Antibodies against erythrocyte Ca2+-transport ATPase specifically inhibit the calmodulin-dependent fraction of the enzyme''s activity.

Antibodies against purified Ca2+-transport ATPase from human erythrocytes were raised in rabbits. Immunodiffusion experiments revealed that precipitating antibodies had been developed. The immunoglobulin fraction inhibited solely the calmodulin-dependent fraction of erythrocyte Ca2+-transport ATPase activity, whereas the basal (in the absence of added calmodulin) activity of the enzyme was not significantly affected by the antibodies. The antibodies produced similar doseresponse curves for the calmodulin- and the oleic acid-stimulated enzyme. However, the immunoglobulin fraction was considerably less effective in inhibiting Ca2+-transport ATPase activated by limited proteolysis. The results obtained with our antibodies are compatible with the interpretation that at least one subpopulation of the antibodies attacks the enzyme at or close to the calmodulin-binding site of the ATPase. The antibodies also inhibited the calmodulin-regulated Ca2+-transport ATPase from pig smooth-muscle plasma membrane, though with lower potency. However, the immunoglobulin fraction failed to suppress pig cardiac sarcoplasmicreticulum Ca2+-transport ATPase activity in the concentration range investigated. In addition, the activity of phosphodiesterase from rat brain, another enzyme modulated by calmodulin, was not at all affected by the immunoglobulin fraction.     

Response of three enzymes to oleic acid, trypsin, and calmodulin chemically modified with a reactive phenothiazine

Calmodulin was covalently modified with 10-(1-propionyloxysuccinimide)-2-trifluoromethylphenothiazine++ + to stoichiometries between 0 and 2 mol/mol in the presence of Ca2+. The modified calmodulins, oleic acid, and trypsin were assayed for their ability to activate pea plant NAD kinase, bovine brain 3',5'-cAMP phosphodiesterase, and human erythrocyte Ca2+-ATPase. All modified calmodulins activated both phosphodiesterase and Ca2+-ATPase; at the highest concentration assayed, calmodulin modified with 2 mol of reagent/mol activated phosphodiesterase and Ca2+-ATPase to 53% and 100%, respectively, of the activation obtained with unmodified calmodulin. However, higher concentrations of the modified calmodulins were required to observe the same activation; at least 900-fold and 100-fold higher concentrations were required for the two enzymes, respectively. NAD kinase was not activated by any calmodulin labeled to a stoichiometry greater than 1 mol/mol even when a concentration equal to 17,000 times the apparent dissociation constant of calmodulin for NAD kinase was assayed. Therefore, the modified protein (and not some fraction resistant to labeling) is active toward the mammalian enzymes but inactive toward plant NAD kinase. The different response of the three enzymes to the chemical modification suggests that the enzymes may utilize different binding domains on calmodulin. NAD kinase also was not activated by other known activators of the two mammalian enzymes, namely lipids and limited proteolysis. In parallel experiments using the same agents on each enzyme, NAD kinase was the only enzyme of the three that was not activated by oleic acid and several other lipids or by limited trypsin digestion. These results show that NAD kinase possesses several attributes which would not be predicted by current models of the mechanism of activation of enzymes by calmodulin.     

Target sizes of human erythrocyte membrane Ca2+-ATPase and Mg2+-ATPase activities in the presence and absence of calmodulin

We have investigated the subunit structure of Ca2+-transport ATPase in human erythrocyte membranes using radiation inactivation analysis. All inactivation data were linear on a semilog plot down to at least 20% of the control activity. We found a target size for the calmodulin-dependent Ca2+-ATPase activity of 331 kDa, consistent with the presence of this enzyme as a dimer in calmodulin-depleted ghosts. Membranes which had been saturated with calmodulin before irradiation yield a a similar size of 317 kDa, implying that activation of Ca2+-transport ATPase by calmodulin does not involve significant change in oligomeric structure. Basal (calmodulin-independent) Ca2+-ATPase activity corresponded to a size of 290 kDa, suggesting that this activity resides in the same, or similar-sized, complex as the calmodulin-dependent activity. Mg2+-ATPase activity, however, was found to reside in a smaller complex of 224 kDa, which proved to be statistically distinct from the target size of Ca2+-ATPase activity. It would appear that Mg2+-ATPase is a distinct entity whose function is likely unrelated to the Ca2+-transport ATPase.     

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.     

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.     

The control by Ca2+ of the polyphosphoinositide phosphodiesterase and the Ca2+-pump ATPase in human erythrocytes

1. Both the Ca(2+)-pump ATPase and the polyphosphoinositide phosphodiesterase of the erythrocyte membrane can, when assayed under appropriate conditions, be activated by Ca(2+) in the micromolar range. We have therefore compared the mechanisms and affinities for Ca(2+) activation of the two enzymes in human erythrocyte membranes, to see whether the polyphosphoinositide phosphodiesterase would be active in normal healthy erythrocytes. 2. At physiological ionic strength and in the presence of calmodulin, the Ca(2+)-pump ATPase was activated by Ca(2+) in a highly co-operative manner, with half-maximal activation occurring at about 0.3mum-Ca(2+). At an optimal Ca(2+) concentration, calmodulin stimulated the Ca(2+)-sensitive ATPase activity about 10-fold. 3. Ca(2+) activated the polyphosphoinositide phosphodiesterase in a non-co-operative manner. The Ca(2+) requirements for breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were identical, which supports our previous conclusion that Ca(2+) activates a single polyphosphoinositide phosphodiesterase that degrades both lipids with equal facility. Added calmodulin did not affect the activity of the polyphosphoinositide phosphodiesterase. 4. At low ionic strength in the absence of Mg(2+), half-maximal activation of the phosphodiesterase was at about 3mum-Ca(2+). The presence of 1mm-Mg(2+) shifted the Ca(2+) activation curve to the right, as did elevation of the ionic strength. When the Ca(2+)-pump ATPase and the polyphosphoinositide phosphodiesterase were assayed in the same incubations and under conditions of intracellular ionic strength and Mg(2+) concentration, the ATPase was fully activated at 3mum-Ca(2+), whereas no polyphosphoinositide phosphodiesterase activity was detected below 100mum-Ca(2+). 5. The Ca(2+)-pump ATPase of the erythrocyte membrane normally maintains the Ca(2+) concentration of healthy erythrocytes below approx. 0.1mum. It therefore seems unlikely that the polyphosphoinositide phosphodiesterase of the erythrocyte membrane ever expresses its activity in a healthy erythrocyte.     

An enzymatic assay for calmodulins based on plant NAD kinase activity

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5's ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5's for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.     

Evidence for the presence in smooth muscle of two types of Ca2+-transport ATPase.

Membrane fractions prepared from smooth muscle of the pig stomach (antral part) contain two Ca2+-dependent phosphoprotein intermediates belonging to different Ca2+-transport ATPases. These alkali-labile phosphoproteins can be separated by electrophoresis in acid medium. The 130 kDa phosphoprotein resembles a corresponding protein in the erythrocyte membrane, whereas the 100 kDa protein resembles that of the Ca2+-transport ATPase in sarcoplasmic reticulum from skeletal muscle. These resemblances are expressed in terms of Mr, reaction to La3+ and in a similar proteolytic degradation pattern. The presence of the calmodulin-stimulated ATPase in mixed membranes from smooth muscle is confirmed by its binding of calmodulin and antibodies against erythrocyte Ca2+-transport ATPase, whereas such binding does not occur with proteins present in the presumed endoplasmic reticulum from smooth muscle.     

Comparison of the calmodulin antagonists compound 48/80 and calmidazolium.

The two presumed calmodulin antagonists calmidazolium and compound 48/80 were compared for their effects on several calmodulin-dependent and calmodulin-independent enzyme systems. Compound 48/80 and calmidazolium were found to be about equipotent in antagonizing the calmodulin-dependent fraction of brain phosphodiesterase and erythrocyte Ca2+-transporting ATPase. Compound 48/80 combines high potency with high specificity in that: (1) the basal, calmodulin-independent, activity of calmodulin-regulated enzymes was not suppressed; (2) calmodulin-independent enzyme activities, such as Ca2+-transporting ATPases of sarcoplasmic reticulum, Mg2+-dependent ATPases of different tissues and Na+/K+-transporting ATPase of cardiac sarcolemma, were far less altered, or not altered at all, by compound 48/80 as compared with calmidazolium; and (3) antagonism of proteolysis-induced stimulation as opposed to calmodulin-induced activation of erythrocyte Ca2+-transporting ATPase required a 32 times higher concentration of compound 48/80. In all these aspects compound 48/80 was found to be a superior antagonist to calmidazolium since inhibition of calmodulin-independent events by the other agent occurred at considerably lower concentrations. Therefore compound 48/80 is proposed to be a much more specific and useful tool for studying the participation of calmodulin in biological processes than the presently used agents.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Stimulation of the erythrocyte Ca2+-ATPase and of bovine brain cyclic nucleotide phosphodiesterase by chemically modified calmodulin

Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+-ATPase whereas the stimulation of the bovine brain cyclic-nucleotide phosphodiesterase was reduced up to 50%. Different species of carbamoylated calmodulin have been isolated but no differences were detected in their interaction with the cyclic-nucleotide phosphodiesterase. Modification of arginine residues by 1,2-cyclohexanedione had no effect of the stimulation of the phosphodiesterase but reduced by 40% the stimulation of the erythrocyte Ca2+ ATPase. Mild oxidation of methionines by N-chlorosuccinimide produced a number of differently modified calmodulins. The different species have been purified and the modified residues have been identified. They affected the two different test enzymes to different extents indicating that methionines in the central helix of calmodulin are of greater importance for the interaction with the phosphodiesterase, whereas methionines located in the C-terminal half of calmodulin are more important for the interaction with the Ca2+-ATPase.     

