Modified procedure for detection of GDP-L-fucose:galactoside 2'-fucosyltransferase.

A modified method was developed for determination of GDP-L-fucose: galactoside 2'-fucosyltransferase in human serum which employed O-nitrophenyl-beta-D-galactopyranoside, phenyl-2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside, or phenyl-beta-D-thiogalactopyranoside as acceptor. Products were identified and characterized by thin layer chromatography with authentic reference compounds and by hydrolysis with alpha-(1 leads to 2)-L-fucosidase. The principal advantages of this method over the previous procedure which used aryl galactoside acceptors, are elimination of the need for a radiochromatogram scanner to locate reaction products and a reduced development time for chromatography. These results in substantial time and cost savings. Moreover, our system can simultaneously monitor possible competing reactions which may interfere with determination of alpha-2-L-fucosyltransferase activity. These include phosphorylase and alpha-L-fucosidase activities and incorporation of [14C]-alpha-L-fucose into endogenous acceptors of enzyme preparations. thus, this modified procedure will facilitate determination of alpha-2-L-fucosyltransferase.