Development of a radiometric spot-wash assay for squalene synthase.
The principle of selective elution from a solid phase has been exploited to develop an assay for the determination of squalene biosynthesis in rat liver homogenates. Using either [1-14C]isopentenyl diphosphate as a precursor for squalene or [2-14C]farnesyl diphosphate as a direct substrate of squalene synthase, the production of radiolabeled squalene is determined after adsorption of assay mixtures onto silica gel thin-layer chromatography sheets and selective elution of the diphosphate precursors into a solution of sodium dodecyl sulfate at alkaline pH. The use of [2-14C]farnesyl diphosphate, and of an endogenous oxygen consumption system (ascorbate/ascorbate oxidase) to prevent further metabolism of squalene, allows the method to be applied as a dedicated assay for squalene synthase activity. The assay has been developed in microtiter plate format and may be deployed either in a quantitative, low-throughout mode or in a qualitative, high-through-put mode. The latter is suitable for screening to aid in the discovery of new inhibitors of squalene synthase.
Tait
R M
RM
Glaxo Group Research, Greenford, Middlesex, United Kingdom.
D000818:Animals; D002855:Chromatography, Thin Layer; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000378:metabolism*; D005260:Female; D045782:Hemiterpenes; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012717:Sesquiterpenes; D013185:Squalene / Q000378:metabolism*
D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004809:isopentenyl pyrophosphate; D009249:NADP; C004808:farnesyl pyrophosphate; D013185:Squalene; D013186:Farnesyl-Diphosphate Farnesyltransferase