Pintschovius J, Fendler K, Bamberg E
Max-Planck-Institut für Biophysik, D-60596 Frankfurt/Main, Germany.
Biophys J. 1999 Feb;76(2):827-36. doi: 10.1016/S0006-3495(99)77246-2.
In the preceding publication (. Biophys. J. 76:000-000) a new technique was described that was able to produce concentration jumps of arbitrary ion species at the surface of a solid supported membrane (SSM). This technique can be used to investigate the kinetics of ion translocating proteins adsorbed to the SSM. Charge translocation of the Na+/K+-ATPase in the presence of ATP was investigated. Here we describe experiments carried out with membrane fragments containing Na+/K+-ATPase from pig kidney and in the absence of ATP. Electrical currents are measured after rapid addition of Na+. We demonstrate that these currents can be explained only by a cation binding process on the cytoplasmic side, most probably to the cytoplasmic cation binding site of the Na+/K+-ATPase. An electrogenic reaction of the protein was observed only with Na+, but not with other monovalent cations (K+, Li+, Rb+, Cs+). Using Na+ activation of the enzyme after preincubation with K+ we also investigated the K+-dependent half-cycle of the Na+/K+-ATPase. A rate constant for K+ translocation in the absence of ATP of 0.2-0.3 s-1 was determined. In addition, these experiments show that K+ deocclusion, and cytoplasmic K+ release are electroneutral.
在前一篇发表的文章（《生物物理杂志》76:000 - 000）中，描述了一种新技术，该技术能够在固体支持膜（SSM）表面产生任意离子种类的浓度跃变。此技术可用于研究吸附在SSM上的离子转运蛋白的动力学。研究了在ATP存在下Na⁺/K⁺ - ATP酶的电荷转运。在此，我们描述了用含有猪肾Na⁺/K⁺ - ATP酶的膜片段且在无ATP情况下进行的实验。快速加入Na⁺后测量电流。我们证明这些电流只能通过细胞质侧的阳离子结合过程来解释，很可能是与Na⁺/K⁺ - ATP酶的细胞质阳离子结合位点结合。仅在加入Na⁺时观察到该蛋白的电致反应，而在加入其他单价阳离子（K⁺、Li⁺、Rb⁺、Cs⁺）时未观察到。在与K⁺预孵育后使用Na⁺激活该酶，我们还研究了Na⁺/K⁺ - ATP酶的K⁺依赖性半周期。测定了在无ATP时K⁺转运的速率常数为0.2 - 0.3 s⁻¹。此外，这些实验表明K⁺解封闭以及细胞质K⁺释放是电中性的。
