Low Concentrations Of Uncouplers Research Articles

Low Concentrations of Uncouplers of Oxidative Phosphorylation Prevent Inflammatory Activation of Endothelial Cells by Tumor Necrosis Factor.

In endothelial cells, mitochondria play an important regulatory role in physiology as well as in pathophysiology related to excessive inflammation. We have studied the effect of low doses of mitochondrial uncouplers on inflammatory activation of endothelial cells using the classic uncouplers 2,4-dinitrophenol and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole, as well as the mitochondria-targeted cationic uncoupler dodecyltriphenylphosphonium (C12TPP). All of these uncouplers suppressed the expression of E-selectin, adhesion molecules ICAM1 and VCAM1, as well as the adhesion of neutrophils to endothelium induced by tumor necrosis factor (TNF). The antiinflammatory action of the uncouplers was at least partially mediated by the inhibition of NFκB activation due to a decrease in phosphorylation of the inhibitory subunit IκBα. The dynamic concentration range for the inhibition of ICAM1 expression by C12TPP was three orders of magnitude higher compared to the classic uncouplers. Probably, the decrease in membrane potential inhibited the accumulation of penetrating cations into mitochondria, thus lowering the uncoupling activity and preventing further loss of mitochondrial potential. Membrane potential recovery after the removal of the uncouplers did not abolish its antiinflammatory action. Thus, mild uncoupling could induce TNF resistance in endothelial cells. We found no significant stimulation of mitochondrial biogenesis or autophagy by the uncouplers. However, we observed a decrease in the relative amount of fragmented mitochondria. The latter may significantly change the signaling properties of mitochondria. Earlier we showed that both classic and mitochondria-targeted antioxidants inhibited the TNF-induced NFκB-dependent activation of endothelium. The present data suggest that the antiinflammatory effect of mild uncoupling is related to its antioxidant action.

Oxidative Stress and Energy Crises in Neuronal Dysfunction

Mitochondrial dysfunction is implicated in many forms of cell death, particularly in the central nervous system. The mitochondria are required at the same time to generate adenosine 5'-triphosphate (ATP) for the cell, sequester excess cytoplasmic Ca(2+), and both produce and detoxify superoxide free radicals. The electron transport chain and proton circuit are central in keeping these three balls in the air at the same time. We have investigated the bioenergetics of the in situ mitochondria in cultured neurons exposed to pathological glutamate concentrations to model glutamate excitotoxicity and have revised the conventional view that mitochondrial calcium loading results in increased oxidative stress that damages the mitochondrion and ultimately the cell. Instead, a central role is played under these conditions by limitations in mitochondrial and cellular ATP generating capacity. Sodium and calcium entering via the N-methyl-D-aspartate receptor impose a large energetic load on cells and can use the entire respiratory capacity of the in situ mitochondria. As a result, even modest restrictions in mitochondrial capacity -- caused by low concentrations of electron transport chain inhibitors such as rotenone, as in models of Parkinson's disease; low concentrations of uncouplers, to test the so-called neuroprotective mild uncoupling hypothesis; or preexisting oxidative stress -- greatly potentiate glutamate excitotoxicity. Our findings may lead to a reevaluation of the potential for mild uncoupling to provide a neuroprotective role in aging-related neurodegenerative disorders because the deleterious consequences of restricting ATP generating capacity greatly outweigh the negligible effects on the levels of mitochondrial superoxide radicals in intact neurons.

The Role of External and Matrix pH in Mitochondrial Reactive Oxygen Species Generation

Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when ΔpH = 0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

The role of respiration in the activation of photosynthesis upon illumination of dark adapted Chlamydomonas reinhardtii

It is reported that O 2 is required for the activation of photosynthesis in dark adapted Chlamydomonas reinhardtii in State 1, under low light intensity. The concentration of dissolved O 2 of ca. 9 µM is sufficient to saturate the requirement. When the concentration of O 2 is 3 μM or below, the activation of photosynthesis is strongly inhibited by myxothiazol, a specific inhibitor of the mitochondrial cytochrome bc 1. The effect of this inhibitor decreases as the O 2 concentration is raised, to disappear completely above 50 μM. Low concentrations of uncouplers delay the activation of photosynthesis, but do not inhibit it when steady state is reached. It is concluded that in State 1 C. reinhardtii mitochondrial respiration is required for the activation of photosynthesis upon illumination of dark adapted cells only when the concentration of O 2 is too low (less than 5 μM) to allow an appreciable activity of the Mehler reaction. The role of respiration does not seem to be due to the synthesis of ATP by oxidative phosphorylation, because photosynthesis activation is not sensitive to oligomycin.

