Macara I G, Kuo S, Cantley L C
J Biol Chem. 1983 Feb 10;258(3):1785-92.
The transport inhibitor, eosin 5-maleimide, reacts specifically at an external site on the membrane-bound domain of the anion exchange protein, Band 3, in the human erythrocyte membrane. The fluorescence of eosin-labeled resealed ghosts or intact cells was found to be resistant to quenching by CsCl, whereas the fluorescence of labeled inside-out vesicles was quenched by about 27% at saturating CsCl concentrations. Since both Cs+ and eosin maleimide were found to be impermeable to the red cell membrane and the vesicles were sealed, these results indicate that after binding of the eosin maleimide at the external transport site of Band 3, the inhibitor becomes exposed to ions on the cytoplasmic surface. The lifetime of the bound eosin maleimide was determined to be 3 ns both in the absence and presence of CsCl, suggesting that quenching is by a static rather than a dynamic (collisional) mechanism. Intrinsic tryptophan fluorescence of erythrocyte membranes was also investigated using anion transport inhibitors which do not appreciably absorb light at 335 nm. Eosin maleimide caused a 25% quenching and 4,4'-dibenzamidodihydrostilbene-2,2'-disulfonate) caused a 7% quenching of tryptophan fluorescence. Covalent labeling of red cells by either eosin maleimide or BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) caused an increase in the susceptibility of membrane tryptophan fluorescence to quenching by CsCl. The quenching constant was similar to that for the quenching of eosin fluorescence and was unperturbed by the presence of 0.5 M KCl. Neither NaCl nor Na citrate produced a large change in the relative magnitude of the tryptophan emission. The tryptophan residues that can be quenched by CsCl appear to be different from those quenched by eosin or BIDS and are possibly located on the cytoplasmic domain of Band 3. The results suggest that a conformational change in the Band 3 protein accompanies the binding of certain anion transport inhibitors to the external transport site of Band 3 and that the inhibitors become exposed on the cytoplasmic side of the red cell membrane.
转运抑制剂 eosin 5-马来酰亚胺特异性地作用于人红细胞膜中阴离子交换蛋白带 3 的膜结合结构域的外部位点。发现用 eosin 标记的重封血影或完整细胞的荧光对 CsCl 的淬灭具有抗性，而在饱和 CsCl 浓度下，标记的内翻囊泡的荧光被淬灭约 27%。由于发现 Cs⁺和 eosin 马来酰亚胺都不能透过红细胞膜且囊泡是封闭的，这些结果表明，在 eosin 马来酰亚胺结合到带 3 的外部转运位点后，抑制剂会暴露于细胞质表面的离子。在不存在和存在 CsCl 的情况下，结合的 eosin 马来酰亚胺的寿命均测定为 3 纳秒，这表明淬灭是通过静态而非动态（碰撞）机制。还使用在 335 nm 处不明显吸收光的阴离子转运抑制剂研究了红细胞膜的固有色氨酸荧光。eosin 马来酰亚胺导致色氨酸荧光淬灭 25%，4,4'-二苯甲酰胺基二氢芪-2,2'-二磺酸盐导致色氨酸荧光淬灭 7%。用 eosin 马来酰亚胺或 BIDS（4-苯甲酰胺基-4'-异硫氰酸根合芪-2,2'-二磺酸盐）对红细胞进行共价标记会导致膜色氨酸荧光对 CsCl 淬灭的敏感性增加。淬灭常数与 eosin 荧光淬灭的常数相似，并且不受 0.5 M KCl 存在的影响。NaCl 和柠檬酸钠都不会使色氨酸发射的相对强度产生大的变化。可被 CsCl 淬灭的色氨酸残基似乎与被 eosin 或 BIDS 淬灭的色氨酸残基不同，并且可能位于带 3 的细胞质结构域上。结果表明，带 3 蛋白的构象变化伴随着某些阴离子转运抑制剂与带 3 的外部转运位点的结合，并且抑制剂会暴露于红细胞膜的细胞质一侧。
