Vishwanatha J K, Wei Z
Department of Biochemistry, University of Nebraska Medical Center, Omaha 68198-4525.
Biochemistry. 1992 Feb 18;31(6):1631-5. doi: 10.1021/bi00121a007.
The ubiquitous dinucleotide P1,P4-di(adenosine-5') tetraphosphate (Ap4A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding specifically to Ap4A is associated with a multiprotein form of DNA polymerase alpha (pol alpha 2) in HeLa cells. The Ap4A binding protein from HeLa cells has been purified to homogeneity starting from pol alpha 2 complex. The Ap4A binding protein is hydrophobic and is resolved from the pol alpha 2 complex by hydrophobic interaction chromatography on butyl-Sepharose and subsequently purified to homogeneity by chromatography on Mono-Q and Superose-12 FPLC columns. The Ap4A binding activity elutes as a single symmetrical peak upon gel filtration with a molecular mass of 200 kDa. Upon polyacrylamide gel electrophoresis under nondenaturing conditions, the purified protein migrates as a single protein of 200 kDa. Upon electrophoresis under denaturing conditions, the binding activity is resolved into two polypeptides of 45 and 22 kDa, designated as A1 and A2, respectively. A1 and A2 can be cross-linked using the homobifunctional cross-linking agent disuccinimidyl suberate. The cross-linked protein migrates as a single protein of 210 kDa on polyacrylamide gels under denaturing conditions, suggesting that these two polypeptides are subunits of a single protein. The purified protein binds Ap4A efficiently, and by Scatchard analysis, we have determined a dissociation constant of 0.25 microM, indicating high affinity of Ap4A binding protein to its ligand. ATP is not required for the binding activity. The nonionic detergent Triton X-100 is necessary for stabilizing the purified protein. Amino acid composition analysis indicates that A1 and A2 are distinct.
普遍存在的二核苷酸P1，P4 - 二（腺苷 - 5'）四磷酸（Ap4A）被认为参与DNA复制与细胞增殖、DNA修复、血小板聚集和血管张力调节。在HeLa细胞中，一种与Ap4A特异性结合的蛋白质与多蛋白形式的DNA聚合酶α（polα2）相关联。从polα2复合物开始，已将HeLa细胞中的Ap4A结合蛋白纯化至同质。Ap4A结合蛋白具有疏水性，通过丁基琼脂糖上的疏水相互作用色谱从polα2复合物中分离出来，随后通过Mono - Q和Superose - 12 FPLC柱色谱纯化至同质。在用凝胶过滤时，Ap4A结合活性以单一对称峰洗脱，分子量为200 kDa。在非变性条件下进行聚丙烯酰胺凝胶电泳时，纯化的蛋白质迁移为单一的200 kDa蛋白质。在变性条件下进行电泳时，结合活性分解为45 kDa和22 kDa的两条多肽，分别命名为A1和A2。A1和A2可以使用同双功能交联剂辛二酸二琥珀酰亚胺酯进行交联。在变性条件下，交联蛋白在聚丙烯酰胺凝胶上迁移为单一的210 kDa蛋白质，表明这两条多肽是单一蛋白质的亚基。纯化的蛋白质能有效结合Ap4A，通过Scatchard分析，我们确定其解离常数为0.25μM，表明Ap4A结合蛋白对其配体具有高亲和力。结合活性不需要ATP。非离子去污剂Triton X - 100对于稳定纯化的蛋白质是必需的。氨基酸组成分析表明A1和A2是不同的。
