A radioimmune assay of ganglioside GM1 synthase using cholera toxin.
A radioimmune assay for uridine 5'-diphosphate-galactose (UDP-Gal):GM2 galactosyltransferase, which synthesizes GM1, has been developed utilizing cholera toxin. This assay is more sensitive and simpler than previously used assays. Radioactive nucleotide substrate and GM2 were incubated with an enzyme sample, and a radiolabeled product, GM1, was reacted with cholera toxin. The GM1-cholera toxin complex was further reacted with anti-cholera toxin and Staphylococcus aureus cell suspension. The resulting complex was transferred onto a nitrocellulose membrane and quantitated by liquid scintillation counting. This assay was found to be sensitive for the detection of 100 pmol of the reaction product, GM1. With this assay method, some properties of the crude enzyme extracts from rat liver were studied. The enzyme had a pH optimum of 6.5-7.0 and required Mn2+. The Km values for UDP-Gal and GM2 were 0.12 mM and 6 microM, respectively.
D000818:Animals; D002772:Cholera Toxin; D002855:Chromatography, Thin Layer; D005700:Galactosyltransferases / Q000378:metabolism*; D051147:Ganglioside Galactosyltransferase; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals; D011485:Protein Binding; D011863:Radioimmunoassay; D051381:Rats
D008670:Metals; D002772:Cholera Toxin; D005700:Galactosyltransferases; D051147:Ganglioside Galactosyltransferase