Abstract
We report here the reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1.cytochrome c complex. Such a complex can be isolated using gel filtration chromatography and is by itself sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1 to 1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphate / metabolism
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Alternative Splicing
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Amino Acid Sequence
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Apoptosis / physiology*
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Apoptotic Protease-Activating Factor 1
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Base Sequence
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Caspase 9
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Caspases / metabolism*
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Cytochrome c Group / physiology*
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DNA Primers
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Enzyme Activation
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Enzyme Precursors / metabolism*
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HeLa Cells
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Humans
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Hydrolysis
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Molecular Sequence Data
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Organelles / physiology*
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Protein Binding
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Proteins / physiology*
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Recombinant Proteins / metabolism
Substances
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APAF1 protein, human
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Apoptotic Protease-Activating Factor 1
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Cytochrome c Group
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DNA Primers
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Enzyme Precursors
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Proteins
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Recombinant Proteins
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Adenosine Triphosphate
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CASP9 protein, human
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Caspase 9
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Caspases