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PubMed2002

A noncontiguous, intersubunit binding site for calmodulin on the skeletal muscle Ca2+ release channel.

Both apocalmodulin (Ca(2+)-free calmodulin) and Ca(2+)-calmodulin bind to and regulate the activity of skeletal muscle Ca(2+) release channel (ryanodine receptor, RYR1). Both forms of calmodulin protect sites after amino acids 3630 and 3637 on RYR1 from trypsin cleavage. Only apocalmodulin protects sites after amino acids 1982 and 1999 from trypsin cleavage. Ca(2+)-calmodulin and apocalmodulin both bind to two different synthetic peptides representing amino acids 3614-3643 and 1975-1999 of RYR1, but Ca(2+)-calmodulin has a higher affinity than apocalmodulin for both peptides. Cysteine 3635, within the 3614-3643 sequence of RYR1, can form a disulfide bond with a cysteine on an adjacent subunit within the RYR1 tetramer. The second cysteine is now shown to be between amino acids 2000 and 2401. The close proximity of the cysteines forming the intersubunit disulfide to the two sites that bind calmodulin suggests that calmodulin is binding at a site of intersubunit contact, perhaps with one lobe bound between amino acids 3614 and 3643 on one subunit and the second lobe bound between amino acids 1975 and 1999 on an adjacent subunit. This model is consistent with the finding that Ca(2+)-calmodulin and apocalmodulin each bind to a single site per RYR1 subunit (Rodney, G. G., Williams, B. Y., Strasburg, G. M., Beckingham, K., and Hamilton, S. L. (2000) Biochemistry 39, 7807-7812).

Amino Acid Sequence↗

PubMed2005

Calmodulin is required for vasopressin-stimulated increase in cyclic AMP production in inner medullary collecting duct.

Calmodulin plays a critical role in regulation of renal collecting duct water permeability by vasopressin. However, specific targets for calmodulin action have not been thoroughly addressed. In the present study, we investigated whether Ca2+/calmodulin regulates adenylyl cyclase activity in the renal inner medullary collecting duct. Rat inner medullary collecting duct suspensions were incubated in the presence or absence of 0.1 nM vasopressin and the calmodulin inhibitors, monodansylcadaverine, W-7, and trifluoperazine, followed by measurement of cAMP. Vasopressin-stimulated cAMP elevation was significantly attenuated in the presence of calmodulin inhibitors. Analysis of transglutaminase 2 knock-out mice confirmed that these compounds were not acting through inhibition of transglutaminase 2 activity. Calmodulin inhibitors also blocked both cholera toxin- and forskolin-stimulated cAMP accumulation. In isolated perfused tubules, W-7 reversibly blocked vasopressin-stimulated urea permeability, a process that requires a rise in intracellular cAMP but does not appear to involve protein trafficking to the apical plasma membrane. These results suggest that calmodulin is required for vasopressin-stimulated adenylyl cyclase activity in the intact inner medullary collecting duct. Reverse transcription-PCR, immunoblotting, and immunohistochemistry revealed the presence of the calmodulin-sensitive adenylyl cyclase type 3 in the rat collecting duct, an isoform previously not known to be expressed in the collecting duct. Long-term treatment of Brattleboro rats with a vasopressin analog markedly decreased adenylyl cyclase type 3 protein abundance, providing an explanation for long-term down-regulation of vasopressin response in the collecting duct. These studies demonstrate the importance of calmodulin in the regulation of collecting duct adenylyl cyclase activity and transport function.

Adenylyl Cyclases↗

PubMed1981

Axonal transport of calmodulin: a physiologic approach to identification of long-term associations between proteins.

Calmodulin is a soluble, heat-stable protein which has been shown to modulate both membrane-bound and soluble enzymes, but relatively little has been known about the in vivo associations of calmodulin. A 17,000-dalton heat-stable protein was found to move in axonal transport in the guinea pig visual system with the proteins of slow component b (SCb; 2 mm/d) along with actin and the bulk of the soluble proteins of the axon. Co-electrophoresis of purified calmodulin and radioactively labeled SCb proteins in two dimensional polyacrylamide gel electrophoresis (PAGE) demonstrated the identity of the heat-stable SCb protein and calmodulin on the basis of pI, molecular weight, and anomalous migration in the presence of Ca2+-chelating agents. No proteins co-migrating with calmodulin in two-dimensional PAGE could be detected among the proteins of slow component a (SCa; 0.3 mm/d, microtubules and neurofilaments) or fast component (FC; 250 mm/d, membrane-associated proteins). We conclude that calmodulin is transported solely as part of the SCb complex of proteins, the axoplasmic matrix. Calmodulin moves in axonal transport independent of the movements of microtubules (SCa) and membranes (FC), which suggests that the interactions of calmodulin with these structures may represent a transient interaction between groups of proteins moving in axonal transport at different rates. Axonal transport has been shown to be an effective tool for the demonstration of long-term in vivo protein associations.

Actins↗

PubMed1983

Analysis of suborganellar fractions from spinach and pea chloroplasts for calmodulin-binding proteins.

Purified chloroplasts from spinach and pea leaves were subfractionated into envelope, thylakoid, and stroma fractions and were analyzed for calmodulin-binding proteins using a 125I-calmodulin gel overlay assay. Calmodulin binding was primarily associated with a major polypeptide (Mr 33,000) in the envelope membrane fraction. In contrast, major calmodulin-binding proteins were not detected in the thylakoid or stroma fractions. Our results provide the first evidence of calmodulin-binding proteins in the chloroplast envelope, and raise the possibility that calmodulin may contribute to the regulation of chloroplast function through its interaction with calmodulin-binding proteins in the chloroplast envelope. In addition, our results combined with those of other investigators support the proposal that subcellular organelles may be a primary site of calmodulin action.

Calmodulin-Binding Proteins↗

PubMed1984

Estrogen stimulates the transient association of calmodulin and myosin light chain kinase with the chicken liver nuclear matrix.

Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.

Animals↗

PubMed1994

Structure-based systematic isolation of conditional-lethal mutations in the single yeast calmodulin gene.

Conditional-lethal mutations of the single calmodulin gene in Saccharomyces cerevisiae have been very difficult to isolate by random and systematic methods, despite the fact that deletions cause recessive lethality. We report here the isolation of numerous conditional-lethal mutants that were recovered by systematically altering phenylalanine residues. The phenylalanine residues of calmodulin were implicated in function both by structural studies of calmodulin bound to target peptides and by their extraordinary conservation in evolution. Seven single and 26 multiple Phe-->Ala mutations were constructed. Mutant phenotypes were examined in a haploid cmd1 disrupted strain under three conditions: single copy, low copy, and overexpressed. Whereas all but one of the single mutations caused no obvious phenotype, most of the multiple mutations caused obvious growth phenotypes. Five were lethal, 6 were lethal only in synthetic medium 13 were temperature-sensitive lethal and 2 had no discernible phenotypic consequences. Overexpression of some of the mutant genes restored the phenotype to nearly wild type. Several temperature-sensitive calmodulin mutations were suppressed by elevated concentration of CaCl2 in the medium. Mutant calmodulin protein was detected at normal levels in extracts of most of the lethal mutant cells, suggesting that the deleterious phenotypes were due to loss of the calmodulin function and not protein instability. Analysis of diploid strains heterozygous for all combinations of cmd 1-ts alleles revealed four intragenic complementation groups. The contributions of individual phe-->ala changes to mutant phenotypes support the idea of internal functional redundancy in the symmetrical calmodulin protein molecule. These results suggest that the several phenylalanine residues in calmodulin are required to different extents in different combinations in order to carry out each of the several essential tasks.

Base Sequence↗

PubMed1993

Requirement of preimplantation human embryos for extracellular calmodulin for development.

The objective of this study was to investigate the requirement for calmodulin in the cell division of the early human preimplantation embryo. Experiments using three agents capable of inhibiting calmodulin activity, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7), W7 linked to agarose beads (W7-agarose) and calmodulin antibody, showed that W7 and calmodulin antibody arrested division of embryos in a dose-dependent manner. As W7 is able to penetrate the zona pellucida and enter the cells, calmodulin antibody can traverse the zona but not enter the cells and W7-agarose cannot traverse the zona, we have deduced that the calmodulin which appears relevant to embryo division may be both intracellular and intrazonal but not extrazonal. We conclude that calmodulin is specifically required for cell division in the early human preimplantation embryo and that the concentration of calmodulin surrounding the embryo within the zona is particularly important to embryo development.

