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.Sc,  A thesis the  University  submitted  of Guelph,  in p a r t i a l  requirement  1984  fulfillment  f o r the degree  Doctor o f  of  of  Philosophy  in The F a c u l t y o f Graduate D i v i s i o n o f Pharmaceutical  Studies  Chemistry o f  F a c u l t y o f Pharmaceutical  We accept t h i s  thesis  to the r e q u i r e d  THE UNIVERSITY  as conforming standard  OF BRITISH COLUMBIA  June, ©  Sciences  1989  Kevin Ka-Wang Wang,  1989  the  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  scholarly purposes may be granted her  representatives.  permission.  The University of British Columbia Vancouver, Canada  for  an advanced  Library shall make it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  DE-6 (2/88)  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  is  by the  understood  that  head of copying  my or  be allowed without my written  ABSTRACT  It  i s well  ATPase  can  ATPase  be a c t i v a t e d  with  activation. present  known t h a t  exogenous Because  proteases  also  cytosol, it  Ca^  +  Ca^ -ATPase during  red blood c e l l  activation  +  formation  bound  and were s t i m u l a t e d  their the  formation  presence o f  transformed  into  kDa and 85 kDa.  kDa and 80  of  of  80 kDa fragments  d i d not  is  a sustained elevation  of  calpain  was i n c r e a s e d and  transformed  fragments  two  of  by  125  I  its  +  the C a ^ - A T P a s e  The  125  the  82  the  stimulated  the  native  calmodulin protects  i i  kDa 124  kDa,  fragments kDa and the  activation.  enzyme was  two phosphoenzyme i n t e r m e d i a t e - f o r m i n g o f these fragments  124  two  by c a l m o d u l i n and  observed p r o t e o l y t i c  however,  and  into  intermediate-forming  kDa and the  by c a l m o d u l i n , whereas,  calpain kDa  phosphoenzyme  bind and were not  calmodulin,  calmodulin. Apparently,  I)  enzyme c o u l d  membranes with p u r i f i e d  Were  kDa.  corresponded t o  Both  (calpain  by c a l p a i n . Both the membrane-bound and the  kDa)  fragments to  82  +  the C a ^ -  calmodulin-1ike  Calmodulin protected  intermediate-forming  the  -pumping  level.  C a ^ - A T P a s e (136  by  a  of  was proposed t h a t t h i s  +  the  to c a l m o d u l i n was l o s t .  phosphoenzyme followed  produce  membrane-bound C a ^ - A T P a s e a c t i v i t y  against p r o t e o l y t i c purified  proteolysis  +  and C a ^ , the sensitivity  can  Ca  a calcium-dependent c y s t e i n e p r o t e a s e  Upon i n c u b a t i o n o f +  plasma membrane  Limited  activate  the c y t o s o l i c f r e e  erythrocyte  by c a l m o d u l i n .  i n the e r y t h r o c y t e  proteolytically  the  sequentially  fragments  of  bound to and were s t i m u l a t e d +  In  127 by  the C a ^ - A T P a s e from c a l p a i n -  mediated  activation  insensitive  by  preventing  Following  limited  Smaller  treatment  +  Ca^ -ATPase,  of  both  and ATP h y d r o l y s i s were i n c r e a s e d t o  those  obtained  ATPase  was  fragments,  upon  addition  transformed  of  identified terminal 50  kDa  mainly  from  into  about  2  sensitive cleavage  teratoma,  initial  rates  near  maximal  levels,  124  The  kDa  the  of  Ca^  and  127  +  Ca^ -  kDa  active  respectively.  +  a plasma membrane C a ^ -  calmodulin-binding  domain  N-terminal  it  was  initially kDa o f 125  the  end.  combining  postulated cleaves o f f  (ii)  further  (iii) the  C-terminal  fragment  which  retained  absence  of  calmodulin,  11 kDa from the domain,  the  C-terminal  producing  absence o f  removes most  (1.5  the  calmodulin, kDa)  of  the  end  the  remaining 124  calpain cleaves off  domain.  and  a second  calmodulin-insensitive  calmodulin-binding  i i i  various  calmodulin-  end and produces a c a l m o d u l i n - s e n s i t i v e most o f  the  the  about  i n the presence o f c a l m o d u l i n ,  9 kDa o f  the  with  of  (i)  in  in  knowledge  about  ability  that  calmodulin-binding  I  this  and c a l m o d u l i n - b i n d i n g  kDa fragment;  by c a l p a i n  By  was  kDa from the C-  o f the enzyme w h i l e the acylphosphate s i t e was l o c a t e d the  +  similar  reconstituted  of calmodulin,  c a l m o d u l i n - b i n d i n g domain and produces the fragment;  fragments  phosphatidylcholine  kDa long and l o c a t e d about 9-10  m o l e c u l a r masses  fragments, I  human  t o be about 3.5  end  estimated  calpain  a  calmodulin-  the  calmodulin.  i n the absence and the presence  from  the  inactive  the  Based on the deduced amino a c i d sequence o f ATPase  of  proteolysis.  calpain  1iposome-reconstituted  to  formation  124 kDa and 80 kDa fragments.  were a l s o produced with f u r t h e r  uptake  the  kDa  about 127  kDa  A slower  cleavage  at  a  site  about  proposed to g e n e r a t e the  127 kDa,  Several proteolyzed membrane  fact  a  as  threonine  characteristic Primary  (T)  I,  revealed shared  structure  proteins  proline  (P),  end  human  at  least  showed date  16  aspartate  be  brain. was  A in  calmodulin-binding the  one  or  (D),  proposed t h a t  to  erythrocyte  to c a l p a i n  that  have  found  from bovine  susceptibility  c a l m o d u l i n - b i n d i n g p r o t e i n s may s e r v e as r e c o g n i t i o n  Signature of t h e s i s co-supervisor  from  from  (E), was  fragments  adducin  analysis  glutamate  also  respectively.  and c a l c i n e u r i n that  was  also  by  It  N-terminal  were  sequenced t o  (PEST s e q u e n c e s ) .  the  proteins  including  neuromodulin  search f u r t h e r  in  from  kDa and 80 kDa a c t i v e  calmodulin-binding  calpain  calmodulin-binding enriched  the 85 kDa, 82  other by  proteins.  kDa  125 kDa and 124 kDa f r a g m e n t s ,  as w e l l  literature  42-44  majority more  serine  PEST  sites  for  of  regions (S)  sequences  and in  calpain.  Signature of t h e s i s c o - s u p e r v i s o r  iv  ACKNOWLEDGEMENTS  I  am w h o l e h e a r t e d l y  and  Dr.  Antonio  inspiration  I  am  I  indebted  Villa!obo  to  Dr.  would  like  my  for  Ph.D.  Frank  to  would  acknowledge  Dr.  also  like  to  Dr.  Dr.  Basil  guidance,  Committee David  D.  Roufogalis  tireless  members:  Godin  would  like  present, Gosh,  in  Dr.  to  Dr.  Mutus  express  particular,  Dorothy  Special strong Dr.  thanks  and  Gilchrist,  for  I would l i k e  it  of  Y.  L i u and D r .  for  providing  support,  Dr.  Dr.  Marc  Sidney  Levine  Katz  for  my  appreciation  Christine Mr.  technical  James G i l c h r i s t  who  preparations D.  R.  in t h i s  Storm f o r  oncomodulin  to  Nichol,  James H a r r i s ,  my  work.  providing  and  Dr.  colleagues,  Dr.  M.  past  and  Jon C h u r c h , D r .  Ms.  Cheryl  a s s i s t a n c e and moral  was needed most.  laboratory,  Mr.  PEST-FIND program.  are g i v e n to Dr. John M c N e i l l  support when Katz's  Dr.  Jeffery,  Bruce Wilson f o r t h e i r  contribution  sarcoplasmic reticulum  thank  B.  the  R e c h s t e i n e r f o r p r o v i d i n g the  of  their  Thesis  Abbott,  junctional  neuromodul i n ,  I  my s u p e r v i s o r s ,  assistance.  s u p p l i e d the I  to  and f r i e n d s h i p .  (Chairman), their  grateful  especially  s h a r i n g experimental  I  and D r .  also l i k e  Mr.  experience  t o acknowledge the f i n a n c i a l  Bruce and  Machan  and  Mr.  support.  S i d n e y Katz f o r to  thank  Allen  and  the  their  members  Mr.  James  inspiration.  support extended by the  Heart F o u n d a t i o n , the B r i t i s h Columbia Heart Foundation and the  v  Shobba  Canadian  University  of  British  Columbia.  I  also  acknowledge  from the  Red Cross o f Canada (Vancouver  Finally,  I wish  graduate  students  British  Columbia  t o thank a l l  the  supply o f  members o f the f a c u l t y ,  making  my  Ph.D.  memorable.  vi  human blood  branch).  i n the F a c u l t y o f Pharmaceutical for  fresh  supporting s t a f f  Sciences,  program  most  University enjoyable  and of and  DEDICATION  To my w i f e  Alice  and to my parent  TABLE OF CONTENTS  CONTENT  page  ABSTRACT  i i  ACKNOWLEDGEMENTS  v  DEDICATION  vii  TABLE OF CONTENTS  viii  LIST OF FIGURES  xiii  LIST OF TABLES  xvii  LIST OF ABBREVIATIONS  xviii  INTRODUCTION  1  I.  Calcium and c e l l  II.  Control of i n t r a c e l l u l a r  III.  functions  1  calcium concentration  7  1.  Mitochondria  7  2.  Endoplasmic and s a r c o p l a s m i c r e t i c u l u m  8  3.  Plasma membrane  9  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) (ii) (iii) (iv) (v) (vi) (vii)  19 20 20 21 26 30 31  Phospholipid ATP Divalent cations C a / " l ° d u l in Monovalent c a t i o n s Phosphorylation Other r e g u l a t o r s 2 +  c a  m  IV.  Calmodulin and c a l m o d u l i n - r e g u l a t e d systems  33  V.  Calpain  39  vi i i  VI.  1.  Overview  39  2.  Regulators  47  3.  Substrate s p e c i f i c i t y  50  4.  S u b s t r a t e s and f u n c t i o n s o f c a l p a i n  53  O b j e c t i v e s o f the  study  58  MATERIALS AND METHODS  60  I.  Materials  60  II.  Methods  65  1.  Protein concentration determination  65  2.  Determination  65  3.  C a l c u l a t i o n s o f the c o n c e n t r a t i o n s o f f r e e Ca and M g  66  Polyacrylamide gradient autoradiography  67  o f i n o r g a n i c phosphate  z +  4.  5.  gel  e l e c t r o p h o r e s i s and  P r e p a r a t i o n o f membrane-free  hemblysate and  c a l m o d u l i n - d e p l e t e d membranes 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  7.  I s o l a t i o n and p u r i f i c a t i o n membrane-free  68 2 +  o f the C a - A T P a s e  of calpain  from  hemolysate  70 z +  8.  Determination  9.  D e t e r m i n a t i o n o f the a c t i v i t y o f the p u r i f i e d Ca -ATPase D e t e r m i n a t i o n o f the p h o s p h o r y l a t e d i n t e r m e d i a t e  o f the membrane-bound C a - A T P a s e  z +  10.  z +  o f the C a - A T P a s e  71  72 72  11.  R e c o n s t i t u t i o n o f the p u r i f i e d  12.  Determination activities  69  2 +  Ca -ATPase  z +  o f the C a - A T P a s e and C a  2 +  73 transport  o f the r e c o n s t i t u t e d c a l c i u m pump  13.  Determination  of calpain a c t i v i t y  (caseinolysis)  14.  Treatment of c a l m o d u l i n - d e p l e t e d membranes w i t h various proteases ix  73 74  75  15.  Treatment  o f the p u r i f i e d  Ca  -ATPase with c a l p a i n  16.  Treatment o f the r e c o n s t i t u t e d C a ^ - A T P a s e with purified calpain  17.  Formation o f the p h o s p b o r y l a t e d i n t e r m e d i a t e the r e c o n s t i t u t e d Ca -ATPase  18.  PEST sequence i d e n t i f i c a t i o n  +  of  and PEST s c o r e  calculation 19.  Data a n a l y s i s  RESULTS I.  II.  III.  Purification  and c h a r a c t e r i z a t i o n  1.  P u r i f i c a t i o n of  2.  Characterization of  of  calpain  calpain calpain  E f f e c t o f c a l p a i n on membrane-bound the Ca^ -ATPase 1.  E f f e c t o f t r y p s i n , papain and c a l p a i n on the a c t i v i t y o f the membrane-bound Ca -ATPase  2.  P r o t e c t i o n by c a l m o d u l i n o f the membrane-bound Ca -ATPase a g a i n s t 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  3.  F u r t h e r c h a r a c t e r i z a t i o n o f the c a l p a i n d i g e s t i o n o f the membrane-bound Ca -ATPase i n the absence and the presence o f c a l m o d u l i n +  P r o t e o l y s i s o f 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 p •  1.  C a l p a i n d i g e s t i o n o f the p u r i f i e d  2.  P u r i f i c a t i o n o f the c a l m o d u l i n - b i n d i n g o f the  3. IV.  Ca^ -ATPase fragments  z +  Ca -ATPase  T r y p s i n fragmentation  o f the  +  Ca^ -ATPase  C h a r a c t e r i z a t i o n o f the u n t r e a t e d and calpain-treated 1 i p o s o m e - r e c o n s i t u t e d Ca -ATPase (Ca pump) 1.  E f f e c t o f A23187, a l a m e t h i c i n the ATP h y d r o l y t i c a c t i v i t y Ca pump  and T r i t o n X-100 on o f the r e c o n s t i t u t e d  2 +  p •  2.  E f f e c t o f c a l p a i n on the Ca*- - t r a n s p o r t  and ATP  hydrolytic activity 3.  o f the Ca^  pump  136  P r o t e c t i v e 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 o f the r e c o n s t i t u t e d C a pump by calpain  149  K i n e t i c p r o p e r t i e s o f the i n t a c t and c a l p a i n - t r e a t e d Ca pump i n p h o s p h a t i d y l c h o l i n e v e s i c l e s  153  z +  4.  2 +  V.  S u s c e p t i b i l i t y of other calmodulin-binding p r o t e i n s  and  t r o p o n i n C s u p e r f a m i l y p r o t e i n s to c a l p a i n  156  1.  P r o t e o l y s i s o f adducin and neuromodulin by c a l p a i n  161  2.  E f f e c t o f c a l p a i n on the j u n c t i o n a l  165  3.  Resistance of troponin C superfamily proteins calpain  SR p r o t e i n s to  DISCUSSION I.  172  D i s c u s s i o n o f the experimental  results  and c h a r a c t e r i z a t i o n  172  1.  Purification  2.  C a l p a i n a c t i v a t e s the A T P - h y d r o l y t i c o f the C a - A T P a s e  of calpain  4.  173  z +  Fragmentation o f 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  Comparison of experimental laboratory  II.  r e s u l t s from  193  this  and from o t h e r l a b o r a t o r i e s  197  S i g n i f i c a n c e o f t h i s work  203  1.  P h y s i o l o g i c a l and p a t h o - p h y s i o l o g i c a l s i g n i f i c a n c e  203  2.  U s i n g c a l p a i n as a t o o l  207  3.  U s i n g the p r o t e o l y t i c  z +  p a t t e r n o f the C a - A T P a s e as  a means o f i d e n t i f i c a t i o n III.  174  z +  C a l p a i n a l s o a c t i v a t e s the C a - t r a n s l o c a t i n g a c t i v i t y o f the C a - A T P a s e z +  5.  172  activity  z +  3.  169  Calmodulin-binding proteins 1. 2.  o f the enzyme  as c a l p a i n s u b s t r a t e s  Calmodulin-dependent enzymes C y t o s k e l e t a l / s t r u c t u r a l and o t h e r  xi  calmodulin-binding  208 208 212  proteins  215  3.  Substrate  s p e c i f i c i t y of c a l p a i n  216  4.  PEST sequences i n c a l m o d u l i n - b i n d i n g p r o t e i n s  218  5.  Recognition of calmodulin-binding proteins  224  6.  PEST sequences i n other c a l p a i n s u b s t r a t e s  230  7.  R e s i s t a n c e o f the t r o p o n i n - C s u p e r f a m i l y  240  8.  Future d i r e c t i o n s  by c a l p a i n  to c a l p a i n  241  CONCLUSIONS  243  BIBLIOGRAPHY  246  APPENDIX  284  xi i  LIST OF FIGURES FIGURE  Page  1  Three-dimensional s t r u c t u r a l  2  Reaction' c y c l e o f the plasma membrane C a ^ - A T P a s e  14  3  Amino a c i d sequence o f the plasma membrane Ca -ATPase from a human teratoma  23  4  5  model o f c a l m o d u l i n +  Schematic f r a g m e n t a t i o n by t r y p s i n  5  +  o f the C a ^ - A T P a s e produced 27  Diagrammatic r e p r e s e n t a t i o n o f the f l i p - f l o p mechanism f o r c a l d e s m o n / c a l m o d u l i n - c a l d e s m o n / a c t i n and t a u / c a l m o d u l i n - t a u / t u b u l i n interaction  37  6  Schematic o f the domain s t r u c t u r e o f c a l p a i n  41  7  Mechanism o f h y d r o l y s i s o f the s u b s t r a t e s by c a l p a i n  44  p e p t i d e bond o f  8  Schematic o f the s t r u c t u r e o f c a l p a s t a t i n  48  9  Schematic p r e s e n t a t i o n o f the i n t e r a c t i o n between the c l e a v a g e s i t e o f s u b s t r a t e and the a c t i v e s i t e of calpain  51  DEAE-Sephacel chromatography o f c a l p a i n I human e r y t h r o c y t e hemolysate  80  10  11  from  P h e n y l - S e p h a r o s e chromatography of DEAESephacel - i s o l a t e d c a l p a i n I  82  Omega-hexylamine agarose chromatography o f p h e n y l Sepharose-purified calpain I  84  Sephacryl S-200 chromatography o f omega-hexylamine agarose-purified calpain I  86  14  P u r i f i c a t i o n o f c a l p a i n from human e r y t h r o c y t e s  88  15  A u t o l y s i s o f p u r i f i e d c a l p a i n (A) and e f f e c t assay temperature on c a l p a i n a c t i v i t y (B)  93  12  13  16  p. A c t i v a t i o n o f c a l p a i n by Ca and e f f e c t v a r i o u s i n h i b i t o r s on c a l p a i n a c t i v i t y  xi i i  of  of 96  T i m e - c o u r s e o f the e f f e c t o f p r o t e a s e s 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 concentration on the membrane-bound Ca -ATPase a c t i v i t y E f f e c t o f c a l m o d u l i n on the a c t i o n o f on the membrane-bound Ca -ATPase  proteases  E f f e c t o f c a l m o d u l i n on c a l p a i n a c t i o n on the membrane-bound Ca -ATPase E f f e c t o f c a l m o d u l i n on the p r o t e o l y t i c a c t i v a t i o n o f the Ca -ATPase a c t i v i t y by c a l p a i n (A) and the C a - A T P a s e a c t i v i t y (B) z +  z +  A c t i v i t y o f the membrane-bound C a - A T P a s e t r e a t e d with c a l p a i n i n the presence and absence o f calmodulin Formation o f the p h o s p h o r y l a t e d i n t e r m e d i a t e o f the n a t i v e membrane-bound Ca -ATPase and the fragments produced by c a l p a i n treatment E f f e c t o f c a l p a i n on the p u r i f i e d Ca activity  -ATPase  Fragmentation o f 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 f o r m a t i o n o f the phosphorylated intermediate z +  S e p a r a t i o n o f the C a - A T P a s e fragments with a c a l m o d u l i n - a g a r o s e column P u r i f i c a t i o n o f the c a l m o d u l i n - b i n d i n g fragments o f the C a - A T P a s e from c a l p a i n - t r e a t e d e r y t h r o c y t e membranes Comparison o f the f r a g m e n t a t i o n p a t t e r n s o f the p u r i f i e d C a - A T P a s e o b t a i n e d by c a l p a i n and trypsin digestion E f f e c t o f A23187, a l a m e t h i c i n and T r i t o n X-100 on the r e c o n s t i t u t e d C a - A T P a s e a c t i v i t y E f f e c t o f c a l p a i n on the i n i t i a l r a t e o f both c a l c i u m uptake and A T P - h y d r o l y s i s of the reconstituted C a pump z +  E f f e c t o f i n c r e a s i n g c a l p a i n c o n c e n t r a t i o n on the i n i t i a l rate of C a 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 r e c o n s t i t u t e d C a pump z  z +  T i m e - c o u r s e o f the e f f e c t o f c a l p a i n d i g e s t i o n on the i n i t i a l r a t e o f Ca uptake and ATP hydrolytic activity Time-course of calpain-induced p r o t e o l y s i s the r e c o n s t i t u t e d C a pump  of  E f f e c t o f c a l m o d u l i n on the p r o t e o l y s i s o f reconstituted C a pump  the  z +  z +  C a l c i u m dependence of the i n i t i a l r a t e o f Ca uptake o f the r e c o n s t i t u t e d C a pump 2 +  +  C a l c i u m dependence o f the C a ^ - A T P a s e a c t i v i t y proteoliposomes Effect of C a  z +  in  o f ATP c o n c e n t r a t i o n on the i n i t i a l r a t e uptake o f the r e c o n s t i t u t e d C a pump 2 +  P r o t e o l y s i s of human e r y t h r o c y t e adducin and bovine b r a i n neuromodulin by c a l p a i n P r o t e o l y s i s of junctional p r o t e i n s by c a l p a i n  sarcoplasmic  reticulum  R e s i s t a n c e o f the t r o p o n i n C s u p e r f a m i l y Ca - b i n d i n g p r o t e i n s to c a l p a i n  of  Proposed model f o r the c o n t r o l o f the human e r t h r o c y t e membrane Ca -ATPase by c a l m o d u l i n and c a l p a i n F l o w - c h a r t f o r the fragmentation o f the e r y t h r o c y t e Ca -ATPase by c a l p a i n i n the presence and absence of calmodulin z +  Schematic o f the p r o t e o l y s i s o f the C a - A T P a s e by trypsin 2 +  Schematic o f the p r o t e o l y s i s o f the C a - A T P a s e by calpain I Schematic o f the formation o f the plasma membrane calpain i n the absence (-) (+) o f c a l m o d u l i n (CaM)  of a c t i v e fragments pump produced by and the presence  Proposed model f o r the dual c o n t r o l o f the plasma membrane C a pump by c a l m o d u l i n and c a l p a i n i n a living cell 2 +  xv  L o c a l i z a t i o n o f PEST r e g i o n s , c a l m o d u l i n - b i n d domain and c a l p a i n cleavage s i t e w i t h i n human brain a-fodrin Model o f c a l p a i n - m e d i a t e d p r o t e o l y s i s calmodulin-binding proteins  xvi  of  LIST OF TABLES  TABLE  page  1  Calpain substrates  54  2  P u r i f i c a t i o n o f c a l p a i n from human e r y t h r o c y t e s  91  3  E f f e c t o f c a l p a i n treatment i n the absence and presence o f c a l m o d u l i n 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  E f f e c t o f c a l p a i n treatment on the degree o f c o u p l i n g and C a * / A T P r a t i o o f the l i p o s o m e reconstituted C a pump  140  Protective e f f e c t of calmodulin against p r o t e o l y t i c activation o f the r e c o n s t i t u t e d Ca -ATPase by c a l p a i n  152  Matching o f c a l m o d u l i n - b i n d i n g p r o t e i n s substrates  210  4  z  z +  5  6  and c a l p a i n  7  E f f e c t o f c a l p a i n on c a l m o d u l i n - d e p e n d e n t enzymes  213  8  PEST sequences o f c a l m o d u l i n - b i n d i n g p r o t e i n s  220  9  PEST sequences o f P E S T - c o n t a i n i n g c a l p a i n s u b s t r a t e s  231  Non-PEST-containing calpain substrates  237  10  xvi i  LIST OF ABBREVIATIONS  %  percent phosphorus-32  A  ampere  a.a.  amino a c i d  ADP  adenosine  Ah  aromatic  AMP  adenosine  5'-monophosphate  ATP  adenosine  5'-triphosphate  9  5'-diphosphate hydrocarbon compound  I  Ca  f r e e c a l c i u m ion 2 +  Ca ATPase  calcium-stimulated,  magnesium-dependent  CaM  calmodulin  CaM-PK II  calmodulin-dependent  cAMP  adenosine 3 ' : 5 ' - c y c l i c  cAMP-PK  c y c l i c AMP-dependent p r o t e i n  CANP  calcium-activated  CBP  calcium binding protein  Ci  Curie  cpm  counts per minute  CRC  calcium release  DAG  Diacylglycerol  DEAE  d i e t h y l aminoethyl  DMD  Duchenne muscular d y s t r o p h y  DTT  dithiothreitol  protein  kinase  II  monophosphate  neutral  kinase  protease (calsequestrin)  channel  xvi i i  ATPase  E  enzyme  EDTA  ethylenediaminetetraacetic  EGF  epidermal  EGTA  ethyleneglycol bis(^-aminoethylether) t e t r a a c e t i c acid  EP  phosphoenzyme  ER  endoplasmic r e t i c u l u m  FPLC  fast  g  gram  Hepes  4-(2-hydroxy  HMG-CoA  3-hydroxyl-3-methyl-glutaryl  IOV  i n s i d e out v e s i c l e  K  Michaelis-Menten constant  m  growth  acid  factor N,N,N',N'-  intermediate  p r o t e i n l i q u i d chromatography  ethyl)-l-piperazineethane-sulfonic coenzyme A  kDa  kilodalton  L  litre  m  milli  M  molar  M.W.  molecular  MAP-2  microtubule associated protein-2  MBP  myelin b a s i c protein  min  minute  MLCK-G  c h i c k e n g i z z a r d myosin l i g h t  chain kinase  MLCK-S  skeletal  chain kinase  mol  mole  MOPS  morpholinopropanesulfonic acid  M  relative  r  PAGE  weight  muscle myosin l i g h t  m o l e c u l a r mass  p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s  xix  acid  PDE  phosphodiesterase  P-j  inorganic  PI  phosphatidyl i n o s i t o l  PS  phosphatidyl serine  PK-C  protein  PM  plasma membrane  PMSF  phenyl m e t h y l s u l f o n y l  RII  regulatory  s  second  S.E.M.  standard e r r o r  SDS  sodium d o d e c y l s u l f a t e  SR  sarcoplasmic  T3  3,3',5'-triiodothyronine  T4  thyroxine  TCA TEMED  phosphate  kinase C  fluoride  s u b u n i t o f type  II  cAMP-PK  o f the mean  reticulum  trichloroacetic  acid  N,N,N',N'-tetramethylethylenediamine  TLCK  N - a - p - t o s y l - L - l y s i n e chloromethyl  Tri s  t r i s(hydroxymethyl)ami nomethane  V  maximum  H  m a x  velocity  micro  xx  ketone  INTRODUCTION  I.  C a l c i u m and c e l l  functions  Calcium i s one o f the e s s e n t i a l elements o f e u k a r y o t i c o r g a n i s m s . vertebrates,  In  i n c l u d i n g man, over 99% o f body c a l c i u m i s immobilized i n  the  bones and t e e t h by complexing w i t h phosphate to form h y d r o x y - a p a t i t e .  The  remaining  the  calcium is d i s t r i b u t e d  intracellular o f the  space.  Extracellular  blood plasma,  controlled  mainly  between  the  extracellular 1987). varies. cells  contain to  intracellular Typically,  level. the  other  is  1.5  contain  the  is  least  2 +  2-orders  This results  plasma membrane  in of  an  of  cells  /iM  (Long  only  Ca  2 +  magnitude  an  and  (Ca )  that  out  of  a  (see  is  bone the  Carafoli,  calcium concentration  and Mouat,  1972),  Carafoli,  fraction  (Hodgkin  concentration  Such  in  have 4 mM (see  pool,  including  2 +  form  intracellular 20  and  In a d d i t i o n , about 50% o f  and  is  between  lower  than  a strong electrochemical cells.  fluid  This calcium level  calcium  ionized  ionized Ca  of  calcium.  only  extracellular  calcium  about 3 mM.  total  mM and heart  cytosolic free at  in  hand, the  Erythrocytes  contrast  which  calcium exists  On the  at  mobilization  d e p o s i t s and the i n t a k e o f d i e t a r y  extracellular  calcium concentration,  i s maintained  by  the  1987). the  Keynes, 10"  5  the  gradient  arrangement  of  brain In  total 1957).  and 1 0 "  8  M,  extracellular of  enables  Ca  across Ca  2 +  to  f u n c t i o n as an i n t r a c e l l u l a r messenger.  A  cell  can  be  considered  1  as  an  entity  in  itself,  using  the  plasma  membrane  important the  to  cell.  effector  as  develop Such  the  on i t s  coupling  systems  effector  (i)  whose  outside by  a  is  known  is  to  their  activity  of  Axelrod,  is  types  growth  and  proteins  C  coupled  of  by  Kahn,  functions  the  both  (Sibley  various  cyclic  dependent p r o t e i n  al., ion  1988).  al..,  as  a  in t u r n , been  Adenylate  cyclase,  and cAMP  in  turn  of  once  1987). and  interact  cAMP  2  is  an  as  1987),  A2  (see  with  the  being cGMP-  (Pl)specific (Jelsema  and  voltage-dependent activated,  activates  the  These k i n a s e s can p h o s p h o r y l a t e a l a r g e is  their  G-proteins  (Levitzki,  a  also  another group o f  identified  including  are  receptor  (ii)  channels,  1987).  with  certain  Carpenter,  phospholipase  One such t a r g e t p r o t e i n et  and  signals  binding  phosphatidyl i n o s i t o l  1987),  AMP (cAMP),  kinases.  proteins. (Hofmann  et  have  exerts  receptors  1986;  cyclase  1986),  of  receptor-effector  signal-transducing  effectors  (Stryer,  (Allende,  to  adenylate  hormones  external  factor  These G - p r o t e i n s ,  several  and  ATP to  of target  be  is  "receptor-  selectively  and  respective  enhanced  White  G-'proteins:  1987)  channel  to  date,  phospholipase  as  it  inside  called  such  "activated"  Two d i f f e r e n t  for a review).  To  and the  so  stimuli,  Yet,  r e s p e c t i v e r e c e p t o r p r o t e i n s . Upon  and epidermal  (see  class  phosphodiesterase  channel  the  achieved  molecule  transduce  insulin  this  1987,  coupled  convert  receptor  ligands  effectors.  +  environment.  and c o u p l i n g i s achieved i n t r a m o l e c u l a r l y .  receptors  Ca^  is  Generally,  effector(s).  tyrosine-kinases  Therefore,  outside  binding s i t e of t h e i r  effect(s)  respective  the  can be regarded as l i g a n d s which i n t e r a c t  binding,  differently:  to  communication between  communication  the e x t r a c e l l u l a r  Gilman,  barrier  c o u p l i n g " mechanism.  growth f a c t o r s ,  ligand  its  will cAMPnumber  a v o l t a g e - d e p e n d e n t Ca^  therefore  regarded  as  an  intracellular  messenger.  Pl-specific  phospholipase  breakdown o f plasma membrane p h o s p h a t i d y l to generate  inositol-triphosphate  turn  enhances  even  at  the  2 +  Ca  submicromolar  kinase a l s o regulates On  the  other  stores  Ca  2 +  IP3  level  below)'  demonstrated  can  al...  will  mobilize  both The  be c o n t r o l l e d  1988).  of G-protein  certain  hormone  c h a n n e l s and a l l o w C a Ca  2 +  2 +  kinase  2 +  C to  from  1984).  1987).  calcium  The  dependent  Ca  elevated  2 +  cause  2 +  systems  regarded  channel  receptors  is  1987).  brief  as also  (Rosenthal  c h a n n e l s which  (Spedding,  will  also  Ca  G-protein-coupled  e n t e r the c e l l  proteins.  2+  are  it  This  intracellular  voltage-dependent  also exist  DAG i n  various Ca -dependent IP3  (PIP2)  activate  (Nishizuka,  (Berridge,  and  stimulation  to  2 +  the  appear  Conceivably,  opening  p a s s i v e l y and e l e v a t e  of  Ca  2 +  cytosolic  concentration.  It concentration  i s thought (Ca  2 +  2 +  intracellular  Ca  (Kretsinger,  1976).  t h a t the t r a n s i e n t l y  transient) receptors, For  functions  which  conformational  indirectly  d i s s o c i a t e s the adjacent  a  facilitated  molecular  changes  actin-myosin  mechanism  of  are  example,  undergoes  is  (ER)  Various non-voltage  of  Ca  Ca  DAG  by  t o be independent types  of  in turn a c t i v a t e  messengers. to  protein  (DAG).  f u n c t i o n by p h o s p h o r y l a t i o n o f t a r g e t  Therefore,  intracellular  et  concentrations  (such as endoplasmic r e t i c u l u m  cytosolic (see  hand,  of  mediates  4,5-bisphosphate  ( I P 3 ) and d i a c y l g l y c e r o l  sensitivity  cell  inositol  C  muscle  to  c y t o s o l i c free  "activate"  a group o f  upon  (see  elevated  binding  Leavis  and  2 +  of  Ca  contraction  3  which (Squire,  2 +  ,  Gergely,  is  proteins  troponin-C 1984)  The end involved  1983).  2 +  so-called  Ca -binding  t r o p o n i n - I from a c t i n .  interaction,  the  Ca  and result  in  Calcium  the ion  also  binds  1982). of  to  In  Ca  an u b i q u i t o u s  fact,  protein  called  c a l m o d u l i n and t r o p o n i n  -binding  proteins.  the  2 +  Ca -binding  Kretsinger, helices (Fig.  1976).  connected  1)  C  Generally, by  a  the  peptide after  functions  via  (Cheung,  1980;  Calmodulin its  (EF  binding proteins w i l l  and  superfamily  and c o n t a i n  loop)  that  appears t o  well  known  with  various  1984).  to  chelates 1973).  be a  regulate  2 to  1973;  the  two Ca  Unlike  2 +  the  multifunctional  various  cellular  calmodulin-binding  proteins  A detailed  be presented  Nockolds,  i s formed by the  and N o c k o l d s ,  calmodulin  and K l e e ,  same  acidic  (Kretsinger  Kretsinger  is  highly  E-F hand s t r u c t u r e  loop  interactions  Manalan  are  and Vanaman,  E-F hands, which i s the name g i v e n  parvalbumin  specific function,  receptor.  and i t s  in  (nomenclature  troponin Ca^  loop  (Klee  C belong t o the  These p r o t e i n s  t o 4 c a l c i u m - b i n d i n g domains, termed  calmodulin  account o f  calmodulin  later.  pi  Another attention binding,  is  the  et  below.  A  lipocortin  of  or c a l p a c t i n  However,  transient  but  short  activated  1987a).  family  it  1988).  is  al.,  Recently,  protein  Ca  2 +  -  and  and  recently  neutral  has  protease  proteolyzes  A detailed  account  of  become  also  its  clear  of  Ca  2 +  that  frequency 2 +  not are  transients  transients  only  important  (for  2 +  Upon  target  calpain  will  be  review  2 +  -  given called  see K l e e ,  unclear.  magnitude (Berridge form o f  oscillations).  Ca  proteins  proteins  are s t i l l  can be i n the  (Ca  4  the  increasing  calpain.  phospholipid-binding  the f u n c t i o n s o f these p r o t e i n s  has  drawn  selected  has been d e s c r i b e d r e c e n t l y  In o t h e r words, C a spikes  that  calcium-activated  calpain  (Suzuki  1988).  Ca*- - b i n d i n g  of  the  and  Ca  2 +  Galione,  a bundle Such  of  Ca  2 +  Fig.  1  Three-dimensional  polypeptide  chain  segment r e p r e s e n t s is  represented  by  of  strutural  calmodulin  is  a  white  of  illustrated  an amino a c i d r e s i d u e . sphere.  connected by an e i g h t - t u r n c r - h e l i x . binding structures.  model  Note  calmodulin. by  the  Each o f the that  ribbon  The and  5  al.  calmodulin  (1988).  each  f o u r c a l c i u m ions has  two  Each lobe c o n t a i n s two E-F hand  Taken from Babu et  main  lobes Ca  2 +  -  gure  1  o s c i l l a t i o n s have a l r e a d y been demonstrated B e r r i d g e and G a l i o n e ,  By now, i t  level  should be q u i t e obvious t h a t C a  controlled.  eventually  N i c o t e r a et  II.  fact,  important  maintenance  of  concentration. involved  in  endoplasmic  a  death  i s an important  al.,  of c y t o s o l i c  1978;  Farber,  Ca  2 +  1981;  2 +  calcium concentration  transient  resting  subcellular  handling  are  (E.R.)  levels  compartments discussed  (including  2 +  cytosolic C a  of  that  here:  elevation  the  sarcoplasmic  is  cytosolic  the Ca  2 +  can p o t e n t i a l l y  be  mitochondria,  the  reticulum  (SR)  of  and the plasma membrane.  Mitochondria  Early amounts o f  studies Ca  2 +  demonstrated  (Vasington  that  mitochondria  and Murphy,  1962;  could  take  cytosolic  free C a  2 +  electrophoretic  .  The uptake uniporter  of C a  2 +  (Vasington  7  by m i t o c h o n d r i a and  up  DeLuca and Engstrom,  T h i s l e d t o the s p e c u l a t i o n t h a t m i t o c h o n d r i a were important  an  signal-  c o n c e n t r a t i o n needs t o be  (Chien et  submicromolar  reticulum  muscle c e l l s )  2 +  2 +  uncontrolled elevation  as c r e a t i n g  Three Ca  (see  1988).  Control of i n t r a c e l l u l a r  As  1.  In  leads to c e l l  al.,  types  1988).  t r a n s d u c i n g messenger and the c y t o s o l i c C a carefully  i n many c e l l  Murphy,  in  large 1961).  regulating  was a c h i e v e d by 1961),  whilst  a  NayCa^  exchanger  mitochondria t h a t the /iM)  protein  (Carafoli  et  was al.,  concentrations  evident  the  that  magnitude present,  below the  important  controlling several  rate that  of  However,  in  the  2 +  (Crompton  of  uptake  calcium  f r e e Ca^  by  in  the  matrix  more r e c e n t  al.,  Ca  m  about  10  2 +  at  is  are  it  one  became  order  1985).  At  believed but  mitochondrial  to  be  rather  in  matrix  calcium-dependent  of  since  (Denton  et  1980).  Endoplasmic and s a r c o p l a s m i c  Sarcoplasmic  reticulum  reticulum  (SR)  of  both  skeletal  muscle c e l l s has been the f o c u s o f r e s e a r c h on C a reasons was the of t o t a l is  Also,  concentration,  i n the are  s t u d i e s showed  (Crompton,  systems  heart  cytosolic C a  mitochondria  2 +  from  low ( K  1976).  reticulum  concentration  Ca^  to r e g u l a t e  transport  cytosolic  release  was r e l a t i v e l y  et  sarcoplasmic  regulating  dehydrogenases  2 +  Ca  mitochondrial  not  to  m i t o c h o n d r i a were u n l i k e l y  submicromolar  2.  1974).  a f f i n i t y of mitochondria f o r C a  and t h e r e f o r e  al.,  demonstrated  protein  a protein  recently  of  been  abundant  amount  in skeletal about  105  sequenced pump, e i t h e r  phospholipid  vesicles,  expense  of  hydrolyzing  review).  The  protein,  phospholamban  Ca -translocating  in  was  Several et  isoforms al.,  right-side-out shown  ATP  Ca*- -ATPase  ATPase  SR and 40-50% i n c a r d i a c S R ) .  kDa.  (see is  (Tada  to  apparently  8  al.,  in  skeletal  Brandl  and  Ca  2 +  1975).  by  One o f  the  (about  90%  al., in  pump  1986).  artificial  inwardly  a  2 +  muscle have  et  Reithmeier,  regulated  cardiac  This C a  SR v e s i c l e s o r  translocate  MacLennan  et  1985;  and  transport.  2 +  of  (MacLennan  The c a l c i u m  2 +  muscle  1985 small  at for  the a  acidic  Interestingly,  phosphorylation  of  phospholamban by  calmodulin-dependent  protein  Remtulla,  Peuch  1978;  Le  and  Ca  hydrolysis  calsequestrin terminal to  (45  et  Ca /mol  of  transverse  out  that  feetlike  are  also  Imagawa et protein the  the  1987;  alkaloid  is  et  2+  much  et  referred  al.,  (Inui  et  al.,  be r e s p o n s i b l e f o r  Katz  rate  of  the  2 +  (up  SR  projections  hexameric (Inui  Each  in  complexing C a cisternae  are  (triadic  SR.  It  turns  proteins  which  et  al.,  identical  1987b).  ATP  protein  e x i s t s mostly  for  a  and  of  2 +  channel  1988).  the  as j u n c t i o n a l  formed by  release  1974;  and  Ca -binding  The  to  kinase  1987a;  subunit  t h a t binds c a l m o d u l i n and a l s o i s the  ryanodine  is  receptor  a  for  The c a l c i u m  release  calcium-induced calcium  release  al1988).  which  similar  increased Another  are  calcium  Endoplasmic r e t i c u l u m Ca~ -ATPase  SR i s  Hymel  believed to  from SR ( L a i  al.,  system by f e e t l i k e  structures  (360-450 kDa)  plant  channel  al.,  as  1979)  calsequestrin).  tubule  This region of  known  al_.,  et  SR and has a high c a p a c i t y  junctions). the  (Kirchberger  protein  (MacLennan and Wong, 1971),  2 +  moles  anchored to the  of  kinase  translocation.  kDa)  cisternae  40-60  2 +  cAMP-dependent  to  (ER) o f nonmuscle c e l l s a l s o c o n t a i n s a s i m i l a r  transports that  of  Ca  SR.  2 +  into  However,  the the  lumen  (Moore  2 +  Ca -ATPase  et_al_., is  only  1978), a  minor  9+ component  of  total  ER  endoplasmic  reticulum  has  messenger  3  IP  (Prentki  b e l i e v e d t h a t the in signal  3.  et  proteins.  been demonstrated al..  IP -induced C a 3  Recently,  1984; 2 +  the to  Muallem  et  be  release  of  Ca  from  induced  by the  second  al..  1985).  It  is  r e l e a s e from ER p l a y s an important  transduction.  Plasma Membrane  9  now role  The  third  membrane. lies  subcellular  An e l e c t r o c h e m i c a l  across t h i s  cell,  at  L-type  least  lipid  channel,  al.,  1987).  is  (eg.  z +  Ca  two  are  of  Ca^  2-3  is  orders  To f a c i l i t a t e p a s s i v e C a 2 +  channels e x i s t  voltage-operated  the  and  2 +  blocked  but  +  Na /Ca tissue  1982,  z +  also  exchanger,  by  kDa  for  two  has  been  The exchanger  2 +  a review).  The p r o t e i n  and i s e l e c t r o g e n i c .  purify  (Hale et  co-workers  the  1987).  agents  The  called  (Hofmann  and N-type c h a n n e l s , which  contains  which  preparations.  a high maximal  attempts t o 70-82  magnitude  entry into  i s the most s t u d i e d  T-type  plasma  et are  systems  for  extruding  Ca  2 +  1987):  one mole C a /iff)  in  (Spedding,  studied  is  mostly  (Soldati  velocity  the  et  (>15  exchanger  al..  1984;  al.,  It  1985)  in  heart other  2 +  affinity  nmol/mg p r o t e i n / s )  revealed et  that  it  al.,  m  in heart.  +  while  and for  0.2-10 Recent  c o u l d be a p r o t e i n  1984)  proposed t h a t i t s  (K  and cell  (Blaustein  exchanges t h r e e moles o f N a  has a low C a  Barzilai  in  also present  types o r t i s s u e s , w i t h the e x c e p t i o n o f mature e r y t h r o c y t e s Nelson,  the  dihydropyridines.  The plasma membrane  nervous  of  handling  dihydropyridines),  The o t h e r  i n s e n s i t i v e to  (i)  gradient  bilayer.  which  antagonists  (Carafoli,  for  three classes of C a  Ca  both  membrane  Carafoli  of and  b a s i c s u b u n i t may be a  33 kDa p r o t e i n .  (ii) from the  The  Ca 2 +  2 +  pumping ATPase o f  SR C a - A T P a s e ,  A detailed  i s present  account o f t h i s  protein  plasma membrane,  i n almost will  10  all  which  is  t i s s u e s and c e l l  be g i v e n below.  distinct types.  This protein  has  a higher (0.5  affinity  nmol  1981).  of In  likely  Ca  2 +  (K  M  about  1 JJM or  Ca*- /mg membrane p r o t e i n / s ) excitable  to  However,  for  be in  cells  (e.g.  complemented  erythrocytes,  by  in  heart  heart  cells)  the  high  the  Na /Ca  where  less),  2 +  lower  (Caroni such  a  low  Na /Ca  exchanger  capacity  and C a r a f o l i ,  +  capacity  +  but  capacity 2 +  is  exchanger.  is  absent,  the  7+ Ca*-  pump alone  cytosolic  Ca  +  2 +  appears  sufficient  concentration.  to  maintain  2 +  invaluable  system to study the f u n c t i o n o f the  plasma membrane o f the  2 +  resting  storing  organelles  erythrocyte  becomes an  2 +  Ca -ATPase.  2+  The plasma membrane C a - p u m p i n q ATPase  III.  1.  the  submicromolar  Due to the l a c k o f C a  and N a / C a  exchanger,  the  Overview  It  was  reported which  almost  three  2+  a  decades ago when Dunham and Glynn  2+  2+  (Mg +Ca )-ATPase  has  activity  Subsequently,  it  (Ca -ATPase)  several-fold  was demonstrated  higher that  in  the  than  the  red  red  the  cell  (1961)  cell  first  membrane,  +  +  (Na +K )-ATPase.  membrane  contains  a  p. Ca^  pumping mechanism which u t i l i z e s  ATP as an energy  calcium against  a chemical and e l e c t r o c h e m i c a l  Schatzmann  Vincenzi,  Ca  2 +  the  and  pumping a c t i v i t y finding  that:  2 +  both  require  both  (Mg +Ca )-ATPase  2+  2+  on,  2 +  activities the  Later  and the C a - A T P a s e  (i) Mg  1969).  Ca  2 +  the  (Watson  11  1969; et  al..,  1971)  2+  and  the  based on  Ca -dependent  Dunham and G l y n n ,  1966;  between  became c l e a r ,  and  extrude  (Schatzmann,  association  activity  transport  (Lee and S h i n ,  gradient  source to  ATPase  1961); active  (ii) Ca  2 +  transport  (Cha  et  triphosphates; +  al.,  (iii)  2+  (Mg +Ca )-ATPase transported  Ca  2 +  (Schatzmann,  activity  in  the  1979).  and  velocity  of  and  the  Ca  2 +  and  TWs  regulation  study and They  a protein  for  of  the  Blostein  (EP)  of  a  3 2  intermediate  p o l y a c r y l a m i d e gel o f about and  is  Ca  2 +  of  Ca  150 kDa. rapidly  that  have  membrane  been  resealed  2 +  )  between  (especially  calmodulin  visualize micromolar  using  vesicles  early with  respect  is  2 +  Mg -and  association in  detail  membrane  later.  the  A different  plasma  et  3 2  as  of  12  et  the  al.,  to  maximum  its fact  al.,  (1974)  the 2 +  ,  1984). to  and  the Katz  2 +  Ca -ATPase. induced  the  acyl-phosphoprotein  substrate.  1980;  that  approach  calcium  at  The EP reaches a steady s t a t e l e v e l (Enyedi  kinetic  et  al.,  hydroxylamine-sensitive  [7- P]ATP  permeable  Roufogalis,  membrane-bound  concentrations  this  i n c l u d i n g ATP, M g  Al-Jobore  by Knauf  of  on the  by the  factors, (see  (see  reports  e l e c t r o p h o r e s i s the EP migrated  over  the  2 +  Sr  including  can now be e x p l a i n e d  discussed  P-labelled  turned  in  and  reviewed  preparations,  by many i n t e r a c t i n g  be  to  1966)  2 +  pumping ATPase from plasma  2 +  (1975)  Ca  Other accounts o f the  C a - A T P a s e was developed  demonstrated  formation  the  activator,  will  for  nucleotide  1979).  Ca^ -ATPase  2 +  can s u b s t i t u t e  Schoffeniels,  1969).  inside-out  Ca -ATPase is regulated  other  g h o s t s by a mechanism which i s  discrepancies  and a f f i n i t y  use ATP over  o f work was done on the c h a r a c t e r i z a t i o n  types  Considerable  properties  and  )  Ca -ATPase  discovery of  fragments  2 +  2 +  the  a g r e a t deal  (Sr  (Wins  Roufogalis,  different  membrane  ion  (Olson and C a z o r t ,  1969;  o f red c e l l , enzyme  Strontium  transport  Since  specifically  from r e s e a l e d red c e l l  ATP-dependent of  1971)  Upon  SDS-  a m o l e c u l a r weight i n seconds at  Allen  et  al.,  4-6°C 1987).  The f o r m a t i o n  of  EP can be understood by examining  s t e p s o f the enzyme. from the work o f Graf  Fig. 2 is a  a number of  and P e n n i s t o n ,  Adamo et  al.,  (starting  1988):  In  from p a r t i a l  pi and Ca  1981;  p Q  phosphate.  Ej  reaction  teams  Kosk-Kosicka e t _ a J L , the model, the 1 )  Ca  (Rega and Garrahan  1986;  Allen  et  al..  c y c l e normally operates 2 +  .j  1975; 1987;  clockwise  represents c y t o s o l i c free  n  Ca  2 +  I  i s Ca^  U t  partial  scheme f o r the r e a c t i o n c y c l e , compiled  investigating  reaction  the  extruded to the e x t r a c e l l u l a r  and  ^  represent  two  medium.  conformational  P^ i s  states  of  inorganic the  Ca  2 +  -  ATPase.  The t r a n s p o r t Ca  2 +  to  a  high  enzyme which surface)  is  cycle  is  initiated  affinity  binding  followed  by the  (reaction  1),  site  an a s p a r t a t e  the ATP i s  formation  of  However, M g Garrahan  CaEjP  2 +  and  reactions  residue  conformational  does  the not  the  1978)  change t o  of  catalytic  probably  binding  split  2 +  into  (EjP)  site  Mg  The  become CaE2P.  the  (Rega  cytosolic  region the  of  the  cytoplasmic  ADP and P^ with  v i a the  and  carboxyl  2).  the  Apparently,  Garrahan,  subsequent  complex  then  This partial  the  group  1975).  (Rega and G a r r a h a n ,  affecting CaEjP  to  (reaction  EP f o r m a t i o n by  of  cytoplasmic  ATP ( a l s o  enzyme  require  cycle.  the  rapidly  appears to a c c e l e r a t e Rega,  of  at  on  binding  i n o r g a n i c phosphate now l i n k e d t o the of  upon the  1975;  partial  undergoes  reaction  is  a  thought  p. t o be concomitant with the t r a n s l o c a t i o n o f Ca^ side Ca  2 +  of  the  enzyme  (Sarkadi,  i s reduced by s e v e r a l  concentrations  of  phosphoprotein  (Lichtner  1980).  At  the  same t i m e ,  o r d e r s o f magnitude  c a l c i u m were  found  and Wolf,  to  1980;  13  towards the  (Sarkadi,  result Allen  in et  a al.,  the  extracellular affinity  1980). large  Excessive  build  1987).  for  up  of  T h i s was  Fig.  2 R e a c t i o n c y c l e o f the  cycle, Ca^  n  the  reactions  and C a ^ .  of  the  represent  ou  respectively.  Ej  enzyme.  indicated  in  proceed Ca^  plasma membrane Ca clockwise.  brackets.  See  Ca r e p r e s e n t s  i n the c y t o s o l and the  and E 2 r e p r e s e n t Requirements  -ATPase.  of  the  other  text  for  reactions.  14  two  different  ligands details  in  the  calcium  extracellular conformational  partial of  During a normal ion.  space, forms  reactions  individual  are  partial  Ca * 2  ATP  in  • 1 ^  A  (1a)  CaEt  (1b)  ADP  Ca^ATP ^  (2)  (Mg ) ?  (Mg )  2 +  /  CaEiP 2 +  (5) (ATP)  EoP Pi  V  (4) Ca  (3)  CaEoP 2+  out  attributed z +  same C a  to  inhibition  of  the  al.,  i n v o l v e s at  affinity  site  reaction of rate  of  (Rega  to  bind  CaE P  steps:  immediately to  E P  is  5)  affinity  for  a c c e l e r a t e s the  E  inhibits  Ca  2 +  rate of E  2  right  Ca  by  (reaction  2 +  from the  (ii)  the  Ca  lanthanum  (La  z +  the C a - A T P a s e  after  2 +  ion  This Ca  2 +  low  affinity)  Schatzmann  o f ATP  )  to  the  and causes EP  released, partial  the  proposed  Ej c o n v e r s i o n s i m i l a r to the  La  3 +  thereby  reaction  back t o  (1985)  The  suggests that  is  The f i n a l  3 +  4  rapid  by mM c o n c e n t r a t i o n s  1978).  2  ). to  al.,  (low  state  2  and i n o r g a n i c phosphate (P^).  E P state.  state  2  E  followed  stimulated  (Szasz et  the  release of  Application of  enzyme i n the the  2  to  the  medium  forming E  (reaction  2  (i)  extracellular  1978).  state  2  side of erythrocytes  conversion of  (high  two  apparently  Garrahan,  s t a b i l i z i n g the the  the  (ii)  extracellular  might  least  2  accumulation  the  i n h i b i t i o n o f C a - A T P a s e was a l s o observed  the  E P with w a t e r ,  step  and  In  1987).  The c o n v e r s i o n between and 5)  CaE2P.  2 +  c o n c e n t r a t i o n range  ( A l l e n et  c o n v e r s i o n from CaEjP t o  Ej  state Mg  2 +  of M g  2 +  of  2 +  that  effect  is  on the CaEjP t o C a E P c o n v e r s i o n . 2  C a l m o d u l i n a p p a r e n t l y enhances the r a t e and the C a transport However, in  the  and  ATP  little  is  reaction  Adamo et which may  al.  hydrolysis  (Al-Jobore  known r e g a r d i n g the  cycle  (Enyedi  et  al.,  et  al..,  regulatory 1980;  2 +  affinity  1984  for  site(s)  Rega  and  16  for  Ca  2 +  .  review).  calmodulin  Garrahan,  (1988) r e c e n t l y suggested t h a t c a l m o d u l i n might  e x p l a i n the enhancement o f a f f i n i t y  for  a  Ca  1980).  stabilize  Ej,  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  solubilize  to  and  further  purify  understand the  the  enzyme.  Ca-ATPase,  Solubilization  from human e r y t h r o c y t e membranes was f i n a l l y (1974).  However,  purification  subjecting  steps r e s u l t e d  the  in  remaining progress  of  99.7%  a  or  slow.  so of  means  other  to  membrane  The d i s c o v e r y t h a t  the  active  enzyme  Ca -ATPase  to  further  T h i s problem was s o l v e d by al.,  1977).  However,  separate  the  enzyme  proteins  made  the  calmodulin  reversibly  affinity  purification  2 +  al.  of  (1980)  -affinity  by the  Ca -ATPase  2 +  almostly using  instead  2 +  Ca -ATPase  suggested the  the C a - A T P a s e .  2+  the  the  activated  thus  this of  a d d i t i o n of calmodulin (Niggli reports  a  affinity  that and  et  Niggli  method.  was  found t o  the  affinity  al.,  state  al.  (1979b)  successful  1981a).  2 +  in  a low  affinity  form  T h i s confirmed  (Schatzmann  existed  and  by  during  exist  high-Ca  2 +  low  et  Subsequently,  the ghost membrane-bound C a - A T P a s e a  (Lynch  phosphatidyl serine  purified  both  use o f c a l m o d u l i n -  s i m u l t a n e o u s l y the  form and c o u l d be c o n v e r t e d t o  earlier high  1979a)  reported  phosphatidylcholine  purification, Ca  al.,  chromatography to p u r i f y  and G i e t z e n e t  using  et  the  purification  2 +  Niggli  the  from  the membrane-bound form and the s o l u b i l i z e d form o f the C a - A T P a s e and Cheung, 1978;  to  and G i e t z e n  2 +  solubilized  inactivation.  specific  of  a c h i e v e d by Wolf  the a d d i t i o n o f s o n i c a t e d p h o s p h o l i p i d s (Wolf e t unavailability  t h e r e was a need  in  Rossi,  the both  1971;  Q u i s t and R o u f o g a l i s , 1975).  Since  Ca^  transport  is  function  of  the  actual  physiological  function  of  the  p• Ca-ATPase,  this  the  enzyme  17  has  long  been  the  focus  of  efforts  in  this  field.  Earlier  work  studying  f u n c t i o n s was a c h i e v e d u s i n g r e s e a l e d e r y t h r o c y t e out v e s i c l e s ( I O V ) . have  studied  great  the  detail.  attempts  highly into  made  1979;  the to  et  et  al.,  2 +  al..  2 +  and  ATPase a c t i v i t y  it  followed  its  as w e l l  as C a  efflux  et the  al., Ca  1977; 2 +  with low C a  2 +  in  a high C a  2 +  Ca  a s s o c i a t e d with c a l m o d u l i n  and Lew,  1976;  Scharff  that  erythrocytes  Taking  advantage  al..  IOV  of  this 2 +  o f the C a  by  Roufogalis  1983).  achieved,  of  of et  this  enzyme  al_.,  1981b;  1986).  2 +  pump i s t o  study the  erythrocytes.  some r e p o r t e d  technique  Ca  t h a t the 1971;  (1988b) demonstrated  b)  and  Ca  calcium e f f l u x  by the  calcium  by  Dagher  a b s o l u t e dependence on mM c o n c e n t r a t i o n  2 +  of M g  and  efflux 2 +  .  Lew  Ca  Ca  (1984) by  2 +  2 +  in  2 +  the  detail Xu  and C a - A T P a s e  The C a - A T P a s e  the  pump.  efflux),  (1988).  2 +  Ferreira  al.  red c e l l s were re-examined  2 +  that  the  passive calcium transport  t h a t both C a  18  et  -  Sarkadi  1982;  Tiffert  2 +  Earlier  suggesting that  (cobalt-exposed  pump i n i n t a c t (1988a,  mode,  Recently,  affecting  in  availability  (Muallem and K a r l i s h ,  ion can a r r e s t  without  properties  Roufogalis  et  cobalt  intact  and  (1982)  and  (Romero and Whittam,  affinity  pump was  Xu  al.  B u r g i n and Schatzmann, 1979), w h i l e o t h e r s demonstrated  pump was  demonstrated  the  (Niggli  from i n t a c t  affinity  inside-  (Haaker  reconstitution  s t u d i e s appeared t o y i e l d c o n f l i c t i n g r e s u l t s : pump f u n c t i o n e d  after  and R o u f o g a l i s ,  2 +  transport  was  proteoliposomes  Soon  the  and Gimble et  2 +  characterization  investigate  +  plasma membrane  Ca -ATPase  into  the  Ca^  by human e r y t h r o c y t e  of  1980).  1982b; V i l l a l o b o  Another approach t o  c)  Ca -transport  solubilization  Ca -ATPase  p r o t e o l iposomes  (1981a, b,  reconstitute  Gietzen  purified  Niggli  al.  mechanism o f  After  were  Racker,  Waisman et  the  and have  activity  showed a K ATP  n  f o r ATP of 0 . 5 mM and K  5  concentration  1989b).  However,  was  between  the  K  significantly  higher  in  cells.  the  for  intact  the  free  2 +  )  for  Ca  for Ca^  1.3  mM (Xu  2 +  The authors  efflux  +  o f 0 . 2 - 0 . 4 /iM when the and  have a  Ca -ATPase  activity  suggested a number o f difficulty  inside  the  Dagher  stoichiometry of  Roufogalis,  (30-40 /imol/L  2 +  for  N  Concurrently,  5  in  cells  and  Lew  during  cells)  was  ( 0 . 2 - 0 . 4 /JM)  estimating  the  (1988)  1989a,  p o s s i b l e reasons  accurately  and calcium-dependent ATP h y d r o l y s i s by the  cells  Ca  showed  2 +  the  efflux  that  cobalt-exposed  Ca  2 +  intact  1:1.  Regulations  (i)  Phospholipid Phospholipid  is  required during p u r i f i c a t i o n  ATPase ( d i s c u s s e d a b o v e ) . 2 +  Ca -ATPase 1977). a  C a  to  K .5(Ca)  concentrations  experiments.  3.  than  (  d i s c r e p a n c y i n c l u d i n g the  Ca  efflux  0 - 5  0.2  Q  activity  Recently,  combination  glycerol,  an  that  Bzdega,  the  detergent  that  1988).  1977;  C^Eg  2 +  shows  seen  in  an the  These authors  a s s o c i a t i o n o f the  al_.,  Ca -ATPase  Interestingly,  2 +  et  phospholipid  K o s k - K o s i c k a and I n e s i  Ca -ATPase  mimics  addition,  (Ronner  active  phospholipid. purified  of  In  this  can  (instead  and  of  Triton in the  the  of  calmodulin the  Ca^ -  effect  for  Schatzmann, that,  using  X-100)  and  absence  solubilized  concentration-dependent  suggested t h a t  19  of  purified  the  i s also required  (1986) demonstrated  be  presence  enzyme.  stabilize  Roelofsen  preparation  enzyme  to  of and  activation  ( K o s k - K o s i c k a and was due to  self-  (ii)  ATP Calcium  transport  by  the  p r o d u c i n g h y d r o l y s i s o f ATP. over o t h e r 1979;  Graff  et  2 +  x  10"  6  al.,  et  (  A  al..,  c o n s i d e r e d to allow  K  T  =  2  be the  Various  Enyedi  et  to  al.,  of  x  ATP  4  M)  while  to  s p e c i f i c i t y f o r ATP  UTP)  (Sarkadi  et  (Muallem  and  binding  the  lower  modulate  the  Karlish,  site  for  affinity  activity  = 11979;  ATP  site  of  al.,  and the  A  affinity  site,  energy-  i n a b i p h a s i c manner ( K ( j p j j  10"  high  the  both c a l c i u m t r a n s p o r t  substrate  Sarkadi  et  al.,  of  the  equally  effective  on  what  as  form  enzyme  1978;  Muallem and K a r l i s h (1981)  exists  (iii)Divalent  actual  1982;  s u b s t r a t e o f the  2 +  The  strict  to  is  could  the  pump  1979).  be the  ATP are  Mg  coupled  forms o f ATP, i n c l u d i n g MgATP, CaATP and f r e e  More r e c e n t l y ,  consensus  1-3  catalytic  mM c o n c e n t r a t i o n s  suggested  ATP s t i m u l a t e s  1986).  (Muallem and K a r l i s h ,  free  j  p  is  (GTP, CTP, ITP,  (Muallem and K a r l i s h , 1979)  M and  Villalobo  1982).  pump  The enzyme has a  nucleoside triphosphates  Ca -ATPase 7  2 +  Ca  Graf  and  et  al.,  been 1977;  Penniston,  1981).  suggested t h a t MgATP, CaATP and  substrate.  of  (Wolf  ATP have  ATP  is  To the  date,  actual  no  clear-cut  physiological  enzyme.  cations  has  several  described before,  Mg  (Sarkadi,  1980).  Mg  ATP might  be the  2 +  z +  regulatory  functions  on  the  2 +  Ca -ATPase.  s t i m u l a t e s the d e p h o s p h o r y l a t i o n s t e p o f the may a l s o be r e q u i r e d  preferred  physiological  As enzyme  t o complex w i t h ATP s i n c e Mgsubstrate  for  the  pump  (Enyedi  p,  et  al.,  1982).  Millimolar  concentrations  20  of  Mg  d e c r e a s e s both  the  V  m a x  and  the  (Klinger form  affinity  for  et  1980;  (Al-Jobore  bound form enzyme have  al.,  Ca  2 +  of  et  al.,  also  showed  that  a  small  found  that  the  2 +  Mg  affinity  accelerates  the  the  (Garrahan  enzyme  for  formation  of  and  2 +  or ATP.  the  purified  al.,  1975).  1978).  the  of  the  thought  ATPase  effect  During  vesicle-  form  enzyme was  Ca -stimulated  stimulatory  solubilized  membrane  (Schatzmann,  form  to  Later activity  These authors  of  Ca  reaction  2 +  without  cycle,  Mg  2 +  p h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n o f  Rega,  1978),  although  ( A l l e n et  al.,  Mg  2 +  is  not  absolutely  1987).  2 +  Ca /calmodulin  t o the of-Ca  extracellular  2 +  and the  (Carafoli  space.  and  Zurini,  2 +  2+  that  1971;  extraction  2+  (Ca +Mg )-ATPase  affinity  state  restored  the  During the  from  1982). has  p Ca*-  has  1976).  of erythrocyte a  high  2 +  Ca  2 +  from the c y t o s o l  cycle this  an  existence  been  Scharff,  Ca  closely coupled.  The  and t h a t the r e a d d i t i o n high  reaction  formation  activities  and R o s s i ,  demonstrated  pump i s t o t r a n s l o c a t e  intermediate  Ca -ATPase  (Schatzmann  2 +  h y d r o l y s i s o f ATP are  phosphoprotein  affinity  the  the  the  2 +  of  the  1981),  and the  activity  portion  Ca  The f u n c t i o n o f the C a  2 +  1982)  for  membrane-bound  inside-out  Initially,  2 +  enhanced  r e q u i r e d f o r the EP formation  Ca  the  i n the absence o f magnesium ( R i c h a r d s et  altering  the  1981),  1986).  ghost  Roufogalis,  (Akyempon and R o u f o g a l i s ,  a b s o l u t e dependence on M g  exists  erythrocyte  A l - J o b o r e and  and R o u f o g a l i s ,  (Villalobo  studies  (iv)  the  Because o f  absolute of  suggested  translocation  dependence on  high in  this,  and  low  early  Ca  2 +  studies  Q u i s t and R o u f o g a l i s  (1975)  membranes with EDTA converted affinity  state  to  a  low  Ca  2 +  o f the E D T A - e x t r a c t o f the membrane  9.  I  affinity  of  21  the  c  Ca  -ATPase  activity.  This  suggested  the  presence  this  conversion.  et  al.  nucleotide  both  the  1970).  Shortly  simultaneously  1977b).  The in  a 3-4  increase  in  Ca  Scharff,  1981;  stimulatory ATPase was  2 +  al.,  of  et  al.,  the C - t e r m i n a l the  enzyme  putative  and  domain  Foder,  velocity  and  and  believed  that  is  solubilized  Roufogalis,  coworkers  near  the  The l o c a t i o n  imposes i n h i b i t i o n the  enzyme.  and  of  the  Ca  z +  -  and P e n n i s t o n , 2 +  Ca -ATPase a  1980;  and  30-fold  Foder a  similar  purified  1981;  and  Niggli  Ca  et  2 +  -  al.,  of  strongly  C-terminal the  suggested of  the  that  the  2 +  the  Ca -ATPase  calmodulin-binding  domain  at  been confirmed from the amino a c i d sequence o f  domain  in  Penniston  Subsequently,  1988;  Verma et  located  next  al., to  1988) and  c a l m o d u l i n - b i n d i n g domain has been suggested ( C a r a f o l i was  and  up t o  Mauldin,  cyclic  Kakiuchi  protein Jarrett  of  membrane-bound  and  the  of  Luthra factor.  activator 1970;  1977;  1982).  on  and  and Greeb,  inhibitory  activating  an  and  for  1986).  has r e c e n t l y  (Shull  the  activator  maximum  (Al-Jobore  Sarkadi  1986).  (1977a)  the  (Roufogalis  calmodulin  demonstrated  studies  in  responsible  Penniston  as  and V i n c e n z i , to  is  laboratories  the  calmodulin  that  (Cheung,  the  that  increase  of  calmodulin-binding (Sarkadi  of  Scharff  1981a; V i l l a l o b o et  The  afterward,  affinity  effect  calmodulin  surfaced  (Gopinath  fold  and  purified  of  realized  binding  factor  Jarrett  partially  discovery  ATPase was c a l m o d u l i n  results  activating  phosphodiesterase  Yamazaki, Vincenzi  an  Subsequently,  (1977)  Independently,  of  absence  of  calmodulin,  on the A T P - h y d r o l y t i c and C a  Upon b i n d i n g of the  2 +  2 +  the  (Fig. upstream et  Also, from  1987).  inhibitory  transport  C a - c a l m o d u l i n complex,  22  al.,  3).  the It  domain  activities  the  a  of  calmodulin-  Fig.  3 Amino a c i d sequence o f the plasma membrane C a - A T P a s e  teratoma.  The  sequence  terminal,  using  residues.  The number at  number o f  the  first  location  of  acylphosphate  identified  the  is  continuous  one-letter the  codes  M o d i f i e d from Verma et  (see  the  site.  that The  N-terminal  appendix)  b e g i n i n g o f each l i n e  amino a c i d o f  as i n d i c a t e d .  from  line.  23  its  represents  The l e t t e r  to  the  amino the  from the  Cacid  residue  P identifies  calmodulin-binding  Amino a c i d sequence s t a r t s  al.(1988).  for  from a human  domain  the is  N-terminal.  Figure 3  1  MGDMANNSVAYSGVKNSLKEANHDGDFGITLAELRALMELRSTDALRKIQ  51  ESYGDVYGICTKLKTSPNEGLSGNPADLERREAVFGKNFIPPKKPKTFLQ  101  LVWEALQDVTLIILEIAAIVSLGLSFYQPPEGDNALCGEVSVGEEEGEGE  151  TGWIEGAAILLSVVCVVLVTAFNDWSKEKQFRGLQSRIEQEQKFTVIRGG  201  QVIQIPVADITVGDIAQVKYGDLLPADGILIQGNDLKIDESSLTGESDHV  251 KKSLDKDPLLLSGTHVREGSGRMVVTAVGVNSQTGIIFTLLGAGGEEEEK 301 KDEKKKEKKNKKQDGAIENRNKAKAQDGAAMEMQPLKSEEGGDGDEKDKK 351  KANLPKKEKSVLQGKLTKLAVQIGKAGLLMSAITVIILVLYFVIDTFWVQ  401 451  KRPWLAECTPIYIQYFVKFFIIGVTVLVVAVPEGLPLAVTISLAYSVKKM P 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 1201  RIEDSEPHIPLIDDTDAEDDAPTKRNSSPPPSPNKNNNAVDSGIHLTIEM NKSATSSSPGSPLHSLETSL  1220 24  binding  domain  undergoes  conformational inhibition  changes  of  functions  at  Roufogalis  conformational  in  activity  is  released  full  capacity.  Niggli  et  al.,  demonstrated but  phosphatidylcholine),  can mimic  significant  difference  activators,  however:  a c i d i c phospholipids s h i f t  of  acidic  found the C a  2 +  Ko.5(Ca)  the  t  o  0  (1981b)  and  Al-Jobore  phospholipids  effect the  2  5  ^  synthetic  hyperbolic.  polyanions  M  2 +  limited  proteolysis  2 +  activates  the  Ca -ATPase  rendered  these  activities  Sarkadi  et  al..  activity  t  (Enyedi  its  by  and  V  m a x  trypsin  Ca  Al-Jobore  (e.g.  2 +  and  (e.g.  o  et  of  the  A two  0 . 5 - 0 . 6 /zM, al.,  1987).  i s sigmoidal while that et  al.  (1982)  acid  also  increased  .  or  chymotrypsin  transport  calmodulin-independent 1980;  action  poly(L)-aspartic  a f f i n i t y o f the C a - A T P a s e but not  and  calmodulin.  KQ 5(ca)  Minocherhomjee  such as  of  the  pump now  phospholipids  the  ,  2 +  Ca  between  shifts  induce  the  -dependence o f c a l m o d u l i n a c t i v a t i o n  Interestingly,  1980;  exist  turn  Subsequently,  acidic  activating  calmodulin  phospholipids is  that  to  in  and  neutral  the  appears  while  that  not  which  domain.  enzymatic  (1981)  the Ca  inhibiting  the  phosphatidyl s e r i n e ) ,  Also,  the  changes,  also  activities  (Taverna  Roufogalis,  and  and  Hanahan,  1981).  The  p r o t e o l y s i s o f the C a - A T P a s e by t r y p s i n has been s t u d i e d i n g r e a t  detail  2 +  (Zurini et  et  al.,  al..  1987;  laboratories still bind  bind but  fragments  of  1984;  Benaim et  Enyedi  et  Carafoli  al.,  not  stimulated  c o u l d not bind  1987;  and S a r k a d i  and be s t i m u l a t e d  is  al.,  1984;  Sarkadi  Olorunsogo found  that  by c a l m o d u l i n w h i l e by,  calmodulin.  or be s t i m u l a t e d  25  et  al.,  1986;  Sarkadi  et  al.,  1988).  The  a 90  kDa  fragment  can  an 85  Smaller  81  by, c a l m o d u l i n .  kDa fragment  can  kDa and 76  kDa  Interestingly,  the  81  kDa  fragment  activation  by a c i d i c  Recently,  a  but  model  of  by C a r a f o l i  cleaves  the  by  calmodulin  cleavage and  al.  removes  the  and  putative  Rossi,  90  kDa  lower  Benaim  last  by  al., to  potassium i o n  Graf  1984).  A  the  formd  active  ghosts  and  an  et  al.,  K  +  and o t h e r monovalent  fragment  Ca -ATPase  Recent  studies  either  side  K  +  from  effects  of  effect  Sodium ion from o u t s i d e  outside  in  high-K  +  (Schatzmann  (Romero  and  (Romero and Romero, 1984).  coupling  2 +  on  Ca  molecular  efflux  and  reactions  26  the  2 +  Romero, that  Na  +  a  depending  in high-choline ghosts  and  produced  extrusion,  to  loaded  elicited  only  The authors c o n c l u d e d t h a t mechanisms: ( i )  external of  membrane  loaded  the pump by two d i s t i n c t  between  the on C a  both i o n s may a f f e c t  influence  (the  c a t i o n s were found  2 +  and p u r i f i e d  1982).  from  concentration.  stimulatory  acidic  4).  (inhibitory-stimulatory)  and  further activated  inside  +  A  activated  from  K  trypsin  calmodulin-binding  u s i n g r e s e a l e d ghosts i n an a l l - c h o l i n e medium demonstrated  on the  ).  was  fragment.  1984)  bifunctional  2 +  Ca  model,  produce  kDa  to  trypsin  active  cleavage  76  for  m  but cannot be  et  domain  K  their  removes the  which binds  calmodulin-independent  both the membrane-bound 1971;  In  responded  cations  Sodium i o n , activate  4). a  that  The  (Fig.  to  Ca -ATPase  (Fig.  inhibitory  still  2 +  the  1984;  form.  fragment  a shift  produces  al.,  EP forming fragment)  Monovalent  of  C-terminal  non-calmodulin-binding  smallest  (i.e.  85 kDa fragment et  kDa  (1987)  and  the  (Zurini  phospholipid-  (v)  et  at  domain produces the  76  proteolysis  N-terminal  subsequent c l e a v a g e  the  phospholipid  presented at  not  cation 2 +  Ca -ATPase  +  (K , (see  the  electric  +  (ii)  Na ),  below).  an The  Fig. The  4 Schematic relative  fragmentation  molecular  number. The d o t t e d shaded area  at  the  molecule.  is  weight  the  of  Ca  each  -ATPase  polypeptide  produced by is  trypsin.  indicated  by  area r e p r e s e n t s the c a l m o d u l i n - b i n d i n g domain w h i l e end of the  calmodulin-activation. acylphosphate  of  The  indicated  Taken from C a r a f o l i  molecule r e p r e s e n t s active  by  site  the  shaded  et a l .  (1987).  27  a domain r e q u i r e d  capable area  in  of the  forming middle  of  a the for the the  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 ' - c a l m o d u l i n . 37°. 1/25 trypsin-enzyme ratio 2  85 short t r y p s i n e x p o s u r e in  •  Y0«"-n<T \ 3 7 ° , 1 / 2 5 J  -JL  trypsin-enzyme  ratio  long t r y p s i n e x p o s u r e in EGTA. 37°. l/25trypsinenzyme ratio  Q  "re;  V  28  )  second mechanism i s supported by t h a t Ca  2 +  affinity  that  high  internal  K  i n h i g h - c h o l i n e ghosts (Romero and Romero, 1984)  i n c r e a s e d both the al.,  fact  level  and t u r n o v e r  o f the  E-P i n t e r m e d i a t e  enhances K  (La Rocca  et  1981).  Initially monovalent the  net  the  Ca  or d i v a l e n t  positive  anion channel evidence  pump  of  carried  the  (band 3)  for  was  thought  c a t i o n s and t h e r e f o r e  charge  electroneutrality  by C a  cell  o f the  this  was  2 +  not  extrusion).  restored  from  counter-transport  would be e l e c t r o g e n i c  by  plasma membrane  came  to  It  net  was thought  CI"  (see  efflux,  Penniston,  reports  (due  that  via  blocked  transport  with  interpreted  similar  these  potency  results  were c l o s e l y c o u p l e d . Niggli  et  as  to  studies  cast  al.  that  SITS, doubt  (1981c).  On the  Since,  significantly close-coupling Ca -ATPase  under  alter exists  catalyzes  its  their  Ca  2 +  originally  :H  +  3  and  exchange.  29  the  it  4,4'-  latter  demonstrated  ATP-dependent  and  Ca  did  suggested  Ca -ATPase  Using  and  inhibitor  treatment was  2 +  processes  These  (1983)  inhibited  activity,  authors  proposed by Waismann  al.  DIDS  anion  and  anion  2 +  )-2-  (1982)  NAP-taurine  Ca -ATPase.  conditions,  band  transport  another  and  The  and R o u f o g a l i s  Smallwood et  Ca^ -ATPase  transport  two  that  the  erythrocytes  between a  inhibit  hand,  whole  the  (DIDS),  interpretation  other  of  showed  disulfonate  directly  on the  DIDS treatment  transport.  2 +  also  that  2 +  a l . , 1981c).  et  Minocherhomjee  subsequently  dfisothiocyano-2-2'-stibene related  indicating  However,  a l . (1982a)  (Waisman  Ca  The  4-acetamido-4-  aminoethylsulfonate  both  the  1983).  (SITS) and N - ( 4 - a z i d o - 2 - n i t r o p h e n y l  (NAP-taurine)  to  that  isothiocyanostilbene-2-disulfonate  et  +  and t h a t  that  that  2 +  not a the  1iposome-reconstituted  Ca  -ATPase, +  (H )  were  Niggli found  et  in  the  assumption t h a t  one  (Niggli  1981a,  as  et  al.,  evidence  protons.  (1986)  Recently,  membrane  vesicles  modulated  2 +  (vi)  Ca  and  et  2 +  also the  ATP  al.,  that  hydrolyzed.  the  extra  protons  Based  on  the  liposomes per ATP h y d r o l y z e d  1981b) they is  two  a  Ca  2 +  interpreted -2H  +  the  antiport  results that  is  Using i o n o p h o r e t i c compounds, V i l l a l o b o  showed t h a t Ca  t T  cardiac  the  pump from sarcolemma  potential  1988).  per  pump  1982b).  by membrane  Romero and O r t i z , Ca  the  demonstrated  i s pumped i n t o  Niggli  (Niggli,  and R o u f o g a l i s  (1982b)  medium  Ca^  that  electroneutral  al.  z +  Ca  pump  both  the  have  (Gassner et  red  been  al.,  counter-transports cell  inside-out  considered  1988;  to  Kuwayama,  1988;  The r e s u l t s o f these s t u d i e s suggested t h a t  pump i s e l e c t r o g e n i c by v i r t u e o f a s t o i c h i m e t r y o f  1 Ca  2 +  :  be  the  +  1H .  Phosphorylation The  laboratory  of  Carafoli  demonstrated  that  the  heart sarcolemma and from e r y t h r o c y t e  membrane are  cAMP-dependent p r o t e i n  and C a r a f o l i ,  1985).  cAMP-dependent  h e a r t and the red c e l l appear  to  affect  Subsequently, muscle (bovine kinase  the  V  m a x  kinase  (Caroni  phosphorylation  of  the  (Lamers  et  al_.,  plasma membrane C a  2 +  1981;  and  Nakamura,  1987;  1981;  2 +  Neyses et  pump  affinity Neyses  pump p u r i f i e d  a o r t a ) was shown t o be s t i m u l a t e d  (Furukawa  2 +  et  from  both  but  et  al., the  does not  al_.,  1985).  from v a s c u l a r smooth  by cGMP-dependent  Furukawa  from  both p h o s p h o r y l a t e d by  Ca  appeared t o enhance the C a  2 +  Ca -ATPase  al_.,  1988).  protein The  7+ p h o s p h o r y l a t i o n was p a r a l l e l e d by an i n c r e a s e i n both Ca^ a f f i n i t y and p, pi maximum Ca^ uptake by the r e c o n s t i t u t e d Ca pump. However, recent s t u d i e s suggested t h a t the s t i m u l a t i o n  30  2 +  o f the C a - A T P a s e  induced by cGMP-  dependent  p h o s p h o r y l a t i o n c o u l d be  V r o l i x et  al.,  purified  forms  stimulated direct  1988).  by  of  Most r e c e n t l y ,  the  protein  kinase  demonstration  of  Other  C  (Baltensperger  et  al.,  both the membrane-bound 2 +  human e r y t h r o c y t e  p r o t e i n kinase C was not  (vii)  indirect  Ca -ATPase  (Smallwood  phosphorylation  et  of  al.,  were  and the  found  1988).  to  be  However,  2 +  the  1988;  Ca -ATPase  protein  a by  reported.  regulators  Besides c a l m o d u l i n , another  2 +  protein activator  o f the C a - A T P a s e was  found i n human e r y t h r o c y t e membranes (Mauldin and R o u f o g a l i s , 1980).  This  p r o t e i n was c h a r a c t e r i z e d 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  elute  1984). that  as a 107-178 kDa p r o t e i n The a c t i v a t o r  the  bound  to  protein it  in  was  a  ATPase was produced by  also  2 +  a  1982). 20  the  endogenous p r o t e i n  been  reported  (Au,  These p r o t e i n s  kDa),  but  their  Ca -independent  inhibitors 1978;  Pedemonte  modes o f  inhibition  Au  et  1985).  The  the  exact  erythrocyte  Since  this  adenylate  function membrane  of  activator  on the this  1981;  inhibitor  cyclase  and  and s p e c i f i c i t y 2 +  Ca -ATPase  31  z +  of  also  activity  have  Wuthrich, kDa t o  differ.  Only  (Lee and A u ,  inhibitor also  -  (6  appeared  was  2 +  protein.  to  1983;  inhibit  cAMP-phosphodiesterase this  is  Ca  2 +  Ca -ATPase  characterized  it  Ca -ATPase  Balegro,  the  with  Therefore,  erythrocyte  and  protein  appeared to be o f small m o l e c u l a r weight  r e p o r t e d by Au was f u r t h e r  1978),  of t h i s  o f the  inhibitor  calmodulin-activated  manner.  al.,  was suggested  c a l m o d u l i n m o l e c u l e s bound t o  the  al..,  ( R o u f o g a l i s et  calmodulin-binding  q u i t e p o s s i b l e t h a t the s t i m u l a t o r y e f f e c t  Several  filtration  p r o t e i n a l s o c o n t a i n e d c a l m o d u l i n . It  activator  calmodulin  on g e l  is  (Au,  unclear.  stimulated  by  thyroid  hormones ( T 3 and T 4 ) ( D a v i s ,  the  basal  and T ^ - s t i m u l a t e d  The  heart  sarcolemma!  methylation  of  activity  2 +  pump was  (Ortega  trisphosphate  by o x i d a t i v e  et  Hebbel et  al.,  1987;  inhibited  the  McDonald,  this  adipocyte  1986).  plasma  enzyme  a c i d can  (Smith et  found  to  be  (Panagia  et  al.,  1986)  (Kuo and T s a n g ,  inhibited  while r e t i n o i c  also  and M a s - O l i v a ,  was p a r t i a l l y al.,  of  phosphatidylethanolamine  by c h o l e s t e r o l inositol  Ca  1981)  agents Also,  al.,  1989).  stimulated 1986)  and by guanine  1988).  inhibit  N-  inhibited  nucleotide  The e r y t h r o c y t e and a c t i v a t e d  by  and  2 +  Ca -ATPase  oxygen  (Leclerc  i n s u l i n and c o n c a n a v a l i n A a l s o  membrane  Ca  -ATPase  (Pershadsingh  and  1981).  The most  controversial  7+ Ca^ -ATPase  plasma membrane  enzymes  appeared 7+  to  be those r e p o r t e d 2+  M g ) - A T P a s e was 1981). of  found  Lotersztajn  phosphoprotein t h a t mediated 2 +  similar  (1984)  pump o f of  et  al_.,  o f the  a type  1987).  2 +  differences  plasma  2 +  2 +  (70  which  (Ca -Mg )-ATPase  al.,  1985)  and  was not  phosphoprotein o f 32  a Ca 2 +  s  (1984) Lin 2 +  kDa,  of  also  (1985a, pump,  transport.  118  fact  the G - m e d i a t e d 7+  inhibition  the  kDa)  be  that  signal  Ca^ -ATPase  isolated b)  a  by glucagon  based on the  activates  Fain  (30  and was suggested t o  is  s  al.,  formed  protein  2 +  G . This  between ATP h y d r o l y s i s and C a  a vanadate-inhibitable  membrane,  liver et  et  -  characterization  An i n h i b i t o r y  Subsequently,  kDa)  on the  2 +  (Ca  (Lotersztajn  kDa.  called  Lin  (Ca -Mg )-ATPase.  (Ca -Mg )-ATPase  a high a f f i n i t y  reported  liver  110  (Lotersztajn to  also  blocked the glucagon-mediated  2 +  affinity  2 +  First,  which n o r m a l l y permanently  transduction, (Lotersztaju  al.  inhibition  was p u r i f i e d  cholera toxin,  this  Ca  hepatocytes.  be c a l m o d u l i n - i n d e p e n d e n t  intermediate the  a G-protein,  to  et  an ATP-dependent  arfd M g  i n the  a  high  claimed  that  based on  kinetic  Lin also  reported  which was  suggested  to  be the (Lin,  authentic 1985a).  non-Ca the  plasma membrane  -transporting  ATPase,  2+  an e c t o - A T P a s e , i s  (Lin  1985a,  b).  2 +  the l i v e r  about  Recently, 2 +  plasma membrane ( C a - M g ) - A T P a s e  membrane  e c t o - A T P a s e which  site  ATP h y d r o l y s i s . B i r c h - M a c h i n and Dawson  liver  for  hepatocytes  possessed  an  10  Lin  times  a  plasma  located  active  reported  that  2 +  from those i n v o l v e d i n  hand,  using  liver  plasma  L o t e r s z t a j n demonstrated t h a t 2 +  both  Ca  al.,  1984;  transport  and  Mall at  al..  et  ATP-dependent C a membrane  2 +  the  2 +  2 +  (Ca -Mg )-ATPase  activity  On the  laboratory  glucagon and a glucagon fragment  the  a  kinetic  transport.  vesicles,  of  reported  was i n f a c t  extracellularly  the  that  (1988)  (1988)  of  plasma membrane C a - A T P a s e t h a t they observed had d i f f e r e n t  properties other  pump o f  She a l s o suggested t h a t the A T P - h y d r o l y z i n g a c t i v i t y  C a - p u m p i n g ATPase  that  ATP-dependent Ca^  of  inhibited  (Lotersztajn  1987). T h i s would suggest t h a t the  (Ca  2 +  -Mg  et 2 +  )-  9+ ATPase and the Therefore,  it  Ca is  c  pump t h a t  p o s s i b l e that  they  characterized 2 +  the  identity  of  the  2 +  (Ca -Mg )-ATPase  group o f L o t e r s z t a j n and t h a t r e p o r t e d by The  are  2 +  Ca -ATPase  the  same  reported  L i n are two d i f f e r e n t  reported  by  protein.  Birch-Machin  by  the  proteins.  and  Dawson  (T988) i s a l s o u n c l e a r .  IV.  C a l m o d u l i n and c a l m o d u l i n - r e g u l a t e d systems  Since pivotal  its  role  d i s c o v e r y (Cheung, of  become apparent  calmodulin (Cheung,  in  1980;  1970; 2 +  Kakiuchi  Ca -mediated Klee  cellular  and Vanaman  33  and Yamazaki,  1982;  1970)  regulation Manalan  and  the has Klee,  1984;  Wang  eukaryotic  et  al..,  1985).  organisms and i n  is a small, acidic protein species  has  been  conservation  was  belongs to the the  E-F  hand  activity lysine et  all (16.5  kDa).  troponin  (see  al..  1980),  phosphorylation demonstrated al.,  1987;  Kubo  and  at  Strott,  including  al.,  N-methylation  1987;  1988).  1983;  Church et  Evidence  in  functions  appear  mitosis,  be  enzymes the  (b)  cytoskeleton  enzymes c a t e g o r y  phosphofructose  are  kinase)  biological  effect  residues  et  al.,  of  a  1986;  can  be  categorized  (structural)  proteins  two m e t a b o l i c (Buschmeier et  34  enzymes al.,  on  and (c)  et  1988;  role  for  1981).  by  (Means  into  et  calmodulin,  three  protein  al.,  these  1982;  cellular Vertebrate  groups:  a)  miscellaneous.  In  (phosphorylase  1987)  al..  exocytosis,  calmodulin  been  Colca  regulatory al.,  and  have  Ghosh e t  regulated  transport  terminal  1981)  v i a various calmodulin-binding p r o t e i n s .  proteins  containing  (Sitaramayya  al.,  metabolism,  of  f u n c t i o n s are e x e r t e d  Calmodulin  its  115  et  (Gagnon et  Manalan  The  Fukami  to  cAMP  lysine  serine  favour  2 +  calmodulin-binding  of  of  post-translational  regulate  a l . . . 1988;  has been demonstrated  1984).  1984).  Several  can p o t e n t i a l l y  threonine  It  degree  2 +  and L a z a r i d e s ,  motility,  Klee,  Klee,  high  enzymatic removal o f the c a r b o x y l  1983),  all  1982).  of C a - b i n d i n g proteins  p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n and C a and  and  and  cellular cell  Manalan  tyrosine,  carboxylmethylation  Many  exceptionally  (Gagnon  Meggio et  virtually  and Vanaman,  c a r b o x y l methyl a t i on  (Plancke  in  amino a c i d sequence from many  (discussed e a r l i e r ) .  have been i d e n t i f i e d :  a]..,  an  present  (Klee  The  C superfamily  calmodulin that  (Murtaugh et  is  types  and  demonstrated  of  cell  determined  structure  modifications  Calmodulin  and two  kinase  key enzymes  and in  the of  control  o f cAMP l e v e l :  cAMP and c y c l i c  breakdown  (Yeager  isozymes  of  nucleotide et  al.,  1985;  1971).  At  PDE has  been  reported  are  also  enzymes  are  Graves,  1985);  (2)  myosin  light  kinase  I  calmodulin-dependent  protein  kinase  II  including tubulin  calmodulin-dependent  1988),  kinase  and a  and  Cheung,  key  demonstrated  (6)  III  to  i n the In binds  microtubule bind  the  of  the  enzymes  and  calmodulin  with  calmodulin  with  associated  protein  calmodulin  (Lee  Six  that  low  low  and W o l f f ,  et  affinity 2  (MAP-2) 1984;  35  protein  1977);  (Klee,  a l . , 1985a);  (3)  phosphorylates  many  1985b);  caldesmon which  (5)  was  also  a l . , 1988).  et  calmodulin-activated  et  1983).  Tau  et  al.,  al-,  while  (a and &)  1982).  also  1981b)  erythroid  1981a)  Tubulin  factor  al.,  are  a phosphorus and an category,  (Kumagai  Sobue et  and  trisphosphate  transduction  (Sobue  and  (4)  (calcineurin)  1,4,5  protein  al.,  1987);  (Scott-Woo  phosphatase  o f a bond between  (Carlin  the  al.,  are  affinity  of  kinase  (Johanson  cytoskeletal/structural  binds with high a f f i n i t y binds  Sharma  in  et  signal  calmodulin-stimulated  forming or b r e a k i n g  also  distinct  kinase  inositol  inositol-phosphate  involved  fodrin  its  (Kennedy et  al.,  Recently,  all  spectrin  (see  i n the presence o f c a l m o d u l i n  Interestingly,  oxygen.  two  in  phosphorylase  (which  calmodulin-dependent  in  be  chain  (Nairn  ( N a i r n et  1986).  enzyme  (1)  and phospholamban)  undergoes a u t o p h o s p h o r y l a t i o n  kinase,  least  involved  including:  protein  (Tallant  formation  involved  calmodulin-activated.  calmodulin-dependent  Walsh,  is  Cheung,  phosphorylation/dephosphorylation,  proteins  (PDE)  i n the  2+  dynein-ATPase,  and  involved  Two ATPases, the plasma membrane C a - p u m p i n g ATPase and the  calmodulin-dependent  (Chan  cyclase is  phosphodiesterase  calmodulin-dependent  Wang, 1988). flagella  adenylate  .  appear  The to  Caldesmon  appears  to  bind  binding  of  calmodulin  to  (Fig.  Tau f a c t o r  caldesmon) same  fashion  protein actin its  in  calmodulin  5).  (Fig.  the  binding  to  1988).  proteins  include  (Seiler  et  1984),  fibre al.,  gap j u n c t i o n 1983),  (Grand  and  domain.  basic protein  Perry,  1980)  luminal  regulated,  membranes  phospholipase Neuromodulin  is  a  Ca  2 +  has  than  in  recently  its  enzymes ( J e f f e r s o n  to  reported  the  actin  and  (i)  the  spectrin inhibited  and upon  calmodul i n -  and  chicken  SR lens  Phospholamban ( M o l l a 1980)  and  histone  et  2 +  more  inhibit  and Schulman, 1988).  2B  calmodulin-binding  Ga -activated  protein  al.,  et  of  has a l s o been suggested to be  (Welsh et  36  the  from j u n c t i o n a l  authentic  membranes  calmodulin  to  of  Other  1984)  Perry,  (Klaerke  growth  in  less s p e c i f i c a l l y via clusters  proteins  (Adreasen  channel  1982).  an  platelet  bind  (i.e.  calmodulin-binding  1988).  al.,  contain  medulla  cell  presence  been  al.,  calmodulin-binding  appears  al..,  and  Several  human  between  o f adducin i s  (Grand  including:  may be i n v o l v e d i n normal neuromodulin  not  renal  binding  association  et  bind c a l m o d u l i n  from °f  et  (Ono  (Welsh et  b a s i c amino a c i d r e s i d u e s . calmodulin  promotes  calspermin  do  mechanism  identified  calcium release  myelin  They p r o b a b l y  newly  (Bennett  the  protein  the  This a c t i v i t y  calmodulin  al.,  a  membrane,  binding  a flip-flop  a l s o binds c a l m o d u l i n and t u b u l i n  Adducin,  al.,  in  caldesmon weakens  5).  plasma  (Bennett et  and a c t i n  et  al.,  K  1987),  (Wong  and  (also  called  al.,  1982).  tightly  1983). several  +  in  channel and  Cheung,  of  (ii)  a  1979).  GAP-43)  that  Interestingly, the  Finally,  absence  of  sphingosine  calmodulin-dependent  Fig. 5  Diagrammatic  presentation  of  caldesmon/calmodulin-caldesmon/actin factor/tubulin  interaction.  represented  CaM,  by  strong i n t e r a c t i o n from Sobue et  al.  CaD,  the and  Calmodulin, A  and  whereas d o t t e d  T, line  (1981c).  37  flip-flop tau  caldesmon,  respectively.  mechanism  for  factor/calmodulin-tau actin, Solid  tubulin  line  are  indicates  i n d i c a t e s weak i n t e r a c t i o n .  Taken  Figure 5  38  V. Calpain  1.  Overview  A  protease  bonds.  (peptide  Proteases  are  hydrolase)  classified  Exopeptidases are r e s t r i c t e d while  endopeptidases  well.  are  Endopeptidases  into  four  these  classes  active  3.4.22),  sites  (also  and  it  highly  extracellular  exported  to  extracellular  years,  or  (ii)  foreign  proteolysis  and  Melloni,  endoplasmic r e t i c u l u m  into  are  serine  (EC  to  cleave these  off  process  further  synthesized protein  peptides  and  after  or  residue  cysteine  at (EC  (EC 3 . 4 . 5 . 2 4 ) .  to  be  the  related  to  it  general  Butler,  their  inside  only  One  example  peptides  (a  signal is  39  for  exert the  protein  is  is  a  are their  cell.  location  in  For  which that  degradation  and  as i n the c y t o s o l  signal  peptidases  The f u n c t i o n o f these  peptide  In  has become obvious  from p r o t e i n s  required  1987).  synthesis  proteases  proteins  More r e c e n t l y ,  1986a).  signal  that  bonds as classified  catalytic 3.4.21),  intracellular  thought  is  (Bond  which  and m i t o c h o n d r i a .  organelles  p e p t i d e bond  peptide  p r o t e o l y s i s a l s o occurs i n o t h e r o r g a n e l l e s as w e l l  (Pontremoli  is  central  essential  peptide  and e n d o p e p t i d a s e s .  and m e t a l l o - p r o t e a s e  spaces,  lysosomes were  lysosomal  cleaves  C- o r N - t e r m i n a l  the  proteases,  c e l l u l a r proteolysis occurred. (i)  the  attacking  on  that  exopeptidases  1986):  controlled  to  on c e l l u l a r  enzyme  has become apparent t h a t c e l l u l a r p r o t e o l y s i s i s  contrast  many  based  an  proteinases)  a s p a r t i c a c i d (EC 3 . 4 . 2 3 ) ,  complex  action  of  called  (Barrett,  In r e c e n t y e a r s , a  into  to e i t h e r  capable  mainly  is  after  proper d e l i v e r y  proteases  their  segment of  of  delivery  of .a the  newly protein  into  these membrane o r g a n e l l e s )  proteases  are  proteins  (Pontremoli  proteinases other  responsible for and  (500-700  tissues  kDa)  have been  (Rechsteiner,  dependent p r o t e a s e . a high m o l e c u l a r ATP-dependent  weight  of  various  ATP/ubiquitin-dependent  identified Upon  in  reticulocytes  and some  an  ATP-dependent  enzymic  t a r g e t p r o t e i n s become s u s c e p t i b l e t o the ATP-  protease  protease  by  Processing  modification  1986a).  1987a).  1982).  Another c y t o s o l i c p r o t e a s e i s macropain which i s a l s o  a mixture  inhibition  and S t e i n e r ,  post-translational  Melloni,  c o n j u g a t i o n with u b i q u i t i n ,  contain  (Docherty  of  (500-700  (Rivett,  1989).  proteases  various  types  kDa)  as  of  This  subunits, protease  and  is  distinct  macromolecule  as e v i d e n t  from  inhibitors.  from  appears  The  first  Guroff  report  (1964).  activating  of  partial  Lastly,  growing  factor  al.,  differing  concentration  of  Ca  Ca 2 +  regulatory Ohno e t  subunit.  al.  (1984)  w i t h domain II  and i t a  pH  is  brain)  This protease  also called  optimum  affinity  for  Calpain i s a heterodimer,  rat  was  between  exist  calpain  I  and  (micromolar calpain  having an 80 kDa c a t a l y t i c  The l a r g e  s u b u n i t was f i r s t  from c h i c k e n .  T h i s 80  being the c a t a l y t i c  calpain 7-8.  (Murachi Two  and  II,  -  is a cysteine et  isozymes millimolar  respectively).  s u b u n i t and a  29 kDa  c l o n e d and sequenced by  kDa p r o t e i n  domain and domain IV  has f o u r  domains  a calmodulin-1ike  p, Ca  by  m u s c l e , which l a t e r turned out to be a Ca  has c  their  (in  1987).  (1964) a l s o d e s c r i b e d a p h o s p h o r y l a s e k i n a s e -  papain,  Calpain in  protease  (Huston and K r e b s , 1968).  homologous t o  1979).  al.  in skeletal  dependent p r o t e a s e protease  Ca -dependent  Meyer et  neutral  mammalian and a v i a n t i s s u e s ( S u z u k i ,  2+  a  to  the  e v i d e n c e has suggested t h a t t h e r e i s a c y t o s o l i c c a l c i u m - d e p e n d e n t p r o t e a s e system p r e s e n t i n a l l  the  p• -binding  domain  (Fig.  6).  It  is  40  believed  that  the  E-F  hand  Ca  -  Fig.  6  Schematic  of  e x p l a n a t i o n see t e x t .  the  domain  Modified  structure  from Suzuki  41  of  (1987).  calpain.  For  detailed  Figure 6  Catalytic 80 kDa subunit cysteine protease domain 100 300 i 1i 1 .i  1 •  = 11 =  1 Cys  Wmm  His  500 . ii  1i  1  calmodulin-like Ca-binding domain 700 i_ . .ii i. »•••• 1 »/'•••••  III  1V  ffi  »•'• •  100  Regulatory 30 kDa subunit  Iff  : • : ' • •'•  ':• ; •  200  ».•.•.•« • • • «  llililll Gly-rich hydrophobic domain  42  calmodulin-like Ca-binding domain  b i n d i n g domain  imposes the  strict  Ca  -dependence o f  At the c a t a l y t i c  domain,  i s a requirement  f o r a reducing environment  keep the papain, the  sulfhydryl  sulfhydryl  amide  Interestingly, activated. (80  was  (Pontremoli appeared  as  calpain  has  found  is  a  both  cysteine  the  transition the  illustrated to  first  The  protease  epoxysuccinyl  inhibitors: peptides  group w h i l e  molecule,  Interestingly,  al_.,  calcium  it  was  E64)  et  al.,  leupeptin  it  as  in  the  case  of  substrate  I  1986).  be  calpain 76  and  II  also and  demonstrated  of can  be  that  et  activated  a]_.,  calpain  inhibited agents  microbial  1981).  papain  peptide  geometry  (Barrett,  inhibition  of  Calpain  activity  is  43  also  dependent  on  to the to  1986). leupeptin  by l e u p e p t i n  by d i a l y s i s i n the presence o f EGTA but not t h a t o f E64  1981).  by  (e.g.  E64 appeared  mimics the  I  kDa  (Pontremoli  (Inomata  and  fully  and  has been demonstrated t h a t the b i n d i n g o f both  and E64 t o c a l p a i n i s calcium-dependent and the  et  kDa  thiol-reactive  (Suzuki  of  to  calpain  activity  (e.g.  abstraction  erythrocyte 78  to in  (Barrett,  intermolecular  Calpain  and a n t i p a i n )  sulphydryl  7  produced i n a c t i v a t i o n  1985).  the  that,  a t t a c k on the  to  for  Recently, and  in  of  autolyzed  requirement  There  as  autolysis  subunit  1986b). lower  believed  Fig.  sequentially  1983).  Melloni,  the  in  to H i s - 2 6 5 .  s t a t e f o r an enzyme-substrate complex and thus b i n d s t i g h t l y  calpain  reversible  is  undergo  catalytic  intramolecular  aldehydes ( l e u p e p t i n alkylate  much  facilitating  activity.  10 mM d i t h i o t h r e i t o l )  It  autolyze  autocatalysis  and  iodoacetate),  to  Murachi,  Further  (Pontremoli  the  Melloni,  have  1986;  autolysis  typical  group,  and  to  Melloni,  1988).  thereby  For example,  kDa)  (e.g.  imidazole ring nitrogens a s s i s t  proton,  carbonyl  Cys-108 i s i n c l o s e p r o x i m i t y  group of Cys-108 r e d u c e d .  the H i s - 2 6 5  catalytic  is  (Suzuki  temperature.  At  Fig.  7 Mechanism o f  Reaction histidine calpain  proceeds (His)  hydrolysis of from  residues  (see F i g . 6)  (1)  to of  peptide (5).  the  The  bond o f  conserved  catalytic  are i l l u s t r a t e d .  44  substrates  domain  by c a l p a i n .  cysteine of  large  M o d i f i e d from B a r r e t t  (Cys) subunit  (1986).  and of  Figure  7  9 C ^ -  S  o  H  /  (Cys - S H - l m H i s , J1  0  JSfHs Cf  0'  R-*. -  R-C-NHjR' S  ' (Cys___Jm-His^  .HN' ^  x  HgNR-  (CyS*  Im-His ^  (3)  It R-C-OH ^Cys  -—>  H*lm-His^  (3)  45  9 R-C-OH  SH  Im HiSp  37°C,  the  remains the  stable  large  fact,  subunit  is  for  inactivated  at  catalytic  an  1987)  enzyme  least  was  (Fig.  It  min  while et  the  al.  activity  1982).  Like 2 +  the small 30 kDa s u b u n i t a l s o b i n d s C a  (Aoki  has  30-40  21°C ( M e l l g r e n  - b i n d i n g domain at  identified 6).  about  50 min at  subunit,  E-F hand Ca  in  et  al..  the C - t e r m i n a l  1986;  Emori  been observed t h a t  et  end o f the  al.,  erythrocyte  1986b;  .  In  small  Suzuki,  calpain  binds  to  7+ the  plasma membrane  80  kDa  subunit  (Pontremoli  et  neutrophil case  the  the  undergo  al_.,  1985a). its  (Pontremoli  2 +  of  Subsequently,  N-terminal  activation  of  et  it  the  responsible  al.,  2.  1985c).  calpain  for  for  a l s o appears  was  calpain  to  Autolytic result  in  bound form a  78  similar  kDa  autolysis  the form  binding that  without  in  (Coolican  of this  undergoing  phosphatidyl i n o s i t o l and  reduced Hathaway,  t h a t the g l y c i n e - r i c h domain near  of  calpain  (Imajoh et  binding  to  active  Also  was i l l u s t r a t e d subunit  a  the  i s observed except  hydrophobic r e g i o n o f the  phosphatidyl i n o s i t o l . II  calpain  al.,  in  activation  plasma membrane  small  and  Interestingly,  by p h o s p h a t i d y l i n o s i t o l  proposed t h a t the  manner  autolytic  membrane-bound  Ca -requirement  1984).  a Ca^ -dependent  can  c a l p a i n to  plasma  autolysis  in  to  al.,  essential  1986).  for  Suzuki  small  s u b u n i t may be  the  plasma  cleavage o f the activation  was  of  small  the  such (1987)  directly  membrane  subunit  protease  of  via  calpain  (DeMartino  et  1986).  Regulators  The most  important  regulation  of c a l p a i n  e x i s t e n c e o f an endogenous c a l p a i n i n h i b i t o r  46  activity  protein  is  perhaps the  (calpastatin)  co-  i n most  tissues types  studied  (liver  (Murachi,  and  1983a).  erythrocyte  It  types)  is  of  now  clear  that  there  are  calpastatin.  The  liver  type  about 107 t o 110 kDa on SDS-PAGE w h i l e the e r y t h r o c y t e kDa.  Each molecule o f the former  t h a t o f the 1987;  Suzuki  found t h a t kDa  latter et  (i)  repeated  only i n h i b i t s  al...  1987b).  the l i v e r  (although  it  (Fig.  as  has  three  repeated  erythrocyte  calpastatin  presence  of  EGTA and  (Melloni  et  al.,  very  similar  likely  that  1982a).  changes  kDa  to  SDS-PAGE)  of  Ca  calpain  is  incapable  If  amount  2 +  (Melloni  of  an  type et  (Imajoh  demonstrated  that  Although  and  all  there  Suzuki,  four  activities  calpain are  in  in  al..  of  was  interacts  1985).  of  has  a  repetitive  reports  are  of  to  to  the  undergo it  it of  in both  is The upon  proteins.  inhibitor  al.,  2 +  calpastatin,  substrate  domains  the Ca  reversed  recently,  that  in  with c a l p a i n .  found mostly  showing  The  Therefore,  however, Most  kDa  appears  and i s  a g a i n s t c a l p a i n (Maki e t  and c a l p a s t a t i n  230  Upon b i n d i n g  proteolysis  excess,  47  68  four  1987b).  presence  1985a).  2+  internally  several  contains  inhibitor  the  Ca -dependent  or  and  al_.,  calpastatin  1982a).  is  et  oligomer  monomers  et  autolysis  it  (Suzuki  is  calpain  proteolyzed  possess i n h i b i t i n g  of  al.,  8)  calpain al.,  proteins,  erythrocyte  the monomeric form o f c a p a s t a t i n  removal  et  appears as 68 kDa on SDS-PAGE)  into  Pontremoli  to  these  (Emori  (it  form  The l i v e r  (see  calpastatin  fractions,  appears  on  the  (Fig.  dissociates  binding of  the  units  m-RNA o f  kDa  (ii) 46  molecules of c a l p a i n while  has a c a l c u l a t e d m o l e c u l a r weight o f  110  8);  4-10  is  i s about 68-70  molecules o f c a l p a i n  From the  c a l c u l a t e d m o l e c u l a r weight o f and  3-5  inhibitor  appears  domains  inhibits  type  two  has  is been  calpastatin  1987).  the  cytosolic  calpain  and  Fig.  8 Schematic of the s t r u c t u r e of c a l p a s t a t i n .  about  14 kDa are  shown. Each repeat  of  inhibition.  Note  that  I.  Taken from Suzuki et  the  al.  unit  erythrocyte  (1987b).  48  I-IV  internal  corresponds to inhibitor  repeats  a functional  lacks  internal  of  unit repeat  Figure 8  100 I  300 I  4 0 0 1  500 I  6 0 0 I  700 L_  718  Primary trwuUtton  2 0 0 I  product  H N 2  1  II  III  IV  1  II  III  IV  II  III  IV  90 L M « T inhibitor  H N3  • COOH  718  290 E r y t h r o c y i o inhibitor  X-HN-  49  -COOM  718  -COOH  calpastatin  are  found  membrane/sarcolemma 1986;  Mellgren  Other erythrocyte  et  in  various  and s a r c o p l a s m i c r e t i c u l u m  al.,  1987a,  protein  regulators  protease  inhibitor  endogenous  protein  including  (Gopalakrishna  plasma  and B a r s k y ,  b).  high m o l e c u l a r weight p r o t e a s e An  subcellular organelles,  of  calpain  protein  (240  and c a l p a i n  activator  also  which  exist,  kDa)  including  that  (Murakami  increases  inhibits  a  human  both  the  and E t l i n g e r ,  1986).  neutrophil  calpain  the  9 .  affinity  for  Ca  c  (Pontremoli  et  al.,  1988)  and  an endogenous  activator  9. that  increases  the  V  but  been  reported  (DeMartino  m a x  not  and  the  (1986) r e p o r t e d  calpain  although  others  in vivo  (Adachi et  3.  Substrate  Calpain  I  and  substrates  usually  very  o r Met  the  poor  failed  protein to  brain  Takeyama  et_al_->  k i n a s e C can  demonstrate  such  calpain  have  1986).  phosphorylate  phosphorylation  1986).  II  appear  (Zimmerman substrates  of cleavage  residue  ?2 p o s i t i o n  have  that  1982;  of  Specificity  protein  Some degree  affinity  Blumenthal,  Hincke and T o l n a i  al.,  Ca  i n the  would  site  Pj  favor  to  hydrolyze  and S c h l a e p f e r , or  only  at  appears to  at  et  1984)  Pj  position  (Sasaki  further  analyzed  in d e t a i l  al.,  the  under the  carboxyl (Fig.  DISCUSSION  50  Small  all  peptides  of  This  section.  the  in  residue  aspect  of are  1983b).  a Lys, Tyr,  a Leu o r Val  side  9).  group  (Murachi,  apply:  p o s i t i o n preceded by e i t h e r cleavage  selective  1984).  not-hydrolyzed  preference  a  will  Arg the in be  Fig. site to  9 of  ?2  Schematic  presentation  substrate at  the to  and the  cleavage  subsites  S3  S '  structure  o f papain  2  on  active  site the  of  of  the  site  interaction  are  molecules  (Schechter and B e r g e r ,  51  the  cleavage  o f c a l p a i n . Amino a c i d r e s i d u e s  substrate  calpain  between  P3  shown w h i l e  corresponding  are  Based  1967).  shown.  on  the  Figure 9  52  4.  S u b s t r a t e s and f u n c t i o n s of  The  physiological  Mellgren  (1987)  physiological Calpain  and  events  substrates  according to proteins;  their (b)  myofibrillar  roles  calpain  of  Suzuki that  can be  al_.  (1987a)  believed  categorized  proteins;  (e)  enzymes;  and (h)  proteins,  as  degradation  o f membrane p r o t e i n s  for  a  contribute 1984).  to  nebulin)  and  assembly  of  receptors: have  Calpain  (f)  calpain  expose the  latent  originally  observed  long-term  proteins  neurofilaments these  three  glucocorticoid,  altered also  hydrolyzes  (vimentin,  of  the  after  in  (a)  al_.,  the  calpain.  general  groups  membrane-bound proteins; factors;  (Table  were  (d) (g)  1).  the  earliest  1976).  Proteolytic  band 3 p r o t e i n  post-synaptic  glutamate  proteins desmin, that  networks  estrogen  properties  probably  (Lynch  and  is  which may  and  Baudry,  (tubulins  and MAP-2),  filamin,  keratin  and  disrupt  the  calpain (Table  can  1).  The  steroid  and p r o g e s t e r o n e r e c e p t o r s  appeared  calpain-fragmentation  soluble,  53  membranes  receptors,  potentiation  suggested  types  by  clotting  peptides  of  o f membrane f u s i o n ( G i l a s a and Kosower, fodrin  filament  following  such as e r y t h r o c y t e  c a l p a i n - h y d r o l y s i s of  the  some  receptor  et  Recently,  influenced  blood  (Dayton  The s u s c e p t i b i l i t y o f m i c r o t u b u l e  intermediate  to  (c)  group,  s p e c t r i n i s l i n k e d to f a c i l i t a t i o n  suggested t o  the  unclear.  reviewed  be  small, natural  substrates  while  to  into  proteins;  identified  1986),  still  and s u b c e l l u l a r l o c a t i o n :  cytoskeletal  Myofibrillar  are  et  are  function  miscellaneous proteins  calpain  steroid  (see  receptor-like  Ah  Table  1).  receptor,  Table 1  Substrates of  Effect  Substrate  (a)  Membrane  calpain  Reference  calpain  proteins  F o d r i n ( a , /J) S p e c t r i n ( a , 0) A n k y r i n (band 2.1) Band 3 Band 4.1 (b)  of  Cvtoskeletal  fragmented fragmented fragmented fragmented fragmented  1  Simon et a l . , 1984 Pant et a l . , 1983 H a l l & Bennet, 1987 Au et a l . , 1988 Pant et a l . , 1983  proteins  T u b u l i n ( a , fl) MAP-1, MAP-2 Nebulin Talin  fragmented degraded degraded fragmented  Vimentin Desmin Keratin Filamin Neurofilament (H,M & L) Adducin ( a , /S) C r y s t a l 1 i n ( a , /}) Lamin ( n u c l e a r )  degraded degraded degraded fragmented degraded  B i l l g e r et a l . , 1988 B i l l g e r et a l . , 1988 G o l l et a l . , 1983 B e c k e r l e et a l . , 1986; O ' H a l l o r a n et a l . , 1985 Nelson & T r a u b , 1986 Nelson & T r a u b , 1986 Ando et a l . , 1988a T r u g l i a & S t r a c h e r , 1981 M e l i k et a l . , 1983  degraded degraded degraged  Wang et a l . , 1989b David & S h e a r e r , 1986 Traub et a l . , 1988  becomes GTP/GDPindependent increased autophosphorylation can e n t e r nucleus l o s t DNA-binding ability fragmented fragmented  Lynch et  (c)  Receptor  2  proteins  aj-adrenergic receptor EGF r e c e p t o r Estrogen receptor Progesterone receptor Ah r e c e p t o r Glucocorticoid receptor  54  al.,  1986  Gates & K i n g ,  1983  Puca et a l . , 1977 V e d e c k i s et a l . , 1980 Poland & G l o v e r , Naray, 1981  1988;  Table 1 (cont.)  Substrate  (d)  Myofibrillar  E f f e c t of  Reference  proteins  Myosin heavy c h a i n Myosin l i g h t c h a i n s C-protein a-actinin Troponin T Troponin I Tropomyosin  (e)  calpain  degraded degraded degraded degraded degraded degraded degraded  Dayton e t I s h i u r a et Dayton e t I s h i u r a et Dayton e t Dayton e t Dayton e t  al., al., al., al., al., al., al.,  1975 1979 1975 1979 1975 1975 1975  activated  Huston & K r e b s ,  1968  activated  M e l l g r e n et  1979  activated  Hiraga & T s u i k i ,  activated  Hamon & B o u r g o i n ,  released a active fragment from ER activated inactivated  L i scum e t  a c t i v a t e d & CaMindependent a c t i v a t e d & CaMindependent a c t i v a t e d & CaMindependent independent o f C a , PS & DAG f i r s t activated then i n a c t i v a t e d activated activated fragmented activated  Ito  Enzymes  Phosphorylase kinase Phosphorylase phosphatase Glycogen synthase (D-form) Tryptophan hydroxylase HMG-CoA r e d u c t a s e Pyruvate k i n a s e Myosin l i g h t c h a i n kinase Cyclic nucleotidephosphodiesterase Calcineurin 2 +  Ca -ATPase Protein  kinase C  Calpain Adenylate c y c l a s e Transglutaminase Pl-phospholipase C Inhibitor-1phosphatase Regulatory subunit (Rj) o f cAMP-PK cAMP-independent p r o t e i n kinases  al,  al.,  1986  1983  Ekman & E r i k s s o n , Ito e t a l . , 1987 et  al.,  T a l l ant et  1979  1980  1987 al.,  1988  Wang e t a l , 1988a, b; A u , 1987 K i s h i m o t o e t a l . , 1980  z +  d i s s o c i a t e d from c a t a l y t i c subunit activated 55  Suzuki et  al,  1987b  Tremblay & Hamet, 1984 Ando e t a l . , 1987 Low e t a l . , 1984 Waelkens et a l . , 1985 Beer e t  al.,  1984  Tahara & T r a u g h ,  1982  Table  1 (cont.)  Substrate  (f)  Effect  Blood c l o t t i n g  Miscellaneous  Calpastatin Casein ( a i , s  &p  Reference  factors  F a c t o r XIII ( t r a n s glutaminase Factor V F i b r i n o g e n (a,/}) Kininogen Von W i l l brand f a c t o r G l y c o p r o t e i n l b (a)  (g)  of c a l p a i n  activated  Ando e t  degraded activated degraded fragmented fragmented  McGowan et a l . , 1983 K u n i c k i et a l . , 1984 Schmaier et a l . , 1986 K u n i c k i et a l . , 1985 Yamamoto et a l . , 1986  fragmented degraded  Suzuki et a l . , 1987b M u r a c h i , 1983b  fragmented degraded  M e l l o n i et a l . , 1984 Sakai et a l . , 1987  degraded  B e l l e s et  al.,  1988  fragmented  Seiler  al.,  1984  fragmented fragmented  Wang et a l . , Haraguchi et  fragmented fragmented fragmented fragmented  I s h i u r a et a l . , 1979 Hayashi et a l . , 1985 Sasaki et a l . , 1984 Sasaki et a l . , 1984  al.,  1987  proteins  s  & p) Hemoglobin ( a , ft) Histone (IIA, IIB & III) C a l c i u m channel (L-type) Calcium release channel Neuromodulin Thyroglobulin  (h)  Natural  Insulin Protamine Glucogen Dynorphin  et  1989b a l . , 1987  peptides  "fragmented" i n d i c a t e s t h a t the o r i g i n a l p r o t e i n undergoes o n l y limited proteolysis "degraded" i n d i c a t e s t h a t the o r i g i n a l p r o t e i n undergoes m u l t i p l e and e x t e n s i v e p r o t e o l y s i s  56  which,  upon  dioxin,  binding  induces  metabolizing  the  upon  also  (Dayton  fibrinogen  (a,  potential  (Poland  proteolysis,  Calpain  turnover  et  0),  al.,  (Table  1).  i s the  "enzymes".  It  enzyme  substrates  are  very  different  enzymes  neutrophils, occur  in  1986a;  such  Ca  involved  Blood  in  interesting  (Lynch  factors:  factors,  be  et  of  al..,  protein  factor  are  released  group  drug  a-adrenergic  myofibrillar  clotting  can  other  The  agonist-independent  calpain  activated, 1).  kinase,  2 +  response t o  although  For  calpain  V,  considered  upon  the  example,  mode o f  platelet substrates  several  phosphodiesterase of  calmodulin.  activation  and  phospholipid  and  stimulus  (Melloni  of et  kinase  et  protein  al.,  can  calcineurin)  Protein  (Kishimoto  activation  the be  calmodulin-dependent  produce a s o - c a l l e d M-form o f the  proteolytic  1986;  C c o u l d be k i n a s e which  al.,  kinase  are  1983).  In  C appears  to  Pontremoli  et  al.,  1988).  Interestingly,  phosphorylation  s u s c e p t i b i l i t y to c a l p a i n et  1988).  and  appears t h a t upon p r o t e o l y s i s by c a l p a i n most o f  (Table  of  Glover,  -450  and von W i l l e b r a n d  The most  by c a l p a i n to  independent  2,3,7,8,-tetrachlorodibenzo-p-  cytochrome  be  1975).  and made independent  proteolyzed is  to  enough  (phosphorylase  activated  of  become  kininogen if  like  and  appeared  substrates  activation  compounds  synthesis  enzymes  receptors, 1986).  to  a_l.,  1987)  susceptible  The  while  (Zhang et  (Toyo-Oka,  of  1982;  phosphorylation  al.,  1988;  calpain-calpastatin  some  Pant,  activity  57  substrates  Parker et renders  increases  al.,  other  1986;  their  Pontremoli  substrates  less  1988).  balance  appears  to  be  altered  in  some  disease  states.  In  Duchenne  muscular  dystrophy  calpain activity  was i n c r e a s e d i n muscle as w e l l  et  Both the  al.,  rats  1984).  had decreased l e v e l s  same as t h a t  erythrocytes  of c a l p a s t a t i n ,  i n normal M i l a n r a t s  As was d i s c u s s e d e a r l i e r , activated  by  and  calmodulin.  (Pontremoli  the  erythrocyte  Alternatively,  limited  2 +  quantity  Therefore, calpain  it  is  proposed  and t h a t  irreversibly. disorders elucidate  that  of  Since DMD  role  calpain and  may  essential  i n the c o n t r o l  Vf.  O b j e c t i v e s o f the  (1)  to p u r i f y  (2)  t o c h a r a c t e r i z e the p u r i f i e d  (3)  to i n v e s t i g a t e  to  in  hypertension), 2 +  in be  activate  involved  o f the C a  1987a).  proteolysis  calpain  C a - A T P a s e may  be  the  pump  with  On the o t h e r hand,  2 +  the  such p r o t e o l y s i s may serve  (e.g. its  large  1986b,  was  2+  erythrocyte  a  hypertensive  C a - p u m p i n g ATPase can be  the C a - A T P a s e .  has  Milan  (Rabbani  calpain level  al.,  exogenous p r o t e a s e a l s o a c t i v a t e s also  of  the  et  patients,  as i n p l a t e l e t s  kidneys  while  (DMD)  the  a  cytosol.  substrate  for  2 +  the C a - A T P a s e  certain it  the  is  diseases important  or to  activity.  study  c a l p a i n I from human e r y t h r o c y t e s .  whether  calpain. 2 +  the C a - A T P a s e i s an i n v i t r o  substrate  of  calpain. (4)  t o examine and t o c h a r a c t e r i z e Ca  2 +  upon  transport  changes i n  f u n c t i o n and k i n e t i c  calpain-treatment.  58  ATP-hydrolytic  activity, 2 +  p r o p e r t i e s o f the C a - A T P a s e  (5)  to  examine  the  structural  changes  of  the  Ca-ATPase  of  the  upon c a l p a i n -  treatment. (6)  to  correlate  the  structural  activity/kinetic  changes  p r o p e r t y changes o f the  mechanism o f a c t i o n o f c a l p a i n  59  in i t s  2 +  Ca -ATPase enzyme and to  e f f e c t s on the  with  the  provide a 2 +  Ca -ATPase.  MATERIALS AND METHODS  I.  Materials  The c h e m i c a l s a n d / o r p r o t e i n s were purchased from the  following  1. Sigma Chemical Co. 2-mercaptoethanol 5'-nucleotidase Activated ADP ( M g  (Crotalus  adamamteus  charcoal  2 +  salt)  Alamethicin Arsenazo  III  Ascorbic  acid  Bovine serum albumin Bromophenol  blue  Calmodulin-agarose cAMP Casein Citric  acid  Dansylated  calmodulin  EDTA EGTA Hepes Histone  ( calf  thymus,  type  11-A)  60  venom, grade  III)  sources:  Hydroxylamine L-lactic  monohydrochloride  dehydrogenase  ( r a b b i t m u s c l e , type  Lanthanum c h l o r i d e Leupeptin Maleic  acid  Manganese  chloride  MOPS NADH ( y e a s t , Nickel  (II)  grade  III)  chloride  o-Phospho-L-tyrosine Ouabain Papain (papaya l a t e x ,  type  III)  Phosphoenolpyruvate PMSF pNPP Pyruvate  kinase  ( r a b b i t muscle, type  S-100 A p r o t e i n  (bovine  brain)  S-100  (bovine  brain)  B protein  Sodium a z i d e Sodium c h o l a t e Sodium d e o x y c h o l a t e Sodium-ortho-vanadate TEMED TLCK Trichloroacetic Tris  acid  base  61  II)  II)  Tris-HCl Triton-XlOO Troponin  (rabbit skeletal  Trypsin  (bovine p a n c r e a s ,  Trypsin  inhibitor  muscle) type  (soybean,  III)  type  Omega-aminohexyl-agarose  2.  BDH Chemicals Calcium  chloride  Chloroform Dimethylformamide Glacial  acetic  acid  Glutaraldehyde Glycerol Glycine Hydrochloric Magnesium  acid  chloride  Methanol Phosphoric Potassium  acid chloride  Potassium hydrogen Potassium  phosphate  hydroxide  Silver  nitrate  Sodium  carbonate  Sodium  chloride  Sodium  hydroxide  62  IV)  3. Amersham [gamma-^P]-ATP  4.  (specific activity  20-40 Ci/mmol)  Calbiochem A23187 Calmodulin (bovine  brain)  Myelin basic protein Parvalbumin  5.  (rat  (porcine spinal  cord)  muscle)  Bio-Rad Laboratories Acrylamide Ammonium p e r s u l f a t e Coomassie b r i l l a n t  blue R-250  N,N'-methylene-bi sacrylamide SDS SDS-PAGE high m o l e c u l a r weight SDS-PAGE low m o l e c u l a r weight  6.  standards standards  Pharmacia DEAE-Sephacel Gel  f i l t r a t i o n m o l e c u l a r weight  standards  Phenyl-Sepharose CL-4B Sephacryl  S-200  Superose-12  63  7.  B o e h r i n q e r Mannheim Dithiothreitol ATP ( M g  8.  2 +  and N a  salts)  L i p i d Products Co. Egg l e c i t h i n  9.  +  ( p h o s p h a t i d y l c h o l i n e , grade  MCB Manufacture C h e m i s t s .  Inc.  Asolectin  10.  New England Nuclear Aquasol  (liquid  scintillation  fluid)  64  I)  Methods  11.  1.  Protein concentration  Protein method o f Briefly,  determination  concentrations  Lowry et up to  al.  100  /il  of  various  (1951), of  u s i n g bovine  M NaOH).  After  10  400  determined  4 mL o f  /zL  of  50  % phenol  c o n t a i n s p h o s p h o m o l y b d i c t u n g s t i c complex,  Li S0 2  4  oxidized  state  increases of  Na C03 2  the  prevented t u r b i d i t y  4  of  the  reagent  and  NaOH  maintained  (unfolding)  60 min  room temperature  read.  If  first  precipitated  sodium protein  2.  protein  the  of p r o t e i n s . or  20  concentrations  of  with 10 % (w/v)  deoxycholate  and  are  (Lowry pH  After  min  2  This  alkaline  Alkaline et  al.,  around  10  allowing 37°C,  copper 1951).  of i n o r g a n i c phosphate  65  to  in  0.1  reagent When  pH. The  and  they  mixture  helped  the  c o l o r development  for  absorbance at  100  the  apparently The  then  /zL  with  660  nm was  proteins  TCA i n the presence o f 0.05  resuspended  3  residues in a p r o t e i n ,  samples were low,  determination.  Determination  at  Na C0  bromine water m a i n t a i n e d  storage.  c o l o r developed markedly  denaturing at  during  while  reagent,  and bromine w a t e r ) .  the p h o s p h o m o l y b d i c t u n g s t i c complex g i v e s a blue c o l o r at 2  the  NaK-tartrate  reagent.  reduced by phenol groups o f t y r o s i n e and tryptophan  a d d i t i o n of L i S 0  copper  and then 80 mL o f 2 % (w/v)  4  by  as a s t a n d a r d .  combining 800 /xL o f 2 % (w/v)  of CuS0  min,  were  serum albumin  sample was mixed with  which was f r e s h l y made by f i r s t and 800 /zL o f 1 % (w/v)  samples  were  % (w/v)  water  of  before  The was  production  measured  Briefly,  at  by  of  the  inorganic method  described  the end o f the r e a c t i o n ,  the r e a c t i o n mixture (400 ascorbic  phosphate  complex  i n 6.5  a result  Raess  H S0 . 2  between  by  molybdate  and  free  Absorbance  development molybdate  at  at  room  also  660  nm  is  temperature.  catalyzes  the  read K2HPO4  was  breakdown  % (w/v)  method  inorganic  after  used  as  in  phosphate.  30 a  of  ammonium  lies  (provided  exactly  of  (1980).  SDS was added to  1.25  this  complex g i v e s a blue c o l o r i n a r e d u c i n g environment acid).  breakdown  200 [xl o f 9 % (w/v)  200 /zL o f of  ATP  Vincenzi  o f 10 % (w/v)  The p r i n c i p l e  4  of  and  /xL) to stop the r e a c t i o n ,  % (v/v)  formation  by  200  a c i d was added and f o l l o w e d  molybdate  as  the The  by a s c o r b i c  min  of  color  standard.  Since  unhydrolyzed  ATP,  blanks  c o n t a i n i n g 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 o f the c o n c e n t r a t i o n s o f f r e e C a ^ and M q  Free Ca*FORTRAN  program  Binding constants Martell which  -  and Mg*  c o n c e n t r a t i o n s were c a l c u l a t e d by computer u s i n g a  (Cations for  Ca  BC), 2 +  ,  Mg  described 2 +  and H  and Smith (1974-1982) except  were  calculated  2 +  as  described  +  by  to  Goldstein  et  al-  (1979).  ATP and EGTA were taken  i n the case o f monoprotonated by  Blinks  et  al.  (1982).  ligands Prior  a p p l i c a t i o n o f the program, c o n s t a n t s were c o r r e c t e d f o r temperature a  BASIC program  (1978) 1982). Smith,  and  using  (LOGTEMP), enthalpy  The c o n s t a n t s  based on the values  were then  1974-1982; B l i n k s et  al.,  formula  tabulated  adjusted 1982).  66  for  by  given  Martell  ionic  by and  strength  from  using  T i n o c o et Smith  to  al.  (1974-  (Martell  and  4.  P o l v a c r y l a m i d e g r a d i e n t gel e l e c t r o p h o r e s i s and a u t o r a d i o g r a p h y  (i)  Sample p r e p a r a t i o n .  with 25 /iL d i g e s t i o n b u f f e r 1 mM 2 - m e r c a p t o e t h a n o l , (pH 6 . 8 ) .  If  20 % (w/v)  0.02  TCA. The p e l l e t  amount o f g  m a x  )  treated  the buffer.  If  (pH 6.8)  instead of  1.35-5.75  %  3.5  6.8),  % (w/v)  %  p r e c i p i t a t e d w i t h 20 % (w/v)  500  phase  /xL  chloroform  to  extract  was c e n t r i f u g e d  was  (v/v)  % (w/v)  slab  removed  (  buffer  for  TCA. The  the  5 min at  before  be analyzed  (w/v))  total  of  of  (1971).  of  gradient  the  excess  10,000 x  addition  EP f o r m a t i o n  mm t h i c k n e s s  The s e p a r a t i n g gel acrylamide  375 mM T r i s - H C l of  glycerol, (see  of  on an  formation  67  of  while  EP,  the  330  contains a  0.1  %  (w/v) gel  mM T r i s - H C l  (pH  at  18 m A / s l a b  SDS (pH 8.3)  "acid"  cast  stacking  (16-18 h)  % (w/v)  were  (acrylamide/N,N'-  (pH 8 . 8 ) ,  above),  was run o v e r n i g h t  mM g l y c i n e and 0.1 the  1.5  total  acrylamide  SDS. The gel  measurement  gels  Laemmli  (30:0.8)),  25 mM T r i s - b a s e , 192 the  with  Tris-HCl.  (5-20  contained  For  precipitated  the d i g e s t i o n b u f f e r c o n t a i n e d 100 mM T r i s - p h o s p h a t e  methylene-bisacrylamide  in  p r o t e i n was f i r s t  samples were to  the method o f  gradient  0.1  with  SDS-polyacrylamide  a c c o r d i n g to  SDS,  was f i r s t  chloroform  (see b e l o w ) ,  linear  bromophenol blue and 100 mM T r i s - H C l  p h o s p h o l i p i d . The mixture  a c i d gel  (ii)  glycerol,  with T r i s - b a s e . When a sample c o n t a i n e d p r o t e o l i p o s o m e s ,  then  and  digestion  % (w/v)  SDS, 10 % ( v / v )  was resuspended with 25 /xL o f d i g e s t i o n  the p r o t e i n / 1 i p i d mixture was  c o n t a i n i n g 5 % (w/v)  sample volume was l a r g e ,  and n e u t r a l i z e d  pellet  Regular p r o t e i n samples (20-40 /xL) were mixed  gel  was  at  4°C.  cast  as  d e s c r i b e d above, g e l s were 7.0 mA/slab Gels  and 6 . 0 ,  the pH values o f the  respectively.  i n 25 mM MOPS (pH 6 . 8 ) ,  were  solution with  except t h a t  stained  with  0.25  solution  then s t o r e d  A until  the  acid  1 paper and a  to  -70°C.  5.  Preparation  erythrocyte All g)  of  of  became  human  red  out  cells  at  (4-5  4°C.  and washed t h r e e times  150  mM KC1  c e l l s were c o l l e c t e d cells  containing  by c e n t r i f u g a t i o n  were  hemolyzed  10 /iM TLCK and 20 m g . L "  Pell icon size)  in  min.  The  Cassette the  approximately  1  The  4°C.  R-250  in  destained gels  were  the g e l s were exposed  calmodulin-depleted  as membrane-free  at  suspended was  (pH  7.4)  volumes  for  m a x  was  (equipped  The f i r s t  hemolyzate  then  with  mode  a  the  the  A-150).  of  0.5  volume  of  The  min.  The buffer  500 /iM PMSF, ( b u f f e r H) the  was  calpain  reduced  (see  for  Millipore  fim diameter  o f f i l t r a t e was c o l l e c t e d  purification  68  through  the  solution  hypotonic  (pH 7.4)  passed  filters  until  5 litres for  inhibitor  from  5-10  250  citrate-  an i s o t o n i c (buffer  of  in  obtained  1 L of  3900 x g  10  soybean t r y p s i n  concentration 1 L.  in  (approximately  5 mM EGTA and 1 mM d i t h i o t h r e i t o l ,  hemolyzate  system  in  old),  adenine,  and 20 mM T r i s - H C l  c o n t a i n i n g 20 mM T r i s - H C l ,  20  and  One u n i t  days  Canadian Red Cross  to  and  transparent.  hemolyzate  medium  15  60 min  30  membranes  packed  red  blue  For a u t o r a d i o g r a p h y ,  phosphate-dextrose  washed  for  at  at  Kodak X-0MAT AR f i l m was then  membrane-free  p r o c e d u r e s were c a r r i e d  containing  SDS, 192 mM g l y c i n e  (5:4:1))  acetic acid.  and s t a c k i n g  was run o v e r n i g h t  Coomassie B r i l l i a n t  background  i n 7.5 % ( v / v )  f o r 36-64 h at  % (w/v)  % (w/v)  d r i e d on Whatmam No. it  The a c i d gel  0.1  A (methanol/water/acetic  separating  pore to  and used below).  The  concentrated  potassium-Hepes w i t h the  membranes (pH 7 . 4 ) ,  cassette  membranes  dithiothreitol, PMSF  in  the  with  IV-E). at  of  The  20,000  x  g  1  membranes was  for  m a x  150-200  mL o f  (600  1000 mg p r o t e i n )  9max  f °  r  determined KC1,  white  min.  L  of  130  amount  of  pellets  buffer  -70T  were  (pH 7 . 4 ) ,  for  15 min at 4°C with r a p i d s t i r r i n g .  (about  30 min and the  in  the  KC1,  mM  7.4)  2  and 500 /xM  concentrated final  by  concentrated  nitrogen  (-196°C)  in  Ca -ATPase,  performed  at  2 +  2 +  o f the  erythrocyte  0.5  membrane  by  % (w/v)  al.  (1986):  suspension  at  125,000 x  resuspension  Triton  X-100,  100 /xM C a C l , 2  150-200 mL). The m i x t u r e  in  300 mM  was  ratio  a s o l e c t i n , 2 % (w/v)  was c a r e f u l l y  X-100  at  c o l l e c t e d . To  volume o f p r e s o n i c a t e d Triton  of  incubated  The s o l u b i l i s a t e was c e n t r i f u g e d  supernatant  a  2 mM DTT,  achieve a d e t e r g e n t / p r o t e i n  69  order  Ca -ATPase  was added a pre-determined  c o n t a i n i n g 4 % (w/v)  20 mM  used.  2  (or  the s u p e r n a t a n t  liquid  1 mM M g C l ,  1/1  for  The  solubilized  containing  to  of  Finally,  as d e s c r i b e d by V i l l a l o b o e t  tiM PMSF and 100 /xM l e u p e p t i n  m a x  (pH  then  min.  until  o f the  500  o c c a s i o n a l l y 1.5/1)  L  mM  were c o n c e n t r a t e d by c e n t r i f u g a t i o n  10 mM potassium-Hepes  125,000 x g  2  in  calmodulin-depleted  The  12  volume mode,  were  20  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 was c a r r i e d out e s s e n t i a l l y  to  soluble proteins.  to  quick-frozen  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  to  constant  membranes  a l i q u o t s o f 2 t o 4 mL and s t o r e d at  4°C,  10  500 /xM PMSF and 10 mM a s c o r b i c a c i d  hemoglobin and o t h e r washed  with  1 mM EDTA and 20 mM potassium-Hepes  centrifugation  6.  washed  1 mM EDTA,  were  (buffer  suspension  then  system o p e r a t i n g  t o remove c a l m o d u l i n , white  were  solution  and 80 mM DTT (TAD  solution)  to  a c h i e v e 0.1  2 mM DTT i n the  final  % (w/v)  a s o l e c t i n , 0.05  solution mixture.  a c a l m o d u l i n - a g a r o s e column (15 bound c a l m o d u l i n e q u i l i b r a t e d % (w/v)  x 1.5  cm)  in buffer  containing  EQ (0.5  (15-17  h)  with  except  that  enzyme  was  buffer  W except t h a t 2 mM EDTA r e p l a c e d the  /xM C a C ^ , 2 mM DTT.  the  700-800 mL o f  eluted  from  divided  Finally,  into  W,  of T r i t o n  column with  loaded onto  Triton  X-100,  aliquots  were  stored  appeared  to  be  a  which  is  X-100  then  E,  which  100 /xM C a C l . 2  freezer  stable  for  at  frozen  in  least  is  and 3 % ( v / v )  acetic acid,  (iii)  the  Isolation  buffer  and p u r i f i c a t i o n  C a l p a i n was prepared  1981; to  et  al.  (1982b)  at 4°C u n t i l  few  and  with  to  use.  months. (i)  90 m i n .  with a c e t i c  a c i d before  following  b a t c h - b i n d i n g to  100 mL 2 M NaCl  sodium a z i d e  (pH  hemolyzate  hemolyzate according  modifications  to  (Waxman, adjusted  150 mL DEAE-Sephacel  The s l u r r y o f r e s i n was washed with 20 mM T r i s - H C l  70  After  200  G o p a l a k r i s h n a and Head, 1985): the pH o f the hemolysate was  6.5  The  used a g a i n .  membrane-free the  as  200 mL o f  o f c a l p a i n from membrane-free  from the  same  nitrogen.  100 mL o f  % (w/v)  The  were pooled  liquid  100 mL o f 6 M urea and ( i v )  mM KC1, 50 mM p o t a s s i u m - H e p e s , 50 jxM C a C ^ , 0.02 and s t o r e d i n t h i s  (ii)  EQ  Two m L - f r a c t i o n s  prior a  buffer  % (w/v).  activity  (-70°C)  o f 20 mM potassium-Hepes and 2 mM EDTA (pH 7.4),  Melloni  0.1  overnight  to  was o n l y 0.05  buffer  were  washed  identical  of C a - A T P a s e  which  in  column was  each u s e , the c a l m o d u l i n - a g a r o s e was t r e a t e d s e q u e n t i a l l y w i t h  7.  and  20-30 mg c o v a l e n t l y  % (w/v)  2 +  peak f r a c t i o n s  small  aliquots  activity  the  The  buffer  concentration  were c o l l e c t e d and the  7.4)  X-100  s o n i c a t e d a s o l e c t i n , 200 mM KC1, 10 mM potassium-Hepes (pH 7.4), 1 100  enzyme  Triton  The s o l u t i o n was then  mM M g C ^ ,  and  % (w/v)  (pH 6 . 6 ) ,  for 1 mM  DTT  and  further  5 mM EGTA washed  (buffer A). NaCl of  packed  with  20  into  A.  mM T r i s - H C l  (pH  mM T r i s - H C l ,  CL-4B  200 mM N a C l ,  (1.5  x 8.5  (pH  7.4).  cm)  The  active  A-200  linear  pooled a c t i v e (30  gradient  filtration  (0-350  x 5.5  mM)  of  i n an u l t r a - f i l t r a t i o n  column  eluted  with buffer  These  preparations  large  and small  (Sephacryl F (50 of  1.5  mM T r i s - H C l  and  1 mM EGTA  (0-350 mM) o f  calpain  subunits of  (5-20  calpain  (80  protein  50-200 iig.mL"*  (3-10  (buffer  buffer  applied  (pH  60  to  a  eluted  7.4).  The  and loaded onto a gel  cm).  The  column was  1 mM DTT and 1 mM EGTA). 1  prot." )  kDa and 29  activity.  unit.mL"*)  with  mL with a PM-30 membrane  (Amicon)  cm x  A  20  A-200).  The p r o t e i n s were  in  and  contain  kDa)  which  using e l e c t r o p h o r e t i c  of calpain a c t i v i t y  with  cm).  unit.mg  2+  used (one u n i t  equilibrated  pooled  (pH 7.4),  do not have C a - i n d e p e n d e n t p r o t e o l y t i c  until  and  cm)  were  NaCl  unit  S-200,  90-95% o f the t o t a l  containing  2.5  and the p r o t e i n s were e l u t e d  f r a c t i o n s were c o n c e n t r a t e d to 5-10  kDa c u t o f f )  more than  1 mM DTT  previously  fractions  omega-hexylamine-agarose column (1.5 a  7.4),  (diameter  1 mM EGTA and 1 mM DTT at pH 7.4  The column was washed with b u f f e r  with  column  A c t i v e f r a c t i o n s were pooled and a p p l i e d onto a column  phenyl-Sepharose  A  glass  The p r o t e i n s were e l u t e d with a l i n e a r g r a d i e n t  in buffer  buffer  a  are  in  criteria,  buffer  in a l a t e r  the  constitute  Preparations of  stored  i s defined  both  and  calpain F at  4°C  section).  7+ 8.  Determination  o f the membrane-bound Ca^ -ATPase  activity  C a l m o d u l i n - d e p l e t e d membranes (50-100 /ig p r o t e i n . m L " * ) at  37°C  maleate  for  30  (pH  calmodulin  min  7.2),  in 66  (when added),  a total mM KC1,  volume 0.1  of  0.4  mL c o n t a i n i n g  mM o u a b a i n ,  2 mM ATP, 0.1  71  were incubated  6.5  55  mM T r i s -  mM M g C l ,  mM EGTA and v a r i o u s  2  120  nM  concentrations  o f C a C ^ to o b t a i n the f r e e Ca" the  and  The M g - A T P a s e a c t i v i t y  in  the  presence o f  activity  assayed  liberated  was determined  9.  concentration  2+  figures.  CaC^  1  Determination  in  the  2.5  indicated  (assayed  mM EGTA) was  presence  of  in  the  absence o f  subtracted  calcium.  colorimetrically  i n the legends  The  from  o f the p u r i f i e d C a - A T P a s e  mL o f 66 mM KC1, 55 mM T r i s - m a l e a t e  0.1%  (w/v)  (when added),  sonicated a s o l e c t i n ,  Determination  150 /iL o f  ouabain, CaCl , 2  7.2).  mM EGTA, /iM L a C l  0.13  4°C f o r  mM EGTA,  mM EDTA,  50  /iM M g C l ,  X-100,  0.1  %  Triton  containing by the 10%  of  2 +  (not  2  essential),  2 +  (5-20  1  2.6  mM C a C l , 2  100  mM (pH  protein)  was  3  The r e a c t i o n  [7- P]ATP  and terminated  e l e c t r o p h o r e s i s at  2.5  32  /iM L a C l ,  (pH 7 . 2 ) .  acid.  mM  [-y- P]ATP  /tg  3 2  [7- P]ATP  The pH 6.6  72  by the  precipitated  and  as d e s c r i b e d above.  4  was  addition protein  15 s  0.1  /iL o f 50 mM KC1, 40 mM T r i s - m a l e a t e ,  (w/v)  3 2  4°C f o r  40 mM T r i s - m a l e a t e ,  Ca -ATPase  2  of  o f the C a - A T P a s e  asolectin  trichloroacetic  f o r a c i d gel  concentrations  sonicated  6 mCi. mmol"*  addition  (w/v)  15 s i n 300  X-100,  concentration.  2 /iM M g C l  purified  incubated at 0.5  Triton  30 120  2  (w/v)  /iM ATP c o n t a i n i n g 6 mCi.mmol"  the  37°C f o r  mM M g C l ,  /ig p r o t e i n ) were i n c u b a t e d at  mM EDTA,  and 2  3  Alternatively,  (w/v)  2 +  50 mM KC1, 5 mM potassium-Hepes,  0.1  100  (50-100  6.5  100 /iM EGTA and v a r i o u s  o f the p h o s p h o r y l a t e d i n t e r m e d i a t e  Plasma membranes in  (pH 7 . 2 ) ,  2 mM ATP, 2 mM DTT, 0.05%  EDTA and C a C ^ to a t t a i n the d e s i r e d f r e e C a  10.  phosphate  2 +  o f the a c t i v i t y 2 +  nM c a l m o d u l i n  total  as d e s c r i b e d above.  P u r i f i e d C a - A T P a s e (1 to 5 /ig p r o t e i n ) was incubated at min i n 0.4  added  the  inorganic  of  of  0.1  0.05 % /iM  ATP  initiated ice-cold  was p r o c e s s e d  11.  R e c o n s t i t u t i o n o f the p u r i f i e d A cholate-dialysis  Villalobo purified  and  Roufogalis  enzyme.  mL  presonicated  final  mixture c o n t a i n e d  CaCl  dialysis 7-11  7.4).  bags  L (5-7  that i t  12.  was  and Racker, used f o r  2 +  Ca -ATPase  the  % (w/v)  The  (50  mixture  kDa c u t o f f )  changes o f  reconstitution  cholate  phosphatidylcholine,  1-2  was  put  inside  and d i a l y z e d  L each)  of  at  the  buffer  reconstituted The  Ca*-  ATP  determined  C a - A T P a s e and C a  hydrolytic  activity  by measuring  method  o f NADH o x i d a t i o n  to  2 +  1 % (w/v)  at  (Raess 340  the  of  the  2  The sodium  and 50 /zM  Spectropore 36  h  against  activities  reconstituted  formation  and V i n c e n z i ,  of  1980)  inorganic or  except  of  the  nm-400 nm i n a dual wavelength  method  of  rate  of  ill  mg p h o s p h o l i p i d s ) were incubated  mM M g C l , 2  concentration 120  containing at  37°C f o r  of  nM c a l m o d u l i n  73  55  was  phosphate  by a  not  determination requirement.  0 . 6 - 1 . 2 /zg p r o t e i n 30 min i n 0.4  (when added),  rate  k i n a s e and l a c t a t e  an e s s e n t i a l  mM T r i s - m a l e a t e  the  spectrophotometer  A) was chosen when  ATP h y d r o l y s i s was  Proteoliposome suspensions (40  a final  (Method  enzyme  by m o n i t o r i n g  The f i r s t  6.5  four  24 t o  transport  dehydrogenase.  containing  the  cholate.  system c o n s i s t i n g o f pyruvate  KC1,  mixture.  d e s c r i b e d above,  u s i n g an ATP r e g e n e r a t i n g  initial  of  by  pump  either  coTorimetric  2 +  o f the  two  4°C f o r  d i d not c o n t a i n p h o s p h a t i d y l c h o l i n e or  Determination  the  as m o d i f i e d  ( 0 . 1 - 0 . 3 mg p r o t e i n ) was added to a  phosphatidylcholine-sodium 1.5  1971),  100 mM KCL, 20 mM potassium-Hepes, 2 mM DTT, 1 mM M g C l  (pH  2  -ATPase  (Kagawa  (1986),  Purified  7-15  cholate,  method  Ca  plus  mL o f (pH  a medium  7.2),  2 mM ATP and  0.65  66 mM various  concentrations of C a C l 0.4  /xM  (Method  free B)  hydrolysis  calcium  ion  was  employed  was  required.  phospholipids) final  (around 70 /xM) and EGTA (around  2  were  20  of  otherwise).  determination  of  Proteoliposomes  73  37°C  mM KC1,  units  pyruvate  in  the  (6-12  1.5  A  t o o b t a i n 0.4  /xM f r e e  90  /xg  mL o f  units  to  attain  second  method  initial  rate  protein  of  plus  (pH  calcium ion.  7 mg  7.4),  a  5 mM  1 /xM c a l m o d u l i n  lactate  dehydrogenase,  (240-280 /xM) and EGTA (around  2  ATP  a medium c o n t a i n i n g  17 mM potassium-Hepes  kinase,  and v a r i o u s c o n c e n t r a t i o n s o f C a C l /xM)  stated  130 txM NADH, 5 mM p h o s p h o e n o l p y r u v a t e ,  2  (when a d d e d ) ,  when  incubated at  concentration  DTT, 730 /xM M g C l ,  (unless  110 xxM)  300-370  The r e a c t i o n was i n i t i a t e d  by the  a d d i t i o n o f 27 jM ATP.  Ca with  2 +  uptake  a dual  above i n the determine  by  the  wavelength  proteoliposomes  was  spectrophotometer  monitored  using  the  presence o f the m e t a l l o c h r o m i c dye Arsenazo III  rates  light  of  ATP h y d r o l y s i s  path o f the  avoid i n t e r f e r e n c e  13.  Determination Calpain  volume o f 0.5  and C a  2 +  transport  in  reaction  a d d i t i o n o f 0.5  a  were  assayed using  and 400  carried  /xM).  1.5-mL  out  mL o f 5% (w/v)  /xM C a C l at  To  total  and the upper p a r t to  interface.  (caseinolysis) casein  mL c o n t a i n i n g 2 mg.mL"^ c a s e i n ,  was  described  spectrophotometer was b l o c k e d w i t h b l a c k tape  of calpain a c t i v i t y  samples  nm-720 nm  (10  by the t u r b u l e n c e produced i n the l i q u i d / a i r  1 mM d i t h i o t h r e i t o l The  650  conditions  volume, a 3-mL c u v e t t e was used under magnetic s t i r r i n g o f the  at  2  37°C  74  substrate  50 mM T r i s - H C l ,  (pH  7.4),  for  30  trichloroacetic  as  in  acid.  and  terminated  After  total  100 /xM EGTA,  unless otherwise min  a  stated. by  the  10 min on i c e ,  the  samples were c e n t r i f u g e d  o f the  supernatant  were run under the absorbance o f  the  blanks was s u b t r a c t e d  of  for  m a x  same c o n d i t i o n s except  A unit  o f one absorbance u n i t  Treatment  4000 x g  5 min and the  was measured s p e c t r o p h o t o m e t r i c a l l y  presence o f c a l c i u m .  14.  at  at  that  at  278 nm.  CaCl2 was not  from the  of calpain a c t i v i t y  total will  absorbance Blanks  added.  The  absorbance i n  the  produce an i n c r e a s e  278 nm under the standard assay c o n d i t i o n s .  calmodulin-depleted  plasma  membranes  with  various  proteases C a l m o d u l i n - d e p l e t e d plasma membranes were t r e a t e d w i t h the i n d i c a t e d with one o f (1-2  mg p r o t e i n )  following MgCl , 2  final  the  following  suspended i n b u f f e r concentrations:  10 mM d i t h i o t h r e i t o l ,  EGTA and C a C l Method b)  to obtain  2  the  indicated  (50-75 ng  protease  55 mM T r i s - m a l e a t e ,  66 mM N a C l , 0.1  the  otherwise. fold  of  i n the  soybean  3  times  for  (1:1)  to o b t a i n  2  Ca  2 +  o f 200 /iM (pH  The r e a c t i o n  25°C f o r  inhibitor  in  60 m i n ,  addition the  case  When r e q u i r e d ,  10 min and c e n t r i f u g e d  at  4000 x g  buffer  IV-E.  The  75  final  IV were  mM M g C l , 2  was  at  of  started  least  the  calmodulin-free  stated a  trypsin,  m a x  of  7.4).  unless  of  /iM  containing  150 /xM EGTA, 6.5  (pH 7 . 4 ) . at  medium  was washed 4 times with 1 mM EGTA, 20 mM T r i s - H C l with  the  and a combination  an i n c u b a t i o n  case o f papain or c a l p a i n .  were c o o l e d on i c e The p e l l e t  in  mM o u a b a i n ,  and i n c u b a t i n g  trypsin  plasma membranes  suspended i n b u f f e r  The p r o t e o l y s i s was stopped by the  excess  leupeptin  protease  protein)  indicated  and 350 /iM C a C l  protease  a)  IV-E were d i l u t e d  a concentration of free  plasma membranes  1 mM d i t h i o t h r e i t o l  Method  65 mM KC1, 19 mM p o t a s s i u m - H e p e s , 250  i n c u b a t e d with the  by adding  methods:  proteases  10 or  membranes for  5 min.  (pH 7.4)  and  pellet  was  resuspended  in  buffer  ATPase a c t i v i t y  o r the  15.  Treatment  3 2  2 +  Ca -ATPase  calpain  at  nM c a l m o d u l i n obtain  time,  aliquots  Treatment  concentration  incubated  with  10 mM d i t h i o t h r e i t o l ,  of  the  300  and a combination o f EGTA and C a C l 200  /zM (pH 7 . 4 ) .  At  the  presence  of  200  /iM  2  desired  2 +  in  0.02  volume o f 55 mM T r i s - m a l e a t e ,  mM EDTA,  of  the  leupeptin  at  25° C f o r  120 min  1  and  Proteolysis  I  of  1  stated  otherwise)  was  0.67  (unless s t a t e d CaCl  2  calpain  p l u s 11 mg p h o s p h o l i p i d s . m L " )  (unless  calpain  concentrations  ion.  1  50 mM potassium-Hepes (pH 7 . 4 ) ,  2 unit.mL"  and v a r i o u s  C a ^ - A T P a s e with p u r i f i e d  (20-60 /ig p r o t e i n . m L "  c o n t a i n i n g 67 mM K C 1 , dtthiothreitol,  was  C a - A T P a s e assay and d e t e r m i n a t i o n  o f the r e c o n s t i t u t e d  were i n c u b a t e d  calcium  65 mM K C 1 ,  for  electophoresis.  Proteoliposomes  free  2  assaying  calpain  protein)  mM M g C l , 0.5  intermediate  s u b j e c t e d t o gel  EGTA  2 +  /ig  before  intermediate.  25°C i n an a p p r o p r i a t e  2 +  volume  C a - A T P a s e with  were taken f o r  phosphorylated  16.  Ca  starting  (40-60  (when added),  a free  the  P-phosphorylated  5 mM p o t a s s i u m - H e p e s , 6.5  to  to  o f the p u r i f i e d  Purified unit.mL"*  IV-E  by  mM M g C l , 10 mM 2  otherwise),  (440-500 /iM)  arrested  i n a medium  the  to  40-100 /iM  attain  addition  of  400  /iM  200  /iM  leupeptin.  17.  Formation  of  the  phosphorylated  intermediate  of  the  reconstituted  2 +  Ca -ATPase P r o t e o l iposomes incubated  at  4°C f o r  (60-120  /tg  protein  15 s i n 400 /iL of  76  plus  32  50 mM K C 1 ,  mg  phospholipid)  were  25 mM p o t a s s i u m - H e p e s ,  pH  7.4,  LaCl ,  mM EGTA,  0.1  mM EDTA,  r e a c t i o n was i n i t i a t e d  spun  at  10,000 g  chloroform to  for  m a x  extract  5 min.  the  phospholipid.  acid-gel  sequences are  developed by Rogers et amino  residues  acid (H,K  containing  residues or  R)  residue  P,  (MPpESj)  0.55 after  " 0-5  al.,  (H )> 0  S  and  and  stretch larger  the  is than  PEST  score  missing either 0 to  qualify  o f the o n e - l e t t e r  was spun  protein  pH 6.6  P,  again and  precipitate  was  as d e s c r i b e d above.  days at  -70°C.  PEST-FIND computer  ending  with  internal  T.  program  A  positively  residues  PEST  score  of  is  has  E/D or  at  to  al., be  S/T,  charged least  8,  calculated  as  PEST s c o r e o f the sequence = E , D, S and T  o f P, E/D and S / T , and H  (Rogers e t  value  by  The mixture was  where MPpr/^j = mole p e r c e n t o f P,  s u b t r a c t i n g one mole e q u i v a l e n t  sequence,  and terminated  PEST sequence was d e f i n e d as a s t r e t c h  (1986); i n b r i e f ,  h y d r o p h o b i c i t y o f the s t r e t c h  19.  The mixture  u s i n g the  a number o f D,  3 2  [7- P]ATP.  and PEST s c o r e c a l c u l a t i o n  beginning  E,  1  was then t r e a t e d with 500 /xL  e l e c t r o p h o r e s i s at  (1986).  with  100 /xM  2  3 2  The remaining  identified  al.  d e s c r i b e d by Rogers et  list  The p e l l e t  PEST sequence i d e n t i f i c a t i o n PEST  of  mM C a C l ,  [7- P]ATP  d r i e d g e l was exposed to X - r a y f i l m f o r 3-6  18.  2.5  2  trichloroacetic acid.  c h l o r o f o r m phase was removed.  then p r o c e s s e d f o r The  mM M g C l ,  by the a d d i t i o n o f  the a d d i t i o n o f i c e - c o l d 10 % (w/v)  the  0.5  300 nM c a l m o d u l i n and 4 /xM ATP c o n t a i n i n g 12 mCi.mmol"  3  The  0.1  1986). larger  then  = average  To q u a l i f y  as a PEST  than  its  as a PEST sequence (see  0  PEST  -5.0.  When  s c o r e has to  the be  legend o f T a b l e 8 f o r a  codes o f amino a c i d r e s i d u e s ) .  Data a n a l y s i s The  fragmentation  patterns  shown in s e v e r a l  77  figures of this  thesis  were a l l of  reproduced i n s e p a r a t e experiments  s e p a r a t e experiments performed i s  these  figures.  Due to  ATPase and c a l p a i n , showing shown.  mean  values  However,  the  the  ±  S.E.M.,  trends  were reproduced i n  separate  S.E.M.  (control)  of untreated  i n d i v i d u a l l y stated  variability  in c e r t a i n  and  (n = 2 to 5 ) .  figures, data  in it  enzyme  experiments  of (n=  enzyme a c t i v i t y  g i v e n i n the legend o f these f i g u r e s .  78  activity  typical the 2 to  of  the  rather  experiments  illustrated 4).  number  i n the legend o f  was d e c i d e d t h a t ,  from  patterns  The t o t a l  Also,  Ca  2 +  -  than  would  be  experiments mean v a l u e ±  from s e p a r a t e experiments  is  RESULTS  I  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  P u r i f i c a t i o n of  In  order  ATPase,  it  source.  to  study  the  was important  fraction.  plasma  effect  to p u r i f y  calpain  I  Briefly,  the  membrane-free  calpain  was  cytosol  purified  protein  chromatography.  Each step e x p l o i t e d  interaction  cassette  with  the  interaction  with omega-hexylamine-agarose  Fig.  14  on  shows the  a gel  (Fig.  filtration  protein  from  profile  and  (b)  human (a)  four  3)  (Fig.  (Sephacryl  mixed  12)  of  large-scale  2)  and 4)  of  -  column  protease:  hydrophobic  ionic/hydrophobic  S-200)  each s t e p  isolation  o f the  10),  2 +  erythrocyte  steps of  property  Ca  from the same  using  (Fig.  11),  after  a  involved  DEAE-Sephacel  phenyl-Sepharose  erythrocyte  preferably  a different  with  size  on the  fraction  system)  interaction  molecular  I  purification  (Pellicon  ionic  of  the p r o t e a s e ,  ultrafiltration  1)  calpain  calpain  Therefore,  cytosolic  of  separation  column  by  (Fig.  13).  purification.  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 a c h i e v e d a f t e r the p h e n y l - S e p h a r o s e s t e p  (Fig.  3).  14,  lane  homogeneity interest  to  hemolysis subunit  of  and  calpain  note  buffer, its  After  that  I  the was  initially  purified activated  last  step  achieved when  calpain fragments  I  (Fig.  only  chromatography, 14,  lane  1 mM EGTA  consistently  (78  79  of  and 75  5).  was  contained  kDa).  apparent It  was  of  used  in  the  the  80  kDa  When 5 mM EGTA was  Fig.  10 DEAE-Sephacel chromatography  hemolysate.  washed with  2 L of of  hemolysate, packed i n t o buffer  NaCl  in  buffer  A (dashed  from human  erythrocyte  as  calcium-dependent  Materials  and Methods ( f i l l e d c i r c l e s ) . fractions  are from a t y p i c a l  are  a g l a s s column and s u b s e q u e n t l y  proteolysis  indicated  preparation  line).  eluted  Protein  280 nm ( s o l i d l i n e ) .  measured  pooled  I  A . The column was then  monitored from the absorbance at  the  calpain  150 mL o f DEAE-Sephacel was s u b j e c t e d to b a t c h - b i n d i n g w i t h 4  L o f membrane-free  (0-350 mM)  of  of  by a s o l i d  of c a l p a i n .  80  bar.  a  gradient  concentration  was  Calpain a c t i v i t y  was  casein,  Each e l u t i o n  with  as  described  in  f r a c t i o n was 7 mL and Data  presented  here  18  (  )  NaCI gradient  O o H  (•  •)  —» O O  rO O O  OJ O O  1  1  1  Calpain activity  o o  p M  o  p  O  (  (mM)  0 ^  (AA278) o o>  0  ^  CO  rO  )  Absorbance  01  3Jn6Lj  A28O  Fig. JL  11  P h e n y l - S e p h a r o s e chromatography  Pooled c a l p a i n f r a c t i o n s  phenyl-Sepharose  (1.5  extensively  buffer  finally  with  eluted  and c a l p a i n  with  activity  x  of  DEAE-Sephacel-isolated calpain  from DEAE-Sephacel were a p p l i e d t o a column of  8.5  cm)  (load),  A containing  buffer (filled  A (elute). circles)  200  the  column  mM NaCl  Protein  was  (wash).  then The  concentration  were measured  as  in  washed  column was  (solid Fig.  10.  line) 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 i n d i c a t e d w i t h a s o l i d b a r . Data p r e s e n t e d here  from a t y p i c a l  calpain  82  preparation.  £8  Fig.  12  Omega-hexylamine-agarose  purified  calpain  I.  Pooled  column were a p p l i e d 5.5 with  directly  chromatography  calpain to  fractions  of  from  a omega-hexylamine  phenyl-Sepharose-  the  phenyl-Sepharose  agarose column  (1.5  cm). The column was washed with 100 mL o f b u f f e r A b e f o r e being a gradient  concentration monitored  as  fractions  are  typical  (0-350  (solid in  Fig.  mM)  line) 10.  and  Each  i n d i c a t e d with  preparation  of  NaCl  in  buffer  calpain fraction  a solid  of c a l p a i n .  84  A  (dashed  activity  (filled  was  bar.  5 mL and the  Data  presented  line).  eluted Protein  circles) pooled here  are  x  were  calpain from a  S8  (  (  )  )  Zl  NaCl gradient  Absorbance  aan6 L J  (mM)  A250  Fig.  13  purified  Sephacryl calpain  I.  S-200  (Amicon)  of  Pooled c a l p a i n f r a c t i o n s  column chromatography unit  chromatography  were c o n c e n t r a t e d  fitted  with  a PM-10  omega-hexylamine-agarose-  from  to  omega-hexylamine-agarose  2 mL with  membrane.  an  ultrafiltration  The c o n c e n t r a t e d  sample  was  then a p p l i e d t o a Sephacryl S-200 column (1.5  x 60 cm) coupled to an FPLC  system.  F and f o r t y  The column was developed with  were c o l l e c t e d determined gel  using  filtration  serum albumin kDa)  a flow Blue  Protein  kDa)  were monitored  as i n  10 m L . h " .  standards: (b),  The e l u t i o n  concentration  1  of  Dextran-2000  protein (67  rate  (43  kDa)  49.5,  volume  (60  mL)  line)  63.2,  Pooled f r a c t i o n s  Data presented here are from a t y p i c a l  86  (40  (160  kDa)  mL)  as  volume  of  (a),  bovine  chymotrypsinogen A 69.5,  of calpain  and c a l p a i n  mL-fractions  Elution  Q  (c),  2  volume  (V ).  human 7 - g l o b u l i n  are  10.  The v o i d  indicated  kDa)  (solid Fig.  is  ovalbumin  and r i b o n u c l e a s e A (14  respectively.  bar.  at  buffer  I  84.2 is  activity are  calpain  and 92.8  indicated (filled  (22 mL,  (V ). e  circles)  i n d i c a t e d with a s o l i d preparation.  Fig.  14  showing  Purification  of  the  pattern  membrane-free Sepharose, 200.  The  (4)  peptide  hemolysate, after  protein  separate c a l p a i n  calpain  (2)  at  from  various  after  omega-hexylamine  patterns  human  presented  preparations.  88  erythrocytes.  stages  of  DEAE-Sephacel, agarose and here  were  (5)  SDS-PAGE  purification: (3) after  routinely  after  gel (1)  phenyl -  Sephacryl  S-  reproduced  in  Figure  2 34  14  5  KDa  89  used,  the  80  kDa s u b u n i t  remained  o f c a l p a i n was a c t i v a t e d calpastatin  recovery  (means  three  only  I  preparations).  but  "calpain" refers  2.  not  200  /zM  It  to c a l p a i n I,  fragment (Fig.  Ca  78  kDa  into  lane  a smaller  itself degree  2 +  ,  of  misconception  2,  the  80  3,  two  that  erythrocytes  the  (Table  2).  Twenty-  300-400  mL o f  4011-fold red  S i n c e human e r y t h r o c y t e s  from t h i s  point  on,  for  cells, contain  convenience,  otherwise.  preparations  followed to  this  The  intact  calpain  subunit the  was  was  analysed  the  transformed  accumulation  activated  small  that  by  subunit 80  of  form  of  was  also  kDa s u b u n i t  the the  into 75  a  kDa  protease  transformed  can  hydrolyze  form o f c a l p a i n must a l s o express some  activity. 80  by  be the  The f a c t  of  t h a t i n the presence o f 10 mM DTT and  kDa c a t a l y t i c  5 and 6 ) .  proteolytic that  with  unless stated  identified  that  II,  illustrated  fragment.  indicates  kDa fragment  of  fragment  previously  15A,  portion  calpain  was c l e a r l y  free  transient  i n human  p r o t e a s e was p u r i f i e d  Starting  calpain  autohydrolysis  SDS-PAGE.  the  I can be p u r i f i e d .  Characterization of  The  a  d e s p i t e the f a c t  was a l s o f o l l o w e d  was found w h i l e  1 mg o f c a l p a i n calpain  that  1989).  two percent  about  indicates  when compared to c a l p a i n  The r e c o v e r y o f c a l p a i n a c t i v i t y  for  This  i n the hemolysate f r a c t i o n ,  was i n excess  (see M u r a c h i ,  intact.  This  KDa s u b u n i t  is  argument the  inactive  settles  form w h i l e  i s the a c t i v e form o f c a l p a i n (see Pontremoli  90  a  et  al.,  common the  76  1985a).  Table 2  P u r i f i c a t i o n of c a l p a i n I from human e r y t h r o c y t e s  STEP  Protein (mg)  3  V  Recovery  4000  DEAE-Sephacel  50  78.1  3.03  240  100  Phenyl-Sepharose  75  13.7  23.87  1894  138  Omega-hexylamineagarose  35  3.6  41.02  3256  62  50.55  4011  22  S-200  assuming f u l l  8  18810  1.03  0.0126  Purification (fold)  Membrane-free hemolysate  Sephacryl  a  Vol (mL)  Specific Activity (unit/mg p r o t . )  recovery from step 1 to step 2  Values presented are averages of three  preparations  100  c  Fig. on  15  A u t o l y s i s of p u r i f i e d  calpain  protein) and to  purified the  incubated for at  0  processed  to  (B). the  Sephacryl i n the  min  25°C.  other  activity  (A),  and e f f e c t  two  calpain  omega-hexylamine-agarose  S-200  ( l a n e 1 and 4 ) ,  step  (lane  4,  5 2 +  ,  for  SDS-PAGE.  in  Materials  of  In  proteolysis  (B),  purified  and Methods  or 37°C ( f i l l e d c i r c l e s ) .  10 % (w/v)  TCA at  the  92  6),  (lane  1,  (1.5  /ig  2 and  3)  respectively,  were  o r 60 min  ( l a n e 3 and 6)  TCA and the  calpain casein  were (4  /tg)  as  a  samples were  reproduced was  in  two  assayed  substrate  at  time.  The f i g u r e  experiments.  in  as  25°C  P r o t e o l y s i s was stopped with  indicated  p r e s e n t s the mean v a l u e s f o r 3 d i f f e r e n t  preparations  50 mM T r i s - H C l  patterns  using  temperature  10 mM DTT,  30 min (lane 2 and 5)  The  o f assay  step  and  P r o t e o l y s i s was stopped with 20 % (w/v)  (open c i r c l e s ) addition  In  (A)  presence o f 200 /iM f r e e C a  experiments.  described  calpain  panel  the B  Figure  KDa  1 2 3  15A  4 5  93  F i g u r e 15 B  B  94  The  effect  (Fig.  15B).  after  30 m i n .  for  at  of  assay  Calpain  activity  whereas  at  least  60  min,  determination  of  calpain  routinely purified  at  on  described  only  Mellgren  2 +  Ca -ATPase,  the  next  preparations Fig.  16A  iiM  calpain  shows  requirement 20  of  series  that  free  fully  Ca  2 +  .  active.  I,  known  highly  leupeptin) the  protease  hand,  in  it  the  37°C linear  For  was  was  assayed  effect  confirmed  purified  calpain  activation protease  agents  inhibited  was  to  of  by these less  of  has  belongs  found  that  as  an  2 +  an  20  increase mM.  I.  absolute  the  class  with  Calpain  is  inhibitors  Fig.  inhibitor,  16B  shows  On the while  observed ( F i g .  membrane-bound C a - A T P a s e  95  the  calpain  to  compounds as e x p e c t e d .  i n c r e a s e d the c a l p a i n a c t i v i t y  E f f e c t o f c a l p a i n on the  to  iodoacetate).  potent  the  o f the enzyme o c c u r r e d at  cysteine-protease  (eg.  the  25°C was chosen s i n c e  of  assay medium up t o  susceptible  considerably  inhibitor  protease  properties  activity  the  alkylating  was  PMSF was  trypsin  II.  and  DTT  at  (1982).  expected  purified  time  which r e q u i r e s micromolar c o n c e n t r a t i o n s o f C a  Calpain  of  be  the  al_.  test at  assessed  relatively  the  activity  Thus  concentration to  the  was  required.  experiments,  exhibited  to  incubation  f o r c a l c i u m . Half-maximal  designated calpain be  of  et the  However,  p r o t e o l y s i s time beyond 30 min was u s u a l l y  In  remains  therefore,  30 min.  activity  decreased with  activity  by  activity,  on the  calpain  progressively  25°C c a l p a i n  as  37°C f o r  calpain  temperature  2 +  to the  also (eg. that other  soybean  16B).  Fig.  16  Activation  inhibitors  of  on c a l p a i n  calpain  by  activity.  Ca^  (A)  The  (B)  The p u r i f i e d  and  the  purified  was assayed w i t h v a r i o u s c o n c e n t r a t i o n s o f and Methods.  +  Ca  2 +  effect  enzyme  (4  as i n d i c a t e d  enzyme (4 /ig p r o t e i n )  of  various  /ig  protein)  in  Materials  was assayed w i t h  100  7+ /iM  free  Ca  (circles), trypsin  iodoacetate  inhibitor  achieved  was  definition) are  typical  (mean  ±  KQ 5 ( c ) a  i n the presence of the (squares),  (triangle).  0.20  unit.h"  and was taken of three  S.E.M.)  of  1  indicated concentrations of PMSF  The (see  (inverted maximum  Materials  as 100% c a l p a i n  and  experiments  o b t a i n e d from the t h r e e  experiments  was  for  presented calpain  ±  soybean  (caseinolysis)  Data  0.34  or  Methods  The u n t r e a t e d  three  96  activity  activity.  separate e x p e r i m e n t s . the  triangle)  leupeptin  0.15  (mean ± S . E . M . )  unit here  activity 1  unit.h" .  i s 20 ± 8 /iM.  Figure  120  16  -i  -10  -8  -6  -4  Log [Inhibitor] (M)  97  -2  1.  Effect  of t r y p s i n ,  papain and c a l p a i n on the  activity  o f the  membrane-  bound C a ^ - A T P a s e  The  plasma  membrane-bound  Ca  -ATPase  activity  p r o g r e s s i v e exposure to c a l p a i n , t r y p s i n or papain and  papain  activated  the  Ca  -ATPase a c t i v i t y  was  (Fig.  tested  17).  maximally  Both t r y p s i n  within  when assayed i n the absence o f c a l m o d u l i n , but the a c t i v a t i o n immediately longer  periods  However,  of  preincubation  with  of  the  of  c o n d i t i o n s was not  treatment  with  the  followed  protease  was t r u e  for  the  Ca  -ATPase by  activated  the  papain.  Therefore,  papain,  might  domain  of  it  activate  the  preincubation  three  enzyme of  the  is the  proteases, a similar conceivable  al.,  mechanism t o  on the  least  assayed  17B).  that  1986).  2 +  Ca -ATPase activity  the  in  120  in  the  may  have  trypsin  or  trypsin  or  calmodulin-binding  Fig. in  of  like  for  increased.  calpain  calpain,  plasma membrane with c a l p a i n  mM EGTA has no e f f e c t  and  was not f u r t h e r  2 +  et  at  When  C a - A T P a s e by c l e a v i n g the  (Sarkadi  17A  at  2 +  suggesting that  that  followed  o f the C a - A T P a s e a c t i v i t y  17C).  z +  This  (Fig.  by i n a c t i v a t i o n  presence o f c a l m o d u l i n the C a - A T P a s e a c t i v i t y  was  min,  Ca -ATPase activity  proteases  (Fig.  10  2 +  the  i n the case o f c a l p a i n the a c t i v a t i o n  under these min  by a p r o g r e s s i v e i n a c t i v a t i o n  after  the  17D  shows  that  presence of presence o r  2.5 in  the absence o f c a l m o d u l i n .  The  effect  of  preincubation  protease concentrations 18).  on the  When assayed i n the  was a c t i v a t e d  maximally  at  of 2 +  the  plasma  Ca -ATPase  membrane  activity  absence o f c a l m o d u l i n , the  with  was examined 2 +  Ca -ATPase  t r y p s i n and papain c o n c e n t r a t i o n s o f  98  different (Fig.  activity  10"^ M and  Fig.  17 T i m e - c o u r s e o f the e f f e c t  ATPase a c t i v i t y . trypsin  (A),  9.1  of p r o t e a s e s on the membrane-bound  Membranes (60 /ig p r o t e i n ) x 10~  7  M papain  a c c o r d i n g to method (b)  (B)  or 6.4  (see M a t e r i a l s  membranes  were  presence  of  Protease  inhibitor  to  the  2.5  incubated  mM EGTA.  in  of  proteases  to  membranes were assayed t h e r e a f t e r min at  100 /iM f r e e  (closed  circles)  typical  experiment.  three  separate  untreated ATPase  (zero  activity  experiments  was  absence was  was added a f t e r the  addition  Ca of  , 120  i n the  experiments  of  obtain  the  zero  2 +  for Ca -ATPase  Data  and p a t t e r n s the  CaCl  start  2  in  time  or  presented  presented  time-courses  200  26.7  ±  /iM 7.2  free  Ca and  2 +  48.5  respectively.  99  A, B  (mean ±  ±  10.1  in the  proteolysis. prior  values. at  37°C f o r  i n the here  The 30  presence  are  from a  were reproduced  varied  S.E.M.) nmol .mg  M  D),  in  slightly.  time) basal and c a l m o d u l i n - s t i m u l a t e d membrane-bound at  7  whereas and  2  the  activity  absence (open c i r c l e s )  but  (C and  except t h a t  added  added t o  x 10~  i n c u b a t i o n was completed and  nM c a l m o d u l i n .  The trends  M calpain  presence o f 250 /iM C a C l  the  Protease  7  x 10~  and Methods),  and C membranes were incubated i n the D,  were t r e a t e d w i t h 8.5  C a -  from  the  prot.  _ 1  in The  Ca  2 +  -  three  .min"*,  F i g u r e 17  100  Fig.  18  ATPase trypsin  Effect  of  activity. (A),  protease The  papain,  concentration  indicated  (B),  (60 fig p r o t e i n ) min  at  20 zzM f r e e  (closed c i r c l e s ) o f two  were then  of  Ca^  in  protease  or c a l p a i n  60 min a c c o r d i n g to method (b)  (C)  during  experiments.  101  membrane-bound  and Methods).  2 +  Data  were  pretreatment  Ca -ATPase activity  absence (open  120 nM c a l m o d u l i n .  the  concentrations  (see M a t e r i a l s  assayed f o r the  on  circles)  or  at  Ca  -  used  for  25°C  for  The membranes at in  37°C f o r  30  the  presence  p r e s e n t e d here are  typical  F i g u r e 18  10"'  M,  respectively.  sensitivity  activation  was  a s s o c i a t e d with  decreased  concentrations.  In  10"^  and  calpain  at  slightly  contrast, the  higher  Ca -ATPase  activation  was  trypsin  in  Ca^ -ATPase  activity  was  or  was maximally  maintained  subsequently  of was  papain  activated  when  c o n c e n t r a t i o n was i n c r e a s e d by two o r d e r s o f magnitude decrease  loss  The C a - A T P a s e a c t i v i t y  2 +  the  the  2 +  to exogenously added c a l m o d u l i n .  dramatically  M  Full  the  (up t o  at  calpain  10"^ M).  observed  at  A  higher  concentrations of c a l p a i n .  2.  Protection  proteolytic  The  by  activation  presence  incubation did activity  (Fig.  a  achieved  concentration percent  in  the  protection  2 +  membrane-bound  the  (0.3  /iM)  Ca -ATPase  proteolytic  However,  the  during  dependent  proteolytic  ranges  manner  from  assay against  (Fig.  67% t o  system  84%  (see  Fig.  proteolytic  trypsin  activation  of  against  or  papain  2 +  the  activation  induced by c a l p a i n was s i g n i f i c a n t l y  concentration  protection  the  calmodulin  prevent  19).  of  by c a l p a i n  of  not  ATPase a c t i v i t y in  calmodulin  Ca -ATPase  of  the  Ca  which  maximum p r o t e c t i o n  20).  The  depending 20).  maximum on  From  activation  was  comparable  c a l m o d u l i n (2.2  ± 0.3  (KQ 5 ) was found  to  the  KQ 5  nM)  (Fig.  for  21B).  103  to  be  1.9  activation  -  reduced by c a l m o d u l i n  of  the a  percent  free  plot  Ca  of  ± 0.4 of  the  2 +  the  z +  the  Ca -ATPase  v e r s u s the c o n c e n t r a t i o n o f added c a l m o d u l i n , the c o n c e n t r a t i o n needed half  2 +  nM ( F i g . 2 +  for  21A),  Ca -ATPase  by  Fig.  19  Effect  of  calmodulin  c  membrane-bound C a - A T P a s e . protease papain  as  (C)  (squares)  follows:  at  (A),  4.3  x  60 min i n the  action  (1.5 10~  7  The  Materials  and  membranes  Methods  activity  at  presence  (filled  37°C  of three  for  and 30  symbols)  were then  min  M trypsin  of  subsequently  120  the  (60  The u n t r e a t e d  2 +  membrane-bound C a - A T P a s e a c t i v i t y  ±  from  the  three  nmol.mg p r o t . ~ * . m i n " * ,  experiments  respectively.  104  was  as  7.2  x  10~  8  M  presence and  described  in  2 +  for  Ca -ATPase  symbols)  Data  the  (see M a t e r i a l s  washed  nM c a l m o d u l i n .  separate experiments.  (a)  (open  9.1  o r i n the  /iM p r o t e i n )  absence  on  were t r e a t e d with (B),  absence ( c i r c l e s )  assayed in  proteases  mg p r o t e i n )  stimulated S.E.M.)  of  o f 300 nM c a l m o d u l i n a c c o r d i n g to method  Methods).  typical  the  Membranes  none  25°C f o r  on  or  presented  in  the  here  are  basal and c a l m o d u l i n -  at  4.3  /iM f r e e  ±  3.7  and  Ca  40.1  2 +  (mean +  13.1  F i g u r e 19  Fig.  20  Effect  Ca-ATPase. triangles)  of  calmodulin  Membranes or  treated  (1.5  on  calpain  mg p r o t e i n )  with  calpain  action  on  were u n t r e a t e d  (6.4  x  10"  7  M)  v a r i o u s c o n c e n t r a t i o n s o f c a l m o d u l i n : none ( f i l l e d M (open  squares),  2.7  x  10"  1 1  M (X),  the  2.7  x  10"  in  membrane-bound  (inverted the  presence  squares), 1 0  M (+),  filled  2.7 2.7  x x  10 10"  _Q (inverted  open  triangles),  (open c i r c l e s ) ,  2.7  Materials  Methods).  described  and in  2 +  Ca -ATPase calmodulin. experiments. 2 +  Ca -ATPase the  the Data  The  10  M (open  absence presented  activity  at  membranes  and Methods  The u n t r e a t e d  legend o f F i g .  x  4.3  (A)  or  in are  triangles),  subsequently  assayed  the  (60  Ca  19.  106  2 +  x  10  M  presence  typical  of  (mean ± S . E . M . )  M (see  washed  /ig p r o t e i n )  basal and calmodul i n - s t i m u l a t e d /iM f r e e  2.7  a c c o r d i n g t o method (a)  were  and then  here  9  _7  x 10"^ M ( f i l l e d c i r c l e s )  Materials in  2.7  of  (B)  of  three  120  as for nM  separate  membrane-bound is  as g i v e n  in  Fig.  21  Effect  o f c a l m o d u l i n on the  ATPase a c t i v i t y  by c a l p a i n  membranes (60 /ig p r o t e i n ) for  60  min  presence  according to  of  the  proteolytic  activation  2 +  (A)  and the C a - A T P a s e a c t i v i t y  were t r e a t e d with c a l p a i n (6.7 method  indicated  (b)  (see  Materials  concentration  of  2 +  for Ca -ATPase activity absence  of  calmodulin.  calpain  in  the  at  absence of  different  concentrations  calculated  as  absence  calmodulin)  of  untreated  the  of  of  the  o f the  of  the  Methods) Then  in  the  membranes  4.3  /iM o f f r e e  Ca  2 +  in  the  Ca -ATPase activity  by  2 +  a i  or  n  presence  (Activation -|p ^ (CaM)) c a  a  Ca -ATPase activity  calpain-treated  membranes  protection  2 +  Ca -ATPase activity  (in  the  of  the  and t h a t  against  of was  n  2 +  the  (%)  calpain-mediated  was then c a l c u l a t e d  c a l p a i n  by  (CaM)  =  x Activation  in  and  c a  Activation  (B),  (A),  equation:  Calmodulin P r o t e c t i o n  In  In  x 10"^ M) at 25°C  (Activation -|p - )  between  1  Ca " "-  and Methods and assayed  the  calmodulin  The c a l m o d u l i n  activation  the f o l l o w i n g  activation  calmodulin  difference  membranes.  proteolytic  37°C f o r 30 min at  The  (B).  calmodulin.  were s u b s e q u e n t l y washed as d e s c r i b e d i n M a t e r i a l s  2  o f the  calpain  the Ca*- -ATPase was assayed as d e s c r i b e d i n absence or  The c a l m o d u l i n  presence o f  activation  ATPase a c t i v i t y  was  the  indicated  calculated  as  100  Materials  concentration the  and Methods  of  difference  calmodulin.  of  the  Ca  i n the presence versus the absence o f c a l m o d u l i n at 4.3  2 +  -  /iM  p. of  free  Ca  .  The  nmol.mg  prot."*.min"  typical  of  three  1  maximum and  separate  was  calmodulin  taken  as  experiments. 108  100%.  activation Data  From these  presented  experiments,  was here the  47 are KQ 5  Fig.  21  (Cont.)  for  p r o t e c t i o n (mean ± S . E . M . )  was found to be 1.9  ± 0.4  for  a c t i v a t i o n (mean ± S . E . M . )  was found to be 2.2  ± 0.3 nM.  109  nM w h i l e the KQ  5  Figure  21  1 1 0  The p o s s i b i l i t y also  excluded  that  calmodulin  by showing t h a t  has a d i r e c t  calmodulin d i d  when c a s e i n was used as a s u b s t r a t e  3.  Further  characterization  (results  modify  not  shown).  ATPase the  activity  protease  capacity  to  is  the  effect  presented  progressively be  in  of  calpain  Fig.  22.  activated  stimulated  c a l m o d u l i n the p r o t e o l y t i c  calpain  o f the c a l p a i n d i g e s t i o n o f the  2 +  of  on c a l p a i n  not  C a - A T P a s e i n the absence and the presence o f  A time-course  effect  by  the  membrane-bound  on the the  membrane-bound absence o f  2 +  C a - A T P a s e and However,  in  the  Therefore,  the  or  induce  a  fragmentation  its of  was p r e s e r v e d .  2 +  proteolysis  -  significantly  B i n d i n g o f c a l m o d u l i n to the membrane-bound C a - A T P a s e c o u l d its  2 +  calmodulin  presence  o f the C a - A T P a s e was  reduced and the c a l m o d u l i n s t i m u l a t i o n  Ca  decreased  z +  activation  activity  calmodulin  In  calmodulin.  was  different of  the  pathway  of  prevent  fragmentation. 2 +  membrane-bound  Ca -ATPase  was  op  examined It  was  ATPase  by f o l l o w i n g found was  calmodulin  that  the  indeed ( Fig.  the  P-phosphorylated  fragmentation  different  in  pattern  the  thereafter molecular  into by  the  weight  of  absence  the  of  the  enzyme.  membrane-bound  versus  the  Ca  presence  2 +  -  of  23).  In the absence o f c a l m o d u l i n , the n a t i v e transformed  intermediate  a  124  kDa  fragment  progressive (80  kDa).  within  formation In  the  111  of  enzyme (136 1-2  min.  a  second  presence  of  kDa) was This  was  fragment  calmodulin,  rapidly followed  of  lower  however,  Fig.  22 A c t i v i t y o f the membrane-bound Ca  the  presence  and absence o f  calmodulin.  -ATPase t r e a t e d with c a l p a i n Membranes  t r e a t e d with c a l p a i n i n the absence ( c i r c l e s ) of  calmodulin  as  described  membrane p r o t e i n )  were taken  washed and t h e r e a f t e r Ca  2 +  i n the  calmodulin three  2 +  17.0  at  Materials the  mg p r o t e i n )  o r i n the presence  and Methods.  (see  separate  z +  and Methods).  experiments.  (mean ± S . E . M . ) nmol.mg p r o t .  _ 1  The  with 4.3 /iM o f  Data  untreated  presented (zero  z +  1  .min" ,  experiments was 14.7  respectively.  112  are  time)  membrane-bound C a - A T P a s e a c t i v i t y from the t h r e e  (56  /ig  i n d i c a t e d time and the membranes were  assayed f o r C a - A T P a s e a c t i v i t y  Materials  were  (squares)  Aliquots  absence (open symbols) o r i n the presence ( f i l l e d  calmodulin-stimulated Ca  in  (1.2  in  at ±6.1  free  symbols)  of  typical  of  basal 4.3  /iM  and free  and 72.1  ±  F i g u r e 22  113  Fig.  23  Formation  absence  or  in  (2.1  the  Materials  and Methods.  protein)  were  containing washed  taken  fragmentation separate  presence At the and  as  patterns  the  (+)  of  intermediate  fragments were  calmodulin  and 200  for  described presented  to  1.5  here  experiments.  114  with  (CaM),  aliquots  /iM l e u p e p t i n .  Materials were  the by  calpain  native calpain in  the  as d e s c r i b e d (100  in  /ig membrane  mL-microcentrifuge  phosphorylation, in  by  produced  treated  indicated times,  transfered IV-E  resuspended  autoradiography,  and  mg p r o t e i n )  1 mL o f b u f f e r  and  phosphorylated  Ca -ATPase  Membranes  (-)  the  2 +  membrane-bound treatment.  of  tubes  The membranes were electrophoresis  and  reproduced  Methods. in  four  and The other  Figure  115  23  the  relative  molecular  masses o f  and 85 kDa, r e s p e c t i v e l y . on the  presence o f C a  (results  not  shown),  2 +  the  ^P-labelled  fragments  The p h o s p h o r y l a t i o n o f the  and they were s e n s i t i v e to hydroxylamine  demonstrating  t h a t they  1.  127  kDa  bands was dependent treatment  indeed r e p r e s e n t e d  o f the Ca^ -ATPase capable o f forming the a c y l p h o s p h a t e  III.  were  fragments  intermediate.  P r o t e o l y s i s o f 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  2 +  C a l p a i n d i g e s t i o n o f the p u r i f i e d  The Fig.  2 +  purified  Ca -ATPase  24A shows a t i m e - c o u r s e  was of  Ca -ATPase  also  subjected  calpain  digestion  to  calpain  on the  digestion.  activity  of  the  p. purified  Ca*- - A T P a s e .  produced a s l i g h t the a c t i v i t y during  the  to  also  Next, after  the a  The  (Fig.  produced (Fig.  effect  the  absence o f  i n the  first  calmodulin  15 min w h i l e  a  the  thereafter.  stimulation 24A).  and  was  Calpain  progressive  calmodulin-stimulated After  decreased digestion  decrease  the same  of  120  from in  the  the  the  min  of  original  presence  of  maximum  Ca  2 +  -  on the  Ca  2 +  -  24A).  of  prolonged  24B shows t h a t  basal  progressively  calmodulin  1.4-fold  ATPase a c t i v i t y  Fig.  o f the C a - A T P a s e  period.  declined  proteolysis,  ATPase  in  2 +  activation  time  activities  calmodulin  proteolysis  i n the presence o f c a l m o d u l i n remained approximately  this  3.3-fold  Calpain  i n the  different  calpain  period  incubation  of  concentrations  absence o f c a l m o d u l i n  116  (2  hours)  increasing  was  followed.  concentrations  Fig.  24  Effect  Purified  of  Ca^ -ATPase  absence ( c i r c l e s ) in M a t e r i a l s added  to  absence  symbols), ATPase  /xg  in  aliquots  as  the  four  taken  at  treated  symbols)  of here  time-course The  of  untreated  2 +  1.1  2 +  are  ±  200  and Methods. with  or  in  to  the  i n the  0.3  117  calmodulin  indicated presence  were  then  (closed Ca  of  four  effect basal  3.6  z +  ±  1.5  -  (squares)  of  2  hours,  assay  medium  for  Ca in  Materials  separate varied  and  Ca  in  2 +  of  assayed  indicated  in  activity  After  mentioned  the  2 +  Purified  Methods.  as  2 /zM f r e e  respectively.  the  Ca  absence (open symbols) o r  typical  and  of  and  2 /iM f r e e  (B)  above  samples  time)  at  the  and  proteolytic  at  the  o f c a l m o d u l i n , as d e s c r i b e d  presence  calmodulin,  (zero  Ca -ATPase activity was  the  Materials  Ca  in  /zM l e u p e p t i n ) ,  The  2 /zM f r e e  calpain  (A)  containing  in  added  with  activity.  a l i q u o t s were taken and  incubated  in  -ATPase  indicated times,  (circles)  and  presented  experiments  was  Ca  z +  or  was  indicated  (closed Data  purified  Ca -ATPase activity  200 /zM l e u p e p t i n .  experiments. purified  symbols)  absence  ATPase a c t i v i t y  However,  (also  assayed f o r  protein)  the  were  containing  Methods.  At the  as i n d i c a t e d in M a t e r i a l s  calmodulin,  presence  the  zzg p r o t e i n )  assay medium  (open  (8  calpain  (6.5  and Methods.  the  on  or i n the presence (squares)  samples were then the  calpain  2 +  -  the and  experiments.  slightly  among  calmodulin-stimulated  (mean ± S . E . M . ) /zmol .mg  from  the 1  prot.'