共查询到20条相似文献,搜索用时 15 毫秒
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Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca2+-activated Mg2+-ATPase and Ca2+ transport activities. Membrane vesicles that were exposed to oxalate as a Ca2+-trapping agent accumulated Ca2+ in the presence of Mg2+ and ATP. The for Ca2+ uptake was 33 nmol/mg protein per h, and the values for Ca2+ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca2+ ionophore A23187, added initially, completely inhibited net Ca2+ uptake and, if added later, caused the release of Ca2+ previously accumulated. A Ca2+-activated ATPase was present in the same membrane vesicles which had a of 1.5 μmol/mg protein per h at free Ca2+ concentration of 10 μM. This Ca2+-ATPase had values of 4.5 μM and 110 μM for Ca2+ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca2+ by the vesicles. The Ca2+-ATPase activity was insensitive to ouabain. Both Ca2+ transport and Ca2+-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca2+ transport activity and a Ca2+-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin. 相似文献
3.
Synexin protein is non-selective in its ability to increase Ca2+-dependent aggregation of biological and artificial membranes 总被引:2,自引:0,他引:2
Synexin, a soluble protein which increases the specificity of Ca2+ to aggregate isolated bovine chromaffin granules was prepared from bovine adrenal medullary tissue by the method of Creutz, Pazoles and Pollard (J. Biol. Chem. , 2858–2866, 1978). We also find that synexin increases both the initial rate and final amplitude of Ca2+-promoted aggregation of granule membranes. This effect is Ca2+-specific. However in contrast to Creutz , we find that synexin also potentiates aggregation of adrenal medulla and liver mitochondria and microsomes as well as phosphatidylserine vesicles. This lack of membrane specificity argues against the suggestion of Creutz that synexin specifically binds the granule to the plasma membrane prior to exocytosis . 相似文献
4.
A Ziegelhoffer M B Anand-Srivastava R L Khandelwal N S Dhalla 《Biochemical and biophysical research communications》1979,89(4):1073-1081
The sarcolemmal membrane obtained from rat heart by hypotonic shock-LiBr treatment method was found to incorporate 32P from [γ-32P] ATP in the absence and presence of cyclic AMP and protein kinase. The phosphorylated membrane showed an increase in Ca2+ ATPase and Mg2+ ATPase activities without any changes in Na+K+ ATPase activity. The observed increase in ATPase activity was found to be associated with an increase in Vmax value of the reaction whereas Ka value for was not altered. These results provide information concerning biochemical mechanism for increased calcium entry due to hormones which are known to elevate cyclic AMP levels in myocardium and produce a positive inotropic effect. 相似文献
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6.
N Narayanan P Lee M Newland R L Khandelwal 《Biochemical and biophysical research communications》1982,108(3):1158-1164
A cytosolic protein fraction, termed CPF-I, derived by (NH4)2 SO4 fractionation of rabbit heart cytosol caused marked inhibition (up to 95%) of ATP-dependent Ca2+ uptake by cardiac sarcoplasmic reticulum. The inhibitory effect of CPF-I was concentration-dependent (50% inhibition with ~ 80–100 μg CPF-I) and heat labile. The inhibitor reduced the velocity of Ca2+ uptake without altering the apparent affinity of the transport system for Ca2+. Concomitant with the inhibition of Ca2+ uptake, Ca2+-sensitive ATP hydrolysis was also inhibited by CPF-I. The inhibitor did not cause release of Ca2+ from Ca2+-preloaded membrane vesicles. The inhibitor activity of CPF-I could be adsorbed to a DEAE cellulose column and could be eluted with a linear gradient of KCl. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum calcium pump in cardiac muscle and raises the intriguing possibility of its participation in the regulation of calcium pump . 相似文献
7.
Tai C. Chen Michael Rosenblatt Jules B. Puschett 《Biochemical and biophysical research communications》1980,94(4):1227-1232
Inhibition of parathyroid hormone (PTH)-sensitive adenylate cyclase by {Nle-8, Nle-18, Tyr-34} bPTH-(3–34) amide was studied in thyroparathyroid-ectomized dogs. The inhibitory effect was shown to be markedly enhanced by the addition of calcium ions into the assay system. At 0.1 mM Ca2+, complete inhibition by the antagonist was obtained. Chelation of exogenous Ca2+ by EGTA eliminated the Ca2+-induced inhibition. Both the basal and hormone-stimulated activities were decreased in the presence of 0.1 mM Ca2+, whereas the addition of EGTA increased both activities. Our results suggest that Ca2+ modulates canine renal PTH-sensitive adenylate cyclase and its inhibition by substituted bPTH-(3–34). 相似文献
8.
A membrane fraction enriched in axolemma was obtained from optic nerves of the squid (Sepiotheutis sepioidea) by differential centrifugation and density gradient fractionation. The preparation showed an oligomycin- and NaN3-insensitive (Ca2+ + Mg2+)-ATPase activity. The dependence of the ATPase activity on calcium concentration revealed the presence of two saturable components. One had a high affinity for calcium () and the second had a comparatively low affinity (). Only the high-affinity component was specifically inhibited by vanadate (K1 = 35 μM). Calmodulin (12.5 μ/ml) stimulated the (Ca2+ + Mg2+)-ATPase by approx. 50%, and this stimulation was abolished by trifluoperazine (10 μM). Further treatment of the membrane fraction with 1% Nonidet P-40 resulted in a partial purification of the ATPase about 15-fold compared to the initial homogenate. This (Ca2+ + Mg2+)-ATPase from squid optic nerve displays some properties similar to those of the uncoupled Ca2+-pump described in internally dialyzed squid axons, suggesting that it could be its enzymatic basis. 相似文献
9.
(1) Vanadate (pentavalent vanadium) inhibits with high affinity () the ATP-dependent Ca2+ efflux in reconstituted ghosts from human red cells. (2) To inhibit Ca2+ efflux vanadate has to have access to the inner surface of the cell membrane. (3) The inhibitory effect of vanadate is potentiated by intracellular Mg2+ and by intracellular K+. (4) Ca2+ in the external medium antagonizes the inhibitory effect of vanadate. 相似文献
10.
Lanthanum (0.25 mM) does not penetrate into fresh or Mg2+-depleted cells, whereas it does into ATP-depleted or , into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca2+-efflux completely and inhibits (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg2+-depleted cells Ca2+-Ca2+ exchange is inhibited by lanthanum. Ca2+-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca2+-leak ± S.D. amounts to of cells per min, whereas in KCl medium to of cells per min at 2.5 mM [Ca2+]e and 0.25 mM [La3+]e pH 7.1.Lanthanum inhibits Ca2+-dependent rapid K+ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K+ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol.Lanthanum at 0.2–0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca2+-loaded cells, however, is blocked by lanthanum owing to Ca2+-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca2+ concentrations. 相似文献
11.
Cholinergic synaptic vesicles from the electric organ of have been subjected to analytical scale separation techniques not utilized in the isolation procedure, and the ATPase activity of separated fractions determined. Most of the ATPase activity migrated with the vesicles. Sensitivity of the ATPase activity to 16 potential inhibitors also was determined. Most of the ATPase activity was inhibited by low concentrations of 4-chloro-7-nitrobenzo-oxadiazole (NBD-C1) and dicyclohexylcarbodiimide (DCCD), but not by a water soluble carbodiimide. The close association of the ATPase with the vesicles and the pattern of inhibition obtained provide further support for the authentic presence of a membrane bound ATPase in the cholinergic synaptic vesicle. 相似文献
12.
D J Hanahan R D Taverna D D Flynn J E Ekholm 《Biochemical and biophysical research communications》1978,84(4):1009-1015
This paper presents the first unambiguous demonstration that a unique protein isolated from the hemolysate of human erythrocytes is responsible for increasing both the apparent Ca2+ ion affinity and maximum rate of ATP hydrolysis of the membrane-bound ATPase. Unlike previous reports where an unpurified extract from red blood cells was used to activate the ATPase, our results clearly demonstrate that a single protein species, whether initially associated with or added back to the membrane is responsible for the observed changes in ATPase activity. 相似文献
13.
Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in a significant (2–3-fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmaksing of a latent Mg2+-dependent Ca2+-stimulated ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+ and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a of 70 μM for ATP and the values for Mg2+ and Ca2+ are about and 10?7 M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed. 相似文献
14.
Amphiphilic, cationic Polymyxin B is shown to displace Ca2+ from ‘gas dissected’ cardiac sarcolemma in a dose-dependent, saturable fashion. The Ca2+ displacement is only partially reversible, 57% and 63%, in the presence of 1 mM or 10 mM Ca2+, respectively. Total Ca2+ displaced by a non-specific cationic probe, lanthanum (La3+), at maximal displacing concentration (1 mM) was membrane protein. At 0.1 mM, Polymyxin B displaced 42% of the total La3+-displaceable Ca2+ or protein. 5 mM Polymyxin displaced Ca2+ in amounts equal to those displaced by 1 mM La3+. Pretreatment of the membranes with neuraminidase (removal of sialic acid) and protease leads to a decrease in La3+-displaceable Ca2+ but to an increase in the fraction displaced by 0.1 mM Polymyxin from 42% to 54%. Phospholipase D (cabbage) treatment significantly increased the La3+-displaceable Ca2+ to protein (), a gain of 0.055 nmol. All of this phospholipid specific increment in bound Ca2+ was displaced by 0.1 mM Polymyxin B. The results suggest that Polymyxin B will be useful as a probe for phospholipid Ca2+-binding sites in natural membranes. 相似文献
15.
Klaus Gietzen Peter Adamczyk-Engelmann Andreas Wüthrich Anka Konstantinova Hermann Bader 《生物化学与生物物理学报:生物膜》1983,736(1):109-118
Compound , a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca2+-transport ATPase, with IC50 values of 0.3 and 0.85 μg/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 μg/ml. Inhibition of Ca2+ transport into inside-out red blood cell vesicles by compound follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca2+-transport ATPase induced by calmodulin is inhibited by compound according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound bind to calmodulin in a Ca2+-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca2+-transport ATPase that has been described hitherto. In addition, compound was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca2+-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound , respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca2+-transport ATPase or calf cardiac sarcolemma (Na+ + K+)-transport ATPase are far less influenced by compound as compared with trifluoperazine and calmidazolium. Because of its high specificity compound is proposed to be a promising tool for studying calmodulin-dependent processes. 相似文献
16.
A chicken pectoralis muscle membrane fraction enriched in a Mg2+- or Ca2+-activated (‘basic’) ATPase was obtained by sucrose gradient centrifugation. Enzymatic properties of the ‘basic’ ATPase were determined and used to localize its enzymatic activity in situ by ultrastructural cytochemistry. The enzyme was activated by Mg2+ or Ca2+ but not by Sr2+, Ba2+, Co2+, Ni2+ or Pb2+. It was present in a membranous fraction with a buoyant density of 1.10-1.12 (24–27.5% () sucrose). ‘Basic’ ATPase activity had a sedimentation pattern similar to the putative plasma membrane enzymes, 5′-nucleotidase and leucyl β-naphthylamidase, but different from that of sarcoplasmic reticulum Ca2+ ATPase. Also unlike sarcoplasmic reticulum Ca2+ ATPase, ‘basic’ ATPase was resistant to N-ethylmaleimide and aldehyde fixatives, was active in a medium containing a high Ca2+ concentration (3 mM), and was lost when exposed to Triton X-100 or deoxycholate. In cytochemical studies, a low Pb2+ concentration was used to capture the enzymatically released phosphate ions. Under conditions which eliminated interfering (Na+ + K+) ATPase and sarcoplasmic reticulum Ca2+ ATPase activities, electron-dense lead precipitates were present at the plasmalemma and T-system membranes. These studies suggest that ‘basic’ ATPase activity is associated with plasmalemma and T-system membranes of skeletal muscle. 相似文献
17.
The uptake and release of Ca2+ by sarcoplasmic reticulum fragments and reconstituted ATPase vesicles was measured by a stopped-flow fluorescence method using chlortetracycline as Ca2+ indicator.Incorporation of the Ca2+ transport ATPase into phospholipid bilayers of widely different fatty acid composition increases their passive permeability to Ca2+ by several orders of magnitude. Therefore in addition to participating in active Ca2+ transport, the ()-activated ATPase also forms hydrophilic channels across the membrane. The relative insensitivity of the permeability effect of ATPase to changes in the fatty acid composition of the membrane is in accord with the suggestion that the Ca2+ channels arise by protein-protein interaction between four ATPase molecules. The reversible formation of these channels may have physiological significance in the rapid Ca2+ release from the sarcoplasmic reticulum during activation of muscle. 相似文献
18.
Although acute alterations in Ca2+ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca2+-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca2+-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca2+ of 280 nM, of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg2+ could replace Ca2+ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg2+ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca2+-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca2+-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca2+ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function. 相似文献
19.
Torben Clausen Tove Lindahl Andersen Marianne Stürup-Johansen Olga Petkova 《生物化学与生物物理学报:生物膜》1981,646(2):261-267
(1) The effects of vanadate of hexose transport, 45Ca-exchange and (Na+, K+)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[14C]glucose as well as the efflux of . (3) Within the same concentration range, vanadate induced an early increase in 45Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of and 45Ca were significantly correlated (, ). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of and this effect was preceded by a rise in the washout of 45Ca. The maximum increases in the fractional losses of and 45Ca were significantly correlated (, ). (5) In extensor digitorum longus and soleus muscles, vanadate increased K+-contents and decreased Na+ contents. (6) The stimulation of 45Ca-washout presumably reflects an increase in the cytoplasmic Ca2+ level, brought about by an inhibitory effect of vanadate on the Ca2+-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca2+ level. 相似文献
20.
M Mason 《Biochemical and biophysical research communications》1974,60(1):64-69
At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl2, apparently because Ca++ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg++, Mn++, Na+, K+, and NH4+ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca++ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca++ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. depends on the ability of that agent to chelate Ca++. 相似文献