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Studies on the reaction cycle of the calcium transport atpase from human erythrocytes Allen, Bruce Gordon 1985

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STUDIES ON THE REACTION CYCLE OF THE CALCIUM TRANSPORT ATPASE FROM HUMAN ERYTHROCYTES by BRUCE GORDON ALLEN B.Sc, The University of British Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Divisions of Pharmaceutical Chemistry and Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1985 © Bruce G. Allen, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. P h a r m a c e u t i c a l C h e m i s t r y and Pharmacology and Department of T o x i c o l o g y * F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s . The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 n a t A October 1 5 , 1985 DE-6(3/81) ABSTRACT The plasma membrane calcium-transport ATPase plays a major role i n maintaining the low c y t o s o l i c calcium concentrations re-quired for normal c e l l u l a r function. Calcium, magnesium, calmo-dul i n and lanthanum have been shown to a l t e r the a c t i v i t y of the calcium-stimulated, magnesium-dependent ATPase a c t i v i t y i n human erythrocytes. In an attempt to examine the reaction sequence of the (Ca 2 + + Mg 2 +)-ATPase, the e f f e c t s of these agents on the k i n e t i c s of calcium dependent phosphoprotein formation, the f i r s t step in the p a r t i a l reaction sequence, were examined. Calmo-dulin-depleted erythrocyte membranes were prepared by hypotonic l y s i s i n the presence of EDTA, according to the method of C a r a f o l i et a l (1980). Calcium-dependent formation of the phosphorylated interme-diate was biphasic; the high c a l c i u m - a f f i n i t y component was associated with low levels of E.Ca.P and a shallow response to changing calcium concentrations, whereas in the region of the low c a l c i u m - a f f i n i t y component, E.Ca.P rose sharply i n response to increasing calcium concentrations. The low a f f i n i t y component of E.Ca.P l i e s i n the range of calcium concentrations which i n h i b i t ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y . When analyzed on LiDS acid PAGE, both components of calcium-dependent phosphoprotein formation were due to hydroxylamine-sensitive phosphorylation of a 135,000-145,000 dalton protein. Hence, the low c a l c i u m - a f f i n i t y compo-nent of phosphoprotein formation and calcium-dependent i n h i b i t i o n of ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y were l i k e l y due to calcium-i n h i b i t i o n of dephosphorylation. Kinetic studies of calcium-dependent phosphoprotein formation, at two d i f f e r e n t calcium concentrations (1.0 pM, 0.4 mM), indicated that a steady-state was reached much sooner at higher calcium concentrations. Lan-thanum, which i s known to block dephosphorylation of the interme-diate complex, increased both the apparent rate of formation and the steady-state level of the phosphorylated intermediate. Calmodulin, which has previously been shown to increase both the maximum v e l o c i t y and the calcium a f f i n i t y of the (Ca^ + + Mg^+)-ATPase, did not a f f e c t either calcium-dependent i n h i b i t i o n of (Ca + Mg )-ATPase a c t i v i t y or the biphasic nature of c a l -cium-dependent phosphoprotein formation. At low calcium concen-tra t i o n s , calmodulin increased the apparent rate of phosphopro-te i n formation, whereas at higher calcium concentrations (0.4 mM) calmodulin reduced the steady-state level of the phosphoprotein; the apparent rate of formation was unaffected. In the presence of lanthanum, calmodulin increased both the.apparent rate of formation and steady-state level of the phosphoprotein, sug-gesting that the true rate of formation was increased by calmodu-l i n at higher calcium concentrations, but t h i s was normally hidden by a simultaneous increase i n the rate of dephosphoryla-t i o n . Removal of endogenous magnesium, using trans-1,2-diamino-cyclohexane t e t r a a c e t i c acid (CDTA) did not a l t e r the calcium s e n s i t i v i t y or rate of formation of the phosphorylated interme-diate, however turnover of the intermediate was markedly reduced. In the absence of free magnesium, both the v e l o c i t y and calcium s e n s i t i v i t y of the (Ca 2 + + Mg 2 +)-ATPase were also found to be lower. The low c a l c i u m - a f f i n i t y component of calcium-dependent phosphoprotein formation, which Schatzmann (1982) has attributed to an action of calcium at a "magnesium-specific" s i t e , was not affected by magnesium concentrations as high as 1 mM. Further-more, t h i s phosphoprotein could be dephosphorylated along either the forward or reverse pathways. These re s u l t s indicate that the transformation from E-^.Ca.P to E 2.Ca.P may not be the s i t e of the calcium-dependent i n h i b i t i o n of dephosphorylation. Calmodulin-depleted membrane fragments were prepared from the erythrocytes of c y s t i c f i b r o s i s patients as well as age- and sex-matched controls. Under conditions i n which dephosphoryla-tion i s i n h i b i t e d , phosphoprotein formation and (Ca^ + Mg" )-ATPase a c t i v i t i e s were determined. Both (Ca^ + Mg^ )-ATPase a c t i v i t y and phoshoprotein formation were found to be s i g n i f i -cantly reduced i n the preparations derived from patients with c y s t i c f i b r o s i s . Turnover of the phosphorylated intermediate did not d i f f e r s i g n i f i c a n t l y between the two groups. A reduction i n (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y and phosphoprotein formation sug-gests that there may be fewer active calcium-pumping s i t e s i n the erythrocyte membranes of c y s t i c f i b r o s i s patients compared to normal subjects. i v TABLE OF CONTENTS page ABSTRACT i i ACKNOWLEDGEMENTS ' DEDICATION ^IV.;. TABLE OF CONTENTS V 1 LIST OF FIGURES Vt if. LIST OF TABLES X . LIST OF ABBREVIATIONS xi' . INTRODUCTION 1 I Role of Calcium i n Modulating I n t r a c e l l u l a r A c t i v i t i e s 1 II Regulation of I n t r a c e l l u l a r Free Calcium Concentrations 3 III The Plasma Membrane Calcium Pump 5 1. Substrates 7 2. Effectors 9 a. Calcium 9 b. Magnesium 11 c. Monovalent Cations 13 3. Modulators 14 a. Calmodulin 14 b. Endogenous Inhibitory Protein 17 4. The Transport Cycle 17 IV Cystic F i b r o s i s 23 AIMS OF THE PRESENT STUDY ' 28 MATERIALS AND METHODS 31 I Materials 31 II Methods 34 1. Preparation of calmodulin-depleted Erythrocyte Membrane Fragments 34 v. ,\. 2. Measurement of ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y 34 3. Phosphorylation of Erythrocyte Membranes 36 a. Dephosphorylation Experiments 38 b. Hydroxylamine Treatment 38 4. Polyacrylamide Gel Electrophoresis and Autoradiography of Phosphorylated Erythrocyte Membrane Fragments 39 a. Polyacrylamide-Gradient Acid-Gel Electrophoresis 39 b. Sample Preparation 40 c. Staining, Destaining and Auto-radiography 41 5. Miscellaneous Methods 41 a. Protein Assay 41 b. Non-Linear Analysis 42 . c. S t a t i s t i c a l Analysis 43 d. Determination of Free Calcium Concentrations 43 RESULTS 44 I E f f e c t of Calcium, Magnesium, Calmodulin and Lanthanum on Calcium Dependent ATP Hydrolysis and Phosphoprotein Formation 44 1. E f f e c t of Calcium Concentration 44 2. E f f e c t of Calmodulin 52 3. E f f e c t of Magnesium 59 II ATP Hydrolysis and Phosphoprotein For-mation i n the Erythrocyte Plasma Membranes of Subjects With Cystic F i b r o s i s 71 DISCUSSION 76 I E f f e c t s of Calcium on ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y and the intermediate reaction sequence 78 II E f f e c t s of Lanthanum on the P a r t i a l Reactions of the ( C a 2 + + Mg 2 +)-ATPase 83 III E f f e c t s of Calmodulin on the Intermediate Reactions of the ( C a 2 + + Mg 2 +)-ATPase 84 IV E f f e c t s of Magnesium on the P a r t i a l Reactions of the ( C a 2 + + Mg 2 +)-ATPase 89 v i V Changes i n the (Ca 2 + + Mg 2 +)-ATPase A c t i v i t y and Phosphoprotein Formation in Erythrocytes from Patients wth Cystic F i b r o s i s 93 CONCLUSIONS 95 BIBLIOGRAPHY 98 LIST OF FIGURES FIGURE page 1. Reaction cycle of the human erythrocyte (Ca + Mg2 + )-ATPase 19 2. E f f e c t of free calcium concentration on (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y and formation of the phosphorylated intermediate 45 3. J^P-Autoradiogram showing hydroxylamine s e n s i t i v i t y of the phosphorylated i n t e r -mediate formed at various free calcium concentrations 48 4. E f f e c t of free calcium concentration on turnover of the phosphorylated intermed-iate of the (Ca 2* + Mg2 + )-ATPase 50 5. (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y and the time course of phosphoprotein formation i n erythrocyte membrane fragments at two calcium concentrations (1 uM, 0.4 mM) 51 6. E f f e c t of lanthanum (0.1 mM) on (C a 2 + + Mg)-ATPase a c t i v i t y and the time course of phosphoprotein formation i n erythrocyte membrane fragments 53 7. E f f e c t of calmodulin (10 ug/ml) on (Ca 2 + + Mg )-ATPase a c t i v i t y i n calmodulin-depleted erythrocyte membrane fragments at various free calcium concentrations 56 8 a. E f f e c t of calmodulin (10 ug/ml) on phos-phoprotein formation i n calmodulin-depleted erythrocyte membrane fragments at various free calcium concentrations 57 b. Eadie-Hoffstee plot of Figure 8a. 9. E f f e c t of calmodulin (10 ug/ml) on turn-over of the phosphorylated intermediate of the (Ca 2 + + Mg 2 +)-ATPase i n the presence of various free calcium concentrations 58 1.0. E f f e c t of calmodulin (10 ug/ml) on (Ca 2 + + Mg)-ATPase a c t i v i t y and the time course of phosphoprotein formation i n the presence of 1 uM free calcium 60 LIST OF FIGURES (cont'd) FIGURE page 11. E f f e c t of calmodulin (10 ug/ml) on (C a 2 + + Mg 2 +)-ATPase a c t i v i t y and the time course of phosphoprotein formation i n the presence of 0.4 mM free calcium 61 12. E f f e c t of calmodulin (10 ug/ml) on (C a 2 + + Mg 2 +)-ATPase a c t i v i t y and the time course of phosphoprotein formation i n the presence of lanthanum (0.1 mM) 62 13. (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y in the presence of either magnesium (25 uM) or CDTA (0.1 mM) at various free calcium concentrations 63 14. a and b. Formation of the phosphorylated intermediate of the (Ca 2 + + Mg 2 +)-ATPase in the presence of either magnesium (25 uM) or CDTA (0.1 mM) at various free calcium concentrations 66 15. Turnover of the phosphorylated intermediate of the (Ca 2 + + Mg 2 +)-ATPase i n the presence of either magnesium (25 pM) or CDTA (0.1 mM) at various free calcium concentations 67 16. ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y and the time course of phosphoprotein formation in the presence of 1.0 uM free calcium and either magnesium (25 uM) or CDTA (0.1 mM) 68 17. ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y and the time course of phosphoprotein formation in the presence of 0.4 mM free calcium and either magnesium (25 uM) or CDTA (0.1 mM) 69 18. E f f e c t of magnesium (0.5 mM) versus CDTA (0.1 mM) on the low calcium a f f i n i t y component of phosphoprotein formation 70 19. E f f e c t of magnesium concentration on formation of the phosphorylated i n t e r -mediate of the (Ca 2 + + Mg 2 +)-ATPase i n the presence of 10 mM free calcium 72 IX LIST OF TABLES TABLE page 1. Kinetic parameters describing the e f f e c t of free calcium concentration on (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y and formation of the phosphorylated intermediate i n the presence and absence of exogenous calmodulin (10 ug/ml) 46 2. E f f e c t of lanthanum (0.2 mM) on formation of the phosphorylated intermediate of the ( C a 2 + + Mg 2)-ATPase i n the presence and absence of 10 mM calcium 54 3. Kinetic parameters for calcium stimulation of ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y i n the presence of either magnesium (25 uM) or CDTA (0.1 mM) 65 4. E f f e c t of ADP (1.0 mM) or ATP (1.0 mM) on the phosphorylated intermediate of the erythrocyte (Ca^ + + Mg 2 +)-ATPase formed in the presence of 10 mM calcium and various concentrations of magnesium 73 5. Phosphoprotein formation, ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y and turnover of the phos-phorylated intermediate i n erythrocyte membranes from normal subjects and c y s t i c f i b r o s i s patients 75 LIST OF ABBREVIATIONS ADP AMP ATP Ca 2+ 2+ 2+ CCa + Mg )-ATPase Ci cAMP CDTA adenosine 5'-diphosphate adenosine 5 *-monophosphate adenosine 5 1-triphosphate degrees Centigrade free (.' ionized) calcium calcium-stimulated, magnesium-dependent ATPase cpm CTP dpm E 1.Ca.P E 2.Ca.P EDTA EGTA et a l f g GTP 3H HEPES Curie adenosine 3 ' : 5 * - c y c l i c monophosphate trans-1,2-diaminocyclohexane-N,N,N',N',-te t r a a c e t i c acid counts per minute cytidine 5'-triphosphate decays per minute phosphorylated enzyme-calcium complex i n the state phosphorylated enzyme-calcium complex i n the E 2 state ethylenediaminetetraacetic acid ethyleneglycolbis-(. beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and others f emto gram guanosine 5 1-triphosphate t r i t i u m (N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid ITP inosine 5'-triphosphate K, d i s s o c i a t i o n constant d 1 l i t e r LiDS lithium dodecylsulphate m m i l l i M molar mA milliamperes min minute mg milligrams mol mole p micro + + Na ,K - sodium-, potassium-stimulated ATPase ATPase 32 P phosphorus-32 PAGE polyacrylamide gel electrophoresis pmol picomoles P^ inorganic phosphate SDS sodium dodecylsulphate S.E.M. standard error of the mean S.R. sarcoplasmic reticulum TCA t r i c h l o r o a c e t i c acid TEMED N,N,N',N*-tetramethylethylenediamine T r i s tris(hydroxymethyl)aminomethane UTP uridine 5 1-triphosphate V maximum v e l o c i t y max J V . observed v e l o c i t y obs J w/v weight per unit volume / per % percent xi7 DEDICATION To my parents. ACKNOWLEDGEMENTS I am deeply grateful to my supervisors, Dr. S. Katz and Dr. B.D. Roufogalis, for t h e i r guidance and support throughout t h i s study. I would l i k e to thank Dr. D. Jeffery, Mrs. L. Hoang and the rest of my laboratory colleagues for th e i r technical assistance and moral support. I would also l i k e to g r a t e f u l l y acknowledge the f i n a n c i a l support extended by both the B r i t i s h Columbia Heart Foundation and the Canadian Heart Foundation. F i n a l l y , I wish to thank a l l members of the faculty, s t a f f and graduate student body in the Faculty of Pharmaceutical Sciences, U.B.C. for making t h i s Masters program enjoyable. INTRODUCTION I. Role of Calcium i n Modulating I n t r a c e l l u l a r A c t i v i t i e s . Recently calcium has been recognized as a key factor i n regulating many c e l l u l a r functions. The action potential i n a nerve c e l l i s triggered and modulated by an in f l u x of calcium across the axonal membrane (Baker, 1972). Muscle contraction i s evoked by calcium release from membrane-associated calcium pools, such as the sarcoplasmic reticulum, into the cytosol (Ebashi, (1976). In mediating c e l l u l a r responses to e l e c t r i c a l or a wide variety of chemical s t i m u l i , calcium may function either; ^ alone as an i n t r a c e l l u l a r "second messenger", or ^ by af f e c t i n g enzyme a c t i v i t i e s such as adenylate cyclase and/or phosphodi-esterase to modulate the second messenger ro l e of c y c l i c AMP (Rasmussen and Goodman, 1977). As a second messenger calcium e l i c i t s a physiological response v i a calcium-modulated proteins. These proteins are not enzymes themselves but serve to transmit the calcium "message" through calcium-dependent interactions with target enzymes and str u c t u r a l proteins. One such protein i s calmodulin, which undegoes a conformational change upon binding calcium, allowing i t to intera c t with, and thereby regulate the a c t i v i t y of a wide variety of target enzymes (for review see Scharff, 1981). Stimulus-evoked r e d i s t r i b u t i o n of i n t r a c e l l u l a r calcium i s known to regulate various c o n t r a c t i l e and secretory systems, membrane and other c e l l u l a r events i n addition to a c t i -vating or i n h i b i t i n g a wide variety of s p e c i f i c enzyme systems (Case, 1980). 1 Due to the s p e c i f i c nature of the interactions involved i n calcium fluxes and binding to target proteins, the b i o l o g i c a l l y active form of calcium i s i t s ionized or "free" state. A l l of the regulatory functions of calcium are based upon the existence of a steep electrochemical gradient for calcium which i s oriented towards the cytoplasm. The concentration of free calcium ions i n the cytoplasm i s t y p i c a l l y less than 1 0 - 1 0 M, whereas much — 3 higher calcium levels (about 10 M) are found i n e x t r a c e l l u l a r f l u i d s and i n i n t r a c e l l u l a r organelles such as the mitochondria and the sarcoplasmic reticulum (SR). The protein targets for calcium are located either i n the cytoplasm or on the membranes exposed to the cytoplasm. Onset of the physiological response i s triggered by an increase i n the cytoplasmic concentration of free calcium ions. The duration of the response i s often dependent on the duration of the calcium "pulse"; for example the contraction of s t r i a t e d muscle termin-ates as the concentration of calcium i n the cytoplasm decreases. In addition, for calcium to act as a second messenger i n more than one response within the same c e l l , changes i n c y t o s o l i c calcium concentration have to be compartmentalized. The stimulus-induced r i s e i n c y t o s o l i c calcium must not r e s u l t i n a uniform increase i n c y t o s o l i c calcium or there would be no s e l e c t i v i t y i n the response e l i c i t e d . Hence a means whereby calcium levels are ra p i d l y restored to t h e i r low resting levels following a stimulus i s essential i n order to terminate calcium-dependent events and to allow calcium to function as a selective second messenger. 