Heyse S, Wuddel I, Apell H J, Stürmer W
Department of Biology, University of Konstanz, Germany.
J Gen Physiol. 1994 Aug;104(2):197-240. doi: 10.1085/jgp.104.2.197.
Experiments were designed to characterize several partial reactions of the Na,K-ATPase and to demonstrate that a model can be defined that reproduces most of the transport features of the pump with a single set of kientic parameters. We used the fluorescence label 5-iodoacetamidofluorescein, which is thought to be sensitive to conformational changes, and the styryl dye RH 421, which can be applied to detect ion-binding and -release reactions. In addition transient electric currents were measured, which are associated mainly with the E1-->E2 conformational transition. Numerical simulations were performed on the basis of a reaction model, that has been developed from the Post-Albers cycle. Analysis of the experimental data allows the determination of several rate constants of the pump cycle. Our conclusions may be summarized as follows: (a) binding of one Na+ ion at the cytoplasmic face is electrogenic. This Na+ ion is specifically bound to a neutral binding site with an affinity of 8 mM in the presence of 10 mM Mg2+. In the absence of divalent cations, the intrinsic binding affinity was found to be 0.7 mM. (b) The analysis of fluorescence experiments with the cardiotonic steroid strophanthidin indicates that the 5-iodoacetamidofluorescein label monitors the conformational transition (Na3)E1-P-->P-E2(Na2), which is accompanied by the release of one Na+ ion. 5-IAF does not respond to the release of the subsequent two Na+ ions, which can be monitored by the RH 421 dye. These experiments indicate further that the conformational transition E1P-->P-E2 is the rate limiting process of the Na+ translocation. The corresponding rate constant was determined to be 22 s-1 at 20 degrees C. From competition experiments with cardiotonic steroids, we estimated that the remaining 2 Na+ ions are released subsequently with a rate constant of at least 5,000 s-1 from their negatively charged binding sites. (c) Comparing the fluorescence experiments with electric current transients, which were performed at various Na concentrations in the absence and presence of strophanthidin, we found that the transition (Na3).E1-P-->P-E2.(Na2) is the major charge translocating step in the reaction sequence Na3.E1-->(Na3).E1-P-->P-E2.(Na2)-->P-E2. The subsequent release of 2 Na+ ions contributed less than 25% to the total electric current transient. (d) The well known antagonism between cardiotonic steroids and K+ binding can be explained by a kinetic model. A quantitative description has been obtained under the assumption that these inhibitors bind only to the states P-E2(Na2) and P-E2(K2).(ABSTRACT TRUNCATED AT 400 WORDS)
实验旨在表征钠钾-ATP酶的几个部分反应，并证明可以定义一个模型，该模型用一组单一的动力学参数就能重现该泵的大部分转运特征。我们使用了荧光标记物5-碘乙酰氨基荧光素，它被认为对构象变化敏感，还有苯乙烯基染料RH 421，可用于检测离子结合和释放反应。此外，还测量了主要与E1→E2构象转变相关的瞬态电流。基于从波斯特-阿尔伯斯循环发展而来的反应模型进行了数值模拟。对实验数据的分析使得能够确定泵循环的几个速率常数。我们的结论可总结如下：(a) 在细胞质面结合一个Na⁺离子是电生的。在10 mM Mg²⁺存在的情况下，这个Na⁺离子特异性地结合到一个中性结合位点，亲和力为8 mM。在没有二价阳离子的情况下，内在结合亲和力为0.7 mM。(b) 用强心甾类毒毛旋花子苷进行的荧光实验分析表明，5-碘乙酰氨基荧光素标记监测构象转变(Na₃)E1-P→P-E2(Na₂)，同时伴随着一个Na⁺离子的释放。5-IAF对随后两个Na⁺离子的释放没有反应，这两个离子的释放可用RH 421染料监测。这些实验进一步表明，构象转变E1P→P-E2是Na⁺转运的限速过程。在20℃时，相应的速率常数确定为22 s⁻¹。从与强心甾类的竞争实验中，我们估计其余两个Na⁺离子随后从其带负电荷的结合位点以至少5000 s⁻¹的速率常数释放。(c) 比较在有无毒毛旋花子苷的情况下，在不同Na浓度下进行的荧光实验和电流瞬变实验，我们发现转变(Na₃).E1-P→P-E2.(Na₂)是反应序列Na₃.E1→(Na₃).E1-P→P-E2.(Na₂)→P-E2中主要的电荷转运步骤。随后两个Na⁺离子的释放对总电流瞬变的贡献小于25%。(d) 强心甾类与K⁺结合之间众所周知的拮抗作用可用动力学模型来解释。在这些抑制剂仅与状态P-E2(Na₂)和P-E2(K₂)结合的假设下，已获得了定量描述。
