Direct measurement of the initial proton extrusion to oxygen uptake ratio accompanying succinate oxidation by rat liver mitochondria.

The problem of obtaining very early ratios for the H+/O stoichiometry accompanying succinate oxidation by rat liver mitochondria was attacked using new techniques for direct measurement rather than extrapolations based on data obtained after mixing and the recovery of the electrode from initial injection of O2. Respiration was quickly initiated in a thoroughly mixed O2-containing suspension of mitochondria under a CO atmosphere by photolysis of the CO-cytochrome c oxidase complex-. Fast responding O2 and pH electrodes were used to collect data every 10 ms. The response time for each electrode was experimentally measured in each experiment and suitable corrections for electrode relaxations were made. With uncorrected data obtained after 0.8 s, the extrapolation back to zero time on the basis of single-exponential curve fitting confirmed values close to 8.0 as previously reported (Costa et al., 1984). The data directly obtained, however, indicate an initial burst in H+/O ratio that peaked to values of approximately 20 to 30 prior to 50 ms and which was no longer evident after 0.3 s. Newer information and considerations that place all extrapolation methods in question are discussed.     

The H+/O ratio of proton translocation linked to the oxidation of succinate by mitochondria. Reply to a commentary

Costa, L.E., Reynafarje, B. and Lehninger, A.L. [(1984) J. Biol. Chem. 259, 4802-4811] have reported 'second-generation' measurements of the H+/O ratio approaching 8.0 for vectorial H+ translocation coupled to succinate oxidation by rat liver mitochondria. In a Commentary in this Journal [Krab, K., Soos, J. and Wikstr?m, M. (1984) FEBS Lett. 178, 187-192] it was concluded that the measurements of Costa et al. significantly overestimated the true H+/O stoichiometry. It is shown here that the mathematical simulation on which Krab et al. based this claim is faulty and that data reported by Costa et al. had already excluded the criticism advanced by Krab et al. Also reported are new data, obtained under conditions in which the arguments of Krab et al. are irrelevant, which confirm that the H+/O ratio for succinate oxidation extrapolated to level flow is close to 8.     

The H+/O ratio of proton translocation linked to the oxidation of succinate by mitochondria

In a recent communication Lehninger and co-workers (Costa, L.E., Reynaferje, B., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 4802-4811) reported values approaching 8 for the H+/O ratio of vectorial proton ejection from rat liver mitochondria respiring with succinate. Here we present a rigorous analysis of these measurements which reveals that they may significantly overestimate the true H+/O stoicheiometry.     

The rapid alteration by tri-iodo-L-thyronine in vivo of both the ADP/O ratio and the apparent H+/O ratio in hypothyroid-rat liver mitochondria.

Mitochondria from the livers of thyroidectomized rats have a lowered ADP/O ratio, which can be restored to normal within 15 min after intravenous injection of a near-physiological dose of tri-iodothyronine. Thyroidectomy lowered the measured delta pH, which appears to be compensated by a rise (not statistically significant) in the membrane potential, so that the protonmotive force is unaltered. A simple simulation technique is described for use in estimating H+/O ratios by the oxygen-pulse technique, which circumvents the problem that this ratio can be seriously underestimated because of re-uptake of protons from the bulk phase by the mitochondria before their expulsion is complete. By this procedure the H+/O ratio of hypothyroid mitochondria is shown to be lowered by the same factor as the ADP/O ratio, and both these ratios are very rapidly restored in parallel by hormone administration. Although these findings could be consistent with a proposal that tri-iodothyronine rapidly modulates by some mechanism the efficiency of the respiratory-chain-linked proton pumps, the kinetic properties of the proton exchange suggest that the bulk-phase protons measured may not reflect faithfully those that drive the ATP synthetase.     

Steady-state H+/O stoichiometry of liver mitochondria.

We have measured the H+/O stoichiometry of rat liver mitochondria respiring in a steady-state, using a novel method. This involves measuring the initial rate of H+ back-flow into mitochondria after respiratory inhibition, with the assumption that this is equal to the steady-state H+-ejection rate. Division by the steady-state O2-consumption rate yields the H+/O ratio. The H+/O values obtained were: 8.3 +/- 1.0 (mean +/- S.E.M.) for 3-hydroxybutyrate: 8.2 +/- 0.7 for glutamate plus malate; 6.0 +/- 0.2 for succinate; 4.1 +/- 0.3 for ascorbate/tetramethylphenylenediamine and 3.0 +/- 0.1 for ascorbate/ferrocyanide. These values correspond to H+/O stoichiometries for electron flow to oxygen from NAD+-linked substrates, succinate and cytochrome c of 8, 6 and 2 (charge/O ratio = 4) respectively.     

The effect of bovine serum albumin on the membrane potential and reactive oxygen species generation in succinate-supported isolated brain mitochondria

Characteristics of the succinate-supported H(2)O(2) formation were compared in mitochondria prepared from guinea-pig brain either by Percoll gradient centrifugation or using digitonin. The high rate of H(2)O(2) generation measured in mitochondria prepared with digitonin (600.6+/-26.8pmol/min/mg protein) was inhibited by rotenone, consistently with a reverse flow of electrons via complex I. The rate of H(2)O(2) formation was significantly smaller in Percoll-purified mitochondria (252.6+/-17.3pmol/min/mg protein) and this was stimulated by rotenone. Since bovine serum albumin (BSA) is usually present in the isolation medium used in the digitonin method, systematic study was performed addressing the effect of BSA on H(2)O(2) formation. Mitochondria prepared by the digitonin method (BSA present in the isolation medium) were highly polarized (185+/-3.2mV) and addition of BSA (0.025%) to the assay medium increased H(2)O(2) generation by only 50%. In Percoll-purified mitochondria DeltaPsim was more depolarized (171+/-2mV) and BSA caused hyperpolarization by 10.7+/-1.9mV. H(2)O(2) formation, which was largely independent of DeltaPsim, was stimulated by 400%, became highly dependent on DeltaPsim and could be inhibited by rotenone in the presence of BSA. This shows that in Percoll-purified mitochondria ROS formation via reverse electron flow is preferred only when BSA is present in the assay medium. It is demonstrated that (i) the presence or absence of BSA could determine the mechanism by which ROS is generated in succinate-supported mitochondria and (ii) depolarization by about 10mV eliminates reverse electron flow and the remaining ROS formation, which is smaller but still significant, is no longer dependent on DeltaPsim.     

Characterization of superoxide-producing sites in isolated brain mitochondria

Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD+/NADH redox couple on H2O2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H2O2 (0.04 +/- 0.02 nmol H2O2/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 +/- 0.25 nmol H2O2/min/mg) or in the presence of the respiratory substrate succinate alone (0.80 +/- 0.27 nmol H2O2/min/mg). The maximal rate of H2O2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 +/- 0.07 nmol H2O2/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to -295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H2O2 induced increased H2O2 production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.     

A new paradigm: manganese superoxide dismutase influences the production of H2O2 in cells and thereby their biological state

The principal source of hydrogen peroxide in mitochondria is thought to be from the dismutation of superoxide via the enzyme manganese superoxide dismutase (MnSOD). However, the nature of the effect of SOD on the cellular production of H(2)O(2) is not widely appreciated. The current paradigm is that the presence of SOD results in a lower level of H(2)O(2) because it would prevent the non-enzymatic reactions of superoxide that form H(2)O(2). The goal of this work was to: a) demonstrate that SOD can increase the flux of H(2)O(2), and b) use kinetic modelling to determine what kinetic and thermodynamic conditions result in SOD increasing the flux of H(2)O(2). We examined two biological sources of superoxide production (xanthine oxidase and coenzyme Q semiquinone, CoQ(*-) that have different thermodynamic and kinetic properties. We found that SOD could change the rate of formation of H(2)O(2) in cases where equilibrium-specific reactions form superoxide with an equilibrium constant (K) less than 1. An example is the formation of superoxide in the electron transport chain (ETC) of the mitochondria by the reaction of ubisemiquinone radical with dioxygen. We measured the rate of release of H(2)O(2) into culture medium from cells with differing levels of MnSOD. We found that the higher the level of SOD, the greater the rate of accumulation of H(2)O(2). Results with kinetic modelling were consistent with this observation; the steady-state level of H(2)O(2) increases if K<1, for example CoQ(*-)+O(2)-->CoQ+O(2)(*-). However, when K>1, e.g. xanthine oxidase forming O(2)(*-), SOD does not affect the steady state-level of H(2)O(2). Thus, the current paradigm that SOD will lower the flux of H(2)O(2) does not hold for the ETC. These observations indicate that MnSOD contributes to the flux of H(2)O(2) in cells and thereby is involved in establishing the cellular redox environment and thus the biological state of the cell.     

Role of antioxidant defences in the species-specific response of isolated atria to menadione

In previous works we demonstrated that 2-methyl-1,4-naphthoquinone (menadione) causes a marked increase in the force of contraction of guinea pig and rat isolated atria. This inotropic effect was significantly higher in the guinea pig than in the rat and was strictly related to the amount of superoxide anion (O(2)(*-)), generated as a consequence of cardiac menadione metabolism through mitochondrial NADH-ubiquinone oxidoreductase. The present study was designed to further elucidate the basis of these quantitatively different positive inotropic responses. To this purpose, we measured O(2)(*-) and hydrogen peroxide (H(2)O(2)) produced by mitochondria isolated from guinea pig and rat hearts in the presence of 20 microM menadione. Moreover, we evaluated the menadione detoxification activity (DT-diaphorase) and the antioxidant defences of guinea pig and rat hearts, namely their GSH/GSSG content, Cu/Zn- and Mn-dependent superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx) activities. Our results indicate that DT-diaphorase activity and glutathione levels were similar in both animal species. By contrast, guinea pig mitochondria produced greater amounts of O(2)(*-) and H(2)O(2) than those of rat heart. This is probably due to both the higher Mn-SOD activity (2.93 +/- 0.02 vs. 1.95 +/- 0.06 units/mg protein; P < 0.05) and to the lower Gpx activity (10.09 +/- 0.30 vs. 32.67 +/- 1.02 units/mg protein; P < 0.001) of guinea pig mitochondria. A lower CAT activity was also observed in guinea pig mitochondria (2.40 +/- 0.80 vs. 6.13 +/- 0.20 units/mg protein; P < 0.01). Taken together, these data provide a rational explanation for the greater susceptibility of guinea pig heart to the toxic effect of menadione: because of the greater amount of O(2)(*-) generated by the quinone and the higher mitochondrial Mn-SOD activity, guinea pig heart is exposed to more elevated concentrations of H(2)O(2) that is less efficiently detoxified, because of lower Gpx and CAT levels of mitochondria.     

Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria

Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 min global ischemia or following 40 min of perfusion alone as controls. H(2)O(2) release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of H(2)O(2) was increased by 178 +/- 14% and 179 +/- 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, H(2)O(2) increased by 272 +/- 22% and 171 +/- 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased H(2)O(2) production by 179 +/- 24% and 155 +/- 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of H(2)O(2) from complex III. Thus ischemic damage to the ETC increased both the capacity and the net production of H(2)O(2) from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury.     

Membrane potential and H2O2 production in duodenal mitochondria from broiler chickens (Gallus gallus domesticus) with low and high feed efficiency

Increased hydrogen peroxide (H2O2) production was observed in duodenal mitochondria obtained from broiler chickens with low feed efficiency (FE). As a decrease in mitochondrial membrane potential (Deltapsi(m)) due to mild uncoupling of oxidative phosphorylation reduces reactive oxygen species production, this study was conducted to evaluate the effect of uncoupling on Deltapsi(m) and H2O2 production in duodenal mitochondria isolated from broilers with low (0.48+/-0.02) and high (0.68+/-0.01) FE. Membrane potential and H2O2 production were measured fluorometrically and in the presence of different levels of an uncoupler, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The Deltapsi(m) was higher (P相似文献   

Alterations in mitochondrial Ca2+ flux by the antibiotic X-537A (lasalocid-A).

A previous communication (Pereira da Silva, L., Bernardes, C.F. and Vercesi, A.E. (1984) Biochem. Biophys. Res. Commun. 124, 80-86) presented evidence that lasalocid-A, at concentrations far below those required to act as a Ca2+ ionophore, significantly inhibits Ca2+ efflux from liver mitochondria. In the present work we have studied the mechanism of this inhibition in liver and heart mitochondria. It was observed that lasalocid-A (25-250 nM), like nigericin, promotes the electroneutral exchange of K+ for H+ across the inner mitochondrial membrane and as a consequence can cause significant alterations in delta pH and delta psi. An indirect effect of these changes that might lead to inhibition of mitochondrial Ca2+ release was ruled out by experiments showing that the three observed patterns of lasalocid-A effect depend on the size of the mitochondrial Ca2+ load. At low Ca2+ loads (5-70 nmol Ca2+/mg protein), under experimental conditions in which Ca2+ release is supposed to be mediated by a Ca2+/2H+ antiporter, the kinetic data indicate that lasalocid-A inhibits the efflux of the cation by a competitive mechanism. The Ca2+/2Na+ antiporter, the dominant pathway for Ca2+ efflux from heart mitochondria, is not affected by lasalocid-A. At intermediate Ca2+ loads (70-110 nmol Ca2+/mg protein), lasalocid-A slightly stimulates Ca2+ release. This effect appears to be due to an increase in membrane permeability caused by the displacement of a pool of membrane bound Mg2+ possibly involved in the maintenance of membrane structure. Finally, at high Ca2+ loads (110-140 nmol Ca2+/mg protein) lasalocid-A enhances Ca2+ retention by liver mitochondria even in the presence of Ca2(+)-releasing agents such as phosphate and oxidants of the mitochondrial pyridine nucleotides. The maintenance of a high membrane potential under these conditions may indicate that lasalocid-A is a potent inhibitor of the Ca2(+)-induced membrane permeabilization. Nigericin, whose chemical structure resembles that of lasalocid-A, caused similar results.     

H2O2 generation is decreased by calcium in isolated brain mitochondria

Release of H(2)O(2) in response to Ca(2+) loads (1-100 microM) was investigated using Amplex red fluorescent assay in isolated guinea-pig brain mitochondria respiring on glutamate plus malate or succinate. In mitochondria challenged with Ca(2+) (10 microM), in the absence of adenine nucleotides and inhibitors of the respiratory chain, the rate of H(2)O(2) release, taken as an indication of H(2)O(2) production, was decreased by 21.8+/-1.6% in the presence of NADH-linked substrates and by 86.5+/-1.8% with succinate. Parallel with this, a Ca(2+)-induced loss in NAD(P)H fluorescence, sustained depolarization, decrease in fluorescent light scattering signal and in calcein fluorescence were detected indicating an increased permeability and swelling of mitochondria, which were prevented by ADP (2 mM). In the presence of ADP H(2)O(2) release from mitochondria was decreased, but Ca(2+) no longer influenced the generation of H(2)O(2). We suggest that the decreased H(2)O(2) generation induced by Ca(2+) is related to depolarization and NAD(P)H loss resulting from a non-specific permeability increase of the mitochondrial inner membrane.     

Upper and lower limits of the proton stoichiometry of cytochrome c oxidation in rat liver mitoplasts

The stoichiometry of vectorial H+ translocation coupled to oxidation of added ferrocytochrome c by O2 via cytochrome-c oxidase of rat liver mitoplasts was determined employing a fast-responding O2 electrode. Electron flow was initiated by addition of either ferrocytochrome c or O2. When the rates were extrapolated to level flow, the H+/O ratios in both cases were less than but closely approached 4; the directly observed H+/O ratios significantly exceeded 3.0. The mechanistic H+/O ratio was then more closely fixed by a kinetic approach that eliminates the necessity for measuring energy leaks and is independent of any particular model of the mechanism of energy transduction. From two sets of kinetic measurements, an overestimate and an underestimate and thus the upper and lower limits of the mechanistic H+/O ratio could be obtained. In the first set, the utilization of respiratory energy was systematically varied through changes in the concentrations of valinomycin or K+. From the slope of a plot of the initial rates of H+ ejection (JH) and O2 uptake (JO) obtained in such experiments, the upper limit of the H+/O ratio was in the range 4.12-4.19. In the second set of measurements, the rate of respiratory energy production was varied by inhibiting electron transport. From the slope of a plot of JH versus JO, the lower limit of the H+/O ratio, equivalent to that at level flow, was in the range 3.83-3.96. These data fix the mechanistic H+/O ratio for the cytochrome oxidase reaction of mitoplasts at 4.0, thus confirming our earlier measurements (Reynafarje, B., Alexandre, A., Davies, P., and Lehninger, A. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7218-7222). Possible reasons for discrepancies in published reports on the H+/O ratio of cytochrome oxidase in various mitochondrial and reconstituted systems are discussed.     

