共查询到20条相似文献,搜索用时 31 毫秒
1.
Alexander Wiedenmann 《BBA》2008,1777(10):1301-1310
The membrane-embedded F0 part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F0 complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F0 part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either synthesis or hydrolysis direction with Δψ or ΔpH as driving forces. The system displayed a high signal-to-noise ratio and can be accurately quantified. In contrast to ATP synthesis in the Escherichia coli F1F0 holoenzyme, no significant difference was observed in the efficiency of ΔpH or Δψ as driving forces for H+-transport through F0. Transport rates showed linear dependency on the driving force. Proton transport in hydrolysis direction was about 2400 H+/(s × F0) at Δψ of 120 mV, which is approximately twice as fast as in synthesis direction. The chloroplast enzyme was faster and catalyzed H+-transport at initial rates of 6300 H+/(s × F0) under similar conditions. The new method is an ideal tool for detailed kinetic investigations of the ion transport mechanism of ATP synthases from various organisms. 相似文献
2.
The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments and, in certain cases, proton transport across the plasma membrane of eukaryotic cells. They are multisubunit complexes composed of a peripheral domain (V(1)) responsible for ATP hydrolysis and an integral domain (V(0)) responsible for proton translocation. Based upon their structural similarity to the F(1)F(0) ATP synthases, the V-ATPases are thought to operate by a rotary mechanism in which ATP hydrolysis in V(1) drives rotation of a ring of proteolipid subunits in V(0). This review is focused on the current structural knowledge of the V-ATPases as it relates to the mechanism of ATP-driven proton translocation. 相似文献
3.
Diez M Zimmermann B Börsch M König M Schweinberger E Steigmiller S Reuter R Felekyan S Kudryavtsev V Seidel CA Gräber P 《Nature structural & molecular biology》2004,11(2):135-141
Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis. 相似文献
4.
The H(+)-ATPases from Escherichia coli, EF(0)F(1), and from chloroplasts, CF(0)F(1), were reconstituted in liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid-base transition and a K(+)/valinomycin diffusion potential and the initial rate of ATP synthesis was measured as a function of the transmembrane pH difference, DeltapH, and the electric potential difference, Deltaφ. With EF(0)F(1), a rate of 80 s(-1) is observed at DeltapH=4.1 and Deltaφ approximately 140 mV. The rate decreases sigmoidally with Deltaφ and at Deltaφ approximately 0 mV, the rate is about 1 s(-1) although DeltapH is still 4.1. Under the same conditions with CF(0)F(1), a rate of 280 s(-1) is observed which decreases to 190 s(-1) when Deltaφ is abolished, i.e. ATP synthesis catalyzed by EF(0)F(1) and CF(0)F(1) depends in a different way on DeltapH and Deltaφ. EF(0)F(1)-catalyzed ATP synthesis was measured as a function of DeltapH at a constant Deltaφ. The rate depends sigmoidally on DeltapH reaching a maximal rate which cannot be further increased by increasing DeltapH. However, this maximal rate depends on Deltaφ, i.e. DeltapH and Deltaφ are not kinetically equivalent in driving ATP synthesis. We assume that EF(0)F(1) must be converted into a metastable, active state before it catalyzes proton transport-coupled ATP synthesis. For EF(0)F(1), this activation step depends only on Deltaφ, whereas for CF(0)F(1), the activation depends on DeltapH and Deltaφ. 相似文献
5.
Recently, a novel molecular mechanism of torque generation in the F(0) portion of ATP synthase was proposed [Rohatgi, Saha and Nath (1998) Curr. Sci. 75, 716-718]. In this mechanism, rotation of the c-subunit was conceived to take place in 12 discrete steps of 30 degrees each due to the binding and unbinding of protons to/from the leading and trailing Asp-61 residues of the c-subunit, respectively. Based on this molecular mechanism, a kinetic scheme has been developed in this work. The scheme considers proton transport driven by a concentration gradient of protons across the proton half-channels, and the rotation of the c-subunit by changes in the electrical potential only. This kinetic scheme has been analyzed mathematically and an expression has been obtained to explain the pH dependence of the rate of ATP synthesis by ATP synthase under steady state operating conditions. For a single set of three enzymological kinetic parameters, this expression predicts the rates of ATP synthesis which agree well with the experimental data over a wide range of pH(in) and pH(out). A logical consequence of our analysis is that DeltapH and Deltapsi are kinetically inequivalent driving forces for ATP synthesis. 相似文献
6.
Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis. 相似文献
7.
Dimroth P 《Biochimica et biophysica acta》2000,1458(2-3):374-386
ATP, the universal carrier of cell energy is manufactured from ADP and phosphate by the enzyme ATP synthase using the energy stored in a transmembrane ion gradient. The two components of the ion gradient (DeltapH or DeltapNa(+)) and the electrical potential difference Deltapsi are thermodynamically but not kinetically equivalent. In contrast to accepted wisdom, the electrical component is kinetically indispensable not only for bacterial ATP synthases but also for that from chloroplasts. Recent biochemical studies with the Na(+)-translocating ATP synthase of Propionigenium modestum have given a good idea of the ion translocation pathway in the F(0) motor. Taken together with biophysical data, the operating principles of the motor have been delineated. 相似文献
8.
Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Cook GM Keis S Morgan HW von Ballmoos C Matthey U Kaim G Dimroth P 《Journal of bacteriology》2003,185(15):4442-4449
We describe here purification and biochemical characterization of the F(1)F(o)-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F(1)F(o)-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F(1) subunits alpha, beta, gamma, delta, and epsilon and F(o) subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F(1)F(o) holoenzyme or the isolated F(1) moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F(1). After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Deltapsi). A transmembrane proton gradient or sodium ion gradient in the absence of Deltapsi was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na(+)/H(+) antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na(+) ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis. 相似文献
9.
Bonnet M Rafi MM Chikindas ML Montville TJ 《Applied and environmental microbiology》2006,72(4):2556-2563
This study examined the bioenergetics of Listeria monocytogenes, induced to an acid tolerance response (ATR). Changes in bioenergetic parameters were consistent with the increased resistance of ATR-induced (ATR(+)) cells to the antimicrobial peptide nisin. These changes may also explain the increased resistance of L. monocytogenes to other lethal factors. ATR(+) cells had lower transmembrane pH (DeltapH) and electric potential (Deltapsi) than the control (ATR(-)) cells. The decreased proton motive force (PMF) of ATR(+) cells increased their resistance to nisin, the action of which is enhanced by energized membranes. Paradoxically, the intracellular ATP levels of the PMF-depleted ATR(+) cells were approximately 7-fold higher than those in ATR(-) cells. This suggested a role for the F(o)F(1) ATPase enzyme complex, which converts the energy of ATP hydrolysis to PMF. Inhibition of the F(o)F(1) ATPase enzyme complex by N'-N'-1,3-dicyclohexylcarbodiimide increased ATP levels in ATR(-) but not in ATR(+) cells, where ATPase activity was already low. Spectrometric analyses (surface-enhanced laser desorption ionization-time of flight mass spectrometry) suggested that in ATR(+) listeriae, the downregulation of the proton-translocating c subunit of the F(o)F(1) ATPase was responsible for the decreased ATPase activity, thereby sparing vital ATP. These data suggest that regulation of F(o)F(1) ATPase plays an important role in the acid tolerance response of L. monocytogenes and in its induced resistance to nisin. 相似文献
10.
Moore TA Moore AL Gust D 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2002,357(1426):1481-98; discussion 1498, 1511
Artificial antenna systems and reaction centres synthesized in our laboratory are used to illustrate that structural and thermodynamic factors controlling energy and electron transfer in these constructs can be modified to optimize performance. Artificial reaction centres have been incorporated into liposomal membranes where they convert light energy to vectorial redox potential. This redox potential drives a Mitchellian, quinone-based, proton-transporting redox loop that generates a Deltamu H(+) of ca. 4.4 kcal mol(-1) comprising DeltapH ca. 2.1 and Deltapsi ca. 70 mV. In liposomes containing CF(0)F(1)-ATP synthase, this system drives ATP synthesis against an ATP chemical potential similar to that observed in natural systems. 相似文献
11.
The energetics of nitrate uptake by intact cells of the halotolerant cyanobacterium Aphanothece halophytica were investigated. Nitrate uptake was inhibited by various protonophores suggesting the coupling of nitrate uptake to the proton motive force. An artificially-generated pH gradient across the membrane (DeltapH) caused an increase of nitrate uptake. In contrast, the suppression of DeltapH resulted in a decrease of nitrate uptake. The increase of external pH also resulted in an enhancement of nitrate uptake. The generation of the electrical potential across the membrane (Deltapsi) resulted in no elevation of the rate of nitrate uptake. On the other hand, the valinomycin-mediated dissipation of Deltapsi caused no depression of the rate of nitrate uptake. Thus, it is unlikely that Deltapsi participated in the energization of the uptake of nitrate. However, Na(+)-gradient across the membrane was suggested to play a role in nitrate uptake since monensin which collapses Na(+)-gradient strongly inhibited nitrate uptake. Exogenously added glucose and lactate stimulated nitrate uptake in the starved cells. N, N'-dicyclohexylcarbodiimide, an inhibitor of ATPase, could alsoinhibit nitrate uptake suggesting that ATP hydrolysis was required for nitrate uptake. All these results indicate that nitrate uptake in A. halophytica is ATP-dependent, driven by DeltapH and Na(+)-gradient. 相似文献
12.
Transport of the amino acids L-valine, L-lysine, and L-glutamic acid and of sucrose was studied in plasma membrane vesicles isolated from developing cotyledons of pea (Pisum sativum L. cv. Marzia). The vesicles were obtained by aqueous polymer two-phase partitioning of a microsomal fraction and the uptake was determined after the imposition of a H(+)-gradient (DeltapH, inside alkaline) and/or an electrical gradient (Deltapsi, inside negative) across the vesicle membrane. In the absence of gradients, a distinct, time-dependent uptake of L-valine was measured, which could be enhanced about 2-fold by the imposition of DeltapH. The imposition of Deltapsi stimulated the influx of valine by 20%, both in the absence and in the presence of DeltapH. Uptake of L-lysine was more strongly stimulated by Deltapsi than by DeltapH, and its DeltapH-dependent uptake was enhanced about 6-fold by the simultaneous imposition of Deltapsi. In the absence of gradients the uptake of L-glutamic acid was about 2-fold higher than that of L-valine, but it was not detectably affected by DeltapH or Deltapsi. Although the transport of sucrose was very low, a stimulating effect of DeltapH could be clearly demonstrated. The results lend further support to the contention that during seed development cotyledonary cells employ H(+)-symporters for the active uptake of sucrose and amino acids. 相似文献
13.
B A Feniouk D A Cherepanov W Junge A Y Mulkidjanian 《Biochimica et biophysica acta》2001,1506(3):189-203
F(0)F(1)-ATP synthase (H(+)-ATP synthase, F(0)F(1)) utilizes the transmembrane protonmotive force to catalyze the formation of ATP from ADP and inorganic phosphate (P(i)). Structurally the enzyme consists of a membrane-embedded proton-translocating F(0) portion and a protruding hydrophilic F(1) part that catalyzes the synthesis of ATP. In photosynthetic purple bacteria a single turnover of the photosynthetic reaction centers (driven by a short saturating flash of light) generates protonmotive force that is sufficiently large to drive ATP synthesis. Using isolated chromatophore vesicles of Rhodobacter capsulatus, we monitored the flash induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in parallel to the transmembrane charge transfer through F(0)F(1) (by following the decay of electrochromic bandshifts of intrinsic carotenoids). With the help of specific inhibitors of F(1) (efrapeptin) and of F(0) (venturicidin), we decomposed the kinetics of the total proton flow through F(0)F(1) into (i) those coupled to the ATP synthesis and (ii) the de-coupled proton escape through F(0). Taking the coupled proton flow, we calculated the H(+)/ATP ratio; it was found to be 3.3+/-0.6 at a large driving force (after one saturating flash of light) but to increase up to 5.1+/-0.9 at a smaller driving force (after a half-saturating flash). From the results obtained, we conclude that our routine chromatophore preparations contained three subsets of chromatophore vesicles. Chromatophores with coupled F(0)F(1) dominated in fresh material. Freezing/thawing or pre-illumination in the absence of ADP and P(i) led to an increase in the fraction of chromatophores with at least one de-coupled F(0)(F(1)). The disclosed fraction of chromatophores that lacked proton-conducting F(0)(F(1)) (approx. 40% of the total amount) remained constant upon these treatments. 相似文献
14.
