Some properties of the purified (Ca2+ + Mg2+-ATPase from human red cell membranes

The (Ca²⁺ + Mg²⁺-ATPase from red cell membranes, purified by means of a calmodulin-containing affinity column according to the method of Gietzen et al. (Gietzen, K., Tej?ka, M. and Wolf, H.U. (1980) Biochem. J. 189, 81–88) with either phosphatidylcholine or phosphatidylserine as phospholipid is characterized. The phosphatidylcholine preparation can be activated by calmodulin, while the phosphatidylserine preparation is fully activated without calmodulin. The enzyme shows a biphasic ATP dependence with two K_m values of 3.5 and 120 μM. The enzyme is phosphorylated by ATP in the presence of Ca²⁺ only.     

Some properties of the purified (Ca2+ + Mg2+)-ATPase from human red cell membranes

The (Ca2+ + Mg2+)-ATPase from red cell membranes, purified by means of a calmodulin-containing affinity column according to the method of Gietzen et al. (Gietzen, K., Tejcka, M. and Wolf, H.U. (1980) Biochem. J. 189, 81-88) with either phosphatidylcholine or phosphatidylserine as phospholipid is characterized. The phosphatidylcholine preparation can be activated by calmodulin, while the phosphatidylserine preparation is fully activated without calmodulin. The enzyme shows a biphasic ATP dependence with two Km values of 3.5 and 120 microM. The enzyme is phosphorylated by ATP in the presence of Ca2+ only.     

Vanadate inhibition of the Ca2+-ATPase from human red cell membranes

(1) VO3(-) combines with high affinity to the Ca2+-ATPase and fully inhibits Ca2+-ATPase and Ca2+-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state of the Ca2+-dependent phosphoenzyme. (2) VO3(-) blocks hydrolysis of ATP at the catalytic site. The sites for VO3(-) also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca2+-ATPase. (3) The sites for VO39-) show positive interaactions in affinity with sites for Mg2+ and K+. This accounts for the dependence on Mg2+ and K+ of the inhibition by VO3(-). Although, with less effectiveness, Na2+ and K+ substitutes for K+ whereas Li+ does not. The apparent affinites for Mg24 and K+ for inhibiton by VO3(-) seem to be less than those for activation of the Ca2+-ATPase. (4) Inhibition by VO3(-) is independent of Ca2+ at concentrations up to 50 microM. Higher concentrations of Ca2+ lead to a progressive release of the inhibitiory effect of VO3(-).     

Modulator binding protein antagonizes activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport of red blood cell membranes

Red blood cells contain a protein that activates membrane-bound (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ transport. The red blood cell activator protein is similar to a modulator protein that stimulates cyclic AMP phosphodiesterase. Wang and Desai [Journal of Biological Chemistry 252:4175–4184, 1977] described a modulator-binding protein that antagonizes the activation of cyclic AMP phosphodiesterase by modulator protein. In the present work, modulator-binding protein was shown to antagonize the activation of (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ transport by red blood cell activator protein. The results further demonstrate the similarity between the activator protein from human red blood cells and the modulator protein from bovine brain.     

Activation of partial reactions of the Ca2+-ATPase from human red cells by Mg2+ and ATP.

(1) At ATP concentrations up to 30 micrometer addition of 0.5 mM MgCl2 in the reaction mixture increases both the rate of formation and the steady-state level of the phosphoenzyme of the Ca2+-ATPase from human red cell membranes. Under these conditions Mg2+ has no effect on the rate of dephosphorylation, which remains slow. (2) In the presence of Mg2+ the rate of dephosphorylation is increased 5 to 10 times by high (1 mM) concentrations of ATP. (3) Provided Mg2+ has reacted with the phosphoenzyme, acceleration of dephosphorylation by ATP takes place in the absence of Mg2+. This suggests that the role of Mg2+ on dephosphorylation is to convert the phosphoenzyme into a form that, after combination with ATP, reacts rapidly with water. (4) The results are consistent with the idea that combination of ATP at a non-catalytic site is needed for rapid dephosphorylation of the Ca2+-ATPase.     

Two classes of site for ATP in the Ca2+-ATPase from human red cell membranes.

(1) The response of the Ca2+-ATPase activity from human red cell membranes to ATP concentrations can be represented by the sum of two Michaelis-like curves: one with a Km of 2.5 micrometer and the other with a Km of 145 micrometer. (2) The maximum Ca2+-ATPase activity elicited by occupation of the site with lower Km represents about 10% of the activity attainable at non-limiting ATP concentrations. (3) 30--50% of the Ca2+-ATPase activity with lower Km remains in the absence of Mg2+ . Mg2+ increases V and the maximum effect of Ca2+, having no effect on the apparent affinities for ATP and Ca2+. (4) The large increase in Ca2+-ATPase activity which results from the occupation of the site with higher Km only takes place when Mg2+ is present. (5) Results are compatible with the idea that the Ca2+-ATPase from human red cell membranes has two classes of site for ATP binding, both of which are occupied when the enzyme catalyzes the hydrolysis of ATP at maximum rate. (6) The properties of the high affinity site suggest that this is the catalytic site of the Ca2+-ATPase. It is proposed that binding of ATP at the low affinity site regulates the turnover of the system.     

Pre-steady-state phosphorylation of the human red cell Ca2+-ATPase

The pre-steady-state kinetics of phosphorylation of the Ca2+-ATPase by ATP was studied at 37 degrees C and in intact red cell membranes to approach physiological conditions. ATP and Ca2+ activate with K0.5 of 4.9 and 26.4 microM, respectively. Preincubation with Ca2+ did not change the K0.5 for ATP. Preincubation with ATP did not alter the initial velocity of phosphorylation suggesting that binding of ATP was not rate-limiting. Mg2+ added at the start of the reaction increased the initial rate of phosphorylation from 4 to 8 pmol/mg/s. With 30 microM Ca2+, the K0.5 for Mg2+ was 60 microM. Mg2+ and Ca2+ added together beforehand accelerated phosphorylation to 70 pmol/mg/s. Phosphorylation of calmodulin-bound membranes was the fastest (280 pmol/mg/s), and its time course showed a neat overshoot before steady state. The results suggest that either preincubation with Ca2+ plus Mg2+ or calmodulin accelerated phosphorylation shifting toward E1 the equilibrium between the E1 and E2 conformers of the enzyme. K+ had no effect on the initial rate of phosphorylation and lowered by 40% the steady-state level of phosphoenzyme in the absence of Mg2+. Phosphorylation is not rate-limiting for the overall reaction since its initial rate was always higher than ATPase activity. In the absence of K+, the turnover of the phosphoenzyme was 2000 min-1, which is close to the values for other transport ATPases.     

The activation of phosphatase activity of the Ca2+-ATPase from human red cell membranes by calmodulin,ATP and partial proteolysis

(1) Depending on the assay conditions, the ability of the Ca²⁺-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg²⁺ and K⁺ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. (2) In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca²⁺ so that the activity becomes negligible above 30 μM Ca²⁺. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca²⁺. (3) In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca²⁺ are needed and phosphatase activity is maximal in media without Ca²⁺. This is the first report of an activity sustained by the Ca²⁺-ATPase of red cell membranes in the absence of Ca²⁺. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca²⁺. These results strongly suggest that during activation by calmodulin, Ca²⁺ is needed only to form the calmodulin-Ca²⁺ complex which is the effective cofactor. (4) Protection by ATP of the inhibitory effects of Ca²⁺ and the induction of phosphatase activity by ATP + Ca²⁺ suggests that activation of the phosphatase by Ca²⁺ in media with ATP requires the combination of the cation at sites in the ATPase. (5) Results can be rationalized assuming that E₂, the conformer of the Ca²⁺-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca²⁺ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E₁ and E₂ towards E₂. On the other hand, ATP + Ca²⁺ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E₂ predominates over other conformers of the ATPase.     

Effects of calmodulin on the phosphoenzyme of the Ca2+-ATPase of human red cell membranes

Comparison of the effects of calmodulin on the Ca2+-ATPase activity and on the steady-state level of the phosphoenzyme, indicates that activation of the Ca2+-ATPase is mainly due to an increase in the turnover of the phosphoenzyme and does not require occupation of the regulatory site of the Ca2+-ATPase by ATP.     

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate. 3. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.     

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.     

The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase from human platelets

The phospholipid requirement of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase activity}$. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.     

Postradiation changes in Ca2+-ATPase and Mg2+-ATPase in thymocyte plasma membranes]

The rats were irradiated in the doses 1, 5, 4, 7 and 10 Gr and on the 1, 8, 15, 22 and 30 day after the irradiation activity of Ca(2+)-ATPase and Mg(2+)-ATPase and peroxidation lipids in the thymocytes was determined. It was found that postradiation changes in activity of Mg(2+)-ATPase were characterized by a higher sensitivity to the processes of lipids peroxidation as compared to Ca(2+)-ATPase.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg2+-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567-576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The Km value for ATP was 1 mM and apparent Km for Mg2+ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd2+ and Zn2+ salts, p-chloromercuribenzoate and N-ethylmaleimide, known inhibitors of membrane endocytosis.     

Isolation and purification of Ca2+,Mg2+-ATPase from plasma membranes of the myometrium

The preparation of the purified Ca2+, Mg2(+)-ATPase has been isolated from triton X-100 solubilizate of plasma membranes of the pig myometrium using the method of affinity chromatography on calmodulin-Sepharose 4B. The specific activity of the enzyme shows its 52-fold purification. The enzymic preparation practically has no Mg2(+)-ATPase activity. By the data of DS-Na-electrophoresis in PAAG the Ca2+, Mg2+ ATPase preparation consists of two polypeptides with Mm 130 and 205 kDa. Autoradiography shows their Ca2(+)-dependent phosphorylation. The purified enzyme is highly sensitive to the inhibitory effect of orthovanadate.     

