Mechanism of proline transport in Escherichia coli K12. III. Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier

Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM. These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.     

Mechanism of proline transport in Escherichia coli K12. II. Effect of alkaline cations on binding of proline to a H+/proline symport carrier in cytoplasmic membrane vesicles

The substrate binding reaction of the proline carrier was investigated in nonenergized conditions using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The binding activity specifically required both alkaline cations (X+), Na+ and Li+, and protons. The Na+-dependent binding activity was dependent on the proline carrier, which is the product of the putP gene, and was not affected by ionophores and energy transduction inhibitors. The parameters of proline binding were determined by double reciprocal plots in reaction media with various combinations of Na+ and H+ concentrations. The apparent dissociation constant was greatly affected by the Na+ and H+ concentrations of the medium and could be expressed as a combination of the reciprocals of the Na+ and H+ concentrations, while the maximum number of binding sites remained constant. The characteristics of proline binding to the carrier can be explained by a mechanism in which the unloaded carrier forms a carrier/H+/X+ (CH+X+) complex by a random equilibrium and only the CH+X+ complex binds substrate in nonenergized conditions, as proposed for the Na+/H+/glutamate symport carrier of E. coli B ( Fujimura , T., Yamato , I., and Anraku , Y. (1983) Biochemistry 22, 1954-1959).     

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.     

Mechanism of the melibiose porter in membrane vesicles of Escherichia coli

The melibiose transport system of Escherichia coli catalyzes sodium--methyl 1-thio-beta-D-galactopyranoside (TMG) symport, and the cation is required not only for respiration-driven active transport but also for binding of substrate to the carrier in the absence of energy and for carrier-mediated TMG efflux. As opposed to the proton--beta-galactoside symport system [Kaczorowski, G. J., & Kaback, H. R. (1979) Biochemistry 18, 3691], efflux and exchange of TMG occur at the same rate, implying that the rates of the two processes are limited by a common step, most likely the translocation of substrate across the membrane. Furthermore, the rate of exchange, as well as efflux, is influenced by imposition of a membrane potential (delta psi; interior negative), suggesting that the ternary complex between sodium, TMG, and the porter may bear a net positive charge. Consistently, energization of the vesicles leads to a large increase in the Vmax for TMG influx, with little or no change in the apparent Km of the process. It is proposed that the sodium gradient (Na+out < Na+in) and the delta psi (interior negative) may affect different steps in the overall mechanism of active TMG accumulation in the following manner: the sodium gradient causes an increased affinity for TMG on the outer surface of the membrane relative to the inside and the delta psi facilitates a reaction involved with the translocation of the positively charged ternary complex to the inner surface of the membrane.     

Effect of membrane potential on the kinetic parameters of the Na+ or H+ melibiose symport in Escherichia coli membrane vesicles

Comparison of the transport properties of the melibiose permease of E. coli acting as a H+-symport or a Na+-symport has been performed by measuring initial rates of [3H]-melibiose transport or its accumulation in isolated membrane vesicles. The results show strikingly that although the membrane potential primarily drives melibiose accumulation by both types of symport, it selectively affects the apparent affinity constant Kt of the H+-melibiose symport while it specifically changes the maximal rate of transport (Vmax) of the Na+-melibiose symport. It is suggested that modification(s) of some partial reaction constants of a given transport cycle might lead to important changes in the kinetic properties of this transport system.     

L-malate transport and proton symport in vesicles prepared from Pseudomonas putida

In vesicles from glucose-grown Pseudomonas putida, L-malate is transported by nonspecific physical diffusion. L-Malate also acts as an electron donor and generates a proton motive force (delta p) of 129 mV which is composed of a membrane potential (delta psi) of 60 mV and a delta pH of 69 mV. In contrast, vesicles from succinate-grown cells transport L-malate by a carrier-mediated system with a Km value of 14.3 mM and a Vmax of 313 nmol X mg protein-1 X min-1, generate no delta psi, delta pH, or delta p when L-malate is the electron donor, and produce an extravesicular alkaline pH during the transport of L-malate. A kinetic analysis of this L-malate-induced proton transport gives a Km value of 16 mM and a Vmax of 667 nmol H+ X mg protein-1 X min-1. This corresponds to a H+/L-malate ratio of 2.1. The failure to generate a delta p in these vesicles is considered, therefore, to be consistent with the induction in succinate-grown cells of an electrogenic proton symport L-malate transport system.     

