Distribution of tumor promoters in lipid membranes and changes in membrane structure

The interaction of tumor promoters differing in molecular structure, namely, 12-O-tetradecanoylphorbol 13-acetate (TPA) and teleocidin, with dipalmitoylphosphatidylcholine (DPPC) vesicles was studied. Investigation by Fourier transform infrared spectroscopy clarified the differences between the tumor promoters in the mode of interaction with lipid bilayer membranes. The temperature dependence of the bandwidth of the C-H or C = O stretching absorption of lipid molecules in the presence of tumor promoters relative to that in pure DPPC vesicles indicated that TPA is incorporated into the hydrophobic core of the lipid bilayer membrane whilst teleocidin binds predominantly to the membrane surface. However, both tumor promoters tend to restrict the motion of lipid molecules in membranes. The same conclusion was derived from measurements of steady-state fluorescence polarization, which showed that tumor promoters decreased the membrane fluidity. On the other hand, carboxyfluorescein (CF) leakage from vesicles was enhanced by the addition of TPA below the phase-transition temperature, whereas the effect of teleocidin on steady-state CF leakage was not as significant. It is considered that the difference in the profile of the TPA-induced increase in CF leakage compared to that of teleocidin might be ascribable to a different binding site for each tumor promoter in the membranes.     

Fluorescence studies of dipalmitoylphosphatidylcholine vesicles reconstituted with the glycoprotein of vesicular stomatitis virus

The vesicular stomatitis virus glycoprotein (G) was reconstituted into dipalmitoylphosphatidylcholine (DPPC) vesicles by detergent dialysis. The DPPC gel to liquid-crystalline phase transition of the DPPC-G protein vesicles was monitored by the fluorescence anisotrophy of trans-paranaric acid, 16-(9-anthroyloxy)palmitoylglucocerebroside, 1,6-diphenyl-1,3,5-hexatriene, and 4-heptadecyl-7-hydroxycoumarin. The DPPC transition temperature measured by all four fluorescent probes was lowered in the presence of the G protein and the DPPC gel state was disordered by the G protein as evidenced by a decreased fluorescence anisotropy for all four probes below the phase-transition temperature. A possible ordering of the DPPC liquid-crystalline state by the G protein was indicated by an increased anisotropy of trans-paranaric acid and 16-(9-anthroyloxy)palmitoylglucocerebroside in the liquid-crystalline state of DPPC-G protein vesicles. The G protein in addition affected the ionization of the 4-heptadecyl-7-hydroxycoumarin in lipid vesicles, increasing the apparent pK of the probe from 9.05 to 9.45.     

Modulation of phospholipase A2 activity by the tumour promoters phorbol esters and teleocidin.

The effects of tumour promoters, namely phorbol esters and teleocidin, on the activity of porcine pancreatic phospholipase A2 (PLA2) was investigated by using a system of small unilamellar vesicles composed of dipalmitoyl-phosphatidylcholine (DPPC). DPPC vesicles encapsulating Quin 2 (Quin 2/DPPC vesicles) were suspended in a medium containing Ca2+. The addition of PLA2 to Quin 2/DPPC vesicles increased the fluorescence intensity of Quin 2. This increase was due to chelation of Quin 2 with Ca2+, which resulted from an increase in the permeability of the phospholipid bilayer caused by the hydrolytic activity of PLA2. The tumour promoters phorbol 12-myristate 13-acetate (PMA) and teleocidin, at low concentrations, enhanced PLA2 activity at temperatures below the phase-transition temperature of the membrane, but, in contrast, high concentrations of the tumour promoters suppressed PLA2 activity. Phorbol 12-myristate (PM) also had a similar effect on PLA2 activity. PMA and PM disturbed the membrane structure markedly, which was indicated by the enhanced leakage of carboxyfluorescein (CF) from DPPC vesicles encapsulating CF. On the other hand, phorbol 12,13-didecanoate and 4 alpha-phorbol 12,13-didecanoate, which did not disturb the membrane structure to the same extent, had an insignificant effect on PLA2 activity. It is therefore concluded that PLA2 catalyses the hydrolysis of phospholipids in bilayer vesicles which contain a moderate degree of structural defects. However, the effects of tumour promoters on PLA2 activity was not related to their potencies as inflammatory and tumour-promoting agents.     

NMR study of the interaction of retinoids with phospholipid bilayers

The interaction of three vitamin A derivatives or retinoids: all-trans-retinoic acid, 13-cis-retinoic acid and retinol with multilamellar phospholipid bilayers was studied using a combination of 2H- and 31P-NMR measurements. The following model membrane systems were used: (1) dipalmitoylphosphatidylcholine (DPPC) bilayers; (2) bilayers composed of a mixture of DPPC and bovine heart phosphatidylcholine (PC); (3) mixed PC/phosphatidylethanolamine (PE) bilayers. Only a weak interaction was observed between 13-cis-retinoic acid and DPPC membranes. Addition of all-trans-retinoic acid at a molar ratio of 1:2 to the lipid causes a small decrease (5 C degrees) in the gel to liquid crystalline phase-transition temperature of DPPC, a small increase in the order parameters of the lipid side-chains of single component bilayers and no measurable effect in the other lipid systems studied. Considerably larger perturbation in the lipid bilayer structure is introduced by addition of retinol which, at a molar ratio of 1:2 to the lipid, lowered the gel to liquid crystalline phase-transition temperature of DPPC by 21 C degrees and caused a decrease of order parameters of the lipid side-chains in all three lipid bilayer systems. These effects are consistent with intercalation of retinol molecules into the bilayer interior. The results for the mixed PC/PE bilayers indicate that the presence of retinol caused lateral separation of PE- and retinol-enriched regions.     

Change in membrane fluidity induced by lectin-mediated phase separation of the membrane and agglutination of phospholipid vesicles containing glycopeptides

Changes in membrane fluidity induced by lectin addition to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles containing synthetic glycopeptides were measured by depolarization of the fluorescent probes 8-anilino-1-naphthalenesulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH). In the present synthesized glycopeptides, N-acetylglucosamine (GlcNAc) and a tripeptide were connected by aliphatic chains of different lengths. A pyrenyl group, which is introduced to the peptide moiety, acted as a probe to characterize the distribution of glycopeptides in the membrane on the basis of its excimer formation. The glycopeptide was shown to be distributed to DPPC vesicles with the peptide moiety buried in the hydrophobic core of the lipid bilayer and the glyco moiety exposed to the outside of the membrane. By the addition of wheat germ agglutinin (WGA) to the vesicles containing the glycopeptides, intravesicular cross-linking of glycopeptides in the membrane and aggregation of vesicles were observed. The intravesicular cross-linking was antagonized by GlcNAc above the phase transition temperature. However, the dissociation of aggregation required the addition of a stronger antagonist, N,N'-diacetylchitobiose. The addition of the glycopeptide to DPPC vesicles above the phase transition temperature decreased the membrane fluidity. However, a succeeding addition of WGA caused a large increase of membrane fluidity at either the surface or the hydrophobic core of the lipid bilayer membrane. This increase of membrane fluidity was attributed to two factors by use of two kinds of antagonists having different potencies: one is a WGA-mediated cross-linking of glycopeptides in the membrane, and the other is a close contact of vesicles on aggregation.     

Action of phospholipases A2 on phosphatidylcholine bilayers. Effects of the phase transition, bilayer curvature and structural defects

We examined the action of porcine pancreatic and bee-venom phospholipase A2 towards bilayers of phosphatidylcholine as a function of several physical characteristics of the lipid-water interface. 1. Unsonicated liposomes of dimyristoyl phosphatidylcholine are degraded by both phospholipases in the temperature region of the phase transition only (cf. Op den Kamp et al. (1974) Biochim. Biophys. Acta 345, 253--256 and Op den Kamp et al. (1975) Biochim. Biophys. Acta 406, 169--177). With sonicates the temperature range in which hydrolysis occurs is much wider. This discrepancy between liposomes and sonicates cannot be ascribed entirely to differences in available substrate surface. 2. Below the phase-transition temperature the phospholipases degrade dimyristoyl phosphatidylcholine single-bilayer vesicles with a strongly curved surface much more effectively than larger single-bilayer vesicles with a relatively low degree of curvature. 3. Vesicles composed of egg phosphatidylcholine can be degraded by pancreatic phospholipase A2 at 37 degrees C, provided that the substrate bilayer is strongly curved. The bee-venom enzyme shows a similar, but less pronounced, preference for small substrate vesicles. 4. In a limited temperature region just above the transition temperature of the substrate the action of both phospholipases initially proceeds with a gradually increasing velocity. This stimulation is presumably due to an increase of the transition temperature, effectuated by the products of the phospholipase action. 5. Structural defects in the substrate bilayer, introduced by sonication below the phase-transition temperature (cf. Lawaczeck et al. (1976) Biochim. Biophys. Acta 443, 313--330) facilitate the action of both phospholipases. The results lead to the general conclusion that structural irregularities in the packing of the substrate molecules facilitate the action of phospholipases A2 on phosphatidylcholine bilayers. Within the phase transition and with bilayers containing structural defects these irregularities represent boundaries between separate lipid domains. The stimulatory effect of strong bilayer curvature can be ascribed to an overall perturbation of the lipid packing as well as to a change in the phase-transition temperature.     

Interactions of phosphorus-containing dendrimers with liposomes

The influence of cationic phosphorus-containing dendrimers generation 3 and 4 on model DMPC or DPPC lipid membranes was studied. Measurements of fluorescence anisotropy and differential scanning calorimetry (DSC) were applied to assess changes in lipid bilayer parameters, including fluidity, anisotropy, and phase-transition temperature. Interaction with both hydrophobic and hydrophilic regions of the bilayer was followed by these methods. Dendrimers of both generations influence lipid bilayers by decreasing membrane fluidity. The results suggest that dendrimers can interact both with the hydrophobic part and the polar head-group region of the phospholipid bilayer. Higher generation dendrimers interact more strongly with model membranes, and the concentration, as well as the generation, is of similar importance.     

Thermodynamic elucidation of solute-induced lipid interdigitation phase: lipid interactions with hydrophobic versus amphipathic species.

Comparative thermodynamic studies on the interactions of aqueous dispersions of dipalmitoyl phosphatidylcholine (DPPC) bilayer vesicles with hydrophobic and amphipathic species were conducted to elucidate the nature of the solute-induced interdigitated lipid phase. Cyclohexanol, a strong hydrophobic species, lowers the temperature (tm) of the lipid main phase transition from the gel to the liquid-crystalline phase. Unlike ethanol (an amphipathic species), as reported previously, cyclohexanol does not exert a biphasic effect on tm (lowering tm at lower concentrations and raising tm at higher concentrations). At cyclohexanol greater than or equal to 15.4 mg/ml or 0.154 M, the thermogram of DPPC vesicles exhibits a small transition adjacent to the main phase transition but at a lower temperature. In contrast, ethanol does not promote such a small transition. Furthermore, the enthalpy (delta H) of the transition is increased in the presence of cyclohexanol. The sign of the enthalpy change (delta H-delta Ho) is positive and that of the free energy change (delta G-delta Go) is negative, a characteristic of solute-solute hydrophobic interaction. In contrast, DPPC bilayer vesicles exhibit both (delta H-delta Ho) and (delta G-delta Go) greater than 0 in the presence of ethanol in a concentration range where lipid vesicles exist in an interdigitated phase. To support the above distinct thermodynamic observations, fluorescence steady-state polarization (P) measurements were also performed. At the temperature below tm, the value of P decreases as cyclohexanol concentration increases, while a biphasic effect on P was found in the presence of ethanol. These findings support the postulation that the solute-induced interdigitated lipid phase requires the solute molecule to be amphipathic in nature.     

The interaction of the antimicrobial peptide gramicidin S with lipid bilayer model and biological membranes

Gramicidin S (GS) is a cyclic decapeptide of primary structure [cyclo-(Val-Orn-Leu-D-Phe-Pro)(2)] secreted by Bacillus brevis. It is a powerful antimicrobial agent with potent cidal action on a wide variety of Gram-negative and Gram-positive bacteria as well as on several pathogenic fungi. Unfortunately, however, GS is rather non-specific in its actions and also exhibits a high hemolytic activity, limiting its use as an antibiotic to topical applications. In a wide variety of environments, the GS molecule exists as a very stable amphiphilic antiparallel beta-sheet structure with a polar and a non-polar surface. Moreover, the large number of structure-activity studies of GS analogs which have been carried out indicate that this 'sidedness' structure is required for its antimicrobial action. In this review, we summarize both published and unpublished biophysical studies of the interactions of GS with lipid bilayer model and with biological membranes. In general, these studies show that GS partitions strongly into liquid-crystalline lipid bilayers in both model and biological membranes, and seems to be located primarily in the glycerol backbone region below the polar headgroups and above the hydrocarbon chains. The presence of GS appears to perturb lipid packing in liquid-crystalline bilayers and GS can induce the formation of inverted cubic phases at lower temperatures in lipids capable of forming such phases at higher temperature in the absence of peptide. The presence of GS at lower concentrations also increases the permeability of model and biological membranes and at higher concentrations causes membrane destabilization. There is good evidence from studies of the interaction of GS with bacterial cells that the destruction of the integrity of the lipid bilayer of the inner membrane is the primary mode of the antimicrobial action of this peptide. The considerable lipid specificity of GS for binding to and destabilization of lipid bilayer model membranes indicates that the design of GS analogs with an improved antimicrobial potency and a markedly decreased toxicity for eukaryotic cell plasma membranes should be possible.     

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.     

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 A height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.     

The effects of ring-size analogs of the antimicrobial peptide gramicidin S on phospholipid bilayer model membranes and on the growth of Acholeplasma laidlawii B.

We have examined the effects of three ring-size analogs of the cyclic beta-sheet antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior and permeability of phospholipid model membranes and on the growth of the cell wall-less Gram-positive bacteria Acholeplasma laidlawii B. These three analogs have ring sizes of 10 (GS10), 12 (GS12) or 14 (GS14) amino acids, respectively. Our high-sensitivity differential scanning calorimetric studies indicate that all three of these GS analogs perturb the gel/liquid-crystalline phase transition of zwitterionic phosphatidylcholine (PtdCho) vesicles to a greater extent than of zwitterionic phosphatidylethanolamine (PtdEtn) or of anionic phosphatidylglycerol (PtdGro) vesicles, in contrast to GS itself, which interacts more strongly with PtdGro than with PtdCho and PtdEtn bilayers. However, the relative potency of the perturbation of phospholipid phase behavior varies markedly between the three peptides, generally decreasing in the order GS14 > GS10 > GS12. Similarly, these three GS ring-size analogs also differ considerably in their ability to cause fluorescence dye leakage from phospholipid vesicles, with the potency of permeabilization also generally decreasing in the order GS14 > GS10 > GS12. Finally, these GS ring-size analogs also differentially inhibit the growth of A. laidlawii with growth inhibition also decreasing in the order GS14 > GS10 > GS12. These results indicate that the relative potencies of GS and its ring-size analogs in perturbing the organization and increasing the permeability of phospholipid bilayer model membranes, and of inhibiting the growth of A. laidlawii B cells, are at least qualitatively correlated, and provide further support for the hypothesis that the primary target of these antimicrobial peptides is the lipid bilayer of the bacterial membrane. The very high antimicrobial activity of GS14 against the cell wall-less bacteria A. laidlawii as compared to various conventional bacteria confirms our earlier suggestion that the avid binding of this peptide to the bacterial cell wall is primarily responsible for its reduced antimicrobial activity against such organisms. The relative magnitude of the effects of GS itself, and of the three ring-size GS analogs, on phospholipid bilayer organization and cell growth correlate relatively well with the effective hydrophobicities and amphiphilicities of these peptides but less well with their relative charge density, intrinsic hydrophobicities or conformational flexibilities. Nevertheless, all of these parameters, as well as others, may influence the antimicrobial potency and hemolytic activity of GS analogs.     

alpha-Tocopherol--a modifier of the phase state of a lipid bilayer

Using 1H-NMR high resolution spectroscopy it was demonstrated that alpha-tocopherol modifies the character of phase transition in the membrane lipid bilayer. Injection of 5 mol% tocopherol into the lipid bilayer from dipalmitoylphosphatidylcholine (DPPC) decreased the temperature and increased the width of the phase transition. Similar action was produced by injection into the bilayer from DPPC of 15-20 mol% linoleic acid. Injection of an equimolar amount of alpha-tocopherol into the bilayer from DPPC predestabilized by linoleic acid exerted a stabilizing action, the mode of phase transition being similar to that observed for pure DPPC. It is assumed that the stabilizing effect of alpha-tocopherol in question is a mechanism via which alpha-tocopherol protects the membrane from the damage-inducing action of free fatty acids.     

Amino acids change solute affinity for lipid bilayers

Time-resolved fluorescence and differential scanning calorimetry (DSC) were used to examine how two amino acids, L-phenylalanine (L-PA) and N-acetyl-DL-tryptophan (NAT), affect the temperature-dependent membrane affinity of two structurally similar coumarin solutes for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles. The 7-aminocoumarin solutes, coumarin 151 (C151) and coumarin 152 (C152), differ in their substitution at amine position—C151 is a primary amine, and C152 is a tertiary amine—and both solutes show different tendencies to associate with lipid bilayers consistent with differences in their respective log-P-values. Adding L-PA to the DPPC vesicle solution did not change C151’s propensity to remain freely solvated in aqueous solution, but C152 showed a greater tendency to partition into the hydrophobic bilayer interior at temperatures below DPPC’s gel-liquid crystalline transition temperature (T_gel-lc). This finding is consistent with L-PA’s ability to enhance membrane permeability by disrupting chain-chain interactions. Adding NAT to DPPC-vesicle-containing solutions changed C151 and C152 affinity for the DPPC membranes in unexpected ways. DSC data show that NAT interacts strongly with the lipid bilayer, lowering T_gel-lc by up to 2°C at concentrations of 10 mM. These effects disappear when either C151 or C152 is added to solution at concentrations below 10 μM, and T_gel-lc returns to a value consistent with unperturbed DPPC bilayers. Together with DSC results, fluorescence data imply that NAT promotes coumarin adsorption to the vesicle bilayer surface. NAT’s effects diminish above T_gel-lc and imply that unlike L-PA, NAT does not penetrate into the bilayer but instead remains adsorbed to the bilayer’s exterior. Taken in their entirety, these discoveries suggest that amino acids—and by inference, polypeptides and proteins—change solute affinity for lipid bilayers with specific effects that depend on individualized amino-acid-lipid-bilayer interactions.     

Effect of acyl chain mismatch on the contact mechanics of two-component phospholipid vesicle during main phase transition

It has been recently demonstrated that acyl chain mismatch of phospholipid bilayer composed of a binary lipid mixture induces component formation on the lateral plane of the bilayer [Biophys. J. 83 (2002) 1820-1883]. In this report, the contact mechanics of unilamellar vesicles composed of binary dimyristoyl-phosphatidylcholine (DMPC)/dipalmitoyl-phosphocholine (DPPC) mixtures on fused silica and amino-modified substrates is simultaneously probed by confocal-reflectance interference contrast microscopy (C-RICM) and cross-polarized light microscopy during gel to liquid crystalline transition of the lipid bilayer. C-RICM results indicate that the average degree of vesicle deformation for DMPC-rich and DPPC-rich vesicles adhering on fused silica substrate is increased by 30% and 14%, respectively, in comparison with that in pure DMPC and DPPC vesicles. Also, lateral heterogeneity induced by acyl chain mismatch increases the average magnitude of adhesion energy in DMPC-rich and DPPC-rich vesicles of all sizes by 6.4 times and 2.3 times, respectively. Similar modulation of adhesion mechanics induced by carbon chain difference is obtained on amino-modified substrate. Most importantly, the thermotropic transition of the mixed bilayer from gel (below T(m)) to fluid phase (above T(m)) further exemplifies the effect of acyl chain mismatch on the increases of degree of vesicle deformation and adhesion energy.     

Effects of lung surfactant proteolipid SP-C on the organization of model membrane lipids: a fluorescence study.

Lipid-protein interactions of pulmonary surfactant-associated protein SP-C in model DPPC/DPPG and DPPC/DPPG/eggPC vesicles were studied using steady-state and time-resolved fluorescence measurements of two fluorescent phospholipid probes, NBD-PC and NBD-PG. These fluorescent probes were utilized to determine SP-C-induced lipid perturbations near the bilayer surface, and to investigate possible lipid headgroup-specific interactions of SP-C. The presence of SP-C in DPPC/DPPG membrane vesicles resulted in (1) a dramatic increase in steady-state anisotropy of NBD-PC and NBD-PG at gel phase temperatures, (2) a broadening of the gel-fluid phase transition, (3) a decrease in self-quenching of NBD-PC and NBD-PG probes, and (4) a slight increase in steady-state anisotropy of NBD-PG at fluid phase temperatures. Time-resolved measurements, as well as steady-state intensity measurements indicate that incorporation of SP-C into DPPC/DPPG or DPPC/DPPG/eggPC vesicles results in a increase in the fraction of the long-lifetime species of NBD-PC. The results presented here indicate that SP-C orders the membrane bilayer surface, disrupts acyl chain packing, and may increase the lateral pressure within the bilayer.     

Molecular details of melittin-induced lysis of phospholipid membranes as revealed by deuterium and phosphorus NMR

Solid-state deuterium and phosphorus-31 nuclear magnetic resonance (2H and 31P NMR) studies of deuterium-enriched phosphatidylcholine [( 3',3'-2H2]DPPC, [sn-2-2H31]DPPC) and ditetradecylphosphatidylglycerol (DMPG-diether), as water dispersions, were undertaken to investigate the action of melittin on zwitterionic and negatively charged membrane phospholipids. When the lipid-to-protein ratio (Ri) is greater than or equal to 20, the 2H and 31P NMR spectral features indicate that the system is constituted by large bilayer structures of several thousand angstrom curvature radius, at T greater than Tc (Tc, temperature of "gel-to-liquid crystal" phase transition of pure lipid dispersions). At T approximately Tc, a detailed analysis of the lipid chain ordering shows that melittin induces a slight disordering of the "plateau" positions concomitantly with a substantial ordering of positions near the bilayer center. At T much greater than Tc, an apparent general chain disordering is observed. These findings suggest that melittin is in contact with the acyl chain segments and that its position within the bilayer may depend on the temperature. On a cooling down below Tc, for Ri greater than 20, two-phase spectra are observed, i.e., narrow single resonances superimposed on gel-type phosphorus and deuterium powder patterns. These narrow resonances are characteristic of small structures (vesicles, micelles, ... of a few hundred angstrom curvature radius) undergoing fast isotropic reorientation, which averages to zero both the quadrupolar and chemical shift anisotropy interactions. On an increase of the temperature above Tc, the NMR spectra indicate that the system returns reversibly to large bilayer structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partition of chlorpromazine into lipid bilayer membranes: the effect of membrane structure and composition

Partition coefficients, kp, of chlorpromazine between the aqueous phase and lipid bilayer vesicles were determined as function of drug concentration, lipid chain length, cholesterol content and temperature encompassing the range of the lipid phase transition. Radioactivity and absorption measurements were performed to determine the kp values. Up to a concentration of 3 . 10(-5) M, the partition coefficient is independent of chlorpromazine concentration, whereas it decreases drastically at higher chlorpromazine concentrations, at which membrane lysis is observed. Membrane structure is not disturbed at less than 3 . 10(-5) M chlorpromazine, as was concluded from electron paramagnetic resonance studies measuring TEMPO partitioning and order degree. However, the lipid phase-transition temperature decreases and is broadened at higher chlorpromazine concentrations. From fluorescence measurements, we conclude the formation of chlorpromazine micelles at concentrations higher than 5 . 10(-5) M in chlorpromazine in the absence of lipids and the formation of mixed micelles in the presence of lipids. The effect of lipid chain length on kp values was investigated. The partition coefficient decreases from 8100 in dilauroyl- to 3400 in dipalmitoylphosphatidylcholine vesicles, both at 50 degrees C, that is, above their corresponding phase-transition temperature tt. At t less than tt the kp values are strongly reduced, by at least a factor of 10, depending on lipid chain length and membrane composition. It is possible to establish a lipid phase-transition curve from the temperature-dependent measurements of the kp values. Cholesterol within the lipid membrane strongly decreases kp. At 20 mol% cholesterol in dipalmitoylphosphatidylcholine membranes, the partition coefficient is reduced from 3400 to 2300. This value is well comparable to the kp value obtained in erythrocyte ghosts. In contradiction to earlier experiments by Conrad and Singer (Biochemistry 20 (1981) 808-818), this value in a biological membrane could be obtained by the hygroscopic desorption as well as the centrifugation method. From our experiments we are justified in further considering artificial bilayer membranes as models for biological membranes.     

Thermotropic and dynamic characterization of interactions of acylated alpha-bungarotoxin with phospholipid bilayer membranes

The interactions of palmitoyl-alpha-bungarotoxin (PBGT) with dipalmitoylphosphatidylcholine (DPPC) bilayers have been studied by using high-sensitivity differential scanning calorimetry together with steady-state and time-resolved phosphorescence and fluorescence spectroscopy. The incorporation of PBGT into large single lamellar vesicles causes a decrease in the phospholipid phase transition temperature (Tm), a broadening of the heat capacity function, and a decrease in the enthalpy change associated with the phospholipid gel to liquid-crystalline transition. Analysis of the dependence of this decreased enthalpy change on the protein/lipid molar ratio indicates that each PBGT molecule exhibits a localized effect upon the bilayer, preventing approximately six lipid molecules from participating in the lipid phase transition. Additional calorimetric experiments indicate that binding to acetylcholine receptor enriched membranes causes a small increase in the Tm of the PBGT/DPPC vesicles. Steady-state fluorescence depolarization measurements employing 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the association of PBGT with the phospholipid bilayer decreases the apparent order of the bulk lipid below Tm while increasing the order above Tm. These results have been further supported by rotational mobility measurements of erythrosin-labeled PBGT associated with giant (about 2-micron) unilamellar vesicles composed of dielaidoylphosphatidylcholine or dioleoylphosphatidylcholine using the time-dependent decay of delayed fluorescence/phosphorescence emission anisotropy. Rotational correlation times in the submillisecond time scale (about 30 microseconds) indicate that the protein is highly mobile in the fluid phase and that below Tm the rotational mobility is only slightly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)     

The polar nature of 7-ketocholesterol determines its location within membrane domains and the kinetics of membrane microsolubilization by apolipoprotein A-I

7-Ketocholesterol is an oxidized derivative of cholesterol with numerous physiological effects. In model membranes, 7-ketocholesterol and cholesterol were compared by physical measures of bilayer order and polarity, formation of detergent resistant domains (DRM), phase separation, and membrane microsolubilization by apolipoprotein A-I. In binary mixtures of a saturated phosphatidylcholine (PC), dipalmitoyl-PC (DPPC), and cholesterol or 7-ketocholesterol, the sterols modulate bilayer order and polarity and induce DRM formation to a similar extent. Cholesterol induces formation of ordered lipid domains (rafts) in tertiary mixtures with dioleoyl-PC (DOPC) and DPPC, or DOPC and sphingomyelin (SM). In tertiary mixtures, cholesterol increased lipid order and reduces bilayer polarity more than 7-ketocholesterol. This effect was more pronounced when the mixtures were in a miscible liquid-disordered (L(d)) phase. Substitution of 7-ketocholesterol for cholesterol dramatically reduced the extent of DRM formation in DOPC/DPPC and DOPC/SM bilayers and ordered lipid phase separation in mixtures of a spin-labeled PC with DPPC and with SM. Compared to cholesterol, 7-ketocholesterol decreased the rate for the microsolubilization of dimyristoyl-PC multilamellar vesicles by apolipoprotein A-I. The membrane effects of 7-ketocholesterol were dependent on the phospholipid matrix. In L(d) phase phospholipids, a model for 7-ketocholesterol indicates that the proximity of the 7-keto and 3beta-OH groups puts both polar moieties at the lipid-water interface to tilt the sterol nucleus to the plane of the bilayer. 7-Ketocholesterol was less effective in forming ordered lipid domains, in decreasing the level of bilayer hydration, and in forming phase boundary bilayer defects. Compared to cholesterol, 7-ketocholesterol can differentially modulate membrane properties involved in protein-membrane association and function.