Koepsell H, Korn K, Ferguson D, Menuhr H, Ollig D, Haase W
J Biol Chem. 1984 May 25;259(10):6548-58.
Brush-border membranes of renal proximal tubules were solubilized with deoxycholate and some proteins were separated and incorporated into proteoliposomes by a reconstitution procedure which was analyzed in detail. The proteoliposomes contained mainly polypeptides with molecular weights of 152,000, 94,000, and 52,000, each of which could be separated further into homologous polypeptides with different isoelectric points. In the proteoliposomes, Na+ cotransport systems for D-glucose, acidic and neutral amino acids, and mono- and dicarboxylic acids were demonstrated by showing that due to an inwardly directed Na+ gradient the substrate concentrations in the proteoliposomes increased significantly over their respective equilibrium values. Using inhibition experiments, selectivity of the different transporters could be demonstrated. Studying the reconstituted L-glutamate transporter in detail, countertransport of L-glutamate and K+ was shown (i) at Na+ equilibrium the intraliposomal L-glutamate concentration increased significantly over the equilibrium value if an outside-directed K+ gradient was applied; (ii) Rb+ influx was significantly stimulated by the outflux of L-glutamate. By applying a K+ diffusion potential across the liposomal membrane by addition of valinomycin it could be shown that during L-glutamate transport in the presence of Na+ and K+ positive charge is transferred together with L-glutamate and Na+. The apparent Km value of L-glutamate uptake driven by concentration differences of 89 mM Na+ (out greater than in) and 89 mM K+ (in greater than out) was 26.3 +/- 1.3 microM. The Vmax value of 70.2 +/- 2.3 pmol X mg of protein-1 X S-1 was half the value measured in intact membranes.
用脱氧胆酸盐溶解肾近端小管的刷状缘膜，通过详细分析的重组程序分离出一些蛋白质并将其整合到蛋白脂质体中。蛋白脂质体主要含有分子量为152,000、94,000和52,000的多肽，每种多肽又可进一步分离为具有不同等电点的同源多肽。在蛋白脂质体中，通过显示由于内向的Na⁺梯度，蛋白脂质体内的底物浓度相对于其各自的平衡值显著增加，证实了D-葡萄糖、酸性和中性氨基酸以及单羧酸和二羧酸的Na⁺共转运系统。通过抑制实验，可以证明不同转运体的选择性。详细研究重组的L-谷氨酸转运体时，发现L-谷氨酸和K⁺的反向转运：(i) 在Na⁺平衡时，如果施加外向的K⁺梯度，脂质体内的L-谷氨酸浓度相对于平衡值显著增加；(ii) L-谷氨酸的外流显著刺激Rb⁺内流。通过添加缬氨霉素在脂质体膜上施加K⁺扩散电位，可以证明在Na⁺和K⁺存在下L-谷氨酸转运过程中，正电荷与L-谷氨酸和Na⁺一起转移。由89 mM Na⁺（外大于内）和89 mM K⁺（内大于外）的浓度差驱动的L-谷氨酸摄取的表观Km值为26.3±1.3 μM。70.2±2.3 pmol·mg蛋白⁻¹·s⁻¹的Vmax值是完整膜中测量值的一半。
