Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A.
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 epsilon(max) = 1.36 x 10(4) 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 K(m) value for 3-hydroxy-3-methylglutaryl CoA was 1.75 x 10(-5) M, and for NADPH the value was 6.81 x 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.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001565:Benzoates; D002021:Buffers; D002250:Carbon Radioisotopes; D003065:Coenzyme A / Q000032:analysis*; D010856:Columbidae; D004220:Disulfides; D004229:Dithiothreitol; D005954:Glucosephosphate Dehydrogenase; D005958:Glucosephosphates; D005977:Glutarates; D007700:Kinetics; D008722:Methods; D008798:Mevalonic Acid; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP; D009574:Nitro Compounds; D012441:Saccharomyces cerevisiae; D013053:Spectrophotometry
D001565:Benzoates; D002021:Buffers; D002250:Carbon Radioisotopes; D004220:Disulfides; D005958:Glucosephosphates; D005977:Glutarates; D009574:Nitro Compounds; D009249:NADP; D000429:Alcohol Oxidoreductases; D005954:Glucosephosphate Dehydrogenase; D008798:Mevalonic Acid; D003065:Coenzyme A; D004229:Dithiothreitol