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Biomedical subjects

J G Demaille

Publications and source records attributed to J G Demaille.

At least 55 records · Page 3Linked to original sources
PubMed1981

Effects of cations on affinity of calmodulin for calcium: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes.

The acid stability of calmodulin has been used to devise a rapid and efficient method of decalcification based on trichloroacetic acid precipitation. Study of the competitive binding of K+, Mg2+, and Ca2+ to the Ca2+-binding sites of calmodulin has allowed determination of the intrinsic binding constants of each of the three cations for the four Ca2+-binding sites. The data are compatible with an ordered binding of Ca2+. If the Ca2+ sites are labeled A, B, C, and D starting at the NH2 terminus, the order of binding is postulated to be B, A, C, and D. The ordered binding properties support the suggestion that calmodulin translates quantitative Ca2+ signals into qualitatively different cellular responses.

Binding Sites↗
PubMed1981

Cyclic adenosine 3',5'-monophosphate-dependent regulation of purified bovine aortic calcium/calmodulin-dependent myosin light chain kinase.

Myosin light chain kinase was extracted from bovine aortic muscularis by a low ionic strength buffer containing 50% glycerol. It was purified 130-fold with a 10% yield by anion-exchange chromatography followed by affinity chromatography on calmodulin-Sepharose. The enzyme was 95% calcium/calmodulin-dependent and exhibited a specific activity of 2-6 mumol/min per mg. It phosphorylated the myosin regulatory light chain exclusively. The apparent Kd for calmodulin was 6.3 nM. Upon phosphorylation of the enzyme by the catalytic subunit of cyclic AMP-dependent protein kinase, its affinity for calmodulin decreased 4-fold, without alteration of the V. When examined by SDS-polyacrylamide gel electrophoresis, the purified enzyme was made up of two major peptides (Mr 142 000 and 131 000, respectively), with a minor 80 000 dalton peptide. All these peptides were 32P-labeled after incubation with [gamma-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. Also, after non-denaturing polyacrylamide gel electrophoresis, they all exhibited myosin light chain kinase activity, suggesting that the 131 000 and 80 000 dalton species are proteolytic products of the native enzyme of Mr 142 000. Vascular smooth muscle myosin light chain kinase is therefore soluble, calcium/calmodulin dependent and phosphorylatable by cyclic AMP-dependent protein kinase with concomitant decrease in its affinity for calmodulin. These features account for the beta-adrenergic relaxation of vascular smooth muscle.

Animals↗
PubMed1981

Immunocytochemical and biochemical evidence for the presence of calmodulin in bull sperm flagellum. Isolation and characterization of sperm calmodulin.

Upon fluorescent staining with a goat antibody anti-ram testis calmodulin, washed bull sperm appears to contain calmodulin in the acrosome, in the post acrosomal region, in the neck region probably associated with the implantation plates and thin laminated fibers, and in a sheath around the upper part of the flagellum. Heads and midpieces + tails were separated by elutriation of sonicated sperm. Immunofluorescent labeling of fragments confirms the presence of calmodulin in implantation plates, where sonication disrupted heads from midpieces, and in a sheath around the midpiece and the upper part of the principal piece. These results were confirmed by electrophoretic and radioenzymatic assays of calmodulin in the fragments, using calmodulin-deficient Ca2+/calmodulin-dependent myosin light chain kinase. Small but significant amounts (approx. 3 micrograms per 10 (10) sperm) are found in midpieces + tails vs. approx. 280 micrograms in the same number of heads. These results are in agreement with a recent report from Jones et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2772-2776. Sperm calmodulin was purified from a whole sperm 1 M KCl extract and found to exhibit the same characteristics as other mammalian calmodulins isolated so far in terms of ultraviolet absorption spectrum and amino acid composition, including one residue of epsilon-N-trimethyllysine. Its behavior upon SDS-polyacrylamide gel electrophoresis was dependent on the presence or absence of Ca2+. The high performance liquid chromatography tryptic peptide maps were similar, if not identical, to mammalian calmodulin maps (Autric et al. (1980) Biochim. Biophys. Acta 631, 139-147). Sperm calmodulin is therefore probably identical to the somatic cell protein.

Amino Acids↗
PubMed1981

Octopus calmodulin. The trimethyllysyl residue is not required for myosin light chain kinase activation.

Octopus calmodulin was purified to homogeneity and shown to contain 0.1 residue each of epsilon-N-monomethyl-lysine, epsilon-N-dimethyllysine, and epsilon-N-trimethyllysine/mol. With the exception of this partial methylation and of a single tyrosyl residue, it shared all the characteristic properties of mammalian calmodulin in terms of molecular weight, amino acid composition, electrophoretic behavior in the presence or absence of Ca2+ ions, and activation of calcium/calmodulin-dependent myosin light chain kinase. In fact, Octopus calmodulin proved to be slightly more effective than ram testis calmodulin in activating both skeletal and smooth muscle myosin light chain kinases in the presence of Ca2+. This provides conclusive evidence that (a) stoichiometric trimethylation of lysine 115 is not required for enzyme activation, and (b) the inability of troponin C to activate myosin light chain kinase (Walsh, M. P., Vallet, B., Cavadore, J. C., and Demaille, J. G.

Adenosine Triphosphatases↗
PubMed1981

[Meiotic maturation of the ovocyte of Xenopus laevis : a 4-step mechanism].

Microinjection of the pure inhibitor (1) of cAMP dependent protein kinases (1.5 muM) into Xenopus oocytes triggered 100% germinal vesicle break down (GVBD), faster than progesterone and slower than the maturation promoting factor (MPF). Cholera toxin does not block (I) induced maturation; however cycloheximide inhibits (I) but not MPF induced GVBD.

Animals↗
PubMed1981

Calmodulin is involved in the first step of oocyte maturation: effects of the antipsychotic drug fluphenazine and of anticalmodulin antibodies on the progesterone-induced maturation of xenopus laevis oocyte.

Specific anticalmodulin antibodies were microinjected into full-grown Xenopus laevis oocyte, and it is shown that in ovo blockade of the complex Ca2+-calmodulin accelerates the kinetics of progesterone-induced maturation, even though the molar ratio of antibody binding sites to total calmodulin was only 0.16. Addition of 200 microM fluphenazine to the oocyte incubation medium resulted in a similar acceleration of steroid-induced maturation. Neither protein kinase inhibitor (PKI) nor maturation promoting factor (MPF)-induced maturation are affected either by the antipsychotic drug or by anticalmodulin antibodies; this result suggests that the adenylate cyclase system may be the target for anticalmodulin antibodies and fluphenazine effects.

Animals↗
PubMed1981

The pure inhibitor of cAMP-dependent protein kinase initiates Xenopus laevis meiotic maturation. A 4-step scheme for meiotic maturation.

The availability of the pure inhibitor of cAMP-dependent protein kinase prompted a re-examination of the inhibitor-induced meiotic maturation of Xenopus laevis oocytes. Injection of the inhibitor (1.5 microM) triggered 100% germinal vesicle breakdown faster than progesterone and slower than the maturation-promoting factor: at 0.15 microM, the inhibitor still triggered 100% meiosis, but with a much slower kinetics. In contrast, injection of 24 microM calmodulin resulted in less than 50% GVBD, and results were variable from female to female. Combined injection of inhibitor and calmodulin failed to show any synergism, which does not favour hypotheses according to which calmodulin acts by activation of cyclic nucleotide phosphodiesterase. The net effect of the inhibitor is to decrease the concentration of the free catalytic sub-unit of cAMP-dependent protein kinase, fully dissociated in the unstimulated oocyte, as shown by the absence of effect of pretreatment with cholera toxin on the inhibitor-induced maturation. After such decrease by about 1 microM, a maturation protein, Mp-P, is dephosphorylated by phosphoprotein phosphatases. Dephospho-Mp triggers the synthesis of MPF in cycloheximide-sensitive steps. Finally, MPF triggers GVBD in steps insensitive to cycloheximide. Evidence for such a 4-step scheme--fall in cAMP levels, then in C sub-unit levels, dephosphorylation of Mp leading to the synthesis of MPF and finally MPF-triggered GVBD--is presented and discussed.

Animals↗
PubMed1981

Calcium-calmodulin-dependent phosphorylations in the control of muscular contraction?

Muscular contraction is triggered by the increase in free calcium concentration and modulated by cyclic nucleotide-dependent phosphorylation. Beside a direct trigger of sarcomeric muscle contraction through binding of troponin C, calcium ions trigger or modulate contractility through calcium-calmodulin-dependent myosin light chain kinases, and increase the rate of relaxation through the calmodulin-dependent phosphorylation of phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump. In both cases, a concerted regulation by calcium and cyclic nucleotides is observed. Hyperactivation of the calcium pump is brought about by additional phosphorylation of phospholamban by cAMP-dependent protein kinase. Similarly myofibrillar myosin light chain kinases from smooth and skeletal muscles are substrates of the cAMP-dependent protein kinase. The calmodulin-dependent protein kinases are probably organized into supramolecular regulatory complexes.

Calcium↗
PubMed1981

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

The complete amino acid sequence of the 349-residue catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle is presented. The sequence of the subunit (Mr 40,580 including phosphate groups at threonine-196 and serine-337) was derived largely by automated Edman degradation of nine fragments generated from the carboxymethylated protein by cleavage of methionyl bonds with cyanogen bromide. These fragments were aligned along the polypeptide chain by analysis of methionine-containing tryptic peptides isolated from protein radiolabeled in vitro by [14C]methyl exchange at methionyl residues. The molecule contains only two cysteinyl residues, at positions 198 and 342. It is relatively polar, containing clusters of cationic residues toward the amino terminus and anionic residues towards the carboxyl terminus. Predictions of secondary structure suggest the presence of three major domains with approximately half of the residues occurring in alpha-helices and 12% in beta-strands.

Amino Acid Sequence↗
PubMed1980

Xenopus laevis oocyte calmodulin in the process of meiotic maturation.

Calcium ions are postulated to be involved in the process of meiotic maturation of amphibian oocytes. Since several of the calcium effects in eukaryotic cells are mediated by calmodulin, the present study was undertaken to assess the presence and level of calmodulin in Xenopus laevis oocytes before and after progesterone treatment. Calmodulin was shown to be present at a concentration of approximately microM in control oocytes cytosol. This level remained stable for 2 h when the maturation promoting factor appeared, and increased to approximately 44 to 59 microM at the time of the germinal vesicle breakdown. Maturation is therefore associated with calmodulin synthesis. Xenopus calmodulin was isolated from oocyte cytosol after heat treatment, anion exchange chromatography, and gel filtration, with a yield of approximately 23%. When compared to mammalian calmodulins, the amphibian protein exhibited the same ultraviolet absorption spectrum, a similar amino acid composition with 1 residue of trimethyllsine, and the same shape conformers in the absence or presence of divalent metals, as shown by different mobilities upon dodecyl sulfate-polyacrylamide gel electrophoresis. The protein migrated faster in the presence than in the absence of Ca2+ ions, Mn2+ and Mg2+ being less effective. It was able to activate calmodulin-deficient myosin light chain kinase. Its high serine content and the tryptic peptide maps obtained after high performance liquid chromatography point, however, to minor differences in the primary structures of mammalian and amphibian calmodulins.

Amino Acids↗