Delta pH Research Articles

Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and countermovement of protons in a SecA- and presecretory protein-dependent manner.

Everted membrane vesicles were prepared from Escherichia coli cells containing either overproduced amounts (OP-membrane vesicles) or normal amounts (normal membrane vesicles) of SecY and SecE, both of which are essential components of the protein translocation apparatus. The rates of translocation of pro-OmpA were similar in the two types of membrane vesicles, whereas translocation ATPase activity, which requires SecA, a precursor protein (pro-OmpA), and membrane vesicles, was appreciably higher with OP-membrane vesicles than with normal membrane vesicles. Since ATP hydrolysis has been shown to take place at an earlier part of the translocation reaction, these results suggest that the overproduction of SecY and SecE enhanced the activity of the earlier process, but not the entire process, of the translocation reaction. The addition of pro-OmpA in the presence of SecA caused the partial collapse of delta pH (inside acidic) generated on OP-membrane vesicles, suggesting that protons come out from the inside of the membrane vesicles in a pro-OmpA-dependent manner. The collapse of delta pH caused by pro-OmpA required SecA, ATP, and SecY and was not detected when normal membrane vesicles were used. These results indicate that the early event of protein translocation, which requires the functioning of SecA, SecY, and SecE, causes the countermovement of protons.

In vitro pore‐forming activity of the lantibiotic nisin

Nisin is a lantibiotic produced by some strains of Lactococcus lactis subsp. lactis. The target for nisin action is the cytoplasmic membrane of Gram-positive bacteria. Nisin dissipates the membrane potential (delta psi) and induces efflux of low-molecular-mass compounds. Evidence has been presented that a delta psi is needed for nisin action. The in vitro action of nisin was studied on liposomes loaded with the fluorophore carboxyfluorescein. Nisin-induced efflux of carboxyfluorescein was observed in the absence of a delta psi from liposomes composed of Escherichia coli lipids or dioleoylglycerophosphocholine (Ole2GroPCho) at low nisin/lipid ratios. The initial rate of carboxyfluorescein efflux is dependent on the nisin/lipid ratio and saturates at high ratios. Both delta psi (inside negative) and delta pH (inside alkaline) enhance the action of nisin, while nisin is more potent at acidic external pH values. Efficient carboxyfluorescein efflux is observed with the zwitterionic phospholipid Ole2GroPCho or mixtures of Ole2GroPCho with dioleoylglycerophosphoethanolamine and neutral glycolipids, while anionic phospholipids are strongly inhibitory. It is concluded that a delta psi is not essential, but that the total protonmotive force stimulates the action of nisin.

Uptake of 4-toluene sulfonate by Comamonas testosteroni T-2

The mechanism of transport of the xenobiotic 4-toluene sulfonate (TS) in Comamonas testosteroni T-2 was investigated. Rapid uptake of TS was observed only in cells grown with TS or 4-methylbenzoate as a carbon and energy source. Initial uptake rates under aerobic conditions showed substrate saturation kinetics, with an apparent affinity constant (Kt) of 88 microM and a maximal velocity (Vmax) of 26.5 nmol/min/mg of protein. Uptake of TS was inhibited completely by uncouplers and only marginally by ATPase inhibitors and the phosphate analogs arsenate and vanadate. TS uptake was also studied under anaerobic conditions, which prevented intracellular TS metabolism. TS was accumulated under anaerobic conditions in TS-grown cells upon imposition of an artificial transmembrane pH gradient (delta pH, inside alkaline). Uptake of TS was inhibited by structurally related methylated and chlorinated benzenesulfonates and benzoates. The results provide evidence that the first step in the degradation of TS by C. testosteroni T-2 is uptake by an inducible secondary proton symport system.

Interactions of bilirubin with isolated presynaptic nerve terminals: functional effects on the uptake and release of neurotransmitters.

1. The functional effects of bilirubin:albumin solutions (10:1, mol/mol) on several synaptosomal functions were investigated using rat cortical, striatal, and hippocampal synaptosomes prepared by iso-osmotic Percoll/sucrose gradient centrifugation. 2. Bilirubin (10-80 microM) depolarized synaptosomes in a tetrodotoxin-insensitive manner as assessed by the equilibrium distribution of tetra-[3H]phenylphosphonium. Depolarization induced by bilirubin was of a lesser magnitude than that caused by KCl or veratridine. Steady-state pH gradients across the synaptosomal membrane were determined using the transmembrane distribution of [14C]methylamine. Bilirubin (20-40 microM) did not modify the intracellular pH in physiological buffers. The pigment effected a 0.14 delta pH change when the synaptosomes were suspended in a Ca2+ and Na+ free choline medium containing ouabain. 3. Bilirubin (20-80 microM) had no effect of its own on [7,8-3H] dopamine release from striatal synaptosomes. In contrast, it inhibited the initial rate of synaptosomal uptake of the catecholamine and its intrasynaptosomal content at 10 min. The pigment (20 and 40 microM) reduced the 35 mM KCl-induced release of endogenous acetylcholine from hippocampal synaptosomes by 20 and 36%, respectively. 4. The association of bilirubin with synaptic plasma membrane vesicles was characterized by a chloroform:methanol 2:1 (v/v) extraction method. At total concentrations of 10 to 80 microM bilirubin, the molar percentage of the pigment in synaptic plasma membrane phospholipids was 1-4%. 5. It is proposed that the two main functional consequences of the bilirubin-nerve ending interaction are an impairment of specific membrane-bound neurotransmitter uptake mechanisms and a reduction of the response to depolarizing stimuli. This may be the basis for rapid alterations in synaptic transmission documented in early reversible bilirubin encephalopathy.

The relation of proton motive force, adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction in Bacillus licheniformis under aerobic growth conditions.

The magnitude of the proton motive force (delta p) and its constituents, the electrical (delta psi) and chemical potential (-Z delta pH), were established for chemostat cultures of a protease-producing, relaxed (rel-) variant and a not protease-producing, stringent (rel+) variant of an industrial strain of Bacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of delta p with the specific growth rate mu was found. The calculated intracellular pH (pHin) was not constant but inversely related to mu. This change in pHin might be related to regulatory functions of metabolism but a regulatory role for pHin itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with mu for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low mu and an increased value at higher mu. For both limitations, the ATP/ADP ratio was directly related to mu. The phosphorylation potential (delta G'p) was invariant with mu. From the values for delta G'p and delta p, a variable -->H+/ATP-stoichiometry was inferred: -->H+/ATP = 1.83 +/- 0.52 mu, so that at a given -->H+/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for mu = 0 to mu max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of delta G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing mu are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible mu with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.

Variability of the calcium ion activity with pH in stone-forming and non-stone-forming urine

In recurrent renal stone-formers (N = 20) and matched healthy adults (N = 20), the actual activity of ionized calcium (alpha Ca2+) and pH were determined in whole urine with an ion-selective electrode. No significant difference was found for the actual median activity of ionized calcium, however the actual median pH was significantly higher in stone-formers compared to healthy adults (pH = 5.57 vs. pH = 5.24; p < 0.005). The relationship between Ca2+ activity and pH was studied in each collected urine by titration with HCl/NaOH. In all urines the Ca2+ activity decreased with increasing pH in a typical bifasic manner. All curves showed a characteristic "breaking point" at a similar median pH in the stone-formers and in the healthy adults (pH = 6.81 vs. pH = 6.77) (NS). However the slope of the curves in the stone-formers and healthy adults changed from a median value of delta lg alpha Ca2+/delta pH of -0.139 and -0.173 (NS) respectively, to a highly significant difference of -1.326 and -1.053 (p < 0.0001) between the groups, indicating increased binding/precipitation of Ca2+ in stone-formers than in healthy adults supporting the theory of the lack of inhibitors in stone-formers. The strong relationship between the activity of ionized calcium and pH, combined with a higher actual pH and a higher decrease of ionized calcium with pH in stone-formers than in healthy adults, indicates hydrogen ion as a major factor in stone-formation. The close relationship between Ca2+ activity and pH indicates the need for simultaneous measurements of the pH in order to interpret data for the Ca2+ activity. In order to preserve a low urinary pH, where Ca2+ is predominantly in a free ionic state, our results suggest that treatment with acidifying salts could be a logical choice in order to prevent stone-formation.

Topography of cat medullary ventral surface hypoxic acidification.

The topographic relationship between previously identified medullary ventral surface respiratory chemosensitive regions and brain surface extracellular fluid (ECF) acid production during acute hypoxia was explored in anesthetized, paralyzed, and artificially ventilated cats. Glass pH electrodes (0.8-mm diam, sheathed in stainless steel tubing) were mounted in mechanical contact with surfaces of medullary surface or adjacent pyramids, pons, spinal cord, or parietal cortex. Isocapnic hypoxia of 5 min [at arterial O2 saturation (SaO2) = 48 +/- 10%] reduced pH over rostral (Mitchell) and caudal (Loeschcke) areas by 0.12 +/- 0.09 and 0.07 +/- 0.04, respectively (n = 10, P < 0.05). Change in pH (delta pH) was proportional to desaturation with slopes 100 delta pH/delta SaO2 of 0.45 (rostral) and 0.20 (caudal) (R = 0.91 and 0.88, respectively). pH drop usually began within 3 min of hypoxia, became stable between 5 and 15 min, began to rise within 2 min of reoxygenation, and returned to control within 10 min. During equally hypoxic tests, intermediate area (Schläfke), pons, and spinal cord surfaces showed no significant acid shift. Parietal cortex ECF pH dropped more slowly but steadily by 0.079 +/- 0.034 during 20 min at SaO2 = 50% after a small but significant initial alkaline shift, and acidification of cortical surface continued for > 5 min after reoxygenation. We conclude that medullary ventral chemosensitive regions produce more lactic acid during hypoxia than neighboring brain surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport of malic acid in the yeast Schizosaccharomyces pombe: evidence for a proton-dicarboxylate symport.

The transport system for malic acid present in Schizosaccharomyces pombe cells, growing in batch culture on several carbon sources, has been studied. It was found that the dicarboxylic acid carrier of S. pombe is a proton-dicarboxylate symporter that allows uphill transport and accumulation as a function of delta pH with the following kinetic parameters at pH 5.0: Vmax = 0.1 nmol of total malic acid s-1 mg (dry weight) of cells-1 and Km = 1.0 mM total malic acid. Malic acid uptake (pH 5.0) was accompanied by disappearance of extracellular protons, the uptake rates of which followed Michaelis-Menten kinetics as a function of the acid concentration. The Km values calculated as the concentrations either of anions or of undissociated acid, at various extracellular pH values, pointed to the monoanionic form as the transported species. Furthermore, accumulated free acid suffered rapid efflux after the addition of the protonophore carbonyl cyanid m-chlorophenyl hydrazone. These results suggested that the transport system was a dicarboxylate-proton symporter. Growth of cells in a medium with glucose (up to 14%, w/v) and malic acid (1.5%, w/v) also resulted in proton-dicarboxylate activity, suggesting that the system, besides being constitutive, was still active at high glucose concentrations. The following dicarboxylic acids acted as competitive inhibitors of malic acid transport at pH 5.0: D-malic acid, succinic acid, fumaric acid, oxaloacetic acid, alpha-ketoglutaric acid, maleic acid and malonic acid. In addition, all of these dicarboxylic acids induced proton movements that followed Michaelis-Menten kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Determination of transmembrane pH gradients and membrane potentials in liposomes

Techniques for determining large transbilayer pH gradients (delta pH) and membrane potentials (delta psi) induced in response to delta pH in large unilamellar vesicle liposomal systems by measuring the transbilayer redistribution of radiolabeled compounds have been examined. For liposomes with acidic interiors, it is shown that protocols using radiolabeled methylamine in conjunction with gel filtration procedures to remove untrapped methylamine provide accurate measures of delta pH in most situations. Exceptions include gel state lipid systems, where transbilayer equilibration processes are slow, and situations where the interior buffering capacity is limited. These problems can be circumvented by incubation at elevated temperatures and by using probes with higher specific activities, respectively. Determination of delta pH in vesicles with a basic interior using weak acid probes such as radiolabeled acetate in conjunction with gel filtration was found to be less reliable, and an alternative equilibrium centrifugation protocol is described. In the case of determinations of the membrane potentials induced in response to these pH gradients, probes such as tetraphenylphosphonium and thiocyanate provide relatively accurate measures of the delta psi induced. It is shown that the maximum transmembrane pH gradient that can be stably maintained by an egg phosphatidylcholine-cholesterol 100-nm-diam large unilamellar vesicle is approximately 3.7 units, corresponding to an induced delta psi of 220 mV or transbilayer electrical field of 5 x 10(5) V/cm.

Kinetics of ATP synthesis catalyzed by the H(+)-ATPase from chloroplasts (CF0F1) reconstituted into liposomes and coreconstituted with bacteriorhodopsin.

The H(+)-ATPase from chloroplasts (CF0F1) was isolated, purified and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. A transmembrane pH difference, delta pH, and a transmembrane electric potential difference, delta psi, were generated by an acid/base transition. The rate of ATP synthesis was measured at constant delta pH and constant delta psi as a function of temperature between 5 degrees C and 45 degrees C. The activation energy was 55 kJ mol-1. CF0F1 was coreconstituted with bacteriorhodopsin at a molar ratio of approximately 1:170 in the same type of liposomes. Illumination of the proteoliposomes leads to proton transport into the vesicles generating a constant delta pH = 1.8. The dependence of the rate of ATP synthesis on ADP concentration was measured with CF0F1 in the oxidized state, E(ox), and in the reduced state, E(red). The results can be described by Michaelis-Menten kinetics with the following parameters: Vmax = 0.5 s-1, Km = 8 microM for E(ox) and Vmax = 2.0 s-1, Km = 8 microM for E(red).

Characterization of an ATP-driven H+ pump in human placental brush-border membrane vesicles.

The presence of an ATP-driven H+ pump as measured by H+ uptake upon addition of ATP was not demonstrable in human placental brush-border membrane vesicles when used in their native form, owing to their right-side-out orientation. However, the presence of the H+ pump in these membranes became evident when the membrane vesicles were transiently exposed to 1% cholate, with subsequent removal of the detergent to re-form the vesicles. Apparently, cholate pretreatment reoriented the H+ pump from an inward-facing configuration to outward-facing. Consequently, H+ uptake in response to externally added ATP was easily demonstrable in these cholate-pretreated vesicles by using the delta pH indicator Acridine Orange. In addition, bafilomycin A1-sensitive ATPase activity was measurable in cholate-pretreated vesicles, but not in native intact vesicles, indicating reorientation of the H+ pump. The reoriented H+ pump was electrogenic because H+ uptake was stimulated by an inside-negative anion-diffusion potential or when the vesicles were voltage-clamped. ATP supported H+ uptake with an apparent Km of 260 microM. ITP and GTP supported the pump activity partially, whereas CTP and UTP did not. Mg2+ and Mn2+ were the most preferred bivalent cations. Co2+ and Zn2+ showed partial activity, whereas Ca2+ and Ba2+ showed little or no activity. The pump was inhibited by nanomolar concentrations of bafilomycin A1 and micromolar concentrations of N-ethylmaleimide, p-chloromercuribenzenesulphonate, NN-dicyclohexylcarbodi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was relatively insensitive to oligomycin, vanadate and NaN3. The inhibition by N-ethylmaleimide was protectable by ATP. It is concluded that human placental brush-border membranes possess an ATP-driven H+ pump and that, on the basis of its characteristics, it belongs to the class of vacuolar (V-type) H+ pumps.

Characteristics of energy-linked proton translocation in liposome reconstituted bovine cytochrome bc1 complex. Influence of the protonmotive force on the H+/e- stoichiometry.

A study is presented on the H+/e- stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level-flow and steady-state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e- ratio of 1 at level-flow and 0.3 at steady-state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine-entrapped bc1 vesicles, a respiration-dependent steady-state delta pH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by subsaturating concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady-state H+/e- ratio appeared to be in linear inverse correlation with the delta pH. This indicates that delta pH exerts a control on the proton pump of the bc1 complex at the steady state. The effect of valinomycin-mediated potassium-diffusion potential on electron-transfer and proton-translocation activities is also shown. The experiments presented show that the H+/e- ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level-flow H+/e- ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre-steady-state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40-50 mV. This activation appears, however, to involve a rate-limiting step which remains normally coupled to proton translocation.

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 ATP-Mg/Pi carrier of rat liver mitochondria catalyzes a divalent electroneutral exchange.

Net transport of ATP-Mg or ADP in exchange for phosphate in isolated rat liver mitochondria has been shown to be an electroneutral process mediated by the ATP-Mg/Pi carrier. We compared the steady state distribution ratios of phosphate, ATP-Mg, and ADP at a pH of 7.4 to determine whether the divalent or monovalent form of these anions is the transported substrate. The log of the divalent ATP-Mg distribution ratio (in/out) approached the log of the divalent phosphate distribution ratio (approximately 0.85), which was approximately twice the value of the delta pH (approximately 0.40) across the inner mitochondrial membrane. This steady state relationship held under several different conditions, e.g. when the medium ATP concentration was varied or if the phosphate gradient was modified by partial uncoupling with the proton ionophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Unidirectional ADP efflux in exchange for external ADP or ATP-Mg was stimulated by an increase in matrix H+. The log of the trivalent ADP distribution ratio (approximately 1.20) approached 3 times the value of delta pH. All these data are consistent with the model of an electroneutral exchange of divalent phosphate (HPO2-4) for divalent ATP-Mg (ATP-Mg2-) or for divalent protonated ADP (HADP2-). We conclude that this transport mechanism accounts for the adenine nucleotide concentration gradient that normally exists between the matrix and external medium.

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

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

Nisin dissipates the proton motive force of the obligate anaerobe Clostridium sporogenes PA 3679.

The influence of nisin on the proton motive force (delta p) generated by glucose-energized cells of the obligate putrefactive anaerobe Clostridium sporogenes PA 3679 was determined. The components of delta p, the transmembrane potential (delta psi) and the pH gradient (delta pH), were determined from the distributions of the lipophilic cation [3H]TPP+ ([3H]tetraphenylphosphonium bromide) and [14C]salicylic acid, respectively. The cells maintained a constant delta p of -111 mV, consisting of a delta pH of 0.4 to 1.0 pH units at an external pH of 5 to 7 and a delta psi of -60 to -88 mV. Nisin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N'-dicyclohexylcarbodiimide (DCCD) at pH 6.0 elicited the complete release of preaccumulated [3H]tetraphenylphosphonium bromide and [14C]salicylic acid, with a concomitant depletion of delta psi and delta pH. Nisin and DCCD caused rapid drops in intracellular ATP levels from 1.2 to 0.01 and 0.06 nmol/mg of cells (dry weight), respectively. Cells exposed to nisin and DCCD lost the ability to form colonies, thus suggesting that delta psi and delta pH are necessary for cell viability. The data suggest that depletion of delta p and exhaustion of cellular ATP reserves are the basis for nisin inhibition of C. sporogenes PA 3679.

Protective effect of the dimer of 16,16-diMePGB1 against KCN-induced mitochondrial failure in hepatocytes.

The dimer and trimer of 16,16-dimethyl-15-dehydroprostaglandin B1 (16,16-diMePGB1) previously have been shown to have protective effects on mitochondrial function. To examine the potential mechanisms involved in protection against mitochondrial failure, we have studied the effects of the dimer of 16,16-diMe-PGB1 (dicalciphor) on mitochondrial function in hepatocytes exposed to KCN. Addition of micromolar concentrations of dicalciphor provided substantial protection against KCN-induced toxicity in a concentration- and time-dependent manner. Dicalciphor, however, had no effect on total or mitochondrial ATP losses in KCN-treated cells. The dimer prevented the marked loss of mitochondrial membrane potential (delta psi) and delta pH that occurs as a result of KCN treatment and prevented KCN-induced loading of phosphate in mitochondria. Furthermore, the dimer of 16,16-diMePGB1 also prevented KCN-induced mitochondrial and cellular swelling. These results demonstrate that dicalciphor protects against KCN-induced damage and that this protection is associated with regulation of specific mitochondrial ion transport functions.

Protonmotive force in freshwater sulfate-reducing bacteria, and its role in sulfate accumulation in Desulfobulbus propionicus.

The protonmotive force in several sulfate-reducing bacteria has been determined by means of radiolabelled membrane-permeant probes (tetraphenylphosphonium cation, TPP+, for delta psi, and benzoate for delta pH). In six of ten freshwater strains tested only the pH gradient could be determined, while the membrane potential was not accessible due to nonspecific binding of TPP+. The protonmotive force of the other four strains was between -110 and -155 mV, composed of a membrane potential of -80 to -140 mV and a pH gradient between 0.25 and 0.8 (inside alkaline) at pH(out) = 7. In Desulfobulbus propionicus the pH gradient decreased with rising external pH values. This decrease, however, was compensated by an increasing membrane potential. Sulfate, which can be highly accumulated by the cells, did not affect the protonmotive force, if added in concentrations of up to 4 mM. The highest sulfate accumulation observed (2500-fold), which occurred at external sulfate concentrations below 5 microM, could be explained by a symport of three protons per sulfate, if equilibrium with the protonmotive force was assumed. At higher sulfate concentrations the accumulation decreased and suggested an electroneutral symport of two protons per sulfate. At sulfate concentrations above 500 microM, the cells stopped sulfate uptake before reaching an equilibrium with the protonmotive force.

Kinetics of leucine transport in brush border membrane vesicles from lepidopteran larvae midgut.

The kinetics of K(+)-leucine cotransport in the midgut of lepidopteran larvae was investigated using brush border membrane vesicles. Initial rate (3 s) of leucine uptake was determined under experimental conditions similar to those occurring in vivo, i.e. in the presence of delta psi much greater than 0 (inside negative) and a delta pH of 1.4 units (7.4in/8.8out). Leucine and K+ bind to the carrier according to a sequential mechanism, and the binding of one substrate changed the dissociation constant for the other substrate by a factor of 0.15. Both trans-K+ and trans-leucine were mixed-type inhibitors of leucine uptake. Moreover, a portion of total leucine uptake was K+ independent, and it was competitively inhibited by trans-leucine. We interpret the trans inhibitory effects to mean that the partially loaded K+ only form is virtually unable to translocate across the membrane, whereas the binary complex carrier, leucine, can isomerize from the trans to the cis side of the membrane. However, the K(+)-independent leucine uptake occurs with a Keq greater than 1, i.e. the efflux route through the partially loaded leucine only form is slower than the rate of isomerization of the unloaded carrier from trans to cis side. Taken together, these results suggest a model in which transport occurs by an iso-random Bi Bi system. Since K+ does not act as a pure competitive activator, this model is different from that proposed for most of the Na(+)-linked solutes transport agencies and may be related to the broadening of the cation specificity of the amino acid transporters in lepidopteran larvae.