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Mitochondrial water spaces were determined by centrifugal filtration, by using 3H2O and [14C]-sucrose, -mannitol, -inulin and -dextran. The volume (in microliter/mg of mitochondrial protein) of each of the spaces was inversely proportional to the amount of mitochondria (mg of protein) centrifuged. The dextran space (representing extramitochondrial water carried down with the mitochondria) decreased the most, and accounted for most of the changes observed in the other spaces. However, the calculated matrix and intermembrane spaces also decreased when increasing amounts of mitochondria were centrifuged. For each space, the same value was obtained when centrifugal filtration was done at 8000 and at 15,600 g, and when the mitochondria were incubated with the markers for 15 s to 5 min, indicating that sucrose, mannitol and inulin do not penetrate the matrix, nor does dextran penetrate the intermembrane space, under the incubation and centrifugation conditions generally used to measure mitochondrial spaces.

Measurements of mitochondrial volumes are affected by the amount of mitochondria used in the determinations