The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase
The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase
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DOI：
10.1007/s00424-008-0467-8
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发表时间：
2009-01-01
影响因子：
4.5
通讯作者：
Koenderink, Jan B.
中科院分区：
文献类型：
--
作者：
De Pont, Jan Joep H. H. M.;Swarts, Herman G. P.;Koenderink, Jan B.
基于对（非）胃H，K-ATP酶和Na，K-ATP酶之间的嵌合体的研究，最近提出了哇巴因结合位点的模型（Qiu等人，J.Biol.Chem. 280（2005）32349）。在该模型中，Na，K-ATPase的特定氨基酸残基与哇巴因的羟基之间的氢键起着至关重要的作用。在本研究中，一系列哇巴因类似物在杆状病毒表达的Na，K-ATP酶和哇巴因敏感的非胃H，K-ATP酶突变体（D312 E/S319 G/A778 P/I795 L/F802 C）上进行了测试。对于每个类似物，通过测量ATP酶抑制和[H-3]哇巴因替代获得的结果相当一致。在Na，K-ATP酶中，毒毛旋花子苷的亲和力比哇巴因高7-10倍，地高辛的亲和力比哇巴因低4-12倍。非胃H，K-ATP酶突变体的结果与Na，K-ATP酶的结果非常相似，除了二氢哇巴因与非胃H，K-ATP酶突变体显示出低得多的亲和力。对接研究表明，所有类似物结合到Na，K-ATP酶的相同口袋。然而，形成氢键的氨基酸不同，取决于哇巴因类似物中羟基或酮基的可用性。
Based on studies with chimeras between (non-)gastric H,K-ATPase and Na,K-ATPase, a model for the ouabain binding site has recently been presented (Qiu et al. J.Biol.Chem. 280 (2005) 32349). In this model, hydrogen bonds between specific amino acid residues of Na,K-ATPase and hydroxyl groups of ouabain play a crucial role. In the present study, a series of ouabain analogues were tested on baculovirus-expressed Na,K-ATPase and an ouabain-sensitive mutant of non-gastric H,K-ATPase (D312E/ S319G/ A778P/ I795L/ F802C). For each analogue, the results obtained by measuring ATPase inhibition and [H-3]ouabain replacement agreed rather well. In Na,K-ATPase, strophanthidin had a 7-10 times higher and digoxin a 4-12 times lower affinity than ouabain. The results of the non-gastric H,K-ATPase mutant were rather similar to that of Na,K-ATPase with exception of dihydro-ouabain that showed a much lower affinity with the non-gastric H,K-ATPase mutant. Docking studies showed that all analogues bind to the same pocket in Na,K-ATPase. However, the amino acids to which hydrogen bonds were formed differed and depended on the availability of hydroxyl or keto groups in the ouabain analogues.