The effect of ionophores on growth and glycosyltransferase production of Streptococcus sanguis

Abstract Batch cultures of Streptococcus sanguis NCTC7865 were grown in complex medium in the presence and absence of the ionophores gramicidin, valinomycin and nigericin, to study their effects on growth and glycosyltransferase production. Growth of S. sanguis was markedly inhibited by nigericin or gramicidin, whereas valinomycin had no significant effect. The presence of ionophores caused only slight decreases in glucosyltransferase activity. Fructosyltransferase activity was however reduced by at least 90%. The results indicate that ΔpH rather than ΔΨ is essential for maintaining normal growth in S. sanguis . However, both ΔpH and ΔΨ are necessary for fructosyltransferase synthesis and secretion, but are not apparently involved in the synthesis and secretion of glucosyltransferase.     

The action of certain antibiotics on mitochondrial, erythrocyte and artificial phospholipid membranes. The role of induced proton permeability

1. The action of the antibiotics enniatin A, valinomycin, the actin homologues, gramicidin, nigericin and dianemycin on mitochondria, erythrocytes and smectic mesophases of lecithin–dicetyl hydrogen phosphate was studied. 2. These antibiotics induced permeability to alkali-metal cations on all three membrane systems. 3. The ion specificity on each membrane system was the same. 4. Enniatin A, valinomycin and the actins did not induce permeability to protons, whereas nigericin and dianemycin rendered all three membrane systems freely permeable to protons. 5. Several differences were noted between permeability induced by nigericin and that induced by gramicidin. 6. The action of all these antibiotics on mitochondrial respiration could be accounted for by changes in passive ion permeability of the mitochondrial membrane similar to those induced in erythrocytes and phospholipid membranes, if it is assumed that a membrane potential is present in respiring mitochondria.     

Interaction of aminoglycosides and ionophores in the killing of Crithidia fasciculata

Crithidia fasciculata was utilized as a prescreen to determine the antiprotozoal action of aminoglycoside antibiotics alone and in combination with surface-altering agents. Paromomycin was tested with the carrier ionophores nigericin and valinomycin, the channel ionophore gramicidin and the polyene antibiotics amphotericin B and nystatin. After exposure to the drugs in suspension, organisms were plated out to determine the survival of C. fasciculata. Killing was time dependent for both the antibiotic and the ionophore. Paromomycin action was found to be potentiated by all the surface altering agents. The aminoglycosides kanamycin, gentamycin and streptomycin were studied alone and in combination with nigericin. Synergistic effects were demonstrated both with kanamycin and gentamycin in combination with nigericin. Streptomycin was ineffective both alone and with surface-altering agents.     

Proton electrochemical gradient and phosphate potential in mitochondria

The paper reports an analysis of the relationship between deltamuH the proton electrochemical potential difference, and deltaGp, the phosphate potential. Depression of deltamuH and deltaGp has been obtained by titration with: (a) carbonylcyanide trifluoromethoxyphenylhydrazone; (b) nigericin (+ valinomycin); (c) KCl (+ valinomycin); and (d) rotenone. The uncoupler depresses deltamuH more than nigericin (+ valinomycin), KCl (+ valinomycin) and rotenone at equivalent deltaGp. The deltaGp/deltamuH ratio is about 3 at high values of deltamuH. When deltaGp and deltamuH are depressed by nigericin (4 valinomycin) the deltaGp/deltamuH ratio remains constant. When deltaGp and deltamuH are depressed by uncouplers, the deltaGp/deltamuH ratio increases hyperbolically tending to infinity while deltamuH tends to zero. The absence of constant proportionality between deltaGp and deltamuH indicates that the proton gradients driving ATP synthesis presumably operate within microscopic environments.     

Proton-coupled calcium transport by intact cells of Azotobacter vinelandii.

Addition of ionophores to resting aerobic cultures of Azotobacter vinelandii OP resulted in 45Ca2+ uptake (Km = 60 microM Ca2+; Vmax 1.1 nmol/min per mg of cell protein) which was inhibited by cations (La3+ greater than Mn2+ greater than Sr2+ greater Ba2+). The rate of Ca2+ entry correlated with the magnitude of a transmembrane proton gradient (inside acid) which developed in the respective order: valinomycin less than tetrachlorosalicylanilide less than nigericin less than gramicidin D less than tetrachlorosalicylanilide plus valinomycin. A process of calcium-proton exchange (antiport) is responsible for calcium accumulation under these conditions.     

Effect of ionophores on intralysosomal pH.

1. The effect of ionophores on the intralysosomal pH (as estimated from the distribution of a weak acid or base), on the distribution of 42K+ across the lysosomal membrane, and on the intralysosomal degradation of 125I-labelled bovine serum albumin has been studied. 2. Nigericin and X537A equilibrate both 42K+ and H+ across the lysosomal membrane. Gramicidin equilibrates H+ across the lysosomal membrane, this equilibration being more effective in a NaCl than in a KCl medium. Thus all three ionophores exhibit the same ion specificity as in other membranes. 3. The effect of the exchange-diffusion ionophores cannot be imitated by the combination of valinomycin with an uncoupler. Valinomycin by itself also has no effect. 4. X537A and nigericin inhibit the intralysosomal degradation of 125I-labelled albumin only when potassium is present. In a sucrose-containing medium no effect is found. Similar results were obtained with gramicidin. 5. These data suggest that the lysosomal membrane is impermeable to monovalent cations at 25 or 37 degrees C, and that the transport of protons is organised in such a way that electroneutrality is maintained.     

Energy coupling of L-glutamate transport and vacuolar H(+)-ATPase in brain synaptic vesicles

Energy coupling of L-glutamate transport in brain synaptic vesicles has been studied. ATP-dependent acidification of the bovine brain synaptic vesicles was shown to require CI-, to be accelerated by valinomycin and to be abolished by ammonium sulfate, nigericin or CCCP plus valinomycin, and K+. On the other hand, ATP-driven formation of a membrane potential (positive inside) was found to be stimulated by ammonium sulfate, not to be affected by nigericin and to be abolished by CCCP plus valinomycin and K+. Like formation of a membrane potential, ATP-dependent L-[3H]glutamate uptake into vesicles was stimulated by ammonium sulfate, not affected by nigericin and abolished by CCCP plus valinomycin and K+. The L-[3H]glutamate uptake differed in specificity from the transport system in synaptic plasma membranes. Both ATP-dependent H+ pump activity and L-glutamate uptake were inhibited by bafilomycin and cold treatment (common properties of vacuolar H(+)-ATPase). ATP-dependent acidification in the presence of L-glutamate was also observed, suggesting that L-glutamate uptake lowered the membrane potential to drive further entry of H+. These results were consistent with the notion that the vacuolar H(+)-ATPase of synpatic vesicles formed a membrane potential to drive L-glutamate uptake. ATPase activity of the vesicles was not affected by the addition of Cl-, glutamate or nigericin, indicating that an electrochemical H+ gradient had no effect on the ATPase activity.     

Inhibitors of energy-dependent efflux of the fungicide fenarimol byAspergillus nidulans

Fenarimol is a fungicide classified as a sterol biosynthesis inhibitor (SBI). Its passive influx in mycelium of wild-type and SBI-resistant strains ofAspergillus nidulans is counteracted by an energy-dependent efflux. Sodium orthovanadate, the ionophores nigericin and valinomycin, and the cationic agents Cu²⁺, cetylpyridinium bromide, and dodine inhibit the energy-dependent efflux of fenarimol and thus allow this fungicide to accumulate in mycelium of both wild-type and SBI-resistant strains. The ionophore gramicidin and anionic and neutral detergents had the opposite effect. It is suggested that fenarimol efflux is mediated by the electrochemical proton gradient across the fungal plasma membrane.     

The effect of valinomycin and nigericin on the efficacy of bacteriophage infection of staphylococcal cells

The effect of ionophore antibiotics, valinomycin and nigericin, on the generation of the membrane potential, the pH gradient and the efficacy of phage infection in tetracycline-resistant staphylococci has been studied. Valinomycin at a concentration of 0.5 microM induces the dissipation of the membrane potential, and nigericin at a concentration of 12.0 microM decreases the value of the pH gradient on the membrane of staphylococci. The separate use of antibiotics has no essential influence on the efficacy of phage infection. The combined use of valinomycin and nigerimycin produces the maximum inhibition of phage infection (64.5%) at the stage of the introduction of DNA into the bacterial cell, which is indicative of a definite role played by the membrane potential and the pH gradient in the transport of phage DNA into staphylococcal cells.     

Acetylcholine Release from Isolated Synaptic Vesicles Related to Ionic Permeability Changes: Continuous Detection with a Chemiluminescent Method

The effect of ionic permeability changes on acetylcholine (ACh) release from isolated cholinergic synaptic vesicles of Torpedo was studied using a chemiluminescent method for continuous ACh detection. Vesicles rendered freely permeable to potassium by valinomycin lost most of their ACh content in K+ media, if the accompanying anion was permeant; it thus appeared that ACh leakage occurred as the result of internal osmotic changes. Upon addition of ionophores that catalyse monovalent cation/H+ exchange (gramicidin D or a mixture of valinomycin plus protonophore FCCP), a rapid but transient ACh release was observed. Surprisingly, nigericin which also catalyses K+/H+ exchange, had no effect on ACh release. The divalent cation ionophore A23187 promoted ACh release only when calcium (and not magnesium) was introduced into the external medium in a millimolar concentration range. As the simultaneous addition of the protonophore FCCP and A23187 decreased this calcium-dependent ACh leakage, a releasing effect of A23187 through Ca2+/H+ exchange is suspected. The present results emphasise the role of internal protons for ACh retention inside synaptic vesicles.     

Effect of inductors of alkali cation permeability on cytochrome c bound to mitochondrial membrane]

The effect of inductors of alkali cation permeability--valinomycin, gramicidin A, gramicidin S and its N,N'-diacetyl derivative--on rat liver mitochondria during respiration has been studied. It is shown that valinomycin, gramycidin A and diacetylgramicidin S at optimal concentration for uncoupling cause two-phase activation of mitochondrial respiration and that this effect results from cytochrome c solubilization. Gramicidin S at optimal concentration cannot remove cytochrome c from the respirating mitochondria. It is suggested that this property of gramicidin S is owned to cytochrome c immobilisation in membrane, due to the effect of this compound.     

Temperature jump as a new technique to study the kinetics of fast transport of protons across membranes

Application of a temperature jump (2.5 degrees C) to a suspension of liposomes, having phosphate (delta pK/delta T approximately 0.005) as the internal buffer and tris(hydroxymethyl)aminomethane (delta pK/delta T approximately 0.031) as the external buffer, created a delta pH (pHin - pHout) of positive sign in ca. 5 microseconds. Decay of this delta pH was monitored by using the fluorescent pH indicator 8-hydroxy-1,3,6-pyrenetrisulfonic acid entrapped inside the liposome. This technique is useful to study transmembrane proton movement in the time range 5 microseconds-10 s at physiological pH values. The kinetics of proton transport aided by ion carriers such as nigericin, monensin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and valinomycin were studied by our method. The electrogenic nature of transport by CCCP and valinomycin and electroneutral ion transport by nigericin and monensin were shown. From the kinetics of proton transport aided by gramicidin, the time-averaged single-channel conductance of gramicidin channels was estimated to be (2.1 +/- 0.5) X 10(-16) S for H+ at pH 7.5.     

An ATPase from dog gastric mucosa: changes of outer pH in suspensions of membrane vesicles accompanying ATP hydrolysis

A microsomal Mg-ATPase from the gastric mucosa of dog, cat and frog has a K_m for ATP in the region 20–25 μM and by the value of this coefficient can be differentiated from the mitochondrial Mg-ATPase. The microsomal Mg-ATPase from dog gastric mucosa can be stimulated by gramicidin, nigericin and valinomycin in a KCl medium. This Mg-ATPase seems to be located in the ion impermeable membrane of microsomal vesicles and ATP hydrolysis driven changes of the outer pH can be observed. The data are consistent with the ATP hydrolysis driven entry of H⁺ ions across the vesicle membrane.     

