Hengst J A, Georgoff I, Isom H C, Jacob S T
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey 17033.
J Biol Chem. 1988 Dec 25;263(36):19270-3.
Nuclear poly(A) polymerase was isolated from [35S]methionine-labeled hepatoma McA-RH 7777 cells and subjected to DEAE-Sephadex chromatography. Flow-through and low salt wash fractions containing poly(A) polymerase activity were pooled and subjected to immunoblot analysis using anti-tumor type poly(A) polymerase antibodies and a biotinylated second antibody. The immune complex contained a single 48-kDa polypeptide band corresponding to the tumor-type enzyme. When immunoprecipitations were carried out using the same fraction and antibodies, at least five 35S-methionine-labeled proteins with approximate molecular masses of 74, 48, 35, 30, and 22 kDa were observed. Pulse-chase studies did not indicate a precursor-product relationship between the immunoprecipitated proteins. Preimmune sera did not react with poly(A) polymerase or other components in the protein complex. These data show that poly(A) polymerase exists as part of a complex with at least four other polypeptides and suggest that these polypeptides may be involved in the cleavage and/or polyadenylation reactions.
从用[35S]甲硫氨酸标记的肝癌McA-RH 7777细胞中分离出核多聚腺苷酸聚合酶，并进行DEAE-葡聚糖凝胶柱层析。将含有多聚腺苷酸聚合酶活性的流出液和低盐洗脱级分合并，并用抗肿瘤型多聚腺苷酸聚合酶抗体和生物素化二抗进行免疫印迹分析。免疫复合物含有一条与肿瘤型酶相对应的单一48 kDa多肽带。当使用相同的级分和抗体进行免疫沉淀时，观察到至少五种分子量约为74、48、35、30和22 kDa的35S-甲硫氨酸标记蛋白。脉冲追踪研究未表明免疫沉淀蛋白之间存在前体-产物关系。免疫前血清不与多聚腺苷酸聚合酶或蛋白质复合物中的其他成分发生反应。这些数据表明多聚腺苷酸聚合酶作为一种复合物的一部分存在，该复合物至少还含有其他四种多肽，并提示这些多肽可能参与切割和/或聚腺苷酸化反应。
