The effect of berbamine derivatives on activated Ca2+-stimulated Mg2+-dependent ATPase in erythrocyte membranes.
ABSTRACT: 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.
Project description:Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.
Project description:1. The heavy microsomal fraction from rat liver apparently has very little Ca2+-stimulated ATPase activity, although it has an active, ATP-driven Ca2+ accumulation system. 2. The addition of ionophore A23187 to the ATPase assay, to allow continuous Ca2+ recycling during the assay time, reveals the presence of a substantial Ca2+-stimulated ATPase with Vmax. 160 nmol of Pi/10 min per mg of protein and Km for Ca2+ 0.19 microM. 3. The Ca2+-stimulated ATPase, but not the basal Mg2+-stimulated ATPase, is potently inhibited by orthovanadate. Both the Ca2+-stimulated ATPase and the vanadate inhibition are enhanced by the presence of Mg2+. 4. Ca2+-stimulated ATPase activity is not responsive to calmodulin or the calmodulin antagonist trifluoperazine.
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.
Project description: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.
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.
Project description:The Ca2+ uptake and the (Ca2+ + Mg2+)-dependent ATPase of the porcine coronary-artery smooth-muscle microsomal fraction ('microsomes') are only slightly stimulated by calmodulin. The Ca2+ uptake after 2 min in the absence of oxalate, corrected for the ATP-independent binding, increased by a factor of 1.44, whereas the (Ca2+ + Mg2+)-dependent ATPase is stimulated 1.39 times. These findings contrast with the effect observed in human erythrocyte 'inside-out' microsomes. In these vesicles calmodulin increases the Ca2+ uptake after 20 min in an oxalate-free medium and the (Ca2+ + Mg2+)-dependent ATPase respectively by a factor of 3.82 and 6.18. The magnitude of the calmodulin stimulation of the Ca2+ transport in coronary-artery microsomes is similar to that observed in heart microsomes.
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.
Project description:The activities of Mg2+-ATPase (Mg2+-activated ATPase), (Ca2+ + Mg2+)-activated ATPase and (Na+ + K+)-activated ATPase have been determined in microsomes (microsomal fractions) obtained from rat myometrium under different hormonal conditions. Animals were either ovariectomized and treated for a prolonged period of time with 17 beta-oestradiol or progesterone, or myometria were obtained at day 21 of pregnancy. In each case the endometrium was carefully removed. The Mg2+-ATPase consists of two components: an inactivating labile component and a second constant component. The rate of ATP hydrolysis by the labile component of the Mg2+-ATPase declines exponentially as a function of time after adding the membranes to the assay medium; this inactivation is caused by the presence of ATP in the medium. This ATPase activity inhibited by ATP is catalysed by a labile enzyme and hence it gradually diminishes within a few hours, even when the microsomes are kept on ice. This labile component has the highest activity in microsomes from pregnant rats, a lower activity in progesterone-treated rats, and the lowest in 17 beta-oestradiol-treated rats. This component of the Mg2+-ATPase is not affected by 90 nM-oxytocin. The constant component of the Mg2+-ATPase must be ascribed to a different enzyme, which, in contrast with the labile component, is very stable and not affected by the hormonal status of the animal. This constant component of the Mg2+-ATPase is inhibited both by Ca2+-calmodulin, and by oxytocin in microsomes from pregnant and from progesterone-treated animals, whereas such inhibition does not occur in microsomes from 17 beta-oestradiol-treated animals. The activity of the (Na+ + K+)-activated ATPase is not dependent on the hormonal status of the animal. Myometrial microsomes present an ATP-dependent Ca2+ transport, irrespective of the hormonal condition, but only in microsomes obtained from rats treated with 17 beta-oestradiol, can a (Ca2+ + Mg2+)-activated ATPase activity be demonstrated. This activity can be stimulated by calmodulin.
Project description:A membrane fraction isolated from lactating murine mammary tissue and enriched for the Golgi membrane marker enzyme galactosyltransferase exhibited Ca2+-stimulated ATPase activity (Ca-ATPase) in 20 microM-free Mg2+ and 10 microM-MgATP, with an apparent Km for Ca2+ of 0.8 microM. Exogenous calmodulin did not enhance Ca2+ stimulation, nor could Ca-ATPase activities be detected in millimolar total Mg2+ and ATP. When assayed with micromolar Mg2+ and MgATP the Ca-ATPases of skeletal-muscle sarcoplasmic reticulum and of calmodulin-enriched red blood cell plasma membranes were half-maximally activated by 0.1 microM- and 0.6 microM-Ca2+ respectively. All three Ca-ATPases were inhibited by similar micromolar concentrations of trifluoperazine, but the Golgi activity was unaffected by quercetin in concentrations which completely inhibited both the sarcoplasmic-reticulum and red-blood-cell enzymes. The results are consistent with the hypothesis that the high-affinity Ca-ATPase is responsible for the ATP-dependent Ca2+ transport exhibited by Golgi-enriched vesicles derived from lactating mammary gland [Neville, Selker, Semple & Watters (1981) J. Membr. Biol. 61, 97-105; West (1981) Biochim. Biophys. Acta 673, 374-386].
Project description:1. The enzymic properties of myosin isolated from chicken gizzard by three different methods have been compared. 2. Although the specific Ca2+-stimulated ATPases of all preparations were similar and high, there were significant differences in the specific activities of the Mg2+-stimulated actomyosin ATPases. 3. There was no direct correlation between the Mg2+-stimulated actomyosin ATPase activity and the extent of P-light-chain phosphorylation in any of the three myosin preparations. 4. A fraction that activates the Mg2+-stimulated actomyosin ATPase of gizzard muscle has been isolated from a gizzard muscle filament preparation. 5. The activator was specific for the Mg2+-activated actomyosin ATPase of smooth muscle. 6. The activator required the addition of calmodulin for full effect.