Cooperative binding of primycin and gramicidin on erythrocyte membranes. A cation transport study

In this paper the authors present a comparative study of the actions of the antibiotics primycin and gramicidin on the erythrocyte membrane permeability. It has been found that both antibiotics have a nonlinear effect on the membrane permeability. Above a threshold antibiotic concentration, which is characteristic of the type of the antibiotic, the cation permeability of the erythrocyte membranes increases sharply. In the range of nonlinearity the transport-kinetic curves level off before achieving the equilibrium radioactive ion distribution between the extra- and intracellular spaces. A stochastic model of the cooperative and aspecific incorporation of antibiotic molecules into the membrane explains the experimental findings. The authors conclude that membrane permeability increases at the places where two or more antibiotic molecules form aggregates in the membrane.     

NMR study of the interactions of polymyxin B, gramicidin S, and valinomycin with dimyristoyllecithin bilayers

The interactions of three polypeptide antibiotics (polymyxin B, gramicidin S, and valinomycin) with artificial lecithin membranes were studied by nuclear magnetic resonance (NMR). Combination of 31P and 2H NMR allowed observation of perturbations of the bilayer membrane structure induced by each of the antibiotics in the regions of the polar headgroups and acyl side chains of the phospholipids. The comparative study of the effects of these membrane-active antibiotics and the lipid bilayer structure demonstrated distinct types of antibiotic-membrane interactions in each case. Thus, the results showed the absence of interaction of polymyxin B with the dimyristoyllecithin membranes. In contrast, gramicidin S exhibited strong interaction with the lipid above the gel to liquid-crystalline phase transition temperature: disordering of the acyl side chains was evident. Increasing the concentration of gramicidin S led to disintegration of the bilayer membrane structure. At a molar ratio of 1:16 of gramicidin S to lecithin, the results are consistent with coexistence of gel and liquid-crystalline phases of the phospholipids near the phase transition temperature. Valinomycin decreased the phase transition temperature of the lipids and increased the order parameters of the lipid side chains. Such behavior is consistent with penetration of the valinomycin molecule into the interior of the lipid bilayers.     

Sterol specific inactivation of gramicidin A induced membrane cation permeability.

Channel inactivation, a time-dependent decrease of the high-cationic permeability induced by gramicidin A, has been found both in cholesterol containing red blood cell membranes and lipid bilayers (Schagina et al., (1989) Biochim. Biophys. Acta 978, 145-150). The rate of channel inactivation strongly depends on the phospholipid to cholesterol molar ratio of the membrane. The channel inactivation is suggested to be the result of an interaction between gramicidin and cholesterol in a stoichiometry of 1:5. Cholesterol dependent inactivation is shown also for gramicidin A analogs: tryptophan-N-formylated gramicidin A, o-pyromellitilgramicidin and malonylbisdesformylgramicidin. When cholesterol in the membrane is substituted by sitosterol, the inactivation of gramicidin-induced cation permeability is preserved, while in the presence of either ergosterol or 7-dehydrocholesterol no indication of the channel inactivation is observed. Thus, the structure of the 'B', ring, not the apolar tail of the sterol molecule, appears to be important in the inactivation process.     

Change in lipid-protein interactions in the membranes of bacteria exposed to gramicidin S]

Effect of cyclopeptide antibiotic gramicidin S on some enzymes and physical state of isolated Micrococcus lysodeikticus membranes is studied. Malate and lactate dehydrogenases were monotonously inhibited under the increase of gramicidin S concentration, while the activity of NADH-dehydrogenase firstly decreased and then reversed to the initial level under further increase of gramicidin S concentration. The oxygen uptake under oxidation of NADH and malate with membranes almost completely inhibited by the antibiotic, while the activity of ascorbate-TMPD-oxidase activity slightly inhibited by the same concentration of gramicidin. The addition of Triton X-100 completely eliminated the inhibitory effect of gramicidin on malate dehydrogenase. The introduction into the membrane of spine probes (2,2,6,6-tetramethyl-4-palmitoylamidopiperidine-1-oxile and 2(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxyazolidinyloxile) revealed that gramicidin caused the condensation of membrane lipid component. It is suggested that ionic interaction of gramicidin S with membrane phospholipids brings to "a freezing" of lipids which is a direct cause of impairing the activity of membrane respiration enzymes and the change of their position in the lipid matrix, thus inhibiting energy-producing processes in cell.     

Low-frequency motion in membranes. The effect of cholesterol and proteins

Nuclear magnetic resonance (NMR) relaxation techniques have been used to study the effect of lipid-protein interactions on the dynamics of membrane lipids. Proton enhanced (PE) 13C-NMR measurements are reported for the methylene chain resonances in red blood cell membranes and their lipid extracts. For comparison similar measurements have been made of phospholipid dispersions containing cholesterol and the polypeptide gramicidin A+. It is found that the spin-lattice relaxation time in the rotating reference frame (T1 rho) is far more sensitive to protein, gramicidin A+ or cholesterol content than is the laboratory frame relaxation time (T1). Based on this data it is concluded that the addition of the second component to a lipid bilayer produces a low-frequency motion in the region of 10(5) to 10(7) Hz within the membrane lipid. The T1 rho for the superimposed resonance peaks derived from all parts of the phospholipid chain are all influenced in the same manner suggesting that the low frequency motion involves collective movements of large segments of the hydrocarbon chain. Because of the molecular co-operativity implied in this type of motion and the greater sensitivity of T1 rho to the effects of lipid-protein interactions generally, it is proposed that these low-frequency perturbations are felt at a greater distance from the protein than those at higher frequencies which dominate T1.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.     

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.     

Cholesterol-dependent gramicidin A channel inactivation in red blood cell membranes and lipid bilayer membranes

The exchange diffusions of tracer cations (22Na+, 86Rb+) are studied on gramicidin-A-treated red blood cell (RBC) membranes. A time-dependent decrease in cation permeability has been observed and has been considered to be the result of a channel inactivation process. The channel inactivation appears at 20 and 30 degrees C but not at a temperature as low as 6 degrees C. The gramicidin A channel inactivation can be monitored by a conductivity decay of molecular lipid membranes (BLM) prepared either from cholesterol or from a mixture of cholesterol and phospholipids but not of pure phosphatidylethanolamine. The role of cholesterol in the channel inactivation is discussed.     

Preparation and properties of O-dansyltyrosine gramicidin C.

Gramicidins A, B, and C are a family of poly-peptide antibiotics which facilitate the passive diffusion of alkali cations and protons through lipid bilayer membranes. It is clear that gramicidin forms a multimeric transmembrane channel and it has been suggested that the channel is an io-conducting dimer in equilibrium on the membrane with non-conducting monomer. We describe the preparation and purification of a derivative of gramicidin C in which the phenolic hydroxyl of the tyrosine at position 11 has been esterified to 8-dimethylaminonaphthalene-1-sulfonate (dansyl). This derivative fluoresces strongly in the visible with an emission maximun in dioxane of 530 nm, an emission lifetime of 16 ns, and a quantum yield of 0.8. Veatch et al. ((1975),J. Mol. Biol. 99, 75) have shown this 0-dansyltyrosine gamicidin C to be a fully active analogue of gramicidin A in artificial lipid bilayer membranes. We here utilize this derivative to further characterize the state of aggregation and rotational mobility of the four interconvertible conformational species formed by gramicidin in nonpolar organic solvents (Veatch et al. (1974), Biochemsitry 13, 5249; Veatch and Blout (1974), Biochemistry 13, 5257). Fluorescence energy transfer from the tryptophans of gramicidin A to the 0-dansyltyrosine of this derivatives supports the conclusion that all of these gramicidin isolated species are aggregates. Decay of fluorescence polarization anisotropy measurements yield a rotational correlation time of 1 ns for the 0-dansyltyrosine chromophore in ethanol in good agreement with the more detailed information previously obtained by 13C-nuclear magnetic resonance for the monomer in dimethyl sulfoxide (Fossel et al. (1974), Biochemistry 13, 5264). However, it is likely that the chromophore has much more rotational mobility than the rest of the gramicidin molecule in the aggregated comformational states.     

Dimerization constant and single-channel conductance of Gramicidin in thylakoid membranes

Summary The effect of the pore-forming antibiotic gramicidin on pure lipid membranes is well characterized. We studied its action in protein-rich thylakoid membranes that contain less than 25% (wt/wt) acyl lipids. A transmembrane voltage was induced by flashing light, and its decay was measured and interpreted to yield the distribution of gramicidin over thylakoids, its dimerization constant and its single-channel conductance in this membrane. The distribution of gramicidin over the ensemble of thylakoids was immediately homogeneous when the antibiotic was added under stirring, while it became homogeneous only after 20 min in a stirred suspension that was initially heterogeneous. The dimerization constant, 5×10¹⁴ cm²/mol, was about 10 times larger than in pure lipid membranes. This was attributed to the upconcentration of gramicidin in the small fractional area of protein free lipid bilayer and further by a preference of gramicidin for stacked portions of the membrane. The latter bears important consequences with regard to bioenergetic studies with this ionophore. As gramicidin was largely dimerized from a concentration of 1 nm (in the suspension) on, the membrane's conductance then increased linearly as a function of added gramicidin. When the negative surface potential at the thylakoid membrane was screened, the conductance of a single gramicidin dimer agreed well with figures reported for bilayers from neutral lipid (about 0.5 pS at 10 mm NaCl). The modulation of the conductance by the surface potential in spinach versus pea thylakoids and between different preparations is discussed in detail.We would like to thank Ms. H. Kenneweg for photographs. financial support by the DFG (SFB 171/B3) is gratefully acknowledged.This paper is dedicated to the Late Prof. Peter Läger.     

Mode of action of gramicidin S on Escherichia coli membrane

The action of a cationic antibiotic gramicidin S on the outer and cytoplasmic membranes of Escherichia coli was studied. It was found that gramicidin S disrupted the permeability barrier of the outer membrane, permitting the permeation of an antibiotic ionophore, this being similar to the action of the dimer in compound 48/80 (Katsu, T., Shibata, M. and Fujita, Y. (1985) Biochim. Biophys. Acta 818, 61-66). However, differently from the dimer, gramicidin S further stimulated the efflux of K+ through the cytoplasmic membrane of E. coli. The time course of K+ permeability change accorded well with that of change in the viability of E. coli cells. These changes occurred at temperatures above the phase transition of the cytoplasmic membrane. This temperature range differed greatly from the case of polymyxin B, a polycationic antibiotic acting at temperatures above the phase transition of the outer membrane. We discuss the mode of gramicidin S action on the cytoplasmic membrane of E. coli, in comparison with the results on red blood cells and liposomes.     

Interrelationships between tyrocidine and gramicidin A' in their interaction with phospholipids in model membranes

(1) The interaction of tyrocidine with different lipids is studied in model membranes and the results are compared to the gramicinid-lipid interaction. (2) The tyrocidine-dielaidoylphosphatidylethanolamine interaction gives rise to a population of phospholipids with a lower gel to liquid-crystalline transition temperature and to an abolition of the bilayer to HII phase transition, resulting in a macroscopic organization with dynamic and structural properties different from those of the pure lipid. (3) Tyrocidine has a strong fluidizing effect on the acyl chains of phosphatidylcholines, manifested by a decrease in enthalpy of the main thermotropic transition. (4) No evidence of a gramicidin A'-like lipid-structure modulating activity was found. However, tyrocidine inhibits the formation by gramicidin of an HII phase in dioleoylphosphatidylcholine model membranes. Instead, a cubic type of lipid organization is observed. (5) Tyrocidine greatly perturbs the barrier properties of dioleoylphosphatidylcholine model membrane. (6) Gramicidin A' reverses the effect of tyrocidine on membrane permeability by forming a complex in the model membrane with an apparent 1:1 stoichiometry. (7) The results suggest that both peptide antibiotics, which are produced by Bacillus brevis ATC 8185 prior to sporulation, show antagonism in their effect on membrane structure similar to their effect on superhelical DNA (Bogh, A. and Ristow, H. (1986) Eur. J. Biochem. 160, 587-591. The possible underlying basic mechanism is indicated.     