Antibodies, Monoclonal↗

PubMed1988

The sites on calmodulin cross-linked to myosin light chain kinase and troponin I with water-soluble carbodiimide.

To elucidate the interaction of calmodulin with calmodulin binding proteins, we studied the location of the interaction sites on calmodulin by using a chemical cross-linking reagent. Calmodulin prepared from wheat germ was cross-linked to myosin light chain kinase and troponin-I with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked products were cleaved partially with cyanogen bromide and cross-linked sites were determined by peptide mapping analysis using SDS-urea polyacrylamide gel electrophoresis. Peptides which contain the cross-linked site were displaced from their position because of the attached fragments of myosin light chain kinase or troponin I. The peptide of calmodulin from the N-terminal to Met-73 in the cross-linked product with myosin light chain kinase had the same mobility as that of uncross-linked calmodulin on the map though the amount of the peptide was decreased in the cross-linked product. The peptide from the N-terminal to Met-110 in the cross-linked product was displaced from its position. Similar change in the mobility of the calmodulin peptides was also observed in the cross-linked products with troponin I. It was concluded, therefore, that at least one cross-linked site for myosin light chain kinase and one for troponin I were located between Met-73 and Met-110 of the wheat germ calmodulin.

Binding Sites↗

PubMed1989

Binding of both Ca2+ and mastoparan to calmodulin induces a large change in the tertiary structure.

The technique of small-angle X-ray scattering has been employed to examine the solution conformation of calmodulin and its complexes with Ca2+ alone, and with both Ca2+ and mastoparan. The radius of gyration decreased by 3.1 +/- 0.3 A upon binding of both 4 mol Ca2+/mol of protein and 1 mol mastoparan/mol of protein to form the ternary complex. A smaller increase was found for the separate binding of 4 mol Ca2+/mol of protein in the absence of mastoparan (0.6 +/- 0.3 A). The analyses of pair distance distribution function showed that the maximal pair distance in calmodulin complex with both Ca2+ and mastoparan decreased by 20-30% in comparison with calmodulin or its complex with Ca2+, and a shoulder near 40 A, which characterizes the dumbbell-shaped molecule of calmodulin, disappeared. These results indicate that the two globular domains of the calmodulin complex with Ca2+ and mastoparan come close together by 8.0-9.5 A on average, if the size and the overall shape of the globular domains are the same in Ca2+-calmodulin-mastoparan complex as in calmodulin or Ca2+-calmodulin complex.

Animals↗

PubMed1988

Expression of human calmodulin cDNA in Escherichia coli and characterization of the protein.

A cDNA clone of human calmodulin, isolated from liver, was subcloned into the expression vector pKK233-2. The resulting expression plasmid, designated pCWCaM1, produced human calmodulin in Escherichia coli SG5. The cDNA was sequenced using novel primers designed for use in plasmid-sequencing protocols with pKK233-2 and pKK223-3. The expressed calmodulin was purified and subjected to NMR analysis which revealed a structure essentially the same as natural calmodulin isolated from human tissue. The activation of myosin light chain kinase by the genetically engineered human calmodulin and bovine brain calmodulin was studied and found to be comparable to a high degree. The expressed calmodulin appears to be comparable to normal calmodulin and can be used for site-directed mutagenesis and structure/function investigations.

Base Sequence↗

PubMed1991

Specific identification and subcellular localization of three calmodulin-binding proteins in the rat gonadotrope: spectrin, caldesmon, and calcineurin.

