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Lipid peroxidation plays an important role in various pathologies and aging, at least partially mediated by ferroptosis. The role of mitochondrial lipid peroxidation during ferroptosis remains poorly understood. We show that supplementation of exogenous iron in the form of ferric ammonium citrate at submillimolar doses induces production of reactive oxygen species (ROS) and lipid peroxidation in mitochondria that precede ferroptosis in H9c2 cardiomyocytes. The mitochondria-targeted antioxidant SkQ1 and the redox mediator methylene blue, which inhibits the production of ROS in complex I of the mitochondrial electron transport chain, prevent both mitochondrial lipid peroxidation and ferroptosis. SkQ1 and methylene blue also prevented accumulation of lipofuscin observed after 24 h incubation of cardiomyocytes with ferric ammonium citrate. Using isolated cardiac mitochondria as an in vitro ferroptosis model, it was shown that rotenone (complex I inhibitor) in the presence of ferrous iron stimulates lipid peroxidation and lipofuscin accumulation. Our data indicate that ROS generated in complex I stimulate mitochondrial lipid peroxidation, lipofuscin accumulation, and ferroptosis induced by exogenous iron.

Ferroptosis is a regulated form of necrotic cell death reliant on iron-catalyzed lipid peroxidation. Although the precise involvement of mitochondria in ferroptosis remains incompletely elucidated, recent research indicates that mitochondrial oxidative events wield a pivotal influence in this mechanism. This article centers on the most recent discoveries, spotlighting the significance of mitochondrial lipid peroxidation in the occurrence of ferroptosis. Modern investigative tools, such as mitochondria-specific dyes responsive to lipid peroxidation and antioxidants targeting mitochondria, have been employed to delve into this phenomenon. The authors' recent empirical evidence demonstrates that mitochondrial lipid peroxidation, quantified using the innovative fluorescent ratiometric probe MitoCLox, takes place prior to the onset of ferroptotic cell death. The mitochondria-targeted antioxidant SkQ1 hinders mitochondrial lipid peroxidation and thwarts ferroptosis, all while leaving unaffected the buildup of reactive oxygen species within the cytoplasm, an antecedent to mitochondrial lipid peroxidation. Similarly, the redox agent methylene blue, impeding the genesis of reactive oxygen species in complex I of the electron transport chain, also imparts a comparable protective effect. These findings collectively imply that reactive oxygen species originating from complex I might hold particular significance in fomenting mitochondrial lipid peroxidation, a pivotal trigger of ferroptosis., Competing Interests: Conflict of interestThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Ferroptosis induced by erastin (an inhibitor of cystine transport) and butionine sulfoximine (an inhibitor of glutathione biosynthesis) was prevented by the mitochondria-targeted antioxidants SkQ1 and MitoTEMPO. These effects correlate with the prevention of mitochondrial lipid peroxidation, which precedes cell death. Methylene blue, a redox agent that inhibits the production of reactive oxygen species (ROS) in complex I of the mitochondrial electron transport chain, also inhibits ferroptosis and mitochondrial lipid peroxidation. Activation of ROS production in complex I with rotenone in the presence of ferrous iron stimulates lipid peroxidation in isolated mitochondria, while ROS produced by complex III are ineffective. SkQ1 and methylene blue inhibit lipid peroxidation. We suggest that ROS formed in complex I promote mitochondrial lipid peroxidation and ferroptosis.

The aim of this pilot study was to examine the effectiveness of a CrossFit-based training program to enhance the general and sport-specific physical fitness of 10−12-year-old judokas. The study was conducted between September 2021 and February 2022. The pedagogical research experiment was designed to be one complete, annual macrocycle (September−June). The current study presents mid-point data. Twenty male participants (3 years average sports experience; age = 11 ± 0.64 years) were randomly assigned to one of two groups: CrossFit-based training (experimental, n = 10) and traditional training (control, n = 10). Baseline testing was conducted by the researcher and included tests for motor skills and general physical fitness domains including Sweden wall pull-ups and leg raises, push-ups, long jump, squats, burpees, shuttle run, and forward rolls. Judo-specific tests included O Soto Gari and O Goshi throws. CrossFit-based training was implemented twice per week for 15−20 min in the experimental group after usual training. The control group completed traditional methods of physical fitness training for judokas with the same training load regarding time. Experimental group participants significantly improved on leg raises (p < 0.01), push-ups (p < 0.05), and shuttle run (p < 0.001); the control group improved their shuttle run (p < 0.001). Only the experimental group improved on the O Soto Gari (p < 0.01) and O Goshi throws (p < 0.05). Results showed that the use of CrossFit-based trainings had a positive effect on 10−12-year-old judokas’ speed-strength abilities, speed-strength endurance, and muscular strength.

Receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of Alzheimer's disease. We have previously revealed that RAGE fragment sequence (60-76) and its shortened analogs sequence (60-70) and (60-65) under intranasal insertion were able to restore memory and improve morphological and biochemical state of neurons in the brain of bulbectomized mice developing major AD features. In the current study, we have investigated the ability of RAGE peptide (60-76) and five shortened analogs to bind beta-amyloid (Aβ) 1-40 in an fluorescent titration test and show that all the RAGE fragments apart from one [sequence (65-76)] were able to bind Aβ in vitro . Moreover, we show that all RAGE fragments apart from the shortest one (60-62), were able to protect neuronal primary cultures from amyloid toxicity, by preventing the caspase 3 activation induced by Aβ 1-42. We have compared the data obtained in the present research with the previously published data in the animal model of AD, and offer a probable mechanism of neuroprotection of the RAGE peptide.

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨ m ), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨ m -sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨ m decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.

Structural and functional impairments of mitochondria in brain tissues in the pathogenesis of Alzheimer's disease (AD) cause energy deficiency, increased generation of reactive oxygen species (ROS), and premature neuronal death. However, the causal relations between accumulation of beta-amyloid (Aβ) peptide in mitochondria and mitochondrial dysfunction, as well as molecular mechanisms underlying deleterious effects of both these factors in sporadic AD, the most common form in humans, remain unknown. Here we used olfactory bulbectomized (OBX) mice of NMRI strain as a model for sporadic AD. Five weeks after surgery, the OBX mice developed major behavioral and biochemical features of AD neurodegeneration, including spatial memory loss, increased brain levels of Aβ, and energy deficiency. Mitochondria isolated from the neocortex and hippocampus of OBX mice displayed severe functional impairments, such as low NADH oxidation rate, reduced transmembrane potential, and decreased cytochrome c oxidase (complex IV) activity that correlated with high levels of soluble Aβ1-40. Mitochondria from OBX mice showed increased contents of lipid peroxidation products, indicative of the development of oxidative stress. We found that neurodegeneration caused by olfactory bulbectomy is accompanied by energy metabolism disturbances and oxidative stress in brain mitochondria similar to those occurring in transgenic animals - familial AD models and patients with sporadic AD. Therefore, OBX mice can serve as a valid AD model for investigating the mechanisms of AD neurodegeneration, drug testing, and development of therapeutic strategies for AD treatment.

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C4R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C12R1). Surprisingly, C12R1 increased H(+) conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C4R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C4R1. In experiments with HeLa cells, C4R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C4R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C12R1. Thus, C4R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease., (© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.)

A conjugate of plastoquinone with decylrhodamine 19 (SkQR1) selectively accumulates in mitochondria of normal and tumor cells. SkQR1 protected the cellular pool of reduced glutathione under oxidative stress. Overexpression of P-glycoprotein (Pgp 170) multidrug resistance pump strongly suppresses accumulation of SkQR1. The inhibitors of Pgp 170 stimulate accumulation of SkQR1 in various cell lines indicating that SkQR1 is a substrate of Pgp 170. The protective effect of SkQR1 against oxidative stress is diminished in the cells overexpressing Pgp 170. It is suggested that mitochondria-targeted antioxidants could selectively protect normal (Pgp 170-negative) cells against the toxic effect of anti-cancer treatments related to oxidative stress., (2009. Published by Elsevier B.V.)

Production of reactive oxygen species (ROS) in mitochondria was studied using the novel mitochondria-targeted antioxidants (SkQ) in cultures of human cells. It was shown that SkQ rapidly (1-2 h) and selectively accumulated in mitochondria and prevented oxidation of mitochondrial components under oxidative stress induced by hydrogen peroxide. At nanomolar concentrations, SkQ inhibited oxidation of glutathione, fragmentation of mitochondria, and translocation of Bax from cytosol into mitochondria. The last effect could be related to prevention of conformational change in the adenine nucleotide transporter, which depends on oxidation of critical thiols. Mitochondria-targeted antioxidants at nanomolar concentrations prevented accumulation of ROS and cell death under oxidative stress. These effects required 24 h or more (depending on the cell type) preincubation, and this was not related to slow induction of endogenous antioxidant systems. It is suggested that SkQ slowly accumulates in a small subpopulation of mitochondria that have decreased membrane potential and produce the major part of ROS under oxidative stress. This population was visualized in the cells using potential-sensitive dye. The possible role of the small fraction of "bad" mitochondria in cell physiology is discussed.

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the "window" between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH*. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C(1/2) values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10(-11) and 8x10(-13) M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Deltapsi values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000 : 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3x10(8) times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

Specific inhibitors of mitochondrial functions were used in studies on the relation between bioenergetics and programmed cell death. The data of the authors are discussed in the review.

In HeLa cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP combined with partial inhibition of glycolysis by DOG resulted in a steady threefold decrease in the intracellular ATP level. The ATP level recovers when the DOG-containing medium was replaced by that with high glucose. In 48 h after a transient (3 h) [ATP] lowering followed by recovery of the ATP level, the majority of the cells commits suicide by means of apoptosis. The cell death does not occur if DOG or an oxidative phosphorylation inhibitor was added separately, treatments resulting in 10-35% lowering of [ATP]. Apoptosis is accompanied by Bax translocation to mitochondria, cytochrome c release into cytosol, caspase activation, reactive oxygen species (ROS) generation, and reorganization and decomposition of chromatin. Apoptosis appears to be sensitive to oncoprotein Bcl-2 and a pancaspase inhibitor zVADfmk. In the latter case, necrosis is shown to develop instead of apoptosis. The cell suicide is resistant to cyclosporine A, a phospholipase inhibitor trifluoroperazine, the JNK and p38 kinase inhibitors, oligomycin, N-acetyl cysteine and mitoQ, differing in these respects from the tumor necrosis factor (TNF)- and H(2)O(2)-induced apoptoses. It is suggested that the ATP concentration in the cell is monitored by intracellular "ATP-meter(s)" generating a cell suicide signal when ATP decreases, even temporarily, below some critical level (around 1 mM).

The effects of specific inhibitors of respiratory chain, F(o)F(1)ATP synthase and uncouplers of oxidative phosphorylation on survival of carcinoma HeLa cells and on the structure of mitochondria in the cells were studied. The inhibitors of respiration (piericidin, antimycin, myxothiazol), the F(1)-component of ATP synthase (aurovertin) and uncouplers (DNP, FCCP) did not affect viability of HeLa cells, apoptosis induced by TNF or staurosporin and the anti-apoptotic action of Bcl-2. Apoptosis was induced by combined action of respiratory inhibitors and uncouplers indicating possible pro-apoptotic action of reactive oxygen species (ROS) generated by mitochondria. Short-term incubation of HeLa cells with the mitochondrial inhibitors and 2-deoxyglucose followed by 24-48 h recovery resulted in massive apoptosis. Apoptosis correlated to transient (3-4 h) and limited (60-70%) depletion of ATP. More prolonged or more complete transient ATP depletion induced pronounced necrosis. The inhibitors of respiration and uncouplers caused fragmentation of tubular mitochondria and formation of small round bodies followed by swelling. These transitions were not accompanied with release of cytochrome c into the cytosol and were fully reversible. The combined effect of respiratory inhibitors and uncouplers developed more rapidly indicating possible involvement of ROS generated by mitochondria. More prolonged (48-72 h) incubation with this combination of inhibitors caused clustering and degradation of mitochondria.

The release of cytochrome c from the intermembrane space of mitochondria into the cytosol is one of the critical events in apoptotic cell death. In the present study, it is shown that release of cytochrome c and apoptosis induced by tumor necrosis factor alpha (TNF) in HeLa cells can be inhibited by (i) overexpression of an oncoprotein Bcl-2, (ii) Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (PTP) or (iii) oligomycin, an inhibitor of H+- ATP-synthase. Staurosporine-induced apoptosis is sensitive to Bcl-2 but insensitive to Cyclosporin A and oligomycin. The effect of oligomycin is not due to changes in mitochondrial membrane potential or to inhibition of ATP synthesis/hydrolysis since (a) uncouplers (CCCP, DNP) which discharge the membrane potential fail to abolish the protective action of oligomycin and (b) aurovertin B (another inhibitor of H+-ATP-synthase, affecting its F1 component) do not affect apoptosis. A role of oligomycin-sensitive F0 component of H+-ATP-synthase in the TNF-induced PTP opening and apoptosis is suggested.

Influence of H2O2 on glycolysis was investigated. A hypothesis previously formulated was tested according to which a mild oxidation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) results in uncoupling of oxidation and phosphorylation at this step of glycolysis due to acylphosphatase activity of the oxidized enzyme. Incubation of a mixture of purified glycolytic enzymes, as well as a muscle extract, in the presence of 10-100 microM H2O2 was shown to result in an increase in the rate of glycolysis. The level of lactate accumulation in the oxidized samples increased by 80-150% compared to the samples containing mercaptoethanol. No ATP was formed by the H2O2-stimulated glycolysis. Thus, H2O2 really caused uncoupling of oxidation and phosphorylation in glycolysis. A role of GAPDH oxidation in regulation of glycolysis is discussed.

Light-driven ATP synthesis is found in cells of the alkalophilic bacterium Natronobacterium pharaonis containing halorhodopsin but deficient in H+-pumping bacteriorhodopsin. Photophosphorylation occurs with cyanide-inhibited respiratory chain as well as without cyanide in conditions with low C1- concentration in the incubation medium. Increase in C1- concentration from 0.1 to 2.35 M in the incubation medium leads to inhibition of photophosphorylation. Continuous illumination increases membrane Delta Psi if respiration is inhibited by cyanide. This effect is stimulated by DCCD, an ATPase inhibitor. These data can be explained if one suggests that halorhodopsin pumps C1- into the cells whereas C1- release from the cells through C1--ATP-synthase is coupled with the ATP synthesis (chloride-ion cycle).

In inverted subcellular vesicles of Escherichia coli grown at high delta mu H+ (neutral pH, no protonophorous uncoupler), ATP-driven Na+ transport and oxidative phosphorylation are completely inhibited by the protonophore CCCP. If E. coli was grown at low delta mu H+, i.e. at high pH or in the presence of uncoupler, some oxidative phosphorylation was observed in the vesicles even in CCCP-containing medium, and Na+ transport was actually stimulated by CCCP. The CCCP-resistant transport and phosphorylation were absent from the unc mutant lacking F0F1 ATPase. Both processes proved to be sensitive to (i) the Na+/H+ antiporter monensin, (ii) the Na+ uniporter ETH 157, (iii) the F0 inhibitors DCCD and venturicidin, and (iv) the F1 inhibitor aurovertin. The CCCP-resistant oxidative phosphorylation was stimulated by Na+ and arrested by oppositely directed delta pNa. These data are consistent with the assumption that, under appropriate growth conditions, the F0F1-type ATPase of E. coli becomes competent in transporting Na+ ions.

An attempt has been made to find out which of the two terminal oxidases, the d-type or the o-type, operates as a Na+ pump in Escherichia coli grown at low delta mu H+ conditions. For this purpose, mutants lacking either d or o oxidase have been studied. It is shown that a d-,o+ mutant grows slowly or does not grow at all under low delta mu H+ conditions (alkaline or protonophore-containing growth media were used). Inside-out subcellular vesicles from the d-,o+ mutant cannot oxidize ascorbate and TMPD, and cannot transport Na+ when succinate is oxidized in the presence of a protonophore. The same vesicles are found to transport Na+ when NADH is oxidized as if the Na(+)-motive NADH-quinone oxidase were operative. On the other hand, a mutant lacking o oxidase (d+,o-) grows at low delta mu H+ conditions as fast as the maternal E. coli strain containing both d and o oxidases. Corresponding vesicles oxidize ascorbate and TMPD as well as succinate, the oxidations being coupled to the protonophore-stimulated Na+ transport. Growth in the presence of a protonophore is found to induce a strong increase in the d oxidase level in the maternal d+,o+ E.coli strain. It is concluded that oxidase of the d-type, rather than of the o-type, operates as a Na+ pump in E. coli grown under conditions unfavorable for the H+ cycle.

Mechanisms of Na+ transport into the inside-out subcellular vesicles of alkalo- and halotolerant Bacillus FTU and of Escherichia coli grown at different pH have been studied. Both microorganisms growing at pH 7.5 are shown to possess a system of the respiration-dependent Na+ transport which (i) is inhibited by protonophorous uncoupler, by delta pH-discharging agent diethylammonium (DEA) acetate, by micromolar cyanide arresting the H(+)-motive respiratory chain, and by amiloride, and (ii) is resistant to the Na+/H+ antiporter monensin and to Ag+, inhibitor of the Na(+)-motive respiratory chain. Growth at pH 8.6 strongly changes the activator and inhibitor pattern. Now (1) protonophore stimulates the Na+ transport, (2) DEA acetate is without effect in the absence of protonophore and is stimulating in its presence, (3) amiloride and low cyanide are ineffective, (4) monensin and Ag+ completely arrest the Na+ accumulation in the vesicles. Independent of pH of the growth medium, (a) valinomycin is stimulatory for the Na+ transport, (b) Na+ ionophore ETH 157 is inhibitory and, (c) Na+ transport can be supported by NADH----fumarate as well as by ascorbate (TMPD)----O2 electron transfers. Growth at alkaline pH results in the appearance of ascorbate (TMPD) oxidation resistant to low and sensitive to high cyanide concentrations. These relationships are in agreement with the concept (Skulachev, V.P. (1984) Trends Biochem. Sci. 9, 483-485) that adaptation to alkaline conditions in bacteria growing in the high [Na+] media causes substitution of Na+ for H+ as a coupling ion. The obtained data indicate that under alkaline conditions, Na+ can be pumped from the cell by the Na(+)-motive respiratory chain with neither H(+)-motive respiration nor the Na+/H+ antiporter involved. In the Na(+)-motive respiratory chain of Bac. FTU or E. coli, two Na+ pumps are localized, one in its initial and the other in its terminal spans.