SEARCH · Search PubMed

Results for “Calmodulin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 793 records · Page 44Linked to original sources

PubMed1990

Calcium-calmodulin-dependent phosphorylation of cytoskeletal proteins from adrenal cells.

We have identified a highly active Ca2+ calmodulin-dependent protein kinase in the cytoskeletons of normal (bovine fasciculata) and transformed (Y-1 mouse tumor) adrenal cells. In view of evidence for the involvement of calmodulin and microfilaments in the regulation of cholesterol transport and hence steroidogenesis, it is likely that this kinase is important in this process. The kinase activity was examined for its capacity to phosphorylate endogenous proteins analyzed by one- and two-dimensional gel electrophoresis, in the presence of saturating amounts of Ca2+ (5 mM) and calmodulin (5 microM). Three inhibitors of calmodulin (trifluoperazine, pimozide and W-7) inhibit steroidogenesis and Ca2(+)-calmodulin-dependent phosphorylation kinase activity with similar values for EC50 for the two processes. All three inhibitors inhibit the increased transport of cholesterol to mitochondria in response to ACTH. Two substrates for the kinase (alpha-spectrin and beta-tubulin) were identified and two others (51,000 and 60,000 molecular weight) were tentatively identified as the subunits of the kinase itself in cytoskeletons of both cell types. Calmodulin-binding proteins analyzed by [125I]iodocalmodulin overlay and calmodulin-Sepharose affinity chromatography were also identified in the same cytoskeletons including alpha-spectrin, the Ca2+ calmodulin-dependent phosphatase calcineurin and three that were tentatively identified as the two subunits of the kinase itself and myosin light chain kinase. It is concluded that calmodulin, by binding to the kinase and phosphatase, is capable of influencing the degree of phosphorylation of specific substrates in the cytoskeleton and of forming complexes with spectrin, actin and tubulin. These events may be involved in the regulation of the rate-limiting step of steroidogenesis, i.e. transport of cholesterol to mitochondria.

Adrenal Gland Neoplasms↗

PubMed1996

HMN-709, a chlorobenzenesulfonamide derivative, is a new membrane-permeable calmodulin antagonist.

Our objective is to describe the basic chemical and biological properties of the new calmodulin antagonist HMN-709 (2-[N-(2-aminoethyl)-N-(4-chlorobenzenesulfonyl)]amino-N-(4-flu orocinnamyl)-N-methylbenzylamine). This newly synthesized compound was found to inhibit the Ca2+/calmodulin-dependent activation of calmodulin kinase I, smooth muscle myosin light chain kinase and Ca2+-phosphodiesterase with IC50 values of 1.57+/-0.21, 2.29+/-0.09 and 0.30+/-0.08 microM (mean+/-S.E.), respectively. This compound showed little or no effect on the Ca2+/calmodulin-independent activation of protein kinase A, protein kinase C and basal phosphodiesterase. In addition, HMN-709 inhibited calmodulin kinase I competitively with respect to calmodulin (Ki=0.88 microM) and non-competitively with respect to ATP. Affinity chromatography, with HMN-709-coupled Sepharose HP, showed that the compound bound to calmodulin in a Ca(2+)-dependent manner and did not bind to calmodulin kinase I. These results suggest that HMN-709 antagonizes calmodulin by binding to Ca2+/calmodulin. HMN-709 inhibited collagen-induced platelet aggregation with an IC50 value of 11.80+/-0.86 microM (mean+/-S.E.) without inhibiting phorbol 12,13-dibutyrate-induced aggregation at doses up to 12 microM. HMN-709 appears to be a new, membrane-permeable calmodulin antagonist that may be used for studying the involvement of calmodulin in cellular processes.

Animals↗

PubMed1980

Isolation and characterization of calmodulin from spinach leaves and in vitro translation mixtures.

Calmodulin, a multifunctional calcium-modulated protein, has been isolated from spinach leaf tissue and from spinach leaf messenger RNA translation products. The translation protein and the spinach leaf protein have been partially characterized and compared to vertebrate calmodulins. Spinach leaf calmodulin will quantitatively activate bovine brain phosphodiesterase and will undergo a calcium-dependent shift in electrophoretic mobility similar to that of bovine brain calmodulin. In the presence of Ca(2+) the spinach and brain proteins comigrate, but in the presence of chelators they do not. A polyadenylylated RNA fraction has been isolated from spinach leaf tissue and translated in a wheat germ cell-free translation system. The calmodulin synthesized in vitro has been isolated by using calcium-dependent affinity chromatography on phenothiazine-Sepharose conjugates. The translation protein comigrates with spinach calmodulin during polyacrylamide gel electrophoresis whether in the presence or the absence of Ca(2+). The translation protein also undergoes a calcium-dependent mobility shift identical to that of spinach calmodulin. Amino acid analysis of the translation calmodulin indicates that it does not contain N(epsilon)-trimethyllysine, an amino acid residue that is characteristic of all calmodulins previously examined. These studies suggest that N(epsilon)-trimethyllysine is not required for the calcium-dependent interaction of calmodulin with phenothiazines and indicate the potential utility of phenothiazine-Sepharose conjugates as affinity-based adsorbents in biological and biochemical investigations.

Journal Article↗

PubMed1990

Analysis of the state of posttranslational calmodulin methylation in developing pea plants.

A specific calmodulin-N-methyltransferase was used in a radiometric assay to analyze the degree of methylation of lysine-115 in pea (Pisum sativum) plants. Calmodulin was isolated from dissected segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by incubation with the calmodulin methyltransferase in the presence of [(3)H]methyl-S-adenosylmethionine. By this approach, the presence of unmethylated calmodulins were demonstrated in pea tissues, and the levels of methylation varied depending on the developmental state of the tissue tested. Calmodulin methylation levels were lower in apical root segments of both etiolated and green plants, and in the young lateral roots compared with the mature, differentiated root tissues. The incorporation of methyl groups into these calmodulin samples appears to be specific for position 115 since site-directed mutants of calmodulin with substitutions at this position competitively inhibited methyl group incorporation. The present findings, combined with previous data showing differences in the ability of methylated and unmethylated calmodulins to activate pea NAD kinase (DM Roberts et al. [1986] J Biol Chem 261: 1491-1494) raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity.

Journal Article↗

PubMed1987

Purification and characterization of calmodulin-dependent functional protein, phosphodiesterase, in the lens.

Calmodulin and calmodulin-dependent functional protein play an important role in the maintenance of lens transparency and homeostasis. In the present study, phosphodiesterase, one of the typical calmodulin-dependent functional proteins, was purified from bovine lens by DEAE-cellulose chromatography, calmodulin-Sepharose 4B chromatography and Superose 12 chromatography. Moreover, calmodulin-dependent phosphodiesterase, and independent phosphodiesterase were separated from crude lens extract using DEAE-cellulose column. The calmodulin-dependent phosphodiesterase was purified 4500-fold with a 0.7% yield; it was a dimer formed with two single polypeptides of 59K as the molecular weight. The enzyme had a higher affinity for cyclic GMP than for cyclic AMP, and functioned at calcium ion concentration above 10(-6) M in the incubation mixture. W-7 as calmodulin antagonist indirectly inhibited the enzyme activity and nifedipine as calmodulin-dependent phosphodiesterase antagonist directly inhibited the enzyme activity. These results suggest that an appearance of calmodulin-dependent phosphodiesterase activity depends on the interrelation between the calcium ion and calmodulin in the lens.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1990

Nuclear localization of 68 kDa calmodulin-binding protein is associated with the onset of DNA replication.

In Chinese hamster embryo fibroblast cells, an increase in intracellular calmodulin levels coincided with the nuclear localization of a calmodulin-binding protein of about 68 kDa as the cells progressed from G1 to S phase. When cells were limited from entering into S phase, by omitting insulin a defined medium, intracellular CaM levels did not increase and the 68 kDa calmodulin-binding protein was completely absent from the nuclei. Corresponding to the nuclear localization of calmodulin and the 68 kDa calmodulin-binding protein in S phase cells, there was a dramatic increase in DNA polymerase and thymidine kinase activities in the nuclei of S phase cells as compared to G1 phase cells. In addition, the 68 kDa calmodulin-binding protein, along with calmodulin, is observed to be an integral component of replitase complex responsible for nuclear DNA replication in S phase cells. These observations point to the association of calmodulin and calmodulin-binding protein(s) with the replication machinery responsible for nuclear DNA replication during S phase. A possible regulatory role of these proteins in the onset of DNA replication and cell proliferation is discussed.

Animals↗

PubMed1999

Processing of the sperm protein Sp17 during the acrosome reaction and characterization as a calmodulin binding protein.

In this study we have demonstrated that the native rabbit sperm protein, Sp17, is a 22- to 24-kDa triplet of proteins in washed ejaculated rabbit spermatozoa and is unaffected by capacitation. However, during the acrosome reaction, Sp17 is processed from a 22- to 24-kDa triplet of proteins to a triplet of proteins at 17-19 kDa by the removal of amino acids from the C-terminal. Recombinant rabbit Sp17 (rRSp17) can also be proteolytically processed by acrosome-reacted spermatozoa in a similar manner. Protease inhibitors prevent the proteolytic processing of Sp17. Both forms of native Sp17 remain associated with acrosome-reacted spermatozoa and are solubilized by ionic detergents. Previously, sequence analysis of Sp17 revealed that Sp17 amino acids 108-137 were 52% identical to the calmodulin binding domain of neuromodulin and contained an IQ motif found in other calmodulin binding proteins. In this study, a truncated recombinant Sp17, rRSp17CB, which lacks amino acids 118-146, including the potential calmodulin binding site, was made. Recombinant rabbit Sp17, but not rRSp17CB, binds to calmodulin in the presence of Ca2+ or EDTA, under reduced or nonreduced conditions in biotinylated-calmodulin overlay assays. In DSS crosslinker experiments, calmodulin bound to rRSp17 in a 1:1 ratio but not to rRSp17CB. Additionally, biotinylated rRSp17 interacts with native sperm calmodulin. We propose that the processing of native Sp17, by removing a C-terminal fragment during the acrosome reaction, might be a mechanism to regulate the calmodulin binding activity of Sp17 and provide calmodulin at specific sites after the acrosome reaction.

Acrosome Reaction↗

PubMed1988

Interaction of alpha-N-Acetyl-beta-endorphin and calmodulin.

Acetylation at the alpha-amino terminal is a common post-translational modification of many peptides and proteins. In the case of the potent opiate peptide beta-endorphin, alpha-N-acetylation is a known physiological modification that abolishes opiate activity. Since there are no known receptors for alpha-N-acetyl-beta-endorphin, we have studied the association of this peptide with calmodulin, a calcium-dependent protein that binds a variety of peptides, phenothiazines, and enzymes, as a model system for studying acetylated endorphin-protein interactions. Association of the acetylated peptide with calmodulin was demonstrated by cross-linking with bis(sulfosuccinimidyl)suberate; like beta-endorphin, adducts containing 1 mol and 2 mol of acetylated peptide per mole calmodulin were formed. Some of the bound peptides are evidently in relatively close proximity to each other since, in the presence of amidated (i.e., lysine-blocked) calmodulin, cross-linking yielded peptide dimers. The acetylated peptide exhibited no appreciable helicity in aqueous solution, but in trifluoroethanol (TFE) considerable helicity was formed. Also, a mixture of acetylated peptide and calmodulin was characterized by a circular dichroic spectrum indicative of induced helicity. Empirical prediction rules, applied earlier to beta-endorphin, suggest that residues 14-24 exhibit alpha-helix potential. This segment has the potential of forming an amphipathic helix; this structural unit is believed to be important in calmodulin binding. The acetylated peptide was capable of inhibiting the calmodulin-mediated stimulation of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity with an effective dose for 50% inhibition of about 3 microM; this inhibitory effect was demonstrated using both an enzyme-enriched preparation as well as highly purified enzyme. Thus, acetylation at the alpha-amino terminal of beta-endorphin, although abolishing opiate activity, does not interfere with the binding to calmodulin. Indeed, beta-endorphin and the alpha-N-acetylated peptide behave very similarly with respect to calmodulin association.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1982

Calmodulin binding to platelet plasma membranes.

Calmodulin copurifies with platelet plasma membranes isolated by glycerol-induced lysis and density gradient centrifugation. These membranes also bind 125I-labeled calmodulin in vitro in the presence of Ca2+. Binding is largely reduced by replacing Ca2+ by Mg2+ or by addition of an excess unlabeled calmodulin. The specific component of binding is saturable, with an apparent Kd of 27 nM and a maximum of 15.9 pmol binding sites per mg of membrane protein. This is equivalent to approx. 4100 binding sites per platelet. Binding was inhibited by addition of phenothiazines, a group of calmodulin antagonists. Half-maximal inhibition was attained with approx. 20 microM trifluoperazine or 50 microM chlorpromazine. In contrast, chlorpromazine-sulfoxide which is inactive towards calmodulin, did not affect the binding. Calmodulin binding polypeptides of the plasma membrane were identified by a gel-overlay technique. A major calmodulin-binding component of molecular weight 149000 was detected. Binding to this band was Ca2+-dependent and inhibited by chlorpromazine. The molecular weight of this polypeptide is similar to that of glycoprotein I and also that of the red cell (Ca2+ + Mg2+)-stimulated ATPase, which is known to bind calmodulin. The possible role of calmodulin in platelet activation is analysed.

Blood Platelets↗

PubMedDate not supplied

Yeast calmodulin: structural and functional elements essential for the cell cycle.

The budding yeast Saccharomyces cerevisiae is a suitable organism for studying calmodulin function in cell proliferation. Genetic studies in yeast demonstrate that vertebrate calmodulin can functionally replace yeast calmodulin. In addition, expression of half of the yeast calmodulin molecule is found to be sufficient for cell growth. Characterization of conditional-lethal mutants of yeast calmodulin as well as the intracellular distribution of calmodulin have suggested that at least two cell cycle steps require calmodulin function. One is nuclear division and the other is the maintenance of cell polarity. A current focus is to understand which kinds of target proteins are involved in mediating the essential functions of yeast calmodulin in these processes. Thus far, three yeast enzymes whose activity is regulated by calmodulin have been identified.

Calmodulin↗

PubMed1991

Variant surface glycoprotein from Trypanosoma brucei clone YTat 1.1 contains a latent calmodulin-binding domain.

Calmodulin affinity chromatography and chromatofocusing were used to purify calmodulin-binding proteins of 32-40-kDa from homogenates of Trypanosoma brucei clone YTat1.1. The trypanosome proteins associated with calmodulins from different sources and reversibly inhibited calmodulin-dependent bovine brain phosphodiesterase. Purified 32-kDa protein bound to calmodulin with an approximate Kd of 1.3 nM. Polyclonal antibodies directed against purified 32-kDa protein and monoclonal antibody ECA6 recognized each of the 32-40-kDa proteins. Immunoprecipitation with biotinylated monoclonal antibody ECA6 (Bio-ECA6) or biotinylated calmodulin (Bio-CaM) identified the 32-40-kDa proteins in phenylmethylsulfonyl fluoride-treated lysates of slender forms of YTat1.1, but not procyclic forms of YTat1.1 or slender forms of EATRO110. In the presence of leupeptin, lysates of slender YTat1.1 contained a single protein of 58 kDa that immunoprecipitated with Bio-ECA6. The 58-kDa protein was exposed to the extracellular space as demonstrated by immunolocalization and sensitivity to pronase treatment in intact cells. The protein was identified as variant surface glycoprotein (VSG) based upon immunolocalization, pattern of expression and cross-reactivity of ECA6 with authentic VSG. The amino-terminal 17 residues of 32-kDa protein were identical with the amino-terminus of YTat1.1 VSG. Putative calmodulin-binding domains were identified in other VSGs by computer modeling. The model was tested with CNBr fragments of VSG 117. The fragments reversibly inhibited calmodulin-dependent activation of phosphodiesterase with approximate Kd of 11 nM. We conclude that endogenously generated proteolytic fragments of VSG from clone YTat1.1, and CNBr fragments of VSG 117 bind with high affinity to calmodulin.

Amino Acid Sequence↗

PubMed1992

Interaction of calmodulin with phospholamban and caldesmon: comparative studies by 1H-NMR spectroscopy.

In order to identify comparative aspects of the interaction of calmodulin with its target proteins, proton magnetic-resonance studies of complex formation between calmodulin and defined segments of phospholamban and caldesmon have been undertaken. Residues 3-15 in the cytoplasmic region of phospholamban, an integral membrane protein of cardiac sarcoplasmic reticulum believed to regulate the calcium pumping ATPase, are shown to contribute to interaction with calmodulin. Using wheat germ calmodulin specifically modified with a spin-label to provide the spectral means for spatial localisation, these residues of phospholamban were correlated with binding in the vicinity of the probe attached to Cys-27 in the N-terminal domain of calmodulin. This interaction, relevant to the mechanism of calmodulin-dependent phosphorylation of phospholamban that relieves its inhibitory influence on the calcium pump, provides a useful model system for comparative study of the properties of calmodulin-binding domains. We contrast here a calmodulin-binding segment in the C-terminal region of caldesmon localised by 1H-NMR study of the interface(s) between the two proteins. These observations are discussed in the context of other calmodulin-binding sequences.

Amino Acid Sequence↗

PubMed1990

Caldesmon, calmodulin and tropomyosin interactions.

Binary complex interactions between caldesmon and tropomyosin, and calmodulin and tropomyosin, and ternary complex interaction involving the three proteins were studied using viscosity, electron microscopy, fluorescence and affinity chromatography techniques. In 10 mM NaCl, caldesmon decreased the viscosity of chicken gizzard tropomyosin by 7-8 fold with a concomitant increase in turbidity (A330nm). Electron micrographs showed spindle-shaped particles in the tropomyosin-caldesmon samples. These results suggest side-by-side aggregation of tropomyosin polymers induced by caldesmon. Binding studies in 10 mM NaCl between caldesmon and chicken gizzard tropomyosin labelled with the fluorescent probe N-(1-anilinonaphthyl-4)maleimide (ANM) gave association constants from 5.3.10(6) to 7.9.10(6) M-1 and stoichiometry from 1.0 to 1.4 tropomyosin per caldesmon. Similar binding was observed for rabbit cardiac tropomyosin and caldesmon. Removal of 18 and 11 residues from the COOH ends of the gizzard and cardiac tropomyosin by carboxypeptidase A, respectively, had no significant effect on their binding to caldesmon. In the presence of Ca2+, chicken gizzard tropomyosin bound to a calmodulin-Sepharose-4B column and was eluted with a salt concentration of 140 mM. This interaction was weakened in the absence of Ca2+, and the bound tropomyosin was eluted by 65 mM KCl. ANM-labelled tropomyosin bound calmodulin in the presence of Ca2+ with a binding constant of 3.5.10(6) M-1 and a binding stoichiometry of 1 to 1.4 tropomyosin per calmodulin. In 10 mM NaCl, calmodulin reduced the specific viscosity of chicken gizzard tropomyosin in the presence of Ca2+ by 5 fold, while a 1.5-fold reduction in viscosity was observed in the absence of Ca2+. In either case, no significant increase in turbidity was observed suggesting that calmodulin reduced head-to-tail polymerization of tropomyosin. The interaction of caldesmon with the calmodulin-ANM-tropomyosin complex in the presence and absence of Ca2+ was also examined. The result is consistent with a model that in the absence of Ca2+, calmodulin binds weakly to either caldesmon or tropomyosin and has little effect on the tropomyosin-caldesmon interaction; whereas, Ca2(+)-calmodulin interacts with caldesmon and reduces its affinity to tropomyosin.

Amino Acid Sequence↗

PubMed1989

Calmodulin-binding proteins are developmentally regulated in gametes and embryos of fucoid algae.

Calcium-binding proteins and calmodulin-binding proteins were identified in gametes and zygotes of the marine brown algae Fucus vesiculosus, Fucus distichus, and Pelvetia fastigiata using gel (SDS-PAGE) overlay techniques. A calcium current appears to be important during cell polarization in fucoid zygotes (K.R. Robinson and L.F. Jaffe, 1975, Science 187, 70-72; K.R. Robinson and R. Cone, 1980, Science 207, 77-78), but there are no biochemical data on calcium-binding proteins in these algae. By using a sensitive 45Ca2+ overlay method designed to detect high-affinity calcium-binding proteins, at least 9-11 polypeptides were detected in extracts of fucoid gametes and zygotes. All samples had calcium-binding proteins with apparent molecular weights of about 17 and 30 kDa. A 17-kDa calcium-binding protein was purified by calcium-dependent hydrophobic chromatography and was identified as calmodulin by immunological and enzyme activator criteria. A 125I-calmodulin overlay assay was used to identify potential targets of calmodulin action. Sperm contained one major calmodulin-binding protein of about 45 kDa. Eggs lacked major calmodulin-binding activity. A 72-kDa calmodulin-binding protein was prominent in zygotes from 1-65 hr postfertilization. Both calmodulin-binding proteins showed calcium-dependent binding activity. Overall, the data suggest that the appearance and distribution of certain calcium-binding and calmodulin-binding proteins are under developmental regulation, and may reflect the different roles of calcium during fertilization and early embryogenesis.

Autoradiography↗

PubMed2003

A model for the activation of plasma membrane calcium pump isoform 4b by calmodulin.

Overexpression of the plasma membrane calcium pump (PMCA) isoform 4b by means of the baculovirus system enabled us, for the first time, to study the kinetics of calmodulin binding to this pump. This was done by stopped-flow fluorescence measurements using 2-chloro-(amino-Lys(75))-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin (TA-calmodulin). Upon mixing with PMCA, the fluorescence of TA-calmodulin changed along a biphasic curve: a rapid and small increase in fluorescence was followed by a slow and large decrease that lasted about 100 s. The experiment was done at several PMCA concentrations. Global fitting nonlinear regression analysis of these results led to a model in which PMCA is present in two forms: a closed conformation and an open conformation. Calmodulin reacts with both conformations but reacts faster and with higher affinity for the open conformation. Measurements of the ATPase activity of PMCA under similar conditions revealed that the open form has higher ATPase activity than the closed one. Contrasting with the reaction with the whole pump, TA-calmodulin reacted rapidly (in about 2 s) with a calmodulin-binding peptide made after the sequence of the calmodulin-binding domain of PMCA (C28). Results of TA-calmodulin binding to C28 are explained by a simpler model, in which only an open conformation exists.

Amino Acid Sequence↗

PubMed1997

Tryptophan residues in caldesmon are major determinants for calmodulin binding.

