Two time-resolved fluorometric high-throughput assays for quantitation of GDP-L-fucose

Two rapid and simple procedures for the quantitative analysis of GDP-L-fuco se (GDP-Fuc) are described. The methods are based on time-resolved fluoresc ence and microplate assay technology. The first assay relies on measuring t he enzyme activity of alpha1,3-fucosyltransferase, In this assay, transfer of fucose from GDP-Fuc converts sialyllactosamine to sialyl Lewis x tetrasa ccharide, which is detected and quantified by relevant antibodies on a micr oplate, The formation of the reaction product is directly dependent on the presence of GDP-Fuc in the concentration range of 10-10,000 nM, In the seco nd method GDP-Fuc inhibits the binding of fucose-specific Aleuria aurantia lectin to fucosylated glycan on a microwell. The lectin-based assay is less sensitive than the enzyme assay, but it is cheaper and faster. We used the se assays in monitoring the amount of GDP-Fuc in crude lysates of transgeni c yeast, which expresses the enzymes producing GDP-Fuc, The newly developed assays are versatile and applicable to measure also other nucleotide sugar s or glycosyltransferase activities in a high-throughput manner. (C) 2000 A cademic Press.