A METHOD FOR DETERMINATION OF GALACTOSYLTRANSFERASE-I ACTIVITY SYNTHESIZING THE PROTEOGLYCAN LINKAGE REGION

An assay method was devised for measuring the activity of galactosyltr ansferase I (UDP-D-galactose:D-xylose galactosyltransferase), which is one of the enzymes synthesizing the linkage region between the core p rotein and glycosaminoglycan chains of proteoglycan. For this method, the reaction mixture contained a fluorescent substrate, 4-methylumbell iferyl-beta-D-xyloside as an acceptor, UDP-galactose as a donor and D- galactal as a competitive inhibitor of endogenous beta-galactosidase i n the enzyme solution. The reaction mixture was incubated at 37 degree s C with enzyme solution prepared from an extract of cultured cells, a nd galactosyl-xylosyl-4-methylumbelliferone was produced as a reaction product. Measurement of galactosyltransferase I activity was performe d by separation and quantitative analysis of this reaction product usi ng high-performance liquid chromatography. Utilizing this method, easi er and more sensitive detection of galactosyltransferase I activity in a cell-free system became possible. Application of the method reveale d that cultured human skin fibroblasts contained galactosyltransferase I activity.