Dimyristoylphosphatidylcholine Large Unilamellar Vesicles Research Articles

The anticancer drug adriamycin interacts with the human erythrocyte membrane.

Adriamycin is an aminoglycosidic anthracycline antibiotic widely used in the treatment of cancer. Increasing reports point to the involvement of cell membranes in its mechanism of action. The interaction of adriamycin with human erythrocytes was investigated in order to determine the membrane binding sites and the resultant structural perturbation. Electron microscopy revealed that red cells incubated with the therapeutical concentration of the drug in human plasma changed their discoid shape to both stomatocytes and echinocytes. According to the bilayer couple hypothesis, this means that adriamycin was incorporated into either the inner or outer leaflets of the erythrocyte membrane. To explain this unusual result, the drug was incubated with molecular models. One of them consisted of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) multilayers, representative of phospholipid classes located in the outer and inner leaflets of the erythrocyte membrane, respectively. X-ray diffraction showed that adriamycin interaction perturbed the polar head and acyl chain regions of both lipids. Fluorescence spectroscopy on another model, consisting of DMPC large unilamellar vesicles (LUV), confirmed the X-ray results in that adriamycin fluidized its hydrophobic moiety. It is concluded that adriamycin incorporates into both erythrocyte leaflets affecting its membrane structure.

The Organochlorine Pesticide Lindane Interacts with the Human Erythrocyte Membrane

Lindane is an organochlorine pesticide widely used in veterinary and human medicine to treat ectoparasites and pediculosis. Given its lipophilic character lipid-rich membranes are a plausible target of its interaction with living organisms. To evaluate its toxic effect on cell membranes lindane was made to interact with human erythrocytes and molecular models of the red cell membrane. These consisted in bilayers of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. Experiments by fluorescence spectroscopy showed that lindane interacted with DMPC large unilamellar vesicles fluidizing both its polar head and its acyl chain regions. These results were confirmed by X-ray diffraction; however, a higher degree of structural perturbation was observed in DMPE bilayers. Electron microscopy of human erythrocytes incubated with lindane revealed that they changed their normal discoid shape to cup-shaped stomatocytes. In accordance with the bilayer couple hypothesis, this result means that lindane inserted in the inner leaflet of the erythrocyte membrane, in agreement with its preferential interaction with DMPE. It is therefore concluded that the toxic effects of the pesticide can be related to its capacity to interact with the lipid moiety of cell membranes.

Interaction of gonadotropin-releasing hormone and its agonist analogs with Ca2+ in a nonpolar milieu. Correlation with biopotencies.

Extracellular Ca2+ is necessary for the action of gonadotropin-releasing hormone (GnRH). Assuming that this partly because of the interaction of the hormone with the relatively abundant extracellular Ca2+ in the low dielectric milieu of the bilayer plasma membrane, we studied the interaction of GnRH and five of its agonist analogs with Ca2+ under membrane-mimetic conditions. The peptides used, in increasing order of their reported gonadotropin-releasing activities, were: des-amide GnRH (or GnRH-OH); [Ala6]GnRH; [D-Ala6]GnRH; des-Gly10[D-Ala6,Pro9-NHEt]GnRH and, des-Gly10[D-Trp6,Pro9-NHEt]GnRH. Changes in the far-UV CD and fluorescence spectra of these peptides in trifluoroethanol were used to monitor conformational changes and obtain the Ca2+-binding isotherms. The data show that GnRH and its active analogs contain two Ca2+ binding sites, whereas the inactive analogs have only one. The extent of Ca2+ binding by the agonist peptides paralleled their biological potency ranking. The superactive analog des-Gly10[D-Trp6,Pro9-NHEt]GnRH exhibited the ability to transport Ca2+ ions across large unilamellar vesicles of dimyristoylphosphatidylcholine. Our study shows that significant differences among the GnRH and its analog peptides, suggestive of differences in their conformations, are manifested only in the presence of Ca2+. This observation would provide a basis for understanding GnRH action in terms of the hormone's interaction with Ca2+ in the lipid milieu.

The organochlorine pesticide heptachlor disrupts the structure of model and cell membranes

Heptachlor is an organochlorine pesticide which is particularly toxic for aquatic life. A significant source of this pesticide for infants is breast milk, where its concentration is considerably higher than in dairy milk. Given the lipophilic character of heptachlor, lipid-rich cell membranes are a very plausible target for its interaction with living organisms. In order to evaluate its toxicity towards cell membranes, heptachlor was made to interact with human erythrocytes and molecular models of the red cell membrane. These consisted of multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), which are types of phospholipids that are respectively located in the outer and inner monolayers of the erythrocyte membrane, and large unilamellar vesicles (LUV) of DMPC. Observations by scanning electron microscopy showed that 10 mM heptachlor produced various degrees of shape alterations to erythrocytes, which ranged from a few blebs in some cells to a great number of protuberances in others. On the other hand, experiments performed by X-ray diffraction on DMPC and DMPE indicated that the bilayer structure of DMPC was much more affected by heptachlor than that of DMPE. Measurements by fluorescence spectroscopy on DMPC LUV confirmed the X-ray diffraction results in that both the hydrocarbon chain and polar head regions of DMPC were structurally perturbed by heptachlor. The results obtained from the model studies could explain the shape changes induced to red cells by heptachlor. According to the bilayer hypothesis, they were due to the preferential interaction of heptachlor with the phosphatidylcholine-rich external moiety of the erythrocyte membrane. It is therefore concluded that toxic effects of this pesticide can be related to its capacity to perturb the phospholipid bilayer structure, whose integrity is essential for cell membrane functions.

Interaction of penicillin G with the human erythrocyte membrane and models.

Penicillin G (PEN) is a widely used antibiotic whose mechanism of action is related to the interference with the synthesis of bacteria cell wall. In order to evaluate its perturbing effect upon human cell membranes PEN was made to interact with human erythrocytes, isolated resealed human erythrocyte membranes and molecular models. The latter were multibilayers of the phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) as well as DMPC large unilamellar vesicles. These studies were performed by scanning electron microscopy, fluorescence spectroscopy and X-ray diffraction methods. The observed results coincide in that PEN did not exert any significant effect upon the structures of the red cell membrane neither on its molecular models. This is in agreement with its reported lack of major toxicity and hematological reactions.

Dynamic heat capacity at the gel to liquid-crystalline phase transition in large unilamellar vesicles of dimyristoylphosphatidylcholine in the ultralow frequency region.

Dynamic heat capacity at the gel to liquid-crystalline phase transition in large unilamellar vesicles of dimyristoylphosphatidylcholine in the ultralow frequency region.

Quantification of the interactions among fatty acid, lysophosphatidylcholine, calcium, dimyristoylphosphatidylcholine vesicles, and phospholipase A 2

The rate of hydrolysis of phosphatidylcholine bilayers by soluble phospholipase A 2 (PLA 2) is greatly enhanced by the presence in the bilayer of a threshold mole fraction of the reaction products: fatty acid and lysophosphatidylcholine (lyso-PC). The threshold requirement of these products appears to vary as a function of vesicle and calcium concentration. To further identify the roles of myristic acid, lyso-PC, and calcium in promoting optimal PLA 2 activity, we have quantified the various interactions among these components and dimyristoylphosphatidylcholine large unilamellar vesicles. The bilayer/water partition coefficient for myristic acid was obtained by competition of vesicles for the binding of the fatty acid to an acrylodan conjugate of an intestinal fatty acid binding protein as monitored by the acrylodan fluorescence emission spectrum. The partition coefficient for lyso-PC was obtained by a similar procedure using the tryptophan emission spectrum of bovine serum albumin. The effect of calcium concentration on these interactions was also quantified. These results were incorporated into an empirical model to describe the threshold requirements for these products in the bilayer. This information is vital for elucidating the mechanism of activation of PLA 2 by the hydrolysis products.

