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 similar to 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 similar to 1.4 MDa and similar to 700 kDa. Kinetic analysis demonstrates that in vitro the similar to 700-kDa complex is produced more rapidly than the similar to 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 similar to 700-kDa Apaf-1 containing apoptosome complex was predominately formed. This complex processed effector caspases. Thus, the similar to 700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis.