Electrochemical Potential Difference Of Protons Research Articles

Bacteriorhodopsin in liposomes. II. Experimental evidence in support of a theoretical model

In the preceding article equations describing relevant ion flows in illuminated suspensions of bacteriorhodopsin liposomes have been derived. Here these equations are subjected to experimental tests. Changes in permeability characteristics of the liposomal membrane are brought about by addition of specific ionophores and change of medium composition. Using light-driven proton uptake and electrochemical potential differences for protons across the membrane as observation parameters, ridig attempts to falsify the derived equations are unsuccessful. Agreement between equations and experimental results is established on the point of: (i) the antagonistic effect of valinomycin and nigericin on the two components of the proton-motive force, (ii) the time dependence of the changes in transmembrane electrical and chemical potential differences after the onset of illumination. In three independent experimental systems evidence was obtained for the correctness of the postulated dependence of the turnover rate of the photochemical cycle on back pressure by the transmembrane electrochemical potential difference for protons.

The influence of thyroxine administered in vivo on the transmembrane protonic electrochemical potential difference in rat liver mitochondria

The influence of thyroxine administered <i>in vivo</i> on the transmembrane protonic electrochemical potential difference in rat liver mitochondria

Gating effects in Halobacterium halobium membrane transport.

The transport of Na(+) via an H(+)/Na(+) antiporter and of aspartate and serine via Na(+)/amino acid symport systems was studied in Halobacterium halobium cell envelope vesicles. Gradients for H(+) were produced by illuminating the bacteriorhodopsin-containing vesicles at different light intensities, and the rate and extent of Na(+) transport were followed as functions of the electrochemical potential difference for protons. The coupling of Na(+) and H(+) gradients suggested a translocation stoichiometry of 2H(+)/Na(+) for the antiporter. The rate of Na(+) transport increases steeply above a critical transmembrane electrochemical proton gradient, and since the electrical and the chemical potentials of H(+) at this threshold point vary with the experimental conditions, while the sum of these potentials is constant, it was concluded that the gating of the Na(+) transport is caused by the total electrochemical gradient.

The influence of thyroxine administered in vivo on the transmembrane protonic electrochemical potential difference in rat liver mitochondria

When mitochondria from normal and thyroxine-treated rats were energized by incubation with succinate, phosphate and MgCl2, it was found that the hormone treatment increased the transmembrane protonic electrochemical potential difference by 16mV and the respiration rate by 46%. Other experiments show these changes to be associated with increases in the intramitochondrial K+ and phosphate concentrations.

83] Incorporation of purple membrane into vesicles capable of light-stimulated ATP synthesis

Purple membranes of Halobacterium halobium catalyze a rapid light-driven proton translocation across the membrane. These membranes contain only one protein, bacteriorhodopsin, which has proved to be a useful tool in studying the mechanism of energy coupling to phosphorylation. Vesicles capable of light-dependent ATP synthesis are reconstituted from mitochondrial ATPase F 1 , crude hydrophobic proteins, purple membranes, and soybean phospholipids. Purified dicyclohexylcarbodiimide (DCCD)-sensitive ATPase of thermophilic bacterium PS3 has also been used instead of F 1 and crude hydrophobic proteins to reconstitute this kind of vesicle. When the reconstituted vesicles are illuminated, the electrochemical potential difference of protons caused by bacteriorhodopsin drives protons through the ATPase, which synthesizes ATP by this energy. The vesicles are reconstituted from a purple membrane, DCCD-sensitive ATPase and phospholipid suspension containing cholate, deoxycholate, ethylenediaminetetraacetic acid (EDTA), dithiothreitol, and Tricine. These components are mixed and the final volume is adjusted to 2.0 ml. The mixture is subjected to sonic oscillation in a Tomy probe sonic oscillator, model 4R-150, at 20 KHz and 150 W in an ice bath for one minute. The mixture is placed in cellophane tubing of 0.25-in. diameter and 0.002-in. thickness and dialyzed against one liter of the dialyzing solution at 30 ° C for 18–20 hours. Therefore, the addition of 100 mM salts is essential to prevent dissociation of purple membranes.

64] The measurement of membrane potential and ΔpH in cells, organelles, and vesicles

The determination of both membrane potential and pH gradient (ΔpH) across a membrane allows the calculation of the proton electrochemical potential difference, or the proton-motive force. The value of the proton-motive force is important for an experimental evaluation of the chemiosmotic hypothesis. It is also an important indicator of the coupling in energy-conversion membrane systems. It is the most sensitive and quantitative indicator of coupling in these systems. As the proton-motive force can be generated by various means, the degree of coupling can be evaluated in systems that lack the complete machinery for oxidative phosphorylation or photophosphorylation. This is in contrast to the more conventional parameters of coupling, such as phosphate/oxygen (P/O), respiratory control, and phosphate potential. The determination of membrane potential or internal pH is not limited to systems of energy conversion; the chapter describes methods for determining the membrane potential in suspensions of cells, organelles, or vesicles.

The rates of onset of photophosphorylation and of the protonic electrochemical potential difference in bacterial chromatophores

We have studied the kinetic and thermodynamic parameters of photophosphorylation in bacterial chromatophores of Rhodopseudomonas capsulata under steady state experimental conditions, as related to the protonic potential difference (Ap) across the membrane [l-4]. The main conclusions of these studies were that under ‘state 4’ conditions, i.e., under conditions of static head for both Ap and phosphorylation, a general agreement exists between the affinity of the reaction of ATP synthesis (A,& and the extent of Ap [ 1,3]. We observed, however, that the steady state rate of ATP synthesis was markedly affected by the rate of electron flow, when this was decreased by antimycin A or by limiting the intensity of actinic light, in spite of the fact that the value of Ap was not largely diminished under these conditions [3,4]. These observations led us to consider the possibility of short range interactions between the ATP synthetase complexes and the electron transport chains, involving the mechanism of energy transduction itself or alternatively only a control of the turnover rate of ATP synthetase. It is clear that more detailed informations on this point would be obtained if the transient rates of ATP synthesis and of Ap formation, upon the onset of illumination, were compared. This approach requires fast and sensitive techniques for the measure of ApH, A$ and ATP concentration. The spectroscopic methods in use in our laboratory for the measurement of ApH and of A$ in chromatophores [3] are fast enough to allow a quantitative evaluation of Ap after a flash, before the relaxation of the protonic gradient reaches a significant extent (in preparation). The luciferine-luciferase assay, recently adapted for the continuous monitoring of photophosphorylation in bacterial chromatophores [S], offers the technical possibility for a comparative study of the transient rates of phosphorylation versus the onset of &. This paper describes some preliminary results of experiments relating Ap and ATP synthesis induced by flashes of light of variable time length.

Proton electrochemical gradient and phosphate potential in submitochondrial particles

1. The aerobic uptake of inorganic ions, such as 86Rb + or 125I −, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of ΔpH, the transmembrane pH differential, and Δψ, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the ΔpH and Δψ values from organic and inorganic ions is large at high but not at low ion/protein ratios. 2. In the absence of weak bases and strong acids the values of Δ \\ ̃ gm H , the proton electrochemical potential difference, are close to 100 mV and the magnitude of ΔpH and Δψ are similar. Weak bases decrease ΔpH and enhance Δψ. Strong acids decrease Δψ and enhance ΔpH. Interchangeability of ΔpH with Δψ occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of Δ \\ ̃ gm H . 3. Concentrations of weak bases capable of abolishing ΔpH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing Δψ affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of ΔpH + Δψ inhibit ATP synthesis. 4. Depression of Δ \\ ̃ gm H is paralleled by inhibition of ATP synthesis and decline of ΔGp, the phosphate potential. Abolition of ATP synthesis occurs only when Δ \\ ̃ gm H is below 20 mV. The ΔG p/Δ \\ ̃ gm H ratio increases hyperbolically with the decrease of Δ \\ ̃ gm H .

Proton electrochemical gradient and rate of controlled respiration in mitochondria

The correlation between Δ \\ ̃ gm H , the proton electrochemical potential difference, and the rate of controlled respiration is analyzed. ΔpH (the proton concentration gradient) is measured on the distribution of [ 3H]acetate, and ΔΨ (the membrane potential) on the distribution of 86Rb +, 45Ca 2+ and [ 3H]triphenylmethylphosphonium used either alone or simultaneously. The effects of the addition of ADP + hexokinase (state-3 ADP) and of carbonylcyanide trifluoromethoxyphenylhydrazone (state-3 uncoupler) on respiration and Δ \\ ̃ gm H are not equivalent: the uncoupler depresses Δ \\ ̃ gm H more than ADP at equivalent respiratory rates. The effects of the additions of nigericin-valinomycin and of ionophore A23187 (state-3 cation transport) and of carbonylcyanide trifluoromethoxyphenylhydrazone (state 3-uncoupler) on respiration and Δ \\ ̃ gm H are also not equivalent: the uncoupler depresses Δ \\ ̃ gm H more than A23187 and nigericin + valinomycin at equivalent respiratory rate. A23187 is very efficient in stimulating respiration with negligible Δ \\ ̃ gm H changes.

Proton electrochemical gradient and phosphate potential in mitochondria

The paper reports an analysis of the relationship between Δ \\ ̃ gm H the proton electrochemical potential difference, and ΔGp, the phosphate potential. Depression of Δ \\ ̃ gm H and ΔGp has been obtained by titration with: (a) carbonylcyanide trifluoromethoxyphenylhydrazone; (b) nigericin (+ valinomycin); (c) KCl (+ valinomycin); and (d) rotenone. The uncoupler depresses Δ \\ ̃ gm H more than nigericin (+ valinomycin), KCl (+ valinomycin) and rotenone at equivalent ΔGp. The ΔG p/Δ \\ ̃ gm H ratio is about 3 at high values of Δ \\ ̃ gm H . When ΔGp and Δ \\ ̃ gm H are depressed by nigericin (+ valinomycin) the ΔG p/Δ \\ ̃ gm H ratio remains constant. When ΔGp and Δ \\ ̃ gm H are depressed by uncouplers, the ΔG p/Δ \\ ̃ gm H ratio increases hyperbolically tending to infinity while Δ \\ ̃ gm H tends to zero. The absence of constant proportionality between ΔGp and Δ \\ ̃ gm H indicates that the proton gradients driving ATP synthesis presumably operate within microscopic environments.

A protonmotive force as the source of energy for galactoside transport in energy depleted Escherichia coli.

An artificially produced electrochemical potential difference for protons (portonmotive force) provided the energy for the transport of galactosides in Escherichia coli cells which were depleted of their endogenous energy reserves. The driving force for the entry of protons was provided by either a transmembrane pH gradient or a membrane potential. The pH gradient across the membrane was created by acidifying the external medium. The membrane potential (inside negative) was established by the outward diffusion of potassium (in the presence of valinomycin) or by the inward diffusion of the permeant thiocyanate ion. The magnitude of the electrochemical potential difference for protons agreed well with magnitude of the chemical potential difference of the lactose analog, thiomethylgalactoside. The observations are consistent with the view that the carrier-mediated entry of each galactoside molecule is accompanied by the entry of one proton.

Obligatory coupling between proton entry and the synthesis of adenosine 5'-triphosphate in Streptococcus lactis.

Proton influx was measured after imposition of an electrochemical potential difference for protons (delta muH+) across the cell membrane of the anaerobe, Streptococcus lactis. As delta muH+ was increased, there was an approximately parallel increase in proton entry, until delta muH+ attained 175 to 200 mV. At this point, a new pathway became available for proton entry, allowing an abrupt increase in both the rate and extent of H+ influx. This gated response depended upon the value of delta muH+ itself, and not upon the value of either the membrane potential or the pH gradient. For delta muH+ above 175 to 200 mV, elevated proton entry occurred only in cells having a functional membrane-bound Ca2+-stimulated, Mg2+stimulated adenosine 5'-triphosphatase (EC 3.6.1.3). When present, elevated proton entry coincided with the appearance of net synthesis of adenosine 5'-triphosphate catalyzed by this adenosine 5'-triphosphatase. These observations demonstrate that membrane-bound adenosine 5'-triphosphatase catalyzes an obligatory coupling between the inward movement of protons and synthesis of adenosine 5'-triphosphate.

Thermodynamics and kinetics of photophosphorylation in bacterial chromatophores and their relation with the transmembrane electrochemical potential difference of protons.

The extent of the protonmotive force (transmembrane ΔH+/F) developed in bacterial chromatophores from Rhodopseudomonas capsulata under continous illumination has been determined with measurements of the quenching of 9-aminoacridine fluorescence and the red band shift of carotenoids. The extent of the membrane potential generated in the dark with K+ pulses in valinomycin-treated chromatophores has been evaluated theoretically and the validity of the calibration curves of the carotenoid signals, obtained by this procedure, has been analyzed consequently. The maximal affinity of the ATP synthesis reaction attainable by chromatophores at high light intensities has been measured and compared with the level of the protonmotive force; this analysis has been extended to conditions of partial uncoupling by a variety of agents with a different mode of action. The results show a parallel decrease of the two parameters and indicate that in most cases a stoichiometry of 2 H+ per ATP is sufficient to account for the force ratios obtained experimentally. However in extensively uncoupled membranes (Δp < 250 mV) photophosphorylation affinity reaches levels incompatible with a minimal stoichiometry of 2 H+ per ATP. The kinetics of light-induced ATP synthesis have been also correlated with the extent of the protonmotive force: a decrease of photophosphorylation linear with Δp is observed in all the uncoupled conditions tested with an apparent threshold of Δp between 120–180 mV, under which no net phosphorylation is detectable. When cyclic electron flow is limited, either by additions of antimycin A or by intensity of actinic light, a correlation between Δp and the rate of photophosphorylation markedly different from that observed in uncoupled conditions is obtained. The results, which are discussed in terms of the degree of coupling of energy transduction between proton gradients and ATP synthesis, using the approach of non-equilibrium thermodynamics, are not completely compatible with the postulates of the chemiosmotic coupling hypothesis in its minimal version.

The generation of the proton electrochemical potential and its role in energy transduction

The evidence that all energy transducing membranes can generate a proton electrochemical potential difference, delta micronH, across the membrane and that this potential can be used to transfer energy among energy transducing units and to generate ATP, has increased the interest for the view that delta micronH plays an obligatory role in energy transduction and ATP synthesis. In the present article we shall concentrate on two experimental questions related with the generation and role of delta micronH: (a) the charge/site ratio; (b) the relation between the proton electrochemical potential on one side and the cation electrochemical potential, the phosphate potential and the redox potential on the other. We shall then discuss the view that energy transduction corresponds to a molecular energy machine rather than to a fuel cell.

ATP-dependent proton translocation in resealed chromaffin granule ghosts.

The catecholamine-storing organelles (chromaffm granules) of the adrenal medulla accumulate the catecholamines by a mechanism that is coupled to ATP hydrolysis [l-3] and inhibited by classical uncouplers of oxidative phosphorylation [4,5] which affect AjiH across the membrane. Evidence has recently been presented that a proton electrochemical potential difference is generated by the ATP hydrolysis [6,7], but the direction of the proton movement in intact chromaffin granules is controversial. Thus, whereas recent studies by Casey et al. [6] indicate that the direction of proton translocation is inward, Pollard et al. [7] have reached the opposite conclusion, i.e., that ATP hydrolysis causes an internal alkalinization. Since the intact granules contain a high concentration of stored compounds (catecholamines, adenine nucleotides and proteins) [8], they have a high internal buffer capacity, which in addition to leaks from the granules during an incubation, may complicate the interpretation of the results [6,9]. We have therefore studied the direction of the proton movement and its stoichiometry to ATP hydrolysis in resealed granule ghosts of low internal buffer capacity derived from bovine adrenal chromaffin granules. Using a

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium

The light-dependent uptake of triphenylmethylphosphonium (TPMP +) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (ΔpH) across the membrane, and TPMP + was used as an index of the electrical potential difference, Δψ. Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, ΔpH amounts to 0.6–0.8 pH unit. ★★ Its value can be increased to 1.5–2.0 by either incubating the cells with TPMP + (10 −3 M) or at low external pH (5.5). — ΔpH can be lowered by uncoupler or by nigericin. The TPMP + uptake by the cells indicates a large Δψ across the membrane, negative inside. It was estimated that in the light, at pH 6.6, Δψ might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, Δ u H + F , amounts under these conditions to about 140 mV. The effects of different ionophores on the light-driven proton extrusion by the cells were in agreement with the effects of these compounds on — ΔpH.

Electrochemical proton gradient and phosphate potential in bacterial chromatophores

One of the obligate and basic requirements of Mitchell’s chemiosmotic hypothesis [ 1 ] is the consistency of the extent of the electrochemical potential difference of protons across the membrane and the amount of energy available for ATP synthesis. An estimation of this amount of energy has been obtained by measuring the maximal affinity of the phosphorylation reaction at which no net ATP synthesis occurs (phosphate potential in state 4). Experiments of this kind, performed in membrane systems as different as mitochondria [2], chloroplasts [3] and membrane fragments from aerobic [4] and photosynthetic bacteria [5], have all yielded values ranging between 14 and 16 Kcal/mole. These values are considered by several authors to be inconsistent with a stoichiometry of 2H’ translocated per ATP synthesized [ 1 ] and with the size of the proton electrochemical gradient, as estimated on the basis of the distribution of permeable ionic and protonable molecules across the energized membrane, for example [6,7]. Fluorimetric and spectrophotometric methods for the evaluation of pH or potential differences across the membranes of some photosynthetic bacteria have been propos d. Specifically, work in this laboratory [8] has indicated that ApH in bacterial chromatophores can be estimated from the extent of the fluorescence quenching of 9-aminoacridine, employing the technique originally proposed by Shuldiner et al. [9] for chloroplasts. In addition, the presence in photosynthetic membranes of pigments undergoing spectral shifts in response to a transmembrane electric field [lo] allows an evaluation of the membrane potential,