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1.
Light-induced proton gradients and internal volumes in chromatophores of Rhodopseudomonas sphaeroides 总被引:1,自引:0,他引:1
To test the predictions of the chemiosmotic hypothesis, it is essential to have sensitive and accurate measures of the aqueous volume and pH within membrane compartments. One unique feature of the present investigation is the application of electron spin resonance probes to determine internal aqueous volume and pH changes in bacterial chromatophores under virtually identical conditions. Volumes of the chromatophores ranged from 6 to 16 microliter/mg bacteriochlorophyll among different preparations, and were sensitive to the osmolarity of the suspending buffer. pH gradients reached two units in illuminated chromatophores as determined with ESR methods, and increased when KCl and valinomycin were added to the assay. Measurements with the fluorescent dye 9-amino-acridine yielded similar pH gradients, provided that an operational vesicle volume, which corrected for the binding of the dye to the membrane, was used in the calculation. The sensitivity of the ESR method allowed the measurement of pH gradients resulting from only a few light flashes. A plot of pH gradients versus number of flashes was linear up to about 30 flashes, and intercepted the origin. This result is consistent with proton release into the bulk aqueous phase after only a single light flash. This ability to measure small pH gradients offers new opportunities for the study of energy-transducing mechanisms. 相似文献
2.
DELAYED light emission from photosynthetic organisms was discovered by Strehler and Arnold1. The emitted light has a spectrum similar to that of chlorophyll a fluorescence and can often persist for minutes after terminating the illumination. In recent years it has been found that the intensity of emission during the first few milliseconds of the decay is sensitive to the high energy state of the chloroplasts2. Wraight and Crofts3 have suggested that this sensitivity is due to the establishment of electrical and pH gradients across the thylakoids during the illumination stage. The linking of the high energy state with these gradients is an essential feature of Mitchell's chemiosmotic hypothesis4. Wraight and Crofts3 have argued that the light-induced pH and electrical gradients act in such a way as to decrease the activation energy necessary to lift electrons from the metastable state, created during the preillumination, to the first singlet of chlorophyll. If this hypothesis is correct, the establishment of an electrical gradient across the thylakoid membranes by some means other than light-induced electron transport should change the intensity of millisecond delayed light emission. One possible way to create membrane potentials is to subject chloroplasts to salt gradients. The magnitude of the potentials developed will be a function of the concentration gradients and the relative rates of penetration of the ions across the thylakoid membranes5,6. 相似文献
3.
Chemiosmotic energy conversion of the archaebacterial thermoacidophile Sulfolobus acidocaldarius: oxidative phosphorylation and the presence of an F0-related N,N'-dicyclohexylcarbodiimide-binding proteolipid 总被引:2,自引:2,他引:0
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The energy-transducing mechanism of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius DSM 639 has been studied, addressing the question whether chemiosmotic proton gradients serve as an intermediate energy store driving an F0F1-analogous ATP synthase. At pH 3.5, respiring S. acidocaldarius cells developed an electrochemical potential of H+ ions, consisting mainly of a proton gradient and a small inside-negative membrane potential. The steady-state proton motive force of 140 to 160 mV was collapsed by protonophores, while N,N'-dicyclohexylcarbodiimide (DCCD) caused a hyperpolarization of the membrane, as expected for a reagent commonly used to inhibit the flux through proton channels of F0F1-type ATP synthases. Cellular ATP content was strongly related to the proton motive force generated by respiration and declined rapidly, either by uncoupling or by action of DCCD, which in turn induced a marked respiratory control effect. This observation strongly supports the operation of chemiosmotic ATP synthesis with H+ as the coupling ion. The inhibition of ATP synthesis by [14C]DCCD was correlated with covalent reactions with membrane proteins. The extraction of labeled membranes with organic solvents specifically yielded a readily aggregating proteolipid of 6 to 7 kilodaltons apparent molecular mass. Its amino acid composition revealed significant similarity to the proteolipid found in eubacteria, such as Escherichia coli, as an extremely hydrophobic constituent of the F0 proton channel. Moreover, the N-terminal amino acid sequence of the Sulfolobus proteolipid displays a high degree of homology to eubacterial sequences, as well as to one derived from nucleic acid sequencing of another Sulfolobus strain (K. Denda, J. Konishi, T. Oshima, T. Date, and M. Yoshida, J. Biol. Chem. 264:7119-7121, 1989). Despite certain structural similarities between eucaryotic vacuolar ATPases and the F1-analogous ATPase from Sulfolobus sp. described earlier, the results reported here promote the view that the archaebacterial ATP-synthesizing complex functionally belongs to the F0F1 class of ATPases. These may be considered as phylogenetically conserved catalysts of energy transduction present in all kingdoms of organisms. 相似文献
4.
