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Journal of Lipid Research, Vol. 14, 625-631, November 1973
Copyright © 1973 by Lipid Research, Inc.

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

Frank H. Hulcher and Wayne H. Oleson

Department of Biochemistry and the Arteriosclerosis Research Center, The Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103

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 _max = 1.36 x 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 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.

Supplementary key words pigeon liver • yeast microsomes • 3-hydroxy-3-methylglutaryl CoA deacylase • sulfhydryl group • arsenite-dithiol complex • enzyme kinetics

Submitted on January 18, 1973
Revised on June 21, 1973
Accepted on July 10, 1973

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