Simplified spectrophotometric assay

A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate: NADP oxidoreductase [acylating CoA] , EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5’-dithiobis(2-nitrobenzoic acid). Sodium arsenite forms a complex with the dithiol, but not with monothiols. Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly. The absorbance due to the coenzyme A4,5’-dithiobis(2nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance-time curve to the time of addition of the reagent. After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from c max = 1.36 x lo4 at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This mahod provides an immediate assay. Recovery of reduced coenzyme A was 98.7%. The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5-50 nmoles of coenzyme A. It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction. The apparent K , value for 3-hydroxy-3-methylglutaryl CoA was 1.75 X M, and for NADPH the value was 6.81 X M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different. Supplementary key words pigeon liver . yeast microsomes . 3-hydroxy-3-mcthylglutaryl CoA deacylase . sulfhydryl group . arsenite-dithiol complex . enzyme kinetics 3-Hydroxy-3-methylglutaryl CoA reductase (mevalonate:NADP oxidoreductase [acylating CoA] EC 1 .l. 1.34) (HMG CoA reductase) is the major regulatory enzyme of hepatic cholesterol biosynthesis (1 -4). The reaction catalyzed involves a two-step, NADPHdependent reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate and formation of NADP and reduced coenzyme A (1, 4, 5). The measurement of activity of this enzyme involves a complex procedure requiring extraction, thin-layer chromatography, and double-label measurement (1, 3, 4, 6, 7-9). The direct spectrophotometric assay of NADPH oxidation is very slow and is subject to interference by other NADPH-requiring reactions. Therefore, it is not considered to be a practical assay for microsomal preparations. In searching for an alternative method to assay the reaction immediately, we have explored the possibility of measuring reduced coenzyme A formed in the reaction. A new spectrophotometric assay based on the stoichiometric formation of coenzyme A during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate has been de-