Abnormal redox status of membrane-protein thiols in sickle erythrocytes.
Abnormal redox status of membrane-protein thiols in sickle erythrocytes.
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镰状红细胞膜蛋白硫醇的氧化还原状态异常。
DOI：
10.1172/jci111857
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Hebbel,RP
中科院分区：
文献类型：
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作者：
Rank,BH;Carlsson,J;Hebbel,RP
虽然镰状红细胞（RBC）经历过度的自氧化，但研究人员尚未发现镰状红细胞膜中蛋白质硫醇异常氧化的证据（例如，由分子间二硫键连接的蛋白质聚集体）。然而，迄今为止使用的传统技术无法检测到硫醇状态的更细微的变化，例如异常的分子内二硫键。我们使用硫醇-二硫交换色谱法检测红细胞膜，该色谱法在活性硫醇的基础上划分十二烷基硫酸钠溶解蛋白，产生凝胶结合（还原硫醇）和滤液（氧化/堵塞硫醇）馏分。来自正常红细胞的膜分裂，因此只有13.6 +/- 1.4%的膜蛋白存在于滤液中。镰状红细胞的膜蛋白异常增加（21.5 +/- 4.3%）进入滤液部分（P < 0.001）。由于镰状红细胞没有高分子量的膜蛋白聚集，这表明镰状红细胞膜中存在异常的分子内硫醇氧化。用巯基阻滞剂和氧化剂处理正常红细胞，模拟了膜蛋白进入滤液部分的这种转移。聚丙烯酰胺凝胶电泳分析显示，正常红细胞的滤液主要由带7和糖蛋白组成，只有微量的其他膜蛋白。在正常背景下，镰状红细胞滤液富含所有蛋白质（包括细胞骨架蛋白带1、2、2.1和4.1），提示镰状红细胞膜普遍氧化摄动。这些观察结果支持了过度红细胞自氧化可能在镰状病病理生理中起作用的概念，并且它们可能有助于解释那些可能反映镰状红细胞细胞骨骼功能障碍的膜异常的发展。图片
Although sickle erythrocytes (RBC) undergo excessive autooxidation, investigators have not found evidence for abnormal oxidation of protein thiols in sickle RBC membranes (e.g., protein aggregates linked by intermolecular disulfide bonds). However, the conventional techniques heretofore used cannot detect more subtle changes in thiol status such as abnormal intramolecular disulfide bonds. We examined RBC membranes using thiol-disulfide exchange chromatography which partitions sodium dodecyl sulfate-solubilized proteins on the basis of reactive thiols, yielding gel-bound (reduced-thiol) and filtrate (oxidized/blocked-thiol) fractions. Membranes from normal RBC partition so that only 13.6 +/- 1.4% of all membrane protein is found in the filtrate fraction. An abnormally increased amount of membrane protein from sickle RBC (21.5 +/- 4.3%) partitions into the filtrate fraction (P less than 0.001). Since sickle RBC do not have high molecular weight aggregates of membrane protein, this indicates abnormal intramolecular thiol oxidation in sickle RBC membranes. Treatment of normal RBC with thiol blockers and oxidants simulates this shift of membrane protein into the filtrate fraction. Analysis using polyacrylamide gel electrophoresis reveals that the filtrate fraction derived from normal RBC consists mostly of band 7 and glycophorins, with only trace amounts of other membrane proteins. Superimposed upon this normal background, sickle RBC filtrates are enriched with all proteins (including cytoskeletal protein bands 1, 2, 2.1, and 4.1), suggesting a generalized oxidative perturbation of sickle RBC membranes. These observations support the concept that excessive RBC autooxidation may play a role in sickle disease pathophysiology, and they perhaps help explain the development of those membrane abnormalities that may reflect cytoskeletal dysfunction in sickle erythrocytes.Images