title: A fluorescence-based assay for Core 1 β3galactosyltransferase (T-synthase) activity.
abstract: Mucin-type O-glycans on glycoproteins in animal cells play important roles in many biological processes. Core 1 β3galactosyltransferase (Core 1 β3GalT, T-synthase) is a key enzyme in the O-glycan biosynthetic pathway. Emerging evidence has shown the importance of O-glycans and the absolute requirement of T-synthase in this pathway. The assessment of the T-synthase activity has historically been conducted using a radioactive method. Here we describe a fluorescence-based assay procedure for T-synthase activity. T-synthase utilizes the acceptor substrate 4-methylumbelliferone-α-GalNAc (GalNAcα-(4-MU)) and the donor substrate UDP-Gal to synthesize the disaccharide product Galβ1,3GalNAcα-(4-MU) structure. This product is specifically hydrolyzed by endo-α-N-acetylgalactosaminidase (O-glycosidase) releasing free 4-MU. Free 4-MU is highly fluorescent at pH 9.6-10 and can be easily measured by a fluorescent detector (Ex: 355 nm; Em: 460 nm). This fluorescence-based T-synthase assay is simple, sensitive, reproducible, not affected by enzyme source, and adaptable for high-throughput assays.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Tongzhong
authors.initials: T
authors.lastname: Ju
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
mesh_terms: D000818:Animals; D057075:Enzyme Assays / Q000379:methods*; D005700:Galactosyltransferases / Q000378:metabolism*; D006801:Humans; D006923:Hymecromone / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
chemical_list: D006923:Hymecromone; D005700:Galactosyltransferases; C076385:glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase