We studied FFA (free fatty acid)-induced uncoupling activity in Acanthamoeba castellanii mitochondria in the non-phosphorylating state. Either succinate or external NADH was used as a respiratory substrate to determine the proton conductance curves and the relationships between respiratory rate and the quinone reduction level. Our determinations of the membranous quinone reduction level in non-phosphorylating mitochondria show that activation of UCP (uncoupling protein) activity leads to a PN (purine nucleotide)-sensitive decrease in the quinone redox state. The gradual decrease in the rate of quinone-reducing pathways (using titration of dehydrogenase activities) progressively leads to a full inhibitory effect of GDP on LA (linoleic acid) induced proton conductance. This inhibition cannot be attributed to changes in the membrane potential. Indeed, the lack of GDP inhibitory effect observed when the decrease in respiratory rate is accompanied by an increase in the quinone reduction level (using titration of the quinol-oxidizing pathway) proves that the inhibition by nucleotides can be revealed only for a low quinone redox state. It must be underlined that, in A. castellanii non-phosphorylating mitochondria, the transition of the inhibitory effect of GDP on LA-induced UCP-mediated uncoupling is observed for the same range of quinone reduction levels (between 50% and 40%) as that observed previously for phosphorylating conditions. This observation, drawn from the two different metabolic states of mitochondria, indicates that quinone could affect UCP activity through sensitivity to PNs.
- Acanthamoeba castellanii
- uncoupling protein
- quinone redox state
Acanthamoeba castellanii is a free-living amoeba found in soil, marine and freshwater environments, and is an important predator of bacteria. A. castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct human diseases. Under axenic non-pathogenic conditions, A. castellanii has been used frequently as a model organism to study mitochondrial energy-dissipating systems, such as a cyanide-resistant alternative oxidase [1,2], an ATP-sensitive potassium channel , and a UCP (uncoupling protein), AcUCP (A. castellanii mitochondrial UCP) [4–8].
UCPs catalyse a proton conductance that dissipates a ΔμH+ (proton electrochemical gradient) built up by the mitochondrial respiratory chain in animals, plants and some fungal and protist mitochondria (for reviews, see [9–15]). UCPs have been proposed to fulfil a physiological function through a ΔμH+ dissipation by a FFA (free fatty acid)-activated, PN (purine nucleotide)-inhibited H+ cycling process driven by the ΔΨ (mitochondrial membrane electrical potential) and pH (both constituting ΔμH+). In unicellular organisms, as well as in non-thermogenic plant and animal tissues, the physiological role of this energy-dissipating system has not yet been established. Recent discussions imply that UCPs (with the exception of thermogenic UCP1 of mammalian brown adipose tissue) may play a central role in the limitation of the production of mitochondrial reactive oxygen species and in the maintenance of the cell energy metabolism balance related to the regulation of ATP production as well as control of the NADH/NAD+ ratio [9–15].
In mitochondria of the amoeboid protozoan A. castellanii, the action of AcUCP has been shown to mediate FFA-activated, PN-inhibited H+ re-uptake driven by ΔμH+, which in state 3 respiration can divert energy from oxidative phosphorylation . The fatty acid efficiency profile in uncoupling of A. castellanii mitochondria has been described . It has also been shown that a cold treatment of amoeba culture increases AcUCP activity and protein level, indicating that UCP could be a cold-response protein in unicellular organisms . Moreover, we have shown that UCPs may play a role in decreasing reactive oxygen species production in unicellular organisms such as the amoeba A. castellanii .
As UCPs are specialized proteins for ΔμH+ dissipation, their activity must be finely regulated. For a long time, an inhibition of H+ conductance by PN has been considered as a diagnostic of UCP activity. However, for UCP1 homologues, conflicting results have been obtained with isolated respiring mitochondria, where the FFA-induced H+ conductance has been shown to be differently sensitive to PN under non-phosphorylating conditions. Insensitivity to PN has been observed for UCPs of amoeboid eukaryotes (A. castellanii and Dictyostelium discoideum), plant UCPs and UCP2 (a UCP ubiquitous in mammalian tissues), as well as UCP3 (a UCP predominantly specific for skeletal muscle) in mitochondria respiring in non-phosphorylating conditions in the absence of superoxide [4,16–21]. On the other hand, under these conditions inhibition by PN has been observed for UCP in isolated mitochondria of Candida parapsilosis  and potato tuber . In the case of mammalian UCPs, quinone has been shown to be an obligatory cofactor for their action [24,25]. Furthermore, during non-phosphorylating respiration, stimulation by superoxide has been shown to be necessary to reveal sensitivity to PN in isolated kidney and skeletal muscle mitochondria [21,26–28] as well as in plant mitochondria . However, in the reconstituted system with heterologously expressed mammalian UCPs, no superoxide activation has been required to demonstrate the FFA-activated PN-sensitive H+ translocation and quinone has no significant activating effect nor any effect on the inhibition by PN [29,30]. Moreover, taking into account the apparent affinity of reconstituted UCPs for PNs [25,30] and the concentration of nucleotides in vivo (2–15 mM, depending on material), UCPs should be almost fully inhibited under in vivo conditions, even in the presence of FFA, unless a regulatory factor or mechanism could modulate (i.e. lower) the inhibition by PN . Prevously, it has been proposed that the membranous quinone redox state could be a metabolic sensor that modulates the PN inhibition of FFA-activated UCPs as observed in isolated skeletal muscle, potato tuber and A. castellanii mitochondria respiring in phosphorylating conditions in the absence of endogenous superoxide production [7,18,31]. Therefore, the question arises as to whether the same regulation takes place in a quite different mitochondrial metabolic state, i.e. under non-phosphorylating conditions, thereby at higher ΔΨ and redox state of quinone (and other respiratory chain components) compared with phosphorylating ones.
The aim of the present study was to examine for the first time the influence of the endogenous quinone redox state on PN inhibition of the FFA-induced UCP activity in non-phosphorylating mitochondria. This study may shed light on differential results obtained so far for isolated mitochondria from various materials under non-phosphorylating conditions.
Cell culture and isolation of mitochondria
The soil amoeba A. castellanii, avirulent strain Neff, was cultured as described by Jarmuszkiewicz et al. . Trophozoites of the amoeba were collected between 44 and 48 h following inoculation at the middle exponential phase (at a density of about 4–5×106 cells/ml). Mitochondria were isolated and purified on a self-generating Percoll gradient (30%) as described previously . The presence of 0.4% BSA in isolation media allowed FFA to be chelated from the mitochondrial suspension and also allowed mitochondria fully depleted of FFA to be obtained. For each mitochondrial preparation, full depletion of FFA was tested by measuring the effect of BSA on LA (linoleic acid)-induced respiration as described by Jarmuszkiewicz et al. . Mitochondrial protein concentration was determined by the biuret method.
Mitochondrial oxygen consumption
Oxygen uptake was measured polarographically with a Clarck type oxygen electrode (Rank Brothers, Cambridge, U.K.) in 2.8 ml of incubation medium (25 °C) with 1 mg of mitochondrial protein. State 3 (phosphorylating) respiration measurements were performed in order to check coupling parameters. Only high quality mitochondria preparations, i.e. with an ADP/O value of around 1.40 and a respiratory control ratio of around 2.5–3.0, were used in all experiments. State 3 respiratory rate was 260±21 nmol of O·min−1 per mg of protein (n=15). Values of O2 uptake are presented as nmol of O·min−1 per mg of protein.
Mitochondrial membrane potential measurements
The ΔΨ was measured using a TPP+ (tetraphenylphosphonium)-specific electrode according to Kamo et al. . The TPP+ electrode was calibrated with four sequential additions (0.4, 0.4, 0.8 and 1.6 μM) of TPP+. After each run, 0.5 mM FCCP [carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone] was added to release TPP+ for base-line correction. For calculation of the ΔΨ value, the matrix volume of amoeba mitochondria was assumed to be 2.0 μl per mg of protein. The calculation assumes that TPP+ distribution between mitochondria and medium followed the Nernst equation. Corrections were made for the binding of TPP+ to mitochondrial membranes. Values of ΔΨ are presented in mV.
Proton leak measurements
The proton conductance response to its driving force can be expressed as the relationship between the oxygen consumption rate and ΔΨ (flux–force relationship) when varying the potential by titration with respiratory chain inhibitors. Respiration rate and ΔΨ were measured simultaneously using electrodes sensitive to oxygen and TPP+. Proton leak rates can be calculated from respiration rates by multiplying by an H+/O ratio of 6. Mitochondria (0.36 mg of protein/ml) were incubated in a standard incubation medium (25 °C) containing 120 mM KCl, 20 mM Tris/HCl (pH 7.2), 3 mM KH2PO4, 0.8 mM MgCl2 and 0.5 mM EGTA. Measurements were performed in the absence of added ADP, i.e. in resting state (state 4). To exclude the activity of an ATP/ADP antiporter and ATP synthase, 1.8 μM CATR (carboxyatractylozide) and 0.5 μg/ml oligomycin were used respectively. BHAM (benzohydroxamate) was used to inhibit alternative oxidase activity. The oxidizable substrates were succinate (5 mM) or external NADH (1 mM or 0.5–0.003 mM) in the presence of rotenone (4 μM) to block electron input from complex I. To activate succinate dehydrogenase, 80 μM ATP was used. To induce AcUCP activity-mediated respiration, measurements were made in the presence of 1.2, 2.4 or 4.8 μM LA. To inhibit the LA-induced AcUCP activity, 1 mM GDP was added.
Respiratory rate, ΔΨ and quinone redox state were varied by modulating the quinone-reducing or QH2 [reduced quinone (quinol)]-oxidizing pathways. To decrease the rate of the quinone-reducing pathway during state 4 respiration (thereby decreasing a steady-state resting respiration), titration of dehydrogenase activity was carried out. To titrate succinate oxidation, an increasing concentration of malonate (up to 5 mM), a competitive inhibitor of succinate dehydrogenase, was used. Titration of NADH oxidation was performed as described previously by varying the NADH concentration (0.5–0.003 mM) in the presence of an enzymatic regenerating system . To decrease the rate of the QH2-oxidizing pathway during state 4 respiration, the bc1 complex (complex III) or complex IV was inhibited with MX (myxothiazol) (up to 9 μM) and cyanide (up to 20 μM) respectively.
Measurements of quinone reduction level
The redox state of quinone in steady-state respiration was determined by an extraction technique followed by HPLC detection according to Van den Bergen et al. . As previously found, endogenous quinone in A. castellanii mitochondria is Q9 (ubiquinone with a side chain composed of 9 isoprene units) . For calibration of the peaks, commercial Q9 (Sigma) was used. A completely oxidized extract was obtained during incubation in the absence of substrate using the evaporation/ventilation step, whereas a completely reduced extract was obtained upon anaerobiosis and in the presence of substrates (5 mM succinate or 1 mM NADH), 1.5 mM KCN and 1.5 mM BHAM. An inactive quinone pool contains quinol that can never be oxidized and quinone that can never be reduced. The presented values of the redox state of quinone deal with the active quinone pool (the difference between the total quinone pool minus the inactive quinone pool) in a given mitochondrial preparation.
Fatty acid-stimulated proton leak and redox state of endogenous quinone in A. castellanii mitochondria
AcUCP-containing mitochondria are activated by FFAs, among which LA is the most efficient [4,8]. So far, to our knowledge, there is no experimental data describing the effect of UCP activity, including FFA-induced UCP activity, on the redox state of endogenous mitochondrial quinone under non-phosphorylating conditions. Figure 1 shows the influence of AcUCP activation upon addition of increasing LA concentration on membranous quinone redox state in a given steady-state of state 4 respiratory rate and ΔΨ with succinate as a respiratory substrate. Additions of LA progressively decreased the quinone redox state together with ΔΨ and stimulated respiratory rate. Moreover, respiratory rates and the quinone redox state values measured with saturating LA (maximal AcUCP activity) and protonophore (FCCP, fully uncoupled state) were almost the same, indicating that maximal electron flux through the respiratory chain was reached at 66–68% of the quinone redox state. Moreover, similar values of quinone reduction level were observed in A. castellanii mitochondria oxidizing succinate under phosphorylating conditions .
Inhibitory effect of GDP on FFA-induced proton leak when the quinone redox state is decreased in A. castellanii mitochondria
As previously reported [4,5], the FFA-induced AcUCP-sustained state 4 respiration was not inhibited (or only weakly inhibited) by the addition of PN (Figure 2A). Since in A. castellanii mitochondria it has been shown that FFA-induced uncoupling during phosphorylating respiration can be inhibited by PN when endogenous quinone is sufficiently oxidized , we investigated the effect of 1 mM GDP on the LA-induced uncoupling when the rate of the quinone-reducing pathway is decreased in non-phosphorylating conditions. In the presence of malonate, when the rate of succinate oxidation and the corresponding quinone redox state were decreased by around 60%, the full inhibition of the LA-induced H+ leak was observed (Figure 2B). The inhibitory effect of GDP was revealed by inhibition of respiratory rate as well as restoration of ΔΨ and the quinone redox state to values observed before LA addition. Thus, our determinations of the quinone reduction level in mitochondria respiring in non-phosphorylating conditions show for the first time that activation of UCP activity leads to a GDP-sensitive decrease in the quinone redox state. This conclusion can be extended to other PNs, since the same results were obtained with 1 mM ATP or GTP (results not shown). In the following experiments, GDP has been used to study the inhibitory effect on the FFA-induced proton leak.
To establish a flux–force relationship to which direct access is given by the simultaneous measurement of oxygen consumption and ΔΨ, after energization of mitochondria with respiratory substrate (succinate or external NADH), the potential was titrated by the addition of a given concentration of inhibitors (malonate, MX or cyanide). In order to avoid possible errors due to non-steady-state conditions (when sequential addition of inhibitors is applied) as well as to assess the quinone redox state for a given steady-state, data from separate measurements with different inhibitor concentrations (as in Figures 2, 4 and 6) were afterwards pooled on common curves (Figures 3, 5 and 7). Figures 3(A), 5(A) and 7(A) show the response of the H+ leak rate to its driving force, ΔΨ. Under control non-phosphorylating conditions, A. castellanii mitochondria exhibited a characteristic non-linear response of H+ leak to ΔΨ similar to animal [21,28] and plant mitochondria [19,20].
Figure 3(A) shows that, when the rate of the quinone-reducing pathway was progressively decreased during succinate oxidation (with increasing concentration of malonate), in the presence of LA, the proton conductance was greater than in the controls, in an LA concentration-dependent manner. This is demonstrated by the upward displacement of the H+ leak curves: the mitochondria required a higher respiration rate to maintain any given ΔΨ when incubated with LA. Below approximately 155 mV, the increase in H+ conductance was diminished and then fully inhibited by addition of GDP. The relationship between respiratory rate and the quinone reduction level [Qr/Qt; where Qr is reduced quinone (quinol), and Qt is the total endogenous pool of quinone in the inner mitochondrial membrane (Qox+Qr) – Qox is oxidized quinone] (Figure 3B) revealed the inhibitory effect of GDP on LA-induced AcUCP-sustained respiration for the quinone redox state below 50%. The full inhibitory effect was reached at approximately 40% of quinone reduction level, when data obtained in the presence of LA and GDP reached the control curve. The respiratory rate versus the quinone reduction level gave a single relationship for all the sets of experimental conditions (Figure 3C). This single relationship, showing that the quinone reduction level declined linearly from 92 to 20%, clearly demonstrates that LA and/or GDP do not affect the activity of the cytochrome pathway during substrate oxidation titration in non-phosphorylating conditions (independently of respiratory substrate, see also Figure 5C).
To establish whether the effect of PNs on LA-induced activity of AcUCP is observed for the same range of respiratory rates, ΔΨ and quinone reduction levels, independently of applied substrate, external NADH was used as a respiratory substrate. Similarly to succinate oxidation (Figure 2), the LA-induced AcUCP-sustained state 4 respiration with saturating NADH concentration (0.5 mM) was not inhibited by the addition of GDP (Figure 4A). However, when the quinone reduction level was decreased (approximately by 70%) by a lower NADH concentration (0.005 mM), the inhibitory effect of GDP was revealed by inhibition of the respiratory rate as well as the restoration of ΔΨ and the quinone redox state to the values observed before LA addition (Figure 4B).
Figures 5(A) and 5(B) show the effect of GDP on the respiration sustained by the LA-induced H+ leak (AcUCP activity) when the rate of the quinone-reducing pathway is gradually decreased in mitochondria oxidizing external NADH or succinate. Determinations of H+ conductance curves (Figure 5A), the relationships between respiratory rate as well as the ΔΨ versus quinone reduction level (Figures 5B and 5C) in the presence or absence of 1.2 μM LA and/or 1 mM GDP indicate that there is no difference between the results obtained with NADH and with succinate. With gradually decreased state 4 respiratory rate, LA-induced uncoupling activity was inhibited by GDP at the ΔΨ below 160 mV and a quinone reduction level below 50%.
No inhibitory effect of GDP on FFA-induced proton leak when the quinone redox state is increased in A. castellanii mitochondria
The above experiments indicate that, in A. castellanii mitochondria under non-phosphorylating conditions, sensitivity to PN of the FFA-induced AcUCP-sustained uncoupling is dependent on the membranous quinone redox state. To exclude the role of respiratory rate and ΔΨ, we progressively decreased them with inhibitors of the QH2-oxidizing pathway (MX or cyanide), leading to an increase in the quinone reduction level. Figure 6 shows an example experiment in which the respiratory rate, ΔΨ and quinone redox state were measured for non-phosphorylating succinate oxidation with a given concentration of MX (5.8 μM) when the effect of PN on the LA-induced mitochondrial uncoupling was studied. In the presence of MX, when the respiratory rate was decreased (by approx. 50%) and the corresponding quinone redox state was increased (by 10%), no inhibition of the LA-induced H+ leak by 1 mM GDP was observed. This results from the absence of a GDP effect on respiratory rate, ΔΨ and quinone redox state in the presence of LA.
Figure 7 presents determinations of H+ conductance curves (Figure 7A), the relationships between respiratory rate as well as the ΔΨ versus quinone reduction level (Figures 7B and 7C) in the presence or absence of 1.2 μM LA and/or 1 mM GDP, when the rate of the QH2-oxidizing pathway (the cytochrome pathway activity) was gradually decreased by increasing concentrations of MX or cyanide during succinate oxidation. Comparison of Figures 7(A) and 5(A) indicates that A. castellanii mitochondria energized with succinate or external NADH and titrated with inhibitors of the quinone-reducing or QH2-oxidizing pathways showed the same H+ leak curves (no GDP, plus or minus FFA conditions). Thus this clearly indicates that the redox state of endogenous quinone does not affect the basal and FFA-induced proton conductance of amoeba mitochondria. Moreover, when the rate of the QH2-oxidizing pathway was gradually decreased (during succinate oxidation), the ΔΨ versus quinone reduction level (Figure 7C) gave a single relationship for all sets of experimental conditions, showing the increase in quinone redox state from 83 to 98% during state 4 titration with both the cytochrome pathway inhibitors (MX and cyanide).
The force–flux relationship established in the presence of 1.2 μM LA to activate AcUCP and in the presence or absence of 1 mM GDP indicates that the nucleotide does not inhibit the LA-induced H+ conductance when the quinone reduction level was gradually increased (Figure 7A). Although titration of succinate oxidation with cyanide or MX comprised the respiratory rate and ΔΨ ranges at which GDP-sensitivity was observed for quinone-reducing pathway titration (with malonate, Figures 3A and 5A), no inhibitory effect of GDP was revealed under these conditions. However, the respiratory rate versus the quinone reduction level relationship is clearly out of the quinone redox state range where sensitivity to PN is revealed under state 4 conditions (Figure 7B). These results clearly indicate that the progressive inhibition of the LA-induced H+ leak in the presence of GDP cannot be attributed to changes in the ΔΨ. The lack of any inhibitory effect of GDP observed when a decrease in state 4 respiration was accompanied by an increase in the quinone reduction level proves that the inhibition by PN can be revealed only for a low quinone redox state. Thus we can conclude that, in A. castellanii mitochondria, the inhibitory effect of GDP on the LA-induced AcUCP-mediated uncoupling during non-phosphorylating respiration depends on the membranous quinone redox state, being the most efficient (full inhibition) when the quinone reduction level reaches approx. 40%.
The results presented in this study obtained with non-phosphorylating A. castellanii mitochondria confirm our previous studies performed on isolated mitochondria (of rat skeletal muscle, potato tubers and A. castellanii cells) respiring in the different metabolic state (phosphorylating conditions), where the ADP/O method was applied to calculate the rate of ADP phosphorylation [7,18,31]. In this approach, we have determined the efficiency of FFA-induced UCP activity in the uncoupling of oxidative phosphorylation when state 3 respiration, within the range where ΔΨ remains constant, is gradually decreased by a lowering rate of the quinone-reducing or QH2-oxidizing pathways. The present work, in which the common approach to study non-phosphorylating mitochondia, i.e. H+ conductance curve determinations (when ΔΨ is varied with respiratory chain inhibitors) has been applied, confirms that the quinone redox state has no effect at the level of FFA-induced UCP activity in the absence of PN and could only regulate this activity through the efficiency of inhibition by PN. It must be underlined that, in A. castellanii non-phosphorylating mitochondria (Figures 3B and 5B), the transition of the GDP inhibitory effect (from 0% to 100%) on LA-induced UCP-mediated uncoupling is observed for the same range of quinone reduction levels (between 50% and 40%) as that observed previously for phosphorylating conditions . This observation drawn from the two different metabolic states of mitochondria and from studies with the application of two different experimental approaches strongly indicates that quinone (oxidized or reduced) affects the affinity of the UCP for GDP. Certainly, further studies are necessary to elucidate the mechanism of such regulation.
In A. castellanii mitochondria, for both respiration states, the full inhibitory effect of PN is observed for a quinone reduction level approx. below 43%. It seems that the range of the quinone redox state when the inhibition by PN occurs could be different for different mitochondria. Indeed, in phosphorylating mitochondria, full sensitivity to PN has been observed for a quinone reduction level below 57% in rat skeletal muscle mitochondria  and below 25% in potato tuber mitochondria . Moreover, the regulation of the PN-sensitivity of FFA-induced UCP activity by the quinone redox state could explain why, in some mitochondria, no or weak inhibition by PN has been observed in non-phosphorylating (state 4) respiration, thereby when the quinone reduction level could be high. Therefore, this phenomenon could account for the insensitivity to PN of FFA-induced uncoupling observed for UCPs of amoeboid eukaryotes (A. castellanii and D. discoideum), some plant UCPs as well as mammalian UCP2 and UCP3 in mitochondria respiring in non-phosphorylating conditions in the absence of superoxide [4,16–21]. Indeed, proton conductance curves established for FFA-induced non-phosphorylating uncoupling have not revealed sensitivity to PN during titration with cyanide when the quinone redox state is probably increased in potato tuber mitochondria [19,20]. On the other hand, as inhibition by PN has been observed for UCPs in isolated mitochondria of C. parapsilosis  and some varieties of potato tubers , it seems that in some organisms (most probably dependently on tissue, physiological state, age and variety) the quinone reduction level of non-phosphorylating state could be sufficiently low to allow inhibition by nucleotides.
Studies with mammalian UCPs have revealed conflicting results concerning the possibility that quinone may be an obligatory cofactor for their action. Namely, oxidized quinone has been shown to activate PN-sensitive FFA-dependent H+ transport through reconstituted UCP1–3 [24,25]. Therefore, it has been proposed that oxidized quinone could facilitate H+ conductance induced by FFAs during UCP action . On the other hand, other studies have shown that quinone has no significant activating effect on the FFA-dependent H+ translocation nor any effect on the inhibition by PN in reconstituted UCP1–3 . Photoaffinity labelling of purified UCP1 with retinoic acid has indicated that quinone increases the binding of activator by UCP1 . From flux-force relationship studies performed with isolated kidney mitochondria, it has been concluded that the redox state of endogenous quinone does not affect mitochondrial proton conductance . The results on isolated A. castellanii mitochondria presented in this study, based on quinone redox determinations, show that the redox state of endogenous quinone does not affect the basal and FFA-induced UCP-mediated H+ conductance but its sensitivity to PNs. This conclusion can be made as determination of membranous quinone reduction level was performed when H+ leak curves were established with inhibitors of the quin-one-reducing or QH2-oxidizing pathways in isolated respiring mitochondria.
It must be underlined that the described regulation of UCP activity by quinone redox state so far has been observed only for the FFA-induced UCP-mediated uncoupling. Indeed, from proton conductance curves established in different mitochondria, under conditions when UCPs are activated by superoxide during non-phosphorylating respiration [19,20,25–27,35,36], it is difficult to estimate unequivocally if sensitivity to PN depends on quinone redox state during titrations of the quinone-reducing pathway (with malonate when succinate is oxidized) and the QH2-oxidizing pathway (with cyanide or MX). However, this cannot be excluded since determinations of quinone reduction level have not been performed in these experiments. Interestingly, addition of reduced quinone to respiring kidney mitochondria has resulted in an increase in FFA-induced GDP-sensitive proton conductance through production of superoxide . Therefore, further studies should explain if regulation of sensitivity of AcUCP to PN through quinone redox state could be also observed under conditions when uncoupling is activated by superoxide. So far, in A. castellanii mitochondria there is no evidence that AcUCP activity is induced by reactive oxygen species.
Taking into account the apparent affinity of reconstituted UCPs for PN [21,30] and the concentration of nucleotides in vivo (2–15 mM, depending on material), UCPs should be almost fully inhibited under in vivo conditions, even in the presence of FFAs, unless a regulatory factor or process could overcome (i.e. lower) the inhibition by PN [14,18]. In the case of UCP1, it has been proposed that FFAs do this in a kinetically simple competitive manner . Moreover, studies with mitochondria isolated from yeast mutant cells lacking quinone and expressing mouse UCP1 have shown that FFA-induced GDP-sensitive proton conductance by UCP1 expressed in yeast mitochondria is not dependent on the presence of quinone in the mitochondrial membrane . However, there are no data excluding the possibility that a highly reduced membranous quinone may lead to overcoming PN-inhibition of UCP1 activity in mammalian mitochondria. The present study shows that, at least in A. castellanii mitochondria, a metabolic sensor that modulates the PN inhibition of FFA-activated UCP activity is the quinone redox state.
In the molecular phylogenetic tree of eukaryotes, A. castellanii, the non-photosynthesizing amoeboid protozoan, appears on a branch basal to the divergence points of plants, animals and fungi [40,41]. Therefore, the evidence of quinone-redox-state-dependent sensitivity to PN of AcUCP could suggest that this phenomenon occurs in UCPs throughout the whole eukaryotic world. However, it seems also probable that, in A. castellanii mitochondria, similar to plant and some fungal mitochondria where a cyanide-resistant alternative oxidase is present, the mechanism of interaction between the quinone pool redox state and UCP activity may be a feedback to prevent the quinone pool from becoming overly reduced. Such a role would not apply to mammals. The fact that, in A. castellanii mitochondria, the transition of the inhibitory effect of PN on FFA-induced AcUCP-mediated uncoupling is observed for the same range of quinone reduction levels in two different metabolic states of mitochondria brings us nearer to elucidating one of the mechanisms of UCP activity regulation in protozoan mitochondria.
This work was supported by grants from the Polish Ministry of Education and Science (3382/B/P01/2007/33, 0252/P01/2007/32) and the Faculty of Biology, Adam Mickiewicz University (PBWB 701/2006).
Abbreviations: AcUCP, uncoupling protein of Acanthamoeba castellanii mitochondria; BHAM, benzohydroxamate; CATR, carboxyatractylozide; FCCP, carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone; FFA, free fatty acid; LA, linoleic acid; MX, myxothiazol; PN, purine nucleotide; Qr, or QH2, reduced quinone (quinol); QT, total endogenous pool of quinone in the inner mitochondrial membrane; TPP+, tetraphenylphosphonium; UCP, uncoupling protein; ΔΨ, mitochondrial membrane electrical potential; ΔμH+, proton electrochemical gradient
- © The Authors Journal compilation © 2008 Biochemical Society