Purification and partial characterization of three forms of α-glucosidase from the fruit fly Drosophila melanogaster

Three forms of β-glucosidase, I, II, and Ill, have been purified from the whole body extract of adult flies of Drosophila melanogaster in yields of 2·1, 5·3, and 6·7%, respectively. The purification procedures involved ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, Sephacryl S-200 gel filtration, and preparative gel electrophoresis. Each purified enzyme showed a single band on polyacrylamide gel on both protein and enzyme activity staining. The molecular weights of α-glucosidases I, II, and liii were estimated to be 200,000, 56,000, and 76,000, respectively, by gel filtration. SDS gels indicated that α-glucosidases II and III were each composed of a single polypeptide chain, whereas α-glucosidase I was composed of two identical subunits. Both α-glucosidases II and III hydrolyzed sucrose and p-nitrophenyl-α-D-glucoside (PNPG), but α-glucosidase I hydrolyzed PNPG to a much lesser extent than sucrose. For sucrose the pH optima of α-glucosidases I, II, and III were pH 6·0, 5·0, and 6·0 and the K_m values were 13·1, 8·9, and 10 nM, respectively. For PNPG the pH optima of a-glucosidases II and III were pH 5·5 and 6·5 and the K_m values were 0·77 and 0·21 mM respectively.