Concentrations Of Valinomycin Research Articles

Localized coupling in oxidative phosphorylation by mitochondria from Jerusalem artichoke (Helianthus tuberosus)

Delocalized chemiosmotic coupling of oxidative phosphorylation requires that a single-value correlation exists between the extent of Δ \\ ̃ gm H + and the kinetic parameters of respiration and ATP synthesis. This expectation was tested experimentally in nigericin-treated plant mitochondria in single combined experiments, in which simultaneously respiration (in State 3 and in State 4) was measured polarographically, FΔψ (which under these conditions was equivalent to Δ \\ ̃ gm H + ) was evaluated potentiometrically from the uptake of tetraphenylphosphonium + and the rate of phosphorylation was estimated from the transient depolarization of mitochondria during State 4-State 3-State 4 transitions. The steady-state rates of the different biochemical reactions were progressively inhibited by specific inhibitors active with different modalities on various steps of the energy-transducing process: succinate respiration was inhibited competitively with malonate or noncompetitively with antimycin A, or by limiting the rate of transport into the mitochondria of the respiratory substrate with phenylsuccinate; Δ \\ ̃ gm H + was dissipated by uncoupling with increasing concentrations of valinomycin; ADP phosphorylation was limited with oligomycin. The results indicate generally that when the rate of respiratory electron flow is decreased, a parallel inhibition of the rate of phosphorylation is also observed, while very limited effects can be detected on the extent of Δ \\ ̃ gm H + . This behavior is in marked contrast to the effect of uncoupling where the decreased rate of ATP synthesis is clearly due to energy limitation. Extending previous observations in bacterial photosynthesis and in respiration by animal mitochondria and submitochondrial particles the results indicate, therefore, that respiration tightly controls the rate of ATP synthesis, with a mechanism largely independent of Δ \\ ̃ gm H + . These data cannot be reconciled with a delocalized chemiosmotic coupling model.

Valinomycin can depolarize mitochondria in intact lymphocytes without increasing plasma membrane potassium fluxes

We have simultaneously measured [ 3H]methyltriphenylphosphonium + and 86Rb + distribution across the mitochondrial and plasma membranes of pig mesenteric lymphocytes. At low concentrations the K + ionophore valinomycin depolarizes the mitochondria but does not increase Rb + fluxes across the plasma membrane. At high concentrations of valinomycin the plasma membrane becomes permeable to Rb + and both TPMP + and Rb + distribution give a measure of the K + diffusion potential.

Studies on valinomycin inhibition of synaptosome-fraction protein synthesis.

The ionophore valinomycin inhibited adult and neonatal synaptosome fraction protein synthesis with half-maximal inhibition at approximately 10nM. Valinomycin had no effect on [3H]leucine uptake into synaptosomes at high or low external [K+]. Synaptosome-fraction protein synthesis was dependent on [K+]e reaching a maximum at 25mM-K+. Valinomycin inhibition of protein synthesis was not reversed at high [K+]e. Valinomycin failed to influence the intrasynaptosomal [K+] even at zero [K+]e. A significant increase in State-4 respiration of synaptosomal fractions was found at 5nM-valinomycin with a decrease in the respiratory control index. At these concentrations of valinomycin there was no inhibition of the ADP-stimulated (State 3) respiration rate. Valinomycin had no effect on cerebral microsomal protein synthesis in vitro, which was inhibited by puromycin (100 micrograms/ml) or the absence of ATP. Valinomycin, 2,4-dinitrophenol and KCN inhibition of protein synthesis was not reversed by added ATP, suggesting impermeability of the membrane to ATP. Valinomycin induced a rapid fall in synaptosome ATP content not observed with atractylate or ouabain. Valinomycin inhibition of protein synthesis under these conditions is secondary to uncoupling of mitochondrial oxidative phosphorylation with a subsequent decrease in intraterminal ATP necessary for translation.

Modulation by small hydrophobic molecules of valinomycin-mediated potassium transport across phospholipid vesicle membranes.

The effects of small hydrophobic molecules on valinomycin-mediated K+ transport in small unilamellar soybean phospholipid vesicles have been studied by using a vesicle-entrapped pH-sensitive hydrophilic fluorescence probe to monitor counterion-limited, passive H+ diffusion into vesicles after an abrupt decrease in external pH [Clement, N. R., & Gould, J. M. (1981) Biochemistry (preceding paper in this issue)]. Under conditions where, even in the absence of valinomycin, transmembrane KL+ movement represented the primary and limiting counterion flux, less than 1 valinomycin molecule/vesicle was sufficient to accelerate the rate of H+ entry into all of the vesicles. Incorporation of the bulkily substituted molecules butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and p-di-tert-butylbenzene into soybean lipid bilayers had no effect upon K+ diffusion in the absence of valinomycin. However, the presence of these hydrophobic molecules increased the apparent efficacy for K+ transport of a given valinomycin concentration by as much as 4-6 fold. The less bulky membrane perturbants tert-butyl alcohol, phenol, and heptane showed very much less dramatic effects. While the rate of valinomycin-mediated K+ transport (in the presence or absence of BHT) was very sensitive to temperature-induced changes in membrane fluidity, the degree of synergistic interaction between valinomycin and BHT was independent of temperature. Furthermore, BHT, BHA, and p-di-tert-butylbenzene, at levels which alter valinomycin-mediated K+ transport, did not by themselves induce changes in membrane fluidity. It is postulated that changes in phospholipid head-group packing and/or surface charge density brought about by the presence of bulky perturber molecules leads to changes in partitioning of valinomycin or the valinomycin-K+ complex between the aqueous and membrane phases.

Ion permeability in the erythrocyte membrane of the dystrophic chicken measured by a fluorometric method.

The relative Cl permeability in the erythrocyte membrane of the dystrophic chicken was estimated from the potential changes measured optically by a fluorescent dye. The reduced Cl conductance relative to the K conductance induced by a constant concentration of valinomycin was suggested in the dystrophic erythrocyte membrane in comparison with that in the control.

Block in the elongation of protein synthesis in rabbit reticulocyte by action of the ionophore valinomycin.

In this paper we show that ribosomes isolated from cells inhibited for protein synthesis by the antibiotic ionophore Valinomycin can still support incorporation of [14C]-leucine into polypeptides in a cell-free system. The extent of this functional integrity depends upon the concentration of Valinomycin used and whether it acted as an ionophore or not. We demonstrate that the antibiotic acts on protein synthesis at the level of elongation and has no action either at the initiation level or as activating an RNase. Moreover, we show that it can inhibit protein synthesis at concentrations where its action as an ionophore cannot be detected.

Interaction between membrane properties and protein synthesis in reticulocytes: influence of trypsinization on [3H]-valinomycin action.

Using [3H]-Valinomycin we show here that two types of sites can be described for this cyclic depsipeptide. A first type is sensitive to low concentration of trypsin while the other, more internal, is uncovered by the use of the protease. Of these two kinds of sites, the more external one seems more concerned with the effect that Valinomycin has on protein synthesis in rabbit reticulocytes. However, when a high concentration of Valinomycin is used, all the sites can be occupied even those which can be revealed only by tripsinization. In this case, even prolonged trypsin action does not result in release of the protein synthesis inhibitory action of Valinomycin. It is concluded that hydrophobic sites are occupied by Valinomycin only after the cell surface has been saturated by hydrophylic bonds with the antibiotic.

The stimulus-secretion coupling of glucose-induced insulin release: Effects of valinomycin upon pancreatic islet function

In isolated rat pancreatic islets, valinomycin (0.01 to 1.0 μ m) caused a dose-related facilitation of 86Rb + outflow and a dose-related inhibition of the glucose-induced changes in both outflow and net uptake of 86Rb +. At high concentrations (0.1–1.0 μ m), the ionophore also inhibited the oxidation of glucose and endogenous nutrients, decreased the adenylate charge, and lowered the concentration of reduced pyridine nucleotides in the islet cells. However, as little at 1.0 to 10.0 n m valinomycin caused anomalies in the handling of 45Ca 2+ (suppression of the early inhibitory effect of glucose upon 45Ca 2+ efflux, and reduction in the amount of 45Ca 2+ recovered in the islets after an extensive washing procedure) and inhibition of insulin release. Moreover, when the effect of glucose upon K + conductance was abolished by high concentrations of valinomycin (0.1–1.0 μ m), the glucose-induced secondary rise in 45Ca 2+ efflux was still observed. These findings suggest that the effects of glucose upon 86Rb + and 45Ca 2+ handling, respectively, although normally concomitant with one another, can be dissociated, in part at least, from one another. It is concluded that the glucose-induced reduction in K + outflow may be unnecessary for the sugar to cause a partial remodeling of Ca 2+ fluxes in the islet cells.

The effect of antibiotics on the photocycle and protoncycle of purple membrane suspensions

The interrelation was studied between the phototransient absorbing maximally at 412 nm (M 412) and light-induced proton release under steady-state conditions in aqueous suspensions of ‘purple membrane’ derived from Halobacterium halobium. The decay of M 412 was slowed down by the simultaneous application of the ionophoric antibiotics valinomycin and beauvericin. The former had only slight activity alone and the latter was effective only in conjunction with valinomycin. The steady-state concentration of M 412 which was formed on illumination was a direct function of the concentration of valinomycin. Maximum stabilization of M 412 was obtained when the valinomycin was approximately equimolar with the bacteriorhodopsin. Addition of salts to the medium increased the number of protons released per molecule of M 412 without affecting the level of M 412 which was produced by continuous illumination. The effectiveness of the salts in this respect depended on the nature of the cation. Ca 2+ and their antagonists La 3+ and ruthenium red were found to have especially high affinity for the system. The extent of light-induced acidification could not be enhanced by increasing the pH of the medium from 6.5 to 7.8. The possible mechanism of action of the ionophores and of the cations on the photocycle and on the proton cycle is discussed.

Protonmotive force and motility of Bacillus subtilis.

Motility of Bacillus subtilis was inhibited within a few minutes by a combination of valinomycin and a high concentration of potassium ions in the medium at neutral pH. Motility was restored by lowering the concentration of valinomycin or potassium ions. The valinomycin concentration necessary for motility inhibition was determined at various concentrations of potassium ions and various pH's. At pH 7.5, valinomycin of any concentration did not inhibit the motility, when the potassium ion concentration was lower than 9 mM. In the presence of 230 mM potassium ion, the motility inhibition by valinomycin was not detected at pH lower than 6.1. These results are easily explained by the idea that the motility of B. subtilis is supported by the electrochemical potential difference of the proton across the membrane, or the protonmotive force. The electrochemical potential difference necessary for motility was estimated to be about -90 mV.

Mechanism of 3,5-di- tert-butyl-4-hydroxybenzylidenemalononitrile-mediated proton uptake in liposomes. Kinetics of proton uptake compensated by valinomycin-induced k +-efflux

1. Kinetic studies were made on proton translocation across the phospholipid bilayer membrane in liposomes; this proton translocation is mediated by the uncoupler 3,5-di- tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847) and compensated by valinomycin-induced potassium efflux. The initial rate of proton uptake was directly proportional to the concentration of SF6847 in the presence of excess valinomycin, indicating that the monomer of SF6847, at least, is a proton carrier and participates in limiting the rate of the proton uptake cycle. 2. The partition constant of SF6847 between the liposomal membranes, prepared from Escherichia coli phospholipids, and the aqueous phase was determined kinetically to be 21 mM −1 in medium of pH 7.0 and 4.5 mM −1 in medium of pH 8.0. The turnover rates of SF6847 across the bilayer membrane at pH 7.0 and 8.0 were determined as 140 and 550 times/molecule per s, respectively. 3. The initial rate of proton uptake depended hyperbolically on the concentration of valinomycin in the presence of a limiting amount of SF6847. It also depended on the concentration of potassium ion (K +) inside the liposomes. These results suggest strongly that a ternary complex valinomycin · K + · SF − (SF −, anionic form of SF6847) is a direct intermediate, participating in limiting the rate of the proton uptake cycle. 4. The equilibrium constants of the following equilibria, valinomycin + K + ▪ valinomycin · K + and valinomycin · K + + SF − ▪ valinomycin · K + · SF − were determined: K 1 was 3.8 · 10 −1M and K 2 A ( A, constant; see text) was 4.0 · 10 −9M under the conditions employed. From these results a model is proposed for the mechanism of the proton uptake cycle mediated by SF6847 in liposomes. 5. The mechanism of proton uptake mediated by carbonylcyanide m-chlorophenylhydrazone (CCCP) was also studied. It was concluded that the mechanism was essentially the same as that mediated by SF6847.

Cholesterol stimulation of penetration of unilamellar liposomes by hydrophobic compounds

The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of negericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution. Cholesterol similarly increases the ability of A-23187, 1799, or NH4SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb+ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca45 trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb+ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.

Laser-induced fluorescence studies of intact biological membranes

Using a pulsed, tunable dye laser details of the fluorescence behavior of single, intact human red blood cells have been studied. The high sensitivity of the laser excited fluorescence allows the detection of potential-dependent uptake and release of dye added to the suspension of living erythrocytes. The membrane potential of the erythrocytes was changed by means of different concentrations of valinomycin and potassium chloride in the suspension medium. Dye concentrations as low as 10 -8 M in water have been detected in a layer of 10 μm thickness and a cross sectional area of 1 μm 2.

The relationship between anion exchange and net anion flow across the human red blood cell membrane.

The conductive (net) anion permeability of human red blood cells was determined from net KCl or K2SO4 effluxes into low K+ media at high valinomycin concentrations, conditions under which the salt efflux is limited primarily by the net anion permeability. Disulfonic stilbenes, inhibitors of anion exchange, also inhibited KCl or K2SO4 efflux under these conditions, but were less effective at lower valinomycin concentrations where K+ permeability is the primary limiting factor. Various concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) had similar inhibitory effects on net and exchange sulfate fluxes, both of which were almost completely DIDS sensitive. In the case of Cl-, a high correlation was also found between inhibition of net and exchange fluxes, but in this case about 35% of the net flux was insensitive to DIDS. The net and exchange transport processes differed strikingly in their anion selectivity. Net chloride permeability was only four times as high as net sulfate permeability, whereas chloride exchange is over 10,000 times faster than sulfate exchange. Net OH-permeability, determined by an analogous method, was over four orders of magnitude larger than that of Cl-, but was also sensitive to DIDS. These data and others are discussed in terms of the possibility that a common element may be involved in both net and exchange anion transport.

Energy-linked potassium uptake by mitochondria from wild-type and poky strains of Neurospora crassa.

Mitochondria from Neurospora crassa, like mammalian mitochondria, carry out rapid, energy-linked K+ uptake and H+ release in the presence of valinomycin. The maximal rate of K+ uptake was about 1.0 mumol/mg of mitochondrial protein per min and was seen at valinomycin concentrations in the range of 100 to 200 mug per mg of mitochondrial protein and at K+ concentrations of 4 mM or above. Uptake could be supported either by substrate oxidation or by adenosine 5'-triphosphate (ATP), and was inhibited in the former case by antimycin or cyanide, in the latter case by oligomycin, and in both cases by 2,4-dinitrophenol. Mitochondria from the cytochrome-deficient mutant poky carried out substrate-driven K+ uptake at reduced rates, but oligomycin-sensitive, ATP-driven K+ uptake at rates about 60% greater than those shown by wild-type mitochondria. This result is consistent with the recent finding (Mainzer and Slayman 1976) that poky contains elevated amounts of oligomycin-sensitive mitochondrial adenosine 5'-triphosphatase activity.

A potassium lonophore inhibits lymphocyte capping: The role of cations in the modulation of membrane receptors

Valinomycin inhibited cap formation induced by antibodies to Ig-molecules in human lymphocytes. At the concentration of valinomycin used in this study the inhibitory effects: (1) could not be explained by impaired cell viability, (2) were reversed by washing the drug from the medium and (3) were prevented by increasing the concentration of K + in the external medium. These results suggest that valinomycin may act, in part, by altering the electrical properties of the cell membrane and suggest a role for membrane potential and cation permeability and flux in the modulation of membrane receptors.

Mechanism of active shrinkage in mitochondria II. Coupling between strong electrolyte fluxes

1. Addition of succinate to valinomycin-treated mitochondria incubated in KCl causes a large electrolyte penetration. The process depends on a steady supply of energy and involves a continuous net extrusion of protons. Rates of respiration and of electrolyte penetration proceed in a parallel manner. 2. A passive penetration of K + salt of permeant anions occurs in respiratory-inhibited mitochondria after addition of valinomycin. Addition of succinate at the end of the passive swelling starts an active extrusion of anions and cations with restoration of the initial volume. The shrinkage is accompanied by a slow reuptake of protons. The initiation of the active shrinkage correlates with the degree of stretching of the inner membrane. The extrusion of electrolytes is inhibited by nigericin, while it is only slightly sensitive to variations of the valinomycin concentration larger than two orders of magnitude. 3. Passive swelling and active shrinkage occurs also when K + is replaced by a large variety of organic cations. The rate of organic cation penetration is enhanced by tetraphenylboron, while the rate of electrolyte extrusion is insensitive to variation of the tetraphenylboron concentration. 4. Active shrinkage, either with K + or organic cation salts, is inhibited by weak acids. The phosphate inhibition is removed by SH inhibitors. The active shrinkage is also inhibited by mersalyl to an extent of about 60%. 5. Three models of active shrinkage are discussed: (a) mechanoprotein, (b) electrogenic proton pump, and (c) proton-driven cation anion pump.

Interaction between membrane properties and protein synthesis in reticulocytes ? A two step inhibition of protein synthesis by Valinomycin

In this work we consider the differential effect of Valinomycin used at different concentrations both on the protein synthesis of reticulocytes and on 42K exchange. We demonstrate that there is a two step action of this antibiotic. At 10(-6)M and below the drug has no effect on the 42K exchange, but it stops, however reversibly, protein synthesis. At 10(-5)M the drug has a very sharp action on the 42K exchange and stops protein synthesis in an irreversible way. Ribosomal population checked by two ways, sucrose gradient and direct counting on E.M. sections shows that at low concentrations of Valinomycin (10(-8)M to 10(-6)M) there is no breakdown of the polysomes which can be detected by either one of these methods. On the contrary, after short incubation with 10(-5)M of Valinomycin the breakdown of ribosomes is very clear, as evidenced by sucrose gradient analysis. By direct ribosomes clusters counting on E.M. sections this breakdown is seen only after long incubation.

Kinetics of the valinomycin-induced potassium ion leak from liposomes with potassium thiocyanate enclosed

The kinetics of the valinomycin-induced K + leak from egg lecithin liposomes with KCNS enclosed were studied by measuring the initial increase of the leak following the addition of the ionophore. Variation of liposome and valinomycin concentrations enabled a discrimination between the affinity of the ionophore for the lipid bilayer and the turnover rate of the transport process. The results indicate that valinomycin is distributed immediately between the outer liposomal bilayer and the aqueous phase. A partition constant of 0.0222 cm at 30°C was obtained for egg lecithin liposomes. By varying the temperature it was shown that the partition constant has a negative temperature coefficient. The Arrhenius activation energy of the transport process itself is 15.7 ± 1.1 kcals/mole. Concerning the mechanism of the valinomycin-mediated transport it was found that the complex formation can be described kinetically by the formation of a ternary complex in which K + and a thiocyanate ion are bound by the ionophore. By varying the fatty acid composition of the membrane constituents it was found that the turnover rate of the transport increased with increasing unsaturation of the phospholipids present in the membrane. This result suggests that the translocation of the complex is the rate-limiting step of the transport cycle. At 30 °C a turnover constant of 328 ± 44 potassium ions/molecule valinomycin per second was obtained for egg lecithin lipsomes.

Studies on the Regulation of Gluconeogenesis in Isolated Rat Liver Cells by Epinephrine and Glucagon

Abstract The addition of glucagon or epinephrine to isolated rat liver parenchymal cells stimulated gluconeogenesis from alanine, lactate, glycerol, xylitol, oxalacetate, pyruvate, and fructose. The stimulation of gluconeogenesis due to maximal concentrations of epinephrine was additive to that of either glucagon or N6, O2'-dibutyryl adenosine 3' : 5'-monophosphate (dibutyryl cyclic AMP). Similarly low concentrations of valinomycin and lysine stimulated gluconeogenesis but the same increases in glucose production due to epinephrine, glucagon, or dibutyryl cyclic AMP were seen in the presence as in the absence of lysine or valinomycin. However, the stimulation of glucose production due to glucagon was not additive to that of dibutyryl cyclic AMP. Gluconeogenesis was reduced by the absence of K+ or Ca2+ from the buffer. The addition of 0.65 mm Ca2+ or 2.4 mm K+ restored basal gluconeogenesis and the response to epinephrine or dibutyryl cyclic AMP. The effect of epinephrine was potentiated by increasing the Ca2+ concentration from 0.65 to 2.6 mm. The addition of ouabain to liver cells had effects similar to those seen when K+ was omitted from the buffer. These results indicate that the stimulation of gluconeogenesis by glucagon or epinephrine is not restricted to substrates which enter the pathways of glucose formation prior to phosphoenolpyruvate, and support the hypothesis that epinephrine mediated gluconeogenesis occurs by a cyclic adenosine 3' : 5'-monophosphate-independent mode of action distinct from that for glucagon.