Ion-dependent generation of the electrochemical proton gradient delta muH+ in reconstituted plasma membrane vesicles from the yeast Metschnikowia reukaufii

Plasma membrane vesicles were reconstituted by freezing and thawing of purified plasma membrane fraction from the yeast Metschnikowia reukaufii and phosphatidylcholine (type II-S from Sigma). The reconstituted plasma membrane vesicles generated a proton gradient (acidic inside) upon addition of ATP in presence of alkali cations. delta pH generation was most efficient when K+ was present both outside and inside the plasma membrane vesicles. Both ATPase activity and proton translocation in plasma membrane vesicles were inhibited by orthovanadate (50% inhibition at 100 microM). Plasma membrane vesicles reconstituted without added phosphatidylcholine generated in addition to delta pH, also an electrical potential difference delta psi (inside positive). Delta psi generation exhibited no K+ specificity. 50 microM dicyclohexylcarbodiimide inhibited completely delta psi generation whereas the K+-channel blocker quinine (5 microM) caused an 8-fold increase of delta psi. The proton gradient was much less affected by the agents. Taking into account the K+-dependent stimulation of the plasma membrane ATPase of M. reukaufii, these results further support the conclusion that the ATPase operates as a partially electrogenic H+/K+ exchanger, as was also suggested for other yeast plasma membrane ATPases.     

Leucine transport in plasma membrane vesicles of Saccharomyces cerevisiae

Yeast plasma membrane vesicles were obtained by the fusion of liposomes with purified yeast membranes by means of the freeze thaw-sonication technique. Beef heart mitochondria cytochrome-c oxidase was incorporated into the vesicles. Addition of substrate (ascorbate/TMPD/cytochrome c) generated a membrane potential negative inside, and an alkaline pH gradient inside the vesicle, that served as the driving force for leucine transport. Both delta pH and delta psi could drive leucine transport. When delta pH was increased in the presence of valinomycin and potassium, at the expense of delta psi, leucine uptake increased by 10%.     

Patterns of electrochemical proton gradient formation by membrane vesicles from an obligately acidophilic bacterium

Isolated membrane vesicles from the obligately acidophilic bacterium Bacillus acidocaldarius generated an electrochemical gradient of protons (delta mu- H+) upon energization with ascorbate-phenazine methosulfate at pH 6.0 or 3.0. At pH 6.0, there was little or no transmembrane pH gradient (delta pH), but a transmembrane electrical potential (delta psi) of ca. -77 mV, positive out, was observed. At pH 3.0, a delta pH equivalent to - 100 mV, acid out, and a delta psi of -73 mV, positive out, were observed upon energization. The total magnitude of the delta mu- H+ was higher than that of whole cells at acid pH, but the very large delta pHs and the reversed delta psi s, i.e., inside positive, that are typical of acidophile cells were not observed in the vesicles. The vesicles exhibited energy-dependent accumulation of alpha-aminoisobutyric acid that was inhibited by both nigericin and valinomycin (plus K+) at pH 3.0 but was inhibited little by nigericin at pH 6.0.     

Influence of ion gradients on the transbilayer distribution of dibucaine in large unilamellar vesicles

The uptake of dibucaine into large unilamellar vesicles in response to proton gradients (delta pH; inside acidic) or membrane potentials (delta psi; inside negative) has been investigated. Dibucaine uptake in response to delta pH proceeds rapidly in a manner consistent with permeation of the neutral (deprotonated) form of the drug, reaching a Henderson-Hasselbach equilibrium where [dibucaine]in/[dibucaine]out = [H+]in/[H+]out and where the absolute amount of drug accumulated is sensitive to the buffering capacity of the interior environment. Under appropriate conditions, high absolute interior concentrations of the drug can be achieved (approximately 120 mM) in combination with high trapping efficiencies (in excess of 90%). Dibucaine uptake in response to delta psi proceeds more than an order of magnitude more slowly and cannot be directly attributed to uptake in response to the delta pH induced by delta psi. This induced delta pH is too small (less than or equal to 1.5 pH units) to account for the transmembrane dibucaine concentration gradients achieved and does not come to electrochemical equilibrium with delta psi. Results supporting the possibility that the charged (protonated) form of dibucaine can be accumulated in response to delta psi were obtained by employing a permanently positively charged dibucaine analogue (N-methyldibucaine). Further, the results suggest that delta psi-dependent uptake may depend on formation of a precipitate of the drug in the vesicle interior. The uptake of dibucaine into vesicles in response to ion gradients is of direct utility in drug delivery and controlled release applications and is related to processes of drug sequestration by cells and organelles in vivo.     

Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane.

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.     

Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea. I. Characterization of proton pumping and ATPase activity

Sealed vesicles were isolated from a plant pathogenic fungus Phytophthora megasperma f. sp. glycinea using a modification of a method previously developed for plant plasma membrane vesicle isolation. Vanadate-sensitive, proton pumping microsomal membrane vesicles were resolved on a linear sucrose density gradient and found to comigrate with a vanadate-sensitive ATPase. Both the proton pumping and ATPase activity of these vesicles had a pH optimum of 6.5 and demonstrated similar properties with respect to substrate specificity and inhibitor sensitivity. These properties were in agreement with previously published data on the Phytophthora plasma membrane ATPase. In contrast with previous reports there was no K+ stimulation of the plasma membrane ATPase and the Km for Mg:ATP (1:1 concentration ratio) was higher (2.5 mM). A comparison of anion (potassium salts) effects upon delta pH and delta psi formation in sealed Phytophthora plasma membrane vesicles revealed a correspondence between the relative ability of anions to stimulate proton transport and to reduce delta psi. The relative order for this effect was KCl greater than KBr much greater than KMes, KNO3, KClO3, K2SO4. This study presents a method for the isolation of sealed vesicles from Phytophthora hyphae. It also provides basic information on the plasma membrane H+-ATPase and its associated proton pumping activity.     

Energy dependence and functional reconstitution of the gamma-aminobutyric acid carrier from synaptic vesicles.

The energy dependence of gamma-aminobutyric acid (GABA) uptake was characterized in rat brain synaptic vesicles and in proteoliposomes reconstituted with a new procedure from vesicular detergent extracts. The proteoliposomes displayed high ATP-dependent GABA uptake activity with properties virtually identical to those of intact vesicles. GABA uptake was similar at chloride concentrations of 0 and 150 mM, i.e. conditions under which either the membrane potential (delta psi) or the pH difference (delta pH) predominates. Delta psi was gradually dissipated by increasing the concentration of SCN-. GABA uptake was reduced by 10 mM SCN-, showing less sensitivity to delta psi reduction than glutamate uptake but more than dopamine uptake. Dissipation of delta pH with NH+4 abolished GABA uptake at pH 7.3, whereas no significant inhibition occurred at pH 6.5. In contrast, dopamine uptake was inhibited more strongly, even at pH 6.5, and glutamate uptake was not reduced in either condition. We conclude that GABA uptake is driven by both components of the proton electrochemical gradient, delta pH and delta psi, and that this is different from the uptake of both dopamine and glutamate, which is more strongly dependent on delta pH and delta psi, respectively. Thus, our data suggest that GABA uptake is electrogenic and occurs in exchange for protons.     

Identification of a Transport Mechanism for NH4+ in the Symbiosome Membrane of Pea Root Nodules

Symbiosome membrane vesicles, facing bacteroid-side-out, were purified from pea (Pisum sativum L.) root nodules and used to study NH4+ transport across the membrane by recording vesicle uptake of the NH4+ analog [14C]methylamine (MA). Membrane potentials ([delta][psi]) were imposed on the vesicles using K+ concentration gradients and valinomycin, and the size of the imposed [delta][psi] was determined by measuring vesicle uptake of [14C]tetraphenylphosphonium. Vesicle uptake of MA was driven by a negative [delta][psi] and was stimulated by a low extravesicular pH. Protonophore-induced collapse of the pH gradient indicated that uptake of MA was not related to the presence of a pH gradient. The MA-uptake mechanism appeared to have a large capacity for transport, and saturation was not observed at MA concentrations in the range of 25 [mu]M to 150 mM. MA uptake could be inhibited by NH4+, which indicates that NH4+ and MA compete for the same uptake mechanism. The observed fluxes suggest that voltage-driven channels are operating in the symbiosome membrane and that these are capable of transporting NH4+ at high rates from the bacteroid side of the membrane to the plant cytosol. The pH of the symbiosome space is likely to be involved in regulation of the flux.     

Serotonin transport in isolated platelet granules. Coupling to the electrochemical proton gradient

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in intact 5-hydroxytryptamine (serotonin) containing dense granules. The granules were isolated and purified from other subcellular organelles under isotonic conditions utilizing a newly developed continuous density gradient of Percoll. As measured by [14C]methylamine distribution, isolated granules suspended in a highly buffered medium at pH 7.0 had an intragranular pH of 5.40, independent of the pH of the external medium. This pH gradient could be collapsed by the addition of 60 mM ammonia. In the presence of Mg-ATP, a transmembrane potential (delta psi) of 30-40 mV, inside positive, was generated and sustained for over 30 min, as measured by [14C]thiocyanate distribution. The addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a proton translocator, resulted in the reversal of the potential to negative values. The Mg-ATP-dependent generation of the delta psi was prevented by addition of dicyclohexylcarbodiimide and trimethyltin, inhibitors of proton-translocating ATPases in this and other subcellular organelles. Ammonia (1-50 mM) addition to highly buffered suspensions of serotonin granules caused a dose-dependent decrease in the delta pH, while thiocyanate added at varying concentrations produced a dose-related collapse of the delta psi and had no effect upon the delta pH. Both the delta pH and delta psi were found to independently drive accumulation of [14C]serotonin into the granules; stepwise collapse of each gradient resulted in a corresponding diminution of [14C]serotonin accumulation. The maximum rate and extent of [14C]serotonin uptake, however, were observed in the presence of both the delta pH and delta psi. The conclusions provide support for the existence of a proton-translocating ATPase in the serotonin granule membrane responsible for the generation of the delta pH and delta psi. Moreover, the results demonstrate a primary role for the electrochemical proton gradient (delta mu H+) in the carrier-mediated active transport of 5-hydroxytryptamine into the platelet granule.     

Quantitative association between electrical potential across the cytoplasmic membrane and early gentamicin uptake and killing in Staphylococcus aureus

The relationship between the magnitude of the transmembrane electrical potential and the uptake of [14C]gentamicin was examined in wild-type Staphylococcus aureus in the logarithmic phase of growth. The electrical potential (delta psi) and the pH gradient across the cell membrane were determined by measuring the equilibrium distribution of [3H]tetraphenyl-phosphonium and [14C]acetylsalicylic acid, respectively. Incubation in the presence of the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) led to an increase in delta psi with no measurable effect on the pH gradient at external pHs ranging from 5.0 to 6.5, and the effect on delta psi was DCCD concentration dependent. In separate experiments, gentamicin uptake and killing were studied in the same cells under identical conditions. At pH 5.0 (delta psi = -140 mV), no gentamicin uptake occurred. In the presence of 40 and 100 microM DCCD, delta psi was increased to -162 and -184 mV, respectively, and gentamicin uptake was observed in a manner that was also dependent on the DCCD concentration. At pH 6.0 (delta psi = -164 mV), gentamicin uptake occurred in the absence of the carbodiimide but was enhanced in a concentration-dependent fashion by 40 and 100 microM DCCD (delta psi = -174 and -216 mV, respectively). In all cases increased gentamicin uptake was associated with an enhanced bactericidal effect. The results indicate that initiation of gentamicin uptake requires a threshold level of delta psi (-155 mV) and that above this level drug uptake is directly dependent on the magnitude of delta psi.     

Sucrose protectable inhibition of the sucrose transporter by N-ethylmaleimide

To understand contradictory data published in the literature,the sensitivity of sucrose and of valine uptake to N-ethylmaleimide(NEM) was reinvestigated in detail with plasma membrane vesiclespurified by phase partitioning from mature sugar beet (Betavulgaris) leaves. Uptake in the vesicles was energized by anartificial proton-motive force combining a pH gradient and anelectrical gradient. Three main parameters were varied in theexperiments: the presence of a reducing agent, dlthiothreitol(DTT) In the medium used to store the vesicles, the temperatureof pretreatment with NEM (12 or 23°C) and the temperatureof incubation with the labelled substrate (12 or 23°C).Sensitivity of sucrose uptake to NEM only appeared with vesiclesthat had been stored in the presence of DTT, and if the pretreatmentwas run at 23°C. The temperature of incubation with labelledsucrose did not affect NEM sensitivity. The NEM sensitivityof valine uptake was not affected in the same way as sucroseuptake by the temperature of preincubation, showing that theeffects observed were specific for a given transporter. Underconditions which normally inhibit sucrose uptake, addition ofsucrose during NEM pretreatment protected the sucrose transporteragainst NEM inhibition. Key words: Sugar transport, plasma membrane, differential labelling, thiol reagents     

Control of proteoliposomal cytochrome c oxidase: the overall reaction

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (delta psi) and pH gradient (delta pH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse delta psi and increase delta pH; the respiration rate decreases. High levels of valinomycin, however, decrease delta pH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases delta psi and collapses delta pH; the respiration rate increases. On a millivolt equivalent basis delta pH is a more effective inhibitor of activity than is delta psi. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both delta pH and delta psi components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing delta pH but in a nonlinear way to delta psi ("non-ohmically"). High levels of both delta psi and delta pH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both delta pH and delta psi components of the proton-motive force, but is more sensitive to delta pH than to delta psi at an equivalent delta mu H+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella.     

Active electrogenic transport H+ in plasma membrane vesicles of cow parsnip phloem cells

Generation of electric (delta psi) and chemical (delta pH) components of electrochemical proton gradient delta muH+, in plasma membrane vesicles of Heracleum sosnovskyi phloem cells was investigated. ATP-dependent generation of delta psi at pH 6.0 in the presence of Mg2+ and K+ was established with the help of fluorescent probes AU+ and ANS-. Protonophore CCCP and proton ATPase inhibitor DCCD suppressed generation, whereas oligomycin, the inhibitor of mitochondrial ATPases did not affect it. Measurings of delta psi value indicated its oscillations within the limits from 10 to 60 mV. ATP-dependent generation of delta pH was established by means of fluorescent probe 9-AA. The effect was eliminated by CCCP and stimulated by K+, that may testify to the transformation of a part of delta psi into delta pH at antiport H+/K+. Existence of H+-ATPase in the plasma membranes of higher plant cells insuring generation of delta muH+ is supposed.     

Electrogenic glutamine uptake by Peptostreptococcus anaerobius and generation of a transmembrane potential.

Peptostreptococcus anaerobius converted glutamine stoichiometrically to ammonia and pyroglutamic acid, and the Eadie-Hofstee plot of glutamine transport was biphasic. High-affinity, sodium-dependent glutamine transport (affinity constant [Kt] of 1.5 microM) could be driven by the chemical gradient of sodium, and more than 20 mM sodium was required for half-maximal velocity. High-affinity glutamine transport was not stimulated or inhibited by a membrane potential (delta psi). Low-affinity glutamine transport had a rate which was directly proportional to the external glutamine concentration, required less than 100 microM sodium, and was inhibited strongly by a delta psi. Cells which were treated with N,N-dicyclohexylcarbodiimide to inhibit the F1F0 ATPase still generated a delta psi but did so only if the external glutamine concentration was greater than 15 mM. Low-affinity glutamine uptake could not be saturated by as much as 200 mM glutamine, but glutamine-1 accounts for only a small fraction of the total glutamine at physiological pH values (pH 6 to 7). On the basis of these results, it appeared that the low-affinity glutamine transport was an electrogenic mechanism which was converting a chemical gradient of glutamine-1 into a delta psi. Other mechanisms of delta psi generation (electrogenic glutamine-pyroglutamate or -ammonium exchange) could not be demonstrated.     

Glutamate transport into synaptic vesicles. Roles of membrane potential, pH gradient, and intravesicular pH.

Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, is transported into bovine synaptic vesicles in a manner that is ATP dependent and requires a vesicular electrochemical proton gradient. We studied the electrical and chemical elements of this driving force and evaluated the effects of chloride on transport. Increasing concentrations of Cl- were found to increase the steady-state ATP-dependent vesicular pH gradient (delta pH) and were found to concomitantly decrease the vesicular membrane potential (delta psi). Low millimolar chloride concentrations, which cause 3-6-fold stimulation of vesicular glutamate uptake, caused small but measurable increases in delta pH and decreases in delta psi, when compared to control vesicles in the absence of chloride. Nigericin in potassium buffers was used to alter the relative proportions of delta pH and delta psi. Compared to controls, at all chloride concentrations tested, nigericin virtually abolished delta pH and increased the vesicle interior positive delta psi. Concomitantly, nigericin increased ATP-dependent glutamate uptake in 0-1 mM chloride but decreased glutamate uptake in 4 mM (45%), 20 mM (80%), and 140 mM (75%) Cl- (where delta pH in the absence of nigericin was large). These findings suggest that either delta psi, delta pH, or a combination can drive glutamate uptake, but to different degrees. In the presence of 4 mM Cl-, where uptake is optimal, both delta psi and delta pH contribute to the driving force for uptake. When the extravesicular pH was increased from 7.4 to 8.0, more Cl- was required to stimulate vesicular glutamate uptake. In the absence of Cl-, as extravesicular pH was lowered to 6.8, uptake was over 3-fold greater than it was at pH 7.4. As extravesicular pH was reduced from 8.0 toward 6.8, less Cl- was required for maximal stimulation. Decreasing the extravesicular pH from 8.0 to 6.8 in the absence of Cl- significantly increased glutamate uptake activity, even though proton-pumping ATPase activity actually decreased about 45% under identical conditions. In the absence of chloride, nigericin increased glutamate uptake at all the pH values tested except pH 8.0. Glutamate uptake at pH 6.8 in the presence of nigericin was over 6-fold greater than uptake at pH 7.4 in the absence of nigericin. We conclude from these experiments that optimal ATP-dependent glutamate uptake requires a large delta psi and a small delta pH.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

Neutral amino acid transport by membrane vesicles of Streptococcus cremoris is subject to regulation by internal pH.

The pH dependence of transport of the neutral amino acids L-serine and L-alanine by membrane vesicles of Streptococcus cremoris have been studied in detail. The rates of four modes of facilitated diffusion (e.g., influx, efflux, exchange, and counterflow) of L-serine and L-alanine increase with increasing H+ concentration. Rates of artificially imposed electrical potential across the membrane (delta psi)-driven transport of L-serine and L-alanine show an optimum at pH 6 to 6.5. Under similar conditions, delta psi- and pH gradient across the membrane (delta pH)-driven transport of L-leucine is observed within the pH range studied (pH 5.5 to 7.5). The effect of ionophores on the uptake of L-alanine and L-serine has been studied in membrane vesicles of S. cremoris fused with proteoliposomes containing beef heart mitochondrial cytochrome c oxidase as a proton motive force (delta p)-generating system (Driessen et al., Proc. Natl. Acad. Sci. USA 82:7555-7559, 1985). An increase in the initial rates of L-serine and L-alanine uptake is observed with decreasing pH, which is not consistent with the pH dependency of delta p. Nigericin, an ionophore that induced a nearly complete interconversion of delta pH into delta psi, stimulated both the rate and the final level of L-alanine and L-serine uptake. Valinomycin, an ionophore that induced a collapse of delta psi with a noncompensating increase in delta pH, inhibited L-alanine and L-serine uptake above pH 6.0 more efficiently than it decreased delta p. Experiments which discriminate between the effects of the internal pH and the driving force (delta pH) on solute transport indicate that at high internal pH the transport systems for L-alanine and L-serine are inactivated. A unique relation exists between the internal pH and the initial rate of uptake of L-serine and L-alanine with an apparent pK of 7.0. The rate of L-alanine and L-serine uptake decreases with increasing internal pH. The apparent complex relation between the delta p and transport of L-alanine and L-serine can be explained by a regulatory effect of the internal pH on the activity of the L-serine and L-alanine carriers.     

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.     

Protonmotive force and catecholamine transport in isolated chromaffin granules.

The effect of the transmembrane potential (delta psi) and the proton concentration gradient (delta pH) across the chromaffin granule membrane upon the rate and extent of catecholamine accumulation was studied in isolated bovine chromaffin granules. Freshly isolated chromaffin granules had an intragranular pH of 5.5 as measured by [14C]methylamine distribution. The addition of ATP to a suspension of granules resulted in the generation of a membrane potential, positive inside, as measured by [14C]thiocyanate (SCN-) distribution. The addition of carboxyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a proton translocator, resulted in a reversal of the potential to negative values (measured by [3H]tetramethylphenylphosphonium (TPMP+)) approaching -90 mV. Changing the external pH of a granular suspension incubated with FCCP produced a linear perturbation in the measured potential from positive to negative values, which can be explained by the distribution of protons according to their electrochemical gradient. When ammonia (1 to 50 mM) was added to highly buffered suspensions of chromaffin granules there was a dose-dependent decrease in the transmembrane proton gradient (delta pH) and an increase in the membrane potential (delta psi). On the other hand, thiocyanate or FCCP, at varying concentration, produced a dose-related collapse of the membrane potential and had no effect upon the transmembrane proton gradient. The addition of larger concentrations of catecholamines caused a decrease in the transmembrane proton gradient and an increase in the membrane potential. Time-resolved influx of catecholamines into the granules was studied radiochemically using low external catecholamine concentrations. The accumulation of epinephrine or norepinephrine was over one order of magnitude greater in the presence of ATP than in its absence. The rate and extent of amine accumulation was found to be related to the magnitude of the membrane potential at fixed transmembrane proton concentration (delta pH) values. Likewise, the accumulation was related to the magnitude of the delta pH at fixed membrane potential values. These results suggest that the existence of both a transmembrane proton gradient and a membrane potential are required for optimal catecholamine accumulation to occur.     

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K⁺. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K⁺-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H⁺/sucrose symporter in PM vesicles isolated from spinach leaves.