Alterations in erythrocyte membrane fluidity in children with trisomy 21: a fluorescence study.

Membrane fluidity of erythrocytes obtained from 15 children with trisomy 21 and 20 healthy controls were studied by measuring steady-state fluorescence anisotropy and fluorescence lifetime of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in hemoglobin-free erythrocyte membranes. Our results demonstrate a significant decrease in DPH fluorescence anisotropy and a significant increase in TMA-DPH fluorescence anistropy in erythrocytes from subjects with trisomy 21. No significant differences between the two groups were observed in the fluorescence lifetime of DPH and TMA-DPH. These data suggest an increase in membrane fluidity in the interior part of the membrane and a decrease in fluidity at the lipid-water interface region. This could be in part attributed to an increased oxidative damage in trisomy 21.     

Partitioning and membrane disordering effects of dopamine antagonists: influence of lipid peroxidation, temperature, and drug concentration.

The effect of four dopamine antagonists (spiperone, haloperidol, pimozide, and domperidone) on the lipid order of caudate nucleus microsomal membranes and on liposomes from membrane lipid extracts was evaluated and related to the partition coefficients (Kp) of the drugs. Lipid membrane order was determined by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. Dopamine antagonists decrease the fluorescence polarization of both probes, indicating that they disorder the membrane lipids at different depths. Pimozide and domperidone, the drugs with higher Kp values, are more effective at decreasing the polarization of DPH, a probe of the membrane core, than that of TMA-DPH. In contrast, spiperone and haloperidol, which have lower values for Kp, induce more significant decreases in TMA-DPH depolarization, a probe of the membrane surface. These findings indicate that higher partition coefficients of the drugs are directly correlated with an increase of fluidity in the hydrophobic core of brain membranes. Ascorbate/Fe(2+)-induced membrane lipid peroxidation increases membrane order. Membrane lipid peroxidation decreases the partition coefficients of the dopamine antagonists tested. Increasing temperature (4-37 degrees C) decreases membrane order, but temperature effect is less evident after lipid peroxidation. The disordering effect of dopamine antagonists increases with increasing drug concentrations (1-15 microM), a maximum being observed at 10 microM. However, this effect is also less evident after membrane lipid peroxidation. We can conclude that dopamine antagonists and membrane lipid peroxidation affect membrane lipid order and that the action of these drugs is dependent on initial bilayer fluidity. Membrane lipid peroxidation increases membrane order while dopamine antagonists show a disordering effect of membrane phospholipids. This disordering effect can indirectly influence the activity of membrane proteins and it is one of the mechanisms through which membrane function can be altered by these drugs.     

Transbilayer effects of ethanol on fluidity of brain membrane leaflets

Previous work on membrane effects of ethanol focused on fluidization of the bulk membrane lipid bilayer. That work was extended in the present study to an examination of ethanol's effect on lipid domains. Two independent methods were developed to examine the effects of ethanol on the inner and outer leaflets of synaptic plasma membranes (SPM). First, differential polarized phase and modulation fluorometry and selective quenching of diphenyl-1,3,5-hexatriene (DPH) were used to examine individual leaflets. Both limiting anisotropy and rotational relaxation time of DPH in SPM indicated that the outer leaflet was more fluid than the inner leaflet. Second, plasma membrane sidedness selective fluorescent DPH derivatives, cationic 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and anionic 3-[p-6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PRO-DPH), confirmed this transmembrane fluidity difference. TMA-DPH and PRO-DPH preferentially localized in the inner and outer leaflets of SPM, respectively. Ethanol in vitro had a greater fluidizing effect in the outer leaflet as compared to the inner leaflet. Thus, ethanol exhibits a specific rather than nonspecific fluidizing action within transbilayer SPM domains. This preferential fluidization of the SPM outer leaflet may have a role in ethanol affecting transmembrane signaling in the nervous system.     

Membrane fluidity changes in P. berghei-infected erythrocytes, investigated with a specific plasma membrane fluorescent probe

Trimethylamino-diphenylhexatriene (TMA-DPH), a novel hydrophobic fluorescent probe with relevant photophysical properties for fluorescence anisotropy measurements in phospholipidic membranes, specifically labels the plasma membranes of whole living-cells, unlike earlier commonly used probes such as 1,6-diphenyl-1,3,5-hexatriene (DPH) and anthroyloxy fatty acids, which invade all hydrophobic regions of the cell. Using TMA-DPH, it was shown that mouse malaria parasite Plasmodium berghei induced a statistically highly significant increase (8%) in the plasma membrane fluidity of the host erythrocyte. The physical factors, which might critically influence the measurements in this study, i.e. the fluorescence lifetime of the probe and the contribution of scattered light, were carefully controlled. The effect observed is discussed on the basis of earlier established metabolic changes in the membrane following infection, namely phospholipidic and cytoskeleton modifications.     

Therapeutic lithium alters polar head-group region of lipid bilayer and prevents lipid peroxidation in forebrain cortex of sleep-deprived rats

Lithium is regarded as a unique therapeutic agent for the management of bipolar disorder (BD). In efforts to explain the favourable effects of lithium in BD, a wide range of mechanisms was suggested. Among those, the effect of clinically relevant concentrations of lithium on the plasma membrane was extensively studied. However, the biophysical properties of brain membranes isolated from experimental animals exposed to acute, short-term and chronic lithium have not been performed to-date. In this study, we compared the biophysical parameters and level of lipid peroxidation in membranes isolated from forebrain cortex (FBC) of therapeutic lithium-treated and/or sleep-deprived rats. Lithium interaction with FBC membranes was characterized by appropriate fluorescent probes. DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulphonate) were used for characterization of the hydrophobic lipid core and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) for the membrane-water interface. Lipid peroxidation was determined by immunoblot analysis of 4-HNE-(4-hydroxynonenal)-protein adducts. The organization of polar head-group region of FBC membranes, measured by Laurdan generalized polarization, was substantially altered by sleep deprivation and augmented by lithium treatment. Hydrophobic membrane interior characterized by steady-state anisotropy of DPH and TMA-DPH fluorescence was unchanged. Chronic lithium had a protective effect against peroxidative damage of membrane lipids in FBC. In summary, lithium administration at a therapeutic level and/or sleep deprivation as an animal model of mania resulted in changes in rat FBC membrane properties.     

Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence anisotropy study

Time-resolved fluorescence anisotropy (TRFA) and steady-state anisotropy measurements and fluorescence intensification microscopic observations were made on RAW264 macrophages labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Microscopic analysis revealed that the fluorescent probe DPH was found in association with plasma membranes and small vesicles. Macrophages treated with immune complexes could not be distinguished from untreated cells, indicating that the same membrane compartments were labeled. The probe TMA-DPH was exclusively localized to the plasma membrane. Steady-state anisotropy measurements indicated that in vitro culture conditions did not significantly affect membrane fluidity. TRFA measurements were conducted to determine the physical properties of macrophage membranes during immune recognition and endocytosis. Data were analyzed by iterative deconvolution to yield phi, the rotational correlation time, and r infinity, the limiting anisotropy. These parameters may be interpreted as the "fluidity" and order parameter of the membrane environment, respectively. Typical values for untreated macrophages were phi = 7.8 ns and r infinity = 0.12. Binding and endocytosis of immune complexes prepared in 4-fold antigen excess increase these values to phi = 22.1 ns and r infinity = 0.15. However, receptor-independent phagocytosis of latex beads decreases these values to phi = 2.2 ns and r infinity = 0.10. Addition of catalase before, but not after, immune complex incubation with cells diminishes the effect upon membrane structure, suggesting that H2O2 participates in fluidity changes. Pretreatment of macrophages with the membrane-impermeable sulfhydryl blocker p-(chloromercuri)benzenesulfonic acid also diminished these effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of senescence on the molecular organization of membranes in ripening tomato fruit

Changes in the molecular organization of membranes in pericarp cells of ripening tomato fruit were examined by fluorescence depolarization after labeling with fluorescent lipid-soluble probes. The fluorescent labels were partitioned into isolated protoplasts and purified plastids from fruit at various stages of senescence. Values for steady-state anisotropy (r_ss) of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled protoplasts rose progressively during the early stages of ripening over a time frame that overlapped the climacteric rise in ethylene production. This can be interpreted as reflecting a decrease in the lipid fluidity of primarily plasma membrane. By contrast, there was no significant change during ripening in r_ss for plastid membranes labeled with DPH, 1-[4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cis- or trans-parinaric acid. Nor was there any change during ripening in the limiting fluorescence anisotropy (r_oo) and order parameter (S) for plastids labeled with DPH or TMA-DPH, parameters that are corrected for any differences in lifetime. Some degree of lifetime heterogeneity, possibly reflecting structurally distinct domains, was discerned in both young and senescent plastids that had been labeled with DPH or TMA-DPH, but this also did not change as ripening progressed. Thus membranes of the pericarp cells sustain different fates as the tomato fruit ripens, implying that there are distinguishable mechanisms of membrane deterioration in senescing tissues.     

The effect of osmotic stress on the biophysical behavior of the Bacillus subtilis membrane studied by dynamic and steady-state fluorescence anisotropy

The thermotropic behavior of intact bacterial membranes and vesicles prepared from total and polar lipids isolated from Bacillus subtilis cultures grown at 37 degrees C in normal (LB) and hyperosmotic (LBN) conditions was studied using 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), and 2-diethylamino-6-lauroyl-naphthalene (Laurdan) as fluorescent probes. No phase transition of bulk lipids was observed in these preparations at the range of temperature studied. The anisotropy values (r(s)) for DPH and TMA-DPH in purified membranes showed significant differences between the LB and LBN conditions, suggesting that there was an increase in membrane packing during the adaptation to osmotic stress. Furthermore, generalized polarization (GP) parameters for Laurdan indicated small but significant changes in water relaxation at the membrane hydrophobic/hydrophilic interface. Membrane preparations showed r(s) higher values than those of lipid vesicles and a higher temperature dependence of the Laurdan GP parameter. This fact indicates that membrane proteins increase the lipid packing and keep the membrane more sensitive to temperature changes.     

Partition coefficients of fluorescent probes with phospholipid membranes

A method for determination of membrane partition coefficients of five fluorescent membrane probes, 1,6-diphenyl-1,3,5-hexatriene (DPH), p-((6-phenyl)-1,3,5-hexatrienyl) benzoic acid (DPH carboxylic acid), 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH propionic acid), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and N-4-(4-didecylaminostyryl)-N-methylpyridinium iodide (4-di-10-ASP), was developed utilizing the fluorescence enhancement of a constant probe concentration by titration with excess phospholipid liposomes. The partition coefficients of DPH, DPH carboxylic acid, DPH propionic acid, TMA-DPH and 4-di-10-ASP into dipalmitoylphosphatidylcholine membranes were determined to be 1.3.10(6), 1.0.10(6), 6.5.10(5), 2.4.10(5) and 2.8.10(6) respectively. Knowledge of the partition coefficients may help select a lipid concentration for membrane studies that necessitate a probe's dominant incorporation into membranes.     

Changes in plasma membrane fluidity of corn (Zea mays L.) roots after Brij 58 treatment

Summary The detergent Brij 58 has been introduced to reverse plasma membrane (PM) vesicles from the right-side-out to the inside-out form. The aim of the present work was to investigate the effect of Brij 58 on the formation of an ATP-dependent proton gradient and on the fluidity of the lipid phase of PM vesicles. PMs of corn (Zea mays L.) roots were isolated by phase-partitioning. The fluidity of PMs was estimated by measurement of fluorescence polarization with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The PMs of corn roots were relatively rigid. The hydrophobic part of the lipid bilayer was more fluid than the hydrophilic part. After intercalation of Brij 58 into the lipid bilayer the membrane fluidity changed in a concentration-dependent manner. Treatment with the detergent Brij 58 increased the degree of fluorescence polarization for TMA-DPH, while it decreased it for DPH. This effect was saturated at a detergent-to-protein ratio of 1 4 for both fluorescence probes. Although the biophysical characteristics of the membrane were changed after Brij 58 treatment, the formation of ATP-dependent proton gradients could still be measured with those vesicles. The generation of an ATP-dependent proton gradient with Brij 58-treated PM vesicles suggests that the detergent treatment indeed turned the originally right-side-out vesicles to sealed inside-out vesicles. The limits of the effect caused by Brij 58 in the context of PM enzyme activities are discussed.Abbreviations Brij 58 polyoxyethylene 20 cetyl ether - DPH 1,6-diphenyl-1,3,5-hexatriene - HCF III hexacyanoferrate (III) - ISO inside-out - PM plasma membrane - RSO right-side-out - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene     

