Hemichrome binding to band 3: nucleation of Heinz bodies on the erythrocyte membrane.
Hemichrome binding to band 3: nucleation of Heinz bodies on the erythrocyte membrane.
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半色素与带 3 结合：亨氏小体在红细胞膜上成核。
DOI：
10.1021/bi00322a006
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Low,PS
中科院分区：
文献类型：
--
作者：
Waugh,SM;Low,PS
摘要：用苯肼处理天然血红蛋白，制备了红细胞Heinz小体的前体Hemichromes，并研究了其与人红细胞膜细胞质表面的相互作用。发现半色体与漏出的红细胞鬼的结合是双相的，具有高亲和力和低亲和力位点。高亲和力位点位于3号带的细胞质结构域，因为(i)已知的3号带配体甘油醛-3-磷酸脱氢酶与半色素体竞争其结合位点，（ii）通过蛋白水解裂解去除3号带的细胞质结构域导致高亲和力位点的丢失，以及（iii）分离的3号带细胞质结构域与半色素体紧密相互作用，迅速形成ph依赖性的，不溶于水的共聚物在水溶液中混合。由于具有带3细胞质结构域的半色原共聚物易于分离，因此对其性质进行了部分表征。该共聚物具有确定的化学计量，每3带二聚体含有~ 2.5个半色四聚体（或~ 5个半色二聚体），与初始反应溶液中半色与3带的比例无关。发现共聚物具有宏观尺寸，产生的颗粒无需使用显微镜就可以很容易地可视化。共沉淀对血红蛋白也具有高度选择性，因为在与天然血红蛋白的混合溶液中，在分离的颗粒中只观察到血红蛋白。此外，将带3的细胞质结构域与含氧血红蛋白、脱氧血红蛋白、（一氧化碳）血红蛋白或高铁血红蛋白混合时，未观察到沉淀。带3的细胞质结构域对血红蛋白的亲和力可能比天然血红蛋白高得多，因为需要20倍摩尔的血红蛋白才能减少50%的共聚。我们认为，带3和血红素在体内的共聚可以解释亨氏小体在红细胞膜上的聚集和在许多血红蛋白病中观察到的溶血现象。天然血红蛋白（Hb）与人红细胞膜的相互作用最近受到了相当大的关注。已经确定了两类结合位点，其中一类是高亲和力的结合位点，位于主要红细胞蛋白的细胞质结构域，带3 （Shaklai等人，1977a, b； Salhany等人，1980;Sayare f）。这项工作得到了美国国立卫生研究院（National Institutes of Health）的Grant GM 24417的部分支持。& Fikiet, 1981)。这种相互作用本质上是静电的，随着pH值和离子强度的降低，Hb对膜的亲和力增加（Shaklai等，1977a, b; Fung, 1981； Fischer等，1975）。在完整的细胞中，也有类似的结合对膜的pH依赖性的报道（Eisinger et al., 1982）。分离的3号带胞质结构域也被证明与两个血红蛋白分子结合，支持3号带作为可溶性蛋白的主要膜附着位点的鉴定
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 Received June 28, 1984 abstract: Hemichromes, the precursors of red cell Heinz bodies, were prepared by treatment of native hemoglobin with phenylhydrazine, and their interaction with the cytoplasmic surface of the human erythrocyte membrane was studied. Binding of hemichromes to leaky red cell ghosts was found to be biphasic, exhibiting both high-affinity and low-affinity sites. The high-affinity sites were shown to be located on the cytoplasmic domain of band 3, since (i) glyceraldehyde-3-phosphate dehydrogenase, a known ligand of band 3, competes with the hemichromes for their binding sites,(ii) removal of the cytoplasmic domain of band 3 by proteolytic cleavage causes loss of the high-affinity sites, and (iii) the isolated cytoplasmic domain of band 3 interacts tightly with hemichromes, rapidly forming a pH-dependent, water-insoluble copolymer upon mixing in aqueous solution. Since the copolymer of hemichromes with the cytoplasmic domain of band 3 was readily isolatable, a partial characterization of its properties was conducted. The copolymer was shown to be of defined stoichiometry, containing~ 2.5 hemichrome tetramers (or~ 5 hemichrome dimers) per band 3 dimer, regardless of the ratio of hemichrome: band 3 in the initial reaction solution. The copolymer was found to be of macroscopic dimensions, generating particles which could be easily visualized without use of a mi-croscope. The coprecipitation was also highly selective for hemichromes, since, in mixed solutions with native hemoglobin, only hemichrome was observed in the isolated pellet. Furthermore, no precipitate was ever observed upon mixing the cytoplasmic domain of band 3 with oxyhemoglobin, deoxyhemoglobin,(carbonmonoxy) hemoglobin, or methemoglobin. The affinity of the cytoplasmic domain of band 3 was likely much higher for hemichromes than for native hemoglobin, since a 20-fold molar excess of hemoglobin was required to reduce copolymerization by 50%. We suggest that the copolymerization of band 3 and hemichromes in vivo can explain the aggregation of Heinz bodies on the erythrocyte membrane and the resulting hemolysis observed in numerous hemoglobinopathies. e interaction of native hemoglobin (Hb) with the human erythrocyte membrane has recently received considerable attention. Two classes of binding sites have been identified, and one of these, the higher affinity class, has been located on the cytoplasmic domain of the major erythrocyte protein, band 3 (Shaklai et al., 1977a, b; Salhany et al., 1980; Sayare f This work was supported in part by Grant GM 24417 from the National Institutes of Health.& Fikiet, 1981). Thisinteraction has been shown to be electrostatic in nature with the affinity of Hb for the mem-brane increasing as pH and ionic strength decrease (Shaklai et al., 1977a, b; Fung, 1981; Fischer et al., 1975). A similar pH dependence of Hbbinding to the membrane has also been reported for intact cells (Eisinger et al., 1982). The isolated cytoplasmic domain of band 3 has also been shown to bind two molecules of hemoglobin, supporting the identification of band 3 as a major membrane attachment site for soluble