KINETIC ANALYSIS OF NA+/K+ ATPASE REACTION MECHANISM

Na+，K+-ATP酶是Na+泵的酶等价物， 耦合ATP水解的三个Na+的运输，和两个K+ 到，细胞。 这种耦合将储存的化学能转化为 可用的电化学能量（在离子梯度中）。 另外这款 蛋白质调节几种生理过程（直接或 间接），包括神经递质的释放和摄取， 静息膜电位，以及血管和内脏肌肉的控制 语气 胰岛素泵活性在体内受胰岛素和甲状腺激素调节 和假定的内源性调节剂。 泵作为受体 洋地黄苷类药物的研究 本项目的长期目标是了解 离子运输和ATP水解释放的能量。 虽然 大量的证据将离子转运与ATP水解联系起来， 配体诱导的构象变化，其确切机制 耦合不清楚。 本提案描述了区分两个 离子传输的替代方案提出：（1）传输是 与构象变化直接相关， （2）构象改变，或（3）构象改变 阳离子结合位点的数目和它们的亲和力， 运输发生在这些变化之前。 瞬态实验， 其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。 建议的实验被设计来测量每个步骤的速率常数 在反应路径中。 具体来说，这些实验将测量 （i）底物结合，（ii）酶 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）构象之间的转换 非磷酸化酶 二价阳离子的影响，替代 还将检查衬底和对这些步骤的其它修改。 技术将包括停流荧光法和分光光度法， 和化学淬火。 这个实验室的许多早期工作 使用用荧光报告基团IAF标记的酶。 这些 研究将扩展到包括其他荧光探针，如 BIPM和FITC、核苷酸二磷酸和三磷酸的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[3 H]-ATP）检查 产物形成

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.