Membrane fluidity as affected by the organochlorine insecticide DDT

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to study the interaction of DDT with model and native membranes. DDT decreases the phase transition midpoint temperature (Tm) of liposomes reconstituted with dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC), and broadens the thermotropic profile of the transition. The effects of DDT are concentration dependent and are more pronounced in bilayers of short-chain lipids, e.g., DMPC. The insecticide fails to alter DPH polarization in the fluid phase of the above lipids. Similar effects were observed in binary mixtures of DMPC plus DPPC. Furthermore, DDT alters the single broad transition of the equimolar mixture of DMPC plus DSPC into a biphasic transition. The lower temperature component has a midpoint at 25 degrees C, i.e., a value close to the Tm of DMPC. DDT inhibits to some extent the cholesterol-induced ordering in DMPC bilayers and high cholesterol concentrations (greater than or equal to 30 mol%) do not prevent insecticide interaction, conversely to the effect observed for lindane (Antunes-Madeira, M.C. and Madeira, V.M.C. (1989) Biochim. Biophys. Acta 982, 161-166). Apparently, the bilayer order is not disturbed by DDT in fluid native membranes of mitochondria and sarcoplasmic reticulum, but moderate disordering effects are noticed in membranes enriched in cholesterol, namely, brain microsomes and erythrocytes.     

Membrane fluidity as affected by the insecticide lindane

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to study the interaction of lindane with model and native membranes. Lindane disorders the gel phase of liposomes reconstituted with dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC), since it broadens and shifts the main phase transition, but no apparent effect is detected in the fluid phase. These effects of lindane are more pronounced in bilayers of short-chain lipids, e.g., DMPC. In equimolar mixtures containing DMPC and DSPC, lindane preferentially interacts with the more fluid lipid species inducing lateral phase separations. However, in mixtures of DMPC and DPPC, the insecticide only broadens and shifts the main phase transition, i.e., an effect similar to that observed in bilayers of pure lipids. Lindane has no apparent effect in DMPC bilayers enriched with high cholesterol content (greater than or equal to 30 mol%), whereas disordering effects can still be detected in bilayers with low cholesterol (less than 30 mol%). Apparently, lindane does not perturb the fluid phase of representative native membranes, namely, mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes in agreement with the results obtained in fluid phospholipid bilayers, despite the reasonable incorporation of the insecticide in these membranes, as previously reported (Antunes-Madeira, M.C. and Madeira, V.M.C. (1985) Biochim. Biophys. Acta 820, 165-172).     

Changes in membrane fluidity evoked by organophosphorus insecticide bromfenvinfos and its methylated analogue

The interaction of organophosphorus insecticides bromfenvinfos and methyl bromfenvinfos with model and native membranes was investigated by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe located in the hydrophobic core of the bilayer and 1,3-bis-(1-pyrene)propane, a probe distributed in the outer region of the bilayer. DPH reported a broadening of the transition profile and solidifying effects in the fluid phase of liposomes formed from dimyristoyl (DMPC), dipalmitoyl (DPPC), and distearoyl (DSPC) phosphatidylcholine in the presence of the insecticides. A shift of the transition temperature towards a lower temperature was observed in DPPC- and DSPC-bromfenvinfos-treated vesicles. Py(3)Py detected an ordering effect of the insecticides in the fluid state of the lipids and abolished pre-transition in DPPC and DSPC vesicles. These results suggest that the insecticides localize in the co-operative region of the bilayer. Cholesterol added to DMPC decreased the influence of the insecticides as reported by both DPH and Py(3)Py. The effect of the insecticides on the fluidity of some native membranes, namely erythrocytes, lymphocytes, brain microsomes, and sarcoplasmic reticulum, depended on the cholesterol content in these membranes, the higher the cholesterol content, the smaller the solidifying effect. The physical mechanism of action of the insecticides on membrane lipids can be similar to that of cholesterol. All observed effects were more pronounced for bromfenvinfos than for its methylated analogue which correlates with the toxicity of these compounds for mammals.     

