3-Hydroxy-3-methylglutaryl-CoA reductase

This chapter presents reactions catalyzed by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and the procedure for the assay of 3-hydroxy-3-methylglutaryl-CoA reductase activity. Two procedures, radioisotopic and spectrophotometric, are used to measure the ability of HMG-CoA reductase to catalyze a particular reaction. The radioisotopic method measures the formation of [14C]mevalonate from [14C]HMG-CoA. The spectrophotometric method measures the disappearance of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm. Each method has advantages and limitations. Advantages of the radioisotopic procedure, an “end-point” assay, include high sensitivity and applicability to simultaneous analysis of large numbers of samples. Ideal for analysis of microsomal preparations or limited quantities of enzyme, its drawbacks include the use of radioisotopes and their relatively high cost. Because radioactive mevaldehyde is not commercially available, the radioisotopic assay also is unsuitable for assay of reaction. The spectrophotometric assay, a kinetic assay that follows the disappearance of NADPH, is most suitable for analysis of enzyme that has been expressed at a high level and that has undergone at least partial purification.