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Kinetic and functional studies of (Mg+Ca)-ATPase activities in human erythrocyte membranes Quist, E. E. 1975

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KINETIC AND FUNCTIONAL STUDIES OF (Mg+Ca)-ATPase ACTIVITIES IN HUMAN ERYTHROCYTE MEMBRANES by E. E. QUIST B.Sc, University of British Columbia, 1971 M.Sc, University of British Columbia, 1973 A t h e s i s s u b m i t t e d i n p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r t h e d e g r e e o f DOCTOR OF PHILOSOPHY i n t h e D i v i s i o n o f P h a r m a c e u t i c a l C h e m i s t r y o f t h e F a c u l t y o f 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 a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1975 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o lumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and stud y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f // j?2frA The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i K INETIC AND FUNCTIONAL STUDIES OF (Mg+Ca)-ATPase ACTIVITIES IN HUMAN ERYTHROCYTE MEMBRANES. by E. E. QUIST ABSTRACT The p r e s e n c e o f b o t h h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s was shown k i n e t i c a l l y i n a number o f membrane p r e p a r a t i o n s d e r i v e d f r o m human e r y t h r o c y t e s . The (Mg+Ca)-ATPase a c t i v i t y i n membranes p r e p a r e d i n b o t h t h e p r e s e n c e and absence o f EDTA had s i m i l a r a f f i n i t y f o r C a ^ + r e f u t i n g t h e hyp-o t h e s i s t h a t l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i s an a r t i f a c t r e s u l t i n g f r o m d e n a t u r a t i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase t o a l o w a f f i n i t y f o r m . I t was f o u n d t h a t h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y was l o s t i n a pH dependent manner f r o m g h o s t membranes i n c u b a t e d i n l o w i o n i c s t r e n g t h m e d i a , ( l e s s t h a n 2 mosm). The l o s s o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y c o r r -esponded t o a l o s s o f a p p r o x i m a t e l y 25% o f t h e membrane p r o t e i n s . The s o l -u b l e p r o t e i n f r a c t i o n i t s e l f had no (Mg+Ca)-ATPase a c t i v i t y . However, i f t h e p r o t e i n f r a c t i o n was c o n c e n t r a t e d and r e c o m b i n e d w i t h t h e r e s i d u a l membranes, h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y was r e s t o r e d . The s o l u b l e component r e q u i r e d f o r r e s t o r a t i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y was f o u n d t o be p r o t e i n c o n s i s t i n g o f two p e p t i d e c h a i n s o f m o l e c u l a r w e i g h t g r e a t e r t h a n 200,000 d a l t o n s when a n a l y z e d by SDS a c r y l a m i d e g e l e l e c t r o p h o r e s i s . A number o f o b s e r v a t i o n s s u g g e s t e d t h a t l o w a f f i n i t y (Mg+Ca)-ATPase i i a c t i v i t y was p h o s p h o l i p i d dependent i n c o n t r a s t t o h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . T r e a t m e n t o f i n t a c t e r y t h r o c y t e s o r r e s e a l e d g h o s t s w i t h t r y p s i n on t h e o u t e r s u r f a c e o f c e l l s r e s u l t e d i n a r e d u c t i o n o f h i g h and lo w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s i n g h o s t membranes p r e p a r e d f r o m t h e s e c e l l s . T h i s r e s u l t was an i n d i c a t i o n t h a t p o l y p e p t i d e s r e q u i r e d f o r b o t h a c t i v i t i e s spanned t h e membrane. Ruthenium r e d and L a C l 3 were f o u n d t o be n o n - s e l e c t i v e i n h i b i t o r s o f h i g h and l o w (Mg+Ca)-ATPase a c t i v i t i e s . B o t h (Mg+Ca)-ATPase a c t i v i t i e s were i n h i b i t e d w i t h s i m i l a r p o t e n c i e s by t h e s e a g e n t s . A s t o i c h i o m e t r y o f two was d e t e r m i n e d f o r c a l c i u m t r a n s p o r t i n r e s e a l e d e r y t h r o c y t e s u s i n g l a n t h a n u m as a s e l e c t i v e i n h i b i t o r . A d d i t i o n o f 0.1 mM lan t h a n u m i n t h e e x t e r n a l medium o f g h o s t s r e s e a l e d w i t h 3 mM C a C ^ 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 100% and t o t a l M g 2 + and C a 2 + dependent ATPase a c t i v i t -i e s by 50%, i n d i c a t i n g t h a t a l l (Mg+Ca)-ATPase a c t i v i t y i n r e s e a l e d g h o s t s was n o t 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 . Schatzmann (1973) p r e v i o u s l y e s t i m a t e d t h e s t o i c h i o m e t r y t o be one. A s t o i c h i o m e t r y o f two was f o u n d h e r e i n r e s e a l e d g h o s t s p r e p a r e d i n t h e p r e s e n c e o r absence o f 1.0 mM EDTA d u r i n g t h e h e m o l y s i s p r e c e d u r e . I n r e s e a l e d v e s i c l e s c o n t a i n i n g o n l y l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y , ATP dependent c a l c i u m u p t a k e i n t o i n s i d e -o u t v e s i c l e s and low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y were s t i m u l a t e d by 2+ e x t e r n a l Ca W i t h s i m i l a r a f f i n i t i e s . . T h i s f i n d i n g s u g g e s t s t h a t c a l c i u m t r a n s p o r t i s a s s o c i a t e d w i t h l ow a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i n c o n -t r a s t t o t h e s u g g e s t i o n by Schatzmann (1973) t h a t c a l c i u m t r a n s p o r t i s a s s o c -i a t e d w i t h h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i n r e s e a l e d g h o s t s . R e s t o r -a t i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y by t h e a d d i t i o n o f s o l u b l e p r o t e i n f r a c t i o n c o n t a i n i n g s p e c t r i n i n c r e a s e d (Mg+Ca)-ATPase a c t i v i t y o v e r i i i two f o l d w i t h o u t a f f e c t i n g t h e v e l o c i t y o f c a l c i u m u p t a k e . T h e r e f o r e , h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y may be a s s o c i a t e d w i t h some f u n c t i o n o t h e r t h a n c a l c i u m t r a n s p o r t . K i n e t i c p l o t s r e v e a l e d t h a t t h e C a ^ + a c t i v -a t i o n o f l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y showed n e g a t i v e c o o p e r a t i v i t y . A n e g a t i v e c o o p e r a t i v e mechanism f o r c a l c i u m t r a n s p o r t would be advantageous f o r e r y t h r o c y t e s u r v i v a l , s i n c e c a l c i u m c o u l d be t r a n s p o r t e d e f f i c i e n t l y o v e r a wide range o f i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n s (0.3 t o 300 urn C a 2 + ) . S i g n a t u r e s o f E x a m i n e r s i v TABLE OF CONTENTS Page ABSTRACT i L I S T OF TABLES v i i LIS T OF FIGURES v i i i L I S T OF ABBREVIATIONS X INTRODUCTION 1 1. (Mg+Ca)-ATPase a c t i v i t y i n Red B l o o d C e l l Membrane F r a g m e n t s . 2 2. P r o p e r t i e s o f t h e ATP Dependent C a l c i u m Pump i n Human 5 E r y t h r o c y t e s . 3. A s s o c i a t i o n o f C a 2 + A c t i v a t e d ATPase A c t i v i t y w i t h C o n t r a c t i l e 6 P r o t e i n s . 4. I n v o l v e m e n t o f I n t r a c e l l u l a r C a 2 + i n Red B l o o d C e l l Shape. 8 5. Aims o f t h e P r e s e n t S t u d y . 12 MATERIALS AND METHODS 13 S e c t i o n I . S t u d i e s w i t h E r y t h r o c y t e G h o s t s . 13 1. P r e p a r a t i o n o f g h o s t s . 13 2. (Mg+Ca)-ATPase a s s a y f o r f r o z e n g h o s t membranes. 13 3. T r e a t m e n t o f g h o s t s w i t h p h o s p h o l i p a s e A^. 14 4. 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 . 15 5. D e t e r m i n a t i o n o f (Mg+Ca)-ATPase a c t i v i t y and Ca t r a n s p o r t 15 i n r e s e a l e d g h o s t s . S e c t i o n I I . S t u d i e s w i t h Membrane F r a g m e n t s . 16 1. P r e p a r a t i o n o f c o n t r o l membrane f r a g m e n t s ( P r e p a r a t i o n A ) . 16 2. P r e p a r a t i o n o f membranes c o n t a i n i n g o n l y l o w a f f i n i t y 16 (Mg+Ca)-ATPase a c t i v i t y ( P r e p a r a t i o n B ) . 3. P r e p a r a t i o n o f s o l u b l e f r a c t i o n C . 17 4. D e t e r m i n a t i o n o f (Mg+Ca)-ATPase a c t i v i t y i n membrane 18 f r a g m e n t s . 5. R e c o n s t i t u t i o n p r o c e d u r e . 19 6. T r e a t m e n t o f p r e p a r a t i o n B and f r a c t i o n C w i t h t r y p s i n . 19 7. T r e a t m e n t o f p r e p a r a t i o n B w i t h p h o s p h o l i p a s e A2. 19 8. Temperature dependence o f l o w a f f i n i t y (Mg+Ca)-ATPase 20 a c t i v i t y . 9. P r e p a r a t i o n o f membranes a c c o r d i n g t o Wolf ( 1 9 7 2 a ) . 20 S e c t i o n I I I . S t u d i e s w i t h R e s e a l e d V e s i c l e s . 20 1. P r e p a r a t i o n o f r e s e a l e d v e s i c l e s . 20 2. D e t e r m i n a t i o n o f (Mg+Ca)-ATPase a c t i v i t y and Ca u p t a k e . 22 3. S e p a r a t i o n o f i n s i d e - o u t v e s i c l e s . 22 4. D e t e r m i n a t i o n o f a c e t y l c h o l i n e s t e r a s e and g l y c e r a l d e h y d e 23 3-phosphate d e h y d r o g e n a s e a c t i v i t y i n r e s e a l e d v e s i c l e s . S e c t i o n I V . T r e a t m e n t o f I n t a c t E r y t h r o c y t e s and R e s e a l e d G h o s t s 24 w i t h P r o t e o l y t i c Enzymes. S e c t i o n V. D e t e r m i n a t i o n o f I n o r g a n i c P h o s p h a t e . 25 S e c t i o n V I . D e t e r m i n a t i o n o f C a l c i u m . 25 S e c t i o n V I I . SDS P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s . 25 1. P r e p a r a t i o n o f p r o t e i n s o l u t i o n s . 26 2. P r e p a r a t i o n o f g e l s . 26 3. S t a i n i n g and d e s t a i n i n g 27 S e c t i o n V I I I . D e t e r m i n a t i o n o f P h o s p h o l i p i d s . 27 S e c t i o n I X . D e t e r m i n a t i o n o f P r o t e i n . 27 S e c t i o n X. T r e a t m e n t o f G h o s t s w i t h N e u r a m i n i d a s e . 27 S e c t i o n XI.> C h e m i c a l s and M a t e r i a l s 28 RESULTS AND DISCUSSION 29 S e c t i o n I . C h a r a c t e r i s t i c s o f H i g h and Low A f f i n i t y (Mg+Ca)-ATP 29 as e A c t i v i t i e s i n E r y t h r o c y t e G h o s t s and Membrane F r a g m e n t s . 1. K i n e t i c s t u d y o f C a ^ a c t i v a t i o n o f (Mg+Ca)-ATPase a c t i v i t y 29 i n e r y t h r o c y t e membrane p r e p a r a t i o n s . v i Page 2. Removal o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y f r o m 37 membrane f r a g m e n t s . 3. R e c o n s t i t u t i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . 42 4. P h o s p h o l i p i d dependence o f h i g h and l o w a f f i n i t y (Mg+Ca) 52 -ATPase a c t i v i t i e s . 5. E f f e c t o f p r e t r e a t m e n t o f i n t a c t e r y t h r o c y t e s and 58 r e s e a l e d g h o s t s w i t h p r o t e o l y t i c enzymes on (Mg+Ca)-ATP-as e a c t i v i t y . 6. E f f e c t o f r u t h e n i u m r e d and L a C l 3 on (Mg+Ca)-ATPase 62 a c t i v i t y i n membrane f r a g m e n t s . S e c t i o n I I . 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 . 65 1. The s t o i c h i o m e t r y o f Ca t r a n s p o r t i n r e s e a l e d human 65 e r y t h r o c y t e g h o s t s u s i n g l anthanum as a s e l e c t i v e i n h i b i t o r . 2. A s s o c i a t i o n between c a l c i u m t r a n s p o r t and (Mg+Ca)-ATPase 72 a c t i v i t y i n r e s e a l e d v e s i c l e s . S e c t i o n I I I . K i n e t i c C h a r a c t e r i s t i c s o f Low A f f i n i t y (Mg+Ca)-ATP 89 ase A c t i v i t y . SUMMARY AND CONCLUSIONS 96 BIBLIOGRAPHY 100 APPENDIX 110 v i i L I ST OF TABLES T a b l e Page 3+ 1 I n h i b i t i o n o f c a l c i u m e f f l u x by L a . 66 v i i i L I ST OF FIGURES F i g u r e Page 1 S c h e m a t i c o f p o s t u l a t e d A T P - C a 2 + membrane i n t e r a c t i o n s 12 c o n t r o l l i n g e r y t h r o c y t e shape and membrane d e f o r m a b i l i t y , 2 The a c t i v a t i o n o f (Mg+Ca)-ATPase by C a 2 + i n g h o s t s p r e p a r e d 30 i n a 40 mosm medium (see M e t h o d s ) , f r o z e n a t -20°C i n t h i s medium and f r e e z e - t h a w e d t h r e e t i m e s b e f o r e u s e . 3 P l o t s o f t h e d a t a i n F i g . 2 a c c o r d i n g t o E a d i e ( 1 9 4 2 ) , i n 32 t h e a b s e n c e and p r e s e n c e o f 1 mM EDTA. 4 The a c t i v a t i o n o f (Mg+Ca)-ATPase by C a 2 + i n membrane f r a g - 33 ments p r e p a r e d u n d e r l o w i o n i c c o n d i t i o n s (12 mosm). 5 P l o t s o f t h e d a t a f r o m F i g . 4 a c c o r d i n g t o E a d i e ( 1 9 4 2 ) . 34 6 The a c t i v a t i o n o f (Mg+Ca)-ATPase a c t i v i t y by C a 2 + i n mem- 38 b r a n e f r a g m e n t s i n c u b a t e d f o r 5, 20, 40, and 60 m i n u t e s a t 37°C i n 0.1 mM EDTA and 1.0 mM T r i s - m a l e a t e pH 8.0. 7 The a c t i v a t i o n o f (Mg+Ca)-ATPase by Ca i n membrane f r a g - 40 ments p r e p a r e d i n 0.2 mM N a C l , 0.5 mM N a C l , 0.2 mM M g C l 2 and d i s t i l l e d w a t e r . 8 E f f e c t o f a d d i t i o n o f s o l u b l e f r a c t i o n C t o membranes d e v o i d 43 o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y a t a C a 2 + c o n c e n t -r a t i o n o f 29.0 jM, 2+ 9 C a a c t i v a t i o n o f t h e ATPase i n r e c o n s t i t u t e d membranes 44 p r e p a r e d by t h e a d d i t i o n o f f r a c t i o n C t o p r e p a r a t i o n B. 10 R e l e a s e o f membrane p o l y p e p t i d e s f r o m e r y t h r o c y t e g h o s t s . 47 11 D i s p o s i t i o n o f p o l y p e p t i d e s i n p r e p a r a t i o n s A and B. 49 12 The a c t i v a t i o n o f (Mg+Ca)-ATPase by C a 2 + i n p r e p a r a t i o n B 51 d e n a t u r e d a t 50°C and 70°C and r e c o n s t i t u t e d by t h e a d d i t i o n o f f r a c t i o n C. 13 E f f e c t o f p h o s p h o l i p a s e A^ on (Mg+Ca)-ATPase a c t i v i t y i n 54 g h o s t s . 14 E f f e c t o f p r e t r e a t m e n t o f p r e p a r a t i o n B w i t h p h o s p h o l i p a s e 55 A 2 on r e c o n s t i t u t i o n o f (Mg+Ca)-ATPase a c t i v i t y by f r a c t i o n C. 15 T e m p e r a t u r e dependence o f l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v - 57 i t y i n p r e p a r a t i o n B a t a C a 2 + c o n c e n t r a t i o n o f 316 yiM. i x F i g u r e Page 16 E f f e c t o f p r e t r e a t m e n t o f i n t a c t e r y t h r o c y t e s w i t h t r y p s i n 60 on (Mg+Ca)-ATPase a c t i v i t y o f g h o s t s p r e p a r e d f r o m t h e t r e a t e d c e l l s . 17 E f f e c t o f p r e t r e a t m e n t o f r e s e a l e d g h o s t s w i t h t r y p s i n on 61 (Mg+Ca)-ATPase a c t i v i t y . 18 I n h i b i t i o n o f (Mg+Ca)-ATPase a c t i v i t y by r u t h e n i u m r e d i n 63 membranes p r e p a r e d a c c o r d i n g t o Wolf ( 1 9 7 2 b ) . 19 I n h i b i t i o n o f (Mg+Ca)-ATPase a c t i v i t y by L a C l i n membrane 64 f r a g m e n t s ( p r e p a r a t i o n A) i n t h e absence o f EGTA. 20 E f f e c t o f La-* + on c a l c i u m e f f l u x f r o m r e s e a l e d g h o s t s . 68 21 E f f e c t o f L a ^ + on ATPase a c t i v i t y i n r e s e a l e d g h o s t s . 69 22 E f f e c t o f r e s e a l i n g v e s i c l e s i n t h e p r e s e n c e o f v a r i o u s 74 c o n c e n t r a t i o n s o f C a C l j on t h e v e l o c i t y o f Ca u p t a k e . 23 ATP dependent c a l c i u m t r a n s p o r t f r o m r e s e a l e d r i g h t - s i d e - o u t 76 v e s i c l e s . 24 A c t i v a t i o n o f l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i n r e s e a l - 79 ed v e s i c l e s b y C a 2 + . * 25 A c t i v a t i o n o f Ca u p t a k e i n r e s e a l e d v e s i c l e s by C a 2 + . 80 26 C a 2 + dependence o f l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y 83 and ATP dependent Ca u p t a k e i n r e s e a l e d v e s i c l e s . 27 Time c o u r s e o f (Mg+Ca)-ATPase a c t i v i t y i n r e s e a l e d v e s i c l e s 85 i n t h e p r e s e n c e o f 28.8 yH C a 2 + , and 229 ^ iM C a 2 + . 28 Time c o u r s e o f Ca u p t a k e i n r e s e a l e d v e s i c l e s i n t h e p r e s e n c e 86 o f 28.8 j i M C a 2 + and 229 ydi C a 2 + . 29 L i n e w e a v e r - B u r k p l o t o f C a 2 + a c t i v a t i o n o f l o w a f f i n i t y 90 (Mg+Ca)-ATPase a c t i v i t y i n p r e p a r a t i o n B (membranes d e v o i d o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y ) . 30 H i l l p l o t o f C a 2 + a c t i v a t i o n o f l o w a f f i n i t y (Mg+Ca)-ATPase 92 a c t i v i t y i n p r e p a r a t i o n B. X LIS T OF ABBREVIATIONS ADP a d e n o s i n e 5 ' - d i p h o s p h a t e ATP a d e n o s i n e 5 1 - t r i p h o s p h a t e EDTA e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d EGTA e t h y l e n e g l y c o l - b i s - ( ^ - a m i n o e t h y l e t h e r ) N , N ' - t e t r a a c e t i c a c i d NEM N - e t h y l m a l e i m i d e P i i n o r g a n i c p h o s p h a t e SDS sodium d o d e c y l s u l f a t e TCA t r i c h l o r o a c e t i c a c i d pKRD ^ - n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e HEPES N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c a c i d UTP u r i d i n e t r i p h o s p h a t e ITP i n o s i n e t r i p h o s p h a t e DPG . 2 , 3 - d i p h o s p h o g l y c e r a t e GTP g u a n o s i n e t r i p h o s p h a t e CTP c y t o s i n e t r i p h o s p h a t e x i ACKNOWLEDGEMENTS I. wish, t o t h a n k D r . B.D, R o u f o g a l i s f o r h i s encouragement and i n t e r e s t t h r o u g h o u t t h i s work. I- w i s h t o t h a n k L o r r a i n e S a t o , Leo Chan and June Lam f o r t h e i r iflQre t h a n competent t e c h n i c a l a s s i s t a n c e . The a u t h o r i s d e e p l y g r a t e f u l t o t h e C a n a d i a n Red C r o s s f o r t h e g e n e r o u s s u p p l y o f b l o o d w h i c h made t h i s work p o s s i b l e . The a u t h o r a l s o w i s h e s t o thank D r. J.H. M c N e i l l , D r . F. A b b o t t i Dr. V. P a l a t y and Dr. B. R i e d e l f o r t h e i r k i n d n e s s and s u p p o r t . D i s s c u s s i o n s w i t h D r. D. G o d i n , D r . E. Watson and D r . F. V i n c e r l g i were o f c o n s i d e r a b l e v a l u e t o t h e a u t h o r . I w i s h t o t h a n k a l l members o f t h e f a c u l t y , s t a f f and g r a d u a t e p t u d e n t body f o r making my s t a y a t UBC v e r y e n i o v a b l e . My w i f e , L a u r i e , f o r d o i n g a n e x c e l l a n t j o b o f t y p i n g t h e f i n a l c o py o f t h e m a n u s c r i p t . x i i DEDICATION To my l o v i n g w i f e and c h i l d r e n f o r making t h i s t h e s i s w o r t h w h i l e . 1 INTRODUCTION The r o l e o f i n t r a c e l l u l a r c a l c i u m i n r e g u l a t i n g a number o f membrane f u n c t i o n s has r e c e n t l y become a s u b j e c t o f w i d e s p r e a d r e s e a r c h . I n t h i s r e g a r d , human e r y t h r o c y t e s have been a p o p u l a r membrane s o u r c e t o s t u d y because o f t h e c o n v e n i e n c e o f o b t a i n i n g t h e s e membranes i n a r e l a t i v e l y p u r e and homogeneous f o r m u s i n g m i l d s e p a r a t i o n p r o c e d u r e s . F o r t h i s r e a s o n a g r e a t d e a l o f o u r knowledge c o n c e r n i n g membrane f u n c t i o n and s t r u c t u r e have been d e r i v e d f r o m s t u d i e s on e r y t h r o c y t e s . I t has been shown t h a t u n d e r n o r m a l p h y s i o l o g i c a l c o n d i t i o n s t h e r e i s a c o n s i d e r a b l e c a l c i u m c o n c e n t r a t i o n g r a d i e n t o f f r e e Ca 2 + i n s i d e t h e c e l l (10~ 6 M) compared t o Ca? + o u t s i d e t h e c e l l (10 " * • % ! ) . I n human r e d b l o o d c e l l s t h e l o w 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 a t t r i b u t e d t o t h e ATP dependent c a l c i u m 2+ pump (Schatzmann 1 9 6 6 ) , t h e h i g h a f f i n i t y o f t h e membrane i t s e l f f o r Ca (Long and Mouat 1 9 7 1 ) , and t h e r e l a t i v e l y l o w p e r m e a b i l i t y o f t h e p l a s m a membrane t o c a l c i u m ( P o r z i g 1 9 7 0 ) . The l e v e l s o f i n t r a c e l l u l a r m e t a b o l i t e s s u c h as ATP a r e a l s o i m p o r t a n t c h e l a t o r s o f i n t r a c e l l u l a r c a l c i u m (Weed ejt a l . 1 9 6 9 ) . 2+ M a i n t e n a n c e o f l o w 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 o f Ca has shown t o be e s s e n t i a l f o r s u r v i v a l o f e r y t h r o c y t e s i n t h e m i c r o c i r c u l a t i o n (La C e l l e 2+ 1 9 6 9 ) . F o r i n s t a n c e e l e v a t i o n s i n t h 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 Ca r e s u l t i n d i s c o i d - e c h i n o c y t i c shape t r a n s f o r m a t i o n s (Weed e t a l . 1 9 6 9 ) , s e l e c t i v e i n c r e a s e s i n p o t a s s i u m p e r m e a b i l i t y (Lew 1971; Blum and Hoffman 1 9 7 2 ) , d e c r e a s e d d e f o r m a b i l i t y (Weed e t a l . 1 9 6 9 ) , i n c r e a s e d v i s c o s i t y (La C e l l e 1 9 7 3 ) , c o n t r a c t i o n ( P a l e k e t a l _ . 1971 a ) , i n c r e a s e d o s m o t i c b e h a v i o u r i n r e c o n s t i t u t e d g h o s t s ( P a l e k e t a l _ . 1971 b) , and i n h i b i t i o n o f enzymes 2 s u c h as Na,K-ATPase ( V i n c e n z i 1 9 7 1 ) . The b i o c h e m i c a l and p h y s i o l o g i c a l mechanisms by w h i c h i n t r a c e l l u l a r Ca m e d i a t e s t h e s e e f f e c t s a r e as y e t n o t c o m p l e t e l y u n d e r s t o o d . However, a r e l a t i o n s h i p between membrane bound (Mg+Ca)-ATPase a c t i v i t y and c a l c i u m t r a n -s p o r t has been r e c o g n i z e d (Schatzmann and V i n c e n z i 1 9 6 9 ) , w h i c h o p e r a t e s a n a l -ogous t o t h e Na,K-pump (Skou 1 9 7 1 ) . On t h e o t h e r v h a n d , o t h e r s have s u g g e s t e d t h a t (Mg+Ca)-ATPase a c t i v i t y may be a s s o c i a t e d w i t h ' c o n t r a c t i l e ' p r o t e i n s i n v o l v e d i n c o n t r o l l i n g t h e volume o f e r y t h r o c y t e s ( O h n i s h i 1961, Wins and S c h o f f e n i e l s 1966 a ) . 1. (Mg+Ca)-ATPase a c t i v i t y i n Red B l o o d C e l l Membrane F r a g m e n t s . Dunham and G l y n n (1961) were t h e f i r s t t o r e p o r t a magnesium d e p e n d e n t c a l c i u m a c t i v a t e d ATPase a c t i v i t y i n e r y t h r o c y t e g h o s t membranes. The s p e c i f i c a c t i v i t y o f t h e (Mg+Ca)-ATPase a c t i v i t y i s s e v e r a l f o l d g r e a t e r t h a n o u a b a i n s e n s i t i v e Na,K-ATPase a c t i v i t y w h i c h i n p a r t may depend on t h e method o f p r e p a r a t i o n o f t h e membranes. B i v a l e n t m e t a l c a t i o n s S r 2 + and B a 2 + (Wins and S c h o f f e n i e l s 1966 b) and t o a l e s s e r e x t e n t M n 2 + , N i 2 + , C o 2 + , C u 2 + , Z n 2 + , . 2+ . and Pb ( P f l e g e r and W o l f 1975) c a n a c t i v a t e o u a b a i n i n s e n s i t i v e ATPase a c t i v i t y . The d e g r e e o f a c t i v a t i o n by d i v a l e n t m e t a l c a t i o n s was f o u n d b y t h e l a t t e r w o r k e r s t o be dependent on t h e i o n i c r a d i u s o f t h e c a t i o n . (Mg+Ca)-ATPase a c t i v i t y i s a l s o enhanced i n t h e p r e s e n c e o f a l k a l i c a t i o n s N a + and K+ (Schatzmann and R o s s i 1971; Bond and G r e e n 1 9 7 1 ) . (Mg+Ca)-ATPase a c t i v i t y i s s p e c i f i c f o r ATP, whereas h y d r o l y s i s o f o t h e r n u c l e o t i d e s CTP, UTP, GTP, and ITP i s n e g l i g i b l e o r n o n - d e t e c t a b l e (Watson e t a l . 1 9 7 1 ) , C a r e f u l s t u d i e s o f t h e Ca a c t i v a t i o n o f (Mg+Ca)-ATPase a c t i v i t y i n e r y t h r o c y t e g h o s t membranes r e v e a l e d t h e p r e s e n c e o f two (Mg+Ca)-ATPase a c t i v i t i e s f r e q u e n t l y r e f e r r e d t o as h i g h and low (Mg+Ca)-ATPase a c t i v i t i e s t o r e f l e c t t h e i r d i f f e r e n t a f f i n i t i e s f o r C a ^ + ( H o r t o n e_t a l . 1970; Schatzmann and R o s s i 1971; Wolf 1 9 7 3 ) . The C a 2 + d i s s o c i a t i o n c o n s t a n t s f o r t h e h i g h and low (Mg+Ca)-ATPase a c t i v i t i e s were r e p o r t e d as 2.3 t o 4yra (Schatzmann and R o s s i 1971; Wolf 1972; Schatzmann 1973) and 100 jim (Schatzmann and R o s s i 1971) r e s p e c t i v e l y . F o r h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y , Mg-ATP has been shown t o be t h e s u b s t r a t e , whereas C a 2 + a c t s a l l o s t e r i c a l l y as an a u t o s t e r i c e f f e c t o r (Wolf 1 9 7 2 ) . More r e c e n t l y however, S c h a r f f (1972) and Schatzmann (1973) c o n c l u d e d t h a t l o w a f f i n i t y (Mg+Ca)-ATP-as e a c t i v i t y was an a r t i f a c t d e r i v e d f r o m p r e p a r a t i o n o f t h e e r y t h r o c y t e g h o s t membranes. These w o r k e r s c o n c l u d e d t h a t t h e p r e s e n c e o f t h e c h e l a t i n g a g e n t s , e i t h e r EDTA o r EGTA, d u r i n g p r e p a r a t i o n o f t h e membranes r e s u l t e d i n c o n v e r s i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y t o a f o r m w i t h a l o w e r a f -2+ f i n i t y f o r Ca [ i e . low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y ] . However, t h i s c o n c l u s i o n does n o t comply w i t h t h e r e s u l t s o f H o r t o n e_t a l . (1970) who u s e d membranes p r e p a r e d i n t h e absence o f e i t h e r EGTA o r EDTA and c l e a r l y showed t h e p r e s e n c e o f h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s . F u r t h e r 2+ s t u d i e s on t h e Ca a c t i v a t i o n o f (Mg+Ca)-ATPase a c t i v i t y i n a number o f membrane p r e p a r a t i o n s w i l l be an i m p o r t a n t p a r t o f t h i s t h e s i s . R e c e n t l y W o l f and G i e t z e n (1974) s o l u b i l i z e d h i g h a f f i n i t y (Mg+Ca)-ATP-as e a c t i v i t y f r o m e r y t h r o c y t e g h o s t s w i t h 0.4% T r i t o n X-100. The s o l u b l e 2+ enzyme has a Ca d i s s o c i a t i o n c o n s t a n t o f 2.0jxM w h i c h c l o s e l y c o r r e s p o n d s t o t h e d i s s o c i a t i o n c o n s t a n t f o u n d f o r t h e membrane bound h i g h a f f i n i t y (Mg+ Ca)-ATPase a c t i v i t y w h i c h i n d i c a t e d t h a t t h e s o l u b i l i z a t i o n p r o c e d u r e d i d n o t d e n a t u r e t h e enzyme. The s o l u b i l i z e d p r o t e i n f r a c t i o n was f o u n d t o c o n t a i n a number o f d i f f e r e n t p r o t e i n bands when a n a l y z e d by SDS a c r y l a m i d e g e l e l e c t r o p h o r e s i s w h i c h s u g g e s t e d o n l y a p a r t i a l p u r i f i c a t i o n o f t h e enzyme was a c h i e v e d . Bond and C l o u g h (1973) r e c e n t l y f o u n d t h a t a s p e c i f i c p r o t e i n a c t i v a t o r o f (Mg+Ca)-ATPase a c t i v i t y c o u l d be r e l e a s e d f r o m r e d b l o o d c e l l g h o s t s by 4 r e p e a t e d w a s h i n g s i n 1.0 mM T r i s (pH 7 . 5 ) . (Mg+Ca)-ATPase a c t i v i t y i n g h o s t s was s t i m u l a t e d a b o u t two f o l d by t h e d i a l y z e d membrane f r e e h e m o l y s a t e , whereas Mg-ATPase and Na,K-ATPase a c t i v i t i e s i n t h e membrane were u n a f f e c t e d . When membranes were e x p o s e d t o h e a t o r N - e t h y l m a l e i m i d e (NEM), membrane (Mg+Ca)-ATPase and t h e h e m o l y s a t e - d e p e n d e n t component were i n a c t i v a t e d a t t h e same r a t e i n d i c a t i n g t h a t a s i n g l e (Mg+Ca)-ATPase i n t h e membrane was a c t i v a t e d by t h e h e m o l y s a t e . P u r i f i e d human h e m o g l o b i n and b o v i n e serum a l b u m i n were a l s o f o u n d t o a c t i v a t e (Mg+Ca)-ATPase a c t i v i t y 1.3 and 1.2 f o l d r e s p e c t i v e l y . The i d e n t i t y o f t h e a c t i v a t o r p r o t e i n was n o t d e t e r m i n e d . 2+ R e c e n t s t u d i e s have d e m o n s t r a t e d a Ca s t i m u l a t e d p h o s p h o r y l a t e d i n t e r -m e d i a t e (E P) i n r e d c e l l g h o s t s a t low ATP and Mg c o n c e n t r a t i o n s (Knauf e t a l . 1974; K a t z and B l o s t e i n 1 9 7 5 ) . The m o l e c u l a r w e i g h t o f E C a P i s a p p r o x i m a t e l y 150,000 d a l t o n s as d e t e r m i n e d by SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . The d e m o n s t r a t i o n t h a t t h e E C a p i s s e p a r a b l e f r o m t h e sod i u m s t i m u l a t e d p h o s p h o p r o t e i n s u g g e s t s t h a t t h e C a - a c t i v a t e d ATPase may be s e p a r a t e and d i s t i n c t f r o m t h e Na,K-ATPase (Knauf e t al_.1974) . The behav-i o u r o f t h e p h o s p h o r y l a t e d component has some p r o p e r t i e s c o n s i s t e n t w i t h i t s 2+ r o l e a s a p h o s p h o r y l a t e d i n t e r m e d i a t e o f Ca -ATPase a c t i v i t y ( K a t z and 2+ B l o s t e i n 1 9 7 5 ) . I n s a r c o p l a s m i c r e t i c u l u m , Ca s t i m u l a t e s t h e p h o s p h o r y l a -t i o n o f a p r o t e i n ( m o l e c u l a r w e i g h t 102,000 d a l t o n s ) and (Mg+Ca)-ATPase a c t -. . . -7 l v i t y w i t h a s i m i l a r a f f i n i t y ( a p p r o x i m a t e l y 10 M) ( I n e s i e t a l _ . 1970; MacLennan e t a l . 1 9 7 1 ) . The (Mg+Ca)-ATPase a c t i v i t y i n t h e s a r c o p l a s m i c r e t -i c u l u m i s a s s o c i a t e d w i t h t h e c a l c i u m pump (Makinose and H a s s e l b a c h 1971), whereas an a s s o c i a t i o n between t h e E P and t h e c a l c i u m t r a n s p o r t s y s t e m i n e r y t h r o c y t e g h o s t s has n o t been shown. T h i s c o r r e l a t i o n may be v e r y d i f f i c u l t 2+ t o d e m o n s t r a t e s i n c e t h e c o n d i t i o n s u s e d t o s t u d y Ca s t i m u l a t i o n o f E C a p and c a l c i u m t r a n s p o r t a r e q u i t e d i f f e r e n t . 5 2. P r o p e r t i e s o f t h e ATP Dependent C a l c i u m Pump i n Human E r y t h r o c y t e s . S i n c e Schatzmann (1966) d e m o n s t r a t e d ATP dependent c a l c i u m t r a n s p o r t i 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 , a number o f s i m i l a r i t i e s between t h i s t r a n -s p o r t s y s t e m and membrane bound (Mg+Ca)-ATPase a c t i v i t y have been r e p o r t e d (Schatzmann and V i n c e n z i 1969; Lee and S h i n 1969; See Schatzmann 1975 f o r r e v i e w ) . B o t h (Mg+Ca)-ATPase a c t i v i t y and c a l c i u m t r a n s p o r t a r e magnesium-2+ d ependent and o u a b a i n - i n s e n s i t i v e . The a c t i v a t i n g s i t e o f Ca i s l o c a t e d on t h e i n n e r membrane s u r f a c e f o r b o t h s y s t e m s (Schatzmann and V i n c e n z i 1 9 6 9 ) . 2+ S r , w h i c h s t i m u l a t e s (Mg+Ca)-ATPase a c t i v i t y i n g h o s t membranes (Wins and S c h o f f e n i e l s 1 9 6 7 ) , i s a c t i v e l y t r a n s p o r t e d o u t o f r e s e a l e d g h o s t s by an ATP-dependent mechanism ( O l s o n and C a z o r t 1 9 6 9 ) . I n r e d b l o o d c e l l membrane 45 f r a g m e n t s , Ca u p t a k e and h y d r o l y s i s were s u p p o r t e d by ATP, b u t n o t by o t h e r n u c l e o t i d e s GTP,ITP and CTP (Cha e t a l . 1 9 7 1 ) . 2+ Ca i s t h o u g h t t o be t r a n s p o r t e d o u t o f t h e c e l l a g a i n s t a c o n c e n t r a t i o n g r a d i e n t w i t h t h e e n e r g y o f t r a n s l o c a t i o n b e i n g s u p p l i e d f r o m a c o n c o m m i t a n t h y d r o l y s i s o f ATP t o ADP and P i (Schatzmann 1 9 7 5 ) . C a l c i u m t r a n s p o r t a c r o s s t h e membrane a p p e a r s t o o c c u r w i t h o u t t h e a i d o f a m e t a b o l i t e o r c a t i o n . V a r i o u s c o n c e n t r a t i o n s o f i n o r g a n i c p h o s p h a t e , ADP, p y r o p h o s p h a t e o r a d e n i n e were f o u n d n o t t o be t r a n s p o r t e d c o n c o m m i t a n t l y w i t h Ca o u t o f r e s e a l e d g h o s t s ( O l s o n and C a z o r t 1 9 7 4 ) . I t was p r e v i o u s l y shown t h a t magnesium i s a l s o n o t t r a n s p o r t e d w i t h c a l c i u m (Schatzmann and V i n c e n z i 1 9 6 9 ) . A l t h o u g h N a + and K + a c t i v a t e (Mg+Ca)-ATPase a c t i v i t y i n e r y t h r o c y t e g h o s t membranes (Schatzmann and R o s s i 1971: Bond and G r e e n 1 9 7 1 ) , n e i t h e r c a t i o n s t i m u l a t e s c a l c i u m t r a n s -p o r t i n r e s e a l e d g h o s t s (Schatzmann and V i n c e n z i 1 9 6 9 ) . C a l c i u m t r a n s p o r t i s v e r y s e n s i t i v e t o changes i n t e m p e r a t u r e , as w o u l d be e x p e c t e d o f a s y s t e m c o u p l e d t o a c h e m i c a l r e a c t i o n . H i g h Q 1 Q v a l u e s o f 3.5 (Schatzmann and V i n c e n z i 1969) and 3.2 (Lee and S h i n 1969) have been r e p o r t e d f o r Ca e f f l u x i n r e s e a l e d g h o s t s . 6 2+ I n t h e p a s t , s t u d i e s c o m p a r i n g t h e a f f i n i t y o f Ca t o a c t i v a t i o n o f ATPase a c t i v i t y and c a l c i u m t r a n s p o r t i n r e s e a l e d g h o s t s have been u n s u c c e s s -f u l s i n c e i t was n o t p o s s i b l e t o d e t e r m i n e t h e 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 -2+ i o n o f Ca a c c u r a t e l y i n r e s e a l e d g h o s t s o r i n t a c t c e l l s . Schatzmann (1973) 2+ s u g g e s t e d t h a t t h e Ca c o n c e n t r a t i o n i n r e s e a l e d g h o s t s c o u l d be c o n t r o l l e d by an EGTA-Ca b u f f e r s y s t e m b e c a u s e EGTA e n t e r s t h e g h o s t d u r i n g h e m o l y s i s and i s r e t a i n e d a f t e r r e s e a l i n g . I n g h o s t s l o a d e d w i t h Ca-EGTA, t h e c o n c e n t -r a t i o n o f C a ^ + r e q u i r e d f o r h a l f s a t u r a t i o n o f t h e C a 2 + s y s t e m was 4.0 x 10~^M o r l e s s . T h e r e f o r e Schatzmann (1973) c o n c l u d e d t h a t h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i s 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 b e c a u s e o f t h e i r s i m i l a r 2+ a f f i n i t i e s f o r Ca . I n c o n t r a s t t o t h e (Mg+Ca)-ATPase a s s o c i a t e d c a l c x u m pump i n s a r c o p l a s m i c r e t i c u l u m (Makinose and H a s s e l b a c k 1 9 7 1 ) , t h e s t o i c h i o m -e t r y o f t h e c a l c i u m pump i n human e r y t h r o c y t e s was e s t i m a t e d t o be one r a t h e r t h a n two (Schatzmann 1 9 7 3 ) . 2+ 3. A s s o c i a t i o n o f Ca A c t i v a t e d ATPase A c t i v i t y w i t h C o n t r a c t i l e P r o t e i n s . Wins and S c h o f f e n i e l s (1966 a) d e m o n s t r a t e d t h a t s h r i n k a g e o f e r y t h r o c y t e g h o s t s ( p o r c i n e ) was l i n k e d t o t h e p r e s e n c e o f ATP and Ca i n t h e g h o s t s . These w o r k e r s s u g g e s t e d t h a t t h e o b s e r v e d g h o s t c o n t r a c t i o n may i n v o l v e t h e ATPase a c t i v i t y o f an a c t o m y o s i n - l i k e s y s t e m i n human r e d c e l l s f i r s t d e s c r i b e d by O h n i s h i ( 1 9 6 1 ) . However, o t h e r s f o u n d t h a t ATP was n o t r e q u i r e d 2+ f o r t h e Ca i n d u c e d c o n t r a c t i o n i n human e r y t h r o c y t e g h o s t s (Weed et^ a l . 1969; P a l e k e t a l . 1971 a ) . R o s e n t h a l e_t a l . (1970) e x t r a c t e d a g r o u p o f s o l u b l e p r o t e i n s u n d e r l o w i o n i c s t r e n g t h c o n d i t i o n s w h i c h c o n t a i n e d o u a b a i n i n s e n s i t i v e ATPase a c t i v i t y . The ATPase was s t i m u l a t e d h a l f m a x i m a l l y w i t h 1.0 mM C a 2 + and i n h i b i t e d by magnesium. I t was s u g g e s t e d t h a t t h e ATPase a c t i v i t y may be a s s o c i a t e d w i t h 7 f i b r i l l a r p r o t e i n s l o c a t e d on the i n n e r "membrane s u r f a c e ( Z e l a n d e r and E r i c s o n 1 9 6 5 ) , s i n c e t h e s e s t r u c t u r e s were a b s e n t i n t h e r e s i d u a l g h o s t membranes a f t e r l o w i o n i c s t r e n g t h e x t r a c t i o n o f t h e s o l u b l e p r o t e i n s ( R o s e n t h a l e t a l . 1 9 7 0 ) . O t h e r s have s u g g e s t e d t h a t t h e ATPase a c t i v i t y may be a s s o c i a t e d w i t h t h e h i g h m o l e c u l a r w e i g h t p r o t e i n s p e c t r i n ( M a r c h e s i and S t e e r s J r . 1968) o r t e t k i n A ( C l a r k 1 9 7 1 ) , w h i c h i s a l s o l o c a t e d on t h e i n n e r s u r f a c e o f t h e membrane ( N i c o l s o n e t a l . 1 9 7 1 ) , and c a n be e x t r a c t e d u n d e r s i m i l a r l o w i o n i c c o n d i t i o n s f r o m e r y t h r o c y t e g h o s t s ( M a r c h e s i and S t e e r s J r . 1 9 6 8 ) . I t has been s u g g e s t e d t h a t s p e c t r i n i s a c o n t r a c t i l e l i k e p r o t e i n s i n c e i t forms f i l a m e n t o u s c o i l s i n t h e p r e s e n c e o f 0.5 mM ATP and 0.1 t o 1.0 mM C a C l 2 o r M g C l 2 ( M a r c h e s i e t a l _ . 1969) . I n t h e p r o t e i n f r a c t i o n e x t r a c t e d by R o s e n t h a l e t a l . (1970) , o n l y Mn2"*" o r Ca 2 - 1" p r o d u c e d a s i g n i f i c a n t s t i m u l a t i o n o f ATPase a c t i v i t y , whereas t h e d i v a l e n t m e t a l c a t i o n s 2+ 2+ 2+ 7+ S r , Cd ,Zn , and. Pb e i t h e r d i d n o t s t i m u l a t e ATPase h y d r o l y s i s o r t h e h y d r o l y s i s was n o t d i s t i n g u i s h a b l e f r o m t h e a c t i v i t y i n t h e abs e n c e o f d i v a l -e n t m e t a l c a t i o n s . S z a s z (1970) s u g g e s t e d t h a t ATPase a c t i v i t y i s n o t a s s o c -i a t e d w i t h c o n t r a c t i l i t y i n g h o s t s membranes s i n c e i n ATP d e p l e t e d g h o s t s , t h e c o n t r a c t i l e e f f e c t o f C a ^ + c o u l d be- p r o d u c e d w i t h C u 2 + o r C d 2 + a l t h o u g h t h e ATPase a c t i v i t y was i n a s t a t e o f c o m p l e t e i n h i b i t i o n . M o r e o v e r , P a l e k e t a l . 2+ (1971 b) were u n a b l e t o d e m o n s t r a t e a c o r r e l a t i o n between Ca s t i m u l a t e d ATPase a c t i v i t y i 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 and volume changes 2+ i n d u c e d by i n t r a c e l l u l a r C a More r e c e n t l y , S t e w a r t e t a l . (1972) r e p o r t e d t h e s o l u b i l i z a t i o n o f a ' c o n t r a c t i l e ' p r o t e i n f r o m human e r y t h r o c y t e g h o s t s w i t h 0.35M KC1-0.15M N a C l (pH 7.5) a t 0° C. The s o l u b i l i z e d p r o t e i n e x h i b i t e d ATPase a c t i v i t y t h a t i s i n h i b i t e d by EDTA b u t n o t by o u a b a i n b u t c o u l d n o t be a c t i v a t e d by 2+ Ca . The p r o t e i n f r a c t i o n formed a f i b r i n o u s p r e c i p i t a t e upon d i l u t i o n o f t h e K C l - N a C l e x t r a c t i n g s o l u t i o n and un d e r g o e s s u p e r p r e c i p i t a t i o n upon a d d i t i o n o f 1.0 mM Mg-ATP. The s o l u b i l i t y and m o l e c u l a r s e i v i n g c h a r a c t e r i s t i c s 8 were v e r y d i f f e r e n t f r o m s p e c t r i n . A t t h e p r e s e n t t i m e , t h e r e f o r e , t h e r e i s no c o n v i n c i n g e v i d e n c e a s s o c -i a t i n g (Mg+Ca)-ATPase a c t i v i t y w i t h ' c o n t r a c t i l e ' p r o t e i n s . The Ca-ATPase 2+ a c t i v i t y d e s c r i b e d by R o s e n t h a l e t a l . (1970) has a v e r y l ow a f f i n i t y f o r Ca 2+ 2+ and was i n h i b i t e d by Mg . Nor was t h e r e a s p e c i f i c i o n r e q u i r e m e n t f o r Ca 2+ and Mg shown f o r t h e ' c o n t r a c t i l e ' p r o t e i n s e x t r a c t e d by S t e w a r t e t a l . 2+ 2+ (1972). S p e c t r i n was shown t o f o r m f i l a m e n t o u s c o i l s w i t h e i t h e r Mg o r Ca , i n d i c a t i n g t h a t t h e i o n r e q u i r e m e n t s f o r t h i s b e h a v i o u r i s d i f f e r e n t f r o m t h o s e f o r (Mg+Ca)-ATPase a c t i v i t y . 2+ 4. I n v o l v e m e n t o f I n t r a c e l l u l a r Ca i n Red B l o o d C e l l Shape. Nakao e t a l . (1960) f o u n d t h a t i n c u b a t i n g whole e r y t h r o c y t e s i n t h e p r e s e n c e o f NaF r e s u l t e d i n a change a f t e r two h o u r s f r o m a n o r m a l d i s c o i d a l shape t o a c r e n a t e d d i s c ( e c h i n o c y t e ) and t h e n t o a c r e n a t e d s p h e r e a f t e r f o u r h o u r s i n c u b a t i o n . A f t e r s i x h o u r s smooth s p h e r e s f o r m e d . The o b s e r v e d shape changes c o r r e l a t e d w i t h a s i m u l t a n e o u s r e d u c t i o n i n i n t r a c e l l u l a r ATP l e v e l s . When t h e c e l l u l a r ATP c o n c e n t r a t i o n s were a b o u t o n e - h a l f t h e no r m a l l e v e l ( 5 0 j ^ m o l e s / 100 ml c e l l s ) , t h e c e l l s r e m a i n e d d i s c o i d a l . However, when t h e ATP l e v e l s were a b o u t o n e - t e n t h n o r m a l (15 j A m o l e s / 100 m l c e l l s ) , t h e c e l l s became smooth s p h e r e s . A t ATP c o n c e n t r a t i o n s o f 1 5 - 5 0 j x m o l e s / 100 ml c e l l s , t h e c e l l s were c r e n a t e d . S i m i l a r shape changes were o b s e r v e d w i t h b l o o d o s t o r e d i n c i t r a t e d e x t r o s e s o l u t i o n a t 4 C where c r e n a t e d c e l l s and smooth s p h e r e s were p r e s e n t a f t e r 12 d a y s and s e v e r a l weeks o f s t o r a g e r e s p e c t i v e l y . These shape changes o c c u r e d w i t h a s i m u l t a n e o u s d e c r e a s e i n c e l l u l a r ATP l e v e l s . I f smooth s p h e r e c e l l s were i n c u b a t e d w i t h g l u c o s e , i n o s i n e and a d -e n i n e , w h i c h r e s u l t i n a r a p i d s y n t h e s i s o f ATP, t h e shape o f t h e c e l l r e t u r n s t o n o r m a l . L a t e r , Weed and B o w d l e r (1966) f o u n d t h a t i n ATP d e p l e t e d b l o o d 9 t h e i r was a l a r g e i n c r e a s e i n v i s c o s i t y a s s o c i a t e d w i t h an i n c r e a s e i n r e d b l o o d c e l l r i g i d i t y . T h e r e was. a l s o a d e c r e a s e i n t h e c r i t i c a l h e m o l y t i c v o l u -t i n : c o n s i s t e n t w i t h a d e c r e a s e i n s u r f a c e a r e a s e c o n d a r y t o a l o s s o f membrane l i p i d (Reed and S w i s h e r 1966; H o r a d i n e t a l . 1 9 6 7 ) . A c o m p r e h e n s i v e s t u d y by Weed ejt a l _ . (1969) r e l a t e d t h e o b s e r v e d . c h a n g e s i n v i s c o s i t y , membrane d e f o r m a b i l i t y , f i l t e r a b i l i t y , and d e c r e a s e d c e l l u l a r 2+ ATP l e v e l s t o i n c r e a s e s i n t h 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 Ca . A p r o g -r e s s i v e i n c r e a s e i n c e l l u l a r c a l c i u m was n o t e d i n s t o r e d r e d b l o o d c e l l s and t h o s e m e t a b o l i c a l l y d e p l e t e d by i n c u b a t i o n a t 37°C. A d i r e c t i n v o l v e m e n t o f i n t r a c e l l u l a r c a l c i u m i n t h e shape changes was t e s t e d by i n c o r p o r a t i n g c a l c i u m i n t o r e c o n s t i t u t e d g h o s t s . Ca p r o d u c e d a c r e n a t e d o r e c h i n o c y t i c shape change w h i c h c o u l d be b l o c k e d by s i m u l t a n e o u s i n c o r p o r a t i o n o f EDTA o r ATP i n t h e r e -s e a l e d g h o s t s . P r e s u m a b l y EDTA o r ATP c h e l a t e d c a l c i u m t h u s p r e v e n t i n g c a l -c i u m f r o m i n t e r a c t i n g w i t h t h e i n n e r membrane s u r f a c e . I n t e r e s t i n g l y , i n t h e o t w e n t y - f o u r h o u r p e r i o d i n w h i c h n o r m a l e r y t h r o c y t e s were ATp d e p l e t e d a t 37 C, changes i n membrane d e f o r m a b i l i t y p r e c e d e d m e a s u r a b l e amounts o f c a l c i u m a c -c u m u l a t i o n b y t h e c e l l s w h i c h s u g g e s t e d t h a t ATP d e p l e t i o n p e r m i t t e d i n t r i n s i c 2+ Ca t o e x e r t a change i n d e f o r m a b i l i t y . T h i s o b s e r v a t i o n was s u p p o r t e d by S z a s z e t a l • ( 1970 ) who f o u n d t h a t t h e d i s c - s p h e r e t r a n s f o r m a t i o n b r o u g h t o n by i n c u b a t i n g c e l l s w i t h NaF c o u l d n o t be p r e v e n t e d by e x t r a c e l l u l a r EGTA. T h e r e f o r e ATP a c t s as an i m p o r t a n t r e g u l a t o r o f t h e f r e e c o n c e n t r a t i o n o f i n t -2+ r a c e l l u l a r Ca . Weed et. a l . (1969) p o s t u l a t e d t h a t t h e ATP dependence o f t h e b i c o n c a v e d i s c c o u l d r e s u l t f r o m t h e c a p a c i t y t o p r e v e n t two c a l c i u m - i n d u c e d p r o c e s s e s : (1) s o l - g e l t r a n s f o r m a t i o n s o f membrane p r o t e i n s and a d j a c e n t p r o t e i n s . (2) c o n f o r m a t i o n a l changes o f membrane c o n t r a c t i l e e l e m e n t s . H emoglobin has a l s o been i m p l i c a t e d i n r e g u l a t i n g t h e f r e e i n t r a c e l l u l a r 2+ Ca c o n c e n t r a t i o n by an i n d i r e c t mechanism. I t has l o n g been known t h a t t h e v i s c o s i t y o f venous b l o o d was g r e a t e r t h a n t h a t o f a r t e r i a l b l o o d a l t h o u g h 10 t h e p h y s i o l o g i c a l mechanism was n o t known ( B e s t and T a y l o r 1 9 5 0 ) . Deoxy-g e n a t i o n and t h e l o w e r i n g o f pH r e s u l t s i n a s i g n i f i c a n t r e v e r s i b l e b i n d i n g o f ATP t o h e m o g l o b i n w i t h a c o n s e q u e n t d e c r e a s e i n p h o s p h o r y l a t i o n o f g l u c o s e (Garby and de V e r d i e r 1 9 6 8 ) . C a l c u l a t i o n s by Chau-Wong and Seeman (1971) i n d i c a t e d t h a t o x y g e n a t e d b l o o d c e l l s w o u l d have a c y t o p l a s m i c f r e e ATP l e v e l o f 1.45 mM and t h a t t h e ATP c o n c e n t r a t i o n i n r e d b l o o d c e l l s i n venous b l o o d (60% o x y g e n a t e d ) w o u l d be 0.94 mM. T h e r e f o r e t h e l e v e l o f membrane bound c a l c i u m w o u l d be e x p e c t e d t o be h i g h e r i n d e o x y g e n a t e d c e l l s s i n c e t h e r e w o u l d be l e s s i n t r a c e l l u l a r c h e l a t i o n o f C a 2 + by ATP. A g r e a t e r amount o f Ca b i n d i n g t o membranes i n venous b l o o d w o u l d e x p l a i n t h e g r e a t e r r i g i d i t y and h i g h e r v i s c o s i t y i n l i n e w i t h t h e model by Weed e t a l . ( 1 9 6 9 ) . The c o n t r a c t i o n o f e r y t h r o c y t e g h o s t s does n o t seem t o be d e p endent on ATP w h i c h one w o u l d e x p e c t i f c o n t r a c t i l e p r o t e i n s were a s s o c i a t e d w i t h ATPase Ox a c t i v i t y . I n c o r p o r a t i o n o f Ca a l o n e i n t o ATP d e p l e t e d r e c o n s t i t u t e d g h o s t s r e s u l t s i n a v e r y l a r g e i n c r e a s e i n membrane r i g i d i t y i n s h a r p c o n t r a s t t o t h e e f f e c t o f M g 2 + (Weed e t aJU 1969) . ATP o r M g ^ p r e v e n t t h e e f f e c t o f Ca p r e s u m a b l y t h r o u g h a r e d u c t i o n i n membrane C a 2 + c o n t e n t by c h e l a t i o n o r mass a c t i o n c o m p e t i t i o n r e s p e c t i v e l y . T h i s f i n d i n g was s u p p o r t e d by P a l e k ejb a l . 2+ (1971 a) who f o u n d t h a t Ca i n c o r p o r a t e d i n t o r e s e a l e d g h o s t s w i l l r e s u l t i n up t o a 50% r e d u c t i o n i n c e l l volume w h i c h c o u l d a l s o be r e p r o d u c e d w i t h 2+ 2+ S r and Ba . I n t r a c e l l u l a r Ca was a l s o f o u n d t o i n c r e a s e t h e membrane perm-e a b i l i t y t o Na + . ATP and o t h e r n u c l e o t i d e s i n h i b i t e d t h e e f f e c t o f Ca i n p r o p o r t i o n t o t h e s t a b i l i t y c o n s t a n t s o f t h e Ca^ + n u c l e o t i d e c o m p l e x e s . 