1997 年 38 巻 1 号 p. 53-59
An elastolytic proteinase was isolated from Aspergillus flavus by column chromatography using diethylaminoethyl (DEAE)-cellulose and carboxymethyl (CM)-Sephadex C-50. The proteinase was found to be homogeneous as indicated by a single band after disc polyacrylamide gel electrophoresis (PAGE). The enzyme had a molecular weight of 40, 000Da as determined by sodium dodecyl sulfate (SDS)-PAGE.
The elastolytic activity was inhibited by leupeptin, diisopropyl fluorophosphate (DFP), α1-antitrypsin, α2-macroglobulin and ethylenediaminetetraacetic acid (EDTA). However, neither N-bromo-succinimide (NBS) nor antithrombin-III showed any inhibitory effect on the enzyme activity. The enzyme contained 414 amino acid residues and exhibited an isoelectric point of 8.6. Its carbohydrate content was calculated to be 3.6% using glucose as a standard, but elastolytic proteinase from A. fumigatus did not contain any carbohydrates. The Aα, Bβ and γ chains of human fibrinogen were cleaved by the enzyme.
Elastolytic proteinase from A. flavus hydrolyzed Ser(9)-His(10), Val(12)-Glu(13), Glu(13)-Ala(14), Ala(14)-Leu(15), Leu(15)-Tyr(16), Tyr(16)-Leu(17), Glu(21)-Arg(22), Phe(25)-Tyr(26), Tyr(26)-Thr(27), Pro(28)-Lys(29) and Lys(29)-Ala(30) bonds of oxidized insulin B chain, showing that enzyme has proteolytic activity.
However, elastolytic proteinase from A. fumigatus had a molecular weight of 32, 000Da, and the enzyme did not contain carbohydrate.
