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Anion regulation of Ca2+ transport ATPase of the human erythrocyte membrane Minocherhomjee, A. M. 1982

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ANION REGULATION OF C  a  2 +  TRANSPORT ATPase OF  THE HUMAN ERYTHROCYTE MEMBRANE  by  A. M.  MINOCHERHOMJEE  M . S c , The U n i v e r s i t y o f T o r o n t o , 1978  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES ( F a c u l t y of Pharmaceutical  Sciences)  We accept t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d standards  THE UNIVERSITY  OF BRITISH COLUMBIA  A p r i l 1982 (c) A . M . Minocherhomjee, 1982  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e for reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s f o r s c h o l a r l y purposes may department or by h i s or her  be  granted by  the head of  representatives.  understood that copying or p u b l i c a t i o n of t h i s f o r f i n a n c i a l gain  s h a l l not be  Department of  Pharmaceutical Sciences  The  U n i v e r s i t y of B r i t i s h Columbia  1956  Main Mall  V6T  1Y3  Vancouver, Canada  Date  DE-6  (3/81)  A p r i l 27, 1982.  my  It is thesis  allowed without my  permission.  thesis  written  ABSTRACT  The mechanism o f r e g u l a t i o n o f t h e Ca  pump ATPase o f t h e human  e r y t h r o c y t e membrane by c a l m o d u l i n , c y c l i c AMP and t h e a n i o n channel was s t u d i e d u s i n g membrane f r a g m e n t s ,  r e s e a l e d "ghosts", i n s i d e - o u t  vesicles  and a T r i t o n X-100 s o l u b i l i z e d enzyme p r e p a r a t i o n . 2+ The (Ca  2+ + Mg )-ATPase a c t i v i t y  i n e r y t h r o c y t e membranes o r a  T r i t o n X-100 s o l u b i l i z e d enzyme p r e p a r a t i o n showed b i p h a s i c ( h i g h and low 2+ affinity)  Ca  activation kinetics.  The a n i o n i c c a l c i u m b i n d i n g p r o t e i n ,  c a l m o d u l i n , i n c r e a s e d both t h e c a l c i u m s e n s i t i v i t y maximum v e l o c i t y  (V  (poly-L-aspartic  acid,  m a x  ) o f the enzyme.  ( 1 ^ 2 + ) and t h e  C e r t a i n polyaniom'c  agents  poly-L-glutamic a c i d ) , a l i c y c l i c sulfonic acids  (HEPES,N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic a c i d , MES,2-N( m o r p h o l i n o e t h a n e s u l f o n i c a c i d ) ) , and a r o m a t i c c a r b o x y l i c a c i d s ( b e n z o i c 2+ but but not not the the VV and s a l i c y l i c a c i d s ) i n c r e a s e d t h e KCa 2+ max. o f (Ca 2+ r  2+  m = v  r  Mg  +  )-ATPase i n e r y t h r o c y t e membranes and T r i t o n X-100 s o l u b i l i z e d enzyme  preparations.  Trifluoperazine  (30 yM) a n t a g o n i z e d a c t i v a t i o n o f  the  enzyme by c a l m o d u l i n and p o l y - L - a s p a r t i c a c i d , but not by sodium-HEPES 2+ 2+ o r sodium-MES.  Limited trypsin  proteolysis of  (Ca  + Mg  )-ATPase  i n t h e e r y t h r o c y t e membrane a b o l i s h e d a c t i v a t i o n by c a l m o d u l i n ,  poly-L-  a s p a r t i c a c i d and sodium-HEPES. These r e s u l t s suggest t h a t t h e modula2+ 2+ 2+ t i o n o f the Ca s e n s i t i v i t y o f (Ca + Mg )-ATPase by c a l m o d u l i n may be a s s o c i a t e d w i t h the a n i o n i c p r o p e r t i e s o f t h i s p r o t e i n , and t h a t property  can be mimicked by some o t h e r a n i o n s , p r o b a b l y by i n t e r a c t i n g  a t an a n i o n - r e g u l a t o r y  s i t e on the enzyme.  C y c l i c AMP (5 yM) was found t o i n h i b i t t h e ( C a activity  this  2 +  + Mg )-ATPase  ( a p p r o x . 20%) i n e r y t h r o c y t e membranes, p r o b a b l y v i a  2+  endogenous  c y c l i c AMP p r o t e i n k i n a s e , s i n c e t h i s e f f e c t c o u l d be b l o c k e d by c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r C.PKI) from t h e r a b b i t s k e l e t a l m u s c l e , By c o n t r a s t ,  bovine heart PKI s t i m u l a t e d (Ca  2+  + Tig  2+  )-ATPase  activity  ( a p p r o x . 100%) by i n c r e a s i n g t h e K 2+ but not the V „ o f t h e enzyme ua max. r  i n membrane o r T r i t o n X - 1 0 0 s o l u b i l i z e d p r e p a r a t i o n s .  At a low c a l c i u m  c o n c e n t r a t i o n the s t i m u l a t i o n by bovine heart PKI and s a t u r a t i n g  levels  o f c a l m o d u l i n was a d d i t i v e , s u g g e s t i n g t h a t the two e f f e c t o r s 2+ 2+  a c t e d by  d i s t i n c t mechanisms.  activity  The s t i m u l a t i o n o f (Ca  + Mg  )-ATPase  by bovine heart PKI was not s o l e l y due to i t s antagonism o f the  protein  k i n a s e because a) m o d i f i c a t i o n o f a r g i n i n e r e s i d u e s o f bovine heart  PKI  a b o l i s h e d i t s i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e , but had no e f f e c t 2+ 2+ on the s t i m u l a t i o n of (Ca + Mg ) - A T P a s e ; b) t r i f l u o p e r a z i n e (20 yM) 2+ 2+ a n t a g o n i z e d the s t i m u l a t i o n o f (Ca  + Mg  )-ATPase by P K I , s i m i l a r l y to  i t s antagonism o f c a l m o d u l i n s t i m u l a t i o n , but i t d i d not a f f e c t i n h i b i t i o n of p r o t e i n k i n a s e by P K I .  It  i s suggested t h a t  the  different  mechanisms a r e i n v o l v e d  i n the i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e 2+ 2+ PKI. the s t i m u l a t i o n o f (Ca and + Mg )-ATPase by bovine heart c y c l i c AMP 2+ 2+ N e x t , the r o l e o f a n i o n channel b l o c k e r s on the (Ca + Mg ) -  ATPase was s t u d i e d .  The p h o t o l a b e l i n g reagent  N-(4-azido-2-m"trophenyl)2+  2 aminoethylsulfonate  (NAP-taurine)  was found to i n h i b i t the (Ca  +  2+ Mg  )-ATPase o f fragmented red c e l l membranes.  inhibition 2+ o c c u r r e d between 25 yM and 50 yM. At t h e s e c o n c e n t r a t i o n s Mg -ATPase and ( N a + K ) - A T P a s e a c t i v i t i e s i n the membranes were not a f f e c t e d . +  H a l f maximal  +  2+ The r e v e r s i b l e  i n h i b i t i o n o f (Ca  2+ + Mg  )-ATPase produced by NAP-  t a u r i n e i n the dark became i r r e v e r s i b l e a f t e r  p h o t o l y s i s i n the  presence  Civ)  ^ of t h i s reagent.  ' 2 + I n c u b a t i o n o f t h e membranes w i t h Ca  c a l m o d u l i n , p r i o r to p h o t o l y s i s  2+  2  Mg  +  , ATP o r  1n t h e presence o f N A P - t a u r i n e , d i d not  p r o t e c t t h e enzyme from I n h i b i t i o n . (Ca  ,  Limited trypsin  proteolysis  of  2+ + Mg  )-ATPase i n fragmented membranes, which a b o l i s h e d a c t i v a t i o n  by c a l m o d u l i n , d i d not a f f e c t t h e i n h i b i t i o n by N A P - t a u r i n e . 2+ N A P - t a u r i n e was found to I n h i b i t  t h e (Ca  2+ + Mg  )-ATPase  activity  from t h e c y t o p l a s m i c s i d e o f t h e membrane, as determined from t h e  follow-  ing e x p e r i m e n t s .  A d d i t i o n o f N A P - t a u r i n e (50 yM) to r e s e a l e d e r y t h r o c y t e 2+ 2+ ghosts i n h i b i t e d l e s s than b% o f t h e (Ca + Mg )-ATPase a c t i v i t y , compared to 50-60°' I n h i b i t i o n 1n ghosts r e s e a l e d i n the presence o f 50 yM 2+ NAP-taurine. F u r t h e r m o r e , N A P - t a u r i n e i n h i b i t e d ATP-dependent Ca transport  into i n s i d e - o u t v e s i c l e s at a s i m i l a r concentration 2+  The i n h i b i t i o n o f t h e (Ca  (.50 yM).  2+ + Mg  )-ATPase a c t i v i t y  o f membranes by NAP-  t a u r i n e appeared to be a d i r e c t a c t i o n on the enzyme, r a t h e r than through 2+ 2+ i n h i b i t i o n o f the a n i o n c h a n n e l , as (Ca  + Mg  )-ATPase a c t i v i t y was not  i n h i b i t e d i n membranes made from red blood c e l l s r e a c t e d  irreversibly  w i t h 50 yM N A P - t a u r i n e or the a n i o n channel b l o c k e r 4 , 4 ' - d i i s o t h i o c y a n o 2 , 2 ' s t i l b e n e d i s u l f o n a t e (DIDS) (5yM) or i n membranes assayed i n the presence o f a n o t h e r a n i o n channel b l o c k e r , p r o b e n e c i d (125 yM). This i s 2+ the f i r s t r e p o r t e d s e l e c t i v e a n t a g o n i s t o f the Ca pump, and i t 1s s u g 2+ gested t h a t N A P - t a u r i n e c o u l d be a u s e f u l t r a n s p o r t ATPase i n a v a r i e t y o f Signature of t h e s i s  supervisor  cells.  t o o l f o r s t u d y i n g t h e Ca  -  TABLE QF CONTENTS page  ABSTRACT  <  .  .  ,  LIST OF TABLES  (i1) (vi11)  LIST OF FIGURES  Ox)  LIST OF ABBREVIATIONS  ( )  INTRODUCTION  1  x i  1.  Regulation of c e l l u l a r calcium  2.  Calcium t r a n s p o r t and ( C a + Mg )-ATPase a c t i v i t y - Basic C h a r a c t e r i s t i c s  3.  R e g u l a t i o n o f t h e c a l c i u m pump ATPase i n human  2 +  .  .  .  .  .  .  .  .  3  2+  6  ^  erythrocytes  4.  .  .  .  .  A)  Role o f c a l m o d u l i n  15  B)  Role o f p r o t e i n k i n a s e s  27  C)  R o l e o f a n i o n channel (Band 3)  .  .  .  .  .  .  30  Aims o f the p r e s e n t study  31  MATERIALS AND METHODS I. II.  33  Materials  33  Methods  36  1. 2.  P r e p a r a t i o n o f e r y t h r o c y t e ghosts by v a r i o u s procedures . . .  36 .  .  .  .  .  .  P r e p a r a t i o n of c a l m o d u l i n d e f i c i e n t  37  (EDTA t r e a t e d ) membranes 3.  Preparation of resealed ghosts  4.  Preparation of inside-out v e s i c l e s  5.  I r r a d i a t i o n o f red c e l l s and membranes with N-(4-Azido-2-nitrophenyl)-2aminoethylsulfonate (NAP-taurine) . . S o l u b i l i z a t i o n of ( C a + M g ) - A T P a s e from e r y t h r o c y t e membranes .  6.  2 +  .  .  .  .  .  .  .  38 .  39 40  .  .  .  .  .  .  41  2+  (vi) page 7.  8.  Limited proteolysis o f membranes  Ctrypsim'zation) 41  P u r i f i c a t i o n o f c y c l i c AMP dependent protein kinase I n h i b i t o r  9.  42  Chemical m o d i f i c a t i o n o f a r g i n y l  side chains  .  .  43  10.  Assay o f ATPases  43  11.  Phosphodiesterase assay  44  12.  P r o t e i n kinase assay  13.  SDS 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  .  .  .  .  .  .  .  .  .  .  45  .  .  .  .  45  .  .  .  .  46  a)  Preparation of protein solutions  b)  Preparation of gels  46  c)  S t a i n i n g and d e s t a i n i n g  47  RESULTS AND DISCUSSION Section I  48  M e c h a n i s t i c s t u d y o f c a l m o d u l i n as an a c t i v a t o r o f c a l c i u m s e n s i t i v i t y and maximum v e l o c i t y o f (Ca2+ + Mg +)-ATPase  48  2  1. 2.  2+ 2+ 2+ K i n e t i c study o f Ca a c t i v a t i o n o f (Ca + Mg ) -ATPase and i t s m o d u l a t i o n by c a l m o d u l i n . .  E f f e c t o f p o l y - L - c a r b o x y l i c a c i d s and o t h e r a n i o n s on the Ca2+ a c t i v a t i o n o f (Ca2+ + Mg2+)-ATPase 51  S e c t i o n I I . E f f e c t o f c y c l i c AMP and c y c l i c AMP dependent p r o t e i n k i n a s e i n h i b i t o r s on (Ca2+ + Mg2+) -ATPase a c t i v i t y . . . . . . . . . . Section IE.  .  R o l e o f a n i o n channel (band 3) i n h i b i t o r s i n t h e r e g u l a t i o n o f (Ca2+ + Mg +)-ATPase a c t i v i t y . . 2  1.  2.  48  E f f e c t o f N A P - t a u r i n e on ( C a activity  2 +  66  83  + Mg )-ATPase 2+  The s i d e d n e s s o f N A P - t a u r i n e i n h i b i t i o n o f (Ca2+ + Mg2+)-ATPase  84  92  (vii) Page 3.  Does NAP-ta.urlne I n h i b i t the ( C a + Mg ) -ATPase and C a t r a n s p o r t by b l o c k i n g a n i o n channel? 2 +  2 +  2 +  CONCLUSIONS BIBLIOGRAPHY APPENDIX  ,  ,  ,  ,  92  ,  ,  104 , 107 123  (vi i i ) LIST OF TABLES page  TABLE  I.  E f f e c t o f a n i o n s on t h e Ca s e n s i t i v i t y of T r i t o n -X-100 s o l u b i l i z e d (Ca2+ + Mg2+)_  II.  Determination of calmodulin in protein preparations by p h o s p h o d i e s t e r a s e a s s a y and c a l m o d u l i n r a d i o immunoassay . . . . . . . . . . . . . .  57  E f f e c t o f a r g i n y l group m o d i f i c a t i o n o f bovine h e a r t c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r and c a l m o d u l i n on m o d u l a t i o n o f ( C a + T1g2+)ATPase a c t i v i t y o r c y c l i c AMP p r o t e i n k i n a s e (C-subunit) a c t i v i t y  78  E f f e c t o f t r i f l u o p e r a z i n e on t h e a b i l i t y o f c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r to s t i m u l a t e (Ca2+ + Mg2+)-ATPase i n 'Dodge g h o s t ' membranes . . . .  79  E f f e c t o f t r i f l u o p e r a z i n e on t h e i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e c a t a l y t i c s u b u n i t (C) c a t a l y s e d p h o s p h o r y l a t i o n o f h i s t o n e by p r o t e i n k i n a s e i n h i b i t o r from bovine h e a r t  80  III.  2 +  IV.  V.  VI.  2+ 2+ E f f e c t o f Mg , Ca , ATP and c a l m o d u l i n on the . i n h i b i t i o n of ( C a + Mg )-ATPase a c t i v i t y of membranes a f t e r p h o t o l y s i s i n the presence o f N A P - t a u r i n e (25 yM) 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y o f ' g h o s t s ' r e s e a l e d i n the presence and absence o f N A P - t a u r i n e . . . 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y o f membranes made from c e l l s t r e a t e d w i t h N A P - t a u r i n e o r D1DS. . . 2+ 2+ E f f e c t o f N A P - t a u r i n e on p u r i f i e d (Ca + Mg ) -ATPase a c t i v i t y 2 +  VII. VIII. IX.  2+  89 93 95 98  Ox)  LIST OF FIGURES page FIGURE  1.  2.  2+ 2+ C a l m o d u l i n a c t i v a t i o n o f (.Ca + Mg )-ATPase a c t i v i t y 1n human e r y t h r o c y t e membranes (Dodge Ghosts)  49  C a l m o d u l i n a c t i v a t i o n o f T r i t o n X-100 s o l u b i l i z e d (Ca + Mg )-ATPase a c t i v i t y  50  2 +  3.  2+  2+ E f f e c t o f p o l y - L - c a r b o x y l i c a c i d s on Ca activation o f human e r y t h r o c y t e membrane ( C a + Mg )ATPase a c t i v i t y 2+ E f f e c t o f p o l y - L - c a r b o x y l i c a c i d s on Ca activation of ( C a + M g ) - A T P a s e i n T r i t o n X-100 s o l u b i l i z e d enzyme from human e r y t h r o c y t e membranes . . . . 2+ E f f e c t o f s u l f o n i c a c i d a n i o n s on t h e Ca activation of ( C a + M g ) - A T P a s e i n human e r y t h r o c y t e membranes 2 +  4.  2 +  5.  2 +  52  2+  2 +  53  2+  54  6A. E f f e c t o f s u l f o n i c a c i d a n i o n s i n the presence and absence o f c a l m o d u l i n on C a a c t i v a t i o n of (Ca + M g ) - A T P a s e i n T r i t o n X-100 s o l u b i l i z e d (Ca + M g ) - A T P a s e from human e r y t h r o c y t e membranes  55  6B. The curves i n F i g u r e 6A were f i t t e d by a n o n - l i n e a r c u r v e - f i t t i n g computer program (see t e x t and T a b l e I)  56  2 +  7.  2 +  2+  2 +  2+  E f f e c t of i n o r g a n i c a n i o n s and a r o m a t i c c a r b o x y l i c a c i d s on C a a c t i v a t i o n o f T r i t o n X-100 s o l u b i l i z e d (Ca + M g ) - A T P a s e from human e r y t h r o c y t e s . . .  61  E f f e c t of p o l y - L - c a r b o x y l i c a c i d s and s u l f o n i c a c i d s on t r y p s i n t r e a t e d membranes .  62  E f f e c t o f t r i f l u o p e r a z i n e on human e r y t h r o c y t e membrane (Ca + M g ) - A T P a s e a c t i v i t y i n t h e presence o f p o l y - L - c a r b o x y l i c a c i d s or sodium-HEPES b u f f e r . .  64  SDS-PAGE o f p r o t e i n k i n a s e i n h i b i t o r and c a l m o d u l i n  69  2 +  2 +  8. 9.  2 +  10.  2+  2+  .  FIGURE 11.  12.  13. 14.  ,  page  E f f e c t o f c y c l i c AMP and PKI on ( C a + Mg )-ATPase a c t i v i t y i n human e r y t h r o c y t e membranes a t v a r i o u s concentrations of free calcium 2 +  2+  2+ E f f e c t o f c a l m o d u l i n and bovine heart PKI on (Ca + Mg2+)-ATPase a c t i v i t y i n e r y t h r o c y t e membranes and T r i t o n X-100 s o l u b i l i z e d enzyme a t 0 . 5 8 yM and 55 yM f r e e c a l c i u m 2+ 2+ A c t i v a t i o n o f (Ca + Mg )-ATPase i n human e r y t h r o c y t e membranes by c a l m o d u l i n and bovine h e a r t PKI . .  72 74  E f f e c t o f c a l m o d u l i n and PKI on c a l c i u m a c t i v a t i o n o f (Ca^+ + Mg2 )-ATPase i n human e r y t h r o c y t e +  membranes  15.  E a d i e - H o f s t e e p l o t o f data i n f i g u r e 14  16.  E f f e c t o f N A P - t a u r i n e on ( C a + Mg )-ATPase a c t i v i t y o f membranes C o n c e n t r a t i o n dependence o f N A P - t a u r i n e i n h i b i t i o n o f (Ca|+ + Mg2+)-ATPase , ( N a + K ) - A T P a s e and M g - A T P a s e a c t i v i t i e s by NAPtaurine  17.  71  2 +  75 76  2+  +  85  +  z+  18. 19.  20. 21. 22.  2+ 2+ E f f e c t o f N A P - t a u r i n e on (Ca + Mg )-ATPase i n membranes w i t h and w i t h o u t p h o t o l y s i s 2+ 2+ D o u b l e - r e c i p r o c o l p l o t o f (Ca + Mg )-ATPase i n h i b i t i o n by N A P - t a u r i n e i n t h e presence o f calmodulin . . . . . . . • 2+ 2+ E f f e c t o f N A P - t a u r i n e on (Ca + Mg )-ATPase i n t r y p s i n t r e a t e d membranes 2+ E f f e c t o f N A P - t a u r i n e on Ca - t r a n s p o r t i n i n s i d e - o u t (1.0.) vesicles Calcium uptake i n i n s i d e - o u t v e s i c l e s made from red blood c e l l s t r e a t e d w i t h DIDS (5 yM) a t 36°C f o r 30 min  86  87  90  91 94  99  LIST OF ABBREVIATIONS  ADP  adenosine 5"-diphosphate  ATP  adenosine  UTP  uridine  triphosphate  ITP  Inosine  triphosphate  GTP  guanosine  CTP  cytosine  DPG  2,3-diphosphoglycerate  P  inorganic  i  5'-triphosphate  triphosphate triphosphate  phosphate  c y c l i c AMP  c y c l i c adenosine 5'-monophosphate  c y c l i c GMP  c y c l i c guanosine  E  enzyme  EP  phosphorylated  ATPase  adenosine t r i p h o s p h a t a s e  (Ca  2 +  PKI DEAEEDTA EGTA  5'-monophosphate  enzyme  intermediate  2+  + Mg )-ATPase 2+  Mg  dependent c a l c i u m s t i m u l a t e d ATPase  protein kinase i n h i b i t o r diethylaminoethylethylenediamine t e t r a a c e t a t e , disodium s a l t ethyleneglycol-bis-(6-aminoethyl  HEPES  N,N -tetraacetic 1  ether)  acid  N-2-hydroxyethylpiperazineMES TES  N'-2-ethanesulfonic  acid  2-N-(morpholinoethanesulfonic Tris(hydroxy-methyl)-methyl  DTT  2-aminoethanesulfonic dithiothreitol  acid  acid)  PMSF  para-methyl  TCA  trichloroacetic acid  SDS  sodium dodecyl  PAGE  p o l y a c r y l amide gel  TFP NAP-taurine  sulfonylfluoride  sulfate electrophorlsis  trifluoperazine N-(4-aztdo-2  nltrophenyl)  2-aminoethyl  sulfonate  DIDS  4,4'-d1isothiocyano-2-2 -stilbene disulfonate  SITS  4-acetamido-4'-isothiocyano  ,  stilbene-2 -disulfonate 1  K^ 2+  a s s o c i a t i o n c o n s t a n t o f the enzyme f o r Ca  g  gram  IOV  inside-out vesicles  K.. diss,  d i s s o c i a t i o n constant  a  Michaelis-Menten constant m  m i11i  y  micro  M  molar  M  m o l e c u l a r weight  mg  milligram  sec  second  min .  minute  ml  milliliter  yl  microliter  mM  millimolar  concentration  yM  micromolar  concentration  nmoles  nanomoles  2+  Uiii) ACKNOWLEDGEMENTS  I would l i k e t o thank my t h e s i s s u p e r v i s o r , f o r h i s a d v i c e , encouragement and help throughout I am g r a t e f u l for  Dr. B,D.  Roufogalis,  t h i s work.  to D r s . D. B r o o k s , D. G o d i n , S . Katz and J ,  being on my a d v i s o r y  McNeill,  committee.  I g i v e s p e c i a l thanks t o Dr. A. A l - J o b o r e and Mr.. D. 'Mauldin f o r many h e l p f u l d i s c u s s i o n s and s u g g e s t i o n s . I am g r a t e f u l  to t h e Canadian Red Cross f o r the s u p p l y o f blood  and to t h e B.C. Heart Foundation f o r Finally,  financial assistance.  I w i s h to thank a l l the members o f t h e f a c u l t y  my program a t U . B . C . v e r y  enjoyable.  f o r making  D E D I C A T I O N  Dedicated t o my parents.  INTRODUCTION  The r o l e o f c a l c i u m as a key m e d i a t o r i n the r e g u l a t i o n o f a variety of c e l l u l a r functions i s well recognized.  For i n s t a n c e , a nerve  a c t i o n p o t e n t i a l t r i g g e r e d by a s t i m u l u s i s modulated by c a l c i u m i n f l u x through the axon membrane (Baker 1972) and muscle c o n t r a c t i o n i s  evoked  2+ by c a l c i u m i n f l u x a c r o s s the plasma membrane and Ca  r e l e a s e from  s a r c o p l a s m i c r e t i c u l u m i n s k e l e t a l muscle o r from membrane bound p o o l s i n smooth muscle ( E b a s h i 1 9 7 6 ) .  I n t r a c e l l u l a r calcium i s also  i n maintenance o f red b l o o d c e l l  shape (Weed and C h a i l l e y 1973) and  c o n t r o l of membrane t r a n s p o r t different cellular  (Porzi'g 1 9 7 2 ) .  involved  In c e l l u l a r responses  to  s t i m u l i , c a l c i u m may f u n c t i o n e i t h e r d i r e c t l y as an i n t r a "second messenger" o r i t may a f f e c t the p r o d u c t i o n o f a n o t h e r  "second messenger" ( c y c l i c AMP) by a l t e r i n g the a c t i v i t i e s o f  adenylate  c y c l a s e and/or p h o s p h o d i e s t e r a s e (Rasmussen and Goodman 1 9 7 7 ) . The r e g u l a t o r y  f u n c t i o n s o f c a l c i u m depend  upon i t s  asymmetric  d i s t r i b u t i o n i n t i s s u e s and o r g a n e l l e s .  The f r e e c o n c e n t r a t i o n o f -5 -7 c a l c i u m i n the c y t o p l a s m i s t y p i c a l l y between 10 and 10 M, i n c o n t r a s t -3 to much h i g h e r l e v e l s o f c a l c i u m ( a p p r o x i m a t e l y  10  M) i n the  extra-  c e l l u l a r f l u i d s and i n i n t r a c e l l u l a r o r g a n e l l e s such as m i t o c h o n d r i a , endoplasmic r e t i c u l u m or s a r c o p l a s m i c r e t i c u l u m i n m u s c l e .  Animal  m a i n t a i n t h i s low l e v e l o f i n t r a c e l l u l a r c a l c i u m by e x t r u d i n g + t h e Na  cells  c a l c i u m by  2+ - Ca  exchange mechanism a n d / o r  by the ATP d r i v e n c a l c i u m pump  o f the plasma membrane.Human red c e l l s s e r v e as a v e r y u s e f u l model system f o r s t u d y i n g the ATP d r i v e n c a l c i u m pump o f the plasma membrane + 2+ f o r several reasons. These c e l l s l a c k the Na - Ca exchange system  ( S c h a t z m a n n l 9 7 5 ; S a r k a d i and Tosteson 1979) and c a l c i u m a c c u m u l a t i n g intracellular organelles. able i n t e r e s t disease states  The e r y t h r o c y t e membrane i s a l s o o f c o n s i d e r -  because i t s s t r u c t u r e (e.g.  and/or f u n c t i o n i s a l t e r e d i n  diabetes m e l l i t u s , hereditary  (Jacob 1 9 7 4 ; M c M i l l a n et_aT  gasses.  spherocytosis)  1978).  The b i c o n c a v e shape o f t h e r e d c e l l volume r a t i o which f a v o u r s  certain  g i v e s i t a high s u r f a c e a r e a /  rapid diffusion e q u i l i b r a t i o n of  respiratory  The d e f o r m a b i 1 i t y o r p l a s t i c i t y o f the red c e l l s promotes  passage through t h e m i c r o v a s c u l a t u r e where i t s f r e i g h t  their  o f oxygen must be  d e l i v e r e d (La C e l l e 1 9 6 9 ) ; the s m a l l e s t v e s s e l s are l e s s than 2JJ i n m e t e r , whereas the red c e l l  has a d i a m e t e r o f about 7JJ.  Both o f  dia-  these  c h a r a c t e r i s t i c s o f the r e d c e l l depend on the mechanical p r o p e r t i e s  of  the red c e l l membrane, the maintenance o f which r e q u i r e s a low l e v e l i n t r a c e l l u l a r calcium.  Small i n c r e a s e s i n i n t r a c e l l u l a r c a l c i u m can  cause c r e n a t i o n , d i s c o c y t e - e c h i n o c y t e shape t r a n s f o r m a t i o n 1969) and i n c r e a s e d r i g i d i t y S a r k a d i e t a i 197.6).  Calcium s e l e c t i v e l y  i n c r e a s e s potassium p e r m e a b i l i t y a c t i v a t e d Ktchannel  Calcium concentrations  the o u a b a i n - s e n s i t i v e ( N a  +  (Weed e t a l  o f red c e l l s (Dunn 1974; Weed e t _aj_ 1969;  o f the red c e l l by s t i m u l a t i o n o f the Ca 1956; Simons 1 9 7 4 ) .  of  (Gardos  above 0.1 mM s t r o n g l y  + K )-ATPase a c t i v i t y  inhibit  i n i s o l a t e d red c e l l  +  membranes, r e s e a l e d - g h o s t s and i n t a c t red c e l l s (Dunham and Glynn 1 9 6 1 ; Davis and V i n c e n z i  1971;  S a r k a d i et_al_ 1 9 7 7 ) .  It  i s e v i d e n t from the  foregoing discussion that accumulation of i n t r a c e l l u l a r calcium leads to s e r i o u s d e l e t e r i o u s e f f e c t s on the c e l l s .  A l t h o u g h the  physico-  chemical mechanisms by which i n t r a c e l l u l a r c a l c i u m mediates t h e s e changes are not c o m p l e t e l y u n d e r s t o o d , i t i s n e v e r t h e l e s s  i m p o r t a n t as a f i r s t  s t e p to understand the fundamental mechanisms by which red c e l l s  regulate  the l e v e l s o f i n t r a c e l l u l a r c a l c i u m .  R e g u l a t i o n o f C e l l u l a r Calcium The normal c o n c e n t r a t i o n o f c a l c i u m i n human red c e l l s i s 10 and 20 ymoles/L packed c e l l s ( H a r r i s o n and Long 1 9 6 8 ; Vincenzi  1969;  extracellular  between  Schatzmann and  Dunn 1 9 7 4 ) , and most o f i t i s bound to i n t r a c e l l u l a r and  b i n d i n g s i t e s on the membrane.  A l t h o u g h the p r e c i s e c o n -  c e n t r a t i o n o f f r e e i n t r a c e l l u l a r c a l c i u m i s not known because o f b i n d i n g to the c e l l membrane (Long and Mouat 1971) and c e l l  constituents  such as h e m o g l o b i n , ATP and 2 , 3 - d i p h o s p h o g l y c e r i c a c i d , i t i s to be i n the low m i c r o m o l a r range (10~^ - 10"^ M) ( H a r r i s o n Schatzmann 1 9 7 3 ;  F e r r e i r a and Lew 1 9 7 6 ) .  its  estimated  and Long 1968;  The c o n c e n t r a t i o n o f  cytoplas-  mic c a l c i u m i s about t h r e e to f o u r o r d e r s o f magnitude s m a l l e r than t h a t present  i n the b l o o d plasma ( a p p r o x . 10  powerful gradient.  defence mechanism(s)  The red c e l l  a g a i n s t the i n w a r d l y  thus needs a  directed calcium  The two main mechanisms seem to be the low p a s s i v e p e r m e a b i l i -  ty o f t h e r e d c e l l extrusion  M).  f o r c a l c i u m and the ATP-dependent,  from the c e l l  interior.  