The structure of the gramicidin A transmembrane channel

Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across lipid bilayer membranes by forming channels. It has been proposed that the conducting channel is a dimer which is in equilibrium with nonconducting monomers in the membrane. To directly test this model in several independent ways, we have prepared and purified a series of gramicidin C derivatives. All of these derivatives are fully active analogs of gramicidin A, and each derivative has a useful chromophore esterified to the phenolic hydroxyl of tyrosine #11. Simultaneous conductance and fluorescence measurements on planar lipid bi-layer membranes containing dansyl gramicidin C yielded four conclusions: (1) A plot of the logarithm of the membrane conductance versus the logarithm of the membrane fluorescence had a slope of 2.0 ± 0.3, over a concentration range for which nearly all the gramicidin was monomeric. Hence, the active channel is a dimer of the nonconducting species. (2) In a membrane in which nearly all of the gramicidin was dimeric, the number of channels was approximately equal to the number of dimers. Thus, most dimers are active channels and so it should be feasible to carry out spectroscopic studies of the conformation of the transmembrane channel. (3) The association constant for dimerization is more than 1,000-fold larger in a glycerolester membrane with 26 Å-hydrocarbon thickness than in a 47 Å-glycerolester membrane. The dimerization constant in a 48 Å-phosphatidyl choline membrane was 200 times larger than in a 47 Å-glycerolester membrane, showing that it depends on the type of lipid as well as on the thickness of the hydrocarbon core. (4) We were readily able to detect 10^?14 mole cm^?2 of dansyl gramicidin C in a bilayer membrane, which corresponds to 60 fluorescent molecules per square μm. The fluorescent techniques described here should be sufficiently sensitive for fluorescence studies of reconstituted gates and receptors in planar bilayer membranes. An alternative method of determining the number of molecules of gramicidin in the channel is to measure the fraction of hybrid channels present in a mixture of 2 chemically different gramicidins. The single-channel conductance of p-phenylazo-benzene-sulfonyl ester gramicidin C (PABS gramicidin C) was found to be 0.68 that of gramicidin A. In membranes containing a mixture of these 2 gramicidins, a hybrid channel was evident in addition to 2 pure channels. The hybrid channel conductance was 0.82 that of gramicidin A. Fluorescence energy transfer from dansyl gramicidin C to diethylamino-phenylazobenzene-sulfonyl ester gramicidin C (DPBS gramicidin C), provided an independent way to measure the fraction of hybrid channels on liposomes. For both techniques the fraction of hybrid channels was found to be 2ad where a² and d² were the fractions of the 2 kinds of pure channels. This result strongly supports a dimer channel and the hybrid data excludes the possibility of a tetramer channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes. The various models which have been proposed for the conformation of the gramicidin transmembrane channel are briefly discussed.     

Voltage-induced thickness changes of lipid bilayer membranes and the effect of an electrin field on gramicidin A channel formation.

The thickness changes of black lipid membranes of different composition after a voltage jump were investigated. In a second series of electrical relaxation experiments the kinetics of channel formation by gramicidin A were measured. The time course of the membrane current was compared with the time course of the thickness change of the membranes. We found that the time course of the current as a consequence of channel formation by gramicidin A did not correlate with the thickness change of the lipid membranes. A possible direct influence of the electric field is discussed.     

Rhodopsin and other proteins in artificial lipid membranes.

Some basic aspects of incorporation of hydrophobic peptides and proteins in artificial lipid membranes are discussed. As examples valinomycin as a carrier model and gramicidin A as a channel former in lipid vesicles and in planar lipid membranes are presented. In the second part of the lecture some examples of incorporation of membrane proteins into lipid vesicles and planar lipid membranes are reported. The interaction with artificial lipid membranes of the Ca++ ATPase from the sarcoplasmic reticulum, of Rhodopsin, and of Bacteriorhodopsin is presented.     

Interactions of pyrethroids with gramicidin-containing liposomal membranes

Fluorescence steady-state anisotropy and phase-modulation lifetime techniques have been utilized to study the interactions of pyrethroid compounds with fluid-phase phosphatidylcholine membranes containing the polypeptide gramicidin. This polypeptide is considered to be a model of hydrophobic regions of cellular integral membrane proteins. The pyrethroids disorder lipid packing in cellular membranes and gel-phase liposomes but do not disorder lipid packing in fluid-phase lipid (Stelzer, K.J. and Gordon, M.A. (1984) J. Immunopharmacol. 6, 381-410; (1985) Biochim. Biophys. Acta 812, 361-368) Irrespective of liposomal size, gramicidin incorporation resulted in a substantial increase in anisotropy of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), in fluid phase lipid. In the absence of gramicidin, permethrin and three other pyrethroids, allethrin, cypermethrin and fenpropathrin, increased DPH anisotropy. In these fluid phase systems, as the protein:lipid ratio was increased, the extent of the pyrethroid-mediated increase in fluorescence anisotropy diminished. Also, the pyrethroids shortened DPH fluorescence lifetimes. At high gramicidin:lipid ratios, permethrin substantially lowered anisotropy in the fluid phase lipid, relative to controls. The data suggest that pyrethroids disturb fluid-phase lipids which have been promoted to a relative state of order by proximity to an integral membrane protein. This type of order is one which is represented by DPH fluorescence anisotropy. A model based on these results is proposed to explain the effects of pyrethroids on lipid packing order in cellular membranes, as determined by DPH fluorescence anisotropy.     

Gramicidin S identified as a potent inhibitor for cytochrome bd-type quinol oxidase

Gramicidin S, a cationic cyclic decapeptide, exhibits the potent antibiotic activity through perturbation of lipid bilayers of the bacterial membrane. From the screening of natural antibiotics, we identified gramicidin S as a potent inhibitor for cytochrome bd-type quinol oxidase from Escherichia coli. We found that gramicidin S inhibited the oxidase with IC(50) of 3.5 microM by decreasing V(max) and the affinity for substrates but showed the stimulatory effect at low concentrations. Our findings would provide a new insight into the development of gramicidin S analogs, which do not share the target and mechanism with conventional antibiotics.     

The effect of hypoxia due to iron-deficiency anemia on the physicochemical properties of biological membranes]

The lipid structure and Ca2+ permeability of red blood cell, hepatocyte and cardiomyocyte membranes were determined while investigating the effect of hypoxia caused by iron deficiency anemia upon the structural and functional state of biological membranes. The lipid composition and barrier characteristics of membranes change under conditions of hypoxia caused by experimental iron deficiency anemia. Quantitative changes in the cell membrane lipids may be considered as an important molecular mechanism of Ca2+ transport disorder in membranes, increase of Ca2+ permeability producing its surplus in the cells and subsequent metabolic homeostatic disturbances.     

The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes

The conformation of gramicidin in diacylphosphatidylcholine model membranes was investigated as a function of the solvent in which peptide and lipid are initially codissolved. By use of circular dichroism it is demonstrated that, upon removal of the solvent and hydration of the mixed gramicidin/lipid film, it is the conformational behavior of the peptide in the organic solvent that determines its final conformation in dimyristoylphosphatidylcholine model membranes. As a consequence, parameters that influence the conformation of the peptide in the solvent also play an essential role, such as the gramicidin concentration and the rate of interconversion between different conformations. Of the various solvents investigated, only with trifluoroethanol is it possible directly to incorporate gramicidin entirely in the beta 6.3-helical (channel) configuration. It is also shown that the conformation of gramicidin in the membrane varies with the peptide/lipid ratio, most likely as a result of intermolecular gramicidin-gramicidin interactions at higher peptide/lipid ratios, and that heat incubation leads to a conformational change in the direction of the beta 6.3-helical conformation. Using lipids with an acyl chain length varying from 12 carbon atoms in dilauroylphosphatidylcholine to 22 carbon atoms in dierucoylphosphatidylcholine, it was possible to investigate the acyl chain length dependence of the gramicidin conformation in model membranes prepared from these lipids with the use of different solvent systems. It is demonstrated for each solvent system that the distribution between different conformations is relatively independent of the acyl chain length but that the rate at which the conformation converts toward the beta 6.3-helical configuration upon heating of the samples is affected by the length of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)