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. 1980 Jul;77(7):3811–3815. doi: 10.1073/pnas.77.7.3811

Dolichyldiphosphoryloligosaccharide—protein oligosaccharyltransferase: Solubilization, purification, and properties

Rathindra C Das 1, Edward C Heath 1
PMCID: PMC349716  PMID: 6933437

Abstract

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 α-lactalbumin as acceptor. The transferase was purified approximately 2000-fold by fractionation on a bovine α-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% α-methyl mannoside; (iii) absence of affinity for ricin-Sepharose and loss of affinity for concanavalin A-agarose after treatment with endo-β-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 μg/ml) and oligosaccharyldiphosphoryldolichol (approximately 10 nmol/ml), and an optimum pH between 7 and 7.5.

Keywords: glycoprotein synthesis, glycosylaminyltransferase, soluble membrane protein

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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