Role of Na+ cycle in cell volume regulation of Mycoplasma gallisepticum.

The mechanism for the extrusion of Na+ from Mycoplasma gallisepticum cells was examined. Na+ efflux from cells was studied by diluting 22Na+-loaded cells into an isoosmotic NaCl solution and measuring the residual 22Na+ in the cells. Uphill 22Na+ efflux was found to be glucose dependent and linear with time over a 60-s period and showed almost the same rate in the pH range of 6.5 to 8.0. 22Na+ efflux was markedly inhibited by dicyclohexylcarbodiimide (DCCD, 10 microM), but not by the proton-conducting ionophores SF6847 (0.5 microM) or carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 microM) over the entire pH range tested. An ammonium diffusion potential and a pH gradient were created by diluting intact cells or sealed membrane vesicles of M. gallisepticum loaded with NH4Cl into a choline chloride solution. The imposed H+ gradient (inside acid) was not affected by the addition of either NaCl or KCl to the medium. Dissipation of the proton motive force by CCCP had no effect on the growth of M. gallisepticum in the pH range of 7.2 to 7.8 in an Na+-rich medium. Additionally, energized M. gallisepticum cells were stable in an isoosmotic NaCl solution, even in the presence of proton conductors, whereas nonenergized cells tended to swell and lyse. These results show that in M. gallisepticum Na+ movement was neither driven nor inhibited by the collapse of the electrochemical gradient of H+, suggesting that in this organism Na+ is extruded by an electrogenic primary Na+ pump rather than by an Na+-H+ exchange system energized by the proton motive force.     

Flagellar motors of marine bacteria Halomonas are driven by both protons and sodium ions

Bacterial cells in aquatic environments are able to reach or stay near nutrient patches by using motility. Motility is usually attained by rotating flagellar motors that are energized by electrochemical potential of H+ or Na+. In this paper, the ion specificity for flagellar rotation of two marine isolates Halomonas spp. strains US172 and US201 was investigated. Both isolates require sodium for growth and possess a respiratory-driven primary sodium pump. They are motile because of lateral flagella regardless of the presence of sodium ions. Their swimming speed under various concentrations of sodium ions with and without carbonylcyanide m-chlorophenylhydrazone, a proton conductor, and with and without phenamil, a specific inhibitor for the sodium-driven flagellar motors, was examined. The effect of carbonylcyanide m-chlorophenylhydrazone on the transmembrane proton gradient was also determined. Our results showed that the flagellar motors of the Halomonas strains were energized by both H+ and Na+ in one cell. The bimodal nature of Halomonas spp. motility with respect to the driving energy source may reflect ecophysiological versatility to adapt to a wide range of salt conditions of the marine environment.     

Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters

Uptake of 22Na+ and 45Ca2+ into everted membrane vesicles from Escherichia coli was measured with imposed transmembrane pH gradients, acid interior, as driving force. Vesicles loaded with 0.5 M KCl were diluted into 0.5 M choline chloride to create a potassium gradient. Addition of nigericin to produce K+/H+ exchange resulted in formation of a pH gradient. This imposed gradient was capable of driving 45Ca2+ accumulation. In another method vesicles loaded with 0.5 M NH4Cl were diluted into 0.5 M choline chloride, creating an ammonium diffusion potential. A gradient of H+ was produced by passive efflux of NH3. With an ammonium gradient as driving force, everted vesicles accumulated both 45Ca2+ and 22Na+. The data suggest that 22Na+ uptake was via the sodium/proton antiporter and 45Ca2+ via the calcium/proton antiporter. Uptake of both cations required alkaline pHout. A minimum pH gradient of 0.9 unit was needed for transport of either ion, suggesting gating of the antiporters. Octyl glucoside extracts of inner membrane were reconstituted with E. coli phospholipids in 0.5 M NH4Cl. NH4+-loaded proteoliposomes accumulated both 22Na+ and 45Ca2+, demonstrating that the sodium/proton and calcium/proton antiporters could be solubilized and reconstituted in a functional form.     

Cation coupling to melibiose transport in Salmonella typhimurium.

Melibiose transport in Salmonella typhimurium was investigated. Radioactive melibiose was prepared and the melibiose transport system was characterized. Na+ and Li+ stimulated transport of melibiose by lowering the Km value without affecting the Vmax value; Km values were 0.50 mM in the absence of Na+ or Li+ and 0.12 mM in the presence of 10 mM NaCl or 10 mM LiCl. The Vmax value was 140 nmol/min per mg of protein. Melibiose was a much more effective substrate than methyl-beta-thiogalactoside. An Na+-melibiose cotransport mechanism was suggested by three types of experiments. First, the influx of Na+ induced by melibiose influx was observed with melibiose-induced cells. Second, the efflux of H+ induced by melibiose influx was observed only in the presence of Na+ or Li+, demonstrating the absence of H+-melibiose cotransport. Third, either an artificially imposed Na+ gradient or membrane potential could drive melibiose uptake in cells. Formation of an Na+ gradient in S. typhimurium was shown to be coupled to H+ by three methods. First, uncoupler-sensitive extrusion of Na+ was energized by respiration or glycolysis. Second, efflux of H+ induced by Na+ influx was detected. Third, a change in the pH gradient was elicited by imposing an Na+ gradient in energized membrane vesicles. Thus, it is concluded that the mechanism for Na+ extrusion is an Na+/H+ antiport. The Na+/H+ antiporter is a transformer which converts an electrochemical H+ gradient to an Na+ gradient, which then drives melibiose transport. Li+ was inhibitory for the growth of cells when melibiose was the sole carbon source, even though Li+ stimulated melibiose transport. This suggests that high intracellular Li+ may be harmful.     

Measurement of intracellular sodium concentration and sodium transport in Escherichia coli by 23Na nuclear magnetic resonance

Escherichia coli is known to actively extrude sodium ions, but little is known concerning the concentration gradient it can develop. We report here simultaneous measurements, by 23Na NMR, of intracellular and extracellular Na+ concentrations of E. coli cells before and after energization. 23Na spectra in the presence of a paramagnetic shift reagent (dysprosium tripolyphosphate) consisted of two resonances, an unshifted one corresponding to intracellular Na+ and a shifted one corresponding to Na+ in the extracellular medium, including the periplasm. Extracellular Na+ was found to be completely visible despite the presence of a broad component in its resonance; intracellular Na+ was only 45% visible. Measurements of Na+ were made under aerobic and glycolytic conditions. Na+ extrusion and maintenance of a stable low intracellular Na+ concentration were found to correlate with the development and maintenance of proton motive force, a result that is consistent with proton-driven Na+/H+ exchange as a means of Na+ transport. In both respiring and glycolyzing cells, at an extracellular Na+ concentration of 100 mM, the intracellular Na+ concentration observed (4 mM) corresponded to an inwardly directed Na+ gradient with a concentration ratio of about 25. The kinetics of Na+ transport suggest that rapid extrusion of Na+ against its electrochemical gradient may be regulated by proton motive force or intracellular pH.     

ATP-dependent cadmium transport by the cadA cadmium resistance determinant in everted membrane vesicles of Bacillus subtilis.

Resistance to cadmium conferred by the staphylococcal plasmid pI258 occurs by means of energy-dependent efflux, resulting in decreased intracellular accumulation of cadmium. Recent sequence information suggested that efflux is mediated by a P-type ATPase. The cadA gene was previously expressed in Bacillus subtilis, conferring resistance to cadmium. Everted membrane vesicles were prepared from B. subtilis cells harboring either a plasmid containing the cadA system or the vector plasmid alone. 109Cd2+ transport into the everted membranes was measured in the presence of various energy sources. Cadmium transport was detected only in the presence of ATP as an energy source. The production of an electrochemical proton gradient (delta mu H+) by using NADH or phenazine methosulfate plus ascorbate was not able to drive transport. Reagents which dissipate delta pH abolished calcium transport due to the Ca2+/H+ antiporter but only partially inhibited cadmium transport. Inhibition of transport by the antibiotic bafilomycin A1 occurred at concentrations comparable to those which inhibit P-type ATPases. A band corresponding to the cadA gene product was identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antibodies to the protein were prepared.     

Bioenergetic properties of alkalophilic Bacillus sp. strain C-59 on an alkaline medium containing K2CO3.

Alkalophilic Bacillus sp. strain C-59 could grow well on an alkaline medium containing K2CO3, as well as Na2CO3, but did not grow on K+-depleted medium. Right-side-out membrane vesicles, energized in the absence of Na+, however, could not take up [14C]methylamine actively, while vesicles equilibrated with 10 mM NaCl actively took up [14C]methylamine. The uptake of [14C]serine was also stimulated by the addition of Na+, and the imposition of a sodium gradient caused transient uptake. These results indicated that an Na+/H+ antiporter was involved in pH homeostasis and generation of an electrochemical sodium gradient in strain C-59 even though a growth requirement for Na+ was not evident. The efflux of 22Na+ from 22Na+-loaded vesicles was more rapid at pH 9.5 than at pH 7 in the presence of an electron donor. On the other hand, vesicles at pH 7 showed more rapid efflux than at pH 9.5 when the antiporter was energized by a valinomycin-mediated K+ diffusion potential (inside negative).     

Reconstitution of a bacterial Na+/H+ antiporter

Membrane proteins from alkalophilic Bacillus firmus RAB were extracted with octylglucoside, reconstituted into liposomes made from alkalophile lipids. The proteoliposomes were loaded with 22Na+. Imposition of a valinomycin-mediated potassium diffusion potential, positive out, resulted in very rapid efflux of radioactive Na+ against its electrochemical gradient. That the Na+ efflux was mediated by the electrogenic Na+/H+ antiporter is indicated by the following characteristics that had been established for the porter in previous studies: dependence upon an electrical potential; pH sensitivity, with activity dependent upon an alkaline pH; inhibition by Li+; and an apparent concentration dependence upon Na+ that correlated well with measurements in cells and membrane vesicles.     

Relationships between the Na+-H+ antiport activity and the components of the electrochemical proton gradient in Escherichia coli membrane vesicles

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)     

Sodium-calcium exchange and calcium-calcium exchange in internally dialyzed squid giant axons.

The influx and efflux of calcium (as 45Ca) and influx of sodium (as 24Na) were studied in internally dialyzed squid giant axons. The axons were poisoned with cyanide and ATP was omitted from the dialysis fluid. The internal ionized Ca2+ concentration ([Ca2+]i) was controlled with Ca-EGTA buffers. With [Ca2+]i greater than 0.5 muM, 45Ca efflux was largely dependent upon external Na and Ca. The Nao-dependent Ca efflux into Ca-free media appeared to saturate as [Ca2+]i was increased to 160 muM; the half-saturation concentration was about 8 muM Ca2+. In two experiments 24Na influx was measured; when [Ca2+]i was decreased from 160 muM to less than 0.5 muM, Na influx declined by about 5 pmoles/cm2 sec. The Nao-dependent Ca efflux averaged 1.6 pmoles/cm2 sec in axons with a [Ca2+]i of 160 muM, and was negligible in axons with a [Ca2+]i of less than 0.5 muM. Taken together, the Na influx and Ca efflux data may indicate that the fluxes are coupled with a stoichiometry of about 3 Na+-to-1 Ca2+. Ca efflux into Na-free media required the presence of both Ca and an alkali metal ion (but not Cs) in the external medium. Ca influx from Li-containing media was greatly reduced when [Ca2+]i was decreased from 160 to 0.23 muM, or when external Li was replaced by choline. These data provide evidence for a Ca-Ca exchange mechanism which is activated by certain alkali metal ions. The observations are consistent with a mobile carrier mechanism which can exchange Ca2+ ions from the axoplasm for either 3 Na+ ions, or one Ca2+ and an alkali metal ion (but not Cs) from the external medium. This mechanism may utilize energy from the Na electrochemical gradient to help extrude Ca against an electrochemical gradient.     

Na+-dependent methyl beta-thiogalactoside transport in Salmonella typhimurium.

We have studied the role of sodium ions in methyl beta-thiogalactoside (TMG) transport via the melibiose permease (TMG II) in Salmonella typhimurium. TMG uptake via TMG II in anaerobic, straved and metabolically poisoned cells is dependent on an inward-directed Na+ gradient. Cells which have been partially depleted of endogenous substrates show H+ extrusion upon sodium-stimulated TMG influx. Measurements of the electrochemical H+ gradient in cells, starved in different ways for endogenous substrates, suggest that this proton extrusion is probably not linked to the actual translocation mechanism but is the result of metabolism induced by TMG plug Na+ uptake.     

Energy transduction in the methanogen Methanococcus voltae is based on a sodium current.

We provide experimental support for the proposal that ATP production in Methanococcus voltae, a methanogenic member of the archaea, is based on an energetic system in which sodium ions, not protons, are the coupling ions. We show that when grown at a pH of 6.0, 7.1, or 8.2, M. voltae cells maintain a membrane potential of approximately -150 mV. The cells maintain a transmembrane pH gradient (pH(in) - pH(out)) of -0.1, -0.2, and -0.2, respectively, values not favorable to the inward movement of protons. The cells maintain a transmembrane sodium concentration gradient (sodium(out)/sodium(in)) of 1.2, 3.4, and 11.6, respectively. While the protonophore 3,3',4',5-tetrachlorosalicylanilide inhibits ATP formation in cells grown at pH 6.5, neither ATP formation nor growth is inhibited in cells grown in medium at pH 8.2. We show that when grown at pH 8.2, cells synthesize ATP in the absence of a favorably oriented proton motive force. Whether grown at pH 6.5 or pH 8.2, M. voltae extrudes Na+ via a primary pump whose activity does not depend on a proton motive force. The addition of protons to the cells leads to a harmaline-sensitive efflux of Na+ and vice versa, indicating the presence of Na+/H+ antiporter activity and, thus, a second mechanism for the translocation of Na+ across the cell membrane. M. voltae contains a membrane component that is immunologically related to the H(+)-translocating ATP synthase of the archaeabacterium Sulfolobus acidocaldarius. Since we demonstrated that ATP production can be driven by an artificially imposed membrane potential only in the presence of sodium ions, we propose that ATP production in M. voltae is mediated by an Na+-translocating ATP synthase whose function is coupled to a sodium motive force that is generated through a primary Na+ pump.     

Active extrusion of potassium in the yeast Saccharomyces cerevisiae induced by low concentrations of trifluoperazine

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.     

Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp.

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.     

Extrusion of sodium ions energized by respiration and glycolysis in Escherichia coli.

Extrusion of sodium ion from cells of Escherichia coli was measured using an Na+ electrode. When oxygen was supplied to an anaerobic cell suspension, extrusion of Na+ was observed. The addition of glucose under anaerobic conditions also caused Na+ efflux. The extrusion of Na+ energized by respiration and glycolysis was completely inhibited by a proton conductor, carbonyl cyanide m-chlorophenylhydrazone. These observations are consistent with the view that Na+ transport occurs secondarily to H+ circulation. Interestgly, induction of the melibiose transport system, which is coupled to Na+, greatly enhanced Na+ transport activity.     

Proton motive force and Na+/H+ antiport in a moderate halophile.

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.     

Energy recycling by lactate efflux in growing and nongrowing cells of Streptococcus cremoris.

Streptococcus cremoris was grown in pH-regulated batch and continuous cultures with lactose as the energy source. During growth the magnitude and composition of the electrochemical proton gradient and the lactate concentration gradient were determined. The upper limit of the number of protons translocated with a lactate molecule during lactate excretion (the proton-lactate stoichiometry) was calculated from the magnitudes of the membrane potential, the transmembrane pH difference, and the lactate concentration gradient. In cells growing in continuous culture, a low lactate concentration gradient (an internal lactate concentration of 35 to 45 mM at an external lactate concentration of 25 mM) existed. The cell yield (Ymax lactose) increased with increasing growth pH. In batch culture at pH 6.34, a considerable lactate gradient (more than 60 mV) was present during the early stages of growth. As growth continued, the electrochemical proton gradient did not change significantly (from -100 to -110 mV), but the lactate gradient decreased gradually. The H+-lactate stoichiometry of the excretion process decreased from 1.5 to about 0.9. In nongrowing cells, the magnitude and composition of the electrochemical proton gradient was dependent on the external pH but not on the external lactate concentration (up to 50 mM). The magnitude of the lactate gradient was independent of the external pH but decreased greatly with increasing external lactate concentrations. At very low lactate concentrations, a lactate gradient of 100 mV existed, which decreased to about 40 mV at 50 mM external lactate. As a consequence, the proton-lactate stoichiometry decreased with increasing external concentrations of protons and lactate at pH 7.0 from 1 mM lactate to 1.1 at 50 mM lactate and at pH 5.5 from 1.4 at l mM lactate to 0.7 at 50 mM lactate. The data presented in this paper suggest that a decrease in external pH and an increase in external lactate concentration both result in lower proton-lactate stoichiometry values and therefore in a decrease of the generation of metabolic energy by the end product efflux process.     

Effects of perturbation of the Na+ electrochemical gradient on influx and efflux of alanine in isolated rat hepatocytes

Transmembrane alanine transport was studied in hepatocytes isolated from 48-h fasted rats. Aminooxyacetate was used to render alanine nonmetabolizable. Gramicidin D eliminated the transmembrane Na+ electrochemical gradient. At 135 mM Na+ and 0.1 mM alanine gramicidin D decreased the steady-state intracellular-to-extracellular alanine distribution ratio from 20.2 to 0.9. The underlying kinetic changes appeared to be a decrease in alanine influx to one-third of the control value and an increase in the rate constant of alanine efflux by a factor of 9. Analogous changes were observed when the Na+ gradient was decreased by ouabain. The inhibitory effect of gramicidin D on alanine influx was confined to the Na+-dependent, saturable component which showed a prominent increase in the apparent Km for alanine and a small decrease in the apparent Vmax. The effect of gramicidin D on alanine efflux was related to the increased cytosolic Na+ concentration: the rate constant of alanine efflux was increased by cytosolic Na+ with half-maximal stimulation at 30 mM; voltage-sensitive alanine efflux could not be demonstrated.     

Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H+ and Na+ from respiring cells

The ejection of protons from oxygen-pulsed cells and the gradients of Na+ concentration (Na+o/Na+i at 150 mM external NaCl) and proton electrochemical potential (delta mu H+) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F0F1-ATPase inhibitors dicyclohexylcarbodiimide (F0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 +/- 0.15 to 0.7 +/- 0.1 nmol/mg dry wt while overall O2 uptake and delta mu H+ were much less affected. H+ efflux was inhibited only 60 to 75%. Aerobic Na+o/Na+i ratios (5.9 +/- 0.6) under these conditions remained 50% above the anaerobic level (2.1 +/- 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H+ efflux and O2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H+/O ratios from 4.0 +/- 0.3 to 1.9 +/- 0.2. Similarly, with KCN and CO the Na+o/Na+i ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 mM KCN. Measurements of the proton motive force (delta mu H+) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na+/H+ antiporter for the active exclusion of Na+ from the cell interior.     

Energization of amino acid uptake by system A in cultured human fibroblasts

The energization of System A in cultured human fibroblasts has been studied by measuring the energy transfer from the electrochemical gradient of Na+ to the chemical gradient of the site A-specific substrate amino acid 2-methylaminoisobutyric acid. The co-transport Na+/amino acid, studied by kinetic analysis and radiochemical measurements, showed a coupling ratio of 1:1. The assessment of the Na+ electrochemical gradient in cultured adherent cells relied on the development of noninvasive procedures as follows: the membrane electrical potential was estimated from the accumulation of L-arginine at equilibrium (Bussolati, O., Laris, P. C., Nucci, F. A., Dall'Asta, V., Longo, N., Guidotti, G. G., and Gazzola, G. C. (1987) Am. J. Physiol. 253, C391-C397); the chemical gradient of Na+ was determined from spectrometric measurements of Na+. The accumulation of 2-methylaminoisobutyric acid was strongly sensitive to changes of Na+ gradient and of membrane electrical potential, indicating that the electrochemical gradient of Na+ contributed energy for the uphill transport of the amino acid through System A. Changes in the Na+ electrochemical gradient were obtained by: (i) alterations of extracellular concentration of Na+; (ii) changes of membrane electrical potential obtained by variation of extracellular [K+]; and (iii) changes of [Na+]in and membrane electrical potential upon incubation of the cells in serum-free saline solutions (Dall'Asta, V., Gazzola, G. C., Longo, N., Bussolati, O., Franchi-Gazzola, R., and Guidotti, G. G. (1986) Biochim. Biophys. Acta 860, 1-8). The correlation between the chemical gradient of 2-methylaminoisobutyric acid and the Na+ electrochemical potential followed a straight line with a yield close to the thermodynamic equilibrium, thus suggesting that the energy stored in the gradient of Na+ electrochemical potential is fully adequate to energize the intracellular accumulation of site A-reactive amino acids in human fibroblasts.