| Popis: |
An extracellular β-glucosidase from Aspergillus fumigatus has been purified to homogeneity as judged by polyacrylamide gel electrophoresis, molecular sieve chromatography, and analytical ultracentrifugation. An average molecular weight of 40,805 was obtained. The enzyme has a pH optimum of 5.0, Km and Vmax values of 20 mm and 5 µmoles per min per mg, respectively, with sophorose as substrate, and 6.3 mm and 1.5 µmoles per min per mg, respectively, using p-nitrophenyl-β-d-glucoside. Specificity studies indicate that the enzyme is specific for the β-configuration and prefers β-1 → 2 and β-1 → 3 linkages. The purified enzyme contained 32.5 moles of glucose, 16.6 moles of mannose, and 1.84 moles of glucosamine per mole of enzyme. However, when the enzyme was precipitated with trichloroacetic acid, all of the glucose was solubilized whereas the mannose and glucosamine remained with the protein, suggesting that glucose was not covalently bound to the enzyme. In order to determine the linkage region of oligosaccharide to protein, the intact enzyme (before treatment with trichloroacetic acid) was treated with mild alkali in the presence of [3H]NaBH4. The only radioactive sugar alcohol formed by this treatment was sorbitol. Since glucose is not covalently bound to the protein and since no radioactive mannitol or glucosaminitol was detected, a hexose linkage to serine or threonine was unlikely. However, hydrolysis of the glycoprotein with stronger alkali in the presence of [3H]NaBH4 yielded an oligosaccharide which gave rise to [3H]glucosaminitol upon acid hydrolysis indicating a glucosamine → asparagine linkage. The β-glucosidase contained 2 moles of glucosamine per mole of protein suggesting at most two oligosaccharide chains. When the protein was digested with Pronase, two major glycopeptides were obtained by Sephadex G-50 chromatography. These contained mannose and aspartic acid in ratios of 10.9 and 4.8, suggesting that there might be two oligosaccharide chains per glycoprotein molecule. |
