Age and growth-related changes in cyclopiazonic acid-potentiated lipophilic cation accumulation by cultured cells and binding to freeze-thaw lysed cells

In a previous study (1) we demonstrated that increased tetraphenylphosphonium (TPP) uptake by renal epithelial cells (LLC-PK₁) exposed to the fungal metabolite cyclopiazonic acid (CPA) was not a result of hyperpolarization across the plasma membrane even though CPA-potentiated TPP uptake could be totally inhibited by the depolarizing agent carbonylcyanide-m-chlorophenylhydrazone (CCCP). We now demonstrate that CPA potentiates TPP accumulation by proliferating skeletal muscle (L6) and LLC-PK₁ cells but not by nonproliferating primary rat hepatocytes. In LLC-PK₁ cells, CPA-potentiated TPP accumulation is observed in cells at all ages. In s cells, CPA-potentiated TPP accumulation is maximal soon after subculturing, and as the cells age they become less sensitive to CPA until TPP accumulation by CPA-treated cells approaches that of untreated cells. The temporal change in sensitivity of L6 cells to CPA may be related to biochemical and/or metabolic changes which occur as the cells age in culture. Hepatocytes, LLC-PK₁ cells, and L6 cells permeabilized by freeze-thaw lysis, all exhibit CPA-potentiated TPP partitioning, even in the presence of CCCP. This result indicates that both TPP and CPA must have access to the intracellular space in order for potentiated TPP partitioning to be observed. We hypothesize that the site of interaction between CPA and TPP is intracellular and probably associated with the cytoplasmic side of the plasma membrane and possibly the mitochondria.     

Effects of medium amino acids on ouabain-sensitive 86Rb+ -uptake and membrane-potential dependent [3H]tetraphenylphosphonium accumulation in Friend erythroleukemia cells

The effects of amino acids present in minimal essential medium were investigated on 86Rb+ -fluxes and on the membrane-potential dependent accumulation of the lipophilic cation [3H]tetraphenylphosphonium (TTP+) in logarithmically growing Friend erythroleukemia cells. The ouabain-sensitive 86Rb+ -uptake measured as well in complete growth medium as in Earle's balanced salt solution (EBSS) with amino acid composition present in growth medium, was 3 to 4-fold increased in comparison to the 86Rb+-uptake measured in pure EBSS only. The Na+,K+,2Cl- -cotransport measured as piretanide-sensitive 86Rb+-uptake was reduced in the presence of amino acids. Stimulation of the ouabain-sensitive 86Rb+ -uptake could be brought about by the addition of alanine alone or of the sodium ionophore monensin. In spite of the activation of the Na+,K+ -pump the membrane-potential dependent accumulation of [3H]TPP+ was about 40 per cent reduced in the presence of medium amino acids indicating a decreased membrane potential under these conditions. On the other hand, monensin which induces an electrically silent Na+ -influx via Na+/H+ -exchange was shown to hyperpolarize the membrane on the basis of [3H]TPP+-accumulation. These results suggest that the intensive uptake of neutral amino acids by Na+-cotransport in rapidly growing cells may be responsible for both stimulation of the Na+,K+ -pump and decrease in the transmembrane potential.     

Tetraphenylphosphonium ion is a true indicator of negative plasma-membrane potential in the yeast Rhodotorula glutinis. Experiments under osmotic stress and at low external pH values.

In the yeast Rhodotorula glutinis, accumulation of the tetraphenylphosphonium ion (TPP+) was increased under conditions of osmotic stress, indicating a hyperpolarization of the negative membrane potential (delta psi). The following observations were consistent with the occurrence of hyperpolarized delta psi: enhanced accumulation of glucosamine, the uptake of which is also driven by delta psi; increased respiratory rate. The accumulation of TPP+ was gradually decreased by lowering the pH of cell suspensions. At pH values below 4.5, no TPP+ was taken up, but instead thiocyanate (SCN-) was accumulated, indicating a positive delta psi. The pH-dependent influx of glucosamine followed the pattern of TPP+ accumulation both in the wild-type and in the nystatin-resistant mutant, M67, which displayed a negative delta psi down to pH 3. Thus TPP+ accumulation in Rh. glutinis reflected actual electrical potential difference across the plasma membrane.     

Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (delta psi, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of delta psi. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 microM), indicating a non mediated uptake. Thus, TPP+ is a suitable delta psi probe for this yeast.     

Differential sensitivity of the cellular compartments of Saccharomyces cerevisiae to protonophoric uncoupler under fermentative and respiratory energy supply.

The effect of a protonophoric uncoupler (CCCP) on the different cellular compartments was investigated in yeast grown aerobically on lactate. These cells were incubated in a resting cell medium under three conditions; in aerobiosis with lactate or glucose or in anaerobiosis with glucose as energetic substrate. For each condition, in vivo 31P NMR was used to measure pH gradients across vacuolar and plasma membrane and phosphorylated compound levels. Respiratory rate (aerobic conditions) and TPP+ uptake were measured independently. Concerning the polyphosphate metabolism, spontaneous NMR-detected polyphosphate breakdown occurred, in anaerobiosis and in the absence of CCCP. In contrast, in aerobiosis, polyphosphate hydrolysis was induced by addition of either CCCP or a vacuolar membrane ATPase-specific inhibitor, bafilomycin A1. Moreover, polyphosphates were totally absent in a null vacuolar ATPase activity mutant. The vacuolar polyphosphate content depended on two factors: vacuolar pH value, strictly linked to the vacuolar H(+)-ATPase activity, and inorganic phosphate concentration. CCCP was more efficient in dissipating the proton electrochemical gradient across vacuolar and mitochondrial membranes than across the plasma membrane. This discrepancy can be essentially explained by a difference of stimulability of each proton pump involved. As long as the energetic state (measured by NDP + NTP content) remains high, the plasma membrane proton ATPase is able to compensate the proton leak. Moreover, this ATPase contributes only partially to the generation of delta pH. The maintenance of the delta pH across the plasma membrane, that of the energetic state, and the cellular TPP+ uptake depend on the nature of the ATP-producing process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the plasma and mitochondrial membrane potentials of alveolar type II cells by the use of ionic probes

The lipophilic cation triphenylmethylphosphonium (TPMP+) and the potassium analog Rb+, were used to monitor the membrane potential (delta psi) of freshly isolated rabbit type II alveolar epithelial cells. Type II cells were found to accumulate TPMP+ rapidly at 37 degrees C in Hanks' balanced-salt solution with 5 microM tetraphenyl boron, but this accumulation was partially due to non-membrane potential dependent binding of TPMP+ to the cell. Lysophosphatidylcholine (lysoPC) was found to abolish delta psi and permitted correction for bound TPMP+ or Rb+. TPMP+ remaining in the cell following correction for binding represents the sum of mitochondrial and plasma membrane potential dependent accumulation. The accumulation of Rb+ by the type II cell was found to be independent of the mitochondrial membrane potential and indicated a trans-plasma membrane Rb+ distribution potential of -62.9 +/- 4 mV. A similar value was obtained by estimating the plasma membrane potential dependent accumulation of TPMP+ in type II cells whose mitochondria were depolarized with carbonylcyanide m-chlorophenylhydrazone (CCCP). The release of TPMP+ due to CCCP treatment also permitted an estimation for the trans-mitochondrial membrane potential of -141.8 +/- 10 mV. These techniques of membrane potential measurements were found to be sensitive to changes in delta psi induced by a number of inhibitors and ionophores. The ability to measure the membrane potential of the type II pneumocyte, and the changes caused by various agents, should be useful in characterizing the functional responses of this pulmonary surfactant producing cell.     

Light-dependent delta mu Na-generation and utilization in the marine cyanobacterium Oscillatoria brevis

Light-dependent Na+ and H+ transports, membrane potential (delta psi) and motility have been studied in the cells of the marine cyanobacterium Oscillatoria brevis. In the presence of a protonophorous uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, the intracellular Na+ level is shown to increase in the dark and decrease in the light. The Na+/H+ antiporter, monensin, stimulates the dark CCCP-dependent [Na+]in increase and abolishes the light-dependent [Na+]in decrease. Na+ ions are necessary for the fast light-induced delta psi generation and H+ uptake by the cells. This uptake is inhibited by monensin being resistant to CCCP. Monensin sensitizes the delta psi level and the motility rate to low CCCP concentrations. The obtained data are consistent with the assumption that O. brevis possesses a primary Na+ pump which utilizes (directly or indirectly) the light energy.     

The sodium cycle. I. Na+-dependent motility and modes of membrane energization in the marine alkalotolerant vibrio Alginolyticus

Respiration, membrane potential generation and motility of the marine alkalotolerant Vibrio alginolyticus were studied. Subbacterial vesicles competent in NADH oxidation and delta psi generation were obtained. The rate of NADH oxidation by the vesicles was stimulated by Na+ in a fashion specifically sensitive to submicromolar HQNO (2-heptyl-4-hydroxyquinoline N-oxide) concentrations. The same amounts of HQNO completely suppressed the delta psi generation. Delta psi was also inhibited by cyanide, gramicidin D and by CCCP + monensin. CCCP (carbonyl cyanide m-chlorophenylhydrazone) added without monensin exerted a much weaker effect on delta psi. Na+ was required to couple NADH oxidation with delta psi generation. These findings are in agreement with the data of Tokuda and Unemoto on Na+-motive NADH oxidase in V. alginolyticus. Motility of V. alginolyticus cells was shown to be (i) Na+-dependent, (ii) sensitive to CCCP + monensin combination, whereas CCCP and monensin, added separately, failed to paralyze the cells, (iii) sensitive to combined treatment by HQNO, cyanide or anaerobiosis and arsenate, whereas inhibition of respiration without arsenate resulted only in a partial suppression of motility. Artificially imposed delta pNa, i.e., addition of NaCl to the K+ -loaded cells paralyzed by HQNO + arsenate, was shown to initiate motility which persisted for several minutes. Monensin completely abolished the NaCl effect. Under the same conditions, respiration-supported motility was only slightly lowered by monensin. The artificially-imposed delta pH, i.e., acidification of the medium from pH 8.6 to 6.5 failed to activate motility. It is concluded that delta mu Na+ produced by (i) the respiratory chain and (ii) an arsenate-sensitive anaerobic mechanism (presumably by glycolysis + Na+ ATPase) can be consumed by an Na+ -motor responsible for motility of V. alginolyticus.     

Intracellular acidosis protects cultured hepatocytes from the toxic consequences of a loss of mitochondrial energization

Cultured rat hepatocytes were treated with potassium cyanide, an inhibitor of cytochrome oxidase; valinomycin, a K+ ionophore; carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophore; and the ATP synthetase inhibitor oligomycin. The effect of these agents on the viability of the cells was related to changes in ATP content and the deenergization of the mitochondria. The ATP content was reduced by over 90% by each inhibitor. All of the agents except oligomycin killed the cells within 4 h. With the exception of oligomycin, the mitochondrial membrane potential as measured by the distribution of [3H]triphenylmethylphosphonium collapsed with each of the agents. Monensin, a H+/Na+ ionophore, potentiated the toxicity of cyanide and CCCP, whereas the toxicity of valinomycin was reduced. The effect of cyanide and monesin on the cytoplasmic pH of cultured hepatocytes was measured with the fluorescent probe, 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Cyanide promptly acidified the cytosol, and the addition of 10 microM monensin caused a rapid alkalinization of the cytosol. A reduction of pH of the culture medium from 7.4 to 6.6 and 6.0 prevented the cell killing both by cyanide alone and by cyanide in the presence of monensin. However, neither monensin nor extracellular acidosis had any effect on the loss of mitochondrial energization in the presence of cyanide. It is concluded that ATP depletion per se is insufficient to explain the cell killing with cyanide, CCCP, and valinomycin. Rather, cell killing is better correlated with a loss of mitochondrial energization. With cyanide an intracellular acidosis interferes with the mechanism that couples collapse of the mitochondrial membrane potential to lethal cell injury.     

Action of monensin, a monovalent cationophore, on cultured human fibroblasts: evidence that it induces high cellular accumulation of glucosyl- and lactosylceramide (gluco- and lactocerebroside)

We have exposed cultured human fibroblasts to micromolar concentrations of the ionophore monensin. A salient result was a rapid accumulation in these cells of glucosylceramide (glucocerebroside, GlcCer) and lactosylceramide (lactocerebroside, LacCer). When we incubated these cells with radioactively labeled galactose, GlcCer and LacCer became highly labeled. These results indicate that monensin greatly increases these simplest glycosphingolipids that are the precursor to the major plasma membrane glycosphingolipids. We observed, simultaneously, a decreased incorporation of labeled galactose into some more highly glycosylated neutral glycosphingolipids and sialoglycosphingolipids (gangliosides), and unlike GlcCer and LacCer, the cellular content of these more highly glycosylated compounds remained the same in the presence or absence of monensin. We have found that cultured Gaucher disease fibroblasts, with genetically impaired lysosomal glucocerebrosidase activity, accumulated even more GlcCer and LacCer than normal cells upon exposure to monensin. This finding shows that monensin affects biosynthesis rather than merely disrupting lysosomal degradation that is already deleted with respect to GlcCer in Gaucher disease cells. These results represent the first indication of an apparently remarkable effect of the monovalent ionophore, monensin, on plasma membrane glycosphingolipid biosynthesis. The evidence suggests a regulatory distinction between initial and higher intracellular glycosylation steps. Monensin does not diminish and may augment initial anabolic mono- and diglycosylations and also appears to inhibit higher glycosylations of glycosphingolipids.     

Active phosphate ion transport in plasma membrane vesicles isolated from mouse fibroblasts.

Inorganic phosphate accumulated 8-fold in plasma membrane vesicles derived from simian virus 40-transformed 3T3 mouse fibroblasts when a NaCl gradient (external greater than internal) was artificially imposed across the membrane. Preincubation with Na+ or addition of monensin markedly reduced phosphate accumulation. Na+-stimulated phosphate transport was not affected by addition of either dicarboxylic acids, antimycin A, or ouabain and persisted after addition of proton ionophores. The coupling of phosphate transport to Na+ gradients was pH-dependent, with maximal stimulation by Na+ below pH 7. These findings suggest that monovalent phosphate anion moves across the plasma membrane in co-transport with sodium ion.     

Interaction between monensin and lysosomotropic amines in the regulation of the processing of epidermal growth factor by BALB/c 3T3 cells

Monensin, like the lysosomotropic amines Chloroquine and methylamine, caused a large accumulation of ¹²⁵I-EGF in BALB/c-3T3 cells that was due to specific increases in the amount of intracellular intact hormone. However using a pulse-chase paradigm of ¹²⁵I-EGF accumulation, marked differences were observed between monensin and the amines. When EGF was accumulated in the presence of monensin, there was a gradual loss of cell-bound radioactivity during a chase in the absence of the drug, and the labeled material recovered in. the medium primarily consisted of degraded hormone. The continued presence of monensin in the chase medium substantively prevented the loss of cell bound material, and what little was recovered in the medium consisted of intact ¹²⁵I-EGF. In contrast, when ¹²⁵I-EGF was accumulated in the presence of methylamine, predominately intact peptide was lost from the cells at a relatively high rate during the chase whether or not methylamine remained in the medium. When monensin was present in the chase medium following accumulation in the presence of either Chloroquine or methylamine, the loss of intracellular ¹²⁵I-EGF was essentially blocked.     

Effect of phytohormones on the transmembrane translocation of protons in plasma membrane vesicles from tubers of Solanum tuberosum L

Effects of phytohormones gibberellic acid (GA) and abscisic acid (ABA) on the ATP-dependent transmembrane transport of protons were studied in plasma membrane vesicles (PMVs) from non-dormant potato tubers. The uptake of H+ into PMVs was assessed by the fluorescence quenching of acridine orange (AO) after the addition of ATP to the incubation medium. Addition of ATP to the incubation medium led to the instantaneous rise of the AO fluorescence intensity followed by its decrease. The fluorescence quenching was not observed in the presence of either protonophore CCCP or inhibitors of the membrane-bound H+-ATPase. It is concluded that the ATP-induced quenching of the AO fluorescence resulted from the accumulation of protons in PMVs due to the function of the plasma membrane-bound H+-ATPase. Depending on their concentrations, GA and ABA either inhibited or stimulated the ATP-driven H+ translocation across the vesicle membrane. The growth-stimulating hormone GA at concentrations of 10(-9)-10(-5) M increased the initial rate of the fluorescence quenching, whereas 10(-4) M GA slightly inhibited the H+ translocation. The growth inhibitor ABA at a concentration of 10(-9) M slightly increased the rate of the proton accumulation in PMVs; at higher concentrations (10(-8)-10(-4) M), ABA inhibited the H+ translocation. Acetic acid, which has pK similar to pK of GA and ABA, did not influence the ATP-dependent H+ accumulation in PMVs, suggesting the hormone-specific action of GA and ABA on the H+-ATPase activity. In the presence of DCC, which completely inhibited the accumulation of H+, GA and ABA did not affect the passive proton efflux from PMVs. It is proposed that the mechanisms of the regulatory effects of phytohormones may involve modification of H+-ATPase activity leading to changes in the electrochemical gradient of H+ across the plasma membrane.     

Potassium transport coupled to ATP hydrolysis in reconstituted proteoliposomes of yeast plasma membrane ATPase

Potassium transport coupled to ATP hydrolysis has been reconstituted in proteoliposomes using a highly purified plasma membrane Mg2+-dependent ATPase of the yeast Schizosaccharomyces pombe. The ATPase activity in the incorporated enzyme was strongly stimulated (2.2-fold) by the H+-conducting agent carbonyl cyanide m-chlorophenylhydrazone (CCCP). The H+/K+ exchanger nigericin (in the presence of K+) stimulated 1.6-fold the ATPase activity. When both ionophores were added together, the stimulation was increased up to 2.7-fold. When a potassium concentration gradient (high K+ in) was applied to the proteoliposome membrane, a significant drop in the CCCP-stimulated ATPase activity was observed. Inversion of the K+ concentration gradient (high K+ out) did not decrease the stimulation by CCCP. High Na+ in also decreased the stimulation induced by CCCP in the absence but not in the presence of external K+. However, high Li+ in had no effect. Direct potassium efflux from the proteolyposomes was detected upon addition of MgATP using a selective K+ electrode. The ATP-dependent potassium efflux was abolished in CCCP and/or nigericin-pretreated proteoliposomes. However, during steady state ATP hydrolysis, a transient and small K+ efflux was observed upon addition of a CCCP pulse. I propose that the plasma membrane Mg2+-dependent ATPase in yeast cells not only carries out electrogenic H+ ejection but also drives the uptake of potassium via a voltage-sensitive gate which is closed in the absence and open in the presence of the membrane potential.     

Uncoupler-induced relocation of elongation factor Tu to the outer membrane in an uncoupler-resistant mutant of Escherichia coli

Escherichia coli UV6, a mutant which is resistant to the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), when grown in the presence of CCCP, but not in its absence, incorporated a new protein (Mr, 42 000) into the cell envelope. This protein was found in both cytoplasmic and outer-membrane fractions. In the outer membrane it was one of three or four most abundant proteins. The protein was tightly bound to the membranes and was not solubilized by several detergents. Solubilization was achieved with sodium lauroylsarcosinate (sarkosyl). The protein was purified close to homogeneity by affinity chromatography on a column of GDP-Sepharose. It was identified as elongation factor Tu (EF-Tu) on the basis of electrophoretic mobility, profiles of peptide fragments produced by proteolysis, and by its ability to bind to GDP-Sepharose. Disruption of cells in the presence of CCCP or incubation of envelopes with EF-Tu did not result in incorporation of EF-Tu into the membranes. It is suggested that this protein is incorporated into the outer membrane as a consequence of an alteration in the normal protein biosynthetic mechanisms of the mutant induced by the presence of CCCP.     

The sodium cycle. I. Na-dependent motility and modes of membrane energization in the marine alkalotolerant Vibrio alginolyticus

Respiration, membrane potential generation and motility of the marine alkalotolerant Vibrio alginolyticus were studied. Subbacterial vesicles competent in NADH oxidation and Δψ generation were obtained. The rate of NADH oxidation by the vesicles was stimulated by Na⁺ in a fashion specifically sensitive to submicromolar HQNO (2-heptyl-4-hydroxyquinoline N-oxide) concentrations. The same amounts of HQNO completely suppressed the Δψ generation. Δψ was also inhibited by cyanide, gramicidin D and by CCCP + monensin. CCCP (carbonyl cyanide m-chlorophenylhydrazone) added without monensin exerted a much weaker effect on Δψ. Na⁺ was required to couple NADH oxidation with Δψ generation. These findings are in agreement with the data of Tokuda and Unemoto on Na⁺-motive NADH oxidase in V. alginolyticus. Motility of V. alginolyticus cells was shown to be (i) Na⁺-dependent, (ii) sensitive to CCCP + monensin combination, whereas CCCP and monensin, added separately, failed to paralyze the cells, (iii) sensitive to combined treatment by HQNO, cyanide or anaerobiosis and arsenate, whereas inhibition of respiration without arsenate resulted only in a partial suppression of motility. Artificially imposed ΔpNa, i.e., addition of NaCl to the K⁺-loaded cells paralyzed by HQNO + arsenate, was shown to initiate motility which persisted for several minutes. Monensin completely abolished the NaCl effect. Under the same conditions, respiration-supported motility was only slightly lowered by monensin. The artificially-imposed ΔpH, i.e., acidification of the medium from pH 8.6 to 6.5 failed to activate motility. It is concluded that Δ _Na⁺ produced by (i) the respiratory chain and (ii) an arsenate-sensitive anaerobic mechanism (presumably by glycolysis + Na⁺ ATPase) can be consumed by an Na⁺-motor responsible for motility of V. alginolyticus.     

Induction of acrosomal reaction and calcium uptake in ram spermatozoa by ionophores

Ram spermatozoa incubated in the presence of Ca2+ and the Ca2+-ionophore A23187 undergo a process which is known as the acrosome reaction. This reaction is characterized by fusion of the outer acrosomal membrane and the overlying plasma membrane to form mixed vesicles which can be seen in the electron microscope. As a result, the trypsin-like acrosin is released from the cells to the medium. The occurrence of the acrosome reaction was determined by following acrosin activity in the medium. After 2 h of incubation of the cells in the presence of ionophore and Ca2+, the released acrosin activity is related to the ionophores according to the sequence: A23187 greater than monensin greater than valinomycin greater than FCCP = without ionophore. The study of Ca2+ uptake by the cells revealed that Ca2+ enters the cell prior to the release of acrosin. Monensin can induce Ca2+ uptake and acrosin release only when Na+ is present in the incubation medium. There is no increase in Ca2+ uptake with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). We suggest that the Na+/H+ exchange induced by monensin causes an increase in intracellular Na which is the driving force for the Ca2+ entry via a Ca2+/Na+ antiporter. Since monensin can induce an increase in Ca2+ uptake only in the presence of Na+, FCCP enhances Ca2+ uptake in the presence of valinomycin, and A23187 is a Ca2+/2H+ exchanger, we suggest that alkalization of the intracellular space is involved in the acrosome reaction. Calcium uptake in the presence of monensin is not affected by the uncoupler FCCP, a result which indicates that Ca2+ is not accumulated in the mitochondria. Incubation of cells for 3 h in the absence of Ca2+ or ionophore caused a 3-fold increase in the rate of acrosin release when monensin and Ca2+ were added together. There was no change in this rate when A23187 was used. We suggest that during the preincubation time (known as capacitation) the permeability of the plasma membrane to Ca2+ is enhanced. This study shows that acrosin release and Ca2+ uptake can be used as a quantitative asay for the determination of the acrosome reaction.     

Generation of a proton potential by succinate dehydrogenase of Bacillus subtilis functioning as a fumarate reductase.

The membrane fraction of Bacillus subtilis catalyzes the reduction of fumarate to succinate by NADH. The activity is inhibited by low concentrations of 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO), an inhibitor of succinate: quinone reductase. In sdh or aro mutant strains, which lack succinate dehydrogenase or menaquinone, respectively, the activity of fumarate reduction by NADH was missing. In resting cells fumarate reduction required glycerol or glucose as the electron donor, which presumably supply NADH for fumarate reduction. Thus in the bacteria, fumarate reduction by NADH is catalyzed by an electron transport chain consisting of NADH dehydrogenase (NADH:menaquinone reductase), menaquinone, and succinate dehydrogenase operating in the reverse direction (menaquinol:fumarate reductase). Poor anaerobic growth of B. subtilis was observed when fumarate was present. The fumarate reduction catalyzed by the bacteria in the presence of glycerol or glucose was not inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or by membrane disruption, in contrast to succinate oxidation by O2. Fumarate reduction caused the uptake by the bacteria of the tetraphenyphosphonium cation (TPP+) which was released after fumarate had been consumed. TPP+ uptake was prevented by the presence of CCCP or HOQNO, but not by N,N'-dicyclohexylcarbodiimide, an inhibitor of ATP synthase. From the TPP+ uptake the electrochemical potential generated by fumarate reduction was calculated (Deltapsi = -132 mV) which was comparable to that generated by glucose oxidation with O2 (Deltapsi = -120 mV). The Deltapsi generated by fumarate reduction is suggested to stem from menaquinol:fumarate reductase functioning in a redox half-loop.     

Changes in host cell energetics in response to bacteriophage PRD1 DNA entry.

Double-stranded DNA bacteriophage PRD1 infects a variety of gram-negative bacteria harboring an IncP-type conjugative plasmid. The plasmid codes for the DNA transfer phage receptor complex in the cell envelope. Our goal was, by using a collection of mutant phage particles for which the variables are the DNA content and/or the presence of the receptor-binding protein, to obtain information on the energy requirements for DNA entry as well as on alterations in the cellular energetics taking place during the first stages of infection. We studied the fluxes of tetraphenylphosphonium (TPP+), phenyldicarbaundecaborane (PCB-), and K+ ions as well as ATP through the envelope of Salmonella typhimurium cells. The final level of the membrane voltage (delta psi) indicator TPP+ accumulated by the infected cells exceeds the initial level before the infection. Besides the effects on TPP+ accumulation, PRD1 induces the leakage of ATP and K+ from the cytosol. All these events were induced only by DNA-containing infectious particles and were cellular ATP and delta psi dependent. PRD1-caused changes in delta psi and in PCB- binding differ considerably from those observed in other bacteriophage infections studied. These results are in accordance with the presence of a specific channel engaged in phage PRD1 DNA transport.     

The sodium cycle. II. Na+-coupled oxidative phosphorylation in Vibrio alginolyticus cells

The role of Na+ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na+-motive respiratory chain of V. alginolyticus. Na+ loaded cells incubated in a K+ or Li+ medium fail to synthesize ATP in response to lactate addition. The addition of Na+ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na+ level. Neither lactate oxidation nor delta psi generation coupled with this oxidation is increased by external Na+ in the Na+-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse delta pNa, i.e., [Na+]in greater than [Na+]out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K+-loaded cells incubated in a high Na+ medium, i.e., when delta pNa of the proper direction [( Na+]in less than [Na+]out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K+ medium (delta pNa is low) is decreased by CCCP even without monensin. Artificial formation of delta pNa by adding 0.25 M NaCl to the K+-loaded cells (Na+ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na+ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na+ instead of H+ as the coupling ion.