Comparison of the energetics of lactose active transport: artificial versus enzyme-associated energy source

To further consider the thermochemical method as a useful approach for active transport research and to investigate the characteristic of a proton electrochemical potential (delta mu H+) across the membrane, the energetics of lactose active transport across Escherichia coli membrane vesicles coupled with an artificial electron donor (phenazine methosulfate-ascorbate) has been investigated. The results were compared with those obtained with an enzyme-associated electron donor (lactate dehydrogenase-D-lactate). The oxidation of an electron donor provided the energy necessary for the transport process. The observed higher heat of ascorbate oxidation reaction in the presence of a proton ionophore (carbonyl cyanide m-chlorophenylhydrazone) further confirmed the formation of delta mu H+ across the membrane. Part of the oxidation energy was utilized to form delta mu H+. Comparison of the energetics revealed that the magnitudes of delta Hox (the enthalpy of the oxidation reaction) and delta Hm (the enthalpy of the formation of delta mu H+) in the two energy sources were comparable (-46 kcal/mol of ascorbate to -40 kcal/mol of D-lactate for delta Hox and 9.6 kcal/mol of ascorbate to 14 kcal/mol of D-lactate for delta Hm). Comparable and low value (about 1%) was also found in the free energy transfer (defined by delta Gm/delta Gox) from the oxidation reaction to the formation of delta mu H+. These results, in combination with the close values of delta mu H+ observed in the two systems, suggested that the characteristic of the created delta mu H+ was independent of the energy source. Examination of delta Hm might provide the information on the ratio of the number of protons produced, as 1 mol of two different electron donors was oxidized. The oxidation reaction in the presence of membrane vesicles was discussed.     

THe proton-per-electron stoicheiometry of ''site 1'' of oxidative phosphorylation at high protonmotive force is close to 1.5.

The maximum redox potential difference between the NAD+/NADH couple and the succinate/fumarate couple generated during ATP-energized reduction of NAD+ by succinate in submitochondrial particles was measured, together with the electrochemical potential difference for protons (delta mu approximately H+). The presence of cyanide, the time-independence of the redox potential difference and the irrelevance of the initial redox state of the NAD+/NADH couple ensured that the experimental situation corresponded to a 'static-head condition' with delta mu approximately H+ as the input force and the redox potential difference as the output force, the flow of electrons having reached dynamic equilibrium. Consequently, the observed value of 1.6 for the ratio delta Ge/delta mu approximately H+ is interpreted as indicating that the leads to H+/e- stoicheiometry at 'site 1' is 1.5 and that therefore the mechanism of the proton pump at 'site 1' is not of the group-translocation type (no direct leads to e - leads to H+ coupling).     

The proton motive force lowers the level of ATP required for the in vitro translocation of a secretory protein in Escherichia coli

The role of the electrochemical potential difference of proton (delta mu H+) in protein translocation across the membrane of Escherichia coli was examined in detail using an efficient in vitro assay system (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). Delta mu H+ reduced the level of ATP necessary for the efficient translocation of OmpF-Lpp, a chimeric model secretory protein. The apparent Km value of the translocation reaction for ATP was lower by 2 orders of magnitude in the presence of delta mu H+ than in its absence. The membrane potential and delta pH, both of which are components of delta mu H+, independently lowered the apparent Km value of the translocation reaction for ATP. An ATP-generating system also lowered the level of ATP required for translocation in the absence of delta mu H+ but not in its presence. It is proposed that ADP formed during protein translocation lowers the affinity of the putative translocation machinery for ATP and that the removal of ADP from the secretory machinery, a possible critical step in the translocation reaction, is stimulated in the presence of either delta mu H+, an ATP-generating system, or a higher concentration of ATP.     

In vivo membrane assembly of the E.coli polytopic protein, melibiose permease, occurs via a Sec-independent process which requires the protonmotive force.

To investigate the mechanism of polytopic membrane protein insertion in Escherichia coli, we have examined the protein and energy requirements for in vivo membrane assembly of the prototypic 12 transmembrane domain sugar co-transporter, melibiose permease (MelB). MelB membrane assembly was analyzed both kinetically, by pulse labeling experiments, and functionally by measuring the activity of the inserted permease. Strikingly, the rate of MelB membrane assembly is decreased approximately 4-fold upon dissipation of the transmembrane electrochemical proton gradient, delta(mu)H+, indicative of a strong requirement for delta(mu)H+. Interestingly, selective dissipation of either the electrical (delta(psi)) or the chemical (delta(pH)) component of delta(mu)H+ demonstrates that either form of energy is required for MelB membrane assembly. In contrast, MelB membrane assembly does not require SecA, SecY or SecE, all three proteins which are strictly required for protein translocation. Neither the rate of MelB membrane assembly nor the amount of functional permease is affected by inactivation or depletion of these Sec proteins. These results strongly suggest that polytopic membrane proteins such as MelB insert into the cytoplasmic membrane by a mechanism fundamentally different from protein translocation.     

Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase.

Preprotein translocation in E. coli requires ATP, the membrane electrochemical potential delta mu H+, and translocase, an enzyme with an ATPase domain (SecA) and the membrane-embedded SecY/E. Studies of translocase and proOmpA binds to the SecA domain. Second, SecA binds ATP. Third, ATP-binding energy permits translocation of approximately 20 residues of proOmpA. Fourth, ATP hydrolysis releases proOmpA. ProOmpA may then rebind to SecA and reenter this cycle, allowing progress through a series of transmembrane intermediates. In the absence of delta mu H+ or association with SecA, proOmpA passes backward through the membrane, but moves forward when either ATP and SecA or a membrane electrochemical potential is supplied. However, in the presence of delta mu H+ (fifth step), proOmpA rapidly completes translocation. delta mu H(+)-driven translocation is blocked by SecA plus nonhydrolyzable ATP analogs, indicating that delta mu H+ drives translocation when ATP and proOmpA are not bound to SecA.     

Some novel aspects of the relationship between the amino acid gradient and the sodium electrochemical gradient in mouse ascites tumour cells

Accumulation of 2-aminoisobutyrate by mouse ascites tumour cells was studied in circumstances where nigericin reversed the normal direction of the Na+ concentration gradient. The membrane potential (delta psi) was assayed using oxonol V as a voltage-sensitive probe. The amino acid gradient (delta mu A) that formed did not significantly exceed the likely magnitude of the Na+ electrochemical gradient when this was in the range 2-6 kJ mol-1. When delta-Na mu increased up to 11 kJ mol-1, delta mu A was almost constant at 7-8 kJ mol-1. The observations indicate that when delta psi is large changes in cellular [Na+] in the range 16-80 mM scarcely affect delta mu A.     

Coupling between the sodium and proton gradients in respiring Escherichia coli cells measured by 23Na and 31P nuclear magnetic resonance

The relationship between the steady-state sodium gradient (delta pNa) and the protonmotive force developed by endogenously respiring Escherichia coli cells has been studied quantitatively, using 23Na NMR for measurement of intracellular and extracellular sodium concentrations, 31P NMR for measurement of intracellular and extracellular pH, and tetraphenylphosphonium distribution for measurement of membrane potential. At constant protonmotive force, the sodium concentration gradient was independent of extracellular concentrations over the measured range of 4-285 mM, indicating that intracellular sodium concentration is not regulated. The magnitude of delta pNa was measured as a function of the composition and magnitude of the protonmotive force. At external pH values below 7.2, delta pNa was parallel to delta pH but showed no simple relationship to the membrane potential; above pH 7.2 the parallel relationship began to diverge, with delta pH continuing to decrease but delta pNa starting to level off or increase. Although plots of delta pNa versus delta pH had slopes of close to 1, the value of delta pNa consistently exceeded that of delta pH by approximately 0.4 units, indicating a partially electrogenic character to the putative H+/Na+ antiport. The apparent stoichiometry was 1.13 +/- 0.01 at external pH below 7.2. The possible significance of this nonintegral stoichiometry is discussed according to a model in which two distinct integral stoichiometries (possibly 1H+/1Na+ and 2H+/1Na+) are available with some relative probability; the model predicts futile cycling of sodium ions and a dissipative proton current. In the course of this study, we discovered that the magnitude of the pH gradient developed by the cells was osmolarity-dependent, yielding steady-state intracellular pH values that varied from 7.1 at 100 mosm to 7.7 at 800 mosm.     

Energetics of ATP-driven reverse electron transfer from cytochrome c to fumarate and from succinate to NAD in submitochondrial particles

The maximum Gibbs free energies of reverse electron transfer from succinate to NAD+ and from cytochrome c to fumarate driven by ATP hydrolysis in submitochondrial particles from beef heart were measured as a function of the Gibbs free energy of ATP hydrolysis. The ratio of the energies delta G'redox/delta G'ATP was 1.40 from succinate to NAD+ and 0.89 from cytochrome c to succinate. The ratio, equivalent to a thermodynamic P/2e-ratio, was dependent on whether the electrochemical proton gradient was primarily a membrane potential or a pH gradient for the cytochrome c to fumarate reaction. The results are consistent with H+/ATP = 3 for F1 ATPase, H+/2e- = 4 for NADH-CoQ reductase, and H+(matrix)/2e- = 2 for succinate-cytochrome c reductase.     

On the relationship between rate of ATP synthesis and H+ electrochemical gradient in rat-liver mitochondria

The relationship between rate of ATP synthesis, JATP, and value of the proton electrochemical gradient, delta mu H, has been analyzed in intact mitochondria. Onset of phosphorylation causes a depression of delta mu H of 1.5 kJ/mol. There is a close parallelism between inhibition of JATP and restoration of delta mu H to its state-4 value during titrations with oligomycin or atractyloside. Titrations with ionophores display the following features: (a) delta mu H can be depressed by 3-4 kJ/mol by valinomycin + K+ without affecting the rate of ATP synthesis; (b) uncouplers abolish JATP completely while depressing delta mu H by 3 kJ/mol; (c) complete abolition of ATP synthesis by inhibitors of electron transport is accompanied by a depression of delta mu H of only 1 kJ/mol. The results indicate that: (a) there is a close functional relationship between redox and ATPase H+ pumps, whereby inhibition of electron transfer is accompanied by simultaneous inhibition of the ATPase H+ pumps; and (b) uncoupling of oxidative phosphorylation is not due to depression of delta mu H per se. The consistence of the present data with either a chemiosmotic model where delta mu H is the sole and obligatory intermediate for energy coupling, or models where there is a direct transfer of energy between the two pumps is discussed.     

The location of redox-sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli

Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane. Treatment of right-side-out membrane vesicles from E. coli ML 308-225 with the membrane-impermeable oxidant ferricyanide resulted in inhibition of L-proline uptake without having significant effect on the magnitude of the delta approximately mu H+. Subsequent addition of reducing agents restored proline transport activity. The membrane-impermeable SH-reagent glutathione hexane maleimide inhibited proline transport in right-side-out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide. Electron transfer in the respiratory chain of right-side-out vesicles led to the generation of a delta approximately mu H+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4-(2-arsonophenyl)azo-3-hydroxy-2,7-naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group. Treatment of inside-out membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside-out vesicles resulted in protection against inhibition by glutathione hexane maleimide. Imposition in these vesicles of a delta approximately mu H+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.     

Cooperative regulation of the Na+/H(+)-antiporter in Halobacterium halobium by delta pH and delta phi

The contributions of the transmembrane pH gradient (delta pH) and electrical potential (delta phi) to the delta mu H(+)-driven Na+ efflux (mediated by the N,N'-dicyclohexylcarbodiimide-sensitive Na+/H(+)-antiporter) were investigated in membrane vesicles of Halobacterium halobium. Kinetic analysis in the dark revealed that two different Na(+)-binding sites are located asymmetrically across the membrane: One, accessible from the external medium, has a Kd (half-maximal stimulation of Na+ efflux) of about less than 50 mM, and the Na+ binding to the site is a prerequisite for the antiporter activation by delta mu H+. The other cytoplasmic site is the Na+ transport site. The Km for the cytoplasmic Na+ decreased as the delta pH increased, while the Vmax remained essentially constant in the presence of defined delta phi (140 mV). On the other hand, delta phi elevation above the gating potential (approximately 100 mV) increased the Vmax without changes in the Km in the presence of a fixed delta pH. It was also noted that the Km value in the absence of delta phi was completely different from and far higher than that observed in the presence of delta phi (greater than 100 mV), indicating the existence of two distinct conformations in the antiporter, resting and delta phi gated; the latter state may be reactive only to delta pH. On the basis of the present data and the previous data on the pH effect (N. Murakami and T. Konishi, 1989 Arch. Biochem. Biophys. 271, 515-523), a model for the delta pH-delta phi regulation of the antiporter activation is proposed.     

A high concentration of SecA allows proton motive force-independent translocation of a model secretory protein into Escherichia coli membrane vesicles

The in vitro translocation of OmpF-Lpp, a model secretory protein, into inverted membrane vesicles of Escherichia coli obligatorily requires the proton motive force (delta mu H+) in the conventional assay system (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). The translocation, however, took place efficiently, even in the absence of delta mu H+, when the system was supplemented with additional SecA. With the stripped membrane vesicles, which are permeable to protons, or in the absence of NADH, the supplementation of SecA remarkably stimulated the translocation activity. The further addition of NADH did not significantly enhance the translocation activity under the SecA-enriched conditions. OmpF-Lpp thus translocated could be recovered from the vesicular lumen by sonication, indicating that complete translocation occurred in the absence of delta mu H+. It is suggested that delta mu H+ is required for high affinity interaction of SecA with the presumed secretory machinery in the cytoplasmic membrane and that a high concentration of SecA modulates the delta mu H+ requirement.     

The electrochemical proton gradient in Mycoplasma cells

The electrochemical proton gradient, delta mu H+ generated upon glycolysis by Mycoplasma mycoides var. Capri cells has been determined. The components, the transmembrane pH gradient, delta pH, and the membrane potential, delta psi, were measured using several methods. The determination of the delta pH was conducted by measuring the transmembrane distribution of weak acids (acetate and butyrate) and of a weak base (methylamine), using flow dialysis and filtration techniques. The transmembrane electrical potential was determined from the distribution of the lipophilic cation Ph3MeP+ and of Rb+ or K+ in the presence of valinomycin. At extra-cellular pH 7.2, glycolyzing Mycoplasma cells maintain an internal pH more alkaline (0.5 pH unit) than that of the milieu and an electrical potential of - 85 mV, interior negative. The delta mu H+ in M. mycoides var. Capri cells is thus about - 115 mV. When the external pH was altered from 7.7 to 5.7 delta psi decreased from - 90 mV to - 60 mV. On other hand although the internal pH decreased, delta pH was found to increase from 0.2 to 1.0 pH unit. Since the changes in delta psi were largely compensated by the changes in delta pH, delta mu H+ remained practically constant at about - 115 mV throughout the pH range tested. Finally, inhibition of delta pH by N,N'-dicyclohexylcarbodiimide, carbonylcyanide-p-trifluoromethoxyphenylhydrazone or nigericin confirmed that chemiosmotic phenomena contribute to energy transduction across the membranes of M. mycoides var. Capri cells.     

Unique relationships between the rates of oxidation and phosphorylation and the protonmotive force in rat-liver mitochondria

The rate of ATP synthesis (JP) in isolated rat-liver mitochondria was strongly dependent on the magnitude of the protonmotive force (delta mu H+) across the mitochondrial inner membrane. Addition of different concentrations of various uncouplers or malonate to mitochondrial incubations in State 3 led to a depression of delta mu H+ and a concomitant decrease in JP. A unique relationship between JP and delta mu H+ was obtained, which was independent of the way in which delta mu H+ was varied. This unique relationship was observed when K+ (in the presence of valinomycin) was used as a probe for delta psi. Different relationships between JP and delta mu H+ were observed when K+ was used as a probe for delta psi and when K+ was measured after separation of the mitochondria by centrifugation without silicone oil. This led to a serious underestimation of delta psi, specifically when uncouplers were present, and non-unique flow-force relationships were thus obtained. Anomalous relationships between JP and delta mu H+ were also found when TPMP+ was used as a probe for delta psi. However, in uncoupler incubations the presence of TBP- strongly affected the TPMP+ accumulation ratio without any effect on the K+ accumulation or on JP and in the presence of TBP- unique relationships between JP and delta mu H+ were again obtained. This indicates that the accumulation of TPMP+ inside the mitochondria is not a straightforward function of delta psi but also depends on conditions like the presence of TBP- or uncouplers. We conclude that there is a unique relationship between the rate of phosphorylation and the protonmotive force in mitochondria and that under some conditions the behaviour of TPMP+ is anomalous.     

Intrinsic characteristics of the proton pump in the luminal membrane of a tight urinary epithelium. The relation between transport rate and delta mu H

A number of tight urinary epithelia, as exemplified by the turtle bladder, acidify the luminal solution by active transport of H+ across the luminal cell membrane. The rate of active H+ transport (JH) decreases as the electrochemical potential difference for H+ [delta mu H = mu H(lumen) - mu H(serosa)] across the epithelium is increased. The luminal cell membrane has a low permeability for H+ equivalents and a high electrical resistance compared with the basolateral cell membrane. Changes in JH thus reflect changes in active H+ transport across the luminal membrane. To examine the control of JH by delta mu H in the turtle bladder, transepithelial electrical potential differences (delta psi) were imposed at constant acid-base conditions or the luminal pH was varied at delta psi = 0 and constant serosal PCO2 and pH. When the luminal compartment was acidified from pH 7 to 4 or was made electrically positive, JH decreased as a linear function of delta mu H as previously described. When the luminal compartment was made alkaline from pH 7 to 9 or was made electrically negative, JH reached a maximal value, which was the same whether the delta mu H was imposed as a delta pH or a delta psi. The nonlinear JH vs. delta mu H relation does not result from changes in the number of pumps in the luminal membrane or from changes in the intracellular pH, but is a characteristic of the H+ pumps themselves. We propose a general scheme, which, because of its structural features, can account for the nonlinearity of the JH vs. delta mu H relations and, more specifically, for the kinetic equivalence of the effects of the chemical and electrical components of delta mu H. According to this model, the pump complex consists of two components: a catalytic unit at the cytoplasmic side of the luminal membrane, which mediates the ATP-driven H+ translocation, and a transmembrane channel, which mediates the transfer of H+ from the catalytic unit to the luminal solution. These two components may be linked through a buffer compartment for H+ (an antechamber).     

Interconversion of components of the bacterial proton motive force by electrogenic potassium transport.

The influence of K+ ions on the components of the transmembrane proton motive force (delta mu H+) in intact bacteria was investigated. In K+-depleted cells of the glycolytic bacterium STreptococcus faecalis the addition of K+ ions caused a depolarization of the membrane by about 60 mV. However, since the depolarization was compensated for by an increase in the transmembrane pH gradient (delta pH), the total proton motive force remained almost constant at about 120 mV. Half-maximal changes in the potential were observed at K+ concentrations at which the cells accumulated K+ ions extensively. In EDTA-treated, K+-depleted cells of Escherichia coli K-12, the addition of K+ ions to the medium caused similar, although smaller changes in the components of delta mu H+. Experiments with various E. coli K-12 K+ transport mutants showed that for the observed potential changes the cells required either a functional TrkA or Kdp K+ transport system. These data are interpreted to mean that the inward movement of K+ ions via each of these bacterial transport systems is electrogenic. Consequently, it leads to a depolarization of the membrane, which in its turn allows the cell to pump more protons into the medium.     

Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro analysis of the process of translocation of OmpA across the Escherichia coli cytoplasmic membrane. A translocation intermediate accumulates transiently in the absence of the proton motive force

The proton motive force (delta mu H+) plays an important role, although it is not absolutely essential, in the in vitro translocation of secretory proteins, such as OmpA, across the cytoplasmic membrane of Escherichia coli (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). The transient accumulation in membrane vesicles of a possible translocation intermediate of OmpA was observed in the absence of delta mu H+. The intermediate was detected on a polyacrylamide gel as a proteinase K-resistant band corresponding to a molecular weight of 26,000. The intermediate did not possess the signal peptide. The appearance of this band was inhibited in the absence of ATP or the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP) and enhanced upon the addition of SecA. Upon the addition of NADH that energizes the membrane, the intermediate was converted to the translocated form of OmpA, even in the presence of AMP-PNP. These results suggest different requirements of ATP and delta mu H+ for the early and late stages of the translocation reaction. The SecA requirement for the early stage of the translocation has also been suggested. In addition to this band, two other bands were observed at higher positions on the gel, when the translocation reaction was performed in the absence of delta mu H+. Although these two bands also represented the mature form of OmpA, which was partly protected from the proteinase K treatment by the membrane vesicles, the accumulation was not transient. These bands did not appear when the translocation reaction was performed in the presence of dithiothreitol. Together with other evidence, the above observations suggest that OmpA, which has an intramolecular disulfide bridge, cannot undergo the translocation unless delta mu H+ is imposed.     

Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.     

The kinetic mechanism of galactoside/H+ cotransport in Escherichia coli

To determine the kinetic mechanism of galactoside active transport by the lactose/H+ cotransporter of Escherichia coli, galactoside binding and transport are studied in the absence and presence of delta mu H+. For several reasons, the substrate beta-D-galactosyl-1-thi-beta-D-galactoside (GalSGal) is preferred over lactose. In the absence of delta mu H+, the cotransporter retains high affinity for GalSGal, and the affinity is the same on both sides of the membrane. At physiological pH, the cotransporter is protonated and the dissociation constant for H+ may be 50 pM. The cosubstrates bind in a random fashion. An isomerization of the cotransporter corresponding to reorientation of the binding sites is rate-determining. When delta mu H+ is imposed, two reorientations become faster, and one becomes slower. The affinity of the cotransporter for GalSGal on both sides of the membrane is unchanged. The inability of the cotransporter to bring the accumulation of galactoside into equilibrium with delta mu H+ at high galactoside concentrations can be explained without postulating uncoupled fluxes of galactoside or H+ across the membrane (leaks). The formation of the ternary carrier-H+-galactoside complex on the cytoplasmic side of the membrane with increasing internal levels of sugar and the rapidity of galactoside exchange inhibit net influx of galactoside and favor exchange. Net transport is slow at high galactoside levels. Thus, the cotransporter can self-regulate transport without uncoupling H+ and galactoside fluxes. Because the values of delta mu H+ during binding and transport studies were measured, these results can be subjected to a quantitative analysis.