Enzymic characterization of murine and human prohormone convertase-1 (mPC1 and hPC1) expressed in mammalian GH4C1 cells
| Autor: | N. G. Seidah, Claude Lazure, François Jean, Michel Chrétien, Normand Rondeau, Ajoy Basak, Suzanne Benjannet, G. N. Hendy |
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| Rok vydání: | 1993 |
| Předmět: |
endocrine system
Genetic Vectors Molecular Sequence Data Prohormone Gene Expression Vaccinia virus Proprotein convertase 1 Biology Transfection Biochemistry Cell Line Substrate Specificity Mice chemistry.chemical_compound medicine Animals Humans Protease Inhibitors Amino Acid Sequence Molecular Biology chemistry.chemical_classification Serine Endopeptidases Biological activity Cell Biology Molecular biology Recombinant Proteins Endopeptidase Enzyme assay Rats Kinetics Pituitary Hormones Enzyme Proprotein Convertase 1 chemistry Enzyme inhibitor biology.protein Electrophoresis Polyacrylamide Gel Proprotein Convertases Oligopeptides Pepstatin Research Article medicine.drug |
| Zdroj: | Biochemical Journal. 292:891-900 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj2920891 |
| Popis: | Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues. To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1). An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography. The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide. mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured. Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes. Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity. In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP. |
| Databáze: | OpenAIRE |
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