It is proven that any model of localized protonmotive energy coupling that relies upon properties of a homogeneous surface phase must, when operated in the steady state, lead to bulk phase electrochemical potentials for protons that are as large as those required by the delocalized chemiosmotic theory. To obtain models consistent with experiments supporting localized energy coupling requires some kind of surface heterogeneity for the proton conducting pathways. Two general classes of heterogeneous surface models are mentioned. One class involves phase-separated lipid domains. The second class involves hydrogen-bonded chains in proteins that traverse the membrane laterally. 相似文献
5.
An evolutionary scheme for the origin of chemiosmotic coupling of redox reactions and ATP synthesis is proposed. It is argued that the primitive heterotroph, which generated ATP by substrate level phosphorylation, used some of this ATP in active proton extrusion to regulate cytoplasmic pH. As fermentation substrates were used up, selection favoured organisms which produced a light-dependent redox pump for proton extrusion. This partly replaced the ATP-dependent proton extrusion, thereby economizing on fermentation substrates. The ATP-requiring mechanism was retained for dark proton extrusion. A further economic advantage would come about if the energy of the light-generated proton gradient were used to reverse the ATP-dependent proton pump, leading to chemiosmotic photophosphorylation. This hypothesis explains the origin of the two kinds of proton pump, and their occurrence in the same membrane; the origin of these two prerequisites of chemiosmotic coupling had not previously been adequately explained. The success of the proton pump based on redox loops of alternating vectorial electron and hydrogen atom carriers, rather than the apparently simpler light-driven proton pump of Halobacterium is explained in terms of the ease of converting the former type of cyclic photophosphorylation, but not the latter, into a system bringing about net redox reactions. 相似文献
6.
Volumes and pH gradients were determined with spin probes in liposomes and zucchini membrane vesicles by quantitating the internal concentrations of probes in the presence of an impermeable line-broadening agent, manganese + EDTA. Volume shrinkage in response to increasing external concentrations of MnEDTA was consistent with perfect osmotic behavior of both vesicle populations. Buffer additions were used to impose pH gradients on the vesicles; liposome gradients measured with a spin-labeled weak acid were slightly smaller than the maximum theoretical imposed gradients, whereas above a threshold magnitude, measured gradients for the plant membranes were significantly smaller than imposed gradients. However, the residual pH gradient in the zucchini vesicles decreased at about the same rate as the liposome gradient. Moreover, this residual gradient was not completely collapsed in the presence of the proton ionophore, FCCP, indicating that the vesicles were impermeable to ions; indeed, ion permeabilities of both vesicle preparations appeared to be similar during the slow phase of the pH gradient collapse. Thus, zucchini membrane vesicles are tightly sealed and appear to have a mechanism for dissipating pH gradients rapidly when these gradients exceed some threshold value. 相似文献
7.
Proton flux in large unilamellar vesicles in response to membrane potentials and pH gradients. 总被引:2,自引:2,他引:0
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The transport of protons across liposomes composed of phosphatidylcholine in response to electrical potentials or pH gradients has been investigated. The results support three major conclusions. The first of these concerns the need for reliable measurements of electrical potentials and pH gradients. It is shown that the potential probe tetraphenylphosphonium and the pH probe methylamine provide accurate and self consistent measures of electrical potentials and pH gradients respectively in these systems. Second, it is shown by two independent techniques that the pH gradients induced in response to valinomycin and potassium dependent electrical potentials are significantly smaller than would be expected for electrochemical equilibrium. The pH gradients observed are stable over an 8 h time course and are sensitive to the ionic composition of the buffers employed, where the presence of external sodium results in the smallest induced pH gradients. These results are discussed in terms of current models of proton conductance across membranes. In a final area of investigation, it is shown that valinomycin and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) can transport sodium ions in a synergistic manner. 相似文献
8.
V P Skulachev 《Bioscience reports》1991,11(6):387-441; discussion 441-4
The development of membrane bioenergetic studies during the last 25 years has clearly demonstrated the validity of the Mitchellian chemiosmotic H+ cycle concept. The circulation of H+ ions was shown to couple respiration-dependent or light-dependent energy-releasing reactions to ATP formation and performance of other types of membrane-linked work in mitochondria, chloroplasts, some bacteria, tonoplasts, secretory granules and plant and fungal outer cell membranes. A concrete version of the direct chemiosmotic mechanism, in which H+ potential formation is a simple consequence of the chemistry of the energy-releasing reaction, is already proved for the photosynthetic reaction centre complexes. Recent progress in the studies on chemiosmotic systems has made it possible to extend the coupling-ion principle to an ion other than H+. It was found that, in certain bacteria, as well as in the outer membrane of the animal cell, Na+ effectively substitutes for H+ as the coupling ion (the chemiosmotic Na+ cycle). A precedent is set when the Na+ cycle appears to be the only mechanism of energy production in the bacterial cell. In the more typical case, however, the H+ and Na+ cycles coexist in one and the same membrane (bacteria) or in two different membranes of one and the same cell (animals). The sets of delta mu H+ and delta mu Na+ generators as well as delta mu H+ and delta mu Na+ consumers found in different types of biomembranes, are listed and discussed. 相似文献
9.
Vladimir P. Skulachev 《Journal of bioenergetics and biomembranes》1994,26(6):589-598
The present state of the chemiosmotic concept is reviewed. Special attention is paid to (i) further progress in studies on the Na+-coupled energetics and (ii) paradoxical bioenergetic effects when protonic or sodium potentials are utilized outside the coupling membrane (TonB-mediated uphill transports across the outer bacterial membrane). A hypothesis is put forward assuming that the same principle is employed in the bacterial flagellar motor. 相似文献
10.
P. Smejtek 《Chemistry and physics of lipids》1974,13(2):141-154
The electrical characteristics of wide membrane channels such as those induced in lipid membranes by alamethicin have been analyzed using an electrodiffusion model. The channel is considered to be a water filled cylinder in which the potential energy barrier is a result of the difference in polarization energy of the ion environment when the ion is located inside as compared to outside of the channel. In addition, an electric field related to the channel structure is assumed. It is shown that without postulating any specific chemical ion-channel interaction one can reproduce experimental membrane potentials for NaCl, KCl, and CaCl2 concentration gradients with a single set of channel parameters. The calculations also yield experimental J-V characteristics of discrete conduction states. In addition, a simple mechanism of interchannel coupling based on the above model is discussed. The model suggests a unifying approach to the problem of the origin of interionic selectivity of membrane channels induced by polyene antibiotics. 相似文献
11.
Spin-labeled secondary amines have been used to measure transmembrane proton gradients in sonicated liposomes. The electron paramagnetic resonance spectra of these probes show changes in the ratio of membrane associated to free aqueous probe as a function of transmembrane pH gradient. As the pH gradient is increased, inside acidic, the amount of membrane associated probe increases. The results are accounted for by a simple thermodynamic theory. 相似文献
12.
Phosphorescent probes are described that are quenchable by dioxygen and that partition into membranes. These probes are derivatives of porphyrin, in which the central metal, either zinc or palladium, induces intersystem crossing to enhance the triplet yield. The location of the probe in a suspension of membranes depends upon the charge distribution of side groups on the porphyrins. Probes that partition into the membrane are sensitive to phase transitions in lecithin artificial membranes. In the mitochondria these membrane probes are within transfer distance from tryptophans in membrane proteins. Although absolute concentrations of oxygen in membranes cannot be determined by this method, by comparing the oxygen dependence of a probe in the aqueous phase with that in the membrane phase under actively respiring and inhibited conditions, it is possible to examine the extent of O2 depletion at the mitochondrial surface. We show that at oxygen tensions of 0.2 microM and higher these gradients are insignificant at usual oxygen consumption rates of mitochondria. 相似文献
13.
