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1.
It was shown that the proton conductivity of Escherichia coli membranes depends on pH and other conditions of bacterial growth. It is considerably lower in cells fermenting glucose and accomplishing the nitrate-nitrite respiration compared with cells accomplishing the oxygen respiration. Proton conductivity increases substantially with decreasing pH of medium. It was found that proton conductivity is related to the redox and membrane potentials of cells. The energy-dependent flux of protons from cells and the ATPase activity of membrane vesicles considerably vary depending on whether bacteria are grown under aerobic or anaerobic conditions. The H+ flux from cells fermenting glucose (pH 7.5) was 1.7 times greater than the H+ flux from cells that accomplish the nitrate-nitrite and oxygen respiration. The N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity increased 2.5 times as K+ concentration increased to 100 mM (including residual K+ in potassium-free medium). The DCCD-sensitive ATPase activity considerably decreased with decreasing pH of medium, whereas the ATPase activity that was not suppressed by DCCD was stimulated. These results can be used for establishing the relationship between membrane proton conductivity and the energy-dependent H+ flux and ATPase activity.  相似文献   

2.
The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient. An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents. The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline). The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate. At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value. The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.  相似文献   

4.
A study is presented of the action of triphenyltin on the kinetics of the anaerobic relaxation of the proton gradient set up by respiration in various type of 'inside-out' inner membrane vesicles obtained by exposure of beef-heart mitochondria to ultrasonic energy. Triphenyltin is shown to act as a powerful inhibitor of the proton conductivity of the H+-ATPase. The inhibition persists after removal of the ATPase protein inhibitor, F1 and the oligomycin-sensitivity conferral protein (OSCP) from the particles. The inhibitory effect of triphenyltin is exerted, as in the case of oligomycin and N,N'-dicyclohexylcarbodiimide, on the F0 moiety of the ATPase complex. Comparison of the characteristics of the effect of triphenyltin on proton translocation in chloride and nitrate media shows that the inhibition of passive proton conductivity studied here is unrelated to the hydroxide/anion exchange induced by the organotin. Lack of additivity of the inhibition of H+ conduction by triphenyltin with that exerted by oligomycin and N,N'-dicyclohexylcarbodiimide and the kinetic pattern of the effect of triphenyltin show that the mechanism of action of the organotin is different from that of the other two inhibitors. The relevance of the results obtained with respect to the subunit location and chemical nature of the reaction site of triphenyltin in the H+-ATPase complex is discussed.  相似文献   

5.
Escherichia coli accumulates K+ by means of multiple uptake systems of which Kup is the major transport system at acidic pH. In cells grown under fermentative conditions at pH 5.5, K+ influx by a wild-type strain upon hyper-osmotic stress at pH 5.5 was accompanied by a marked decrease in H+ efflux, with a 1:1 ratio of K+ to H+ fluxes. This was observed with cells treated with N,N'-dicyclohexylcarbodiimide. Similar results with a mutant defective in Kdp and TrkA but with a functional Kup system but not in a mutant defective in Kdp and Kup but having an active TrkA system suggest that Kup operates as a H+ -K+ -symporter.  相似文献   

6.
Exposure to electromagnetic irradiation (EMI) of 51.8 and 53.0?GHz and low intensity (flux capacity of 0.06?mW?cm(-2) ) for 1?h markedly decreased the energy-dependent H(+) and K(+) transport across membranes of Enterococcus hirae ATCC 9790. After EMI, there was also a significant decrease of overall and N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity of the membrane vesicles. These measures were considerably lower at 53.0 GHz. EMI in combination with different antibiotics, such as ceftriaxone and kanamycin at their minimal inhibitory concentrations (100 and 200?μM, respectively), enhanced bacterial cell growth and altered their membrane transport properties. Total H(+) efflux was most sensitive to ceftriaxone but DCCD-inhibited H(+) efflux and total K(+) influx were sensitive to kanamycin. The results indicate that cell membrane proteins could be a target in the action of EMI and enhanced antibacterial effects in combination with antibiotics. The DCCD-sensitive F(0) F(1) -ATPase or this ATPase in combination with K(+) uptake protein probably plays a key role in these effects.  相似文献   

7.
Trifluoperazine (TFP), the antipsychotic drug, induces substantial K+ efflux, membrane hyperpolarization and inhibition of H+-ATPase in the yeast Saccharomyces cerevisiae. Investigations on the mechanism of these effects revealed two different processes observed at different incubation conditions. At an acidic pH of 4.5 and an alkaline pH of 7.5, K+ efflux was accompanied by substantial proton influx which led to intracellular acidification and dissipation of delta psi formed by cation efflux. The results indicated nonspecific changes in membrane permeability. Similar results were also observed when cells were incubated at pH 5.5-6.0 with higher concentrations of TFP (above 75 microM). On the other hand, low concentrations of TFP (30-50 microM) at pH 5.5-6.0 caused marked membrane hyperpolarization and K+ efflux unaccompanied by the efflux of other cations and by H+ influx. Our experiments indicate that under these conditions K+ efflux was an active process. (1) K+ efflux proceeded only in the presence of a metabolic substrate and was inhibited by metabolic inhibitors. (2) When 0.3-0.9 mM-KCl was present in the medium at pH 6.0, the concentration of K+ within the cells (measured at the end of the incubation with TFP) was much lower than the theoretical concentration of Kin+ if the distribution of K+ between medium and cell water was at equilibrium (at zero electrochemical gradient). (3) Valinomycin decreased the net K+ efflux and decreased the membrane hyperpolarization induced by TFP, probably by increasing the flux of K+ into the cells along its electrochemical gradient. (4) Conditions which led to active K+ efflux also led to a marked decrease in cellular ATP level. The results indicate that under a specific set of conditions TFP induces translocation of K+ against its electrochemical gradient.  相似文献   

8.
Proton motive force and Na+/H+ antiport in a moderate halophile.   总被引:4,自引:3,他引:1       下载免费PDF全文
The influence of pH on the proton motive force of Vibrio costicola was determined by measuring the distributions of triphenylmethylphosphonium cation (membrane potential, delta psi) and either dimethyloxazolidinedione or methylamine (osmotic component, delta pH). As the pH of the medium was adjusted from 5.7 to 9.0, the proton motive force steadily decreased from about 170 to 100 mV. This decline occurred, despite a large increase in the membrane potential to its maximum value at pH 9.0, because of the loss of the pH gradient (inside alkaline). The cytoplasm and medium were of equal pH at 7.5; membrane permeability properties were lost at the pH extremes of 5.0 and 9.5. Protonophores and monensin prevented the net efflux of protons normally found when an oxygen pulse was given to an anaerobic cell suspension. A Na+/H+ antiport activity was measured for both Na+ influx and efflux and was shown to be dissipated by protonophores and monensin. These results strongly favor the concept that respiratory energy is used for proton efflux and that the resulting proton motive force may be converted to a sodium motive force through Na+/H+ antiport (driven by delta psi). A role for antiport activity in pH regulation of the cytosol can also explain the broad pH range for optimal growth, extending to the alkaline extreme of pH 9.0.  相似文献   

9.
Calcium transport in membrane vesicles of Streptococcus cremoris   总被引:2,自引:0,他引:2  
Rightside-out membrane vesicles of Streptococcus cremoris were fused with proteoliposomes containing the light-driven proton pump bacteriorhodopsin by a low-pH fusion procedure reported earlier [Driessen, A.J.M., Hellingwerf, K.J. & Konings, W.N. (1985) Biochim. Biophys. Acta 808, 1-12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing delta psi with a concomitant increase in delta pH stimulates Ca2+ uptake while dissipation of the delta pH results in a reduced rate of Ca2+ uptake. Also an artificially generated delta pH, interior acid, can drive Ca2+ uptake in S. cremoris membrane vesicles. Ca2+ uptake depends strongly on the presence of external phosphate while Ca2+-efflux-induced proton flux is independent of the presence of external phosphate. Ca2+ accumulation is abolished by the divalent cation ionophore A23187. Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the membrane-bound ATPase by N,N'-dicyclohexylcarbodiimide strongly inhibits Ca2+ release. Further studies on Ca2+ efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+ exit from intact cells is an electrogenic process. It is concluded that Ca2+ efflux in S. cremoris is mediated by a secondary transport system catalyzing exchange of calcium ions and protons.  相似文献   

