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 orN-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.