In vitro model for regulation of apoptosis and its study

摘要

(57) [Summary]A cell-free system using the cytosol of normally growing cells is provided that reproduces the measurable characteristics of an apoptosis program. The apoptosis program is started by adding dATP, as a specific example, to 100,000 G supernatant of HeLa cells. A 15 kDa protein was obtained by fractionation of this cytosol, and it was identified as cytochrome c by determination of an absorption spectrum and an amino acid sequence. Cytochrome c is required for apoptosis in vitro. Apoptotic activity was lost by immunological removal of cytochrome c from the cytosol or by the addition of sucrose to stabilize mitochondria in the preparation of the cytosol. Addition of exogenous cytochrome c to the extract from which cytochrome c had been removed restored the apoptotic activity. Cells that undergo apoptosis in vivo have increased cytochrome c release into the cytosol, indicating that mitochondria are involved in apoptosis by releasing cytochrome c.