Reconstitution and assay of the activity of the caspase-9±Apaf-1 holoenzyme complexes

Recombinant WT and mutant caspase-9 proteins (20 nM) were incubated in the presence or absence of recombinant Apaf-1 (20 nM) in buffer A. The reaction mixtures were stimulated with cytochrome c (5 ng ml) and dATP (1 mM) and incubated for 0±60 min at 30 8C with puri®ed procaspase-3 C163A, and the processing of procaspase-3 was analysed by western blot analysis with anti-caspase-3 antibody (Fig. 1c). The holoenzyme complexes were also reconstituted as above in caspase-9-depleted S100 extracts from Apaf1-de®cient mouse embryonic ®broblasts (Fig. 1d). The DEVD-AMC cleaving activity of the reconstituted extracts were measured over a time of 0±120 min by luminescence spectrometry and represented in arbitrary spectrometric units. In some experiments the WT and the unprocessed triple-mutant (E306/D315/330A) caspase-9 proteins (speci®c activity ,10 ̄uorogenic units per s per ng) were reconstituted with Apaf-1 and cytochrome c plus dATP in the presence of increasing amounts of XIAP, and the cleavage of S-labelled procaspase-3 by the complexes was analysed by SDS-PAGE and autoradiography (Fig. 2a).