Biochemical and functional differences in rat liver mitochondrial subpopulations obtained at different gravitational...

Previous studies have reported that liver mitochondria may be fractionated into different subpopulations. However, no careful studies have been performed to exclude mitochondrial damage and to investigate more thoroughly the possible biochemical differences existing between the subpopulations. In this study, we analysed the integrity and the biochemical properties of rat liver mitochondria. Mitochondrial fractions were obtained by differential centrifugation at different gravitational forces: 1000 g (M1 fraction), 3000 g (M3 fraction) and 10,000 g (M10 fraction). The integrity of these organelles was checked by measuring citrate synthase activity both in the presence and absence of Triton X-100 detergent. Biochemical analyses included polarographic determination of cytochrome oxidase activity and respiratory parameters and spectrophotometric determination of cytochrome content. (1) The integrity of mitochondria was almost homogeneous between fractions (88.5, 80 and 78.3% in M1, M3 and M10 fractions, respectively). (2) The heaviest M1 fraction contains mitochondria which are on average twice as large as M3 and about three times as large as M10. (3) The M1 fraction exhibited the highest specific cytochrome oxidase activity (1040 +/- 20 n Atoms O/min x mg protein) and the highest respiratory rates (72 +/- 3 n Atoms O/min x mg protein and 526 +/- 45 n Atoms O/min x mg protein for States 4 and 3, respectively). Oxidative capacity and respiratory rates decreased as the size of the organelles decreased, reaching values of 1/5 and 1/14 in the M3 and M10 fractions as compared to the M1. (4) These changes are accompanied by a change in the respiratory control ratio (RCR), which varies from 7.3 in M1 to about 2.0 in M10. A similar trend was observed in cytochrome contents but the differences were not as great as cytochrome oxidase activity and State 3 respiration. These results, as a whole, show that a mitochondrial heterogeneity exists in rat liver cell. We suggest that the above-mentioned differences might represent steps of mitochondrial maturation. The maturation would be fundamentally based on the increase of efficiency of the mechanism for ATP synthesis.