The affinity of rodent Na+,K+-ATPase α1-subunit to cardiotonic steroids (CTS) is known to be approximately 1000-fold less than the affinity of Na+,K+-ATPase α1-subunit from other mammals. The CTS-resistant isoform of Na+,K+-ATPase α1-subunit (α1R) is expressed in rodent cells, in contrast to the CTS-sensitive isoform of the α1-subunit (α1S), which is expressed in cells of other mammals. Earlier we have established that incubation with ouabain in concentrations that completely suppressed the activity of Na+,K+-ATPase α1-isoform (α1S-Na+,K+-ATPase) led to a death of human endothelial and smooth muscle cells but did not affect the survival of rat cells expressing α1R-Na+,K+-ATPase. Conformational transitions that are induced by CTS binding to Na+,K+-ATPase play a key role in the process of cell survival. To reveal differences in the CTS-induced conformational changes of α1R- and α1S-Na+,K+-ATPase isoforms, we used three different CTS (two cardenelids, ouabain and digoxin, and one bufadienolid, marinobufagenin) and analyzed the trypsinolysis products of α1-subunits in two main conformations of Na+,K+-ATPase (E1 and E2-P). The treatment of the pig kidney α1S-Na,K-ATPase in E1 conformation by trypsin results in a significant decrease of the amount of α1S-subunit and in the appearance of protein fragments with molecular masses of about 40, 35, 23, and 19 kDa. Preincubation of Na+,K+-ATPase in E1 conformation with ouabain or with digoxin (1 mM) leads to a decrease of the amount of α1S-subunit, increase of the amount of polypeptide fragment with molecular mass of about 40 kDa, and a significant rise of the amount of fragment with molecular mass of about 35.5 kDa, which was not found after the preincubation of the Na+,K+-ATPase in E1 conformation with marinobufagenin (1 mM). In the absence of CTS the trypsinolysis of α1S-Na+,K+-ATPase in E2-P conformation results in a decrease of the amount of α1S-subunit and an increase of the amount of proteolytic products with molecular mass of about 40 and 35.5 kDa. Preincubation of the Na+,K+-ATPase in E2-P conformation in the presence of any of the CTS studied induces the appearance of big amount of an additional peptide fragment with molecular mass of about 45 kDa. Preincubation of α1R-Na+,K+-ATPase from rat kidney with any of the CTS does not change the composition of proteolytic products and their molecular masses in either E1 or E2-P conformation. The results suggest that the structure of the CTS-binding site and a conformational response of α1-Na+,K+-ATPase to binding of CTS is mainly determined by the primary structure of the CTS-resistant and CTS-sensitive α1-subunits of the Na+,K+-AТРase.

中文翻译：

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哇巴因、地高辛或海蟾蜍精的结合诱导肾脏 α1-Na+,K+-ATPase 异构体的不同构象变化，对强心类固醇具有抗性和敏感性

已知啮齿动物 Na+,K+-ATPase α1-亚基对强心类固醇 (CTS) 的亲和力比其他哺乳动物的 Na+,K+-ATPase α1-亚基的亲和力低约 1000 倍。Na+,K+-ATPase α1-亚基 (α1R) 的 CTS 抗性同种型在啮齿动物细胞中表达，而 CTS 敏感的 α1-亚基 (α1S) 同种型在其他哺乳动物细胞中表达。早些时候我们已经确定，在完全抑制 Na+,K+-ATPase α1-异构体（α1S-Na+,K+-ATPase）活性的浓度下与哇巴因孵育会导致人内皮细胞和平滑肌细胞死亡，但不影响存活表达 α1R-Na+,K+-ATPase 的大鼠细胞。CTS 与 Na+,K+-ATPase 结合诱导的构象转变在细胞存活过程中起关键作用。为了揭示 CTS 诱导的 α1R- 和 α1S-Na+,K+-ATPase 异构体构象变化的差异，我们使用了三种不同的 CTS（两种cardenelids，哇巴因和地高辛，以及一种蟾蜍二烯酸，marinobufagenin）并分析了α1-的胰蛋白酶解产物Na+,K+-ATPase 的两种主要构象（E1 和 E2-P）中的亚基。胰蛋白酶处理E1构象的猪肾α1S-Na,K-ATPase导致α1S-亚基数量显着减少，出现分子量约为40、35、23和19的蛋白质片段千达。将 E1 构象的 Na+,K+-ATPase 与哇巴因或地高辛 (1 mM) 预温育导致 α1S-亚基数量减少，分子量约为 40 kDa 的多肽片段数量增加，分子量约为 35.5 kDa 的片段数量显着增加，这在 E1 构象的 Na+,K+-ATPase 与海蟾蜍毒配基 (1 mM) 预温育后未发现。在没有 CTS 的情况下，E2-P 构象中的 α1S-Na+,K+-ATPase 的胰蛋白酶水解导致 α1S-亚基的数量减少，而分子量约为 40 和 35.5 kDa 的蛋白水解产物的数量增加。在所研究的任何 CTS 存在下，E2-P 构象的 Na+,K+-ATPase 的预温育诱导大量额外的肽片段的出现，其分子量约为 45 kDa。将来自大鼠肾脏的 α1R-Na+,K+-ATPase 与任何 CTS 预温育不会改变蛋白水解产物的组成及其 E1 或 E2-P 构象的分子量。