共查询到20条相似文献,搜索用时 46 毫秒
1.
Görlach M Dieter P Seydewitz HH Kaiser C Witt I Marme D 《Biochimica et biophysica acta》1985,832(2):228-232
Calmodulin from Drosophila heads has been purified to apparent electrophoretic homogeneity. It has the same characteristics as bovine brain calmodulin with respect to the migration upon polyacrylamide gel electrophoresis and maximal activation of a calmodulin-deficient cAMP phosphodiesterase. The amino acid composition resembles bovine brain calmodulin with the exception that trimethyllysine is absent and that it contains only one tyrosine. The tryptic peptide map of Drosophila calmodulin suggests some differences in the amino acid sequence as compared to bovine brain calmodulin. These proposed differences in the primary structure may explain why Drosophila calmodulin is less potent than bovine brain calmodulin in the activation of a cAMP phosphodiesterase from bovine brain. 相似文献
2.
O Hatase M Tokuda T Itano H Matsui A Doi 《Biochemical and biophysical research communications》1982,104(2):673-679
Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca2+-dependent phosphodiesterase of bovine brain in the presence of Ca2+. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane. 相似文献
3.
In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [gamma-32P]ATP as phosphate donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the Ca2+-calmodulin-dependent enzyme, (3':5'AMP)-phosphodiesterase, by the EGTA extract, and (iii) electrophoretic comigration of EGTA-extracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32P-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis. 相似文献
4.
The calcium-dependent regulatory proteins, calmodulins, have been isolated from human blood platelets and guinea pig peritoneal polymorphonuclear leucocytes using the urea methanol procedure of Grand et al. [Biochem. J. 177, 521-529 (1978)]. The calmodulins were purified to homogeneity as indicated by polyacrylamide gel electrophoresis and both proteins comigrated with bovine brain calmodulin with mobilities corresponding to molecular weights of 16 000-17 000. The yield of calmodulin from platelets was higher on a wet weight basis than the yield from leucocytes but the former compared favourably with yields reported for brain and other tissues. Both calmodulin preparations significantly stimulated brain cyclic nucleotide phosphodiesterase, erythrocyte ghost Ca2+ ATPase and platelet phosphorylase kinase activities at the microgram level. Stimulation of Lubrol-solubilised brain adenylate cyclase was only marginally significant with platelet calmodulin and rarely demonstrable with the leucocyte preparations. Although biological activities of both proteins were retained during storage at -20 degrees C, higher-molecular-weight aggregates slowly formed which could not be dissociated during dodecylsulphate/mercaptoethanol denaturation. 相似文献
5.
Truncated mutants of the colicin A lysis protein are acylated and processed when overproduced 总被引:2,自引:0,他引:2
Danièle Cavard 《FEMS microbiology letters》1994,117(2):169-174
Abstract Bordetella calmodulin-like protein was purified from culture supernatant fluid of B. pertussis, B. parapertussis and B. bronchiseptica by successive chromatography on hydroxyapatite, Toyopearl HW-50F and QAE-Toyopearl 550C columns. The purified calmodulin-like protein appeared to be homogeneous by SDS-polyacrylamide gel electrophoresis. The apparent molecular mass of calmodulin-like protein on SDS-polyacrylamide gel electrophoresis was 10 kDa, which was smaller than bovine brain calmodulin (17 kDa). The purified calmodulin-like protein activated both Bordetella adenylate cyclase and mammalian phosphodiesterase in a Ca2+ -dependent manner. This activation was inhibited by calmodulin antagonists. The calmodulin-like protein, like calmodulin, was retained by a hydrophobic resin in the presence of Ca2+ and eluted by the addition of EDTA. These results indicated that the Bordetella calmodulin-like protein is closely related to calmodulin. As a putative calmodulin the extracellular calmodulin may be involved in Bordetella pathogenesis. 相似文献
6.
Production and specificity of monoclonal antibodies against calmodulin from Dictyostelium discoideum 总被引:10,自引:0,他引:10
Monoclonal antibodies were raised against calmodulin purified from Dictyostelium discoideum. To increase its antigenicity, the calmodulin was conjugated to keyhole limpet hemocyanin; mice were immunized with the conjugate. Hybridomas producing antibodies against calmodulin were identified by screening culture supernatants with calmodulin coupled to bovine serum albumin. The specificity of antibodies from hybridoma culture supernatants was tested by Western blot of Dictyostelium cell lysates. For the purpose, methods were developed that permitted sensitive detection of calmodulin bound to membranes. The key elements of the blotting protocol were used of PVDF membrane, transfer conducted in phosphate buffer, and glutaraldehyde fixation after transfer. These methods permitted detection of as little as 0.1 ng of calmodulin spotted directly onto the membrane, or 10 ng transferred from an SDS polyacrylamide gel. Ten calmodulin-specific antibodies were identified; most of these reacted preferentially with the calcium-containing form of Dictyostelium calmodulin. Several of the monoclonal antibodies cross-reacted with calmodulin from bovine brain. 相似文献
7.
H Ohshima S Oguchi H Adachi S Iida H Suzuki T Sugimura H Esumi 《Biochemical and biophysical research communications》1992,183(1):238-244
Nitric oxide (NO) synthase (EC 1.14.23) was purified to homogeneity from bovine cerebrum. The molecular weight of NO synthase was estimated to be 150 kDa by both SDS/PAGE and gel filtration at high salt concentration. For activity, the enzyme required NADPH, Ca2+, calmodulin and tetrahydrobiopterin as cofactors. Rabbit polyclonal antibody to bovine brain NO synthase reacted with 150 kDa NO synthase in various bovine and rat organs, including the brain, pituitary and adrenal glands, but not with that in stimulated macrophages, indicating that there are at least two immunologically distinct NO synthases. 相似文献
8.
Calmodulin-binding proteins from brain and other tissues. 总被引:8,自引:1,他引:7
The calmodulin contents of rabbit brain, lung, kidney and liver, of bovine aorta and uterus, and of chicken gizzard have been determined. 2. The calmodulin in all of these tissues has been shown to be present in the form of very stable complexes with several other proteins. 3. A calmodulin-binding protein of mol.wt. 22 000 has been purified in high yield from bovine brain. It has been shown to interact with calmodulin and rabbit skeletal-muscle troponin C in a Ca2+-dependent manner. 4. The 22 000-mol.wt. protein inhibits the activation of bovine brain phosphodiesterase by calmodulin, but has very little affect on the activation of myosin light-chain kinase. 5. Calmodulin-binding proteins of mol.wts. 140000, 77000 and 61000 have also been partially purified from rabbit brain by affinity chromatography and have been shown to interact in a Ca2+-dependent manner with calmodulin. 6. The apparent molecular weights of the calmodulin-calmodulin-binding protein complexes, determined by gel filtration in the presence of 6M-urea, have been shown to be similar for most of the mammalian tissues examined. 7. By using 125I-labelled calmodulin, similar complexes have been demonstrated in rabbit skeletal muscle, although they are present at much lower concentrations. 相似文献
9.
Calmodulin in starfish oocytes. I. Calmodulin antagonists inhibit meiosis reinitiation 总被引:2,自引:0,他引:2
A heat-stable factor has been found in starfish (Patiria miniata and Marthasterias glacialis) oocytes that activates two calmodulin-dependent enzymes: bovine brain phosphodiesterase (10-fold increase) and sea urchin egg NAD-kinase (10- to 50-fold increase). The dose-response curves for activation of these enzymes were found to be parallel for the starfish egg extract and pure mammalian brain calmodulin. The active factor was purified by chromatography on DE 52 cellulose to which it remained bound and was eluted by 0.225 M ammonium sulfate. Active fractions were pooled, dialyzed, and run on a polyacrylamide gel. The starfish active factor comigrated with pure bovine brain calmodulin. A radioimmunoassay was performed on the purified factor; it cross-reacted with antibodies against pure calmodulin. That calmodulin may play a role in hormonally induced maturation of starfish oocytes is suggested by the fact that two calmodulin antagonists (trifluoperazine and vinblastine), which are also inhibitors of NAD-kinase, were found to block 1-methyladenine-induced oocyte maturation. The inhibition could be reversed by increasing the hormone concentration. Oocytes were sensitive to trifluoperazine only during the hormone-dependent period. 相似文献
10.
