Warnock D G, Reenstra W W, Yee V J
Am J Physiol. 1982 Jun;242(6):F733-9. doi: 10.1152/ajprenal.1982.242.6.F733.
Fluorescence quenching of acridine orange was used to characterize the generation and collapse of pH gradients by the Na+/H+ antiporter of brush border membrane vesicles prepared from rabbit renal cortex. Quenching was observed when acridine orange, a weak base, was taken up to an acidic intravesicular space. Na+/H+ exchange was examined with both Na+ uptake and efflux studies. Acridine orange fluorescence quenching demonstrated the cation specificity of the Na+/H+ antiporter (i.e., sodium and lithium) and was inhibited by amiloride. Parallel studies with nigericin, a K+/H+ antiporter, demonstrated that acridine orange responded very rapidly to pH gradients. Therefore, acridine orange equilibration was not rate limiting in our studies of the Na+/H+ antiporter. Initial rate measurements were made to obtain kinetic parameters for the Na+/H+ antiporter. In sodium influx studies, the half-maximal rate of acridine orange fluorescence change was obtained with an external sodium concentration of 13.3 +/- 0.5 mM.
利用吖啶橙的荧光猝灭来表征由兔肾皮质制备的刷状缘膜囊泡的Na⁺/H⁺逆向转运蛋白产生和破坏pH梯度的情况。当弱碱吖啶橙被摄取到酸性的囊泡内空间时，可观察到猝灭现象。通过Na⁺摄取和外流研究来检测Na⁺/H⁺交换。吖啶橙荧光猝灭证明了Na⁺/H⁺逆向转运蛋白的阳离子特异性（即钠和锂），并被氨氯吡脒抑制。用缬氨霉素（一种K⁺/H⁺逆向转运蛋白）进行的平行研究表明，吖啶橙对pH梯度反应非常迅速。因此，在我们对Na⁺/H⁺逆向转运蛋白的研究中，吖啶橙平衡不是限速因素。进行初始速率测量以获得Na⁺/H⁺逆向转运蛋白的动力学参数。在钠内流研究中，当外部钠浓度为13.3±0.5 mM时，获得了吖啶橙荧光变化的半数最大速率。
