1,721,075 research outputs found
H+/ATP ratio of proton transport-coupled ATP synthesis and hydrolysis catalysed by CF0F1-liposomes
No full textThe H+/ATP ratio and the standard Gibbs free energy of ATP synthesis were determined with a new method using a chemiosmotic model system. The purified H+-translocating ATP synthase from chloroplasts was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. During reconstitution, the internal phase was equilibrated with the reconstitution medium, and thereby the pH of the internal liposomal phase, pHin, could be measured with a conventional glass electrode. The rates of ATP synthesis and hydrolysis were measured with the luciferin/luciferase assay after an acid-base transition at different [ATP]/([ADP][Pi]) ratios as a function of ΔpH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and ΔpH at equilibrium, ΔpH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][Pi]) ratio as a function of ΔpH (eq) and of the transmembrane electrochemical potential difference, Δμ̃H+ (eq), resulted in H+/ATP ratios of 3.9 ± 0.2 at pH 8.45 and 4.0 ± 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 ± 2 kJ/mol at pH 8.45 and 36 ± 3 kJ/mol at pH 8.05
The H+/ATP ratio of H+-ATPsynthases from chloroplasts, E. coli and mitochondria
No full textThe H+/ATP ratio is an important parameter for energy balance in cells and for the mechanism of coupling between proton transport and ATP synthesis. Rotational catalysis predicts that the H+/ATP coincides with the ratio of the c-subunits to the β-subunits, implying that a value of 4.7 is expected in the chloroplast ATPsynthase and a value of 3.3 is expected in the mitochondria and Escherichia coli enzyme. This ratio can be determined based on the energetics given by the chemiosmotic theory. The isolated enzymes were reconstituted into liposomes. The internal phase of the liposomes was equilibrated with the acidic medium during reconstitution, so that the internal pH could be measured with a glass electrode. An acid-base transition was carried out and the initial rates of ATP synthesis or ATP hydrolysis were measured with luciferin/luciferase as a function of ΔpH at constant Q = [ATP] / ([ADP] [Pi]). From the shift of the equilibrium ΔpH as a function of Q, the standard Gibbs free energy for phosphorylation and the H+/ATP ratios were determined
ATP synthesis in chromatophores driven by artificially induced ion gradients
No full textAn electrochemical potential difference for protons (μH+) across the membrane of bacterial chromatophores was induced by an artificially generated pH difference (pH) and a K+/valinomycin diffusion potential, φ. The initial rate of ATP synthesis was measured with a rapid‐mixing quenched‐flow apparatus in the time range between 70 ms and 30 s after the acid–base transition. The rate of ATP synthesis depends exponentially on pH;. Increasing diffusion potentials shift the pH dependency to lower pH values. Diffusion potentials were calculated from the Goldman equation. Using estimated permeability coefficients, the rate of ATP synthesis depends only on the electrochemical potential difference of protons irrespective of the relative contribution of pH and φ. Copyright © 1991, Wiley Blackwell. All rights reserve
The activity of the ATP synthase from Escherichia coli is regulated by the transmembrane proton motive force
No full textThe ATP synthase from Escherichia coli was reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid-base transition and a K+/valinomycin diffusion potential, and one second after energization, the electrochemical proton gradient was dissipated by uncouplers, and the ATP hydrolysis measurement was started. In the presence of ADP and P(i), the initial rate of ATP hydrolysis was up to 9-fold higher with pre-energized proteoliposomes than with proteoliposomes that had not seen an electrochemical proton gradient. After dissipating the electrochemical proton gradient, the high rate of ATP hydrolysis decayed to the rate without pre-energization within about 15 s. During this decay the enzyme carried out approximately 100 turnovers. In the absence of ADP and P(i), the rate of ATP hydrolysis was already high and could not be significantly increased by pre-energization. It is concluded that ATP hydrolysis is inhibited when ADP and P(i) are bound to the enzyme and that a high Δμ(H+) is required to release ADP and P(i) and to convert the enzyme into a high activity state. This high activity state is metastable and decays slowly when Δμ(H+) is abolished. Thus, the proton motive force does not only supply energy for ATP synthesis but also regulates the fraction of active enzymes
Activation of the H+-ATP synthase in the photosynthetic bacterium Rhodobacter capsulatus
No full textThe regulation of the membrane-bound H+-ATPase from the photosynthetic bacterium Rhodobacter capsulatus was investigated. In the presence of uncouplers the rate of ATP hydrolysis was about 40 mM ATP/M bacteriochlorophyll (Bchl)/s. Without uncouplers this rate increased and if, additionally, the chromatophores were illuminated, it was almost doubled. If uncouplers were added shortly after illumination, the rate increased to 300- 350 mM ATP/M Bchl/s. Obviously, energization of the membrane leads to the formation of a metastable, active state of the H+-ATPase. The maximal rate of ATP hydrolysis can be measured only when first all H+-ATPases are activated by Δμ(H+) and when the Δμ(H+) is abolished in order to release its back pressure on the hydrolysis rate. The half-life time of the metastable state in the absence of Δμ(H+) is about 30 s. It is increased by 3 mM P(i) to about 80 s and it is decreased by 1 mM ADP to about 15 s. Quantitatively, the fraction of active H+-ATPases shows a sigmoidal dependence on pH(in) (at constant pH(out)) and the magnitude of Δφ determines the maximal fraction of enzymes which can be activated: ΔpH and Δφ are not equivalent for the activation process
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Quantitative estimation of the H+-storage capacity of chromatophores and comparison with acid-base induced ATP synthesis
No full text1. The kinetics of decay in the dark of the transmembrane pH difference (ApH) induced by light in non- phosphorylating chromatophores of Rhodobacter capsulatus were studied using the fluorescent probe 9- aminoacridine, in the presence of 50 mM KCl and 2 pM valinomycin. The transient fluorescence changes induced by acid to base transitions of chromatophore suspensions were used as an empirical calibration [Casadio, R. & Melandri, B. A. (1985) Arch. Biophys. Biochem. 238, 219-2281, The kinetic competence of the probe response was tested by accelerating the ApH decay with the ionophore nigericin. 2. The time course in the dark of the increase in the internal pH in pre-illuminated chromatophores was analyzed on the basis of a model which assumes a certain number of internal buffers in equilibrium with the free protons and a diffusion-controlled H+ efflux [Whitmarsh, J. (1987) Photosynt. Res. 12, 43-62]. This model was extended to include the effects of the transmembrane electric potential difference on the H + efflux. 3. The diffusion constant for proton efflux was measured at different values of the internal pH by evaluating the frequency of trains of single-turnover flashes capable of maintaining different ApH in a steady state. The steady-state equation derived from the model does not include any parameter relative to the internal buffers and allows unequivocal determination of the diffusion constant on the basis of the known H'je- ratio (equal to two) for the active proton translocation by the bacterial photosynthetic chain. A value for the first-order diffusion constant corresponding to a permeability coefficient, PH = 0.2 pm . s-', was obtained at an external pH of 8.0; this value was constant for an internal pH ranging over 7.0-4.7. 4. Using the value of the diffusion constant determined experimentally, a satisfactory fitting of the kinetics of ApH decay in the dark could be obtained when the presence of two internal buffers (with pK values of 3.6 and 6.7, respectively) was assumed. For these calculations, the time course of the transmembrane electric potential difference was evaluated from the electrochromic signal of carotenoids, calibrated with K+-induced diffusion potentials. The two internal buffers, suitable for modelling the behaviour of the system, were at concentrations of 250 mM (pK = 3.6) and 24 mM (pK = 6.7) respectively. 5. The ability of the chromatophore suspension to store free and bound protons in the internal compartment, evaluated theoretically from the model on the basis of the above concentrations and pK values of the internal buffers, was compared with the quantity of protons disappearing from the outer suspending buffer measured with a glass electrode. Different steady states for the internal pH and for proton uptake were obtained in continuous light, adding subsaturating concentrations of nigericin. A close agreement between these two sets of data could be demonstrated. 6. From the results of this study, a complete phenomenological description of the properties of the H+-binding groups present in the internal compartment of chromatophores can be obtained. This allows a quaniitative estimate of the quantify of inner bound protons as a function of the internal pH, information useful for evaluating the storage capacity for chemiosmotic energy of this system. In photosynthetic membranes the redox reactions cata- lyzed by the electron-transfer chains are coupled to an active translocation of protons, which leads to the formation of a large difference in the proton electrochemical potential be- tween the two sides of the coupling membrane. This difference in proton potential supplies the driving force for ATP synthesis catalyzed by the H+-ATPase, and in bacteria for other energy- requiring processes such as proton-coupled secondary active transport and flagellar motion [I]. While the essential features of this chemiosmotic description of the bioenergetics of photo- synthesis are universally accepted, debate still continues on the exact number and nature of the compartments to be in- Correspondence to B. A. Melandri, Dipartimento di Biologia, Universita di Bologna, via Irnerio 42, 1-40126 Bologna, Italy cluded in a reliable chemiosmotic model [2-51. The eluci- dation of the pathways for proton fluxes during the chemiosmotic coupling of the proton pumps is experimentally very difficult. The exact modelling of the proton gradient, in all its intensive, extensive and kinetic aspects is one of the fundamental aspects of this problem. Beyond the measure of the electrostatic and concentration terms of the protonic electrochemical potential, for which an extensive literature exists, other parameters of the proton gradient are equally important. These parameters include the concentrations and pKvalues of the endogenous H +-binding groups present in the membrane, and the kinetic constants for passive and mediated proton flows across the membrane. The determination of the buffering capacity of the cou- pling membrane is fundamental for evaluating quantitativel
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
VARIATIONAL MEAN FIELD GAMES FOR MARKET COMPETITION
No full textInternational audienceIn this paper, we explore Bertrand and Cournot Mean Field Games models for market competition with reflection boundary conditions. We prove existence, uniqueness and regularity of solutions to the system of equations, and show that this system can be written as an optimality condition of a convex minimization problem. We also provide a short proof of uniqueness to the system addressed in [Graber, P. and Ben-soussan, A., Existence and uniqueness of solutions for Bertrand and Cournot mean field games, Applied Mathematics & Optimization (2016)], where uniqueness was only proved for small parameters . Finally, we prove existence and uniqueness of a weak solution to the corresponding first order system at the deterministic limit
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