期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1980
卷号：77
期号：7
页码：3811-3815
DOI：10.1073/pnas.77.7.3811
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Dolichyldiphosphoryloligosaccharide--protein oligosaccharyltransferase was solubilized from hen oviduct rough endoplasmic reticulum by extraction with 0.2% Nonidet P40. Oligosaccharyltransferase activity was assayed in an incubation mixture containing Glcn-Manx-GlcNAc2-diphosphoryldolichol as an oligosaccharyl donor and the 125I-labeled tryptic peptide consisting of residues 29-58 from bovine -lactalbumin as acceptor. The transferase was purified approximately 2000-fold by fractionation on a bovine -lactalbumin-Sepharose column; the active material bound quantitatively to the gel and was eluted by removal of divalent cation from the wash buffer. The product of the transferase activity, 125I-glycopeptide, was determined as concanavalin A-agarose-adsorbed radioactivity by a filter disc assay method. 125I-Labeled concanavalin A-agarose-bound product was characterized as a glycopeptide as follows: (i) gel filtration behavior on Sephadex G-50; (ii) elution from concanavalin A-agarose with 1% -methyl mannoside; (iii) absence of affinity for ricin-Sepharose and loss of affinity for concanavalin A-agarose after treatment with endo-{beta}-N-acetylglucosaminidase H; (iv) enzymatic synthesis of identical product upon using [3H]oligosaccharyldiphosphoryldolichol and unlabeled peptide acceptor; and (v) digestion of 3H-labeled peptide with Pronase, resulting in the formation of lower molecular weight glycopeptide. Oligosaccharyltransferase activity exhibited an absolute requirement for divalent cations (3 mM Mn2+; Mg2+ was 30% as effective), complete dependence on exogenously supplied peptide acceptor (1.33 {micro}g/ml) and oligosaccharyldiphosphoryldolichol (approximately 10 nmol/ml), and an optimum pH between 7 and 7.5.
关键词：glycoprotein synthesis ; glycosylaminyltransferase ; soluble membrane protein
