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

• Published in: Journal of biochemistry (Tokyo) Vol. 85; no. 1; pp. 123 - 130
• Main Authors: TANIMURA, Teiichi, KITAMLIRA, Keisuke, FUKUDA, Tomoko, KIKUCHI, Toshihide
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 1979
• Subjects: alpha-Glucosidases - isolation & purification; alpha-Glucosidases - metabolism; Animals; Drosophila melanogaster - enzymology; Glucosidases - isolation & purification; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Molecular Weight
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a132301

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 Km 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 Km values were 0·77 and 0·21 mM respectively.