Clark R E, Martin G G, Barton M C, Shapiro D J
Proc Natl Acad Sci U S A. 1982 Jun;79(12):3734-8. doi: 10.1073/pnas.79.12.3734.
Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase; mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], the key regulatory enzyme in cholesterol biosynthesis, has been purified to apparent homogeneity. Purified HMG-CoA reductase yields a single diffuse band when NaDodSO4/polyacrylamide gels are stained with Coomassie blue and yields two adjacent bands when gels are stained with silver. Purified reductase was used to elicit the production of monoclonal antibodies. Spleen cells from BALB/c mice immunized with purified HMG-CoA reductase were fused with Sp-2/0 myeloma cells. Clones producing monoclonal antibodies to HMG-CoA reductase were identified by using a solid-phase radioimmunoassay and were subcloned in soft agar. The three relatively stable hybridoma lines isolated secrete different Igs as judged by their antibody subclasses and differing abilities to inhibit HMG-CoA reductase in solution. Efficient precipitation of solubilized HMG-CoA reductase was achieved with the two IgG antibodies but not with the IgM. A mixture of all three monoclonal antibodies immunoprecipitates more than 90% of the HMG-CoA reductase activity in solubilized rat liver extracts. These monoclonal antibodies should be useful probes for investigation of the regulation of HMG-CoA reductase and cholesterol synthesis.
大鼠肝脏微粒体3-羟基-3-甲基戊二酰辅酶A还原酶[HMG-CoA还原酶；甲羟戊酸:NADP+氧化还原酶(辅酶A酰化)，EC 1.1.1.34]是胆固醇生物合成中的关键调节酶，已被纯化至表观均一。纯化的HMG-CoA还原酶在用考马斯亮蓝染色的NaDodSO4/聚丙烯酰胺凝胶上产生一条单一的弥散带，而在用银染色的凝胶上产生两条相邻的带。纯化的还原酶被用于诱导单克隆抗体的产生。用纯化的HMG-CoA还原酶免疫的BALB/c小鼠的脾细胞与Sp-2/0骨髓瘤细胞融合。通过使用固相放射免疫测定法鉴定产生针对HMG-CoA还原酶的单克隆抗体的克隆，并在软琼脂中进行亚克隆。根据抗体亚类和在溶液中抑制HMG-CoA还原酶的不同能力判断，分离出的三个相对稳定的杂交瘤细胞系分泌不同的免疫球蛋白。两种IgG抗体能有效沉淀溶解的HMG-CoA还原酶，而IgM则不能。所有三种单克隆抗体的混合物能免疫沉淀溶解的大鼠肝脏提取物中90%以上的HMG-CoA还原酶活性。这些单克隆抗体应是研究HMG-CoA还原酶和胆固醇合成调节的有用探针。
