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Purification and characterization of a fibrinogenolytic serine proteinase from Aspergillus fumigatus culture filtrate
Author(s) -
Larcher Gérald,
Bouchara Jean-Philippe,
Annaix Véronique,
Symoens Françoise,
Chabasse Dominique,
Tronchin Guy
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)81052-n
Subject(s) - isoelectric point , pmsf , chemistry , biochemistry , ammonium sulfate precipitation , chromatography , aspergillus fumigatus , enzyme , sephadex , serine , polyacrylamide gel electrophoresis , enzyme assay , size exclusion chromatography , biology , microbiology and biotechnology
A fibrinogenolytic proteinase has been isolated from Aspergillus fumigatus culture filtrate by ammonium sulfate precipitation followed by successive chromatographics on Sephadex G‐75 and immobilized phenylalanine. The purified proteinase exhibited a molecular weight of about 33 kDa. When analysed by SDS‐polyacrylamide gels containing co‐polymerized fibrinogen, the proteinase appeared as a broad band at the top of the gels, which could correspond to polymerization of the enzyme, as suggested by SDS‐PAGE analysis of the unboiled eluate. The isoelectric point was 8.75 and the enzyme was not glycosylated. Proteinase activity was optimum at pH 9 and between 37 and 42°C, although a decrease in activity was observed above 37°C. PMSF and chymostatin markedly inhibited the proteinase activity, and good kinetic constants were obtained for the synthetic substrate, N ‐Suc‐Ala‐Ala‐Pro‐Phe‐pNA. These results provide direct evidence that this enzyme belongs to the chymotrypsin‐like serine proteinase group.