Mitochondrial uncouplers with an extraordinary dynamic range

We have discovered that some weak uncouplers (typified by butylated hydroxytoluene) have a dynamic range of more than 10(6) in vitro: the concentration giving measurable uncoupling is less than one millionth of the concentration causing full uncoupling. They achieve this through a high-affinity interaction with the mitochondrial adenine nucleotide translocase that causes significant but limited uncoupling at extremely low uncoupler concentrations, together with more conventional uncoupling at much higher concentrations. Uncoupling at the translocase is not by a conventional weak acid/anion cycling mechanism since it is also caused by substituted triphenylphosphonium molecules, which are not anionic and cannot protonate. Covalent attachment of the uncoupler to a mitochondrially targeted hydrophobic cation sensitizes it to membrane potential, giving a small additional effect. The wide dynamic range of these uncouplers in isolated mitochondria and intact cells reveals a novel allosteric activation of proton transport through the adenine nucleotide translocase and provides a promising starting point for designing safer uncouplers for obesity therapy.

In vivo changes of the oxidation-reduction state of NADP and of the ATP/ADP cellular ratio linked to the photosynthetic activity in Chlamydomonas reinhardtii.

The ATP/ADP and NADP/NADPH ratios have been measured in whole-cell extract of the green alga Chlamydomonas reinhardtii, to understand their availability for CO(2) assimilation by the Calvin cycle in vivo. Measurements were performed during the dark-light transition of both aerobic and anaerobic cells, under illumination with saturating or low light intensity. Two different patterns of behavior were observed: (a) In anaerobic cells, during the lag preceding O(2) evolution, ATP was synthesized without changes in the NADP/NADPH ratio, consistently with the operation of cyclic electron flow. (b) In aerobiosis, illumination increased the ATP/ADP ratio independently of the intensity used, whereas the amount of NADPH was decreased at limiting photon flux and regained the dark-adapted level under saturating photon flux. Moreover, under these conditions, the addition of low concentrations of uncouplers stimulated photosynthetic O(2) evolution. These observations suggest that the photosynthetic generation of reducing equivalents rather than the rate of ATP formation limits the photosynthetic assimilation of CO(2) in C. reinhardtii cells. This situation is peculiar to C. reinhardtii, because neither NADPH nor ATP limited this process in plant leaves, as shown by their increase upon illumination in barley (Hordeum vulgare) leaves, independent of light intensity. Experiments are presented and were designed to evaluate the contribution of different physiological processes that might increase the photosynthetic ATP/NADPH ratio-the Mehler reaction, respiratory ATP supply following the transfer of reducing equivalents via the malate/oxaloacetate shuttle, and cyclic electron flow around PSI-to this metabolic situation.

Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells

Mitochondrial membrane potential, in situ, is an important indicator of mitochondrial function and dysfunction. Because of recent interest in the role of mitochondria in signaling, cell injury and cell death, there is a need for a convenient, sensitive and accurate method for the measurement of the mitochondrial membrane potential, Δψ m, in situ, in a heterogeneous cell population. We have adapted a flow cytometry method for the quantitative measurement of ΔΨ m which utilizes the lipophilic, cationic, fluorescent probe 3,3′-dihexyloxacarbocyanine iodide (DiOC 6(3)). We developed a new protocol in which cells are equilibrated with very low dye concentrations (<1 nM). Only under these condition, the cell fluorescence appears to be correlated with the magnitude of ΔΨ m, as evident from the sensitivity of the fluorescence to low concentrations of uncouplers, ionophores and inhibitors of the mitochondrial proton pumps. The magnitude of the plasma membrane potential, ΔΨ p, also affects cell fluorescence, and a procedure that corrects for this effect is outlined. This method offers a distinct advantage over existing methods for estimation of Δψ m by flow cytometry.

