A coupled assay for UDP-GlcNAc:Galβ1-3GalNAc-R β1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc)
Copyright policy )A coupled assay for UDP-GlcNAc:Galβ1-3GalNAc-R β1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc)
UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-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 Gal beta 1-3GalNAc alpha-paranitrophenyl (pNp) as acceptor. The product, Gal beta 1-3(GlcNAc beta 1-6)GalNAc alpha-pNp was then further reacted with purified bovine beta 1-4Gal-T and UDP-[3H]Gal to produce Gal beta 1-3([3H]Gal beta 1-4GlcNAc beta 1-6) GalNAc alpha-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.
- Lunenfeld-Tanenbaum Research Institute Canada
- University of Perugia Italy
- University of Toronto Canada
- Mount Sinai Hospital Canada
- Mount Sinai Health System United States
Radioisotope Dilution Technique, Molecular Sequence Data, Teratoma, Oligosaccharides, CHO Cells, N-Acetylglucosaminyltransferases, Tritium, Kinetics, Mice, Carbohydrate Sequence, Cricetinae, Chromatography, Gel, Tumor Cells, Cultured, Animals, Chromatography, High Pressure Liquid
Radioisotope Dilution Technique, Molecular Sequence Data, Teratoma, Oligosaccharides, CHO Cells, N-Acetylglucosaminyltransferases, Tritium, Kinetics, Mice, Carbohydrate Sequence, Cricetinae, Chromatography, Gel, Tumor Cells, Cultured, Animals, Chromatography, High Pressure Liquid
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