GLYCOSYLTRANSFERASE ASSAY SYSTEM UTILIZING AN ACYLATED GLYCOPEPTIDE ACCEPTOR

A glycosyltransferase assay system was devised utilizing as acceptor a purified glycopeptide which was acylated at its N-terminus using capr ylic (C-s) anhydride. The glycopeptide contained five amino acids and an N-linked biantennary oligosaccharide, and it was purified from a pr onase digest of bovine fibrinogen. Desialylation and beta-galactosidas e digestion conditions were developed to produce asialo- and asialo-ag alacto glycopeptides. Using fatty acid anhydrides, N-acylation conditi ons for these glycopeptides were then optimized. The products formed w hen the appropriate acylated glycopeptide was incubated with either of two N-acetylglucosaminyltransferases and UDP-[H-3]N-acetylglucosamine were easily separated from unused sugar nucleotide and breakdown prod ucts by exploiting the affinity of the radiolabeled acylated glycopept ide products for pellicular C-18 cartridges. The products of the enzym atic reactions bound quantitatively to the cartridges and could be elu ted in small amounts of methanol. The K-m values for the unacylated an d acylated glycopeptide accepters were similar when measured using eit her N-acetylglucosaminyltransferase V or the N-acetylglycosaminyltrans ferase which transfers N-acetylglucosamine in beta(1,3) linkage to N-a cetyllactosamine (or lactose). This assay system can be used to measur e many glycosyltransferases and other enzymes which transfer to N-link ed biantennary oligosaccharides and is applicable to additional glycos yltransferases that transfer to other oligosaccharides which can be pr epared as glycopeptides. (C) 1995 Academic Press, Inc.