Liscum L, Luskey K L, Chin D J, Ho Y K, Goldstein J L, Brown M S
J Biol Chem. 1983 Jul 10;258(13):8450-5.
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.
一种针对3-羟基-3-甲基戊二酰辅酶A还原酶的单克隆抗体和还原酶mRNA的cDNA被用于研究该酶的亚基结构及其在大鼠肝脏中mRNA的调控。尽管单克隆抗体和cDNA是用培养的仓鼠细胞材料制备的，但这两种试剂与大鼠肝脏中的还原酶蛋白和mRNA发生交叉反应。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和用单克隆抗体进行免疫印迹分析，大鼠肝脏还原酶的亚基分子量为90,000。当通过冻融从微粒体中溶解该酶时，由于蛋白水解作用，亚基分子量降至52,000 - 58,000。这种蛋白水解作用受到乙二醇双四乙酸（EGTA）和亮抑酶肽的抑制。用32P放射性标记的还原酶cDNA探针与大鼠肝脏基因组DNA的限制性片段杂交，通过Southern印迹分析可视化。在对照大鼠的肝脏中，当将32P - cDNA与聚腺苷酸加尾（poly(A+)）RNA进行印迹杂交时，未检测到还原酶mRNA。当给大鼠喂食消胆胺和洛伐他汀时，肝脏还原酶活性增加了45倍。在这些条件下，可免疫检测到的还原酶蛋白量增加了33倍，并且通过印迹杂交可见还原酶mRNA为一条长度约为4千碱基的条带。当给用洛伐他汀/消胆胺处理的大鼠喂食胆固醇时，还原酶活性和可免疫检测到的蛋白明显下降，还原酶mRNA降至几乎检测不到的水平。我们得出结论，用消胆胺和洛伐他汀处理通过提高其mRNA量来增加大鼠肝脏中还原酶蛋白的量，并且给这种诱导的大鼠喂食胆固醇通过降低其mRNA水平来降低肝脏还原酶蛋白的量。
