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H Charbonneau

Publications and source records attributed to H Charbonneau.

At least 55 records · Page 3Linked to original sources
PubMed1989

The amino acid sequence of the sex steroid-binding protein of rabbit serum.

The amino acid sequence of the sex steroid-binding protein (SBP or SHBG) of rabbit serum, specific for binding testosterone and 5 alpha-dihydrotestosterone, was determined using a complementary combination of mass spectrometric and Edman degradation techniques. The monomeric unit of the homodimeric protein is a single chain glycopeptide of 367 amino acid residues, with N-linked oligosaccharide side chains at Asn-345 and Asn-361 and disulfide bonds connecting Cys-158 to Cys-182 and Cys-327 to Cys-355. The polypeptide molecular weight of the monomer calculated from the sequence is 39,769. The molecular weight of the homodimer including 9% carbohydrate is 87,404. The sequence contains a relatively hydrophobic segment between Trp-241 and Leu-282, which includes many leucine residues in an alternating pattern. An amino acid sequence repeat is also located within that segment. Both of these patterns are present in human SBP and in the androgen-binding protein of rat epididymis. The sequence data indicate that the previously reported microheterogeneity of rabbit SBP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reflects variants generated by differential glycosylation of the monomer rather than different gene products. Seventy-nine percent of the amino acids of rabbit SBP are identical to those of human SBP; rabbit SBP thus joins human SBP and rat androgen-binding protein in one gene family that is distinct from the steroid hormone receptor superfamily. It appears that the problem of binding sex steroid hormones has been solved independently in two different gene families that contain completely different steroid-binding domains. Since the nonhomologous steroid-binding domains of both families of proteins recognize essentially the same steroid structure, it will be interesting to determine the structural basis of the two different protein designs that lead to similar steroid-binding specificity.

Amino Acid Sequence↗
PubMed1989

Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase.

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

mRNA abundance, rather than differences in subunit assembly, determine differential expression of HLA-DR beta 1 and -DR beta 3 molecules.

The class II major histocompatibility molecules HLA-DR are formed by the association of a single DR alpha chain with two nonallelic DR beta chains. In DR3 cells one DR beta chain is severalfold more abundant than the other. We have studied the mechanism that controls the differential expression of these DR beta genes. We determined the amino-terminal sequences of the two expressed DR beta chains. Comparison of these sequences with the nucleotide sequences of the DR3B1 and DRB3a genes indicates that the abundant chain is the B1 gene product. Supporting this conclusion, an informative mutant, 9.4.3, was found to have lost the abundant beta chain and beta 1 mRNA. This mutant expresses normal cell surface levels of the DR beta 3 chain and exhibits no significant dosage compensation of its beta 3 chain. The unchanged level of DR beta 3 dimer on the cell surface suggests that free DR alpha chains are not the limiting factor in the surface expression of the beta 3 chain and, further, that the differential regulation of the surface expression of the two DR beta chains occurs at a step prior to DR assembly. Quantitation of DR beta mRNAs by locus-specific oligonucleotide probes showed that beta 1 mRNA is 4.5-fold more abundant than beta 3 mRNA, strongly indicating that the greater surface expression of DR beta 1 is a direct consequence of greater beta 1 mRNA abundance.

Cell Line↗
PubMed1989

Amino acid sequence of a mouse mucosal mast cell protease.

The amino acid sequence has been determined of a mouse mucosal mast cell protease isolated from the small intestines of mice infected with Trichinella spiralis. The active protease contains 226 residues. Those corresponding to the catalytic triad of the active site of mammalian serine proteases (His-57, Asp-102, and Ser-195 in chymotrypsin) occur in identical positions. A computer search for homology indicates 74.3% and 74.1% sequence identity of the mouse mast cell protease compared to those of rat mast cell proteases I and II (RMCP I and II), respectively. The six half-cystine residues in the mouse mast cell protease are located in the same positions as in the rat mast cell proteases, cathepsin G, and the lymphocyte proteases, suggesting that they all have identical disulfide bond arrangements. At physiological pH, the mouse and rat mucosal mast cell proteases have net charges of +3 and +4, respectively, as compared to +18 for the protease (RMCP I) from rat connective tissue mast cells. This observation is consistent with the difference in solubility between the mucosal and connective tissue mast cell proteases when the enzymes are extracted from their granules under physiological conditions.

Amino Acid Sequence↗
PubMed1989

Protein tyrosine dephosphorylation and signal transduction.

The protein tyrosine phosphatases comprise a family of enzymes that specifically dephosphorylate tyrosyl residues. Determination of the amino acid sequence of a major low molecular mass form isolated from human placenta (PTPase 1B) provided the basis for the first identification of transmembrane proteins that bear intracellular phosphatase domains. The existence of such molecules, bearing the hallmarks of receptors, raises the exciting possibility of a novel mechanism of signal transduction in which the early events involve the ligand-induced dephosphorylation of tyrosyl residues in proteins.

Female↗
PubMed1989

Human placenta protein-tyrosine-phosphatase: amino acid sequence and relationship to a family of receptor-like proteins.

