FIGURE 4: An incompletely released, MAMs-associated, pool of cytochrome c is decreased in κmdm34 cells. (A) The fraction of NADH-reducible cytochrome c was measured by measuring reduction after adding 1 mM NADH, followed by measuring the reduction after adding dithionite on the same sample. Typically, two redox spectra with NADH and two redox spectra with dithionite were acquired sequentially, to ensure that redox ratios did not change during the time of the experiment. Each point is a different mitochondria preparation (**: significantly different p<0.01). (B) The ratio (reduction by NADH/reduction by dithionite) was measured for cytochrome c and for cytochrome b. The graph shows the ratio (% reduction cytochrome c) / (% reduction of cytochrome b). The values were corrected to remove the contribution of cytochrome c1, considering that the molecular ratio cyt.b/cyt.c1 is 2 and that the reduction of cytochrome c1 was proportionnal to that of cytochrome b (as they belong to the same respiratory complex). Each point is a different mitochondria preparation. (*: p<0.05; **: p<0.01). (C) Mitochondria were fractionated on a sucrose density gradient. A small pool of cytochrome c was found at a lower density than that of the main fraction, and may represent partly released cytochrome c, not reducible by NADH in experiments (A) and (B). The figure is representative of 3 independent experiments.