a c y l p h o s p h a t e formation by the p u r i f i e d nat ive or fragmented C a 2 + - A T P a s e i l l u s t r a t e d in panel B. Lanes 1, 2 and 3 correspond to the p u r i f i e d ATPase preparat ions shown in lanes 1, 2 and 3 of panel B, r e s p e c t i v e l y . The prote in pat terns presented here were reproduced in another separate experiment. 126 F i g u r e 27 ?! ^ SS V if o CO 1 1 CM J r 1 CD CO m co CM 4 1 J 1 1 i (0 co co co CO CM ?! 2S§ v \/ i i CD CO 127 of calmodul in ( F i g . 27A, lane 2) were not re ta ined by the ca lmodul in-agarose column. Table 3 presents the recovery of the to ta l C a 2 + - A T P a s e a c t i v i t y in the experiment shown in F i g . 27. The untreated membranes show a 5 . 8 - f o l d s t i m u l a t i o n of the Ca -ATPase a c t i v i t y induced by ca lmodul in , and the 9 I Ca -ATPase p u r i f i e d from these membranes a lso shows a 3 . 1 - f o l d s t i m u l a t i o n . However, the c a l p a i n - t r e a t e d membranes ( in the absence of calmodulin) had increased C a 2 + - A T P a s e a c t i v i t y when assayed in the absence of calmodul in and the s t imulat ion induced by calmodul in was reduced to 1 . 5 - f o l d . However, the C a 2 + - A T P a s e fragments p u r i f i e d from these membranes by calmodulin a f f i n i t y chromatography (most prominently the 125 kDa fragments) s t i l l show a 2 . 5 - f o l d s t imu la t ion of the ATPase a c t i v i t y induced by calmodulin which compares to a 3 . 1 - f o l d s t imu la t ion of the 9 . p u r i f i e d i n t a c t Ca^ -ATPase. On the other hand, membranes t reated with c a l p a i n in the presence of calmodulin show only a s l i g h t increase in the 9 • basal Ca*- -ATPase a c t i v i t y (assayed in the absence of calmodulin) and a 4.4-fold s t imu la t ion induced by ca lmodul in . The preparat ion of C a 2 + -ATPase fragments (127 kDa plus 85 kDa) p u r i f i e d from these membranes show a 2 . 2 - f o l d s t imula t ion of a c t i v i t y by ca lmodul in . When the recovery of the t o t a l C a 2 + - A T P a s e a c t i v i t y was examined, a lso shown in Table 1, i t becomes c l e a r that in the case of the membranes t reated with ca lpa in in the absence of calmodulin only a small por t ion of the fragmented C a 2 + -ATPase was re ta ined by the calmodul in-agarose column, 10% compared to 59% and 66% in the case of membranes t reated with c a l p a i n in the presence of calmodulin or without ca lpa in treatment, r e s p e c t i v e l y . 128 Table 3 E f fec t of ca lpa in treatment in the absence and presence of calmodul in on C a - A T P a s e a c t i v i t y before and a f te r p u r i f i c a t i o n Addi t ion ,2+ Total Ca -ATPase a c t i v i t y (/imol .min" 1 ) membrane-bound -CaM +CaM p u r i f i e d -CaM +CaM Fold s t imu la t ion by calmodulin membrane-bound p u r i f i e d Recovery of p u r i f i e d C a - A T P a s e a c t i v i t y from the membranes None Calpain Calpain plus calmodulin 2, .1 12, .2 2, .6 8.1 5.8 3.1 66 6, .8 10. ,4 0, .4 1.0 1.5 2.5 10 2. .6 11, .4 3, .1 6.7 4.4 2.2 59 Membranes (130 mg protein) were treated with ca lpa in (0.07 u n i t . m L " 1 ) e i t h e r in the absence or presence of 650 /ig calmodul in, as i n d i c a t e d . Untreated membranes were used as c o n t r o l . The nat ive or fragmented Ca 2 + -ATPase was then s o l u b i l i z e d and p u r i f i e d using three separate calmodulin-agarose columns. The to ta l a c t i v i t y of both the membrane-bound, and the p u r i f i e d Ca 2 + -ATPase a c t i v i t i e s were determined using a l i q u o t s assayed at 4.3 /iM and 2 /iM free C a 2 + , r e s p e c t i v e l y , in the absence or in the presence of calmodulin (CaM) as ind ica ted . The f o l d s t imulat ion by calmodulin was c a l c u l a t e d as the r a t i o of the C a 2 + -ATPase a c t i v i t y in the presence of calmodulin versus the , C a 2 + - A T P a s e a c t i v i t y in the absence of calmodul in . Percent recovery of C a 2 + - A T P a s e a c t i v i t y from the membranes was ca lcu la ted using the Ca 2 + -ATPase a c t i v i t y assayed in the presence o f ca lmodul in . Values presented here are t y p i c a l of two separate experiments. 3. Tryps in fragmentation of the C a - A T P a s e The fragmentation patterns of the C a - A T P a s e produced by ca lpa in and by t r y p s i n were a lso compared.Calpain treatment of the p u r i f i e d C a 2 + -ATPase in the absence of calmodulin produced the c h a r a c t e r i s t i c 124 kDa, 80 kDa, 55 kDa and 39-37 kDa fragments ( F i g . 28 B, lane 1) . On the other hand, short time exposure to t r y p s i n (3 min) in the absence of calmodulin produced a 124 kDa fragment and smal ler fragments o f 86 kDa, 82 kDa, 34 kDa and 32 kDa ( F i g . 28 B, lane 2) . Longer exposure to t r y p s i n (30 min) resu l ted in the accumulation of 82 kDa, 77 kDa and 32 kDa fragments ( F i g . 28B, lane 3 ) . Calpa in treatment in the presence of calmodul in led to the formation of 127 kDa, 85 kDa, 55 kDa and 39-37 kDa fragments ( F i g . 28C, lane 1), whereas short time exposure to t r y p s i n in the presence of calmodulin produced a 127 kDa fragment and smal ler fragments of 86 kDa, 82 kDa, 34 kDa and 32 kDa ( F i g . 28C, lane 2) . Longer exposure to t r y p s i n in the presence of calmodulin led to the accumulation of the 82 kDa and 32 kDa fragments ( F i g . 28C, lane 3) . F i g . 28A shows the p u r i f i e d nat ive C a 2 + -ATPase as a c o n t r o l . IV. Charac te r i za t ion of the untreated and c a l p a i n - t r e a t e d l iposome- r e c o n s t i t u t e d C a 2 + - A T P a s e ( C a 2 + pump) 130 F i g . 28 Comparison of the fragmentation patterns of the p u r i f i e d C a - ATPase obtained by ca lpa in and t r y p s i n d i g e s t i o n . (A) Untreated p u r i f i e d C a - A T P a s e (60 /Ltg p r o t e i n ) . P u r i f i e d C a - A T P a s e (60 /ig prote in) was incubated at 25°C with ca lpa in (0.02 uni t .mL"*) fo r 60 min ( lane 1) or with t r y p s i n (5 / ig.mL" 1 ) f o r 3 min (lane 2) or 30 min (lane 3) in the absence (B) or the presence (C) of 300 nM calmodulin (CaM) in medium conta in ing 55 mM T r i s - m a l e a t e , 5 mM potassium-Hepes, 6.5 mM M a C ^ , 0.5 mM EDTA, 10 mM d i t h i o t h r e i t o l , 65 mM KC1 and 200 /iM f ree C a 2 + (pH 7 .4 ) . The p r o t e o l y s i s was stopped with 10% (w/v) i c e - c o l d t r i c h l o r o a c e t i c a c i d . The p r e c i p i t a t e d prote in was subjected to gel e l e c t r o p h o r e s i s (pH 8 .3 ) . Calpa in fragments are ind icated by s o l i d t r i a n g l e s and t r y p s i n i s ind ica ted by open t r i a n g l e s . The fragmentation patterns presented here were reproduced in two other separate experiments. 131 F i g u r e 28 CO CO CN If (OCM COCO V 9 ' 1 I ! Ik co CO CM l A LO 00 CO<t CO CM CM h» co if I IT) i mm • i if 1 i 1 i K h co o CO CM 00 *" T" CO CO T _ *t CM CO CO If I It COCO 132 1. E f f e c t of A23187. a lamethicin and T r i t o n X-100 on the ATP h y d r o ! v t i c a c t i v i t y of the reconst i tu ted Ca^ pump It was of great in te res t to examine the e f f e c t of c a l p a i n on the C a 2 + - t r a n s l o c a t i n g funct ion of the C a 2 + - A T P a s e . Therefore the l a t t e r enzyme was reconst i tu ted into liposomes fo r these s t u d i e s . In order to observe p r o t e o l y t i c ac t i va t ion of the recons t i tu ted C a 2 + pump and the lack of calmodulin s t imu la t ion , as pred ic ted from the above experimental r e s u l t s on the membrane-bound and p u r i f i e d enzyme, a c a l m o d u l i n - s e n s i t i v e recons t i tu ted C a 2 + pump preparat ion was r e q u i r e d . The major i ty of previous work on the reconst i tu ted Ca^ -ATPase was c a r r i e d out on a s o l e c t i n v e s i c l e s (Haaker and Racker, 1979; N i g g l i et a l . , 1981b, 1982b; Benaim et a l . , 1986; V i l l a l o b o and Roufoga l is , 1986). Due to the presence of a c i d i c phosphol ip id components in a s o l e c t i n , however, the ca lmodul in -s t imula tory pi e f f e c t on Ca^ t ransport has not been repor ted . This problem was resolved by using h igh ly p u r i f i e d egg yo lk phosphat idy lcho l ine (N igg l i et a l - , 1981b) fo r r e c o n s t i t u t i o n of the enzyme, but the v e s i c l e s so formed could be more Ca 2 + -permeab le . In the l i g h t of t h i s , i t was necessary to pi f i r s t e s t a b l i s h the degree of coupl ing and the sidedness of the Ca pump in the phosphat idy lchol ine l iposomes. F i g . 29 shows that the p. recons t i tu ted Ca -ATPase a c t i v i t y was st imulated about 3 - f o l d by A23187, pi i n d i c a t i n g s a t i s f a c t o r y impermeabil i ty of the proteoliposomes to Ca^ . The r e l a t i v e l y low degree of A23187 s t imula t ion with h igh ly p u r i f i e d phosphat idy lchol ine was noted prev ious ly ( V i l l a l o b o and R o u f o g a l i s , 1986). Under s i m i l a r c o n d i t i o n s , using a s o l e c t i n instead of phospha t idy lcho l ine , the C a 2 + - A T P a s e a c t i v i t y was st imulated 6-7 f o l d by A23187 ( r e s u l t s not 133 F i g . 29 E f f e c t of A23I87, a lamethicin and T r i t o n X-100 on the 9 , r e c o n s t i t u t e d Ca^ -ATPase a c t i v i t y . Proteoliposomes (0.8 ng p ro te in and 0.65 mg phosphol ip id) were incubated at 37° C f o r 30 min with 4.3 /zM of f ree C a 2 + , 2 mM ATP, 120 nM calmodul in , 66 mM KC1, 6.5 mM M g C l 2 , 5 mM potassium-Hepes, 55 mM Tr is -ma lea te , pH 7.2 in the absence (open symbols) or the presence (c losed symbols) of 120 nM A23187. Var ious amounts of a lamethic in (A) or T r i t o n X-100 (B) were a lso added, as i n d i c a t e d . Resul ts presented here are t y p i c a l of two separate experiments. 134 2 + C a - A T P a s e a c t i v i t y ( n m o l - m g p r o t . \ m i n ^ ^ 09 o o C a - A T P a s e a c t i v i t y ( n m o l - m g p r o t . ' ' . m i n o o shown). F i g . 29A a lso i l l u s t r a t e s that a lameth ic in , at concentrat ions higher than 0.5 / jg .ml"* , fur ther increased the C a 2 + - A T P a s e a c t i v i t y (assayed in the presence of A23187) by about 25%. Alameth ic in i s known to increase the permeabi l i ty of phosphol ip id membranes (Muel ler and Rudin, 1968) and there fore i t appeared that about 20% of the C a 2 + pump molecules were or iented with the c a t a l y t i c s i t e in the 'lumen of the l iposomes, whereas the major i ty of the enzyme had the c a t a l y t i c s i t e ( s ) or iented to the outs ide (inward Ca pumping o r i e n t a t i o n ) . At higher concentra t ions of a lameth ic in (> 10 /jg.mL"*) the proteoliposomes appeared to be completely permeable to ATP and C a 2 + , s ince the C a 2 + - A T P a s e a c t i v i t y was maximal and no longer required A23187 to express f u l l a c t i v i t y . S i m i l a r l y , C a 2 + - A T P a s e a c t i v i t y in the presence of A23187 was increased about 25% by T r i t o n X-100 at concentrat ions greater than 0.005% (w/v) , while higher concentrat ions (> 0.1% (w/v)) T r i t o n X-100 a lso abol ished the requirement of A23187 for f u l l C a 2 + - A T P a s e a c t i v i t y ( F i g . 29B), fu r ther support ing the predominance of the inwardly C a 2 + - t r a n s p o r t i n g C a 2 + pump o r i e n t a t i o n . 2. E f f e c t o f c a l p a i n on the C a 2 + - t r a n s p o r t and ATP h v d r o l v t i c a c t i v i t y of  the C a 2 + pump In t h i s s e r i e s of experiments, proteoliposomes were preincubated e i t h e r with or without c a l p a i n . A f t e r the treatment, leupept in was added to the mixture to ar rest p r o t e o l y s i s . Leupeptin alone d id not a f f e c t e i t h e r C a 2 + uptake or the ATP hydro lys is of the C a 2 + pump ( r e s u l t s not shown). F i g . 30A shows that the i n i t i a l rate of C a 2 + uptake (measured 136 F i g . 30 E f f e c t of ca lpa in on the i n i t i a l rate of both calc ium uptake and  ATP h y d r o l y s i s of the reconst i tu ted C a 2 + pump. Proteoliposomes (55 /ig p ro te in and 42 mg phosphol ipid) were incubated at 25° C f o r 120 min in a medium conta in ing 67 mM KC1, 50 mM potassium-Hepes (pH 7 .4 ) , 0.67 mM M g C l 2 , 10 mM d i t h i o t h r e i t o l , 400 /iM f ree C a 2 + in the absence or the presence of c a l p a i n (as i n d i c a t e d ) . Leupeptin (200 tiM) was added at the end of the incubat ion and the proteoliposome suspensions were assayed f o r : (A) ca lc ium uptake and (8) ATP-hydro ly t i c a c t i v i t y measurement. In (A) , the t ime-course of calcium uptake was monitored as in Mate r ia ls and Methods, in the absence or the presence of 1 /iM calmodul in (as i n d i c a t e d ) . Add i t ions of 27 /iM ATP and 60 nM A23187 were as ind ica ted on the t r a c e s . Numbers beside the t races represent i n i t i a l rates of C a z + uptake in nmol.mg p r o t . _ 1 . m i n " ^ . In (B) , the t ime-course of ATP h y d r o l y s i s was monitored according to method A in Mater ia ls and Methods, in the absence or the presence o f 1 /iM calmodulin (as i n d i c a t e d ) . Add i t ions of 27 /iM ATP and 60 nM A23187 are shown on the t r a c e s . Numbers beside the t races represent i n i t i a l rates of A T P - h y d r o l y s i s . Numbers in brackets are the ATP h y d r o l y s i s contro l r a t i o ( A T P H C R ) . The pat terns of t races presented here were e s s e n t i a l l y reproduced in two other separate experiments. 137 Figure 30 -Calpain +Calpain 2min - C a , P d n +Calpain 2 m i n 138 with the Ca - s e n s i t i v e dye Arsenazo III) into the recons t i tu ted C a ^ + pump proteol ipsomes i s st imulated 5 - fo ld by calmodulin at 0.4 /JM f ree C a 2 + . A f t e r c a l p a i n treatment the i n i t i a l rate of C a 2 + uptake was a lso increased about 3 - f o l d (299 compared to 85 nmol.mg p r o t . - 1 . m i n " 1 ) and became almost i n s e n s i t i v e to calmodulin ( F i g . 30A). In p a r a l l e l , the i n i t i a l rate of ATP hydro lys is was a lso measured under almost i d e n t i c a l c o n d i t i o n s , except that Arsenazo III was omitted ( F i g . 30B). The i n i t i a l rate of ATP hydro lys is by the untreated C a Z + pump (127 nmol.mg p r o t . - 1 . m i n " 1 ) was st imulated 7 - f o l d by calmodulin (to 886 nmol.mg p r o t . _ 1 . m i n " 1 ) ( F i g . 30B). The addi t ion of A23187 increased the rate o f ATP h y d r o l y s i s by 2 . 0 - f o l d and 2 . 6 - f o l d in the absence or the presence of ca lmodul in , r e s p e c t i v e l y . Calpain treatment increased the i n i t i a l ra te of ATP h y d r o l y s i s in the absence of calmodulin to 562 nmol.mg p r o t . _ 1 . m i n " 1 , approaching the ca lmodul in -s t imula ted i n i t i a l rate of 731 nmol.mg p r o t . _ 1 . m i n " 1 . Furthermore, A23187 increased the rate of ATP h y d r o l y s i s in the absence or the presence of calmodulin by 2 . 2 - f o l d and 2 . 4 - f o l d , r e s p e c t i v e l y . Table 4 summarizes the r e s u l t s of several such experiments. It was shown that the ATP h y d r o l y s i s contro l r a t i o ( A T P H Q R ) (the r a t i o of the rate of ATP hydro lys is in the presence versus the absence of A23187) was maintained between 2 and 3, both in the absence and in the presence of ca lmodul in . Calpain treatment d id not appear to a l t e r t h i s r a t i o . From the A T P ^ Q R va lues , fo l lowing non-e q u i l i b r i u m thermodynamic formulat ions (Rottenberg, 1971), the degree of coupl ing (q) was c a l c u l a t e d , which was expressed as fo l lows: 139 Table 4 E f f e c t of c a l p a i n treatment on the degree o f coupl ing and C a 2 + / A T P r a t i o of the 1 iposome-reconst i tuted C a 2 + pump Treatment CaM A T P H C R 3 Degree of coupl ing ( q ) b Experimental M e c h a n i s t i c 0 C a 2 + / A T P r a t i o C a 2 + / A T P r a t i o None 2.0 ± 0.1 0.71 + 0.04 2.2 ± 0.3 0.73 ± 0.04 0.60 ± 0.10 1.20 ± 0.17 0.44 ± 0.08 0.83 ± 0.16 Calpain - 2.0 ± 0.2 0.71 ± 0.02 + 2.0 ± 0.4 0.70 ± 0.05 0.52 ± 0.03 1 . 0 4 . ± 0.07 0.41 ± 0.05 0.84 ± 0.15 ATP HCR rate of ATP h y d r o l y s i s (+A23187) rate of ATP h y d r o l y s i s (-A23187) q = 1 ATP HCR „ 1/2 c Mechanist ic C a 2 + / A T P r a t i o = Experimental C a 2 + / A T P r a t i o Liposomes (54 /ig prote in plus 49 mg phosphol ip id) were incut without c a l p a i n , as descr ibed in F i g . 30. Ca uptake and jbated with or ATP-hydro lys is measurements were c a r r i e d out as descr ibed in the Mater ia ls and Methods. The values (means ± S .E .M . ) in t h i s tab le were obtained from three separate experiments. 1 4 0 or rate of ATP h y d r o l y s i s (-A23187) rate of ATP h y d r o l y s i s (+A23187) 1/2 ATP HCR 1/2 [1] [2] The c a l c u l a t e d value of q was maintained at about 0.7 in the absence or the presence of ca lpa in treatment, both with and without ca lmodul in . Table 4 a lso shows that the C a 2 + t ranspor t to ATP h y d r o l y s i s s t o i c h i o m e t r i c r a t i o ( C a 2 + / A T P r a t i o ) under these experimental cond i t ions was around 0.60 ± 0.10 and 0.44 ± 0.08, in the absence or the presence of 7+ ca lmodul in , r e s p e c t i v e l y . However, due to the high o v e r a l l Ca^ permeabi l i ty of these v e s i c l e s , a s i g n i f i c a n t f r a c t i o n of the to ta l ATP P i p h y d r o l y s i s was uncoupled to C a d t r a n s l o c a t i o n . By d e f i n i t i o n , q^ equals the f r a c t i o n of the C a 2 + - A T P a s e molecules that are coupled (equation [1] ) . Therefore , the experimental C a 2 + / A T P r a t i o was corrected (to c a l c u l a t e the mechanist ic C a 2 + / A T P ra t io ) using the fo l lowing express ion: Experimental C a 2 + / A T P r a t i o Mechanist ic C a 2 + / A T P r a t i o [3] The corrected C a 2 + / A T P r a t i o was 1.20 ± 0.17 and 0.83 ± 0.16, in the absence and the presence of ca lmodul in , r e s p e c t i v e l y , and was not s i g n i f i c a n t l y a l te red by ca lpa in treatment. 141 The e f f e c t of ca lpa in concentrat ion on the recons t i tu ted Ca*1 pump was determined at a constant incubat ion time (120 min) ( F i g . 31) . The i n i t i a l rates of C a 2 + uptake and C a 2 + - A T P a s e a c t i v i t y were f u l l y act iva ted and became almost i n s e n s i t i v e to calmodulin at about 1 to 5 un i t .mL" 1 c a l p a i n . Another approach taken was to fo l low the t ime-course of ca lpa in treatment. F i g . 32 shows that the i n i t i a l rates of both C a 2 + uptake and the Ca -ATPase a c t i v i t y were p rogress ive ly increased fo l lowing ca lpa in treatment ( in the absence of calmodulin) and became l a r g e l y calmodulin i n s e n s i t i v e a f t e r 120 min. On the other hand, i r r e s p e c t i v e of whether calmodul in was absent or present dur ing c a l p a i n treatment, the i n i t i a l ra te o f C a 2 + uptake and C a z + - A T P a s e a c t i v i t y measured in the presence of calmodul in slowly dec l ined with time of p r o t e o l y s i s by about 17-19% and 26-28%, r e s p e c t i v e l y , of t h e i r i n i t i a l (zero-t ime) values ( F i g . 32) . To determine the molecular changes accompanying the a c t i v a t i o n by c a l p a i n , the fragmentation of the recons t i tu ted C a 2 + pump, both in the absence and the presence of ca lmodul in , was examined. F i g . 33A shows that with increas ing time of p r o t e o l y s i s , the recons t i tu ted 136 kDa C a 2 + - A T P a s e was transformed mainly into 124 kDa and 127 kDa fragments, in the absence or the presence of ca lmodul in , r e s p e c t i v e l y . A f t e r 120 min, a por t ion of unfragmented i n t a c t enzyme (136 kDa) was s t i l l present , which l i k e l y c o n s i s t e d of outwardly C a 2 + pumping or ienta ted C a 2 + - A T P a s e molecules and C a 2 + - A T P a s e molecules not reconst i tu ted in the liposome membranes. Acylphosphoprotein intermediate (EP) formation by the contro l enzyme gave a s i n g l e band of 136 kDa ( F i g . 33B, lane 1). A f t e r ca lpa in treatment (120 142 F i g . 31 E f f e c t of increas ing ca lpa in concentrat ion on the i n i t i a l rate of  C a 2 + uptake and ATP hydro lv t i c a c t i v i t y of the recons t i tu ted C a z + pump. Proteoliposomes (128 /ig prote in and 112 mg phosphol ip id) were incubated at 25° C with the ind icated amount of ca lpa in f o r 60 min, as descr ibed in F i g . 30. In (A) , c a l p a i n - t r e a t e d proteoliposomes were used to determine the i n i t i a l rate of Ca -uptake in the absence (open c i r c l e s ) or the presence (c losed c i r c l e s ) of 1 /iM ca lmodul in , as descr ibed in F i g . 30. In (B) , t reated proteoliposomes were assayed fo r C a z + - A T P a s e a c t i v i t y (Method A) in the absence (open c i r c l e s ) or the presence (c losed c i r c l e s ) of ca lmodul in . Values presented here are means of two separate experiments. 143 2 + C a - A T P a s e a c t i v i t y ( n m o l - m g p r o t . . m i n ) ( n m o l -o Q_ *Q Q 3 C 3 y . O H 3 .^ en o o o o o 1 o • n C a 2 + U p t a k e m g p r o t . " ' ' • m i n ~ ~ 1 ) Ol o o _ J O Q_ Q . O H ro o o o o c 2H 3 1 1 o • 0) O O -5 a> F i g . 32 Time-course of the e f f e c t of ca lpa in d i g e s t i o n of recons t i tu ted Ca2"1" pump on the i n i t i a l rate of Ca2"1" uptake and ATP h y d r o l y t i c a c t i v i t y . Proteoliposomes (156 /xg prote in plus 112 mg phosphol ip id) were t reated with 2 u n i t . m L " 1 of c a l p a i n in the absence ( c i r c l e s ) or the presence (squares) of calmodulin f o r the ind ica ted per iod of t ime, as in Mater ia l and Methods; c a l p a i n was not added fo r the zero time va lues . A f t e r the add i t ion of 200 /iM l e u p e p t i n , the proteoliposomes were then used f o r Ca uptake measurements (A) or C a 2 + - A T P a s e a c t i v i t y determinations (B) in the absence (open symbols) or the presence of 300 nM calmodulin (c losed symbols) , as descr ibed in F i g . 30. Data presented are t y p i c a l o f three separate ' experiments. However, the t ime-course of p r o t e o l y t i c e f f e c t var ied s l i g h t l y among experiments. The untreated (zero time) basal and p i ca lmodul in -s t imula ted calc ium uptake by the recons t i tu ted Ca^ -ATPase a c t i v i t y 0.4 /iM f ree C a 2 + and 27 /iM ATP (mean ± S .E .M. ) from the three experiments was 77 ± 37 and 610 ± 278 nmol.mg p r o t . " 1 . m i n " 1 , r e s p e c t i v e l y . The untreated basal and ca lmodul in-s t imulated recons t i tu ted C a 2 + - A T P a s e a c t i v i t y at 0.4 /zM f ree C a 2 + (mean ± S .E .M. ) from the three experiments was 270 ± 110 and 1040 ± 430 nmol.mg p r o t . " 1 . m i n " 1 , r e s p e c t i v e l y . 145 2 + C a - A T P a s e a c t i v i t y •1 . -1 C a 2 + U p t a k e ( n m o l . m g p r o t . . m i n ) ( n m o l . m g p r o t . ~ 1 - m i n ~ 1 ) 4=» 3 CD 3 3* O O). O O N> O O o o \ CD CO O 0 ) J o (0 o' ro-o O o o o 0> o o rmx--5 CD O J F i g . 33 Time-course of ca lpa in - induced p r o t e o l y s i s of the reconst i tu ted  Ca pump. Proteoliposomes (156 fig prote in plus 112 mg phosphol ip id) were t reated with c a l p a i n fo r the ind icated per iod of time in the absence (-) or the presence (+) of 130 nM calmodul in , as descr ibed in F i g . 32. A f t e r the p r o t e o l y s i s was arrested with 200 /*M of l e u p e p t i n , the proteoliposomes were subjected to (A) a l k a l i n e gel e lec t rophores is (pH 8.3) fol lowed by s i l v e r s t a i n i n g or (B) 3 2P-phosphoenzyme formation fol lowed by ac id gel e l e c t r o p h o r e s i s (pH 6.8) and autoradiography. The numbers on the s ides of the lanes represent molecular masses of prote in standards in kDa. The open t r i a n g l e ind ica tes ca lpa in and/or i t s fragment. In (B), proteol iposomes were t reated with 0 (lane 1) or 2 u n i t . m L " 1 ca lpa in in the absence ( lane 2) or the presence (lane 3) of 130 nM ca lmodul in . Fragmentation pat terns presented here were reproduced in four separate experiments. 147 F i g u r e 33 CO CM ™ V ^ C O CO I CD CO + + + + + O CD CM r - O) CO » 1 1 Vt f t CO If 11 I I I o CM o CO o CO LO CO o 148 min) in the absence of ca lmodul in , a major EP band corresponding to the major 124 kDa band s t a i n i n g with Coomassie blue was observed, as well as minor bands of 136 kDa and 80 kDa ( F i g . 33B, lane 2) . Calpa in treatment in the presence of ca lmodul in , however, produced a major acylphosphoprotein band corresponding to the major 127 kDa band s t a i n i n g with Coomassie blue and l e s s prominent bands of 136 kDa and 85 kDa ( F i g . 33B, lane 3 ) . 3. P ro tec t ive e f f e c t of calmodulin against p r o t e o l y t i c a c t i v a t i o n of the  r econs t i tu ted C a 2 + pump by c a l p a i n In another s e r i e s of experiments, methods were developed to 9+ determine the e f f e c t of calmodulin preincubat ion on the Ca^ -ATPase and Ca -uptake a c t i v i t y fo l lowing ca lpa in treatment of the proteol iposomes, which requi red p r i o r separat ion of the calmodulin and c a l p a i n from the recons t i tu ted enzyme. The proteoliposomes were f i r s t pre- incubated with ca lpa in in the absence or the presence of ca lmodul in , while contro l proteoliposomes were pre- incubated in the absence of c a l p a i n . In order to separate calmodulin and ca lpa in from the proteoliposomes a f t e r treatment, i n d i v i d u a l samples were passed through a gel f i l t r a t i o n column (Sephacryl S-200, 1.5 x 60 cm) in the presence of EGTA. The proteoliposomes e l u t i n g in the void volume were c o l l e c t e d . Figure 34 shows that these proteol iposomes were calmodul i n - f r e e (lanes b and d) and a lso f ree of a u t o l y t i c fragments of c a l p a i n (lane a to d ) . Calpain treatment in the absence of calmodulin produced the 124 kDa major fragment and l i g h t e r bands of 80 kDa, 55 kDa, 39 and 37 kDa, as descr ibed above (lane c ) . In 149 F i g . 34 E f f e c t of calmodulin on the p r o t e o l y s i s of the reconst i tu ted Cac+  pump. Proteol iposomes (129 /ig prote in plus 120 mg phosphol ipid) were incubated at 25°C fo r 90 min in a medium (2.7 mL) conta in ing 75 mM KC1, 50 mM potassium-Hepes (pH 7 .4 ) , 10 mM d i t h i o t h r e i t o l , 400 /iM f ree C a 2 + in the presence of (a) no add i t ion (b) 500 nM calmodulin (c) 2 u n i t . m L " 1 ca lpa in or (d) 500 nM calmodulin and 2 u n i t . m L - 1 c a l p a i n . At the end of the incuba t ion , 5 mM EGTA was added to each sample. A f t e r c h i l l i n g on ice fo r 10 min, 2 mL of each sample was separate ly loaded onto a Sephacryl S-200 column (60 x 1.5 cm). The column was e q u i l i b r a t e d and e luted with 100 mM KC1, 200 /iM M g C l 2 , 200 /tM EGTA, 20 mM potassium-Hepes, pH 7.4 and 2 mM d i t h i o t h r e i t o l at 20 m L . h " 1 . Two mL-Fract ions were c o l l e c t e d . The liposome peak f r a c t i o n s (e lu t ion volume 38-41 mL) were pooled, assayed fo r C a 2 + - A T P a s e a c t i v i t y (see Table 5) and subjected to SDS-PAGE (lanes a -d ) . The gel was sta ined with Coomassie Blue R-250. Numbers shown are apparent molecular mass in kDa. Fragmentation patterns presented here were reproduced in another separate experiment. 150 F i g u r e 34 a b e d 1 5 1 Table 5 Protect ive e f f e c t of calmodulin against p r o t e o l y t i c a c t i v a t i o n of the reconst i tu ted Ca -ATPase by ca lpa in Ca^ -ATPase a c t i v i t y ( n m o l . m i n _ i . m L _ i ) Fold s t imula t ion by calmodulin Addi t ion - A23187 +A23187 -A23187 +A23187 -CaM +CaM -CaM +CaM None 1.36 4.55 4.95 14.94 3.4 3.0 Calmodulin 1.37 4.99 5.04 15.86 3.6 3.1 Calpain 3.66 4.13 11.43 12.58 1.1 1.1 Calpain + calmodulin 1.70 4.86 5.34 . 13.26 2.9 2.5 Proteoliposomes (129 /ig prote in plus 120 mg phosphol ipid) were incubated in the presence of 10 mM d i t h i o t h r e i t o l and 40 /iM free C a 2 + and one of the add i t ions i n d i c a t e d , as descr ibed in Figure 34. A f te r gel f i l t r a t i o n (see Mater ia ls and Methods), the calmodul in-free proteoliposomes were assayed for Ca -ATPase a c t i v i t y , as descr ibed in Mater ia ls and Methods. Ca -ATPase a c t i v i t y is given in nmol per min per mL of pooled liposome peak f rac t ions (void volume). Values presented are t y p i c a l of two separate experiments. the presence of ca lmodul in , ca lpa in fragmented the Ca -ATPase into a 127 kDa major fragment and minor fragments of 85 kDa, 55 kDa, 39 kDa and 37 kDa (lane d ) . When the C a 2 + - A T P a s e a c t i v i t y of the proteol iposomes, pre -incubated with or without calmodulin a lone , as measured, the s t imu la t ion by calmodulin was found to be about 3 . 0 - f o l d and 3 . 1 - f o l d , r e s p e c t i v e l y ( in the presence of A23187) (Table 5 ) . Th is again i n d i c a t e s that the calmodulin present in the pre - incubat ion medium had been completely separated from the proteol iposomes. Proteoliposomes pre - t rea ted with c a l p a i n alone had increased basal C a 2 + - A T P a s e a c t i v i t y (11.43 compared to 4.95 n m o l . m i n " 1 . m L " 1 ) , while the s t imula t ion by calmodulin was reduced to 1 .1 - fo ld ( in the presence of A23187). On the other hand, proteoliposomes pretreated with ca lpa in in the presence of calmodulin maintained a 2 .5-f o l d calmodulin s t imula t ion of the C a 2 + - A T P a s e a c t i v i t y ( in the presence o f A 2 3 1 8 7 ) . 4. K i n e t i c proper t ies of the i n t a c t and c a l p a i n - t r e a t e d C a 2 + pump in  phosphat idy lchol ine v e s i c l e s The in tac t and the c a l p a i n - t r e a t e d reconst i tu ted C a 2 + pump was studied with respect to i t s dependence on C a 2 + and ATP. F i g . 35A i l l u s t r a t e s c l e a r l y that the a f f i n i t y f o r C a 2 + of the i n i t i a l rate of ca lc ium uptake by the in tac t C a 2 + pump was g r e a t l y increased by calmodulin (KQ 5 around 4 /iM and 0.2 /xM in the absence and the presence of ca lmodul in , r e s p e c t i v e l y ) . Furthermore, calmodulin increased V m a x from 400 to 850 nmol.mg p r o t . _ 1 . m i n " 1 . A f t e r ca lpa in treatment in the absence of ca lmodul in , however, the C a 2 + pump appeared to have high a f f i n i t y f o r C a 2 + 153 F i g . 35 Calcium dependence of the i n i t i a l rate of Ca uptake of the 9 i r e c o n s t i t u t e d Ca pump. Proteoliposomes (237 /xg prote in plus 105 mg phosphol ip id) were t reated with 0 (A) or 2 u n i t . m L - 1 c a l p a i n (B) at 25°C f o r 120 min, as in the Mater ia ls and Methods. The proteol iposomes were then subjected to C a 2 + uptake measurements in the absence (open c i r c l e s ) or the presence (c losed c i r c l e s ) of 1 /iM calmodulin at the ind ica ted concentra t ions of f ree C a 2 + . . Values presented are the means of two separate experiments. 154 Figure 35 155 (KQ_5 around 0.3 /iM) even when assayed in the absence of calmodul in ( F i g . 35B), and the add i t ion of calmodulin d id not fur ther increase the a f f i n i t y fo r Ca^ and only s l i g h t l y increased V M A X (from 550 to 650 nmol.mg p r o t . " * . m i n " 1 ) . The Ca 2 + -dependence of the A T P - h y d r o l y t i c a c t i v i t y of the r e c o n s t i t u t e d enzyme was a lso examined ( F i g . 36) . The i n t a c t C a 2 + pump exh ib i ted the low C a 2 + a f f i n i t y (KQ 5 around 2 /iM), low V M A X mode t y p i c a l of the enzyme in the absence of calmodulin and the high C a 2 + a f f i n i t y (KQ 5 around 0.2 /iM), high V M A X mode when assayed in the presence of calmodulin ( F i g . 36A). The treatment with ca lpa in again converted the enzyme in to the predominantly high C a 2 + a f f i n i t y (KQ 5 around 0.3 /iM), high V M A X mode even when i t was assayed in the absence of calmodul in ( F i g . 36B). The e f f e c t of ATP on the i n i t i a l rate of C a 2 + uptake by the r e c o n s t i t u t e d C a ^ T pump was a lso i n v e s t i g a t e d . F i g . 37A demonstrates c l e a r l y that the enzyme has two d i s t i n c t a f f i n i t i e s f o r ATP at 0.4 /iM f ree C a 2 + . The apparent Kg 5(ATP) v a l u e s a r e 1-4 /iM and 289 /iM in the absence of calmodul in and 1.1 /iM and 205 /iM in i t s presence. The c a l p a i n -fragmented Ca pump expressed comparable apparent KQ 5 values f o r ATP (0.9 /iM and 245 /iM), both in the absence and in the presence of calmodulin ( F i g . 37B). Therefore ca lpa in treatment d id not s i g n i f i c a n t l y modify the b iphas ic ATP-dependence of the Ca^ pump. V . S u s c e p t i b i l i t y of other ca lmodul in-b inding prote ins and the t roponin C  super fami ly prote ins to ca lpa in 156 F i g . 36 Calcium dependence of the Ca -ATPase a c t i v i t y in proteol iposomes. Proteoliposomes (50 tig prote in plus 22 mg phosphol ip id) were t reated with 0 (A) or 2 un i t .mL" 1 ca lpa in (B) at 25°C fo r 120 min, as in the Mater ia ls and Methods. The proteoliposomes were then assayed f o r C a 2 + - A T P a s e a c t i v i t y (Method A) in the absence (open c i r c l e s ) or the presence (c losed c i r c l e s ) of 120 nM calmodulin at the ind ica ted concentrat ions of f ree Ca . Values presented are the means o f two separate experiments. 157 Figure 36 158 F i g . 37 E f f e c t of ATP concentrat ion on the i n i t i a l rate of Ca^ uptake of  the recons t i tu ted Ca2"*" pump . Proteol iposomes (466 tig p ro te in p lus 210 mg phosphol ip id) were t reated with 0 (A) or 2 u n i t . m L " 1 c a l p a i n (B) at 25°C fo r 120 min as ind ica ted in Mater ia ls and Methods. The proteoliposomes were then subjected to C a 2 + uptake measurement in the absence (open c i r c l e s ) or the presence (c losed c i r c l e s ) of 1 /iM calmodulin at var ious concentrat ions of ATP (0.5 to 2000 /iM). Values presented are the means of two separate experiments. 159 Figure 37 1 6 0 1. P r o t e o l y s i s of adducin and neuromodulin by ca lpa in Based on the s u s c e p t i b i l i t y of the Ca -ATPase to c a l p a i n , i t was of i n t e r e s t to f i n d out whether other ca lmodul in-b inding prote ins or enzymes are a lso c a l p a i n subs t ra tes . Adducin i s a plasma membrane-associated pro te in present in human ery throcy tes . It has two n o n - i d e n t i c a l subuni ts : an a -subuni t (103 kDa) and a /J-subunit (97 kDa) which binds ca lmodul in . Adducin was sometimes found to c o - p u r i f y (as a minor component) with the ery throcyte plasma membrane C a 2 + - A T P a s e on calmodulin a f f i n i t y chromatography. When such adducin-conta in ing C a 2 + - A T P a s e preparat ions were t reated with c a l p a i n , i t was found that both a- and z3-subunits of adducin were r a p i d l y degraded to smal ler fragments between 80-95 kDa ( F i g . 38A). Longer incubat ion with ca lpa in resu l ted in fur ther degradation of these fragments ( r e s u l t s not shown). This r e s u l t i s cons is ten t with the report (Gardner and Bennett , 1986) that adducin was very prone to p r o t e o l y s i s dur ing p u r i f i c a t i o n . Another ca lmodul in -b ind ing prote in tested was neuromodulin from bovine b r a i n , which binds calmodulin more t i g h t l y in the absence of Ca than in i t s presence (Andreasen et a l . . , 1983). Although i t s true molecular weight i s 24 kDa, i t appears as a 57 kDa band on SDS-PAGE, probably due to i t s high content of basic amino ac id res idues (Wakim et a l . , 1987). Upon c a l p a i n treatment, neuromodulin was found to be p r o g r e s s i v e l y transformed into several fragments ( F i g . 38B). Due to the unusual m o b i l i t y of neuromodulin on SDS-PAGE, i t was impossible to 161 F i g . 38 P r o t e o l y s i s of human erythrocyte adducin and bovine bra in neuromodulin by c a l p a i n . (A) Adducin (97 kDa and 103 kDa) was s o l u b l i z e d and c o - p u r i f i e d with the C a c r pump from human ery throcytes as descr ibed in M a t e r i a l s and Methods. The adducin preparat ion (100 ttg prote in ) was then subjected to e i t h e r no treatment (lane 2) or c a l p a i n treatment f o r 15 min ( lane 3) or 30 min (lane 4) at 25°C in 50 mM sodium-Hepes (pH 7 .4 ) , 10 mM d i t h i o t h r e i t o l , 0.5 mM EDTA, 0.7 mM C a C l 2 , 65 mM KG1, 0.06% (w/v) sonicated a s o l e c t i n , 0.03% (w/v) T r i ton X-100 and 2 /ug of c a l p a i n I. P r o t e o l y s i s was terminated with 20% (w/v) i c e - c o l d t r i c h l o r o a c e t i c ac id before being subjected to gel e lec t rophores is (Laemmli, 1970). The open arrow i n d i c a t e s the bands of adducin and s o l i d arrows i n d i c a t e fragments p i of adducin . The Ca pump (136 kDa) and i t s fragment (124 kDa) are ind ica ted by the s o l i d t r i a n g l e s , while the large subunit o f c a l p a i n (76 kDa) i s ind ica ted by the open t r i a n g l e . Molecular weight standards used in lane 1 are: myosin, 200 kDa (a) , / J -ga lac tos idase , 116 kDa (b) , phosphorylase b, 97 kDa (c ) , bovine serum albumin, 66 kDa (d) , ovalbumin, 43 kDa (e ) , carbonic anhydrase, 31 kDa (f) and soybean t r y p s i n i n h i b i t o r , 21.5 kDa (g) . (B) Neuromodulin p u r i f i e d from bovine bra in was a g i f t from Dr. Yuechung L i u , Dr. Edwin R. Chapman and Dr. Daniel R. Storm, U n i v e r s i t y of Washington, U .S .A . Neuromodulin (4 /tg) was subjected to e i t h e r no treatment (lane 2) or ca lpa in treatment f o r 15 min ( lane 3) or 30 min ( lane 4) at 25°C in 50 mM-Hepes (pH 7 .4) , 10 mM d i t h i o t h r e i t o l , 0.5 mM EDTA, 0.7 mM C a C l 2 and 0.5 /xg ca lpa in I. P r o t e o l y s i s was terminated as in (A) . The open arrow indicates the band of neuromodulin (appearing as a 57 kDa band) while s o l i d arrows ind ica te fragments of neuromodulin. P u r i f i e d c a l p a i n I (0.5 /tg) from human erythrocyte was appl ied to lane 5. 162 F i g . 38 (Cont. ) Open t r i a n g l e s i n d i c a t e the large subunit of c a l p a i n (80 kDa) and i t s a c t i v e fragment (76 kDa). Molecular weight standards app l ied in lane 1 are as descr ibed in (A) . SDS-PAGE gels in panel A and B were s ta ined with Coomassie Blue R-250. The p r o t e o l y s i s patterns presented were reproduced in two other separate experiments. 163 F i g u r e 38 CO co CM - I! w I I n o I I TJ 0) * • O ) < CO CM • i I CO f / U O "TJ CD H -164 determine the true molecular weight of the fragments d i r e c t l y from the g e l . 2. E f f e c t of c a l p a i n on the .junctional SR prote ins The e f f e c t of ca lpa in I on the prote in components o f the junc t iona l SR f r a c t i o n (from rabbi t ske le ta l muscle) was a lso examined, with p a r t i c u l a r regard to the calmodul in-binding calc ium re lease channe l . It was observed that the calcium re lease channel (410 kDa) was r a p i d l y degraded by c a l p a i n (ca lpa in : SR prote in r a t i o = 1:186) ( F i g . 39A), as was reported by S e i l e r et a l . (1984) using chicken c a l p a i n II as the protease. In the present study, mul t ip le cleavage s i t e s occurred to the calc ium re lease channel producing fragments of 370, 340, 280, 165, 155, 147, 142, 140 and 137 kDa, r e s p e c t i v e l y . In F i g . 39B, i t i s seen that with a c a l p a i n : SR prote in r a t i o (1:63), most of the l a r g e r fragments disappeard with time and only the 137 kDa fragment accumulated and there fore may represent a l i m i t fragment (see lane 8, F i g . 39B). In f a c t , the 340 kDa fragment i s commmonly observed in untreated junc t iona l SR, most l i k e l y as a r e s u l t of p r o t e o l y s i s by endogenous c a l p a i n during i s o l a t i o n ( S e i l e r et a l . , 1984). The content of t h i s 340 kDa endogenous fragment could be l a r g e l y reduced i f leupept in was inc luded in the buf fers -used during i s o l a t i o n of the junct iona l SR. In a d d i t i o n , two other pept ides ( 82 kDa, 42 kDa) of junct iona l SR were found to be proteolyzed by c a l p a i n treatment ( F i g . 39). In sharp contrast to the plasma membrane C a 2 + - A T P a s e , the SR Ca 2 + -ATPase was not fragmented by c a l p a i n at a l l ( F i g . 39) . Calpa in treament of the junct iona l SR under s i m i l a r c o n d i t i o n s a lso 165 F i g . 39 P r o t e o l y s i s of . junctional sarcoplasmic re t icu lum prote ins by  c a l p a i n . Junct iona l SR was prepared according to Inui et a l . (1987) and was subjected to c a l p a i n treatment using a ca lpa in : SR prote ins r a t i o of 1 : 186 (A) or 1: 63 (B). In (A), junc t iona l SR (840 /xg) was incubated in 600 nl with none (lane 1 and 12) or 4.5 /ig c a l p a i n I (0.35 un i t .mL" 1 ) ( lane 2-11) at 25°C in 50 mM potassium-Hepes (pH 7 .4 ) , 15 mM d i t h i o t h r e i t o l , 280 /iM C a C l 2 , 80 /iM EGTA. At time 0 ( lane 1) , 0.5 ( lane 2 ) , 1 ( lane 3 ) , 2 ( lane 4) , 5 (lane 5) , 7 ( lane 6 ) , 10 ( lane 7 ) , 20 (lane 8 ) , 30 ( lane 9 ) , 45 (lane 10), 60 min (lane 11 & 12), a l i q u o t s (32 /iL) were taken and added to 20 /iL of sample d i g e s t i o n buf fe r (see Methods) conta in ing 500 /iM leupept in before being subjected to SDS-PAGE. In (B) , j unc t iona l SR (63 /tg protein) was incubated in 60 /iL at 25°C with none ( lane 1) or 1 /ig ca lpa in I (0.7 un i t .mL" 1 ) in 15 mM d i t h i o t h r e i t o l , 50 mM K-Hepes (pH 7 .4 ) , 370 /iM C a C l 2 , 170 /iM EGTA (from c a l p a i n ) . At time 0 ( lane 1) , 1 ( lane 2) , 5 (lane 3) , 10 (lane 4 ) , 15 (lane 5) , 30 (lane 6) , 45 ( lane 7) and 60 min (lane 8 ) , p r o t e o l y s i s was stopped by the add i t ion of 30 /tL sample d iges t ion buf fer conta in ing 500 /iM leupept in before being subjected to SDS-PAGE. In both panel A and B, the numbers on the r igh t s ide of the ge ls are the apparent molecular weights o f the ca lc ium re lease channel (410 kDa) and i t s p r o t e o l y t i c fragments. The open arrow ind ica tes the la rge subunit of ca lpa in I whi ls t the open t r i a n g l e s i n d i c a t e the two add i t iona l peptides that are a lso suscept ib le to c a l p a i n treatment. CRC, CPP and CBP represent the calcium re lease channel , the calc ium pump pro te in (105 kDa), ca lseques t r in (calcium binding p r o t e i n , 58 kDa), r e s p e c t i v e l y . S o l i d t r i a n g l e s and the l e t t e r s i n d i c a t e the p o s i t i o n of the fo l lowing molecular weight markers: the o r i g i n (a ) , a-macroglobul i n , 360 166 F i g . 39 (Cont. ) kDa (b) , myosin, 200 kDa (c ) , / J -ga lac tos idase , 116 kDa (d) , phosphorylase b, 97 kDa (e ) , bovine serum albumin, 66 kDa ( f ) , ovalbumin, 43 kDa (g) , carbonic anhydrase, 31 kDa (h) and soybean t r y p s i n i n h i b i t o r , 21.5 kDa ( i ) and egg white lysozyme, 14.4 kDa ( j ) . Both ge ls were s ta ined with Coomassie Blue as d e s c r i b e d . P r o t e o l y s i s pat terns presented were reproduced in two other separate experiments. 167 1 6 8 did not a f f e c t i t s Ca2+-ATPase a c t i v i t y ( r e s u l t s not shown). Ca lsequest r in a lso was not suscept ib le to c a l p a i n . 3. Resistance of t roponin C superfamily prote ins to ca lpa in Since several ca lmodul in -b inding prote ins are shown to be c a l p a i n substrates while calmodulin i t s e l f i s not , i t was of i n t e r e s t to see i f other t roponin C superfamily members of C a 2 + - b i n d i n g prote ins share t h i s c h a r a c t e r i s t i c with ca lmodul in . It was found that besides ca lmodul in , t roponin C, S-lOOa p r o t e i n , S-100/3 p r o t e i n , oncomodulin, parvalbumin and c a l c i n e u r i n B ( regulatory subunit of c a l c i n e u r i n ) d id not appear to be fragmented by ca lpa in ( F i g . 40) . I n t e r e s t i n g l y , two binding prote ins of these C a 2 + - b i n d i n g p r o t e i n s , namely, t roponin I, which binds t roponin C, and c a l c i n e u r i n A (60 kDa, c a t a l y t i c subunit) which binds c a l c i n e u r i n B, were fragmented by ca lpa in ( F i g . 40) . 169 F i g . 40 Resistance of the troponin C superfamily of C a - b i n d i n g prote ins  to c a l p a i n . Troponin I and C mixture (2 /tg, rabb i t ske le ta l muscle) , calmodul in (1 / ig, bovine b r a i n ) , S-lOOa prote in (1 / ig , bovine b r a i n ) , S-100/3 p ro te in (1 / ig , bovine b r a i n ) , oncomodulin (1 / ig , ra t hepatoma, a g i f t from Dr. B. Mutus, Un ivers i ty of Windsor, Canada), parvalbumin (1 /tg, ra t muscle) and c a l c i n e u r i n (3 /tg, bovine b r a i n , p u r i f i e d e s s e n t i a l l y according to T a l l a n t and Cheung, 1986) were subjected separate ly to e i t h e r no treatment (-) or ca lpa in treatment (+) at 25°C fo r 60 min in 50 mM T r i s - H C l , 10 mM d i t h i o t h r e i t o l , 0.3 mM EGTA, 0.5 mM C a C l 2 and 0.2 /tg p u r i f i e d c a l p a i n I (see F i g . 38). Termination of p r o t e o l y s i s and gel e l e c t r o p h o r e s i s are as descr ibed in F i g . 39. The l e t t e r I and C on the l e f t i n d i c a t e the bands of troponin I and C, r e s p e c t i v e l y , whi le the l e t t e r A , A ' and B on the r igh t ind ica te the subunit A (60 kDa), major fragment of subunit A (43 kDa) and subunit B (17 kDa) of c a l c i n e u r i n , r e s p e c t i v e l y . The 80 kDa subunit of ca lpa in and i t s major a c t i v e fragment (76 kDa) are ind ica ted with an open t r i a n g l e . Molecular weight standards: 200 kDa (a ) , 116 kDa (b) , 97 kDa (c ) , 66 kDa (d) , 43 kDa (e ) , 31 kDa (f) and 21.5 kDa (g) were as descr ibed in F i g . 38 as well as egg white lysozyme, 14.4 kDa (h) . Protein patterns presented here were reproduced in two other separate experiments. 170 Ill M . W . S t d . T r o p o n i n C a l m o d u l i n S - 1 0 0 d S - 1 0 0 p O n c o m o d u l i n P a r v a l b u m i n C a l c i n e u r i n O -W \ I i t CO CD Q . O CT D) • lit • 1 1 3> > O "D 0) 3 I + I + I + I + I + I + I + 0> aunS . L j DISCUSSION I. D iscuss ion o f the experimental r e s u l t s 1. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of c a l p a i n In the f i r s t se r ies of experiments, i t was c l e a r l y demonstrated that c a l p a i n was p u r i f i e d to apparent homogeneity ( F i g . 14) using a s e r i e s of chromatographic steps ( F i g . 10, 11, 12 and 13). Next, the p u r i f i e d c a l p a i n was c h a r a c t e r i z e d . F i r s t , the a u t o l y s i s of c a l p a i n was studied and i t was found that the 80 kDa subunit of c a l p a i n was indeed autolyzed to a c t i v e fragments of 78 and 76 kDa ( F i g . 15A), as reported in the l i t e r a t u r e (see Pontremoli and M e l l o n i , 1986b). It was a lso found that ca lpa in a c t i v i t y was more s tab le at 25°C than at 3 7 ° C , as reported by others (Mellgren et a l . , 1982). The p u r i f i e d c a l p a i n preparat ions showed a half-maximal a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y at about 20 /xM f ree C a 2 + ( F i g . 16A). Thus, the protease belongs to the c l a s s designated 9+ c a l p a i n I, which requires micromolar concentra t ions of Ca to be f u l l y a c t i v e (Murachi, 1983b). The ca lpa in preparat ions a lso showed s e n s i t i v i t y towards cyste ine proteinase i n h i b i t o r y agent's ( leupept in and iodoace ta te ) . This is again confirmed in t h i s work ( F i g . 16B). In b r i e f , the c a l p a i n preparat ions show apparent homogeneity and a l l the proper t ies of c a l p a i n I. Therefore , at t h i s p o i n t , i t was decided that these ca lpa in preparat ions could be used to study the e f f e c t of ca lpa in I on the Ca -172 ATPase. 2. Ca lpa in ac t iva tes the ATP-hydro lv t ic a c t i v i t y o f the Ca -ATPase The f i r s t step was to look fo r an e f f e c t of c a l p a i n on the C a 2 + -ATPase a c t i v i t y by pre t rea t ing plasma membranes i s o l a t e d from human ery throcytes with the protease. It was subsequently found that in the presence of C a 2 + and c a l p a i n , the C a z + - A T P a s e a c t i v i t y was ac t iva ted and became c a l m o d u l i n - i n s e n s i t i v e in a time-dependent manner ( F i g . 17C). In p a r a l l e l , t r y p s i n and papain treatments of the plasma membrane were a lso found to ac t i va te the C a 2 + - A T P a s e ( F i g . 17A, B ) . When the C a 2 + a f f i n i t y of the membrane-bound C a 2 + - A T P a s e was examined, i t was confirmed that with the cont ro l membranes the enzyme had low a f f i n i t y f o r Ca in the absence of added calmodul in but high a f f i n i t y f o r C a 2 + in the presence of calmodul in ( F i g . 19A). Calpain treatment apparently act iva ted the C a 2 + - A T P a s e by conver t ing the enzyme in to the high a f f i n i t y mode even in the absence of calmodulin ( F i g . 20). A l s o , the a c t i v a t i o n was accompanied by a l o s s o f fu r ther ca lmodul in-s t imu la t ion ( i . e . i t became calmodul i n - i n s e n s i t i v e ) ( F i g . 20). These e f f e c t s , in f a c t , can a lso be demonstrated with t r y p s i n or papain treatment ( F i g . 19B, C) as descr ibed by others (Sarkadi et a l . , 1986). I n t e r e s t i n g l y , i t was found that calmodulin could protect the membrane-pi bound Ca -ATPase against calpain-mediated a c t i v a t i o n over a s i m i l a r concentra t ion range that i s required to a c t i v a t e the C a 2 + - A T P a s e ( F i g . 20 and 21). This p ro tec t ive e f fec t of calmodulin was not observed with 173 t r y p s i n or papain treatment ( F i g . 19B, C ) . In F i g . 41, a model i s proposed to i l l u s t r a t e the e f f e c t s of calmodulin and c a l p a i n on the a c t i v i t y of the C a 2 + - A T P a s e . F i r s t l y , calmodulin ac t i va tes the C a 2 + -ATPase r e v e r s i b l y while ca lpa in ac t iva tes the C a 2 + - A T P a s e i r r e v e r s i b l y . Secondly, the two high a c t i v i t y forms of the C a 2 + - A T P a s e appear to be non-i n t e r c o n v e r t i b l e based on the fac t that the c a l p a i n - a c t i v a t e d C a 2 + - A T P a s e i s no longer st imulated by calmodulin and that the calpain-mediated p r o t e o l y t i c a c t i v a t i o n of the C a 2 + - A T P a s e i s l a r g e l y prevented by ca lmodul in . A l s o , reduct ion of i n t r a c e l l u l a r C a 2 + concentrat ion by the ca lmodul in -s t imula ted Ca^ pump w i l l tend to fu r ther minimize the calpain-mediated i r r e v e r s i b l e a c t i v a t i o n under normal p h y s i o l o g i c a l c o n d i t i o n s . pi 3. Fragmentation of the C a - A T P a s e by c a l p a i n : comparison with t r y p s i n At t h i s point i t appeared that the calpain-mediated a c t i v a t i o n of the C a 2 + - A T P a s e could be a complex event. It was therefore decided that the p r o t e o l y t i c a c t i v a t i o n as well as the p ro tec t i ve e f f e c t of calmodulin should be fu r ther character ized by fo l lowing the fragmentation of the p. p. Ca^ -ATPase by c a l p a i n . F i r s t , the membrane-bound Ca^ -ATPase was v i s u a l i z e d by the 3 2 P-phosphoenzyme intermediate formed. It was found that in the absence of ca lmodul in , the 136 kDa in tac t EP-forming enzyme was fragmented sequent ia l l y by c a l p a i n in to two EP-forming fragments (about 124 kDa and 80 kDa) ( F i g . 23), with a t ime-course p a r a l l e l to that of p r o t e o l y t i c a c t i v a t i o n ( F i g . 22). However, in the presence of 174 F i g . 41 Proposed model for the contro l of the human er throcyte membrane  C a c -ATPase by calmodulin and c a l p a i n . (x) means reac t ion does not proceed. For a d e t a i l e d exp lanat ion , see t e x t . 175 Ca 2 + -ATPase low activity Calmodulin reversible irreversible Calpain Calmodulin - activated Ca 2 + -ATPase high activity Proteolytically-activated Ca 2 + -ATPase high activity ca lmodul in , d i f f e r e n t EP-forming fragments of 127 kDa and 85 kDa were observed ( F i g . 23). Under these c o n d i t i o n s , i t was confirmed that very l i t t l e p r o t e o l y t i c a c t i v a t i o n occurred . Since EP-formation i s a r e f l e c t i o n of an ac t ive ATPase molecule ( in tac t enzyme or fragment), these r e s u l t s suggest the fo l low ing : ( i ) one or both of the 124 kDa and 80 kDa fragments was (were) the calmodul in- independent ac t ive fragment(s) ; ( i i ) one or both of the 127 kDa or 85 kDa fragments was (were) the ac t ive fragment(s) which reta ined the c a l m o d u l i n - s e n s i t i v i t y . In the l i g h t of these r e s u l t s , the ca lpain-mediated fragmentation of the s o l u b l i z e d and p u r i f i e d C a 2 + - A T P a s e , in the absence versus the presence of calmodulin was studied ( F i g . 25). A scheme of the fragmentation sequence i s suggested in F i g . 42. In the absence of ca lmodul in , the C a 2 + - A T P a s e was sequent ia l l y proteolyzed to a 125-124 kDa heterogeneous fragment (more than one component) and therea f te r to a 82-80 kDa heterogeneous fragment, both capable of forming the acylphosphate intermediate ( F i g . 25B). However, only the minor 125 kDa and 82 kDa components of these fragments re ta ined the capac i ty to bind and to be st imulated by calmodul in , whereas the major fragments formed, namely the 124 kDa and 80 kDa components, l o s t the capac i ty to bind calmodulin ( F i g . 26). When the fragmentation pattern of the membrane-bound Ca^ -ATPase was O 9 s t u d i e d , one had to re ly on the formation of J < 1 P - a c y l phosphate intermediate to v i s u a l i z e the d i f f e r e n t fragments. With t h i s technique i t becomes d i f f i c u l t to i d e n t i f y whether the 124 kDa and the 80 kDa fragments a lso contain the minor 125 kDa and 82 kDa components ( F i g . 23 and F i g . 177 F i g . 42 Flow-chart fo r the fragmentation of the erythrocyte C a - A T P a s e bv  ca lpa in in the presence and absence of ca lmodul in . Numbers in the boxes ind ica tes the apparent molecular weight (kDa) of e i t h e r the nat ive C a 2 + -ATPase or i t s fragments. (+) ind ica tes the capac i ty to form 3 2 P - l a b e l l e d acylphosphate intermediate and (*) ind ica tes ca lmodul in -b ind ing c a p a c i t y . 178 Figure 42 179 27A). However, the Ca -ATPase fragments, p u r i f i e d from membranes pretreated with ca lpa in in the absence of calmodulin by re tent ion on a c a l m o d u l i n - a f f i n i t y column, have apparent molecular masses 125 kDa and 82 kDa ( F i g . 27B, C ) . Therefore , i t appears that the remaining 124 kDa and 80 kDa fragments which were not re ta ined by the calmodul in-agarose column are c a l m o d u l i n - i n s e n s i t i v e , p r o t e o l y t i c a l l y - a c t i v a t e d forms of the C a 2 + -ATPase. On the other hand, in the presence of ca lmodul in , the enzyme was proteolyzed to d i f f e r e n t higher molecular mass fragments of 127 kDa and 85 kDa, r e s p e c t i v e l y ( F i g . 23 and 25B). Both of these fragments were capable of forming a J t P - p h o s p h o r y l a t e d intermediate and were re ta ined by the calmodul in-agarose column in the presence of calc ium ( F i g . 25C and 27B). Th is i s cons is ten t with the fac t that calmodulin prevents the p r o t e o l y t i c a c t i v a t i o n and the l o s s of calmodulin s t imu la t ion of the enzyme. Calpain a lso produces other fragments of the p u r i f i e d C a 2 + - A T P a s e with r e l a t i v e molecular mass of 55 kDa, 39 kDa, 37 kDa and 32 kDa ( F i g . 25B). In contrast to the l a r g e r fragments d iscussed above, none of these smal ler fragments form a phosphorylated intermediate ( F i g . 25C). There fore , i t seems that the 80 kDa fragment i s the smal lest form of the Ca^ -ATPase s t i l l capable of forming the acylphosphate intermediate a f t e r p r o t e o l y s i s by c a l p a i n . This i s s i m i l a r to the smal lest 3 2 P - l a b e l l e d fragments (81 kDa and 76 kDa) obtained by t r y p s i n i z a t i o n of both the membrane-bound (Sarkadi et a l . . , 1986; Enyedi et aj_., 1987), and the p u r i f i e d C a 2 + - A T P a s e (Zur in i et a l . , 1984). I n t e r e s t i n g l y , the C a 2 + -ATPase a c t i v i t i e s of both the 81 kDa and 76 kDa t r y p t i c fragments were reported to be i n s e n s i t i v e to s t imu la t ion by calmodulin (Sarkadi et a l . , 180 1986; Benaim et a l . , 1984). Therefore , the fragmentation patterns o f the C a - A T P a s e obtained by ca lpa in and by t r y p s i n were compared ( F i g . 28). I n t e r e s t i n g l y , t rans ien t formation of 124 kDa and 127 kDa fragments produced by t r y p s i n in the absence and presence o f ca lmodul in , r e s p e c t i v e l y , was observed, as in the case o f c a l p a i n . This suggests that both proteases have s i m i l a r or i d e n t i c a l i n i t i a l s i t e s of cleavage in the Ca^ -ATPase molecule. However, longer t r y p s i n treatment (30 min) of the p u r i f i e d C a 2 + - A T P a s e caused accumulation of major fragments between 77 kDa and 86 kDa, while fragments between 124 kDa and 127 kDa remain prominent even a f te r prolonged c a l p a i n treatment (120 min) ( F i g . 28) . E a r l i e r r e s u l t s in t h i s t h e s i s demonstrated that calmodulin f a i l e d to prevent p r o t e o l y t i c a c t i v a t i o n of the C a 2 + - A T P a s e by t r y p s i n , in cont ras t to the case of ca lpa in ( F i g . 19). In agreement with these r e s u l t s , F i g . 28 shows that even in the presence of ca lmodul in , t r y p s i n produced large amounts of an 82 kDa fragment, p r e v i o u s l y reported to be of high a c t i v i t y and i n s e n s i t i v e to calmodulin (Zur in i et a l . , 1984; Benaim et a l . , 1984; Enyedi et a l . , 1987). However, calmodul in appeared to protect against fur ther t r y p t i c fragmentation to the 76 kDa fragment seen in the absence of calmodulin ( F i g . 28). The 86 kDa, 82 kDa and 77 kDa fragments that were observed here very l i k e l y to be the 85 kDa, 81 kDa and 76 kDa fragments, r e s p e c t i v e l y , reported by others (Zur in i et a l . , 1984; Benaim et a l . , 1984; Sarkadi et a l . , 1986; Enyedi et a l . , 1987). The small d i f f e r e n c e in r e l a t i v e molecular mass of the t r y p t i c fragments reported in t h i s t h e s i s and that of others i s probably due to the d i f f e r e n t gel systems used. F ig 43 provides a schematic of the fragmentation of the pi Ca*- -ATPase obtained with t r y p s i n . It i s proposed that the 90 kDa, 86 kDa, / 181 F i g . 43 Schematic of the p r o t e o l y s i s of the Ca -ATPase by t r y p s i n . The nat ive i n t a c t enzyme and i t s fragments are represented by the opened bars . The number in the middle of a bar i s the molecular mass of that po lypept ide . The l e t t e r P represent the l o c a t i o n of the acylphosphate s i t e , while the l e t t e r s N and C represent the N-terminal and the C-terminus, r e s p e c t i v e l y . The darkened area was the i d e n t i f i e d ca lmodul in-b inding domain. See text f o r d e t a i l s . 182 Figure 43 183 82 kDa, 77 kDa fragments a l l have the same or s i m i l a r N-terminal end which i s generated by a cleavage about 34 kDa from the N-terminal of the in tac t p r o t e i n . This cleavage produces a prominent 34 kDa fragment and the 90-95 kDa fragment. The 34 kDa fragment i s fur ther cleaved to produce a 32 kDa fragment with time (compare lane 3 to lane 2, F i g . 28B and 28C) (see a lso Zur in i et a l . , 1984; Papp et a l . , 1989). The 90 kDa i n i t i a l fragment i s fu r ther cleaved several times at the C-terminal end to produce the ca lmodul in -b ind ing 86 kDa fragment, and the non-calmodul in-b inding 82 kDa and the 77 kDa fragment ( F i g . 288 and 28C; see a lso Zur in i et a l . , 1984; Benaim et a l . , 1984; Sarkadi et a l . . 1986; Enyedi et a l . . 1987). Note a lso that a l l of the 90 kDa, 86 kDa, 82 kDa and 77 kDa fragments are EP-forming ac t ive fragments. The i r formation i s cons is ten t with the f a c t that the ca lmodul in -b ind ing domain i s about 9-10 kDa from the C-terminal end and the acylphosphate s i t e i s about 50 kDa from the N-terminal of the i n t a c t C a 2 + - A T P a s e (Shul l and Greeb, 1988; Verma et a l . . 1988). Based on k i n e t i c s t u d i e s , a 76 kDa fragment (77 kDa in F i g . 43) was l a t e r found to be i n s e n s i t i v e to a c i d i c phosphol ip id s t imula t ion while a 82 kDa fragment (80 kDa in F i g . 43) re ta ined such c a p a b i l i t y (Enyedi et a l . , 1987). There fore , a 5 kDa fragment removed from the 82 kDa region appears to be the a c i d i c p h o s p h o l i p i d - i n t e r a c t i n g domain. The a c i d i c p h o s p h o l i p i d - i n t e r a c t i n g domain could have two poss ib le l o c a t i o n s : The f i r s t would be r i g h t next to and upstream from the N-terminal s ide of the calmodulin b inding domain ( i . e . at the C-terminal end of the 82 kDa fragment): t h i s region c o n s i s t s of a high content of negat ive ly charged res idues (Shul l and Greeb, 1988; Verma et a l . , 1988) which can be viewed 184 to bind and l o c a l i z e Ca^ ions . These l o c a l i z e d calc ium ions could then conceivabley bind negat ive ly charged a c i d i c p h o s p h o l i p i d , which in turn ac t i va tes the enzyme. An a l t e r n a t i v e l o c a t i o n would be about 45-50 kDa from the N-terminal of the in tac t prote in ( i . e . at the N-terminal end of the 82 kDa fragment). Papp et a l . (1989) favor the second p o s s i b i l i t y . They argue that in t h i s region (about 45-50 kDa from the N-terminal) there are a number of p o s i t i v e l y charged res idues (Verma et a l . , 1988) which may i n t e r a c t with a c i d i c phospho l ip id . However, besides the fac t that a c i d i c phospho l ip id -ac t i va ted C a 2 + - A T P a s e has a somewhat higher C a 2 + a f f i n i t y than the ca lmodul in -ac t iva ted enzyme (Enyedi et a l . , 1987), the a c t i v a t i o n of the C a - A T P a s e by a c i d i c phosphol ip id and that by calmodulin are very s i m i l a r (N igg l i et al_., 1981a; A l - J o b o r e and Roufoga l i s , 1981). Th is suggests a s i m i l a r mode of a c t i v a t i o n by both types of a c t i v a t o r s . There fore , i t i s reasonable to p red ic t s i m i l a r or adjacent s i t e s of ac t ion of both calmodulin and a c i d i c phosphol ip id on the C a 2 + - A T P a s e . The fac t that the presence of calmodulin prevented the formation of the 77 kDa fragment ( F i g . 28) a lso supports the f i r s t p o s s i b i l i t y . It appears, the re fo re , that the f i r s t p o s s i b i l i t y i s more compatible with the a v a i l a b l e experimental data and i s there fore chosen in the scheme ( F i g . 43) . Based on the proper t ies of the fragments of the C a 2 + - A T P a s e produced by ca lpa in and the homology to the t r y p s i n - p r o t e o l y s i s , a scheme of p j . calpain-mediated fragmentation of the Ca^ -ATPase can be constructed ( F i g . 44) . From the r e s u l t s that ( i ) the 127 kDa and the 125 kDa fragments can s t i l l bind calmodulin while the 124 kDa does not ( F i g . 26 and 27), and 185 F i g . 44 Schematic of the p r o t e o l y s i s of the Ca -ATPase by c a l p a i n I. The in tac t Ca -ATPase and i t s fragments are represented by opened bars . The number in the middle of a bar i s the r e l a t i v e molecular mass of that po lypept ide . The l e t t e r P represents the l o c a t i o n of the aspar ta te -acylphosphate s i t e , while the l e t t e r N and C represent the amino- and carboxy l - te rmina l ends, r e s p e c t i v e l y . The darkened area i s the i d e n t i f i e d calmodulin binding domain. See text fo r d e t a i l s . 186 Figure 4 4 187 ( i i ) that the 127 kDa fragment i s produced in the presence of calmodulin while the 125 kDa and 124 kDa fragments were produced in the absence of ca lmodul in , i t i s obvious that calmodulin in f luences the cleavage s i t e s from which these fragments are produced. In other words, the 127 kDa, 125 kDa and 124 kDa fragments are produced by m u l t i p l e cleavages near or at the ca lmodul in -b inding domain at the C-terminal end of the enzyme. Transformation from the 136 kDa i n t a c t p ro te in to the 127 kDa fragment means a t runcat ion of about 9 kDa from the o r i g i n a l C - t e r m i n a l . Since the ca lmodul in -b ind ing domain s t a r t s about 9 kDa from the C-terminal end and occupies a 3.5 kDa region (Verma et a l . , 1988), the 127 kDa fragment would have i t s ca lmodul in-b inding domain l a r g e l y i n t a c t ( F i g . 45) , which i s cons is ten t with the fac t that i t binds calmodulin ( F i g . 27 and 28). On the other hand, the 125 kDa component w i l l have l o s t about 11 kDa from the C-terminal end or a fur ther por t ion of about 2 kDa from the N-terminal end of the ca lmodul in -b inding domain ( F i g . 45) . I n t e r e s t i n g l y , the 125 kDa can s t i l l bind calmodulin ( F i g . 27). In the case of the 124 kDa fragment, i t would have l o s t about 12 kDa from the C-terminal and about 3 kDa of the 3."5 kDa calmodul in -b ind ing domain ( F i g . 45) . With most of the calmodul i n -binding domain t runcated , the 124 kDa does not bind calmodul in ( F i g . 27 and 28). It was observed that with longer times o f p r o t e o l y s i s , besides fragments of 124-127 kDa, small EP-forming ac t ive fragments (of 80-85 kDa) were a lso produced ( F i g . 23 and 25). I n t e r e s t i n g l y , the 85 kDa fragment, l i k e the 127 kDa fragment, was produced only in the presence of calmodul in ( F i g . 23 and 25). On the other hand, the 80-82 kDa fragments, l i k e the 124-125 kDa fragments, were produced only in the absence of calmodul in ( F i g . 25). Furthermore, the 80 kDa fragment, as well as the 124 188 F i g . 45 Schematic of the formation of a c t i v e fragments of the plasma  membrane Ca^ pump produced by ca lpa in I in the absence (-) and the  presence (+) of calmodulin (CaM). The l e t t e r s N and C represent the amino and carboxyl - terminus of the amino ac id sequence, r e s p e c t i v e l y . The numbers beside the polypeptide represent the approximate amino ac id res idue numbers. The large number in the centre of each polypept ide represents i t s r e l a t i v e molecular mass. The l e t t e r "P" represents the acylphosphate s i t e and the darkened region represents the ca lmodul in-binding domain. The calmodulin molecule i s represented by a c i r c l e . 189 Figure 45 190 kDa fragment, do not bind calmodul in-agarose while the 82 kDa can be co-p u r i f i e d with the 125 kDa fragment using a calmodul in-agarose column ( F i g . 26 and 27). A l l these f ind ings point to s i m i l a r i t i e s between the two components from each of the three pa i rs of fragments (127 kDa/85 kDa, the 125 kDa/82 kDa and 124 kDa/80 kDa). A simple c a l c u l a t i o n revea ls that the d i f f e r e n c e in s i z e between the two components of each p a i r of fragments are: 42 kDa, 43 kDa and 44 kDa r e s p e c t i v e l y . There fore , i t i s tempting to suggest that the 85 kDa, 82 kDa and 80 kDa fragments o r i g i n a t e from the 127 kDa, 125 kDa and 124 kDa fragments, r e s p e c t i v e l y and are produced by an i d e n t i c a l or s i m i l a r cleavage about 42-44 kDa from the N-terminal end ( F i g . 44 and 45). Together, these cons idera t ions would a lso suggest tha t , by binding to the ca lmodul in -b inding domain, calmodulin can p h y s i c a l l y or otherwise protect t h i s 3-5 kDa region (127 kDa - 124 kDa or 85 kDa - 80 kDa) from the at tack of c a l p a i n , which i s cons is ten t with the d i f f e r e n t i a l fragmentation produced in the absence versus the presence of calmodulin ( F i g . 23 and 25). A l s o , two fragments of 37 and 39 kDa, r e s p e c t i v e l y , are always observed during p r o t e o l y s i s both in the absence and the presence of calmodulin ( F i g . 25). These 39-37 kDa doublet fragments can be in terpre ted as being fu r ther cleavage products of the i n i t i a l fragment (40-44 kDa) from the N- terminal , as d iscussed above. However, the 39-37 kDa doublet was found to c o - p u r i f y with the l a r g e r fragments (eg. 82 kDa and 125 kDa) on a calmodul in-agarose column (see F i g . 27). There are two p o s s i b i l i t i e s : ( i ) the 39-37 doublet d id not come from the N-terminal c leavage, but rather o r ig ina ted from a region cover ing the ca lmodul in -191 binding domain at the C-terminus, ( i i ) the 39-37 kDa doublet came from the N-terminal cleavage and reta ined a sequence which, although lack ing a t y p i c a l ca lmodul in -b inding domain, a lso binds ca lmodul in ; ( i i i ) the 39-37 kDa doublet o r i g i n a t e s from the N-terminal cleavage and r e t a i n s a domain which assoc ia tes with the 80-85 kDa fragments. In t h i s c a s e , the doublet fragment w i l l have been reta ined by the calmodul in-agarose i n d i r e c t l y . If the f i r s t p o s s i b i l i t y i s c o r r e c t , one would expect more accumulation of the 39-37 kDa doublet from p r o t e o l y s i s in the presence of calmodul in than in i t s absence, s ince in the absence of calmodulin the ca lmodul in -b ind ing domain i s r e a d i l y dest royed. However, i t was observed that the 39-37 kDa fragments were produced in equal quant i ty during p r o t e o l y s i s in the absence and in the presence of calmodulin ( F i g . 25). In support of the second p o s s i b i l i t y , Zur in i et a l . (1984) have suggested that the C a 2 + -ATPase may bind a second molecule of ca lmodul in , based on t h e i r r e s u l t s with [ 1 2 ^ I ] iodoaz idoca lmodu l in l a b e l l i n g of the enzyme. I n t e r e s t i n g l y , in the same paper, a 33.5 kDa fragment of the C a 2 + - A T P a s e produced by t r y p s i n ( l i k e l y to be the same as the 32 kDa fragment in F i g . 28 and 43) o c c a s i o n a l l y c o - p u r i f i e d with a l a r g e r ca lmodul in -b ind ing fragment (90 kDa) (see F i g . 8A of Zur in i et a l . , 1984). Since p o s s i b i l i t y ( i i ) was most cons is ten t with the a v a i l a b l e experimental r e s u l t s , i t was chosen in the model ( F i g . 44) . However, i t must be s t ressed that p o s s i b i l i t y ( i i i ) could not be e l im ina ted . To extend the s p e c u l a t i o n , i t i s suggested that the 55 kDa and 32 kDa fragments o r ig ina ted from a major cleavage o f the 80-85 kDa fragments. Note that the l o c a t i o n of the acylphosphate s i t e (on e i t h e r the 55 kDa or the 32 kDa fragment) i s not ass igned . It must again be s t ressed that the proposed o r i g i n s of the 55 kDa, 39-37 kDa and 32 kDa 192 fragments are c l e a r l y p r o v i s i o n a l . D i rec t evidence from f u r t h e r work should c l a r i f y these p o i n t s . In t h i s t h e s i s i t was demonstrated that t r y p s i n treatment of the p u r i f i e d Ca^ -ATPase, besides producing major fragments of 77-86 kDa, a lso produced two la rger but more t rans ien t fragments: a 124 kDa fragment in the absence of calmodulin and a 127 kDa fragment in i t s presence ( F i g . 28). Since ca lpa in treatment of the C a 2 + - A T P a s e a lso produced major fragments of 124 kDa and 127 kDa, in the absence and the presence of ca lmodul in , r espec t i ve ly ( F i g . 25 and 28), It i s l i k e l y that the c a l m o d u l i n - s e n s i t i v i t y of 124 kDa and 127 kDa fragments produced with t r y p s i n resemble t h e i r counterparts produced with c a l p a i n . In other words, the 127 kDa t r y p t i c fragment would bind calmodul in whi le the 124 kDa t r y p t i c fragment would have l o s t such a b i l i t y . It i s fu r ther postu la ted that t r y p s i n might i n i t i a l l y at tack the C-terminal end of the C a z + - A T P a s e . In the absence of ca lmodul in , such at tack would remove the C-terminal segment, inc lud ing most or a l l of the ca lmodul in -b ind ing domain, arid produce the 124 kDa fragment. In the presence o f ca lmodul in , a s i m i l a r cleavage which removes the C-terminal segment would spare the ca lmodul in -b ind ing domain and produce the s l i g h t l y l a r g e r 127 kDa fragment ( F i g . 43) . Papp et a l . (1989) recent ly suggested that a 125 kDa t r y p t i c fragment of the C a 2 + - A T P a s e that they observed was produced by a s i n g l e cleavage near the N-terminal end rather than the C - t e r m i n a l . However, no d i r e c t evidence supporting t h i s c la im was provided (Papp et a l . , 1989). 4. Ca lpa in a lso ac t iva tes the Ca - t r a n s l o c a t i n g a c t i v i t y of the Ca -193 ATPase Since the phys io log ic funct ion of the C a - A T P a s e i s to pump Ca (from the c y t o s o l ) out of the c e l l , i t was important to e s t a b l i s h whether c a l p a i n treatment would act iva te the C a 2 + - t r a n s l o c a t i n g func t ion in the same way that i t ac t iva ted the ATP h y d r o l y t i c a c t i v i t y . In recent years the p u r i f i e d C a 2 + - t r a n s l o c a t i n g ATPase from human erythrocytes has been extensive ly character i zed (see A l - J o b o r e et a j . . , 1981; C a r a f o l i and Z u r i n i , 1982; N igg l i et a l . . 1987; A l - J o b o r e et a l . , 1984; Roufogal is and V i l l a l o b o , 1989). Fewer s tudies have been repor ted , however, on the 1iposome-reconst i tuted enzyme (Haaker and Racker, 1979; N i g g l i et a l . , 1981b; Benaim et a l . , 1986; V i l l a l o b o and R o u f o g a l i s , 1986), and in most studies the r e c o n s t i t u t i o n was performed with a s o l e c t i n as the source o f phosphol ip id . Due to the presence of a c i d i c phosphol ip id components in a s o l e c t i n , however, the calmodul i n - s t i m u l a t o r y e f f e c t on C a 2 + t r a n s l o c a t i o n has not been reported to our knowledge. In order f o r us" to observe p r o t e o l y t i c a c t i v a t i o n of the recons t i tu ted C a 2 + pump and the l o s s of calmodulin s t i m u l a t i o n , as predicted from e a r l i e r r e s u l t s on membrane-bound ( F i g . 17, 18, 20, and 22) and p u r i f i e d enzymes ( F i g . 24), a 7+ c a l m o d u l i n - s e n s i t i v e reconst i tu ted Ca pump preparat ion was r e q u i r e d . Th is problem was resolved by using h ighly p u r i f i e d egg yo lk phosphat idy lcho l ine (Niggl i et al_., 1981b) fo r r e c o n s t i t u t i o n of the enzyme. With the phosphat idy lcho l ine - reconst i tu ted C a c pump, the fo l low ing was demonstrated: ( i ) the i n i t i a l rates of both ca lc ium uptake and A T P - h y d r o l y t i c a c t i v i t y were st imulated at l e a s t 3- to 4 - f o l d by 194 calmodul in at 0.4 /iM f ree Ca^ ( F i g . 30) . Treatment of proteol iposomes with c a l p a i n increased these rates to l e v e l s c lose to those obtained upon add i t ion of calmodulin before ca lpa in treatment ( F i g . 30) . ( i i ) the i n i t i a l ra te of both C a 2 + uptake and A T P - h y d r o l y t i c a c t i v i t y of the in tac t C a 2 + pump exhib i ted two a f f i n i t i e s fo r C a 2 + : low a f f i n i t y in the absence o f calmodulin and high a f f i n i t y in i t s presence ( F i g . 35A and F i g . 36A). Ca lpa in treatment s h i f t e d the enzyme into the high a f f i n i t y mode regard less of whether calmodulin was absent or present in the assay medium ( F i g . 35B and F i g . 36B). ( i i i ) The i n i t i a l rate of Ca uptake exhib i ted two a f f i n i t i e s fo r ATP (approx. 1.1-1.4 /iM and 245-289 /iM), both in the absence and in the presence of calmodulin ( F i g . 37A). Th is b iphas ic dependence on ATP was not a l te red by ca lpa in treatment ( F i g . 37B). Th is i s the f i r s t report where the 1 iposome-reconst i tu ted plasma membrane C a 2 + pump was subjected to l i m i t e d p r o t e o l y s i s . The success of the approach used in the present study was in part due to the f a c t that the fragmentation by ca lpa in was more s e l e c t i v e than that of t r y p s i n or other proteases . The technique employed in t h i s repor t can be appl ied to f u r t h e r s tudies on the proper t ies of the Ca pump in i t s p u r i f i e d and r e c o n s t i t u t e d s t a t e . A study of the fragmentation pat tern coupled with 3 2 P - a c y l p h o s p h o p r o t e i n formation ( F i g . 33) revealed that the 124 kDa and the 127 kDa forms of the reconst i tu ted C a 2 + pump were the major ac t ive ATPase fragments produced by ca lpa in in the absence or the presence of ca lmodul in , r e s p e c t i v e l y . The formation of the 124 kDa fragment occurred concomitant ly with the p r o t e o l y t i c a c t i v a t i o n of the i n i t i a l ra tes of both the C a 2 + uptake and the ATP h y d r o l y t i c a c t i v i t y of the enzyme ( F i g . 195 31 and 33). The formation of the 124 kDa and 127 kDa fragments were observed p r e v i o u s l y when the membrane-bound or the p u r i f i e d Ca^ -ATPase was t reated with ca lpa in I ( F i g . 23 and 25). In t h i s t h e s i s , i t was observed that the presence of calmodulin protected the membrane-bound C a 2 + - A T P a s e from p r o t e o l y t i c a c t i v a t i o n of the A T P - h y d r o l y t i c a c t i v i t y ( F i g . 20 and 21). It was a lso found that the 127 kDa fragment was s t i l l s e n s i t i v e to ca lmodul in , whereas the 124 kDa fragment ne i the r bound, nor was st imulated by, calmodulin ( F i g . 25 and 27). There fo re , i t was of i n t e r e s t to examine whether such a p ro tec t ive e f f e c t of calmodulin occurred on the reconst i tu ted C a 2 + pump. By g e l - f i l t r a t i o n chromatography (Sephacryl S-200) , i t was poss ib le to separate c a l p a i n and calmodul in from the proteol iposomes a f te r they were pretreated with c a l p a i n in the absence or in the presence of ca lmodul in . Besides the expected 124 kDa fragment formed in the absence of ca lmodul in , l e s s prominent bands of 80 kDa, 55 kDa, 39 kDa and 37 kDa were a lso observed ( F i g . 34, lane c ) . In the presence o f ca lmodul in , in add i t ion to the prominent 127 kDa fragment, l e s s prominent bands of 85 kDa, 55 kDa, 39 kDa and 37 kDa were a lso i d e n t i f i e d ( F i g . 34, lane d ) . These smal ler fragments were a lso observed p r e v i o u s l y when the p u r i f i e d C a 2 + - A T P a s e was proteolyzed by c a l p a i n ( F i g . 24) . I t was observed that ca lpa in alone produced p r o t e o l y t i c a c t i v a t i o n of the r e c o n s t i t u t e d C a 2 + - A T P a s e a c t i v i t y and decreased the f o l d - s t i m u l a t i o n by calmodul in from 3.4 to 1.1 (Table 5) , whereas the ca lmodul in-s t imu la t ion a f t e r ca lpa in treatment in the presence of calmodul in remained prominent ( 2 . 9 - f o l d ) (Table 5) . Therefore , the p r o t e c t i v e e f f e c t of calmodul in against p r o t e o l y t i c a c t i v a t i o n of the r e c o n s t i t u t e d C a c -ATPase was c l e a r l y demonstrated. Furthermore, s ince unl ike the plasma membrane-195 bound or p u r i f i e d enzyme ( F i g . 23 and 25), the 80 kDa ( in the absence of calmodulin) and the 85 kDa act ive fragments ( in the presence of calmodulin) were only formed in small quant i ty from p r o t e o l y s i s of the 1 iposome-reconst i tuted enzyme ( F i g . 33), i t may be concluded that the 124 kDa fragment formed in the absence of calmodul in was the major p r o t e o l y t i c a l l y - a c t i v a t e d and c a l m o d u l i n - i n s e n s i t i v e C a 2 + - A T P a s e fragment, whereas the 127 kDa fragment formed in the presence of calmodul in was the 9+ major C a t T - A T P a s e fragment which reta ined c a l m o d u l i n - s e n s i t i v i t y . 5. Comparison of experimental r e s u l t s from t h i s labora tory and from other  1 aborator ies The laboratory of C a r a f o l i has i d e n t i f i e d an 85 kDa fragment of the C a 2 + - A T P a s e produced by t r y p s i n ( l i k e l y to be equiva lent to the 86 kDa in t h i s work, see F i g . 28 and 43) . From c i r c u m s t a n t i a l ev idence, they concluded that although t h i s fragment binds ca lmodul in , i t i s not ac t i va ted by i t (Benaim et a l . , 1984). On the other hand, they found that a"90 kDa fragment can bind and be st imulated by ca lmodul in , while an 81 kDa fragment ( l i k e l y to be the same as the 82 kDa fragment in t h i s work) ne i ther bound nor was st imulated by calmodulin (Zur in i et al_. , 1984; Benaim et a l . , 1984) Therefore , they proposed that a 9 kDa sequence at the C-terminal end i s important fo r ca lmodu l in -s t imu la t ion (Cara fo l i et a l . , 1987). They a lso proposed that the 9 kDa segment conta ins a 5 kDa sequence which binds calmodulin (ca lmodul in-b inding domain) and a 4 kDa sequence at the C-terminal s ide of the ca lmodul in -b ind ing domain, which is e s s e n t i a l fo r the expression of the calmodulin s t i m u l a t i o n . By comparing 197 the fragmentation models fo r t r y p s i n ( F i g . 43) and fo r c a l p a i n ( F i g . 44), t h e i r proposal would mean that the 127 kDa and 85 kDa fragments of the C a - A T P a s e (produced by ca lpa in ) have l o s t the 4 kDa sequence e s s e n t i a l f o r calmodulin s t i m u l a t i o n . However, experimential r e s u l t s from t h i s t h e s i s do not support t h i s : F i g . 27, Table 3, F i g . 34 and Table 5 c l e a r l y show that the 127 kDa and 85 kDa fragments do bind and are st imulated by ca lmodul in . In f a c t , the 125 kDa and 82 kDa fragments, having the ca lmodul in -b inding domain h a l f - t r u n c a t e d , a lso bind and i s st imulated by calmodulin ( F i g . 27 and Table 3 ) . I n t e r e s t i n g l y , C a r a f o l i et a l . (1987) a lso reported pre l iminary r e s u l t s on the p r o t e o l y s i s of the Ca^- -ATPase by c a l p a i n in the same r e p o r t . Since they d id not observe rap id l o s s of ca lmodul in -s t imula t ion by ca lpa in treatment and as they d id not observe accumulation of fragments beyond the 124 kDa fragment, they concluded that ( i ) ca lpa in cleavage of the C a 2 + - A T P a s e produced a l i m i t peptide (124 kDa) and ( i i ) such cleavage i s at the N-terminus (s ince the ca lmodul in -b ind ing domain i s at the C-terminal end). From the fragmentation o f the C a 2 + - A T P a s e found in the present work ( F i g . 44 and 45") i t i s c l e a r both of t h e i r conclusions about the e f f e c t of c a l p a i n on the C a 2 + - A T P a s e are l i k e l y i n c o r r e c t because: ( i ) even in t h e i r work, the ca lmodu l in -s t imu la t ion was a c t u a l l y decreased from the o r i g i n a l 6 - f o l d to 2 - f o l d a f t e r 240 min of ca lpain- t reatment ( F i g . 7 of C a r a f o l i et a l . , 1987). Th is drop of ca lmodul in -s t imula t ion was l i k e l y to have been due to the formation of an ac t iva ted 124 kDa fragment, the remaining 2 - f o l d ca lmodul in -s t imula t ion being l i k e l y contr ibuted by the presence of a l e s s prominent c a l m o d u l i n - s e n s i t i v e 125 kDa fragment, which would not be resolved from the 124 kDa fragment e a s i l y on t h e i r SDS-PAGE system; and 198 ( i i ) in t h e i r work, although fragmentation beyond the 124 kDa fragment was not obv ious , the to ta l amount of the 124 kDa fragment produced was fa r l e s s than the s t a r t i n g amount of the 136 kDa i n t a c t p ro te in ( F i g . 7 of C a r a f o l i et a l . , 1987), i n d i c a t i n g that the 124 kDa fragment was not a l i m i t fragment and was fur ther degraded. The reason that they d id not observe the formation of these small fragments was l i k e l y the s i l v e r -impregnation method fo r g e l - s t a i n i n g that they used. Since t h i s method invo lves a continuous development of c o l o r (analogous to photographic development), one can e a s i l y miss f a i n t e r peptide bands i f the development i s stopped prematurely. In t h i s t h e s i s , most of the ge ls were sta ined with Comassie B lue , which does not have the potent ia l problem mentioned above, and consequently the smal ler fragments were e a s i l y observed (see F i g . 25 and 28). It i s worth noting that immediately before the r e s u l t s on the a c t i v a t i n g e f f e c t of ca lpa in on the membrane-bound C a z + - A T P a s e were publ ished (Wang et al_. , 1988a), Au (1987) reported that the p ig ery throcyte C a 2 + - A T P a s e was act iva ted by c a l p a i n a lso from pig e r y t h r o c y t e s . Th is author a lso claimed that the calpain-mediated p r o t e o l y t i c a c t i v a t i o n of the membrane-bound C a 2 + - A T P a s e was best seen a f t e r the membranes were f i r s t t reated with the detergents saponin or g lycodeoxychola te . This could have been because calmodul in was not exhaust ive ly removed from the membranes used in that work and there fore the remaining calmodulin could have s i g n i f i c a n t l y protected the C a 2 + -ATPase against p r o t e o l y t i c a c t i v a t i o n . The subsequent treatment of the membranes with the detergents could have therea f te r removed the remaining 199 calmodulin and enhanced the s u s c e p t i b i l i t y of the Ca*- -ATPase to c a l p a i n . A l t e r n a t i v e l y , the d i f fe rence in s u s c e p t i b i l i t y could be due to a species d i f f e r e n c e (pig versus human)- i .e . the phosphol ip id composit ion and/or o rgan iza t ion of the enzyme in the l i p i d b i l a y e r could be d i f f e r e n t ) . A f t e r the r e s u l t s of the molecular c h a r a c t e r i z a t i o n of the calpain-mediated fragmentation and p r o t e o l y t i c a c t i v a t i o n of the C a 2 + - A T P a s e were publ ished (Wang et a l . , 1988b), Au et a l . (1989) publ ished another report , t h i s time using human erythrocyte membrane as the source of C a 2 + - A T P a s e . They found that by incubating the erythrocyte membrane at 30°C f o r 30 min in the presence of 5 mM cyste ine and 0.4 mM free C a 2 + , the C a 2 + - A T P a s e a c t i v i t y could be s t imula ted . They a t t r ibu ted t h i s a c t i v a t i o n t o t a l l y to membrane-bound c a l p a i n . They a lso suggested that the a c t i v a t i n g e f f e c t on the membrane-bound C a 2 + - A T P a s e that we observed (Wang et a l . , 1988a) might be due to membrane-bound ca lpa in and be independent of the p u r i f i e d c a l p a i n added. While i t i s true that ca lpa in i s l i k e l y to be both plasma membrane-bound as well as in the cytosol (Gopalakrishna and Barsky, 1986), i t was demonstrated that so lub le ca lpa in can be r e a d i l y attached to the pTasma membrane in a Ca 2 + -dependent manner (Pontremoli et a l . . 1985b). There fore , the importance of d i s t i n g u i s h i n g membrane-bound versus c y t o s o l i c c a l p a i n , as suggested by Au et a l . (1989) i s obscure. A l s o , these authors f a i l e d to observe that in our work ( F i g . 3 of Wang et a l . , 1988a; or F i g . 18C of t h i s t h e s i s ) , the a c t i v a t i o n of the C a 2 + - A T P a s e was dependent on the concentrat ion of added ca lpa in during the incubat ion ( in the presence of ca lc ium) . Furthermore, the membranes are washed with EDTA-containing buf fer during i s o l a t i o n , and c a l p a i n i s apparently depleted from these membranes. Therefore , by incubat ing the membranes 200 without added protease at 25°C for 30-60 min in the presence of d i t h i o t h r e i t o l and 200 11M f ree C a 2 + , the membrane-bound C a 2 + - A T P a s e expressed the t y p i c a l low C a 2 + a f f i n i t y , low V m a x mode when assayed in the absence of ca lmodul in , and was sh i f ted to the high C a 2 + a f f i n i t y , high vmax m o c ' e ky the add i t ion of calmodulin ( F i g . 4A of Wang et a l . , 1988a or F i g . 19A, t h i s t h e s i s ) . Therefore , in the membrane preparat ions used in t h i s t h e s i s , the con t r ibu t ion of c o - p u r i f i e d membrane-bound c a l p a i n to the p r o t e o l y t i c a c t i v a t i o n that was observed would be n e g l i g i b l e . On the other hand, the a c t i v a t i n g e f f e c t that Au et a l . (1989) observed can be in terpreted as a combination of ( i ) res idua l ca lpa in bound to the membrane that they i s o l a t e d and ( i i ) calc ium-induced a c t i v a t i o n of the enzyme which i s d i s t i n c t from the e f f e c t of c a l p a i n . Our l abora tory has been c h a r a c t e r i z i n g t h i s type of C a 2 + a c t i v a t i o n (Roufoga l is , Brzuszczak, Xu, Conigrave, Machan and Wang, manuscript in p repara t ion ) . T y p i c a l l y , t h i s a c t i v a t i o n was observed a f t e r membranes were t reated f o r 30-60 min at 37°C in the presence of 0 .5 -2 .0 mM f ree C a 2 + . Th is a c t i v a t i o n was not blocked by l e u p e p t i n , d id not requi re d i t h i o t h r e i t o l and was r e a d i l y reversed by subsequent so lub i l i za t ion of the C a 2 + - p r e t r e a t e d C a 2 + - A T P a s e . A f t e r the experimental work of t h i s t h e s i s was completed, using i n s i d e - o u t v e s i c l e s (I0V) made from human erythrocyte membranes, Papp et al, . (1989) showed that c a l p a i n treatment ac t iva ted the C a 2 + - t r a n s l o c a t i n g funct ion and rendered i t calmodul in- independent . Th is report thus confirmed the r e s u l t with the 1 iposome-reconst i tuted C a 2 + - A T P a s e presented in t h i s t h e s i s ( F i g . 30 to 36; see a lso Wang et a l . , 1989a; 1989d). These authors a lso found that a 125 kDa fragment ( l i k e l y to be 201 equiva lent to the 124 kDa fragment in t h i s work) of the enzyme was s t i l l responsive to a c i d i c phosphol ip id a c t i v a t i o n , although i t was i n s e n s i t i v e to calmodulin a c t i v a t i o n and f a i l e d to bind a monoclonal antibody apparent ly d i rec ted to the ca lmodul in -b inding domain (Papp et a l . , 1989). T h i s , aga in , i s cons is tent with the proposed model of fragmentation ( F i g . 44) , which suggests that the 124 kDa fragment w i l l have most of i t s ca lmodul in -b ind ing domain truncated but would leave the a c i d i c p h o s p h o l i p i d - i n t e r a c t i n g domain in tac t ( F i g . 43 and see DISCUSSION e a r l i e r ) . Papp et a l . (1989) a lso observed a ca lpa in -produced 81 kDa EP-forming fragment of the C a 2 + - A T P a s e , which they in terpre ted as o r i g i n a t i n g from a 90 kDa fragment that was a l ready present in the untreated membranes ( F i g . 4A of Papp e t _ a l _ . , 1989). However, in t h i s t h e s i s , s t a r t i n g with a s i n g l e 136 kDa EP-forming C a 2 + - A T P a s e prote in (both the membrane-bound form and the s o l u b l i z e d and p u r i f i e d form), s i g n i f i c a n t formation of the 80 kDa fragment was s t i l l observed a f t e r c a l p a i n treatment ( F i g . 22, 25 and 27). There fore , t h e i r specula t ions are not supported by t h i s t h e s i s . Most r e c e n t l y , James et a l . (1989) reported that in the absence of ca lmodul in , the membrane-bound and the p u r i f i e d C a 2 + - A T P a s e (138 kDa) were converted by c a l p a i n to two fragments of about 124 kDa. They a lso found t h a t , in the absence of ca lmodul in , c a l p a i n f i r s t c leaved the C a 2 + - A T P a s e in the middle of the ca lmodul in -b inding domain and thereby produced a calmodul i n - b i n d i n g 124 kDa fragment and a 14 kDa fragment. A second cleavage c lose to the N-terminal of the ca lmodul in -b ind ing domain produced a non-calmodul in-b inding 124 kDa fragment. Using a syn the t ic peptide 202 corresponding to the ca lmodul in-b inding domain, they a lso i d e n t i f i e d that the major cleavage s i t e s ( ind icated by arrows) were d i f f e r e n t in the absence and in the presence of calmodulin (James et a l . , 1989): ± CaM - CaM + CaM ' L R R G Q I L W F R G L N R I Q T Q I K V V N A F S S S These three cleavages at the ca lmodul in -b inding domain observed by them are l i k e l y the same cleavages which produce the 127 kDa, 125 kDa and 124 kDa fragments, as observed in t h i s t h e s i s : 124 kDa 125 kDa 127 kDa si/ \iv v]/ L R R G Q I L H F R G L N R I Q T - Q I K V V H A F S S S — There fore , i t appears that the work of James et a l . (1989) i s genera l ly in agreement with the scheme of fragmentation of the C a 2 + - A T P a s e by c a l p a i n I proposed in t h i s t h e s i s ( F i g . 44 and 45). V incenzi and Hinds (1988) have a lso independently proposed that s e l e c t i v e p r o t e o l y s i s of the C a 2 + - A T P a s e by c a l p a i n could cause the l o s s of Ca^- -ATPase a c t i v i t y associated with erythrocyte ag ing. I I . S i g n i f i c a n c e of t h i s work 1. P h y s i o l o g i c a l and pa tho-phys io log ica l s i g n i f i c a n c e 203 Since both the C a - A T P a s e and ca lpa in are Ca -dependent enzymes present in e ry th rocy tes , the calpain-mediated a c t i v a t i o n of the C a 2 + -ATPase and the loss of i t s calmodulin s e n s i t i v i t y may indeed occur j_n v i v o . It was demonstrated here that p u r i f i e d c a l p a i n I can e f f e c t i v e l y a c t i v a t e the C a 2 + t ransport funct ion and ATP h y d r o l y t i c a c t i v i t y of the Ca*- -pumping ATPase of human erythrocytes and render these a c t i v i t i e s calmodul in- independent . Such i r r e v e r s i b l e a c t i v a t i o n was prevented by the presence o f ca lmodul in . If these events can be ext rapolated to the l i v i n g c e l l ( F i g . 46) , at a r e s t i n g state the c e l l pumps C a 2 + outward and hydrolyzes ATP at a slow r a t e , well below i t s maximum c a p a c i t y . In response to c e r t a i n s t i m u l i , the i n t r a c e l l u l a r calc ium concent ra t ion may be e l e v a t e d , and would normally t r i g g e r the ca lc ium/ca lmodul in complex to bind and a c t i v a t e the C a 2 + pump. However, i f the e levated ca lc ium leve l p e r s i s t s , perhaps in a s s o c i a t i o n with mechanical s t r e s s on the red c e l l s (Larsen et a l . , 1981), aging of the red c e l l s , or other pa thophys io log ica l c o n d i t i o n s , c a l p a i n may be ac t iva ted and subsequently 9+ a c t i v a t e the C a t T pump i r r e v e r s i b l y , p o s s i b l y as a defense mechanism against the u n c o n t r o l l e d , e levated i n t r a c e l l u l a r ca lc ium concentra t ion which would otherwise be l e tha l to the c e l l s . I n t r a c e l l u l a r calmodul in might a lso prevent the i r r e v e r s i b l e act ion of c a l p a i n i f s u f f i c i e n t amounts of i t are a c c e s s i b l e to the C a 2 + pump at these e levated C a 2 + c o n c e n t r a t i o n s . Moreover, the act ion of ca lpa in on the C a 2 + pump could exp la in the absence or decrease of calmodulin s t imula t ion observed in the plasma 9+ membrane Ca^ -ATPase of some c e l l s , such as dog ery throcytes (Schmidt et 204 F i g . 46 Proposed model fo r the dual contro l of the plasma membrane Ca - pump by calmodulin and ca lpa in in a l i v i n g c e l l . Calmodulin i s represented by the f i l l e d c i r c l e . For de ta i l ed exp lanat ion , see t e x t . 205 a l . , 1985), or erythrocytes from hypertensive pa t ien ts or animals (Olorunsogo et a l . , 1985; Vezzol i et a l . , 1985; Orlov et a l . , 1983). I n t e r e s t i n g l y , both the erythrocytes and kidneys of Mi lan hypertensive ra ts had decreased l e v e l s of c a l p a s t a t i n (an endogenous c a l p a i n i n h i b i t o r ) whi le the c a l p a i n l eve l was the same as normal Mi lan (cont ro l ) ra ts (Pontremoli et a l . , 1986b; Pontremoli et a l . , 1986c; Pontremoli et a l . , 1987b). Th is imbalance of c a l p a i n - c a l p a s t a t i n might r e s u l t in an overexpressed c a l p a i n a c t i v i t y , which might produce s i g n i f i c a n t p r o t e o l y s i s of the C a 2 + - A T P a s e in these c e l l s . In Duchenne muscular dystrophy (DMD) p a t i e n t s , ca lpa in a c t i v i t y was higher in muscle as well as in p l a t e l e t s (Rabbani et al_., 1984). In f a c t , c a l p a i n has long been impl ica ted in DMD pathogenesis (see Sugita et a l . , 1980; Imahori, 1985). A recent p re l iminary report demonstrated that C a 2 + - A T P a s e a c t i v i t y was s i g n i f i c a n t l y higher in erythrocytes from DMD pat ien ts than in normal ery throcytes (Moses et a l . , 1987). Such e levated C a 2 + - A T P a s e a c t i v i t y could be a r e s u l t of ca lpa in a c t i v a t i o n . In f a c t , the C a 2 + - A T P a s e i s not unique to e r y t h r o c y t e s ; a s i m i l a r plasma membrane C a 2 + - A T P a s e has been demonstrated in hear t , b ra in and smooth muscle among others (see Penniston, 1983). There fo re , the c a l p a i n -mediated p r o t e o l y i s of the C a 2 + pump could conce ivab ly occur during c e r t a i n s ta te (s ) invo lv ing these t i s s u e s or c e l l s as w e l l . 2. Using c a l p a i n as a tool Due to the c h a r a c t e r i s t i c fragmentation pat tern of the C a 2 + - A T P a s e 207 produced by c a l p a i n , in p a r t i c u l a r , the d i f f e r e n t i a l fragmentation in the absence versus the presence of ca lmodul in , ca lpa in has proved to be an important t o o l , along with other proteases (eg. t r y p s i n and chymotyrpsin) , in understanding the overa l l o rgan iza t ion of the C a 2 + -ATPase (see F i g . 44 and 45). It a lso helps s e t t l e some ambigui t ies or cont rovers ies about the s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s o f the enzyme in t r y p s i n i z a t i o n s tudies (eg. the 85 kDa fragment) (see above). 3. Using the p r o t e o l y s i s pat tern of the C a 2 + - A T P a s e by c a l p a i n as a  means of i d e n t i f i c a t i o n of the enzyme The unique, calmodul in- induced d i f f e r e n t i a l fragmentation pat tern of p r o t e o l y s i s of the plasma membrane C a 2 + - A T P a s e produced by c a l p a i n ( F i g . 23 and 25) and the f a c t that the SR C a z + - A T P a s e i s not hydrolyzed by c a l p a i n ( F i g . 39) leads to the th ink ing that c a l p a i n treatment might be useful in d i s t i n g u i s h i n g these two major C a 2 + - A T P a s e s . For ins tance , in the case of a newly d iscovered enzyme which resembles one or both of these two C a 2 + - A T P a s e s , a quick tes t with c a l p a i n - p r o t e o l y s i s (which can be v i s u a l i z e d with 3 2 P - E P formation) would help c l a r i f y the s i t u a t i o n . For example, t h i s method might help s e t t l e the i d e n t i t y of the c o n t r o v e r s i a l , calmodulin- independent plasma membrane C a 2 + pump in l i v e r which a lso has a reported molecular weight of only 110 kDa (Lotersz ta jn et a l . , 1984). III. Calmodul in-b inding prote ins as c a l p a i n substrates 208 Kosaki et a l . (1983) reported that in p l a t e l e t s , several ca lmodul in-binding pro te ins (100 kDa, 90 kDa, 60 kDa and 40 kDa) were degraded by endogenous c a l p a i n I. Wallace et a l . (1987) confirmed t h i s study by showing that a s i m i l a r set of ca lmodul in-b inding prote ins in p l a t e l e t s were degraded by c a l p a i n upon p l a t e l e t a c t i v a t i o n . While studying the ca lpa in-mediated p r o t e o l y s i s of the ca lmodul in -ac t iva ted C a 2 + - A T P a s e from erythrocyte membrane, It was observed that another ca lmodul in -b ind ing pro te in adducin (103 + 97 kDa), which co -e lu ted as a minor component in the preparat ions of the C a 2 + - A T P a s e from a c a l m o d u l i n - a f f i n i t y column, was a lso fragmented by ca lpa in treatment ( F i g . 38A, lanes 2, 3 and 4) . Subsequently, the ca lmodul in -b ind ing calcium re lease channel of junc t iona l SR was a lso found to be proteolyzed by ca lpa in ( F i g . 39) , as reported by S e i l e r et aj_. (1984). Therefore , a systematic search f o r other ca lmodul in -b ind ing prote ins which might a lso be c a l p a i n - s e n s i t i v e was made (Table 6 ) . Of the 30 ca lmodul in-b inding prote ins l i s t e d in Table 6, s ix teen have a l ready been reported to be substrates f o r c a l p a i n i n v i t r o (Wang et a l . (1989b)). So f a r , no calmodul i n - b i n d i n g pro te ins have been reported to be r e s i s t a n t to c a l p a i n , although i t was found that one form of calmodulin-dependent c y c l i c nuc leot ide phospho-diesterase was c a l p a i n -r e s i s t a n t (Wang, V i l l a l o b o , and Roufoga l is , unpublished r e s u l t s ) . Never the less , t h i s i s a rather s t r i k i n g f i n d i n g given the f a c t that c a l p a i n has a narrow range of substrate s p e c i f i c i t y . Many prote ins are c a l p a i n - r e s i s t a n t , inc lud ing bovine serum albumin, ovalbumin, sarcoplasmic 9+ re t icu lum C a - A T P a s e , c a l s e q u e s t r i n , ca lmodul in , a c t i n , u b i q u i t i n , 209 Table 6 Matching of CaM-bindinq prote ins and c a l p a i n subst ra tes Reference* (as CaM-binding Protein)  Protein Subunit M.W. (kDa) Calpain-sensitivity (Ref) (a) Enzymes: Adenylate cyclase 150 (1,2) N-D. b Phosphorylase kinase 145(a),128(8), (3) + (34,35) 45 (7), 17(5) + (36,37) Myosin light chain kinase Ca2 +-ATPase (plasma membrane) 150 (4) 136 (5,6) + (38-40) Phosphofructose kinase. 80 (7) N.D. Phosphodiesterase (cyclic 61,63 (8-10) + (37) nucleotide) Calcineurin 60(a),19(B) (11) + (41,42) Inositol 1,4,5 trisphosphate 53 (12) N.D. ' kinase CaM-dependent protein kinase I 37-39 (13) N.D. CaM-dependent protein kinase I I 50(a),58(B),60(B') (14) N.D. CaM-dependent protein kinase I I I 140 (15) N.D. Dynein ATPase 400 (16) N.D. (b) Cvtoskeleton and structural proteins: Nebulin 300 (17) MAP-2 270 (18) Fodrin (brain spectrin) 240(a),235(B) (19) Spectrin 240(a),220(B) (20) Caldesmon 150,75 (21) Microvillus 110 kDa protein 110 (22) Tubulin 50,55 (23) Tau factor 55-62 (24) Adducin 97(a), 100(B) (25) (c) Others: 350 50 Calcium release channel Regulatory subunit (type I I cAMP-dependent protein kinase) Calspermin 32 Gap junction protein (lens) 26 Phospholamban 5 Neuromodulin (P-57, GAP-43) 24 Myelin basic protein 22 Histone 2B 19 (26,27) (28) (29) (30) (31) (32) (33) (33) (43) (44,45) (46,47) (47,48) (30) N.D. + (45) N.D. + (49) + (26,27) N.D. N.D. N.D. N.D. + (50) + (51) + (52) 2 1 0 Table 6 (Cont.) a (1) Yeager et a l . , 1985; (2) Shattuck et a l . , 1987; (3) Chan & Graves, 1984; (4) Klee, 1977; (5) Lynch & Cheung, 1979; (6) Niggli et a l . , 1979; (7) Mayr & Heilmeyer, 1983; (8) Cheung, 1970; (9) Kakiuchi & Yamazaki, 1970; (10) Sharma et a l . , 1980; (11) Tallant & Cheung, 1986; (12) Johanson et a l . , 1988; (13) Nairn et a l . , 1985a; (14) Kennedy et a l . , 1987; (15) Nairn et a l . , 1985b; (16) Blum et a l . , 1980; (17) Patel et a l . , 1988; (18) Lee & Wolff, 1984; (19) Carl in et a l . , 1983; (20) Sobue et a l . , 1981a; (21) Sobue et a l . , 1981b; (22) Glenney & Weber, 1980; (23) Kumagai et a l . , 1982; (24) Sobue et a l . , 1981c; (25) Gardner & Bennett, 1986; (26) Seller et a l . , 1984; (27) Wang, Gilchrist, Roufoglis, Katz and Belcastro, unpublished results; (28) Hathaway et a l . , 1981; (29) Ono et a l . , 1984; (30) Welsh et a l . , 1982; (31) Molla et a l . , 1983; (32) Andreasen et a l . , 1983; (33) Grand and Perry, 1980; (34) Meyer et a l . , 1964; (35) Huston & Krebs, 1968; (36) Kosaki et a l . , 1983; (37) Ito et a l . , 1987; (38) Au, 1987; (39) Wang et a l . , 1988a; (40) Wang et a l . , 1988b; (41) Tallant et a l . , 1988; (42) Wang et a l . , 1989c; (43) Goll et a l . , 1983; (44) Kubota et a l . , 1986; (45) Billger et a l . , 1988; (46) Siman et a l . , 1984; (47) Seubert et a l . , 1987; (48) Pant et a l . , 1983; (49) See Fig. 38A; (50) See Fig. 38B; (51) Banik et a l . , 1985; (51) (52) Sakai et a l . , 1987. b not determined. 211 phosphorylase and cytochrome c (Zimmerman and Sch laepfe r , 1984; Pontremoli et a l . , 1985a; F i g . 25 and 39, t h i s t h e s i s ) . Of the 16 i d e n t i f i e d c a l p a i n - s e n s i t i v e ca lmodul in-b inding p r o t e i n s , 5 are enzymes, 7 are c y t o s k e l e t a l / s t r u c t u r a l prote ins and the other 4 represent a miscel laneous group o f pro te ins (Table 6 ) . 1. Calmodulin-dependent enzymes Among the f i v e c a l p a i n - s e n s i t i v e calmodulin-dependent enzymes (phosphorylase k inase , myosin l i g h t chain k inase , plasma membrane C a 2 + -ATPase, phosphodiesterase and c a l c i n e u r i n ) , s i m i l a r i t i e s in t h e i r p r o t e o l y s i s by ca lpa in are found (Table 7): ( i ) the fragmentation patterns of myosin l i g h t chain kinase (Kosaki et a l . , 1983; Ito et a l . , 1987), C a 2 + - A T P a s e ( F i g . 23 and 25) and c a l c i n e u r i n (Ta l lan t et a l . . 1988; Wang et a j . . , 1989c) are d i f f e r e n t in the absence and the presence of ca lmodul in ; ( i i ) p r o t e o l y s i s of four out of the f i v e enzymes in the absence o f calmodulin ac t iva tes t h e i r respec t ive enzymatic a c t i v i t i e s (Table 7 ) . In f a c t , e a r l i e r s tudies revealed that phosphodiesterase (Cheung, 1971; K inca id et a l . , 1985), phosphorylase kinase (Depaoli-Roach et a l . , 1979), myosin l i g h t chain kinase (Walsh et a l . , 1982; Foyt et a l . , 1985), c a l c i n e u r i n (Manalan and K lee , 1983; T a l l a n t and Cheung, 1984), plasma membrane C a z + - A T P a s e (Zur in i et a l . , 1984) and calmodulin-dependent p ro te in kinase II (Levine and Sahyoun, 1987) are a l l ac t iva ted and become c a l m o d u l i n - i n s e n s i t i v e a f te r l im i ted p r o t e o l y s i s by e x t r a c e l l u l a r p ro teases , such as t r y p s i n or chymotrypsin. It seems l i k e l y that these 212 I. Table 7 Effect of calpain on calmodulin-dependent enzymes Enzymes E f f e c t of ca lpa in on a c t i v i t y " E f f e c t of CaM on ca lpa in a c t i o n " Phosphorylase kinase Myosin l i g h t chain kinase Ca 2 + -ATPase (plasma membrane) Phosphodiesterase Ca lc ineur in ac t i va ted i n a c t i v a t e d & independent of CaM ac t iva ted & independent of CaM ac t iva ted & independent of CaM ac t iva ted & independent of caM N.D. prevents l o s s of CaM-s e n s i t i v i t y prevents a c t i v a t i o n and l o s s of C a M - s e n s i t i v i t y No e f f e c t slows down the l o s s of C a M - s e n s i t i v i t y not determined see references in Table 6 e x t r a c e l l u l a r proteases mimic Ca -dependent p r o t e o l y s i s by endogenous c a l p a i n . The mechanism of a c t i v a t i o n of these calmodulin-dependent enzymes by l i m i t e d p r o t e o l y s i s i s envisaged as f o l l o w s : ca lmodul in-dependent enzymes contain a ca lmodul in-b inding domain with an adjacent or over lapping i n h i b i t o r y domain. Upon binding of ca lmodul in , the ca lmodul in -b ind ing and i n h i b i t o r y domains undergo conformational changes, which re lease the i n h i b i t i o n of enzymatic a c t i v i t y . L imi ted p r o t e o l y s i s e i t h e r removes both the ca lmodul in-b inding and i n h i b i t o r y domains or only the former. In e i t h e r case , the i n h i b i t i o n i s r e l i e v e d and the enzyme expresses i t s f u l l a c t i v i t y , which i s no longer c a l m o d u l i n - s e n s i t i v e . In the absence of ca lmodul in , ca lpa in p r o t e o l y s i s of plasma membrane C a 2 + -ATPase and c a l c i n e u r i n produces ac t ive c a l m o d u l i n - i n s e n s i t i v e fragments. On the other hand, in the presence of ca lmodul in , p r o t e o l y s i s produces ac t ive enzyme fragments which re ta in t h e i r ca lmodul in -b ind ing capac i ty (Wang et a l . . 1988a; 1988b; T a l l a n t et a l . , 1988). There fo re , i t seems that calmodulin can funct ion to modulate calpain-mediated p r o t e o l y s i s of ca lmodul in -b inding p r o t e i n s . The e f f e c t of calmodulin on ca lpa in p r o t e o l y s i s of ca lmodul in -b ind ing prote ins i s in some ways analogous to the e f f e c t of u b i q u i t i n on the ATP-dependent protease system (Rechsteiner , 1987a). Thus u b i q u i t i n , when conjugated to target p r o t e i n s , s igna ls the ATP-dependent protease to hydrolyze the conjugated p r o t e i n , whereas ca lmodul in , when bound to the ca lmodul in -b ind ing p r o t e i n , modif ies the pattern of p r o t e o l y s i s . The d i f f e r e n c e i s that whereas ATP-dependent p r o t e o l y s i s occurs only when substrates are conjugated to u b i q u i t i n , ca lpa in p r o t e o l y s i s occurs both in 2 1 4 the absence and in the presence of ca lmodul in . I n t r i g u i n g l y , both calmodul in and u b i q u i t i n are small prote ins (148-residues f o r calmodulin and 76 res idues fo r ub iqu i t in ) present in a l l eukaryot ic c e l l s and conserved to an exceptional degree (Manalan and K l e e , 1984; F i n l e y and Varshavsky, 1985). Both prote ins are m u l t i - f u n c t i o n a l and are involved in var ious biochemical processes in c e l l s . Furthermore, u b i q u i t i n i t s e l f i s not proteolyzed by ATP-dependent protease and, l i k e w i s e , calmodul in i s not proteolyzed by c a l p a i n . 2. C v t o s k e l e t a l / s t r u c t u r a l and other ca lmodul in -b ind ing prote ins When the calpain-mediated p r o t e o l y s i s of ca lmodul in -b ind ing prote ins that are not enzymes was fur ther examined, i t was observed that many of these are cy toske le ta l p r o t e i n s , inc lud ing s p e c t r i n , which forms the cy toske le ton backbone and components of microtubules ( tubu l in and MAP-2). The e f f e c t o f calmodulin on the p r o t e o l y s i s of these ca lmodul in -b ind ing pro te ins i s l e s s c l e a r . No apparent d i f f e r e n c e in fragmentation pattern iff the absence and the presence of calmodulin was found f o r caldesmon (Kosaki et al_. , 1983), e ry thro id s p e c t r i n (Seubert et al_. , 1987) and adducin (Wang, V i l l a l o b o and Roufoga l is , unpublished r e s u l t s ) . However, calmodul in increases the rate of degradation of bra in s p e c t r i n ( fodr in ) by c a l p a i n (Seubert et a l . , 1987). The funct ion of the p r o t e o l y s i s of cy toske le ta l or membrane prote ins might be to induce cy toske le ta l or c e l l membrane remodel ing. For i n s t a n c e , c e l l membrane fus ion requi res removal of some membrane prote ins 215 to provide a l i p i d enriched environment at the s i t e of fus ion (Kosower et a l . , 1983). Lynch and Baudry (1984) hypothesized that as a r e s u l t of ca lc ium i n f l u x upon neuronal s t i m u l a t i o n , fodr in i s degraded l o c a l l y by c a l p a i n and induces remodeling of the postsynapt ic membrane. Consequently, the i n i t i a l l y l a tent glutamate receptors in the postsynapt ic membrane are exposed, which might cont r ibute to the long-term po ten t i a t ion observed in that study. Besides these passive ro les of the c y t o s k e l e t o n , microtubule prote ins may a lso be involved in the r e p e r t o i r e of movements of the eukaryot ic c e l l , i nc lud ing movements of i n d i v i d u a l c e l l s or of organe l les wi th in the cytosol ( i . e . m i t o s i s , axonal t ranspor t or movements of pigment or secretory granules) (Bryan, 1974). There fore , these processes may be viewed as being p o t e n t i a l l y d u a l l y c o n t r o l l e d by calmodulin and c a l p a i n . 3. Substrate s p e c i f i c i t y of c a l p a i n Using n a t u r a l l y occurr ing and a r t i f i c i a l peptides as substrates f o r c a l p a i n , Sasaki et a l . (1984) formulated a general preference r u l e fo r c a l p a i n p r o t e o l y s i s at the cleavage s i t e : a Lys , T y r , Arg or Met res idue in the Pj p o s i t i o n preceded by a hydrophobic amino ac id res idue (Leu or Val) in the P2 p o s i t i o n would favor cleavage at the carboxyl s ide of the res idue in the Pj p o s i t i o n (nomenclature of Schechter and Berger , 1967). Very s i m i l a r preference was observed using neuropeptides as substrates (Hirao and Takahashi , 1984). However, t h i s pattern does not exp la in a l l the cases (Sasaki et a l . , 1984). Furthermore, small peptides are known to be ra ther poor substrates fo r c a l p a i n as compared to pro te in substrates 216 (Murachi, 1983b), and there fo re , the preference ru le der ived from peptide substrates may not apply to prote in subst ra tes . Recent ly , Sakai et a l . (1987) studied the s p e c i f i c i t y of ca lpa in cleavage s i t e s in h istones and found that the P2-P1 preference ru le was not n e c e s s a r i l y fo l lowed. Instead, they found that suscept ib le bonds were never located in the midst of e i t h e r hydrophobic or hydroph i l i c amino acid c l u s t e r s , but in the v i c i n i t y of the boundary between hydroph i l i c and hydrophobic c l u s t e r s . Th is cleavage s i t e s p e c i f i c i t y could account fo r the fac t that p r o t e o l y s i s induced by c a l p a i n usua l l y produces l a r g e r l i m i t fragments which are not fu r ther s u s c e p t i b l e to ca lpa in cleavage. For ins tance , Sakai et al_. (1987) observed that a l l the fragments re leased from histones underwent no fur ther degradat ion , although many of them s t i l l contained bonds which would be s u s c e p t i b l e to ca lpa in i f they were present in i n t a c t h i s t o n e s . Th is f i n d i n g suggests that ca lpa in recognizes c e r t a i n higher order s t ruc ture in i t s substrates before cleavage i s induced. Th is would expla in why small pept ides are in general poor substrates f o r c a l p a i n , s ince they are not large enough to contain the required recogn i t ion s t t e ( s ) . When t h i s " recogni t ion s i t e " concept i s appl ied to the ca lmodul in -binding p r o t e i n s , which are often substrates f o r c a l p a i n , i t fo l lows that there may be c e r t a i n common region(s) or features on these p ro te ins that are recognized by c a l p a i n . The most obvious region would be the ca lmodul in -b ind ing domain of these p r o t e i n s . It has been demonstrated that the ca lmodul in -b ind ing domains of many d i f f e r e n t ca lmodul in -b ind ing prote ins share a high degree of homology (Blumenthal et a l . , 1985; Lukas 217 et a l . , 1986; Wakim et a l . . 1987; Bennett and Kennedy, 1987; L in et a l . , 1987; James et a l . . 1988, Shul l and Greeb, 1988). A t y p i c a l ca lmodul in-binding domain i s thought to be a short amphipathic a l p h a - h e l i x peptide segment with a high percentage of bas ic amino ac id res idues (a rg in ine , h i s t i d i n e and l y s i n e ) (Blumenthal and Krebs, 1988). It i s be l ieved that the r e s u l t i n g net p o s i t i v e charge favors the i n t e r a c t i o n with the s t rongly a c i d i c ca lmodul in . As d iscussed e a r l i e r , the la rge subunit of c a l p a i n (80 kDa) has i t s c a t a l y t i c domain near the N-terminal region and has a p, calmodul in-1 ike E-F hand C a c -b ind ing domain at the C-terminal region (Ohno et a l . , 1984). It i s conceivable that the ca lmodul in -1 ike domain of c a l p a i n can bind to the ca lmodul in -b ind ing domain of the substrate p r o t e i n s . Once binding i s achieved, the c a l p a i n c a t a l y t i c domain proceeds to c leave i t s bound subst ra te . However, t h i s seemingly a t t r a c t i v e mechanism cannot account f o r the f a c t that some ca lmodul in -b inding p r o t e i n s , a f t e r l o s i n g t h e i r ca lmodul in -b ind ing domain, are s t i l l s u s c e p t i b l e to fu r ther p r o t e o l y s i s (Kosaki et a l . , 1983; Ito et a l . , 1987; Wang et al_., 1988b). This prompted us to look f o r an a l t e r n a t i v e mechanism. 4. PEST sequences in ca lmodul in -b inding pro te ins Recent ly , Rogers et a l . (1986) found that each of ten s h o r t - l i v e d pro te ins examined ( h a l f - l i v e s l e s s than 2 h) conta in one or more regions enr iched in p r o l i n e (P) , glutamic ac id (E ) , aspartate (D), ser ine (S) and threonine (T) (PEST r e g i o n s ) . S i m i l a r inspec t ion of 35 more stable pro te ins ( h a l f - l i v e s between 20 and 220 h) revealed that only 15 of these 218 contained PEST reg ions . The strength of a PEST sequence can be evaluated by i t s PEST score (which i s a r e f l e c t i o n of the to ta l mole percent of P ,E ,D ,S and T and the average h y d r o p h i l i c i t y of the s t r e t c h , see Methods fo r d e f i n i t i o n ) (Rogers et a l . , 1986). While PEST scores t h e o r e t i c a l l y can range from -45 to +50, two types of PEST regions e x i s t : those with a PEST score > 0, i n d i c a t i n g a strong PEST r e g i o n , and those with a PEST score < 0 but > - 5 , i n d i c a t i n g a weak PEST reg ion . S t r i k i n g l y , in the o r i g i n a l study 9 of the 10 s h o r t - l i v e d prote ins have at l e a s t 1 strong PEST r e g i o n , whereas only 3 of the 35 s tab le prote ins have a strong PEST r e g i o n . These authors concluded that these PEST regions confer the property of rap id degradation to the prote ins conta in ing them. They a lso hypothesized that s ince the E and D residues in the PEST sequence are both negat ive ly charged, and S and T are p o t e n t i a l l y phosphory latable , phosphorylat ion of such PEST sequences would produce very negat ive ly charged regions which may bind ca lc ium. In t u r n , t h i s l o c a l calc ium concentrat ion may a c t i v a t e ca lpa ins (Rogers et a l . , 1986). Therefore , in r e l a t i o n to the f i n d i n g that many ca lmodul in -b inding prote ins are ca lpa in subst ra tes , i t was postu la ted that many ca lmodul in -b inding prote ins may share sequence homology in add i t ion to the ca lmodul in -b ind ing domain, namely, PEST reg ions , and that i t may be these PEST regions that are commonly recognized by c a l p a i n . In order to t e s t t h i s hypothesis , potent ia l PEST sequences in ca lmodul in -b inding prote ins that have been sequenced were searched f o r , using a PEST sequence-searching computer program 'PEST-FIND' (Rogers et a l . . 1986). It was found that a l l the 2 1 9 Table 8 PEST sequences of ca lmodul in -b inding prote ins PEST Seauence d > b PEST Score Ref*" Protein Residues Tau Factor (mouse brain) Neuromodulin (bovine brain) CaM-PK II (a) (rat brain) (B) Tubulin (a) (rat brain) Tubulin (fl) (human brain) Ca 2 +-ATPase (Human, plasma membrane) Myelin basic protein (bovine brain) 111- 121 KTPPGSGEPPK 10.6 (1) 121-140 RSGYSSPGSPGTPGSR 5.1 307-338 HLSNVSSTGSIDMVDSPQLATL -4.3 ADEVSASLAK 112- 135 KGEGAPDAATEQAAPQAPAPSEEK 8.1 (2) 167-209 KQADVPAAVTAAAATAPAAEDAAAMATAQ 3.5 PPTETAESSQAEEK 225- 245 KAGAYDFPSPEWDTVTPEAK 2.7 (3) 226- 246 KAGAYDFPSPEWDTVTPEAK 2.7 (4) 430-451 KDYEEVGVDSVEGEGEEEGEEYend 13.7 (5) 396-443 HWYTGEGMDEMEFTEAESNMNDLV 10.5 (6) SEYQQYQDATAEEEEDFGEEAEEEEAend 64-80 KTSPNEGLSGNPADLER 2.4 (7) 1078-1095 KEEIPEEELAEDVEEIDH 14.8 1158-1174 HIPLIDDTDAEDDAPTK 8.3 1175-1185 RNSSPPPSPNK 7.1 1202-1214 KSATSSSPGSPLH 1.6 (NO PEST) - (8) Hi stone 2B (calf thymus) (NO PEST) (9) RII of cAMP-PK (bovine heart) a - Fodrin 45-89 93-103 RASTPPAAPPSGSQDFDPGAGLVADAVAD SESEDEEDLVPIPGR RVSVCAETYNPDEEEEDTDPR (+101)-(+lll) e HPESAEDLQEK (human brain a-spectrin) 8.9 15.2 6.8 (10) (11) 220 T a b l e 8 ( C o n t . ) Protein (Ref) Residues PEST Sequence d > ° PEST score Ref. d MLCK-G (chicken gizzard) 80-94 94-127 KPDPPAGTPCASDIR RSSSLLSWYGSSYDGGSAVQSYTVEIWNS VDNK 1.4 -3.8 (12) 371-445 KVLFGTPEFVAPEVINYEPIGYETDMWSI GVICYILVSGLSPFMGDNDNETLANVTSA TWDFDDEAFDEISDDAK -2.3 602-627 HFQIDYDEEGNCSLTISEVCGDDDAK 0.1 MLCK-S (rabbit skeletal muscle) 50-59 175-201 KQDPDPSTPK . KPLSEASELIFEGVPATPGPTEPGPAK 13.8 4.5 (13) 473-525 KTDMWSLGVITYMLLSGLSPFLGDDDTETL NNVLSGNWYFDEETFEAVSDEAK -1.9 PhosDhorylase kinase subunit (rabbit skeletal muscle) 239-261 RMIMSGNYQFGSPEWDDYSDTVK -5.1 (14) PhosDholamban (rabbit skeletal muscle) (NO PEST) - (15) Gap Junction Protein (bovine lens) 241-260 RPSESNGQPEVTGEPVELK 7.4 (16) a Part ia l sequences are presented with amino acid letter code: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine. bPEST sequences are identified using the PEST-FIND computer program developed by Rogers et a l . (1986). PEST sequence was defined as a stretch of amino acid residues beginning and ending with positively charged residues (H,K or R) with a number of internal residues of at least 8. CPEST score is calculated as described by Rogers et a l . , (1986); in brief, PEST score = 0.55 (MPpEST^ - 0 - 5 (H 0 ) , where MP p p S T = mole percent of P,E,D,S and T after subtracting one mole equivalent of P,E and S, and H Q = average hydrophobicity of the stretch (Rogers et a l . , 1986). To qualify as a PEST sequence, the PEST score value has to be larger than -5.0. When the stretch is missing either P, E/D or S/T, then i ts PEST score has to be larger than 0 to qualify as a PEST sequence. ^References of amino acid sequences are as follows: (1) Lee et a l . , 1988; (2) Wakim et a l . , 1987; (3) Bennett & Kennedy, 1987; (4) Bulleit et a l . , 1988; (5) Lemischka et a l . , 221 Table 8 (Cont . ) 1981; (6) Hall et a l . , 1983; (7) Verma et a l . , 1988; (8) Eylor et a l . , 1971; (9) Iwai et a l . , 1972; (10) Takio et a l . , 1984; (11) McMahon & Moon, 1987, (12) Guerrieno et a l . , 1986; (13) Kennelly et a l . , 1987; (14) Reimann et a l . , 1984; (15) F u j i i , et a l . , 1987; (16) Gorin et a l . , 1984. e Residue numbers arbi trari ly assigned, for detai l , see legend of Fig.4-7. 222 ca lmodul in -b ind ing prote ins but three sequenced to date conta in one or more PEST sequences (Table 8 ) . Furthermore, the PEST scores of these regions are genera l l y high (> ze ro ) . In f a c t , a l l of these calmodul in-binding p ro te ins but one (phosphorylase k inase , 7 - s u b u n i t ) have at l eas t one strong PEST region (with PEST score > 0) (Table 8 ) . Of the PEST-conta in ing ca lmodul in -b inding prote ins in Table 8, t u b u l i n (a , / 3 ) , e ry throcyte C a c -ATPase, a - s p e c t r i n and myosin l i g h t chain kinase (chicken g izzard ) have been demonstrated to be fragmented by c a l p a i n (Table 6 ) . The three ca lmodul in -b inding prote ins that do not have PEST sequences are myelin bas ic p r o t e i n , histone I IB and phosphol amban (Table 8 ) . I n t e r e s t i n g l y , none of the three prote ins appear to have " t y p i c a l " ca lmodul in -b ind ing domains (Ey lar et a l . , 1971; Iwai et a l . , 1970; F u j i i et a l . . 1987). Worth mentioning i s that the plasma membrane, ca lmodul in-st imulated Ca*- -ATPase shares a high degree- of s t r u c t u r a l homology with the sarcoplasmic re t icu lum (SR) C a 2 + - A T P a s e (Verma et a l . , 1988; MacLennan et a l . , 1985) which, however, lacks a t y p i c a l ca lmodul in -b ind ing domain. Whereas the former has f i v e strong PEST sequences (PEST scores 2 .4 , 14.8, 8 .3 , 7.1 and 1.6) ( four of them near the C-terminal and one near the N-t e r m i n a l ) , the l a t t e r has only three weak PEST regions (PEST scores : - 3 . 4 , -4 .0 and - 4 . 9 ) . In support of the observed c o r r e l a t i o n , the plasma membrane C a 2 + - A T P a s e i s r a p i d l y hydrolyzed at 4-5 s i t e s (Wang et a l . , 1988b), whi le the SR C a 2 + - A T P a s e is r e s i s t a n t to c a l p a i n ( S e i l e r et a l . 1984; F i g . 39, t h i s t h e s i s ) . Furthermore, the f i r s t few cleavage s i t e s are a lso loca ted at the C-terminal end of the plasma membrane C a 2 + - A T P a s e , corresponding to the region where many of the PEST sequences are found. 223 Thus, the strong PEST regions on the plasma membrane C a - A T P a s e may be the r e c o g n i t i o n s i t e s fo r c a l p a i n . The ca lmodul in -b ind ing prote in neuromodulin was the f i r s t tes t of the hypothes is . From the known sequence of neuromodulin (Wakim et a l . , 1987), two strong PEST reg ions , with PEST scores o f 8.1 and 3 .5 , r e s p e c t i v e l y , were i d e n t i f i e d (Table 3 ) . It was p r e d i c t e d , the re fo re , that neuromodulin would be a good substrate f o r c a l p a i n . Subsequently, neuromodulin was indeed found to be hydrolyzed by c a l p a i n in to several fragments ( F i g . 38B). Another tes t of the hypothesis was human a - f o d r i n (brain s p e c t r i n ) , whose molecular s t ruc ture has r e c e n t l y been e l u c i d a t e d . Based on the p a r t i a l amino ac id sequence, a - f o d r i n , l i k e the e ry th ro id a - s p e c t r i n , appears to have twenty-two 106-residue repeat s t ruc tures (McMahon and Moon, 1987). Human a - f o d r i n possesses a h igh ly basic ca lmodul in -b ind ing region at the junc t ion between the 11th and 12th repeats , which accounts fo r i t s high a f f i n i t y f o r calmodul in (Harr is and Morrow, 1988; Har r is et a l . , 1988) ( F i g . 47). Ca lpa in I and II appear to cTeave a - f o d r i n (240 kDa) s e l e c t i v e l y in the middle o f the molecule , to produce a 150 kDa ca lmodul in -b inding fragment (Harr is et a l . , 1988). Very r e c e n t l y , the cleavage s i t e was i d e n t i f i e d to be 10 res idues upstream from the ca lmodul in -b ind ing domain (see F i g . 47) (Harr is et a l . , 1988), which i s cons is ten t with the f ind ing that calmodulin s t imulates the degradation of bra in s p e c t r i n by ca lpa in (Seubert et a l . , 1987). A search fo r PEST sequences in a - f o d r i n based on the p a r t i a l amino ac id sequence covering the 7th to 15th repeats (McMahon and Moon, 1987), revealed that the only 224 F i g . 47 L o c a l i z a t i o n of PEST reg ions , the ca lmodul in -b ind ing domain and  the c a l p a i n cleavage s i t e wi thin human brain a - f o d r i n . Of the proposed 22 repeats , only the tenth , e leventh , twel f th and t h i r t e e n t h (represented by Roman numerals) are i l l u s t r a t e d (McMahon and Moon, 1987; Har r is et a l . , 1988). Since the complete amino ac id sequence of a - f o d r i n i s not a v a i l a b l e , the f i r s t residue of the XII repeat i s a r b i t r a r i l y assigned residue +1 and i s used as the reference r e s i d u e . Residues moving downstream (towards the C - te rmina l ) , and away from res idue 1, have increas ing p o s i t i v e residue numbers, whereas res idues moving upstream and away from residue 1 have increas ing negative res idue numbers ( s t a r t s from -1 and on) . The PEST region (residue number +101 to +111) i s i l l u s t r a t e d by the dotted area. The ca lmodul in-b inding domain ( res idue number -18 to +5) i s shown by the shaded area. A ca lpa in cleavage s i t e (between residue -28 and -27) i s i l l u s t r a t e d by the arrow and the dotted l i n e . 225 Figure 47 X X I X I I X I I I (-28)<->(-27) calpain cleavage -145 | +1 +108 i i • CaM-binding PEST (-18)+-+(+5) (+101)+-*-(+111) 226 PEST sequence (PEST score 6.8) was located in c lose proximity (128 res idues downstream) to the ca lpa in cleavage s i t e ( F i g . 47) . 5. Recognit ion of ca lmodul in-b inding by c a l p a i n A schematic drawing of how ca lmodul in -b ind ing pro te ins may be recognized by ca lpa in i s presented in F i g . 48. Since PEST regions are h igh ly h y d r o p h i l i c and therefore are very l i k e l y to form surface loops (Rechste iner , 1987b), they would be e a s i l y a c c e s s i b l e to ca lpa in molecules . In the model, the negative charges of E/D res idues ins ide the PEST regions of ca lmodul in-b inding prote ins l o c a l i z e calc ium ions at the negat ive ly -charged ca lc ium-binding domain of c a l p a i n (C-terminal end of the 80 kDa subun i t ) . Upon i n t e r a c t i o n with s u b s t r a t e - a s s o c i a t e d ! C a 2 + , the c a t a l y t i c domain of ca lpa in (near the N-terminal end of the 80 kDa subunit ) i s ac t iva ted and at tacks the adjacent cleavage s i t e ( s ) , which are of course d i s t i n c t from the PEST r e g i o n s . A l s o , because of the complex t e r t i a r y s t ructure of p r o t e i n s , the PEST regions and cleavage s i t e s need not be c lose by in the primary amino ac id sequence in order to be" in s p a t i a l prox imi ty . In some ca lmodul in -b ind ing pro te ins the binding o f calmodulin might induce conformational changes which protect c e r t a i n cleavage s i t e s from ca lpa in at tack ( F i g . 48B), as seen with the ery throcyte C a 2 + - A T P a s e ( F i g . 23, 25 and 45) . A l t e r n a t i v e l y , such conformational changes may expose new cleavage s i t e s that are otherwise i n a c c e s s i b l e to c a l p a i n . Consequently, fragmentation of ca lmodul in-binding prote ins by ca lpa in could be qui te d i f f e r e n t in the absence versus the presence of calmodulin ( F i g . 23 and 25, t h i s t h e s i s ; Ito et a l . . 1987; T a l l a n t et a l . , 1988). For some ca lmodul in -b ind ing prote ins (eg. a-227 F i g . 48 Model of calpain-mediated p r o t e o l y s i s of ca lmodul in -b ind ing  p r o t e i n . Schematic drawing i l l u s t r a t i n g that c a l p a i n recognizes ca lmodul in -b ind ing prote in PEST regions with i t s Ca2+-binding domain (A) and subsequently attacks cleavage s i t e s 1 and 2. (B) Upon calmodulin i n t e r a c t i o n with the ca lmodul in-b inding domain (shadowed r e g i o n ) , there is a conformational change or phsyica l occ lus ion which prevents the adjacent cleavage s i t e ( s i t e 2) from attack by c a l p a i n . Note that the s i z e of c a l p a i n , i t s substrate and calmodulin are not drawn to the same p r o p o r t i o n s . 228 Figure 48 229 f o d r i n ) , the binding of calmodulin can simply modify the rate of p r o t e o l y s i s of the molecule by ca lpa in (Seubert et a l . , 1987), probably by c o n t r o l l i n g the a c c e s s i b i l i t y of c e r t a i n cleavage s i t e s v i a conformational changes. It i s p o s s i b l e that the PEST regions in ca lmodul in -b ind ing prote ins funct ion s o l e l y in ca lpa in r e c o g n i t i o n . A l t e r n a t i v e l y , the PEST regions might have other funct ions as yet unknown. In t h i s case , i t w i l l be important to de f ine such f u n c t i o n s , fo r t h i s would most c e r t a i n l y help in the f u r t h e r understanding o f the funct ion or regu la t ion o f ca lmodul in -binding pro te ins in genera l . 6. PEST sequences in other ca lpa in substrates A f t e r the hypothesis that ca lmodul in-b inding prote ins are recognized by c a l p a i n through t h e i r PEST sequences was formulated, the quest ion whether other c a l p a i n substrates a lso contain PEST sequences was asked. To answer t h i s ques t ion , the known amino acid sequences o f 41 pro te in substra tes f o r c a l p a i n were analyzed. Of these 41 p r o t e i n s , 29 have one or more PEST sequences (Table 9 ) , while 12 do not (Table 10). Of the 29 PEST-conta in ing subs t ra tes , 26 contain one or more strong PEST sequences. The other three substrates which contain only a weak PEST sequence are v iment in , pyruvate kinase (L-type) and a - a c t i n i n . Among the PEST-conta in ing pro te ins of known amino acid sequence are f i v e ca lmodul in -binding enzymes (as descr ibed above), the large subunit of c a l p a i n I and c a l p a i n II, c a l p a i n i n h i b i t o r prote in ( c a l p a s t a t i n ) , the p ro te in kinase C fami ly and three receptor prote ins (EGF receptor , progesterone receptor 230 Table 9 PEST sequences of PEST-containinq calpain substrates Species used Species for Protein as calpain a.a.sequence Residues substrate (Ref. a) (Ref. a)  PEST sequence PEST SCORE Calpain I (large subunit) human human (27) (1R) & others 295-322 RNPWGEVEWTGAWSDSSSEWNN 4.2 VDPYER 517-548 KSAGTVELDDQIQANLPDEQVL 3.3 SEEEIDENFK Calpain II (large subunit) chicken chicken (28) 288-315 (1R) & others 508-539 RNPWGQVEWTGAWSDGSSEWDN 4.1 IDPSDR KQSDTAELDEEISADLADEEEI 14.8 TEDDIEDGFK Calpastatin rabbit rabbit (29) (1R) & others 150-197 KSGMDAALDDUDTLGEPSETQ 9.5 EDSTAYTGPEISDPMSSTYIEE LGK 212-249 KTGVAGPPPDSVTPLGPDDAID 4.4 ALSSDFTCSSPVASGK 496-533 KPLLPSEPTAQLPALSEDLLLD 2.7 ALSEDFSGPSSASSLK MLCK-G chicken (2,3) chicken (30) (See Table 8) Ca 2 +-ATPase (plasma membrane) human (4-6) human (31) (See Table 8) Neuromodulin bovine (7) bovine (32) (See Table 8) 231 Table 9 (Cont.) Species used Species for Protein as calpain a.a.sequence Residues substrate (Ref.) (Ref.) a  PEST sequence PEST SCORE Tubulin (a) Tubulin (B) (a)-Fodrin PK-C (a isozyme) (BI isozyme) (BII isozyme) (Y isozyme) HMG-CoA cattle (8) cattle (8) human (9,10) & rat rat (11) rat (33) human (34) human (35) rat (36) C h i n e s e C h i n e s e 276-302 276-302 276-302 316-334 381-395 reductase hamster (12) hamster (37) 429-442 442-456 Casein ( a s l ) cattle (13R) cattle (38) 58-79 (See Table 8) (See Table B ) (See Table 8) KLLNQEEGEYYNVPIPEGDEEG 0.7 NVELR KLLSQEEGEYFNVPVPPEGSEG 3.5 NEELR KLLSQEEGEYFNVPVPPEGSEG 3.5 NEELR RMGPSSSPIPSPSPSPTDSK 15.8 KLSSVEEEPGVSQDR 4.2 RTQELEIELPSEPR 5.5 RPNEECLQILESAEK -4.4 KEMEAESISSSEEIVPNSVEQK 6.6 151-193 Casein (032) cattle (13R) cattle (39) 6-16 45-70 Casein (B) cattle (13R) cattle (40) 1-25 Vimentin 113-134 human (14) hamster (41) 406-419 RQFYQLDAYPSGAWYYVPLGTQ -1.9 YTDAPSFSDIPNPIGSENSEK HVSSSEESIISQETYK 2.2 RNANEEEYSIGSSSEESAEVAT 11.0 EEVK RELEELNVPGEIVESLSSSEES 6.6 ITR KYPVQPFTESQSLTLTDVENLH -2.8 RETNLESLPLVDTH -2.8 232 Table 9 (Cont.) Protein Species used Species for as calpain substrate (Ref.) a a.a.sequence Residues (Ref.) PEST sequence PEST SCORE Band 3 rat (15) (anion channel) von Willebrand human factor (16) Factor V mouse (42) 72-105 245-272 362-383 910-929 human 463-505 (43) 687-728 905-948 1622-1666 1947-1967 Pyruvate rat (17-18) rat (44) kinase (L-type) human (19) human (45) 104-118 18-34 420-439 712-757 799-820 877-892 894-907 1044-1072 RDLTIPVTEMQDPEALPTEQTA 11.1 TDYVPSSTSTPH HQPSLETQLYCGQAEGGSEGPS 1.1 TSGTLK RSLESFIDCSIVLPPTDAPSEK 0.4 KVTFDEENGLDEYDEVPMPVend 2.4 HCDVVNLTCEAC 0.6 QEPGGIVVPP TDAPVSPTTLY VEDISEPPLH ROEIVSYLCOLAPE -3.9 APPPTLPPDMAQV TVGPGLLGVST LGPK RCCSGEGLQIPTLS -3.7 PAPDCSQPLDVILLLDG SSSFPASYFDEMK KTQCCDEYECACNCVN -2.9 STVSCPLGYLASTATND CGCTTTTCLPDK KIPGTCCDTCEE 1.5 PECNDITAR REATESFATSPLSYR -0.9 RPEPTNSSLNLSVTSFK -2.3 HGVTFSPYEDEVNSSFTSGR 0.0 RNSSLNQEEEEFNLTALALENGT 0.7 EFVSSNTD1IVGSNYSSPSNISK HSSPYSEDPIEDPLQPDVTGIR 8.2 RPWFDLPSQDTGSPSR 10.2 RPV1EDPPSDLLLLK -5.0 KSNETSLPTDLNQTLPSMDFGWI -0.1 ASLPDH 233 Table 9 (Cont.) Species used Species for Protein as calpain a.a.sequence Residues PEST sequence PEST substrate (Ref.) SCORE (Ref.) a  1072-1098 HNQNSSNDTGQASCPPGLYQTVP 3.8 PEEH 1136-1151 KSFPTDISQMSPSSEH 4.4 1151-1182 HEVWQTVISPOLSQVTLSPELSQ 1.9 TNLSPDLSH 1299-1335 HTTLSLDFSQTNLSPELSQTNLS 2.3 PALGQMPLSPDPSH 1335-1477 HTTLSLDLSQTNLSPELSQTNLS 6.4 POLSEMPLFADLSQIPLTPDLDQ MTLSPDLGETDLSPNFGQMSLSP OLSQVTLSPDISDTTLLPBLSQI SPPPDLDQIFYPSESSQSLLLQE -FNESFPYPDIGQMPSPSSPTLND TFLSK 1572-1588 RETDIEDSDDIPEDTTYK 19.3 Fibrinogen human (20) human (46) 29-45 KDSDWPFCSDEDWNYK 1.7 (a) 252-268 RGGSTSYGTGSETESPR 11.0 461-491 KEVvTSEDGSDCPEAMDLGTLSG 0.0 IGTLDGFR Fibrinogen human (20) human (47) 217-234 KGGETSEMYLIQPDSSVK -3.5 (B) 380-391 RDNDGWLTSDPR 0.3 Kininogen human (21) human (48) 129-143 HPISTQSPDLEPILR -2.9 251-266 RDIPTNSPELEETLTH 9.2 379-388 KEETTVSPPH 10.7 505-523 HLASSSEDSTTPSAQTQEK 12.3 523-535 KTEGPTPIPSLAK -2.3 535-595 KPGVTVTFSDFQDSDLIATMMP 5.0 PISPAPIQSDDDWIPDIQTDPN GLSFNPISDFPDTTSPK EGF receptor human and human (49) 237-260 KDTCPPLMLYNPTTYQMDVNPE -2.9 rat (22,23) GK 234 Table 9 (Cont.) Protein Species used as calpain substrate (Ref.) a Species for a.a.sequence (Ref.) Residues PEST sequence PEST SCORE 359-374 HTPPLDPQELDILK -3.2 889-905 KPYDGIPASEISSILEK -4.9 975-1008 RALMDEEDMDOVVDADEYLIPQ 2.6 QGFFSSPSTSR 1044-1075 RYSSDPTGALTEDSIDDTFLPV 4.4 PEYINQSVPK Progesterone chicken (24) chicken (50) 45-69 RSSDEEEEEEEQEEEEEEEEPQ 34.4 receptor QR 169-179 RPGPEDASENR 8.7 257-278 RSSPSVPAAOLAEYGYPPPOGK 3.1 335-374 KAEPPLLPGAYGPPAAPDSLPS -0.8 TSAAPPGLYSPLGLNGH 400-419 RPDTETSQSSQYSFESIPQK 8.7 560-573 KPETPSSLLTSLNH -1.0 Estrogen cattle (25) human (51) 303-335 KNSIALSLTADQMVSALLDAEP -3.8 receptor PILYSEY0PTR 555-567 RGGASVEETDQSH 1.1 a-Actinin chicken (26) chicken (52) 137-153 KDDPLTNLNTAFDVAEK -4.7 Neurofilament human cattle 53-82 RSYSSSSGSLMPS -4.9 (light) (54) (53) LENLDLSQVAAIS NDLK 456-472 HVQEEQTEVEETIEASK 8.2 477-491 KDEPPSEGEAEEEEK 28.4 516-531 KEEEEGGEGEEGEETK 26.2 Neurofilament human cattle 495-509 KEEEPEAEEEEVAAK 18.4 (medium) (54) (55) 235 Table 9 (Cont.) Protein Species used as calpain substrate (Ref.) a Species for a.a.sequence (Ref.) Residues PEST sequence PEST SCORE 544-555 KSDQAEEGGSEK 8.4 561-583 KEEGEQEEGETEAEAEGEEAEAK 22.4 776-790 KAGGEGGSEEEGSDK 10.3 Neurofilament human cattle 66-95 RGAGAASSTDSL -4.4 (heavy) (54) (56) DTLSNGPEGC MVAVATSR 452-476 KETVIVEEQT 17.7 EETQVTEEV TEEEEK 502-514 KSPPAEEAASPEK 12.1 a ( l ) Suzuki et a l . , 1987b; (2) Kosaki et a l . , 1983; (3) Ito et a l . , 1987; (4) Au, 1987; (5) Wang et a l . , 1988a; (6) Wang et a l . , 1988b, (7) See Fig . IB; (8) Bil lger et a l . , 1988; (9) Seubert et a l . , 1987; (10) Siman et a l . 1984; (11) Kishimoto et a l . , 1983; (12) Liscum et a l . , 1983; (13) Murachi, 1983a; (14) Nelson. & Traub, 1981; (15) Croall et a l . , 1986; (16) Kunicki et a l . , 1986; (17) Ekman & Eriksson, 1980; (18) Dahlqvist-Edberg & Ekman, et a l . , 1981; (19) McGowan et a l . , 1983; (20) Kunicki et a l . , 1984; (21) Schmaier et a l . , 1986; (22) Cassel & Glaser, 1982; (23) Gates & King, Or. 1983; (24) Vedeckis et a l . , 1980; (25) Puca et a l . , 1977; (26) Ishiura et a l . , 1980; (27) Aoki et a l . , 1986; (28) Ohno et a l . , 1984; (29) Emori et a l . , 1987; (30) Guerriero, et a l . , 1986; (31) Verma et a l . , 1988; (32) Wakim et a l . , 1987; (33) Lemischka et a l . , 1981; (34) Hall et a l . , 1983; (35) McMahon & Moon, 1987; (36) Kikkawa et a l . , 1987; (37) Chin et a l . , 1984; (38) Mercier et a l . , 1971; (39) Brignon, e t . a l . , 1977; (40) Ribadeau-Dumas et a l . , 1972; (41) Quax-Jeuken et a l . , 1983; (42) Kopito & Lodish, 1986; (43) Titani et a l . , 1986 (44) Inoue et a l . , 1986; (45) Jenny et a l . , 1987; (46) Rixon et a l . , 1983; (47) Chung et a l . , 1983; (48) Kellermann et a l . , 1986; (49) Ullr ich et a l . , 1984; (50) Gronemeyer et a l . , 1987; (51) Green et a l . , 1986; (52) Baron et a l . , 1987. (53) Julien et a l . , 1987; (54) Malik et a l ; 1983; (55) Myers et a l . , 1987; (56) Lees et a l . , 1988. b residue numbers arbitrari ly assigned, for detai l , see legend of Fig. 4-7. R review article 236 Table 10 Non-PEST-containing c a l p a i n substra tes Pro te in Species used as Species f o r a . a . ca lpa in substrate sequence ( R e f . a ) ( R e f . a ) c a l p a i n (small subunit ) t roponin I t roponin T tropomyosin ( a ) tropomyosin (B) hemoglobin ( a ) hemoglobin (B) myel in bas ic p ro te in h is tone IIA h is tone IIB h is tone III desmin rabb i t & others rabb i t (2) rabb i t (2) rabb i t (2) rabb i t (2) human (3) human (3) c a t t l e (4) c a t t l e (5) c a t t l e (5) c a t t l e (5) human (6) p ig & human (7,8) r a b b i t (9) r a b b i t (10-11) rabb i t (12) r a b b i t (13) human (14) human (15) c a t t l e (16) c a t t l e (17) c a t t l e (18) c a t t l e (19) chicken (20) a (1) Suzuki et a l . , 1987b; (2) Dayton et a l . , 1975; (3) Mel loni et a l . , 1984;' (4) Banik et a l . , 1985; (5) Sakai et a l . , 1987; (6) Nelson & Traub, 1981; (7) Sakihama et a l . , 1985; (8) Emori et a l . , 1986a; (9) Wilkinson & Grand, 1975; (10) Pear lstone et a l . , 1977a; (11) Pear lstone et a l . , 1977b; (12) Stone & S m i l l e , 1978; (13) Mak et a l . , 1980; (14) Konigsberg & H i l l , 1962; (15) Brauni tzer et a l . , 1961; (16) Ey la r et a l . , 1971; (17) Yeoman et a l . , 1972; (18) Iwai et a l . , 1970; (19) DeLange et a l . , 1972; (20) G e i s l e r & Weber, 1982. ^ review a r t i c l e 237 and estrogen recep tor ) . The number of PEST regions of these substrate pro te ins v a r i e s from 1 to 12 ( for Factor V ) . The PEST scores are as high as 34.6 ( fo r the progesterone receptor ) . Of the four cloned isozymes of p ro te in kinase C, three {a, Bl and /JII) have a s i n g l e h igh ly conserved PEST region (residue 276-302) located at the end of the C-2 region of the regu la tory domain (Kikkawa et a l . , 1987, Ohno et al_. , 1987). The y-isoenzyme has a higher degree of v a r i a t i o n in that r e g i o n , which causes the PEST score to f a l l below - 5 . However, about 15 res idues downstream there i s a PEST sequence (residue 316-334, at the V 3 region) unique to t h i s isoenzyme, with a PEST score of 14.0 (Table 9 ) . Very r e c e n t l y , the cleavage s i t e s on three isozymes (a , B and y) of prote in kinase C produced by ca lpa in I and II have been i d e n t i f i e d (Kishimoto et al_. , 1989). Importantly, ca lpa in cleavage s i t e s of prote in kinase C isozymes were in proximity to the PEST sequences found. Furthermore, Kishimoto et a l . (1989) found that the r e l a t i v e rates of cleavage of a, B and y isozymes were about 2:16:100 fo r ca lpa in I and 23:48:100 f o r c a l p a i n II. Since the respec t ive PEST score f o r the s i n g l e PEST sequence f o r a , B and 7 isozymes i s 0 .7 , 3.5 and 15.8, r e s p e c t i v e l y (Table 9 ) , the strength of the PEST score var ies in the same order as the rate of p r o t e o l y s i s for these isozymes. I n te res t ing ly , HMG-CoA reductase , the r a t e - l i m i t i n g enzyme of cho les te ro l b iosyn thes is , has i t s two PEST-sequences and two c a l p a i n cleavage s i t e s in adjacent regions (the PEST sequences being about 40 kDa and 47 kDa, r e s p e c t i v e l y , from the N-terminus) (Chin et a l . , 1984), whi le the ca lpa in cleavage s i t e s are about 35 kDa and 44 kDa from the N-terminus (Liscum et a l . , 1985). The high conservat ion of PEST sequences among isoenzymes and the proximity of PEST sequences to ca lpa in cleavage 238 s i t e s suggest that PEST regions are recogn i t ion s i t e s on ca lmodul in-binding prote ins and some other ca lpa in subs t ra tes . I n t e r e s t i n g l y , the non-PEST-containing substrates are mostly small p ro te ins (Table 10). There are at l eas t two explanat ions as to why these non-PEST-conta in ing prote ins a lso serve as c a l p a i n subs t ra tes : (a) the PEST-conta in ing prote ins and non-PEST-containing prote ins may represent two separate groups of ca lpa in subst ra tes . The non-PEST pro te ins may be recognized at other sequence(s) , perhaps analogous to PEST. (b) The d e f i n i t i o n of PEST regions and the PEST score c a l c u l a t i o n may have minor f laws which might a f f e c t i d e n t i f i c a t i o n of some po ten t ia l PEST sequences and a lso t h e i r PEST scores . The f i r s t p o s s i b i l i t y seems to be supported by the f a c t that many small peptides that d e f i n i t e l y do not have PEST sequences s t i l l serve as ca lpa in subst ra tes . Among these are i n s u l i n /5-c h a i n , protamine, glucagon and dynorphin (Sasaki et a l . , 1984). On the other hand, in support of the second p o s s i b i l i t y , i t was observed that many non-PEST-containing substrates had a high number of bas ic amino ac id r e s i d u e s : h i s t i d i n e (H), l y s i n e (K) and arg in ine (R), ( e . g . myelin bas ic p r o t e i n ) , which, by d e f i n i t i o n , precludes the ex is tence of "PEST" reg ions . A l s o , the s t r i c t requirement for s t a r t i n g and ending a PEST sequence with H, K or R sometimes s i g n i f i c a n t l y reduces the PEST scores of a h igh ly concentrated PEST-enriched region which lacks a p o s i t i v e l y charged residue to mark i t s boundary. The 30 kDa subunit of c a l p a i n i s a good example of t h i s . It has regions which are qui te r i c h in P, D/E and S/T but which are e i t h e r d is rupted by H, K or R or are lack ing these to s t a r t or end the po ten t ia l PEST region proper ly . Taken together , t h i s suggests that the 239 r e s t r i c t i o n of the requirement of p o s i t i v e l y charged amino ac id residues to s t a r t or end a sequence important for ca lpa in recogn i t ion may not always be j u s t i f i e d . Another important point i s that not a l l PEST-conta in ing prote ins are c a l p a i n s u b s t r a t e s . For ins tance , c a l s e q u e s t r i n , which i s i n s e n s i t i v e to c a l p a i n ( S e i l e r et a l . , 1984; Wang, V i l l a l o b o and R o u f o g a l i s , unpublished r e s u l t s ) , i s a PEST-containing prote in according to i t s amino ac id sequence (Reithmeier et a l . , 1987). Such cases could be expla ined by a dual requirement f o r p r o t e o l y s i s by c a l p a i n : ( i ) the requirement o f the PEST-region as a recogn i t ion s i t e and ( i i ) the ex is tence of a s u s c e p t i b l e cleavage s i t e ( e . g . f u l f i l l i n g the P 2 - P 1 requirement or another as yet undefined r e s t r i c t i o n ) which i s in c lose proximity to the PEST r e g i o n . In other words, PEST-containing prote ins which do not have s u s c e p t i b l e cleavage s i t e s w i l l not be attacked by c a l p a i n . 7. Resistance of the t roponin-C superfamily to ca lpa in During the study of the e f f e c t of ca lpa in on the plasma membrane C a 2 + - A T P a s e and c a l c i n e u r i n , i t was observed that both calmodul in (Wang et a l . , 1988b) and the calmodul in-1 ike small subunit o f c a l c i n e u r i n ( c a l c i n e u r i n B) (Wang et a l . , 1989c) were r e s i s t a n t to c a l p a i n . There fore , the c a l p a i n - s u s c e p t i b i l i t y of other members of the t roponin C superfami ly of calc ium binding prote ins was invest iga ted (F ig 40) . There has been some controversy about the s u s c e p t i b i l i t y of t roponin C to c a l p a i n (Dayton et a l . , 1975; Ishiura et a l . , 1979; Murakami and Uchida, 240 1979). In order to c l a r i f y t h i s d iscrepancy, a mixture of t roponin I and t roponin C was t reated with ca lpa in I from human e r y t h r o c y t e s . It was found that c a l p a i n I r a p i d l y cleaved troponin I while leav ing t roponin C i n t a c t ( F i g . 40) . To expand t h i s study, the c a l p a i n - s e n s i t i v i t y of other members of the t roponin C superfamily was a lso i n v e s t i g a t e d , i nc lud ing oncomodulin, S-lOOa p r o t e i n , S-100/J prote in and parvalbumin ( F i g . 40) . Consis tent with the p r e d i c t i o n , none of these prote ins i s c leaved by c a l p a i n I ( F i g . 40) . Th is suggests that the members of the t roponin C superfamily are in general c a l p a i n - r e s i s t a n t . To take t h i s hypothesis one step f u r t h e r , i t i s proposed that the var ious b inding prote ins ( target prote ins) of the t roponin C superfami ly of C a 2 + - b i n d i n g prote ins are ca lpa in subst ra tes . The evidence comes from the fo l lowing observat ions: ( i ) many ca lmodul in -b inding pro te ins are c a l p a i n s u b s t r a t e s ; ( i i ) c a l c i n e u r i n A, which binds c a l c i n e u r i n B, i s hydrolysed by c a l p a i n ; ( i i i ) t roponin I, which binds t roponin C i s c a l p a i n - s e n s i t i v e ; and ( iv ) prote in k i n a s e - C , a w e l l - e s t a b l i s h e d c a l p a i n subs t ra te , appears to bind to an 21 kDa i n h i b i t o r y prote in which belongs to the t roponin C superfamily (McDonald and Walsh, 1985; McDonald et a l . , 1987). I f t h i s hypothesis i s c o r r e c t , the target pro te ins of these C a 2 + -b inding prote ins may be considered to belong to a large subgroup made up of substrates of c a l p a i n . In a d d i t i o n , the troponin C - l i k e Ca -b ind ing prote ins might contro l the hydro lys is of t h e i r respect ive target prote ins by c a l p a i n at the substrate l e v e l . 8. Future D i r e c t i o n s 241 In order to tes t the hypotheses presented, at l eas t two d i r e c t i o n s can be taken: (a) obtain amino acid sequences of ca lmodul in -b inding prote ins and other potent ia l substrates fo r ca lpa in and to i d e n t i f y t h e i r PEST reg ions ; (b) determine whether the remaining ca lmodul in -b ind ing prote ins are hydrolyzed by c a l p a i n . Subsequently, e f f o r t s can be d i r e c t e d to determining whether the ca lpa in cleavage s i t e s and the PEST regions are in prox imi ty . A l s o , the loss of PEST region(s) by i n i t i a l cleavage with c a l p a i n or other proteases should render the remaining fragment i n s e n s i t i v e to fu r ther cleavage by c a l p a i n . S i t e - d i r e c t e d mutagenesis of "important" res idue(s) in the PEST sequence of a prote in should p red ic tab ly a f f e c t i t s p r o t e o l y s i s by c a l p a i n . It would a lso be o f i n t e r e s t to f i n d out whether ca lmodul in -b inding prote ins and other PEST-conta in ing substrates fo r ca lpa in have short h a l f - l i v e s j_n v i v o . I f the proposed hypotheses are c o r r e c t , they would form the f i r s t cons is ten t molecular bas is fo r substrate s p e c i f i c i t y of c a l p a i n and eventua l ly might help in the fur ther understanding of prote in metabolism in the c e l l . 242 CONCLUSIONS (1) Calpain I, the micromolar Ca - r e q u i r i n g c a l c i u m - a c t i v a t e d neutral protease from the human erythrocyte c y t o s o l i c f r a c t i o n , was p u r i f i e d to apparent homogeneity. The p u r i f i e d protease showed the c h a r a c t e r i s t i c molecular mass fo r both the large and small subuni ts , high a f f i n i t y C a ^ + -dependence, t h i o l - r e d u c i n g agent requirement and protease i n h i b i t o r s p e c i f i c i t y of ca lpa in I. (2) Treatment of the i s o l a t e d human erythrocyte plasma membrane-bound C a c -ATPase with p u r i f i e d ca lpa in I (from the same c e l l s ) i r r e v e r s i b l y ac t iva ted the basal Ca 2 + -dependent ATP-hydro ly t i c (ATPase) a c t i v i t y in two ways: ( i ) by increas ing the K m ( C a ) by about 2-orders of magnitude; and ( i i ) by increas ing the V m a x of the enzyme. This a c t i v a t i o n was k i n e t i c a l l y i n d i s t i n g u i s h a b l e to the a c t i v a t i o n produced by calmodulin b inding to the C a 2 + - A T P a s e . (3) The calpain-mediated a c t i v a t i o n of membrane-bound C a 2 + - A T P a s e was concomitant with the loss of c a l m o d u l i n - a c t i v a t i o n o f the C a 2 + - A T P a s e . (4) The presence of calmodulin dur ing ca lpa in- t reatment of the membrane-bound C a 2 + - A T P a s e l a r g e l y prevented the calpain-mediated a c t i v a t i o n . (5) Studies on the fragmentation of ( i ) the membrane-bound form, ( i i ) 243 the solubi l ized and p u r i f i e d form and ( i i i ) phosphat idy lchol ine l iposome-r e c o n s t i t u t e d form of C a z + - A T P a s e revealed that (a) in the absence of ca lmodul in , ca lpa in transformed the 136 kDa enzyme into two major non-ca lmodul in -b ind ing act ive fragments (124 kDa and 80 kDa) and two minor calmodul i n - b i n d i n g ac t ive fragments (125 kDa and 82 kDa); and (b) in the presence of ca lmodul in , the C a z + - A T P a s e was proteolyzed by c a l p a i n into two major calmodul in -b ind ing ac t ive fragments of 127 kDa and 85 kDa, r e s p e c t i v e l y . (6) The 124 kDa and 80 kDa fragments of the C a z + - A T P a s e have calmodul in-9 • i n s e n s i t i v e C a c -ATPase a c t i v i t y and cont r ibuted to the p r o t e o l y t i c a c t i v a t i o n of the enzyme by c a l p a i n . (7) C a l p a i n - p r o t e o l y s i s of the C a z + - A T P a s e i n the presence of calmodulin produces only ca lmodu l in -sens i t i ve ac t ive fragments (127 kDa and 85 kDa) but not the calmodulin-independent ac t ive fragments (124 kDa and 80 kDa), which explained the pro tec t ive e f f e c t of calmodulin against p r o t e o l y i c a c t i v a t i o n of the Ca*- -ATPase. (8) From the publ ished primary s t ructure (amino ac id sequence) of a human plasma membrane C a z + - A T P a s e , the i d e n t i f i c a t i o n of the l o c a t i o n of the ca lmodul in -b ind ing domain and the acylphosphate s i t e , together with estimated molecular masses and ca lmodul in -b inding a b i l i t y of the var ious fragments, i t was poss ib le to postulate that c a l p a i n I f i r s t c leaved o f f about 9 kDa from the C-terminal end of the C a 2 + - A T P a s e , inc lud ing about 2 kDa o f the ca lmodul in-b inding domain (corresponding to approximately 3.5 244 kDa), producing the 125 kDa fragment which i s s t i l l c a l m o d u l i n - s e n s i t i v e . A f u r t h e r cleavage removed most (1 kDa) of the remaining calmodul in-b inding domain and produced the c a l m o d u l i n - i n s e n s i t i v e 124 kDa fragment. The a s s o c i a t i o n of calmodulin with the ca lmodul in -b ind ing domain protected the l a t t e r from p r o t e o l y t i c at tack by c a l p a i n . Under t h i s c o n d i t i o n , a c a l m o d u l i n - s e n s i t i v e 127 kDa fragment was produced. A slow cleavage at a region about 42-44 kDa from the C-terminal end was proposed to produce the 85 kDa, 82 kDa and 80 kDa a c t i v e fragments from the 127 kDa, 125 kDa and 124 kDa fragments, r e s p e c t i v e l y . (9) Calpain- t reatment of the phosphat idy lchol ine 1 iposome-reconst i tuted C a 2 + - A T P a s e increased the i n i t i a l ra tes of C a 2 + uptake and ATP h y d r o l y s i s to l e v e l s s i m i l a r to those obtained upon add i t ion of ca lmodul in . This a c t i v a t i o n was concomitant with the formation of mainly the 124 kDa fragment of the enzyme, while the presence of calmodulin l a r g e l y prevented p r o t e o l y t i c a c t i v a t i o n of the C a 2 + - A T P a s e a c t i v i t y by forming the 127 kDa act ive fragment. (10) The s u s c e p t i b i l i t y of the C a 2 + - A T P a s e to c a l p a i n was in fac t not unique but a c h a r a c t e r i s t i c shared by 15 other calmodulin-dependent enzymes and p r o t e i n s . Furthermore, primary s t ruc tu re a n a l y s i s showed that the major i ty of ca lmodul in-b inding prote ins have one or more "PEST" sequences, which are proposed to serve as recogn i t ion s i t e s fo r c a l p a i n . 245 BIBLIOGRAPHY Adach i , Y . , Kopayashi , N . , Murachi, T. and Hatanaka, M. (1986) C a 2 + -dependent cyste ine prote inase , ca lpa ins I and II are not phosphorylated i n v i v o . Biochem. Biophys. Res. Commun. 136, 1090-1096. Adamo, H . P . , Rega, A . F . and Garrahan, P . J . ? (1988) P re -s teady -s ta te phosphorylat ion of the human red c e l l Ca -ATPase. J . B i o l . Chem. 263, 17548-17554. 2+ and Akyempon, C .K . and Roufoga l is , B.D. (1982) The s to ich iometry o f the Ca pump in human erythrocyte v e s i c l e s : modulation by Ca , Mg  ca lmodul in . Ce l l Calcium 3, 1-17. A l - J o b o r e , A . and Roufoga l is , B.D. (1981) Phosphol ip id and calmodulin a c t i v a t i o n of s o l u b i l i z e d ca lc ium- t ranspor t ATPase from human eryhrocytes: regula t ion by magnesium. Can. J . Biochem. 59, 880-888. A l - J o b o r e , A . , Mauldin, D. , Minocherhomjee, A. and R o u f o g a l i s , B.D. (1981) Regulat ion of the calcium pump ATPase in human ery throcytes In : Erythrocyte Membranes 2: Recent C l i n i c a l and Experimental Advances (Kruckeberg, W.C. , Eaton, J.W. and Brewer, G . J . , eds . ) pp. 57-73, Alan R. L i s s , New York. A l - J o b o r e , A . , Minocherhomjee, A . , V i l l a l o b o , A . and R o u f o g a l i s , B.D. (1984) Ac t ive calcium transport in normal and abnormal human erythrocytes i n : Erythrocyte Membranes 3: Recent C l i n i c a l and Experimental Advances (Kruckeberg, W.C. , Eaton, J.W. and Brewer, G . J . , eds . ) pp. 243-292, Alan R. L i s s , New York. A l l e n , B . G . , Katz , S. and Roufoga l is , B.D. (1987) E f f e c t s o f C a 2 + , M g 2 + and calmodulin on the formation and decomposit ion of the phosphorylated intermediate o f the ery throcyte C a - s t i m u l a t e d ATPase. Biochem. J . 244, 617-623. A l l e n d e , J . E . (1988) GTP-mediated macromolecular i n t e r a c t i o n s : the common features of d i f f e r e n t systems. FASEB J . 2, 2356-2367. Ando, Y . , Imamura, S . , Yamagata, Y . , K i tahara , A . , S a j i , H . , Murachi , T. and Kannagi, R. (1987) P l a t e l e t f a c t o r XIII i s ac t iva ted by c a l p a i n . Biochem. Biophys. Res. Commun. 144, 484-490. Ando, Y . , Imamura, S . , Murachi, T. and Kannagi, R. (1988b) Calpain ac t i va tes two transglutaminases from procine s k i n . Arch . Dermatol. Res. 280, 380-384. Ando, Y . , M iyach i , Y . , Imamura, S . , Kannagi, R. and Murachi , T. (1988a) 246 P u r i f i c a t i o n and charac te r i za t ion of ca lpa ins from pig epidermis and t h e i r ac t ion on epidermal k e r a t i n . J . Invest . Dermatol. 90, 26-30. Andreasen, T . J . , Luet je , C.W., Heideman, W. and Storm, D.R. (1983) P u r i f i c a t i o n of a novel calmodulin binding pro te in from bovine cerebra l cortex membranes. Biochemistry 22, 4615-4618. A o k i , K. , Imajoh, S . , Ohno, S . , Emori, Y . , Koike , M. , K o s a k i , G. and Suzuk i , K. (1986) Complete amino ac id sequence o £ the large subunit of the low-Ca - r e q u i r i n g form o f human Ca - a c t i v a t e d neutral protease (/tCANP) deduced from i t s cDNA sequence. FEBS l e t t . 205, 313-317. Au, K .S . (1978) An endogenous i n h i b i t o r of e ry throcyte membrane (Ca z + +Mg z + ) -ATPase . Int. J . Biochem. 9, 477-480. Au, K . S . (1987) A c t i v a t i o n of erythrocyte membrane C a z + - A T P a s e by c a l p a i n . Biochim. Biophys. Acta 905, 273-278. Au, K . S . , Cho, K . L . , Lee, K .S . and L a i , K.M. (1985) An endogenous i n h i b i t o r prote in of synapt ic plasma membrane ( C a z + + M g z + ) - A T P a s e . Biochim. Biophys. Acta 821, 348-354. Au, K . S . , Hsu, L. and Morr ison, M. (1988) C a z + - m e d i a t e d catabol ism of human erythrocyte band 3 p r o t e i n . Biochim. Biophys. Acta 946, 113-118. Au, K . S . , Lee, M.F. and S i u , Y . L . (1989) Ca Z + -med ia ted a c t i v a t i o n o f human erythrocyte membrane C a - A T P a s e . Biochim. Biophys. Acta 978, 197-202. Babu, Y . S . , Bugg, C E . and Cook, W.J . (1988) Three-dimensional s t ruc ture of ca lmodul in , i n : Calmodulin (Cohen, P. and K l e e , C . E . , eds . ) pp. 83-89, E l s e v i e r , Amsterdam. Bal tensperger , K . , C a r a f o l i , E. and C h i e s i , M. (1988) The Ca Z + -pumping ATPase and the major substrates of the cGMP-dependent prote in kinase in smooth muscle sarcolemma a r e . d i s t i n c t e n t i t i e s . Eur. J . Biochem. 172, 7-16. Banik, N . L . , McAlhaney, W.W. and Hogan, E . L . (1985J Calc ium-st imulated p r o t e o l y s i s in myel in: evidence f o r a Ca - a c t i v a t e d neutral prote inase associated with p u r i f i e d myelin of ra t CNS. J . Neurochem. 45, 581-588. Baron, M.D. , Davison, M.D., Jones, P . , P a t e l , B. and C r i t c h l e y , D.R. (1987) I so la t ion and c h a r a c t e r i z a t i o n of a cDNA encoding a ch ick a - a c t i n i n . J . B i o l . Chem. 262, 2558-2561. B a r r e t t , A . J . (1986) An in t roduct ion to the prote inases i n : Proteinase Inh ib i tors (Bar re t t , A . J . and Salvesen, G . , eds) pp. 3-22, 247 E l s e v i e r , Amsterdam. B a r z i l a i , A . , Spanier , R. and Rahamimoff, H. (1984) I s o l a t i o n , p u r i f i c a t i o n , and r e c o n s t i t u t i o n of the Na + gradient-dependent C a z + t ranspor ter (Na + -Ca exchanger) from bra in synapt ic plasma membrane. Proc. N a t l . Acad. S c i . USA 81, 6521-6525. Becker le , M. , O ' H a l l o r a n , T . , C r o a l l , D. and Burr idge , K. (1986) The adhesion plaque p r o t e i n , t a l i n , i s re la ted to p235, a major substrate of the calcium-dependent protease in p l a t e l e t s . J . C e l l . Biochem. S10A, 259. Beer, D . G . , But ley , S.M. and J la lk inson , A .M. (1984) Developmental changes in the endogenous C a - s t i m u l a t e d p r o t e o l y s i s of mouse lung cAMP-dependent prote in k inases . Arch . Biochem. Biophys. 228, 207-219. B e l l e s , B . , Hescheler , J . , Trautwein, W., Blomgren, K. and K a r l s s o n , J . O . (1988) A poss ib le phys io log ica l r o l e of the Ca-dependent protease c a l p a i n and i t s i n h i b i t o r c a l p a s t a t i n on the Ca current in guinea p ig myocytes. Pf lugers Arch . 412, 554-556. Benaim, G . , C l a r k , A. and C a r a f o l i , E. (1986) ATPase a c t i v i t y and C a z + t ranspor t by reconst i tu ted t r y p t i c fragments of the C a z + pump of the erythrocyte plasma membranes. Ce l l Calcium 7, 175-186. Benaim, G . , Z u r i n i , M. and C a r a f o l i * E. (1984) D i f f e r e n t conformational s ta tes of the p u r i f i e d C a - A T P a s e of the erythrocyte plasma membrane revealed by c o n t r o l l e d t r y p s i n p r o t e o l y s i s . J . B i o l . Chem. 259, 8471-8477. Bennett , M.K. and Kennedy, M.B. (198J) Deduced primary s t ruc ture of the B subunit of bra in type II Ca z + / ca lmodul in -dependent pro te in kinase determined by molecular c l o n i n g . Proc. N a t l . Acad. S c i . USA 84, 1794-1798. Bennett, V . , Gardner, K. and S t e i n e r , J . P . (1988) Brain adducin: a prote in kinase C substrate that mediate s i t e - d i r e c t e d assembly at the s p e c t r i n - a c t i n j u n c t i o n . J . B i o l . Chem. 263, 5860-5869. Ber r idge , M . J . (1987) Inos i to l t r isphosphate and d i a c y l g l y c e r o l : two i n t e r a c t i n g second messengers. Ann. Rev. Biochem. 56, 159-193. Ber r idge , M .J . and Gal ione , A . , (1988) C y t o s o l i c calc ium o s c i l l a t o r s . FASEB J . 2, 3074-3082. B i l l g e r , M. , W a l l i n , M. and K a r l s s o n , J . - 0 . (1988) P r o t e o l y s i s of tubu l in and microtubule -assoc ia ted prote ins 1 and 2 by ca lpa in I and II. D i f fe rence in s e n s i t i v i t y of assembled and disassembled microtubules . C e l l Calcium 9, 33-44. B i rch-Machin , M.A. and Dawson, A . P . (1988) C a z + t ranspor t by rat l i v e r plasma membranes: the t ranspor ter and the prev ious ly reported 248 C a - A T P a s e are d i f f e r e n t enzymes. Biochim. Biophys. Acta 944, 308-314. B l a u s t e i n , M.P. and Nelson, M. (1982) N a + - C a 2 + exchange: i t s r o l e in the regu la t ion of c e l l ca lc ium Ln: Membrane Transport of Calcium. ( C a r a f o l i , E . , ed.) Academic Press . Inc . , New York, 217-236. B l i n k s , J . , Wier, W., Hess, P. and Prendergast , F. (1982) Measurement of C a z + concentrat ion in l i v i n g c e l l s . Prog. Biophys. Mol . B i o l . 40, 1-114. Blum, J . J . , Hayes, A . , Jamieson, G.A. J r . and Vanaman, T . C . (1980) Calmodulin confers calc ium s e n s i t i v i t y on c i l i a r y dynein ATPase. J . C e l l . B i o l . 8_7, 386-397. Blumenthal, D.K. and Krebs, E . G . (1988) Calmodul in-b inding domains on target prote ins I n : Calmodulin (Cohen, P. and K lee , C . B . , eds. ) pp. 341-355, E l s e v i e r , Amsterdam. Blumenthal, D .K . , Tak io , K . , Edelman. A . M . , Charbonneau, H . , T i t a n i , K . , Walsh, K.A. and Krebs, E .G . (1985) I d e n t i f i c a t i o n of the ca lmodul in -b inding domain of ske le ta l myosin l i g h t chain k inase. Proc. N a t l . Acad. S c i . USA 82, 3187-3191. Bond, J . S . and B u t l e r , P . E . (1987) I n t r a c e l l u l a r proteases. Ann. Rev. Biochem. 56> 333-364. Brand l , C . J . , Green, N.M. , Korczak, B. and MacLennan, D.H. (1986) Two C a 2 + ATPase genes: homologies and mechanist ic imp l ica t ions of deduced amino ac id sequences. C e l l 44> 597-607. Braun i tzer , G . , Gehr ing-Mul le r , R., Hilschmann, N . , H i l s e , K . , Hobom, G . , Rudlo f f , V. and Wittmann-Liebold, B. (1961) The c o n s t i t u t i o n of normal adult haemoglobin. Hoppe-Seyler 's Z . P h y s i o l . Chem. 325, 283-286. Br ignon, G . , Ribadeau-Dumas, B . , Merc ier , J . and P e l i s s i e r , J . (1977) Complete amino ac id sequence of bovine a s o - c a s e i n . FEBS l e t t . 76, 274-279. Bryan, J . (1974) Biochemical p roper t ies of microtubules . Fed. Proc. 33, 152-157. B u l l e i t , R . F . , Bennett, M.K. , Mol loy , S . S . , Hur ley , J . B . and Kennedy, M.B. (1988) Conserved and v a r i a b l e regions in the subunits of bra in type II C a 2 + / c a l m o d u l i n - d e p e n d e n t prote in k inase. Neuron I , 63-72. Burgin , H . , Schatzmann, H . J . (1979) The r e l a t i o n between net ca lc ium, a l k a l i ca t ion and c h l o r i d e movements in red c e l l s exposed to s a l i c y l a t e . J . P h y s i o l . 287_> 15-32. 249 J C a r a f o l i , E. (1987) I n t r a c e l l u l a r calcium homeostasis. Ann. Rev. Biochem. 56, 395-433. C a r a f o l i , E. and Z u r i n i , M. (1982) The Ca 2 + -pumping ATPase of plasma membranes: p u r i f i c a t i o n s , r e c o n s t i t u t i o n , and p r o p e r t i e s . Biochim. Biophys. Acta 683, 279-301. C a r a f o l i , E . , F i s c h e r , R., James, P . , Krebs, J . , Maeda, M. , Enyedi , A . , M o r e l l i , A. and d e F l o r a , A. (1987) The calc ium pump o f the plasma membrane: recent studied on the p u r i f i e d enzyme and on i t s p r o t e o l y t i c fragments, with p a r t i c u l a r a t tent ion to the calmodulin binding domain In : Calc ium-Binding Prote ins in Health and Diseases (Means, A . N . , ed.) pp. 78-91, Academic Press , New York. C a r a f o l i , E . , T iozzo , R., L u g l i , G . , C r o v e t t i , F. and K r a t z i n g , C. (1974) The re lease of calcium from heart mitochondria by sodium. J . Mol . C e l l . C a r d i o l . 6, 361-371. Car l i n , R .K . , B a r t e l t , D.C. and S i e k e v i t z , P. (1983) I d e n t i f i c a t i o n of fodr in as a major ca lmodul in -b inding pro te in in postsynapt ic densi ty preparat ions . J . C e l l B i o l . 96, 443-448. C a r o n i , P. and C a r a f o l i , E. (1981) Regulat ion o f C a 2 + pumping ATPase of heart sarcolemma by a phosphory la t ion-dephory la t ion process . J . B i o l . Chem. 256, 9371-9373. Carpenter G. (1987) Receptors f o r epidermal growth f a c t o r and other polypept ide mitogens. Ann. Rev. Biochem. 56, 881-914. C a s s e l , D. and Glaser , L. (1982) P r o t e o l y t i c cleavage of epidermal growth f a c t o r receptor . J . B i o l . Chem. 257, 9845-9848. Cha, Y . N . , S h i n , B .C . and Lee, K .S . (1971) Ac t i ve uptake of C a + + and C a + + -ac t iva ted M g + + ATPase in red c e l l membrane fragments. J . Gen. P h y s i o l . 5Z, 202-215. Chan, K . - F . J . and Graves, D . J . (1984) Molecular proper t ies of phosphorylase kinase In: Calcium and C e l l Functions (Cheung, W.Y., ed.) V o l . V, pp. 1-31, Academic Press , New York. Cheung, W.Y. (1970) C y c l i c 3 ' , 5 ' - n u c l e o t i d e phospho-diesterase. Demonstration of an a c t i v a t o r . Biochem. Biophys. Res. Commun. 38, 533-538. Cheung, W.Y. (1971) C y c l i c 3 ' , 5 ' - n u c l e o t i d e phosphodiesterase. J . B i o l . Chem. 246, 2859-2869. Cheung, W.Y. (1980) Calmodulin plays a p ivo ta l ro le in c e l l u l a r r e g u l a t i o n . Science 207, 19-27. Chien , K . R . , Abrams, J . , F e r r o n i , A . , Mar t in , T . J . and Farber , J . L . (1978) 250 Accelerated phosphol ip id degradation and assoc ia ted membrane dysfunct ion in i r r e v e r s i b l e ischemic l i v e r c e l l i n j u r y . J . B i o l . Chem. 253, 4809-4817. C h i n , D . J . , G i l , G . , R u s s e l l , D.W., Liscum, L . , Luskey, K . L . , Basu, S . K . , Okayama, H . , Berg, P . , G o l d s t e i n , J . L . and Brown, M.S. (1984) Nucleot ide sequence o f 3 -hydroxy-3 -methy l -g lu tary l coenzyme A reductase, a g lycopro te in of endoplasmic r e t i c u l u m . Nature 308, 613-617. Chung, D.W., Que, B . G . , Rixon, M.W., Mace, M. J r . and Davie , E.W. (1983) Charac te r i za t ion of the complementary deoxyr ibonuc le ic ac id and genomic deoxyr ibonucle ic ac id f o r the /J-chain of human f i b r i n o g e n . Biochemistry 22, 3244-3250. Church, J . G . , Ghosh, S . , Roufoga l i s , B.D. and V i l l a l o b o , A. (1988) Endogenous hyperphosphorylat ion in plasma membrane from an a s c i t e hepatocarcinomera c e l l l i n e . Biochem. C e l l . B i o l . 66, 1-12. C o l c a , J . R . , DeWald, D . B . , Pearson, J . D . , Palazuk, B . J . , Laur ino , J . P . and McDonald, J . M . (1987) Insul in st imulates the phosphorylat ion o f calmodulin in i n t a c t ad ipocytes . J . B i o l . Chem. 262, 11399-11402. C o o l i c a n , S . A . and Hathaway, D.R. (1984) E f f e c t of L -a -phospha t idy l inos i to l on a vascu lar smooth muscle Ca -dependent protease. J . B i o l . Chem. 259, 11627-11630. C r o a l l , D . E . , Morrow, J . S . and DeMartino, G.N. (1986) Limited p r o t e o l y s i s of the erythrocyte membrane skeleton by calcium-dependent p ro te inases . Biochim. Biophys. Acta 882, 287-296. Crompton M. (1985) The regu la t ion of mitochondrial calc ium t ransport in hear t . Curr . Top. Membr. Transp. 25, 231-276. Crompton, M. , S i g e l , E . , Salzmann, M. and C a r a f o l i , E. (,1976) A k i n e t i c study of the energy- l inked i n f l u x of C a ^ + in to heart mi tochondr ia . Eur. J . Biochem. 69, 429-434. Dagher G. and Lew V . L . (1988) Maximal calc ium ext rus ion capaci ty and stoichiometry of the human red c e l l calc ium pump. J . P h y s i o l . 407, 596-586. Dah lqv is t -Edberg , U. and Ekman, P. (1981) P u r i f i c a t i o n of a C a 2 + - a c t i v a t e d protease from rat erythrocytes and i t s p o s s i b l e e f f e c t on pyruvate kinase i n v i v o . Biochim. Biophys. Acta 660, 96-101. David , L . L . and Shearer, T .R . (1986) P u r i f i c a t i o n of ca lpa in II from rat lens and determination of endogenous subs t ra tes . Exp. Eye Res. 42, 227-238. Dayton, W.R., G o l l , D . E . , Stromer, M.H. , R e v i l l e , W.J . Zeece, M.G. and 251 Robson, R.M. (1975) Some proper t ies of a Ca - a c t i v a t e d protease that may be involved in m y o f i b r i l l a r p ro te in turnover In : Proteases and B i o l o g i c a l Control (Re ich, E . , R i f k i n , D.B. and Shawn, E . , eds. ) V o l . 2, pp. 551-577, Cold Spring Harbour Laboratory, U .S .A . Dayton, W.R., G o l l , D.EL, Zeece, M.G. , Robson, R.M. and R e v i l l e , W.J . (1976) A Ca - a c t i v a t e d protease p o s s i b l y involved in m y o f i b r i l l a r prote in turnover . P u r i f i c a t i o n from porcine muscle. Biochemistry 15, 2150-2167. DeLuca, H .F . and Engstrom, G.W. (1961) Calcium uptake by ra t kidney mi tochondr ia . Proc. N a t l . Acad. S c i . USA 4_Z, 1744-1750. DeLange, R . J . , Hooper, J . A . and Smith, E . L . (1972) Complete amino ac id sequence of cal f - thymus hi stone III. Proc. N a t l . Acad. S c i . , USA 69, 882-884. DeMartino, G.N. and Blumenthal, D.K. (1982) I d e n t i f i c a t i o n of a f a c t o r that st imulates calcium-dependent pro teases . Biochemistry 21, 4297-4303. DeMartino, G . N . , Huff , C A . and C r o a l l , D .E . (1986) Autopro teo lys is of the small subunit of calcium-dependent protease II ac t i va tes and regulates protease a c t i v i t y . J . B i o l . Chem. 261, 12047-12052. Denton, R .M. , McCormack, J . G . and E d g e l l , N . J . (1980) Role of calc ium ions in the regu la t ion of in t rami tochondr ia l metabolism. Biochem. J . 190, 107-117. Depaol i -Roach, A . A . , Gibbs, J . B . and Roach, P . J . (1979) Calcium and calmodulin a c t i v a t i o n of muscle phosphorylase k inase: e f f e c t of t r y p t i c p r o t e o l y s i s . FEBS l e t t . 105, 321-324. Docherty, K . , S t e i n e r , D.F. (1982) P o s t - t r a n s l a t i o n a l p r o t e o l y s i s in polypept ide hormone b i o s y t h e s i s . Ann. Rev. P h y s i o l . 44, 625-638. Dunham, E . T . and Glynn, I.M. (1961) Adenosine t r iphosphatase a c t i v i t y and the ac t ive movements of a l k a l i metal i o n s . J . P h y s i o l . 156, 274-293. Ekman, P. and E r i k s s o n , I. (1980) The j n v i t r o mod i f i ca t ion of phosphorylated pyruvate kinase by a C l r + - a c t i v a t e d protease from rat l i v e r . A c t a . Chemica. Scand. B34, 419-422. Emori, Y . , Kawasaki, H . , Imajoh, S. Imahori, K. and Suzuk i , K. (1987) Endogenous i n h i b i t o r fo r calcium-dependent cyste ine protease conta ins four internal repeats that could be responsib le for i t s mul t ip le reac t ive s i t e s . Proc. N a t l . Acad. S c i . USA 84, 3590-3594. Emori, Y . , Kawasaki, H . , Suz ihara , H . , Imajoh, S . , Kawashima, S. and 252 Suzuk i , K. (1986a) Iso la t ion and sequence a n a l y s i s of cDNA clones f o r the large subunits of two isozymes of rabb i t ca lc ium-dependent protease. J . B i o l . Chem. 261, 9465-9471. Emori , Y . , Ohno, S . , T o b i t a , M. and Suzuki , K. (1986b) Gene s t ruc ture of calcium-dependent protease re ta ins the ancestra l organiza t ion of the ca lc ium-b ind ing prote in gene. FEBS L e t t . 194, 249-252. Enyedi , A . , F l u r a , M. , Sarkad i , B . , Gardos, G. and C a r a f o l i , E. (1987) The maximal v e l o c i t y and the calcium a f f i n i t y of the red c e l l ca lc ium pump may be regulated independently. J . B i o l . Chem. 262, 6425-6430. Enyedi , A . , Sa rkad i , B. and Gardos, G. (1982) On the substrate s p e c i f i c i t y of the red c e l l calcium pump. Biochim. Biophys. Acta 687, 109-112. Enyedi , A . , S a r k a d i , B . , Szasz , B . , Bot. G. and Gardos, _G. (1980) Molecular proper t ies of red c e l l calcium pump. C e l l Calcium 1, 291-310. E y l a r , E . H . , B r o s t o f f , S . , Hashim, G . , Caccam, J . and Burnet t , P. (1971) Basic A l prote in of the myelin membrane. The complete amino acid sequence. J . B i o l . Chem. 246, 5770-5784. Farber , J . L . (1981) The r o l e of calcium in c e l l death. L i f e S c i . 29, 1289-1295. F e r r e i r a , H . G . , Lew, V . L . (1976) Use of ionophore A23187 to measure cytoplasmic Ca buf fe r ing and a c t i v a t i o n of the Ca pump by in te rna l Ca. Nature 259, 47-49. F i n l e y , D. and Varshavsky, A. (1985) The u b i q u i t i n system: funct ions and mechanism. Trends Biochem. S c i . 10, 343-349. Foder, B. and S c h a r f f , 0. (1981) Decrease of apparent calmodulin a f f i n i t y of ery throcyte (Ca z + +Mg 2 + ) -ATPase at low C a 2 + concent ra t ions . Biochim. Biophys. Acta 649, 367-376. Foyt , H . L . , G u e r r i e r o , V. J r . and Means, A .R . (1985) Funct ional domains of chicken g i zza rd myosin l i g h t chain k inase. J . B i o l . Chem. 260, 7765-7774. F u j i i , J . , Veno, A . , K i tano , K . , Tanalea, S . , Kadoma, M. and Tada, M. (1987) Complete complementary DNA-derived amino ac id sequence of canine card iac phospholamban. J . C l i n . Invest . 79, 301-304. Fukami, Y . , Nakamura, T . , Nakayama, A. and Kanehisa, T. (1986) Phosphorylat ion of ty ros ine residues of calmodulin in Rous sarcoma v i rus- t ransformed c e l l s . Proc. N a t l . Acad. S c i . , USA 83, 4190-4193. 253 Furukawa, K. I . and Nakamura. H. (1987) C y c l i c GMP regu la t ion of the plasma membrane (Ca -Mg z + ) -ATPase in vascular smooth muscle. J . Biochem. 101, 287-290. Furukawa, K . I . Tawada, Y . , Shigekawa, M. (1988) Regulat ion of the plasma membrane C a 2 + pump by c y c l i c nucleot ides in cu l tu red vascular smooth muscle c e l l s . J . B i o l . Chem. 263, 8058-8065. Gagnon, C , K e l l y , S . , Manganiel lo , V . , Vaughn, M. , Odya, C. and S t r i t tmather , W. (1981) Mod i f i ca t ion of calmodulin funct ion by enzymatic carboxyl methylat ion. Nature 29_I, 515-516. Gardner, K. and Bennett, V. (1986) A new erythrocyte membrane-associated prote in with calmodulin binding a c t i v i t y . J . B i o l . Chem. 261, 1339-1348. Garrahan, P . J . and Rega, A . F . (1978) A c t i v a t i o n of the p a r t i a l reac t ions of the Ca -ATPase from human red c e l l s by M g 2 + and ATP_. Biochim. Biophys. Acta 513, 59-65. Gassner, B . , Luterbacher , S . , Schatzmann, H . J . and Wuthrich, A . (1988) Dependence of the red blood c e l l calc ium pump on the membrane p o t e n t i a l . C e l l Calcium 9, 95-103. Gates, R . E . and K ing , L . E . J r . (1983) P r o t e o l y s i s of the epidermal growth f a c t o r receptor by endogenous ca lc ium-ac t iva ted neutral protease from ra t l i v e r . Biochem. Biophys. Res. Commun. 113, 255-261. G e i s l e r , N. and Weber, K. (1982) The amino ac id sequence of chicken muscle desmin provides a common s t ruc tu ra l model f o r intermediate f i lament p r o t e i n s . EMB0 J . 1, 1649-1656. Ghosh, S . , Church, J . G . , Roufoga l is , B.D. and V i l l a l o b o , A . (1988) Phosphorylat ion of l i v e r plasma membrane-bound ca lmodul in . Biochem. C e l l B i o l . 66, 922-927. G ie tzen , K . , S e i l e r , S . , F l e i s c h e r , S. and Wolf, H.W. (1980) Reconst i tu t ion of the C a 2 + t ransport system of human e ry th rocy tes . Biochem. J . 188, 47-54. Gilman, A . G . (1987) G p r o t e i n s . Transducers of receptor-generated s i g n a l s . Ann. Rev. Biochem. 56, 615-649. Gimble, J . M . , Waisman, D.M. , G u s t i n , M. , Goodman, D .B .P . and Rasmussen, H. (1982) Studies of the C a 2 t ransport mechanism of human erythrocyte i n s i d e - o u t membrane v e s i c l e s . Evidence of the development of a p o s i t i v e i n t e r i o r membrane p o t e n t i a l . J . B i o l . Chem. 257, 10781-10788. G l a s e r , T. and Kosower, N.S. (1986) C a l p a i n - c a l p a s t a t i n and f u s i o n . FEBS l e t t . 206, 115-120. 254 Glenney, J . R . and Weber, K. (1980) Calmodul in-binding prote ins of the microf i laments present in i s o l a t e d brush borders and m i c r o v i l l i of i n t e s t i n a l e p i t h e l i a l c e l l s . J . B i o l . Chem. 255, 10551-10554. G o l d s t e i n , D. (1979) C a l c u l a t i o n of the concentrat ion of the f ree ca t ions and cat ion-1 igand complexes in so lu t ions conta in ing mul t ip le d i v a l e n t - c a t i o n s and l i g a n d s . Biophys. J . 26, 235-242. G o l l , D . E . , Shannon, J . D . , Edmunds, T . , Sathe, S . K . , K leese , W.C. and Naga in is , P.A. (1983) Proper t ies and regu la t ion o f the Ca -dependent prote inase In: Calc ium-Binding Prote ins 1983 (de Bernad, B . , Sco t tocasa , G . L . , Sandr i , G . , C a r a f o l i , E . , Tayor , A . N . , Vanaman, T . C . and Wi l l i ams, R . J . P . , eds . ) pp. 19-35. E l s e v i e r Sc ience , Amsterdam. Gopalakr ishna, R. and Head, J . F . (1985) Rapid p u r i f i c a t i o n o f ca lc ium-ac t iva ted protease by calcium-dependent hydrophob ic - in te rac t ion chromatography. FEBS l e t t . 186, 246-250. Gopalakr ishna, R. and Barsky, S . H . (1986) Hydrophobic a s s o c i a t i o n of ca lpa ins with s u b c e l l u l a r o r g a n e l l e s . J . B i o l . Chem. 261, 13936-13942. Gopinath, R.M. and V i n c e n z i , F . F . (1977) Phosphodiesterase pro te in a c t i v a t o r mimics red blood c e l l cytoplasmic a c t i v a t o r of (Ca -M g z + ) - A T P a s e . Biochem. Biophys. Res. Commun. 77, 1203-1209. G o r i n , M .B . , Yancey, S . B . , C l i n e , J . , Revel , J . - P . and Horwitz, J . (1984) The major i n t r i n s i c pro te in (MIP) of the bovine lens f i b e r membrane: c h a r a c t e r i z a t i o n and s t ructure based on cDNA c l o n i n g . C e l l 39, 49-59. Graf , E. and Penniston, J . I . (1981) Ca ATP: The subs t ra te , at low ATP concentrat ion o f C a - A T P a s e from human erythrocyte membranes. J . B i o l . Chem. 256, 1587-1592. Graf , E . , Verma, A . K . , G o r s k i , J . P . , Lopaschuk, G . , N i g g l i , V . , Z u r i n i , M. C a r a f o l i , E. and Penniston, J . T . (1982) Molecular proper t ies of ca lc ium pumping ATPase from human e ry th rocy tes . Biochemistry 21, 4511-4516. Grand, R . J . A . and Perry , S .V . (1980) The binding of calmodulin to myelin bas ic prote in and histone H2B. Biochem. J . 189, 227-240. Green, S . , Walter , P . , Kumar, V . , Krus t , A . , Bornert , J . - M . , Argos, P. and Chambon, P. (1986) Human oestrogen receptor cDNA: sequence, expression and homology to v - e r b - A . Nature 320, 134-140. Gronemeyer, H . , Turco t te , B . , Q u i r i n - S t r i c k e r , C , Bocquel , M.T. , Meyer, M . E . , Krozowski, Z . , J e l t s c h , J . M . , Lerouge, T . , G a m i e r , J . M . , Lerouge, T . , G a m i e r , J . M . and Chambon, P. (1987) The chicken progesterone receptor : sequence, expression and funct iona l 255 a n a l y s i s . EMBO J . 6, 3985-3994. Guer r i e ro , V. J r . , Russo, M.A. , O lson , N . J . , Putkey, J . A . and Means, A .R . (1986) Domain organiza t ion of chicken g izzard myosin l i g h t chain kinase deduced from a cloned cDNA. Biochemistry 25, 8372-8381. Gurof f , G. (1964) A neutral ca lc ium-ac t iva ted proteinase from the soluble f r a c t i o n of rat b r a i n . J . B i o l . Chem. 239, 149-155. Haaker, H. and Racker, E. (1979) P u r i f i c a t i o n and r e c o n s t i t u t i o n of the Ca -ATPase from plasma membranes of p ig e ry throcy tes . J . B i o l . Chem. 254, 6598-6602. Hale , C . C . , S laughter , R . S . , Ahrens, D.C. and Reeves, J . P . (1984) I d e n t i f i c a t i o n and p a r t i a l p u r i f i c a t i o n of the card iac sodium-calc ium exchange p r o t e i n . Proc. N a t l . Acad S c i . USA 81, 6569-6573. H a l l , T . G . and Bennett, V. (1987) Regulatory domains of erythrocyte ankyr in . J . B i o l . Chem. 262, 10537-10545. H a l l , J . L . , Dudley, L . , Dobner, P . R . , Lewis, S . A . and Cowan, N . J . (1983) I d e n t i f i c a t i o n o f two human ^ - t u b u l i n i s o t y p e s . Mol . C e l l . B i o l . 3, 854-862. Hamon, M. and Bourgoin, S. (1979) Charac te r i za t ion of the C a 2 + - i n d u c e d p r o t e o l y t i c a c t i v a t i o n o f tryptophan hydroxylase from the rat bra in system. J . Neurochem. 32, 1837-1844. Haraguchi , K . , Akasu, F . , Endo, T. and Onaya, T. (1987) Demonstration of calcium-dependent proteases (ca lpa ins) and thyrog lobu l in p r o t e o l y s i s in hog thyro id c y t o s o l . Endocr ino logica Japonica 34, 1-8. H a r r i s , A . S . and Morrow, J . S . (1988) P r o t e o l y t i c processing of human brain alpha spec t r in ( f o d r i n ) : i d e n t i f i c a t i o n of a hypersens i t ive s i t e . J . Neuroscience 8, 2640-2651. H a r r i s , A . S . , C r o a l l , D .E . and Morrow, J . S . (1988) The calmodul in-b inding s i t e in a - f o d r i n i s near the calcium-dependent protease- I cleavage s i t e . J . B i o l . Chem. 263, 15754-15761. Hathaway, D .R. , A d e l s t e i n , R.S. and K lee , C . B . (1981) Interact ion of calmodulin with myosin l i g h t chain kinase and cAMP-dependent prote in kinase in bovine b r a i n . J . B i o l . Chem. 256, 8183-8189. Hayashi , M. , Inomata, M., Nakamura, M. , Imahori, K. and Kawashima, S. (1985) Hydro lys is of protamine by ca lc ium-ac t iva ted neutral protease. J . Biochem. 97, 1363-1370. Hebbel, R . P . , Shalev, 0 „ , Fokar, W. and Rank, B.H. (1986) Inh ib i t ion of erythrocyte Ca -ATPase by ac t iva ted oxygen through t h i o l and 256 l i p i d dependent mechanism. Biochim. Biophys. Acta 862, 8-16. Hincke, M.T. and T o l n a i , S. (1986) Phosphorylat ion of bovine card iac ca lc ium-ac t iva ted neutral protease by prote in k inase -C . Biochem. Biophys. Res. Commun. 137, 559-565. H i raga , A. and T s u i k i , S. (19861 A c t i v a t i o n of a D-form of rabbi t muscle glycogen synthase by Ca - a c t i v a t e d protease. FEBS l e t t . 205, 1-H i r a o , T . and Takahashi , K. (1984) P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a ca lc ium-ac t iva ted neutral protease from monkey bra in and i t s ac t ion on neuropeptides. J . Biochem. 96, 775-784. Hodgkin, A . L . and Keynes, R.D. (1957) Movements of l a b e l l e d calcium in squid g iant axons. J . P h y s i o l . 138, 253-281. Hoffman, F . , Nasta i rczyk , W., Rohrkasten, A . , Schneider , T . and ,S ieber , M. (1987) Regulation of the L-type calc ium channel . Trends Pharmacol. S c i . 8, 393-398. Huston, R.B. and Krebs, E .G . (1968) A c t i v a t i o n of ske le ta l muscle phosphorylase kinase by Ca . II. I d e n t i f i c a t i o n of the kinase a c t i v a t i n g f a c t o r as a p r o t e o l y t i c enzyme. Biochemistry 7, 2116-2121. Hymel, L . , Inu i , M., F l e i s c h e r , S. and S c h i n d l e r , H. (1988) P u r i f i e d ryanodine receptor of ske le ta l muscle sarcoplasmic . re t iculum forms Ca - a c t i v a t e d o l igomer ic C a z + channels in planar b i l a y e r . Proc. N a t l . Acad. S c i . USA 85, 441-445. Imagawa, T . , Smith, J . S . , Coronado, R. and Campbell , K .P . (1987) P u r i f i e d ryanodine receptor from ske le ta l muscle sarcoplasmic ret iculum i s the Ca -permeable pore of the calcium re lease channel . J . B i o l . Chem. 262, 16636-16643. Imahori, K. (1985) Calcium-dependent neutral protease: i t s c h a r a c t e r i z a t i o n and regu la t ion In : Calcium and Ce l l Functions (Cheung, W.Y. , ed.) V o l . I l l , pp. 473-485, Academic Press , New York. Imajoh, S. and Suzuk i , K. (1985) Revers ib le i n t e r a c t i o n between C a 2 + -ac t iva ted neutral protease (CANP) and i t s endogenous i n h i b i t o r . FEBS l e t t . 187, 47-50. Imajoh, S . , Kawasaki, H. and Suzuki , K. (1986) The amino-terminal hydrophobic region of the small subunit of ca lc ium-ac t iva ted neutral protease (CANP) i s essen t ia l fo r i t s a c t i v a t i o n by phosphatidyl i n o s i t o l . J . Biochem. 99, 1281-1284. Inomata, M. , K a s a i , Y . , Nakamura, M. and Kawashima, S. (1988) A c t i v a t i o n mechanism of ca lc ium-ac t iva ted neutral protease. J . B i o l . Chem. 257 263, 19783-19787. Inoue, H . , Noguchi, T . and Tanaka, T . (1986) Complete amino ac id sequence of rat L-type pyruvate kinase deduced from the cDNA sequence. Eur. J . Biochem. 154, 465-469. Inu i , M. , S a i t o , A. and F l e i s c h e r , S. (1987a) P u r i f i c a t i o n of the ryanodine receptor and i d e n t i t y with fee t s t ruc tures of junc t iona l c is te rnae of sarcoplasmic re t icu lum from fas t ske le ta l muscle. J . B i o l . Chem. 262, 1740-1747. Inu i , M. , S a i t o , A. and F l e i s c h e r , S. (1987b) I s o l a t i o n of the ryanodine receptor from card iac sarcoplasmic re t icu lum and i d e n t i f y with the feet s t r u c t u r e s . J . B i o l . Chem. 262, 15637-15642. I s h i u r a , S . , S u g i t a , H . , Suzuki , K. Y. and Imahori, K. (1979) Studies of a ca lc ium-ac t iva ted neutral protease from chicken ske le ta l muscle. J . Biochem. 86, 579-581. I to , M. , Tanaka, T . , Nunoki, K . , Hidaka, H. and Suzuk i , IC (1987) The Ca - a c t i v a t e d protease (ca lpain) modulates Ca z + / c 3 l m o d u l in dependent a c t i v i t y of smooth muscle myosin l i g h t chain k inase. Biochem. Biophys. Res. Commun. 145, 1321-1328. Iwai, K . , Hayashi , H. and Ishikawa, K. (1972) C a l f thymus l y s i n e - and s e r i n e - r i c h histone III. Complete amino ac id sequence and i t s imp l i ca t ion f o r i n t e r a c t i o n s of h is tones with DNA. J . Biochem. 72, 357-367. Iwai, K . , Ishikawa, K. and Hayashi , H. (1970) Amino ac id sequence of s l i g h t l y l y s i n e - r i c h h is tone . Nature 226, 1056-1058. James, P . , Maeda, M. , F i s c h e r , R., Verma, A . , Krebs, J . , Penniston, J . T . and C a r a f o l i , E. (1988) I d e n t i f i c a t i o n and primary s t ruc ture of a calmodulin binding domain of the C a 2 + pump of human ery throcy tes . J . B i o l . Chem. 263, 2905-2910. James, P . , Vorherr , T . , Krebs, J . , M o r e l l i , A . , C a s t e l l o , G . , McCormick, D . J . , Penniston, J . T . , De F l o r a , A. and C a r a f o l i , E. (1989) Modulation of erythrocyte C a - A T P a s e by s e l e c t i v e ca lpa in cleavage of the ca lmodul in-b inding domain. J . B i o l . Chem. 264, 8289-8296. J a r r e t t , H.W. and Penniston, J . T . (1977a) P u r i f i c a t i o n of a C a 2 + - A T P a s e a c t i v a t o r from human erythrocyte membranes. Fed. Proc. 36, 642. J a r r e t t , H.W. and Penniston, J . T . (1977b) P a r t i a l p u r i f i c a t i o n of the Ca - M g z + ATPase a c t i v a t o r from human e ry throcy tes : Its s i m i l a r i t y to the a c t i v a t o r of 3 ' - 5 ' - c y c l i c nucleot ide phosphodiesterase Biochem. Biophys. Res. Commun. 77, 1210-1216. J e f f e r s o n , A . B . and Schulman, H. (1988) Sphingosine i n h i b i t s calmodul in-258 dependent enzymes. J . B i o l . Chem. 263, 15241-15244. Jelsema, C . L . and Axe l rod , J . (1987) St imulat ion of phospholipase Ap a c t i v i t y in bovine rod outer segments by By subunits of t ransducin and i t s i n h i b i t i o n by the a: subuni t . Proc . N a t l . Acad. S c i . USA 84, 3623-3627. Jenny, R . J . , Pit tman, D .D . , T o o l e , J . J . , K r i z , R.W., Aldape, R . A . , Hewick, R .M. , Kaufman, R . J . and Mann, K.G. (1987) Complete cDNA and der ived amino ac id sequence of human f a c t o r V. Proc. N a t l . Acad. S c i . USA 84, 4846-4850. Johanson, R . A . , Hansen, C A . and Wi l l iamson, J . R . (1988) P u r i f i c a t i o n of D-myo- inosi to l 1 ,4 ,5 - t r isphosphate 3-k inase from ra t b r a i n . J . B i o l . Chem. 263, 7465-7471. J u l i e n , J . - P . Grosve ld , F . , Yazdanbaksh, K . , F l a v e l l , D . , M e i j e r , D. and Mushynski, W. (1987) The s t ructure o f a human neurofi lament gene (NF-L) : a unique exon- intron organiza t ion in the intermediate f i lament gene fami ly . Biochim. Biophys. Acta 909, 10-20. Kagawa, Y. and Racker, E. (1971) Par t i a l r e s o l u t i o n of the enzyme c a t a l y z i n g ox ida t ive phosphory lat ion. J . B i o l . Chem. 246, 5477-5487. K a k i u c h i , S . and Yamazaki, R. (1970) Calcium dependent phosphodiesterase a c t i v i t y and i t s a c t i v a t i n g f a c t o r (PAF) from b r a i n . Biochem. Biophys. Res. Commun. 41, 1104-1110. Katz , S. and B l o s t e i n , R. (1975) Calcium st imulated membrane phosphorylat ion of ATPase a c t i v i t y of the human e ru throcy te . Biochim. Biophys. Acta 389, 314-324. Katz , S. and Remtul la , M.A. (1978) Phosphodiesterase p ro te in a c t i v a t o r s t imulates calc ium transport in card iac microsomal preparat ions enr iched in sarcoplasmic re t icu lum. Biochem. Biophys. Res. Commun. 83, 1373-1379. Kel lermann, J . , L o t t s p e i c h , F . , Henschen, A. and M u l l e r - E s t e r l , W. (1986) Completion of the primary s t ructure of human high-molecular-mass k in inogen. Eur. J . Biochem. 154, 471-478. Kennedy, M . B . , Bennett , M.K. , Erondu, N .E . and M i l l e r , S . G . (1987) Calcium/calmodulin-dependent prote in kinases i n : Calcium and C e l l Functions (Cheung, W.Y., ed.) V o l . VI I , pp. 61-107, Academic P r e s s , New York. Kenne l ly , P . J . , Tak io , K . , Blumenthal, D .K . , Edelman, A . M . , Glaccum, M.B . , K l e v i t , R . E . , Roush, C . L . , Sco t t , J . O . , T a k i o , K . , T i t a n i , K . , Walsh, K.A. and Krebs, E .G . (1987) Organizat ion of myosin l i g h t chain kinase from rabbi t ske le ta l muscle i n : Calcium Binding Prote ins in Health and Diseases (Means, A . R . , ed.) pp. 494-504, 259 Academic Press , New York. Kikkawa, U . , O g i t a , K . , Ono, Y . , Asaoka, Y . , Shearman, M .S . , F u j i i , T . , Ase, K . , Sek iguch i , K . , Igarashi , K. and N ish izuka , Y. (1987) The common s t ruc ture and a c t i v i t i e s of subspecies of ra t bra in pro te in kinase C fami ly . FEBS l e t t . 223, 212-216. K i n c a i d , R., S t i th -Coleman, I .E . and Vaughan, M. (1985) P r o t e o l y t i c a c t i v a t i o n of calmodulin-dependent c y c l i c nuc leot ide phosphodiesterase. J . B i o l . Chem. 260, 9009-9015. K i rchberger , M.A. , Tada, M. and Katz , A.M. (1974) Adenosine 3 ' : 5 ' -monophosphate-dependent prote in k inase-ca ta lyzed phosphorylat ion reac t ion and i t s r e l a t i o n s h i p to calcium t ranspor t in card iac sarcoplasmic re t i cu lum. J . B i o l . Chem. 249, 6166-6173. Kishimoto, A . , Kajikawa, N . , S h i o t a , M. and N ish izuka , Y. (1983) P r o t e o l y t i c a c t i v a t i o n of c a l c i u m - a c t i v a t e d , phospho l ip id -dependent prote in kinase by calcium-dependent neutral protease. J . B i o l . Chem. 258, 1156-1164. Kishimoto, A . , Mikawa, K . , Hashimoto, K. , Yasudo, I., Tanaka, S . , Tominaga, M. , Kuroda, T . and Nish izuka , Y. (1989) Limited p r o t e o l y s i s of pro te in kinase C subspecies by ca lc ium dependent neutral protease (ca lpa in) J . B i o l . Chem. 264, 4088-4092. K laerke , D . A . , Peterson, J . and Jorgensen, P .L . (1987) P u r i f i c a t i o n of Ca - a c t i v a t e d K + channel prote in on calmodulin a f f i n i t y columns a f t e r detergent s o l u b i l i z a t i o n of luminal membranes from outer renal medul la . FEBS l e t t . 216, 211-216. K lee , C . B . (1977) Conformational t r a n s i t i o n accompanying the binding of Ca to the prote in a c t i v a t o r of 3 ' . 5 ' - c y c l i c adenosine monophosphate phosphodiesterase. Biochemistry 16, 1017-1026. K lee , C . B . (1988) Ca 2 + -dependent phosphol ip id - (and membrane-) binding p r o t e i n s . Biochemistry 27, 6645-6653. K lee , C . B . and Vanaman, T . C . (1982) Calmodulin. Adv. Prote in Chem. 357, 213-321. K l i n g e r , R., Wetzker, R . . F l e i s c h e r , I. and Frunder, H. (1980) E f f e c t of ca lmodul in , C a z + and M g z + on the (Ca z + +Mg^ + ) -ATPase of erythrocyte membranes. C e l l Calcium 1_, 229-240. Knauf, P . A . , Proverb io , F. and Hoffman, J . T . (1974) E lec t rophore t i c separat ion of d i f f e r e n t phosphoproteins a s s o c i a t i o n with C a -ATPase and Na,K-ATPase in human red c e l l ghosts . J . Gen. P h y s i o l . 63, 324-336. Konigsberg, W. and H i l l , R . J . (1962) The s t ructure of human hemoglobin. J . B i o l . Chem. 237, 3157-3162. 260 Kopi to , R.R. and Lod ish , H.F. (1985) Structure of the murine anion exchange p r o t e i n . J . C e l l . Biochem. 29, 1-17. Kosak i , G . , Tsu j inaka , T . , Kambayashi, J . , Morimoto, K. , Yamamoto, K. , Yamagami, K . , Sobue, K. and K a k i u c h i , S. (1983) S p e c i f i c cleavage of ca lmodul in -b inding prote ins by low Ca - r e q u i r i n g form of Ca - a c t i v a t e d neutral protease in human p l a t e l e t s . Biochem. Int. 6, 767-775. Kosk-Kos icka , D. S c a i l l e t , S. and Ines i , G. (1986) The p a r t i a l react ions in the c a t a l y t i c c y c l e o f the calcium-dependent adenosine t r iphosphatase p u r i f i e d from erythrocyte membranes. J . B i o l . Chem. 261, 3333-3338. Kosk-Kos icka , D. and Bzdega, T . (1988) A c t i v a t i o n of the erythrocyte C a z + -ATPase by e i t h e r s e l f - a s s o c i a t i o n or i n t e r a c t i o n with ca lmodul in . J . B i o l . Chem. 263, 18184-18189. Kosower, N . S . , G l a s e r , T. and Kosower, E.M. (1983) Membrane-mobility agent-promoted fus ion of e ry throcy tes : f u s i b i l i t y i s cor re la ted with at tack by ca lc ium-ac t iva ted cytoplasmic proteases on membrane p r o t e i n s . Proc. N a t l . Acad. S c i . USA 80, 7542-7546. K r e t s i n g e r , R.H. (1976) Calc ium-binding p r o t e i n s . Ann. Rev. Biochem. 45, 239-266. Kre ts inger R.H. and Nockolds, C . E . (1973) Carp muscle ca lc ium-b ind ing p r o t e i n . II. S t ructure determinat ion and general d e s c r i p t i o n . J . B i o l . Chem. 248, 3313-3326. Kubo, M. and S t r o t t , C A . (1988) Phosphorylat ion of calmodulin on threonine res idue(s) by cytosol prepared from adrenal cor tex . Biochem. Biophys. Res. Commun. 156, 1333-1339. Kubota, S . , Onaka, T . , Murofushi , H . , Ohsawa, N. and Takaku, F. (1986) P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of high Ca - r e q u i r i n g neutral proteases from porcine and bovine b r a i n s . Biochemistry 25, 8396-8402. Kumagai, H . , N i s h i d a , E. and Saka i , H. (1982) The i n t e r a c t i o n between calmodulin and microtubule p r o t e i n s . IV. Quant i ta t ive ana lys is of the binding between calmodulin and tubu l in dimer. J . Biochem. 91, 1329-1336. K u n i c k i , T . J . Montgomery, R.R. and Schu l lek , J . (1985) Cleavage of human von Wil lebrand f a c t o r by p l a t e l e t ca lc ium-ac t iva ted protease. Blood 65, 352-356. K u n i c k i , T . J . , Mosesson, M.W. and P idard , D. (1984) Cleavage of f ib r inogen by human p l a t e l e t ca lc ium-ac t iva ted protease. Thromb. Res. 35, 169-182. 261 Kuo, T . H . and Tsang W. (1988) Guanine nuc leo t ide - and i n o s i t o l t r iphosphate- induced i n h i b i t i o n of the C a z + pump in rat heart sarcolemmal v e s i c l e s . Biochem. Biophys. Res. Commun. 152, 1111-1116. Kuwayama, ft. (1988) The membrane potent ia l modulates the ATP-dependent C a ^ + pump of card iac sarcolemma. Biochim. Biophys. Acta 940, 295-299. La Rocca, J . N . , Rega, A . F . and Garrahan, P . J . (1981) Phosphorylat ion and dephosphorylat ion of the C a z + pump of human red c e l l s in the presence of monovalent c a t i o n s . Biochim. Biophys. Acta 645, 10-16. Laemmli, U.K. (1970) Cleavage of s t r u c t u r a l pro te ins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. L a i , F . A . , E r i c k s o n , H . P . , Rousseau, E . , L i u , A . Y . and Meissner , G. (1988) P u r i f i c a t i o n and r e c o n s t i t u t i o n o f the ca lc ium re lease channel from ske le ta l muscle. Nature 331,, 315-319. Larsen , F . L . , Katz , S . , Roufoga l i s , B.D. and Brooks, D .E . (1981) Phys io log ica l shear s t resses enhance the C a z + permeabi l i ty of human e ry throcy tes . Nature 294, 667-668. Lamers, J . M . J . , S t i n i s , H . J . and De Jonge, H.R. (1981) On the ro le of c y c l i c AMP a n i Ca^-ca lmodul in -dependent phosphorylat ion in the cont ro l of (Ca z + +Mg 2 + ) -ATPase of ca rd iac sarcolemma. FEBS l e t t . 127, 139-143. L e a v i s , P .C . and Gergely, J . (1984) Thin f i lament prote ins and th in f i lament l inked regu la t ion of ver tebrate muscle c o n t r a c t i o n . CRC c r i t . Rev. Biochem. 16, 235-305. L e c l e r c , L . , G i raud, F . , Ga lac teros , F. and Poyant, C. (1987) The ca lmodul in -s t imulated (Ca z + +Mg z + ) -ATPase in human erythrocyte membranes: e f f e c t s of s i c k l i n g and ox ida t i ve agents. Biochim. Biophys. Acta 89_7, 33-40. Lee, G . , Cowan, N. and K i rschner , M. (1988) The primary s t ructure and heterogeneity of tau prote in from mouse b r a i n . Science 239, 285-292. Lee, K . S . and Au, K .S . (1983) A prote in i n h i b i t o r of erythrocyte membrane (Ca z + +Mg z + ) -ATPase . Biochim. Biophys. Acta 742, 54-62. Lee, Y . C . and Wolf f , J . (1984) Calmodulin binds to both microtubule-associa ted prote in 2 and tau p r o t e i n s . J . B i o l . Chem. 259, 1226-1230. Lee, K.W. and Sh in , B .C . (1969) Studies on the ac t ive t ransport of calcium 262 of human red c e l l s . J . Gen. P h y s i o l . 54> 713-729. Lees, J . F . , Schneidman, P . S . , Skuntz, S . F . , Carden, M . J . and L a z z a r i n i , R.A. (1988) The s t ructure and organiza t ion of the human heavy neurofi lament subunit (NF-H) and the gene encoding i t . EMBO J . 7, 1947-1955. Lemischka, I .R., Farmer, S . , R a c a n i e l l o , V .R. and Sharp, P.A. (1981) Nucleot ide sequence and evo lu t ion of a mammalian a - tubu l in messenger RNA. J . Mol . B i o l . 151, 101-120. LePeuch, C . J . and Demai l le , H . J . (1979) Concerted regu la t ion of card iac sarcoplasmic re t icu lum calcium t ranspor t by c y c l i c adenosine monophosphate dependent and calcium-calmodulin-dependent phosphory la t ion . Biochemistry 18, 5150-5157. Lev ine , H. I l l and Sahyoun, N.E. (1987) Charac te r i z a t ion o f a so lub le M r -30000 c a t a l y t i c fragment of the neuronal calmodulin-dependent prote in kinase II. Eur. J . Biochem. 168, 481-486. L e v i t z k i , A. (1987) Regulat ion of adenylate cyc lase by hormones and G-p r o t e i n s . FEBS l e t t . 2 H , 113-118. L i c h t n e r , R. and Wolf. H.V ? (1980) Phosphorylat ion of the i s o l a t e d h igh-a f f i n i t y (Ca z + +Mg 2 + ) -ATPase of the human erythrocyte membrane. Biochim. Biophys. Acta 598, 472-485. L i n , C . R . , K a p i l o f f , M .S . , Durger ian, S . , Tatemoto, K . , Russo, A . F . , Hanson, P . , Schulman, H. and Rosenfe ld , M.G. (1987) Molecular c lon ing of a b r a i n - s p e c i f i c calcium/calmodul in-dependent prote in k inase. Proc. N a t l . Acad. S c i . USA 84, 5962-5966. L i n , S . - H . (1985a) Novel ATP-dependent calc ium t ranspor t component from rat l i v e r plasma membranes. The t ranspor te r and prev ious ly reported (Ca -Mg z + ) -ATPase are d i f f e r e n t p r o t e i n s . J . B i o l . Chem. 260, 7850-7856. L i n , S . - H . (1985b) The ra t l i v e r plasma membrane high a f f i n i t y ( C a 2 + -M g ) - A T P a s e i s not a calcium pump. Comparison with ATP-dependent calc ium t ranspor te r . J . B i o l . Chem. 260, 10976-10980. L i n , S . - H . and F a i n , J . N . (1984) P u r i f i c a t i o n of ( C a 2 + - M g 2 + ) - A T P a s e from rat l i v e r plasma membranes. J . B i o l . Chem. 259, 3016-3020. L i n , S . - H . and R u s s e l l , W.E. (1988) Two Ca 2 + -dependent ATPases in rat l i v e r plasma membrane. The prev ious ly p u r i f i e d ( C a - M g ) - A T P a s e i s not a C a - p u m p but an ecto-ATPase. J . B i o l . Chem. 263, 12253-12258. Liscum, L . , Cummings, R .D . , Anderson, R.G.W., DeMartino, G . N . , G o l d s t e i n , J . L . and Brown, M.S. (1983) 3-hydroxy-3-methylg lutary l -CoA reductase: A transmembrane g lycopro te in of the endoplasmic 263 re t icu lum with N- l inked "high-mannose" o l i g o s a c c h a r i d e s . Proc. N a t l . Acad. S c i . USA 80, 7165-7169. Liscum, L . , F iner-Moore, J . , St roud, R.M. , Luskey, K . L . , Brown, M.S. and G o l d s t e i n , J . L . (1985) Domain s t ruc tu re of 3-hydroxy-3-methy lg lu l ta ry l coenzyme A reductase , a g l y c o p r o t e i n of the endoplasmic re t icu lum. J . B i o l . Chem. 260, 522-530. Long, C. and Mouat, B. (1973) The binding of ca lc ium ions by erythrocytes and ' g h o s t ' - c e l l membranes. Biochem. J . 123, 829-836. L o t e r s z t a j n , S . , Epand, R.M. , M a l l a t , A . and Pecker, F. (1984) Inh ib i t ion by glucagon of the calcium pump in l i v e r plasma membranes. J . B i o l . Chem. 259, 8195-8201. L o t e r s z t a j n , S . , Hanoune, J . and Pecker, F. (1981) A high a f f i n i t y ca lc ium-st imula ted magnesium-dependent ATPase in ra t l i v e r plasma membranes. Evidence on an endogenous p ro te in a c t i v a t o r d i s t i n c t from calmodul in . J . B i o l . Chem. 256, 11209-11215. L o t e r s z t a j n , S . , M a l l a t , A . , Pavo l ine , C. and Pecker, F. (1985) The i n h i b i t o r of l i v e r plasma membrane (Ca - M g z + ) - A T P a s e . J . B i o l . Chem. 260, 9692-9698. L o t e r s z t a j n , S . , Povoi re , C , M a l l a t , A . , S t e n z e l , D . , I n s e l , P.A. and Pecker, F. (1987) Cholera Toxin Blocks Glucagon-mediated i n h i b i t i o n of the l i v e r plasma membrane (Ca - M g z + ) - A T P a s e . J . B i o l . Chem. 262, 3114-3117. Low, M . G . , C a r r o l l , R.C. and W e g l i c k i , W.B. (1984) M u l t i p l e forms of p h o s p h o i n o s i t i d e - s p e c i f i c phospholipase C o f d i f f e r e n t r e l a t i v e molecular masses in animal t issues. Evidence f o r modi f ica t ion o f the p l a t e l e t enzyme by Ca z + -dependent p r o t e i n a s e . Biochem. J . 221, 813-820. Lowry, O . H . , Rosebrough, N . J . , F a r r , A .L . and R a n d a l l , R . J . (1951) Prote in measurement with f o l i n phenol reagent . J . B i o l . Chem. 193, 265-275. Lukas, T . J . , Burgers, W.H. , Prendergast, F . G . , Lau, W. and Watterson, D.M. (1986) Calmodulin binding domains: C h a r a c t e r i z a t i o n of a phosphorylat ion and calmodulin b inding s i t e from myosin l i g h t chain k inase. Biochemistry 25, 1458-1464. Luthra , M .G . , Au, K .S . and Hanahan, D . J . (19771 P u r i f i c a t i o n of an a c t i v a t o r of human erythrocyte membrane (Ca^ + +Mg z + ) -ATPase, Arch . Biochem. Biophy. 77, 678-687. Lynch, C . G . , Sobo, G . E . and Exton, J . H . (1986) An endogenous C a 2 + -s e n s i t i v e proteinase converts the hepat ic a j - a d r e n e r g i c receptor to guanine n u c l e o t i d e - i n s e n s i t i v e forms. Biochim. Biophys. Acta 885, 110-120. 264 Lynch, G. and Baudry, M. (1984) The biochemistry of memory: a new and s p e c i f i c hypothesis . Science 224, 1057-1063. Lynch, T . J . and Cheung, W.Y. (1979) Human erythrocyte C a 2 + - M g 2 + - A T P a s e : mechanism of s t imula t ion by C a 2 + . A r c h . Biochem. Biophys. 194, 165-170. MacLennan, D .H . , Brandl , C . J . , Korczak, B. and Green, M.N. (1985) Amino acid sequence of a Ca 2 ++Mg -dependent ATPase from rabbi t muscle sarcoplasmic re t icu lum, deduced from i t s complementary DNA sequence. Nature 316, 696-700. MacLennan, D.H. and Wong, P . T . S . (1971) I so la t ion of a ca lc ium-sequester ing prote in from sarcoplasmic re t i cu lum. Proc. N a t l . Acad. S c i . USA 68, 1231-1235. MacLennen, D.H. and Reithmeier , R . A . F . (1985) Structure of c a l s e q u e s t r i n . i n : St ructure and funct ion of sarcoplasmic ret iculum ( F l e i s c h e r , S. and Tonomura, Y . , eds . ) pp. 99-100, Academic Press , Or land, FL . Mak, A . S . , S m i l l i e , L . B . and Stewart, G.R. (1980) A comparison of the amino ac id sequences of rabb i t ske le ta l muscle a- and B-tropomyosins. J . B i o l . Chem. 255, 3647-3655. Maki, M. , Takano, E . , Mor i , H . , Sato, A . , Murachi , T. and Hatanaka, M. (1987) A l l four i n t e r n a l l y r e p e t i t i v e domain of p ig c a l p a s t a t i n possess i n h i b i t o r y a c t i v i t i e s against c a l p a i n I and II. FEBS l e t t . 223, 174-180. Mal ik , M.N. , Fenko, M.D., Iqba l , K. and Wisniewski , H.M. (1983) P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of two forms o f Ca - a c t i v a t e d neutral protease from c a l f b r a i n . J . B i o l . Chem. 258, 8955-8962. M a l l a t , A . , Pavoine, C , Dufour, M. , L o t e r s z t a j n , S . , B a t a i l l e , D. and Pecker, F. (1987) A glucagon fragment i s respons ib le f o r the i n h i b i t i o n of the l i v e r C a ^ + pump by g lucagon. Nature 325, 620-622. Manalan, A . S . and K lee , C . B . (1983) A c t i v a t i o n of c a l c i n e u r i n by l i m i t e d p r o t e o l y s i s . Proc. N a t l . Acad. S c i . USA 80, 4291-4295. Manalan, A . S . and Klee , C . B . (1984) Calmodulin In : Advances in C y c l i c Nucleot ide and Protein phosphorylat ion Research (Greengard, P. and Robison, G . A . , eds. ) V o l . 18, pp. 227-278, Raven Press , New York. M a r t e H , A. and Smith R. (1974-1982) C r i t i c a l S t a b i l i t y Constants , V o l s . 1-5, Plenum Press , New York. Mauldin, D. and Roufoga l is , B.D. (1980) A pro te in a c t i v a t o r of M g 2 + -265 dependent Ca' 1 -s t imulated ATPase in human erythrocyte membranes d i s t i n c t from calmodul in . Biochem. J . 187, 501-513. Mayr, G.W. and Heilmeyer, L .M.G. J r . (1983) Phosphofructokinase is a calmodulin binding p r o t e i n . FEBS l e t t . 159, 51-57. McDonald, J . F . , Groschel -Stewart , U. and Walsh, M.P. (1987) Proper t ies and d i s t r i b u t i o n of the prote in i n h i b i t o r (M r 17000) of prote in kinase C. Biochem. J . 242, 695-705. McDonald, J . R . and Walsh, M.P. (1985) C a 2 + - b i n d i n g prote ins from bovine bra in inc lud ing a potent i n h i b i t o r of p ro te in kinase C. Biochem. J . 232, 559-567. McGowan, E . B . , Yeo, K-T and Detwi ler , T . C . (1983) The ac t ion of ca lc ium-dependent protease on p l a t e l e t surface g l y c o p r o t e i n s . A r c h . Biochem. Biophys. 227, 287-301. McMahon, A . P . and Moon, R.T. (1987) St ructure and evo lu t ion of a non-ery thro id s p e c t r i n , human a - f o d r i n . Biochem. Soc. Trans. 15, 804-807. Means, A . R . , Tash, J . S . and Chafouleas, J . G . (1982) Phys io log ica l Impl icat ions of the presence, d i s t r i b u t i o n , and regu la t ion of calmodulin in eukaryot ic c e l l s . P h y s i o l . Reviews 62, 1-39. Meggio, F . , B runa t i , A.M. and Pinna, L .A . (1987) Polycat ion-dependent , Ca -antagonized phosphorylat ion of calmodulin by case in kinase-2 and a spleen ty ros ine prote in k inase . FEBS l e t t . 215, 241-246. Me l lg ren , R .L . (1987) Calcium-dependent proteases: an enzyme system ac t ive at c e l l u l a r membranes? FASEB J . 1, 110-115. Me l lg ren , R . L . , Aylward, J . H . , K i l l i l e a , S .D . and Lee, E . Y . C . (1979) The a c t i v a t i o n and d i s s o c i a t i o n of a nat ive high molecular weight form of rabbjt ske le ta l muscle phosphorylase phosphatase by endogenous Ca -dependent proteases. J . B i o l . Chem. 254, 648-652. Me l lg ren , R . L . , Lane, R.D. and Kakar, S . S . (1987a) A sarcolemma-associated i n h i b i t o r i s capable of modulating calcium-dependent prote inase a c t i v i t y . Biochim. Biophys. Acta 930, 370-377. Me l lg ren , R . L . , Lane, R.D. and Kakar, S . S . (1987b) Isolated bovine myocardial sarcolemma and sarcoplasmic re t icu lum v e s i c l e s contain t i g h t l y bound calcium-dependent protease i n h i b i t o r . Biochem. Biophys. Res. Commun. 1_42, 1025-1031. Me l lg ren , R . L . , Repe t t i , A . , Muck, T . C . and E a s l y , J . (1982) Rabbit ske le ta l muscle calcium-dependent protease r e q u i r i n g m i l l i m o l a r C a z + . J . B i o l . Chem. 257, 7203-7209. 266 M e l l o n i , E . , Pontremol i , S . , M i c h e t t i , M. , Sacco, 0 . , Sparatore , B. and Horecker B . L . (1986) The involvement of c a l p a i n in the a c t i v a t i o n of prote in kinase C in neut roph i ls st imulated by phorbol m y r i s t i c a c i d . J . B i o l . Chem. 261, 4101-4105. M e l l o n i , E . , Sal ami no, F . , Sparatore , B . , M i c h e t t i , M. and Pontremol i , S. (1984) Charac te r i za t ion of the s i n g l e peptide generated from the amino-terminus end of a - and ^-hemoglobin chains by the Ca -dependent neutral p ro te inase . Biochim. Biophys. Acta 788, 11-16. M e l l o n i , E . , Sparatore B . , Salamino, F . , M i c h e t t i , M. and Pontremol i , S. (1982a) C y t o s o l i c calc ium dependent prote inase of human e ry throcy tes : formation of an enzyme-natural i n h i b i t o r complex induced by Ca . Biochem. Biophys. Res. Commun. 106, 731-740. M e l l o n i , E . , Sparatore , B . , Salamino, F . D . , M i c h e t t i , M. and Pontremol i , S. (1982b) C y t o s o l i c calc ium dependent neutral prote inase of human ery throcy tes : the r o l e o f calc ium ions on the molecular and c a t a l y t i c proper t ies of the enzyme. Biochem. Biophys. Res. Commun. 107, 1053-1059. Merc ier , J . - C , Grosclaude, F. and Ribadeau-Dumas, B. (1971) The primary s t ructure of bovine a s j c a s e i n . Eur. J . Biochem. 23, 41-51. Meyer, W . L . , F i s c h e r , E .H. and Krebs, E . G . J1964) A c t i v a t i o n of ske le ta l muscle phosphorylase b kinase by Ca . Biochemistry 3, 1033-1039. Minocherhomjee, A. and Roufoga l i s , B.D. (1982) S e l e c t i v e antagonism o f the C a z + t ranspor t ATPase of the red c e l l membrane by N- (4 -az ido-2 -n i t ropheny l ) -2 -aminoethy l -su l fona te (NAP-taurine) J . B i o l . Chem. 257. 5426-5430. Minocherhomjee, A . , A l - J o b o r e , A . and Roufoga l i s , B.D. (1982) Modulation of the ca lc ium- t ranspor t ATPase in human erythrocytes by anions. Biochim. Biophys. Acta 690, 8-14. M o l l a , A . , Capony, J . - P . and Demai l le , J . G . (1983) Regulat ion of ca rd iac sarcoplasmic re t icu lum calc ium pumping In : Calc ium-Binding Prote ins 1983 (deBernad, B . , Scot tocasa , G . L . , S a n d r i , G . , C a r a f o l i , E . , T a y l o r , A . N . , Vanaman, T . C . and Wi l l i ams , R . J . P . , eds . ) pp.393-399, E l s e v i e r Sc ience , Amsterdam. Moore, L . , Chen, T . and Knapp, H.R (1978) Energy-dependent calcium sequestrat ion a c t i v i t y in ra t l i v e r microsomes. J . B i o l . Chem. 250. 4562-4568. Moses, L. and Anandaraj , M . P . J . S . (1987) Membrane C a 2 + M g 2 + - A T P a s e a c t i v a t i o n by calcium ac t iva ted neutral protease (CANP) in condi t ions where there is e levated i n t r a c e l l u l a r Ca . an ins igh t from erythrocytes of Duchenne muscular dystrophy (DMD). Seventh Internat ional Washington Spring Symposium. C e l l Calcium Metabolism. Washington, abs. 132. 267 Muallem, S. and K a r l i s h , S . J . D . (1979) Is the red c e l l calcium pump regulated by ATP? Nature 277, 238-240. Muallem, S. and K a r l i s h , S . J . D . (1982) Regulat ion of the Ca 2 + -pump by calmodulin in in tac t c e l l s . Biochim. Biophys. Acta 687, 329-332. Muallem, S . , S c h o e f i e l d , M., Pandol, S . , Sachs, G. (1985) Inos i to l t r isphosphate modi f ica t ion of ion t ranspor t in rough endoplasmic re t icu lum. Proc. N a t l . Acad. S c i . USA 82, 4433-4437. Mue l le r , P. and Rudin, D.O. (1968) Act ion p o t e n t i a l s induced in bimolecular l i p i d membranes. Nature 217, 713-719. Murachi , T. (1983a) I n t r a c e l l u l a r C a 2 + protease and i t s i n h i b i t o r p r o t e i n : Calpain and c a l p a s t a t i n I n : Calcium and C e l l Functions (Cheung, W.Y. , ed.) V o l . IV, pp. 377-410, Academic Press , New York. Murachi , T . (1983b) Calpain and c a l p a s t a t i n . Trends Biochem. S c i . 8, 167-169. Murachi , T . (1989) I n t r a c e l l u l a r regula tory system i n v o l v i n g c a l p a i n and c a l p a s t a t i n . Biochem. Int. 18, 263-294. Murachi , T . , Tanaka, K . , Hatanaka, M. and Murakami, T . (1979) I n t r a c e l l u l a r Ca -dependent protease (ca lpa in) and i t s h igh-molecular-weight endogenous i n h i b i t o r ( ca lpas ta t in ) I n : Advance in Enzyme Regulat ion (Wiker, G . , ed.) V o l . 19, 407-423. Pergamon Press , Oxford. Murakami, K. and E t l i n g e r , J . D . (1986) Endogenous i n h i b i t o r of nonlysosomal high molecular weight protease and calcium-dependent protease Proc. N a t l . Acad. S c i . USA 83, 7588-7592. Murakami, U. and Uchida, K. (1979) Degradation of ra t c a r d i a c m y o f i b r i l s and m y o f i b r i l l a r prote ins by a myosin-c leav ing protease . J . Biochem. 86, 553-562. Murtaugh, T . J . , Rowe P .M. , V incent , P . L . , Wright, L . S . and S i e g e ! , F . L . (1983) Pos t t rans la t iona l mod i f i ca t ions of ca lmodul in . Methods Enzymol. 102, 158-170. Myers, M.W,., L a z z a r i n i , R . A . , Lee, V. M . - Y . , Sch laep fe r , W.W. and Nelson, D .L . (1987) The human mid -s i ze neurofi lament subuni t : a repeated prote in sequence and the r e l a t i o n s h i p of i t s gene to the intermediate f i lament gene fami ly . EMB0 J . 6, 1617-1626. Na i rn , A . C . , Hemmings, H.C. J r . and Greengard, P. (1985a) Prote in kinases in the b r a i n . Ann. Rev. Biochem. 54, 931-976. Na i rn , A . C . , Bhagat, B. and P a l f r e y , H.C. (1985b) I d e n t i f i c a t i o n of calmodulin-dependent prote in kinase III and i t s major M r 100,000 268 substrate in mammalian t i s s u e s . Proc. N a t l . Acad. S c i . USA 82, 7939-7943. Naray, A. (1981) The e f f e c t of a C a 2 + - a c t i v a t e d protease on the g l u c o c o r t i c o i d receptor of lymphoid t i s s u e s . J . S te ro id Biochem. 14, 71-76. Nelson, W.J . and Traub, P. (1981) Preper t ies of a C a z + - a c t i v a t e d protease s p e c i f i c fo r the in termedia te -s ized f i lament prote in vimentin in Ehr ich-asc i tes - tumour c e l l s . Eur. J . Biochem. 1_16, 51-57. Neyses, L . , R e i n l i b , L. and C a r a f o l i , E. (1985) P h o s p h o r y l a t i o n of the C a z + pumping ATPase of heart sarcolemma and erythrocyte plasma membrane by the cAMP-dependent prote in k inase . J . B i o l . Chem. 260, 10283-10287. N i c o t e r a , P . , McConkey, D. , Svenson, S . A . , Bellomo^ G. and Or ren ius , S. (1988) Cor re la t ion between c y t o s o l i c Ca concentra t ion and c y t o t o x i c i t y in hepatocytes exposed to ox ida t i ve s t r e s s . Toxicology 52, 55-63. N i g g l i , V . , Adunyah, E . S . and C a r a f o l i , E. (1981a) A c i d i c phospho l ip ids , unsaturated fa t ty acids and l i m i t e d p r o t e o l y s i s mimic the e f f e c t of calmodulin on the p u r i f i e d erythrocyte Ca -ATPase. J . B i o l . Chem. 256, 8588-8592. N i g g l i , V . , Adunyah, pE.S . * , Penniston, J . T . and C a r a f o l i , E. (1981b) P u r i f i e d (Ca z + -Mg^ + ) -ATPase of the erythrocyte membrane. Reconst i tu t ion and e f f e c t of calmodulin and phospho l ip ids . J . B i o l . Chem. 256, 395-401. N i g g l i , V . , Penn is ton , J . T . and C a r a f o l i , E. (1979b) P u r i f i c a t i o n of the (Ca z + +Mg 2 + ) -ATPase from human erythrocyte membranes using a calmodulin a f f i n i t y column. J . B i o l . Chem. 254, 9955-9958. N i g g l i , V . , Ronner, P . , C a r a f o l i , E and Penniston J . T . (1979a) E f f e c t s of calmodulin on the ( C a - M g -ATPase p a r t i a l l y p u r i f i e d from erythrocyte membrane. Arch . Biochem. Biophys. 198, 124-130. N i g g l i , V . , S i g e l , E. and C a r a f o l i , E. (1982a) Inh ib i t ion of the p u r i f i e d and reconst i tu ted calcium pump of erythrocytes by /iM concentrat ions of DIDS and NAP-taur ine. FEBS l e t t . 138, 164-166. N i g g l i , V . , S i g e l , E. and C a r a f o l i , E. (1982b) The p u r i f i e d C a 2 + pump of human erythrocyte membranes ca ta lyzes an e lec t roneut ra l Ca - H + exchange in reconst i tu ted l iposomal system. J . B i o l . Chem. 257, 2350-2356. N i g g l i , V . , Z u r i n i , M. and C a r a f o l i , E. (1987) P u r i f i c a t i o n , r e c o n s t i t u t i o n , and molecular c h a r a c t e r i z a t i o n of the C a z + pump of the plasma membranes. Methods Enzymol. 139, 791-809. 269 Nish izuka , Y. (1984) The r o l e of pro te in kinase C in c e l l surface s ignal t ransduct ion and tumor promotion. Nature 308, 693-698. O ' H a l l o r a n , T . , Becker le , M.C. and Burr idge , K. (1985) I d e n t i f i c a t i o n of t a l i n as a major cytoplasmic prote in impl icated in p l a t e l e t a c t i v a t i o n . Nature 317, 449-451. Ohno, S . , Emori , T . , Imajoh, S . , Kawasaki, H . , K i s a r a g i , M. and Suzuk i , K. (1984) Evolut ionary o r i g i n of a calcium-dependent protease by fus ion of genes f o r a t h i o l protease and a ca lc ium-binding p r o t e i n . Nature 312, 566-570. Ohno, S . , Kawasaki, H . , Imajoh, S . , Suzuk i , K . , Inagaki , M., Yokokura, H . , Sakoh, T . and Hidaka, H. (1987) T i s s u e - s p e c i f i c expression of three d i s t i n c t types of rabb i t prote in kinase C. Nature 325, 161-166. Olorunsogo, 0 . 0 . , Okudolo, B . E . . Lawal, S . O . A . and F a l a s e , A . O . (1985) Erythrocyte membrane Ca -pumping ATPase of hypertensive humans: reduced s t imula t ion by ca lmodul in . B iosc ience Reports 5, 525-531. Olorunsogo, 0 . 0 . , V i l l a l o b o , A . , Wang, K.K.W. and Roufoga l i s , B.D. (1988) The e f f e c t of calmodulin on the i n t e r a c t i o n o f carbodi imides with the p u r i f i e d human erythrocyte (Ca z + +Mg 2 + ) -ATPase . Biochim. Biophys. Acta 945, 33-40. O l s o n , I . J . and Cazor t , R . J . (1969) A c t i v e calc ium and stront ium t ranspor t in human erythrocyte ghosts . J . Gen. P h y s i o l . 53, 311-322. Ono, T . , Koide, Y . , A r a i , Y. and Yamashita, K. (1984) Heat -s tab le ca lmodul in-b inding prote in in ra t t e s t i s . J . B i o l . Chem. 259, 9011-9016. Or-lov, S.N. . . Pokudin, N.I. and Postnov, V . Y . (1983) Calmodul in-dependent C a z + t ransport in erythrocytes of spontaneously hypertensive r a t s . Pf lugers Arch . 397, 54-56. Ortega, A. and Mas-O l iva , J . (1986) D i rec t regula tory e f f e c t of cho les te ro l on the calmodulin st imulated calc ium pump of card iac sarcolemma Biochem. Biophys. Res. Commun. 139, 868-874. Panagia, V . , OJaimura, K . , Makino, N. and D h a l l a , N.S. (1986) St imulat ion of C a 2 + pump in rat heart sarcolemma by phosphatidyl-ethanolamine N-methylat ion. Biochem. Biophys. Acta 856, 383-387. Pant, H.C. (1988) Dephosphorylation of neurofi lament prote ins enhances t h e i r s u s c e p t i b i l i t y to degradation by c a l p a i n . Biochem. J . 256, 665-668. Pant, H . C , Vi rmani , M. and G a l l a n t , P . E . (1983) Calcium-induced p r o t e o l y s i s of spec t r in and band 3 prote in in rat erythrocyte 270 membranes. Biochem. Biophys. Res. Commun. 117, 372-377. Papp, B . , Sarkad i , B . , Enyedi , A . , Car ide , A . J . , Penniston, J . T . and Gardos, G. (1989) Funct ional domains of the jn s i t u red c e l l membrane calc ium pump revealed by p r o t e o l y s i s and monoclonal an t ibod ies . Poss ib le s i t e s f o r regu la t ion by c a l p a i n and a c i d i c l i p i d s . J . B i o l . Chem. 264, 4577-4582. Parker, R . A . , M i l l e r , S . J . and G i l s o n , D.M. (1986) Phosphorylat ion state of HMG CoA reductase a f f e c t s i t s c a t a l y t i c a c t i v i t y and degradat ion. Adv. Enzym. Regul. 25, 329-343. P a t e l , K. , Strong, P . N . , Dubowitz, V. and Dunn, M .J . (1988) Calmodul in-binding p r o f i l e s f o r nebul in and dystrophin in human ske le ta l muscle. FEBS l e t t . 234, 267-271. Pear ls tone , J . R . , Carpenter , M.R. and S m i l l i e , L .B . (1977a) Primary s t ruc ture of rabb i t ske le ta l muscle t r o p o n i n - T . P u r i f i c a t i o n of cyanogen bromide fragments and the amino ac id sequence of fragment CB2. J . B i o l . Chem. 252, 971-977. Pear ls tone , J . R . , Carpenter , M.R. and S m i l l i e , L .B . (1977b) Primary s t ruc ture of rabb i t ske le ta l muscle t roponin T . J . B i o l . Chem. 252, 978-982. Pedemonte, C .H . and Balegro , H .F . (1981) I s o l a t i o n of a thermostable modi f ie r o f Ca -s t imula ted ATPase from human red c e l l s Biochem. Biophys. Acta 99, 994-1000. Penniston, J . T . (1983) Plasma membrane C a 2 + - A T P a s e s as C a 2 + pumps. In: Calcium and C e l l Functions (Cheung, W.Y. , ed.) V o l . IV, pp. 99-149, Academic Press , New York. Pershadsingh, H.A. and McDonald, J . M . (1981) (Ca 2 + +Mg 2 + ) -ATPase in adipocyte plasmalemma: i n h i b i t i o n by i n s u l i n and concanaval in A in the i n t a c t c e l l . Biochem. Int. 2, 243-248. Plancke, Y .D. and Lazar ides , E. (1983) Evidence f o r a phosphorylated form of calmodulin in chicken bra in and muscle. Mol . C e l l . B i o l . 3, 1412-1420. Poland, A. and Glover , E. (1988) Ca 2 + -dependent p r o t e o l y s i s o f the Ah receptor . A r c h . Biochem. Biophys. 261, 103-111. Pontremol i , S. and M e l l o n i , E. (1986a) Extralysosomal pro te in degradat ion. Ann. Rev. Biochem. 55, 455-481. Pontremol i , S. and M e l l o n i , E. (1986b) Regulat ion of Ca 2 + -dependent proteinase of human ery throcy tes . In: Calcium and C e l l Funct ions , (Cheung, W.Y. , ed.) V o l . VI , pp. 159-183, Academic Press , New York. 271 Pontremol i , S . , M e l l o n i , E. and Horecker, B . L . (1985a) Regulat ion of mammalian c y t o s o l i c Ca - r e q u i r i n g neutral prote inases In: Current Topics in C e l l u l a r Regulat ion ( S h a l t i e l , S. and Chock, P . B . , eds . ) V o l . 27, pp. 293-304, Academic Press , New York. Pontremol i , S . , M e l l o n i , E . , M i c h e t t i , M. , Sacco, 0 . , Salamino, F . , Sparatore , B. and Horecker, B . L . (1986a) Biochemical responses in ac t iva ted human neut rophi ls mediated by pro te in kinase C and a C a z + - r e q u i r i n g pro te inase . J . B i o l . Chem. 261, 8309-8313. Pontremol i , S . , M e l l o n i , E . , M i c h e t t i , M. , Salamino, F . , Sparatore , B. and Horecker, B .L . (1988) An endogenous a c t i v a t o r o f the Ca -dependent prote inase of human neut rophi ls that increases i t s a f f i n i t y fo r C a z + . Proc. N a t l . Acad. S c i . USA 85, 1740-1743. Pontremol i , S . , M e l l o n i , E . , M i c h e t t i , M. , Sparatore , B . , Salamino F, Sacco, 0. and Horecker, B . L . (1987b) Phosphorylat ion by prote in kinase C of a 20 kDa cy toske le ta l polypept ide enhances i t s s u s c e p t i b i l i t y to d i g e s t i o n by c a l p a i n . Proc. N a t l . Acad. S c i . 84, 398-401. Pontremol i , S . , M e l l o n i , E . , Salamino, F . , Sparatore , B . , V i o t t i , P . , M i c h e t t i , M., Duzz i , L. and B i a n c h i , G. (1986b) Decreased leve l of ca lpa in i n h i b i t o r a c t i v i t y in red blood c e l l s from Milan hypertensive r a t s . Biochem. Biophys. Res. Commun. 138, 1370-1375. Pontremol i , S . , M e l l o n i , E . , Salamino, F . , Sparatore , B . , M i c h e t t i , M. , Sacco, 0. and B i a n c h i , G. (1987b) Decreased l eve l o f ca lpa in i n h i b i t o r a c t i v i t y in kidney from Milan hypertensive r a t s . Biochem. Biophys. Res. Commun. 145, 1287-1294. Pontremol i , S . , M e l l o n i , E . , Sparatore , B y M i c h e t t i , M. and Horecker, B .L . (1984) A dual r o l e f o r the Ca - r e q u i r i n g prote inase in the degradation of hemoglobin by erythrocyte membrane pro te inases . Proc. N a t l . Acad. S c i . USA 81_, 6714-6717. Pontremol i , S . , M e l l o n i , E . , Sparatore , B . , Salamino, F . , M i c h e t t i , M. , Sacco, 0. and Horecker, B . L . (1985b) Binding to erythrocyte membrane i s the p h y s i o l o g i c a l mechanism f o r a c t i v a t i o n of Ca -dependent neutral p ro te inase . Biochem. Biophys. Res. Commun. 128, 331-338. Pontremol i , S . , Sparatore, B . , Salamino, F . , M i c h e t t i , M. , Sacco, 0. and M e l l o n i , E. (1985c) Revers ib le a c t i v a t i o n of human neutrophi l ca lpa in promoted by i n t e r a c t i o n with plasma membranes. Biochem. Int . ]_1, 35-44. P r e n t k i , M. , Biden, T . J . , J a n j i c , D. , I rv ine , R . F . , Ber r idge , M .J . and Wolheim, C .B . (1984) Rapid mob i l i za t ion of Ca from rat insulinoma microsomes by i n o s i t o l - 1 , 4 , 5 - t r i s p h o s p h a t e . Nature 309, 562-564. 272 Puca, G . A . , Nola , E . , S i c a , V. and B r e s c i a n i , F. (1977) Estrogen binding prote ins of c a l f u terus. Molecular and funct iona l c h a r a c t e r i z a t i o n of the receptor transforming f a c t o r : a r e -ac t iva ted protease. J . B i o l . Chem. 252, 1358-1366. Quax-Jeuken, Y . E . F . M . , Quax, W.J . and Bloemendal, H. (1983) Primary and secondary s t ruc ture of hamster vimentin pred ic ted from the nuc leot ide sequence. Proc. N a t l . Acad. S c i . USA. 80, 3548-3552. Q u i s t , E . E . and Roufoga l i s , B.D. (1975) Calcium t ranspor t in human ery throcy te : separat ion and r e c o n s t i t u t i o n of high and low Ca a f f i n i t y (Mg z + +Ca z + ) -ATPase in membranes prepared at low i o n i c s t rength . A r c h . Biochem. Biophys. 168, 240-251. Rabbani, N . , Moses, L . , A n a n d a v a l l i , T . E . and Anandaraj , M . P . J . S . (1984) Ca lc ium-act iva ted neutral protease from muscle and p l a t e l e t s of Duchenne muscular dystrophy cases . C l i n i c a Chimica Acta 143, 163-168. Raess, B .U. and V i n c e n z i , F . F . (1980) A Semi-automated method f o r the determinat ion of mul t ip le membrane ATPase a c t i v i t i e s . J . Pharmacol. Methods 4, 273-283. Rechste iner , M. (1987a) Ubiqui t in-mediated pathways f o r i n t r a c e l l u l a r p r o t e o l y s i s . Ann. Rev. C e l l B i o l . 