Concentrations Of Uncouplers Research Articles

The calcium-binding glycoprotein and mitochondrial calcium movements

The calcium-binding glycoprotein isolated from mitochondria can be shown to move from one mitochondrial compartment to another as a function of calcium and magnesium presence as well as calcium transport. The movement is reversible in vitro and the possibility is therefore considered that the glycoprotein may behave as a mobile calcium-carrier. In the presence of acetate and phosphate, calcium-pre-loaded mitochondria release the cation upon addition of uncoupling concentrations of pentachlorophenol. The rate of calcium efflux can be modulated either by changing pentachlorophenol or phosphate concentrations. Simultaneously a release of calcium-binding glycoprotein can be detected and a negative linear relation has been found between amount of glycoprotein released and rate of calcium passive efflux. The data are interpreted to indicate that calcium efflux occurs only when the glycoprotein is bound to the mitochondrial membranes.

Changes in E. coli cell envelope structure caused by uncouplers of active transport and colicin E1.

It is of interest to inquire whether agents that uncouple or deenergize membranes cause concomitant structural changes. The agents considered here are the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and the bacteriocidal protein colicin E1, agents for which there is some precedent for believing that they interact with membranes. In intact E. coli ML 308-225 cells the inhibition of [14C]-PROLINE ACtive transport by FCCP increases with uncoupler concentration from approximately 20% at 2 muM to approximately 100% at 5 muM. The increase in the rotational relaxation time (rho) of the cell-bound fluorescent probe N-phenyl-1-naphthylamine (PhNap)1 and 8-anilino-1-naphthalene-sulfonate (ANS) under these conditions shows the same dependence on FCCP concentration. For cells treated with EDTA to remove part of the outer lipopolysaccharide layer, inhibition of proline transport and the increase in rho value of ANS show the same dependence on FCCP concentration with saturation at 0.3 muM. EDTA treatment causes a large increase in the binding and rotational relaxation time of PhNap, the latter quantity approaching a value obtained with purified inner membrane. Similar effects are produced in untreated cells by 5muM FCCP...

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.

The minimum effective amount of uncouplers for rat liver mitochondria

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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.

The effects of phosphate and electron transport on the carbonyl cyanide m-chlorophenylhydrazone-induced ATPase of rat-liver mitochondria

1. The effects of phosphate and electron transport on the ATPase induced in ratliver mitochondria by the uncoupler carbonyl cyanide m-chlorophenylhydrazone have been measured at different uncoupler concentrations and compared with those of ATP, oligomycin and aurovertin. 2. The inhibitory action of respiratory-chain inhibitors on the ATPase activity, which is independent of the actual inhibitor used, is greatly delayed or prevented by the presence of uncoupler, and, in the case of rotenone, can be reversed completely by the subsequent addition of succinate (in the absence of uncoupler). These results can be explained on the basis of the proposal previously made by others that coupled electron transfer causes a structural change in the ATPase complex that results in a decreased affinity of the ATPase inhibitor for the mitochondrial ATPase. 3. Inorganic phosphate specifically stimulates the ATPase activity at high uncoupler concentrations (> 0.2 μM), but has no effect at low concentrations. The stimulation is prevented or abolished by sufficiently high concentrations of aurovertin. 4. Aurovertin prevents the inhibition of the uncoupler-induced ATPase by high uncoupler concentrations. 5. It is proposed that the steady-state concentration of endogenous P i may be an important regulator of the turnover of the ATPase in intact mitochondria and that the inhibition of ATPase activity by high concentrations of uncoupler is at least partially mediated via changes in the concentration of endogenous P i.

Conductance and voltage-current relationship of lipid bilayers in the presence of TTFB

The aim of this work was to discover the mechanism of lipid bilayer conductance in the presence of one of the most effective uncouplers of oxidative phosphorylation, TTFB. Current clamp data and dependences of membrane conductance on pH and on uncoupler concentration were used for the determination of the charge-transfer-limiting-step. It was proved that the uncoupler dimer-anion takes partin the charge transfer through the inner hydrophobic layer of the membrane. In the presence of Cu 2+ ions in aqueous solutions, lipid bilayers with TTFB show N-shaped steady state voltage-current characteristics. It was shown that changes in pH and in uncoupler concentration in unstirred aqueous layers near the membrane, may be a reason for the development of this shape of the voltage-current curves.

Proton evolution associated with the photooxidation of water in photosynthesis

1. We have used a fast and sensitive glass electrode to measure the pH changes associated with single turnovers of photosynthetic electron transport induced by short flashes in spinach chloroplasts. 2. In dark-adapted chloroplast suspensions, containing uncoupling concentrations of gramicidin D or methylamine, the flashyield of proton release oscillated in the same way as the flash yield of O 2; with a periodicity of four, the yield of the third flash being maximal. 3. Based upon the similar behavior of the flash yields of proton and O 2 liberation we conclude that, following the accumulation of 4 oxidizing equivalents, water is decomposed in a final concerted reaction. 4. Because these oscillations can only be seen in the presence of uncouplers we conclude that the O 2 evolving system is located inside the thylakoid membrane. 5. In contrast to the relaxation of System II and O 2 release, which occur in < 1 ms after a flash, the efflux time of the protons released in the decomposition of water was approx. 80 ms. From this we conclude that even in the presence of gramicidin or methylamine, there is a diffusion barrier to the movement of these protons. 6. We also observed the proton influx associated with electron transport from Photosystem II to System I. The half-time of this influx was approx. 30 ms, again slower than the electron transfer time. This half-time was not influenced by gramicidin D. 7. In contrast, methylamine abolished this influx. At the same time, there occurred at net acidification of the external medium during illumination. 8. The proton uptake associated with the reoxidation of the endogenous photo-reductant of System I had a half-time of about 0.3 s. The proton uptake associated with the reoxidation of photo-reduced methylviologen showed a half-time of 8 ms.

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.

Trinitrophenol: a membrane-impermeable uncoupler of oxidative phosphorylation.

Picrate (trinitrophenol) is a unique uncoupler of oxidative phosphorylation. Unlike the commonly used uncouplers (e.g., 2,4-dinitrophenol, pentachlorophenol, m-chlorocarbonylcyanide phenylhydrazone, and 5-chloro-3-t-butyl-2'-chloro-4'-nitrosalicylanilide), picrate seems to penetrate the mitochondrial inner membrane very slowly. Consequently, it is ineffective when added to intact mitochondria or to mitochondria depleted of their outer membranes. In contrast, when added to phosphorylating submitochondrial particles prepared by sonication in which the inner membrane orientation is inside-out, picrate binds to the uncoupler-binding sites and uncouples oxidative phosphorylation. This unique property of picrate has made it possible to compare the potencies of picrate and dinitrophenol for (a) uncoupling and (b) increasing the proton permeability of submitochondrial particle vesicles. At 50% uncoupling concentration, dinitrophenol increased the proton permeability of submitochondrial particle vesicles by 9- to 12-fold. In contrast, at 100% uncoupling concentrations or higher, picrate augmented the proton permeability of the particles by only about 3-fold. These results indicate that facilitation of transmembrane proton equilibration does not determine the degree of uncoupling, and lead to the corollary conclusion that the magnitude of transmembrane proton gradient need not be the quantitative driving force for ATP synthesis.

The binding of uncouplers of oxidative phosphorylation to rat-liver mitochondria

The binding of different uncouplers of oxidative phosphorylation to rat-liver mitochondria was measured. At pH 7.2 and about 0.7 mg mitochondrial protein/ml the percentage bound of the uncoupler added was 84% for 2,3,4,5,6-pentachlorophenol (PCP), 40% for carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 35% for 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB), 4% for α′,α′-bis (hexafluoroacetonyl)acetone (1799), and less than 4% for 2,4-dinitrophenol. These percentages are constant up to amounts of uncoupler added several times the one needed for maximal uncoupling. The values found for FCCP and TTFB are in contradiction to the proposed stoichiometric interaction of uncouplers with the coupling sites of the mitochondrial membrane. From titration experiments of the rate of O 2 uptake by rat-liver mitochondria in State 4 as a function of the uncoupler concentration in the presence of albumin or of different types of liposomes the conclusion is drawn that the negative surface charge of the mitochondrial phospholipids may be an important parameter in determining the binding of anionic uncouplers to rat-liver mitochondria.

An adenosine triphosphate-phosphate exchange catalyzed by a soluble enzyme couple inhibited by uncouplers of oxidative phosphorylation.

The sulfenic acid form of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), which is an acyl phosphatase, will catalyze an acetyl phosphate-Pi exchange reaction. This exchange reaction is reversibly inhibited by the uncouplers of oxidative phosphorylation, 2,4-dinitrophenol, m-Cl carbonylcyanide-phenylhydrazone, pentachlorophenol, and 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanalide, and is irreversibly inhibited by cyanide and dicumarol. An ATP-Pi exchange reaction similar to that catalyzed by mitochondria can be simulated by a system composed of oxidized glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase (EC 2.7.1.28), 3-phosphoglycerate, ATP, (32)Pi, and appropriate cofactors. The ATP-Pi exchange is inhibited by uncouplers of oxidative phosphorylation. Higher concentrations of uncouplers will also inhibit the ATPase reaction catalyzed by the coupled enzyme system. The exchange reactions catalyzed by the sulfenic acid form of glyceraldehyde-3-phosphate are consistent with a sulfenyl carboxylate intermediate. On the basis of these observations, a reaction scheme has been postulated for covalent coupling in oxidative phosphorylation that includes a sulfenyl carboxylate as a nonphosphorylated, high energy intermediate and an acyl phosphate as a phosphorylated, high energy intermediate.

Kinetic study of the effect of uncouplers on substrate uptake by rat-liver mitochondria.

The initial rate of the uptake of Pi, succinate, citrate and ketoglutarate, even at low concentrations, is not affected by uncouplers, when added simultaneously with the substrate at concentrations which uncouple oxidative phosphorylation and decrease substrate uptake at equilibrium. The first-order constant of succinate uptake is significantly increased by uncouplers. If, however, mitochondria are preincubated with uncouplers in order to collapse the ΔpH, the rate of substrate uptake is lowered. At high concentrations, of the same order of those used for the substrates, uncouplers have an inhibitory effect also when added together with the substrate. It is concluded that (a) the uncoupling effect is not related to the interaction of uncouplers with substrate carriers or to their entry on these transport systems, (b) the inhibition of substrate uptake at equilibrium can be accounted for only by the collapse of the ΔpH, (c) the interaction of high uncoupler concentrations with the substrate carriers is a secondary and unspecific effect.

Release of respiratory control by uncouplers: The question of stoichiometry

An alternative kinetic model of uncoupler action is envisaged in which the observed stoichiometry of uncoupler and titratable factor do not reflect chemical binding reactions. This model assumes (a) that highly effective uncouplers dissolve in the membrane, whereas less effective uncouplers remain largely in aqueous solution; (b) that a high affinity exists between respiratory chains and “low energy states”; (c) that maximal respiration requires the presence of only a small number of low-energy states and forms high-energy from low-energy states; (d) that uncouplers act by a bimolecular “collision” process without binding. In this model, respiratory chains must be capable of driving energy equivalents into a storage mode, with no effect on respiration rate until the store is almost filled. Results obtained with 2,4-dinitrophenol and S-13 show that the shapes of curves obtained when uncoupler concentration is plotted against respiration rate are the same for both “stoichiometric” and classical uncouplers. Mouse liver mitochondria, in the absence of added cations, showed a “titration” response to both dinitrophenol and S-13. Differences are: (1) the dinitrophenol titration point (approximately 35 μ m) is less dependent on mitochondrial concentration than that of S-13 (approximately 0.09 μ m) (for 1 mg of protein/ml); (2) a “lag” phase of some 30 sec occurs with S-13 at concentrations (approximately 0.1 μ m) giving the same uncoupling effect as 50 μ m-dinitrophenol (which shows no lag phase). Cytochrome b reduction level continues to fall upon DNP addition, and the rapid decline in per cent reduction of cytochrome b by a given increment of DNP ceases at approximately 35 μ m DNP, the concentration required for maximal respiration rate. Under some circumstances the release of respiration by uncoupler in the presence of azide shows an apparent titration independent of the final rate of respiration. This is true for DNP as well as for the apparent “stoichiometric” uncouplers. It is suggested that this does not necessarily identify a more specific uncoupling reaction than that observed in the absence of azide. The apparent release of azide inhibition by uucouplers is attributed to changes in the steady state level of cytochrome reduction on passing from state 3 to the uncoupled state, and to an oligomycin-like effect of azide on the mitochondrial ATPase.

Effects of ring substituents on the activity of phenols as inhibitors and uncouplers of mitochondrial respiration.