2 I I . Regulation of I n t r a c e l l u l a r Free Calcium Concentrations A large electrochemical gradient for calcium i s thermodyna-mically unfavorable and therefore must be maintained at the expense of energy by the c e l l . Low cytoplasmic calcium concen-trations are achieved by binding of calcium to s i t e s within the c e l l and by energy-dependent calcium transporting systems i n both the plasma membrane and i n t r a c e l l u l a r organelles. I n t r a c e l l u l a r calcium binding s i t e s include negatively charged phospholipids associated with the various membrane systems and a group of macromolecules referred to as calcium binding proteins. There are two types of active transport mechanisms associated with calcium metabolism: ^ Na +-Ca 2 + exchange, (Schatzmann, 1975; Sarkadi and Tosteson, 1979), where calcium i s extruded i n exchange for i n -wardly moving sodium ions thereby e x p l o i t i n g energy o r i g i n a l l y invested i n the sodium gradient by the Na +,K +-ATPase; and, 2^ Calcium pumping, catalyzed by a s p e c i f i c family of enzymes, at the expense of metabolic energy i n the form of ATP. Sodium-calcium exchange occurs i n plasma membranes (with the exception of the erythrocyte) whereas, i n addition, calcium pumping en-zymes are found i n the plasma membrane, sarcoplasmic reticulum, and mitochondrial membranes. These are independent calcium transport systems, molecularly and possibly mechanistically d i f -ferent from one another. I n t r a c e l l u l a r organelles are well suited for rapid regula-t i o n of c y t o s o l i c calcium levels since t h e i r t o t a l calcium-transporting membrane area i s generally much greater than that of 3 the plasma membrane. Although estimates of free calcium concen-trations i n these organelles range from 10"^ - 10~ 5 M, t h e i r t o t a l calcium content i s i n the millimolar range due to extensive calcium buffering c a p a b i l i t i e s . In the mitochondria, calcium combines with inorganic phosphate to form an osmotically inactive calcium phosphate complex. In the sarcoplasmic reticulum, calcium appears to bind to calsequestrin, a low - a f f i n i t y (K d = 0.8 mM) high capacity calcium binding protein (Ostwald and MacLennan, 1974). This process increases the capacity of the organelle for calcium uptake. Calcium transport into the mitochondria repre-sents a special case with respect to ATP-driven calcium fluxes. The actual dr i v i n g force for calcium movement appears to be the membrane potential created by the extrusion of protons by an ATPase (Car a f o l i , 1974; Bygrave, 1978; C a r a f o l i and Crompton, 1978). The calcium "pump" of mitochondrion has a K d for calcium of roughly 10" 5 M (Crompton et a l , 1976; C a r a f o l i , 1982) whereas for the (Ca 2 + + Mg 2 +)-ATPase in S.R. i n skeletal muscle the K d has been estimated to be 3 - 5 x 10~ 7 M (deMeis, 1971; Kanazawa et a l , 1971; Meisner, 1973; Ikemoto, 1974) and 1 - 2 x 10~ 6 M i n cardiac muscle (Shikegawa et a_l, 1976) . In view of i t s low a f f i n i t y for calcium there i s some question as to the extent to which calcium uptake into the mitochondria i s involved i n main-taining low c y t o s o l i c calcium concentrations i n the unstimulated c e l l . There have been suggestions that the function of the mitochondrial calcium transport system i s to regulate the calcium concentration within the mitochondria i t s e l f , not that of the cytoplasm (Denton and McCormack, 1980). However, Penniston 4 (1983) has suggested that the mitochondria may primarily be a back-up system designed to l i m i t large increases i n cytoplasmic free calcium l e v e l s . Though mechanisms such as calcium binding and calcium storage organelles are suitable for rapid removal of small amounts of calcium from the cytoplasm they would not be able to maintain a low calcium concentration i n the cytoplasm i n d e f i n i t e l y . The e x t r a c e l l u l a r calcium which enters the c e l l i n response to each stimulus or action potential would ra p i d l y saturate these s i t e s . Penniston (1983) has suggested that the role of the plasma membrane calcium transport mechanisms i s to recycle that portion of the cytoplasmic calcium that i s extracel-l u l a r i n o r i g i n , so as to prevent a build-up of calcium within the c e l l . There i s s t i l l some controversy regarding the r e l a t i v e im-portance of the calcium pump with respect to Na ,Ca -exchange m maintaining a low cytoplasmic free calcium concentration. Stud-ies conducted i n both the squid axon (DiPolo and Beauge, 1979) and sarcolemmal v e s s i c l e s i s o l a t e d from dog heart (Caroni and C a r a f o l i , 1981) have suggested that ATP-dependent calcium e f f l u x i s dominant at the physiological r e s t i n g calcium concentration, whereas the Na +,Ca 2 +-exchange i s e f f e c t i v e only at much higher concentrations, such as might occur during stimulation. I l l The Plasma Membrane Calcium Pump The plasma membrane calcium pump and i t s associated (Ca^ + Mg 2 +)-ATPase a c t i v i t y have been p a r t i c u l a r l y well characterized in the human erythrocyte, as these c e l l s provide a unique system 5 for studying the regulation of t h i s enzyme. Erythrocytes do not contain calcium-accumulating organelles or a Na ,Ca -exchange mechanism, yet they maintain a low i n t r a c e l l u l a r free calcium concentration and they can be obtained i n a r e l a t i v e l y pure and homogeneous form using mild i s o l a t i o n procedures. For these reasons erythrocytes provide a r e a d i l y available source of plasma membrane (C a 2 + + Mg 2 +)-ATPase which i s free from contamination by other species of calcium ATPases. Dunham and Glynn (1961) were the f i r s t to demonstrate the presence of a ouabain-insensitive magnesium-dependent, calcium-stimulated adenosine triphosphatase a c t i v i t y i n the plasma membrane of human erythrocytes. This ATP-hydrolytic a c t i v i t y was several-fold more active than the ouabain-sensitive Na +,K +-ATPase a c t i v i t y i n these membranes. A calcium transport a c t i v i t y was reported i n the erythrocyte plasma membrane f i v e years l a t e r by Schatzmann (1966) and i t was shown that t h i s calcium pumping mechanism was c l o s e l y coupled to the (Ca + Mg )-ATPase a c t i v i t y (Schatzmann and Vincenzi, 1969; Lee and Shin,1969). It i s now well established that the s p l i t t i n g of ATP to ADP and P^ i s necessary to produce active calcium transport (Schatzmann, 1975; Sarkadi and Tosteson, 1979). The plasma membrane calcium pump exists as an i n t r i n s i c membrane protein with a molecular weight of 135,000-145,000 d a l -tons (Knauf et a l , 1974; Katz and Blostein, 1975; Schatzmann and Burgin, 1978; Lichtner and Wolf, 1979; C a r a f o l i et. aj., 1982) which, i n l i g h t of i t s function, probably spans the plasma mem-brane. Studies of the p u r i f i e d enzyme using polyacrylamide gel 6 electrophoresis also show evidence of a higher molecular weight peptide (Niggli et al_, 1979; Schatzmann, 1982) which i s thought to represent a dimer of the enzyme (Hinds et a_l, 1982) . In addition, r e s u l t s from radiation i n a c t i v i a t i o n studies indicated that the enzyme functions as a dimer i n the plasma membrane (Minocherhomjee et a_l, 1983). The significance of these observa-tions, regarding the true functional unit of the calcium pump, i s s t i l l unknown. The molecular weight of the calcium pump protein from sarcoplasmic reticulum has been estimated to be between 100,000 and 115,000 daltons (Inesi et a_l, 1970; MacLennan, 1970; Louis and Shooter, 1972; Thorley-Lawson and Green, 1975; LeMaire et a_l, 1976; Rizzolo e_t a_l, 1976), emphasizing the fact that the calcium transport mechanisms found i n these two membrane systems are d i f f e r e n t . The measured a c t i v i t y and calcium s e n s i t i v i t y of the (Ca^ + Mg 2 +)-ATPase varies depending upon the method of membrane prepar-ation, the nature of the substrate and cofactors added to the assay medium and the presence of regulators (Roufogalis, 1979). The substrate of the calcium pump i s ATP and i t s cofactors include magnesium, calcium and possibly potassium. The best known regulator of the erythrocyte calcium pump i s calmodulin, however Au (1978) has recently discovered a protein which i n h i -b i t s the a c t i v i t y of the calcium pump. 1. Substrates Active transport of calcium requires ATP as a substrate. Other nucleoside triphosphates (CTP, GTP, ITP, UTP) may sub s t i -7 tute for ATP (Lee and Shin, 1969; Olson and Cazort, 1969), how-ever the enzyme demonstrates a higher s p e c i f i c i t y for ATP (Cha et a l , 1971; Sarkadi et al_, 1979). Other phosphate esters, such as ADP, AMP, pyrophosphate, acetyl phosphate, or nitrophenyl phos-phate cannot support active calcium transport (Olson and Cazort, 1969; Schatzmann and Vincenzi, 1969; Sarkadi et a_l, 1979; Caride et a_l, 1983) . There i s some controversy as to the actual species of ATP which provides the energy for calcium transport. The proposed substrates include: Mg-ATP (Wolf, 1972; Wolf et a l , 1977); Ca-ATP (Penniston et al^, 1980; Graf and Penniston, 1981a); and free ATP (Rega and Garrahan, 1975; Schatzmann, 1977; Richards et a_l, 1978; Sarkadi et a_l, 1978) . More recent studies tend to suggest that more than one of these forms of ATP may act as the substrate. Muallem and Karl i s h (1981) have suggested that Mg-ATP, Ca-ATP and free ATP are equally e f f e c t i v e whereas Penniston (1982b) proposed that Mg-ATP i s the substrate but that Ca-ATP can also be u t i l i z e d i n the absence of magnesium. In short, there i s not a good consensus on the true physiological substrate(s) for calcium transport. ATP° a c t i v a t i o n of both calcium transport (Muallem and Kar l i s h , 1979; Mollman and Pleasure, 1980) and i t s associated ( C a 2 + + Mg2 + )-ATPase a c t i v i t y (Richards et a_l, 1978; Muallem and Kar l i s h , 1979, 1980; Steige and Luterbacher, 1981) follows a biphasic curve, showing both a "high-" and a "l o w - a f f i n i t y " component for ATP ( K A T p ^ 1 = 1-2 x 10~ 6 M, K A T p ^ 2 = 2-3 x 10" 4 M). These r e s u l t s have been taken to mean that there are two binding 8 s i t e s for ATP of d i f f e r e n t a f f i n i t i e s . Muallem and Kar l i s h (1978) suggested that at the h i g h - a f f i n i t y binding s i t e ATP acts as the substrate for transport, while higher ATP concentrations may regulate the a c t i v i t y of the enzyme through an a l l o s t e r i c e f f e c t at the low-a f f i n i t y binding s i t e . As an alternate i n t e r -pretation, Neet and Green (1977) showed that these r e s u l t s could equally well be explained i n terms of a single s i t e having d i f -ferent a f f i n i t i e s in two states of the reaction cycle. In both int a c t erythrocytes and resealed ghosts active c a l -cium extrusion requires i n t r a c e l l u l a r ATP, whereas calcium uptake into inside-out v e s i c l e s requires the presence of ATP i n the incubation medium (Sarkadi, 1980). These r e s u l t s show that ATP interacts at a s i t e on a region of the enzyme, normally facing the cytoplasm, which does not cross the membrane during the translocation of calcium. The inorganic phosphate (P^) produced from the enzymatic hydrolysis of ATP to ADP i s not co-transported with calcium, but released back into the cytoplasm (Olson and Cazort, 1974; Schatzmann and Roelofsen, 1977). 2. Effectors a. Calcium. As the ro l e of the calcium pump i s to move calcium from a region of low calcium concentration (the cyto-plasm) to a region of considerably higher calcium concentration (the e x t r a c e l l u l a r medium) the pump should have a high a f f i n i t y for calcium on the i n t r a c e l l u l a r side of the membrane and a much lower a f f i n i t y on the outer face of the membrane. At the i n t e r -nal membrane surface, calcium activates the calcium transport 9 system and i t s associated (Ca* + Mg^ )-ATPase a c t i v i t y while showing almost no e f f e c t at the external surface (Sarkadi, 1980) . To overcome d i f f i c u l t i e s i n c o r r e c t l y estimating the free calcium concentraion, EGTA i s added as a calcium buffer. The free calcium concentration i s calculated from the d i s s o c i a t i o n constants of a l l possible chelators of calcium present i n the assay medium aft e r correction for assay conditions such as temperature and ionic strengh. When measured i n the presence of a Ca-EGTA buf-f e r i n g system the a f f i n i t y for calcium at the internal surface i s roughly 1 - 5 x 10~ 6 M (Schatzmann, 1973; Schatzmann, 1982; Penniston, 1983). There have been some doubts regarding the v a l i d i t y of d i s s o c i a t i o n constants for calcium which are esimated in the presence of EGTA. This anion has been reported to i n -crease the calcium s e n s i t i v i t y of calcium uptake into inside-out v e s i c l e s (Sarkadi et a l , 1973) and on the (C a 2 + + Mg 2 +)-ATPase a c t i v i t y in resealed ghosts (Schatzmann, 1973), i n i n t a c t ery-throcyte membranes (Orlov and Schevchenko, 1978; Al-Jobore and Roufogalis, 1981a) and i n Triton X-100 s o l u b i l i z e d preparations (Al-Jobore and Roufogalis, 1981a; Al-Jobore et al_, 1981). Re-cently, however, Muallem and Kar l i s h (1982) demonstrated that the a f f i n i t y of the (Ca^ + Mg^ )-ATPase i n in t a c t erythrocytes i s high ( Kd,Ca = 2 x 10"^ M ) • This finding corroborates previous determinations of calcium a f f i n i t y which employed EGTA, but leaves open questions concerning the nature of the low calcium s e n s i t i v i t y expressed i n other preparations i n the absence of EGTA. In addition to i t s stimulatory action upon the (Ca 2 + + 10 Mg )-ATPase calcium, i n excess amounts (above 1 x 10~ 4 - 5 x 10 4 M), w i l l i n h i b i t the a c t i v i t y of t h i s enzyme (Schatzmann, 1982); th i s e f f e c t i s thought to occur at an i n h i b i t o r y s i t e on the enzyme (Klinger et a_l, 1980; Vincenzi et a_l, 1980; Graf and Penniston, 1981) . P a r t i a l digestion of the enzyme with trypsin, which cleaves a regulatory domain off of the enzyme, does not a f f e c t calcium-dependent i n h i b i t i o n (Steiger and Schatzmann, 1981; Roufogalis et a_l, 1982) . These res u l t s indicate that the s i t e of i n h i b i t i o n i s l i k e l y to be i n the c a t a l y t i c domain. A possible explanation for t h i s e f f e c t of calcium i s discussed below. There i s some controversy i n the l i t e r a t u r e concerning the true stoichiometry of the plasma membrane calcium pump. H i l l c o e f f i c i e n t s for calcium range from around 1 (Schatzmann, 1973; Schatzmann and Roelofsen, 1977) to around 2 (Quist and Roufogalis, 1977; Scharff, 1978; Akyempon and Roufogalis, 1982). F e r r i e r r a and Lew (1976) have shown that 2 calcium ions p a r t i c i -pate i n the reaction cycle. Direct measurement of the s t o i c h i o -metry reconstituted v e s i c l e s (Niggli et a_l, 1982) suggest a Ca/ATP r a t i o of 1/1. Schatzmann (1985) has pointed out that a stoichiometry of 2 i s not thermodynamically feasable whereas a one to one r a t i o would be acceptable. However, t h i s assumes an electrogenic mode of operation of the pump, whereas the counter-transport of one or two protons per calcium ion would a l t e r t h i s r e s t r i c t i o n (Tanford, 1984). b. Magnesium. Magnesium has several roles i n regulating ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y . Although not transported by the 11 calcium pump, magnesium must be present i n in t a c t c e l l s and resealed ghosts to observe optimum active calcium extrusion (Schatzmann and Vincenzi, 1969; Lee and Shin, 1969; Olson and Cazort, 1969). In inside-out vesi c l e s active calcium uptake requires magnesium on the c y t o s o l i c side of the v e s i c l e s (Weiner and Lee, 1972; Maclntyre and Green, 1977; Sarkadi et a]_, 1978; Cha et a_l, 1971) . These re s u l t s suggest that of the ATP- and Mg 2 +-binding s i t e s are located on the same side of the membrane, namely on the c y t o s o l i c surface of the membrane. Although t h i s enzyme was o r i g i n a l l y thought to be absolutly dependent upon the presence of magnesium for a c t i v i t y (Dunham and Glynn, 1961; Lee and Shin, 1969; Shatzmann, 1975) more recent reports indicate that t h i s i s not the case. Richards et a_l (1978) found that a small calcium-stimulated ATPase a c t i v i t y remained i n the absence of magnesium and that while magnesium enhanced the stimulatory e f f e c t of calcium, i t had no e f f e c t on the a f f i n i t y for calcium or ATP. The stimulatory action of magnesium on the ATPase f o l -lowed simple saturation k i n e t i c s (K d^ M g= 0.33 mM) (Richards et a l , 1978) . Magnesium concentrations i n the millimolar range compete with calcium, s h i f t i n g the calcium concentration which produces 1/2 maximum ac t i v a t i o n of the ATPase to higher values i n erythro-cyte ghosts (Klinger et a_l,1980; Al-Jobore and Roufogalis, 1981b; Scharff, 1980), Tri t o n X-100 s o l u b i l i z e d membranes (Al-Jobore and Roufogalis, 1981b), i n inside-out v e s i c l e s (Akyempon and Roufogalis, 1982) and i n the p u r i f i e d enzyme (V i l l a l o b o et a l , 1985) . Klinger et a_l (1980) found that magnesium also antag-12 onized calcium-dependent i n h i b i t i o n of the (Ca* + Mg )-ATPase. Al-Jobore and Roufogalis (1981b) suggested that magnesium allows the binding of activating calcium but prevents the binding of calcium at an i n h i b i t o r y s i t e on the enzyme. Schatzmann (1982) proposed a sim i l a r idea, suggesting that at high concentrations, calcium became i n h i b i t o r y by way of a competition at a magnesium-s p e c i f i c s i t e . c. Monovalent Cations. Although the calcium pump i s not dependent on the presence of monovalent cations for a c t i v i t y (Schatzmann, 1975) , both calcium transport and i t s associated (Ca + Mg* )-ATPase a c t i v i t y are 30-50% higher when measured in sodium or potassium containing solutions than i n choline, t r i s or sucrose media (Schatzann and Rossi, 1971; Sarkadi et a_l, 1978). A si m i l a r e f f e c t has also been shown i n p u r i f i e d enzyme prepara-tions (Graf et a_l, 1982) . Rb + or NH 4 + w i l l mimic the stimulatory e f f e c t s of Na + and K + but L i + w i l l not (Wolf et a l , 1977). The a f f i n i t i e s for sodium and potassium d i f f e r markedly for t h i s e f f e c t ( K N a = 20-30 mM, K K = 4-6 mM) (Schatzmann and Rossi, 1971; Scharff, 1978; Kratje et a l , 1983). Kratje and coworkers (1983) recently showed that monovalent cations appear to exert t h e i r e f f e c t s on the internal side of the membrane. This observation makes i t u n l i k e l y that t h i s stimulation of calcium transport i s due to a counter-transport mechanism. Although calmodulin has been shown to enhance the stimulatory e f f e c t of potassium on the rate of ATP hydrolysis by the (Ca 2 + + Mg 2 +)-ATPase, (Scharff, 1978), t h i s stimulatory e f f e c t of monovalent cations i s indepen-dent of that produced by calmodulin (Sarkadi et a_l, 1980) . It i s 13 questionable that t h i s represents a regulatory mechanism for the calcium pump, as the c y t o s o l i c concentrations of potassium and sodium exceed that required to obtain t h i s e f f e c t . 3. Modulators a. Calmodulin. Calmodulin, a heat-stable, a c i d i c , calcium binding protein with a molecular weight of approximatly 16,800 daltons (Klee and Vanaman, 1982), has been is o l a t e d from a number of tissues, including brain, heart and t e s t i s . I n i t i a l l y charac-terized as an activator of phosphodiesterase, calmodulin has since been shown to be an i n t r a c e l l u l a r modulator of a number of calcium-dependent enzymes. Each calmodulin molecule can bind 3-4 calcium ions with d i s s o c i a t i o n constants i n the range of 10"^ -10~ 4 M (Wolff et a_l, 1972) . The binding of calcium a l t e r s the conformation of calmodulin, increasing i t s h e l i c a l content (Ded-bman et a_l, 1977) and exposing hydrophobic regions (LaPorte et a l , 1980). In t h i s conformation the calcium-calmodulin complex i s able to bind to target enzymes (Niggli et a_l, 1979) and i n -crease or decrease t h e i r a c t i v i t y . The f i r s t observation that calmodulin was involved i n regu-l a t i n g calcium fluxes across membranes was that of Bond and Clough (1973), who reported that a factor i n hemolysate from human erythrocytes enhanced the a c t i v i t y of the erythrocyte (Ca* + Mg 2 +)-ATPase. Kinetic analysis of the calcium a c t i v a t i o n of the ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y i n i s o l a t e d erythrocyte mem-branes revealed the presence of two (Ca + Mg* )-ATPase a c t i v i -14 t i e s . Scharff (1972) had shown that erythrocyte "ghosts" pre-pared i n the presence of calcium had higher ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y and a f f i n i t y for calcium (Scharff, 1976) than those prepared i n the presence of the calcium chelator, EDTA. These two a c t i v i t i e s were referred to as high and low a f f i n i t y ( C a 2 + + 2 + Mg* )-ATPase, r e f l e c t i n g t h e i r d i f f e r e n t a f f i n i t i e s for calcium (Horton et a l , 1970; Schatzmann and Rossi, 1971; Scharff, 1972). Quist and Roufogalis (1975) showed that incubation of erythrocyte membranes i n the presence of EDTA converted the ( C a 2 + + Mg 2 +)-ATPase from a h i g h - a f f i n i t y state to a l o w - a f f i n i t y state; read-d i t i o n of the extract restored h i g h - a f f i n i t y ATPase a c t i v i t y . These r e s u l t s suggested the action of a regulatory factor instead of two d i s t i c t types of ( C a 2 + + Mg 2 +)-ATPase. A c y t o s o l i c factor was i d e n t i f i e d as calmodulin (Gopinath and Vincenzi, 1977; Jarret and Penniston, 1977) and shown to regulate a revers i b l e s h i f t of the ( C a 2 + + Mg 2 +)-ATPase between low a f f i n i t y / l o w a c t i v i t y and h i g h - a f f i n i t y / h i g h a c t i v i t y states of the enzyme (Scharff and Foder, 1978). Calmodulin binding to the enzyme results i n a 3-4 f o l d increase i n the apparent maximum v e l o c i t y and a 30 f o l d increase i n the a f f i n i t y for calcium at the transport s i t e (Roufogalis and Mauldin, 1980; Foder and Scharff, 1981; Scharff and Foder, 1978, 1982). In the presence of calcium, calmodulin binds very t i g h t l y to human erythrocyte membranes (Graff et a_l, 1980) . The d i s s o c i a -t i o n constant for calmodulin i s 25 uM i n the presence of 0.1 uM calcium (Scharff and Foder, 1982). Increasing the calcium con-centration to 20 pM reduces the d i s s o c i a t i o n constant (increases the a f f i n i t y ) of calmodulin to 2.5 nM (Scharff and Foder, 1982). 15 These authors found that the rate constant for the association of calmodulin with the (C a 2 + + Mg 2 +)-ATPase i s dependent on the calcium concentration. Hence, at low calcium concentrations a delay i n the act i v a t i o n of the calcium pump may be expected. This delay has been observed experimentally (Vincenzi et a l , 1980; Scharff and Foder, 1982). Scharff and coworkers (1983) suggested that such a mechanism provides a system whereby an abrupt r i s e of the calcium concentration within a c e l l w i l l induce a slow association between calmodulin and the calcium pump, r e s u l t i n g i n an act i v a t i o n with a lag which allows short pulses of calcium entry into a c e l l to produce a prolonged r i s e i n c y t o s o l i c calcium. Without t h i s delay, pulses of calcium entry would be terminated so rapidly that no effe c t o r mechanisms would be activated. Brief exposure of human erythrocyte membranes to trypsin activates both the (C a 2 + + Mg 2 +)-ATPase (Taverna and Hanahan, 1980) and calcium transport a c t i v i t y (Enyedi et a l , 1980; Sarkadi et al_, 1980) . This e f f e c t resembles the e f f e c t of calmo-dul i n i n that both maximum v e l o c i t y and calcium s e n s i t i v i t y are increased. Following t r y p t i c digestion, these membranes are no longer activated by the addition of exogenous calmodulin. Sarkadi and coworkers (1980) proposed that calmodulin binds to a "subunit" of the enzyme, on the cytoplasmic surface of the mem-brane, and that t h i s subunit of the enzyme functions as an i n h i -b i t o r of the calcium pump. Tryptic cleavage or calmodulin bind-ing relax the constraining e f f e c t of t h i s i n h i b i t o r y subunit, thereby increasing both the calcium a f f i n i t y and the maximum 16 v e l o c i t y of the calcium pump. b. Endogenous Inhibitory Protein. Evidence now suggests that i n addition to a stimulator of the ( C a 2 + + Mg 2 +)-ATPase there i s also an i n h i b i t o r present i n the cytosol of erythro-cytes. Concentrated membrane-free erythrocyte cytoplasm has been shown to i n h i b i t both ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y (Au, 1978) and calcium transport (Sarkadi et a_l, 1980) i n erythrocyte mem-branes. Lee and Au (1981) have recently i s o l a t e d a protein from the hemolysate of pig erythrocytes which demonstrates these properties. This protein has an apparent molecular weight of 7,500 daltons and i s p a r t i a l l y inactivated by trypsin, l y o p h y l i -zation and heat. Calcium concentrations up to 5.0 x 10~ 4 M do not i n t e r f e r e with the i n h i b i t o r y actions of t h i s protein. The physiological role of t h i s i n h i b i t o r i n the regulation of the calcium pump in unknown. 3. The Transport Cycle. Although i t i s now well documented that active calcium transport requires the s p l i t t i n g of ATP to ADP and P^ (Schatmann, 1975; Sarkadi and Tosteson, 1979) the exact mechanism by which energy from the hydrolysis of ATP i s u t i l i z e d to transport c a l -cium against an electrochemical gradient i s not c l e a r l y under-stood. The reaction mechanism of the (Ca* + Mg* )-ATPase may be broken down into a series of steps or " p a r t i a l reactions". Con-siderable progress has been made i n the elucidation of the par-t i a l reactions of the (Ca* + Mg* )-ATPase i n erythrocyte mem-17 y + y + branes. The (Ca* + Mg* )-ATPase reaction proceeds through a phosphorylated intermediate. The phosphorylated intermediate i s a 135,000-145,000 dalton protein (Knauf et al., 1974; Katz and Blostein, 1975; Schatzmann and Burgin, 1978; Lichtner and Wolf, 1979; C a r a f o l i et a_l, 1982) which i s chemically d i s t i n c t from the Na +,K +-ATPase phosphoenzyme of molecular weight around 100,000 daltons (Knauf et a_l, 1974; Katz and Blostein, 1975). Schatzmann (1985) recently presented a k i n e t i c model, adapted from that proposed by Rega and Garrahan (1975), shown in Figure 1. This model incorporates most of the present knowlege concerning the y + y + reaction scheme of the (Ca* + Mg* )-ATPase. The transport cycle i s i n i t i a t e d when calcium and ATP i n t e r -act at s p e c i f i c s i t e s on the cytoplasmic face of the enzyme, re s u l t i n g i n formation of a phosphorylated intermediate of the enzyme. The enzyme demonstrates an absolute requirement for calcium at t h i s step (Katz and Blostein, 1975; Rega and Garrahan, 1975; Schatzmann and Burgin, 1978; N i g g l i et a_l, 1979; Steiger and Luterbacher, 1981a). The calcium dependency of phosphoryla-t i o n (K d = 1-6 pM) i s s i m i l a r to that of the ( C a 2 + + Mg 2 +)-ATPase (K d = 2-5 ]M) (Knauf et a l , 1974; Katz and Blostein, 1975; Rega and Garrahan, 1975; Richards et a_l, 1975) . This form of the enzyme, exhibiting high a f f i n i t y calcium binding s i t e s oriented towards the cytoplasm, i s commonly referred to as the E-^  state. The phosphorylated enzyme complex, E-^.Ca.P, which re s u l t s from the transfer of the gamma phosphate group of ATP to a s i t e on the enzyme, i s sensitive to hydroxylamine and basic conditions (Katz and Blostein, 1975; Rega and Garrahan, 1975; Lichtner and Wolf, 18 Figure 1. The reaction cycle of the human erythrocyte calcium pump. In the normal mode the cycle turns i n a clock-wise d i r e c t i o n . Ca^ means ionized calcium i n the cytosol, Ca 0 means ionized calcium i n the ex t r a c e l l u l a r medium and P means inorganic phosphate. and E 2 are two conformational forms of the protein. Requirements of the reactions are indicated by aster i s k s . Taken from Schatzmann (1985) . 19 19a 1980b). These are properties associated with an acyl-phosphate bond, ind i c a t i n g that the phosphate group was transfered from ATP to a carboxylic acid group on the enzyme. In the absence of calcium, magnesium w i l l not i n i t i a t e phosphorylation, however magnesium greatly accelerates formation of the phosphorylated intermediate (Rega and Garrahan, 1975; Garrahan and Rega, 1978) . Schatzmann (1982) recently suggested that formation of the phosphoprotein may be a magnesium-dependent process, although there i s evidence to the contrary i n the l i t e r a t u r e (Rega and Garrahan, 1975). Monovalent cations, such as sodium and potassium, are also able to stimulate phosphoryla-t i o n of the enzyme (Larocca et a_l, 1981) . Once formed, the intermediate complex E-^.Ca.P undergoes a conversion to a second state, E 2.Ca.P. This step i s thought to r e s u l t i n calcium translocation by a reorientation of the calcium-binding s i t e towards the e x t r a c e l l u l a r side of the mem-brane and a reduction i n the a f f i n i t y of t h i s s i t e for calcium by several orders of magnitiude (Sarkadi, 1980). Rega and Garrahan (1978) demonstrated that t h i s step preceeded dephosphorylation and was stimulated by magnesium (K^ M g = 8 x 10"^ M, Garrahan and Rega, 1978). In the absence of magnesium, transformation from the E^Ca.P state to the E 2.Ca.P state i s believed to be very slow. Sarkadi (1980) has proposed that, i n the absence of magne-sium t h i s step becomes r a t e - l i m i t i n g . Lichtner and Wolf (1980a) have demonstrated that, analogous to calcium-dependent i n h i b i t i o n of ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y , excessive concentrations of calcium r e s u l t i n a large buildup of the phosphoprotein. This e f f e c t was attributed to i n h i b i t i o n of the conversion from 20 E-^.Ca.P into E 2.Ca.P. In the E 2 state, the phosphorylated intermediate i s more reactive than i n the E^ state, and can undergo rapid hydrolysis. Rega and Garrahan (1975) suggested that when the enzyme i s i n the E 2 state the phosphate group i s more accessible to water, hence more re a d i l y hydrolysed. In a recent study, Chiesi and coworkers (1983) showed that the binding s i t e for P^ becomes less polar when the enzyme i s i n the E 2 state as compared to the E-^  state. This finding suggests that the water for hydrolysis may be d i s -placed from another s i t e within the binding pocket, rather than enter the pocket due to an increase i n a c c e s s i b i l i t y . In the model shown i n Figure 1, Schatzmann (1985) has implied that the hydrolysis step i s accelerated by both magnesium and ATP. Rega and Garrahan (1978) showed that, i n the absence of magnesium, ATP could not stimulate decomposition of the phosphorylated i n t e r -mediate. However, i f the complex was formed i n the presence of magnesium, and then the magnesium subsequently removed, ATP was able to accelerate decomposition of the phosphoprotein. These re s u l t s suggest that magnesium was required at a step prior to dephosphoryation whereas free ATP stimulated the actual decom-posi t i o n of the complex. The monovalent cations, sodium and potassium, are also able to stimulate dephosphorylation of the phosphorylated intermediate, either by promoting conversion of the intermediate complex into the E 2 state or by d i r e c t l y stimu-l a t i n g breakdown of the phosphoprotein (Enyedi et a_l, 1980b; Larocca et a_l, 1981). A very slow backward operation of the reaction cycle (ATP synthesis from ADP and P i) has been demon-21 strated (Rossi et a_l, 1978; Wuthrich et a_l, 1979) . hence, th i s reaction step i s r e v e r s i b l e . However, a very large calcium gra-dient i s required, suggesting that reversal of dephosphorylation i s thermodynamically unfavorable (Schatzmann, 1985). The f i n a l step i n the transport cycle i s t r a n s i t i o n of the enzyme from the E 2 state back into the E^ ^ state. This allows the enzyme to begin a new reaction cycle. Although i t was mentioned previously that magnesium stimulates formation of the E^.Ca.P complex, Schatzmann (1985) has pointed out that the observed increase i n the rate of phosphorylation could as e a s i l y be ex-plained by accelerating the t r a n s i t i o n of the enzyme from E 2 back into the E-^  state. This proposed mechanism involves magnesium stimulation of the enzyme in a symmetrical fashion (E^ to E 2 ; E 2 to E-^ ) , however, i t assumes that the equilibrium between E 2 and E^ favors the E 2 state. The s i t e s of the regulatory e f f e c t s of calmodulin on the calcium pump transport cycle include both phosphorylation (Enyedi et a l , 1980; Muallem and K a r l i s h , 1980; Rega and Garrahan, 1980) and dephosphorylation (Rega and Garrahan, 1980; J e f f e r y et a l , 1981). However, there i s very l i t t l e known with regards to how these e f f e c t s are produced. Application of lanthanum to the external surface of erythro-cytes i n h i b i t s calcium transport and (Ca" + Mg )-ATPase a c t i v -i t y (Quist and Roufogalis, 1975; Szasz et a l , 1978b). This treatment also causes accumulation of the phosphorylated i n t e r -mediate (Szasz et a_l, 1978a; Schatzmann and Burgin, 1978) . These re s u l t s suggest that lanthanum i s reacting with the phosphory-lated intermediate while the enzyme i s i n the E 2 state. Lantha-22 num w i l l also stimulate phosphoenzyme formation i n erythrocyte membrane fragments, i n the absence of calcium (Szasz et a l , 1978a). In addition, Szasz and coworkers (1978a) found that membrane fragments and inside-out v e s i c l e s were affected by lower concentrations of lanthanum than were int a c t erythrocytes. These res u l t s suggest a possible i n t e r a c t i o n with the E.^  state of the enzyme as well as the E 2 state, leading to increased phospho-protein formation. Luterbacher and Shatzmann (1983) have sug-gested that lanthanum i n h i b i t s the conformational transformation of the phosphorylated enzyme from the E^ state into the E 2 state. This mechanism does not explain how lanthanum acts from the e x t r a c e l l u l a r surface, nor does i t explain how the intermediate complex i s formed i n the absence of calcium. To explain the a b i l i t y of lanthanum to i n h i b i t the pump from the e x t r a c e l l u l a r medium, Sarkadi (1980) has proposed that lanthanum binds to the phosphoprotein i n the E 2 state following the d i s s o c i a t i o n of calcium, thereby s t a b i l i z i n g the enzyme i n the phosphorylated form. IV Cystic F i b r o s i s Cystic F i b r o s i s (CF) i s a c l i n i c a l disorder characterized by chronic obstructive lung disease, exocrine pancreatic i n s u f f i -ciency and elevated sweat e l e c t r o l y t e s (di Sant 1 Agnese. and Talamo, 1967). Genetic evidence suggests tha CF i s transmitted as an autosomal recessive t r a i t (Lobeck, 1972; Sant' Agnese and Davis, 1976; Nader et a l , 1978). The incidence of CF among white 23 populations i s approximately 1 i n 2000 l i v e b i r t h s (Nadler et a l , 1978) , while 1 i n 20 individuals i s a healthy c a r r i e r (Danks et a l , 1965; Lobeck, 1972). The primary lesion(s) responsible for producing CF have not yet been i d e n t i f i e d . Biochemical and h i s t o l o g i c a l evidence shows that a l l exo-crine glands are affected i n c y s t i c f i b r o s i s . As a rule, the secretions of exocrine glands i n CF subjects behave i n abnormal ways; they are thicker than normal (Wood et a_l, 1976), tending to p r e c i p i t a t e and obstruct the ducts or passageways into which they are discharged. Sites of obstruction include lung airways, para-nasal sinuses, mucus-secreting submandibular glands, small int e s -t i n e , pancreas, b i l i a r y t r a c t , and male and female reproductive tracts (Wood et al_, 1976). This ducal obstruction can lead to f i b r o s i s of cer t a i n exocrine glands or a deficiency of the exo-crine secretion at i t s physiologic target organ. The end-results of t h i s obstruction process include chronic pulmonary i n s u f f i -ciency, hepatic c i r r h o s i s , pancreatic i n s u f f i c i e n c y , i n t e s t i n a l obstruction, mal-absorption and mal-digestion. A l l patients who are homozygous for CF also show a marked increase i n the level of sodium, chloride and, to a lesser ex-tent, potassium i n t h e i r sweat (di Sant' Agnese et a_l, 1953). Morphologically, no difference has been seen between the eccrine sweat glands of patients with CF and those i n control subjects (Munger et a_l, 1961) . Using i n v i t r o microperfusion, Mangos (1973) demonstrated that when normal sweat was perfused into CF sweat gland ducts, normal levels of sodium reabsorption were recorded, but when CF sweat was perfused into normal or CF sweat gland ducts, decreased levels of sodium reabsorption were obser-24 ved. These re s u l t s suggested that the abnormal e l e c t r o l y t e lev-els found i n CF sweat are due to the presence of an i n h i b i t o r y "factor" i n CF sweat and not a defect i n the ductal tissue. Increased concentrations of sodium and chloride i n sweat, without a p a r a l l e l a l t e r a t i o n i n precursor f l u i d (Schultz, 1969) suggests that the plasma membrane transport of e l e c t r o l y t e s by duct epithelium may be abnormal. Erythrocytes taken from CF patients have been used as a simple model system to examine plasma membrane transport of e l e c t r o l y t e s as a possible defect. Balfe et a_l (1968) found that sodium transport was abnormal i n erythrocytes from CF patients who were either homozygotes or heterozygotes. These authors suggested that i f the postulated defect of sodium transport observed i n CF patients represents a primary defect i n the disease state, a study of thi s abnormality in erythrocytes may help to determine the pathogenic e f f e c t . Subsequently, workers showed that only the ouabain-insensitive component of sodium e f f l u x (Lapey and Gardner, 1971) and ATPase a c t i v i t y (Cole and Dirks, 1972) were decreased in erythrocytes from CF patients. These findings implied that an ATPase a c t i v i t y other than the one coupled to sodium transport was affected i n the disease state. Horton et a_l (1970) measured the (C a 2 + + Mg )-ATPase a c t i v i t y of erythrocyte membranes from both normal subjects and CF patients and found a decreased a c t i v i t y i n the erythrocytes from CF subjects. Recently, these r e s u l t s have been confirmed (Katz, 1978a; Ansah and Katz, 1980; Foder et a l , 1980; Gietzen et a_l, 1984) and have been correlated with a decrease i n calcium uptake into inside-out v e s i c l e s prepared from CF erythro-25 cytes (Ansah and Katz, 1980). A similar reduction i n (Ca* + Mg 2 +)-ATPase a c t i v i t y has been found i n the plasma membranes of cultured skin f i b r o b l a s t s from CF patients (Katz, 1978b; Katz and Ansah, 1980). Katz (1978a) reported that the decrease i n ( C a 2 + + Mg 2 +)-ATPase was due to a reduction i n maximal ac t i v a t i o n and not a change i n the a f f i n i t y of the enzyme for calcium. These studies do not indicate whether the reduction i n plasma membrane (Ca* + Mg* )-ATPase a c t i v i t y observed i n CF subjects i s a primary defect or whether i t i s a secondary e f f e c t due to some other i n vivo or i n v i t r o a l t e r a t i o n i n the plasma membrane of these c e l l s . The defect could be i n the structure of the calcium-transporting enzyme i t s e l f . A l t e r n a t i v e l y , the changes i n (Ca + Mg )-ATPase a c t i v i t y may r e f l e c t some regula-tory or environmental modification which has occurred i n vivo or was induced during i n v i t r o handling of these CF c e l l s . This second e f f e c t , namely an a l t e r a t i o n induced during processing of the samples, may explain why several groups (Duffy et a_l, 1974; Feig et a_l, 1974; McEvoy et a_l, 1974) f a i l e d to observe any a l t e r a t i o n i n the a c t i v i t y of the (Ca + Mg )-ATPase i n eryth-rocytes taken from CF patients. Recently, Miner et a_l (1983) observed a s i g n i f i c a n t reduction i n formation of the calcium-dependent phosphorylated intermediate of the calcium pump in erythrocytes taken from CF patients, suggesting a reduction i n the number of active calcium-pumping s i t e s per c e l l . Glaubensklee and Galey (1984) reported a 40% reduction i n erythrocyte membrane associated immunoreactive calmodulin, suggesting a regulatory defect. However, addition of exogenous calmodulin to membrane fragments made from CF erythrocytes did not return ( C a 2 + + Mg 2 +)-26 ATPase a c t i v i t y to control values (Katz and Emery, 1981; Gietzen et a l , 1984). Other groups have shown differences i n the l i p i d composition of CF erythrocyte membranes with respect to normal subjects (McEnvoy et a_l, 1974; Rogiers et a l , 1980). Any of these changes could r e s u l t i n the deficiency i n (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y observed i n c y s t i c f i b r o s i s . 27 AIMS OF THE PRESENT STUDY A recently reported property of the calcium-transport ATPase in erythrocyte membranes concerns i t s i n h i b i t i o n by calcium (Klinger et a l , 1980; Vincenzi et a_l, 1980; Graf and Penniston, 1981) , which i s reportedly antagonized by magnesium (Klinger et a l , 1980). Lichtner and Wolf (1980a) showed that excess calcium produced a buildup of the phosphorylated intermediate. To ex-pl a i n these r e s u l t s , Schatzmann (1982) has proposed that excess calcium (greater that 5.0 x 10~ 5 M) in h i b i t e d the transformation of the intermediate complex from E-^.Ca.P to E2«Ca.P, by competing with magnesium at a "magnesium-specific" s i t e , r e s u l t i n g in i n h i -b i t i o n of dephosphorylation of the intermediate complex. The objective of the present study was to further characterize the in h i b i t o r y e f f e c t of calcium upon the transport cycle of the calcium transport ATPase. This required the determination of the effects of magnesium on the a b i b i l t y of calcium to i n h i b i t de-phosphorylation and the s e n s i t i v i t y of the phosphoprotein to dephosphorylation induced by either ATP or ADP. Calmodulin, which i s known to increase both the maximum ve l o c i t y and calcium a f f i n i t y of the (Ca* + Mg )-ATPase, has also been reported to stimulate the rate of formation (Muallem and K a r l i s h , 1980) and breakdown (Rega and Garrahan, 1980; Jef f e r y et a_l, 1981) of the phosphorylated intermediate of t h i s enzyme. Je f f e r y et a l (1981) found that magnesium and calmodulin both stimulated dephosphorylation on t h e i r own, however in the presence of magnesium (0.25 mM) calmodulin had no additional stimulatory effects and vice versa. These re s u l t s suggest that 28 these two activators may be acting at the same k i n e t i c step. Magnesium i s believed to enhance the rate of dephosphorylation by f a c i l i t a t i n g the transformation from E-^.Ca.P to E2.Ca.P (Rega and Garrahan, 1975) , hence th i s may also be the s i t e at which c a l -modulin stimulates dephosphorylation. The ef f e c t of calmodulin on the rate of phosphoenzyme formation, reported by Muallem and Kar l i s h (1980) , could be explained by either a d i r e c t e f f e c t on the formation of the phosphoenzyme or by increasing the rate at which E 2 returns to the E-^ conformation following dephosphory-l a t i o n . One aim of the present study was to further investigate the e f f e c t s of calmodulin on the formation of the intermediate complex in order to determine the actual step at which calmodulin exerts i t s e f f e c t s . Recently, Schatzmann (1982) suggested that magnesium may be required for the f i r s t step i n the transport cycle of the calcium pump, formation of a phosphorylated intermediate. Rega and Garrahan (1975) previously showed that phosphorylation of the enzyme did not require magnesium, however i t i s possible that there was s u f f i c i e n t magnesium contamination i n t h e i r preparation to s a t i s f y any magnesium requirements for phosphorylation. In the present study the magnesium chelator CDTA was employed to bind any remaining endogenous magnesium and es t a b l i s h whether or not there i s i n fact a magnesium requirement at t h i s stage of the reaction cycle. Decreased a c t i v i t y of the plasma membrane calcium trans-porting ATPase has been found i n association with c y s t i c f i b r o s i s . Previous reports have revealed a s p e c i f i c decrease i n 29 the ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y of erythrocyte membrane prepar-ations (Katz, 1978a). This finding has been supported by re-ports of a s i m i l a r reduction i n calcium uptake into inside-out ve s i c l e s prepared from the erythrocytes of subjects with c y s t i c f i b r o s i s (Ansah and Katz, 1980). Recently, Miner and coworkers (1983) demonstrated that, i n the presence of lanthanum, phospho-protein formation was less i n the erythrocyte membranes from subjects with c y s t i c f i b r o s i s than those of normal subjects. It remained to be determined whether or not t h i s change was due to a change i n the enzyme i t s e l f versus a change i n the number of active pumping s i t e s present. This question was therefore inves-tigated in t h i s study. 30 MATERIALS AND METHODS I. Materials The chemicals and/or proteins were purchased from the following sources: 1. Sigma Chemical Co. EGTA EDTA CDTA Tris-ATP 2-Mercaptoethanol T r i s base T r i s hydrochloride Lithium dodecylsulphate Bovine serum albumin Tr i c h l o r o a c e t i c acid Hydroxylamine monohydrochloride Bromophenol blue F o l i n and Ciocalteu's phenol reagent Glycerol HEPES Potassium chloride (KC1) Magnesium chloride (MgC^) Sodium carbonate Sucrose TEMED C i t r i c acid 31 Tris-ADP Potassium hydroxide (KOH) Potassium phosphate (KH-pPO^ ) Activated charcoal (Norit-A) Phosphoric acid C i t r i c acid (monohydrate) Sodium c i t r a t e Adenine hydrochloride 2. Amersham [gamma-32P]-ATP (Specific a c t i v i t y = 20-40 Ci/mmol) 3. ICN [2,8-3H]-ATP (Specific a c t i v i t y = 20-40 Ci/mmol) 4. Calbiochem Calmodulin (Bovine brain) 5. BDH Biochemicals Calcium chloride (CaCl 2) Sodium dodecylsulphate 6. Bio-Rad Aerylamide N,N'-methylene-bisaerylamide Coomassie B r i l l i a n t Blue R-250 Ammonium persulphate 32 SDS-PAGE High Molecular Weight Standards SDS-PAGE Low Molecular Weight Standards 7 . Fisher S c i e n t i f i c Co. Sodium chloride (NaCl) Lanthanum chloride (LaCl^) G l a c i a l Acetic acid Cello-Seal 8. New England Nuclear Aquasol l i q u i d s c i n t i l a t i o n f l u i d 33 II. Methods 1. Preparation of Calmodulin-Depleted Erythrocyte Membrane Fragments Human blood, not more than four days old, was either ob-tained from the Red Cross or c o l l e c t e d into EDTA-vacutainers and stored overnight before use. Erythrocytes were coll e c t e d by centrifugation at 2500 g for 5 minutes and red c e l l membranes prepared by the method of C a r a f o l i et a_l (1980) with some modifi-cations: Erythrocytes were washed three times i n f i v e volumes of 130 mM KC1 and 20 mM t r i s - C l pH 7.4. The c e l l s were then hemolysed i n f i v e volumes of 1 mM EDTA, 10 mM t r i s - C l pH 7.4 with s t i r r i n g for 10 minutes on ice followed by centrifugation at 18,000 g and 4°C for 10 minutes. This step was repeated f i v e more times after which the membranes were washed f i v e times i n 10 mM K-HEPES, pH 7.4. The ghosts were resuspended i n a storage buffer containing 20 mM K-HEPES pH 7.4, 130 mM KC1, 0.5 mM MgCl 2, 0.05 mM CaCl 2 and 2 mM d i t h i o t h r e i t o l . The membrane suspension was quick-frozen i n l i q u i d nitrogen and stored for up to 5 days at -80°C. Immediately before use, the membranes were thawed out, resuspended and washed three times i n a solution containing 40 mM K-HEPES pH 7.4 at 10°C. 2. Measurement of (Ca* + Mg* )-ATPase A c t i v i t y Ca* -ATPase a c t i v i t y was measured by a modification of the 34 method of Katz and Blostein (1975) i n 0.2 ml of a medium con-taining ( f i n a l concentration) 2 uM tris-ATP (containing [gamma-3 2P]-ATP; 0.056 Ci/mmol), 0.1 mM EGTA, 40 mM K-HEPES pH 7.4, 0.05 mM ouabain and the desired free calcium and magnesium concentra-tions. Calmodulin, lanthanum chloride, and/or CDTA were added to t h i s medium when required. After preincubation for f i v e minutes at 10°C, the assay was started by the addition of 0.1 ml of membrane suspension (1-2 mg/ml). In experiments where deter-mination of phosphoprotein formation and ATPase a c t i v i t y were performed simultaneously, a more concentrated membrane suspen-sion was used (2-4 mg/ml). After ten minutes the reaction was terminated by the rapid addition of 0.4 ml of an ice-cold stop solution containing 5% t r i c h l o r o a c e t i c acid (w/v), 2.5 mM ATP (disodium salt) and 5 mM potassium phosphate. The membranes were then pelleted by centrifugation at 1500 g and 4°C for 10 minutes and 0.3 ml of the supernatant was combined with 0.6 ml of charcoal suspension (0.19 g of Fisher Norit-A/ml 5% TCA). After f i v e minutes of vigorous mixing the samples were centrifuged for f i v e minutes at 3000 g and 0.45 ml of the clear supernatant transfered to counting v i a l s , 5 ml of Aquasol added and mixed. The v i a l s were counted for J*P and the amount of ATP hydrolysis present expressed as pmol ATP hydrolysed per mg protein per minute as determined by the following equations: s p e c i f i c a c t i v i t y = (media counts - background) t o t a l ATP ATPase = (sample counts - b l a n k ) ( d i l ' n factor) ( s p e c i f i c a c t i v i t y ) ( r x n vol)(rxn time)(protein cone) 35 where: s p e c i f i c a c t i v i t y = P counts (dpm) per pmol ATP. media counts = P counts (dpm) present i n 0.02 ml of media, t o t a l ATP = t o t a l amount of ATP present i n the incubation medium (400 pmol). background = P counts (dpm) in f l u o r alone, sample counts = J*P counts (dpm) obtained per sample, blank = P counts (dpm) obtained i n the absence of membranes, d i l u t i o n factor = correction for reaction volume sampled, reaction volume = f i n a l volume of reaction medium (0.2 ml), reaction time = period of enzyme-catalyzed hydrolysis, protein cone. = concentration (mg/ml) of erythrocyte membrane protein present i n the reaction medium. Ca* -dependent ATPase a c t i v i t y was determined by subtracting the a c t i v i t y obtained i n the presence of EGTA alone from that obtained when calcium was added to the reaction medium. 3. Phosphorylation of Erythrocyte Membranes Phosphorylation of the membranes was performed at 10°C i n 0.2 ml of a medium consisting of ( f i n a l concentration) 40 mM K-HEPES pH 7.4, 2 uM tris-ATP (containing [gamma-32P]-ATP, f i n a l a c t i v i t y 1.13 Ci/mmol; [2,8-3H]-ATP, f i n a l a c t i v i t y 1.13 Ci/mmol), 0.1 mM tris-EGTA, 0.05 mM ouabain, and the desired free calcium and magnesium concentrations. Calmodulin, lanthanum chloride and/or CDTA were added to t h i s medium when required. After f i v e minutes of preincubation at 10°C the assay was started 36 by the a d d i t i o n of 0.1 ml of membrane suspension (2-4 mg/ml). In experiments where phosphoprotein formation was determined a t v a r i o u s time p o i n t s , the membrane suspension contained a l l reagents, except ATP, a t t h e i r f i n a l c o n c e n t r a t i o n s . The r e a c t i o n was terminated a f t e r v a r i o u s time i n t e r v a l s by the r a p i d a d d i t i o n of 0.4 ml of an i c e - c o l d s o l u t i o n c o n t a i n i n g 5% t r i c h l o r o a c e t i c a c i d (w/v), 2.5 mM ATP (disodium s a l t ) and 5 mM potassium phosphate. The membranes were then p e l l e t e d by cen-t r i f u g a t i o n a t 1500 g and 4°C f o r 10 minutes. The supernatant was decanted and the p e l l e t a p p l i e d onto g l a s s m i c r o - f i b e r f i l -t e r s (Whatman GF/A), washed e x t e n s i v e l y w i t h an i c e - c o l d s o l u t i o n c o n t a i n i n g 5% t r i c h l o r o a c e t i c a c i d (w/v), a i r d r i e d and counted f o r J*P and JH. The amount of phosphoprotein formed, expressed as fmol per mg p r o t e i n , was determined by the f o l l o w i n g c a l c u l a t -i o ns . s p e c i f i c a c t i v i t y = (media counts - background) t o t a l ATP phosphoprotein = ( sample counts - background) _ ( s p e c i f i c a c t i v i t y ) ( r e a c t i o n v o l ) ( p r o t e i n cone) ( sample 3H counts - background) ( s p e c i f i c a c t i v i t y ) ( r e a c t i o n v o l ) ( p r o t e i n cone) where: s p e c i f i c a c t i v i t y = J*P/ JH (dpm) counts per pmol ATP. media counts = J*P/ JH counts (dmp) present i n 20 p i of media. t o t a l ATP = t o t a l amount of ATP present i n the medium (400 pmol). background = J*P/ JH counts (dpm) i n f l u o r a l o n e . sample counts = P/ H counts (dpm) o b t a i n e d i n sample. 37 reaction volume = f i n a l volume of the reaction medium (0.2 ml), protein concentration = concentration (mg/ml) of red c e l l membrane protein present i n the reaction medium. a. Dephosphorylation Experiments. Erythrocyte membrane fragments (1-2 mg/ml) were incubated as described previously, for 15 seconds at 10°C i n phosphorylation medium containing the desired cations, then 20 p i of a solution containing either 11 mM ADP or 11 mM cold ATP was added. After either 5 or 15 seconds the reaction was terminated by rapid addition of 0.4 ml of an ice cold solution containing 5% t r i c h l o r o a c e t i c acid (w/v), 2.5 mM ATP (disodium salt) and 5 mM potassium phosphate. The membranes were pelleted, washed and counted as described above. Total phosphorylation was that amount of phosphoprotein formed a f t e r a 15 second reaction time. The f r a c t i o n of the t o t a l phospho-protein remaining a f t e r the 'chase' was determined separately for each experiment then the mean and the standard error of the mean was calculated for each condition. b. Hydroxy1amine Treatment: Erythrocyte membrane fragments were incubated for 15 seconds at 10°C i n phosphorylation medium containing the desired cations, as described previously, and then the reaction stopped by adding 0.4 ml of an i c e - c o l d solution containing 15% t r i c h l o r o a c e t i c acid (w/v). The membranes were then pelleted by centrifugation at 1500 g and 4°C for 10 minutes and the supernatant decanted. Membranes were resuspended i n 0.5 ml of either 0.6 M hydroxylamine/ 0.8 M sodium acetate pH 5.2 or 38 0.6 M sodium chloride/ 0.8 M sodium acetate pH 5.2 (control). After 10 minutes at room temperature, 2 ml of ice-cold 15% t r i -chloroacetic acid (w/v) was added to the sample and the membranes pelleted by centrifugation at 1500 g and 4°C for 10 minutes, the supernatant removed and the p e l l e t resuspended i n either 5% t r i c h l o r o a c e t i c acid (for f i l t r a t i o n ) or sample buffer (for acid LiDS-PAGE). 4. Polyacrylamide Gel Electrophoresis and Autoradiography  of Phosphorylated Erythrocyte Membrane Fragments a. Polyacry1amide-Gradient Acid-Gel Electrophoresis. Poly-acrylamide gradient (5-15%) gels of 1.5 mm thickness were cast according to the method of Laemmli and Favre (1973) . Buffers and running conditions which' would s t a b i l i z e the acy1-phosphate i n -termediate of the erythrocyte membrane (C a 2 + + Mg 2 +)-ATPase were modified from those described by Lichtner and Wolfe (1979). The 5% "resolving" gel consists of 2.5 ml of an acrylamide-methylene-bisacrylamide (30%: 0.8%) mixture, 0.1 M tris-phosphate buffer pH 7.0, 1.35% g l y c e r o l , 0.2% lithium dodecylsulphate (LiDS), 0.025% tetraethyl-methylenediamine (TEMED) and 0.3 ml ammonium persul-phate (7.5 mg/ml) in a t o t a l volume of 15 ml. The 15% "re-solving" gel contained 7.5 ml of aerylamide-methylene-bisacry1-amide (30%: 0.8%), 0.1 M tris-phosphate buffer pH 7.0, 5.75% g l y c e r o l , 0.2% LiDS, 0.05% TEMED and 0.225 ml ammonium persul-phate (7.5 mg/ml) i n a t o t a l volume of 15 ml. The 5% and the 15% "resolving" gel solutions were immediatly added to the gel chamber with the aid of a gradient former. About 0.5 ml of 39 d i s t i l l e d water was c a r e f u l l y layered on top of the gel and the gel was allowed to polymerize for 3-4 hours at room temperature. The d i s t i l l e d water was decanted p r i o r to the addition of the "stacking" g e l . The "stacking" gel (3.5%) consisted of 0.855 ml of a c r y l -amide-methylene-bisacrylamide (30%: 0.8%) mixture, 0.125 mM t r i s -phosphate buffer pH 6.5, 0.2% LiDS, 0.136% TEMED and 0.2 ml ammonium persulphate (15 mg/ml) in a t o t a l volume of 7.33 ml. After mixing, the 'stacking' gel solution was gently poured into the gel chamber over the polymerized 'resolving' g e l . A t e f l o n 'comb' was then inserted into the stacking gel and the gel a l -lowed to polymerize for at least 2 hours. The gel buffer contained 20 mM tris-phosphate pH 7.0 and 0.2% LiDS. Gels were run at 4°C under constant current (40 mA/ slab) for 7 hours. The protein standards used for estimation of molecular weight (in daltons) were; myosin (200,000), B-galactosidase (116,250), phosphorylase b (92,500), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin i n h i b i t o r (21,500) and lysozyme (14,400). b. Sample Preparation. When phosphorylated erythrocyte membranes were to be analysed on an acid gel the the reaction volume was reduced to 75 p i , JH-ATP was omitted from the medium the s p e c i f i c a c t i v i t y of the *P-ATP was increased to 6.01 Ci/mmol ( f i n a l a c t i v i t y ) and the reaction was stopped with 0.4 ml 40 of an ice-cold solution containing 15% t r i c h l o r o a c e t i c acid (w/v). After stopping the reaction, the samples were centrifuged at 1500 g and 4°C for 10 minutes and the supernatant removed. The p e l l e t was then resuspended i n 25 p i of sample buffer con-taining ( f i n a l concentration); 50 mM tris-phosphate pH 6.5, 2.5% LiDS, 0.12 M sucrose, 0.5 M 2-mercaptoethanol and 0.01% bromo-phenol blue. Samples were heated at 60°C for 10 seconds, the pH neutralized with 2 M tris-base and 50 pi of each sample was applied per well. c. Staining, Destaining and Autoradiography. The gels were stained with 0.25% Coomassie B r i l l i a n t Blue R-250 in methanol/acetic acid/water (5:1:4) for 30 minutes at room temper-ature, followed by a f i r s t destaining procedure i n methanol/acetic acid/water (45:1:15) for 1 hour (3 changes) and a second destaining procedure i n methanol/acetic acid/water (4:1:15) u n t i l the background became transparent. Prior to drying, the gels were fixed i n acetic acid/glycer-ol/water (10:3:87) for 30 minutes, then 70% methanol for 20 minutes. The gels were immediatly dried under vacuum at 80°C for 90 minutes. The dried gels were exposed to X-ray f i l m (Kodak Min-R) along with an i n t e n s i f y i n g screen (Cronex Lightning Plus, Dupont) for 7-10 days at -80°C following which the films were developed. 5. Miscellaneous Methods a. Protein Assay. Erythrocyte membranes (10-80 pg) were 41 assayed for protein using the standard protein assay of Lowry et al (1951). Bovine serum albumin was used as the standard protein. b. Non-linear Analysis. Data was analysed using a FORTRAN non-linear reagression package 'NON-LIN' which f i t the data to pre-chosen k i n e t i c models. The models chosen were; i . For ATPase data: Model 1 Vobs = ( V m a x ) (S)/ (S + Kj_ + S 2/K 2) Model 2 Vobs = ( V m a x> (S)/ (S + K 1) where; V"obs = observed reaction v e l o c i t y . V"max = maximum reaction v e l o c i t y . S = substrate (free calcium) concentration. = f i r s t d i s s o c i a t i o n constant for the substrate. K 2 = second d i s s o c i a t i o n constant for the substrate, i i . For Phosphorylation data: vobs = (V 1)(S)/(S + K x) + (V 2)(S)/(S + K 2) where: vobs = observed phosphoprotein formation. V-^  = maximum E.P formation for the f i r s t component. S = substrate (free calcium) concentration. K-^  = f i r s t d i s s o c i a t i o n constant for the substrate. V 2 = maximum E.P formation for the second component. K 0 = second d i s s o c i a t i o n constant for the substrate. 42 c. S t a t i s t i c a l Analysis. S t a t i s t i c a l analysis of a l l data was performed using a general least squares analysis of variance package (UBC:GENLIN). The v a r i a b i l i t y i n the mean values was expressed as the standard error of the mean (S.E.M.). Where mean values representing two or more experimental conditions were to be compared, the n u l l hypothesis (no difference) was adopted. In order to test t h i s hypothesis, a Newman-Keuls multiple range test was used to determine confidence i n t e r v a l s , at both the 0.05 and the 0.01 significance l e v e l s , for each sample mean. If two means being tested were found to occupy the same confidence i n t e r v a l , the n u l l hypothesis was accepted. However, i f the means did not occupy the same confidence i n t e r v a l the n u l l hypothesis was rejected and the means values were said to be s i g n i f i c a n t l y d i f f e r e n t at the given significance l e v e l . Inter-assay v a r i a -b i l i t y was compensated for by performing an analysis of covariance. d. Determination of Free Calcium Concentration. Free c a l -cium concentrations were determined using the Fortran program 'CATIONS' written by Goldstein (1979). Equilibrium constants for cations and ligands were obtained fron Martell and Smith (1979) and were corrected for io n i c strength, pH and temperature ac-cording to the methods described by these authors. Equilibrium constants for monoprotonated species were calculated according to the procedure of Blinks et a_l (1982) . 43 RESULTS 1. E f f e c t of Calcium, Magnesium, Calmodulin and Lanthanum on Calcium-Dependent ATP Hydrolysis and Phosphoprotein  Formation. In these studies, calmodulin-depleted erythrocyte membranes were prepared by the method of C a r a f o l i et a_l (1980) , stored at -80°C and used within 5 days. Unless stated otherwise assays of (C a 2 + + Mg 2 +)-ATPase a c t i v i t y and calcium-dependent phospho-protein (E.Ca.P) formation were carr i e d out at 10°C i n the pre-sence of 40 mM K-HEPES pH 7.4, 25 pM MgCl 2 and 2 pM ATP, to reduce the turnover of the enzyme to f a c i l i t a t e determination of E.Ca.P and to minimize kinase-mediated phosphorylation. a. E f f e c t of Calcium Concentration. Figure 2 shows calcium dependent ATP hydrolysis and E.Ca.P formation as a function of o + p + free calcium (Ca ) concentration. The e f f e c t of Ca concen-t r a t i o n on ATPase a c t i v i t y was two-fold; i t stimulated hydrolysis at low concentrations but i n h i b i t e d a c t i v i t y at concentrations greater than 4-6 x 10 J M. Maximum v e l o c i t y , from the mean of a number of experiments, under the assay conditions used was 49.4 +_ 3.7 pmol/mg/min (Table 1). Analysis of the ac t i v a t i o n curve by non-linear regression, as described i n Methods, revealed that C a 2 + stimulation of (Ca 2 ++Mg 2 +)-ATPase a c t i v i t y , i n these c a l -modulin-depleted membranes, was characterized by a d i s s o c i a t i o n constant (K^) of 4.24 x 10 -^ M and calcium-dependent i n h i b i t i o n of the enzyme was characterized by a d i s s o c i a t i o n constant (K 2) of 1.68 x 10 M (Table 1). When determining phosphoprotein 44 Figure 2. y + y + • • E f f e c t of calcium concentration on (Ca* + Mg )-ATPase a c t i v i t y and calcium-dependent phosphoprotein formation in erythrocyte membrane fragments at various calcium concentrations. ATPase a c t i v i t y (• •) and phosphoprotein formation (• •) were deter-mined as described i n Methods. ATPase and phosphoprotein refer to the calcium component afte r subtraction of the values measured in the presence of EGTA (0.1 mM) alone. The res u l t s shown here are the mean +_ S.E.M. of observations from at least f i v e membrane preparations with each membrane preparation assayed i n t r i p l i c a t e . 4 5 45a Table 1 Kinetic parameters of the ef f e c t of calcium and calmodulin on (C a 2 + + Mg 2 +)-ATPase a c t i v i t y and the formation of the phosphory-lated intermediate of the calcium pump from human erythrocyte membrane fragments. 1. ATPase a c t i v i t y : control + calmodulin (n=6) a (n=6) K l (x 10" 7 M) c 4.24 + 0.98b 0.84 + 0.25 K2 (x 10~ 3 M) c 1.68 + 0.31 0.72 + 0.18 V m a x (pmol/mg/min)c 49.4 + 3.7 93.9 + 6.0 2. Phosphoprotein formation: control (n=5) + calmodulin (n=5) K l (x 10" 8 M) d 5.06 + 1.51 4.72 + 0.82 K 2 (x 10~ 4 M) d 7.16 + 0.27 8.89 + 0.82 V l (fmol/mg) d 400 + 18 641 + 45 V2 (fmol/mg) d 4020 + 210 3835 +_ 221 1n' refers to the number of membrane preparations assayed; assay was performed i n t r i p l i c a t e . Results are expressed as the mean _+ SEM. Kinetic constants were estimated by model 1 described i n Methods Kinetic constants were estimated as described i n Methods 46 9 + formation as a function of Ca* concentration, a 15 second reac-t i o n time was chosen i n order to allow measurement of the steady-state E.Ca.P levels while minimizing the contribution from kinase-mediated phosphorylation. As shown i n Figure 2, the calcium-dependent E.Ca.P formation was biphasic, with a high calcium a f f i n i t y component, = 5.1 x 10 M and a low calcium a f f i n i t y component, K 2 = 7.2 x 10~ 4 M (Table 1). The high a f f i -n i t y component was associated with low levels of E.Ca.P and a very shallow response to changing calcium concentration, whereas in the region of the low a f f i n i t y component E.Ca.P rose sharply to reach plateau levels which were 10 times higher than observed in the high a f f i n i t y region of the calcium curve. The low a f f i -n i t y component of E.Ca.P and the calcium-dependent i n h i b i t i o n of (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y demonstrated very similar calcium d i s s o c i a t i o n constants, 7.2 x 1 0 - 4 M and 1.9 x 10~ 3 M (Table 1), respectively. Autoradiograms of phosphorylated erythrocyte membranes, as shown i n Figure 3, reveal that at a l l calcium concentrations T 9 tested the majority of the J*P lable was incorporated into a protein with an apparent molecular weight of approximatly 140,000 daltons. Phosphorylation of t h i s protein was calcium-dependent and sensitive to treatment with hydroxylamine. This confirmed that the high level's of phosphoprotein formed i n the presence of millimolar concentrations of calcium did, i n f a c t , represent the acy1-phosphate intermediate of the calcium pump (E.Ca.P) and not a kinase-mediated phosphorylation of other membrane proteins. The r a t i o of ATPase a c t i v i t y to E.Ca.P formation, referred 47 Figure 3 . Acid-Polyacrylamide gel electrophoresis and autoradiography of calcium dependent acy1-phosphate formation and hydroxylamine s e n s i t i v i t y . 3 2 P was incorporated at the indicated free calcium concentrations followed by treatment with either 0.6 M hydroxyl-amine/ 0.8 M Na-acetate, pH 5.2 (+) or 0.6 M NaCl/ 0.8 M Na-acetate, pH 5.2 (-). Proteins were denatured with LiDS then separation on a 5-15% acrylamide-gradient slab g e l . Phosphory-l a t i o n , hydroxylamine treatment, electrophoresis, autoradio-graphy, and determination of free calcium concentrations were as described in Methods. 48 MWt 1 MWt KD KD o r i g i n 200 116 - w " 116 66.2 - • 66.2 45 — o r i g i n 200 45 31 - " 31 14.4 - " 14.4 hydroxylamine - + - + — + -log [Ca] 8 7 6 — + — + — + hydroxylamine 5 4 3 -log [Ca] M to as the turnover number of the enzyme, i s an index of the rate at which the individual pump molecules are cyclin g under the various assay conditions u t i l i z e d . Figure 4 shows that turnover of the enzyme was highest at calcium concentrations of 1-5 x 10"^ M. At t h i s calcium concentration ATPase a c t i v i t y was reaching maximum ve l o c i t y whereas very l i t t l e E.Ca.P was accumulated. To study the eff e c t of free calcium concentration on the rate of formation and steady-state level of E.Ca.P two calcium concentrations were chosen for further investigation from the curve of ATPase a c t i v i t y versus calcium concentration shown i n Figure 2. The free calcium values chosen were 1.0 x 10 -^ M and 4.0 x 1 0 - 4 M. At both of these calcium concentrations ATPase a c t i v i t y was approximatly 70-80% of maximum v e l o c i t y , however 1.0 — ft y + x 10 M Ca* i s i n the range where calcium activates ATPase a c t i v i t y whereas 4.0 x 10~ 4 M C a 2 + i s i n the range of calcium-i n h i b i t i o n . Determination of E.Ca.P formation over time in the presence of 1.0 x 10"^ M or 4.0 x 10~ 4 M C a 2 + , as shown i n Figure 5 indicates that in the presence of 4.0 x 10~ 4 M C a 2 + , E.Ca.P reached steady-state levels within 15 to 20 seconds whereas i n the presence of 1.0 x 10 -^ M C a 2 + steady state was not reached even afte r 30 seconds. Under these conditions ATPase a c t i v i t y was greater at the lower calcium concentration. Lanthanum i s a t r i v a l e n t cation known to i n h i b i t calcium transport (Quist and Roufogalis 1975, Sarkadi et a_l 1977) and calcium-dependent ATPase a c t i v i t y (Weiner and Lee 1972) i n eryth-rocyte membranes. Several authors (Szasz et a l 1978, Schatzmann and Burgin 1978, Muallem and K a r l i s h 1982) have also found that lanthanum increased the steady-state level of phosphoprotein. 49 Figure 4 . E f f e c t of calcium concentration on the turnover of the (Ca + Mg* )-ATPase i n erythrocyte membrane fragments. The turnover number i s the r a t i o of ATPase a c t i v i t y to phosphoprotein forma-t i o n . The r e s u l t shown i s the mean _+ S.E.M. of observations fron f i v e membrane preparations; each membrane preparation was assayed i n t r i p l i c a t e . 50 300 5oa Figure 5. Eff e c t of calcium concentration on (Ca* + Mg* )-ATPase a c t i v i t y and the time course of calcium-dependent phosphoprotein formation in erythrocyte membrane fragments. ATPase a c t i v i t y and phospho-protein formation were measured in the presence of either 1 pM (• •) or 0.4 mM (• •) free calcium. ATPase a c t i v i t y and phosphoprotein formation were determined as described i n Methods. The res u l t shown i s the mean _+ S.E.M. of observations from f i v e membrane preparations; each membrane preparation was assayed i n t r i p l i c a t e . ATPase a c t i v i t y and phosphoprotein formation were determined i n p a r r a l l e l for each membrane preparation. Single asterisk indicates s i g n i f i c a n t l y d i f f e r e n t from control at p < 0.05. Double asterisk indicates s i g n i f i c a n t l y d i f f e r e n t from control at p < 0.01. 51 3000 > —I 13 t i me (sec) 51a Lanthanum i s now thought to i n h i b i t dephosphorylation of the phosphorylated intermediate. As the rate of formation and steady state level of E.Ca.P observed i n a time-course study actually represents the net e f f e c t of phosphorylation and dephosphoryla-t i o n reactions, the e f f e c t of lanthanum on the time-course was studied in order to determine the rate of formation and steady-state level of phosphoprotein when i t s breakdown was i n h i b i t e d . In t h i s study, shown i n Figure 6, lanthanum (0.1 mM) inhibited ATPase a c t i v i t y by 68% while i t stimulated the rate of phospho-protein formation and increased the steady-state level of phos-phoprotein by 141%. In a recent a r t i c l e Luterbacher and Schatzmann (1983) suggested that lanthanum blocked dephosphoryla-tion by i n h i b i t i n g the conformational change from E-^.Ca.P to E 2.Ca.P. This theory was tested by examining the e f f e c t of lanthanum (0.2 mM) on the amount of phosphoprotein formed i n the presence of 10 mM calcium. The buildup of phosphoprotein in the presence of 10 mM calcium i s also believed to be due to an i n h i b i t i o n of dephosphorylation either at t h i s same step or at the subsequent step, by l i m i t i n g the d i s s o c i a t i o n of calcium from the low a f f i n i t y binding s i t e on E 2.Ca.P. In the presence of both lanthanum and calcium, phosphoprotein formation was re-duced by 71% compared to calcium alone (Table 2). Thus, lan-thanum appeared to compete with the a b i l i t y of calcium to accumu-late phosphoprotein. b. E f f e c t of Calmodulin. Calmodulin was added to c a l -modulin-depleted erythrocyte membranes i n order to study i t s 52 Figure 6. Eff e c t of lanthanum on (C a 2 + + Mg 2 +)-ATPase a c t i v i t y and the time course for calcium-dependent phosphoprotein formation i n erythro-cyte membrane fragments. ATPase a c t i v i t y and phosphoprotein formation, measured i n the presence of either 0.15 mM t o t a l added CaCl 2 (• • ) (control) or 0.15 mM CaCl 2 plus 0.1 mM L a C l 3 (• • ) , were determined as described i n Methods. The r e s u l t shown i s the mean _+ S.E.M. of observations from four membrane preparations; each membrane preparation was assayed i n t r i p l i -cate. ATPase a c t i v i t y and phosphoprotein formation were deter-mined simultaneously for each membrane preparation. Asterisk indicates s i g n i f i c a n t l y d i f f e r e n t from control, p < 0.01. 53 Table 2. The e f f e c t of LaCl-j on formation of the phosphorylated i n t e r -mediate of the (Ca 2 + Mg 2 +)-ATPase in the presence and absence of 10 mM Ca C l 2 . Condition Phosphoprotein formation % change (fmol/mg) 0.2 mM L a C l 3 429 10 mM CaCl 2 4462 0.2 mM L a C l 3 + 1300 -71% 10 mM CaCl 2 Phosphoprotein formation was measured as described i n methods. Results shown represent a single experiment. 54 e f f e c t on both the rate of formation and the steady-state levels of E.Ca.P. The e f f e c t s of calmodulin on the (Ca 2 + + Mg 2 +)-ATPase i n erythrocyte plasma membranes are well documented: they are, ^ an increase i n the observed maximum ve l o c i t y ; and 2^ an increase in the a f f i n i t y of the enzyme for calcium. This i s demonstrated in Figure 7, showing the effects of exogenously added calmodulin (0.01 mg/ml) on ATPase a c t i v i t y at various C a 2 + concentrations. Calmodulin increased the V m a x for hydrolysis of ATP by 90% (from 49.4 to 93.9 pmol/mg/min), produced a small but not s i g n i f i c a n t decrease in the d i s s o c i a t i o n constant for calcium (from 4 x 1 0 - 7 to 0.8 x 1 0 - 7 M; Table 1), but had no e f f e c t on the calcium-dependent i n h i b i t i o n of ATPase a c t i v i t y . In contrast to i t s e f f e c t s on ATPase a c t i v i t y , calmodulin addition had no s i g n i f i -cant e f f e c t on E.Ca.P levels when assayed at 15 seconds. In the presence of calcium concentrations ranging from 10"^ to 10 - 4 M, exogenously added calmodulin produced a small, but not s i g n i -f i c a n t increase i n E.Ca.P levels (Table 1). However, at higher C a 2 + concentrations exogenous calmodulin had no e f f e c t on E.Ca.P (Table 1, Figure 8a). Analysis of t h i s data by either non-linear regression (Table 1) or by an Eadie-Hoffstee plot (Figure 8b) did not reveal any calmodulin-induced changes i n either of the two calcium d i s s o c i a t i o n constants. Turnover of the enzyme (Figure 9) was increased by calmodulin at low calcium concentra-tions, however t h i s e f f e c t was only s i g n i f i c a n t at the lowest — fi calcium concentration used (5.08 x 10 M). To examine the e f f e c t of calmodulin on E.Ca.P formation as a function of time, two calcium concentrations were chosen from Figure 1 as i n the previous section, one i n the range of calcium 55 Figure 7. E f f e c t of calmodulin on (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y i n calmo-dulin-depleted erythrocyte membrane fragments at various calcium concentrations. Calmodulin-stimulated (10 ug/ml) (• • ••) and basal (• •) ATPase a c t i v i t y was determined as described in Methods. ATPase refers to the calcium component of hydrolysis after subtraction of the values measured in the presence of EGTA (0.1 mM) alone. The r e s u l t shown i s the mean + S.E.M. of obser-vations from six membrane preparations; each membrane preparation was assayed i n t r i p l i c a t e . Asterisk indicates s i g n i f i c a n t l y d i f f e r e n t from basal a c t i v i t y , p < 0.01. 