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1.
《生物化学与生物物理学报:生物膜》1985,816(2):379-386
(1) 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. (2) 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. (3) The effects of compound 48/80 are independent of the concentration of Ca2+ between 30 and 200 μM. (4) 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. (5) 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. 相似文献
2.
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. 相似文献
3.
Fred L. Larsen Beat U. Raess Thomas R. Hinds Frank F. Vincenzi 《Journal of cellular biochemistry》1978,9(2):269-274
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. 相似文献
4.
In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin. 相似文献
5.
The activity of the solubilized and purified (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes was inhibited by N,N'-dicyclohexylcarbodiimide in a concentration-dependent manner. The carbodiimide prevented formation of the phosphorylated intermediate during the catalytic cycle of the enzyme. Treatment of the enzyme with N,N'-dicyclohexyl[14C]carbodiimide resulted in the formation of a 14C-labelled polypeptide corresponding to the enzyme monomer (molecular weight 136,000). The tryptic fragmentation of this 14C-labelled enzyme resulted in the formation of three major 14C-labelled fragments with molecular weights of 58,000, 36,500 and 23,000, the latter two probably representing transmembrane and calmodulin-binding domains of the enzyme, respectively. In the absence of calmodulin, 6.7 molecules of N,N'-dicyclohexyl[14C]carbodiimide covalently bound to each molecule of Ca2+-ATPase; in the presence of calmodulin, the number of molecules of carbodiimide bound was 13.1. The binding of N,N'-dicyclohexylcarbodiimide to the (Ca2+ + Mg2+)-ATPase greatly reduced its ability to bind to a calmodulin-agarose gel. 相似文献
6.
The interactions between calmodulin, ATP and Ca2+ on the red cell Ca2+ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of by 5–10-fold and the rate of Ca2+-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates along a biphasic concentration curve (), but in stripped membranes the curve is essentially hyperbolic (). In calmodulin-bound membranes Ca2+ activates at low concentrations () in stripped membranes the apparent Ca2+ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E2 and E1 forms of the Ca2+ pump protein. 相似文献
7.
Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA 总被引:4,自引:0,他引:4
Islet cell plasma membranes contain a calcium-stimulated and magnesium-dependent ATPase (Ca2+ + Mg2+)-ATPase) which requires calmodulin for maximum enzyme activity (Kotagal, N., Patke, C., Landt, M., McDonald, J., Colca, J., Lacy, P., and McDaniel, M. (1982) FEBS Lett. 137, 249-252). Investigations indicated that exogenously added calmodulin increases the velocity and decreases the Km for Ca2+ of the high affinity (Ca2+ + Mg2+)-ATPase. These studies routinely employed the chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to maintain Ca2+ concentrations in the submicromolar range. During the course of these investigations, it was found unexpectedly that increasing the concentrations of EGTA (0.1-4 mM) and total calcium in the media, while maintaining constant free Ca2+ levels, increased the velocity of the high affinity (Ca2+ + Mg2+)-ATPase. The free calcium concentrations under these conditions were verified by a calcium-sensitive electrode. The (Ca2+ + Mg2+)-ATPase maximally activated by 2-4 mM EGTA was not further stimulated by calmodulin, whereas camodulin stimulation increased as the concentration of EGTA in the media was decreased. A similar enhancement by Ca-EGTA was observed on active calcium transport by the plasma membrane-enriched fraction. Moreover, Ca-EGTA had a negligible effect on both active calcium transport as well as Ca2+-stimulated ATPase activity by the islet cell endoplasmic reticulum, processes which are not stimulated by calmodulin. The results indicate that stimulation by Ca-EGTA may be used to differentiate calcium transport systems by these subcellular organelles. Furthermore, the concentration of EGTA routinely employed to maintain free Ca2+ levels may itself obscure effects of calmodulin and other physiological agents on calcium-dependent activities. 相似文献
8.
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 Km values of 3.5 and 120 μM. The enzyme is phosphorylated by ATP in the presence of Ca2+ only. 相似文献
9.
The fluorescence quenching properties of a brominated derivative of androstenol 5 alpha,6 beta-dibromoandrostan-3 beta-ol have been used to study binding to phospholipid bilayers and to the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum of rabbit skeletal muscle. It is shown that androstenol is excluded from the phospholipid/protein interface of the ATPase but can bind to other (non-annular sites) on the ATPase. Binding to these sites increases in strength with decreasing chain length for the phospholipids present in the system. Binding is also stronger in the presence of phospholipids in the gel phase than in the liquid crystalline phase. Androstenol increases the ATPase activity of the ATPase reconstituted with phosphatidylcholines of chain lengths less than C18, but has no effect on activity for the ATPase reconstituted with phosphatidylcholines of chain lengths C18 or greater. The effects of cholestanols on the activity of the ATPase reconstituted with dimyristoleoylphosphatidylcholine depend on the configuration of the sterol, with 5 alpha-cholestan-3 alpha-ol having little effect but the other isomers causing a marked stimulation. 相似文献
10.
Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations 总被引:2,自引:0,他引:2
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. 相似文献
11.
12.
The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+
Media prepared with CDTA and low concentrations of Ca2+, as judged by the lack of Na+-dependent phosphorylation and ATPase activity of (Na+ +K+)-ATPase preparations are free of contaminant Mg2+. In these media, the Ca2+-ATPase from human red cell membranes is phosphorylated by ATP, and a low Ca2+-ATPase activity is present. In the absence of Mg2+ the rate of phosphorylation in the presence of 1 microM Ca2+ is very low but it approaches the rate measured in Mg2+-containing media if the concentration of Ca2+ is increased to 5 mM. The KCa for phosphorylation is 2 microM in the presence and 60 microM in the absence of Mg2+. Results are consistent with the idea that for catalysis of phosphorylation the Ca2+-ATPase needs Ca2+ at the transport site and Mg2+ at an activating site and that Ca2+ replaces Mg2+ at this site. Under conditions in which it increases the rate of phosphorylation, Ca2+ is without effect on the Ca2+-ATPase activity in the absence of Mg2+ suggesting that to stimulate ATP hydrolysis Mg2+ accelerates a reaction other than phosphorylation. Activation of the E1P----E2P reaction by Mg2+ is prevented by Ca2+ after but not before the synthesis of E1P from E1 and ATP, suggesting that Mg2+ stabilizes E1 in a state from which Mg2+ cannot be removed by Ca2+ and that Ca2+ stabilizes E1P in a state insensitive to Mg2+. The response of the Ca2+-ATPase activity to Mg2+ concentration is biphasic, activation with a KMg = 88 microM is followed by inhibition with a Ki = 9.2 mM. Ca2+ at concentration up to 1 mM acts as a dead-end inhibitor of the activation by Mg2+, and Mg2+ at concentrations up to 0.5 mM acts as a dead-end inhibitor of the effects of Ca2+ at the transport site of the Ca2+-ATPase. 相似文献
13.
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. 相似文献
14.
Chengjing Cao Timothy Lockwich Terrence L. Scott Robert Blumenthal Adil E. Shamoo 《Molecular and cellular biochemistry》1991,103(2):97-111
Summary In reconstituted rabbit skeletal muscle (Ca2+ + Mg2+)-ATPase proteoliposomes, Ca2+-uptake is decreased by more than 90% with T2 cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T3 cleavage, which represents the cleavage of A1 fragment to A1a + A1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca2+ efflux between control SR and tryptically digested SR in the absence of Mg+ ruthenium red or in the presence of ATP. However, the passive Ca2+ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg2+ + ruthenium red. These results show that the Ca2+ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca2+ channel inhibitors, Mg2+ and ruthenium red. In the reconstituted ATPase proteoliposomes, the Ca2+ efflux rates are the same regardless of digestion (T2); also, efflux is not affected by the presence or absence of Mg2+ + ruthenium red. These results indicate that T2 cleavage causes uncoupling of the Ca2+-pump from ATP hydrolytic activity.A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca2+ + Mg2+)-ATPase polypeptide with the loss of Ca2+-transport. Fits of the theoretical equations to the data are consistent with that Ca2+-transport system appears to require a dimer of the polypeptide (Ca2+ + Mg2+)-ATPase. 相似文献
15.
Benzenemethane Sulfonylfluoride (329-98-6) is an irreversible inactivator of many esterases including mammalian acetylcholinesterases. However, previous reports indicated that acetylcholinesterase from the electric eel, Electrophorus electricus (EC 3.1.1.7) failed to react with benzenemethane sulfonylfluoride at measurable rates. We report here that eel acetylcholinesterase reacts with this inactivator at a low rate. Hydrolysis of the sulfonylating agent is so much faster than enzyme inactivation that, under most conditions, there will be only slight inactivation. Like the reaction of other active site acylating agents with this enzyme, inactivation can be accelerated in the presence of certain organic cations. We introduce a rate equation for enzyme sulfonylation which incorporates both the hydrolysis of the inactivator and the complication that fluoride resulting from hydrolysis of the inactivator is a potent competitive inhibitor of this enzyme. This rate equation accurately describes the time course of enzyme inactivation. 相似文献
16.
17.
M G Luthra 《Biochimica et biophysica acta》1982,692(2):271-277
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
18.
Protein-lipid interaction. Biophysical studies of (Ca2+ + Mg2+)-ATPase reconstituted systems 总被引:5,自引:0,他引:5
J C Gomez-Fernandez F M Goni D Bach C J Restall D Chapman 《Biochimica et biophysica acta》1980,598(3):502-516
Differential scanning calorimetry, fluorescence spectroscopy and freeze-fracture electron microscopy have been applied to a study of the reconstituted Ca2+-ATPase proteins from sarcoplasmic reticulum when they are incorporated into pure lipid/water systems. The results obtained with these techniques have been used to examine the effects of this intrinsic protein upon the surrounding lipid at temperatures above and below the main lipid solid-fluid phase transition temperature (Tc). 1. Above this Tc value, the freeze-fracture data show that the proteins are randomly distributed within the plane of the bilayer. The fluorescence data show that as the protein content in the bilayer increases, so does the 'microviscosity'. 2. Below Tc the proteins occur in high protein to lipid patches, separate from the remaining crystalline lipid. The fluorescence data indicate that at these temperatures the presence of the protein causes a decrease in microviscosity, whilst the calorimetric data indicate a decrease in enthalpy of the main lipid transition. 3. A premelting of the high protein to lipid patches formed by phase separation within the lipid bilayers is indicated by the calorimetric and fluorescence data. This observation is used to rationalise the 'anomalous' properties of the dipalmitoyl phosphatidylcholine-ATPase of exhibiting activity at temperatures well below the lipid phase transition at 41 degrees C. 相似文献
19.
《生物化学与生物物理学报:生物膜》1986,858(1):21-30
(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. 相似文献
20.
J Mas-Oliva 《Biochimica et biophysica acta》1985,812(1):163-167
The (Ca2+ + Mg2+)-ATPase from erythrocyte ghosts catalyzed the hydrolysis of ATP together with the synthesis of ATP or ATP in equilibrium 'Pi exchange. The modulation of the ATPase reaction cycle was controlled by high- and low-affinity calcium-binding sites asymmetrically located on the enzyme. Calmodulin accelerated the reaction cycle in both directions, stimulating the overall turnover of the enzyme. Calcium transport was achieved utilizing optimal conditions for the expression of the ATP in equilibrium Pi exchange system. 相似文献