Isolation and immunochemical characterization of fractions from membranes of Aspergillus fumigatus with protease activity.
Two fractions exhibiting acid protease activity (AFPI and AFPII) were isolated by extraction of membrane vesicles of Aspergillus fumigatus with Triton X-100. These two fractions produced single bands in both polyacrylamide and sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed apparent molecular weights of 73,000 and 43,000, respectively. Molecular weights determined by gel filtration in the absence and presence of Triton X-100 and sedimentation velocities in analytical ultracentrifugation indicated hydrophobic characteristics, since both fractions readily aggregated and complexed with Triton X-100; both exhibited elevated enzyme activities in the presence of Triton X-100. Carbohydrate content was 93% for AFPI and 85% for AFPII. The enzymatic fractions demonstrated different pH optima in the acid range as well as different temperature stabilities. Both protease fractions cross reacted in double immunodiffusion, while in crossed immunoelectrophoresis both demonstrated five precipitin peaks, each with similar patterns. AFPI demonstrated two additional precipitin peaks in crossed immunoelectrophoresis. As determined by crossed immunoaffinoelectrophoresis, the protease fractions demonstrated galactose and mannose residues. In biotin-avidin enzyme-linked immunosorbent assay both fractions reacted with allergic bronchopulmonary aspergillosis and aspergilloma sera. It can be concluded that two fractions with protease activity of A. fumigatus reported here may be of significance in Aspergillus-induced diseases.
Department of Biological Sciences, University of Wisconsin, Milwaukee 53201.
J E
JE
Piechura
V P
VP
Kurup
L J
LJ
Daft
D001229:Aspergillosis, Allergic Bronchopulmonary / Q000201:enzymology*; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000276:immunology; D002462:Cell Membrane / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007123:Immunoelectrophoresis, Two-Dimensional; D008055:Lipids / Q000302:isolation & purification; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000276:immunology / Q000378:metabolism*; D013696:Temperature
D008055:Lipids; D010447:Peptide Hydrolases