2+ More r e c e n t l y P a l e k e t a l . (1974) e v a l u a t e d t h e r e l a t i o n s h i p between Ca 2+ Mg and ATP on d i s c o c y t e - e c h i n o c y t e t r a n s f o r m a t i o n s i n r e c o n s t i t u t e d g h o s t s . F o u r d i f f e r e n t c o n f i g u r a t i o n s were f o u n d when r e c o n s t i t u t e d g h o s t s were exam-i n e d u n d e r t h e e l e c t r o n m i c r o s c o p e d e p e n d i n g on t h e i o n s o r n u c l e o t i d e s i n c o r p o r a t e d : (1) f l a t , d i s c o i d a l e l e c t r o n p e n e t r a b l e g h o s t s were p r o d u c e d 11 when g h o s t s were p r e p a r e d w i t h o u t a d d i t i v e s o r w i t h ATP, Mg o r w i t h EDTA a l o n e . (2) b i c o n c a v e and cup-shaped l e s s e l e c t r o n p e n e t r a b l e g h o s t s p r e -d o m i n a t e d when e q u i m o l a r c o n c e n t r a t i o n s o f M g 2 + and ATP (0.5 t o 2.0 mM) were i n t r o d u c e d i n t o t h e g h o s t s d u r i n g h e m o l y s i s . (3) s p h e r i c a l , s p i c u l a t e d 2+ o r m a r k e d l y e l e c t r o n dense g h o s t s o f s m a l l e r volume were p r e s e n t when Ca a l o n e was i n t r o d u c e d d u r i n g h e m o l y s i s . (4) s m a l l , smooth e l e c t r o n - d e n s e s p h e r o i d s were p r e s e n t when b o t h Ca and Mg-ATP (2 mM) were added d u r i n g h e m o l y s i s . These shapes were i n d e p e n d e n t o f o u a b a i n , m o n o v a l e n t c a t i o n s ( N a C l , K C l o r c h o l i n e c h l o r i d e ) , transmembrane o s m o t i c g r a d i e n t and t h e g h o s t s volume. I f t h e g h o s t s were r e p e a t e d l y washed w i t h t h e a s s o c i a t e d r e m o v a l o f w a t e r s o l u b l e f i b r i n o u s p r o t e i n s f r o m t h e membranes ,the shape e f f e c t s o f C a 2 + and Mg-ATP were p r e v e n t e d . The a u t h o r s c o n c l u d e t h a t C a 2 + , Mg 2* and ATP i n t e r a c t w i t h i n t e r n a l l y l o c a t e d f i b r i l l a r ( c o n t r a c t i l e - l i k e ) p r o t e i n s w h i c h a r e i n v o l v e d i n m a i n t e n a n c e o f t y p i c a l shape o f r e d c e l l s . T h i s s y s t e m a p p e a r s t o be o r g a n i z e d as a l a y e r a s s o c i a t e d w i t h b u t n o t an i n t e g r a l p a r t o f t h e r e d c e l l membranes. A l t h o u g h P a l e k e t a l . ( 1 9 7 4 ) s u g g e s t t h e shape i s de p e n d e n t o n M g 2 t , c o n t r o l e x p e r i m e n t s w i t h C a 2 + and ATP were n o t c a r r i e d o u t . Weed e t a l _ . (1969) f o u n d t h a t t h e r e was no d i f f e r e n c e between t h e shape o f g h o s t s i n c o r p o r a t e d w i t h Ca and ATP, Ca and Mg, o r Ca and EDTA. The c a l c i u m m e d i a t e d c h a n g e s d i s c u s s e d s i g n i f i c a n t l y a f f e c t t h e l i f e s p a n o f t h e e r y t h r o c y t e i n t h e c i r c u l a t i o n ( L a C e l l e 1969; Weed and C h a i l l e y 1 9 7 3 ) . D e c r e a s e s i n c e l l u l a r d e f o r m a b i l i t y a d v e r s e l y a f f e c t t h e t h e c a p a c i t y o f t h e e r y t h r o c y t e t o s u r v i v e t h e s e v e r e f l o w r e q u i r e m e n t s o f t h e m i c r o c i r c u l a t i o n and t h e p h a g o c y t i c e l e m e n t s o f t h e r e t i c u l o -e n d o t h e l i a l s y s t e m . T h e r e i s e v i d e n c e t h a t aged c e l l s o r c e l l s w i t h l o w e r : pH l o s e t h e i r d e f o r m a b i l i t y and t h e r e f o r e w o u l d l e s s e a s i l y t r a n s v e r s e t h r o u g h t h e m i c r o c a p i l l a r i e s i n t h e s p l e e n ( L a C e l l e 1973)„ The p h y s i o l o g i c a l mechanisms known t o be i n v o l v e d i n r e g u l a t i n g t h 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 o f C a 2 + have been summarized i n F i g . l . F i g . l . S c h e m a t i c o f p o s t u l a t e d A T P - C a ^ membrane i n t e r a c t i o n s c o n t r o l l i n g e r y t h r o c y t e shape and membrane d e f o r m a b i l i t y . E f f e c t i v e ATP c o n c e n t r a t i o n a v a i l a b l e t o c h e l a t e C a 2 + t o p r e v e n t i t s i n t e r a c t i o n a t h i g h a f f i n i t y , i n n e r membrane p r o t e i n s i t e X may be r e d u c e d as t h e t o t a l ATP i s l o w e r e d and a l s o by i n c r e a s e d ATP-Hb b i n d i n g as t h e DPG c o n c e n t r a t i o n d e c r e a s e s . M g 2 + may compete w i t h C a 2 * f o r s i t e X. Taken f r o m L a C e l l e e t a l . ( 1 9 7 3 ) . 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 ^ i s r e g u l a t e d by c h e l a t i o n t o 2+ ATP, b i n d i n g t o membrane s i t e s X and a l s o by d i f f u s i o n o f Ca i n t o t h e 2+ 2+ c e l l . The ATP d e p e n d e n t Ca pump l o w e r s t h e i n t r a c e l l u l a r C a c o n c e n t r a t i o n 2+ by pumping Ca o u t o f t h e c e l l . Mg-ATP may a l s o be bound t o s i t e s o n t h e membranes 5. Aims o f t h e P r e s e n t S t u d y . 2+ 1. To s t u d y t h e Ca a c t i v a t i o n o f (Mg+Ca)-ATPase a c t i v i t y i n a number o f membrane p r e p a r a t i o n s t o d e t e r m i n e i f b o t h h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s a r e p r e s e n t . 2. To s e p a r a t e h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s . 3. To d e t e r m i n e w h i c h (Mg+Ca)-ATPase a c t i v i t y o r i f b o t h (Mg+Ca)-ATPase a c t i v i t i e s a r e a s s o c i a t e d w i t h ATP dependent c a l c i u m t r a n s p o r t i n v e s i c l e s . 4. To s t u d y t h e e f f e c t o f l a n t h a n u m on c a l c i u m t r a n s p o r t and (Mg+Ca)-ATPase a c t i v i t y i n r e s e a l e d g h o s t s and on membrane f r a g m e n t s . 13 MATERIALS AND METHODS S e c t i o n I . S t u d i e s w i t h E r y t h r o c y t e G h o s t s . 1. P r e p a r a t i o n o f e r y t h r o c y t e g h o s t s . Whole human b l o o d p r e s e r v e d i n a c i d - c i t r a t e - d e x t r o s e s o l u t i o n was o b t a i n e d f r o m t h e l o c a l Red C r o s s B l o o d Bank and u s e d w i t h i n 20 da y s o f c o l l e c t i o n . The r e d c e l l s were washed t w i c e i n i s o t o n i c s a l i n e s o l u t i o n a t 2,000 x g f o r 10 m i n u t e s . The b u f f y c o a t was c a r e f u l l y removed by s u c t i o n w i t h l o s s o f a b o u t one t h i r d o f t h e r e d c e l l s . 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 method o f s t e p w i s e h e m o l y s i s d e s c r i b e d by S c h r i e r ( 1 9 6 7 ) . The washed c e l l s were c o n s e c u t i v e l y h e m o l y z e d i n 10 volumes o f 0.08 0.06 and 0.04 M N a C l and c e n t r i f u g e d a t 8,000, 13,000 and 15,000 x g f o r 10 m i n u t e s a t 2-4 C, r e s p e c t i v e l y . The c e l l s were f i n a l l y h e m o l y z e d i n 0.015 M N a C l and 0.005 M T r i s - m a l e a t e (pH 7.1) a t 2-4°C and washed t w i c e i n t h i s s o l u t i o n . One m i l l i l i t e r o f g h o s t s was f o u n d t o c o n t a i n 3.5 t o 4.4 mg o f p r o t e i n . These g h o s t s were u s e d i m m e d i a t e l y f o r t h e p r e p a r a t i o n o f r e s e a l e d g h o s t s o r v e s i c l e s o r were s t o r e d a t -20 C f o r up t o 10 d a y s . 2. (Mg+Ca)-ATPase a s s a y f o r f r o z e n g h o s t membranes. The i n c u b a t i o n medium (3 ml) f o r t h e a s s a y o f (Mg+Ca)-ATPase a c t i v i t y c o n t a i n e d 55 mM T r i s - m a l e a t e b u f f e r (pH 7 . 2 ) , 66mM N a C l , 0.1 mM EGTA, 6.5 mM MgCl2» 2 mM ATP, v a r i o u s c o n c e n t r a t i o n s o f C a C ^ and 0.2 ml o f membranes. F r o z e n g h o s t membranes were p r e p a r e d by f r e e z i n g and t h a w i n g t h r e e t i m e s a t -20 C. The r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f ATP and t h e r e a c t i o n m i x t u r e was i n c u b a t e d f o r one hou r a t 37 C. The r e a c t i o n was s t o p p e d w i t h t h e a d d i t i o n o f 1 ml o f c o l d t r i c h l o r o a c e t i c a c i d (20 % ) . The Ca i o n c o n c e n t r a t i o n s were c a l c u l a t e d a c c o r d i n g t o e q u a t i o n s g i v e n by K a t z e t a l . (1 9 7 0 ) . The CaEGTA s t a b i l i t y c o n s t a n t o f 1 0 1 0 * 6 5 r e p o r t e d by Schatzmann (1973) 14 was u s e d i n t h e s e e q u a t i o n s . T a b l e s A and B, A p p e n d i x , l i s t s t h e v a l u e s o f o t h e r s t a b i l i t y c o n s t a n t s u s e d i n t h e c a l c u l a t i o n s and t h e c a l c u l a t e d v a l u e s f o r C a 2 + a t pH 7.2 u s e d i n t h i s s t u d y . (Mg+Ca)-ATPase a c t i v i t y was d e t e r m i n e d by m e a s u r i n g i n o r g a n i c p h o s p h a t e ( P i ) r e l e a s e d f r o m ATP. The r e l e a s e o f P i was f o u n d t o be l i n e a r w i t h t i m e ( u p t o 60 m i n u t e s ) and p r o t e i n c o n c e n t r a t i o n . (Mg+Ca)-ATPase a c t i v i t y was c o r r e c t e d f o r Mg-ATPase a c t i v i t y and non-enzymic h y d r o l y s i s o f ATP by s u b t r a c t i o n o f t h e a c t i v i t y o b t a i n e d i n t h e absence o f added C a C l 2 . ATPase a c t i v i t y was e x p r e s s e d p e r m i l l i l i t e r o f g h o s t s (umoles P i / h r / m l g h o s t s ) r a t h e r t h a n mg o f p r o t e i n i n o r d e r t o a l l o w c o m p a r i s o n between a c t i v i t i e s o b t a i n e d i n membrane f r a g m e n t s o f d i f f e r e n t p r o t e i n c o n t e n t s due t o t h e t r e a t m e n t o f g h o s t s a t v a r i o u s low i o n i c s t r e n g t h c o n d i t i o n s ( See S e c t i o n I I , M a t e r i a l s and M e t h o d s ) . 3. T r e a t m e n t o f g h o s t s w i t h p h o s p h o l i p a s e A^. The p r o c e d u r e u s e d was a m o d i f i c a t i o n o f t h e method o f R o e l o f s e n and v a n Deenen ( 1 9 7 3 ) . Two m i l l i l i t e r s o f g h o s t s ( f r e e z e - t h a w e d t h r e e t i m e s a t -20°C) were suspended i n 4 ml o f 110 mM T r i s - m a l e a t e (pH 7.2) , 0.5% b o v i n e serum a l b u m i n and 5 mM C a C ^ . 100 IU o f p u r i f i e d p h o s p h o l i p a s e f r o m p o r c i n e p a n c r e a s ( a g i f t f r o m D r s . B. R o e l o f s e n and L.L.M. va n Deenen) was added. I n c u b a t i o n was c a r r i e d o u t i n e r l e n m e y e r f l a s k s i n a s h a k i n g w a t e r b a t h a t 37°C f o r 60 m i n u t e s . A f t e r 60 m i n u t e s , t h e m i x t u r e was c o o l e d i n i c e and c e n t r i f u g e d a t 20,000 x g f o r 10 m i n u t e s . I n o r d e r t o remove Ca, t h e p e l l e t was suspended i n 12 ml o f i c e c o l d s o l u t i o n c o n t a i n i n g 15 mM N a C l , 5 mM T r i s - m a l e a t e (pH 7.2) and 2 mM EGTA. The membranes were r e c e n t r i f u g e d and suspended i n 15 ml o f i c e c o l d 15 mM N a C l , 5 mM T r i s - m a l e a t e (pH 7.2) and 0.5% serum a l b u m i n . The m i x t u r e was c e n t r i f u g e d and t h e p e l l e t was r e s u s p e n d e d t o a f i n a l volume o f 2 ml i n 15 mM N a C l and 5 mM T r i s - m a l e a t e (pH 7 . 2 ) . T h i s p r e p a r a t i o n was t h e n a s s a y e d f o r (Mg+Ca)-ATPase a c t i v i t y . C o n t r o l 15 g h o s t s membranes were t r e a t e d s i m i l a r l y i n t h e absence o f p h o s p h o l i p a s e A 2 and a s s a y e d f o r (Mg+Ca)-ATPase a c t i v i t y . 4. 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 . One ml o f f r e s h l y p r e p a r e d g h o s t s was suspended i n 3 ml o f a l o a d i n g medium w h i c h c o n t a i n e d i n f i n a l c o m p o s i t i o n 4 mM M g C ^ , 4 mM Na^ATP and 10 mM T r i s - m a l e a t e (pH 7.1) and v a r i o u s c o n c e n t r a t i o n s o f C a C l 2 . The s u s p e n s i o n was a l l o w e d t o e q u i l i b r a t e a t 2-4 C f o r 10 m i n u t e s . R e s e a l i n g was a c h i e v e d by r e s t o r a t i o n o f i s o t o n i c i t y w i t h 0.2 m l o f 2.88 M N a C l and i n c u b a t i o n f o r 10 o m i n u t e s i n a w a t e r b a t h a t 25 C. The t e m p e r a t u r e o f i n c u b a t i o n was f o u n d t o . be v e r y c r i t i c a l i n d e t e r m i n i n g t h e f i n a l Ca c o n t e n t i n t h e r e s e a l e d g h o s t s ( Q u i s t and R o u f o g a l i s 1975) and t h e r e f o r e t h e r e s e a l i n g t e m p e r a t u r e was c a r e f u l l y c o n t r o l l e d . The t u b e s were r e t u r n e d t o t h e i c e b a t h and washed t w i c e w i t h 5 ml o f 2 mM M g C l 2 , 125 mM N a C l and 20 mM T r i s - m a l e a t e (pH 7.1) a t 5,000 x g f o r 10 m i n u t e s a t 2-4°C. The r e s e a l e d g h o s t s were suspended t o a f i n a l volume o f 3 m l w i t h t h e same s o l u t i o n w h i c h a l s o c o n t a i n e d 1 mM C a C ^ . When L a C l ^ was i n v e s t i g a t e d , i t was added i n t h e e x t e r n a l medium o f t h e r e s e a l e d g h o s t s and i n c u b a t e d a t 2-4 C f o r a p p r o x i m a t e l y 10 m i n u t e s . 5. D e t e r m i n a t i o n o f (Mg+Ca)-ATPase a c t i v i t y and Ca t r a n s p o r t i n r e s e a l e d  g h o s t s . To i n i t i a t e b o t h ATPase a c t i v i t y and Ca t r a n s p o r t , t h e r e s e a l e d g h o s t s were p l a c e d i n a w a t e r b a t h a t 37 C. To d e t e r m i n e ATPase a c t i v i t y t h e r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f 1 ml o f i c e c o l d TCA (20%) a t a p p r o p r i a t e t i m e i n t e r v a l s . ATPase a c t i v i t y was d e t e r m i n e d by m e a s u r i n g t h e r e l e a s e o f P i f r o m ATP. I n o r d e r t o e s t i m a t e t h e c o n t r i b u t i o n o f l e a k y g h o s t s t o t o t a l ATPase a c t i v i t y , t h e g h o s t s were r e s e a l e d as above b u t i n t h e absence o f ATP. The f o r m a t i o n o f P i a f t e r t h e a d d i t i o n o f 1 mM ATP t o t h e e x t e r n a l i n c u b a t i o n medium a t 37 C was u s e d t o e s t i m a t e t h e ATPase a c t i v i t y due t o l e a k y o r n o n - s e a l e d g h o s t s . 16 Mg-ATPase a c t i v i t y was d e t e r m i n e d i n t h e absence o f C a C l ^ by d i r e c t i n c u b -a t i o n o f h e m o l y z e d g h o s t s w i t h ATP a t 37°C. O u a b a i n s e n s i t i v e Na,K-ATPase a c t i v i t y was n o t p r e s e n t i n g h o s t s r e s e a l e d w i t h N a C l i n t h e absence o f added K C l and u n d e r t h e s e c o n d i t i o n s does n o t c o n t r i b u t e t o t o t a l ATPase a c t i v i t y ( Q u i s t 1 9 7 3 ) . Ca e f f l u x i n r e s e a l e d g h o s t s was measured as t h e l o s s o f c e l l u l a r Ca w i t h t i m e a t 37°C. The r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f 6 ml o f i c e c o l d 119 mM N a C l and 6 mM L a C l ^ . L a C l 3 was i n c l u d e d i n t h i s s o l u t i o n t o d i s p l a c e l o o s e l y bound Ca f r o m t h e e x t e r n a l s u r f a c e o f r e s e a l e d g h o s t s (van Breeman 1 9 6 9 ) . The t u b e s were t h e n c e n t r i f u g e d a t 5,000 x g f o r 10 m i n u t e s a t 2-4°C. The s u p e r n a t a n t was a s p i r a t e d o f f and t h e p e l l e t was wash-ed once more i n 6 ml o f t h e same s o l u t i o n . Ca was e x t r a c t e d f r o m t h e p e l l e t and measured by a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y (see S e c t i o n V I , M a t e r i a l s & M e t h o d s ) . S e c t i o n I I . S t u d i e s w i t h Membrane F r a g m e n t s . 1. P r e p a r a t i o n o f c o n t r o l membrane f r a g m e n t s ( P r e p a r a t i o n A ) . One volume o f g h o s t s ( s t o r e d f r o z e n a t -20°C) was d i l u t e d w i t h f i v e volumes o f 0.5 mM N a C l . The pH was q u i c k l y a d j u s t e d t o pH 6.5 by t i t r a t i o n w i t h 10 mM m a l e i c a c i d . T h i s s u s p e n s i o n was t h e n i m m e d i a t e l y c e n t r i f u g e d a t 20,000 x g f o r 15 m i n u t e s a t 2-4°C. The s u p e r n a t a n t was d i s c a r d e d and t h e p e l l e t was s u s p e n d e d t o t h e o r i g i n a l volume o f t h e s t a r t i n g g h o s t p r e p a r a t i o n w i t h 0.015 M N a C l and 0.005 M T r i s - m a l e a t e (pH 7 . 1 ) . T h i s p r e p a r a t i o n c o n t a i n s b o t h h i g h and l o w Ca a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s and i s r e f e r r e d t o as p r e p a r -a t i o n A. 2. P r e p a r a t i o n o f membranes c o n t a i n i n g o n l y low a f f i n i t y (Mg+Ca)-ATPase  a c t i v i t y ( P r e p a r a t i o n B ) .  Membranes d e v o i d o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y were p r e p a r e d 17 by s u s p e n d i n g one volume o f g h o s t s i n f i v e volumes o f 0.1 mM EDTA and 1.0 mM T r i s - m a l e a t e (pH 8.0) , o r o t h e r low i o n i c s t r e n g t h s o l u t i o n s , and i n c u b a t e d a t 37*C f o r v a r i o u s t i m e i n t e r v a l s , u s u a l l y 30 m i n u t e s . The o t h e r l o w i o n i c s t r e n g t h media s t u d i e d were 0.2 mM N a C l , 0.5 mM N a C l , 0.2 mM M g C l 2 and d i s t i l l e d w a t e r . The pH o f t h e s e l a t t e r s u s p e n s i o n s r a n g e d f r o m 7.2 t o 7.5 as a r e s u l t o f b u f f e r i n g by t h e membranes. The s u s p e n s i o n s were t h e n c e n t r i f u g e d f o r 15 m i n u t e s a t 20,000 x g a t 2-4°C. The s u p e r n a t a n t was e i t h e r c o l l e c t e d f o r p r e p a r a t i o n o f f r a c t i o n C (see below) o r d i s c a r d e d and t h e p e l l e t was r e s u s p e n d e d i n 15 mM N a C l and 5 mM T r i s - m a l e a t e (pH 7.1) t o t h e o r i g i n a l volume o f g h o s t s u s e d . These membranes were a s s a y e d f o r (Mg+Ca)-ATPase a c t i v i t y i m m e d i a t e l y a f t e r p r e p a r a t i o n as t h e (Mg+Ca)-ATPases a r e r e l a t i v e l y l a b i l e u n d e r t h e s e c o n d i t i o n s . 3. P r e p a r a t i o n o f s o l u b l e f r a c t i o n C. The s u p e r n a t a n t c o l l e c t e d f r o m t h e f i n a l c e n t r i f u g a t i o n s t e p i n t h e above p r o c e d u r e was c o n c e n t r a t e d 6 f o l d by u l t r a f i l t r a t i o n (Amicon: model 52) u n d e r 10 p s i o f n i t r o g e n u s i n g XM-100 u l t r a f i l t r a t i o n membranes ( D i a f l o R ) . T h i s f r a c t i o n c o n t a i n s a p p r o x i m a t e l y 25% o f t h e o r i g i n a l membrane p r o t e i n s and i s r e f e r r e d t o as f r a c t i o n C. T r a c e s o f h e m o g l o b i n c o u l d be v i s u a l i z e d i n t h i s f r a c t i o n . I n some e x p e r i m e n t s , f r a c t i o n C was f u r t h e r washed by u l t r a f i l t r a t i o n w i t h two 25 ml a l i q u o t s o f 0.5 mM N a C l u n t i l a w h i t e opaque s o l u t i o n was o b t a i n e d . T h i s p r o t e i n f r a c t i o n was r e c o n s t i t u t e d w i t h p r e p a r a t i o n B membranes (see r e c o n s t i t u t i o n p r o c e d u r e S e c t i o n I I . 5 , M a t e r i a l s and Methods) o r f u r t h e r c o n c e n t r a t e d t o a p r o t e i n c o n t e n t o f 1 mg/ml and a n a l y z e d f o r p r o t e i n s by SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s as d e s c r i b e d i n Method V I I . The p r o t e i n s i n f r a c t i o n C were a l s o a n a l y z e d by t h e same p r o c e d u r e . 18 4. D e t e r m i n a t i o n o f (Mg+Ca)-ATPase a c t i v i t y i n membrane f r a g m e n t s . The i n c u b a t i o n m i x t u r e (3 ml) f o r t h e a s s a y o f (Mg+Ca)-ATPase a c t i v i t y c o n t a i n e d 55 mM T r i s - m a l e a t e (pH 7 . 2 ) , 66 mM N a C l , 0.1 mM EGTA, 6.5 mM MgCl,, and 2 mM ATP. 0.2 ml o f e i t h e r p r e p a r a t i o n A o r p r e p a r a t i o n B was added. I n r e c o n s t i t u t i o n e x p e r i m e n t s v a r i a b l e amounts o f f r a c t i o n C, u s u a l l y 0.2 m l , were added t o g e t h e r w i t h p r e p a r a t i o n B. The c o n c e n t r a t i o n o f C a C l ^ was v a r i e d i n t h i s m i x t u r e and t h e c o n c e n t r a t i o n o f f r e e Ca i o n s i n t h e p r e s e n c e o f EGTA were c a l c u l a t e d as p r e v i o u s l y d e s c r i b e d ( T a b l e A, A p p e n d i x ) . I n a few e x p e r -i m e n t s HEPES ( N - 2 - h y d r o x y m e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c a c i d ) b u f f e r was s u b s t i t u t e d f o r T r i s b u f f e r . HEPES b u f f e r , as w e l l as T r i s b u f f e r , c h e l a t e s n e g l i g b l e amounts o f d i v a l e n t m e t a l c a t i o n s and t h e r e f o r e does n o t a f f e c t t h e 2+ f r e e c o n c e n t r a t i o n o f Ca (Good e t a l . 1 9 6 6 ) . .When L a C l ^ w a s i n c l u d e d i n t h e 2+ i n c u b a t i o n medium EGTA was e x c l u d e d , as t h e Ca c o n c e n t r a t i o n was a l t e r e d i n 3+ 3+ t h e p r e s e n c e o f L a due t o t h e h i g h a f f i n i t y o f L a f o r EGTA ( O h n i s h i e t a l . 1963; Ogawa 1968; S i l l e n e t a l . 1 9 6 4 ) . The r e a c t i o n was s t a r t e d by t h e a d d i t -i o n o f ATP and t h e t u b e s were i n c u b a t e d f o r one ho u r i n a s h a k e r w a t e r b a t h a t 37°C. The r e a c t i o n was u s u a l l y s t o p p e d by t h e a d d i t i o n o f 1 ml o f c o l d TCA ( 2 0 % ) . When r u t h e n i u m r e d was p r e s e n t , t h e r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f 1 ml o f c o l d s i l i c o t u n g s t i c a c i d (8%) i n p e r c h l o r i c a c i d (1.2 M) t o p r e c i p i t a t e t h e r u t h e n i u m r e d w h i c h w o u l d o t h e r w i s e i n t e r f e r e w i t h t h e c o l o r i m e t r i c a s s a y f o r P i . The r e l e a s e o f P i f r o m ATP was f o u n d t o be l i n e a r i n b o t h t i m e ( t o 120 m i n u t e s ) and p r o t e i n c o n c e n t r a t i o n . ATPase a c t i v i t y was e x p r e s s e d a s ^ i m o l e s P i / h r / m l g h o s t s as t h e p r o t e i n c o n c e n t r a t i o n o f g h o s t s membranes v a r i e d w i t h t h e p r e p a r a t i o n c o n d i t i o n s . A c t i v i t i e s e x p r e s s e d r e l a t i v e t o t h e volume o f g h o s t s were c o n s t a n t and a l l o w c o m p a r i s o n o f s p e c i f i c a c t i v i t i e s o f membranes s u b j e c t e d t o t h e d i f f e r e n t t r e a t m e n t s u s e d i n t h e s t u d y o f membrane f r a g m e n t s . (Mg+Ca)-ATPase a c t i v i t i e s 19 were c o r r e c t e d f o r b a s a l Mg-ATPase a c t i v i t y and n o n - c a t a l y t i c h y d r o l y s i s o f ATP by s u b t r a c t i o n o f t h e a c t i v i t y o b t a i n e d i n t h e absence o f added C a C l 2 . 5. R e c o n s t i t u t i o n p r o c e d u r e . 0.2 ml e a c h o f p r e p a r a t i o n B and f r a c t i o n C were combined i n a f i n a l v o l -ume o f 3 ml (normal i n c u b a t i o n medium) a t room t e m p e r a t u r e f o r 10 m i n u t e s b e f o r e a s s a y i n g f o r (Mg+Ca)-ATPase a c t i v i t y . However, r e c o n s t i t u t i o n d i d n o t app e a r t o be t i m e dependent. The c o n c e n t r a t i o n o f f r a c t i o n C was a l s o v a r i e d i n t h e p r e s e n c e o f 0.2 ml o f p r e p a r a t i o n B [and a s s a y e d f o r (Mg+Ca)-ATPase a c t i v i t y ] . I n some r e c o n s t i t u t i o n e x p e r i m e n t s , p r e p a r a t i o n B was h e a t e d i n a w a t e r b a t h a t 50°C and 70°C f o r 30 m i n u t e s b e f o r e r e c o m b i n a t i o n w i t h f r a c -t i o n C. 6. T r e a t m e n t o f p r e p a r a t i o n B and f r a c t i o n C w i t h t r y p s i n . Two ml o f p r e p a r a t i o n B o r f r a c t i o n C was i n c u b a t e d f o r 30 m i n u t e s a t 37°C w i t h 1 mg o f t r y p s i n . The r e a c t i o n was s t o p p e d w i t h t h e a d d i t i o n o f 1 mg o f t r y p s i n i n h i b i t o r ( s o y b e a n ) . C o n t r o l s were r u n i n t h e absence o f t r y p -s i n and i n t h e p r e s e n c e o f 1 mg o f t r y p s i n and t r y p s i n i n h i b i t o r . (Mg+Ca) -ATPase a c t i v i t y was a s s a y e d b o t h i n t h e t r y p s i n t r e a t e d p r e p a r a t i o n B o r i n t r y p s i n - t r e a t e d p r e p a r a t i o n B r e c o n s t i t u t e d w i t h 0.2 ml o f n o n - t r e a t e d f r a c t i o n C. 0.2 ml o f t r y p s i n - t r e a t e d f r a c t i o n C was a l s o r e c o n s t i t u t e d w i t h 0. 2 ml o f n o n - t r e a t e d p r e p a r a t i o n B by t h e same p r o c e d u r e and a s s a y e d f o r (Mg+Ca)-ATPase a c t i v i t y . 7. T r e a t m e n t o f p r e p a r a t i o n B w i t h p h o s p h o l i p a s e A ? . P r e p a r a t i o n B was t r e a t e d w i t h 100 IU and 200 IU o f p h o s p h o l i p a s e A 2 by an i d e n t i c a l p r o c e d u r e u s e d f o r t h e p h o s p h o l i p a s e t r e a t m e n t o f g h o s t s ( s e c t i o n 1. 3 M a t e r i a l s and M e t h o d s ) . C o n t r o l s were r u n i n t h e absence o f added p h o s -p h o l i p a s e A 2 . The c o n t r o l and p h o s p h o l i p a s e A 2 t r e a t e d membranes were a s s a y e d i m m e d i a t e l y f o r (Mg+Ca)-ATPase a c t i v i t y o r a f t e r r e c o n s t i t u t i o n w i t h u n t r e a t e d 20 f r a c t i o n C. 8. T e m p e r a t u r e dependence o f low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . 18 ml o f i n c u b a t i o n medium c o n t a i n i n g 1.2 ml o f f r e s h l y p r e p a r e d p r e p -a r a t i o n B, 55 mM T r i s - m a l e a t e (pH 7 . 2 ) , 66 mM N a C l , 0.1 mM EGTA, 6.5 mM M g C l 2 2+ and 316 mM Ca was added t o j a c k e t e d v e s s e l s u n d e r c o n s t a n t s t i r r i n g . The t e m p e r a t u r e was c o n t r o l l e d t o w i t h i n +_ 0.5°C. A f t e r a t e n m i n u t e p r e i n c u b -a t i o n p e r i o d , t h e r e a c t i o n was s t a r t e d w i t h t h e a d d i t i o n o f 0.6 ml o f 60 mM Na 2ATP (2 mM f i n a l c o n c e n t r a t i o n ) . 3 ml a l i q u o t s were t a k e n e v e r y 20 m i n u t e s f o r 100 m i n u t e s f o r P i a n a l y s i s . I n o r d e r t o d e t e r m i n e t h e c o n t r i b u t i o n o f n o n - c a t a l y t i c ATP h y d r o l y s i s and Mg-ATPase a c t i v i t y t o t o t a l ATPase a c t i v i t y , t h e a s s a y was a l s o , made i n t h e absence o f added C a C l 2 . The pH o f t h e i n c u b a t i o n medium was a d j u s t e d t o pH 7.2 a t e v e r y t e m p e r a t u r e s t u d i e d as t h e pH o f T r i s b u f f e r i s t e m p e r a t u r e d e p e n d e n t . 9. P r e p a r a t i o n o f membranes a c c o r d i n g t o Wolf ( 1 9 7 2 a ) . Membranes were p r e p a r e d a c c o r d i n g t o p r o c e d u r e d e s c r i b e d by Wolf ( 1 9 7 2 a ) . B r i e f l y , p a c k e d e r y t h r o c y t e s were l y s e d i n 10 v o l u m e s o f a medium c o n t a i n i n g ( f i n a l c o n c e n t r a t i o n ) a b o u t 14 mM N a C l , 10 mM KC1, 20 mM T r i s - H C l b u f f e r (pH 7.4) and 20 mM s u c r o s e . The h e m o l y z e d c e l l s were c e n t r i f u g e d and washed 5 t o 6 t i m e s i n 1 mM KC1, 2mM T r i s -HC1 b u f f e r (pH 7.7) and 2 mM s u c r o s e u n t i l a s l i g h t l y p i n k g h o s t s u s p e n s i o n was o b t a i n e d . The g h o s t s u s p e n s i o n was f r o z e n a t -80°C a f t e r a d d i t i o n o f an e q u a l volume o f 0.5 M s u c r o s e and s t o r e d a t -20°C. 0.2 m l o f t h i s g h o s t p r e p a r a t i o n was a s s a y e d f o r (Mg+Ca)-ATPase a c t i v i t y by t h e same p r o c e d u r e as f o r p r e p a r a t i o n A. S e c t i o n I I I . S t u d i e s w i t h R e s e a l e d V e s i c l e s . 1. P r e p a r a t i o n o f r e s e a l e d v e s i c l e s . V e s i c l e s were p r e p a r e d f r o m g h o s t s u n d e r low i o n i c c o n d i t i o n s by a method a n a l o g o u s t o t h e p r o c e d u r e d e s c r i b e d by S t e c k ejt a l . ( 1 9 7 0 ) . S t e c k and cowork-e r s f o u n d t h a t g h o s t s suspended i n 10 volumes o f 0.1 mM EDTA and 1.0 mM T r i s - H C l (pH 8.0) bud s p o n t a n e o u s l y by an e n d o c y t i c p r o c e s s t o f o r m i n s i d e -o u t v e s i c l e s . R o u g h l y 45-50% o f t h e v e s i c l e s formed a r e i n s i d e - o u t w i t h t h e r e m a i n i n g v e s i c l e s b e i n g r i g h t - s i d e o u t . I n t h i s s t u d y one volume o f g h o s t s was suspended i n f i v e volumes o f 0.5 mM N a C l and i n c u b a t e d f o r 30 m i n u t e s a t 37°C. The t u b e s were t h e n c e n t r i f u g e d a t 20,000 x g f o r 15 m i n u t e s a t 2-4°C. The p e l l e t was suspended t o t h e o r i g i n a l volume o f g h o s t s w i t h 15 mM N a C l and 5 mM T r i s - M a l e a t e (pH 7.1). I n some e x p e r i m e n t s a p r e p a r a t i o n c o n s i s t i n g s o l e l y o f r i g h t - s i d e v e s i c l e s was p r e p a r e d by s u s p e n d i n g one volume o f g h o s t s i n 5 volumes 1 yM C a C l 2 , 0.2 mM M g C l 2 and 1.0 mM T r i s - m a l e a t e (pH 8.0) and i n c u b a t i n g t h e s u s p e n s i o n s a t 37°C f o r 30 m i n u t e s . These t u b e s were a l s o c e n t r i f u g e d a t 20,000 x g f o r 15 m i n u t e s a t 4°C and suspended t o t h e o r i g i n a l volume o f g h o s t s . To r e s e a l t h e v e s i c l e s , r o u t i n e l y , 20 ml o f e i t h e r v e s i c l e p r e p a r a t i o n was suspended i n 60 ml o f medium w h i c h c o n t a i n e d i n f i n a l c o m p o s i t i o n 10 mM T r i s - m a l e a t e CpH 7 . 2 ) , 4 mM M g C l 2 and 0.5 mM C a C l 2 i n a 125 ml e r l e n m e y e r f l a s k i n i c e . I n c e r t a i n e x p e r i m e n t s 4 mM ATP was i n c l u d e d i n t h i s medium and t h e c o n c e n t r a t i o n o f C a C l 2 was v a r i e d . The s u s p e n s i o n was a l l o w e d t o e q u i l i b r a t e f o r 5 m i n u t e s a t 2-4°C w i t h s u b s e q u e n t r e s t o r a t i o n o f i s o t o n i c i t y w i t h 4 m l o f 2.88 M N a C l . The e r l e n m e y e r f l a s k s were t h e n i n c u b a t e d f o r 10 m i n u t e s a t 37°C t o f a c i l i t a t e r e s e a l i n g . The s u s p e n s i o n was c e n t r i f u g e d i n 50 ml p o l y -c a r b o n a t e t u b e s a t 7000 x g f o r 10 m i n u t e s a t 4°C. The p e l l e t was washed t w i c e w i t h 60 ml o f s o l u t i o n c o n t a i n i n g 55 mM T r i s - m a l e a t e (pH 7 . 2 ) , 6.4 mM M g C l 2 and 66 mM N a C l a t 7,000 x g f o r 10 m i n u t e s a t 0°C. The p e l l e t was suspended i n 20 ml o f t h e same s o l u t i o n . R o u t i n e l y 1 volume (6 ml) o f v e s i c l e s u s p e n s i o n was d i l u t e d i n two v o l -umes (12 ml) o f 55 mM T r i s - m a l e a t e (pH 7 . 2 ) , 6.4 mM M g C l 2 , 100 mM N a C l and 0.15 mM EGTA. The c o n c e n t r a t i o n o f C a C l 2 was v a r i e d i n t h i s medium. I n r e c o n -s t i t u t i o n e x p e r i m e n t s 1.2 ml o f c o n c e n t r a t e d f r a c t i o n C was a l s o i n c l u d e d . 22 2. D e t e r m i n a t i o n o f (Mg+Ca)-ATPase a c t i v i t y and Ca u p t a k e . To i n i t i a t e e i t h e r A T P a s e a c t i v i t y o r Ca t r a n s p o r t , t h e f l a s k s were i n c u b -a t e d f o r 10 m i n u t e s a t 37°C and 0.6 ml o f 60 mM ATP ( f i n a l c o n c e n t r a t i o n 2 mM) was added t o t h e f l a s k s . A t a p p r o p r i a t e t i m e i n t e r v a l s , 2 ml a l i q u o t s o f t h e i n c u b a t i o n s u s p e n s i o n were removed f o r e i t h e r P i o r Ca a n a l y s i s . To d e t e r m i n e ATPase a c t i v i t y , t h e a l i q u o t was q u i c k l y m i x e d w i t h 1 m l o f c o l d TCA (20%) and t h e c o n c e n t r a t i o n o f P i was d e t e r m i n e d . R e l e a s e o f P i f r o m ATP was f o u n d t o be l i n e a r f o r up t o a t l e a s t 15 m i n u t e s . (Mg+Ca)-ATP-ase a c t i v i t y was c o r r e c t e d f o r Mg-ATPase a c t i v i t y and non-enzymic h y d r o l y s i s by s u b t r a c t i o n o f a c t i v i t y o b t a i n e d i n t h e absence o f added C a C ^ f r o m t h e t o t a l ATPase a c t i v i t y . ATPase a c t i v i t i e s were e x p r e s s e d i n ^ i m o l e s P i / h r / m g o f p r o t e i n as some o f t h e membranes were l o s t d u r i n g w a s h i n g o f t h e r e s e a l e d v e s i c l e s . The amount l o s t d u r i n g w a s h i n g v a r i e d w i t h d i f f e r e n t b l o o d samples and i t was t h e r e f o r e n e c e s s a r y t o d e t e r m i n e t h e p r o t e i n c o n t e n t i n t h e f i n a l i n c u b a t i o n . m e d i u m i n e a c h s t u d y . To d e t e r m i n e Ca u p t a k e i n v e s i c l e s , two ml a l i q u o t s o f i n c u b a t i o n m i x t u r e were removed a t r e g u l a r t i m e i n t e r v a l s and suspended i n 8 ml o f c o l d 119 mM N a C l and 6 mM L a C l . j . The t u b e s were t h e n c e n t r i f u g e d a t 15,000 x g f o r 15 m i n u t e s and t h e s u p e r n a t a n t was c o m p l e t e l y removed by s u c t i o n . Ca was e x t r a c t -ed f r o m t h e p e l l e t and measured by a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y (see S e c t i o n V I , M a t e r i a l s & M e t h o d s ) . 3. S e p a r a t i o n o f i n s i d e - o u t v e s i c l e s . I n two e x p e r i m e n t s t h e r e s e a l e d v e s i c l e p r e p a r a t i o n d e s c r i b e d i n ( s e c t i o n I I I . 1, M a t e r i a l s & Methods) was l a y e r e d upon an e q u a l volume o f d e x t r a n b a r r i e r s o l u t i o n ( d e n s i t y 1.1 g/ml d e x t r a n T-110) and c e n t r i f u g e d f o r two h o u r s a t 100,000 x g i n a Beckman SW 41 r o t o r . T h i s p r o c e d u r e i s r e p o r t e d t o s e p -a r a t e i n s i d e - o u t v e s i c l e s f r o m r i g h t - s i d e o u t v e s i c l e s ( S t e c k and K a n t 1 9 7 4 ) . The t o p band m a t e r i a l was c o l l e c t e d and washed w i t h 40 ml o f 55 mM T r i s -23 m a l e a t e (pH 7 . 2 ) , 6.4 mM M g C l 2 and 100 mM N a C l a t 7000 x g f o r 15 m i n u t e s . The p e l l e t was s uspended t o t h e o r i g i n a l volume o f v e s i c l e p r e p a r a t i o n . 2+ The v e s i c l e s u s p e n s i o n was a s s a y e d f o r ATPase a c t i v i t y a t d i f f e r e n t Ca c o n c e n t r a t i o n s , as above. 4. D e t e r m i n a t i o n o f a c e t y l c h o l i n e s t e r a s e and g l y c e r a l d e h y d e 3-phosphate  d e h y d r o g e n a s e a c t i v i t y i n r e s e a l e d v e s i c l e s . To d e t e r m i n e t h e s i d e d n e s s o f t h e v e s i c l e s and t h e e f f i c i e n c y o f r e s e a l -i n g , t h e v e s i c l e s were a s s a y e d f o r a c e t y l c h o l i n e s t e r a s e a c t i v i t y ( E l l m a n e t a l . 1961) and g l y c e r a l d e h y d e 3-phosphate d e h y d r o g e n a s e a c t i v i t y ( C o r i e t a l . 1 9 4 8 ) i n t h e p r e s e n c e and a bsence o f 0.1% T r i t o n X-100 by a s l i g h t 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 d e s c r i b e d by S t e c k and K a n t ( 1 9 7 4 ) . D e t e r m i n a t i o n o f t h e l a t e n t a c t i v i t i e s i n t h e p r e s e n c e o f d e t e r g e n t o f an o u t s i d e membrane mark e r ( a c e t y l -c h o l i n e s t e r a s e ) arid an i n s i d e membrane mark e r enzyme ( g l y c e r a l d e h y d e 3-p h o s p h a t e d e h y d r o g e n a s e ) a l l o w s e s t i m a t i o n o f t h e r e l a t i v e p r o p o r t i o n s o f i n s i d e - o u t , r i g h t - s i d e - o u t and u n s e a l e d membranes. I n b r i e f , t h e m a r k e r e n z -yme a s s a y s were p e r f o r m e d as f o l l o w s . To a s s a y a c e t y l c h o l i n e s t e r a s e a c t i v i t y , 0.1 ml o f v e s i c l e p r e p a r a t i o n was b r i e f l y p r e i n c u b a t e d a t room t e m p e r a t u r e w i t h an e q u a l volume o f 0.155 M N a C l o r 0.155 M N a C l p l u s 0.2% T r i t o n X-100 ( v / v ) . 2.8 ml o f 110 mM T r i s -m a l e a t e (pH 8.0) and .04 M N a C l and 0.1 ml o f 10 mM DTNB [ 5 , 5 ' - d i t h i o b i s - ( 2 -n i t r o b e n z o i c a c i d ) ] were added t o e a c h t u b e . 0.1 ml o f 0.03 M a c e t y l t h i o c h o -l i n e was added t o s t a r t t h e r e a c t i o n and t h e s o l u t i o n was q u i c k l y t r a n s f e r r e d t o a c u v e t and t h e r e a c t i o n was f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y a t 412 nm a t room t e m p e r a t u r e . To a s s a y g l y c e r a l d e h y d e 3-phosphate d e h y d r o g e n a s e a c t i v i t y , 0.1 ml o f i n c u b a t i o n medium was i n c u b a t e d f o r 1 m i n u t e i n s e m i - m i c r o c u v e t t e s w i t h e i t h e r 0.1 ml o f 0.155 M N a C l o r 0.2% T r i t o n X-100 i n 0.155 M N a C l . 0.82 ml o f 30 mM sodium p y r o p h o s p h a t e and 4 mM c y s t i n e HC1 were added f o l l o w e d by 24 0.03 ml o f 0.4 M sodium a r s e n a t e and 0.05 ml o f 20 mM J3NAD. 0.01 ml o f 0.1 mM g l y c e r a l d e h y d e 3-phosphate was added t o s t a r t t h e r e a c t i o n and t h e r e a c t -i o n was f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y a t 340 nm a t room t e m p e r a t u r e . The i n c r e a s e i n a b s o r b a n c e between t h e f i r s t and s e c o n d m i n u t e was u s e d as a meas-u r e o f t h e enzyme a c t i v i t y . S e c t i o n I V . T r e a t m e n t o f I n t a c t E r y t h r o c y t e s and R e s e a l e d G h o s t s w i t h  P r o t e o l y t i c Enzymes. Three ml o f washed e r y t h r o c y t e s were suspended i n 13 ml o f 0.155 M N a C l and 2 m l o f 110 mM T r i s - m a l e a t e (pH 7.2) i n t h e p r e s e n c e o f v a r i o u s c o n c e n t r a -t i o n s o f t r y p s i n and c h y m o t r y p s i n . These t u b e s were i n c u b a t e d f o r 1 h o u r a t 37°C i n a w a t e r b a t h . The t u b e s were t h e n removed f r o m t h e b a t h and c e n t r i f -uged a t 5,000 x g f o r 10 m i n u t e s a t 4°C. The s u p e r n a t a n t r e m a i n e d c l e a r i n d i c -a t i n g t h a t t h e t r e a t m e n t o f t h e c e l l s w i t h p r o t e o l y t i c enzymes d i d n o t h e m o l -y z e t h e c e l l s . The s u p e r n a t a n t was removed and g h o s t s were p r e p a r e d f r o m t h e c e l l s by s t e p w i s e h e m o l y s i s . The g h o s t s were f r o z e n a t -20°C and s t o r e d f o r 12 h o u r s . These f r o z e n g h o s t s were f r e e z e - t h a w e d 2 t i m e s and a s s a y e d f o r (Mg+ Ca)-ATPase a c t i v i t y a s d e s c r i b e d i n Methods I . One ml o f f r e s h l y p r e p a r e d g h o s t s was suspended i n t h r e e ml o f l o a d i n g medium w h i c h c o n t a i n e d i n f i n a l c o m p o s i t i o n 4 mM M g C l ^ , .5 mM C a C l 2 and 10 mM T r i s - m a l e a t e (pH 7 . 1 ) . I s o t o n i c i t y was r e s t o r e d w i t h 0.2 ml o f 2.88 M N a C l and t h e t u b e s were i n c u b a t e d a t 37°C f o r 10 m i n u t e s . The r e s e a l e d g h o s t s were i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f t r y p s i n . C o n t r o l s were a l s o made b o t h i n t h e absence o f enzyme and w i t h enzyme p l u s t r y p s i n i n h i b i t o r ( s o y b e a n ) . The t u b e s were i n c u b a t e d f o r 30 m i n u t e s a t 37°C and t h e r e a c t i o n was s t o p p e d by a d d i n g 1 mg t r y p s i n i n h i b i t o r a t a c o n c e n t r a t i o n e q u a l t o t h e amount o f t r y p s i n u s e d and by p l a c i n g t h e t u b e s i n i c e . The t u b e s were c e n t r i f u g e d a t 8,000 x g f o r 10 m i n u t e s a t 2-4°C. The p e l l e t was t h e n washed t w i c e w i t h 10 ml 25 o f 15 mM N a C l and 5 mM T r i s - m a l e a t e (pH 7.1) a t 15,000 x g f o r 10 m i n u t e s a t 4°C. The g h o s t s were suspended t o a f i n a l volume o f 5 ml w i t h t h e same s o l u t i o n and f r o z e n a t -20°C. The g h o s t s were f r e e z e - t h a w e d t h r e e t i m e s and a s s a y e d f o r (Mg+Ca)-ATPase a c t i v i t y . S e c t i o n V. D e t e r m i n a t i o n o f I n o r g a n i c P h o s p h a t e . I n o r g a n i c p h o s p h a t e ( P i ) r e l e a s e d f r o m ATP was d e t e r m i n e d by t h e method o f F i s k e and SubbaRow (1925) o r by a s l i g h t m o d i f i c a t i o n o f t h a t p r o c e d u r e ( R o u f o g a l i s 1 9 7 1 ) . The c o l o r was r e a d i n a s p e c t r o p h o t o m e t e r (Coleman-H i t a c h i 124) a t 660 nm a g a i n s t a d i s t i l l e d w a t e r r e f e r e n c e i n a 1.0 ml c u v e t . KH^PO^ ( a n a l y t i c a l r e a g e n t ) (100-500yiH) was u s e d as a p r i m a r y s t a n d a r d . S e c t i o n V I . D e t e r m i n a t i o n o f C a l c i u m . Ca was e x t r a c t e d f r o m r e s e a l e d g h o s t s o r v e s i c l e s 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 Sparrow and J o h n s t o n e ( 1 9 6 4 ) . The membrane p e l l e t s were d i s s o l v e d i n 1 ml o f 3 M TCA: g l a c i a l a c e t i c a c i d (1:1) w i t h v o r t e x i n g . Two ml o f d i s t i l l e d w a t e r was added t o t h e t u b e s . The t u b e s were v o r t e x e d and made up t o a f i n a l volume o f 5 ml w i t h 30 mM L a C l ^ and c e n t r i f u g e d a t 15,000 x g f o r 10 m i n u t e s a t room t e m p e r a t u r e . The c o n c e n t r a t i o n o f Ca i n t h e s u p e r n a t a n t was d e t e r m i n e d by a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y ( T e c h t r o n , model AA-5) a t 422.7 nm. S t a n d a r d C a C 0 3 r e f e r e n c e s o l u t i o n s were o b t a i n e d f r o m F i s h e r S c i e n t i f i c Company. A s t a n d a r d c u r v e o b e y i n g B e e r ' s Law was o b t a i n e d between 10 t o 100 /IM Ca. S e c t i o n V I I . SDS P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s . P o l y a e r y 1 a m i d e g e l e l e c t r o p h o r e s i s was p e r f o r m e d i n an E-C c o n t i n u o u s g e l s l a b e l e c t r o p h o r e s i s s y s t e m . The o v e r a l l a p p r o a c h was t h a t o f Weber and Osbo r n (1969) who r e p o r t e d t h a t s e p a r a t i o n o f p r o t e i n s by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n t h e p r e s e n c e o f t h e a n i o n i c d e t e r g e n t sodium d o d e c y l s u l f a t e 26 (SDS) i s dependent on t h e m o l e c u l a r w e i g h t s o f t h e i r p o l y p e p t i d e c h a i n s . F a i r b a n k s e t a l _ . (1971) have d e m o n s t r a t e d t h a t membrane bound p o l y p e p t i d e s can be c o n v e n i e n t l y c l a s s i f i e d by t h i s p r o c e d u r e . 1. P r e p a r a t i o n o f p r o t e i n s o l u t i o n s . S o l u b l e p r o t e i n f r a c t i o n s (1 mg/ml) were h e a t e d a t 100°C f o r 5 m i n u t e s t o d e s t r o y t h e a c t i v i t y o f t r a c e amounts o f endogenous p r o t e o l y t i c enzymes ( H u l l a 1 9 7 4 ) . 1.0 ml o f p r o t e i n s o l u t i o n was mixed w i t h 0.5 ml o f s o l u t i o n c o n t a i n -i n g 30 mM T r i s - m a l e a t e (pH 8 . 0 ) , 0.3% m e r c a p t o e t h a n o l and 3% SDS, 10% s u c r o s e , 3 mM EDTA, and 10 ug/ml p y r a n Y, and i n c u b a t e d f o r 20 m i n u t e s a t 37°C t o r e d u c e s u l f h y d r y l g r o u p s . A p r o t e i n s o l u t i o n c o n t a i n i n g 1 mg/ml o f Chymotryp-s i n o g e n A ( P h a r m a c i a ) , o v a l b u m i n (Pharmacia) and c a t a l a s e (Sigma) was u s e d t o c a l i b r a t e t h e g e l s . 2. P r e p a r a t i o n o f g e l s . G e l s were p r e p a r e d by a m i n o r m o d i f i c a t i o n o f t h e method o f N e l s o n e t a l . ( 1 9 7 4 ) . P r e p a r a t i o n o f g e l s i n v o l v e d m i x i n g 11.1 ml o f 0.1 M T r i s - m a l e a t e (pH 8 . 0 ) , 4.77 ml o f d i s t i l l e d w a t e r , 5.0 ml o f a c r y l a m i d e s o l u t i o n (22.2 g o f a c r y l a m i d e and 0.6 g o f m e t h y l e n e b i s a c r y l a m i d e i n a f i n a l volume o f 100 ml o f w a t e r ) , 0.23 m l o f 10% SDS, 1.1 ml o f f r e s h l y p r e p a r e d ammonium p e r -s u l f a t e (15 mg/ml) and 30 u l o f t e t r a e t h y l m e t h y l e n e d i a m i n e (TEMP). 160 m l o f t h i s g e l was s u f f i c i e n t t o p r e p a r e a g e l w i t h a t h i c k n e s s o f 3 mm. A f t e r m i x i n g , t h i s s o l u t i o n was i m m e d i a t e l y p o u r e d i n t o t h e g e l chamber o f t h e a p p a r -a t u s and c o v e r e d w i t h a few ml o f d i s t i l l e d w a t e r . The g e l was a l l o w e d t o p o l y m e r i z e f o r a t l e a s t 12 h o u r s a t 20°C. A f t e r t h i s t i m e t h e d i s t i l l e d w a t e r was removed w i t h a p a s t e U r p i p e t . B o t h chambers o f t h e a p p a r a t u s were f i l l e d w i t h 50 mM T r i s - m a l e a t e (pH 8.0) and . 1 % SDS. The g e l was p r e r u n a t 150 V and 90 mA f o r 30 m i n u t e s a t 2 0 b C . A t t h e end o f t h e p r e r u n , 25 t o 50 u l o f p r o t e i n sample were a p p l i e d t o t h e g e l s and r u n a t t h e same v o l t a g e as t h e p r e r u n f o r a b o u t 2 h o u r s o r u n t i l t h e t r a c k i n g dye had moved 10 cm. 3. S t a i n i n g and d e s t a i n i n g . The g e l s were s t a i n e d i n .4 g c o o m a s s i e b r i l l i a n t b l u e , 450 ml m e t h a n o l , 100 ml g l a c i a l a c e t i c a c i d and 450 m l o f d i s t i l l e d w a t e r f o r a t l e a s t 2 h o u r s a t room t e m p e r a t u r e . The g e l s were d e s t a i n e d o v e r n i g h t i n a d i f f u s i o n d e s t -a i n e r ( H o e f e r S c i e n t i f i c I n s t r u m e n t s ) c o n t a i n i n g 75 m l o f g l a c i a l a c e t i c a c i d , 50 ml o f m e t h a n o l and 875 ml o f w a t e r . S e c t i o n V I I I . D e t e r m i n a t i o n o f P h o s p h o l i p i d s . The t o t a l p h o s p h o l i p i d c o n t e n t o f membrane p r e p a r a t i o n s was d e t e r m i n e d by t h e method o f Chen e t a l . ( 1 9 5 6 ) . S e c t i o n I X . D e t e r m i n a t i o n o f P r o t e i n . The c o n c e n t r a t i o n o f p r o t e i n was d e t e r m i n e d by t h e method o f Lowry et_ a l . (1951) f o r i n s o l u b l e and s o l u b l e p r o t e i n s . B o v i n e serum a l b u m i n (3 X r e c r y -s t a l l i z e d was u s e d as a p r o t e i n s t a n d a r d . S t a n d a r d c u r v e s were l i n e a r f r o m 50 t o 250 ug o f p r o t e i n ; a t 500 nm. S e c t i o n X. T r e a t m e n t o f G h o s t s w i t h N e u r a m i n i d a s e . Two ml o f g h o s t s were s u s p e n d e d i n 6 ml o f 60 mM sodium a c e t a t e b u f f e r (pH 5.5) c o n t a i n i n g 1.0 mM C a C l ^ . 50 j i l o f n e u r a m i n i d a s e (sigma) ( V i b r i o comma) was added and t h e t u b e was i n c u b a t e d f o r 15 m i n u t e s a t 37°C. The r e a c t i o n was t h e n s t o p p e d i n i c e . The s u s p e n s i o n was added t o 18 m l o f 15 mM N a C l and 5 mM T r i s - m a l e a t e (pH 7.1) and 2.0 ml o f 7.5 mM EGTA and c e n t r i f u g e d a t 15,000 x g f o r 10 m i n u t e s a t 4°C. The s u p e r n a t a n t was a s p i r -a t e d and t h e p e l l e t was r e s u s p e n d e d i n 18 ml o f 15 mM N a C l and 5 mM T r i s -m a l e a t e (pH 7.1) and c e n t r i f u g e d as above. The p e l l e t was r e s u s p e n d e d t o a f i n a l volume o f 2 m l i n t h e same s o l u t i o n and t h e membranes were a s s a y e d f o r 28 (Mg+Ca)-ATPase a c t i v i t y a s i n S e c t i o n I . 2, M a t e r i a l s & Methods. S e c t i o n X I . C h e m i c a l s and M a t e r i a l s . I n o r g a n i c s a l t s were a n a l y t i c a l r e a g e n t g r a d e . Na2ATP ( S i g m a ) , L a C l 3 ( F i s h e r ) , EGTA (Sigma) were u s e d as r e c e i v e d . T r y p s i n (Type I I I , 2x c r y s t a -l l i z e d f r o m b o v i n e p a n c r e a s ) , t r y p s i n i n h i b i t o r (soybean Type 1-S) and r u t h e n i u m r e d were o b t a i n e d f r o m t h e Sigma Chem. Co. C e r t i f i e d a t o m i c a b s o r p t i o n c a l c i u m r e f e r e n c e s o l u t i o n was u s e d as a s t a n d a r d f o r a t o m i c a b s o r p t i o n s p e c t r o s c o p y . A l l c h e m i c a l s u s e d were a n a l y z e d f o r c a l c i u m c o n t a m i n a t i o n by t h e above method. P u r i f i e d p h o s p h o l i p a s e A 2 was a ge n e r o u s g i f t f r o m B. R o e l o f s e n and L.L.M. Van Deenen. C h e m i c a l s f o r a c r y l a m i d e g e l e l e c t r o p h o r e s i s : O r t e c i n c o r p o r a t e d a c r y l a m i d e , enzyme g r a d e (Eastman), N , N ' - m e t h y l e n e - b i s a c r y l a m i d e (Eastman), ammonium p e r s u l f a t e (E. C. A p p a r a t u s C o r p . ) , N,N,N' , _ N ' - t e t r a m e t h y l - e t h y l e n e — d i a m i n e (Eastman), p y r a r i Y ( F i s h e r ) , c o o m a s s i e b l u e (Sigma) and l a u r y l s u l f a t e (Sigma) were u s e d as r e c e i v e d . 29 RESULTS AND DISCUSSION S e c t i o n I . C h a r a c t e r i s t i c s o f H i g h and Low A f f i n i t y (Mg+Ca)-ATPase  A c t i v i t i e s i n E r y t h r o c y t e G h o s t s and Membrane F r a g m e n t s . 1. K i n e t i c s t u d y o f Ca a c t i v a t i o n o f (Mg+Ca)-ATPase a c t i v i t y i n e r y t h r o - c y t e membrane p r e p a r a t i o n s . The v a r i a b i l i t y i n 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)-ATPase and o t h e r ATPase a c t i v i t i e s i n e r y t h r o c y t e membrane p r e p a r a t i o n s i s w e l l documented (Bramley e t a l . 1 9 7 2 ; Hanahan 1 9 7 3 a ) . T h i s v a r i a b i l i t y i s a f u n c t i o n o f t h e method o f t h e p r e p a r a t i o n o f t h e membranes and s t o r a g e c o n d i t i o n s . G h o s t s ) p r e p a r e d i n i n t e r m e d i a t e i o n i c s t r e n g t h media (<"o 40 mosm) have l a t e n t ATPase a c t i v i t i e s due t o t h e i r l ow p e r m e a b i l i t i e s t o ATP (Bramley e t a l . 1 9 7 1 ) . L a t e n t ATPase a c t i v i t i e s c a n be exp o s e d i n t h e s e g h o s t s by s o n i c a t i o n ( Bramley e t a l . 1971) , d e t e r g e n t s (Bramley et^ a l . 1 9 7 1 ) , f r e e z i n g - t h a w i n g (Hanahan e t a l . 1 9 7 3 b ) and t r e a t m e n t w i t h d i v a l e n t m e t a l i o n c h e l a t i n g a g e n t s ( Q u i s t 1 9 7 3 ) . F r e e z e - t h a w i n g o f g h o s t s t o expose (Mg+Ca)-ATPase a p p e a r s p r e f e r a b l e s i n c e s o n i c a t i o n and d e t e r g e n t s (Wins 1969; B r a m l e y e t a l . 1971; W o l f 1972a; Hanahan e t ad.1973) and under some c o n d i t i o n s EGTA and EDTA (Wolf 1972a; S c h a r f f 1972; Schatzmann 1973) a l t e r t h e p r o p e r t i e s o f t h i s enzyme s y s t e m . The k i n e t i c p r o p e r t i e s o f (Mg+Ca)-ATPase a c t i v i t i e s i n a number o f e r y t h -r o c y t e membrane p r e p a r a t i o n s were i n v e s t i g a t e d h e r e t o d e t e r m i n e w h e t h e r t h e 2+ d i f f e r e n t p r e p a r a t i o n c o n d i t i o n s w o u l d a f f e c t t h e V and K o f Ca a c t i v -max A 2+ a t i o n . T y p i c a l Ca a c t i v a t i o n c u r v e s o f (Mg+Ca)-ATPase a c t i v i t y i n r e d b l o o d c e l l g h o s t s p r e p a r e d i n i n t e r m e d i a t e i o n i c m e d i a (<^> 40 mosm) a r e i l l u s t r a t e d i n F i g . 2. Cu r v e a was o b t a i n e d from g h o s t s (see S e c t i o n I . 1, M a t e r i a l s & M e t h o d s ) , f r o z e n f o r t h r e e d a y s a t -20°C and f r e e z e - t h a w e d t h r e e t i m e s i n 30 Figure 2. The activation of (Mg+Ca)-ATPase by Ca in ghosts prepared in a 40 mosm medium ( see Methods), frozen at -20 C in this medium and freeze-thawed three times before use. Ghosts were prepared in the absence, curve a ( © ) and in the presence, curve b (O) of 1.0 mM EDTA. Data in curve a represent the means of two separate experiments. 31 0.015 M N a C l and 0.005 M T r i s - m a l e a t e (pH 7.1) b e f o r e u s e . C u r v e b was o b t a i n e d f r o m g h o s t s t r e a t e d s i m i l a r l y t o t h o s e i n c u r v e a , e x c e p t t h a t 1 mM EDTA was i n c l u d e d i n t h e 0.08 M, 0.06 M and 0.04 M N a C l h e m o l y z i n g s o l u t i o n s u s e d i n 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 . The b i p h a s i c shape o f t h e C a 2 + a c t i v -a t i o n c u r v e s s u g g e s t s t h e p r e s e n c e o f h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s . I n o r d e r t o e s t i m a t e t h e V_,„ and K, t h e d a t a f r o m c u r v e s a and max A b ( F i g . 3) were p l o t t e d a c c o r d i n g t o E a d i e ( 1 9 4 2 ) . From t h e s l o p e s o f t h e c u r -ves a and b ( F i g . 3) t h e a p p a r e n t Ca d i s s o c i a t i o n c o n s t a n t s (K ) were e s t i m -2+ a t e d t o be 1.4 y.M and 1.0 y.H f o r t h e h i g h a f f i n i t y Ca b i n d i n g s i t e s and a p p r o x i m a t e l y 100 ^ (M f o r t h e l o w a f f i n i t y C a 2 + b i n d i n g s i t e s , r e s p e c t i v e l y . The f o r l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y c a n o n l y be a p p r o x i m a t e d f r o m t h e s l o p e s o f t h e s e c u r v e s s i n c e i t w i l l be shown l a t e r t h a t l ow a f f i n i t y ?+ (Mg+Ca)-ATPase does n o t y i e l d l i n e a r k i n e t i c p l o t s w i t h r e s p e c t t o Ca a c t i v a t i o n ( F i g . 2 9 ) . The s i g n i f i c a n c e o f t h i s f i n d i n g w i l l be d i s c u s s e d i n d e t a i l i n s e c t i o n I I I . 2+ The shape o f t h e Ca a c t i v a t i o n c u r v e s o f (Mg+Ca)-ATPase a c t i v i t y i n membrane f r a g m e n t s p r e p a r e d i n r e l a t i v e l y l o w e r i o n i c s t r e n g t h c o n d i t i o n s (below 20 mosm) a l s o s u g g e s t s t h e p r e s e n c e o f two d i s t i n c t (Mg+Ca)-ATPase 2+ a c t i v i t i e s ( F i g . 4 ) . C u r v e a ( F i g . 4) shows t h e Ca a c t i v a t i o n c u r v e s i n a membrane f r a g m e n t p r e p a r a t i o n ( P r e p a r a t i o n A) o b t a i n e d by s u s p e n d i n g g h o s t s i n f i v e volumes o f 0.5 mM N a C l a t pH 6.5. C u r v e b ( F i g . 4) i l l u s t -2+ r a t e s t h e Ca a c t i v a t i o n c u r v e s f o r g h o s t s membranes p r e p a r e d by t h e method o f W olf (1972a) a t an i o n i c s t r e n g t h o f 12 mosm and pH 7.7. B o t h c u r v e s were comp a r a b l e i n shape and t h e amount o f (Mg+Ca)-ATPase a c t i v i t y . A p l o t o f t h e d a t a f r o m c u r v e s a and b ( F i g . 4) a c c o r d i n g t o E a d i e (1942) y i e l d e d two d i s t i n c t s l o p e s f o r b o t h p r e p a r a t i o n s ( F i g . 5 ) . The a p p a r e n t K f t f o r t h e h i g h a f f i n i t y p a r t o f b o t h c u r v e s i s 1.92 ^ M whereas K B v a l u e s o f 32 Figure 3. Plots of the data in Fig. 2 according to Eadie (1942), in the absence {•) and presence (O) of 1 mM EDTA. 33 6r p C a 2 + 2+ Figure 4. The activation of (Mg+Ca)-ATPase by Ca in membrane fragments prepared under low ionic conditions ( 12 mosm). Curve a (• ) was obtained in fragments prepared in 0.5 mM NaCl, pH 6.5, (preparation A) and curve b (O) was obtained from fragments prepared according to Wolf (1972). Data in curve a represent the means + standard errors of four to six separate experiments. Data in curve b represent the means of two separate experiments. 34 6h Figure 5. Plots of the data from Fig. 4 according to Eadie (1942), 35 2+ a p p r o x i m a t e l y 100.0 |*M were o b t a i n e d f o r t h e l o w a f f i n i t y C a b i n d i n g s i t e s . A V m a x o f 5.5 jamoles P i / h r / m l o f g h o s t s was o b t a i n e d f o r t o t a l (Mg+Ca)-ATPase a c t i v i t y f r o m t h e s e p l o t s , w h i c h c o r r e s p o n d s t o a p p r o x i m a t e l y 1.5 t o 1.8 j i m o l e s P i / h r / m g o f p r o t e i n . 2+ I n a l l membrane p r e p a r a t i o n s s t u d i e d , t h e r e i s a b i p h a s i c Ca a c t i v -a t i o n dependence f o r (Mg+Ca)-ATPase a c t i v i t y , c o n s i s t e n t w i t h p r e v i o u s s u g -2+ g e s t i o n s o f t h e p r e s e n c e o f h i g h and l o w Ca a f f i n i t i e s (Wins 1969; H o r t o n e t a l . 1970; Schatzmann and R o s s i 1971; Wolf 1 9 7 2 ) . T h i s b e h a v i o u r was f o u n d i n f r e e z e d - t h a w e d g h o s t s p r e p a r e d i n t h e p r e s e n c e and absence o f EDTA d u r i n g t h e h e m o l y s i s p r o c e d u r e ( F i g 2 ) , and i n membranes p r e p a r e d a c c o r d i n g t o Wol f (1972a) a t pH 7.7 a t an i n t e r m e d i a t e i o n i c s t r e n g t h o f 12 mosm ( F i g . 4, c u r v e b) o r a t l o w i o n i c s t r e n g t h ( p r e p a r a t i o n A) ( F i g . 4, c u r v e a ) . The 2+ a p p a r e n t d i s s o c i a t i o n c o n s t a n t s (K^) f o r Ca d e t e r m i n e d i n membranes p r e p a r e d by t h e f o u r methods was s i m i l a r and were i n agreement w i t h t h o s e p r e v i o u s l y p u b l i s h e d (Schatzmann and R o s s i 1971; W o l f 1 9 7 2 a ) . T h i s s u g g e s t s t h a t t h e p r o c e d u r e s u s e d t o expose l a t e n t ATPase a c t i v i t y d i d n o t a l t e r t h e a f f i n i t y 2+ o f C a " f o r b i n d i n g s i t e s on (Mg+Ca)-ATPase(s) i n t h e membranes. However, t h e maximum ATPase a c t i v i t y o f t h e p r e p a r a t i o n s d i f f e r e d i n a manner p r e s u m a b l y r e f l e c t i n g t h e amount o f enzyme e x p o s e d , as f o r m e r l y s u g g e s t e d by Br a m l e y e t a l . (1971; 1 9 7 2 ) . I n c o n t r a s t t o a p r e v i o u s r e p o r t by Bramley e t a l . ( 1 9 7 1 ) , membranes p r e p a r e d by w a s h i n g g h o s t s i n 0.4 t o 12 mosm s o l u t i o n s r e t a i n a h i g h l e v e l o f (Mg+Ca)-ATPase a c t i v i t y . T h i s d i f f e r e n c e may be e x p l a i n e d by t h e f i n d -i n g t h a t (Mg+Ca)-ATPase a c t i v i t y i n membranes p r e p a r e d a t l o w i o n i c s t r e n g t h must be a s s a y e d t h e same day o f p r e p a r a t i o n and c a n n o t be s t o r e d a t -20°C w i t h o u t a l o s s o f (Mg+Ca)-ATPase a c t i v i t y . 2+ The f i n d i n g o f two Ca a c t i v a t e d (Mg+Ca)-ATPase a c t i v i t i e s c o n f l i c t s 2+ w i t h a r e c e n t r e p o r t by Schatzmann (1973) s t a t i n g t h a t Ca a c t i v a t i o n o f 36 (Mg+Ca)-ATPase a c t i v i t y i n g h o s t s p r e p a r e d by t h e methods o f Wolf (1972) 2+ gave a l i n e a r d o u b l e r e c i p r i c a l p l o t w i t h a s i n g l e K A f o r Ca o f 2.35 uM, c o r r e s p o n d i n g t o t h e h i g h a f f i n i t y (Mg+Ca)-ATPase component. I n o r d e r t o r e s o l v e t h e c o n f l i c t between t h i s r e s u l t and a p r e v i o u s r e p o r t by Schatzmann and R o s s i (1971) i n d i c a t i n g t h e p r e s e n c e o f h i g h and l o w (Mg+Ca)-ATPase a c t i v -i t i e s , Schatzmann (1973) s u g g e s t e d t h a t t h e l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v -i t y was an a r t i f a c t r e s u l t i n g f r o m t h e use o f EDTA i n t h e p r e p a r a t i o n o f t h e membranes by t h e p r o c e d u r e o f G a r r a h a n and Rega ( 1 9 6 7 ) . I t i s p o s s i b l e t h a t i n t h e r e c e n t s t u d y o f Schatzmann (1973) s u f f i c i e n t l y h i g h c o n c e n t r a t i o n s o f 2+ C a * T were n o t a c h i e v e d t o r e v e a l t h e low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y o r a l t e r n a t i v e l y , t h a t t h i s a c t i v i t y was l o s t by d e n a t u r a t i o n . I t has been f o u n d i n t h e p r e s e n t s t u d y t h a t r e p e a t e d w a s h i n g s o f g h o s t s i n 40 mosm media c o n s i d -e r a b l y r e d u c e d l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y b u t n o t t h e h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . T h e r e f o r e t h e l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y may be more l a b i l e . H o r t o n e t a l . ( 1 9 7 0 ) , u s i n g membranes p r e p a r e d by t h e method o f P o s t e t a l . (1960) w h i c h does n o t i n c l u d e e i t h e r EGTA o r EDTA i n 2+ t h e p r o c e d u r e , a l s o o b t a i n e d a Ca a c t i v a t i o n c u r v e c l e a r l y s howing t h e p r e s e n c e o f h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s . However, i t a p p e a r s t h a t t r e a t m e n t o f g h o s t s w i t h EGTA ( S c h a r f f 1972) o r EDTA (Schatzmann 1973) f o r p r o l o n g e d p e r i o d s b o t h r e d u c e s t o t a l (Mg+Ca)-ATPase a c t i v i t y 2+ ( S c h a r f f 1972) and i n c r e a s e s t h e K A f o r Ca a c t i v a t i o n o f t h e h i g h a f f i n i t y component some two f o l d (Wolf 1 9 7 2 a ) . The k i n e t i c e v i d e n c e p r e s e n t e d c l e a r l y shows t h e p r e s e n c e o f h i g h and 2+ lo w Ca a f f i n i t y s i t e s f o r (Mg+Ca)-ATPase(s), b u t does n o t i n d i c a t e w h e t h e r 2+ t h e two Ca a f f i n i t y s i t e s a r e a s s o c i a t e d w i t h two d i s t i n c t enzymes o r a r e a s s o c i a t e d w i t h one enzyme w i t h two c a t e g o r i e s o f s i t e s o r w i t h i n t e r a c t i o n s 37 between t h e Ca s i t e s (Schatzmann and R o s s i 1 9 7 1 ) . T h e r e f o r e f u r t h e r s t u d i e s were u n d e r t a k e n t o d i s t i n g u i s h between t h e above p o s s i b i l i t i e s . 2. Removal o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y f r o m membrane f r a g m e n t s . F a i r b a n k s e_t a l . (1971) f o u n d t h a t a number o f e r y t h r o c y t e membrane pep-t i d e s , a r b i t r a r i l y c l a s s i f i e d as Bands 1, 2 and 5 by t h e i r r e l a t i v e m o b i l i t y on SDS a c r y l a m i d e g e l s , c o u l d be removed f r o m g h o s t s under m i l d c o n d i t i o n s . T h i s was a c h i e v e d by s u s p e n d i n g g h o s t s i n 10 volumes o f 0.1 mM EDTA (pH 8 . 0 ) . I t seemed p o s s i b l e t h a t one o r more o f t h e s e e l u t e d p r o t e i n s c o u l d be r e q u i r e d f o r e i t h e r h i g h o r l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i f t h e two a c t i v i t i e s were a s s o c i a t e d w i t h d i s t i n c t enzymes. T h e r e f o r e , g h o s t s were s u b j e c t e d t o s i m i l a r l ow i o n i c c o n d i t i o n s as d e s c r i b e d by F a i r b a n k s et_ a l . ( 1 9 7 1 ) . Membrane f r a g m e n t s were p r e p a r e d by i n c u b a t i n g g h o s t s a t 37°C f o r s p e c i f i e d t i m e p e r -i o d s i n 5 volumes o f 0.1 mM EDTA and 1 mM T r i s - m a l e a t e (pH 8 . 0 ) . A t i m e dep-e n d e n t l o s s o f (Mg+Ca)-ATPase a c t i v i t y o c c u r e d d u r i n g t h e i n c u b a t i o n ( F i g . 6 ) . A f t e r 5 m i n u t e s , t h e r e was a 36% r e d u c t i o n i n maximal (Mg+Ca)-ATPase a c t i v i t y a s compared t o c o n t r o l ( p r e p a r a t i o n A) membranes. I n c u b a t i o n f o r l o n g e r t i m e p e r i o d s r e s u l t e d i n a f u r t h e r l o s s o f (Mg+Ca)-ATPase a c t i v i t y . I t was a p p a r e n t t h a t i ( M g + C a ) - A T P a s e a c t i v i t y a t l o w c o n c e n t r a t i o n s o f C a 2 + , i n t h e h i g h a f f i n i t y 2+ r e g i o n o f t h e Ca a c t i v a t i o n c u r v e , was p r e f e r e n t i a l l y l o s t , whereas t h e a c t i v i t y i n t h e low a f f i n i t y p a r t o f t h e c u r v e was n o t g r e a t l y a f f e c t e d . A f t e r 40 m i n u t e s , r e m o v a l o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y a p p e a r e d c o m p l e t e . I t s h o u l d be n o t e d however t h a t (Mg+Ca)-ATPase a c t i v i t y was s t i l l p r e s e n t a t 2+ _q c o n c e n t r a t i o n s o f Ca l e s s t h a n 10 J M. A f t e r 60 m i n u t e s , t h e (Mg+Ca)-ATPase a c t i v i t y i n t h e l o w a f f i n i t y r e g i o n o f t h e c u r v e a l s o began t o d e c r e a s e ; p o s -s i b l y t h e r e s u l t o f d e n a t u r a t i o n by p r o l o n g e d i n c u b a t i o n . The C a 2 + a c t i v a t i o n c u r v e s f o r (Mg+Ca)-ATPase a c t i v i t y i n a number o f 38 Figure 6. The activation of (Mg+Ca)-ATPase activity by Ca in membrane fragments incubated for 5 ( © ) , 20 ( O ) , 40 (O) and 60 (• ) minutes at 37°C in 0.1 mM EDTA and 1.0 mM Tris-maleate, pH 8.0. A control curve (&) showing C a 2 + activation of (Mg+Ca)-ATPase activity in fragments prepared in the same medium but at pH 6.5 is shown. 39 o t h e r low i o n i c s t r e n g t h media were a l s o s t u d i e d ( F i g . 7 ) . I n t h e s e e x p e r -i m e n t s b u f f e r s were n o t u s e d , b u t t h e pH o f t h e s u s p e n s i o n was 7.2 t o 7.5 due t o b u f f e r i n g by t h e membranes. A t v e r y low c o n c e n t r a t i o n s o f C a 2 + (below l O - ^ M) t h e r e was a v e r y s h a l l o w a c t i v a t i o n o f (Mg+Ca)-ATPase a c t i v i t y by C a 2 + , whereas a c t i v a t i o n by C a 2 + o f (Mg+Ca)-ATPase i n c r e a s e d i n a n o r m a l f a s h i o n above t h i s c o n c e n t r a t i o n . As was n o t e d i n ( F i g . 6) , c l o s e r examin-2+ a t i o n o f t h e Ca a c t i v a t i o n c u r v e s o f t h e s e membranes ( F i g . 7) i n d i c a t e d t h a t t h e h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y was p r e f e r e n t i a l l y l o s t by t h e s e p r o c e d u r e s . T h i s was shown by t h e d e c r e a s e i n t h e r a t i o o f a c t i v i t i e s a t 1 0 ~ 5 M and 1 0 ~ 4 M C a 2 + f r o m 0.5 i n p r e p a r a t i o n A t o 0.2 t o 0.3 i n t h e p r e p a r a t i o n s i n F i g s . 6 and 7. The l o s s o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y f r o m membranes does n o t depend on t h e p r e s e n c e o f EDTA a s i t o c c u r -ed i n a number o f l o w i o n i c m edia n o t c o n t a i n i n g c h e l a t i n g a g e n t s . I t was f o u n d t h a t a p p r o x i m a t e l y 24% o f t h e membrane p r o t e i n s were l o s t f r o m t h e membranes u n d e r t h e s e l o w i o n i c c o n d i t i o n s , i n agreement w i t h p r e -v i o u s r e p o r t s ( M a r c h e s i e t a l . 1968; F a i r b a n k s e t a l ^ . 1971) . F a i r b a n k s e t  a l . (1971) f o u n d t h a t bands 1, 2 and 5 were n o t l o s t f r o m g h o s t s p r e p a r e d a t lo w i o n i c s t r e n g t h c o n d i t i o n s a t a l o w e r pH (pH 5 . 8 ) . S i m i l a r l y , h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y was r e t a i n e d i n membrane f r a g m e n t s p r e p a r e d u n d e r l o w i o n i c c o n d i t i o n s i f t h e pH was 6.5 o r l e s s . T h e r e f o r e , a pH-dependent l o s s o f h i g h a f f i n i t y (Mg+Ca)-ATPase f r o m membranes o c c u r s a t pH 6.5 - 8.0 o r h i g h e r . The l o s s o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y seems t o o c c u r concomm-i t a n t l y w i t h a l o s s o f s o l u b l e membrane p r o t e i n . The t i m e c o u r s e o f p r o t e i n l o s s c o r r e s p o n d s t o t h e l o s s o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y f r o m t h e membranes. F a i r b a n k s e t a l . (1971) f o u n d t h a t p e p t i d e s c h a r a c t e r i z e d as bands 1, 2 and 5 on SDS p o l y a c r y l a m i d e g e l s were c o m p l e t e l y removed i n 15 m i n u t e s . I n t h e p r e s e n t s t u d y , l o s s o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y was a l s o 40 Figure 7. The activation of (Mg+Ca)-ATPase by Ca in membrane fragments prepared in 0.2 mM NaCl (O ) , 0.5 mM NaCl ( © ) , 0.2 mM MgClo (•) and disti l led water (• ). The pH was 7.2 to 7.5. 41 42 r a p i d . 90% o f t h e a c t i v i t y was l o s t i n 20 m i n u t e s and 100% was l o s t w i t h i n 40 m i n u t e s ( F i g . 6 ) . I t was a l s o n o t e d t h a t r e m o v a l o f membrane p r o t e i n s and h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y d i d n o t o c c u r a t pH l e s s t h a n 6.5. T h e r e f o r e , i t i s c o n c e i v a b l e t h a t t h e h i g h a f f i n i t y (Mg+Ca)-ATPase, a s u b u n i t o f t h a t enzyme o r a s e p a r a t e p r o t e i n n e c e s s a r y f o r i t s a c t i v i t y may be e l u t e d f r o m t h e membranes under t h e c o n d i t i o n s u s e d . The e l u t e d p r o t e i n s a p p e a r t o be w e a k l y bound t o t h e membrane by e l e c t r o s t a t i c f o r c e s o r may r e m a i n i n p l a c e b e c a u s e o f e x t e n s i v e s e l f a s s o c i a t i o n w i t h i n t h e membrane as s u g g e s t e d by S t e c k ( 1 9 7 4 ) . (Mg+Ca)-ATPase a c t i v i t y c o u l d n o t be d e t e c t e d i n t h e s o l u b l e p r o t e i n f r a c t i o n ( f r a c t i o n C) by t h e c o l o r i m e t r i c a s s a y u s e d i n t h i s s t u d y . P r e v i o u s l y R o s e n t h a l e t a l . (1970) f o u n d v e r y l ow l e v e l s o f a C a 2 + a c t i v a t e d , 2+ Mg i n d e p e n d e n t ATPase a c t i v i t y i n a s i m i l a r s o l u b l e p r o t e i n f r a c t i o n w h i c h was d i a l y z e d e x t e n s i v e l y . However, t h e s e a u t h o r s u s e d a more s e n s i t i v e i s o -t o p e method f o r m e a s u r i n g ATPase a c t i v i t y . 3. R e c o n s t i t u t i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . A l t h o u g h f r a c t i o n C d i d n o t c o n t a i n m e a s u r a b l e amounts o f (Mg+Ca)-ATPase a c t i v i t y , r e c o m b i n a t i o n o f t h i s f r a c t i o n w i t h r e s i d u a l membranes ( p r e p a r a t i o n B) u n d e r i s o t o n i c c o n d i t i o n s r e s u l t e d i n an i n c r e a s e i n (Mg+Ca)-ATPase a c t i v i t y . The e l u t e d p r o t e i n f r a c t i o n was c o n c e n t r a t e d 6 f o l d by u l t r a f i l t r a t i o n i n o r d e r t o p r o d u c e f r a c t i o n C. R e c o n s t i t u t i o n w i t h i n c r e a s i n g volumes o f f r a c t i o n C 2+ a t a Ca c o n c e n t r a t i o n o f 28.8 ^iM l e d t o an i n c r e a s i n g a c t i v a t i o n o f (Mg+Ca) -ATPase a c t i v i t y ( F i g . 8 ) , w h i c h began t o l e v e l o f f above 0.08 ml o f f r a c t i o n C. 2+ The Ca dependence o f t h e a c t i v a t i o n was s t u d i e d f u r t h e r i n t h e p r e s e n c e o f 0.2 ml o f p r e p a r a t i o n B and 0.2 ml o f f r a c t i o n C. The r e c o n s t i t u t i o n p r o c e d u r e r e s u l t e d i n a p a r t i c u l a r l y marked i n c r e a s e i n (Mg+Ca)-ATPase a c t i v i t y b e l o w 10"^ M C a 2 + i n t h e h i g h a f f i n i t y r e g i o n o f t h e a c t i v a t i o n c u r v e ( F i g . 8 ) . F o r i n s t -— 5 A 2+ a n c e , t h e r e was an i n c r e a s e i n t h e r a t i o o f a c t i v i t i e s a t 10 M and 10"^M Ca 43 01 1 1 1 I i 0 .04 .08 .12 .16 .20 ml of fraction C Figure 8. Effect of addition of soluble fraction C to membranes devoid of high affinity (Mg+Ca)-ATPase activity at a C a 2 + concentration of 29.0 ^M. Each point represents the mean of duplicate determinations. 44 Figure 9. Ca activation of the ATPase in reconstituted membranes prepared by the addition of fraction C to preparation B ( see section 11.5, Materials and Methods). 46 f r o m 0.3 ( F i g . 3) t o 0.5 ( F i g . 8 ) . T h i s r e s u l t s u g g e s t s t h a t b i n d i n g o f a component i n f r a c t i o n C t o s i t e ( s ) on t h e o r i g i n a l membranes u n d e r i s o t o n i c c o n d i t i o n s a r e r e q u i r e d f o r e x p r e s s i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v -i t y . S t u d i e s were t h e n u n d e r t a k e n t o i n v e s t i g a t e t h e n a t u r e o f t h e component i n f r a c t i o n C r e q u i r e d f o r e x p r e s s i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i n t h e membranes. P r e l i m i n a r y s t u d i e s i n d i c a t e d t h a t t h e component was a p r o t e i n s i n c e h e a t i n g f r a c t i o n C a t 70°C f o r 30 m i n u t e s o r i n c u b a t i o n w i t h t r y p s i n (0.5 mg/ml) d e s t r o y e d t h e a b i l i t y o f f r a c t i o n C t o a c t i v a t e (Mg+Ca) -ATPase a c t i v i t y i n p r e p a r a t i o n B. The component i s r e l a t i v e l y h e a t s t a b l e , however, s i n c e h e a t i n g f r a c t i o n C f o r 5 m i n u t e s a t 100°C r e s u l t e d i n o n l y a l o s s o f 20% o f i t s a c t i v a t i n g a b i l i t y when r e c o m b i n e d w i t h p r e p a r a t i o n B. The p r o t e i n may have a m o l e c u l a r w e i g h t o f g r e a t e r t h a n 100,000 s i n c e i t d i d n o t p a s s t h r o u g h a XM 100 D i a f l o R u l t r a f i l t r a t i o n membrane d u r i n g t h e c o n c e n t -r a t i o n p r o c e d u r e . When f r a c t i o n C was a n a l y z e d by SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i t was f o u n d t o c o n t a i n a number o f p r o m i n e n t p r o t e i n b a n d s , i n c l u d i n g bands 1, 2 and 5 d e s c r i b e d by F a i r b a n k s e t a l . (1971) and h e m o g l o b i n ( F i g . 1 0 ) . I f f r a c t i o n C was washed f r e e o f a l l v i s u a l t r a c e s o f h e m o g l o b i n by r e p e a t e d w a s h i n g s and u l t r a f i l t r a t i o n w i t h an XM 100 u l t r a f i l t r a t i o n membrane (see M e t h o d s ) , e s s e n t i a l l y o n l y bands 1 and 2 w h i c h c o r r e s p o n d t o s u b u n i t s o f s p e c -t r i n w i t h m o l e c u l a r w e i g h t s g r e a t e r t h a n 200,000 d a l t o n s were r e t a i n e d . Thus r e p e a t e d u l t r a f i l t r a t i o n o f f r a c t i o n C by t h i s p r o c e d u r e r e s u l t e d i n a p u r i f -i c a t i o n o f s p e c t r i n s i n c e o t h e r p r o t e i n s components i n f r a c t i o n C h a v i n g m o l -e c u l a r w e i g h t s o f l e s s t h a n 100,000 p a s s e d t h r o u g h t h e f i l t e r . I t was f o u n d t h a t c o n c e n t r a t e d f r a c t i o n s o f s p e c t r i n a g g r e g a t e i f s t o r e d o v e r n i g h t a t 4°C and t h e r e f o r e must be p r e p a r e d f r e s h f o r r e c o n s t i t u t i o n w i t h p r e p a r a t i o n B. 7 Figure 1 0 . Release of membrane polypeptides from erythrocyte ghosts. One volume of ghosts was diluted with 5 volumes of O.lmM EDTA and 1 . 0 mM Tris-maleate (pH 8 . 0 ) , incubated for 3 0 minutes at 37 C and centrifuged. The supernatant was concent^ rated 6 fold by ultrafiltration to yield fraction C, and 2 5 J J 1 ( I ) was electrophoresed and stained with coomassie blue. Fraction C was further washed and reconcentrated by ultra-f i l tration and 2 5 j i l ( I I ) was electrophoresed. A standard protein solution containing catalase ( MW 2 1 0 , 0 0 0 ) , Ovalbumin (MW 4 5 , 0 0 0 ) and chymotrypsinogen A (MW 2 5 , 0 0 0 ) were also electro-phoresed in order to estimate molecular weights of the polypeptides ( I I I ) . The bands were labelled 1 to 7 according to Fairbanks et a l . ( 1 9 7 1 ) . 48 F u r t h e r m o r e s p e c t r i n w i l l d e g r a d e t o s m a l l e r p e p t i d e s as a r e s u l t o f t r a c e amounts o f endogenous p r o t e o l y t i c enzymes ( H u l l a 1 9 7 4 ) . An e q u a l volume o f t h e s o l u b l e f r a c t i o n c o n t a i n i n g p r e d o m i n a n t l y s p e c t r i n was f o u n d t o e f f e c t i v e l y a c t i v a t e h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i n p r e p a r a t i o n B. T h e r e f o r e i t i s l i k e l y t h a t s p e c t r i n i s r e s p o n s i b l e f o r c o n f e r r i n g h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y when bound t o some component i n p r e p a r a t i o n B membranes. S p e c t r i n a p p e a r s t o be s p e c i f i c i n a c t i v a t i n g (Mg+Ca)-ATPase a c t i v i t y i n p r e p a r a t i o n B as serum a l b u m i n (1 rag/ml) was i n e f f e c t i v e o v e r a Ca c o n c e n t r a t i o n r a n g e o f 0.3 t o 300 uM. The d i s p o s i t i o n o f t h e m a j o r p o l y p e p t i d e s i n p r e p a r a t i o n A and B were a l s o s t u d i e d by s o l u b i l i z i n g t h e membrane bound p r o t e i n s w i t h SDS and a p p l y i n g t h e s o l u b i l i z e d p o l y p e p t i d e s on SDS a c r y l a m i d e g e l s ( F i g . 1 1 ) . P r e p a r a t i o n A, as e x p e c t e d c o n t a i n e d a f a r g r e a t e r number o f p r o t e i n bands t h a n p r e p a r a t i o n B. Most s i g n i f i c a n t l y , f o r t h e p u r p o s e s o f t h i s s t u d y , p r e p a r a t i o n A c o n t a i n s v e r y p r o m i n e n t bands 1 and 2 o r s p e c t r i n , whereas o n l y t r a c e amounts o f t h e s e bands a r e d e t e c t a b l e i n p r e p a r a t i o n B. However, i n some e l e c t r o p h o r e t i c s t u d i e s o f d i f f e r e n t b l o o d samples s p e c t r i n was n o n - d e t e c t a b l e i n p r e p a r a t i o n B . P r e p a r a t i o n B was f o u n d t o c o n t a i n m a i n l y band 3 p o l y p e p t i d e s . The r e s u l t s shown i n F i g . 11 were i n c l o s e agreement w i t h t h e p r e v i o u s r e p o r t by F a i r b a n k s e t a l . 