Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Calmodulin”

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

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

At least 775 records · Page 43Linked to original sources
PubMed1988

[Calmodulin and neuron: immunohistochemical studies].

Calmodulin is present in higher concentrations in brain tissues. The content rapidly increased during the 2nd postnatal week in rat brain. Although the protein is ubiquitous in all eukaryotic cells, immunohistochemical studies have revealed that calmodulin is mainly localized in the neurons, exhibiting a similar distribution to that of gamma-type neuron-specific enolase. In the mouse retina, both calmodulin and gamma-enolase were found to be localized in optic nerves, ganglion cells, and inner and outer plexiform layers. The development study showed that gamma-enolase increased in the 2nd postnatal week and that the levels of calmodulin did not significantly change in that stage. In the mouse retina with an inherited retinal dysplasia (C3H), in which all the photoreceptor cells degenerate during the 2nd and 3rd postnatal weeks, calmodulin-specific staining decreased in the residual layers. Calmodulin is also enriched in mammalian testes. In the mouse testis, levels of calmodulin were high in the spermatocytes and in the spermatids, as compared to the level in spermatozoa. This suggests that the large amount of calmodulin in the testis may be associated with miotic divisions and/or spermatogenesis. Immunocytochemical staining of calmodulin in C6 glioma cells and PC12 pheochromocytoma cells showed a high level of calmodulin to be localized on the half spindles between poles and chromosomes in mitotic cells. The protein was also shown to be localized on fibrous structures in the interphase of those cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Amino acid sequence of a novel calmodulin from Paramecium tetraurelia that contains dimethyllysine in the first domain.

A class of Paramecium behavioral mutants called pantophobiacs have a deficiency in calcium-dependent potassium efflux, and this deficiency can be corrected by the microinjection of wild-type Paramecium calmodulin (Hinrichsen, R. D., Burgess-Cassler, A., Soltvelt, B. C., Hennessey, T., and Kung, C. (1986) Science 232, 503-506). As a starting point in investigations of which features allow wild-type Paramecium calmodulin to fully restore this behavior while other calmodulins are inactive or poorly effective, we elucidated the amino acid sequence of the wild-type calmodulin. We utilized an approach that combined Edman chemistry with mass spectrometry. This approach resulted in the identification of a new post-translational modification in calmodulin: N epsilon,N epsilon-dimethyllysine at residue 13. This particular modification has not been described for calmodulins studied previously. The only other first-domain modification that has been described for any calmodulin is acetylation of the amino terminus (Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) J. Biol. Chem. 255, 962-975). These results along with analyses of pantophobiac calmodulin and calmodulin binding proteins will provide insight into calmodulin's role in a well-defined behavioral mutant.

Acetylation↗
PubMed1987

Calmodulin stimulation of the rat cerebral cortical adenylate cyclase is required for the detection of guanine nucleotide- or hormone-mediated inhibition.

Functional interactions between the inhibitory guanine nucleotide-regulatory component (Ni) and the adenylate cyclase catalytic subunit (C) from cerebral cortex have been investigated. The inhibition of adenylate cyclase activity by guanosine 5'-(beta, gamma-imido)triphosphate [Gpp(NH)p] was used as a functional measurement of Ni-C interactions in membranes and cholate extracts. Calmodulin stimulation of C activity was required for the detection of Gpp(NH)p inhibition in these preparations. A similar calmodulin requirement was observed for adenosine receptor-mediated inhibition in membranes. The requirement for calmodulin was demonstrated directly in membranes from which calmodulin had been removed by washing and centrifugation. Adenylate cyclase activity in these preparations was not stimulated by free Ca2+ (1 microM). However, upon the readdition of calmodulin (1 microM), these preparations were stimulated 4-fold by Ca2+. Under these assay conditions, Gpp(NH)p- and adenosine receptor-mediated inhibition was absolutely dependent on Ca2+-calmodulin stimulation. However, forskolin stimulation of activity also restored Gpp(NH)p-mediated inhibition. The following experiments were used to implicate the role of calmodulin in detergent-solubilized preparations: (i) by demonstrating that free Ca2+ was required to observe Gpp(NH)p-mediated inhibition, and (ii) by demonstrating that the calmodulin antagonist, calmidazolium, abolished Gpp(NH)p-mediated inhibition while concomitantly decreasing basal activity. As observed in membranes, detection of guanine nucleotide-mediated inhibition required calmodulin stimulation of the detergent-solubilized adenylate cyclase. These results suggest that stimulation of cerebral cortical C activity by either calmodulin or forskolin is required for Ni-mediated inhibition.

Adenylyl Cyclase Inhibitors↗
PubMed1988

Modification of calmodulin on Lys-75 by carbamoylating nitrosoureas.

This paper describes characterization of the reaction of calmodulin with a series of nitrosoureas which are capable of releasing amine-reactive isocyanates of varying hydrophobic character. The site of calcium-dependent carbamoylation on calmodulin by the antineoplastic agent 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (methyl CCNU) was determined to be Lys-75 as demonstrated using [ring-14C]methyl CCNU and sequence analysis of the sole labeled peptide obtained from tryptic digestion of reversed-phase high pressure liquid chromatography (HPLC)-purified radiolabeled calmodulin. CCNU, the 4-desmethylcyclohexyl derivative of methyl CCNU, and its reactive hydrolysis product, cyclohexyl isocyanate, were also determined to modify calmodulin in a similar manner and at the same site, as demonstrated by specific blockade of modification by the calmodulin antagonist calmidazolium. Nitrosoureas which release the less hydrophobic 4-hydroxy- and 4-carboxycyclohexyl isocyanates are unable to modify calmodulin at 25-fold higher concentrations than those required for modification with methyl CCNU, CCNU, or cyclohexyl isocyanate. With this monomodified Lys-75 derivative, purified to homogeneity by HPLC, differential effects of modification on the activation of bovine brain 3',5'-cyclic nucleotide phosphodiesterase (phosphodiesterase) and human erythrocyte Ca2+,Mg2+-ATPase were observed. Compared to the amounts of native calmodulin needed, phosphodiesterase required 7-fold higher amounts of this derivative to reach maximal activation, whereas the activation of the ATPase was unaffected. Clearly, different regions of calmodulin are responsible for the activation of phosphodiesterase and the ATPase. We conclude that Lys-75 is not essential for the function of calmodulin but is in a region of the molecule involved in interaction with phosphodiesterase as well as the binding of certain hydrophobic calmodulin antagonists.

Amino Acid Sequence↗
PubMed1985

Characterization of calmodulin-dependent and cyclic-AMP-dependent protein kinase stimulation of cardiac sarcoplasmic reticulum calcium transport.

The mechanism by which calmodulin stimulates Ca2+ transport in cardiac microsomal preparations enriched in sarcoplasmic reticulum (SR) was investigated. Under incubation conditions in which the majority of the phosphoprotein formed was Ca2+-dependent and no phospholamban phosphorylation was observed (10 degrees C, 15-sec incubations in the presence of 2 microM ATP), calmodulin was found to have no effect on the steady-state level of the acylphosphate phosphorylation site of Ca2+-ATPase. A significant stimulation of Mg2+, Ca2+-ATPase activity by calmodulin and a 3-fold increase in the turnover of the Ca2+ pump were, however, observed. As the ATP concentration in the incubation media was elevated (20 and 200 microM ATP), a significant degree of phosphoprotein formed was observed to be cyclic AMP (cAMP)-dependent. The degree of Ca2+-dependent phosphorylation remained constant. Under these conditions, calmodulin had no effect on the degree of phosphoprotein formed. However, when the experiments were conducted at 30 degrees C for 5 min in the presence of 500 microM ATP, a significant amount of the phosphoprotein formed was calcium-calmodulin-dependent and was additive to phosphoprotein formation observed in the presence of cAMP-dependent protein kinase. The ratio of calcium-calmodulin-dependent to cAMP-dependent phosphorylation was 1:1. K+ (110 mM) decreased the levels of phosphorylation observed in the presence of calcium and calmodulin, but had less of an effect on the levels observed in the presence of cAMP-dependent protein kinase. Autoradiographic analysis of SR membranes labeled with [32P]-ATP revealed two protein bands (24,500 and 40,000 daltons) phosphorylated in the presence of added calcium and calmodulin that were not observed in the absence of either of these additions to the reaction media. These results suggest that calmodulin stimulates Ca2+ transport by a direct effect on the Mg2+, Ca2+-ATPase. An indirect effect on Ca2+ transport via a calcium-calmodulin-dependent protein kinase, though, cannot be ruled out.

Adenosine Triphosphatases↗
PubMed1987

Calmodulin activates adenylate cyclase from rat anterior pituitary.

Bovine brain calmodulin activated adenylate cyclase in calmodulin-deficient rat anterior pituitary membranes. This activation appeared to be specific by the following criteria: 1) calmodulin activation was Ca2+ dependent and responded biphasically to calcium, displaying activation at low and inhibition at higher concentrations; 2) calmidazolium, a potent calmodulin antagonist, inhibited calmodulin activation of adenylate cyclase; 3) activation of the enzyme occurred in a dose-dependent manner, at calmodulin concentrations normally found in most cells (1- to 20-microM range). However, this response was not saturated using calmodulin concentrations as high as 50 microM. The data suggest that endogenous calmodulin can be dissociated from normal anterior pituitary adenylate cyclase, that the enzyme can be subsequently stimulated by addition of micromolar concentrations of calmodulin, and that this enzyme appears to be at least 50-fold less sensitive to calmodulin than is the brain adenylate cyclase.

Adenylyl Cyclases↗
PubMed1986

Effect of cholera toxin on the activation of adenylate cyclase by calmodulin in bovine striatum.

The effect of cholera toxin on activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, GTP, dopamine, and forskolin was investigated in bovine striatum. Adenylate cyclase activity was measured in washed membrane fractions prepared from homogenates that had been preincubated with cholera toxin. Pretreatment of striatal membranes with cholera toxin increased the response of adenylate cyclase to GTP, calmodulin, and forskolin as compared to vehicle controls. After cholera toxin pretreatment, the maximal response of adenylate cyclase to GTP was increased 4.7-fold and the apparent Ka for GTP was reduced 3-fold. The apparent Vmax for calmodulin was doubled after cholera toxin pretreatment. The activation of adenylate cyclase by forskolin was increased by cholera toxin, but the effect on kinetic parameters was not determined due to solubility considerations. In contrast, dopamine-stimulated adenylate cyclase activity was abolished after cholera toxin pretreatment. Examination of a concentration-response curve for cholera toxin in altering these activities revealed that calmodulin-stimulated adenylate cyclase was maximally affected at lower concentrations of cholera toxin than was activation by GTP and forskolin. Cholera toxin also affected the interaction between calmodulin and GTP. In the absence of cholera toxin, calmodulin decreased the apparent Ka for GTP nearly 10-fold. After cholera toxin pretreatment, however, calmodulin could not further decrease the apparent Ka for GTP but increased the maximal response to GTP by 30%. Calmodulin could potentiate GTP activation by stabilizing the interaction between Ns and the catalytic subunit, an action which could be negated by prior treatment with cholera toxin. ADP-ribosylation of the striatal homogenates with [32P]NAD demonstrated predominant labeling of a band of Mr 45,000 which corresponds to the known molecular weight of the alpha-subunit of the stimulatory GTP-binding protein, Ns. These results suggest that the activational state of Ns can affect the stimulation of adenylate cyclase by calmodulin and forskolin. Calmodulin and forskolin may act at separate sites on the catalytic subunit that can allosterically interact with Ns.

Adenosine Diphosphate Ribose↗
PubMed1984

Specific acylation of calmodulin. Synthesis and adduct formation with a fluorenyl-based spin label.

The spin-labeling reagent, N4-(9'-fluorenylmethyloxycarbonyl)-4-amino-1-oxyl-4-succinimidyloxyca rbonyl- 2,2,6,6-tetramethylpiperidine, and the same enriched in 14C at the 4-formyl group, were synthesized as new acylating compounds for protein amino groups that can preserve charge. Porcine testicular calmodulin was modified with this reagent at pH 7.8 in the presence of Ca2+ under conditions that yielded a fairly homogeneous derivative as judged by electrophoretic analysis and tryptic digestion patterns. The tryptic peptides were separated by gel filtration and reverse-phase high-performance liquid chromatography, and the resulting, highly purified 14C-labeled peptides were hydrolyzed and their amino acid compositions determined. The results indicate that at least 87% of the modifications occur at lysyl residues 75 and 148, and the former appears to be the most reactive. This bilabeled calmodulin adduct does not activate a bovine brain cyclic nucleotide phosphodiesterase preparation. The fluorenylmethyloxycarbonyl portion of this inactive calmodulin derivative can, however, be removed by conditions that do not diminish native calmodulin activity in the phosphodiesterase assay. The resulting calmodulin adduct is active in the enzymic assay, although with diminished potency compared to calmodulin. The specificity of the reaction of this acylating reagent with calmodulin may be due to recognition of the tricyclic fluorene ring by the phenothiazine-binding sites since it was found that trifluoperazine inhibited the labeling reaction. Also, calmodulin was far more reactive to this reagent than were several other proteins. This is the first report of a specific, characterized lysine modification on calmodulin, and it is possible that other phenothiazine-binding proteins may also exhibit similar selectivity for acylation. Electron paramagnetic resonance spectra of the calmodulin adducts suggest a high degree of spin immobilization in both the Ca2+-free and Ca2+-saturated states.

Acylation↗
PubMed1981

Studies of the Ca2+ transport mechanism of human erythrocyte inside-out plasma membrane vesicles. I. Regulation of the Ca2+ pump by calmodulin.

Calcium accumulation by human erythrocyte inside-out vesicles was linear for at least 30 min in the presence of ATP. In untreated inside-out vesicles, 3.76 +/- 1.44 nmol of calcium/min/unit of acetylcholinesterase were transported, compared with 10.57 +/- 2.05 (+/- S.D.; n = 11) in those treated with calmodulin. The amount of calmodulin necessary for 50% activation of Ca2+ accumulation was 60 +/- 22 ng/ml (+/- S.D.; n = 4). The Km (Ca2+) for calmodulin-stimulated accumulation was 0.8 +/- 0.05 microM (+/- S.D.; n = 5) using Ca2+ /ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) buffers, or 25 microM with direct addition of unbuffered calcium. In the absence of calmodulin, these values were 0.4 and 60 microM, respectively, Km (ATP) values of 90 and 60 microM in the presence and absence of calmodulin, respectively, were measured at constant magnesium concentration (3 mM). In the presence of calmodulin, a broad pH profile is exhibited from pH 6.6 to 8.2. Maximal calcium accumulation occurs at pH 7.8. In the absence of calmodulin, the pH profile exhibits a linear upward increase from pH 7.0 to 8.2. The (Ca2+-Mg2+)-ATPase activity, measured under identical conditions, was 2.40 +/- 0.72 nmol of Pi/min/unit of acetylcholinesterase in the untreated vesicles and 11.29 +/- 2.87 nmol of Pi/min/unit of acetylcholinesterase (+/- S.D.; n = 4) in calmodulin-treated vesicles. A stoichiometry of 1.6 Ca2+/ATP hydrolyzed was determined in the absence of calmodulin; in the presence of calmodulin, this ratio was decreased to 0.94 Ca2+/ATP hydrolyzed.

Adenosine Triphosphatases↗
PubMed1982

Presence of calmodulin in parathyroid adenomas.

A significant percent of parathyroid adenomas demonstrate decreased sensitivity to the suppressive effects of calcium on parathyroid hormone secretion. The recognition that calmodulin mediates a large number of calcium-dependent cellular events suggests that calmodulin may play a key role in the normal regulation by calcium of parathyroid activity. We therefore undertook to determine if parathyroid adenomas contained calmodulin and if so, if the calmodulin content of adenomas could be correlated with the serum concentrations of calcium and immunoreactive parathyroid hormone (IPTH) in hyperparathyroid patients prior to surgery. Calmodulin was assayed by activation of a calmodulin-stimulatable phosphodiesterase (PDE) obtained from rat brain. We found PDE-stimulating activity in each of six adenoma extracts examined. The stimulation had the following properties, which are characteristic of calmodulin-mediated processes: (1) it required the presence of calcium; (2) the dose response to adenoma extract and human red blood cell calmodulin were parallel, and (3) the stimulation was phenothiazine-inhibitable. The amount of calmodulin present in the adenoma extracts ranged from 0.4 to 1.25 units/micrograms protein. No correlation was found between the calmodulin content of the adenomas and the presurgical levels of either serum calcium or serum IPTH.

Adenoma↗
PubMed1983

Direct effect of insulin on the binding of calmodulin to rat adipocyte plasma membranes.

The interaction of calmodulin with its binding proteins on the adipocyte plasma membrane has previously been described (Goewert, R. R., Landt, M., and McDonald, J. M. (1982) Biochemistry 21, 5310-5315). In this paper we report that insulin directly affects specific calcium-dependent calmodulin binding to adipocyte plasma membranes. The direct effect of insulin on total calcium-dependent 125I-calmodulin binding was studied using 0.8 microM calmodulin. Insulin (100 microunits/ml) directly stimulated the binding of calmodulin by 19.6 +/- 2.3% (n = 6, p less than 0.001) at steady state, whereas the relatively inactive insulin analogue, desoctapeptide insulin, at equimolar concentrations had no effect. Analysis of Scatchard plots indicated that insulin increased the number of high affinity binding sites on the membrane without altering the affinity of these sites. The effect of insulin on the high affinity calmodulin binding was dependent upon increasing concentrations of insulin between 0 and 60 microunits/ml. A maximum stimulation of 75 +/- 17% (n = 4) of calcium-dependent calmodulin binding was observed at 40 microunits/ml of insulin. Effects of insulin were observed within 5 min of initiating 125I-calmodulin binding. These effects of insulin were most prominent above the K0.5 for calcium (approximately 2.0 microM). These direct effects of insulin on high affinity calmodulin binding suggest that the intracellular redistribution of calmodulin may play an important role in the early regulatory events directed by insulin on cellular metabolism.

Adipose Tissue↗
PubMed1984

Quantitation of energy coupling between Ca2+, calmodulin, skeletal muscle myosin light chain kinase, and kinase substrates.

Interactions between Ca2+, calmodulin, rabbit skeletal muscle myosin light chain kinase, and kinase substrates were investigated by microequilibrium dialysis using 45CaCl2 and by fluorescence anisotropy using fluorescent labeled calmodulin. We have determined the free energy coupling for the interaction of Ca2+ and skeletal muscle myosin light chain kinase with calmodulin (delta GCM), and the free energy coupling for the interaction of calmodulin and enzyme substrates with myosin light chain kinase (delta GSCaM). The mean dissociation constants for Ca2+ interaction with calmodulin in the presence and absence of enzyme were 0.40 and 14 microM, respectively, yielding a minimal estimate for delta GCM of -2.11 kcal/mol of Ca2+ (-8.44 kcal/4 mol of Ca2+). Dissociation constants for the interaction of fluorescent 5-iodo-amino-ethyl-amino-naphthalene-1-sulfonic acid-labeled calmodulin and native calmodulin with skeletal muscle myosin light 100 and 15 nM, respectively. In the presence of substrates (phosphorylatable skeletal muscle myosin light chain and App(NH)p) these dissociation constants were 30 and 3 nM, respectively. The free energy coupling for binding of calmodulin and substrates to myosin light chain kinase was -0.95 kcal/mol of calmodulin. These data confirm that interaction of Ca2+ with calmodulin is highly cooperative in the presence of myosin light chain kinase with the degree of cooperativity dependent upon the concentrations of Ca2+, enzyme, and substrates.

Animals↗
PubMed1980

Enzymatic methylation of calmodulin in rat brain cytosol.

The methylation of brain peptides was investigated by incubating brain high speed supernatant fractions with S-adenosyl-L-[methyl-3H]methionine and resolving the radioactive peptide products by polyacrylamide gradient gel electrophoresis. Peptides of 17,000, 21,000, 29,000, and 34,000 daltons were methylated in vitro; the 17,000-dalton peptide had the same electrophoretic mobility as authentic calmodulin and showed the shift in electrophoretic mobility in the presence of EGTA which is characteristic of calmodulin. The identification of the 17,000-dalton peptide as calmodulin was confirmed by electrophoresis under nondenaturing conditions and by two-dimensional isoelectric focusing electrophoresis. The enzyme responsible for calmodulin methylation and its substrate, the putative desmethylcalmodulin, were separated from one another by chromatography on DEAE-cellulose. When this partially purified enzyme was incubated with the substrate-containing fraction, calmodulin was the only methylated product. A test tube assay for calmodulin N-methyltransferase was developed and validated by comparison with calmodulin methylation assessed by gel electrophoresis. Both procedures showed that calmodulin methyltransferase is inhibited by EGTA and stimulated by divalent cations, with manganese giving the highest activity. Similar values for ph optimum and kinetic constants were obtained when the electrophoresis and test tube assays were compared. A rapid chromatographic procedure for the separation of methylated lysines and arginines was developed and used to demonstrate that epsilon-trimethyllysine is the radioactive amino acid formed when calmodulin is methylated in vitro. The methyltransferse, which is distinguished from previously described methylase III enzymes by its divalent cation requirement and substrate specificity, is thus designated as S-adenosyl-L-methionine calmodulin lysine methyltransfease.

Animals↗
PubMed1981

Fluorescence studies of the interaction of calmodulin with myosin light chain kinase.

The interaction of calmodulin with myosin light chain kinase produces an approximately 30% increase in myosin light chain kinase tryptophan fluorescence. This represents the first report of calmodulin-induced structural changes in a protein which it activates. We fund that the calmodulin-myosin light chain kinase interaction is: 1) dependent on [Ca2+] (half-maximal binding at pCa 6.2) and essentially independent of [Mg2+], 2) occurs before saturation of all four reported Ca2+-specific sites on calmodulin. 3) saturates with 1 mol of calmodulin bound per mol of kinase with an apparent affinity of approximately 2.0 X 10(7) M-1, 4) is specific for calmodulin over troponin-C, 5) is directly related to the activation of myosin light chain kinase for phosphorylation of myosin light chain. Fluorescence stopped flow studies of these calmodulin-induced fluorescence changes in myosin light chain kinase indicate that Ca2+ binding to calmodulin occurs very rapidly and is not rate-limiting while the calmodulin-induced fluorescence increase in myosin light chain kinase occurs as a biphasic process with rates of approximately 65 s-1 and 6 s-1. The fluorescence increase produced by calmodulin binding to myosin light chain kinase is completely reversed by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at a rate of approximately 2 s-1.

Animals↗
PubMed1980

Calmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of intestinal epithelial cells.

Isolated microfilament cores of intestinal microvilli are known to contain actin and four major associated proteins among which is calmodulin. Immunofluorescence microscopy reveals that calmodulin is present in the microvilli prior to biochemical fractionation of intestinal cells and thus is not bound artifactually during the isolation procedure. Identification of the major microvillus calmodulin-binding protein was achieved by the use of an [125I]calmodulin gel overlay technique. Proteins of microvilli or brush borders were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After removal of sodium dodecyl sulfate, direct binding of radiolabeled calmodulin to the separated polypeptides was assayed by autoradiography. Three calmodulin-binding polypeptides are detected in brush borders. Two polypeptides (apparent Mr = 280,000 and 140,000) show Ca2+ -dependent binding, whereas the third polypeptide (Mr = 110,000) can bind calmodulin in the presence or absence of Ca2+. Microvillus core filaments contain only the latter species. Microvillus cores treated with 25 mM Mg2+ retain calmodulin and the 110,000 polypeptide, whereas the other two associated proteins are greatly reduced, consistent with the hypothesis that the 110,000 protein is the major calmodulin-binding protein of the core filament structure. We discuss the currently documentable structure of the core filaments and evaluate the general usefulness of the calmodulin gel overlay technique.

Animals↗
PubMed1994

Ethanol modulates [125I]calmodulin binding to synaptic plasma membranes from rat brain.

