Dolichyldiphosphoryloligosaccharide--protein oligosaccharyltransferase; solubilization, purification, and properties.
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 Glc(n)-Man(x)-GlcNAc(2)-diphosphoryldolichol as an oligosaccharyl donor and the (125)I-labeled tryptic peptide consisting of residues 29-58 from bovine alpha-lactalbumin as acceptor. The transferase was purified approximately 2000-fold by fractionation on a bovine alpha-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, (125)I-glycopeptide, was determined as concanavalin A-agarose-adsorbed radioactivity by a filter disc assay method. (125)I-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% alpha-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 [(3)H]oligosaccharyldiphosphoryldolichol and unlabeled peptide acceptor; and (v) digestion of (3)H-labeled peptide with Pronase, resulting in the formation of lower molecular weight glycopeptide. Oligosaccharyltransferase activity exhibited an absolute requirement for divalent cations (3 mM Mn(2+); Mg(2+) was 30% as effective), complete dependence on exogenously supplied peptide acceptor (1.33 mug/ml) and oligosaccharyldiphosphoryldolichol (approximately 10 nmol/ml), and an optimum pH between 7 and 7.5.
D000818:Animals; D002645:Chickens; D004288:Dolichol Phosphates / Q000378:metabolism*; D005260:Female; D006020:Glycopeptides / Q000096:biosynthesis; D006023:Glycoproteins / Q000096:biosynthesis*; D006602:Hexosyltransferases; D008345:Manganese / Q000378:metabolism; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology; D010057:Oviducts; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D012995:Solubility; D014166:Transferases / Q000302:isolation & purification* / Q000378:metabolism
D004288:Dolichol Phosphates; D006020:Glycopeptides; D006023:Glycoproteins; D008565:Membrane Proteins; D011106:Polyisoprenyl Phosphates; D008345:Manganese; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase