A method for the determination of N-acetylglucosaminyltransferase III activity in rat tissues involving HPLC

A fluorescence assay method for UDP-GlcNAc:glycopeptide β1-4 N-acetylglucosaminyltransferase (GnT-III) has been developed involving a pyridylaminated sugar as a substrate. A fluorescent sugar chain, in which the reducing end of the GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc has been aminated with 2-aminopyridine, is incubated with an enzyme sample, and then the fluorescent product with a bisecting N-acetylglucosamine is separated by reverse-phase high performance liquid chromatography and quantitated with a fluorescence detector. This assay method was found to be sensitive enough for the detection of 0.1 pmol of a reaction product. This assay is a reliable alternative to the use of a radiolabeled substrate and can be used for assaying N-acetylglucosaminyltransferase activity in crude extracts of various rat tissues. The kinetic experiments were carried out using crude enzyme extracts from the rat kidney. The enzyme has a pH optimum of 6.25 and requires Mn 2+. The K m values for UDP-GlcNAc and a sugar acceptor substrate were found to be 3.1 m m and 190 μ m, respectively. The enzyme activity in the rat kidney was higher than those in the other tissues examined.