Enfuvirtide effects on human erythrocytes and lymphocytes functional properties.

Enfuvirtide (T-20) is the first inhibitor of human Immunodeficiency Virus type-1 (HIV-1) entrance on a target cell approved for clinical use. Recent studies indicated that its action mechanism involves the interaction with the membrane surface, increasing the concentration in the site of action. In the present study, the in vitro interaction between enfuvirtide and blood cells of healthy human donors, namely erythrocytes and lymphocytes, and the peptide effect on plasma and lymphocyte suspensions supernatant ions were evaluated, in order to better characterize the action of this peptide. Enfuvirtide causes a decrease in the concentration of hemoglobin and in the percentages of methemoglobin and carboxyhemoglobin, together with increased values of P50, pCO2, and [HCO3-], and significant decreases of pO2 and pH, in blood plasma. The supernatants of lymphocyte suspensions derived from blood incubated with enfuvirtide presented a decrease in pH and [HCO3-]. Fluorescence anisotropy measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), used to assess erythrocyte and lymphocyte membrane fluidity, did not yield enfuvirtide-induced changes (an effect could be expected due to peptide partition to lipid bilayers). Erythrocytes incubated with high enfuvirtide concentrations showed a significant decrease in osmotic fragility. As for erythrocyte deformability, enfuvirtide leads to increased elongation indexes for low shear stress values, whereas for high shear stress values it has the opposite effect. Despite the observed statistically significant variations in several parameters, these enfuvirtide-induced changes are not expected to lead to any detectable biomedical outcome for enfuvirtide-treated patients.     

Molecular order and dynamics in planar lipid bilayers: effects of unsaturation and sterols

Angle-resolved fluorescence depolarization experiments were carried out on 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules embedded in macroscopically oriented multilayers of saturated [dimyristoylphosphatidylcholine (DMPC)] and unsaturated [palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dilineoylphosphatidylcholine (DLPC), plant digalactosyldiglyceride (DGDG)] lipids with and without cholesterol. In all the lipid systems studied the order parameter (P2) of TMA-DPH molecules was found to be higher than that for DPH. Considerations of the order parameter (P4), however, indicate that DPH molecules have a heterogeneous distribution in bilayers of unsaturated lipids, with a significant fraction of the molecules lying with their long axes parallel to the bilayer planes. Both the DPH and TMA-DPH molecules exhibit a decrease in the molecular order as well as a decrease in their rates of motion on increasing the unsaturation of the hydrocarbon chains. The addition of cholesterol tends to reverse this effect, with an increase in both the order and dynamics. Bilayers of DOPC, however, exhibit a somewhat different result. It is suggested that the discrepancies between these observations and findings with lipid vesicle systems simply reflect the effects of curvature on the behavior of the probe molecules. The results indicate that the concept of membrane fluidity must be used with great caution.     

Effects of non-electrolyte molecules with anesthetic activity on the physical properties of DMPC multilamellar liposomes

The effects of 13 non-electrolytes with moderate anesthetic potency on the order of DMPC liposomes were examined. Changes in order were monitored by steady-state fluorescence polarization techniques using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPG). At 30 degrees C, all of the compounds tested decreased the DPH steady-state anisotropy (rs), with potencies highly correlated to their oil/water partition coefficients. However, only the most hydrophobic anesthetics decreased TMA-DPH RS. Some of the most hydrophilic compounds, including ethanol and urethane, actually increased TMA-DPH rs, suggestive of an increase in membrane order. The concept of selectivity was borrowed from partitioning theory and used to explain some effects on anesthetic potency of decreasing temperature to 18 degrees C. In the gel as opposed to the liquid crystalline phase, selectivity for decreasing membrane order (as monitored by DPH) markedly increased, suggesting that anesthetic partitioning and/or the site of anesthetic action was occurring in a more hydrophobic domain. The solute-independent difference (or capacity) between two membranes for perturbation was defined as membrane sensitivity. Sensitivity appeared to also decrease with decreasing temperature, despite the decrease in membrane partitioning. This effect is thought to result from the selective delivery of the anesthetic solute to the membrane interior and away from more hydrophilic domains where anesthetics may order membrane structure.     

Tamoxifen perturbs lipid bilayer order and permeability: comparison of DSC, fluorescence anisotropy, laurdan generalized polarization and carboxyfluorescein leakage studies

The perturbation of the lipid bilayer structure by tamoxifen may contribute to its multiple mechanisms of anticancer action not related to estrogen receptors. This study evaluates the effect of tamoxifen on structural characteristics of model membranes using differential scanning calorimetry (DSC), fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-[trimethylammonium)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), as well as 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) generalized polarization. The comparative measurements in multilammelar vesicles (MLV) prepared from dipalmitoylphosphatidylcholine (DPPC) revealed that tamoxifen decreases the phase transition temperature (Tm) paralleled by a broadening of the phase transition profile. In large unilamellar vesicles (LUV) prepared from egg yolk phosphatidylcholine (EPC), tamoxifen increased the lipid bilayer order predominantly in the outer bilayer region. From membrane permeability measurements, we conclude that the tamoxifen-induced release of entrapped carboxyfluorescein (CF) results from a permanent bilayer disruption and the formation of transient holes in the lipid bilayer.     

Effect of nedocromil sodium on polymorphonuclear leukocyte plasma membrane

The effect of nedocromil sodium on the plasma membrane fluidity of polymorphonuclear leukocytes (PMNs) was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results show that nedocromil sodium 300 muM significantly decreased membrane fluidity of PMNs. The decrease in membrane fluidity of PMNs induced by fMLP was abolished in the presence of nedocromil sodium. These data suggest that nedocromil sodium interferes with the plasma membranes of PMNs and modulates their activities.     

Comparison between flow cytometry and fluorometry for the kinetic measurement of membrane fluidity parameters

Steady-state fluorescence polarization measurements obtained with a flow cytometer were compared with those obtained with an SLM subnanosecond fluorometer. Measurements were made over time after exposure of HeLa cells to the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), or [12-(9:anthroyloxy) stearate (12-AS). After 1 min, anisotropy values of 0.28 (DPH), 0.28 (TMA-DPH), and 0.21 (12-AS) were obtained. Thereafter, the anisotropy of DPH- and 12-AS-labelled cells rapidly decreased (0.18 and 0.12 after 5 min), while that of TMA-DPH-labelled cells changed only slightly (0.27 after 30 min), suggesting that DPH and 12-AS, unlike TMA-DPH, do not remain anchored in the HeLa plasma membrane, but translocate to more fluid environments inside the cell. These suggestions were confirmed by visual observation with fluorescence microscopy. There was no significant difference between the results obtained with the flow cytometer and those obtained with the fluorometer.     

Membrane oxidative damage induced by ionizing radiation detected by fluorescence polarization

Effects of ionizing radiation on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbations of the lipid bilayer polarity. We have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization on two different cell lines (Chinese hamster ovary CHO-K1 and lymphoblastic RPMI 1788 cell lines). γ-Irradiation was performed using a ⁶⁰Co source with dose rates of 0.1 and 1 Gy/min for final doses of 4 and 8 Gy. Irradiation induced a decrease of fluorescence intensity and anisotropy of DPH and TMA-DPH in both cell lines, which was dose-dependent but varied inversely with the dose rate. Moreover, the fluorescence anisotropy measured in lymphoblastic cells using TMA-DPH was found to decrease as early as 1 h after irradiation, and remained significantly lower 24 h after irradiation. This study indicates that some alterations of membrane fluidity are observed after low irradiation doses and for some time thereafter. The changes in membrane fluidity might reflect oxidative damage, thus confirming a radiation-induced fluidization of biological membranes. The use of membrane fluidity changes as a potential biological indicator of radiation injury is discussed. Received: 14 May 1996 / Accepted in revised form: 30 September 1996     

Regulation of chloride transport in parotid secretory granules by membrane fluidity.