In an effort to characterize the second messenger system for LH release, we have previously identified five calmodulin-binding proteins in rat gonadotropes of Mr greater than 205,000, 200,000, 135,000, 60,000, and 52,000. In the present study, we have used a calmodulin overlayer assay combined with Western blotting to determine the molecular identity of three calmodulin-binding proteins in rat gonadotropes: the alpha subunit of spectrin (Mr greater than 205,000), caldesmon (Mr 84,000), and the alpha subunit of calcineurin (Mr 60,000). The Mr greater than 205,000 and Mr 60,000 components or rat pituitary which bind calmodulin are immunoreactive with spectrin and calcineurin antisera, respectively. Rat pituitary also contains an Mr 84,000 component, which is immunoreactive with polyclonal sera and monoclonal antibody raised to chicken gizzard caldesmon (Mr 150,000). Like caldesmon from other sources, the Mr 84,000 component remains soluble after heat treatment and preferentially binds either filamentous actin or calmodulin, depending on the Ca2+ concentration. The three calmodulin-binding proteins were localized specifically in gonadotropes using indirect immunofluorescence microscopy or by Western-blotting cell fractions enriched for gonadotropes. After differential centrifugation of pituitary homogenate, spectrin immunoreactivity was found associated with the nuclear and secretory granule fractions, whereas caldesmon immunoreactivity was seen in the cytosolic fraction and calcineurin in the cytosolic and nuclear fractions. Although the precise role for these proteins remains unknown, the apparent requirement for calmodulin and the small number of calmodulin-binding proteins in the gonadotrope suggest their involvement in mediating GnRH actions.

Animals↗

PubMed1995

Halothane and isoflurane alter the Ca2+ binding properties of calmodulin.

BACKGROUND: Ca2+ plays an important role in signal transduction and anesthetic mechanisms. To date, no one has observed a direct effect of volatile anesthetics on a Ca(2+)-binding protein. We therefore examined the effects of halothane and isoflurane on the Ca(2+)-binding properties of bovine brain calmodulin. METHODS: The fluorescence emission of calmodulin was obtained over a range of Ca2+ concentrations (10(-7)-10(-4)M) in the presence and absence of halothane and isoflurane. The intrinsic tyrosine fluorescence of calmodulin was measured at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. Fluorescence measurements were carried out in 50 mM hydroxyethylpiperazineethane sulfonic acid, 100 mM KC1, and 2 mM ethyleneglycol-bis-(beta-aminoethyl ether) tetraacetic acid at pH 7.0 and 37 degrees C. Experiments were performed in polytetrafluorethylene-sealed cuvettes so that the volatile anesthetic concentrations remained constant. The titration data were analyzed in two ways. The data were fit to the Hill equation by using nonlinear regression analysis to derive the Hill coefficient and the dissociation constant. The data were also analyzed by two-way analysis of variance with multiple comparisons to determine statistically significant effects. Volatile anesthetic concentrations were measured by gas chromatography. RESULTS: The presence of volatile anesthetics altered the Ca(2+)-binding affinity of calmodulin in a dose-dependent fashion. At 0.57% (0.25 mM) halothane and 1.7% (0.66 mM) isoflurane, the affinity of calmodulin for Ca2+ relative to control was decreased. However, at higher concentrations of both anesthetics, the affinity for Ca2+ was increased. When the volatile anesthetics were allowed to evaporate from the experimental solutions, the observed rightward shift of the calmodulin-Ca2+ binding curve for Ca2+ at low concentrations of the anesthetics returned to the control position. The leftward shift seen at high concentrations of the anesthetics was irreversible after evaporation of 8.7% (3.3 mM) isoflurane and 5.7% (2.5 mM) halothane. CONCLUSIONS: These data demonstrate a complex interaction of two hydrophobic volatile anesthetics with calmodulin. A biphasic effect was observed both for halothane and for isoflurane. Calmodulin, an EF-hand Ca(2+)-binding protein, undergoes a conformational shift when binding Ca2+, exposing several hydrophobic residues. These residues may be sites at which the anesthetics act.

Animals↗

PubMedDate not supplied

Aprindine inhibits calmodulin-stimulated phosphodiesterase and Ca-ATPase activities.

Aprindine, an antiarrhythmic agent with structural similarities to lidocaine and procainamide, has proved effective in treatment of patients with ventricular premature depolarizations, ventricular tachycardia, and supraventricular arrhythmias. While its effects at an electrophysiologic level have been elucidated, its mechanism of action at a biochemical level has remained largely undefined. The data in this communication demonstrate that aprindine inhibits the activation of bovine brain cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) by calmodulin. This inhibition is specific for the calmodulin-stimulated enzyme, as no effect of aprindine is seen when phosphodiesterase is assayed in the absence of calmodulin. The inhibition is competitive with respect to substrate (cyclic AMP) and calmodulin concentrations. In the presence of 10 nM calmodulin, the ID50 for aprindine is 18 microM. This inhibition is not the result of aprindine acting as a calcium chelator because increasing the calcium concentration does not reverse the inhibitory effect. Aprindine also inhibits calmodulin-stimulated Ca-ATPase (ATP phosphohydrolase EC 3.6.1.3) activity, but again has no effect on the enzyme in the absence of calmodulin. Aprindine has hydrophobic properties which may be responsible for the inhibitory effect. Sufficient concentrations of aprindine are achieved in myocardial tissues to interfere with the ability of calmodulin to stimulate a number of enzymes present in the heart.

Animals↗

PubMed2002

Regulatory implications of a novel mode of interaction of calmodulin with a double IQ-motif target sequence from murine dilute myosin V.

Apo-Calmodulin acts as the light chain for unconventional myosin V, and treatment with Ca(2+) can cause dissociation of calmodulin from the 6IQ region of the myosin heavy chain. The effects of Ca(2+) on the stoichiometry and affinity of interactions of calmodulin and its two domains with two myosin-V peptides (IQ3 and IQ4) have therefore been quantified in vitro, using fluorescence and near- and far-UV CD. The results with separate domains show their differential affinity in interactions with the IQ motif, with the apo-N domain interacting surprisingly weakly. Contrary to expectations, the effect of Ca(2+) on the interactions of either peptide with either isolated domain is to increase affinity, reducing the K(d) at physiological ionic strengths by >200-fold to approximately 75 nM for the N domain, and approximately 10-fold to approximately 15 nM for the C domain. Under suitable conditions, intact (holo- or apo-) calmodulin can bind up to two IQ-target sequences. Interactions of apo- and holo-calmodulin with the double-length, concatenated sequence (IQ34) can result in complex stoichiometries. Strikingly, holo-calmodulin forms a high-affinity 1:1 complex with IQ34 in a novel mode of interaction, as a "bridged" structure wherein two calmodulin domains interact with adjacent IQ motifs. This apparently imposes a steric requirement for the alpha-helical target sequence to be discontinuous, possibly in the central region, and a model structure is illustrated. Such a mode of interaction could account for the Ca(2+)-dependent regulation of myosin V in vitro motility, by changing the structure of the regulatory complex, and paradoxically causing calmodulin dissociation through a change in stoichiometry, rather than a Ca(2+)-dependent reduction in affinity.

Amino Acid Motifs↗

PubMed1999

Conformational and metal-binding properties of androcam, a testis-specific, calmodulin-related protein from Drosophila.

Androcam is a testis-specific protein of Drosophila melanogaster, with 67% sequence identity to calmodulin and four potential EF-hand calcium-binding sites. Spectroscopic monitoring of the thermal unfolding of recombinant calcium-free androcam shows a biphasic process characteristic of a two-domain protein, with the apo-N-domain less stable than the apo-C-domain. The two EF hands of the C-domain of androcam bind calcium cooperatively with 40-fold higher average affinity than the corresponding calmodulin sites. Magnesium competes with calcium binding [Ka(Mg) approximately 3 x 10(3) M(-1)]. Weak calcium binding is also detected at one or more N-domain sites. Compared to apo-calmodulin, apo-androcam has a smaller conformational response to calcium and a lower alpha-helical content over a range of experimental conditions. Unlike calmodulin, a tryptic cleavage site in the N-domain of apo-androcam remains trypsin sensitive in the presence of calcium, suggesting an altered calcium-dependent conformational change in this domain. The affinity of model target peptides for androcam is 10(3)-10(5) times lower than for calmodulin, and interaction of the N-domain of androcam with these peptides is significantly reduced. Thus, androcam shows calcium-induced conformational responses typical of a calcium sensor, but its properties indicate calcium sensitivity and target interactions significantly different from those of calmodulin. From the sequence differences and the altered calcium-binding properties it is likely that androcam differs from calmodulin in the conformation of residues in the second calcium-binding loop. Molecular modeling supports the deduction that there are significant conformational differences in the N-domain of androcam compared to calmodulin, and that these could affect the surface, conferring a different specificity on androcam in target interactions related to testis-specific calcium signaling functions.

Amino Acid Sequence↗

PubMed1985

Increased calmodulin levels in psoriasis and low Ca++ regulated mouse epidermal keratinocyte cultures.

