MODES OF OPERATION OF THE SODIUM PUMP

该项目的长期目标是提供一个完整的， 钠-钾反应的可能动力学和热力学描述 泵或（Na+K）ATP酶。 钠钾泵存在于 几乎所有动物细胞的膜，在那里它负责 维持跨膜Na和K梯度，这是神经的基础， 肌肉兴奋，容量和pH调节，液体分泌，感觉 转导、营养物质和其他离子的次级运输等。 泵的产电性质（排出的钠多于输入的钠 钾）在感觉适应、起搏器 生成和突触调制。 洋地黄类药物的作用 心脏几乎肯定是通过钠泵的 钠泵 人类消耗体内所有ATP的20-50%。 为此选择的细胞 工作是一个巨大的神经纤维的鱿鱼，因为它非常大， 大小，允许实验性的访问和控制，这是其他 细胞 从乌贼的大脑中，（Na+K）ATP酶将被 提取，其酶性质与运输相比， 原位轴突泵的性质。 这项建议的具体目标是尽可能建立， 由泵介导的“生物物理”事件之间的动力学相关性， 如各种运输模式称为Na：K交换，K：Na交换 或反向泵送、Na：Na交换、K：K交换和“非偶联”Na 外排，由相同的酶催化的“生物化学”事件，如ATP 水解，ADP：ATP交换，ATP增强的“封闭钾”的去封闭， 酶磷酸化和去磷酸化。 轴突运输 膜将使用内部透析技术进行研究， 细胞内部和外部处于完全的实验控制之下， 可以测量任一方向上的同位素通量。 膜电位 将通过一个非常稳定的（正负40 MuV） 电压箝位电路 由泵产生的电流将 测量为强心类固醇引起的保持电流的变化。 一次一个变量的系统变化（外部或内部Na， K、Mg或代谢物浓度、pH或膜电位）将产生 大量的动力学数据曲线，然后必须通过最少的 在已知的生物化学的框架内， (Na+K）ATP酶。

The long-term objective of this project is to provide an as complete as possible kinetic and thermodynamic description of the sodium-potassium pump, or (Na+K)ATPase. The sodium-potassium pump is present in the membrane of nearly all animal cells, where it is responsible for maintaining the transmembrane Na and K gradients which underlie nerve and muscle excitation, volume and pH regulation, fluid secretion, sensory transduction, secondary transport of nutrients and other ions, etc. The electrogenic nature of the pump (it expels more sodium than it imports potassium) plays a central role in sensory adaptation, in pacemaker generation, and in synaptic modulation. The action of digitalis drugs on the heart is almost certainly through the sodium pump. The sodium pump in man consumes 20-50% of all ATP made in the body. The cell chosen for this work is a giant nerve fiber of the squid which, because of its very large size, allows experimental access and control not available with other cells. From the brain of the squid, the (Na+K)ATPase enzyme will be extracted, and its enzymic properties compared with the transport properties of the in situ axon pump. The specific aims for this proposal are to establish, wherever possible, kinetic correlations between "biophysical" events mediated by the pump, such as the various transport modes known as Na:K exchange, K:Na exchange or reverse pumping, Na:Na exchange, K:K exchange, and "uncoupled" Na efflux, with "biochemical" events catalyzed by the same enzyme, such as ATP hydrolysis, ADP:ATP exchange, ATP-enhanced de-occlusion of "occluded K", enzyme phosphorylation and dephosphorylation. Transport across the axon membrane will be studied using the internal dialysis technique, whereby the cell interior and exterior are put under complete experimental control, and isotope fluxes in either direction can be measured. Membrane potential will be controlled by means of a very stable (plus and minus 40 MuV) voltage-clamp circuit. The electric current generated by the pump will be measured as the cardiotonic steroid induced change in holding current. Systematic variations of one variable at a time (external or internal Na, K, Mg or metabolite concentration, pH, or membrane potential) will yield extensive sets of kinetic data curves which must then be analyzed by least squares methods within the framework of the known biochemistry of the (Na+K)ATPase.