Biophysical perturbations induced by ethylazinphos in lipid membranes

Perturbations induced by ethylazinphos on the physical organization of dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol membranes were studied by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6-, 12-(9-anthroyloxy) stearic acids and 16-(9-anthroyloxy) palmitic acid. Ethylazinphos (50 and 100 microM) increases the fluorescence polarization of the probes, either in the gel or in the fluid phase of DPPC bilayers, and this concentration dependent effect decreases from the surface to the bilayer core. Additionally, the insecticide displaces the phase transition to a lower temperature range and broadens the transition profile of DPPC. A shifting and broadening of the phase transition is also observed by DSC. Furthermore at insecticide/lipid molar ratios higher than 1/7, DSC thermograms, in addition to the normal transition centered at 41 degrees C, also display a new phase transition centered at 45.5 degrees C. The enthalpy of this new transition increases with insecticide concentration, with a corresponding decrease of the main transition enthalpy. Ethylazinphos in DPPC bilayers with low cholesterol (< or = 20 mol%) perturbs the membrane organization as described above for pure DPPC. However, cholesterol concentrations higher than 20 mol% prevent insecticide interaction, as revealed by fluorescence polarization and DSC data. Apparently, cholesterol significantly modulates insecticide interaction by competition for similar distribution domains in the membrane. The present results strongly support our previous hypothesis that ethylazinphos locates in the cooperativity region, i.e. the region of C1-C9 atoms of the acyl chains, and extends to the lipid-water interface, where it increases lipid packing order sensed across all the thickness of the bilayer. Additionally, and, on the basis of DSC data, a lateral regionalization of ethylazinphos is here tentatively suggested.     

Effects of alcohols on lipid bilayers with and without cholesterol: the dipalmitoylphosphatidylcholine system

Differential scanning calorimetry is a useful method to study the thermotropic phase transitions of a phospholipid bilayer. In the present study DSC is used to determine the effects of methanol and ethanol on DPPC and DPPC/2 mol% cholesterol bilayers. The biphasic effect of the main transition and the presence of an extra peak on the DSC cooling scans were observed above certain alcohol concentrations. In the presence of 2% cholesterol, the concentration at which the biphasic effect occurs is increased by both short-chain alcohols. 1,6-Diphenyl-1,3,5-hexatriene (DPH) is used as a fluorescent probe to directly determine the onset of interdigitation in these systems as reflected by a drop in the DPH fluorescence intensity.     

Bilayer polarity and its thermal dependency in the l(o) and l(d) phases of binary phosphatidylcholine/cholesterol mixtures

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (l(o)) and liquid-disordered (l(d)) phases display significantly different polarities. Moreover, in the l(o) phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 degrees C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.     

Effect of cholesterol on the formation of an interdigitated gel phase in lysophosphatidylcholine and phosphatidylcholine binary mixtures

We previously reported that 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) forms an interdigitated gel phase in the presence of 1-palmitoyl-sn-glycero-3-phosphocholine (16:0LPC) at concentrations below 30 mol%. In the present investigation, fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), X-ray diffraction, and differential scanning calorimetry (DSC) were used to investigate the effect of cholesterol on the phase behavior of 16:0LPC/DPPC binary mixtures. At 25 degrees C, 30 mol% 16:0LPC significantly decreases the DPH fluorescence intensity during the transition of DPPC from the L(beta') phase to the L(betaI) phase. However, the addition of cholesterol to 16:0LPC/DPPC mixtures results in a substantial increase in fluorescence intensity. The changes in DPH fluorescence intensity reflect the probe's redistribution from an orientation parallel to the acyl chain to the center of the bilayer, suggesting a bilayer structure transition from interdigitation to noninterdigitation. The normal repeat period of small angle X-ray diffraction patterns can be restored and a reflection appears at 0.42 nm with a broad shoulder around 0.41 nm in wide angle X-ray diffraction patterns when 10 mol% cholesterol is incorporated into 30 mol% 16:0LPC/DPPC vesicles, indicating that the mixtures are in the gel phase (L(beta')). Moreover, DSC results demonstrate that 10 mol% cholesterol is sufficient to significantly decrease the main enthalpy, cooperativity and lipid chain melting of 30 mol% 16:0LPC/DPPC binary mixtures, which are L(betaI), indicating that the transition of the interdigitated phase is more sensitive to cholesterol than that of the noninterdigitated phase. Our data imply that the interdigitated gel phase induced by 16:0LPC is prevented in the presence of 10 mol% cholesterol, but unlike ethanol, an increasing concentration of 16:0LPC is not able to restore the interdigitation structure of the lipid mixtures.     

A fluorescence anisotropy study on the phase behavior of dimyristoylphosphatidylcholine/cholesterol mixtures

The phase behavior of L-alpha-dimyristoylphosphatidylcholine/cholesterol mixtures was studied in multilamellar vesicles by fluorescence polarization of the sterol molecule dehydroergosterol and of the polyene molecule alpha-parinaric acid. In the absence of cholesterol, dehydroergosterol exhibited an increase in polarization as DMPC vesicles were heated through the phase transition. This rise in polarization anisotropy was observed over a 0.6-1.0 degrees C increase in temperature with the midpoint of the phase transition occurring at 23.6 degrees C. Addition of 5 mol% cholesterol completely obliterated this change in polarization anisotropy through the phase transition of DMPC. alpha-Parinaric acid underwent a characteristic decrease in polarization anisotropy through the phase transition of DMPC. The change in anisotropy through the phase transition was over 4-fold greater than the values observed with dehydroergosterol. Vesicles containing 5 mol% cholesterol in the presence of alpha-parinaric acid underwent a decrease in polarization anisotropy that was over 75% of the original decrease in amplitude observed in the absence of any membrane cholesterol. The difference in sensitivity of the two fluorescent probes to the phase transition of DMPC as a function of membrane cholesterol content may be explained by a preferential partitioning of dehydroergosterol (and cholesterol) into a sterol-rich phase at low sterol concentrations. This partitioning allows dehydroergosterol to detect sterol-rich regions in the membrane bilayer.     

Perturbation of phospholipid bilayers by DDT

The localization of the effects of DDT (5–50 mol%) addition on the acyl chain dynamics in unilamellar vesicles of two phosphatidylcholines (DPPC and egg PC) has been investigated by steady-state fluorescence polarization of a series of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12 and 16) whose fluorophore is located at a graded series of depths from the surface to the centre of the bilayer. The results show that DDT is a fluidizer of DPPC and egg PC bilayers. The increase in microviscosity of DPPC bilayers at 23°C begins at the centre of the bilayer (5 mol% DDT) and proceeds outward to the surface with increasing concentration of DDT (17 mol%). This pattern of effects is not evident in fluid bilayers of DPPC at 54°C or egg PC at 23°C. DDT (33 mol%) also lowers the phase transition temperature of DPPC bilayers by approximately 2 Cdeg. DDT (17 mol%) had no effect on the mean excited fluorescence life-time of 2-AP and 12-AS in DPPC, DOPC and egg PC bilayers. No quenching of 2-AP fluorescence was evident.     

Phosphatidylinositol induces fluid phase formation and packing defects in phosphatidylcholine model membranes

Liposomes consisted of phosphatidylinositol (PI) and phosphatidylcholine (PC) have been utilized as delivery vehicle for drugs and proteins. In the present work, we studied the effect of soy PI on physical properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes such as phase state of lipid bilayer, lipid packing and phase properties using multiple orthogonal biophysical techniques. The 6-dodecanoyl-2-dimethylamino naphthalene (Laurdan) fluorescence studies showed that presence of PI induces the formation of fluid phases in DMPC. Differential scanning calorimetry (DSC), temperature dependent fluorescence anisotropy measurements, and generalized polarization values for Laurdan showed that the presence of as low as 10mol% of PI induces substantial broadening and shift to lower temperature of phase transition of DMPC. The fluorescence emission intensity of DPH labeled, PI containing DMPC lipid bilayer decreased possibly due to deeper penetration of water molecules in lipid bilayer. In order to further delineate the effect of PI on the physico chemical properties of DMPC is due to either significant hydrophobic mismatch between the acyl chains of the DMPC and that of soy PI or due to the inositol head group, we systematically replaced soy PI with PC species of similar acyl chain composition (DPPC and 18:2 (Cis) PC) or with diacylglycerol (DAG), respectively. The anisotropy of PC membrane containing soy PI showed largest fluidity change compared to other compositions. The data suggests that addition of PI alters structure and dynamics of DMPC bilayer in that it promotes deeper water penetration in the bilayer, induces fluid phase characteristics and causes lipid packing defects that involve its inositol head group.     

Bilayer polarity and its thermal dependency in the ?o and ?d phases of binary phosphatidylcholine/cholesterol mixtures

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (?_o) and liquid-disordered (?_d) phases display significantly different polarities. Moreover, in the ?_o phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 °C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.     

Effect of cholesterol content on the structural and dynamic membrane properties of DMPC/DSPC large unilamellar bilayers

In this study, we report the effect of cholesterol content on the dynamic and structural properties of a dimyristoyl-phosphatidylcholine and distearoyl-phosphatidylcholine mixture in large unilamellar vesicles. The range of cholesterol concentrations studied varied around approximately 33.3 mol%, where it has been postulated that an abrupt change in bilayer organization occurs. Steady-state fluorescence measurements demonstrated a typical behavior; at low temperatures in the main phase transition, the cholesterol concentration did not affect the gel phase, but at 37.5 °C (phase coexistence) and in the liquid crystalline phase, the presence of cholesterol produced an increase in the fluorescence anisotropy of DPH and the generalized polarization of Laurdan. The greater effect was observed in the liquid crystalline phase, in which the bilayer became a mixture of fluid-like and liquid-ordered phases. The results obtained at approximately 33.3 mol% of Cholesterol demonstrated that the Generalized Polarization of Laurdan, the DPH lifetime, the limiting anisotropy and the rotational correlation time, as well as the fluorescence quenching of DPH by TEMPO, are at maxima, while the fluorescence intensity of dehydroergosterol and the lipid solubility in TritonX-100 are at minima. These results correlate well with the hypothesis of domain segregation in the DMPC/DSPC/Cholesterol LUV system. In this context, we postulate that at 33.3 mol% of Cho, the proportion of ordered domains reaches a maximum.     

Partition of lindane in synthetic and native membranes

Partition coefficients of the insecticide gamma-1,2,3,4,5,6-hexachlorocyclohexane (trivially, lindane) were determined in model and native membranes. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10-40 degrees C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol dramatically decreases partition (2100 falls to 100, at 10 degrees C) and abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in lindane partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 2450, 600 and 50 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. The lindane partition sequence in native membranes is as follows: mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes. This sequence correlates reasonably well with the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values. Therefore, the presence of proteins in native membranes contributes to the insecticide partition, probably by favouring its interaction with lipids.     

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.     

Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner.     

Dynamic fluorescence measurements on the main phase transition of dipalmytoylphosphatidylcholine vesicles

The kinetics of the main phase transition in dipalmytoylphosphatidylcholine (DPPC) vesicles have been investigated using our iodine laser-Tjump technique with fluorescence detection. A set of three fluorescent probes has been used to sense different parts of the bilayer hydrocarbon chain region. The well established membrane probes DPH and TMADPH as well as DPHPC, a labelled DPPC molecule. We report three relaxation signals in the s and ms time range, which are detected with all three probes. This result supports our model of the main phase transition in DPPC vesicles.Abbreviations DMPC Dimyristoylphosphatidylcholine - DPPC Dipalmytoylphosphatidylcholine - DPH 1,6-Diphenylhexa-1,3,5-triene - TMADPH 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene - DPHPC Diphenylhexatriene-phosphatidylcholine - T_m Temperature of the main phase transition     

Perturbations induced by alpha- and beta-endosulfan in lipid membranes: a DSC and fluorescence polarization study.

The interaction of alpha- and beta-endosulfan isomers with lipid bilayers was searched by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6- and 12-(9-anthroyloxy) stearic acids (2-AS, 6-AS and 12-AS) and 16-(9-anthroyloxy) palmitic acid (16-AP). Both endosulfan isomers, at insecticide/lipid molar ratios ranging from 1/40 to 1/1, shift the phase transition midpoint to lower temperature values and broaden the transition profile of dipalmitoylphosphatidylcholine (DPPC) bilayers. At insecticide/lipid molar ratios of 1/40, the isomers fully abolish the bilayer pretransition. Conversely to beta-endosulfan, alpha-endosulfan promotes a new phase transition, centered at 35.4 degrees C, in addition to the main phase transition of DPPC. Therefore, the alpha-isomer may undergo a heterogeneous distribution in separate domains in the plane of the membrane, whereas the beta-isomer may undergo a homogeneous distribution. Fluorescence polarization data indicate that alpha-endosulfan increases the lipid structural order in the regions probed by 2-AS and decreases it in the regions probed by 6-AS, 12-AS and 16-AP. On the other hand, the beta-isomer produces disordering effects in the upper regions of the bilayers, probed by 2-AS, and ordering in deeper regions, probed by 6-AS, 12-AS and 16-AP, mainly in the gel phase. The incorporation of cholesterol into DPPC bilayers progressively decreases the effects of beta-isomer which are vanished at 20 mol% cholesterol. However, this and higher cholesterol concentrations did not prevent alpha-endosulfan membrane interaction, as revealed by DSC and fluorescence polarization. The distinct effects promoted by alpha- and beta-endosulfan are discussed in terms of molecular orientation and positioning within the bilayer. Apparently, the alpha-isomer preferentially locates closer to the phospholipid headgroups whereas the beta-isomer distributes in deeper domains of the bilayer.     

The effect of cholesterol on measured interaction and compressibility of dipalmitoylphosphatidylcholine bilayers

We have examined the phase diagram of dipalmitoylphosphatidylcholine (DPPC)--cholesterol-water mixtures at low cholesterol content, and report phase separation between 3 and 10 mol% cholesterol. The two lamellar phases at equilibrium in this region appear to be pure DPPC and 11 mol% cholesterol in DPPC. For these two lamellar phases, which are made up of alternating layers of water and bimolecular lipid leaflets, we have measured the forces of interaction between leaflets and the lateral pressure and compressibility of the leaflets. Both bilayers experience a strong repulsive force when forced together only a few ?ngstr?ms (1 A = 0.1 nm) closer than their maximum separation in excess water. However, the presence of 11 mol% cholesterol causes the bilayers to move apart of 35-A separation from the 19-A characteristic of pure DPPC in excess water. This swelling may result from a decrease in van der Waals attraction between bilayers or from an increase in bilayer repulsion. Differences in bilayer interaction can be a cause for phase separation. More importantly these differences can cause changes in the composition of regions of membranes approaching contact. At 11 mol%, cholesterol substantially increases the lateral compressibility of DPPC bilayers leading to higher lateral density fluctuations and potentially higher bilayer permeability.     

Electron spin resonance and steady-state fluorescence polarization studies of lipid bilayers containing integral proteins

We derive equations that describe changes in the steady-state fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) or in the spectrum of electron spin resonance (ESR) nitroxide spin-labeled lipid probes as a function of the intrinsic molecule concentration in lipid bilayer membranes. We make use of an assumption used by us in an earlier paper. The equations are independent of any membrane model. They are valid when a DPH probe or a spin-labeled chain is equivalent to an unlabeled lipid hydrocarbon chain only as far as their general space-filling properties are concerned. We consider cases where the bilayer is either in a single homogeneous phase or in a two-phase region. We apply our equations to analyze ESR data from delipidated sarcoplasmic reticulum membranes and from egg yolk phosphatidylcholine bilayers containing Ca2+-ATPase, and DPH data from dipalmitoylphosphatidylcholine (DPPC) bilayers containing Ca2+-ATPase, both for T greater than Tc. The following conclusions were derived: (i) Ca2+-ATPase oligomers are "randomly" distributed, for the concentrations studied, in the fluid phase. (ii) There is no fixed stoichiometric ratio of "boundary" lipids and oligomers. (iii) Between 24k and 28k lipid molecules are able to surround each isolated oligomer composed of k Ca2+-ATPase monomers. Finally, we apply our equations to analyze DPH studies on DPPC bilayers containing Ca2+-ATPase for T less than Tc. We find that the results reported are in accord with the predictions of the model. In the Appendix, we show that an analytical expression for probabilities used by us is in very good agreement with the results of computer simulation.     

Partition of lindane in synthetic and native membranes

Partition coefficients of the insecticide γ-1,2,3,4,5,6-hexachlorocyclohexane (trivially, lindane) were determined in model and native membranes. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10–40°C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol dramatically decreases partition (2100 falls to 100, at 10°C) and abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in lindane partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 2450, 600 and 50 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. The lindane partition sequence in native membranes is as follows: mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes. This sequence correlates reasonably well with the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values. Therefore, the presence of proteins in native membranes contributes to the insecticide partition, probably by favouring its interaction with lipids.