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At least 163 records · Page 9Linked to original sources
PubMed1986

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists.

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.

Animals↗
PubMed1985

Calmodulin in interferon preparations: effect of interferon on calmodulin bioactivity.

Heat-stable calmodulin immunoreactivity and bioactivity were detected in crude preparations of various types of human, murine and chicken interferons (IFNs). Calmodulin containing HuIFN-alpha was retained on a trifluorophenothiazine-Sepharose column. The two activities were separated by serial elutions with 50 microM Ca2+ (HuIFN-alpha) followed by 2 mM EGTA (calmodulin). While maintaining its full antiviral activity, calmodulin free HuIFN-alpha inhibited enhancement of Ca2+-ATPase activity in vitro by authentic purified eukaryote calmodulin. These results indicate that IFNs are calmodulin-binding proteins and that the secretion of both IFNs and calmodulin occurs from IFN-induced cells.

Animals↗
PubMed1987

Calmodulin modulates prolactin secretion in vitro: studies with calmodulin containing liposomes.

The control of prolactin secretion by Ca calmodulin and cyclic AMP was studied. Ca++ ionophore A23187 stimulated both cyclic AMP accumulation and prolactin release by primary culture of anterior pituitary cells in vitro. The increase of cyclic AMP formation by A23187 preceded that of prolactin release. To test the calmodulin involvement in these processes we used either selective calmodulin antagonist, the naphthalene sulphonamide derivative W7, or calmodulin containing liposomes. W7 dose dependently inhibited both basal or A23187 stimulated cyclic AMP accumulation and prolactin secretion. Insertion of Ca calmodulin within the cells stimulated prolactin secretion without modifying cyclic AMP accumulation. W7 inhibited the Ca calmodulin containing liposomes stimulation of prolactin release. These results suggest that calmodulin participates to the process of prolactin release.

Animals↗
PubMed1986

Isolation of the yeast calmodulin gene: calmodulin is an essential protein.

Calmodulin was purified from Saccharomyces cerevisiae based on its characteristic properties. Like other calmodulins, the yeast protein is small, heat-stable, acidic, retained by hydrophobic matrices in a Ca2+-dependent manner, exhibits a pronounced Ca2+-induced shift in electrophoretic mobility, and binds 45Ca2+. Using synthetic oligonucleotide probes designed from the sequences of two tryptic peptides derived from the purified protein, the gene encoding yeast calmodulin was isolated. The gene (designated CMD1) is a unique, single-copy locus, contains no introns, and resides on chromosome II. The amino acid sequence of yeast calmodulin shares 60% identity with other calmodulins. Disruption or deletion of the yeast calmodulin gene results in a recessive-lethal mutation; thus, calmodulin is essential for the growth of yeast cells.

Amino Acid Sequence↗
PubMed2004

Target recognition by calmodulin: the role of acid region contiguous to the calmodulin-binding domain of calcineurin A.

Small-angle X-ray scattering was used to investigate the role of acid region contiguous to the calmodulin-binding domain (391-414) of calcineurin in the target recognition by calmodulin. Three synthetic peptides with the residues 385-414, 380-414 and 374-414 of calcineurin A were used for this aim. The X-ray data are consistent with the fact that calmodulin binds all three peptides with or without Ca2+. Without Ca2+, the whole peptide including acid residues interacts with dumbbell shaped calmodulin, while the acid region is extruded from globular shaped calmodulin with Ca2+. Consequently, a conformation of sequence 374-414 in calcineurin might be changed by Ca2+-signal via calmodulin, suggesting the consequence of this region with acid residues in the full activation mechanism of calcineurin by Ca2+-bound calmodulin.

Acids↗
PubMed1991

Structure-activity relationships of retinoids as inhibitors of calmodulin-dependent human erythrocyte Ca(2+)-ATPase activity and calmodulin binding to membranes.

All-trans retinoic acid displaces the binding of radiolabelled calmodulin to human erythrocyte membranes, and inhibits the activity of plasma membrane Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase; EC 3.6.1.3). This enzyme is dependent upon the action of calmodulin. In this study we explored the structural attributes of the retinoids which confer this ability to inhibit enzyme activity and calmodulin binding. With respect to the fatty acid side-chain, a clear requirement for inhibition is a trans-configuration of the polar end-group. The importance of the ring structure is indicated by the ineffectiveness of polyprenoic acid and a benzene ring retinoid analogue as inhibitors of enzyme activity and calmodulin binding. There was good correlation between the relative potencies of the analogues as enzyme inhibitors and as inhibitors of calmodulin binding. The ability of selected retinoid analogues, at physiological concentrations with respect to all-trans retinoic acid, to inhibit erythrocyte Ca(2+)-ATPase activity and membrane binding of calmodulin underscores the structurally specific effects of these compounds on the interaction of calmodulin with the membrane-bound enzyme.

Calcium-Transporting ATPases↗
PubMed1980

Immunocytochemical localization of calmodulin and a heat-labile calmodulin-binding protein (CaM-BP80) in basal ganglia of mouse brain.

Antisera to calmodulin, a Ca2%-dependent modulator protein, and a heat-labile calmodulin-binding protein have been used to localize these proteins in mouse caudate-putamen. The two proteins appear to be located at identical sites in this brain area. At the light microscopic level, calmodulin and calmodulin-binding protein are found within the cytoplasm and processes of large cells. At the electron microscopic level the proteins are associated with neuronal elements only, primarily at postsynaptic sites within neuronal somata and dendrites. Within the dendrites the immunocytochemical label is associated predominantly with the postsynaptic density and dendritic microtubules. These results are in accord with recent biochemical and immunihistochemical studies of calmodulin in brain and in dividing cells. Thus, calmodulin and the heat-labile calmodulin-binding protein may play a role in the nervous system at the site of neurotransmitter action and at the level of microtubular function.

Animals↗
PubMed1982

Pig skin epidermal calmodulin: effect on calmodulin deficient phosphodiesterase.

Calmodulin, a calcium-dependent modulator protein, is known to mediate a great number of Ca++-dependent processes in various tissues. Although it was originally described as a protein activator of cyclic nucleotide phosphodiesterase, the sensitivity of phosphodiesterases to this compound are suggested to be variable from tissue to tissue. In order to determine whether there was calmodulin-like activity in pig skin epidermis and to see its relationship to epidermal phosphodiesterase, we used an established calmodulin deficient phosphodiesterase system prepared from bovine heart. Calmodulin deficient phosphodiesterase prepared from bovine heart was markedly stimulated by the addition of pig skin (epidermal) boiled extract in the presence of calcium. Boiled skin extract alone had only little phosphodiesterase activity by itself. This effect of boiled skin extract on bovine heart phosphodiesterase was inhibited by the addition of EGTA, a divalent metal ion chelator of relative Ca++ specificity. At a fixed concentration of EGTA, increasing the Ca++ concentration counteracted the effect of EGTA. Pure pig skin epidermis (separated by trypsinization, NaBr, CaCl2-sucrose or NH4Cl treatment) was also shown to have heat-stable calmodulin activity. In contrast to the bovine heart phosphodiesterase, epidermal phosphodiesterase was only partially inhibited when Ca++ was removed by EGTA. The addition of boiled skin extract on the crude extract of epidermal phosphodiesterase had minimal effect on the enzyme activity. Overall results indicate that although pig skin epidermis contains significant amount of calmodulin, the regulation of phosphodiesterase may not be the main biological activity of epidermal calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Calmodulin-sensitive and calmodulin-insensitive components of adenylate cyclase activity in rat striatum have differential responsiveness to guanyl nucleotides.