^.min" ,  C a - A T P a s e Activity  C a - A T P a s e Activity  (pmol'mg prof." * min" )  (fimol-mg prof." * min" )  2+  2+  1  1  ^  ro  O) H i - * -  i  r 4  ^  rr  o  •  //  \  o,  / 0  •  • •  1/  *  •  CD  '  1  of calpain activate  the basal Ca  of  Furthermore,  calmodulin).  produced  a  activity  gradual  decline  -ATPase a c t i v i t y increasing  of  the  (assayed i n the absence  the  calpain  concentration  calmodulin-stimulated  when the p u r i f i e d enzyme was t r e a t e d e i t h e r  Ca  -ATPase  i n the absence or  the  presence o f c a l m o d u l i n .  The  fragmentation  pattern  c a l p a i n was a l s o examined  125-124  (Fig.  2 +  purified  C a - A T P a s e band kDa  band.  of  (136  T h i s was  the  25B). kDa)  In  In  contrast,  fragments smaller  have  in  relative  fragments  of  the  the  C a - A T P a s e produced  absence o f  was q u i c k l y  followed  by  band o f 82-80 kDa, and s m a l l e r fragments kDa.  2 +  purified  presence  molecular  the  calmodulin,  transformed  formation  of  kDa,  27  kDa  of  calmodulin,  masses  55 kDa, 39 kDa, 37  18  2 +  purified  C a - A T P a s e are  intermediate. and  and  the  127  of  127  kDa and 32  Fig.  capable  of  25C  shows  kDa  and  of  85  kDa,  Therefore, the  purified  in  the  the  a broad  another  kDa  and  kDa.  which  i n the of  P-labelled  2 +  Ca -ATPase  most  broad  were  prominent  85  kDa,  and  F i g . 25A shows, (76  kDa, 46 kDa,  fragments  P-labelled  presence 3 2  the  3 2  forming  The 125-124 kDa and 82-80 kDa,  intermediates. fragments  kDa).  the  o f 55 kDa, 39 kDa, 37 kDa and 32  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 36  into  by  of  the  phosphorylated  absence o f c a l m o d u l i n , calmodulin,  formed  EP  acylphosphate-forming  similar  to  those  of  the  membrane-bound enzyme.  It  was o f  hydrolytic the  interest  activity  purified  2 +  to  e s t a b l i s h the  o f the fragmented  C a - A T P a s e was  effect  of  c a l m o d u l i n on the ATP 2 +  form o f the C a - A T P a s e .  treated  119  with  calpain  in  the  Therefore, absence  of  Fig.  25  Fragmentation  c a l p a i n and calpain  (pH 7.4) cold  the  /zg p r o t e i n ) (pH 7 . 4 ) ,  (lane 2).  (lane  presence At  the  with  with  (+)  (w/v)  time,  (0.5  (pH  triangles  8.3).  and  the  0.5  (CaM)  protein  30 min  2 +  in  as d e s c r i b e d were taken  trichloroacetic Calpain of  was  in  acid  mM C a C l  autolytic  in  Materials  is  (-)  2  ice-  protein) or  in  the  and Methods.  protein  precipitated  being  subjected  to  are  indicated  by  fragments  calmodulin  (47 /zg  absence  before  50  processed f o r  the  and the  by  Purified  and 0.7  Ca -ATPase  /zg p r o t e i n )  band  -ATPase  (A)  25°C f o r  mM EGTA,  precipitated Purified  aliquots  ice-cold  electrophoresis solid  Ca  r e a c t i o n was stopped w i t h 10% (w/v)  (B)  calmodulin  indicated  10%  purified  was incubated at  a c i d and the  calpain  of  1)  The a u t o l y t i c  trichloroacetic  treated  and  10 mM d i t h i o t h r e i t o l ,  e l e c t r o p h o r e s i s at pH 8 . 3 . was  solublized  f o r m a t i o n o f the p h o s p h o r y l a t e d i n t e r m e d i a t e .  (10  mM T r i s - H C l  of  indicated  by  the  open  7+ triangle. (0')  or  (C)  Ca^ -ATPase (20  i n c u b a t e d with c a l p a i n  absence Materials The  Purified  (-)  or  in  and Methods.  samples were  Materials  the  then  acid  subjected  and  under  The  patterns  separate  (+)  of  for  to  60 min  calmodulin,  phosphorylation  (60')  as  200 with  untreated in  the  indicated  in  /zM l e u p e p t i n . 32  [-y- P]ATP  The r e a c t i o n was stopped w i t h 10 % (w/v)  electrophoresis proteolysis  presence  /zg p r o t e i n )  was e i t h e r  P r o t e o l y s i s was stopped with  and Methods).  trichloracetic  (0.5  /zg p r o t e i n )  the  acidic  precipitated conditions  presented  here  experiments.  120  protein (pH were  6.6),  was  ice-cold  subjected  and  reproduced  (see  to  gel  autoradiography. in  three  other  B CaM  -  + -+-  1 3 6 — —  C  + - + -+-+  _  _  caM  -=^127  136—  -  + -  ^  W  +  ^  ^125-124 76  —82-80  46  —55  15'  30' 60'  90'  120'  2  7  125-124 =^85 82-80  18 0'  1  0'  60'  calmodulin were  and  passed  calcium.  the  resulting  through  a  heterogeneous fragments  calmodulin-agarose  column  (Fig.  in  the  peak  2 +  of  Ca -ATPase  s t i m u l a t e d by c a l m o d u l i n ) was o b t a i n e d i n the f l o w - t h r o u g h  contains  124 kDa and  Ca-ATPase the  peak  analysis of t h i s kDa  component  Therefore,  (stimulated  identified  a  that  column  with  peak  band ( F i g .  by c a l m o d u l i n )  fainter  heterogeneous  could  be  calmodulin-independent  2.  of  of  EGTA  (Fig.  82  kDa  125-124  further  it  protein  fractions  (Fig.  band  into  that  2).  it  A second elution  of  Electrophoretic  c o n t a i n s a prominent  kDa and 82-80  resolved  (not  lane  26A).  of  activity  demonstrated  26B,  before  profile  was o b t a i n e d a f t e r  second peak demonstrated t h a t  and  the  analysis  80 kDa p r o t e i n  calmodulin-agarose  presence  F i g . 26A p r e s e n t s the  A first  electrophoretic  1)  washed with a C a - c o n t a i n i n g b u f f e r  the column f r a c t i o n a t i o n .  and  lane  2 +  The column was then  e l u t i o n with an E G T A - c o n t a i n i n g b u f f e r .  26A)  26B,  (Fig.  26B,  lane  125 3).  kDa bands p r e v i o u s l y  calmodulin-dependent  and  fragments.  2  P u r i f i c a t i o n o f the calmodul i n - b i n d i n g fragments o f the Ca ~*"-ATPase  In were  these  treated  presence  of  experiments, with  two  calpain,  saturating  one  washed  with  an  in  the  concentrations  exogenously added c a l m o d u l i n . were  batches  After  of  calmodulin-depleted  absence (5  /jg.mg  treatment,  EDTA-containing buffer  to  and  the  membranes  other  membrane  in  prot."*)  the of  both batches o f membranes remove  calmodulin  and/or  p.  calpain,  solubilized  with  p u r i f i e d with two i n d i v i d u a l  Triton  X-100  and  the  Ca^ -ATPase  c a l m o d u l i n - a g a r o s e columns.  122  fragments  As a c o n t r o l , a  Fig.  26 S e p a r a t i o n  column.  (A)  unit.mL"!  o f the 2 +  Purified  calpain  C a - A T P a s e fragments  C a - A T P a s e (85  for  120  min  as  /tg  (1.2  mL) was then  pre-equilibrated CaCl , (w/v)  with  with  fractions a  2 mM EGTA,  (w/v)  sonicated effluent  fractions absence circles). 1),  mM KC1, 20  and  fractions apparent bands  of  1.5  The mixture  collected.  mM KC1,  20  0.05%  (w/v)  as  Both the  the  presence  of  at  X-100  50 /iM  and  column  was  1.5  X-100  4  120  EGTA  /iM f r e e C a  nM  ( l a n e 3) molecular  the  unbound  (lane  were s u b j e c t e d masses  relevant  (kDa)  part  presented here were e s s e n t i a l l y  of  to  of  2)  gel the  the  gel  and EGTA e l u t e d  Commassie are  2 +  in  123  (pH 8 . 3 ) .  Blue-stained  separate  The  of  the  (close  activity  indicated.  reproduced i n another  0.1%  elution  calmodulin  electrophoresis  (pH  mL f r a c t i o n s  and the  and then  and  2 +  both  0.1%  same b u f f e r  Triton  flow-through  2 +  7.4),  cm)  mM potassium-Hepes  "EGTA") and  Ca -ATPase activity  or  The  cm x 1.5  (pH  Triton  1.3  Methods.  A l i q u o t s o f 100 /iL o f the t r e a t e d C a - A T P a s e samples  1 mL o f  the  300  (indicated  were assayed f o r circles)  (w/v)  with  and  /iM l e u p e p t i n .  0.05%  2 mM d i t h i o t h r e i t o l ,  were c o l l e c t e d .  (B)  Materials  mM potassium-Hepes  mL were  containing  asolectin  (open  treated  The column was washed with the  of  buffer  7.4),  the  300  sonicated a s o l e c t i n .  eluted  o f 200  2 mM d i t h i o t h r e i t o l ,  2  flow-through  in  was  loaded onto a c a l m o d u l i n - a g a r o s e column (5  1 mM M g C l ,  2  protein)  described  P r o t e o l y s i s was stopped by the a d d i t i o n  with a c a l m o d u l i n - a g a r o s e  (lane peak The  protein results  experiment.  batch o f u n t r e a t e d as  membranes was used and the  native  Ca-ATPase  purified  above.  Fig.  27A i l l u s t r a t e s  intermediate  the f o r m a t i o n o f the  P - l a b e l l e d phosphorylated 2 +  (EP)  o f the n a t i v e membrane-bound C a - A T P a s e  1),  the  the  absence o f c a l m o d u l i n (lane 2 ) ,  (136  kDa)  (lane  124 kDa and 80 kDa membrane-bound fragments produced by c a l p a i n  bound fragments 3).  Panel  kDa)  plus  purified  a  smaller the  major  contaminant  of  both  fragments  the  kDa and 85  presence o f  o f the p u r i f i e d  100  kDa  (lane  127  purified  from the  calmodulin-saturated,  and  calpain  native  and the  membranes  (lane  enzyme  (136  fragments  treated  calmodulin-depleted,  125 kDa fragment,  kDa  kDa membrane-  calmodulin  1),  in  with  o r i n the presence ( l a n e 3.) o f c a l m o d u l i n .  and the 39-37 kDa d o u b l e t .  However,  the  127  c a l m o d u l i n - a g a r o s e column from  membranes were the  fragment,  in  B shows the p r o t e i n p a t t e r n  with  The  and the  produced by c a l p a i n  c a l p a i n i n the absence (lane 2)  treated  3 2  85  a small  No fragment kDa  treated  calpain-  amount o f the 82 kDa  o f 80 kDa was o b s e r v e d .  fragments  were  membranes.  purified  In  from  addition,  the  larger  amounts o f the 39-37 kDa d o u b l e t were a l s o p r e s e n t .  When the incubated lane  1)  above mentioned p u r i f i e d  with  and the  [T-  3 2  P]ATP,  127 kDa,  only  the  native intact  and fragmented enzyme  125 kDa, 85 kDa and 82  (136  enzymes were  kDa)  kDa fragments  (Fig.  27C,  (Fig.  27C,  oo  l a n e s 2 and 3) was  formed the  reconfirmed that  the  J d  P-phosphorylated intermediate.  124  and 80  kDa fragments  125  formed  Therefore, in  the  it  absence  Fig.  27  P u r i f i c a t i o n o f the c a l m o d u l i n - b i n d i n g fragments  ATPase  from  depleted in  the  absence o r  ography  presence o f  3 2  showing  /xg p r o t e i n )  absence  130  (lane  mg  either  2)  membrane  agarose column. from  the  (C)  native  untreated  or  in  of  the  the  or  in  shown  in  fragmented  2 and 3 correspond to  1,  2 and 3 o f panel  Materials  the  3 2  separate  The p r o t e i n experiment.  Ca  2 +  -  3).  (B)  2 +  A using  a calmodulin2 +  the 1,  (lane  purified Ca -ATPase 2  and  3  in  of  panel  ATPase p r e p a r a t i o n s  126  Autoradi-  panel  P - a c y l p h o s p h a t e formation  2 +  purified  (A)  and  Ca -ATPase purified  Ca -ATPase illustrated  B, r e s p e c t i v e l y .  were reproduced i n another  panel  lanes  calpain  o r t r e a t e d with c a l p a i n  fragmented in  1  membrane-bound  calmodulin  2 and 3 correspond to  Autoradiogram showing  1,  the  (lane 1),  mentioned  preparations  or  of  presence o f  native  protein  Lanes 1,  membrane  respectively. purified  unit.mL"  as d e s c r i b e d  formation  -  Calmodulin-  60 min with 0.07  calmodulin,  P-acylphosphate  Coomassie Blue s t a i n i n g from  for  membranes.  Membranes without treatment were used as c o n t r o l s .  ATPase (18 the  erythrocyte  membranes were incubated  Methods.  in  calpain-treated  o f the Ca  patterns  A,  by the  B.  shown i n presented  Lanes lanes here  Figure  27  ?! ^ SS V  o  if  11  CO  J r  CM  1 CD CO  co co  4 1  co m  J  CM  1  1  i (0  co  ?! 2S§ v \/ CO CM  i i CD CO  127  of  calmodulin  (Fig.  27A,  lane  2)  were  not  retained  by  the  calmodulin-  agarose column.  Table  3 presents  the experiment stimulation 9  Ca  recovery  shown i n F i g . 27.  of  the  Ca  of  the  The u n t r e a t e d  -ATPase a c t i v i t y  2 +  total  Ca -ATPase activity  in  membranes show a 5 . 8 - f o l d  induced  by  calmodulin,  and  the  I  -ATPase  purified  stimulation.  from  However,  these  the  calpain-treated  1.5-fold.  and  the  However,  stimulation 2 +  the  Ca -ATPase  membranes by c a l m o d u l i n a f f i n i t y kDa fragments) by  still  induced  which  by  a  the  3.1-fold absence  was  of  reduced  purified  (most  stimulation to  (in  calmodulin  fragments  compares  shows  when assayed i n the absence  chromatography  show a 2 . 5 - f o l d  calmodulin  also  membranes  had i n c r e a s e d C a - A T P a s e a c t i v i t y  calmodulin  induced  membranes  2 +  calmodulin) of  the  of  from  to  these  prominently  the  the  activity  a 3.1-fold  ATPase  stimulation  125  of  the  9 .  purified calpain  intact in  the  Ca^ -ATPase.  On the  other  presence o f c a l m o d u l i n  hand,  membranes  show o n l y a s l i g h t  treated  increase  with  in  the  9 •  basal  Ca*- -ATPase a c t i v i t y  4.4-fold  stimulation  ATPase fragments a 2.2-fold the  total  (127  by  of  of  the  calmodulin  ATPase was r e t a i n e d the  in  was  case o f only  the  absence o f  calmodulin.  activity  2 +  that  in  kDa plus 85 kDa)  Ca -ATPase activity  absence  and 66% i n  induced  stimulation  becomes c l e a r the  (assayed  The  purified  the  a small  preparation  also  membranes portion  When  the  shown  in  treated of  the  by the c a l m o d u l i n - a g a r o s e column,  case o f membranes t r e a t e d with c a l p a i n  c a l m o d u l i n or without c a l p a i n t r e a t m e n t , 128  of  and a Ca  2 +  -  from these membranes show  by c a l m o d u l i n . examined,  calmodulin)  respectively.  recovery Table  with  1,  it  calpain  in  fragmented  Ca  10% compared to in  the  of  presence  2 +  -  59% of  Table 3  E f f e c t o f c a l p a i n treatment in the absence and presence o f on C a - A T P a s e 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  Total ,2+ Ca -ATPase a c t i v i t y (/imol . m i n " )  calmodulin  Fold stimulation by c a l m o d u l i n  1  Recovery o f p u r i f i e d C a - A T P a s e activity from the membranes  Addition membrane-bound -CaM +CaM  purified -CaM +CaM  membrane-bound  purified  None  2,.1  12, .2  2,.6  8.1  5.8  3.1  66  Calpain  6,.8  10.,4  0,.4  1.0  1.5  2.5  10  C a l p a i n plus calmodulin  2..6  11,.4  3,.1  6.7  4.4  2.2  59  Membranes  (130  mg p r o t e i n )  were t r e a t e d with c a l p a i n  absence or presence o f 650 /ig c a l m o d u l i n , control. three  The  native  fragmented  2 +  C a - A T P a s e was  separate c a l m o d u l i n - a g a r o s e columns.  and the  2 +  purified  and 2 /iM f r e e C a indicated. ATPase  or  ,  respectively,  The f o l d  activity  absence of  Ca -ATPase activities 2 +  in  stimulation the  presence  calmodulin.  c a l c u l a t e d using the  Percent  The t o t a l  unit.mL" )  Untreated  then  either  in  the  membranes were used as  solubilized  activity  were determined  and  purified  using  o f both the membrane-bound,  using a l i q u o t s  assayed at  4.3  /iM  in the absence or i n the presence o f c a l m o d u l i n (CaM)  as  by calmodulin was c a l c u l a t e d  2 +  of  calmodulin  recovery  2 +  Ca -ATPase activity  presented here are t y p i c a l  as i n d i c a t e d .  1  (0.07  of two separate  of  versus 2 +  as  the  experiments.  ratio  of  the  2 +  the , C a - A T P a s e a c t i v i t y  Ca -ATPase activity  assayed i n  the  from the  presence o f  Ca  -  in  the  membranes  was  calmodulin.  Values  3.  T r y p s i n fragmentation  The fragmentation  o f the C a - A T P a s e  patterns  o f the C a - A T P a s e produced by c a l p a i n and  by t r y p s i n were a l s o c o m p a r e d . C a l p a i n ATPase  in  the  absence o f  treatment  c a l m o d u l i n produced the  80 kDa, 55 kDa and 39-37 kDa fragments hand,  short  time exposure to t r y p s i n  produced a 124  kDa fragment  kDa and 32 kDa ( F i g . resulted  28 B, lane  127  kDa,  whereas  85  short  produced a 127 kDa  and 32  kDa  presence o f fragments  (3 min)  2).  the  purified  characteristic  28 B, l a n e i n the  fragments  1).  of  86  124  On the  absence o f kDa,  2 +  Ca  -  kDa, other  calmodulin 82  kDa,  Longer exposure to t r y p s i n  (30  82 kDa, 77 kDa and 32 kDa fragments  34  min) (Fig.  3).  C a l p a i n treatment of  (Fig.  and s m a l l e r  i n the accumulation o f  28B, l a n e  of  kDa, time  i n the presence o f c a l m o d u l i n l e d to the  55  kDa and 39-37  exposure  kDa fragment (Fig.  28C,  calmodulin led  (Fig.  28C,  to  kDa fragments  trypsin  and s m a l l e r lane to  lane  3).  of  the  2).  the  in  the  of  exposure  accumulation of  Fig.  28C,  presence  fragments  Longer  (Fig.  28A shows the  the  86  formation  of  kDa,  lane  1),  calmodulin 82  kDa,  34  to  trypsin  in  the  82  kDa and 32  kDa  purified  native  Ca  2 +  -  ATPase as a c o n t r o l .  IV.  Characterization 2 +  reconstituted Ca -ATPase ( C a  untreated 2 +  pump)  130  and  calpain-treated  liposome-  Fig.  28  Comparison o f  the  fragmentation  patterns  ATPase o b t a i n e d by c a l p a i n and t r y p s i n d i g e s t i o n . Ca-ATPase incubated with  at  trypsin  absence  (B)  (60 /Ltg p r o t e i n ) . 25°C  calpain 1  (5 or  with  /ig.mL" ) the  Purified  for  presence  c o n t a i n i n g 55 mM T r i s - m a l e a t e , EDTA,  10 mM d i t h i o t h r e i t o l ,  (C)  of  unit.mL"*) (lane 300  Calpain  protein  fragments  was  are  or  subjected  indicated  purified  Untreated (60  for  /ig  60  C a -  purified  protein)  min  (lane  was 1)  (lane  3)  in  nM c a l m o d u l i n  (CaM)  in  medium  Ca  by  gel  solid  2 +  (pH 7 . 4 ) .  electrophoresis triangles  The f r a g m e n t a t i o n  reproduced i n two o t h e r s e p a r a t e  131  patterns  experiments.  the  mM M a C ^ , 0.5 mM  ice-cold trichloroacetic acid. to  or  30 min  65 mM KC1 and 200 /iM f r e e  i n d i c a t e d by open t r i a n g l e s . were  2)  the  5 mM p o t a s s i u m - H e p e s , 6.5  p r o t e o l y s i s was stopped with 10% (w/v) precipitated  (A)  Ca-ATPase  (0.02 3 min  of  and  (pH trypsin  presented  The The 8.3). is here  Figure  28  CO CO CN (OCM COCO  If 1  V 9' I  !  Ik l LO  A  co  00 CO CM CO<t CM h» CO CM  co  if i if •1 i i 1 i K h  mm  co  CO CM  *" "  o  00  T  CO CO  T _  132  *t CM CO CO  If I  I It IT) COCO  1.  Effect  activity  of  A23187.  alamethicin  o f the r e c o n s t i t u t e d Ca^  It  was  of  great  2 +  Ca -translocating enzyme was  interest  function  reconstituted  of  activation  lack  calmodulin  stimulation,  results  on  vesicles  the  2 +  ATP h y d r o ! v t i c  2 +  of  the  calpain  Therefore  these  Ca  from the  enzyme,  2 +  on  the  studies.  reconstituted  as p r e d i c t e d  of  In  the  latter order  pump and  above  to the  experimental  a calmodulin-sensitive  pump p r e p a r a t i o n was r e q u i r e d . The m a j o r i t y o f p r e v i o u s  reconstituted  (Haaker and Racker,  1986;  on the  effect  Ca -ATPase.  on the membrane-bound and p u r i f i e d  reconstituted C a  al.,  the  liposomes f o r  proteolytic  of  examine  the  into  X-100  pump  to  observe  work  and T r i t o n  Ca^ -ATPase  was  1979;  et  Niggli  V i l l a l o b o and R o u f o g a l i s ,  p h o s p h o l i p i d components i n  1986).  asolectin,  carried al.,  out  1981b,  Due to the  however,  the  on  asolectin  1982b; Benaim et  presence o f  acidic  calmodulin-stimulatory  pi  effect by  on Ca^  using  1981b) could  transport  highly  for  has not been r e p o r t e d .  purified  egg  reconstitution  of  2+  be more C a - p e r m e a b l e .  yolk the In  T h i s problem was r e s o l v e d  phosphatidylcholine  enzyme, the  but  light  the  of  (Niggli  vesicles  this,  it  et so  al-, formed  was n e c e s s a r y  to  pi first in  e s t a b l i s h the degree o f c o u p l i n g and the the  phosphatidylcholine  liposomes.  s i d e d n e s s o f the Fig.  29  Ca  shows  pump  that  the  p.  reconstituted  Ca  -ATPase a c t i v i t y  was s t i m u l a t e d  about  3-fold  by  A23187, pi  indicating The  satisfactory  relatively  low  impermeability  degree  of  A23187  of  the  proteoliposomes  stimulation  p h o s p h a t i d y l c h o l i n e was noted p r e v i o u s l y ( V i l l a l o b o Under s i m i l a r the  2 +  c o n d i t i o n s , using a s o l e c t i n  Ca -ATPase  activity  was  stimulated  133  with  fold  Ca^ .  purified  and R o u f o g a l i s ,  instead of 6-7  highly  to  1986).  phosphatidylcholine,  by A23187  (results  not  Fig.  29  Effect  of  A23I87,  alamethicin  and  Triton  X-100  on  the  protein  and  9 ,  reconstituted  Ca^ -ATPase  activity.  0.65  mg p h o s p h o l i p i d ) were  free  Ca  2 +  ,  2 mM ATP,  120  incubated at  55 mM T r i s - m a l e a t e ,  or  (closed  presence  alamethicin  (A)  or  37° C f o r  nM c a l m o d u l i n ,  potassium-Hepes, the  Proteoliposomes  symbols)  Triton  X-100  R e s u l t s p r e s e n t e d here are t y p i c a l  66  pH 7.2  of  120  (B)  mM KC1, 6.5 i n the  also  o f two s e p a r a t e  134  ng  30 min w i t h  4.3  mM M g C l , 2  /zM o f 5 mM  absence (open symbols)  nM A23187. were  (0.8  Various added,  as  experiments.  amounts  of  indicated.  2+  Ca  -ATPase activity  ( n m o l - m g p^ r o t . 09 \ min ^ o  o  Ca ( nmol-mg  -ATPase activity prot.  ''.min  o  o  shown).  Fig.  higher  than  29A 0.5  also  illustrates  /jg.ml"*,  that  further  alamethicin,  increased  increase 1968)  the  permeability  and t h e r e f o r e  were  oriented  whereas  the  with  outside  of  alamethicin  maximal  the  (inward Ca  and  no  site  /jg.mL"*)  longer  the  Ca -ATPase  about  25%  by  while  h i g h e r c o n c e n t r a t i o n s (> 0.1%  requirement supporting  of the  activity  X-100  A23187  2 +  required  Similarly,  Triton  the  enzyme had the  to ATP and C a  2 +  in  for  at  since  the  to  2 +  of  the  than  activity  inwardly  be  activity  was  full  (w/v)) T r i t o n X-100  Ca -ATPase  to  activity.  A23187 was  greater  to  concentrations  appeared  express of  liposomes, oriented  Ca -ATPase  presence  concentrations  the  higher  Rudin,  pump m o l e c u l e s  2 +  the  and  site(s)  proteoliposomes  A23187  in  full  predominance  ,  of  catalytic At  2 +  activity  i s known to  (Mueller  'lumen  pumping o r i e n t a t i o n ) .  10  permeable  Alamethicin  p h o s p h o l i p i d membranes  catalytic  of  (>  Ca -ATPase  appeared t h a t about 20% o f the C a  the  majority  the  completely  it  of  concentrations  2 +  the  (assayed i n the presence o f A23187) by about 25%.  at  increased  0.005%  (w/v),  a l s o a b o l i s h e d the (Fig.  29B),  2 +  Ca -transporting  further  Ca  2 +  pump  orientation.  2.  the C a  2 +  In either to  2 +  Effect  the  either shown).  o f c a l p a i n on the C a - t r a n s p o r t  and ATP h v d r o l v t i c  activity  of  pump  this  series  of  experiments,  with o r without c a l p a i n . mixture Ca  2 +  to  uptake  Fig.  30A  After  proteoliposomes the  treatment,  arrest  proteolysis.  or  ATP h y d r o l y s i s o f  the  shows t h a t  the  initial  136  Leupeptin the  rate  of  were  leupeptin  alone Ca  preincubated  2 +  Ca  did  pump 2 +  was added not  affect  (results  uptake  not  (measured  Fig. ATP  30  Effect  of  hydrolysis  protein  of  containing  MgCl ,  10  2  end o f t h e  calpain  time-course  2 +  pump.  Proteoliposomes  mM KC1,  (as  50  25° C f o r  mM potassium-Hepes  400  /iM  indicated).  free  Ca  Leupeptin  2 +  in  (pH  tiM)  nmol.mg  prot.  monitored  and  of  (8)  ATP-hydrolytic a c t i v i t y  calcium  uptake  was  _ 1  the  traces  .min"^.  represent  120 min  /ig in a mM  absence  or  the  was added at  the  measurement.  monitored  In  (B),  initial  the  as  a c c o r d i n g to method A i n M a t e r i a l s  nM A23187 initial  are  shown  rates  of  on  the  in  traces.  ATP h y d r o l y s i s c o n t r o l  ratio  here were e s s e n t i a l l y  reproduced i n two o t h e r  H C R  ).  137  of  In  (A),  Materials  and  of  ATP  indicated).  on the Ca  and Methods,  indicated).  ATP-hydrolysis. (ATP  rates  time-course  presence o f 1 /iM c a l m o d u l i n (as  represent  (55  0.67  the  (200  and  7.4),  27 /iM ATP and 60 nM A23187 were as i n d i c a t e d  beside  60  Ca  both c a l c i u m uptake  i n the absence o r the presence o f 1 /iM c a l m o d u l i n (as  Numbers  the  rate of  i n c u b a t i o n and the p r o t e o l i p o s o m e s u s p e n s i o n s were assayed f o r :  Additions of  and  67  c a l c i u m uptake  Methods,  or  reconstituted  mM d i t h i o t h r e i t o l ,  presence of  the  the  initial  and 42 mg p h o s p h o l i p i d ) were incubated at  medium  (A)  c a l p a i n on the  z +  traces.  uptake  hydrolysis in  the  in was  absence  A d d i t i o n s o f 27 /iM ATP  Numbers Numbers  The p a t t e r n s  in  beside  brackets  of traces  separate  the  traces are  the  presented  experiments.  Figure  30  -Calpain  +Calpain  2min  -  C  a  ,  P  d  n  +Calpain  2min  138  with the Ca  proteolipsomes  is  stimulated  A f t e r c a l p a i n treatment the about 3 - f o l d  5-fold  initial  almost  (Fig.  30B).  (127  of  initial  - 1  1  1  .min" )  initial  of  rate  was  initial  nmol.mg  rate of  731  /JM f r e e  by  2.0-fold  1  that  and became  was  Arsenazo  _ 1  and  in 1  .min" ,  nmol.mg p r o t .  7-fold  The a d d i t i o n  also  2 +  .  almost  III  Calpain the  _ 1  o f A23187 in  the  was  1  .min" .  omitted  Ca  pump 886  increased  treatment  the  Z +  (to  absence  absence  approaching  measured  by c a l m o d u l i n  2.6-fold  respectively.  prot.  Ca  uptake was a l s o i n c r e a s e d  ATP h y d r o l y s i s  except  30B).  calmodulin,  562  of  stimulated  r a t e o f ATP h y d r o l y s i s  to  0.4  30A).  (Fig.  ATP h y d r o l y s i s  presence  2 +  at  r a t e o f ATP h y d r o l y s i s by the u n t r e a t e d  prot. .min" ) _ 1  calmodulin  rate of C a  conditions,  The i n i t i a l  nmol.mg p r o t . rate  the  identical  nmol.mg  by  compared to 85 nmol.mg p r o t . . m i n " )  parallel,  under  i n t o the r e c o n s t i t u t e d C a ^ pump  - 1  (299  i n s e n s i t i v e to c a l m o d u l i n ( F i g .  In  +  - s e n s i t i v e dye Arsenazo III)  the  or  the  increased  the  of  calmodulin  calmodulin-stimulated  Furthermore,  A23187 i n c r e a s e d  the r a t e o f ATP h y d r o l y s i s i n the absence or the presence o f c a l m o d u l i n by 2.2-fold  and  several ratio  such  absence  (the  ratio  It of  was the  Table  shown t h a t rate  of  to  equilibrium (q)  in alter  the  presence  this  ratio.  thermodynamic  of  calmodulin. From  formulations  the  4 summarizes the  between  (Rottenberg,  in  values,  presence in  the  did  not  following  1971),  the  of  control  both  treatment  was c a l c u l a t e d , which was expressed as f o l l o w s :  139  results  the  2 and 3,  Calpain  ATP^QR  the  ATP h y d r o l y s i s  ATP h y d r o l y s i s  absence o f A23187) was maintained  and  coupling  respectively.  experiments.  (ATPHQR)  v e r s u s the  appear  2.4-fold,  non-  degree  of  2 +  T a b l e 4 E f f e c t o f c a l p a i n treatment on the degree o f c o u p l i n g and C a / A T P r a t i o o f the 1 i p o s o m e - r e c o n s t i t u t e d C a pump 2 +  Treatment  CaM  A T P  HCR  Degree o f coupling (q)  3  Experimental 2 +  Ca /ATP ratio  b  None  Calpain  Mechanistic  0  2 +  Ca /ATP ratio  2.0  ± 0.1  0.71  +  0.04  0.60  ± 0.10  1.20  ±  0.17  2.2  ± 0.3  0.73  ±  0.04  0.44  ± 0.08  0.83  ±  0.16  -  2.0  ± 0.2  0.71  ±  0.02  0.52  ± 0.03  1.04.±  0.07  +  2.0  ± 0.4  0.70  ±  0.05  0.41  ± 0.05  0.84  0.15  ±  r a t e o f ATP h y d r o l y s i s (+A23187) ATP HCR  r a t e o f ATP h y d r o l y s i s (-A23187) 1/2  1 q = ATP HCR  „ Experimental  c  2 +  Mechanistic C a / A T P r a t i o  2 +  Ca /ATP  ratio  =  Liposomes (54 /ig p r o t e i n p l u s 49 mg p h o s p h o l i p i d ) were incut jbated with or without c a l p a i n , as d e s c r i b e d i n F i g . 30. Ca uptake and A T P - h y d r o l y s i s measurements were c a r r i e d out as d e s c r i b e d i n the M a t e r i a l s and Methods. The v a l u e s (means ± S . E . M . ) i n this table were o b t a i n e d from three separate experiments.  140  r a t e o f ATP h y d r o l y s i s  (-A23187)  1/2  [1] r a t e o f ATP h y d r o l y s i s (+A23187) or 1/2 [2] ATP HCR  The c a l c u l a t e d value o f q was maintained or  the  Table  presence  of  4  shows  also  stoichiometric  calpain  ratio  was around 0.60  treatment,  that  the  Ca  2 +  (Ca /ATP ratio)  ± 0.10  and 0.44  both 2 +  at  about 0.7  with  and without  transport  under these  ± 0.08,  i n the  i n the  to  absence  calmodulin.  ATP  hydrolysis  experimental  conditions  absence or the  presence  of  7+ calmodulin,  respectively.  permeability  of  these  Therefore,  vesicles,  P uncoupled to C a  due  the  experimental  2 +  Mechanistic C a / A T P  corrected  significantly  fraction  overall  of  the  i  translocation.  By d e f i n i t i o n  2 +  C a / A T P r a t i o was c o r r e c t e d u s i n g the f o l l o w i n g  Ca^  total  2 +  p , q^ e q u a l s  ratio  presence  altered  [1]).  calculate  Ca /ATP  the  ratio  ratio  Ca /ATP the  (to  ATP  expression:  2 +  Experimental  and  high  2 +  2 +  absence  the  o f the C a - A T P a s e molecules t h a t are coupled (equation  mechanistic C a / A T P r a t i o )  The  to  a significant  d  h y d r o l y s i s was the f r a c t i o n  However,  [3]  was of  by c a l p a i n  1.20  ±  calmodulin, treatment.  141  0.17  and  0.83  respectively,  ±  0.16, and  in was  the not  The was  effect  of  determined  initial and  rates  became  calpain.  Ca  a constant  of C a  2 +  (in  insensitive calmodulin Ca  calmodulin  after was 2 +  calmodulin  the  the  or  initial  min)  present  during  z +  of t h e i r  the  initial  molecular  fragmentation  of  of  became  2 +  calpain  treatment,  measured i n by  accompanying Ca  2 +  calpain  of  whether  the  initial  presence  about  32).  activation  pump,  of  17-19% and  (Fig.  the  and  calmodulin  the  (zero-time) values  reconstituted  uptake  largely  1  calpain  following  irrespective  proteolysis  changes  the  both C a  The  unit.mL"  time-course of of  pump  activated  5  hand,  activity  time  1 to  1  31).  were f u l l y  increased  and  other  and C a - A T P a s e  the  Ca*  (Fig.  about  rates  calmodulin) On the  at  follow  progressively  d e c l i n e d with  determine  calpain,  of  min.  absent  26-28%, r e s p e c t i v e l y ,  To  to  were  absence 120  slowly  (120  2 +  shows t h a t  uptake  time  reconstituted  uptake and C a - A T P a s e a c t i v i t y  activity  the  on the  incubation  insensitive  F i g . 32  -ATPase  of  concentration  Another approach taken was to  treatment  rate  at  almost  treatment. the  calpain  both  in  by the  absence and the presence o f c a l m o d u l i n , was examined.  F i g . 33A shows t h a t  with i n c r e a s i n g time o f p r o t e o l y s i s , the r e c o n s t i t u t e d  136 kDa C a - A T P a s e  was t r a n s f o r m e d mainly or  the  124  kDa and 127  kDa f r a g m e n t s ,  presence o f c a l m o d u l i n , r e s p e c t i v e l y .  unfragmented consisted of 2 +  into  Ca -ATPase  intact  enzyme  outwardly molecules  Acylphosphoprotein a s i n g l e band o f  Ca not  (136 2 +  kDa)  was  2 +  After still  i n the absence  120 min,  present,  a portion  which  of  likely  2 +  pumping o r i e n t a t e d  C a - A T P a s e molecules and  reconstituted  the  liposome  by the  control  intermediate  136 kDa ( F i g .  (EP)  in  formation  33B, lane  142  1).  After  membranes. enzyme gave  c a l p a i n treatment  (120  Fig. Ca  2 +  31  Effect  uptake  of  increasing calpain concentration  and ATP h y d r o l v t i c  P r o t e o l i p o s o m e s (128 25° Fig. the  C with 30.  the In  initial  /ig p r o t e i n  indicated  (A), rate  A)  of  the  initial  reconstituted  amount  of  calpain  for  60  min,  of  in  -uptake  the  absence  o f 1 /iM c a l m o d u l i n ,  (open  circles)  calmodulin.  absence  (open  circles)  or  the  Values presented here are means  143  at in  determine or  the  as d e s c r i b e d i n F i g . 30.  In  z +  the  of  pump.  as d e s c r i b e d  t r e a t e d p r o t e o l i p o s o m e s were assayed f o r C a - A T P a s e a c t i v i t y in  z +  and 112 mg p h o s p h o l i p i d ) were incubated  p r o t e o l i p o s o m e s were used to  Ca  rate  Ca  calpain-treated  presence (closed c i r c l e s ) (B),  activity  on the  presence  (closed  o f two s e p a r a t e  (Method  circles)  of  experiments.  Ca  2+  -ATPase activity  ( nmol-mg prot.  . min o o o  en  o o  o  Q_ *Q Q. y  3  C a )  (  n  2  +  U p t a k e  mol-m g p r o t . " ' ' • m i n ~ ~ ) 1  Ol  o o  ro  o o  o o  0) O O  _ J  O  -5  a>  Q_ OH  Q .  1  C 3  c  3 ^.  1  O H  1  2H  3 o  n  • o •  Fig. 2  32  Time-course of  1  Ca " " pump on the  2  (156  unit.mL"  (squares)  of  of  of  absence  plus  calpain  the  the  indicated  added f o r the  or  as d e s c r i b e d i n  separate'  experiments.  the  reconstituted  mg p h o s p h o l i p i d ) (circles)  period of  the  zero  or  time,  activity  F i g . 30.  of  Data  the  the as  nM  The  untreated  Material After  (B)  of  proteolytic time)  the  (closed  typical  (zero  the  in  calmodulin  presented are of  presence  used f o r Ca  determinations  300  treated  in  time v a l u e s .  time-course  among e x p e r i m e n t s .  activity.  were  p r o t e o l i p o s o m e s were then  presence  However,  of  and ATP h y d r o l y t i c  absence  or C a - A T P a s e  symbols)  slightly  112  2 +  (A)  symbols),  varied  in  200 /iM l e u p e p t i n ,  (open  calpain digestion  1  /xg p r o t e i n  c a l p a i n was not  uptake measurements  of  r a t e o f Ca " " uptake  calmodulin f o r  and Methods; addition  1  effect 2  initial  Proteoliposomes with  the  three effect  basal  and  p i  calmodulin-stimulated activity  0.4  /iM f r e e  experiments  was  respectively. 2 +  Ca -ATPase  77  Ca ±  2 +  was  270  uptake  by  and 27 /iM ATP 37  The u n t r e a t e d  activity  experiments  calcium  at  0.4  ±  110  and basal  610  the  reconstituted  (mean ±  278  ± S.E.M.) nmol.mg  and c a l m o d u l i n - s t i m u l a t e d  /zM f r e e and  Ca  2 +  1040  respectively.  145  (mean ±  430  ± S.E.M.) nmol.mg  Ca^ -ATPase  from the 1  three 1  prot." .min" , reconstituted from the 1  three 1  prot." .min" ,  Ca  2+  ( nmol.mg  C a  -ATPase activity prot.  •1 . 1 .min ) (  nmol.mg  O  2  +  prot. ~ -min  ~ )  1  O  o  o  U p t a k e  o o  1  0>  o o rmx-  -5  4=»  3  O  CD  O). O  3 3*  O  N> O  CD  o  \  CO O  CD 0 ) J  o (0  o' ro-  o  OJ  Fig.  33  Time-course  Ca  pump.  the were  calpain  presence  proteolysis subjected  silver  staining  open  lanes  (+)  for of  triangle  fig p r o t e i n  indicated  32  (pH 6.8)  plus  period  gel  in  the  as d e s c r i b e d  in  F i g . 32.  and/or  (lane  Fragmentation  2)  or  patterns  the  presence  presented  here  experiments.  147  (lane were  the (pH  8.3)  3)  by  in  fragment.  of  1  130  reproduced  After  followed  followed  or 2 u n i t . m L "  (-)  proteoliposomes  standards  its  were  absence  The numbers on the  protein  p r o t e o l i p o s o m e s were t r e a t e d with 0 (lane 1) absence  of  formation  masses o f  calpain  reconstituted  time  and a u t o r a d i o g r a p h y .  molecular  the  112 mg p h o s p h o l i p i d )  electrophoresis  P-phosphoenzyme  indicates  of  with 200 /*M o f l e u p e p t i n ,  alkaline  (B)  represent  proteolysis  130 nM c a l m o d u l i n ,  (A)  or  (156  the  was a r r e s t e d to  electrophoresis the  calpain-induced  Proteoliposomes  treated with o r the  of  gel  sides of kDa.  The  In  (B),  calpain nM  in  acid  by  in  the  calmodulin.  four  separate  Figure  ™  33  V ^ C O CO  CO CM  I CD CO  + + + + +  O  CD  CM  r - O)  CO  »  1 1  Vt  CO  If  o  CM  ft  o  CO  o  CO  11  I  I  LO  I  CO  o  148  min)  in  major  the  124  absence o f  kDa band  minor bands o f in  the  136  kDa and 80  presence  3.  EP band c o r r e s p o n d i n g to  the  33B,  calmodulin,  lane  2).  however,  and l e s s prominent  the  treatment  produced  major  bands o f  Calpain  127  136  as  a  major  kDa band  kDa and 85  staining kDa  (Fig.  3).  Protective  effect  reconstituted C a  In  of  kDa ( F i g .  band c o r r e s p o n d i n g to  with Coomassie blue lane  a major  s t a i n i n g with Coomassie blue was o b s e r v e d , as w e l l  acylphosphoprotein  33B,  calmodulin,  another  2 +  of  calmodulin  against  proteolytic  activation  of  the  developed  to  pump by c a l p a i n  series  of  experiments,  methods  were  9+ determine Ca  the  -uptake  which  effect  activity  required  reconstituted calpain  in  of  following  prior  calpain  separation  enzyme.  the  calmodulin preincubation  of  absence  calmodulin  the  calmodulin  or  the  presence  and c a l p a i n  of  from the  a gel  S-200,  x 60 cm)  EGTA.  in  the  void  volume  p r o t e o l iposomes were autolytic absence bands o f  fragments of  presence o f collected.  calmodul i n - f r e e of  calmodulin  80 kDa,  were  calpain  (lane  produced  the  55 kDa,  and  first  calpain  calmodulin,  while  a to 124  39 and 37 kDa,  149  b  d).  kDa  and  34 d)  Calpain major  and  as d e s c r i b e d above  control to  (Sephacryl eluting  that also  treatment  fragment  with  treatment,  column  shows  the  In o r d e r  The p r o t e o l i p o s o m e s  Figure  (lanes  from  pre-incubated  filtration  and  proteoliposomes,  proteoliposomes a f t e r  samples were passed through i n the  the  Ca^ -ATPase  in the absence o f c a l p a i n .  individual 1.5  of  The p r o t e o l i p o s o m e s were  p r o t e o l i p o s o m e s were p r e - i n c u b a t e d separate  treatment  on the  and  these free  of  in  the  lighter  (lane c ) .  In  Fig.  34  pump.  Effect  o f c a l m o d u l i n on the p r o t e o l y s i s o f the r e c o n s t i t u t e d  P r o t e o l iposomes  i n c u b a t e d at  (129  (pH 7 . 4 ) ,  presence o f  (a)  or  nM c a l m o d u l i n  500  incubation, 10 m i n ,  KC1,  200  no a d d i t i o n  cm).  /iM M g C l , 2  (b) and  500 2  - 1  at  200  /tM EGTA, 1  20  mL.h" .  2 +  Ca -ATPase activity  in  kDa.  reproduced i n another  After  At  Two  mL-Fractions  the  2 +  in  1  end  of  a Sephacryl  and e l u t e d  were  experiment.  150  the  calpain the for  S-200  with 100 mM  pH 7.4  and 2 mM  collected.  The  mL) were p o o l e d , assayed f o r  and s u b j e c t e d t o SDS-PAGE ( l a n e s  Fragmentation  were  c h i l l i n g on i c e  20 mM p o t a s s i u m - H e p e s ,  T a b l e 5)  separate  2 unit.mL"  loaded onto  was s t a i n e d with Coomassie Blue R-250. mass  (c)  calpain.  ( e l u t i o n volume 38-41  (see  phospholipid)  400 /iM f r e e C a  nM c a l m o d u l i n  unit.mL  mg  c+  mL) c o n t a i n i n g 75 mM KC1, 50  The column was e q u i l i b r a t e d  liposome peak f r a c t i o n s  molecular  120  10 mM d i t h i o t h r e i t o l ,  each sample was s e p a r a t e l y  x 1.5  dithiothreitol  The gel  plus  5 mM EGTA was added t o each sample.  2 mL o f  column (60  protein  25°C f o r 90 min i n a medium (2.7  mM potassium-Hepes  (d)  /ig  Ca  Numbers shown are  patterns  presented  a-d).  apparent  here  were  Figure  34  a b e d  151  Table 5  Protective e f f e c t of calmodulin against p r o t e o l y t i c of the r e c o n s t i t u t e d Ca -ATPase by c a l p a i n  Ca^ -ATPase a c t i v i t y  _ i  _ i  (nmol.min .mL )  activation  Fold stimulation by c a l m o d u l i n  Addition - A23187  +A23187  -CaM  -A23187  +A23187  -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 p r o t e i n plus 120 mg p h o s p h o l i p i d ) were i n c u b a t e d i n the presence o f 10 mM d i t h i o t h r e i t o l and 40 /iM f r e e Ca and one o f the a d d i t i o n s i n d i c a t e d , as d e s c r i b e d in F i g u r e 34. A f t e r gel f i l t r a t i o n (see M a t e r i a l s and M e t h o d s ) , the c a l m o d u l i n f r e e proteoliposomes were assayed f o r Ca -ATPase a c t i v i t y , as d e s c r i b e d i n Materials and Methods. Ca -ATPase a c t i v i t y i s given i n nmol per min per mL o f pooled liposome peak f r a c t i o n s ( v o i d volume). Values presented are t y p i c a l o f two separate experiments. 2 +  the  presence o f c a l m o d u l i n ,  kDa major  fragment  and minor  When the  incubated with  o r without  (in  the  was  presence  calmodulin  present  separated  from  c a l p a i n alone  A23187)  1  (in  pretreated fold  the  with  (Table  the  5).  the  stimulation  kDa,  2 +  stimulation  again  a  the  had  127  kDa and 37 pre-  stimulation respectively  indicates  activity  that  been  the  completely  pre-treated  with  (11.43 compared  to  by c a l m o d u l i n was reduced  to  On the of  39  and 3 . 1 - f o l d ,  medium  into  proteoliposomes,  as measured,  Ca -ATPase  the  55  This  presence  of  kDa,  -ATPase  Proteoliposomes  presence o f A23187). in  Ca  o f the  pre-incubation  w h i l e the  calpain  calmodulin  85  the  3.0-fold  proteoliposomes.  nmol.min" .mL" ),  1.1-fold  of  alone,  be about  had i n c r e a s e d basal 1  4.95  calmodulin  to  in  the  fragments  Ca -ATPase activity  found of  fragmented  2 +  kDa (lane d ) .  by c a l m o d u l i n  calpain  other  hand,  calmodulin  2 +  Ca -ATPase  proteoliposomes  maintained  activity  (in  the  a  2.5-  presence  ofA23187).  4.  Kinetic  properties  of  the  intact  and  calpain-treated  Ca  2 +  pump  in  phosphatidylcholine vesicles  The studied  intact with  illustrates  and  respect  clearly  c a l c i u m uptake  by the  (KQ 5  4  around  calmodulin, to  850  /iM  to  that  however,  calpain-treated its the  intact and  respectively).  nmol.mg p r o t .  calmodulin,  the  _ 1  dependence affinity  Ca  0.2  2 +  /xM  1  the C a  2 +  on  Ca  Ca  2 +  2 +  in  the  After  and  of  pump was g r e a t l y  Furthermore,  .min" .  for  reconstituted  absence  Ca  2 +  ATP.  the  initial  and  the  Fig.  35A  rate  of  presence  of  from  400  treatment  in  the  m a x  absence  pump appeared to have high a f f i n i t y  153  was  i n c r e a s e d by c a l m o d u l i n  calmodulin increased V  calpain  pump  for  of  Ca  2 +  Fig.  35  Calcium  dependence  of  the  initial  rate  of  Ca  uptake  of  the  105  mg  9 i  reconstituted  Ca  pump.  Proteoliposomes  phospholipid)  were t r e a t e d with 0 (A)  for  as  120  then or  min,  subjected  the  to  presence  concentrations separate  in  of  the  Ca  2 +  Materials uptake  (closed free  2 +  .  of  Values  experiments.  154  protein  2 unit.mL  measurements  .  /xg  and Methods.  circles) Ca  or  (237  1 /iM  - 1  calpain  (B)  at  25°C  The p r o t e o l iposomes were  i n the  absence  calmodulin  presented  plus  are  at the  (open the  circles) indicated  means  of  two  F i g u r e 35  155  (KQ_5  around 0.3  35B),  and the  for  Ca^  /iM)  even when assayed in the  a d d i t i o n o f c a l m o d u l i n d i d not f u r t h e r  and  only  1  slightly  increased  enzyme was a l s o  exhibited  low C a  the  (KQ 5  enzyme around  calmodulin enzyme high V  M  A  X  in  the  2 +  the  0.2  (Fig.  into  (from  M A X  550  to  examined  affinity  high  36A).  V  The  M  A  The  650  X  intact  low V  and  mode when  assayed  in  calpain  again  high  with  Ca  2 +  the  M  calmodulin  treatment  predominantly  mode even when i t  36).  (KQ 5 around 2 /iM),  absence o f  /iM),  (Fig.  affinity  A  nmol.mg  high  Ca  the  (KQ 5  of  Ca  mode  X  (Fig.  affinity  The C a - d e p e n d e n c e of the A T P - h y d r o l y t i c a c t i v i t y  reconstituted the  V  i n c r e a s e the  2+  prot."*.min" ).  of  absence o f c a l m o d u l i n  2 +  2 +  the pump  typical affinity  presence converted  around  of the  0.3  /iM),  was assayed i n the absence o f c a l m o d u l i n  (Fig.  36B).  The  effect  reconstituted clearly Ca  2 +  of  .  Ca^  The apparent  fragmented  (Fig.  T  ATP pump  on was  the  initial  also  Ca  and  1.1  /iM  v  a  and  pump expressed  /iM and 245 /iM), 37B).  Kg 5(ATP)  l  u  e  s  205  of  a  r  e  /iM  Ca Fig.  affinities  2 +  uptake 37A  f o r ATP at 0.4  superfamily proteins to  /iM f r e e  /iM and 289 /iM i n the  absence  in  its  calpain-  presence.  apparent  c a l p a i n treatment d i d not  S u s c e p t i b i l i t y o f other  the  demonstrates  KQ 5  The values  both i n the absence and in the presence o f  Therefore  by  1-4  comparable  b i p h a s i c ATP-dependence o f the Ca^  V.  rate  investigated.  t h a t the enzyme has two d i s t i n c t  calmodulin  (0.9  of  significantly  for  ATP  calmodulin modify  the  pump.  calmodulin-binding proteins  calpain  156  and t h e  troponin C  Fig.  36  Calcium  proteoliposomes.  dependence  Proteoliposomes  were t r e a t e d with 0 (A) in  the  Materials  2 +  Ca -ATPase presence  (closed  concentrations separate  of  (50  (Method  free  Ca  of .  Ca  1  calpain  -ATPase  plus  (B)  at  activity  in  the  120 Values  experiments.  157  absence  nM  25°C f o r  (open  calmodulin  presented  are  in  22 mg p h o s p h o l i p i d )  The p r o t e o l i p o s o m e s were then A)  circles)  the  tig p r o t e i n  or 2 u n i t . m L "  and Methods.  activity  of  120 m i n , assayed  circles)  at the  the  or  as for the  indicated  means  of  two  F i g u r e 36  158  Fig. the  37  Effect  reconstituted  phospholipid) for were  o f ATP c o n c e n t r a t i o n  120  min  then  circles)  2  indicated  subjected the  concentrations two separate  initial  Ca "*" pump . P r o t e o l iposomes (466  were t r e a t e d with 0 (A) as  or  on the  to  presence  o f ATP (0.5  in Ca  Materials 2 +  uptake  (closed  experiments.  159  tig p r o t e i n  or 2 u n i t . m L "  1  and Methods. measurement  circles)  to 2000 /iM).  r a t e o f Ca^  of  in  (B)  at  25°C  proteoliposomes  the  absence  1 /iM c a l m o d u l i n  Values presented  of  p l u s 210 mg  calpain The  uptake  are  at  (open various  the means  of  Figure 37  160  1.  P r o t e o l y s i s o f adducin and neuromodulin by c a l p a i n  Based on the i n t e r e s t to are  also  protein  susceptibility  find  out whether o t h e r  calpain  present  an a - s u b u n i t  substrates.  kDa)  plasma  to  degraded  incubation  during  (97  kDa)  or  2 +  which  on  was found t h a t both asmaller  calpain  not  shown).  Bennett,  1986)  fragments resulted  binds  enzymes  subunits:  calmodulin. with  calmodulin  further  This result  that  adducin  is was  kDa  very  with  prone  to  were  adducin  (Fig.  degradation  consistent  the  affinity  and z3-subunits o f  between 80-95  in  of  membrane-associated  (as a minor component)  Ca -ATPase  with  (results  38A).  of  these  the  report  proteolysis  purification.  Another  calmodulin-binding  protein  bovine b r a i n ,  which binds c a l m o d u l i n  than  presence  in  molecular probably al.,  plasma  was  When such a d d u c i n - c o n t a i n i n g C a - A T P a s e p r e p a r a t i o n s  were r a p i d l y  and  a  proteins  has two n o n - i d e n t i c a l  co-purify  membrane  it  (Gardner  It  and a / J - s u b u n i t  t r e a t e d with c a l p a i n ,  fragments  is  it  2 +  chromatography.  Longer  -ATPase to c a l p a i n ,  calmodulin-binding  i n human e r y t h r o c y t e s .  (103  Ca  Adducin  Adducin was sometimes found to erythrocyte  o f the  its  weight due  1987).  progressively unusual  to  is  24  its  Upon  (Andreasen  high  of  it  et  of  al..,  several  neuromodulin  as  basic  treatment,  into  was  more t i g h t l y  appears  content  calpain  transformed  mobility  kDa,  tested  on  161  i n the  1983). a  57  amino  kDa acid  SDS-PAGE,  (Fig. it  band  true  SDS-PAGE, (Wakim  found  38B). was  its  on  residues was  from  absence o f Ca  Although  neuromodulin fragments  neuromodulin  Due  et  to  be  to  the  impossible  to  Fig.  38  Proteolysis  of  neuromodulin by c a l p a i n .  human (A)  Materials  and Methods.  subjected  to  ( l a n e 3)  either  sonicated  asolectin,  Proteolysis  arrow  mM  was  being  the  kDa and 103  (lane 2)  kDa)  bands o f  as d e s c r i b e d  ttg p r o t e i n )  was  in  then  10 mM  mM  CaCl ,  20%  65  2  Triton  with gel  solublized  25°C i n 50 mM sodium-Hepes (pH 7 . 4 ) , 0.7  or c a l p a i n  was  brain  15 min  (w/v)  to  (100  bovine  treatment f o r  EDTA,  0.03%  subjected  indicates  at  terminated  and  pump from human e r y t h r o c y t e s  no treatment  0.5  adducin  The adducin p r e p a r a t i o n  o r 30 min (lane 4)  dithiothreitol,  before  Adducin (97 c r  and c o - p u r i f i e d with the C a  erythrocyte  X-100  (w/v)  mM  and  ice-cold  electrophoresis  KG1,  2  /ug  0.06%  of  (w/v)  calpain  trichloroacetic  (Laemmli,  adducin and s o l i d arrows  I. acid  1970).  The open  indicate  fragments  p i  of  adducin.  indicated kDa) in  is  The  by the  Ca  1  by the  are:  p h o s p h o r y l a s e b, 43 kDa ( e ) ,  (B)  from  Dr.  Yuechung of  no treatment  30 min  ( l a n e 4)  as  in  (A).  as a 57 Purified  Dr.  Washington,  either  0.7  (lane  kDa  its  2  31  Edwin  U.S.A. 2)  (a),  kDa band)  while I  (0.5  /xg  s o l i d arrows /tg)  kDa  (b),  inhibitor,  from  bovine  Daniel  (4  /tg)  was  for  R.  a  gift  Storm,  subjected  to  ( l a n e 3)  or  15 min  10 mM d i t h i o t h r e i t o l , P r o t e o l y s i s was  band o f  indicate  was  Dr.  treatment  I.  brain  and  neuromodulin  fragments  from human e r y t h r o c y t e  162  116  used  and soybean t r y p s i n  Neuromodulin  the  standards  (76  ovalbumin,  Chapman  calpain  are  66 kDa ( d ) ,  purified  indicates  weight  kDa)  of calpain  /J-galactosidase,  kDa (f)  R.  (124  subunit  Molecular  or c a l p a i n  and 0.5  The open arrow  fragment  large  at 25°C i n 50 mM-Hepes (pH 7 . 4 ) ,  mM C a C l  calpain  w h i l e the  Neuromodulin Liu,  and  bovine serum a l b u m i n ,  c a r b o n i c anhydrase,  (g).  mM EDTA,  200  97 kDa ( c ) ,  kDa  kDa)  open t r i a n g l e .  myosin,  21.5  University  (136  solid triangles,  indicated  lane  pump  of  0.5  terminated (appearing  neuromodulin.  was a p p l i e d  to  lane  5.  Fig.  Open  38  (Cont.)  triangles  active  fragment  indicate (76  are as d e s c r i b e d i n Coomassie  Blue  i n two o t h e r  kDa). (A).  R-250.  separate  the  large  subunit  Molecular  weight  of  calpain  standards  (80  kDa)  applied  and in  lane  its 1  SDS-PAGE g e l s  i n panel  A and B were s t a i n e d with  The p r o t e o l y s i s  patterns  p r e s e n t e d were  experiments.  163  reproduced  Figure  38  w  CO  c o CM  I II  - I!  o  n  I  TJ  0)  * •  O)  f /  <  CO  CM  • i  I CO  U  164  O  "TJ  CD  H-  determine  the  true  molecular  weight  of  the  fragments  directly  from  the  gel.  2.  Effect  o f c a l p a i n on the .junctional  The e f f e c t SR  fraction  particular was  of  calpain  (from  regard  observed  reported  to  that  protease.  by  In  calcium release 147, a  142,  the  :  disappeard therefore  most  340  kDa  et  muscle)  release  (1984)  study,  protein time  and  fragment a  ratio  a limit is  fragments  ( 82  kDa,  42  treatment the  of  al.,  isolation  the  of kDa)  (Fig.  the  137  (see  observed  junctional  In  the  larger  340  SR C a - A T P a s e was not fragmented  C a l p a i n treament o f the j u n c t i o n a l  165  SR  to  to  the 155,  fragments  In  calpain  the  SR,  during  kDa endogenous  be  buffers-  two  other  proteolyzed  plasma  by c a l p a i n at  under s i m i l a r  and fact,  junctional  addition,  SR were found to  sharp c o n t r a s t  2+  In  the  accumulated  endogenous  SR.  as  165,  F i g . 39B).  this  as  i s seen t h a t with  untreated  of  II  occurred  fragment  in  by  it  39A),  280,  It  rapidly  (Fig.  340,  lane 8,  was  l e u p e p t i n was i n c l u d e d i n the  of junctional 39).  of  kDa  The content  reduced i f  370,  most  proteolysis  1984).  of  with  channel.  calpain  sites  In F i g . 39B,  fragment  commmonly  result et  only  = 1:186)  cleavage  (1:63),  kDa)  chicken  junctional  examined,  release  (410  ratio  the  also  calcium  using  multiple  producing  was  channel  : SR p r o t e i n  al.  c o u l d be l a r g e l y  Ca -ATPase, 39).  SR  (Seiler  by c a l p a i n 2 +  (calpain  channel  as  during  peptides  calcium  may r e p r e s e n t  isolation  used  skeletal  components o f  calmodulin-binding  present  with  likely  fragment  protein  140 and 137 kDa, r e s p e c t i v e l y .  calpain  the  the  Seiler  the  on the  rabbit  degraded by c a l p a i n was  I  SR p r o t e i n s  membrane all  conditions  (Fig. also  Fig.  39  Proteolysis  calpain.  Junctional  of  .junctional  sarcoplasmic  SR was prepared a c c o r d i n g to  s u b j e c t e d to c a l p a i n treatment using a c a l p a i n 186  (A)  or  600  nl  with  (lane  1:  63  (B).  none  2-11)  at  In  (lane  (A),  1 and 12)  25°C  in  280 /iM C a C l ,  2),  1 (lane 3),  2 (lane 4),  8),  30  (lane  were taken  and added to  containing  500  junctional  SR (63  (lane  1)  or  K-Hepes (lane 45  (pH  1),  370  7)  subjected  to  1  170  5 (lane 3 ) ,  10  (lane 8 ) ,  SDS-PAGE.  In  buffer both  the  (410  large  subunit  additional CPP  and  protein  kDa)  /iM  CBP (105  that  represent kDa),  are  the  in  in  molecular  7.4), 1),  15  (lane  20  (lane  aliquots  (32  (see  at  (from  (lane 4),  15  panel  In  (B),  25°C with  none  calpain).  (lane 5),  500 /iM l e u p e p t i n  A and  B,  the  fragments.  I whilst  the  30  release (calcium  and the l e t t e r s  weight markers:  the  166  time  (lane  channel,  0 6),  addition  before  being  on the  right  o f the c a l c i u m  release  indicates  indicate  calpain  50 mM  At  The open arrow  a l s o s u s c e p t i b l e to calcium  numbers  open t r i a n g l e s  /iL)  Methods)  SDS-PAGE.  60 /iL  mM  0.5  (lane 7),  buffer  in 1  15 mM d i t h i o t h r e i t o l ,  EGTA  containing  calsequestrin  Solid triangles  (pH  1 :  unit.mL" )  p r o t e o l y s i s was stopped by the  proteolytic  of calpain  peptides  respectively. following  and i t s  10  and was  incubated  (0.35  (lane  to  s i d e o f the g e l s are the apparent m o l e c u l a r weights channel  I  by  r a t i o of  was  11 & 12),  subjected  unit.mL" )  /iM C a C l ,  o f 30 /tL sample d i g e s t i o n  (lane  incubated  2  and 60 min  At time 0  being  was  (0.7  calpain  sample d i g e s t i o n  before  I  /ig  /xg)  7 (lane 6),  60 min  /tg p r o t e i n )  1 (lane 2),  (lane  10),  (840  proteins  al.(1987)  potassium-Hepes  5 (lane 5 ) ,  /iM l e u p e p t i n  7.4),  mM  20 /iL o f  1 /ig c a l p a i n  4.5  Inui et  : SR p r o t e i n s  SR  80 /iM EGTA.  2  45  or  50  dithiothreitol,  (lane 9),  junctional  reticulum  the  two  t r e a t m e n t . CRC,  the  calcium  binding  protein,  indicate  the p o s i t i o n o f  the  a-macroglobul i n ,  360  origin  (a),  58  pump kDa),  Fig.  39  (Cont.)  kDa ( b ) , b,  97  myosin,  kDa  (e),  200  bovine  c a r b o n i c anhydrase, and  egg  white  kDa ( c ) ,  serum albumin,  31 kDa (h)  lysozyme, as  /J-galactosidase,  Coomassie  Blue  reproduced  i n two o t h e r  66  kDa  116 (f),  and soybean t r y p s i n  14.4  described. separate  kDa  (j).  Both  Proteolysis experiments.  167  kDa  (d),  phosphorylase  ovalbumin,  43  inhibitor,  21.5  gels  were  patterns  kDa kDa  stained  presented  (g), (i) with were  168  did  not  affect  its  Ca2+-ATPase  activity  (results  not  shown).  C a l s e q u e s t r i n a l s o was not s u s c e p t i b l e to c a l p a i n .  3.  R e s i s t a n c e o f t r o p o n i n C s u p e r f a m i l y p r o t e i n s to  Since substrates other  several while  troponin  characteristic troponin  C,  calcineurin fragmented these  calmodulin-binding  calmodulin  C superfamily with  B (regulatory by  calpain  subunit  Ca -binding proteins,  fragmented  kDa,  by c a l p a i n  not,  It  was  it  40).  of  are  was o f  found  that  Interestingly,  interest  besides  did  two  troponin  I,  catalytic  subunit)  which  169  be to  calpain see  share  not  if  this  calmodulin,  oncomodulin, parvalbumin  calcineurin)  40).  to  2 +  namely,  (Fig.  shown  C a - b i n d i n g proteins  S-100/3 p r o t e i n ,  (Fig.  2 +  is  members o f  calmodulin.  S-lOOa p r o t e i n ,  and c a l c i n e u r i n A (60 were  itself  proteins  calpain  to  be  proteins  of  binds t r o p o n i n  C,  binds c a l c i n e u r i n  B,  binding  which  appear  and  Fig.  40  to  R e s i s t a n c e of the t r o p o n i n C s u p e r f a m i l y o f C a - b i n d i n g  calpain.  calmodulin  Troponin (1  100/3  protein  gift  from D r .  rat  I  and  / i g , bovine (1  /ig,  C mixture  brain),  bovine  and  calcineurin  (3  and Cheung,  no  calpain  treatment  (-)  10  calpain  indicate  letter  A,  fragment  or  kDa)  200  kDa ( a ) ,  and  21.5  (see  are  Fig.  bands  subunit  are  /tg,  of  A  (43  bovine  1986)  0.3  skeletal  /ig, (1  bovine  /ig,  rat  muscle), brain),  kDa)  (+)  at  F i g . 39. I  and  indicate  60  of  C,  either  in  50 mM 0.2  /tg  and  gel  and C on  the  respectively,  B (17  to  proteolysis  subunit  A (60  kDa)  while kDa),  of  116  kDa ( b ) ,  kDa  (g)  were  lysozyme,  14.4  kDa ( h ) .  two o t h e r  separate  97 kDa ( c ) , as  described  43  in  as  Protein patterns  experiments.  170  M o l e c u l a r weight  66 kDa ( d ) , Fig.  38  kDa ( e ) , well  the major  calcineurin,  The 80 kDa subunit o f c a l p a i n and i t s major a c t i v e i n d i c a t e d with an open t r i a n g l e .  (1 /tg,  and  2  I  a  essentially  min  mM C a C l  The l e t t e r  the  subunit  for  0.5  Termination  and  purified  25°C  S-  hepatoma,  were s u b j e c t e d s e p a r a t e l y  troponin right  brain,  mM EGTA,  38).  as d e s c r i b e d i n  and B on the  respectively. (76  I  the  A' of  (1  oncomodulin  treatment  mM d i t h i o t h r e i t o l ,  electrophoresis left  rabbit  S-lOOa p r o t e i n  brain),  according to T a l l a n t  purified  /tg,  B. Mutus, U n i v e r s i t y o f Windsor, Canada), parvalbumin  muscle)  Tris-HCl,  (2  proteins  as  fragment  standards: 31  kDa  egg  (f)  white  p r e s e n t e d here were reproduced i n  Ill  O  O  CD  M.W.  W  Std.  Troponin  I  \  Q.  O  CT  D)  "D 0)  • lit • 3  I  + I  Calmodulin  + S-100  I  d  + S-100  p  Oncomodulin Parvalbumin  i  1  Calcineurin  1  t  3>  CO  0>  aunS.Lj  >  I + I + I + I  +  DISCUSSION  I.  D i s c u s s i o n o f the experimental  1.  Purification  In  the  first  and c h a r a c t e r i z a t i o n  series  c a l p a i n was p u r i f i e d chromatographic  of to  steps  was  active  found t h a t  the  fragments  of  literature calpain others  (see  apparent (Fig.  (Mellgren  a half-maximal Ca  2 +  (Fig.  80 78  and  al.,  stable  was  homogeneity 11,  12  the  (Fig.  and  of  demonstrated  14)  13).  kDa  calpain  (Fig.  that  using a s e r i e s  Next,  at  was  15A),  1986b).  25°C  the  indeed as  It  than  of  purified  at  autolyzed  reported  the that  was  also  found  37°C,  as  reported  protease  activity  belongs  to  at the  to  in  The p u r i f i e d c a l p a i n p r e p a r a t i o n s  o f the p r o t e o l y t i c  Thus,  clearly  the a u t o l y s i s o f c a l p a i n was s t u d i e d and  and M e l l o n i ,  1982).  activation  16A).  76  calpain  it  kDa s u b u n i t  was more et  10,  First,  Pontremoli  activity  of  experiments,  c a l p a i n was c h a r a c t e r i z e d . it  results  by  showed  about 20 /xM f r e e class  designated  9+ calpain active  I,  which  (Murachi,  towards  This  is  again  I.  preparations  Therefore,  concentrations  calpain preparations  proteinase  calpain preparations  of c a l p a i n  micromolar  1983b). The  cysteine  iodoacetate). the  requires  inhibitory  confirmed  show apparent at  this  c o u l d be used to  in  study the  it  Ca  to  a l s o showed  agent's work  homogeneity  point,  172  this  of  be  sensitivity  (leupeptin  (Fig.  16B).  and a l l  the  In  of calpain  and brief,  properties  was d e c i d e d t h a t these  effect  fully  calpain  I on the Ca  -  ATPase.  2.  Calpain activates  The ATPase  first  step  activity  erythrocytes  2 +  examined,  the it  the  Ca  2 +  Ca  2 +  in  (i.e.  in  treatment  Interestingly,  a  time-dependent of  of  the  became also  C)  that  was a c t i v a t e d  and  (Fig.  17C).  In also  2 +  (Fig.  of  but  19A).  further  high Calpain  the  enzyme  (Fig.  20).  calmodulin-  (Fig.  20).  These  trypsin  or  papain  al.,  1986).  (Sarkadi  could  was  enzyme had  calmodulin  with  others  Ca -ATPase  converting  loss  demonstrated  calmodulin  human the  calmodulin  absence o f  as d e s c r i b e d by  found  added  calmodul i n - i n s e n s i t i v e ) be  -  in  membranes the  2 +  a  2 +  B).  calmodulin  by  Ca  from  found t h a t  manner  C a - A T P a s e by  accompanied  can  was  the  of  the  plasma membrane were  control of  on  isolated  membrane-bound  absence  presence  the  17A,  mode even i n the was  19B, it  the  it  fact,  (Fig.  the  activated  activation  stimulation  in  membranes  z +  in  -ATPase  calpain  the C a - A T P a s e a c t i v i t y  affinity  Ca  of  subsequently  2 +  high a f f i n i t y  effects,  was  the C a - A T P a s e ( F i g .  apparently  the  It  o f the Ca  effect  plasma  and papain treatments  for  for  treatment  an  was confirmed t h a t with the  affinity  affinity  for  protease.  and c a l p a i n ,  trypsin  When  Also,  look  pretreating  the  found t o a c t i v a t e  into  to  calmodulin-insensitive  parallel,  low  was  by  with  presence of C a became  the A T P - h y d r o l v t i c a c t i v i t y  et  protect  the  membrane-  pi bound  Ca  -ATPase  against  c o n c e n t r a t i o n range t h a t and  21).  This  is  protective  calpain-mediated required to effect  of  173  activation  activate calmodulin  the was  over  a  similar  2 +  Ca -ATPase (Fig. not  observed  20  with  trypsin  or  papain  proposed  to  activity ATPase  of  illustrate  the  while  proteolytic  stimulated  activation  calmodulin.  Also,  41,  and  model  calpain  activates  2 +  the  a  Ca -ATPase  is  on  the  the Ca  2 +  -  irreversibly.  forms o f the C a - A T P a s e appear t o be non-  the  Ca^  calmodulin  2 +  t h a t the c a l p a i n - a c t i v a t e d C a - A T P a s e  calmodulin  reduction  calmodulin-stimulated calpain-mediated  by  Fig.  calmodulin  activates  the f a c t  of  of  In  2 +  the two high a c t i v i t y  longer  C).  Firstly,  calpain  based on  19B,  effects  Ca -ATPase.  interconvertible no  (Fig.  2 +  the  reversibly  Secondly,  is  treatment  of  that  2 +  Ca -ATPase  is  intracellular  pump  irreversible  and  will  tend  activation  the  calpain-mediated  largely  Ca  2 +  to  under  prevented  concentration  by  by  the  further  minimize  the  normal  physiological  conditions.  pi Fragmentation o f the C a - A T P a s e by c a l p a i n : comparison with  3.  At t h i s p o i n t 2 +  it  appeared t h a t the c a l p a i n - m e d i a t e d a c t i v a t i o n  Ca -ATPase  c o u l d be a complex e v e n t .  proteolytic  activation  should  be  further  p. Ca^ -ATPase visualized that was  in  124  the  32  by  following  activation  by  the  intermediate the  23),  two  decided that  the  of  calmodulin  fragmentation  of  the  p. Ca^ -ATPase  was  formed.  kDa i n t a c t  into  the  It  was  However,  EP-forming  in  found  E P - f o r m i n g enzyme  with a t i m e - c o u r s e p a r a l l e l  22).  174  136  of  effect  membrane-bound  calpain  (Fig.  therefore  protective  the  calmodulin,  (Fig.  was  the  First,  sequentially  It  as  P-phosphoenzyme  kDa and 80 kDa)  proteolytic  well  calpain.  absence o f  fragmented  (about of  the  by  as  characterized  by  trypsin  the  fragments to  presence  that of  Fig. Ca  c  41  Proposed model  -ATPase  proceed.  by  for  calmodulin  For a d e t a i l e d  the and  control  of  calpain.  explanation,  the (x)  see t e x t .  175  human e r t h r o c y t e means  reaction  membrane does  not  Ca -ATPase 2+  low  Calmodulin  Calmodulin - activated Ca -ATPase 2+  high  activity  reversible  activity  irreversible  Calpain  Proteolytically-activated Ca -ATPase 2+  high  activity  calmodulin, observed little  different  (Fig.  23).  results  the  was  (were)  light  of  solublized of  the  (i)  it  was  confirmed  Since  kDa were that  very  EP-formation  one o r both o f  the  calmodul i n - i n d e p e n d e n t  127 kDa o r  is  124  kDa and  active  a  these 80  fragment(s);  85 kDa fragments was (were) the  calmodulin,  these r e s u l t s ,  and  sequence  the  is  calpain-mediated  Ca -ATPase, studied  suggested  in  Fig.  2+  heterogeneous  active  25B).  these  the  fragmentation  absence  both  capable  A  scheme  42.  In  the  p r o t e o l y z e d to  However,  components  of  fragments  stimulated  by c a l m o d u l i n , whereas  only  retained the  of the  versus  25).  forming minor  the  125  of  the  absence  of  t o a 82-80  acylphosphate  kDa  the  capacity  to  major  fragments  formed,  124 kDa and 80 kDa components, l o s t the c a p a c i t y  the  a 125-124 kDa  (more than one component) and t h e r e a f t e r  fragment,  (Fig.  in  (Fig.  C a - A T P a s e was s e q u e n t i a l l y  heterogeneous fragment  intermediate  was  the  2 +  purified  calmodulin  fragmentation  kDa  kDa and 85  which r e t a i n e d the c a l m o d u l i n - s e n s i t i v i t y .  the  presence  occurred.  following:  one o r both o f the  fragment(s)  of  127  conditions,  activation  suggest the  In  these  of  o f an a c t i v e ATPase molecule ( i n t a c t enzyme o r f r a g m e n t ) ,  kDa fragments (ii)  Under  proteolytic  reflection  EP-forming fragments  bind  and  82  and  kDa  to  namely  to b i n d c a l m o d u l i n  be the  (Fig.  26). When  the fragmentation p a t t e r n  o f the membrane-bound Ca^ -ATPase was O 9  studied,  one  intermediate  had  to  rely  on  the  formation  to v i s u a l i z e the d i f f e r e n t  fragments.  becomes d i f f i c u l t  to i d e n t i f y whether the  also  minor  contain  the  125  kDa and 82  177  of  J < 1  P - a c y l phosphate  With t h i s t e c h n i q u e  it  124 kDa and the 80 kDa fragments  kDa components  (Fig.  23  and F i g .  Fig.  42 F l o w - c h a r t  calpain  in  the  f o r the  presence  indicates  the  apparent  ATPase o r  its  fragments.  fragmentation  and absence o f  molecular  acylphosphate i n t e r m e d i a t e  (+)  weight  indicates  and (*)  o f the e r y t h r o c y t e calmodulin. (kDa) the  of  Numbers  either  capacity  Ca-ATPase  to  the form  in  native 3 2  indicates calmodulin-binding  178  the  bv  boxes Ca  2 +  -  P-labelled capacity.  F i g u r e 42  179  27A).  However,  pretreated  with  the calpain  calmodulin-affinity kDa  (Fig.  Ca  -ATPase  in  the  fragments,  absence o f  column, have apparent  27B, C ) . T h e r e f o r e ,  it  calmodulin-insensitive,  calmodulin  from  by  membranes  retention  on a  m o l e c u l a r masses 125 kDa and 82  appears t h a t  80 kDa fragments which were not r e t a i n e d are  purified  the remaining  124 kDa and  by the c a l m o d u l i n - a g a r o s e column  proteolytically-activated  forms  of  the  Ca  2 +  -  ATPase. On  the  other  hand,  in  p r o t e o l y z e d to d i f f e r e n t kDa, of  respectively forming  J t  a  the  ( F i g . 23 and 25B).  P-phosphorylated  i s c o n s i s t e n t with the  activation  also  relative  25B).  In  smaller  to the  fragments  form  it  proteolysis  seems t h a t  by (81  membrane-bound  ATPase  and  presence o f  enzyme was  were  retained  calcium ( F i g .  by  the  25C and  t h a t c a l m o d u l i n prevents the  a  kDa  and  the  (Sarkadi  2 +  of  55  80  27B).  proteolytic  the  kDa,  37  the  be i n s e n s i t i v e to  81  Ca -ATPase (Fig.  fragments d i s c u s s e d above, none o f  these  similar  1986; al., kDa  intermediate is  to by  the  the  (Fig.  smallest  1984). and  76  smallest  trypsinization  Enyedi  stimulation  180  kDa and 32  25C).  form o f  acylphosphate intermediate  obtained  et  2 +  purified  kDa  is  al..,  (Zurini both  39  forming the  kDa)  et  kDa,  of  kDa fragment  This 76  fragments  phosphorylated  capable of  Ca -ATPase  to  other  larger  calpain.  activities  reported  the  m o l e c u l a r mass o f  Ca^ -ATPase s t i l l  purified  the  Both o f these fragments were c a p a b l e  intermediate  fact  produces  contrast  Therefore,  fragments  calmodulin,  and the l o s s o f c a l m o d u l i n s t i m u l a t i o n o f the enzyme.  Calpain with  of  h i g h e r m o l e c u l a r mass fragments o f 127 kDa and 85  c a l m o d u l i n - a g a r o s e column i n This  presence  et  aj_.,  tryptic  by c a l m o d u l i n  after  P-labelled  of  1987),  Interestingly, kDa  3 2  the  both  the  and  the  the  fragments (Sarkadi  et  Ca  2 +  -  were al.,  1986;  Benaim et  Ca-ATPase  al.,  obtained  Interestingly, produced  by  trypsin  in  both  the  min)  was  prominent  28).  Earlier  to  prevent to  results,  Fig.  28  produced l a r g e high et  activity  al.,  case  Enyedi  in  absence o f  case o f  identical  However,  in t h i s  of  fragments  between  calpain  (Fig.  even  calmodulin  in  the  to  (Zurini  However,  28).  al.,  difference  respectively,  1984;  Sarkadi et  in r e l a t i v e  thesis used.  and Fig  that 43  1986;  to  The  86  the  others  provides  a  is  kDa  trypsin, with  calmodulin,  in  these trypsin  et  al.,  1984;  Benaim  appeared  76 kDa fragment 82  kDa  and  to  seen  77  kDa  to be the 85 kDa, 81 kDa and  Enyedi et  probably  schematic  by  agreement  kDa,  m o l e c u l a r mass o f the t r y p t i c of  (120  calmodulin  r e p o r t e d by o t h e r s al.,  kDa and 127  p r e v i o u s l y r e p o r t e d to be o f  fragmentation (Fig.  fragments  failed  presence o f  calmodulin  1987).  (30  that calmodulin  In  t h a t were observed here very l i k e l y  Benaim et  treatment  (Fig.  Ca -ATPase  19).  cleavage  min)  2 +  the  suggests  of  major  treatment  28).  calmodulin,  sites  124  the  fragments  This  trypsin  t h e s i s demonstrated of  tryptic  initial  of  (Fig.  kDa  of  caused a c c u m u l a t i o n o f  activation  al.,  127  calpain.  longer  prolonged c a l p a i n  et  further  76 kDa f r a g m e n t s ,  systems  or  and  presence  after  and i n s e n s i t i v e  against  this  the  and  amounts o f an 82 kDa fragment,  1984;  fragments  in  patterns  were compared  kDa  while  shows t h a t  protect the  124  kDa,  results  the  as  2+  even  of  similar  proteolytic  contrast  the f r a g m e n t a t i o n  absence  Ca -ATPase  kDa and 86  remain  the  molecule.  purified  77  in  have  Ca^ -ATPase  between  formation  observed,  proteases  o f the  Therefore,  by c a l p a i n and by t r y p s i n  transient  respectively, that  1984).  of  due the  (Zurini al.,  et  1984;  1987). The small  fragments to  al.,  the  reported  different  fragmentation  of  in gel the  pi Ca*- -ATPase o b t a i n e d with t r y p s i n .  It  i s proposed t h a t the 90 kDa, 86 kDa,  / 181  Fig.  43  native bars.  Schematic intact  The  of  the  enzyme  number  in  and  the  polypeptide.  The  letter  site,  the  letters  while  terminus,  respectively.  proteolysis its  middle P  are  of  is  a bar the  area  details.  182  Ca  -ATPase  was  by t r y p s i n .  represented the  the the  by  molecular  location  C represent  The darkened  b i n d i n g domain. See t e x t f o r  the  fragments  represent  N and  of  of  the  N-terminal identified  the  The  opened  mass o f  that  acylphosphate and  the  C-  calmodulin-  Figure 43  183  82 kDa, 77 kDa fragments a l l  have the same or s i m i l a r N - t e r m i n a l  end which  i s generated by a c l e a v a g e about 34 kDa from the N-terminal  o f the  protein.  and the  T h i s c l e a v a g e produces a prominent  kDa fragment.  The 34  kDa fragment  is  34 kDa fragment  further  c l e a v e d to  fragment w i t h time (compare l a n e 3 to l a n e 2, Zurini  et  further  al.,  1984;  cleaved  Papp et  several  calmodulin-binding  al.,  that a l l active  1984;  (Fig.  fragments.  2 +  Ca -ATPase  Their  (Shull  later  found  a  kDa  (Enyedi  82 et  C-terminal  end  also  fragment  to  produce  is the  1986;  also  Zurini  Enyedi et a l . .  et  al.,  1984;  1987). Note a l s o  al.,  is  is  is  about  about  9-10  50  to  be  studies,  1987).  (80  Verma et  a 76  kDa  al..  acidic  Fig.  43)  the  fact  that  C-terminal  N-terminal  the  end and  o f the  intact  1988).  kDa fragment  to  in  Therefore, be the  kDa from the  kDa from the  insensitive  fragment  c o n s i s t e n t with  (77  kDa  phospholipid retained  a 5 kDa fragment  such  removed  in  Fig.  43)  stimulation capability from the  82  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  p h o s p h o l i p i d - i n t e r a c t i n g domain c o u l d have two p o s s i b l e l o c a t i o n s :  The f i r s t the  al..  and Greeb, 1988;  kDa r e g i o n appears to acidic  kDa i n i t i a l  (see  and the n o n - c a l m o d u l i n - b i n d i n g 82 kDa  formation  domain  Based on k i n e t i c  while  the  288 and 28C; see  Sarkadi et  acylphosphate s i t e  was  at  produce a 32 kDa  F i g . 28B and 28C)  The 90  90-95  o f the 90 kDa, 86 kDa, 82 kDa and 77 kDa fragments are E P - f o r m i n g  calmodulin-binding the  times  1989).  86 kDa fragment,  and the 77 kDa fragment Benaim et  al.,  intact  would be  right  next to and upstream from the N - t e r m i n a l  c a l m o d u l i n b i n d i n g domain  fragment): residues  this (Shull  region  (i.e.  at  the  C-terminal  c o n s i s t s o f a high content  and Greeb,  1988;  Verma et  184  al.,  1988)  end o f  the  of n e g a t i v e l y  side of 82  kDa  charged  which can be viewed  to  bind  and l o c a l i z e  conceivabley activates  the  from the the  82  bind  i o n s . These l o c a l i z e d  negatively  enzyme.  N-terminal kDa  Ca^  charged  acidic  An a l t e r n a t i v e  of  the  fragment).  intact  Papp et  They argue t h a t i n t h i s  region  al.  phospholipid,  location  protein  would  (i.e.  (1989)  c a l c i u m ions c o u l d  be  at  favor  the  the  about  second  al.,  interact  besides  the  with a c i d i c p h o s p h o l i p i d . However, 2 +  C a - A T P a s e has  than the c a l m o d u l i n - a c t i v a t e d  (Niggli  suggests  a  et  similar  Therefore,  fact  somewhat  enzyme (Enyedi  et  al.,  it  is  al_.,  1981a;  mode  of  Al-Jobore  activation  reasonable  to  and  by  predict  fact  end  which may  Ca  2 +  1987), the  acidic  affinity activation  by c a l m o d u l i n are  both  types or  1981).  of  very This  activators.  adjacent  sites  2 +  o f both c a l m o d u l i n and a c i d i c p h o s p h o l i p i d on the C a - A T P a s e . that  the  presence o f c a l m o d u l i n  kDa fragment  (Fig.  28)  therefore,  that  the  available  also  experimental  prevented  s u p p o r t s the  first  first  possibility  data  is  the  formation  possibility. more  chosen i n the  o f the  of  of It  compatible  and i s t h e r e f o r e  of  there  that  Roufogalis,  similar  kDa  possibility.  1988)  higher  C a - A T P a s e by a c i d i c p h o s p h o l i p i d and t h a t  similar  action  a  turn  45-50  (about 45-50 kDa from the N - t e r m i n a l ) (Verma et  o f the  in  N-terminal  are a number o f p o s i t i v e l y charged r e s i d u e s  phospholipid-activated  which  then  of The  the  77  appears, with  scheme  the (Fig.  43) .  Based on the by  calpain  and  properties the  homology  to  fragments the  44) . From the still  bind  fragmentation  results  that  calmodulin while  (i)  2 +  C a - A T P a s e produced  trypsin-proteolysis, p  calpain-mediated  the  a  scheme  j.  o f the Ca^ -ATPase can be c o n s t r u c t e d the  the  127  124  kDa and the  kDa does not  185  of  125 kDa fragments (Fig.  26  and 27),  (Fig. can and  Fig.  44 Schematic o f the  i n t a c t Ca  -ATPase  number  the  in  polypeptide. acylphosphate  and i t s  middle The  fragments  of  a  bar  letter  P  represents  site,  carboxyl-terminal  p r o t e o l y s i s o f the  while  the  is  the  letter  ends, r e s p e c t i v e l y .  c a l m o d u l i n b i n d i n g domain.  are  -ATPase by c a l p a i n  represented  relative the N and  molecular  location  mass  of  the  C represent  the  details.  I.  by opened b a r s .  The darkened area i s the  See t e x t f o r  186  Ca  of  The The that  aspartateamino-  and  identified  Figure  44  187  (ii) while  that  the  127  kDa fragment  the  125  kDa and 124  it  from which  these fragments  the  124  obvious t h a t  Transformation  from  means a t r u n c a t i o n  would  a  3.5  have  other  at  the  intact  starts  about  (Verma  calmodulin-binding  hand, the  the C - t e r m i n a l  produced by m u l t i p l e  kDa  region  i s c o n s i s t e n t with the the  are  136  domain kDa  its  fact  protein  the  that  al.,  domain  it  end o r a f u r t h e r  would have l o s t about  3."5  it  (Fig.  domain  truncated,  with  of  124-127 kDa, small  were a l s o  produced  like  127  the  calmodulin the  calmodulin  (Fig.  kDa  (Fig. 124-125 (Fig.  observed  25).  the  127  kDa  fragment  127  S i n c e the  45).  45),  about  Furthermore,  124  11 kDa  the  kDa  the  of  proteolysis,  fragments  Interestingly,  85  in  the  only  produced  the  80-82  only  the 80 kDa fragment,  188  (of  the  in  the  calmodul i n -  times  were  125 kDa  fragment,  longer  hand,  from  N-terminal  calmodulin  other  On  and about 3 kDa o f  With most o f  produced  which  27 and 28).  bind  On the  fragments,  fragment  not  was  end and  kDa  (Fig.  (Fig.  the  at  enzyme.  Interestingly,  case o f  or  the  kDa does  23 and 2 5 ) .  and 25).  kDa  45).  125  of  intact  have l o s t  EP-forming a c t i v e  fragment,  23  In  (Fig.  that  fragments  like  27).  124  was  near  of  sites  127 kDa,  C-terminal  the  binds c a l m o d u l i n  the  It  the  C-terminal.  1988),  12 kDa from the C - t e r m i n a l  and 2 8 ) .  cleavage  p o r t i o n o f about 2 kDa from the  kDa calmodul i n - b i n d i n g domain  binding  to  largely  absence  cleavages  9 kDa from the  et  calmodulin  the  the  end  125 kDa component w i l l  bind c a l m o d u l i n  in  C-terminal  end o f the c a l m o d u l i n - b i n d i n g domain ( F i g . can s t i l l  influences  o f about 9 kDa from the o r i g i n a l  calmodulin-binding occupies  presence o f  were produced  calmodulin  domain  the  the  are p r o d u c e d . In o t h e r words,  kDa fragments  calmodulin-binding  produced i n  kDa fragments  calmodulin,  kDa and  is  is  (Fig.  besides  80-85  kDa  27  kDa)  fragment,  presence  kDa the  as w e l l  of  fragments, absence  of  as the  124  Fig.  45  Schematic o f the  membrane  Ca^  presence  (+)  and  pump  produced  by  o f c a l m o d u l i n (CaM).  carboxyl-terminus  numbers  beside  residue  numbers.  represents  formation  its  acylphosphate  the  of  relative  b i n d i n g domain.  and  large  the  I  in  fragments the  o f the  absence  (-)  The l e t t e r s N and C r e p r e s e n t acid  sequence,  represent  number  molecular  active  calpain  amino  polypeptide The  site  the  of  in  mass.  darkened  the  the The  region  letter  and the  of  each  "P"  represents  amino  acid  the  calmodulin-  The c a l m o d u l i n molecule i s r e p r e s e n t e d by a c i r c l e .  189  The  polypeptide  represents the  the  amino  respectively.  approximate  centre  plasma  F i g u r e 45  190  kDa fragment, purified 26  do not  with the  and 2 7 ) .  bind c a l m o d u l i n - a g a r o s e  125 kDa fragment  All  these  findings  components from each o f the  are:  in  point  to  three pairs  between  the  kDa,  of  fragments  an i d e n t i c a l (Fig.  44  or  similar  and 4 5 ) .  kDa fragments,  Together,  about  these  between  A simple c a l c u l a t i o n  two components o f  cleavage  kDa can be c o -  (127  Therefore,  respectively  kDa)  protect  from  the  differential calmodulin  (Fig.  attack  of  23 and  two  during (Fig.  interpreted  as  (40-44  from  the  reveals  that  the  it  kDa)  kDa)  possibilities: cleavage,  calpain,  25).  the  but  of  37  and  39  both  in  the  These  further  39-37  N-terminal,  the  rather  124  is  from  N-terminal  a l s o suggest  to the  and are produced  by end  that,  p h y s i c a l l y or  kDa o r 85 kDa -  consistent  absence v e r s u s the  kDa,  kDa  cleavage  80  with  the  presence  of  respectively,  doublet  fragments  can  the  fragment  of  above.  c o - p u r i f y with the l a r g e r  39-37  column  doublet  originated  from  191  did a  always  and  products  (see not  region  presence  initial  However,  fragments  Fig.  come  the  are  absence  as d i s c u s s e d  on a c a l m o d u l i n - a g a r o s e (i)  which  produced i n the  fragments  being  kDa -  fragments  originate  c o n s i d e r a t i o n s would  (127  of  i s tempting  42-44 kDa from the  region  two  25).  d o u b l e t was found to  and 125  kDa  proteolysis  calmodulin  kDa  3-5  fragmentation  Also, observed  this  the kDa,  by b i n d i n g to the c a l m o d u l i n - b i n d i n g domain, c a l m o d u l i n can otherwise  (Fig.  kDa/85  each p a i r  the 85 kDa, 82 kDa and 80 kDa fragments  125 kDa and 124  82  similarities  42 kDa, 43 kDa and 44 kDa r e s p e c t i v e l y .  suggest t h a t 127  size  the  u s i n g a c a l m o d u l i n - a g a r o s e column  125 kDa/82 kDa and 124 kDa/80 kDa). difference  while  27).  from  covering  the (eg.  There the  the  of be  39-37 82 kDa  are  two  N-terminal calmodulin-  b i n d i n g domain at the C - t e r m i n u s , N-terminal typical  cleavage  and  retained  originates  from the  which a s s o c i a t e s with the fragment  will  first  the 39-37 kDa d o u b l e t in  its  readily  fragments  were  absence and i n  is  second  ATPase may bind  (likely  the  to  be  occasionally kDa)  (see  same  8A o f  could 55  (Fig.  44).  kDa and 32  the  the  et  However,  it  be s t r e s s e d t h a t the  32  more  the  doublet  during  (Fig. have  o f the  the  In  than  39-37 in  support  suggested  calmodulin,  of  calmodulin-binding  proteolysis  25).  If  accumulation  was observed t h a t  kDa  that  enzyme.  kDa the  of  the  based on t h e i r  fragment  the  Ca  2 +  -  results  Interestingly,  in  Fig.  calmodulin-binding 1984).  results,  must be s t r e s s e d t h a t  it  in  from a major  l o c a t i o n o f the  kDa fragment)  is  proposed o r i g i n s o f the  192  and  43)  fragment  (90  not  (ii)  was  was chosen i n  possibility  the s p e c u l a t i o n , i t  originated  28  Since p o s s i b i l i t y  experimental  To extend  case,  a domain  2 +  al.,  Note t h a t the  55 kDa o r the  this  39-37  of the C a - A T P a s e produced by t r y p s i n 32  available  kDa fragments  80-85 kDa f r a g m e n t s . either  as  Zurini  not be e l i m i n a t e d .  it  (1984) of  the  presence o f c a l m o d u l i n  quantity  c o - p u r i f i e d with a l a r g e r  Fig.  i n the  labelling  kDa fragment  most c o n s i s t e n t w i t h the model  al.  (iii)  a  calmodulin-agarose i n d i r e c t l y .  calmodulin  et  [ ^I]iodoazidocalmodulin a 33.5  the  equal  a second molecule  the same p a p e r ,  the  in  Zurini  In  lacking  and r e t a i n s  one would expect  However,  1 2  with  cleavage  absence o f c a l m o d u l i n the  presence o f  possibility,  by the  from p r o t e o l y s i s  produced  although  a l s o binds c a l m o d u l i n ;  correct,  destroyed.  the  which,  80-85 kDa fragments.  absence, s i n c e i n the  domain i s  sequence  N-terminal  have been r e t a i n e d  possibility  the 39-37 kDa d o u b l e t came from the  a  c a l m o d u l i n - b i n d i n g domain,  kDa d o u b l e t  the  (ii)  (iii)  i s suggested t h a t cleavage  of  the  acylphosphate s i t e  (on  assigned.  It  must  again  55 kDa, 39-37 kDa and 32 kDa  fragments  are  clearly  should c l a r i f y  In  also  Direct  evidence  from  thesis  it  Ca^ - A T P a s e ,  produced  two  was  demonstrated  besides  larger  fragment  in  presence  (Fig.  28).  produced  major  fragments  the  producing  but  absence of  more  calpain  of  124  trypsin  major  and  127  treatment  kDa  of  kDa,  words, kDa  the  127  tryptic  postulated  that  z +  terminal arid  counterparts  kDa t r y p t i c  fragment  would  have  t r y p s i n might  segment, the  cleavage  43).  fragment cleavage  It  is  initially  in  its  i n c l u d i n g most or a l l  o f the  124  the  kDa  which  Papp et  calpain  .  In  the that  the  It  is  C-terminal  other  the  124  further  end o f  the  fragment.  removes  the  In  c a l m o d u l i n - b i n d i n g domain,  presence  C-terminal  of  segment  al.  (1989) r e c e n t l y  suggested t h a t  calmodulin, would  spare  a 125 kDa  the  N-terminal  end r a t h e r  than  the  also  activates  the  Ca  -translocating  193  the  tryptic  2+  near  a  127 kDa fragment  o f the C a - A T P a s e t h a t they observed was produced by a  Calpain  also  fragments produced with  ability.  attack  kDa  likely  would bind c a l m o d u l i n w h i l e such  124  absence and  C-terminal.  d i r e c t e v i d e n c e s u p p o r t i n g t h i s c l a i m was p r o v i d e d (Papp e t  4.  a  kDa,  Ca -ATPase  c a l m o d u l i n - b i n d i n g domain and produce the s l i g h t l y l a r g e r (Fig.  77-86  2 +  the  produced w i t h  lost  the  In the absence o f c a l m o d u l i n , such a t t a c k would remove the C-  produce  similar  their  fragment  Ca -ATPase.  work  of  fragment  i n the  the c a l m o d u l i n - s e n s i t i v i t y o f 124 kDa and 127 kDa resemble  of  fragments:  a  kDa and 127  treatment  fragments  transient  calmodulin  Since  that  presence o f c a l m o d u l i n , r e s p e c t i v e l y ( F i g . 25 and 2 8 ) ,  trypsin  further  these p o i n t s .  this  purified  provisional.  activity  single  However,  al.,  of  no  1989).  the  Ca  -  ATPase  Since (from the calpain  the  physiologic function  cytosol)  out of the c e l l ,  treatment  same way t h a t  In  it  recent  erythrocytes  would  activate  activated  years  has  the  been  extensively  1984;  R o u f o g a l i s and V i l l a l o b o ,  Niggli  et  and Z u r i n i ,  the  al.,  2 +  1989).  Benaim et  et  pump Ca  function  al..  (see  ATPase  Al-Jobore  source o f p h o s p h o l i p i d .  components Ca  2 +  asolectin,  translocation  us" to the  in  1987;  enzyme 1986;  (Haaker  observe p r o t e o l y t i c  loss  of  calmodulin  membrane-bound ( F i g .  17,  the  aj..,  A l - J o b o r e et  al.,  and  Villalobo  reported,  Racker,  and  activation  of  stimulation, 20,  1979;  Roufogalis,  calmodul i n - s t i m u l a t o r y  been r e p o r t e d  18,  human  asolectin  Due t o the presence o f a c i d i c p h o s p h o l i p i d  however,  has not  the  et  1986), and i n most s t u d i e s the r e c o n s t i t u t i o n was performed w i t h as the  in  from  Fewer s t u d i e s have been  al.,  whether  activity.  Ca -translocating  Niggli  to  to e s t a b l i s h  2 +  characterized  1982;  is  Ca -translocating  1iposome-reconstituted 1981b;  Ca-ATPase  was important  the  purified  Carafoli  on  it  the  the ATP h y d r o l y t i c  1981;  however,  of  to the  our knowledge. reconstituted  as p r e d i c t e d  and 22)  In Ca  on  order  for  2 +  from e a r l i e r  and p u r i f i e d  effect  pump and results  enzymes ( F i g .  24),  on a  7+ calmodulin-sensitive  reconstituted  This  resolved  problem  was  phosphatidylcholine enzyme.  With  the  (Niggli  ATP-hydrolytic  by  et  pump  using  al_.,  1981b)  preparation  highly for  activity  (i) were  the  initial  stimulated  194  rates at  was  purified  required. egg  reconstitution  phosphatidylcholine-reconstituted  f o l l o w i n g was demonstrated: and  Ca  Ca  c  yolk of  pump,  the the  o f both c a l c i u m uptake least  3-  to  4-fold  by  calmodulin  at  0.4  /iM f r e e  Ca^  (Fig.  with c a l p a i n  i n c r e a s e d these r a t e s  addition  calmodulin  initial Ca  2 +  of  of  calmodulin  Calpain  shifted  absence  and  for  in  for C a  in  the  (iii)  is  membrane C a  the  2 +  presence  first  approach used i n  the  fragmentation  other  of  further  studies  reconstituted 3 2  127  ATPase  on the  state.  kDa forms fragments  calmodulin,  both the  Ca  where  formation the  the  (Fig.  uptake  the  calpain  part  The f o r m a t i o n  proteolytic  Ca  Ca  2 +  than  both  in  report  biphasic  absence o r  o f the  and the ATP h y d r o l y t i c  124 of  plasma  The success the of  fact  of  that  trypsin  or  can be a p p l i e d  to  its  pattern that  the  37B).  that  pump were  the  mode  exhibited  This  (Fig.  pump i n  activation  195  uptake  due t o  revealed  36A).  i n the assay medium  proteolysis. in  33)  in  absence  affinity  37A).  fragmentation  (Fig.  intact  1iposome-reconstituted  selective  reconstituted  produced by  the  the  o f the  /iM and 245-289 /iM),  limited  of  the  i n the  high  r a t e o f Ca  study was  A study o f  of  with 2 +  present  (ii)  35A and F i g .  by c a l p a i n treatment  properties  respectively.  concomitantly  the  The technique employed i n t h i s  P-acylphosphoprotein  the  report  the  30).  (Fig.  into  calmodulin  by c a l p a i n was more  proteases.  (Fig.  p r o t e o l iposomes  low a f f i n i t y  presence  1.1-1.4  pump was s u b j e c t e d t o  the  :  The i n i t i a l  dependence on ATP was not a l t e r e d  This  its  2 +  enzyme  ATP (approx.  the  c l o s e t o t h o s e o b t a i n e d upon  c a l m o d u l i n was absent o r p r e s e n t  35B and F i g . 36B). affinities  levels  treatment  of  uptake and A T P - h y d r o l y t i c a c t i v i t y  and high a f f i n i t y  treatment  Treatment  calpain  two a f f i n i t i e s  r e g a r d l e s s o f whether  two  2 +  r a t e o f both C a  pump e x h i b i t e d  (Fig.  before  to  30).  the the the  purified  and  c o u p l e d with 124  kDa and  major  active  presence  of  kDa fragment  occurred  the  rates  activity  initial  o f the  enzyme  of  (Fig.  31  and  33).  observed was  The  p r e v i o u s l y when  treated  observed  with  that  2 +  Ca -ATPase (Fig.  the  from  to  I  (Fig. of  proteolytic was  to  by,  in  the  formed kDa,  39  presence less  the  the  absence o f  kDa and 37 of  such 2 +  identified  bands  ( F i g . 34,  p r e v i o u s l y when the  a  kDa  purified  In  this  protected the  reconstituted  by  calmodulin  stimulation prominent  after  bound, nor it  effect  calmodulin  of  expected  c a l m o d u l i n , l e s s prominent  in  a l s o observed addition  85  lane d ) .  kDa,  to  55  (  the  kDa,  of  of  chromatography  Fig.  kDa  kDa  bands o f 34,  prominent  39  124  lane 127  and  37  fragment  80  kDa,  55  In  the  c). kDa  kDa  fragment, were  2 +  p u r i f i e d C a - A T P a s e was p r o t e o l y z e d by c a l p a i n  3.4  to  1.1  c a l p a i n treatment (Table  demonstrated.  also  5).  and decreased the  (Table  5),  whereas  (Fig.  activation  of  fold-stimulation the  calmodulin-  i n the presence o f c a l m o d u l i n remained Therefore,  the  protective  effect  c a l m o d u l i n a g a i n s t p r o t e o l y t i c a c t i v a t i o n o f the r e c o n s t i t u t e d C a was c l e a r l y  was  These s m a l l e r fragments were a l s o observed  2 +  (2.9-fold)  still  Therefore,  By g e l - f i l t r a t i o n  B e s i d e s the  Ca -ATPase activity  from  activity was  neither  2 4 ) . I t was observed t h a t c a l p a i n alone produced p r o t e o l y t i c the  was  membrane-bound  kDa fragment  27).  protective  pump.  it  ATP-hydrolytic  127  were  Ca^ -ATPase  thesis,  the  kDa fragment and  fragments  they were p r e t r e a t e d with c a l p a i n i n the absence  kDa were  calmodulin,  prominent  25  the  25).  of  124  (Fig.  presence of calmodulin.  in  and  127  was p o s s i b l e to s e p a r a t e c a l p a i n and c a l m o d u l i n from  the p r o t e o l i p o s o m e s a f t e r or  and  a l s o found t h a t the  whether  it  23  calmodulin  o c c u r r e d on the r e c o n s t i t u t e d C a (Sephacryl S - 2 0 0 ) ,  kDa  activation  calmodulin  examine  124  membrane-bound or  presence  It  the  c a l m o d u l i n , whereas  stimulated  interest  of  the  calpain  20 and 2 1 ) .  sensitive was  formation  Furthermore,  195  since unlike  the  c  of  -ATPase  plasma membrane-  bound o r p u r i f i e d  enzyme  calmodulin)  and  the  85  calmodulin)  were  only  formed  in  1iposome-reconstituted  enzyme  (Fig.  kDa  fragment  formed  (Fig.  127  kDa  in  active  80  kDa  fragments  small  the  the  quantity  33),  it  absence  (in  (in from  the  the  absence o f  presence  proteolysis  may be c o n c l u d e d t h a t  of  calmodulin  was  and c a l m o d u l i n - i n s e n s i t i v e C a - A T P a s e  kDa fragment  formed i n the  of  of  the  the  124  the  2 +  proteolytically-activated whereas the  23 and 25),  major  fragment,  presence o f c a l m o d u l i n was  the  9+ t T  major C a - A T P a s e fragment which r e t a i n e d  5.  Comparison o f experimental  results  calmodulin-sensitivity.  from t h i s  laboratory  and from  other  1 aboratories  The l a b o r a t o r y  of  Carafoli  2 +  C a - A T P a s e produced by t r y p s i n this  work,  see  concluded  that  activated  by i t  a"90  neither  Benaim et  al.,  al.,  C-terminal 1987).  sequence at essential  43).  this al.,  was  From  binds  stimulated  end i s  important  by they  for  calmodulin  (Zurini  proposed t h a t  et  the  9  kDa  (calmodulin-binding  not  found t h a t while  al_.,  an  1984;  (Carafoli  contains  domain)  calmodulin s t i m u l a t i o n .  197  at et  a 5 kDa  and a 4 kDa  s i d e of the c a l m o d u l i n - b i n d i n g domain, which  the e x p r e s s i o n of the  81  work)  a 9 kDa sequence  segment  in  they  is  in t h i s  calmodulin-stimulation  proposed t h a t  it  hand, they  by c a l m o d u l i n ,  calmodulin  the  evidence,  calmodulin,  other  of  to the 86 kDa  circumstantial  1984). On the  Therefore,  binds  kDa fragment  be e q u i v a l e n t  fragment  1984)  also  an 85  to be the same as the 82 kDa fragment  the C - t e r m i n a l  for  ( l i k e l y to  can bind and be s t i m u l a t e d  nor  They  sequence which  and  (Benaim et  ( likely  bound  28  although  kDa fragment  kDa fragment  the  Fig.  has i d e n t i f i e d  is  By comparing  the  fragmentation  their  proposal  Ca-ATPase for  (produced  thesis  do not  clearly  show  stimulated  al.  rapid loss not  the  kDa In  reported calpain  (i)  and  kDa)  fragmentation  fact,  in  45")  it  clear  the  Ca -ATPase  2 +  of  kDa  the  domain  domain  the  report.  are  such is  their  likely  kDa  34 do  and  82  Since  from  and  kDa  bind  did  beyond the  124  2-fold  after  240 min o f  1987). T h i s drop o f the  formation  of  Ca -ATPase  the found  C-terminal  and  is  Carafoli  et  not  of  i n the  c o n c l u s i o n s about incorrect  because:  calmodulin-stimulation prominent resolved  calpain-treatment  activated  being l i k e l y  calmodulin-sensitive from the  124  124  125  kDa fragment  the  observe  present the  work  effect  (i)  even  in  limit  (since  the  From  the  (Fig. of  (Fig.  7 of  kDa  fragment,  contributed kDa easily  198  to the  did they  produced a  end).  c a l m o d u l i n - s t i m u l a t i o n was l i k e l y  an  are  kDa fragment,  2 +  the  5  fragments,  44  and  c a l p a i n on  their  work,  the c a l m o d u l i n - s t i m u l a t i o n was a c t u a l l y d e c r e a s e d from the o r i g i n a l to  this  bind  c l e a v a g e i s at the N-terminus at  essential  proteolysis  they  the  Table  also  the  of  and  Interestingly, on  44),  by c a l p a i n treatment and as they  fragments  Ca -ATPase  both o f  125  results  same  Fig.  fragments  27 and T a b l e 3 ) .  2 +  the  3,  results  half-truncated,  preliminary  and ( i i )  of  85  c a l p a i n cleavage of  calmodulin-binding  is  127  27,  (Fig.  4 kDa sequence  Table  this:  accumulation  (124  the  calpain  kDa fragments  Fig.  concluded t h a t peptide  have l o s t  of calmodulin-stimulation  observe  kDa and 85  support  calmodulin.  by  and f o r  experimential  also  Ca^- -ATPase  127  43)  However,  by c a l m o d u l i n ( F i g .  (1987)  the  by c a l p a i n )  calmodulin-binding  stimulated  (Fig.  stimulation.  that  by  the  trypsin  would mean t h a t  calmodulin  having  models f o r  Carafoli  6-fold et  al.,  have been due remaining  to  2-fold  by the presence o f a l e s s  fragment, on t h e i r  which  would  SDS-PAGE  not  be  system;  and  (ii)  in t h e i r  not  obvious,  less  than  Carafoli limit  work, the  the et  involves  the  124  kDa fragment  starting  amount  of  the  136  kDa  1987),  a  further of  for  continuous  Fig.  25 and  28).  It  worth  activating published  (Wang  the  small  of et  This  the  that  al_.,  membranes  that  color  glycodeoxycholate. removed  remaining  author  against  also  could  membranes w i t h the d e t e r g e n t s  (1987)  treated  activation.  by  with been  not  did  the  of a  not  silver-  this  to  method  photographic  the development  gels  were  problem  the  stained  mentioned  results  (see  on  z +  Ca -ATPase  the were  reported  that  the  pig  calpain  also  from  pig  that  the  calpain-mediated  2 +  Ca -ATPase the  was  detergents  because that  significantly The  was  were e a s i l y observed  best  calmodulin work  and  protected  removed the  seen  saponin was  or not  therefore the  subsequent treatment  c o u l d have t h e r e a f t e r  199  the  before  claimed  have  Since  of  membranes used i n  could  used.  membrane-bound  Au  have  likely  potential  membrane-bound  first  from the  proteolytic  most  7  they  was  far  (Fig.  kDa fragment  (analogous  have the  the  protein  p e p t i d e bands i f  activated  the  This  calmodulin  on  produced was  reason t h a t  they  immediately  was  were  The  thesis,  1988a),  of  124  s m a l l e r fragments  calpain  activation  exhaustively  this  intact  fragments  of  which does not  Ca -ATPase  erythrocytes.  ATPase  In  2 +  erythrocyte  after  these  noting  effect  proteolytic  degraded.  development  and c o n s e q u e n t l y  is  the  one can e a s i l y miss f a i n t e r  Comassie B l u e ,  above,  that  gel-staining  stopped p r e m a t u r e l y .  with  the  indicating  and was  method  development), is  of  formation  impregnation  124 kDa fragment was  amount  al.,  the  beyond the  total  fragment  observe  although fragmentation  Ca of  2 +  -  the  remaining  c a l m o d u l i n and enhanced the Alternatively, difference  susceptibility  the d i f f e r e n c e  (pig  versus  of the  Ca*- -ATPase to  calpain.  i n s u s c e p t i b i l i t y c o u l d be due to  human)-i.e.  the  phospholipid  a species  composition  and/or  organization  o f the enzyme i n the l i p i d b i l a y e r c o u l d be d i f f e r e n t ) .  the  of  results  fragmentation (Wang et  the  molecular  and p r o t e o l y t i c  al.,  1988b),  Au  the  by i n c u b a t i n g  presence  activity  of  activation  et  time u s i n g human e r y t h r o c y t e found t h a t  characterization  the  al.  p u b l i s h e d another  erythrocyte  c o u l d be s t i m u l a t e d .  membrane-bound  at  mM f r e e  They a t t r i b u t e d  might  2 +  Ca -ATPase that  Ca  this  observed  30°C f o r 2 +  ,  it  membrane-bound as w e l l was demonstrated  pTasma membrane Therefore, cytosolic  in  the  that  et  2+  a  Ca -dependent  importance  calpain,  t h e s e authors f a i l e d  as  of  suggested  1988a; or F i g . 18C o f t h i s t h e s i s ) ,  presence  of  EDTA-containing depleted  from  calcium). buffer  et  al.  these  membranes.  et  purified  both plasma  isolation, Therefore,  the and by  1986), to  the  1985b).  membrane-bound  versus  is  (Fig.  the a c t i v a t i o n  200  1988a)  al..  (1989)  i n our work  Furthermore,  during  on  attached  of  obscure.  Also,  3 o f Wang et  al.,  2 +  the C a - A T P a s e was  dependent on the c o n c e n t r a t i o n o f added c a l p a i n d u r i n g the the  effect  o f the  (Pontremoli  distinguishing  t o observe t h a t  to  ( G o p a l a k r i s h n a and B a r s k y ,  manner  by Au  totally  al.,  to be  s o l u b l e c a l p a i n can be r e a d i l y  in  Ca -ATPase  activating (Wang  They  30 min  activation  is true that calpain i s l i k e l y as i n the c y t o s o l  this  2 +  the  be due t o membrane-bound c a l p a i n and be independent  c a l p a i n added. While  it  we  ,  2 +  membrane  0.4  report  source o f C a - A T P a s e .  membrane-bound c a l p a i n . They a l s o suggested t h a t the the  calpain-mediated  2 +  (1989)  and  the  o f the C a - A T P a s e were p u b l i s h e d  membrane as the  5 mM c y s t e i n e  of  After  membranes  incubation  are  calpain incubating  washed  is the  (in with  apparently membranes  without  added  dithiothreitol  protease  at  and  11M f r e e  200  expressed the t y p i c a l absence v  m o c  max  of '  e  Fig.  19A,  this  thesis,  the  this  activation  of  they  which  is  activating  isolated  distinct  characterizing Conigrave,  min  the  this  type  high  Ca  2 +  2 +  Ca -ATPase  affinity,  4A o f Wang et  membrane  co-purified  effect (i)  (ii) the  t h a t Au et residual  al.,  of  Ca  1988a or in  membrane-bound c a l p a i n  to  preparations  activation Our  of  the  laboratory  (Roufogalis,  did  not  require  2 +  .  This activation  dithiothreitol  has  been  B r z u s z c z a k , Xu,  s o l u b i l i z a t i o n o f the C a - p r e t r e a t e d  After  the  experimental  i n s i d e - o u t v e s i c l e s (I0V)  work  of  made from  this  and  confirmed presented 1989d).  rendered  the in  this  result thesis  These authors  it  thesis  was  human e r y t h r o c y t e  (Fig.  also  the 30  completed,  membranes,  the  found t h a t  201  36;  see a l s o  a 125  kDa  by  using  Papp  et  2 +  Ca -translocating This  report  Ca -ATPase  Wang et  fragment  thus  2 +  1iposome-reconstituted to  reversed  2 +  calmodulin-independent.  with  blocked  Ca -ATPase.  (1989) showed t h a t c a l p a i n treatment a c t i v a t e d  function  was not  and was r e a d i l y  2 +  subsequent  al,.  Ca  preparation).  enzyme  37°C  o f 0 . 5 - 2 . 0 mM f r e e  in  observed can be  a c t i v a t i o n was observed a f t e r membranes were t r e a t e d f o r 30-60 min at  by l e u p e p t i n ,  manuscript  (1989)  On the  c a l p a i n bound t o the membrane  calpain.  activation  high  this  presence  and Wang,  2 +  of  the  used  al.  calcium-induced  effect  of  Typically,  i n the  Machan  and  from  presence  mode when assayed i n  m a x  the  i n the  the  membrane-bound  low V to  in  t h a t was observed would be n e g l i g i b l e .  as a combination o f  that  ,  shifted  Therefore,  contribution  the  interpreted  2 +  Ca  30-60  affinity,  and was  thesis). the  hand,  2 +  for  a d d i t i o n of calmodulin ( F i g .  proteolytic  other  low C a  calmodulin,  ky the  25°C  al.,  (likely  1989a; to  be  equivalent  to the  124  kDa fragment  in t h i s  work)  o f the  r e s p o n s i v e to a c i d i c p h o s p h o l i p i d a c t i v a t i o n  , although  to  bind  calmodulin  apparently This,  directed  again,  (Fig.  activation  44),  is  to  the  Papp et  domain  untreated  (both  al.  fragment  originating  thesis,  from  a  starting the  the  with  a  of  (Fig.  not supported by t h i s  recently,  but  intact  43  4A  of  Papp  that, in  the  form  and  the  solublized  kDa fragment  was  James et  al.  (1989)  reported  calmodul i n - b i n d i n g  However,  kDa EP-  2 +  and  their  as  in  the  in  Ca -ATPase  to  two  fragments  of  that  this  protein  purified  form),  observed  after  speculations  the  calmodulin-binding  124  kDa  fragment  and  the  are  absence  2 +  about  domain a  in  C a - A T P a s e (138 124  i n the absence o f c a l m o d u l i n , c a l p a i n f i r s t of  DISCUSSION  present  still  Therefore,  acidic  thesis.  by c a l p a i n  middle  the  its  interpreted  1989).  EP-forming  1989).  fragmentation  see  already  c a l m o d u l i n , the membrane-bound and the p u r i f i e d converted  al.,  leave  was  et_al_.,  25 and 2 7 ) .  antibody  have most o f  they  kDa  22,  will  still  insensitive  c a l p a i n - p r o d u c e d 81  136  80  of  and  single  the  (Papp et  which  that  was  monoclonal  would  (Fig.  fragment  it  model  kDa fragment  Ca -ATPase,  kDa  (Fig.  formation  c a l p a i n treatment  124  2 +  the  90  a  proposed  (1989) a l s o observed a  membrane-bound  significant  to  truncated domain  of  membranes  Most  with  which suggests t h a t the  phospholipid-interacting  forming  failed  c a l m o d u l i n - b i n d i n g domain  consistent  calmodulin-binding  earlier).  and  enzyme was  14  kDa.  They  of  kDa) were also  found  2 +  c l e a v e d the C a - A T P a s e and  thereby  kDa  produced  fragment.  A  a  second  c l e a v a g e c l o s e to the N-terminal o f the c a l m o d u l i n - b i n d i n g domain produced a  non-calmodulin-binding  124  kDa  fragment.  202  Using  a  synthetic  peptide  c o r r e s p o n d i n g to the  major  the  calmodulin-binding  cleavage  absence and i n the  sites  (indicated  by  presence o f c a l m o d u l i n  ± CaM  were  (James et  cleavages  are  the  at  the  calmodulin-binding which  as observed i n t h i s  produce  the  si/  \iv  v]/  L H F R G L N R  I QT-Q I K V V H  agreement  calpain  I  appears with  that  the  selective  and  Hinds  proteolysis  o f Ca^- -ATPase a c t i v i t y  the  scheme  I proposed i n t h i s  Vincenzi  the  of  kDa,  kDa  A F S S S  al.  fragmentation  thesis  (Fig.  (1988)  have  the  by them  125  and  127 kDa  work o f James et of  127  observed  thesis:  125 kDa  it  in  1989):  domain  124 kDa  Therefore,  different  that  + CaM '  same cleavages  124 kDa f r a g m e n t s ,  L R R G Q  al.,  identified  L W F R G L N R I Q T Q I K V V N A F S S S  These t h r e e likely  they a l s o  arrows)  - CaM  L R R G Q I  in  domain,  44 and  also  of  independently  2 +  a s s o c i a t e d with e r y t h r o c y t e  S i g n i f i c a n c e o f t h i s work  1.  P h y s i o l o g i c a l and p a t h o - p h y s i o l o g i c a l  203  the  is  generally  2 +  Ca -ATPase  by  45).  C a - A T P a s e by c a l p a i n  II.  (1989)  —  proposed  that  c o u l d cause the  loss  aging.  significance  Since present  both  in  ATPase  and  vivo.  It  activate  the  Ca-ATPase  erythrocytes, the  loss  the  of  Ca  Ca*- -pumping  2 +  calmodulin here  transport  ATPase  of  that  function  human  erythrocytes  If  hydrolyzes response  ATP  to  at at  a a  certain  be e l e v a t e d ,  state  rate,  stimuli,  2 +  persists,  i n a s s o c i a t i o n with  et  al.,  pathophysiological  pump.  1981),  the  However,  aging  conditions,  below  trigger  the  (Larsen  render  cell  pumps  its  intracellular  bind and a c t i v a t e perhaps  can  occur  2 +  -  j_n  effectively  activity these  Ca  of  the  activities  t o the  2 +  maximum  living  outward  and  capacity.  In  calcium concentration  may  c a l c i u m / c a l m o d u l i n complex if  the  mechanical of  Ca  a c t i v a t i o n was p r e v e n t e d by the  the  well  the  and would normally Ca  I  enzymes  the  indeed  calpain  and  of  these events can be e x t r a p o l a t e d  resting  slow  may  and ATP h y d r o l y t i c  presence o f c a l m o d u l i n . 46),  -dependent  activation  purified  Such i r r e v e r s i b l e  (Fig.  Ca  sensitivity  calmodulin-independent.  cell  are  calpain-mediated  its  was demonstrated the  and c a l p a i n  the  elevated  calcium  s t r e s s on the  red  cells,  c a l p a i n may be a c t i v a t e d  red  to  level cells  or  other  and s u b s e q u e n t l y  9+ activate against  the the  Ca  t T  pump  uncontrolled,  which would o t h e r w i s e might  also  amounts  of  irreversibly,  prevent it  elevated  be l e t h a l the  to  a c c e s s i b l e to  the  action  as  intracellular the  irreversible  are  possibly  the  a  defense  calcium  concentration  cells.  Intracellular  action  of  Ca  2 +  pump  calpain at  these  mechanism  calmodulin if  sufficient  elevated  Ca  2 +  concentrations.  Moreover, absence  or  decrease  of  9+ membrane Ca^ -ATPase o f  of  calpain  calmodulin some c e l l s ,  on the  Ca  stimulation such as  204  2 +  pump c o u l d e x p l a i n observed  in  dog e r y t h r o c y t e s  the  the  plasma  (Schmidt  et  Fig.  46  Proposed model  for  the  pump by c a l m o d u l i n and c a l p a i n by the  filled  circle.  dual  control  in a l i v i n g  For d e t a i l e d  of  cell.  explanation,  205  the  plasma membrane  Calmodulin i s see t e x t .  Ca  -  represented  al.,  1985),  (Olorunsogo  or et  al.,  Interestingly, rats  erythrocytes 1985;  both  the  Vezzoli  the  calpain  (Pontremoli 1987b).  et  al.,  erythrocytes  et  level  al.,  This  imbalance calpain  proteolysis  the  of  d y s t r o p h y (DMD) platelets  implicated A recent  and  of  2 +  preliminary  al_.,  higher (Moses  c o u l d be a r e s u l t  fact,  report  et  al.,  certain  state(s)  in  1984).  1983).  hypertensive inhibitor)  (control)  Pontremoli  might  In  rats  et  result  produce  cells.  al.,  in  an  significant  Duchenne  muscular  was h i g h e r i n muscle as w e l l  In  fact,  from  calpain  al.,  1980;  has  Ca -ATPase  DMD p a t i e n t s elevated  long  Imahori,  2 +  that  Such  i s not  2 +  involving  al.,  Milan  1986c;  animals  been 1985).  activity  than  in  2 +  Ca -ATPase  as  was  normal activity  activation.  Ca -ATPase  has  (see  the  or  et  Milan  might  these  1987).  2 +  of  al.,  (see S u g i t a et  Ca -ATPase  proteolyis  of  normal  which  erythrocytes  smooth muscle among o t h e r s mediated  et  demonstrated  of calpain  the  membrane  in  Orlov  kidneys  as  calpain activity  et  1985;  calpain-calpastatin  Ca -ATPase  patients,  patients  (an endogenous c a l p a i n  same  Pontremoli  i n DMD p a t h o g e n e s i s  erythrocytes  plasma  the  activity,  (Rabbani  significantly  In  was  1986b;  overexpressed  2.  hypertensive  had d e c r e a s e d l e v e l s o f c a l p a s t a t i n  while  in  from  Ca  unique  been  to  erythrocytes;  demonstrated  P e n n i s t o n , 1983). 2 +  pump  could  in  a  heart,  Therefore,  conceivably  similar  brain  the  and  calpain-  occur  during  these t i s s u e s o r c e l l s as w e l l .  U s i n g c a l p a i n as a t o o l  Due t o  the  characteristic  fragmentation  207  pattern  of  the  2 +  Ca -ATPase  produced by c a l p a i n , absence  versus  important  the  presence  tool,  chymotyrpsin), ATPase  in p a r t i c u l a r ,  (see  along  in  Fig.  44  and  about the  trypsinization  studies  Using  the  of  23  and  and  25)  calpain useful  (Fig. in  45).  other  the  It  calpain  overall  also  o f the  of  helps  39)  fact leads  distinguishing  that to  the  the  these  some  in  to  be  trypsin of  the  2 +  Ca -ATPases,  v i s u a l i z e d with example,  3 2  a P-EP  t h i s method  calmodulin-independent a reported  III.  quick  and  ambiguities  (see  2 +  -  or  r e l a t i o n s h i p s o f the enzyme  in  above).  2 +  the  Ca -ATPase  by  calpain  two  fragmentation  2 +  Ca -ATPase  as  z +  that  is  calpain  not  a  formation)  with  (Fig. by  treatment might  be  Ca -ATPases.  