A 2-phase liquid scintillation assay for glycolipid synthetases.
Glycolipid synthetases can be assayed conveniently by incubating the lipid substrate with the radiosugar-labeled nucleotide in a small plastic scintillation vial. At the end of the incubation period, water and perchloric acid are added, then n-butanol, then a toluene-based scintillation cocktail. The radioactive lipid partitions into the scintillation fluid, leaving excess sugar nucleotide in the aqueous phase. Only a small fraction of the total radioactivity in the aqueous layer is detectable. This method is illustrated for ceramide:UDP-glucose glucosyltransferase. The approach should be applicable to other lipid synthetases that can be assayed with radioactive hydrophilic substrate.
D000818:Animals; D005964:Glucosyltransferases / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009929:Organ Size; D012588:Scintillation Counting / Q000379:methods*
D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase