A TIME-RESOLVED IMMUNOFLUOROMETRIC METHOD FOR THE MEASUREMENT OF SIALYL-LEWIS-X-SYNTHESIZING ALPHA-1,3-FUCOSYL-TRANSFERASE ACTIVITY

We describe here an assay that employs a highly sensitive nonradioacti ve method, time-resolved fluorometry, for measuring the activity of th e enzyme GDP-Fuc:Neu-NAc alpha 2-3Gal beta 1-4GlcNAc-R (Fuc to GlcNAc) alpha 1,3-fucosyltransferase (alpha 1,3FT). In this assay, a neoglyco protein substrate of alpha 1,3FT is immobilized on a microtiter plate, Incubation with the fucose donor GDP-fucose and enzyme source convert s the acceptor NeuNAc alpha 2-3Gal beta 1-4GlcNAc-R to the product Neu NAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-R, which is quantified using a product-specific (antisialyl Lewis x) primary antibody and eur opium chelate-labeled secondary antibody, In the development of the as say, we used extracts of alpha 1,3FT-transfected insect cells as the s pecific enzyme source, The reaction product formation was proportional to time of incubation (0-2 h) and the extract added (0.1-10 mu U of e nzyme) and was dependent on the GDP-fucose and glycoconjugate acceptor , We have also demonstrated with different cultured cancer cell lines that this time-resolved immunofluorometric assay allows rapid measurem ent of alpha 1,3FT activity from a large number of crude cell lysate s amples. Our results indicated that cell Lines which expressed more sia lyl Lewis x determinant on their surfaces had higher levels of alpha 1 ,3FT activity, The advantages of this new assay are high sensitivity a nd a wide linear range of measurement. The assay is expected to be use ful in the determination of regulation mechanisms of sialyl Lewis x-sy nthesizing alpha 1,3-fucosyltransferases. (C) 1997 Academic Press.