Toon M R, Solomon A K
Biochim Biophys Acta. 1986 Aug 21;860(2):361-75. doi: 10.1016/0005-2736(86)90533-x.
The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.
已确定对氯汞苯磺酸盐（pCMBS）抑制人红细胞水转运的结合常数为160±30微摩尔，对尿素转运抑制的结合常数为0.09±0.06微摩尔，这表明这两个抑制过程存在不同的位点。反应动力学表明，这两个过程均由pCMBS与膜位点之间的双分子缔合以及随后的构象变化组成。两个过程都非常缓慢，水抑制过程的结合速率常数比抑制剂与膜转运蛋白结合的通常速率常数慢约10^5倍。pCMBS与水转运抑制位点的结合可被半胱氨酸逆转，而与尿素转运抑制位点的结合则不能被逆转。特异性的芪类阴离子交换抑制剂4,4'-二苯甲酰胺芪-2,2'-二磺酸盐（DBDS）会使pCMBS抑制水转运的时间进程发生显著变化，这与阴离子交换和水转运之间的联系一致。对带3上可用巯基的考虑表明，尿素转运抑制位点在带3上，但不是巯基，并且，如果水转运抑制位点是巯基，则它位于与带3复合的另一种蛋白质上，可能是带4.5。
