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

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THE RELATIONSHIP BETWEEN Mg+Ca-ATPase AND ACTIVE CALCIUM TRANSPORT IN RESEALED HUMAN ERYTHROCYTE GHOSTS by E. E. QUIST A thesis submitted i n p a r t i a l f u l f i l l m e n t of the requirements f o r the degree of MASTER OP SCIENCE In the D i v i s i o n of Pharmaceutical Chemistry of the Faculty of Pharmaceutical Sciences We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1973 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements 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 Columbia, 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 s t u d 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 purposes may be g r anted by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or 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 allowed without my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8 , Canada Date THE RELATIONSHIP BETWEEN Mg+Ca-ATPase AND ACTIVE CALCIUM TRANSPORT IN RESEALED HUMAN ERYTHROCYTE GHOSTS by E.E. q u i S T ABSTRACT Human red blood c e l l ghosts were prepared by a m o d i f i c a t i o n of the procedure of stepwise hemolysis (57)• EDTA (1 . 0 mM) was in c l u d e d i n the washing procedure t o remove endogenous ATP and d i v a l e n t c a t i o n s . Ghosts r e s e a l e d w i t h a p p r o p r i a t e amounts of ATP, calcium and magnesium were found t o have Mg+Ca-ATPase a c t -i v i t y and l i n e a r i t y was maintained up t o t h i r t y minutes. A c t i v e c a l c i u m t r a n s p o r t could be s t u d i e d i n these ghosts by measuring the change l n the c e l l u l a r c o n c e n t r a t i o n of calci u m over time by atomic a b s o r p t i o n spectrophotometry. V a r i a t i o n i n the c o n c e n t r a t i o n of calci u m i n the l o a d i n g medium r e s u l t e d i n an a c t i v a t i o n of Mg+Ca-ATPase and two peaks were evident on the a c t i v a t i o n curve.The hig h and low a f f i n i t y Mg+Ca-ATPase were maximally s t i m u l a t e d a t 0 .25 and 5«0 mM calcium l n the l o a d i n g medium,respectively. The v e l o c i t y of calci u m t r a n s p o r t was a l s o found t o be dep-endent on the c o n c e n t r a t i o n of ca l c i u m i n the l o a d i n g medium i i and was a c t i v a t e d over the c o n c e n t r a t i o n range of 0 . 1 t o 5«0 mM calcium. A change i n the c o n c e n t r a t i o n of c e l l u l a r c a l c i u m was not evident i n the absence of added ATP. I n c o n t r a s t t o the a c t i v a t i o n of Mg+Ca-ATPase two peaks were not obtained, and the a c t i v a t i o n curve had a si g m o i d a l appearance. Comparison of the calc i u m a c t i v a t i o n curves of Mg+Ca-ATPase and calcium t r a n s p o r t revealed, a s i m i l a r i t y i n the shape and p o s i t i o n of the low a f f i n i t y p a r t of the Kg+Ca-ATPase and calc i u m t r a n s p o r t a c t i v a t i o n curves. A s t o i c h i o m e t r y of two (Ca :ATP) was obtained, i n the low a f f i n i t y a c t i v i t y range. Ruthenium red (0 .05 t o 0.4 mM) s e l e c t i v e l y I n h i b i t e d the low a f f i n i t y Mg+Ca-ATPase and i n h i b i t e d c a l c i u m t r a n s p o r t over the same c o n c e n t r a t i o n range to a s i m i l a r degree. Both low a f f i n i t y Mg+Ca-ATPase and calcium t r a n s p o r t were I n h i b i t e d by e x t e r n a l ruthenium red w i t h an I ^ 0 of 0 .2 mM. P r o p r a n o l o l , q u l n i d i n e and q u i n i n e (10~^ to 10~^M) were found t o be I n e f f e c t i v e i n s t i m u l a t i n g or I n h i b i t i n g Mg+Ca-ATPase when added t o the I n t e r n a l and e x t e r n a l aspects of the ghosts. Manganese, added t o the l o a d i n g medium over a wide concent-r a t i o n range, was unable t o s u b s t i t u t e f o r ca l c i u m i n a c t i v a t i n g Mg+Ca-ATPase. E x t e r n a l d i v a l e n t c a t i o n s c a l c i u m and magnesium f u r t h e r i n c r -eased Mg+Ca-ATPase a c t i v i t i e s when added to the e x t e r n a l medium. Maximal s t i m u l a t i o n occurred a t a c o n c e n t r a t i o n of approximately 3 . 0 mM and calcium was almost twice as e f f e c t i v e as magnesium. Signatures of Examiners Iv TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES v i LIST OF FIGURES v i i INTRODUCTION 1 LITERATURE REVIEW 3 Homeostatlc Mechanisms C o n t r o l l i n g The 3 I n t r a c e l l u l a r C o n c e n tration Of Calcium A c t i v e Transport I n Human Red Blood C e l l s 6 Mg+Ca-ATPase A c t i v i t y I n Human Red Blood C e l l 14 Membrane Fragments I n h i b i t o r s E f f e c t i n g . Mg+Ga-ATPase And A c t i v e C a 2 + 19 Transport I n Red Blood C e l l s A c t i v e Calcium Transport I n Other Tissues 21 METHODS AND MATERIALS 25 P r e p a r a t i o n Of Red Blood C e l l Ghosts 25 Loading Procedure 28 Re s e a l i n g And Washing Procedure 28 Incubation Procedures 29 Determination Of ATPase A c t i v i t y 29 Determination Of The V e l o c i t y Of Calcium 31 Transport P r o t e i n Assay 32 Washing Procedure 33 RESULTS AND DISCUSSION 34 P r o p e r t i e s Of The Red Blood C e l l Ghost 34 P r e p a r a t i o n Page Ion Requirements For Resealing 36 Na.K-ATPase A c t i v i t y In Resealed Red Blood 37 C e l l Ghosts Mg+Ca-ATPase A c t i v i t y In Resealed Red Blood 4 l C e l l Ghosts The E f f e c t of External Divalent Cations On 43 ATPase A c t i v i t y The E f f e c t Of Internal Calcium On Calcium E f f l u x 49 The E f f e c t Of Ruthenium Red On Mg+Ca-ATPase 57 A c t i v i t i e s The E f f e c t Of Ruthenium Red On Calcium Transport 6 l The E f f e c t Of Drugs (Quinine, Quinidine, and 66 Propranolol) On Mg+Ca-ATPase The E f f e c t Of Manganese On Mg+Ca-ATPase A c t i v i t y 67 CONCLUSIONS 69 BlBLIOGRA PHY 73 APPENDIX 80 LIST OF TABLES Table I Ion Antagonisms. II E f f e c t s Of Ruthenium Red On ATPases Of Erythrocyte Membranes. II I Features Of The Na-Ca Exchange Mechanism In Cyanide Poisoned Squid Axons. IV Preparation Of RBC Ghosts, Loading And Assay Procedure. v l l LIST OF FIGURES F i g u r e Page 1. Changes i n Ca c o n c e n t r a t i o n i n c e l l s and 9 medium and P i r e l e a s e from ATP i n r e s e a l e d c e l l s . 2. E f f e c t of p r e i n c u b a t i o n w i t h i o d o a c e t a t e . 12 3 . The s t i m u l a t i o n of calcium t r a n s p o r t by 13 i n t e r n a l calcium. 2+ 4. (Ca )-dependent ATPase a c t i v i t y i n human 15 red c e l l s . 5 . ATPase a c t i v i t y as a f u n c t i o n of C a * + 18 co n c e n t r a t i o n l n the presence and absence of 80mM K +. 6. E f f e c t of N a + and K + on ATPase a c t i v i t y l n 18 hemoglobin-free r e d c e l l membranes, prepared by f r e e z i n g and thawing. 7 . Time course of ATPase a c t i v i t y i n the absence 38 of e x t e r n a l ouabain and i n the presence of 0.2mM e x t e r n a l ouabain. 8. A c t i v a t i o n of Na,K-ATPase a c t i v i t y by e x t e r n a l 40 potassium l n the presence of l.OmM e x t e r n a l calcium, zero e x t e r n a l calcium and O.lmM e x t e r n a l ouabain. v i i l Figure Page 9. E f f e c t of varying the concentration of 42 calcium l n tne loading medium on Mg+Ca-ATPase a c t i v i t y . 10. Eadie plo t of calcium a c t i v a t i o n of 44 Mg+Ca-ATPases i n ghosts. 11. The e f f e c t of external d i v a l e n t cations on 46 ATPase a c t i v i t y . 12. E f f e c t of varying the concentration of 48 calcium i n the loading medium on Mg+Ca-ATPase a c t i v i t y i n the absence of external calcium and l n the presence of l.OmM external calcium. 13 . Changes i n the concentration of c e l l u l a r 50 calcium with time. 1 4 . Relationship between c e l l u l a r calcium and 52 calcium l n the loading medium. 15. Comparison of the v e l o c i t y of active calcium 54 transport and Mg+Ca-ATPase a c t i v i t y as a function of calcium i n the loading medium. 16. H i l l p l o t of calcium a c t i v a t i o n of calcium 56 e f f l u x l n ghosts. 17. Effect of ruthenium red ln the external medium on Mg+Ca-ATPase ac t i v i t y . 18. The effect of ruthenium red on the activation of Mg+Ca-ATPase. 19. Effect of ruthenium red in the external medium on the velocity of active calcium transport in ghosts loaded with 3.0mM calcium. 20. The effect of ruthenium red on the velocity of active calcium transport. 21. The effect of ruthenium red (0.2mM) i n the external medium on Mg+Ca-ATPase act i v i t y and active calcium transport as a function of the concentration of calcium l n the loading medium. 22. Effect of varying the concentration of manganese in the loading medium on ATPases activ i t y i n the absence of external calcium and ln the presence of l.OmM external calcium. X ACKNOWLEDGEMENTS I wish to express my gratitude to Dr. B.D. Roufogalis f o r his excellent guidance and assistance throughout the course of t h i s work and to Dr. D. Godin f o r his helpful suggestions l n the early stages of t h i s study. DEDICATION To Laurie and J u l i a n 1 INTRODUCTION Human e r y t h r o c y t e s m a i n t a i n a low i n t r a c e l l u l a r concent-r a t i o n of calcium by means of an a c t i v e calcium t r a n s p o r t system r e q u i r i n g ATP ( 1 ) . Schatzmann proposed that a c t i v e calcium t r a n s p o r t was a s s o c i a t e d w i t h Mg+Ca-rATPase, analogous to the Na, K pump system (11). Some s i m i l a r i t i e s between the p r o p e r t i e s of Mg+Ca-ATPase i n red blood c e l l membrane fragments and c a l c i u m t r a n s p o r t l n r e s e a l e d ghosts have been re p o r t e d as I n d i r e c t evidence supporting t h i s hypothesis (12,26,30). An a s s o c i a t i o n between these systems was a l s o demonstrated when both a c t i v i t i e s were s t u d i e d simultaneously i n r e s e a l e d human e r y t h r o c y t e ghosts loaded w i t h a s i n g l e c o n c e n t r a t i o n (ImM) of calci u m ( 3 0 ) . However, w i t h the f i n d i n g of more thafeone Mg+Ca-ATPase i n red blood c e l l membrane fragments (32,33,34) i t became apparent t h a t f u r t h e r d i r e c t evidence showing an a s s o c i a t i o n between Mg+Ca-ATPase was r e q u i r e d . Therefore, i n the present t h e s i s the a s s o c i a t i o n of Mg+Ca-ATPase a c t i v i t y and a c t i v e calcium t r a n s p o r t has been r e i n v e s t i g a t e d i n the same p r e p a r a t i o n of r e s e a l e d ghosts over a wide range of ca l c i u m c o n c e n t r a t i o n s . Ruthenium red which has been re p o r t e d 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 i n red blood c e l l membrane fragments (40) was t e s t e d on r e s e a l e d ghosts t o determine whether t h i s dye would a l s o I n h i b i t c a l c i u m t r a n s p o r t . Ruthenium red was a l s o 2 used as a t o o l f o r i n v e s t i g a t i n g the a s s o c i a t i o n between the calcium t r a n s p o r t system and a Mg+Ca-ATPase. I t was considered t h a t t h i s t r a n s p o r t system may be a important s i t e of drug a c t i o n , p a r t i c u l a r l y f o r drugs i n v o l v e d l n the m o b i l i z a t i o n of calcium, because of the mult i t u d e of p h y s i o l o g i c a l e f f e c t s r e g u l a t e d by i n t e r n a l c a l c i u m . Drugs of i n t e r e s t which were s t u d i e d Include q u i n i n e , q u i n i d i n e , propran-o l o l and t e t r a c a i n e . The red blood c e l l ghost was used as a model membrane system l n t h i s study s i n c e r e d blood c e l l s can be obtained i n l a r g e q u a n t i t i e s and ghosts f r e e of the i n t r a -c e l l u l a r contents can be prepared by the method of r e v e r s a l of hemolysis. The red blood c e l l has p r e v i o u s l y proven to be Inv a l u a b l e f o r studying the mechanism of c a t i o n t r a n s p o r t and asymmetric ouabain i n h i b i t i o n of Na,K-ATPase. Many s i m i l a r i t i e s have been shown between mechanisms o p e r a t i n g on t h i s system and those on the plasma membrane of other t i s s u e s . o 3 LITERATURE REVIEW Homeostatlc Mechanisms C o n t r o l l i n g The I n t r a c e l l u l a r  C oncentration Of Calcium The homeostatlc mechanisms by which c e l l s m a i n t a i n a low I n t r a c e l l u l a r c o n c e n t r a t i o n of calcium has been of i n t e r e s t to p h y s i o l o g i s t s f o r the past seventy years. I n mammalian c e l l s , the i n t r a c e l l u l a r c o n c e n t r a t i o n of calcium has been estimated -7 -5 to be between 10 M and 10 M i n c o n t r a s t t o a calc i u m concent-r a t i o n of 10"*^ M l n the e x t r a c e l l u l a r f l u i d (1,2). Maintenance of a low I n t r a c e l l u l a r c o n c e n t r a t i o n of calc i u m i s of v i t a l import-ance t o the s u r v i v a l of the c e l l as an i n c r e a s e over the normal l e v e l has a profound e f f e c t on c e l l u l a r and enzyme f u n c t i o n s . For i n s t a n c e , i n human e r y t h r o c y t e s i n t r a c e l l u l a r c o n c e n t r a t i o n s of calcium g r e a t e r than lO'^M i n h i b i t s Na,K-ATPase which c o n t r o l s the d i s t r i b u t i o n of sodium and potassium across the plasma mem-brane (3»4). A number of other enzymes i n h i b i t e d by calcium are shown i n Table I . I t has a l s o been accepted t h a t i n t r a c e l l -u l a r calcium i s a c o u p l i n g f a c t o r i n e x c i t a t i o n - c o n t r a c t i o n coup-l i n g i n muscle c e l l s ( 6 ) . I n human red blood c e l l s , i n t r a c e l l u l a r c a lcium has been shown to r e g u l a t e the pa s s i v e p e r m e a b i l i t y of the plasma membrane t o N a + and K + (7,8), the volume of the c e l l (9) and membrane d e f o r m a b l l i t y (10). 4 Table I . Ion Antagonisms (5) Enzyme A c t i v a t i n g Ions I n h i b i t i n g Ions Methionine adenosyl- Mg or Mn + (K, Ca or Zn t r a n s f e r a s e NHit.Rb) Pantothenate Mg or Mn Ca or Zn synthetase Mg + (K.Rb.Cs) Pyruvate kinase 5-nucleotidase Ca and Na or L i Mg Ca A r g l n l n o s u c c i n a t e Mg Ca or Mn synthetase Glutamine synthetase Mg or Mn Ca R i b o f l a v i n k i n a se Mg.Zn.Co or Mn Ca Inorganic Mg Ca or Zn pyrophosphatase Phosphopyruvate Mg.Zn.Mn or Gd Ca or Sr hydratase Ca or (K or NH k) Myosin ATPase Mg G l y c y l - l e u c l n e Zn or Mn * Ca d i p e p t i d a s e A number of w e l l - e s t a b l i s h e d homeostatlc mechanisms by which c e l l s c o n t r o l the d i s t r i b u t i o n of ca l c i u m across the plasma membrane have been summarized as f o l l o w s : 1, P a s s i v e p e r m e a b i l i t y ; Plasma membranes i n the r e s t i n g s t a t e are r e l a t i v e l y Impermeable t o calcium under p h y s i o l o g i c a l c o n d i t i o n s . However a slow exchange of calc i u m across the plasma membrane i s evident (12,13). The d i s t r i b u t i o n of calc i u m across the plasma membrane i s a l s o not ac c o r d i n g t o the e q u i l i b -rium p o t e n t i a l as c a l c u l a t e d by the Nernst Equation ( i i ) . I n red blood c e l l s the r e l a t i v e l y impermeable nature of the plasma membrane does not account f o r the low i n t r a c e l l u l a r c o ncentrat-i o n of calcium s i n c e calcium i s taken up i n c e l l s d epleted of 5 ATP or stored i n the c o l d ( 7 ) . This o b s e r v a t i o n i n d i c a t e s t h a t the d i s t r i b u t i o n of calcium i s maintained o n l y i n a metabol-l c a l l y a c t i v e c e l l . 2. A c t i v e calcium t r a n s p o r t ; I n human e r y t h r o c y t e s (12,13), He La c e l l s (44) and L c e l l s (45), c a l c i u m has been shown to be t r a n s p o r t e d across the plasma membrane a g a i n s t an e l e c t r o -chemical p o t e n t i a l by a mechanism r e q u i r i n g ATP. 3. Na-Ca Coupling; I n nerve (14,15) and c a r d i a c muscle (47)• one i n t r a c e l l u l a r c a l c i u m i o n i s exchanged f o r two t o three sod-ium Ions by a system not coupled to a chemical r e a c t i o n . 4. I n t r a c e l l u l a r o r g a n e l l e s ; (a) Sarcoplasmic r e t i c u l u m - The sarcoplasmic r e t i c u l u m i s a network of t u b u l e s , v e s i c l e s , and cysternae surrounding the m y o f i b r i l s ( 9 ) . This s t r u c t u r e r a p i d l y accumulates cal c i u m by an a c t i v e t r a n s p o r t system c l o s e l y a s s o c i a t e d w i t h a magnes-ium dependent ATPase (54). These s t r u c t u r e s are extremely impor-t a n t f o r r e l a x i n g the c o n t r a c t u r e of s k e l e t a l and c a r d i a c muscle c e l l s . (b) Mitochondria - M i t o c h o n d r i a a c t i v e l y accumulate calcium by an a c t i v e c a l c i u m t r a n s p o r t system r e q u i r i n g ATP. Mitochondria may be important i n m a i n t a i n i n g a low i n t r a c e l l u l a r c o n c e n t r a t i o n of c a l c i u m i n nerve or i n muscle having a p o o r l y developed sarcoplasmic r e t i c u l u m (16,17 , 6 4 , 6 9 ) . 5. I n t r a c e l l u l a r b i n d i n g and c h e l a t i o n ; Calcium i s a l s o bound t o a n i o n i c s i t e s (such aa on p r o t e i n s ) and c h e l a t e d to molecules such as c a r b o x y l l c a c i d s and n u c l e o t i d e s . The r e l a t i v e importance of calcium b i n d i n g t o these s i t e s i s not known. 6 Active Transport In Human Red Blood C e l l s Red blood c e l l s have proved to be a useful model system f o r studying active transport of cations and solutes. These c e l l s are av a i l a b l e i n large quantities l n a homogeneous form and the plasma membranes can be Isolated by r e l a t i v e l y gentle means f o r enzyme studies. Perhaps f o r t h i s reason the coup-l i n g of a transport process to a chemical reaction was f i r s t i d e n t i f i e d to occur across the plasma membrane of the red blood c e l l . Wilbrandt l n 1939 (19) was the f i r s t to demonstrate that the d i s t r i b u t i o n of sodium and potassium was re l a t e d to g l y -c o l y s i s . I n h i b i t i o n of g l y c o l y s i s by iodoacetate and sodium f l u o r i d e l e d to a loss of i n t r a c e l l u l a r potassium and a change i n the osmotic resistance of the c e l l . This study stimulated other workers to speculate that ATP was d i r e c t l y involved i n the d i s t r i b u t i o n of potassium, as ATP i s synthesized s o l e l y by g l y c o l y s i s l n the human red blood c e l l . The importance of ATP l n maintaining the I n t r a c e l l u l a r l e v e l s of potassium was v e r i f i e d l n experiments with reconstituted or resealed ghosts (22). Red blood c e l l s may be p a r t i a l l y hemolyzed by exposure to hypotonic solutions, usually with i o n i c strengths of not l e s s than 0.02 M (20,21). The c e l l s become leaky due to osmotic shock and ATP and other ions may be introduced into the c e l l . With the additi o n of a concentrated s a l t s o l u t i o n to t h i s 7 hemolysate, the l s o t o n l c i t y can be r e s t o r e d and the r e s t o r a t i o n of low c a t i o n p e r m e a b i l i t y thus obtained i s adequate f o r s t u d -y i n g a c t i v e c a t i o n movements. Gardos (22) i n t r o d u c e d ATP i n t o r e s e a l e d ghosts and demonstrated an accumulation of potassium by a system which r e q u i r e d ATP. Schatzmann (23) demonstrated t h a t t h i s ATP dependent system was i n h i b i t e d by ouabain. A l i n k between potassium t r a n s p o r t and the membrane bound enzyme Na,K-ATPase was recognized when Na, K-iVTPase was found i n crab nerve by Skou (24). S i n c e then, Na.KsATPase has been found l n the plasma membrane of most c e l l s , i n c l u d i n g the human e r y t h r o -cyte (3) . I n red blood c e l l s Na,K-ATPase and the a c t i v e t r a n s -p o r t of sodium and potassium were I n h i b i t e d by approximately 10~->gm m l " 1 of oubaln (23). The s t o i c h i o m e t r y and other prop-e r t i e s of Na, K-ATPase system have now been w e l l reviewed (2ff). On the other hand, the f i n d i n g of an energy dependent ca l c i u m t r a n s p o r t system has o n l y r e c e n t l y been demonstrated l n red blood c e l l s and the s t u d i e s have been r e l a t i v e l y few. H i s t o r i c a l l y , Dunham and Glynn (3) i n 1961 were the f i r s t t o r e p o r t the e x i s t -ence of a ouabain i n s e n s i t i v e c a l c i u m a c t i v a t e d ATPase i n red blood c e l l membrane fragments (RBCMF). This enzyme which was found l n these s t u d i e s t o have a s p e c i f i c a c t i v i t y two t o three times as great as Na,K-iiTPase was a l s o found t o be magnesium dependent. This enzyme w i l l be r e f e r r e d to henceforth as Mg+Ca-ATPase. Schatzmann (11) reasoned t h a t Mg+Ca-ATPase may be a s s o c i a t e d w i t h a t r a n s p o r t system which maintains a low c o n c e n t r a t i o n of I n t r a c e l l u l a r c a l c i u m i n red blood c e l l s , a n a l -ogous to the Na,K-ATPase system. Resealed ghosts loaded w i t h 8 ATP, magnesium and calcium were found to r a p i d l y lose c e l l u l a r calcium when incubated at 37°C ( F i g . 1,Panel A) (12). In ghosts loaded with 1.0 or 2.0 mM calcium, tiOfi of the c e l l u l a r calcium was l o s t within ten minutes. In agreement with the hypothesis that Mg+Ca-ATPase was linked to t h i s transport process, a con-comittant increase i n the concentration of inorganic phosphate (Fi g . l.Panal C) due to ATP hydrolysis was evident during the course of the calcium transport. In a previous study, Schatzmann (11) found that resealed ghosts l o s t calcium at a slow rate i n the absence of added ATP. This r e s u l t was a t t r i b u t e d to an incomplete removal of endogenous ATP during the hemolysis pro-cedure used l n the preparation of the tthosts. This explanation was shown to be v a l i d since c e l l s depleted of energy by means of a 1? hr incubation i n glucose f r e e medium at 37*C showed no l o s s of c e l l u l a r calcium unless ATP was Included i n the loading medium (F i g . 1,Panel B). Ghosts resealed i n the presence of 0.1 mM calcium ' l o s t calcium i n t o an external medium containing 1.0 mM calcium. Therefore the loss of c e l l u l a r calcium demonstrated by Schatzmann l n resealed ghosts (11) appears to be due to an a c t i v e transport system since the process was found to be dependent on ATP and operated against a concentration gradient. However, the evidence l i n k i n g calcium e f f l u x to a Mg+Ca-ATPase was not d i r e c t . In resealed ghosts only i n t r a c e l l u l a r calcium stimulated ATPase a c t i v i t y (12). This asymmetrical stimulation of Mg+Ca-ATPase a c t i v i t y by i n t e r n a l calcium Is analogous to the Na,K-ATPase transport system where only external potassium 1.5 30 60 Time (min) Fig. 1 Changes of Ca concentration in cells and medium and P, release from ATP in re.-sealed cells. Haemolysis in water containing 2 mM-Tris-ATP, 5 nwt-Tris-Cl, 4 mM-MgCl, and 1 or 2 mM (in three expts. in tho ATP free sample) CaCI,. Reversal "f haemolysis in presence of KC1. Previous to haemolysis starvation during 17 hr at 37'C in glucose-free solution- (130 mM-Na, 5 mJ i-K, 20 m.M-Tn's, 155mM-Cl). -Milium: 130 mM-Xa, 5m>i-K, 20 mM-Tr,is, 1 rnit-Ca, 157 mu-Cl, 10-«g/ml. ••'laljain. Temp. 37° C. Haematocrit: ATP samplo 0-249, ATP-free sample 0-253. • with ATP in tho-cells, O without ATP. Arrow in panel A : mean concentration i:K-asured in whole suspension. Four experiments, vertical bar* 2x S.E. of mean^)2)» 10 and Internal sodium w i l l stimulate Na,K-ATPase ( 6 5 ) . Schatzmann also f e l t that the Mg+Ca-ATPase present i n red blood c e l l s was s e n s i t i v e enough to be part of a calcium transport system which maintains a low I n t r a c e l l u l a r concentration of calcium^whlch i n the case of the human red blood c e l l has been estimated to be l e s s than 5 X110"^M. In RBCMF Mg+Ca-ATPase was found to be -4 optimally stimulated at 10 M calcium and had a threshold at 10"7M. Other evidence associating Mg+Ca-ATPase with a c t i v e calcium transport was as follows: (a) Ionic Requirements; Shin and Lee (26) were able to show that active calcium transport was dependent on i n t r a c e l l u l a r magnesium. Since a l l the endogenous magnesium was not removed during the preparation of t h e i r ghosts, (ethylene dlamlne)-t e t r a a c e t i c acid (EDTA) (a di v a l e n t metal ion chelating agent), was used to chelate the endogenous magnesium. Calcium transport was abolished l n ghosts resealed i n 1.0 mM EDTA, 2 . 0 mM C a C l 2 , and 2 .0 mM ATP. (b) I t has also been shown that strontium can substitute f o r calcium l n a c t i v a t i n g Mg+Ca-ATPase i n RBCMF (31) and that strontium i s transported out of asesealed ghosts by a mechanism requi r i n g ATP ( 2 7 ) . (c) In resealed ghosts, a c t i v e calcium transport does not depend on a l k a l i metal ions f o r e i t h e r stimulation of the v e l -o c i t y of calcium e f f l u x or f o r a jja-Ca exchange re a c t i o n . In ghosts resealed with choline or l n T r i s buffer, rather than sodium or potassium, the v e l o c i t y of calcium transport was not changed ( 2 6 , 1 2 ) . 11 (d) Concomitant inward transport of magnesium was not observed in cel l s extruding calcium, nor did the addition of magnesium to the external medium stimulate the ATPase a c t i v i t y of resealed ghosts (79). Therefore the p o s s i b i l i t y that c a l -cium transport i s coupled to the inward movement of magnesium seems unlikely. (e) Lastly, hydrogen Ion does not seem to be coupled to calcium transport since the pH of a non-buffered medium did not change during active calcium transport ( 2 6 ) . 2 .Nucleotide s p e c i f i c i t y ; Studies undertaken to determine the nucleotide requirement for calcium transport ln resealed ghosts have been inconclusive. Lee and Shin reported that u r i -dine triphosphate (UTP) and cytldlne triphosphate (CTP) were as effective as ATP l n supporting calcium transport In resealed ghosts (26). Guanoslne triphosphate (GTP) was found to be less effective and inoslne triphosphate (ITP) the poorest substrate. In another study, GTP and ITP were reported to be as effective as ATP ( 2 7 ) . These facts do not support the contention that Mg+Ca-ATPase and calcium transport are closely associated since Mg+Ca-ATPase i n RBCMF i s specific for ATP ( 2 9 ) . However, the conflicting reports on nucleotide s p e c i f i c i t y l n resealed ghosts may be a result of incomplete removal of nucleoside dlphosphokinase which can catalyze the synthesis of ATP by tran-sfer of the phosphate from nucleoside triphosphates to ADP during preparation of the ghosts ( 3 5 ) . Calcium efflux i s very dependent on the intracellular con-centration of ATP ( 2 6 ) . In c e l l s pretreated with iodoacetate, 12 no s i g n i f i c a n t t r a n s p o r t o f c a l c i u m was observed l n the absence of ATP ( F i g . 2 ) . However, t h e r e was about 6y/> t r a n s p o r t i n ghosts not p r e t r e a t e d w i t h i o d o a c e t a t e In the absence of added ATP. I t appears t h a t ghosts c o n t a i n a p p r o x i m a t e l y 0 . 3 mM of endogenous ATP, s i n c e a p p r o x i m a t e l y the same degree (58;S) of c a l c i u m was t r a n s p o r t e d i n l o d a c e t a t e t r e a t e d c e l l s when 0 . 3 mM ATP was added. I n ghosts loaded w i t h 1.0 mM ATP a more complete and f a s t e r removal of c e l l u l a r c a l c i u m o c c u r r e d . 3 . Temperature dependence; 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 temperature, as would be expected o f a system cou p l e d t o a ch e m i c a l r e a c t i o n . High Q 1 0 v a l u e s of 3 . 5 (12) and 3 .16 (26) have been r e p o r t e d f o r c a l c i u m e f f l u x from r e s e a l e d g h o s t s . AT? LO FICURE 2. Effect of preincu-bation with iodoacetate. None + A T P (1 mit), tie initial preincubation with iodoacetate was omitted. Other conditions, standard. None — A T P , no preincubation with iodoace-tate and no A T P in lysing solution. Other conditions, standard. Iodoacetate — A T P , standard conditions except' omission of A T P from solution. Iodoacetate + A T P (0.3 mit), standard conditions, except 0.3 mil A T P in lysing solution. Each point represents average of four experiments. Bars indi-cate standard error (%z)(Zi>). a LO o tt 20 TIME IN MINUTES 13 4 . The e f f e c t of I n t r a c e l l u l a r calcium on the v e l o c i t y of calcium e f f l u x ; Romero and Whittam demonstrated that the v e l -o c i t y of calcium e f f l u x was dependent on the i n t r a c e l l u l a r concentration of calcium i n red blood c e l l s (7) . C e l l s were loaded to give a range of i n t e r n a l calcium from 0.4 to 2.7 pimoles calcium per ml of c e l l s , by Incubating ATP depleted c e l l s f o r various time i n t e r v a l s i n a Ringer s o l u t i o n containing 10 mM calcium at 37 C. The c e l l s were enriched with ATP by incubation with adenine and inoslne and the rate of calcium l o s s was det-ermined ( F i g . 3)« The rate of extrusion increased with i n c r e a s -ing calcium concentration, reaching a plateau of about 2.6 yamole calcium per ml X hr. The i n t e r n a l calcium concentration g i v i n g h a l f maximal a c t i v a t i o n was about 0.9 /imole per ml c e l l s . 3 r 1 2 3 Ca content (^mole/ml. cells) Fig. 3.The stimulation of calcium transport by internal calcium. Cells washed free of glucose were loaded with calcium by incubation at 37° C for periods up to 3 hr in Ringer solution containing (mil): NaCI, 150; CaCI2, 10; Tris-HCl, 20, p H 7-6. Adenine (5 mn) and inosine (10 mil) were added, the incubation was continued and samples wero taken at 10 min intervals. The rate of calcium extrusion (/mnolo calcium/ml. cells x hr) was calculated from the change in 10 min, and has been plotted against the mean cell cal-cium concentration during this timo. Results are shown from three experiments ("J), 14 Hg+Ca-ATPase A c t i v i t y In Human Red Blood C e l l Membrane Fragments Since the discovery of an ouabain i n s e n s i t i v e calcium stim-ulated ATPase l n red blood c e l l membrane fragments (RBCMF), in v e s t i g a t o r s have attempted to characterize t h i s enzyme. The p h y s i o l o g i c a l r o l e of Mg+Ca-ATPase however has not been complet-e l y agreed upon. As previously mentioned i t was suggested that Mg+Ca-ATPase was associated with a c t i v e calcium transport ( 1 2 , 2 6 ) , whereas other i n v e s t i g a t o r s have f e l t that t h i s enzyme may be responsible f o r r e g u l a t i n g the c e l l volume ( 3 1 ) . The Mg+Ca-ATPase reported by these l a t t e r workers i s thought to be associated with a m y o f i b r i l l a r p r o t e i n c a l l e d s p e c t r i n (55)• Recently data has been obtained which may r e c o n c i l e t h i s d i f f e r e n c e i n opinion of the f u n c t i o n a l r o l e of Mg+Ca-ATPase. Care f u l studies have revealed that more than one Mg+Ca-ATPase i s present i n RBCMF, as d i f f e r e n t i a t e d by t h e i r a f f i n i t i e s f o r calcium ( 3 0 , 3 2 , 3 3 , 3 4 ) . Figure 4 i l l u s t r a t e s the a c t i v a t i o n of Mg+Ca-ATPase l n RBCMF found by Horton et a l . ( 3 2 ) . A c t i v a t i o n by calcium appears at 10~aM calcium and increases to 3 . 0 10""''M calcium, at which point the rate of increase slows and a plateau i s observed up to 3 X. 10~^M. The a c t i v i t y then r a p i d l y increases to an optimum at 3 X i u M calcium. The enzyme, maximally stim-ulated at 3 X 10~^M calcium i s r e f e r r e d to as the high a f f i n i t y Mg+Ca-ATPase due to i t s higher a f f i n i t . y f o r calcium. The enzyme —4 maximally stimulated at 3 A. 10 M calcium i s re f e r r e d to as the low a f f i n i t y Mg+Ca-ATPase. Qther values reported i n the l i t e r a t -ure Indicate that the high a f f i n i t y Mg+Ca-ATPase i s optimally 15 stimulated from 10 to 50 uM calcium and the low a f f i n i t y Mg+Ca-ATPase anywhere from 0.1 to 0.5 mM calcium (33,34,78,43,32). The wide v a r i a t i o n of the reported values i s a t t r i b u t e d to the d i f f e r e n t methods used f o r the i s o l a t i o n of the RBCMF. Generally the high a f f i n i t y Mg+Ca-ATPase has a s p e c i f i c a c t i v i t y of 0.3 to 0.5 umoles Pi mg-^hr""1 compared to a s p e c i f i c a c t i v i t y of 0.6 to 0.88 umoles P i mg^ta 1" 1 f o r the low a f f i n i t y Mg+Ca-ATPase. The presence of two Kg+Ca-ATPases has not been shown previously i n whole red blood c e l l s or i n resealed ghosts. The f i n d i n g of two Mg+Ca-ATPases i n RBCMF f u r t h e r complicates studies attempt-ing to r e l a t e active calcium transport and Mg+Ca-ATPase i n r e -sealed ghosts. Few k i n e t i c studies have been done on these enzyme systems. The properties which have been determined are as follows t V| 1 1 1 1 1 1 1 1 ' I O IO"9 IO"7 IO"5 IO"3 IO"' Co**Molar Fig.4 (Ca + +)-dependent ATPase a c t i v i t y l n human red cells.Each point represents the mean of four experiments with the standard error of the mean (32.). 16 1. Ion requirements; Both high and low a f f i n i t y Mg+Ca-ATPases require magnesium. Optimal concentrations of magnesium for these enzymes have been estimated to be between 3 to 8 mM(33).* 2. Mg-ATP was shown kinetically to be the substrate for high a f f i n i t y Mg+Ca-ATPase 133). Calcium acts a l l o s t e r l c a l l y as a positive autosteric effector ( 3 3 ) . Whether or not Mg-ATP is the substrate for low a f f i n i t y Mg+Ca-ATPase has not been determined• 3. Effects of other cations on Mg+Ca-ATPase activity; Both strontium and barium have been shown to be activators of Mg+Ca-ATPase ( 3 1 ) . Strontium activates more strongly than calcium, but has a lower a f f i n i t y . Careful kinetic studies were not done to determine which Mg+Ca-ATPase was activated by barium or strontium, but judging from the low a f f i n i t y of these cations, the low a f f i n i t y Mg+Ca-ATPase i s activated at least. This finding i s of special interest since strontium i s also actively transported out of resealed ghosts, presumably by the same system as calcium ( 2 7 ) . There i s evidence that Mg+Ca-ATPase l n RBCMF i s stimulated + + + by univalent cations i n the order of K > Na > Rb (30,36). Pot-assium activates Mg+Ca-ATPase act i v i t y approximately 100^ by a mechanism which i s non-competitive with calcium {Fig. 5 ) . The presence of both magnesium and calcium i s required for this activation. Cesium and lithium were reported to have no stim-ulating effect, indicating that the activation by potassium, sodium and rubidium may not be duejjust to a change i n ionic 1? strength ( 3 6 ) . However, i n a study done by Schatzmann and Rossi ( 3 0 ) l i t h i u m was shown to be about one-third as e f f e c t i v e as potassium i n a c t i v a t i n g Mg+Ca-ATPase. Sodium and potassium appear to act at the same s i t e since no a d d i t i o n a l a c t i v a t i o n occurs when these ions are added together ( 3 6 ) . A c t i v a t i o n by Na + or K + of Mg+Ca-ATPase occurs over a wide range of calcium concentrations (Fig. 6 ) . A c t i v a t i o n i s s t i l l apparent at 1 mM, i n d i c a t i n g that low a f f i n i t y Mg+Ca-ATPase a c t i v i t y i s activated by these cations. Schatzmann et a l . (30) a r b i t r a r i l y c a l l e d the a c t i v i t y i n the absence of added a l k a l i metal cations alkali-cation-independent Mg+Ca-ATPase a c t i v i t y and l n the presence of a l k a l i metal cations, a l k a l i - c a t i o n -dependent Mg+Ca-ATPase a c t i v i t y . Since a l k a l i metal ions do not have any e f f e c t on active calcium transport l n resealed ghosts, Schatzmann et a l . (30) proposed that the extra a c t i v i t y seen l n the presence of a l k a l i metal cations was due to an uncoupling a c t i o n of calcium on the Na,K-ATPase responsible f o r the trans-port of sodium and potassium. This hypothesis however, seems very u n l i k e l y . Bond and Green (36) f e l t that t h i s e f f e c t may be a t r i v i a l property of RBCMF. For Instance, these ions may expose l a t e n t Mg+Ca-ATPase a c t i v i t y by a membrane e f f e c t expos-ing the c a t a l y t i c s i t e of the enzyme. A s i m i l a r e f f e c t i s observed l n RBCMF by freeze-thawlng or detergents (34). I 1 • 1 • 1 1 1 O 0.0J 0.04 0.05 0.03 0.10 032 [Co 2 *], mM F i g . 5 A T P a s e a c t i v i t y as a ( u n c t i o n of C a 2 - c o n c e n t r a t i o n i n the presence a n d absence of So m M K+. O r d i n a t e : t o t a l A T P a s e a c t i v i t y . T h e C a 1 ^ c o n c e n t r a t i o n g i v e n o n the abscissa is the c o n c e n -t r a t i o n a d d e d , w i t h o u t c o r r e c t i o n for b i n d i n g b y A T P or b y m e m b r a n e s . T h e zero C a 5 " p o i n t s m a y be s l i g h t l y h i g h , since E G T A (ethy)eneglycol-bis(/J-aniinoethyl ester)-A' , .V'-tetraacetic acid) was n o t a d d e d to chelate possible traces of e n d o g e n o u s C a J , f . T h e inset shows a W o o l f p l o t o f the d a t a , f r o m w h i c h A',„ values were o b t a i n e d . O t h e r c o n d i t i o n s are g i v e n i n METHODS ( 3 b ) . o.s 1.1 3 4 i 6 7 S • 10 30 110" C o 2 * concn. (M) Fig.6 E f f e c t of Na+ and K on ATPase a c t i v i t y i n hemoglobin f r e e red blood o e l l membranes prepared by f r e e z i n g and thawing (30). 19 2+ I n h i b i t o r s E f f e c t i n g Mg+Ca-ATPase And Active Ga Transport In Red Blood C e l l s In recent years numerous Investigators have sought a s p e c i f i c I n h i b i t o r of Mg+Ca-ATPase, as ouabain has been i n v a l -uable i n e l u c i d a t i n g the mechanism of Na,K-ATPase. A s p e c i f i c i n h i b i t o r of Mg+Ca-ATPase and a c t i v e calcium transport would be useful i n providing d i r e c t evidence that calcium transport and Mg+Ca-ATPase are intimately associated. A number of drugs have been tested on t h i s system. Among those without any e f f e c t on Mg+Ca-ATPase or calcium transport were ollgomycln and ouabain (1,11) which are s p e c i f i c Na,K-ATPase i n h i b i t o r s (37). Caffeine (lmg/ml) had no e f f e c t on active calcium transport i n resealed ghosts (12). A number of agents have been shown to be non-specific -4 i n h i b i t o r s of Mg+Ca-ATPase. Mersalyl (5.0 X 10 M)(11) and ethacrynic a c i d (10 M) (23) both I n h i b i t ATP dependent calcium transport l n resealed ghosts. However, these drugs have also been shown to equally I n h i b i t Mg+Ca-ATPase and Na,K-ATPase a c t i v i t y i n RBCMF i n a concentration range of 10~ to 10 M ( 5 ) . Ethacrynic a c i d and mersalyl are thought to I n h i b i t enzyme a c t -i v i t y by binding to protein s u l f h y d r y l groups (5). The lantha-nides have been found to be useful agents f o r studying calcium i n t e r a c t i o n s since they possess a s i m i l a r r a d i i to calcium but have much higher a f f i n i t i e s f o r anionic s i t e s due to a higher charge density (56). Holminlum and praesodynium i n h i b i t Mg+Ca-ATPase a c t i v i t y l n RBCMF and ac t i v e calcium transport i n —4 resealed ghosts, with an Icn of approximately 10 M (39). 20 However, these ions are non-specific as Na,K-ATPase and Mg-ATPase a c t i v i t i e s were also i n h i b i t e d . Lanthanum has been useful f o r e s t a b l i s h i n g the mechanism f o r calcium trans-port i n mitochondria ( 7 0 ) . Watson and Vincenzl ( 4 0 ) r e c e n t l y reported that the inorg-anic dye ruthenium red s e l e c t i v e l y i n h i b i t s Mg+Ca-ATPase a c t i v i t y In RBCMF. Ruthenium red has previously been shown to react with carboxyl and sulphate groups on mucopolysacchar-ides ( 4 1 ) . Table II i l l u s t r a t e s the e f f e c t of ruthenium red on the ATPases of RBCMF. Mg-ATPase and Na,K-ATPase were not s i g n i f i c a n t l y Inhibited by ruthenium red. However, both the a l k a l i dependent and the a l k a l i independent f r a c t i o n s of Mg+Ca-ATPase were almost completely i n h i b i t e d by 6 X 1 0 M ruthenium red. E f f e c t s Of Ruthenium Red On ATPases Of Erythrocyte Membranes (k0) (Values are expressed as ^ imoles of inorganic phosphate l i b e r a t e d per mg pr o t e i n i n 1 h.) Table II ATPase a c t i v i t y ATPases Ruthenium red concentration (M): 0 6*10 -6 6 « 1 0 -5 Mg+Ca-ATPase alkali-cation-dependent alkali-cation-independent Na,K-ATPase Mg-ATPase ~Q~7M 0.32 0.56 0.28 0.11 0.22 0.33 0.26 0.11 0733" 0.09 0 . 0 4 0.05 0.25 0.10 21 This study d i d not d i s t i n g u i s h between i n h i b i t i o n of high or low a f f i n i t y Mg+Ca4ATPase as 0.12mM calcium was used i n a l l studies. Considering the high concentration of calcium used, the low a f f i n i t y Mg+Ca-ATPase, at l e a s t , was i n h i b i t e d . The mechanism of a c t i o n of t h i s i n h i b i t o r also remains undet-ermined as the concentration of ATP, calcium and magnesium was not altered l n i t s presence. The s i t e of action of t h i s drug also remains unknown, as the studies were done on RBCMF. However, ruthenium red shows p o t e n t i a l as a tool f o r studying a c t i v e calcium transport, since t h i s drug has been shown to block a c t i v e calcium transport i n mitochondria (42). 2+ Bader (43) recently reported that Mn s p e c i f i c a l l y a c t i v -ated low a f f i n i t y Mg+Ca-ATPase, without a f f e c t i n g the high a f f i n i t y Mg+Ca-ATPase i n RBCMF. Bader also reported that low a f f i n i t y Mg+Ca-ATPase a c t i v i t y was s e l e c t i v e l y i n h i b i t e d by 10"3M methylhydroxylamlne and 5.0 X 10*5M C u 2 + (EGTA; C u 2 + at a r a t i o of 10:1). This was the f i r s t report of s e l e c t i v e l n h l b -2+ i t l o n of a Mg+Ca-ATPase. However, Cu i s a s u l f h y d r y l group reagent, which renders membranes permeable to cations. There-2+ fore t h i s i n h i b i t o r would not be useful to study a c t i v e Ca transport i n resealed ghosts or i n whole red blood c e l l s . Active Calcium Transport In Other Tissues Besides human erythrocytes,cultured He La c e l l s (44) and L c e l l s (45), ATP dependent calcium transport across the plasma membrane of other tissues has not been demonstrated. However, 22 a calcium or magnesium stimulated ATPase has been located l n the sarcolemma of s k e l e t a l muscle (46)% cardiac muscle and i n guinea pig ileum (48)• Hurwitz (48) suggests that ATP depend-ent active calcium transport may be important i n lowering the i n t r a c e l l u l a r concentration of calcium i n guinea pig ileum, due to the poorly developed sarcoplasmic reticulum i n t h i s t i s s u e . His hypothesis i s supported by the f a c t that the ileum r e l i e s heavily on e x t r a c e l l u l a r calcium f o r c o n t r a c t i l e a c t i v i t y . There i s some evidence that calcium extrusion from squid axon may be ATP dependent (17) but the Na-Ca exchange mechanism may be more important across t h i s plasma membrane. There i s some evidence that the Na-Ca exchange mechanism evident i n nerve, may also operate i n s k e l e t a l muscle (49), cardiac muscle (50) and smooth muscle ( 5 1 ) . The requirement of external sodium f o r calcium e f f l u x appears to be very s p e c i f i c , since external lithium, choline or potassium cannot substitute f o r sodium. I t has been estimated that two to three external sodium ions are exchanged f o r one Internal calcium ion by t h i s mechanism ( 1 7 ) . The d i r e c t i o n of the Na-Ca exchange mechanism can be reversed l n d i r e c t i o n l n nerves bathed i n solutions def-i c i e n t l n external sodium. Other features of the Na-Ca exchange mechanism i n cyanide poisoned squid axons are shown i n Table I I I , taken from Baker ( 1 7 ) . Cyanide was added i n t h i s preparation to release calcium from mitochondria and thus prevent i n t e r f e r -ence of calcium uptake by mitochondria when making calcium f l u x measurements. 23 Table I I I Features Of The Na-Ca Exchange Mechanism In Cyanide Poisoned Squid Axons(17) . 1. I t i s temperature s e n s i t i v e and has a ^ 0 of about three. 2. I t i s i n s e n s i t i v e to high concentrations of the cardiac glycoside ouabain. 3 . I t i s i n h i b i t e d by externally applied lanthanum Ions (80). 4 . I t i s very dependent on the i o n i c composition of the external medium. Removal of external calcium reduces the e f f l u x to a variable extent. Baker and Crawford (81); found no e f f e c t whereas Blaustein and Hodgkin (82) found that removal of external calcium reduced the e f f l u x to about half ( l e . 2 pmoles/cm sec to 1 pmole/cm^sec). In the absence of external calcium, complete replacement of sodium by lithium,potassium, choline or sugar causes a further reduction of e f f l u x to about one-twentieth of i t s o r i g i n a l value, 0.1 pmole/cm sec. Nerve and muscle c e l l s also maintain a low i n t r a c e l l u l a r calcium concentration by means of the sarcoplasmic reticulum and mitochondria (17*54) . Both of these structures can accum-ulate calcium by an a c t i v e ATP dependent system. Estimates o f f the calcium sequestering capacity of sarcoplasmic reticulum l n v i t r o suggests that t h i s membrane system i s able to lower the cytoplasmic calcium concentration below the In vivo l e v e l required f o r contraction (approx. 10~ 6M ) (54) . I t has also been shown that the speed of calcium accumulation by sarcoplasmic reticulum i s compatible with the time of tension decay i n muscle ( 5 2 ) . Calcium i s accumulated against an a c t i v i t y grad-i e n t by an a c t i v e transport system which i s coupled to a Mg+Ca-ATPase ( 5 3 ) . This enzyme has a low Km f o r calcium, (approx. - 7 10 M) and a requirement f o r magnesium, optimal at 5 . 0 mM. A number of models describing t h i s transport system have been reported ( 5 4 ) . Since t h i s transport system shares a number of 24 p r o p e r t i e s t o the Mg+Ca-ATPase l n human e r y t h r o c y t e s , a scheme taken from Martonosl (54) showing the molecular mechanism r e l a t i n g ATP h y d r o l y s i s to calc i u m t r a n s p o r t i s o u t l i n e d as f o l l o w s : 1. E+ATP s E-ATP 2. E-ATP+2Ca 3. E Ca - ATP Ca E .Ca - ATP Ca EoJ p +ADP ^ C a Outside Ca 4 . E*<0P' Ca E*+PA + 2Ca I n s i d e 5. E* „ w E E=enzyme, E*=the c o n f o r m a t i o n a l l y / a l t e r e a form of the c a r r i e r . I n t h i s scheme ATP binds t o the enzyme l n the absence of c a l c i u m (step 1 ) , which promotes the b i n d i n g of calc i u m (step 2 ) , The bound calc i u m then a c t i v a t e s the phosphate t r a n s f e r from ATP l e a d i n g t o the forma t i o n of a phosphoprotein in t e r m e d i a t e (step 3)» The c a r r i e r than undergoes a conformational change w i t h the t r a n s l o c a t i o n of calc i u m from the outer to the i n n e r s u r f a c e of the sarcoplasmic r e t i c u l u m , Release of calcium from t h i s low a f f i n i t y c a r r i e r (step 4 ) , f o l l o w e d by h y d r o l y s i s of the phosphoprotein,completes the c y c l e (step 5 ) . 25 METHODS AND MATERIALS Outdated c l t r a t e d human blood (0 p o s i t i v e ) was obtained from the Canadian Red Cross and s t o r e d f o r not more than two o weeks a t 5 C. Blood was used w i t h i n twenty days of c o l l e c t i o n . P r e p a r a t i o n Of Red Blood C e l l Ghosts Red blood c e l l (RBC) ghosts were prepared by a m o d i f i c a t -i o n of the procedure of stepwise hemolysis o u t l i n e d by S c h r l e r (57)• Whole blood (150 ml) was suspended l n one volume of 0 .155 M NaCl l n e i g h t 40 ml polypropylene c e n t r i f u g e tubes. The tubes o were c e n t r i f u g e d a t 4 C f o r ten minutes a t 2,000 X g l n a r e f -r i g e r a t e d c e n t r i f u g e (eg. I n t e r n a t i o n a l model, B-20, 870 angle head). The supernatant and white b u f f y l a y e r were removed by s u c t i o n . Considerable care was taken to remove the top b u f f y coat as completely as p o s s i b l e , d e s p i t e a c o n s i d e r a b l e l o s s of red c e l l s (approx. o n e - t h i r d ) . The red c e l l s were resuspended w i t h a g l a s s s t i r r i n g rod and washed again i n the same tubes w i t h t e n volumes of 0 .155 M NaCl w i t h subsequent removal of the supernatant and any remaining white c e l l s . For a summary of the p r e p a r a t i o n , l o a d i n g and assay procedures to be o u t l i n e d r e f e r to Table IV. The c e l l s were then p a r t i a l l y hemolyzed by suspension i n ten volumes of 0.08 M NaCl c o n t a i n i n g 1.0 mM EDTA (disodium s a l t ) and c e n t r i f u g e d a t 8,000 X g f o r ten minutes. Temperature 26 Table IV Pr e p a r a t i o n Of RBC Ghosts. Loading. And Assay Procedure I P r e p a r a t i o n : 1. Wash w i t h 0.155M NaCI (twice) 2. 0.08M NaCI + l.OmM EDTA 3. 0.06M NaCI + l.OmM EDTA 4. 0.04M NaCI + l.OmM EDTA 5 . 0.015M NaCI + 5.0mM TRIS-MALEATE, pH 7.1 I I Loading: 1.0ml ghosts + 3.0ml of s o l u t i o n c o n t a i n i n g ; 4.0mM Na2ATP, 4.0mM MgCl 2, lOmM THIS, pH7.1 and v a r i o u s concentrations of C a C l 2 I I I R e s e a l i n g : 1. Add 0.2ml of 2.b76M NaCI 2. Stand a t R.T., 10 min. 3. Wash w i t h 2.0mM MgCl 2, 0 . 1 2 5 M NaCI, and 20mM TRIS-MALEATE (twice) 4. Suspend to 3»0ml l n assay medium (as above) + ImM C a C l 9 27 o was maintained a t 2-4 C d u r i n g hemolysis procedures t o ensure th a t the ghosts d i d not r e s e a l Immediately a f t e r hemolysis ( 5 8 ) . EDTA was i n c l u d e d i n the hemolysis medium to remove endogenous d i v a l e n t metal c a t i o n s such as c a l c i u m and magnesium. C h e l a t -i n g agents have a l s o been found to be u s e f u l l n f a c i l i t a t i n g the removal of s o l u b l e enzymes and hemoglobin from the c e l l u l a r i n t e r i o r (59) without a f f e c t i n g r e s e a l i n g ( 5 8 ) . The ghosts were then suspended l n ten volumes of 0.06 M NaCl c o n t a i n i n g 1.0 mM EDTA and were c e n t r i f u g e d at 13,000 X g 0 f o r t e n minutes a t 2-4 C. At t h i s step the c e l l s l o s t a c o n s i d -e r a b l e amount of hemoglobin as evidenced by a very dark supernat-ant. A much higher c e n t r i f u g a l f o r c e i s r e q u i r e d a t t h i s step to s p i n down the red c e l l s as the c e l l s have become s w o l l e n and t h e r e f o r e have much l e s s d e n s i t y than the whole BBC. The ghosts were hemolyzed i n ten volumes of 0.04 M NaCl c o n t a i n i n g 1.0 mM EDTA and c e n t r i f u g e d a t 15 ,000 X g f o r ten minutes a t 2-4* 0 . The ghosts were f i n a l l y resuspended i n ten volumes of 0 . 0 5 M NaCl and 0.005 M T r l s - M a l e a t e (pH 7.1) and c e n t r i f u g e d a t 20,000 o X g f o r ten minutes a t 2-4 C. The p e l l e t a t t h i s stage was pink i n c o l o r . The p e l l e t was a l s o Viscous i n nature and could be poured i n t o a 40 ml polypropylene c e n t r i f u g e tubes. The s o l i d red button remaining i n the bottom of the c e n t r i f u g e tubes c o n s i s t s of c e l l s which would not hemolyze under these c o n d i t i o n s or type I I I ghosts (58) and was d i s c a r d e d . These ghosts were used w i t h i n f i f t e e n minutes i n the l o a d i n g s t e p . 28 Loading Procedure 1 .0 ml of ghosts (approx. 10 ghosts or 5*0 mg of membrane protein) was pipeted into a 15 ml polypropylene t e s t tube ( c a l i -brated ) containing 3 . 0 ml of solution,to give a f i n a l concen-t r a t i o n of 4 . 0 mM Na2ATP, 4 . 0 mM MgCl 2 and iO.O mM Tris-maleate (pH 7 . 1 ).Unless otherwise stated ,the concentration of calcium was varied i n t h i s medium. When required, monovalent cations and drugs were also Included. The tbes were allowed to stand f i v e to ten minutes to allow the Ions to e q u i l i b r l a t e before o r e s e a l l n g at 2-4 C ( 1 2 ) . Reseallng And Washing Procedure P a r t i a l l y hemolyzed red blood c e l l ghosts can r e t a i n t h e i r o r i g i n a l shape and permeability by r e s t o r a t i o n of l s o t o n i c i t y ( 2 0 , 2 1 ) . The RBC ghosts were resealed by the a d d i t i o n of 0 , 2 ml of 2 . 8 7 6 M NaCI s o l u t i o n . The tubes were well shaken and placed i n a water bath at room temperature f o r exactly ten min-utes. Restoration of l s o t o n i c i t y and the Increase i n temperature both f a c i l i t a t e r e s e l l i n g ( 5 8 ) . Ghosts not allowed to stand f o r as long as ten minutes at room temperature were found to take up l e s s calcium as measured by atomic absorption spectrophotom-etry since they did not r e s e a l as completely and calcium was l o s t during the washing. The tubes were returned to the i c e bath a f t e r ten minutes. 29 The resealed ghosts were washed with 5»5 ml of an i s o t o n i c s o l u t i o n containing 125.0 mM NaCl, 2.0 mM MgCl 2 and 20.0 mM Tris-Maleate (pH 7.1) at 5,000 X g f o r ten minutes at 2 - V c . The r e s u l t i n g p e l l e t forms a compact p e l l e t at t h i s stage. The resealed ghosts were washed once more i n 10.0 ml of the above s o l u t i o n . The c l e a r supernatant was aspirated o f f leaving approximately 2.5 ml of s o l u t i o n . In most experiments 0.1 ml of 30 mM C a C l 2 was added to the tubes to give a f i n a l concent-r a t i o n of 1C0 mM calcium. Required amounts of ruthenium red and other drugs were also added at t h i s stage. The resealed ghosts were f i n a l l y brought to a f i n a l volume of 3.0 ml l n the previously graduated t e s t tubes with the addit i o n of the i s o t o n i c washing s o l u t i o n . Incubation Procedures In a l l experiments the r e a c t i o n was started by Immersing 0 the tubes i n a shaking water bath at 37 C. A short delay of three minutes i n reaching the temperature of 37°C was i n e v i t -able i n these experiments as was evident l n the e f f l u x measure-ments ( F i g . 13) . Determination of ATPase A c t i v i t y - ATPase a c t i v i t y was determined by measuring the inorganic phosphate released from ATP by a modification of the method of Flske and SubbaRow (60,63). The procedure i s as follows: 30 The enzyme reaction was terminated by the addition of 1.0 ml of cold sillcotungstic acid (8.0$) in perchloric acid (1.2 M), usually after a thirty minute incubation period. The tubes were placed in ice, to minimize ATP hydrolysis, for at least one o hour. The tubes were then centrifuged at 20,000 X g at 0-4 C in a refrigerated centrifuge (eg. International model, B-20, 874 angle head). 3.0 ml of the supernatant was transfered into a second test tube (eg. 20 X 150 mm) containing 1.4 ml d i s t i l l e d water and kept i n ice. At zero time, 0 .4 ml of molybdate reagent was added followed immediately by the addition of 0.2 ml of amlnonaptholsulfonlc acid reagent and stirred on a vortex for fi f t e e n seconds. The color was allowed to develop for thirty minutes at room temperature. After thirty minutes the color was read in a spectrophotometer (eg. Coleman-Hitachi 124) at 660 nm against a d i s t i l l e d water reference i n a 1.0 cm light path cuvet. KH2P0^ (analytical reagent) was used as the primary standard. ATPase ac t i v i t y was found to be linear up to thirty minutes in resealed ghosts (Fig. 7 ) . Mg+Ca-ATPase act i v i t y was deter-mined by measuring the difference i n ATPase ac t i v i t y in the abs-ence and presence of calcium ln the loading medium. Since the ATPase activity in the absence of added calcium can not be estimated (eg. ghosts do not reseal under this condition) the ac t i v i t y ln the absence of added calcium was estimated by extrap-olation of the calcium activation curve to zero." calcium. Na,K-ATPase activity was determined by measuring the a c t i v i t y 31 i n ghosts resealed with NaCl (2.876 M) with the a d d i t i o n of external potassium (0-15.0 mM). Ouabain (0 . 1 mM) abolished the a c t i v i t y stimulated by external potassium. Na,K-ATPase and Mg+Ca-ATPase a c t i v i t y was reported as umoles of Pi released from ATP per mg of membrane protein per hour (umoles PJL mg'-^hr"1). Determination Of The V e l o c i t y Of Calcium Transport Calcium transport or e f f l u x was measured as the change i n c e l l u l a r calcium with time at 37°C. The incubation was termin-ated by the a d d i t i o n of 6.0 ml of an ice cold s o l u t i o n contain-ing NaCl (119 mM) and lanthanum (6.0mM). The tubes were immed-i a t e l y placed l n a ice bath. LaCl^ was included l n t h i s s o l u t i o n to displace l o o s e l y bound external calcium and to i n h i b i t the calcium transport system ( 8 3 ) . This procedure was Invoked i n order to obtain a more accurate determination of i n t r a c e l l u l a r calcium and calcium t i g h t l y bound i n the membrane. In p r a c t i c e , l i t t l e d i f f e r e n c e was found when the ghosts were washed with i c e cold NaCl s o l u t i o n (0 .155 M) alone. The tubes were centrifuged o f o r ten minutes at 5.000 X g at 2-4 C. The supernatant was rem-oved by a s p i r a t i o n and the p e l l e t was washed once more l n 6.0 ml of the same s o l u t i o n . Calcium was extracted from the p e l l e t by a s l i g h t modificat-ion of the wet ashing procedure reported by Sparrow et a l . ( 6 l ) . B r i e f l y , 1.0 ml of 3.0 M t r i c h l o r a c e t i c a c i d : g l a c i a l a c e t i c a c i d , 1:1, was added to d i s s o l v e the p e l l e t . The p e l l e t was 32 resuspended with a vortex mixer and the tubes were placed f o r f i f t e e n minutes i n a water bath at 70°C. A f t e r t h i s time, 2 .0 ml of d i s t i l l e d water was added. The tubes were mixed by vortex and were placed i n the water bath f o r ten minutes at 70°C. They were then allowed to cool to room temperature, allowing coagulation of the protein, and made up to a f i n a l volume of 5 . 0 ml with 3 0 . 0 mM LaCl-j (approx. 2 . 0 ml) l n a c a l i b r a t e d test tube. LaCl^ was added to prevent Interference from phosphates and Inorganic ions l n preparation f o r atomic absorption spectrophotometry. The p r e c i p i t a t e was removed by ce n t r i f u g a t i o n at 7.500 X g f o r ten minutes. The concentration of calcium i n the protein free supernatant was determined by measuring the absorption at the 4227 A l i n e from a hollow cathode calcium lamp on a Varian-Techtron atomic absorption spectrophotometer, model AA-5. A nitrous oxide-acetylene flame was used. A lamp current (hollow calcium cathode lamp) was set at 4 . 0 mA and the s l i t width at 125 p. Standard CaCO^ solutions were prepared from standard reference solutions obtained from Fisher S c i e n t i f i c Company. A standard curve obeying Beers law was obtained between 10-100 uM calcium. Protein Assay The concentration of protein was determined by the method of Lowry (62) f o r insoluble proteins. Bovine serum albumin (Sigma, 3X r e c r y s t a l l i z e d ) was used as a standard. Standard curves were l i n e a r from 50 to 250 ug of protein when read at 33 500 nm. 1 . 0 ml of the hemoglobin fr e e ghost preparation was found to contain 4 . 4 mg of membrane protein. Hemoglobin free ghosts were prepared by washing the ghosts three a d d i t i o n a l times i n 0.015 M NaCI and 0.005mM T r i s -Maleate (pH 7 . 1 ) f o r ten minutes at 2 0 , 0 0 0 X g. Temperature o was cont r o l l e d at 2 - 4 C during these washings. No measurable change In the s p e c i f i c a c t i v i t y of Mg+Ca-ATPase between hemog-l o b i n containing and hemoglobin free ghosts was obtained. Washing Procedure During the course of t h i s work, i t was found that the method f o r washing the polypropylene test tubes was very c r i t i c a l f o r obtaining maximal ATPase a c t i v i t i e s . Therefore the following washing procedure was adopted. The te s t tubes were soaked overnight i n tap water containing 0.5% Liqulnox detergent. The tubes were rinsed with tap water and soaked l n 10$ HNO^ f o r at least, one hour i n 5 . 0 mM EDTA (tetrasodium s a l t ) . The tubes were well rinsed with tap water and then rinsed ten times with glass d i s t i l l e d water and allowed to dry l n a i r . A loss of low a f f i n i t y Mg+Ca-ATPase a c t i v i t y r e s u l t s l n test tubes soaked i n 0.5% Liqulnox alone, presumably due to a bu i l d up of detergent i n the tubes. 0 34 RESULTS AND DISCUSSION Properties Of The Red Blood C e l l Ghost Preparation Of fundamental importance to t h i s study was the prepar-a t i o n of a red blood c e l l ghost population which could be made 'leaky' and then resealed to regain the o r i g i n a l cation permeability c h a r a c t e r i s t i c s of the whole c e l l . I t was also important that the a c t i v i t y of the various ATPase systems were not destroyed during the preparation. In previous studies, Schatzmann et a l . (12) and others (26 ,27) prepared ghosts by a method Involving a one step hemolysis, followed by r e s e a l l n g . This procedure did not allow a complete removal of endogenous enzymes which can synthesize ATP ( 7 0 ) . Further-more, endogenous di v a l e n t metal cations and ATP were not completely removed by t h i s method. Therefore i n order to reduce the endogenous cations and ATP concentrations i n these preparations, the c e l l s were prelncubated at 37°C f o r 17 hr l n glucose free medium (12) or were incubated i n the pres-ence of iodoacetate (26). These two methods are undesirable since some destruction of the plasma membrane must occur as evidenced by hemolysis during incubation. In t h i s study, therefore, a modification of the procedure of stepwise hemolysis as outlined by Schrler (57) was adopted as described l n the Methods. EDTA (dlsodium s a l t ) (1.0 mM) 35 was Included i n the washing s o l u t i o n s to Insure removal of 2+ 2 + Mg , Ca , ATP and s o l u b l e enzymes (74). Care, however, had to be taken i n using EDTA i n membrane p r e p a r a t i o n s , s i n c e under c e r t a i n c o n d i t i o n s EDTA s o l u b l l i z e s membrane bound p r o t -e i n s ( 6 7 ) . Furthermore, t o t a l removal of membrane d i v a l e n t 2 + 2+ c a t i o n s , Mg and Ca , could r e s u l t i n a ghost p o p u l a t i o n which may not r e s e a l , as these ions are thought to be s t r u c t -u r a l components of the plasma membrane ( 6 7 ) . Under the c o n d i t i o n s used i n t h i s study (see Methods), the p r e p a r i o n remained r e l a t i v e l y impermeable to d i v a l e n t and monovalent c a t i o n s and r e t a i n e d ATPase a c t i v i t y . E v i d -ence supporting the v i a b i l i t y of t h i s p r e p a r a t i o n f o r t r a n s -p o r t s t u d i e s was as f o l l o w s : 1. Under the l i g h t microscope, the ghosts regained t h e i r o r i g i n a l biconcave d i s c shape a f t e r r e s e a l l n g ( m a g n i f i c a t i o n 500X). 2. The s p e c i f i c a c t i v i t i e s obtained f o r Na,K-ATPase and Mg+Ca-ATPase were comparable to those r e p o r t e d i n other s t u d i e s (40 , 6 5 ) . The Na, K-ATPase and Mg+Ca-ATPase a c t i v i t i e s were shown t o be asymmetrically s t i m u l a t e d by e x t e r n a l potas-sium and I n t e r n a l c a l c i u m , r e s p e c t i v e l y . Such c h a r a c t e r -i s t i c s can o n l y be demonstrated l n c e l l s w i t h an I n t a c t plasma membrane. 3 . A l o s s of c e l l u l a r c a l c i u m 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 was demonstrated ( F i g . 1 3 ) , whereas no l o s s of c e l l -u l a r c a l c i u m occurs i n f r o z e n r e d blood c e l l membrane fragments used i n our s t u d i e s . I n a d d i t i o n , no change l n the c e l l u l a r c o n c e n t r a t i o n of magnesium occurred l n the r e s e a l e d ghosts, 36 i n d i c a t i n g that tne membrane i s also not f r e e l y permeable to magnesium. 4.No v i s u a l l o s s of hemoglobin occurred during washing subsequent to r e s e a l i n g . Ion Requirements For Reseallng Maizels et al. ( 7 5 )»in an e a r l y study .reported that calcium was an e s s e n t i a l requirement f o r maintaining red blood c e l l membrane permeability..Recently, however, Bramely et al. ( 7 6 ) claimed magnesium could substitute f o r calcium i n r e s e a l l n g , though l e s s e f f e c t i v e l y . However, i n the present study, i n c l u s i o n of at l e a s t 0 .05 mM calcium i n the loading medium was found to be an absolute requirement f o r r e s e a l i n g . This was demonstrated i n an experiment where ghosts were loaded i n a medium containing no added calcium, followed by the normal procedure f o r r e s e a l i n g . Subsequent suspension of these ghosts i n a medium containing 1 .0 mM calcium r e s u l t e d l n an uptake of c e l l u l a r calcium approximately equivalent to the values norm-s'- 1 a l l y obtained with 'leaky g h o s t s ' (I.e. 0 . 0 6 jimoles Ca mg ). o On incubation at 37 C, loss of c e l l u l a r calcium was observed. This r e s u l t c l e a r l y i n dicates that the ghosts had not resealed i n the loading medium devoid of calcium and suspension l n a medium containing calcium allowed entry of calcium i n t o the c e l l i n t e r i o r and subsequent r e s e a l i n g . The presence of 4 . 0 mM magnesium was thus not s u f f i c i e n t f o r complete r e s e a l i n g i n these preparations. The di f f e r e n c e between these r e s u l t s and 37 the r e s u l t s r e p o r t e d by Bramely et a l . i?6) may be due to the use of EDTA i n our s t u d i e s , which reduces endogenous calcium to very low l e v e l s . Na.K-ATPase A c t i v i t y I n Resealed Red Blood C e l l Ghosts I n v e s t i g a t i o n of Na.K-ATPase a c t i v i t y was undertaken to determine whether the i n t e g r i t y of t h i s t r a n s p o r t enzyme had been preserved. I n ghosts loaded w i t h 0 . 5 mM c a l c i u m and 0 . 1 mM EGTA, l i n e a r i t y l n ATP s p l i t t i n g was observed f o r t h i r t y minutes ( F i g . 7 ) . D e v i a t i o n from l i n e a r i t y occurred a f t e r t h i r t y minutes, presumably due t o a d e p l e t i o n of ATP. Calcium was i n c l u d e d i n the l o a d i n g medium to f a c i l i t a t e r e s e a l l n g of the ghosts, and d i d not appear t o i n h i b i t the Na,K-ATPase a c t i v i t y . T h i r t y minutes was t h e r e f o r e chosen as the i n c u b a t i o n time i n subsequent Na,K-ATPase experiments. F i g u r e 7 shows th a t 0 . 2 mM ouabain, when a p p l i e d e x t e r n a l l y , reduced t o t a l ATPase a c t i v i t y from 0.525 t o 0 .36 ^nmoles P i mg^hr"* 1. The d i f f e r e n c e accounts f o r the Na.K-ATPase present. I n an experiment t o determine whether Na.K-ATPase could be a c t i v a t e d a s y m m e t r i c a l l y , the c o n c e n t r a t i o n of potassium was v a r i e d e x t e r n a l l y i n gnosts r e s e a l e d w i t h NaCI ( F i g . b). Maximal s t i m u l a t i o n of ATPase a c t i v i t y occured a t 8 . 0 mM e x t e r n a l potassium. I n the presence of 0 . 1 MM e x t e r n a l ouabain the ATP s p l i t t i n g s t i m u l a t e d by e x t e r n a l potassium was a b o l -i s h e d . Since the t o t a l ATPase a c t i v i t y of the ghosts l n the p r e s -ence of 0 . 1 mM ouabain was equal t o the a c t i v i t y l n the absence 38 1 J I I I 0 10 20 30 40 Time (min) Figure 7. Time course of ATPase a c t i v i t y in the absence of external ouabain (O) and i n the presence of 0.2 mM external ouabain . (®).The concentration of calcium in the loading medium v/as 0.5 mM. 39 of added potassium, no Na, K-ATPase a c t i v i t y was present i n ghosts r e s e a l e d w i t h NaCI, unless e x t e r n a l potassium was added. In f u t u r e determinations of Mg+Ca-ATPase a c t i v i t y , i n t e r f e r e n c e from ATP s p l i t t i n g due t o Na.K-ATPase l n c e l l s r e s e a l e d w i t h NaCI would not be expected, s i n c e e x t e r n a l potassium was not used. A d d i t i o n of 15.0 mM potassium i n the l o a d i n g medium, i n the absence of e x t e r n a l potassium, d i d not i n c r e a s e the b a s a l r a t e of ATPase a c t i v i t y (0.3 jimoles mg" 1hr" 1), i n d i c a t -i n g t h a t only e x t e r n a l potassium a c t i v a t e s Na, K-ATPase. A d d i t i o n of 0.1 mM ouabain i n the l o a d i n g medium d i d not I n h i b i t s t i m u l a t i o n due to e x t e r n a l potassium. These two f i n d i n g s demonstrate th a t both oubain and potassium have e x t e r n a l binding s i t e s , i n agreement w i t h r e s u l t s r e p o r t e d by previous workers (65). The r e s u l t a l s o supports the view th a t the c e l l s have r e s e a l e d , s i n c e i n t e r n a l potassium does not have access to the o u t s i d e of the membrane. In the presence of 1.0 mM e x t e r n a l calcium ( P i g . 8 ) , the a c t i v a t i o n of Na,K-ATPase by e x t e r n a l potassium was e s s e n t i a l l y unchanged. The s p e c i f i c a c t i v i t y of Na,K-ATPase (at 10.0 mM e x t e r n a l potassium) was approximately 0.35 umoles P i mg""*hr~* i n the presence or absence of 1.0 mM e x t e r n a l calcium. Therefore e x t e r n a l calcium does not a f f e c t the s p e c i f i c a c t i v i t y of Na, K-ATPase. As a comparison, the s p e c i f i c a c t i v i t y of Na,K-ATPase obtained l n a p r e p a r a t i o n of f r o z e n RBCMF by Watson et a l . ( 4 0 ) was 0.28 pmoles P i mg~*hr" 1. The maximal value obtained l n t h i s study was 0.J5 /imoles P i mg" 1hr" 1. 40 2.0 4.0 6.0 8.0 10.0 12.0 14.0 [K+] (mM) Figure 8. Activation of Na,K-ATPase a c t i v i t y by external potassium i n the presence of 1.0 mM external calcium (•Kzero external calcium (O) and 0.1 mM external ouabain (#).The concentration of calcium i n the loading medium was 0.5 mM.Other conditions were standard.Each point represents the mean of two experiments. 41 Mg+Ca-ATPase A c t i v i t y In Resealed Red Blood C e l l Ghosts V a r i a t i o n of the concentration of calcium l n the loading medium led to a marked increase i n ATPase a c t i v i t y ( F i g . 9 ) . Two peaks were obtained, possibly representing two calcium stimulated Mg+Ca-ATPases. The a c t i v i t y was corrected f o r Mg-ATPase and non-enzymatic hydrolysis as l n the Methods. The f i n d i n g of two Mg+Ca-ATPases i n resealed ghosts was i n agreement with e a r l i e r studies on frozen RBCMF, where the high a f f i n i t y Mg+Ca-ATPase was optimally activated by approx-imately 10*^ to 10~^M calcium, and a low a f f i n i t y Mg+Ca-ATPase -5 k with optimal a c t i v a t i o n at approximately 10 ^ to lO'^M calcium. In resealed ghosts (Fig. 9 ) , high a f f i n i t y Mg+Ca-ATPase and low a f f i n i t y Mg+Ca-ATPase were maximally stimulated by 0.25mM and 5»0mM calcium l n the loading medium, r e s p e c t i v e l y . The marked differences i n a f f i n i t y f o r calcium between RBCMF and resealed ghosts le d to the conclusion that the concentrations of calcium i n the loading medium were much higher (^ 10 f o l d ) than the actual free I n t r a c e l l u l a r concentrations of calcium at the beginning of the incubation. For Instance, i n ghosts loaded with 5«0mM calcium, there may not be an e f f i c i e n t 1:1 loading r a t i o between the loading medium and the c e l l I n t e r i o r ( 3 8 ) . Furthermore, some of the loaded calcium may have been l o s t during the r e s e a l l n g procedure, which required an incub-a t i o n f o r ten min. at room temperature Isee Methods). A consid-erable amount of calcium may be chelated by binding s i t e s that are not a v a i l a b l e l n RBCMF, e s p e c i a l l y since such s i t e s may 42 Figure 9. Effect of varying the concentration of calcium i n the loading medium on Mg+Ca-ATPase activity.Each point represents the mean of three experiments(+ standard e r r o r s ) . 43 have been depleted by EDTA. Therefore the co n c e n t r a t i o n s of c a l c i u m i n d i c a t e d on the a b c i s s a are not t r u e i n t r a c e l l u l a r c o n c e n t r a t i o n s of calcium, but represent a r e l a t i v 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 of calcium added l n the l o a d i n g medium. An Eadie p l o t (77) of the calcium a c t i v a t i o n data, r e v e a l e d two s t r a i g h t l i n e s ( P i g . 10). The Vmax and the calcium d i s s o c -i a t i o n constant f o r the low a f f i n i t y Mg+Ca-iiTPase were c a l c u l -ated to be 0.690 ^imoles PI mg ''"hr"1 and 1 .23 mM r e s p e c t i v e l y . A Vmax and Ka of 0.340 p i o l e s PI mg" 1hr" 1 and 1.26 X 10"^M were obtained f o r the high a f f i n i t y Mg+Ca-ATPase. Values reported by Bader (78) f o r the s p e c i f i c a c t i v i t i e s of Mg+Ca-ATPases In RBCMF were 0 . 5 and 0.8 jimoles P i mg" 1hr" 1 f o r the h i g h and low a f f i n i t y Mg+Ca-ATPase, r e s p e c t i v e l y . The s l i g h t l y lower values r e p o r t e d here f o r the r e s e a l e d ghosts may be due t o assaying a t s l i g h t l y l e s s than o p t i m a l c o n d i t i o n s . There are p r e l i m i n a r y i n d i c a t i o n s t h a t an Increase i n the c o n c e n t r a t i o n of magnesium i n the l o a d i n g medium w i l l f u r t h e r enhance Mg+Ca-ATPase a c t i v i t y i n t h i s p r e p a r a t i o n . The E f f e c t Of E x t e r n a l D i v a l e n t Cations On ATPase A c t i v i t y P r e v i o u s l y , Schatzmann and V l n c e n z l (12) reported t h a t o n l y I n t r a c e l l u l a r calcium s t i m u l a t e d ATPase a c t i v i t y l n r e s e a l e d ghosts, w h i l e e x t e r n a l c a l c i u m had no s i g n i f i c a n t e f f e c t . However, e x t e r n a l d i v a l e n t c a t i o n s were found to s i g n -i f i c a n t l y s t i m u l a t e ATPase l n t h i s ghost p r e p a r a t i o n ( F i g . 11). The o r d i n a t e represents the i n c r e a s e l n ATPase a c t i v i t i e s over 4 4 Figure 10. Eadie plot of calcium a c t i v a t i o n of Mg+Ca-ATPases i n ghosts. 45 ( Ul| 6 l U LfJ S9[0Uirf)A 46 Figure 11. The effect of external divalent cations on ATPase a c t i v i t y . The ghosts were loaded with 0.5 mM Calcium and 0.4 mM EGTA. Other conditions were standard. Ca (O), Mg (•). 4? basal l e v e l s obtained l n the absence of added external d i v a l -ent cations. The maximal increase i n ATPase a c t i v i t y occured at 3.0 mM external calcium, with an increase over basal a c t i v -i t y of 0.140 /imoles Pi mg'-'-hr"1. External magnesium was only about one-half as e f f e c t i v e as external calcium l n increasing ATPase a c t i v i t y , with a maximal increase of 0.06 pjnoles Pi mg~^ h r " 1 . Since increasing the external potassium concentration ( i n the presence of 0.1 mM ouabain) did not a f f e c t Mg+Ca-ATPase a c t i v i t y , stimulation of ATPase a c t i v i t y appears to require d i v a l e n t metal cations. To determine i f magnesium and calcium were entering the c e l l or were stimulating an external ATPase such as Na,K-ATPase or Mg+Ca-ATPase, the concentration of calcium was varied i n the loading medium of resealed ghosts suspended i n external medium containing zero calcium and 1.0 mM calcium ( F i g . 12). Since there i s no apparent s h i f t i n the p o s i t i o n of the calcium a c t i v a t i o n curve l n the presence of 1.0 mM calcium, external calcium does not have access to the c e l l u l a r I n t e r i o r . The stim-u l a t i o n must therefore be due to calcium a c t i n g on the external surface of the membrane. Since external calcium did not a f f e c t the Na,K-ATPase a c t i v i t y as shown previously ( F i g . 8) the extra ATPase a c t i v i t y seen l n the presence of 1.0 mM external calcium i s not due to stimulation of Na.K-ATPase at an external binding s i t e . The p o s s i b i l i t y that some ghosts were present which did not r e s e a l and were being activated i s u n l i k e l y because of the high 4.0 3.0 2.0 pCa gure 12. Effect of varying the concentration of calcium in the loading medium on Mg+Ca-ATPase act iv i ty in the absence of external calcium (#) and in the presence of 1.0 mM external calcium (OJ.0.4 mM E6TA was included in the loading medium.Other conditions were standard.Each point represents the mean of at least two experiments. 49 concentrations of divalent cations required f o r t h i s a c t i v a t i o n , r e l a t i v e to the concentrations required to stimulate membrane fragments. I t was speculated that calcium and magnesium may be replac-ing endogenous magnesium and calcium removed from the membrane by EDTA. The binding of calcium or magnesium may restore the conformation Mg+Ca-ATPase, r e s u l t i n g i n an increased a c t i v i t y . > The E f f e c t Of Internal Calcium On Calcium E f f l u x Q u a l i t a t i v e studies i n d i c a t i n g that the v e l o c i t y of calcium e f f l u x i s dependent on the Internal concentration of calcium have been previously reported l n resealed ghosts (30) and i n whole red blood c e l l s ( 7 ) . Schatzmann and Bossi ( 3 0 ) , however, were unable to demonstrate a c o r r e l a t i o n between a c t i v a t i o n of Mg+Ca-ATPase and the v e l o c i t y of calcium e f f l u x i n t h e i r preparations of resealed ghosts. In t h i s study ghosts were loaded with d i f f e r e n t concent-rations of calcium and resealed as i n the Methods. The loss o of c e l l u l a r calcium was determined as a f u n c t i o n of time at 37 C. (Pig. 13)• In ghosts loaded over the concentration range of 0 . 1 to 5 . 0 mM calcium, a rapid l o s s of c e l l u l a r calcium was apparent. In most of the ghosts an equilibrium l e v e l of c e l l -u l a r calcium was reached within ten minutes. The concentration of calcium l n the ghosts at time zero was d i r e c t l y proportional 50 0 10 20 30 Time (min) Figure 13. Changes in the concentration of c e l l u l a r calcium with time. Each point represents the mean of two to three experiments (+ standard errors) .except where no bars are shown. The concentration of calcium in the loading medium was 0.5 mM ( • ) , 1.0 mM (A), 2.0 mM (O) . 3.0 mM (@) and 5.0 mM ( A ) . 51 to the c o n c e n t r a t i o n of calc i u m l n the l o a d i n g medium. T h i s 2+ -1 i s shown l n f i g u r e 14 where a p l o t of jimoles Ca mg a g a i n s t the c o n c e n t r a t i o n of calcium i n the l o a d i n g medium y i e l d e d a s t r a i g h t l i n e . Evidence was obtained showing t h a t the l o s s of c e l l u l a r calcium was due to a c t i v e c a l c i u m t r a n s p o r t . For i n s t a n c e , i f ATP was not i n c l u d e d l n the l o a d i n g medium, a l o s s of c e l l u l a r calcium over time d i d not occur, i n d i c a t i n g t h a t the ca l c i u m l o s s was an ATP dependent process. This f i n d i n g a l s o shows t h a t endogenous ATP was removed d u r i n g the p r e p a r a t i o n of the ghosts. Furthermore, a l o s s of c e l l u l a r c a l c i u m occurred i n ghosts loaded w i t h l e s s than 1.0 mM calcium, showing t h a t i n t r a c e l l u l a r c a l c i u m was t r a n s p o r t e d a g a i n s t an e l e c t r o c h e m i c a l p o t e n t i a l . As the c o n c e n t r a t i o n of calcium i n the l o a d i n g medium was i n c r e a s e d from 0.1 mM t o 5 . 0 mM, there i s an Increase l n the steepness of the slopes of the curves, i n d i c a t i n g t h a t the v e l o c i t y of t r a n s p o r t i s i n c r e a s i n g as the c o n c e n t r a t i o n of calcium I s in c r e a s e d i n the l o a d i n g medium. The e f f l u x curves obtained conform t o a f i r s t order process, i n t h a t f o r each curve the v e l o c i t y of e f f l u x decreases e x p o n e n t i o n a l l y w i t h time. Thus a p l o t of l o g ca l c i u m content from the data i n f i g u r e 13 a g a i n s t time y i e l d e d s t r a i g h t l i n e s . From these s t r a i g h t l i n e s e x t r a p o l a t e d p o i n t s f o r the f i r s t ten minutes were obtained. I n i t i a l v e l o c i t i e s could then be a c c u r a t e l y determined by r e p l o t t i n g the data on a p l o t of c o n c e n t r a t i o n of c a l c i u m a g a i n s t time. The f i r s t t h r e e minutes were not used l n the c a l c u l a t i o n s of i n i t i a l v e l o c i t i e s s i n c e t h i s time 52 Figure 14. Relationship between ce l lu lar calcium and calcium in the loading medium.Each point represents the mean of three experimental determinations (+ standard errors). 5 3 represents the time required f o r the tubes to warm up from 0°C to 37°C (see F i g . 13). A p l o t of the i n i t i a l v e l o c i t i e s of calcium e f f l u x against the concentration of calcium i n the loading medium yielded a slgmoldal curve ( F i g . 15» open c i r c l e s ) . Increasing the concen-t r a t i o n of calcium from 0.1 mM to 1.0 mM d i d not r e s u l t i n a large increase l n the v e l o c i t y of calcium e f f l u x . A more marked stimulation of calcium e f f l u x occurred i n ghosts loaded with concentrations of calcium greater than 1.0 mM. In contrast to the a c t i v a t i o n of Mg+Ca-ATPase a c t i v i t y two peaks were not obtained suggesting that there i s only one calcium transport system present. Figure 15 indicates that calcium stimulation of both ATPase a c t i v i t y and calcium e f f l u x are p a r a l l e l i n the calcium concentration range of 1.0 to 5«0 mM, where the low a f f i n i t y Mg+Ca-ATPase i s thought to operate. A comparison of the curves i n t h i s region Indicates a stoichiometry of two calcium ions transported per molecule of ATP hydrolyzed. At calcium concentrations of l e s s that 1.0 mM, where the high a f f i n i t y Mg+Ca-ATPase i s thought to operate, the stoichiometry measured appears to be l e s s than one, and the shapes of the calcium e f f l u x and the ATPase calcium a c t i v a t i o n curves do not coincide. This suggests that at low calcium concentrations there Is an ATPase s p l i t t i n g (high a f f i n i t y Mg+Ca-ATPase) which i s not coupled to calcium transport. The stoichiometry of two (Ca:ATP) contrasts to the prev-i o u s l y reported value of 0.77 (12), but agrees with the s t o i c h i o -metry found f o r caloium transport l n muscle sarcoplasmic 54 pCa Figure 15. Comparison of the velocity of active calcium transport and Mg+Ca-ATPase act iv i ty as a function of calcium in the loading mediumjhe Mg+Ca-ATPase data represents: the mean of three experiments and the standard errors are smaller than the circ les drawn.The calcium transport data represent the mean of three determinations (_+ standard errors).except where no bars are shown.where points represent the mean of duplicate determinations. 55 reticulum (5*0 • I t seems more reasonable that the calcium pump l n red blood c e l l s would also have a high stoichiometry, since l e s s energy would have to be expended i n extruding a given amount of I n t r a c e l l u l a r calcium. The e f f l u x data was plotted according to H i l l (68) l n order to Investigate the meaning of the sigmoldal shape of the calcium e f f l u x curve ( F i g . 1 6 ) . At l e a s t two s t r a i g h t l i n e s were obtained with H i l l c o e f f i c i e n t s (n) of approxim-a t e l y 0 . 3 9 f o r concentrations of calcium l e s s than 1.0 mM and 1.0 f o r ghosts loaded with greater than 1 .0 mM calcium. This change i n n values may mean that the calcium transport system has a cooperative component. Binding of one molecule of calcium may increase the a f f i n i t y of the transport system f o r the binding of another molecule of calcium, at a thresh-old value of approximately 1 .0 mM calcium In the loading medium. Changes i n n values often r e f l e c t a change i n the conformation or" s p a c i a l arrangement of enzyme subunlts. A sub-unit structure f o r Mg+Ca-aTPase l n red blood c e l l s has not been reported. However, Kyte (71) r e c e n t l y reported that the Na,>K-pump i n the plasma membrane of re n a l cortex appears to have a subunlt structure. An a l t e r n a t i v e explanation i s that the break l n the slope of f i g u r e 16 may be due to the presence of two calcium activated calcium transport systems. The r e s u l t s presented above do not r u l e out t h i s p o s s i b i l i t y . However, r e s u l t s obtained using ruthenium red (to be discussed l a t e r ) make t h i s explanation l e s s l i k e l y . -4.0 •3.0 -2.0 Log [Ca2+](M) Figure 16. Hill plot of calcium activation of calcium efflux in ghosts.v is the velocity of calcium efflux and V is the maximum velocity of this efflux,as determined from a Lineweaver-Burk plot. 5 7 The E f f e c t Of Ruthenium Red On Mg+Ca-ATPase A c t i v i t i e s In the previous section, a s i m i l a r i t y between the shape of the calcium a c t i v a t i o n curves f o r the low a f f i n i t y Mg+Ca-ATPase and the calcium transport system was noted. This a s s o c i a t i o n was fu r t h e r Investigated using ruthenium red as a t o o l . Watson et a l . (40) reported that ruthenium red, an Inorganic dye used l n st a i n i n g mucopolysaccharides, s e l e c t i v e l y i n h i b i t e d Mg+Ca-ATPase a c t i v i t y l n RBCMF. No d i s t i n c t i o n , however, was made between I n h i b i t i o n of high or low a f f i n i t y Mg+Ca-ATPase a c t i v i t y or whether calcium transport was also i n h i b i t e d . In t h i s study ruthenium red, added externally, was found to i n h i b i t Mg+Ca-ATPase i n a dose dependent manner i n ghosts loaded with 3 . 0 and 5 . 0 mM calcium.* ( F i g . 1 7 ) . In the ghosts loaded with 3 . 0 and 5*0 mM calcium, the i n h i b i t i o n curves were found to be p a r a l l e l . Since the I ^ Q value of ruthenium red at both concentrations of Internal calcium was 0 .2 mM, the mechanism of I n h i b i t i o n would not be expected to be competitive with respect to calcium. This f i n d i n g i s not su r p r i s i n g l n view of the f a c t s which suggest that ruthenium red I n h i b i t s by binding to anionic s i t e s on the outside of membranes. Calcium has previously been shown here and elsewhere (12) to ac t i v a t e Mg+Ca-ATPase on the Internal surface of the membrane. Evidence supporting an external binding s i t e f o r ruthenium red are as follows: (1) A preincubation time was not required. Ghosts preIn-cubated i n 0 . 2 mM ruthenium red f o r 0 , 10, 20 and 30 minutes at 58 Figure 17. Effect of ruthenium red in the external medium on Mg+Ca-ATPase activity.The concentration of ruthenium red was varied in ghosts loaded with o.5 mM calcium ( D ^ . O mM calcium (O) and 5.0 mM calcium (•). 59 2-1°C. i n h i b i t e d low a f f i n i t y Mg+Ca-ATPase by 50#. I f ruth-enium red was penetrating Into the i n t e r i o r of the ghosts,and then i n h i b i t i n g Mg+Ca-ATPase, greater i n h i b i t i o n would be exp-ected f o r ghosts preincubated f o r longer periods of time, (2) Ruthenium red does not penetrate through the plasma membrane of Intact c e l l s as evidenced from electron microscopy ( 7 2 ) . (3) Penetration of ruthenium red through the plasma membrane of resealed ghosts would be very slow since ruthen-ium red possesses a hexavalent p o s i t i v e charge (40). Ruthenium red, therefore, probably exerts I t s I n h i b i t i o n by binding to mucopolysaccharides on the outside surface of the ghost. Binding at these s i t e s may induce an unfavourable conformational perturbation of the membrane bound Mg+Ca-ATPase, leading to a reduction i n the c a t a l y t i c e f f i c i e n c y of t h i s enzyme. Binding to an a l l o s t e r i c s i t e would also favour a non-competitive type of i n h i b i t i o n by ruthenium red. In ghosts resealed with 0 .5 mM calclum y 0 . 2 mM external ruthenium red d i d not a f f e c t Mg+Ca-ATPase a c t i v i t y ( Fig. 17). In order to explain t h i s analomous r e s u l t , the concentration of calcium was varied l n the loading medium, the ghosts were resealed and suspended i n a medium containing 0,2mM ruthenium red (Fig.18). Ruthenium red had e s s e n t i a l l y no e f f e c t on Mg+Ca-ATPase a c t i v i t y at concentrations of calcium lower than l.OmM, where the high a f f i n i t y Mg+Ca-ATPase i s considered to operate. However, the low a f f i n i t y Mg+Ca-ATPase was markedly i n h i b i t e d by ruthenium red. The % i n h i b i t i o n was dependent on 60 CD to CU o +-> o <C 01 CO ro ex. t— I « + 4.0 3.0 2.0 pCa Figure 18. The effect of ruthenium red on the activation of Mg+Ca-ATPase. The calcium concentration in the loading medium was varied in the absence of ruthenium red ( •) and in the presence of 0.2 mM ruthenium red (O)in the external medium.Where bars are shown the data represent the mean of three experiments (+ standard errors).Other points represent the mean of duplicate determinations. 61 the concentration of calcium l n the loading medium. For example, ghosts loaded with 2 . 0 mM calcium were i n h i b i t e d by £1% whereas ghosts loaded with 3 . 0 mM calcium were i n h i b i t e d by WIJ> (see F i g . 2 1 ) . The E f f e c t Of Ruthenium Red On Calcium Transport Since ruthenium red was shown to s e l e c t i v e l y I n h i b i t low a f f i n i t y Mg+Ca-ATPase, t h i s dye showed promise as a t o o l f o r i n v e s t i g a t i n g whether or not a Mg+Ca-ATPase i s associated with calcium transport. In ghosts loaded with 3 . 0 mM calcium, external ruthenium red i n h i b i t e d the v e l o c i t y of active calcium transport i n a dose dependent manner (F i g . 1 9 ) . The I ^ Q value was estimated to be 0 . 2 mM, which i s the same value estimated f o r the i n h i b i t i o n of the low a f f i n i t y Mg+Ca-ATPase (Fig. 1 7 ) . Thus both the a c t i v e calcium transport system and low a f f i n i t y Mg+Ca-ATPase have a s i m i l a r a f f i n i t y f o r ruthenium red. The e f f e c t o f 0 . 2 mM external ruthenium red on the a c t i v a -t i o n of a c t i v e calcium transport by Internal calcium was also determined (Fig. 2 0 ) . The pattern of ruthenium red i n h i b i t i o n of calcium transport was e s s e n t i a l l y the same as that on low a f f i n i t y Mg+Ca-ATPase. The % i n h i b i t i o n by ruthenium red increased with an increase i n the concentration of calcium from 1.0 mM to 3 . 0 mM and then leveled o f f ( F i g . 2 1 ) . Thus the degree of I n h i b i t i o n by 0 .2 mM ruthenium red depends on the concentra-t i o n of calcium l n the loading medium (see F i g . 2 0 ) . There are 6 2 [Ruthenium Red] (mM) Figure 19. Effect of ruthenium red in the external medium on the velocity of active calcium transport in ghosts loaded with 3.0 mM calcium. 63 1.4 1.2 1.0 .8 .4 4.0 3.0 2.0 pCa Figure 20. The effect of ruthenium red on the velocity of active calcium transport.The calcium concentration in the loading medium was varied in the absence of ruthenium red ( •) and in the presence of 0-2 mM ruthenium red (O).The data indicated by the open c i rc les represent the mean of two determinations. 64 Figure 21. The effect of ruthenium red (0.2 mM) in the external medium on Mg+Ca-ATPase activity (O) and active calcium transport (•) as a function of the concentration of calcium in tne loading medium. 65 two possible explanations f o r t h i s behaviour. I t Is poss-i b l e that there are more binding s i t e s a v a i l a b l e f o r ruthen-ium red at higher concentrations of i n t e r n a l calcium, since the volume of the red c e l l i s regulated by the Internal concen-t r a t i o n of calcium (10). A more l i k e l y explanation becomes apparent upon reexamination of figur e 16. It was previously noted that there was a change l n the H i l l c o e f f i c i e n t s from .39 to 1.0 as the concentration of calcium was varied i n the loading medium. I t was suggested that t h i s change may be due to a change l n the subunit structure of the enzyme. I f low a f f i n i t y Mg+Ca-ATPase and the calcium transport system are indeed associated, the same explanation would hold here too. Thus the binding of ruthenium red to external mucopolysaccha-rides could prevent the a b i l i t y of calcium to a f f e c t an i n t e r -a ction of the transport enzyme subunlts or an Increase i n cooperativity. Ruthenium red could thus bind to and s t a b i l i z e the l e s s a c t i v e form of the transport system,so that only a ce r t a i n maximal v e l o c i t y of calcium e f f l u x and Mg+Ca-ATPase a c t i v i t y would be obtainable. Support of t h i s hypothesis awaits i s o l a t i o n of the low a f f i n i t y Mg+Ca-ATPase and i d e n t i f i c a t i o n of a subunit structure. Preliminary experiments also indicate that i n h i b i t i o n by ruthenium red may be temperature dependent. For instance, 0.4 .mM external ruthenium red Increased the l a g time f o r calcium e f f l u x i n ghosts loaded with 3.0 mM calcium from three to four minutes. Furthermore, membranes must be preincubated at 2 to 4 C. i n order f o r i n h i b i t i o n to be obtained. The possib-i l i t y thus exists that ruthenium red can only bind to one 66 form of the transport enzyme. This form may be the more e f f i c i e n t transport form, induced by high concentrations of i n t r a c e l l u l a r calcium and possibly by low temperatures. The E f f e c t Of Drugs (Quinine. Quinldine. and Propranolol) On Mg+Ca-ATPase Quinldine has been shown to i n h i b i t the binding of calcium i n i s o l a t e d sarcoplasmic reticulum (79)• The p o s s i b i l i t y was considered that t h i s drug may have e f f e c t s on other systems c o n t r o l l i n g the d i s t r i b u t i o n of calcium, such as active calcium transport across the plasma membrane. The human erythrocyte was used as a model system to determine whether quinldine or quinine affected low a f f i n i t y Mg+Ca-ATPase i n resealed ghosts or RBCMF. Both quinldine and quinine were found to be l n e f f e c -- 5 - 3 t l v e over a concentration range of 10 M to 10 M i n both prep-arations. Interference by these drugs on the inorganic phosp-hate assay (63) at concentrations greater than 10 M^ was observed. Propranolol was also without e f f e c t on Mg+Ca-ATPase a c t i v i t y i n resealed ghosts l n the concentration range of 10 J to 10 M. The e f f e c t of these drugs on ac t i v e calcium transport was not determined. 67 The E f f e c t Of Manganese On Mg+Ca-ATPase A c t i v i t y Bader ( 4 3 ) recently reported that manganese can substitute f o r calcium l n a c t i v a t i n g low a f f i n i t y Mg+Ca-ATPase i n RBCMF. Manganese was reported to act i v a t e low a f f i n i t y Mg+Ca-ATPase with the same potency as calcium and to have no e f f e c t on high a f f i n i t y Mg+Ca-ATPase. Therefore manganese was considered as a p o t e n t i a l t o o l f o r d i f f e r e n t i a t i n g enzyme function. The concentration of manganese was varied l n the loading medium In the presence or absence of 1.0 mM external calcium (Fig. 2 2 ) . Calcium was not Included i n the loading medium i n these experiments. I n h i b i t i o n of ATPase a c t i v i t y was observed at concentrations of manganese les s than 1.0 mM. The a c t i v i t y returned to the control l e v e l at 5 « 0 mM manganese. The concent-rations of manganese was also varied i n the absence of 1.0 mM external calcium to ensure that the s l i g h t a c t i v a t i o n occurring between 3 « 0 and 5.0 mM manganese was due to manganese and not external calcium leaking i n t o the ghosts. Since t h e i r was no appreciable change i n the two r e s u l t s , manganese may be able to substltue f o r calcium i n re s e a l i n g the ghosts, however, i t s e f f e c t on a c t i v a t i n g Mg+Ca-ATPase seems n e g l i g i b l e . Manganese did not ac t i v a t e calcium activated ATPase i n RBCMF. It i s possible that contamination of the manganese with calcium could account f o r the diffe r e n c e In r e s u l t s obtained here and those reported by Bader ( 4 3 ) . 68 Figure 22. Effect of varying the concentration of manganese in the loading medium on ATPases act iv i ty in the absence of external calcium (O) and in the presence of 1.0 mM external cal cium(#) .Each point represents a single experiment. CONCLUSIONS 69 A suit a b l e preparation of resealed erythrocyte ghosts was developed i n t h i s study. The use of 1.0 mM EDTA i n the washing procedure resulted i n a ghost population e s s e n t i a l l y free of endogenous ATP and dival e n t cations. This method of preparation has advantages over previous methods used f o r preparing ATP free ghosts since long preincubation periods i n metabolically depleted media or iodoacetate treatment of the whole c e l l s p r i o r to preparation of the ghosts i s not required. Ghosts pre-pared by t h i s method required the addition of calcium (> .05 mM) to the loading medium i n order to r e s e a l . Magnesium was unable to substitute f o r calcium l n t h i s respect. The use of EDTA d i d not r e s u l t i n removal or denaturation of membrane ATPase(s) since s p e c i f i c a c t i v i t i e s comparable to those reported i n the l i t e r a t u r e were obtained f o r both Na,K-ATPase and Mg+Ca-ATPase. Further evidence was given i n d i c a t i n g that the ghosts had resealed (p 35) and that Na,K-ATPase and Mg+Ca-ATPase could be asymmetrically stimulated by cations. In contrast to a report by Schatzmann and Vincenzi (12) i t was found that external d i v a l e n t cations (Mg and Ca) further increased Mg+Ca-ATPase a c t i v i t y i n t h i s ghost preparation. The mechanism of a c t i o n was a t t r i b u t e d to the replacement of membrane di v a l e n t cations removed by EDTA (not used by Schatzmann and V i n c e n z i ) . Presumably calcium or magnesium are required to maintain the most favourable conformation of t h i s enzyme. I t was of Interest that the s p e c i f i c a c t i v i t y of Na.K-ATPase was 70 not in c r e a s e d by these e x t e r n a l d i v a l e n t metal c a t i o n s . This study was the f i r s t to show the. presence of a high and low a f f -i n i t y Mg+Ca-ATPase l n r e s e a l e d human e r y t h r o c y t e ghosts. Previous s t u d i e s had shown a high and low a f f i n i t y Mg+Ca-ATPase l n RBCMF. However, the co n c e n t r a t i o n s of calcium i n the l o a d -i n g medium were more than ten times higher than the concentrat-ions of ca l c i u m r e q u i r e d to achieve optimal a c t i v a t i o n l n RBCMF. I t was suggested t h a t the co n c e n t r a t i o n s of calcium i n the l o a d i n g medium do not rep r e s e n t the t r u e i n t r a c e l l u l a r c o n c e n t r a t i o n of f r e e calcium, f o r reasons o u t l i n e d on p 42 and ref e r e n c e 30. The present s t u d i e s provide s t r o n g evidence t h a t o n l y the h y d r o l y s i s of ATP due t o low a f f i n i t y Mg+Ca-ATPase i s coup-l e d t o calcium t r a n s p o r t . A s t o i c h i o m e t r y of two was found i n c o n t r a s t to a previous estimate by Schatzmann and V l n c e n z l (12), who reported a s t o i c h i o m e t r y of 0.77. However, t h e i r study (12) was done w i t h only one i n t e r n a l calcium c o n c e n t r a t i o n (1.0 mM). I n a d d i t i o n , the present study has shown t h a t a s t o i c h i o m e t r y of l e s s than one i s obtained a t low co n c e n t r a t i o n s of calcium where the h i g h a f f i n i t y Mg+Ca-ATPase i s a l s o f u n c t -i o n i n g . The r e s u l t s f u r t h e r i n d i c a t e t h a t the o p e r a t i o n of the calcium pump i s a cooperative process; the pump becoming more e f f i c i e n t as the c o n c e n t r a t i o n of calc i u m i s i n c r e a s e d . This mechanism may be very important to the s u r v i v a l of the c e l l i n s i t u a t i o n s i n which the i n t e r n a l c a l c i u m c o n c e n t r a t i o n becomes h i g h . Ruthenium red may prove to be a u s e f u l t o o l f o r i n v e s t -i g a t i n g the cooperative aspects of the mechanism of calcium 71 transport, since t h i s dye i n h i b i t s the more e f f i c i e n t form of the transport system. The f i n d i n g that the i n h i b i t i o n by ruthenium red increased with increasing concentrations of calcium l n resealed ghosts was not found i n RBCMF where ruthenium red Inhibited equally at a l l concentrations of calcium (76). This f i n d i n g Implies that i n RBCMF only one state of the enzyme can e x i s t , i n contrast to the enzyme l n the resealed ghost. The f i n d i n g that low a f f i n i t y Mg+Ca-ATPase may be associated with calcium transport was at f i r s t somewhat su r p r i s i n g since one might expect that the Mg+Ca-ATPase with the highest a f f i n i t y f o r calcium might be best suited f o r maintaining a low i n t r a c e l l u l a r calcium. However the low a f f i n i t y Mg+Ca-ATPase possesses a higher s p e c i f i c a c t i v i t y than the high a f f i n i t y Mg+Ca-ATPase and may possess a threshold s u f f i c i e n t l y low to maintain a low I n t r a c e l l u l a r concentration of calcium. In conditions of low pH and low oxygen tension which can occur l n the spleen (84,85). e x t r a c e l l u l a r calcium can enter the red blood c e l l . In such cases the low a f f i n i t y Mg+Ca-ATPase may be well adapted to handling high i n t e r n a l calcium conditions. The function of the high a f f i n i t y Mg+Ca-ATPase was not determined l n t h i s study, but may be associated with the a c t l n -l i k e protein (spectrin) i s o l a t e d from the Inner aspect of the RBC ( 5 5 ) . Quinine, quinldine and propranolol were found to have no e f f e c t on Mg+Ca-ATPase l n resealed ghosts when added either externally or I n t e r n a l l y . These drugs are thought to be membrane s t a b i l i z i n g agents and i t was anticipated that rtg+Ca-/iTPase a c t i v i t y might be blocked i f these drugs act by expansion of the membrane and by producing unfavourable conformational perturbations i n the membrane microenvironment. 73 BIBLIOGRAPHY 1. Vincenzi, F., C e l l u l a r Mechanisms f o r Calcium Transfer and Homeostasis, pp 135-1^9, E d i t o r s , Nichols, G., and Wasserman, R.H., Academic Press, New York, 1971. 2. Rasmussen, H., C e l l communication, calcium ion, and c y c l i c adenosine monophosphate. Science, 170. 404-412, 1971. 3. Dunham, E.T. and Glynn, I.M., Adenosine triphosphatase a c t i v i t y and the active movements of a l k a l i metal ions. J . P h y s i o l . . 1£6, 274-^93,1961. 4. Epstein, F.H. and Whlttam, R., The mode of i n h i b i t i o n by calcium of cell-membrane adenosine triphosphatase a c t i v i t y . Biochem. J . , 22, 232-238, 1966. 5. Dixon, M. and Webb, E., Enzymes, p 422, Academic Press, New York, 1964. 6. Bianchl, CP., C e l l Calcium, p 37, Butterworth and Co. Ltd., London, 1968. 7. Romero, P.J. and Whlttam, R., The c o n t r o l by i n t e r n a l calcium of membrane permeability to sodium and potassium. J . Physiol., 214, 481-507, 1971. 8. Blum, R.M. and Hoffman, J.F., Ca induced K transport l n human red c e l l s ; L o c a l i z a t i o n of the Ca-sensitive s i t e to the Inside of the membrane. Biochem. Biophys. Res. Commun., 46, 1146-1152, 1972. 9. weed, R.I., L a c e l l e , P.L., and M e r r i l l , E.W., Metabolic dependence of red c e l l deformabllity. J . C l i n . Invest. i*8, 795 - 0 - 0 9 . 1969. 10. Palek, J . , Curby, W.A., and L l o n e t t i , F.J., E f f e c t s of calcium and adenosine triphosphate on the volume of human red c e l l ghosts. Am. J . Physiol., 220. 19-26, 1971. 2+ 11. Schatzmann, H.J., ATP-dependent Ca -extrusion from human red c e l l s . Experlentla, 22, 364-365, 1966. 12. Schatzmann, H.J. and Vincenzi, F.F., Calcium movements across the membrane of human red c e l l s . J . Physiol., 221. 369-395. 1969. 13. Manery, J.F., E f f e c t s of Ca Ions on membranes. Fed. P r o c , 2£, 1804-1810, 1966„ 74 Blaustein, M.P. and Hodgkln, A.L., The e f f e c t of cyanide on the e f f l u x of calcium squid axons. J . P h y s i o l . , 2 0 0 . . 497-527. 1969. Baker, P.F.,and Blaustein, M.P., The influence of calcium on sodium e f f l u x l n squid axons. J . P h y s i o l . , 200, 431-4-38, 1969. Lehnlnger, A.L., Mitochondria and calcium ion transport. Biochem. J . , 112, 129-138, 1970. Baker, P.F., Transport and metabolism of calcium ions l n nerves. Progress i n Biophysics, pp 177-223, Pergamon Press, Toronto, 1972. I n e s i , G., Annual Review of Biophysics and Bioenglneering, 1 , pp 1 9 1 - 2 1 0 , 1972. Wilbrandt, W., Die permeabilitat der roten blutorperchen f u r einfache zucker. Pfug. Arch, ges P h y s i o l . , 241. 302-311, 1939. Szelesky, M., Manyal, S., and Straub, F.B., Uber den mechanismus der osmotishen hamolyse. Acta P h y s i o l . Hung., 2, (suppl.) 571-584, 1952. T e o r e l l , T., Permeability properties of erythrocyte ghosts. J . Gen. P h y s i o l . , 21* 669-701, 1952. Gardos, G., Akkumulation der kaiiumlonen durch menschliche blutkorperchen. Acta P h y s i o l . Acad. S c i . Hung., 6, 191-2ia, 1954. Schatzmann, H.J., Herzglykoside a i s hemmstoffe f u r den aktiven kalium-und natriumtransport durch die eryth-rocetenmembran.. Helv.- P h y s i o l . Pharmacol. Acta, 1 1 , 346-354, 1953. Skou, J.C., The Influence of some cations on an adenosine triphosphatase from p e r i p h e r a l nerves. Bxochim. Biophys. Acta, 23. 394-401, 1957. Vincenzi, F.F. and Schatzmann, H.J., Some properties of Ca-actlvated ATPase i n human red c e l l membranes. Helv. P h y s i o l . Acta, 25_, C R 2 3 3 - 2 3 4 , I967. Lee, K.S. and Shin, B.C., Studies on the a c t i v e transport of calcium l n human red c e l l s . J . Gen. P h y s i o l . , i i t , 713-729, 1969. Olson, E.J. and Cazort, R.J., Active calcium and strontium transport i n human erythrocyte ghosts. J . Gen. P h y s i o l . , i l , 311-315. 1969. 75 28. Hoffman, J.P., The red c e l l membrane and the transport of sodium and potassium. Am. J . Med., 41, 666-680, 1966. 2 9 . Watson, E.L., Vincenzl, F.F. and Davis, P.W., Nucleotides as substrates of Ca-ATPase and Na,K-ATPase l n Isolated red c e l l membranes. L i f e S c i . , 10, 1399-1404, 1971. 2+ 2+ 3 0 . Schatzmann, H.J. and Rossi, G.L., (Ca +Hg )-activated membrane ATPases i n human red c e l l s and t h e i r possible r e l a t i o n s to cation transport. Biochim. Biophys. Acta, 241, 379-392, 1971. 31. Wins, P. and Sc h o f f e n l e l s , E., Studies on red c_ell ghost ATPase systems: Properties of (Mg + + Ca 2 ) dependent ATPase. Biochim. Biophys. Acta, 120, 341-349, 1966. 2+ 3 2 . Horton, C.R., Cole, W.H., and Bader, H., Depressed (Ca )-transport ATPase i n c y s t i c f i b r o s i s erythrocytes. Biochem. Biophys. Res. Commun., 40, 505-509, 1970. 2+ 33. Wolf, H.U., Studies on a Ca -dependent ATPase of human erythrocyte membranes. Biochim. Biophys. Acta, 266, 361-375. 1972. 34. Wins, P., The i n t e r a c t i o n of red c e l l membrane ATPase with calcium. Arch. Int. P h y s i o l . Biochim., 77, 245-250, 1969. 35. Mourad, N. and Parks, J r . R.E., Erythrocyte nucleoside diphosphoklnase. J . B i o l . Chem., 24l, 271-278, 1966. 3 6 . Bond, G.H. and Green, J.W., E f f e c t s of monovalent cations on the (Mg 2 ++Ca' i +)-dependent ATPase of the red c e l l membrane. Biochim. Biophys. Acta, 241, 393-398, 1971. 37. Wheeler, K.P. and Whittam, R., S t r u c t u r a l and enzymatic aspects of the hydrolysis of ATP by membranes of kidney cortex and erythrocytes. Biochem. J . , 93. 349-363, 1964. 38. Porzig, H., Calcium e f f l u x i n human erythrocyte ghosts. J . Mem. B i o l . , 2, 324-340, 1970. 39. Schatzmann. +H.J. and Tschbold, M., The lanthanldes Ho^ + and P r J as I n h i b i t o r s of calcium transport l n human red c e l l s . Experientla, 2£, 59-61, 1971. 40. Watson, E.L., Vincenzl, F.F. and Davis, P.W., Ca 2*-activated membrane ATPase : Selective i n h i b i t i o n by ruthenium red. Biochim. Biophys. J i c t a , 249. 606-6iU, i 9 7 i . 76 41. Gustafson, G.T. and Pike, E., Histochemlcal a p p l i c a t i o n of ruthenium red i n the study of mast c e l l u l t r a -structure. Acta Pathol. Microbiol Scand., 69, 393-403, 1967. 42. Moore, C.L., S p e c i f i c i n h i b i t i o n of mitochondrial C a 2 + transport by ruthenium red. Biochem. Biophys. Res. Commun., 42, 298-305, 1971. 43. Bader, H., Separation of (High-Ca 2 +) and (low-Ca 2*)-ATPase In human erythrocytes. #70, 5th Int. Congress Pharmacol. Abs.Volunt.Papers,San Francisco, 19?2. 44. Borle, A.B., Ki n e t i c analysis of calcium movements i n HeLa c e l l cultures: II.calcium e f f l u x . J . Gen. Phy s i o l . , 52t 57-69, 1969. 45. Lamb, J.F. and Lindsay, R., E f f e c t of Na, metabolic i n h i b -i t o r s and ATP on calcium movements i n L c e l l s . J . P hysiol., 218. 691-708, 1971. 46. Ferdman, D.L., Glmmelielkh, N.G., and Dyadyusha, G.P., Enzyme a c t i v i t y of the sarcolemma membrane of rabbit s k e l e t a l muscles. Biokhlmiya, ^4, 507-510, 1968. 47. Dletze, G. and Hepp, K., Calcium stimulated ATPase of cardiac sarcolemma. Biochem. Biophys. Res. Commun., 44, 1041-1049, 1971. 48. Hurwltz, L., F l t z p a t r l c k , D.F., Bebbas, G., and Landon, E.J., L o c a l i z a t i o n of the calcium pump l n smooth muscle. Science, 179. 384-386, 1973. 4 9 . Cosmos, E. and Harris, E.J., In v i t r o studies of the gain and exchange of calcium i n fr o g s k e l e t a l muscle. J . Gen. Physiol., 44, 1121-1130. 1961. 50. Reuter, H. and S e i t z , N., The dependence of calcium e f f l u x from cardiac muscle on temperature and external ion composition. J . Physiol., 1^1, 451-470, 1968. 51. Goodford, P.J., The calcium content of the smooth muscle of guinea-pig taenia c o l i . J . Physiol., 192. 145-157, 1967. 52. Ohnishl, T. and Ebashi, S., Spectrophotometrical measure-ment of instantaneous calcium binding of the relax -ing f a c t o r of muscle. J . Biochem., £4, 506-511, I 9 6 3 . 53* Hasselbach, W., Relaxing f a c t o r and the rel a x a t i o n of muscle. Prog. Biophys. Mol. B i o l . . 14, 167-222, 1964. 77 54. Martonosl , A., M e t a b o l i c Pathways, V o l . V I. Academic Pr e s s , New York, pp 317-346, 1972. 55. Ohnlshl.T., E x t r a c t i o n of a c t l n - a n d a y o s l n - l l k e p r o t e i n s from the e r y t h r o c y t e membrane. J . Biochem. Tokyo, £2, 307-308, 1962. 56. L e t t v i n , J.Y. and P i c k a r d , W.F., A theory of pa s s i v e Ion f l u x through axon membranes. Nature, 202, 1338-1341, 1964. 57. S h r l e r , S.L., ATP s y n t h e s i s I n human e r y t h r o c y t e membranes. Biochim. Biophys. A c t a , l ^ i . 591-598, 1967. 58. Bodeman, H. and Passow, H., F a c t o r s c o n t r o l l i n g the r e s e a l l n g of the membrane of human e r y t h r o c y t e ghosts a f t e r hypotonic hemolysis. J . Mem. B i o l . , 8, 1-26, 1972. 59. Bramley, T.A, and Coleman, R., E f f e c t s of i n c l u s i o n of Ca2+,Mg , EDTA, or EGTA d u r i n g the p r e p a r a t i o n of e r y t h r o c y t e ghosts by hem o l y s i s . Biochim. Biophys. A c t a , 2 3 0 , 219-228, 1972. 6 0 . F i s k e , C H . and Subba Row, Y.J., The c o l o r i m e t r i c d e t e r -m i n a t i o n of phosphorus. J . B i o l . Chem. 66, 375-400, 1925. 61. Sparrow, M.P. and Johnstone, 3.M., A r a p i d micromethod f o r e x t r a c t i o n of Ca and Hg from t i s s u e . B iochim. Biophys. A c t a , £0, 426-427, 1964. 62. Lowry, O.H., Rosenbrough, N.J., F a r r , A.L. and R a n d a l l , R . J . , P r o t e i n measurement w i t h the f o l l n phenol reagent. J . B i o l Chem., 1<£, 265-275, 1951. 63. R o u f o g a l l s , B.D., De t e r m i n a t i o n of I n o r g a n i c phosphate i n the presence of l n t e r f e r r l n g amines and n o n l o n l c d e t e r g e n t s . A n a l . Biochem., 44, 325-328, 1971. 64. Baker, P.F., Hodgkln, A.L. and Ridgway, E.B., D e p o l a r i z a t i o n and c a l c i u m e n t r y I n sq u i d axons. J . P h y s i o l . , 218, 709-755, 1971. 65. Whlttam, R., The asymmetrical s t i m u l a t i o n of a membrane adenosine t r i p h o s p h a t a s e i n r e l a t i o n to a c t i v e c a t i o n t r a n s p o r t . Biochem. J . , 84, 110-118, 1962. 66. Green, D.E., Membrane p r o t e i n s : A p e r s p e c t i v e . Annal. N. Y. Acad. S c i . , 150-172, 1972. 78 67. Reynolds, J.A., Are Inorganic cations e s s e n t i a l Tor the s t a b i l i t y of b i o l o g i c a l membranes. Annal.N. Y. Acad. S c i . , 12£, 75-85. 1972. 68. H i l l , A.V., The combinations of haemoglobins with oxygen and carbon dioxide. Biochem. J . , 2* 471-460, 1913. 69. A z z i , A. and Chance, B., The "energized state" of mito-chondria: l i f e t i m e and ATP equivalence. Biochim. Biophys. Acta, 182, 141-151, 1969. 70. Spencer, T. and Bygrave, P.A., The Influence of lanthanum on calcium-stimulated ATP t r a n s l o c a t i o n i n r a t l i v e r mitochondria. FEBS Lett e r s , Zb, 225-227, 1972. 71.. Kyte, J . , P u r i f i c a t i o n of the sodium-and potassium-dependent adenosine triphosphatase from canine renal medulla. J . B i o l . Chem., 1£, 4157-4165, 1971. 72. Luft, J.H., Ruthenium red and v i o l e t . I . Chemistry p u r i f i c a t i o n , methods of use f o r electron microscopy and mechanism of a c t i o n . Anat. R e c , 17JL, 347-368, 1971. 73. Isaacson, A. and Sandow, A., quinine and caffeine e f f e c t s on^5ca movements i n f r o g s a r t o r l u s muscle. J . Gen. Physiol., £0, 2109-2128, 1967. 74. Dodge, J.T., M i t c h e l l , C , and Hanahan, D.D., The prepar-a t i o n and chemical c h a r a c t e r i s t i c s of hemoglobin free ghosts of human erythrocytes. Arch. Biochem. Biophys., 100. 114-130, 1963. 75. Bolingbroke, V. and Malzeis, M., Calcium ions and the permeability of human erythrocytes. J . Physiol., 142, 563-585. 1959. 76. Roufogalis, B.D. and Sato, L., unpublished observations. 77* Eadie, G.S., The i n h i b i t i o n of cholinesterase by physostlg-mine and prostigmine. J . B i o l . Chem., 146. 85-93. 1942. 78. Bader, H., Two. (Ca^*)-activated ATPases i n human erythro-cytes. Fed. P r o c , JO, 545. 1971. 79* Schatzmann. H.J., Ca-activated membrane ATPase i n human red c e l l s and Its possible r o l e In active calcium transport. Protlde Of The B i o l o g i c a l F l u i d s , Vol. PP 251-255, Amsterdam, E l s v i e r , 1967. 80. van Breeman, C. ;and de Weer, P., Lanthanum i n h i b i t i o n of ^ C a e f f l u x from squid giant axon. Nature, 2_26, 760-761, 1970. 81. Baker, P.F. and Crawford, A.C, Sodium-dependent transport of magnesium Ions l n giant squid axons of l o l l g o f o r b e s i . J . Physiol., 216. 38P, 1971. 7 9 B l a u s t e i n , M.P. and Hodgkln, A.L., The e f f e c t of cyanide on the e f f l u x of calcium from squid axons. J . P h y s i o l . , 400, 497 - 5 2 7 . 1 9 6 9 . van Breeman, C., Blockade of membrane calcium fluxes by lanthanum i n r e l a t i o n to vascular smooth muscle cont-r a c t i l i t y . Arch. Int. Phys. Biochlmle, 7_2» 7 1 0-7i6, 1 9 6 9 . Weed, R.I., The Importance of erythrocyte d e f o r m a b l l l t y . Am. J . Med., 4£, 147 - 1 5 0 , 1 9 7 0 . L a c e l l e , P.L., A l t e r a t i o n of erythrocyte membrane deform-a b l l i t y i n stored blood. Transfusion, 238-245, 1 9 6 9 . 80 APPENDIX 81 Table A Effect of internal calcium on the velocity of calcium efflux. r - i -1 -1 -log [GaJ i n the mnolesCa mg; hr loading medium (M) mean + SE n 4.0 .279 1 3.6 .291 . 0 4 2 2 3.3 .337 .027 3 3.0 .427 .031 3 2.7 . 7 4 2 .072 3 2.5 1.035 .021 3 2.3 1.15 .076 3 Table B Effect of internal calcium on the velocity of Mg+Ca-ATPase. -log fCaJ l n the umoles PI mp; hr -loading medium (M) mean + SE n 4.0 .296 .005 3 3.6 .333 . 0 0 7 3 3.3 .316 .006 3 3.0 .350 .007 3 2.7 . 4 1 1 . 0 0 4 3 2.52 .4926 .020 4 2.39 .5525 1 2.3 .577 . 0 1 4 3 82 Table C E f f e c t of external ruthenium red on the v e l o c i t y of calcium transport. [Ca] i n the [Ruthenium Red] umoles Ca^mg'^hr--loadlng medium (mM) (mM) 3.0 0 1.074, 1.012, 1.02 3.0 .1 .610, .635 3.0 .2 .475. .565 3.0 .4 .341, .385 Table D Changes i n the calcium concentration of resealed ghosts with time. 2+ —1 [Ca] i n the Time umoles Ca me; loading medium (mM) (min.) mean + SE n 0.1 0 .443 1 0.1 5 .223 1 0.1 10 .176 1 0.1 20 .202 1 0 .25 2 .455 .001 2 0 .25 4 .330 1 0 .25 6 .261 1 0 .25 8 .197 .004 2 Table D (cont'd) [Ca) I n the Time umoles n 2+ -: Ca mg l o a d i n g medium (mM) (min.) mean + SE n 0.5 0 .474 .006 4 0.5 2 .432 .045 2 0.5 3 .366 .025 2 0.5 4 .364 0.5 5 .252 .002 2 0.5 6 .258 .042 2 0.5 7 .184 0.5 8 .188 0.5 10 .152 .016 3 0.5 20 .136 1 1.0 0 .585 .027 4 1.0 2 .398 1 1.0 3 .398 .022 2 1.0 5 .269 .026 3 1.0 6 .239 1 1.0 7 .193 1 1.0 10 .173 .002 2 1.0 20 .215 .011 2 2.0 0 .807 .007 3 2.0 3 .6965 .014 2 2.0 4 .621 .039 2 2.0 5 .454 1 1 Table D (cont'd) [Ca] i n the Time umoles Ca^mg*"1 loading medium (mM) (min.) mean + SE n 2.0 6 .412 .039 3 2 .0 7 .307 - 1 2 . 0 8 .273 1 2 . 0 10 .229 .003 3 3 . 0 0 1.017 ' .009 3 3 . 0 2 .98 1 3 . 0 3 .797 1 3 . 0 5 .624 .075 3 3 . 0 7 .284 1 3 . 0 10 .202 .025 3 3 . 0 20 .612 .007 3 5 . 0 0 1.425 .036 3 5 . 0 2 1.44 5 . 0 3 1 .445. 5 . 0 5 1-19. .089 3 5 . 0 7 0.930 1 5 . 0 10 .489 . 0 3 ^ 2 5 . 0 20 .296 1 5 . 0 30 .250 85 Table E E f f e c t of ruthenium red ( 0 . 2 mM) on a c t i v a t i o n of Mg+Ca-ATPase i n the loading medium. [Ca] i n the {Ruthenium Red"] umoles PI mg'^hr""1 loading medium (mM) (mM) mean + SE n 0.23 0 . 2 .301 .023 3 .5 0 . 2 .321 .010 3 1.0 0 . 2 .356 .012 3 3 . 0 1.0 .301 .009 3 5 . 0 1.0 .3076 .025 3 Table P E f f e c t of varying the concentration of ruthenium red on Mg+Ca-ATPase a c t i v i t y . ECa] i n the [Ruthenium Red] umoles P i mg'^hr"1 loading medium (mM) (mM) mean + SE n 0 . 5 0 .3166 .006 3 0 . 5 .2 .321 .011 3 3 . 0 0 .4926 .020 3 3 . 0 . 0 5 .391 1 3 . 0 .10 .352 1 Table P (cont'd) 86 [Ca} loac i n the l i n g medium (mM) [Ruthenium Red! U (mM) J uraoles mean + P i mg' SE • i h r - 1 n 3.0 .20 .301 .009 3 3.0 .40 .210 1 5.0 0 .577 .014 3 5.0 .05 .501 .026 3 5.0 .1 .406 .023 3 5.0 .2 .307 .025 3 5.0 .4 .232 .011 3 Table G E f f e c t of 0.2 mM e x t e r n a l ruthenium r e d on the a c t i v a t i o n of c a l c i u m t r a n s p o r t by i n t e r n a l c a l c i u m . [Calcium) l n the [Ruthenium Red"] umoles Ca 2 +mg" 1hr" 1 l o a d i n g medium (1.0 mM) (mM) 1.0 0.® .425, .440 2.0 0.2 .479, .466 3.0 0.2 .475, .565 5.0 0.2 .430, .540 Table H 87 E f f e c t of varying the concentration of calcium i n the loading medium l n the presence and absence of external calcium. - a l l conditions are standard except 0 . 4 mM EGTA was included i n the loading medium. -l o g jCa] l n the [Ca] i n the umoles Pi rng'-^hr loading medium (M) external medium (mM) 9 . 0 1.0 .310,.318 3.52 1.0 .313,.325 3 .30 1.0 . 3 6 0 , .342 3.125 1.0 . 3 4 0 , .346 3 . 0 1.0 .349,.367 2.699 1.0 .400..406 2.523 1.0 .462,.473 2.30 1.0 .586,.574 2.125 1.0 .594,.606 2 . 0 1.0 . 4 9 1 , . 4 9 9 2.22 1.0 .495,-504 ^.30 0 . 2 5 4 , .248 ^ . 0 0 .272, .282 3.699 0 . 2 9 0 , . 2 9 8 3.30 0 .295. .304 3.125 0 .310..318 2.823 0 .358,.364 2.602 0 .384,.396 2.426 0 .423,.480 88 Table H (cont'd) - l o g (ca] i n the [c&] l n the umoles Pi mg" 1hr" 1 loading medium (M) external medium (mM) 2 . 3 0 0 .490, .460 2.20 0 . 5 0 , .516 2;125 0 .541, ;553 2 . 0 0 . 4 9 8 , .518 1.9 0 .440, .468 Table I E f f e c t of manganese on Mg+Ca-ATPase a c t i v i t y i n resealed ghosts. - l o g |Wj l n the jca] i n the umoles P i mg-'Hir"1 loading medium external medium $.0 1.0 .318 3 . 3 1.0 .302 3 . 0 1.0 .296 3.52 1.0 .288 3.30 1.0 .324 4 . 0 0 .306 3 . 3 0 .296 3 . 0 0 .278 3 . 7 0 .281 3.52 0 .302 3.30 0 .314 3.126 0 .290 Table J 89 E f f e c t of ouabain on ATPase a c t i v i t y on ghosts resealed with K G 1 (2 .8?6 M) - Ca i n the loading medium was 0 .5 mM. Time [ouabain] In the umole P i mg~1hr (min) external medium (mM) 0 0 .156 10 0 .203 30 0 .535 40 0 .605 0 0 . 2 .156 10 0 .2 .225 20 0 .2 .306 30 0 . 2 .392 40 0 . 2 .44? 90 Table K A c t i v a t i o n of Na,K-ATPase by external potassium i n resealed ghosts, - Ca^ + i n the loading medium was 0,5 mM and calcium was not present l n the loading medium when indicated, [K^] i n the [ouabain]In the [ c a 2 H ] i n the umoles P i external medium (mM) external medium external medium mg^hr" 1 0 1.5 4 .5 10.0 0 1.5 4 .5 10.0 15.0 O.L 0.1 0.1 0.1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .302 .J00 .298 .300 .303. .297 , .337 .447..460 .550, .553 .638..646 .636,.645 0 1.5 4 .5 10.0 15.0 0 O 0 0 0 1.0 1.0 1.0 1.0 1.0 .365.-373 .562,.570 .643,.656 .676,.682 .648,.660 Table L E f f e c t of external calcium on ATPase a c t i v i t y . -the ghosts were loaded with 0.5 mM calcium and .4 mM EGTA. Other conditions were standard. l n the umoles Pi mg'^hr" external medium (mM) 1.0 . 0 7 3 , .070 1.5 .098,.104 2.0 .118,.130 3.0 .340..335 5.0 .146,.140 Table M E f f e c t of external magnesium on ATPase a c t i v i t y , -conditions same as Table L. [Mg 2 H3 In the umoles P i mg'^hr"1 external medium 1.0 .02,.03 2.0 .044,.050 3 . 0 .062,.055 4.0 .048,.052 

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