Ouabain binding to renal tubules of the rabbit.
Ouabain binding to renal tubules of the rabbit.
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DOI：
10.1083/jcb.76.2.278
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发表时间：
1978-02
期刊：
影响因子：
--
通讯作者：
Stirling C
中科院分区：
文献类型：
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作者：
Shaver JL;Stirling C
众所周知，哇巴因是 Na-K ATP 酶依赖性转运的特异性抑制剂，会干扰肾小管盐的重吸收。在这项研究中，我们采用放射化学方法来测量[3H]哇巴因与兔肾髓质切片结合的动力学，并采用高分辨率定量放射自显影来确定细胞结合位点的位置和数量。动力学遵循简单的双分子反应，缔合常数为 2.86 +/- 0.63 SD x 10(3) M-1 min-1，解离常数为 1.46 x 10(-3) min-1，产生的平衡结合常数为 0.51 x 10(-6) M。结合高度依赖于温度。在10(-6)M浓度下，25℃至35℃之间的积累速率表现出Q10为1.8。在 0 摄氏度时，哇巴因解离速率可以忽略不计。随着钾浓度的增加，证明了结合的特异性。在 1 µM、6 mM 和 50 mM K+ 浓度下，哇巴因积累速率分别比零 K+ 时观察到的降低了 2.5 倍和 7 倍。 1 mM 毒毛花素 K 完全抑制结合。哇巴因结合的主要部位是粗升肢；在细肢和集合管中观察到很少或没有结合。此外，结合仅限于基底外侧膜。根据放射自显影颗粒密度测量，估计每个细胞含有超过 4 x 10(6) 哇巴因结合位点或 Na-K ATP 酶分子。这些结果与生理和生化观察相结合表明 Na-K ATP 酶在该片段的盐重吸收中起着关键作用。
It is well known that ouabain, a specific inhibitor of Na-K ATPase-dependent transport, interferes with renal tubular salt reabsorption. In this study, we employed radiochemical methods to measure the kinetics of [3H]ouabain binding to slices of rabbit renal medulla and high resolution quantitative autoradiography to determine the location and number of cellular binding sites. The kinetics obeyed a simple bimolecular reaction with an association constant of 2.86 +/- 0.63 SD x 10(3) M-1 min-1 and a dissociation constant of 1.46 x 10(-3) min-1, yielding an equilibrium binding constant of 0.51 x 10(-6) M. Binding was highly dependent upon temperature. At a concentration of 10(-6) M, the rate of accumulation between 25 degrees C and 35 degrees C exhibited a Q10 of 1.8. At 0 degree C the rate of ouabain dissociation was negligible. The specificity of binding was demonstrated with increasing potassium concentrations. At a concentration of 1 microM, 6 mM, and 50 mM K+ produced a 2.5- and 7-fold decrease, respectively, in the rate of ouabain accumulation observed at zero K+. Binding was completely inhibited by 1 mM strophanthin K. The major site of ouabain binding was the thick ascending limb; little or no binding was observed in thin limbs and collecting ducts. Moreover, binding was confined to the basolateral membranes. From autoradiographic grain density measurements, it was estimated that each cell contains over 4 x 10(6) ouabain binding sites or Na-K ATPase molecules. These results taken together with physiological and biochemical observations suggest that Na-K ATPase plays a key role in salt reabsorption by this segment.