Intracellular pH of Thermoplasma acidophila

Thermoplasma acidophila, a mycoplasma-like organism, was grown at 56 °C and pH 2. The intracellular pH of this organism is close to neutral as measured by the distribution of a radioactive weak organic acid, 5,5-dimethyl-2,4-oxazolidinedione, across the plasma membrane. The cell can maintain the pH gradient when subjected to heat or to metabolic inhibitors. Our experiments seem to indicate that a major portion of the pH gradient is not maintained by active processes, but rather by a Donnan potential across the cell membrane.     

Trehalase activation in yeasts is mediated by an internal acidification

It has been reported that the addition of glucose, uncouplers and nystatin to yeast cells grown in a sugarfree medium causes trehalase activation; it has been postulated that this activation might be mediated by the depolarization of the plasma membrane. In this article the values of membrane potential and pH gradient across the plasma membrane of Saccharomyces cerevisiae have been determined under the same conditions as those in which trehalase is activated. Membrane potential was evaluated from the distribution of triphenylmethylphosphonium, the pH gradient from the distribution of benzoic acid across the plasma membrane. When the effect of several agents on the two components of the electrochemical proton gradient across the plasma membrane of ethanol-grown yeast cells were studied, under trehalase activation conditions, the following observations were made. (a) The addition of glucose activated trehalase and caused internal acidification of the cells, but had practically no effect on the membrane potential. (b) The addition of 200 mM KCl depolarized the cell membrane but did not affect the internal pH, nor trehalase activity. (c) Although carbonyl cyanide m-chlorophenylhydrazone depolarized the cells at external pH 6.0 and 7.0, it only activated trehalase at an external pH 6.0, leading to the acidification of the internal medium at this pH. (d) Nystatin caused an increase in the triphenylmethylphosphonium accumulation at external pH 6.0 and 7.0, but only activated trehalase at external pH 6.0, causing acidification of the cell interior at this pH. (e) Activation of trehalase was also observed when the internal acidification was caused by addition of a weak acid such as acetate. It is concluded that trehalase activation is mediated by an intracellular acidification and is independent of the membrane potential.     

Anaerobic transport of serine and 2-aminoisobutyric acid by Staphylococcus epidermidis.

A membrane-bound ATPase detected in extracts of anaerobically grown Staphylococcus epidermidis was inhibited by a variety of compounds which inhibit ATPases in other organisms. Serine and 2-aminoisobutyric acid (AIB) were shown to enter the organism via the same transport system. The transport of AIB, the membrane potential and the transmembrane pH gradient were partially or completely abolished by the same inhibitors and also by uncoupling agents and lipid-soluble ions. It is proposed therefore that this ATPase generates and maintains an electrochemical gradient of protons across the cytoplasmic membrane of S. epidermidis capable of driving AIB uptake. Studies of AIB-induced proton movements suggested that AIB enters via a proton symport mechanism.     

31P and 13C NMR analyses of the energy metabolism of the thermophilic anaerobe Clostridium thermocellum

The energy metabolism of an anaerobic obligate thermophile, Clostridium thermocellum, has been examined as a function of incubation temperature using 31P NMR spectroscopy. Specifically investigated were the generation and availability of ATP as a function of temperature, activation energies for key processes in energy metabolism including formation of a pH gradient across the cell membrane, transport of key nutrients, and initial steps in glycolysis, and the existence of a membrane phase transition in the intact organism. Cells generate ATP via glycolysis at all temperatures examined; hence, limitation of the energy supply is not directly responsible for the lack of growth of this organism at low temperatures. Estimations of activation energies show a distinct hierarchy in the ATP-utilizing reactions examined. Conservation of ATP hydrolysis energy as delta pH has the lowest activation energy (less than or equal to 4 kcal/mol), two transport processes exhibit 10 kcal/mol activation energies, and early phosphorylation steps in glycolysis have significantly higher activation energies (approximately 25 kcal/mol). Neither the membrane-bound ATPase responsible for formation of the pH gradient nor the permease involved in phosphate transport shows evidence of a change in behavior around the phase transition temperature determined for extracted lipids of C. thermocellum. Line widths of inorganic phosphate do show a break in behavior around 35-40 degrees C. Possible explanations for this behavior are discussed.     

The effect of hormones on proton compartmentation in hepatocytes

Liver mitochondria isolated from rats treated acutely with glucagon exhibit higher respiration-dependent H+ ion gradients across the mitochondrial inner membrane than mitochondria from control rats. It has been suggested that similar increases in mitochondrial delta pH in situ could stimulate gluconeogenesis, chiefly because the transport of pyruvate into mitochondria would increase in response to the increase in mitochondrial matrix pH. In order to determine whether the increased delta pH observed in vitro in isolated mitochondria also occurs in situ, the effect of glucagon on the pH in the cytosol and mitochondria matrix spaces of isolated hepatocytes was determined. For qualitative results, the spectral responses of intracellularly trapped 6-carboxyfluorescein was used to monitor cytosol pH, while fluorescein-loaded hepatocytes were used to monitor the mitochondrial pH. Hepatocytes were incubated with the diacetate ester derivatives of these dyes. The esters are permeable to the cell membranes, but are rapidly hydrolyzed in the cells. The free unesterified dyes are relatively impermeable to the cell membranes. After being trapped in the cell, 6-carboxyfluorescein remains localized in the cell cytosol, whereas fluorescein is taken up by the mitochondria as a function of the mitochondrial delta pH. In order to quantitate the actual pH in these compartments, the spectral responses (490-465 nm) of 6-carboxyfluorescein-loaded hepatocytes were used to determine the cytosolic pH. Calibration of these responses was obtained within the cell by determination of the dye's differential absorption coefficient (epsilon 490-465 nm) in various high K+ buffers after equilibration of the internal and external pH with valinomycin and the uncoupler 1799. All absorbance values were corrected for dye leakage. Equal hematocrits of unloaded cells were used to correct for absorbance contributions from cellular constituents. The mitochondrial pH was determined by a combination of the indicator dye and [14C]5,5'-demethyloxazolidine-2, 4-dione (DMO) distribution ratio methods. The weak acid DMO freely distributes across the plasma membrane and mitochondrial membrane in whole cells according to the pH gradient across each membrane. Knowledge of the cytoplasmic pH from the 6-carboxyfluorescein data allows the expected distribution of DMO across the plasma membrane to be calculated. The excess accumulation of DMO in intact hepatocytes over that predicted from the plasma membrane pH gradient alone was then used to calculate the pH gradient across the mitochondrial inner membrane. The effects of valinomycin, uncouplers, and hormones on the pH in cytosolic and mitochondrial compartm     

Role of scalar protons in metabolic energy generation in lactic acid bacteria

Lactic acid bacteria are able to generate a protonmotive force across the cytoplasmic membrane by various metabolic conversions without involvement of substrate level phosphorylation or proton pump activity. Weak acids like malate and citrate are taken up in an electrogenic process in which net negative charge is translocated into the cell thereby generating a membrane potential. The uptake is either an exchange process with a metabolic end-product (precursor/ product exchange) or a uniporter mechanism. Subsequent metabolism of the internalized substrate drives uptake and results in the generation of a pH gradient due to the consumption of scalar protons. The generation of the membrane potential and the pH gradient involve separate steps in the pathway. Here it is shown that they are nevertheless coupled. Analysis of the pH gradient that is formed during malolactic fermentation and citrate fermentation shows that a pH gradient, inside alkaline, is formed only when the uptake system forms a membrane potential, inside negative. These secondary metabolic energy generating systems form a pmf that consists of both a membrane potential and a pH gradient, just like primary proton pumps do. It is concluded that the generation of a pH gradient, inside alkaline, upon the addition of a weak acid to cells is diagnostic for an electrogenic uptake mechanism translocating negative charge with the weak acid.     

Lactic acid translocation: terminal step in glycolysis by Streptococcus faecalis

Streptococcus faecalis obtains metabolic energy chiefly from the conversion of glucose to lactic acid; the present experiments deal with the mechanism of lactic acid translocation across the cytoplasmic membrane. Efflux of [(14)C]lactate from preloaded cells was accelerated by raising the external pH, and also by the ionophores nigericin and valinomycin. These results suggest that lactate leaves the cell by an electroneutral process, presumably as lactic acid. Further evidence was obtained by studying the entry of [(14)C]lactate into nonmetabolizing cells. It appears that the membrane is essentially impermeable to the lactate anion, but allows passage of lactic acid. The most persuasive evidence is that, upon establishment of a pH gradient such that the cytoplasm was alkaline, l-[(14)C]lactate accumulated in the cells against the concentration gradient. Accumulation was transient, and dissipated in parallel with the collapse of the pH gradient. The concentration gradient attained at the peak was a function of the pH difference. Ionophores which are known to collapse a pH gradient, such as nigericin and valinomycin, abolished accumulation of l-lactate. We infer that lactic acid translocation, whether into the cells or outward, is an electroneutral process and for that reason the distribution of lactic acid across the membrane is a function of the pH of cytoplasm and medium. The specificity of translocation and its kinetic parameters suggest that it is mediated by a carrier of low specificity.     

Membrane-related processes and overall energy metabolism inTrypanosoma brucei and other kinetoplastid species

An electrochemical proton gradient exists across the plasma membrane and the mitochondrial membrane of the bloodstream form ofTrypanosoma brucei. The membrane potential across the plasma membrane and the regulation of the internal pH depend on the temperature.Leishmania donovani regulates its internal pH and maintains a constant electrochemical proton gradient across its plasma membrane under all conditions examined. The mitochondrion of theT. brucei bloodstream form is energized, even though the reactions taking place in it do not result in net ATP synthesis and the Kreb's cycle and the respiratory chain are absent. Glucose is transported across the plasma membrane ofT. brucei by a facilitated diffusion carrier, that can transport a wider range of substrates than its mammalian counterparts. Pyruvate exits the cell via a facilitated diffusion transporter as well. Conflicting evidence exists for the mechanism of glucose transport inL. donovani; biochemical evidence suggests proton/glucose symport, while facilitated diffusion is indicated by physiological data.     

Red cell pH of lamprey (Lampetra fluviatilis) is actively regulated

Summary The red cell pH of lamprey (Lampetra fluviatilis) was measured using the DMO method (based on the passive distribution of the wead acid, DMO, across the red cell membrane). The measured red cell pH was higher than the pH of the incubation medium throughout the pH range (7.2–8.2) studied, and higher than the red cell pH calculated from the chloride distribution ratio. Treatment of cells with the metabolic inhibitors 2,4-dinitrophenol or KCN caused a drop in the red cell pH to values lower than the pH of incubation medium, and abolished the difference between the measured red cell pH and the pH calculated from the chloride distribution ratio. These data strongly suggest that the proton gradient across lamprey red cell membrane is actively maintained. Acid extrusion from lamprey red cells may require sodium, as indicated by the observation that when choline was substituted for sodium in the incubation medium, the intracellular pH decreased significantly.     

A variable stoichiometry model for pH homeostasis in bacteria.

The composition of the proton-motive force of a hypothetical bacterial cell of wide pH tolerance is analyzed according to a model whereby the electron transport chain and various proton-linked sodium and potassium ion transporting modes are responsible for the development of the membrane potential and the chemical potentials of the three cations. Simultaneous use of two or more modes employing the same metal cation, but at a different stoichiometric ratio with respect to protons, produces nonintegral stoichiometry; the modes could represent either different devices or different states of a single device. Cycling of the cation, driven by proton-motive force, results. The relative conductances of the various modes are postulated to be pH-dependent. The pattern of potentials that results is qualitatively in accord with current knowledge and may reflect the mechanism of pH homeostasis in bacteria. The membrane potential is outwardly directed (positive inside) at extremely acid pH, becoming inwardly directed as the pH increases; the pH gradient across the membrane is large and inwardly directed (alkaline inside) at acid pH, becoming smaller and eventually inverting at alkaline pH values; the transmembrane potassium gradient is outwardly directed (high concentration inside) at all pH values; the transmembrane sodium gradient is inwardly directed at all pH values, following the pH gradient from acid through neutral pH, but then diverging at alkaline pH.     

