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H Charbonneau

Publications and source records attributed to H Charbonneau.

At least 73 records · Page 4Linked to original sources
PubMed1985

Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase.

In the course of determining the primary structure of rabbit skeletal muscle myosin light chain kinase (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) a peptide fragment was obtained that appears to represent the calmodulin-binding domain of this enzyme. Low concentrations of the peptide inhibited calmodulin activation of MLCK (Ki congruent to 1 nM). The peptide was not associated with a catalytically active, calmodulin-independent form of MLCK that was obtained by limited proteolysis. The peptide is 27 residues in length and represents the carboxyl terminus of MLCK. The sequence of the peptide shows no significant homology with any known protein sequence. The peptide contains one tryptophanyl residue and a high percentage of basic and hydrophobic residues, but no acidic or prolyl residues. Much of the sequence has a high probability of forming alpha helix. A chemically synthesized peptide has been prepared to study the interactions of the peptide and calmodulin in more detail. The intrinsic tryptophan fluorescence of the synthetic peptide shows a significant enhancement (approximately equal to 45%) in the presence of Ca2+ and calmodulin; fluorescence enhancement is maximal at a peptide:calmodulin stoichiometry of 1:1. Calmodulin-Sepharose affinity chromatography in the presence of 2 M urea indicates that the interaction of peptide and calmodulin is Ca2+-dependent. The results of these studies indicate that the catalytic and calmodulin-binding domains of MLCK represent distinct and separable regions of the protein. In addition, the results provide a basis for future studies of the molecular and evolutionary details of calmodulin-dependent enzyme regulation.

Amino Acid Sequence↗
PubMed1985

[Not Available].

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Canada↗
PubMed1985

[Not Available].

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Canada↗
PubMed1984

[Not Available].

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Demography↗
PubMed1983

Preparation and properties of calcium-dependent resins with increased selectivity for calmodulin.

Calmodulin from both animal and plant sources is known to bind a number of hydrophobic compounds with resultant inhibition of calmodulin function. Some of these compounds, including certain phenothiazine and naphthalene sulfonamide derivatives, have been previously shown to be useful in the chromatographic isolation of calmodulin, when covalently linked to a solid support. With the exception of fluphenazine linked to epoxide-activated Sepharose, these resins have the undesirable characteristics of requiring high salt concentrations in the elution buffer for efficient elution of calmodulin, thus decreasing the selectivity for this protein. The synthesis of nine Sepharose-ligand affinity resins is reported. Some of the ligands are newly synthesized naphthalene sulfonamide and phenothiazine derivatives. The synthetic ligands have been coupled to three types of Sepharose: epoxide-activated, CNBr-activated, and carbodiimide-activated. The properties of these resins are reported and their relative abilities to act selectively in the isolation of calmodulin are compared. 2-Trifluoromethyl-10-aminopropyl phenothiazine (TAPP), when linked to epoxide-activated Sepharose, was found to be the most useful for calmodulin isolation in terms of its combined stability, capacity, and ability to select for calmodulin. This resin was found to behave as a true affinity resin. A quantitative evaluation of its affinity behavior was consistent with the presence of two high-affinity Ca2+-dependent phenothiazine binding sites on calmodulin, in apparent agreement with previous reports which involved the use of different methods.

Animals↗
PubMed1983

Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.

The initiation of DNA replication of plasmid R6K is triggered by a 35-kilodalton initiator protein. The initiator protein had been elusive because of its lability and the lack of a convenient assay procedure to aid its purification. Using recombinant DNA techniques, we have fused the cistron of the initiator near its COOH-terminal end, in the correct reading frame, to the lacZ cistron of Escherichia coli at the ninth codon from the NH2 terminus. The fused cistron yielded a protein that was not only stable in vivo but also had dual activities: initiation of DNA replication in vivo and in vitro and hydrolysis of beta-galactoside. Using an affinity column that is specific for beta-galactosidase, we have demonstrated the rapid purification of the hybrid protein to near homogeneity. Exploiting the polymeric structure of the initiator, we have also isolated the nonfused form of the initiator protein, associated through subunit interaction with the beta-galactosidase-fused protein, which permits its purification by affinity chromatography. NH2-terminal amino acid sequence analysis of the heteropolymer has not only shown that the fused and nonfused initiators have the same sequence but also confirmed the protein sequence of the initiator as predicted from its nucleotide sequence. The techniques described here should be generally useful for the isolation of other proteins that are difficult to purify by conventional procedures.

Bacterial Proteins↗
PubMed1981

Plant and fungal calmodulin: Ca2+-dependent regulation of plant NAD kinase.

Although little is known about the role(s) of second messengers, including free Ca2+, in plant cells there has been increasing evidence for a role for Ca2+ in metabolic regulation in plants. The recent demonstration that the Ca2+-binding protein, calmodulin exists in extracts of higher plants and basidiomycete fungi provides a basis for understanding Ca2+-dependent metabolic regulation in plant cells. In this review we summarize the similarities and differences of plant, fungal and mammalian calmodulin. We also discuss the known in vitro functions of calmodulin in higher plants. A Ca2+-calmodulin-dependent NAD kinase has been purified to homogeneity from extracts of pea seedlings and shown to be absolutely dependent upon calmodulin and microM levels of free Ca2+ for activity. The available evidence suggest that this Ca2+-calmodulin-dependent NAD kinase is the major form of plant NAD kinase and that this regulatory enzyme is localized in the chloroplast. A model is presented which predicts that the rate of photosynthesis is regulated by a receptor-mediated change in the level of chloroplastic free Ca2+ upon illumination. Free Ca2+, acting as a second messenger, forms a Ca2+-calmodulin complex thus converting calmodulin to its active conformation. This Ca2+-calmodulin complex then activates chloroplastic NAD kinase resulting in an increased NADP/NAD ratio.

Adenylyl Cyclases↗
PubMed1980

Characterization of the plant nicotinamide adenine dinucleotide kinase activator protein and its identification as calmodulin.

A protein activator of plant NAD kinase has been extracted from plant sources (peanuts and peas), purified to homogeneity, characterized, and identified as calmodulin. A comparison of the properties of calmodulin isolated from either plant or animal sources shows that they are strikingly similar proteins. The similarities include molecular weight, Stokes radii, amino acid composition, Ca2+-dependent enhancement of tyrosine fluorescence, Ca2+-dependent interaction with troponin I, equal abilities to activate cyclic nucleotide phosphodiesterase, Ca2+-dependent inhibition of calmodulin action by the phenothiazine drugs, and electrophoretic mobility. We discuss the possibility that plant cells may undergo Ca2+-dependent regulatory events that are mediated by calmodulin in a manner similar to those found in animals.

Animals↗
PubMed1979

Ca2+-induced bioluminescence in Renilla reniformis. Purification and characterization of a calcium-triggered luciferin-binding protein.

A Ca2+-triggered luciferin-binding protein (BP-LH2) from the bioluminescent marine coelenterate, Renilla reniformis, has been purified by conventional methods. One kilogram of processed animals yields approximately 2.7 mg of pure protein with an overall yield of 55%. Physicochemical studies show that BP-LH2 is a globular protein containing one single polypeptide chain with one disulfide bond. Ultracentrifugation studies, amino acid analysis, and sodium dodecyl sulfate-gel electrophoresis show that BP-LH2 has an average molecular weight of 18,500. BP-LH2 has a Stokes radius of 23 A, a sedimentation coefficient, S020,w, of 2.3 S, and an isoelectric point of 4.3. The acidic nature of the protein was confirmed by amino acid analysis, which showed that 27% of the residues are acidic. The protein contains no carbohydrate, phosphate, or tryptophan. There is one noncovalently bound molecule of coelenterate type luciferin resulting in distinct protein spectral properties with absorption maxima at 276 nm (epsilon 0.1% 276 = 1.31) and 446 nm (episoln 0.1% 446 = 0.47) and a fluorescence emission at 520 nm (uncorrected). In the presence of Ca2+, BP-LH2 will react with Renilla luciferase to give the characteristic in vitro blue bioluminescence. Ca2+ binding produces a distinct change in the spectral properties of BP-LH2 including a 4-fold enhancement of tyrosine fluorescence at 332 nm and a 5-fold fluorescence enhancement at 520 nm. In addition, the visible absorption maximum shifts from 446 nm to 420 nm. The fluorescence enhancement at 320 nm occurs over the range from 1 to 10 micrometer Ca2+. BP-LH2 has two Ca2+-binding sites with an estimated Kd of 0.02 micrometer, in 10 muM Tris at pH 7.2. BP-LH2 was compared to several well studied Ca2+-binding proteins and was found to possess similar Ca2+-binding and physicochemical properties. This study clearly demonstrates that BP-LH2 is capable of triggering a bioluminescent flash in response to an intracellular Ca2+ transient.

Amino Acids↗
PubMed1978

[Not Available].

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Canada↗
PubMed1977

Structure of native Renilla reinformis luciferin.

The structure of native luciferin from the bioluminescent coelenterate Renilla reniformis is shown to be 3,7-dihydro-2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzylimidazo[1,2-a]pyrazin-3-one by mass spectral analysis of synthetic luciferin and the luciferin derived from a protein directly involved in the bioluminescent system. A previous report of the molecular weight of luciferin is shown to be incorrect by reexamination of the spectral data and by synthesis of two derivatives. Detailed analysis of kinetic, emission, and quantum yield data for the isolated and synthetic luciferins confirms this structure. Confirmation of this structure in a number of species from different phyla suggests a common substrate for a variety of bioluminescent marine organisms.

Journal Article↗
PubMed1977

[Not Available].

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Canada↗
PubMedDate not supplied

Glycosylation reduces the bioactivity of calcitonin.

The biological significance of peptide hormone glycosylation is uncertain. To examine the effect of Asn-linked glycosylation on calcitonin's bioactivity we purified glycosylated calcitonin from a transplantable rat medullary thyroid carcinoma. Glycosylated calcitonin constituted 2.3% of the total extracted immunoreactive calcitonin. The structure of this peptide differed from nonglycosylated calcitonin only by the oligosaccharide modification of asparagine 3. Affinity of glycosylated calcitonin for lentil lectin indicated that the oligosaccharide was a complex processed form. In a standard in vivo bioassay glycosylated calcitonin had a markedly reduced hypocalcemic activity compared to nonglycosylated calcitonin, an effect most likely due to the presence of the oligosaccharide.

Amino Acids↗