A fluorometric assay for glycosyltransferase activities using sugars aminated and tagged with 7-hydroxycoumarin-3-carboxylic acid as substrates and high performance liquid chromatography.
We developed a novel fluorometric assay method for the measurement of glycosyltransferase activities using mono- and di-saccharides aminated and tagged with 7-hydroxycoumarin-3-carboxylic acid (coumarin) as substrates, N-acetylglucosamine (GlcNAc)-coumarin for beta1,4-galactosyltransferase from bovine milk and Gal beta1-4GlcNAc-coumarin for alpha2,3- and alpha2,6-sialyltransferases from rat liver. Using Gal beta1-3GlcNAc and Gal beta1-4Glc-NAc-coumarin, alpha1,3/4- and alpha1,3-fucosyltransferase activities were also determined, respectively. These enzymatic products liberated by the reactions of glycosyltransferases in the presence of sugar nucleotides, were separated by a normal phase or an ion-pair reversed phase HPLC with an isocratic elution and fluorescence detection. We applied this assay method to determine the glycosyltransferase activities in 8 kinds of human tumor cell lines, including the cell lines derived from hepatocytes (HuH-7, HepG2), colonic cells (Colo205, HT-29), myelocytes (HL-60, U-937), B-lymphocytes (Daudi) and T-lymphocytes (Jurkat). This assay method is accurate and easy compared with other isotopic and non-isotopic assay methods, and is sensitive enough to measure glycosyltransferase activities in cell homogenates.
D000588:Amines / Q000737:chemistry; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D016695:Glycosyltransferases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014407:Tumor Cells, Cultured; D014468:Umbelliferones / Q000737:chemistry*