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The relationship between Mg+Ca-AtPase and active calcium transport in researled human erythrocyte ghosts Quist, E. E. (Eugene Edwin) 1973

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THE RELATIONSHIP BETWEEN Mg+Ca-ATPase  AND  ACTIVE CALCIUM TRANSPORT IN RESEALED HUMAN ERYTHROCYTE GHOSTS by E . E. QUIST  A t h e s i s submitted i n p a r t i a l f u l f i l l m e n t o f the requirements f o r the degree o f MASTER OP SCIENCE In the D i v i s i o n o f P h a r m a c e u t i c a l Chemistry of the F a c u l t y  of P h a r m a c e u t i c a l S c i e n c e s  We a c c e p t t h i s t h e s i s as conforming required  t o the  standard  THE UNIVERSITY OF BRITISH COLUMBIA September  1973  In p r e s e n t i n g t h i s t h e s i s  in partial  f u l f i l m e n t of  an  University  of B r i t i s h  advanced degree at  the  Library  I further for  the  s h a l l make i t f r e e l y a v a i l a b l e  agree that  his  of  this thesis  written  representatives.  be  g r a n t e d by  Columbia, I agree  for reference  for f i n a n c i a l gain  permission.  The U n i v e r s i t y o f B r i t i s h Vancouver 8 , Canada  the  Head o f my  It i s understood that  Department  Date  requirements  and  permission f o r extensive copying of  s c h o l a r l y p u r p o s e s may  by  the  Columbia  shall  not  be  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  THE RELATIONSHIP BETWEEN Mg+Ca-ATPase AND ACTIVE CALCIUM TRANSPORT IN RESEALED HUMAN ERYTHROCYTE GHOSTS  by E.E. q u i S T  ABSTRACT  Human r e d b l o o d c e l l g h o s t s were p r e p a r e d of the procedure o f stepwise hemolysis  (57)•  by a m o d i f i c a t i o n EDTA ( 1 . 0 mM) was  i n c l u d e d i n t h e washing p r o c e d u r e t o remove endogenous ATP and divalent cations. ATP,  G h o s t s r e s e a l e d w i t h a p p r o p r i a t e amounts o f  c a l c i u m and magnesium were f o u n d t o have Mg+Ca-ATPase a c t -  i v i t y and l i n e a r i t y was m a i n t a i n e d  up t o t h i r t y m i n u t e s .  Active  c a l c i u m t r a n s p o r t c o u l d be s t u d i e d i n t h e s e g h o s t s by m e a s u r i n g t h e change l n 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 c a l c i u m o v e r time by a t o m i c a b s o r p t i o n  spectrophotometry.  V a r i a t i o n i n the concentration o f calcium i n the loading medium r e s u l t e d i n a n a c t i v a t i o n o f Mg+Ca-ATPase and two peaks were e v i d e n t on t h e a c t i v a t i o n curve.The h i g h and l o w a f f i n i t y Mg+Ca-ATPase were m a x i m a l l y s t i m u l a t e d a t 0 . 2 5 and 5«0 mM c a l c i u m l n the loading  medium,respectively.  The v e l o c i t y o f c a l c i u m t r a n s p o r t was a l s o found t o be dependent on 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 i n t h e l o a d i n g medium  ii and was a c t i v a t e d over t h e c o n c e n t r a t i o n range o f 0 . 1 t o 5«0 mM c a l c i u m .  A change i n t h e c o n c e n t r a t i o n o f c e l l u l a r  was n o t e v i d e n t i n t h e absence o f added ATP.  calcium  In contrast to  t h e a c t i v a t i o n o f Mg+Ca-ATPase two peaks were n o t o b t a i n e d , and t h e a c t i v a t i o n curve had a s i g m o i d a l appearance. Comparison o f t h e c a l c i u m a c t i v a t i o n c u r v e s o f Mg+Ca-ATPase and c a l c i u m t r a n s p o r t r e v e a l e d , a s i m i l a r i t y i n t h e shape and p o s i t i o n o f t h e low a f f i n i t y p a r t o f t h e Kg+Ca-ATPase and c a l c i u m transport a c t i v a t i o n curves.  A s t o i c h i o m e t r y o f two (Ca :ATP)  was obtained, i n t h e low a f f i n i t y a c t i v i t y r a n g e . Ruthenium r e d ( 0 . 0 5 t o 0.4 mM) s e l e c t i v e l y I n h i b i t e d t h e low a f f i n i t y Mg+Ca-ATPase and i n h i b i t e d c a l c i u m t r a n s p o r t o v e r the same c o n c e n t r a t i o n r a n g e t o a s i m i l a r d e g r e e .  Both low  a f f i n i t y Mg+Ca-ATPase and c a l c i u m t r a n s p o r t were I n h i b i t e d by e x t e r n a l r u t h e n i u m r e d w i t h an I ^ o f 0 . 2 0  mM.  P r o p r a n o l o l , q u l n i d i n e and q u i n i n e (10~^ t o 10~^M) were f o u n d t o be I n e f f e c t i v e i n s t i m u l a t i n g o r I n h i b i t i n g Mg+Ca-ATPase when added t o t h e I n t e r n a l and e x t e r n a l a s p e c t s o f t h e g h o s t s . Manganese, added t o t h e l o a d i n g medium o v e r a wide  concent-  r a t i o n r a n g e , was u n a b l e t o s u b s t i t u t e f o r c a l c i u m i n a c t i v a t i n g Mg+Ca-ATPase. E x t e r n a l d i v a l e n t c a t i o n s c a l c i u m and magnesium f u r t h e r i n c r eased Mg+Ca-ATPase a c t i v i t i e s when added t o t h e e x t e r n a l medium. Maximal s t i m u l a t i o n o c c u r r e d a t a c o n c e n t r a t i o n o f a p p r o x i m a t e l y 3 . 0 mM and c a l c i u m was a l m o s t t w i c e a s e f f e c t i v e as magnesium.  S i g n a t u r e s of Examiners  Iv TABLE OF CONTENTS Page ABSTRACT  i  LIST OF TABLES  vi  LIST OF FIGURES  v i i  INTRODUCTION  1  LITERATURE REVIEW  3  Homeostatlc  3  Mechanisms C o n t r o l l i n g The  I n t r a c e l l u l a r C o n c e n t r a t i o n Of C a l c i u m A c t i v e T r a n s p o r t I n Human Red B l o o d C e l l s  6  Mg+Ca-ATPase A c t i v i t y I n Human Red B l o o d C e l l  14  Membrane Fragments I n h i b i t o r s E f f e c t i n g . Mg+Ga-ATPase And A c t i v e C a  2 +  19  T r a n s p o r t I n Red B l o o d C e l l s A c t i v e Calcium Transport I n Other Tissues METHODS AND MATERIALS  21 25  P r e p a r a t i o n Of Red B l o o d C e l l G h o s t s  25  Loading Procedure  28  R e s e a l i n g And Washing P r o c e d u r e  28  I n c u b a t i o n Procedures  29  D e t e r m i n a t i o n Of ATPase A c t i v i t y  29  D e t e r m i n a t i o n Of The V e l o c i t y Of C a l c i u m  31  Transport P r o t e i n Assay  32  Washing P r o c e d u r e  33  RESULTS AND DISCUSSION P r o p e r t i e s Of The Red B l o o d C e l l Ghost Preparation  34 34  Page Ion Requirements F o r R e s e a l i n g  36  Na.K-ATPase A c t i v i t y I n Resealed Red Blood  37  Cell  Ghosts  Mg+Ca-ATPase A c t i v i t y I n Resealed Red Blood Cell  4l  Ghosts  The E f f e c t of E x t e r n a l D i v a l e n t C a t i o n s On  43  ATPase A c t i v i t y The E f f e c t Of I n t e r n a l C a l c i u m On C a l c i u m E f f l u x  49  The E f f e c t Of Ruthenium Red On Mg+Ca-ATPase  57  Activities The E f f e c t Of Ruthenium Red On Calcium T r a n s p o r t  6l  The E f f e c t Of Drugs (Quinine, Q u i n i d i n e , and  66  P r o p r a n o l o l ) On Mg+Ca-ATPase The E f f e c t Of Manganese On Mg+Ca-ATPase A c t i v i t y  67  CONCLUSIONS  69  BlBLIOGRA PHY  73  APPENDIX  80  LIST OF TABLES Table I  Ion Antagonisms.  II  E f f e c t s Of Ruthenium Red On ATPases Of E r y t h r o c y t e Membranes.  I I I F e a t u r e s Of The Na-Ca Exchange Mechanism In Cyanide Poisoned Squid Axons. IV  P r e p a r a t i o n Of RBC Ghosts, Loading And Assay Procedure.  vll  LIST OF FIGURES Figure 1.  Page Changes i n Ca c o n c e n t r a t i o n i n c e l l s and  9  medium and P i r e l e a s e from ATP i n r e s e a l e d cells. 2.  E f f e c t of preincubation with iodoacetate.  12  3.  The s t i m 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 by  13  internal  calcium.  2+ 4.  (Ca )-dependent ATPase a c t i v i t y i n human red  5.  15  cells.  ATPase a c t i v i t y a s a f u n c t i o n o f C a *  +  18  c o n c e n t r a t i o n l n t h e p r e s e n c e and absence o f 80mM K . +  6.  E f f e c t o f N a and K +  +  on ATPase a c t i v i t y l n  h e m o g l o b i n - f r e e r e d c e l l membranes,  18  prepared  by f r e e z i n g and t h a w i n g . 7.  Time c o u r s e o f ATPase a c t i v i t y i n t h e absence  38  of e x t e r n a l o u a b a i n and i n t h e p r e s e n c e o f 0.2mM e x t e r n a l o u a b a i n . 8.  A c t i v a t i o n o f Na,K-ATPase a c t i v i t y by e x t e r n a l p o t a s s i u m l n t h e p r e s e n c e o f l.OmM e x t e r n a l c a l c i u m , z e r o e x t e r n a l c a l c i u m and O.lmM e x t e r n a l ouabain.  40  viil Figure 9.  Page E f f e c t o f v a r y i n g the c o n c e n t r a t i o n o f  42  c a l c i u m l n tne l o a d i n g medium on Mg+Ca-ATPase a c t i v i t y . 10.  Eadie p l o t of c a l c i u m a c t i v a t i o n of  44  Mg+Ca-ATPases i n g h o s t s . 11.  The e f f e c t of e x t e r n a l d i v a l e n t c a t i o n s on  46  ATPase a c t i v i t y . 12.  E f f e c t o f v a r y i n g the c o n c e n t r a t i o n o f  48  c a l c i u m i n t h e l o a d i n g medium on Mg+Ca-ATPase a c t i v i t y i n the absence o f e x t e r n a l c a l c i u m and 13.  l n the presence of l.OmM e x t e r n a l c a l c i u m .  Changes i n the c o n c e n t r a t i o n o f c e l l u l a r  50  calcium with time. 14.  R e l a t i o n s h i p between c e l l u l a r c a l c i u m and  52  c a l c i u m l n the l o a d i n g medium. 15.  Comparison o f the v e l o c i t y of a c t i v e c a l c i u m  54  t r a n s p o r t and Mg+Ca-ATPase a c t i v i t y as a f u n c t i o n o f c a l c i u m i n the l o a d i n g medium. 16.  H i l l p l o t o f c a l c i u m a c t i v a t i o n of c a l c i u m e f f l u x l n ghosts.  56  17.  E f f e c t of ruthenium red l n the external medium on Mg+Ca-ATPase a c t i v i t y .  18.  The e f f e c t of ruthenium red on the a c t i v a t i o n of Mg+Ca-ATPase.  19.  E f f e c t of ruthenium red i n the external medium on the v e l o c i t y of active calcium transport i n ghosts loaded with 3.0mM calcium.  20.  The e f f e c t of ruthenium red on the v e l o c i t y of a c t i v e calcium  21.  transport.  The e f f e c t of ruthenium red (0.2mM) i n the external medium on Mg+Ca-ATPase a c t i v i t y and active calcium transport as a function of the concentration of calcium l n the loading medium.  22.  E f f e c t of varying the concentration of manganese i n the loading medium on ATPases a c t i v i t y i n the absence of external  calcium  and l n the presence of l.OmM external  calcium.  X  ACKNOWLEDGEMENTS  I wish t o express my g r a t i t u d e t o Dr. B.D. R o u f o g a l i s f o r h i s e x c e l l e n t guidance and a s s i s t a n c e throughout the course of t h i s work and t o Dr. D. Godin f o r h i s h e l p f u l suggestions l n the e a r l y stages of t h i s study.  DEDICATION  To L a u r i e and J u l i a n  1  INTRODUCTION  Human e r y t h r o c y t e s m a i n t a i n a l o w i n t r a c e l l u l a r r a t i o n o f c a l c i u m by means o f a n a c t i v e c a l c i u m system r e q u i r i n g ATP ( 1 ) .  concent-  transport  Schatzmann p r o p o s e d 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 was a s s o c i a t e d w i t h Mg+Ca-rATPase, a n a l o g o u s t o t h e Na, K pump system ( 1 1 ) .  Some s i m i l a r i t i e s between t h e  p r o p e r t i e s o f Mg+Ca-ATPase i n r e d b l o o d and  c e l l membrane f r a g m e n t s  c a l c i u m t r a n s p o r t l n r e s e a l e d g h o s t s have been r e p o r t e d a s  I n d i r e c t evidence supporting t h i s hypothesis  (12,26,30).  An  a s s o c i a t i o n between t h e s e systems was a l s o d e m o n s t r a t e d when b o t h a c t i v i t i e s were s t u d i e d s i m u l t a n e o u s l y  i n r e s e a l e d human  e r y t h r o c y t e ghosts loaded w i t h a s i n g l e c o n c e n t r a t i o n calcium  (30).  (ImM) o f  However, w i t h t h e f i n d i n g o f more t h a f e o n e  Mg+Ca-ATPase i n r e d b l o o d c e l l membrane f r a g m e n t s (32,33,34) i t became a p p a r e n t t h a t f u r t h e r d i r e c t e v i d e n c e showing an a s s o c i a t i o n between Mg+Ca-ATPase was r e q u i r e d . the present  Therefore, i n  t h e s i s t h e a s s o c i a t i o n o f Mg+Ca-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 has been r e i n v e s t i g a t e d i n t h e same 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 o v e r a wide r a n g e o f c a l c i u m concentrations. Ruthenium r e d w h i c h has been r e p o r t e d t o s e l e c t i v e l y Mg+Ca-ATPase a c t i v i t y i n r e d b l o o d  inhibit  c e l l membrane f r a g m e n t s (40)  was t e s t e d on r e s e a l e d g h o s t s t o d e t e r m i n e whether t h i s dye would a l s o I n h i b i t c a l c i u m t r a n s p o r t .  Ruthenium r e d was a l s o  2 used as a t o o l f o r i n v e s t i g a t i n g t h e a s s o c i a t i o n between t h e c a l c i u m t r a n s p o r t system and a Mg+Ca-ATPase. I t was c o n s i d e r e d important  t h a t t h i s t r a n s p o r t s y s t e m may be a  s i t e o f d r u g a c t i o n , p a r t i c u l a r l y f o r drugs i n v o l v e d  l n t h e m o b i l i z a t i o n o f c a l c i u m , because o f t h e m u l t i t u d e o f p h y s i o l o g i c a l e f f e c t s r e g u l a t e d by i n t e r n a l c a l c i u m .  Drugs o f  i n t e r e s t w h i c h were s t u d i e d I n c l u d e q u i n i n e , q u i n i d i n e , p r o p r a n o l o l and t e t r a c a i n e .  The r e d b l o o d  model membrane system l n t h i s s t u d y be o b t a i n e d  c e l l g h o s t was used a s a s i n c e r e d blood  c e l l s can  i n l a r g e q u a n t i t i e s and g h o s t s f r e e o f t h e i n t r a -  c e l l u l a r contents of h e m o l y s i s .  c a n be p r e p a r e d by t h e method o f r e v e r s a l  The r e d b l o o d c e l l has p r e v i o u s l y p r o v e n t o be  I n v a l u a b l e f o r s t u d y i n g t h e mechanism o f c a t i o n t r a n s p o r t and asymmetric o u a b a i n i n h i b i t i o n o f Na,K-ATPase.  Many s i m i l a r i t i e s  have been shown between mechanisms o p e r a t i n g on t h i s system and  t h o s e on t h e plasma membrane o f o t h e r t i s s u e s .  o  3  LITERATURE REVIEW  H o m e o s t a t l c Mechanisms C o n t r o l l i n g The I n t r a c e l l u l a r C o n c e n t r a t i o n Of C a l c i u m  The  homeostatlc  mechanisms by w h i c h c e l l s m a i n t a i n a low  I n t r a c e l l u l a r c o n c e n t r a t i o n o f c a l c i u m has been o f i n t e r e s t t o p h y s i o l o g i s t s f o r the past seventy  years.  I n mammalian c e l l s ,  the i n t r a c e l l u l a r c o n c e n t r a t i o n o f c a l c i u m has been -7 -5 t o be between 10  M and 10  estimated  M i n c o n t r a s t t o a calcium concent-  r a t i o n o f 10"*^M l n t h e e x t r a c e l l u l a r f l u i d  (1,2).  Maintenance o f  a low I n t r a c e l l u l a r c o n c e n t r a t i o n of c a l c i u m i s of v i t a l  import-  ance t o t h e s u r v i v a l o f t h e c e l l a s a n i n c r e a s e o v e r t h e normal l e v e l has a p r o f o u n d e f f e c t on c e l l u l a r and enzyme f u n c t i o n s . F o r i n s t a n c e , i n human e r y t h r o c y t e s i n t r a c e l l u l a r  concentrations  o f c a l c i u m g r e a t e r t h a n lO'^M i n h i b i t s Na,K-ATPase w h i c h c o n t r o l s t h e d i s t r i b u t i o n o f sodium and p o t a s s i u m a c r o s s t h e plasma membrane (3»4).  A number o f o t h e r enzymes i n h i b i t e d by c a l c i u m  a r e shown i n T a b l e I .  I t has a l s o been a c c e p t e d  that  intracell-  u l a r c a l c i u m i s a c o u p l i n g f a c t o r i n e x c i t a t i o n - c o n t r a c t i o n coupl i n g i n muscle c e l l s  (6).  I n human r e d b l o o d c e l l s ,  intracellular  c a l c i u m has been shown t o r e g u l a t e t h e p a s s i v e p e r m e a b i l i t y o f t h e plasma membrane t o N a  +  and K  +  ( 7 , 8 ) , t h e volume o f t h e c e l l  (9) and membrane d e f o r m a b l l i t y ( 1 0 ) .  4 Table I .  Enzyme  I o n Antagonisms  A c t i v a t i n g Ions  Methionine adenosyltransferase Pantothenate synthetase Pyruvate kinase 5-nucleotidase Arglnlnosuccinate synthetase Glutamine synthetase R i b o f l a v i n kinase Inorganic pyrophosphatase Phosphopyruvate hydratase M y o s i n ATPase Glycyl-leuclne dipeptidase  (5)  Inhibiting  Ions  Mg o r Mn + (K, NHit.Rb) Mg o r Mn  Ca o r Zn  Mg + (K.Rb.Cs) Mg Mg  Ca and Na o r L i Ca Ca o r Mn  Mg o r Mn Mg.Zn.Co o r Mn Mg  Ca Ca Ca o r Zn  Mg.Zn.Mn o r Gd  Ca o r S r  Ca o r Zn  Ca o r (K o r NH ) Zn o r Mn * k  Mg Ca  A number o f w e l l - e s t a b l i s h e d h o m e o s t a t l c mechanisms by which c e l l s c o n t r o l the d i s t r i b u t i o n of calcium across the plasma membrane have been summarized a s f o l l o w s : 1, P a s s i v e p e r m e a b i l i t y ; P l a s m a membranes i n t h e r e s t i n g s t a t e a r e r e l a t i v e l y Impermeable t o c a l c i u m under p h y s i o l o g i c a l conditions.  However a slow exchange o f c a l c i u m a c r o s s t h e  plasma membrane i s e v i d e n t ( 1 2 , 1 3 ) .  The d i s t r i b u t i o n o f c a l c i u m  a c r o s s t h e plasma membrane i s a l s o n o t a c c o r d i n g t o t h e e q u i l i b r i u m p o t e n t i a l a s c a l c u l a t e d by t h e N e r n s t E q u a t i o n  (ii).  In  r e d b l o o d c e l l s t h e r e l a t i v e l y impermeable n a t u r e o f t h e plasma membrane does n o t a c c o u n t f o r t h e l o w i n t r a c e l l u l a r  concentrat-  i o n o f c a l c i u m s i n c e c a l c i u m i s t a k e n up i n c e l l s d e p l e t e d o f  5 ATP  o r s t o r e d i n the c o l d ( 7 ) .  This observation i n d i c a t e s that  the d i s t r i b u t i o n of c a l c i u m i s maintained  only i n a metabol-  lcally active cell. 2. A c t i v e c a l c i u m t r a n s p o r t ; I n human e r y t h r o c y t e s He La c e l l s  (44) and L c e l l s  ( 4 5 ) , c a l c i u m has  been shown t o  be t r a n s p o r t e d a c r o s s t h e plasma membrane a g a i n s t an c h e m i c a l p o t e n t i a l by a mechanism r e q u i r i n g 3. Na-Ca C o u p l i n g ; I n n e r v e (14,15) and one  electro-  ATP. c a r d i a c muscle ( 4 7 ) •  i n t r a c e l l u l a r c a l c i u m i o n i s exchanged f o r two  ium Ions by a system n o t c o u p l e d  (12,13),  to three sod-  to a chemical r e a c t i o n .  4. I n t r a c e l l u l a r o r g a n e l l e s ; (a) S a r c o p l a s m i c  r e t i c u l u m - The  sarcoplasmic  i s a network o f t u b u l e s , v e s i c l e s , and c y s t e r n a e the m y o f i b r i l s ( 9 ) .  reticulum  surrounding  This s t r u c t u r e r a p i d l y accumulates calcium  by an a c t i v e t r a n s p o r t system c l o s e l y a s s o c i a t e d w i t h a magnesium dependent ATPase ( 5 4 ) .  These s t r u c t u r e s a r e e x t r e m e l y  t a n t f o r r e l a x i n g t h e c o n t r a c t u r e o f s k e l e t a l and  impor-  c a r d i a c muscle  cells. (b) M i t o c h o n d r i a - M i t o c h o n d r i a a c t i v e l y a c c u m u l a t e c a l c i u m by an a c t i v e c a l c i u m t r a n s p o r t s y s t e m r e q u i r i n g ATP. M i t o c h o n d r i a may  be i m p o r t a n t  i n m a i n t a i n i n g a low  intracellular  c o n c e n t r a t i o n o f c a l c i u m i n n e r v e o r i n muscle h a v i n g a p o o r l y developed sarcoplasmic r e t i c u l u m (16,17,64,69). 5. I n t r a c e l l u l a r b i n d i n g and bound t o a n i o n i c s i t e s  c h e l a t i o n ; Calcium  i s also  (such a a on p r o t e i n s ) and c h e l a t e d t o  m o l e c u l e s s u c h as c a r b o x y l l c a c i d s and n u c l e o t i d e s .  The  relative  i m p o r t a n c e o f c a l c i u m b i n d i n g t o t h e s e s i t e s i s n o t known.  6  A c t i v e T r a n s p o r t In Human Red Blood  Red for  blood c e l l s have proved  Cells  t o be a u s e f u l model system  s t u d y i n g a c t i v e t r a n s p o r t o f c a t i o n s and s o l u t e s .  These  c e l l s a r e a v a i l a b l e i n l a r g e q u a n t i t i e s l n a homogeneous form and  the plasma membranes can be I s o l a t e d by r e l a t i v e l y g e n t l e  means f o r enzyme s t u d i e s .  Perhaps f o r t h i s reason the coup-  l i n g o f a t r a n s p o r t p r o c e s s t o a chemical r e a c t i o n was  first  i d e n t i f i e d t o occur a c r o s s the plasma membrane o f t h e r e d blood  cell. l n 1939  Wilbrandt  (19)  was the f i r s t  t o demonstrate t h a t  the d i s t r i b u t i o n o f sodium and potassium  was r e l a t e d t o g l y -  colysis.  I n h i b i t i o n o f g l y c o l y s i s by i o d o a c e t a t e and sodium  f l u o r i d e l e d t o a l o s s o f i n t r a c e l l u l a r potassium i n the osmotic  r e s i s t a n c e of the c e l l .  and a change  T h i s study s t i m u l a t e d  o t h e r workers t o s p e c u l a t e t h a t ATP was d i r e c t l y i n v o l v e d i n the d i s t r i b u t i o n o f potassium,  as ATP i s s y n t h e s i z e d  by g l y c o l y s i s l n the human r e d blood c e l l . ATP  The importance o f  l n m a i n t a i n i n g the I n t r a c e l l u l a r l e v e l s o f potassium  verified (22). to  solely  was  l n experiments w i t h r e c o n s t i t u t e d o r r e s e a l e d ghosts  Red blood c e l l s may be p a r t i a l l y hemolyzed by exposure  hypotonic  l e s s than  s o l u t i o n s , u s u a l l y w i t h i o n i c s t r e n g t h s o f not  0.02 M (20,21).  The c e l l s become l e a k y due t o osmotic  shock and ATP and o t h e r i o n s may be i n t r o d u c e d i n t o the c e l l . With the a d d i t i o n o f a c o n c e n t r a t e d s a l t s o l u t i o n t o t h i s  7 h e m o l y s a t e , t h e l s o t o n l c i t y c a n be r e s t o r e d and t h e r e s t o r a t i o n of  low c a t i o n p e r m e a b i l i t y t h u s o b t a i n e d i s adequate Gardos (22)  y i n g a c t i v e c a t i o n movements.  f o r stud-  i n t r o d u c e d ATP  into  r e s e a l e d g h o s t s and d e m o n s t r a t e d  an a c c u m u l a t i o n o f p o t a s s i u m  by a system w h i c h r e q u i r e d ATP.  Schatzmann (23)  t h a t t h i s ATP dependent system was  demonstrated  i n h i b i t e d by o u a b a i n .  A  link  between p o t a s s i u m t r a n s p o r t and t h e membrane bound enzyme Na,K-ATPase was r e c o g n i z e d when Na, K-iVTPase was found i n c r a b n e r v e by Skou ( 2 4 ) . the  S i n c e t h e n , Na.KsATPase has been found l n  plasma membrane o f most c e l l s , i n c l u d i n g the human e r y t h r o -  c y t e (3).  I n r e d b l o o d c e l l s Na,K-ATPase and the a c t i v e  trans-  p o r t o f sodium and p o t a s s i u m were I n h i b i t e d by a p p r o x i m a t e l y 10~->gm m l "  1  o f o u b a l n (23).  The s t o i c h i o m e t r y and o t h e r p r o p -  e r t i e s o f Na, K-ATPase system have now  been w e l l r e v i e w e d  (2ff).  On t h e o t h e r hand, t h e f i n d i n g o f an energy dependent c a l c i u m t r a n s p o r t system has o n l y r e c e n t l y been d e m o n s t r a t e d c e l l s and t h e s t u d i e s have been r e l a t i v e l y few. Dunham and G l y n n (3)  i n 1961  l n red blood  Historically,  were t h e f i r s t t o r e p o r t t h e e x i s t -  ence o f a o u a b a i n i n s e n s i t i v e c a l c i u m a c t i v a t e d ATPase i n r e d b l o o d c e l l membrane f r a g m e n t s  (RBCMF).  T h i s enzyme w h i c h  was  found l n t h e s e s t u d i e s t o have a s p e c i f i c a c t i v i t y two t o t h r e e t i m e s as g r e a t as Na,K-iiTPase was a l s o f o u n d t o be magnesium dependent.  T h i s enzyme w i l l be r e f e r r e d t o h e n c e f o r t h as  Mg+Ca-ATPase.  Schatzmann (11)  r e a s o n e d t h a t Mg+Ca-ATPase  may  be a s s o c i a t e d w i t h a t r a n s p o r t system w h i c h m a i n t a i n s a low c o n c e n t r a t i o n of I n t r a c e l l u l a r c a l c i u m i n red blood c e l l s , ogous t o t h e Na,K-ATPase system.  anal-  Resealed ghosts loaded w i t h  8 ATP,  magnesium and  c a l c i u m were found to r a p i d l y l o s e  c a l c i u m when incubated  a t 37°C ( F i g . 1,Panel A)  loaded w i t h 1.0  mM  was  or 2.0  c a l c i u m , tiOfi of the c e l l u l a r  l o s t w i t h i n t e n minutes.  t h a t Mg+Ca-ATPase was comittant  In agreement w i t h the  ghosts  calcium  hypothesis  l i n k e d to t h i s t r a n s p o r t p r o c e s s , a con-  t o ATP  h y d r o l y s i s was  of the c a l c i u m t r a n s p o r t .  (11) found t h a t r e s e a l e d ghosts i n the absence of added ATP. incomplete  study,  the  Schatzmann  l o s t c a l c i u m a t a slow r a t e  T h i s r e s u l t was  removal of endogenous ATP  shown t o be v a l i d  evident during  In a p r e v i o u s  a t t r i b u t e d to  d u r i n g the hemolysis  cedure used l n the p r e p a r a t i o n of the was  In  i n c r e a s e i n the c o n c e n t r a t i o n of i n o r g a n i c phosphate  ( F i g . l . P a n a l C) due course  (12).  cellular  hosts.  tt  since c e l l s depleted  This  pro-  explanation  of energy by means  of a 1? hr i n c u b a t i o n i n g l u c o s e f r e e medium a t 37*C showed l o s s of c e l l u l a r c a l c i u m u n l e s s ATP medium ( F i g . 1,Panel  was  Included  mM  c a l c i u m i n t o an e x t e r n a l medium c o n t a i n i n g 1.0  (11) appears t o be due  system s i n c e the p r o c e s s  was  calcium ' l o s t mM  calcium.  t o an a c t i v e t r a n s p o r t  found to be dependent on ATP  and  against a concentration gradient.  However, the evidence was  i n the l o a d i n g  the l o s s o f c e l l u l a r c a l c i u m demonstrated by Schatzmann  l n r e s e a l e d ghosts  operated  no  B).  Ghosts r e s e a l e d i n the presence of 0.1  Therefore  an  not d i r e c t .  l i n k i n g c a l c i u m e f f l u x t o a Mg+Ca-ATPase  In r e s e a l e d g h o s t s  s t i m u l a t e d ATPase a c t i v i t y  (12).  only i n t r a c e l l u l a r  T h i s asymmetrical  calcium  stimulation  of Mg+Ca-ATPase a c t i v i t y by i n t e r n a l c a l c i u m Is analogous to the Na,K-ATPase t r a n s p o r t system where o n l y e x t e r n a l potassium  1.5  30  60  Time (min) Fig. 1 Changes of Ca concentration in cells and medium and P, release from A T P in re.-sealed cells. Haemolysis in water containing 2 mM-Tris-ATP, 5 nwt-Tris-Cl, 4 mM-MgCl, and 1 or 2 mM (in three expts. in tho A T P free sample) CaCI,. Reversal "f haemolysis in presence of KC1. Previous to haemolysis starvation during 17 hr at 37'C in glucose-free solution- (130 mM-Na, 5 m J i - K , 20 m.M-Tn's, 1 5 5 m M - C l ) . -Milium: 130 mM-Xa, 5m>i-K, 20 mM-Tr,is, 1 rnit-Ca, 157 mu-Cl, 10-«g/ml. ••'laljain. Temp. 37° C. Haematocrit: A T P samplo 0-249, ATP-free sample 0-253. • with ATP in tho-cells, O without ATP. Arrow in panel A : mean concentration i:K-asured in whole suspension. Four experiments, vertical bar* 2x S.E. of mean^)2)»  10 I n t e r n a l sodium w i l l s t i m u l a t e Na,K-ATPase ( 6 5 ) .  and  a l s o f e l t t h a t the Mg+Ca-ATPase present  i n red blood  s e n s i t i v e enough to be p a r t of a c a l c i u m m a i n t a i n s a low  be l e s s than 5 X110"^M.  cells  was  t r a n s p o r t system which  I n t r a c e l l u l a r concentration  i n the case of the human red blood  Schatzmann  of calcium^whlch  c e l l has been estimated  In RBCMF Mg+Ca-ATPase was  to  found to  be  -4 o p t i m a l l y s t i m u l a t e d a t 10  M c a l c i u m and  had  a threshold at  10" M. 7  Other evidence a s s o c i a t i n g Mg+Ca-ATPase w i t h a c t i v e t r a n s p o r t was  as f o l l o w s :  (a) I o n i c Requirements; S h i n and that a c t i v e calcium magnesium.  calcium  t r a n s p o r t was  (26)  Lee  dependent on  were a b l e to show intracellular  S i n c e a l l the endogenous magnesium was  d u r i n g the p r e p a r a t i o n of t h e i r ghosts,  (ethylene  not removed dlamlne)-  t e t r a a c e t i c a c i d (EDTA) (a d i v a l e n t metal i o n c h e l a t i n g was  used to c h e l a t e the endogenous magnesium.  was  abolished  and  2 . 0 mM  l n ghosts r e s e a l e d  i n 1.0  mM  EDTA, 2 . 0  calcium  strontium  transport  mM  CaCl , 2  ATP.  (b) I t has a l s o been shown t h a t s t r o n t i u m for  Calcium  agent),  can s u b s t i t u t e  l n a c t i v a t i n g Mg+Ca-ATPase i n RBCMF (31)  i s transported  r e q u i r i n g ATP  and  that  out of asesealed ghosts by a mechanism  (27).  (c) I n r e s e a l e d ghosts,  a c t i v e calcium  t r a n s p o r t does not  depend on a l k a l i metal ions f o r e i t h e r s t i m u l a t i o n of the  vel-  o c i t y of c a l c i u m e f f l u x or f o r a jja-Ca exchange r e a c t i o n .  In  g h o s t s r e s e a l e d w i t h c h o l i n e o r l n T r i s b u f f e r , r a t h e r than sodium or potassium, the v e l o c i t y of c a l c i u m t r a n s p o r t was changed  (26,12).  not  11  (d) Concomitant inward transport of magnesium was not observed i n c e l l s extruding calcium, nor d i d the a d d i t i o n of magnesium to the external medium stimulate the ATPase a c t i v i t y of resealed ghosts (79).  Therefore  cium transport i s coupled  to the inward movement of magnesium  the p o s s i b i l i t y that c a l -  seems u n l i k e l y . (e) L a s t l y , hydrogen Ion does not seem to be coupled to calcium transport since the pH of a non-buffered medium d i d not change during a c t i v e calcium transport  (26).  2 . N u c l e o t i d e s p e c i f i c i t y ; Studies undertaken to determine the nucleotide requirement f o r calcium transport l n resealed ghosts have been i n c o n c l u s i v e . dine triphosphate  Lee and Shin reported that u r i -  (UTP) and c y t l d l n e triphosphate  as e f f e c t i v e as ATP l n supporting  calcium transport In resealed  ghosts (26). Guanoslne triphosphate e f f e c t i v e and inoslne triphosphate  (GTP) was found to be l e s s  (ITP) the poorest  In another study, GTP and ITP were reported as ATP ( 2 7 ) .  (CTP) were  substrate.  to be as e f f e c t i v e  These f a c t s do not support the contention  that  Mg+Ca-ATPase and calcium transport are c l o s e l y associated since Mg+Ca-ATPase  i n RBCMF i s s p e c i f i c f o r ATP ( 2 9 ) .  However, the  c o n f l i c t i n g reports on nucleotide s p e c i f i c i t y l n resealed ghosts may be a r e s u l t of incomplete removal of nucleoside dlphosphokinase which can catalyze the synthesis of ATP by t r a n s f e r of the phosphate from nucleoside preparation of the ghosts  triphosphates  to ADP during  (35).  Calcium e f f l u x i s very dependent on the i n t r a c e l l u l a r conc e n t r a t i o n of ATP ( 2 6 ) .  In c e l l s pretreated with  iodoacetate,  12 no  significant  t r a n s p o r t o f c a l c i u m was o b s e r v e d  o f ATP ( F i g . 2 ) . ghosts ATP.  However, t h e r e was a b o u t  not pretreated with I t appears  l n the absence  6y/> t r a n s p o r t i n  i o d o a c e t a t e I n t h e absence o f added  that ghosts  contain approximately  0.3  of  e n d o g e n o u s ATP, s i n c e a p p r o x i m a t e l y  of  c a l c i u m was t r a n s p o r t e d i n l o d a c e t a t e t r e a t e d c e l l s  0 . 3 mM ATP was a d d e d .  I n ghosts  more c o m p l e t e a n d f a s t e r 3. ive  loaded  t o changes i n temperature, t o a chemical  3.16  (26)  resealed  w i t h 1.0  removal of c e l l u l a r  Temperature dependence; C a l c i u m  coupled and  t h e same d e g r e e  reaction.  occurred.  Q  be e x p e c t e d 1 0  values  of 3.5 from  AT?  a  o  tt  20  TIME IN MINUTES  sensit-  o f a system  ghosts.  LO  when  ATP a  calcium  have been r e p o r t e d f o r c a l c i u m e f f l u x  LO  (58;S)  transport i s very  a s would High  mM  mM  FICURE 2. Effect of preincubation with iodoacetate. None + A T P (1 mit), tie initial preincubation with iodoacetate was omitted. Other conditions, standard. None — A T P , no preincubation with iodoacetate and no A T P in lysing solution. Other conditions, standard. Iodoacetate — A T P , standard conditions except' omission of A T P from solution. Iodoacetate + A T P (0.3 mit), standard conditions, except 0.3 mil A T P in lysing solution. Each point represents average of four experiments. Bars indicate standard error (%z)(Zi>).  (12)  13 4.  The e f f e c t o f I n t r a c e l l u l a r c a l c i u m on t h e v e l o c i t y o f  c a l c i u m e f f l u x ; Romero and Whittam demonstrated t h a t the v e l o c i t y o f c a l c i u m e f f l u x was dependent on the i n t r a c e l l u l a r c o n c e n t r a t i o n o f c a l c i u m i n r e d blood c e l l s loaded  t o g i v e a range o f i n t e r n a l  c a l c i u m p e r ml o f c e l l s , v a r i o u s time i n t e r v a l s c a l c i u m a t 37 C.  (7).  C e l l s were  c a l c i u m from 0 . 4 t o 2.7 pimoles  by I n c u b a t i n g ATP d e p l e t e d  i n a Ringer s o l u t i o n  cells for  c o n t a i n i n g 10 mM  The c e l l s were e n r i c h e d w i t h ATP by i n c u b a t i o n  w i t h adenine and i n o s l n e and t h e r a t e o f c a l c i u m l o s s was d e t ermined  ( F i g . 3)«  The r a t e o f e x t r u s i o n i n c r e a s e d w i t h i n c r e a s -  i n g c a l c i u m c o n c e n t r a t i o n , r e a c h i n g a p l a t e a u o f about 2 . 6 yamole c a l c i u m p e r ml X h r .  The i n t e r n a l c a l c i u m c o n c e n t r a t i o n  h a l f maximal a c t i v a t i o n was about 0 . 9 /imole p e r ml c e l l s . 3r  1  2  3  Ca content (^mole/ml. cells) Fig. 3.The stimulation of calcium transport by internal calcium. Cells washed free of glucose were loaded with calcium by incubation at 37° C for periods up to 3 hr in Ringer solution containing (mil): NaCI, 150; CaCI 2 , 10; Tris-HCl, 20, p H 7-6. Adenine (5 mn) and inosine (10 mil) were added, the incubation was continued and samples wero taken at 10 min intervals. The rate of calcium extrusion (/mnolo calcium/ml. cells x hr) was calculated from the change in 10 min, and has been plotted against the mean cell calcium concentration during this timo. Results are shown from three experiments ("J),  giving  14 Hg+Ca-ATPase A c t i v i t y I n Human Red Blood  C e l l Membrane Fragments  S i n c e t h e d i s c o v e r y o f an ouabain i n s e n s i t i v e c a l c i u m u l a t e d ATPase l n r e d blood  stim-  c e l l membrane fragments (RBCMF),  i n v e s t i g a t o r s have attempted t o c h a r a c t e r i z e t h i s enzyme.  The  p h y s i o l o g i c a l r o l e of Mg+Ca-ATPase however has not been complete l y agreed upon.  As p r e v i o u s l y mentioned i t was suggested t h a t  Mg+Ca-ATPase was a s s o c i a t e d w i t h a c t i v e c a l c i u m t r a n s p o r t  (12,26),  whereas o t h e r i n v e s t i g a t o r s have f e l t t h a t t h i s enzyme may be r e s p o n s i b l e f o r r e g u l a t i n g the c e l l volume  (31).  The Mg+Ca-ATPase  r e p o r t e d by these l a t t e r workers i s thought t o be a s s o c i a t e d with a m y o f i b r i l l a r p r o t e i n c a l l e d s p e c t r i n (55)• R e c e n t l y d a t a has been o b t a i n e d  which may r e c o n c i l e t h i s  d i f f e r e n c e i n o p i n i o n o f the f u n c t i o n a l r o l e o f Mg+Ca-ATPase. C a r e f u l s t u d i e s have r e v e a l e d t h a t more t h a n one Mg+Ca-ATPase i s present f o r calcium  i n RBCMF, as d i f f e r e n t i a t e d by t h e i r (30,32,33,34).  affinities  F i g u r e 4 i l l u s t r a t e s the a c t i v a t i o n  of Mg+Ca-ATPase l n RBCMF found by Horton e t a l . ( 3 2 ) . by c a l c i u m appears a t 10~ M  Activation  c a l c i u m and i n c r e a s e s t o 3 . 0 10""''M  a  c a l c i u m , a t which p o i n t the r a t e o f i n c r e a s e slows and a p l a t e a u i s observed up t o 3 X. 10~^M. t o an optimum a t 3 X i u u l a t e d a t 3 X 10~^M  The a c t i v i t y  M calcium.  then r a p i d l y i n c r e a s e s  The enzyme, maximally  c a l c i u m i s r e f e r r e d t o a s the h i g h  Mg+Ca-ATPase due t o i t s h i g h e r a f f i n i t . y f o r c a l c i u m .  stim-  affinity The enzyme  —4 maximally s t i m u l a t e d a t 3 A. 10 low a f f i n i t y Mg+Ca-ATPase.  M calcium  i s r e f e r r e d t o as t h e  Qther values reported  i n the l i t e r a t -  ure I n d i c a t e t h a t the h i g h a f f i n i t y Mg+Ca-ATPase i s o p t i m a l l y  15 stimulated  from 10  t o 50  uM c a l c i u m and the low a f f i n i t y  Mg+Ca-ATPase anywhere from 0.1 t o 0.5 mM The wide v a r i a t i o n of the r e p o r t e d  values  calcium  (33,34,78,43,32).  i s a t t r i b u t e d to the  d i f f e r e n t methods used f o r the i s o l a t i o n of the RBCMF.  Generally  the h i g h a f f i n i t y Mg+Ca-ATPase has a s p e c i f i c a c t i v i t y of t o 0.5 0.6  umoles  t o 0.88  P i mg-^hr"" compared to a s p e c i f i c a c t i v i t y o f 1  umoles P i m g ^ t a " 1  1  f o r the low a f f i n i t y  The presence o f two Kg+Ca-ATPases i n whole r e d blood  c e l l s or i n resealed ghosts.  ing to r e l a t e a c t i v e calcium sealed ghosts.  Mg+Ca-ATPase.  has n o t been shown p r e v i o u s l y  two Mg+Ca-ATPases i n RBCMF f u r t h e r c o m p l i c a t e s  The f i n d i n g o f studies  attempt-  t r a n s p o r t and Mg+Ca-ATPase i n r e -  Few k i n e t i c s t u d i e s have been done on these  enzyme systems.  The p r o p e r t i e s which have been determined a r e  as f o l l o w s t  V| 1 1 1 1 1 1 1 1 ' I O Fig.4  0.3  IO"9  IO"7 IO"5 Co**Molar  IO"3  IO"'  ( C a ) - d e p e n d e n t ATPase a c t i v i t y l n human r e d c e l l s . E a c h p o i n t r e p r e s e n t s the mean of f o u r experiments w i t h the standard e r r o r o f the mean (32.). ++  16  1. Ion requirements; require magnesium.  Both high and low a f f i n i t y Mg+Ca-ATPases  Optimal concentrations of magnesium f o r  these enzymes have been estimated to be between 3 to 8 mM(33).* 2 . Mg-ATP was shown k i n e t i c a l l y to be the substrate f o r high a f f i n i t y Mg+Ca-ATPase 1 3 3 ) . Calcium acts a l l o s t e r l c a l l y as a p o s i t i v e a u t o s t e r i c e f f e c t o r ( 3 3 ) .  Whether or not Mg-ATP  i s the substrate f o r low a f f i n i t y Mg+Ca-ATPase has not been determined• 3 . E f f e c t s of other cations on Mg+Ca-ATPase a c t i v i t y ; Both strontium and barium have been shown to be a c t i v a t o r s of Mg+Ca-ATPase ( 3 1 ) .  Strontium a c t i v a t e s more strongly than  calcium, but has a lower a f f i n i t y .  Careful k i n e t i c studies  were not done to determine which Mg+Ca-ATPase was a c t i v a t e d by barium or strontium, but judging from the low a f f i n i t y of these cations, the low a f f i n i t y Mg+Ca-ATPase i s activated a t l e a s t . This f i n d i n g i s of s p e c i a l i n t e r e s t since strontium i s also a c t i v e l y transported out of resealed ghosts, presumably by the same system as calcium ( 2 7 ) . There i s evidence that Mg+Ca-ATPase l n RBCMF i s stimulated  +  +  +  by univalent cations i n the order of K > Na > Rb  (30,36).  assium a c t i v a t e s Mg+Ca-ATPase a c t i v i t y approximately a mechanism which i s non-competitive  Pot-  100^ by  with calcium {Fig. 5 ) .  The presence of both magnesium and calcium i s required f o r t h i s activation.  Cesium and l i t h i u m were reported to have no stim-  u l a t i n g e f f e c t , i n d i c a t i n g that the a c t i v a t i o n by potassium, sodium and rubidium may not be duejjust to a change i n i o n i c  1? strength  (36).  Rossi(30)  However,  i n a study done by Schatzmann and  l i t h i u m was shown t o be about o n e - t h i r d as e f f e c t i v e  as potassium i n a c t i v a t i n g Mg+Ca-ATPase.  Sodium and potassium  appear t o a c t a t the same s i t e s i n c e no a d d i t i o n a l a c t i v a t i o n occurs when these i o n s a r e added t o g e t h e r A c t i v a t i o n by N a  +  or K  +  o f Mg+Ca-ATPase occurs  range o f c a l c i u m c o n c e n t r a t i o n s apparent a t 1 mM,  i n d i c a t i n g t h a t low a f f i n i t y  Mg+Ca-ATPase  Schatzmann e t a l . (30)  c a l l e d the a c t i v i t y i n the absence of added a l k a l i  metal c a t i o n s a l k a l i - c a t i o n - i n d e p e n d e n t and  over a wide  (Fig. 6 ) . Activation i s s t i l l  a c t i v i t y i s a c t i v a t e d by these c a t i o n s . arbitrarily  (36).  Mg+Ca-ATPase  activity  l n the presence o f a l k a l i metal c a t i o n s , a l k a l i - c a t i o n -  dependent Mg+Ca-ATPase a c t i v i t y .  S i n c e a l k a l i metal i o n s do  not have any e f f e c t on a c t i v e c a l c i u m t r a n s p o r t l n r e s e a l e d Schatzmann e t a l . (30) proposed t h a t the e x t r a a c t i v i t y ln  the presence o f a l k a l i metal c a t i o n s was due t o an  ghosts,  seen uncoupling  a c t i o n o f c a l c i u m on t h e Na,K-ATPase r e s p o n s i b l e f o r the t r a n s p o r t of sodium and potassium. very u n l i k e l y .  This hypothesis  Bond and Green (36) f e l t  be a t r i v i a l p r o p e r t y  o f RBCMF.  however, seems  t h a t t h i s e f f e c t may  F o r Instance,  these i o n s may  expose l a t e n t Mg+Ca-ATPase a c t i v i t y by a membrane e f f e c t ing  the c a t a l y t i c s i t e o f the enzyme.  observed l n RBCMF by f r e e z e - t h a w l n g  expos-  A similar effect i s  or detergents ( 3 4 ) .  I O  1  0.0J  • 0.04  •  1  0.05  0.03  1  0.10  1 1  032  [Co *], mM F i g . 5 A T P a s e a c t i v i t y as a ( u n c t i o n of C a c o n c e n t r a t i o n i n the presence a n d absence of So m M K . O r d i n a t e : t o t a l A T P a s e a c t i v i t y . T h e C a ^ c o n c e n t r a t i o n g i v e n o n the a b s c i s s a is the c o n c e n t r a t i o n a d d e d , w i t h o u t c o r r e c t i o n for b i n d i n g b y A T P o r b y m e m b r a n e s . T h e zero C a " p o i n t s m a y be s l i g h t l y h i g h , since E G T A (ethy)eneglycol-bis(/J-aniinoethyl e s t e r ) - A ' , . V ' - t e t r a a c e t i c acid) was n o t a d d e d to chelate possible traces of e n d o g e n o u s C a . T h e i n s e t shows a W o o l f p l o t o f t h e d a t a , f r o m w h i c h A',„ v a l u e s were o b t a i n e d . O t h e r c o n d i t i o n s are g i v e n i n METHODS ( 3 b ) . 2  2 -  +  1  5  J , f  o.s  1.1 3 4 i 6 7 S • 10 30 110" C o * concn. (M) 2  F i g . 6 E f f e c t of Na+ and K on ATPase a c t i v i t y i n hemoglobin f r e e r e d b l o o d o e l l membranes p r e p a r e d by f r e e z i n g and thawing (30).  19 I n h i b i t o r s E f f e c t i n g Mg+Ca-ATPase And A c t i v e Ga In Red Blood C e l l s  In recent  2+  Transport  years numerous I n v e s t i g a t o r s have sought a  s p e c i f i c I n h i b i t o r of Mg+Ca-ATPase, as ouabain has been i n v a l uable i n e l u c i d a t i n g the mechanism o f Na,K-ATPase. i n h i b i t o r o f Mg+Ca-ATPase and a c t i v e c a l c i u m  t r a n s p o r t would  be u s e f u l i n p r o v i d i n g d i r e c t evidence t h a t c a l c i u m and  Mg+Ca-ATPase a r e i n t i m a t e l y a s s o c i a t e d .  A specific  transport  A number o f drugs  have been t e s t e d on t h i s system.  Among those w i t h o u t any  e f f e c t on Mg+Ca-ATPase o r c a l c i u m  t r a n s p o r t were o l l g o m y c l n and  ouabain (1,11) which a r e s p e c i f i c Na,K-ATPase i n h i b i t o r s ( 3 7 ) . Caffeine  (lmg/ml) had no e f f e c t on a c t i v e c a l c i u m  r e s e a l e d ghosts  transport i n  (12).  A number o f agents have been shown t o be n o n - s p e c i f i c  -4 i n h i b i t o r s o f Mg+Ca-ATPase. e t h a c r y n i c a c i d (10  Mersalyl  (5.0 X 10  M)(11) and  M) (23) both I n h i b i t ATP dependent  transport l n resealed ghosts.  calcium  However, these drugs have a l s o  been shown t o e q u a l l y I n h i b i t Mg+Ca-ATPase and Na,K-ATPase a c t i v i t y i n RBCMF i n a c o n c e n t r a t i o n Ethacrynic  range of 10~  t o 10  M (5).  a c i d and m e r s a l y l a r e thought t o I n h i b i t enzyme a c t -  i v i t y by b i n d i n g t o p r o t e i n s u l f h y d r y l groups ( 5 ) .  The l a n t h a -  n i d e s have been found t o be u s e f u l agents f o r s t u d y i n g  calcium  i n t e r a c t i o n s s i n c e they possess a s i m i l a r r a d i i t o c a l c i u m but have much h i g h e r a f f i n i t i e s f o r a n i o n i c s i t e s due t o a h i g h e r charge d e n s i t y  (56).  Holminlum and praesodynium  inhibit  Mg+Ca-ATPase a c t i v i t y l n RBCMF and a c t i v e c a l c i u m r e s e a l e d g h o s t s , w i t h an Icn o f a p p r o x i m a t e l y 10  transport i n  —4  M (39).  20 However, these i o n s a r e n o n - s p e c i f i c as Na,K-ATPase and Mg-ATPase a c t i v i t i e s were a l s o i n h i b i t e d .  Lanthanum has  been u s e f u l f o r e s t a b l i s h i n g the mechanism f o r c a l c i u m  trans-  port i n mitochondria ( 7 0 ) . ( 4 0 ) recently reported  Watson and V i n c e n z l  that  the i n o r g -  a n i c dye ruthenium r e d s e l e c t i v e l y i n h i b i t s Mg+Ca-ATPase a c t i v i t y I n RBCMF.  Ruthenium r e d has p r e v i o u s l y been shown  to r e a c t w i t h c a r b o x y l ides  and s u l p h a t e  ( 4 1 ) . Table I I i l l u s t r a t e s  on t h e ATPases of RBCMF.  groups on mucopolysacchar-  the e f f e c t o f ruthenium r e d  Mg-ATPase and Na,K-ATPase were n o t  s i g n i f i c a n t l y I n h i b i t e d by ruthenium r e d . a l k a l i dependent and t h e a l k a l i  However, both the  independent f r a c t i o n s o f  Mg+Ca-ATPase were almost c o m p l e t e l y i n h i b i t e d by 6 X 1 0  M  ruthenium r e d .  Table I I E f f e c t s Of Ruthenium Red On ATPases Of E r y t h r o c y t e  Membranes (k0)  (Values a r e expressed as ^imoles o f i n o r g a n i c phosphate l i b e r a t e d per mg p r o t e i n i n 1 h.) ATPase a c t i v i t y ATPases  Ruthenium r e d c o n c e n t r a t i o n (M):  0 Mg+Ca-ATPase alkali-cation-dependent alkali-cation-independent Na,K-ATPase Mg-ATPase  ~Q~7M 0.32 0.56 0.28 0.11  6*10-6  6«10  0733"  0.09  0.22 0.33 0.26 0.11  0.04  0.05 0.25 0.10  -5  21 T h i s study d i d not d i s t i n g u i s h between i n h i b i t i o n o f o r low a f f i n i t y Mg+Ca4ATPase as 0.12mM c a l c i u m was all  studies.  used i n  C o n s i d e r i n g the h i g h c o n c e n t r a t i o n of  used, the low a f f i n i t y Mg+Ca-ATPase, a t l e a s t , was  high  calcium inhibited.  The mechanism of a c t i o n of t h i s i n h i b i t o r a l s o remains undetermined as the c o n c e n t r a t i o n of ATP, not a l t e r e d l n i t s presence.  The  c a l c i u m and magnesium  was  s i t e of a c t i o n of t h i s drug  a l s o remains unknown, as the s t u d i e s were done on RBCMF. However, ruthenium red shows p o t e n t i a l as a t o o l f o r s t u d y i n g a c t i v e c a l c i u m t r a n s p o r t , s i n c e t h i s drug has block a c t i v e calcium transport i n mitochondria  been shown to (42).  2+ Bader (43)  r e c e n t l y r e p o r t e d t h a t Mn  a t e d low a f f i n i t y Mg+Ca-ATPase, without a f f i n i t y Mg+Ca-ATPase i n RBCMF.  methylhydroxylamlne and  a r a t i o o f 10:1).  5.0  a f f e c t i n g the  activ-  high  Bader a l s o r e p o r t e d t h a t  a f f i n i t y Mg+Ca-ATPase a c t i v i t y was 10"3M  specifically  selectively inhibited X 10* M C u 5  2 +  (EGTA; C u  low by  2 +  at  T h i s was  the f i r s t r e p o r t of s e l e c t i v e l n h l b 2+ i t l o n of a Mg+Ca-ATPase. However, Cu i s a s u l f h y d r y l group reagent, which renders membranes permeable to c a t i o n s . There2+ f o r e t h i s i n h i b i t o r would not be u s e f u l t o study a c t i v e Ca t r a n s p o r t i n r e s e a l e d ghosts o r i n whole r e d blood  cells.  A c t i v e Calcium T r a n s p o r t In Other T i s s u e s  Besides human e r y t h r o c y t e s , c u l t u r e d He La c e l l s cells  (45),  ATP  (44)  and  L  dependent c a l c i u m t r a n s p o r t a c r o s s the plasma  membrane of o t h e r t i s s u e s has not been demonstrated.  However,  22  a c a l c i u m or magnesium s t i m u l a t e d ATPase has the sarcolemma of s k e l e t a l muscle (46)% guinea p i g i l e u m  (48)•  Hurwitz (48)  ent a c t i v e c a l c i u m t r a n s p o r t may  been l o c a t e d l n  c a r d i a c muscle and suggests  t h a t ATP  be important  t o the p o o r l y developed  tissue.  His h y p o t h e s i s  sarcoplasmic  i s supported  depend-  i n lowering  i n t r a c e l l u l a r c o n c e n t r a t i o n of c a l c i u m i n guinea p i g due  may  be ATP  reticulum i n this  by the f a c t t h a t the  be more important  activity.  but the Na-Ca exchange mechanism  e v i d e n t i n nerve, may c a r d i a c muscle (50)  t h a t the Na-Ca exchange mechanism  a l s o operate  and  (49),  i n s k e l e t a l muscle  smooth muscle ( 5 1 ) .  The  requirement  o f e x t e r n a l sodium f o r c a l c i u m e f f l u x appears t o be v e r y s i n c e e x t e r n a l l i t h i u m , c h o l i n e o r potassium I t has been e s t i m a t e d  sodium i o n s a r e exchanged f o r one mechanism ( 1 7 ) .  may  a c r o s s t h i s plasma membrane.  There i s some evidence  f o r sodium.  ileum  t h a t c a l c i u m e x t r u s i o n from s q u i d axon  (17)  dependent  the  ileum,  r e l i e s h e a v i l y on e x t r a c e l l u l a r c a l c i u m f o r c o n t r a c t i l e There i s some evidence  in  t h a t two  specific,  cannot s u b s t i t u t e to three external  I n t e r n a l c a l c i u m i o n by  this  The d i r e c t i o n o f the Na-Ca exchange mechanism  can be r e v e r s e d l n d i r e c t i o n l n nerves bathed i n s o l u t i o n s d e f i c i e n t l n e x t e r n a l sodium.  Other f e a t u r e s o f the Na-Ca exchange  mechanism i n cyanide poisoned taken from Baker ( 1 7 ) .  s q u i d axons a r e shown i n T a b l e I I I ,  Cyanide was  t o r e l e a s e c a l c i u m from m i t o c h o n d r i a  added i n t h i s and  ence of c a l c i u m uptake by m i t o c h o n d r i a measurements.  preparation  thus prevent  interfer-  when making c a l c i u m  flux  23 Table I I I F e a t u r e s Of The Na-Ca Exchange Mechanism In Cyanide Poisoned Squid A x o n s ( 1 7 ) . 1. I t i s temperature s e n s i t i v e and has a ^ of about t h r e e . 2. I t i s i n s e n s i t i v e to h i g h c o n c e n t r a t i o n s of the c a r d i a c g l y c o s i d e ouabain. 3 . I t i s i n h i b i t e d by e x t e r n a l l y a p p l i e d lanthanum Ions ( 8 0 ) . 4 . I t i s very dependent on the i o n i c composition of the e x t e r n a l medium. Removal of e x t e r n a l c a l c i u m reduces the e f f l u x t o a v a r i a b l e e x t e n t . Baker and Crawford (81); found no e f f e c t whereas B l a u s t e i n and Hodgkin (82) found t h a t removal o f e x t e r n a l c a l c i u m reduced the e f f l u x t o about h a l f ( l e . 2 pmoles/cm sec t o 1 pmole/cm^sec). I n the absence of e x t e r n a l c a l c i u m , complete replacement of sodium by l i t h i u m , p o t a s s i u m , c h o l i n e o r sugar causes a f u r t h e r r e d u c t i o n of e f f l u x to about one-twentieth of i t s o r i g i n a l v a l u e , 0.1 pmole/cm s e c . 0  Nerve and muscle c e l l s a l s o m a i n t a i n a low  intracellular  c a l c i u m c o n c e n t r a t i o n by means o f the s a r c o p l a s m i c r e t i c u l u m (17*54).  and m i t o c h o n d r i a  Both of these s t r u c t u r e s can accum-  u l a t e c a l c i u m by an a c t i v e ATP  dependent system.  Estimates o f f  the c a l c i u m s e q u e s t e r i n g c a p a c i t y of s a r c o p l a s m i c r e t i c u l u m l n v i t r o suggests  t h a t t h i s membrane system i s a b l e t o lower  the c y t o p l a s m i c c a l c i u m c o n c e n t r a t i o n below the I n v i v o l e v e l r e q u i r e d f o r c o n t r a c t i o n (approx. 1 0 ~ M ) ( 5 4 ) .  I t has a l s o been  6  shown t h a t the speed of c a l c i u m accumulation r e t i c u l u m i s compatible muscle ( 5 2 ) .  Calcium  by  sarcoplasmic  w i t h the time of t e n s i o n decay i n  i s accumulated a g a i n s t an a c t i v i t y  grad-  i e n t by an a c t i v e t r a n s p o r t system which i s coupled to a Mg+Ca-ATPase ( 5 3 ) .  T h i s enzyme has a low Km  f o r calcium,  (approx.  -7 10  M) and a requirement  f o r magnesium, o p t i m a l a t 5 . 0  mM.  A  number of models d e s c r i b i n g t h i s t r a n s p o r t system have been reported  (54).  Since t h i s t r a n s p o r t system shares a number of  24 p r o p e r t i e s t o t h e Mg+Ca-ATPase l n human e r y t h r o c y t e s , a scheme taken from Martonosl  (54) showing t h e m o l e c u l a r  mechanism  r e l a t i n g ATP h y d r o l y s i s t o c a l c i u m t r a n s p o r t i s o u t l i n e d a s follows: 1. E+ATP  s  E-ATP  2. E-ATP+2Ca  3. E  Ca - ATP Ca  EoJ p  Ca 4.  .Ca - ATP Ca  E  E*+P  E*<0P'  A  ^Ca  Outside  +ADP  + 2Ca  Inside  Ca  5. E*  „  w  E  E=enzyme, E*=the c o n f o r m a t i o n a l l y / a l t e r e a f o r m o f t h e c a r r i e r .  I n t h i s scheme ATP b i n d s t o t h e enzyme l n t h e absence o f c a l c i u m ( s t e p 1 ) , w h i c h promotes t h e b i n d i n g o f c a l c i u m  (step 2 ) ,  The  bound c a l c i u m t h e n a c t i v a t e s t h e phosphate t r a n s f e r f r o m ATP leading t o the formation of a phosphoprotein ( s t e p 3)»  intermediate  The c a r r i e r t h a n undergoes a c o n f o r m a t i o n a l  change  w i t h the t r a n s l o c a t i o n of calcium from the outer t o the i n n e r surface of the sarcoplasmic t h i s low a f f i n i t y c a r r i e r  reticulum,  Release of calcium from  ( s t e p 4 ) , f o l l o w e d by h y d r o l y s i s o f  the phosphoprotein,completes the c y c l e (step 5 ) .  25 METHODS AND  Outdated  MATERIALS  c l t r a t e d human b l o o d (0 p o s i t i v e ) was  obtained  from the Canadian Red C r o s s and s t o r e d f o r n o t more t h a n  two  o  weeks a t 5 C.  B l o o d was  used w i t h i n twenty days of c o l l e c t i o n .  P r e p a r a t i o n Of Red B l o o d C e l l  Red b l o o d c e l l  (RBC)  Ghosts  g h o s t s were p r e p a r e d by a m o d i f i c a t -  i o n o f t h e p r o c e d u r e o f s t e p w i s e h e m o l y s i s o u t l i n e d by S c h r l e r (57)•  Whole b l o o d (150 ml) was  suspended l n one volume o f  M NaCl l n e i g h t 40 ml p o l y p r o p y l e n e c e n t r i f u g e t u b e s .  The  0.155 tubes  o  were c e n t r i f u g e d a t 4 C f o r t e n minutes a t 2,000 X g r i g e r a t e d c e n t r i f u g e (eg. I n t e r n a t i o n a l model, B-20, head).  l n a ref870  angle  The s u p e r n a t a n t and w h i t e b u f f y l a y e r were removed by  suction.  C o n s i d e r a b l e c a r e was  t a k e n t o remove the t o p b u f f y  c o a t as c o m p l e t e l y as p o s s i b l e , d e s p i t e a c o n s i d e r a b l e red c e l l s  (approx. o n e - t h i r d ) .  The r e d c e l l s were  l o s s of  resuspended  w i t h a g l a s s s t i r r i n g r o d and washed a g a i n i n t h e same tubes w i t h t e n volumes o f 0.155  M NaCl w i t h subsequent  the s u p e r n a t a n t and any r e m a i n i n g w h i t e c e l l s .  removal  of  F o r a summary  o f t h e p r e p a r a t i o n , l o a d i n g and a s s a y p r o c e d u r e s t o be o u t l i n e d r e f e r t o T a b l e IV. The c e l l s were t h e n p a r t i a l l y hemolyzed by s u s p e n s i o n i n t e n volumes o f 0.08  M N a C l c o n t a i n i n g 1.0 mM  EDTA ( d i s o d i u m  s a l t ) and c e n t r i f u g e d a t 8,000 X g f o r t e n m i n u t e s .  Temperature  26  Table IV  P r e p a r a t i o n Of RBC G h o s t s . L o a d i n g . And A s s a y  Procedure  I Preparation: 1.  Wash w i t h 0.155M NaCI  (twice)  2.  0.08M NaCI + l.OmM EDTA  3.  0.06M NaCI + l.OmM EDTA  4.  0.04M NaCI + l.OmM EDTA  5.  0.015M NaCI + 5.0mM TRIS-MALEATE, pH 7.1  I I Loading: 1.0ml g h o s t s + 3.0ml o f s o l u t i o n c o n t a i n i n g ; 4.0mM Na2ATP, 4.0mM M g C l , 2  lOmM THIS, pH7.1 and v a r i o u s concentrations of C a C l  III  2  Resealing: 1.  Add 0.2ml o f 2.b76M NaCI  2.  Stand a t R.T., 10 m i n .  3.  Wash w i t h 2.0mM M g C l , 0 . 1 2 5 M NaCI, 2  and 20mM TRIS-MALEATE 4.  (twice)  Suspend t o 3»0ml l n a s s a y medium (as above) + ImM C a C l  9  27 o  was m a i n t a i n e d a t 2-4 C d u r i n g h e m o l y s i s p r o c e d u r e s t o ensure (58).  t h a t the ghosts d i d not r e s e a l Immediately a f t e r hemolysis  EDTA was i n c l u d e d i n t h e h e m o l y s i s medium t o remove endogenous d i v a l e n t m e t a l c a t i o n s s u c h as c a l c i u m and magnesium.  Chelat-  i n g a g e n t s have a l s o been found t o be u s e f u l l n f a c i l i t a t i n g t h e r e m o v a l o f s o l u b l e enzymes and hemoglobin from t h e i n t e r i o r (59)  without a f f e c t i n g r e s e a l i n g  The g h o s t s were t h e n suspended NaCl c o n t a i n i n g 1.0  cellular  (58).  l n t e n volumes o f 0.06  M  mM EDTA and were c e n t r i f u g e d a t 1 3 , 0 0 0 X g 0  f o r t e n minutes a t 2-4 C.  A t t h i s s t e p the c e l l s l o s t a c o n s i d -  e r a b l e amount o f hemoglobin as e v i d e n c e d by a v e r y d a r k s u p e r n a t ant.  A much h i g h e r c e n t r i f u g a l f o r c e i s r e q u i r e d a t t h i s s t e p  t o s p i n down t h e r e d c e l l s as t h e c e l l s have become s w o l l e n and t h e r e f o r e have much l e s s d e n s i t y t h a n t h e whole BBC. were hemolyzed  The  ghosts  i n t e n volumes o f 0.04 M NaCl c o n t a i n i n g 1.0  mM  EDTA and c e n t r i f u g e d a t 1 5 , 0 0 0 X g f o r t e n m i n u t e s a t 2 - 4 * 0 . The g h o s t s were f i n a l l y resuspended  i n t e n volumes o f 0 . 0 5  N a C l and 0.005 M T r l s - M a l e a t e (pH 7.1)  M  and c e n t r i f u g e d a t 2 0 , 0 0 0  o  X g f o r t e n m i n u t e s a t 2-4 C. in color.  The p e l l e t a t t h i s s t a g e was p i n k  The p e l l e t was a l s o V i s c o u s i n n a t u r e and c o u l d be  poured i n t o a 40 ml p o l y p r o p y l e n e c e n t r i f u g e t u b e s .  The  solid  r e d b u t t o n r e m a i n i n g i n t h e bottom o f t h e c e n t r i f u g e t u b e s c o n s i s t s o f c e l l s w h i c h would n o t hemolyze under t h e s e c o n d i t i o n s o r t y p e I I I g h o s t s (58)  and was d i s c a r d e d . These g h o s t s were  used w i t h i n f i f t e e n m i n u t e s i n t h e l o a d i n g s t e p .  28 Loading  Procedure  1.0  ml of ghosts  p r o t e i n ) was brated  pipeted into  ) containing 3.0  t r a t i o n of 4 . 0 (pH 7 . 1 was  (approx. 10 a 15  ml p o l y p r o p y l e n e  mM  2  ).Unless  or 5*0  mg  of membrane  t e s t tube  ml of s o l u t i o n , t o g i v e a f i n a l  Na ATP, 4 . 0  mM  ghosts  otherwise  MgCl  2  and  iO.O  mM  (cali-  concen-  Tris-maleate  s t a t e d ,the c o n c e n t r a t i o n of  calcium  v a r i e d i n t h i s medium. When r e q u i r e d , monovalent c a t i o n s  and drugs were a l s o I n c l u d e d . The  tbes were a l l o w e d  to  f i v e t o t e n minutes t o a l l o w the Ions to e q u i l i b r l a t e  stand before  o  r e s e a l l n g a t 2-4  R e s e a l l n g And  C  (12).  Washing Procedure  P a r t i a l l y hemolyzed r e d b l o o d o r i g i n a l shape and (20,21).  The  RBC  c e l l ghosts  can r e t a i n  p e r m e a b i l i t y by r e s t o r a t i o n of  ghosts  lsotonicity  were r e s e a l e d by the a d d i t i o n of  ml of 2 . 8 7 6 M NaCI s o l u t i o n . The  their  0,2  tubes were w e l l shaken and  p l a c e d i n a water bath a t room temperature f o r e x a c t l y t e n minu t e s . R e s t o r a t i o n of l s o t o n i c i t y and both f a c i l i t a t e r e s e l l i n g  (58).  the I n c r e a s e  i n temperature  Ghosts not allowed  to stand f o r  as l o n g as t e n minutes a t room temperature were found  to  take  up l e s s c a l c i u m as measured by atomic a b s o r p t i o n  spectrophotom-  e t r y s i n c e they d i d not r e s e a l as c o m p l e t e l y  calcium  l o s t d u r i n g the washing. The a f t e r t e n minutes.  and  tubes were r e t u r n e d  was  to the i c e bath  29 The r e s e a l e d ghosts were washed w i t h 5»5 s o l u t i o n c o n t a i n i n g 125.0 Tris-Maleate  (pH 7.1)  mM  NaCl, 2.0  mM  ml of an  MgCl  2  isotonic  and 20.0  mM  a t 5,000 X g f o r t e n minutes a t 2 - V c .  The r e s u l t i n g p e l l e t forms a compact p e l l e t a t t h i s s t a g e . The r e s e a l e d ghosts were washed once more i n 10.0 above s o l u t i o n .  The c l e a r s u p e r n a t a n t was  a p p r o x i m a t e l y 2.5 of  30 mM  CaCl  r a t i o n of 1C0  2  ml of s o l u t i o n .  was mM  added  ml of the  aspirated o f f leaving  I n most experiments 0.1  to the tubes t o g i v e a f i n a l  calcium.  concent-  Required amounts o f ruthenium r e d  and o t h e r drugs were a l s o added a t t h i s s t a g e .  The  resealed  g h o s t s were f i n a l l y brought t o a f i n a l volume o f 3.0 the  ml  ml l n  p r e v i o u s l y graduated t e s t tubes w i t h the a d d i t i o n o f the  i s o t o n i c washing  solution.  I n c u b a t i o n Procedures  In a l l experiments the r e a c t i o n was  s t a r t e d by Immersing 0  the  tubes i n a shaking water bath a t 37 C.  A s h o r t d e l a y of  t h r e e minutes i n r e a c h i n g the temperature of 37°C was a b l e i n these experiments as was  inevit-  e v i d e n t l n the e f f l u x measure-  ments ( F i g . 1 3 ) . D e t e r m i n a t i o n o f ATPase A c t i v i t y  -  ATPase a c t i v i t y was  determined by measuring the i n o r g a n i c  phosphate r e l e a s e d from ATP by a m o d i f i c a t i o n o f the method of  F l s k e and SubbaRow ( 6 0 , 6 3 ) .  The procedure i s as f o l l o w s :  30 The enzyme reaction was terminated by the addition of 1.0  ml  of cold s i l l c o t u n g s t i c acid (8.0$) i n p e r c h l o r i c acid (1.2 M), usually a f t e r a t h i r t y minute incubation period.  The tubes were  placed i n i c e , to minimize ATP hydrolysis, f o r at l e a s t one o  hour.  The tubes were then centrifuged at 20,000 X g at 0-4 C  i n a r e f r i g e r a t e d centrifuge (eg. International model, 874 angle head).  3.0  B-20,  ml of the supernatant was transfered into  a second test tube (eg. 20 X 150 mm)  containing 1.4 ml d i s t i l l e d  water and kept i n i c e . At zero time, 0.4 ml of molybdate reagent was added followed immediately by the addition of 0.2 ml of amlnonaptholsulfonlc acid reagent and s t i r r e d on a vortex f o r f i f t e e n seconds.  The color was allowed to develop f o r t h i r t y  minutes at room temperature.  A f t e r t h i r t y minutes the color  was read i n a spectrophotometer  (eg. Coleman-Hitachi 124) at  660 nm against a d i s t i l l e d water reference i n a 1.0 cm l i g h t path cuvet.  KH P0^ ( a n a l y t i c a l reagent) was used as the 2  primary standard. ATPase a c t i v i t y was found to be l i n e a r up to t h i r t y i n resealed ghosts ( F i g . 7 ) .  minutes  Mg+Ca-ATPase a c t i v i t y was deter-  mined by measuring the difference i n ATPase a c t i v i t y i n the absence and presence of calcium l n the loading medium.  Since the  ATPase a c t i v i t y i n the absence of added calcium can not be estimated (eg. ghosts do not r e s e a l under t h i s condition) the a c t i v i t y l n the absence of added calcium was estimated by extrapo l a t i o n of the calcium a c t i v a t i o n curve to zero." calcium. Na,K-ATPase a c t i v i t y was determined by measuring the a c t i v i t y  31 i n ghosts r e s e a l e d with NaCl ( 2 . 8 7 6 M) with the a d d i t i o n of Ouabain ( 0 . 1  e x t e r n a l potassium (0-15.0 mM). the a c t i v i t y s t i m u l a t e d Na,K-ATPase and  Mg+Ca-ATPase a c t i v i t y was per mg  reported  as  of membrane p r o t e i n per  mg'-^hr" ). 1  JL  D e t e r m i n a t i o n Of The  V e l o c i t y Of C a l c i u m  Calcium t r a n s p o r t or e f f l u x was  ated by the a d d i t i o n of 6.0 ing NaCl (119  mM)  i a t e l y placed  l n a i c e bath.  and  Transport  measured as the change i n  c e l l u l a r c a l c i u m with time a t 37°C.  The  i n c u b a t i o n was  lanthanum (6.0mM). L a C l ^ was  The  tubes were immed-  included l n t h i s solution to i n h i b i t  c a l c i u m t r a n s p o r t system ( 8 3 ) .  T h i s procedure was  order to o b t a i n a more a c c u r a t e  determination  calcium  of  the  Invoked i n intracellular  t i g h t l y bound i n the membrane.  l i t t l e d i f f e r e n c e was  termin-  ml of an i c e c o l d s o l u t i o n c o n t a i n -  to d i s p l a c e l o o s e l y bound e x t e r n a l c a l c i u m and  c a l c i u m and  abolished  by e x t e r n a l potassium.  umoles of P i r e l e a s e d from ATP hour (umoles P  mM)  In p r a c t i c e ,  found when the ghosts were washed w i t h i c e  c o l d NaCl s o l u t i o n (0.155 M)  alone.  The  tubes were c e n t r i f u g e d  o  f o r t e n minutes a t 5.000 X g a t 2-4 oved by a s p i r a t i o n and  C.  the p e l l e t was  The  supernatant was  rem-  washed once more l n 6.0  ml  of the same s o l u t i o n . Calcium was i o n of the wet B r i e f l y , 1.0 a c i d , 1:1,  e x t r a c t e d from the p e l l e t by a s l i g h t ashing  ml of 3.0  was  procedure r e p o r t e d  modificat-  by Sparrow e t  M trichloracetic acid:  added to d i s s o l v e the p e l l e t .  al.(6l).  glacial acetic The  pellet  was  32 resuspended w i t h a v o r t e x mixer and the tubes were p l a c e d f o r f i f t e e n minutes i n a water bath a t 70°C. ml o f d i s t i l l e d water was  added.  A f t e r t h i s time,  The tubes were mixed  2.0  by  v o r t e x and were p l a c e d i n the water bath f o r t e n minutes a t 70°C.  They were then allowed t o c o o l t o room temperature,  a l l o w i n g c o a g u l a t i o n of the p r o t e i n , and made up to a volume of 5 . 0  ml w i t h 3 0 . 0 mM  c a l i b r a t e d t e s t tube.  LaCl-j (approx. 2 . 0  L a C l ^ was  added  final  ml) l n a  to prevent I n t e r f e r e n c e  from phosphates and I n o r g a n i c i o n s l n p r e p a r a t i o n f o r atomic a b s o r p t i o n spectrophotometry.  The p r e c i p i t a t e was  c e n t r i f u g a t i o n a t 7.500 X g f o r t e n minutes.  The  of c a l c i u m i n the p r o t e i n f r e e s u p e r n a t a n t was  removed by concentration  determined by  measuring the a b s o r p t i o n a t the 4227 A l i n e from a hollow cathode c a l c i u m lamp on a V a r i a n - T e c h t r o n atomic a b s o r p t i o n spectrophotometer, model AA-5. was  used.  A lamp c u r r e n t  s e t a t 4 . 0 mA  A n i t r o u s o x i d e - a c e t y l e n e flame  (hollow c a l c i u m cathode lamp)  and the s l i t w i d t h a t 125 p.  Standard CaCO^  s o l u t i o n s were prepared from s t a n d a r d r e f e r e n c e o b t a i n e d from F i s h e r S c i e n t i f i c Company. obeying Beers law was  was  solutions  A standard curve  o b t a i n e d between 10-100 uM c a l c i u m .  P r o t e i n Assay  The c o n c e n t r a t i o n o f p r o t e i n was of Lowry (62)  f o r insoluble proteins.  (Sigma, 3X r e c r y s t a l l i z e d ) was  determined by the method Bovine serum albumin  used as a s t a n d a r d .  Standard  c u r v e s were l i n e a r from 50 t o 250 ug of p r o t e i n when r e a d a t  33 500 nm.  1 . 0 ml o f the hemoglobin f r e e ghost p r e p a r a t i o n was  found t o c o n t a i n 4 . 4 mg of membrane p r o t e i n . Hemoglobin f r e e ghosts were prepared by washing the ghosts t h r e e a d d i t i o n a l times i n 0.015 Maleate  (pH 7 . 1 )  M NaCI and 0.005mM T r i s -  f o r t e n minutes a t 2 0 , 0 0 0 X g.  Temperature  o  was c o n t r o l l e d a t 2 - 4 C d u r i n g these washings.  No measurable  change In the s p e c i f i c a c t i v i t y of Mg+Ca-ATPase between hemogl o b i n c o n t a i n i n g and hemoglobin f r e e ghosts was o b t a i n e d . Washing  Procedure  During the course o f t h i s work, i t was found t h a t the method f o r washing t h e p o l y p r o p y l e n e t e s t tubes was v e r y c r i t i c a l f o r o b t a i n i n g maximal ATPase a c t i v i t i e s . washing procedure was adopted.  T h e r e f o r e the f o l l o w i n g  The t e s t tubes were  soaked  o v e r n i g h t i n t a p water c o n t a i n i n g 0.5% L i q u l n o x d e t e r g e n t . tubes were r i n s e d w i t h t a p water and soaked l e a s t , one hour i n 5 . 0 mM EDTA  l n 1 0 $ HNO^  (tetrasodium s a l t ) .  The  f o r at  The tubes  were w e l l r i n s e d w i t h t a p water and then r i n s e d t e n times w i t h g l a s s d i s t i l l e d water and a l l o w e d t o d r y l n a i r . A l o s s of low a f f i n i t y Mg+Ca-ATPase a c t i v i t y r e s u l t s l n t e s t tubes soaked  i n 0.5% L i q u l n o x a l o n e , presumably due t o a  b u i l d up of d e t e r g e n t i n the t u b e s .  0  34  RESULTS AND  P r o p e r t i e s Of The Red  DISCUSSION  Blood C e l l Ghost  Preparation  Of fundamental importance to t h i s study was  the  prepar-  a t i o n of a r e d blood c e l l ghost p o p u l a t i o n which c o u l d made 'leaky' and  then r e s e a l e d t o r e g a i n the o r i g i n a l c a t i o n  p e r m e a b i l i t y c h a r a c t e r i s t i c s of the whole c e l l . important  be  I t was  also  t h a t the a c t i v i t y of the v a r i o u s ATPase systems  were not d e s t r o y e d  d u r i n g the p r e p a r a t i o n .  by r e s e a l l n g .  previous  (26,27)  prepared  step hemolysis,  followed  s t u d i e s , Schatzmann e t a l . (12) and g h o s t s by a method I n v o l v i n g a one  In  others  T h i s procedure d i d not a l l o w a complete removal (70).  of endogenous enzymes which can s y n t h e s i z e ATP more, endogenous d i v a l e n t metal c a t i o n s and completely  removed by t h i s method.  reduce the endogenous c a t i o n s and preparations, l n glucose  ATP  Therefore ATP  These two  i n order  to  i n these  a t 37°C f o r 17  f r e e medium (12) o r were i n c u b a t e d (26).  were not  concentrations  the c e l l s were p r e l n c u b a t e d  ence of i o d o a c e t a t e  Further-  i n the  methods a r e  pres-  undesirable  s i n c e some d e s t r u c t i o n of the plasma membrane must o c c u r evidenced  by hemolysis d u r i n g  In t h i s study, of stepwise  hr  as  incubation.  t h e r e f o r e , a m o d i f i c a t i o n of the procedure  hemolysis as o u t l i n e d by S c h r l e r (57) was  as d e s c r i b e d l n the Methods.  EDTA (dlsodium  salt)  adopted  (1.0  mM)  35  was I n c l u d e d 2+ 2  i n t h e washing s o l u t i o n s t o I n s u r e r e m o v a l o f  +  Mg  , Ca  , ATP and s o l u b l e enzymes ( 7 4 ) .  C a r e , however, had  t o be t a k e n i n u s i n g EDTA i n membrane p r e p a r a t i o n s , s i n c e under c e r t a i n c o n d i t i o n s EDTA s o l u b l l i z e s membrane bound p r o t eins (67).  F u r t h e r m o r e , t o t a l r e m o v a l o f membrane d i v a l e n t 2  c a t i o n s , Mg  2+  +  and Ca  , could r e s u l t i n a ghost p o p u l a t i o n  w h i c h may n o t r e s e a l , as these i o n s a r e t h o u g h t t o be s t r u c t u r a l components o f t h e plasma membrane ( 6 7 ) . Under t h e c o n d i t i o n s used i n t h i s s t u d y  (see M e t h o d s ) ,  t h e p r e p a r i o n remained r e l a t i v e l y impermeable t o d i v a l e n t and monovalent c a t i o n s and r e t a i n e d ATPase a c t i v i t y .  Evid-  ence s u p p o r t i n g t h e v i a b i l i t y o f t h i s p r e p a r a t i o n f o r t r a n s p o r t s t u d i e s was a s f o l l o w s : 1. Under t h e l i g h t m i c r o s c o p e , t h e g h o s t s t h e i r o r i g i n a l biconcave  regained  d i s c shape a f t e r r e s e a l l n g  ( m a g n i f i c a t i o n 500X). 2. The s p e c i f i c a c t i v i t i e s o b t a i n e d f o r Na,K-ATPase and Mg+Ca-ATPase were comparable t o t h o s e r e p o r t e d i n o t h e r studies (40,65).  The Na, K-ATPase and Mg+Ca-ATPase a c t i v i t i e s  were shown t o be a s y m m e t r i c a l l y  s t i m u l a t e d by e x t e r n a l p o t a s -  s i u m and I n t e r n a l c a l c i u m , r e s p e c t i v e l y .  Such c h a r a c t e r -  i s t i c s c a n o n l y be d e m o n s t r a t e d l n c e l l s w i t h an I n t a c t plasma membrane. 3.  A l o s s of c e l l u l a r calcium against a concentration  g r a d i e n t was d e m o n s t r a t e d ( F i g . 1 3 ) , whereas no l o s s o f c e l l u l a r c a l c i u m o c c u r s i n f r o z e n r e d b l o o d c e l l membrane f r a g m e n t s used i n o u r s t u d i e s .  I n a d d i t i o n , no change l n 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 magnesium o c c u r r e d l n t h e r e s e a l e d  ghosts,  36 i n d i c a t i n g t h a t tne membrane i s a l s o not f r e e l y permeable t o magnesium. 4.No v i s u a l l o s s o f hemoglobin o c c u r r e d d u r i n g washing subsequent t o r e s e a l i n g .  Ion Requirements For R e s e a l l n g  M a i z e l s e t a l . ( 7 5 ) » i n an e a r l y study was an e s s e n t i a l requirement  .reported that calcium  f o r maintaining red blood  cell  membrane p e r m e a b i l i t y . . R e c e n t l y , however, Bramely e t a l . ( 7 6 ) claimed magnesium c o u l d s u b s t i t u t e f o r c a l c i u m i n r e s e a l l n g , though l e s s e f f e c t i v e l y .  However, i n the p r e s e n t  study,  i n c l u s i o n o f a t l e a s t 0 . 0 5 mM c a l c i u m i n the l o a d i n g medium was found  t o be an a b s o l u t e requirement  for resealing.  This  was demonstrated i n a n experiment where ghosts were l o a d e d i n a medium c o n t a i n i n g no added c a l c i u m , f o l l o w e d by the normal procedure  f o r resealing.  Subsequent suspension o f these  ghosts  i n a medium c o n t a i n i n g 1 . 0 mM c a l c i u m r e s u l t e d l n an uptake o f c e l l u l a r calcium approximately a l l y obtained with  e q u i v a l e n t t o the v a l u e s norm-  'leaky g h o s t s '  o  s'1 ( I . e . 0 . 0 6 jimoles Ca mg ) .  On i n c u b a t i o n a t 37 C, l o s s o f c e l l u l a r c a l c i u m was  observed.  T h i s r e s u l t c l e a r l y i n d i c a t e s t h a t the ghosts had n o t r e s e a l e d i n the l o a d i n g medium d e v o i d o f c a l c i u m and s u s p e n s i o n  ln a  medium c o n t a i n i n g c a l c i u m allowed e n t r y o f c a l c i u m i n t o the c e l l i n t e r i o r and subsequent r e s e a l i n g .  The presence  of 4 . 0  magnesium was thus n o t s u f f i c i e n t f o r complete r e s e a l i n g i n these p r e p a r a t i o n s .  The d i f f e r e n c e between these r e s u l t s and  mM  37  the  r e s u l t s r e p o r t e d by Bramely e t a l . i?6)  the  use o f EDTA i n o u r s t u d i e s , w h i c h r e d u c e s endogenous  may be due t o  c a l c i u m t o v e r y low l e v e l s .  Na.K-ATPase A c t i v i t y  I n R e s e a l e d Red B l o o d C e l l  Ghosts  I n v e s t i g a t i o n o f Na.K-ATPase a c t i v i t y was u n d e r t a k e n t o d e t e r m i n e whether t h e i n t e g r i t y been p r e s e r v e d .  o f t h i s t r a n s p o r t enzyme had  I n g h o s t s l o a d e d w i t h 0 . 5 mM c a l c i u m and 0 . 1  mM EGTA, l i n e a r i t y l n ATP s p l i t t i n g was o b s e r v e d f o r t h i r t y minutes ( F i g . 7 ) .  D e v i a t i o n from l i n e a r i t y occurred a f t e r  t h i r t y m i n u t e s , presumably due t o a d e p l e t i o n o f ATP. C a l c i u m was i n c l u d e d i n t h e l o a d i n g medium t o f a c i l i t a t e r e s e a l l n g o f t h e g h o s t s , and d i d n o t appear t o i n h i b i t t h e Na,K-ATPase a c t i v i t y .  T h i r t y m i n u t e s was t h e r e f o r e  chosen  as t h e i n c u b a t i o n t i m e i n subsequent Na,K-ATPase e x p e r i m e n t s . F i g u r e 7 shows t h a t 0 . 2 mM o u a b a i n , when a p p l i e d  externally,  r e d u c e d t o t a l ATPase a c t i v i t y f r o m 0 . 5 2 5 t o 0 . 3 6 ^nmoles P i mg^hr"* . 1  The d i f f e r e n c e a c c o u n t s f o r t h e Na.K-ATPase p r e s e n t .  I n a n e x p e r i m e n t t o d e t e r m i n e whether Na.K-ATPase c o u l d be a c t i v a t e d a s y m m e t r i c a l l y , t h e c o n c e n t r a t i o n o f p o t a s s i u m was v a r i e d e x t e r n a l l y i n g n o s t s r e s e a l e d w i t h NaCI ( F i g . b). Maximal s t i m u l a t i o n o f ATPase a c t i v i t y o c c u r e d a t 8 . 0 mM e x t e r n a l potassium. the  I n t h e p r e s e n c e o f 0 . 1 MM e x t e r n a l o u a b a i n  ATP s p l i t t i n g s t i m u l a t e d by e x t e r n a l p o t a s s i u m was a b o l -  ished. S i n c e t h e t o t a l ATPase a c t i v i t y o f t h e g h o s t s l n t h e p r e s ence o f 0 . 1 mM o u a b a i n was e q u a l t o t h e a c t i v i t y l n t h e absence  38  1  J  0  10  I 20  I  I  30  40  Time (min) Figure 7. Time course o f ATPase a c t i v i t y i n the absence o f external ouabain (O) and i n the presence o f 0.2 mM external ouabain . ( ® ) . T h e concentration of calcium i n the loading medium v/as  0.5 mM.  39 of added p o t a s s i u m , no Na,  K-ATPase a c t i v i t y was  present  i n g h o s t s r e s e a l e d w i t h NaCI, u n l e s s e x t e r n a l p o t a s s i u m added.  In future determinations  i n t e r f e r e n c e from ATP  of Mg+Ca-ATPase a c t i v i t y ,  s p l i t t i n g due  t o Na.K-ATPase l n c e l l s  r e s e a l e d w i t h NaCI would n o t be e x p e c t e d , s i n c e p o t a s s i u m was  not  external  used.  A d d i t i o n o f 15.0  mM  p o t a s s i u m i n the l o a d i n g medium,  i n the absence o f e x t e r n a l p o t a s s i u m , d i d not i n c r e a s e b a s a l r a t e of ATPase a c t i v i t y ing  (0.3 jimoles m g " h r " ) , 1  Inhibit  mM  K-ATPase.  o u a b a i n i n the l o a d i n g medium d i d  s t i m u l a t i o n due  to e x t e r n a l potassium.  f i n d i n g s d e m o n s t r a t e t h a t b o t h o u b a i n and  The  not  These  two  p o t a s s i u m have  e x t e r n a l b i n d i n g s i t e s , i n agreement w i t h r e s u l t s by p r e v i o u s w o r k e r s ( 6 5 ) .  the indicat-  1  t h a t o n l y e x t e r n a l p o t a s s i u m a c t i v a t e s Na,  A d d i t i o n of 0.1  was  reported  r e s u l t a l s o s u p p o r t s the  t h a t t h e c e l l s have r e s e a l e d , s i n c e i n t e r n a l  view  p o t a s s i u m does  not have a c c e s s t o the o u t s i d e of t h e membrane. I n t h e p r e s e n c e of 1.0  mM  external calcium  (Pig. 8),  t h e a c t i v a t i o n of Na,K-ATPase by e x t e r n a l p o t a s s i u m essentially  unchanged.  (at 10.0  e x t e r n a l p o t a s s i u m ) was  mM  The  specific  a c t i v i t y o f Na,K-ATPase a p p r o x i m a t e l y 0.35  P i mg""*hr~* i n the p r e s e n c e o r absence o f 1.0 calcium. specific  mM  umoles  external  T h e r e f o r e e x t e r n a l c a l c i u m does not a f f e c t a c t i v i t y o f Na,  was  the  K-ATPase.  As a c o m p a r i s o n , the s p e c i f i c  a c t i v i t y o f Na,K-ATPase  obtained  l n a p r e p a r a t i o n o f f r o z e n RBCMF by Watson e t  was  pmoles P i mg~*hr" .  0.28  t h i s s t u d y was  1  0.J5  The  maximal v a l u e o b t a i n e d  /imoles P i m g " h r " . 1  1  al.(40) ln  40  2.0  4.0  6.0  8.0  10.0  12.0  14.0  [ K + ] (mM)  Figure 8. A c t i v a t i o n of Na,K-ATPase a c t i v i t y by external potassium i n the presence of 1.0 mM external calcium (•Kzero external calcium (O) and 0.1 mM external ouabain (#).The concentration of calcium i n the loading medium was 0.5 mM.Other conditions were standard.Each point represents the mean of two experiments.  41 Mg+Ca-ATPase A c t i v i t y In Resealed Red  V a r i a t i o n of the c o n c e n t r a t i o n  Blood C e l l Ghosts  of c a l c i u m  l n the  loading 9).  medium l e d to a marked i n c r e a s e i n ATPase a c t i v i t y  (Fig.  Two  calcium  peaks were obtained,  possibly representing  s t i m u l a t e d Mg+Ca-ATPases. Mg-ATPase and The  finding  The  a c t i v i t y was  two  corrected f o r  non-enzymatic h y d r o l y s i s as l n the Methods. of two  Mg+Ca-ATPases i n r e s e a l e d ghosts was  in  agreement w i t h e a r l i e r s t u d i e s on f r o z e n RBCMF, where the h i g h a f f i n i t y Mg+Ca-ATPase was  o p t i m a l l y a c t i v a t e d by approx-  i m a t e l y 10*^  and  to 10~^M  calcium,  a low a f f i n i t y Mg+Ca-ATPase  -5  k  w i t h o p t i m a l a c t i v a t i o n a t a p p r o x i m a t e l y 10 ^ t o lO'^M In r e s e a l e d ghosts ( F i g . 9 ) ,  h i g h a f f i n i t y Mg+Ca-ATPase and  low a f f i n i t y Mg+Ca-ATPase were maximally s t i m u l a t e d and  5»0mM c a l c i u m  calcium.  by 0.25mM  l n the l o a d i n g medium, r e s p e c t i v e l y .  The  marked d i f f e r e n c e s i n a f f i n i t y f o r c a l c i u m between RBCMF and r e s e a l e d ghosts l e d to the c o n c l u s i o n t h a t the of c a l c i u m  concentrations  i n the l o a d i n g medium were much h i g h e r  than the a c t u a l f r e e I n t r a c e l l u l a r c o n c e n t r a t i o n s a t the b e g i n n i n g loaded  of the i n c u b a t i o n .  with 5«0mM c a l c i u m ,  t h e r e may  For Instance,  of  Furthermore, some of the loaded  calcium  i n ghosts  the c e l l  c a l c i u m may  a t room temperature  e r a b l e amount of c a l c i u m may  1:1  Interior  have been  l o s t d u r i n g the r e s e a l l n g procedure, which r e q u i r e d an a t i o n f o r t e n min.  fold)  not be an e f f i c i e n t  l o a d i n g r a t i o between the l o a d i n g medium and (38).  (^ 10  Isee Methods).  incubA  consid-  be c h e l a t e d by b i n d i n g s i t e s  are not a v a i l a b l e l n RBCMF, e s p e c i a l l y s i n c e such s i t e s  that  may  42  Figure 9. E f f e c t of varying the concentration of calcium i n the loading medium on Mg+Ca-ATPase a c t i v i t y . E a c h point represents the mean of three experiments(+ standard e r r o r s ) .  43 have been d e p l e t e d by EDTA.  Therefore  the concentrations  o f c a l c i u m i n d i c a t e d on t h e a b c i s s a a r e n o t t r u e  intracellular  concentrations of calcium, but represent a r e l a t i v e  intra-  c e l l u l a r c o n c e n t r a t i o n o f c a l c i u m added l n t h e l o a d i n g medium. An E a d i e p l o t (77) o f t h e c a l c i u m a c t i v a t i o n d a t a , two s t r a i g h t l i n e s  revealed  ( P i g . 1 0 ) . The Vmax and t h e 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 f o r t h e l o w a f f i n i t y Mg+Ca-iiTPase were  calcul-  a t e d t o be 0.690 ^imoles P I mg ''"hr" and 1 . 2 3 mM r e s p e c t i v e l y . 1  A Vmax and Ka o f 0.340 p i o l e s P I m g " h r " 1  1  and 1.26 X 10"^M  were o b t a i n e d f o r t h e h i g h a f f i n i t y Mg+Ca-ATPase.  Values  r e p o r t e d by Bader (78) f o r t h e s p e c i f i c a c t i v i t i e s o f Mg+Ca-ATPases I n RBCMF were 0 . 5 and 0.8 jimoles P i m g " h r " 1  low a f f i n i t y Mg+Ca-ATPase, r e s p e c t i v e l y .  1  f o r t h e h i g h and  The s l i g h t l y  lower  v a l u e s r e p o r t e d h e r e f o r t h e r e s e a l e d g h o s t s may be due t o assaying a t s l i g h t l y l e s s than optimal c o n d i t i o n s .  There a r e  p r e l i m i n a r y i n d i c a t i o n s t h a t an Increase i n the c o n c e n t r a t i o n o f magnesium i n t h e l o a d i n g medium w i l l f u r t h e r enhance Mg+Ca-ATPase a c t i v i t y i n t h i s p r e p a r a t i o n .  The E f f e c t Of E x t e r n a l D i v a l e n t C a t i o n s On ATPase A c t i v i t y  P r e v i o u s l y , Schatzmann and V l n c e n z l (12) r e p o r t e d  that  o n l y I n t r a c e l l u l a r c a l c i u m s t i m u l a t e d ATPase a c t i v i t y l n r e s e a l e d g h o s t s , w h i l e e x t e r n a l c a l c i u m had no s i g n i f i c a n t effect.  However, e x t e r n a l d i v a l e n t c a t i o n s were found t o s i g n -  i f i c a n t l y s t i m u l a t e ATPase l n t h i s g h o s t p r e p a r a t i o n ( F i g . 1 1 ) . The o r d i n a t e r e p r e s e n t s t h e i n c r e a s e l n ATPase a c t i v i t i e s  over  44  F i g u r e 10.  Eadie plot of calcium a c t i v a t i o n of Mg+Ca-ATPases i n g h o s t s .  45  (  Ul|  6lU  LfJ S9[0Uirf)A  46  Figure 11. The e f f e c t of external d i v a l e n t cations on ATPase a c t i v i t y . The ghosts were loaded with 0.5 mM Calcium and 0.4 mM EGTA. Other conditions were standard. Ca (O), Mg ( • ) .  4? b a s a l l e v e l s obtained ent c a t i o n s . a t 3.0  mM  The  l n the absence of added e x t e r n a l  maximal i n c r e a s e i n ATPase a c t i v i t y  e x t e r n a l calcium,  i t y of 0.140  occured  w i t h an i n c r e a s e over b a s a l  /imoles P i mg'-'-hr" .  about one-half  dival-  1  activ-  E x t e r n a l magnesium was  as e f f e c t i v e as e x t e r n a l c a l c i u m  only  l n increasing  ATPase a c t i v i t y , w i t h a maximal i n c r e a s e of 0.06  pjnoles P i  mg~^  hr" . 1  Since  i n c r e a s i n g the e x t e r n a l potassium  ( i n the presence of 0.1  mM  concentration  ouabain) d i d not a f f e c t Mg+Ca-ATPase  a c t i v i t y , s t i m u l a t i o n of ATPase a c t i v i t y appears to r e q u i r e d i v a l e n t metal c a t i o n s . To determine i f magnesium and  c a l c i u m were e n t e r i n g  the  c e l l or were s t i m u l a t i n g an e x t e r n a l ATPase such as Na,K-ATPase o r Mg+Ca-ATPase, the c o n c e n t r a t i o n  of c a l c i u m was  varied i n  the l o a d i n g medium of r e s e a l e d g h o s t s suspended i n e x t e r n a l medium c o n t a i n i n g zero c a l c i u m and  1.0  mM  calcium  ( F i g . 12).  S i n c e t h e r e i s no apparent s h i f t i n the p o s i t i o n of the a c t i v a t i o n curve l n the presence of 1.0  mM  calcium,  calcium  external  c a l c i u m does not have a c c e s s  t o the c e l l u l a r I n t e r i o r .  u l a t i o n must t h e r e f o r e be due  t o c a l c i u m a c t i n g on the  The  stim-  external  s u r f a c e of the membrane. S i n c e e x t e r n a l c a l c i u m d i d not a f f e c t the Na,K-ATPase a c t i v i t y as shown p r e v i o u s l y ( F i g . 8) the e x t r a ATPase a c t i v i t y seen l n the presence of 1.0  mM  external calcium  i s not  to s t i m u l a t i o n o f Na.K-ATPase a t an e x t e r n a l b i n d i n g The  p o s s i b i l i t y t h a t some ghosts were p r e s e n t  due  site.  which d i d  not  r e s e a l and were being a c t i v a t e d i s u n l i k e l y because of the  high  4.0  3.0  2.0  pCa  gure 12.  Effect of varying the concentration of calcium in the loading medium on Mg+Ca-ATPase a c t i v i t y in the absence of external calcium (#) and in the presence of 1.0 mM external calcium (OJ.0.4 mM E6TA was included in the loading medium.Other conditions were standard.Each point represents the mean of at least two experiments.  49 concentrations of d i v a l e n t cations required f o r t h i s  activation,  r e l a t i v e t o the c o n c e n t r a t i o n s r e q u i r e d t o s t i m u l a t e membrane fragments. I t was s p e c u l a t e d t h a t c a l c i u m and magnesium may be r e p l a c i n g endogenous magnesium and c a l c i u m removed from the membrane by EDTA.  The b i n d i n g o f c a l c i u m o r magnesium may r e s t o r e the  conformation  Mg+Ca-ATPase, r e s u l t i n g i n an i n c r e a s e d a c t i v i t y . >  The E f f e c t Of I n t e r n a l Calcium  On Calcium  Efflux  Q u a l i t a t i v e s t u d i e s i n d i c a t i n g t h a t the v e l o c i t y of c a l c i u m e f f l u x i s dependent on the I n t e r n a l c o n c e n t r a t i o n of c a l c i u m have been p r e v i o u s l y r e p o r t e d l n r e s e a l e d g h o s t s whole r e d blood c e l l s  (7).  (30) and i n  Schatzmann and B o s s i ( 3 0 ) ,  however,  were unable t o demonstrate a c o r r e l a t i o n between a c t i v a t i o n of Mg+Ca-ATPase and the v e l o c i t y of c a l c i u m e f f l u x i n t h e i r preparations of resealed  ghosts.  I n t h i s study ghosts were loaded w i t h d i f f e r e n t r a t i o n s o f c a l c i u m and r e s e a l e d as i n the Methods.  concentThe l o s s o  of c e l l u l a r c a l c i u m was determined as a f u n c t i o n of time a t 37 C. ( P i g . 13)• 0.1  I n ghosts  t o 5 . 0 mM  apparent.  l o a d e d over the c o n c e n t r a t i o n range o f  c a l c i u m , a r a p i d l o s s of c e l l u l a r c a l c i u m  was  I n most o f the ghosts an e q u i l i b r i u m l e v e l o f c e l l -  u l a r c a l c i u m was reached  w i t h i n t e n minutes.  The c o n c e n t r a t i o n  of c a l c i u m l n the ghosts a t time z e r o was d i r e c t l y p r o p o r t i o n a l  50  0  10  20  30  Time (min)  Figure 13.  Changes i n the concentration of c e l l u l a r calcium with time. Each point represents the mean of two to three experiments (+ standard e r r o r s ) .except where no bars are shown. The concentration of calcium i n the loading medium was 0.5 mM ( • ) , 1.0 mM (A), 2.0 mM ( O ) . 3.0 mM (@) and 5.0 mM ( A ) .  51 to  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 l n t h e l o a d i n g medium. 2+ -1  i s shown l n f i g u r e 14 where a p l o t o f jimoles Ca  mg  This against  the c o n c e n t r a t i o n o f c a l c i u m i n t h e l o a d i n g medium y i e l d e d a straight  line.  E v i d e n c e was o b t a i n e d  showing t h a t t h e l o s s o f c e l l u l a r  c a l c i u m was due t o a c t i v e c a l c i u m t r a n s p o r t .  For instance, i f  ATP was n o t i n c l u d e d l n t h e l o a d i n g medium, a l o s s o f c e l l u l a r c a l c i u m o v e r time d i d n o t o c c u r , i n d i c a t i n g t h a t t h e c a l c i u m l o s s was a n ATP dependent p r o c e s s .  T h i s f i n d i n g a l s o shows t h a t  endogenous ATP was removed d u r i n g t h e p r e p a r a t i o n o f t h e g h o s t s . Furthermore, a l o s s of c e l l u l a r calcium occurred  i n ghosts  l o a d e d w i t h l e s s t h a n 1.0 mM c a l c i u m , showing t h a t i n t r a c e l l u l a r c a l c i u m was t r a n s p o r t e d a g a i n s t an e l e c t r o c h e m i c a l p o t e n t i a l . As 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 i n t h e l o a d i n g medium was i n c r e a s e d f r o m 0.1 mM t o 5 . 0 mM, t h e r e i s a n I n c r e a s e l n the steepness o f the slopes of the curves, i n d i c a t i n g t h a t the v e l o c i t y of t r a n s p o r t i s i n c r e a s i n g as the concentration of c a l c i u m I s i n c r e a s e d i n t h e l o a d i n g medium. obtained  The e f f l u x  conform t o a f i r s t o r d e r p r o c e s s , i n t h a t f o r each  curve t h e v e l o c i t y o f e f f l u x decreases e x p o n e n t i o n a l l y time.  curves  with  Thus a p l o t o f l o g c a l c i u m c o n t e n t f r o m t h e d a t a i n  f i g u r e 13 a g a i n s t t i m e y i e l d e d s t r a i g h t l i n e s .  From t h e s e  s t r a i g h t l i n e s e x t r a p o l a t e d p o i n t s f o r the f i r s t t e n minutes were o b t a i n e d .  I n i t i a l v e l o c i t i e s c o u l d t h e n be a c c u r a t e l y  d e t e r m i n e d by r e p l o t t i n g t h e d a t a on a p l o t o f c o n c e n t r a t i o n of c a l c i u m a g a i n s t t i m e .  The f i r s t t h r e e m i n u t e s were n o t used  l n the c a l c u l a t i o n s o f i n i t i a l v e l o c i t i e s s i n c e t h i s time  52  Figure 14.  Relationship between c e l l u l a r calcium and calcium in the loading medium.Each point represents the mean of three experimental determinations (+ standard errors).  5 3  r e p r e s e n t s the time r e q u i r e d f o r the tubes to warm up from 0°C  t o 37°C (see F i g . 1 3 ) . A p l o t of the i n i t i a l v e l o c i t i e s of c a l c i u m e f f l u x a g a i n s t  the c o n c e n t r a t i o n of c a l c i u m i n the l o a d i n g medium y i e l d e d s l g m o l d a l curve  ( F i g . 15»  open c i r c l e s ) .  t r a t i o n of c a l c i u m from 0.1 mM  t o 1.0  I n c r e a s i n g the  mM  s t i m u l a t i o n of c a l c i u m e f f l u x o c c u r r e d i n ghosts c o n c e n t r a t i o n s of c a l c i u m g r e a t e r than 1.0  mM.  the a c t i v a t i o n of Mg+Ca-ATPase a c t i v i t y two suggesting  system p r e s e n t .  t h a t t h e r e i s o n l y one  F i g u r e 15  o f both ATPase a c t i v i t y and  A more marked loaded  In contrast to  peaks were not calcium transport  c a l c i u m e f f l u x are p a r a l l e l i n the t o 5«0 mM,  a f f i n i t y Mg+Ca-ATPase i s thought to o p e r a t e .  