Purification and properties of glucosidase I from mung bean seedlings.
The microsomal enzyme fraction from mung bean seedlings was found to contain glucosidase activity capable of releasing [3H]glucose from the glucose-labeled Glc3Man9GlcNAc. The enzymatic activity could be released in a soluble form by treating the microsomal particles with 1.5% Triton X-100. When the solubilized enzyme fraction was chromatographed on DE-52, it was possible to resolve glucosidase I activity (measured by the release of [3H]glucose from Glc3Man9GlcNAc) from glucosidase II (measured by release of [3H]glucose from Glc2Man9GlcNAc). The glucosidase I was purified about 200-fold by chromatography on hydroxylapatite, Sephadex G-200, dextran-Sepharose, and concanavalin A-Sepharose. The purified enzyme was free of glucosidase II and aryl-glucosidase activities. Only a single glucose residue could be released from the Glc3Man9GlcNAc by this purified enzyme and the other product was the Glc2Man9GlcNAc. Furthermore, this enzyme was inhibited in a dose-dependent manner by kojibiose, an alpha-1,2-linked glucose disaccharide, but not by other alpha-linked glucose disaccharides. These data indicate that this glucosidase is a specific alpha-1,2-glucosidase. The pH optimum for the glucosidase I was about 6.3 to 6.5, and no requirements for divalent cations were observed. The enzyme was inhibited strongly by the glucosidase processing inhibitors, castanospermine and deoxynojirimycin, and less strongly by the plant pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. However, the enzyme was not inhibited by the mannosidase processing inhibitors, swainsonine, deoxymannojirimycin or 1,4-dideoxy-1,4-imino-D-mannitol. The stability of the enzyme under various conditions and other properties of the enzyme were determined.
D002845:Chromatography / Q000379:methods; D004187:Disaccharides / Q000494:pharmacology; D007887:Fabaceae / Q000201:enzymology*; D005947:Glucose / Q000378:metabolism; D005959:Glucosidases / Q000302:isolation & purification*; D065089:Glycoside Hydrolase Inhibitors; D006863:Hydrogen-Ion Concentration; D017830:Octoxynol; D010946:Plants, Medicinal; D011092:Polyethylene Glycols; D012639:Seeds / Q000201:enzymology; D013696:Temperature; D000520:alpha-Glucosidases / Q000302:isolation & purification* / Q000378:metabolism
D004187:Disaccharides; D065089:Glycoside Hydrolase Inhibitors; D011092:Polyethylene Glycols; D017830:Octoxynol; D005959:Glucosidases; C042059:glucosidase I; D000520:alpha-Glucosidases; D005947:Glucose