hymecromone and galactal

An assay method was devised for measuring the activity of galactosyltransferase I (UDP-D-galactose:D-xylose galactosyltransferase), which is one of the enzymes synthesizing the linkage region between the core protein and glycosaminoglycan chains of proteoglycan. For this method, the reaction mixture contained a fluorescent substrate, 4-methylumbelliferyl-beta-D-xyloside as an acceptor, UDP-galactose as a donor and D-galactal as a competitive inhibitor of endogenous beta-galactosidase in the enzyme solution. The reaction mixture was incubated at 37 degrees C with enzyme solution prepared from an extract of cultured cells, and galactosyl-xylosyl-4-methylumbelliferone was produced as a reaction product. Measurement of galactosyltransferase I activity was performed by separation and quantitative analysis of this reaction product using high-performance liquid chromatography. Utilizing this method, easier and more sensitive detection of galactosyltransferase I activity in a cell-free system became possible. Application of the method revealed that cultured human skin fibroblasts contained galactosyltransferase I activity.

Topics: Binding, Competitive; Carbohydrate Sequence; Cells, Cultured; Chromatography, High Pressure Liquid; Fibroblasts; Galactose; Galactosyltransferases; Glycosaminoglycans; Humans; Hymecromone; Indicators and Reagents; Molecular Sequence Data; Oligosaccharides; Proteoglycans; Skin; Spectrometry, Fluorescence; Uridine Diphosphate Galactose