Kuroda K, Kagiyama-Takahashi R, Shinomiya T
Mitsubishi Kasei Institute of Life Sciences, Tokyo.
J Biochem. 1990 Dec;108(6):926-33. doi: 10.1093/oxfordjournals.jbchem.a123316.
Two hybrid cell lines (DM88-5E12 and DM88-4C9) secreting monoclonal antibodies against DNA polymerase alpha-primase complex from Drosophila melanogaster Kc cells were established by immunizing mice with the complex partially purified by a conventional method. The IgG subclasses of both antibodies were IgG1. Both antibodies immunoprecipitated the DNA polymerase alpha-primase complex from D. melanogaster Kc cells. The DNA-polymerizing activity was neutralized by 4C9 antibody, but not by 5E12 antibody. The DNA priming activity was not neutralized by either antibody. These antibodies did not cross-react to HeLa DNA polymerase alpha-primase complex. A rapid, two-step purification of DNA polymerase alpha-primase complex from D. melanogaster Kc cell was carried out by 5E12 antibody column chromatography followed by single-stranded DNA cellulose column chromatography. The immunoaffinity-purified enzyme had both DNA-polymerizing and DNA-priming activities with the specific activities of 50,000 and 2,000 units/mg, respectively. The effects of aphidicolin, NEM, ddTTP, BuPdGTP, and DMSO on the enzyme activity showed that the purified enzyme was DNA polymerase alpha, but not DNA polymerase beta, gamma, or delta. The purified enzyme consisted of polypeptides with apparent molecular weights of 180 (and 145, 140, 130 kDa), 72, 63, 51, and 49 kDa. The 5E12 antibody was shown to bind to all the high-molecular-weight polypeptides, 180, 145, 140, and 130 kDa, by immuno-Western blotting analysis.
通过用常规方法部分纯化的来自黑腹果蝇Kc细胞的DNA聚合酶α-引发酶复合物免疫小鼠，建立了两种分泌抗该复合物单克隆抗体的杂交细胞系（DM88-5E12和DM88-4C9）。两种抗体的IgG亚类均为IgG1。两种抗体均能从黑腹果蝇Kc细胞中免疫沉淀DNA聚合酶α-引发酶复合物。4C9抗体可中和DNA聚合活性，但5E12抗体不能。两种抗体均不能中和DNA引发活性。这些抗体与HeLa DNA聚合酶α-引发酶复合物无交叉反应。通过5E12抗体柱层析，随后进行单链DNA纤维素柱层析，对来自黑腹果蝇Kc细胞的DNA聚合酶α-引发酶复合物进行了快速两步纯化。免疫亲和纯化的酶具有DNA聚合和DNA引发活性，比活性分别为50,000和2,000单位/毫克。阿非科林、NEM、ddTTP、BuPdGTP和DMSO对酶活性的影响表明，纯化的酶是DNA聚合酶α，而不是DNA聚合酶β、γ或δ。纯化的酶由表观分子量为180（以及145、140、130 kDa）、72、63、51和49 kDa的多肽组成。通过免疫蛋白质印迹分析表明5E12抗体可与所有高分子量多肽（180、145、140和130 kDa）结合。