The effects of ethanol in vitro on Ca(++)-dependent binding of [125I] calmodulin to brain synaptic plasma membranes (SPM) from control and chronically ethanol-treated rats were studied. In SPM from control animals, ethanol at 50 to 200 mM inhibited [125I] calmodulin binding; the inhibition was correlated with a decreased membrane affinity for [125I]calmodulin as shown by Scatchard analysis, and an increased dissociation of [125I]calmodulin-membrane complexes as shown by kinetic analysis. Arrhenius analysis indicates that [125I]calmodulin binding was influenced by lipid transition of the membrane, and that ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were found to be resistant to the inhibitory effect of ethanol on binding. The resistance to ethanol inhibition was correlated with a higher membrane affinity for [125I]calmodulin and a higher transition temperature, as compared with control SPM. Because a variety of membrane-bound processes are regulated by calmodulin or calmodulin-dependent processes, the inhibitory effect of ethanol on membrane binding of calmodulin could lead to a cascade of consequences in synaptic function. Moreover, the resistance of the membranes to ethanol inhibition after chronic ethanol treatment implies that membrane binding of calmodulin is part of the mechanism underlying alcohol tolerance and dependence.

Animals↗
PubMed1993

Glycated calmodulin from platelets as an index of glycemic control.

In an effort to test whether a significant fraction of calmodulin would become glycated within the life span of the platelet (10-14 days), we monitored the kinetics of calmodulin glycation in vitro. Under the conditions we used, the fraction of glycated calmodulin reached a maximum (approximately 21%) within 10 days. We then extended the studies to human subjects. The intraplatelet concentrations of calmodulin and glycated calmodulin from age-matched type I diabetic subjects were monitored by a combination of m-aminophenylboronate affinity chromatography and enzyme-linked immunosorbent assay. The results indicate that the concentrations of total intraplatelet calmodulin (nonglycated plus glycated) were not dependent on the glycemic state of the subjects. Data from control and diabetic subjects showed a poor correlation between the concentrations of glycohemoglobin and of glycated calmodulin. However, a better correlation was obtained when glycated calmodulin concentrations were compared with those of serum fructosamine. The fraction of glycated calmodulin in the control population (7.71% +/- 0.75%) was significantly (P < 0.05) different from that of the diabetic population (21.6% +/- 1.26%). Given that the clinical role of the fructosamine assay remains controversial, estimation of glycated calmodulin in platelets might be useful as a short time-window index of glycemic control.

Adolescent↗
PubMed1997

Proliferative stimulation of lymphocytes by calmodulin binding proteins isolated from amniotic fluid.

In view of the role of calmodulin and calmodulin binding proteins in modulating the second messenger functions of Ca2+, we studied the presence of such proteins in amniotic fluid, which may be considered as a dynamic medium for promoting foetal growth. Affinity chromatography of amniotic fluid proteins revealed the presence of calmodulin binding proteins in samples obtained either at 28 or 36 wk of pregnancy. The relative content of these proteins increased in amniotic fluid from 1.5 mg/g total protein at 28 wk to 3.6 mg/g at full term of pregnancy. Culturing murine splenocytes in presence of the isolated calmodulin binding proteins (10 micrograms protein/10(6) cells) resulted in nearly 4-fold enhancement of 3H-thymidine incorporation into them as compared to controls. In comparison, similar incorporation of the radiolabel into lymphocytes obtained from cord blood was enhanced only by 2 fold in presence of calmodulin binding proteins, though at a much lower protein concentration (50 ng/10(6) cells). SDS-PAGE on 12.5 per cent gels of eluates obtained from calmodulin-agarose columns, followed by overlay of corresponding western blots with biotinylated calmodulin revealed the presence of a 68 kDa calmodulin binding protein in samples collected either at 28 wk or at full-term of pregnancy. In addition, the amniotic fluid also contained 83 kDa calmodulin binding protein at 28 wk. This first-time demonstration of mitogenic, calmodulin binding proteins in amniotic fluid suggests that such mitogens may participate in promoting foetal growth.

Amniotic Fluid↗