Improved methods for the assay and activation of 3 - h y d roxy - 3 - m et h y I g I ut a ry I coe nzy m e A reductase Barbara

A simple and rapid mixed-phase method for the quantitative assay of 3-hydroxy-3-methylglutaryl (HMG)CoA reductase and a procedure for the efficient reactivation of Mg-ATP-inactivated microsomal HMG-CoA reductase by potato acid phosphatase are described. The mixed-phase assay entails the direct addition of the acidified, deproteinized incubation mixture to a toluene-based scintillation fluor. The enzymatic reaction product [3H]mevalonolactone partitions into the toluene while unreacted 3H-labeled HMG-CoA substrate remains in the aqueous phase and is not detected on scintillation counting. The accuracy and reproducibility of this method are compared to a thin-layer chromatographic assay for HMG-CoA reductase. Microsomal and solubilized HMG-CoA reductase inactivated by incubation with Mg-ATP is reactivated by purified potato acid phosphatase. Under appropriate conditions quantitative reactivation of HMG-CoA reductase is achieved, indicating that endogenous inhibitory and activating proteins regulate HMG-CoA reductase via a kinase-phosphatase system.-Philipp, B. W., and D. J. Shapiro. Improved methods for the assay and activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.]. Lipid Res. 1979. 20: 588-593. Supplementary key words cholesterol biosynthesis . kinase . phosphatase The hepatic synthesis of cholesterol is regulated primarily at the reductive conversion of HMG-CoA to mevalonic acid (1 -3). This reaction is catalyzed by the microsomal enzyme HMG-CoA reductase. Most assays for HMG-CoA reductase are based on the conversion of isotopically labeled HMG-CoA to mevalonic acid and the separation of the biosynthesized mevalonic acid (MVA) from the more polar HMG-CoA. While many approaches to this separation have been described (414), most assays have employed thin-layer chromatography to separate MVA and HMG-CoA. Extraction of MVA into an organic solvent is a prerequisite for most, but not all (5), of these assays. In this paper we describe a simple, rapid, one-step procedure for the separation of the [3H]mevalonolactone synthesized by HMG-CoA reductase from the more polar HMG-CoA. Addition of an aliquot of the acidified and deproteinized reaction mixture to a toluene-based scintillation fluor results in partition of the [3H]mevalon~lactone into the lowpolarity toluene phase in which it can be counted. The 3H-labeled HMG-CoA and the HMG-CoA breakdown products formed during incubations are sufficiently polar so that they are quantitatively retained in the aqueous phase and are therefore not detected on scintillation counting. We describe the application of this method to quantitation of the reactivation of Mg-ATP-inactivated reductase by potato acid phosphatase. Reactivation of HMG-CoA reductase by a well-characterized purified phosphatase suggests that liver proteins which activate and inactivate HMG-CoA reductase ( 5 , 1517) function through a kinasephosphatase mechanism. MATERIALS AND METHODS