Magnetic alignment and orientational order of dipalmitoylphosphatidylcholine bilayers containing palmitoyllysophosphatidylcholine

Mixed bilayers of 1-palmitoyl-sn-glycero-3-phosphocholine (palmitoyllysophosphatidylcholine; PaLPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dipalmitoyl phosphatidylcholine; DPPC) have been investigated by 2H-NMR and 31P-NMR spectroscopy. Binary phospholipid mixtures were studied in which the acyl chains of one or the other component were perdeuterated. At temperatures below the main order-disorder phase transition, the mixed PaLPC/DPPC bilayers appear to coexist with PaLPC micelles. The micelles disappear at temperatures above the phase transition, where mixed bilayers in the liquid-crystalline state are formed. The orientational order of the alkyl chains of the PaLPC component is essentially identical to that of the DPPC component in the mixed bilayers, both in the low temperature and liquid-crystalline phases. However, the presence of PaLPC perturbs the segmental ordering of DPPC as compared to the pure system. The order is increased in the low-temperature phase, where effective diffusion of the chains about their long axes occurs, but is decreased in the liquid-crystalline phase compared to pure DPPC bilayers. The mixed liquid-crystalline bilayers orient preferentially with their director axes perpendicular to the magnetic field. This alignment is easily observed in 31P- and 2H-NMR spectra, where the intensity of the perpendicular edges of the lineshapes is pronounced. One possible explanation of the magnetic alignment involves alteration of the curvature free energy of the DPPC bilayer due to incorporation of PaLPC in the mixed membranes.     

Effects of pH and cholesterol on DMPA membranes: a solid state 2H- and 31P-NMR study.

The effect of pH and cholesterol on the dimyristoylphosphatidic acid (DMPA) model membrane system has been investigated by solid state 2H- and 31P-NMR. It has been shown that each of the three protonation states of the DMPA molecule corresponds to a 31P-NMR powder pattern with characteristic delta sigma values; this implies additionally that the proton exchange on the membrane surface is slow on the NMR time scale (millisecond range). Under these conditions, the 2H-labeled lipid chains sense only one magnetic environment, indicating that the three spectra detected by 31P-NMR are related to charge-dependent local dynamics or orientations of the phosphate headgroup or both. Chain ordering in the fluid phase is also found to depend weakly on the charge at the interface. In addition, it has also been found that the first pK of the DMPA membrane is modified by changes in the lipid lateral packing (gel or fluid phases or in the presence of cholesterol) in contrast to the second pK. The incorporation of 30 mol% cholesterol affects the phosphatidic acid bilayer in a way similar to what has been reported for phosphatidylcholine/cholesterol membranes, but to an extent comparable to 10-20 mol % sterol in phosphatidylcholines. However, the orientation and molecular order parameter of cholesterol in DMPA are similar to those found in dimyristoylphosphatidylcholine.     

High-pressure 31P NMR study of dipalmitoylphosphatidylcholine bilayers.

High-pressure 31P NMR was used for the first time to investigate the effects of pressure on the structure and dynamics of the phosphocholine headgroup in pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar aqueous dispersions and in DPPC bilayers containing the positively charged form of the local anesthetic tetracaine (TTC). The 31P chemical shift anisotropies, delta sigma, and the 31P spin-lattice relaxation times, T1, were measured as a function of pressure from 1 bar to 5 kbar at 50 degrees C for both pure DPPC and DPPC/TTC bilayers. This pressure range permitted us to explore the rich phase behavior of DPPC from the liquid-crystalline (LC) phase through various gel phases such as gel I (P beta'), gel II (L beta'), gel III, gel IV, gel X, and the interdigitated, Gi, gel phase. For pure DPPC bilayers, pressure had an ordering effect on the phospholipid headgroup within the same phase and induced an interdigitated Gi gel phase which was formed between the gel I (P beta') and gel II (L beta') phases. The 31P spin-lattice relaxation time measurements showed that the main phase transition (LC to gel I) was accompanied by the transition between the fast and slow correlation time regimes. Axially symmetric 31P NMR lineshapes were observed at pressures up to approximately 3 kbar but changed to characteristic axially asymmetric rigid lattice lineshapes at higher pressures (3.1-5.1 kbar).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of stigmastanol and stigmastanyl-phosphorylcholine, two plasma cholesterol lowering substances, on synthetic phospholipid membranes. A 2H- and 31P-NMR study.

Cholesterol, stigmastanol, and stigmastanyl-phosphorylcholine (ST-PC) were incorporated into model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). POPC and ST-PC were deuterated at the lipid headgroup, DOPC at the cis-double bonds. The influence of the three sterols on the motion and conformation of the lipid headgroups and the hydrocarbon chains was monitored with 2H- and 31P-NMR. All three sterols were freely miscible with the lipid matrix in concentrations of up to 50 mol% without inducing phase separations or nonbilayer structures. However, the molecules exert quite different effects on the phospholipid bilayer. Cholesterol and stigmastanol are largely buried in the hydrocarbon part of the membrane, distinctly restricting the flexing motions of the fatty acyl chains whereas the conformation of the phospholipid headgroups is little affected. In contrast, ST-PC is anchored with its headgroup in the layer of phospholipid dipoles, preventing an extensive penetration of the sterol ring into the hydrocarbon layer. Hence ST-PC has almost no effect on the hydrocarbon chains but induces a characteristic conformational change of the phospholipid headgroups. The 2H- and 31P-NMR spectra of mixed phospholipid/ST-PC membranes further demonstrate that the PC headgroup of ST-PC has a similar orientation as the surrounding phosphatidylcholine headgroups. For both types of molecules the -P-N+ dipole is essentially parallel to the membrane surface. Addition of ST-PC induces a small rotation of the POPC headgroup towards the water phase.     

Effects of alcohols on lipid bilayers with and without cholesterol: the dipalmitoylphosphatidylcholine system

Differential scanning calorimetry is a useful method to study the thermotropic phase transitions of a phospholipid bilayer. In the present study DSC is used to determine the effects of methanol and ethanol on DPPC and DPPC/2 mol% cholesterol bilayers. The biphasic effect of the main transition and the presence of an extra peak on the DSC cooling scans were observed above certain alcohol concentrations. In the presence of 2% cholesterol, the concentration at which the biphasic effect occurs is increased by both short-chain alcohols. 1,6-Diphenyl-1,3,5-hexatriene (DPH) is used as a fluorescent probe to directly determine the onset of interdigitation in these systems as reflected by a drop in the DPH fluorescence intensity.     

The effect of cholesterol on measured interaction and compressibility of dipalmitoylphosphatidylcholine bilayers

We have examined the phase diagram of dipalmitoylphosphatidylcholine (DPPC)--cholesterol-water mixtures at low cholesterol content, and report phase separation between 3 and 10 mol% cholesterol. The two lamellar phases at equilibrium in this region appear to be pure DPPC and 11 mol% cholesterol in DPPC. For these two lamellar phases, which are made up of alternating layers of water and bimolecular lipid leaflets, we have measured the forces of interaction between leaflets and the lateral pressure and compressibility of the leaflets. Both bilayers experience a strong repulsive force when forced together only a few ?ngstr?ms (1 A = 0.1 nm) closer than their maximum separation in excess water. However, the presence of 11 mol% cholesterol causes the bilayers to move apart of 35-A separation from the 19-A characteristic of pure DPPC in excess water. This swelling may result from a decrease in van der Waals attraction between bilayers or from an increase in bilayer repulsion. Differences in bilayer interaction can be a cause for phase separation. More importantly these differences can cause changes in the composition of regions of membranes approaching contact. At 11 mol%, cholesterol substantially increases the lateral compressibility of DPPC bilayers leading to higher lateral density fluctuations and potentially higher bilayer permeability.     

Effects of cholesterol on conformational disorder in dipalmitoylphosphatidylcholine bilayers. A quantitative IR study of the depth dependence

A method originally proposed by Snyder and Poore [(1973) Macromolecules 6, 708-715] as a specific probe of trans-gauche isomerization in hydrocarbon chains and recently applied [Mendelsohn et al. (1989) Biochemistry 28, 8934-8939] to the quantitative determination of phospholipid acyl chain conformational order is utilized to monitor the effects of cholesterol at various depths in dipalmitoylphosphatidylcholine (DPPC) bilayers. The method is based on the observation that the CD2 rocking modes from the acyl chains of specifically deuterated phospholipids occur at frequencies in the Fourier transform infrared spectrum which depend upon the local geometry (trans or gauche) of the C-C-C skeleton surrounding a central CD2 group. Three specifically deuterated derivatives of DPPC, namely, 4,4,4',4'-d4 DPPC (4-d4 DPPC), 6,6,6',6'-d4 DPPC (6-d4 DPPC), and 12,12,12',12'-d4 DPPC (12-d4 DPPC), have been synthesized, and the effects of cholesterol addition at 2:1 DPPC/cholesterol (mol:mol) on acyl chain order at various temperatures have been determined. At 48 degrees C, cholesterol inhibits gauche rotamer formation by factors of approximately 9 and approximately 6 at positions 6 and 4, respectively, of the acyl chains, thus demonstrating a strong ordering effect in regions of the bilayer where the sterol rings are presumed to insert parallel to the DPPC acyl chains. In contrast, the ability of the sterol to order the acyl chains is much reduced at the 12-position. The sterol demonstrates only a slight disordering of phospholipid gel phases. Finally, the contributions of different classes of gauche conformers to the spectra have been determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H-, 13C- and 31P-NMR studies of dioctanoylphosphatidylcholine and dioctanoylthiophosphatidylcholine

Coupling constants and chemical shifts were measured for dioctanoylphosphatidylcholine and its thio analogue in a CDCl3/CD3OD solvent mixture. Replacing the bridging oxygen atom of the CH-CH2-O-P portion of the phosphatidylcholine molecule with a sulfur atom affects chemical shifts and coupling constants in the glycerol backbone portion of the molecule as well as in the choline head group region. Preferred conformations about selected bonds in the phospholipids were determined from the vicinal 1H-1H, 31P-1H and 31P-13C coupling constants. A reduction of the 31P T2* (effective spin-spin relaxation time) for the thio analogue, as well as changes in the relative chemical shifts of 13C nuclei in the acyl chains, suggest a somewhat greater degree of aggregation for the thio analogue. The quadrupolar coupling constant 1J(14N-13C) for the choline methyls of either analogue, however, indicates that aggregation of these phospholipids in the CDCl3/CD3OD solvent mixture is not significant. Differences in conformation between dioctanoylphosphatidylcholine and its thio analogue may be responsible for their differences in chemical and physical properties.     

1H- and 31P-NMR studies on smooth muscle of bullfrog stomach

³¹P-NMR spectra of bullfrog stomach smooth muscle showed peaks for creatine phosphate (4.8 μmol·g⁻¹ wet wt.), ATP (3.6), inorganic phosphate (P_i, 2.4), phosphomonoesters (3.0) and phosphodiesters (3.3). The intracellular pH was 7.3, and calculated from the chemical shift of P_i. ¹H-NMR spectra of smooth muscle yielded peaks of 2.9 for lactate, 6.6 for total creatine (creatine phosphate + creatine) and methyl protons of choline tentatively assigned to glycerolphosphorylcholine or to membrane phospholipids. Creatine phosphate and ATP decreased under anaerobic conditions, and intracellular acidification was observed with the concomitant increase in lactate. ³¹P saturation transfer studies showed that saturation of the γ-ATP resonance reduced the intensity of creatine phosphate to 60% of its control value, and the measured T₁ value of creatine phosphate was 2.4 s with saturation. The calculated forward flux of the creatine kinase reaction (decomposition direction of creatine phosphate) was 0.77 μmol·g⁻¹ wet wt.·s⁻¹. The creatine kinase flux was approx. 100-times larger than the ATP turnover rate, calculated from the oxygen consumption rate with the assumption, P/O = 3. In conclusion, the creatine kinase reaction is at equilibrium in resting smooth muscle of bullfrog stomach.     

Bisintercalation of ditercalinium into a d(CpGpApTpCpG)2 minihelix: a 1H- and 31P-NMR study

The structure of the complex formed in aqueous solution between ditercalinium, a bisintercalating drug, and the self-complementary hexanucleotide d(CpGpApTpCpG)2 is investigated by 400-MHz 1H-nmr and 162-MHz 31P-nmr. Whatever the drug to helix ratio, ditercalinium occurred in the bound form, whereas free and complexed hexanucleotide are in slow exchange. This allows unambiguous resonance assignment through two-dimensional chemical exchange experiments. The strong upfield shifts measured on most aromatic protons on both drug and bases as well as on DNA imino protons are consistent with bisintercalation of the dimer. Nuclear Overhauser effects observed between drug and nucleotide protons give a defined geometry for complexation, and suggest a DNA conformational change upon drug binding.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

Head-group conformation in phospholipids: a phosphorus-31 nuclear magnetic resonance study of oriented monodomain dipalmitoylphosphatidylcholine bilayers

Angular-dependent 31P NMR spectra of oriented biaxial monodomain DPPC.H2O multilayers are employed to study head-group conformation in this phospholipid. The results indicate that the O-P-O plane of the phosphate, where the O's are the nonesterified oxygens of the phosphodiester, is tilted at 47 +/- 5 degrees with respect to the bilayer normal. This PO4 orientation could result in the choline moiety being extended parallel to the bilayer plane, and it will explain the breadth of the axially symmetric 31P powder spectrum observed for DPPC in excess water. This work is the first direct observation of this conformation for lecithins and it illustrates the utility of high-resolution solid-state NMR in structural studies of disordered systems.     

31P-NMR assignment and conformational study of NADPH bound to Lactobacillus casei dihydrofolate reductase based on two-dimensional 1H-31P-heteronuclear and 1H-detected 1H-31P-shift-correlation experiments.

For any detailed NMR conformational study of a protein-ligand complex it is essential to have specific resonance assignments. We have now assigned the pyrophosphate 31P resonances in spectra of NADPH bound to Lactobacillus casei dihydrofolate reductase (DHFR) by using a combination of 1H-31P-heteronuclear shift-correlation (HETCOR), 1H-31P-heteronuclear multiple-quantum-coherence correlation spectroscopy (HMQC-COSY), 1H-1H COSY, homonuclear Hartmann-Hahn (HOHAHA) and NOE spectroscopy (NOESY) experiments. The nicotinamide pyrophosphate phosphorus, P(n), has been unequivocally assigned to a signal (-14.07 ppm) which shows a large 3JP-O-C-H coupling constant. Such a coupling constant when combined with the appropriate Karplus relationship provides conformational information about the P-O-C-H torsion angle. The torsion angle changes by 65 degrees +/- 10 degrees for the binary complex compared with the value in free NADPH. The observed coupling constants for the binary (DHFR--NADPH) and ternary (DHFR--NADPH--methotrexate) complexes (12.3 and 10.5 +/- 0.6 Hz, respectively) indicate that the pyrophosphate group has a similar conformation in the two complexes.     

Conformation and dynamic structure of poly(inosinic acid) in neutral aqueous solution by 1H-, 2H-, 13C-, and 31P-NMR spectroscopy

The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: ¹H (90 and 250 MHz), ²H (13.8 MHz), ¹³C (75.4 MHz), and ³¹P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the ¹H and ¹³C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T₁ ratios between the sugar protons, T₁ (H₁′)/T₁ (H₃′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T₂. For a given temperature between 22 and 62°C, the ¹³C-{¹H} NOE value is practically the same for seven protonated carbons (C₂, C₈, C_1′, C_2′, C_3′, C_4′, C_5′). This is also true for the T₁ ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τ_c, for all protons and carbons. The τ_c values determined from either the ¹³C-{¹H} NOE or proton T₁ ratios, T₁(90 MHz)/T₁(250 MHz), and/or deuterium transverse relaxation time T₂ agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T₁ (³¹P) and ³¹P-{¹H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data.     

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.     

The influence of n-alkanols and cholesterol on the duration and conductance of gramicidin single channels in monoolein bilayers

The mean lifetime of gramicidin A channels in bilayers formed from monoolein and squalane was sharply reduced by the absorption of a range of n-alkanols and cholesterol. Results are shown for n-hexanol, n-octanol, n-decanol, n-dodecanol, n-tetradecanol, n-hexadecanol, n-octadecanol and cholesterol. The longer chain n-alkanols were apparently more effective than the shorter members and cholesterol was the most effective of the substances examined. The single channel conductance was also affected, though to a much lesser extent than the mean channel lifetime, the n-alkanols producing increases and cholesterol a decrease. It is suggested that membrane fluidity changes are not likely to be primarily responsible for the reductions in channel lifetimes but that the bilayer tension, which is known to be increased by n-octanol, could be significant.     

31P-NMR spectroscopy and the metabolic properties of different muscle fibers

To study the in vivo recruitment of different fiber types and their metabolic properties, 31P-nuclear magnetic resonance spectroscopy (31P-NMRS) of the human calf muscle was performed in seven normal sedentary subjects. In the exhaustive exercise protocol used, the work load was increased every minute during 5 min. This resulted in a prominent split of the Pi resonance in all subjects, indicating pH compartmentation in the muscles studied. From the chemical shift of the Pi peaks relative to phosphocreatine (PCr) at the end of the exercise, intracellular pH (pHi) averaged 6.92 +/- 0.05 (SD) in compartment 1 and 6.23 +/- 0.15 in compartment 2. The recovery of both Pi resonances after exercise could be followed easily in five of these subjects. The recovery rate of the Pi peak is a good estimate of the oxidative metabolism at the end of the exercise. A monoexponential regression analysis showed that the mean initial recovery rate S0 was 2.49 +/- 0.17%/s in compartment 1 and only 0.87 +/- 0.12%/s in compartment 2, indicating aerobic function three times higher in compartment 1 at the end of exercise. The mean relative ATP fraction dropped significantly (P less than 0.001), from 20.0 +/- 1.0% of the total 31P signal integral before exercise to 14.0 +/- 1.6% at the end of exercise. The simultaneous visualization of two compartments, in good order, one with high pHi and fast recovery and another with low pHi and slow recovery, is rationalized by the different metabolic behavior of type I and II fibers in human calf muscle in response to exhaustive exercise. This study demonstrates that 31P-NMRS is an excellent noninvasive procedure to quantify aerobic metabolism in both fiber types simultaneously.     

31P-NMR study of the orotate phosphoribosyltransferase equilibrium with thiopyrophosphate as substrate

31P-nuclear magnetic resonance spectroscopy was used to directly determine the equilibrium of the reaction catalysed by yeast orotate phosphoribosyltransferase, using orotidine monophosphate and inorganic pyrophosphate as substrates. A Keq value of 0.71 was determined, in good agreement with that of 0.49 calculated by Victor, Greenberg and Sloan (J. Biol. Chem. 254 (1979) 2647-2655), from kinetic data. Substitution of thiopyrophosphate as the substrate shifted the position of the equilibrium 55-fold, to yield a Keq value of 39. Only the beta S analogue of 5-phosphoribosyl 1-diphosphate appeared to be synthesized in this reaction.     

Cation permeability induced by valinomycin,gramicidin D and amphotericin B in large lipidic unilamellar vesicles studied by 31P-NMR

Permeability induced by mobile carriers and channel-forming compounds in large unilamellar lipidic vesicles (LUV) has been studied by the proteon-cation exchange method. Proton movement has been monitored by pH-stat and ³¹P-NMR techniques. pH-stat measurements indicate that, in the presence of valinomycin, the proton efflux develops with a rate dependent upon valinomycin concentration, until equilibrium is reached. ³¹P-NMR spectra, monitoring pH-dependent intravesicular phosphate ionization, show that after addition of valinomycin the initial pH peak (pH 5.5; =0.25) shifts progressively to the position corresponding to the pH at equilibrium (pH 7.4; =2.20).In the presence of the channel-forming compounds, gramicidin D or amphotericin B, permeability developed in a few minutes whatever the concentration used. The percentage of total titratable proton released depends upon the antibiotic concentration. ³¹P-NMR spectra shows two signals from internal phosphate: one signal corresponding to the initial pH and a second signal corresponding to the pH at equilibrium indicating an all-or-none mode of action; just after addition of the antibiotic, two populations of vesicles coexist in proportions that depend on ionophore concentration; after longer incubation times all vesicles are permeabilized.The results obtained primarily reflect the differences in the mode of interaction with the membrane, of valinomycin as compared to the channel-forming reagents, gramicidin D or amphotericin B.