10.
Proton circulation in Vibrio costicola.   总被引:3,自引:2,他引:1       下载免费PDF全文
The importance of proton movements was assessed in the moderate halophile Vibrio costicola. When anaerobic cells in acidic buffer (pH 6.5) were given an O2 pulse, protons were extruded regardless of the presence of Na+. At pH 8.5, however, V. costicola produced an acidic response to an O2 pulse in the absence of Na+ and an alkaline response when Na+ was present. An Na+/H+ antiport activity was confirmed at pH 8.5. All of these effects were prevented by protonophores or butanol treatment. Growth in complex medium at pH 8.5 was prevented by a high concentration (50 microM) of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or a low concentration (5 microM) of another protonophore, 3,3',4',5-tetrachlorosalicylanilide (TCS). The relative ineffectiveness of the former protonophore was caused by the proteose peptone and tryptone ingredients of the complex medium, since 5 microM completely prevented growth in their absence. The results are explained by a primary respiratory-linked proton efflux coupled to a secondary Na+/H+ antiport operating at alkaline pH. Evidence was seen for a role of Na+ in stimulating proton influx at alkaline pH, presumably via the pH homeostasis mechanism.  相似文献   

11.
Effects of butanol on Clostridium acetobutylicum   总被引:3,自引:0,他引:3  
The internal pH of Clostridium acetobutylicum was determined at various stages during the growth of the organism. Even in the presence of significant quantities of acetic, butyric, and lactic acids, an internal pH of 6.2 was maintained. Experiments using N,N'-dicyclohexylcarbodiimide indicated that a functioning H+-ATPase is necessary for internal pH control. Butanol, one of the end products of the fermentation, had numerous harmful effects on C. acetobutylicum. At a concentration high enough to inhibit growth, butanol destroyed the ability of the cell to maintain internal pH, lowered the intracellular level of ATP, and inhibited glucose uptake. Experiments done at two different external pH values suggested that the butanol-mediated decrease in ATP concentration was independent of the drop in internal pH. Glucose uptake was not affected by arsenate, suggesting that uptake was not ATP dependent. The effects of butanol on C. acetobutylicum are complex, inhibiting several interrelated membrane processes.  相似文献   

12.
Effects of butanol on Clostridium acetobutylicum.   总被引:6,自引:5,他引:1       下载免费PDF全文
The internal pH of Clostridium acetobutylicum was determined at various stages during the growth of the organism. Even in the presence of significant quantities of acetic, butyric, and lactic acids, an internal pH of 6.2 was maintained. Experiments using N,N'-dicyclohexylcarbodiimide indicated that a functioning H+-ATPase is necessary for internal pH control. Butanol, one of the end products of the fermentation, had numerous harmful effects on C. acetobutylicum. At a concentration high enough to inhibit growth, butanol destroyed the ability of the cell to maintain internal pH, lowered the intracellular level of ATP, and inhibited glucose uptake. Experiments done at two different external pH values suggested that the butanol-mediated decrease in ATP concentration was independent of the drop in internal pH. Glucose uptake was not affected by arsenate, suggesting that uptake was not ATP dependent. The effects of butanol on C. acetobutylicum are complex, inhibiting several interrelated membrane processes.  相似文献   

