KINETIC ANALYSIS OF NA+/K+ ATPASE REACTION MECHANISM

Na+，K+-ATPase是酶作用下的Na+泵，它 将三磷酸腺苷的水解作用与三个钠离子和两个钾离子的转运相结合 进入牢房。这种耦合将储存的化学能转化为 可用电化学能(以离子梯度表示)。此外，这一点 蛋白质调节几个生理过程(直接或 间接)，包括神经递质的释放和摄取，产生 静息膜电位与血管和内脏肌的控制 语气。体内泵活动受胰岛素和甲状腺激素的调节 以及推定的内源性调节因子。泵起着受体的作用 用于洋地黄苷类药物。 该项目的长期目标是了解 离子传输和三磷酸腺苷水解释放的能量。尽管一个 大量证据表明，离子转运与三磷酸腺苷通过 配体诱导的构象变化，其确切机制 目前尚不清楚两者之间的联系。 本提案描述了区分两种方法的实验 离子传输的替代方案，其提出：(1)传输是 与构象变化直接相关，离子的释放 遵循构象变化，或(2)构象变化改变 阳离子结合位点数及其亲和力，以及该离子 传输发生在这些变化之前。暂态实验， 它测量酶循环中不同步骤的速率常数， 将用于区分这两种可能性。 建议的实验旨在测量每一步的速率常数 在反应途径中。具体地说，这些实验将测量 (I)底物结合，(Ii)酶的正向和反向速率常数 磷酸化，(Iii)ADP和P[i]释放，(Iv)构象 磷酸化酶形式之间的转换，(V)电荷 易位和(Vi)构象转换 非磷酸化的酶。二价阳离子的影响，替代 基材，以及在这些步骤上的其他修改也将被检查。 技术将包括停流荧光法和分光光度法， 和化学猝灭。这个实验室早期的许多工作已经 用荧光报告剂组IAF标记的酶。这些 研究将扩大到包括其他荧光探针，如 二磷酸和三磷酸的BIPM和FITC，TNP类似物，以及pH 敏感染料，如BCECF和SNARF。化学淬火实验将 使用放射性底物(例如[~(32)P]-和[~3H]-ATP)检查 产品形成。

The Na+,K+-ATPase is the enzymatic equivalent of the Na+ pump, which couples ATP hydrolysis to the transport of three Na+ out of, and two K+ into, the cell. This coupling converts stored chemical energy into usable electrochemical energy (in the ion gradients). In addition, this protein regulates several physiological processes (directly or indirectly), including neurotransmitter release and uptake, generation of resting membrane potential, and control of vascular and visceral muscle tone. Pump activity is regulated in vivo by insulin and thyroid hormone and by putative endogenous regulators. The pump serves as the receptor for digitalis glycoside drugs. The long-term goal of this project is to understand the coupling between ion transport and the energy released by ATP hydrolysis. Although a great deal of evidence links ion transport with ATP hydrolysis through ligand-induced conformational changes, the exact mechanism of this coupling is unclear. The present proposal describes experiments to distinguish between two alternate schemes for ion transport which propose that: (1) transport is linked directly with conformational change, and that the release of ions follows the conformational change, or (2) conformational changes alter the number of cation binding sites and their affinities, and that ion transport occurs prior to these changes. Transient state experiments, which measure rate constants for different steps in the enzyme cycle, will be used to distinguish between these two possibilities. Proposed experiments are designed to measure rate constants for each step in the reaction pathway. Specifically, these experiments will measure forward and reverse rate constants for (i) substrate binding, (ii) enzyme phosphorylation, (iii) ADP and P[i] release, (iv) conformational transitions between the phosphorylated enzyme forms, (v) charge translocation, and (vi) conformational transition between the nonphosphorylated enzyme. The effect of divalent cations, alternative substrates, and other modifications on these steps will also be examined. Techniques will include stopped-flow fluorimetry and spectrophotometry, and chemical quench. Much of the earlier work in this laboratory has used enzyme labeled with the fluorescent reporter group IAF. These studies will be extended to include other fluorescence probes, such as BIPM and FITC, TNP-analogs of nucleotide di- and tri-phosphates, and pH sensitive dyes such as BCECF and SNARF. Chemical quench experiments will use radioactive substrates (e.g. [32P]- and [3H]-ATP) to examine rates of product formation.