Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A

• Main Authors: Frank H. Hulcher, Wayne H. Oleson
• Format: Article
• Language: English
• Published: Elsevier 1973-11-01
• Series: Journal of Lipid Research
• Subjects: pigeon liver; yeast microsomes; 3-hydroxy-3-methylglutaryl CoA deacylase; sulfhydryl group; arsenite-dithiol complex; enzyme kinetics
• Online Access: http://www.sciencedirect.com/science/article/pii/S0022227520368437

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 A–5,5′-dithiobis(2-nitrobenzoic 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 εmax = 1.36 × 104 at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This method 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 Km value for 3-hydroxy-3-methylglutaryl CoA was 1.75 × 10–5 M, and for NADPH the value was 6.81 × 10–4 M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different.