A FLUORESCENT ASSAY-METHOD FOR GDP-L-FUC-N-ACETYL-BETA-D-GLUCOSAMINIDE ALPHA-1-6FUCOSYLTRANSFERASE ACTIVITY, INVOLVING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

An assay method for GDP-L-Fuc: N-acetyl-beta-D-glucosaminide alpha 1-6 fucosyltransferase (alpha 1-GFucT; EC 2.4.1.68) activity has been deve loped, involving a fluorescent pyridylaminated substrate. A glycopepti de derived from bovine gamma-globulin was coupled with 4-(2-pyridylami no)butylamine (PABA) through the peptide bond, and the following subst rate was obtained. [GRAPHICS] The substrate and guanosine diphospho-fu copyranoside (GDP-Fuc) were incubated with a crude enzyme extract for 2 h, and then the enzymatic product was separated by reversed phase HP LC. Quantitation of the product involved measurement of the fluorescen ce intensity of the fucosylated pyridylaminated sugar. The structures of both synthesized GnGn-bi-Asn-PABA (substrate), and synthesized GnGn F-bi-Asn-PABA (product) were analyzed by H-1 NMR. The enzymatic produc t was also analyzed by H-1 NMR and was found to have alpha 1-glucose a t the reducing end GlcNAc. This method is highly specific for alpha 1- GFucT and is applicable for various experiments, including purificatio n and cell culture ones.