1971. T h i s r e s u l t was a l s o c o n s i s t e n t w i t h t h e s u g g e s t i o n t h a t r e m o v a l o f s p e c t r i n f r o m p r e p a r a t i o n A membranes c o r r e s p o n d s t o a l o s s o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . I t was t h o u g h t t h a t f r a c t i o n C c o u l d a c t i v a t e (Mg+Ca)-ATPase a c t i v i t y by c o n v e r t i n g l o w a f f i n i t y (Mg+Ca)-ATPase t o a more a c t i v e enzyme w i t h a h i g h e r a f f i n i t y f o r C a 2 + . T h e r e f o r e , f r a c t i o n C was r e c o n s t i t u t e d w i t h II lb la III A Figure 11. Disposition of polypeptides in preparations A and B. 25 ul (la) and 50 Ll (lb) of preparation A and 25 jil (II) of preparation B were electrophoresed and stained with coomassie blue. A standard protein solution containing catalase (MW 210,000), ovalbumin (MW 45,000) and chymo-trypsinogen A (25,000) was also electrophoresed (III), to compare the molecular weights of the bands. Minor bands tn III represent subunits of catalase. The bands we labelled 1 to 7 according to Fairbanks et aTL (1971). 50 membranes d e v o i d o f ATPase a c t i v i t y . . Low a f f i n i t y (Mg+Ca)-ATPase and Mg -ATPase a c t i v i t i e s were d e s t r o y e d i n p r e p a r a t i o n B by p r e h e a t i n g t h e s e mem-b r a n e s f o r 30 m i n u t e s a t 50° and 70°C. R e c o m b i n a t i o n o f t h e s e membranes w i t h t h e s o l u b l e f r a c t i o n C r e s t o r e d h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y , a l t h o u g h o n l y t o 33% o f t h e c o n t r o l a c t i v i t y a t a C a 2 + c o n c e n t r a t i o n o f 3.53 uM. T h i s c o r r e s p o n d e d t o a p p r o x i m a t e l y 46% o f c o n t r o l h i g h a f f i n i t y (Mg+Ca)-ATPase a c t -i v i t y a f t e r c o r r e c t i o n f o r t h e c o n t r i b u t i o n o f l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y a t t h i s C a 2 + c o n c e n t r a t i o n ( F i g . 1 1 ) . The C a 2 + a c t i v a t i o n c u r v e was h y p e r b o l i c and l e v e l e d o f f above 3.5 ^iM C a 2 + . The K A f o r C a 2 + i n t h e s e mem-b r a n e s was a p p r o x i m a t e l y 1.6 j i M , s i m i l a r t o t h e a p p a r e n t K^- f o r h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y f o u n d i n p r e p a r a t i o n A membranes. I f p r e p a r a t i o n B was t r e a t e d w i t h t r y p s i n (2 mg/ml f o r 30 m i n u t e s a t 37°C) ATPase a c t i v i t y was c o m p l e t e l y l o s t and f r a c t i o n C c o u l d n o t r e s t o r e h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y t o t h i s p r e p a r a t i o n . T h i s r e s u l t s u g g e s t s t h a t t h e membrane compon-e n t t o w h i c h s p e c t r i n b i n d s t o c o n f e r h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i s a p r o t e i n . The l a c k o f c o m p l e t e r e c o n s t i t u t i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y when added t o h e a t d e n a t u r e d membranes o f p r e p a r a t i o n B f u r -t h e r s u g g e s t s t h a t r e c o m b i n a t i o n o f s p e c t r i n t o t h e membranes r e q u i r e s some i n t e g r i t y o f membrane p r o t e i n s . Bond and C l o u g h (1973) r e p o r t e d t h a t an a c t i v a t o r p r o t e i n o f (Mg+Ca)-ATPase a c t i v i t y c o u l d be removed f r o m e r y t h r o c y t e g h o s t s on r e p e a t e d w a s h i n g w i t h 1 mM T r i s (pH 8 . 0 ) . The p r o t e i n s s o l u b i l i z e d f r o m g h o s t s u n d e r t h e s e c o n d i t i o n s were d i a l y z e d f o r a t l e a s t one week. A l t h o u g h t h e a c t i v a t o r was e x t r a c t e d a t low i o n i c s t r e n g t h i t was p r o b a b l y n o t s p e c t r i n , s i n c e s p e c t r i n w o u l d be c o m p l e t e l y h y d r o l y z e d i n t o l o w m o l e c u l a r w e i g h t p e p t i d e s by endogen-ous p r o t e o l y t i c enzymes d u r i n g t h e t i m e used f o r d i a l y s i s ( M a r c h e s i e t a l . 51 Figure 12. The activation of (Mg+Ca)-ATPase by Ca in preparation B denatured at 50 C (•) and 70°C ( O) and reconstituted by the addition of fraction C. 52 1970; H u l l a 1 9 7 4 ) . A l s o t h e r e were d i f f e r e n c e s i n t h e h e a t s t a b i l i t y o f t h e a c t i v a t o r r e p o r t e d by Bond and C l o u g h (1973) and t h e s o l u b l e a c t i v a t o r r e -p o r t e d i n t h i s s t u d y . F o r i n s t a n c e f r a c t i o n C c o u l d be h e a t e d f o r 10 m i n u t e s a t 100°C w i t h o n l y a l o s s o f 20% o f i t s a c t i v a t i n g a b i l i t y whereas t h e p r o t e i n a c t i v a t o r was c o m p l e t e l y i n a c t i v a t e d by h e a t i n g a t 54°C f o r 10 m i n u t e s . F u r t h e r m o r e , (Mg+Ca)-ATPase a c t i v i t y was n o t a c t i v a t e d by a l b u m i n i n t h i s s t u d y whereas b o t h a l b u m i n and h e m o g l o b i n a c t i v a t e d (Mg+Ca)-ATPase a c t i v i t y . " 1.2 f o l d i n t h e o t h e r s t u d y . Bond and C l o u g h (1973) c o n c l u d e t h a t t h e i r a c t i v a t o r p r o t e i n a c t i v a t e d a s i n g l e (Mg+Ca)-ATPase a c t i v i t y i n t h e membrane. As t h e s e a u t h o r s s t u d i e d C a 2 + a c t i v a t i o n o f ATPase a c t i v i t y a t 10~^M C a C l 2 t h e s p e c i f i c i t y o f t h e a c t i v a t o r f o r e i t h e r h i g h o r l o w (Mg+Ca) -ATPase a c t i v i t y was n o t shown.. I t i s q u i t e p o s s i b l e t h a t t h e r e i s more t h a n one a c t i v a t o r o f (Mg+Ca)-ATPase a c t i v i t y i n e r y t h r o c y t e membranes and t h e one r e p o r t e d by Bond and C l o u g h (1973) may have a m o l e c u l a r w e i g h t o f l e s s t h a n 100,000 and t h e r e f o r e may have been l o s t d u r i n g t h e u l t r a f i l t r a t i o n p r o c e d u r e u s e d f o r c o n c e n t r a t i n g f r a c t i o n C i n t h i s s t u d y . 4. P h o s p h o l i p i d dependence o f h i g h and low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s . The a c t i v i t i e s o f a number o f membrane enzymes s u c h as Na,K-ATPase f r o m v a r i o u s membrane s o u r c e s ( R o e l o f s e n and Van Deenen 1973; T a n i g u c h i and I i d a 1972; K i m e l b e r g and P a p a h a d j o p o u p o l o s 1974) and (Mg+Ca)-ATPase i n s a r c o p l a s m i c r e t i c u l u m ( I n e s i e t a l . 1973) have been shown t o be p h o s p h o l i p i d d ependent. I n human e r y t h r o c y t e g h o s t s p u r i f i e d p h o s p h o l i p a s e A 2 ( p a n c r e a s ) and p h o s p h o l -i p a s e C ( B a c i l l u s c e r e u s ) c o m p l e t e l y i n a c t i v a t e d Na,K-ATPase a c t i v i t y w i t h an a l m o s t c o m p l e t e breakdown o f p h o s p h a t i d y l c h o l i n e , p h o s p h a t i d y l e t h a n o l a -mine and p h o s p h a t i d y l s e r i n e ( R o e l o f s e n and Van Deenen 1973) . The above a u t h o r s a l s o showed t h a t Na,K-ATPase a c t i v i t y c o u l d be c o m p l e t e l y r e c o n s t i t -u t e d i n t h e s e t r e a t e d g h o s t s by t h e a d d i t i o n o f p h o s p h a t i d y l s e r i n e and l e s s 53 c o m p l e t e l y w i t h a number o f o t h e r p h o s p h o l i p i d s . I n o r d e r t o i n v e s t i g a t e t h e p h o s p h o l i p i d dependence o f h i g h and low (Mg+Ca)-ATPase a c t i v i t i e s , g h o s t s were p r e t r e a t e d w i t h 100 I.U. o f a p u r i f i e d p h o s p h o l i p a s e A 2 ( p a n c r e a s ) and a s s a y e d f o r (Mg+Ca)-ATPase a c t i v i t y . P h o s p h o l i p a s e A 2 h y d r o l y z e s t h e f a t t y -a c i d e s t e r l i n k a g e a t o f p h o s p h o l i p i d s b u t does n o t a t t a c k s p h i n g o m y e l i n (Zwaal e_t a l ^ . 1973) . The p h o s p h o l i p a s e A 2 t r e a t m e n t a p p e a r e d t o p r e f e r e n t i a l -l y i n a c t i v a t e l ow a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i n t h e g h o s t membranes s i n c e (Mg+Ca)-ATPase a c t i v i t y was g r e a t l y r e d u c e d a t C a 2 + c o n c e n t r a t i o n s g r e a t e r t h a n 10~^M whereas a c t i v a t i o n a t joM c o n c e n t r a t i o n s was e s s e n t i a l l y u n a f f e c t e d ( F i g . 1 3 ) . I n a n o t h e r e x p e r i m e n t , p r e p a r a t i o n B was p r e t r e a t e d w i t h 100 and 200 I.U. o f p h o s p h o l i p a s e A^ and f r a c t i o n C was r e c o n s t i t u t e d w i t h t h e s e membranes ( F i g . 1 4 ) . As shown i n t h i s f i g u r e , p h o s p h o l i p a s e A 2 i n a c t i v a t e d (Mg+Ca)-ATPase a c t i v i t y i n t h e membranes b u t d i d n o t r e d u c e t h e a b i l i t y o f f r a c t i o n C t o r e s t o r e (Mg+Ca)-ATPase a c t i v i t y as compared t o t h e u n t r e a t e d membranes. I n b o t h g h o s t and p r e p a r a t i o n B membranes, t r e a t m e n t w i t h 100 I.U. o f p h o s p h o l i p a s e A^ r e d u c e d t o t a l membrane p h o s p h o l i p i d s 73% i n c l o s e agreement w i t h r e s u l t s o b t a i n e d by Zwaal e t a l . ( 1 9 7 3 ) . These s t u d i e s p r o -v i d e e v i d e n c e t h a t l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i s p h o s p h o l i p i d dependent i n c o n t r a s t t o t h e h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . A s t u d y o f t h e t e m p e r a t u r e dependence o f l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i n p r e p a r a t i o n B a l s o s u g g e s t e d a c l o s e dependence o f t h i s enzyme a c t i v i t y on t h e p h o s p h o l i p i d e n v i r o n m e n t i n t h e membrane. A b r e a k i n t h e A r r h e n i u s p l o t was n o t e d a t 16°C ( F i g . 1 5 ) . The e n e r g i e s o f a c t i v a t i o n as c a l c u l a t e d from t h e s l o p e s o f t h i s c u r v e were 14,400 and 27,600 c a l o r i e s / mole above and below t h e t r a n s i t i o n t e m p e r a t u r e , r e s p e c t i v e l y . The e x p l a n -a t i o n f o r b r e a k s i n A r r h e n i u s p l o t s r a n g i n g f r o m 15° t o 20°C have been a t t -r i b u t e d t o changes i n t h e p h y s i c a l s t a t e o f t h e p h o s p h o l i p i d s i n t h e 54 i.5r (J) o en 1.0 -co 0.5 o o E 0 -O-O 1 pCa 5 2+ 4 Figure 13. Effect of phospholipase A2 on (Mg+Ca)-ATPase activity in ghosts. Ca^+ activation curves were obtained for ghosts treated with 100 IU of pancreas phospholipase A2 (O) and non-treated ghosts ( • ) . Each point represents the mean of at least duplicate determinations. 55 Figure 14. Effect of. pretreatment of preparation B v/ith phospho-lipase on reconstitution of (Mg+Ca)-ATPase activity by fraction C. A. Ca^ "1" activation curves were obtained for untreated ed membranes ( © ) and membranes treated with 100 IU (A) and 200 III (B) of phospholipase A 2 . Also shown are Ca^+ activation curves obtained after reconstitution of fraction C with untreated membranes (O) and membranes pretreated with 100 IU (a ) and 200 IU (•) of. phospholipase A~. ? + B. Comparison of extra Ca activated (Mg+Ca)-ATPase activity obtained after reconstitution of soluble fraction C with untreated membranes (O) and membranes pretreated with 100 IU (a ) and 200 IU ( •) of phospholipase A 2 . Each point represents the mean of at least duplicate determinations. Id seioujrf 57 3.0r 2.0 X > o 1.0 32 3.3 34 35 l /T x 104deg"1 36 Figure 15. Temperature dependence of low affinity (Mg+Ca)-ATPase activity in preparation B at a C a 2 + concentration of of 316 uM.. Each point represents the mean of duplicate determinations. 58 membranes r a t h e r t h a n t h e p r o t e i n components (Charnock ejt a l _ . 1971; I n e s i ejt a l _ . 1 9 7 3 ) . F o r i n s t a n c e , t h e t e m p e r a t u r e e f f e c t s o f Na,K-ATPase a c t i v i t y were f o u n d t o depend on t h e l i p i d m o i e t y and t o be c o n s t a n t w i t h a g i v e n l i p i d r e g a r d l e s s o f t h e enzyme s o u r c e (Tanaka and T e r u y a 1973; K i m e l b e r g and P a p a h a d j o p o u l o s 1 9 7 4 ) . T h e r e f o r e t h e s e s t u d i e s i n d i c a t e d t h a t low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y may be s t r o n g l y a f f e c t e d by t h ermo-t r o p i c g e l l i q u i d c r y s t a l l i n e p hase t r a n s i t i o n s o f p h o s p h o l i p i d s a n a l o g o u s t o Na,K-ATPase a c t i v i t y ( T a n a g u c h i and I i d a 1 9 7 2 ) . 5. E f f e c t o f p r e t r e a t m e n t o f i n t a c t e r y t h r o c y t e s and r e s e a l e d g h o s t s w i t h  p r o t e o l y t i c enzymes on (Mg+Ca)-ATPase a c t i v i t y . D i f f e r e n c e s i n t h e p r o p e r t i e s o f h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s n o t e d h e r e s u g g e s t t h a t t h e two a c t i v i t i e s may be a s s o c i a t e d w i t h d i s t i n c t enzymes. I t w o u l d be u s e f u l t o d e t e r m i n e t h e symmetry o f a r r a n g e -ment o f t h e p r o t e i n s i n t h e membrane w h i c h a r e r e q u i r e d f o r h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s . L a b e l l i n g and enzyme d e g r a d a t i o n t e c h -n i q u e s have shown membrane p r o t e i n s t o be a s y m m e t r i c a l l y a r r a n g e d w i t h i n e r y t h r o c y t e membranes ( S t e c k 1 9 7 4 ) . F o r i n s t a n c e m a j o r g l y c o p r o t e i n s and band 3 p r o t e i n s have been shown t o s p a n t h e membrane ( M a r c h e s i e t a l _ . 1973; F a i r b a n k s e t a l . 1971) whereas bands 1, 2, 5 and 6 p r o t e i n s a r e o n l y a c c e s -s i b l e on t h e i n n e r s u r f a c e o f t h e membrane ( S t e c k 1 9 7 4 ) . P r e v i o u s s t u d i e s have shown t h a t t r y p s i n d i g e s t i o n o f i n t a c t human e r y t h r o c y t e s c l e a v e s o n l y t h e m a j o r g l y c o p r o t e i n whereas c h y m o t r y p s i n and p r o n a s e a r e l e s s s e l e c t i v e and a l s o d i g e s t p o l y p e p t i d e component 3 ( T r i p l e t t e t a l _ . 1972; T a s h i m a 1973) . O t h e r p o o r l y r e s o l v e d g l y c o p r o t e i n s have a l s o been r e p o r t e d t o be d i g e s t e d by p r o t e o l y t i c enzymes i n i n t a c t e r y t h r o c y t e s ( S t e c k 1 9 7 4 ) . 59 Assuming t h a t p r o t e i n s c o n f e r r i n g t h e s e ATPase a c t i v i t i e s have d i f f e r e n t s p a t i a l a r r a n g e m e n t s i n t h e membranes i t seemed p o s s i b l e t h a t e i t h e r l ow o r h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y c o u l d be s p e c i f i c a l l y i n h i b i t e d w i t h p r o t e o l y t i c enzymes by c o n t r o l l e d t r e a t m e n t o f t h e o u t s i d e s u r f a c e o f i n t a c t e r y t h r o c y t e s o r r e s e a l e d g h o s t s . However, i n c u b a t i o n o f washed e r y t h r o c y t e s w i t h t r y p s i n r e s u l t e d i n i n h i b i t i o n o f b o t h h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s i n f r o z e n g h o s t s mem-b r a n e s ( F i g . 1 6 ) . The C a 2 + a c t i v a t i o n c u r v e s showed t h a t t h e i n h i b i t i o n was dependent on t h e c o n c e n t r a t i o n o f p r o t e o l y t i c enzyme and t h a t b o t h t h e h i g h and l o w C a 2 + a f f i n i t y p o r t i o n s o f t h e a c t i v a t i o n c u r v e were r e d u c e d . I n h i b -i t i o n o f (Mg+Ca)-ATPase a c t i v i t y by t r y p s i n was f o u n d t o be q u i t e v a r i a b l e and depended on t h e b l o o d sample s t u d i e d . I n h i b i t i o n was u s u a l l y l e s s w i t h f r e s h l y o b t a i n e d b l o o d compared t o b l o o d s a m p l e s s t o r e d f o r 20 d a y s o r more. I n c u b a t i o n o f r e s e a l e d g h o s t s w i t h v a r y i n g c o n c e n t r a t i o n s o f t r y p s i n a l s o r e s u l t e d i n a c o n c e n t r a t i o n d e p endent i n h i b i t i o n o f (Mg+Caj-ATP ase . a c t i v i t y c o m p a r a b l e t o t h e e f f e c t s e e n i n i n t a c t e r y t h r o c y t e s ( F i g . 1 7 ) . A g a i n b o t h h i g h a n d l o w (Mg+Ca)-ATPase a c t i v i t i e s were i n h i b i t e d b y t r y p s i n t r e a t m e n t . As p r o t e o l y t i c enzymes a r e t o o l a r g e t o p e n e t r a t e t h r o u g h t h e membrane and do n o t l y s e t h e c e l l ( S t e c k 1 9 7 4 ) , t h e s e r e s u l t s s u g g e s t t h a t p r o t e i n s a s s o c i a t e d w i t h h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y s p a n t h e membrane, as t h e y a r e s u s c e p t i b l e t o a t t a c k by e x t e r n a l l y a p p l i e d p r o t e o l -y t i c enzymes. I n t h e c a s e o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y t h e d i g -e s t e d p r o t e i n w o u l d n o t be e x p e c t e d t o be s p e c t r i n , b u t some o t h e r p r o t e i n o r p r o t e i n s a s s o c i a t e d w i t h s p e c t r i n i n t h e membrane. However, t h i s a s p e c t was n o t p u r s u e d f u r t h e r h e r e . T r e a t m e n t o f membrane f r a g m e n t s w i t h n e u r a m i n i d a s e d i d n o t a f f e c t e i t h e r 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 , as was a l s o f o u n d f o r 60 a .2+ Effect of pretreatment of intact erythrocytes with trypsin on (Mg+Ca)-ATPase activity of ghosts prepared from the treated cel ls . Washed erythrocytes were incubated for one hour at 37 C in the presence of 0 ( © ) , 1 mg (O) , 2.5 mg( A) and 5 mg {&) of trypsin. The reaction was stopped with trypsin inhibitor and ghosts were prepared from the cells by stepwise hemolysis. The ghosts were freeze-thawed three times and assayed for Ca^ + activated ATPase activity. 61 00 00 o CD 0) O E =1. pCa 2-h Figure 17. Effect of pretreatment of resealed ghosts with trypsin on (Mg+Ca)-ATPase activity. Resealed ghosts were incubated for 30 minutes at 37 C with 0 ( • ) , 1 mg (O) , 3 mg ( * ) , 5 mg (a ) and 7 mg ( •) of trypsin. The reaction was stopped with trypsin inhibitor and the ghosts were washed twice in 15 mM NaCl and 5 mM Tris-maleate (pH 7.1) and frozen at - 20 C. The ghosts were freeze-thawed three times and assayed for Ca^+ activated ATPase activity. 62 Na,K-ATPase a c t i v i t y i n human e r y t h r o c y t e g h o s t s by G o d i n e_t a l . ( 1 9 7 0 ) . As n e u r a m i n i d a s e c l e a v e s o f f a p p r o x i m a t e l y 90% o f s i a l i c a c i d a s s o c i a t e d w i t h g l y c o p r o t e i n s ( E y l a r e_t a l _ . 1 9 6 2 ) , s i a l i c a c i d a s s o c i a t e d w i t h g l y c o p r o -t e i n s may n o t be i m p o r t a n t f o r m a i n t e n a n c e o f ATPase a c t i v i t y . P r o t e o l y t i c enzymes a l s o c l e a v e o v e r 90% o f s i a l i c a c i d r e s i d u e s , and t h e r e f o r e p r o b a b l y do n o t i n h i b i t ATPase a c t i v i t y by t h i s mechanism ( T r i p l e t t and C a r r a w a y 1 9 7 2 ) . 6. E f f e c t o f r u t h e n i u m r e d and L a C l ^ on (Mg+Ca)-ATPase a c t i v i t y i n membrane  f r a g m e n t s . I t was o f i n t e r e s t t o examine t h e e f f e c t o f c e r t a i n compounds as p o t e n t -i a l s e l e c t i v e i n h i b i t o r s o f h i g h and low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s . R u thenium r e d , a h e x a v a l e n t dye u s e d t o s t a i n m u c o p o l y s a c c h a r i d e s , was shown' by Watson e t a l . ( 1 9 7 1 ) t o s e l e c t i v e l y i n h i b i t (Mg+Ca)-ATPase a c t i v i t y w i t h o u t a f f e c t i n g e i t h e r Na,K-ATPase o r Mg-ATPase a c t i v i t i e s . I n t h i s s t u d y r u t h e n -ium r e d was f o u n d t o i n h i b i t b o t h h i g h and l o w (Mg+Ca)-ATPase a c t i v i t i e s w i t h IJ .Q v a l u e s o f 9 0 ^iM and 5 4 ^iM r e s p e c t i v e l y ( F i g . 1 8 ) . I n r e s e a l e d g h o s t s , r u t h e n i u m r e d a p p l i e d t o t h e e x t e r n a l s u r f a c e i n h i b i t e d b o t h Ca e f f l u x and (Mg+Ca)-ATPase a c t i v i t y t o a maximum o f 60% ( Q u i s t 1 9 7 3 ) , b u t o n l y i n g h o s t s l o a d e d w i t h g r e a t e r t h a n 1 . 0 mM C a C l 2 . I n c o n t r a s t i n h i b i t i o n by r u t h e n i u m 2+ r e d was e v i d e n t a t a l l c o n c e n t r a t i o n s o f Ca s t u d i e d i n membrane f r a g m e n t s . L a C l ^ , when v a r i e d a t 1 0 ^iM C a C l 2 [ t h e r e g i o n o f o p t i m a l h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y ] and a t 4 7 0 jaM [ t h e r e g i o n o f low a f f i n i t y (Mg+Ca) -ATPase a c t i v i t y ) i n h i b i t e d b o t h h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v -i t i e s , w i t h I ^ 0 v a l u e s o f 2 5 ^iM and 1 8 j i M , r e s p e c t i v e l y ( F i g . 1 9 ) . Thus L a C l ^ , l i k e r u t h e n i u m r e d , i s n o t a u s e f u l a g e n t f o r d i f f e r e n t i a t i n g b etween ?+ t h e two Ca a c t i v a t e d ATPase a c t i v i t i e s i n membrane f r a g m e n t s . A more s e l -e c t i v e i n h i b i t i o n o f L a C l ^ added a s y m m e t r i c a l l y t o t h e e x t e r n a l s u r f a c e o f r e s e a l e d g h o s t s w i l l be d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n . 63 100 > • mm o < 80 o a. < 60 o tion 40 • mm n m mm 20 C Ruthenium Red (10 5 M ) Figure 18. Inhibition of (Mg+Ca)-ATPase activity by ruthenium red in membranes prepared according to Wolf (1972a). Curves show percept inhibition of (Mg+Ca)-ATPase activities at 3 jiM Ca^+ ( • ) and 200 JJM C a z + ( 0 ) . 64 0 2 4 6 8 10 LaCI (10" 5M) 3 Figure 19. Inhibition of (Mg+Ca)-ATPase activity by LaClo in membrane fragments (preparation A) in the absence of EGTA. Curves show percent inhibition of enzyme activity by LaCI3 at 10 uM CaCl2 ( © ) and 466 uM CaCl 2 (O) . Points represent means from duplicate determinations. 65 S e c t i o n I I . 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 . 1. The s t o i c h i o m e t r y o f Ca t r a n s p o r t i 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  u s i n g l anthanum as a s e l e c t i v e i n h i b i t o r . S i n c e t h e d i s c o v e r y o f ATP dependent 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 g h o s t s by Schatzmann (1966) and i t s p r o p o s e d a s s o c i a t i o n w i t h (Mg+Ca) -ATPase a c t i v i t y (Schatzmann 1966;" Schatzmann and V i n c e n z i 1969; Lee and S h i n 1969; O l s o n and C a z o r t 1 9 6 9 ) , few a t t e m p t s have been made t o d e t e r m i n e t h e s t o i c h i o m e t r y o f t h e c a l c i u m t r a n s p o r t s y s t e m . The l a c k o f a s p e c i f i c i n h i b i t o r o f t r a n s p o r t (Mg+Ca)-ATPase, s u c h as o u a b a i n i n t h e c a s e o f t h e Na,K-ATPase a s s o c i a t e d w i t h t h e Na-pump (Skou 1 9 7 1 ) , made t h e d e t e r m i n a t i o n o f t h e s t o i c h i o m e t r y d i f f i c u l t , s i n c e ATPase a c t i v i t y r e l a t e d t o c a l c i u m t r a n s p o r t c o u l d n o t r e a d i l y be s e p a r a t e d f r o m t o t a l ATPase a c t i v i t y p r e s e n t . Schatzmann and T s c h a b o l d (1971) r e p o r t e d t h a t t h e l a n t h a n i d e s praesodymium ( P r 3 + ) and holmium ( H o 3 + ) c o m p l e t e l y i n h i b i t e d Ca t r a n s p o r t i n r e s e a l e d g h o s t s and (Mg+Ca)-ATPase a c t i v i t y i n f r o z e n g h o s t s membranes a t a c o n c e n t -r a t i o n o f 10 JM. Werner and Lee (1972) f o u n d t h a t l o w c o n c e n t r a t i o n s o f t h e 3+ l a n t h a n u m (La ) a l s o s p e c i f i c a l l y i n h i b i t e d C a - a c t i v a t e d ATPase a c t i v i t y i n membrane f r a g m e n t s w i t h o u t a f f e c t i n g e i t h e r Mg-ATPase o r Na,K-ATPase a c t i v -i t i e s . I t has been shown h e r e t h a t L a i n h i b i t e d b o t h h i g h and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s i n membrane f r a g m e n t s w i t h a s i m i l a r p o t e n c y ( F i g . 1 9 ) . However, i t was o f i n t e r e s t t o d e t e r m i n e t h e e f f e c t o f L a 3 + a p p l i e d on t h e e x t e r n a l s i d e o f r e s e a l e d g h o s t s . V a r i o u s c o n c e n t r a t i o n s o f L a C l - j were added i n t h e e x t e r n a l medium o f g h o s t s r e s e a l e d i n t h e p r e s e n c e o f 3 mM C a C l 2 . T a b l e 1 shows t h e e f f e c t o f v a r y i n g t h e c o n c e n t r a t i o n o f L a C I , i n t h e e x t e r n a l medium on Ca e f f l u x . An 66 T a b l e 1 I n h i b i t i o n o f c a l c i u m e f f l u x by L a 3 * . L a C l was a p p l i e d i n t h e e x t e r n a l medium ( s e e M e t h o d s ) . J L a C a l c i u m E f f l u x % (mM) (umoles/hr/mg p r o t e i n ) I n h i b i t i o n 0 0.474 0 0.01 0.474 0 0.03 0.368 22 0.06 0.236 50 0.10 0 100 0.50 0 100 67 I v a l u e o f 60 uM was o b t a i n e d . F i g . 20A shows t h e t i m e c o u r s e o f c a l c i u m e f f l u x i n t h e p r e s e n c e and a b s e n c e o f 0.1 mM L a C l . j i n r e s e a l e d g h o s t s p r e -p a r e d by s t e p w i s e h e m o l y s i s i n t h e p r e s e n c e o f 1.0 mM EDTA. The same e x p e r -i m e n t i s shown i n F i g . 20B, e x c e p t t h a t the g h o s t s were p r e p a r e d i n t h e a b s -ence o f EDTA (see M a t e r i a l s and M e t h o d s ) . I n t h e a b s e n c e o f L a C l 3 , a f t e r a s h o r t l a g p e r i o d p r o b a b l y due t o warming t h e t u b e f r o m 2 t o 37°C, t h e r e i s a r a p i d l o s s o f c a l c i u m w h i c h l e v e l e d o f f a f t e r a b o u t 14 m i n u t e s . I n t h e a b s e n c e o f ATP i n t h e l o a d i n g medium, t h e r e was no s i g n i f i c a n t l o s s o f c a l c i u m f r o m r e s e a l e d g h o s t s i n t h e p r e s e n c e and absence o f 0.1 mM L a C l ^ . The r a t e s o f Ca e f f l u x i n F i g . 20A and 20B, d e t e r m i n e d f r o m t h e s l o p e s o f t h e c u r v e s between 4 and 10 m i n u t e s , were 0.474 and 0.440 ^imoles Ca/hr/mg p r o t e i n , r e s p e c t i v e l y . I n t h e p r e s e n c e o f 0.1 mM L a C l ^ , c a l c i u m e f f l u x was c o m p l e t e l y a b o l i s h e d i n g h o s t s p r e p a r e d i n b o t h t h e p r e s e n c e and a b s e n c e o f EDTA. The t i m e c o u r s e o f ATP h y d r o l y s i s i n r e s e a l e d g h o s t s p r e p a r e d w i t h EDTA, i n t h e p r e s e n c e and absence o f 0.1 mM L a C l ^ i n t h e e x t e r n a l medium, i s shown i n F i g . 21A. F i g . 21B shows a s i m i l a r e x p e r i m e n t w i t h g h o s t s p r e p a r e d i n t h e a b s e n c e o f EDTA. A f t e r a b r i e f l a g t i m e , t h e r a t e o f ATPase a c t i v i t y i n c r e a s e d e x p o n e n t i a l l y and began t o l e v e l o f f a f t e r a p p r o x i m a t e l y 14 m i n u t e s , s i m i l a r t o t h e Ca e f f l u x c u r v e s . The r a t e s o f ATP h y d r o l y s i s were a l s o e s t i m a t e d by t h e d e t e r m i n a t i o n o f t h e s l o p e s o f t h e c u r v e s between 4 and 10 m i n u t e s . I n t h e a b s e n c e o f L a C l ^ t h e r a t e s o f ATP h y d r o l y s i s i n F i g . 21A and 21B were 0.520 and 0.480 j i m o l e s P i / h r / m g o f p r o t e i n , r e s p e c t i v e l y . The a d d i t i o n o f 0.1 mM L a C l 3 i n t h e e x t e r n a l medium r e d u c e d t o t a l ATPase a c t i v i t y t o 0.285 and 0.250 ^lmoles P i / h r / m g o f p r o t e i n ^ r e s p e c t i v e l y . I n t e r e s t i n g l y , t h e a d d i t i o n o f 0.1 mM L a C l ^ t o t h e e x t e r n a l medium c o m p l e t -e l y a b o l i s h e d ATP d e p e n d e n t c a l c i u m e f f l u x ( F i g s . 20A and 2 0 B ) , whereas t o t a l 68 Time(min) Figure 20. Effect of La J on calcium efflux from resealed ghosts. A, 1 mM EDTA was used during the stepwise hemolysis procedure. B, in the absence of EDTA. Ghosts were loaded in 3 mM CaCl 2 medium as described in section 1.4, Materials and Methods. LaCl3 was applied in the external medium during incubation. The data represent the means + standard errors of three separate experiments. (©—©), control; (O—O), in the presence of 0.1 mM LaCl3; (B - f l ) , in the absence of ATP in the loading medium. 69 01 1 1 1 I I I i • 0 2 4 6 8 10 12 14 16 Time (min) 01 i i i i i i i l 0 2 4 6 8 10 12 14 16 Time (min) Figure 21. Effect of L a 3 + on ATPase activity in resealed ghosts. A, 1 mM EDTA was used during the stepwise hemolysis procedure. B, in the absence of EDTA. Ghosts were loaded with 3 mM CaCl 2 as described in section 1.4, Materials and Methods. LaCI3 was applied to the external medium during incubation. The data are means + standard errors of three separate experiments. ( © - © ) , control; (O—o), in the presence of 0.1 mM LaCl 3 . 70 ATPase a c t i v i t y was i n h i b i t e d by o n l y 50% ( F i g s . 21A and 21D). T h i s i n d i c a t e s t h a t a s i g n i f i c a n t p r o p o r t i o n o f t h e ATPase a c t i v i t y may n o t be a s s o c i a t e d w i t h Ca t r a n s p o r t . Of t h e r e m a i n i n g ATPase a c t i v i t y 50% was e s t i m a t e d t o be Mg-ATPase a c t i v i t y , w h i c h i s n o t s e n s i t i v e t o l a n t h a n u m a t t h e c o n c e n t r a t i o n s u s e d i n t h i s s t u d y (see W einer and Lee 1 9 7 2 ) . The r e m a i n -i n g a c t i v i t y (25% o f t o t a l ATPase) may be a s s o c i a t e d w i t h a (Mg+Ca)-ATPase n o t a s s o c i a t e d w i t h Ca t r a n s p o r t . (Ouabain s e n s i t i v e Na,K-ATPase was i n a c t -i v e i n g h o s t s r e s e a l e d w i t h N a C l i n t h e absence o f added KC1 ( O u i s t 1973) and t h e r e f o r e u n d e r t h e s e c o n d i t i o n s does n o t c o n t r i b u t e t o t o t a l ATPase a c t i v i t y ) . A s s uming t h a t L a C l ^ i n h i b i t e d o n l y t h e ATPase a c t i v i t y a s s o c i a t e d w i t h Ca t r a n s p o r t , t h e s t o i c h i o m e t r y o f t h e Ca t r a n s p o r t s y s t e m may be e s t i m -a t e d . S u b t r a c t i o n o f t h e ATP h y d r o l y s i s i n t h e p r e s e n c e o f 0.1 mM L a C ^ f r o m t o t a l ATPase a c t i v i t y gave r a t e s o f 0.235 and 0.230 umoles P i / h r / m g o f p r o t e i n i n t h e a b s e n c e o r p r e s e n c e o f 0.1 mM EDTA d u r i n g h e m o l y s i s , r e s p e c t -i v e l y . D i v i d i n g t h e s e r a t e s i n t o t h e r a t e s o f Ca t r a n s p o r t gave s t o i c h i o -m e t r i c v a l u e s ( moles o f c a l c i u m i o n s t r a n s p o r t e d p e r mole o f ATP h y d r o l y -zed) o f 2.02 and 1.91. These e s t i m a t e s w o u l d be l o w i f ATPase a c t i v i t y o t h e r t h a n t h a t a s s o c i a t e d w i t h Ca t r a n s p o r t was i n h i b i t e d by l a n t h a n u m . A l t e r n a t i v e l y , i f l a n t h a n u m p a r t i a l l y u n c o u p l e d c a l c i u m t r a n s p o r t f r o m ATP ase a c t i v i t y , a h i g h e r v a l u e f o r t h e s t o i c h i o m e t r y w o u l d be c a l c u l a t e d . I t i s u n l i k e l y t h a t l a n t h a n u m p e n e t r a t e d t h e membrane i n t h e s h o r t t i m e i n w h i c h t h e e x p e r i m e n t was c a r r i e d o u t and t h e r e s u l t s u g g e s t s t h a t l a n t h -anum i n h i b i t e d b o t h (Mg+Ca)-ATPase and c a l c i u m t r a n s p o r t by b i n d i n g t o e x t e r n a l s i t e s o f t h e membrane. R e c e n t l y Schatzmann (1973) c o n c l u d e d t h a t t h e s t o i c h i o m e t r y o f t h e c a l c i u m pump i n r e s e a l e d e r y t h r o c y t e s was c l o s e r t o one t h a n two. The 71 r e a s o n s f o r t h e d i f f e r e n c e s between t h e two e s t i m a t e s a r e n o t e n t i r e l y c l e a r . However, o u r e s t i m a t e r e s u l t s f r o m t h e use o f lan t h a n u m as a t o o l f o r e s t i m a t i n g o n l y t h e ATPase a c t i v i t y 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 . On t h e o t h e r hand, Schatzmann c o r r e c t e d f o r Mg-ATPase a c t i v i t y o n l y and a s -sumed t h a t o n l y one (Mg+Ca)-ATPase a c t i v i t y was p r e s e n t i n t h e p l a s m a membrane o f e r y t h r o c y t e s . D i f f e r e n c e s i n t h e d e g r e e o f f u n c t i o n a l r e c o n s t i t u t i o n o f l e a k y c e l l s may a l s o c o n t r i b u t e t o t h e d i s c r e p a n c y . Schatzmann a l l o w e d g h o s t s t o r e s e a l a t "room t e m p e r a t u r e " . Our s t u d i e s show t h a t c o m p l e t e n e s s o f r e s e a l i n g g h o s t s t o c a l c i u m was v e r y d e p endent on b o t h t i m e and t e m p e r -a t u r e ( Q u i s t and R o u f o g a l i s 1 9 7 5 ) , and r e s e a l i n g was c a r r i e d o u t a t 25°C f o r 10 m i n u t e s . R e c o n s t i t u t i o n o f g h o s t s a f t e r one s t e p h e m o l y s i s u s e d by Schatzmann e_t a l . (Schatzmann and V i n c e n z i 1969; Schatzmann 1973) may n o t be as c o m p l e t e as t h e s t e p w i s e h e m o l y s i s p r o c e d u r e u s e d i n t h i s s t u d y . I n t h i s s t u d y c o m p a r a b l e r a t e s o f ATPase a c t i v i t y and c a l c i u m e f f l u x were o b t a i n e d i n g h o s t s p r e p a r e d w i t h o r w i t h o u t EDTA. The s t o i c h i o m e t r i e s were a l s o c o m p a r a b l e . P r e v i o u s t o t h i s s t u d y S c h a r f f (1972) and Schatzmann (1973) c o n c l u d e d t h a t l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y may be an a r t i f -a c t r e s u l t i n g f r o m t h e d e n a t u r a t i o n by EDTA o f h i g h a f f i n i t y (Mg+Ca)-ATPase t o an enzyme w i t h l o w e r a f f i n i t y f o r c a l c i u m . T h i s i n t e r p r e t a t i o n i s n o t c o n s i s t e n t w i t h t h e p r e s e n t r e s u l t s , w h i c h show t h a t t h e use o f EDTA unde r t h e s e c o n d i t i o n s has no e f f e c t on e i t h e r c a l c i u m t r a n s p o r t o r ATPase a c t i v i t y . * The s t o i c h i o m e t r y o b t a i n e d h e r e i n human e r y t h r o c y t e g h o s t s c o r r e s p o n d s 1 The i n i t i a l t o t a l c a l c i u m c o n t e n t o f g h o s t s p r e p a r e d i n t h e p r e s e n c e o f EDTA was somewhat h i g h e r t h a n f o r g h o s t s p r e p a r e d i n the abs e n c e o f EDTA (compare v a l u e s f o r c a l c i u m c o n t e n t a t z e r o t i m e i n F i g s . 20A and 2 0 B ) . Thus t r e a t m e n t w i t h EDTA may r e s u l t i n e x p o s u r e o f a g r e a t e r number o f mem-br a n e a n i o n i c s i t e s t o w h i c h c a l c i u m c a n b i n d . 72 t o t h a t f o u n d f o r t h e c a l c i u m pump i n s a r c o p l a s m i c r e t i c u l u m ( M a r t o n o s i 1 9 7 2 ) , and c u l t u r e d L c e l l s (Lamb and L i n d s a y 1 9 7 1 ) . 2. A s s o c i a t i o n between c a l c i u m t r a n s p o r t and (Mg+Ca)-ATPase a c t i v i t y i n  r e s e a l e d v e s i c l e s . S t u d i e s w i t h r e s e a l e d v e s i c l e s p r e p a r e d f r o m g h o s t s under low i o n i c c o n d i t i o n s a l l o w e d a more q u a n t i t a t i v e e s t i m a t i o n o f t h e r e l a t i o n s h i p b e t ^ ween c a l c i u m t r a n s p o r t and (Mg+Ca)-ATPase a c t i v i t y . I n s i d e - o u t v e s i c l e s c a n be p r o d u c e d f r o m g h o s t s under l o w i o n i c and s l i g h t l y a l k a l i n e c o n d i t -i o n s by an i n w a r d b u d d i n g o r e n d o c y t i c p r o c e s s o f t h e g h o s t membrane as f i r s t d i s c o v e r e d by S t e c k e t a l . ( 1 9 7 0 ) . R e p o r t e d l y , i n s i d e - o u t v e s i c l e s c a n be s e p a r a t e d f r o m r i g h t s i d e - o u t v e s i c l e s on a d e x t r a n g r a d i e n t . Ca t r a n s p o r t s t u d i e s w i t h i n s i d e - o u t v e s i c l e s a r e advantageous o v e r r e s e a l e d g h o s t s i n t h a t t h e c o n c e n t r a t i o n o f C a 2 + can be a c c u r a t e l y r e g u l a t e d by i n c l u d i n g EGTA i n t h e e x t e r n a l medium. P r e v i o u s l y W einer and Lee (1972) f o u n d ATP dependent u p t a k e o f ^ C a i n t o i n s i d e - o u t v e s i c l e s , b u t t h e dependence o f ?+ t h e r a t e o f u p t a k e on Ca was n o t s t u d i e d . F u r t h e r m o r e , i t was n o t c l e a r l y d e m o n s t r a t e d t h a t t h e v e s i c l e s were r e s e a l e d e f f i c i e n t l y . The v e s i c l e s u s e d i n t h i s s t u d y 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 method o f S t e c k e t a l . ( 1 9 7 0 ) . F u r t h e r m o r e p r e c a u t i o n s were t a k e n t o e n s u r e t h a t t h e v e s -i c l e s were r e s e a l e d by u s i n g t h e method d e v e l o p e d f o r r e s e a l i n g g h o s t s . The s e p a r a t i o n o f i n s i d e - o u t v e s i c l e s f r o m r e s e a l e d r i g h t s i d e - o u t v e s i c l e s was a t t e m p t e d i n o n l y a few e x p e r i m e n t s . As t h e s e v e s i c l e s a r e i n i t i a l l y p r e p a r e d by t h e same method used t o p r e p a r e p r e p a r a t i o n B membranes, t h e y a r e d e v o i d o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . T h i s a c t i v i t y c a n be r e s t o r e d , however, by r e c o n s t i t u t i n g t h e r e s e a l e d v e s i c l e s w i t h f r a c t i o n C o r s p e c t r i n , and t h u s a l l o w d e t e r m i n a t i o n o f w h i c h o f t h e ATPases i s 73 a s s o c i a t e d w i t h a c t i v e Ca t r a n s p o r t . P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e t h a t t h e v e s i c l e s r e s e a l e d by t h e p r o c e d u r e s u s e d i n t h i s s t u d y a r e r e l -2+ a t i v e l y impermeable t o Ca and ATP. V e s i c l e s were u s u a l l y p r e p a r e d by i n -c u b a t i n g f r e s h l y p r e p a r e d g h o s t s i n 5 volumes o f 0.5 mM N a C l f o r 30 m i n u t e s a t 37°C. The v e s i c l e s were t h e n i m m e d i a t e l y r e s e a l e d i n 4 mM M g C l 2 and 10 mM T r i s - m a l e a t e (pH 7.1) w i t h v a r y i n g c o n c e n t r a t i o n s o f C a C l 2 , u s u a l l y 0.5 mM. A f t e r r e s t o r a t i o n o f i s o t o n i c i t y t h e v e s i c l e s 2+ were i n c u b a t e d f o r 10 m i n u t e s a t ' 37°C and washed t w i c e t o remove e x c e s s Ca The v e s i c l e s were i n c u b a t e d f o r 10 m i n u t e s i n 106 jM C a 2 + and t h e Ca u p t a k e r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f ATP. C a l c i u m u p t a k e was l i n e a r up t o 12 m i n u t e s ( F i g . 2 2 ) . I t was n o t e d t h a t t h e s l o p e s o f t h e c a l c i u m u p t a k e c u r v e s were e s s e n t i a l l y e q u i v a l e n t i n v e s i c l e s r e s e a l e d i n t h e p r e s e n c e o f d i f f e r e n t c o n c e n t r a t i o n s o f C a C l 2 , s u g g e s t i n g t h a t Ca u p t a k e i s i n d e p e n d e n t o f i n t e r n a l Ca c o n c e n t r a t i o n and t h a t t h e e x c e s s C a C l 2 u s e d i n t h e r e s e a l i n g p r o c e d u r e was s u c c e s s f u l l y removed by w a s h i n g . As e x p e c t e d , t h e i n i t i a l c a l c i u m c o n t e n t o f t h e v e s i c l e s i n c r e a s e d as v e s i c l e s were r e s -e a l 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 o f C a C l 2 . A t i m e dependent l o s s o f c a l c i u m d i d n o t o c c u r i n t h e absence o f ATP and i n t h e p r e s e n c e o f 0.1 mM EGTA i n t h e e x t e r n a l medium i n d i c a t i n g t h a t t h e r e s e a l e d v e s i c l e s a r e imp-ermeable t o c a l c i u m and need n o t be c o r r e c t e d f o r c a l c i u m l e a k a g e . The ATP d e p e ndent c a l c i u m u p t a k e p r o b a b l y r e p r e s e n t s c a l c i u m t r a n s p o r t e d i n t o t h e v e s i c l e s , as w a s h i n g t h e v e s i c l e s w i t h 6 mM L a C l 3 d i d n o t d e c r e a s e t h e amount o f Ca t a k e n up. L a C l 3 r e a d i l y d i s p l a c e s c a l c i u m bound on t h e e x t e r n a l s u r -f a c e o f membranes (Van Breeman 1 9 6 9 ) . I t has a l s o been p r e v i o u s l y r e p o r t e d t h a t v e s i c l e s r e s e a l e d u n d e r l e s s i d e a l c o n d i t i o n s t h a n u s e d i n t h i s s t u d y (Bodemann and Passow 1972) a r e r e l a t i v e l y impermeable t o Na,K and a c e t y l t h -i o c h o l i n e (Kant and S t e c k 1 9 7 2 ) . 74 I ! L 0 4 8 12 Time (min) Figure 22. Effect of resealing vesicles in the presence of various concentrations of CaCl 2 on the velocity of Ca uptake. Vesicles were resealed in the presence of 0.1 mM CaCl ? ( © ) , 0.5 mM CaCl 2 ( O ) , 1.0 mM CaCl 2 ( A ) , 3.0 mM CaCl 2 ( a)and 5.0 mM CaCl 2 ( • )• After the resealing procedure, the vesicles were washed with isotonic medium containing EDTA to remove extravesicular calcium. Calcium uptake was then measured at a EGTA regulated C a 2 + concentration of 106^iM. 75 ATP dependent c a l c i u m e f f l u x , a n a l o g o u s t o c a l c i u m e f f l u x i n r e s e a l -ed g h o s t s , c o u l d a l s o be measured i n r i g h t - s i d e - o u t v e s i c l e s p r e p a r e d i n 1 yM C a C l 2 , 0.2 mM M g C l 2 and 1.0 mM T r i s - m a l e a t e (pH 8.0) and r e s e a l e d i n C a C l 2 and ATP ( F i g . 2 3 ) . I f t h e v e s i c l e s were r e s e a l e d i n t h e absence o f ATP, t h e s u b s e q u e n t a d d i t o n o f e x t r a v e s i c u l a r c a l c i u m d i d n o t s u p p o r t Ca e f f l u x , i n d i c a t i n g t h a t t h e v e s i c l e s were impermeable t o ATP i n t h e t i m e p e r i o d s t u d i e d . I n agreement w i t h t h e f i n d i n g o f S t e c k e_t a l _ . (1970) t h e p r e s e n c e o f 0.1 mM C a C l 2 d i d n o t a l l o w v e s i c l e f o r m a t i o n . The r e s e a l e d v e s i c l e s p r e p a r e d w i t h 0.5 mM N a C l were s p h e r i c a l and l e s s t h a n 1 yH i n d i a m e t e r when examined by phase c o n t r a s t m i c r o s c o p y . The absence o f v e s i c l e s o r g h o s t s w i t h d i a m e t e r s g r e a t e r t h a n 1 j i M i n d i c a t e d c o m p l e t e d i s r u p t i o n o f g h o s t s u n d e r t h e l o w i o n i c s t r e n g t h c o n d i t i o n s . The r e l a t i v e p r o p o r t i o n o f i n s i d e - o u t , r i g h t - s i d e - o u t and u n s e a l e d membranes was e s t i m a t e d by d e t e r m i n i n g t h e a c t i v i t y o f enzymes l o c a t e d on t h e i n n e r and o u t e r s u r f a c e s o f t h e e r t h r o c y t e membrane. S i d e d n e s s a s s a y s o f a c e t y l -c h o l i n e s t e r a s e ( o u t s i d e membrane marker) and g l y c e r a l d e h y d e 3-phosphate dehydrogenase ( i n s i d e membrane m a r k e r ) , i n d i c a t e d t h a t t h e p r e p a r a t i o n c o n s i s -t e d o f 44-48% r e s e a l e d i n s i d e - o u t v e s i c l e s , 39% r i g h t - s i d e - o u t v e s i c l e s and 15% u n s e a l e d membranes. T h i s i s c o n c l u d e d f r o m e x p e r i m e n t s showing t h a t 46% o f t h e t o t a l a c e t y l c h o l i n e s t e r a s e and 39% o f t o t a l g l y c e r a l d e h y d e 3-phosphate dehydrogenase a c t i v i t i e s a r e l a t e n t as d e t e r m i n e d by t h e i n c r e a s e i n t h e i r a c t i v i t i e s . I n t h e p r e s e n c e o f 0.1% t r i t o n X-100. The r e m a i n i n g a c t i v i t y , 1 5 % j c o u l d be due t o t h e p r e s e n c e o f u n s e a l e d membranes i n w h i c h t h e s u b s t r a t e s f o r t h e mar k e r enzymes a r e r e a d i l y a c c e s s i b l e t o b o t h s i d e s o f t h e membrane. L a y e r i n g o f r e s e a l e d v e s i c l e s on a d e x t r a n g r a d i e n t r e s u l t e d i n s l i g h t p u r i f -i c a t i o n o f i n s i d e - o u t v e s i c l e s t o 60% o f t h e t o t a l membranes as j u d g e d by 76 F i g u r e 23. ATP dependent c a l c i u m t r a n s p o r t f r o m r e s e a l e d r i g h t -s i d e - o u t v e s i c l e s . V e s i c l e s were r e s e a l e d i n t h e p r e s e n c e (O ) and Absence ) o f ATP. 77 70 t h e amount o f l a t e n t a c e t y l c h o l i n e s t e r a s e a c t i v i t y i n t h e s e v e s i c l e s . T h i s p r e p a r a t i o n was n o t u s e d i n ATPase o r c a l c i u m t r a n s p o r t s t u d i e s as i s o l a t i o n o f t h e v e s i c l e s by t h i s p r o c e d u r e l e d t o a c o m p l e t e l o s s o f ATPase a c t i v i t y under t h e e x p e r i m e n t a l c o n d i t i o n s u s e d . One a i m o f s t u d y i n g c a l c i u m t r a n s p o r t i n v e s i c l e s was t o compare 2+ t h e Ca dependence o f ATP d e p e n d e n t Ca u p t a k e and ATPase a c t i v i t y . T h e r e f o r e , c a l c i u m u p t a k e and ATPase a c t i v i t y were d e t e r m i n e d s i m u l t a n -2+ e o u s l y o v e r a w i d e r a n g e o f Ca i n t h e e x t r a v e s i c u l a r medium. 2+ Ca a c t i v a t i o n o f ATPase a c t i v i t y was l i n e a r f r o m 0 t o a t l e a s t 10 2+ m i n u t e s a t a l l c o n c e n t r a t i o n s o f Ca s t u d i e d ( F i g . 2 4 ) . The s l o p e s o r v e l o c i t i e s o f (Mg+Ca)-ATPase a c t i v i t y i n c r e a s e d as t h e C a 2 + c o n c e n t r a t i o n 2+ was i n c r e a s e d f r o m 0.3 j i M , r e a c h i n g a maximal v e l o c i t y a t 330 ^iM Ca . The v e l o c i t i e s d e t e r m i n e d f r o m t h e s l o p e s were c o r r e c t e d f o r Mg-ATPase a c t i v i t y 2+ and p l o t t e d v e r s u s t h e c o n c e n t r a t i o n o f Ca i n F i g . 26. The t i m e c o u r s e o f ATP d e p e n d e n t Ca u p t a k e a t v a r i o u s c o n c e n t r a t i o n s 2+ o f Ca i s shown i n F i g . 25. Ca u p t a k e i s l i n e a r up t o 10 m i n u t e s a t a l l t h e Ca c o n c e n t r a t i o n s s t u d i e d . A l t h o u g h n o t shown, t h e c a l c i u m u p t a k e c u r v e s l e v e l e d o f f a f t e r 15 m i n u t e s i n t h e p r e s e n c e o f 106 jaM C a 2 + . Ca 2+ u p t a k e d i d n o t o c c u r a t any o f t h e Ca c o n c e n t r a t i o n s s t u d i e d i n t h e a b s e n c e o f added ATP. A d d i t i o n o f 0.5 mM o x a l a t e t o t h e medium d i d n o t i n f l u e n c e t h e r a t e o r t o t a l c a l c i u m u p t a k e i n t h i s p r e p a r a t i o n . The change i n t h e c a l c i u m c o n t e n t was n e g l i g i b l e i n t h e p r e s e n c e o f 0.1 mM EGTA and i n t h e 2+ a b s e n c e o f added Ca f u r t h e r i n d i c a t i n g t h a t t h e w a s h i n g p r o c e d u r e was a d e q u a t e f o r r e m o v i n g e x t r a v e s i c u l a r Ca p r e s e n t a f t e r t h e r e s e a l i n g s t e p . Some v a r i a t i o n i n t h e s p e c i f i c a c t i v i t y o f (Mg+Ca)-ATPase a c t i v i t y and 79 C '5 •*-» o CL U) E CO o £ 8 10 Time (min) Figure 24. Activation of low affinity (Mg+Ca)-ATPase activity in resealed vesicles by C a 2 + . The time course for ATPase activity was determined in the absence of added CaClo ( • ) , 0.297 uM C a 2 + ( • ) , 3.5 LIM C a 2 + (O ) , 28.8 jiM Ca2+ (x ), 86.3 JJM Ca2+ ( a ) , 229 >iM C a 2 + (A) and 330 J J M C a 2 + ( £ ) . The concentration of C a 2 + was regulated in the presence of EGTA as described in the Methods. Each point represents the mean of triplicate determinations. ) 80 Figure 25. Activation of Ca uptake in resealed vesicles by C a c 1 . The time course of ATP dependent Ca uptake was determined in the absence of added CaCl? ( D ) , 0.297 uM Ca2+ ( • ) , 1.63 >iM C a 2 + (O ) , 3.5 uM Ca2+ ( A ) , 28.8>iM Ca2+ ( A ) , 172.39JJM Ca2+ ( ffl) anci 330 JJM Ca2+ ( x ) . The concentration of C a 2 r was regulated in the presence of 0.1 mM EGTA as described in the Methods. Each point represents the mean of at least duplicate determinations. jjrnoles C a / m g protein 82 c a l c i u m u p t a k e was n o t e d i n v e s i c l e s p r e p a r e d from d i f f e r e n t b l o o d s a m p l e s , 2+ a l t h o u g h t h e r e l a t i v e Ca dependence o f t h e a c t i v i t i e s d i d n o t change. The v e l o c i t i e s o f c a l c i u m u p t a k e v e r s u s t h e p C a 2 + i s shown i n F i g . 26. 2+ A c o m p a r i s o n o f t h e Ca dependence c u r v e s f o r low a f f i n i t y (Mg+Ca) -ATPase a c t i v i t y and ATP dependent c a l c i u m u p t a k e i n d i c a t e s t h a t b o t h 2+ s y stems have a s i m i l a r a f f i n i t y f o r Ca ( F i g . 2 6 ) . The c a l c i u m u p t a k e and (Mg+Ca)-ATPase c u r v e s a r e p a r a l l e l o v e r t h e C a 2 + range s t u d i e d and b o t h a r e c o n c a v e up when p l o t t e d i n t h i s manner. The s t o i c h i o m e t r y (jimoles o f c a l c i u m t a k e n up p e r j i m o l e o f ATP h y d r o l y z e d ) i s a p p r o x i m a t e l y 1.5 o v e r 2+ t h e e n t i r e C a " c o n c e n t r a t i o n r a n g e . The d i f f e r e n c e w i t h t h e s t o i c h i o m e t r y o f 2.0 f o u n d i n r e s e a l e d g h o s t s c o u l d be a c c o u n t e d f o r i n t h e f o l l o w i n g way. I n s i d e - o u t v e s i c l e s and u n s e a l e d v e s i c l e s w o u l d c o n t r i b u t e t o (Mg+Ca)-ATP-ase a c t i v i t y whereas o n l y i n s i d e - o u t v e s i c l e s w o u l d be e x p e c t e d t o c o n t r i b u t e t o c a l c i u m u p t a k e b e c a u s e o f t h e l a n t h a n u m u s e d i n t h e w a s h i n g f o r t h e a s s a y o f c a l c i u m u p t a k e . T h e r e f o r e , t o e s t i m a t e (Mg+Ca)-ATPase a c t i v i t y r e l a t e d t o i n s i d e - o u t v e s i c l e s , t h e t o t a l (Mg+Ca)-ATPase a c t i v i t y s h o u l d be m u l t i p l i e d by a f a c t o r o f 46/61 (% i n s i d e - o u t v e s i c l e s / % i n s i d e - o u t v e s i c l e s + % u n s e a l e d membranes) w h i c h w o u l d b r i n g t h e s t o i c h i o m e t r y t o 2+ a l m o s t 2 a t a l l c o n c e n t r a t i o n s o f Ca s t u d i e d . R i g h t - s i d e - o u t v e s i c l e s w o u l d n o t c o n t r i b u t e t o e i t h e r c a l c i u m u p t a k e o r (Mg+Ca)-ATPase. As shown above, i t i s a l s o u n l i k e l y t h a t t h e c a l c i u m u p t a k e measured may be u n d e r e s t -i m a t e d due t o p a s s i v e d i f f u s i o n o f c a l c i u m f r o m l e a k y v e s i c l e s . The s i m i l a r 2+ C a " dependence o f b o t h ATP dependent c a l c i u m u p t a k e and ATPase a c t i v i t y s u g g e s t s a c l o s e a s s o c i a t i o n between c a l c i u m t r a n s p o r t and t h e low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y p r e s e n t i n t h e s e v e s i c l e s . As shown p r e v i o u s l y ( F i g . 9 ) , r e c o n s t i t u t i o n o f f r a c t i o n C w i t h p r e p a r -a t i o n B membranes r e s u l t e d i n an a l m o s t c o m p l e t e r e s t o r a t i o n o f h i g h a f f i n i t y 83 Figure 26. C a 2 + dependence of low affinity (Mg+Ca)-ATPase activity and ATP dependent Ca uptake in resealed vesicles. Each point represents the mean of at least duplicate determinations. (O-O), Ca uptake; (•—*), ATPase activity. 84 (Mg+Ca)-ATPase a c t i v i t y . I t was e s s e n t i a l t o d e t e r m i n e i f r e s t o r a t i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y a f f e c t e d t h e 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 . T h e r e f o r e f r a c t i o n C was r e c o n s t i t u t e d w i t h r e s e a l e d v e s i c l e s u nder i s o t o n i c c o n d i t i o n s and ATP dependent c a l c i u m u p t a k e and ATPase a c t i v i t y were measured s i m u l t a n e o u s l y . R e c o n s t i t u t i o n o f f r a c t i o n C r e s u l t e d i n a 2.5 and 2.1 f o l d a c t i v a t i o n o f (Mg+Ca)-ATPase a c t i v i t y a t 2+ Ca c o n c e n t r a t i o n s o f 28.8 jiM and 229 pM, r e s p e c t i v e l y ( F i g . 2 7 ) . I n c o n t r a s t , t h e v e l o c i t y o f c a l c i u m u p t a k e was n o t s i g n i f i c a n t l y a l t e r e d 2+ i n v e s i c l e s r e c o n s t i t u t e d w i t h f r a c t i o n C a t 28.8 J J M and 229 ^iM Ca ( F i g . 2 8 ) . As r e c o n s t i t u t i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y by t h e a d d i t i o n o f s o l u b l e f r a c t i o n C t o t h e v e s i c l e s enhanced (Mg+Ca)-ATPase a c t i v i t y o v e r two f o l d w i t h o u t a f f e c t i n g t h e v e l o c i t y o f ATP dependent c a l c i u m u p t a k e , h i g h a f f i n i t y (Mg+Ca)-ATPase does n o t appear t o be i n v o l v e d i n a c t i v e c a l c i u m t r a n s p o r t . H i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y must t h e r e f o r e be i n v o l v e d i n some f u n c t i o n o t h e r t h a n c a l c i u m t r a n s p o r t . E v i d e n c e has been g i v e n h e r e t h a t t h e p r o t e i n i n f r a c t i o n C c o n f e r r i n g h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y c o r r e s p o n d e d t o t h e t h e p r o t e i n commonly r e f e r r e d t o as s p e c t r i n ( M a r c h e s i e t a l . 1968) o r t e t k i n A ( C l a r k e 1 9 7 1 ) . N i c o l s o n and P a i n t e r ( 1 9 7 3 ) s u g g e s t e d a s t r u c t u r a l transmembrane l i n k a g e between i n n e r s u r f a c e s p e c t r i n and t h e i n t e g r a l p r o t e i n component g l y c o p h o r i n . I n t h i s s t u d y , i t was found t h a t an i s o l a t e d f r a c t i o n c o n t a i n i n g s p e c t r i n was d e v o i d o f (Mg+Ca)-ATPase a c t i v i t y , b u t a d d i t i o n o f t h i s f r a c t i o n t o membranes d e p l e t e d o f s p e c t r i n r e s u l t e d i n an a l m o s t c o m p l e t e r e s t o r a t i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . I t i s q u i t e p o s s i b l e t h a t s p e c t r i n b i n d s w i t h g l y c o p h o r i n o r some s i m i l a r p r o t e i n t o r e s t o r e h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . I n t h e p r e s e n c e o f ATP, Ca, and Mg, s p e c t r i n may i n f l u e n c e t h e 05 I I : L ; I I „ > „ 0 2 4 6 8 10 Time (min) Figure 27. Time course of (Mg+Ca)-ATPase activity in resealed vesicles in the presence of 28.8 pM C a 2 + ( A ) , a n d 229 >iM Ca2+ ( © ) . Time course of (Mg+Ca)-ATPase activity in resealed vesicles after reconstitution with fraction p in the presence of 28.8>iM C a z + ( A ) and 229^M C a 2 + ( 0 ) . Each point represents the mean of three determinations. 86 Time (min) Figure 2 8 . Time course of Ca uptake in resealed vesicles in the presence of 2 8 . 8 u^M C a 2 + (*) and 2 2 9 C a 2 + ( • ) . Time course of Ca uptake in resealed vesicles after reconstitution with fraction C in the presence of 28.8 jiH Ca2+ ( &) and 2 2 9 J J M C a 2 + ( O ) . Each point represents the mean of triplicate determinations. 87 l a t e r a l movement o f g l y c o p h o r i n and s u b s e q u e n t d e f o r m a b i l i t y o r shape c h a n g e s o f t h e membrane. E v i d e n c e f o r t h i s p r o p o s a l w o u l d h e l p e x p l a i n c o n f l i c t i n g r e p o r t s i n t h e l i t e r a t u r e s u g g e s t i n g t h a t (Mg+Ca)-ATPase a c t i v i t y i s a s s o c i a t e d w i t h b o t h c a l c i u m t r a n s p o r t (Schatzmann and V i n c e n z i 1969) and c o n t r a c t i l i t y (Wins and S c h o f f e n i e l s 1967; P a l e k ejb a_l_. 1 9 71b). E v i d e n c e p r e s e n t e d h e r e s u p p o r t s t h e h y p o t h e s i s t h a t t h e p r o t e i n t o w h i c h s p e c t r i n b i n d s spans t h e membrane and may be one and t h e same as g l y c o p h o r i n . F o r i n s t a n c e , p r e t r e a t m e n t o f i n t a c t e r y t h r o c y t e s and r e s e a l e d g h o s t s w i t h t r y p s i n r e s u l t e d i n a l o s s o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i n g h o s t membranes p r e p a r e d f r o m t h e s e c e l l s ( F i g s . 16 & 1 7 ) . T h i s r e s u l t c o u l d be e x p l a i n e d i f s p e c t r i n w h i c h i s a s s o c i a t e d w i t h i n n e r membrane s u r -f a c e was a s s o c i a t e d w i t h a p r o t e i n s p a n n i n g t h e membrane. " T r i p p l e t t e t a l . (1972) f o u n d t h a t t r e a t m e n t o f i n t a c t e r y t h r o c y t e s w i t h e i t h e r t r y p s i n o r c h y m o t r y p s i n r e s u l t e d i n an a l m o s t c o m p l e t e d e g r a d a t i o n o f g l y c o p h o r i n . O t h e r o b s e r v a t i o n s s u p p o r t t h e e v i d e n c e a s s o c i a t i n g l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y w i t h ATP d e p e n d e n t c a l c i u m t r a n s p o r t . F o r i n s t a n c e , l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i s d e p e n d e n t on p h o s p h o l i p i d s i n c o n t r a s t t o t h e h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y ( F i g s . 13 & 1 4 ) . 45 Lee and S h i n (1972) a l s o f o u n d t h a t (Mg+Ca)-ATPase a c t i v i t y and Ca u p t a k e i n e r y t h r o c y t e membrane f r a g m e n t s c a n be i n h i b i t e d by p r e t r e a t m e n t o f t h e membranes w i t h p h o s p h o l i p a s e A o r C. I n a d d i t i o n t h e r e i s a d i s c o n t -o i n u i t y a t 16 C i n t h e A r r h e n i u s p l o t f o r low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i n p r e p a r a t i o n D ( F i g . 1 5 ) . S i m i l a r d i s c o n t i n u t i e s have been n o t e d f o r o t h e r membrane bound t r a n s p o r t enzymes s u c h as Na ,'K-ATPase (Tanaka and T e r u y a 1974; C h a r n o c k e t a l ^ 1971) o r (Mg+Ca)-ATPase i n t h e s a r c o p l a s m i c r e t i c u l u m ( I n e s i e t a l . 1 9 7 3 ) . The e n e r g y o f a c t i v a t i o n (14,400 c a l / m o l e ) c a l c u l a t e d 88 above t h e t r a n s i t i o n t e m p e r a t u r e f o r low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y ( F i g . 15) c o r r e l a t e d c l o s e l y t o t h e e n e r g y o f a c t i v a t i o n (13,500 c a l / m o l e ) f o r ATP dependent c a l c i u m t r a n s p o r t i n r e s e a l e d g h o s t s (Lee and S h i n 1 9 6 9 ) . 09 S e c t i o n I I I . K i n e t i c C h a r a c t e r i s t i c s o f Low A f f i n i t y (Mg+Ca)-ATPase  A c t i v i t y . 1 I n t h e p r e v i o u s s e c t i o n , i t was shown t h a t l o w a f f i n i t y (Mg+Ca)-ATPase 2+ and c a l c i u m t r a n s p o r t c o u l d be a c t i v a t e d o v e r a wide r a n g e o f Ca c o n c e n t -r a t i o n s (0.3 yM. t o 330 ^iM) . T h i s p r o p e r t y w o u l d be v e r y a d v a n t a g e o u s f o r 2+ t h e s u r v i v a l o f t h e c e l l i f t h 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 Ca f l u c t u a t e d g r e a t l y . T h i s b e h a v i o u r i s u n u s u a l i n t h e s e n s e t h a t most enzymes' a r e a c t i v a t e d o v e r a much n a r r o w e r r a n g e o f a c t i v a t o r c o n c e n t r a t i o n . T h e r e -2+ f o r e t h e k i n e t i c s o f Ca a c t i v a t i o n o f (Mg+Ca)-ATPase a c t i v i t y were f u r t h e r examined i n p r e p a r a t i o n B. 2+ Ca a c t i v a t i o n o f low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y d e t e r m i n e d i n p r e p a r a t i o n B, when p l o t t e d by t h e L i n e w e a v e r - B u r k method (1934) y i e l d e d a smooth c u r v e w h i c h was c o n c a v e down ( F i g . 2 9 ) . The i n s e t o f t h e same 2+ f i g u r e shows t h e M i c h a e l i s - M e n t e n p l o t o v e r t h e same Ca c o n c e n t r a t i o n . A smooth c u r v e d e v i a t i n g f r o m a s i m p l e h y p e r b o l a by f a i l i n g t o r e a c h s a t u r a t i o n was o b t a i n e d . N o n - l i n e a r r e c i p r o c a l p l o t s w h i c h a r e c o n c a v e down have been o b t a i n e d f o r a number o f s o l u b l e r e g u l a t o r y enzymes s u c h as c i t r a t e c l e a v a g e enzyme and g l u t a m a t e d e h y d r o g e n a s e (Plowman and C l e l a n d 1967; D a l z i e l and E n g l e 1 9 6 9 ) . These p l o t s i n d i c a t e n e g a t i v e c o o p e r a t i v i t y . The H i l l p l o t ( H i l l 1 9 1 3 ) , a d i a g n o s t i c t e s t f o r n e g a t i v e c o o p e r a t i v i t y ( K o s h l a n d J r . 1 9 7 0 ) , i s c o n c a v e up w i t h e s t i m a t e d s l o p e s o f 0.4 and 1.1 a t l o w and h i g h c o n c e n t r a t i o n s o f C a 2 + r e s p e c t i v e l y ( F i g . 3 0 ) . T h e r e f o r e , i t i s q u i t e p o s s i b l e t h a t l o w a f f i n i t y (Mg+Ca)-ATPase i s r e g u l a t e d by a n e g -a t i v e c o o p e r a t i v e mechanism. A n o t h e r p o s s i b i l i t y f o r w h i c h no e v i d e n c e has been o b t a i n e d , i s t h e p r e s e n c e - o f two c a l c i u m t r a n s p o r t s y s t e m s w i t h d i f f e r -e n t K's f o r C a 2 + . T h i s w o u l d t h e r e f o r e i m p l y t h e e x i s t e n c e o f t h r e e (Mg+Ca) -ATPase a c t i v i t i e s . The k i n e t i c e v i d e n c e f i t s t h e s u g g e s t i o n o f n e g a t i v e c o o p e r -90 Figure 29. Lineweaver-Burk plot of activation of low affinity (Mg+Ca)-ATPase activity in preparation B ( membranes devoid of high affinity (Mg+Ca)-ATPase activity). Inset shows Ca2+ activation of low affinity (Mg+Ca)-ATPase in the same preparation. Each point represents the mean of three determinations. velocity T6 92 Figure 30. Hill plot of Ca activation of low affinity (Mg+Ca)-ATPase activity in preparation B. 93 94 a t i v i t y more c l o s e l y s i n c e one w o u l d n o t e x p e c t s u c h a smooth c u r v a t u r e i n t h e r e c i p r i c o l p l o t w i t h d i f f e r e n t enzymes u n l e s s t h e y have s i m i l a r K A v a l u e s . The a p p a r e n t K A v a l u e s were e s t i m a t e d t o be 0.5 ^iM and 100 yoM as d e t e r m i n e d f r o m t h e a s y m p t o t e s o f t h e d o u b l e r e c i p r o c a l p l o t . A n e g a t i v e c o o p e r a t i v e mechanism i s c o n s i s t e n t w i t h t h e p h y s i o l o g i c a l r o l e o f t h i s enzyme. R e g u l a t i o n o f c a l c i u m t r a n s p o r t by a n e g a t i v e l y c o o p e r a t i v e mechanism w o u l d be a d v a n t a g e o u s i n e r y t h r o c y t e s as t h e t r a n s p o r t 2+ s y s t e m c o u l d o p e r a t e e f f i c i e n t l y o v e r a w i d e r a n g e o f i n t r a c e l l u l a r Ca c o n c e n t r a t i o n s . N o r m a l l y t h 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 c a l c i u m i s l e s s t h a n 1 0 " ^ M (Schatzmann 1 9 7 3 ) . However, under c o n d i t i o n s o f l o w pH and l o w oxygen t e n s i o n w h i c h o c c u r s p h y s i o l o g i c a l l y i n t h e s p l e e n ( L a C e l l e 1 9 6 9 ) , l a r g e i n c r e a s e s i n t h 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 o f 2+ . . . Ca may o c c u r . N e g a t i v e c o o p e r a t i v i t y p e r m i t s t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t t o be a d j u s t e d t o t h e p h y s i o l o g i c a l c o n c e n t r a t i o n . As t h e d i s s o c -i a t i o n c o n s t a n t i s r a i s e d , t h e maximum v e l o c i t y ( o r c a p a c i t y ) w i l l n o r m a l l y be i n c r e a s e d i n o r d e r t o make f u l l u s e o f t h e c a t a l y t i c p o t e n t i a l . The d a t a ( F i g . 26) i n d i c a t e s t h a t t h e v e l o c i t y b u t n o t t h e s t o i c h i o m e t r y o f c a l c i u m t r a n s p o r t i s i n c r e a s e d a t h i g h e r 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 2 t (£10 yH) The s u g g e s t i o n t h a t l o w a f f i n i t y (Mg+Ca)-ATPase d i s p l a y s n e g a t i v e c o o p e r a t i v i t y w o u l d i m p l y a s u b u n i t s t r u c t u r e f o r t h i s enzyme w i t h a minimum o f two s u b u n i t s . N e g a t i v e c o o p e r a t i v i t y i s i n t e r p r e t e d t o mean 2+ t h a t t h e b i n d i n g o f e a c h m o l e c u l e o f a c t i v a t o r (Ca ) d e c r e a s e s t h e a f f i n i t y 2+ o f v a c a n t s i t e s on n e i g h b o u r i n g s u b u n i t s f o r Ca ( K o s h l a n d J r . 1 9 7 0 ) . A s u b u n i t s t r u c t u r e has been s u g g e s t e d f o r membrane bound N a , K - t r a n s p o r t ATPase i s o l a t e d f r o m a number o f membrane s o u r c e s . ( K y t e 1972; I l o k i n 1974; Nakao e_t a l _ . 1974) and e v i d e n c e has been p r e s e n t e d ( S c h n e i d e r 1974) t h a t t h e a c t i v e sodium t r a n s p o r t s y s t e m i n v o l v e s an a l l o s t e r i c p r o t e i n d i s p l a y i n g p o s i t i v e c o o p e r a t i v i t y . SUMMARY AND CONCLUSIONS K i n e t i c s t u d i e s o f t h e C a 2 + a c t i v a t i o n o f (Mg+Ca)-ATPase a c t i v i t y i n a number o f membrane p r e p a r a t i o n s p r e p a r e d f r o m human e r y t h r o c y t e s showed t h e p r e s e n c e o f h i g h and low Ca a f f i n i t y (Mg+Ca)-ATPase a c t i v -i t i e s . The h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y and t h e l o w a f f i n i t y 2+ (Mg+Ca)-ATPase a c t i v i t i e s h ad a p p a r e n t K A v a l u e s f o r Ca o f 1.0 t o 1.92 |iM and a p p r o x i m a t e l y 100 jiM, r e s p e c t i v e l y . The p r e s e n c e o f l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y was c l e a r l y shown i n membrane p r e p a r a t i o n s p r e p a r e d i n t h e a b s e n c e o f EDTA, i n d i c a t i n g t h a t l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i s n o t an a r t i f a c t r e s u l t i n g f r o m t h e c o n v e r s i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase t o a l o w e r Ca a f f i n i t y f o r m by EDTA o r EGTA u s e d i n p r e p a r a t i o n o f e r y t h r o c y t e g h o s t s , as s u g g e s t e d by Schatzmann ( 1 9 7 3 ) . H i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y was l o s t f r o m g h o s t membranes suspended i n l o w i o n i c m edia a t e l e v a t e d pH. The l o s s o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y c o r r e s p o n d e d t o t h e l o s s o f a number o f s o l u b l e p r o t e i n s ( a p p r o x i m a t e l y 24% o f t o t a l membrane p r o t e i n ) . H i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y o r membrane p r o t e i n s were n o t e l u t e d f r o m t h e mem-b r a n e s i f t h e pH o f t h e l o w i o n i c medium was a d j u s t e d t o pH 6.5 o r l e s s . I f t h e p r o t e i n s w h i c h were e l u t e d f r o m g h o s t membranes were c o n c e n t r a t e d by u l t r a f i l t r a t i o n and r e c o m b i n e d w i t h t h e membrane r e s i d u e , h i g h a f f i n -i t y (Mg+Ca)-ATPase a c t i v i t y was a l m o s t c o m p l e t e l y r e s t o r e d . (Mg+Ca)-ATPase a c t i v i t y c o u l d n o t be d e t e c t e d i n t h e c o n c e n t r a t e d s o l u b l e p r o t e i n f r a c t i o n , i n d i c a t i n g t h a t some component i n t h e s o l u b l e p r o t e i n f r a c t i o n r e c o m b i n e s 97 w i t h a component i n t h e membrane t o r e s t o r e h i g h a f f i n i t y (Mg+Ca)-ATP-ase a c t i v i t y . A n a l y s i s o f t h e s o l u b l e p r o t e i n f r a c t i o n by SDS a c r y l a m i d e g e l e l e c t r o -p h o r e s i s i n d i c a t e d t h e p r e s e n c e o f a number o f p r o t e i n bands. By r e p -e a t e d w a s h i n g s and f i l t r a t i o n w i t h an XM 100 u l t r a f i l t r a t i o n membrane, a l l p r o t e i n s e x c e p t t h o s e a s s o c i a t e d w i t h h i g h m o l e c u l a r w e i g h t p r o t e i n bands (bands I and I I ) were l o s t . The r e m a i n i n g p r o t e i n s a r e s u b u n i t s o f t h e p r o t e i n commonly r e f e r r e d t o as s p e c t r i n o r t e t k i n A. S p e c t r i n a l o n e c o u l d a c t i v a t e h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y e q u a l l y a s e f f i c i e n t l y as p r o t e i n f r a c t i o n C c o n t a i n i n g o t h e r l o w m o l e c u l a r w e i g h t p r o t e i n s i n a d d i t i o n t o s p e c t r i n . P h o s p h o l i p a s e A 2 t r e a t m e n t o f g h o s t s s e l e c t i v e l y i n a c t i v a t e d l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y s u g g e s t i n g t h a t t h e l o w a f f i n i t y a c t i v i t y i s p h o s p h o l i p i d d e p e n d e n t , i n c o n t r a s t t o t h e h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y . T h i s c o n c l u s i o n was p a r t i a l l y s u p p o r t e d by t h e f i n d i n g t h a t t h e A r r h e n i u s p l o t f o r l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y was d i s c o n -t i n u o u s w i t h a t e m p e r a t u r e b r e a k a t 16°C c h a r a c t e r i s t i c o f o t h e r w e l l s t u d i e d ATPases w h i c h a r e a l s o p h o s p h o l i p i d d e p e n d e n t . Low and h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t i e s a r e p r o b a b l y a s s o c i a t e d w i t h d i f f e r e n t " e n z y m e s . Heat t r e a t m e n t o f p r e p a r a t i o n B membranes d e s t r o y e d a l l ATPase a c t i v i t y . However, 46% o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v -i t y c o u l d be r e c o n s t i t u t e d by r e c o m b i n a t i o n o f t h e s o l u b l e p r o t e i n f r a c -t i o n w i t h t h e h e a t t r e a t e d membranes. I t i s a l s o u n l i k e l y t h a t one enzyme w o u l d have a f f i n i t y s i t e s w h i c h have d i f f e r e n t p h o s p h o l i p i d dep-e n d e n c i e s . T r e a t m e n t o f i n t a c t e r y t h r o c y t e s and r e s e a l e d g h o s t s w i t h t r y p s i n 00 i n h i b i t e d b o t h h i g h and low a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y as meas-u r e d i n g h o s t s p r e p a r e d f r o m t h e s e t r e a t e d c e l l s . As t r y p s i n was added i n t h e e x t e r n a l medium o f b o t h p r e p a r a t i o n s i n d i c a t e s t h a t (Mg+Ca)-ATPases a p p e a r t o be a s s o c i a t e d w i t h p r o t e i n s w h i c h span t h e membrane. T r e a t -ment o f g h o s t s w i t h n e u r a m i n i d a s e had no e f f e c t on (Mg+Ca)-ATPase a c t i v i t y . R uthenium r e d and L a C l 3 , s p e c i f i c i n h i b i t o r s o f (Mg+Ca)-ATPase a c t i v i t y i n g h o s t s , d i d n o t show s p e c i f i c i t y f o r e i t h e r 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 . These a g e n t s i n h i b i t e d b o t h (Mg+Ca)-ATPase a c t i v i t i e s i n membrane f r a g m e n t s w i t h s i m i l a r p o t e n c i e s . The s t o i c h i o m e t r y o f c a l c i u m t r a n s p o r t was d e t e r m i n e d t o be two i n r e s e a l e d e r y t h r o c y t e g h o s t s . T h i s was a c h i e v e d by a d d i n g l a n t h a n u m t o t h e e x t e r n a l medium o f t h e r e s e a l e d g h o s t s where l a n t h a n u m a p p e a r e d t o be s p e c i f i c f o r (Mg+Ca)-ATPase a c t i v i t y 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 . The p r e v i o u s r e p o r t e d v a l u e o f one f o r t h e s t o i c h i o m e t r y (Schatzmann 1 9 7 3 ) , p r o b a b l y r e s u l t e d f r o m f a i l u r e t o s u b t r a c t (Mg+Ca)-ATPase a c t i v i t y w h i c h i s n o t 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 . The r a t e o f c a l c i u m t r a n s p o r t and i t s s t o i c h i o m e t r y , was a p p r o x i m a t e l y t h e same i n g h o s t s p r e p a r e d i n t h e p r e s e n c e and ab s e n c e o f EDTA. I n a p r e p a r a t i o n c o n s i s t i n g o f 46% r e s e a l e d i n s i d e - o u t v e s i c l e s , and 2+ w h i c h c o n t a i n s o n l y l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y , t h e Ca dependence o f ATP dependent c a l c i u m u p t a k e and (Mg+Ca)-ATPase a c t i v i t y were compared. The s t u d y i n d i c a t e d t h a t t h e c a l c i u m t r a n s p o r t s y s t e m and l o w a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y a r e c l o s e l y a s s o c i a t e d . R e c o n s t i t u t i o n o f h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y i n r e s e a l e d v e s i c l e s i n c r e a s e d (Mg+Ca)-ATPase a c t i v i t y two f o l d w i t h o u t s i g n i f i c a n t l y 99 a f f e c t i n g t h e v e l o c i t y o f c a l c i u m u p t a k e . T h e r e f o r e i t was s u g g e s t e d t h a t h i g h a f f i n i t y (Mg+Ca)-ATPase a c t i v i t y may be a s s o c i a t e d w i t h some f u n c t i o n o t h e r t h a n c a l c i u m t r a n s p o r t . S i n c e h i g h a f f i n i t y (Mg+Ca)-ATPase r e q u i r e s s p e c t r i n f o r i t s a c t i v i t y i t i s p o s s i b l e t h a t t h i s enzyme a c t i v i t y may be a s s o c i a t e d w i t h volume o r d e f o r m a b i l i t y changes c o n t r o l l e d by i n t r a c e l l u l a r c a l c i u m . i A s t u d y o f t h e k i n e t i c s o f C a 2 + a c t i v a t i o n o f low a f f i n i t y (Mg+Ca)-ATP-ase a c t i v i t y r e v e a l e d t h a t t h i s enzyme, and t h u s c a l c i u m t r a n s p o r t , a r e r e g u l a t e d by a n e g a t i v e c o o p e r a t i v e mechanism. 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Biochem., 11_: 2897-2903, 1972. 108 Van Breeman, C : B l o c a d e o f membrane c a l c i u m f l u x e s by lanthanum i n r e l a t i o n t o v a s c u l a r smooth muscle c o n t r a c t i l i t y . A r c h . I n t . Phys . B i o c h i m i e , 7_7: 710-716, 1969. V i n c e n z i , F.F.: A c a l c i u m pump i n r e d c e l l membranes. I n " C e l l u l a r Mechanisms f o r C a l c i u m T r a n s f e r and H o m o s t a s i s " (G. N i c o l s and R.H. Wasserman, e d s . ) , pp. 135-149, Academic P r e s s , New Y o r k , 1 9 7 1 . 2+ Watson, E.L., V i n c e n z i , F.F. and D a v i s , P.W.: Ca - a c t i v a t e d membrane ATPase ATPase: S e l e c t i v e i n h i b i t i o n by r u t h e n i u m r e d . B i o c h i m . B i o p h y s . A c t a . , 249: 606-610, 1971. Watson, E.L., V i n c e n z i , F.F. and D a v i s , P.W.: N u c l e o t i d e s as s u b s t r a t e s o f Ca-ATPase and Na,K-ATPase i n i s o l a t e d r e d c e l l membranes. L i f e S c i . , 10: 1399-1404, 1971. 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No. 2, p. 9, 1973. 2+ Wolf, H.U. and Gietzen, K. : The s o l u b i l i z a t i o n of high a f f i n i t y Ca ATPase of human erythrocyte membranes. Hoppe-Seylers Z. P h y s i o l o g i c a l Chem., 355:1272, 1974. Zelander, T. and Ericsson, E.: The plasmamembrane of the red blood c e l l , J . U l t r a s t r u c t . Res., 12: 240, 1965. Zwaal, R.F.A., Roelofsen, B. and C o l l e y , CM. : L o c a l i z a t i o n of red c e l l membrane constituents. Biochim. Biophys. Acta., 300: 159-182, 1973. 1.10 APPENDIX I l l TABLE A C a l c u l a t e d v a l u e s f o r f r e e c a l c i u m i o n c o n c e n t r a t i o n s a c c o r d i n g t o K a t z e t a l _ . (1970) . C a 2 + c o n c e n t r a t i o n s were c a l c u l a t e d i n t h e p r e s e n c e o f 6.4 mM M g C l 2 , 2.0 mM ATP, 0.1 mM EGTA, 66 mM N a C l and 55 mM T r i s - m a l e a t e o r HEPES b u f f e r , pH 7.2.  [Ca] tyiM) uM C a 2 + , p C a 2 + t o t a l 66.6 0.297 6.527 80.0 0.580 6.237 93.4 1.629 5.788 100.0 3.530 5.452 116.0 14.627 4.936 133.0 28.804 4.541 166.0 56.938 4.250 200.0 86.309 4.064 300.0 172.393 3.703 366.0 229.238 3.639 466.0 316.240 3.500 666.0 490.346 TABLE B C a l c u l a t e d a p p a r e n t s t a b i l i t y c o n s t a n t s a t pH 7.2 E q u a t i o n (1) [MgATP 2 -] = 8.8 x 10 (2) 2+ [Mg ] [ATP] [CaATP 2"] = 3.15 x 10 (3) [ C a 2 + ] [ATP 4~] [CaEGTA 2"] 2+ [Ca ] [EGTA] = 6.61 x 10 R e f e r e n c e : 0 ' S u l l i v a n and P e r r i n (1964) Ogawa (1968) Schatzmann (1973) 

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