The quantitative assay of 3-hydroxy-3-methylglutaryl coenzyme A reductase: comparison of a thin-layer chromatographic assay with a rapid chloroform extraction assay

Abstract

Two assays for the quantitative measurement of HMG-CoA reductase, the major regulatory enzyme in hepatic cholesterol biosynthesis, are described. The first of these procedures employs thin-layer chromatography (TLC) of the reaction product on plastic-backed silica gel G strips impregnated with ammonium carbonate. The TLC strip is then cut into 14 segments and each segment is assayed for radioactivity. Extraction efficiency and exact chromatographic mobility are monitored by the use of authentic [4-(3)H]mevalonolactone as an internal reference. A wide and complete separation is achieved between HMG and mevalonolactone. Also the radiochemical purity of biosynthetic [3-(14)C]mevalonolactone can be assessed by measurement of the (3)H/(14)C ratio across the mevalonolactone peak. While the TLC assay is accurate and sensitive, it is laborious. Therefore a second assay procedure was developed using chloroform extraction of an incubation mixture saturated with solid equal molar KH(2)PO(4)-K(2)HPO(4) at pH 6.8. Extraction efficiency was monitored by the addition of authentic [4-(3)H]mevalonolactone as an internal reference. The chloroform assay procedure was compared with the TLC procedure over a wide range of enzyme activities, both for rat liver microsomal HMG-CoA reductase and for solubilized enzyme. Excellent correspondence (r = 0.999) between the two assays was observed. TLC performed on the chloroform extract demonstrated that biosynthetic [3-(14)C]mevalonolactone was the only (14)C-labeled compound present in the extract. The chloroform extraction assay is rapid; 20-30 samples can be processed in 2-3 hr. This procedure should facilitate studies concerning the nature and regulation of HMG-CoA reductase.