A rapid, highly reproducible, micro-incubation, direct thin-layer chromatographic assay for 3-hydroxy-3-methylglutaryl-CoA reductase is described. The isolation and quantitation of mevalonic acid is simplified by direct application of the deproteinized, acidified incubation mixture to thin-layer chromatography sheets. The tedious and variable extraction of mevalonic acid into ether is eliminated by this procedure. Background levels are lower than those obtained with comparable assays and permit quantitation of as little as 30 pmoles of mevalonic acid. The method is illustrated by analysis of 3-hydroxy-3-methylglutaryl CoA reductase from rat liver, cultured L cells, and solubilized microsomal reductase. The rapid inactivation of reductase by MgATP and the low levels of reductase activity in cholesterol-treated L cells are accurately determined by using the micro-incubation, direct thin-layer chromatography method.
|Original language||English (US)|
|Number of pages||9|
|Journal||BBA - Enzymology|
|State||Published - Dec 29 1974|
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