Determination of liver microsomal HMGCoA reductase

One gram of liver was placed in 5 ml cold buffer (−4°C) at pH 7.4. The buffer contained 0.1 M triethanolamine. HCl, 0.02 M EDTA, and 2.0mM dithiothreitol, homogenized using glass homogenizer. The homogenate was centrifuged for 10 min at 12,000 g to remove mitochondria. Supernatant was centrifuged at 20,000 g for 30 min. The microsomes obtained were used for the assay of protein concentration and HMGCoA reductase. For assay 0.5–1 mg of microsomal protein, 100 nmoles of HMG CoA, and 2 µmoles of NADPH, 2 units of glucose-6-phosphate dehydrogenase, and 3 µmoles of glucose-6-phosphate. These components are added to 0.8 ml of 0.1 M triethanolamine-0.02 M EDTAbuffer at pH 7.4 without dithiothreitol. The concentration of monothiol is determined by reacting DTNB with the reaction mixture in a cuvette placed in a recording spectrophotometer⁹.

The unit of HMGCoA reductase is defined as the micromole of mevalonate produced per unit of enzyme per second.