Absorbance- and light-based solid-phase assays for CMPNeuAc:Galbeta1-4GlcNAc-R alpha-2,3-sialyltransferase.
We now report a solid-phase assay for CMPNeuAc: Galbeta1-3/4GlcNAc-R alpha-2,3-sialyltransferase (alpha2,3ST) that is nonradioactive and allows specific identification of the sialylated product. An acceptor glycoprotein, desialylated fetuin, is immobilized on a microtiter plate. The transfer of sialic acid from CMPNeuAc generates the product NeuAc alpha2-3Gal beta1-4GlcNAc-R that is specifically bound by biotinylated Maackia amurensis leukoagglutinin (MAL). The binding of biotinylated MAL is measured with either an absorbance-based reagent (streptavidin conjugated to alkaline phosphatase) or a light-based reagent (streptavidin conjugated to the bioluminescent protein aequorin, Aqualite). The rat liver alpha2,3ST was used to optimize the assay. The formation of product is linear with respect to time and dependence on the amounts of CMPNeuAc, enzyme, and acceptor coated on the plates. As little as 0.2 microU of enzyme can be measured using the streptavidin-aequorin reagent. The assay is useful with crude tissue extracts, as demonstrated by the determination of the alpha2,3ST activity in human serum and in microsomes of HL-60 and Chinese hamster ovary cells.
Yeh
J C
JC
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, 73190, USA.
Cummings
R D
RD
D000331:Aequorin; D000818:Animals; D001212:Asialoglycoproteins / Q000737:chemistry; D001426:Bacterial Proteins; D016466:CHO Cells; D006224:Cricetinae; D060748:Fetuins; D006367:HeLa Cells; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D010835:Phytohemagglutinins / Q000737:chemistry*; D011137:Polystyrenes; D051381:Rats; D012799:Sialyltransferases / Q000032:analysis*; D013053:Spectrophotometry; D019809:Streptavidin; D000509:alpha-Fetoproteins / Q000737:chemistry
D001212:Asialoglycoproteins; D001426:Bacterial Proteins; D060748:Fetuins; D010835:Phytohemagglutinins; D011137:Polystyrenes; D000509:alpha-Fetoproteins; C017029:asialofetuin; C100156:leukoagglutinins, plants; D000331:Aequorin; D019809:Streptavidin; D012799:Sialyltransferases