Cobb C E, Beth A H
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Biochemistry. 1990 Sep 11;29(36):8283-90. doi: 10.1021/bi00488a012.
The anion-exchange protein (band 3) reaction site in human erythrocytes for the fluorescent/phosphorescent probe eosinyl-5-maleimide (EMA) has been identified. Proteolytic dissection of band 3 in situ indicated that EMA reacts with the membrane-spanning Mr 17K peptide produced by chymotrypsin cleavage of band 3 in intact erythrocytes followed by removal of the cytoplasmic domain by mild trypsin digestion of ghost membranes. Sequencing of the major eosin-labeled peptide obtained from HPLC purification of an extensive chymotrypsin digest of purified Mr 17K peptide allowed assignment of the covalent reaction site for EMA to lysine-430 of the human erythrocyte protein [Tanner et al. (1988) Biochem. J. 256, 703-712]. Hydropathy plots based upon the primary structure of the protein [Lux et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9089-9093] suggest that this residue is in an extracellularly accessible loop connecting membrane-spanning segments 1 and 2 of native band 3 in the erythrocyte membrane. Inhibition of sequential labeling of intact erythrocytes by pairs of chemical probes including EMA, the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2-DIDS), and the reactively bifunctional spin-label bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA) has also been investigated. Each of these reagents affinity labels band 3 when added separately to a suspension of intact human erythrocytes by formation of one or more stable covalent bonds. Prelabeling of intact erythrocytes with EMA reduced subsequent labeling of band 3 by H2-DIDS by approximately 95% and by BSSDA by 90%. Similarly, prelabeling with H2-DIDS reduced subsequent labeling of band 3 by EMA by over 90%, and BSSDA prelabeling reduced EMA labeling by approximately 95%. Therefore, though having widely divergent chemical structures and protein modification reactivities, each of these negatively charged reagents may be competing for reaction with spatially overlapping sites on band 3 which are accessible from the extracellular space.
已确定人红细胞中用于荧光/磷光探针曙红基-5-马来酰亚胺（EMA）的阴离子交换蛋白（带3）反应位点。对完整红细胞中带3进行原位蛋白酶解分析表明，EMA与胰凝乳蛋白酶切割完整红细胞中的带3所产生的跨膜Mr 17K肽发生反应，随后通过对血影膜进行温和的胰蛋白酶消化去除细胞质结构域。对从纯化的Mr 17K肽的广泛胰凝乳蛋白酶消化产物经HPLC纯化得到的主要曙红标记肽进行测序，确定了EMA与人红细胞蛋白赖氨酸-430的共价反应位点[ Tanner等人（1988年），《生物化学杂志》256, 703 - 712]。基于该蛋白一级结构的亲水性图谱[ Lux等人（1989年），《美国国家科学院院刊》86, 9089 - 9093]表明，该残基位于红细胞膜中天然带3的跨膜片段1和2之间的一个细胞外可及环中。还研究了包括EMA、阴离子转运抑制剂4,4'-二异硫氰酸二氢芪-2,2'-二磺酸盐（H2-DIDS）和反应性双功能自旋标记双（磺基-N-琥珀酰亚胺基）二氧杂环戊烯-2-螺-5'-氮杂环庚烷（BSSDA）在内的成对化学探针依次标记完整红细胞的抑制作用。当分别添加到完整人红细胞悬液中时，这些试剂中的每一种都通过形成一个或多个稳定的共价键来亲和标记带3。用EMA对完整红细胞进行预标记可使随后H2-DIDS对带3的标记减少约95%，使BSSDA对带3的标记减少90%。同样，用H2-DIDS进行预标记可使随后EMA对带3的标记减少超过90%，而BSSDA预标记可使EMA标记减少约95%。因此，尽管这些带负电荷的试剂具有广泛不同的化学结构和蛋白质修饰反应性，但它们中的每一种可能都在竞争与带3上可从细胞外空间接近的空间重叠位点发生反应。
