Measurement of alpha(1-3) fucosyltransferase activity using scintillation proximity.
The alpha 3 fucosyltransferases are a family of glycosyltransferases involved in the addition of fucose onto glycoproteins and glycolipids. One of the best defined roles for the alpha 3 fucosyltransferases is in the biosynthesis of the carbohydrate antigen sialyl Lewis X, the minimal ligand for the selectin family of adhesion molecules. We describe here the development of a single-step assay for the measurement of alpha 3 fucosyltransferase activity based on the principle of scintillation proximity. The fucosyltransferase catalyses the transfer of [3H]fucose, from GDP-[3H]fucose, onto the sugar chains of a glycoprotein acceptor noncovalently bound to a scintillant-impregnated microsphere (SPA bead). The resultant signal can be used as a measure of enzyme activity. Due to the nature of this assay no steps are required to separate unused substrate from product. Kinetic data from the assay compare favorably with those obtained from assays currently used for the alpha 3 fucosyltransferases. This SPA-based assay appears generic for the alpha 3 fucosyltransferases and readily adaptable for other glycosyltransferases. The particular advantage of the assay is anticipated to be found in the simple, routine testing of a large number of samples.
Hood
C M
CM
Glycobiology Research Unit, Glaxo Wellcome Medicines Research Centre, Stevenage, Hertfordshire, United Kingdom.
Kelly
V A
VA
Bird
M I
MI
Britten
C J
CJ
D000837:Anion Exchange Resins; D001212:Asialoglycoproteins / Q000378:metabolism; D060748:Fetuins; D005643:Fucose / Q000378:metabolism; D005647:Fucosyltransferases / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008863:Microspheres; D011994:Recombinant Proteins / Q000378:metabolism; D012588:Scintillation Counting / Q000379:methods; D014316:Tritium; D014909:Wheat Germ Agglutinins / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000378:metabolism
D000837:Anion Exchange Resins; D001212:Asialoglycoproteins; D060748:Fetuins; D011994:Recombinant Proteins; D014909:Wheat Germ Agglutinins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D014316:Tritium; D006154:Guanosine Diphosphate Fucose; D005643:Fucose; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase