Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c.
A cell-free system based on cytosols of normally growing cells is established that reproduces aspects of the apoptotic program in vitro. The apoptotic program is initiated by addition of dATP. Fractionation of cytosol yielded a 15 kDa protein that is required for in vitro apoptosis. The absorption spectrum and protein sequence revealed that this protein is cytochrome c. Elimination of cytochrome c from cytosol by immunodepletion, or inclusion of sucrose to stabilize mitochondria during cytosol preparation, diminished the apoptotic activity. Adding back cytochrome c to the cytochrome c-depleted extracts restored their apoptotic activity. Cells undergoing apoptosis in vivo showed increased release of cytochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.
Liu
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X
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Kim
C N
CN
Yang
J
J
Jemmerson
R
R
Wang
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X
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases; D002457:Cell Extracts / Q000737:chemistry / Q000494:pharmacology*; D002474:Cell-Free System; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003574:Cytochrome c Group / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D006367:HeLa Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D003574:Cytochrome c Group; D004792:Enzyme Precursors; D000255:Adenosine Triphosphate; D004247:DNA; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases