Verfahren zur Bestimmung der 3‐Hydroxy‐3‐methylglutaryl‐Coenzym A‐Reduktase‐Aktivität in Rattenleber

1 A new assay procedure for the enzyme 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase in rat liver microsomal preparations has been developed. Highly labeled [5‐ 14 C]3‐hydroxy‐3‐methylglutaryl‐CoA is incubated with rat liver microsomal suspensions in the presence of a NADPH regenerating system. After addition of carrier mevalonate the reaction is stopped by heating and mevalonate transformed into the lactone. After extraction the latter is converted into the more volatile trimethylsilyl ether, which is separated by gas liquid chromatography from accompanying radioactive materials and from an internal standard employed for quantitative mass determination. The eluate from the column after mass detection is continuously combusted to 14 CO 2 , the radioactivity of which is detected in the flow‐through cell of a scintillation spectrometer, i.e. mass and radioactivity of mevalolactonetrimethylsilyl ether are recorded almost synchronously. A formula is given for the calculation of specific activities from the large number of variables involved in the procedure. 2 A method is described for the determination of the specific radioactivities of the 3‐hydroxy‐3‐methylglutaryl‐CoA preparations used. It mainly consists of (a) a gaschromatographic procedure for determination of the ratio of the radioactivities of 3‐methylglutaconic and 3‐hydroxy‐3‐methylglutaric acids in a solution containing their enzymatically synthesized CoA‐esters, and (b) an optical assay for hydroxymethylglutaryl‐CoA, which involves the cleavage of that compound by hydroxymethyl‐glutaryl‐CoA acetoacetate lyase to acetoacetate and acetyl‐CoA. The latter is determined in a citrate synthase test system. 3 Up to a certain enzyme concentration mevalonate formation from 3‐hydroxy‐3‐methyl‐glutaryl‐CoA is proportional to the amount of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase used in the assay. The K m for 3‐hydroxy‐3‐methylglutaryl‐CoA is determined to be 4.7 × 10 −5 M. The assay system must include a thiolgroup carrier to protect enzyme sulfhydryl groups. 4 It is shown that the microsomal fraction also contains 3‐hydroxy‐3‐methylglutaryl‐CoA acetoacetate lyase which consumes the same substrate as the reductase and remains active in absence of Mg 2+ . In this way the lyase may interfere with the test for 3‐hydroxy‐3‐methylglutaryl‐CoA reductase, if the concentration of microsomal protein is too high. The microsomal protein concentration is low enough, if one obtains a linear relationship between mevalonate formation and the concentration of microsomal protein.