J. C. Smith S. J. Frank C. L. Bashford B. Chance B. Rudkin 《The Journal of membrane biology》1980,54(2):127-139
Summary The second-order rate constants characterizing the association of potential-sensing dyes of the cyanine, merocyanine, and oxonol classes with glycerylmonooleate suspensions, azolectin vesicles, or submitochondrial particles have been measured and the implications for redistribution type mechanisms proposed to explain the potential-dependent optical signals of these probes considered. The second-order rate constants obtained for the cyanines and oxonols are compatible with microsecond probe response times only on the assumption that a high local dye concentration exists in the aqueous phase immediately adjacent to the membrane surface. Calculations based on a surface charge density induced by a bias potential suggest that the necessary local concentration cannot be attained by a diffusion polarization mechanism. A model based on the rapid recombination of ejected dye with the membrane bilayer seems capable of explaining microsecond probe response times in systems where the potential is rapidly changing polarity; calculations suggest that an ejected dye molecule would not diffuse out of an unstirred layer of 100 microns thickness on a millisecond time scale. Microsecond probe responses are also compatible with a first-order potential-dependent dye ejection from the membrane with no rapid recombination when the potential is not changing polarity. The apparent first-order rate constants describing the interaction of merocanine M-540 with a glycerylmonooleate suspension are independent of dye concentration; the reaction may be diffusion limited. The high local dye concentration need not be met in this case for a mechanism based on the transfer of dye onto the membrane from the aqueous phase to describe the microsecond signals of this dye, but other mechanisms have been proposed to explain such signals. The mechanism leading to potentialdependent signals from optical probes appear to differ substantially between suspensions of energy-transducing biological membranes and those involving excitable membranes such as the squid giant axon or model black lipid membranes. 相似文献
14.
The relationship between the electrochemical proton gradient and active transport in Escherichia coli membrane vesicles. 总被引:27,自引:0,他引:27
In the previous paper [ramos, S., and Kaback, H.R. (1977), Biochemistry 16 (preceding paper in this issue)], it was demonstrated that Escherichia coli membrane vesicles generate a large electrochemical proton gradient (delta-muH+) under appropriate conditions, and some of the properties of delta-muH+ and its component forces [i.e., the membrane potential (delta psi) and the chemical gradient of protons (deltapH)] were described. In this paper, the relationship between delta-muH+, delta psi, and deltapH and the active transport of specific solutes is examined. Addition of lactose or glucose 6-phosphate to membrane vesicles containing the appropriate transport systems results in partial collapse of deltapH, providing direct evidence for the suggestion that respiratory energy can drive active transport via the pH gradient across the membrane. Titration studies with valinomycin and nigericin lead to the conclusion that, at pH 5.5, there are two general classes of transport systems: those that are driven primarily by delta-muH+ (lactose, proline, serine, glycine, tyrosine, glutamate, leucine, lysine, cysteine, and succinate) and those that are driven primarily by deltapH (glucose 6-phosphate, D-lactate, glucuronate, and gluconate). Importantly, however, it is also demonstrated that at pH 7.5, all of these transport systems are driven by delta psi which comprises the only component of delta-muH+ at this external pH. In addition, the effect of external pH on the steady-state levels of accumulation of different solutes is examined, and it is shown that none of the pH profiles correspond to those observed for delta-muH+, delta psi, or deltapH. Moreover, at external pH values above 6.0-6.5, delta-muH+ is insufficient to account for the concentration gradients established for each substrate unless the stoichiometry between protons and accumulated solutes is greater than unity. The results confirm many facets of the chemiosmotic hypothesis, but they also extend the concept in certain important respects and allow explanations for some earlier observations which seemed to preclude the involvement of chemiosmotic phenomena in active transport. 相似文献
15.
Resolution of phospholipid conformational heterogeneity in model membranes by spin-label EPR and frequency-domain fluorescence spectroscopy.
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We have utilized both fluorescent and nitroxide derivatives of stearic acid as probes of membrane structural heterogeneity in phospholipid vesicles under physiological conditions, as well as conditions of varying ionic strengths and temperatures where spectral heterogeneity has been previously observed and attributed to multiple ionization states of the probes. To identify the source of this spectral heterogeneity, we have utilized complimentary measurements of the relaxation properties (lifetimes) and motion of both (a) spin labeled and anthroyloxy derivatives of stearic acid (i.e., SASL and AS) and (b) a diphenylhexatriene derivative of phosphatidylcholine (DPH-PC) in single component membranes containing dimyristoylphosphatidylcholine (DMPC). We use an 15N stearic-acid spin label for optimal sensitivity to membrane heterogeneity. The lifetime and dynamics of the fluorescent phospholipid analogue DPH-PC (with no ionizable groups over this pH range) were compared with those of AS, allowing us to discriminate between changes in membrane structure and the ionization of the label. The quantum yield and rotational dynamics of DPH-PC are independent of pH, indicating that changes in pH do not affect the conformation of the host phospholipids. However, both EPR spectra of SASL and the lifetime or dynamics of AS are affected profoundly by changes in solution pH. The apparent pKa's of these two probes in DMPC membranes were determined to be near pH 6.3, implying that at physiological pH and ionic strength these stearic-acid labels exist predominantly as a single ionized population in membranes. Therefore, the observed temperature- and ionic-strength-dependent alterations in the spectra of SASL as well as the lifetime or dynamics of AS in DMPC membranes at neutral pH are due to changes in membrane structure rather than the ionization of the probes. The possibility that ionic gradients across biological membranes induce alterations in phospholipid structures, thereby modulating lipid-protein interactions is discussed. 相似文献
16.
Techniques for determining large transbilayer pH gradients (delta pH) and membrane potentials (delta psi) induced in response to delta pH in large unilamellar vesicle liposomal systems by measuring the transbilayer redistribution of radiolabeled compounds have been examined. For liposomes with acidic interiors, it is shown that protocols using radiolabeled methylamine in conjunction with gel filtration procedures to remove untrapped methylamine provide accurate measures of delta pH in most situations. Exceptions include gel state lipid systems, where transbilayer equilibration processes are slow, and situations where the interior buffering capacity is limited. These problems can be circumvented by incubation at elevated temperatures and by using probes with higher specific activities, respectively. Determination of delta pH in vesicles with a basic interior using weak acid probes such as radiolabeled acetate in conjunction with gel filtration was found to be less reliable, and an alternative equilibrium centrifugation protocol is described. In the case of determinations of the membrane potentials induced in response to these pH gradients, probes such as tetraphenylphosphonium and thiocyanate provide relatively accurate measures of the delta psi induced. It is shown that the maximum transmembrane pH gradient that can be stably maintained by an egg phosphatidylcholine-cholesterol 100-nm-diam large unilamellar vesicle is approximately 3.7 units, corresponding to an induced delta psi of 220 mV or transbilayer electrical field of 5 x 10(5) V/cm. 相似文献
17.
Kalyan C. Kondapalli Laura M. Kallay Melanie Muszelik Rajini Rao 《The Journal of biological chemistry》2012,287(43):36239-36250
Human NHA2, a newly discovered cation proton antiporter, is implicated in essential hypertension by gene linkage analysis. We show that NHA2 mediates phloretin-sensitive Na+-Li+ counter-transport (SLC) activity, an established marker for hypertension. In contrast to bacteria and fungi where H+ gradients drive uptake of metabolites, secondary transport at the plasma membrane of mammalian cells is coupled to the Na+ electrochemical gradient. Our findings challenge this paradigm by showing coupling of NHA2 and V-type H+-ATPase at the plasma membrane of kidney-derived MDCK cells, resulting in a virtual Na+ efflux pump. Thus, NHA2 functionally recapitulates an ancient shared evolutionary origin with bacterial NhaA. Although plasma membrane H+ gradients have been observed in some specialized mammalian cells, the ubiquitous tissue distribution of NHA2 suggests that H+-coupled transport is more widespread. The coexistence of Na+ and H+-driven chemiosmotic circuits has implications for salt and pH regulation in the kidney. 相似文献
18.