Zymogen granule membranes contain Cl- conductance and Cl/anion exchange activities that become important for primary fluid production after fusion with the apical plasma membrane of the acinar cell. We have used steady-state fluorescence anisotropy of diphenylhexatriene derivatives and measurements of Cl- transport in isolated secretory granules to determine the contribution of membrane fluidity to the regulation of transport across the granule membrane. Secretory granules from several unstimulated glands (rat pancreas and parotid, rabbit gastric glands) were shown to have low membrane fluidity compared to plasma membranes. In addition, Cl- transport activity in different granule preparations showed a strong correlation to the membrane fluidity when measured with 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), but not with 3-[p-(6-phenyl)-1,3,5-hexatrienyl)-phenyl]propionic acid (PA-DPH). These data suggest that TMA-DPH preferentially partitions into a specific lipid environment associated with, or which exerts an influence on, the Cl- transport proteins and that increases in the fluidity of this environment are associated with higher transport rates. Data from other types of plasma membranes indicate that TMA-DPH partitions much more than PA-DPH into the cytoplasmic leaflet, suggesting that this part of the granule membrane is involved in the observed fluidity changes. Furthermore, increasing the bulk membrane fluidity with the local anesthetics benzyl alcohol and n-alkanols increased the Cl- transport rates up to 10-fold. This increase was apparently through specific transporters as anion selectivity was maintained in spite of the higher absolute rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

A decrease of lipid fluidity of the porcine intestinal brush-border membranes by treatment with malondialdehyde.

The effect of treatment of the porcine intestinal brush-border membranes with malondialdehyde (MDA) on their lipid fluidity was examined using a fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). When the membranes were treated with MDA, the fluorescence anisotropy of DPH-labeled membranes increased and the amount of DPH molecules incorporated into the membranes decreased from 3.25 to 2.23 nmol/mg protein. In addition, the response of the fluorescence anisotropy of DPH-labeled membranes to benzyl alcohol, a well-known fluidizer, was markedly suppressed by treatment of the membranes with MDA. These results suggest that treatment of the membranes with MDA causes a decrease of the membrane lipid fluidity. This interpretation was further supported by the increase observed in the fluorescence anisotropy of DPH-labeled liposomes prepared from the extracted lipids of MDA-treated membranes. The results of SDS-polyacrylamide gel electrophoresis suggested that the formation of high-molecular-weight aggregates of the membrane proteins is not involved in the increase of the fluorescence anisotropy of DPH-labeled membranes by treatment with MDA. On the basis of these results, changes in the physical properties of the intestinal brush-border membranes by treatment with MDA are discussed.     

Effects of lactose permease of Escherichia coli on the anisotropy and electrostatic surface potential of liposomes

The membrane transport protein lactose permease (LacY), a member of the Major Facilitator Superfamily (MFS) containing twelve membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes of: (i) 1,2-dimyristoyl-sn-glycero-3-phosphocoline (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in equimolar proportions; and (ii) Escherichia coli total lipid extract. The structural order of the lipid membranes, in the presence and absence of LacY, was investigated using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene sulfonate (TMA-DPH) evidenced: (i) the insertion of LacY into the bilayer; and (ii) a surface effect on the membranes. The most dramatic effects were observed when LacY was reconstituted in the E. coli lipid matrix. The effect of the protein on the electrostatic surface potential of each bilayer was also examined using a fluorescent pH indicator, 4-Heptadecyl-7-hydroxycoumarin (HHC). Changes in surface potential were enhanced in the presence of the substrate (i.e. lactose) only when the lipid matrices were charged. These results suggest a role for charged phospholipids (i.e. phosphatidylethanolamine or phosphatidylglycerol) in proton transfer to the amino acids involved in substrate translocation.