Calmodulin has been shown to interact with the COOH-terminal domain of gizzard h-caldesmon at three sites, A (residues 658-666), B (residues 687-695), and B' (residues 717-725), each of which contains a Trp residue [Zhan et al. (1991) J. Biol. Chem. 266, 21810-21814; Marston et al. (1994) J. Biol. Chem. 296, 8134-8139; Mezgueldi et al. (1994) J. Biol. Chem. 269, 12824-12832]. To determine the contribution of each of the three Trp residues in the calmodulin-caldesmon interaction, we have mutated the Trp residues to Ala in the COOH-terminal domain of fibroblast caldesmon (CaD39) and studied the effects on calmodulin binding by fluorescence measurements and using immobilized calmodulin. Wild-type CaD39 binds with a Kd of 0.13 x 10(-6) M and a stoichiometry of 1 mol of calmodulin per mol of caldesmon. Replacing Trp 659 at site A or Trp 692 at site B to Ala reduces binding by 22- and 31-fold (Kd = 2.9 x 10(-6) and 4.0 x 10(-6) M), respectively, and destabilizes the CaD39-calmodulin complex by 1.75 and 1.94 kcal mol-1, respectively. Mutation of both Trp 659 and Trp 692 to Ala further reduces binding with a Kd of 6.1 x 10(-6) M and destabilizes the complex by 2.17 kcal mol-1. On the other hand, mutation of Trp 722 at site B' to Ala causes a much smaller decrease in affinity (Kd = 0.6 x 10(-6) M) and results in a destabilization energy of 0.87 kcal mol-1. To investigate the relative importance of the amino acid residues near each Trp residue in the caldesmon-calmodulin interaction, deletion mutants were constructed lacking site A, site B, and site A + B. Although deletion of site A decreases binding of CaD39 to calmodulin by 13-fold (Kd = 1.7 x 10(-6) M), it results in tighter binding than mutation of Trp 659 to Ala at this site, suggesting that the residues neighboring Trp 659 may contribute negatively to the interaction. Deletion of site B causes a similar reduction in binding (Kd = 4.1 x 10(-6) M) as observed for replacing Trp 692 to Ala at this site, indicating that Trp 692 is the major, if not the only, binding determinant at site B. Deletion of both site A and site B drastically reduces binding by 62-fold. Taken together, these results suggest that Trp 659 and Trp 692 are the major determinants in the caldesmon-calmodulin interaction and that Trp 722 in site B' plays a minor role.

Amino Acid Sequence↗

PubMed1980

The binding of calmodulin to myelin basic protein and histone H2B.

1. A calmodulin-binding protein of apparent mol.wt. 19 000 has been purified from chicken gizzard. Similar proteins have been isolated from bovine uterus, rabbit skeletal muscle and rabbit liver. 2. These proteins migrated as an equimolar complex with bovine brain calmodulin on electroporesis on polyacrylamide gels in the presence of Ca2+ and 6M-urea. The complex was dissociated in the presence of EGTA. 2. The chicken gizzard calmodulin-binding protein has been shown to be identical with chicken erythrocyte histone H2B on the basis of partial amino acid sequence determination. 4. The calmodulin-binding proteins of apparent mol.wt. 22 000 isolated previously from bovine brain [Grand & Perry (1979) Biochem. J. 183, 285-295] has been shown, on the basis of partial amino-acid-sequence determination, to be identical with myelin basic protein. 5. The activation of bovine brain phosphodiesterase by calmodulin is inhibited by excess bovine uterus calmodulin-binding protein (histone H2B). 6. The phosphorylation of myelin basic protein by phosphorylase kinase is partially inhibited, whereas the phosphorylation of uterus calmodulin-binding protein (histone H2B) is unaffected by calmodulin or troponin C. 7. The subcellular distribution of myelin basic protein and calmodulin suggests that the two proteins do not exist as a complex in vivo.

Amino Acid Sequence↗

PubMed1989

Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments.

We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).

Actins↗