3-Hydroxy-3-methylglutaryl coenzyme A reductase from rat liver. Its purification, properties, and immunochemical studies.
3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) was purified from rat liver microsomes after solubilization by a slow freezing and thawing method. The purification was accomplished by a five-step procedure involving incubation at 37 degrees, ammonium sulfate fractionation, ultrafiltration, and column chromatography on Bio-Gel A-0.5m and Sephadex G-200. The specific activities of the purified enzyme preparations were up to 480 nmol of mevalonate formed/min/mg of protein, which represented an increase of 350-fold above that of the microsomes. The purified enzyme was found to be essentially homogeneous as evidences by the usual criteria. A subunit molecular weight of 120,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On gel filtration a number of different molecular weight forms were observed which seem to be influenced by temperature, method of purification, and possibly an enzyme-Bio-Gel A-0.5m interaction. The solubilized enzyme consisted predominantly of a species with a molecular weight slightly greater than 200,000 by gel filtration and may be a dimer of two 120,000 subunits. The purified preparation contained lipids; the total cholesterol content was 18 mug/mg of enzyme protein and corresponds to a ratio of 5 mol of cholesterol/enzyme subunit of 120,000 daltons. The specificity of rabbit antiserum prepared against the purified enzyme was demonstrated by double diffusion analysis and quantitative precipitin reactions with solubilized enzyme. The antiserum, in addition to inhibiting the activity of solubilized enzyme also blocked the activity of intact microsomes. The microsomal HMG-CoA reductase is accessible to the antibody, indicating a localization of the enzyme on the outer cytoplasmic surface of the membranes. Intestinal microsomal HMG-CoA reductase was shown to cross-react with antibody to the liver enzyme.
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D006903:Hydroxymethylglutaryl CoA Reductases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D005779:Immunodiffusion; D008055:Lipids / Q000032:analysis; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D011233:Precipitin Tests; D051381:Rats
D000596:Amino Acids; D008055:Lipids; D046911:Macromolecular Substances; D000429:Alcohol Oxidoreductases; D006903:Hydroxymethylglutaryl CoA Reductases