For  calpain-proteolysis  would help  might h e l p s e t t l e  the  plasma membrane C a  2 +  clarify identity pump  instance,  208  (which  the o f the  in l i v e r  m o l e c u l a r weight o f o n l y 110 kDa ( L o t e r s z t a j n  as c a l p a i n  substrates  of  hydrolyzed  2 +  major  pattern  produced by c a l p a i n  SR C a - A T P a s e  thinking  test  Calmodulin-binding proteins  an  Ca  the case o f a newly d i s c o v e r e d enzyme which resembles one o r both o f two  the  enzyme  plasma membrane  the  proved  (eg.  settle  calmodulin-induced d i f f e r e n t i a l  the  has  organization  the 85 kDa fragment)  pattern  fragmentation  proteases  structure-function  (eg.  means o f i d e n t i f i c a t i o n  proteolysis  with  proteolysis  The u n i q u e ,  calmodulin,  understanding  controversies  3.  of  the d i f f e r e n t i a l  et  in  these  can  situation.  be For  controversial, which a l s o has al.,  1984).  Kosaki et binding  al.  proteins  endogenous showing  (100  calpain  that  a  were degraded  erythrocyte  the was  I.  90  kDa,  Wallace set  by c a l p a i n  of  (103  preparations  the by  al.  (1987)  the  which c o - e l u t e d  2 +  from  Ca -ATPase  treatment  study  in  While  another  kDa),  this  proteins  Ca -ATPase  was a l s o found t o be p r o t e o l y z e d by c a l p a i n  lanes  Seiler  et  aj_.  (1984).  calmodulin-binding made ( T a b l e 6 ) . sixteen  have  (Wang e t  al.  reported  to  of  proteins  Of the  already  So f a r ,  be r e s i s t a n t  Nevertheless, calpain  has  reticulum  is  a narrow  calpain-resistant,  might  systematic  also  be  2,  3  to  to  calpain,  cyclic  be s u b s t r a t e s  although  nucleotide and  a  rather  range  of  it  4).  junctional  search  for  for  calpain i n  proteins  by  other was  in Table  was found t h a t  6,  vitro  have  been  one  form  p h o s p h o - d i e s t e r a s e was c a l p a i n -  Roufogalis,  striking  substrate  and  as r e p o r t e d  listed  in  column,  calpain-sensitive  no calmodul i n - b i n d i n g  Villalobo,  this  a  39),  30 c a l m o d u l i n - b i n d i n g p r o t e i n s  (1989b)).  (Wang,  which  been r e p o r t e d  calmodulin-dependent  resistant  Therefore,  (Fig.  from  component  S u b s e q u e n t l y , the c a l m o d u l i n - b i n d i n g c a l c i u m r e l e a s e channel o f SR  the  calmodulin-binding  as a minor  38A,  by  studying  a calmodulin-affinity (Fig.  by  platelets  2 +  calmodulin-activated that  calmodulin-  were degraded  confirmed  activation.  observed  calpain  several  kDa)  calmodulin-binding  was  + 97  fragmented  kDa and 40  upon p l a t e l e t  It  of  in p l a t e l e t s ,  60  et  proteolysis of  membrane,  adducin  also  kDa,  similar  calpain-mediated  protein  (1983) r e p o r t e d t h a t  finding  unpublished given  specificity.  the  results). fact  Many p r o t e i n s  that are  i n c l u d i n g bovine serum a l b u m i n , o v a l b u m i n , s a r c o p l a s m i c  9+ Ca-ATPase,  calsequestrin,  209  calmodulin,  actin,  ubiquitin,  Table 6  Matching o f CaM-bindinq p r o t e i n s and c a l p a i n  Protein  (a)  Myosin light chain kinase Ca -ATPase (plasma membrane) Phosphofructose kinase. Phosphodiesterase (cyclic nucleotide) Calcineurin Inositol 1,4,5 trisphosphate kinase CaM-dependent protein kinase I CaM-dependent protein kinase I I CaM-dependent protein kinase I I I Dynein ATPase 2+  Calpainsensitivity (Ref)  b  150 145(a),128(8), 45 (7), 17(5) 150 136 80 61,63  (1,2) (3)  N-D. + (34,35)  (4) (5,6) (7) (8-10)  + (36,37) + (38-40) N.D. + (37)  60(a),19(B) 53  (11) (12)  + (41,42) N.D. '  37-39 50(a),58(B),60(B') 140 400  (13) (14) (15) (16)  N.D. N.D. N.D. N.D.  (17) (18) (19) (20) (21) (22) (23) (24) (25)  (43) (44,45) (46,47) (47,48) (30) N.D. + (45) N.D. + (49)  (26,27) (28)  + (26,27) N.D.  (29) (30) (31) (32) (33) (33)  N.D. N.D. N.D. + (50) + (51) + (52)  Cvtoskeleton and structural proteins: Nebulin MAP-2 Fodrin (brain spectrin) Spectrin Caldesmon Microvillus 110 kDa protein Tubulin Tau factor Adducin  (c)  Reference* (as CaM-binding Protein)  Enzymes: Adenylate cyclase Phosphorylase kinase  (b)  Subunit M.W. (kDa)  substrates  300 270 240(a),235(B) 240(a),220(B) 150,75 110 50,55 55-62 97(a), 100(B)  Others: Calcium release channel 350 Regulatory subunit (type I I 50 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  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  p h o s p h o r y l a s e and cytochrome c (Zimmerman et  al.,  1985a;  Fig.  calpain-sensitive  25  group o f p r o t e i n s  the  five  (phosphorylase ATPase,  proteins  (Table  Calmodulin-dependent  Among  39,  this  thesis).  calmodulin-binding  cytoskeletal/structural  1.  and  kinase,  light  2 +  Ca -ATPase et  aj..,  chain  (Fig.  23  1989c)  calmodulin;  (ii)  absence  calmodulin  of  (Table  7).  (Cheung, et  al.,  1985),  1971;  fact,  myosin  light  and  kinase  kinase  II  (Levine  calmodulin-insensitive proteases,  such  in  of  al.,  et  identified  enzymes,  7  are  a miscellaneous  (i)  four  studies 1985),  and  as t r y p s i n  or  et  limited  the  revealed  (Walsh et 1983;  al.,  et  al..  that  al.,  are  and all  proteolysis It  -  patterns 1987),  1988;  Wang  presence  enzymes  enzymatic  2 +  their  al.,  the  five  Tallant  1984)  chymotrypsin.  212  et  and  Ca  in  Ito  (Tallant  respective  and Sahyoun, 1987) after  1983;  in  of the  activities  phosphodiesterase  phosphorylase k i n a s e  Klee,  (Zurini  of  membrane  the f r a g m e n t a t i o n  absence  out  their  plasma  enzymes  similarities  al.,  the  chain kinase  (Manalan z +  7):  and c a l c i n e u r i n  earlier  1979), myosin l i g h t  kinase,  calcineurin),  activates  plasma membrane C a - A T P a s e protein  16  calmodulin-dependent  chain  (Kosaki  different  K i n c a i d et  calcineurin  are  and the o t h e r 4 r e p r e s e n t  calpain-sensitive  proteolysis  In  the  Pontremoli  enzymes  and 25)  are  5  1984;  6).  phosphodiesterase  myosin  Of  proteins,  p r o t e o l y s i s by c a l p a i n are found (Table of  and S c h l a e p f e r ,  (Depaoli-Roach  1982; and  Foyt et  Cheung,  al.,  1984),  calmodulin-dependent activated by  and become  extracellular  seems l i k e l y  that  these  I.  Table 7  Effect of calpain on calmodulin-dependent enzymes  Effect of calpain on a c t i v i t y "  Enzymes  Phosphorylase  kinase  Myosin l i g h t c h a i n kinase  2+  Ca -ATPase  (plasma membrane)  N.D.  activated inactivated of CaM activated o f CaM  E f f e c t o f CaM on c a l p a i n a c t i o n "  & independent  & independent  p r e v e n t s l o s s o f CaMsensitivity p r e v e n t s a c t i v a t i o n and loss of C a M - s e n s i t i v i t y  Phosphodiesterase  a c t i v a t e d & independent o f CaM  No e f f e c t  Calcineurin  a c t i v a t e d & independent of caM  slows down the l o s s o f CaM-sensitivity  not  determined  see r e f e r e n c e s  i n Table 6  extracellular calpain.  proteases  The  enzymes  by  dependent  mimic  mechanism  limited  of  inhibitory  is  of  Upon  which  inhibition  of  the  case,  expresses the  its  and  other  active  enzyme  (Wang et  activity,  calmodulin,  calcineurin  On the  that  full  absence o f  ATPase  either  hand,  al..  calmodulin  active  Limited  is  relieved  is  dependent  of  of  calmodulin, their  et  al.,  modulate  conjugated hydrolyze  to the  difference substrates  is  system  target  that  (Rechsteiner,  proteins,  conjugated  calmodulin-binding  the  changes,  domains o r and  the  only  enzyme  plasma membrane  In Ca  2 +  -  fragments.  proteolysis  produces  calmodulin-binding  capacity  1988).  it  Therefore,  calpain-mediated  seems  proteolysis  of  proteins.  i n some ways analogous t o the  protease  or  proteolysis  calmodulin-insensitive  retain  1988b; T a l l a n t to  calmodulin,  and i n h i b i t o r y  proteolysis  presence  which  of  The e f f e c t o f c a l m o d u l i n on c a l p a i n p r o t e o l y s i s o f proteins  calmodulin-  i s no l o n g e r c a l m o d u l i n - s e n s i t i v e .  calpain  can f u n c t i o n  calmodulin-binding  follows:  activity.  inhibition  which  the  fragments 1988a;  the  produces  in  endogenous  calmodulin-dependent  binding  enzymatic  calmodulin-binding  In  as  by  domains undergo c o n f o r m a t i o n a l  e i t h e r removes both the former.  these  envisaged  domain.  and i n h i b i t o r y  the  proteolysis  a c a l m o d u l i n - b i n d i n g domain w i t h an a d j a c e n t  calmodulin-binding release  -dependent  activation  proteolysis  enzymes c o n t a i n  overlapping  Ca  protein,  protein, whereas  whereas the  ATP-dependent  are conjugated to u b i q u i t i n ,  2 1 4  of  1987a).  signals  modifies  effect  the  calmodulin-binding  ubiquitin Thus  on the ATP-  ubiquitin,  ATP-dependent  protease  calmodulin,  when bound  pattern  proteolysis.  of  proteolysis  when  occurs  to  to  only  the The when  c a l p a i n p r o t e o l y s i s o c c u r s both  in  the  absence  calmodulin and  76  and  in  and u b i q u i t i n  residues  conserved  the  to  Varshavsky,  for  an  presence are  1985).  small  ubiquitin)  exceptional  of  calmodulin.  proteins present  degree  Intriguingly,  (148-residues in  (Manalan  all and  calmodulin  eukaryotic Klee,  Both p r o t e i n s are m u l t i - f u n c t i o n a l  various biochemical processes in c e l l s .  for  1984;  and are  Furthermore,  cells  and  Finley  and  involved  ubiquitin  not p r o t e o l y z e d by ATP-dependent p r o t e a s e and, l i k e w i s e ,  both  itself  calmodulin is  in is not  p r o t e o l y z e d by c a l p a i n .  2.  Cvtoskeletal/structural  When that  are  these  not  are  enzymes was f u r t h e r  cytoskeletal  effect  proteins iff  the  proteins  the c a l p a i n - m e d i a t e d p r o t e o l y s i s o f c a l m o d u l i n - b i n d i n g  cytoskeleton The  and o t h e r c a l m o d u l i n - b i n d i n g  proteins,  it  including  was observed spectrin,  backbone and components o f m i c r o t u b u l e s  of  is  examined,  calmodulin  less clear.  absence  (Kosaki  et  adducin  (Wang,  and  al_.,  the  1983),  Villalobo  on the  proteolysis  No apparent presence erythroid  of  of  difference  in  calmodulin  was  spectrin  and R o u f o g a l i s ,  t h a t many o f  which  (tubulin  these  proteins  forms  and MAP-2).  calmodulin-binding  fragmentation found  (Seubert  et  for al_.,  pattern  caldesmon 1987)  unpublished r e s u l t s ) .  (Seubert  The f u n c t i o n might  be  instance,  to cell  et  al.,  o f the  induce  and  However,  c a l m o d u l i n i n c r e a s e s the r a t e o f d e g r a d a t i o n o f b r a i n s p e c t r i n ( f o d r i n ) calpain  the  by  1987).  proteolysis of cytoskeletal  cytoskeletal  or  membrane f u s i o n r e q u i r e s  215  cell  o r membrane  membrane  removal  of  proteins  remodeling.  some membrane  For  proteins  to p r o v i d e a l i p i d e n r i c h e d al.,  1983).  Lynch and  calcium  influx  calpain  and  environment  Baudry  upon neuronal induces  C o n s e q u e n t l y , the  (1984)  hypothesized  stimulation,  remodeling  initially  at the s i t e o f f u s i o n (Kosower  fodrin  of  the  l a t e n t glutamate  that  is  as  a result  of  locally  by  degraded  postsynaptic  membrane.  r e c e p t o r s i n the p o s t s y n a p t i c  membrane are exposed, which might c o n t r i b u t e t o the l o n g - t e r m observed i n t h a t microtubule of  the  study.  proteins  eukaryotic  B e s i d e s t h e s e p a s s i v e r o l e s o f the  may a l s o be i n v o l v e d  cell,  including  o r g a n e l l e s w i t h i n the c y t o s o l of  pigment  processes  or may  secretory be  as  of  potentiation cytoskeleton,  repertoire  of  individual  cells  m i t o s i s , axonal t r a n s p o r t  granules)  viewed  i n the  movements  (i.e.  (Bryan,  being  et  1974).  potentially  movements or  or movements  Therefore,  dually  of  these  controlled  by  as s u b s t r a t e s  for  c a l m o d u l i n and c a l p a i n .  3.  Substrate s p e c i f i c i t y of  Using calpain,  naturally  Sasaki  et  o c c u r r i n g and a r t i f i c i a l al.  c a l p a i n p r o t e o l y s i s at in  the  Val)  position  i n the  residue Very  Pj  in  (Hirao  (1984)  formulated  the  Pj  position  and T a k a h a s h i , (Sasaki  be r a t h e r  poor  preference  a Lys, Tyr,  et  cleavage at  (nomenclature  of  was  observed  using  1984).  However,  this  al.,  substrates  1984). for  216  residue  carboxyl  neuropeptides pattern small  does  as  not  for  residue (Leu  side of  S c h e c h t e r and B e r g e r ,  Furthermore,  calpain  the  rule  Arg o r Met  preceded by a hydrophobic amino a c i d  preference  the cases  peptides  a general  the c l e a v a g e s i t e :  p o s i t i o n would f a v o r  P2  similar  calpain  or the  1967).  substrates explain  all  p e p t i d e s are known t o  as compared to  protein  substrates  (Murachi,  1983b),  substrates  may not apply  (1987)  studied  found  that  Instead, of  vicinity  substrates.  of calpain  preference  P2-P1  hydrophobic the  or  boundary  This cleavage s i t e  rule  hydrophilic between  usually  susceptible  to  degradation,  calpain  although  This  finding  suggests  in  its  are  that  Sakai  et  peptides  not  if  i n h i s t o n e s and  are  large  to  i n t h e midst  clusters,  but  and hydrophobic that  in  the  clusters.  proteolysis  which  For  instance,  Sakai  still  contained  were p r e s e n t  are not et  certain  cleavage  induced.  is  poor  contain  bonds  in intact  recognizes  i n general  enough  followed.  fragments  them  they  before  al.  al_.  r e l e a s e d from h i s t o n e s underwent no  of  calpain  substrates  why small they  peptide  limit  cleavage.  many  be s u s c e p t i b l e t o c a l p a i n  since  from  necessarily  acid  hydrophilic  produces l a r g e r  would  explain  sites  not  amino  (1987) observed t h a t a l l the fragments  structure  Recently,  cleavage was  derived  s p e c i f i c i t y c o u l d account f o r the f a c t  induced by c a l p a i n  further  rule  found t h a t s u s c e p t i b l e bonds were never l o c a t e d  of  further  the p r e f e r e n c e  to protein  the s p e c i f i c i t y  the  they  either  and t h e r e f o r e ,  histones.  higher  order  This  would  substrates  the  which  for  required  calpain,  recognition  stte(s).  When t h i s  "recognition  binding p r o t e i n s ,  which are o f t e n  t h e r e may be c e r t a i n are  recognized  by  calmodulin-binding that  share  concept  calpain. domain  of  a high degree  is  substrates  common r e g i o n ( s )  the c a l m o d u l i n - b i n d i n g  proteins  site"  The these domains  applied  for calpain,  or features most  obvious  proteins.  to the  It  on these region  217  (Blumenthal  follows  that  proteins  that  would  has been  o f many d i f f e r e n t  o f homology  it  calmodulin-  be  the  demonstrated  calmodulin-binding et  a l . , 1985; Lukas  et  al.,  1987;  1986;  Wakim et  James et  al..  b i n d i n g domain segment  with  histidine  is  a  al..  1988,  Shull  thought  high  1987;  to  and Kennedy,  and Greeb,  be  percentage  and l y s i n e )  Bennett  a short of  (Blumenthal  1988).  amino  and K r e b s ,  a c i d i c calmodulin. kDa)  has  its  As d i s c u s s e d e a r l i e r ,  catalytic  domain  near  acid  is  interaction  the l a r g e  the  residues It  al.,  calmodulin-  alpha-helix  1988).  the r e s u l t i n g net p o s i t i v e charge f a v o r s the  L i n et  A typical  amphipathic  basic  1987;  peptide  (arginine,  believed  with the  that  strongly  subunit of c a l p a i n  N-terminal  region  and  has  (80 a  p, calmodulin-1ike  E-F  (Ohno et  al.,  1984).  calpain  can  bind  proteins. to  hand It to  Ca  domain  at  the  C-terminal  its  the  bound  calmodulin-binding  substrate.  cannot  account  proteins,  after  losing  s u s c e p t i b l e to f u r t h e r al_.,  region  i s c o n c e i v a b l e t h a t the c a l m o d u l i n - 1 i k e domain o f  mechanism  et  -binding  domain  of  Once b i n d i n g i s a c h i e v e d , the c a l p a i n c a t a l y t i c  cleave  Wang  c  for  However,  the  their  fact  that  This  some  prompted  us  al., to  attractive  calmodulin-binding domain,  1983;  look  substrate  domain proceeds  seemingly  calmodulin-binding  p r o t e o l y s i s (Kosaki et  1988b).  this  the  Ito  et  are  still  al.,  1987;  for  an  alternative  of  ten  short-lived  mechanism.  4.  PEST sequences i n c a l m o d u l i n - b i n d i n g p r o t e i n s  Recently, proteins  Rogers  examined  proteins  (T)  al.  (half-lives  enriched in proline threonine  et  (PEST  (half-lives  (P),  (1986)  l e s s than  glutamic  regions). between  found  acid  2 h) (E),  Similar  20 and 220  218  that  h)  each  contain aspartate  inspection revealed  one o r more (D), of  serine 35  regions (S)  more  t h a t o n l y 15 o f  and  stable these  c o n t a i n e d PEST r e g i o n s . by  its  PEST  P,E,D,S  score  definition)  can  range  PEST score  (which  and T and the  for  s c o r e > 0,  original  PEST r e g i o n , region. property  9 of  a strong  charged,  and  phosphorylation  of  charged  which  regions  the  such  t o the  substrates, share  it  was  sequence  namely,  PEST  to the  S  and  PEST  commonly  in  and by  that  many  it  sequences  in  sequenced  were  searched  for,  (Rogers  to  may  calpain.  PEST  'PEST-FIND'  that  addition  potential  program  PEST  least  regions  turn, al.,  In  be  order  to  calmodulin-binding using et  al..  2 1 9  a  PEST  1986).  very  this  the  both  negatively  local  1986).  are  test  this  may  domain, that  are  hypothesis,  that  found  in  calpain  proteins  regions  proteins  calcium  Therefore,  PEST  was  PEST  They a l s o  have  sequence-searching It  strong  confer  calmodulin-binding  these  the  phosphorylatable,  calmodulin-binding the  PEST  in  PEST sequence are  produce  In  a  1  c o n t a i n i n g them.  would  (Rogers et  with  have a s t r o n g  potentially  calcium.  those with a  t h a t many c a l m o d u l i n - b i n d i n g p r o t e i n s  homology  recognized  these  are  at  proteins  proteins  T  theoretically  Strikingly,  have  of  see Methods  and those  proteins  sequences  bind  postulated  regions,  region,  evaluated  percent  stretch,  PEST s c o r e s  E and D r e s i d u e s i n the  may  finding  PEST  that  c o n c e n t r a t i o n may a c t i v a t e c a l p a i n s relation  the  mole  PEST r e g i o n s e x i s t :  35 s t a b l e  concluded  h y p o t h e s i z e d t h a t s i n c e the  of  total  a weak PEST r e g i o n .  only 3 of  authors  the  While  short-lived  of rapid degradation  negatively  1986).  two types o f  10  of  hydrophilicity  indicating the  o f a PEST sequence can be  a reflection  al.,  indicating  whereas  These  et  t o +50,  > -5,  study  is  average  (Rogers  from -45  < 0 but  The s t r e n g t h  that  been  computer all  the  Table 8  PEST sequences  of calmodulin-binding  d  proteins  b  Protein  Residues  PEST Seauence >  Tau Factor (mouse brain)  111- 121  KTPPGSGEPPK  121-140  RSGYSSPGSPGTPGSR  307-338  HLSNVSSTGSIDMVDSPQLATL ADEVSASLAK  112- 135  KGEGAPDAATEQAAPQAPAPSEEK  8.1  167-209  KQADVPAAVTAAAATAPAAEDAAAMATAQ PPTETAESSQAEEK  3.5  225- 245  KAGAYDFPSPEWDTVTPEAK  2.7  (3)  226- 246  KAGAYDFPSPEWDTVTPEAK  2.7  (4)  430-451  KDYEEVGVDSVEGEGEEEGEEYend  13.7  (5)  396-443  HWYTGEGMDEMEFTEAESNMNDLV SEYQQYQDATAEEEEDFGEEAEEEEAend  10.5  (6)  2.4  (7)  Neuromodulin (bovine brain)  CaM-PK II (a) (rat brain) (B) Tubulin (a) (rat brain) Tubulin (fl) (human brain)  2+  Ca -ATPase (Human, plasma membrane)  64-80  KTSPNEGLSGNPADLER  10.6  (1)  5.1 -4.3  KEEIPEEELAEDVEEIDH  1158-1174  HIPLIDDTDAEDDAPTK  8.3  1175-1185  RNSSPPPSPNK  7.1  1202-1214  KSATSSSPGSPLH  1.6  (NO  PEST)  Hi stone 2B (calf thymus)  (NO  PEST)  a - Fodrin  Ref*"  1078-1095  Myelin basic protein (bovine brain)  RII of cAMP-PK (bovine heart)  PEST Score  45-89  RASTPPAAPPSGSQDFDPGAGLVADAVAD SESEDEEDLVPIPGR  93-103  RVSVCAETYNPDEEEEDTDPR e  (+101)-(+lll) HPESAEDLQEK  (human brain a-spectrin)  220  (2)  14.8  -  (8)  (9)  8.9  (10)  15.2 6.8  (11)  Table 8 (Cont.) Protein (Ref) MLCK-G (chicken gizzard)  MLCK-S (rabbit skeletal muscle)  PhosDholamban (rabbit skeletal  PEST Sequence  80-94  KPDPPAGTPCASDIR  94-127  RSSSLLSWYGSSYDGGSAVQSYTVEIWNS VDNK  -3.8  371-445  KVLFGTPEFVAPEVINYEPIGYETDMWSI GVICYILVSGLSPFMGDNDNETLANVTSA TWDFDDEAFDEISDDAK  -2.3  602-627  HFQIDYDEEGNCSLTISEVCGDDDAK  50-59  KQDPDPSTPK  175-201  PhosDhorylase kinase subunit (rabbit skeletal muscle)  >°  PEST score  . KPLSEASELIFEGVPATPGPTEPGPAK  1.4  Ref.  d  (12)  0.1 13.8  (13)  4.5  473-525  KTDMWSLGVITYMLLSGLSPFLGDDDTETL NNVLSGNWYFDEETFEAVSDEAK  -1.9  239-261  RMIMSGNYQFGSPEWDDYSDTVK  -5.1  (14)  (NO PEST)  -  (15)  RPSESNGQPEVTGEPVELK  7.4  (16)  muscle)  Gap Junction Protein (bovine lens)  d  Residues  241-260  a  P a r t i a l sequences are presented with amino acid l e t t e r code: A, alanine; C, cysteine; D, aspartic acid; E , glutamic acid; F, phenylalanine; G, glycine; H, h i s t i d i n e ; I, isoleucine; K, lysine; L , leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, v a l i n e ; W, tryptophan; and Y, tyrosine. b  PEST 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. C  PEST score is calculated as described by Rogers et a l . , (1986); in b r i e f , PEST score = 0.55 (MPpEST^ - 5 ( H ) , where M P p = mole percent of P,E,D,S and T after subtracting one mole equivalent of P,E and S, and H = 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 t s PEST score has to be larger than 0 to qualify as a PEST sequence. - 0  0  p  ST  Q  ^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) B u l l e i t 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 a r b i t r a r i l y assigned, for d e t a i l , see legend of Fig.4-7.  222  calmodulin-binding more  PEST  regions  proteins  sequences  are  (Table  generally  high  binding proteins  but  one  region  strong  PEST  but 8). (>  Furthermore, In  the  fact,  (with  PEST  erythrocyte  Ca  score  proteins  contain  PEST of  scores these  7-subunit)  > 0) in  date  all  one (phosphorylase k i n a s e ,  calmodulin-binding  gizzard)  sequenced t o  zero).  containing  c  three  (Table  Table  - A T P a s e , a - s p e c t r i n and myosin l i g h t  of  calmodulin-  Of  least  the  tubulin  PEST-  (a,  chain kinase  have been demonstrated to be fragmented by c a l p a i n  or  these  have at  8).  8,  one  /3),  (chicken  (Table  6).  The t h r e e c a l m o d u l i n - b i n d i n g p r o t e i n s t h a t do not have PEST sequences are  myelin  basic  Interestingly,  none  calmodulin-binding et  al..  1987).  stimulated  of  histone  the  domains  three  (Eylar  Worth mentioning  Ca*- -ATPase  shares  I IB  and  proteins  et  al.,  i s that  a high  1985)  Whereas the 8.3,  7.1  membrane 1988b), 1984;  the  and  (four  In  2 +  Ca -ATPase while 39,  of  to  Iwai  et  have al.,  8).  "typical"  1970;  Fujii  degree- o f  structural  (Verma et  lacks a typical  al.,  homology with 1988; MacLennan  c a l m o d u l i n - b i n d i n g domain.  s t r o n g PEST sequences (PEST s c o r e s 2 . 4 , them near  the  C-terminal  and one near  14.8, the  l a t t e r has o n l y t h r e e weak PEST r e g i o n s (PEST s c o r e s :  -4.9).  Fig.  however,  former has f i v e  and 1.6)  terminal), -4.0  which,  appear  (Table  the plasma membrane, c a l m o d u l i n -  s a r c o p l a s m i c r e t i c u l u m (SR) C a - A T P a s e al.,  phosphol amban  1971;  2 +  the et  protein,  the  support is  of  rapidly 2 +  SR C a - A T P a s e  this  thesis).  the  observed  hydrolyzed is  at  resistant  Furthermore,  the  correlation, 4-5  to  sites  calpain  first  few  (Wang  corresponding  many of  the  r e g i o n where  223  the  PEST  et  cleavage  end o f the plasma membrane  -3.4, plasma  (Seiler  are a l s o l o c a t e d at the C - t e r m i n a l to  the  N-  al.,  et  al.  sites  2 +  Ca -ATPase,  sequences are  found.  Thus,  the  strong  PEST r e g i o n s  the r e c o g n i t i o n s i t e s f o r  The the  1987),  two  respectively, that  From the  strong  neuromodulin  neuromodulin fragments  (Fig.  a-spectrin, (McMahon  38B).  repeats,  which  regions,  1988;  cTeave  a-fodrin  PEST  3).  It  to  be  Another t e s t  the  have  for  region  Harris  et  (240  Human at its  al., kDa)  the  the  c a l m o d u l i n - b i n d i n g domain  i s c o n s i s t e n t w i t h the of  brain  spectrin  sequences the  7th  to  in  by c a l p a i n  a-fodrin  15th  finding  (see  between for  based on the  repeats  the  Calpain I  (Harris  highly 11th  the al.,  basic  and  12th  (Harris  and II  of et  erythroid structures  calmodulin  the middle  and  appear  to  molecule,  to  1988).  Very  t o be 10 r e s i d u e s upstream from (Harris  al.,  partial  et  1987).  amino  (McMahon and Moon, 1987),  224  the  a  al.,  that calmodulin stimulates et  3.5,  elucidated.  repeat  possesses  47).  F i g . 47)  (Seubert  and  therefore,  been  like  106-residue  the c l e a v a g e s i t e was i d e n t i f i e d  al.,  Subsequently,  calpain  a-fodrin,  in  8.1  calpain.  has r e c e n t l y  junction  selectively  of  of  a-fodrin  a-fodrin  (Fig.  (Wakim et  predicted,  by  test  h y p o t h e s i s was human  high a f f i n i t y  1988)  first  several  produce a 150 kDa c a l m o d u l i n - b i n d i n g fragment recently,  may be  into  structure  twenty-two  1987).  the  scores  hydrolyzed of  was  was  a good s u b s t r a t e  accounts f o r  Morrow,  with  (Table  found  to  Moon,  Ca-ATPase  neuromodulin  amino a c i d sequence,  appears  calmodulin-binding  neuromodulin  sequence o f  whose m o l e c u l a r  partial  and  known  be  indeed  (brain s p e c t r i n ) ,  protein  identified would  was  Based on the  PEST  were  plasma membrane  calpain.  calmodulin-binding  hypothesis.  on the  acid  1988),  the  degradation  A search f o r sequence  revealed  which  that  PEST  covering the  only  Fig.  47  Localization  of  the c a l p a i n c l e a v a g e s i t e repeats,  o n l y the  tenth,  PEST r e g i o n s ,  eleventh,  are  1988).  the  complete  first  residue  available, residue  +1  downstream  the and  is  (towards  increasing positive away from r e s i d u e -1  and o n ) .  by the d o t t e d  illustrated  used the  as  twelfth  amino of the  acid  the  XII  sequence repeat  reference  C-terminal),  and  r e s i d u e numbers, whereas  1 have i n c r e a s i n g n e g a t i v e  The PEST r e g i o n area.  (residue  is  of  away  Harris  arbitrarily  et  by  al.,  is  not  assigned  Residues  from  and  proposed 22  a-fodrin  residue  moving 1,  have  r e s i d u e s moving upstream and r e s i d u e numbers  number +101  to +111)  by the arrow and the d o t t e d  (starts  is  (residue  A c a l p a i n cleavage s i t e  225  domain  (represented  1987;  residue.  The c a l m o d u l i n - b i n d i n g domain  is illustrated  Of the  and t h i r t e e n t h  (McMahon and Moon,  +5) i s shown by the shaded a r e a . -28 and -27)  calmodulin-binding  w i t h i n human b r a i n a - f o d r i n .  Roman numerals) Since  the  from  illustrated  number -18  to  (between r e s i d u e  line.  F i g u r e 47  X  XI  XII  XIII  (-28)<->(-27)  calpain cleavage -145  | i i  •  +1  +108  CaM-binding  PEST  (-18)+-+(+5)  (+101)+-*-(+111)  226  PEST  sequence  (PEST  r e s i d u e s downstream)  5.  score  6.8)  was  schematic  recognized highly  to the c a l p a i n c l e a v a g e s i t e  drawing  by c a l p a i n  hydrophilic  (Rechsteiner, molecules.  is  and  In  of  how  close  (Fig.  proximity  (128  47).  calmodulin-binding  presented  therefore  1987b),  PEST r e g i o n s  they  in  are  of  would  be  calcium-binding  catalytic  calpain  of  domain  of  activated  course  complex  easily  and a t t a c k s  distinct  tertiary  (near  from  structure  the  of  spatial  proximity.  calmodulin  cleavage  might  sites  erythrocyte  induce  2 +  binding proteins  calpain.  et  al.,  calcium  calpain  the  N-terminal  (Fig.  end  of  adjacent  cleavage  PEST  regions.  Also,  attack 23,  the  ions  25  PEST  at  the  end  the  (Fig. and  23 and 25,  site(s),  regions  48B),  as  45).  of  and  kDa which  of  the  cleavage  sites  this  binding  protect  certain  seen  that  fragmentation  to  the  with  the  Alternatively, are of  such  otherwise  calmodulin-  i n the absence versus  thesis;  calmodulin-binding  227  80  because  changes which  new c l e a v a g e  some  the  amino a c i d sequence i n o r d e r  Consequently,  For  calpain  (C-terminal  the  by c a l p a i n c o u l d be q u i t e d i f f e r e n t  1988).  to  loops  2 +  conformational  the presence o f c a l m o d u l i n ( F i g . Tallant  surface  are  with s u b s t r a t e - a s s o c i a t e d ! C a , the  proteins,  changes may expose to  form  some c a l m o d u l i n - b i n d i n g p r o t e i n s  calpain  Ca -ATPase  conformational inaccessible  from  In  be  E/D r e s i d u e s i n s i d e  localize  of  s i t e s need not be c l o s e by i n the primary be" i n  to  may  PEST r e g i o n s  accessible  charges o f  domain  proteins  Since  likely  calmodulin-binding proteins  Upon i n t e r a c t i o n  is  48.  very  the 80 kDa s u b u n i t ) .  subunit)  Fig.  the model, the n e g a t i v e  negatively-charged  of  in  R e c o g n i t i o n of c a l m o d u l i n - b i n d i n g by c a l p a i n A  are  located  Ito  et  al..  proteins  1987;  (eg.  a-  Fig.  48  Model  protein.  of  Schematic  calmodulin-binding and  calpain-mediated  subsequently  interaction  drawing  protein attacks  proteolysis  illustrating  PEST r e g i o n s cleavage  that  with  sites  1  of  its and  calmodulin-binding calpain  recognizes  Ca2+-binding 2.  (B)  domain  Upon  calmodulin  with the c a l m o d u l i n - b i n d i n g domain (shadowed r e g i o n ) ,  a conformational cleavage  site  calpain,  its  change or p h s y i c a l  (site  2)  substrate  from and  attack  o c c l u s i o n which p r e v e n t s by  calpain.  calmodulin  proportions.  228  are  Note not  that  drawn  (A)  there  is  the  adjacent  the  size  to  the  of  same  F i g u r e 48  229  fodrin),  the  binding  of  calmodulin  can  p r o t e o l y s i s o f the molecule by c a l p a i n controlling  the  simply  (Seubert  a c c e s s i b i l i t y of c e r t a i n  et  modify al.,  the  rate  of  1987), p r o b a b l y by  cleavage s i t e s v i a  conformational  changes. It  is  function might  solely  have  important the  p o s s i b l e that in  other  calpain  whether To  recognition.  in  as  yet  such f u n c t i o n s , of  for  the  substrates  their  calpain  this for  calmodulin-binding  Alternatively, In  this  function  this  the  PEST  case,  it  or  regulation  the  also  known  9),  was  amino  acid  Of these  while  41  12 do not  proteins, (Table  b i n d i n g enzymes calpain family  II, and  of  (L-type)  known  amino  and acid  (as d e s c r i b e d above),  calpain three  kinase  contain  inhibitor  receptor  protein  proteins  the  only  a weak  a-actinin. sequence large  are  (EGF r e c e p t o r ,  the  asked.  41  protein  29 have Of the  one 29  PEST s e q u e n c e s .  PEST  sequence  Among  subunit  (calpastatin),  230  question  10).  which  substrates  the  sequences o f  The  proteins  in  recognized  PEST sequences was  26 c o n t a i n one o r more s t r o n g  containing  help  be  calmodulin-  are  formulated,  contain  c a l p a i n were a n a l y z e d .  pyruvate  of  proteins  PEST-containing substrates,  vimentin,  will  substrates  sequences  substrates  question,  three  regions  would most c e r t a i n l y  calmodulin-binding  PEST  PEST sequences (Table  other  proteins  general.  hypothesis that  through  other  answer  o r more  the  in  unknown.  PEST sequences i n o t h e r c a l p a i n  After by  PEST r e g i o n s  functions  understanding  binding proteins  6.  calpain  to d e f i n e  further  the  five of  the  are  PEST-  calmodulin-  calpain  I  and  protein  kinase C  progesterone  receptor  Table 9  Protein  PEST sequences of PEST-containinq calpain substrates  Species used as calpain substrate (Ref. )  Species for a.a.sequence (Ref. )  human (1 ) & others  human (27)  Residues  PEST sequence  a  PEST SCORE  a  Calpain I (large subunit)  Calpain II (large subunit)  Calpastatin  295-322  RNPWGEVEWTGAWSDSSSEWNN VDPYER  517-548  KSAGTVELDDQIQANLPDEQVL SEEEIDENFK  3.3  288-315  RNPWGQVEWTGAWSDGSSEWDN IDPSDR  4.1  508-539  KQSDTAELDEEISADLADEEEI TEDDIEDGFK  14.8  150-197  KSGMDAALDDUDTLGEPSETQ EDSTAYTGPEISDPMSSTYIEE LGK  9.5  212-249  KTGVAGPPPDSVTPLGPDDAID ALSSDFTCSSPVASGK  4.4  496-533  KPLLPSEPTAQLPALSEDLLLD ALSEDFSGPSSASSLK  2.7  R  chicken (1 ) & others  chicken (28)  R  rabbit (1 ) & others  rabbit (29)  R  MLCK-G  chicken (2,3)  chicken  Ca -ATPase (plasma membrane)  human (4-6)  human (31)  (See Table 8)  Neuromodulin  bovine (7)  bovine  (See Table 8)  2+  (30)  (See Table 8)  (32)  231  4.2  Table 9 (Cont.) Species used as calpain substrate (Ref.)  Species for a.a.sequence (Ref.)  Tubulin (a)  cattle (8)  rat (33)  (See Table 8)  Tubulin (B)  cattle (8)  human (34)  (See Table B )  (a)-Fodrin  human (9,10) & rat  human (35)  (See Table 8)  PK-C  rat (11)  rat (36)  Protein  Residues  PEST sequence  PEST SCORE  a  (a isozyme)  (BI isozyme)  (BII isozyme) (Y isozyme) HMG-CoA reductase  Casein ( a ) s l  Casein (032)  Casein (B)  Vimentin  Chinese  Chinese  hamster (12)  hamster (37)  R  cattle (13 )  R  cattle (13 )  R  cattle (13 )  human (14)  cattle (38)  cattle (39)  cattle (40)  hamster (41)  276-302  KLLNQEEGEYYNVPIPEGDEEG NVELR  0.7  276-302  KLLSQEEGEYFNVPVPPEGSEG NEELR  3.5  276-302  KLLSQEEGEYFNVPVPPEGSEG NEELR  3.5  316-334  RMGPSSSPIPSPSPSPTDSK  381-395  KLSSVEEEPGVSQDR  4.2  429-442  RTQELEIELPSEPR  5.5  442-456  RPNEECLQILESAEK  58-79  KEMEAESISSSEEIVPNSVEQK  151-193  RQFYQLDAYPSGAWYYVPLGTQ YTDAPSFSDIPNPIGSENSEK  6-16  HVSSSEESIISQETYK  45-70  RNANEEEYSIGSSSEESAEVAT EEVK  11.0  1-25  RELEELNVPGEIVESLSSSEES ITR  6.6  113-134  KYPVQPFTESQSLTLTDVENLH  -2.8  406-419  RETNLESLPLVDTH  -2.8  232  15.8  -4.4 6.6  -1.9  2.2  Table 9 (Cont.)  Protein  Species used as calpain substrate (Ref.) rat (15)  Species for a.a.sequence (Ref.)  Residues  mouse (42)  72-105  RDLTIPVTEMQDPEALPTEQTA TDYVPSSTSTPH  11.1  245-272  HQPSLETQLYCGQAEGGSEGPS TSGTLK  1.1  362-383  RSLESFIDCSIVLPPTDAPSEK  0.4  910-929  KVTFDEENGLDEYDEVPMPVend  2.4  463-505  HCDVVNLTCEAC QEPGGIVVPP TDAPVSPTTLY VEDISEPPLH  0.6  687-728  ROEIVSYLCOLAPE APPPTLPPDMAQV TVGPGLLGVST LGPK  -3.9  905-948  RCCSGEGLQIPTLS PAPDCSQPLDVILLLDG SSSFPASYFDEMK  -3.7  1622-1666  KTQCCDEYECACNCVN STVSCPLGYLASTATND CGCTTTTCLPDK  -2.9  1947-1967  KIPGTCCDTCEE PECNDITAR  PEST sequence  PEST SCORE  a  Band 3 (anion channel)  von Willebrand human factor (16)  human (43)  1.5  Pyruvate rat (17-18) kinase (L-type)  rat (44)  104-118  REATESFATSPLSYR  -0.9  Factor V  human (45)  18-34  RPEPTNSSLNLSVTSFK  -2.3  420-439  HGVTFSPYEDEVNSSFTSGR  0.0  712-757  RNSSLNQEEEEFNLTALALENGT EFVSSNTD1IVGSNYSSPSNISK  0.7  799-820  HSSPYSEDPIEDPLQPDVTGIR  8.2  877-892  RPWFDLPSQDTGSPSR  10.2  894-907  RPV1EDPPSDLLLLK  -5.0  1044-1072  KSNETSLPTDLNQTLPSMDFGWI ASLPDH  -0.1  human (19)  233  Table 9 (Cont.)  Protein  Species used as calpain substrate (Ref.)  Species for a.a.sequence (Ref.)  Residues  PEST sequence  PEST SCORE  a  Fibrinogen (a)  Fibrinogen (B)  Kininogen  EGF receptor  human (20)  human (20)  human (21)  human and rat (22,23)  human (46)  human (47)  human (48)  human (49)  1072-1098  HNQNSSNDTGQASCPPGLYQTVP PEEH  3.8  1136-1151  KSFPTDISQMSPSSEH  4.4  1151-1182  HEVWQTVISPOLSQVTLSPELSQ TNLSPDLSH  1.9  1299-1335  HTTLSLDFSQTNLSPELSQTNLS PALGQMPLSPDPSH  2.3  1335-1477  HTTLSLDLSQTNLSPELSQTNLS POLSEMPLFADLSQIPLTPDLDQ MTLSPDLGETDLSPNFGQMSLSP OLSQVTLSPDISDTTLLPBLSQI SPPPDLDQIFYPSESSQSLLLQE FNESFPYPDIGQMPSPSSPTLND TFLSK  6.4  -  1572-1588  RETDIEDSDDIPEDTTYK  29-45  KDSDWPFCSDEDWNYK  252-268  RGGSTSYGTGSETESPR  461-491  KEVvTSEDGSDCPEAMDLGTLSG IGTLDGFR  217-234  KGGETSEMYLIQPDSSVK  380-391  RDNDGWLTSDPR  129-143  HPISTQSPDLEPILR  251-266  RDIPTNSPELEETLTH  379-388  KEETTVSPPH  10.7  505-523  HLASSSEDSTTPSAQTQEK  12.3  523-535 535-595  KTEGPTPIPSLAK KPGVTVTFSDFQDSDLIATMMP PISPAPIQSDDDWIPDIQTDPN GLSFNPISDFPDTTSPK  -2.3 5.0  237-260  KDTCPPLMLYNPTTYQMDVNPE GK  -2.9  234  19.3 1.7 11.0 0.0  -3.5 0.3 -2.9 9.2  Table 9 (Cont.)  Protein  Species used as calpain substrate (Ref.)  Species for a.a.sequence (Ref.)  Residues  PEST sequence  PEST SCORE  a  Progesterone receptor  Estrogen receptor  chicken (24)  cattle (25)  chicken (50)  human (51)  359-374  HTPPLDPQELDILK  -3.2  889-905  KPYDGIPASEISSILEK  -4.9  975-1008  RALMDEEDMDOVVDADEYLIPQ QGFFSSPSTSR  2.6  1044-1075  RYSSDPTGALTEDSIDDTFLPV PEYINQSVPK  4.4  45-69  RSSDEEEEEEEQEEEEEEEEPQ QR  34.4  169-179  RPGPEDASENR  8.7  257-278  RSSPSVPAAOLAEYGYPPPOGK  3.1  335-374  KAEPPLLPGAYGPPAAPDSLPS TSAAPPGLYSPLGLNGH  -0.8  400-419  RPDTETSQSSQYSFESIPQK  560-573  KPETPSSLLTSLNH  -1.0  303-335  KNSIALSLTADQMVSALLDAEP PILYSEY0PTR  -3.8  555-567  RGGASVEETDQSH  8.7  1.1  a-Actinin  chicken (26)  chicken (52)  137-153  KDDPLTNLNTAFDVAEK  -4.7  Neurofilament (light)  human (54)  cattle (53)  53-82  RSYSSSSGSLMPS LENLDLSQVAAIS NDLK  -4.9  456-472  HVQEEQTEVEETIEASK  477-491  KDEPPSEGEAEEEEK  28.4  516-531  KEEEEGGEGEEGEETK  26.2  495-509  KEEEPEAEEEEVAAK  18.4  Neurofilament (medium)  human (54)  cattle (55)  235  8.2  Table 9 (Cont.)  Protein  Species used as calpain substrate (Ref.)  Species for a.a.sequence (Ref.)  Residues  PEST sequence  PEST SCORE  a  Neurofilament (heavy)  human (54)  cattle (56)  544-555  KSDQAEEGGSEK  561-583  KEEGEQEEGETEAEAEGEEAEAK 22.4  776-790  KAGGEGGSEEEGSDK  10.3  66-95  RGAGAASSTDSL DTLSNGPEGC MVAVATSR  -4.4  452-476  KETVIVEEQT EETQVTEEV TEEEEK  17.7  502-514  KSPPAEEAASPEK  12.1  a  8.4  ( 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 F i g . IB; (8) B i l l g e r 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) DahlqvistEdberg & 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) U l l r i c h 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 a r b i t r a r i l y assigned, for d e t a i l ,  R  review a r t i c l e  236  see legend of F i g . 4-7.  T a b l e 10  Non-PEST-containing c a l p a i n  Protein  S p e c i e s used as calpain substrate (Ref. ) a  calpain (small  rabbit  & others  substrates  Species f o r a . a . sequence (Ref. ) a  p i g & human  (7,8)  subunit)  troponin  rabbit  (2)  rabbit  (9)  rabbit  (2)  rabbit  (10-11)  rabbit  (2)  rabbit  (12)  rabbit  (2)  rabbit  (13)  I  troponin T tropomyosin ( a ) tropomyosin (B) human  (3)  human (14)  hemoglobin (B)  human  (3)  human (15)  myelin basic protein  cattle  (4)  cattle  (16)  histone  IIA  cattle  (5)  cattle  (17)  histone  IIB  cattle  (5)  cattle  (18)  histone  III  cattle  (5)  cattle  (19)  hemoglobin ( a )  human  desmin  chicken  (6)  a  (20)  (1) Suzuki e t a l . , 1987b; (2) Dayton e t a l . , 1975; (3) M e l l o n i e t a l . , 1 9 8 4 ; ' (4) Banik et a l . , 1985; (5) Sakai e t a l . , 1987; (6) Nelson & T r a u b , 1981; (7) Sakihama e t a l . , 1985; (8) Emori e t a l . , 1986a; (9) W i l k i n s o n & Grand, 1975; (10) P e a r l s t o n e e t a l . , 1977a; (11) P e a r l s t o n e e t a l . , 1977b; (12) Stone & S m i l l e , 1978; (13) Mak e t a l . , 1980; (14) Konigsberg & H i l l , 1962; (15) B r a u n i t z e r et a l . , 1961; (16) E y l a r e t a l . , 1971; (17) Yeoman et a l . , 1972; (18) Iwai et a l . , 1970; (19) DeLange e t a l . , 1972; (20) G e i s l e r & Weber, 1982. ^ review  article  237  and  estrogen  proteins  receptor).  varies  as 34.6  (for  protein  kinase  PEST r e g i o n  isoenzyme the  PEST  there this  from 1 to  the C,  a  PEST  produced  by  on  (1989)  found  S i n c e the 7  PEST  these enzyme  II  the  have  single  the  a  end o f  Ohno  et  in  that  316-334,  at  14.0 (a,  relative  of  rates  calpain  the  of  of  I  which  score varies  and 15.8,  downstream unique  protein  kinase C  in  the  same o r d e r  Interestingly,  cholesterol  calpain  (Liscum et  among isoenzymes  HMG-CoA  biosynthesis, in adjacent  cleavage al.,  and the  sites  1985).  as  has  kinase et  isozymes  cleavage  a,  of  regions  rate  (the  two  about  The high  proximity  of  238  35  the  of  reductase,  B  strength  (Chin et  conservation  PEST sequences to  of for  and  al.,  two  about 1984),  kDa from the  of  II.  rate-limiting  PEST sequences being  kDa and 44  y  and  B  proteolysis  the  et  and  calpain  PEST-sequences  from the N-terminus) are  9),  C  al_.,  Kishimoto  the  its  the  Furthermore,  (Table  to  recently,  and 23:48:100 f o r  respectively  y-  causes  r e s p e c t i v e PEST score f o r the s i n g l e PEST sequence f o r a , 3.5  the  The  (Kishimoto  protein  of  1987).  Very  of  conserved  region)  3  9). y)  isozymes  residues  V  as high  C-2 r e g i o n  region,  identified  PEST sequences f o u n d .  the  substrate  highly  al_.,  the  B and  been  these  cloned  15  (Table  cleavage s i t e s  40 kDa and 47 kDa, r e s p e c t i v e l y ,  terminus  four  about  have  2:16:100 f o r  c a l p a i n cleavage s i t e s  w h i l e the  Of the  However,  isozymes  of  The PEST s c o r e s are  variation  (residue  and  that  isozymes. of  I  to  isozymes i s 0 . 7 ,  the  -5.  calpain  isozymes were about  1987,  a PEST s c o r e o f  calpain  proximity  al.,  below  three  Importantly,  were i n al.  with  at  of  regions  Factor V).  located  degree  fall  PEST  and /JII)  Bl  et  sequence  isoenzyme, sites  {a,  of  receptor).  276-302)  a higher  cleavage  12 ( f o r  (Kikkawa  score to  is  1989).  three  domain has  number  progesterone  (residue  regulatory  The  N-  PEST sequences  calpain  cleavage  sites  suggest  that  binding proteins  (Table  the  10).  non-PEST-containing PEST-containing two  separate at  definition  of  proteins  the  fact  sequences chain, other  that  in  sites  calpain  many  as  mostly  small  as t o why  these  substrates:  peptides  of  (a)  may  to  some p o t e n t i a l  that  definitely  substrates.  (Sasaki  et  The minor  PEST sequences be supported  not  have  are  i n s u l i n /5-  1984).  On  the  many n o n - P E S T - c o n t a i n i n g s u b s t r a t e s had a high number o f b a s i c amino  acid  protein),  w h i c h , by d e f i n i t i o n ,  Also,  strict  H,  the  K or  (H),  lysine  requirement  R sometimes  for  (K)  and a r g i n i n e  was  PEST  that  histidine  it  (b)  observed  residues:  second p o s s i b i l i t y ,  al.,  may be  may have  do  Among t h e s e  the  represent  PEST.  p o s s i b i l i t y seems t o  glucagon and dynorphin the  analogous  PEST s c o r e c a l c u l a t i o n  The f i r s t  calmodulin-  The non-PEST p r o t e i n s  perhaps  identification  of  calpain  substrates.  and the  small  support  are  two e x p l a n a t i o n s  serve  sequence(s),  affect  on  substrates.  least  serve as c a l p a i n  protamine,  recognition  non-PEST-containing proteins  PEST s c o r e s .  still  hand,  at  also  and  PEST r e g i o n s  f l a w s which might  by  are  proteins  other  and a l s o t h e i r  are  non-PEST-containing substrates  There  groups o f  recognized  regions  and some other c a l p a i n  Interestingly, proteins  PEST  (R),  (e.g.  p r e c l u d e s the e x i s t e n c e o f starting  significantly  myelin  basic  "PEST" r e g i o n s .  and ending a PEST sequence with  reduces  the  PEST  scores  of  a  highly  c o n c e n t r a t e d P E S T - e n r i c h e d r e g i o n which l a c k s a p o s i t i v e l y charged r e s i d u e to mark i t s  boundary.  this.  It  either  disrupted  potential  The 30 kDa subunit o f c a l p a i n  is  a good example  has r e g i o n s which are q u i t e r i c h i n P, D/E and S / T but which by H,  PEST r e g i o n  K or  R or  properly.  are Taken  239  lacking  these  together,  to  this  start  or  of are  end  the  suggests t h a t  the  restriction to  start  of  or  always be  the  end  requirement  a  sequence  important  calpain substrates. (Seiler  results),  dual  important  a  point  al.,  1984;  requirement  et  for  al.,  site  (e.g.  words,  fulfilling  recognition  During  PEST-containing proteins  are  according  Such cases  and ( i i )  the  the  study  of  the  2 +  the  1988b)  (calcineurin Therefore,  and B) the  the (Wang  acid  c o u l d be e x p l a i n e d  by a  the  its  requirement  or  t o the  do  not  calpain  some  of  (Dayton  et  al.,  as  yet  PEST r e g i o n .  In  susceptible  on  the  plasma  membrane  was observed t h a t both c a l m o d u l i n (Wang et  al.,  about  1975;  the  calpain  calpain  small  1989c)  subunit  were  c a l p a i n - s u s c e p t i b i l i t y of other  controversy  of  another  have  the  Ishiura  members o f  240  susceptibility et  al.,  of  resistant  s u p e r f a m i l y o f c a l c i u m b i n d i n g p r o t e i n s was i n v e s t i g a t e d been  unpublished  existence of a susceptible  which  calmodulin-1ike et  to  by c a l p a i n .  effect  it  insensitive  amino  (i)  to  P 2 - P 1 requirement  proteins  is  and R o u f o g a l i s ,  by c a l p a i n :  not be a t t a c k e d  C a - A T P a s e and c a l c i n e u r i n ,  has  residues not  R e s i s t a n c e o f the t r o p o n i n - C s u p e r f a m i l y to  al.,  acid  may  which i s i n c l o s e p r o x i m i t y  PEST-containing  cleavage s i t e s w i l l  protein  1987).  proteolysis  undefined r e s t r i c t i o n )  7.  calpain  Wang, V i l l a l o b o  P E S T - r e g i o n as a r e c o g n i t i o n s i t e  other  charged amino  c a l s e q u e s t r i n , which  PEST-containing  (Reithmeier  cleavage  for  i s t h a t not a l l  For i n s t a n c e ,  et  is  sequence  positively  justified.  Another  calpain  of  1979;  of  calcineurin to  the  (Fig  calpain. troponin C  40).  troponin  Murakami  There C  to  and U c h i d a ,  1979).  In  troponin  C was  found t h a t intact  o r d e r to c l a r i f y  calpain  (Fig.  members  of  calpain  I  the  with  the  (Fig.  40).  the  this  This  by  calpain; and  substrate,  to  1987).  appears  this  none  (target are  calpain (i)  many  calcineurin (iii)  (iv)  protein  bind to  8.  Future  the  substrate  In  A,  troponin  leaving  parvalbumin  these the  proteins  members  of  other  including (Fig.  is  of  was  troponin C  investigated,  and  of  it  is  the  40).  cleaved  the  by  troponin C  troponin  which I,  binds  which  belong to  Directions  241  protein 1985;  a large  troponin  h y d r o l y s i s of t h e i r  proteins  are  which  is  C  is  calpain belongs  McDonald et o f these  al., Ca  2 +  -  subgroup made up  C-like  respective  B,  troponin  the t a r g e t p r o t e i n s  the  from  a well-established  (McDonald and Walsh,  addition,  comes  calcineurin  binds  the  C superfamily  The e v i d e n c e  an 21 kDa i n h i b i t o r y  level.  proposed t h a t  calmodulin-binding  kinase-C,  calpain.  by c a l p a i n at the  also  substrates.  of  control  of  proteins)  may be c o n s i d e r e d to  p r o t e i n s might  while  further,  hypothesis i s c o r r e c t ,  of  was  protein  binding proteins substrates  I  and  It  the c a l p a i n - s e n s i t i v i t y  suggests t h a t  troponin C superfamily If  human e r y t h r o c y t e s .  I  calpain-resistant.  (ii)  calpain-sensitive;  t o the  S-100/J  observations:  substrates;  hydrolysed  study,  h y p o t h e s i s one step  proteins  from  C superfamily  prediction,  2 +  calpain  I  cleaved troponin  protein,  C a - b i n d i n g proteins following  calpain  rapidly  i n general  various binding  d i s c r e p a n c y , a mixture o f t r o p o n i n  To expand t h i s  S-lOOa  To take  with  troponin  s u p e r f a m i l y are  of  I  40).  oncomodulin, Consistent  treated  this  Ca  target  -binding proteins  In can  be  order taken:  proteins PEST  to  (a)  and o t h e r  regions;  proteins  test  potential  (b)  Also,  or  insensitive "important"  to  interest  find  to  containing  help  for  whether  the  loss of  in  its  out  the  and t o  whether  hypotheses  are  correct,  have  short  they  specificity  would  242  PEST r e g i o n s cleavage  half-lives the  are with  fragment  mutagenesis protein  would  proteins  of c a l p a i n  understanding o f p r o t e i n  a  It  form  directed  remaining  of  calpain.  calmodulin-binding  calpain  substrate  by  can be  Site-directed  sequence  their  calmodulin-binding  efforts  the  directions  identify  by i n i t i a l  render  PEST  proteolysis  for  in the f u r t h e r  PEST r e g i o n ( s )  two  calmodulin-binding  remaining  by c a l p a i n .  substrates  basis for  least of  calpain  Subsequently,  should  cleavage  residue(s) affect  sequences  substrates  proteases  further  predictably  at  the c a l p a i n c l e a v a g e s i t e s and the the  other  presented,  acid  are h y d r o l y z e d by c a l p a i n .  in proximity.  molecular  amino  determine  whether  proposed  hypotheses  obtain  to determining  calpain  the  also  should be  and o t h e r j_n v i v o . first  of  PESTIf  the  consistent  and e v e n t u a l l y  metabolism i n the  of  cell.  might  CONCLUSIONS  (1)  Calpain  I,  the  micromolar  protease  from the  apparent  homogeneity.  Ca  -requiring  human e r y t h r o c y t e The  cytosolic fraction,  purified  protease  m o l e c u l a r mass f o r both the l a r g e and small dependence,  thiol-reducing  agent  s p e c i f i c i t y of calpain  I.  (2)  isolated  Ca  Treatment c  -ATPase  with  activated  the  two ways:  (i)  and  (ii)  of  by  The  human  calpain  I  2+  i n c r e a s i n g the the  indistinguishable  V  to  subunits, and  erythrocyte (from  the  same  m  C a  )  of the  by  the  about enzyme.  activation  neutral  was p u r i f i e d  the  +  high a f f i n i t y C a ^ protease  inhibitor  membrane-bound  cells)  irreversibly  (ATPase) 2-orders This  activity of  activation  produced  by  was  calmodulin  2 +  Ca -ATPase.  calpain-mediated  activation  of  membrane-bound  2 +  Ca -ATPase  (4)  of  The presence o f c a l m o d u l i n d u r i n g c a l p a i n - t r e a t m e n t 2 +  bound C a - A T P a s e l a r g e l y  on the  prevented the c a l p a i n - m e d i a t e d  fragmentation  of  243  (i)  the  was  2 +  Ca -ATPase.  Studies  in  magnitude;  concomitant with the l o s s o f c a l m o d u l i n - a c t i v a t i o n o f the  (5)  to  characteristic  plasma  ATP-hydrolytic  K (  m a x  showed  requirement  Ca -dependent  increasing  b i n d i n g to the  (3)  purified  basal  by  kinetically  the  calcium-activated  the  membrane-  activation.  membrane-bound  form,  (ii)  the  s o l u b i l i z e d and p u r i f i e d  reconstituted calmodulin,  form  of  calpain  form and  z +  Ca -ATPase  transformed  (iii)  phosphatidylcholine liposome-  revealed  the  136  that  (a)  in  the  absence  kDa enzyme  into  two  major  kDa)  calmodulin-binding  active  fragments  (124  kDa and 80  calmodul i n - b i n d i n g  active  fragments  (125  kDa and 82  presence two  of  major  calmodulin,  z +  the  Ca -ATPase  calmodul i n - b i n d i n g  active  was  and two  kDa);  and  proteolyzed  fragments  of  127  by kDa  of  nonminor  (b)  in  the  calpain  into  and  kDa,  85  respectively.  The 124  (6)  kDa and 80 kDa fragments  o f the  z +  C a - A T P a s e have c a l m o d u l i n -  9 •  insensitive activation  (7)  Ca  -ATPase  activity  C a l p a i n - p r o t e o l y s i s of  but not which  z +  calmodulin-sensitive active  explained  the  protective  p u b l i s h e d primary  membrane  z +  Ca -ATPase,  calmodulin-binding  domain  m o l e c u l a r masses  fragments,  the  the  presence o f  fragments  active  effect  fragments  of  it  the  structure the  and  (127  proteolytic  (124  calmodulin  calmodulin  kDa and 85 kDa and 80  against  (amino  kDa) kDa),  proteolyic  identification the  a c i d sequence) o f a human of  acylphosphate  the  location  site,  and c a l m o d u l i n - b i n d i n g a b i l i t y  was p o s s i b l e to p o s t u l a t e  about 9 kDa from the C - t e r m i n a l kDa o f  to  o f the Ca*- -ATPase.  From the  estimated  contributed  the C a - A T P a s e i n  the c a l m o d u l i n - i n d e p e n d e n t  activation  plasma  and  o f the enzyme by c a l p a i n .  produces o n l y  (8)  c  that  calpain  I  2 +  end o f the C a - A T P a s e ,  c a l m o d u l i n - b i n d i n g domain  244  (corresponding  to  of  together of  first  the  with  various  cleaved  including  the  off  about 2  approximately  3.5  kDa),  p r o d u c i n g the  A further binding The  cleavage  domain  protected  the  condition,  to  kDa fragment  removed  of  (1  from  produce  a region the  85  kDa,  82  kDa and 80  Calpain-treatment  increased to  calmodulin.  This  the  124  kDa  largely  prevented  The  unique  levels  of  majority  of  characteristic  of  remaining 124  calmodulin-  kDa  fragment.  by  calpain.  domain  Under  was p r o d u c e d .  C-terminal  this  A slow  end was proposed  fragments  from  the  127  respectively.  phosphatidylcholine  to  rates  those  enzyme,  of  with  while  activation  1iposome-reconstituted Ca  obtained  was concomitant  the  127 kDa a c t i v e  a  attack  kDa a c t i v e  initial  proteolytic  enzymes and p r o t e i n s . the  the  activation  susceptibility  but  the  similar  fragment  forming the  (10)  of  the  calmodulin-binding  about 42-44 kDa from the  (9)  hydrolysis  of  calmodulin-sensitive.  127 kDa fragment  125 kDa and 124 kDa fragments,  2 +  still  the  proteolytic  kDa,  Ca -ATPase  kDa)  with  a calmodulin-sensitive  at  is  calmodulin-insensitive  calmodulin  latter  which  most  and produced the  association  cleavage  125  the  of  the  the  2 +  uptake  upon  and  ATP  addition  of  formation  presence  of  of  mainly  calmodulin  2 +  Ca -ATPase  activity  by  fragment.  the  2 +  Ca -ATPase shared  Furthermore,  by  primary  calmodulin-binding  to 15  calpain other  structure  proteins  have  s e q u e n c e s , which are proposed to serve as r e c o g n i t i o n  245  was  in  fact  not  calmodulin-dependent analysis one  or  sites  showed t h a t more  for  "PEST"  calpain.  BIBLIOGRAPHY  2 +  Adachi,  Y . , K o p a y a s h i , N . , M u r a c h i , T . and Hatanaka, M. (1986) Ca dependent cysteine proteinase, calpains I and II are not p h o s p h o r y l a t e d i n v i v o . Biochem. B i o p h y s . R e s . Commun. 136, 10901096.  Adamo,  H . P . , Rega, A . F . and Garrahan, P . J . (1988) Pre-steady-state p h o s p h o r y l a t i o n o f the human r e d c e l l Ca - A T P a s e . J . B i o l . Chem. 263, 17548-17554. ?  Akyempon,  C . K . and R o u f o g a l i s , B . D . (1982) The s t o i c h i o m e t r y o f the Ca 2+ pump i n human e r y t h r o c y t e v e s i c l e s : modulation by Ca , Mg a and c a l m o d u l i n . C e l l Calcium 3 , 1-17.  A l - J o b o r e , A . and R o u f o g a l i s , B . D . (1981) P h o s p h o l i p i d and c a l m o d u l i n activation of s o l u b i l i z e d calcium-transport ATPase from human e r y h r o c y t e s : r e g u l a t i o n by magnesium. C a n . J . Biochem. 59, 880888. A l - J o b o r e , A . , M a u l d i n , D . , Minocherhomjee, A . and R o u f o g a l i s , B . D . (1981) R e g u l a t i o n o f the c a l c i u m pump ATPase i n human e r y t h r o c y t e s I n : Erythrocyte Membranes 2: Recent Clinical and Experimental Advances (Kruckeberg, W . C . , E a t o n , J . W . and Brewer, G . J . , e d s . ) pp. 5 7 - 7 3 , Alan R. L i s s , New Y o r k . Al-Jobore, 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) A c t i v e c a l c i u m t r a n s p o r t i n normal and abnormal human e r y t h r o c y t e s i n : E r y t h r o c y t e Membranes 3 : Recent C l i n i c a l and Experimental Advances (Kruckeberg, W . C . , E a t o n , J . W . and Brewer, G.J., e d s . ) pp. 243-292, Alan R. L i s s , New Y o r k . Allen,  2 +  2 +  B . G . , K a t z , S . and R o u f o g a l i s , B . D . (1987) E f f e c t s o f C a , M g and calmodulin on the formation and d e c o m p o s i t i o n o f the p h o s p h o r y l a t e d i n t e r m e d i a t e o f the e r y t h r o c y t e Ca-stimulated ATPase. Biochem. J . 244, 617-623.  Allende,  J . E . (1988) GTP-mediated macromolecular i n t e r a c t i o n s : the f e a t u r e s o f d i f f e r e n t systems. FASEB J . 2, 2356-2367.  Ando,  Imamura, S . , Yamagata, Y . , K i t a h a r a , A . , S a j i , H . , M u r a c h i , and K a n n a g i , R. (1987) P l a t e l e t f a c t o r XIII i s a c t i v a t e d c a l p a i n . Biochem. B i o p h y s . Res. Commun. 144, 484-490.  Ando,  Ando,  Y.,  Y . , Imamura, S . , M u r a c h i , T . and activates two transglutaminases D e r m a t o l . Res. 280, 380-384. Y . , Miyachi,  Y.,  Imamura,  T. by  Kannagi, R. (1988b) Calpain from procine skin. Arch.  S . , Kannagi,  246  common  R.  and M u r a c h i ,  T.  (1988a)  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 o f c a l p a i n s from p i g e p i d e r m i s and t h e i r a c t i o n on epidermal k e r a t i n . J . I n v e s t . D e r m a t o l . 90, 26-30. Andreasen, T . J . , Luetje, C . W . , Heideman, W. and Storm, D.R. P u r i f i c a t i o n o f a novel c a l m o d u l i n b i n d i n g p r o t e i n from c e r e b r a l c o r t e x membranes. B i o c h e m i s t r y 2 2 , 4615-4618. Aoki,  (1983) bovine  K . , Imajoh, S . , Ohno, S . , E m o r i , Y . , K o i k e , M . , K o s a k i , G. and S u z u k i , K. (1986) Complete amino a c i d sequence o £ the l a r g e s u b u n i t o f the low-Ca - r e q u i r i n g form o f human Ca -activated n e u t r a l p r o t e a s e (/tCANP) deduced from i t s cDNA sequence. FEBS l e t t . 205, 313-317.  Au,  K . S . (1978) An endogenous inhibitor of erythrocyte ( C a + M g ) - A T P a s e . I n t . J . Biochem. 9 , 477-480. z +  membrane  z +  z +  A u , K . S . (1987) A c t i v a t i o n o f e r y t h r o c y t e membrane C a - A T P a s e by c a l p a i n . B i o c h i m . B i o p h y s . A c t a 905, 273-278. Au,  K . S . , Cho, K . L . , L e e , K . S . and L a i , K.M. (1985) An endogenous i n h i b i t o r p r o t e i n o f s y n a p t i c plasma membrane ( C a + M g ) - A T P a s e . B i o c h i m . B i o p h y s . A c t a 821, 348-354. z +  z +  z +  Au,  K . S . , H s u , L. and M o r r i s o n , M. (1988) human e r y t h r o c y t e band 3 p r o t e i n . 113-118.  Au,  K . S . , L e e , M . F . and S i u , Y . L . (1989) C a - m e d i a t e d a c t i v a t i o n o f human e r y t h r o c y t e membrane C a - A T P a s e . B i o c h i m . B i o p h y s . A c t a 978, 197-202.  Babu,  Ca -mediated catabolism of B i o c h i m . B i o p h y s . A c t a 946,  Z +  Y . S . , Bugg, C E . and Cook, W . J . (1988) T h r e e - d i m e n s i o n a l s t r u c t u r e o f c a l m o d u l i n , i n : Calmodulin (Cohen, P. and K l e e , C . E . , e d s . ) pp. 8 3 - 8 9 , E l s e v i e r , Amsterdam. Z+  B a l t e n s p e r g e r , K . , C a r a f o l i , E. and C h i e s i , M. (1988) The C a - p u m p i n g ATPase and the major s u b s t r a t e s o f the cGMP-dependent p r o t e i n kinase i n 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 C a l c i u m - s t i m u l a t e d p r o t e o l y s i s i n m y e l i n : evidence f o r a Ca - a c t i v a t e d neutral p r o t e i n a s e a s s o c i a t e d with p u r i f i e d m y e l i n o f r a t CNS. J. Neurochem. 45, 581-588.  Baron,  M . D . , D a v i s o n , M . D . , J o n e s , P . , P a t e l , B. and C r i t c h l e y , D.R. (1987) I s o l a t i o n and c h a r a c t e r i z a t i o n o f a cDNA encoding a c h i c k a-actinin. J . B i o l . Chem. 262, 2558-2561.  Barrett,  A . J . (1986) An i n t r o d u c t i o n t o I n h i b i t o r s ( B a r r e t t , A . J . and  247  the p r o t e i n a s e s i n : S a l v e s e n , G . , eds)  Proteinase pp. 3-22,  Elsevier, Barzilai,  Amsterdam.  A., Spanier, R. and Rahamimoff, H. (1984) Isolation, purification, and r e c o n s t i t u t i o n o f the N a gradient-dependent Ca transporter (Na -Ca exchanger) from b r a i n s y n a p t i c plasma membrane. P r o c . N a t l . A c a d . S c i . USA 8 1 , 6521-6525. +  z +  Beckerle,  Beer,  +  M . , O ' H a l l o r a n , T . , C r o a l l , D. and B u r r i d g e , K. (1986) The adhesion plaque p r o t e i n , talin, is related t o p235, a major substrate o f the calcium-dependent p r o t e a s e i n p l a t e l e t s . J. C e l l . Biochem. S10A, 259.  D . G . , B u t l e y , S . M . and J l a l k i n s o n , A . M . (1984) Developmental changes i n 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 o f mouse lung cAMPdependent p r o t e i n k i n a s e s . A r c h . Biochem. B i o p h y s . 228, 207-219.  Belles,  B . , H e s c h e l e r , J . , T r a u t w e i n , W., Blomgren, K. and K a r l s s o n , J . O . (1988) A p o s s i b l e p h y s i o l o g i c a l r o l e o f the Ca-dependent p r o t e a s e 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 t h e Ca c u r r e n t in guinea p i g myocytes. P f l u g e r s A r c h . 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 t r a n s p o r t by r e c o n s t i t u t e d t r y p t i c fragments o f the C a pump o f the e r y t h r o c y t e plasma membranes. C e l l C a l c i u m 7, 175-186.  z +  z +  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 c o n f o r m a t i o n a l s t a t e s o f the p u r i f i e d C a - A T P a s e o f the e r y t h r o c y t e plasma membrane r e v e a l e d 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 . Biol. Chem. 259, 8471-8477.  Bennett,  M.K. and Kennedy, M.B. (198J) Deduced primary s t r u c t u r e o f the B s u b u n i t o f b r a i n type II C a / c a l m o d u l i n - d e p e n d e n t p r o t e i n k i n a s e determined by m o l e c u l a r c l o n i n g . P r o c . N a t l . A c a d . S c i . USA 84, 1794-1798. z+  Bennett,  V . , Gardner, K. and S t e i n e r , J . P . (1988) B r a i n a d d u c i n : a p r o t e i n k i n a s e C s u b s t r a t e t h a t 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.  Berridge,  M . J . (1987) Inositol trisphosphate 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. A n n . Rev. Biochem. 56, 159-193.  Berridge,  M . J . and G a l i o n e , A . , FASEB J . 2, 3074-3082.  Billger,  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 o f t u b u l i n and m i c r o t u b u l e - a s s o c i a t e d p r o t e i n s 1 and 2 by c a l p a i n I and II. Difference in sensitivity of assembled and disassembled m i c r o t u b u l e s . C e l l Calcium 9, 33-44.  (1988)  Cytosolic  B i r c h - M a c h i n , M.A. and Dawson, A . P . (1988) plasma membranes: the t r a n s p o r t e r  248  z +  calcium  oscillators.  Ca t r a n s p o r t by r a t l i v e r and the p r e v i o u s l y reported  Ca-ATPase 308-314.  are d i f f e r e n t  enzymes.  +  Biochim.  Biophys.  Acta  944,  2 +  B l a u s t e i n , M.P. and N e l s o n , M. (1982) N a - C a exchange: i t s r o l e i n the r e g u l a t i o n o f c e l l c a l c i u m L n : Membrane T r a n s p o r t o f C a l c i u m . ( C a r a f o l i , E . , e d . ) Academic P r e s s . I n c . , New York, 217-236. Blinks,  J . , W i e r , W., H e s s , P. and P r e n d e r g a s t , F. (1982) Measurement o f Ca c o n c e n t r a t i o n i n l i v i n g c e l l s . P r o g . B i o p h y s . M o l . B i o l . 40, 1-114. z +  Blum,  J . J . , Hayes, A . , J a m i e s o n , G . A . J r . and Vanaman, T . C . (1980) Calmodulin c o n f e r s c a l c i u m s e n s i t i v i t y on c i l i a r y d y n e i n ATPase. J . C e l l . B i o l . 8_7, 386-397.  Blumenthal, D . K . and K r e b s , E . G . (1988) C a l m o d u l i n - b i n d i n g domains on t a r g e t p r o t e i n s I n : C a l m o d u l i n (Cohen, P. and K l e e , C . B . , e d s . ) pp. 341-355, E l s e v i e r , Amsterdam. B l u m e n t h a l , D . K . , T a k i o , K . , Edelman. A . M . , Charbonneau, H . , T i t a n i , K . , Walsh, K . A . and K r e b s , E . G . (1985) Identification of the c a l m o d u l i n - b i n d i n g domain o f s k e l e t a l myosin l i g h t c h a i n k i n a s e . P r o c . N a t l . A c a d . S c i . USA 8 2 , 3187-3191. Bond,  J . S . and B u t l e r , P . E . (1987) Biochem. 56> 333-364.  Intracellular  proteases.  A n n . Rev.  2 +  Brandl,  C . J . , G r e e n , N . M . , K o r c z a k , B. and MacLennan, D . H . (1986) Two C a ATPase genes: homologies and m e c h a n i s t i c i m p l i c a t i o n s o f deduced amino a c i d sequences. C e l l 44> 597-607.  B r a u n i t z e r , G . , G e h r i n g - M u l l e r , R . , H i l s c h m a n n , N . , H i l s e , K . , Hobom, G . , R u d l o f f , V. and W i t t m a n n - L i e b o l d , B. (1961) The c o n s t i t u t i o n o f normal a d u l t haemoglobin. H o p p e - S e y l e r ' s Z . P h y s i o l . Chem. 325, 283-286. Brignon,  G . , Ribadeau-Dumas, B . , M e r c i e r , J . and P e l i s s i e r , J . (1977) Complete amino a c i d sequence o f bovine a o - c a s e i n . FEBS l e t t . 76, 274-279. s  Bryan,  J . (1974) Biochemical 152-157.  Bulleit,  of microtubules.  Fed. Proc.  33,  R . F . , B e n n e t t , M . K . , M o l l o y , S . S . , H u r l e y , J . B . and Kennedy, M.B. (1988) Conserved and v a r i a b l e r e g i o n s i n the s u b u n i t s o f b r a i n type II C a / l o d u l i n - d e p e n d e n t p r o t e i n k i n a s e . Neuron I , 6372. 2 +  Burgin,  properties  c a  m  H . , Schatzmann, H . J . (1979) The r e l a t i o n between net c a l c i u m , alkali c a t i o n and c h l o r i d e movements i n 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  Carafoli,  E . (1987) I n t r a c e l l u l a r 56, 395-433.  c a l c i u m h o m e o s t a s i s . A n n . Rev. Biochem.  Carafoli,  E . and Z u r i n i , M. (1982) The C a - p u m p i n g membranes: purifications, reconstitution, B i o c h i m . B i o p h y s . A c t a 683, 279-301.  Carafoli,  E . , F i s c h e r , R . , James, P . , K r e b s , J . , Maeda, M . , E n y e d i , A . , M o r e l l i , A . and d e F l o r a , A . (1987) The c a l c i u m pump o f the plasma membrane: recent s t u d i e d on the p u r i f i e d enzyme and on i t s proteolytic fragments, with particular attention to the c a l m o d u l i n b i n d i n g domain I n : C a l c i u m - B i n d i n g P r o t e i n s i n Health and Diseases (Means, A . N . , e d . ) p p . 7 8 - 9 1 , Academic P r e s s , New York.  Carafoli,  E . , T i o z z o , 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 r e l e a s e o f c a l c i u m from h e a r t m i t o c h o n d r i a by sodium. J . M o l . C e l l . C a r d i o l . 6, 361-371.  2+  ATPase o f plasma and properties.  Carl i n ,  R.K., Bartelt, 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 o f f o d r i n as a major c a l m o d u l i n - b i n d i n g p r o t e i n in postsynaptic d e n s i t y p r e p a r a t i o n s . J . C e l l B i o l . 9 6 , 443-448.  Caroni,  P. and C a r a f o l i , E . (1981) R e g u l a t i o n o f C a pumping ATPase o f h e a r t sarcolemma by a p h o s p h o r y l a t i o n - d e p h o r y l a t i o n p r o c e s s . J . B i o l . Chem. 256, 9371-9373.  2 +  Carpenter  Cassel,  G. (1987) Receptors f o r epidermal growth factor p o l y p e p t i d e mitogens. A n n . Rev. Biochem. 56, 881-914.  and  D. and G l a s e r , L. (1982) P r o t e o l y t i c c l e a v a g e o f epidermal f a c t o r r e c e p t o r . J . B i o l . Chem. 257, 9845-9848.  other  growth  + +  + +  Cha, Y . N . , S h i n , B . C . and L e e , K . S . (1971) A c t i v e uptake o f C a and C a activated M g ATPase i n r e d c e l l membrane f r a g m e n t s . J . Gen. P h y s i o l . 5Z, 202-215. + +  Chan,  Cheung,  Cheung,  Cheung,  K.-F.J. and Graves, D.J. (1984) Molecular properties of phosphorylase k i n a s e In: C a l c i u m and C e l l F u n c t i o n s (Cheung, W . Y . , e d . ) V o l . V, pp. 1-31, Academic P r e s s , New York. W.Y. (1970) Cyclic 3',5'-nucleotide phospho-diesterase. Demonstration o f an a c t i v a t o r . Biochem. B i o p h y s . Res. Commun. 38, 533-538. W.Y. (1971) C y c l i c 3 ' , 5 ' - n u c l e o t i d e Chem. 246, 2859-2869. W.Y. (1980) Calmodulin plays regulation. S c i e n c e 207, 19-27.  a  C h i e n , K . R . , Abrams, J . , F e r r o n i , A . , M a r t i n ,  250  phosphodiesterase.  pivotal  role  T . J . and F a r b e r ,  in  J.  Biol.  cellular  J . L . (1978)  Accelerated phospholipid degradation and a s s o c i a t e d dysfunction 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 . Chem. 253, 4809-4817. Chin,  membrane J . Biol.  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 . , B e r g , P . , G o l d s t e i n , J . L . and Brown, M . S . (1984) Nucleotide sequence o f 3 - h y d r o x y - 3 - m e t h y l - g l u t a r y l coenzyme A r e d u c t a s e , a g l y c o p r o t e i n o f endoplasmic r e t i c u l u m . Nature 308, 613-617.  Chung,  D.W., Que, B . G . , R i x o n , M.W., Mace, M. J r . and D a v i e , E.W. (1983) C h a r a c t e r i z a t i o n o f the complementary d e o x y r i b o n u c l e i c a c i d and genomic deoxyribonucleic acid for the /J-chain of human f i b r i n o g e n . B i o c h e m i s t r y 22, 3244-3250.  Church,  Colca,  J . G . , Ghosh, S . , Roufogalis, B . D . and V i l l a l o b o , A. (1988) Endogenous h y p e r p h o s p h o r y l a t i o n i n 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 . 6 6 , 1-12. J . R . , DeWald, D . B . , P e a r s o n , J . D . , P a l a z u k , B . J . , L a u r i n o , J . P . and McDonald, J . M . (1987) I n s u l i n s t i m u l a t e s the p h o s p h o r y l a t i o n o f calmodulin in i n t a c t adipocytes. J . B i o l . Chem. 262, 1139911402.  Coolican,  Croall,  S.A. and Hathaway, D.R. (1984) Effect p h o s p h a t i d y l i n o s i t o l on a v a s c u l a r smooth muscle Ca p r o t e a s e . J . B i o l . Chem. 259, 11627-11630.  of L-a-dependent  D . E . , Morrow, J . S . and DeMartino, G . N . (1986) L i m i t e d p r o t e o l y s i s of the erythrocyte membrane skeleton by calcium-dependent p r o t e i n a s e s . B i o c h i m . B i o p h y s . A c t a 882, 287-296.  Crompton  M. (1985) The r e g u l a t i o n o f m i t o c h o n d r i a l c a l c i u m h e a r t . C u r r . T o p . Membr. T r a n s p . 25, 231-276.  transport  Crompton,  M . , S i g e l , E . , Salzmann, M. and C a r a f o l i , E. (,1976) A study of the energy-linked influx of Ca^ into m i t o c h o n d r i a . E u r . J . Biochem. 6 9 , 429-434. +  Dagher  in  kinetic heart  G. and Lew V . L . (1988) Maximal c a l c i u m e x t r u s i o n c a p a c i t y and s t o i c h i o m e t r y o f the human red c e l l c a l c i u m pump. J . P h y s i o l . 407, 596-586. 2 +  D a h l q v i s t - E d b e r g , U. and Ekman, P. (1981) P u r i f i c a t i o n o f a C a - a c t i v a t e d protease from rat erythrocytes and i t s possible e f f e c t on pyruvate kinase i n v i v o . B i o c h i m . B i o p h y s . A c t a 660, 96-101. David,  Dayton,  L . L . and S h e a r e r , T . R . (1986) P u r i f i c a t i o n o f c a l p a i n II from r a t l e n s and d e t e r m i n a t i o n o f endogenous s u b s t r a t e s . Exp. Eye Res. 42, 227-238. W.R., G o l l ,  D . E . , Stromer,  M.H., R e v i l l e ,  251  W . J . Zeece,  M.G. and  Robson, R.M. (1975) Some p r o p e r t i e s o f a Ca - a c t i v a t e d protease that may be i n v o l v e d in m y o f i b r i l l a r protein turnover In: P r o t e a s e s and B i o l o g i c a l C o n t r o l ( R e i c h , E . , R i f k i n , D . B . and Shawn, E . , e d s . ) V o l . 2 , p p . 551-577, Cold Spring Harbour Laboratory, U.S.A. Dayton,  W . R . , G o l l , D . E L , 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 possibly involved in m y o f i b r i l l a r p r o t e i n t u r n o v e r . P u r i f i c a t i o n from p o r c i n e m u s c l e . B i o c h e m i s t r y 1 5 , 2150-2167.  DeLuca,  H . F . and Engstrom, G.W. (1961) C a l c i u m uptake by r a t m i t o c h o n d r i a . P r o c . N a t l . A c a d . S c i . USA 4_Z, 1744-1750.  DeLange,  R . J . , Hooper, J . A . and S m i t h , E . L . (1972) Complete amino a c i d sequence o f c a l f - t h y m u s hi stone III. P r o c . N a t l . A c a d . S c i . , USA 69, 882-884.  kidney  DeMartino, G . N . and Blumenthal, D.K. (1982) I d e n t i f i c a t i o n o f a f a c t o r that stimulates calcium-dependent proteases. B i o c h e m i s t r y 21, 4297-4303. DeMartino, G . N . , H u f f , C A . and C r o a l l , D . E . (1986) A u t o p r o t e o l y s i s o f the small s u b u n i t o f calcium-dependent p r o t e a s e II a c t i v a t e s and r e g u l a t e s p r o t e a s e 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 o f c a l c i u m ions i n the r e g u l a t i o n o f i n t r a m i t o c h o n d r i a l m e t a b o l i s m . Biochem. J . 190, 107-117.  D e p a o l i - R o a c h , A . A . , G i b b s , J . B . and Roach, P . J . (1979) Calcium and c a l m o d u l i n a c t i v a t i o n o f muscle p h o s p h o r y l a s e k i n a s e : e f f e c t o f t r y p t i c p r o t e o l y s i s . FEBS l e t t . 1 0 5 , 321-324. Docherty,  K., Steiner, D . F . (1982) Post-translational p r o t e o l y s i s in p o l y p e p t i d e hormone b i o s y t h e s i s . A n n . Rev. P h y s i o l . 44, 625-638.  Dunham, E . T . and G l y n n , I.M. (1961) Adenosine t r i p h o s p h a t a s e a c t i v i t y and the a c t i v e movements o f a l k a l i metal i o n s . J . P h y s i o l . 156, 274293. Ekman,  P. and E r i k s s o n , I. (1980) The j n vitro modification of p h o s p h o r y l a t e d pyruvate kinase by a C l r - a c t i v a t e d p r o t e a s e from r a t l i v e r . A c t a . Chemica. Scand. B34, 419-422. +  Emori,  Y . , Kawasaki, H . , Imajoh, S . Imahori, K. and S u z u k i , K. (1987) Endogenous inhibitor f o r calcium-dependent cysteine protease c o n t a i n s f o u r i n t e r n a l r e p e a t s t h a t c o u l d be r e s p o n s i b l e f o r i t s multiple reactive s i t e s . P r o c . N a t l . A c a d . S c i . USA 84, 35903594.  Emori,  Y.,  Kawasaki,  H.,  Suzihara,  H.,  252  Imajoh,  S.,  Kawashima,  S. and  S u z u k i , K. (1986a) I s o l a t i o n and sequence a n a l y s i s o f cDNA c l o n e s for the l a r g e subunits o f two isozymes o f r a b b i t calciumdependent p r o t e a s e . J . B i o l . Chem. 261, 9465-9471. Emori,  Y . , Ohno, S . , T o b i t a , M. and S u z u k i , K. (1986b) Gene s t r u c t u r e calcium-dependent protease r e t a i n s the a n c e s t r a l o r g a n i z a t i o n the c a l c i u m - b i n d i n g p r o t e i n gene. FEBS L e t t . 194, 249-252.  of of  Enyedi,  A . , F l u r a , M . , S a r k a d 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 c a l c i u m a f f i n i t y o f the red c e l l c a l c i u m pump may be r e g u l a t e d i n d e p e n d e n t l y . J . B i o l . Chem. 262, 6425-6430.  Enyedi,  A . , Sarkadi, B. and Gardos, G. (1982) On the substrate s p e c i f i c i t y o f the r e d c e l l c a l c i u m pump. B i o c h i m . B i o p h y s . A c t a 687, 109-112.  Enyedi,  A . , Sarkadi, B . , S z a s z , B . , B o t . G. and G a r d o s , M o l e c u l a r p r o p e r t i e s o f red c e l l c a l c i u m pump. Cell 291-310.  Eylar,  E . H . , B r o s t o f f , S . , Hashim, G . , Caccam, J . and B u r n e t t , P. (1971) B a s i c A l p r o t e i n o f the myelin membrane. The complete amino a c i d sequence. J . B i o l . Chem. 246, 5770-5784.  Farber,  J . L . (1981) 1289-1295.  Ferreira,  Finley,  Foder,  The r o l e  of  calcium  in c e l l  death.  Life  S c i . 29,  H . G . , Lew, V . L . (1976) Use o f ionophore A23187 t o measure c y t o p l a s m i c Ca b u f f e r i n g and a c t i v a t i o n o f the Ca pump by i n t e r n a l C a . Nature 259, 47-49.  D. and V a r s h a v s k y , A . (1985) The u b i q u i t i n system: mechanism. Trends Biochem. S c i . 10, 343-349.  f u n c t i o n s and  B. and S c h a r f f , 0 . (1981) Decrease o f apparent c a l m o d u l i n a f f i n i t y of erythrocyte (Ca +Mg )-ATPase at low C a concentrations. B i o c h i m . B i o p h y s . A c t a 649, 367-376. z+  Foyt,  _G. (1980) Calcium 1,  2+  2 +  H . L . , G u e r r i e r o , V. J r . and Means, A . R . (1985) F u n c t i o n a l domains o f c h i c k e n g i z z a r d myosin l i g h t c h a i n k i n a s e . J . B i o l . Chem. 260, 7765-7774.  Fujii,  Fukami,  J . , Veno, A . , K i t a n o , K . , T a n a l e a , S . , Kadoma, M. and Tada, M. (1987) Complete complementary DNA-derived amino a c i d sequence o f c a n i n e c a r d i a c phospholamban. J . C l i n . I n v e s t . 79, 301-304. Y., Nakamura, T., Nakayama, A. and K a n e h i s a , T. (1986) Phosphorylation of tyrosine residues of calmodulin i n Rous sarcoma v i r u s - t r a n s f o r m e d c e l l s . P r o c . N a t l . A c a d . S c i . , USA 83, 4190-4193.  253  Furukawa,  K . I . and Nakamura. H. (1987) C y c l i c GMP r e g u l a t i o n membrane (Ca - M g ) - A T P a s e in vascular smooth Biochem. 101, 287-290. z+  Furukawa,  o f the plasma muscle. J.  K . I . Tawada, Y . , Shigekawa, M. (1988) R e g u l a t i o n o f t h e plasma membrane C a pump by c y c l i c n u c l e o t i d e s i n c u l t u r e d v a s c u l a r smooth muscle c e l l s . J . B i o l . Chem. 263, 8058-8065. 2 +  Gagnon,  C , Kelly, S . , Manganiello, V . , Vaughn, M . , Odya, C . and S t r i t t m a t h e r , W. (1981) M o d i f i c a t i o n o f c a l m o d u l i n f u n c t i o n by enzymatic c a r b o x y l m e t h y l a t i o n . Nature 29_I, 515-516.  Gardner,  K. and B e n n e t t , V . (1986) A new e r y t h r o c y t e membrane-associated p r o t e i n with c a l m o d u l i n b i n d i n g 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 o f t h e p a r t i a l reactions o f the Ca -ATPase from human red c e l l s by M g and ATP_. B i o c h i m . B i o p h y s . A c t a 513, 5 9 - 6 5 . 2 +  Gassner,  Gates,  B., L u t e r b a c h e r , S . , Schatzmann, H . J . and W u t h r i c h , A . (1988) Dependence o f the r e d b l o o d c e l l c a l c i u m pump on t h e membrane p o t e n t i a l . C e l l C a l c i u m 9 , 95-103.  R . E . and K i n g , L . E . J r . (1983) P r o t e o l y s i s o f the epidermal growth f a c t o r r e c e p t o r by endogenous c a l c i u m - a c t i v a t e d n e u t r a l p r o t e a s e from r a t l i v e r . Biochem. B i o p h y s . R e s . Commun. 1 1 3 , 255-261.  Geisler,  Ghosh,  Gietzen,  N. and Weber, K. (1982) The amino a c i d sequence o f c h i c k e n muscle desmin p r o v i d e s a common s t r u c t u r a l model for intermediate filament proteins. EMB0 J . 1, 1649-1656. S . , Church, J . G . , R o u f o g a l i s , B . D . and V i l l a l o b o , Phosphorylation of liver plasma membrane-bound Biochem. C e l l B i o l . 66, 922-927. K., Seiler, S., Fleischer, S. and Reconstitution of the Ca transport e r y t h r o c y t e s . Biochem. J . 188, 47-54. 2 +  Gilman,  A . G . (1987) G proteins. Transducers signals. Ann. Rev. Biochem. 56, 615-649.  A. (1988) calmodulin.  Wolf, H.W. system of  of  (1980) human  receptor-generated  Gimble, J . M . , Waisman, D . M . , G u s t i n , M . , Goodman, D . B . P . and Rasmussen, H. (1982) Studies of the Ca transport mechanism of human erythrocyte inside-out membrane vesicles. Evidence of the development o f 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. 2  Glaser,  T . and Kosower, N . S . (1986) l e t t . 206, 115-120.  Calpain-calpastatin  254  and f u s i o n .  FEBS  Glenney,  J . R . and Weber, K. (1980) C a l m o d u l i n - b i n d i n g p r o t e i n s o f the m i c r o f i l a m e n t s p r e s e n t i n i s o l a t e d brush borders and m i c r o v i l l i o f 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 o f the c o n c e n t r a t i o n o f the f r e e c a t i o n s and c a t i o n - 1 i g a n d complexes i n s o l u t i o n s c o n t a i n i n g multiple d i v a l e n t - c a t i o n s and l i g a n d s . B i o p h y s . J . 26, 235-242. Goll,  D . E . , Shannon, J . D . , Edmunds, T . , Sathe, S . K . , K l e e s e , W.C. and Nagainis, P.A. (1983) P r o p e r t i e s and r e g u l a t i o n o f the Ca dependent proteinase In: Calcium-Binding Proteins 1983 (de Bernad, B . , S c o t t o c a s a , G . L . , S a n d r i , G . , C a r a f o l i , E . , T a y o r , A.N., Vanaman, T . C . and W i l l i a m s , R . J . P . , eds.) pp. 19-35. E l s e v i e r S c i e n c e , Amsterdam.  G o p a l a k r i s h n a , R. and Head, J . F . (1985) Rapid p u r i f i c a t i o n o f c a l c i u m a c t i v a t e d p r o t e a s e by calcium-dependent hydrophobic-interaction chromatography. FEBS l e t t . 186, 246-250. G o p a l a k r i s h n a , R. and B a r s k y , S . H . (1986) Hydrophobic a s s o c i a t i o n o f c a l p a i n s 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, 1393613942. Gopinath,  R.M. and Vincenzi, F.F. (1977) Phosphodiesterase protein a c t i v a t o r mimics red blood c e l l c y t o p l a s m i c a c t i v a t o r o f (Ca Mg )-ATPase. Biochem. B i o p h y s . Res. Commun. 77, 1203-1209. z +  Gorin,  M . B . , Yancey, S . B . , C l i n e , J . , R e v e l , J . - P . and H o r w i t z , J . (1984) The major intrinsic protein (MIP) o f the bovine l e n s fiber membrane: c h a r a c t e r i z a t i o n and s t r u c t u r e based on cDNA c l o n i n g . C e l l 39, 4 9 - 5 9 .  Graf,  E.  and P e n n i s t o n , J . I . (1981) Ca ATP: The s u b s t r a t e , at low ATP c o n c e n t r a t i o n o f C a - A T P a s e from human e r y t h r o c y t e 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 P e n n i s t o n , J . T . (1982) M o l e c u l a r p r o p e r t i e s o f c a l c i u m pumping ATPase from human e r y t h r o c y t e s . B i o c h e m i s t r y 21, 4511-4516.  Grand,  R . J . A . and P e r r y , S . V . (1980) The b i n d i n g o f c a l m o d u l i n to b a s i c p r o t e i n and h i s t o n e H2B. Biochem. J . 189, 227-240.  myelin  Green, S . , W a l t e r , P . , Kumar, V . , K r u s t , A . , B o r n e r t , J . - M . , A r g o s , P. and Chambon, P. (1986) Human oestrogen r e c e p t o r cDNA: sequence, e x p r e s s i o n and homology to v - e r b - A . Nature 320, 134-140. Gronemeyer, H . , T u r c o t t e , B . , Q u i r i n - S t r i c k e r , C , B o c q u e l , M . T . , Meyer, M . E . , K r o z o w s k i , 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 c h i c k e n progesterone receptor: sequence, expression and functional  255  analysis.  EMBO J . 6, 3985-3994.  G u e r r i e r o , V. J r . , Russo, M . A . , O l s o n , N . J . , Putkey, J . A . and Means, A . R . (1986) Domain o r g a n i z a t i o n o f c h i c k e n g i z z a r d myosin l i g h t c h a i n k i n a s e deduced from a c l o n e d cDNA. B i o c h e m i s t r y 25, 8372-8381. Guroff,  G. (1964) A n e u t r a l c a l c i u m - a c t i v a t e d p r o t e i n a s e from the s o l u b l e f r a c t i o n o f r a t 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 o f the Ca -ATPase from plasma membranes o f p i g e r y t h r o c y t e s . J . B i o l . Chem. 254, 6598-6602.  Hale,  C . C . , Slaughter, 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 o f the c a r d i a c sodiumc a l c i u m exchange p r o t e i n . P r o c . N a t l . Acad S c i . USA 8 1 , 65696573.  Hall,  T . G . and B e n n e t t , ankyrin. J . B i o l .  Hall,  V. (1987) Regulatory domains Chem. 262, 10537-10545.  of  erythrocyte  J . L . , D u d l e y , L . , Dobner, P . R . , L e w i s , 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,  2 +  M. and B o u r g o i n , S . (1979) C h a r a c t e r i z a t i o n o f the C a - 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 h y d r o x y l a s e from the r a t b r a i n system. J . Neurochem. 32, 1837-1844.  H a r a g u c h i , K . , A k a s u , F . , Endo, T . and Onaya, T . (1987) Demonstration o f calcium-dependent proteases (calpains) and thyroglobulin p r o t e o l y s i s i n hog t h y r o i d c y t o s o l . E n d o c r i n o l o g i c a J a p o n i c a 34, 1-8. Harris,  A . S . and Morrow, J . S . (1988) P r o t e o l y t i c alpha s p e c t r i n ( f o d r i n ) : i d e n t i f i c a t i o n J . Neuroscience 8 , 2640-2651.  p r o c e s s i n g o f human b r a i n of a hypersensitive s i t e .  Harris,  A . S . , C r o a l l , D . E . and Morrow, J . S . (1988) The c a l m o d u l i n - b i n d i n g site in a - f o d r i n is near the calcium-dependent protease-I c l e a v a g e 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 l e e , C . B . (1981) I n t e r a c t i o n o f c a l m o d u l i n with myosin l i g h t c h a i n kinase and cAMP-dependent p r o t e i n k i n a s e i n 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) Hydrolysis o f protamine by c a l c i u m - a c t i v a t e d neutral p r o t e a s e . J . Biochem. 97, 1363-1370.  Hebbel,  R . P . , S h a l e v , 0 „ , F o k a r , W. and Rank, B . H . (1986) I n h i b i t i o n of e r y t h r o c y t e Ca -ATPase by a c t i v a t e d oxygen through t h i o l and  256  l i p i d dependent mechanism.  B i o c h i m . B i o p h y s . A c t a 862,  8-16.  Hincke,  M.T. and T o l n a i , S . (1986) P h o s p h o r y l a t i o n o f bovine c a r d i a c calcium-activated neutral protease by protein kinase-C. Biochem. B i o p h y s . Res. Commun. 137, 559-565.  Hiraga,  A . and T s u i k i , S. (19861 A c t i v a t i o n o f a D-form o f r a b b i t muscle g l y c o g e n synthase by Ca - a c t i v a t e d p r o t e a s e . FEBS l e t t . 205, 1-  Hirao,  T.  and T a k a h a s h i , 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 o f a c a l c i u m - a c t i v a t e d neutral p r o t e a s e from monkey b r a i n and its a c t i o n on n e u r o p e p t i d e s . J . Biochem. 96, 775-784.  Hodgkin,  A . L . and Keynes, R.D. (1957) Movements o f s q u i d g i a n t axons. J . P h y s i o l . 138, 253-281.  Hoffman,  F . , N a s t a i r c z y k , W., R o h r k a s t e n , A . , S c h n e i d e r , T . a n d , S i e b e r , M. (1987) Regulation of the L-type calcium channel. Trends Pharmacol. S c i . 8, 393-398.  Huston,  R.B. and K r e b s , E.G. (1968) A c t i v a t i o n of skeletal muscle phosphorylase k i n a s e by Ca . II. I d e n t i f i c a t i o n o f 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. B i o c h e m i s t r y 7, 21162121.  Hymel,  labelled  calcium  in  L., I n u i , M . , F l e i s c h e r , S . and S c h i n d l e r , H. (1988) Purified ryanodine r e c e p t o r o f s k e l e t a l muscle s a r c o p l a s m i c . r e t i c u l u m forms Ca - a c t i v a t e d oligomeric Ca channels in planar bilayer. P r o c . N a t l . A c a d . S c i . USA 85, 441-445. z +  Imagawa, T . , S m i t h , J . S . , Coronado, R. and C a m p b e l l , K . P . (1987) P u r i f i e d ryanodine r e c e p t o r from s k e l e t a l muscle s a r c o p l a s m i c r e t i c u l u m i s the Ca -permeable pore o f the calcium release channel. J . B i o l . Chem. 262, 16636-16643. Imahori,  K. (1985) Calcium-dependent neutral protease: its c h a r a c t e r i z a t i o n and r e g u l a t i o n I n : Calcium and C e l l F u n c t i o n s (Cheung, W . Y . , e d . ) Vol. Ill, pp. 473-485, Academic P r e s s , New York.  Imajoh,  S . and S u z u k i , K. (1985) R e v e r s i b l e i n t e r a c t i o n between Ca a c t i v a t e d n e u t r a l p r o t e a s e (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 S u z u k i , K. (1986) The amino-terminal hydrophobic r e g i o n o f the small subunit of calcium-activated neutral protease (CANP) is essential for its activation by p h o s p h a t i d y l 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 o f c a l c i u m - a c t i v a t e d n e u t r a l p r o t e a s e . J . B i o l . Chem.  2 +  257  263,  19783-19787.  Inoue, H . , N o g u c h i , T . and Tanaka, T . (1986) Complete amino a c i d sequence o f r a t L-type pyruvate kinase deduced from the cDNA sequence. E u r . J . Biochem. 154, 465-469. Inui,  M., Saito, A . and F l e i s c h e r , S . (1987a) Purification of the ryanodine receptor and identity with feet structures of j u n c t i o n a l c i s t e r n a e o f s a r c o p l a s m i c r e t i c u l u m from f a s t s k e l e t a l m u s c l e . J . B i o l . Chem. 262, 1740-1747.  Inui,  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 o f the ryanodine r e c e p t o r from c a r d i a c s a r c o p l a s m i c r e t i c u l u m and i d e n t i f y with the f e e t s t r u c t u r e s . J . B i o l . Chem. 262, 15637-15642.  Ishiura,  S . , S u g i t a , H . , S u z u k i , K. Y. and Imahori, K. (1979) S t u d i e s o f a c a l c i u m - a c t i v a t e d n e u t r a l protease from c h i c k e n s k e l e t a l m u s c l e . J . Biochem. 86, 579-581.  Ito,  Tanaka, T . , Nunoki, K . , H i d a k a , H. and S u z u k i , IC (1987) The Ca - a c t i v a t e d protease (calpain) modulates Ca / 3lmodulin dependent a c t i v i t y o f smooth muscle myosin l i g h t c h a i n k i n a s e . Biochem. B i o p h y s . Res. Commun. 145, 1321-1328.  M.,  z +  c  Iwai,  K.,  H a y a s h i , 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 h i s t o n e III. Complete amino a c i d sequence and i t s i m p l i c a t i o n f o r i n t e r a c t i o n s o f h i s t o n e s w i t h DNA. J . Biochem. 72, 357-367.  Iwai,  K.,  Ishikawa, K. and H a y a s h i , H. (1970) Amino a c i d s l i g h t l y l y s i n e - r i c h h i s t o n e . Nature 226, 1056-1058.  James,  sequence  of  P . , Maeda, M . , F i s c h e r , R., Verma, A . , K r e b s , J . , P e n n i s t o n , 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 r u c t u r e o f a c a l m o d u l i n b i n d i n g domain o f the C a pump o f human e r y t h r o c y t e s . J . B i o l . Chem. 263, 2905-2910. 2 +  James,  P . , V o r h e r r , T . , K r e b s , J . , M o r e l l i , A . , C a s t e l l o , G . , McCormick, D.J., P e n n i s t o n , 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 selective calpain c l e a v a g e o f the c a l m o d u l i n - b i n d i n g domain. J . B i o l . Chem. 264, 8289-8296. 2 +  Jarrett,  H.W. and P e n n i s t o n , J . T . (1977a) P u r i f i c a t i o n o f a C a - A T P a s e a c t i v a t o r from human e r y t h r o c y t e membranes. F e d . P r o c . 36, 642.  Jarrett,  H.W. and P e n n i s t o n , J . T . (1977b) P a r t i a l p u r i f i c a t i o n o f the Ca - M g ATPase activator from human erythrocytes: Its similarity to the activator of 3'-5'-cyclic nucleotide p h o s p h o d i e s t e r a s e Biochem. B i o p h y s . Res. Commun. 77, 1210-1216. z +  Jefferson,  A . B . and Schulman, H.  (1988)  258  Sphingosine i n h i b i t s  calmodulin-  dependent Jelsema,  enzymes.  J . Biol.  Chem. 263, 15241-15244.  C . L . and A x e l r o d , J . (1987) S t i m u l a t i o n o f p h o s p h o l i p a s e Ap activity in bovine rod o u t e r segments by By s u b u n i t s of t r a n s d u c i n and i t s i n h i b i t i o n by the a: s u b u n i t . P r o c . N a t l . A c a d . S c i . USA 84, 3623-3627.  J e n n y , R . J . , P i t t m a n , D . D . , T o o l e , J . J . , K r i z , R.W., A l d a p e , R . A . , Hewick, R . M . , Kaufman, R . J . and Mann, K . G . (1987) Complete cDNA and d e r i v e d amino a c i d sequence o f human f a c t o r V . Proc. N a t l . Acad. S c i . USA 84, 4846-4850. Johanson,  Julien,  R . A . , Hansen, C A . and W i l l i a m s o n , J . R . (1988) P u r i f i c a t i o n D-myo-inositol 1,4,5-trisphosphate 3 - k i n a s e from r a t b r a i n . B i o l . Chem. 263, 7465-7471.  of J.  J . - P . G r o s v e l d , F . , Yazdanbaksh, K . , F l a v e l l , D . , M e i j e r , D. and M u s h y n s k i , W. (1987) The s t r u c t u r e o f a human n e u r o f i l a m e n t gene ( N F - L ) : a unique e x o n - i n t r o n o r g a n i z a t i o n i n the intermediate f i l a m e n t gene f a m i l y . B i o c h i m . B i o p h y s . A c t a 909, 10-20.  Kagawa,  Y . and Racker, E. catalyzing oxidative 5487.  Kakiuchi,  S . and Yamazaki, R. (1970) Calcium dependent p h o s p h o d i e s t e r a s e activity 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. B i o p h y s . R e s . Commun. 4 1 , 1104-1110.  Katz,  Katz,  (1971) Partial resolution o f t h e enzyme p h o s p h o r y l a t i o n . J . B i o l . Chem. 246, 5477-  S. and Blostein, R. (1975) Calcium stimulated membrane p h o s p h o r y l a t i o n o f ATPase a c t i v i t y o f the human eruthrocyte. B i o c h i m . B i o p h y s . A c t a 389, 314-324. S.  and R e m t u l l a , M.A. (1978) P h o s p h o d i e s t e r a s e p r o t e i n activator s t i m u l a t e s c a l c i u m t r a n s p o r t i n c a r d i a c microsomal preparations enriched in sarcoplasmic reticulum. Biochem. B i o p h y s . R e s . Commun. 8 3 , 1373-1379.  K e l l e r m a n n , 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 o f the primary s t r u c t u r e o f human h i g h - m o l e c u l a r - m a s s k i n i n o g e n . E u r . J . Biochem. 154, 471-478. Kennedy,  M . B . , Bennett, M . K . , Erondu, N . E . and M i l l e r , S . G . (1987) C a l c i u m / c a l m o d u l i n - d e p e n d e n t p r o t e i n k i n a s e s i n : C a l c i u m and C e l l F u n c t i o n s (Cheung, W . Y . , e d . ) V o l . V I I , pp. 61-107, Academic P r e s s , New Y o r k .  Kennelly,  P . J . , T a k i o , K . , Blumenthal, D . K . , Edelman, A . M . , Glaccum, M . B . , K l e v i t , R . E . , Roush, C . L . , S c o t t , J . O . , T a k i o , K . , T i t a n i , K . , Walsh, K . A . and K r e b s , E . G . (1987) O r g a n i z a t i o n o f myosin l i g h t c h a i n k i n a s e from r a b b i t skeletal muscle i n : Calcium B i n d i n g P r o t e i n s i n Health and D i s e a s e s (Means, A . R . , e d . ) p p . 494-504,  259  Academic P r e s s ,  New York.  Kikkawa,  U . , O g i t a , K . , Ono, Y . , Asaoka, Y . , Shearman, M . S . , F u j i i , T . , A s e , K . , S e k i g u c h i , K . , I g a r a s h i , K. and N i s h i z u k a , Y. (1987) The common s t r u c t u r e and a c t i v i t i e s of subspecies of r a t brain p r o t e i n k i n a s e C f a m i l y . FEBS l e t t . 223, 212-216.  Kincaid,  R., Stith-Coleman, I.E. and Vaughan, M. (1985) activation of calmodulin-dependent cyclic phosphodiesterase. J . B i o l . Chem. 260, 9009-9015.  Proteolytic nucleotide  Kirchberger, M . A . , T a d a , M. and K a t z , A . M . (1974) Adenosine 3':5'monophosphate-dependent p r o t e i n k i n a s e - c a t a l y z e d p h o s p h o r y l a t i o n r e a c t i o n and i t s r e l a t i o n s h i p t o c a l c i u m t r a n s p o r t in cardiac sarcoplasmic reticulum. J . B i o l . Chem. 249, 6166-6173. Kishimoto, A . , Kajikawa, N., Shiota, M. and N i s h i z u k a , Y. (1983) Proteolytic activation of calcium-activated, phospholipiddependent p r o t e i n k i n a s e by calcium-dependent n e u t r a l p r o t e a s e . J . B i o l . Chem. 258, 1156-1164. Kishimoto, A . , Mikawa, K., Hashimoto, K., Yasudo, I., Tanaka, S., Tominaga, M., Kuroda, T. and N i s h i z u k a , Y. (1989) Limited p r o t e o l y s i s o f p r o t e i n k i n a s e C s u b s p e c i e s by c a l c i u m dependent n e u t r a l p r o t e a s e ( c a l p a i n ) J . B i o l . Chem. 264, 4088-4092. Klaerke,  D . A . , P e t e r s o n , J . and J o r g e n s e n , P . L . (1987) P u r i f i c a t i o n o f Ca - a c t i v a t e d K channel p r o t e i n on c a l m o d u l i n a f f i n i t y columns a f t e r d e t e r g e n t s o l u b i l i z a t i o n o f luminal membranes from o u t e r renal m e d u l l a . FEBS l e t t . 216, 211-216. +  Klee,  C . B . (1977) Conformational t r a n s i t i o n accompanying the b i n d i n g o f Ca to the protein activator of 3'.5'-cyclic adenosine monophosphate p h o s p h o d i e s t e r a s e . B i o c h e m i s t r y 16, 1017-1026.  Klee,  C . B . (1988) C a - d e p e n d e n t p h o s p h o l i p i d p r o t e i n s . B i o c h e m i s t r y 27, 6645-6653.  Klee,  C . B . and Vanaman, 213-321.  2+  Klinger,  T . C . (1982)  Adv. P r o t e i n  binding  Chem. 357,  R . , Wetzker, R . . F l e i s c h e r , I. and F r u n d e r , H . (1980) E f f e c t calmodulin, Ca and Mg on the (Ca +Mg^ )-ATPase e r y t h r o c y t e membranes. C e l l Calcium 1_, 229-240. z +  Knauf,  Calmodulin.  (and membrane-)  z +  z+  +  of of  P . A . , Proverbio, F. and Hoffman, J . T . (1974) Electrophoretic s e p a r a t i o n o f 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 i n human red c e l l g h o s t s . J . Gen. P h y s i o l . 63, 324-336.  K o n i g s b e r g , W. and H i l l , R . J . (1962) The s t r u c t u r e B i o l . Chem. 237, 3157-3162.  260  o f human hemoglobin. J .  Kopito,  R.R. and L o d i s h , exchange p r o t e i n .  H . F . (1985) S t r u c t u r e of J . C e l l . Biochem. 29, 1-17.  the  murine  Kosaki,  G . , T s u j i n a k a , 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 calmodulin-binding proteins by low Ca - r e q u i r i n g form o f Ca - a c t i v a t e d n e u t r a l p r o t e a s e i n human p l a t e l e t s . Biochem. I n t . 6, 767-775.  K o s k - K o s i c k a , D. S c a i l l e t , S . and I n e s i , G. (1986) The p a r t i a l in the c a t a l y t i c cycle of the calcium-dependent triphosphatase purified from e r y t h r o c y t e membranes. Chem. 261, 3333-3338.  anion  reactions adenosine J . Biol.  z +  K o s k - K o s i c k a , D. and Bzdega, T . (1988) A c t i v a t i o n o f the e r y t h r o c y t e C a ATPase by e i t h e r s e l f - a s s o c i a t i o n o r i n t e r a c t i o n with c a l m o d u l i n . 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 f u s i o n o f e r y t h r o c y t e s : f u s i b i l i t y i s c o r r e l a t e d with attack by c a l c i u m - a c t i v a t e d cytoplasmic proteases on membrane p r o t e i n s . P r o c . N a t l . A c a d . S c i . USA 80, 7542-7546.  Kretsinger, R . H . (1976) 45, 239-266.  Calcium-binding  proteins.  Ann.  K r e t s i n g e r R . H . and N o c k o l d s , C . E . (1973) Carp muscle p r o t e i n . II. S t r u c t u r e d e t e r m i n a t i o n and general B i o l . Chem. 248, 3313-3326. Kubo,  M.  Kubota,  Rev.  Biochem.  calcium-binding description. J .  and S t r o t t , C A . (1988) Phosphorylation o f c a l m o d u l i n on threonine residue(s) by c y t o s o l prepared from adrenal cortex. Biochem. B i o p h y s . R e s . Commun. 156, 1333-1339.  S . , Onaka, T . , M u r o f u s h i , 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 o f high Ca - r e q u i r i n g n e u t r a l p r o t e a s e s from p o r c i n e and bovine b r a i n s . B i o c h e m i s t r y 25, 83968402.  Kumagai,  H . , N i s h i d a , E. and S a k a i , H. (1982) The i n t e r a c t i o n between c a l m o d u l i n and m i c r o t u b u l e p r o t e i n s . IV. Q u a n t i t a t i v e a n a l y s i s o f the b i n d i n g between c a l m o d u l i n and t u b u l i n dimer. J . Biochem. 9 1 , 1329-1336.  Kunicki,  T . J . Montgomery, R.R. and S c h u l l e k , von W i l l e b r a n d factor by p l a t e l e t Blood 65, 352-356.  Kunicki,  T . J . , Mosesson, M.W. and P i d a r d , D. (1984) Cleavage o f f i b r i n o g e n by human p l a t e l e t c a l c i u m - a c t i v a t e d p r o t e a s e . Thromb. Res. 35, 169-182.  261  J . (1985) Cleavage o f human calcium-activated protease.  Kuo,  T.H. and Tsang W. (1988) Guanine nucleotideand inositol t r i p h o s p h a t e - i n d u c e d i n h i b i t i o n o f the C a pump i n r a t h e a r t sarcolemmal v e s i c l e s . Biochem. B i o p h y s . R e s . Commun. 152, 11111116. z +  Kuwayama,  La R o c c a ,  ft. Ca^ 299.  +  (1988) The membrane p o t e n t i a l modulates the ATP-dependent pump o f c a r d i a c sarcolemma. B i o c h i m . B i o p h y s . A c t a 940, 295-  J . N . , Rega, A . F . and Garrahan, P . J . (1981) P h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n o f the C a pump o f human red c e l l s i n the presence o f monovalent c a t i o n s . B i o c h i m . B i o p h y s . A c t a 645, 10-16. z +  Laemmli,  Lai,  U . K . (1970) Cleavage o f s t r u c t u r a l p r o t e i n s d u r i n g the assembly o f the head o f b a c t e r i o p h a g e T 4 . Nature 227, 680-685.  F . A . , E r i c k s o n , H . P . , Rousseau, E . , L i u , A . Y . and M e i s s n e r , 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 c a l c i u m r e l e a s e channel from s k e l e t a l m u s c l e . Nature 331,, 315-319.  Larsen,  Lamers,  F . L . , Katz, S . , Roufogalis, B . D . and P h y s i o l o g i c a l shear s t r e s s e s enhance t h e human e r y t h r o c y t e s . Nature 294, 667-668.  Brooks, D.E. (1981) Ca permeability of z +  J . M . J . , S t i n i s , H . J . and De J o n g e , H.R. (1981) On the r o l e o f c y c l i c AMP a n i C a ^ - c a l m o d u l i n - d e p e n d e n t p h o s p h o r y l a t i o n i n the c o n t r o l o f ( C a + M g ) - A T P a s e o f c a r d i a c sarcolemma. FEBS l e t t . 127, 139-143. z+  2+  Leavis,  P . C . and G e r g e l y , J . (1984) T h i n f i l a m e n t p r o t e i n s and t h i n f i l a m e n t l i n k e d r e g u l a t i o n o f v e r t e b r a t e muscle c o n t r a c t i o n . CRC c r i t . Rev. Biochem. 16, 235-305.  Leclerc,  L . , G i r a u d , F . , G a l a c t e r o s , F. and Poyant, C . (1987) The calmodulin-stimulated (Ca +Mg )-ATPase i n human erythrocyte membranes: e f f e c t s o f s i c k l i n g and o x i d a t i v e a g e n t s . Biochim. B i o p h y s . A c t a 89_7, 33-40. z+  Lee,  z+  G . , Cowan, N. and K i r s c h n e r , M. (1988) The primary s t r u c t u r e and h e t e r o g e n e i t y o f tau p r o t e i n from mouse b r a i n . S c i e n c e 239, 285292.  L e e , K . S . and A u , K . S . (1983) A protein i n h i b i t o r of erythrocyte ( C a + M g ) - A T P a s e . B i o c h i m . B i o p h y s . A c t a 742, 54-62. z+  Lee,  membrane  z+  Y . C . and W o l f f , J . (1984) Calmodulin binds a s s o c i a t e d p r o t e i n 2 and tau p r o t e i n s . 1226-1230.  L e e , K.W. and S h i n , B . C . (1969) S t u d i e s on the a c t i v e  262  t o both m i c r o t u b u l e J . B i o l . Chem. 259,  transport  of calcium  o f human red c e l l s . J . Gen. P h y s i o l . 54> Lees,  713-729.  J . F . , Schneidman, P . S . , S k u n t z , S . F . , C a r d e n , M . J . and L a z z a r i n i , R.A. (1988) The s t r u c t u r e and o r g a n i z a t i o n o f the human heavy n e u r o f i l a m e n t 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 S h a r p , P . A . (1981) Nucleotide sequence and e v o l u t i o n of a mammalian a-tubulin messenger RNA. J . M o l . B i o l . 151, 101-120. LePeuch,  Levine,  C . J . and D e m a i l l e , H . J . (1979) Concerted r e g u l a t i o n o f c a r d i a c sarcoplasmic reticulum calcium transport by c y c l i c adenosine monophosphate dependent and calcium-calmodulin-dependent phosphorylation. B i o c h e m i s t r y 18, 5150-5157. H. I l l and Sahyoun, N . E . (1987) C h a r a c t e r i z a t i o n o f a s o l u b l e M 30000 c a t a l y t i c fragment o f the neuronal calmodulin-dependent p r o t e i n kinase II. E u r . J . Biochem. 168, 481-486. r  Levitzki,  A . (1987) R e g u l a t i o n o f a d e n y l a t e p r o t e i n s . FEBS l e t t . 2 H , 113-118.  cyclase  Lichtner,  R. and W o l f . H.V (1980) P h o s p h o r y l a t i o n a f f i n i t y (Ca +Mg )-ATPase o f the human B i o c h i m . B i o p h y s . A c t a 598, 472-485. ?  z+  2+  by  hormones  and G-  o f the i s o l a t e d h i g h erythrocyte membrane.  Lin,  C.R., Kapiloff, M.S., Durgerian, S . , Tatemoto, K., Russo, A . F . , Hanson, P . , Schulman, H. and R o s e n f e l d , M.G. (1987) M o l e c u l a r cloning of a b r a i n - s p e c i f i c calcium/calmodulin-dependent protein k i n a s e . P r o c . N a t l . A c a d . S c i . USA 84, 5962-5966.  Lin,  S . - H . (1985a) Novel ATP-dependent c a l c i u m t r a n s p o r t component from rat liver plasma membranes. The t r a n s p o r t e r and previously reported (Ca - M g ) - A T P a s e are d i f f e r e n t proteins. J. Biol. Chem. 260, 7850-7856. z+  2 +  Lin,  S.-H. (1985b) The r a t l i v e r plasma membrane high a f f i n i t y ( C a M g ) - A T P a s e i s not a c a l c i u m pump. Comparison w i t h ATP-dependent c a l c i u m t r a n s p o r t e r . J . B i o l . Chem. 260, 10976-10980.  Lin,  S . - H . and F a i n , J . N . (1984) P u r i f i c a t i o n o f ( C a - M g ) - A T P a s e r a t l i v e r plasma membranes. J . B i o l . Chem. 259, 3016-3020.  Lin,  S . - H . and R u s s e l l , W.E. (1988) Two C a - d e p e n d e n t ATPases i n rat l i v e r plasma membrane. The p r e v i o u s l y p u r i f i e d (Ca-Mg)-ATPase i s not a C a - p u m p but an e c t o - A T P a s e . J . B i o l . Chem. 263, 1225312258.  2 +  Liscum,  2 +  from  2+  L . , Cummings, R . D . , Anderson, R . G . W . , DeMartino, G . N . , Goldstein, J.L. and Brown, M.S. (1983) 3-hydroxy-3-methylglutaryl-CoA reductase: A transmembrane glycoprotein of the endoplasmic  263  r e t i c u l u m with N - l i n k e d "high-mannose" N a t l . A c a d . S c i . USA 8 0 , 7165-7169. Liscum,  oligosaccharides.  Proc.  L . , F i n e r - M o o r e , J . , S t r o u d , R . M . , L u s k e y , K . L . , Brown, M.S. and Goldstein, J.L. (1985) Domain structure of 3-hydroxy-3methylglultaryl coenzyme A r e d u c t a s e , a glycoprotein o f the endoplasmic r e t i c u l u m . J . B i o l . Chem. 260, 522-530.  Long, C . and Mouat, B. (1973) The b i n d i n g o f c a l c i u m i o n s by e r y t h r o c y t e s 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 P e c k e r , F . (1984) I n h i b i t i o n by glucagon o f the c a l c i u m pump i n l i v e r plasma membranes. J . B i o l . Chem. 259, 8195-8201. Lotersztajn, S . , Hanoune, J . and P e c k e r , F. (1981) A high affinity c a l c i u m - s t i m u l a t e d magnesium-dependent ATPase i n r a t l i v e r plasma membranes. Evidence on an endogenous p r o t e i n a c t i v a t o r distinct from c a l m o d u l i n . J . B i o l . Chem. 256, 11209-11215. Lotersztajn, S . , M a l l a t , A . , P a v o l i n e , C . a n d P e c k e r , F. (1985) The i n h i b i t o r o f l i v e r plasma membrane (Ca - M g ) - A T P a s e . J . Biol. Chem. 260, 9692-9698. z +  L o t e r s z t a j n , S . , P o v o i r e , 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 inhibition o f the l i v e r plasma membrane (Ca - M g ) - A T P a s e . J . B i o l . Chem. 262, 3114-3117. z +  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 o f 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 m o l e c u l a r masses i n animal t i s s u e s . Evidence f o r m o d i f i c a t i o n o f the p l a t e l e t enzyme by C a - d e p e n d e n t p r o t e i n a s e . Biochem. J . 221, 813-820. z+  Lowry,  O . H . , Rosebrough, N . J . , F a r r , A . L . and R a n d a l l , R . J . (1951) P r o t e i n measurement with f o l i n phenol r e a g e n t . J . B i o l . Chem. 193, 265-275.  L u k a s , T . J . , B u r g e r s , W . H . , P r e n d e r g a s t , F . G . , Lau, W. and W a t t e r s o n , D.M. (1986) Calmodulin binding domains: Characterization of a p h o s p h o r y l a t i o n and c a l m o d u l i n b i n d i n g s i t e from myosin l i g h t chain kinase. B i o c h e m i s t r y 25, 1458-1464. Luthra,  M . G . , A u , K . S . and Hanahan, D . J . (19771 P u r i f i c a t i o n o f an a c t i v a t o r o f human e r y t h r o c y t e membrane ( C a ^ + M g ) - A T P a s e , A r c h . Biochem. Biophy. 77, 678-687. +  Lynch,  z+  2 +  C . G . , Sobo, G . E . and E x t o n , J . H . (1986) An endogenous Ca s e n s i t i v e p r o t e i n a s e c o n v e r t s the h e p a t i c 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 f o r m s . Biochim. Biophys. Acta 885, 110-120.  264  Lynch,  G. and Baudry, M. (1984) The b i o c h e m i s t r y o f memory: s p e c i f i c h y p o t h e s i s . S c i e n c e 224, 1057-1063.  a new and  Lynch,  T . J . and Cheung, W.Y. (1979) Human e r y t h r o c y t e C a - M g - A T P a s e : mechanism o f s t i m u l a t i o n by C a . A r c h . Biochem. B i o p h y s . 194, 165-170.  2 +  2 +  2 +  MacLennan, D . H . , B r a n d l , C . J . , K o r c z a k , B. and Green, M.N. (1985) Amino a c i d sequence o f a Ca +Mg -dependent ATPase from r a b b i t muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature 316, 696-700. 2+  MacLennan, D.H. and Wong, P.T.S. (1971) Isolation of s e q u e s t e r i n g p r o t e i n from s a r c o p l a s m i c r e t i c u l u m . A c a d . S c i . USA 68, 1231-1235. MacLennen, D.H. and Reithmeier, R.A.F. (1985) calsequestrin. in: Structure and f u n c t i o n r e t i c u l u m ( F l e i s c h e r , S . and Tonomura, Y . , Academic P r e s s , O r l a n d , F L . Mak,  a calciumProc. Natl.  Structure of of sarcoplasmic e d s . ) p p . 99-100,  A . S . , S m i l l i e , L . B . and S t e w a r t , G . R . (1980) A comparison o f the amino a c i d sequences o f r a b b i t skeletal muscle a- and Btropomyosins. J . B i o l . Chem. 255, 3647-3655.  Maki,  M . , Takano, E . , M o r i , H . , S a t o , A . , M u r a c h i , T . and Hatanaka, M. (1987) A l l f o u r i n t e r n a l l y r e p e t i t i v e domain o f p i g 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 a g a i n s t c a l p a i n I and II. FEBS l e t t . 223, 174-180.  Malik,  M.N., Fenko, M.D., Iqbal, K. and W i s n i e w s k i , 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 o f two forms o f Ca -activated n e u t r a l protease from c a l f b r a i n . J . B i o l . Chem. 258, 8955-8962.  Mallat,  A . , P a v o i n e , C , D u f o u r , M . , L o t e r s z t a j n , S . , B a t a i l l e , D. and P e c k e r , F. (1987) A glucagon fragment i s r e s p o n s i b l e f o r the i n h i b i t i o n o f the l i v e r C a ^ pump by g l u c a g o n . Nature 325, 620622. +  Manalan,  A . S . and K l e e , C . B . (1983) A c t i v a t i o n o f c a l c i n e u r i n by proteolysis. P r o c . N a t l . A c a d . S c i . USA 8 0 , 4291-4295.  Manalan,  A . S . and K l e e , C . B . (1984) C a l m o d u l i n I n : Advances i n C y c l i c N u c l e o t i d e and P r o t e i n p h o s p h o r y l a t i o n Research (Greengard, P. and R o b i s o n , G . A . , e d s . ) V o l . 18, p p . 227-278, Raven P r e s s , New York.  MarteH,  A . and Smith R. (1974-1982) 1-5, Plenum P r e s s , New York.  Mauldin,  D.  and R o u f o g a l i s ,  B.D.  Critical  (1980)  265  Stability  A protein  limited  Constants,  activator  of  Vols.  Mg  2 +  -  1  dependent Ca' - s t i m u l a t e d ATPase i n human e r y t h r o c y t e d i s t i n c t from c a l m o d u l i n . Biochem. J . 187, 501-513. Mayr,  membranes  G.W. and Heilmeyer, L.M.G. J r . (1983) P h o s p h o f r u c t o k i n a s e calmodulin binding p r o t e i n . FEBS l e t t . 159, 51-57.  is  a  McDonald, J . F . , G r o s c h e l - S t e w a r t , U. and Walsh, M.P. (1987) P r o p e r t i e s and distribution o f the p r o t e i n inhibitor (M 17000) o f protein k i n a s e C. Biochem. J . 242, 695-705. r  2 +  McDonald,  J . R . and Walsh, M.P. (1985) C a - b i n d i n g p r o t e i n s from bovine b r a i n i n c l u d i n g a potent i n h i b i t o r o f p r o t e i n k i n a s e C . Biochem. J . 232, 559-567.  McGowan,  E . B . , Yeo, K-T and D e t w i l e r , T . C . (1983) dependent protease on p l a t e l e t s u r f a c e Biochem. B i o p h y s . 227, 287-301.  McMahon,  A . P . and erythroid 804-807.  Means,  The a c t i o n o f c a l c i u m glycoproteins. Arch.  Moon, R . T . (1987) S t r u c t u r e and e v o l u t i o n o f a nons p e c t r i n , human a - f o d r i n . Biochem. S o c . T r a n s . 15,  A.R., Tash, J.S. and Chafouleas, J.G. (1982) Physiological Implications o f the p r e s e n c e , d i s t r i b u t i o n , and r e g u l a t i o n of c a l m o d u l i n i n e u k a r y o t i c c e l l s . P h y s i o l . Reviews 62, 1-39.  Meggio,  F . , B r u n a t i , A . M . and P i n n a , L . A . (1987) Polycation-dependent, Ca - a n t a g o n i z e d p h o s p h o r y l a t i o n o f c a l m o d u l i n by c a s e i n k i n a s e - 2 and a s p l e e n t y r o s i n e p r o t e i n k i n a s e . FEBS l e t t . 215, 241-246.  Mellgren,  R . L . (1987) Calcium-dependent p r o t e a s e s : an enzyme system a c t i v e at c e l l u l a r membranes? FASEB J . 1, 110-115.  Mellgren,  R . L . , A y l w a r d , J . H . , K i l l i l e a , S . D . and L e e , 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 o f a n a t i v e high m o l e c u l a r weight form o f r a b b j t skeletal muscle p h o s p h o r y l a s e phosphatase by endogenous Ca -dependent p r o t e a s e s . J . B i o l . Chem. 254, 648652.  Mellgren,  R . L . , Lane, R.D. and Kakar, S . S . (1987a) A s a r c o l e m m a - a s s o c i a t e d i n h i b i t o r i s capable o f modulating c a l c i u m - d e p e n d e n t p r o t e i n a s e activity. B i o c h i m . B i o p h y s . A c t a 930, 370-377.  Mellgren,  R.L., Lane, R.D. and Kakar, S.S. (1987b) Isolated bovine myocardial sarcolemma and s a r c o p l a s m i c r e t i c u l u m v e s i c l e s c o n t a i n tightly bound calcium-dependent protease inhibitor. Biochem. B i o p h y s . Res. Commun. 1_42, 1025-1031.  Mellgren,  R.L., Repetti, A . , Muck, T . C . and E a s l y , J . (1982) Rabbit s k e l e t a l muscle calcium-dependent p r o t e a s e r e q u i r i n g millimolar C a . J . B i o l . Chem. 257, 7203-7209. z +  266  Melloni,  E . , P o n t r e m o l i , S . , M i c h e t t i , M . , S a c c o , 0 . , S p a r a t o r e , B. and Horecker B . L . (1986) The involvement o f c a l p a i n i n the a c t i v a t i o n of p r o t e i n k i n a s e C i n n e u t r o p h i l s s t i m u l a t e d by phorbol m y r i s t i c a c i d . J . B i o l . Chem. 261, 4101-4105.  Melloni,  E . , Sal ami n o , F . , S p a r a t o r e , B . , M i c h e t t i , M. and P o n t r e m o l i , S . (1984) C h a r a c t e r i z a t i o n o f the s i n g l e p e p t i d e generated from the amino-terminus end o f a - and ^-hemoglobin c h a i n s by the Ca dependent n e u t r a l p r o t e i n a s e . B i o c h i m . B i o p h y s . A c t a 788, 11-16.  Melloni,  E . , S p a r a t o r e B . , S a l a m i n o , F . , M i c h e t t i , M. and P o n t r e m o l i , S . (1982a) Cytosolic calcium dependent proteinase of human e r y t h r o c y t e s : f o r m a t i o n o f an enzyme-natural inhibitor complex induced by Ca . Biochem. B i o p h y s . R e s . Commun. 106, 731-740.  Melloni,  E . , S p a r a t o r e , B . , S a l a m i n o , F . D . , M i c h e t t i , M. and P o n t r e m o l i , S. (1982b) C y t o s o l i c c a l c i u m dependent n e u t r a l proteinase of human e r y t h r o c y t e s : the r o l e o f c a l c i u m i o n s on t h e m o l e c u l a r and catalytic properties o f the enzyme. Biochem. B i o p h y s . R e s . Commun. 107, 1053-1059.  Mercier,  J . - C , G r o s c l a u d e , F . and Ribadeau-Dumas, B. (1971) The primary s t r u c t u r e o f bovine a j c a s e i n . E u r . J . Biochem. 2 3 , 4 1 - 5 1 . s  Meyer,  W . L . , F i s c h e r , E . H . and K r e b s , E . G . J 1 9 6 4 ) A c t i v a t i o n o f s k e l e t a l muscle phosphorylase b k i n a s e by Ca . B i o c h e m i s t r y 3 , 1033-1039.  Minocherhomjee, A . and R o u f o g a l i s , B . D . (1982) S e l e c t i v e antagonism o f the Ca t r a n s p o r t ATPase o f the r e d c e l l membrane by N - ( 4 - a z i d o - 2 nitrophenyl)-2-aminoethyl-sulfonate ( N A P - t a u r i n e ) J . B i o l . Chem. 257. 5426-5430. z +  Minocherhomjee, A . , A l - J o b o r e , A . and R o u f o g a l i s , B . D . (1982) o f t h e c a l c i u m - t r a n s p o r t ATPase i n human e r y t h r o c y t e s B i o c h i m . B i o p h y s . A c t a 690, 8 - 1 4 .  Modulation by a n i o n s .  Molla,  A . , Capony, J . - P . and D e m a i l l e , J . G . (1983) R e g u l a t i o n o f c a r d i a c sarcoplasmic reticulum calcium pumping In: Calcium-Binding P r o t e i n s 1983 (deBernad, B . , Scottocasa, G . L . , Sandri, G . , C a r a f o l i , E . , T a y l o r , A . N . , Vanaman, T . C . and W i l l i a m s , R . J . P . , e d s . ) p p . 3 9 3 - 3 9 9 , E l s e v i e r S c i e n c e , Amsterdam.  Moore,  L . , Chen, T . and Knapp, H.R (1978) Energy-dependent calcium s e q u e s t r a t i o n a c t i v i t y i n r a t l i v e r microsomes. J . B i o l . Chem. 250. 4562-4568.  Moses,  L. and A n a n d a r a j , M.P.J.S. (1987) Membrane Ca Mg -ATPase activation by c a l c i u m a c t i v a t e d neutral protease (CANP) in c o n d i t i o n s where t h e r e i s e l e v a t e d i n t r a c e l l u l a r Ca . an i n s i g h t from e r y t h r o c y t e s o f Duchenne muscular d y s t r o p h y (DMD). Seventh International Washington Spring Symposium. Cell Calcium M e t a b o l i s m . Washington, a b s . 132.  2 +  267  2 +  Muallem,  S . and K a r l i s h , S . J . D . (1979) Is the r e g u l a t e d by ATP? Nature 277, 238-240.  red  cell  calcium  pump  Muallem,  S. and K a r l i s h , S . J . D . (1982) R e g u l a t i o n of the C a - p u m p by c a l m o d u l i n i n i n t a c t c e l l s . B i o c h i m . B i o p h y s . A c t a 687, 329-332.  Muallem,  S., S c h o e f i e l d , M., Pandol, S . , Sachs, G. (1985) Inositol t r i s p h o s p h a t e m o d i f i c a t i o n o f ion t r a n s p o r t i n rough endoplasmic r e t i c u l u m . P r o c . N a t l . A c a d . S c i . USA 82, 4433-4437.  Mueller,  P. and Rudin, D.O. (1968) Action b i m o l e c u l a r l i p i d membranes. Nature 217,  2+  potentials 713-719.  induced  in  2 +  M u r a c h i , T . (1983a) I n t r a c e l l u l a r C a p r o t e a s e and i t s i n h i b i t o r p r o t e i n : C a l p a i n and c a l p a s t a t i n I n : Calcium and C e l l F u n c t i o n s (Cheung, W . Y . , e d . ) V o l . IV, pp. 377-410, Academic P r e s s , New Y o r k . M u r a c h i , T . (1983b) C a l p a i n and c a l p a s t a t i n . 169.  Trends Biochem. S c i . 8,  involving  calpain  167-  Murachi,  T . (1989) I n t r a c e l l u l a r r e g u l a t o r y system c a l p a s t a t i n . Biochem. Int. 18, 263-294.  and  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 p r o t e a s e ( c a l p a i n ) and i t s highm o l e c u l a r - w e i g h t endogenous i n h i b i t o r ( c a l p a s t a t i n ) I n : Advance i n Enzyme R e g u l a t i o n (Wiker, G . , e d . ) V o l . 19, 407-423. Pergamon Press, Oxford.  Murakami,  K. and Etlinger, J.D. (1986) Endogenous inhibitor of nonlysosomal high m o l e c u l a r weight p r o t e a s e and c a l c i u m - d e p e n d e n t p r o t e a s e P r o c . N a t l . A c a d . S c i . USA 83, 7588-7592.  Murakami,  U. and U c h i d a , K. (1979) Degradation o f r a 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 p r o t e i n s by a m y o s i n - c l e a v i n g p r o t e a s e . J. Biochem. 86, 553-562.  Murtaugh,  T . J . , Rowe P . M . , V i n c e n t , P . L . , W r i g h t , L . S . and S i e g e ! , F . L . (1983) P o s t t r a n s l a t i o n a l modifications of calmodulin. Methods Enzymol. 102, 158-170.  Myers, M.W,., L a z z a r i n i , R . A . , Lee, V. M . - Y . , S c h l a e p f e r , W.W. and N e l s o n , D . L . (1987) The human m i d - s i z e n e u r o f i l a m e n t s u b u n i t : a repeated protein sequence and the relationship of its gene to the i n t e r m e d i a t e f i l a m e n t gene f a m i l y . EMB0 J . 6, 1617-1626. Nairn,  Nairn,  A . C . , Hemmings, H . C . J r . and Greengard, P. (1985a) i n the b r a i n . Ann. Rev. Biochem. 54, 931-976.  Protein  kinases  A . C . , Bhagat, B. and P a l f r e y , H.C. (1985b) Identification of calmodulin-dependent p r o t e i n k i n a s e III and i t s major M 100,000 r  268  s u b s t r a t e i n mammalian 7939-7943.  tissues.  Proc.  Natl.  Acad.  S c i . USA 82,  2 +  Naray,  A. (1981) The effect g l u c o c o r t i c o i d receptor 14, 71-76.  of a Ca -activated protease on the o f lymphoid t i s s u e s . J . S t e r o i d Biochem.  Nelson,  W . J . and T r a u b , P. (1981) P r e p e r t i e s o f a C a - a c t i v a t e d p r o t e a s e s p e c i f i c f o r the i n t e r m e d i a t e - s i z e d f i l a m e n t p r o t e i n v i m e n t i n i n E h r i c h - a s c i t e s - t u m o u r c e l l s . E u r . 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 o f the Ca pumping ATPase o f heart sarcolemma and e r y t h r o c y t e plasma membrane by the cAMP-dependent p r o t e i n k i n a s e . J . B i o l . Chem. 260, 10283-10287.  z +  z +  Nicotera,  P . , McConkey, D . , Svenson, S . A . , B e l l o m o ^ G. and O r r e n i u s , S . (1988) Correlation between c y t o s o l i c Ca concentration and cytotoxicity in hepatocytes exposed to oxidative stress. T o x i c o l o g y 52, 55-63.  Niggli,  V . , Adunyah, E . S . and C a r a f o l i , E. (1981a) A c i d i c p h o s p h o l i p i d s , unsaturated f a t t y a c i d s 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 c a l m o d u l i n on the p u r i f i e d e r y t h r o c y t e Ca - A T P a s e . J . B i o l . Chem. 256, 8588-8592.  Niggli,  V., Adunyah, p E . S . * , P e n n i s t o n , J . T . and C a r a f o l i , E. (1981b) Purified (Ca -Mg^ )-ATPase of the erythrocyte membrane. R e c o n s t i t u t i o n and e f f e c t o f c a l m o d u l i n and p h o s p h o l i p i d s . J . B i o l . Chem. 256, 395-401. z+  Niggli,  +  V . , P e n n i s t o n , 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 o f the (Ca +Mg )-ATPase from human e r y t h r o c y t e membranes using a c a l m o d u l i n a f f i n i t y column. J . B i o l . Chem. 254, 9955-9958. z+  2+  Niggli,  V . , Ronner, P . , C a r a f o l i , E and Penniston J . T . (1979a) E f f e c t s o f calmodulin on the (Ca-Mg -ATPase p a r t i a l l y purified from e r y t h r o c y t e membrane. A r c h . Biochem. B i o p h y s . 198, 124-130.  Niggli,  V . , S i g e l , E. and C a r a f o l i , E. (1982a) I n h i b i t i o n o f the and reconstituted calcium pump of erythrocytes c o n c e n t r a t i o n s o f DIDS and N A P - t a u r i n e . FEBS l e t t . 138,  Niggli,  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 pump o f human e r y t h r o c y t e membranes c a t a l y z e s an e l e c t r o n e u t r a l Ca - H exchange i n r e c o n s t i t u t e d l i p o s o m a l system. J . B i o l . Chem. 257, 2350-2356.  purified by /iM 164-166.  2 +  +  Niggli,  V., Zurini, M. and Carafoli, E. (1987) Purification, r e c o n s t i t u t i o n , and m o l e c u l a r c h a r a c t e r i z a t i o n o f the C a pump o f the plasma membranes. Methods Enzymol. 139, 791-809. z +  269  N i s h i z u k a , Y . (1984) The r o l e o f p r o t e i n k i n a s e C i n c e l l s u r f a c e t r a n s d u c t i o n and tumor p r o m o t i o n . Nature 308, 693-698. O ' H a l l o r a n , T . , B e c k e r l e , M.C. and B u r r i d g e , K. t a l i n as a major c y t o p l a s m i c p r o t e i n a c t i v a t i o n . Nature 317, 449-451.  signal  (1985) I d e n t i f i c a t i o n o f implicated in p l a t e l e t  Ohno, S . , E m o r i , T . , Imajoh, S . , Kawasaki, H . , K i s a r a g i , M. and S u z u k i , K. (1984) E v o l u t i o n a r y o r i g i n o f a c a l c i u m - d e p e n d e n t p r o t e a s e by f u s i o n o f genes f o r a t h i o l p r o t e a s e and a c a l c i u m - b i n d i n g protein. Nature 312, 566-570. Ohno, S . , Kawasaki, H . , Imajoh, S . , S u z u k i , K . , I n a g a k i , M . , Yokokura, H . , Sakoh, T . and H i d a k a , H. (1987) T i s s u e - s p e c i f i c e x p r e s s i o n o f t h r e e d i s t i n c t types o f r a b b i t p r o t e i n k i n a s e C . Nature 325, 161-166. O l o r u n s o g o , 0 . 0 . , Okudolo, B . E . . Lawal, S . O . A . and F a l a s e , A . O . (1985) E r y t h r o c y t e membrane Ca -pumping ATPase o f h y p e r t e n s i v e humans: reduced s t i m u l a t i o n by c a l m o d u l i n . B i o s c i e n c e Reports 5, 525-531. O l o r u n s o g o , 0 . 0 . , V i l l a l o b o , A . , Wang, K.K.W. and R o u f o g a l i s , B . D . (1988) The e f f e c t o f c a l m o d u l i n on the i n t e r a c t i o n o f c a r b o d i i m i d e s with the purified human erythrocyte (Ca +Mg )-ATPase. Biochim. B i o p h y s . A c t a 945, 3 3 - 4 0 . z +  Olson,  Ono,  I.J. and C a z o r t , R . J . (1969) A c t i v e t r a n s p o r t i n human e r y t h r o c y t e g h o s t s . 322. T.,  Or-lov,  2 +  calcium and s t r o n t i u m J . Gen. P h y s i o l . 53, 311-  Koide, Y . , A r a i , Y. and Yamashita, K. (1984) calmodulin-binding protein in rat t e s t i s . J . B i o l . 9011-9016.  Heat-stable Chem. 259,  S . N . . . P o k u d i n , N . I . and P o s t n o v , V . Y . (1983) Calmodul in-dependent Ca transport in erythrocytes o f spontaneously hypertensive r a t s . P f l u g e r s A r c h . 397, 5 4 - 5 6 . z +  Ortega,  A. and M a s - O l i v a , J. (1986) Direct regulatory effect of c h o l e s t e r o l on the c a l m o d u l i n s t i m u l a t e d c a l c i u m pump o f c a r d i a c sarcolemma Biochem. B i o p h y s . R e s . Commun. 1 3 9 , 868-874.  Panagia,  V . , OJaimura, K . , Makino, N. and D h a l l a , N . S . (1986) S t i m u l a t i o n of C a pump i n r a t heart sarcolemma by p h o s p h a t i d y l - e t h a n o l a m i n e N - m e t h y l a t i o n . Biochem. B i o p h y s . A c t a 856, 383-387. 2 +  Pant,  Pant,  H . C . (1988) D e p h o s p h o r y l a t i o n o f n e u r o f i l a m e n t p r o t e i n s enhances t h e i r s u s c e p t i b i l i t y t o d e g r a d a t i o n by c a l p a i n . Biochem. J . 256, 665-668. H.C, Virmani, M. and proteolysis of spectrin  Gallant, and band  270  P . E . (1983) 3 protein in  Calcium-induced rat erythrocyte  membranes. Papp,  372-377.  B . , S a r k a d i , B . , E n y e d i , A . , C a r i d e , A . J . , P e n n i s t o n , J . T . and Gardos, G. (1989) F u n c t i o n a l domains o f the j n s i t u red c e l l membrane c a l c i u m pump r e v e a l e d by p r o t e o l y s i s and monoclonal a n t i b o d i e s . P o s s i b l e s i t e s f o r r e g u l a t i o n 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,  Patel,  Biochem. B i o p h y s . Res. Commun. 117,  R . A . , M i l l e r , S . J . and G i l s o n , D.M. (1986) P h o s p h o r y l a t i o n s t a t e of HMG CoA r e d u c t a s e affects its catalytic activity and d e g r a d a t i o n . Adv. Enzym. R e g u l . 25, 329-343. K . , S t r o n g , P . N . , Dubowitz, V. and Dunn, M . J . (1988) Calmodulinb i n d i n g p r o f i l e s f o r n e b u l i n and d y s t r o p h i n i n human s k e l e t a l muscle. FEBS l e t t . 234, 267-271.  Pearlstone, J.R., Carpenter, M.R. and S m i l l i e , L.B. (1977a) Primary s t r u c t u r e o f r a b b i t s k e l e t a l muscle t r o p o n i n - T . P u r i f i c a t i o n o f cyanogen bromide fragments and the amino acid sequence of fragment CB2. J . B i o l . Chem. 252, 971-977. Pearlstone, J.R., Carpenter, M.R. and S m i l l i e , L.B. (1977b) Primary s t r u c t u r e o f r a b b i t s k e l e t a l muscle t r o p o n i n T . J . B i o l . Chem. 252, 978-982. Pedemonte, C . H . and B a l e g r o , H . F . (1981) I s o l a t i o n o f a thermostable m o d i f i e r o f Ca - s t i m u l a t e d ATPase from human red c e l l s Biochem. B i o p h y s . A c t a 99, 994-1000. 2 +  P e n n i s t o n , J . T . (1983) Plasma membrane C a - A T P a s e s as C a C a l c i u m and C e l l F u n c t i o n s (Cheung, W . Y . , e d . ) V o l . 149, Academic P r e s s , New Y o r k .  2 +  pumps. IV, pp.  2+  In: 99-  2+  Pershadsingh, H.A. and McDonald, J.M. (1981) (Ca +Mg )-ATPase in a d i p o c y t e plasmalemma: i n h i b i t i o n by i n s u l i n and c o n c a n a v a l i n A i n the i n t a c t c e l l . Biochem. I n t . 2, 243-248. Plancke,  Poland,  Y . D . and L a z a r i d e s , E. (1983) Evidence f o r a p h o s p h o r y l a t e d form o f c a l m o d u l i n i n c h i c k e n b r a i n and m u s c l e . Mol. C e l l . B i o l . 3, 1412-1420. A . and G l o v e r , receptor. Arch.  2+  E. (1988) C a - d e p e n d e n t p r o t e o l y s i s Biochem. B i o p h y s . 261, 103-111.  P o n t r e m o l i , S . and M e l l o n i , E. (1986a) E x t r a l y s o s o m a l p r o t e i n Ann. Rev. Biochem. 55, 455-481.  of  the  Ah  degradation.  2+  Pontremoli, S. and M e l l o n i , E. (1986b) Regulation of Ca -dependent p r o t e i n a s e of human e r y t h r o c y t e s . In: Calcium and C e l l F u n c t i o n s , (Cheung, W . Y . , e d . ) V o l . V I , pp. 159-183, Academic P r e s s , New York.  271  P o n t r e m o l i , S . , M e l l o n i , E . and H o r e c k e r , B . L . (1985a) R e g u l a t i o n o f mammalian cytosolic Ca - r e q u i r i n g neutral proteinases In: Current T o p i c s i n C e l l u l a r R e g u l a t i o n ( S h a l t i e l , S . and Chock, P . B . , e d s . ) V o l . 27, pp. 293-304, Academic P r e s s , New Y o r k . Pontremoli, S . , Melloni, E . , Michetti, M., Sacco, 0 . , Salamino, F., S p a r a t o r e , B. and H o r e c k e r , B . L . (1986a) B i o c h e m i c a l responses i n a c t i v a t e d human n e u t r o p h i l s mediated by p r o t e i n k i n a s e C and a C a - r e q u i r i n g p r o t e i n a s e . J . B i o l . Chem. 261, 8309-8313. z +  P o n t r e m o l i , S . , M e l l o n i , E . , M i c h e t t i , M . , Salamino, F . , S p a r a t o r e , B. and Horecker, B . L . (1988) An endogenous a c t i v a t o r o f the Ca dependent p r o t e i n a s e o f human n e u t r o p h i l s t h a t i n c r e a s e s i t s a f f i n i t y f o r C a . P r o c . N a t l . A c a d . S c i . USA 8 5 , 1740-1743. z +  Pontremoli, S . , M e l l o n i , E . , M i c h e t t i , M., Sparatore, B . , Salamino F, S a c c o , 0. and H o r e c k e r , B . L . (1987b) P h o s p h o r y l a t i o n by p r o t e i n kinase C o f a 20 kDa c y t o s k e l e t a l polypeptide enhances its s u s c e p t i b i l i t y t o d i g e s t i o n by c a l p a i n . P r o c . N a t l . A c a d . S c i . 84, 398-401. P o n t r e m o l i , S . , M e l l o n i , E . , Salamino, F . , S p a r a t o r e , B., Viotti, P., M i c h e t t i , M . , D u z z i , L. and B i a n c h i , G. (1986b) Decreased l e v e l of calpain inhibitor a c t i v i t y i n red b l o o d c e l l s from M i l a n hypertensive rats. Biochem. B i o p h y s . Res. Commun. 138, 13701375. Pontremoli, S . , M e l l o n i , E . , Salamino, F . , Sparatore, B . , M i c h e t t i , M., S a c c o , 0. and B i a n c h i , G. (1987b) Decreased l e v e l o f c a l p a i n inhibitor activity in kidney from M i l a n hypertensive rats. Biochem. B i o p h y s . Res. Commun. 145, 1287-1294. Pontremoli, S . , M e l l o n i , E . , Sparatore, B M i c h e t t i , M. and H o r e c k e r , B . L . (1984) A dual r o l e f o r the Ca - r e q u i r i n g p r o t e i n a s e i n the d e g r a d a t i o n o f hemoglobin by e r y t h r o c y t e membrane p r o t e i n a s e s . P r o c . N a t l . A c a d . S c i . USA 81_, 6714-6717. y  Pontremoli, S . , M e l l o n i , E . , Sparatore, B . , Salamino, F . , M i c h e t t i , M., S a c c o , 0. and H o r e c k e r , 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 o f Ca dependent neutral p r o t e i n a s e . Biochem. B i o p h y s . Res. Commun. 128, 331-338. P o n t r e m o l i , S . , S p a r a t o r e , B . , Salamino, F . , M i c h e t t i , M . , S a c c o , 0. and M e l l o n i , E . (1985c) R e v e r s i b l e a c t i v a t i o n o f human n e u t r o p h i l calpain promoted by i n t e r a c t i o n with plasma membranes. Biochem. I n t . ]_1, 35-44. Prentki,  M . , B i d e n , T . J . , J a n j i c , D . , I r v i n e , R . F . , B e r r i d g e , M . J . and Wolheim, C . B . (1984) Rapid m o b i l i z a t i o n o f Ca from rat i n s u l i n o m a 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 . , N o l a , E . , S i c a , V. and B r e s c i a n i , F. (1977) Estrogen b i n d i n g proteins of calf uterus. Molecular and functional c h a r a c t e r i z a t i o n o f the r e c e p t o r t r a n s f o r m i n g f a c t o r : a r e activated 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 structure o f hamster vimentin predicted from the n u c l e o t i d e sequence. P r o c . N a t l . A c a d . S c i . USA. 8 0 , 35483552. Quist,  E . E . and R o u f o g a l i s , B . D . (1975) Calcium t r a n s p o r t i n human e r y t h r o c y t e : s e p a r a t i o n and r e c o n s t i t u t i o n o f high and low Ca a f f i n i t y ( M g + C a ) - A T P a s e i n membranes prepared at low i o n i c s t r e n g t h . A r c h . Biochem. B i o p h y s . 168, 240-251. z+  Rabbani,  Raess,  z+  N . , Moses, L . , A n a n d a v a l l i , T . E . and A n a n d a r a j , M . P . J . S . (1984) C a l c i u m - a c t i v a t e d n e u t r a l protease from muscle and p l a t e l e t s o f Duchenne muscular dystrophy c a s e s . C l i n i c a Chimica A c t a 143, 163-168.  B . U . and V i n c e n z i , F . F . (1980) A Semi-automated determination of multiple membrane ATPase Pharmacol. Methods 4 , 273-283.  R e c h s t e i n e r , M. (1987a) U b i q u i t i n - m e d i a t e d p r o t e o l y s i s . A n n . Rev. C e l l B i o l . 3 ,  pathways 1-30.  method f o r activities.  for  the J.  intracellular  R e c h s t e i n e r , M. (1987b) R e g u l a t i o n o f enzyme l e v e l s by p r o t e o l y s i s : r o l e o f PEST r e g i o n s . A d v . Enzym. R e g u l . 27, 135-151.  the  Rega, A . F . and G a r r a h a n , P . J . (1975) Calcium ion-dependent p h o s p h o r y l a t i o n o f human e r y t h r o c y t e membranes. J . Membrane. B i o l . 22, 313-327. Rega, A . F . and G a r r a h a n , P . J . (1978) Calcium independent d e p h o s p h o r y l a t i o n o f the C a - A T P a s e o f human red c e l l s by ADP. B i o c h i m . B i o p h y s . A c t a 507, 182-184. + +  Rega,  A . F . and G a r r a h a n , P . J . (1980) E f f e c t s o f c a l m o d u l i n on the phosphoenzyme o f the Ca -ATPase o f human r e d c e l l membranes. Biochem. B i o p h y s . A c t a 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 o f the 7 s u b u n i t o f p h o s p h o r y l a s e b k i n a s e with cAMP-dependent p r o t e i n kinase. B i o c h e m i s t r y 23, 4185-4192.  R e i t h m e i e r , R . A . F . , O h n i s h i , M . , C a r p e n t e r , M . R . , S l u p s k y , J . R . , Gounden, K., Fliegel, L., Khanna, V . K and MacLennan D.H. (1987) C a l s e q u e s t r i n I n : C a l c i u m - B i n d i n g P r o t e i n s i n H e a l t h and D i s e a s e (Means, A . R . , e d . ) p p . 6 2 - 7 1 , Academic P r e s s , New Y o r k .  273  Ribadeau-Dumas, B . , B r i g n o n , G . , G r o s c l a u d e , F. and M e r c i e r , J . - C . (1972) The complete primary s t r u c t u r e o f / J - c a e i n . E u r . J . Biochem. 25, 505-514. Richards  Rivett,  Rixon,  , D . E . , Rega, A . F . arid G a r r a h a n , P . J . (1978) Two c l a s s a s o f s i t e for ATP i n the C a - A T P a s e from human red c e l l membranes. B i o c h i m . B i o p h y s . A c t a 5_n, 194-201. A . J . (1989) The m u l t i c a t a l y t i c Biochem. B i o p h y s . 268, 1-8.  protease  o f mammalian  cells.  Arch.  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 o f a complementary d e o x y r i b o n u c l e i c a c i d coding f o r the a c h a i n o f human f i b r i n o g e n . B i o c h e m i s t r y 22, 3238-3244.  R o e l o f s e n , B and Schatzmann, H . J . (1977) The l i p i d requirement o f the (Ca^ +Mg^ )-ATPase i n the human e r y t h r o c y t e membrane, as s t u d i e d by various highly p u r i f i e d phospholipases. Biochim. Biophys. A c t a 464, 17-36. ?  +  +  Rogers,  S . , W e l l s , R. and R e c h s t e i n e r , M. (1986) Amino a c i d sequences common t o r a p i d l y degraded p r o t e i n s : the PEST h y p o t h e s i s . S c i e n c e 234, 364-368.  Romero,  P . J . and Whittam, R.(1971) The c o n t r o l by i n t e r n a l c a l c i u m o f membrane p e r m e a b i l i t y t o sodium and p o t a s s i u m . J . P h y s i o l . 214, 481-507.  Romero,  P . J . - a n d Romero, E. (1984) The Modulation o f the c a l c i u m pump o f human r e d c e l l s by N a and K . B i o c h i m . B i o p h y s . A c t a 778, 245252. +  Romero,  +  P . J . and O r t i z , C E . (1988) E l e c t r o g e n i c b e h a v i o r o f t h e r e d c e l l Ca^ pump r e v e a l e d by d i s u l f o n i c s t i l b e n e s . J . Membrane Biol. 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 ( C a ^ + M g ^ ) - a c t i v a t e d ATPase o f e r y t h r o c y t e membranes. Arch. Biochem. B i o p h y s . 179, 578-583. +  +  R o s e n t h a l , W., H e s c h e l e r , J . , T r a u t w e i n , W, a n d S c h u l t z , G. (1988) C o n t r o l + of voltage-dependent c G-protein-coupled 0 Ca? receptors. F A S E B J . 2, 2784-2790. n  f  i  a  n  n  e  l  s  b  y  R o t t e n b e r g , H. (1979) N o n - e q u i l i b r i u m thermodynamics o f energy c o n v e r s i o n i n b i o e n e r g e t i c s . B i o c h i m . B i o p h y s . A c t a 549, 225-253. R o u f o g a l i s , B . D . (1979) R e g u l a t i o n red blood c e l l membrane. 1349.  o f the c a l c i u m t r a n s l o c a t i o n a c r o s s the C a n . J . P h y s i o l . Pharmacol. 57, 1331-  274  R o u f o g a l i s , B . D . and M a u l d i n , D. (1980) R e g u l a t i o n by c a l m o d u l i n o f the calcium a f f i n i t y o f the c a l c i u m t r a n s p o r t ATPase i n human erythrocytes. Can. J . Biochem. 58, 922-926. 2 +  2 +  Roufogalis, B.D. and V i l l a l o b o , A. (1989) The (Ca +Mg )-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 f o r S o l u t e T r a n s p o r t (Raess, B. and T u n n i c l i f f , G . , e d s . ) Humana Press I n c . , New J e r s e y , i n p r e s s . R o u f o g a l i s , B . D . , E l l i o t t , C T . and R a l s t o n , G . B . (1984) C h a r a c t e r i z a t i o n of a (Ca +Mg )-ATPase activator bound t o human e r y t h r o c y t e membranes. C e l l Calcium 5, 77-88. z+  Sakai,  2+  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 activated neutral protease indicated in histone h y d r o l y s i s . J . Biochem. i p _ l , 911-918.  Sakihama,  T . , K a k i d a n 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 . , K a n n a g i , R . , N a k a n i s h i , S . , Ohmori, M . , T a k i o , K . , T i t a n i , K. and M u r a c h i , T . (1985) A p u t a t i v e Ca - b i n d i n g p r o t e i n : s t r u c t u r e o f t h e l i g h t s u b u n i t o f p o r c i n e c a l p a i n e l u c i d a t e d by m o l e c u l a r c l o n i n g and p r o t e i n sequence a n a l y s i s . Proc. N a t l . Acad. S c i . USA 8 2 , 6075-6079.  Sarkadi,  B. (1980) A c t i v e c a l c i u m t r a n s p o r t B i o p h y s . A c t a 604, 159-190.  Sarkadi,  B . , E n y e d i , A . and Gardos, G. (1980) M o l e c u l a r p r o p e r t i e s o f the r e d c e l l c a l c i u m pump. I. Effects of calmodulin, proteolytic d i g e s t i o n and drugs on k i n e t i c s o f a c t i v e c a l c i u m uptake i n i n s i d e - o u t r e d c e l l membrane v e s i c l e s . C e l l C a l c i u m 1 , 287-310.  Sarkadi,  B . , E n y e d i , A . , F o l d e s - P a p p , Z . and G a r d o s , G. (1986) M o l e c u l a r c h a r a c t e r i z a t i o n o f the i n s i t u r e d c e l l membrane c a l c i u m 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.  Sarkadi,  B . , E n y e d i , A . and Gardos, G. (1987) C o n f o r m a t i o n a l changes o f the in situ red c e l l membrane calcium pump affects its p r o t e o l y s i s . B i o c h i m . B i o p h y s . A c t a 899, 129-133.  Sarkadi,  B . , M a c l n t y r e , J . D . and Gardos, t r a n s p o r t i n i n s i d e - o u t red c e l l 89, 7 8 - 8 2 .  Sarkadi,  B . , S c h u b e r t , A . and Gardos, G. (1979) E f f e c t s o f calcium-EGTA buffers on a c t i v e calcium transport in inside-out red c e l l membranes. E x p e r i e n t i a 35, 1045-1047.  Sarkadi,  B . , S z a s z , I., G e r l o c z y , A . and G a r d o s , G . , (1977) T r a n s p o r t parameters and s t o i c h i o m e t r y o f a c t i v e c a l c i u m i o n e x t r u s i o n i n i n t a c t human red cells. B i o c h i m . B i o p h y s . A c t a 464, 92-107.  275  i n human r e d c e l l s .  Biochim.  G. (1978) K i n e t i c s o f a c t i v e membrane v e s i c l e s . FEBS l e t t .  Sasaki,  T . , K i k u c h i , T . , Yumoto, N . , Yoshimura, N. and M u r a c h i , T . (1984) Comparative s p e c i f i c i t y and k i n e t i c s t u d i e s on p o r c i n e c a l p a i n I and c a l p a i n II with n a t u r a l l y o c c u r r i n g p e p t i d e s and s y n t h e t i c f l u o r o g e n i c s u b s t r a t e s . J . B i o l . Chem. 259, 12489-12494. 2 +  2 +  2 +  Scharff,  0. (1976) Ca -activation of membrane-bound (Ca +Mg )dependent ATPase from human e r y t h r o c y t e s prepared i n the presence or absence o f Ca . B i o c h i m . B i o p h y s . A c t a 43, 206-218.  Scharff,  0. and F o d e r , B. (1982) Rate c o n s t a n t s C a - A T P a s e i n e r y t h r o c y t e membranes. 691, 133-143.  Scharff,  0 . , F o d e r , B. and S k i b s t e d , U. (1983) H y s t e r e t i c the C a pump r e v e a l e d by c a l c i u m t r a n s i e n t s c e l l s . Biochim. B i o p h y s . Acta 730, 295-305.  f o r calmodulin binding to Biochim. Biophys. Acta  z +  Schatzmann, H . J . (1966) ATP-dependent C a E x p e r i e n t i a 22, 364-365.  + +  extrusion  activation of i n human red  from human red  cells.  Schatzmann, H . J . (1969) Transmembrane c a l c i u m movements i n r e s e a l e d human red c e l l s I n : Calcium and C e l l F u n c t i o n s ( C u t h b e r t , A . W . , e d . ) pp. 8 5 - 9 5 , Cuthbert Macmillan C o . , London. 2 +  Schatzmann, H . J . (1978) A c t i v e c a l c i u m t r a n s p o r t and C a - a c t i v a t e d ATPase i n human red c e l l s . I n : Current t o p i c s i n membranes and t r a n s p o r t (Bronner, F. and Klemzeller, A . , eds) V o l . 6, pp. 126-168, Academic P r e s s , New Y o r k . Schatzmann, H . J . (1985) Calcium e x t r u s i o n a c r o s s the plasma membrane by the c a l c i u m pump and the Ca - N a exchange system I n : C a l c i u m and Cell P h y s i o l o g y (Marme, D . , e d . ) pp. 19-52, S p r i n g e r - V e r l a g , New York. +  2 +  2 +  Schatzmann, H . J . and R o s s i , G . L . (1971) ( C a + M g ) - a c t i v a t e d membrane ATPase i n 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 t o c a t i o n t r a n s p o r t : B i o c h i m . B i o p h y s . A c t a 241, 379-392. Schatzmann, H . J . and V i n c e n z i , F . F . (1969) Calcium movements a c r o s s membrane o f human red c e l l s . J . P h y s i o l . 201, 369-395. S c h e c h t e r , I. and B e r g e r , A . p r o t e a s e s . I. P a p a i n .  (1967) On the s i z e o f the a c t i v e Biochem. B i o p h y s . Res. Commun. 27,  the  site in 157-162.  Schmaier,  A . H . , S m i t h , P . A . , Purdon, A . D . , White, J . G . and Colman, R.W. (1986) High m o l e c u l a r weight k i n i n o g e n : localization in the u n s t i m u l a t e d and a c t i v a t e d 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 calpain(s). Blood 67, 119-130.  Schmidt,  J . W . , H i n d s , T . R . and V i n c e n z i , F . F . (1985) On the f a i l u r e o f c a l m o d u l i n to a c t i v a t e C a pump ATPase o f dog red blood c e l l s . Comp. Biochem. P h y s i o l . 82A, 601-607. 2 +  276  Scott-Woo, G . C . and Walsh, M.P. (1988) Characterization of the a u t o p h o s p h o r y l a t i o n o f c h i c k e n g i z z a r d caldesmon. Biochem. J . 255, 817-824. Seiler,  S . , Wegener, A . D . , Whang, D . D . , Hathaway, D.R. and J o n e s , L . R . (1984) High m o l e c u l a r weight p r o t e i n s i n c a r d i a c and s k e l e t a l muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are p h o s p h o r y l a t e d , and a r e degraded by Ca a c t i v a t e d p r o t e a s e . J . B i o l . Chem. 259, 8550-8557.  Seubert,  P . , Baudry, M . , Dudek, S . and L y n c h , G . (1987) Calmodulin s t i m u l a t e s the d e g r a d a t i o n o f b r a i n 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 s o l a t i o n o f bovine b r a i n c a l m o d u l i n dependent c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e i s o z y m e s . Methods. Enzymol. 159, 582-595.  Sharma, R . K . , Wang, T . H . , W i r c h , E . and Wang, J . H . (1980) P u r i f i c a t i o n and properties of brain calmodulin-dependent cyclic nucleotide p h o s p h o d i e s t e r a s e . J . B i o l . Chem. 255, 5916-5923. Shattuck,  Shull,  R . L . , Yeager, R . E . and Storm, D.R. (1987) C a l m o d u l i n - s t i m u l a t e d a d e n y l a t e c y c l a s e I n : Calcium and C e l l F u n c t i o n s (Cheung, W . Y . , e d . ) V o l . V I I , p p . 3 9 - 6 0 , Academic P r e s s , New Y o r k .  G . E . and Greeb, J . (1988) M o l e c u l a r c l o n i n g o f t w o - i s o f o r m s o f the plasma membrane C a - t r a n s p o r t i n g ATPase from r a t b r a i n . J. B i o l . Chem. 263, 8646-8657. z +  Sibley,  Siman,  D . R . , B e n o v i c , J . L . , C a r o n , M.G. Regulation of transmembrane p h o s p h o r y l a t i o n . C e l l 48, 913-922.  and L e f k o w i t z , R . J . (1987) signalling by receptor  R . , Baudry, M. and L y n c h , G. (1984) B r a i n f o d r i n : s u b s t r a t e f o r c a l p a i n I, an endogenous c a l c i u m - a c t i v a t e d p r o t e a s e . P r o c . N a t l . A c a d . S c i . USA 8 1 , 3572-3576.  Sitaramayya, A., Wright, L.S. and S i e g e l , methylation of calmodulin i n r a t brain 255. 8894-8900.  F.L. (1980) Enzymatic cytosol. J . B i o l . Chem.  Smallwood, J . I . , Waisman, D . M . , L a f r e n i e r e , D. and Rasmussen, H. (1983) Evidence t h a t the e r y t h r o c y t e c a l c i u m pump c a t a l y z e s a Ca .nH exchange. J . B i o l . Chem. 258, 11092-11097. Smallwood, J.I., Giigi, B. and Rasmussen, H. (1988) Regulation of erythrocyte Ca pump a c t i v i t y by p r o t e i n k i n a s e C . J . Biol. Chem. 262, 2195-2202. z +  Smith,  T . J . , Davis,  F.B.  and D a v i s ,  277  P.J.  (1989)  Retinoic  acid  is  a  modulator o f t h y r o i d hormone human e r y t h r o c y t e membrane.  a c t i v a t i o n o f Ca -ATPase i n J . B i o l . Chem. 262, 687-689.  the  Sobue,  K., Fugita, M., Muramoto, Y. and K a k i u c h i , S. (1981c) The c a l m o d u l i n - b i n d i n g p r o t e i n i n m i c r o t u b u l e s i s tau f a c t o r . FEBS l e t t . 132, 137-140.  Sobue,  K . , Muramoto, Y . , F u g i t a , M. and K a k i u c h i , S . (1981a) C a l m o d u l i n binding protein of erythrocyte c y t o s k e l e t o n . Biochem. B i o p h y s . Res. Commun. 100, 1063-1070.  Sobue, K . , Muramoto, Y . , F u g i t a , 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 calmodulin-binding protein from chicken gizzard that i n t e r a c t s with F - a c t i n . P r o c . N a t l . A c a d . S c i . USA 78, 5652-5655. Soldati,  L., L o n g o n i , S . and C a r a f o l i , E. (1985) r e c o n s t i t u t i o n o f the N a / C a exchanger o f J . B i o l . Chem. 260, 13321-13327. +  2 +  Spedding,  Squire,  Stone,  Stryer,  z +  M. (1987) Three types o f C a channel T r e n d s . Pharmacol. S c i . 8, 117-119.  J . M . (1983) M o l e c u l a r mechanisms Neuro. S c i . 6, 409-413. D.  explain  muscular  discrepancies.  contraction.  and S m i l l i e , L . B . (1978) The amino a c i d sequence s k e l e t a l or-tropomyosin. J . B i o l . Chem. 253, 1137-1148.  L . (1986) 87-119.  Sugita,  in  Solubilization and c a r d i a c sarcolemma.  C y c l i c GMP cascade o f  vision.  Ann. Rev.  of  Trends  rabbit  Neurosci.  9,  H., Ishiura, S., Nonaka, I. and S u g i t a , H. (1980) Calcium a c t i v a t e d protease (CANP) and i t s i n h i b i t o r i n muscular d y s t r o p h y I n : Muscular Dystrophy ( E b a s h i , S . and Ozawa, E . , e d s . ) pp. 265282, U n i v e r s i t y o f Tokyo P r e s s , Tokyo.  Suzuki,  K. (1987) Calcium a c t i v a t e d n e u t r a l p r o t e a s e : domain s t r u c t u r e activity regulation. Trends Biochem. S c i . H , 103-105.  Suzuki,  K . , Imajoh, S . , E m o r i , Y . , Kawasaki, H . , Minami, Y. and Ohno, S . (1987a) C a l c i u m - a c t i v a t e d n e u t r a l p r o t e a s e and i t s endogenous inhibitor. FEBS l e t t . 220, 271-277.  Suzuki,  K . , Imajoh, S . , E m o r i , Y . , Kawasaki, H . , Minamik, Y. and Ohno, S . (1987b) R e g u l a t i o n of a c t i v i t y of calcium activated neutral p r o t e a s e . Adv. Enzyme R e g u l . 27, 153-169.  Suzuki,  K., T s u y i , S . and I s h i u r a , S. (1981) E f f e c t o f C a on the i n h i b i t i o n o f c a l c i u m - a c t i v a t e d n e u t r a l p r o t e a s e by l e u p e p t i n , a n t i p a i n and e p o x y s u c c i n a t e d e r i v a t i v e s . FEBS l e t t . 136, 119-122.  Szasz,  and  2 +  I.,  Hasito,  M.,  Sarkadi,  B.  and Gardos, G.  278  (1978)  Phosphorylation  1  o f the Ca* pump i n t e r m e d i a t e i n i n t a c t c e l l s , and i n s i d e - o u t v e s i c l e s (1978) M o l e c . C e l l . 152. Tada,  i s o l a t e d membranes Biochem. 22, 147-  M., Kirchberger, M.A. and Li, H.C. (1975) Phosphoprotein p h o s p h a t a s e - c a t a l y z e d d e p h o s p h o s p h o r y l a t i o n o f the 22,000 d a l t o n phosphoprotein of cardiac sarcoplasmic reticulum. J. Cyclic N u c l e o t i d e s Res. 1, 329-338.  Tahara,  S . M . and Traugh, J . A . (1982) D i f f e r e n t i a l activation of two p r o t e a s e - a c t i v a t e d p r o t e i n k i n a s e s from r e t i c u l o c y t e s by a Ca stimulated protease and identification of phosphorylated t r a n s l a t i o n a l components. E u r . J . Biochem. 126, 395-399.  Takeyama,  Y . , N a k a n i k s h i , H . , U r a t s u j i , Y . , K i s h i m o t o , A . and N i s h i z u k i , Y. (1986) A c a l c i u m - p r o t e a s e a c t i v a t o r a s s o c i a t e d with brain microsomal i n s o l u b l e elements. FEBS l e t t . 194, 110-114.  Takio,  K . , S m i t h , S . B . , K r e b s , E . G . , Walsh, K . A . and T i t a n i , K. (1984) Amino a c i d sequence o f the r e g u l a t o r y s u b u n i t o f bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase. B i o c h e m i s t r y 23, 4200-4206.  Tallant,  E.A. and Cheung, W.Y. (1984) Activation of calmodulin-dependent protein phosphatase trypsinization. B i o c h e m i s t r y 23, 973-979.  bovine brain by limited  Tallant,  E.A. and Cheung, W.Y. (1986) Calmodulin-dependent protein phosphatase i n : Calcium and C e l l F u n c t i o n s (Cheung, W . Y . , ed.) V o l . V I , pp. 71-112, Academic P r e s s , New Y o r k .  Tallant,  E . A . , Brumley, L.M. and W a l l a c e , R.W. (L988) A c t i v a t i o n o f a calmodulin-dependent phosphatase by a Ca -dependent p r o t e a s e . B i o c h e m i s t r y 2_7, 2205-2211.  Taverna,  R.D. and Hanahan, D . J . (1980) M o d u l a t i o n o f human e r y t h r o c y t e Ca /Mg ATPase a c t i v i t y by p h o s p h o l i p a s e A and p r o t e a s e s . A comparison with c a l m o d u l i n . B i o c h i m . B i o p h y s . Res. Commun. 94, 652-659. z +  2  Tiffert,  T., G a r c i a - S a n c h o , J . and Lew, V . L . (1984) I r r e v e r s i b l e ATP d e p l e t i o n caused by low c o n c e n t r a t i o n s o f formaldehyde and o f calcium-chelator esters in intact human red c e l l s . Biochim. B i o p h y s . A c t a 773, 143-156.  Tinoco,  J . , S a v i r , K. and Wong, J . (1978) and A p p l i c a t i o n s in B i o l o g i c a l Jersey.  Titani,  K . , Kumar, S . , Tako, K . , E r i c s s o n , L . H . , Wade, R . D . , A s h i d a , K . , Walsh, K . A . , Chopek, M.W., S a d l e r , J . E . and F u j i k a w a , K. (1986) Amino a c i d sequence of human von W i l l e b r a n d f a c t o r . Biochemistry  279  Physical Chemistry: S c i e n c e s , p. 124,  Principles Hall, New  25, Toyo-Oka,  Traub,  3171-3184.  T. (1982) Phosphorylation with cyclic adenosine 3'5' Monophosphate-dependent p r o t e i n k i n a s e r e n d e r s bovine t r o p o n i n sensitive to the degradation by calcium-activated neutral p r o t e a s e . Biochem. B i o p h y s . Res. Commun. 1 0 7 , 4 4 - 5 0 .  P . , S c h e r b a r t y h , A . , W i l l i n g a l e - T h e u n e , J . and P a u l i n - L e v a s s e u r , 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 o f v i m e n t i n and n u c l e a r 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 r o t e i n a s e . E u r . J . C e l l B i o l . 46, 478-490.  Tremblay,  J. and Hamet, P. (1984) Calcium-dependent proteolytic s t i m u l a t i o n o f adenylate c y c l a s e i n p l a t e l e t s from spontaneously hypertensive r a t s . Metabolism 3 3 , 689-695.  T r u g l i a , J . A . and S t r a c h e r , 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 o f a c a l c i u m dependent s u l f h y d r y l p r o t e a s e from human p l a t e l e t s . Biochem. B i o p h y s . Res. Commun. 100, 814-822. Ullrich,  A . , Coussens, L . , H a y f l i c k , J . S . , D u l l , T . J . , G r a y , A . , Tarn, A . W . , L e e , 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 . , W h 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 r e c e p t o r cDNA sequence and aberrant e x p r e s s i o n o f the a m p l i f i e d gene i n A431 epidermoid carcinoma c e l l s . Nature 309, 418-425.  Vasington, F . D . and Murphy, J . (1961) A c t i v e m i t o c h o n d r i a . F e d . P r o c . 20, 146.  binding  of  calcium  by  2 +  V a s i n g t o n , F . D . and Murphy, J . C . (1962) Ca uptake by r a t kidney mitochondria and its dependence on respiration and p h o s p h o r y l a t i o n . J . B i o l . Chem. 237, 2670-2772. Vedeckis,  W . Y . , Freeman, M . R . , S c h r a d e r , W.T. and 0 ' M a l l e y , B.W. (1980) Progesterone-binding components of chick oviduct: partial 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 o f a c a l c i u m - a c t i v a t e d p r o t e a s e which h y d r o l y z e s the progesterone r e c e p t o r . B i o c h e m i s t r y 19, 335-349.  Verma, A . K . , F i l o t e o , A . G . , S t a n d o r d , D . R . , Wieben, E . D . , P e n n i s t o n , J . T . , S t r e h l e r , E . E . , F i s c h e r , R . , Heim, R . , V o g e l , G . , Mathews, S . , Strehler-Page M.-A., James P . , Vorherr, T., K r e b s , J . and C a r a f o l i , E . (1988) Complete primary s t r u c t u r e o f a human plasma membrane C a pump. J . B i o l . Chem. 263, 14152-14159. z +  Vezzoli,  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 i n e r y t h r o c y t e s o f s p o n t a n e o u s l y h y p e r t e n s i v e r a t s of the M i l a n s t r a i n . J . Hypertension 3 , 645-648.  V i l l a l o b o , A . and R o u f o g a l i s , B . D . (1986) Proton c o u n t e r t r a n s p o r t by the reconstituted erythrocyte Ca - t r a n s l o c a t i n g ATPase: Evidence  280  using ionophoretic  compounds.  J . Membrane B i o l .  93,  249-258.  V i l l a l o b o , A . , Brown, L. and R o u f o g a l i s , B.D. (1986) K i n e t i c p r o p e r t i e s o f 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. B i o c h i m . B i o p h y s . A c t a 854, 9-20. Vincenzi,  Vrolix,  F . F . and H i n d s , T . R . (1988) Decreased Ca pump ATPase a c t i v i t y a s s o c i a t e d with i n c r e a s e d d e n s i t y i n human red blood c e l l s . Blood C e l l s 14, 139-159.  M . , Raeymaekers, L . , Wuytack, F . , Hofmann, F. and C a s t e e l s , R. (1988) Cyclic GMP-dependent protein kinase stimulates the plasmalemmal Ca pump o f smooth muscle v i a p h o s p h o r y l a t i o n o f p h o s p h a t i d y l i n o s i t o l . Biochem. J . 255, 855-863. z +  Waelkens,  E . , G o r i s , J . and Merlevede, W. (1985) A c t i v a t i o n o f the PCSyp r o t e i n phosphatase by a Ca -dependent p r o t e a s e . FEBS l e t t . 192, 317-320.  Waisman,  D.M., Gimble, J . M . , Goodman, D.B. and Rasmussen, H. (1981a) S t u d i e s on the C a transport mechanism o f human erythrocyte inside out plasma membrane v e s i c l e s . I. R e g u l a t i o n o f the C a pump by c a l c i u m , c a l m o d u l i n , ATP and pH. J . B i o l . Chem. 256, 409-414. z  z +  Waisman,  D . M . , Gimble, J . M . . Goodman, D . B . and Rasmussen, H. (1981b) S t u d i e s on the C a transport mechanism o f human erythrocyte inside out plasma membrane v e s i c l e s . II. Stimulation o f the Ca pump by phosphate. J . B i o l . Chem. 256, 415-419. z +  z +  Waisman,  D.M., Gimble, J . M . , Goodman, D . B . P . , Rasmussen, S t u d i e s o f the C a transport mechanism o f Human I n s i d e - o u t plasma membrane v e s i c l e s . III. Stimulation pump by a n i o n s . J . B i o l . Chem. 256, 420-424. z +  Wakim,  Wang,  2 +  B . T . , A l e x a n d e r , K . A . , Masure, H . R . , C i m l e r , B . M . , Storm, D.R. and Walsh, K . A . (1987) Amino a c i d sequence o f P-57, a n e u r o s p e c i f i c calmodulin-binding protein. B i o c h e m i s t r y 26, 7466-7470.  Wallace,  Walsh,  H. (1981c) Erythrocyte o f the C a  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 calmodulin-binding proteins: 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 . B i o c h e m i s t r y 26, 27662773. M.P., Dabrowska, R., Hinkins, S. and H a r t s h o r n e , D.J. (1982) Calcium-independent myosin l i g h t c h a i n k i n a s e o f smooth m u s c l e . P r e p a r a t i o n by l i m i t e d c h y m o t r y p t i c d i g e s t i o n o f the c a l c i u m ion dependent enzyme, purification, and characterization. B i o c h e m i s t r y 21, 1919-1925.  J . H . , P a l l e n , C . J . , Sharma, R . K . , (1985) The c a l m o d u l i n r e g u l a t o r y  281  Adachi, system.  A . - M . and A d a c h i , K. Current Topics Cell.  Regul. Wang,  27,  419-469.  K.K.W., Roufogalis, B.D. and Villalobo, A. (1988b) Further c h a r a c t e r i z a t i o n o f the c a l p a i n - m e d i a t e d p r o t e o l y s i s o f the human e r y t h r o c y t e plasma membrane C a - A T P a s e . A r c h . Biochem. B i o p h y s . 267, 317-327.  Wang, K . K . W . , V i l l a l o b o , A . and R o u f o g a l i s , B.D. (1988a) A c t i v a t i o n o f the C a - A T P a s e o f human e r y t h r o c y t e membrane by an endogenous Ca dependent n e u t r a l p r o t e a s e . A r c h . Biochem. B i o p h y s . 260, 696704. Wang,  K.K.W., Roufoaalis, B.D. and V i l l a l o b o , A. (1989a) Calpain I activates C a t r a n s p o r t by the human e r y t h r o c y t e plasma membrane calcium pump I n : Calcium Binding Proteins in Normal and Transformed C e l l s (Lawson, D . E . M . and P o c h e t , R., e d s . ) Plenum P r e s s , London, i n p r e s s . z +  Wang,  K.K.W., Villalobo, binding proteins press.  A. as  and R o u f o g a l i s , B.D. (1989b) calpain substrates. Biochem.  CalmodulinJ . 262, in  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 . (1989c) C h a r a c t e r i z a t i o n o f the fragmented forms o f 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, i n p r e s s .  Wang,  K.K.W., Roufogalis, B.D. and V i l l a l o b o , A . . (1989d) Calpain I a c t i v a t e s C a ^ t r a n s p o r t by the r e c o n s t i t u t e d e r y t h r o c y t e Ca pump. J . Membrane B i o l . , i n p r e s s . +  z +  Watson,  E.L., Vincenzi, F.F. and D a v i s , P.W. (1971) N u c l e o t i d e s as s u b s t r a t e s o f C a - A T P a s e and NaK-ATPase i n i s o l a t e d red c e l l membranes. L i f e S c i . 1 0 , 1399-1404.  Warxman,  L. (1981) Calcium-activated Methods Enzymol. 80, 664-680.  proteases  in  mammalian  tissues.  Welsh,  M . J . , A s t e r , J . C . , I r e l a n d , M . , A l c a l a , J . and M a i s e l , H. (1982) Calmodulin binds to c h i c k l e n s gap j u n c t i o n p r o t e i n i n a c a l c i u m independent manner. S c i e n c e 2_16, 642-644.  White,  M.F. and Kahn, C R . (1986) The i n s u l i n r e c e p t o r and tyrosine p h o s p h o r y l a t i o n . I n : The Enzymes (Boyer, P.D. and K r e b s , E . G . , e d s . ) V o l . 17, pp. 247-310, Academic P r e s s , New Y o r k .  W i l k i n s o n , J . M . and Grand, R . J . A . (1975) The amino a c i d sequence o f t r o p o n i n I from r a b b i t s k e l e t a l m u s c l e . Biochem. J . 149, 493-496. Wins,  P.  and S c h o f f e n i e l s (19,66) S t u d i e s on r e d - c e l l ghost ATPase systems. P r o p e r t i e s o f a ( M g + C a ) - d e p e n d e n t ATPase. Biochim. Biophys. A c t a 120, 341-350. z+  z+  282  Wolf,  H . U . and G i e t z e n , K. (1974) The s o l u b i l i z a t i o n Ca -ATPase o f human e r y t h r o c y t e membranes P h y s i o l . 335, 1272.  o f high a f f i n i t y Hoppe-Seyler's Z.  Wolf,  H . U . , D i e c k v o s s , G. and L i c h t n e r R. (1977) P u r i f i c a t i o n and properties of high-affinity Ca -ATPase o f human erythrocyte membranes. A c t a B i o l . Med. Germ. 3 6 , 847-858.  Wong,  P . Y . K . and Cheung, W.Y. (1979) Calmodulin s t i m u l a t e s human p l a t e l e t p h o s p h o l i p a s e A 2 . Biochem. B i o p h y s . R e s . Commun. 90, 473-480.  Wuthrich,  A . (1982) I s o l a t i o / i from haemolysate o f a p r o t e i n a c o u s i n h i b i t o r o f the red c e l l Ca -pump ATPase. I t s a c t i o n on t h e k i n e t i c s o f the enzyme. C e l l Calcium 3 , 201-214.  Xu,  and R o u f o g a l i s , B . D . (1988a) Asymmetric e f f e c t s of divalent c a t i o n s and protons on a c t i v e C a e f f l u x and C a - A T P a s e i n i n t a c t r e d blood c e l l s . J . Membrane. B i o l . 105, 155-164.  Y.H.  z +  Xu,  z +  Y . H . and R o u f o g a l i s , B . D . (1988b) ATP dependence o f a c t i v e c a l c i u m transport in r e d blood cells In: Progress in Biochemical Pharmacology. Symposium on Circulating Sodium Transport Inhibitors (Paoletti, R., e d . ) p p . 107-118, S . K a r g e r , A . G . Basel.  Yamamoto,  K . , K o s a k i , G . , S u z u k i , K . , Tanoue, K. and Yamazaki, H. (1986) Cleavage s i t e o f calcium-dependent p r o t e a s e i n human p l a t e l e t membrane g l y c o p r o t e i n l b . Thrombosis Res. 43, 4 1 - 5 5 .  Yeager,  R . E . , Heideman, W., Rosenberg, G . B . and Storm, P u r i f i c a t i o n o f the c a l m o d u l i n - s e n s i t i v e a d e n y l a t e bovine c e r e b r a l c o r t e x . B i o c h e m i s t r y 24, 3776-3783.  Yeoman,  L . C . , O l s o n , M . O . J . , Sugano, N . , J o r d a n , J . J . , T a y l o r , C . W . , S t a r b u c k , W.C. and Busch, H. (1972) Amino a c i d sequence o f the c e n t e r o f 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 S t r a c h e r , A . (1988) Phosphorylation of p l a t e l e t a c t i n b i n d i n g p r o t e i n p r o t e c t s a g a i n s t p r o t e o l y s i s by c a l c i u m dependent s u l f h y d r y l protease. Biochem. B i o p h y s . R e s . Commun. 151, 355-360.  Zimmerman, U.J. and Schlaepfer, W.W. (1984) neutral p r o t e a s e (CANP) i n b r a i n and o t h e r N e u r o b i o l . 23, 63-78. Zurini,  D.R. (1985) c y c l a s e from  M . , K r e b s , J . , P e n n i s t o n , J . T . and C a r a f o l i , proteolysis o f the p u r i f i e d Ca-ATPase membrane. J . B i o l . Chem. 259, 618-627.  283  Calcium tissues.  activated Progress  E . (1984) C o n t r o l l e d o f the erythrocyte  APPENDIX  L i s t o f amino a c i d s and t h e i r t h r e e - l e t t e r  amino a c i d Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic a c i d Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine  t h r e e - l e t t e r code  and o n e - l e t t e r codes  o n e - l e t t e r code  Ala Arg Asn Asp Cys Glu Gin Gly His lie Leu Lys Met Phe Pro Ser Thr Trp Tyr Val  A R N D C E Q G H I L K M F P S T W Y V  284  PUBLICATIONS Papers 1. Wang. K . K . W . . V i l l a l o b o , A . and R o u f o g a l i s , B . D . (1988) A c t i v a t i o n o f the C a - A T P a s e o f human e r y t h r o c y t e membrane by an endogenous Ca -dependent neutral protease. A r c h . Biochem. B i o p h y s . 260, 696-704. 2 +  2+  2.  O l o r u n s o g o , 0 . 0 . , V i l l a l o b o , A . , Wang, K.K.W. and R o u f o g a l i s , B . D . (1988) The e f f e c t o f c a l m o d u l i n on the i n t e r a c t i o n o f c a r b o d i i m i d e s with the p u r i f i e d human e r y t h r o c y t e ( C a + M g ) ATPase. B i o c h i m . B i o p h y s . A c t a 945, 3 3 - 4 0 . 2 +  3.  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) F u r t h e r c h a r a c t e r i z a t i o n o f the c a l p a i n - m e d i a t e d p r o t e o l y s i s o f t h e human e r y t h r o c y t e plasma membrane C a - A T P a s e . A r c h . Biochem. B i o p h y s . 26_7_, 317-327. 2 +  4.  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) C a l p a i n I a c t i v a t e s C a * t r a n s p o r t by the human e r y t h r o c y t e plasma membrane c a l c i u m pump I n : Calcium B i n d i n g P r o t e i n s i n Normal and Transformed C e l l s (Lawson, D . E . M . and Pochet, R . , e d s . ) Plenum P r e s s , London, i n p r e s s . 2  5.  Wang. K . K . W . . V i l l a l o b o . A . and R o u f o g a l i s , B . D . (1989) C a l m o d u l i n b i n d i n g p r o t e i n s as c a l p a i n s u b s t r a t e s . Biochem. J . 262, i n press.  6.  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) C h a r a c t e r i z a t i o n o f the fragmented forms o f 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 p r e s s .  7.  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) C a l p a i n a c t i v a t e s C a * t r a n s p o r t by the r e c o n s t i t u t e d e r y t h r o c y t e pump. J . Membrane B i o l . , i n p r e s s . 2  8.  I Ca  2 +  R o u f o g a l i s , B . D . , B r z u s z c z a k , I., Xu, Y . - H . , C g n i g r a v e , A . D . , Machan, C. and Wang, K.K.W. (1989) P e r s i s t e n t C a - i n d u c e d a c t i v a t i o n of e r y t h r o c y t e membrane C a - A T P a s e u n r e l a t e d to c a l p a i n p r o t e o l y s i s . , submitted. z +  2 +  9.  Wang, K . K . W . , Machan, C , A l l a n , B . G . and R o u f o g a l i s , B . D . (1989) P r o t e i n k i n a s e C p h o s p h o r y l a t e s the c a r b o x y l - t e r m i n a l o f t h e human e r y t h r o c y t e C a - A T P a s e . , s u b m i t t e d . 2 +  10.  Wang, K . K . W . , Machan, C , V i l l a l o b o , A . and R o u f o g a l i s , B . D . (1989) 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 o f two novel c a l m o d u l i n - and p h o s p h o l i p i d - b i n d i n g p r o t e i n s from human e r y t h r o c y t e membrane., in p r e p a r a t i o n .  11.  G i l c h r i s t . J . S . C . , Wang. K . K . W . , K a t z , 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 j u n c t i o n a l s a c r o p l a s m i c r e t i c u l u m c a l c i u m r e l e a s e channel by c a l p a i n I and II., in p r e p a r a t i o n .  PUBLICATIONS  (cont.)  Abstracts 2 +  1. Wang. K.K.W. and R o u f o g a l i s , B . D . (1987) A c t i v a t i o n o f the C a ATPase o f the human red c e l l membrane by endogenous c a l p a i n . V l l t h I n t e r n a t i o n a l Washington Spring Symposium. Washington, D . C . , USA. A b s . 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 o f the e r y t h r o c y t e C a - A T P a s e by c a l p a i n . S i x t h I n t e r n a t i o n a l Symposium on C a l c i u m - B i n d i n g P r o t e i n s in H e a l t h and D i s e a s e . Nagoya, J a p a n . A b s . 167. 2 +  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) C a l p a i n I a c t i v a t e s C a * t r a n s p o r t by the r e c o n s t i t u t e d human e r y t h r o c y t e plasma membrane C a - A T P a s e . F i r s t European Symposium on C a l c i u m B i n d i n g P r o t e i n s i n Normal and Transformed C e l l s . B r u x e l l e s , B e l g i u m . A b s . H2. 2  2 +  4.  G i l c h r i s t , J . S . C . , Wang, K . K . W . . K a t z , S. and B e l c a s t r o , A . N . (1989) C a t i o n - d e p e n d e n t p u r i f i c a t i o n of the 360 kDa channel p r o t e i n from d e t e r g e n t s o l u b i l i s e d j u n c t i o n a l t e r m i n a l c i s t e r n a e sarcoplasmic reticulum using calmodulin-agarose a f f i n i t y chromatography. F i r s t European Symposium on C a l c i u m B i n d i n g P r o t e i n s i n Normal and Transformed C e l l s . B r u x e l l e s , B e l g i u m . A b s . 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:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098268/manifest

Comment

Related Items