6-Ketocholestanol abolishes the effect of the most potent uncouplers of oxidative phosphorylation in mitochondria

The effect of a keto-derivative of cholesterol, namely, 6-ketocholestanol (5α-cholestan-3β-ol-6-one; kCh) on the uncoupling of oxidation and phosphorylation by various uncouplers was studied in rat heart mitochondria. kCh was found to completely abolish the uncoupling effect (the increase in the respiration rate under the respiratory control conditions and the decrease in the membrane potential) caused of FCCP, CCCP and SF6847 and partially by TTFB at low concentrations of uncouplers. It was without effect on the uncoupling by PCP, DNP and palmitate. Carboxyatractylate, a specific inhibitor of the ADP/ATP-antiporter, was shown to almost completely abolish the uncoupling induced by palmitate and partially by low concentration of TTFB, PCP and DNP. Effects of high concentrations of all these uncouplers as well as of any concentrations of gramicidin proved to be kCh- and carboxyatractilate-insensitive. The data are discussed in terms of the hypothesis on the protein-mediated mechanism of the protonophorous uncoupling.

Early metabolic effects and mechanism of ammonium transport in yeast

Studies were performed to define the effects and mechanism of NH 4 + transport in yeast. The following results were obtained. Glucose was a better facilitator than ethanol-H 2O 2 for ammonium transport; low concentrations of uncouplers or respiratory inhibitors could inhibit the transport with ethanol as the substrate. With glucose, respiratory inhibitors showed only small inhibitory effects, and only high concentrations of azide or trifluoromethoxy carbonylcyanide phenylhydrazone could inhibit ammonium transport. Ammonium in the free state could be concentrated approximately 200-fold by the cells. Also, the addition of ammonium produced (a) stimulation of both respiration and fermentation; (b) an increased rate of H + extrusion and an alkalinization of the interior of the cell; (c) a decrease of the membrane potential, as monitored by fluorescent cyanine; (d) an immediate decrease of the levels of ATP and an increase of ADP, which may account for the stimulation of both fermentation and respiration; and (e) an increase of the levels of inorganic phosphate. Ammonium was found to inhibit 86Rb + transport much less than K +. Also, while K + produced a competitive type of inhibition, that produced by NH 4 + was of the noncompetitive type. From the distribution ratio of ammonium and the pH gradient, an electrochemical potential gradient of around −180 mV was calculated. The results indicate that ammonium is transported in yeast by a mechanism similar to that of monovalent alkaline cations, driven by a membrane potential. The immediate metabolic effects of this cation seem to be due to an increased [H +]ATPase, to which its transport is coupled. However, the carriers seem to be different. The transport system studied in this work was that of low affinity.

Relationships between rates of steady-state ATP synthesis and the magnitude of the proton-activity gradient across thylakoid membranes

Rates of ATP synthesis by illuminated spinach chloroplast thylakoids in the steady state have been compared to the magnitude of the pH difference across the thylakoid membrane (ΔpH). The apparent dependence of the rate of photophosphorylation on ΔpH was not affected by the method used to vary these parameters. ΔpH generated by electron flow through each photosystem alone or through both photosystems was equivalent in its ability to drive ATP synthesis. The uncouplers, gramicidin and carbonylcyanide m-chlorophenylhydrazone, affected the relationship between phosphorylation and ΔpH in the same way as alteration in the rate of electron transport by changes in light intensity. Also, N,N′-dicyclohexylcarbodiimide, an inhibitor of phosphorylation, increased ΔpH as phosphorylation is inhibited. The Km of ADP for photophosphorylation was previously reported to increase when uncouplers were used to decrease the rate, but to decrease when the light intensity was lowered (Vinkler, C. (1981) Biochem. Biophys. Res. Commun. 99, 1095–1100). We show that higher uncoupler concentrations decrease the Km for ADP and provide an explanation for the increase in the Km caused by low uncoupler concentrations. These results are consistent with the concept that steady state ATP synthesis in thylakoids is driven by delocalized pH gradients.

The effect of low concentrations of uncouplers on the detectability of proton deposition in thylakoids. Evidence for subcompartmentation and preexisting pH differences in the dark

Flash-induced pH changes inside thylakoids were measured with neutral red as an indicator in the presence and absence of low concentrations of uncouplers. We found that both carrier-type and pore-forming uncouplers caused the rapidly rising phase of the neutral red signal, previously attributed to proton deposition by water oxidation, to disappear. Gramicidin was particularly efficient in this respect, requiring only one molecule of uncoupler per 10 4 chlorophyll molecules to render the rapid proton deposition undectectable. This suggests that gramicidin did not act on each water-oxidizing enzyme individually, but rather at the level of the thylakoid membrane. In contrast to the effect on water-derived protons, the appearance of protons from plastoquinol was unaffected by gramicidin. Nor did gramicidin affect the rise of the neutral red signal due to proton deposition during two Photosystem I partial reactions with artificial donors. At the low gramicidin concentrations used, its effect on the neutral red signal could not be attributed to a general increase in proton permeability of the thylakoid membrane (acceleration of half decay from 9 to 0.8 s). The extent of alkalinization of the external medium during the first few hundred milliseconds following a light flash was unaltered by gramicidin, and we did not observe a kinetic correlation between the disappearance of the water proton and the decay of the transmembrane electric field. The last two findings suggest that the undetected protons had not crossed the thylakoid membrane, but instead were buffered away by some gramicidin-induced extra buffering power. pH titration of this extra buffering power revealed an apparent p K ranging between 7.2 and 7.7 and a stoichiometry of 2 H + site . The rapid phase of the neutral red signal regained 90% of its original amplitude after seven flashes were applied at 6.7 Hz repetition rate to a sample containing gramicidin. This suggests limits to the extra buffering power. One possible interpretation of our experiments is the following: Protons derived from water oxidation are initially deposited into extended and highly buffering special domains, and only escape into the common internal phase when the buffering capacity of the domains is saturated. As an alternative one may consider that the thylakoid lumen is partitioned into at least two domains, each dominated by different photosystems and with slow proton equilibration between them. Either view requires internal subcompartmentation. The consequences of such subcompartmentation for chloroplast bioenergetics are still obscure.

Initiation of flagella rotation in Rhodopseudomonas sphaeroides

Flagellate bacteria move by rotating semi-rigid helical flagella in either a clockwise or anticlockwise direction, using the frequency of switching between the two directions of rotation to bias their overall movement in a favourable direction (Macnab, 1978). It is probable that flagellar rotation involves the movement of protons down the membrane proton gradient, between two complex protein rings in the cytoplasmic membrane. One protein ring appears to be fixed to the flagellum and free to rotate, and the other is fixed in the membrane. The movement of protons via an unidentified proton pore in the ring system causes the two rings to move against each other. This rotation can use either the electrical or chemical potential to power the rotation (Khan & Macnab, 1980). Although the energetics of flagellar rotation have been investigated, the mechanism by which flagellar rotation is initiated, or the rotation in one direction is stopped and restarted in the opposite direction, by using a unidirectional proton gradient, is unknown. Photosynthetic bacteria become non-motile if incubated anaerobically in the dark, but regain motility upon actinic illumination. If a useful protonmotive force is the only requirement for initiation of flagellar rotation, as is assumed for its continued rotation, then the time taken for motility to resume upon illumination after incubation in the dark should be similar to the time for the creation of a stable increased membrane potential. Rhodopseudomonas sphaeroides possesses an endogenous indicator of membrane potential, the carotenoids. When there is a change in the potential across the cytoplasmic membrane, there is an electrochromic shift in the absorbance of the carotenoids (Jackson & Crofts, 197 1). Photosynthetically grown R . sphaeroides were isolated into either Hepes [4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid1 or potassium phosphate buffer (lmM, pH7.0) with IOpM-disodium EDTA. After several hours anaerobic incubation in the dark, the bacteria were actinically illuminated and the carotenoid bandshift was measured in an Aminco dual-wavelength spectrophotometer as previously described (Armitage & Evans, 1979). There was a very fast rise in membrane potential as measured by the carotenoid bandshift (half-time, ti 5 5 ms). Similarly, if the bacteria were incubated in 1 O p ~ buffer for several hours in the dark in an anaerobic stirred vessel there was a decrease of about 0.1 pH unit within 15 s of illumination. However, when R. sphaeroides, isolated under similar conditions and sealed into micro-slides (50mm, with O.lmm pathlength), was examined with similar illumination conditions on the microscope, the first bacterium did not start to move for several seconds and the normal percentage of motile bacteria was not observed until more than 60s illumination. The effect could be enhanced by the addition of low concentrations of uncouplers. If 1 ~ M C C C P ' (final concn.) was added to the bacteria in the dual-wavelength spectrophotometer, there was a gradual blue shift in carotenoid absorbance taking several minutes. When the new baseline absorbance stabilized (the decrease in potential being presumably the result of residual respiration in the dark), the actinic light-induced bandshift remained unaltered or increased. The bacteria therefore retained their ability to maintain a light-induced membrane potential. Measurement of external pH under similar conditions showed the same result as for untreated bacteria. However, examination microscopically of the CCCP-treated cells showed an increase in the time taken for motility to resume. It was now several minutes before the first bacterium moved, and many more before 50% were motile. When motility restarted, individual bacteria moved at normal speed and their behaviour was similar to that of untreated bacteria. The length of the delay was related to the concentration of uncoupler, which could also be related to the decrease in the size of the bandshift as the concentration of uncoupler was increased. As the light-induced membrane potential, measured by the bandshift, decreased, the time taken for motility to resume increased (16 s for first motile bacterium at 0.5 ~ M C C C P , 30s at l p ~ , 65s at 5pM). However, the time taken for motility to resume at each uncoupler concentration was independent of the length of dark incubation after uncoupler addition, the maximum delay remaining constant after 12 min dark incubation. The length of the delay was also pH-dependent; at pH6.5 O. I~M-CCCP caused a delay of 2min, whereas at pH7.5 the same concentration only delayed the onset of motility by 7 s. These results suggest that the protonmotive force alone is not enough for the initiation of flagellar rotation, but there is an additional potential-dependent component, possibly a protein modification. It is possible that this is also required for the switching mechanism, as bacteria with artificially low membrane potentials have been shown to be unable to change direction (Khan & Macnab, 1980).

The effect of the uncoupler carbonyl cyanide m-chlorophenylhydrazone on K + transport, ATP level and intracellular pH of Chlorella fusca

1. Low concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced net K + uptake by Chlorella fusca, optimal concentrations being 3 μM CCCP in the light and 1 μM CCCP in the dark. Higher concentrations increasingly stimulated K + release. 2. Measurements of the unidirectional K + fluxes showed that CCCP-induced net K + uptake in the light was mainly a consequence of an inhibition of efflux. In the dark, influx was slightly stimulated in addition. 3. In conditions of CCCP-induced net K + uptake, the ATP level was decreased by less than 10%. With higher CCCP concentrations it fell drastically. 4. By means of the 5,5-dimethyloxazolidine-2,4-dione distribution technique, an acidification of the cell interior on the addition of CCCP was found. 5. It is concluded that uncoupler-induced net K + uptake is due to an enhanced proton leakage into the cell across the plasmalemma. Intracellular acidification by this process stimulates ATP-dependent K +/H + exchange which, in itself, is not affected at low uncoupler concentrations.

Cyanide-insensitive oxidation of ascorbate + NNN'N'-tetramethyl-p-phenylenediamine mixture by mung-bean (Phaseolus aureus) mitochondria. An energy-linked function.

