Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu).
Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu).
复制标题
二苯乙烯二磺酸盐与带 3 HT（Pro-868-->Leu）反应的不同灵敏度。
DOI：
10.1073/pnas.92.25.11844
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发表时间：
1995
影响因子：
11.1
通讯作者：
Lawrence,C
中科院分区：
文献类型：
--
作者：
Salhany,JM;Schopfer,LM;Kay,MM;Gamble,DN;Lawrence,C
Band 3 HT (Pro-868-->Leu) 是一种突变阴离子交换蛋白，具有多种表型特征，包括 2 至 3 倍大的 Vmax，以及阴离子转运抑制剂 4,4'-二异硫氰基二氢芪-2,2'-二磺酸盐 (H2DIDS) 的共价结合减少。我们使用荧光动力学方法来研究与带 3 结合的抑制剂，以确定带 3 HT 中的点突变是否在该转运蛋白的膜结合域内产生局部或广泛的构象变化。我们的结果表明，与野生型蛋白相比，带 3 HT 与 H2DIDS 的共价结合慢了 10 至 20 倍。相比之下，4,4'-二异硫氰芪-2,2'-二磺酸盐 (DIDS) 的共价结合没有观察到动力学差异。此外，与野生型蛋白相比，带3 HT的H2DIDS释放动力学异常，而4,4'-二苯甲胺二苯乙烯-2,2'-二磺酸盐(DBDS)释放动力学没有显示差异。我们得出的结论是，在第 868 位用亮氨酸取代脯氨酸不会扰乱带 3 的膜结合结构域中“赖氨酸 A”的结构，而是会在蛋白质的 C 端亚结构域中产生明显的局部构象变化，从而改变 H2DIDS 亲和力。当与 Vmax 增加的观察相结合时，这些结果表明第 868 位的蛋白质结构变化影响运输循环中的周转步骤。
Band 3 HT (Pro-868-->Leu) is a mutant anion exchange protein which has several phenotypic characteristics, including a 2- to 3-fold larger Vmax, and reduced covalent binding of the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS). We have used fluorescence kinetic methods to study inhibitor binding to band 3 to determine if the point mutation in band 3 HT produces localized or wide-spread conformational changes within the membrane-bound domain of this transporter. Our results show that covalent binding of H2DIDS by band 3 HT is slower by a factor of 10 to 20 compared with the wild-type protein. In contrast, no such difference in the kinetics was observed for covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In addition, the kinetics of H2DIDS release from band 3 HT was abnormal, while the kinetics of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) release showed no difference when compared with the wild-type protein. We conclude that substitution of leucine for proline at position 868 does not perturb the structure of "lysine A" in the membrane-bound domain of band 3 but rather produces an apparently localized conformational change in the C-terminal subdomain of the protein which alters H2DIDS affinity. When combined with the observation of an increased Vmax, these results suggest that protein structural changes at position 868 influence a turnover step in the transport cycle.