Calcium has been shown to regulate the proliferation of epidermal keratinocytes in vitro. We became interested in the role of the calcium binding protein, calmodulin, in hyperproliferative, low calcium regulated keratinocytes in vitro and in the in vivo hyperproliferative state, psoriasis. Calmodulin levels were measured by radioimmune assay in neonatal mouse keratinocytes grown in 0.02 mM calcium (hyperproliferative) and 1.2 mM calcium (normal) media, and in cells that had been grown in low calcium medium and then switched to normal calcium. On a whole culture basis the normal cells had more calmodulin than the low calcium cells. However, when low calcium monolayers were compared to the normal basal monolayer, the low calcium hyperproliferative cells had more calmodulin. Cells that were switched from 0.02 mM calcium to 1.2 mM calcium showed increasing calmodulin levels over time. Psoriatic plaques contained 2-3 times more calmodulin than the skin of normal controls when examined on a per micrograms of DNA, per micrograms of protein, and per gram of wet weight basis. Adjacent uninvolved psoriatic skin also had significantly elevated calmodulin levels in all data bases except per microgram of protein/cm2. These data suggest that increased calmodulin levels are associated with epidermal hyperproliferation and/or with the state of differentiation.

Animals↗

PubMed1981

Altered calmodulin activity in fluphenazine-resistant mutant strains. Pleiotropic effect on development and cellular organization in Volvox carteri.

Genetically altered calmodulin activity in spontaneously derived mutant strains, which were selected for resistance to the toxic effect of a specific inhibitor, the phenothiazine drug fluphenazine, is demonstrated. Partially purified calmodulin preparations from wild-type and fluphenazine-resistant strains of the multicellular alga Volvox carteri, were tested for the ability to activate Ca2+-ATPase of the erythrocyte membranes, and the inhibition of this stimulatory activity by fluphenazine. Unlike the preparation obtained from wild-type cells, mutant calmodulin is shown to be insensitive to fluphenazine inhibition, in one case, and calmodulin from another strain was found to be inactive in vitro, i.e. it did not activate Ca2+-ATPase. The pleiotropic phenotype of the spontaneously derived mutant strains involved aberrant multicellular organization and hormone-independent commitment of the multipotent asexual reproductive cells, gonodia, to sexual development. These results clearly implicate calmodulin in the control of development and morphogenesis in this simple multicellular eukaryote. In addition, intracellular inhibition of calmodulin in wild-type cells is shown to block the morphogenic process of embryo inversion and to arrest motility. The availability of mutant calmodulin will facilitate further investigation of the role of this ubiquitous regulatory protein in the control of development and differentiation in multicellular eukarytes, as well as the fine structure/function relationship with regard to calmodulin modulation of a wide variety of cellular processes.

Calcium-Binding Proteins↗

PubMed1985

Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A stopped-flow fluorescence study using Quin 2 reveals a two-domain structure.

The kinetics of calcium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by fluorescence stopped-flow methods, using the calcium indicator Quin 2. Two distinct rate processes, each corresponding to the release of two calcium ions are resolved for calmodulin at both low and high ionic strength. The effect of 0.1 M KCl is to accelerate the slow process from 9.1 +/- 1.5 s-1 to 24 +/- 6.0 s-1 and to reduce the rate of the fast process from 650 s-1 to 240 +/- 50 s-1 at 25 degrees C. In the presence of 0.1 M KCl it was possible to determine activation parameters for the fast process: delta H# = 41 +/- 5 kJ mol-1 and delta S# = -63 +/- 17 J K-1 mol-1. These values are in good agreement with those obtained by 43Ca NMR. Studies of the tryptic fragments TR1C and TR2C, comprising the N-terminal or C-terminal half of calmodulin, clearly identified Ca2+-binding sites I and II as the low-affinity (rapidly dissociating) sites and sites III and IV as the high-affinity (slowly dissociating) sites. The kinetic properties of the two proteolytic fragments are closely similar to the fast and slowly dissociating sites of native calmodulin, supporting the idea that calmodulin is constructed from two largely independent domains. The presence of the calmodulin antagonist trifluoperazine markedly decreased the Ca2+ dissociation rates from calmodulin. One of the two high-affinity trifluoperazine-binding sites was found to be located on the N-terminal half and the other on the C-terminal half of calmodulin. The affinity of the C-terminal site is at least one order of magnitude greater.

Aminoquinolines↗