title: Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and its mRNA in rat liver as studied with a monoclonal antibody and a cDNA probe.
abstract: A monoclonal antibody directed against 3-hydroxy-3-methylglutaryl Coenzyme A reductase and a cDNA to reductase mRNA were used to study the subunit structure of the enzyme and the regulation of its mRNA in rat liver. Although the monoclonal antibody and the cDNA were made with materials from cultured hamster cells, the two reagents cross-reacted with reductase protein and mRNA from rat liver. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibody, the subunit molecular weight of rat liver reductase was 90,000. When the enzyme was solubilized from microsomes by freeze-thawing, the subunit molecular weight was reduced to 52,000-58,000, owing to proteolysis. This proteolysis was inhibited by EGTA and leupeptin. The cDNA probe for reductase, radiolabeled with 32P, hybridized to restriction fragments of genomic DNA from rat liver, as visualized by Southern blot analysis. In the livers of control rats, no reductase mRNA was detected when the 32P-cDNA was blot-hybridized to poly(A+) RNA. Hepatic reductase activity was increased 45-fold when rats were fed cholestyramine and mevinolin. Under these conditions, the amount of immunodetectable reductase protein rose by 33-fold, and the reductase mRNA became visible by blot hybridization as a band of approximately 4 kilobases in length. When the mevinolin/cholestyramine-treated rats were fed cholesterol, reductase activity and immunodetectable protein declined markedly and the reductase mRNA was reduced to barely detectable levels. We conclude that treatment with cholestyramine and mevinolin increases the amount of reductase protein in rat liver by elevating the amount of its mRNA and that cholesterol feeding to such induced rats lowers the amount of hepatic reductase protein by decreasing the level of its mRNA.
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism*; D005260:Female; D005796:Genes; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics* / Q000276:immunology; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D010053:Ovary; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D012333:RNA, Messenger; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases