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At least 235 records · Page 13Linked to original sources
PubMed1983

Production of a high affinity antibody specific to the calcium-free-form of calmodulin, using N-acetyl-muramyl-L-alanyl-D-isoglutamine-calmodulin conjugate.

Covalent coupling of N-acetyl-muramyl-L-alanyl-D--isoglutamine (MDP) to calmodulin was used in order to enhance its immunogenicity. Rabbit antibodies against calmodulin were obtained, interacting with the calcium--free-form of calmodulin. The radioimmunoassay was developed using the whole sera (titre 1:20,000) having a sensitivity of 20 pg per tube (50% displacement 500 pg/assay tube). The radioimmunoassay shows the same concentrations of calmodulin in rat brain homogenate (4.9 +/- 0.86 micrograms/mg protein), bovine neurosecretosomes (0.77 +/- 0.10 microgram/mg protein), and bovine neurohypophysial secretory vesicles (0.05 +/- 0.01 microgram/mg protein) as the phosphodiesterase activation method.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1981

Subcellular distribution of calmodulin and calmodulin-binding sites in Tetrahymena pyriformis.

The subcellular distribution of calmodulin and particulate calmodulin-binding activity was studied in a eukaryotic protozoan, Tetrahymena pyriformis NT-1. The particulate calmodulin-binding activity was found to be localized principally in microsomes and to some extent in cilia and surface membranes called pellicles. Nearly all (93%) of the total amount of calmodulin was recovered in two soluble compartments, the ciliary and postmicrosomal supernatant fractions.

Animals↗
PubMed1991

Specific inositol phosphates inhibit basal and calmodulin-stimulated Ca(2+)-ATPase activity in human erythrocyte membranes in vitro and inhibit binding of calmodulin to membranes.

D-Myo-inositol 1,4,5-trisphosphate (Ins[1,4-,5]P3) inhibits rat heart sarcolemmal Ca(2+)-ATPase activity (T. H. Kuo, Biochem. Biophys. Res. Commun. 152: 1111, 1988). We have studied the effect and mechanism of action of Ins(1,4,5)P3 and related inositol phosphates on human red cell membrane Ca(2+)-ATPase (EC 3.6.1.3) activity in vitro. At 10(-6) M, Ins(1,4,5)P3 and D-myo-inositol 4,5-bisphosphate (Ins[4,5]P2) inhibited human erythrocyte membrane Ca(2+)-ATPase activity in vitro by 42 and 31%, respectively. D-Myo-inositol 1,3,4,5-tetrakisphosphate, D-myo-inositol 1,4-bisphosphate, and D-myo-inositol 1-phosphate were not inhibitory. Enzyme inhibition by Ins(1,4,5)P3 was blocked by heparin. Exogenous purified calmodulin also stimulated red cell membrane Ca(2+)-ATPase activity; this stimulation was inhibited by Ins(1,4,5)P3. Ins(4,5)P2 and Ins(1,4,5)P3, but not Ins(1,4)P2, inhibited the binding of [125I]calmodulin to red cell membranes. Thus, specific inositol phosphates reduce plasma membrane Ca(2+)-ATPase activity and enhancement of the latter in vitro by purified calmodulin. The mechanism of these effects may in part relate to inhibition by inositol phosphates of binding of calmodulin to erythrocyte membranes.

Calcium-Transporting ATPases↗
PubMed1984

Effects of extracellular calmodulin and calmodulin antagonists on B16 melanoma cell growth.

Two drugs known to inhibit the action of calmodulin, prochlorperazine offP) and N-(6-aminohexyl)-5-chloro-1-napthalene sulfonamide (W7), were investigated for their ability to control cell proliferation in murine B16 melanoma cells in culture. PCP and W7 inhibited [3H]thymidine uptake in these cells, 50% inhibition occurring with 13 microM PCP and 40 microM W7. In the presence of relatively high concentrations of fetal calf serum (FCS), cells withstood high concentrations of both drugs (100 microM PCP and 200 microM W7) and showed increased pigment production. Drug-inhibited DNA synthesis could be reversed by the addition of fresh medium containing FCS or by the addition of exogenous pure calmodulin. Extracellular calmodulin itself stimulated DNA synthesis. FCS was found to contain calmodulin-like activity at concentrations that may be relevant to the stimulation of [3H]thymidine uptake by cells in culture.

Animals↗
PubMed1989

The role of calmodulin in rat parotid amylase secretion: effects of calmodulin antagonists on secretion and acinar cell structure.

Using dispersed rat parotid cells, the effects of three calmodulin antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide (W-7), and N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), on amylase release and acinar cell structure were examined. TFP and W-7 strongly inhibited both isoproterenol (ISO)- and dibutyryl cyclic AMP-stimulated amylase release at a concentration of 50 or 100 microM, while W-5, a weak calmodulin antagonist, had only little effect. Cyclic AMP level was markedly elevated by ISO even in the presence of TFP or W-7. These results indicate that the calmodulin antagonists affect amylase release at steps distal to cyclic AMP metabolism. Electron micrographs demonstrated that treatment of parotid cells with either TFP or W-7 caused a loss of luminal microvilli and surface folds. When cells were stimulated by ISO in the presence of TFP or W-7, the enlarged lumina did not recover to their original size and the discharged secretory material was retained in the lumina. Numerous secretory granules remained in the acinar cytoplasm. W-5 affected the acinar cell structure only a little. These observations lead to the assumption that TFP and W-7 interfered with the normal functions of the cytoskeletal system. It is proposed that calmodulin may be involved in the exocytosis of parotid amylase through the regulation of the cytoskeletal system.

Amylases↗
PubMed1992

The effects of calcium site occupancy and reagent length on reactivity of calmodulin lysyl residues with heterobifunctional aryl azides. Mapping interaction domains with specific calmodulin photoprobe derivatives.

The relationship of structural and functional moieties on calmodulin is important in all venues of cell activity. In this study, we investigate the effect of lysine modification on calmodulin function. Azidosalicylate reagents containing different "linker arm" lengths, between the photoactive terminus and an amine-reactive N-hydroxysuccinimidyl ester moiety were used to modify calmodulin lysines at three different positions in a calcium-dependent manner. The short cross-linker, (ASNE-2 (where ASNE represents azidosalicylate N-hydroxysuccinimidyl ester), modifies Lys-75, whereas the longer reagent, ASNE-6, modifies lysines 21, 75, and 94. The modification of these different lysines is shown to be calcium-dependent. At 1-100 microM levels of calcium, only Lys-94 is modified, suggesting that modification of this residue is directed by both the binding of calcium to calcium-binding loops III and IV and the hydrophobic pocket exposed between these two loops as a result of calcium binding. At higher calcium concentrations (> 200 microM), where sites I and II become filled, modification of Lys-21 or Lys-75 also was observed. All the modified calmodulins were able to stimulate 3',5'-cyclic-nucleotide phosphodiesterase fully although the Kact for the Lys-75 and Lys-21 derivatives increased 10- and 50-fold, respectively. None of the modifications affected the activation of erythrocyte plasma membrane Ca(2+)-ATPase. Only the ASNE-6 Lys-75 derivative showed efficient (40%) photocross-linking to the Ca(2+)-ATPase. The ASNE-2 Lys-75 derivative as well as the ASNE-6 Lys-21 and Lys-94 derivatives did not show efficient calcium-dependent photocross-linking to this enzyme.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Calmodulin sensitive phosphodiesterase of porcine cerebral cortex: kinetic behavior, calmodulin activation, and stability.

The calmodulin sensitive phosphodiesterase of porcine cerebral cortex was characterized in terms of kinetic behavior, calmodulin activation, and stability. This enzyme displayed non-Michaelis-Menten kinetics in the presence or absence of calmodulin. The apparent affinity for cyclic GMP was higher than that for cyclic AMP but at saturating levels of substrate, this enzyme catalyzed the hydrolysis of cyclic AMP at a greater rate than it did cyclic GMP. The affinity of this enzyme for calmodulin was about 20-fold lower than usually reported. The apparent loss of phosphodiesterase activity after storage was found to be due to a strong association with container surfaces and could be prevented or reversed by the presence of 0.1% Triton X-100.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

p-Benzoyl-L-phenylalanine, a new photoreactive amino acid. Photolabeling of calmodulin with a synthetic calmodulin-binding peptide.

A new photoreactive amino acid analog, p-benzoyl-L-phenylalanine, is described. Convenient methods for the preparation of this amino acid and its subsequent incorporation into synthetic peptides by the solid-phase technique are outlined. To illustrate its utility, p-benzoyl-L-phenylalanine was substituted in place of tryptophan in a 17-residue calmodulin-binding peptide. The substitution did not measurably affect the affinity of this peptide for calmodulin. When this peptide was photolyzed at 350 nm in a 1:1 molar ratio with calmodulin in the presence of 500 microM CaCl2, 70% of the calmodulin was derivatized. The specificity of the reaction was investigated by photolysis in the absence of CaCl2 where little binding occurs; under these conditions little or no photolabeling occurred.

Affinity Labels↗
PubMed1995

Touch-inducible genes for calmodulin and a calmodulin-related protein are located in tandem on a chromosome of Arabidopsis thaliana.

Genes for calmodulin and calmodulin-related proteins in Arabidopsis are up-regulated by a variety of physical stimuli, which include rain, wind and touch [Braam and Davis (1990) Cell 60: 357]. We have isolated five genes for calmodulin (AtCAL1, 2, 3, 5, 6) and one gene for a calmodulin-related protein (AtCAL4) from an Arabidopsis genomic library. Touch stimulus of Arabidopsis plants induces the accumulation of mRNA transcribed from AtCAL4 and AtCAL5, but not from the other isolated genes. The two touch-inducible genes are arrayed in tandem with a short intergenic region of 700 bp but they show different organ-specific patterns of expression.

Arabidopsis↗
PubMed1998

Sequences of cDNAs encoding calmodulin, and partial structures of calmodulin kinase, and a calcium channel of kdr-resistant and -susceptible German cockroaches, Blattella germanica.

Complementary DNA sequences of genes encoding calmodulin, partial structures of calmodulin-dependent protein kinase II (CaM-kinase II) and an L-type-like calcium channel al subunit (IVS5-IVS6-EF hand region) were identified and compared between susceptible and kdr strains of German cockroach, Blattella germanica. For this purpose, polymerase chain reactions (PCR) were used to obtain their sequences using cDNA from poly(A) + RNA isolated from their heads and thoraces. No mutation differences were found in all three sequences of calcium-regulating proteins between susceptible and strain. Northern blot analysis, however, showed reduced expressions of CaM-kinase II mRNA in two kdr strains. Western blot analysis with an antibody preparation against CaM-kinase II on protein levels confirmed the above strain difference in the titer of this enzyme. In contrast, the levels of calmodulin as well as that of an L-type-like calcium channel gene expression were not different between susceptible and kdr strains.

Amino Acid Sequence↗
PubMed2000

Chronic elevation of calmodulin in the ventricles of transgenic mice increases the autonomous activity of calmodulin-dependent protein kinase II, which regulates atrial natriuretic factor gene expression.

Although isoforms of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been implicated in the regulation of gene expression in cultured cells, this issue has yet to be addressed in vivo. We report that the overexpression of calmodulin in ventricular myocytes of transgenic mice results in an increase in the Ca2+/calmodulin-independent activity of endogenous CaMKII. The calmodulin transgene is regulated by a 500-bp fragment of the atrial natriuretic factor (ANF) gene promoter which, based on cell transfection studies, is itself known to be regulated by CaMKII. The increased autonomous activity of CaMKII maintains the activity of the transgene and establishes a positive feed-forward loop, which also extends the temporal expression of the endogenous ANF promoter in ventricular myocytes. Both the increased activity of CaMKII and transcriptional activation of ANF are highly selective responses to the chronic overexpression of calmodulin. These results indicate that CaMKII can regulate gene expression in vivo and suggest that this enzyme may represent the Ca2+-dependent target responsible for reactivation of the ANF gene during ventricular hypertrophy.

Age Factors↗
PubMed1988

Analysis of calmodulin acceptor proteins and the influence of calmodulin antagonists on human spermatozoa.

The possible role of calmodulin in regulating a number of calcium-dependent functions exhibited by human spermatozoa was investigated by using the antagonists trifluoperazine and calmidazolium. At high doses both antagonists inhibited the motility of human spermatozoa and induced a concomitant rise in [Ca2+]i and a decline in cAMP. Lower doses of these antagonists, particularly calmidazolium, suppressed the ability of human spermatozoa to generate reactive oxygen species and exhibit sperm-oocyte fusion, without influencing [Ca2+]i, cAMP, or motility. This inhibition of sperm-oocyte fusion was effective even if the spermatozoa were subsequently exposed to A23187, suggesting that calmodulin may regulate this aspect of human sperm function at a point downstream from calcium influx. Both radiolabelling and affinity chromatography techniques were used to detect a number of calcium-dependent and calcium-independent calmodulin acceptor proteins in the human spermatozoon. The major calcium-dependent acceptor proteins exhibited Mr values of 32,000 and 22,000-27,000, respectively, and did not appear to be associated with the sperm plasma membrane.

Calcium↗
PubMed1998

Cell separation based on the reversible interaction between calmodulin and a calmodulin-binding peptide.

A cell separation system based on the calcium-dependent interaction of calmodulin (CM) with a calmodulin-binding peptide (CBP) has been developed. The prototype of this system utilizes an indirect method to label the target cell population. Cells are first labeled with a primary monoclonal antibody directed to a specific cell surface antigen, then with a secondary affinity reagent, consisting of a polyclonal goat anti-mouse IgG (GAM-IgG) that has been cross-linked to a CBP derived from the sequence of the rabbit skeletal muscle myosin light chain kinase. In the presence of Ca2+, the CBP on the cells labeled with GAM-IgG-CBP binds to biotinylated calmodulin (CM-Biotin) with high affinity. The target cells are then captured with a solid-phase streptavidin. The unbound non-target cells are washed away and the immobilized target cells are released by chelating Ca2+ with EGTA. The specificity of the GAM-IgG-CBP and CM-Biotin and the feasibility of using this system to separate cells was demonstrated using the KG-1 human acute myelogenous leukemia cell line. KG-1 cells were fractionated on the basis of cell surface expression of HLA-DR. The cell selection reagents and the cell separation process did not affect KG-1 cell viability while cells selected by this procedure were 90% pure with a yield of 75%. This cell separation system also was used for rare cell isolation from normal human peripheral blood mononuclear cells. T cells expressing the Vbeta5 T cell receptor, which represent < 5% of the unfractionated cells, were isolated with 89% viability, 72% purity, 80% yield, and retained the ability to respond to activation signals as measured by blast transformation. The results from this study show that a cell selection system based on the reversible interaction between CM and a CBP can be applied to gently and efficiently isolate cells from a heterogeneous starting population that are free of the solid matrix without exposure to the stresses of mechanical or enzymatic release.

Biotin↗
PubMed1991

Presence of calmodulin and calmodulin-binding proteins in the nuclei of brain cells.

The nuclear calmodulin levels have been measured in rat neurons and glial cells. The values are 1.0 and 1.1 micrograms/mg of protein, respectively. These levels are about threefold higher than those in the nuclei of rat liver cells. We have also investigated the presence of several calmodulin-binding proteins in the nuclei of both brain cellular types. As similarly observed in the nuclei of liver cells, we detected the presence of alpha-spectrin and a 62-kDa calmodulin-binding protein (p62) in the nuclei of neurons and glial cells by immunoblotting and immunocytochemical methods. Both proteins are enriched in the purified nuclear matrix samples from both cellular types. In contrast to that occurring in rat hepatocytes, we have not been able to detect, by immunoblotting methods, caldesmon in the nuclear matrices of neurons and glial cells. The immunocytochemical studies suggest, however, that caldesmon can be present in the nuclei but in a fraction distinct from the nuclear matrices.

Animals↗
PubMed1988

Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein.

Neuromodulin (formerly designated P-57) is an abundant, neural specific, calmodulin-binding protein which exhibits higher affinity for calmodulin in the absence of free Ca2+ than in the presence of free Ca2+. In this study a series of proteolytic fragments of neuromodulin were systematically screened for calmodulin-Sepharose binding activity. A 9-amino acid fragment, designated M1-C1 and having the sequence RGHITRKKL, was identified as the putative CaM-binding domain of neuromodulin. Two heptadecapeptides, designated FP57-Phe and FP57-Trp, were synthesized, each containing the M1-C1 sequence and the four flanking amino acids from each site. The FP57-Trp peptide contained a tryptophan residue in place of the native phenylalanine. Anti-FP57-Phe antibody binding to neuromodulin was inhibited by preincubation of antibodies with excess FP57-Phe. 125I-CaM gel overlay of neuromodulin was inhibited by anti-FP57-Phe antibodies. Addition of CaM to FP57-Trp increased peptide tryptophanyl fluorescence. In the presence of Ca2+, the stoichiometry of the FP57-Trp.CaM complex was 1:1, FP57-Trp binding to CaM was competitive with neuromodulin. The Ca2+-independent dissociation constant of the FP57-Phe.CaM complex was 0.41 microM. The Ca2+-dependent affinity of the complex could not be measured directly but appeared to be significantly greater than the Ca2+-independent affinity.

Amino Acid Sequence↗
PubMed1986

Purification and characterization of bovine lung calmodulin-dependent cyclic nucleotide phosphodiesterase. An enzyme containing calmodulin as a subunit.

A rabbit lung cyclic nucleotide phosphodiesterase (PDE) prepared by successive chromatography on DEAE-cellulose and G-200 Sephadex columns in the presence of EGTA was activated by Ca2+ and contained calmodulin (CaM), suggesting that the enzyme exists as a stable CaM X PDE complex (Sharma, R. K., and Wirch, E. (1979) Biochem. Biophys. Res. Commun. 91, 338-344). An enzyme with similar properties was demonstrated to exist in bovine lung extract. C1, a monoclonal antibody previously shown to react with the 60-kDa subunit of bovine brain PDE isozymes (Sharma, R. K., Adachi, A.-M., Adachi, K., and Wang, J. H.) (1984) J. Biol. Chem. 259, 9248-9254), cross-reacted with the lung enzyme. Purification of the lung enzyme by C1 antibody immunoaffinity chromatography rendered the enzyme dependent on exogenous CaM for Ca2+ stimulation. Further purification was achieved by CaM affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed a predominant polypeptide of Mr 58,000 and a minor band of about 50,000. The purified enzyme could be reconstituted into a PDE X CaM complex upon incubation with CaM in the presence of either Ca2+ or EGTA. The reconstituted protein complex did not dissociate in buffers containing 0.1 mM EGTA. Analysis of the purified and reconstituted lung phosphodiesterase by Sephacryl S-300 gel filtration indicated that the lung enzyme is a dimeric protein and that the reconstituted enzyme contained two molecules of calmodulin. Analysis of the reconstituted phosphodiesterase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also showed it to contain equimolar calmodulin and the enzyme subunit. The CaM antagonists, fluphenazine, compound 48/80, and calcineurin at concentrations abolishing CaM stimulation of bovine brain PDE had little effect on the activity of reconstituted bovine lung phosphodiesterase.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1999

Structural features underlying the unusual mode of calmodulin phosphorylation by protein kinase CK2: A study with synthetic calmodulin fragments.

To shed light on the paradoxical behaviour of calmodulin, whose phosphorylation is inhibited by the regulatory beta-subunit of protein kinase CK2, a series of peptides encompassing the phosphoacceptor sites of calmodulin have been synthesized and assayed as substrates of CK2 alpha-subunit either alone or combined with the beta-subunit. The shortest peptide whose phosphorylation is reduced instead of being enhanced by the beta-subunit encompasses the sequence 68-106, including the central helix and the Ca2+-binding loop-III. In contrast, the phosphorylation of a peptide encompassing loop II and the central helix (54-92) is stimulated, like that of several shorter peptides, by the beta-subunit. Our data localize to the C-terminal domain of calmodulin the structural elements that are responsible for inverted susceptibility to beta-subunit regulation.

Amino Acid Sequence↗
PubMed1999

Association of calmodulin with cytoskeletal structures at different stages of HeLa cell division, visualized by a calmodulin-EGFP fusion protein.

The fusion protein of calmodulin (CaM) with the enhanced green fluorescent protein EGFP has been expressed in a stably transfected HeLa cell line in order to visualise the localisation of calmodulin during the cell cycle on a continuous basis in live cells, and for immunofluorescence colocalisation with cytoskeletal structures. High-resolution images of CaM-EGFP in the mitotic apparatus show the characteristic strongly convoluted structure of the centrosome. CaM-EGFP also apparently associates with both polar and mitotic microtubules, and with a specific intracentrosomal structure. During cytokinesis, CaM-EGFP is also found decorating selected oriented filaments in close proximity to microtubules in the midbody region. In interphase cells, it is seen with filamentous and punctuate localisation at the nuclear envelope. The intensity and continuity of the CaM-EGFP images suggest that a significant fraction of the cellular calmodulin remains attached to cytoplasmic structures during the cell cycle.

Calmodulin↗