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At least 19 recordsLinked to original sources

PubMed1998

Inhibition of calmodulin-activated smooth-muscle myosin light-chain kinase by calmodulin-binding peptides and fluorescent (phosphodiesterase-activating) calmodulin derivatives.

Aspects of the biochemistry of calmodulin have been addressed that bear on its cell biological role as a mediator of Ca2+ regulation. Calmodulin-binding peptides derived from the amino acid sequence of smooth-muscle myosin light-chain kinase (MLCK) were characterized as inhibitors of calmodulin activation of MLCK-catalyzed phosphorylation of the smooth-muscle regulatory light chain (MLC). MLCK activity was determined by measuring the rate of formation of one of the reaction products, ADP, in a coupled enzymatic assay by continuous fluorimetric monitoring of NADH removal in 100 microM CaCl2 at ionic strength 0.15 M, pH 7.0 and 21 degreesC. The Km value of calmodulin was 3.5 nM, a value 16-35-fold greater than the Kd value of calmodulin for MLCK [Török, K., and Trentham D. R. (1994) Biochemistry 33, 12807-12820]. The different Km and Kd values are most likely associated with the rate-limiting step in MLC phosphorylation being associated with product release from MLCK. The values of the inhibition constants, Ki, were the following: Ac-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-CONH2 (Trp peptide), 8.6 (+/-1. 4 sd) pM; Y4-analogue of Trp peptide (Tyr peptide), 7.3 (+/-0.1) nM; and A-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-S-S (RS20-like peptide), 0. 11-0.39 nM. The Ki values were consistent with kinetically determined Kd values of the peptides to calmodulin. Kinetic determination of Kd values required the use of a fluorescently labeled calmodulin, 2-chloro-(epsilon-amino-Lys75)-[6-(4-N, N-diethylamino-phenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-calmodulin).1 Since, as here, Lys75 is a convenient labeling site on calmodulin for the introduction of fluorescent probes, the biological activity of the Lys-modified calmodulins was evaluated. TA-calmodulin and calmodulin selectively modified by 1-N, N-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-C1) at Lys75 (dansyl-calmodulin) were characterized as activators of cyclic AMP phosphodiesterase (PDE) and inhibitors of MLCK. The Km value for dansyl-calmodulin was equal to that of calmodulin, and that of TA-calmodulin was 3.5-fold greater. TA-calmodulin and Lys75-labeled dansyl-calmodulin thus distinguish between PDE and MLCK being agonists to the former and antagonists to the latter.

Amino Acid Sequence↗

PubMed1992

Modulation of calmodulin levels, calmodulin methylation, and calmodulin binding proteins during carrot cell growth and embryogenesis.

Carrot cell cultures were used to study the dynamics of calmodulin protein levels, calmodulin methylation, and calmodulin-binding proteins during plant growth and development. Comparisons of proliferating and nonproliferating wild carrot cells show that, while calmodulin protein levels does not vary significantly, substantial variation in post-translational methylation of calmodulin on lysine-115 is observed. Calmodulin methylation is low during the lag and early exponential stages, but increases substantially as exponential growth proceeds and becomes maximal in the postexponential phase. Unmethylated calmodulin quickly reappears within 12 h of reinoculation of cells into fresh media, suggesting that the process is regulated according to the cell growth state. Calmodulin and calmodulin-binding proteins were also analyzed during the formation and germination of domestic carrot embryos in culture. Neither calmodulin methylation nor calmodulin protein levels varied significantly during somatic embryogenesis. However, upon germination of embryos, the level of calmodulin protein doubled. By calmodulin overlay analysis, we have detected a major 54,000 M(r) calmodulin-binding protein that also increased during embryo germination. This protein was purified from carrot embryo extracts by calmodulin-Sepharose chromatography. Overall, the data suggest that calmodulin methylation is regulated depending upon the state of cell growth and that calmodulin and its target proteins are modulated during early plant development.

Calmodulin↗

PubMed1984

Calmodulin X (Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump.

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM X (Ca2+)4 is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E X CaM X (Ca2+)4 complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01-1 microM) was 0.9 nM; the respective apparent dissociation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1-50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by 125I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM X (Ca2+)4. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E X CaM) is at least 100-fold greater than the apparent dissociation constant of the E X CaM X (Ca2+)4 complex, as judged from non-saturation 125I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.

Animals↗

PubMed1986

The insulin receptor and calmodulin. Calmodulin enhances insulin-mediated receptor kinase activity and insulin stimulates phosphorylation of calmodulin.

Despite intensive research efforts, the functional role and regulation of the insulin receptor kinase remain enigmatic. In this investigation, we demonstrate that calmodulin enhances insulin-stimulated phosphorylation of the beta subunit of the insulin receptor and histone H2b and that insulin also stimulates phosphorylation of calmodulin. Using wheat germ lectin-enriched insulin receptor preparations obtained from rat adipocyte plasma membranes, calmodulin stimulated the rate and increased the amount of 32P incorporated predominantly into tyrosine residues of the beta subunit of the receptor when assayed in the presence of insulin. The stimulatory effect of calmodulin was both dose-dependent and saturable with half-maximal and maximal phosphorylation of the beta subunit occurring at 0.4 and 2.0 microM calmodulin, respectively. Ca2+ enhanced the ability of calmodulin to stimulate insulin-mediated phosphorylation of the beta subunit with an apparent K0.5 of approximately 0.6 microM. Calmodulin also induced an approximately 2-fold increase in both the rate and amount of insulin-mediated incorporation of 32P into histone H2b. The stimulatory effect of calmodulin was only observed in the presence of insulin and was concentration-dependent (K0.5 approximately 3.0 microM calmodulin), saturable (at 5 microM calmodulin), and Ca2+-dependent (K0.5 = 0.2 microM free Ca2+). Insulin also induced phosphorylation of a 17-kDa protein. On the basis of its molecular weight and purification via immunoadsorption with protein A-Sepharose-bound anti-calmodulin IgG, this phosphoprotein was identified as a phosphorylated form of calmodulin. Phosphorylation of calmodulin was only observed in the presence of insulin and was both Ca2+- and insulin concentration-dependent with half-maximal effects observed at 0.1 microM free Ca2+ and 350 microunits/ml insulin. Collectively, these results support the hypothesis that Ca2+ and calmodulin participate in the molecular mechanism whereby binding of insulin to its receptor is coupled to changes in cellular metabolism.

Adipose Tissue↗

PubMed1983

Correlation between calmodulin-dependent increase in the rate of calcium transport and calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum. Characterization of calmodulin-dependent phosphorylation.

The aim of the present study was to prove a correlation between the calmodulin-dependent increase in the rate of calcium transport by dog cardiac sarcoplasmic reticulum and calmodulin-dependent phosphorylation. The dependence of phosphorylation on the total calmodulin concentration at 75 microM and 1 microM free calcium gave apparent calmodulin half-saturation constants Km (CaM) of 9.4 nM and 181 nM, respectively, whilst the apparent Km (CaM) for the rate of calmodulin-stimulated calcium transport carried out at 1 microM calcium, but phosphorylated prior to the calcium uptake at 75 microM or 1 microM calcium, were 12.5 nM and 127 nM, respectively. A positive correlation was obtained between calmodulin-dependent increase in the rate of calcium transport and hydroxylamine-insensitive phosphoester formed by the calcium/calmodulin-regulated, membrane-bound protein kinase. More than 90% of incorporated [32P]phosphate is confined to a 26-28-kDa or 9-11-kDa protein as determined by polyacrylamide gel electrophoresis following solubilization in sodium dodecyl sulfate at 37 degrees C and at 100 degrees C, respectively, similar to the results obtained by phosphorylation with cAMP-dependent protein kinase. The data indicate that calmodulin-dependent phosphorylation of the above protein(s) is causally related to the stimulation of the rate of calcium transport by cardiac sarcoplasmic reticulum, which is at least partially due to a shift in the calcium dependence of the rate of calcium transport to lower free calcium concentrations, K(Ca), of 1.25 microM and 0.61 microM in controls and calmodulin-dependent phosphorylation, respectively. Activation of calmodulin-dependent phosphorylation by free calcium at total calmodulin concentrations of 300 nM, 100 nM and 30 nM gave apparent K(Ca) values of 0.83 microM, 1.44 microM and 2.3 microM and Hill coefficients of 4.13, 3.76 and 3.79, respectively, indicating that all four calcium binding sites of calmodulin have to be saturated to obtain activation of the calcium/calmodulin-regulated protein kinase. The calmodulin-dependent modulation of calcium transport in vivo is, therefore, determined to great extent by the total calmodulin concentration present in the sarcoplasm.