a c t i v e calcium  The p a s s i v e p e r m e a b i l i t y o f  fresh  normal human r e d c e l l s i s almost u n d e t e c t a b l e when they are i n c u b a t e d i n i s o - o s m o t i c s a l i n e c o n t a i n i n g 2-10 mM c a l c i u m .  T h i s low p e r m e a b i l i t y  of  the membrane f o r c a l c i u m was demonstrated by b l o c k i n g a c t i v e c a l c i u m extrusion  by energy d e p l e t i o n o r i n c u b a t i n g the c e l l s i n the c o l d , when  c a l c i u m uptake of 1 - 10 ytnoles/L c e l l s per hr was observed and V i n c e n z i  1969; Lew and F e r r e i r a  1978).  Exhaustive  (Schatzmann  d e p l e t i o n o f ATP  and 2 , 3 - d i p h o s p h o g l y c e r i c a c i d causes i n c r e a s e i n c a l c i u m uptake to l e v e l s a p p r o x i m a t e l y 100 pmoles/L c e l l s per h r . ,  i n d i c a t i n g a probable  r o l e o f ATP i n m a i n t a i n i n g the low p a s s i v e p e r m e a b i l i t y o f the membrane  for  c a l c i u m (Szasz and Gardos 1 9 7 4 ) , o r an e f f e c t o f the d e p l e t i n g agents  on the membrane.  However, the p e r m e a b i l i t y o f the membrane to c a l c i u m  can be i n c r e a s e d i n c e r t a i n d i s e a s e Gill  s t a t e s (Eaton e t a\_ 1 9 7 3 ; W i l e y and  1976) o r by b i o c h e m i c a l / p h a r m a c o l o g i c a l m a n i p u l a t i o n s , i n c l u d i n g  i n c r e a s i n g pH (Romero and Whittam 1971) and o s m o t i c c o n c e n t r a t i o n i n the medium ( P l i s h k e r and G i t e l m a n 1 9 7 6 ) , and by exposure o f c e l l s to membrane a c t i v e drugs such as p r o p r a n o l o l  (Szasz e t a_l_ 1 9 7 7 ) ,  p-chloromercury-  b e n z e n e s u l f o n i c a c i d (Garrahan  and Rega 1976) o r by the c a l c i u m ionophore  A 23187 (Reed 1973; 1 9 7 6 ) .  i s of p a r t i c u l a r i n t e r e s t that  It  p e r m e a b i l i t y o f red c e l l s to c a l c i u m under s t a t i c c o n d i t i o n s  the (i.e.  suspending the c e l l s i n i s o t o n i c s a l i n e w i t h c a l c i u m ) may not  represent  p h y s i o l o g i c a l c o n d i t i o n s o f shear s t r e s s which the c e l l s are s u b j e c t e d to upon t h e i r passage through the m i c r o c a p i l l a r i e s (which are o f t e n than h a l f the d i a m e t e r o f the red c e l l s ) .  less  I t was r e c e n t l y shown by  Larsen et a]_ (1981) t h a t m e t a b o l i c a l l y d e p l e t e d red c e l l s s u b j e c t e d to shear r a t e  (1200 sec"^)  conditions.  Whatever the cause o f i n c r e a s e d c a l c i u m i n f l u x ,  flux  take up 10 f o l d more c a l c i u m than under s t a t i c  i s n o t compensated f o r by a c t i v e c a l c i u m e x t r u s i o n ,  i n t r a c e l l u l a r calcium w i l l  eventually  i f the i n -  the i n c r e a s e d  l e a d to p a t h o l o g i c a l  consequences.  The p r e c i s e r e l a t i o n s h i p between i n c r e a s e d c a l c i u m and red c e l l physiology  patho-  i s not c l e a r , but a r o l e f o r " c o n t r a c t i l e p r o t e i n s " o f  s p e c t r i n - a c t i n network a t t a c h e d to the c y t o p l a s m i c s u r f a c e o f membranes has been i m p l i c a t e d .  the  the  The presence o f c a l c i u m i n m i c r o m o l a r  c o n c e n t r a t i o n s may a l t e r the c o n f i g u r a t i o n o f t h i s network,  thereby  c a u s i n g changes i n c e l l shape ( P a l e k et_ a]_ 1 9 7 5 , K i r k p a t r i c k 1974) o r may cause c r o s s - l i n k i n g o f s p e c t r i n w i t h a c t i n and o t h e r producing i r r e v e r s i b l e  s t i f f e n i n g and s p h e r o e c h i n o c y t e  it  proteins,  formation  (Lorand  e_t aj_ 1 9 7 6 ) .  The i r r e v e r s i b l e  c r o s s - l i n k i n g o f p r o t e i n s by c a l c i u m i s 2+  caused by a c t i v a t i o n o f a c y t o p l a s m i c t r a n s g l u t a m i n a s e ( a t A l l a n and M i c h e l l  Ca  > 0 . 5 mM).  ( 1 9 7 5 ; 1977) have suggested a r o l e f o r c a l c i u m - d e p e n -  dent changes i n the l i p i d c o m p o s i t i o n o f red c e l l membranes, r e s u l t i n g in accumulation of 1 , 2 - d i a c y l g l y c e r o l  i n the plasma membrane, which may  l e a d to e c h i n o c y t e t r a n s f o r m a t i o n and m i c r o v e s i c u l a t i o n o f e r y t h r o c y t e s . Thus, c a l c i u m may r e g u l a t e the r o l e o f both p r o t e i n s and l i p i d s r e g u l a t i o n of c e l l  shape and f u n c t i o n .  the red c e l l s have a mechanism f o r their v i a b i l i t y ,  It  i n the  is imperative, therefore,  r a p i d e x t r u s i o n o f c a l c i u m to  that  ensure  p a r t i c u l a r l y under adverse p a t h o l o g i c a l c o n d i t i o n s .  Calcium T r a n s p o r t and (Ca  + Mg  ) ATPase a c t i v i t y  -  Basic Characteristics  The m o l e c u l a r b a s i s o f a c t i v e c a l c i u m t r a n s p o r t ( e x t r u s i o n ) human red b l o o d c e l l s i s a Ca 2+ ((Ca  2+  in  2+ s t i m u l a t e d , Mg dependent ATPase  2+ + Mg  )-ATPase) w h i c h uses energy from the h y d r o l y s i s  o f ATP to  a c t i v e l y e x t r u d e c a l c i u m a g a i n s t a chemical and e l e c t r o c h e m i c a l g r a d i e n t (Schatzmann 1 9 6 6 ; Schatzmann and V i n c e n z i 1 9 6 9 ) . Dunham and Glynn (1961) 2+ 2+ f i r s t demonstrated the presence o f a (Ca + Mg )-ATPase a c t i v i t y which was s e v e r a l f o l d h i g h e r than, the ouabain s e n s i t i v e ( N a + K ) - A T P a s e +  +  2+ 2+ S e v e r a l l i n e s o f e v i d e n c e i n d i c a t e t h a t (Ca + Mg )-ATPase 2+ 2+ i s the enzymatic e x p r e s s i o n o f the "Ca -pump", e x t r u d i n g Ca from t h e activity.  red c e l l  cytoplasm. 2+  1)  C a l c i u m s t i m u l a t e s (Ca  2+ + Mg  2+ )-ATPase a c t i v i t y and Ca  trans-  p o r t o n l y a t the i n t r a c e l l u l a r membrane s u r f a c e (Schatzmann and V i n c e n z i 1969).  Addition of external  c a l c i u m (up to 2 - 5 mM) does not a f f e c t  the r a t e o f c a l c i u m e f f u x e i t h e r i n r e s e a l e d ghosts o r i n t a c t red cells,  i n d i c a t i n g t h a t the u n i d i r e c t i o n a l c h a r a c t e r o f c a l c i u m 2+  e x t r u s i o n i s p r e s e r v e d even a g a i n s t o p p o s i t e l y d i r e c t e d Ca t i o n r a t i o s of several  concentra-  thousand (Schatzmann and V i n c e n z i 1969; Lee and  S h i n 1969; Schatzmann 1 9 7 3 ) . 2+ 2)  Both a c t i v e c a l c i u m - t r a n s p o r t and (Ca  2+ + Mg  )-ATPase  activity  2+ r e q u i r e Mg  and ATP w i t h i n the c e l l  (Lee and S h i n 1 9 6 9 ) .  The red c e l l  c a l c i u m pump a c c e p t s v a r i o u s m e t a l - l i g a n d e d forms o f ATP o r f r e e  ATP  as s u b s t r a t e s , depending on the e x p e r i m e n t a l c o n d i t i o n s ( d e s c r i b e d i n d e t a i l e l s e w h e r e ) . . B a s e d on k i n e t i c c a l c u l a t i o n s S a r k a d i et aj_ (1981)  proposed t h a t t h e t r u e p h y s i o l o g i c a l s u b s t r a t e i n s i t u i s most p r o b a b l y Mg  2+  ATP  4-  .  In a d d i t i o n to its r o l e as a MgATP s u b s t r a t e , f r e e Mg  required for active calcium transport.  than Ca  but s m a l l e r  2+ .  Thus, Mg  Mn  2 +  ( 0 . 0 8 nm), C o  Ca  2 +  ( 0 . 0 9 9 nm), S r  2 +  2 +  ( i o n i c r a d i u s = 0.065 nm) can be s u b s t i t u t e d by ( 0 . 0 7 8 nm), F e  2 +  ( 0 . 1 1 2 nm), B a  2 +  ( 0 . 0 7 6 nm), N i  2 +  ( 0 . 0 7 8 nm).  ( 0 . 1 3 4 nm) are not accepted a t  t h i s s i t e (Sarkadi et aj_1981). I n o r g a n i c phosphate i s r e l e a s e d i n the c e l l  i  interior. +  It 2+  i m p o r t a n t t o note at t h i s p o i n t t h a t no c o - t r a n s p o r t o f H , Mg m e t a b o l i t e s was d e t e c t e d t o accompany c a l c i u m e f f l u x 1974).  is  2+ Mg i n t h i s r o l e can be s u b s t i 2+  t u t e d by m e t a l s which have an i o n i c r a d i u s s i m i l a r t o Mg 2+  2+  GTP,  ITP,  is  , or  (Olson and C a z o r t  CTP or OTP can s u b s t i t u t e f o r ATP when i n c o r p o r a t e d  i n t o r e s e a l e d ghosts  (Lee and S h i n 1969; Olson and C a z o r t 1969) a l t h o u g h  a h i g h e r s p e c i f i c i t y was found f o r ATP i n membrane fragments 1971) and i n i n s i d e - o u t v e s i c l e s  (Cha e t a l  ( S a r k a d i e t a]_ 1 9 7 9 ) . ADP, AMP,  pyrophosphate and a c e t y l phosphate cannot support a c t i v e c a l c i u m t r a n s port  (Schatzmann and V i n c e n z i  1969; Olson and Cazort 1969; S a r k a d i e t a l  1979b) . 2+ 3)  S t r o n t i u m can s u b s t i t u t e f o r c a l c i u m f o r a c t i v a t i n g  (Ca  2+ + Mg  ATPase i n red c e l l membranes (Wins and S c h o f f e n i e l s 1966;  P f l e g e r and  Wolf 1 9 7 5 ) .  (Olson and  Cazort  Strontium i s transported  from r e s e a l e d ghosts  )-  1969, Olson 1979) and i n t o i n s i d e - o u t red c e l l v e s i c l e s and c a l -  m o d u l i n s t i m u l a t e s t h i s uptake ( S a r k a d i e t al_ 1980b)by a mechanism 2+ 2+ 2+ r e q u i r i n g Mg ( o r Mn ) and ATP on the c y t o p l a s m i c s i d e . A l t h o u g h Sr 2+ 2+ 2+ and Ca have s i m i l a r a f f i n i t i e s f o r (Ca + Mg )-ATPase (Wins and S c h o f f e n i e l s 1 9 6 6 ) , o n l y Ca  2+  i s t r a n s p o r t e d when both Ca  2+  and S r  2+  are p r e s e n t s i m u l t a n e o u s l y i n e r y t h r o c y t e g h o s t s (Schatzmann and V i n c e n z i 1969).  Calcium was shown t o i n h i b i t a c t i v e s t r o n t i u m t r a n s p o r t n o n -  c o m p e t i t i v e l y by o c c u p y i n g a s i t e o t h e r than t h e t r a n s p o r t s i t e 1979).  M o r e o v e r , t h i s i n h i b i t i o n c o u l d be p a r t i a l l y r e v e r s e d  (Olson  by i n -  2+ c r e a s i n g t h e c o n c e n t r a t i o n o f Mg from 1 mM t o 4 mM e i t h e r d u r i n g l y s i s 2+ or a t the t i m e o f i n c u b a t i o n . Mn , however, was i n e f f e c t i v e i n r e v e r s 2+ 2+ ing the Ca induced i n h i b i t i o n o f Sr t r a n s p o r t (Olson 1 9 7 9 ) . B a r i u m , magnesium, manganese, c o p p e r , c o b a l t , and i r o n a r e not t r a n s p o r t e d by t h e c a l c i u m pump, but t h e s e metal i o n s seem t o  interact  w i t h t h e c a l c i u m - s i t e and i n h i b i t enzyme a c t i v i t y ( S a r k a d i ejt al_ 1 977, 1980, 1 9 8 1 ; Schatzmann and V i n c e n z i 1969; Schatzmann 1975).  4)  A c t i v e c a l c i u m e x t r u s i o n from human red c e l l s does not depend  upon a c t i v e sodium and potassium t r a n s p o r t , nor upon t h e transmembrane gradient of N a  +  or K  +  ( S a r k a d i and Tosteson 1979).  However, N a  +  and  + + + 2+ 2+ K , as compared t o L i , c h o l i n e or Cs , s t i m u l a t e (Ca + Mg )ATPase activity efflux  ( S a r k a d i 1980a,b).Both ( C a  2 +  + M g ) - A T P a s e a c t i v i t y and C a 2+  from r e s e a l e d g h o s t s was maximum when K  i n s i d e and N a  +  +  2 +  (=25 mM) was a t t h e  (=130 mM) a t t h e o u t s i d e o f t h e r e s e a l e d ghost membrane  ( W i e r i c h s and Bader 1980).  5)  Calcium t r a n s p o r t  has a high Q-j  0  v a l u e (=3.5) (Schatzmann and  V i n c e n z i 1 9 6 9 , Schatzmann 1973, Lee and S h i n 1 9 6 9 ) , as expected t r a n s p o r t coupled to a chemical r e a c t i o n .  for  The e n e r g i e s o f a c t i v a t i o n  2+ f o r Ca  -transport  (13.5 - 25.0 K cal/(mol)  1969; Lee and S h i n 1969) and ( C a mol)  2 +  (Schatzmann and V i n c e n z i  + Mg )-ATPase 2+  ( Q u i s t and R o u f o g a l i s 1975a,b) a r e s i m i l a r .  (14.4 - 27.6 K c a l /  R e a c t i o n sequence of (Ca  + Mg  )-ATPase proposed by Muallem and  K a r l i s h (1980, 1981)  A new approach t o t h e study o f t h e m o l e c u l a r b a s i s o f t h e r e d c e l l c a l c i u m pump was used by Katz and B l o s t e i n (1975) and Knauf e t a l (1974) who demonstrated c a l c i u m dependent membrane p h o s p h o r y l a t i o n by 32 [y-  P]ATP.  I t was shown t h a t m i c r o m o l a r c o n c e n t r a t i o n s o f c a l c i u m  induced the f o r m a t i o n o f a p h o s p h o p r o t e i n [EP] which i s s e n s i t i v e to hydroxylamine  (a l a b i l e  acyl-phosphate  and b a s i c c o n d i t i o n s ) o f m o l e c u l a r  weight =150,000 [the sodium s t i m u l a t e d p h o s p h o p r o t e i n was around m o l e c u l a r weight  1 0 0 , 0 0 0 ] . The p h o s p h o p r o t e i n i s produced i n seconds a t 0°C  [Tj = 4 0 - 6 0 s e c ] (Enyedi e t a l 1980) and shows a r a p i d t u r n o v e r upon a d d i t i o n o f excess n o n - r a d i o a c t i v e ATP. 2+ the (Ca 1975;  Its  formation c o r r e l a t e s  with  2+ + Mg  )-ATPase a c t i v i t y  Rega and Garrahan 1975).  i n t h e membrane ( K a t z and B l o s t e i n T h i s new approach p r o v i d e s  valuable  i n f o r m a t i o n on t h e p a r t i a l r e a c t i o n sequence o f the red c e l l c a l c i u m pump.  I t was f i r s t  shown by Rega and Garrahan (1975) and l a t e r  by  Schatzmann and Bu'rgin (1978) and Szasz e_t aj_ (1978) t h a t f o r m a t i o n  of 2+  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 d i d not r e q u i r e t h e presence o f Mg  and  t h a t the b a s i c r o l e o f magnesium i s i n the d e p h o s p h o r y l a t i o n o f t h e c a l c i u m - t r a n s p o r t ATPase.  However, c a l c i u m dependent f o r m a t i o n o f t h e 2+ phosphoenzyme i s a c c e l e r a t e d by Mg (T < 5 sec) (Schatzmann and B u r g i n t  1978; Rega and Garrahan 1978; Enyedi et a l 1980) p o s s i b l y by t h e a c t i o n 2+ o f Mg  a t a l a t e r s t a g e i n the r e a c t i o n sequence of the pump.  Garrahan  and Rega (1978) p o s t u l a t e d a magnesium dependent c o n f o r m a t i o n a l change of the i n i t i a l  phosphoenzyme  (E^P ) i n t o a form ^ P )  r e a c t w i t h water t o r e l e a s e P^.  which can r a p i d l y  Monovalent c a t i o n s have no e f f e c t on  t h e f o r m a t i o n o f phosphoenzyme but the r a t e o f d e p h o s p h o r y l a t i o n o f c a l c i u m pump i n t e r m e d i a t e i s i n c r e a s e d by N a  +  and K  +  the  i n t h e presence o f  2+ Mg (Enyedi ejt a]_ 1 9 8 0 ) . Whereas t h e s u b s t r a t e f o r t h e c a l c i u m pump was i n i t i a l l y proposed t o be f r e e ATP (Rega and Garrahan 1975; Schatzmann 1 9 7 7 ) , Graf  and P e n n i s t o n (1981) proposed t h a t a t low ATP  c o n c e n t r a t i o n s , CaATP was the s u b s t r a t e .  R e c e n t l y , Muallem and K a r l i s h (.19.81)  have made a s y s t e m a t i c s t u d y o f t h e e f f e c t s o f nucleotide s p e c i e s and proposed t h a t a t low c o n c e n t r a t i o n s o f t h e n u c l e o t i d e (1 - 10 y M ) , MgATP, 2+ 2+ CaATP or f r e e ATP s u s t a i n (Ca + Mg )-ATPase a c t i v i t y e f f e c t i v e l y ,  whereas a t h i g h e r c o n c e n t r a t i o n s o f t h e n u c l e o t i d e (100 - 2 0 0 0 y M) MgATP a c c e l e r a t e d ATP h y d r o l y s i s an i n h i b i t o r (K.  (K  = 165 yM)  360 yM) and CaATP a c t e d as (MgATP) by competing w i t h MgATP f o r t h e  (Ca-ATP) regulatory  site.  MgATP was found t o enhance (Ca  enhancing h y d r o l y s i s o f E £ P ,  + Mg  )-ATPase by  by i n t e r a c t i n g a t the n o n - c a t a l y t i c  site  (regulatory s i t e ) .  ATP i s not h y d r o l y s e d a t t h e r e g u l a t o r y s i t e and 2+ 2+ 2+ h y d r o l y s i s o f E 2 - P by ATP does not r e q u i r e Ca or Mg . However, Mg i s needed i n d i r e c t l y f o r t h e f o r m a t i o n o f E 2 - P , which r a p i d l y r e a c t s water  with  i n the presence o f mM c o n c e n t r a t i o n s o f ATP (Rega and Garrahan  1978; R i c h a r d s e t al_ 1978).  It  has a l s o been suggested t h a t high c o n c e n -  t r a t i o n s o f MgATP (K  = 0.143 mM) s t i m u l a t e t h e maximum v e l o c i t y o f d 2+ 2+ 2+ the (Ca + Mg )-ATPase i n a Mg -dependent f a s h i o n by promoting t h e  c o n v e r s i o n o f E£ t o E^ i n the f i n a l r e a c t i o n sequence ( R i c h a r d s et a l 1978). A l t h o u g h t h e a s s o c i a t i o n between (Ca  2+  2+) 2+ + Mg -ATPase and Ca -  2+ 2+ has been c l e a r l y demonstrated and t h e r o l e o f Ca , Mg , and 2+ 2+ ATP i n t h e r e g u l a t i o n o f (Ca + Mg )-ATPase u n c o v e r e d , c o n s i d e r a b l e transport  discrepancies exist 2+ (Ca  i n t h e l i t e r a t u r e on t h e k i n e t i c p r o p e r t i e s  2+ + Mg )-ATPase i n human red c e l l membranes, p a r t i c u l a r l y 2+ 2+  r e g a r d t o t h e c a l c i u m s e n s i t i v i t y o f t h e pump/(Ca  + Mg  a c t i v i t i e s and t h e s t o i c h i o m e t r y o f the c a l c i u m pump.  of with  )-ATPase  Whereas  and Whittam (1971) found t h a t t h e ATP dependent c a l c i u m e f f l u x  Romero from  r e s e a l e d ghosts was h a l f - m a x i m a l l y a c t i v a t e d a t a c a l c i u m c o n t e n t  of  0.9  mmol/L packed e e l 1s,Schatzmann (1973) r e p o r t e d t h a t h a l f maximal 2+ 2+ s t i m u l a t i o n o f Ca e f f l u x o c c u r r e d a t l e s s than 4 yM Ca i n ghosts loaded w i t h calcium-EGTA  buffers.  h a l f maximal a c t i v a t i o n o f (Ca  2+  Schatzmann (1973) a l s o showed t h a t  2+ + Mg )-ATPase a c t i v i t y appeared a t  1000 f o l d lower Ca  2+  c o n c e n t r a t i o n than t h a t i n g h o s t s loaded w i t h 2+ c a l c i u m i n the absence o f EGTA. The f a c t o r s a f f e c t i n g t h e Ca -affinity 2+ 2+ 2+ o f (Ca + Mg )-ATPase and Ca - t r a n s p o r t have been reviewed r a t h e r extensively  by R o u f o g a l i s (1979) and w i l l  be summarized b r i e f l y  below.  The d i s c r e p a n c y between t h e k i n e t i c s o f c a l c i u m a c t i v a t i o n o f 2+  2+ + Mg )-ATPase a l s o e x i s t s i n e r y t h r o c y t e ghost membranes. 2+ 2+  (Ca  e t al_ (1970) showed the presence of two (Ca  + Mg  i n e r y t h r o c y t e ghost membranes, f r e q u e n t l y r e f e r r e d Ca  2 +  affinity  vities  2 +  2  t o as t h e high and low  2+  for calcium.  affinity  )-ATPase a c t i v i t i e s  ( C a + M g ) - ATPase a c t i v i t i e s to r e f l e c t t h e i r d i f f e r e n t  sensiti-  The c a l c i u m d i s s o c i a t i o n c o n s t a n t s f o r the high and low  2+ Ca  Horton  2+  (Ca  + Mg  )-ATPase a c t i v i t i e s were found t o be a p p r o x i m a t e l y  4 yM and 100 y M , r e s p e c t i v e l y  (Schatzmann and R o s s i 1 9 7 1 ; Wolf 1972).  S c h a r f f (1972) and Schatzmann ( 1 9 7 3 ) , however, c o n c l u d e d t h a t the low 2+ affinity  (Ca  2+ + Mg  )-ATPase a c t i v i t y was an a r t i f a c t due t o  conversion  2+ of t h e high Ca  affinity  form o f t h e enzyme t o a form w i t h low  affinity  f o r c a l c i u m , d e r i v e d from p r e p a r a t i o n o f e r y t h r o c y t e ghost membranes i n the presence o f c h e l a t i n g agents such as EGTA or EDTA. c o n c l u s i o n d i d not concur w i t h the r e s u l t s o f Horton  However, t h i s  ( 1 9 7 0 ) , who used  membranes prepared i n t h e absence o f EGTA/EDTA and demonstrated the 2+ 2+ presence o f h i g h and low a f f i n i t y Furthermore,  (Ca  + Mg  )-ATPase a c t i v i t i e s .  t h e pump seems to demonstrate a low a f f i n i t y  when the enzyme a c t i v i t y was s t u d i e d i n whole c e l l s  for calcium  ( S a r k a d i e t al_ 1 9 7 9 ) .  Q u i s t and R o u f o g a l i s ( 1 9 7 5 ) , u s i n g a v a r i e t y o f membrane p r e p a r a t i o n s , a l s o showed b i p h a s i c ( h i g h and low) c a l c i u m a c t i v a t i o n k i n e t i c s and suggested t h a t the " l o w - c a l c i u m a f f i n i t y " enzyme played a fundamental role in active calcium transport. As knowledge o f the r e g u l a t i o n o f 2+ 2+ 2+ c a l c i u m a f f i n i t y o f the Ca -pump/(Ca + Mg )-ATPase i s germane t o  u n d e r s t a n d i n g i t s p h y s i o l o g i c a l r o l e i n human e r y t h r o c y t e s , t h i s t o p i c will  form an i m p o r t a n t p a r t o f t h i s  thesis. 2+  Efforts  t o c h a r a c t e r i z e the s t o i c h i o m e t r y  also yielded c o n f l i c t i n g results.  o f t h e Ca  Schatzmann and V i n c e n z i  pump have (1969) and  Schatzmann (1973) a r r i v e d a t a s t o i c h i o m e t r i c r a t i o o f 1:1 f o r t h e human red c e l l c a l c i u m pump.  These a u t h o r s compared the r a t i o o f  inorganic  phosphate l i b e r a t e d from ATP to the r a t i o o f c a l c i u m e x t r u s i o n  in calcium  loaded r e s e a l e d g h o s t s . (1978).  S i m i l a r r e s u l t s were o b t a i n e d by Larsen et a l 2+ In c o n t r a s t , when lanthanum was used t o b l o c k Ca transport in 2+  c a l c i u m - l o a d e d r e s e a l e d g h o s t s , t h e r a t i o o f lanthanum s e n s i t i v e Ca efflux 1 975b).  t o lanthanum s e n s i t i v e ATP s p l i t t i n g was 2:1 ( Q u i s t  and R o u f o g a l i s  S i m i l a r l y , i n i n t a c t c e l l s t h e molar r a t i o o f lanthanum  t i v e calcium transport  -  sensi-  t o lanthanum s e n s i t i v e ATP s p l i t t i n g was 2:1  ( S a r k a d i e_t aj_ 1 9 7 7 ) . These l a t t e r a u t h o r s used ionophore A23187 to l o a d 2+ the c e l l s w i t h Ca  and ouabain  t o i n h i b i t the sodium pump. Q u i s t and  R o u f o g a l i s (1975b) used f r e s h red c e l l  ghosts and found lanthanum t o  c o m p l e t e l y i n h i b i t t h e c a l c i u m t r a n s p o r t but o n l y 50% o f t h e ATPase a c t i 2+ 2+ v i t y measured i n the presence o f Ca + Mg . Larsen et al_ (1978) used o u t d a t e d blood i n a c i d / c i t r a t e / d e x t r o s e to make g h o s t s and d i d not f i n d 2+ 2+ a s i g n i f i c a n t lanthanum i n s e n s i t i v e  (Ca  + Mg  )-ATPase a c t i v i t y ;  hence  t h e i r e s t i m a t e o f t h e s t o i c h i o m e t r y u s i n g t h e lanthanum method was 1 : 1 . The reason f o r t h i s d i f f e r e n c e  i s not c l e a r , but i t has been suggested  t h a t the p a s s i v e p e r m e a b i l i t y o f the membrane to both c a l c i u m and lanthanum i n c r e a s e s c o n s i d e r a b l y i n ghosts made from outdated (Szasz ejt aj_ 1978 a , b, c ) .  Thus, e x t e r n a l l y  cells  added lanthanum,  which n o r m a l l y does not p e n e t r a t e the membrane g h o s t s , may have penet r a t e d the ghosts from o u t d a t e d blood and i n h i b i t e d t h e c a l c i u m - s t i m u l a ted-ATPase a c t i v i t i e s not a s s o c i a t e d w i t h c a l c i u m pumping ( S a r k a d i e t al_  1979), Recently,  i n s i d e - o u t red c e l l membrane v e s i c l e s have been used 2+  to study t h e c a l c i u m pump s t o i c h i o m e t r y .  In such v e s i c l e s t h e (Ca  +  2+ Mg  )-ATPase shows an " u p h i l l " c a l c i u m t r a n s p o r t .  I t was shown by  S a r k a d i (1980) t h a t the s t o i c h i o m e t r y o f t h e c a l c i u m pump depended on the c o n c e n t r a t i o n o f c a l c i u m and ranged from 0 . 3 : 1 t o 2:1 a t i n c r e a s i n g calcium concentrations.  Recently,  Larsen et al_ (1981) and Akyempong  and R o u f o g a l i s (1981) showed t h a t t h e s t o i c h i o m e t r y o f the pump can v a r y depending on the c o n c e n t r a t i o n o f the e f f e c t o r s u s e d . T h i s may be 2+ 2+ due i n p a r t to t h e complex k i n e t i c s o f (Ca + Mg )-ATPase observed a t 2+ 2+ v a r i o u s c o n c e n t r a t i o n s o f Ca , Mg and ATP used 1n t h e assay medium 2+ (Katz et al_ 1 9 7 9 ) . The v a r i a b i l i t y i n the apparent a f f i n i t y o f (Ca + 2+ 2+ Mg )-ATPase f o r Ca has been suggested to be a f f e c t e d by t h e method o f membrane p r e p a r a t i o n , which i n t u r n may govern whether c a l m o d u l i n i s a s s o c i a t e d or d i s s o c i a t e d from  t h e Ca  2+  pump due t o t h e presence o r  absence o f c a l c i u m i n the hemolysing b u f f e r . 2+ a f f e c t i n g t h e k i n e t i c s o f t h e (Ca experimental  Additional  factors  2+ + Mg  )-ATPase and v a r i a t i o n i n the  f i n d i n g s may i n c l u d e t h e f o l l o w i n g .  1) The use o f c a l c i u m - c h e l a t i n g s u b s t a n c e s , such as EDTA o r EGTA ( S a r k a d i et aj_ 1979; A l - J o b o r e e t aj[ 1981) or t h e C a  2 +  ionophore A23187  (Scharff  and Foder 1978; Haaker and Racker 1979) d u r i n g the ( C a + Mg )-ATPase 2+ 2+ or Ca - t r a n s p o r t a s s a y . I t was observed t h a t the k i n e t i c s o f (Ca + 2+ 2+ Mg )-ATPase o r Ca - t r a n s p o r t depended on the t o t a l c o n c e n t r a t i o n o f 2 +  Ca  2+  i n t h e Ca-EGTA  alone.  buffer  r a t h e r than the c o n c e n t r a t i o n o f f r e e Ca  T h u s , an i n t e r a c t i o n o f Ca  2+  i n the Ca  2+  -EGTA  e t a i 1979) or EGTA i n the Ca-EGTA ' form ( A l - J o b o r e 2  was p o s t u l a t e d .  2+  4-  form  2+  / (Sarkadi  and R o u f o g a l i s 1981)  2} Human e r y t h r o c y t e membranes c o n t a i n s i g n i f i c a n t amounts o f  protein  k i n a s e and phosphatase a c t i v i t i e s CAvruch and F a i r b a n k s 1 9 7 4 ; G r e e n q u i s t and Shohet 1 9 7 5 ; Hosey and Tao 1977) which can be s t i m u l a t e d by m i c r o 2+ m o l a r c o n c e n t r a t i o n s o f Ca ( F a i r b a n k s and Avruch 1974; Q u i s t 1 9 8 0 ) . 2+ 2+ Thus, when measuring (Ca  + Mg  )-dependent i n o r g a n i c phosphate  (.P.)  l i b e r a t i o n from ATP, t h e c o n t r i b u t i o n o f P. from k i n a s e o r phosphatase activities  cannot be d i s t i n g u i s h e d .  T h i s problem may be s o l v e d a t l e a s t 2 + 2 +  i n p a r t by u s i n g p u r i f i e d p r e p a r a t i o n s o f (Ca  +Mg  )-ATPase r e c o n s t i t u -  t e d i n l i p o s o m a l systems o r w i t h the use o f a s p e c i f i c i n h i b i t o r o f the 2+ 2+ Ca -pump ATPase to c h a r a c t e r i z e ATP s p l i t t i n g not a s s o c i a t e d w i t h Ca 2+ transport.  