Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine-linked oligosaccharides.
Trimming glucosidase I and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific alpha-glucosidase(s) were inhibited by 1-deoxynojirimycin with median inhibitory concentration of 3 microM, 20 microM, 12 microM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of 1-deoxynojirimycin and formed the basis for the design of the affinity ligand. Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a subunit molecular mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was approximately equal to 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 degrees C in the presence of detergent (t 1/2 approximately equal to 20 days) and showed high specificity for the hydrolysis of the terminal (alpha 1,2)-linked glucose residue in the natural substrate Glc3-Man9-(GlcNAc)2.
D017485:1-Deoxynojirimycin; D000818:Animals; D001216:Asparagine / Q000032:analysis; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D005959:Glucosidases / Q000302:isolation & purification*; D065089:Glycoside Hydrolase Inhibitors; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis; D012995:Solubility; D000520:alpha-Glucosidases / Q000302:isolation & purification*
D065089:Glycoside Hydrolase Inhibitors; D009844:Oligosaccharides; D017485:1-Deoxynojirimycin; D001216:Asparagine; C034251:4-nitrophenyl-alpha-glucosidase; D005959:Glucosidases; C042059:glucosidase I; D000520:alpha-Glucosidases; D005944:Glucosamine