共查询到20条相似文献,搜索用时 15 毫秒
1.
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
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.
Jeffrey M. Gimble Michael Gustin David B.P. Goodman Howard Rasmussen 《生物化学与生物物理学报:生物膜》1982,685(3):253-259
The measurement of chlortetracycline fluorescence was employed as a probe for measuring the process to calcium transport by human erythrocyte inside-out vesicles. Chlortetracycline is a divalent metal chelator which increases its fluorrescence when bound to calcium in the presence of a membrane. Addition of calcium and ATP to inside out vesicles in the presence of chlortetracycline increased the chlortetracycline fluorescence as a function of time following an initial delay. Only after a threshold level of calcium had been accumulated did the fluorescence increase. The presence of both ATP and calcium were required. The addition of calmodulin increased the rate and absolute magnitude of the chlortetracycline fluorescence change. Similarly, calmodulin stimulated the rate and extent of 45Ca transport by inside-out vesicles. Moreover, the presence of saponin abolished both chlortetracycline fluorescence change and 45Ca uptake; a non-hydrolyzable ATP analog would not substitute for ATP in either 45Ca transport or chlortetracycline fluorescence experiments. Comparison between the slopes of the linear portions of chlortetracycline fluorescence change and calcium transport time courses at varied free calcium concentrations showed a consistent ratio between the slopes. This suggests that calcium transport change can be calibrated by employing chlortetracycline fluorescence. Based on this data, it is concluded that chlortetracycline fluorescence is a rapid and accurate method for monitoring calcium transport by human erythrocyte inside-out vesicles. 相似文献
4.
Menno De Metz Jocelyne Enouf Marilyne Lebret Sylviane Lévy-Tolédano 《生物化学与生物物理学报:生物膜》1984,773(2):325-328
Several nucleotide triphosphates (NTPs) were tested as energy source for the Ca2+ uptake by human platelet membrane vesicles. The Ca2+ uptake by these membranes was driven by ATP, GTP, ITP, UTP and CTP. The steady-state level of accumulated Ca2+ was equal with the different NTPs. The highest uptake velocity was found with ATP, but about 40–80% of the velocity with ATP could be accomplished with the other nucleotides. The highest affinity was also found with ATP (Km apparent 15 μM). The liberation of Pi from the various NTPs was measured simultaneously with the Ca2+ uptake. The coupling ratio (moles of Ca2+ taken up/moles of Pi liberated) varied from 0.4 for ATP to 2.3 for UTP and was almost independent of the NTP concentration. The enzyme activity with ATP as substrate is strongly dependent on the Ca2+ concentration in contrast to the activity with GTP, ITP, UTP or CTP. 相似文献
5.
Toshihide Kobayashi Hajime Okamoto Jun-Ichi Yamada Morio Setaka Takao Kwan 《生物化学与生物物理学报:生物膜》1984,778(1):210-218
Incubation of washed rabbit platelets with suspensions of dilauroylglycerophosphocholine resulted in the shedding of vesicles without causing any appreciable leakage of cytoplasmic marker (lactate dehydrogenase) or organelle marker ([14C]serotonin). The response was dependent on incubation time, concentration of dilauroylglycerophosphocholine and reaction temperature. Vesicles were separated from platelets and exogenous dilauroylglycerophosphocholine by a series of centrifugation steps. An average diameter of vesicles was 100–200 nm on scanning electron microscopy. Vesicles were enriched 5-fold in plasma membrane marker enzyme, acetylcholinesterase, whereas specific activities of lactate dehydrogenase and intracellular membrane marker enzyme, NADH-cytochrome c reductase were decreased in vesicles. Protein analysis by SDS-polyacrylamide gel electrophoresis showed that actin and actin-binding protein were present, while myosin was barely detectable in vesicles. Vesicles contained all phospholipid species of intact platelets and cholesterol but almost 50% of phospholipids in vesicles was dilauroylglycerophosphocholine. The phospholipid to protein ratio in vesicles was about 6.5-times higher than in intact platelets. 相似文献
6.
ATP promotes 45Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca2+-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the 45Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca2+-transport activity elicited by oxalate behaved like NADH-cytochrome reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca2+-storage capacity was not detectable in the absence of Ca2+-trapping agent. Low digitonin concentrations selectively increased the Ca2+ permeability of the plasmalemmal vesicles. The two Ca2+-transport activities were further differentiated by their distinct sensitivities to K+, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca2+-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca2+ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation. 相似文献
7.
