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C Lazure

Publications and source records attributed to C Lazure.

At least 73 records · Page 4Linked to original sources
PubMed1992

Purification of cationic cystine-rich peptides from rat bone marrow. Primary structures and biological activity of the rat corticostatin family of peptides.

Seven cationic, cystine-rich peptides of 29 to 32 amino acid residues have been purified from extracts of rat bone marrow (R-1, R-1a, R-1b, R-2, R-3, R-4 and R-5). Structural analysis clearly indicated that all seven peptides belong to the corticostatin/defensin family of leukocyte-derived peptides known to participate in oxygen-independent killing of phagocytosed bacteria. For R-1 to R-5, six cysteine residues were found at characteristic and highly conserved positions. R-1a and R-1b were partially characterized and appear to be structural variants of R-1. Aside from the conserved cysteines, there is a remarkable degree of structural diversity evident within the sequences of those members of the corticostatin/defensin family characterized so far. The structures of the peptides that we have purified can be compared directly with the sequences obtained for rat defensins isolated from extracts of peritoneal neutrophils (Lehrer, Ganz and Selsted, Cell, 64 (1991) 229-230). Some discrepancies are apparent which can be explained in terms of proteolytic cleavage of several of these peptides at both amino- and carboxyl-termini. The corticostatins owe their bioactivity to their ability to compete with corticotropin for occupancy of the corticotropin receptor (Zhu, Hu, Mulay, Esch, Shimasaki and Solomon, Proc. Natl. Acad. Sci. USA, 85 (1988) 592-596). The potency of these peptides can be expressed in terms of their capacity to inhibit the steroidogenic response of isolated rat adrenocrotical cells half-maximally stimulated by corticotropin (i.e., at the ED50 concentration for corticotropin in this assay, namely 33 pM). In this assay, the rat peptides R-1, R-2 and R-3 were shown to be inactive. In contrast, the more cationic peptides R-4 and R-5 were found to inhibit steroidogenesis. R-4 was somewhat less active than rabbit corticostatin (IC50 25 nM) showing an IC50 value of 50 nM. R-5 appeared to be significantly less potent than R-4. The lower yield of R-5 precluded an accurate estimate of the corticostatic potency of this peptide. R-4 differs in structure from R-5 in having an arginine to serine substitution at position 7. It can be concluded that an arginine at this position accounts, at least in part, for the corticostatic activity of R-4.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗
PubMed1992

The isolation and partial characterization of trypsinogen, pancreatic secretory trypsin inhibitor and multiple forms of chymotrypsinogen and trypsin from the pancreas of the ostrich (Struthio camelus).

1. PSTI, two chymotrypsinogens and two trypsins were purified to homogeneity by acid extraction, salt fractionation, SP-Sephadex C-50 chromatography and RP-HPLC. 2. A third chymotrypsinogen, a trypsinogen and another trypsin were purified using an alkaline extraction procedure, followed by Trasylol- and Benzamidine-Sepharose affinity chromatography and hydroxylapatite chromatography. 3. The enzymes differed in amino acid composition as well as in specific activities towards synthetic amidase and esterase substrates. 4. N-terminal amino acid sequences were determined for one chymotrypsinogen and one trypsin.

Amino Acid Sequence↗
PubMed1992

Release of a 22-kDa protein derived from the amino-terminal domain of the 49-kDa NIa of turnip mosaic potyvirus in Escherichia coli.

The coding region for the precursor 6K-small nuclear inclusion a (NIa) protein and for the NIa protein of turnip mosaic potyvirus (TuMV) were introduced into the plasmid pET-11d for high-level expression in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses of E. coli proteins showed that the NIa protein underwent endoproteolysis and released a 22-kDa polypeptide. NH2-terminal amino acid sequencing of the recombinant 22-kDa protein was performed and was identical to the predicted amino end of the NIa protein. Site-directed mutagenesis confirmed that the hydrolysis was associated with the NIa proteolytic activity and that the proteinase recognized a Glu residue within an amino acid sequence found in the NIa protein which fitted the TuMV consensus cleavage site sequence. Fusion of the 6K protein with the NIa protein partially inhibited the hydrolytic reaction. The recombinant 22-kDa protein is likely the VPg of TuMV.

Base Sequence↗
PubMed1991

Immunological identification and sequence characterization of a peptide derived from the processing of neuroendocrine protein 7B2.

A newly raised antiserum against the C-terminal region of neuroendocrine protein 7B2 was used to purify a novel peptide from the culture media of the mouse corticotroph cell line AtT-20. Based on partial sequencing, this peptide, which we call Cter-7B2, begins at Ser156 and appears to result from the cleavage of pro7B2 after a five-basic-residue sequence. Thus, 7B2 processing may contribute to the diversity of peptides found in neuronal and endocrine cells.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of corticostatic peptides from guinea pig bone marrow.

Three corticostatic peptides (GP-CS1, GP-CS2 and GP-CS3) were purified from extracts of guinea pig bone marrow. Each was identified on the basis of their ability to inhibit the secretion of corticosterone by isolated rat adrenal cells stimulated by ACTH. GP-CS1 and GP-CS2 were found to be 31 residues in length, rich in arginine and to have six cysteines typical of the corticostatin/defensin family of peptides previously purified from phagocytic cells of the immune system. GP-CS1 was found to be identical to GP-CS2 except for having a leucine at position 21 instead of isoleucine. GP-CS3 was also found to be rich in arginine and cysteine but structurally distinct from the other peptides. A combination of endoprotease mapping, ion-spray mass spectrometry and gas-phase sequencing revealed that GP-CS3 was a novel homo-dimer consisting of two 13 amino acid residue subunits cross-linked through eight cysteines in an anti-parellel configuration.

Adrenal Glands↗
PubMed1991

Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay.

A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human alpha 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.

Amino Acid Sequence↗
PubMed1991

The production by alternate splicing of two mRNAs differing by one codon could be an intrinsic property of neuroendocrine protein 7B2 gene expression in man.

Two types of mRNAs for neuroendocrine protein 7B2 were deduced from the sequence of cDNAs clones isolated from a human pituitary cDNA library. One type lacks an Ala100 codon present in the other. The difference is located at an intron site within the human 7B2 gene and can be explained by the transcriptional utilization of two alternate acceptor splice sites, three nucleotides apart. Heteroduplex analysis of DNA fragments amplified by the polymerase chain reaction indicated that this 7B2 mRNA dimorphism occurs in several human endocrine tissues as well as in other species, suggesting that the alternate processing of 7B2 gene transcripts may be an intrinsic mechanism of its expression and could underlie some yet unknown biological functions.

Animals↗
PubMed1991

Purification and primary structure of glucagon from ostrich pancreas splenic lobes.

Glucagon is a highly conserved polypeptide hormone which appears to play a more important role in regulation of glycaemia in birds than insulin. Ostrich glucagon was isolated and purified from ostrich pancreas splenic lobes using an adapted acid ethanol extraction procedure, gel filtration, ion exchanges, and HPLC steps. The purified glucagon fraction appeared to contain small quantities of a more acidic contaminant (polyacrylamide gel isoelectric focussing, PAGE) but appeared homogeneous on SDS-PAGE. Amino acid analysis and sequence analysis showed identity with the duck hormone. Identity with the duck hormone was confirmed by liquid phase as well as gas phase sequencing. The ostrich glucagon preparation seemed to have a higher Km than the porcine homologue in stimulating glycerol release from isolated chicken adipocytes.

Adipose Tissue↗
PubMed1991

Processed forms of neuroendocrine proteins 7B2 and secretogranin II are found in porcine pituitary extracts.

The complete structure of the novel polypeptide 7B2 recently deduced from cDNA clones has been reported to be highly conserved in a variety of species. The deduced amino acid sequence of the mature protein is predicted to be 185 or 186 amino acids long. While its biological role is still unknown, its occurrence in neuroendocrine secretory granules has been largely documented. This report shows: (i) that the protein, isolated from a large quantity of porcine pituitary glands, does not correspond to the full predicted cDNA structure but, on the contrary, to a truncated form; (ii) that the latter could arise from proteolytic cleavage at position 150 following pairs of basic residues; (iii) that it contains an extra residue at position 100 which is absent in the cDNA sequence; and, finally, (iv) that it displays a higher than expected molecular weight on SDS-polyacrylamide gel electrophoresis. In addition, a copurifying peptide was identified as an NH2-terminal related fragment of the secretogranin II molecule. Protein sequencing of the latter demonstrates (i) that the correct amino terminus of mature porcine secretogranin II is an Ala residue and not the previously proposed Gln residue and (ii) that this fragment could also arise from proteolytic cleavage at a pair of basic residues located within the secretogranin II sequence.

Amino Acid Sequence↗
PubMed1991

Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2.

Using a 796-basepair cDNA fragment obtained from a mouse pituitary library we have screened two mouse insulinoma libraries and isolated a full-length cDNA clone (2516 basepairs; 753 amino acids), designated mPC1. The cDNA sequence of mPC1 codes for a protein containing 753 amino acids and three potential N-glycosylation sites. This cDNA encodes a putative novel subtilisin-like proteinase, exhibiting within its presumed catalytic domain 64%, 55%, and 47% amino acid sequence identity to the recently characterized candidate prohormone convertases human Furin, mouse PC2, and yeast Kex2 gene products, respectively. An identical sequence to mPC1 was derived from a cDNA library of mouse corticotroph AtT-20 tumor cells. An ArgGlyAsp tripeptide identical to the recognition sequence of integrins was observed in the structures of the mammalian PC1, PC2, and Furin. In situ hybridization results demonstrated a distinct localization of the mPC1 and mPC2 transcripts in pituitary and brain. Thus, whereas both mPC1 and mPC2 are found in the intermediate lobe of the pituitary, only mPC1 is easily detected in the anterior lobe. In extrahypothalamic regions of the brain, including cortex, hippocampus, thalamus, and spinal cord, mPC2 transcripts predominate over mPC1. Both mRNAs are found in only a fraction of hypothalamic neurons, with greater abundance of mPC1 over mPC2 in the supraoptic nucleus. The genes coding for mPC1 and mPC2 map to the murine chromosomes 13 (band 13c) and 2 (2F3-2H2 region), respectively.

Amino Acid Sequence↗
PubMed1990

Granulins, a novel class of peptide from leukocytes.

We report the isolation and characterization of a novel class of leukocyte peptides with possible cytokine-like activities which we call granulins. They are cystine-rich with molecular weights of approximately 6 Kda, except for granulin D, which appears to be a dimer. We present the sequence of one member of this family, a 56 residue peptide, granulin A, and amino-terminal sequences for three other granulins from human peripheral leukocytes. A fifth related peptide was isolated and partially sequenced from rat bone marrow, suggesting that at least some of the granulin in peripheral leukocytes is preformed in the marrow. Rat granulin, and human granulin A, are closely related, showing that the granulin structures are highly conserved between species.

Amino Acid Sequence↗
PubMed1990

Fragmentation of bovine chromogranin A by plasma kallikrein.

Chromogranin A has been reported to be processed in vivo by an as yet undefined proteinase(s) suggesting that it is a precursor of biologically active peptides such as pancreastatin. In this study, plasma kallikrein was used as a model proteinase to identify the cleavage sites exposed in bovine parathyroid chromogranin A. Purified bovine parathyroid chromogranin A was digested with human plasma kallikrein. The proteolytic fragments produced were isolated by HPLC and chemically characterized by amino acid composition and sequence analysis. The combined results indicate that the enzyme has preference for specific single Arg residues, cutting C-terminal to this amino acid, although certain pairs of basic sites were also cleaved. The characterized fragments were released in a selective manner from the whole molecule with rapid production of the fragments covering positions 1-247 and 352-358.

Amino Acid Sequence↗
PubMed1990

Sequence of the 3'-terminal region of turnip mosaic virus RNA and the capsid protein gene.

A sequence of 1801 nucleotides originating from the 3' end region of turnip mosaic virus (TuMV) RNA was cloned using the polymerase chain reaction and found to contain one long open reading frame (ORF). The amino acid sequence of three different regions of the isolated TuMV capsid protein (including the NH2 terminus) was determined and these partial sequences were found in the translation product predicted to be encoded by the large ORF. The data suggested that the TuMV capsid protein was a product arising from the maturation of a larger polyprotein, as observed for other potyviruses. Furthermore, the putative cleavage site corresponded to a glutamine-alanine dipeptide, a site commonly used in plant virus polyprotein processing. The capsid protein cistron was composed of 864 nucleotides and corresponded to a region encoding 288 amino acids with a calculated Mr of 33,186; the adjacent 3' non-coding region was 667 nucleotides long. The deduced amino acid sequence of the TuMV capsid protein is closely related to other potyvirus capsid proteins, with most of the variation being found within the NH2-terminal region.

Amino Acid Sequence↗
PubMed1990

Syntheses of argininal semicarbazone containing peptides and their applications in the affinity chromatography of serine proteinases.

Eight argininal semicarbazone containing peptides prepared by liquid phase synthesis were all found to be reversible inhibitors of model serine proteinases including trypsin and plasma kallikrein (PK). Among the peptides tested, those having a Lys residue at position P2 displayed the maximum binding potency towards PK. One of the peptides, Leu-enkephalin-argininal semicarbazone, a comparatively weak inhibitor, was chosen in order to develop an affinity-based purification protocol for PK. The affinity column was prepared by covalent attachment of the NH2-terminal moiety of the peptidyl semicarbazone to a solid-phase matrix bearing a spacer group. For efficient binding of PK, it was found necessary to optimize parameters like the concentration of inhibitor linked to the solid matrix, the ionic strength of the buffer used, the temperature and the pH. The majority of the bound enzyme could be recovered following elution with guanidine hydrochloride or benzamidine hydrochloride in a high salt buffer at pH 6.0. The usefulness of the affinity procedure towards the purification of other serine proteinases is also discussed.

Amino Acid Sequence↗
PubMed1989

Rat plasma kallikrein: purification, NH2-terminal sequencing and development of a specific radioimmunoassay.

Rat plasma kallikrein (rPK) was purified to homogeneity form plasma using affinity and high-performance liquid chromatography techniques, and subjected to NH2-terminal sequencing. The data showed that the sequenced segments of the regulatory (heavy) and catalytic (light) chains of the proteinase, respectively, display 73 and 91% sequence similarity with their counterpart in human plasma kallikrein. This sequence homology in conjunction with the determined molecular structure and inhibitor sensitivity support the identity of the isolated enzyme as plasma kallikrein. A polyclonal antiserum against rPK was obtained after immunization of rabbits with the purified enzyme, and a specific radioimmunoassay was developed. Since Tyr-iodinated rPK was not recognized by the antiserum, two alternative approaches were found to be successful. These included the use of a tracer consisting of rPK modified with either the affinity reagent 125I-labeled DTyr-Glu-Phe-Lys-Arg chloromethyl ketone or with the Bolton Hunter reagent. The usable range of the assay is between 15-150 fmol per tube. The antibody was shown to bind both monomeric and dimeric forms of rPK. Denaturation of the enzyme in sodium dodecyl sulfate does not abolish immune recognition only as long as the regulatory subunit is attached to the catalytic chain. Oxidation or reduction of rPK results in complete loss of immunoreactivity. This observation suggests that perhaps the disulfide linkage of the catalytic and regulatory polypeptides somehow helps to protect the antigenic epitope from denaturation. Alternatively, the epitope(s) recognized by the antibody spans a domain which includes both Tyr and Cys residues necessary for immune recognition.

Amino Acid Sequence↗
PubMed1989

Molecular heterogeneity of pro-atrial natriuretic factor.

Analysis by two-dimensional gel electrophoresis and Western blotting of the atrial natriuretic factor (ANF) content of atrial granules revealed the presence of at least 15 immunoreactive spots whose molecular mass distribution ranged from 16.8 to 35 kDa and their pI values from 5.12 to 5.98. About 90% of the immunoreactive ANF material was contained within four spots (spot 1: 34.8 kDa, pI 5.67; spot 5: 16.8 kDa, pI 5.50; spot 6: 16.8 kDa, pI 5.67; spot 7: 16.8 kDa, pI 5.98). Investigation of the molecular nature of spot 1 indicated that it is a dimer of pro-ANF since it possesses the same immunoreactivity, the same charge, double its mass, and can be converted with dithiothreitol into a 16.8-kDa pro-ANF form. Alkaline phosphatase and protein kinase A treatments indicated that spots 5, 6, and 7 are probably not phosphorylated forms of pro-ANF. Carboxypeptide A and B treatments in conjunction with amino acid analysis suggested that spot 7 is ANF-(1-128); spot 6, the major one, ANF-(1-126); and spot 5, ANF-(1-123) or ANF-(1-124). Water deprivation or morphine injection, two maneuvers which are known to influence ANF secretion and atrial ANF content, failed to affect the molecular heterogeneity of pro-ANF except for spot 1. The formation of the dimer appeared to be time-dependent. These results emphasize the heterogeneity of the pro-ANF molecule stored in atrial granules. We suggest that this heterogeneity may be due, in part, to the action of some proteases, such as carboxypeptidase E or a tripeptidyl carboxyhydrolase.

Alkaline Phosphatase↗
PubMed1989

Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications.

Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle. Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture. The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively. Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence. Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases. Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases. Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme. Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology. The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.

Amino Acid Sequence↗