The effect of cardiotoxin on (Ca2+ + Mg2+)-ATPase of the erythrocyte and sarcoplasmic reticulum

The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases.     

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

The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 microM 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 microM. 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 (CaiZ) decreases in the order of Ca3Z greater than Ca4Z greater than Ca2Z greater than or equal to 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 microM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 microM, corresponding to 50-80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/h membrane protein. We therefore conclude that most of the calmodulin is 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.     

Calmodulin binding proteins in human erythrocyte membranes

Human erythrocyte membranes reveal different calmodulin-binding proteins determined by a 125I-calmodulin gel overlay procedure. Beside the well-established Ca2+-transport ATPase, other proteins (205, 91, 72 and 42 kDa) bind calmodulin in a Ca2+-dependent manner. Two proteins of the human erythrocyte membrane are able to bind calmodulin only in the absence of Ca2+. One of them (76 kDa) is probably an integral, the other (240 kDa) a peripheral protein.     

Elucidation of conservative elements of calmodulin-dependent enzymes with the use of monoclonal antibodies

Monoclonal antibodies against human erythrocyte membrane Ca2+-ATPase were obtained. The binding of monoclonal antibodies to the enzyme resulted in a decrease in the enzyme sensitivity to calmodulin (CaM). The effects of monoclonal antibodies on other CaM-dependent enzymes, namely, on the phosphodiesterase of cAMP, phosphorylase kinase, and Ca2+-CaM-dependent protein kinase II (PK II), were studied. It was found that all four enzymes contain a common antigenic site. However, the inhibitory effect of antibodies was observed only with respect to Ca2+-ATPase and PK II. The kinetics of the binding of monoclonal antibodies and their inhibitory action were investigated. It was shown that the antigenic site is confined to the calmodulin-binding portion of Ca2+-ATPase and PK II.     

Topographical mapping of calmodulin-target enzyme interaction domains

Calmodulin derivatives, specifically biotinylated in domains I and III, were synthesized to address the structures of calmodulin necessary for binding to its target enzymes in active conformations. By binding avidin to these biotinylated calmodulins, the role of specific sequences of the calmodulin molecule in target enzyme interactions could then be evaluated. The role of domain I in these interactions was assessed by biotinylation of Cys-27 of wheat germ calmodulin with N-ethylmaleimidobiotin. This modification did not affect the ability of this calmodulin to activate 3'-5'-cyclic nucleotide phosphodiesterase (PDE) or human erythrocyte Ca2+-Mg2+ ATPase. The addition of avidin to form a stable calmodulin-avidin complex also did not affect activation. Bovine testes calmodulin was biotinylated on Lys-94 by calcium-dependent reaction with N-hydroxysuccinimido ester-biotin at pH 6.0. This derivative was used to probe the Ca+2 binding region of domain III. The incorporation of biotin at Lys-94 of bovine calmodulin did not affect calmodulin activation of PDE. However, compared to unmodified calmodulin, a 4-fold higher concentration of this derivative was required to fully activate the ATPase. The addition of excess avidin to this derivative abolished all activation for both PDE and the ATPase. Sites of modification were determined by sequence analysis of labeled peptides.     

Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins

1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C. 2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity. 3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations. 4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase. 5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.     

Calcium-independent bacterial activator of cyclic nucleotide phosphodiesterase and Ca2+/Mg2+-ATPase

Ca2+-independent protein-modulator (BacM) was found in the culture medium of Staphylococcus aureus. BacM activated calmodulin-dependent cyclic nucleotide phosphodiesterase and Ca2+/Mg2+-ATPase in the same way as calmodulin. BacM was shown to be a proteolytic fragment of the exotoxin secreted by the S. aureus strain under study. The kinetic analyses of the ATPase activation by BacM and CaM were performed. These studies demonstrated that the enzyme molecule contains at least two activator-sensitive sites. Experiments on the ATPase activation by Ca2+ both in the presence and in the absence of BacM and CaM documented that CaM-ATPase and BacM-ATPase complexes can exist at low concentrations of calcium. Analysis of activation curves of ATPase by Ca2+ revealed three Ca2+-binding sites in the enzyme-activator complex.     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.