where the  c a l c i u m i o n s t r a n s p o r t e d per molecule of ATP c a l c i u m c o n c e n t r a t i o n s of l e s s t h a t 1.0  mM,  measured appears t o be l e s s than one,  t h a t a t low  t h e r e I s an ATPase s p l i t t i n g  The  the  At  high  stoichiometry  the shapes of  the  the ATPase c a l c i u m a c t i v a t i o n curves do  T h i s suggests  i s not coupled  and  two  hydrolyzed. where the  a f f i n i t y Mg+Ca-ATPase i s thought to operate,  low  A comparison of  the curves i n t h i s r e g i o n I n d i c a t e s a s t o i c h i o m e t r y of  coincide.  with  indicates that calcium s t i m u l a t i o n  c a l c i u m c o n c e n t r a t i o n range of 1.0  c a l c i u m e f f l u x and  concen-  d i d not r e s u l t i n a  l a r g e i n c r e a s e l n the v e l o c i t y of c a l c i u m e f f l u x .  obtained  a  calcium  not  concentrations  ( h i g h a f f i n i t y Mg+Ca-ATPase) which  to calcium t r a n s p o r t .  s t o i c h i o m e t r y of two  (Ca:ATP) c o n t r a s t s to the  prev-  i o u s l y r e p o r t e d v a l u e of 0.77  ( 1 2 ) , but agrees w i t h the  stoichio-  metry found f o r caloium t r a n s p o r t l n muscle  sarcoplasmic  54  pCa  Figure 15.  Comparison of the velocity of active calcium transport and Mg+Ca-ATPase a c t i v i t y as a function of calcium in the loading mediumjhe Mg+Ca-ATPase data represents: the mean of three experiments and the standard errors are smaller than the c i r c l e s drawn.The calcium transport data represent the mean of three determinations (_+ standard errors).except where no bars are shown.where points represent the mean of duplicate determinations.  55 (5*0 •  reticulum  I t seems more r e a s o n a b l e  t h a t the  pump l n r e d blood c e l l s would a l s o have a h i g h  calcium  stoichiometry,  s i n c e l e s s energy would have to be expended i n e x t r u d i n g g i v e n amount of I n t r a c e l l u l a r The  e f f l u x d a t a was  a  calcium. (68)  p l o t t e d a c c o r d i n g to H i l l  ln  o r d e r t o I n v e s t i g a t e the meaning of the s i g m o l d a l shape o f the c a l c i u m e f f l u x curve  ( F i g . 16).  l i n e s were o b t a i n e d w i t h H i l l a t e l y 0.39 and 1.0  At l e a s t two  coefficients  (n) of approxim-  f o r c o n c e n t r a t i o n s of c a l c i u m l e s s t h a n 1.0  f o r ghosts  loaded w i t h g r e a t e r than 1.0  T h i s change i n n v a l u e s may  c a l c i u m may  mM  mM  calcium.  mean t h a t the c a l c i u m t r a n s p o r t  system has a c o o p e r a t i v e component. of  straight  B i n d i n g of one  molecule  i n c r e a s e the a f f i n i t y of the t r a n s p o r t system  for  the b i n d i n g of another  molecule of c a l c i u m , a t a t h r e s h -  old  v a l u e of a p p r o x i m a t e l y  1.0  medium.  mM  c a l c i u m In the l o a d i n g  Changes i n n v a l u e s o f t e n r e f l e c t a change i n the  conformation  A  sub-  u n i t s t r u c t u r e f o r Mg+Ca-aTPase l n r e d blood c e l l s has not  been  reported.  or" s p a c i a l arrangement o f enzyme s u b u n l t s .  However, Kyte (71)  r e c e n t l y r e p o r t e d t h a t the Na,>K-  pump i n t h e plasma membrane of r e n a l c o r t e x appears to have a subunlt s t r u c t u r e .  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 t h a t the  break l n the s l o p e o f f i g u r e 16 may two  be due  t o the presence of  c a l c i u m a c t i v a t e d c a l c i u m t r a n s p o r t systems.  presented  above do not r u l e out t h i s p o s s i b i l i t y .  r e s u l t s o b t a i n e d u s i n g ruthenium r e d make t h i s e x p l a n a t i o n l e s s  likely.  The  results  However,  ( t o be d i s c u s s e d  later)  -4.0  •3.0  -2.0  Log [Ca2+](M)  Figure 16.  Hill plot of calcium activation of calcium efflux in ghosts.v is the velocity of calcium efflux and V is the maximum velocity of this efflux,as determined from a Lineweaver-Burk plot.  57  The E f f e c t Of Ruthenium Red On Mg+Ca-ATPase  Activities  I n the p r e v i o u s s e c t i o n , a s i m i l a r i t y between the shape of the c a l c i u m a c t i v a t i o n curves f o r the low a f f i n i t y Mg+CaATPase and the c a l c i u m t r a n s p o r t system was noted.  This a s s o c i a t i o n  was f u r t h e r I n v e s t i g a t e d u s i n g ruthenium r e d as a t o o l .  Watson  et a l . (40) r e p o r t e d t h a t ruthenium r e d , an I n o r g a n i c dye used l n s t a i n i n g mucopolysaccharides, Mg+Ca-ATPase a c t i v i t y l n RBCMF.  selectively  inhibited  No d i s t i n c t i o n , however, was  made between I n h i b i t i o n o f h i g h o r low a f f i n i t y  Mg+Ca-ATPase  a c t i v i t y o r whether c a l c i u m t r a n s p o r t was a l s o i n h i b i t e d . In t h i s study ruthenium r e d , added e x t e r n a l l y , was  found  t o i n h i b i t Mg+Ca-ATPase i n a dose dependent manner i n ghosts loaded w i t h 3 . 0 and 5 . 0 mM calcium.* ( F i g . 1 7 ) .  I n the ghosts  loaded w i t h 3 . 0 and 5*0 mM c a l c i u m , the i n h i b i t i o n curves were found  t o be p a r a l l e l .  S i n c e the I ^ Q v a l u e o f ruthenium r e d  a t both c o n c e n t r a t i o n s o f I n t e r n a l c a l c i u m was 0 . 2 mM, the mechanism o f I n h i b i t i o n would not be expected with r e s p e c t t o c a l c i u m .  t o be c o m p e t i t i v e  T h i s f i n d i n g i s not s u r p r i s i n g l n  view o f the f a c t s which suggest  t h a t ruthenium r e d I n h i b i t s by  b i n d i n g t o a n i o n i c s i t e s on t h e o u t s i d e o f membranes. has p r e v i o u s l y been shown here and elsewhere (12)  Calcium  to a c t i v a t e  Mg+Ca-ATPase on the I n t e r n a l s u r f a c e o f the membrane.  Evidence  s u p p o r t i n g an e x t e r n a l b i n d i n g s i t e f o r ruthenium r e d a r e as follows: (1) cubated  A p r e i n c u b a t i o n time was n o t r e q u i r e d .  Ghosts p r e I n -  i n 0 . 2 mM ruthenium r e d f o r 0 , 1 0 , 20 and 30 minutes a t  58  Figure 17.  Effect of ruthenium red in the external medium on Mg+Ca-ATPase activity.The concentration of ruthenium red was varied in ghosts loaded with o.5 mM calcium ( D ^ . O mM calcium (O) and 5.0 mM calcium (•).  59 2-1°C. i n h i b i t e d  low a f f i n i t y Mg+Ca-ATPase by 50#.  I f ruth-  enium r e d was p e n e t r a t i n g I n t o the i n t e r i o r o f the ghosts,and then i n h i b i t i n g Mg+Ca-ATPase, g r e a t e r i n h i b i t i o n would be expected f o r ghosts p r e i n c u b a t e d f o r l o n g e r p e r i o d s o f time, (2) Ruthenium r e d does not p e n e t r a t e through the plasma membrane o f I n t a c t c e l l s as evidenced from e l e c t r o n  microscopy  (72). (3)  P e n e t r a t i o n o f ruthenium r e d through the plasma  membrane o f r e s e a l e d ghosts would be v e r y slow s i n c e r u t h e n ium r e d possesses a hexavalent p o s i t i v e  charge ( 4 0 ) .  Ruthenium r e d , t h e r e f o r e , p r o b a b l y e x e r t s I t s I n h i b i t i o n by b i n d i n g t o mucopolysaccharides the g h o s t .  on the o u t s i d e s u r f a c e o f  B i n d i n g a t these s i t e s may induce an u n f a v o u r a b l e  c o n f o r m a t i o n a l p e r t u r b a t i o n o f the membrane bound Mg+Ca-ATPase, l e a d i n g t o a r e d u c t i o n i n the c a t a l y t i c e f f i c i e n c y enzyme.  of t h i s  B i n d i n g t o an a l l o s t e r i c s i t e would a l s o f a v o u r a  non-competitive type o f i n h i b i t i o n by ruthenium r e d . In ghosts r e s e a l e d w i t h 0 . 5 mM c a l c l u m 0 . 2 y  ruthenium  mM  r e d d i d n o t a f f e c t Mg+Ca-ATPase a c t i v i t y  In o r d e r t o e x p l a i n t h i s analomous r e s u l t ,  external ( F i g . 17).  the c o n c e n t r a t i o n  of c a l c i u m was v a r i e d l n t h e l o a d i n g medium, the ghosts were r e s e a l e d and suspended i n a medium c o n t a i n i n g 0,2mM ruthenium red  (Fig.18).  Ruthenium r e d had e s s e n t i a l l y  no e f f e c t on  Mg+Ca-ATPase a c t i v i t y a t c o n c e n t r a t i o n s o f c a l c i u m lower  than  l.OmM, where the h i g h a f f i n i t y Mg+Ca-ATPase i s c o n s i d e r e d t o operate.  However, the low a f f i n i t y Mg+Ca-ATPase was  inhibited  by ruthenium r e d .  markedly  The % i n h i b i t i o n was dependent on  60  CD  to CU  o  +-> o <C  CroO 01  ex.  t— I  « +  4.0  3.0  2.0  pCa  Figure 18.  The effect of ruthenium red on the activation of Mg+Ca-ATPase. The calcium concentration in the loading medium was varied in the absence of ruthenium red ( •) and in the presence of 0.2 mM ruthenium red (O)in the external medium.Where bars are shown the data represent the mean of three experiments (+ standard errors).Other points represent the mean of duplicate determinations.  61 the c o n c e n t r a t i o n o f c a l c i u m l n the l o a d i n g medium. example, ghosts  loaded w i t h 2 . 0  mM  £1% whereas ghosts  loaded w i t h 3 . 0  by WIJ> (see F i g .  21).  The E f f e c t Of Ruthenium Red  mM  by  c a l c i u m were i n h i b i t e d  Transport  shown to s e l e c t i v e l y  low a f f i n i t y Mg+Ca-ATPase, t h i s dye for  c a l c i u m were i n h i b i t e d  On Calcium  S i n c e ruthenium r e d was  For  Inhibit  showed promise as a  tool  i n v e s t i g a t i n g whether o r not a Mg+Ca-ATPase i s a s s o c i a t e d  with calcium transport.  I n ghosts loaded w i t h 3 . 0  mM  calcium,  e x t e r n a l ruthenium r e d i n h i b i t e d the v e l o c i t y of a c t i v e c a l c i u m t r a n s p o r t i n a dose dependent manner ( F i g . 1 9 ) . to be 0 . 2  mM,  The I ^  which i s the same v a l u e  value  Q  was  estimated  estimated  for  the i n h i b i t i o n of the low a f f i n i t y Mg+Ca-ATPase ( F i g .  Thus both the a c t i v e c a l c i u m t r a n s p o r t system and low  17).  affinity  Mg+Ca-ATPase have a s i m i l a r a f f i n i t y f o r ruthenium r e d . The  effect of0.2  mM  e x t e r n a l ruthenium r e d on the  activa-  t i o n of a c t i v e c a l c i u m t r a n s p o r t by I n t e r n a l c a l c i u m was determined of  (Fig. 20).  The  c a l c i u m t r a n s p o r t was  a f f i n i t y Mg+Ca-ATPase.  p a t t e r n of ruthenium r e d  also  inhibition  e s s e n t i a l l y the same as t h a t on  low  The % i n h i b i t i o n by ruthenium r e d  i n c r e a s e d w i t h an i n c r e a s e i n the c o n c e n t r a t i o n of c a l c i u m from 1.0 of  mM  to 3.0  mM  and  I n h i b i t i o n by 0 . 2  then l e v e l e d o f f ( F i g . 2 1 ) . mM  Thus the degree  ruthenium r e d depends on the  t i o n of c a l c i u m l n the l o a d i n g medium (see F i g . 2 0 ) .  concentraThere are  62  [Ruthenium Red] (mM)  Figure 19. Effect of ruthenium red in the external medium on the velocity of active calcium transport in ghosts loaded with 3.0 mM calcium.  63  1.4  1.2  1.0  .8  .4  4.0  3.0  2.0  pCa  Figure 20.  The effect of ruthenium red on the velocity of active calcium transport.The calcium concentration in the loading medium was varied in the absence of ruthenium red ( •) and in the presence of 0-2 mM ruthenium red (O).The data indicated by the open c i r c l e s represent the mean of two determinations.  64  Figure 21.  The effect of ruthenium red (0.2 mM) in the external medium on Mg+Ca-ATPase activity ( O ) and active calcium transport (•) as a function of the concentration of calcium in tne loading medium.  65 two  p o s s i b l e e x p l a n a t i o n s f o r t h i s behaviour.  I t I s poss-  i b l e t h a t t h e r e a r e more b i n d i n g s i t e s a v a i l a b l e f o r r u t h e n ium r e d a t h i g h e r c o n c e n t r a t i o n s of i n t e r n a l c a l c i u m ,  since  the volume o f the r e d c e l l i s r e g u l a t e d by the I n t e r n a l concent r a t i o n of calcium  (10).  A more l i k e l y e x p l a n a t i o n becomes  apparent upon r e e x a m i n a t i o n noted  o f f i g u r e 16.  I t was p r e v i o u s l y  t h a t t h e r e was a change l n the H i l l c o e f f i c i e n t s from  .39 t o 1.0 as the c o n c e n t r a t i o n o f c a l c i u m was v a r i e d i n the l o a d i n g medium.  I t was suggested  t h a t t h i s change may be due  to a change l n the s u b u n i t s t r u c t u r e o f the enzyme.  I f low  a f f i n i t y Mg+Ca-ATPase and t h e c a l c i u m t r a n s p o r t system a r e indeed a s s o c i a t e d , the same e x p l a n a t i o n would h o l d here t o o . Thus the b i n d i n g o f ruthenium r e d t o e x t e r n a l mucopolysacchar i d e s c o u l d prevent  the a b i l i t y o f c a l c i u m t o a f f e c t an i n t e r -  a c t i o n o f the t r a n s p o r t enzyme s u b u n l t s o r an Increase i n cooperativity.  Ruthenium r e d c o u l d thus bind t o and s t a b i l i z e  the l e s s a c t i v e form o f the t r a n s p o r t system,so t h a t o n l y a c e r t a i n maximal v e l o c i t y o f c a l c i u m e f f l u x and Mg+Ca-ATPase a c t i v i t y would be o b t a i n a b l e .  Support o f t h i s h y p o t h e s i s  awaits  i s o l a t i o n o f the low a f f i n i t y Mg+Ca-ATPase and i d e n t i f i c a t i o n of a s u b u n i t s t r u c t u r e . P r e l i m i n a r y experiments a l s o i n d i c a t e t h a t i n h i b i t i o n by ruthenium r e d may be temperature dependent. 0.4 .mM  e x t e r n a l ruthenium r e d Increased  e f f l u x i n ghosts minutes.  For instance,  the l a g time f o r c a l c i u m  loaded w i t h 3.0 mM c a l c i u m from t h r e e t o f o u r  Furthermore, membranes must be p r e i n c u b a t e d a t  2 t o 4 C. i n o r d e r f o r i n h i b i t i o n t o be o b t a i n e d .  The p o s s i b -  i l i t y thus e x i s t s t h a t ruthenium r e d can o n l y b i n d t o one  66 form o f the t r a n s p o r t enzyme.  T h i s form may be the more  e f f i c i e n t t r a n s p o r t form, induced  by h i g h c o n c e n t r a t i o n s o f  i n t r a c e l l u l a r c a l c i u m and p o s s i b l y by low  temperatures.  The E f f e c t Of Drugs (Quinine. Q u i n l d i n e . and P r o p r a n o l o l ) On Mg+Ca-ATPase  Q u i n l d i n e has been shown to i n h i b i t the b i n d i n g of c a l c i u m i n i s o l a t e d s a r c o p l a s m i c r e t i c u l u m (79)•  The p o s s i b i l i t y was  c o n s i d e r e d t h a t t h i s drug may have e f f e c t s on o t h e r systems c o n t r o l l i n g the d i s t r i b u t i o n o f c a l c i u m , such as a c t i v e c a l c i u m t r a n s p o r t a c r o s s the plasma membrane.  The human e r y t h r o c y t e  was used as a model system t o determine whether q u i n l d i n e o r q u i n i n e a f f e c t e d low a f f i n i t y Mg+Ca-ATPase i n r e s e a l e d o r RBCMF.  Both q u i n l d i n e and q u i n i n e were found  t o be l n e f f e c -  -3  -5  t l v e over a c o n c e n t r a t i o n range o f 10  ghosts  M t o 10  M i n both  prep-  arations.  I n t e r f e r e n c e by these drugs on t h e i n o r g a n i c phosp-  hate assay  (63) a t c o n c e n t r a t i o n s g r e a t e r than 10 ^M was  observed. P r o p r a n o l o l was a l s o without i n r e s e a l e d ghosts The  e f f e c t on Mg+Ca-ATPase  l n the c o n c e n t r a t i o n range o f 10  J  activity  t o 10  M.  e f f e c t o f these drugs on a c t i v e c a l c i u m t r a n s p o r t was  not determined.  67  The E f f e c t Of Manganese On Mg+Ca-ATPase  Activity  Bader ( 4 3 ) r e c e n t l y r e p o r t e d t h a t manganese can s u b s t i t u t e f o r c a l c i u m l n a c t i v a t i n g low a f f i n i t y Mg+Ca-ATPase i n RBCMF. Manganese was r e p o r t e d t o a c t i v a t e low a f f i n i t y  Mg+Ca-ATPase  w i t h t h e same potency as c a l c i u m and t o have no e f f e c t on h i g h a f f i n i t y Mg+Ca-ATPase.  T h e r e f o r e manganese was c o n s i d e r e d  as a p o t e n t i a l t o o l f o r d i f f e r e n t i a t i n g enzyme f u n c t i o n . The  c o n c e n t r a t i o n o f manganese was v a r i e d l n the l o a d i n g  medium I n t h e presence o r absence o f 1 . 0 mM e x t e r n a l c a l c i u m (Fig. 2 2 ) .  Calcium was n o t Included  these experiments.  i n t h e l o a d i n g medium i n  I n h i b i t i o n o f ATPase a c t i v i t y was observed  a t c o n c e n t r a t i o n s o f manganese l e s s than 1.0 mM.  The a c t i v i t y  r e t u r n e d t o t h e c o n t r o l l e v e l a t 5 « 0 mM manganese.  The concent-  r a t i o n s o f manganese was a l s o v a r i e d i n the absence o f 1 . 0 mM e x t e r n a l c a l c i u m t o ensure t h a t the s l i g h t a c t i v a t i o n o c c u r r i n g between 3 « 0 and 5 . 0 mM manganese was due t o manganese and n o t external calcium leaking i n t o the ghosts.  S i n c e t h e i r was no  a p p r e c i a b l e change i n the two r e s u l t s , manganese may be a b l e t o s u b s t l t u e f o r c a l c i u m i n r e s e a l i n g t h e g h o s t s , however, i t s e f f e c t on a c t i v a t i n g Mg+Ca-ATPase seems n e g l i g i b l e . Manganese d i d n o t a c t i v a t e c a l c i u m a c t i v a t e d ATPase i n RBCMF. I t i s p o s s i b l e that contamination  o f t h e manganese w i t h  calcium  c o u l d account f o r t h e d i f f e r e n c e I n r e s u l t s o b t a i n e d here and those r e p o r t e d by Bader ( 4 3 ) .  68  Figure 22.  Effect of varying the concentration of manganese in the loading medium on ATPases a c t i v i t y in the absence of external calcium (O) and in the presence of 1.0 mM external cal cium(#) .Each point represents a single experiment.  69 CONCLUSIONS  A suitable preparation developed i n t h i s s t u d y .  of r e s e a l e d e r y t h r o c y t e  The use of 1.0  procedure r e s u l t e d i n a ghost p o p u l a t i o n endogenous ATP and d i v a l e n t c a t i o n s . has  advantages over p r e v i o u s  media o r i o d o a c e t a t e  p r i o r to preparation  mM EDTA i n the washing e s s e n t i a l l y f r e e of  T h i s method o f p r e p a r a t i o n  methods used f o r p r e p a r i n g  f r e e ghosts s i n c e l o n g p r e i n c u b a t i o n depleted  periods  treatment o f the whole c e l l s  pared by t h i s method r e q u i r e d  Ghosts  the a d d i t i o n of c a l c i u m  t o t h e l o a d i n g medium i n o r d e r  to r e s e a l .  resealed  (> . 0 5  o f membrane ATPase(s) i n the  f o r both Na,K-ATPase and Mg+Ca-ATPase.  evidence was g i v e n i n d i c a t i n g t h a t the ghosts had (p 35) and t h a t Na,K-ATPase and Mg+Ca-ATPase  asymmetrically  stimulated  c o u l d be  by c a t i o n s .  In c o n t r a s t t o a r e p o r t by Schatzmann and V i n c e n z i i t was found t h a t e x t e r n a l d i v a l e n t c a t i o n s  preparation.  mechanism o f a c t i o n was a t t r i b u t e d t o the replacement o f  membrane d i v a l e n t c a t i o n s removed by EDTA and  (12)  (Mg and Ca) f u r t h e r  i n c r e a s e d Mg+Ca-ATPase a c t i v i t y i n t h i s ghost The  mM)  The use o f EDTA d i d  s i n c e s p e c i f i c a c t i v i t i e s comparable t o those r e p o r t e d  Further  pre-  Magnesium was unable  l n t h i s respect.  not r e s u l t i n removal o r d e n a t u r a t i o n  l i t e r a t u r e were o b t a i n e d  ATP  i n metabolically  o f the ghosts i s not r e q u i r e d .  to s u b s t i t u t e f o r calcium  ghosts was  Vincenzi).  Presumably c a l c i u m  m a i n t a i n the most f a v o u r a b l e  (not used by Schatzmann  o r magnesium a r e r e q u i r e d t o  c o n f o r m a t i o n o f t h i s enzyme.  It  was o f I n t e r e s t t h a t t h e s p e c i f i c a c t i v i t y o f Na.K-ATPase was  70 n o t i n c r e a s e d by t h e s e e x t e r n a l d i v a l e n t m e t a l c a t i o n s .  This  s t u d y was t h e f i r s t t o show the. p r e s e n c e o f a h i g h and low a f f i n i t y Mg+Ca-ATPase l n r e s e a l e d human e r y t h r o c y t e g h o s t s . P r e v i o u s s t u d i e s had shown a h i g h and low a f f i n i t y Mg+Ca-ATPase l n RBCMF.  However, t h e c o n c e n t r a t i o n s o f c a l c i u m i n t h e l o a d -  i n g medium were more t h a n t e n t i m e s h i g h e r t h a n t h e c o n c e n t r a t ions of calcium r e q u i r e d t o achieve optimal a c t i v a t i o n l n RBCMF.  I t was s u g g e s t e d t h a t t h e c o n c e n t r a t i o n s o f c a l c i u m  i n t h e l o a d i n g medium do n o t r e p r e s e n t t h e t r u e i n t r a c e l l u l a r c o n c e n t r a t i o n o f f r e e c a l c i u m , f o r r e a s o n s o u t l i n e d on p 42 and r e f e r e n c e 30. The  present s t u d i e s provide strong evidence that only  t h e h y d r o l y s i s o f ATP due t o low a f f i n i t y Mg+Ca-ATPase i s coupled to calcium transport.  A s t o i c h i o m e t r y o f two was f o u n d  i n c o n t r a s t t o a p r e v i o u s e s t i m a t e by Schatzmann and V l n c e n z l ( 1 2 ) , who r e p o r t e d a s t o i c h i o m e t r y o f 0.77. study  However, t h e i r  (12) was done w i t h o n l y one i n t e r n a l c a l c i u m c o n c e n t r a t i o n  (1.0 mM).  I n a d d i t i o n , t h e p r e s e n t s t u d y has shown t h a t a  s t o i c h i o m e t r y o f l e s s t h a n one i s o b t a i n e d a t low c o n c e n t r a t i o n s o f c a l c i u m where t h e h i g h a f f i n i t y Mg+Ca-ATPase i s a l s o f u n c t ioning.  The r e s u l t s f u r t h e r i n d i c a t e t h a t t h e o p e r a t i o n o f  t h e c a l c i u m pump i s a c o o p e r a t i v e p r o c e s s ; t h e pump becoming more e f f i c i e n t as 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 i s i n c r e a s e d . T h i s mechanism may be v e r y i m p o r t a n t t o t h e s u r v i v a l o f t h e c e l l i n s i t u a t i o n s i n w h i c h t h e i n t e r n a l c a l c i u m c o n c e n t r a t i o n becomes high.  Ruthenium r e d may prove t o be a u s e f u l t o o l f o r i n v e s t -  i g a t i n g t h e c o o p e r a t i v e a s p e c t s o f t h e mechanism o f c a l c i u m  71 t r a n s p o r t , s i n c e t h i s dye i n h i b i t s the more e f f i c i e n t of  the t r a n s p o r t system.  form  The f i n d i n g t h a t the i n h i b i t i o n by  ruthenium r e d i n c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s of c a l c i u m l n r e s e a l e d ghosts was n o t found  i n RBCMF where  ruthenium r e d I n h i b i t e d e q u a l l y a t a l l c o n c e n t r a t i o n s o f calcium  (76).  T h i s f i n d i n g I m p l i e s t h a t i n RBCMF o n l y one  s t a t e o f the enzyme can e x i s t , i n c o n t r a s t t o the enzyme l n the r e s e a l e d  ghost.  The f i n d i n g t h a t low a f f i n i t y Mg+Ca-ATPase may be a s s o c i a t e d w i t h c a l c i u m t r a n s p o r t was a t f i r s t s u r p r i s i n g s i n c e one might expect  somewhat  t h a t the Mg+Ca-ATPase w i t h  the h i g h e s t a f f i n i t y f o r c a l c i u m might be best s u i t e d f o r m a i n t a i n i n g a low i n t r a c e l l u l a r c a l c i u m . a f f i n i t y Mg+Ca-ATPase possesses  However the low  a higher s p e c i f i c a c t i v i t y  the h i g h a f f i n i t y Mg+Ca-ATPase and may possess  a threshold  s u f f i c i e n t l y low t o m a i n t a i n a low I n t r a c e l l u l a r of  calcium.  than  concentration  I n c o n d i t i o n s of low pH and low oxygen t e n s i o n  which can o c c u r l n the s p l e e n can e n t e r the r e d blood c e l l .  (84,85). extracellular  calcium  I n such cases the low a f f i n i t y  Mg+Ca-ATPase may be w e l l adapted t o h a n d l i n g h i g h  internal  calcium conditions. The f u n c t i o n of t h e h i g h a f f i n i t y Mg+Ca-ATPase was not determined l n t h i s study,  but may be a s s o c i a t e d w i t h the a c t l n -  l i k e p r o t e i n ( s p e c t r i n ) i s o l a t e d from the Inner a s p e c t of the RBC ( 5 5 ) . Q u i n i n e , q u i n l d i n e and p r o p r a n o l o l were found  t o have  no e f f e c t on Mg+Ca-ATPase l n r e s e a l e d ghosts when added externally or I n t e r n a l l y .  either  These drugs a r e thought t o be membrane  s t a b i l i z i n g agents and i t was a n t i c i p a t e d t h a t a c t i v i t y might  rtg+Ca-/iTPase  be blocked i f these drugs a c t by expansion of  the membrane and by producing unfavourable c o n f o r m a t i o n a l p e r t u r b a t i o n s i n the membrane  microenvironment.  73 BIBLIOGRAPHY  1.  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T r a n s f u s i o n , 238-245, 1 9 6 9 .  80  APPENDIX  81 Table A E f f e c t of i n t e r n a l calcium on the v e l o c i t y of calcium efflux. r -i -log [GaJ i n the loading medium (M)  mnolesCa mg-1 ; hr-1 mean + SE n  4.0  .279  3.6  .291  .042  2  3.3  .337  .027  3  3.0  .427  .031  3  2.7  .742  .072  3  2.5  1.035  .021  3  2.3  1.15  .076  3  1  Table B  E f f e c t of i n t e r n a l calcium on the v e l o c i t y of Mg+Ca-ATPase. -log fCaJ l n the loading medium (M)  umoles PI mp; hr mean + SE n  4.0  .296  .005  3  3.6  .333  .007  3  3.3  .316  .006  3  3.0  .350  .007  3  2.7  .411  .004  3  2.52  .4926  .020  4  2.39  .5525  2.3  .577  1 .014  3  82 Table C  E f f e c t o f e x t e r n a l ruthenium r e d on the v e l o c i t y of calcium t r a n s p o r t . [Ca] i n the l o a d l n g medium (mM)  [Ruthenium Red] (mM)  umoles Ca^mg'^hr--  3.0  0  1.074, 1.012, 1.02  3.0  .1  .610, .635  3.0  .2  .475. .565  3.0  .4  .341, .385  Table D  Changes i n the c a l c i u m c o n c e n t r a t i o n o f r e s e a l e d ghosts with time.  2+ [Ca] i n the l o a d i n g medium (mM)  Time (min.)  —1  umoles Ca me; mean + SE n  0.1  0  .443  1  0.1  5  .223  1  0.1  10  .176  1  0.1  20  .202  1  0.25  2  .455  .001 2  0.25  4  .330  1  0.25  6  .261  1  0.25  8  .197  .004 2  Table D (cont'd) [Ca) I n t h e l o a d i n g medium (mM)  Time (min.)  1 umoles n 2+ -: mean +Ca SE mg n  0.5  0  .474  .006  4  0.5  2  .432  .045  2  0.5  3  .366  .025  2  0.5  4  .364  0.5  5  .252  .002  2  0.5  6  .258  .042  2  0.5  7  .184  0.5  8  .188  0.5  10  .152  .016  3  0.5  20  .136  1.0  0  .585  1.0  2  .398  1.0  3  .398  .022  2  1.0  5  .269  .026  3  1.0  6  .239  1  1.0  7  .193  1  1.0  10  .173  .002  2  1.0  20  .215  .011  2  2.0  0  .807  .007  3  2.0  3  .6965  .014  2  2.0  4  .621  .039  2  2.0  5  .454  1  .027  4 1  1  Table D  [Ca] i n the l o a d i n g medium (mM)  Time (min.)  (cont'd)  umoles Ca^mg*" mean + SE n 1  2.0  6  .412  .039 3  2.0  7  .307  -  2.0  8  .273  1  2.0  10  .229  .003 3  3.0  0  1.017 '  .009 3  3.0  2  .98  1  3.0  3  .797  1  3.0  5  .624  .075 3  3.0  7  .284  1  3.0  10  .202  .025 3  3.0  20  .612  .007 3  5.0  0  1.425  .036 3  5.0  2  1.44  5.0  3  1.445.  5.0  5  1-19.  .089 3  5.0  7  0.930  1  5.0  10  .489  .03^ 2  5.0  20  .296  1  5.0  30  .250  1  85 Table E  E f f e c t o f ruthenium r e d ( 0 . 2 mM) on a c t i v a t i o n o f Mg+Ca-ATPase i n the l o a d i n g medium. [Ca] i n the l o a d i n g medium (mM)  {Ruthenium Red"] (mM)  umoles PI mg'^hr"" mean + SE n  1  0.23  0.2  .301  .023  3  .5  0.2  .321  .010  3  1.0  0.2  .356  .012  3  3.0  1.0  .301  .009  3  5.0  1.0  .3076  .025  3  Table P  E f f e c t o f v a r y i n g the c o n c e n t r a t i o n Mg+Ca-ATPase a c t i v i t y . ECa] i n the l o a d i n g medium (mM)  [Ruthenium Red] (mM)  o f ruthenium r e d on umoles P i mg'^hr" mean + SE n  0.5  0  .3166  .006  3  0.5  .2  .321  .011  3  3.0  0  .4926  .020  3  3.0  .05  .391  1  3.0  .10  .352  1  1  86  Table P (cont'd)  [Ca} i n t h e l o a c l i n g medium  [Ruthenium Red! (mM)  (mM)  U  J  uraoles P i mg'• i h r mean + SE n  3.0  .20  .301  3.0  .40  .210  5.0  0  .577  .014  3  5.0  .05  .501  .026  3  5.0  .1  .406  .023  3  5.0  .2  .307  .025  3  5.0  .4  .232  .011  3  .009  1  3 1  Table G  E f f e c t o f 0.2 mM e x t e r n a l r u t h e n i u m r e d on t h e a c t i v a t i o n of c a l c i u m t r a n s p o r t by i n t e r n a l c a l c i u m . [Calcium) l n t h e l o a d i n g medium (1.0 mM)  [Ruthenium Red"] (mM)  umoles C a m g " h r " 2 +  1.0  0.®  .425, .440  2.0  0.2  .479, .466  3.0  0.2  .475, .565  5.0  0.2  .430, .540  1  1  87 Table H  E f f e c t o f v a r y i n g the c o n c e n t r a t i o n of c a l c i u m i n the l o a d i n g medium l n the presence and absence of e x t e r n a l calcium. - a l l c o n d i t i o n s a r e standard except 0 . 4 mM EGTA was i n c l u d e d i n the l o a d i n g medium. - l o g jCa] l n the l o a d i n g medium (M)  [Ca] i n the e x t e r n a l medium  (mM)  umoles P i rng'-^hr  9.0  1.0  .310,.318  3.52  1.0  .313,.325  3.30  1.0  .360,.342  3.125  1.0  .340,.346  3.0  1.0  .349,.367  2.699  1.0  .400..406  2.523  1.0  .462,.473  2.30  1.0  .586,.574  2.125  1.0  .594,.606  2.0  1.0  .491,.499  2.22  1.0  .495,-504  ^.30  0  .254,.248  ^.0  0  .272,.282  3.699  0  .290,.298  3.30  0  .295..304  3.125  0  .310..318  2.823  0  .358,.364  2.602  0  .384,.396  2.426  0  .423,.480  88 Table H - l o g (ca] i n the l o a d i n g medium (M)  (cont'd)  [c&] l n the e x t e r n a l medium  umoles P i m g " h r " 1  (mM)  2.30  0  .490, .460  2.20  0  . 5 0 , .516  2;125  0  .541, ;553  2.0  0  . 4 9 8 , .518  1.9  0  .440, .468  Table I  E f f e c t o f manganese on Mg+Ca-ATPase a c t i v i t y i n r e s e a l e d ghosts. - l o g |Wj l n the l o a d i n g medium  jca] i n the e x t e r n a l medium  umoles P i  mg-'Hir"  $.0  1.0  .318  3.3  1.0  .302  3.0  1.0  .296  3.52  1.0  .288  3.30  1.0  .324  4.0  0  .306  3.3  0  .296  3.0  0  .278  3.7  0  .281  3.52  0  .302  3.30  0  .314  3.126  0  .290  1  1  89 Table J  E f f e c t of ouabain on ATPase a c t i v i t y on ghosts w i t h K G 1 ( 2 . 8 ? 6 M) - Ca i n the l o a d i n g medium was 0 . 5 Time (min)  [ouabain] I n the e x t e r n a l medium (mM)  resealed mM.  umole P i mg~ hr 1  0  0  .156  10  0  .203  30  0  .535  40  0  .605  0  0.2  .156  10  0.2  .225  20  0.2  .306  30  0.2  .392  40  0.2  .44?  90  Table K  A c t i v a t i o n of Na,K-ATPase by e x t e r n a l potassium i n r e s e a l e d ghosts, - Ca^ i n the l o a d i n g medium was 0,5 mM and c a l c i u m was not present l n the l o a d i n g medium when indicated, +  [ K ^ ] i n the e x t e r n a l medium (mM)  [ouabain]In the [ c a ] i n the e x t e r n a l medium e x t e r n a l medium 2 H  umoles P i mg^hr"  1  0  O.L  0  .302  1.5  0.1  0  .J00  4.5  0.1  0  .298  10.0  0.1  0  .300  0  0  0  .303..297,.337  1.5  0  0  .447..460  4.5  0  0  .550,.553  10.0  0  0  .638..646  15.0  0  0  .636,.645  0  0  1.0  .365.-373  1.5  O  1.0  .562,.570  4.5  0  1.0  .643,.656  10.0  0  1.0  .676,.682  15.0  0  1.0  .648,.660  Table L  E f f e c t o f e x t e r n a l c a l c i u m on ATPase a c t i v i t y . -the ghosts were loaded w i t h 0.5 mM c a l c i u m and .4 mM EGTA. Other c o n d i t i o n s were s t a n d a r d . l n the e x t e r n a l medium  umoles P i mg'^hr"  (mM)  1.0  .073,.070  1.5  .098,.104  2.0  .118,.130  3.0  .340..335  5.0  .146,.140  Table M E f f e c t o f e x t e r n a l magnesium on ATPase a c t i v i t y , - c o n d i t i o n s same as T a b l e L. [Mg 3 I n t h e e x t e r n a l medium 2H  umoles P i mg'^hr"  1.0  .02,.03  2.0  .044,.050  3.0  .062,.055  4.0  .048,.052  1  

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