Görlach M, Burd C G, Dreyfuss G
Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia 19104-6148.
Exp Cell Res. 1994 Apr;211(2):400-7. doi: 10.1006/excr.1994.1104.
The poly(A)-binding protein (PABP) binds to the messenger (mRNA) 3'-poly(A) tail found on most eukaryotic mRNAs and together with the poly(A) tail has been implicated in governing the stability and the translation of mRNA. In order to further understand the role of the PABP in these processes, we have undertaken a detailed analysis of the cellular localization, the abundance, and the RNA-binding properties of the human PABP (hPABP). We raised monoclonal antibodies against the 70-kDa hPABP and confocal immunofluorescence microscopy with these antibodies reveals that it is localized exclusively to the cytoplasm. The hPABP exhibits a very low turnover rate in these cells and quantitative immunoblotting experiments demonstrated that growing HeLa cells contain a surprisingly high number of approximately 8 x 10(6) PABP molecules per cell, which corresponds to an intracellular concentration of about 4 microM. In an in vitro selection/amplification assay from random sequence oligonucleotide pools the hPABP selects oligo(rA)-rich sequences and it binds oligo(rA)25 with an apparent Kd of 7 nM. The hPABP binds to unrelated RNA sequences with an about 100-fold lower affinity (Kd > or = 0.5 microM). The abundance of the hPABP indicates that there is an approximately three-fold excess of the protein over binding sites on cytoplasmic poly(A). This excess and the high concentration of the hPABP, which is three orders of magnitude above its Kd for oligo(rA)25, suggest that the hPABP may bind to additional, lower affinity binding sites in vivo.
聚腺苷酸结合蛋白（PABP）可与大多数真核生物信使核糖核酸（mRNA）上的3'-聚腺苷酸尾相结合，并且已证实它与聚腺苷酸尾共同作用于mRNA的稳定性和翻译过程。为了进一步了解PABP在这些过程中的作用，我们对人PABP（hPABP）的细胞定位、丰度及RNA结合特性进行了详细分析。我们制备了针对70 kDa hPABP的单克隆抗体，利用这些抗体进行的共聚焦免疫荧光显微镜检测显示，hPABP仅定位于细胞质中。hPABP在这些细胞中的周转率非常低，定量免疫印迹实验表明，处于生长状态的HeLa细胞中每个细胞含有数量惊人的约8×10⁶个PABP分子，这相当于细胞内浓度约为4 μM。在从随机序列寡核苷酸库进行的体外筛选/扩增试验中，hPABP选择富含寡聚（rA）的序列，并且它以7 nM的表观解离常数（Kd）结合寡聚（rA）₂₅。hPABP以大约低100倍的亲和力（Kd≥0.5 μM）结合不相关的RNA序列。hPABP的丰度表明，该蛋白相对于细胞质聚腺苷酸上的结合位点有大约三倍的过量。这种过量以及hPABP的高浓度（比其对寡聚（rA）₂₅的Kd高三个数量级）表明，hPABP在体内可能结合其他亲和力较低的结合位点。
