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.
D002855:Chromatography, Thin Layer; D005260:Female; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000097:blood / Q000302:isolation & purification; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D006602:Hexosyltransferases / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D013379:Substrate Specificity
D006023:Glycoproteins; D015636:Magnesium Chloride; D006154:Guanosine Diphosphate Fucose; D005647:Fucosyltransferases; D006602:Hexosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase; D008274:Magnesium