Control processes in oxidative phosphorylation: kinetic constraints and stoichiometry

Control processes in oxidative phosphorylation have been studied in three experimental models. (1) In isolated yeast mitochondria, external ATP is a regulatory effector of cytochrome-c oxidase activity. In phosphorylating or uncoupling states, the relationships between respiratory rate and delta mu H+, and the respiratory rate and cytochrome-c oxidase reduction level are dependent on this kinetic regulation. (2) In rat liver mitochondria, the response of the respiratory rate to uncoupler addition is age-dependent: liver mitochondria isolated from young rats maintain a greater delta mu H+ than liver mitochondria isolated from adults, with the same respiratory rate obtained with the same concentration of uncoupler. This behaviour is linked to redox proton pump properties, i.e., to the degree of intrinsic uncoupling induced by uncoupler addition. (3) The effect of almitrine, a new kind of ATPase/ATPsynthase inhibitor, was studied in mammalian mitochondria. (i) Almitrine inhibits oligomycin-sensitive ATPase - it decreases the ATPase/O value without any change in delta mu H+; (ii) almitrine increased the mechanistic H+/ATP stoichiometry of ATPase/ATPsynthase; (iii) almitrine-induced changes in H+/ATPase stoichiometry depend on the flux magnitude through ATPase. These results are discussed in terms of the following interdependent parameters; flux value, force, pump efficiency and control coefficient.     

A kinetic study on the suicide inactivation of peroxidase by hydrogen peroxide

In the absence of reductant substrates, and with excess H2O2, peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) shows the kinetic behaviour of a suicide inactivation, H2O2 being the suicide substrate. From the complex (compound I-H2O2), a competition is established between two catalytic pathways (the catalase pathway and the compound III-forming pathway), and the suicide inactivation pathway (formation of inactive enzyme). A kinetic analysis of this system allows us to obtain a value for the inactivation constant, ki = (3.92 +/- 0.06) x 10(-3) x s-1. Two partition ratios (r), defined as the number of turnovers given by one mol of enzyme before its inactivation, can be calculated: (a) one for the catalase pathway, rc = 449 +/- 47; (b) the other for the compound III-forming pathway, rCoIII = 2.00 +/- 0.07. Thus, the catalase activity of the enzyme and, also, the protective role of compound III against an H2O2-dependent peroxidase inactivation are both shown to be important.     

Reconstitution and partial purification of the Na(+)-selective Na+/H+ antiporter of beef heart mitochondria.

Na+/H+ antiporters play important physiological roles in most biological membranes. Although they were first discovered in mitochondria (Mitchell, P., and Moyle, J. (1969) Eur. J. Biochem. 9, 149-155), the mitochondrial Na+/H+ antiporter has not yet been reconstituted nor has the protein responsible for its activity been identified. We used detergents to extract proteins from beef heart mitochondria and reconstituted these proteins into lipid vesicles loaded with the fluorescent probe, sodium-binding benzofuran isophthalate. The vesicles exhibited spontaneous, electroneutral Na+ transport that was inhibited by Li+ and Mn2+ with appropriate kinetic constants. These protocols were then used to follow fractionation of the solubilized proteins with DEAE-cellulose columns. We obtained a fraction that catalyzed Na+/H+ antiport with Vmax values of 75-120 mumol/mg protein/min, 500-700 times faster than observed in intact mitochondria. Na+ transport was inhibited by Li+ with I50 values of 0.5-1.0 mM and by Mn2+ with I50 value of 0.5 mM. The Km for Na+ was 31 mM. These values correspond to those found in intact mitochondria, and we conclude that the solubilized mitochondrial Na+/H+ antiporter has been partially purified in a reconstitutively active state.     

On the mechanism by which dicyclohexylcarbodiimide and quinine inhibit K+ transport in rat liver mitochondria

Passive uptake of potassium acetate into the mitochondrial matrix can be induced by nigericin, a K+/H+ antiporter, or by A23187, a Mg2+/2H+ antiporter. The latter process is thought to reflect operation of the Mg2+-dependent, endogenous K+/H+ antiporter, but there is ambiguity with respect to the mechanism of K+ transport in this assay (Nakashima, R.A., and Garlid, K.D. (1982) J. Biol. Chem. 257, 9252-9254). Kinetic analysis of potassium acetate transport provides verification that Mg2+ depletion 1) unmasks the K+/H+ antiporter, 2) opens up an intrinsic anion uniporter, 3) has no effect on acetic acid transport, and 4) does not induce high K+ uniport conductance. Mg2+-dependent uptake of potassium acetate is thereby shown to be mediated specifically by operation of the endogenous K+/H+ antiporter, as previously proposed. An extension of this analysis confirms that N,N'-dicyclohexylcarbodiimide and quinine block potassium acetate uptake via specific action on the K+/H+ antiporter. These findings support those of a previous study (Martin, W.H., Beavis, A.D., and Garlid, K.D. (1984) J. Biol. Chem. 259, 2062-2065) in which binding of [14C]N,N'-dicyclohexylcarbodiimide to membrane proteins under selective conditions was used to identify an 82,000-dalton band as the protein responsible for K+/H+ antiport in mitochondria.     

On the effects of thiocyanate and venturicidin on respiration-driven proton translocation in Paracoccus denitrificans

A fast-responding O2 electrode has been used to confirm and extend observations of a significant kinetic discrepancy between O2 reduction and consequent proton translocation in 'O2-pulse' experiments in intact cells of P. denitrificans. The permeant, chaotropic SCN- ion abolishes this discrepancy, and greatly increases the observable----H+/O ratio, to a value approaching its accepted, true, limiting stoichiometry. The observable H+ decay rates are very slow, particularly in the absence of SCN-. The submaximal----H+/O ratios observed in the absence of SCN- are essentially independent of the size of the O2 pulse, in a manner not easily explained by a delocalised chemiosmotic energy-coupling scheme. Osmotically active protoplasts of P. denitrificans do not show a significant kinetic discrepancy between O2 reduction and H+ translocation, even in the the absence of SCN-. However, the submaximal----H+/O ratios observed in the absence of SCN- are again essentially independent of the size of the O2 pulse. As in intact cells, the observable H+ decay rates are very slow. The energy-transfer inhibitor venturicidin causes a significant increase in the----H+/O ratio observed in protoplasts of P. denitrificans in the absence of SCN-; the decay kinetics of the H+ translocation process are also somewhat modified. Nevertheless, the----H+/O ratio observed in the presence of venturicidin is also independent of the size of the O2 pulse. This observation militates further against arguments in which (a) a non-ohmic leak of protons from the bulk aqueous phase might alone be the cause of the low----H+/O ratios observed in the absence of SCN-, and (b) in which there might be a delta p-dependent change ('redox slip') in the actual----H+/O ratio. It is concluded that the observable protonmotive activity of the respiratory chain of P. denitrificans in the absence of SCN- is directly influenced by the state of the H+-ATP synthetase in the cytoplasmic membrane of this organism. We are unable to explain the data in terms of a model in which the putative protonmotive force may be acting to affect the----H+/O ratio. The possibility is considered that the delocalised bulk-to-bulk phase membrane potential set up in response to protonmotive activity is energetically insignificant.     

Fusicoccin affects cytochrome c leakage and cytosolic 14-3-3 accumulation independent of H-ATPase activation

Fusicoccin (FC) is a well known toxin acting as a 14-3-3 protein-mediated activator of the plasma membrane H(+)-ATPase and the biochemical and physiological changes induced in the cell by this toxin have, up to now, been ascribed to the increased rate of proton extrusion by this pump leading to external acidification and cell hyperpolarization. In a recent work (Malerba M et al. 2003, Physiologia Plantarum, 119: 480-488) it was shown that, besides the previously well studied changes, FC induces a large stimulation of H(2)O(2) production, an activation of alternative respiration and a leakage of cytochrome c from mitochondria. In this article further studies on the relation between the H(2)O(2) overproduction and medium acidification are reported. The increase in the rate of H(2)O(2) accumulation is particularly evident when high concentrations of the toxin ensure a rapid acidification of the medium, but it is not obtained when the time-course of acidification is reproduced by external acid additions. The FC-dependent H(2)O(2) overproduction is strongly inhibited by inhibitors of the H(+)-ATPase activity, such as vanadate and erythrosin B, and it does not occur when the activation of the H(+)-ATPase is prevented by phenylarsine oxide (PAO), an inhibitor of the activating interaction between the enzyme and its regulative 14-3-3 protein. Interestingly, all these inhibitors only partially prevent the leakage of cytochrome c from the mitochondria. A kinetic analysis of FC-dependent changes of 14-3-3s shows that the initial increase in the plasma membrane level of these proteins, presumably due to translocation of free cytosolic forms, is followed by a remarkable increase in the level of the 14-3-3 proteins located in the cytosol. This latter change is not prevented by inhibitors of the activity or activation of the H(+)-ATPase. These results suggest that, besides the H(+)-ATPase activation, FC can induce other cell changes possibly mediated by changes of the regulative 14-3-3 proteins.