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

Ronald Jemmerson, Jiang Liu, Danielle Hausauer, Kong Peng Lam, Anna Mondino, R. David Nelson

Research output: Contribution to journalArticle

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 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.

Original languageEnglish
Pages (from-to)3599-3609
Number of pages11
JournalBiochemistry
Volume38
Issue number12
DOIs
Publication statusPublished - Mar 23 1999

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Synthetic Vaccines
Cytochromes c
Phospholipids
Monoclonal Antibodies
Association reactions
Antigens
Hybridomas
Caspases
Membranes
Detergents
Cells
Apoptosis
Immunosorbents
Phosphatidic Acids
Mitochondria
Flow cytometry
Confocal microscopy
Intracellular Membranes
Cell death
Confocal Microscopy

ASJC Scopus subject areas

  • Biochemistry

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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. / Jemmerson, Ronald; Liu, Jiang; Hausauer, Danielle; Lam, Kong Peng; Mondino, Anna; Nelson, R. David.

In: Biochemistry, Vol. 38, No. 12, 23.03.1999, p. 3599-3609.

Research output: Contribution to journalArticle

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