| Abstrakt: |
We describe here an assay that employs a highly sensitive nonradioactive method, time-resolved fluorometry, for measuring the activity of the enzyme GDP-Fuc:NeuNAcα2–3Galβ1–4GlcNAc-R (Fuc to GlcNAc) α1,3-fucosyltransferase (α1,3FT). In this assay, a neoglycoprotein substrate of α1,3FT is immobilized on a microtiter plate. Incubation with the fucose donor GDP-fucose and enzyme source converts the acceptor NeuNAcα2–3Galβ1–4GlcNAc-R to the product NeuNAcα2–3Galβ1–4(Fucα1–3)GlcNAc-R, which is quantified using a product-specific (antisialyl Lewis x) primary antibody and europium chelate-labeled secondary antibody. In the development of the assay, we used extracts of α1,3FT-transfected insect cells as the specific enzyme source. The reaction product formation was proportional to time of incubation (0–2 h) and the extract added (0.1–10 μU of enzyme) 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 measurement of α1,3FT activity from a large number of crude cell lysate samples. Our results indicated that cell lines which expressed more sialyl Lewis x determinant on their surfaces had higher levels of α1,3FT activity. The advantages of this new assay are high sensitivity and a wide linear range of measurement. The assay is expected to be useful in the determination of regulation mechanisms of sialyl Lewis x-synthesizing α1,3-fucosyltransferases. |
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