title: An electrophoresis-based assay for glycosyltransferase activity.
abstract: Polyacrylamide gel electrophoresis (PAGE) and capillary zone electrophoresis (CZE) were used to measure the activity of glycosyltransferases. Acceptor molecules were prepared by reductive amination of the monopotassium 7-amino-1,3-naphthalenedisulfonic acid (AGA) Schiff base with sugars. The resulting sugar conjugates were purified by gradient PAGE and recovered using semidry electrotransfer into a positively charged nylon membrane. The beta(1----4)galactosyltransferase was shown, by PAGE analysis, to transfer a beta-galactosyl residue to the AGA conjugate of beta-D-GlcNAc-(1----4)-beta-D-GlcNAc-(1----4)-D-GlcNAc (compound 4). Similarly, alpha(1----2)fucosyltransferase isolated from porcine submaxillary glands was shown to transfer fucose from GDP-fucose to the AGA conjugate of beta-D-Gal-(1----4)-beta-D-GlcNAc-(1----6)-D-Gal (compound 5). This conjugate (compound 5) was also an acceptor for the alpha(1----3/4)fucosyltransferase partially purified from human milk. The latter reaction was followed by both gradient PAGE and CZE, having sensitivities of 200 pmol and 80 fmol, respectively.
authors.affiliation: Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242.
authors.forename: K B
authors.initials: KB
authors.lastname: Lee
authors.forename: U R
authors.initials: UR
authors.lastname: Desai
authors.forename: M M
authors.initials: MM
authors.lastname: Palcic
authors.forename: O
authors.initials: O
authors.lastname: Hindsgaul
authors.forename: R J
authors.initials: RJ
authors.lastname: Linhardt
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives; D002240:Carbohydrate Sequence; D004586:Electrophoresis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005647:Fucosyltransferases / Q000378:metabolism; D016695:Glycosyltransferases / Q000032:analysis*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008895:Milk, Human / Q000201:enzymology; D008969:Molecular Sequence Data; D016339:Spectrometry, Mass, Fast Atom Bombardment
chemical_list: C033153:amido-G-acid; D015081:2-Naphthylamine; D016695:Glycosyltransferases; D005647:Fucosyltransferases