Biological implication of conformational flexibility in ouabain: observations with two ouabain phosphate isomers.
Biological implication of conformational flexibility in ouabain: observations with two ouabain phosphate isomers.
复制标题
哇巴因构象灵活性的生物学意义：两种磷酸哇巴因异构体的观察。
DOI：
10.1021/bi0101751
复制
发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Hamlyn,JM
中科院分区：
文献类型：
--
作者：
Kawamura,A;Abrell,LM;Maggiali,F;Berova,N;Nakanishi,K;Labutti,J;Magil,S;HaupertJr,GT;Hamlyn,JM
哇巴因是一种高极性和异常有效的钠泵抑制剂，在其类固醇A环部分具有罕见的构象灵活性。强心类固醇中环状屈曲的生物学意义尚未被描述。因此，我们制备了哇巴因1，5，19-和1，11，19-磷酸。前者稳定类固醇A环椅构象，后者将A环锁定在半船构象中，减少ABC环部分的挠曲。用狗肾细胞系在pH微生理仪(细胞传感器)上，哇巴因及其1，5，19-磷酸在10-5M处使胞外酸化速率降低15−20%。在缓蚀剂洗脱过程中，1，5，19-磷酸类似物的回收率比哇巴因快3倍∼。在10-4M时，1，11，19-磷酸引起微弱的(∼7%)反应，其逆转∼的速度比哇巴因快44倍。对纯化的Na+，K+-ATPase的研究表明，哇巴因及其1，5，19-磷酸类似物的药效相似(EC50分别为1.1和5.2×10-7M)，其效价是1，11，19-磷酸类似物的100倍。结合动力学研究表明，1，5，19-磷酸类似物与纯化的Na+，K+-ATPase结合和解离的速度分别是哇巴因的3倍和16倍。这两个类似物都是~3H-哇巴因结合的竞争性抑制物。综上所述，这些结果表明哇巴因中A环显著的构象灵活性通常会减缓这种类固醇与钠泵的初始结合。然而，一旦哇巴因被结合，甾体A环和BC环的弯曲对于维持高亲和力结合是至关重要的。我们的结果表明哇巴因结合部位是由结构上可移动的元件组成的，并强调了受体和配基动力学之间的同步性在该系统中作为生物活性决定因素所起的作用。
Ouabain is a highly polar and unusually potent sodium pump inhibitor that possesses uncommon conformational flexibility in its steroid A-ring moiety. The biological significance of ring flection in the cardiotonic steroids has not been described. Accordingly, we prepared ouabain 1,5,19- and 1,11,19-phosphates. The former stabilizes the steroid A-ring chair conformation and the latter locks the A-ring in the half-boat conformation and decreases flection of the ABC-ring moiety. Using a dog kidney cell line (MDCK) in a pH microphysiometer (Cytosensor), ouabain and its 1,5,19-phosphate at 10-5M reduced the rate of extracellular acidification by 15−20%. During inhibitor washout, the rate of recovery from the 1,5,19-phosphate analogue was ∼3 times faster than ouabain. The 1,11,19-phosphate at 10-4M elicited a weak (∼7%) response, and the effects reversed ∼44-fold faster than ouabain. Studies with purified Na+,K+-ATPase showed that ouabain and its 1,5,19-phosphate analogue were of similar efficacy (EC50= 1.1 and 5.2 × 10-7M, respectively) and >100-fold more potent than the 1,11,19-phosphate analogue. Studies of the binding kinetics showed that the 1,5,19-phosphate analogue bound 3-fold and dissociated 16-fold faster from the purified Na+,K+-ATPase than ouabain. Both analogues were competitive inhibitors of3H-ouabain binding. Taken together, these results suggest that the marked conformational flexibility of the A-ring in ouabain ordinarily slows the initial binding of this steroid to the sodium pump. However, once ouabain is bound, flection of the steroidal A- and BC-rings is critical for the maintenance of high-affinity binding. Our results indicate that the ouabain-binding site is comprised of structurally mobile elements and highlight the roles that synchronization between receptor and ligand dynamics play as determinants of biological activity in this system.