Thiol-disulfide-dependent interconversion of active and latent forms of rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme a reductase

Abstract

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase, EC 1.1.1.34) in preparations of thiol-deficient rat liver microsomes and microsomes containing thiols have been compared. Unlike microsomes containing thiols, which possess an active hydroxymethylglutaryl-CoA reductase (E a), thiol-deficient microsomes contain an inactive, latent enzyme (E 1) which can be activated by addition of thiols. E a can be converted to E 1 by dialysis. The maximal degree of activation of E 1 depends on the activating thiol with the order of effectiveness: dithioerythritol = dithiothreitol > glutathione ( GSH) > cysteine. E a is inhibited by oxidized glutathione (GSSG). The degree of the inhibition of E a by GSSG is proportional to the ratio GSSG/thiol in the reaction. E 1 was solubilized from microsomes and purified. Its molecular weight is estimated to be 104000 by gel filtration chromatography on Sepharose 6B. The reducing agents NaBH 4, dithionite and ascorbate failed to activate E 1. NaBH 4 did not inhibit E a whereas only partial inhibition was caused by ascorbate and dithionite. Soluble E a binds to both blue dextran/Sepharose 4B and agarose/hexane-3-hydroxy-3-methylglutaryl Coenzyme A affinity resins at low-salt concentrations. By contrast, soluble E 1 did not bind to agarose/hexane-hydroxymethylglutaryl-CoA whereas quantitative binding of E 1 to blue dextran/Sepharose 4B was still observed at low salt concentrations. These results indicate that thiols are necessary cofactors for hydroxymethylglutaryl-CoA reductase reaction. Their effect on the activation of E 1 is not caused by change in the state of aggregation of the enzyme. Rather, the reversible change of the enzyme from E 1 to E a is affected by increasing the affinity of the enzyme to the substrate hydroxymethylglutaryl-CoA.