Fig. 7. P49 is a substrate inhibitor of human caspase-9. (A) Dose-dependent inhibition. Purified recombinant caspase-9 (40 pmol) was incubated with the indicated amounts (pmol) of purified P49-His₆, D94A-mutated P49-His₆, P35-His₆ or D87A-mutated P49-His₆. After 30 min, residual caspase activity was measured by using the substrate LEHD-afc as described in the legend to Figure 2. (B) P49 cleavage. In vitro synthesized, ³⁵S-radiolabeled, wild-type (wt) or D94A-mutated P49 was incubated with buffer alone (lanes 1 and 2), caspase-9 (lanes 3 and 4) or human caspase-3 (lanes 5 and 6) and analyzed by SDS–PAGE. The 40 kDa (*) and 9 kDa (*′) cleavage fragments are indicated. (C) Inhibition of procaspase-3 processing. Purified caspase-9 was mixed with buffer alone (–), wild-type (wt) or D94A-mutated P49-His₆ (lanes 3–11), or wild-type or D87A-mutated P49-His₆ (lanes 12–19). After 30 min, in vitro synthesized, ³⁵S-labeled, procaspase-3 was added, incubated for 6 h and analyzed by SDS–PAGE. Input procaspase-3 is shown (lane 1). Caspase-3 p19 and p12 subunits are indicated (top).