A Conformational Change in Cytochrome<i>c</i>of Apoptotic and Necrotic Cells Is Detected by Monoclonal Antibody Binding and Mimicked by Association of the Native Antigen with Synthetic Phospholipid Vesicles

Abstract

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 cbound 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.