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1.
The process of proton transfer along a proton channel is considered using bacteriorhodopsin as a model system, for which a large body of experimental data is available. The possible amino acid composition of the external proton half-channel of bacteriorhodopsin and the stepwise scheme of proton transfer consistent with experimental data are proposed. The rate of proton transfer between fixed centers is assessed for certain regions of this channel for which spectroscopic data are available. 相似文献
2.
Hervé Cretin 《The Journal of membrane biology》1982,65(3):175-184
Summary Lutoids (vacuo-lysosomal particles) were isolated from the latex ofHevea brasiliensis. Using flow dialysis with14C-methylamine uptake as a pH probe and86Rb rubidium+valinomycin distribution for estimations of transmembrane electrical potential, intact lutoids exhibited a pH of 1 unit (interior more acid) and a of –70 mV (interior negative), when suspended in an isotonic medium at physiological concentration of potassium (30mm) and pH 7.0, in the absence of ATP. In most cases, the Donnan potential was shown to fully account for pH in nonenergized lutoids. The addition of Mg-ATP (5mm) resulted in a marked acidification of the lutoidic internal space (0.7 to 1 pH unit) depending on the composition of the medium, and in a membrane depolarization by 60 mV (interior becoming less negative). The resulting electrochemical potential of protons (
) increased by a hundred millivolts when lutoids were energized by ATP. These data strongly support an inward electrogenic proton translocating function for the ATPase of the vacuo-lysosomal membrane of lutoids. Results are discussed in terms of thein vivo maintenance of large lutoids/cytoplasm proton gradients, and of the rôle of these vacuo-lysosomes in the homeostasis of the cytoplasmic metabolism. 相似文献
3.
Flash-driven ATP formation by spinach chloroplast thylakoids, using the luciferin luminescence assay to detect ATP formed in single turnover flashes, was studied under conditions where a membrane protein amine buffering pool was either protonated or deprotonated before the beginning of the flash trains. The flash number for the onset of ATP formation was delayed by about 10 flashes (from 15 to about 25) when the amine pool was deprotonated as compared to the protonated state. The delay was substantially reversed again by reprotonating the pool upon application of 20–30 single-turnover flashes and 8 min of dark before addition of ADP, Pi, and the luciferin system. In the case of deprotonation by desaspidin, the uncoupler was removed by binding to BSA before the reprotonating flashes were given. Reprotonation was carried out before addition of ADP and Pi, to avoid a possible interference by the ATP-ase, which can energize the system by pumping protons. The reprotonated state, as indicated by an onset lag of about 15 flashes rather than 25 for the deprotonated state, was stable in the dark over extended dark times. The number of protons released by 10 flashes is approximately 30 nmol H+ (mg chl)–1, an amount similar to the size of the reversibly protonated amine group buffering pool. The data are consistent with the hypothesis that the amine buffering groups must be in the protonated state before any protons proceed to the coupling complex and energize ATP formation. Other work has suggested that the amine buffering pool is sequestered within membrane proteins rather than being exposed directly to the inner aqueous bulk phase. Therefore, it is possible that the sequested amine group array may provide localized association-dissociation sites for proton movement to the coupling complex. 相似文献
4.
Unraveling the mechanism of proton translocation in the extracellular half‐channel of bacteriorhodopsin
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Bacteriorhodopsin, a light activated protein that creates a proton gradient in halobacteria, has long served as a simple model of proton pumps. Within bacteriorhodopsin, several key sites undergo protonation changes during the photocycle, moving protons from the higher pH cytoplasm to the lower pH extracellular side. The mechanism underlying the long‐range proton translocation between the central (the retinal Schiff base SB216, D85, and D212) and exit clusters (E194 and E204) remains elusive. To obtain a dynamic view of the key factors controlling proton translocation, a systematic study using molecular dynamics simulation was performed for eight bacteriorhodopsin models varying in retinal isomer and protonation states of the SB216, D85, D212, and E204. The side‐chain orientation of R82 is determined primarily by the protonation states of the residues in the EC. The side‐chain reorientation of R82 modulates the hydrogen‐bond network and consequently possible pathways of proton transfer. Quantum mechanical intrinsic reaction coordinate calculations of proton‐transfer in the methyl guanidinium‐hydronium‐hydroxide model system show that proton transfer via a guanidinium group requires an initial geometry permitting proton donation and acceptance by the same amine. In all the bacteriorhodopsin models, R82 can form proton wires with both the CC and the EC connected by the same amine. Alternatively, rare proton wires for proton transfer from the CC to the EC without involving R82 were found in an O′ state where the proton on D85 is transferred to D212. Proteins 2016; 84:639–654. © 2016 Wiley Periodicals, Inc. 相似文献
5.
The mechanism of proton exclusion in the aquaporin-1 water channel 总被引:11,自引:0,他引:11
Aquaporins are efficient, yet strictly selective water channels. Remarkably, proton permeation is fully blocked, in contrast to most other water-filled pores which are known to conduct protons well. Blocking of protons by aquaporins is essential to maintain the electrochemical gradient across cellular and subcellular membranes. We studied the mechanism of proton exclusion in aquaporin-1 by multiple non-equilibrium molecular dynamics simulations that also allow proton transfer reactions. From the simulations, an effective free energy profile for the proton motion along the channel was determined with a maximum-likelihood approach. The results indicate that the main barrier is not, as had previously been speculated, caused by the interruption of the hydrogen-bonded water chain, but rather by an electrostatic field centered around the fingerprint Asn-Pro-Ala (NPA) motif. Hydrogen bond interruption only forms a secondary barrier located at the ar/R constriction region. The calculated main barrier height of 25-30 kJ mol(-1) matches the barrier height for the passage of protons across pure lipid bilayers and, therefore, suffices to prevent major leakage of protons through aquaporins. Conventional molecular dynamics simulations additionally showed that negatively charged hydroxide ions are prevented from being trapped within the NPA region by two adjacent electrostatic barriers of opposite polarity. 相似文献
6.
A highly sensitive quantitative method was developed to detect protons released or taken up upon ligand binding. A small change in pH due to proton release or uptake was detected by measuring the difference in the absorbance of a pH indicator upon ligand addition. Owing to the difference detection of protons, the uncertainty of pH due to CO2 dissolution and unknown buffering capacities of sample solutes could be compensated with easy manipulations. Precise calibration of the absolute amount of protons could also be made very easily. The amount of protons measurable by the method is as small as 0.5 nmol that is 10 to 30 times more sensitive than the pH-stat method. We measured the Mg2+ ion-induced proton releases of ADP to confirm the accuracy and reliability of the method and of Escherichia coli ribosomes to show the improvement in sensitivity. The method is useful for protometric studies of biomolecules that are difficult to obtain in large amount. 相似文献
7.
Histidine scanning mutagenesis of basic residues of the S4 segment of the shaker k+ channel 总被引:3,自引:0,他引:3
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The voltage sensor of the Shaker potassium channel is comprised mostly of positively charged residues in the putative fourth transmembrane segment, S4 (Aggarwal, S.K., and R. MacKinnon. 1996. Neuron. 16:1169-1177; Seoh, S.-A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Neuron. 16:1159-1167). Movement of the voltage sensor in response to a change in the membrane potential was examined indirectly by measuring how the accessibilities of residues in and around the sensor change with voltage. Each basic residue in the S4 segment was individually replaced with a histidine. If the histidine tag is part of the voltage sensor, then the gating charge displaced by the voltage sensor will include the histidine charge. Accessibility of the histidine to the bulk solution was therefore monitored as pH-dependent changes in the gating currents evoked by membrane potential pulses. Histidine scanning mutagenesis has several advantages over other similar techniques. Since histidine accessibility is detected by labeling with solution protons, very confined local environments can be resolved and labeling introduces minimal interference of voltage sensor motion. After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible. Histidine replacement of residues R365, R368, and R371, on the other hand, showed that each of these residues traverses entirely from internal exposure at hyperpolarized potentials to external exposure at depolarized potentials. This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient. In the case of 371H, depolarization drives the histidine to a position that forms a proton pore. Kinetic models of titrateable voltage sensors that account for proton transport and conduction are presented. Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure. 相似文献
8.
Sergio Paoletti Roberto Gilli Luciano Navarini Vittorio Crescenzi 《Glycoconjugate journal》1997,14(4):513-517
The study of the proton dissociation process of weak polyacids (eg α carboxylic poly(monoprotic)acid) is based on the knowledge
of the change in electrostatic free energy, G(el), as a function of the variation of the number of charges on the polymer
chain. The original treatment proposed by Manning can be used to describe the proton dissociation process of weak poly(monoprotic)acids,
in the absence of pH-induced conformational transitions. In order to describe the alpha dependence of pKa of weak co-poly(monoprotic)acids
containing two different acidic groups in different amounts along the polymer chain, a simple modification of the model is
proposed. Abbreviations used: Due to the difficulty of using non-ASCI characters in the main text, we have used the following
abbreviations: G(el) Gel 10-pH 10-pH pKa pKa alpha α alphan αn dG(el)/dalpha δGel/δα KA and KB KA and KB CA and CB CA and
CB βA and βB βA and βB δpK δpK §max ξmax
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
9.
10.
David G. Nicholls 《Bioscience reports》1997,17(3):251-257
The proton conductance of the mitochondrial inner membrane can be quantified by applying Ohm's law to the experimentally determined protonmotive force and the proton current flowing around the proton circuit in the absence of ATP synthesis or ion transport. This last parameter is derived from the rate of State 4 respiration multiplied by the H+/O stoichiometry for the substrate. When the activity of the dehydrogenase supplying electrons to the respiratory chain is progressively increased the proton conductance increases rapidly when the protonmotive force is greater than 220 mV. The consequences of this non-ohmic relationship are discussed. 相似文献
11.
John Gutknecht 《The Journal of membrane biology》1988,106(1):83-93
Summary Mechanisms of proton conductance (G
H) were investigated in phospholipid bilayer membranes containing long-chain fatty acids (lauric, myristic, palmitic, oleic or phytanic). Membranes were formed from diphytanoyl phosphatidylcholine in decane plus chlorodecane (usually 30% vol/vol). Fatty acids were added either to the aqueous phase or to the membrane-forming solution. Proton conductance was calculated from the steadystate total conductance and the H+ diffusion potential produced by a transmembrane pH gradient. Fatty acids causedG
H to increase in proportion to the first power of the fatty acid concentration. TheG
H induced by fatty acids was inhibited by phloretin, low pH and serum albumin.G
H was increased by chlorodecane, and the voltage dependence ofG
H was superlinear. The results suggest that fatty acids act as simple (A– type) proton carriers. The membrane: water partition coefficient (K
p
) and adsorption coefficient () were estimated by finding the membrane and aqueous fatty acid concentrations which gave identical values ofG
H. For palmitic and oleic acidsK
p
was about 105 and was about 10–2 cm. The A– translocation or flip-flop rate (k
a
) was estimated from the value ofG
H and the fatty acid concentration in the membrane, assuming that A– translocation was the rate limiting step in H+ transport. Thek
A
's were about 10–4 sec–1, slower than classical weak-acid uncouplers by a factor of 105. Although long-chain fatty acids are relatively inefficient H+ carriers, they may cause significant biological H+ conductance when present in the membrane at high concentrations, e.g., in ischemia, hypoxia, hormonally induced lipolysis, or certain hereditary disorders, e.g., Refsum's (phytanic acid storage) disease. 相似文献
12.
Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonates during the photocycle 总被引:15,自引:0,他引:15
M S Braiman T Mogi L J Stern N R Hackett B H Chao H G Khorana K J Rothschild 《Proteins》1988,3(4):219-229
The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr----Phe substitution, difference spectra have been obtained for the bR----K and bR----M photoreactions. Only the Tyr-185----Phe mutation results in the disappearance of a set of bands that were previously shown to be due to the protonation of a tyrosinate during the bR----K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United States of America 83:347, (1986]). The Tyr-185----Phe mutation also eliminates a set of bands in the bR----M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr----Phe mutations. Thus, tyrosinate-185 gains a proton during the bR----K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C = NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185----Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation. 相似文献
13.
A. N. WEBBER N. R. BAKER CANDIDA D. PAIGE M. F. HIPKINS 《Plant, cell & environment》1986,9(3):203-208
Abstract Photosynthetic electron transport activities and the ability to generate and maintain a trans-thylakoid proton electrochemical gradient were examined during chloroplast development in 4-day-old wheat leaves grown under a diurnal light regime. Polarographic and spectropholometric studies on leaf tissue demonstrated that poorly developed chloroplasls at the leaf base could photo-oxidize water and transfer electrons from photosystem 2 to photosystem 1. The capacity for non-cyclic whole-chain electron transport increased during chloroplast development. Thylakoids isolated from the leaf base, although capable of pumping protons into the inlrathylakoid space, could not maintain a trans-membrane proton electrochemical gradient; this ability developed at later stages of chloroplast biogenesis in the leaf. The implications of these results for the energetics of the developing leaf are discussed. 相似文献
14.
Junji Hyodo Koki Kitabayashi Kenta Hoshino Yuji Okuyama Yoshihiro Yamazaki 《Liver Transplantation》2020,10(25)
The environmental benefits of fuel cells and electrolyzers have become increasingly recognized in recent years. Fuel cells and electrolyzers that can operate at intermediate temperatures (300–450 °C) require, in principle, neither the precious metal catalysts that are typically used in polymer‐electrolyte‐membrane systems nor the costly heat‐resistant alloys used in balance‐of‐plant components of high‐temperature solid oxide electrochemical cells. These devices require an electrolyte with high ionic conductivity, typically more than 0.01 S cm?1, and high chemical stability. To date, however, high ionic conductivities have been found in chemically unstable materials such as CsH2PO4, In‐doped SnP2O7, BaH2, and LaH3?2xOx. Here, fast and stable proton conduction in 60‐at% Sc‐doped barium zirconate polycrystal, with a total conductivity of 0.01 S cm?1 at 396 °C for 200 h is demonstrated. Heavy doping of Sc in barium zirconate simultaneously enhances the proton concentration, bulk proton diffusivity, specific grain boundary conductivity, and grain growth. An accelerated stability test under a highly concentrated and humidified CO2 stream using in situ X‐ray diffraction shows that the perovskite phase is stable over 240 h at 400 °C under 0.98 atm of CO2. These results show great promises as an electrolyte in solid‐state electrochemical devices operated at intermediate temperatures. 相似文献
15.
Summary To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO3-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO3-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO3-ATPase in kidney membrane fractions. HCO3-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO3-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO3-ATPase to vanadate indicates that it belongs to the F0–F1 class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO3-ATPase activity at the sites of urinary acidification. 相似文献
16.
A One-Dimensional (Proton and Phosphorus) and Two-Dimensional (Proton) In Vivo NMR Spectroscopic Study of Reversible Global Cerebral Ischemia 总被引:5,自引:1,他引:4
S. Brulatout Ph. Méric I. Loubinoux J. Borredon J. L. Corrèze P. Roucher B. Gillet G. Bérenger J. C. Beloeil †B. Tiffon †J. Mispelter J. Seylaz 《Journal of neurochemistry》1996,66(6):2491-2499
Abstract: The suitability of two-dimensional (2D) proton spectroscopy for monitoring, in vivo, the changes in levels of brain metabolites induced by cerebral ischemia was investigated in an experimental model of 30-min reversible ischemia induced by four-vessel occlusion in the rat. The resulting data were compared with those obtained by one-dimensional (1D) proton and phosphorus spectroscopy. Phosphorus spectra obtained during ischemia showed significant drops in levels of phosphocreatine (−73%), β-ATP (−60%), and intracellular pH (to 6.30) and an increase in inorganic phosphate level (905%). 1D and 2D proton spectra showed decreases in the N -acetylaspartate/creatine-phosphocreatine ratio that were not significantly different [−21% (1D) and −32% (2D)]. Similarly, the increases in lactate/creatine-phosphocreatine ratio were not significantly different [2,546% (1D) and 3,020% (2D)]. 2D spectroscopy also indicated a decrease in aspartate (−66%) and an increase in the inositol-choline derivative (+124%) pools during ischemia and an increase in alanine pool (+516%) during reperfusion. The glutamate-glutamine pool and taurine content did not change significantly during ischemia but decreased during reperfusion. The glucose level transiently decreased (−67%) during ischemia and increased immediately after (+261%). The levels of all the metabolites investigated returned to control values within 175 min after ischemia. 2D spectroscopy seems to be a reliable method of monitoring the changes in levels of cerebral compounds known to be involved in ischemia. 相似文献
17.
