PHOSPHORYLATION OF NATIVE 97-KDA 3-HYDROXY-3-METHYLGLUTARYL-COENZYME-A REDUCTASE FROM RAT-LIVER - IMPACT ON ACTIVITY AND DEGRADATION OF THE ENZYME

  • 25 March 1989
    • journal article
    • research article
    • Vol. 264  (9) , 4877-4887
Abstract
Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [.gamma.-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state is isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 6-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA membrane spanning and cytoplasmic domains of the native 97-kDa protein.