The amino acid sequence of the cytosolic human placenta protein-tyrosine-phosphatase 1B (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) has been determined. It consists of a single chain of 321 residues with an N-acetylated N-terminal methionine and an unusually proline-rich C-terminal region. The enzyme is structurally related to the two cytoplasmic domains of both the leukocyte common antigen CD45 and LAR, a CD45-like molecule with an external segment that resembles a neural cell adhesion molecule. A low molecular weight protein encoded by a cDNA clone from T cells also shows extensive sequence similarities. The present study defines homologous domains common to this diverse family of PTPases that includes both soluble and receptor-like transmembrane forms. The cysteinyl residues 121 and 215 of PTPase 1B are conserved among all members of the family and are candidates for involvement in catalysis since PTPase 1B is inactivated by thiol modifying reagents. Two segments rich in positively charged residues (residues 33-47 and 227-238) may provide sites of interaction with inhibitory anionic polymers such as heparin or poly(Glu/Tyr).

Amino Acid Sequence↗
PubMed1989

cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family.

A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). One positive clone was isolated and the nucleotide sequence was determined. It contained 1305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3' untranslated end, although a poly(A)+ tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues (Mr, 48,400). This was supported by the synthesis of a Mr 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low Mr PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes.

Amino Acid Sequence↗
PubMed1989

Osteogenesis imperfecta. The position of substitution for glycine by cysteine in the triple helical domain of the pro alpha 1(I) chains of type I collagen determines the clinical phenotype.

Skin fibroblasts grown from three individuals with osteogenesis imperfecta (OI) each synthesized a population of normal type I collagen molecules and additional molecules that had one or two alpha 1(I) chains that contained a cysteine residue within the triple-helical domain, a region from which cysteine normally is excluded. The patients had very different phenotypes. One patient with OI type I had a population of alpha 1(I) chains in which glycine at position 94 of the triple helix was substituted by cysteine; a patient with OI type III had a population of alpha 1(I) chains in which glycine at position 526 of the triple helix was substituted by cysteine; and the third patient, with OI type II, had a cysteine for glycine substitution at position 718 of the alpha 1(I) chain. From all three patients, molecules that contained two mutant chains formed interchain, intramolecular disulfide bonds, and although less stable to thermal denaturation than normal molecules, they were more stable than molecules that contained only a single mutant chain. These findings indicate that substitutions for glycine within the triple-helical domain of the alpha 1(I) chain are not invariably lethal and that their phenotypic effect largely depends on the nature of the substituting residue and its location in the chain.

Adult↗
PubMed1988

Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase.

It has been proposed on the basis of amino acid sequence homology that the leukocyte common antigen CD45 represents a family of catalytically active, receptor-linked protein tyrosine phosphatases [Charbonneau, H., Tonks, N. K., Walsh, K. A., & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7182-7186]. The present study confirms that CD45 possesses intrinsic protein tyrosine phosphatase (PTPase) activity. First, a mouse monoclonal antibody to CD45 (mAb 9.4) specifically eliminated, by precipitation, PTPase activity from a high Mr fraction containing CD45, prepared by gel filtration (Sephacryl S200) of a Triton X-100 extract of human spleen. Second, PTPase activity was demonstrated in a highly purified preparation of CD45 that was eluted with a high pH buffer from an affinity column, constructed from the same antibody. Third, on sucrose density gradient centrifugation, PTPase activity was only found in those fractions that contained CD45 as determined by Western analysis. When CD45 was caused to aggregate, first by reacting it with mAb 9.4 and then adding a secondary, cross-linking anti-mouse mAb, the PTPase activity shifted to the same higher Mr fractions that contained CD45. No shift in CD45 or PTPase was observed following addition of a control IgG2a. On this basis, it is concluded that CD45 is a protein tyrosine phosphatase.

Antigens, Differentiation↗
PubMed1988

Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin-dependent enzymes.

Nanomolar concentrations of synthetic peptides corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase were found to inhibit calmodulin activation of seven well-characterized calmodulin-dependent enzymes: brain 61 kDa cyclic nucleotide phosphodiesterase, brain adenylate cyclase, Bordetella pertussis adenylate cyclase, red blood cell membrane Ca++-pump ATPase, brain calmodulin-dependent protein phosphatase (calcineurin), skeletal muscle phosphorylase b kinase, and brain multifunctional Ca++ (calmodulin)-dependent protein kinase. Inhibition could be entirely overcome by the addition of excess calmodulin. Thus, the myosin light chain kinase peptides used in this study may be useful antagonists for studying calmodulin-dependent enzymes and processes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta.

We have characterized a mutation that produces mild, dominantly inherited osteogenesis imperfecta. Half of the alpha 1 (I) chains of type I collagen synthesized by cells from an affected individual contain a cysteine residue in the 196-residue carboxyl-terminal cyanogen bromide peptide of the triple-helical domain (Steinmann, B., Nicholls, A., and Pope, F. M. (1986) J. Biol. Chem. 261, 8958-8964). Unexpectedly, sequence determined from a proteolytic fragment of the alpha 1 (I) chain derived from procollagen molecules synthesized in the presence of both [3H]proline and [35S]cysteine indicated that the cysteine is located at the third residue carboxyl-terminal to the triple-helical domain, normally a glycine. The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele. This represents a new class of mutations, point mutations outside the triple-helical domain of the chains of type I collagen, that produce the osteogenesis imperfecta phenotype.

Alleles↗
PubMed1988

Purification of calmodulin-stimulated cyclic nucleotide phosphodiesterase by monoclonal antibody affinity chromatography.

Immobilized ACC-1 and ACAP-1 antibodies are effective tools for the purification of active calmodulin-dependent phosphodiesterases. ACC-1 antibody binds all bovine and rat brain isozymes in a Ca2+-dependent manner and has been used for their purification. Since ACC-1 binds both bovine brain isozymes (61- and 63-kDa forms) and ACAP-1 recognizes only the 61-kDa isozyme, ACAP-1 can be used to separate and purify the two brain isozymes. The procedures described here for phosphodiesterase isolation from brain are rapid and require few enzymatic assays, resulting in preparations of good purity, specific activity, and yield (Tables II, III). The procedures for brain tissue can be easily adapted for use with larger amounts of tissue. The cross-reactivity of ACP-1 for rat brain phosphodiesterase suggests that this antibody may recognize isozymes from other mammalian tissues.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase.

A major protein tyrosine phosphatase (PTPase 1B) has been isolated in essentially homogeneous form from the soluble and particulate fractions of human placenta. Unexpectedly, partial amino acid sequences displayed no homology with the primary structures of the protein Ser/Thr phosphatases deduced from cDNA clones. However, the sequence is strikingly similar to the tandem C-terminal homologous domains of the leukocyte common antigen (CD45). A 157-residue segment of PTPase 1B displayed 40% and 33% sequence identity with corresponding regions from cytoplasmic domains I and II of human CD45. Similar degrees of identity have been observed among the catalytic domains of families of regulatory proteins such as protein kinases and cyclic nucleotide phosphodiesterases. On this basis, it is proposed that the CD45 family has protein tyrosine phosphatase activity and may represent a set of cell-surface receptors involved in signal transduction. This suggests that the repertoire of signal transduction mechanisms may include the direct control of an intracellular protein tyrosine phosphatase, offering the possibility of a regulatory balance with those protein tyrosine kinases that act at the internal surface of the membrane.

Amino Acid Sequence↗
PubMed1986

Identification of a conserved domain among cyclic nucleotide phosphodiesterases from diverse species.

Partial amino acid sequences have been determined for the Ca2+/calmodulin-stimulated cyclic nucleotide phosphodiesterase from bovine brain and the cGMP-stimulated cyclic nucleotide phosphodiesterase from bovine heart. Examination of these sequences for homologous segments and comparison with protein sequences derived from the nucleotide sequences of the yeast PDE2 gene and the Drosophila dunce+ gene [Chen, C.-N., Denome, S. & Davis, R. L. (1986) Proc. Natl. Acad. Sci. USA 83, 9313-9317; Sass, P., Field, J., Nikawa, J., Toda, T. & Wigler, M. (1986) Proc. Natl. Acad. Sci. USA 83, 9303-9307] reveal a 200- to 270-residue segment in each that is homologous to the others. The molecular masses of the four proteins vary from 40 kDa to 105 kDa, and the structural resemblance appears to be constrained to a single segment of each protein. These related segments are proposed to comprise the catalytic domains in this set of enzymes. The lack of absolute sequence identity between the two bovine enzymes shows that they are unique gene products that are not produced by alternative processing of a larger protein or of a single mRNA precursor. The data also strongly support the conclusion that the dunce+ gene locus of Drosophila and the PDE2 gene locus in yeast code for structural genes of cyclic nucleotide phosphodiesterases.

Amino Acid Sequence↗
PubMed1985

Amino acid sequence of the calcium-dependent photoprotein aequorin.

The Ca(II)-dependent photoprotein aequorin produces the luminescence of the marine coelenterate Aequorea victoria. The complete amino acid sequence of aequorin has been determined. A complete set of nonoverlapping peptides was produced by cyanogen bromide cleavage. These peptides were aligned by using the amino-terminal sequence of the intact protein and the sequences of selected arginyl and lysyl cleavage products. Although the aequorin preparations employed in these studies were homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the presence of a minimum of 3 isotypes was demonstrated by the location of 17 sites of sequence microheterogeneity. Two amino acid variants were observed at each of 16 positions while 1 position had 3 different replacements. The protein as isolated has 189 amino acids with an unblocked amino terminus. According to the sequence reported here, the molecular weight of the apoprotein is 21 459 while that of the holoprotein is 21 914. The molecule possesses three internally homologous domains which were judged to be EF-hand Ca(II) binding domains by several different criteria. Aequorin is homologous to troponin C and to calmodulin. These findings demonstrate that aequorin is a member of the Ca(II) binding protein superfamily.

Aequorin↗