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1.
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.  相似文献   

2.
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.  相似文献   

3.
4.
Phosphorylation of solubilized and purified high-affinity (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of human erythrocyte membranes shows no dependence on cyclic AMP concentration in the range 0.1–1000 μM.Ca2+-dependent phosphoprotein is sensitive to hydroxylamine and molybdate treatment. The phosphate linkage shows maximum stability at low pH values, which is progressively lost as the pH rises, with a shoulder around pH 6. SDS gel electrophoresis of the phosphorylated protein yields a peak which shows relative mobility corresponding to a molecular weight of 145 000 and sensitivity to MgATP-chase and hydroxylamine treatment. This indicates that the phosphoprotein represents the phosphorylated intermediate of the high-affinity (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes.  相似文献   

5.
The correlation between the ATP-dependent Ca2+ binding and the phosphorylation of the membranes from swine and bovine erythrocytes was studied. The Ca2+ binding was measured by using 45CaCl2, and the phosphorylation by [γ-32P]ATP was studied with the technique of SDS polyacrylamide gel electrophoresis. 200 mM NaCl and KCl markedly repressed the Ca2+ binding of swine erythrocyte membranes. The radioactivity of 32P-labelled membranes was revealed mainly in 250 000 dalton protein and a lipid fraction. NaCl and KCl also repressed the phosphorylation of the lipid which was identified as triphosphoinositide by paper chromatography. The membranes prepared from trypsin-digested erythrocytes completely retained the Ca2+-binding activity, and lost 30% of (Ca2+ + Mg2+)-ATPase activity. The Ca2+-binding and ATPase activity of isolated membranes decreased to 55% and to 0%, respectively, by tryptic digestion. Neither the Ca2+ binding nor the phosphorylation of polyphosphoinositides were detected in bovine erythrocyte membranes.These results suggest that the formation of triphosphoinositide rather than the (Ca2+ + Mg2+)-ATPase of membranes is linked to the ATP-dependent Ca2+ binding of erythrocyte membranes.  相似文献   

6.
In the absence of Mg2+, the observed activity of the erythrocyte plasma membrane Ca2+-ATPase is due to the hydrolysis of CaATP at a low rate. In the presence of Mg2+, the activity of the enzyme is much higher, but it is inhibited by high levels of free Mg2+. This inhibition appears to be due to competition of Mg2+ and Ca2+ for a site on the enzyme, rather than for ATP.  相似文献   

7.
A ouabain-insensitive ATPase activity associated with the water-soluble proteins of the human and bovine erythrocyte membrane is demonstrated by means of activity-staining in polyacrylamide gels. The ATPase activity from both sources had an absolute requirement for Mg2+, activity becoming easily detectable at 0.2 mM Mg2+. At low Mg2+ concentrations added Ca2+ appeared to decrease the intensity of the ATPase stain. The activity is unaffected by monovalent cations, does not hydrolyse p-nitrophenyl phosphate and is not inhibited by 2 : 4 dinitrophenol. The ATPase has an apparent molecular weight of approximately 100 000 as determined by electrophoresis in acrylamide gels containing dodecyl sulphate.  相似文献   

8.
The presence of Ca2+-ATPase activities with high-affinity sites for Ca2+ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca2+, the substrate specificity of Ca2+-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca2+-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca2+-dependent ATP-hydrolysis was greater than that of ADP, AMP and p-nitrophenylphosphate at Ca2+ concentrations below 25 μM. At 0.2 mM Ca2+ the rates of ATP, ADP, AMP and p-nitrophenylphosphate hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca2+, but at 0.2 mM Ca2+, Ca2+-induced hydrolysis of ADP and AMP was greater than either ATP or p-nitrophenylphosphate. Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca2+-stimulated ATP hydrolysis at 1 μM Ca2+ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca2+, Ca2+-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca2+-stimulated ATP hydrolysis, either at low or at high Ca2+ concentrations. Chlorpromazine fully inhibited Ca2+-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca2+, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca2+-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca2+-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca2+ concentrations, but this Ca2+-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.  相似文献   

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建立了一种亲和层析纯化肌质网Ca2+-ATP酶的方法.用非离子型去污剂C12E8 溶解肌质网,再通过反应红-120琼脂糖亲和层析柱使肌质网Ca2+-ATP酶纯度从粗品中的65%提高到99%,并具有较高ATP水解活性.经SDS-聚丙烯酰胺凝胶电泳检测,为电泳纯.  相似文献   

11.
Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca2+-ATPase and calmodulin-insensitive (Na+ + K+)- and Mg2+-ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca2+-ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca2+-ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na+ + K+)-ATPase and Mg2+-ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.  相似文献   

12.
13.
Specific activity and Ca2+-affinity of (Ca2++Mg2+)ATPase of calmodulin-depleted ghosts progressively increase during preincubation with 0.1–2 mM Ca2+. Concomitantly, the increment in ATPase activity caused by calmodulin and the binding of calmodulin to ghosts decrease. The effects of calcium ions are abolished by the addition of calmodulin. ATP protects the enzyme from a Ca2+-dependent decrease of the maximum activity but does not seem to influence the Ca2+-dependent transformation of the low Ca2+-affinity enzyme into a high Ca2+-affinity form.  相似文献   

14.
1. In the presence of ATP, the Ca2+ pump of human red cell membranes catalyzes the hydrolysis of p-nitrophenyl phosphate. The requirement for ATP of the Ca2+-p-nitrophenylphosphatase activity was studied in relation to the two classes of site for ATP that are apparent during Ca2+ -ATPase activity. 2. (a) The K0.5 for ATP as activator of the Ca2+ -p-nitrophenylphosphatase extrapolated at 0 mM PNPP is equal to the Km of the Ca2+ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca2+ -ATPase or as activator of the Ca2+ -p-nitrophenylphosphatase. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca2+ -p-nitrophenylphosphatase. 4. At ATP concentrations that almost saturate the high-affinity site, Ca2+ -p-nitrophenylphosphatase activity increases as a function of PNPP along an S-shaped curve, while Ca2+ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca2+ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca2+ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca2+ -p-nitrophenylphosphatase activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca2+ -p-nitrophenylphosphatase activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca2+ -ATPase activity at the high-affinity size persists during Ca2+ -p-nitrophenylphosphatase activity.  相似文献   

15.
The (Ca2+ + Mg2+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) from human erythrocytes occurred in two different states, A-state and B-state, depending on the membrane preparation.The A-state showed low maximum activity (V) and the Ca2+ activation was characterized by a Hill coefficient, nH, of about 1 and a Michaelis constant, KCa, about 30 μM.The B-state showed high V, a nH above 1, which indicates positive cooper-activity of Ca2+ activation, and a KCa of about 1 μM.With varying ATP concentrations, both the A-state and the B-state showed negative cooperativity and slightly different values of Km.The B-state was shifted to the A-state when the membranes were exposed to low Ca2+ concentrations. The shift reached 50% at approx. 0.5 μM Ca2+. At the low Ca2+ concentrations an activator was released from the membranes.The A-state was shifted to the B-state when the membranes were exposed to Ca2+ in the presence of the activator. The shift reached 50% at about 30 μM Ca2+. The recovery of high V was time dependent and lasted several minutes. Increasing concentrations of Ca2+ and activator accelerated the recovery.It is suggested that the A-state and the B-state correspond to enzyme free of activator and enzyme associated with activator, respectively. Furthermore, the two states may represent a resting and an active state, respectively, of the calcium pump.  相似文献   

16.
17.
The interaction between calmodulin and the pure, solubilized Ca2+ ATPase from human erythrocyte membranes was examined by kinetic titration. The data indicated that the two proteins interacted in a molar ratio of 1:1 with a Kd of 4.2 nm. The dependence of enzyme activity on calmodulin concentration agreed quantitatively with that predicted by kinetic theory.  相似文献   

18.
Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca2+-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH2)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent Km of 0.2 μM Ca2+ and a Vmax of 5.3 nmol Ca2+/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca2+-ATPase are: Km = 0.25 μM Ca2+ and Vmax = 19.2 nmol Pi/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the Vmax of ATP-dependent calcium uptake amounts to 25 nmol Ca2+/min per mg protein and of the Ca2+-ATPase to 23 nmol Pi/min per mg protein. The close correlation between kinetic parameters of Ca2+-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca2+-pump located in duodenal basolateral plasma membranes.  相似文献   

19.
Calmodulin-depleted isotonic erythrocyte ghosts contain 200 ng residual calmodulin/mg protein which is not removed by extensive washings at pCa2+ > 7. Specific activity and Ca2+-affinity of the (Ca2+ + Mg2+)ATPase increase at increasing calmodulin, with K0.5 Ca of 0.38 μM at calmodulin concentrations corresponding to that in erythrocytes. High Ca2+ concentrations inhibit the enzyme. Specific activity and Ca2+-affinity of the enzyme decrease at increasing Mg2+ concentrations. The Ca2+ ? Mg2+ antagonism is likewise observed at inhibitory Ca2+ concentrations.  相似文献   

20.
The structural preferences of soya phosphatidylinositol in isolation and in mixtures with soya phosphatidylethanolamine, and the influence of Ca2+ and Mg2+ on these preferences, have been examined employing 31P-NMR and freeze-fracture techniques. It is shown that phosphatidylinositol assumes the bilayer organization on hydration both in the presence and absence of Ca2+ and Mg2+. In mixed systems with HII phase) phosphatidylethanolamine, phosphatidylinositol induces lipidic particle structure at low (<10 mol%) concentrations and bilayer structure at higher levels. In systems containing 15 or 20 mol% phosphatidylinositol, Ca2+ (but not Mg2+) can induce HII phase structure. The results indicate that phosphatidylinositol is a more effective agent than other acidic phospholipids for stabilizing bilayer structure, particularly when high levels of divalent cations are present. These findings are discussed in terms of functional roles of phosphatidylinositol and mechanisms whereby Ca2+ induces structural reorganization in mixed systems containing acidic phospholipids and phosphatidylethanolamine.  相似文献   

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