A 2‐phase liquid scintillation assay for glycolipid synthetases

Abstract

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, thenn‐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 a radioactive hydrophilic substrate.