Purification and characterization of glucosidase II, an endoplasmic reticulum hydrolase involved in glycoprotein biosynthesis.
Rat liver glucosidase II, an endoplasmic reticulum hydrolase involved in the biosynthesis of the N-linked class of glycoproteins, has been purified in good yield to a state approaching homogeneity. The purified enzyme hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, 4-methylumbelliferyl-alpha-D-glucopyranoside, maltose, and the precursor oligosaccharides glucose1-2mannose9N-acetylglucosamine, but it does not act on glucose3mannose9N-acetylglucosamine or p-nitrophenyl-beta-D-glucopyranoside. The ratio of the rate at which glucose is released from p-nitrophenyl-alpha-D-glucopyranoside to that from glucose2mannose9N-acetylglucosamine or glucose1mannose9N-acetylglucosamine remains constant throughout the 8-step purification procedure; thus it appears that a single enzyme is responsible for the activities toward both the artificial and oligosaccharide substrates. The fact that the enzyme cleaves both of the inner 1,3-linked glucosyl residues from the precursor oligosaccharides supports the view that they are linked in the alpha-configuration. The pH dependence of enzymatic activity is quite similar for different substrates, showing a broad optimum between pH 6 and 7.5. Activity toward p-nitrophenyl-alpha-D-glucopyranoside is enhanced by 12 mM 2-deoxy-D-glucose (260-300% activation) and 25 mM mannose (150% activation), but these two compounds inhibit the action of the enzyme toward the precursor oligosaccharides. By isoelectrofocusing the purified enzyme exhibits one form, which has a pI of 3.5-3.8. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicates that glucosidase II has a subunit molecular weight of 65,000. Ferguson plot analysis of the behavior of native enzyme in polyacrylamide gels indicates that it is a 262,000-dalton tetramer. Gel filtration gives a molecular weight of 288,000. Several lines of evidence indicate that the enzyme is a glycoprotein.
D000818:Animals; D002645:Chickens; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005959:Glucosidases / Q000302:isolation & purification*; D006023:Glycoproteins / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D000520:alpha-Glucosidases / Q000302:isolation & purification* / Q000378:metabolism
D006023:Glycoproteins; D046911:Macromolecular Substances; D009844:Oligosaccharides; C034251:4-nitrophenyl-alpha-glucosidase; D005959:Glucosidases; D000520:alpha-Glucosidases