title: Enzymatic assay of galactosyltransferase by capillary electrophoresis.
abstract: The kinetic parameters of a galactosyltransferase-catalyzed reaction were determined for the first time using capillary zone electrophoresis (CZE) using the methylumbelliferyl (MU) glycoside of N-acetylglucosamine as the acceptor molecule. The CZE was performed using borate buffer and the enzymatic transformations were monitored at 214 nm. The kinetic parameters obtained for MU-GlcNAc were Km = 35.9 microM and Vmax = 7.5 micromol/min/mg, and those for UDP-Gal were Km = 115.3 microM and Vmax = 12.4 micromol/min/mg. A representative inhibition assay was also carried out using UDP as an inhibitor to give the Ki value of 83.9 microM against MU-GlcNAc. The structure of the synthetic product was also confirmed using 1H NMR spectroscopies after isolation by simple chromatography.
authors.affiliation: Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Saitama, Wako-shi, 351-01, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kanie
authors.forename: A
authors.initials: A
authors.lastname: Kirsch
authors.forename: O
authors.initials: O
authors.lastname: Kanie
authors.forename: C H
authors.initials: CH
authors.lastname: Wong
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D007700:Kinetics; D007788:Lactose Synthase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data
chemical_list: D007788:Lactose Synthase