S i n c e a s p e c i f i c i n h i b i t o r o f the Ca  r e p o r t e d so f a r ,  considerable effort  -pump has not been  has been expended i n the s t u d y o f  the mechanism o f r e g u l a t i o n o f the c a l c i u m pump. R e g u l a t i o n of Calcium Pump ATPase i n human  erythrocytes  A) Role o f C a l m o d u l i n I t was f i r s t  demonstrated by G o p i n a t h and V i n c e n z i  (1977) and  J a r r e t t and P e n n i s t o n (1977) t h a t a p u r i f i e d b r a i n p h o s p h o d i e s t e r a s e or adenylate cyclase a c t i v a t o r 2+ 1978) s t i m u l a t e s (Ca membranes.  (termed " c a l m o d u l i n " by Cheung et a l  in  2+ + Mg  )-ATPase a c t i v i t y  i n i s o l a t e d red c e l l  Hinds et a l ( 1 9 7 8 ) , Larsen and V i n c e n z i  (1979) and M a c l n t y r e  and Green (1978) l a t e r demonstrated t h a t c a l m o d u l i n a l s o s t i m u l a t e s c a l c i u m uptake i n i n s i d e - o u t red c e l l  vesicles. It 2+  c a l m o d u l i n i s i n v o l v e d i n many p r o c e s s e s o f Ca  i s known now t h a t  transport in  membranes and t h a t i t may p l a y a d i f f e r e n t r o l e i n d i f f e r e n t systems ( C a r a f o l i  1981).  various transport  The a r e a o f c a l m o d u l i n b i o c h e m i s t r y and p h a r -  macology i s one o f t h e f a s t e s t growing i n b a s i c s c i e n c e s .  Some o f  its  properties  and c h a r a c t e r i s t i c s a r e d i s c u s s e d i n the e n s u e i n g s e c t i o n .  C a l m o d u l i n i s a low m o l e c u l a r w e i g h t ( M  r  = 16,700), a c i d i c  (iso-  2+ e l e c t r i c p o i n t = 4 . 2 ) , h e a t - s t a b l e , Ca  binding protein (Scharff,  C a l m o d u l i n i s u b i q u i t o u s l y d i s t r i b u t e d i n most e u c a r y o t i c  1981).  c e l l s and shows  l a c k of t i s s u e or s p e c i e s s p e c i f i c i t y (Stevens e t a l 1 9 7 6 ) .  In  a number o f o t h e r c a l c i u m b i n d i n g p r o t e i n s such as t r o p o n i n  contrast,  C, p a r a l b u -  m i n , c a l s e q u e s t r i n , v i t a m i n D - i n d u c i b l e p r o t e i n , show l i m i t e d t i s s u e d i s t r i b u t i o n and/or appear to be concerned w i t h s p e c i f i c t i s s u e (Brostrom and W o l f f 1 9 8 1 ) .  Mammalian b r a i n and t e s t i s  1976; Dedman et aj_ 1977) and the e l e c t r o p l a x  processes  (Watterson et a l  of Electrophorus  electricus  ( C h i l d e r s and S i e g e ! 1975) a r e very r i c h sources o f c a l m o d u l i n . c e l l u l a r and f r a c t i o n a t i o n s t u d i e s have l o c a l i z e d c a l m o d u l i n  Sub-  predominant-  l y i n the c y t o p l a s m i c o r s o l u b l e f r a c t i o n s , a l t h o u g h a s i g n i f i c a n t i s associated with p a r t i c u l a t e f r a c t i o n s 2+ to p a r t i c u l a t e f r a c t i o n s (Vandermeers e t al_ 1 9 7 8 ) . temperature  requires  Ca  as w e l l .  Calmodulin b i n d i n g  and o c c u r s a t s p e c i f i c s i t e s  Binding i s also saturatable,  and t r y p s i n s e n s i t i v e  amount  (Brostrom  reversible,  and W o l f f 1 9 8 1 ) .  C a l m o d u l i n has been shown to p l a y a r o l e i n r e g u l a t i o n o f a variety of c e l l u l a r functions.  It  i s involved  in synaptic  transmission  (De Lorenzo e t al_ 1 9 7 9 ) , c a l c i u m i n d u c e d i n s u l i n r e l e a s e and i n t e s t i n a l secretion involved  (Richman and Klee 1 9 7 9 ) .  C a l m o d u l i n was a l s o shown to be  i n the r e g u l a t i o n o f c a l c i u m t r a n s p o r t  in sarcoplasmic  reticulum  ( K a t z and R e m t u l l a 1978) and i n synaptosomes  (Sobue et^ aj_ 1 9 7 9 ) , i n  regulation of cell multiplication (Whitfield  e t al^ 1979) and f e r t i l i z a -  t i o n o f oocytes  by spermatozoa  (Jones e t al_ 1 9 7 8 ) .  by c a l m o d u l i n i n c l u d e the c a l c i u m t r a n s p o r t ATPases,  The enzymes  regulated  phosphodiesterase,  a d e n y l a t e c y c l a s e , myosin l i g h t c h a i n k i n a s e i n muscle/non-muscle  cells,  p h o s p h o r y l a s e B k i n a s e and s e v e r a l o t h e r p r o t e i n k i n a s e s . ( T h e referred  t o a r e c e n t r e v i e w by Brostrom and W o l f f  c a l m o d u l i n i n r e g u l a t i n g t h e s e enzyme s y s t e m s ) .  reader i s  (1981) f o r t h e r o l e o f C a l m o d u l i n thus seems  t o possess t h e i n h e r e n t a b i l i t y t o modulate a l a r g e number o f c e l l u l a r f u n c t i o n s and e s p e c i a l l y t h o s e r e g u l a t e d by c a l c i u m . C a l m o d u l i n from animal sources has o n l y one h i s t i d i n e , no t r y p t o phan or c y s t e i n e , but one t r i m e t h y l l y s i n e a c i d i c over b a s i c amino a c i d r e s i d u e s .  r e s i d u e and a predominance o f  R e c e n t l y , chemical m o d i f i c a t i o n  s t u d i e s have been used t o p r o v i d e an o v e r v i e w o f t h e amino a c i d s which 2+ may be i n v o l v e d  i n t h e b i n d i n g o f Ca  (Walsh and Stevens 1977). t i o n of the s i n g l e  - c a l m o d u l i n complex to t h e enzymes  I t was shown by T h i r y et aj_ (1980) t h a t m o d i f i c a -  h i s t i d i n e r e s i d u e or both t y r o s i n e r e s i d u e s had no  e f f e c t on the a c t i v a t i o n p r o p e r t i e s o f c a l m o d u l i n u s i n g a d e n y l a t e c y c l a s e and p h o s p h o d i e s t e r a s e .  The c a r b o x y m e t h y l a t i o n  residues or the amidation of several  carboxyl  o f two m e t h i o n i n e  groups reduced t h e a c t i -  v a t i o n p r o p e r t i e s o f c a l m o d u l i n on a d e n y l a t e c y c l a s e and p h o s p h o d i e s t e r ase.  Modification of arginyl  t h a t shows 1.3 f o l d h i g h e r  groups  (4 o f t h e 6) r e s u l t s i n a d e r i v a t i v e  for adenylate c y c l a s e .  However, t h i s  i n c r e a s e d K f o r c a l m o d u l i n i s a l s o accompanied by a 50% i n c r e a s e i n t h e V , of adenylate c y c l a s e . In c o n t r a s t , m o d i f i c a t i o n o f a r g i n y l r e s i d u e s ma x m  m  v  o f c a l m o d u l i n had no e f f e c t on i t s a b i l i t y t o a c t i v a t e p h o s p h o d i e s t e r a s e ( T h i r y et a l 1 9 8 0 ) .  R e c e n t l y , Gagnon e_t aj_ (1981) showed t h a t  m e t h y l a t e d c a l m o d u l i n was l e s s e f f e c t i v e  in s t i m u l a t i n g phosphodiesterase,  In our l a b o r a t o r y we found t h a t c a r b o x y m e t h y l a t e d 2+ ineffective  i n s t i m u l a t i n g (Ca  c y t e membranes ( A . A l - J o b o r e  carboxy-  c a l m o d u l i n was a l s o  2+ + Mg  )-ATPase a c t i v i t y o f t h e e r y t h r o -  and B.D. R o u f o g a l i s - u n p u b l i s h e d r e s u l t s ) .  These s t u d i e s prompted f u r t h e r  c h a r a c t e r i z a t i o n of t h e r o l e o f  free  carboxyl  and a r g i n y l  2+ on (Ca  groups i n the a c t i v a t i o n p r o p e r t i e s  of calmodulin  2+ + Mg  )-ATPase o f red c e l l membranes.  c e r t a i n p o l y a n i o n i c compounds ( v i z . poly-L-glutaraic-acid M t y o f the ( C a  2 +  r  Our s t u d i e s i n d i c a t e d t h a t  poly-L-aspartic acid M  f  = 20,000 and  = 14,000 and 26,000) i n c r e a s e the Ca  + M g ) - A T P a s e wihout a f f e c t i n g the V  sensitivi-  (Al-Jobore et al  2+  max 1980) .  In c o n t r a s t , b l o c k i n g a r g i n y l 2+  on i t s a c t i v a t i o n o f (Ca 1981) .  2+ + Mg )-ATPase (Minocherhom.jee and R o u f o g a l i s ,  In a d d i t i o n t o i t s r a t h e r dulin also exhibits  groups o f c a l m o d u l i n had no e f f e c t  unique a m i n o - a c i d c o m p o s i t i o n , c a l m o -  f o u r i n t e r n a l l y homologous sequences o r d o m a i n s ,  each o f which c o n t a i n s t h e a p p r o p r i a t e amino a c i d s forming the h e l i x - l o o p helix,  calcium binding structure  P a t t e r s o n e t a]_ 1 9 8 0 ) .  This  latter  property  i s a l s o common to o t h e r c a l c i u m b i n d i n g p r o t e i n s such as p a r v a l 2+ bumin and t r o p o n i n C ( R e i d and Hodges 1 9 8 0 ) . C a l m o d u l i n b i n d s f o u r Ca 2+  i o n s and the f o u r domains.  Ca  b i n d i n g s i t e s appear to c o r r e s p o n d to the  S t a r t i n g a t the amino terminus c a l m o d u l i n shows t h e  four  highest  degree o f homology between the f i r s t and t h i r d and between the second and f o u r t h domains.  D e s p i t e t h e general 2+  t h a t c a l m o d u l i n b i n d s 4 Ca  agreement i n the  literature  i o n s , t h e r e appears to be o n l y poor  agree-  ment r e g a r d i n g the a f f i n i t i e s o f these s i t e s f o r c a l c i u m ( B r o s t r o m and Wolff 1981).  P a r t o f the d i s c r e p a n c i e s has  c o n d i t i o n s and procedures  been a t t r i b u t e d to  the  employed i n d e t e r m i n i n g the b i n d i n g d a t a ,  e s p e c i a l l y t h e use of EGTA to c o n t r o l  the l e v e l of free c a l c i u m .  et aj_ (1977) w i t h r e a g e n t s f r e e d o f c o n t a m i n a t i n g d i v a l e n t  Wolff  cations,  used e q u i l i b r i u m d i a l y s i s and determined t h e c o n c e n t r a t i o n o f bound and t o t a l c a l c i u m by atomic a b s o r p t i o n s p e c t r o p h o t o m e t r y . e l i m i n a t e s t h e need f o r EGTA to c o n t r o l  the level  This  technique 2+ o f f r e e Ca . I t was  2+ shown t h a t o f t h e f o u r Ca affinity  f o r calcium (K  binding s i t e s , three s i t e s exhibited - 0,2 yM) but low a f f i n i t y  d  for Mg  2 +  (K  high = 140 n M ) .  d  The f o u r t h s i t e has s i m i l a r a f f i n i t i e s f o r both c a l c i u m and magnesium (K  d j C g  2+ =  1 M and K P  rf  M g  2 + = 20 yM) ( W o l f f e t aj_ 1 9 7 7 ) .  f o r e suggested t h a t the f u n c t i o n a l  I t was  there-  i n t r a c e l l u l a r form o f c a l m o d u l i n i s  2+ 2+ p r o b a b l y a 3 Ca : 1 Mg m o l e c u l e . A v a r i e t y o f data a l s o s u p p o r t s 2+ ?+ the s u g g e s t i o n t h a t Mg and o t h e r d i v a l e n t metal ions compete w i t h the Ca^ 2+ 2+ b i n d i n g s i t e on c a l m o d u l i n , a) C a l m o d u l i n binds 4 moles o f Mg o r Mn 2+ per mole o f p r o t e i n and these i o n s can be d i s p l a c e d by Ca (Wolff et al 2+ 1977). b) An i n c r e a s e i n the apparent K f o r Ca of v a r i o u s c a l m o d u l i n m  dependent enzymes ( e . g .  ATPase, a d e n y l a t e c y c l a s e ,  phosphodiesterase)  2+ o c c u r s w i t h i n c r e a s i n g Mg  concentrations  ( K a t z ejt al_ 1979; Brostrom  and W o l f f 1976; Brostrom e t a]_ 1 9 7 7 ) . Thus b i n d i n g o f c a l m o d u l i n to various  enzymes i n the presence o f d i f f e r e n t  complex  kinetics. R e g u l a t i o n o f enzyme (E)  by c a l m o d u l i n (C)  to proceed by 2 s e q u e n t i a l , f u l l y r e v e r s i b l e n Ca  2 +  x(Ca  2 +  d i v a l e n t c a t i o n s may i n v o l v e  + C^==i ( C a  2 +  )  n  mass a c t i o n  :  equations (1)  2 +  L  J  x  E «. active  v  where n = number o f b i n d i n g s i t e s which must be o c c u p i e d by Ca c o n v e r t c a l m o d u l i n to an a c t i v a t i n g 2+ (Ca  believed  C  ) C + E. . . ' ^ ^ [ ( C a ) C] 'n inactive ^ 'n 4  i s generally  form f o r the enzyme.  For  2+  (2) '  to  the  2+ + Mg  )-ATPase i t was suggested t h a t n = 3 > 4 > 2 > l , a n d  that  c a l m o d u l i n d i s s o c i a t e s from the c a l m o d u l i n s a t u r a t e d enzyme i n the range o f 10"^ - 10" M even a t c a l m o d u l i n c o n c e n t r a t i o n s 6  Scharff 1981), while n = 4 for  o f 5^.M (Foder and  p h o s p h o d i e s t e r a s e (Huang e t al_ 1981) and  myosin l i g h t c h a i n k i n a s e (Blumenthal  and S t u l l 1 9 8 0 ) .  However, i t  is  not known i f Ca  2+  binds s e q u e n t i a l l y o r randomly t o t h e v a r i o u s domains  (Brostrom and W o l f f  1981); 2+  x = unknown number o f (Ca  ) ~ c a l m o d u l i n complexes.  Saturation of c a l -  n  2+ modulin w i t h Ca  r e s u l t s i n a c o n f o r m a t i o n a l change i n v o l v i n g an i n c r e a -  se i n t h e a - h e l i c a l c o n t e n t from 28% t o 42%, and a c o n f o r m a t i o n a l change to a more compact f o r m .  The i n c r e a s e i n a - h e l i c a l c o n t e n t i s  by an i n c r e a s e i n e l l i p t i c i t y , an enhanced t y r o s i n e negative tyrosine Hidaka  reflected  f l u o r e s c e n c e and a  d i f f e r e n c e spectrum ( W o l f f e_t al_ 1 9 7 7 ) .  Tanaka and  (1980) have shown t h a t t h e c o n f o r m a t i o n a l change produced i n 2+  c a l m o d u l i n by b i n d i n g o f Ca  to the high a f f i n i t y  s i t e s exposes  hydro-  phobic groups which i n t u r n appear t o be i n v o l v e d i n a c t i v a t i n g c a l m o d u l i n - d e p e n d e n t enzymes.  Thus, both the a c i d i c n a t u r e o f c a l m o d u l i n and  2+ i t s Ca  - i n d u c i b l e hydrophobic  r e g u l a t i n g c a l m o d u l i n dependent It  p r o p e r t i e s seem t o be i m p o r t a n t  in  enzymes.  i s i m p o r t a n t t o note from e q u a t i o n (1) t h a t t h e s e n s i t i v i t y  of  the enzyme f o r c a l c i u m w i l l v a r y as a f u n c t i o n o f c a l m o d u l i n c o n c e n t r a t i o n , a c c o r d i n g t o the law o f mass a c t i o n .  S e c o n d l y , c a t i o n s which com-  2+ pete f o r Ca  b i n d i n g s i t e s may a l s o a f f e c t the s e n s i t i v i t y  o f t h e enzyme  2+ f o r Ca  .  i n d i c a t e s that the calcium s e n s i t i v i t y a l s o 2+ depends on t h e a f f i n i t y o f t h e i n a c t i v e enzyme f o r Ca - c a l m o d u l i n 2+ complex.  E q u a t i o n (2)  Thus, enzymes w i t h r e l a t i v e l y  high a f f i n i t y  f o r t h e Ca  -  c a l m o d u l i n - c o m p l e x may be a c t i v a t e d p r e f e r e n t i a l l y over t h o s e w i t h affinities  i n c e l l s exposed t o s t i m u l i .  f o r c a l m o d u l i n e x i s t among v a r i o u s  lower  Indeed, a range o f a f f i n i t i e s  proteins  (Brostrom and W o l f f  1981).  Based on k i n e t i c d a t a , Graf and P e n n i s t o n (1981) and Foder and S c h a r f f 2+ (1S81) showed t h a t c a l m o d u l i n i n t e r a c t s w i t h p u r i f i e d s o l u b i l i z e d Ca ATPase from human e r y t h r o c y t e i n a m o l a r r a t i o o f 1 : 1 .  Furthermore,  the  2+ apparent a f f i n i t y o f c a l m o d u l i n f o r  (Ca  2+ + Mg  )-ATPase i s  strongly  dependent on the c o n c e n t r a t i o n o f c a l c i u m and decreased a p p r o x i m a t e l y 1000 t i m e s when t h e c o n c e n t r a t i o n o f c a l c i u m was reduced from 112 to 0.5 yM ( F o d e r  and S c h a r f f 1981).  The c o n c e n t r a t i o n o f c a l m o d u l i n i n  the e r y t h r o c y t e c y t o s o l  i s a p p r o x i m a t e l y 5 yM, c o r r e s p o n d i n g t o 50-80 2+ 2+ times t h e c e l l u l a r c o n c e n t r a t i o n o f (Ca + Mg )-ATPase(approximately 10 ymol/g membrane p r o t e i n ) . S i n c e the c o n c e n t r a t i o n o f i n t r a c e l l u l a r c a l c i u m i s n o r m a l l y l e s s than 10~^M, i t was proposed by Foder and S c h a r f f (1981) t h a t i n v i v o most o f t h e c a l m o d u l i n i s d i s s o c i a t e d from t h e Ca transport-ATPase i o n o f (Ca  2+  and t h a t c a l m o d u l i n b i n d i n g and i t s subsequent  2+ 2+ + Mg )-ATPase r e q u i r e s t h a t the Ca concentration  2+  -  activatrises  to 1 0 " - 1 0 " M . 6  5  It  i s g e n e r a l l y a c c e p t e d t h a t i n human red c e l l s , c a l m o d u l i n 2+  affects  (Ca  2+ + Mg )-ATPase a c t i v i t y and a c t i v e c a l c i u m t r a n s p o r t  through a c a l c i u m dependent b i n d i n g to t h e enzyme (Hanahan  et a l 1978; 2+  N i g g l i et aj_ 1 9 7 9 ) . T h i s p r o p e r t y Mg )-ATPase 2+  has been u t i l y s e d to p u r i f y t h e (Ca  ( G i e t z e n e t a]_ 1980; N i g g l i et a l 1981). 2+  The r o l e o f c a l m o d u l i n i n t h e r e a c t i o n sequence o f (Ca ATPase i s summarized 1)  +  2+ + Mg  )-  below:  2+ 2+ C a l m o d u l i n b i n d i n g t o the (Ca + Mg )-ATPase i n c r e a s e s the apparent 2+ c a l c i u m a f f i n i t y o f t h e system and i n c r e a s e s t h e V o f t h e Ca max transport-ATPase  ( M a c l n t y r e and Green 1976; Hinds e t a l 1981; S a r k a d i  et a l 1980). 2)  C a l m o d u l i n has no apparent e f f e c t on the a f f i n i t y 2+ the Ca  -transport-ATPase  the regulatory 1980).  2+ o f ATP or Mg for  ( S a r k a d i et a]_ 1978) a l t h o u g h i t may a l t e r  r o l e o f high ATP c o n c e n t r a t i o n s (Muallem and K a r l i s h  3) In the absence o f Mg  , but i n the presence o f Ca  , calmodulin  i n c r e a s e s the r a t e and t h e s t e a d y s t a t e l e v e l o f t h e (EP c o m p l e x ) .  phosphoenzyme  (Muallem and K a r l i s h 1 9 8 0 , 1 9 8 1 ; Rega and Garrahan 1 9 8 0 ) . 2+  4) In t h e presence o f Mg  2+ + Ca  , c a l m o d u l i n does not a f f e c t t h e  s t a t e l e v e l o f EP a t low c o n c e n t r a t i o n s but decreases t h e l e v e l high c o n c e n t r a t i o n s ( J e f f e r y et aj_ 1 9 8 0 ) ,  steady at  and a c c e l e r a t e s t h e r a t e  of  EP f o r m a t i o n (Muallem and K a r l i s h 1980) and d e c o m p o s i t i o n (Muallem and K a r l i s h 1 9 8 1 ) . 5) C a l m o d u l i n has a l s o been suggested t o s t i m u l a t e EP d e p h o s p h o r y l a t i o n 2+ when measured i n the absence o f Mg (Rega and Garrahan 1 9 8 0 ) . 2+ 6) J e f f e r y e t al_ (1980) have shown t h a t both Mg  and c a l m o d u l i n s t i m u -  l a t e the d e c o m p o s i t i o n o f EP complex and t h a t the e f f e c t s o f t h e s e two e f f e c t o r s was a n t a g o n i s t i c , s u g g e s t i n g a s i m i l a r mechanism o f EP d e c o m p o s i t i o n . 7) Muallem and K a r l i s h (1980) proposed t h a t t h e £2 s t a t e o f t h e enzyme i s c o n v e r t e d t o E-| by c a l m o d u l i n i . e .  c o n v e r s i o n o f the enzyme from a 2+ s t a t e o f low a f f i n i t y to a s t a t e o f high a f f i n i t y f o r Ca A l t h o u g h r e l a t i v e l y much i s known about the r o l e o f c a l m o d u l i n i n  different  enzyme/protein  which c o n t r o l  systems, l i t t l e i s known about t h e  the l e v e l s or f u n c t i o n s o f c a l m o d u l i n .  factors  In 1977, Wang and  Desai i s o l a t e d and c h a r a c t e r i z e d a c a l m o d u l i n - b i n d i n g p r o t e i n  (CaM-BP)  from t h e bovine b r a i n which e x h i b i t e d c a l c i u m dependent b i n d i n g t o calmodulin.  L a r s e n et a]_ (1978) showed t h a t t h i s p r o t e i n  a n t a g o n i z e d t h e a c t i v a t i o n o f (Ca o f red blood c e l l membranes. have been i d e n t i f i e d .  2+  (CaM-BP)  2+ 2+ + Mg )-ATPase and Ca -transport  Since then, several  types o f CaM-BP's  Klee e t al_ (1 979) demonstrated the presence o f a  heat l a b i l e CaM-BP i n nerve t i s s u e , w h i c h they termed c a l c i n e u r i n .  Sharma  et a]_ (1978) r e p o r t e d the i s o l a t i o n o f heat s t a b l e CaM-BP from b o v i n e brain.  Au (1978) and S a r k a d i et aj_ (1980) showed t h a t a c o n c e n t r a t e d  heat l a b i l e p r o t e i n f r a c t i o n o f red c e l l a c t i v a t i o n o f t h e red c e l l membrane (Ca transport.  c y t o p l a s m prevents  2+  2+ 2+ + Mg )-ATPase and Ca -  L i t t l e i s known about the f a c t o r s g o v e r n i n g  f u n c t i o n s 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 i n the c e l l s . the o r i g i n a l i n t r a c e l l u l a r e n v i r o n m e n t ,  calmodulin  the l e v e l s  or  Furthermore,  t h e s e p r o t e i n s may n u l l i f y  in the  e f f e c t o f c a l m o d u l i n ( S a r k a d i et a ! J 9 8 0 ; P e n n i s t o n 1980; Wang 1981) and the a c t u a l c o n t r o l o f enzyme a c t i v i t y may be a complex f u n c t i o n 2+ Ca  2+ , Mg , ATP and f r e e c a l m o d u l i n c o n c e n t r a t i o n s .  of  S a r k a d i (1980)  s u g g e s t t h a t the c a l m o d u l i n e f f e c t s i n t h e i n t a c t red c e l l s may be largely  suppressed because the Ca  both a low a f f i n i t y  2+  pump i n i n t a c t c e l l s 2+  f o r c a l c i u m and low Ca  demonstrates  t r a n s p o r t r a t e s as compared  to ghosts r e s e a l e d i n the presence o f p u r i f i e d c a l m o d u l i n o r i n i n s i d e out v e s i c l e s i n the presence o f c a l m o d u l i n i n the i n c u b a t i o n medium. The f u n c t i o n o f c a l m o d u l i n as an a c t i v a t o r o f s e v e r a l  enzymes was  shown to be b l o c k e d n o t o n l y by CaM-BP's but a l s o by p h e n o t h i a z i n e s . L e v i n and Weiss (1977)  first  reported that c e r t a i n a n t i p s y c h o t i c drugs,  such as t r i f l u o p e r a z i n e and r e l a t e d p h e n o t h i a z i n e s , b l o c k e d c a l m o d u l i n a c t i v a t i o n o f phosphodiesterase. the b i n d i n g o f p h e n o t h i a z i n e s  These a u t h o r s a l s o demonstrated t h a t  t o c a l m o d u l i n was c a l c i u m dependent and  t h a t t h e r e appeared to be a c o r r e l a t i o n between n e u r o l e p t i c potency and anticalmodulin a c t i v i t y .  I t was t h e r e f o r e  i m p l i e d t h a t antagonism o f  c a l m o d u l i n formed the b a s i s o f n e u r o l e p t i c a c t i v i t y .  R o u f o g a l i s (1981)  Raess and V i n c e n z i (1980) and G i e t z e n e t al_ (19.80) demonstrated t h a t 2+ 2+ p h e n o t h i a z i n e s a l s o a n t a g o n i z e d c a l m o d u l i n a c t i v a t i o n o f (Ca + Mg )-  ATPase and Ca little  2+  - t r a n s p o r t i n human e r y t h r o c y t e membranes.  However,  o r no c o r r e l a t i o n was found between c a l m o d u l i n antagonism and  antipsychotic activity  e . g . h a l o p e r i d o l , a member o f t h e  butyrophenone  c l a s s o f c l i n i c a l a n t i p s y c h o t i c s i s 50 f o l d more p o t e n t than c h l o r p r o mazine c l i n i c a l l y , y e t i t b i n d s t o c a l m o d u l i n w i t h lower a f f i n i t y chlorpromazine  ( L e v i n and Weiss 1979) and has a s i m i l a r o r  than  slightly  lower potency i n a n t a g o n i z i n g c a l m o d u l i n - a c t i v a t e d p h o s p h o d i e s t e r a s e ( L e v i n and Weiss 1979) o r  i s i n e f f e c t i v e against calmodulin a c t i v a t i o n 2+  o f membrane bound e r y t h r o c y t e (Ca promazine (Raess and V i n c e n z i  1980).  2+ + Mg  )-ATPase compared t o c h l o r r  Another problem i n r e l a t i n g  c a l m o d u l i n i n h i b i t i o n w i t h c l i n i c a l a n t i p s y c h o t i c potency i s the l a c k of s t e r e o s p e c i f i c i t y of calmodulin i n h i b i t i o n i n c o n t r a s t to a n t i p s y c h o tic activity.  R o u f o g a l i s (1981) r e p o r t e d t h a t a l l o f a s e r i e s o f  promazine a n a l o g s , i n which t h e c h l o r i n e s u b s t i t u t i o n was v a r i e d  chlorfrom  p o s i t i o n 1 to 4 on the A r i n g o f t h e t r i c y c l i c nucleus, i n h i b i t e d calmodu2+ 2+ Tin a c t i v a t i o n o f e r y t h r o c y t e (Ca  + Mg  )-ATPase,  y e t o n l y the compound  w i t h c h l o r i n e s u b s t i t u t e d on p o s i t i o n 2 shows t r a n q u i l i z e r a c t i v i t y v i v o . S i m i l a r l y , Raess  and V i n c e n z i  (1980) showed t h a t both (+)-  ( - ) - i s o m e r s o f b u t a c l a m o l (a n o n - p h e n o t h i a z i n e n e u r o l e p t i c ) 2+ 2+ ineffective  i n b l o c k i n g c a l m o d u l i n - s e n s i t i v e (Ca  though ( + ) - b u t a c l a m o l  has t r a n q u i l i z e r a c t i v i t y .  t h a t the potency o r s t e r e o c h e m i s t r y  + Mg  in  and  were  )-ATPase,  even  I t was thus c o n c l u d e d  of calmodulin antagonists  either  w i t h i n a d i v e r s e s e r i e s o f a n t i p s y c h o t i c agents o r w i t h i n a subgroup o f p h e n o t h i a z i n e analogs does not c o r r e l a t e w e l l w i t h the c l i n i c a l experimental 1981).  efficacy  o f compounds as a n t i - p s y c h o t i c agents  or  (Roufogalis  I t was i n d i c a t e d i n the above s e c t i o n t h a t p h e n o t h i a z i n e s b i n d t o c a l m o d u l i n i n a c a l c i u m dependent manner.  I t was shown t h a t the  trifluo-  2+ p e r a z i n e s i t e s are d i s t i n c t from the Ca  binding s i t e s C K l e v i t et a l  2+ 1981) and t h a t Ca  dependent exposure o f hydrophobic s i t e s i s  for binding of t r i f l u o p e r a z i n e to calmodulin.  important  I t may be noted t h a t  2+ a l t h o u g h Ca  i n d u c e s t h e exposure o f hydrophobic s i t e s i n two o t h e r  calcium binding proteins v i z .  t r o p o n i n C and S - 1 0 0 , o n l y t r o p o n i n C b i n d s  t r i f l u o p e r a z i n e and i n a c a l c i u m dependent manner ( L e v i n and Weiss 1 9 7 8 ; Kobayashi et al_ 1 9 7 9 ) .  However, r e c e n t l y S-100 was found to 2+  w i t h i m m o b i l i z e d p h e n o t h i a z i n e i n a Ca  -dependent manner.  interact Phenothiazi-  nes are not the o n l y compounds c a p a b l e o f i n h i b i t i n g c a l m o d u l i n - d e p e n d e n t a c t i v a t i o n o f t h e enzyme s y s t e m s , as a v a r i e t y o f compounds a r e a b l e to a n t a g o n i z e the a c t i o n o f c a l m o d u l i n .  These agents i n c l u d e a n t i p s y c h o t i c  agents, a n t i d e p r e s s a n t s , l o c a l a n e s t h e t i c s , v i n c r i s t i n e , r a u w a l f i a l o i d s , anti-hypertensives , sulfonamide  N-(6-aminohexyl)-5-chloro-1-naphtha!ene-  (W-7),l-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-e-(2,4-  dichlorobenzyloxy)phenethyl]  i m i d a z o l i n i u m c h l o r i d e ( R 2 4 5 7 1 ) , and  T r i t o n X-100 ( R o u f o g a l i s 1 9 8 2 ) .  A f e a t u r e common to t h e s e agents i s  they are hydrophobic o r a m p h i p a t h i c and g e n e r a l l y pH.  alka-  These compounds have o t h e r w i s e  cationic at physiological  l i t t l e similarity in their  and p h a r m a c o l o g i c a l a c t i o n s ( V i n c e n z i  that  1981; Roufogalis 1982).  structures Although  the p h e n o t h i a z i n e s are u s e f u l t o o l s to s t u d y the c a l m o d u l i n a c t i v a t i o n of various  enzyme systems i n v i t r o , i t  i s i m p o r t a n t to note t h a t  agents do not b i n d t o c a l m o d u l i n a l o n e .  these  I n d e e d , p h e n o t h i a z i n e s have been  shown to b i n d some " c a l m o d u l i n - l i k e " p r o t e i n s i s o l a t e d from Chlamydompnas f l a g e l l a (Van E l d i k e t aj_ 1980) and r a t h e a r t azines a l s o antagonize  (Na  +  (MacManus, 1 9 8 1 ) , P h e n o t h i -  + K ) - A T P a s e and the a c t i v a t i o n o f a number +  o f enzymes fay l i p i d s , and they a l s o e x h i b i t r e c e p t o r b l o c k i n g a c t i o n s ( a - a d r e r t e r g i c , m u s c a r i n i c , dopamine and s e r o t o n i n ) .  For a c r i t i c a l  dis-  c u s s i o n on the s p e c i f i t y and s e l e c t i v i t y o f p h e n o t h i a z i n e s the r e a d e r i s r e f e r r e d t o a r e c e n t r e v i e w by R o u f o g a l i s ( 1 9 8 2 ) .  It  i s , however, c l e a r  t h a t p h e n o t h i a z i n e s are f a i r l y n o n - s p e c i f i c i n t h e i r a c t i o n , a t l e a s t when used i n v i v o .  We have shown t h a t t r i f l u o p e r a z i n e i n h i b i t s 2+  i o n o f e r y t h r o c y t e (Ca  2+ + Mg  ) - A T P a s e , both membrane bound and T r i t o n  X - 1 0 0 s o l u b i l i z e d , by two a c i d i c p r o t e i n s ( v i z . (M  r  activat-  poly-L-aspartic acid  = 20,000) and cAMP p r o t e i n k i n a s e i n h i b i t o r from bovine  a p p a r e n t l y u n r e l a t e d to c a l m o d u l i n . concentration of trifluoperazine  The a c t i v a t i o n was i n h i b i t e d a t a  (30 yM) s i m i l a r to t h a t r e q u i r e d  i n h i b i t i o n of calmodulin a c t i v a t i o n . be d i s c u s s e d l a t e r under r e s u l t s  heart)  for  The mechanism o f i n h i b i t i o n w i l l  and d i s c u s s i o n . 2+  An i n t e r e s t i n g o b s e r v a t i o n was made t h a t (Ca  2+ + Mg  )-ATPase  2+ a c t i v i t y and Ca  - t r a n s p o r t c o u l d be s t i m u l a t e d not o n l y by c a l m o d u l i n  but a l s o by a number o f a c i d i c p h o s p h o l i p i d s ( N i g g l i  e t al_ 1980;  A l - J o b o r e and R o u f o g a l i s 1 9 8 1 ) , b y l o n g c h a i n u n s a t u r a t e d (Vincenzi  fatty-acids  1 9 8 1 ; A l - J o b o r e and R o u f o g a l i s 1 9 8 1 ) , c e r t a i n p o l y anions  (Minocherhomjee and R o u f o g a l i s 1981) and by l i m i t e d p r o t e o l y s i s  (Taverna  and Hannahan 1980; N i g g l i  trifluo-  e t aj_ 1 9 8 1 ) .  I t was a l s o shown t h a t  p e r a z i n e ( a p p r o x . 100 uM) was a b l e to a n t a g o n i z e t h e a c t i v a t i o n produced by a c i d i c p h o s p h o l i p i d s , f a t t y - a c i d s , p o l y a n i o n s , and a l t h o u g h  only  p a r t i a l l y , by l i m i t e d p r o t e o l y s i s  (see C a r a f o l i 1 9 8 1 ) . I t was proposed 2+ by S a r k a d i (1980) t h a t a subunit o f the Ca t r a n s p o r t enzyme i s e a s i l y d i g e s t e d a t the i n t e r n a l membrane s u r f a c e and p o s s i b l y the f u n c t i o n i n g 2+ of t h i s calmodulin-binding regulatory affinity  o f the pump.  s u b u n i t determines t h e Ca  -  B) Role o f P r o t e i n K i n a s e s I t was shown i n the p r e y i o u s s e c t i o n t h a t many membrane f u n c t i o n s are r e g u l a t e d by the c a l c i u m - c a l m o d u l i n complex.  However, i n a d d i t i o n t o  c a l c i u m , membrane p h o s p h o r y l a t i o n may a l s o r e g u l a t e c e r t a i n c e l l u l a r functions.  Membrane p h o s p h o r y l a t i o n can be c a t a l y s e d hy p r o t e i n k i n a s e  enzymes a s s o c i a t e d w i t h the membrane o r by c y t o p l a s m i c p r o t e i n k i n a s e s . S e v e r a l p r o t e i n k i n a s e a c t i v i t i e s a r e p r e s e n t i n the r e d c e l l al 1981).  (Boivin  et  C y c l i c n u c l e o t i d e dependent hi s t o n e k i n a s e s and c y c l i c n u c l e o -  t i d e independent c a s e i n k i n a s e s have been i d e n t i f i e d i n the human e r y t h r o c y t e (Rubin e t aj_ 1 9 8 2 ; F a i r b a n k s and Avruch 1 9 7 4 ; Avruch and Fairbanks 1974).  Both c y c l i c AMP dependent and independent p r o t e i n  k i n a s e s were shown to be c o m p a r t m e n t a l i z e d between the membrane and c y t o s o l i n human e r y t h r o c y t e s 1980).  (Drey fuss et aj_ 1 9 7 8 ; B o i v i n and Galand  Although the r o l e o f these k i n a s e s i n membrane f u n c t i o n s remains  t o be e s t a b l i s h e d , i t has been known f o r q u i t e some time t h a t red c e l l membrane c o n t a i n s s e v e r a l p r o t e i n s which a r e p h o s p h o r y l a t e d by t h e s e k i n a s e s (Rubi n et aj_ 1 9 7 2 ; Avruch and F a i r b a n k s 1 9 7 4 ) .  In membranes  from v a r i o u s mammalian t i s s u e s and c e l l s , p h o s p h o r y l a t i o n o f membrane p r o t e i n s was shown to c o r r e l a t e w i t h changes i n p e r m e a b i l i t y to i o n s 2+ (Greengard 1 9 7 6 ) , drugs ( C a r l s e n e t al_ 1 9 7 7 ) , Ca 1977), glucose transport proteins (Gazitt 1975).  ( W e l l e r and Morgan  (Chang et al_ 1 9 7 4 ) , arrangement o f membrane  e t aj_ 1 9 7 6 ) , and membrane f l u i d i t y (Kury and McConnel  In the case o f human e r y t h r o c y t e membranes, i t has been suggested  t h a t the degree o f p h o s p h o r y l a t i o n c o u l d r e g u l a t e t h e  interaction  between c e r t a i n p r o t e i n s which may be c r u c i a l i n m a i n t a i n i n g e r y t h r o c y t e shape and deformabi 1 i t y  (Yawata e t al_ 1976; Kury and .McConnel 1 1 9 7 5 ) .  In p a r t i c u l a r , i n the p a s t few y e a r s  i n c r e a s i n g i n t e r e s t has been  d i r e c t e d toward the s t u d y o f the p h o s p h o r y l a t i o n o f s p e c t r i n , the roost abundant membrane p r o t e i n l o c a t e d on t h e c y t o s o l i c s u r f a c e , s i n c e i t s p h o s p h o r y l a t i o n s t a t e may modulate i t s i n t e r a c t i o n w i t h a c t i n and o t h e r membrane components, which i n t u r n may c o n t r o l the s h a p e , d e f o r m a b i l i t y and f u n c t i o n a l p r o p e r t i e s o f the membrane.  Although c o n t r o v e r s i a l ,  it  i s b e l i e v e d t h a t s p e c t r i n (band 2) and band 3 ( t h e most abundant i n t e g r a l membrane p r o t e i n ) k i n a s e (Type I)  a r e poor s u b s t r a t e s f o r c y c l i c AMP dependent  hi stone  ( S u z u k i et al_ 1 9 8 1 ; C l a r i e t al_ 1 9 8 1 ) , whereas both t h e s e  p r o t e i n s are good s u b s t r a t e s f o r c y c l i c AMP independent p r o t e i n k i n a s e (casein-kinase)  (Suzuki  Simkowski and Tao 1 9 8 0 ) .  e t a l 1 9 8 1 ; C l a r i e t a l 1 9 8 1 ; V i c k e r s e t aj_ 1979; C y c l i c AMP dependent h i s t o n e k i n a s e , however,  has been shown to p h o s p h o r y l a t e membrane p r o t e i n s , e s p e c i a l l y (band 2 . 1 ) , bands 4 . 5 and 4 . 8 ( B o i v i n et a l 1 9 8 1 ) . A n k y r i n ( M  ankyrin r  = 215,000)  i s l o c a l i z e d on the c y t o p l a s m i c s u r f a c e o f t h e membrane and may be r e s p o n s i b l e f o r the attachment o f s p e c t r i n to the membrane through i t s high a f f i n i t y  b i n d i n g s i t e s f o r s p e c t r i n ( B e n n e t t and Stenbuck  1979).  A n k y r i n a l s o i n t e r a c t s d i r e c t l y w i t h band 3 ( B e n n e t t and Stenbuck 1 9 8 0 ) . A s s o c i a t i o n o f s p e c t r i n w i t h a n k y r i n has been demonstrated t o  restrict  the l a t e r a l m o b i l i t y o f band 3 and o t h e r i n t e g r a l membrane p r o t e i n s the e r y t h r o c y t e ghost ( F o w l e r and B e n n e t t 1 9 7 8 ) .  in  Thus, although s p e c t r i n  may not be a very good s u b s t r a t e f o r c y c l i c nucleotide dependent h i s t o n e kinase,  i t i s p o s s i b l e t h a t i t s s t a t e can be a l t e r e d i n d i r e c t l y t h r o u g h  c y c l i c AMP-dependent p h o s p h o r y l a t i o n o f It  ankyrin.  i s r a t h e r i n t r i g u i n g t h a t the human r e d c e l l  adenylate c y c l a s e or phosphodiesterase a c t i v i t y c y c l i c AMP dependent h i s t o n e k i n a s e a c t i v i t y Recent r e p o r t s ,  has l i t t l e o r no  but a v e r y high p o t e n t i a l  ( B o i v i n and Galand 1 9 7 8 ) .  however, i n d i c a t e t h a t d e s p i t e the absence o f a c t i v e  c y c l i c AMP m e t a b o l i s m i n the human e r y t h r o c y t e . c y c l i c AMP can e n t e r e r y t h r o c y t e by  p a s s i v e uptake tn s u f f i c i e n t amounts t o a l t e r t h e  the  activity  o f c y c l i c AMP dependent p r o t e i n k i n a s e s o r t o a l t e r t h e r a t e o f t u r n o v e r o f c e r t a i n p h o s p h o r y l a t e d membrane p e p t i d e s (Thomas et aj_ 1 9 7 9 ; Tsukamato e t a]_ 1 9 8 0 ) . The l e v e l s o f c y c l i c AMP thus appear t o be r e g u l a t e d by d i f f u s i o n a c r o s s t h e red c e l l membrane. A recent report  by Varghese and Cunningham (1980) i n d i c a t e d t h a t 2+  m i c r o m o l a r c o n c e n t r a t i o n s o f c y c l i c AMP I n h i b i t red c e l l membrane f r a g m e n t s , protein kinase.  (Ca  2+ + Mg  )-ATPase i n  p r o b a b l y v i a membrane a s s o c i a t e d c y c l i c AMP  S i n c e c y c l i c AMP dependent p r o t e i n k i n a s e mediated  p h o s p h o r y l a t i o n can be s p e c i f i c a l l y b l o c k e d by c y c l i c AMP dependent p r o t e i n k i n a s e i n h i b i t o r s (PKI)  which have been i s o l a t e d from  various  t i s s u e s (Whitehouse et a l 1 9 8 0 ) , the e f f e c t ( s ) o f PKI's on t h e k i n e t i c s o f 2+ 2+ Ca a c t i v a t i o n o f Ca - t r a n s p o r t ATPase has been i n v e s t i g a t e d i n t h i s thesis. It  i s i m p o r t a n t to note t h a t i n c o n t r a s t to c a l m o d u l i n , which  shows l a c k o f s p e c i e s o r t i s s u e s p e c i f i c i t y , the PKI's i s o l a t e d from d i f f e r e n t animal t i s s u e s such as t h e r a b b i t s k e l e t a l m u s c l e , bovine or r a t t e s t i s a r e not i d e n t i c a l .  However, a l l the PKI's a r e  heat s t a b l e p r o t e i n s (Walsh et aj_ 1 9 7 1 ; al_ 1977; D e m a i l l e e t a l 1978; F u r s t e n t h a l  acidic,  Weber and Rosen 1977; 1980).  heart  Beal e t  S i n c e both PKI's and  c a l m o d u l i n a r e h e a t - s t a b l e a c i d i c p r o t e i n s , the e f f e c t o f PKI on 2+ e r y t h r o c y t e (Ca  2+ + Mg  )-ATPase was i n v e s t i g a t e d i n f u r t h e r  compared to t h e a c t i v a t i o n o f t h i s enzyme by c a l m o d u l i n .  d e t a i l and  C) Role o f Anion Channel (Band  HI)  In the model f o r c a l c i u m e x t r u s i o n from human red c e l l s ,  i t was  shown t h a t a c t i v e c a l c i u m t r a n s p o r t i s not c o u p l e d t o c o - o r c o u n t e r t r a n s p o r t o f monovalent o r d i v a l e n t c a t i o n s .  E l e c t r o - n e u t r a l i t y during  2+ a c t i v e Ca  extrusion  i s p r o b a b l y m a i n t a i n e d by net CI  efflux,  since  t h e CI" conductance o f t h e human red c e l l membrane i s h i g h enough t o 2+ permit Ca  movement (Gunn 1978). Waisman e_t a l _ ( l 9 8 1 ) , u s i n g i n s i d e - o u t  v e s i c l e s , r e p o r t e d t h a t band 3 ( a n i o n c h a n n e l ) i n h i b i t o r s such as 4 - a c e t a m i d o - 4 ' - i s o t h i o cyano s t i l b e n e - 2 ' - d i s u l f o n a t e (SITS) and N - ( 4 - a z i d o - 2 - n i t r o p h e n y l ) - 2 - a m i n o e t h y l s u l f o n a t e (NAP-taurine) 2+ c a l m o d u l i n s t i m u l a t e d Ca potency.  blocked  t r a n s p o r t and anion t r a n s p o r t w i t h s i m i l a r  T h i s o b s e r v a t i o n l e d t h e s e a u t h o r s t o suggest t h a t t h e s e two  t r a n s p o r t p r o c e s s e s were c l o s e l y c o u p l e d .  Furthermore,  they suggested  t h a t a t low c o n c e n t r a t i o n s (.K. = 45 - 80 y M ) , N A P - t a u r i n e b l o c k e d c a l m o d u l i n dependent c a l c i u m t r a n s p o r t a c t i v i t y v i a blockade o f a n i o r i t r a n s p o r t through t h e anion c h a n n e l . The band 3 p r o t e i n , which i s t h e major transmembrane p r o t e i n , n o r m a l l y f a c i l i t a t e s the e l e c t r o n e u t r a l exchange o f anions a c r o s s the e r y t h r o c y t e membrane ( C a b a n t c h i k and R o t h s t e i n 1974; Ho and G u i d o t t i , 1975).  Band 3 p r o t e i n i s a l s o known t o f u n c t i o n as an a n i o n u n i p o r t  i n response to an e l e c t r o c h e m i c a l g r a d i e n t a c r o s s the membrane (Knauf e t a i l 971; W o l o s i n e t a l 1 9 7 7 ) .  G r i n s t e i n et a l (1979) showed t h a t  4 , 4 ' - d i i s o t h i o c y a n o - 2 , 2 ' - s t i l b e n e d i s u l f o n a t e (DIDS) i s a t r a n s p o r t  site  s p e c i f i c , non-penetrating i r r e v e r s i b l e  it  i n h i b i t o r o f band 3 and t h a t  i s e f f e c t i v e as an i n h i b i t o r o f band 3 o n l y when a p p l i e d e x t r a c e l l u l a r l y . Knauf e t aj_ (1978) showed t h a t N A P - t a u r i n e a c t s as a r e v e r s i b l e  inhibi-  t o r o f band 3 ( i n t h e dark) when i t i s p r e s e n t e i t h e r w i t h i n t h e c e l l  o r i n t h e e x t e r n a l medium. in  However, t h e r e i s a s u b s t a n t i a l  t h e c o n c e n t r a t i o n o f N A P - t a u r i n e r e q u i r e d t o b l o c k t h e a n i o n channel  from the two s i d e s o f the membrane. is and  difference  a relatively  low a f f i n i t y  On t h e c y t o p l a s m i c s i t e , N A P - t a u r i n e  i n h i b i t o r o f c h l o r i d e exchange (K. = 370 yM)  competes w i t h CI" f o r the s u b s t r a t e s i t e o f t h e a n i o n exchange  system.  On t h e o t h e r hand, e x t e r n a l  N A P - t a u r i n e i s a f a r tnore p o t e n t  i n h i b i t o r o f anion-exchange (K. - 20 yM) and causes n o n - c o m p e t i t i v e i n h i b i t i o n o f t h e a n i o n - c h a n n e l by b i n d i n g t o a " m o d i f i e r " s i t e .  I t was  suggested by t h e s e a u t h o r s t h a t the m o d i f i e r s i t e i s a c c e s s i b l e t o NAPt a u r i n e o n l y from t h e o u t s i d e , whereas t h e t r a n s p o r t s i t e may be a c c e s s i b l e from e i t h e r  side.  S t a r o s and R i c h a r d s (.1974) showed t h a t i n e r y t h r o c y t e membrane p r e p a r a t i o n s N A P - t a u r i n e , upon p h o t o a c t i v a t i o n , l a b e l s s e v e r a l bands i n a d d i t i o n to band 3 .  These o b s e r v a t i o n s prompted  s t u d i e s i n the p r e s e n t t h e s i s on (a) t a u r i n e as an i n h i b i t o r o f Ca  protein  further  t h e s i d e d n e s s o f a c t i o n o f NAP-  2+  - t r a n s p o r t ATPase, (b) t h e mechanism and 2+ s p e c i f i c i t y o f N A P - t a u r i n e i n i n h i b i t i o n o f Ca - t r a n s p o r t , and (c) a 2+ comparison between DIDS and N A P - t a u r i n e as i n h i b i t o r s o f Ca in  -transport  human e r y t h r o c y t e i n s i d e out v e s i c l e s .  O b j e c t i v e s o f the P r e s e n t Study 1) To s t u d y t h e i n f l u e n c e o f c a l m o d u l i n on the and  V of ( C a max.  2 +  Kc ^ a  +  + Mg )-ATPase. 2+  3  2) To d e t e r m i n e t h e r o l e o f cAMP mediated p r o t e i n p h o s p h o r y l a t i o n on t h e 2+ (Ca  2+ + Mg  )-ATPase a c t i v i t y ,  u s i n g cAMP dependent p r o t e i n  kinase i n h i b i t o r s . 3) To d e t e r m i n e the r o l e o f heat s t a b l e a c i d i c p r o t e i n s , o t h e r  than  2t c a l m o d u l i n , and o t h e r anions, i n the r e g u l a t i o n .Qf (Ca. ase  t Mg  )-ATP-  activity,  4) To s t u d y t h e r o l e o f the anion channel 2+ Ca  2+  -transport  i n t h e red  (Band 3) i n t h e r e g u l a t i o n  of  cell.  5) To s e p a r a t e t h e e f f e c t s o f N A P - t a u r i n e as an i n h i b i t o r o f t h e a n i o n channel and c a l c i u m - t r a n s p o r t ; and i t s comparison w i t h t h e a c t i o n o f t h e a n i o n channel b l o c k e r ,  DIDS.  MATERIALS AND flETHQPS  [I]  Materials: The c h e m i c a l s and/or p r o t e i n s were purchased from the  following  sources: 1) Sigma Chemical  Company:  EDTA EGTA Trizma base Imidazole  (grade  I)  HEPES MES Maleic acid PMSF Tris-ATP T r i t o n X-100 Poly-L-carboxylic Histone(type  acids  11-A)  C y c l i c AMP dependent p r o t e i n k i n a s e ( b o v i n e Protein kinase c a t a l y t i c  subunit  (bovine heart)  C y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r skeletal  ( b o v i n e heart and r a b b i t  muscle)  Calmodulin depleted  phosphodiesterase  C y c l i c AMP Bovine serum albumin ( f a t t y - a c i d Sodium  tungstate  Boric acid  heart)  free)  Taurine DIDS T r y p s i n (Type  I)  Soya bean t r y p s i n Lauryl  i n h i b i t o r (Type 1-S)  sulfate  Coomassie blue  2)  J.T.  Baker Chemical Company:  1,2-cyclohexanedione TCA Sodium  hydroxide  Sodium c h l o r i d e  3)  Monosodium dihydrogen  phosphate (NaHgPQ^)  Disodium monohydrogen  phosphate (J^HPO^)  F i s h e r S c i e n t i f i c Company: Calcium c h l o r i d e Carbowax  (CaC^)  (PEG 20,000)  Lanthanum c h l o r i d e  4)  Pierce NAP-taurine SITS DIDS  5)  Amersham: [ _ P]_ATP ( s p e c i f i c a c t i v i t y Y  32  = 5-10x10  6  DPM/nmole)  CaCl  6)  2  (specific activity  = l,5j*10  6  CPM/^mole),  Whatman: DE-52 a n i o n exchange r e s i n ,  7)  A l l i e d Chemicals: H y d r o c h l o r i c a c i d (HC1) G l a c i a l A c e t i c A c i d (CHg COOH) S u l f u r i c Acid  8)  (H S0 ) 2  4  Biorad Laboratories: SDS  9)  Merck, Sharp and Dohme P r o b e n e c i d (a  10)  S m i t h , K l i n e and French Co. Trifluoperazine  11)  gift)  hydrochloride  (Montreal): (a  gift).  Calbiochem; Calmodulin.  12)  Eastman: Acrylamide(enzyme  grade)  N,N'-methylene-bisacrylamide N,N,N',N"-tetramethyl-ethylene-diamine.  [II]  Methods 1) P r e p a r a t i o n o f E r y t h r o c y t e Ghosts Human blood preserved  i n a c i d - c i t r a t e - d e x t r o s e s o l u t i o n was  o b t a i n e d from t h e l o c a l Red Cross Blood Bank and used w i t h i n 5 days o f c o l l e c t i o n f o r p r e p a r a t i o n o f e r y t h r o c y t e ghosts  (membranes).  Membranes were prepared by t h e methods o u t l i n e d by a) Dodge et a l ( 1 9 6 3 ) , b) S c h r i e r (1967) and c ) C a r a f o l i et a l  (1980).  a) Membrane p r e p a r a t i o n by t h e procedure o f Dodge et a l : Erythrocytes  c o l l e c t e d by c e n t r i f u g a t i o n a t 2,500 x ^ were  washed t h r e e t i m e s w i t h 310 i d e a l mosm-sodium phosphate b u f f e r , 7.4 (P 310 b u f f e r ) .  pH  The washed c e l l s were suspended i n an equal  volume o f P 310 b u f f e r and 10 ml o f t h i s s u s p e n s i o n was r a p i d l y p i p e t t e d i n t o 140 mis o f 20 i d e a l mosm-sodium phosphate pH. 7.4 (P 20 b u f f e r ) ,  f o l l o w e d by s w i r l i n g .  The  hemolysed  c e l l s were c e n t r i f u g e d a t 22,000 x £ f o r 40 m i n . and the was c a r e f u l l y d e c a n t e d . p o r t i o n s o f P 20 b u f f e r .  buffer,  hemolysate  The c e l l s were washed t w i c e more i n 140 ml The  ' g h o s t s ' from t h e i n d i v i d u a l  centri-  fuge b o t t l e s were pooled and washed once more by c e n t r i f u g a t i o n 40,000 x £ f o r 20 mins i n an equal volume o f P 20 b u f f e r .  at  One  m i l l i l i t e r of ghosts was found to c o n t a i n between 3 . 8 - 4 . 2 mg o f p r o t e i n (determined  by an automated Lowry procedure (1951) w i t h a  Technicon a u t o a n a l y s e r ) .  These ghosts were w h i t e and s t o r e d a t  - 2 0 ° C f o r ! 6 h r b e f o r e use and used w i t h i n 4 days o f  preparation.  b) Membrane p r e p a r a t i o n by s t e p w i s e hemolysis procedure as o u t l i n e d by S c h r i e r : The red c e l l s  were washed t h r i c e i n i s o t o n i c s a l i n e s o l u t i o n  a t 2500 x g_. The washed c e l l s were c o n s e c u t i v e l y hemolysed 1n 10 volumes o f 0.08M, 0.06M and 0.04M NaCl and c e n t r i f u g e d a t 8 , 0 0 0 , 13,000 and 15,000 x £ f o r 10 m i n . a t 4 ° C , r e s p e c t i v e l y .  The  cells  were f i n a l l y hemolysed i n 0.015M NaCl and 0.005M t r i s - m a l e a t e (pH 7.1) a t 2 - 4°C and washed t w i c e i n t h i s s o l u t i o n .  These ghosts were  s l i g h t l y p i n k i n c o l o r and were used i m m e d i a t e l y f o r t h e p r e p a r a t i o n o f r e s e a l e d ghosts or i n s i d e - o u t v e s i c l e s .  c ) Membrane p r e p a r a t i o n by t h e procedure d e s c r i b e d by C a r a f o i l ' et a l : The r e d c e l l s were washed t h r i c e i n 5 volumes o f 130mM 20mM T r i s - C l , pH 7 . 4 .  KC1,  The c e l l s were hemolysed i n 5 volumes o f ImM  Na-EDTA,  lOmM T r i s - C l , pH 7 . 4 , and c e n t r i f u g e d a t 18,000 x £ f o r  10 m i n .  The ghosts were then washed f i v e times i n the h e m o l y s i s  buffer,  and f o u r more times i n lOmM HEPES, pH 7 . 4 , w i t h o u t  EDTA.  The ghosts were w h i t e and were f i n a l l y resuspended i n 130mM KC1, 20mM HEPES, pH 7 . 4 , 500yM M g C l , 50yM C a C l , 2mM d i t h i o t h r e i t o l and 2  2  s t o r e d a t - 8 0 ° C and used w i t h i n 3-4 weeks.  2) P r e p a r a t i o n o f C a l m o d u l i n d e f i c i e n t (EDTA-treated) membranes: One volume o f Dodge ' g h o s t s ' was suspended i n f i v e volumes o f O.lmM EDTA and 1.OmM T r i s - m a l e a t e f o r 15 m i n .  (pH 8 . 0 ) and i n c u b a t e d a t 37°C  The s u s p e n s i o n was then c e n t r i f u g e d a t 2 0 , 0 0 0 x £ a t  4°C f o r 15 m i n .  The s u p e r n a t e n t was c a r e f u l l y d i s c a r d e d and the  p e l l e t was resuspended i n 15mM NaCl and 5mM T r i s - m a l e a t e t h e o r i g i n a l volume o f ghosts u s e d .  (pH 7.1)  to  The EDTA-treated membranes were  2+ used i m m e d i a t e l y f o r a s s a y Of (Ca t h e s e membranes a r e r e l a t i v e l y  2+ t Mg  )-ATPase a c t i y i t y ,  as  l a b i l e under t h e s e c o n d i t i o n s .  3) P r e p a r a t i o n o f r e s e a l e d g h o s t s Resealed ghosts were prepared e s s e n t i a l l y as d e s c r i b e d by Q u i s t and R o u f o g a l i s ( 1 9 7 5 ) . follows.  Red c e l l  The main s t e p s i n t h e procedure were as  "ghosts" were prepared from human b l o o d , s t o r e d  acid-citrate-dextrose  in  (Canadian Red C r o s s ) f o r no more than 4 days  at 4 ° C , by a s t e p w i s e h e m o l y s i s procedure as o u t l i n e d by S c h r i e r (1967).  These ghosts were used i m m e d i a t e l y f o r r e s e a l i n g .  Ghosts  (.1 ml) were suspended i n the "loading-medium" (3 m l ) , which c o n t a i n e d ( i n f i n a l c o m p o s i t i o n ) 4mM M g C I , 3mM C a C l , 4mM T r i s - A T P , O.lmM 2  o u a b a i n , lOmM T r i s - m a l e a t e ,  2  pH 7.1 at 4°C ± N A P - t a u r i n e .  s u s p e n s i o n was e q u i l i b r a t e d f o r 10 min a t 2 - 4 ° C .  The  R e s e a l i n g was  a c h i e v e d by r e s t o r a t i o n o f i s o t o n i c i t y w i t h N a C l , f o l l o w e d by i n c u b a t i o n i n a water bath at 25°C f o r 10 m i n . returned  The samples were  to t h e i c e - b a t h and washed t w i c e w i t h 5 ml o f 2mM M g C ^ ,  125mM N a C l , O.lmM ouabain,' 20mM T r i s - m a l e a t e , f o r 10 min a t 4 ° C .  pH 7.5 a t 5,000 x g_  The r e s e a l e d ghosts were then suspended i n t h e  same s o l u t i o n which c o n t a i n e d i n a d d i t i o n ImM C a C ^ . taurine  When NAP-  (50 yM) was added t o r e s e a l e d ghosts the sample was a l l o w e d  to i n c u b a t e i n the absence o f l i g h t a t 4°C f o r 10 min p r i o r i n i t i a t i o n o f the a s s a y .  The ATP h y d r o l y s i s  the tubes i n a bath a t 37°C.  to  was i n i t i a t e d by p l a c i n g  The ATPase a c t i v i t y was stopped by  a d d i n g SDS t o a f i n a l c o n c e n t r a t i o n o f 2%.  The ATPase a c t i v i t y  measured by d e t e r m i n i n g t h e r e l e a s e o f i n o r g a n i c phosphate from ATP by t h e method o f F i s k e and SubbaRow ( 1 9 2 5 ) .  was  (P..)  2+ Mg -ATPase  activity  was determined i n the absence o f C a C l . b y d i r e c t  incubation  2  o f ghosts  ( p r i o r to re sea. l i n g w i t h NaCl) w i t h ATP a t 3 7 ° C ,  The c o n t r i b u t i o n o f " l e a k y - g h o s t s " to t o t a l ATPase a c t i v i t y e s t i m a t e d by r e s e a l i n g ghosts i n the absence o f ATP.  The  was  formation  o f P.. a f t e r a d d i t i o n of ATP to the e x t e r n a l medium a t 37°C g i v e s ATPase a c t i v i t y sis,  due to "leaky g h o s t s " .  U s i n g t h i s method o f  analy-  a p p r o x i m a t e l y 60-65% o f t h e ghosts were found to be r e s e a l e d . 2+  The Mg  -ATPase a c t i v i t y  (17%) was s u b t r a c t e d from t o t a l  a c t i v i t y to obtain the calcium s t i m u l a t a b l e  ATPase  activity.  4) P r e p a r a t i o n o f i n s i d e - o u t v e s i c l e s I n s i d e - o u t v e s i c l e s were prepared from ghosts as described (Quist  and R o u f o g a l i s 1977).  dure were as f o l l o w s .  The main steps i n the p r o c e -  On t h e same day o f p r e p a r a t i o n , t h e ghosts  ( p r e p a r e d a c c o r d i n g to t h e method o f S c h r i e r ) o r i g i n a l volume o f the packed c e l l s pH 7 . 1 , d i l u t e d w i t h 5 volumes and ImM T r i s - m a l e a t e , constant shaking.  previously  were suspended to t h e  i n 15mM NaCl and 5mM T r i s - m a l e a t e ,  (25 ml) o f a s o l u t i o n o f O.lmM EGTA  pH 8 and i n c u b a t e d a t 37°C f o r 30 min w i t h  After  centrifugation  a t 20,000 x £ a t 4°C f o r  10 m i n , t h e p e l l e t s were resuspended to 1 volume (5 -ml) i n 15mM NaCl and 5mM T r i s - m a l e a t e ,  pH 7 . 1 .  The v e s i c l e p r e p a r a t i o n was suspended  i n 3 volumes o f lOmM T r i s - m a l e a t e ,  4mM M g C l  pH 7.1 and i n c u b a t e d f o r 5 min a t 0 - 4 ° C .  2  and 0.5mM CaClg a t  Isotonicity  was  restored  by t h e a d d i t i o n o f 1 ml o f 2.9M NaCl and t h e v e s i c l e s i n c u b a t e d a t 30°C f o r  10 m i n .  The v e s i c l e s were p e l l e t e d a t 20,000 x £ f o r  10 min and washed t w i c e  i n 66mM NaCl and 55mM T r i s - m a l e a t e  at pH 7 . 1 .  The f i n a l p e l l e t s were combined and suspended to 4 volumes i n the  same washing medium,  S i d e d n e s s p f t h e y e s i c l e s was determined by  t h e a s s a y o f a c e t y l c h o l i n e s t e r a s e i n the absence and presence o f T r i t o n X-100 (0.2%).  Approximately  50-55% o f t h e v e s i c l e s were  inside-out.  5) I r r a d i a t i o n  o f c e l l s and membranes w i t h N A P - t a u r i n e  Whole c e l l s , washed w i t h 140mM KC1 and 20mM T r i s - H C l , 2+ were suspended i n the b u f f e r used f o r assay o f (Ca a t 10% h e m a t o c r i t  pH 7 . 4 ,  2+ + fig  )-ATPase  i n the absence and presence o f N A P - t a u r i n e .  The  sample was i r r a d i a t e d f o r 40 min a t 4°C w i t h a S y l v a n i a 3M lamp (500 W) f o c u s s e d on t h e sample.  After  i r r a d i a t i o n the c e l l s were  c e n t r i f u g e d at 3,000 x £ and washed t w i c e w i t h t h e above c o n t a i n i n g 0.5% bovine serum albumin and one more time albumin. above.  buffer  without  Membranes were then prepared from t h e s e c e l l s as d e s c r i b e d Membranes o b t a i n e d from DIDS t r e a t e d c e l l s were processed  similarly.  The c e l l s were a l l o w e d to r e a c t w i t h DIDS (5 yM) a t 37°C  f o r 30 min p r i o r to  hemolysis.  For i r r a d i a t i o n o f membranes i n t h e presence o f  NAP-taurine,  the membranes ( a p p r o x . 4 mg p r o t e i n / m l f i n a l c o n c e n t r a t i o n ) were 2+ 2+ i n c u b a t e d i n the (Ca + Mg )-ATPase assay b u f f e r ( w i t h o u t ATP) i n the absence and presence o f N A P - t a u r i n e and p h o t o l y s e d f o r 40 min a t 4 ° C , as above.  50 y l  o f the p h o t o l y s e d membrane p r e p a r a t i o n was then  2+ assayed f o r  (Ca  a s s a y medium.  2+ + Mg  )-ATPase a c t i v i t y  i n 0 . 6 ml o f the s t a n d a r d  As t h e enzyme p r e p a r a t i o n was d i l u t e d 1 2 - f o l d ,  the  c o n c e n t r a t i o n o f u n r e a c t e d N A P - t a u r i n e was reduced to a c o n c e n t r a t i o n ( a p p r o x . 2 yM) which was shown i n c o n t r o l the ( C a  2 +  + Mg )ATPase 2+  activity.  experiments  not t o  inhibit  In experiments t o p r o t e c t a g a i n s t N A P - t a u r i n e  inhibition,  membranes were p h o t o l y s e d w i t h N A P - t a u r i n e (25 jiM) as d e s c r i b e d  2+ above, but i n t h e presence o f v a r i o u s 2+ ATPase v i z .  e f f e c t o r s o f (Ca  2+ + Mg  )-  2+  Ca  , Mg  , ATP and c a l m o d u l i n .  C o n t r o l a c t i v i t i e s were  e s t a b l i s h e d i n the same c o n d i t i o n s w i t h i r r a d i a t i o n i n t h e absence of NAP-taurine. 2+ 6) S o l u b i l i z a t i o n o f (Ca  2+ + Mg  )-ATPase from human e r y t h r o c y t e mem-  branes 2+ (Ca  2+ + Mg  )-ATPase was s o l u b i l i z e d  nes as d e s c r i b e d by C a r a f o l i e t al_ ( 1 9 8 0 ) .  from e r y t h r o c y t e membraE s s e n t i a l l y , t h e membrane  (4 - 4 . 5 mg p r o t e i n / m l ) was t r e a t e d w i t h T r i t o n X-100 (.1 -mg T r i t o n / mg membrane p r o t e i n ) at  a t 4°C f o r 10 m i n , f o l l o w e d by c e n t r i f u g a t i o n  100,000 x £ f o r 30 m i n .  The s u p e r n a t e n t was c a r e f u l l y  and used as a s o u r c e o f enzyme. p r o t e i n was s o l u b i l i z e d by t h i s 7) L i m i t e d p r o t e o l y s i s  Approximately,  removed  10-12% o f membrane  procedure.  (trypsinization)  o f membranes  To 1 ml o f membranes (4 mg/ml) prepared by t h e procedure o f C a r a f o l i et_ al_ ( 1 9 8 0 ) , was added 1 ml o f lOmM sodium-HEPES pH 7 . 4 , c o n t a i n i n g t r y p s i n sample t r y p s i n was o m i t t e d . 3 min.  ( 0 . 2 yg/mg p r o t e i n ) .  buffer,  In t h e c o n t r o l  The samples were i n c u b a t e d a t 37°C f o r  P r o t e o l y s i s was stopped by a d d i t i o n o f 1 ml o f a soyabean  trypsin-inhibitor  ( 0 . 4 yg/mg p r o t e i n )  i n the above b u f f e r .  samples were c e n t r i f u g e d a t 20,000 x £ f o r 10 m i n .  The  The membranes  were washed one more t i m e i n lOmM sodium-HEPES b u f f e r and r e c o n s t i t u t e d t o t h e o r i g i n a l volume (1 m l ) . led  Trypsinization  o f membranes 2+ t o a l o s s o f a p p r o x i m a t e l y 22% o f membrane p r o t e i n s . (Ca +  2+ Mg  )-ATPase a c t i v i t y was measured w i t h i n 30 min a f t e r  trypsinizat-  ion. 8) P u r i f i c a t i o n o f c y c l i c AMP dependent p r o t e i n k i n a s e i n h i b i t o r The PKI from bovine h e a r t o b t a i n e d from Sigma Chemical Company, a l t h o u g h r e p o r t e d t o be f r e e o f "heat s t a b l e modulator"  (calmodulin),  was found to c o n t a i n s m a l l but s i g n i f i c a n t c o n t a m i n a t i o n by calmodulin,  as determined by t h e p h o s p h o d i e s t e r a s e a s s a y and by r a d i o -  immunoassay. column.  The PKI p r e p a r a t i o n was p u r i f i e d on a Whatman DE 52  The PKI p r e p a r a t i o n from Sigma (5 mg/5 ml) was  dialyzed  a g a i n s t 1OmM i m i d a z o l e - H C l , pH 6 . 8 , a n d a p p l i e d to a DE 52 column (2.1 x 20 cm) p r e v i o u s l y  e q u i l i b r a t e d i n t h e same b u f f e r .  The column  was washed w i t h 25 ml o f t h e b u f f e r and e l u t e d w i t h 200 ml o f a l i n e a r g r a d i e n t o f 10 to 300mM NaCl i n lOmM i m i d a z o l e - H C l , pH 6 . 8 . F r a c t i o n s o f 2 - 3 ml  were c o l l e c t e d .  T h i s was f o l l o w e d by washing  w i t h 100 ml o f 1M NaCl i n t h e i m i d a z o l e b u f f e r .  The  c o n t a i n i n g t h e c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r by t h e i r lytic  fractions  (as  determined  i n h i b i t i o n o f t h e p h o s p h o r y l a t i o n o f hi stone by t h e  subunit)  were pooled and c o n c e n t r a t e d by d i a l y s i s (M  3,000) a g a i n s t p o l y e t h y l e n e t e d to a p p r o x .  glycol.  b u f f e r c o n t a i n i n g 5mM EDTA, f o l l o w e d by e x t e n s i v e to remove EDTA.  i n an i d e n t i c a l manner.  cut  off  The p r o t e i n s o l u t i o n , c o n c e n t r a -  5 m l , was then d i a l y z e d a g a i n s t lOmM  imidazole buffer  cata-  imidazole  d i a l y s i s against  The 1M NaCl f r a c t i o n was  treated  Both t h e pooled g r a d i e n t f r a c t i o n s and t h e  1M NaCl f r a c t i o n were t e s t e d f o r t h e presence o f c a l m o d u l i n by c a l modulin  radioimmunoassay and by t h e i r a b i l i t y t o  calmodulin-depleted  phosphodiesterase.  stimulate  9) Chemical m o d i f i c a t i o n o f a r g i n y l - s i d e c h a i n ? A r g i n i n e r e s i d u e s were m o d i f i e d by t r e a t m e n t o f t h e (protein  k i n a s e i n h i b i t o r and c a l m o d u l i n ) w i t h 0.05M  d i o n e i n 0.15M b o r a t e b u f f e r ,  proteins  1,2-cyclohexane-  pH 9 f o r 3hours a t 3 7 ° C , e s s e n t i a l l y by  t h e method o f D e m a i l l e e t al_ (1 9 7 7 ) .  Control  samples were t r e a t e d  i n t h e same way i n t h e absence o f 1 , 2 - d i c y c l o h e x a n e d i o n e . T r i s buffers  Since  were shown t o d e c r e a s e t h e s t a b i l i t y o f t h e m o d i f i e d  a r g i n i n e product was performed  ( P a t t h y a n d S m i t h , 1 9 7 5 ) , assay o f ATPase  i n sodium maleate b u f f e r ,  activities  pH 6 . 9 before and a f t e r  arginine modification.  10) Assay o f  ATPases  (Ca  2+  2+ + Mg )-ATPase a c t i v i t y  32  was determined from  P^ r e l e a s e d  32 from [y-  P]ATP ( K a t z and B l o s t e i n , 1975). The i n c u b a t i o n medium  contained (unless  i n d i c a t e d o t h e r w i s e ) 66mM NaCl (or t h e  a n i o n ) , 6.5 mM M g C l ^ , O.lmM o u a b a i n , O.lmM EGTA, v a r i o u s  appropriate concentra-  t i o n s o f f r e e c a l c i u m and 55mM o f e i t h e r T r i s , HEPES, MES o r TES a d j u s t e d to the r e q u i r e d pH (pH 6 . 9 o r 7.2)  i n t h e a s s a y medium a t  37°C by a d d i t i o n o f maleate ( T r i s - m a l e a t e ) ,  HC1 ( T r i s - C l ) o r sodium  hydroxide  (sodium-HEPES, sodium-MES, sodium-TES and s o d i u m - m a l e a t e ) .  The e f f e c t s o f p o l y - L - c a r b o x y l i c studied in a Tris-maleate  buffer.  a c i d s and v a r i o u s 2+ Free Ca  a n i o n s were  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 s o l v i n g a s e r i e s o f q u a d r a t i c e q u a t i o n s as d e s c r i b e d previously  (Akyempon and R o u f o g a l i s 1981) and checked d i r e c t l y  by  a c a l c i u m s e l e c t i v e e l e c t r o d e a t a p p r o p r i a t e pH ( 6 . 9 o r 7.2) as previously (0.18  d e s c r i b e d ( A l - J o b o r e and R o u f o g a l i s 1981).  Dodge  mg p r o t e i n / 0 . 6 ml a s s a y ) , T r i t o n X-100 s o l u b i l i z e d ( C a  'ghosts' 2 +  + Mg ) 24  -ATPase ( 0 . 0 2 mg p r o t e i n / 0 . 6 ml a s s a y ) o r p u r i f i e d ( C a ase  2 +  + Mg )-ATP2 +  ( 1 . 8 yg p r o t e i n / 0 . 6 ml assray) were assayed w i t h 2mM ATP  salt)  f o r 60 min ( / g h o s t s ' ) o r 30 min ( . p u r i f i e d  (Ca  2 +  (Tris-  + Mg )-ATPase) 2+  o r w i t h 20 TJM ATP ( ' g h o s t s ' ) were assayed f o r 3 m i n and w i t h 0.5mM ATP 2+ (Tris-salt) at  37°C.  s o l u b i l i z e d CCa  2+ + Mg  )-ATPase was assayed f o r 10 min  P u r i f i e d human e r y t h r o c y t e c a l m o d u l i n o r o t h e r e f f e c t o r s  the enzyme were added to the enzyme p r e p a r a t i o n 10 min p r i o r t o ing  of  start-  the r e a c t i o n w i t h ATP. 2+ Mg  in  -ATPase a c t i v i t y was determined by measuring ATP h y d r o l y z e d  the above i n c u b a t i o n medium i n t h e presence o f O.lmM ouabain but  2+ w i t h o u t added Ca and s u b t r a c t e d from the t o t a l ATPase a c t i v i t y t o 2+ + + o b t a i n the Ca stimulatable a c t i v i t y . Ouabain s e n s i t i v e (Na + K ) ATPase a c t i v i t y was measured i n a medium c o n t a i n i n g 55mM T r i s - m a l e a t e (pH 7 . 2 ) , 66mM N a C l , 5mM KC1, 2mM M g C l , O.lmM EGTA w i t h o r 2  without  O.lmM ouabain and 2mM T r i s - A T P , and i n c u b a t e d f o r 60 min a t 3 7 ° C .  11)  P h o s p h o d i e s t e r a s e assay P h o s p h o d i e s t e r a s e a c t i v i t y was determined by the method o f Sharma e t al_ ( 1 9 7 8 ) .  The procedure i n v o l v e d  c o u p l i n g o f t h e phospho-  d i e s t e r a s e r e a c t i o n to a 5 ' - n u c l e o t i d a s e r e a c t i o n which was  followed  by a n a l y s i n g the r e s u l t i n g i n o r g a n i c phosphate by an automated F i s k e and Subbarow assay as d e s c r i b e d by Raess and V i n c e n z i  (1980).  Cal-  m o d u l i n s t i m u l a t i o n o f the enzyme a c t i v i t y was measured a t 3 0 ° C , pH 7 . 5 , i n a r e a c t i o n m i x t u r e c o n t a i n i n g 40mM T r i s - H C l , z o l e , 5mM M g C l » 1.2mM c y c l i c AMP, 0 . 0 5 u n i t s o f 2  40mM i m i d a -  phosphodiesterase, 2+  0 . 3 u n i t s o f 5 ' - n u c l e o t i d a s e and e i t h e r O.lmM Ca kinase i n h i b i t o r (or  and p r o t e i n  c a l m o d u l i n ) o r O.lmM EGTA ( n o n - a c t i v a t e d  acti-  vity).  The r e a c t i o n was a l l o w e d t o proceed f o r 15 min and stopped by  the a d d i t i o n o f SDS to a f i n a l c o n c e n t r a t i o n o f 2%.  Inorganic  phos-  phate r e l e a s e d was determined as d e s c r i b e d above.  12) P r o t e i n k i n a s e a s s a y I n h i b i t i o n o f p r o t e i n k i n a s e by p r o t e i n k i n a s e i n h i b i t o r was determined w i t h e i t h e r p r o t e i n k i n a s e and c y c l i c AMP o r w i t h f r e e c a t a l y t i c subunit.  The r e a c t i o n m i x t u r e c o n t a i n e d 24 mg/ml  histone  i n 50mM MES, pH 6 . 9 , 5 yM c y c l i c AMP, 5 yg c y c l i c AMP p r o t e i n  kinase  ( o r 5 yg p r o t e i n k i n a s e c a t a l y t i c s u b u n i t ) , 6.5mM M g C ^ , O.lmM EGTA, 5mM DTT,  and [y-  P] ATP (100 dpm/pmol) a t a f i n a l ATP  o f 100 yM i n a t o t a l volume o f 200 y l .  concentration  The r e a c t i o n m i x t u r e was  i n c u b a t e d f o r 10 min i n a s h a k i n g water bath a t 3 0 ° C .  The  reaction  was t e r m i n a t e d by t h e a d d i t i o n o f 4 ml o f i c e c o l d 5% TCA and 0.25% sodium t u n g s t a t e , pH 2 , f o l l o w e d i m m e d i a t e l y by 0.2 ml o f 0.63% bov i n e serum albumin as a c a r r i e r  protein.  The m i x t u r e was a l l o w e d to  s t a n d f o r 5 min i n i c e and c e n t r i f u g e d f o r 15 m i n .  The  supernatant  was c a r e f u l l y removed by a s p i r a t i o n and t h e p r e c i p i t a t e d i s s o l v e d i n 0.1 ml o f 1 N NaOH, f o l l o w e d by 2 ml o f 5% TCA-0.25% sodium t u n g s t a t e , pH 2 .  The p r o t e i n was r e p r e c i p i t a t e d by adding 1.1 N ^ S O ^ .  p r e c i p i t a t e was washed two more times as above and f i n a l l y i n 0.1 ml o f 1 N NaOH.  The r a d i o a c t i v i t y  in a l i q u i d s c i n t i l l a t i o n  13) SDS P o l y a c r y ! a m i d e  Gel  The  dissolved  i n t h i s sample was measured  counter.  Electrophoresis  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 was performed i n a gel s l a b electrophoresis  system.  The procedure used was e s s e n t i a l l y as o u t -  l i n e d hy Weber and Osborn Cl969), by p o l y a c r y l a m i d e gel  electrophore-  s i s i n the presence o f the a n i o n i c d e t e r g e n t sodium dodecyl  sulfate  CSDS).  a) P r e p a r a t i o n o f p r o t e i n s o l u t i o n s : To 20 y l o f the p r o t e i n sample 0 2 - 1 0 yg ) was added 30 y l the sample b u f f e r  containing ( i n a f i n a l concentration)  of  62.5mM T r i s - C l  (pH 6 . 8 ) , 3% SDS, 10% g l y c e r o l , 5% B-mercaptoethanol 0.001% bromophenol b l u e .  The sample was b o i l e d f o r 2 min and c o o l e d .  m o l e c u l a r weight p r o t e i n s o l u t i o n  A standard  (Pharmacia) was t r e a t e d s i m i l a r l y  and used to c a l i b r a t e the g e l s .  b) P r e p a r a t i o n o f g e l s : The  ' r e s o l v i n g ' or  1 0 . 5 ml o f 1.5M T r i s - C l  ' r u n n i n g ' gel (12.5%) was p r e p a r e d by m i x i n g (pH 8 . 8 ) , 0 . 4 2 ml o f 10% SDS, 1 7 . 5 ml 30%  a c r y l a m i d e , 0.8% m e t h y l e n e - b i s a c r y l a m i d e and 6.4 ml d i s t i l l e d w a t e r . A f t e r degassing f o r 5 m i n , 2 . 9 4 mis o f 0.5% ammonium p e r s u l f a t e and 4 . 2 mis o f 0.5% t e t r a e t h y l m e t h y l e n e - d i a m i n e  (TEMED) were a d d e d .  42 ml o f t h i s gel was s u f f i c i e n t to produce good r e s o l u t i o n o f the p r o t e i n bands.  A f t e r m i x i n g , t h i s s o l u t i o n was i m m e d i a t e l y poured  i n t o the gel chamber c a r e f u l l y w i t h a 10 ml p i p e t t e . covered v e r y c a r e f u l l y w i t h 2 ml o f 0.375 M T r i s - C l  The gel was b u f f e r (pH 8 . 8 ) .  The gel was a l l o w e d to p o l y m e r i z e f o r 12 hours a t room t e m p e r a t u r e . After  t h i s time the  ' b u f f e r ' was c a r e f u l l y removed w i t h a P a s t e u r  pipette. The Tris-Cl  ' s t a c k i n g g e l ' (.3%) was prepared by m i x i n g 1 ml o f 1.25 M  (pH 6 . 8 ) , 0.1 ml o f 10% SDS, 1 ml o f 30% a c r y l a m i d e - 0.8%  methylene h i s - a c r y l a m i d e and 6.2 jnl o f d i s t i l l e d w a t e r .  After  d e g a s i n g f o r 5 jni.n, 0 . 7 ml o f 0.5% ammonium p e r s u l f a t e and 1 ml TEMED were added.  A f t e r m i x i n g , s t a c k i n g g e l s o l u t i o n was poured  w i t h a 5 ml p i p e t t e i n t o t h e gel chamber o v e r t h e resolving-gel.  polymerized  This was f o l l o w e d by i n s e r t i n g a t e f l o n "comb" i n t o  the s t a c k i n g gel making s u r e t h a t no a i r bubbles were t r a p p e d the comb's t e e t h and the s t a c k i n g - g e l s o l u t i o n . was a l l o w e d to p o l y m e r i z e f o r a t l e a s t 12 h o u r s .  The  Tris-192mM G l y c i n e )  between  stacking-gel  Both chambers o f  the e l e c t r o p h o r e s i s c e l l were f i l l e d w i t h T r i s - G l y c i n e  carefully  of  b u f f e r (25mM  (pH 8 . 3 ) c o n t a i n i n g 0 . 1 % SOS and the "comb" was  removed. 25 - 50 pi o f the p r o t e i n sample was a p p l i e d to  the  gel w i t h a 50 pi H a m i l t o n s y r i n g e and run a t 400 V and 25 mA ( c o n stant current) u n t i l  the t r a c k i n g dye had moved a p p r o x i m a t e l y  10 cm.  c) S t a i n i n g and D e s t a i n i n g : The g e l s were s t a i n e d w i t h 0.2% Coomassie B r i l l i a n t B l u e i n 50% t r i c h l o r o a c e t i c a c i d f o r 15 min a t room t e m p e r a t u r e .  The  gels  were then washed w i t h d i s t i l l e d water and d e s t a i n e d w i t h 7.5% a c e t i c a c i d and 5% methanol u n t i l  the background was  transparent.  To photograph the g e l , the gel was soaked i n 1% g l y c e r o l a c e t i c a c i d s o l u t i o n f o r 30 m i n .  I t was t r a n s f e r r e d  g l a s s p l a t e and any excess l i q u i d was  removed.  - 10%  onto a p l a i n  RESULTS AND DISCUSSION  S e c t i o n I.  M e c h a n i s t i c study o f c a l m o d u l i n as an a c t i v a t o r o f sensitivity  and maximum v e l o c i t y o f  (Ca * + 2  Ca  2 +  Mq " ")2  1  ATPase  1.  K i n e t i c study o f C a  +  a c t i v a t i o n o f (Ca * + Mg )-ATPase 2  and  2+  i t s m o d u l a t i o n by c a l m o d u l i n . Calcium a c t i v a t i o n of ( C a  2 +  + Mg )-ATPase 2+  i n 'Dodge g h o s t s ' and  T r i t o n X-100 s o l u b i l i z e d enzyme p r e p a r a t i o n s was b i p h a s i c when the enzyme activity  was measured i n the absence o f c a l m o d u l i n ( F i g s . 1 and 2 ) .  The  k i n e t i c s o f c a l c i u m a c t i v a t i o n can be d e s c r i b e d by assuming a mixed popul a t i o n o f high and low c a l c i u m a f f i n i t y and R o u f o g a l i s 1 9 8 1 ) . affinity  s t a t e s o f the enzyme  (Al-Jobore  C a l m o d u l i n i n c r e a s e d both the apparent  and maximum v e l o c i t y  o f the ( C a  + Mg )-ATPase  2 +  ( F i g . 1) and T r i t o n X-100 s o l u b i l i z e d ( C a  2+  2 +  Ca  i n ghosts  + Mg )-ATPase 2+  (Fig. 2).  the presence o f c a l m o d u l i n , both enzyme p r e p a r a t i o n s d i s p l a y e d high a f f i n i t y activate  (Ca  Ca 2 +  2 +  activation kinetics.  + Mg )ATPase 2+  In  single  The a b i l i t y o f c a l m o d u l i n to  o f e r y t h r o c y t e membranes was l o s t upon c a r -  boxymethyl a t i o n o f c a l m o d u l i n ( A l - J o b o r e observation).  2 +  and R o u f o g a l i s , u n p u b l i s h e d  Moreover, carboxymethylated  c a l m o d u l i n a l s o showed reduced  a c t i v a t i o n p r o p e r t i e s on another c a l m o d u l i n s e n s i t i v e enzyme, p h o s p h o d i esterase  (Gagnon et a l . 1 9 8 1 ) .  These o b s e r v a t i o n s  suggested t h a t  a n i o n i c nature o f c a l m o d u l i n due t o the presence o f s e v e r a l  free  groups may be important i n the r e g u l a t i o n o f c a l m o d u l i n s e n s i t i v e  the carboxyl enzymes.  cm  (3  t  3  -LogCC^reetM) 2+ F i g u r e 1.  C a l m o d u l i n a c t i v a t i o n o f (Ca  2+ + Mg  )-ATPase a c t i v i t y  human e r y t h r o c y t e membranes (Dodge G h o s t s ) . v i t y was measured i n t h e absence (•)  in  The enzyme a c t i -  and i n the presence  (o)  o f c a l m o d u l i n (2 y g / 0 . 6 m l ) . The i n c u b a t i o n medium c o n t a i n e d 55 mM T r i s - m a l e a t e ,  pH 7 . 2 , 0.1 mM EGTA, 0.1 mM o u a b a i n ,  6.5 mM M g C I £ » 66 mM NaCl and v a r i o u s c o n c e n t r a t i o n s o f calcium.  The enzyme a c t i v i t y was assayed witff 2 mM T r i s - A T P  f o r 60 min at 3 7 ° C .  TJie r e s u l t s shown are means ± S . E . M ,  four separate experiments. Ca  2 +  free  concentrations.  P <  0.01 compared t o c o n t r o l  for at a l l  T  -LogCCa?*]^ (M) 2+ Figure 2.  C a l m o d u l i n a c t i v a t i o n o f T r i t o n X-100 s o l u b i l i z e d (Ca 2+ Mg  )-ATPase a c t i v i t y .  i n the absence (•) (2 yg/0.6 m l ) .  +  The enzyme a c t i v i t y was measured  and i n t h e presence (o)  of calmodulin  The i n c u b a t i o n medium was i d e n t i c a l  t h a t d e s c r i b e d i n F i g u r e 1.  The enzyme a c t i v i t y  to  was  assayed w i t h 0 . 5 mM ATP f o r 15 min at 3 7 ° C . The r e s u l t s are means ± S . E . M . f o r at. l e a s t t h r e e s e p a r a t e P < 0 . 0 1 , compared to c o n t r o l .  shown  experiments.  2.  Effect of poly-L-carboxylic activation of To  (Ca  a c i d s and o t h e r a n i o n s on t h e  Ca  2 +  + Mg +)-ATPase  2 +  2  study the e f f e c t  of free carboxyl  calmodulin in regulation of ( C a  2 +  groups and the a n i o n i c nature  + Mg )-ATPase,  certain  2+  and a n i o n i c compounds were s t u d i e d .  The  (Ca  2 +  poly-anionic  + Mg )-ATPase 2+  activity  i n e r y t h r o c y t e membranes was a c t i v a t e d at low c a l c i u m c o n c e n t r a t i o n s s h i f t e d to a p r e d o m i n a n t l y (M  r  high a f f i n i t y  = 20,000) and p o l y - L - g l u t a m i c  (Fig.  3).  r  = 20,000 and M  membranes, t h e i r was s t u d i e d .  = 26,000)  r  solubilized  acids (M  (Ca  2 +  + Mg )-ATPase  2 +  r  poly-L-aspartic  2 +  2+  o f the pump f o r C a  2 +  (Fig.  of ( C a  2 +  2 +  Ca  2 +  2 +  )-  acids  In the  a c t i v a t i o n o f the enzyme was  55 mM T r i s - H C l  o r 55 mM sodium m a l e a t e , pH  s u l f o n i c a c i d s a l s o i n c r e a s e d the C a  o f the T r i t o n X - 1 0 0 s o l u b i l i z e d enzyme ( F i g . 6 ) . the C a  2 +  s u l f o n i c a c i d s , i n c l u d i n g sodium-HEPES  absence o f s u l f o n i c a c i d s b i p h a s i c C a  The  + Mg  i n the presence o f p o l y - L - c a r b o x y l i c  observed i n 55 mM T r i s - m a l e a t e ,  sen-  4).  (55 mM) or sodium-MES (55 mM) i n e r y t h r o c y t e g h o s t s ( F i g . 5 ) .  5).  detergent  a c t i v i t i e s as w e l l , by a f f e c t i n g the  but a l s o i n the presence o f c e r t a i n  7.2 ( F i g .  2+  r  An i n c r e a s e i n the apparent c a l c i u m s e n s i t i v i t y ATPase was observed not o n l y  + Mg )-ATPase  a c i d ( M = 20,000) and  = 20,000 and 2 6 , 0 0 0 ) i n c r e a s e d the  + Mg )-ATPase  the  in erythrocyte  2+  e f f e c t ( s ) on T r i t o n X-100 s o l u b i l i z e d ( C a  It was shown t h a t  poly-L-glutamic  (Ca  and  acid  In an attempt to d e l i n e a t e the mechanism and k i n e t i c s o f  a c t i v a t i o n by these p o l y a n i o n s on the  sitivity  form by p o l y - L - a s p a r t i c  acid (M  sensitivity  2 +  In T r i s - m a l e a t e  buffer  a c t i v a t i o n was best f i t t e d to the e q u a t i o n f o r b i p h a s i c  a c t i v a t i o n described previously  and i n T a b l e  I,  with a  of  ( A l - J o b o r e and R o u f o g a l i s , 1981)  o f 2 . 2 ± 0 . 7 uM f o r the high C a  component and a K,j o f 40 uM o r more f o r the low C a  2 +  2 +  affinity  affinity component.  >»  5  6  Figure 3.  E f f e c t of p o l y - L - c a r b o x y l i c 2+ e r y t h r o c y t e membrane (Ca enzyme a c t i v i t y  5  a c i d s on Ca 2+ + Mg  2+  4  a c t i v a t i o n o f human  )-ATPase a c t i v i t y .  The  was measured i n a medium c o n t a i n i n g 55 mM T r i s -  m a l e a t e , pH 7 . 2 , 0.1 mM EGTA, 0.1 mM o u a b a i n , 6.5 mM M g C l , 2  66 mM N a C l , 2 mM ATP and v a r i o u s c o n c e n t r a t i o n s o f f r e e c a l c i u m as i n d i c a t e d .  Enzyme a c t i v i t y was measured i n t h e absence  and i n the presence o f 81 yM p o l y - L - a s p a r t i c a c i d , M 57 yM p o l y - L - g l u t a m i c poly-L-glutamic a c i d , M  acid, M  = 20,000 (•),  = 2 6 , 0 0 0 (•).  = 20,000  and 44 yM  The r e s u l t s shown are  means ± S . E . M . o f at l e a s t 3 s e p a r a t e e x p e r i m e n t s . compared t o c o n t r o l .  f  (•)  *P < 0 . 0 5  6  5  -LogCCa 3 2+  free  4 (M)  2+ F i g u r e 4.  E f f e c t of p o l y - L - c a r b o x y l i c 2+ 2+ (Ca  + Mg  a c i d s on Ca  measured i n the absence (•) poly-L-aspartic acid, M 4  = 20,000 (•),  26,000 (•).  of  )-ATPase i n T r i t o n X-100 s o l u b i l i z e d enzyme  from human e r y t h r o c y t e membranes.  acid, M  activation  The enzyme a c t i v i t y  was  and i n t h e presence o f 81 yM  = 20,000 (*),  57 yM p o l y - L - g l u t a m i c  and 44 yM p o l y - L - g l u t a m i c a c i d , M =  The a s s a y medium was i d e n t i c a l  f  to t h a t  in  F i g u r e 3 except t h a t the c o n c e n t r a t i o n o f ATP was 0 . 5 mM and t h e assay t i m e 10 m i n . Data r e p r e s e n t the means o f three separate  experiments.  re £. CO  is  & .co CO  I  ~  +  CM  + CM  re O  E  co o E a.  -LogCCa J 2+  free  (M) 2+  ure 5.  E f f e c t o f s u l f o n i c a c i d a n i o n s on the Ca 2+ 2+ (Ca  + Mg  activation  )-ATPase i n human e r y t h r o c y t e membranes.  were assayed i n 55 mM T r i s - maleate b u f f e r (•), (•),  55 mM sodium-maleate (•),  55 mM sodium-MES ( ) . A  37°C.  Ghosts  55 mM T r i s - H C l  55 mM sodium-HEPES (^)  The pH o f t h e a s s a y m i x t u r e s  The a s s a y m i x t u r e c o n t a i n e d the b u f f e r  of  and  was 6 . 9 a t  (as i n d i c a t e d ,  0.1 mM EGTA, 0.1 mM o u a b a i n , 66 mM N a C l , 6.5 mM M g C l , and 2  v a r i o u s c o n c e n t r a t i o n s o f f r e e c a l c i u m . Data r e p r e s e n t means o f t h r e e s e p a r a t e  experiments.  the  T  F i g u r e 6A.  E f f e c t o f s u l f o n i c a c i d a n i o n s i n t h e presence and absence o f 2+ 2+ 2+ c a l m o d u l i n on Ca a c t i v a t i o n o f (Ca + Mg )-ATPase i n 2+ 2+ T r i t o n X-100 s o l u b i l i z e d (Ca e r y t h r o c y t e membranes. 55 mM T r i s - m a l e a t e sodium-KES ( ) A  0 . 6 ml ( o ) .  + Mg  )-ATPase from human  The s o l u b i l i z e d enzyme was assayed  buffer  (•),  55 mM sodium-HEPES (*),  55 mM  and 55 mM sodium-HEPES p l u s 0 . 6 ug c a l m o d u l i n / The pH o f t h e a s s a y m i x t u r e was 7.2 at 3 7 ° C .  The o t h e r components o f t h e a s s a y m i x t u r e were i d e n t i c a l t h o s e shown i n F i g u r e 5 . Each p o i n t r e p r e s e n t s four  experiments.  Appendix  I),  in  to  the means o f  An a n a l y s i s o f v a r i a n c e was performed  (see  V/Ca (xiO) 2H  (n moles Pj. mg-" mirr ) 1  LCa 3 2+  F i g u r e 6B.  1  OjM)  The c u r v e s i n F i g u r e 6A were f i t t e d by a n o n - l i n e a r c u r v e f i t t i n g computer program (see t e x t and T a b l e I ) . a c t i v i t y in Tris-maleate (o), i n Na-HEPES  (•).  Enzyme  Na-HEPES( ) and c a l m o d u l i n A  In sodium-HEPES o r sodium-MES (55 mM i n anion) t h e C a b e s t f i t t e d by a s i n g l e h i g h C a 0.08 yM). linear  program,  g h o s t s , the maximum v e l o c i t y  assay medium)  2 +  component (K^ = 0.35 ±  i n the form o f E a d i e - H o f s t e e  affinity  2 +  + Mg )-ATPase 2+  without  to the e f f e c t o f c a l m o d u l i n i n T r i s - m a l e a t e 2 +  affinity  in  i n c r e a s e d the maximum  further affecting  (Kd = 0 . 3 ± 0 . 0 4 uM) ( F i g . 6 A , B ) .  both the apparent C a  As i n  The a d d i t i o n o f c a l m o d u l i n (0.6 yg/0.6 ml  i n the presence o f sodium-HEPES b u f f e r  o f the ( C a  plots.  non-  o b t a i n e d was not s i g n i f i c a n t l y d i f f e r e n t  T r i s or s u l f o n i c a c i d b u f f e r s .  Ca  affinity  a c t i v a t i o n was  F i g . 6B shows the f i t o f the data by a computer generated  curve f i t t i n g  velocity  2 +  2 +  This i s in  buffer,  2 +  apparent  contrast  where oalmodulin i n c r e a s e s  and the maximum v e l o c i t y  ATPase, both i n ghosts and s o l u b i l i z e d ( C a  its  of ( C a  + Mg2 )-ATPase  2 +  + Mg^" ")1  preparations  +  ( F i g u r e s 1 and 2 ) . The  shift  in C a  2 +  affinity  o f the enzyme i n the presence  of  sodium-HEPES or sodium-MES cannot be a t t r i b u t e d to a change i n the c o n c e n t r a t i o n o f sodium (the c o u n t e r i o n i n the s u l f o n i c a c i d b u f f e r s ) , no a c t i v a t i o n was o b t a i n e d i n sodium maleate b u f f e r concentration  (see F i g . 5 ) .  2 +  k i n e t i c p a t t e r n observed  parations (results affinity  not shown).  ly  b u f f e r d i d not a l t e r  the  i n 'ghosts' o r s o l u b i l i z e d p r e -  i s a l s o u n l i k e l y t h a t the low C a  The  f a c t t h a t these anions s h i f t e d the  o f s o l u b i l i z e d enzyme p r e p a r a t i o n s  on the C a - t r a n s p o r t  effect  It  from  2 +  component r e s u l t e d from the presence o f T r i s , as i t was a l s o seen  i n sodium maleate b u f f e r . affinity  at a s i m i l a r sodium  In a d d i t i o n , d e l e t i o n o f sodium c h l o r i d e  t h e assay medium i n the presence o f T r i s - m a l e a t e biphasic C a  because  2 +  Ca  suggests t h a t they act  2 +  direct-  ATPase complex, r a t h e r than by an i n d i r e c t  on membrane s t r u c t u r e .  In support o f t h i s was the f i n d i n g t h a t  anions also a c t i v a t e d a preparation of p u r i f i e d i n a s o l e c t i n , o b t a i n e d from Dr. J . T .  (Ca  2 +  + Mg )-ATPase 2+  P e n n i s t o n , Mayo C l i n i c , M i n n e s o t a  the  (results  not shown).  Furthermore,  may have s h i f t e d the a f f i n i t y mobilizing tightly  to t e s t the p o s s i b i l i t y t h a t  o f the ( C a  2 +  + Mg )-ATPase 2+  polyanions  i n d i r e c t l y by  bound c a l m o d u l i n ( i . e . c a l m o d u l i n not removed by hypo-  t o n i c l y s i s i n the absence o f C a  2 +  ) , membranes were p r e i n c u b a t e d w i t h  p o l y a n i o n s and washed t w i c e w i t h 20 mM sodium phosphate, pH 7.4 to any l o o s e l y bound c a l m o d u l i n . The washed membranes s t i l l sensitivity  to p o l y a n i o n s t i m u l a t i o n o f ( C a  the u n t r e a t e d membranes ( r e s u l t s  retained their  + Mg )-ATPase,  2 +  2+  not shown).  remove  s i m i l a r to  It was determined t h a t  n e i t h e r p o l y a n i o n s nor s u l f o n i c a c i d s a l t e r e d the c a l c i u m s e n s i t i v i t y a l t e r i n g the s e n s i t i v i t y  o f the enzyme to MgCl2 o r ATP ( r e s u l t s  by  not  shown). To study the nature o f anion a f f e c t s on the m o d u l a t i o n of the Ca " " s e n s i t i v i t y 2  1  o f the c a l c i u m t r a n s p o r t ATPase, v a r i o u s  a n i o n s were used (Table I ) .  Of the range o f i n o r g a n i c and o r g a n i c a n i o n s  t e s t e d , o n l y the a r o m a t i c c a r b o x y l i c acid,  a c i d s , b e n z o i c a c i d and s a l i c y l i c  i n c r e a s e d the c a l c i u m s e n s i t i v i t y  t o a p r e d o m i n a n t l y s i n g l e high C a  2 +  and s h i f t e d the b i p h a s i c k i n e t i c s  affinity  s t a t e (Table I,  A l i p h a t i c c a r b o x y l i c and s u l f o n i c a c i d s (sodium g l u c o n a t e , a s p a r t a t e , g l u t a m a t e and TES) were i n e f f e c t i v e (Table I ) .  Fig. 7). hippurate,  at the same c o n c e n t r a t i o n s  S i m i l a r l y , none o f the i n o r g a n i c anions t e s t e d , which i n c l u d e  both water sitivity  c l a s s e s of  structure  'toakers" and "breakers", i n c r e a s e d the C a  o f the enzyme at 66 mM, w h i l e F" and to a l e s s e r  NO3" s l i g h t l y  i n h i b i t e d the enzyme a c t i v i t y  (Table  2 +  sen-  extent  I).  In an attempt to i n v e s t i g a t e t h e s i t e and mechanism o f a c t i o n o f t h e s e anions and p o l y a n i o n s i n r e l a t i o n to c a l m o d u l i n , two types o f iments were performed.  Firstly,  the enzyme was t r e a t e d w i t h  trypsin.  T r y p s i n treatment o f ghosts i n c r e a s e d both the maximum v e l o c i t y calcium s e n s i t i v i t y  o f the  (Ca  2 +  + Mg )-ATPase 2+  exper-  and the  ( F i g . 8 ) , p r o b a b l y by  2+ E f f e c t o f a n i o n s on the Ca  s e n s i t i v i t y o f T r i t o n X-100 s o l u b i l i z e d  2 + 2 + (Ca  +Mg  )-ATPase.  The assay-medium c o n t a i n e d 55 mM T r i s - m a l e a t e  (pH 7.2 a t 3 7 ° C ) , 0.1 mM EGTA, 6.5 mM MgCl mM, 0.1 mM o u a b a i n , v a r i o u s 2  c o n c e n t r a t i o n s o f f r e e c a l c i u m and 66 mM i n a n i o n , t e s t e d as t h e sodium 2+ salts.  The k i n e t i c s o f t h e Ca  a c t i v a t i o n were examined over a range  2+ o f Ca c o n c e n t r a t i o n s i n experiments s i m i l a r t o t h o s e shown i n F i g . 2 . 2+ v a t i o n 2+ The Ca a c t i v a t i o n was f i t t e d t o t h e e q u a t i o n f o r b i p h a s i c Ca actiV  l  tCa  2 +  ]  2  K  J  K  1  + rca^ ] +  ?  1  [Ca  +  ]  1  2+ where V-| and K-j are t h e maximum v e l o c i t y and Ca d i s s o c i a t i o n constant 2+ ^ of t h e high Ca a f f i n i t y component and K ' = - 2 - ( t h e c o r r e s p o n d i n g ?  maximum v e l o c i t y and Ca component).  2+  d i s s o c i a t i o n c o n s t a n t o f t h e low Ca  The c r i t e r i o n used f o r d e c i d i n g whether  2+  affinity  the k i n e t i c s  the b i p h a s i c a c t i v a t i o n o r a h y p e r b o l i c f u n c t i o n ( l e f t  fitted  hand term o f  e q u a t i o n o n l y ) was t h e c o r r e l a t i o n c o e f f i c i e n t o f the f i t  (r)  and the  magnitude o f t h e s t a n d a r d d e v i a t i o n o f t h e e s t i m a t e s o f t h e c o n s t a n t s . V-| i s i n nmoles Pyminper mg p r o t e i n and K-j i s i n yM. V  2  and K 2  K " i s the r a t i o 2  of  K i n e t i c s o f Ca  2+  Activation  Anion C a  2  Activation  K i n e t i c Constants ^1  ^1  ^2*  CI"  Biphasic  322 + 27  2.2  + 0.7  0.30 + 0,02  Br'  Biphasic  315 + 25  2.1  + 0,6  0.32 + 0.022  r  Biphasic  320 + 26  2.1  + 0.65  0 , 3 0 + 0.02  F"  Biphasic  308 + 18  2.5  + 0.72  0.27 + 0.019  Bi p h a s i c  315 + 26  2.3  + 0.66  0,29 + 0.02  Biphasic  318 + 29  2.1  + 0,65  0.29 + 0.02  HCO3"  Biphasic  315 + 25  2.1  + 0.66  0,33 + 0.03  CH C00"  Biphasic  320 + 26  2.3  + 0.67  0 , 3 0 + 0,025  NO3-  Biphasic  305 + 20  2.6  + 0.78  0.26 + 0,023  Gluconate"  Bi phasic  325 + 26  2.3  + 0.69  0 . 2 9 + 0.03  Hippurate"  Bi p h a s i c  318 + 18  2.2  + 0.71  0,31 + 0,032  Salicylate"  Hyperbo1ic  340 + 32  0.60 + 0.08  —  Benzoate"  Hyperbolic  330 + 29  0.62 + 0.08  --  Aspartate"  Biphasic  315 + 23  2.2  + 0.08  0,29 + 0.021  Glutamate"  Biphasic  320 + 26  2,0  + 0.85  0.28 + 0.019  Taurine*  Bi p h a s i c  315 + 25  2.2  + 0.68  0.30 + 0.022  TES*  Bi p h a s i c  309 + 18  2.1  + 0.71  0,32 + 0.023  2-  SO4 C0  3  2  -  3  •Taurine and *TES were used a t a c o n c e n t r a t i o n o f 55 mM  I _ 400  6  5  4  -LogCCaP^tM) F i g u r e 7.  E f f e c t o f i n o r g a n i c a n i o n s and a r o m a t i c c a r b o x y l i c Ca  2 +  a c t i v a t i o n o f T r i t o n X-100 s o l u b i l i z e d ( C a 2+ 2+  ATPase from human e r y t h r o c y t e s . activity  (Ca  + Mg  2 +  a c i d s on + Mg )2 +  )-ATPase  was assayed i n a medium c o n t a i n i n g 55 mM T r i s - m a l e a t e ,  pH 7 . 2 , 0.1 mM EGTA, 0.1 mM o u a b a i n , 6.5 mM M g C l » v a r i o u s 2  c e n t r a t i o n s o f f r e e c a l c i u m and 66 mM NaCl (•), b i c a r b o n a t e (•), benzoate  66 mM sodium carbonate (*),  (A) or 66 mM sodium s a l i c y l a t e (•).  the means o f at l e a s t t h r e e s e p a r a t e  con-  66 mM sodium  66 mM sodium Data  experiments.  represent  6  5  -LogCCa D 2+  Figure 8.  free  (M)  E f f e c t o f p o l y - L - c a r b o x y l i c a c i d s and s u l f o n i c a c i d s on 2+ 2+ trypsin  t r e a t e d membranes,  (Ca  + Mg  )-ATPase  activity  o f n o n - t r y p s i n t r e a t e d ( c o n t r o l ) membranes was assayed i n Tris-maleate buffer  (55 mM) (•).  were assayed i n T r i s - m a l e a t e (55 mM)  (A).  acid (M  = 20 000) ( C a  similar  (55 mM) (o)  membranes  o r sodium-HEPES  In the presence o f 56 uM p o l y - L - a s p a r t i c  assayed b e f o r e (•) Tris-maleate  Trypsin-treated  2 +  + M g ) - A T P a s e a c t i v i t y was 2+  and a f t e r t r y p s i n t r e a t m e n t  (•)  (55 mM). The data shown i s t y p i c a l o f  experiments.  in three  c l e a v i n g the c a l m o d u l i n b i n d i n g s i t e on the (Ca " " + M g ) - A T P a s e 2  et a l 1 9 8 0 ; Enyedi  1  et a l 1 9 8 0 ; N i g g l i et a l 1 9 8 1 ) .  (Sarkadi  2+  Addition of  either  p o l y - L - a s p a r t i c a c i d or sodium-HEPES to the t r y p s i n t r e a t e d membranes f a i l e d to f u r t h e r  a f f e c t the C a  2 +  sensitivity  or a c t i v i t y  enzyme compared to t h a t i n T r i s - m a l e a t e b u f f e r a l o n e . gest that pretreatment  o f the  These r e s u l t s  o f the membrane w i t h t r y p s i n l e a d s to a change i n  t h e enzyme s i m i l a r t o t h a t produced by b i n d i n g o f the a n i o n s .  However,  u n l i k e a n i o n s , but s i m i l a r l y to c a l m o d u l i n , t r y p s i n treatment t h e r e f f e c t o f i n c r e a s i n g the maximum v e l o c i t y o b t a i n e d . results  sug-  has the  fur-  While t h e s e  show t h a t the anions t e s t e d have s i m i l a r e f f e c t s to c a l m o d u l i n and  t r y p s i n treatment,  i t cannot be c o n c l u d e d from the k i n e t i c a n a l y s i s t h a t  the v a r i o u s anions and c a l m o d u l i n n e c e s s a r i l y bind at the same s i t e . S e c o n d l y , the e f f e c t o f t r i f l u o p e r a z i n e on the a c t i v a t i o n by a n i o n s was examined, s i n c e i t  i s known to b l o c k the c a l m o d u l i n a c t i v a t i o n  of  (Ca  Whereas 30 uM t r i f l u o p e r a z i n e b l o c k e d ( C a  +  2 +  + Mg )-ATPase. 2+  Mg )-ATPase 2+  a c t i v a t i o n by p o l y - L - a s p a r t i c a c i d , i t d i d not antagon-  i z e the e f f e c t o f sodium-HEPES on the enzyme a c t i v i t y known at present (Ca  2 +  i f trifluoperazine  It  i s not  i n h i b i t s the p o l y a n i o n a c t i v a t i o n of ionic  inter-  a c t i o n i n the absence or presence o f c a l c i u m o r to the enzyme.  Until  2 +  + Mg )-ATPase  (Fig. 9).  2+  by b i n d i n g to the p o l y a n i o n through  mechanism o f the c a l m o d u l i n blockade by t r i f l u o p e r a z i n e  is established, it  i s not p o s s i b l e t o a n a l y s e the p r e c i s e mechanism by which 1 i c a c i d s and s u l f o n i c a c i d s a l t e r the C a (Ca  2 +  2 +  sensitivity  the  poly-L-carboxy-  o f the  + Mg )-ATPase. 2+  These s t u d i e s suggest t h a t d i s t i n c t s t r u c t u r a l may c o n t r i b u t e to the i n c r e a s e i n the C a velocity  of ( C a  2 +  + Mg )-ATPase. 2+  2 +  features of calmodulin  sensitivity  and maximum  The r e s u l t s o b t a i n e d w i t h  poly-anions  and some o f the o t h e r anions examined suggest t h a t the a c i d i c nature  of  F i g u r e 9.  E f f e c t o f t r i f l u o p e r a z i n e on human e r y t h r o c y t e 2+ 2+ (Ca  + Mg  )-ATPase a c t i v i t y  membrane  i n t h e presence o f  c a r b o x y l i c a c i d s or sodium-HEPES b u f f e r .  The  poly-L-  enzyme  a c t i v i t y was measured i n sodium-HEPES b u f f e r e i t h e r t h e absence (n) (•).  in  o r i n the presence o f 30 yM t r i f l u o p e r a z i n e  The enzyme was assayed i n 55 mM T r i s - m a l e a t e  buffer  (•)  and i n the presence o f 56 yM p o l y - L - a s p a r t i c a c i d  (M  = 20,000) w i t h o u t t r i f l u o p e r a z i n e  t r i f l u o p e r a z i n e (*). similar  experiments.  (A) o r w i t h 30 yM  The data shown i s t y p i c a l o f  three  c a l m o d u l i n may be i n v o l v e d i n a l t e r i n g the C a tion).  2 +  sensitivity  (K-activa-  The mechanism o f K - a c t i v a t i o n by the anions may be through  s t e r i c m o d u l a t i o n o f the enzyme c o n f o r m a t i o n to a high C a s t a t e , through a r e g u l a t o r y  s i t e on the enzyme.  2 +  allo-  affinity  It was proposed t h a t  cal-  m o d u l i n and p h o s p h a t i d y l s e r i n e bind to o v e r l a p p i n g but d i s t i n c t r e g i o n s on the regulatory  s i t e , thereby  c o n s t r a i n t on C a Roufogalis 1981). velocity  2 +  a l t e r i n g i t s c o n f o r m a t i o n , and removing  a c t i v a t i o n o f the  (Ca  2 +  + Mg )-ATPase 2+  (Al-Jobore  the and  The a b i l i t y o f c a l m o d u l i n t o a l s o i n c r e a s e the maximum  (V-activation)  may be due to i t s hydrophobic n a t u r e , as demon-  s t r a t e d by s i m i l a r (K + V) a c t i v a t i o n by c e r t a i n a c i d i c p h o s p h o l i p i d s (Al-Jobore  and R o u f o g a l i s 1 9 8 1 ; 1982) which are both n e g a t i v e l y  and h y d r o p h o b i c . red c e l l  It  charged  remains to be determined i f endogenous anions i n t h e  a f f e c t the s t a t e o f the c a l c i u m pump i n v i v o .  Section II,  E f f e c t o f c y c l i c AMP and c y c l i c AMP dependent kinase i n h i b i t o r s  The i n t e r d e p e n d e n c y  ( P K I ' s ) on ( C a  of various  2 +  protein  + t)g )^ATPase  activity  2+  c y c l i c AMP and c a l c i u m - c a l m o d u l i n  dependent c e l l u l a r events i s not c l e a r l y u n d e r s t o o d .  C a l m o d u l i n has been 2+  shown to s t i m u l a t e b r a i n a d e n y l a t e c y c l a s e , p h o s p h o d i e s t e r a s e and (Ca  +  2+ Mg  )-ATPase enzymes, among o t h e r s  (Cheung 1 9 8 0 ) .  have been shown t o i n h i b i t p h o s p h o r y l a t i o n by i n t e r a c t i n g w i t h the f r e e c a t a l y t i c (Ashby and Walsh 1 9 7 3 ) .  The c y c l i c AMP PKI's  by c y c l i c AMP p r o t e i n  kinases  s u b u n i t s o f t h e s e enzymes  They a l s o i n h i b i t c a l m o d u l i n  -  stimulated  phos-  p h o d i e s t e r a s e i n S e r t o l i c e l l s o f t e s t e s , which c o n t a i n both c a l m o d u l i n and PKI ( B e a l e e_t al_. 1 9 7 7 ) .  In the e r y t h r o c y t e plasma membrane, 2+  AMP has been shown under c e r t a i n c o n d i t i o n s t o i n h i b i t t h e (Ca ATPase a c t i v i t y  p r o b a b l y y_1_a an endogenous c y c l i c AMP p r o t e i n  (Varghese and Cunningham 1 9 8 0 ) . characterize  i o n on (Ca  + Mg  to  phosphorylat-  2+ + Mg  )-ATPase a c t i v i t y  o f c y c l i c AMP p r o t e i n Bovine h e a r t  using the s p e c i f i c protein  inhibitors  kinases.  PKI from Sigma, f u r t h e r p u r i f i e d on a Whatman DE 52  column, was shown to be f r e e o f c a l m o d u l i n by a number o f c r i t e r i a II).  )-  kinase  I t was t h e purpose o f t h i s study  the r o l e o f c y c l i c AMP dependent p r o t e i n k i n a s e  2+  cyclic 2+  Pure PKI d i d not s t i m u l a t e c a l m o d u l i n - d e f i c i e n t  (Table  phosphodiesterase,  whereas the PKI p r e p a r a t i o n from Sigma b e f o r e p u r i f i c a t i o n and pure c a l m o d u l i n s t i m u l a t e d t h i s enzyme.  The absence of any s i g n i f i c a n t  levels  o f c a l m o d u l i n i n t h e p u r i f i e d PKI was c o n f i r m e d by c a l m o d u l i n radioimmunoa s s a y ( 1 . 3 ng o f c a l m o d u l i n which was found per 2 ug o f PKI ( T a b l e was shown to be w e l l  below t h e t h r e s h o l d  (Ca  activity).  2 +  + Mg )-ATPase 2+  (50 ng)  Moreover,  r e q u i r e d to  II),  stimulate  0.1% - SDS-12.5%-polyacrylamide  D e t e r m i n a t i o n o f c a l m o d u l i n tn v a r i o u s  protein preparations,  Calmodulin  was e s t i m a t e d u s i n g t h e p h o s p h o d i e s t e r a s e a s s a y and c a l m o d u l i n r a d i o immunoassay, as d e s c r i b e d 1n t h e t e x t . -1 was 100 nmoles P^ min  activity  -1 mg p r o t e i n  The c a l m o d u l i n c o n t e n t o f v a r i o u s total  The p h o s p h o d i e s t e r a s e  i n t h e absence o f added p r o t e i n s . p r e p a r a t i o n s i s expressed per 2 yg  of  protein.  Preparation  Bovine heart PKI (Sigma)  Phosphodiesterase a c t i v a t i o n (%)  Calmodulin radioimmunoassay  75%  75 "ng/2  y  g  Rabbit s k e l e t a l muscle PKI (Sigma)  none  0.16 ng/2 yg  P u r i f i e d bovine h e a r t PKI  none  1.33 ng/2 yg  Calmodulin (400 ng)  230%  420 ng  C a l m o d u l i n immunoassays were k i n d l y performed by Dr. C h a f o l e a s i n the l a b o r a t o r y o f Dr. Means, B a y l o r C o l l e g e o f M e d i c i n e , T e x a s .  gel e l e c t r o p h o r e s i s showed a s i n g l e p r o t e i n band o f M = 15,500 ( f i g .  10).  f  The p u r i f i e d p r o t e i n k i n a s e I n h i b i t o r s t a l muscle were used i n f u r t h e r  from bovine h e a r t and r a b b i t s k e l e -  studies.  In agreement w i t h t h e r e p o r t o f Varghese and Cunningham (.1980),  It  was found t h a t c y c l i c AMP (5 yM) produced an i n h i b i t i o n ( a p p r o x . 20%) o f the(Ca (Fig.  2 +  + Mg )-ATPase 2+  11).  activity  1n 'Dodge g h o s t s ' assayed a t 20 yM ATP  C y c l i c AMP (5 y M ) , however, had no e f f e c t on t h e enzyme a c t i -  v i t y when i t was assayed a t 2 mM ATP ( r e s u l t s 2+ o f (Ca  The  inhibition  2+ + Mg  )-ATPase a c t i v i t y  c y c l i c AMP mediated  2+  o f bovine h e a r t  because t h e  via  inhibi-  produced by c y c l i c AMP c o u l d be a b o l i s h e d by  r a b b i t s k e l e t a l muscle PKI ( F i g .  AMP (5 yM) on (Ca  produced by c y c l i c AMP was p r o b a b l y  protein kinase phosphorylation,  t i o n o f ATPase a c t i v i t y  approx.  not shown).  11).  However, when the e f f e c t o f c y c l i c  2+ + Mg  PKI,  )-ATPase a c t i v i t y  was s t u d i e d i n t h e  i t was found t h a t t h e enzyme a c t i v i t y  100% i n the presence o r absence o f c y c l i c AMP ( F i g .  the p r o t e i n kinase i n h i b i t o r s are small a c i d i c p r o t e i n s  presence  was s t i m u l a t e d 11).  Since  (Ashby and Walsh  1973; Whitehouse and McPherson 1 9 8 0 ) , t h e e f f e c t ( s ) o f t h e s e p r o t e i n s on 2+ 2+ the k i n e t i c s o f (Ca  + Mg  )-ATPase was f u r t h e r i n v e s t i g a t e d and compared  to t h a t o f c a l m o d u l i n . 2+ 2+ Bovine h e a r t PKI a l s o s t i m u l a t e d (Ca + Mg )-ATPase a c t i v i t y i n T r i t o n X-100 s o l u b i l i z e d p r e p a r a t i o n (assayed a t 0 , 5 mM ATP) ( F i g . 1 2 ) . 2+ 2+ However, whereas c a l m o d u l i n s t i m u l a t e d (Ca + Mg )-ATPase a c t i v i t y a t 2+ both low ( 0 . 5 8 yM) and high (55 yM) f r e e Ca , bovine h e a r t PKI s t i m u l a t e d 2+ o n l y a t the low Ca c o n c e n t r a t i o n , both i n ' g h o s t s ' ( F i g . 12) and i n T r i t o n X-100 s o l u b i l i z e d p r e p a r a t i o n s ( r e s u l t not shown). At 0.58 yM 2+ 2+ 2+ Ca bovine h e a r t PKI s t i m u l a t e d (Ca + Mg )-ATPase i n t h e presence o f a s a t u r a t i n g l e v e l . (3 y g / 0 . 6 ml) o f c a l m o d u l i n , w h i l e a t 55 yM Ca  2+  , PKI  69  Molecular Weight 94,000  67,000  43,000  30,000  21,000  14,000 F i g u r e 10. SDS-PAGE o f p r o t e i n k i n a s e i n h i b i t o r and c a l m o d u l i n .  The  p r o t e i n s were s e p a r a t e d on a 1 2 . 5 % - a c r y l a m i d e - 0 . 1 % - S D S s l a b gel as d e s c r i b e d i n the Methods. loaded on t h e gel ( s t a r t i n g weight  from l e f t ) ;  Lane 1, s t a n d a r d m o l e c u l a r  p r o t e i n s , 3 yg each o f p h o s p h o r y l a s e B ( M  c a r b o n i c anhydrase  (M  r  r  = 94,000),  = 6 7 , 0 0 0 ) , ovalbumin (M  43,000),  = 3 0 , 0 0 0 ) , soyabean t r y p s i n  inhibitor  bovine serum albumin ( M  (M  The f o l l o w i n g p r o t e i n s were  r  = 21,000) and lysozyme (M  = 1 4 , 3 0 0 ) ; Lane 2 , 10 yg calmo-  d u l i n ; Lane 3 , 2 yg r a b b i t s k e l e t a l muscle PKI c o n t a m i n a n t ;  Lane 4 , 10 yg PKI ( b o v i n e heart);  heart);  Lane 6 , 2 p g - c a l m o d u l i n .  Lane 5 , 5 ^ g PKI  (bovine  C o * C o n c e n t r a t i o n (M' ?  ure 1 1 .  E f f e c t o f c y c l i c AMP and PKI on (Ca activity  + Mg  )-ATPase  i n human e r y t h r o c y t e membranes a t v a r i o u s 2+ 2+  concentrations of free calcium.  (Ca  + Mg  )-ATPase  was determined i n 'Dodge g h o s t ' membranes i n the absence (*)  and i n t h e presence o f 5 yM c y c l i c AMP ( o ) , 5 yM  c y c l i c AMP p l u s 2 yg/0.6 ml o f r a b b i t s k e l e t a l muscle PKI ( • ) » 2 y g / 0 . 6 ml o f b o v i n e h e a r t PKI ( a ) , and 5 yM c y c l i c AMP p l u s 2 yg bovine h e a r t PKI (•).  (Ca  2 +  + Mg )-ATPase  a c t i v i t y was measured a t 20 yM ATP f o r 3 m i n . of the experimental  2+  The  rest  p r o c e d u r e was the same as t h a t d e s c r i b e d  i n t h e Methods. Each p o i n t r e p r e s e n t s the means o f d u p l i c a t e o r t r i p l i c a t e determinations. similar  The curves shown are t y p i c a l o f  experiments.  three  F i g u r e 1.2.  E f f e c t of c a l m o d u l i n and bovine h e a r t PKI on (Ca ATPase a c t i v i t y i n e r y t h r o c y t e  + Mg  )-ATPase a c t i v i t y  )-  membranes and T r i t o n X-100  s o l u b i l i z e d enzyme a t 0.58 yM and 55 uM f r e e 2+ 2+ (Ca  + Mg  calcium.  i n 'Dodge g h o s t ' membranes  (DG)  was measured a t 2 mM ATP f o r 1 h a t 37°C and s o l u b i l i z e d enzyme a c t i v i t y  (SE) a t 0 . 5 mM ATP f o r 10 min ( f o r d e t a i l s o f  incubation mixture  see M a t e r i a l s and Methods) . A , P K I  (2 y g / 0 , 6  m l ) ; B, C a l m o d u l i n (.3 pg/0.6 m l ) ; C, PKI (2 p g / 0 . 5 ml) + c a l m o d u l i n (3 y g / 0 . 6 m l ) . Data shown i s t y p i c a l o f similar  experiments.  three  d i d not f u r t h e r s t i m u l a t e a.boye t h a t i n t h e presence o f c a l m o d u l i n  (Fig.  12) , The c o n c e n t r a t i o n - d e p e n d e n c e o f a c t i v a t i o n o f (Ca by bovine heart 13) .  2+  + Wg  2+  )-ATPase 2+  PKI and c a l m o d u l i n was i n v e s t i g a t e d a t 0.58 yM Ca  (Fig.  While t h e enzyme a c t i v i t y was a c t i v a t e d m a x i m a l l y a t a lower c o n c e n -  t r a t i o n o f PKI (2 yg/0.6 ml) than o f c a l m o d u l i n (3 yg/0.6 m l ) , the maximum a c t i v a t i o n by c a l m o d u l i n was h i g h e r than t h a t o f PKI. The e f f e c t o f PKI 2+ 2+ 2+ on t h e k i n e t i c s o f Ca a c t i v a t i o n o f (Ca + Mg )-ATPase was i n v e s t i g a 2+ ted ( F i g . 1 4 ) . In the absence o f t h e a c t i v a t o r s t h e Ca a c t i v a t i o n was 2+ b i p h a s i c , due to a mixed p o p u l a t i o n o f h i g h and low Ca a f f i n i t y states 2+ o f the enzyme ( A l - J o b o r e and R o u f o g a l i s 1 9 8 1 ) . The K^ o f t h e high Ca 2+ a f f i n i t y i s approx. 2 yM and t h e K^ f o r t h e low Ca a f f i n i t y component i s 40 yM or more.  In t h e presence o f e i t h e r c a l m o d u l i n or P K I , t h e low  2+ Ca  affinity  fitted  component was v i r t u a l l y  by a s i n g l e high C a  2 +  affinity  a b o l i s h e d , and the a c t i v a t i o n was (K  d  = 0 . 4 yM) ( F i g .  14).  However,  2+ 2+ u n l i k e c a l m o d u l i n , PKI d i d not i n c r e a s e the v" o f t h e (Ca + Mg ) 2+ ATPase a c t i v i t y . The s h i f t i n t h e Ca s e n s i t i v i t y o f t h e enzyme by PKI max  and c a l m o d u l i n and the i n c r e a s e i n V _ by c a l m o d u l i n i s seen more max in Eadie-Hofstee p l o t s o f t h e data ( F i g . 1 5 ) . v  clearly  The PKI from r a b b i t s k e l e t a l muscle d i d not s t i m u l a t e t h e (Ca  2+  +  2+ Mg  )-ATPase ( F i g .  weight  1 1 ) , even though i t s i s o - e l e c t r i c p o i n t and m o l e c u l a r  are s i m i l a r t o the bovine h e a r t PKI (Whitehouse  ejt al_. 1 9 8 0 ) .  The reason f o r t h i s i s not known, but i t may be due to s p e c i e s ces i n the amino a c i d c o m p o s i t i o n and o t h e r s t r u c t u r a l proteins.  differen-  properties of  Whitehouse «rt aj_. (1980) have r e c e n t l y demonstrated  the  three  charge isomers o f PKI from bovine h e a r t , a l l o f which were c a p a b l e o f i n h i b i t i n g the c y c l i c AMP p r o t e i n k i n a s e .  Because a l l t h e charge  isomers  Protein (jig/assay)  Figure 13.  A c t i v a t i o n o f (Ca  + Mg  )-ATPase i n human  erythrocyte 2+ membranes by c a l m o d u l i n and bovine heart P K I , (Ca + 2+ 2+ Mg )-ATPase a c t i v i t y was determined a t 0 , 5 8 -\ffl Ca , w i t h PKI ( A ) and c a l m o d u l i n ( p ) . Each p o i n t r e p r e s e n t s means o f t h r e e e x p e r i m e n t s .  the  F i g u r e 14.  E f f e c t o f c a l m o d u l i n and PKI on c a l c i u m a c t i v a t i o n o f 2+ 2+ (Ca + Mg )-ATPase i n human e r y t h r o c y t e membranes. 2+ 2+ (Ca  + Mg  absence (*) ml (•)  )-ATPase a c t i v i t y was determined i n t h e and i n the presence o f 3 ug c a l m o d u l i n / 0 . 6  and 2 yg bovine heart PKI/0.6 ml ( o ) .  s e n t the means o f t h r e e s e p a r a t e e x p e r i m e n t s .  Data r e p r e -  0  ±  2  JL  4  -L  6  8  -L  10  -L  12  14  V/ri+Ojmoles Pi/h per ml ghost/pM) F i g u r e 15.  E a d i e - H o f s t e e p l o t o f data i n f i g u r e 14. o b t a i n e d i n t h e absence (*) 3 yg c a l m o d u l i n / 0 . 6 ml (•) 0 . 6 mis (o).  Curves were  and i n t h e presence o f and 2 yg bovine h e a r t  PKI/  were presumably p r e s e n t i n t h i s s t u d y ,  i t i s not known i f any s p e c i f i c 2+  charge Isomer  2+  i s more p o t e n t than the o t h e r s i n s t i m u l a t i n g (Ca  + Mg  )-  ATPase. 2+ B o v i n e h e a r t PKI a l s o s t i m u l a t e d (Ca  2+ + Mg  )-ATPase i n  'Dodge  g h o s t s ' t r e a t e d w i t h EDTA ( E D T A - t r e a t e d membranes), which a r e d e p l e t e d o f endogenous c a l m o d u l i n ( R o u f o g a l i s and M a u l d i n 1980) ( r e s u l t s  not  shown).  These membranes a l s o l a c k t h e endogenous c y c l i c AMP p r o t e i n k i n a s e and Galand 1977) which f u r t h e r  (Boivin 2+  suggests t h a t PKI d i d not s t i m u l a t e (Ca  +  2+ Mg  )-ATPase by an a c t i o n on c y c l i c AMP p r o t e i n k i n a s e . D e m a i l l e ejt al_. (1979) showed t h a t m o d i f i c a t i o n o f t h e  arginyl  groups o f PKI a b o l i s h e d i t s i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e . M o d i f i c a t i o n of arginyl  groups o f bovine h e a r t PKI o r c a l m o d u l i n had no 2+ 2+  e f f e c t on t h e i r a b i l i t y t o s t i m u l a t e (Ca III).  Arginyl  + Mg  )-ATPase a c t i v i t y  group m o d i f i c a t i o n o f P K I , however, a n t a g o n i z e d  (Table  its  i n h i b i t i o n o f t h e p h o s p h o r y l a t i o n o f hi stone by c y c l i c AMP p r o t e i n k i n a s e . 2+ TFP has been shown t o i n h i b i t c a l m o d u l i n s t i m u l a t i o n o f (Ca + Mg )-ATPase  ( L e v i n and Weiss 1979).  2+  2+ (Ca  TFP (20 yM) i n h i b i t e d a c t i v a t i o n  of  2+ + Mg  )-ATPase by PKI i n t h e presence o r absence o f c a l m o d u l i n  (Table I V ) .  By c o n t r a s t , t h e a b i l i t y o f bovine h e a r t PKI to i n h i b i t  c y c l i c AMP p r o t e i n k i n a s e was not a f f e c t e d by TFP (Table V ) , that d i f f e r e n t  s i t e s or c o n f o r m a t i o n s o f t h e PKI m o l e c u l e may be i n v o l v e d 2+ 2+  i n s t i m u l a t i o n o f (Ca protein kinase.  suggesting  It  + Mg  )-ATPase and i n h i b i t i o n o f c y c l i c AMP  i s a l s o o f i n t e r e s t t h a t i n h i b i t i o n o f c y c l i c AMP  p r o t e i n k i n a s e by PKI does not r e q u i r e c a l c i u m , as i n h i b i t i o n o f  histone  p h o s p h o r y l a t i o n o c c u r s even i n t h e presence o f EGTA ( r e s u l t not shown). 2+ 2+ Thus PKI s t i m u l a t i o n o f (Ca + Mg )-ATPase resembles t h a t o f 2+ c a l m o d u l i n , as both i n c r e a s e the s e n s i t i v i t y f o r Ca . However, u n l i k e  E f f e c t of a r g i n y l  group m o d i f i c a t i o n o f bovine h e a r t c y c l i c AMP p r o t e i n k i n a s e 2+  and c a l m o d u l i n on m o d u l a t i o n of (Ca (C-subunit)  activity.  Inhibitor  2+ + Mg  )-ATPase a c t i v i t y or c y c l i c AMP p r o t e i n k i n a s e  M o d i f i c a t i o n o f PKI and c a l m o d u l i n a r g i n y l  groups and the assay o f  h i s t o n e p h o s p h o r y l a t i o n and ATPase a c t i v i t y a r e d e s c r i b e d i n Methods, Residue M o d i f i e d  C-subunit catalyzed t r a n s f e r o f 32p t o h i s t o n e (pmoles P-j t r a n s f e r r e d per 10 min)  Reagent Unmodified 1,2-Cyclohexanedione  Arginine  (Ca + Mg )-ATPase a c t i v i t y (ymoles P^ per ml ' g h o s t s ' ) 2 +  2+  PKI  PKI  38  3.5  4.5  3.5  4.3  130 ( 1 4 0 )  a  Calmodulin  The v a l u e i n p a r e n t h e s i s r e p r e s e n t s c o n t r o l v a l u e w i t h o u t p r o t e i n k i n a s e i n h i b i t o r . Data r e p r e s e n t the means o f two ( h i s t o n e p h o s p h o r y l a t i o n )  o r t h r e e ( ( C a + M g ) - A T P a s e ) s e p a r a t e experiments 2+  2+  E f f e c t of t r i f l u o p e r a z i n e  (TFP)  on t h e a b i l i t y o f c y c l i c AMP 2+  p r o t e i n k i n a s e i n h i b i t o r t o s t i m u l a t e (Ca i n 'Dodge g h o s t ' membranes (j)G).  2+ + Mg  )-ATPase  When u s e d , TFP was added to  the r e a c t i o n mixture  i n t h e dark 10 min b e f o r e t h e r e a c t i o n 2+ 2+ was s t a r t e d w i t h ATP, (Ca + Mg )-ATPase a c t i v i t y was 2+ assayed a t 2 mM ATP and 0 , 5 8 yM Ca  , as d e s c r i b e d i n Methods,  Data r e p r e s e n t the means o f a t l e a s t t h r e e s e p a r a t e Enzyme P r e p a r a t i o n  Dodge ghosts  TFP (20 yM)  )-ATPase  1.4  3.2  yg)  +  1.6  4.0  DG + c a l m o d u l i n (3 yg)  + Mg  1.5  (DG)  +  DG + PKI (2  (Ca  experiments.  +  1,75  Effect of t r i f l u o p e r a z i n e  (JFP)  AMP p r o t e i n k i n a s e c a t a l y t i c (X)  on the i n h i b i t i o n o f c y c l i c s u b u n i t c a t a l y z e d phospho-  r y l a t i o n o f h i s t o n e by p r o t e i n k i n a s e I n h i b i t o r heart.  from bovine  H i s t o n e p h o s p h o r y l a t i o n was performed as d e s c r i b e d  i n Methods.  TFP and PKI were p r e i n c u b a t e d w i t h the  mixture for  10 min i n t h e dark p r i o r to s t a r t i n g the  w i t h ATP.  Data shown i s t y p i c a l o f t h r e e s e p a r a t e  Assay C o n d i t i o n s  PKI (2  38  yg)  140  20 yM TFP PKI (2 yg)  + TFP (20 yM)  reaction  experiments.  Histone Phosphorylation (pmoles 32p t r a n s f e r r e d / 1 0 min)  140  Control  reaction  40  c a l m o d u l i n , PKI does not i n c r e a s e the maximum v e l o c i t y ,  The s t i m u l a t i o n  2+ by PKI a t low Ca  c o n c e n t r a t i o n s i s not due s o l e l y to  c y c l i c AMP p r o t e i n k i n a s e p h o s p h o r y l a t i o n because a) c o m p a r a t i v e l y s m a l l I n h i b i t i o n o f the enzyme a c t i v i t y  an e f f e c t on ,the  c y c l i c AMP caused a  Capprox.  20%)  at  a l l c a l c i u m c o n c e n t r a t i o n s u s e d , w h i l e t h e s t i m u l a t i o n by PKI i s about 100% and independent o f added c y c l i c AMP.  Furthermore,  PKI s t i m u l a t i o n  o c c u r r e d a t 2 mM ATP, c o n d i t i o n s under which c y c l i c AMP I n h i b i t i o n  of  2+  2+ (Ca + Mg )-ATPase was not o b s e r v e d , b) M o d i f i c a t i o n o f a r g i n y l groups o f PKI l e d t o a l o s s i n t h e i n h i b i t i o n by PKI o f h l s t o n e p h o s p h o r y l a t i o n by c y c l i c AMP p r o t e i n k i n a s e , but had no e f f e c t on the s t i m u l a t i o n o f 2+ (Ca  2+ + Mg 2+  w i t h (Ca  )-ATPase.  T h i s suggests t h a t t h e s i t e s o f i n t e r a c t i o n o f  PKI  2+ + Mg  )-ATPase and p r o t e i n k i n a s e a r e d i f f e r e n t  or that  argi-  n i n e r e s i d u e s a r e not r e q u i r e d f o r a c t i v a t i o n o f ATPase, as i s a l s o t h e case w i t h c a l m o d u l i n ( T a b l e I I I ) ,  c) PKI s t i m u l a t e d EDTA-treated  membra-  nes which a r e d e p l e t e d o f c y c l i c AMP p r o t e i n k i n a s e (.Boivin and Galand 1977). Khandelwal e t al_ (1980)  have r e p o r t e d t h a t under c e r t a i n c o n d i t -  ions the " p h o s p h o p r o t e i n - p h o s p h a t a s e " a c t i v i t y o f l i v e r  i s i n c r e a s e d by  c a l m o d u l i n and t r o p o n i n - C and they suggested t h a t t h i s e f f e c t may be due to t h e n e g a t i v e charge of t h e s e compounds, as the e f f e c t showed l a c k o f 2+ c a l c i u m dependency.  A l t h o u g h i t i s p o s s i b l e t h a t t h e i n c r e a s e i n (Ca  +  2+ Mg  )-ATPase a c t i v i t y  by PKI observed i n t h i s study c o u l d be due to  i n c r e a s e d phosphatase a c t i v i t y ,  as a r e s u l t o f i t s n e g a t i v e charge a l o n e ,  t h i s i s l e s s l i k e l y because r a b b i t s k e l e t a l muscle P K I , which has a s i m i l a r c h a r g e , was i n e f f e c t i v e and the e f f e c t  i s observed i n  membranes which l a c k t h e c y c l i c AMP p h o s p h o r y l a t i n g system.  EDTA-treated The  inhibi2+ 2+ t o r y e f f e c t o f TFP on both PKI and c a l m o d u l i n s t i m u l a t i o n o f (Ca + Mg )  -ATPase suggests t h a t  caution  s h o u l d be used i n i n v o k i n g  a  r o l e f o r calmo-  d u l i n 1n a c e l l u l a r p r o c e s s on t h e b a s i s o f t h i s e v i d e n c e a l o n e .  The l a c k  o f TFP e f f e c t on c y c l i c AMP p r o t e i n k i n a s e p h o s p h o r y l a t i o n 1n t h e presence (or absence) o f PKI f u r t h e r  s u p p o r t s t h e c o n c l u s i o n t h a t d i f f e r e n t mecha-  nisms or i n t e r a c t i o n s a r e i n v o l v e d 1n t h e two a c t i o n s o f In summary, 2+ (Ca  PKI.  i t was shown t h a t PKI from b o v i n e h e a r t s t i m u l a t e s  2+ + Mg )-ATPase a t low c a l c i u m c o n c e n t r a t i o n s by i n c r e a s i n g the s e n 2+  sitivity  f o r f r e e Ca  , w i t h o u t a f f e c t i n g t h e maximum v e l o c i t y o f  enzyme.  The s t i m u l a t i o n o f ( C a  2 +  + Mg )-ATPase 2+  a t low C a  2 +  the  by PKI  is  seen even i n t h e presence o f s a t u r a t i n g 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 . 2+ 2+ was found t h a t a number o f a n i o n s s t i m u l a t e (Ca Jobore e t al_. 1 9 8 1 ) , and i t  + Mg  )-ATPase  It  (Al-  i s p o s s i b l e t h a t the s t i m u l a t i o n by bovine  heart PKI i s r e l a t e d , a t l e a s t i n p a r t , to i t s a n i o n i c n a t u r e . 2+ 2+ s p e c i f i c p a r t i a l r e a c t i o n s o f the (Ca  + Mg  The  )-ATPase r e a c t i o n sequence  a f f e c t e d by t h i s c l a s s o f a c t i v a t o r s remains to be e s t a b l i s h e d .  Section III.  R o l e o f a n i o n channel of  (Ca  (band 3) i n h i b i t o r s i n t h e  + Mg )-ATPase  2 +  activity  2+  Waisman et a l . (1981) and Gimble et a l . (1981) r e c e n t l y band 3 ( a n i o n channel)  inhibitors,  stilbene-2'-disulfonate sulfonate  (NAP-taurine),  p o r t and anion t r a n s p o r t This observation  (SITS) and  regulation  reported  such as 4 - a c e t a m i d o - 4 ' - i s o t h i o c y a n o N-(4-azido-2-nitrophenyl)-2-aminoethyl  b l o c k e d both 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 t r a n s in inside-out vesicles with s i m i l a r  l e d these a u t h o r s to suggest t h a t these two  p r o c e s s e s were c l o s e l y coupled and t h a t the C a  2 +  pump i s  F u r t h e r m o r e , they suggested t h a t at low c o n c e n t r a t i o n s  potency. transport  electrogenic.  (K^ = 45-80 uM),  N A P - t a u r i n e b l o c k e d the c a l m o d u l i n dependent c a l c i u m t r a n s p o r t v i a blockade of anion-transport  activity  t h r o u g h the anion c h a n n e l .  In t h i s t h e s i s the a c t i o n o f anion channel  i n h i b i t o r s was  studied  f u r t h e r u s i n g fragmented membranes, r e s e a l e d ghosts and i n s i d e - o u t c l e s , to t e s t  i f the anion channel  p o r t ATPase a c t i v i t y  primarily  e c t a c t i o n on the ( C a o f the anion channel (Na  +  2 +  b l o c k e r s i n h i b i t e d the  vesi-  calcium-trans-  by b l o c k i n g the anion channel or by a d i r -  + Mg )-ATPase 2+  enzyme.  b l o c k e r s on M g - A T P a s e 2+  +  F u r t h e r m o r e , the  and ouabain  + K ) - A T P a s e a c t i v i t i e s was a l s o s t u d i e d .  SITS, NAP-taurine  that  The  sensitive  structures  and DIDS ( 4 , 4 ' - d i i s o t h i o c y a n o - 2 - 2 ' - s t i l b e n e  are shown below.  NAP-taurine  effect  of disulfonate)  »  3  SDj  DIDS  1.  SITS  E f f e c t o f N A P - t a u r i n e on (Ca** + h V ) - A T P a s e +  The  (Ca  2 +  + Mg )-ATPase  activity  2+  activity  o f red c e l l membranes was i n h i b i t e d  i n the presence o f N A P - t a u r i n e i n t h e absence o f i r r a d i a t i o n or d i r e c t daylight  (Fig. 16).  The i n h i b i t i o n o f enzyme a c t i v i t y  by 50 \tl N A P - t a u r -  i n e was seen at a l l c o n c e n t r a t i o n s o f c a l c i u m examined ( F i g . shows the c o n c e n t r a t i o n - r e s p o n s e curves ATPase, M g - A T P a s e and ( N a 2+  i n h i b i t i o n of ( C a and ( N a  +  2 +  for i n h i b i t i o n of ( C a  + K ) - A T P a s e by N A P - t a u r i n e .  +  +  + Mg )-ATPase 2+  16). 2 +  F i g . 17  + Mg  2 +  )-  H a l f maximal  o c c u r r e d at around 50 yM.  Mg -ATPase 2+  + K ) - A T P a s e a c t i v i t i e s were not i n h i b i t e d over the same c o n +  c e n t r a t i o n range.  At higher c o n c e n t r a t i o n s (>200 yM), N A P - t a u r i n e began  t o i n h i b i t the o t h e r ATPases,  p r o b a b l y by a n o n - s p e c i f i c a c t i o n on the  membranes. The p h o t o l a b e l i n g reagent N A P - t a u r i n e o f f e r s reversible  i n h i b i t o r of ( C a  irreversible reversible  2 +  + Mg )-ATPase 2+  inhibitor after light  i n h i b i t i o n of ( C a  2 +  the advantage o f being a  b e f o r e i r r a d i a t i o n and an  induced p h o t o a c t i v a t i o n ( F i g . 1 8 ) .  + Mg )-ATPase 2+  by N A P - t a u r i n e was shown by  i n c u b a t i n g membranes i n the absence and presence o f N A P - t a u r i n e t h e dark ( i n a p r e i n c u b a t i o n medium i d e n t i c a l to t h a t ATPase a c t i v i t y  but from which ATP was e x c l u d e d )  2+  (25 yM)  in  used f o r measuring  and d i l u t i n g 50 y l o f  i n c u b a t i o n m i x t u r e to 600 y l o f the assay medium ( F i g . 1 8 ) . + Mg )-ATPase  The  The  (Ca  2 +  a c t i v i t y was u n a f f e c t e d by these c o n d i t i o n s , i n d i c a t i n g  t h a t t h e b i n d i n g o f N A P - t a u r i n e t o the enzyme was r e v e r s i b l e  (Fig.  18).  the  o  y-  6  5  -Log|Can  free  2+ F i g u r e 16.  E f f e c t o f N A P - t a u r i n e on (Ca o f membranes.  4  („) 2+  + Mg  )-ATPase  activity  The enzyme a c t i v i t y was measured i n t h e  dark i n t h e absence (•)  and i n t h e presence (o) o f NAP2+  t a u r i n e (50 yM) a t v a r i o u s c o n c e n t r a t i o n s o f f r e e Ca Data r e p r e s e n t the means o f two s e p a r a t e e x p e r i m e n t s .  o  40  Figure 17.  80 NAP-taurine (uM)  120  200  C o n c e n t r a t i o n dependence o f N A P - t a u r i n e i n h i b i t i o n o f (Ca + M g ) - A T P a s e (•), ( N a + K ) - A T P a s e (A) and 2+ (Ca Mg*"'-ATPase (o) a c t i v i t i e s by N A P - t a u r i n e . 9i 2 +  2+  +  +  2 +  Mg  +  )-ATPase a c t i v i t y was measured a t 0.58 yM f r e e c a l c i u m .  Each p o i n t r e p r e s e n t s t h e means o f t r i p l i c a t e d e t e r m i n a t i o n s , The curves shown a r e t y p i c a l o f t h r e e s i m i l a r e x p e r i m e n t s .  =C l . 5  r  'o> E  •5  1  0  E  a> oo  D Q_  < 0.5  D  u 0.0 0  6 -Log[Ca  5 2 +  ]  f  (M)  ree 2+  Figure 18.  E f f e c t o f N A P - t a u r i n e on (Ca membranes w i t h and w i t h o u t  2+ + Mg  )-ATPase i n  photolysis.  Membranes (4 mg/ml)  i n t h e presence o f N A P - t a u r i n e (25 yM) were e i t h e r kept i n t h e dark (o) or i r r a d i a t e d f o r 15 min (•)  o r 30 min  (o).  Membranes were a l s o i r r a d i a t e d i n t h e absence o f N A P - t a u r i n e (•).  50 y l  o f t h e s e membranes were d i l u t e d to 0.6 ml i n 2+ 2+ the s t a n d a r d a s s a y medium t o measure (Ca + Mg )-ATPase 2+ 2+ activity.  ( ) A  Represents (Ca  + Mg  )-ATPase a c t i v i t y  of  c o n t r o l membranes i n t h e presence o f N A P - t a u r i n e (25 yM) i n t h e assay medium i n t h e d a r k . three s i m i l a r  experiments.  Data shown i s t y p i c a l  of  However, p h o t o l y s i s o f membranes w i t h 25 uM N A P - t a u r i n e f o r 15 min or 30 min r e s u l t e d i n r e t e n t i o n o f the i n h i b i t i o n o f the ( C a a f t e r 5 0 - f o l d d i l u t i o n i n the assay medium ( F i g . 1 8 ) . irreversible  i n h i b i t i o n of ( C a  s i m i l a r to the r e v e r s i b l e  2 +  + Mg )-ATPase  + Mg )-ATPase  2 +  2+  The e x t e n t  of  seen upon p h o t o l y s i s was  2+  i n h i b i t i o n o f t h e enzyme assayed i n the  o f N A P - t a u r i n e i n the d a r k .  presence  These r e s u l t s suggest t h a t N A P - t a u r i n e c o u l d  be used as a p h o t o l a b e l i n g probe because t h e i r r e v e r s i b l e  i n h i b i t i o n upon  p h o t o l y s i s c l o s e l y r e f l e c t s the r e v e r s i b l e  dark.  b i n d i n g i n the  In an attempt to d e f i n e the s i t e o f a c t i o n o f N A P - t a u r i n e on t h e (Ca  + M g ) - A T P a s e , membranes were exposed to l i g h t  2 +  2+  NAP-taurine  (25 yM) i n the absence and presence o f v a r i o u s  o f C a , M g , ATP and c a l m o d u l i n . +  i n the presence o f  2 +  It  was f i r s t  concentrations  established that  irradia-  t i o n o f c a l m o d u l i n i n the presence o f 25 yM N A P - t a u r i n e d i d not a f f e c t activity, (results  as shown by i t s a b i l i t y to s t i m u l a t e ( C a not shown).  2 +  its  + Mg )-ATPase 2+  None o f the agents p r o t e c t e d a g a i n s t i n h i b i t i o n by  N A P - t a u r i n e (Table V I ) .  These r e s u l t s suggest t h a t N A P - t a u r i n e i n h i b i t e d  the  n o n - c o m p e t i t i v e l y w i t h r e s p e c t to C a  ATP,  (Ca  2 +  + Mg )-ATPase 2+  and c a l m o d u l i n .  2 +  ,  Mg , 2 +  The n o n - c o m p e t i t i v e nature o f i n h i b i t i o n by NAP-  t a u r i n e w i t h r e s p e c t to c a l m o d u l i n i s a l s o shown i n a d o u b l e - r e c i p r o c o l plot (Fig. 19). v a t i o n of ( C a  2 +  S i n c e t r y p s i n t r e a t m e n t o f membranes a b o l i s h e s the + Mg )-ATPase 2+  by c a l m o d u l i n , probably by l o s s of a r e g u l a -  t o r y component o f the enzyme ( N i g g l i Enyedi  acti-  et a l . 1 9 8 0 ; S a r k a d i et a l . 1980;  et a l . 1 9 8 0 ) , the e f f e c t o f N A P - t a u r i n e on t r y p s i n - t r e a t e d mem-  b r a n e s was i n v e s t i g a t e d . Mg )-ATPase  activity  2+  2 +  reports  + Mg )-ATPase 2+  s t i m u l a t e d the ( C a  2 +  +  and a b o l i s h e d i t s s t i m u l a t i o n by c a l m o d u l i n , i n  concurrence with previous t e d the ( C a  T r y p s i n treatment  (Fig.  activity  20).  N A P - t a u r i n e (50 yM)  of t r y p s i n - t r e a t e d  inhibi-  membranes to  the  E f f e c t o f Mg 2+ (Ca  , Ca  , ATP and c a l m o d u l i n on t h e i n h i b i t i o n o f  2+ + tig  )-ATPase a c t i v i t y o f membranes a f t e r p h o t o l y s i s  in  t h e presence o f N A P - t a u r i n e (25 yM). The membranes (~4 mg/ml f i n a l c o n c e n t r a t i o n ) medium c o n t a i n i n g 55 mM T r i s - m a l e a t e  were i n c u b a t e d i n a  (pH 7 . 2 ) , 66 mM N a C l , 0,1 mM o u a b a i n ,  N A P - t a u r i n e (25 yM) and v a r i o u s e f f e c t o r s o f t h e enzyme, as I n d i c a t e d . 2+ 2+ 2+ When Mg was v a r i e d , t h e c o n c e n t r a t i o n o f f r e e Ca was 10 yM. When Ca 2+ was v a r i e d , t h e c o n c e n t r a t i o n o f Mg was kept a t 6.5 mM, and when ATP o r 2+ 2+ c a l m o d u l i n were v a r i e d , t h e c o n c e n t r a t i o n s o f Ca and Mg were 10 yM and 6.5 mM, r e s p e c t i v e l y .  The membranes were p h o t o l y s e d a t 4°C f o r 40 m i n ; 2+  50 y l o f t h i s enzyme p r e p a r a t i o n was used f o r measurement o f (Ca -ATPase a c t i v i t y  a t 55 yM f r e e  calcium)  Medium o f P h o t o l y s i s  (Ca  2+  2+ + Mg )-ATPase  (ymoles Control  (No  P i mg"^  0.45  N A P - t a u r i n e (25 yM) N A P - t a u r i n e (25 yM) + M g  2 +  hr"^) 0.75  NAP-taurine)  NAP-taurine + C a  activity  2 +  ( 1 mM) (10 mM)  ( 5 yM) ( 55 yM)  0.47 0.46 0.46 0.48  N A P - t a u r i n e + ATP ( 0 . 5 mM) ( 2 . 0 mM)  0.43 0.44  N A P - t a u r i n e + c a l m o d u l i n (0.1 y g ) ( 1 . 2 y g) •  0.46  Data r e p r e s e n t the means of a t 1 e a s t t h r e e s e p a r a t e  experiments.  2+ + Mg  )  F i g u r e 19.  D o u b l e - r e c i p r o c a l p l o t o f CCa  + Mg  )-ATPase i n h i b i t i o n  by N A P - t a u r i n e i n t h e presence o f c a l m o d u l i n . (•)  Represents  enzyme a c t i v i t y  i n t h e presence o f c a l m o d u l i n ; (o) Represents  enzyme a c t i v i t y  i n t h e presence o f c a l m o d u l i n and N A P - t a u r i n e  (50 yM) i n t h e a s s a y medium.  N A P - t a u r i n e was added t o membra-  nes i n the a s s a y medium ( 0 . 6 ml) and i n c u b a t e d i n t h e d a r k .  B D  E  1.5  O  E  1.0  CD  or> O  ^  0.5  D u  0.0  Figure 20,  2+ 2+ E f f e c t o f N A P - t a u r i n e on (Ca + Mg )-ATPase i n t r y p s i n t r e a t e d membranes. 2+ 2+ A: (Ca + Mg )-ATPase a c t i v i t y o f n o n - t r y p s i n t r e a t e d membranes determined a t 55 yM f r e e Ca 2+ 2+ 2+ B: C a l m o d u l i n (2 yg)  a c t i v a t i o n o f (Ca  + Mg  )-ATPase  a c t i v i t y o f n o n - t r y p s i n t r e a t e d membranes. C: ( C a + M g ) - A T P a s e a c t i v i t y a f t e r t r e a t m e n t w i t h 0.2 (Ca + Mg )-ATPase a 2 +  2+  2 +  2+  yg t r y p s i n / m g p r o t e i n . D: E f f e c t o f c a l m o d u l i n (2 yg) Effect of calmodulin (  on ( C a  2 +  + Mg )-ATPase 2+  a c t i v i t y o f t r y p s i n t r e a t e d membranes, E: E f f e c t o f N A P - t a u r i n e (50 yM) on ( C a + Mg )-ATPase E f f e c t o f N A P - t a u r i n e (50 yM) on ( C a 2 +  2 H  a c t i v i t y o f t r y p s i n t r e a t e d membranes.  2+  same extent  as i n n a t i v e membranes.  These r e s u l t s f u r t h e r  s u g g e s t i o n t h a t N A P - t a u r i n e i n h i b i t s the ( C a  + Mg )-ATPase  2 +  2+  d i s t i n c t from t h a t o f the c a l m o d u l i n r e g u l a t o r y 2.  support the at a s i t e  site.  The s i d e d n e s s o f N A P - t a u r i n e i n h i b i t i o n o f ( C a  + Mq )-ATPase  2 +  2+  A number o f e x p e r i m e n t s were performed to determine from which s i d e o f the membrane N A P - t a u r i n e i n h i b i t e d the ( C a  2 +  + Mg )-ATPase. 2+  Direct  a c c e s s t o t h e c y t o p l a s m i c face o f the membrane was a c h i e v e d by r e s e a l i n g g h o s t s i n the presence o f N A P - t a u r i n e (Table V I I ) . ally  no e f f e c t on the ( C a  2 +  + Mg )-ATPase 2+  r e s e a l e d i n the absence o f N A P - t a u r i n e .  a c t i v i t y when added to ghosts  However, when g h o s t s were r e -  s e a l e d i n the presence o f 50 uM N A P - t a u r i n e , (Ca  2 +  + Mg )-ATPase 2+  the s i t e ( s )  membrane.  was o b s e r v e d .  60% i n h i b i t i o n o f the These r e s u l t s suggested t h a t  at which N A P - t a u r i n e b i n d s to cause i n h i b i t i o n o f ( C a  Mg )-ATPase 2+  activity  N A P - t a u r i n e had v i r t u -  2 +  +  i s a c c e s s i b l e from the i n n e r o r c y t o p l a s m i c s i d e o f the  This conclusion i s further  t a u r i n e on C a - t r a n s p o r t 2 +  supported by the a c t i o n o f NAP-  in inside-out v e s i c l e s ( F i g . 2 1 ) .  seen t h a t 50 yM N A P - t a u r i n e i n h i b i t e d the C a - t r a n s p o r t  F i n a l l y , the ( C a  2 +  to i t s i n h i b i t i o n o f ( C a  + Mg )-ATPase 2+  activity  2 +  can be  in inside-out  2 +  v e s i c l e s to a s i m i l a r e x t e n t  It  + Mg )-ATPase. 2+  i n membranes prepared from red  c e l l s p h o t o l y s e d i n the presence o f 50 yM N A P - t a u r i n e was s i m i l a r to t h a t from u n t r e a t e d c e l l s ,  s u g g e s t i n g t h a t N A P - t a u r i n e d i d not i n h i b i t the  enzyme from the o u t e r membrane s u r f a c e (see T a b l e V I I I ) .  At t h i s c o n c e n -  t r a t i o n N A P - t a u r i n e b i n d s to a m o d i f i e r s i t e on the o u t e r membrane s u r f a c e (Knauf et a l . 1978) and i t does not permeate the red c e l l  under these c o n -  d i t i o n s ( S t a r o s et a l . 1974; Cabantchik et a l . 1 9 7 8 ) . 3.  Does N A P - t a u r i n e i n h i b i t t h e ( C a  2 +  + M g ) - A T P a s e and 2+  Ca " "- t r a n s p o r t by b l o c k i n g t h e a n i o n channel ? 2  1  A number o f compounds have been r e p o r t e d to i n h i b i t band 3 ( a n i o n  CCa  + Mg  )-ATPase a c t i v i t y  and absence o f  o f ghosts r e s e a l e d i n the  NAP-taurine,  The r e s e a l i n g medium c o n t a i n e d (.in f i n a l c o n c e n t r a t i o n ) MgCl , 2  2  Data r e p r e s e n t  the means o f  three  experiments.  (Ca  )-ATPase a c t i v i t y -1 -1 (umoles P. mg hr )  Composition of Loading Medium  Control  4 mM  3 mM C a C l , 4 mM T r i s - A T P , 0.1 mM o u a b a i n , 10 mM T r i s - m a l e a t e ,  pH 7.1 ± N A P - t a u r i n e (50 y M ) . separate  presence  (no  NAP-taurine)  Control (NAP-taurine t h e assay medium)  added to  N A P - t a u r i n e (50yM) i n r e s e a l i n g medi urn  + Mg  0.63 0.58 0.25  Time (min) Figure 21.  E f f e c t of N A P - t a u r i n e on Ca 2+ (1.0.) vesicles. o f ATP (•),  Ca  -transport  in inside-out  - t r a n s p o r t was measured i n t h e absence  i n the presence o f 2 mM ATP (•)  and i n t h e p r e -  sence o f ATP p l u s N A P - t a u r i n e (.50 yM) i n t h e dark ( o ) .  The  r e a c t i o n medium c o n t a i n e d 55 mM T r i s - m a l e a t e , pH 7 . 2 , 66 mM 45 N a C l , 6.4 mM M g C l , 0.1 mM EGTA and 2  c a l c i u m c o n c e n t r a t i o n o f 55 yM. concentration).  similar  experiments.  C a C l , to give a f r e e 2  The r e a c t i o n was i n i t i a t e d  a f t e r a 5 min p r e i n c u b a t i o n ( 3 7 ° C ) final  3  by a d d i t i o n o f ATP (2 mM,  The data shown i s t y p i c a l o f  three  2+ (Ca  2+ + Mg  )-ATPase a c t i v i t y o f membranes made from c e l l s  t r e a t e d w i t h NAP-taur1ne o r DIDS Human e r y t h r o c y t e s  (.10% h e m a t o c r i t ) were e i t h e r i n c u b a t e d w i t h  N A P - t a u r i n e (25 yM) and p h o t o l y s e d a t 4°C o r i n c u b a t e d a t 37°C w i t h DIDS (5 yM) f o r 30 m i n .  The c e l l s were washed and then hemolysed f o r  p r e p a r a t i o n o f membranes. a t 55 yM f r e e  The enzyme a c t i v i t y o f membranes was measured  calcium.  Membrane p r e p a r a t i o n  (Ca  2+  2+ + Mg )-ATPase  (ymoles  0.62  50 yM N A P - t a u r i n e  0.57  5 yM DIDS  Data r e p r e s e n t the means o f four experiments  activity  P^ mg~^ hr~^  Control  experiments  the  0.62  f o r the c o n t r o l and t h r e e  f o r the N A P - t a u r i n e and DIDS t r e a t e d  cells.  channel).  N A P - t a u r i n e was shown to be a r e v e r s i b l e  inhibitor  (in  the  d a r k ) o f the a n i o n channel when i t was present e i t h e r i n s i d e or o u t s i d e the c e l l (15-fold) either  (Knauf et a l . 1 9 7 8 ) .  However, t h e r e  i s a substantial  i n the c o n c e n t r a t i o n r e q u i r e d to i n h i b i t t h e anion channel  side.  On the i n s i d e , N A P - t a u r i n e has a r e l a t i v e l y  band 3 (K-j = 370 uM), whereas e x t e r n a l i n h i b i t o r o f band 3 (Ki  = 20 uM) (Knauf et a l . 1 9 7 8 ) .  o f membranes d i r e c t l y  a n i o n channel  potent  + Mg )-ATPase 2+  than i n d i r e c t l y by i n h i b i t i n g t h e were indeed  coupled).  Whereas 50 uM N A P - t a u r i n e and 5 pM DIDS (Knauf et a l . 1978) i n h i b i t  t h e anion channel ATPase a c t i v i t y  from the e x t e r n a l  membrane s u r f a c e , ( C a  i n membranes made e i t h e r  DIDS t r e a t e d c e l l s was comparable to t h a t tions b)  for  Four l i n e s o f  (assuming t h a t the ATPase and the anion channel  i n some way t i g h t l y a)  rather  2 +  from  low a f f i n i t y  N A P - t a u r i n e i s a f a r more  e v i d e n c e i n d i c a t e t h a t N A P - t a u r i n e i n h i b i t s the ( C a activity  difference  of i r r e v e r s i b l e  band 3  2 +  + Mg^ )+  from 50 pM N A P - t a u r i n e o r 5 pM from c o n t r o l  c e l l s under  condi-  i n h i b i t i o n (Table V I I I ) .  P r o b e n e c i d (125 uM) has a l s o been r e p o r t e d to be an anion channel  b l o c k e r ( M o t a i s and Cousin 1 9 7 6 ) .  However, 125 pM p r o b e n e c i d had no  effect  activity  on the ( C a  2 +  + Mg )-ATPase 2+  not  shown).  c)  P r e - i r r a d i a t e d NAP-taurine  (i.e.  i n unsealed membranes  NAP-taurine  a l o n e ) was a b l e to i n h i b i t the C a - t r a n s p o r t 2 +  as n o n i r r a d i a t e d N A P - t a u r i n e i b l e nor i r r e v e r s i b l e  (results  photolysed  in  (results  buffer  ATPase to the same extent  not shown), whereas  neither  revers-  blockade o f the anion channel was observed w i t h NAP-  t a u r i n e which was p r e - i r r a d i a t e d (Knauf et a l . 1978; R o t h s t e i n et a l . 1977). d)  The most c o n v i n c i n g e v i d e n c e came from experiments  i n e at c o n c e n t r a t i o n s  i n which  between 25 pM and 50 pM i n h i b i t e d p u r i f i e d  NAP-taur(Ca  2 +  + Mg )-ATPase  (Table I X ) .  2+  The i n h i b i t i o n by 50 uM N A P - t a u r i n e o f the  p u r i f i e d enzyme i s more pronounced than t h a t o f the enzyme i n membranes. The e f f e c t o f pretreatment b l o c k e r on c a l c i u m t r a n s p o r t cells  i s shown i n F i g . 2 2 .  o f i n t a c t red c e l l s w i t h an anion channel  i n t o i n s i d e - o u t v e s i c l e s prepared from t h e s e Treatment o f red c e l l s w i t h 5 \M DIDS l e d to  an i n h i b i t i o n o f c a l c i u m t r a n s p o r t l a c k of i n h i b i t i o n of ( C a DIDS t r e a t e d c e l l s  2 +  i n i n s i d e - o u t v e s i c l e s , d e s p i t e the  + Mg )-ATPase  activity  2+  (Table V I I I ) .  i n membranes from  These r e s u l t s , t h e r e f o r e ,  concur  with  t h e o b s e r v a t i o n o f Waisman e t al_. (1981) t h a t the i n h i b i t i o n o f c a l c i u m transport  in inside-out v e s i c l e s after  a n i o n channel b l o c k e r (these o f anion t r a n s p o r t  p r e i n c u b a t i o n o f c e l l s w i t h an  a u t h o r s used SITS) i s a r e s u l t o f i n h i b i t i o n  through band 3 .  T h e r e f o r e , w h i l e net uptake o f c a l c i u m  can be i n h i b i t e d by b l o c k i n g band 3 (Waisman et a l . 1 9 8 1 ) , the situation,  converse  i n which the net uptake o f anions may be reduced due t o b l o c k -  ade o f net C a - u p t a k e by the i n h i b i t i o n o f the 2 +  Ca -transport 2 +  ATPase, must a l s o be c o n s i d e r e d . In c o n c l u s i o n , the present i n h i b i t s the ( C a  2 +  results  + Mg )-ATPase 2+  show t h a t N A P - t a u r i n e  activity  o f red c e l l  selectively  membranes.  The  o u a b a i n - s e n s i t i v e ( N a , K ) - A T P a s e and M g - A T P a s e are i n h i b i t e d o n l y at +  +  much h i g h e r c o n c e n t r a t i o n s .  2+  The  i n h i b i t i o n of ( C a  2 +  + Mg )-ATPase 2+  membranes by N A P - t a u r i n e appears to be independent o f anion channel b i t i o n , as determined by a number of c r i t e r i a .  The f a c t t h a t  at c o n c e n t r a t i o n s between 25-50 uM i n h i b i t e d a pure ( C a preparation provides effect  further  bit  NAP-taurine 2+  inhibitory  available inhibitors  l a n t h a n u m , ruthenium red)  the c a l m o d u l i n a c t i v a t i o n o f the  et a l . 1 9 8 1 ) ) .  Previously  inhi-  + Mg )-ATPase  compelling evidence for a d i r e c t  o f N A P - t a u r i n e on the enzyme.  either nonspecific (e.g.  2 +  of  or predominantly  pump ( e . g .  R 24571  are  inhi-  (Gietzen  The i n h i b i t i o n produced by N A P - t a u r i n e c o u l d not be p r o -  '  E f f e c t o f N A P - t a u r i n e on p u r i f i e d C . C a  2+ P u r i f i e d (Ca  2+  +Wg )-ATPa?e 2+  2+ + Mg  )-ATPase a c t i v i t y  i n t h e absence o r  o f N A P - t a u r i n e 1n the a s s a y medium was measured i n t h e d a r k ,  presence  1,8 yg o f  enzyme p r o t e i n was i n c u b a t e d i n a -medium c o n t a i n i n g 55 mM T r i s - m a l e a t e , 66 mM N a C l , 0.1 mM o u a b a i n , 6.5 mM t l g C l » 2  10 yM f r e e c a l c i u m , 2 mM ATP,  w i t h and w i t h o u t N A P - t a u r i n e , f o r 30 min a t 3 7 ° C . P u r i f i e d enzyme was k i n d l y s u p p l i e d by Dr. J . T . P e n n i s t a n , Mayo C l i n i c , R o c h e s t e r . 2+  Additions  2+ + Mg )-ATPase a c t i v i t y -1 -1 (ymoles P* mg min )  (Ca  Control  4.7  + 25 yM N A P - t a u r i n e  2,0  + 50 yM N A P - t a u r i n e  1,0  Time (min) Figure 22.  Calcium uptake i n i n s i d e - o u t v e s i c l e s made from red blood c e l l s t r e a t e d w i t h DIDS (5 yM) a t 37°C f o r 30 m i n .  After  t r e a t m e n t c e l l s were washed and ghosts prepared by h e m o l y s i s . Fresh ghosts were used f o r the p r e p a r a t i o n o f i n s i d e - o u t v e 2+ sides.  Ca  - t r a n s p o r t was measured 1n v e s i c l e s from u n t r e a t e d  c e l l s i n the absence of ATP (•),  and i n the presence o f 2 mM  ATP i n v e s i c l e s from u n t r e a t e d c e l l s c e l l s t r e a t e d w i t h 5 yM DIDS ( o ) . of three s i m i l a r experiments.  (•)  and i n v e s i c l e s  from  The d a t a shown i s t y p i c a l  TOO  t e c t e d by p r e i n c u b a t i o n o f the enzyme w i t h C a  2 +  , M g , ATP o r c a l m o d u l i n . 2 +  Indeed, t r y p s i n t r e a t e d membranes which l o s e t h e i r  sensitivity  to s t i m u l a -  t i o n by c a l m o d u l i n , due p r o b a b l y to a l o s s o f the c a l m o d u l i n b i n d i n g comp o n e n t , were i n h i b i t e d by N A P - t a u r i n e at s i m i l a r c o n c e n t r a t i o n s to the non-trypsin the ( C a  treated preparations.  + Mg2 )-ATPase  2 +  s i t e o f a c t i o n o f N A P - t a u r i n e on  i s u n c l e a r at p r e s e n t , but i s p r o b a b l y an  +  allosteric  The  s i t e on the enzyme d i s t i n c t from the Ca " ", M g , ATP and 2  1  2 +  calmodulin s i t e s . The (Ca  2 +  f i n d i n g t h a t DIDS i n h i b i t e d C a  + Mg )-ATPase  activity  2+  (1981) t h a t  2 +  t r a n s p o r t without  supports the s u g g e s t i o n o f Waisman et a l .  net movement o f anions may occur d u r i n g C a  inside-out vesicles.  inhibiting  transport  2 +  However, the present r e s u l t s show t h a t  (25-50 yM) i n h i b i t s ( C a  2 +  + Mg )-ATPase 2+  in  NAP-taurine  by a d i r e c t a c t i o n on the enzyme,  r a t h e r than by the i n h i b i t i o n o f band 3 suggested by these a u t h o r s . t h e r e appear to be d i s t i n c t d i f f e r e n c e s net t r a n s p o r t results  of C a  2 +  ATPase a c t i v i t y  i n the mechanism o f i n h i b i t i o n of  and anions by DIDS and N A P - t a u r i n e .  indicate that NAP-taurine i n c a n i n e heart  b r a n e s , but not the C a  2 +  Preliminary  (25 yM) i n h i b i t s the ( C a  2 +  + Mg  2 +  -ATPase a c t i v i t y  A study o f the s t r u c t u r e  i n the p a n c r e a t i c a c i n a r  activity  (Ca  2 +  2+  i n h i b i t i o n by N A P - t a u r i n e .  2+  relationships is  N A P - t a u r i n e may be a general a variety of t i s s u e s .  These r e s u l t s  cells.  + Mg )-ATPase currently  under way to o b t a i n i n f o r m a t i o n on the m o l e c u l a r mechanisms o f ( C a Mg )-ATPase  )-  sarcolemma and s a r c o p l a s m i c r e t i c u l u m mem-  T a u r i n e (up to 55 mM) had no e f f e c t on the red c e l l activity.  Thus,  suggest  s e l e c t i v e a n t a g o n i s t o f the C a  2 +  2 +  +  that pump i n  General D i s c u s s i o n  The Ca cytes  2+  a f f i n i t y o f t h e Ca  2+  t r a n s p o r t ATPase i n i n t a c t  erythro-  2+ 2+ In i s o l a t e d membranes t h e (Ca + Mg )2+ 2+ can be i s o l a t e d i n a low Ca a f f i n i t y s t a t e , a h i g h Ca  in vivo is uncertain.  ATPase a c t i v i t y affinity  s t a t e or w i t h both components depending on t h e procedures u s e d .  A number o f f a c t o r s can i n f l u e n c e t h e r e l a t i v e d i s t r i b u t i o n o f t h e two states.  C a l m o d u l i n was shown to i n c r e a s e both the apparent  the Vmax. o f t h e Ca  2+  transport  ATPase. 2+  a n i o n i c n a t u r e o f c a l m o d u l i n and Ca  1^2+ and  I t was suggested t h a t both the  induced exposure o f  hydrophobic  r e s i d u e s may be i m p o r t a n t i n r e g u l a t i o n o f the enzyme by c a l m o d u l i n . The c a r b o x y l groups o f c a l m o d u l i n are i n v o l v e d not o n l y the b i n d i n g o f 2+ 2+ Ca a t the 4 s p e c i f i c Ca b i n d i n g s i t e s but i t i s c o n c e i v a b l e t h a t t h e y 2+ a l s o make t h e b i n d i n g o f Ca no reason a p r i o r i  at these s i t e s e a s i e r .  Although t h e r e  is  to b e l i e v e t h a t the a n i o n i c nature of calmodulin i s  i m p o r t a n t f o r o t h e r a s p e c t s o f c a l m o d u l i n ' s f u n c t i o n s , the s t u d i e s w i t h poly-L-carboxylic  a c i d s suggest t h a t the a n i o n i c n a t u r e o f these compounds  may be i m p o r t a n t i n m e d i a t i n g changes i n t h e K^ 2+ o f the ATPase. fl  Because p o l y - L - c a r b o x y l i c  acids, s u l f o n i c acid buffers  and Na-MES) and a r o m a t i c c a r b o x y l i c 1(^2+ o f the ATPase,  a c i d produce s i m i l a r e f f e c t s on the  i n s p i t e of t h e i r very d i f f e r e n t  hence p h y s i c o - c h e m i c a l p r o p e r t i e s ) ,  (Na-HEPES  structures  (and  alternative explanations for  their  2+ e f f e c t s on t h e Ca  transport  ATPase must a l s o be c o n s i d e r e d .  s t a n c e , t h e s e agents may i n c r e a s e the l o c a l  For i n 2+  c o n c e n t r a t i o n o f f r e e Ca  at  2+ the Ca b i n d i n g s i t e on t h e enzyme o r enhance a p o s s i b l e r a t e l i m i t i n g 2+ 2+ d i f f u s i o n o f Ca t o t h e a c t i v e s i t e ( o r an a c c e s s o r y Ca binding site)  2+ on t h e Ca  transport  ATPase.  Furthermore,  at the pH o f a s s a y , the  HEPES and MES c o n t a i n a m i x t u r e o f z w i t t e r i o n s i o n s which v a r y due to the d i f f e r e n c e s zwitterionic  and s i n g l y charged  i n t h e i r pK ' s . a  on t h e ATPase.  w i t h the ATPase i s done, i t  Until  negative  Thus, the  form o f t h e s e compounds may a l s o c o n t r i b u t e t o  observed e f f e c t s  buffers  their  b i n d i n g s t u d i e s o f t h e s e agents  i s not p o s s i b l e t o d e f i n e the s i t e  (regulatory  component)  or the p r e c i s e mechanism o f a c t i o n o f t h e s e compounds 2+ Although t h e mechanism o f r e g u l a t i o n o f the Ca pump o f human e r h t h r o c y t e membrane i s b e g i n n i n g t o emerge, i t i s f a r from c l a r i f i e d . 2+ For i n s t a n c e , 1t i s not e x a c t l y c l e a r at present whether the Ca pump i s e l e c t r o g e n i c o r o b l i g a t o r i l y compensated by c o - o r c o u n t e r t r a n s p o r t 2+ of o t h e r i o n .  Waisman et a l (1981) p o s t u l a t e d t h a t t h e Ca  pump i s  e l e c t r o g e n i c , r e s u l t i n g i n c r e a t i o n o f a p o s i t i v e membrane p o t e n t i a l i n s i d e the 1 . 0 . v e s i c l e s which serves uptake.  as t h e d r i v i n g  force of  S i n c e t h i s process i s i n h i b i t e d by the anion channel  anion blocker  N A P - t a u r i n e o r by p r i o r treatment o f c e l l s w i t h SITS, they suggested 2+ t h a t t h e s e compounds blocked Ca t r a n s p o r t by b l o c k i n g t h e o b l i g a t o r y movement o f anions through the a n i o n c h a n n e l . However, t h e r e s u l t s shown 2+ i n t h i s t h e s i s i n d i c a t e t h a t N A P - t a u r i n e b l o c k e d Ca t r a n s p o r t by a 2+ 2+ d i r e c t a c t i o n on t h e Ca t r a n s p o r t ATPase. The i n h i b i t i o n o f Ca transport  o b t a i n e d w i t h DIDS t r e a t e d c e l l s ,  however, suggests t h a t  this  i n h i b i t i o n was p r o b a b l y mediated t h r o u g h b l o c k a d e o f t h e anion c h a n n e l . However, an a l t e r n a t i v e  e x p l a n a t i o n i s a l s o worth c o n s i d e r i n g .  example, DIDS may have r e a c t e d w i t h some amino groups on t h e  For external  2+ membrane s u r f a c e i n some u n s p e c i f i e d way i n h i b i t i n g Ca  transport  without  2+ i n h i b i t i n g Ca  transport  ATPase a c t i v i t y .  R e c e n t l y , N i g g l i , V . , S i g e l , E.  and C a r a f o l i , E. ( J .  Biol.  Chem. 2 5 7 , 2350-2356, 1982) have shown t h a t  t h e c a l c i u m pump c a t a l y s e s an e l e c t r o n e u t r a l exchange o f Ca It  2+  + - 2H .  i s hoped t h a t f u t u r e work i n t h i s and r e l a t e d areas w i l l c l a r i f y 2+  fundamental mechanism(s) o f the Ca e r y t h r o c y t e membrane.  t r a n s p o r t ATPase o f t h e human  the  CONCLUSIONS  1)  2+ 2+ The (Ca + Mg )-ATPase a c t i v i t y  fn e r y t h r o c y t e  membranes o r  T r i t o n X - 1 0 0 s o l u b i l i z e d enzyme p r e p a r a t i o n showed b i p h a s i c (.high and low a f f i n i t y )  2)  2+ Ca activation kinetics,  C a l m o d u l i n i n c r e a s e d both t h e c a l c i u m s e n s i t i v i t y and t h e maximum velocity of (Ca  3)  2 +  + Mg )-ATPase. 2+  Certain poly-L-carboxyl i c acids glutamic a c i d ) , a l i c y c l i c carboxylic acids  ( . p o l y - L - a s p a r t i c a c t d and p o l y - L -  s u l f o n i c a c i d s (HEPES, MES) and a r o m a t i c  ( b e n z o i c a c i d and s a l i c y l i c a c t d ) i n c r e a s e d t h e  2+ 2+ c a l c i u m s e n s i t i v i t y but not the maximum v e l o c i t y o f t h e (Ca + flg ) -ATPase i n e r y t h r o c y t e  membranes and T r i t o n X-100 s o l u b i l i z e d p r e p a 2+  rations. 2+ (Ca  I t i s suggested t h a t m o d u l a t i o n o f t h e Ca  sensitivity of  2+ + Mg  )-ATPase  by c a l m o d u l i n 1s d u e , a t l e a s t i n p a r t t o i t s  a n i o n i c p r o p e r t i e s (low I s o e l e c t r i c pH) and t h a t t h i s p r o p e r t y can be mimicked by some o t h e r a n i o n s , p r o b a b l y by t h e i r i n t e r a c t i o n a t an a n i o n - r e g u l a t o r y s i t e on t h e enzyme. 2+ 2+ 4)  C y c l i c AMP (5 yM) i n h i b i t e d (Ca 20%) i n e r y t h r o c y t e  + Mg  )-ATPase a c t i v i t y  (approx.  membranes and t h i s e f f e c t c o u l d be b l o c k e d by  c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r from the r a b b i t s k e l e t a l m u s c l e . 2+ 2+ I t i s suggested t h a t c y c l i c AMP r e g u l a t e s  (Ca  + Mg  a c t i v i t y v i a an endogenous c y c l i c AMP p r o t e i n k i n a s e .  )-ATPase  5)  B o v i n e h e a r t c y c l i c AMP p r o t e i n k i n a s e i n h i b i t o r * on t h e o t h e r hand, stimulated ( C a Ca  2 +  + Tlg )-ATPase a c t i v i t y  2 +  2+  s e n s i t i v i t y o f t h e enzyme,  (."100.%) by i n c r e a s i n g the  Stimulation of ( C a  2 +  + Mg )-ATPase 2+  a c t i v i t y by bovine h e a r t PKI (.approx, 100%) o c c u r r e d i n t h e presence o r absence o f c y c l i c AMP and tinder c o n d i t i o n s (2 mM ATP) where c y c l i c AMP had no e f f e c t .  The s u g g e s t i o n t h a t the s t i m u l a t i o n was a d i r e c t  2+ e f f e c t on the Ca  t r a n s p o r t ATPase was supported by a r g l n y l  modification studies.  It  group  1s suggested t h a t d i f f e r e n t mechanisms a r e  i n v o l v e d i n the i n h i b i t i o n o f c y c l i c AMP p r o t e i n k i n a s e and s t i m u l a 2+ t i o n o f (Ca  6)  2+ + Mg )-ATPase a c t i v i t i e s by bovine h e a r t c y c l i c AMP PKI  In agreement w i t h t h e s u g g e s t i o n o f Waisman et_ al_ (.1981) and Gimble et al_(1981) i t was found t h a t t h e a n i o n channel b l o c k e r  4,4'-diiso-  t h i o c y a n o - 2 , 2 ' s t i l b e n e d i s u l f o n a t e (DIDS) (5 yM) I n h i b i t e d Ca transport  in inside-out v e s i c l e s .  o f DIDS on the e l e c t r o g e n i c Ca  2+  It  2+  i s concluded that the e f f e c t  pump o c c u r s v i a i t s i n h i b i t i o n o f 2+ 2+  band 3 , as DIDS (5 yM) had no e f f e c t on (Ca  + Mg  )-ATPase i n  frag-  mented membranes. 7)  N - ( 4 - a z i d o - 2 - n i t r o p h e n y l ) - 2 aminoethyl s u l f o n a t e (a p h o t o l a b e l i n g 2+ reagent),  was found t o i n h i b i t d i r e c t l y t h e Ca  out v e s i c l e s .  -transport  in  inside-  D e s p i t e i t s known e f f e c t s as an a n i o n channel b l o c k e r ,  under t h e c o n d i t i o n s o f t h i s s t u d y , N A P - t a u r i n e (50 yM) was found t o 2+ 2+ 2+ i n h i b i t d i r e c t l y (Ca + Mg )-ATPase a c t i v i t y . Mg -ATPase and (Na  +  + K ) - A T P a s e a c t i v i t i e s were not i n h i b i t e d a t t h e s e c o n c e n t r a +  t i o n s of NAP-taurine.  T 06 2+ N A P - t a u r i n e i n h i b i t s t h e (Ca  2+ t Mg  )-ATPa?e a c t i v i t y  from t h e  cyto-  p l a s m i c s i d e o f the membrane and the i n h i b i t i o n c o u l d not be p r o t e c t e d 2+ by Ca  2+ , Mg  , ATP o r c a l m o d u l i n .  It  i s c o n c l u d e d t h a t a) t h e  inhlbi-  2+ t i o n o f Ca t r a n s p o r t 1n 1ns1de-out v e s i c l e s by N A P - t a u r i n e i s not v i a I n h i b i t i o n o f t h e a n i o n c h a n n e l , as proposed by Walsman et a l (1981);  and b) NAP-taur1ne c o u l d be used as s e l e c t i v e 2+  o f the Ca  -pump ATPase.  antagonist  BIBLIOGRAPHY  107  Akyempon, C. and R o u f o g a l i s , B . D . 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Med. 8 8 , 5 5 5 - 5 6 2 .  APPENDIX  A n a l y s i s o f V a r i a n c e on Data i n F i g u r e 6  A t y p i c a l example o f t h e a n a l y s i s o f v a r i a n c e o f the d a t a i s shown f o r 0.58 yM f r e e  Ca  2 +  .  Control 86  280  450  K = 3  95  300  447  n  105  310  418  I  110  305  435  S  "A"" V S  Calmodulin  HEPES  4  9  V  n  B  X  9  S  114  B  =  n  4  = 298  V  173  c  =  T  = 12 = 278  2  = 166  4  X , = 437 S = 211 2  C  where n^, n , n^ r e p r e s e n t the number o f r e p l i c a t i o n s f o r t r e a t m e n t ;  in  g  t h i s case they are each f o u r .  The t o t a l sample (12) i s denoted by n-p  The mean o f the t h r e e t r e a t m e n t means i s r e p r e s e n t e d by X;  also called  the grand mean. The v a r i a n c e w i t h i n each t r e a t m e n t group has been computed by d i v i d i n g the sum o f squared d e v i a t i o n s from each t r e a t m e n t mean by (n^ - 1 ) , ( n  g  - 1) and n  c  - 1), respectively.  S i n c e , the t r e a t m e n t  n-  numbers are a l l the same ( 4 ) , t h e mean o f the group v a r i a n c e i s c a l c u l a t e d s i m p l y as the mean o f 1 1 4 , 1 7 2 . 9 and 2 1 1 , which i s 166.  This i s  repre-  sented by S  and i s c a l l e d w i t h i n t h e group o r e r r o r v a r i a n c e .  number o f t r e a t m e n t groups 1s denoted by the symbol Since within-groups  variance  The  i n t h i s case 3 .  (S ) i s 166, t h i s becomes an e s t i m a t e  2 o f o , which i s the random v a r i a t i o n  in the p o p u l a t i o n .  Now, the sum o f squared d e v i a t i o n s squares"  (SS)  SS = (99 - 2 7 8 )  H  Q  a n d  a  n  S  _  2  I  from t h e grand mean or "sum o f  SS  _  " -RTT -  + (299 - 2 7 8 )  2  2  + (437 - 2 7 8 )  2  SS T  = 28,861  Therefore, 2 ^2  =  n  ^X  = 4 x 28,861 = 115,444.  2 2 T h e r e f o r e , S which i s an e s t i m a t e o f a i s o b t a i n e d from t h e  variability  among the t r e a t m e n t means. The p r i n c i p l e o f v a r i a n c e s t a t e s t h a t , any v a r i a b i l i t y  among t h e  2 t r e a t m e n t means c o n s i s t o f an e s t i m a t e o f a t h a t i s due t o and o r i g i n a t i n g effects ie.  F =  a + K a 2  p l u s any e x t r a  from d i f f e r e n c e s  produced by  variality treatment  where, K i s t h e v a r i a b i l i t y i n the t r e a t m e n t means beyond t h a t which i s expected as an e s t i m a t e o f a  h  (2,9)  2  alone.  115,444 166  ~  = 695 ( > 8 . 0 2 a t 0.01 l e v e l o f  significance)  The s u b s c r i p t s , 2 and 9 r e p r e s e n t t h e t r e a t m e n t and w i t h i n group degrees o f freedom. Thus F i s s i g n i f i c a n t beyond 0.01 l e v e l .  The Student-Neumann-Kuels T e s t The h i g h l y s i g n i f i c a n t F - v a l u e , tical  significance exists  and p o s s i b l y b o t h .  (695, P < 0.001), indicates s t a t i s -  between a t l e a s t one p a i r o f t r e a t m e n t means  We can now compare t h e v a r i o u s mean p a i r s t o see  which are s t a t i s t i c a l l y d i f f e r e n t  from w h i c h .  F i r s t , we can c a l c u l a t e an e s t i m a t e o f t h e s t a n d a r d e r r o r mean as an e s t i m a t e o f a S X  2  n  166 4  2  = 6.44  Next we f i n d the e r r o r degrees o f freedom a s s o c i a t e d w i t h analysis.  q  (0.01  qS  Y  level)  of  4.6  5.43  29.62  34.97  the  the  The top row c o n t a i n s K v a l u e s r e l e v a n t t o our e x p e r i m e n t .  For  example, K = 2 i s a s s o c i a t e d w i t h any two means t h a t are a d j a c e n t , K = 3 refers means.  to comparison o f two means t h a t are a t extremes o f  qSy i s the minimum d i f f e r e n c e t h a t must be found between  means a s s o c i a t e d w i t h a s p e c i f i c v a l u e  three two  K.  Thus,  X  I  A  X  c  - X  A  = 338 (>34.97)*  X  B  - X  A  = 199 (>29.62)*  X  c  - X  B  = 139 (>29.62)*  B 11  X  X  ,  C l  Thus, t h e r e s u l t s suggest t h a t HEPES and c a l m o d u l i n a r e somehow a s s o c i a t e d w i t h an i n c r e a s e i n t h e enzyme a c t i v i t y F v a l u e s s i g n i f i c a n t a t 0.01 l e v e l free C a  2 +  2+ a t 0.58 yM f r e e Ca . 2+  up t o 25 yM f r e e Ca  .  Beyond 100 yM  , no s t a t i s t i c a l s i g n i f i c a n c e e x i s t s between c o n t r o l  However, s t a t i s t i c a l s i g n i f i c a n c e e x i s t s membrances a t a l l v a l u e s o f f r e e c a l c i u m .  and HEPES.  between c o n t r o l c a l m o d u l i n  

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