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.
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.
Jemmerson
R
R
Department of Microbiology and Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Liu
J
J
Hausauer
D
D
Lam
K P
KP
Mondino
A
A
Nelson
R D
RD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000737:chemistry* / Q000276:immunology; D004789:Enzyme Activation; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D006825:Hybridomas; D051379:Mice; D018716:Molecular Mimicry; D009336:Necrosis; D010455:Peptides / Q000276:immunology; D010743:Phospholipids / Q000276:immunology; D011233:Precipitin Tests; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D003574:Cytochrome c Group; D010455:Peptides; D010743:Phospholipids; D020169:Caspases; D016650:Fluorescein-5-isothiocyanate