Freshly prepared washed or purified mung-bean (Phaseolus aureus) mitochondria utilize oxygen with ascorbate/tetramethyl-p-phenylenediamine mixture as electron donor in the presence of KCN. ATP control of the oxygen uptake can be observed with very fresh mitochondria. The electron flow, which is inhibited by antimycin A, salicylhydroxamic acid or octylguanidine, takes place by reversed electron transport through phosphorylation site II and thence to oxygen through the cyanide-insensitive pathway. Oligomycin and low concentrations of uncoupler partially inhibit the oxygen uptake in a manner similar to that observed for other energy-linked functions of plant mitochondria. An antimycin A-insensitive oxygen uptake occurs if high concentrations of uncoupler are used, indicating that the pathway of electron flow has been altered. The process of cyanide-insensitive ascorbate oxidation is self-starting, and, since it occurs in the presence of oligomycin, it is concluded that the reaction can be energized by a single energy-conservation site associated with the cyanide-insensitive oxidase pathway.

Different sensitivities of chloroplasts to uncouplers when ATP formation is induced by continuous illumination, by brief illumination, by pre-illumination, or by acid-base transitions.

All of the uncouplers tested are more effective in decreasing ATP formation at the beginning of the illumination period than they are during continuous illumination. The difference is quite small with the uncouplers methylamine and atebrin but very large with the uncouplers octylamine and carbonylcyanide p‐fluoromethoxyphenylhydrazone (FCCP). The illumination time required to initiate photophosphorylation in the absence of permeant ions is less than 5 ms at high light intensities and the short delay is not much affected by methylamine and atebrin. The delay on the onset of ATP formation is increased to about 40 ms by permeant ions such as K+ in the presence of valinomycin, although the same permeant ions have little or no effect on steady‐state phosphorylation [Ort and Dilley (1976) Biochim. Biophys. Acta, 449, 95–107]. Octylamine and FCCP also delay the onset of phosphorylation for about 40 ms and they do so at concentrations too low to cause significant inhibition of steady‐state phosphorylation. This may be the reason that they are much more inhibitory to phosphorylation during short illumination periods than during longer illumination periods. The delays in phosphorylation introduced by permeant ions and by low concentrations of the uncouplers octylamine and FCCP are not additive. Higher concentrations of all of the uncouplers which are capable of causing major inhibitions of steady‐state phosphorylation increase the delay in the onset of phosphorylation, whether or not permeant ions are present. The increase in delay caused by these higher concentrations is a function of the degree of uncoupling and of the extent of the delay already present at low uncoupler concentrations. For reasons which remain obscure, post‐illumination ATP formation is much more sensitive to the uncoupler methylamine than is ATP formation induced by either continuous illumination or acid‐base transitions. It is concluded that for the first 40 ms of illumination a transmembrane electric potential gradient is required for photophosphorylation and that permeant ions prevent the formation of such a potential by carrying charges across the lamellar membrane. Presumably the protonated form of octylamine, the octylammonium ion, is itself a permeant cation and therefore functions like K+ in the presence of valinomycin, whereas methylamine once protonated is probably trapped inside the lamellar vesicle, thus preserving the membrane potential. On the other hand, FCCP may destroy any developing membrane potential by carrying outside a small proportion of the hydrogen ions formed inside during electron transport. During the initial 40 ms of illumination, phosphorylation depends on the chemical potential of the hydrogen ions even though a transmembrane electric potential gradient seems also to be required.

The control of the adenosine triphosphatase of Rhodospirillum rubrum chromatophores by divalent cations and the membrane high energy state.

1. The rate of ATP hydrolysis, catalysed by Rhodospirilum rubrum chromatophores is accelerated by low concentrations and inhibited by high concentrations of uncoupling agent. 2. The inhibition at high concentrations of uncoupling agent is potentiated by the presence of free magnesium ions. At low uncoupler concentrations magnesium has no effect on the rate of ATP hydrolysis. 3. Inhibition of ATP hydrolysis by high concentrations of uncoupling agent and free magnesium ions is reversed by illumination. Illumination has less effect at low magnesium concentrations. 4. Free calcium ions inhibit ATP hydrolysis independently of the coupled state of the membrane. 5. Under coupled conditions, magnesium ions can overcome the inhibition induced by calcium. The two ions complete for the same site on the enzyme. 6. Inhibition by free magnesium in highly uncoupled chromatophores and inhibition by free calcium are both non-competitive with respect to the divalent cation-ATP substrate. 7. These data are consistent with a model in which divalent cations can bind to a site on the enzyme which is distinct from the substrate site. The regulation of the enzyme activity by the high energy state of the membrane is dependent on the occupant of this site.

Light-dependent proton and rubidium translocation in membrane vesicles from [formula omitted

A procedure for the isolation of membrane vesicles after sonication of Halobacterium halobium is described. Upon illumination these vesicles took up rubidium. This process was stimulated 3 to 7 fold by valinomycin, and inhibited by uncouplers of oxidative phosphorylation or by nigericin. In the light, these vesicles extruded protons. However, on addition of low concentrations of uncoupler the direction of proton movement was reversed. All proton movements were abolished by high concentrations of uncoupler or by nigericin. These observations suggest that part of the vesicle population was inverted and less sensitive to uncouplers.

Respiration-dependent uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitochondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATPase activity was stimulated at higher concentrations of uncouplers. 2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide- p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response. 3. The addition of succinate, NADH or ascorbate N,N,N′,N′- tetramethyl-p- phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin. 4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide- p-trifluoromethoxyphenylhydrazone at 10 -8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin. 5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity. 6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.

Chemical modification of mitochondria: Uncoupler binding by mitochondria in different metabolic states

At low uncoupler concentrations the binding of carbonyl-cyanide- m-chlorophenyl-hydrazone to mitochondria was found to depend sensitively on the metabolic state of mitochondria. The binding data are consistent with the assumption that at low concentrations and pH 7.4 the uncoupler is bound mainly in anionic form to the inner mitochondrial membrane and that upon energization the inner membrane undergoes conformation change, exposes buried ionizable groups and hence acquires a negative net membrane charge. Deenergization of the inner membrane by a small amount of uncoupler removes the negative net membrane charge and consequently increases the apparent binding constants. Based upon the present results on uncoupler binding and previous observations on the physiological properties of alkylating uncouplers, a possible molecular mechanism involving electron carriers and coupling factors is suggested for coupling electron transport to phosphorylation.

Inhibition of respiration in submitochondrial particles by uncouplers of oxidative phosphorylation

High concentrations of uncouplers of oxidative phosphorylation inhibit the oxidation of succinate and NADH catalyzed by phosphorylating beef heart submitochondrial particles. Inhibition is reversed by bovine serum albumin but not by ATP. ATP does not influence either the stimulation of respiration at low uncoupler concentrations or the inhibition of respiration at high uncoupler concentrations. The cytochromes of the electron transfer chain appear to be in the oxidized state during the oxidation of NADH catalyzed by submitochondrial particles partially inhibited by uncoupler, indicating that the inhibitory site resides at, or prior to, the substrate dehydrogenase. These data are consistent with a recent hypothesis which attempts to account for uncoupling action on the basis of competition between uncoupler and substrate molecules for an energy-requiring transport system in the mitochondrial membrane. However, not supportive of such a hypothesis are data indicating that in submitochondrial particles the inhibition does not appear to be of the competitive type and, furthermore, agents and procedures which destroy permeability barriers in mitochondria, and preparations of “open” submitochondrial particles, do not interfere with the uncoupler-mediated inhibition of substrate oxidation by the submitochondrial particle. It is suggested that high uncoupler concentrations interfere with the function of the nonheme iron moieties of the succinic and NADH dehydrogenases. Consistent with this suggestion are the findings that dithiothreitol partially reverses the uncoupler-mediated inhibition of substrate oxidations in submitochondrial particles and also protects against the inhibitory effects of uncoupler. In addition, the nonheme iron components of the succinic and NADH dehydrogenases were reducible by their respective substrates, while the characteristic electron paramagnetic resonance signal of the succinic dehydrogenase nonheme iron moiety was not observed in the uncoupler-inhibited submitochondrial particle upon the addition of NADH.