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-fucose (GDP-Fuc) are described. The methods are based on time-resolved fluorescence and microplate assay technology. The first assay relies on measuring the enzyme activity of alpha1, 3-fucosyltransferase. In this assay, transfer of fucose from GDP-Fuc converts sialyllactosamine to sialyl Lewis x tetrasaccharide, which is detected and quantified by relevant antibodies on a microplate. 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 second 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 these assays in monitoring the amount of GDP-Fuc in crude lysates of transgenic yeast, which expresses the enzymes producing GDP-Fuc. The newly developed assays are versatile and applicable to measure also other nucleotide sugars or glycosyltransferase activities in a high-throughput manner.
Räbinä
J
J
Department of Bacteriology and Immunology, Haartman Institute, Haartmaninkatu 3, FIN-00014, Finland. jarkko.rabina@helsinki.fi
Mattila
P
P
Renkonen
R
R
D002624:Chemistry, Clinical / Q000191:economics / Q000379:methods; D004305:Dose-Response Relationship, Drug; D005643:Fucose / Q000378:metabolism; D005647:Fucosyltransferases / Q000378:metabolism; D006154:Guanosine Diphosphate Fucose / Q000032:analysis*; D006155:Guanosine Diphosphate Mannose / Q000032:analysis; D037102:Lectins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014170:Transformation, Genetic
D037102:Lectins; D006154:Guanosine Diphosphate Fucose; D005643:Fucose; D006155:Guanosine Diphosphate Mannose; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase