A coupled assay for UDP-GlcNAc:Galβ1-3GalNAc-R β1,6- N-acetylglucosaminyltransferase (GlcNAc to GalNAc)

Abstract

UDP-GlcNAc:Galβ1-3GalNAc-R β1,6- N-acetylglucosaminyltransferase (GlcNAc to GalNAc) (i.e., core 2 GlcNAc-T) is a developmentally regulated enzyme of the O-linked oligosaccharide biosynthesis pathway. We have developed a coupled-enzyme assay for core 2 GlcNAc-T that is approximately 100 times more sensitive than the standard assay using UDP-[ 3H]GlcNAc as a sugar donor. Core 2 GlcNAc-T reactions were performed using unlabeled UDP-GlcNAc donor and Galβ1-3GalNAcα-paranitrophenyl (pNp) as acceptor. The product, Galβ1-3(GlcNAcβ1-6)GalNAcα-pNp was then further reacted with purified bovine β1-4Gal-T and UDP-[ 3H]Gal to produce Galβ1-3([ 3H]Galβ1-4GlcNAcβ1-6)GalNAcα-pNp, which was separated on an Ultrahydrogel HPLC column. Approximately 10% of the available GlcNAc-terminating acceptor was substituted in the Gal-T reaction, allowing 1 pmol of product to be readily detected. The increased sensitivity of the coupled assay should facilitate studies of core 2 GlcNAc-T activity where material is limiting or specific activity is low.