A solid-phase assay for the activity of CMPNeuAc:Gal beta 1-4GlcNAc-R alpha-2,6-sialyltransferase.
A solid-phase assay for the activity of CMPNeuAc:Gal beta 1-4GlcNAc-R alpha-2,6-sialyltransferase (2,6ST) has been developed. In the assay an acceptor glycoprotein is immobilized onto microtiter plate wells. The two glycoprotein acceptors used were asialofetuin (ASF), which contains oligosaccharides terminating in the sequence Gal beta 1-4GlcNAc-R, and a neoglycoprotein of bovine serum albumin containing covalently attached Gal beta 1-4GlcNAc-R units. Samples containing the donor CMPNeuAc and the 2,6ST were incubated with the immobilized acceptor to generate the product NeuAc alpha 2-6Gal beta 1-4GlcNAc-R. The product was detected by a biotin-streptavidin system using the biotinylated plant lectin Sambucus nigra agglutinin (SNA), which binds to sialic acid in alpha-2,6, but not in alpha-2,3, linkage. The biotinylated SNA bound to the product was then detected with streptavidin and biotinylated forms of either alkaline phosphatase or the recombinant bioluminescent protein aequorin. The assay was optimized with respect to the commercially available 2,6ST and shown to be dependent on the concentration of acceptor and CMPNeuAc and proportional to the 2,6ST activity in the range of 20 to 400 microU in a 1-h assay. The solid-phase assay also allows for the selective detection of 2,6ST activity in human and fetal bovine serum, where the activity was proportional in the range of 0.1 to 2 microliters of serum.
D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D002240:Carbohydrate Sequence; D002417:Cattle; D060748:Fetuins; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D012710:Serum Albumin, Bovine; D012799:Sialyltransferases / Q000032:analysis / Q000097:blood / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods; D000509:alpha-Fetoproteins / Q000378:metabolism; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D001212:Asialoglycoproteins; D060748:Fetuins; D006023:Glycoproteins; D007202:Indicators and Reagents; D000509:alpha-Fetoproteins; C017029:asialofetuin; D012710:Serum Albumin, Bovine; D012799:Sialyltransferases; D006026:Glycoside Hydrolases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase