Interestingly, addition of free cytochrome c to extracts containing mitochondria also greatly accelerates apoptosis, consistent with the idea that release of cytochrome c from mitochondria is a rate limiting step in this process. Taken together these data suggest that Reaper might induce downstream release of cytochrome c from mitochondria, thereby triggering caspase activation. To determine whether Reaper can indeed induce mitochondrial cytochrome c release, Reaper was incubated in unfractionated Xenopus egg extracts. Aliquots were assayed at various times for cytochrome c release and caspase activity. Although the exact timing of apoptotic events varies between extracts, Reaper accelerates both the release of cytochrome c from mitochondria and the consequent activation of DEVD-cleaving caspases.
Many questions remain to be resolved.