The photosynthetic electron transfer chain generates proton motive force (pmf), composed of both electric field (Deltapsi) and concentration (DeltapH) gradients. Both components can drive ATP synthesis, whereas the DeltapH component alone can trigger feedback regulation of the antenna. It has often been suggested that a relatively large pmf is needed to sustain the energetic contributions of the ATP synthase reaction (DeltaG(ATP)), and that the Deltapsi component is dissipated during illumination, leading to an acidic lumen in the light. We suggest that this is incompatible with the stabilities of lumenal components and the observed activation of downregulation. Recent work on the chloroplast ATP synthase suggests that a more moderate pmf can sustain DeltaG(ATP). In addition, in vivo probes suggest that a substantial fraction of pmf can be stored as Deltapsi. Together, these factors should allow sufficient DeltaG(ATP) to maintain lumen pH in a range where lumenal enzyme activities are nearly optimal, and where the level of NPQ is regulated. 相似文献
15.
The most prominent residue of subunit a of the F(1)F(o) ATP synthase is a universally conserved arginine (aR227 in Propionigenium modestum), which was reported to permit no substitution with retention of ATP synthesis or H(+)-coupled ATP hydrolysis activity. We show here that ATP synthases with R227K or R227H mutations in the P.modestum a subunit catalyse ATP-driven Na(+) transport above or below pH 8.0, respectively. Reconstituted F(o) with either mutation catalysed 22Na(+)(out)/Na(+)(in) exchange with similar pH profiles as found in ATP-driven Na(+) transport. ATP synthase with an aR227A substitution catalysed Na(+)-dependent ATP hydrolysis, which was completely inhibited by dicyclohexylcarbodiimide, but not coupled to Na(+) transport. This suggests that in the mutant the dissociation of Na(+) becomes more difficult and that the alkali ions remain therefore permanently bound to the c subunit sites. The reconstituted mutant enzyme was also able to synthesise ATP in the presence of a membrane potential, which stopped at elevated external Na(+) concentrations. These observations reinforce the importance of aR227 to facilitate the dissociation of Na(+) from approaching rotor sites. This task of aR227 was corroborated by other results with the aR227A mutant: (i) after reconstitution into liposomes, F(o) with the aR227A mutation did not catalyse 22Na(+)(out)/Na(+)(in) exchange at high internal sodium concentrations, and (ii) at a constant (Delta)pNa(+), 22Na(+) uptake was inhibited at elevated internal Na(+) concentrations. Hence, in mutant aR227A, sodium ions can only dissociate from their rotor sites into a reservoir of low sodium ion concentration, whereas in the wild-type the positively charged aR227 allows the dissociation of Na(+) even into compartments of high Na(+) concentration. 相似文献
16.
Nakano M Imamura H Toei M Tamakoshi M Yoshida M Yokoyama K 《The Journal of biological chemistry》2008,283(30):20789-20796
Vacuolar-type H(+)-ATPase (V-ATPase) catalyzes ATP synthesis and hydrolysis coupled with proton translocation across membranes via a rotary motor mechanism. Here we report biochemical and biophysical catalytic properties of V-ATPase from Thermus thermophilus. ATP hydrolysis of V-ATPase was severely inhibited by entrapment of Mg-ADP in the catalytic site. In contrast, the enzyme was very active for ATP synthesis (approximately 70 s(-1)) with the K(m) values for ADP and phosphate being 4.7 +/- 0.5 and 460 +/- 30 microm, respectively. Single molecule observation showed V-ATPase rotated in a 120 degrees stepwise manner, and analysis of dwelling time allowed the binding rate constant k(on) for ATP to be estimated ( approximately 1.1 x 10(6) m(-1) s(-1)), which was much lower than the k(on) (= V(max)/K(m)) for ADP ( approximately 1.4 x 10(7) m(-1) s(-1)). The slower k(on)(ATP) than k(on)(ADP) and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses. 相似文献
17.
