A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles
A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles
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DOI：
10.1021/bi9809268
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发表时间：
1999-03-23
期刊：
影响因子：
2.9
通讯作者：
Nelson, RD
中科院分区：
文献类型：
--
作者：
Jemmerson, R;Liu, J;Nelson, RD
通过流式细胞术，使用单克隆抗体（mAb）检测小鼠细胞色素c （cyt c）在凋亡和坏死T杂交瘤细胞中的构象变化，该单克隆抗体识别非天然形式的蛋白质氨基酸残基44周围的区域。cyt c的构象改变是凋亡的早期事件，可以在其他凋亡指标阴性的凋亡前细胞中发现。由于mAb不能结合固定和渗透活细胞，也不能免疫沉淀从死细胞中提取的可溶性cyt c，它似乎可以识别与其他细胞成分结合在洗涤剂敏感复合物中的cyt c。巧合的是，竞争性酶联免疫吸附试验也显示单抗可以结合与合成磷脂酸囊泡相关的cyt - c。这表明，死亡细胞中cyt - c的构象变化可能是由于它与细胞死亡时改变的细胞膜有关。免疫荧光共聚焦显微镜观察，凋亡后T杂交瘤细胞构象改变的cyt c呈点状分布，表明其仍与线粒体有关。此外，凋亡后细胞的重膜部分而不是活细胞的重膜部分在caspase活化中起作用。这表明膜结合细胞c是T杂交瘤细胞中相关的caspase共激活因子。
By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody (mAb) that recognizes the region around amino acid residue 44 on a non-native form of the protein. The conformational change in cyt c is an early event in apoptosis, which can be identified in pre-apoptotic cells that are negative for other indicators of apoptosis. Since the mAb did not bind fixed and permeabilized live cells and did not immunoprecipitate soluble cyt c extracted with detergent from dead cells, it appears to recognize cyt c bound in a detergent-sensitive complex to other cellular components. Coincidentally, the mAb was also shown by competitive enzyme-linked immunosorbent assay to bind cyt c associated with synthetic phosphatidic acid vesicles. This suggests that the conformational change of cyt c in dying cells could be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent confocal microscopy, conformationally altered cyt c in post-apoptotic T hybridoma cells showed a punctate distribution, indicating that it remained associated with mitochondria. Furthermore, the heavy membrane fraction of post-apoptotic cells but not of live cells was functional in caspase activation. This suggests that membrane-bound cyt c is the relevant caspase coactivation factor in the T hybridoma cells.