Kannan R, Labotka R, Low P S
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 1988 Sep 25;263(27):13766-73.
Because the interaction of denatured hemoglobins (i.e. hemichromes) with the red cell membrane has been associated with several abnormalities commonly observed in hemichrome-containing erythrocytes, we have undertaken to isolate and characterize the hemichrome-rich membrane protein aggregates from sickle cells. The aggregates were isolated by two procedures: one at low ionic strength by centrifugation of detergent-solubilized spectrin-depleted inside-out vesicles, and the other at physiological ionic strength by detergent solubilization of whole cells followed by cytoskeletal disruption and centrifugation. The extensively washed aggregates obtained by both methods yielded similar results. These insoluble complexes were found to be highly cross-linked by predominantly intermolecular disulfide bonds; however, other nonreducible covalent linkages were also observed. Both in the presence and absence of reducing agents, the aggregate disintegrated when the hemichromes were removed by high ionic strength, suggesting that the aggregate depended heavily on the cohesive properties of the hemichromes for stability. Protein assays demonstrated that the aggregates comprised approximately 1.3% of the total membrane protein, roughly two-thirds of which appeared to be globin chains. Other major components identified in the aggregate were band 3, ankyrin, bands 4.1, 4.9, and 5, glycophorins A and B, and autologous IgG. Quantitative analysis of the IgG content demonstrated that three-fourths of the surface-bound IgG on washed sickle cells was clustered at these aggregate sites, representing an enrichment of approximately 250-fold over nonaggregated regions of the membrane. Since clustered cell surface IgG is thought to trigger removal of erythrocytes from circulation, the hemichrome-induced membrane reorganization at these aggregate sites may be an important cause of the greatly shortened life span of sickle cells.
由于变性血红蛋白（即高铁血红蛋白）与红细胞膜的相互作用与含高铁血红蛋白的红细胞中常见的几种异常情况有关，我们着手从镰状细胞中分离并鉴定富含高铁血红蛋白的膜蛋白聚集体。通过两种方法分离聚集体：一种是在低离子强度下，对用去污剂溶解并去除血影蛋白的内向外囊泡进行离心；另一种是在生理离子强度下，先用去污剂溶解全细胞，然后破坏细胞骨架并进行离心。通过这两种方法获得的经过充分洗涤的聚集体产生了相似的结果。发现这些不溶性复合物主要通过分子间二硫键高度交联；然而，也观察到了其他不可还原的共价键。无论是否存在还原剂，当通过高离子强度去除高铁血红蛋白时，聚集体都会解体，这表明聚集体的稳定性在很大程度上依赖于高铁血红蛋白的内聚特性。蛋白质分析表明，聚集体约占总膜蛋白的1.3%，其中约三分之二似乎是球蛋白链。在聚集体中鉴定出的其他主要成分是带3、锚蛋白、带4.1、4.9和5、血型糖蛋白A和B以及自身IgG。对IgG含量的定量分析表明，洗涤后的镰状细胞表面结合的IgG中有四分之三聚集在这些聚集体部位，比膜的非聚集区域富集了约250倍。由于聚集在细胞表面的IgG被认为会触发红细胞从循环中清除，因此这些聚集体部位由高铁血红蛋白诱导的膜重组可能是镰状细胞寿命大大缩短的一个重要原因。
Zotero 插件