A time-resolved immunofluorometric method for the measurement of sialyl Lewis x-synthesizing alpha1,3-fucosyltransferase activity.
We describe here an assay that employs a highly sensitive nonradioactive method, time-resolved fluorometry, for measuring the activity of the enzyme GDP-Fuc:NeuNAcalpha2-3Galbeta1-4GlcNAc-R (Fuc to GlcNAc) alpha1,3-fucosyltransferase (alpha1,3FT). In this assay, a neoglycoprotein substrate of alpha1,3FT is immobilized on a microtiter plate. Incubation with the fucose donor GDP-fucose and enzyme source converts the acceptor NeuNAcalpha2-3Galbeta1-4GlcNAc-R to the product NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)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 alpha1,3FT-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 microU 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 alpha1,3FT 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 alpha1,3FT 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 alpha1,3-fucosyltransferases.
Räbinä
J
J
Haartman Institute, Department of Bacteriology and Immunology, FIN-00014 University of Helsinki, Helsinki, Finland.
Smithers
N
N
Britten
C J
CJ
Renkonen
R
R
D005434:Flow Cytometry; D005470:Fluorometry / Q000379:methods*; D005643:Fucose / Q000378:metabolism; D005647:Fucosyltransferases / Q000378:metabolism*; D006801:Humans; D009844:Oligosaccharides / Q000032:analysis; D012015:Reference Standards; D000080506:Sialyl Lewis X Antigen; D013997:Time Factors
D009844:Oligosaccharides; D000080506:Sialyl Lewis X Antigen; D005643:Fucose; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase