A FLUORESCENT ASSAY FOR THE DETERMINATION OF UDP-GLCNAC - GAL-BETA-1,3GALNAC-R (GLCNAC TO GALNAC) BETA-1,6 N-ACETYLGLUCOSAMINYLTRANSFERASE ACTIVITY

A fluorescent assay for UDP-GlcNAc: Gal beta 1,3Gal-Ac-N-R beta 1,6-N- acetylglucosaminyltransferase (core 2 GlcNAc-T) activity has been deve loped involving dansylation of the enzyme reaction product. Core 2 Glc NAc-T detection was performed using unlabeled UDP-GlcNAc as the donor and Gal beta 1,SGalNAc alpha-pAp as the acceptor. The product, Gal bet a 1,3(GlcNAc beta 1,6)-GalNAc alpha-pAp, was guantitatively derivatize d with dansyl chloride at the NH2 moiety of the pAp group and the resu ltant fluorescent trisaccharide was separated on a Spherisorb ODS2 HPL C column. This method, rapid and economical, was found to be sensitive enough for the detection of 1 pmol of reaction product and therefore represents a reliable alternative to assays which use radiolabeled sub strates. Additionally, the approach described here can be adapted for the assay of other glycosyltransferases, where the acceptor substrate has a pAp group as a hydrophobic aglycon linker. (C) 1995 Academic Pre ss, Inc.