内毒素休克大鼠肝线粒体质子跨膜转运的改变 总被引:6,自引:0,他引:6
用稳态荧光探针标记技术动态观察内毒素休克大鼠肝亚线粒体质子跨膜转运的变化.发现,休克5 h ATP、NADH和琥珀酸钠所致的9-氨基-6-氯-2-甲氧基吖啶(ACMA)最大荧光淬灭值(ΔAmax)显著低于对照组(P<0.05)、最大荧光淬灭时间(TΔAmax)、半数荧光淬灭时间(T1/2ΔAmax)非常显著延长(P<0.01),肝线粒体质子跨膜转运能力下降;膜脂分子烃链和膜脂深层次流动性下降;线粒体膜PLA2活性增加;血浆脂质过氧化产物MDA和线粒体MDA含量均显著增加.可能膜脂质过氧化和磷脂酶A2的水解是引起内毒素休克肝线粒体质子转运功能改变的重要因素. 相似文献
18.
Photosynthetic and respiratory electron transport and their interplay with ion transport have been studied in Arthrospira platensis, a filamentous alkaliphilic cyanobacterium living in hypersaline lakes. As typical for alkaliphiles, A. platensis apparently does not maintain an outward positive pH gradient at its plasma membrane. Accordingly, sodium extrusion occurs
via an ATP-dependent primary sodium pump, in contrast to the Na+/H+ antiport in most cyanobacteria. A. platensis is strongly dependent on sodium/bicarbonate symport for the uptake of inorganic carbon. Sodium extrusion in the presence
of the Photosystem II inhibitor diuron indicates that a significant amount of ATP is supplied by cyclic electron transport
around Photosystem I, the content of which in A. platensis is exceptionally high. Plastoquinol is oxidized by two parallel pathways, via the cytochrome b
6
f complex and a putative cytochrome bd complex, both of which are active in the light and in the dark.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
19.
20.
BaZr0.7Sn0.1Y0.2O3–δ (BZSY) is developed as a novel chemically stable proton conductor for solid oxide fuel cells (SOFCs). BZSY possesses the same cubic symmetry of space group Pm‐3m with BaZr0.8Y0.2O3‐δ (BZY). Thermogravimetric analysis (TGA) and X‐ray photoelectron spectra (XPS) results reveal that BZSY exhibits remarkably enhanced hydration ability compared to BZY. Correspondingly, BZSY shows significantly improved electrical conductivity. The chemical stability test shows that BZSY is quite stable under atmospheres containing CO2 or H2O. Fully dense BZSY electrolyte films are successfully fabricated on NiO–BZSY anode substrates followed by co‐firing at 1400 °C for 5 h and the film exhibits excellent electrical conductivity under fuel cell conditions. The single cell with a 12‐μm‐thick BZSY electrolyte film outputs by far the best performance for acceptor‐doped BaZrO3‐based SOFCs. With wet hydrogen (3% H2O) as the fuel and static air as the oxidant, the peak power density of the cell achieves as high as 360 mWcm?2 at 700 °C, an increase of 42% compared to the reported highest performance of BaZrO3‐based cells. The encouraging results demonstrate that BZSY is a good candidate as the electrolyte material for next generation high performance proton‐conducting SOFCs. 相似文献