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PubMed1991

Three amino acid substitutions in domain I of calmodulin prevent the activation of chicken smooth muscle myosin light chain kinase.

TaM-BMI is a genetically engineered chimeric protein consisting of the first 55 amino acids of cardiac troponin C (but with the normally inactive first Ca2+ binding domain reactivated by site- directed mutagenesis) ligated to the last three domains of chicken calmodulin (George, S.E., VanBerkum, M.F., Ono, T., Cook, R., Hanley, R.M., Putkey, J.A., and Means, A. R. (1990) J. Biol. Chem. 265, 9228-9235). This protein binds chicken smooth muscle myosin light chain kinase (smMLCK) but fails to activate the enzyme, thus functioning as a potent competitive inhibitor (Ki = 66 nM). We have created 29 mutants of calmodulin designed to identify the minimal number of alterations which must be introduced in the first domain to convert the protein to a competitive inhibitor of smMLCK. Alterations of three amino acids predicted to lie on the external surface of calmodulin (E14A, T34K, S38M) recapitulated the phenotype of TaM-BMI and exhibited a Ki of 38 nM. Both the triple mutant and TaM-BMI activated phosphodiesterase and bound a synthetic peptide analog of the calmodulin binding region of smMLCK with an affinity similar to that of native calmodulin (Kact and Kd values of approximately 2 and 3 nM respectively). When a synthetic peptide analog of the myosin light chain phosphorylation site was used as substrate rather than the 20-kDa light chains, TaM-BMI and the triple mutant were partial agonists: the Km for peptide substrate was increased 100- and 60-fold, and catalytic activity was 45 and 60%, respectively, relative to calmodulin. These data suggest TaM-BMI and E14A/T34K/S38M may interact with the calmodulin binding domain of smMLCK in a manner similar to calmodulin. However, alterations in electrostatic and hydrophobic interactions created by the three amino acid substitutions prevent the conformational change in the enzyme usually produced by calmodulin binding. Lack of such changes results in loss of catalytic activity and light chain binding. Additionally, our results show that altering only 3 amino acids residues converts calmodulin to an enzyme-selective antagonist, thus demonstrating the ability to separate calmodulin binding to smMLCK from calmodulin-induced activation of the enzyme.

Animals↗
PubMed1991

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1.

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.

Actins↗
PubMed1990

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes.

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.

Animals↗
PubMed1990

Studies on the interaction of daurisoline alkaloid derivatives and calmodulin by fluorescence spectroscopy.

A new kind of bisbenzylisoquinoline compounds, daurisoline alkaloid derivatives, has been found to be very potent calmodulin antagonists. The fluorescence spectra of interaction between these derivatives and calmodulin have been studied. The experimental results showed that these derivatives could interact with calmodulin resulting in forming a complex and diminishing fluorescence intensity. The process was Ca2(+)-dependent. These derivatives can bind to calmodulin and result in conformational change of calmodulin. But the binding site of these derivatives on calmodulin may be different from that of trifluoperazine. These derivatives can not displace all Ca2+ on calmodulin like trifluoperazine can do. Their abilities of antagonizing calmodulin to stimulate calmodulin-dependent cyclic nucleotide phosphodiesterase and the affinities of binding to calmodulin were related to hydrophobicity of substituting groups in side chain of these derivatives. Increase in hydrophobicity of these substituting groups increased binding of the derivatives and generally increased the inhibition of calmodulin stimulation of calmodulin-dependent cyclic nucleotide phosphodiesterase.

Alkaloids↗
PubMed1988

Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin.

The octaethyleneglycol mono-n-dodecyl ether solubilized Ca2+-ATPase purified from human erythrocytes has been studied to determine the physical mechanism of its activation by calmodulin. The dependence of Ca2+-ATPase activity on the enzyme concentration shows a transformation from a calmodulin-dependent to a fully active calmodulin-independent form. The transformation is cooperative with a half-maximal activation at 10-20 nM enzyme. This suggests that at higher enzyme concentrations interactions between Ca2+-ATPase polypeptide chains substitute for calmodulin-enzyme interactions, resulting in activation. In support of this interpretation, the inclusion of higher octaethyleneglycol mono-n-dodecyl ether concentrations shifts the half-maximal transformation to higher enzyme concentrations. Regardless of the detergent concentration, calmodulin decreases by about 2-fold the enzyme concentration required to observe half-maximal Ca2+-ATPase activation, without affecting the maximal velocity or cooperativity. This indicates that calmodulin facilitates interactions between enzyme molecules. The fluorescein-5'-isothiocyanate-modified Ca2+-ATPase shows an increase in fluorescence polarization which occurs over the same narrow concentration range that is seen with the Ca2+-ATPase activity, confirming association of enzyme molecules. Stimulation of the Ca2+-ATPase activity by calmodulin has revealed a stoichiometry of 0.73, with a dissociation constant of 1.6 nM calmodulin. We have demonstrated by use of calmodulin-Sepharose chromatography that both the calmodulin-dependent and independent Ca2+-ATPase forms bind calmodulin, even though stimulation of activity is seen only with the former one. Our data suggest the following two mechanisms for the Ca2+-ATPase activation: self-association of enzyme molecules or interaction with calmodulin.

Calcium-Transporting ATPases↗
PubMed1987

Bacterially synthesized vertebrate calmodulin is a specific substrate for ubiquitination.

Calmodulin purified from bacteria which express a cloned chicken calmodulin gene can be selectively conjugated with ubiquitin, using enzymes present in reticulocyte extracts. Analyses of peptide products generated from limited proteolytic digestion of the calmodulin conjugate containing a single ubiquitin indicate that lysine 115 on calmodulin is the site of linkage. This linkage site is identical to that previously reported for calmodulin purified from Dictyostelium discoideum. Substrate-dependent ATP hydrolysis by a partially purified ubiquitin conjugation enzyme system from reticulocyte extracts was used to determine the enzyme affinity to calmodulin. Km values of 7 and 9 microM were determined for dictyostelium and the bacterially expressed calmodulin, respectively. The bacterially expressed calmodulin, unlike the Dictyostelium protein, can also form conjugates containing a 2-5 molar ratio of ubiquitin but at a slower rate than that observed for conjugation at lysine 115. Results from these studies further support our hypothesis that the post-translational methylation of lysine 115 found in most forms of calmodulin serves the important function of protecting calmodulin from ubiquitination and from degradation by the cytoplasmic ubiquitin-dependent proteolytic pathway. The capability of the bacterially expressed calmodulin to form conjugates with a high molar ratio of ubiquitin suggests that the post-translational acetylation of the N terminus of calmodulin may serve a similar function.

Adenosine Triphosphate↗
PubMed1987

The interaction of calmodulin with human erythrocyte spectrin. Inhibition of protein 4.1-stimulated actin binding.

The functional significance of calmodulin binding to human erythrocyte spectrin has been investigated under native conditions. Both native calmodulin and calmodulin derivatized with the photoactivable cross-linker methyl 4-azidobenzimidate (azidocalmodulin) have been used. When azidocalmodulin is photolyzed in the presence of erythrocyte ghosts, ghost extracts, or purified protein, it cross-links predominately to the beta subunit of erythrocyte spectrin. This cross-linking is calcium-dependent, requires photolysis, and is inhibited by 100 microM trifluoperazine or unlabeled calmodulin. Calmodulin labeled spectrin exhibits a specific and non-calcium-dependent inhibition of its ability to bind actin, even in the presence of protein 4.1. Its ability to self-associate or to bind spectrin-depleted membrane vesicles is unperturbed. Native calmodulin also inhibits protein 4.1-stimulated spectrin-actin binding, but unlike that of covalently bound calmodulin, inhibition by the uncross-linked calmodulin requires calcium. The degree of inhibition of spectrin-actin-4.1 binding induced by native calmodulin is significant since 109 microM calmodulin inhibits over 63% of the spectrin-actin binding induced by 4.5 microM protein 4.1. These results demonstrate a specific effect of calmodulin on erythroid spectrin function and suggest that calmodulin may influence the binding of protein 4.1 and actin to spectrin within the cytoskeleton.

