通过铷离子内流研究HeLa细胞中活性阳离子转运的动力学参数和机制。

Kinetic parameters and mechanism of active cation transport in HeLa cells as studied by Rb+ influx.

作者信息

Ikehara T, Yamaguchi H, Sakai T, Miyamoto H

出版信息

Biochim Biophys Acta. 1984 Sep 5;775(3):297-307. doi: 10.1016/0005-2736(84)90184-6.

Abstract

On incubation of HeLa cells in chilled isotonic medium, intracellular Na+ (Nac+) increased and K+ (Kc+) decreased with time, reaching steady levels after 3 h. The steady levels varied in parallel with the extracellular cation concentrations ([Na+]e, [K+]e). The cell volumes and the protein and water contents, respectively, of cells kept for 3 h in chilled media of various [Na+]e and [K+]e were not significantly different. Ouabain-sensitive Rb+ influx took place at the initial rate for a certain period which depended on [Na+]c at the beginning of the assays. The existence of two external K+ loading sites per Na+/K+-pump was demonstrated. The affinities of the sites for Rb+ as a congener of K+ were almost the same. Na+e inhibited ouabain-sensitive Rb+ influx competitively, whereas K+ was not inhibitory. Kinetic parameters were determined: the K 1/2 for Rbe+ in the absence of Na+e was 0.16 mM and th Ki for Na+e was 36.8 mM; the K 1/2 for Na+c was 19.5 mM and the Ki for K+c seemed to be extremely large. The rate equation of the ouabain-sensitive Rb+ influx suggests that Na+ and K+ are exchanged alternately through the pump by a binary mechanism.

摘要

在将HeLa细胞置于冰冷的等渗培养基中培养时，细胞内的Na⁺（Nac⁺）随时间增加，而K⁺（Kc⁺）随时间减少，3小时后达到稳定水平。稳定水平与细胞外阳离子浓度（[Na⁺]e、[K⁺]e）平行变化。在含有不同[Na⁺]e和[K⁺]e的冰冷培养基中保存3小时的细胞，其细胞体积、蛋白质含量和水分含量分别无显著差异。哇巴因敏感的Rb⁺内流在一定时期内以初始速率发生，该速率在测定开始时取决于[Na⁺]c。证明每个Na⁺/K⁺泵存在两个外部K⁺加载位点。这些位点对作为K⁺同系物的Rb⁺的亲和力几乎相同。Na⁺e竞争性抑制哇巴因敏感的Rb⁺内流，而K⁺无抑制作用。测定了动力学参数：在无Na⁺e时Rb⁺e的K 1/2为0.16 mM，Na⁺e的Ki为36.8 mM；Na⁺c的K 1/2为19.5 mM，K⁺c的Ki似乎极大。哇巴因敏感的Rb⁺内流的速率方程表明，Na⁺和K⁺通过泵以二元机制交替交换。