title: Preparation of cytosolic extracts and activation of caspases by cytochrome c.
abstract: It can be useful to explore the caspase activation process in an in vitro setting. In this protocol, cytosolic extracts prepared from cell culture are incubated with cytochrome c and adenosine triphosphate (dATP), leading to the oligomerization of apoptotic protease activating factor-1 (APAF-1) and the formation of the apoptosome. The apoptosome serves as an activation platform for caspase-9, which binds to the apoptosome through heterodimeric caspase recruitment domain (CARD) interactions and then dimerizes. This leads to cleavage of the executioner, caspase-3. These extracts contain highly active caspases that can be analyzed using a variety of biochemical assays.
authors.affiliation: Department of Life Sciences, New York Institute of Technology, Old Westbury, New York 11568;
authors.forename: Gavin P
authors.initials: GP
authors.lastname: McStay
authors.affiliation: Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Green
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism / Q000494:pharmacology*; D003600:Cytosol / Q000737:chemistry*; D004789:Enzyme Activation / Q000187:drug effects
chemical_list: D000255:Adenosine Triphosphate; D045304:Cytochromes c; D020169:Caspases