Pinner E, Padan E, Schuldiner S
Division of Microbial and Molecular Ecology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
J Biol Chem. 1994 Oct 21;269(42):26274-9.
NhaB, a Na+/H+ antiporter from Escherichia coli, was overproduced, purified, and reconstituted in a functional state, demonstrating that a single polypeptide, the product of the nhaB gene, can catalyze full activity. NhaB is a minor protein that accounts for less than 0.1% of the total membrane protein. The use of proteoliposomes made possible the determination of important kinetic and pharmacological properties in the absence of passive and mediated leaks. The activity of NhaB was found to have some pH dependence; the apparent Km for Na+ changes by 10-fold from 1.55 mM at pH 8.5 to 16.66 mM at pH 7.2, while the Vmax remains constant. It was demonstrated that NhaB is electrogenic and translocates more H+ than Na+ per cycle; the rate of sodium efflux from proteoliposomes was accelerated by a membrane potential, negative inside, and NhaB activity generated a membrane potential as monitored by two techniques. The stoichiometry of NhaB was estimated by a thermodynamic method in which the magnitude of delta psi generated by NhaB was measured at various Na+ gradients. A kinetic method, in which the electrophoretic movement of 86Rb+ (in the presence of valinomycin) was monitored in parallel with measurements of NhaB-mediated 22Na+ uptake, allowed us to determine a stoichiometry of 3H+/2Na+. The significance of the existence of two antiporters with different stoichiometries, NhaA and NhaB, active in the same cell, is discussed.
NhaB是一种来自大肠杆菌的Na⁺/H⁺逆向转运蛋白，经大量表达、纯化并重构为功能状态，这表明nhaB基因的单一多肽产物可催化完整活性。NhaB是一种次要蛋白质，占总膜蛋白的比例不到0.1%。利用蛋白脂质体能够在不存在被动和介导泄漏的情况下测定重要的动力学和药理学特性。发现NhaB的活性具有一定的pH依赖性；Na⁺的表观Km值在pH 8.5时为1.55 mM，在pH 7.2时为16.66 mM，变化了10倍，而Vmax保持恒定。已证明NhaB是生电的，每个循环转运的H⁺比Na⁺多；蛋白脂质体中钠外流的速率因膜电位（膜内为负）而加快，并且通过两种技术监测发现NhaB活性产生了膜电位。通过一种热力学方法估算了NhaB的化学计量，该方法在不同的Na⁺梯度下测量NhaB产生的Δψ大小。一种动力学方法，在监测NhaB介导的²²Na⁺摄取的同时平行监测⁸⁶Rb⁺（在缬氨霉素存在下）的电泳迁移，使我们能够确定化学计量为3H⁺/2Na⁺。文中讨论了在同一细胞中具有不同化学计量的两种逆向转运蛋白NhaA和NhaB存在的意义。
