Figure 5.

Proteolytic activity of caspase-2 is dispensable for its ability to engage mitochondria. (A) Enzyme activity of wild-type caspase-2 (200 ng), either untreated (Casp-2), treated with 10 mM IAA (+IAA) for 60 min at 37°C or treated with z-VDVAD-fmk (250 μM), as monitored by the release of AMC from VDVAD-AMC. (B) Wild-type Jurkat cells (10⁶) were permeabilized with digitonin and incubated in the absence or presence of 100 ng of wild-type caspase-2 (Casp-2), inactivated processed caspase-2 or IAA-based inactivation buffer alone for 10 min at 22°C. (C) Isolated liver mitochondria (0.5 mg of protein) were incubated under the same conditions as described for (B) except that digitonin was omitted and the amount of recombinant caspase-2 was 150 ng. For (B,C), supernatant and pellet fractions were separated by SDS–PAGE and western blotted. (D) RCR measurement of mitochondrial suspensions incubated under the same conditions as described for (C).