Ives H E, Yee V J, Warnock D G
J Biol Chem. 1983 Nov 25;258(22):13513-6.
The subcellular distribution of the Na+/H+ antiporter in renal proximal tubule cells was studied with differential and density gradient centrifugation. Enzyme markers for basolateral membranes [Na+/K+)-ATPase), brush border membranes (maltase), and a variety of intracellular organelles (NADPH cytochrome c reductase, thiamine pyrophosphatase, acid phosphatase, and succinate cytochrome c reductase) were simultaneously assayed in sucrose density gradients. Basolateral membranes (median rho = 1.150) were well separated from brush border membranes (median rho = 1.165) by this technique. Markers for other cellular organelles had intermediate or bimodal distributions. To determine the cellular location of the Na+/H+ antiporter, Na+-dependent collapse of preformed pH gradients was assayed in the sucrose density gradient fractions using acridine orange. Na+/H+ antiporter activity paralleled the distribution of the brush border membrane fractions; activity in the peak basolateral membrane fraction was less than 5% of that in the peak brush border fraction. To determine whether antiporter activity was potentially detectable in all cell fractions, nigericin was added to each fraction and K+/H+ exchange was assayed with acridine orange. Activity was present in all sucrose density gradient fractions. In addition, there was no alteration in Na+/H+ exchange activity measured in brush border membranes after mixing with cell sol or basolateral membranes, showing that neither inhibitors nor activators of the Na+/H+ antiporter were present in any of the cell fractions. These controls confirmed the finding that Na+/H+ antiporter activity was absent from basolateral membranes. The presence of the Na+/H+ antiporter in brush border membranes and its absence from basolateral membranes is consistent with its playing an important role in the vectorial transport of H+ from blood to tubular lumen in the renal proximal tubule.
利用差速离心和密度梯度离心法研究了肾近端小管细胞中Na⁺/H⁺逆向转运体的亚细胞分布。在蔗糖密度梯度中同时检测了基底外侧膜（[Na⁺/K⁺]-ATP酶）、刷状缘膜（麦芽糖酶）以及多种细胞内细胞器（NADPH细胞色素c还原酶、硫胺素焦磷酸酶、酸性磷酸酶和琥珀酸细胞色素c还原酶）的酶标志物。通过该技术，基底外侧膜（中位密度=1.150）与刷状缘膜（中位密度=1.165）得到了很好的分离。其他细胞器的标志物具有中间分布或双峰分布。为了确定Na⁺/H⁺逆向转运体在细胞内的位置，使用吖啶橙在蔗糖密度梯度级分中检测预先形成的pH梯度的Na⁺依赖性塌陷。Na⁺/H⁺逆向转运体活性与刷状缘膜级分的分布平行；基底外侧膜峰值级分中的活性不到刷状缘膜峰值级分中活性的5%。为了确定在所有细胞级分中是否都能潜在检测到逆向转运体活性，向每个级分中加入尼日利亚菌素，并用吖啶橙检测K⁺/H⁺交换。所有蔗糖密度梯度级分中均存在活性。此外，刷状缘膜与细胞溶胶或基底外侧膜混合后，所测的Na⁺/H⁺交换活性没有改变，这表明任何细胞级分中都不存在Na⁺/H⁺逆向转运体的抑制剂或激活剂。这些对照证实了基底外侧膜中不存在Na⁺/H⁺逆向转运体活性这一发现。刷状缘膜中存在Na⁺/H⁺逆向转运体而基底外侧膜中不存在，这与其在肾近端小管中H⁺从血液向肾小管腔向量转运中发挥重要作用是一致的。
