A fluorescence study of the interaction and location of (+)-totarol, a diterpenoid bioactive molecule, in model membranes

(+)-Totarol, a diterpene extracted from Podocarpus totara, has been reported as a potent antioxidant and antibacterial agent. Although the molecular mechanism of action of this hydrophobic molecule remains unknown, recent work made in our laboratory strongly suggests that it could be lipid-mediated. Since (+)-totarol contains a phenolic ring, we have studied the intrinsic fluorescent properties of this molecule, i.e., quantum yield, lifetime, steady-state anisotropy and emission spectra, both in aqueous and in phospholipid phases, in order to obtain information on the interaction and location of (+)-totarol in biomembrane model systems. The phospholipid/water partition coefficient of (+)-totarol was found to be very high (K(p)=1.8x10(4)), suggesting that it incorporates very efficiently into membranes. In order to estimate the transverse location (degree of penetration) of the molecule in the fluid phase of DMPC model membranes, the spin labelled fatty acids 5-NS and 16-NS were used in differential quenching experiments. The results obtained show that (+)-totarol is located in the inner region of the membrane, far away from the phospholipid/water interface. Since (+)-totarol protects against oxidative stress, its interaction with an unsaturated fatty acid, trans-parinaric acid, was studied using fluorescence resonance energy transfer. No significant interactions were observed, molecules of trans-parinaric acid distributing themselves randomly amongst those of (+)-totarol in the phospholipid membrane.     

Effects of (+)-totarol, a diterpenoid antibacterial agent, on phospholipid model membranes

(+)-Totarol, a highly hydrophobic diterpenoid isolated from Podocarpus spp., is inhibitory towards the growth of diverse bacterial species. (+)-Totarol decreased the onset temperature of the gel to liquid-crystalline phase transition of DMPC and DMPG membranes and was immiscible with these lipids in the fluid phase at concentrations greater than 5 mol%. Different (+)-totarol/phospholipid mixtures having different stoichiometries appear to coexist with the pure phospholipid in the fluid phase. At concentrations greater than 15 mol% (+)-totarol completely suppressed the gel to liquid-crystalline phase transition in both DMPC and DMPG vesicles. Incorporation of increasing amounts of (+)-totarol into DEPE vesicles induced the appearance of the H(II) hexagonal phase at low temperatures in accordance with NMR data. At (+)-totarol concentrations between 5 and 35 mol% complex thermograms were observed, with new immiscible phases appearing at temperatures below the main transition of DEPE. Steady-state fluorescence anisotropy measurements showed that (+)-totarol decreased and increased the structural order of the phospholipid bilayer below and above the main gel to liquid-crystalline phase transition of DMPC respectively. The changes that (+)-totarol promotes in the physical properties of model membranes, compromising the functional integrity of the cell membrane, could explain its antibacterial effects.     

HPLC study on the 'history' dependence of gramicidin A conformation in phospholipid model membranes

A novel HPLC methodology for the study of gramicidin A reconstituted in model membranes has been tested in comparison with circular dichroism data. It is shown that this chromatographic technique not only corroborates most of the recent spectroscopic results but allows one to explain them in terms of mass fractions of different actual conformational species of GA in the phospholipid assemblies. In particular, the dependence of the inserted peptide configuration on the organic solvent and other parameters involved in the 'history' of the sample preparation and handling has been analyzed by HPLC in two phospholipid model systems: small unilamellar vesicles and micelles. Moreover, a slow conformational transition of GA towards a beta 6.3-helical configuration, accelerated by heat incubation, has been also chromatographically visualized and quantitatively interpreted.     

Electron spin resonance study of phospholipid membranes employing a comprehensive line-shape model

The electron spin resonance spectra of the 1-myristoyl-2-[6-(4,4-dimethyloxazolidine-N-oxyl)myristoyl]-sn-glycero- 3-phosphocholine spin-label in highly oriented, fully hydrated bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine have been studied as a function of temperature and magnetic field orientation. The oriented spectra show clear indications of slow motional components (rotational correlation times greater than 3 ns) even in the fluid phase (T greater than 23 degrees C), indicating that motional narrowing theory is not applicable to the spectral analysis. The spectra have been simulated by a comprehensive line-shape model that incorporates trans-gauche isomerization in addition to restricted anisotropic motion of the lipid long molecular axis and that is valid in all motional regimes. In the gel (L beta') phase the spin-label chains are found to be tilted at 28 degrees with respect to the normal of the orienting plane. In the intermediate (P beta') phase there is a continuous distribution of tilt angles between 0 degrees and 25 degrees. In fluid (L alpha) phase there is no net tilt of the lipid chains. The chains rotate at an intermediate rate about their long axis in the fluid phase (tau R,parallel = 1.4-6.6 ns for T = 50-25 degrees C), but the reorientation of the chain axis is much slower (tau R, perpendicular= 13-61 ns for T = 50-25 degrees C), whereas trans-gauche isomerization (at the C-6 position) is rapid (tau J less than or equal to 0.2 ns). Below the chain melting transition both chain reorientation and chain rotation are at the ESR rigid limit (tau R greater than or equal to 100 ns), and trans-gauche isomerization is in the slow-motion regime (tau J = 3.7-9.5 ns for T = 22-2 degrees C). The chain order parameter increases continuously with decreasing temperature in the fluid phase (SZZ = 0.47-0.61 for T = 50-25 degrees C), increases abruptly on going below the chain melting transition, and then increases continuously in the intermediate phase (SZZ = 0.79-0.85 for T = 22-14 degrees C) to an approximately constant value in the gel phase (SZZ congruent to 0.86 for T = 10-2 degrees C).(ABSTRACT TRUNCATED AT 400 WORDS)     

A theoretical model for surface polarizability in phospholipid membranes

A simple theoretical model based on statistical thermodynamics is proposed in order to correlate surface polarizability in membranes with frequency shifts in the electronic transitions of fluorescence probes. This model predicts a different behaviour for the absorption and emission processes of probes in lipid environments and suggests that as a first approximation these spectroscopic variations can be linearly related to the deformability (polarizability) of the ionic heads of the phospholipids. Some preliminary spectroscopic measurements suggest that this hypothesis might be a correct interpretation of the electronic shift observed in the spectral analysis of phospholipid membranes.     

Ultrasonic study of melittin effects on phospholipid model membranes.

Low dose effects of melittin on dilute suspensions of dipalmitoylphosphatidylcholine multilamellar vesicles are investigated by studying the acoustic properties of the system. The temperature dependencies of sound velocity and absorption have been measured at 7.2 MHz in the temperature range of 20-55 degrees C, for different peptide/lipid molar ratios, R. The most pronounced effects were observed at R = 5 x 10(-3), in the vicinity of the pretransition, with a simultaneous increase in sound absorption and velocity. This indicates that melittin affects the polar head group region of the bilayer resulting in a decrease in mobility of the polar head groups. A nonmonotonic dependence of the main transition temperature, with an initial decrease followed by an increase as melittin is added, is interpreted as a consequence of a destabilizing action of the interfaces between mellitin-affected clusters and the unaffected phase.     

A comparative study of the effects of cholesterol and sclareol, a bioactive labdane type diterpene, on phospholipid bilayers

Sclareol (labd-14-ene-8,13-diol) is a highly water-insoluble molecule that belongs to the labdane type diterpenes and is characterized as a biologically active molecule, due to its cytotoxic and cytostatic effects against human leukemic cell lines. A superimposition study between sclareol and cholesterol, based on their corresponding hydrophobic and polar molecular segments calculated from their lipophilic profiles, revealed their spatial similarities. This structural similarity between the two molecules prompted us to compare their effects on the structure and stability of phospholipid dipalmitoylphosphatidylcholine (DPPC) membranes. Differential scanning calorimetry (DSC) was applied to compare the thermal changes caused by either cholesterol or sclareol when are incorporated in DPPC bilayers. The results showed that sclareol is incorporated into phospholipid model membranes and mimics the thermal effects of cholesterol especially at concentrations up to X(sclareol)=9.1 mol%. These effects can be summarized as the abolition of pre-transition, lowering of the main phase transition and reduction of the enthalpy change (DeltaH) of the gel to liquid-crystalline phase transition of DPPC bilayers. At concentrations X> or =16.7 mol%, sclareol and cholesterol caused different heterogeneity in lipid bilayers or a reversible transition from a vesicular suspension to an extended peak bilayer network. This different fluidization, exerted by the two molecules at high concentration, may be related to their different stability and the z-average mean diameter of the liposomes they form. Small unilamellar vesicles, prepared by the thin film hydration method showed that DPPC bilayers containing a high concentration of sclareol in equimolar ratio sclareol:cholesterol were unstable, in contrast to the ones containing only cholesterol.     

Destabilization of phospholipid model membranes by YplA, a phospholipase A2 secreted by Yersinia enterocolitica

Yersinia enterocolitica produces a virulence-associated phospholipase A(2) (YplA) that is secreted via its flagellar type-III secretion apparatus. When the N-terminal 59 amino acids of YplA are removed (giving YplA(S)), it retains phospholipase activity; however, it is altered with respect to the apparent kinetics of hydrolysis using fluorescent phospholipid substrates in micellar form. To explore the physical properties of YplA more carefully, Langmuir phospholipid monolayers were used to study the association of YplA with biological membranes. YPlA and YplA(S) both associate with Langmuir monolayers, but YplA(S) appears to interact better at low initial lipid densities while YplA interacts better at higher densities. This may indicate that the N-terminus of YplA has a role in mediating its initial interaction with compact cellular membranes, which is consistent with spectroscopic observations that fluorescein-labeled YplA may interact more readily with the nonpolar region of liposomes than does YplA(S).     

Possible role of phospholipid molecule reorientation in ion transport through membranes]

A theory of ionic transport through membranes in which the phospholipid molecules play the role of ion-carriers is developed. The transport of ions is accompanied by a membrane asymmetry. Expressions for stationary-state ion-fluxes in the case of one sort of ions transport, and in the case of coupled transport of ions of two sorts, are obtained. In the latter case near equilibrium the cross-coefficients are equal in accordance with Onsager's law. The possible role of phospholipids as ioncarriers in native membranes is discussed.     

Interactions of inorganic mercury with phospholipid micelles and model membranes. A31P-NMR study

The binding of inorganic mercury Hg(II) to phospholipid headgroups has been investigated by phosphorus-31 nuclear magnetic resonance of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC) in water micellar and multilamellar phases. HgCl₂ triggers the aggregation of phospholipid micelles, leading to a lipid-mercury precipitate that is no longer detectable by high-resolution³¹P-NMR. The remaining signal area corresponds to micelles in the soluble fraction and is a non-linear function of the initial mercury-to-lipid molar ratio. Kinetics of micelle aggregation are exponential for the first 15 min and show a plateau tendency after 120 min. Apparent Hg(H) affinities for phospholipid headgroups are in the order: PE > PS > PC. The same binding specificity is observed when HgCl₂ is added to (1:1) mixtures of different micelles (PE + PC; PS + PC). However, mercury binding to mixed micelles prepared with two lipids (PE/PC or PS/PC) induces the aggregation of both lipids. Hg(II) also leads to a³¹P-NMR chemical shift anisotropy decrease of PC, PS and mixed (1:1) PE/PC multilamellar vesicles and markedly broadens PS spectra. This indicates that HgCl₂ binding forces phospholipid headgroups to reorient and that the concomitant network formation leads to a slowing down of PS membrane collective motions. Formation of a gel-like lamellar phase characterized by a broad NMR linewidth is also observed upon HgCl₂ binding to PE samples both in fluid (L) or hexagonal (H_II) phases. The PE hexagonal phase is no longer detected in the presence of HgCl₂. Mixed PE/PC dispersions remain in the fluid phase upon mercury addition, indicating that no phase separation occurs. Addition of excess NaCl leads to the appearance of the non-reactive species HgCl _inf4 ^sup2– and induces the reversal of all the above effects.Abbreviations A(t) time-dependence of peak area - A₄₀ peak area at t=40 min - 1/ rate of peak area decrease - isotropic chemical shift - isotropic chemical shift change - chemical shift anisotropy - DPPC dipalmitoylphosphatidylcholine - Hg(II) inorganic mercury - NMR nuclear magnetic resonance - pCl –log [Cl^–] - PC phosphatidylcholine - PE phosphatidylethanolamine - PL phospholipid - PS phosphatidylserine - R_i mercury-to-lipid molar ratio - MLV multilamellar vesicles - SUV small unilamellar vesicles     

Electron spin resonance study of the interaction of alpha-tocopherol with phospholipid model membranes.

The effect of up to 20 mol% incorporation of alpha-tocopherol on acyl chain order and dynamics in liquid crystalline phosphatidylcholine (PC) membranes was studied as a function of acyl chain unsaturation by electron spin resonance (ESR) of 5-, 7-, 12- and 16-doxyl spin labelled stearic acids intercalated into the membrane. Order parameters S in the upper portion of the chain (positions 5 and 7) and correlation times tau C in the lower portion (positions 12 and 16) determined from the ESR spectra indicate that in general alpha-tocopherol restricts acyl chain motion within the membrane. The magnitude of the increases in order appears to be dependent upon phospholipid molecular area, being the greatest (up to 15%) in saturated dimyristoylphosphatidylcholine (14:0-14:0 PC) which possesses a relatively small area per molecule as opposed to much smaller increases (less than 3%) in unsaturated PC membranes of larger molecular area. This behavior is interpreted as incompatible with the hypothesis of Lucy and coworkers (A.T. Diplock and J.A. Lucy (1973) FEBS Lett. 29, 205-210), who proposed that membranes are structurally stabilized by interactions between the phytyl side chain of alpha-tocopherol and the polyunsaturated chains of phospholipids.     

Effects of verbascoside,a phenylpropanoid glycoside from lemon verbena,on phospholipid model membranes

Phenylpropanoid glycosides are water-soluble compounds widely distributed, most of them deriving from medicinal herbs. Among them, verbascoside or acteoside has exhibited a wide biological activity, being free radical scavenging the most representative one. Moreover, antitumor, antimicrobial, anti-inflammatory, anti-thrombotic and wound healing properties have been previously described. Herein, the interaction of verbascoside with phospholipid membranes has been studied by means of differential scanning calorimetry, fluorescence anisotropy and dynamic light scattering. Verbascoside showed stronger affinity for negatively charged membranes composed of phosphatidylglycerol (PG) than for phosphatidylcholine (PC) membranes. This compound promoted phase separation of lipid domains in PC membranes and formed a stable lipid complex with and approximate phospholipid/verbascoside ratio of 4:1. Despite its hydrophilic character, verbascoside's caffeoyl moiety was located deep into the hydrophobic core of PC membranes and was almost inaccessible to spin probes located at different depths in PG membranes. This compound affected the ionization behavior of the PG phosphate group and most likely interacted with the vesicles surface. The presence of verbascoside decreased the particle size in PG unilamellar vesicles through the increase of the phospholipid head group area. A localization of verbascoside filling the upper region of PG bilayers close to the phospholipid/water interface is proposed. These effects on membranes may help to understand the mechanism of the biological activity of verbascoside and other similar phenylpropanoid glycosides.     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Location and orientation of Triclosan in phospholipid model membranes

Triclosan is a hydrophobic antibacterial agent used in dermatological preparations and oral hygiene products. Although the molecular mechanism of action of this molecule has been attributed to inhibition of fatty acid biosynthesis, earlier work in our laboratories strongly suggested that the antibacterial action of Triclosan is mediated at least partly through its membranotropic effects. In order to assess its location in phospholipid membranes, high-resolution magic-angle spinning natural abundance ¹³C NMR of Triclosan embedded within egg yolk lecithin model membranes has been used to obtain ¹³C spin–lattice relaxation times for both Triclosan and lecithin carbon atoms in the presence of Gd³⁺ ions. The results indicate that Triclosan is localized in the upper region of the phospholipid membrane, its hydroxyl group residing in the vicinity of the C=O/C2 carbon atoms of the acyl chain of the phospholipid, and the rest of the Triclosan molecule is probably aligned in a nearly perpendicular orientation with respect to the phospholipid molecule. Intercalation of Triclosan into bacterial cell membranes likely compromises the functional integrity of those membranes, thereby accounting for at least some of this compounds antibacterial effects.Abbreviations COLOC correlation by long-range coupling - EYL egg yolk lecithin - HETCOR heteronuclear chemical-shift correlation - MAS magic-angle spinning - MLV multilamellar vesicles     

Fluorescence study of the divalent cation-transport mechanism of ionophore A23187 in phospholipid membranes

The mechanism for transport of divalent cations across phospholipid bilayers by the ionophore A23187 was investigated. The intrinsic fluorescence of the ionophore was used in equilibrium and rapid-mixing experiments as an indicator of ionophore environment and complexation with divalent cations. The neutral (protonated) form of the ionophore binds strongly to the membrane, with a high quantum yield relative to that in the aqueous phase. The negatively charged form of the ionophore binds somewhat less strongly, with a lower quantum yield, and does not move across the membrane. Complexation of the negatively charged form with divalent cations was measured by the decrease in fluorescence. An apparent rate constant (kapp) for transport of the ionophore across the membrane was determined from the rate of fluorescence changes observed in stopped-flow rapid kinetic experiments. The variation of kapp was studied as a function of pH, temperature, ionophore concentration, membrane lipid composition, and divalent cation concentration and type. Analysis and comparison with equilibrium constants for protonation and complexation show that A23187 and its metal:ionophore complexes bind near the membrane-water interface in the lipid polar-head region. The interfacial reactions occur rapidly, compared with the transmembrane reactions, and are thus in equilibrium during transport. The transport cycle can be described as follows: a 1:1 complex is formed between the membrane bound A23187-(Am-) and the aqueous divalent cation with dissociation constant K1 approximately 4.6 x 10(-4) M. This is in equilibrium with a 1:2 (metal:ionophore) complex (K2 approximately 3.0 x 10(-4) [ionophore/lipid]) that is responsible for transporting the divalent cations across the membrane. The rate constant for translocation of the 1:2 complex is 0.1-0.3 s-1. Dissociation of the complex of the trans side and protonation occur rapidly. The rate constant for translocation of H+ . A23187- is 28 s-1. A theory is presented that is capable of reproducing the kinetic data at any calcium concentration. The cation specificity for ionophore complex transport (kapp), determined at low ionophore concentration for a series of divalent cations, was found to be proportional to the equilibrium constant for 1:1 complexation. The order of ion specificity for these processes was found to be Ca2+ greater than Mg2+ greater Sr2+ greater than Ba2+. Interactions with Na+ were not observed. Maximal values of kapp were observed for vesicles prepared from pure dimyristoyl phosphatidylcholine. Inclusion of phosphatidyl ethanolamine, phosphatidic acid, or dipalmatoyl phosphatidylcholine resulted in lower values of kapp. Calcium transport by A23187 is compared with that of X537A, and it is shown that the former is 67-fold faster. The difference in rates is due to differences in the ability of each ionophore to form a 1:2 complex from a 1:1 complex.