Azides↗
PubMed1985

Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase.

Limited proteolysis has been utilized to study the structural organization of rabbit skeletal muscle myosin light chain kinase. The enzyme (Mr approximately 89,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) consists of an amino-terminal, protease-susceptible region of unidentified function and a carboxyl-terminal, protease-resistant region of Mr approximately 40,000 containing the catalytic and calmodulin-binding domains. Partial digestion with trypsin produced an intermediate 56,000-dalton fragment and a stable 38,000-dalton fragment, both of which were catalytically active and calmodulin-dependent. Chymotryptic digestion yielded three catalytically active fragments of about 37,000, 36,000, and 35,000 daltons. The Mr = 37,000 fragment was calmodulin-dependent with an apparent affinity equivalent to that of the native enzyme (approximately 1 nM). The 36,000-dalton fragment was also calmodulin-dependent but had a approximately 200-fold lower apparent affinity. The Mr = 35,000 fragment was calmodulin-independent. These three chymotryptic fragments, had identical amino termini. Nineteen residues were missing from the carboxyl terminus of the calmodulin-independent chymotryptic fragment whereas only 8 or 9 carboxyl-terminal residues were missing from the calmodulin-dependent tryptic fragments. These results suggest that the 11-residue sequence (IAVSAANRFKK) in the carboxyl-terminal region of myosin light chain kinase contributes directly to the binding of calmodulin. This conclusion is in accord with data (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3187-3191) that the carboxyl-terminal, 27-residue CNBr peptide of the native enzyme shows Ca2+-dependent, high affinity binding to calmodulin and that similar calmodulin-binding activity, although detectable in unfractionated CNBr digests of calmodulin-dependent enzyme forms, is much reduced in a CNBr digest of the calmodulin-independent, Mr = 35,000 chymotryptic fragment.

Animals↗
PubMed1984

Inhibitor peptide of mitochondrial proton adenosine triphosphatase. Neutralization of its inhibitory action by calmodulin.

In the presence of ATP and Mg2+, the homogeneous ATPase peptide inhibitor of rat liver mitochondria markedly inhibits the proton ATPase from this source (Cintrón N. M., and Pedersen, P. L. (1979) J. Biol. Chem. 254, 3439-3443). Under these conditions, calmodulin prevents the inhibitor peptide from inhibiting the liver H+-ATPase. About 1.5 mol of calmodulin/mol of inhibitor is necessary to effect a half-maximal response (apparent Km = 0.5 microM calmodulin). The capacity of calmodulin to neutralize the action of the ATPase inhibitor peptide appears highly specific. This effect is not produced by insulin, trypsin inhibitor, lysozyme, ribonuclease, myoglobin, cytochrome c, ovalbumin, or bovine albumin. Only polyglutamate was found to mimic the action of calmodulin. However, when added together with calmodulin, polyglutamate failed to elicit an additive effect indicating that its site of interaction on the ATPase inhibitor peptide differs from that of calmodulin. Calcium is not essential in the assay medium for calmodulin to neutralize the action of the ATPase inhibitor peptide. The neutralization effect produced by calmodulin is also source-independent, with preparations of calmodulin from bovine brain and rat testes being equally competent. Calmodulin has no direct effect on the ATPase activity of the proton ATPase, nor does it affect the capacity of the enzyme to participate in either ATP synthesis or the ATP-dependent transhydrogenase reaction. Moreover, calmodulin fails to reverse inhibition of the H+-ATPase to which ATPase inhibitor peptide is already bound. Overall, these results indicate that calmodulin interacts in a direct and highly specific manner with the "free" ATPase peptide inhibitor of rat liver mitochondria.

Animals↗
PubMed1980

Hydrophobic regions function in calmodulin-enzyme(s) interactions.

Certain naturally occurring lipids (phosphatidylinositol, phosphatidylserine, arachidonic acid) and sodium dodecyl sulfate activate at least two calmodulin-dependent enzymes, bovine brain 3':5'-cyclic nucleotide phosphodiesterase and chicken gizzard myosin light chain kinase in the absence of Ca2+. 2-p-Toluidinyl-naphthalene-6-sulfonate (TNS), which is often used as a probe for hydrophobic groups of proteins, inhibits these two calmodulin-dependent enzymes. Kinetic analysis of inhibition of chicken gizzard myosin kinase by TNS revealed a competitive fashion against calmodulin-induced activation. The interaction between TNS and purified bovine brain calmodulin as demonstrated in the appearance of TNS fluorescence in the presence of 3 microM or more of calcium ion was not observed in the presence of 2 mM EGTA. This suggests that TNS is able to bind to calmodulin in the presence of Ca2+. Moreover, a calmodulin-interacting agent N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide suppressed the TNS fluorescence induced by complex formation with calmodulin in the presence of Ca2+. These results suggest that when Ca2+ binds to the high affinity sites of calmodulin, it induces a conformational change which exposes hydrophobic groups, and the calmodulin is then capable of activating calmodulin-dependent enzymes. We propose that hydrophobic properties of Ca2+-calmodulin are important for the activation of Ca2+-calmodulin-dependent enzymes.

Adenosine Triphosphatases↗
PubMed1984

The synthesis and reaction of a specific affinity label for the hydrophobic drug-binding domains of calmodulin.

An affinity-labeling reagent for the two hydrophobic drug-binding domains of calmodulin has been prepared and its reaction with calmodulin characterized. The reagent, 10-(3-propionyloxysuccinimide)-2-(trifluoromethyl)phenothiazine, was shown to be very specific labeling reagent for these domains. Its specificity was demonstrated by the following observations. 1) Previous reports have shown that Ca2+ is required for phenothiazine binding to calmodulin, and here we show that the affinity-labeling reagent reacts with and inactivates calmodulin in the presence of Ca2+, but not in its absence. 2) Inclusion of trifluoperazine, fluphenazine, W-7, or 10-(3-aminopropyl)-2-(trifluoromethyl)phenothiazine in the reaction mixture protected calmodulin from inactivation by the reagent. 3) Inactivation by the reagent yielded calmodulin that was no longer retained on a phenothiazine-Sepharose column under conditions in which unreacted calmodulin was retained. 4) The measured stoichiometry of the reaction in the presence of excess reagent was 2.1 mol of reagent per mol of calmodulin which agrees well with previous reports of two high-affinity phenothiazine-binding sites on calmodulin. 5) The stoichiometry of the reaction was further confirmed by tryptic peptide maps which show two phenothiazine-labeled peptides unique to the fully reacted protein. 6) The spectral properties of the reagent, while attached to calmodulin, change in the presence of Ca2+ in a manner consistent with the known effects of Ca2+ binding by calmodulin on these hydrophobic domains. The specificity of the reagent makes it useful for further characterization of these hydrophobic binding domains on calmodulin.

Affinity Labels↗
PubMed1985

Amino Acid sequence of a novel calmodulin from the unicellular alga chlamydomonas.

An amino acid sequence for a Chlamydomonas calmodulin has been elucidated with emphasis on the characterization of differences that are unique to Chlamydomonas and Dictyostelium calmodulin. While the concentration of calmodulin required for half-maximal activation of plant NAD kinase varies among vertebrate, higher plant, algal, and slime mold calmodulins, only calmodulins from the unicellular alga Chlamydomonas and the slime mold Dictyostelium show increased maximal activation of NAD kinase (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798; Marshak, Clarke, Roberts, Watterson 1984 Biochemistry 23: 2891-2899). The same preparations of calmodulin do not show major differences in phosphodiesterase or myosin light chain kinase activator activity.We report here that a Chlamydomonas calmodulin has four primary structural features similar to Dictyostelium that are not found in other calmodulins characterized to date: an altered carboxy terminus including a novel 11-residue extension for Chlamydomonas calmodulin, unique residues at positions 81 and 118, and an unmethylated lysine at position 115. The only amino acid sequence identity unique to Chlamydomonas and Dictyostelium calmodulin is the presence of a lysine at position 115 instead of a trimethyllysine. These studies indicate that the methylation state of lysine 115 may be important in the maximal NAD kinase activator activity of calmodulin and support the concept that calmodulin has multiple functional domains in addition to multiple structural domains.