Purification of a low affinity Mg2+ (Ca2+)-ATPase from the plasma membranes of a human oat cell carcinoma

Plasma membranes of many mammalian cells contain a Mg2+-dependent ATPase activity which is easily inactivated by detergents. This activity is the combined expression of at least two ATP-hydrolyzing enzymes (Knowles, A.F., Isler, R.E., and Reece, J.F. (1983) Biochim. Biophys. Acta 731, 88-96). We have purified one of these enzymes from the plasma membranes of a human oat cell carcinoma xenograft. The enzyme was extracted from the membranes by 0.5% digitonin and purified on a DE52 column. The purified enzyme contained a major protein band of Mr = 30,000 when dissociated by sodium dodecyl sulfate. It hydrolyzed all nucleoside triphosphates in the presence of Mg2+ or Ca2+, but showed little activity toward nucleoside diphosphates. The enzyme was inhibited by p-chloromercuriphenyl sulfonate, slowly inactivated by p-fluorosulfonylbenzoyl-5'-adenosine and dithiothreitol at room temperature, and lost activity readily in solutions containing low concentrations of several detergents. This knowledge of the macromolecular structure of the Mg2+(Ca2+)-ATPase and its catalytic properties is important in determining the orientation of the enzyme in the membrane and its physiological function.     

Comparison of the red blood cell Ca2+-ATPase in ghost membranes and after purification

We have compared properties of the red blood cell Ca²⁺-ATPase in two types of preparations: red cell membrane ghosts (enzyme in unfractionated membranes) and after purification (detergent-soluble enzyme). The Ca²⁺-ATPase activity was studied with respect to its requirement for: calmodulin, calcium, magnesium, monovalent cations, ionic strength, pH, and temperature. Sensitivity of the Ca²⁺-ATPase activity in the two preparations to anticalmodulin drugs and to engineered calmodulins with amino acid substitutions was determined. Finally, stoichiometry of the formation of phosphorylated enzyme intermediate (EP) and titrations of the ATP binding region with fluorescein 5-isothiocyanate (FITC) were characterized. For the first time a high phosphorylation level of 2.0–2.4 mmol EP/mg of purified enzyme is reported.The two enzyme preparations have been found to be very similar with respect to the dependency of all the regulating factors described here. These results complement findings reported from various laboratories on the similarity of other kinetic properties as well as the similarity of modulation of the Ca²⁺-ATPase activity by phospholipids and proteolysis in the membranous and purified enzyme. Thus, the purified detergent-soluble enzyme is very well suited for kinetic characterization of the red cell Ca²⁺-ATPase.     

Characterization of a (Ca2+ + Mg2+)-ATPase activator bound to human erythrocyte membranes

Incubation of human erythrocyte ghosts with an equal volume of 0.2 mM EDTA in isotonic KCl decreased both the activity and Ca2+ sensitivity of the (Ca2+ + Mg2+)-ATPase remaining associated with the membrane. Readdition of the EDTA-extract activated the (Ca2+ + Mg2+)-ATPase activity. The activator activity was trypsin sensitive, heat stable and retained by a phenothiazine affinity column, consistent with properties expected of calmodulin. However, unlike calmodulin, the activity was not retained by DEAE Sephadex A-50 and it eluted from Sephacryl S-200 as heterogeneous peaks of activator activity of apparent molecular weight between 107,000 and 178,000. Nevertheless, the activator in the EDTA extract both before and after gel filtration contained calmodulin, as determined by radioimmunoassay and by its activation of calmodulin - deficient phosphodiesterase. SDS-gel electrophoresis of the activator isolated by gel filtration showed a protein of Mr 56,000 in addition to a low molecular weight protein corresponding to calmodulin. It is suggested that the red cell membrane contains a calmodulin binding protein which tightly binds calmodulin as a polymeric complex in a Ca2+-independent manner.     

In vitro stimulation of human red blood cell Ca2+-ATPase by thyroid hormone

Ca²⁺-ATPase activity in human erythrocyte ghosts previously washed to remove endogenous thyroid hormone is stimulated $\text{in}\text{vitro}$ by physiologic concentrations of thyroxine (T₄) and triiodothyronine (T₃). Two- to three-fold increases (P <0.005) in Ca²⁺-ATPase activity occurred after 60–120 minutes' exposure of membranes to iodothyronines at concentrations of T₄ and T₃ of 10^?8 M to 10^?12 M. T₄ was more active than T₃ and its activity did not depend upon prior conversion to T₃. The Ca²⁺-ATPase effect represents an extranuclear action of thyroid hormone in a human cell model.