IDENTIFICATION OF THE EOSINYL-5-MALEIMIDE REACTION SITE ON THE HUMAN ERYTHROCYTE ANION-EXCHANGE PROTEIN - OVERLAP WITH THE REACTION SITES OF OTHER CHEMICAL PROBES
IDENTIFICATION OF THE EOSINYL-5-MALEIMIDE REACTION SITE ON THE HUMAN ERYTHROCYTE ANION-EXCHANGE PROTEIN - OVERLAP WITH THE REACTION SITES OF OTHER CHEMICAL PROBES
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DOI：
10.1021/bi00488a012
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发表时间：
1990-09-11
期刊：
影响因子：
2.9
通讯作者：
BETH, AH
中科院分区：
文献类型：
--
作者：
COBB, CE;BETH, AH
已确定人红细胞中荧光/磷光探针 eosinyl-5-maleimide (EMA) 的阴离子交换蛋白（带 3）反应位点。带 3 的原位蛋白水解解剖表明 EMA 与完整红细胞中带 3 的染色蛋白酶裂解产生的跨膜 Mr 17K 肽发生反应，然后通过轻度胰蛋白酶消化鬼膜去除胞质结构域。对纯化的 Mr 17K 肽的大量胰凝乳蛋白酶消化物进行 HPLC 纯化获得的主要伊红标记肽进行测序，可以将 EMA 的共价反应位点分配给人红细胞蛋白的赖氨酸 430 [Tanner 等人，2015]。 (1988)生物化学。 J.256, 703-712]。基于蛋白质一级结构的水路图 [Lux 等人。 (1989) 程序。国家。阿卡德。科学。 U.S.A. 86, 9089-9093]表明该残基位于连接红细胞膜中天然带3的跨膜片段1和2的细胞外可接近的环中。还研究了成对的化学探针对完整红细胞的连续标记的抑制作用，这些探针包括 EMA、阴离子转运抑制剂 4,4''-二异硫氰基二氢芪-2,2''-二硫化物 (H2-DIDS) 和反应性双功能自旋标记双(磺基-N-琥珀酰亚胺基) doxyl-2-spiro-5''-壬二酸 (BSSDA)。这些试剂中的每一种在单独添加到完整人红细胞的悬浮液中时通过形成一个或多个稳定的共价键而亲和标记条带3。用 EMA 对完整红细胞进行预标记，使 H2-DIDS 对带 3 的后续标记减少了约 95%，对 BSSDA 对带 3 的标记减少了 90%。同样，使用 H2-DIDS 预标记将 EMA 对条带 3 的后续标记减少了 90% 以上，而 BSSDA 预标记将 EMA 标记减少了约 95%。因此，尽管具有广泛不同的化学结构和蛋白质修饰反应性，但这些带负电荷的试剂中的每一种都可能与带3上可从细胞外空间接近的空间重叠位点竞争反应。
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 chromotrypsin 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 segment 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''-disfulfonate (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.