3, 1-30. Rechste iner , M. (1987b) Regulat ion o f enzyme l e v e l s by p r o t e o l y s i s : the r o l e of PEST r e g i o n s . Adv. Enzym. Regul . 27, 135-151. Rega, A . F . and Garrahan, P . J . (1975) Calcium ion-dependent phosphorylat ion of human erythrocyte membranes. J . Membrane. B i o l . 22, 313-327. Rega, A . F . and Garrahan, P . J . (1978) Calcium independent dephosphorylat ion o f the C a + + - A T P a s e of human red c e l l s by ADP. Biochim. Biophys. Acta 507, 182-184. Rega, A . F . and Garrahan, P . J . (1980) E f f e c t s of calmodulin on the phosphoenzyme of the Ca -ATPase of human red c e l l membranes. Biochem. Biophys. Acta 596, 487-489. Reimann, E . M . , T i t a n i , K. , E r i c s s o n , L . H . , Wade, R .D . , F i s c h e r , E .H . and Walsh, K.A. (1984) Homology of the 7 subunit of phosphorylase b kinase with cAMP-dependent prote in k inase. Biochemistry 23, 4185-4192. Reithmeier , R . A . F . , O h n i s h i , M., Carpenter , M.R., Slupsky, J . R . , Gounden, K . , F l i e g e l , L . , Khanna, V.K and MacLennan D.H. (1987) Ca lsequest r in I n : Calcium-Binding Prote ins in Health and Disease (Means, A . R . , ed. ) pp. 62-71, Academic Press , New York. 273 Ribadeau-Dumas, B . , Br ignon, G . , Grosclaude, F. and Merc ie r , J . - C . (1972) The complete primary s t ructure of / J -cae in . Eur. J . Biochem. 25, 505-514. Richards , D .E . , Rega, A . F . arid Garrahan, P . J . (1978) Two c l a s s a s of s i t e fo r ATP in the C a - A T P a s e from human red c e l l membranes. Biochim. Biophys. Acta 5_n, 194-201. R i v e t t , A . J . (1989) The m u l t i c a t a l y t i c protease of mammalian c e l l s . A r c h . Biochem. Biophys. 268, 1-8. Rixon, M.W., Chan, W . - Y . , Davie , E.W. and Chung, D.W. (1983) C h a r a c t e r i z a t i i o n of a complementary deoxyr ibonucle ic ac id coding fo r the a chain of human f i b r i n o g e n . Biochemistry 22, 3238-3244. Roelofsen, B ? and Schatzmann, H . J . (1977) The l i p i d requirement of the (Ca^ ++Mg^ +)-ATPase in the human erythrocyte membrane, as studied by var ious h igh ly p u r i f i e d phosphol ipases. Biochim. Biophys. Acta 464, 17-36. Rogers, S . , We l ls , R. and Rechste iner , M. (1986) Amino ac id sequences common to r a p i d l y degraded p r o t e i n s : the PEST hypothes is . Science 234, 364-368. Romero, P . J . and Whittam, R.(1971) The contro l by in te rna l calc ium of membrane permeabi l i ty to sodium and potassium. J . P h y s i o l . 214, 481-507. Romero, P . J . - a n d Romero, E. (1984) The Modulation of the calc ium pump of human red c e l l s by Na + and K + . Biochim. Biophys. Acta 778, 245-252. Romero, P . J . and O r t i z , C E . (1988) E lec t rogen ic behavior o f the red c e l l C a ^ + pump revealed by d i s u l f o n i c s t i l b e n e s . J . Membrane B i o l . 101, 237-246. Ronner, P . , G r a z z o t t i . P. and C a r a f o l i , E. (1977) A L i p i d requirement f o r the (Ca^ + +Mg^ + ) -act ivated ATPase of erythrocyte membranes. A r c h . Biochem. Biophys. 179, 578-583. Rosenthal , W., Hescheler , J . , Trautwein, W, a n d n S c h u l t z , G. (1988) Contro l o f voltage-dependent 0Ca?+ c f i a n n e l s b y G -pro te in -coupled r e c e p t o r s . F A S E B J . 2, 2784-2790. Rottenberg, H. (1979) Non-equi l ibr ium thermodynamics of energy conversion in b i o e n e r g e t i c s . Biochim. Biophys. Acta 549, 225-253. Roufoga l i s , B.D. (1979) Regulat ion of the calc ium t r a n s l o c a t i o n across the red blood c e l l membrane. Can. J . P h y s i o l . Pharmacol. 57, 1331-1349. 274 Roufoga l is , B.D. and Mauldin, D. (1980) Regulat ion by calmodul in of the calc ium a f f i n i t y of the calcium t ranspor t ATPase in human e ry th rocy tes . Can. J . Biochem. 58, 922-926. Roufoga l is , B.D. and V i l l a l o b o , A. (1989) The (Ca 2 + +Mg 2 + ) -ATPase : P u r i f i c a t i o n and r e c o n s t i t u t i o n . i n : the Red C e l l Membrane: A Model fo r Solute Transport (Raess, B. and T u n n i c l i f f , G . , eds. ) Humana Press Inc . , New Jersey , in p ress . Roufoga l i s , B . D . , E l l i o t t , C T . and Ra ls ton , G .B. (1984) Charac te r i z a t io n of a (Ca z + +Mg 2 + ) -ATPase a c t i v a t o r bound to human erythrocyte membranes. C e l l Calcium 5, 77-88. Saka i , K . , Akanuma, H . , Imahori, K. and Kawashima, S. (1987) A unique s p e c i f i c i t y of a calcium act iva ted neutral protease ind ica ted in histone h y d r o l y s i s . J . Biochem. ip_l , 911-918. Sakihama, T . , Kak idan i , H . , Z e n i t a , K . , Yumoto, N . , K i k u c h i , T . , S a s a k i , T . , Kannagi, R., Nakanishi , S . , Ohmori, M. , T a k i o , K . , T i t a n i , K. and Murachi , T . (1985) A putat ive Ca -b ind ing p r o t e i n : s t ruc ture o f the l i g h t subunit of porcine ca lpa in e l u c i d a t e d by molecular c lon ing and prote in sequence a n a l y s i s . Proc. N a t l . Acad. S c i . USA 82, 6075-6079. Sarkad i , B. (1980) Ac t ive calcium t ransport in human red c e l l s . Biochim. Biophys. Acta 604, 159-190. Sarkad i , B . , Enyedi , A . and Gardos, G. (1980) Molecular p roper t i es o f the red c e l l ca lc ium pump. I. E f f e c t s of ca lmodul in , p r o t e o l y t i c d i g e s t i o n and drugs on k i n e t i c s of a c t i v e ca lc ium uptake in i n s i d e - o u t red c e l l membrane v e s i c l e s . C e l l Calcium 1, 287-310. Sarkad i , B . , Enyedi , A . , Foldes-Papp, Z . and Gardos, G. (1986) Molecular c h a r a c t e r i z a t i o n of the in s i t u red c e l l membrane ca lc ium pump by l i m i t e d p r o t e o l y s i s . J . B i o l . Chem. 261, 9552-9557. Sarkad i , B . , Enyedi , A. and Gardos, G. (1987) Conformational changes of the in s i t u red c e l l membrane calc ium pump a f f e c t s i t s p r o t e o l y s i s . Biochim. Biophys. Acta 899, 129-133. Sarkad i , B . , Maclntyre , J . D . and Gardos, G. (1978) K i n e t i c s o f a c t i v e t ranspor t in ins ide -ou t red c e l l membrane v e s i c l e s . FEBS l e t t . 89, 78-82. Sarkad i , B . , Schubert , A. and Gardos, G. (1979) E f f e c t s of calcium-EGTA buf fers on ac t ive calcium t ransport in i n s i d e - o u t red c e l l membranes. Exper ient ia 35, 1045-1047. Sarkad i , B . , Szasz , I., Ger loczy , A. and Gardos, G . , (1977) Transport parameters and stoichiometry of ac t ive calc ium ion ext rus ion in i n t a c t human red c e l l s . Biochim. Biophys. Acta 464, 92-107. 275 Sasak i , T . , K i k u c h i , T . , Yumoto, N . , Yoshimura, N. and Murachi , T. (1984) Comparative s p e c i f i c i t y and k i n e t i c s tudies on porc ine c a l p a i n I and ca lpa in II with na tu ra l l y occurr ing pept ides and synthe t ic f luorogen ic subst ra tes . J . B i o l . Chem. 259, 12489-12494. Schar f f , 0. (1976) C a 2 + - a c t i v a t i o n of membrane-bound ( C a 2 + + M g 2 + ) -dependent ATPase from human erythrocytes prepared in the presence or absence of Ca . Biochim. Biophys. Acta 43, 206-218. S c h a r f f , 0. and Foder, B. (1982) Rate constants f o r calmodul in b inding to C a - A T P a s e in erythrocyte membranes. Biochim. Biophys. Acta 691, 133-143. S c h a r f f , 0 . , Foder, B. and Sk ibs ted , U. (1983) H y s t e r e t i c a c t i v a t i o n of the C a z + pump revealed by calcium t r a n s i e n t s in human red c e l l s . Biochim. Biophys. Acta 730, 295-305. Schatzmann, H . J . (1966) ATP-dependent C a + + ext rus ion from human red c e l l s . Exper ien t ia 22, 364-365. Schatzmann, H . J . (1969) Transmembrane calcium movements in resea led human red c e l l s In : Calcium and C e l l Functions (Cuthbert , A .W. , ed.) pp. 85-95, Cuthbert Macmillan C o . , London. Schatzmann, H . J . (1978) Ac t ive calcium t ransport and C a 2 + - a c t i v a t e d ATPase in human red c e l l s . In : Current t o p i c s in membranes and t ranspor t (Bronner, F. and K lemze l le r , A . , eds) V o l . 6, pp. 126-168, Academic Press , New York. Schatzmann, H . J . (1985) Calcium extrusion across the plasma membrane by the calc ium pump and the Ca - N a + exchange system In : Calcium and C e l l Physiology (Marme, D. , ed.) pp. 19-52, S p r i n g e r - V e r l a g , New York. Schatzmann, H . J . and R o s s i , G .L . (1971) ( C a 2 + + M g 2 + ) - a c t i v a t e d membrane ATPase in human red c e l l s and t h e i r p o s s i b l e r e l a t i o n to ca t ion t ranspor t : Biochim. Biophys. Acta 241, 379-392. Schatzmann, H . J . and V i n c e n z i , F . F . (1969) Calcium movements across the membrane of human red c e l l s . J . P h y s i o l . 201, 369-395. Schechter , I. and Berger, A. (1967) On the s i z e of the a c t i v e s i t e in proteases . I. Papain. Biochem. Biophys. Res. Commun. 27, 157-162. Schmaier, A . H . , Smith, P . A . , Purdon, A . D . , White, J . G . and Colman, R.W. (1986) High molecular weight k in inogen: l o c a l i z a t i o n in the unstimulated and act iva ted p l a t e l e t and a c t i v a t i o n by a p l a t e l e t c a l p a i n ( s ) . Blood 67, 119-130. Schmidt, J . W . , Hinds, T .R . and V i n c e n z i , F . F . (1985) On the f a i l u r e of calmodulin to ac t iva te C a 2 + pump ATPase of dog red blood c e l l s . Comp. Biochem. P h y s i o l . 82A, 601-607. 276 Scott-Woo, G . C . and Walsh, M.P. (1988) C h a r a c t e r i z a t i o n of the autophosphorylat ion of chicken g i z z a r d caldesmon. Biochem. J . 255, 817-824. S e i l e r , S . , Wegener, A . D . , Whang, D .D . , Hathaway, D.R. and Jones, L.R. (1984) High molecular weight prote ins in c a r d i a c and ske le ta l muscle junct iona l sarcoplasmic re t icu lum v e s i c l e s bind ca lmodul in , are phosphorylated, and are degraded by Ca -act iva ted protease. J . B i o l . Chem. 259, 8550-8557. Seubert , P . , Baudry, M., Dudek, S. and Lynch, G. (1987) Calmodulin st imulates the degradation of bra in s p e c t r i n by c a l p a i n . Synapse I, 20-24. Sharma, R.K. and Wang, J . H . (1988) I so la t ion of bovine bra in ca lmodul in-dependent c y c l i c nucleot ide phosphodiesterase isozymes. Methods. Enzymol. 159, 582-595. Sharma, R . K . , Wang, T . H . , Wirch, E. and Wang, J . H . (1980) P u r i f i c a t i o n and proper t ies of brain calmodulin-dependent c y c l i c nuc leot ide phosphodiesterase. J . B i o l . Chem. 255, 5916-5923. Shattuck, R . L . , Yeager, R .E . and Storm, D.R. (1987) Calmodul in-st imulated adenylate cyc lase In : Calcium and C e l l Funct ions (Cheung, W.Y. , ed. ) V o l . VI I , pp. 39-60, Academic P r e s s , New York. S h u l l , G . E . and Greeb, J . (1988) Molecular c lon ing of two-isoforms of the plasma membrane C a z + - t r a n s p o r t i n g ATPase from ra t b r a i n . J . B i o l . Chem. 263, 8646-8657. S i b l e y , D.R. , Benovic, J . L . , Caron, M.G. and Lefkowi tz , R . J . (1987) Regulat ion of transmembrane s i g n a l l i n g by receptor phosphory la t ion . C e l l 48, 913-922. Siman, R., Baudry, M. and Lynch, G. (1984) Bra in f o d r i n : substrate fo r c a l p a i n I, an endogenous ca lc ium-ac t iva ted protease . Proc. N a t l . Acad. S c i . USA 81, 3572-3576. Sitaramayya, A . , Wright, L . S . and S i e g e l , F . L . (1980) Enzymatic methylat ion of calmodulin in ra t bra in c y t o s o l . J . B i o l . Chem. 255. 8894-8900. Smallwood, J . I . , Waisman, D.M., L a f r e n i e r e , D. and Rasmussen, H. (1983) Evidence that the erythrocyte calc ium pump ca ta lyzes a Ca .nH exchange. J . B i o l . Chem. 258, 11092-11097. Smallwood, J . I . , G i i g i , B. and Rasmussen, H. (1988) Regulat ion of erythrocyte C a z + pump a c t i v i t y by pro te in kinase C. J . B i o l . Chem. 262, 2195-2202. Smith, T . J . , Davis , F .B . and Davis , P . J . (1989) Ret ino ic ac id i s a 277 modulator of thyro id hormone a c t i v a t i o n of Ca -ATPase in the human erythrocyte membrane. J . B i o l . Chem. 262, 687-689. Sobue, K . , F u g i t a , M., Muramoto, Y. and K a k i u c h i , S. (1981c) The ca lmodul in-b inding prote in in microtubules i s tau f a c t o r . FEBS l e t t . 132, 137-140. Sobue, K . , Muramoto, Y . , Fug i ta , M. and K a k i u c h i , S. (1981a) Calmodul in-binding prote in of erythrocyte c y t o s k e l e t o n . Biochem. Biophys. Res. Commun. 100, 1063-1070. Sobue, K . , Muramoto, Y . , Fug i ta , M. and K a k i u c h i , S. (1981b) P u r i f i c a t i o n of a ca lmodul in-b inding prote in from chicken g i z z a r d that i n t e r a c t s with F - a c t i n . Proc. N a t l . Acad. S c i . USA 78, 5652-5655. S o l d a t i , L . , Longoni, S. and C a r a f o l i , E. (1985) S o l u b i l i z a t i o n and r e c o n s t i t u t i o n of the N a + / C a z + exchanger of c a r d i a c sarcolemma. J . B i o l . Chem. 260, 13321-13327. Spedding, M. (1987) Three types of C a 2 + channel exp la in d i s c r e p a n c i e s . Trends. Pharmacol. S c i . 8, 117-119. S q u i r e , J . M . (1983) Molecular mechanisms in muscular c o n t r a c t i o n . Trends Neuro. S c i . 6, 409-413. Stone, D. and S m i l l i e , L .B . (1978) The amino ac id sequence o f rabb i t ske le ta l or-tropomyosin. J . B i o l . Chem. 253, 1137-1148. S t r y e r , L. (1986) C y c l i c GMP cascade of v i s i o n . Ann. Rev. Neurosc i . 9, 87-119. S u g i t a , H . , I sh iura , S . , Nonaka, I. and S u g i t a , H. (1980) Calcium ac t iva ted protease (CANP) and i t s i n h i b i t o r in muscular dystrophy In : Muscular Dystrophy (Ebashi , S. and Ozawa, E . , eds . ) pp. 265-282, Un ive rs i t y of Tokyo Press , Tokyo. Suzuk i , K. (1987) Calcium act iva ted neutral protease: domain s t ruc tu re and a c t i v i t y r e g u l a t i o n . Trends Biochem. S c i . H , 103-105. Suzuk i , K . , Imajoh, S . , Emori, Y . , Kawasaki, H . , Minami, Y. and Ohno, S. (1987a) Calc ium-act ivated neutral protease and i t s endogenous i n h i b i t o r . FEBS l e t t . 220, 271-277. Suzuk i , K . , Imajoh, S . , Emori, Y . , Kawasaki, H . , Minamik, Y. and Ohno, S. (1987b) Regulation of a c t i v i t y of ca lc ium ac t iva ted neutral protease . Adv. Enzyme Regul. 27, 153-169. Suzuk i , K . , T s u y i , S. and I sh iu ra , S. (1981) E f f e c t of C a 2 + on the i n h i b i t i o n of ca lc ium-ac t iva ted neutral protease by l e u p e p t i n , an t ipa in and epoxysuccinate d e r i v a t i v e s . FEBS l e t t . 136, 119-122. Szasz , I., H a s i t o , M. , Sarkad i , B. and Gardos, G. (1978) Phosphorylat ion 278 of the Ca*1 pump intermediate in i n t a c t c e l l s , i s o l a t e d membranes and ins ide -out v e s i c l e s (1978) Molec. C e l l . Biochem. 22, 147-152. Tada, M., K i rchberger , M.A. and L i , H.C. (1975) Phosphoprotein phosphatase-catalyzed dephosphosphorylation of the 22,000 dal ton phosphoprotein of card iac sarcoplasmic re t i cu lum. J . C y c l i c Nucleot ides Res. 1, 329-338. Tahara, S.M. and Traugh, J . A . (1982) D i f f e r e n t i a l a c t i v a t i o n of two protease-act iva ted prote in kinases from r e t i c u l o c y t e s by a Ca -st imulated protease and i d e n t i f i c a t i o n of phosphorylated t r a n s l a t i o n a l components. Eur. J . Biochem. 126, 395-399. Takeyama, Y . , Nakanikshi , H . , U r a t s u j i , Y . , K ishimoto, A . and N i s h i z u k i , Y. (1986) A calc ium-protease a c t i v a t o r assoc ia ted with brain microsomal inso lub le elements. FEBS l e t t . 194, 110-114. T a k i o , K . , Smith, S . B . , Krebs, E . G . , Walsh, K.A. and T i t a n i , K. (1984) Amino acid sequence of the regula tory subunit o f bovine type II adenosine c y c l i c 3 ' , 5 ' -phosphate dependent p ro te in k inase. Biochemistry 23, 4200-4206. T a l l a n t , E .A. and Cheung, W.Y. (1984) A c t i v a t i o n of bovine bra in calmodulin-dependent prote in phosphatase by l i m i t e d t r y p s i n i z a t i o n . Biochemistry 23, 973-979. T a l l a n t , E .A. and Cheung, W.Y. (1986) Calmodulin-dependent prote in phosphatase i n : Calcium and C e l l Funct ions (Cheung, W.Y. , ed.) V o l . V I , pp. 71-112, Academic P r e s s , New York. T a l l a n t , E . A . , Brumley, L.M. and Wal lace, R.W. (L988) A c t i v a t i o n of a calmodulin-dependent phosphatase by a Ca -dependent protease. Biochemistry 2_7, 2205-2211. Taverna, R.D. and Hanahan, D . J . (1980) Modulation of human erythrocyte C a z + / M g ATPase a c t i v i t y by phospholipase A 2 and proteases . A comparison with calmodul in . Biochim. Biophys. Res. Commun. 94, 652-659. T i f f e r t , T . , Garcia-Sancho, J . and Lew, V . L . (1984) I r r e v e r s i b l e ATP deple t ion caused by low concentrat ions of formaldehyde and of ca lc ium-che la tor esters in in tac t human red c e l l s . Biochim. Biophys. Acta 773, 143-156. T inoco , J . , S a v i r , K. and Wong, J . (1978) Physica l Chemistry: P r i n c i p l e s and App l i ca t ions in B i o l o g i c a l Sc iences , p. 124, H a l l , New J e r s e y . T i t a n i , K. , Kumar, S . , Tako, K. , E r i c s s o n , L . H . , Wade, R .D . , Ash ida , K. , Walsh, K . A . , Chopek, M.W., Sad le r , J . E . and Fujikawa, K. (1986) Amino acid sequence of human von Wil lebrand f a c t o r . Biochemistry 279 25, 3171-3184. Toyo-Oka, T . (1982) Phosphorylat ion with c y c l i c adenosine 3 ' -5' Monophosphate-dependent prote in kinase renders bovine troponin s e n s i t i v e to the degradation by ca lc ium-ac t i va ted neutral protease. Biochem. Biophys. Res. Commun. 107, 44-50. Traub, P . , Scherbartyh, A . , Wi l l inga le -Theune, J . and Pau l in -Levasseur , M. (1988) D i f f e r e n t i a l s e n s i t i v i t y of vimentin and nuclear lamins from E h r l i c h a s c i t e s tumor c e l l s toward Ca - a c t i v a t e d neutral p ro te inase . Eur. J . C e l l B i o l . 46, 478-490. Tremblay, J . and Hamet, P. (1984) Calcium-dependent p r o t e o l y t i c s t imula t ion of adenylate cyc lase in p l a t e l e t s from spontaneously hypertensive r a t s . Metabolism 33, 689-695. T r u g l i a , J . A . and Stracher , A. (1981) P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a calc ium dependent su l fhydry l protease from human p l a t e l e t s . Biochem. Biophys. Res. Commun. 100, 814-822. U l l r i c h , A . , Coussens, L . , H a y f l i c k , J . S . , D u l l , T . J . , Gray, A . , Tarn, A.W. , Lee, J . , Yarden, Y . , Libermann, T . A . , S c h l e s s i n g e r , J . , Downward, J . , Mayes, E . L . V . , Wh i t t l e , N . , W a t e r f i e l d , M.D. and Seeburg, P.H. (1984) Human epidermal growth f a c t o r receptor cDNA sequence and aberrant expression of the ampl i f i ed gene in A431 epidermoid carcinoma c e l l s . Nature 309, 418-425. Vas ington , F.D. and Murphy, J . (1961) Ac t i ve binding of calc ium by mitochondr ia . Fed. Proc. 20, 146. Vas ington , F .D. and Murphy, J . C . (1962) C a 2 + uptake by ra t kidney mitochondria and i t s dependence on r e s p i r a t i o n and phosphory la t ion . J . B i o l . Chem. 237, 2670-2772. Vedeck is , W.Y. , Freeman, M.R., Schrader, W.T. and 0 'Ma l l ey , B.W. (1980) Progesterone-binding components of ch ick ov iduc t : p a r t i a l p u r i f i c a t i o n and charac te r i za t ion of a ca lc ium-ac t i va ted protease which hydrolyzes the progesterone recep to r . Biochemistry 19, 335-349. Verma, A . K . , F i l o t e o , A . G . , Standord, D.R. , Wieben, E . D . , Penniston, J . T . , S t r e h l e r , E . E . , F i s c h e r , R., Heim, R., Voge l , G . , Mathews, S . , St rehler -Page M . -A . , James P . , Vorherr , T . , Krebs, J . and C a r a f o l i , E. (1988) Complete primary s t ruc ture of a human plasma membrane C a z + pump. J . B i o l . Chem. 263, 14152-14159. V e z z o l i , G . , E l l i , A . A . , T r i p o d i , G . , B i a n c h i , G. and C a r a f o l i , E. (1985) Calcium ATPase in erythrocytes of spontaneously hypertensive ra ts of the Milan s t r a i n . J . Hypertension 3, 645-648. V i l l a l o b o , A. and Roufoga l is , B.D. (1986) Proton counter t ransport by the reconst i tu ted erythrocyte Ca - t r a n s l o c a t i n g ATPase: Evidence 280 using ionophoret ic compounds. J . Membrane B i o l . 93, 249-258. V i l l a l o b o , A . , Brown, L. and Roufoga l is , B.D. (1986) K i n e t i c proper t ies of the p u r i f i e d Ca - t r a n s l o c a t i n g ATPase from human erythrocyte plasma membrane. Biochim. Biophys. Acta 854, 9-20. V i n c e n z i , F . F . and Hinds, T .R. (1988) Decreased Ca pump ATPase a c t i v i t y associated with increased densi ty in human red blood c e l l s . Blood C e l l s 14, 139-159. V r o l i x , M. , Raeymaekers, L . , Wuytack, F . , Hofmann, F. and C a s t e e l s , R. (1988) C y c l i c GMP-dependent prote in kinase st imulates the plasmalemmal C a z + pump of smooth muscle v i a phosphorylat ion of phosphatidyl i n o s i t o l . Biochem. J . 255, 855-863. Waelkens, E . , G o r i s , J . and Merlevede, W. (1985) A c t i v a t i o n of the PCSy-prote in phosphatase by a Ca -dependent protease . FEBS l e t t . 192, 317-320. Waisman, D.M. , Gimble, J . M . , Goodman, D.B. and Rasmussen, H. (1981a) Studies on the C a z t ransport mechanism of human erythrocyte ins ide out plasma membrane v e s i c l e s . I. Regulat ion of the C a z + pump by ca lc ium, ca lmodul in , ATP and pH. J . B i o l . Chem. 256, 409-414. Waisman, D.M. , Gimble, J . M . . Goodman, D.B. and Rasmussen, H. (1981b) Studies on the C a z + t ranspor t mechanism of human erythrocyte i n s i d e out plasma membrane v e s i c l e s . II. S t imula t ion of the C a z + pump by phosphate. J . B i o l . Chem. 256, 415-419. Waisman, D.M. , Gimble, J . M . , Goodman, D . B . P . , Rasmussen, H. (1981c) Studies of the C a z + t ranspor t mechanism of Human Erythrocyte Inside-out plasma membrane v e s i c l e s . III. S t imula t ion of the C a 2 + pump by anions. J . B i o l . Chem. 256, 420-424. Wakim, B . T . , Alexander, K . A . , Masure, H.R. , C imle r , B . M . , Storm, D.R. and Walsh, K.A. (1987) Amino acid sequence of P-57, a n e u r o s p e c i f i c ca lmodul in-b inding p r o t e i n . Biochemistry 26, 7466-7470. Wal lace, R.W., T a l l a n t , E .A. and McManus, M.C. (1987) Human p l a t e l e t ca lmodul in-b inding p r o t e i n s : I d e n t i f i c a t i o n and Ca -dependent p r o t e o l y s i s upon p l a t e l e t a c t i v a t i o n . Biochemistry 26, 2766-2773. Walsh, M .P . , Dabrowska, R., H ink ins , S. and Hartshorne, D . J . (1982) Calcium-independent myosin l i g h t chain kinase of smooth muscle. Preparat ion by l im i ted chymotryptic d i g e s t i o n of the calc ium ion dependent enzyme, p u r i f i c a t i o n , and c h a r a c t e r i z a t i o n . Biochemistry 21, 1919-1925. Wang, J . H . , P a l l e n , C . J . , Sharma, R . K . , Adach i , A . - M . and Adach i , K. (1985) The calmodulin regula tory system. Current Topics C e l l . 281 Regul . 27, 419-469. Wang, K.K.W. , Roufoga l is , B.D. and V i l l a l o b o , A. (1988b) Further c h a r a c t e r i z a t i o n of the calpain-mediated p r o t e o l y s i s of the human erythrocyte plasma membrane C a - A T P a s e . A r c h . Biochem. Biophys. 267, 317-327. Wang, K.K.W. , V i l l a l o b o , A. and Roufoga l i s , B.D. (1988a) A c t i v a t i o n of the C a - A T P a s e of human erythrocyte membrane by an endogenous Ca -dependent neutral protease. A r c h . Biochem. Biophys. 260, 696-704. Wang, K.K.W. , Roufoaa l is , B.D. and V i l l a l o b o , A . (1989a) Calpain I ac t i va tes C a z + t ransport by the human erythrocyte plasma membrane calc ium pump I n : Calcium Binding Prote ins in Normal and Transformed C e l l s (Lawson, D.E.M. and Pochet, R., eds . ) Plenum Press , London, in p ress . Wang, K.K.W. , V i l l a l o b o , A. and Roufoga l i s , B.D. (1989b) Calmodulin-binding prote ins as c a l p a i n subs t ra tes . Biochem. J . 262, in p ress . Wang, K.K.W. , Roufoga l is , B.D. and V i l l a l o b o , A . (1989c) Charac te r i za t ion of the fragmented forms of c a l c i n e u r i n produced by c a l p a i n I. Biochem. C e l l B i o l . 67, in p r e s s . Wang, K.K.W. , Roufoga l is , B.D. and V i l l a l o b o , A. . (1989d) Calpain I ac t i va tes C a ^ + t ransport by the recons t i tu ted ery throcyte C a z + pump. J . Membrane B i o l . , in p r e s s . Watson, E . L . , V i n c e n z i , F . F . and Dav is , P.W. (1971) Nucleot ides as substrates of C a - A T P a s e and NaK-ATPase in i s o l a t e d red c e l l membranes. L i f e S c i . 10, 1399-1404. Warxman, L. (1981) Ca lc ium-act iva ted proteases in mammalian t i s s u e s . Methods Enzymol. 80, 664-680. Welsh, M . J . , A s t e r , J . C . , I re land, M. , A l c a l a , J . and M a i s e l , H. (1982) Calmodulin binds to ch ick lens gap junc t ion prote in in a ca lc ium-independent manner. Science 2_16, 642-644. White, M.F. and Kahn, C R . (1986) The i n s u l i n receptor and ty ros ine phosphory la t ion . I n : The Enzymes (Boyer, P.D. and Krebs, E . G . , eds . ) V o l . 17, pp. 247-310, Academic Press , New York. Wi lk inson , J . M . and Grand, R . J . A . (1975) The amino ac id sequence of t roponin I from rabbi t ske le ta l muscle. Biochem. J . 149, 493-496. Wins, P. and Schof fen ie ls (19,66) Studies on r e d - c e l l ghost ATPase systems. Proper t ies of a (Mg z + +Ca z + ) -dependent ATPase. Biochim. Biophys. Acta 120, 341-350. 282 Wolf, H.U. and Gie tzen , K. (1974) The s o l u b i l i z a t i o n of high a f f i n i t y -Ca -ATPase of human erythrocyte membranes Hoppe-Seyler 's Z. P h y s i o l . 335, 1272. Wolf, H . U . , Dieckvoss, G. and Lichtner R. (1977) P u r i f i c a t i o n and proper t ies of h i g h - a f f i n i t y Ca -ATPase of human erythrocyte membranes. Acta B i o l . Med. Germ. 36, 847-858. Wong, P .Y .K . and Cheung, W.Y. (1979) Calmodulin st imulates human p l a t e l e t phospholipase A2. Biochem. Biophys. Res. Commun. 90, 473-480. Wuthrich, A. (1982) Iso la t io / i from haemolysate of a proteinacous i n h i b i t o r of the red c e l l Ca -pump ATPase. Its ac t ion on the k i n e t i c s of the enzyme. C e l l Calcium 3, 201-214. Xu, Y . H . and Roufoga l is , B.D. (1988a) Asymmetric e f f e c t s of d iva len t ca t ions and protons on act ive C a z + e f f l u x and C a z + - A T P a s e in i n t a c t red blood c e l l s . J . Membrane. B i o l . 105, 155-164. Xu, Y . H . and Roufoga l is , B.D. (1988b) ATP dependence of ac t ive calc ium t ransport in red blood c e l l s In : Progress in Biochemical Pharmacology. Symposium on C i r c u l a t i n g Sodium Transport Inh ib i tors ( P a o l e t t i , R., ed.) pp. 107-118, S. Karger , A . G . B a s e l . Yamamoto, K. , Kosak i , G . , Suzuk i , K . , Tanoue, K. and Yamazaki, H. (1986) Cleavage s i t e of calcium-dependent protease in human p l a t e l e t membrane g lycoprote in l b . Thrombosis Res. 43, 41-55. Yeager, R . E . , Heideman, W., Rosenberg, G.B. and Storm, D.R. (1985) P u r i f i c a t i o n of the c a l m o d u l i n - s e n s i t i v e adenylate c y c l a s e from bovine cerebral cor tex . Biochemistry 24, 3776-3783. Yeoman, L . C . , O lson, M . O . J . , Sugano, N . , Jordan, J . J . , T a y l o r , C.W., Starbuck, W.C. and Busch, H. (1972) Amino ac id sequence o f the center of the a r g i n i n e - l y s i n e - r i c h hi stone from c a l f thymus: the t o t a l sequence. J ; B i o l . Chem. 247, 6018-6023. Zhang, Z . , Lawrence, J . and Stracher , A. (1988) Phosphorylat ion of p l a t e l e t a c t i n binding prote in protects against p r o t e o l y s i s by calcium dependent su l fhydry l protease. Biochem. Biophys. Res. Commun. 151, 355-360. Zimmerman, U . J . and Sch laepfe r , W.W. (1984) Calcium ac t iva ted neutral protease (CANP) in bra in and other t i s s u e s . Progress Neurob io l . 23, 63-78. Z u r i n i , M., Krebs, J . , Penniston, J . T . and C a r a f o l i , E . (1984) Cont ro l led p r o t e o l y s i s of the p u r i f i e d C a - A T P a s e of the erythrocyte membrane. J . B i o l . Chem. 259, 618-627. 283 APPENDIX L i s t of amino acids and t h e i r t h r e e - l e t t e r and o n e - l e t t e r codes amino ac id t h r e e - l e t t e r code o n e - l e t t e r code Alanine A la A Arg in ine Arg R Asparagine Asn N A s p a r t i c ac id Asp D Cysteine Cys C Glutamic ac id Glu E Glutamine Gin Q Glyc ine Gly G H i s t i d i n e His H Iso leucine l i e I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F P r o l i n e Pro P Ser ine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Va l ine Val V 284 PUBLICATIONS Papers 1. Wang. K.K.W. . V i l l a l o b o , A. and Roufoga l is , B.D. (1988) A c t i v a t i o n of the C a 2 + - A T P a s e of human erythrocyte membrane by an endogenous Ca 2 + -dependent neutral protease. Arch . Biochem. Biophys. 260, 696-704. . 2. Olorunsogo, 0 . 0 . , V i l l a l o b o , A . , Wang, K.K.W. and Roufoga l i s , B.D. (1988) The e f f e c t of calmodulin on the in te rac t ion of carbodi imides with the p u r i f i e d human erythrocyte ( C a 2 + + M g 2 + ) -ATPase. Biochim. Biophys. Acta 945, 33-40. 3. Wang, K.K.W. , Rou foga l i s , B.D. and V i l l a l o b o , A. (1988) Further c h a r a c t e r i z a t i o n of the calpain-mediated p r o t e o l y s i s of the human erythrocyte plasma membrane C a 2 + - A T P a s e . Arch . Biochem. Biophys. 26_7_, 317-327. 4. Wang, K.K.W. . Rou foga l i s , B.D. and V i l l a l o b o , A. (1989) Calpain I ac t i va tes C a 2 * t ranspor t by the human erythrocyte plasma membrane calc ium pump In : Calcium Binding Prote ins in Normal and Transformed C e l l s (Lawson, D.E.M. and Pochet, R. , eds . ) Plenum Press , London, in p ress . 5. Wang. K.K.W. . V i l l a l o b o . A. and Roufoga l i s , B.D. (1989) Calmodul in-binding prote ins as ca lpa in subst ra tes . Biochem. J . 262, in press . 6. Wang, K.K.W. . Rou foga l i s , B.D. and V i l l a l o b o , A . (1989) Charac te r i za t ion of the fragmented forms of c a l c i n e u r i n produced by c a l p a i n I. Biochem. C e l l B i o l . , in press . 7. Wang, K.K.W. . Rou foga l i s , B.D. and V i l l a l o b o , A. (1989) Calpain I ac t i va tes C a 2 * t ranspor t by the reconst i tu ted erythrocyte C a 2 + pump. J . Membrane B i o l . , in press . 8. Roufoga l is , B . D . , Brzuszczak, I., Xu, Y . - H . , Cgnigrave, A . D . , Machan, C. and Wang, K.K.W. (1989) Pers is ten t C a z + - i n d u c e d a c t i v a t i o n of erythrocyte membrane C a 2 + - A T P a s e unrelated to ca lpa in p r o t e o l y s i s . , submitted. 9. Wang, K.K.W. , Machan, C , A l l a n , B .G. and Roufoga l i s , B.D. (1989) Prote in kinase C phosphorylates the carboxyl - terminal of the human erythrocyte C a 2 + - A T P a s e . , submitted. 10. Wang, K.K.W. , Machan, C , V i l l a l o b o , A. and Roufoga l is , B.D. (1989) P u r i f i c a t i o n and Charac te r i za t ion of two novel ca lmodul in- and phospho l ip id -b ind ing prote ins from human erythrocyte membrane., in prepara t ion . 11. G i l c h r i s t . J . S . C . , Wang. K.K.W. , Katz , S. and B a l c a s t r o , A . N . (1989) P r o t e o l y s i s of the junct ional sacroplasmic re t icu lum calcium re lease channel by ca lpa in I and II., in prepara t ion . PUBLICATIONS (cont . ) Abst rac ts 1. Wang. K.K.W. and Roufoga l i s , B.D. (1987) A c t i v a t i o n of the C a 2 + -ATPase of the human red c e l l membrane by endogenous c a l p a i n . V l l t h Internat ional Washington Spring Symposium. Washington, D . C . , USA. Abs. 116. 2. Wang, K .K .W. , R o u f o g a l i s , B.D. and V i l l a l o b o , A. (1988) P r o t e o l y s i s of the erythrocyte C a 2 + - A T P a s e by c a l p a i n . S ix th In ternat ional Symposium on Calcium-Binding Proteins in Health and Disease . Nagoya, Japan. Abs. 167. 3. Wang, K .K.W. . R o u f o g a l i s , B.D. and V i l l a l o b o , A. (1989) Ca lpa in I a c t i v a t e s C a 2 * t ransport by the reconst i tu ted human erythrocyte plasma membrane C a 2 + - A T P a s e . F i r s t European Symposium on Calcium Binding Prote ins in Normal and Transformed C e l l s . B r u x e l l e s , Belgium. Abs. H2. 4. G i l c h r i s t , J . S . C . , Wang, K.K.W. . Katz , S. and B e l c a s t r o , A . N . (1989) Cation-dependent p u r i f i c a t i o n of the 360 kDa channel p ro te in from detergent s o l u b i l i s e d junct ional terminal c i s t e r n a e sarcoplasmic re t icu lum using calmodulin-agarose a f f i n i t y chromatography. F i r s t European Symposium on Calcium Binding Prote ins in Normal and Transformed C e l l s . B r u x e l l e s , Belgium. Abs. H6. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0098268/manifest

Comment

Related Items