Baxi M D, McLennan A G, Vishwanatha J K
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha 68198-4525.
Biochemistry. 1994 Dec 6;33(48):14601-7. doi: 10.1021/bi00252a028.
The ubiquitous dinucleotide diadenosine tetraphosphate (Ap4A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding to Ap4A is associated with a multiprotein form of DNA polymerase alpha (pol alpha 2) in HeLa cells. We have purified the pol alpha-associated Ap4A binding protein to homogeneity. The Ap4A binding protein is resolved into two polypeptides of 45 and 22 kDa, designated as A1 and A2, respectively. We have utilized [alpha-32P]8-N3-Ap4A to label the purified binding protein, and by cross-linking the photoaffinity label we have determined that Ap4A binds to the A1 subunit. No binding to the ligand is observed with the A2 subunit. Photoaffinity labeling is saturated with approximately 0.4 microM photolabel, with a half-maximal binding at 0.15 microM. The labeling is UV-dependent and is competed by both 8-N3-Ap4A and Ap4A. Photoaffinity labeling is not affected in the presence of dATP and dGTP and is reduced only in the presence of excess of ATP indicating the specificity of the protein for Ap4A. Of the diadenosine polyphosphates, Ap4A and Ap5A competed for binding, while Ap2A and Ap3A did not compete for binding. Further, the presence of at least one adenosine may be necessary since Ap4G competes but Gp4G does not compete for binding to the protein. Various methylene bisphosphonate and thiophosphate analogs of Ap4A were tested to see their effect on photoaffinity labeling with 8-N3-Ap4A. Significant differences were observed among the various analogs in their ability to prevent the photoaffinity labeling of the ligand to the binding protein.
普遍存在的二核苷酸四磷酸二腺苷（Ap4A）被认为参与DNA复制和细胞增殖、DNA修复、血小板聚集及血管张力调节。在HeLa细胞中，一种与Ap4A结合的蛋白质与多蛋白形式的DNA聚合酶α（pol α 2）相关。我们已将与pol α相关的Ap4A结合蛋白纯化至同质。Ap4A结合蛋白可分解为分别为45 kDa和22 kDa的两条多肽，分别命名为A1和A2。我们利用[α-32P]8-N3-Ap4A标记纯化的结合蛋白，通过对光亲和标记进行交联，我们确定Ap4A与A1亚基结合。未观察到A2亚基与配体的结合。光亲和标记在约0.4 μM光标记时达到饱和，半最大结合浓度为0.15 μM。标记依赖紫外线，且可被8-N3-Ap4A和Ap4A竞争。光亲和标记在dATP和dGTP存在时不受影响，仅在过量ATP存在时减少，表明该蛋白质对Ap4A具有特异性。在二腺苷多磷酸中，Ap4A和Ap5A竞争结合，而Ap2A和Ap3A不竞争结合。此外，由于Ap4G竞争而Gp4G不竞争与该蛋白质的结合，因此至少存在一个腺苷可能是必需的。测试了Ap4A的各种亚甲基双膦酸盐和硫代磷酸盐类似物对用8-N3-Ap4A进行光亲和标记的影响。在各种类似物阻止配体与结合蛋白进行光亲和标记的能力方面观察到显著差异。