本文报导了一种能同时纯化钙调神经磷酸酶和钙调素的有效方法。牛脑粗提液经DE-52纤维素层析分段洗脱:0.5mol/L NaCl缓冲液洗脱峰经phenyl-sepharose亲和柱和G75 sephadex制得电泳纯钙调素。0.18mol/L KCl缓冲液洗脱峰经Affigel-Blue层析,硫酸铵盐析,钙调素亲和层析,G-200 Sephadex凝胶过滤制得电泳纯钙调神经磷酸酶。 相似文献
11.
A calmodulin-dependent protein phosphatase has been identified in human platelets by its cross-reactivity with an antibody developed against a bovine brain calmodulin-dependent protein phosphatase and by its calmodulin-stimulated dephosphorylation of 32P-labeled substrates. The platelet enzyme was partially purified to separate it from calmodulin and calmodulin-independent phosphatases. The partially purified enzyme was stimulated by calmodulin, requiring 15 nM calmodulin for half-maximal activation. Calmodulin increased the Vmax of the phosphatase, with no significant effect on its Km. The enzyme was stimulated irreversibly and made calmodulin-independent by limited proteolysis. The optimal pH for the phosphatase was 7.5. After partial purification, phosphatase activity was significantly increased in the presence of Mn2+ and Ca2+ over that observed in the presence of Ca2+ alone. The enzyme effectively dephosphorylated casein, histone, protamine, and platelet actin. The holophosphatase was estimated to have a molecular weight of 76,900 as determined by sedimentation on sucrose gradients. Immunoblotting techniques using an antibody against the brain phosphatase suggests that the enzyme consists of 2 subunits of 60,000 and 16,500 daltons; the 60,000-dalton subunit co-migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 60,000-dalton calmodulin-binding protein in the platelet suggesting that it is the calmodulin-binding subunit of the enzyme. The identification of a calmodulin-dependent protein phosphatase in human platelets suggests a role for Ca2+-dependent dephosphorylation in platelet activation. 相似文献
12.
A cDNA clone of human calmodulin, isolated from liver, was subcloned into the expression vector pKK233-2. The resulting expression plasmid, designated pCWCaM1, produced human calmodulin in Escherichia coli SG5. The cDNA was sequenced using novel primers designed for use in plasmid-sequencing protocols with pKK233-2 and pKK223-3. The expressed calmodulin was purified and subjected to NMR analysis which revealed a structure essentially the same as natural calmodulin isolated from human tissue. The activation of myosin light chain kinase by the genetically engineered human calmodulin and bovine brain calmodulin was studied and found to be comparable to a high degree. The expressed calmodulin appears to be comparable to normal calmodulin and can be used for site-directed mutagenesis and structure/function investigations. 相似文献
13.
