Talor Z, Ng S C, Cragoe E J, Arruda J A
Section of Nephrology, University of Illinois, Chicago.
Life Sci. 1989;45(6):517-23. doi: 10.1016/0024-3205(89)90102-1.
We measured the binding of [3H]-5-(N-methyl-N-isobutyl) amiloride (MIA) to purified rabbit renal brush border membranes. MIA binding was protein, temperature and time dependent with optimal binding at pH 8.0 or above. At low pH MIA binding was inhibited, suggesting competition between H+ ions and MIA for the MIA binding site. There was 70-80% specific binding which reached a plateau at 30 min and remained stable thereafter for 150 min. Scatchard analysis revealed one family of binding sites with Bmax of 3.4 +/- 0.4 pmoles/mg protein and Kd of 30.5 +/- 2.3 nM. MIA inhibited the Vmax of the Na-H antiporter (assessed by acridine orange quenching) in a dose dependent fashion with 100% inhibition at MIA concentration of 10(-3) M and this inhibition was greater than that of amiloride. We conclude that MIA, a potent inhibitor of the Na-H antiporter, displays a high percentage of specific binding to renal brush border membranes and can be used to assess the number of the Na-H antiporters.
我们测定了[3H]-5-(N-甲基-N-异丁基)氨氯吡脒（MIA）与纯化的兔肾刷状缘膜的结合情况。MIA结合具有蛋白质依赖性、温度依赖性和时间依赖性，在pH 8.0及以上时结合最佳。在低pH值下，MIA结合受到抑制，这表明H+离子与MIA在MIA结合位点上存在竞争。特异性结合率为70 - 80%，在30分钟时达到平台期，此后150分钟内保持稳定。Scatchard分析显示存在一类结合位点，Bmax为3.4±0.4 pmol/mg蛋白质，Kd为30.5±2.3 nM。MIA以剂量依赖性方式抑制钠-氢反向转运体的Vmax（通过吖啶橙淬灭评估），在MIA浓度为10(-3) M时抑制率达100%，且这种抑制作用大于氨氯吡脒。我们得出结论，作为钠-氢反向转运体的强效抑制剂，MIA与肾刷状缘膜具有较高比例的特异性结合，可用于评估钠-氢反向转运体的数量。
