Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second beta-glucosidase.
A second extracellular beta-glucosidase (betalarge) of Aspergillus fumigatus was purified to homogeneity and shown to be a glycoprotein, as determined by polyacrylamide gel electrophoresis followed by staining for protein and for carbohydrate. Its molecular weight was approximately 340,000 by gel filtration, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave an apparent molecular weight of 170,000, suggesting that the enzyme has two subunits. The glucosidase contained covalently bound sugars consisting of about 2 mol of glucosamine and 16 mol of mannose per mol of protein. The carbohydrate was found to be attached to the peptide via glucosaminyl leads to peptide linkage, possibly to asparagine residues. At pH 4.5 this enzyme readily hydrolyzed p-nitrophenyl-beta-D-glucopyranoside (Km = 0.88 mM) and cleaved two glucose disaccharides: gentiobiose (beta,1 leads to 6; Km = 0.75 mM) and cellobiose (beta,1 leads to 4; Km = 0.84 mM). Although its activity is similar to that of a previously purified beta-glucosidase (betasmall), the two enzymes differ with respect to their pH activity profiles, substrate specificities, and molecular weights. Also double diffusion tests with anti-betasmall antiserum and both purified beta-glucosidases revealed a nonidentical cross-reaction. Microcomplement fixation of native and periodate-oxidized betasmall suggested that the oligosaccharide chain(s) was not a major antigenic site.
D000645:Ammonium Sulfate; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000276:immunology; D011232:Chemical Precipitation; D002845:Chromatography; D004187:Disaccharides / Q000378:metabolism; D000939:Epitopes; D005944:Glucosamine / Q000032:analysis; D005947:Glucose; D005959:Glucosidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis / Q000096:biosynthesis*; D006027:Glycosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D008358:Mannose / Q000032:analysis; D008970:Molecular Weight; D009578:Nitrobenzenes
D004187:Disaccharides; D000939:Epitopes; D006023:Glycoproteins; D006027:Glycosides; D006882:Hydroxyapatites; D009578:Nitrobenzenes; D005959:Glucosidases; D005947:Glucose; D005944:Glucosamine; D008358:Mannose; D000645:Ammonium Sulfate