Journal Article↗
PubMed1989

A calmodulin-binding peptide relaxes skinned muscle from guinea-pig taenia coli.

During smooth muscle activation the calcium calmodulin complex interacts with myosin light chain kinase (MLCK) whereby activating it. A synthetic peptide analogue (RS20) corresponding to the calmodulin recognition sequence of MLCK has been synthesized and previously found to inhibit the calmodulin stimulated light chain kinase activity. Here we studied the effect of this peptide on skinned fibers from guinea pig taenia coli. Maximal contractions induced by 30 microM Ca2+ at 0.1 microM calmodulin could be completely relaxed by the peptide at 1 microM. The inhibitory effect was accompanied by partial dephosphorylation only of the regulatory myosin light chain. Relaxation could be reversed by addition of calmodulin which also increased the extent of light chain phosphorylation. The calmodulin concentration required for reversing the inhibition depended on the concentration of the inhibitory peptide suggesting that the peptide competed with MLCK for the calmodulin binding site. As the calcium-calmodulin-peptide mixture constitutes a calmodulin buffer, our results suggest, that the peptide is a calmodulin antagonist unique in terms of its potency and that less than nanomolar concentrations of free calmodulin may be required for inducing smooth muscle contractions.

Animals↗
PubMed1984

Characterization of bovine brain calmodulin-dependent protein phosphatase.

Calmodulin-dependent protein phosphatase of bovine brain exhibited a pH optimum of 7 and appeared to require sulfhydryl groups for activity. Phosphatase activity was inhibited by both NaF and ZnCl2, but was stimulated approximately 2-fold by MnCl2. The enzyme exhibited broad substrate specificity, dephosphorylating casein, troponin I, protamine, histone, and phosvitin, and was not phosphorylated by cAMP-dependent protein kinase. With 32P-labeled casein as a substrate, phosphatase was activated 15-fold by calmodulin; the dissociation constant of phosphatase for calmodulin was 30 nM. Activation of the enzyme by calmodulin as a function of Ca2+ was highly cooperative; the Hill coefficient was 4.9. At a saturating concentration of calmodulin, half-maximal activation of phosphatase was obtained at 0.3 microM Ca2+. Calmodulin increased the Vmax from 1.7 to 41 nmol mg protein-1 min-1 with no significant change in its Km. Formation of a Ca2+-dependent complex between calmodulin and the phosphatase was demonstrated by a calmodulin-Sepharose affinity column, gel-filtration chromatography, and sedimentation on a sucrose density gradient. The rate of formation and dissociation of the calmodulin X phosphatase complex was rapid and readily reversible in response to changes in Ca2+ concentration. The calmodulin X phosphatase complex consists of 1 mol of calmodulin and 1 mol of phosphatase.

Animals↗
PubMed1982

Ca2+ and calmodulin-regulated endogenous tubulin kinase activity in presynaptic nerve terminal preparations.

