3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a transmembrane glycoprotein, located on the endoplasmic reticulum.1 This enzyme catalyzes the four-electron reduction of HMG-CoA to coenzyme A and mevalonate, which is the ratelimiting step in sterol biosynthesis.2 The activity of HMGR is controlled through synthesis, degradation, and phosphorylation in order to maintain the concentration of mevalonate derived products. In addition to the physiological regulation of HMGR, the human enzyme has been targeted successfully by drugs in the clinical treatment of high serum cholesterol levels.3,4 Controlling serum cholesterol levels has an important therapeutic role as hypercholesterolemia often leads to the development of atherosclerosis and consequently to cardiovascular pathologies, which might result in myocardial infarction and stroke. Recent evidence suggests that a disturbance of cholesterol homeostasis contributes to the development of a chronic inflammatory state.5
The assay is based on a spectrophotometric measurement of the decrease in absorbance, which represents the oxidation of NADPH by the catalytic subunit of HMGR in the presence of the substrate HMG-CoA.
Figure 1.Inhibition of HMG-CoA reductase (HMGR) activity by Pravastatin.
HMG-CoA reductase activity and inhibition assay was performed in a UV compatible 96 well plate, using the HMG-CoA Reductase Assay Kit. Approximately 6 µg of the enzyme were incubated at 37 °C with 400 µM NADPH, 0.3 mg/mL HMG-CoA and different concentrations of Pravastatin that is a specific inhibitor of HMG-CoA reductase.