Miller C A, Cohen M D, Costa M
Institute of Environmental Medicine, New York University Medical Center, NY 10016.
Carcinogenesis. 1991 Feb;12(2):269-76. doi: 10.1093/carcin/12.2.269.
Actin was found to be the major protein crosslinked to the DNA of intact Chinese hamster ovary cells that were treated with either potassium chromate (hexavalent) or cis-diamminedichloroplatinum(II) (cis-platinum). This protein was identified as actin by its mol. wt (45 kd), its isoelectric point (pI = 5.4), positive reactivity with an actin antibody, and by protease mapping. Additionally, a purified actin standard migrated to the same location in a two-dimensional gel system as p45. Actin comprised approximately 20% of the protein component in chromate-induced DNA-protein crosslinks. In addition, to actin, several other major proteins (e.g. 53 kd, pI = 5.2, 50 kd, pI = 9) were crosslinked to DNA following exposure to cis-platinum or chromate. These proteins were abundant in the nuclear matrix fraction. Hexavalent chromate is the toxicologically active form because it is readily taken up into cells by an anion transport system. In contrast, trivalent chromium is considerably less toxic because it cannot enter the cell; however, most of the hexavalent form is eventually reduced to the trivalent form inside the cell. Previous studies have suggested that the trivalent form of chromium participates in complexing DNA with proteins. DNA-protein crosslinks were formed in isolated nuclei or in mixtures of purified DNA and protein incubated with trivalent chromium. However, the formation of these complexes required at least 16 h of incubation to exchange the parent compound ligands. Hexavalent chromate did not form these complexes in vitro under similar conditions. Incubation of trivalent chromium with purified actin and DNA resulted in DNA-actin crosslinks as detected by an electrophoretic mobility different from that of either free actin or DNA when the complex was transferred from a gel to nitrocellulose and stained for protein. These studies describe a new technique for detecting DNA-protein complexes and demonstrate that actin-DNA structures in intact cells create sites that selectively react with metal DNA-protein crosslinking agents.
研究发现，肌动蛋白是完整的中国仓鼠卵巢细胞DNA交联的主要蛋白质，这些细胞用铬酸钾（六价）或顺二氯二氨铂（II）（顺铂）处理。通过其分子量（45kd）、等电点（pI = 5.4）、与肌动蛋白抗体的阳性反应以及蛋白酶图谱分析，该蛋白质被鉴定为肌动蛋白。此外，纯化的肌动蛋白标准品在二维凝胶系统中迁移到与p45相同的位置。在铬酸盐诱导的DNA - 蛋白质交联中，肌动蛋白约占蛋白质成分的20%。此外，除了肌动蛋白，暴露于顺铂或铬酸盐后，还有几种其他主要蛋白质（如53kd，pI = 5.2；50kd，pI = 9）与DNA交联。这些蛋白质在核基质部分含量丰富（大量存在）。六价铬是具有毒理学活性的形式，因为它很容易通过阴离子转运系统进入细胞。相比之下，三价铬的毒性要小得多，因为它不能进入细胞；然而，大部分六价形式最终在细胞内被还原为三价形式。先前的研究表明，三价铬参与DNA与蛋白质的络合。在分离的细胞核中或在与三价铬孵育的纯化DNA和蛋白质混合物中形成了DNA - 蛋白质交联。然而，这些复合物的形成需要至少16小时的孵育以交换母体化合物配体。在类似条件下，六价铬在体外不形成这些复合物。用纯化的肌动蛋白和DNA孵育三价铬，当复合物从凝胶转移到硝酸纤维素膜上并进行蛋白质染色时，通过电泳迁移率检测到DNA - 肌动蛋白交联，其电泳迁移率与游离肌动蛋白或DNA不同。这些研究描述了一种检测DNA - 蛋白质复合物的新技术，并证明完整细胞中的肌动蛋白 - DNA结构产生了与金属DNA - 蛋白质交联剂选择性反应的位点。
