A fluorometric microassay procedure for monitoring the enzymatic activity of GM1-ganglioside beta-galactosidase by use of high-performance liquid chromatography.
For the measurement of the enzymatic activity of GM1-ganglioside (II3 NeuAcGgOse4Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl) galactosyl-glucosylceramide) beta-galactosidase in crude enzyme samples, a microassay using nonradioisotopic GM1-ganglioside was devised. To reduce the volume of the reaction mixture and eliminate the interferences due to the fluorescent contaminants in the reaction mixture, NADH, a product after the oxidation of the released galactose with NAD and beta-galactose dehydrogenase, was fluorometrically estimated by use of high-performance liquid chromatography. By this method, as little as 10 pmol of galactose can be detected. Using rat brain homogenates as an enzyme sample, the several parameters were reexamined to define the optimal conditions for the assay. This assay method was also applied to human cultured skin fibroblast homogenates, and it was found that this method can be used for the diagnosis of GM1-gangliosidosis, instead of the usual method using the radioisotope-labeled natural substrate.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005347:Fibroblasts / Q000201:enzymology; D005696:Galactosidases / Q000032:analysis*; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008832:Microchemistry; D009243:NAD / Q000032:analysis; D051381:Rats; D013050:Spectrometry, Fluorescence; D001616:beta-Galactosidase / Q000032:analysis*
D009243:NAD; D005696:Galactosidases; C021502:acid beta-galactosidase; D001616:beta-Galactosidase