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.     

Proton motive force generation by citrolactic fermentation in Leuconostoc mesenteroides.

In Leuconostoc mesenteroides subsp. mesenteroides 19D, citrate is transported by a secondary citrate carrier (CitP). Previous studies of the kinetics and mechanism of CitP performed in membrane vesicles of L. mesenteroides showed that CitP catalyzes divalent citrate HCit2-/H+ symport, indicative of metabolic energy generation by citrate metabolism via a secondary mechanism (C. Marty-Teysset, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Biol. Chem. 270:25370-25376, 1995). This study also revealed an efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate cometabolism, suggesting that under physiological conditions, CitP may function as a precursor/product exchanger rather than a symporter. In this paper, the energetic consequences of citrate metabolism were investigated in resting cells of L. mesenteroides. The generation of metabolic energy in the form of a pH gradient (delta pH) and a membrane potential (delta psi) by citrate metabolism was found to be largely dependent on cometabolism with glucose. Furthermore, in the presence of glucose, the rates of citrate utilization and of pyruvate and lactate production were strongly increased, indicating an enhancement of citrate metabolism by glucose metabolism. The rate of citrate metabolism under these conditions was slowed down by the presence of a membrane potential across the cytoplasmic membrane. The production of D-lactate inside the cell during cometabolism was shown to be responsible for the enhancement of the electrogenic uptake of citrate. Cells loaded with D-lactate generated a delta psi upon dilution in buffer containing citrate, and cells incubated with citrate built up a pH gradient upon addition of D-lactate. The results are consistent with an electrogenic citrate/D-lactate exchange generating in vivo metabolic energy in the form of a proton electrochemical gradient across the membrane. The generation of metabolic energy from citrate metabolism in L. mesenteroides may contribute significantly to the growth advantage observed during cometabolism of citrate and glucose.     

Interaction of benzene with bilayers. Thermal and structural studies

The thermal and structural properties of saturated phosphatidylcholine liposomes are significantly altered by benzene. Upon the addition of benzene, the liposomes first swell and then disperse into small multilamellar vesicles. At 20 degrees C these vesicles contain striations or ripples in the plane of the bilayer. Major changes in the thermal behavior of DSPC-benzene liposomes occur near mole ratios of 2:1 and 1:1. At a 2:1 mole ratio, the area under the main endothermic peak, delta Hm, essentially disappears; however, the total heat absorbed, delta Hf, remains approximately equal to that of the control. This occurs because for benzene mole fractions 0.12 less than x less than 0.50, benzene increases the apparent molar heat capacity, Cp, of the gel phase to about 1.2 kcal/(mol . deg). We interpret this increase in heat capacity to be due to an increase in the concentration of defects (or disorder) in the gel phase. At mole fractions of benzene between 0.5 and 0.9, the transition temperature decreases by 20-30 degrees C, and broad, multiple transitions are observed. From 0.5 less than or equal to x less than or equal to 0.9, the apparent molar heat capacity of the liquid-crystal phase increases to that of the defected rippled gel phase. The value of delta Hf approaches the heat of fusion for 2 mol of n-octadecane, suggesting that benzene uncouples the liquid-crystalline acyl chains. The lipids affected by benzene or "boundary lipids" have higher heat capacity than nonperturbed lipids. The apparent molar specific heat, Cp, of 1,2-distearoyl-sn-glycero-3-phosphorylcholine (and 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine) multilamellar vesicles is 0.20 +/- 0.05 kcal/(mol. deg) in the L beta', P beta, and L alpha phases. Cp fluctuates about this value in all three phases upon repeated phase transitions in the same sample. However, the value of Cp in the P beta (rippled) phase exhibits much greater fluctuations in Cp than that in the L alpha phase. We attribute these fluctuations to crystal packing defects.     

The electrochemical proton gradient in Escherichia coli membrane vesicles.

Membrane vesicles isolated from Escherichia coli grown under various conditions generate a transmembrane pH gradient (delta pH) of about 2 pH units (interior alkaline) under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure delta pH and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential (delta psi) across the membrane, the vesicles are demonstrated to develop an electrochemical proton gradient (delta-muH+) of almost - 200 mV (interior negative and alkaline) at pH 5.5 in the presence of reduced phenazine methosulfate or D-lactate, the major component of which is a deltapH of about - 120 mV. As external pH is increased, deltapH decreases, reaching 0 at about pH 7.5 and above, while delta psi remains at about - 75 mV and internal pH remains at pH 7.5-7.8. The variations in deltapH correlate with changes in the oxidation of reduced phenazine methosulfate or D-lactate, both of which vary with external pH in a manner similar to that described for deltapH. Finally, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin with little change in delta-muH+ and no change in respiratory activity. These data and those presented in the following paper (Ramos and Kaback 1976) provide strong support for the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.     

Thermodynamics of stacking-interactions in polyA and polyC

Thermodynamics of stacking-contacts breakdown in single chains of polyA and polyC was studied by scanning microcalorimetry. Temperature relationships of partial heat capacities of polymers within 5 divided by 130 degrees C were measured for the first time and contributions to heat capacity of corresponding nucleosidemonophosphates were taken into account. As a result of the data analysis on the basis of Ising linear model entalpies and entropies of stacking-interactions were determined, as well as the values of the cooperativity factor, which in the scale of mole contacts equaled: delta Hm = 14.5 +/- 8 kJ/mole, delta Sm = 46.3 +/- 3 J/K.mole, delta Cp,m = 110 J/K.mole, delta = 0.18 and delta Hm = 13.8 +/- 0.8 kJ/mole, delta Sm = 41.6 +/- 3 J/K.mole, delta Cp,m = 80 J/K.mole, sigma = 0.13 for polyA and polyC respectively in solution with 0.12 M NaCl and pH 7.0.     

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.     

Enthalpy changes in the formation of the proton electrochemical potential and its components

Enthalpy changes in the formation of a proton electrochemical potential (Delta mu H+) and its components, DeltapH (proton gradient) and Deltapsi (electrical potential), across two types of E. coli membrane vesicles were investigated. Flow dialysis experiments showed that in 0.1 M KPi, pH 6.6, E. coli GR19N membrane vesicles coupled with d-lactate exhibited 57 mV for DeltapH, 70 mV for Deltapsi, and 127 mV for Delta mu H+. Microcalorimetric measurements revealed that the corresponding enthalpy changes (DeltaH(pH), DeltaH(psi) and DeltaHm) were 3.5, 3.3 and 6.9 kcal/mole, respectively. Moreover, in E. coli ML 308-225 membrane vesicles across which 120mV of Delta mu H+ was generated, values of DeltaH(pH) and DeltaH(psi) were determined as 7.0 and 6.6 kcal/mole, as compared with the previously reported 14.1 kcal/mole for DeltaH(m). Comparisons of these enthalpy data revealed that component enthalpies (DeltaH(pH) and DeltaH(psi)) essentially added up to the total enthalpy (DeltaHm), providing a self-consistent test for the obtained data. In both membranes, the ratio ofDeltaH(psi) to Deltapsi was comparable to that of DeltaH(pH) to DeltapH in the formation of Delta mu H+. These observations indicated that the process of the movement of H+ across the membranes was the major contributor to the observed energetic changes. Moreover, the enthalpy change in the formation of Delta mu H+ was compared with the membranes derived from GR19N and ML 308-225 and coupled with NADH and d-lactate. The results were discussed in terms of trans-membrane phenomena.     

Temperature dependence of enthalpy changes for ethidium and propidium binding to DNA: effect of alkylamine chains

Calorimetric titrations have been performed on the binding of ethidium and propidium to calf thymus DNA at temperatures in the 15-60 degrees C range. Enthalpy changes (delta HB) derived from these experiments performed with the new Omega reaction calorimeter have a precision of +/- 0.10 kcal/mol or less at all temperatures. For ethidium (a monocation), delta HB varies little with temperature, and the heat capacity change (delta CP) for the binding reaction derived from these parameters is 10 cal/deg/mol. In contrast, delta HB changes from -6.5 to -8.1 kcal/mol for DNA binding of propidium (a dication due to a charged amine group at the end of an alkyl chain attached to the phenanthridine ring nitrogen), and delta CP is -57 cal/deg/mol. At 21 degrees C a plot of delta HB vs mole ratio is curved downward for propidium in the 0.08-0.25 range, whereas the same plot at 45 degrees C is a straight line from 0.05 to 0.15 and sharply downward thereafter. Similar plots for ethidium follow the latter pattern between 25 and 50 degrees C. These observations and our analyses of delta HB and delta SB are consistent with the hypothesis that the location in the DNA complex and the rotational motion of the alkylamine chain change substantially over the temperature range in this study. Only near 50 degrees C is delta HB equal for the binding of these two cations to DNA, and caution must be used in analyses of enthalpic effects when the temperature dependence for delta HB is not available.     

The relationship between the electrochemical proton gradient and active transport in Escherichia coli membrane vesicles.

In the previous paper [ramos, S., and Kaback, H.R. (1977), Biochemistry 16 (preceding paper in this issue)], it was demonstrated that Escherichia coli membrane vesicles generate a large electrochemical proton gradient (delta-muH+) under appropriate conditions, and some of the properties of delta-muH+ and its component forces [i.e., the membrane potential (delta psi) and the chemical gradient of protons (deltapH)] were described. In this paper, the relationship between delta-muH+, delta psi, and deltapH and the active transport of specific solutes is examined. Addition of lactose or glucose 6-phosphate to membrane vesicles containing the appropriate transport systems results in partial collapse of deltapH, providing direct evidence for the suggestion that respiratory energy can drive active transport via the pH gradient across the membrane. Titration studies with valinomycin and nigericin lead to the conclusion that, at pH 5.5, there are two general classes of transport systems: those that are driven primarily by delta-muH+ (lactose, proline, serine, glycine, tyrosine, glutamate, leucine, lysine, cysteine, and succinate) and those that are driven primarily by deltapH (glucose 6-phosphate, D-lactate, glucuronate, and gluconate). Importantly, however, it is also demonstrated that at pH 7.5, all of these transport systems are driven by delta psi which comprises the only component of delta-muH+ at this external pH. In addition, the effect of external pH on the steady-state levels of accumulation of different solutes is examined, and it is shown that none of the pH profiles correspond to those observed for delta-muH+, delta psi, or deltapH. Moreover, at external pH values above 6.0-6.5, delta-muH+ is insufficient to account for the concentration gradients established for each substrate unless the stoichiometry between protons and accumulated solutes is greater than unity. The results confirm many facets of the chemiosmotic hypothesis, but they also extend the concept in certain important respects and allow explanations for some earlier observations which seemed to preclude the involvement of chemiosmotic phenomena in active transport.     

Simultaneous reconstitution of Escherichia coli membrane vesicles with D-lactate and D-amino acid dehydrogenases

Purified preparations of D-amino acid dehydrogenase [Olsiewski, P.J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 225, 4487] and D-lactate dehydrogenase [Kohn, L.D., & Kaback, H.R. (1973) J. Biol. Chem. 248, 7012] bind independently to right-side-out and inverted Escherichia coli vesicles and to phosphatidylcholine liposomes without detectable competition. The reconstituted vesicles catalyze D-lactate- and D-alanine-dependent respiration (O2 uptake), proton translocation, and proton/lactose symport. The enzymes do not share common sites of association on either face of the E. coli membrane, and binding of both enzymes to the bilayer appears to be due to nonspecific affinity for the surface rather than specific binding to proteinaceous receptors. Each enzyme, however, appears to reduce a common proton translocating step in the membrane-bound respiratory chain, and substrate-derived electrons are transferred through a common rate-determining redox component that precedes the site of proton translocation. The results suggest that although binding is nonspecific, there is a common site for proton translocation in the membrane between the flavin-linked dehydrogenases and the cytochromes and that this site is accessible by distinct routes of electron transfer from primary dehydrogenases on either surface of the membrane.     

Mechanism of proline transport in Escherichia coli K12. I. Effect of a membrane potential on the kinetics of 2H+/proline symport in cytoplasmic membrane vesicles

The stoichiometric coupling mechanism of the membrane potential (delta psi) in the reaction of H+/proline symport was investigated kinetically, using cytoplasmic membrane vesicles of the proline carrier-overproducing strain of Escherichia coli MinS/ pLC4 -45. When a delta psi was imposed across the cytoplasmic membrane by respiration, the Michaelis constant of transport (Kt) was lowered to about 1 microM, which was 2 orders of magnitude smaller than that of passive influx and efflux, and the maximum velocity (Vmax) was concomitantly enhanced as an exponential function of delta psi. Thermodynamically, the carrier translocated proline with a stoichiometry of 2 mol of protons versus 1 mol of substrate when driven by a delta psi at pH 8.0. Data on the delta psi dependence of Vmax of proline transport could be explained quantitatively by the Geck-Heinz hypothesis (Geck, P., and Heinz, E. (1976) Biochim, Biophys. Acta 443, 49-63). A symmetrical model of the 2H+/proline symport via formation of a carrier/H+/substrate (CH+H+S) intermediate is proposed. In this model, the effect of delta psi on the Kt was resolved as stimulation of formation of a transport intermediate, whereas the effect of delta psi on the Vmax was explained by enhancement of translocation of loaded carriers between the two sides of the membrane.     

Purification of the lactose:H+ carrier of Escherichia coli and characterization of galactoside binding and transport

The lactose carrier, a galactoside:H+ symporter in Escherichia coli, has been purified from cytoplasmic membranes by pre-extraction of the membranes with 5-sulfosalicylate, solubilization in dodecyl-O-beta-D-maltoside, Ecteola-column chromatography, and removal of residual impurities by anti-impurity antibodies. Subsequently, the purified carrier was reincorporated into E. coli phospholipid vesicles. Purification was monitored by tracer N-[3H]ethylmaleimide-labeled carrier and by binding of the substrate p-nitrophenyl-alpha-D-galactopyranoside. All purified carrier molecules were active in substrate binding and the purified protein was at least 95% pure by several criteria. Substrate binding to the purified carrier in detergent micelles and in reconstituted proteoliposomes yielded a stoichiometry close to one molecule substrate bound per polypeptide chain. Large unilamellar proteoliposomes (1-5-micron diameter) were prepared from initially small reconstituted vesicles by freeze-thaw cycles and low-speed centrifugation. These proteoliposomes catalyzed facilitated diffusion and active transport in response to artificially imposed electrochemical proton gradients (delta mu H+) or one of its components (delta psi or delta pH). Comparison of the steady-state level of galactoside accumulation and the nominal value of the driving gradients yielded cotransport stoichiometries up to 0.7 proton/galactoside, suggesting that the carrier protein is the only component required for active galactoside transport. The half-saturation constants for active uptake of lactose (KT = 200 microM) or beta-D-galactosyl-1-thio-beta-D-galactoside (KT = 50-80 microM) by the purified carrier were found to be similar to be similar to those measured in cells or cytoplasmic membrane vesicles. The maximum rate for active transport expressed as a turnover number was similar in proteoliposomes and cytoplasmic membrane vesicles (kcat = 3-4 s-1 for lactose) but considerably smaller than in cells (kcat = 40-60 s-1). Possible reasons for this discrepancy are discussed.     

Direct measurement of lactose/proton symport in Escherichia coli membrane vesicles: further evidence for the involvement of histidine residue(s)

Addition of lactose to Escherichia coli ML 308-225 membrane vesicles under nonenergized conditions induces transient alkalinization of the medium, and the initial rate of proton influx is stimulated by valinomycin and abolished by nigericin or carbonyl cyanide m-chlorophenylhydrazone. A functional lac y gene product is absolutely required as the effect is not observed in ML 308-225 vesicles treated with N-ethylmaleimide nor with vesicles from uninduced Escherichia coli ML 30. Furthermore, the magnitude of the phenomenon is enhanced about 3-fold in vesicles from Escherichia coli T206, which contain amplified levels of the lac carrier protein. Kinetic parameters for lactose-induced proton influx are the same as those determined for lactose-facilitated diffusion, and quantitative comparison of the initial rates of the two fluxes indicates that the stoichiometry between protons and lactose is 1:1. Treatment of ML 308-225 vesicles with diethyl pyrocarbonate causes inactivation of lactose-induced proton influx. Remarkably, however, treatment with the histidine reagent enhances the rate of lactose-facilitated diffusion in a manner suggesting that the altered lac carrier catalyzes lactose influx without the symport of protons. The results are consistent with the hypothesis that acylation of a histidyl residue(s) in the lac carrier protein dissociates lactose influx from proton influx and indicate that this residue(s) play(s) an important role in the pathway of proton translocation.     

Thermodynamics of glucagon aggregation.

Heats of dilution of concentrated glucagon solutions have been measured calorimetrically at 10 and 25 degrees C in 0.2 M potassium phosphate buffer of pH 10.6. Analysis of the data in terms of a monomer-trimer equilibrium gives the following thermodynamic parameters for the association reaction at 25 degrees C: delta G degrees = 7.34 kcal/mol of trimer, delta H degrees = -31.2 kcal/mol, deltaS degrees = -80 cal/(K mol), deltaCp = 430 cal/(K mol). The sensitivity of heat of dilution data to the association constant and stoichiometry of the reaction is discussed.     

Oxidative phosphorylation in right-side-out membrane vesicles from Escherichia coli.

Oxidative phosphorylation in Escherichia coli membrane vesicles with a right-side-out orientation and loaded with ADP was investigated. Substrates of the electron transport chain could energize the phosphorylation of ADP, with the order of effectiveness being D-lactate greater than reduced phenazinemethosulfate greater than succinate greater than reduced nicotinamide adenine dinucleotide. Inhibitors of D-lactate oxidation, proton conductors, and inhibitor of the Mg2+ATPase (EC 3.6.1.3) all inhibited oxidative phosphorylation when coupled to D-lactate oxidation. ATP synthesis was absent in membrane vesicles prepared from a mutant strain lacking the Mg2+ATPase. Valinomycin or nigericin partially inhibited oxidative phosphorylation in the presence of potassium. Valinomycin plus nigericin completely inhibited ATP synthesis. The effect of various agents on the respiration-dependent establishment of a transmembrane pH gradient was also examined. NaCN and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the establishment of a pH gradient while dicyclohexylcarbodiimide had no effect. These results are in good agreement with a chemiosmotic model for oxidative phosphorylation.     

Conformational stability and mechanism of folding of ribonuclease T1

Urea and thermal unfolding curves for ribonuclease T1 (RNase T1) were determined by measuring several different physical properties. In all cases, steep, single-step unfolding curves were observed. When these results were analyzed by assuming a two-state folding mechanism, the plots of fraction unfolded protein versus denaturant were coincident. The dependence of the free energy of unfolding, delta G (in kcal/mol), on urea concentration is given by delta G = 5.6 - 1.21 (urea). The parameters characterizing the thermodynamics of unfolding are: midpoint of the thermal unfolding curve, Tm = 48.1 degrees C, enthalpy change at Tm, delta Hm = 97 kcal/mol, and heat capacity change, delta Cp = 1650 cal/mol deg. A single kinetic phase was observed for both the folding and unfolding of RNase T1 in the transition and post-transition regions. However, two slow kinetic phases were observed during folding in the pre-transition region. These two slow phases account for about 90% of the observed amplitude, indicating that a faster kinetic phase is also present. The slow phases probably result from cis-trans isomerization at the 2 proline residues that have a cis configuration in folded RNase T1. These results suggest that RNase T1 folds by a highly cooperative mechanism with no structural intermediates once the proline residues have assumed their correct isomeric configuration. At 25 degrees C, the folded conformation is more stable than the unfolded conformations by 5.6 kcal/mol at pH 7 and by 8.9 kcal/mol at pH 5, which is the pH of maximum stability. At pH 7, the thermodynamic data indicate that the maximum conformational stability of 8.3 kcal/mol will occur at -6 degrees C.     

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.     

pH-dependent changes in proton:substrate stoichiometries during active transport in Escherichia coli membrane vesicles.

Experiments are presented in which the proton electrochemical gradient (deltamuH+) IN Escherichia coli membrane vesicles (interior negative and alkaline) was measured under a variety of conditions and compared with steady-state levels of accumulation of lactose, proline, D-lactate, and glucose-6-P measured under identical conditions. Accumulation of lactose and proline is proportional to the magnitude of deltamuH+ at pH 5.5, where the pH gradient (deltapH) and the electrical potential (deltapsi) both contribute to deltamuH+, and at pH 7.5, where deltapsi represents the only component of deltamuH+. Moreover, the proportionality constants between deltamuH+ and lactose or proline accumulation indicate that the proton:substrate stoichiometries are 1:1 at pH 5.5 and 2:1 at pH 7.5. Evidence is also presented which indicates that the functional group responsible for the increase in proton:proline stoichiometry has a pK of approximately 6.8. Accumulation of D-lactate and glucose-6-P is directly related to the magnitude of deltapH at pH 5.5, and stoichiometry values of one and approximately 1.7 are obtained for D-lactate and glucose-6-P, respectively, at this pH. At pH 7.5, on the other hand, accumulation of each organic acid bears a linear relationship to deltapsi, and proton:substrate stoichiometries of unity are observed in both instances. The results are consistent with the models discussed by Rottenberg (Rottenberg, H. (1976), FEBS Lett. 66, 159).     

Lactose transport system of Streptococcus thermophilus. Functional reconstitution of the protein and characterization of the kinetic mechanism of transport.

The kinetic mechanism of the lactose transport system of Streptococcus thermophilus was studied in membrane vesicles fused with cytochrome c oxidase containing liposomes and in proteoliposomes in which cytochrome c oxidase was coreconstituted with the lactose transport protein. Selective manipulation of the components of the proton (and sodium) motive force indicated that both a membrane potential and a pH gradient could drive transport. The galactoside/proton stoichiometry was close to unity. Experiments which discriminate between the effects of internal pH and delta pH as driving force on galactoside/proton symport showed that the carrier is highly activated at alkaline internal pH values, which biases the transport system kinetically toward the pH component of the proton motive force. Galactoside efflux increased with increasing pH with a pKa of about 8, whereas galactoside exchange (and counterflow) exhibited a pH optimum around 7 with pKa values of 6 and 8, respectively. Imposition of delta pH (interior alkaline) retarded the rate of efflux at any pH value tested, whereas the rate of exchange was stimulated by an imposed delta pH at pH 5.8, not affected at pH 7.0, and inhibited at pH 8.0 and 9.0. The results have been evaluated in terms of random and ordered association/dissociation of galactoside and proton on the inner surface of the membrane. Imposition of delta psi (interior negative) decreased the rate of efflux but had no effect on the rate of exchange, indicating that the unloaded transport protein carries a net negative charge and that during exchange and counterflow the carrier recycles in the protonated form.     

Quantitative analysis of proton-linked transport systems. The lactose permease of Escherichia coli.

Evidence is presented that lactose uptake into whole cells of Escherichia coli occurs by symport with a single proton over the range of external pH 6.5--7.7. The proton/lactose stoicheiometry has been measured directly over this pH range by comparison of the initial rates of proton and lactose uptake into anaerobic resting cell suspensions of E. coli ML308. Further, the relationship between the protonmotive force and lactose accumulation has been studied in E. coli ML308-225 over the range of external pH 5.9--8.7. At no point was the accumulation of the beta-galactoside in thermodynamic equilibrium with the protonmotive force. It is concluded that the concentration of lactose within the cell is governed by kinetic factors rather than pH-dependent changes in the proton/substrate stoicheiometry. The relevance of these findings to the model of pH-dependent proton/substrate stoicheiometries derived from studies with E. coli membrane vesicles is discussed.