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.     

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.     

Sodium-dependent transport of branched-chain amino acids by a monensin-sensitive ruminal peptostreptococcus.

A recently isolated ruminal peptostreptococcus which produced large amounts of branched-chain volatile fatty acids grew rapidly with leucine as an energy source in the presence but not the absence of Na. Leucine transport could be driven by an artificial membrane potential (delta psi) only when Na was available, and a chemical gradient of Na+ (delta uNa+) also drove uptake. Because Na+ was taken up with leucine and a Z delta pH could not serve as a driving force (with or without Na), it appeared that leucine was transported in symport with Na+. The leucine carrier could use Li as well as Na and had a single binding site for Na+. The Km for Na was 5.2 mM, and the Km and Vmax for leucine were 77 microM and 328 nmol/mg of protein per min, respectively. Since valine and isoleucine competitively inhibited (Kis of 90 and 49 microM, respectively) leucine transport, it appeared that the peptostreptococcus used a common carrier for branched-chain amino acids. Valine or isoleucine was taken up rapidly, but little ammonia was produced if they were provided individually. The lack of ammonia could be explained by an accumulation of reducing equivalents. The ionophore, monensin, inhibited growth, but leucine was taken up and deaminated at a slow rate. Monensin caused a loss of K, an increase in Na, a slight increase in delta psi, and a decrease in intracellular pH. The inhibition of growth was consistent with a large decrease in ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and reconstitution of Escherichia coli proline carrier using a site specifically cleavable fusion protein

We previously constructed a bifunctionally active membrane-bound fusion protein, in which Escherichia coli proline carrier (the product of the putP gene) was linked with beta-galactosidase (the product of the lacZ gene) through a collagen linker (Hanada, K., Yamato, I., and Anraku, Y. (1987) J. Biol. Chem. 262, 14100-14104). The proline carrier was purified from this site specifically cleavable fusion protein. Cytoplasmic membranes overproducing the fusion protein were solubilized with dodecylmaltoside, and the solubilized fraction was subjected to anti-beta-galactosidase IgG-Sepharose chromatography. The fusion protein was specifically adsorbed to the immunoaffinity resin and then treated with collagenase for splitting the proline carrier moiety of the fusion protein from the beta-galactosidase moiety. The collagenase used for the collagenolysis was then removed by anti-collagenase IgG-Sepharose chromatography. In this way, the proline carrier was purified to more than 95% homogeneity of the protein. Proline transport in proteoliposomes reconstituted with the purified carrier was dependent on the membrane potential and the chemical gradient of Na+ across the membrane with apparent Michaelis constants for proline and for Na+ stimulation of 3.6 microM and 31 microM, respectively. These results indicated that the proline carrier mediates electrogenic Na+/proline symport.     

Sodium-dependent transport of branched-chain amino acids by a monensin-sensitive ruminal peptostreptococcus

A recently isolated ruminal peptostreptococcus which produced large amounts of branched-chain volatile fatty acids grew rapidly with leucine as an energy source in the presence but not the absence of Na. Leucine transport could be driven by an artificial membrane potential (delta psi) only when Na was available, and a chemical gradient of Na+ (delta uNa+) also drove uptake. Because Na+ was taken up with leucine and a Z delta pH could not serve as a driving force (with or without Na), it appeared that leucine was transported in symport with Na+. The leucine carrier could use Li as well as Na and had a single binding site for Na+. The Km for Na was 5.2 mM, and the Km and Vmax for leucine were 77 microM and 328 nmol/mg of protein per min, respectively. Since valine and isoleucine competitively inhibited (Kis of 90 and 49 microM, respectively) leucine transport, it appeared that the peptostreptococcus used a common carrier for branched-chain amino acids. Valine or isoleucine was taken up rapidly, but little ammonia was produced if they were provided individually. The lack of ammonia could be explained by an accumulation of reducing equivalents. The ionophore, monensin, inhibited growth, but leucine was taken up and deaminated at a slow rate. Monensin caused a loss of K, an increase in Na, a slight increase in delta psi, and a decrease in intracellular pH. The inhibition of growth was consistent with a large decrease in ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy-dependent uptake of 4-chlorobenzoate in the coryneform bacterium NTB-1.

The uptake of 4-chlorobenzoate (4-CBA) in intact cells of the coryneform bacterium NTB-1 was investigated. Uptake and metabolism of 4-CBA were observed in cells grown in 4-CBA but not in glucose-grown cells. Under aerobic conditions, uptake of 4-CBA occurred with a high apparent affinity (apparent Kt, 1.7 microM) and a maximal velocity (Vmax) of 5.1 nmol min-1 mg of protein-1. At pH values below 7, the rate of 4-CBA uptake was greatly reduced by nigericin, an ionophore which dissipates the pH gradient across the membrane (delta pH). At higher pH values, inhibition was observed only with valinomycin, an ionophore which collapses the electrical potential across the membrane (delta psi). Under anaerobic conditions, no uptake of 4-CBA was observed unless an alternative electron acceptor was present. With nitrate as the terminal electron acceptor, 4-CBA was rapidly accumulated by the cells to a steady-state level, at which uptake of 4-CBA was balanced by excretion of 4-hydroxybenzoate. The mechanism of energy coupling to 4-CBA transport under anaerobic conditions was further examined by the imposition of an artificial delta psi, delta pH, or both. Uptake of 4-CBA was shown to be coupled to the proton motive force, suggesting a proton symport mechanism. Competition studies with various substrate analogs revealed a very narrow specificity of the 4-CBA uptake system. This is the first report of carrier-mediated transport of halogenated aromatic compounds in bacteria.     

Kinetics of the intestinal brush border proline (Imino) carrier

The kinetics of L-proline transport across intestinal brush borders via the Imino carrier were studied using membrane vesicles. The Imino carrier is defined as the agent responsible for L-alanine insensitive. Na+-dependent uptake of L-proline. Initial rate measurements were made under voltage clamped conditions (pD = 0) to investigate L-proline transport as a function of cis and trans Na+ and proline concentrations. Under zero-trans conditions, increasing cis Na+ activated proline uptake with a Hill coefficient of 1.7 and decreased the apparent Kt with no change in Jimax. The Jimax was approximately 60 pmol mg-1 s-1 and the apparent Kt ranged from 0.25 mM at cis Na = 100 to 1.0 mM at cis Na+ = 30 mM. Trans Na inhibited proline uptake via a reduction in Jimax. Trans proline had no significant effect in the absence of trans Na+, but it relieved the trans Na+ inhibition. Under equilibrium exchange conditions, the Jimax was twice that observed under zero-trans conditions. These kinetics of L-proline transport suggest a model in which uptake occurs by a rapid equilibrium iso-ordered ter ter system. Two Na+ ions bind first to the carrier on the cis face of the membrane to increase the affinity of the carrier for proline. The fully loaded complex then isomerizes to release the substrates to the trans side. The partially loaded Na+-only forms are unable to translocate across the membrane. A rate-limiting step appears to be the isomerization of unloaded carrier from the trans to the cis side of the membrane.     

Solubilization and reconstitution of proline carrier in Escherichia coli; quantitative analysis and optimal conditions

Proline carrier of Escherichia coli was extracted from the carrier-overproducing membranes with dodecylmaltoside in the presence of phospholipid. The solubilized carrier showed the same proline binding activity as that in normal membranes. As judged from determinations of the binding activity in the micellar state as a marker of active carrier and the radioactivity of N-[ethyl-2-3H]ethylmaleimide-labeled carrier as a marker of carrier polypeptide, 80% of the carrier molecules in the membranes were extracted. Optimal conditions for reconstitution of the solubilized carrier were established. By a combination of freeze-thawing, sonication and dilution procedures, 70% of the solubilized carrier molecules were incorporated into proteoliposomes and the restored active transport of proline showed an apparent Kt of 1 microM and turnover number of 0.6 s-1. The transport of proline was driven by a membrane potential in a Na+ (or Li+)-dependent manner.     

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).     

The high and low affinity transport systems for dipeptides in kidney brush border membrane respond differently to alterations in pH gradient and membrane potential

The principal aim of the present study was to investigate the effects of variation in proton gradient and membrane potential on the transport of glycyl-L-glutamine (Gly-Gln) by renal brush border membrane vesicles. Under our conditions of transport assay, Gly-Gln was taken up by brush border membrane vesicles almost entirely as intact dipeptide. This uptake was mediated by two transporters shared by other dipeptides and characterized as the high affinity (Kt = 44.1 +/- 11.2 microM)/low capacity (Vmax = 0.41 +/- 0.03 nmol/mg protein/5 s) and low affinity (Kt = 2.62 +/- 0.50 mM)/high capacity (Vmax 4.04 +/- 0.80 nmol/mg protein/5 s) transporters. In the absence of a pH gradient, only the low affinity system was operational, but with a reduced transport capacity. Imposing a pH gradient of 1.6 pH units increased the Vmax of both transporters. Kinetic analysis of the rates of Gly-Gln uptake as a function of external pH revealed Hill coefficients of close or equal to 1, indicating that transporters contain only one binding site for the interaction with external H+. The effects of membrane potential on Gly-Gln uptake were investigated with valinomycin-induced K+ diffusion potentials. The velocity of the high affinity system but not of the low affinity system increased linearly with increasing inside-negative K+ diffusion potentials (p less than 0.01). The Kt of neither system was affected by alterations in either pH gradient or membrane potential. We conclude that (a) the high affinity transporter is far more sensitive to changes in proton gradient and membrane potential than the low affinity transporter and (b) in the presence of a pH gradient, transport of each dipeptide molecule requires cotransport of one hydrogen ion to serve as the driving force.     

Changes in membrane ionic conductance, but not changes in slip, can account for the non-linear dependence of the electrochemical proton gradient upon the electron-transport rate in chromatophores

Decrease in the rate of cyclic electron transport (JE) measured from the absorbance changes associated with reaction centre bacteriochlorophyll led to a less than proportionate decrease in the membrane potential (delta psi) measured by electrochromism. In principle this result can be explained either by a delta psi-dependent slip in the H+/e- coupling ratio (nE) or by a delta psi-dependent change in the membrane ionic conductance. Simultaneous measurement of the membrane ionic current (JDIS) did not reveal any significant changes in the H+/e- ratio (JDIS/JE) and showed that conductance changes (JDIS/delta psi) account quantitatively for the curved dependence of delta psi on JE. Simultaneous recordings of JDIS and the extravesicular pH from cresol-red absorbance changes, suggest that protons are the main current-carrying species across the chromatophore membrane at high values of delta psi in the presence and absence of Fo-ATPase inhibitor. At reduced delta psi the flux of other ions outweighs the hydrogen ion current.     

Tobramycin uptake in Escherichia coli is driven by either electrical potential or ATP.

Aminoglycoside antibiotics such as streptomycin and tobramycin must traverse the bacterial cytoplasmic membrane prior to initiating lethal effects. Previous data on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis have demonstrated that transport of aminoglycosides is regulated by delta psi, the electrical component of the proton motive force. However, several laboratories have observed that growth of bacterial cells can occur in the apparent absence of delta psi, and we wished to confirm these studies with E. coli and further investigate whether transport of aminoglycosides could occur in the absence of a membrane potential. Treatment of acrA strain CL2 with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) dissipated delta psi, decreased intracellular ATP levels, and resulted in cessation of growth; after a variable period of time (3 to 7 h), growth resumed, ultimately achieving growth rates comparable to those of untreated cells. Absence of delta psi in these cells was confirmed by absence of [3H]tetraphenyl phosphonium+ uptake as measured by membrane filtration, lack of flagellar motion, and inability of these cells to transport proline (but not methionine). Regrowth was associated with restoration of normal intracellular ATP as measured by luciferin-luciferase bioluminescence assay. Unlike unacclimatized CL2 cells treated with CCCP, these cells transported [3H]tobramycin similarly to untreated cells; aminoglycoside-induced killing was seen in association with transport. These studies suggest that under certain circumstances aminoglycoside transport can be driven by ATP (or other high-energy activated phosphate donors) alone, in the absence of a measurable delta psi. delta uncBC mutants of CL2 incapable of interconverting delta psi and ATP were treated with CCCP, resulting in dissipation of delta psi but no alteration in ATP content. Despite maintenance of normal ATP, there was no transport of [3H] bramycin, confirming that under normal growth conditions ATP has no role in the transport of aminoglycosides.     

A proton gradient is the driving force for uphill transport of lactate in human placental brush-border membrane vesicles

The characteristics of lactate transport in brush-border membrane vesicles isolated from normal human full-term placentas were investigated. Lactate transport in these vesicles was Na+-independent, but was greatly stimulated when the extravesicular pH was made acidic. In the presence of an inwardly directed H+ gradient ([H+]o greater than [H+]i), transient uphill transport of lactate could be demonstrated. This H+ gradient-dependent stimulation was not a result of a H+ diffusion potential. Transport of lactate in the presence of the H+ gradient was not inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or by furosemide, ruling out the participation of an anion exchanger in placental lactate transport. Many monocarboxylates strongly interacted with the lactate transport system, whereas, with the single exception of succinate, dicarboxylates did not. The monocarboxylates pyruvate and lactate, but not the dicarboxylate succinate, when present inside the vesicles, were able to exert a trans-stimulatory effect on the uptake of radiolabeled lactate. Kinetic analyses provided evidence for a single transport system with a Kt of 4.1 +/- 0.4 mM for lactate and a Vmax of 54.2 +/- 9.9 nmol/mg of protein/30 s. Pyruvate inhibited lactate transport competitively, by reducing the affinity of the system for lactate without altering the maximal velocity. It is concluded that human placental brush-border membranes possess a transport system specific for lactate and other monocarboxylates and that this transport system is Na+-independent and is energized by an inwardly directed H+ gradient. Lactate-H+ symport rather than lactate-OH- antiport appears to be the mechanism of the H+ gradient-dependent lactate transport in these membranes.     

pH dependence of the Coxiella burnetii glutamate transport system.

The transport of glutamate, apparently a primary energy source for Coxiella burnetii, has been examined. C. burnetii is shown to possess a pH-dependent active transport system for L-glutamate with an apparent Kt of 61.1 microM and Vmax of 8.33 pmol/s per mg at pH 3.5. Both L-glutamine and L-asparagine competitively inhibited transport of glutamate, but D-glutamate, L-aspartate, L-glutamate-gamma-methyl ester, methionine sulfoximine, or alpha-ketoglutarate did not compete. This transport system is both temperature and energy dependent. Uptake of glutamate is highly sensitive to uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol and carbonyl cyanide-m-chlorophenyl hydrazone that decrease the proton motive force across the cytoplasmic membrane. ATPase inhibitors such as dicyclohexylcarbodiimide or metabolic poisons such as KCN, NaF, or arsenite were much less effective as inhibitors of glutamate transport. Uptake of glutamate did not appear to be coupled to Na+ symport as in Escherichia coli since no monovalent cation requirement could be demonstrated. Instead, the Vmax of glutamate transport showed good correlation with the transmembrane pH gradient (delta pH). From these results, we propose that L-glutamate transport by C. burnetii is energized via a proton motive force.