The interaction between the Ca2+-binding protein, calmodulin, and guanyl nucleotides was investigated in a rat striatal particulate fraction. We found that the ability of calmodulin to stimulate adenylate cyclase in the presence of guanyl nucleotides depends upon the type and concentration of the guanyl nucleotide. Adenylate cyclase activity measured in the presence of calmodulin and GTP reflected additivity at every concentration of these reactants. On the contrary, when the activating guanyl nucleotide was the nonhydrolyzable analog of GTP, guanosine-5'-(beta,gamma-imido)triphosphate (GppNHp), calmodulin could further activate adenylate cyclase only at concentrations less than 0.2 microM GppNHp. Kinetic analysis of adenylate cyclase by GppNHp was compatible with a model of two components of adenylate cyclase activity, with over a 100-fold difference in sensitivity for GppNHp. The component with the higher affinity for GppNHp was competitively stimulated by calmodulin. The additivity between calmodulin and GTP in the striatal particulate fraction suggests that they stimulate different components of cyclase activity. The calmodulin-stimulatable component constituted 60% of the total activity. Our two-component model does not delineate, at this point, whether there are two separate catalytic subunits or one catalytic subunit with two GTP-binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1983

The interaction of dihydropyridine calcium channel blockers with calmodulin and calmodulin inhibitors.

The calmodulin inhibitors R24571 and trifluoperazine were found to inhibit competitively the binding of [3H]nitrendipine to a 48,000 X g particulate fraction of rat brain with IC50 values of 1.0 and 18.8 microM, respectively. Equilibrium dialysis was used to test the ability of the dihydropyridines nitrendipine, felodipine, and nicardipine to inhibit the binding of [3H]chlorpromazine, [14C]pimozide, and 45Ca2+ to calmodulin. At dihydropyridine concentrations near the limit of solubility (10 microM), the only significant effect in these three binding experiments was a 26% inhibition of [14C]pimozide binding to calmodulin by nicardipine, indicating that the dihydropyridines do not bind to the same site on calmodulin as chlorpromazine, pimozide, or calcium. Equilibrium dialysis was also used to determine the ability of the dihydropyridines to interact directly with calmodulin. [3H] Nitrendipine bound to calmodulin in a calcium-dependent manner; however, this binding was of a low-affinity, unsaturable nature. These results suggest that the dihydropyridine drugs do not interact with calmodulin at concentrations that are pharmacologically significant.

Animals↗
PubMed1996

Ca2+-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2+-calmodulin-dependent protein kinase.

The cmd1-6 allele contains three mutations that block Ca2+ binding to calmodulin from Saccharomyces cerevisiae. We find that strains containing cmd1-6 lose viability during cell cycle arrest induced by the mating pheromone alpha-factor. The 50% lethal dose (LD50) of alpha-factor for the calmodulin mutant is almost fivefold below the LD50 for a wild-type strain. The calmodulin mutants are not more sensitive to alpha-factor, as measured by activation of a pheromone-responsive reporter gene. Two observations indicate that activation of the Ca2+-calmodulin-dependent protein phosphatase calcineurin contributes to survival of pheromone-induced arrest. First, deletion of the gene encoding the calcineurin regulatory B subunit, CNB1, from a wild-type strain decreases the LD50 of alpha-factor but has no further effect on a cmd1-6 strain. Second, a dominant constitutive calcineurin mutant partially restores the ability of the cmd1-6 strain to survive exposure to alpha-factor. Activation of the Ca2+-calmodulin-dependent protein kinase (CaMK) also contributes to survival, thus revealing a new function for this enzyme. Deletion of the CMK1 and CMK2 genes, which encode CaMK, decreases the LD50 of pheromone compared with that for a wild-type strain but again has no effect in a cmd1-6 strain. Furthermore, the LD50 of alpha-factor for a mutant in which the calcineurin and CaMK genes have been deleted is the same as that for the calmodulin mutant. Finally, the CaMK and calcineurin pathways appear to be independent since the ability of constitutive calcineurin to rescue a cmd1-6 strain is not blocked by deletion of the CaMK genes.

Base Sequence↗
PubMed1993

Calmodulin and calmodulin-binding proteins during cell fusion in Dictyostelium discoideum: developmental regulation by calcium ions.

The calcium-dependent regulatory protein calmodulin (CaM) mediates diverse cellular functions via a large number of calmodulin-binding and -dependent proteins (CaMBPs). The use of [35S]calmodulin, labeled during its expression (VU-1-CaM) in Escherichia coli, visualized over 25 CaMBPs in Dictyostelium discoideum. Seven, with M(r)s of 155,000, 91,000, 85,000, 48,000, 46,000, 38,000, and 28,000, were present only during sexual development. In addition, intracellular calmodulin levels were low during gamete formation but rose during cell fusion in response to the presence of extracellular calcium. Thus, calmodulin appears to mediate gamete formation and fusion through two distinct mechanisms: first, via unique developmentally regulated CaMBPs, and, second, via the regulation of intracellular calmodulin levels. The identification of the CaMBP spectrin in sexually developing Dictyostelium cells suggests that this cytoskeleton/plasma membrane, crosslinking protein may function during biomembrane fusion in D. discoideum as it does in other organisms.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Novel bovine heart calmodulin-dependent protein kinase which phosphorylates a high molecular weight calmodulin-binding protein.

A novel calmodulin-dependent protein kinase has been isolated from bovine cardiac muscle by successive chromatography on DEAE-Sepharose 6B, Calmodulin-Sepharose 4B affinity and Sepharose 6B chromatography columns. The protein kinase was shown by gel filtration chromatography to have a molecular mass of 36,000 daltons. The highly purified protein kinase stoichiometrically phosphorylated the high molecular weight calmodulin-binding protein from cardiac muscle [Sharma RK (1990) J Biol Chem 265, 1152-1157] in a Ca2+/calmodulin-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the high molecular weight calmodulin-binding protein. Other Ca2+/calmodulin-dependent protein kinases failed to phosphorylate the high molecular weight calmodulin-binding protein. The distinct substrate specificity of this protein kinase indicates that it is not related to the known calmodulin-dependent protein kinases and therefore constitutes a novel protein kinase.

Animals↗
PubMed1986

Further comparison of calmodulin-dependent protein kinase II from brain and calmodulin-dependent glycogen synthase kinase from skeletal muscle.

Calmodulin-dependent protein kinase II was purified from rabbit brain and its properties were compared with those of calmodulin-dependent protein kinase II from rat brain and calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Rabbit brain calmodulin-dependent protein kinase II was clearly distinguished from rabbit skeletal muscle glycogen synthase kinase with respect to size, behavior on autophosphorylation, immunological cross-reactivity and peptide mapping, but was indistinguishable from rat brain calmodulin-dependent protein kinase II in all respects examined. Thus, differences between calmodulin-dependent protein kinase II and glycogen synthase kinase appear not to reflect a species difference but to reflect a tissue difference.

Animals↗
PubMed1988

A monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca2+-calmodulin.

A mouse monoclonal IgG1 antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca2+ (EC50 = 20 microM) for the interaction. Other divalent cations such as Zn2+, Mn2+, Cd2+, Co2+, and Ni2+ will substitute for Ca2+, while Mg2+ and Ba2+ will not. The antibody reacts with both the alpha- and beta-subunits on Western blots in a similar Ca2+-dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of 125I Bolton-Hunter-labeled kinase with unlabeled enzyme. A variety of other proteins including tubulin do not compete for antibody binding. The Mr 30,000 catalytic fragment obtained by proteolysis of either the soluble or the cytoskeletal form of the kinase fails to react with the antibody. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the Kmapp for calmodulin and an increase in the Vmax for the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since the Mr 30,000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca2+-dependent conformational change.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin.

We have recently identified a novel 190-kD calmodulin-binding protein (p190) associated with the actin-based cytoskeleton from mammalian brain (Larson, R. E., D. E. Pitta, and J. A. Ferro. 1988. Braz. J. Med. Biol. Res. 21:213-217; Larson, R. E., F. S. Espindola, and E. M. Espreafico. 1990. J. Neurochem. 54:1288-1294). These studies indicated that p190 is a phosphoprotein substrate for calmodulin-dependent kinase II and has calcium- and calmodulin-stimulated MgATPase activity. We now have biochemical and immunological evidence that this protein is a novel calmodulin-binding myosin whose properties include (a) Ca2+ dependent action activation of its Mg-ATPase activity, which seems to be mediated by Ca2+ binding directly to calmodulin(s) associated with p190 (maximal activation by actin requires the presence of Ca2+ and is further augmented by addition of exogenous calmodulin); (b) ATP-sensitive cross-linking of skeletal muscle F-actin, as demonstrated by the low-speed actin sedimentation assay; and (c) cross-reactivity with mAbs specific for epitopes in the head of brush border myosin I. We also show that p190 has properties distinct from conventional brain myosin II and brush border myosin I, including (a) separation of p190 from brain myosin II by gel filtration on a Sephacryl S-500 column; (b) lack by p190 of K(+)-stimulated EDTA ATPase activity characteristic of most myosins; (c) lack of immunological cross-reactivity of polyclonal antibodies which recognize p190 and brain myosin II, respectively; (d) lack of immunological recognition of p190 by mAbs against an epitope in the tail region of brush border myosin I; and (e) distinctive proteolytic susceptibility to calpain. A survey of rat tissues by immunoblotting indicated that p190 is expressed predominantly in the adult forebrain and cerebellum, and could be detected in embryos 11 d post coitus. Immunocytochemical studies showed p190 to be present in the perikarya and dendritic extensions of Purkinje cells of the cerebellum.

Adenosine Triphosphatases↗
PubMed1992

Calmodulin trapping by calcium-calmodulin-dependent protein kinase.

Multifunctional calcium-calmodulin-dependent protein kinase (CaM kinase) transduces transient elevations in intracellular calcium into changes in the phosphorylation state and activity of target proteins. By fluorescence emission anisotropy, the affinity of CaM kinase for dansylated calmodulin was measured and found to increase 1000 times after autophosphorylation of the threonine at position 286 of the protein. Autophosphorylation markedly slowed the release of bound calcium-calmodulin; the release time increased from less than a second to several hundred seconds. In essence, calmodulin is trapped by autophosphorylation. The shift in affinity does not occur in a site-directed mutant in which threonine at position 286 has been replaced by a non-phosphorylatable amino acid. These experiments demonstrate the existence of a new state in which calmodulin is bound to CaM kinase even though the concentration of calcium is basal. Calmodulin trapping provides for molecular potentiation of calcium transients and may enable detection of their frequency.

Amino Acid Sequence↗
PubMed1984

Heat-stable calmodulin-binding protein in rat testis. Inhibition of calmodulin-stimulated cyclic nucleotide phosphodiesterase activity.

An inhibitor of procine brain calmodulin-dependent cyclic nucleotide phosphodiesterase was purified about 940-fold from rat testis. This inhibitor inhibited the calmodulin-induced activation of the enzyme without affecting its basal activity. The inhibitor activity was counteracted by a high concentration of calmodulin, but was not by a high concentration of Ca2+. The analysis on polyacrylamide disc gel electrophoresis demonstrated that the inhibitor and calmodulin form a complex in the presence of Ca2+ but not in the presence of excess amount of EGTA. This inhibitor also inhibited the calmodulin-induced activation of Ca2+, Mg2+ -ATPase of human erythrocytes. The inhibitor appeared to be a heat-stable protein, since the inhibitor activity was not attenuated by boiling up to 9 min but was completely abolished by tryptic or chymotryptic digestion. The molecular weights of the inhibitor determined by linear polyacrylamide gradient gel electrophoresis under nondenaturing conditions and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 40,000 and 32,000, respectively. Thus, the inhibitor is suggested to be a calmodulin-binding protein composed of a monomer which has unique properties different from those of other tissues.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