Lukacovic M F, Verkman A S, Dix J A, Solomon A K
Biochim Biophys Acta. 1984 Dec 5;778(2):253-9. doi: 10.1016/0005-2736(84)90366-3.
The human red cell anion transport protein, band 3, contains six pCMBS (p-chloromercuribenzene sulfonate) reactive SH groups, five of which react with N-ethylmaleimide. We have carried out equilibrium binding experiments using N-ethylmaleimide-treated red cell ghosts and found that the sulfhydryl reactive water transport inhibitor, pCMBS, inhibits the binding to band 3 of the specific anion exchange inhibitor DBDS (4,4'-dibenzoamido-2,2'-disulfonic stilbene) in a non-competitive manner. Stopped-flow kinetic studies, in which DBDS is mixed with ghosts in the presence of pCMBS, show that pCMBS slows the DBDS induced conformational change in band 3. A non-competitive reaction scheme has been developed which incorporates the quantitative results of equilibrium and kinetic studies. The pCMBS effect on DBDS binding and kinetics is reversed with 5 mM cysteine suggesting a sulfhydryl bond is involved in pCMBS binding to band 3. These data suggest that pCMBS has a specific binding site on band 3, consistent with the hypothesis that band 3 mediates red cell water transport.
人类红细胞阴离子转运蛋白即带3蛋白，含有6个对pCMBS（对氯汞苯磺酸盐）有反应的巯基，其中5个可与N - 乙基马来酰亚胺反应。我们使用经N - 乙基马来酰亚胺处理的红细胞血影进行了平衡结合实验，发现对巯基有反应的水转运抑制剂pCMBS以非竞争性方式抑制特异性阴离子交换抑制剂DBDS（4,4'-二苯甲酰胺基 - 2,2'-二磺酸芪）与带3蛋白的结合。在pCMBS存在的情况下将DBDS与血影混合的停流动力学研究表明，pCMBS减缓了DBDS诱导的带3蛋白构象变化。已制定了一个非竞争性反应方案，该方案纳入了平衡和动力学研究的定量结果。5 mM半胱氨酸可逆转pCMBS对DBDS结合和动力学的影响，这表明巯基键参与了pCMBS与带3蛋白的结合。这些数据表明pCMBS在带3蛋白上有一个特异性结合位点，这与带3蛋白介导红细胞水转运的假设一致。