We have purified calmodulin from the eukaryotic microorganism Dictyostelium discoideum (Clarke, M., Bazari, W. L., and Kayman, S. C. (1980) J. Bacteriol. 141, 397-400) and have compared it to calmodulin purified from bovine brain. The two proteins behaved almost identically during fractionation on ion exchange and gel filtration columns and on isoelectric focusing gels. Dictyostelium calmodulin had one-third the specific activity of brain calmodulin in the Ca2+-dependent activation of brain cyclic nucleotide phosphodiesterase; this activation was inhibited for both proteins by 25 microM trifluoperazine. Dictyostelium calmodulin also activated erythrocyte (Ca2+ + Mg2+)-ATPase and interacted with the inhibitory subunit of skeletal muscle troponin. Competition radioimmune assays showed that Dictyostelium calmodulin could compete with brain calmodulin for antibodies to brain calmodulin. These similarities indicate a close relationship between Dictyostelium and brain calmodulin and suggest that the functional capabilities of the protein have been conserved even among evolutionarily distant species. However, substantial differences in primary structure were detected by amino acid analyses and peptide mapping. Most interesting is the lack of trimethyllysine in Dictyostelium calmodulin. This unusual amino acid, which is commonly found in calmodulins, is therefore not essential for interaction between calmodulin and the calmodulin-regulated proteins tested here. 相似文献
14.
Dephosphorylation of neuromodulin by calcineurin 总被引:8,自引:0,他引:8
Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes. Phosphorylation of neuromodulin by protein kinase C causes a significant reduction in its affinity for calmodulin (Alexander, K. A., Cimler, B. M., Meirer, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). It has been proposed that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons and that activation of protein kinase C causes the release of free calmodulin at high concentrations near its target proteins. It was the goal of this study to determine whether bovine brain contains a phosphoprotein phosphatase that will utilize phosphoneuromodulin as a substrate. Phosphatase activity for phosphoneuromodulin was partially purified from a bovine brain extract using DEAE-Sephacel and Sephacryl S-200 gel filtration chromatography. The neuromodulin phosphatase activity was resolved into two peaks by Affi-Gel Blue chromatography. One of these phosphatases, which represented approximately 60% of the total neuromodulin phosphatase activity, was tentatively identified as calcineurin by its requirement for Ca2+ and calmodulin (CaM) and inhibition of its activity by chlorpromazine. Therefore, bovine brain calcineurin was purified to homogeneity and examined for its phosphatase activity against bovine phosphoneuromodulin. Calcineurin rapidly dephosphorylated phosphoneuromodulin in the presence of micromolar Ca2+ and 3 microM CaM. The apparent Km and Vmax for the dephosphorylation of neuromodulin, measured in the presence of micromolar Ca2+ and 2 microM CaM, were 2.5 microM and 70 nmol Pi/mg/min, respectively, compared to a Km and Vmax of 4 microM and 55 nmol Pi/mg/min, respectively, for myosin light chain under the same conditions. Dephosphorylation of neuromodulin by calcineurin was stimulated 50-fold by calmodulin in the presence of micromolar free Ca2+. Half-maximal stimulation was observed at a calmodulin concentration of 0.5 microM. We propose that phosphoneuromodulin may be a physiologically important substrate for calcineurin and that calcineurin and protein kinase C may regulate the levels of free calmodulin available in neurons. 相似文献
15.
NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5's ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5's for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 相似文献
16.
Rapid purification of calmodulin and S-100 protein by affinity chromatography with melittin immobilized to sepharose 总被引:2,自引:0,他引:2
Melittin-Sepharose was prepared for Ca2+-dependent affinity chromatography of calmodulin and S-100 protein. This matrix exhibits extremely high capacity (approximately 10 mg calmodulin/ml gel), low nonspecific binding, and excellent recovery (greater than 90%) under optimal conditions. Recovery of calmodulin from melittin-Sepharose was related to the degree of saturation of column capacity with lower yields when only partial saturation was achieved. Large-scale, simultaneous purification of calmodulin and S-100 protein from brain was carried out using selective adsorption to organomercurial agarose followed by melittin-Sepharose chromatography; yields were 250-300 mg of calmodulin and 200-300 mg of S-100 per kg tissue. Calmodulin also was purified in a single step from bovine testis supernatant using melittin-Sepharose in yields comparable to those from brain. 相似文献
17.
A rabbit lung cyclic nucleotide phosphodiesterase (PDE) prepared by successive chromatography on DEAE-cellulose and G-200 Sephadex columns in the presence of EGTA was activated by Ca2+ and contained calmodulin (CaM), suggesting that the enzyme exists as a stable CaM X PDE complex (Sharma, R. K., and Wirch, E. (1979) Biochem. Biophys. Res. Commun. 91, 338-344). An enzyme with similar properties was demonstrated to exist in bovine lung extract. C1, a monoclonal antibody previously shown to react with the 60-kDa subunit of bovine brain PDE isozymes (Sharma, R. K., Adachi, A.-M., Adachi, K., and Wang, J. H.) (1984) J. Biol. Chem. 259, 9248-9254), cross-reacted with the lung enzyme. Purification of the lung enzyme by C1 antibody immunoaffinity chromatography rendered the enzyme dependent on exogenous CaM for Ca2+ stimulation. Further purification was achieved by CaM affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed a predominant polypeptide of Mr 58,000 and a minor band of about 50,000. The purified enzyme could be reconstituted into a PDE X CaM complex upon incubation with CaM in the presence of either Ca2+ or EGTA. The reconstituted protein complex did not dissociate in buffers containing 0.1 mM EGTA. Analysis of the purified and reconstituted lung phosphodiesterase by Sephacryl S-300 gel filtration indicated that the lung enzyme is a dimeric protein and that the reconstituted enzyme contained two molecules of calmodulin. Analysis of the reconstituted phosphodiesterase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also showed it to contain equimolar calmodulin and the enzyme subunit. The CaM antagonists, fluphenazine, compound 48/80, and calcineurin at concentrations abolishing CaM stimulation of bovine brain PDE had little effect on the activity of reconstituted bovine lung phosphodiesterase. 相似文献
18.
Evidence for the presence of calmodulin in fish mucus 总被引:5,自引:0,他引:5
Partly purified mucus collected from the skin of three species of fish contains a protein that, on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, comigrates with bovine brain calmodulin and shows the same calcium-dependent shift in electrophoretic mobility as calmodulin. Fish mucus contains a heat-stable activator of cyclic nucleotide phosphodiesterase; activation is concentration dependent and sensitive to the specific calmodulin inhibitor calmidazolium (R 24571). The presence of calmodulin in fish mucus is further indicated by means of a specific radioimmunoassay. A drop in the calcium concentration of the water induces an increase in the immunoassayable calmodulin concentration of mucus, which indicates that the function of calmodulin in mucus is related to control of permeability of the skin epithelium to water and ions. 相似文献
19.
H Nakabayashi H Komada T Yoshida H Takanari K Izutsu 《Biology of the cell / under the auspices of the European Cell Biology Organization》1992,75(1):55-59
Calmodulin was purified from human tonsillar lymphocytes utilizing calcium-dependent binding of calmodulin to fluphenazine-Sepharose. The molecular weight and phosphodiesterase activation of the lymphocyte calmodulin were very similar to those of purified bovine brain calmodulin. Trifluoperazine (TFP), a calmodulin inhibitor, suppressed lymphocyte stimulation as assessed by 3H-thymidine incorporation into DNA of lectin-stimulated lymphocytes. TFP had no effect on the early 45Ca2+ uptake induced by mitogenic lectins, although this latter was inhibited by verapamil which also suppressed the 3H-thymidine incorporation. The results are in keeping with the interpretation that the inhibition of T cell stimulation by TFP was not due to suppression of Ca2+ uptake, but due to inactivation of Ca(2+)-calmodulin complex which might be formed subsequent to Ca2+ entry into the cell. 相似文献
20.
Calmodulin was isolated and purified from shrimp abdominal muscle by heat precipitation, ion exchange and hydrophobic interaction chromatography. The purified calmodulin was homogeneous when evaluated by polyacrylamide gel electrophoresis. A still remaining contaminant was eliminated by high performance liquid chromatography on a phenyl column. The biological and physicochemical properties of shrimp calmodulin such as amino acid composition, molecular weight and the ability to activate calmodulin-deficient bovine heart phosphodiesterase were compared to those of other invertebrate calmodulins. 相似文献