The Journal of Membrane Biology ( IF 2.3 ) Pub Date : 2020-11-13 , DOI: 10.1007/s00232-020-00153-y Hans-Jürgen Apell 1 , Milena Roudna 1
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Abstract
Kinetic experiments were performed with preparations of kidney Na,K-ATPase in isolated membrane fragments or reconstituted in vesicles to obtain information of the activation energies under turnover conditions and for selected partial reactions of the Post-Albers pump cycle. The ion transport activities were detected with potential or conformation sensitive fluorescent dyes in steady-state or time-resolved experiments. The activation energies were derived from Arrhenius plots of measurements in the temperature range between 5 °C and 37 °C. The results were used to elaborate indications of the respective underlying rate-limiting reaction steps and allowed conclusions to be drawn about possible molecular reaction mechanisms. The observed consequent alteration between ligand-induced reaction and conformational relaxation steps when the Na,K-ATPase performs the pump cycle, together with constraints set by thermodynamic principles, provided restrictions which have to be met when mechanistic models are proposed. A model meeting such requirements is presented for discussion.
Graphic Abstract
中文翻译：
Na,K-ATPase 的部分反应：活化能的测定和机理方法
摘要
用分离的膜碎片或在囊泡中重组的肾 Na, K-ATPase 制剂进行动力学实验，以获得在周转条件下的活化能信息和后阿尔伯斯泵循环的选定部分反应。在稳态或时间分辨实验中用电位或构象敏感的荧光染料检测离子转运活性。活化能来自在 5 °C 和 37 °C 之间的温度范围内测量的 Arrhenius 图。结果用于详细说明各自潜在的限速反应步骤的指示，并允许得出关于可能的分子反应机制的结论。当 Na、K-ATPase 执行泵循环以及由热力学原理设置的约束，提供了在提出机械模型时必须满足的限制。提出了满足此类要求的模型以供讨论。
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