Effects of double-layer polarization on ion transport.

It has been proposed that changes in ionic strength will alter the shape of current-voltage relations for ion transport across a lipid membrane. To investigate this effect, we measured currents across glyceryl monooleate membranes at applied potentials between 10 and 300 mV using either gramicidin and 1 mM NaCl or valinomycin and 1 mM KCl. A bridge circuit with an integrator as null detector was used to separate the capacitative and ionic components of the current. The changes in the current-voltage relations when ionic strength is varied between 1 and 100 mM are compared with predictions of Gouy-Chapman theory for the effects of these variations on polarization of the electrical diffuse double-layer. Double-layer polarization accounts adequately for the changes observed using membranes made permeable by either gramicidin or valinomycin.     

Gramicidin S: A Potent Inhibitor of Membrane-bound Epidennal Adenosine Triphosphatase from Nicotiana tabacum L. Leaves

The effect of ionophores and tyrocidine on membrane-bound adenosinetriphosphatase (ATPase) activity in epidermal cells from tobacco(Nicotiana tabacum L. cv. Samsun) leaves was investigated. GramicidinS inhibited Mg^2$-K^$-ATPase activity in the epidermal membraneof tobacco leaves. Its half-maximal inhibition was found at2.4?10^–5 M (under conditions of 370 µg membraneprotein per 2 ml reaction mixture). The degree of inhibitionof the epidermal ATPase was in the following order: tyrocidine>gramicidinS>DCCD>vanadate>DES>gramicidin D, all at 10^–4M. The ionophores, valinomycin, nigericin and salinomycin, inhibitedthe epidermal ATPase activity only slightly or not at all. TheATPase solubilized from the membrane with detergents was negligiblyinhibited by gramicidin S and tyrocidine. Thus, gramicidin Sacts in the manner of tyrocidine rather than as an ionophoreand may disturb the organization of the lipoprotein membrane,which in turn inactivates the membranebound epidermal ATPase. (Received July 13, 1981; Accepted December 4, 1981)     

Effect of metabolic inhibitors on entry of exogenous deoxyribonucleic acid into Ca2+-treated Escherichia coli cells.

The effect of various metabolic inhibitors (carbonylcyanid-m-chlorophenylhydrazone, nigericin, valinomycin, dicyclocarbodiimide, arsenate, NaF, etc.) and lipid-soluble synthetic ions (tetraphenylphosphonium bromide and tetraphenylboron sodium) on deoxyribonucleic acid (DNA) entry during transformation of Ca2+-treated Escherichia coli cells with plasmid DNA and on cell viability was investigated. In contrast to intact cells, Ca2+-treated E. coli cells were permeable to nigericin, valinomycin, and the other drugs tested. The inhibitors differentially affected [14C]proline active transport, and whereas some drugs inhibited transformation, the effects did not correlate with the effects on transport. The most potent inhibitors of transformation were nigericin, dicyclocarbodiimide, and tetraphenylboron sodium. Carbonylcyanid-m-chlorophenylhydrazone, tetraphenylphosphonium bromide, and valinomycin were relatively inactive. Tetraphenylboron sodium- and nigericin-treated cells bound were plasmid [14C]DNA in the deoxyribonuclease-resistant form than the control and other sample cells. Nevertheless, te penetration of exogenous plasmid DNA into the cell was greatly reduced, at least in case of nigericin. Unlike the other drugs, nigericin and dicyclocarbodiimide drastically affected the cell viability, the former within very short times of interaction. It is concluded that proton motive force does not play any significant role in DNA entry into Ca2+-treated E. coli cells. The results also suggest that adenosine 5'-triphosphate is not required for DNA entry either. The inhibitory effect of certain drugs is discussed in terms of structural perturbations induced by the drugs in cell envelope membranes.     

Effect of 2,4-dinitrophenol and ionophores on growth and methanogenesis inMethanobacterium thermoautotrophicum

2,4-Dinitrophenol and gramicidin D completely inhibited growth and methanogenesis inMethanobacterium thermoautotrophicum. At low K⁺ concentrations valinomycin inhibited growth and methanogenesis relatively slightly, at high K⁺ concentrations (0.1m KCl) growth was inhibited completely and methanogenesis by about 50%. Monensin and nigericin inhibited growth completely; methanogenesis was inhibited like with valinomycin at high K⁺ concentrations. The results can be interpreted in terms of Mitchell’s chemiosmotic theory as follows. The protonmotive force inM. thermoautotrophicum is the basic source of energy for endergonic processes. Dissipation of the electrical component of protonmotive force may probably be compensated by an increased generation of the proton gradient. However, the osmotic component is essential for growth ofM. thermoautotrophicum.     

Staining of mitochondrial membranes with 10-nonyl acridine orange, MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs

BACKGROUND: We set out to develop an assay for the simultaneous analysis of mitochondrial membrane potential and mass using the probes 10-nonyl acridine orange (NAO), MitoFluor Green (MFG), and MitoTracker Green (MTG) in HL60 cells. However, in experiments in which NAO and MFG were combined with orange emitting mitochondrial membrane potential (DeltaPsi(m)) probes, we found clear responses to DeltaPsi(m) altering drugs for both probes. METHODS: The three probes were titrated to determine whether saturation played a role in the response to drugs. The effects of a variety of DeltaPsi(m) altering drugs were tested for MFG and MTG at probe concentrations of 20 nM and 200 nM and for NAO at 0.1 microM and 5 microM, using rhodamine 123 at 0.1 microM as a reference probe. RESULTS: Incubation of GM130, HL60, and U937 cells with 2,3-butanedione monoxime (BDM), nigericin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), 2,4-dinitrophenol (DNP), gramicidin, ouabain, and valinomycin resulted in increases of the fluorescence intensity for MFG or MTG with only a few exceptions. The fluorescence intensity of cells stained with 0.1 microM NAO increased following incubation with BDM, nigericin, and decreased for FCCP, CCCP, DNP, gramicidin, and valinomycin. The results with 5 microM NAO were similar. CONCLUSIONS: MFG, MTG, and NAO appeared poor choices for the membrane potential independent analysis of mitochondrial membrane mass. Considering the molecular structure of these probes that favor accumulation in the mitochondrial membrane because of a positive charge, our results are not surprising. Cytometry 39:203-210, 2000. Published 2000 Wiley-Liss, Inc.     

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase exhibit proton translocating activity in the presence of gramicidin.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) were characterized for electron transfer and proton translocating activities in the presence of the mobile potassium ionophore, valinomycin, and the channel-forming ionophore, gramicidin, in order to determine if the ionophores modify the functional properties of the enzyme. In agreement with previous work, incubation of COV with valinomycin resulted in a perturbation of the absorbance spectrum of oxidized heme aa3 in the Soret region (430 nm); gramicidin had no effect on the heme aa3 absorbance spectrum. Different concentrations of the two ionophores were required for maximum respiratory control ratios in COV; 40- to 70-fold higher concentrations of valinomycin were required to completely uncouple electron transfer activity when compared to gramidicin. The proton translocating activity of COV incubated with each inophore gave a similar apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) of 0.66 +/- 0.10. However, COV treated with low concentrations of gramicidin (0.14 mg/g phospholipid) exhibited 1.5- to 2.5-fold higher rates of alkalinization of the extravesicular media after the initial proton translocation reaction than did COV treated with valinomycin, suggesting that gramicidin allows more rapid equilibration of protons across the phospholipid bilayer during the proton translocation assay. Moreover, at higher concentrations of gramicidin (1.4 mg/g phospholipid), the observed H+/e- ratio decreased to 0.280 +/- 0.020, while the rate of alkalinization increased an additional 2-fold, suggesting that at higher concentrations, gramicidin acts as a proton ionophore. These results support the hypothesis that cytochrome c oxidase is a redox-linked proton pump that operates at similar efficiencies in the presence of either ionophore. Low concentrations of gramicidin dissipate the membrane potential in COV most likely by a channel mechanism that is different from the carrier mechanism of valinomycin, yet does not make the phospholipid bilayer freely permeable to protons.