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Isolation and characterization of a secreted metalloprotease of Aspergillus fumigatus.

Service de Dermatologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.

ABSTRACT

A metalloprotease (MEP) secreted by Aspergillus fumigatus was isolated from an alkaline protease-deficient mutant after the fungus was cultivated in the presence of collagen as the sole nitrogen and carbon source. The enzyme was purified 50-fold from the culture supernatant after adsorption to hydroxylapatite and carboxy-methyl-Sephadex and after gel filtration. The molecular mass was determined to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was estimated at pH 5.5 by isoelectric focusing. Reducing agents and divalent cations strongly inhibited enzyme activity, whereas nonionic detergents had no effect. A. fumigatus MEP was totally inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. MEP is not able to cleave elastin and is thermosensitive. Sera from patients suffering from aspergilloma reacted with MEP in Western blotting (immunoblotting) analyses, suggesting that MEP promotes an antigenic response in these patients.

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