Animals↗

PubMed1998

Modulation of calmodulin function by ubiquitin-calmodulin ligase and identification of the responsible ubiquitylation site in vertebrate calmodulin.

Calmodulin is the universal calcium modulator in eukaryotic cells. Its biological activity is closely regulated by the second messenger Ca2+. Previous studies in cell-free extracts [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191] have shown that calmodulin is reversibly ubiquitylated by ubiquityl-calmodulin synthetase (ubiquitin-calmodulin ligase, EC 6.3.2.21) in the presence of Ca2+ without being channeled to degradation by the 26S proteasome. As shown here monoubiquitylation strongly decreases the biological activity of calmodulin towards phosphorylase kinase by reducing its affinity approximately threefold and the maximal degree of activation approximately twofold. Thus, a structural clarification of the ubiquitylation site on calmodulin has become crucial for advancing our knowledge in this field on a molecular level. As demonstrated by sequence analysis and mass spectrometry of conjugates, the ubiquitylation site is located in the first Ca2+-binding loop of calmodulin and has the octapeptide structure -L-F-D-K21-D-G-D-G- with Lys21 being the ubiquitylated residue in vertebrate and other calmodulins. This catalytic recognition sequence is, however, not the only structural requirement for calmodulin ubiquitylation by ubiquityl-calmodulin synthetase. Removal of the 41 C-terminal amino acids (fourth Ca2+-binding loop) separated by several nanometers from Lys21 drastically decreases the affinity and reactivity of the synthetase for calmodulin, indicating a more extensive structural requirement for the substrate binding site i.e. binding recognition. This allows the enzyme to discriminate in a site-specific manner between two nearly identical catalytic recognition sites in vertebrate calmodulin of which the second site -V-F-D-K94-D-G-N-G- in the third Ca2+-binding loop is apparently not ubiquitylated by the synthetase.

Amino Acid Sequence↗

PubMed1997

Circular dichroism and 1H NMR studies on the structures of peptides derived from the calmodulin-binding domains of inducible and endothelial nitric-oxide synthase in solution and in complex with calmodulin. Nascent alpha-helical structures are stabilized by calmodulin both in the presence and absence of Ca2+.

There exist two types of nitric-oxide synthase (NOS); constitutive isozymes that are activated by binding calmodulin in response to elevated Ca2+ and an inducible isozyme that binds calmodulin regardless of Ca2+. To study the structural basis of the difference in Ca2+ sensitivity, we have designed synthetic peptides of minimal lengths derived from the calmodulin-binding domain of endothelial NOS (eNOS) and that of macrophage NOS (iNOS). A peptide, KRREIPLKVLVKAVLFACMLMRK, derived from human iNOS sequence, retained the ability to bind to calmodulin both in the presence and absence of Ca2+, while a peptide derived from human eNOS sequence, RKKTFKEVANAVKISASLMG, bound to calmodulin only in the presence of Ca2+. Circular dichroism and two-dimensional 1H nuclear magnetic resonance studies suggested that both peptides assume nascent alpha-helical structures in aqueous solution. When mixed with calmodulin, both peptides showed circular dichroism spectra characteristic for alpha-helix. In contrast to other target proteins, the addition of iNOS peptide to calmodulin did not affect the Ca2+ binding of calmodulin appreciably. The peptide derived from the calmodulin-binding domain of iNOS, therefore, binds in alpha-helical structures both to Ca2+-calmodulin and apo-calmodulin, which is unique among various target proteins of calmodulin.

Amino Acid Sequence↗

PubMed1986

Calcium release from calmodulin and its C-terminal or N-terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped-flow fluorescence with Quin 2 and intrinsic tyrosine. Inhibition of calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum.

Calcium dissociation from the C-terminal and N-terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped-flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA. Calcium dissociation from the C-terminal half of calmodulin, which contains only the two high-affinity calcium-binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s-1 and 12.8 s-1, respectively, in the C-terminal half and 10.5 s-1 and 10.8 s-1, respectively, in intact calmodulin (pH 7.0, 25 degrees C, 100 mM KCl). Alkylation of the C-terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s-1 and 0.73 s-1 respectively; tyrosine: kobs 1.65 s-1 and 0.61 s-1 respectively). Alkylation of intact calmodulin resulted in a four-phase calcium dissociation measured with Quin 2 (kobs 85.3 s-1, 11.1 s-1, 1.92 s-1 and 0.59 s-1); the latter two phases are assumed to represent calcium release from high-affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s-1 and 0.53 s-1 respectively) and the rate parameters determined in the C-terminal half. Evidently perturbation of the calcium-binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high-affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N-terminal half resulted in biphasic calcium release with rates (kobs 153 s-1 and 10.9 s-1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin-melittin or fragment-melittin complexes, measured with Quin 2, were slower and monophasic in the C-terminal half (kobs 1.12 s-1), biphasic in the N-terminal half (kobs 140 s-1 and 26.8 s-1 respectively) and triphasic in intact calmodulin (kobs 126 s-1, 12.1 s-1 and 1.38 s-1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low-affinity sites mainly due to a reduced calcium 'off rate', presumably because of conformation restrictions.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoquinolines↗

PubMed1983

Antigenic structure of calmodulin: production and characterization of antisera specific for plant calmodulins or Ca2+-replete vs. Ca2+-free calmodulins.

Calmodulin was purified from bovine testis and soybean seed, using affinity chromatography on (6-amino-hexyl)-5-chloro-1-naphthylene sulfonamide (W7)-Sepharose. The simple preparation of this affinity medium is presented. Although bovine and soybean calmodulins share affinity for W7-Sepharose, share biological activity, and have similar amino acid compositions, antisera raised against soybean calmodulin recognize animal calmodulins with 20,000-fold less avidity than they do plant calmodulins. We have also raised antisera which recognize only Ca2+-calmodulin; they are incapable of recognizing the Ca2+-free conformation of calmodulin. Two classes of such antisera were obtained. One class exhibited half-maximal binding of [125I]-calmodulin with 0.5 microM Ca2+ while the other required 2.5 microM ionic Ca2+ for the same degree of antigen binding. These studies together indicate that plant calmodulins possess an epitope not found on animal calmodulins, and that the conformational alterations calmodulin undergoes upon binding Ca2+ can be exploited to raise antisera against those conformations.

Amino Acids↗

PubMed1992

Effects of calmodulin antagonists on antibody binding to calmodulin. Distinct conformers of calmodulin induced by the binding of drugs.

An indirect enzyme-linked immunosorbent assay has been used to study the interactions between calmodulin and two calmodulin antagonists, trifluoperazine and a neuropeptide isolated from the hypothalamus. The binding of a monospecific anti-calmodulin antibody, raised in rabbit against dinitrophenylated calmodulin, to calmodulin was tested at various concentrations of these drugs under equilibrium conditions. Trifluoperazine at low concentrations stimulated, but at relatively high concentrations inhibited, immunocomplex formation. The neuropeptide displaced the antibody from calmodulin at nanomolar concentrations. Enzyme-linked immunosorbent assays were also carried out with the large tryptic fragments of calmodulin. The results suggest that (i) the C-terminal fragment binds the antibody with an affinity which is comparable with that of intact calmodulin; (ii) the neuropeptide can form complexes with both N- and C-terminal fragments, but with two orders of magnitude less activity in case of the C-terminal fragment; and (iii) trifluorperazine does not stimulate antibody binding to the C-terminal fragment. Therefore the tertiary structure of calmodulin must be intact to ensure long-distance interactions between the binding sites of trifluoperazine, the neuropeptide and the antibody. These interactions may produce distinct conformers of calmodulin which may exhibit altered potency, not only for antibody binding but also for stimulation/inhibition of target enzymes.

Animals↗

PubMed1992

Mechanism for ganglioside-mediated modulation of a calmodulin-dependent enzyme. Modulation of calmodulin-dependent cyclic nucleotide phosphodiesterase activity through binding of gangliosides to calmodulin and the enzyme.

Gangliosides were recently shown to bind to calmodulin (Higashi, H., Omori, A., and Yamagata, T. (1992) J. Biol. Chem. 267, 9831-9838). This prompted us to investigate the effects of gangliosides on the calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. Several species of gangliosides competitively inhibited calmodulin-stimulated phosphodiesterase activity, with GD1b, GT1b, and GD1a being noted to do so particularly (group 1). GM1, GQ1b, and GM2 (group 2) were less inhibitory, and GM3, GM3(NeuGc), GalCer, sulfatide, GgOse4Cer, and oligosaccharide portions of inhibitory gangliosides showed no inhibition in accordance with the binding specificity of calmodulin to gangliosides. Trypsin-activated phosphodiesterase was inhibited by gangliosides with similar specificity, indicating interactions of gangliosides with the enzyme. Inhibition, however, was less than that of calmodulin-dependent activity by these compounds and, in both cases, was eliminated by excess calmodulin. In the absence of calmodulin, group 1 gangliosides at lower concentrations activated the intact enzyme but inhibited it over a certain range of increase in concentration. Ganglioside-dependent modulation of calmodulin-dependent phosphodiesterase activity is thus shown to be due to interactions of gangliosides with both calmodulin and the enzyme, and consequently, ganglioside-calmodulin binding is likely the mechanism for regulation of the enzyme.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1983

Production of antibodies to calmodulin in rabbits and enzyme immunoassays for calmodulin and anti-calmodulin.

The production of anti-calmodulin antibodies in rabbit was examined with the use of performic acid-oxidized calmodulin from bovine brain or an emulsion of native calmodulin and methylated bovine serum albumin as the antigen. The antibodies in rabbit sera were determined serially by means of a sensitive enzyme immunoassay method that uses a mini-column of goat (anti-rabbit IgG) IgG-coupled Sepharose 4B for separation. The antibodies could be produced in rabbits with either antigen. Although the native calmodulin with methylated bovine serum albumin seemed to be a better antigen, the titer of antiserum was not raised to levels detectable in the double immunodiffusion test. Monospecific antibodies were purified from the antiserum, and a competitive enzyme immunoassay method for the assay of calmodulin was developed with the use of the column-separation technique employed in the assay of anti-calmodulin. The method could determine from 10 ng to 10 micrograms (0.6 pmol to 0.6 nmol) of rat calmodulin and gave no cross-reaction with S-100 protein. Calmodulin contents in rat brain determined by the present method were consistent with those measured by the bioassay using calmodulin-deficient phosphodiesterase.

Animals↗

PubMed1985

Calcium release from intact calmodulin and calmodulin fragment 78-148 measured by stopped-flow fluorescence with 2-p-toluidinylnaphthalene sulfonate. Effect of calmodulin fragments on cardiac sarcoplasmic reticulum.

