Dolichylpyrophosphate oligosaccharides: large-scale isolation and evaluation as oligosaccharyltransferase substrates.
Oligosaccharyltransferase (OST) catalyzes the transfer of a branched oligosaccharide from a dolichylpyrophosphate oligosaccharide (Dol-PP-OS) to the asparagine of a nascent polypeptide chain in vivo and peptide substrates in vitro. Here we report the isolation and purification of Dol-PP-OS from bovine pancreas and thyroid. Steady-state kinetic parameters comparing the two Dol-PP-OS to a shorter dolichylpyrophosphate disaccharide (DolPP-DS) previously synthesized in our laboratory are reported. These were determined for Dol-PP-OS, Dol-PP-DS, and the tripeptide Bz-Asn-Leu-Thr-NH2 with solubilized OST and, for the first time, saturation kinetics were observed for all substrates. The kinetic data provide a basis for analyzing quantitatively the individual contributions of oligosaccharide donor and peptide acceptor substrates to OST-catalyzed glycosylation.
Gibbs
B S
BS
College of Pharmacy, Department of Chemistry, University of Michigan, Ann Arbor 48109, USA.
Coward
J K
JK
D000818:Animals; D002417:Cattle; D005069:Evaluation Studies as Topic; D007700:Kinetics; D010194:Pancreatin / Q000737:chemistry; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013961:Thyroid Gland / Q000737:chemistry
D011104:Polyisoprenyl Phosphate Oligosaccharides; D010194:Pancreatin