Improved methods for the assay and activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.
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.
D000135:Acid Phosphatase / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000494:pharmacology; D000818:Animals; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation; D004793:Enzyme Reactivators; D005260:Female; D006903:Hydroxymethylglutaryl CoA Reductases / Q000032:analysis / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D012995:Solubility
D004793:Enzyme Reactivators; D006903:Hydroxymethylglutaryl CoA Reductases; D000135:Acid Phosphatase; D000251:Adenosine Triphosphatases