H S Van Walraven H J Marvin E Koppenaal R Kraayenhof 《European journal of biochemistry》1984,144(3):555-561
ATP hydrolysis-induced proton translocation and electric potential generation have been studied in ATPase proteoliposomes by means of various optical probes. The proteoliposomes consisted of reconstituted ATPase complex and native lipid mixture isolated from the thermophilic cyanobacterium Synechococcus 6716 [Van Walraven et al. (1983) Eur. J. Biochem. 137, 101-106]. The native cartenoids and added oxonol VI served as probes for the electric membrane potential generated by the net charge separation (negative outside, positive inside). Their responses, with similar half-times as 9-tetradecylamino-6-chloro-2-methoxyacridine, are sensitive to valinomycin and stimulated by nigericin, as expected. The proton concentrations of extraliposomal and intraliposomal aqueous spaces were monitored by neutral red and cresol red; for internal measurements these pH indicators were trapped inside the vesicles during detergent dialysis. Internal acidification and external alkalinization induced by ATP hydrolysis are inhibited by nigericin and enhanced by valinomycin; at the commonly used higher valinomycin concentrations the neutral red response becomes transient, while the much slower cresol red response is diminished right from its onset. At smaller preset pH gradients both ATP hydrolysis activity and neutral red response are diminished. At increasing MgCl2 concentrations the neutral red responses are slowed down and the cresol red responses are slightly enhanced; this is observed for both internal and external dye responses. Neutral red permeation through the membrane is insignificant under our experimental conditions but is enhanced at temperatures below the lipid-phase transition. In the case of externally added neutral red the non-permeant buffer Hepes is only effective at high MgCl2 concentration, whereas some external cresol red response is visible only at high MgCl2 concentration in the presence of Hepes. The kinetics of the pH indicator and electric potential probe responses clearly distinguish fast interfacial and intra-membrane proton displacements from slow bulk proton equilibration. The data are summarized in a model that supports the importance of localized proton displacements for the primary energy-transducing events. 相似文献
19.
The rate of halobacterial photophosphorylation was found to be a linear function of light intensity over a wide range (between 1 and 20 mW/cm2). At higher light intensities (above 25 mW/cm2) the ATP-synthesizing system itself limits the maximal rate of photophosphorylation. The optimal external pH range for this type of photophosphorylation is between pH 6.2 and 7.2 external. The photophosphorylation rate is directly proportional to the bacteriorhodopsin content of the cells. The quantum requirement for photophosphorylation was found to be 22 +/- 5 photons per ATP molecule synthesized. According to Mitchell's chemiosmotic hypothesis of energy coupling phosphorylation can be driven by a membrane potential or a pH gradient or a combination of both. From the results of experiments with drugs which abolish or reduce either one of the two components we conclude that the major driving force for photophosphorylation above an external pH value of 6.5 is the membrane potential, while at more acidic pH value the pH gradient becomes dominating. We did not observe a correlation between a transient alkalinization of the medium and ATP-synthesis upon illumination under certain conditions. 相似文献
20.
The culture of Chinese hamster ovary (CHO) cells to produce monoclonal antibodies (MAb) requires accurate measurement and control of pH. Unwanted pH drifts in cell culture can adversely affect process performance, product quality, and product yield. To measure and control pH throughout the length of a culture, most cell culture processes use traditional glass pH probes. Several variables can affect the design and performance of glass pH electrodes and lead to drift in the measurement. Understanding these variables and their effects on pH performance can lead to design improvements and potentially reduce the drift. In this study, a set of Rosemount Analytical glass pH probes was investigated in cell culture operations. Electrochemical properties of the probes were monitored throughout the experiments. Experimental results show that the glass membrane potential experiences the biggest change during cell culture operations. Changes in the reference electrode potential are small compared with the changes in glass membrane potential. The glass membranes are affected by the steam sterilization process and this is the main cause for drift in the probe sensing signal during cell culture operations. Steam sterilization can cause the potential of glass membranes to change by up to 15 mV (~ 0.25 pH units). This change in membrane potential can be observed as an undesirable pH drift in bioreactors. 相似文献