56 Figure 8 a and b. Effec t of calmodulin on calcium-dependnet phosphoprotein forma-tion i n calmodulin-depleted erythrocyte membrane fragments at various free calcium concentrations. Calmodulin-stimulated (10 pg/ml) (• •) and basal (• •) phosphoprotein formation were determined as described i n Methods. Phosphoprotein refers to the calcium component of phosphorylation after subtraction of the values measured i n the presence of EGTA (0.1 mM) alone. The re s u l t shown i s the mean +_ S.E.M. of observations from f i v e membrane preparations; each membrane preparation was assayed in t r i p l i c a t e . Figure 8b i s an Eadie-Hoffstee plot of the same data. 57 4500 -8 -7 -6 -5 -4 -3 -2 -1 l o g [ C a + + J CM) 57a 4500 b 3600H 57b Figure 9. Turnover of the (Ca* + Mg )-ATPase i n erythrocyte membrane fragments i n the absence (• •) (control) and presence (• •) of exogenous calmodulin (10 ug/ml) at various free calcium con-centrations. The turnover number i s the r a t i o of ATPase a c t i v i t y to phosphoprotein formation. Free calcium concentrations were determined as described i n Methods. The r e s u l t shown i s the mean _+ S.E.M. of observations from f i v e membrane preparations; each membrane preparation was assayed in t r i p l i c a t e . Asterisk i n d i -cates s i g n i f i c a n t l y d i f f e r e n t from control, p < 0.01. 58 4D0 C 3 0 G -l o g ( C a + +| CM) 58a a c t i v a t i o n of the ATPase (1.0 x 10~ D M) and the other i n the range of calcium-dependent i n h i b i t i o n of ATPase a c t i v i t y (4.0 x 10~ 4 M). In the presence of 1.0 x 10~ 6 M C a 2 + (Figure 10), calmodulin stimulated ATPase a c t i v i t y by 64% and increased the rate of formation of E.Ca.P and i t s level at a l l time points up to 30 seconds. Under these conditions steady-state was not reached. When the calcium concentration was increased to 4.0 x 10~ 4 M (Figure 11), calmodulin enhanced (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y by 48%; i t did not e f f e c t the rate of formation of E.Ca.P but i t reduced the steady-state for E.Ca.P by 27%. As discussed i n the previous section, lanthanum i s thought to block dephosphorylation of the phosphorylated intermediate. In order to observe the e f f e c t of calmodulin on the formation of the phosphoprotein with minimal interference from dephosphoryla-ti o n , lanthanum (0.1 mM) was added to the reaction medium. In the presence of 0.1 M L a C l 3 and 0.15 mM CaCl 2 (Figure 12), calmo-dul i n stimulated ATP hydrolysis and i t increased the apparent rate of formation and steady-state level of E.Ca.P. c. E f f e c t s of Magnesium. In order to determine i f magne-sium i s a pre-requisite for formation of the ternary complex E-^.Ca.P, the magnesium chelator CDTA was added to the assay medium in place of magnesium. Figure 13 shows (Ca + Mg )-ATPase a c t i v i t y , at various free calcium concentrations, i n the presence of either 0.025 mM Mg* (the standard assay condition) or 0.1 mM CDTA. In the absence of free magnesium, the V m a x for ATP hydrolysis was reduced from 25.0 to 17.4 pmol/mg/min and the 59 Figure 10. Ef f e c t of calmodulin on (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y and the time course of calcium-dependent phosphoprotein formation i n erythrocyte membrane fragments i n the presence of 1 uM free calcium. Calmodulin-stimulated (10 ug/ml) (• •) and basal (• •) ATPase a c t i v i t y and phosphoprotein formation were deter-mined as described in Methods. The re s u l t shown i s the mean _+ S.E.M. of f i v e membrane preparations; each membrane preparation was assayed i n t r i p l i c a t e . ATPase a c t i v i t y and phosphoprotein formation were determined simultaneously for each membrane pre-paration. Asterisk indicates s i g n i f i c a n t l y d i f f e r e n t from basal a c t i v i t y , p < 0.01. 60 (Buu/Touij.) u i a ^ o j d o L | d s o L | d Figure 11. Eff e c t of calmodulin on (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y and the time course of calcium-dependent phosphoprotein formation i n erythrocyte membrane fragments i n the presence of 0 . 4 mM free calcium. Calmodulin-stimulated (10 pg/ml) (• •) and basal (• •) ATPase a c t i v i t y and phosphoprotein formation were deter-mined as described i n Methods. The r e s u l t shown i s the mean +_ S.E.M. of observations from f i v e membrane preparations; each membrane preparation was assayed i n t r i p l i c a t e . ATPase a c t i v i t y and phosphoprotein formation were determined simultaneously for each membrane preparation. Asterisk indicates s i g n i f i c a n t l y d i f f e r e n t from basal a c t i v i t y , p < 0.01. 61 ( B O J / T O U J J - ) u i a ^ o j d o L j d s o L j d Figure 12. Ef f e c t of calmodulin on (C a 2 + + Mg 2 +)-ATPase a c t i v i t y and the time course of calcium-dependent phosphoprotein formation in erythrocyte membrane fragments in the presence of lanthanum. Calmodulin-stimulated (10 pg/ml) (• •) and basal (• •) ATPase a c t i v i t y and phosphoprotein formation, measured i n the presence of 0.15 mM CaCl 2 and 0.1 mM LaC^, were determined as described in Methods. The re s u l t shown i s the mean +_ S.E.M. of f i v e mem-brane preparations; each membrane preparation was assayed i n t r i p l i c a t e . ATPase a c t i v i t y and phosphoprotein formation were determined simultaneously for each membrane preparation. Single asterisk indicates s i g n i f i c a n t l y d i f f e r e n t from basal a c t i v i t y at p < 0.05. Double asterisk indicates s i g n i f i c a n t l y d i f f e r e n t from basal a c t i v i t y at p < 0.01. 62 ATPase a c t i v i t y (pmol/mg/min) ( B U J / T o u i - j . ) u i B ^ o j d o L | d s o q d Figure 13. Eff e c t of magnesium on (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y i n erythro-cyte membrane fragments at various free calcium concentrations. ATPase a c t i v i t y was assayed i n the presence of either 25 pM magnesium (• •) or 0.1 mM CDTA (• •) . ATPase a c t i v i t y was determined as described i n Methods. ATPase refers to the calcium component of hydrolysis after subtraction of the values measured in the presence of EGTA (0.1 mM) alone. The r e s u l t shown i s the mean _+ S.E.M. of observations from f i v e membrane preparations; each membrane preparation was assayed i n t r i p l i c a t e . 63 of the enzyme for calcium was increased s l i g h t y from 0.60 x 10 -^ to 3.1 x 10"^ M (Table 3). Phosphoprotein formation, shown in Figures 14 a and b, was unaffected by chelation of magnesium — R — 9 at a l l calcium concentrations from 5.0 x 10 to 1.0 x 10 M. Turnover of the enzyme (Figure 15) was considerably depressed with respect to the control condition. In a time-course study where the calcium concentration was 1.0 x 10"^ M, chelation of magnesium produced a marked reduction i n ATPase a c t i v i t y , but only a small depression i n E.Ca.P levels at a l l time points studied (Figure 16). When the calcium concentration was i n -creased to 4.0 x 10 - 4 M, the rate of formation and the steady state level of E.Ca.P were unaffected by chelation of free magne-sium, while (Ca + Mg )-ATPase a c t i v i t y was only s l i g h t l y depressed, as shown i n Figure 17. Micromolar concentrations of magnesium stimulate a conforma-ti o n a l change of the enzyme from the high-energy E-^.Ca.P complex to the lower energy form E2-Ca.P. The E2«Ca.P form of the enzyme demonstrates a lower a f f i n i t y for calcium and a much higher r e a c t i v i t y towards water. In response to suggestions by several authors that the calcium-dependent i n h i b i t i o n of (Ca + Mg )-ATPase a c t i v i t y and concomitant buildup of phosphoprotein was due to calcium competing with magnesium at t h i s s i t e , thereby r e s u l -ting i n i n h i b i t i o n of the conversion of E-^.Ca.P to E2»Ca.P, the low a f f i n i t y component of phosphoprotein formation was studied i n the presence of either 0.1 mM CDTA or 0.5 mM Mg* . Though forma-tio n of E.Ca.P was stimulated s l i g h t l y by 0.5 mM Mg at lower calcium concentrations (Figure 18), the buildup of phosphoprotein at higher calcium concentrations was not affected by magnesium 64 Table 3. Kinetic parameters of the (Ca 2 + + Mg 2 +)-ATPase i n the presence of either 25 uM Mg2 + or 0.1 mM CDTA. 25 pM Mg 2 + 0.1 mM CDTA (n=5) a (n=5) K m (x 10~ 7 M) c 5.97 + 2.26b 25.7 + 10.3 vmax (pmol/mg/min)c 25.0 _+ 3.61 17.4 _+ 2.9 a 'n' refers to the number of membrane preparations assayed; each assay was performed i n t r i p l i c a t e . k Results are expressed as the mean +_ SEM. c Kinetic constants were estimated by model 2 as described i n Methods. 65 Figure 14 a and b. Eff e c t of magnesium on calcium-dependent phosphoprotein formation in erythrocyte membrane fragments at various free calcium concen-t r a t i o n s . Phosphoprotein formation was assayed i n the presence of either 25 pM magnesium (• •) or 0.1 mM CDTA (• • ) . ATPase a c t i v i t y was determined as described i n Methods. Phosphoprotein refers to the calcium component of phosphorylation after subtrac-t i o n of the values measured i n the presence of EGTA (0.1 mM) alone. The r e s u l t shown i s the mean _+ S.E.M. of observations from four membrane preparations; each membrane preparation was assayed in t r i p l i c a t e . 66 1500 a 66a BDDD b 66b Figure 15. Turnover of the (Ca + Mg* )-ATPase i n erythrocyte membrane fragments i n the presence of either 25 uM magnesium (• HI) or 0.1 mM CDTA (• •) at various free calcium concentrations. The turnover number i s the r a t i o of ATPase a c t i v i t y to phosphoprotein formation. Free calcium concentrations were determined as des-cribed i n Methods. The r e s u l t shown was determined from the means of f i v e ATPase assays and four phosphorylation assays. 67 3 0 0 67a Figure 16. Eff e c t of magnesium on (Ca* + Mg )-ATPase a c t i v i t y and the time course of calcium-dependent phosphoprotein formation i n erythro-cyte membrane fragments i n the presence of 1.0 pM free calcium. ATPase a c t i v i t y and phosphoprotein formation i n the presence of either 25 pM magnesium (• •) or 0.1 mM CDTA (• •) were deter-mined as described i n Methods. The r e s u l t shown i s the mean +_ S.E.M. of observations from four membrane preparations; each membrane preparation was assayed i n t r i p l i c a t e . ATPase a c t i v i t y and phosphoprotein formation were determined simultaneously for each membrane preparation. Asterisk indicates s i g n i f i c a n t l y d i f f e r e n t from 25 pM magnesium condition, p < 0.01. 68 ATPase a c t i v i t y (pmol/mg/min) ( B U J / T O O I J . ) u T a ^ o j d o i _ | d s o L | c l Figure 17. Eff e c t of magnesium on (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y and the time course of calcium-dependent phosphoprotein formation in erythro-cyte membrane fragments i n the presence of 0.4 mM free calcium. ATPase a c t i v i t y and phosphoprotein formation in the presence of either 25 p.M magnesium (• • ) or 0.1 mM CDTA (• •) were deter-mined as described i n Methods. The re s u l t shown i s the mean +_ S.E.M. of observations from four membrane preparations; each membrane preparation was assayed in t r i p l i c a t e . ATPase a c t i v i t y and phosphoprotein formation were determined simultaneously for each membrane preparation. 69 3000 69a Figure 18. Eff e c t of magnesium on the low calcium a f f i n i t y component of calcium-dependent phosphoprotein formation. Phosphoprotein f o r -mation i n the presence of either 0.5 mM magnesium (• •) or 0.1 mM CDTA (• •) were determined as described i n Methods. Phos-phoprotein refers to the calcium component afte r subtraction of the values measured i n the presence of EGTA (0.1 mM) alone. The res u l t shown i s the mean +_ S.E.M. of observations from at least three membrane preparations; each membrane preparation was as-sayed in t r i p l i c a t e . The asterisk indicates the r e s u l t i s s i g n i f i c a n t l y d i f f e r e n t from the CDTA condition, p < 0.01. 70 7oa (Figure 18). In addition, E.Ca.P formation in the presence of 10 p + . mM Ca (Figure 19) was not affected by magnesium concentrations as high as 1 mM. Table 4 shows that E.Ca.P produced i n the presence of 10 mM Ca was rapidly degraded i n the presence of either 1 mM ADP or 1 mM cold ATP, and that concentrations of magnesium up to 1 mM had no s i g n i f i c a n t e f f e c t on the s e n s i t i v i t y of E.Ca.P to hydrolysis by either ATP or ADP. Following a 5 second exposure to 1 mM ADP, 22% of the phosphoprotein remained, which was s i g n i f i c a n t l y less than the 39% which remained f o l -lowing 5 second exposure to ATP. After a 15 second exposure time however, there was no s i g n i f i c a n t difference i n residual E.Ca.P following ADP or ATP treatment. Therefore both ADP and ATP appear to dephosphorylate E.Ca.P, i n a largely magnesium-independent manner (Table 4 ) . I I . ATP Hydrolysis and Phosphoprotein Formation i n the Erythrocyte Plasma Membranes of Subjects with Cystic  F i b r o s i s The possible role of the ( C a 2 + + Mg 2 +)-ATPase i n the defect in calcium handling associated with c y s t i c f i b r o s i s was further studied under conditions designed to accumulate phosphoprotein, by i n h i b i t i n g dephosphorylation of the enzyme. Both 10 mM CaCl 2 and 0.1 mM L a C ^ caused a large buildup i n the phosphorylated intermediate such that [E-j^  + E 21 << [E-j^ P + E 2P] . This provided a means of comparing the number of functional enzyme units i n erythrocyte plasma membrane from c y s t i c f i b r o s i s subjects com-pared to those from age- and sex-matched controls. In the pres-71 Figure 19. Ef f e c t of magnesium on phosphoprotein formation i n the presence of 10 mM calcium. Phosphoprotein formation, determined as des-cribed i n Methods, refers to t o t a l phosphorylation. The r e s u l t shown i s the mean +_ S.E.M. of observations from at least three membrane preparations; each membrane preparation was assayed in t r i p l i c a t e . 72 9 0 0 0 cn E \ Q 6 0 0 0 E 01 •P 0 L Q_ 0 r D_ (0 0 £. D_ 3 0 0 0 H r o ~ ~ i — 2 5 -\V T 5 0 0 1 0 0 0 |magnes l um) CuM) 72a Table 4. Ef f e c t of exposure to either 1 mM ADP or 1 mM cold ATP on the acyl-phosphate intermediate of the erythrocyte (Ca+Mg)-ATPase formed in the presence of 10 mM C a 2 + and various concentrations of magnesium. Membrane fragments were phosphorylated for 15 seconds i n the presence of various magnesium concentrations, then dephosphorylation was i n i t i a t e d by addition of either ADP or ATP (1 mM f i n a l concentration) and the reaction terminated after either 5 or 15 seconds by the rapid addition of ice cold 5% TCA solution. Percentage of the Total Phosphoprotein Remaining ADP ATP 5 sec 15 sec 5 sec 15 sec (n=3) (n=4) (n=3) (n=4) 0.1 mM CDTA 25% +3% 9% + 3% 38% + 3%* 11% + 5%** 25 uM Mg 21% + 2% 12% + 2% 36% + 3%* 16% + 4%** 0.5 mM Mg 18% +2% 9% + 2% 39% + 4%* 9% + 1%** 1.0 mM Mg 24% + 2% 18% + 4% 45% + 7%* 11% + 4%** note: the ef f e c t s of magnesium were found to be non-significant. S i g n i f i e s difference from 5 second treatment with ADP ^ (p < 0.01). S i g n i f i e s difference from 5 second treatment with ATP (p _< 0.01) . 'n' refers to the number of membrane preparations assayed; each preparation was assayed i n t r i p l i c a t e . Results are expressed as the mean + SEM. 73 ence of 10 mM C a 2 + a 16% drop i n the steady-state level of E.Ca.P was noted in CF compared to the control group (Table 5). (Ca + Mg )-ATPase a c t i v i t y (Table 5) was also lower i n CF membranes however there was no difference in the turnover number of the enzyme between the two groups. When 0.1 mM LaCl-j was used there was less buildup of the steady-state level of phosphoprotein, however CF samples again had a s i g n i f i c a n t l y lower steady-state level of the phosphorylated enzyme and ATP hydrolysis with res-pect to control values (Table 5). Turnover numbers did not d i f -f e r . Phosphoprotein formation and ATPase a c t i v i t y i n the pres-ence of 0.025 mM MgCl 2 alone (Table 5) were not altered i n the CF membranes. 74 Table 5. 7 + 7 + Calcium-dependent phosphoprotein formation, (Ca + Mg )-ATPase a c t i v i t y and turnover in c y s t i c f i b r o s i s (CF) and control erythrocyte membranes in the presence of 25 pM magnesium and either 10 mM calcium chloride or 0.1 mM lanthanum chloride. ATPase and phosphoprotein refer to the components remaining after subtraction of values measured i n the presence of 25 uM Mg and 0.1 mM EDTA (basal a c t i v i t y ) . Turnover i s the r a t i o of ATPase a c t i v i t y to phosphoprotein l e v e l . 'n' refers to the number of subjects sampled; each sample was assayed i n t r i p l i c a t e . The re s u l t shown i s the mean +_ S.E.M. Asterisk indicates s i g n i f i c a n -t l y d i f f e r e n t from control, p < 0.01. 10.mM CaCl 2 control (n=7) CF (n=8) Phosphoprotein (fmol/mg) ATPase (pmol/mg/min) Turnover (min - 1) 6080 + 69 21.4 + .7 3.46 + 12 5110 + 66 17.1 + .8 3.42 + 11 0.1 mM LaCl-Phosphoprotein (fmol/mg) 1498 _+ 70 ATPase (pmol/mg/min) 25.7 + .9 Turnover (min - 1) 64.1 + 12 1114 +_ 66 21.4 + .9 48.6 + 11 0.025 mM MgCl 2 Phosphoprotein (fmol/mg) ATPase (pmol/mg/min) 209 + 70 6.90 + .89 186 + 66 7.72 + .84 75 DISCUSSION ATP- and magnesium-stimulated active calcium extrusion i s a basic plasma membrane function i n most l i v i n g c e l l s and i t s general c h a r a c t e r i s t i c s seem to be preserved i n the human erythrocyte membrane (Schatzmann, 1975; Sarkadi, 1980). Further-more, due to the lack of subcellular membrane elements i n the erythrocyte i t i s possible to i s o l a t e a plasma membrane (Ca* + Mg 2 +)-ATPase which i s free from contamination by other species of Ca* +-ATPase. These q u a l i t i e s make the erythrocyte an ideal sys-tem i n which to study the ki n e t i c s of the plasma membrane calcium pump enzyme. Calcium-dependent formation of a phosphorylated intermediate of the erythrocyte calcium pump from gamma-J*P-ATP has been shown by several authors (Katz and Blostein, 1973; Knauf et a_l, 1974; Katz and Blostein, 1975; Rega and Garrahan, 1975; Wolf et a l , 1977; Schatzmann and Burgin, 1978; Szasz et a l , 1978; N i g g l i et a l , 1979) and i t i s now accepted that hydrolysis of ATP by the calcium pump i n human erythrocytes proceeds through a series of p a r t i a l reactions involving formation of a phosphorylated i n t e r -mediate. Investigation of the ef f e c t s of regulatory proteins and various modifications to the io n i c environment on phosphoprotein formation has and w i l l continue to contribute to our better understanding of the molecular properties of the calcium pump enzyme. A special advantage i n using membrane fragments i s that changes i n phosphoprotein formation can be d i r e c t l y compared to alte r a t i o n s i n ATPase a c t i v i t y . This allows the rate of forma-76 t i o n , steady-state level and rate of decay of the E.Ca.P complex to be compared with the net v e l o c i t y of the enzyme through i t s entire sequence of p a r t i a l reactions. From such comparisons a model of the reaction sequence has been developed, indicating the order of the various p a r t i a l reactions and the s i t e s where regu-latory proteins and ions may be exerting t h e i r e f f e c t ( s ) . The current understanding of the p a r t i a l reaction sequence for t h i s enzyme, previously described i n the Introduction, i s depicted i n 9 + Figure 1. B r i e f l y : Enzyme state E-^  i s considered to bind Ca* at the c y t o s o l i c membrane surface, thereby promoting reaction with ATP, forming the intermediate complex E^.Ca.P. The conforma-t i o n a l t r a n s i t i o n of the "high energy" phosphoprotein E-^.Ca.P to i t s "lower energy" form E 2.Ca.P. i s believed to r e s u l t i n a decreased a f f i n i t y of the binding s i t e for calcium and i t s re-orientation to face the exterior surface of the erythrocyte membrane. This conformational change appears to be stimulated by micromolar concentrations of magnesium. Translocation of calcium i s thought to r e s u l t from displacement of calcium from t h i s low a f f i n i t y state. Rega and Garrahan (1975) have suggested that i n the E 2.Ca.P form the acy1-phosphate bond i s more accessible to water and i s therefore rapidly hydrolysed to E 2.Ca plus inorganic phosphate. E 2.Ca then dissociates to E 2 plus Ca and E 2 under-goes a conformational t r a n s i t i o n back to E-^ . In the following discussion I w i l l attempt to explain the e f f e c t s of calcium, lanthanum, calmodulin and magnesium on c a l -9 + 9 J . cium-dependent phosphoprotein formation and (Ca + Mg )-ATPase a c t i v i t y i n terms of the various steps i n the reaction sequence 77 of t h i s enzyme. I. E f f e c t s of Calcium on (Ca2"1" + Mg2"1")-ATPase A c t i v i t y and the  Intermediate Reaction Sequence One in t e r e s t i n g c h a r a c t e r i s t i c of the erythrocyte calcium-transport ATPase i s that t h i s enzyme, which requires calcium to function, i s also i n h i b i t e d by calcium. The i n h i b i t o r y action of calcium on the a c t i v i t y of the (Ca + Mg )-ATPase has been reported by several authors (Vincenzi et a_l, 1980; Klinger et a l , 1980; Graf and Penniston, 1981; Al-Jobore and Roufogalis, 1981). We found that ATPase a c t i v i t y was inh i b i t e d by calcium concentra-tions greater than 10~ 4 M (K i = 1.7 x 10~ 3 M), which agrees with the finding of Schatzmann (1982) that calcium concentrations — s greater than 1 - 5 x 10 M are i n h i b i t o r y . When the e f f e c t of calcium concentration on formation of the phosphorylated intermediate (E.Ca.P) was studied a d i s t i n c t l y biphasic r e l a t i o n s h i p (K-j^  = 5 x 10~ 8 M; K 2 = 7.2 x 10~ 4 M) was observed, i n which levels of the E.Ca.P complex were maximal i n the presence of 10 mM C a 2 + . Lichtner and Wolf (1980a) obtained a simi l a r response to calcium concentration using a p u r i f i e d enzyme preparation. The low a f f i n i t y component of E.Ca.P was of special i n t e r e s t as i t occurred within the same range of free calcium concentrations where calcium-dependent i n h i b i t i o n of Ca* -ATPase a c t i v i t y occurred. One possible explaination for t h i s observa-tion i s that either the pump protein or an associated protein was being phosphorylated by a calcium-dependent protein kinase, re-su l t i n g i n i n h i b i t i o n of the (C a 2 + + Mg 2 +)-ATPase. Autoradio-78 grams of phosphorylated membranes indicate that, at a l l calcium concentrations tested, most of the J*P was incorporated into a 135,000-145,000 dalton protein and i t was rapidly released upon exposure to hydroxylamine, as previously shown by Lichtner and Wolf (1980b). The molecular weight of the phosphoprotein mea-sured here i s similar to that of 138,000 daltons reported by C a r a f o l i et a_l (1982) for the erythrocyte membrane Ca 2 + -ATPase. The s i m i l a r i t y i n molecular weight, as well as the s e n s i t i v i t y of the phosphorylated enzyme to hydroxylamine. are consistent with formation of an acy1-phosphate bond on the calcium pump protein rather than a kinase-mediated phosphorylation. Calcium-dependent i n h i b i t i o n of ATPase a c t i v i t y and the low a f f i n i t y component of E.Ca.P formation have been attributed to i n h i b i t i o n of the dephosphorylation pathway by calcium (Lichtner and Wolf, 1980a; Schatzmann, 1982). These authors have proposed that at high concentrations, calcium competes with magnesium at the 'magnesium-specific' s i t e , thereby i n h i b i t i n g the conforma-t i o n a l change from E-^.Ca.P to E 2.Ca.P. A similar biphasic rela t i o n s h i p between calcium concentration and E.Ca.P formation has been demonstrated for the Ca 2 +-ATPase i n sarcoplasmic r e t i c u -lum (S.R.) v e s i c l e s prepared from rabbit s k e l e t a l muscle (Almeida and de Meis, 1977). These workers found that the low a f f i n i t y component resulted from a high calcium concentration on the luminal side of the S.R. membrane but not on the c y t o s o l i c side, and therefore they suggested the low a f f i n i t y component was associated with the presence of a low a f f i n i t y calcium binding s i t e on the region of the enzyme exposed to the lumen (ie. 79 E 2.Ca.P). It should be pointed out here that i n the sarcoplasmic reticulum, in contrast to the erythrocyte, the binding s i t e s for ATP and Mg 2 +, as well as the high a f f i n i t y calcium s i t e s , are found on the c y t o s o l i c side of the membrane. Ikemoto (1974) has also presented evidence that the binding of Ca* to a lower a f f i n i t y s i t e (association constant 1.0-1.6 x 10 3 M - 1) could be involved i n the i n h i b i t i o n of the ATPase by excess Ca* . In addition to i n h i b i t i n g dephosphorylation, binding of calcium to the "low a f f i n i t y " s i t e enhanced phosphorylation of the enzyme by 3 2P^; t h i s phosphate could subsequently be transfered to ADP, forming ATP under conditions which reverse the calcium pump mechanism (de Meis and Caravalho, 1976). Although much of thi s work has been done i n the S.R. system of skeletal muscle, similar r e s u l t s have been obtained from erythrocytes. In resealed ghosts, i n the presence of a steep inwardly oriented calcium gradient, Rossi et a_l (1978) observed 3 2 P ^ incorporation into ATP. Wuthrich et a_l (1979) showed picomolar amounts of net ATP synthesis by running the calcium pump backwards in inside-out membrane v e s i c l e s . Thus, r e s u l t s obtained i n both erythrocyte plasma membranes and skel e t a l S.R. are not entirely, consistent with the viewpoint of how calcium i n h i b i t s dephosphorylation by i n h i b i t i n g the conformational change from E-^.Ca.P to E 2.Ca.P. An alternative explanation for these re s u l t s involves an inte r a c t i o n between calcium and the E 2 state of the enzyme, possibly the E 2.P complex. Such an in t e r a c t i o n could maintain the enzyme i n a phosphorylated state (E2.Ca.P) drive the dephosphorylation path-way i n the opposite d i r e c t i o n . In addition, these r e s u l t s sug-gest a p a r a l l e l between the S.R. system and the erythrocyte 80 plasma membrane system i n terms of the mechanism of calcium-dependent i n h i b i t i o n of dephosphorylation. In the presence of 10~ 6 M C a 2 + , formation of the E.Ca.P complex approached steady-state with a ^-1/2 greater than 15 seconds. Lichtner and Wolf (1980a), working at the same calcium concentration, found slower apparent rates of formation (^-\/2 = 30-60 seconds). However, t h e i r assays were carr i e d out at a lower temperature (0° vs 10°C). These re s u l t s suggest that the rate of the f i r s t reaction: E± + C a 2 + + ATP =^=^= EjL-Ca.P + ADP was slow at t h i s calcium concentration. However, under the same conditions turnover number was maximal and ATPase a c t i v i t y was 70-80% of i t s maximum v e l o c i t y ( V m a x ) . Taken together, these results imply the following: ^ In the presence of 10 -^ M C a 2 + and the assay conditions employed for the present study, forma-ti o n of the Ej^.Ca.P complex i s the r a t e - l i m i t i n g step i n the reaction cycle either d i r e c t l y or by virt u e of a r a t e - l i m i t i n g E 2 - E l t r a n s i t i o n ; 2^ as the phosphorylation reached steady-state slowly, even though turnover of the phosphorylated intermediate was high, the rate of formation of the E.Ca.P complex must have been very similar to the rate of dephosphorylation; and, 3^ a l l p a r t i a l reactions which occur between the formation of the phos-phorylated intermediate and i t s decay, including the conforma-ti o n a l change between E-^.Ca.P and E 2.Ca.P, were at least as rapid as the formation and breakdown of the intermediate. Sarkadi (1980) has proposed that, i n the presence of magnesium, the rate l i m i t i n g step i n the pumping cycle i s either the formation of the 81 E-^.Ca complex or the calcium-translocation step. In l i g h t of these r e s u l t s , the "slow" step would appear to be the calcium-dependent phosphorylation step, either d i r e c t l y or due to a l i m i t i n g amount of available free enzyme (E^). When the calcium concentration was increased to 4.0 x 10~ 4 M, both the steady-state level and the apparent rate of formation (t-^/2 = ^ seconds) of the E.Ca.P complex were increased compared to that observed at 10~ 6 M C a 2 + . At t h i s C a 2 + concentration (4.0 x 10~ 4 M) (C a 2 + + Mg 2 +)-ATPase a c t i v i t y was inh i b i t e d by 20-30% (with respect to V m a x ) and turnover of the phosphorylated intermediate was inh i b i t e d by more than 90%. These observations are consistent with r e s u l t s , discussed above, showing that at concentrations greater than 5 x 10 M, calcium i n h i b i t s dephos-phorylation of the phosphoprotein complex. When the dephosphory-l a t i o n pathway i s in h i b i t e d , the rate of formation of E.Ca.P exceeds the rate of breakdown. As a re s u l t of t h i s , the steady-state level of E.Ca.P must r i s e to a point where dephosphoryla-tion i s 'driven' at a rate equal to formation of the complex, and a new equilibrium i s established. There are at least two factors which may be contributing to the increase i n the apparent rate of formation observed. B r i e f l y , these are: ^ The high a f f i n i t y calcium binding s i t e s on the enzyme, which have a d i s s o c i a t i o n constant for calcium of approximately 10 -^ M (Schatzmann, 1973; Fer r e i r a and Lew, 1976; Larsen et a_l, 1978), would not be satur-ated at a calcium concentration of 10 -^ M. Increasing the c a l -cium concentration to 4.0 x 10~ 4 M saturates the high a f f i n i t y calcium binding s i t e s , which would have a stimulatory e f f e c t on the apparent rate of formation. 2^ As dephosphorylation becomes 82 i n h i b i t e d by calcium and higher steady-state levels of E.Ca.P are required to drive the dephosphorylation pathway, the apparent rate of formation of the E.Ca.P complex begins to approximate the true rate of formation. One additional consequence of the i n h i -b i t i o n of dephosphorylation i s a s h i f t i n the equilibrium of the enzyme from the dephosphorylated states (E-^  and E 2) towards the phosphorylated states (E^.Ca.P and E2.Ca.P) re s u l t i n g i n less E-^  available to bind to Ca and ATP, accounting for at least part of the observed reduction i n steady-state a c t i v i t y of the enzyme. ? + I I . E f f e c t of Lanthanum on the P a r t i a l Reactions of the (Ca +  Mg2*)-ATPase Lanthanum produced e f f e c t s which were v i r t u a l l y i d e n t i c a l to those of high (4.0 x 10~ 4 M) C a 2 + , namely; (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y was greatly reduced while the apparent rate of formation and steady-state level of the E.Ca.P complex were increased markedly. These ef f e c t s are consistent with previous reports that lanthanum i n h i b i t s the dephosphorylation pathway of the erythro-cyte ( C a 2 + + Mg 2 +)-ATPase (Szasz et a_l, 1978; Schatzmann and Burgin, 1978; Muallem and K a r l i s h , 1982). Recently Luterbacher and Schatzmann (1983) suggested that lanthanum i n h i b i t s the con-formational t r a n s i t i o n required for dephosphorylation, whereas Sarkadi (1980) has proposed that lanthanum binds to the E 2.P complex following the d i s s o c i a t i o n of calcium, thereby s t a b i l i -zing the enzyme i n a phosphorylated form. Both of these mecha-nisms imply that lanthanum acts on the phosphorylated interme-83 diate, however, Szasz et a_l (1978) have shown that lanthanum w i l l stimulate phosphoprotein formation even in the absence of c a l -cium. In the present study lanthanum appeared to antagonize the calcium-dependent i n h i b i t i o n of dephosphorylation. Together, these two res u l t s suggest a possible in t e r a c t i o n at the high-s' + o + a f f i n i t y calcium binding s i t e on the (Ca + Mg )-ATPase, a l -though they do not rule out other possible s i t e s of action for lanthanum. I l l E f f e c t of Calmodulin on the Intermediate Reactions of the  (Ca 2 + + Mg2*)-ATPase Previously, i t was thought that the number of calmodulin binding s i t e s exceeded the number of pump s i t e s by about f i v e f o l d (see Al-Jobore et a_l, 1984). These r e s u l t s were based on ^ dir e c t calmodulin binding studies (Graf et a_l, 1980) and k i n e t i c t i t r a t i o n of (C a 2 + + Mg 2 +)-ATPase a c t i v a t i o n (Jarret and Kyte, 1979), which showed that there were about 4000 - 6000 calmodulin binding s i t e s per erythrocyte, and 2^ phosphorylation studies (Knauf et a_l, 1974; Rega and Garrhan, 1975) which suggested there were approximately 700 calcium pumping s i t e s per erythrocyte. It i s now known, however, that the levels of phosphoprotein used to determine t h i s value were not maximum values and therefore do not provide an accurate means of determining the t o t a l number of pump sit e s per c e l l . From r e s u l t s obtained i n the present study, assuming 10 -^ g of membrane protein per erythrocyte ghost (Lichtner and Wolf, 1979), we have estimated that there are roughly 2400-3600 pump s i t e s per c e l l . This value agrees well 84 with that of Schatzmann (1982) , who estimates at least 2700 s i t e s per erythrocyte. Thus, the ( C a 2 + + Mg 2 +)-ATPase does, i n fact, appear to represent a major f r a c t i o n of the t o t a l number of calmodulin binding s i t e s within the erythrocyte. 2 + The e f f e c t s of calmodulin on the k i n e t i c s of the (Ca* + Mg 2 +)-ATPase, namely an increase i n the maximum rate of hydro-l y s i s of ATP and an increase i n the apparent calcium a f f i n i t y , have been well documented (for reviews see Roufogalis, 1979; Sarkadi, 1980; Al-Jabore et a l , 1984; Schatzmann, 1982; Penniston, 1983; Schatzmann, 1985). There i s now convincing evidence that both formation (Muallem and K a r l i s h , 1980) and breakdown (Jeffery et a_l, 1981; Rega and Garrahan, 1980) of the calcium-dependent phosphorylated intermediate are accelerated by calmodulin, enhancing the turnover of the enzyme. Klinger et a_l (1980) and Schatzmann (1982) have presented evidence suggesting that calmodulin does not influence calcium-dependent i n h i b i t i o n of ATPase a c t i v i t y . S i m i l a r l y , we have found that calmodulin does not a l t e r the apparent a f f i n i t y of calcium-dependent i n h i -b i t i o n of ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y or the biphasic r e l a t i o n -ship between calcium concentration and E.Ca.P formation. These studies allow i d e n t i f i c a t i o n of which p a r t i c u l a r sequences of steps within the reaction cycle are accelerated by calmodulin, but they do not define the p a r t i c u l a r reaction steps affected by calmodulin. They do , however, suggest that the step at which calmodulin acts to stimulate dephosphorylation i s not the same step with which calcium interferes to i n h i b i t dephosphorylation. In a study by Muallem and K a r l i s h (1980), calmodulin was shown to increase the apparent rate of formation of the E.Ca.P 85 complex without a f f e c t i n g the steady-state l e v e l . In the present study, however, the e f f e c t of calmodulin on the k i n e t i c s of E.Ca.P formation d i f f e r e d greatly depending on the calcium con-centration i n the assay medium. When the calcium concentration was maintained at 10 -^ M, calmodulin increased the apparent rate of formation of the E.Ca.P complex, however steady-state was not reached within the range of reaction times examined (1-30 se-conds) . At a higher C a 2 + concentration (4.0 x 10~ 4 M) calmodulin had no e f f e c t on the apparent rate of formation, but i t reduced the steady-state level of E.Ca.P by more than 25%. The reduction by calmodulin of the steady-state level has been reported pre-viously by other investigators (Jeffery et a_l, 1981; Rega and Garrahan, 1980) at lower calcium concentrations (5-6 x 10"^ M). The increase i n apparent rate of phosphoprotein formation observed in the presence of 10 M Ca upon addition of calmodu-l i n could be achieved i n at least three ways. These are: ^ an increase i n the calcium a f f i n i t y of the h i g h - a f f i n i t y calcium binding s i t e s , such that a higher degree of