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.
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CMPNeuAc:Gal beta 1-4GlcNAc-R α-2,6-唾液酸转移酶活性的固相测定。
DOI：
10.1016/0003-2697(92)90389-o
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发表时间：
1992
影响因子：
2.9
通讯作者：
Cummings,RD
中科院分区：
文献类型：
--
作者：
Mattox,S;Walrath,K;Ceiler,D;Smith,DF;Cummings,RD
建立了固相法测定CMPNeuAc：Gal β1-4GlcNAc-R α-2，6-唾液酸转移酶（2，6ST）活性的方法。在该测定中，将受体糖蛋白固定在微量滴定板威尔斯孔上。所用的两种糖蛋白受体是无唾液酸胎球蛋白（ASF），其含有以序列Gal β1-4GlcNAc-R终止的寡糖，以及含有共价连接的Gal β1-4GlcNAc-R单元的牛血清白蛋白的新糖蛋白。将含有供体CMP NeuAc和2，6ST的样品与固定化受体一起孵育，以生成产物NeuAc α2- 6 Gal β1-4GlcNAc-R。使用生物素化的植物凝集素黑接骨木凝集素（SNA）通过生物素-链霉亲和素系统检测产物，SNA与α-2，6键而非α-2，3键的唾液酸结合。然后用链霉亲和素和碱性磷酸酶或重组生物发光蛋白水母发光蛋白的生物素化形式检测与产物结合的生物素化SNA。针对市售2，6 ST优化了测定，并显示在1小时测定中，在20至400 μU范围内，测定依赖于受体和CMPNeuAc的浓度，并与2，6 ST活性成比例。固相测定还允许选择性检测人和胎牛血清中的2，6 ST活性，其中活性在0.1至2 μl血清范围内成比例。
A solid-phase assay for the activity of CMPNeuAc:Gal β1-4GlcNAc-R α-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 β1-4GlcNAc-R, and a neoglycoprotein of bovine serum albumin containing covalently attached Gal β1-4GlcNAc-R units. Samples containing the donor CMPNeuAc and the 2,6ST were incubated with the immobilized acceptor to generate the product NeuAc α2-6Gal β1-4GlcNAc-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 α-2,6, but not in α-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 μU 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 μl of serum.