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

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

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

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

The use of carotenoids and oxonol VI as probes for membrane potential in proteoliposomes

Carotenoids present in lipids extracted from the cyanobacterium Synechococcus 6716 indicate trans-membrane potential in proteoliposomes reconstituted from these lipids and the ATPase complex isolated from the same organism. A carotenoid absorbance band shift to a longer wavelength is obtained with valinomycin-induced potassium ion diffusion potentials, irrespective of the polarity of the potassium gradient. In contrast to this, the (externally added) probe oxonol VI only shows an absorbance band shift when the external potassium ion concentration is higher than the internal one. In liposomes without ATPase complex, no carotenoid absorbance band shifts were observed.     

A PVC-based electrode sensitive to DDA+ as a device for monitoring the membrane potential in biological systems

A polyvinyl chloride (PVC)-based membrane electrode sensitive to dibenzyldimethyl ammonium cation (DDA⁺) was constructed, and operational parameters such as the selectivity coefficients, the detection limit, and the response time were obtained. In comparison with the selectivity coefficients obtained with the previous liquid-membrane electrode, significant improvement was not obtained, but the response time became pronouncedly shorter. Furthermore, the electrode lifetime was remarkably prolonged. With the electrode developed, the change in the membrane potential of liposomes containing dibutyl ferrocene which separated oxidizing and reducing agent solutions was measured. The DDA⁺ uptake, U, and the membrane potential estimated from U changed in accordance with the redox potential in the medium when the concentration of internal ferricyanide was kept constant. The membrane potential collapsed when the uncoupler of oxidative phosphorylation was added. The ANS fluorescence measurement indicated that negative charges appeared on energization with oxidizing-reducing agent. The change in membrane potential of mitochondria during energization was also measured. It was found that the liposome described above is a good model for the generation of membrane potentials in mitochondria.     

Amino acid transport in membrane vesicles from CHO-K1 and alanine-resistant transport mutants

Membrane vesicles were prepared from CHO-K1 and alanine-resistant transport mutants, alar4 and alar4-H3.9. Alar4 is a constitutive mutant of the A system, and alar4-H3.9, derived from alar4, may be the result of amplification of a gene coding for an A-system transporter. Under conditions in which the same membrane potential (interior negative) and Na+ gradient were employed, the mutant vesicles show increases in the A system over that of the parental CHO-K1 cell line, paralleling, but not equivalent to, that found in whole cells. L-system and 5'-nucleotidase activities of these vesicles were similar, indicating that the increased A-system activity of the mutant vesicles is not due to the differential enrichment of the A system in these vesicles. The membrane potential was produced by a K+ diffusion gradient (internal greater than external) in the presence of valinomycin or by the addition of a Na+ salt of a highly permeant anion such as SCN-. Monensin was employed to study the effect of the Na+ gradient on transport and membrane potential. The latter was determined by measuring the uptake of tetraphenylphosphonium ion. A negative membrane potential determines the concentrative ability and the initial velocity of the A system in these vesicles. The concentration of external Na+ has a stimulatory effect on the initial velocity of this system. However, the Na+ gradient (external greater than internal) has no effect on the initial velocity or the membrane potential when the potential is set by valinomycin and high internal K+. Little if any ASC system could be detected in vesicles from CHO-K1.     

Membrane potential difference of isolated plant vacuoles: positive or negative? I. Evidence for membrane binding of cationic probes

The binding of membrane potential cationic probes was studied on phospholipidic liposomes by equilibrium dialysis and microelectrophoresis. Surface binding of lipophilic cations (benzyltributylammonium or tetraphenylphosphonium) appears to be the major accumulation mechanism in liposomes and simulates the existence of a negative transmembrane potential (E_m), in absence of any transmembrane ionic gradient. Furthermore, this apparent negative potential has a classical response with regard to common E_m effectors, namely a depolarization induced by KCl or FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone). The relevance of these results to the study of transtonoplast potential difference on isolated vacuoles was investigated. Tetraphenylphosphonium was shown to bind to the tonoplast, the essential features of binding and interaction with E_m effectors being similar in vacuoles and liposomes. Therefore the assumption of negligible binding of cationic probe to vacuoles, classically admitted in determinations of vacuolar E_m using lipophilic cations, is untenable.     

Liposomes as model for taste cells: receptor sites for bitter substances including N-C=S substances and mechanism of membrane potential changes

Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.     

Ion transport and the vibrating probe.

The theory of ion transport in the vicinity of a vibrating probe is developed. It is shown that the convection loops produced by the probe will not affect the electrical current density, assuming that the action of the probe does not affect the sources of the current in the biological system. However, the convection loops will significantly alter the ion concentration gradients in the unstirred layer near a tissue or cell surface. The concentration gradients within each convection loop will be reduced, while the concentration gradients between the loops and outside of the loops will be increased relative to the gradients existing without the probe. As a consequence, the electrical potential gradients can be changed relative to the potential gradients existing in the absence of the convection caused by the probe. If the mobility of the ion species carrying the electrical current is greater than the average ion mobility in the medium, then a decrease in ion concentration gradient will be accompanied by an increase in electrical potential gradient, while an increase in concentration gradient will be accompanied by a decrease or even a reversal of electrical potential gradient. Thus, the electrical potential gradient measured by the probe will depend on the concentration gradient in the vicinity of the probe, which will depend in turn on the spatial relation of the convection loops to the probe. An example of the effect of the convection loops on ion concentration and electrical potential is obtained from the theory via a numerical computer calculation. Experimental tests of this theory are discussed.     

Determination of osmotic volumes and pH gradients of plant membrane and lipid vesicles using ESR spectroscopy

Volumes and pH gradients were determined with spin probes in liposomes and zucchini membrane vesicles by quantitating the internal concentrations of probes in the presence of an impermeable line-broadening agent, manganese + EDTA. Volume shrinkage in response to increasing external concentrations of MnEDTA was consistent with perfect osmotic behavior of both vesicle populations. Buffer additions were used to impose pH gradients on the vesicles; liposome gradients measured with a spin-labeled weak acid were slightly smaller than the maximum theoretical imposed gradients, whereas above a threshold magnitude, measured gradients for the plant membranes were significantly smaller than imposed gradients. However, the residual pH gradient in the zucchini vesicles decreased at about the same rate as the liposome gradient. Moreover, this residual gradient was not completely collapsed in the presence of the proton ionophore, FCCP, indicating that the vesicles were impermeable to ions; indeed, ion permeabilities of both vesicle preparations appeared to be similar during the slow phase of the pH gradient collapse. Thus, zucchini membrane vesicles are tightly sealed and appear to have a mechanism for dissipating pH gradients rapidly when these gradients exceed some threshold value.     

Depolarization of the Internal Membrane System in the Activation of Frog Skeletal Muscle

"Skinned" muscle fibers, single fibers from the frog semitendinosus muscle in which the sarcolemma had been removed, could be reversibly activated by electrical stimulation. Electrical responsiveness was abolished when the skinned fiber was prepared from a muscle exposed to a cardiac glycoside, and the development of responsiveness was delayed when the muscle was bathed in high potassium solution. The findings were taken as evidence that active sodium-potassium exchange across the internal membranes restored electrical excitability, after the sarcolemma had been removed, by establishing a potential gradient across the internal membranes. In general, the contractions were graded with the strength of the applied current. On occasion, however, "all-or-none" type responses were seen, raising the possibility that the internal membranes were capable of an electrically regenerative response. Activation could also be produced by an elevation of the intracellular chloride ion concentration or a decrease in the intracellular potassium, ion concentration, suggesting that depolarization of some element of the internal membrane system, that is, a decrease in the potential of the lumen of the internal membrane system relative to the potential of the myofibrillar space, was responsible for activation in these experiments. The distribution of both the electrically induced contractions and those produced by changes in the intracellular ion concentrations indicated that the responsive element of the internal membrane system was electrically continuous over many sarcomeres.     

Preparation and characterization of large dioctadecyldimethylammonium chloride liposomes and comparison with small sonicated vesicles

Dioctadecyldimethylammonium chloride (DODAC) unilamellar liposomes with a mean external diameter of 0.5 μm and sharp gel-to-liquid-crystalline phase transition temperatures (T_c) were obtained by chloroform vaporization and compared with small sonicated DODAC vesicles. Sucrose, impermeant through large DODAC liposomes and sonicated vesicles, was used for internal volume determinations. The internal volumes for large DODAC liposomes and sonicated DODAC vesicles were 9.0 ± 1.3 and 0.13 ± 0.2 l/mol, respectively. Ideal osmometer behaviour, towards KCl (0–50 mM) and sucrose, was observed only for large DODAC liposomes. Sonicated DODAC vesicles were osmotically non-responsive towards sucrose and flocculated upon addition of KCl. At temperatures near the T_c, a steep increase in the initial shrinkage rate and a minimum for the total extent of shrinkage were observed for large DODAC liposomes. Large DODAC liposomes are proposed as an adequate synthetic membrane model.     

Measurement of the electrochemical proton gradient in submitochondrial particles

The pH gradient and membrane potential of submitochondrial particles from bovine heart were estimated by the uptake of [14C]ethylamine and [36Cl]perchlorate, using filtration through a glass fiber prefilter and Millipore filter without washing to separate the vesicles from the medium. An external volume probe of [3H] sucrose was also used. Internal volume of the vesicles was measured by the extent of uptake of glucose, which equilibrates slowly across the membrane. The electrochemical potential gradient of H+ (delta micro H+) calculated from uptake of ethylamine and perchlorate, assuming the ions taken up were free in solution inside the vesicles, was 23 to 24 kJ/mol of H+ (240-250 mV) during respiration in the absence of ATP. The ratio of the free energy of ATP synthesis (delta GATP) to delta micro H+ was 2.2 to 2.3 during oxidative phosphorylation and only slightly higher during ATP hydrolysis indicating that the H+-translocating ATPase is close to equilibrium under both conditions. The nonintegral ratio suggests there is a systematic error in the measurement of delta micro H+. The value of delta micro H+ calculated from ion uptake could be too high if some of the ions taken up are bound to the membrane or concentrated into the electric double layer at the inner membrane-water interface. The effects of vesicle volume (varied osmotically) and permeant ions (which affect internal ionic strength and pH) on the ratio of delta GATP to delta micro H+ suggested that ion association with the membrane in fact caused significant overestimation of delta micro H+. Association of ethylammonium and perchlorate ions with unenergized submitochondrial particles was measured by centrifugation, in the presence of a high concentration of impermeant salt to minimize association with the external surface. The results were used to estimate the extent of binding during the ion uptake assays, and delta micro H+ was recalculated taking this binding into account. The resulting values were between 19 and 20 kJ/mol of H+ (197-207 mV) during respiration in the absence of ADP, and the ratio of delta GATP to delta micro H+ was about 3 during oxidative phosphorylation.     

Proton decay kinetics for vesicles containing buffers—an analytical solution

The problem of predicting the kinetics of proton efflux and the decay of the internal proton concentration for vesicles containing one or more buffers for which the internal proton concentration is initially higher than that of the surrounding medium is examined. An analytical solution is derived that describes the time course of the proton efflux from vesicles and the decay of the internal proton concentration under conditions of zero transmembrane electric potential. The effect of the internal buffers is to increase the time required for the proton concentration gradient to equilibrate across the membrane. To simplify the analysis we assume that the equilibration of the internal and external proton activity is due primarily to proton diffusion through the membrane, and not to hydroxyl ion flux. For a vesicle containing a single buffer the solution requires six independent physical parameters: the initial internal proton concentration, the external proton concentration, the ratio of the vesicle surface area to the internal volume, the permeability coefficient of the membrane for protons, the total concentration of the internal buffer, and the equilibrium constant for the dissociation of the internal buffer. Determination of these physical values is sufficient to predict the time dependence of the internal proton concentration and of the proton efflux. Over a pH range that is below or near the pK of the internal buffer the solution is complex. However, if the initial pH is one unit or more higher than the pK of the internal buffer the kinetics of the internal proton concentration and proton efflux can be described by a pseudo first order reaction. In this case the apparent rate constant depends linearly on the permeability coefficient and is dominated by the total internal buffer concentration and its pK. For example, increasing the internal buffer concentration inside a vesicle by 10-fold results in an approximately 10-fold increase in the half-time of the proton efflux kinetics. The theoretical analysis is applied to thylakiod vesicles using experimentally determined values for the physical parameters. The predictions of the analysis are compared to experimentally observed kinetics.     

Membrane inlet ion trap mass spectrometry for the direct measurement of dissolved gases in ecological samples

The use of an ion trap mass spectrometer with three different membrane inlet probes is described. Two methods of removing water from the sample are compared. One is the use of a PTFE-silicone rubber double membrane, PTFE is relatively impermeable to water and so reduces the amount entering with the gas sample (Probe A). The second is the use of a silicone rubber membrane covered long probe, which condenses water out of the sample (Probe B). Response times (100%) for dissolved N2O, O2, Ar and CO2 without He in the chamber vary from between 158 and 684 s with Probe A. For the same probe with He, the response times were between 283 and 551 s. In the gas phase response times were between 99 and 153 s with He and 117 and 122 s without He. Probe B had 100% response of between 122 and 152 s for dissolved gases. Further extension of the probe by 2 m slowed response times as did increasing the ionisation time. Response times for Probe B increased to between 99 and 340 s when ionisation time increased from 1000 to 24,930 microseconds. Plots of output against concentration showed the steepest line of response for the short single membrane covered probe with 1000 microseconds ionisation time. Increasing the ionisation time, extending the probe and the use of a double membrane all reduced the gradient of output against concentration for every gas tested. In an intact sediment core, concentrations of O2, N2O and CO2 rose at the start and the concentration of N2 fell. As the disturbed sediment settled, this was reversed. The initial increase in O2 concentration stimulated respiration and inhibited the final pathway in dentrification producing higher concentrations of N2O and reducing the concentration of N2.     

Internal volume determination of liposomes by a rapid equilibrium technique using hydrogen iodide as a marker

Summary A new method is described for determining the aqueous internal volume of small unilamellar liposomes. The method is based on an equilibrium uptake of hydrogen iodide by liposomes. After the chromatographical separation of liposomes from external solution, the membrane is solubilized and the amount of captured iodide determined using an ion selective electrode.     

The dependence of the rate of transhydrogenase on the value of the protonmotive force in chromatophores from photosynthetic bacteria

In conditions where the pH gradient is negligible, the rate of the pyridine nucleotide transhydrogenase in chromatophores of Rhodobacter capsulatus has a threshold dependence on membrane potential. The relationship is similar when either antimycin or myxothiazal or carbonyl cyanide p-trifluoromethoxyphenylhydrazone is used to depress the membrane potential. The relationship is distorted when membrane potential is reduced by lowering the photosynthetic light intensity.     

Synchronization modulation of Na/K pump molecules can hyperpolarize the membrane resting potential in intact fibers

Previously, we have theoretically studied the possibility of electrical rhythmic entrainment of carrier-mediated ion transporters, and experimentally realized synchronization and acceleration of the Na/K pumping rate in the cell membrane of skeletal muscle fibers by a specially designed synchronization modulation electric field. In these studies we either used cut fibers under a voltage clamp or intact fibers, but in the presence of ion channels blockers. A question remained as to whether the field-induced activation observed in the pump molecules could effectively increase the intracellular ionic concentration and the membrane potential at physiological conditions. In this paper, we studied the effects of the field on intact fibers without any channel blockers. We monitored the field-induced changes in the ionic concentration gradient across the cell membrane and the membrane potential non-invasively by using a fluorescent probe and confocal microscopic imaging techniques. The results clearly show that the entrainment of the pump molecules by the synchronization modulation electric field can effectively increase the ionic concentration gradient, and hence, hyperpolarize the membrane potential.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3--5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 micrometer diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration.     

Theoretical study of flow,pressure and solute concentration in double membrane systems

A model system consisting of two rigidly held membranes in series was investigated through the application of the Kedem and Katchalsky thermodynamic single membrane flow equations. This analysis results in predictions of the steady state flow properties as well as values for the solute concentration and pressure of the internal compartment when the system is under the influence of a constant solute concentration or hydrostatic pressure gradient. It is demonstrated that although the flow properties and internal compartment pressure are complicated functions of the membrane permeability coefficients and driving gradient across the system, the relationships are greatly simplified by the explicit appearance of the internal compartment steady state solute concentration in the equations. It is shown that the steady state volume flow rate depends on the absolute value of the solute concentration in the external compartments, as well as the solute concentration gradient across the system. The properties of non-linear dependence of volume flow on concentration gradient, and rectification of volume flow are discussed and shown to be independent properties of the system. For the system under the influence of a solute concentration gradient, the internal compartment pressure can be greater or less than the ambient pressure, and depends mainly on the order in which the membranes are encountered by the volume flow. These properties are qualitatively correlated with certain available experimental observations in biological systems.     

Anion transport in dog, cat, and human red cells. Effects of varying cell volume and Donnan ratio

Membrane potential and the rate constants for anion self-exchange in dog, cat, and human red blood cells have been shown to vary with cell volume. For dog and cat red cells, the outward rate constants for SO4 and Cl increase while the inward rate constant for SO4 decreases as cells swell or shrink. These changes coincide with the membrane potential becoming more negative as a result of changes in cell volume. Human red cells exhibit a similar change in the rate constants for SO4 and Cl efflux in response to cell swelling, but shrunken cells exhibit a decreased rate constant for SO4 efflux and a more positive membrane potential. Hyperpolarization of shrunken dog and cat red cells is due to a volume-dependent rate constant for SO4 efflux and a more positive membrane potential. Hyperpolarization of shrunken dog and cat red cells is due to a volume-dependent increase in PNa. If this increase in PNa is prevented by ATP depletion or if the outward Na gradient is removed, the response to shrinking is identical to human red cells. These results suggest that the volume dependence of anion permeability may be secondary to changes in the anion equilibrium ratio which in red cells is reflected by the membrane potential. When the membrane potential and cell volume of human red cells were varied independently by a method involving pretreatment with nystatin, it was found that the rate of anion transport (for SO4 and Cl) does not vary with cell volume but rather with membrane potential (anion equilibrium ratio); that is, the rate constant for anion efflux is decreased and that for influx is increased as the membrane potential becomes more positive (internal anion concentration increases) while the opposite is true with membrane hyperpolarization (a fall in internal anion concentration).