Apaf-1 oligomerizes into biologically active ~700-kDa and inactive ~1.4-MDa apoptosome complexes

Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large in the order of ~1.4 MDa. We now demonstrate that dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M(r) values of ~1.4 MDa and ~700 kDa. Kinetic analysis demonstrates that in vitro the ~700-kDa complex is produced more rapidly than the ~1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide or N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the ~700-kDa Apaf-1 containing apoptosome complex was predominately formed. This complex processed effector caspases. Thus, the ~700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis.