The localization of galactosyltransferases in polyacrylamide gels by a coupled enzyme assay

Abstract

A coupled enzyme assay for GlcNAc 1 1 Abbreviations used: GlcNAc, N-acetylglucosamine-(2-acetamido-2-deoxy- d-glucose); PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis[2-(5-phenyloxazolyl)]benzene; INT, p-iodonitrotetrazolium. : UDP-galactose galactosyltransferase has been developed that allows this enzyme to be assayed spectrophotometrically and in nondenaturing polyacrylamide gels. Utilizing three, intermediate enzymes, galactosyltransferase activity has been coupled to the production of NADH with a stoichiometry of 2 mol of NADH produced for each mol of galactose transferred to GlcNAc. The enzyme reactions coupled to the production of UDP by galactosyltransferase can be summarized as follows: ▪ The activities of partly purified bovine milk galactosyltransferase and galactosyltransferase in dialyzed fetal calf serum have been determined spectrophotometrically by measuring NADH production at 340 nm. The reaction is dependent on N-acetylglucosamine, UDP-galactose, and Mn 2+. For both enzyme sources, activities calculated from NADH production are similar to those determined from assays that use radioactive sugar nucleotide substrates. Both galactosyltransferase activities have been localized on 7.5% nondenaturing polyacrylamide gels after electrophoresis by incubating the gel with an agarose indicator gel containing the coupled enzyme system. Enzyme activity is marked by NADH fluorescence, which is dependent on the presence of N-acetylglucosamine in the indicator gel. The intensity of fluorescence increases with increasing galactosyltransferase activity applied to the gel.