Jennings M L, Monaghan R, Douglas S M, Nicknish J S
J Gen Physiol. 1985 Nov;86(5):653-69. doi: 10.1085/jgp.86.5.653.
The extracellular lysine residues in the human erythrocyte anion transport protein (band 3) have been investigated using chemical modification with the impermeant homobifunctional active ester bis(sulfosuccinimidyl)-suberate (BSSS). This agent forms covalent intra- and intermolecular cross-links in human band 3 in intact cells (Staros and Kakkad. 1983. J. Membr. Biol. 74:247). We have found that the intermolecular cross-link has no detectable effect on the anion transport function of band 3. The intramolecular cross-link, however, causes major changes in the characteristics of the anion transport. These functional alterations are caused by the modification of lysine residues at the stilbene disulfonate binding site. BSSS pretreatment at pH 7.4 irreversibly inhibits Cl-Br exchange by at least 90% when the transport is assayed at extracellular pH above 8. In the same BSSS-pretreated cells, however, the Cl-Br exchange rate is activated by lowering the pH of the flux medium (intracellular pH fixed at 7). The flux is maximal at pH 5-6; a further lowering of the extracellular pH inhibits the anion exchange. This acid-activated Cl-Br exchange in the BSSS-treated cells is mediated by band 3, as indicated by phenylglyoxal and phloretin inhibition of the flux. Thus, the BSSS pretreatment has little effect on the maximal Cl-Br exchange flux catalyzed by band 3, but it shifts the alkaline branch of its extracellular pH dependence by approximately 5 pH units. BSSS also eliminates the self-inhibition of Cl-halide exchange by high extracellular Br or I concentrations. These results indicate that the BSSS-modified lysines do not participate directly in anion translocation, but that one of the lysines normally provides a positive charge that is necessary for substrate anion binding. This positive charge is removed by the BSSS treatment but can be replaced by lowering the extracellular pH. The results also provide insight regarding the halide selectivity of the maximal rate of chloride-halide exchange: the native selectivity (Br much greater than I) is nearly abolished by BSSS treatment, which suggests that the selectivity results from the very strong binding of iodide to an outward-facing modifier site.
利用不可渗透的同双功能活性酯双（磺基琥珀酰亚胺基）辛二酸酯（BSSS）进行化学修饰，对人红细胞阴离子转运蛋白（带3蛋白）的细胞外赖氨酸残基进行了研究。该试剂在完整细胞的人带3蛋白中形成共价分子内和分子间交联（Staros和Kakkad，1983年，《膜生物学杂志》74:247）。我们发现分子间交联对带3蛋白的阴离子转运功能没有可检测到的影响。然而，分子内交联会导致阴离子转运特性发生重大变化。这些功能改变是由二苯乙烯二磺酸盐结合位点处的赖氨酸残基修饰引起的。当在细胞外pH高于8时测定转运时，在pH 7.4下用BSSS预处理会不可逆地抑制Cl-Br交换至少90%。然而，在相同的经BSSS预处理的细胞中，通过降低通量介质的pH（细胞内pH固定为7）可激活Cl-Br交换速率。通量在pH 5-6时最大；细胞外pH进一步降低会抑制阴离子交换。如苯乙二醛和根皮素对通量的抑制所示，BSSS处理细胞中的这种酸激活的Cl-Br交换是由带3蛋白介导的。因此，BSSS预处理对带3蛋白催化的最大Cl-Br交换通量影响很小，但它将其细胞外pH依赖性的碱性分支移动了约5个pH单位。BSSS还消除了高细胞外Br或I浓度对Cl-卤化物交换的自我抑制。这些结果表明，经BSSS修饰的赖氨酸不直接参与阴离子转运，但其中一个赖氨酸通常提供底物阴离子结合所需的正电荷。该正电荷通过BSSS处理被去除，但可通过降低细胞外pH来替代。这些结果还提供了关于氯化物-卤化物交换最大速率的卤化物选择性的见解：BSSS处理几乎消除了天然选择性（Br远大于I），这表明选择性源于碘化物与外向修饰位点的非常强的结合。