Amino acid sequence of a non-specific wheat phospholipid transfer protein and its conformation as revealed by infrared and Raman spectroscopy. Role of disulfide bridges and phospholipids in the stabilization of the α-helix structure

A wheat non specific phospholipid transfer protein has been isolated from wheat seeds and its amino acid sequence reveals that it is composed of 90 residues for a molecular weight of 9607. From the comparison of its sequence with those of the eight known proteins of the same family, hypotheses on the role of some conserved residues in the transfer activity can be made. The conformation of this protein has been studied by Raman and Fourier transform infrared spectroscopy and this is the first report on the structure of non specific plant phospholipid transfer proteins. As opposed to previous studies on the structure prediction from the amino acid sequence, the results obtained show that plant non specific phospholipid transfer proteins are not almost entirely composed of beta-sheets. Instead, infrared results show that the wheat protein contains 41% alpha-helix and 19% beta-sheet structures, while 40% of the conformation is undefined or composed of turns. Raman spectroscopy shows that three disulfide bridges adopt a gauche-gauche-gauche conformation while the other exhibits a gauche-gauche-trans conformation, and that the two tyrosine residues are hydrogen bonded to water molecules. The cleavage of the disulfide bonds affects significantly the conformation of the protein, the extended confirmation being increased by 15% at the expense of the alpha-helix content. On the other hand, the binding of 1-palmitoyllysophosphatidylcholine to the protein leads to an increase of 8% of the alpha-helix content compared to the free protein. Secondary structure predictions from the amino acid sequence suggest that the binding of a phospholipid stabilizes helicity of the amphipathic helices while the reduction of disulfide bonds would affect the stability of the N-terminal helix. The extended structure located at the C-terminus is not affected. Finally, the wheat phospholipid transfer protein has no effect on the thermotropic behavior of large unilamellar vesicles of dimyristoylphosphatidylcholine while it increases the conformational order of the acyl chains of large unilamellar vesicles of dimyristoylphosphatidylglycerol in the liquid-crystalline state. No major conformational changes of the protein are observed when it is adsorbed to phospholipid vesicles. These results suggest that the helical structure is essential for the transfer activity without excluding a possible role of the C-terminal extended structure on the adsorption to phospholipid vesicles.

The interaction of d-propranolol and dimyristoyl phosphatidylcholine large unilamellar vesicles investigated by quasielastic light scattering and Fourier-transform infrared spectroscopy

The effect of d-propranolol on dimyristoylphosphatidylcholine large unilamellar vesicles has been investigated by quasielastic light scattering and Fourier transform infrared spectroscopy. The results indicate that the drug interacts with the phosphate groups of the polar heads, shifts the phase transition point and alters the low temperature phase. A second transition has been observed, and the new phase of the vesicles induced by the presence of the drug might be an interdigitated one.

Spontaneous incorporation of bacteriorhodopsin into large preformed vesicles

Bacteriorhodopsin, either as purple membrane sheets or as detergent-solubilized protein, was found to incorporate spontaneously into both large unilamellar vesicles (LUVs) and sized multilamellar vesicles (MLVs) under either gel-phase or liquid-phase conditions. These results were obtained with LUVs of various lipid compositions, including dimyristoylphosphatidylcholine (DMPC), DMPC and cholesterol, dioleoylphosphatidylcholine (DOPC), and DOPC and cholesterol. The lipid to protein (L/P) ratio of all proteoliposomes arising from these preformed vesicles continually increased in the presence of protein-free vesicles. Under fluid-phase conditions, the mixing of LUVs of DMPC with proteoliposomes showed vesicle growth independent of lipid concentration, suggesting that growth was due to lipid transfer. However, under gel-phase conditions a more rapid, concentration-dependent increase in the L/P ratio of the proteoliposome was observed, suggesting that growth occurred by a mechanism other than lipid transfer from vesicles to proteoliposomes. The use of the proteoliposome as an artificial lipid-protein membrane model is discussed.

Effect of proteins on fluorophore lifetime heterogeneity in lipid bilayers

The effect of three different membrane proteins on the fluorescence lifetime heterogeneity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in phospholipid vesicle systems was investigated. For large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) at 37 degrees C, the fluorescence decay was essentially monoexponential (8.6 and 8.2 ns, respectively) except for a minor component typical of DPH. For gramicidin D reconstituted into DMPC vesicles at a protein/lipid molar ratio of 1/7, the most appropriate analysis of the data was found to be in the form of a bimodal Lorentzian distribution. Centers of the major lifetime components were almost identical with those recovered for vesicles without proteins, while broad distributional widths of some 4.0 ns were recovered. Variation of the protein/lipid molar ratio in sonicated POPC vesicles revealed an abrupt increase in distributional width at ratios approximating 1/15-1/20, which leveled off at about 2.5 ns. For bacteriorhodopsin in DMPC vesicles and cytochrome b5 in POPC, the most appropriate analysis of the data was again found to be in the form of a bimodal Lorentzian also with broad distributional widths in the major component. Lifetime centers were decreased for these proteins due to fluorescence energy transfer to the retinal of the bacteriorhodopsin and heme of the cytochrome b5. Fluorescence energy transfer is distance dependent, and since a range of donor-acceptor distances would be expected in a membrane, lifetime distributions should therefore be recovered independently of other effects for proteins possessing acceptor chromophores.(ABSTRACT TRUNCATED AT 250 WORDS)

Exchange and flip-flop of dimyristoyl phosphatidylcholine in liquid-crystalline, gel and two-component, two-phase large unilamellar vesicles

The rate and extent of spontaneous exchange of dimyristoylphosphatidylcholine (DMPC) from large unilamellar vesicles (LUV) composed of either DMPC or mixtures of DMPC/distearoylphosphatidylcholine (DSPC) have been examined under equilibrium conditions. The phase state of the vesicles ranged from all-liquid-crystalline through mixed gel/liquid-crystalline to all-gel. The exchange rate of DMPC between liquid-crystalline DMPC LUV, measured between 25 and 55 degrees C, was found to have an Arrhenius activation energy of 24.9 +/- 1.4 kcal/mol. This activation energy and the exchange rates are very similar to those obtained for the exchange of DMPC between DMPC small unilamellar vesicles (SUV). The extent of exchange of DMPC in LUV was found to be approximately 90%. This is in direct contrast to the situation in DMPC SUV where only the lipid in the outer monolayer is available for exchange. Thus, transbilayer movement (flip-flop) is substantially faster in liquid-crystalline DMPC LUV than in SUV. Desorption from gel-phase LUV has a much lower rate than gel-phase SUV with an activation energy of 31.7 +/- 3.7 kcal/mol compared to 11.5 +/- 2 kcal/mol reported for SUV. A defect-mediated exchange in gel-phase SUV, which is not the major pathway for exchange in LUV, is proposed on the basis of the thermodynamic parameters of the activation process. Surprisingly, the rates of DMPC exchange between DMPC/DSPC two-component LUV, measured over a wide range of compositions and temperatures, were found to exhibit very little dependence on the composition or phase configuration of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Competition between cholesterol and phospholipids for binding to filipin

The interaction of the polyene antibiotic filipin with large unilamellar vesicles (LUV) has been studied by circular dichroism (CD). (1) On the one hand, the interaction has been followed with preformed LUV. In the presence of cholesterol-free dimyristoylphosphatidylcholine (DMPC) LUV, monomeric filipin, above a critical concentration of about 1·10 −6 mol/1 and up to a DMPC/filipin molar ratio of approximately 4, bound to LUV and self-associated in the membranes as demonstrated by the presence of an excitonic doublet. For increasing DMPC/filipin molar ratios, filipin remained bound to membranes but became more and more under monomeric form. In the presence of preformed cholesterol-containing DMPC LUV, for increasing concentrations of filipin, the formation of a 1:1 filipin-cholesterol complex was first observed; then, for an antibiotic/cholesterol molar ratio higher than 1 an interaction between filipin and phospholipid, identical to the one observed with cholesterol-free LUV, was observed. (2) On the other hand, the interaction with cholesterol-containing model membranes has been followed in the same experimental conditions as those used in recent studies by 2H-NMR (Dufourc, E.J. and Smith, I.C.P. (1985) Biochemistry 24, 2420–2424) and by ESR (Maurin, L., Bancel, F., Morin, P. and Bienvenue, A. (1988) Biochim. Biophys. Acta 939, 102–110), that is with filipin incorporated into membranes during their preparation. For preparations with filipin/cholesterol molar ratios of about 1:1 we confirm the results obtained by both methods. In the presence of an excess of filipin (filipin/cholesterol molar ratio 10:1), the CD spectrum observed with egg-yolk PC at 6°C (conditions used in the ESR study) is identical to that observed in the presence of pure DMPC above the transition temperature (Milhaud, J., Mazerski, J., Bolard, J. and Dufourc, E.J. (1989) Eur. Biophys. J. 17, 151–158). We conclude that in these conditions a filipin-phospholipid association occurs once the sterolic sites of fixation have been occupied.

The effect of loperamide on the thermal behavior of dimyristoylphosphatidylcholine large unilamellar vesicles

The effect of loperamide, a drug belonging to the opiate family, on dimyristoyl phosphatidylcholine large unilamellar vesicles (DMPC LUV) was investigated by quasielastic light scattering (QLS) and Fourier transform infrared spectroscopy (FT-IR).Both techniques show that, in the presence of loperamide, DMPC LUV undergoes a two step transition in cooling: one step around the transition point of pure lipid vesicles, the other at a lower temperature. The temperature of the latter step transition is different for the head and tail regions of the drug-containing vesicles: FT-IR spectra demonstrate that the hydrophobic acyl chains transition starts at a temperature well above that of the interfacial region whereas the transition of the entire vesicle, explored by QLS, is broad and covers both temperature ranges. These transitions are thermally reversible in the FT-IR which measures local order but aggregation effects prevent the thermal reversibility of the QLS results. The nature of the drug-lipid interaction is also discussed.

Interaction of filipin with dimyristoylphosphatidylcholine membranes studied by 2H-NMR, circular dichroism, electronic absorption and fluorescence.

Interaction of the pentene antibiotic filipin with dimyristoylphosphatidylcholine (DMPC) membranes has been monitored by 2H-NMR, circular dichroism (CD), electronic absorption and fluorescence in the temperature range 10 degrees to 60 degrees C. Interaction appears to depend on whether filipin is added before or after membrane formation and also upon the temperature of the system. When filipin is added to preformed DMPC large unilamellar vesicles (LUV), the association constants, as determined by electronic absorption are 39 x 10(3) M-1, 15 x 10(3) M-1 and 0.6 x 10(3) M-1 at 15 degrees, 30 degrees and 50 degrees C, respectively. Under identical conditions, CD spectra of bound filipin exhibit features characteristic of an aggregation over the whole temperature range. When filipin is incorporated in membranes during their preparation, the 2H-NMR spectra of deuterated DMPC indicate that the drug has a slight disordering effect on the lipid matrix below the temperature, Tc, of the gel-to-fluid phase transition and above Tc + 11 degrees C. Between these two temperature boundaries the system consists of two lipid regions of very different dynamic properties. One of the regions, which is attributed to a filipin-lipid complex, has the properties of gel-like lipids whereas the other has those of fluid-like lipids. The latter domain is however more ordered than the pure lipid at corresponding temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of the polyene antibiotic amphotericin B with model membranes: differences between small and large unilamellar vesicles

The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membrannes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation to cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 ×10 −3, a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca 2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 × 10 −4M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of ≈ 10 −5M. These results are compared with recent data concerning the interaction of LUV with the pantaenic antibiotic filipin (Fig. 1) (J. Milhaud, J. Mazerski, E. Dufourc and J. Bolard, submitted; J. Milhaud, J. Bolard, P. Benveniste and M.A. Hartmann [1988] Biochim. Biophys. Acta 943, 315–325).

Effect of ethanol on dimyristoylphosphatidylcholine large unilamellar vesicles investigated by quasi-elastic light scattering and vibrational spectroscopy

The gel-like liquid phase transition of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles prepared by reverse phase evaporation has been investigated in buffers containing ethanol by quasi-elastic light scattering (QLS) and vibrational (infrared and Raman) spectroscopy. With the QLS technique, the relative change in the vesicles area (which is related to the molecular cross-sectional area of lipid molecules) was followed versus both temperature and ethanol concentration. When the latter was low, the depression of the transition point was a linear function of the alcohol concentration, c, but the vesicles area was practically unmodified. At alcohol concentration 10% v/v, an abrupt change of the vesicles area was observed and for c > 10% the depression of the transition point was a non-linear function of c. The infrared and Raman spectra showed a perturbation of the hydrophobic regions, including the terminal methyl groups of the acyl tails.