3-hydroxy-3-methylglutaryl-CoA reductase: solubilization in the presence of proteolytic inhibitors, partial purification, and reversible phosphorylation-dephosphorylation.
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.
D000818:Animals; D002792:Cholestyramine Resin / Q000494:pharmacology; D005260:Female; D006903:Hydroxymethylglutaryl CoA Reductases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D012995:Solubility
D011480:Protease Inhibitors; D002792:Cholestyramine Resin; D006903:Hydroxymethylglutaryl CoA Reductases; D010749:Phosphoprotein Phosphatases