13.
The effects of N,N'-dicyclohexylcarbodiimide (DCCD) and various ionophores on light-induced 22Na+-transport were studied in right-side-out membrane vesicles from Halobacterium halobium R1M1. The light-induced Na+ efflux was inhibited at the same DCCD concentration (greater than 40 nmol/mg protein) as required for inhibition of the Na+-dependent membrane potential (delta phi) formation. This supports our previous indication that the DCCD-sensitive, Na+-dependent transformation of pH-gradient (delta pH) into delta phi is mediated by Na+/H+-antiporter (Murakami, N. and Konishi, T. (1985) J. Biochem. 98, 897-907). FCCP or a combination of valinomycin and triphenyltin (TPT) inhibits the light-induced Na+ efflux in accordance with the notion of protonmotive force (delta mu H+)-driven antiporter. However, a marked lag in initiation of the Na+ efflux occurred in the presence of valinomycin, TPMP+, or a small amount of FCCP, suggesting that a gating step is involved in the Na+ efflux. On the other hand, the delta pH-dissipating ionophore TPT did not cause the lag. A simultaneous determination of delta phi, delta pH, and Na+ efflux rate at the initial stage of illumination revealed that the antiporter is gated by delta phi rather than delta mu H+.  相似文献   

14.
ATPase activity sensitive to N,N'-dicyclohexylcarbodiimide and dependent on K+ content in medium is observed only in anaerobically grown Escherichia coli and as the analysis of mutants with defects in different subunits of (F0F1) H+-ATPase and in potassium transport shows only under the structural integrity of both F0F1 and K+-ionophore (the Trk system). The obtained results confirm the data on the H+/K+-exchange and indicate that the F0F1 and Trk systems in anaerobically grown bacteria unite into the same membrane supercomplex inside which the direct energy transfer occurs without a mediation of delta-mu H+.  相似文献   

15.
The coherent electromagnetic radiation (EMR) of the frequency of 51.8 and 53 GHz with low intensity (the power flux density of 0.06 mW/cm(2)) affected the growth of Escherichia coli K12(lambda) under fermentation conditions: the lowering of the growth specific rate was considerably (approximately 2-fold) increased with exposure duration of 30-60 min; a significant decrease in the number of viable cells was also shown. Moreover, the enforced effects of the N,N'-dicyclohexylcarbodiimide (DCCD), inhibitor of H(+)-transporting F(0)F(1)-ATPase, on energy-dependent H(+) efflux by whole cells and of antibiotics like tetracycline and chloramphenicol on the following bacterial growth and survival were also determined after radiation. In addition, the lowering in DCCD-inhibited ATPase activity of membrane vesicles from exposed cells was defined. The results confirmed the input of membranous changes in bacterial action of low intensity extremely high frequency EMR, when the F(0)F(1)-ATPase is probably playing a key role. The radiation of bacteria might lead to changed metabolic pathways and to antibiotic resistance. It may also give bacteria with a specific role in biosphere.  相似文献   

16.
Carboxyfluorescein diacetate is a nonfluorescent compound which can be used in combination with flow cytometry for vital staining of yeasts and bacteria. The basis of this method is the assumption that, once inside the cell, carboxyfluorescein diacetate is hydrolyzed by nonspecific esterases to produce the fluorescent carboxyfluorescein (cF). cF is retained by cells with intact membranes (viable cells) and lost by cells with damaged membranes. In this report, we show that Saccharomyces cerevisiae extrudes cF in an energy-dependent manner. This efflux was studied in detail, and several indications that a transport system is involved were found. Efflux of cF was stimulated by the addition of glucose and displayed Michaelis-Menten kinetics. A Km for cF transport of 0.25 mM could be determined. The transport of cF was inhibited by the plasma membrane H(+)-ATPase inhibitors N,N'-dicyclohexylcarbodiimide and diethylstilbestrol and by high concentrations of tetraphenylphosphonium ions. These treatments resulted in a dissipation of the proton motive force, whereas the intracellular ATP concentration remained high. Transport of cF is therefore most probably driven by the membrane potential and/or the pH gradient. The viability of S. cerevisiae was determined by a two-step procedure consisting of loading the cells with cF followed by incubation at 40 degrees C in the presence of glucose. Subsequently, the fluorescence intensity of the cells was analyzed by flow cytometry. The efflux experiments showed an excellent correlation between the viability of S. cerevisiae cells and the ability to translocate cF. This method should prove of general utility for the rapid assessment of yeast vitality and viability.  相似文献   

17.
The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in delta pH-driven Na+ efflux experiments. When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.  相似文献   

18.
Carbonylcyanide m-chlorophenylhydrazone (CCCP) or nigericin induced translocation of H+ In the dark across the cell membrane of blue-green algae Plectonema boryanum and Anacystis nidulans. The direction of the H+ flux depended on the pH of the suspending medium. At acidic pH, an influx of H+ and at alkaline pH an efflux of H+ were observed. It is suggested that the influx takes place at pH'S higher than the "internal" pH and the efflux at pH's lower than that. The internal pH was estimated to be 7.4+/-0.2 for Plectonema boryanum and 7.5+/-0.1 for Anacystis nidulans. Similar H+ changes due to CCP were observed under illumination, where the light induced efflux of H+ was limited by the counter-flux of cations. The internal pH of cells in the light, estimated from the pH-dependent reversion in the rate of the H+ change, was about 8.5.  相似文献   

19.
Roles of the respiratory Na+ pump in bioenergetics of Vibrio alginolyticus   总被引:3,自引:0,他引:3  
Bioenergetic characteristics of Na+ pump-defective mutants of a marine bacterium Vibrio alginolyticus were compared with those of the wild type and revertant. Generation of membrane potential and motility at pH 8.5 in the mutants were completely inhibited by a proton conductor, carbonylcyanide m-chlorophenylhydrazone, whereas those in the wild type or revertant were resistant to the inhibitor. Motility and amino acid transport were driven by the electrochemical potential of Na+ not only in the wild type or revertant but also in the mutants. In the absence of the proton conductor, motility and amino acid transport of the mutants did not significantly differ from those of the wild type or revertant even at pH 8.5, where the Na+ pump has maximum activity. Therefore, the electrochemical potential of Na+ in the mutants seemed to be maintained at a normal level by a respiration-dependent H+ pump and Na+/H+ antiporter. On the other hand, growth of the mutants became defective as the medium pH increased, especially on minimal medium. These results indicate that the Na+ pump is an important energy-generating mechanism when nutrients are limited at alkaline pH.  相似文献   

20.
Ca2+ accumulation in energized rat liver mitochondria has been studied after the blockage of mitochondrial permeability transition pore (MPTP) by cyclosporin A. It is shown that Ca2+ transport is coupled to the countertransport of protons: from the matrix of mitochondria in the medium in the course of Ca2+ accumulation, and, on the contrary, from the medium to mitochondrial matrix after membrane depolarization. In standard incubation medium containing K+, Cl-, oxidation substrate (glutamate) and inorganic phosphate (H2PO4(-)) the observed stoichiometry of the exchange is 1Ca2+ : 1H+. In accordance with this exchange ratio, proton, as well as cation, transport follows the same first-order kinetics, which is characterized in both cases by very close values of reaction half-times and rate constants. It is shown that reversion of Ca2+ -uniporter, sensitive to ruthenium red, is necessary for Ca2+ - efflux from the matrix ofdeenergized mitochondria when MPTP is blocked by cyclosporin A. It is also shown that Ca2+ -uniporter reversion takes place only after membrane depolarization and permeabilization by protonophore CCCP. Calcium release from mitochondria in the presence of CCCP is accompanied by proton flow into the matrix. Both calcium and proton fluxes are sensitive to Ca2+ uniporter blocker, ruthenium red, which gives the evidence of the identity of Ca2+ -efflux and influx pathways. The data obtained lead to the conclusion that calcium-proton exchange is necessary for Ca2+ -uniporter reversion and the reversibility of energy-dependent Ca2+ -uptake in mitochondria.  相似文献   

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