3-hydroxy-3-methylglutaryl-CoA reductase: solubilization in the presence of proteolytic inhibitors, partial purification, and reversible phosphorylation-dephosphorylation

Abstract

A growing body of evidence indicates that 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34, reductase) is degraded by proteolytic enzymes during solubilization by traditional freeze-thaw techniques. We have solubilized reductase in an active, stable form with nonionic detergents [Lubrol WX or poly(oxyethylene) ether type W-1]. Solubilization proceeded in high (greater than 70%) yield in the presence of the proteolytic inhibitors leupeptin, phenylmethanesulfonyl fluoride, and ethylene glycol bis(beta-aminoethyl ether)-N,N,-N',N'-tetraacetic acid and was independent of prior freeze-thawing of the microsomes. We have purified detergent-solubilized reductase 40-fold in high yield by means of sucrose density gradient centrifugation and dye-ligand chromatography. Detergent-solubilized reductase is heat labile, unlike reductase solubilized by the freeze-thaw method. Detergent-solubilized reductase can be inactivated up to 90% by use of reductase kinase. This inactivation requires both adenosine 5'-triphosphate and adenosine 5'-diphosphate, as has been previously observed for both microsomal and freeze-thaw solubilized reductase. Inactivation is reversed by subsequent treatment with a phosphoprotein phosphatase.