Electrical capacitance of lipid bilayer membranes of hydrogenated egg lecithin at the temperature phase transition

Electrical capacitance of the planar bilayer lipid membrane (BLM) formed from hydrogenated egg lecithin (HEL) has been studied during many passages through the phase transition temperature. In contrast to the BLM from individual synthetic phospholipids, membranes from HEL did not demonstrate any capacitance change at the phase transition temperature maximum, as measured by differential scanning calorimeter at 52 degrees C. Instead, two temperatures have been discerned by capacitance records: thickening at 42-43 degrees C and thinning at 57-59 degrees C. The first temperature region is close to the transition temperature of dipalmitoyllecithin, whereas the second is close to that of distearoyllecithin, two main components of the HEL. It was suggested that capacitance measurements were able to reveal a phase separation in the BLM from HEL which was not detected by differential scanning calorimetry. The phase transition of the BLM from the liquid crystal state to the gel state is followed by thickening of the bilayer structure, partly due to a gauche- trans transition of lipid molecules but mainly due to redistribution of the solvent n-decane.     

Effect of polymyxin B on the conductivity of negatively charged bilayer lipid membranes

Effect of polymyxin B on the planar bilayer lipid membranes (BLM) formed from synthetic phosphatidic acid has been studied. The addition of cholesterol to phospholipid in molar ratio 1 : 2 was followed by an increase of BLM conductance from 2 x 10(-8) to 3 x 10(-7) Ohm-1 cm-2. It was suggested that the observed increase of conductance was due to the fluidity of the membrane matrix in the presence of cholesterol. It was shown that 10(-6)--10(-5) M polymyxin slightly affected the conductance of BLM from phosphatidic acid. It was found that polymyxin increased conductance of negatively charged BLM modified by palmitic acid from 10(-8) to 10(-6) Ohm-1 cm-2.     

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum.

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.     

Changes in thermal phase transition of various membranes during temperature acclimation in Tetrahymena

Changes in the thermal phase transition temperature of membrane lipids were studied by X-ray wide-angle diffraction during adaptation of Tetrahymena pyriformis to a lower growth temperature. After a shift in growth temperature from 39 to 15 degrees C, the phase transition temperature was lowered gradually in microsomal and pellicular phospholipids, whereas that in mitochondrial phospholipids was unchanged for 10 h after the temperature shift. Only a small decrease in the transition temperature of mitochondrial phospholipids was observed, even after 24 h following the shift. Transition temperatures of microsomal, pellicular and mitochondrial phospholipids reached the growth temperature (15 degrees C) about 6, 10 and 24 h after the temperature shift. The temperature dependence of the solid phase in membrane phospholipids was estimated from the 4.2 A peak of the X-ray diffraction pattern. In the case of the phospholipids extracted from cells grown at 39 degrees C, the solid phase was increased upon lowering temperature in a similar manner in all three membrane fractions: mitochondria, pellicles and microsomes. However, in the case of the phospholipids from cells exposed to a lower growth temperature (15 degrees C) for 10 h, the increase in the solid phase was significantly smaller in mitochondrial phospholipids than in two other membrane fractions. The difference in the thermal behaviour of mitochondrial lipid from pellicular and microsomal lipids is discussed in terms of phase transition and phase separation.     

The use of the fluorescent probe alpha-parinaric acid to determine the physical state of the intracytoplasmic membranes of the photosynthetic bacterium, Rhodopseudomonas sphaeroides.

alpha-Parinaric acid has been used to determine the degree of ordering of the hydrocarbon region of purified intracytoplasmic membranes of Rhodopseudomonas sphaeroides. The usefulness of alpha-parinaric acid as a probe of membrane fluidity was established by comparison of its fluorescent properties in phosphatidylcholine vesicles with those of the more commonly used fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Both fluorescent probes were shown to monitor similar environments in the phosphatidylcholine vesicles when the phospholipids were maintained at temperatures above their phase transition temperature. The rotational mobility of alpha-parinaric acid in the intracytoplasmic membranes was determined from 0 to 50 degrees C, a region where no phase transitions were detectable. The rotational mobility of alpha-parinaric acid dissolved in vesicles formed from total extracted intracytoplasmic membrane phospholipids, was 2--3-fold greater than that measured in the intact intracytoplasmic membranes; demonstrating that the presence of protein greatly reduces the mobility of the phospholipid acyl chains of the intracytoplasmic membranes. Due to the high protein content of these membranes, the perturbing effect of protein on acyl chain mobility may extend to virtually all the intracytoplasmic membrane phospholipid.     

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.     

Temperature-induced Phase Changes in the Membranes of Glyoxysomes, Mitochondria, and Proplastids from Germinating Castor Bean Endosperm

Sucrose-gradient purified mitochondria, glyoxysomes, and proplastids from germinating castor bean (Ricinus communis L.) endosperm were examined by electron-spin resonance spectroscopy. A temperature-induced phase change was demonstrated in all of these organelles, their derivative membranes, and in micelles formed from the membrane phospholipids. The apparent transition temperature of the membrane lipids varied slightly between the samples, but in all cases, fell within the temperature range around 10 C where physiological and biochemical changes in the response to temperature for most chilling-sensitive plants occur.     

Studies of Electric Capacitance of Membranes: I. A Model Membrane Composed of a Filter Paper and a Lipid Analogue

A hydrophobic filter paper of a given pore size containing a synthetic lipid, i.e. dioleyl phosphate, was interposed between aqueous electrolyte solutions having the same chemical composition and temperature. The electric capacitance and conductance of the membrane immersed in various concentrations of KCl were measured in the frequency range from 20 to 3 × 10⁶ cycle/sec. The observed capacitance and conductance were found to be strongly dependent on the applied frequency. A theory is proposed to account for this dispersion of impedance observed in the present membrane-electrolyte system. The dispersion is attributed to the formation of bilayer membranes of the lipid inside the filter paper. The effects of the salt concentration, the adsorbed quantity of the lipid, and the pore size of the filter paper on the capacitance and conductance of the membrane are discussed in terms of the distribution function of bilayers formed within the filter paper.     

Stable phospholipid membrane supported on porous filter paper

A novel method to construct a stable and uniform phospholipid membrane of large area and good manipulability is reported. Using the Langmuir-Blodgett (LB) technique, a monolayer of phospholipid can be transferred to filter paper. The electrical conductance across the pores of the lipid membrane is about 1.8 X 10(-9) S/cm2, corresponding to the conductance of 10(-7)-10(-10) S/cm2 reported for bilayer lipid membranes (BLM) of phospholipids. A scanning electron micrograph demonstrated that the phospholipid membrane on the filter paper was uniform.     

Nuclear magnetic resonance and thermal studies on the interaction between salicylic acid and model membranes

DSC and (1H and 31P) NMR measurements are used to investigate the perturbation caused by the keratolytic drug, salicylic acid (SA) on the physicochemical properties of the model membranes. Model membranes (in unilamellar vesicular (ULV) form) in the present studies are prepared with the phospholipids, dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidic acid (DPPA) and mixed lipid DPPC-DPPE (with weight ratio, 2.5:2.2). These lipids have the same acyl (dipalmitoyl) chains but differed in the headgroup. The molar ratio of the drug to lipid (lipid mixture), is in the range 0 to 0.4. The DSC and NMR results suggest that the lipid head groups have a pivotal role in controlling (i) the behavior of the membranes and (ii) their interactions with SA. In the presence of SA, the main phase transition temperature of (a) DPPE membrane decreases, (b) DPPA membrane increases and (c) DPPC and DPPC-DPPE membranes are not significantly changed. The drug increases the transition enthalpy (i.e., acyl chain order) in DPPC, DPPA and DPPC-DPPE membranes. However, the presence of the drug in DPPC membrane formed using water (instead of buffer), shows a decrease in the transition temperature and enthalpy. In all the systems studied, the drug molecules seem to be located in the interfacial region neighboring the glycerol backbone or polar headgroup. However, in DPPC-water system, the drug seems to penetrate the acyl chain region also.     

Spectrophotometric analysis of organisation of dipalmitoylphosphatidylcholine bilayers containing the polyene antibiotic amphotericin B

Amphotericin B (AmB) is a polyene antibiotic widely used in the treatment of deep-seated fungal infections. The mode of action of AmB is directly related to the effect of the drug on the lipid phase of biomembranes. In the present work the effect of AmB on the properties of lipid bilayers formed with dipalmitoylphosphatidylcholine (DPPC) and the effect of the lipid phase on the molecular organisation of AmB were studied with application of spectrophotometry in the UV-Vis region. The absorption spectra of AmB in lipid membranes display a complex structure with hypsochromically and bathochromically shifted bands indicative of formation of molecular aggregates of the drug. Formation of molecular aggregates was analysed at different concentrations of the drug in the lipid phase in the range 0.05--5 mol% and at different temperatures in the range 5--55 degrees C. The aggregation level of AmB in the ordered phase of DPPC displayed a minimum corresponding to a concentration of 1 mol% with respect to the lipid. An increase in the aggregation level was observed in the temperature region corresponding to the main phase transition. The structure of molecular aggregates of AmB is analysed on the basis of spectroscopic effects in terms of the exciton splitting model. Analysis of the position of the absorption maximum of AmB in the lipid phase of DPPC in terms of the theory of solvatochromc effects makes it possible to ascribe the refractive indices n=1.40 and n=1.49 to the hydrophobic core of the membrane in the L(alpha) and the P(beta)' phase respectively. Analysis of the aggregation of AmB in the lipid phase in relation to the physical state of the membrane reveals that the temperature range of the main phase transition of a lipid cluster in the immediate vicinity of AmB depends on its concentration. The termination of the phase transition temperature, as read from the AmB aggregation, varies between 42 degrees C at 1 mol% AmB in DPPC and 49 degrees C at 5 mol% AmB in DPPC. The exciton splitting theory applied to the analysis of the spectroscopic data makes it possible to calculate the diameter of the AmB pore as 2.8 A in the gel phase and 3.6 A in the fluid phase of the DPPC membrane, on the assumption that the pore is formed by nine AmB molecules.     

Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes.

PDC-109 is the main component of bovine seminal plasma and has been suggested to play an important role in the genesis of bovine sperm cells. Here, the effect of binding of PDC-109 to membranes on the structure and physical properties of the lipid phase was investigated. For that, ESR measurements were undertaken on model membranes (lipid vesicles) and on biological membranes (epididymal spermatozoa) by employing various spin-labeled phospholipids. We found that PDC-109 alters the membrane structure of lipid vesicles as well as of bovine epididymal spermatozoa in that the mobility of spin-labeled phospholipids was reduced in the presence of the protein. This immobilizing effect of the protein was not restricted to analogues of phosphatidylcholine but was also detected with spin-labeled phosphatidylethanolamine. However, the extent of immobilization was lower for phosphatidylethanolamine compared with phosphatidylcholine, supporting the lipid headgroup specificity of the protein. Besides phospholipid headgroups, the physical state of membrane lipids is also important for the interaction of PDC-109 with membranes, in that, e.g., the immobilizing effect of the protein on labeled lipids was larger in membranes above the phase transition temperature compared with the effect below this temperature. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.     

Subzero temperature study of the inner mitochondrial membrane and related phospholipid membrane systems with the fluorescent probe, trans-parinaric acid

The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0°C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospholipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at ?25°C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.     

Compartmentation of gramicidin 5 in membranes of sensitive bacteria and protein-lipid interactions]

Gramicidin S is sorbed on the isolated membranes of granicidin-sensitive Micrococcus lysodeikticus strain. The antibiotic inhibits the membrane malate dehydrogenase within the temperature range of 9--42 degrees C, i.e. under conditions of gel and liquid-crystalline lipid state; however its effect at 10 degrees C is 10 times as low as is observed at 42 degrees C. The inhibitory effect of gramicidin S on malate dehydrogenase can be eliminated and the antibiotic can be removed from the membrane by an excess of different phospholipids. No transfer of the membrane components on exogenous phospholipids is observed. A prolonged (about 2 hrs, 30 degrees C) incubation of the membranes with gramicidin S results in irreversible inactivation of malate dehydrogenase, although the antibiotic can be still eliminated by an addition of phospholipid emulsions. It is suggested that gramicidin S forms complexes with phospholipids, in which the antibiotic is oriented to water. These complexes disturb the lipid-protein interactions, resulting in relaxation of the binding between the boundary phospholipids and proteins, in the loosening of near-protein lipid zones and simultaneous condensation of acid phospholipids in the whole membrane. Destruction of the lipid zone is accompanied by changes in the enzyme activity, by separation of lipid and protein regions and by transphase enzyme transitions (expulsion or immersion). A slow formation of secondary protein-protein associates may be irreversible.     

Order-disorder phase transition and lipid dynamics in rabbit small intestinal brush border membranes. Effect of proteins

The total lipids extracted from brush border membranes form smectic lamellar phases when dispersed in water. 31P broad-band nuclear magnetic resonance (NMR) shows that between body temperature (37 degrees C) and freezing of the solvent, the extracted lipids form bilayers with the lipid molecules undergoing fast anisotropic motion. This is also true for the lipids present in the brush border membrane. The electron spin resonance (ESR) results obtained with various hydrophobic spin probes incorporated in either brush border vesicle membranes or their extracted lipids are consistent with this interpretation. By use of a variety of chemically different spin-labels, the temperature dependence of brush border membranes and their extracted lipids was probed. The temperature dependence of various ESR spectral parameters shows discontinuities that, by comparison with differential scanning calorimetry, are assigned to a lipid thermotropic phase transition. Differential scanning calorimetry shows that the lipid in brush border membranes undergoes a broad, reversible phase transition of low enthalpy between 10 and 30 degrees C, with a peak temperature of about 25 degrees C. Hence, the brush border membrane of rabbit small intestine functions in the liquid-crystalline state, well above the peak temperature and also above the upper limit of the lipid phase transition. Therefore, in itself, the thermotropic lipid phase transition is unlikely to play a physiological role. The low enthalpy of the lipid phase transition, indicative of a lack of cooperativity, is primarily attributed to the relatively high cholesterol content and to heterogeneity in the lipid composition of this membrane [Hauser, H., Howell, K., Dawson, R. M. C., & Bowyer, D. E. (1980) Biochim. Biophys. Acta 602, 567-577].(ABSTRACT TRUNCATED AT 250 WORDS)     

Antioxidant levels in germinating soybean seed axes in relation to free radical and dehydration tolerance

The axis of soybean seeds suffer dehydration injury if they are dried to 10% moisture at 36 hours of imbibition, but tolerate this stress if dried at 6 hours of imbibition. Deesterification of membrane phospholipids has been correlated with the increased permeability and increased lipid phase transition temperatures of membranes from dehydration injured tissues. Deesterification, measured as increased free fatty acid:phospholipid and decreased phospholipid:sterol ratios, occurred primarily when the tissue was in the dry state and did not change significantly (P ≤ 0.05) with increasing imbibition time.

When liposomes were exposed to free radicals in vitro, wide angle x-ray diffraction indicated that the phase transition temperature of liposomes prepared from membrane lipid from 36-hour axes (susceptible) increased from 6 to 31°C. In contrast, those from membrane lipid from 6-hour axes (tolerant) increased from 3 to only 8°C, indicating that the tolerance of free radicals previously observed in these membranes was due to a lipid-soluble component.

Lipid-soluble antioxidants were detected in 6-hour imbided axes in much greater quantities than in the 36-hour imbibed axes. The presence of lipid-soluble antioxidants in the membrane apparently contributes to the free radical tolerance of seed membranes observed during the early stages of germination, and these antioxidants may contribute to the dehydration tolerance of this tissue.

     

The action of membranotropic compounds with various structures on the parameters of the phase transition of dimyristoylphosphatidylcholine

The effect of substances of different nature on the thermodynamic characteristics of dimyristoylphosphatidylcholine (DMPC) phase transition by the differential scanning microcalorimetry has been studied. The substances disposed in hydrophobic part of membrane--alpha-tocopherol, ubiquinone Q10, ionol and vitamin K3 cause the decrease of enthalpy and cooperativity of phase transition. The substances which have the side hydrocarbon chain (tocopherol and ubiquinone Q10) compared with ones without it (ionol and vitamin K3) and reduced quinones (Q10 and vitamin K3) compared with the oxidized ones have stronger influence on the enthalpy and cooperativity of transition. The inclusion of the local anesthetic dicaine disposed mainly in the zone of polar heads of phospholipids into DMPC membranes decreases the temperature of phase transition considerably and practically does not change the cooperativity. A possibility to use the method of differential scanning microcalorimetry to estimate the localization of membrane tropic substances within lipid bilayer is under discussion.     

Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I. Membrane stability in cells of differing growth phase

Physical properties of Escherichia coli membrane lipids in logarithmic- and stationary-phase cells were studied by measuring the fluorescence polarization change of cis- and trans-parinaric acid as a function of temperature. In aqueous dispersions of phospholipids extracted from cytoplasmic and outer membranes of cells of differing growth phase, a similar polarization increase was observed over the range from physiological temperature to below 0 degrees C, and nearly the same transition ratios were obtained in all samples. The cytoplasmic membrane of both of the growth-phase cells showed a higher polarization ratio above the transition temperatures, compared to that in the aqueous dispersion of phospholipids. The polarization ratios below the transition temperatures of these specimens were lower than the value obtained with the lipids, especially in the stationary-phase specimens. The outer membrane specimens showed a similar polarization change but the transition temperature ranges were considerably higher both in the logarithmic- and the stationary-phase specimens, compared to those in the cytoplasmic membrane specimens. Freeze-thawing of logarithmic-phase cells showed the emergence of activity of certain enzymes which are known to be located in the membranes. The stationary-phase cells did not suffer from any such deleterious effect and maintained a high level of cell viability in a similar treatment. These results indicate that in the stationary-phase cell membranes lipids are in a highly ordered state, and the lipid state causes a membrane stability which results in the high resistance of the cell to freeze-thawing.     

The effects of bovine myelin basic protein on the phase transition properties of sphingomyelin

The basic protein of myelin can spontaneously associate with the synthetic phospholipid N-palmitoyl-sphingosinephosphatidylcholine. The protein alters the phase transition properties of the lipid from a single transition at 41.5 degrees C to two overlapping transitions, one being slightly above and the other slightly below the transition temperature of the pure lipid. The effect was not seen upon the addition of poly(L-lysine) to this lipid nor does the myelin basic protein alter the phase transition properties of dimyristoylphosphatidylcholine. The results thus demonstrate that the myelin basic protein can interact with a major zwitterionic lipid component of myelin in addition to acidic phospholipids.     

Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids

The formability, current-voltage characteristics and stability of the planar lipid bilayer membranes from the synthetic phytanyl-chained glycolipids, 1, 3-di-O-phytanyl-2-O-(beta-glycosyl)glycerols (Glc(Phyt)(2), Mal(N)(Phyt)(2)) were studied. The single bilayer membranes were successfully formed from the glycolipid bearing a maltotriosyl group (Mal(3)(Phyt)(2)) by the folding method among the synthetic glycolipids examined. The membrane conductance of Mal(3)(Phyt)(2) bilayers in 100 mM KCl solution was significantly lower than that of natural phospholipid, soybean phospholipids (SBPL) bilayers, and comparable to that of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers. From the permeation measurements of lipophilic ions through Mal(3)(Phyt)(2) and DPhPC bilayers, it could be presumed that the carbonyl groups in glycerol backbone of the lipid molecule are not necessarily required for the total dipole potential barrier against cations in Mal(3)(Phyt)(2) bilayer. The stability of Mal(3)(Phyt)(2) bilayers against long-term standing and external electric field change was rather high, compared with SBPL bilayers. Furthermore, a preliminary experiment over the functional incorporation of membrane proteins was demonstrated employing the channel proteins derived from octopus retina microvilli vesicles. The channel proteins were functionally incorporated into Mal(3)(Phyt)(2) bilayers in the presence of a negatively charged glycolipid. From these observations, synthetic phytanyl-chained glycolipid bilayers are promising materials for reconstitution and transport studies of membrane proteins.