Tang S S, Rogg H, Schumacher R, Dzau V J
Molecular and Cellular Vascular Research Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Endocrinology. 1992 Jul;131(1):374-80. doi: 10.1210/endo.131.1.1612017.
Although the action of angiotensin-II (Ang-II) is believed to be mediated by a transmembrane signal transduction mechanism, accumulating evidence suggests that Ang-II may also have a direct nuclear action. We have characterized the nuclear Ang-II-binding site in purified nuclei preparation from rat liver and compared it to plasma membrane Ang-II receptors. [125I]Ang-II binding to isolated nuclei reached equilibration in 30 min at 25 C, slower than binding to plasma membrane, which reached equilibration within 10 min. Scatchard analysis of [125I]Ang-II binding to isolated nuclei revealed a single class of binding sites (Kd = 1.4 nM; binding capacity = 10 fmol/mg protein or 460 sites/nucleus). In the nuclear preparation, Ang-II and its fragments competed for binding a potency order of Ang-III = Ang-II greater than Ang-II-(1-7) greater than Ang-II-(1-6) greater than Ang-II-(1-5). Losartan potassium (DuP 753), a selective blocker of the Ang-II receptor subtype I, fully inhibits nuclear Ang-II binding with affinity similar to that in plasma membrane. The pH optimum for [125I]Ang-II binding to nuclei was 7.0, while binding to plasma membrane was optimal at pH 8.0. Low concentrations (0.05-0.1 mM) of dithiothreitol increased [125I]Ang-II binding to nuclei, but not to plasma membrane. In the absence of detergent, Ang-II-binding sites appear to consist of soluble protein releasable from nuclei by freezing and thawing, hence distinct in physicochemical properties from the membrane-bound receptor. Size-exclusion HPLC estimated the mol wt of the soluble Ang-II-binding sites to be 66 kilodaltons. These nuclear Ang-II-binding sites have some similarities to but also show notable physicochemical differences from plasma membrane Ang-II receptors, and they may play a role in mediating the intracellular action of Ang-II.
尽管人们认为血管紧张素 II（Ang-II）的作用是通过跨膜信号转导机制介导的，但越来越多的证据表明，Ang-II 可能也具有直接的核作用。我们已经对大鼠肝脏纯化细胞核制剂中的核 Ang-II 结合位点进行了表征，并将其与质膜 Ang-II 受体进行了比较。[125I]Ang-II 与分离细胞核的结合在 25℃下 30 分钟达到平衡，比与质膜的结合慢，质膜结合在 10 分钟内达到平衡。对[125I]Ang-II 与分离细胞核结合的 Scatchard 分析显示出一类单一的结合位点（解离常数 Kd = 1.4 nM；结合容量 = 10 fmol/mg 蛋白质或 460 个位点/细胞核）。在细胞核制剂中，Ang-II 及其片段竞争结合的效力顺序为：Ang-III = Ang-II ＞ Ang-II-(1-7) ＞ Ang-II-(1-6) ＞ Ang-II-(1-5)。氯沙坦钾（DuP 753），一种 Ang-II 受体 I 亚型的选择性阻滞剂，以与质膜中相似的亲和力完全抑制核 Ang-II 结合。[125I]Ang-II 与细胞核结合的最适 pH 为 7.0，而与质膜结合在 pH 8.0 时最佳。低浓度（0.05 - 0.1 mM）的二硫苏糖醇增加了[125I]Ang-II 与细胞核的结合，但不增加与质膜的结合。在没有去污剂的情况下，Ang-II 结合位点似乎由通过冻融可从细胞核中释放的可溶性蛋白质组成，因此在物理化学性质上与膜结合受体不同。尺寸排阻高效液相色谱法估计可溶性 Ang-II 结合位点的分子量为 66 千道尔顿。这些核 Ang-II 结合位点与质膜 Ang-II 受体有一些相似之处，但也表现出显著的物理化学差异，它们可能在介导 Ang-II 的细胞内作用中发挥作用。
