Rate constants for calmodulin binding to Ca2+-ATPase in erythrocyte membranes

The Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes, which is part of the Ca2+ pump, can be activated by binding of calmodulin. Rate constants (k1) for association of calmodulin and enzyme, which depends on the Ca2+ concentration, have been determined by the aid of an enzyme model. k1 increased from 0.25 . 10(6) to 17.3 . 10(6) M-1 . min-1 (70 times) when the free Ca2+ concentration was raised from 0.7 to 20 microM. The binding of calmodulin to the Ca2+-ATPase is reversible. The rate constants (k-1) for dissociation of enzyme-calmodulin complex decreased from 6.0 to 0.044 min-1 (135 times) when the free Ca2+ concentration was increased from 0.1 to 2-20 microM. The apparent dissociation constant Kd = k-1/k1 accordingly increased from 2.5 nM to 25 microM (or higher) when the Ca2+ concentration was reduced from 20 to 0.1 microM. Therefore, at 10(-7) M free Ca2+ most of the Ca2+-pump enzyme will not bind calmodulin. For the intact cell the time dependences of activation and deactivation of the Ca2+-pump enzyme have been estimated from the rate constants above. The results suggest that the Ca2+ pump is well suited to maintain a cytosolic concentration of 10(-7) M free Ca2+ (or lower) in the unstimulated cell and, when the cell is stimulated, to allow transient Ca2+ signals up to approx. 10(-5) M in the cytosol.     

Purification, characterization, and reconstitution of the Ca2+-transport system (high-affinity Ca2+, Mg2+-ATPase) of the human erythrocyte membrane

The Ca2+-transport system of human erythrocyte membranes was solubilized by deoxycholate in the presence of the nonionic detergent Tween 20 and was purified by calmodulin affinity chromatography. The method yields a functional enzyme, which as compared with the erythrocyte membrane was purified 207-fold based on specific activity, and about 330-fold based on protein content. The activity of the isolated enzyme can be increased about 9-fold by the addition of calmodulin, resulting in a specific activity of 10.1 mumoles/mg . min at 37 degrees C. Triton X-100 and deoxycholate stimulate the calmodulin-deficient Ca2+-ATPase in a concentration dependent manner, which results in a loss of the calmodulin-sensitivity. The Ca2+-transport ATPase could be reconstituted after solubilization of the ATPase by deoxycholate and controlled dialysis near room temperature. The system was reconstituted to form membraneous vesicles capable of energized Ca2+ accumulation. The membrane vesicles showed a protein to lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original erythrocyte membrane. The stimulation by calmodulin of the calmodulin-depleted membrane-bound and partially purified Ca2+-ATPase is strongly time dependent. At a Ca2+-concentration of 40 microM and low calmodulin concentrations, approx. 120 min are required to regain full activity. This time period is decreased to about 15 min in the presence of a high excess of calmodulin. Vice versa, at fixed concentrations of calmodulin, the time necessary for regain of full activity is decreased as the Ca2+ concentrations is increased. The dependence of the Ca2+-ATPase activity on the calmodulin concentration shows strong deviation from Michaelis-Menten kinetics at Ca2+ concentrations below (4--10 microM) and above (200 microM) the optimum concentration of 40 microM. Mathematical analysis of the results at 200 microM Ca2+ leads to the assumption that 4 calmodulin molecules interact with one oligomer of Ca2+-ATPase consisting of 4 identical subunits.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca²⁺ and decreased approx. 1000-times when the Ca²⁺ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (Ca_iZ) decreases in the order of $\text{Ca}{}_3\text{Z}\text{>}\text{Ca}{}_4\text{Z}\text{>}\text{Ca}{}_2\text{Z}\text{?}\text{CaZ}$. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca²⁺-ATPase in the range of 10^?7–10^?6 M Ca²⁺, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca²⁺-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca²⁺-transport ATPase in erythrocytes at the prevailing Ca²⁺ concentration (probably 10^?7 – 10^?8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca²⁺-ATPase requires that the Ca²⁺ concentration rises to 10^?6 – 10^?5 M.     

Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Novel effects of calmodulin and calmodulin antagonists on the plasma membrane (Ca2+ + Mg2+)-ATPase from rabbit kidney proximal tubules.

In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted of calmodulin is characterized by a high Ca2+ affinity (Km = 2.2-3.4 microM) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (Km less than 0.05 microM). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the Km for Ca2+ to 5.7 microM and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30 microM) and ATP (5 mM) concentrations, Pi protected against 48/80 and potentiated inhibition by calmidazolium. At 25 microM ATP, Pi protected against calmidazolium inhibition. We propose that the effects of ATP and Pi arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.     

The effect of berbamine derivatives on activated Ca2+-stimulated Mg2+-dependent ATPase in erythrocyte membranes.

Ca2+-stimulated Mg2+-dependent ATPase (Ca2+ + Mg2+-ATPase) stimulated by calmodulin, by partial proteolysis or by oleic acid in erythrocyte membranes was inhibited by various derivatives of the naturally occurring alkaloid berbamine. The ability of these derivatives to inhibit trypsin-activated Ca2+ + Mg2+-ATPase correlated well with their ability to inhibit the calmodulin-stimulated enzyme. Inhibition of the trypsin-activated Ca2+ + Mg2+-ATPase by O-4-(ethoxybutyl)berbamine (EBB) was competitive with respect to ATP. The Ki for inhibition was about 8 microM. These results suggest that the binding site of EBB on the activated Ca2+ + Mg2+-ATPase may bear structural similarity to that on calmodulin, and may be closely related to the ATP-binding site on the enzyme.     

Activation of erythrocyte membrane Ca2+-ATPase by calpain

Ca2+-ATPase of erythrocyte membranes, prepared from erythrocytes substantially removed of contaminating leukocytes, was found to be activated by calpain isolated from the same source. Saponin or glycodeoxycholate treatment of membranes was essential for elicitation of the calpain response. Unlike the membrane bound ATPase, solubilized ATPase was inactivated by calpain. Digestion of membranes with the protease did not affect the Km (ATP) of Ca2+-ATPase though stimulation of the membrane ATPase by calmodulin could be partially substituted by calpain treatment. As compared with control, Ca2+-ATPase of calpain-digested membranes attained maximal activity at a lower free Ca2+ concentration.     

Further characterization and reconstitution of the purified Ca2+-pumping ATPase of heart sarcolemma

The Ca2+-pumping ATPase has been isolated from calf heart sarcolemma by calmodulin affinity chromatography (Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256, 3263-3270) as a polypeptide of Mr about 140,000. The purified enzyme has high affinity for Ca2+ in the presence of calmodulin (Km about 0.4 microM) but shifts to a low affinity state (Km about 20 microM) in its absence. Calmodulin increases also the Vmax of the enzyme. The effects of calmodulin are mimicked by phosphatidylserine and by a limited proteolytic treatment of the enzyme with trypsin. The purified ATPase can be reconstituted in asolectin liposomes, where it pumps Ca2+ with an approximate stoichiometry to ATP of 1. The purified (and reconstituted) enzyme is not phosphorylated by added ATP and cAMP-dependent protein kinase under conditions where the enzyme in situ is stimulated concomitant with the phosphorylation of the sarcolemmal membrane (Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256, 9371-9373). Hence, the target of the regulatory phosphorylation system is not the ATPase molecule. The purified ATPase cross-reacts with an antibody raised against the erythrocyte Ca2+-pumping ATPase. Under the same conditions, the purified sarcoplasmic reticulum Ca2+-ATPase does not react. The proteolytic splitting pattern of the purified heart sarcolemma and erythrocyte enzymes are similar but not identical.     

Calmodulin-dependent Ca2+-pump ATPase of human smooth muscle sarcolemma

An enzymatically active Ca2+-stimulated ATPase has been isolated from the sarcolemmal sheets of human smooth muscle (myometrium). Ca2+-ATPase activity was quantitated in an assay medium which simulated the characteristic free ionic concentrations of the cytosol. New computer programs for calculating the composition of solutions containing metals (Ca, Mg, Na, K) and ligands (EGTA, ATP), based on the updated stability constants, were used. In detergent-soluble form the enzyme has a high Ca2+-affinity expressed by an apparent Km (Ca2+) of 0.25 +/- 0.04 microM. The maximum specific activity (about 20 nmol of Pi/mg protein/min) was found in the micromolar domain of free-Ca2+ concentrations, the same levels required for normal maximal contractions in smooth muscle. The variation of free-Ca2+ concentration in the assay medium over 4 orders of magnitude (pCa 9 to pCa 5) resulted in a sigmoidal dependence of enzymatic activity, with a Hill coefficient of 1.4, which suggested the regulation of Ca2+-ATPase by allosteric effectors. The presence and the activator role of endogenous calmodulin in smooth muscle sarcolemma was proved by calmodulin-depletion experiments and by using suitable anticalmodulinic concentrations of trifluoperazine. The addition of exogenous calmodulin restored the enzyme activity. Apparently, the concentration of calmodulin in isolated smooth muscle sarcolemma is about 0.1% of sarcolemmal proteins, as deduced from the comparison of calmodulin-depletion and calmodulin-readdition experiments. Calmodulin increased significantly the enzyme Ca2+-affinity and Vmax (by a factor of about 10). At variance with the sarcoplasmic reticulum Ca2+-ATPase, the sarcolemmal Ca2+-ATPase is extremely sensitive to orthovanadate, half-maximal inhibition being observed at 0.8 microM vanadate. In conclusion, the Ca2+-ATPase isolated from smooth muscle sarcolemma appears very similar to the well-known Ca2+-pump ATPases of erythrocyte membrane, heart sarcolemma or axolemma. We suggest that this high-affinity Ca2+-ATPase represents the calmodulin-regulated Ca2+-extrusion pump of the smooth muscle sarcolemma.     

Persistent Ca2(+)-induced activation of erythrocyte membrane Ca2(+)-ATPase unrelated to calpain proteolysis

Preincubation of human erythrocyte membranes with calcium in the submillimolar to millimolar concentration range resulted in an increase of the Ca2+ affinity and apparent maximum velocity of the Ca2(+)-stimulated Mg2(+)-dependent ATPase (Ca2(+)-ATPase). The activation was persistent, as it was not reversed when the Ca2(+)-preincubated membranes were washed with ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid-containing buffers. Magnesium was not required for the activation, whereas greater than 2 mM Mg2+ partially antagonized the activation by Ca2+. In some membrane preparations ATP was required in addition to Ca2+ for activation of the Ca2(+)-ATPase, but nonhydrolyzable analogs of ATP had the same effect. Calmodulin prevented the activation by Ca2+ over the same concentration range in which it interacts with the Ca2(+)-ATPase. Taken together the results obtained provided strong evidence that the Ca2+ activation of the enzyme was not due to proteolytic cleavage by endogenous calpain. Thus, activation by Ca2+ was not blocked by leupeptin (100-200 microM), did not require dithiothreitol, and occurred at Ca2+ concentrations greater than those required for activation of calpain I. Furthermore, Ca2+ activation did not result in change in the mobility the native 136-kDa species of the Ca2(+)-ATPase on SDS-gel electrophoresis. Moreover, solubilization of the Ca2(+)-pretreated membranes with Triton X-100 reversed the Ca2+ activation of the Ca2(+)-ATPase. On the other hand, Ca2(+)-pretreatment of the membranes modified the susceptibility of the Ca2(+)-ATPase to both cleavage and activation by exogenously added calpain I. We conclude that pretreatment of Ca2(+)-ATPase in erythrocyte membranes with millimolar Ca2+ activates the enzyme by inducing a persistent conformational change of the enzyme which is, however, subsequently reversed by detergent solubilization.     

Studies of the Ca2+ transport mechanism of human erythrocyte inside-out plasma membrane vesicles. I. Regulation of the Ca2+ pump by calmodulin

Calcium accumulation by human erythrocyte inside-out vesicles was linear for at least 30 min in the presence of ATP. In untreated inside-out vesicles, 3.76 +/- 1.44 nmol of calcium/min/unit of acetylcholinesterase were transported, compared with 10.57 +/- 2.05 (+/- S.D.; n = 11) in those treated with calmodulin. The amount of calmodulin necessary for 50% activation of Ca2+ accumulation was 60 +/- 22 ng/ml (+/- S.D.; n = 4). The Km (Ca2+) for calmodulin-stimulated accumulation was 0.8 +/- 0.05 microM (+/- S.D.; n = 5) using Ca2+ /ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) buffers, or 25 microM with direct addition of unbuffered calcium. In the absence of calmodulin, these values were 0.4 and 60 microM, respectively, Km (ATP) values of 90 and 60 microM in the presence and absence of calmodulin, respectively, were measured at constant magnesium concentration (3 mM). In the presence of calmodulin, a broad pH profile is exhibited from pH 6.6 to 8.2. Maximal calcium accumulation occurs at pH 7.8. In the absence of calmodulin, the pH profile exhibits a linear upward increase from pH 7.0 to 8.2. The (Ca2+-Mg2+)-ATPase activity, measured under identical conditions, was 2.40 +/- 0.72 nmol of Pi/min/unit of acetylcholinesterase in the untreated vesicles and 11.29 +/- 2.87 nmol of Pi/min/unit of acetylcholinesterase (+/- S.D.; n = 4) in calmodulin-treated vesicles. A stoichiometry of 1.6 Ca2+/ATP hydrolyzed was determined in the absence of calmodulin; in the presence of calmodulin, this ratio was decreased to 0.94 Ca2+/ATP hydrolyzed.     

Enhanced activation of human erythrocyte Ca2+-ATPase by calmodulin after storage or brief exposure to disulfite

Ca2+-ATPase of human erythrocyte membranes which are prepared from freshly drawn human blood can be activated by the calmodulin present in the hemolysate to 1.5-times the basal level. However, when the membranes are prepared from blood stored for 5-14 days the activation by calmodulin reaches 2.5-times the basal level. An enhanced reactivity to calmodulin of similar magnitude was produced by brief exposure of fresh erythrocytes to 25 mM Na2S2O5 prior to isolation of the membranes. Reincubation of the activated cells in a disulfite-free medium restored the membrane-bound Ca2+-ATPase to a state of normal reactivity to calmodulin. It is hypothesized that these results are related to the level of cytoplasmic Ca2+ which is partly controlled by complex formation with 2,3-diphosphoglycerate, the concentration of which is diminished when its specific phosphatase is activated by Na2S2O5.     

The effect of calmodulin on the phosphoprotein intermediate of Mg2+-dependent Ca2+-stimulated adenosine triphosphatase in human erythrocyte membranes.

The effect of calmodulin on the formation and decomposition of the Ca2+-dependent phosphoprotein intermediate of the (Mg2+ + Ca2+)-dependent ATPase in erythrocyte membranes was investigated. In the presence of 60 microM-Ca2+ and 25 microM-MgCl2, calmodulin (0.5-1.5 microgram) did not alter the steady-state concentration of the phosphoprotein, but increased its rate of decomposition. Higher calmodulin concentrations significantly decreased the steady-state concentration of phosphoprotein. Calmodulin (0.5-1.7 microgram) increased Ca2+-transport ATPase activity by increasing the turnover rate of its phosphoprotein intermediate. Increasing the MgCl2 concentration from 25 microM to 250 microM increased the (Mg2+ + Ca2+)-dependent ATPase activity, but decreased the concentration of the phosphoprotein intermediate. Similarly to calmodulin, MgCl2 increased the turnover rate of the Ca2+-transport ATPase complex (about 3-fold). At the higher MgCl2 concentration calmodulin did not further affect the decomposition of the phosphoprotein intermediate. It was concluded that both calmodulin and MgCl2 increase the turnover of the Ca2+-pump by enhancing the decomposition of the Ca2+-dependent phosphoprotein intermediate.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

Compound 48/80 and calmodulin modify the interaction of ATP with the (Ca2+ + Mg2+)-ATPase of red cell membranes

Compound 48/80, an anti-calmodulin agent, reduces the maximum effect of ATP and does not affect the apparent affinity for ATP of the high-affinity site of the Ca2+-ATPase from calmodulin-bound membranes of human red cells. In the same preparation, 48/80 reduces more than 50-times the apparent affinity for ATP of the low-affinity site with little change in the maximum effect of the nucleotide at this site of the Ca2+-ATPase. The effects of compound 48/80 are independent of the concentration of Ca2+ between 30 and 200 microM. The apparent affinity of the low-affinity site of the Ca2+-ATPase for ATP is almost 100-fold less in calmodulin-stripped membranes than in calmodulin-bound membranes. In calmodulin-stripped membranes, exogenous calmodulin increases the apparent affinity for ATP up to the control values. These results indicate that apart from increasing the apparent affinity of the transport site for Ca2+, calmodulin also increases the apparent affinity of the regulatory site of the Ca2+-ATPase for ATP. Since this effect is exerted within the physiological ranges of ATP concentrations, it may participate in the physiological regulation of Ca2+ pumping by calmodulin.     

Fluorescence energy transfer studies of purified erythrocyte Ca2+-ATPase. Ca2+-regulated activation by oligomerization

Fluorescence resonance energy transfer has been used to study oligomerization of the purified erythrocyte Ca2+-ATPase. The energy transfer efficiency has been measured at different enzyme concentrations, from fluorescein 5'-isothiocyanate attached on one enzyme molecule to eosin 5-maleimide or tetramethylrhodamine 5-isothiocyanate attached on another enzyme molecule. The energy transfer efficiency showed a sigmoid dependence on enzyme concentration and was half-maximal at 10-12 nM enzyme; this dependence on enzyme concentration closely resembled previously demonstrated dependence of Ca2+-ATPase activity and polarization of the fluorescein 5'-isothiocyanate enzyme (Kosk-Kosicka, D., and Bzdega, T. (1988) J. Biol. Chem. 263, 18184-18189). Thus, the three independent methods establish that enzyme concentration-dependent oligomerization is a mechanism of activation of the erythrocyte Ca2+-ATPase. Further energy transfer studies demonstrated that enzyme oligomerization required calcium. This calcium dependence was characterized by high affinity (half-maximal energy transfer at pCa 7.15) and cooperativity (Hill coefficient of 2.36), being very similar in both respects to the Ca2+ dependence of the Ca2+-ATPase activity. The data indicated that the oligomerization process produced a highly cooperative, Ca2+-regulated activation of the enzyme at physiologically relevant Ca2+ concentrations. These studies show that the Ca2+-ATPase can be fully activated by a Ca2+-dependent oligomerization mechanism, which is independent of the previously described activation by calmodulin. We propose two pathways for the activation of the Ca2+-ATPase, taking into account the interdependencies between the Ca2+, calmodulin, and enzyme concentrations.     

Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Calmodulin regulation of Ca2+ transport in human erythrocytes.

Inside-out vesicles of human erythrocytes took up Ca2+ against an electrochemical gradient. This Ca2+ uptake was dependent on ATP and was stimulated by calmodulin. Treatment of vesicles with 1 mM-EDTA exposed an apparent low-CA2+-affinity Ca2+-transport component with Kd of about 100 microM-Ca2+ or more. This was converted into a single high-Ca2+-affinity transport activity of Kd about 2.5 microM-Ca2+ in the presence of 2 micrograms of calmodulin/ml, showing that the decrease in transport activity after EDTA treatment was reversible. Vesicles not extracted with EDTA showed mainly apparent high-Ca2+-affinity kinetics even in the absence of added calmodulin. Trifluoperazine (30 microM) and calmodulin-binding protein (20 micrograms/ml) inhibited about 50% of the high-affinity Ca2+ uptake and (Ca2+ + Mg2+)-ATPase (Ca2+-activated, Mg2+-dependent ATPase) activity of these vesicles, indicating that the vesicles isolated by the procedure used retained some calmodulin from the erythrocytes. Comparison of Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities in inside-out vesicles yielded a variable Ca2+/P1 stoichiometric ratio. At low free Ca2+ concentrations (below 20 micro-Ca2+), a Ca2+/P1 ration of about 2 was found, whereas at higher Ca2+ concentrations the stoichiometry was approx. 1. The stoichiometry was not significantly altered by calmodulin.