Absence Of Oligomycin Research Articles

Sensory neurons derived from diabetic rats exhibit deficits in functional glycolysis and ATP that are ameliorated by IGF-1

ObjectiveThe distal dying-back of the longest nerve fibres is a hallmark of diabetic neuropathy, and impaired provision of energy in the form of adenosine triphosphate (ATP) may contribute to this neurodegenerative process. We hypothesised that energy supplementation via glycolysis and/or mitochondrial oxidative phosphorylation is compromised in cultured dorsal root ganglion (DRG) sensory neurons from diabetic rodents, thus contributing to axonal degeneration. Functional analysis of glycolysis and mitochondrial respiration and real-time measurement of ATP levels in live cells were our specific means to test this hypothesis. MethodsDRG neuron cultures from age-matched control or streptozotocin (STZ)-induced type 1 diabetic rats were used for in vitro studies. Three plasmids containing ATP biosensors of varying affinities were transfected into neurons to study endogenous ATP levels in real time. The Seahorse XF analyser was used for glycolysis and mitochondrial respiration measurements. ResultsFluorescence resonance energy transfer (FRET) efficiency (YFP/CFP ratio) of the ATP biosensors AT1.03 (low affinity) and AT1.03YEMK (medium affinity) were significantly higher than that measured using the ATP-insensitive construct AT1.03R122/6K in both cell bodies and neurites of DRG neurons (p < 0.0001). The ATP level was homogenous along the axons but higher in cell bodies in cultured DRG neurons from both control and diabetic rats. Treatment with oligomycin (an ATP synthase inhibitor in mitochondria) decreased the ATP levels in cultured DRG neurons. Likewise, blockade of glycolysis using 2-deoxy-d-glucose (2-DG: a glucose analogue) reduced ATP levels (p < 0.001). Cultured DRG neurons derived from diabetic rats showed a diminishment of ATP levels (p < 0.01), glycolytic capacity, glycolytic reserve and non-glycolytic acidification. Application of insulin-like growth factor-1 (IGF-1) significantly elevated all the above parameters in DRG neurons from diabetic rats. Oligomycin pre-treatment of DRG neurons, to block oxidative phosphorylation, depleted the glycolytic reserve and lowered basal respiration in sensory neurons derived from control and diabetic rats. Depletion was much higher in sensory neurons from diabetic rats compared to control rats. In addition, an acute increase in glucose concentration, in the presence or absence of oligomycin, elevated parameters of glycolysis by 1.5- to 2-fold while having no impact on mitochondrial respiration. ConclusionWe provide the first functional evidence for decreased glycolytic capacity in DRG neurons derived from type 1 diabetic rats. IGF-1 protected against the loss of ATP supplies in DRG cell bodies and axons in neurons derived from diabetic rats by augmenting various parameters of glycolysis and mitochondrial respiration.

Red LED Light Acts on the Mitochondrial Electron Chain of Donkey Sperm and Its Effects Depend on the Time of Exposure to Light.

This work aimed to investigate how stimulation of donkey sperm with red LED light affects mitochondrial function. For this purpose, freshly diluted donkey semen was stimulated with red light for 1, 5, and 10 min, in the presence or absence of oligomycin A (Omy A), a specific inhibitor of mitochondrial ATP synthase, or FCCP, a specific disruptor of mitochondrial electron chain. The results obtained in the present study indicated that the effects of red LED light on fresh donkey sperm function are related to changes in mitochondria function. In effect, irradiation of donkey sperm resulted in an increase in mitochondrial membrane potential (MMP), the activity of cytochrome C oxidase and the rate of oxygen consumption. In addition, in the absence of oligomycin A and FCCP, light-stimulation augmented the average path velocity (VAP) and modified the structure of motile sperm subpopulations, increasing the fastest and most linear subpopulation. In contrast, the presence of either Omy A or FCCP abolished the aforementioned effects. Interestingly, our results also showed that the effects of red light depend on the exposure time applied, as indicated by the observed differences between irradiation protocols. In conclusion, our results suggest that exposing fresh donkey sperm to red light modulates the function of their mitochondria through affecting the activity of the electron chain. However, the extent of this effect depends on the irradiation pattern and does not exclude the existence of other mechanisms, such as those related to thermotaxis.

Red LED Light Acts on the Mitochondrial Electron Chain of Mammalian Sperm via Light-Time Exposure-Dependent Mechanisms.

This work analyzes the effects of red LED light on mammalian sperm mitochondrial function, using the pig as an animal model. Liquid-stored pig semen was stimulated with red-light for 1, 5 and 10 min in the presence or absence of oligomycin A, a specific inhibitor of mitochondrial ATP synthase, or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a specific disruptor of mitochondrial electron chain. Whereas exposure for 1 and 5 min significantly (p < 0.05) decreased total motility and intracellular ATP levels, irradiation for 10 min induced the opposite effect. Oligomycin A abolished the light-effects on intracellular ATP levels, O2 consumption and mitochondrial membrane potential, whereas compared to non-irradiated samples, FCCP significantly (p < 0.05) increased O2 consumption when sperm were irradiated for 1 min. Both oligomycin A and FCCP significantly (p < 0.05) decreased total motility. Red-light increased cytochrome c oxidase activity with a maximal effect after 5 min of irradiation, which was abolished by both oligomycin A and FCCP. In conclusion, red-light modulates sperm mitochondrial function via electron chain activity in an exposition, time-dependent manner.

SERCA, complex I of the respiratory chain and ATP-synthase inhibition are involved in pleiotropic effects of NS1619 on endothelial cells

A large conductance potassium (BKCa) channel opener, NS1619 (1,3-dihydro-1- [2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one), is well known for its protective effects against ischemia-reperfusion injury; however, the exact mode of its action remains unclear. The aim of this study was to characterize the effect of NS1619 on endothelial cells.The endothelial cell line EA.hy926, guinea pig hearts and submitochondrial particles isolated from the heart were used. In the isolated guinea pig hearts, which were perfused using the Langendorff technique, NS1619 caused a dose-dependent increase in coronary flow that was inhibited by L-NAME. In EA.hy926 cells, NS1619 also caused a dose-dependent increase in the intracellular calcium ion concentration [Ca2+]i, as measured using the FURA-2 fluorescent probe. Moreover, NS1619 decreased the oxygen consumption rate in EA.hy926 cells, as assessed using a Clark-type oxygen electrode. However, when NS1619 was applied in the presence of oligomycin, the oxygen consumption increased. NS1619 also decreased the mitochondrial membrane potential, as measured using a JC-1 fluorescent probe in the presence and absence of oligomycin. Additionally, the application of NS1619 to submitochondrial particles inhibited ATP synthase.In summary, NS1619 has pleiotropic actions on EA.hy926 cells and acts not only as an opener of the BKCa channel in EA.hy926 cells but also as an inhibitor of the respiratory chain component, sarcoplasmic reticulum ATPase, which leads to the release of Ca2+ from the endoplasmic reticulum. Furthermore, NS1619 has the oligomycin-like property of inhibiting mitochondrial ATP synthase.

Activating oxidative phosphorylation by a pyruvate dehydrogenase kinase inhibitor overcomes sorafenib resistance of hepatocellular carcinoma

Background:Sorafenib is the only drug approved for the treatment of hepatocellular carcinoma (HCC). The bioenergetic propensity of cancer cells has been correlated to anticancer drug resistance, but such correlation is unclear in sorafenib resistance of HCC.Methods:Six sorafenib-naive HCC cell lines and one sorafenib-resistant HCC cell line (Huh-7R; derived from sorafenib-sensitive Huh-7) were used. The bioenergetic propensity was calculated by measurement of lactate in the presence or absence of oligomycin. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, and siRNA of hexokinase 2 (HK2) were used to target relevant pathways of cancer metabolism. Cell viability, mitochondrial membrane potential, and sub-G1 fraction were measured for in vitro efficacy. Reactive oxygen species (ROS), adenosine triphosphate (ATP) and glucose uptake were also measured. A subcutaneous xenograft mouse model was used for in vivo efficacy.Results:The bioenergetic propensity for using glycolysis correlated with decreased sorafenib sensitivity (R2=0.9067, among sorafenib-naive cell lines; P=0.003, compared between Huh-7 and Huh-7 R). DCA reduced lactate production and increased ROS and ATP, indicating activation of oxidative phosphorylation (OXPHOS). DCA markedly sensitised sorafenib-resistant HCC cells to sorafenib-induced apoptosis (sub-G1 (combination vs sorafenib): Hep3B, 65.4±8.4% vs 13±2.9% Huh-7 R, 25.3± 5.7% vs 4.3±1.5% each P<0.0001), whereas siRNA of HK2 did not. Sorafenib (10 mg kg−1 per day) plus DCA (100 mg kg−1 per day) also resulted in superior tumour regression than sorafenib alone in mice (tumour size: −87% vs −36%, P<0.001).Conclusion:The bioenergetic propensity is a potentially useful predictive biomarker of sorafenib sensitivity, and activation of OXPHOS by PDK inhibitors may overcome sorafenib resistance of HCC.

Control of Respiration by Cytochrome c Oxidase in Intact Cells

Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Delta psi) and chemical (Delta pH) component of the mitochondrial transmembrane proton electrochemical gradient (Delta mu(H(+))) was investigated. The overall O(2) consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Delta psi and Delta pH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (C(IV) = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (C(IV) = 0.85) was also measured in the presence of nigericin, i.e. when Delta psi is maximal, and in the presence of nigericin and valinomycin (C(IV) = 0.77), when Delta mu(H(+)) is abolished. In contrast, CcOX displayed a markedly lower control coefficient (C(IV) = 0.30) upon addition of valinomycin, when Delta psi is converted into Delta pH. These results show that Delta psi is responsible for the tight control of CcOX over respiration in actively phosphorylating cells.

A re‐evaluation of the role of matrix acidification in uncoupler‐induced Ca2+ release from mitochondria

Massive amounts of Ca(2+) can accumulate in mitochondria, owing to its complexation with matrix phosphate. Under conditions in which the mitochondrial uniporter is the foremost pathway for Ca(2+) efflux, the release of sequestered Ca(2+) by protonophoric uncouplers is invariably demonstrated. This has been recently ascribed to matrix acidification, which results in the dissociation of the Ca(2+)-phosphate complex. In the present study, we compared the effect of stepwise depolarization on Ca(2+) release induced by either the complex III inhibitor stigmatellin or an uncoupler in energized Ca(2+)-loaded rat liver mitochondria in the presence of phosphate, at extramitochondrial pH (pH(o)) 6.8 and pH(o) 7.8. Both poisons were examined in the presence and absence of oligomycin. Prior to Ca(2+) loading, mitochondria were allowed to phosphorylate 0.5 mm ADP. Opening of the permeability transition pore was additionally hampered by cyclosporin A, and was monitored by changes in light scattering. Na(+) was excluded from the medium, preventing Na(+)/Ca(2+) exchange. At both pH(o) values, Delta pH was in the range 0.11-0.15. Complete depolarization by uncoupling with or without oligomycin resulted in an approximately pH 0.05 acidic shift, but there was none in the case of stigmatellin plus oligomycin. At pH(o) 6.8 and in the presence of oligomycin, the uncoupler-induced Ca(2+) release started in the -80 to -50 mV range, whereas in the absence of oligomycin, the release occurred at approximately -15 mV. Stigmatellin induced minor Ca(2+) release only in the presence of oligomycin, starting at approximately -4 mV. At pH(o) 7.8, the uncoupler-induced Ca(2+) release started at approximately -11 mV, irrespective of the presence or absence of oligomycin. Unexpectedly, at this alkaline pH and in the presence of oligomycin, stigmatellin induced substantial Ca(2+) release, starting at approximately -10 mV. From the above findings, we conclude that matrix acidification cannot be the sole explanation for uncoupler-induced Ca(2+) release from mitochondria.

Uncoupler induced graded mitochondrial depolarization and attenuated matrix calcium uptake are enhanced by complex V inhibition indicative of blocked ATP hydrolysis

Mitochondrial (m) Ca2+ uptake occurs mainly via the Ca2+ uniporter and is highly dependent on the membrane potential (Δ Ψ m). We measured Ca2+ uptake at decreasing ΔΨ m in isolated mitochondria in the presence and absence of oligomycin (OMN) to test the effect of complex V reversal. [Ca2+]m, ΔΨm, pHm, and NADHm were measured spectrophotometrically using indo‐1, rhodamine 123, BCECF fluorescence and autofluorescence, respectively, in guinea pig heart isolated mitochondria. After energizing with pyruvic acid, 0, 10, 20, 30 or 100 µM dinitrophenol (DNP) was added to stepwise reduce Δ?m, after which 80 and 130 nM [Ca2+]e was added. OMN was added in some experiments to block complex V. The effect of DNP to decrease Δ?m, was exaggerated in the presence of OMN. Partial depolarization decreased the rate of matrix Ca2+ uptake, while uptake of Ca2+ resulted in further depolarization. With partial depolarization [Ca2+]m did not reach steady state but increased despite further depolarization. OMN reversed this increase and actually caused a decrease in [Ca2+]m with greater depolarization. pHm and NADHm were also lower with OMN. Our study demonstrates the importance of 1) electrical and chemical gradients for Ca2+ uptake, and 2) a likely effect of ATP hydrolysis at complex V to support ΔΨ m even during mild, graded uncoupling. Proton leaks may lead to reversal of ATP synthesis to maintain membrane potential. NIH, AHA, VA Merit grants.

Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion

Among eight components of avermectin, B1 fractions have the most effective antiparasitic activities and the lowest level of toxic side-effects and are used widely in veterinary and agricultural fields. Intraspecific protoplast fusion between two strains of Streptomyces avermitilis, one an avermectin high producer (strain 76-05) and the other a genetically engineered strain containing the mutations aveD− and olmA− (strain 73-12) was performed for enhancement and selective production of avermectin B in the absence of oligomycin. Two recombinant strains (F23 and F29) were isolated and characterized with regards to the parental merits. F23 and F29 produced only the four avermectin B components with high yield and produced no oligomycin. The avermectin production of F23 and F29 was about 84.20% and 103.45% of the parental strain 76-05, respectively, and increased about 2.66-fold and 3.50-fold, respectively, compared to that of parental strain 73-12. F23 and F29 were genetically stable prototrophic recombinants and F29 was quite tolerant of fermentation conditions compared to avermectin high producer parental strain 76-05. The ability to produce avermectin B with high yield without the production of other avermectin components and oligomycin will make F23 and F29 useful strains for avermectin production. Strain F29’s tolerance of fermentation conditions will also make it suitable for industrial applications.

ATP from Subplasmalemmal Mitochondria Controls Ca2+-dependent Inactivation of CRAC Channels

A sustained Ca2+ entry is the primary signal for T lymphocyte activation after antigen recognition. This Ca2+ entry mainly occurs through store-operated Ca2+ channels responsible for a highly selective Ca2+ current known as I(CRAC). Ca2+ ions act as negative feedback regulators of I(CRAC), promoting its inactivation. Mitochondria, which act as intracellular Ca2+ buffers, have been proposed to control all stages of CRAC current and, hence, intracellular Ca2+ signaling in several types of non-excitable cells. Using the whole-cell configuration of the patch clamp technique, which allows control of the intracellular environment, we report here that respiring mitochondria located close to CRAC channels can regulate slow Ca2+-dependent inactivation of I(CRAC) by increasing the Ca2+-buffering capacity beneath the plasma membrane, mainly through the release of ATP.

Functional identification of ATP-sensitive K+ uniporter in mitochondria from sugar beet taproot

Mitochondria isolated from sugar beet (Beta vulgaris L.) taproot were shown to swell spontaneously after the transfer from a sucrose-containing isolation medium to isoosmotic potassium chloride solutions. The kinetics of this process was strongly retarded after the replacement of potassium with sodium in the incubation medium and was substantially stimulated by the electron-transport chain activity and valinomycin. At neutral pH of the incubation medium, the rate of K+-dependent swelling of mitochondria decreased by 30–50% after adding 1 mM ATP but was insensitive to other nucleotides (GTP, UTP, and CTP). In the medium acidified to pH 6.0, the addition of ATP caused shrinkage of mitochondria that had been swollen in the KCl medium. In the absence of this nucleotide, the kinetics of K+-dependent swelling of mitochondria was considerably decelerated upon the acidification of the incubation medium. The effects of ATP were independent of the presence or absence of oligomycin and atractyloside. However, the ATP-dependent shrinkage of mitochondria was inhibited in the presence of quinine, and this agent also inhibited K+-dependent swelling of organelles in potassium acetate solutions. The presence of K+ ions in the incubation medium caused a rapid dissipation of the mitochondrial membrane potential (Δψ) that was generated during succinate oxidation. The addition of ATP to the reaction medium resulted in the oligomycin-insensitive restoration of Δψ. The results are regarded as evidence that the membrane of taproot mitochondria is endowed with functionally active ATP-sensitive K+ uniporter. This system is likely to represent a K+ channel that catalyzes the electrogenic transfer of potassium ions to the mitochondrial matrix. It is supposed that the membrane of taproot mitochondria also contains a quinine-sensitive K+/H+ antiporter that catalyzes the efflux of potassium from the matrix or, on the contrary, the accumulation of K+ in the presence of potassium acetate.

Oligomycin and antimycin A prevent nitric oxide–induced apoptosis by blocking cytochrome C leakage

Nitric oxide (NO) is a potent inducer of apoptosis, and its cytotoxicity is closely related to mitochondrial dysfunction. In this study we investigated the effects of a F0F1-ATPase inhibitor, oligomycin, and a mitochondrial respiratory chain complex III inhibitor, antimycin A, on NO-induced apoptosis. We used a normal rat gastric-epithelium cell line, RGM-1, treated with a pure NO donor, NOC-1 —1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene — in the presence or absence of oligomycin or antimycin A. Changes in the expressions of Bax or Bcl-2 proteins, release of cytochrome C from mitochondria into the cytosol, activation of caspase-3, and changes in the mitochondrial membrane potential (ΔΨ) were measured with the use of Western blotting, c43 lorimetric assays, and a mitochondrial potential sensor, JC-1 dye. Treatment with NOC-18 induced dose-dependent apoptotic cell death in RGM-1 cells. Cell death was accompanied by mitochondrial depolarization, increases in Bax protein expression and cytochrome C leakage, and, subsequently, caspase-3 activation. Oligomycin and antimycin A prevented NO-induced apoptosis in a dose-dependent fashion by preventing cytochrome C release independent of Bcl-2 expression. However, neither compound affected the up-regulation of Bax protein. On the one hand, oligomycin treatment was not accompanied by a decline in ΔΨ. On the other hand, antimycin A treatment decreased ΔΨ regardless of NOC-18 treatment. The findings of this study suggest that various functional molecules that constitute the mitochondrial respiratory chain may contribute to cytochrome C release that occurs during NO-induced apoptosis.

Carvedilol in heart mitochondria: Protonophore or opener of the mitochondrial K ATP channels?

Carvedilol ({1–[carbazolyl–(4)-oxy]-3-[2-methoxyphenoxyethyl) amino]-propanol-(2)}) has been shown to protect cardiac mitochondria from oxidative stress. In this work we examined the mechanisms responsible for an observed depressive effect in the mitochondrial transmembrane potential (ΔΨ). Two possible mechanisms were considered: a protonophoretic activity and the opening of mitochondrial ATP-sensitive potassium channels. We show that carvedilol increases mitochondrial inner membrane permeability to protons, but not to potassium, causing an increase in state IV respiration in the presence and absence of oligomycin. By contrast, a K ATP-channel inhibitor, 5-hydroxydecanoic acid, did not affect carvedilol-induced depolarizations. Hence, our results suggest that carvedilol depresses mitochondrial ΔΨ by a weak protonophoretic mechanism.

Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

The relationship between changes in mitochondrial membrane potential (Deltapsi(m)) and the failure of cytoplasmic Ca(2+) homeostasis, delayed Ca(2+)deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetramethylrhodamine methyl ester (TMRM(+)) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of Deltapsi(m) and plasma membrane potential (Deltapsi(P)). Glutamate usually caused an immediate decrease in Deltapsi(m) of <10 mV, attributable to Ca(2+) accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca(2+) that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca(2+) ([Ca(2+)](c)) has risen to the set point at which mitochondria retain Ca(2+). In the presence of oligomycin no increase in [Ca(2+)](c) occurs during this depolarization. We conclude that transient Ca(2+) loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca(2+) extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.

Biotransformation of saturated monohydroxyl fatty acids to 2‐tetrahydrofuranyl acetic acid derivatives: Mechanism of formations and the biological activity of 5‐n‐hexyl‐tetrahydrofuran‐2‐acetic acid

AbstractTransformation of 12‐hydroxyoctadecanoic acid (12‐HOA) to 5‐n‐hexyl‐tetrahydrofuran‐2‐acetic acid (5‐HTFA) byBacillus lentus NRRL B‐14864 (B‐14864) was carried out in the presence or absence of oligomycin, 2‐bromooctanoic acid (2‐BA), or sodium azide. In addition, several saturated and monounsaturated monohydroxyfatty acids, saturated monooxofatty acids, and monounsaturated fatty acid were used as substrates for transformation reactions by B‐14864 or corynebacterium FUI‐2. Methyl esters of the transformation products were analyzed by gas chromatography and gas chromatography/mass spectroscopy. Various γ‐lactones and tetrahydrofuran fatty acid derivatives were conversion products when saturated monohydroxyfatty acids were used as substrates; the production of 5‐HTFA from 12‐HOA by B‐14864 cells was completely inhibited in the presence of high concentration of oligomycin, 2‐BA, or sodium azide; and fatty acid β‐oxidation metabolic intermediates, 6‐hydroxydodecanoic, 4‐oxododecanoic, and 4‐oxodecanoic acids were products when 12‐HOA, 10‐oxo‐, and 12‐oxooctadecanoic acids were used as substrates. Our results suggest that the production of 5‐HTFA from 12‐HOA by B‐14864 was through the fatty acid β‐oxidation pathway. Three‐day‐old driedfruit beetle pupae were topically treated with 5‐HTFA to test for juvenile hormone activity, and 5‐HTFA was found to possess juvenile hormone‐like activity in pure form but not when it was diluted to 10%.

Mechanistic stoichiometry of yeast mitochondrial oxidative phosphorylation.

This study investigates the relationships between the efficiency of oxidative phosphorylation (ATP/O) and respiratory flux in yeast mitochondria. To manipulate the electron flux through the respiratory chain, different substrates leading to NAD(P)H were used. By testing the effect of ADP either on respiratory rate in the presence or absence of oligomycin or on the level of NAD(P)H, on one hand, and the effects of uncouplers on respiration, on the other, we distinguished several categories of substrates: those for which the low respiration rate was mainly controlled by dehydrogenase activities and others for which the respiration was high and controlled downstream from the dehydrogenases. By using these different substrates, we observed that the ATP/O ratio decreased irrespective of the proton-motive force when the electron flux increased, unlike the situation when the respiratory rate was modulated by addition of the respiratory inhibitor. This result suggests that the oxidative phosphorylation efficiency depends on the value of the flux crossing the proton pumps. This relationship between efficiency (ATP/O) and electron flux was linked to a main control upstream from the respiratory chain. Such changes in the ATP/O ratio at least involved changes in the stoichiometry (H+/2e-) of the respiratory chain. Indeed, in non-phosphorylating mitochondria, the ratio of stoichiometries at site 2+3 over site 3 varied according to the proton-motive force. This cannot be explained by a variation in proton leak alone but involved both (i) a variable stoichiometry (H+/2e-) in relation to the electron flux value and (ii) different relationships between the variation in stoichiometry and the flux value at each coupling site.

Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes.

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.

Energy-storage capacity of the mitochondrial proton-motive force

Resting state respiration of rat-liver mitochondria in the presence of oligomycin was rapidly blocked with cyanide and the dissipation of the membrane potential was followed with a tetraphenylphosphonium-sensitive electrode. From the rate of this dissipation and the electric capacitance of the mitochondrial membrane the energy stored in form of the membrane potential was calculated as about 7 μJ/mg protein. In the absence of oligomycin, dissipation of the membrane potential was slower, as it was partly compensated by proton ejection by mitochondrial ATPase hydrolyzing endogenous ATP. This allowed to calculate the total energy storage capacity of the proton-motive force. It amounted to the equivalence of 3.3 nmol ATP/mg protein or about 130 μJ/mg protein. The stoichiometry of proton-pumping ATPase utilizing endogenous ATP was estimated as three protons per molecule ATP.

Steady-state levels of phosphorylated intermediates of (Na,K)-ATPase monitored with oligomycin and anthroylouabain.

We present experiments designed to test whether or not the ADP-sensitive (E1P) and the K-sensitive (E2P) phosphoenzyme forms of (Na,K)-ATPase are intermediates of ATP hydrolysis in the presence of Na and K. We have used the apparent rate constant of anthroylouabain binding, k' on, and its inhibition by oligomycin to monitor the steady-state levels of E2P and E1P, respectively. We have measured k' on in purified dog kidney (Na,K)-ATPase at 37 degrees C as a function of Na, K, and MgATP or Mg plus inorganic phosphate in the presence and absence of oligomycin. In the presence of 35 mM Na and no K, k' on was saturated with [ATP] greater than 25 microM, and oligomycin (10 microM) inhibited less than 10%. Inhibition by oligomycin increased when [Na] was increased. At 1.9 M Na oligomycin inhibited k' on and equilibrium binding of anthroylouabain with Ki = 136 nM. Oligomycin had no effect on k' on in the presence of Mg + inorganic phosphate. In the presence of 35 mM Na and 4.5 mM K, k' on was much slower, inhibition by oligomycin increased, and ATP stimulated both k' on and inhibition by oligomycin with K0.5 greater than 0.1 mM. From the k' on measurements in the presence and absence of K, we estimated that the steady-state level of E2P was between 4 and 15% of the enzyme with 25 microM to 6.5 mM ATP, 35 mM Na, and 4.5 mM K at 37 degrees C. For these conditions we estimated the rate constant of E2P hydrolysis to be 46,000 (+/- 1,000) min-1. These results are contrary to the predictions derived from a model in which acid-stable phosphoenzymes are not intermediates of ATP hydrolysis in the presence of Na and K. They are fully consistent with a model in which E1P and E2P are intermediates of ATP hydrolysis coupled to transport of Na and K.

Genetics of the mammalian oxidative phosphorylation system: characterization of a new oligomycin-resistant Chinese hamster ovary cell line.

The properties of a new type of oligomycin-resistant Chinese hamster ovary (CHO) cell line (Olir 2.2) are described in this paper. Olir 2.2 cells were approximately 50,000-fold more resistant to oligomycin than were wild-type CHO cells when tested in glucose-containing medium, but only 10- to 100-fold more resistant when tested in galactose-containing medium. Olir 2.2 cells grew with a doubling time similar to that of wild-type cells both in the presence or absence of oligomycin. Oligomycin resistance in Olir 2.2 cells was stable in the absence of drug. In vitro assays indicated that there was approximately a 25-fold increase in the resistance of the mitochondrial ATPase to inhibition by oligomycin in Olir 2.2 cells, with little change in the total ATPase activity. The electron transport chain was shown to be functional in Olir 2.2 cells. Olir 2.2 cells were cross-resistant to other inhibitors of the mitochondrial ATPase (such as rutamycin, ossamycin, peliomycin, venturicidin, leucinostatin, and efrapeptin) and to other inhibitors of mitochondrial functions (such as chloramphenicol, rotenone, and antimycin). Oligomycin resistance was expressed codominantly in hybrids between Olir 2.2 cells and wild-type cells. Cross-resistance to ossamycin, peliomycin, chloramphenicol, antimycin, venturicidin, leucinostatin, and efrapeptin was also expressed codominantly in hybrids. Fusions of enucleated Olir 2.2 cells with wild-type cells and characterization of the resulting cybrid clones indicated that resistance to oligomycin and ossamycin results from a mutation in both a nuclear gene and a cytoplasmic gene. Cross-resistance to efrapeptin, leucinostatin, venturicidin, and antimycin results from a mutation in only a nuclear gene.