The purpose of this study was establishing the basic energetic parameters of amoeba Acanthamoeba castellanii mitochondria respiring with malate and their response to oxidative stress caused by hydrogen peroxide in the presence of Fe2+ ions. It appeared that, contrary to a previous report (Trocha LK, Stobienia O (2007) Acta Biochim Polon 54: 797), H2O2-treated mitochondria of A. castellanii did not display any substantial impairment. No marked changes in cytochrome pathway activity were found, as in the presence of an inhibitor of alternative oxidase no effects were observed on the rates of uncoupled and phosphorylating respiration and on coupling parameters. Only in the absence of the alternative oxidase inhibitor, non-phosphorylating respiration progressively decreased with increasing concentration of H2O2, while the coupling parameters (respiratory control ratio and ADP/O ratio) slightly improved, which may indicate some inactivation of the alternative oxidase. Moreover, our results show no change in membrane potential, Ca2+ uptake and accumulation ability, mitochondrial outer membrane integrity and cytochrome c release for 0.5-25 mM H2O2-treated versus control (H2O2-untreated) mitochondria. These results indicate that short (5 min) incubation of A. castellanii mitochondria with H2O2 in the presence of Fe2+ does not damage their basic energetics.
Mitochondria of the amoeba Acanthamoeba castellanii possess a free fatty acid-activated uncoupling protein (AcUCP) that mediates proton re-uptake driven by the mitochondrial proton electrochemical gradient. We show that AcUCP activity diverts energy from ATP synthesis during state 3 mitochondrial respiration in a fatty acid-dependent way. The efficiency of AcUCP in mitochondrial uncoupling increases when the state 3 respiratory rate decreases as the AcUCP contribution is constant at a given linoleic acid concentration while the ATP synthase contribution decreases with respiratory rate. Respiration sustained by this energy-dissipating process remains constant at a given linoleic acid concentration until more than 60% inhibition of state 3 respiration by n-butyl malonate is achieved. The present study supports the validity of the ADP/O method to determine the actual contributions of AcUCP (activated with various linoleic acid concentrations) and ATP synthase in state 3 respiration of A. castellanii mitochondria fully depleted of free fatty acid-activated and describes how the two contributions vary when the rate of succinate dehydrogenase is decreased by succinate uptake limitation.
The purpose of this study was to examine the effects of oxidative stress caused by hydroperoxide (H2O2) in the presence of iron ions (Fe2+) on mitochondria of the amoeba Acanthamoeba castellanii. We used isolated mitochondria of A. castellanii and exposed them to four levels of H2O2 concentration: 0.5, 5, 15, and 25 mM. We measured basic energetics of mitochondria: oxygen consumption in phosphorylation state (state 3) and resting state (state 4), respiratory coefficient rates (RC), ADP/O ratios, membrane potential (ΔΨm), ability to accumulate Ca2+ , and cytochrome c release. Our results show that the increasing concentrations of H2O2 stimulates respiration in states 3 and 4. The highest concentration of H2O2 caused a 3-fold increase in respiration in state 3 compared to the control. Respiratory coefficients and ADP/O ratios decreased with increasing stress conditions. Membrane potential significantly collapsed with increasing hydroperoxide concentration. The ability to accumulate Ca2+ also decreased with the increasing stress treatment. The lowest stress treatment (0.5 mM H2O2) significantly decreased oxygen consumption in state 3 and 4, RC, and membrane potential. The ADP/O ratio decreased significantly under 5 mM H2O2 treatment, while Ca2+ accumulation rate decreased significantly at 15 mM H2O2. We also observed cytochrome c release under increasing stress conditions. However, this release was not linear. These results indicate that as low as 0.5 mM H2O2 with Fe2+ damage the basic energetics of mitochondria of the unicellular eukaryotic organism Acanthamoeba castellanii.
Mitochondria of amoeba Acanthamoeba castellanii in addition to the conventional cytochrome pathway possess, like plant mitochondria, a cyanide-resistant alternative quinol oxidase. In mitochondria isolated from amoeba batch culture grown temporarily at low temperature (6°C), higher respiration was accompanied by lower coupling parameters as compared to control culture (grown at 28°C). In the presence of benzohydroxamate, respiratory rates and coupling parameters were similar in both types of mitochondria indicating that growth in cold conditions did not disturb the cytochrome pathway. Increased contribution of alternative oxidase in total mitochondrial respiration in low-temperature-grown amoeba cells was confirmed by calculation of its contribution using ADP/O measurements. Furthermore, in mitochondria from low-temperature- grown cells the content of the alternative oxidase was increased and correlated with the increase in the unstimulated and GMP-stimulated cyanide-resistant respiratory activity. A possible physiological role of higher activity of alternative oxidase as response to growth at a low temperature in unicellular organisms, such as amoeba, is discussed.
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