A conformational change in cytochrome c of apoptotic and necrotic cells isdetected by monoclonal antibody binding and mimicked by association of thenative antigen with synthetic phospholipid vesicles

By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal a ntibody (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 ea rly 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 c yt c extracted with detergent from dead cells, it appears to recognize cyt c bound in a detergent-sensitive complex to other cellular components. Coin cidentally, the mAb was also shown by competitive enzyme-linked immunosorbe nt assay to bind cyt c associated with synthetic phosphatidic acid vesicles . This suggests that the conformational change of cyt c in dying cells coul d be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent confocal microscopy, conformat ionally altered cyt c in post-apoptotic T hybridoma cells showed a punctate distribution, indicating that it remained associated with mitochondria. Fu rthermore, the heavy membrane fraction of post-apoptotic cells but not of l ive cells was functional in caspase activation. This suggests that membrane -bound cyt c is the relevant caspase coactivation factor in the T hybridoma cells.