Calcium release from high and low-affinity calcium-binding sites of intact bovine brain calmodulin (CaM) and from the tryptic fragment 78-148, purified by high-pressure liquid chromatography, containing only the high-affinity calcium-binding sites, was determined by fluorescence stopped-flow with 2-p-toluidinylnaphthalene sulfonate (TNS). The tryptic fragments 1-77 and 78-148 each contain a calcium-dependent TNS-binding site, as shown by the calcium-dependent increase in TNS fluorescence. The rate of the monophasic fluorescence decrease in endogenous tyrosine on calcium dissociation from intact calcium-saturated calmodulin (kobs 10.8 s-1 and 3.2 s-1 at 25 degrees C and 10 degrees C respectively) as well as the rate of equivalent slow phase of the biphasic decrease in TNS fluorescence (kobsslow 10.6 s-1 and 3.0 s-1 at 25 degrees C and 10 degrees C respectively) and the rate of the solely monophasic decrease in TNS fluorescence, obtained with fragment 78-148 (kobs 10.7 s-1 and 3.5 s-1 at 25 degrees C and 10 degrees C respectively), were identical, indicating that the rate of the conformational change associated with calcium release from the high-affinity calcium-binding sites on the C-terminal half of calmodulin is not influenced by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed with intact calmodulin only (kobsfast 280 s-1 at 10 degrees C) but not with fragment 78-148, is most probably due to the conformational change associated with calcium release from low-affinity sites on the N-terminal half. The calmodulin fragments 1-77 and 78-148 neither activated calcium/calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum nor inhibited calmodulin-dependent activation at a concentration approximately 1000-fold greater (5 microM) than that of the calmodulin required for half-maximum activation (5.9 nM at 0.8 mM Ca2+ and 5 mM Mg2+) of calmodulin-dependent phosphoester formation.

Animals↗

PubMed2006

Conformational dynamics of calmodulin in complex with the calmodulin-dependent kinase kinase alpha calmodulin-binding domain.

As the primary intracellular calcium sensor, calcium-saturated calmodulin (CaM) regulates numerous and diverse proteins. Several mechanisms, including tissue-specific expression, localization, and sequestration, work in concert to limit the total number of available targets of calmodulin within a cell. While the free energies of binding of calmodulin-binding domains of regulated proteins by CaM have been shown to be highly similar, they result from vastly different enthalpic and entropic contributions. Here, we report the backbone and side-chain methyl dynamics of calcium-activated calmodulin in complex with a peptide corresponding to the CaM-binding domain of calmodulin kinase kinase, along with the thermodynamic underpinnings of complex formation. The results show a considerable reduction in side-chain mobility throughout CaM upon binding the CaMKKalpha peptide, which is consistent with the enthalpically driven nature of the binding. Site-specific comparison to another kinase-derived peptide complex with similar thermodynamic values reveals significant differences in dynamics largely localized to the hydrophobic binding sites.

Animals↗

PubMed1983

Inhibitory effects of calmodulin antagonists on plasma membrane cyclases in Tetrahymena: calmodulin-dependent guanylate cyclase and calmodulin-independent adenylate cyclase.

Trifluoperazine was shown previously to inhibit the activation of Tetrahymena guanylate cyclase activity by calmodulin [S. Nagao, S. Kudo and Y Nozawa, Biochem. Pharmac. 19, 2709 (1981)]. The present paper reports that N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), another representative calmodulin inhibitor, inhibited the calmodulin-induced activation of the guanylate cyclase, and that trifluoperazine and W-7 also inhibited Tetrahymena adenylate cyclase. The adenylate cyclase activity was found to be present in a membrane-bound form and not to be influenced by calmodulin. The inhibitions of the adenylate cyclase activity by these agents were dose-dependent and not Ca2+-dependent. These findings suggest that the inhibitory actions of these drugs may not necessarily be specific for calmodulin-dependent enzymes in T. pyriformis.

Adenylyl Cyclases↗

PubMed1987

Comparative effects of calmodulin inhibitors on calmodulin's hydrophobic sites and on the activation of cyclic nucleotide phosphodiesterase by calmodulin.

Experiments were designed to investigate the effect of inhibitors on calmodulin's hydrophobic sites and their consequences on the activation of a target enzyme, cyclic nucleotide phosphodiesterase. Two fluorescent probes, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS) and 9-anthroylcholine (9AC) were used to study the interactions with calmodulin of inhibitors devoid of direct effect on the probes. Contrary to W-7, nicergoline, nicardipine and quercetin, which decreased the fluorescence of the two probes bound to calmodulin, bepridil only decreased 9AC fluorescence but increased the fluorescence intensity at the wavelength of the emission maximum of TNS. In spite of this difference, bepridil as well as W-7 and nicergoline competitively inhibited calmodulin activation of phosphodiesterase. In addition, nicergoline also inhibited phosphodiesterase activity competitively to cyclic GMP. These results show differences in the interactions of inhibitors with calmodulin; these differences are not detected in functional studies of the effect of inhibitors on phosphodiesterase activation.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1989

Expression of calmodulin-dependent phosphodiesterase, calmodulin-dependent protein phosphatase, and other calmodulin-binding proteins in human SMS-KCNR neuroblastoma cells.

Calmodulin (CaM)-dependent enzymes, such as CaM-dependent phosphodiesterase (CaM-PDE), CaM-dependent protein phosphatase (CN), and CaM-dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM-dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS-KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS-KCNR cells expressed CN and CaM-PDE, but did not appear to have other neuronal CaM-binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB-14 glioma cells did not markedly alter the expression of CaM-binding proteins; 21-day treatment with retinoic acid, however, did induce expression of novel CaM-binding proteins of 74 and 76 kilodaltons. Using affinity-purified polyclonal antibodies, CaM-PDE immunoreactivity was detected as a 75-kilodalton peptide in undifferentiated cells, but as a 61-kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite-bearing cells; however, CaM-dependent phosphatase activity was seen. Immunoblot analysis with affinity-purified antibodies against CN indicated that this enzyme was present in SMS-KCNR cells regardless of their state of differentiation. Although SMS-KCNR cells did not show a complete pattern of neuronal CaM-binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM-PDE and CN expression. It is possible that CaM-dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.

Avidin↗

PubMed1997

Calmodulin content, Ca2+-dependent calmodulin binding proteins, and testis growth: identification of Ca2+-dependent calmodulin binding proteins in primary spermatocytes.

In contrast with the transient pre-replicative increase in calmodulin (CaM) level observed in proliferative activated cells, postnatal development of rat testis was paralleled by 3 specific rises in CaM. The first one occurred between 5 and 10 days, coincident with the appearance and proliferation start of spermatogonia and Sertoli cells. Meiosis accomplishment and spermatid differentiation were paralleled by 2 additional rises, at 24 and 32 days, respectively. The plateau phase of testis growth was coincident with the appearance of maturating spermatids and spermatozoa in the germinal epithelium, and with a decrease in CaM content. Testicular DNA:g wet tissue ratio reached the highest level in 15-day-old rats and gradually decreased up to 35 days, when a constant level was reached. A similar level of Ca2+-CaMBPs was observed in 5- and 20-day-old rat testis. Although all subcellular fractions showed the ability to bind CaM in a Ca2+-dependent manner, CaM was mainly recovered in the nuclear and soluble fractions of adult and immature rat testis. Several Ca2+-CaMBPs with an apparent M(r) of 82, 75, 64, 19, and 14 kD were purified by affinity chromatography from pachytene primary spermatocyte nuclear matrix. Ca2+-CaMBPs showing an M(r) of 120, 78, 72, and 66 kD were also purified from the supernatant obtained after DNA and RNA hydrolysis of meiotic nuclei. Major cytosolic Ca2+-CaMBPs of primary spermatocytes showed an M(r) of 120, 84, 44, and 39 kD. The functions that these Ca2+-CaMBPs might have during the first meiotic prophase is discussed.

Animals↗