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Effects of Ca2+, Mg2+ and calmodulin on the formation and decomposition of the phosphorylated intermediate of the erythrocyte Ca2+-stimulated ATPase.

ABSTRACT: Formation of the phosphorylated intermediate (ECaP) of the human erythrocyte Ca2+-stimulated ATPase (Ca2+-ATPase) was more rapid and reached steady state sooner at 400 microM-Ca2+ than at 1 microM-Ca2+. Calmodulin increased the apparent rate of ECaP formation at 1 microM-Ca2+, whereas at 400 microM-Ca2+, calmodulin decreased the steady-state level of the ECaP without affecting its apparent rate of formation. Removal of endogenous Mg2+ with trans-1,2-diaminocyclohexane-NNN'N'-tetra-acetic acid, which decreased both the velocity and Ca2+-sensitivity of the Ca2+-ATPase, did not alter the Ca2+-sensitivity or the apparent rate of formation of ECaP. ECaP formation at high Ca2+ concentrations was not affected by Mg2+ concentrations as high as 1 mM, and the ECaP could be dephosphorylated by ADP and ATP along either the forward or reverse pathways. The results suggest that high Ca2+ concentrations inhibit Ca2+-ATPase activity by preventing dephosphorylation of the E2P complex, rather than by inhibition of the transformation from E1CaP ('high-Ca2+-affinity' ECaP) to E2CaP ('lower-energy' ECaP).

SUBMITTER: Allen BG

PROVIDER: S-EPMC1148041 | biostudies-other | 1987 Jun

REPOSITORIES: biostudies-other

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A protein activator of Mg2+-dependent, Ca2+-stimulated ATPase in human erythrocyte membranes distinct from calmodulin.

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| S-EPMC1161817 | biostudies-other

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

Project description: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.

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| S-EPMC1148650 | biostudies-other

Activation of erythrocyte Ca2+-plus-Mg2+-stimulated adenosine triphosphatase by protein kinase (cyclic AMP-dependent) inhibitor. Comparison with calmodulin.

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Luminal dissociation of Ca2+ from the phosphorylated Ca2+-ATPase is sequential and gated by Mg2+.

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

Project description: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.

| S-EPMC1147468 | biostudies-other

The (Ca2+ + Mg2+)-stimulated ATPase of the rat parotid endoplasmic reticulum.

Project description:A membrane fraction enriched in endoplasmic reticulum was prepared from rat parotid glands by using sucrose-gradient centrifugation. The fraction showed a 10-fold increase in specific activity of NADPH: cytochrome c reductase activity over that of tissue homogenates and minimal contamination with plasma membranes or mitochondria. The endoplasmic reticulum fraction possessed both Mg2+ -stimulated ATPase as well as Ca2+, Mg2+-ATPase [( Ca2+ + Mg2+)-stimulated ATPase]activity. The Ca2+, Mg2+-ATPase required 2-5 mM-Mg2+ for optimal activity and was stimulated by submicromolar concentrations of free Ca2+. The Km for free Ca2+ was 0.55 microM and the average Vmax. was 60 nmol/min per mg of protein. The Km for ATP was 0.11 mM. Other nucleotides, such as GTP, CTP or ADP, could not substitute for ATP in supporting the Ca2+-activated nucleotidase activity. Increasing the K+ concentration from 0 to 100 mM caused a 2-fold activation of the Ca2+, Mg2+-ATPase. Trifluoperazine, W7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide] and vanadate inhibited the enzyme. The concentration of trifluoperazine and vanadate required for 50% inhibition of the ATPase were 52 microM and 28 microM respectively. Calmodulin, cyclic AMP, cyclic AMP-dependent protein kinase and inositol 1,4,5-trisphosphate had no effect on the ATPase. The properties of the Ca2+, Mg2+ -ATPase were distinct from those of the Mg2+-ATPase, but comparable with those reported for the parotid endoplasmic-reticulum Ca2+-transport system [Kanagasuntheram & Teo (1982) Biochem. J. 208, 789-794]. The results suggest that the Ca2+, Mg2+-ATPase is responsible for driving the ATP-dependent Ca2+ accumulation by this membrane.

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Erythrocyte-ghost Ca2+-stimulated Mg2+-dependent adenosine triphosphatase in Duchenne muscular dystrophy.

Project description:The Ca2+-stimulated Mg2-dependent ATPase activities (Ca2+-ATPase) of erythrocyte-ghost membranes from patients with Duchenne muscular dystrophy (DMD) and carriers of DMD were compared with activities of normal controls. The Ca2+-ATPase activity of DMD-patient ghost preparations was found to follow the same pattern of activation by Ca2+ as the control membranes. However, the Ca2+-ATPase activity in DMD and some DMD-carrier preparations was substantially elevated compared with controls. To characterize further the elevated Ca2+-ATPase activity found in DMD-patient ghost membrane preparations, we estimated kinetic parameters using both fine adjustment and weighting methods to analyse our experimental data. It was established that in both DMD and DMD-carrier preparations the increase in Ca2+-ATPase activity was reflected by a significant increase in Vmax. rather than by any change in Km. The response of the membrane Ca2+-ATPase activity to changes in temperature was also investigated. In all preparations a break in the Arrhenius plot occurred at 20 degrees C, and in DMD and DMD-carrier preparations an elevated Ca2+-ATPase activity was detected at all temperatures. Above 20 degrees C the activation energy for all types of preparation was the same, whereas below this temperature there appeared to be an elevated activation in DMD and DMD-carrier preparations compared with normal controls. The concept that a generalized alteration in the physicochemical nature of the membrane lipid domain may be responsible for the many abnormal membrane properties reported in DMD is discussed.

| S-EPMC1163668 | biostudies-other

CaMKII autonomy is substrate-dependent and further stimulated by Ca2+/calmodulin.

Project description:A hallmark feature of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca(2+)-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active ( approximately 70-100%), implicating that it is no longer regulated and that its dramatically increased Ca(2+)/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15-25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca(2+)/CaM, both in vitro and within cells. Altered K(m) and V(max) made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca(2+) signals, but prevents complete uncoupling from subsequent cellular stimulation.

| S-EPMC2878555 | biostudies-literature

Does a calmodulin-dependent Ca2+-regulated Mg2+-dependent ATPase contribute to hepatic microsomal calcium uptake?

Project description:Solubilization of microsomal proteins followed by calmodulin affinity chromatography resulted in the separation of two distinct Ca2+-Mg2+-ATPases (Ca2+-regulated Mg2+-dependent ATPases), one being insensitive to calmodulin (ATPase-1), the other being stimulated about 5-fold by calmodulin (ATPase-2). ATPase-2 accounts for only 8% of total microsomal Ca2+-Mg2+-ATPase-activity. ATPase-1 and -2 can also be distinguished by different pH optima, different sensitivity towards inhibition by vanadate and LaCl3, and different apparent Mr values of the phosphoenzyme intermediates (115,000 and 150,000 for ATPase-1 and ATPase-2 respectively). ATPase-1 from liver co-migrated with Ca2+-Mg2+-ATPase from rat skeletal-muscle sarcoplasmic reticulum, whereas ATPase-2 from liver co-migrated with calmodulin-dependent Ca2+-Mg2+-ATPase derived from rat skeletal-muscle sarcolemma. After separation of parenchymal and nonparenchymal liver cells, a calmodulin-dependent Ca2+-Mg2+-ATPase of Mr 150,000 was found only in the non-parenchymal cells. The kinetic parameters of ATPase-2 and the similarity of the apparent Mr of its phosphoenzyme intermediate to that of skeletal-muscle sarcolemma Ca2+-Mg2+-ATPase makes it likely that the calmodulin-sensitive Ca2+-Mg2+-ATPase found in rat liver microsomal fractions reflects a contamination with plasma membranes (possibly from non-parenchymal cells) rather than a true location in the endoplasmic reticulum of parenchymal liver cells.

| S-EPMC1147919 | biostudies-other

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