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-β-d-galactopyranoside, phenyl-2-acetamido-2-deoxy-3-O-β-d-galacto-pyranosyl-α-d-galactopyranoside, or phenyl-β-d-thiogalactopyranoside as acceptor. Products were identified and characterized by thin layer chromatography with authentic reference compounds and by hydrolysis with α-(1 → 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 result in substantial time and cost savings. Moreover, our system can simultaneously monitor possible competing reactions which may interfere with determination of α-2-l-fucosyltransferase activity. These include phosphorylase and α-l-fucosidase activities and incorporation of [¹⁴C]-α-l-fucose into endogenous acceptors of enzyme preparations. Thus, this modified procedure will facilitate determination of α-2-l-fucosyltransferase.