The effects of substituents on the activity of phenols as uncouplers and inhibitors of mitochondrial respiration have been examined. These activities have been correlated with constants describing the effects of the substituents on the electronic and hydrophobic‐bonding properties of the phenol. The coefficients in these correlation equations have been compared with each other and with the coefficients in similar correlations for the effects of phenols on the solubilised ATPase from mitochondria, and their activity in mediating proton conduction across artificial lipid membranes and as inhibitors of a variety of enzymes.Differences in the coefficients in the correlation equations for uncoupling and inhibition of respiration indicate that these depend on different properties of the phenol and hence that they are separate effects and that inhibition of substrate accumulation is not a satisfactory explanation for the inhibition of respiration.High concentrations of uncouplers induce swelling of mitochondria. In the case of 2,4‐dibromophenol, or dicoumarol, this occurs at concentrations similar to those producing inhibition of respiration. With other phenols inhibition of respiration is observed at concentrations lower than those required to induce swelling.The observations reported here support the hypothesis that phenols uncouple by mediating proton conduction across the inner mitochondrial membrane and that, in general, the inhibition of respiration by high concentrations of phenols is due to direct action on a protein component of the respiratory chain.

A possible biochemical mode of action of the fasciolicides nitroxynil, hexachlorophene and oxyclozanide

AbstractThe commercial fasciolicides nitroxynil, hexachlorophene and oxyclozanide have the following minimum uncoupling concentrations when tested with rat liver mitochondria by an oxygen electrode assay: 27‐33 μ, 0.6‐0.8 μ and 0.3‐0.4 μM, respectively. The compounds stimulated the rate of oxygen uptake of intact flukes in Warburg flasks 20‐40% over the untreated rate at 10‐100 μ, 1‐10 μM and 0.1‐1 μM, respectively. Apparent death (i.e. lack of movement after 24 h exposure) was caused by 170‐350 μM, 3.5‐7 μm and 3.5‐7 μM, respectively. The approximate correlation between the concentrations necessary to cause uncoupling in isolated mitochondria, stimulation of oxygen uptake by whole flukes, and apparent death, suggest that the fasciolicidal effect of all three compounds may be due to uncoupling.

Studies of the Energy‐Transfer System of Submitochondrial Particles

Submitochondrial particles elicit a fluorescence of 8‐anilino‐naphthalene‐1‐sulfonate which is increased when the particles are brought into the energized state. Various parameters of the anilino‐naphthalene sulfonate response were investigated in “EDTA particles” from beef‐heart mitochondria in the energized and non‐energized states. The results reveal striking differences between teh two types of anilino‐naphthalene sulfonate response in several respects, including the effects of pH and ionic strength, the number of binding sites, the dissociation constant, and the quantum yield. Conditions are established which result in a maximal ratio of energy‐dependent to non‐energy‐dependent anilino‐naphthalene sulfonate response and a virtually linear relationship of both types of response to particle concentration. It is shown that, under such conditions, there is a linear inverse relationship between the steady state of energy‐dependnet anilino‐naphthalene sulfonate response and the respiratory rate of the particles as measured with succinate as substrate in the presence of oligomycin and increasing concentrations of an uncoupler. Extrapolation of this straight line to respiratory rate = 0 allows an estimation of the extent of anilinonaphthalene sulfonate response at complete respiratory control, i.e., in the maximally energized state. Experiments with different substrates and electron‐transport inhibitors reveal that the linear relationship between the extent of energy‐dependent anilino‐naphthalene sulfonate response and the respiratory rate in the presence of increasing concentrations of uncoupler holds only when the cytochrome b‐c1 step of the respiratory chain is rate‐limiting. The available evidence indicates that the energy‐dependent anilino‐naphthalene sulfonate response originates mainly or exclusively from a restricted region or locus of the membrane, which is closely related to energy‐coupling site II of the respiratory chain. With ATP as the source of energy, the anilino‐naphthalene sulfonate response increases with low, and decreases with high, concentrations of oligomycin, in a fashion similar to that earlier found for oxidative phosphorylation and its reversal.

The effect of the polar moiety of lipids on bilayer conductance induced by uncouplers of oxidative phosphorylation

Bilayer membranes were formed from decane, cholesterol, and three lipids isolated fromStaphylococcus aureus: positively charged lysyl phosphatidylglycerol (LysPG), negatively charged phosphatidylglycerol (PG), and neutral diglucosyldiglyceride (DiGluDiGly). The uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and 3-t-butyl,5-chloro,2'-chloro,4'-nitrosalicylanilide (S 13), increased the electrical conductance of all three differently charged bilayers. S 13 was found to be the most effective reagent of the known uncouplers in increasing conductance of the bilayers. The conductance induced by uncouplers was investigated as a function of pH and uncoupler concentration. The pH of maximum conductance for each uncoupling agent was dependent on both the uncoupler and the lipid; it was lower for each uncoupler in LysPG and higher in PG compared to DiGluDiGly bilayers. At a pH below the optimum for LysPG, the conductance of the positively charged membrane was 500 times and of the neutral one 10 times higher than that of the negatively charged bilayer at equal uncoupler concentration and pH. Above the pH optimum for DiGluDiGly, the conductance was approximately equal for the positive and neutral membranes, but was lower in PG bilayers. Conductance depended linearly on uncoupler concentration. The bilayer conductance induced by S 13 was entirely due to increased proton permeability in all three lipids. The findings are consistent with the role of uncouplers as "carriers" for protons across the hydrocarbon interior of lipid membranes. The differences in conductance of differently charged lipid bilayers at equal uncoupler concentration, as well as the change of pH optimum of conductance with lipid charge, can be explained in terms of an electrostatic energy contribution of the fixed lipid charges to the distribution of the uncoupler anion between the aqueous and the membrane phases.

Blocking phenomena and charge transport through membranes.

Substances which uncouple oxidative phosphorylation in mitochondrial membranes usually increase the electrical conductivity of synthetic bimolecular phospholipid membranes. Among these uncouplers is a group characterized chemically as weak acids. For this group the conductivity of synthetic membranes, when measured versus pH at fixed uncoupler concentration, shows a maximum at a pH approximately equal to the pK value of the uncoupler used. Corresponding maxima in membrane electrical potential arising from ion concentration gradients are also observed. To explain such phenomena a model is proposed which assumes charge transport by the direct transfer of either protons or anions of the uncoupler between binding sites located on the membrane boundaries. A fixed surface density of such sites is assumed. The transfer of an ion requires both its presence on an initiating site and the availability of a terminal site which is not already occupied by an ion of the same species. Failure to satisfy both criteria leads to blockage of current flow at both low and high concentrations of the transported ion.

Interactions of uncoupling agent and of antimycin A with cytochrome b in yeast and heart muscle preparation under anaerobic conditions

When added to an anaerobic suspension of aerobically grown yeast, of mitochondria isolated from it, or of Keilin-Hartree heart muscle preparation, uncoupling agents elicited a considerable oxidation of cytochrome b. For a maximum effect, about three orders of magnitude higher concentrations were required than those effective in uncoupling oxidative phosphorylation, but an extrapolation to zero of the curve relating oxidation of cytochrome b to uncoupler concentration indicates that a slight effect may also occur at uncoupling concentrations. Ubiquinone was also partly oxidized, while cytochromes a·a 3 and c + c 1 remained unaffected. An unknown component of mitochondria may accept electrons from cytochrome b under these conditions. The effect was rather specific for uncoupling agents and was not displayed by a number of compounds devoid of uncoupling capacity. Antimycin A added to intact yeast cells or to isolated yeast mitochondria under anaerobic conditions also induced the anaerobic oxidation of cytochrome b. Under aerobic conditions, however, antimycin A prevented the cytochrome b oxidation via the respiratory chain and caused an additional slight reduction of cytochrome b to a form whose absorption maximum in the α-region was shifted to 565 nm. In the heart muscle preparation, antimycin A did not induce cytochrome b oxidation under anaerobic conditions but did cause an additional reduction by succinate of cytochrome b to the form absorbing at 565 nm. In normal ubiquinone-containing heart muscle particles, uncoupling agents did not induce the anaerobic oxidation of that part of cytochrome b which wits reducible by succinate in the presence of antimycin A. However, in ubiquinone-depleted particles, this part of cytochrome b became accessible to the uncoupler-induced anaerobic oxidation. The antimycin A block preventing oxidation of cytochrome b by air via the respiratory chain was relieved by some uncouplers at very high concentrations.