Abstract
Apoptosis in metazoans is executed by a group of intracellular proteases named caspases. One of the caspase-activating pathways in mammals is initiated by the release of cytochrome c from mitochondria to cytosol, where it binds to Apaf-1 to form a procaspase-9-activating heptameric protein complex named apoptosome. We report here the reconstitution of this pathway with purified recombinant Apaf-1, procaspase-9, procaspase-3, and cytochrome c from horse heart. Apaf-1 contains a dATP as a cofactor. Cytochrome c binding to Apaf-1 induces hydrolysis of dATP to dADP, which is subsequently replaced by exogenous dATP. The dATP hydrolysis and exchange on Apaf-1 are two required steps for apoptosome formation.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Apoptosis*
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Apoptotic Protease-Activating Factor 1
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Caspase 3
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Caspases / metabolism
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Cytochromes c / metabolism*
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Deoxyadenine Nucleotides / metabolism*
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Enzyme Activation
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Horses
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Hydrolysis
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Intracellular Membranes / metabolism*
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism*
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Organelles / metabolism*
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Proteins / genetics
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Proteins / metabolism*
Substances
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Apoptotic Protease-Activating Factor 1
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Deoxyadenine Nucleotides
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Intracellular Signaling Peptides and Proteins
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Proteins
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Cytochromes c
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Caspase 3
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Caspases
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2'-deoxyadenosine triphosphate