Radiation inactivation of ion channels formed by gramicidin A. Protection by lipid double bonds and by alpha-tocopherol.

The conductance induced by the channel-forming peptide gramicidin A in lipid membranes is reduced by many orders of magnitude on exposure of the membrane and its aqueous environment to ionizing radiation. This results from an interaction of free radicals of water radiolysis with the tryptophan residues of gramicidin A. The sensitivity of the ion channels towards irradiation is strongly reduced in the presence of either vitamin E or of highly unsaturated lipids. An increase of the D37 dose up to a factor of 50 was found. The phenomena are interpreted via a reduction of the effective concentration of free radicals (such as OH.) in the membrane by reaction with unsaturated fatty acid residues or with vitamin E.     

A fluorescence approach of the gamma radiation effects on gramicidin A inserted in liposomes.

The fluorescence of tryptophan residues of gramicidin A (gA), bound to phosphatidylcholine liposomes contains valuable information about local changes in the environment of the molecule induced by gamma radiation. With this work, we aim to demonstrate that the gamma radiation effect on the peptide involves the action of free radicals, derived from water radiolysis and the process of lipid peroxidation. Basically, the methodology consists of the analysis of UV and fluorescence emission spectra of the peptide liposome complexes under control conditions and upon gamma irradiation. Free radical production was impaired by the removal of molecular oxygen or the presence of ethanol in the liposome suspension. The intensity of the tryptophan fluorescence was recorded as a function of the gamma radiation dose in the range of 0-250 Gy and the data were fitted with a single decay exponential function containing an additional constant term (named residual fluorescence). The correlation between the decrease in tryptophan fluorescence emission (D(c) = 80 +/- 10 Gy) and increase in gamma radiation dose indicates the partial damage of the tryptophan side chains of gA. O(2) removal or ethanol addition partially reduced the decay of the tryptophan fluorescence emission involving an indirect action of gamma radiation via a water radiolysis mechanism. The residual fluorescence emission (A(0)) increases in O(2)-free buffer (98 +/- 13) and in 10% ethanol-containing buffer (74 +/- 34) compared to control conditions (23 +/- 5). Varying the dose rate between 1-10 Gy/min at a constant dose of 50 Gy, an inverse dose-rate effect was observed. Thus, our study provides evidence for the lipid peroxidation effect on the tryptophan fluorescence. In conclusion, this article sustains the hypothesis of the connection between the lipid peroxidation and structural changes of membrane proteins induced by gamma radiation. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of ionizing radiation on artificial (planar) lipid membranes. I. Radiation inactivation of the ion channel gramicidin A

The ion channel formed by the pentadecapeptide gramicidin A in planar lipid membranes is extremely sensitive to ionizing radiation. The membrane conductance may drop by several orders of magnitude under appropriate experimental conditions (low pH and presence of oxygen). The radiation sensitivity is strongly reduced for gramicidin M-. This analogue has the four tryptophan residues replaced by phenylalanines. Experiments performed in the presence of various radical scavengers suggest that the inactivation of the channel is due to a combined action of OH and of HO2 radicals at the tryptophan residues. The shape of the inactivation curves following continuous radiolysis or pulse radiolysis were found to be in fair agreement with a simple model which assumes that the damage of a single tryptophan residue is sufficient for channel inactivation. The conductance of inactivated channels could not be resolved within the experimental accuracy. This is contrary to photolysis of gramicidin channels found by Busath and Waldbilling (1983), where a broad distribution of low conductance states was observed. The inactivation by radiolysis seems to represent an 'all-or-none-process' of the channel conductance.     

Pulse radiolysis at planar lipid membranes doped with ion carriers or pore formers

The effect of 14 MeV electrons on ion transport through planar lipid membranes was investigated. The membranes were formed in the presence of well defined ion carriers or pore forming substances. In the presence of the ion carriers valinomycin or nonactin or in the presence of the pore formers nystatin or amphotericin B, irradiation produced a transient increase of the membrane conductance followed by a long lasting decrease. The effects are interpreted on the basis of a time-dependent chemical modification of the membrane structure caused by exposure to high energy radiation. The pore former gramicidin A shows an exponential inactivation with increasing dose. At pH 3 and in the presence of oxygen the pore is highly sensitive to radiation ($\text{D}{}_{37}\text{≈ 10}\text{Gy}$) whereas at pH 9.5 a considerably lower radiation sensitivity ($\text{D}{}_{37}\text{≈ 1000}\text{Gy}$), was found. In the absence of oxygen, gramicidin A is virtually insensitive to irradiation. This is considered an evidence that the inactivation of this ion channel is primarily caused by the perhydroxyl radical HO₂.     

Membrane deterioration in senescing carnation flowers : coordinated effects of phospholipid degradation and the action of membranous lipoxygenase

The lipid fluidity of microsomal membranes from the petals of cut carnation flowers decreases as the flowers senesce. A comparable change in fluidity was induced by in vitro aging of microsomal membranes from young flowers under conditions in which membranous lipoxygenase-like activity was active. There was no change in fluidity when the membranes were aged in the presence of inhibitors of lipoxygenase or were heat-denatured prior to aging. Membranes from naturally senesced flowers and membranes that had been aged in vitro both sustained an increase in saturated:unsaturated fatty acid ratio that accounted for the decrease in lipid fluidity, and in both instances there was evidence for depletion of the unsaturated fatty acids, linoleic acid, and linolenic acid, which are substrates for lipoxygenase. Loss of lipid phosphate reflecting breakdown of membrane phospholipids preceded the depletion of unsaturated fatty acids attributable to the lipoxygenase-like activity. The data have been interpreted as indicating that fatty acid substrates for membrane-associated lipoxygenase-like activity are made available by the initiation of phospholipid degradation, and that the utilization of these substrates results in a selective depletion of unsaturated fatty acids from the membrane and an ensuing decrease in bulk lipid fluidity.     

An evaluation of the contribution of membrane lipids to protection against ultraviolet radiation

Using dioleoylphosphatidylcholine liposomes incorporating various fatty acids and neutral lipids, we have examined the ability of such lipids to provide protection of Escherichia coli and vesicular stomatitis virus (VSV) against the lethal effect of ultraviolet (254 nm) radiation. While the presence of varying amounts of saturated (palmitic) or polyunsaturated (arachidonic) fatty acids or the lipid antioxidant, alpha-tocopherol, had little effect on killing by ultraviolet radiation, considerable radioprotection was observed with beta-carotene, retinal and vitamin K-1 at final concentrations of 1 mg/ml. In another approach, vesicular stomatitis virus grown under conditions in which its envelope fatty acid composition was substantially modified, showed little change in its sensitivity to inactivation by ultraviolet radiation. The results provide strong evidence for a radioprotective role of certain, relatively rare natural lipid components with conjugated polyene systems, but not of the more ubiquitous and abundant membrane fatty acids.     

Photodynamic activation of ion transport through lipid membranes and its correlation with an increased dielectric constant of the membrane

Illumination of biological membranes with visible light in the presence of membrane-active sensitizers (e.g. rose bengal) is known to inactivate transport proteins such as ion channels and ion pumps. In some cases, however, illumination gives rise to an activation of transport. This is shown here for ion channels formed by alamethicin in lipid membranes, and for porin channels, which were isolated from the outer membrane of E. coli (OmpC) and from the outer membrane of mitochondria (VDAC) and were reconstituted in lipid membranes. An activation (in the form of an increased conductance) was also observed in the presence of the cation carriers valinomycin and nonactin. The activation phenomena were only present, if the membranes were made from lipids containing unsaturated double bonds. Activation was reduced in the presence of the antioxidant vitamin E.We suggest that the activation of the different transport systems has a common physical basis, namely an increase of the dielectric constant, epsilon(m), of the membrane interior by the presence of polar oxidation products of photodynamically induced lipid peroxidation. Experimental evidence for an enhanced dielectric constant was obtained from the finding of a light-induced increase of the membrane capacitance in the presence of rose bengal.     

Fatty Acids are Key in 4-Hydroxy-2-Nonenal-Mediated Activation of Uncoupling Proteins 1 and 2

The production of reactive oxygen species (ROS) in mitochondria is very sensitive to the proton motive force and may be decreased by mild uncoupling, mediated e.g. by mitochondrial uncoupling proteins (UCPs). UCPs were conversely hypothesized to be activated by ROS. Conclusions from experiments studying the reactive product of lipid peroxidation 4-hydroxy-2-nonenal (HNE) in isolated mitochondria and UCP knock-out mice are highly controversial. Here we investigated the molecular mechanism of HNE action by evaluating the separate contributions of lipid and protein phases of the membrane and by comparing UCP1 and UCP2, which were reconstituted in planar lipid bilayers. We demonstrated that aldehyde does not directly activate either UCP1 or UCP2. However, HNE strongly potentiated the membrane conductance increase (G_m) mediated by different long-chain fatty acids in UCP-containing and in UCP-free membranes and this suggest the involvement of both lipid-mediated and protein-mediated mechanisms with FA playing the central role. G_m increase was concentration-dependent and exhibited a typical saturation kinetic with the binding constant 0.3 mM. By using Electron Paramagnetic Resonance, membrane fluidity change could be excluded as a cause for the HNE-mediated increase in the presence of FA. The impact of the HNE binding to definite positively charged UCP amino acid residues is discussed as a possible protein-mediated mechanism of the UCP activation.     

Inactivation by ionizing radiation of ion channels formed by polyene antibiotics amphotericin B and nystatin in lipid membranes: an inverse dose-rate behavior.

The phenomena reported are part of a study about the effects of ionizing radiation on membrane transport. We found that the conductance of lipid membranes in the presence of the polyene-antibiotics nystatin or amphotericin B is reduced to virtually zero following irradiation. Ion channels formed by these substances seem to represent extremely sensitive structures being inactivated by radiation doses in the range of a few Centigray (1 cGy = 1 rad) at sufficiently small dose rates. Inactivation shows a so-called inverse dose-rate behavior, i.e., at constant radiation dose the effect increases with decreasing dose rate. Similar to radiation-induced lipid peroxidation the phenomenon may be understood on the basis of a radical chain mechanism initiated by free radicals of water radiolysis. The process--via peroxidation of the polyene part of the molecules--is suggested to modify the hydrophobic exterior and to destabilize the barrel-like structure of the ion channels.     

Photodynamic activation of ion transport through lipid membranes and its correlation with an increased dielectric constant of the membrane

Illumination of biological membranes with visible light in the presence of membrane-active sensitizers (e.g. rose bengal) is known to inactivate transport proteins such as ion channels and ion pumps. In some cases, however, illumination gives rise to an activation of transport. This is shown here for ion channels formed by alamethicin in lipid membranes, and for porin channels, which were isolated from the outer membrane of E. coli (OmpC) and from the outer membrane of mitochondria (VDAC) and were reconstituted in lipid membranes. An activation (in the form of an increased conductance) was also observed in the presence of the cation carriers valinomycin and nonactin. The activation phenomena were only present, if the membranes were made from lipids containing unsaturated double bonds. Activation was reduced in the presence of the antioxidant vitamin E.We suggest that the activation of the different transport systems has a common physical basis, namely an increase of the dielectric constant, ε_m, of the membrane interior by the presence of polar oxidation products of photodynamically induced lipid peroxidation. Experimental evidence for an enhanced dielectric constant was obtained from the finding of a light-induced increase of the membrane capacitance in the presence of rose bengal.     

Effect of membrane fatty acid changes on the radiation sensitivity of human lymphoid cells

To test the influence of changes in membrane fatty acid composition on the radiation response of mammalian cells, human LDV cells were cultured in medium containing delipidated serum supplemented with either oleic or linoleic acid. Analysis of lipid extracts of the cells by gas liquid chromatography showed that, after 3 or more days growth in oleic or linoleic acid supplemented media, there were substantial overall increases in the proportion of oleate and linoleate, respectively, in the cellular lipid. Smaller absolute changes were measured in nuclear phospholipid, which was found to be low in unsaturated phospholipid compared to the rest of the cell. Fluorescence polarization measurements using diphenylhexatriene indicated an increased membrane fluidity in cells grown in the presence of excess linoleic acid and to a lesser extent for oleic acid. The clonogenic capacity of the cells after irradiation in air or nitrogen was not altered by any of these membrane compositional changes. The lack of effect on radiation sensitivity, in contrast to that reported for bacteria (E. coli K1060), is consistent with the fact that little or no change was brought about in the nuclear membrane composition, since evidence from partial cell irradiation experiments indicates that the cell nucleus is the sensitive target for cell killing by ionizing radiation. The ability of the cell to maintain a low level of unsaturated phospholipids in its nuclear membrane may be an important general defence mechanism against free radical damage to chromatin mediated by lipid peroxidation.     

The effect of variations in growth temperature, fatty acid composition and cholesterol content on the lipid polar head-group composition of Acholeplasma laidlawii B membranes

We have systematically investigated the effect of variations in growth temperature, fatty acid composition and cholesterol content on the membrane lipid polar headgroup composition of Acholeplasma laidlawii B. Two important lipid compositional parameters have been determined from such an analysis. The first parameter studied was the ratio of the two major neutral glycolipids of this organism, monoglucosyldiacylglycerol (MGDG) and diglucosyldiacylglycerol (DGDG). As the former lipid prefers to exist in a reversed hexagonal phase at higher temperatures, with unsaturated fatty acyl chains or in the presence of cholesterol, the ratio of these two lipids reflects the phase state preference of the total A. laidlawii membrane lipids. Although we find that the MGDG/DGDG ratio is reduced in response to an increase in fatty acid unsaturation, increases in growth temperature or cholesterol content reduce this ratio only in cells enriched in a saturated but not an unsaturated fatty acid. The second parameter studied was the ratio of these neutral glycolipids to the only phosphatide in the A. laidlawii membrane, phosphatidylglycerol (PG); this parameter reflects the relative balance of uncharged and charged lipids in the membrane of this organism. We find that the MGDG + DGDG/PG ratio is lowest in cells enriched in the saturated fatty acid even though these cells already have the highest lipid bilayer surface charge density. Moreover, this ratio is not consistently related to growth temperature or changes in cholesterol levels, as expected. We therefore conclude that A. laidlawii strain B, apparently unlike strain A, does not possess coherent regulatory mechanisms for maintaining either the phase preference or the surface charge density of its membrane lipid constant in response to variations in growth temperature, fatty acid composition or cholesterol content.     

The effect of dietary polyenylphosphatidylcholine on microsomal delta-6-desaturase activity, fatty acid composition, and microviscosity in rat liver under oxidative stress

Polyenylphosphatidylcholine is a choline-glycerophospholipid containing up to 80% of total fatty acids as linoleic acid and may be an important factor in ensuring normal functioning of cell membranes. We tested the effect of a polyenylphosphatidylcholine-supplemented diet and compared it with both a trilinolein-supplemented and a laboratory chow diet on the fatty acid composition, microviscosity, and delta-6-desaturase activity of liver microsomal membranes of 12-month-old rats, in the absence or presence of oxidative stress induced by adriamycin. Polyenylphosphatidylcholine- and trilinolein-supplemented diets showed a similar increase in linoleic acid content and delta-6-desaturase activity in liver microsomes, indicating that low amounts of linoleic acid are able to partially restore the enzyme activity in old rats, independent of the source of linoleic acid. After adriamycin treatment, delta-6-desaturase activity increased in polyenylphosphatidylcholine and trilinolein groups, indicating a protective mechanism against the damage induced by polyunsaturated fatty acid peroxidation. The measurement of malondialdehyde production showed a protective effect on adriamycin-induced lipid peroxidation by polyenylphosphatidylcholine supplementation only. Microsomal membrane microviscosity did not change independent of diet and adriamycin treatment, suggesting that the response of microsomes to lipid peroxidation might be the maintenance of a given membrane order. Administration of polyenylphosphatidylcholine can prevent or minimize the liver damage induced by adriamycin treatment.     

A confirmation of the phase behavior of Escherichia coli cytoplasmic membrane lipids by X-ray diffraction.

The lipid fatty acid composition of the cytoplasmic membranes of Escherichia coli can be varied by growing an unsaturated fatty acid auxotroph in the presence of different fatty acid supplements. Electron spin resonance (ESR) studies of spin-label partitioning into the cytoplasmic membranes of different lipid fatty acid compositions as a function of temperature have been interpreted as indicating a broad order-to-disorder transition in the membrane lipids, the end points of the transition depending upon the fatty acid composition. We have utilized x-ray diffraction to confirm the ESR studies for three different fatty acid supplements (oleic, elaidic, and bromostearic). We found that the characteristic end-point temperatures detected by ESR were indeed the end-point temperatures of a broad order-to-disorder transition of the cytoplasmic membrane lipids. In addition, Patterson functions calculated from lamellar x-ray diffraction from partially oriented cytoplasmic membranes indicate a decrease in average membrane thickness upon fatty acid chain melting.     

Involvement of membrane lipids in radiation damage to potassium ion permeability of Escherichia coli.

Radiation damage to K+ permeability of an unsaturated fatty acid auxotroph of E. coli grown with oleate or linolenate was investigated at different temperatures. A remarkable effect of radiation was observed at 0 degrees C with cells that had been grown with linolenate at 42 degrees C. This indicates that, besides protein, membrane lipids at least are involved in the radiation damage. The damage also seems to be affected by the fluidity of membrane lipids.     

Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal regulation of the fatty acid composition of lipopolysaccharides and phospholipids of Proteus mirabilis.

The fatty acid composition of the lipid A moiety of the lipopolysaccharide and phospholipid fractions of Proteus mirabilis changed significantly on varying the growth temperature. A decrease in the growth temperature from 43 degrees C to 15 degrees C resulted in a decrease in the palmitic acid content of the lipopolysaccharide from 19.4% of total fatty acids at 43 degrees C to 1.4% at 15 degrees C, and by the appearance of an unsaturated fatty acid residue, hexadecenoic acid. Changes in the 3-hydroxy-myristic acid content of the lipid A were minimal. The decrease in the growth temperature also resulted in a decrease in the saturated fatty acid content of the phospholipid fraction, which was accompanied by an increase in their fluidity, as measured by the freedom of motion of spin-labeled fatty acids incorporated into dispersions made of the phospholipids. Nevertheless, the fluidity obtained with membrane phospholipids extracted from the cells grown at various temperatures were essentially the same when fluidity was determined at the growth temperature, supporting the hypothesis that variations in the fatty acid composition of membrane phospholipids serve to produce membranes having a constant fluidity at different temperatures of growth.     

Effects of UV-C radiation on membrane potential and electric conductance in internodal cells of Nitellopsis obtusa

Effects of 253.7 nm ultraviolet radiation on membrane potential and conductance in internodal cells of Nitellopsis obtusa were studied. The radiation caused transient depolarization of plasmalemma and tonoplast and simultaneous increase in electric conductance. These effects were partly reversible and the degree of the recovery depended on the duration of the exposure. In cells with potential difference (between vacuole and external medium) more negative than – 140 mV, the radiation induced an action potential. The hyperpolarized state created by visible light and indole-3-acetic acid was fully suppressed by the radiation. The results are discussed taking into account the data for Chara corallina obtained by C. J. Doughty and A. B. Hope. It is suggested that 253.7 nm radiation inhibits electrogenic proton pumps in the plasmalemma and activates the Cl⁻ channels.     

Implications of modifying membrane fatty acid composition on membrane oxidation,integrity, and storage viability of freeze‐dried probiotic,Lactobacillus acidophilus La‐5

The aim of this study was to investigate the effect of altering the fatty acid profile of the lipid membrane on storage survival of freeze‐dried probiotic, Lactobacillus acidophilus La‐5, as well as study the membrane integrity and lipid oxidation. The fatty acid composition of the lipid membrane of L. acidophilus La‐5 was significantly different upon growth in MRS (containing Tween 80, an oleic acid source), or in MRS with Tween 20 (containing C12:0 and C14:0), linoleic, or linolenic acid supplemented. Bacteria grown in MRS showed the highest storage survival rates. No indications of loss of membrane integrity could be found, and membrane integrity could therefore not be connected with loss of viability. Survival of bacteria grown with linoleic or linolenic acid was more negatively affected by the presence of oxygen, than bacteria grown in MRS or with Tween 20 supplemented. A small, but significant, loss of linolenic acid during storage could be identified, and an increase of volatile secondary oxidation products during storage was found for bacteria grown in MRS, or with linoleic, or linolenic acid supplemented, but not for bacteria grown with Tween 20. Overall, the results indicate that lipid oxidation and loss of membrane integrity are not the only or most important detrimental reactions which can occur during storage. By altering the fatty acid composition, it was also found that properties of oleic acid gave rise to more robust bacteria than more saturated or unsaturated fatty acids did. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:799–807, 2015     

Effect of photoperiod and gibberellic acid on the plasma membrane H+ATPase activity of spinach leaves

Plasma membrane H⁺ATPase extracted from leaves of spinach plants induced to flower by gibberellic acid treatments or by a transfer to a photoperiod of 24 h had a lower Km_app than that from vegetative plants grown in short days. The Km_app obtained after inhibition by vanadate was decreased in vegetative plants and increased in induced ones showing a differential effect of this inhibitor on the kinetic properties of the enzyme between vegetative and induced plants. The phospholipid fatty acid analysis of the purified plasma membrane showed an increase of C18:1/C18:2 fatty acid ratio upon induction by light or by gibberellic acid treatments, whereas the saturated to unsaturated fatty acid ratio was kept constant. The decrease in the Km_app observed after induction may be thus interpreted in terms of the observed changes in lipid environment.