Carroll S B, Stollar B D
Proc Natl Acad Sci U S A. 1982 Dec;79(23):7233-7. doi: 10.1073/pnas.79.23.7233.
Monoclonal antibodies were prepared against purified calf thymus RNA polymerase II (RNA nucleotidyltransferase, EC 2.7.7.6). Two distinct antibodies were identified on the basis of their ability to bind RNA polymerase II in a solid-phase assay and to remove enzyme activity from solution in complexes precipitated by anti-immunoglobulin antibodies. One monoclonal antibody directly inhibited enzyme activity in solution. Detailed study of the mechanism of enzyme inhibition by this antibody revealed that it prevented the formation of enzyme-DNA complexes and had no effect on chain elongation. Inhibition of DNA binding and catalytic activity depended on preincubation of antibody with enzyme and was irreversible. Without preincubation, the enzyme readily bound to DNA in the presence of antibody, indicating that the binding of the free enzyme to DNA is faster than that of the antibody to the enzyme. In a competitive immunoassay enzyme-DNA complexes were much less reactive with antibody than was free enzyme, suggesting that DNA either blocks the antigenic site or alters the enzyme's conformation, preventing antibody recognition. These antibodies may serve as useful reagents for analysis of RNA polymerase structure and function.
制备了针对纯化的小牛胸腺RNA聚合酶II（RNA核苷酸转移酶，EC 2.7.7.6）的单克隆抗体。根据它们在固相分析中结合RNA聚合酶II以及从抗免疫球蛋白抗体沉淀的复合物溶液中去除酶活性的能力，鉴定出两种不同的抗体。一种单克隆抗体直接抑制溶液中的酶活性。对该抗体抑制酶活性机制的详细研究表明，它阻止了酶-DNA复合物的形成，对链延伸没有影响。DNA结合和催化活性的抑制取决于抗体与酶的预孵育，并且是不可逆的。没有预孵育时，酶在抗体存在下很容易与DNA结合，这表明游离酶与DNA的结合比抗体与酶的结合更快。在竞争性免疫分析中，酶-DNA复合物与抗体的反应性比游离酶低得多，这表明DNA要么阻断抗原位点，要么改变酶的构象，从而阻止抗体识别。这些抗体可作为分析RNA聚合酶结构和功能的有用试剂。