saturation i s achieved at t h i s low calcium concentration, which could, i n theory, "drive" the rate of formation to a higher value (assuming that the E-^  - E 2 t r a n s i t i o n i s not rate-limiting) , 2^ an a l t e r a -t i o n i n the equilibrium between the E-^  and E 2 forms of the enzyme towards the E-^  form, r e s u l t i n g i n more high a f f i n i t y s i t e s avai-lable to interact with C a 2 + and ATP (Muallem and K a r l i s h , 1980), or 3) an a l t e r a t i o n i n the equilibrium between E-^  + C a 2 + + ATP and E-^.Ca.P i n favor of the intermediate complex. At a higher calcium concentration (4.0 x 10~ 4 M) calmodulin did not a l t e r the 86 apparent rate of formation of the E.Ca.P complex, which supports the f i r s t mechanism, that the increased rateof E.Ca.P formation i s due to an increase in a f f i n i t y of the calcium binding s i t e s and once the calcium concentration i s high enough to saturate these s i t e s , no further e f f e c t i s seen. In addition, the obser-vation that calmodulin lowered the steady-state amount of E.Ca.P, as others have also shown (Jeffery et a_l, 1981; Rega and Garrahan, 1980) , indicates that the rate of dephosphorylation was increased. Jeffery et al_ (1981) found that both magnesium (0.25 mM) and calmodulin stimulated the rate of dephosphorylation, however in the presence of high magnesium, calmodulin did not produce an additional increase i n dephosphorylation, and vice versa. From these r e s u l t s the authors concluded that calmodulin and magnesium may be stimulating the rate of dephosphorylation by acting on the same k i n e t i c step, namely the conformational trans-formation from the form of the enzyme with a low r e a c t i v i t y to water (E-^.Ca.P) to that conformation characterized by a high r e a c t i v i t y to water (E 2.Ca.P). In the presence of lanthanum (0.1 mM), which i n h i b i t s dephosphorylation and at a r e l a t i v e l y high free calcium concentration of 1-2 x 10~ 4 M, calmodulin increased the apparent rate of formation and the steady-state l e v e l of E.Ca.P. This r e s u l t indicates that the true rate of formation of the E.Ca.P complex i s , i n f a c t , increased by calmodulin even at calcium concentrations i n excess of those s u f f i c i e n t to saturate the high a f f i n i t y binding s i t e s . Therefore, t h i s rate increase i s not simply due to an increase i n calcium a f f i n i t y . An alternate mechanism was suggested by Muallem and K a r l i s h (1980) , who considered that since the rate of transformation from 87 E 2 to E-L i s slow, by accelerating t h i s step with calmodulin both the rate of formation of E.Ca.P and the apparent calcium a f f i n i t y of the enzyme are increased. Although one can e a s i l y see how such an action would increase the rate of phosphorylation, i t i s d i f f i c u l t to see how i t w i l l account for the change i n calcium a f f i n i t y . The conformational change from E 2 to E-^  involves a re-orientation of the calcium binding s i t e to the c y t o s o l i c side of the membrane and an increase i n calcium a f f i n i t y by several orders of magnitude. As the high and low a f f i n i t y s i t e s d i f f e r by several orders of magnitude, i t i s un l i k e l y that both c o n t r i -bute to the apparent a f f i n i t y for calcium of the (Ca* + Mg* )-ATPase a c t i v i t y , as may happen with two or more s i t e s having similar a f f i n i t i e s . Therefore, according to Muallem and Karlish's theory, the increased a f f i n i t y i s due to an increase i n the number of s i t e s with which calcium can i n t e r a c t . Such an eff e c t would uniformly increase the rate of ATP hydrolysis but should not a l t e r i t s calcium dependence. Schatzmann (1982) has questioned the notion that calmodulin increases the maximum velo-c i t y ( V m a x ) , noting that i n calmodulin-depleted enzyme the c a l -cium a c t i v a t i o n curve moves towards the region of i n h i b i t o r y calcium concentrations, as the a f f i n i t y of calcium-dependent i n h i b i t i o n i s not affected by calmodulin. This, then, would be responsible for the lower values of V m a x estimated i n the absence of calmodulin. Such ideas suggest that calmodulin produces a single change i n the enzyme which manifests i t s e l f as an a l t e r a -t i o n i n both calcium a f f i n i t y and V m a x . Contrary to t h i s idea, Minocherhomjee et a_l (1982) showed that polyanions could mimic 88 the e f f e c t of calmodulin on calcium a f f i n i t y but not on V m a x , and as i n the case of calmodulin, t h i s a c t i v a t i o n could be antago-nized by t r i f l u o p e r a z i n e . Thus calmodulin may produce more than one e f f e c t upon the enzyme, allowing more rapid conformational changes i n both the phosphorylated states and the non-phosphory-lated states, and also increasing the a f f i n i t y of the high a f f i -n i ty calcium binding s i t e s . IV E f f e c t of Magnesium on the Intermediate Reactions of the  ( C a 2 + + Mg 2 +)-ATPase In addition to other activators, the (Ca* + Mg* )-ATPase requires micromolar amounts of magnesium i n order to function at i t s maximum rate. Several authors (Rega and Garrahan, 1975; Schatzmann and Burgin, 1978; Szasz et a_l, 1978) have demonstrated that formation of the E.Ca.P complex does not require the pres-ence of magnesium, however i n a recent review a r t i c l e Schatzmann o + (1982) suggested that Mg i s probably required at the phos-phorylation step. As the previous studies did not employ a magnesium-specific chelator i n the assay systems used, there i s a p o s s i b i l i t y that endogenous levels of Mg were high enough to meet any requirements for phosphorylation. In the present study, membranes were washed i n a magnesium-free buffer and assayed i n the presence of the magnesium chelator, CDTA, to bind any r e s i -dual magnesium. In t h e i r "magnesium-free" condition (no added magnesium) , Richards et a_l (1978) found that the V m a x for ATP hydrolysis was depressed, whereas with CDTA in the medium both the apparent calcium a f f i n i t y and the V m a were reduced. There-89 fore small amounts of magnesium do maintain the enzyme in the h i g h - a f f i n i t y E.^  form. In addition, the calcium activation of ATPase a c t i v i t y appears to be biphasic, whereas i t i s hyperbolic in the presence of magnesium (0.025 mM). The difference between our findings and those of Richards et a l may be attributable to contamination by residual free magnesium i n the i r assay, or perhaps to some ef f e c t of CDTA upon the enzyme i t s e l f i n our study. When the formation of the E.Ca.P complex i n the absence of magnesium was compared to that with 25 pM magnesium, we found; no change i n the calcium-dependency of E.Ca.P formation at _o calcium concentrations within the range of 5.0 x 10 to 1.0 x 10~ 2 M (free), and 2^ no s i g n i f i c a n t change i n the rate of formation of the E.Ca.P complex at either low (10~ 6 M) or high (4.0 x 1 0 - 4 M) calcium concentrations. Hence these results confirm those of previous investigators, demonstrating that mag-nesium i s not required for the formation of the E.Ca.P complex. On the other hand, Larocca et a_l (1981) found that magnesium (0.5 mM) increased the rate of formation of the phosphorylated i n t e r -mediate, while steady-state levels were independent of magnesium concentration. In contrast to t h i s , J e f f e r y et a_l (1981) found that i n addition to enhancing dephosphorylation of E.Ca.P, magne-sium (0.25 mM) decreased the steady-state l e v e l s . These discrep-ancies i n r e s u l t s concerning the e f f e c t s of magnesium on steady-state levels of the E.Ca.P complex are d i f f i c u l t to r a t i o n a l i z e and may i n fact be highly dependent on other factors, such as temperature or ATP concentration, which are known to have a profound e f f e c t on the a c t i v i t y of the enzyme. Although these 90 other reports appear contradictory i n nature, stating that eiher phosphorylation or dephosphorylation was affected, they a l l sup-port the r e s u l t s of the present study which suggest that magne-sium stimulates the turnover of the enzyme by simultaneously increasing both the rate of formation and the rate of breakdown of the phosphorylated intermediate. Hence no change i s detected in either the apparent rate of formation or i n the calcium-dependency of phosphoprotein formation, but a large e f f e c t i s seen i n the v e l o c i t y of ATP hydrolysis. Rega and Garrahan (1975) showed that magnesium stimulated a t r a n s i t i o n of the intermediate complex E.Ca.P from the E^ state of the enzyme to the E 2 confor-mation, which then rapidly dephosphorylates. It i s possible that magnesium may also stimulate the reverse of t h i s change, namely the conversion from E 2 to E-^ . As with calmodulin, magnesium appears to in some way f a c i l i t a t e a conformational change i n the enzyme in both the phosphorylated and the non-phosphorylated states. The actual mechanism of t h i s action i s not known. As described previously, the i n h i b i t i o n of dephosphorylation by calcium has been attributed to calcium blocking the conforma-t i o n a l change from E^.Ca.P to E 2.Ca.P by competing at a magnesium-specific binding s i t e (Schatzmann, 1982). This theory implies two things, namely; there i s a magnesium-specific s i t e involved, and 2^ the buildup i n E.Ca.P observed i n the presence of high concentrations of calcium represents the E-^.Ca.P form of the enzyme. Considering the very low turnover number observed i n the presence of millimolar concentrations of calcium (1-5 m i n - 1 ) , conversion to E 2.Ca.P and or subsequent hydrolysis proceed very slowly. In regards to the magnesium-specificity of the s i t e , 91 varying the magnesium concentration from zero to 1.0 x 10~ J M had no e f f e c t on E.Ca.P formation i n the presence of 10 mM calcium. Other investigators have demonstrated that magnesium w i l l compete with calcium to antagonize the buildup of E.Ca.P (Lichtner and Wolf, 1980a) and the i n h i b i t i o n of (Ca 2 + + Mg 2 +)-ATPase a c t i v i t y by calcium (Klinger et a_l, 1980). These studies, however, u t i -l i z e d equimolar or greater amounts of magnesium to "compete" with calcium. This i s not consistent with the concept of a mag-nesium-specific s i t e . Either the s i t e at which the e f f e c t takes place does not discriminate between magnesium and calcium, or what i s seen represents magnesium antagonizing the binding of calcium at a low - a f f i n i t y calcium binding s i t e . To determine whether calcium blocks the equilibrium between E-^.Ca.P and E 2«Ca.P or s t a b i l i z e s E 2.Ca.P, the nucleotide s e n s i t i v i t y of the phosphoprotein formed at high calcium concentrations was exa-mined. Dephosphorylation of the phosphorylated intermediate was i n i t i a t e d using either ATP (1 mM) or ADP (1 mM). ATP i s known to stimulate dephosphorylation by dr i v i n g the reaction i n the f o r -ward d i r e c t i o n whereas, ADP i s believed to stimulate dephosphory-l a t i o n i n both the forward d i r e c t i o n and by reversing the phos-phorylation by reacting with the high energy phosphate i n E-^.Ca.P (Rega and Garrahan, 1978; Schatzmann, 1982). In the presence of 10 mM Ca , increasing the free magnesium concentration from zero (0.1 mM CDTA) up to 1 mM had no e f f e c t upon the s e n s i t i v i t y of the phosphorylated intermediate to dephosphorylation by either ADP or ATP. Garrahan and Rega (1978) previously demonstrated that in the presence of high concentrations of ATP (1.0 mM), magnesium 92 stimulated dephosphorylation by promoting the conformational change from E-^.Ca.P to E 2.Ca.P. Theoretically, i f t h i s conforma-ti o n a l change was blocked by calcium, magnesium should have enhanced the s e n s i t i v i t y of the phosphorylated intermediate to dephosphorylation by ATP i n the foreward d i r e c t i o n . Although dephosphorylation was greater i n the presence of ADP after a f i v e second chase than for ATP, after f i f t e e n seconds, dephosphoryla-ti o n by ATP was 80-90% complete and equal to that of ADP. From these r e s u l t s , i n h i b i t i o n of .the conformational change from E-^.Ca.P to E 2.Ca.P by high calcium concentrations does not appear to be s u f f i c i e n t to account for the accumulation (low turnover) of the phosphorylated intermediate observed in the presence of high calcium concentrations. V Changes i n the (Ca2"1" + Mg2"1")-ATPase A c t i v i t y and Phosphoprotein Formation i n Erythrocytes from Patients  with Cystic F i b r o s i s The methodology developed to estimate maximum levels of phosphorylated enzyme intermediate, which i s a measure of the available functional enzyme a c t i v i t y , was used to examine the functional level of the calcium pump i n a disease state thought to involve a defect i n calcium handling by the c e l l . Previous 7 + 2 + studies have indicated that there i s a decrease i n (Ca* + Mg )-ATPase a c t i v i t y i n erythrocyte membranes of patients with c y s t i c f i b r o s i s (CF) (Horton et a_l, 1970; Katz, 1978; Ansah and Katz, 1980; Foder et a l , 1980; Gietzen et a_l, 1980; for review see Katz et a l , 1984). This decrease i n ATPase a c t i v i t y has been cor-93 related with a decrease in calcium transport a c t i v i t y i n inside-out v e s i c l e preparations of erythrocyte membranes (Ansah and Katz, 1980). Recently, Miner et a_l (1983) presented a report in d i c a t i n g that in the presence of lanthanum, which blocks de-phosphorylation, s i g n i f i c a n t l y less phosphoprotein was formed i n CF erythrocytes. We found that i n the presence of lanthanum (0.1 mM) or high calcium concentrations (10 mM), compared to age and sex-matched controls, there was a s i g n i f i c a n t reduction i n the phosphorylated intermediate formed i n erythrocytes from CF patients, and t h i s reduction was correlated with a similar reduction i n ATPase a c t i v i t y . The turnover number of the i n t e r -mediate, however, did not d i f f e r s i g n i f i c a n t l y from controls. There was no difference in the "basal" levels of ATPase a c t i v i t y or phosphoprotein formation assayed i n the presence of magnesium (2.5 x 10 -^ M) alone. These re s u l t s suggest that the number of active calcium pumping s i t e s may be lower i n CF membranes due to either the presence of non-functional Ca-ATPase molecules, or ^ fewer Ca-ATPase molecules i n the CF erythrocyte membranes. The p o s s i b i l i t y of decreased enzyme a c t i v i t y due to impaired function of the enzyme seems doubtful, as the turnover number of the ex i s t i n g enzyme was not altered. 94 CONCLUSIONS 1. At low calcium concentrations (1.0 uM) , calmodulin stimulated the apparent rate of formation of the phosphorylated intermediate, whereas at higher concentrations of calcium (0.4 mM) calmodulin did not a l t e r the apparent rate of formation but decreased the steady-state l e v e l s . These r e s u l t s are consistent with previous studies which found that calmodulin stimulated both formation and breakdown of the intermediate complex. These re-sults also suggest that at the lower calcium concentrations phosphorylation was the r a t e - l i m i t i n g step, whereas at higher calcium concentrations dephosphorylation became r a t e - l i m i t i n g . The steps stimulated by calmodulin l i k e l y represent those which are constrained by the " i n h i b i t o r y subunit". Although there i s s t i l l no d i r e c t evidence as to the step at which calmodulin acts to produce the observed increase i n the rate of phosphoprotein formation, current knowlege concerning the e f f e c t s of calmodulin may be best explained by suggesting that camodulin produces a change i n the enzyme which has more than one e f f e c t on i t s reac-t i o n cycle. 2. At concentrations greater than 5.0 x 10 M, calcium appeared to block dephosphorylation of the intermediate complex. Previous reports stated that t h i s i n h i b i t o r y e f f e c t of calcium could be antagonized by magnesium and was l i k e l y due to calcium antagonizing the magnesium-stimulated t r a n s i t i o n from E-^.Ca.P to E 2.Ca.P (Schatzmann, 1982). Results from the present study were not consistent with t h i s idea: ^ phosphoprotein formed due to 95 calcium-dependent i n h i b i t i o n of dephosphorylation could be de-phosphorylated i n either the foreward or backward d i r e c t i o n ; 2^ the absence of magnesium i n the reaction medium did not simulate the effects of millimolar concentratons of calcium; and 3^ magnesium did not appear to antagonize the i n h i b i t i o n of dephos-phorylation by calcium. An alternate mechanism was proposed where calcium interacts with the E2.P complex to produce E 2.Ca.P, thereby maintaining the enzyme in the phosphorylated state. This proposal assumes that calcium d i s s o c i a t i o n precedes the actual dephosphorylation step. 3. The presence of magnesium does not appear to be an absolute requirement for phosphorylation. However, since magne-sium does increase the apparent calcium s e n s i t i v i t y of the (Ca + Mg 2 +)-ATPase i n addition to increasing the turnover of the phosphorylated intermediate, i t i s possible that magnesium stimu-lates the rate of transformation from E 2 to E-^ , as suggested by Sarkadi (1980) i n addition to i t s stimulatory e f f e c t s on the t r a n s i t i o n from E^Ca.P to E 2.Ca.P. 4. Lanthanum has been shown to i n h i b i t the ( C a 2 + + Mg 2 +)-ATPase i n intact red c e l l s when present i n the e x t r a c e l -l u l a r medium. Sarkadi (1980) proposed that lanthanum binds to the E 2.P complex, once calcium has dissociated, s t a b i l i z i n g the enzyme in the phosphorylated state. Results from the present study, as well as others suggest that lanthanum may stimulate phosphorylation of the enzyme in the absence of calcium. These r e s u l t s suggest that i n addition to i t s e f f e c t s on E 2-Pf lantha-96 num may substitute for calcium i n promoting phosphorylation. The phosphorylated intermediate formed i n the presence of lanthanum i s rapidly dephosphorylated by ADP but decomposes slowly i n the foreward d i r e c t i o n (Shatzmann and Burgin, 1978), suggesting that the phosphoprotein formed i n the presence of lanthanum converts to the E 2 state very slowly. 5 . Reduced levels of both ( C a 2 + + Mg 2 +)-ATPase a c t i v i t y and of the phosphorylated intermediate were observed i n membrane fragments from patients with c y s t i c f i b r o s i s with respect to normal subjects. 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