The antibiotics venturicidin, oligomycin and ossamycin were investigated as potential inhibitors of the Escherichia coli H+-ATPase. It was found that venturicidin strongly inhibited ATP-driven proton transport and ATP hydrolysis, while oligomycin weakly inhibited these functions. Inhibition of the H+-ATPase by venturicidin and oligomycin was correlated with inhibition of F0-mediate proton transport. Both inhibitors were found to interfere with the covalent reaction between dicyclohexyl[14C]carbodiimide and the F0 subunit c (uncE protein). Ossamycin had no direct inhibitory effect on E. coli F0 or F1; rather, it was found to uncouple ATP hydrolysis from proton transport. 相似文献
18.
ATP synthesis by F-type ATP synthase is obligatorily dependent on the transmembrane voltage.
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
ATP synthase is the universal enzyme that manufactures cellular ATP using the energy stored in a transmembrane ion gradient. This energy gradient has two components: the concentration difference (DeltapH or DeltapNa(+)) and the electrical potential difference DeltaPsi, which are thermodynamically equivalent. However, they are not kinetically equivalent, as the mitochondrial and bacterial ATP synthases require a transmembrane potential, DeltaPsi, but the chloroplast enzyme has appeared to operate on DeltapH alone. Here we show that, contrary to the accepted wisdom, the 'acid bath' procedure used to study the chloroplast enzyme develops not only a DeltapH but also a membrane potential, and that this potential is essential for ATP synthesis. Thus, for the chloroplast and other ATP synthases, the membrane potential is the fundamental driving force for their normal operation. We discuss the biochemical reasons for this phenomenon and a model that is consistent with these new experimental facts. 相似文献
19.
Enthalpy changes in the formation of a proton electrochemical potential (Delta mu H+) and its components, DeltapH (proton gradient) and Deltapsi (electrical potential), across two types of E. coli membrane vesicles were investigated. Flow dialysis experiments showed that in 0.1 M KPi, pH 6.6, E. coli GR19N membrane vesicles coupled with d-lactate exhibited 57 mV for DeltapH, 70 mV for Deltapsi, and 127 mV for Delta mu H+. Microcalorimetric measurements revealed that the corresponding enthalpy changes (DeltaH(pH), DeltaH(psi) and DeltaHm) were 3.5, 3.3 and 6.9 kcal/mole, respectively. Moreover, in E. coli ML 308-225 membrane vesicles across which 120mV of Delta mu H+ was generated, values of DeltaH(pH) and DeltaH(psi) were determined as 7.0 and 6.6 kcal/mole, as compared with the previously reported 14.1 kcal/mole for DeltaH(m). Comparisons of these enthalpy data revealed that component enthalpies (DeltaH(pH) and DeltaH(psi)) essentially added up to the total enthalpy (DeltaHm), providing a self-consistent test for the obtained data. In both membranes, the ratio ofDeltaH(psi) to Deltapsi was comparable to that of DeltaH(pH) to DeltapH in the formation of Delta mu H+. These observations indicated that the process of the movement of H+ across the membranes was the major contributor to the observed energetic changes. Moreover, the enthalpy change in the formation of Delta mu H+ was compared with the membranes derived from GR19N and ML 308-225 and coupled with NADH and d-lactate. The results were discussed in terms of trans-membrane phenomena. 相似文献
20.
Sulfate is a partial inhibitor at low and a non-essential activator at high [ATP] of the ATPase activity of F(1). Therefore, a catalytically-competent ternary F(1) x ATP x sulfate complex can be formed. In addition, the ANS fluorescence enhancement driven by ATP hydrolysis in submitochondrial particles is also stimulated by sulfate, clearly showing that the ATP hydrolysis in its presence is coupled to H(+) translocation. However, sulfate is a strong linear inhibitor of the mitochondrial ATP synthesis. The inhibition was competitive (K (i) = 0.46 mM) with respect to Pi and mixed (K (i) = 0.60 and K'(i) = 5.6 mM) towards ADP. Since it is likely that sulfate exerts its effects by binding at the Pi binding subdomain of the catalytic site, we suggest that the catalytic site involved in the H(+) translocation driven by ATP hydrolysis has a more open conformation than the half-closed one (beta(HC)), which is an intermediate in ATP synthesis. Accordingly, ATP hydrolysis is not necessarily the exact reversal of ATP synthesis. 相似文献