A coupled assay for UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-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.
Datti
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A
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Orlacchio
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Siminovitch
K A
KA
Dennis
J W
JW
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis* / Q000378:metabolism; D009844:Oligosaccharides / Q000302:isolation & purification; D011865:Radioisotope Dilution Technique; D013724:Teratoma; D014316:Tritium; D014407:Tumor Cells, Cultured
D009844:Oligosaccharides; D014316:Tritium; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase