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 caprylic (C8) anhydride. The glycopeptide contained five amino acids and an N-linked biantennary oligosaccharide, and it was purified from a pronase digest of bovine fibrinogen. Desialylation and beta-galactosidase digestion conditions were developed to produce asialo- and asialo-agalacto glycopeptides. Using fatty acid anhydrides, N-acylation conditions for these glycopeptides were then optimized. The products formed when the appropriate acylated glycopeptide was incubated with either of two N-acetylglucosaminyltransferases and UDP-[3H]N-acetylglucosamine were easily separated from unused sugar nucleotide and breakdown products by exploiting the affinity of the radiolabeled acylated glycopeptide products for pellicular C18 cartridges. The products of the enzymatic reactions bound quantitatively to the cartridges and could be eluted in small amounts of methanol. The Km values for the unacylated and acylated glycopeptide acceptors were similar when measured using either N-acetylglucosaminyltransferase V or the N-acetylglycosaminyltransferase which transfers N-acetylglucosamine in beta(1,3) linkage to N-acetyllactosamine (or lactose). This assay system can be used to measure many glycosyltransferases and other enzymes which transfer to N-linked biantennary oligosaccharides and is applicable to additional glycosyltransferases that transfer to other oligosaccharides which can be prepared as glycopeptides.
Wilkes
C
C
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602.
Pierce
M
M
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D006020:Glycopeptides / Q000378:metabolism*; D016695:Glycosyltransferases / Q000032:analysis*; D008969:Molecular Sequence Data
D006020:Glycopeptides; D016695:Glycosyltransferases