hexacyanoferrate-iii and malic-acid

Phosphorylation in isolated Ascaris suum mitochondria was much greater in the presence of malate than succinate, but, in the absence of added adenine nucleotides, incubations in succinate resulted in substantial elevations in intramitochondrial ATP levels. Succinate-dependent phosphorylation was stimulated aerobically and this stimulation was due almost entirely to a site I, rotenone-sensitive, phosphorylation. Increased substrate level phosphorylation, coupled to propionate formation, or additional sites of electron-transport associated ATP synthesis were not significant. Under aerobic conditions, 14CO2 evolution from 1,4-[14C]succinate was stimulated and NADH/NAD+ ratios were elevated, but the formation of [14C]propionate was unchanged. It appears that succinate was metabolized to pyruvate and acetate, and NADH, generated from the decarboxylations of malate and pyruvate, was the primary source of reducing power fueling electron-transport. The terminal oxidase and final electron-acceptor are still not clearly defined. However, ferricyanide, H2O2, and 100% oxygen all stimulated succinate-dependent phosphorylation. A possible role for cytochrome c peroxidase in A. suum mitochondrial metabolism is discussed.

Topics: Adenine Nucleotides; Animals; Ascaris; Energy Metabolism; Ferricyanides; Hydrogen Peroxide; Malates; Mitochondria; Models, Biological; Phosphorylation; Succinates; Succinic Acid

P/2e- stoichiometries in six assay systems spanning different portions of the respiratory chain were estimated by direct determinations of Pi uptake in suspensions of bovine heart mitochondria containing a hexokinase trap. The electron donors were malate + pyruvate, succinate, and ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine, and the electron acceptors were ferricyanide (Site 1, Site 2, and Sites 1 + 2) and O2 (Sites 1 + 2 + 3, Sites 2 + 3, and Site 3). A major objective was to find conditions in which the six systems yield results in sufficiently good agreement to allow confidence as to their reliability. This objective was achieved, and maximum values of 1.1, 0.5, and 1.0 were observed in the Sites 1, 2, and 3 systems, respectively. This required that the energy-conserving reactions be relatively nonlimiting and that the P/2e- ratios be estimated from the slopes of plots of respiration rate versus phosphorylation rate obtained by inhibiting oxidative phosphorylation with respiratory chain inhibitors. The latter requirement allows avoidance of the effect of an apparent endogenous uncoupler and is based on the observation (Tsou, C. S., and Van Dam, K. (1969) Biochim. Biophys. Acta 172, 174-176) that uncoupling agents at low concentrations decrease the rate of phosphorylation nearly as much in absolute amount at low rates of respiration as at high rates. The maximum P/2e- stoichiometry at Site 1 is considered to be 1.0, and the value observed in the Site 1 system is suggested to be higher as a result of H+ ejection at the transhydrogenase level. Respiratory control due to carboxyatractyloside inhibition was examined and found to differ greatly among the systems. It is pointed out that this observation is not consistent with the lack of complete control being due primarily to ion cycling and that, in view of this, the relatively meager control at Site 3 is not consistent with O2 being reduced on the matrix side of the coupling membrane.

Topics: Animals; Ascorbic Acid; Atractyloside; Cattle; Electron Transport; Ferricyanides; Malates; Mitochondria, Heart; Oxidative Phosphorylation; Oxygen Consumption; Phosphates; Pyruvates; Pyruvic Acid; Succinates; Succinic Acid; Tetramethylphenylenediamine; Uncoupling Agents