Does cat B induce release of cytochrome c from mitochondria?
The preceding results place cat B upstream of mitochondria in the TNF-α/AcD–induced apoptotic cascade in mouse hepatocytes. Although more complicated models are possible, the most direct model would have cat B or a substrate of cat B inducing cytochrome c release from mitochondria. To assess this possibility, purified cat B was incubated with isolated mitochondria in the presence and absence of cytosol. At the end of the incubation, the mitochondria were sedimented and the resulting supernatants analyzed for cytochrome c. In the absence of cytosol, active cat B directly induced a moderate release of cytochrome c from mitochondria (Figure 7a, compare lanes 1–3 with lane 7) corresponding to about 8% of the maximum release as obtained treating the mitochondria with the detergent Triton X-100 (Figure 7a, lane 9). However, cytochrome c–releasing activity of cat B was fivefold greater in the presence of cytosol (Figure 7a, lanes 4–6). Addition of calpain to the cell-free system did not induc