8.
JoséD. Cavieres 《生物化学与生物物理学报:生物膜》1984,771(2):241-244
An average target size of 251 kDa has been obtained for the (Ca2+ + Mg2+)-ATPase of calmodulin-depleted erythrocyte ghosts by radiation inactivation with 16 MeV electrons. This is close to twice the size of the purified calcium-pump polypeptide. When calmodulin was included during the ATPase assay, a component of about 1 MDa appeared in addition to the activated dimer. 相似文献
9.
The characteristics of folate binding by brush border membranes from rat kidney homogenates were investigated. At pH 7.4, binding of [3′, 5′, 9-3H]-pteroylglutamic acid to membranes containing endogenous folate is inhibited by anions, with chloride being most effective followed by bromide, thiocyanate, iodide, phosphate and sulfate. A maximum inhibition of 70–75% is attained at a concentration of 0.1 M chloride and an incubation time of 30 min. The inhibition diminishes with increased incubation time and at 24 h is negligible. The binding of [3′,5′,9-3H]pteroylglutamic acid to brush border membranes stripped of endogenous folate by acid treatment is not inhibited by anions. Anion sensitivity can be restored to these treated membranes by reconstitution with membrane-derived folate, particularly 5-methyltetrahydropteroylglutamic acid, or by preincubation with synthetic 5-methyltetrahydropteroylglutamic acid. Inhibition of [3′,5′,9-3H]pteroylglutamic acid binding by anions in membranes with endogenous folate is best explained by an anion-induced stabilization of endogenous folate-binding protein complex resulting in a decreased rate of exchange with exogenous [3′,5′,9-3H]pteroylglutamic acid. 相似文献
10.
Calcium efflux and EGTA-induced calcium release from an internal platelet membrane fraction have been studied after the oxalate-supported calcium uptake had reached steady state. Increasing external calcium concentrations stimulate the calcium efflux velocity, with an apparent half-maximal stimulation at about 5 μM outside calcium concentration and a maximal velocity of calcium efflux of . Moreover, the ratio of the liberated calcium on the loaded calcium seems to be independent of the increasing external calcium concentration. Increasing the calculated internal calcium concentration by varying the oxalate potassium concentration from 10 mM to 1 mM results in an increase of the liberated calcium from the membrane vesicles from 7.4% to 63%, respectively, without changing the calcium efflux velocity. Similar conclusions can be drawn from the observation of results from the calcium efflux and EGTA-induced calcium release methods. Moreover, calcium pump reversal does not seem to be responsible for the calcium efflux or calcium release. All these different points added to the previously described regulation of calcium efflux by the catalytic subunit of cAMP protein kinase suggest us that the mechanism of calcium liberation by the platelet membranes is different from the calcium uptake. 相似文献
11.
Sergio Grinstein Wendy Furuya 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1984,803(4):221-228
The site and mechanism of accumulation of acridine derivatives into platelets and their isolated organelles were investigated. In addition, their suitability as indicators of cytoplasmic pH was analysed. Direct microscopic observation showed that quinacrine and 9-aminoacridine are concentrated inside organelles in platelets. Using fractionation studies, the acridine derivatives were found to accumulate particularly in dense and α-granules. Uptake into these organelles is driven by a pH differential across their membrane (acidic inside). Because of their cellular distribution, acridine derivatives were found to be poor indicators of cytoplasmic pH. In contrast, a poorly permeant dicarboxylated fluorescein derivative, generated in situ by cytosolic enzymes, is shown to be a more reliable probe of intracellular pH. The results are compared with previous reports of the use of 9-aminoacridine as a cytoplasmic pH probe in platelets and of quinacrine as a selective dense-granule marker. 相似文献
12.
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
13.
The ouabain-insensitive, Mg2+-dependent, Na+-stimulated ATPase activity present in fresh basolateral plasma membranes from guinea-pig kidney cortex cells (prepared at pH 7.2) can be increased by the addition of micromolar concentrations of Ca2+ to the assay medium. The Ca2+ involved in this effect seems to be associated with the membranes in two different ways: as a labile component, which can be quickly and easily ‘deactivated’ by reducing the free Ca2+ concentration of the assay medium to values lower than 1 μM; and as a stable component, which can be ‘deactivated’ by preincubating the membranes for periods of 3–4 h with 2 mM EDTA or EGTA. Both components are easily activated by micromolar concentrations of Ca2+. The of the system for Na+ is the same, 8 mM, whether only the stable component or both components, stable and labile, are working. In other words, the activating effect of Ca2+ on the Na+-stimulated ATPase is on the , and not on the of the system for Na+. The activating effect of Ca2+ may be related to some conformational change produced by the interaction of this ion with the membranes, since it can also be obtained by resuspending the membranes at pH 7.8 or by ageing the preparations. Changes in the Ca2+ concentration may modulate the ouabain-insensitive, Na+-stimulated ATPase activity. This modulation could regulate the magnitude of the extrusion of Na+ accompanied by Cl? and water that these cells show, and to which the Na+-ATPase has been associated as being responsible for the energy supply of this mode of Na+ extrusion. 相似文献
14.
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
15.
Paul K. Schick Barbara P. Schick Gabriel Brandeis David C.B. Mills 《生物化学与生物物理学报:生物膜》1981,643(3):659-662
Arachidonic acid (20:4) and other fatty acids and aldehydes in phosphatidylethanolamine (PE) present on the platelet surface was determined. Surface-exposed PE was isolated by using 2,4,6-trinitrobenzenesulfonate, a nonpenetrating probe (Schick, P.K., Kurica, K.B. and Chacko, G.K. (1976) J. Clin. Invest. 57, 1221–1226). PE contains 50% total platelet arachidonic acid. Approx. 16% platelet PE is present on the platelet surface. The study showed that the fatty acid and aldehyde composition of PE on the platelet surface is virtually identical to that in PE present inside the platelet. Therefore, 8 nmol arachidonic acid are present in PE in the outer layer of the plasma membrane in 109 platelets. 相似文献
16.
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
17.
Several characteristics of calmodulin association with brain synaptic and coated vesicles were analyzed and compared. Radioimmunoassay revealed that both classes of vesicles contain approx. 1 μg of calmodulin per mg of vesicle protein. Discontinuous sucrose gradients revealed that coated and synaptic vesicles preparations were homogeneous and had different sedimentation properties. Binding of 125I-labeled calmodulin to synaptic and coated vesicles was Ca2+ dependent and displaced by unlabeled calmodulin but not by troponin-C. Scatchard analysis revealed the presence of two binding sites. In both vesicle types there was one high-affinity, low-binding-capacity site () and one low-affinity, high-binding-capacity site (). (Ca2+ + Mg2+)-ATPase activity was stimulated in both synaptic and coated vesicles by calmodulin. Thus synaptic and coated vesicles may possess similar calmodulin binding sites. 相似文献
18.
Insulin and its analogues displaced membrane-bound calcium within a physiological range of insulin concentration, in proportion to both biological potency and ability to displace porcine 125I-labelled insulin from the insulin receptor. Mild tryptic digestion of the membrane reduced insulin binding but did not reduce specific calcium binding. Displacement of membrane-bound calcium by insulin was dependent on insulin binding to its intact receptor. These studies suggest that Ca2+ may exert a controlling influence on insulin-receptor binding in vivo. 相似文献
19.
Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na+-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of Mr ? 165000 which is apparently a dimer of Mr ? 85 000. When reconstituted and tested for transport, this protein showed Na+-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals. 相似文献
20.
Yoshio Okada 《生物化学与生物物理学报:生物膜》1981,648(2):120-128
Interactions of concanavalin A with human erythrocytes were studied using 125I-labelled concanavalin A and a centrifugal technique with dibutyl phthalate which permitted complete separation of bound and free concanavalin A. Binding of 125I-labelled concanavalin A to human erythrocytes was dependent on cell concentration, pH and temperature. Specificity of binding was confirmed by inhibition and dissociation studies with sugars and native concanavalin A. Positive cooperative binding of concanavalin A to human erythrocytes was observed at low concanavalin A concentrations (less than 1 μ/ml) in both buffers studied. Positive cooperativity at higher concanavalin A concentrations (more than 100 μ/ml) was seen in Tris-Hepes buffer but not in phosphate-buffered saline. Consistent with this cooperative effect was the observation that although dissociation of 125I-labelled concanavalin A from the erythrocytes was complete in the presence of 1 mg/ml of the native lectin, release was inhibited by low concentrations (1 μ/ml). A comparison of concanavalin A binding with hemagglutination studies suggest that the amount of concanavalin A bound determines the rate of erythrocyte agglutination and the size of the aggregates formed. 相似文献