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1.
The calmodulin activation of the ( (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM 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 μM. 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 . 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 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id 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
2.
Evelyn T. Maizels Richard A. Jungmann 《Biochemical and biophysical research communications》1982,107(1):32-37
A 105,000 × g supernatant fraction from prepubertal rat ovaries was incubated in the presence of [γ-32P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by autoradiography. Inclusion of Ca2+ in the phosphorylation reaction promoted a selective 32p incorporation into two proteins of Mr = 95,000 and 50,000. Inclusion of chlorpromazine with Ca2+ blocked the Ca2+-stimulated increase of 32p incorporation. Our results demonstrate the presence of Ca2+-stimulated protein phosphorylation system capable of recognizing endogenous substrate proteins in the prepubertal rat ovary. 相似文献
3.
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 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 of membranes is linked to the ATP-dependent Ca2+ binding of erythrocyte membranes. 相似文献
4.
The calcium dependence and the time course of phosphatidylethanolamine and phosphatidylcholine degradation by sheep erythrocyte membrane suspensions in presence of Triton X-100 were investigated. One enzyme with phospholipase A2 specificity was found to be responsible for both phosphatidylethanolamine and phosphatidylcholine degradation.The localization of this enzyme in the membrane of the sheep erythrocyte was investigated by proteolytic treatment of sealed erythrocyte ghosts from the outside and of ghosts which had both sides of the membrane exposed to chymotrypsin. The inability of sealed ghosts to take up chymotrypsin was followed by flux measurements of [14C]dextran carboxyl previously trapped in the ghosts. No efflux of the marker was found during the proteolytic treatment. By comparing the residual phospholipase activities in the membranes from both ghost preparations, we concluded that the phospholipase is oriented to the exterior of the sheep erythrocyte. 相似文献
5.
6.
An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at followed by sedimentation of a microsomal fraction at . The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ( activity and [3H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca2+ concentration for enzyme activity was 5–10 μM. Both Na+ and K+ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca2+ from the cardiac cell. 相似文献
7.
The temperature-dependent relationship between K+ active influx, Mg2+-ATPase activity, transmembrane potential (ΔΨ) and the membrane lipid composition has been investigated in mycoplasma PG3. Native organisms were grown in a medium containing 10 μg/ml cholesterol and either oleic plus palmitic (chol (+), O + P) or elaidic (chol (+), E) acids. Adapted cells were grown in a medium free of exogenous cholesterol and supplemented with elaidic acid (chol (?), E).Arrhenius plots of 42K+ active influx gave a linear relationship for (chol (+), O + P) cells (). On the other hand, when oleic plus palmitic acids are replaced by elaidic acid, an upward discontinuity appears between 28 and 30°C, which is associated with a large increase in the apparent activation energy of the process ().Finally, a biphasic response with a break at approx. 23°C (; ) is observed for (chol (?), E) organisms. From the lack of correspondence between these effects on the K+ influx and the temperature dependence of both the Mg2+-ATPase activity and ΔΨ, it is suggested that changes in the membrane lipid composition affect the K+ transport at the level of the K+ carrier itself.Differential scanning calorimetry, steady-state fluorescence polarization of diphenylhexatriene and freeze-fracture electron microscopy experiments further suggest that the effect is largely due to modifications of the membrane microviscosity and that the K+ carrier is associated with the most fluid lipid species present in the membrane. 相似文献
8.
Nirmala Kotagal Christina Patke Michael Landt Jay McDonald Jerry Colca Paul Lacy Michael McDaniel 《FEBS letters》1982,137(2):249-252
The carboxyl group reagent dicyclohexylcarbodiimide inhibits the electrogenic entry of Cl? and NO3? into rat liver mitochondria at alkaline pH. The inhibition is time dependent and 50% inhibition is obtained by the addition of 3–4 nmol DCCD/mg protein. The blockage of the pH-dependent anion-conducting pore appears to be unrelated to the other known actions of DCCD on rat liver mitochondria but seems similar to its effect on the uncoupling protein of brown adipose tissue. 相似文献
9.
Madan G. Luthra Richard P. Watts Karen L. Scherer 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,633(2):299-304
The effect of purified calmodulin on the calcium-dependent phosphorylation of human erythrocyte membranes was studied. Under the conditions employed, only one major peak of phosphorylation was observed when solubilized membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this phosphorylated protein band was estimated to be 130 000 and in the presence of purified red blood cell calmodulin, the rate of phosphorylation of this band was increased. These data suggest that calmodulin activation of (Ca2+ + Mg2+)-ATPase could be a partial reflection of an increased rate of phosphorylation of the (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes. 相似文献
10.
Albert W. Girotti 《生物化学与生物物理学报:生物膜》1980,602(1):45-56
Exposure of human erythrocyte ghosts (pH 8, 10°C) to visible light in the presence of the photosensitizer, methylene blue, results in a relatively rapid loss of spectrin (bands 1 and 2 on sodium dodecyl sulfate gel electropherograms) and the appearance of high molecular weight cross-linked derivatives. Isolated spectrin also undergoes photosensitized cross-linking, indicating that the reaction is not lipid-dependent.Extensive cross-linking was neither reversed by dithiothreitol nor prevented by prior blocking of SH groups with , suggesting that cysteine residues are not crucial bridging sites. The possible requirement for NH2 groups, as suggested by previous model studies (Dubbelman, T.M.A.R., de Goeij, A.F.P.M. and van Steveninck, J. (1978) Biochim. Biophys. Acta 511, 141–151), was tested. Succinylation of spectrin protected against cross-linking, but this effect is attributed to the disruption of quaternary structure, as deduced from sedimentation measurements. However, virtually complete blocking of NH2 groups by amidination perturbed overall structure relatively little, and had no effect on cross-linking. Moreover, exogenous amines such as ethylamine, added in large excess to spectrin prior to irradiation, did not interfere with cross-link formation. These results suggest that NH2 groups are not involved in the reaction. 相似文献
11.
Ken-Ichi Yoshida 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,801(2):290-297
We found that a small, reproducible amount of calmodulin is present in the cytoskeleton of human platelets. Triton-insoluble materials (cytoskeletons), which were prepared by cetrifugation at 1000 × g for 10 min of platelets after lysis by Triton X-100, stimulated cyclic AMP phosphodiesterase activity in the presence of Ca2+ but not in the presence of the calcium chelator, EGTA, or the calmodulin antagonist, trifluoperazine. The activation of the enzyme was also obtained after heating Triton-insoluble materials. An alkaline glycerol polyacrylamide gel electrophoresis of fractions obtained after gel fitration of solubilized Triton residues showed a protein band which had a faster electrophoretic mobility in the absence than in the presence of Ca2+. Upon thrombin activation of platelets, calmodulin in the Triton-insoluble cytoskeletons increased rapidly parallel to actin, actin-binding protein and myosin. With other stimulants such as collagen, epinephrine and ADP, similar results were obtained but with slower association of these proteins with cytoskeletons. However, after treatment with the Ca2+-inophore A23187, calmodulin, actin and actin-binding protein in Triton residues decreased rapidly, whereas the association of myosin increased. Thus, calmodulin seems to be associated with actin filaments rather than myosin filaments, and may be involved in the generation of contractile force in the cell. 相似文献
12.
Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [γ-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 μM) in a similar way as the from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte . In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal . In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes. 相似文献
13.
The possible presence and properties of the Ca2+-dependent K+ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187+Ca2+, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K+ (balanced by the uptake of Na+) and a stimulation of both the influx and the efflux of 86Rb. These effects were antagonized by quinine, a known inhibitor of the Ca2+-dependent K+ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca2+-dependent K+ channel whose characteristics are similar to those described in other cell systems. 相似文献
14.
Conventional freeze-fracture electron microscopy was used to study water-soluble proteins and different forms of Ca2+-ATPase-detergent complexes. Freeze-fracture images of solutions containing proteins larger than myoglobin showed the presence of distinct, randomly dispersed particles on smooth fracture surfaces. The distribution of sizes of these particles was close to Gaussian, with a mean size which was correlated to the Stokes diameter. Monomeric Ca2+-ATPase from sarcoplasmic reticulum, solubilized by deoxycholate or a non-ionic detergent, showed a bimodal distribution of particles sizes. Even more complex distributions were found for dimeric and trimeric preparations of Ca2+-ATPase. The results can be interpreted on the assumption that the Ca2+-ATPase molecule is elongated, with an overall length of about 110 Å and a width in its largest part of about 75 Å. It is concluded on the basis of the presented results that freeze-fracture electron microscopy can be successfully used for morphological studies of protein molecules in solution. 相似文献
15.
Pigeon erythrocyte membrane was solubilized partially, but relatively unselectively by Triton X-100. Vesicles were reconstituted from mixtures of Triton-solubilized membrane and lipid (phosphatidylcholine plus phosphatidylethanolamine plus cholesterol) by addition of bovine high-density lipoprotein. This efficiently removed the Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electropherograms of reconstituted vesicles showed band patterns resembling those of the original membrane. The reconstituted vesicles showed ATP-dependent active accumulation of 45Ca2+. ATP-dependent 45Ca2+ uptake by the reconstituted vesicles resembled the corresponding activity of the original membrane vesicles; in both preparations the Ca2+ uptake rate depended on the square of the Ca2+ concentration and had similar values, 0.16 μM and 0.18 μM, respectively. 相似文献
16.
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
17.
Bulbs of Crocus sativus, variety Cartwrightianus contain a protein factor with aggregating properties on human platelets. This factor was purified by different chromatographic techniques and shows a molecular weight of 42 000, as it was estimated by Sephadex G-75 column chromatography and sodium dodecyl sulfate (SDS) polyacrylamide slab gel electrophoresis. 相似文献
18.
Gerhard Kopperschläger Elke Usbeck Eberhard Hofmann 《Biochemical and biophysical research communications》1976,71(1):371-378
Intracellular cross-linking of yeast phosphofructokinase with a series of diimidoesters of different chain length resulted in the appearance of tetramers as largest cross-linked product of the enzyme subunits. The native enzyme is evidently composed of eight subunits being arranged in two tetramers α4β4. In the tetramers the monomers are probably assembled in tetrahedral geometry. 相似文献
19.
(1) The effects of calmodulin binding on the rates of Ca2+-dependent phosphorylation and dephosphorylation of the red-cell Ca2+ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg2+-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg2+-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca2+ + Mg2+)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca2+ affinity for sustaining ATP hydrolysis is raised at least 20-fold, Km(Ca) ? 10 μM (—calmodulin) and Km(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca2+ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (Km(Ca) ? 3–4 · 10-8 M). (Ca2+ + Mg2+)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca2+ + Mg2+)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca2+ + Mg2+)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (Km(MgATP) ? 360 μM), and CaATP is an inhibitor (Ki(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca2+- pump active site, producing a high (Ca2+ + Mg2+)-ATPase activity, high Ca2+ affinity and regulation of activity by MgATP. 相似文献
20.
B. Vestergaard-Bogind 《生物化学与生物物理学报:生物膜》1983,730(2):285-294
Ionophore A23187-mediated Ca2+-induced oscillations in the conductance of the Ca2+-sensitive K+ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K+ activity were followed with a K+-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular 45Ca content. An increased cellular 45Ca content at the successive minima of the oscillations where the K+ channels are closed indicates that the activation of the channels might be a process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K+ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about ?65 mV was observed. At a membrane potential of about ?70 mV and an intracellular concentration of about 2·10?4M no inactivation of K+ channels took place. Inactivation of the K+ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular 45Ca content support the notion that a necessary component of the oscillatory system is a Ca2+ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium. 相似文献