Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002792:Cholestyramine Resin / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000302:isolation & purification / Q000378:metabolism*; D007106:Immune Sera; D007118:Immunoassay; D005779:Immunodiffusion; D007700:Kinetics; D008148:Lovastatin / Q000031:analogs & derivatives*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D009281:Naphthalenes / Q000494:pharmacology; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011487:Protein Conformation; D051381:Rats
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D007106:Immune Sera; D009281:Naphthalenes; D002792:Cholestyramine Resin; C012258:mevastatin; D011092:Polyethylene Glycols; D017830:Octoxynol; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases; D005459:Fluorides