Light-driven proton translocations in Halobacterium halobium.

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating electrochemical proton gradient across the cell membrane. However, the type response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium. The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow. The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N'-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor. Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.     

Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation.

The mitochondrial membrane potential (delta psi m) in intact lymphocytes was calculated by measuring the distribution of radiolabelled methyltriphenylphosphonium cation. The value obtained was 120 mV. The pH gradient across the mitochondrial membrane in situ (delta pH m) was estimated to be 73 mV (1.2 pH units). Thus the electrochemical gradient of protons was about 190 mV. Addition of the mitogen concanavalin A did not alter delta psi m, showing that, if movement of Ca2+ across the inner membrane of lymphocyte mitochondria occurs when concanavalin A is added, it is accompanied by charge-compensating ion movements.     

The influence of a transmembrane pH gradient on protonation probabilities of bacteriorhodopsin: the structural basis of the back-pressure effect

Bacteriorhodopsin pumps protons across a membrane using the energy of light. The proton pumping is inhibited when the transmembrane proton gradient that the protein generates becomes larger than four pH units. This phenomenon is known as the back-pressure effect. Here, we investigate the structural basis of this effect by predicting the influence of a transmembrane pH gradient on the titration behavior of bacteriorhodopsin. For this purpose we introduce a method that accounts for a pH gradient in protonation probability calculations. The method considers that in a transmembrane protein, which is exposed to two different aqueous phases, each titratable residue is accessible for protons from one side of the membrane depending on its hydrogen-bond pattern. This method is applied to several ground-state structures of bacteriorhodopsin, which residues already present complicated titration behaviors in the absence of a proton gradient. Our calculations show that a pH gradient across the membrane influences in a non-trivial manner the protonation probabilities of six titratable residues which are known to participate in the proton transfer: D85, D96, D115, E194, E204, and the Schiff base. The residues connected to one side of the membrane are influenced by the pH on the other side because of their long-range electrostatic interactions within the protein. In particular, D115 senses the pH at the cytoplasmic side of the membrane and transmits this information to D85 and the Schiff base. We propose that the strong electrostatic interactions found between D85, D115, and the Schiff base as well as the interplay of their respective protonation states under the influence of a transmembrane pH gradient are responsible for the back-pressure effect on bacteriorhodopsin.     

Diphtheria toxin at low pH depolarizes the membrane, increases the membrane conductance and induces a new type of ion channel in Vero cells.

Receptor-dependent translocation of diphtheria toxin across the surface membrane of Vero cells was studied using patch clamp techniques. Translocation was induced by exposing cells with surface-bound toxin to low pH. Whole cell current and voltage clamp recordings showed that toxin translocation was associated with membrane depolarization and increased membrane conductance. The conductance increase was voltage independent, with a reversal potential of approximately 15 mV. This value was unaffected by changing the Cl- gradient across the membrane and microfluorometric measurements showed that the cytosolic Ca2+ concentration was only marginally elevated by the translocation. The conductance increase is thus mainly due to monovalent cations. Exposing outside-out and cell-attached patches with bound toxin to low pH induced a new type of ion channel in the membrane. The channel current was inward at negative membrane potentials and the single channel conductance was approximately 30 pS. This value is about three times larger than for receptor-independent channels induced by diphtheria toxin or toxin fragments in artificial lipid membranes.     

Light-driven proton translocations in Halobacterium halobium

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating an electrochemical proton gradient across the cell membrane. However, the typical response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light is turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium.The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow.The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N′-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor.Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.     

Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes.

The interaction of the peptide antibiotic nisin with liposomes has been studied. The effect of this interaction was analyzed on the membrane potential (inside negative) and the pH gradient (inside alkaline) in liposomes made from Escherichia coli phosphatidylethanolamine and egg phosphatidylcholine (9:1, wt/wt). The membrane potential and pH gradient were generated by artificial ion gradients or by the oxidation of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and cytochrome c by the beef heart cytochrome c oxidase incorporated in the liposomal membranes. Nisin dissipated the membrane potential and the pH gradient in both types of liposomes and inhibited oxygen consumption by cytochrome c oxidase in proteoliposomes. The dissipation of the proton motive force in proteoliposomes was only to a minor extent due to a decrease of the oxidase activity by nisin. The results in these model systems show that a membrane potential and/or a pH gradient across the membrane enhances the activity of nisin. Nisin incorporates into the membrane and makes the membrane permeable for ions. As a result, both the membrane potential and pH gradient are dissipated. The activity of nisin was found to be influenced by the phospholipid composition of the liposomal membrane.     

Properties of the Peribacteroid Membrane ATPase of Pea Root Nodules and Its Effect on the Nitrogenase Activity

Peribacteroid membrane vesicles from pea (Pisum sativum) root nodules were isolated from membrane-enclosed bacteroids by an osmotic shock. The ATPase activity associated with this membrane preparation was characterized, and its electrogenic properties were determined. The pH gradient was measured as a change of the fluorescence intensity of 9-amino-6-chloro-2-methoxyacridine and the membrane potential as a shift of absorbance of bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. It was demonstrated that the ATPase generates a pH gradient as well as a membrane potential across the peribacteroid membrane. The reversibility of the ATPase was demonstrated by a light-dependent ATP synthesis by peribacteroid membrane vesicles fused with bacteriorhodopsin-phospholipid vesicles. The light-driven ATP synthesis by the peribacteroid membrane ATPase was completely inhibited by a proton-conducting ionophore. The proton-pumping activity of the peribacteroid membrane ATPase could also be demonstrated with peribacteroid membrane-enclosed bacteroids, and effects on nitrogenase activity were established. At pH values below 7.5, an active peribacteroid membrane ATPase inhibited the nitrogenase activity of peribacteroid membrane-enclosed bacteroids. At pH values above 8, at which whole cell nitrogenase activity was inhibited, the protonpumping activity of the peribacteroid membrane ATPase could partially reverse the pH inhibition. Vanadate, an inhibitor of plasma membrane and peribacteroid membrane ATPases, stimulated nodular nitrogenase activity. It will be proposed that the proton-pumping activity of the peribacteroid membrane ATPase in situ is a possible regulator of nodular nitrogenase activity.     

Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes.

The interaction of the peptide antibiotic nisin with liposomes has been studied. The effect of this interaction was analyzed on the membrane potential (inside negative) and the pH gradient (inside alkaline) in liposomes made from Escherichia coli phosphatidylethanolamine and egg phosphatidylcholine (9:1, wt/wt). The membrane potential and pH gradient were generated by artificial ion gradients or by the oxidation of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and cytochrome c by the beef heart cytochrome c oxidase incorporated in the liposomal membranes. Nisin dissipated the membrane potential and the pH gradient in both types of liposomes and inhibited oxygen consumption by cytochrome c oxidase in proteoliposomes. The dissipation of the proton motive force in proteoliposomes was only to a minor extent due to a decrease of the oxidase activity by nisin. The results in these model systems show that a membrane potential and/or a pH gradient across the membrane enhances the activity of nisin. Nisin incorporates into the membrane and makes the membrane permeable for ions. As a result, both the membrane potential and pH gradient are dissipated. The activity of nisin was found to be influenced by the phospholipid composition of the liposomal membrane.     

The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.     

Proton motive force across the membrane of Mycoplasma gallisepticum and its possible role in cell volume regulation.

A proton motive force (delta (-) microH+) of 70 to 130 mV was measured across the membrane of Mycoplasma gallisepticum cells. The membrane potential was measured utilizing the lipid-soluble cation tetraphenylphosphonium. The method was validated by showing that in the presence of valinomycin the ratio of the concentrations (in/out) of tetraphenylphosphonium agreed well with those for K+ and Rb+. The pH gradient was calculated from the measured distribution ratio of benzoic acid. The proton motive force was approximately the same in cells harvested at early exponential, midexponential, and stationary phases of growth. The proportion of pH gradient to membrane potential varied with external pH. In the absence of glucose, cells incubated in an isosmotic NaCl solution showed low adenosine triphosphate and delta (-) microH+ levels and a tendency to swell and lyse compared with cells incubated with added glucose. It is concluded that energy is required for normal cell volume regulation.