Synaptosomal tubulin was shown to be the major substrate for a Ca2+-calmodulin regulated protein kinase in synaptosome soluble fractions as determined by two-dimensional gel electrophoresis and peptide mapping. Ca2+ activated this endogenous tubulin kinase system in presynaptic nerve terminal preparations. The Ca2+-dependent activation of the tubulin kinase system was mediated by the Ca2+ binding protein, calmodulin. Trifluoperazine, a known inhibitor of calmodulin, significantly blocked the calmodulin-stimulated [32P]phosphate incorporation into synaptic tubulin. This inhibition of endogenous tubulin phosphorylation could be reversed by addition of exogenous calmodulin to the reaction mixture. The concentrations of Ca2+ and calmodulin required to produce a half-maximal stimulation of the tubulin kinase were 0.8 microM and 0.3 microM respectively. Greater than 70% of soluble tubulin present in the nerve terminal was phosphorylated in less than 50 s by this kinase system. Evidence is presented indicating that the synaptic Ca2+-calmodulin tubulin kinase is a distinct enzyme system from the previously described cyclic AMP microtubule-associated kinase. The anticonvulsant phenytoin inhibited the Ca2+-calmodulin stimulated phosphorylation of tubulin, and alpha- and beta-tubulin were identified as major components of previously designated synaptic phosphoprotein bands of DPH-L and DPH-M. Existence of the kinase as a calmodulin-tubulin-kinase complex is suggested from kinetic studies. The Ca2+-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. The activation of the tubulin kinase by Ca2+ and calmodulin may play a role in the functional utilization of tubulin in the nerve terminal and may mediate some of the effects of Ca2+ on synaptic function.

Animals↗
PubMed1988

Characterization of calmodulin-binding components in the pituitary gonadotrope.

We have used an 125I-calmodulin gel overlayer assay to identify calmodulin-binding component in the rat pituitary. Tissue-specific and Ca2+ -dependent patterns of 125I-calmodulin binding were observed, with five major Ca2+-dependent 125I-calmodulin-labeled components of subunit Mr greater than 205,000, 200,000, 135,000, 60,000, and 52,000. Ca2+-dependent binding was defined as that which was abolished in the presence of 1 mM EGTA. Calmodulin binding was inhibited by calmodulin antagonists such as penfluridol (1 microM) or pimozide (1 microM). Some Ca2+-independent binding was observed and appears to be due to (nonspecific) hydrophobic interaction of calmodulin with acid-soluble proteins, principally histones. Subcellular fractionation revealed that the Ca2+-dependent calmodulin-binding components are localized primarily in the cytosolic fraction. Separation of dispersed anterior pituitary cells by a linear metrizamide gradient yielded gonadotrope-enriched fractions; these contained all five 125I-calmodulin-binding components corresponding to the major bands in the pituitary homogenate. Studies with ovariectomized and steroid-replaced animals indicated that the tissue content of calmodulin-binding components, like those of calmodulin itself, did not appear to be differentially regulated by steroids. A comparison of rat and bovine pituitary tissue homogenates revealed that binding components migrating at the same apparent Mrs were found for four of the components (the largest component is lacking in the bovine system).

Animals↗
PubMed1995

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.

Animals↗
PubMed1997

DY-9760e, a novel calmodulin antagonist with cytoprotective action.

We report the pharmacological characterization and cytoprotective effect of DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-( 4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, a novel antagonist of calmodulin. DY-9760e inhibited calmodulin-dependent enzymes, including calmodulin-dependent protein kinase II and IV, calcineurin, [corrected] calmodulin-dependent phosphodiesterase and myosin light chain kinase with Ki values of 1.4, 12, 2.0, 3.8 and 133 microM, respectively. These antagonistic effects of DY-9760e were more potent than those of W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, another calmodulin antagonist. This compound showed little or no effect on calmodulin-independent enzymes, such as protein kinase A and C and calpain I and II. Analysis of the hydrophobic interaction of DY-9760e with calmodulin by using 2-p-toluidinylnaphthalene-6-sulfonate and 9-anthroylcholine revealed that, like W-7, DY-9760e bound to the hydrophobic regions of calmodulin. The [14C]DY-9760e binding assay indicated that DY-9760e bound to calmodulin at one class of binding site. Finally, DY-9760e substantially protected N1E-115 neuroblastoma cells from cytotoxicity induced by the Ca2+ ionophore, A23187. These results indicate that DY-9760e, a novel calmodulin antagonist, possesses a cytoprotective action and suggest that calmodulin plays a critical role in mediating some of the biochemical events leading to cell death following Ca2+ overload.

Calcimycin↗