Rat liver mitochondria isolated in 0.25 M sucrose were osmotically lysed with distilled water. The effect of osmotic lysis on mitochondrial compartmentation was monitored by following the changes in the specific Mg++-ATPase and the stimulation of this activity by DNP. Each resuspension in distilled water caused a progressive increase in the specific Mg++-ATPase and a decrease in DNP-stimulation. Lysed mitochondria yielded P:O ratios of slightly less than 1.0 when each of the "site-specific" substrates, NADH, D-ß-hydroxybutyrate, succinate, and ascorbate, were oxidized. These data indicate that only site 3 phosphorylation remained undiminished. The crude, lysed mitochondria were subfractionated by centrifugation on linear sucrose density gradients. Assays for protein, malate dehydrogenase, D-ß-hydroxybutyrate dehydrogenase, and succinate dehydrogenase indicated that the inner compartment could be clearly separated from the outer membrane vesicles. The results also suggested that the small vesicle fraction contained a small proportion of vesiculated inner membranes. Inner mitochondrial compartments, "contracted" by preincubation in the presence of ATP, sedimented to a markedly lower density on the gradients than did the unincubated preparations and about 50% of the ghosts showed a highly condensed morphology. In the contracted preparations, relatively low malate dehydrogenase and D-ß-hydroxybutyrate dehydrogenase activities were found in the fractions comprised of the inner compartments. The specific activity and distribution of succinate dehydrogenase were about the same as were found with the unincubated, lysed mitochondria.
THE EFFECTS OF OSMOTIC LYSIS ON THE OXIDATIVE PHOSPHORYLATION AND COMPARTMENTATION OF RAT LIVER MITOCHONDRIA
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Arnold I. Caplan, John W. Greenawalt; THE EFFECTS OF OSMOTIC LYSIS ON THE OXIDATIVE PHOSPHORYLATION AND COMPARTMENTATION OF RAT LIVER MITOCHONDRIA . J Cell Biol 1 January 1968; 36 (1): 15–31. doi: https://doi.org/10.1083/jcb.36.1.15
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