Structural and dynamic effects of oxidatively modified phospholipids in unsaturated lipid membranes.

Phospholipid hydroperoxides and phospholipid alcohols are two of the major forms of oxidatively modified phospholipids produced during oxidant stress and lipid peroxidation. The process of lipid peroxidation is known to affect the physiological function of membranes. We, therefore, investigated the effects of lipid peroxidation products on the molecular interactions in membranes. Our study was specifically focused on the effects of lipid peroxidation products on static membrane structure (molecular orientational order) and on the reorientational dynamics of the probe molecules in lipid bilayers. The study was done by performing angle-resolved fluorescence depolarization measurements (AFD) on the fluorescent probe diphenylhexatriene (DPH) and by performing angle-resolved electron spin resonance (A-ESR) measurements on cholestane (CSL) nitroxide spin probes embedded in macroscopically oriented planar bilayers consisting of 2-10% 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)phosphatidylcholine (PLPC-OOH) or 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) in 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC) or dilinoleoylphosphatidylcholine (DLPC). Both probe molecules have rigid cylindrical geometries and report on the overall molecular order and dynamics. However, being more polar, the nitroxide spin probe CSL is preferentially located near the surface of the membrane, while the less polar fluorescent probe DPH reports preferentially near the central hydrophobic region of the lipid bilayers. The results show that the presence of relatively small amounts of oxidatively modified phospholipids within the PLPC or DLPC membranes causes pronounced structural effects as the molecular orientational order of the probe molecules is strongly decreased. In contrast, the effect on membrane reorientational dynamics is minimal.     

Fusion of Spiroplasma floricola cells with small unilamellar vesicles is dependent on the age of the culture.

Small unilamellar vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride and mixed with intact Spiroplasma floricola cells. The increase in fluorescence observed was interpreted as a result of the dilution of the probe in the unlabeled S. floricola membranes because of lipid mixing upon fusion. The progression of S. floricola cultures to the stationary phase of growth was accompanied by a sharp decrease in the ability of the cells to fuse with small unilamellar vesicles. Low fusogenic activity was also detected in cells from cultures that were aged in a growth medium maintained at pH 7.5 throughout the growth cycle. Chemical analysis of the cell membrane preparations isolated from cells harvested at the various phases of growth revealed that the phospholipid content and composition and the cholesterol/phospholipid molar ratio were changed very little upon aging of the cultures. Likewise, no changes in the fatty acid composition of membrane lipids were detected, with palmitic and oleic acids predominating throughout the cycle. Nonetheless, upon aging of S. floricola cultures, a pronounced increase in the levels of both cholesteryl esters, incorporated from the growth medium, and organic peroxides was observed. A decrease in both fluorescence anisotropy of diphenylhexatriene and merocyanine 540 binding to membranes of aged cells was also detected. The possible influence of these changes on the fusogenic activity of the cells is discussed.     

Effects of alcohols on fluorescence anisotropies of diphenylhexatriene and its derivatives in bovine blood platelets: relationships of the depth-dependent change in membrane fluidity by alcohols with their effects on platelet aggregation and adenylate cyclase activity.

The effects of three short-chain alkyl alcohols and benzyl alcohol on the membrane fluidity of bovine blood platelets were investigated by studies on the fluorescence anisotropies of diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and its anionic propionic acid derivative (DPH-PA). These alcohols decreased the fluorescence anisotropy of DPH, which is thought to be located within the hydrophobic core of the membrane, in concentration ranges that inhibited platelet aggregation. On the other hand, they had little or no effects on the fluorescence anisotropy of DPH-PA which is thought to be located in the interfacial region of the lipid bilayer. Likewise, they had little or no effects on the fluorescence anisotropy of TMA-DPH, which is also thought to be located in the interfacial region of the lipid bilayer, either when the probe was located in the outer layer of the plasma membrane or when the probe was located in the inner membrane compartment. These results suggest that alcohols mainly increase the fluidity in the central region of the lipid bilayer. Consistent with their effects on the fluorescence anisotropy of DPH, these alcohols increased the intracellular cyclic AMP concentration. Thus alcohols may inhibit platelet function due to stimulation of adenylate cyclase, which is mediated by perturbation of the central region of the membrane lipid bilayer.     

Changes in the fluorescence parameters of bound N-(1-pyrene) maleimide by lipid peroxidation of intestinal brush-border membranes

Using a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM), we have examined of lipid peroxidation on the microenvironment around SH groups of the membrane proteins in porcine intestinal brush-border membrane vesicles. The lipid peroxidation of the membranes was performed with various concentrations of t-butylhydroperoxide (t-BuOOH) in the presence of 100 microM ascorbic acid and 10 microM Fe2+. Treatment of NPM-labeled membranes with these oxidizing agents resulted in a decrease of the fluorescence lifetime, suggesting modification of the environmental properties around the bound dye. Measurement of the steady-state fluorescence anisotropy of the labeled membranes indicated restriction of the motion of the bound dye by the lipid peroxidation membranes. This interpretation was further supported by an elevation of the transition temperature of the anisotropy, a decrease in the quenching rate constant of the fluorescence with acrylamide and a decrease in the SH reactivity of the membrane proteins for NPM by lipid peroxidation. Based on these results, the possibility of conformation changes in the vicinity of SH groups in the membrane proteins associated with lipid peroxidation has been discussed.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15'-phenylpentadeca-8',10',12',14'-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.     

Biosynthetic incorporation of fluorescent carbazolylundecanoic acid into membrane phospholipids of LM cells and determination of quenching constants and partition coefficients of hydrophobic quenchers

A fluorescence quenching method was developed for determining partition coefficients and diffusional rates of small molecules in cell membranes. This method involves quenching the fluorescence of carbazole-labeled membranes by hydrophobic molecules that partition into membranes. Cell membrane phospholipids of mouse LM cells in tissue culture were biosynthetically labeled with the carbazole moiety by supplementing the growth media with 11-(9-carbazolyl)undecanoic acid. Plasma membranes, microsomes, and mitochondria were isolated free of nonmembranous neutral lipids, and the incorporation of the fluorescent probe was characterized. Quenching studies of the carbazole moiety by a series of N-substituted picolinium perchlorate salts showed that the carbazole moiety was located in the hydrophobic interior of the membrane bilayer. The carbazole fluorescence also was quenched by the hydrophobic quenchers lindane, methoxychlor, and 1,1-dichloro-2,2-bis(rho-chlorophenyl)ethylene, indicating that these compounds partitioned into the membrane. Stern-Volmer quenching constants determined by fluorescence lifetime and intensity measurements were identical, as expected for dynamic quenching. The effects of different lipid compositions on quenching constants and partition coefficients were determined by comparing different membrane fractions. These parameters also were measured in membranes from cells in which the phospholipid composition was altered by substituting ethanolamine for choline in the growth medium. Changes in the lipid composition produced changes in the bimolecular quenching constants. For example, bimolecular quenching constants for 1,1-dichloro-2,2-bis(rho-chlorophenyl)ethylene were higher in mitochondrial membranes than in plasma membranes and microsomes. They were also higher in dispersions made from membrane phospholipids as compared with intact membranes or total lipid dispersion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.     

Steady-state fluorescence anisotropy changes of 1,6-diphenyl-1,3,5,-hexatriene in membranes from Bacillus megaterium spores

The steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene incorporated into isolated Bacillus megaterium spore membranes was measured. Compounds capable of triggering spore germination in vivo caused an increase in the anisotropy of diphenylhexatriene. These increases in anisotropy of diphenylhexatriene in spore membranes are likely to represent at least a portion of the trigger mechanism for spore germination based on the following observations. First, there was an exceptional positive correlation between compounds that both triggered germination in vivo and caused changes in anisotropy in vitro. Second. the capacity of membranes to respond to germinants by increases in anisotropy was unique to membranes from spores but disappeared after germination. Third, alteration of spores chemically or genetically to block the in vivo triggering of germination by l-proline also blocked the in vitro anisotropy change with l-proline but not d-glucose. Finally, there was no correlation between the transport activities of specific compounds and the ability of these compounds to either trigger germination or alter the anisotropy of diphenylhexatriene in the membranes. Although we do not known the nature of the molecular interactions giving rise to the anisotropy changes, we hypothesize that they are due to changes in protein conformation that alter protein-protein and/or protein-lipid interactions. Such modifications of membrane structures could account for the rapid release of small molecular weight compounds such as K⁺ and Ca²⁺ early in germination.     

Effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane of S49 lymphoma cells

The effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane were examined with cultured S49 lymphoma cells. The beta-adrenergic receptor-coupled adenylate cyclase system was used as a probe of the functional properties of the plasma membrane. Steady-state fluorescence anisotropy of diphenylhexatriene and the lipid composition of the plasma membrane were used as probes of the physical properties of the membrane. Cells were grown under conditions such that the concentration of ethanol in the growth medium remained stable and oxidation of ethanol to acetaldehyde was not detected. Chronic exposure of S49 cells to 50 mM ethanol or growth of cells at elevated temperature resulted in a decrease in adenylate cyclase activity. There were no changes in the density of receptors or in the affinity of beta-adrenergic receptors for agonists or antagonists following chronic exposure to ethanol. The fluorescence anisotropy of diphenylhexatriene was lower in plasma membranes prepared from cells that had been treated with 50 mM ethanol than in membranes prepared from control cells. However, this change was not associated with changes in the fatty acid composition or the cholesterol to phospholipid ratio of the plasma membrane. There was a small but statistically significant decrease in the amount of phosphatidylserine and an increase in the amount of phosphatidylethanolamine. These changes cannot account for the decrease in anisotropy. In contrast to the effect of ethanol, a decrease in adenylate cyclase activity following growth of S49 cells at 40 degrees C was not associated with a change in anisotropy.     

Liquid ordered phase in cell membranes evidenced by a hydration-sensitive probe: Effects of cholesterol depletion and apoptosis

Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells.     

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15′-phenylpentadeca-8′,10′,12′,14′-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.     

Fluorescent Probes DPH,TMA-DPH and C17-HC Induce Erythrocyte Exovesiculation

An experimental approach has been developed to study human erythrocyte vesiculation, using the fluorescent probes diphenylhexatriene (DPH), trimethylamino-diphenylhexatriene (TMA-DPH) and heptadecyl-hydroxycoumarin (C17-HC). Acetylcholinesterase (AChE) enzyme activity measurements confirmed the presence of exovesicles released from erythrocyte membranes labeled with DPH, TMA-DPH or C17-HC. The fluorescence intensity and anisotropy values obtained showed that the amphiphilic probes TMA-DPH and C17-HC are preferentially incorporated in the exovesicles (when compared with DPH). There is a significant decrease of the cholesterol content of the exovesicle suspensions with time, independently of the fluorescence probe used, reaching undetectable cholesterol levels for the samples incubated for 48 hr. The ratios between the concentration of cholesterol released in the exovesicles after 1 hr incubation with DPH, TMA-DPH or C17-HC and the probe concentration used in the incubation were 84.7, 3.82 and 0.074, respectively. The size of the released vesicles was evaluated by dynamic light scattering spectroscopy. Some hypotheses are proposed that could explain the resemblance and differences between the results obtained for erythrocytes labeled with each probe, considering the present knowledge of membrane vesiculation mechanisms, lipid microdomains (rafts), erythrocyte membrane phospholipid asymmetry and AChE inhibition by TMA-DPH and C17-HC. This work demonstrates that the fluorescent probes DPH, TMA-DPH and C17-HC induce rapid erythrocyte exovesiculation; their use can lead to new methodologies for the study of this still poorly understood mechanism.     

Lateral pressures in biomembranes estimated from the dynamics of fluorescent probes

A theoretical model is proposed which states that the time-independent fluorescence anisotropy of the rod-shaped molecule diphenylhexatriene incorporated into lipid bilayers is a direct result of forces constraining the diphenylhexatriene molecule. These forces are postulated as equating with the lateral pressure operating within the bilayer independently of the probe molecule.Insertion into the model of experimental observations (recorded in the literature) on anisotropy of diphenylhexatriene in lipid bilayers as a function of temperature yielded values of lateral pressure, which decreased with temperature, and sharply at the temperature defining the transition from gel phase to fluid phase. The values so predicted for the mid-point of the transition and for the entirely fluid phase, respectively, compared favourably with estimates of the lateral pressures in these physical states, that have been reported elsewhere and arrived at either from theories describing lipid chain behaviour or from lipid monolayer compression experiments. Previously documented effects on anisotropy induced by incorporation of cholesterol into fluid lipid bilayers have been interpreted as reflections of rises in intramembranal lateral pressure.     

Use of Steady-State Laurdan Fluorescence to Detect Changes in Liquid Ordered Phases in Human Erythrocyte Membranes

In artificial phospholipid bilayers, dual measurements of laurdan steady-state anisotropy and emission spectra can be used to identify the presence of liquid ordered phases. Human erythrocytes were used as a model to test whether similar measurements could be applied to biological samples. Specifically, laurdan anisotropy and emission spectra were obtained from native erythrocytes before and after treatment with calcium ionophore and from the microvesicles (known to be enriched in liquid ordered domains) shed from the cells during calcium entry. Spectral and anisotropy data were consistent with an increased order and reduced fluidity of erythrocyte membrane lipids upon ionophore treatment. Microvesicle membranes appeared more ordered than native erythrocytes and similar to ionophore-treated cells based on laurdan emission. In contrast, the anisotropy value was lower in microvesicles compared to ionophore-treated cells, suggesting greater probe mobility. Parallel measurements of diphenylhexatriene anisotropy corroborated the laurdan data. These results were consistent with the liquid ordered property of microvesicle membranes based on comparisons to behavior in artificial membranes. Two-photon microscopy was used to examine the distribution of laurdan fluorescence along the surface of erythrocyte membranes before and after ionophore treatment. A dual spatial analysis of laurdan anisotropy, as revealed by the distribution of laurdan emission spectra, and intensity excited by polarized light suggested that the plasma membranes of ionophore-treated erythrocytes may also exhibit elevated numbers of liquid ordered domains.     

Antioxidant Activity of Vitamin E and Trolox: Understanding of the Factors that Govern Lipid Peroxidation Studies In Vitro

Peroxidation of lipids is of significant interest owing to the evidence that peroxyl radicals and products of lipid peroxidation may be involved in the toxicity of compounds initiating a deteriorative reaction in the processing and storage of lipid-containing foods. In view of the significance of the antioxidant role of the dietary compound vitamin E and its water-soluble analogue Trolox in research of lipid-containing foods, it is desirable to determine more specifically how and where they operate its antioxidant activity in lipid membranes. In this study, unilamellar liposomes of phosphatidylcholine were used as membrane mimetic systems to estimate the antioxidant properties of vitamin E and Trolox and establish a relationship between their interactions with the membrane and their consequent antioxidant activity. Lipid peroxidation was initiated by the peroxyl radical (ROO^•) in lipid and aqueous media by the thermal decomposition of azocompounds and was assessed by the fluorescence intensity decay of the fluorescent probe diphenylhexatriene propionic acid. Results obtained showed that membrane lipoperoxidation is related not only to the scavenging characteristics of the compounds studied but also to their ability to interact with the lipid bilayers, and consequently liposomes provide additional information to that obtained currently from assays performed in aqueous buffer media.     

Effect of lipid composition on rat liver nuclear membrane fluidity

Nuclear membrane fluidity is measured in rat liver by use of the fluorescence anisotropy of two probes: diphenylhexatriene and its cationic derivative trimethylammonium-diphenylhexatriene. It has been shown that, in 2-month-old rat liver cells, the bilayer surface is less fluid than the hydrophobic core. The fluidity was higher in 6-day-old rat liver nuclei, in which both the amount of cholesterol and the cholesterol/phospholipid ratio decreased. The influence of the single phospholipids, and in particular of phosphatidylcholine, has been studied by increasing the phosphatidylcholine with a choline base exchange reaction in isolated nuclear membranes. After this reaction, the fluorescence anisotropy of the bilayer surface increased, whereas at the hydrophobic core it decreased. Analysis of fatty acid composition shows an increase of phosphatidylcholine unsaturated fatty acids. The results show that the fluidity of nuclear membranes changes in relation to the lipid content and to the fatty acid composition. The role of nuclear membrane fluidity in cell function is discussed. © 1997 John Wiley & Sons, Ltd.     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.     

Interactions of pyrethroids with gramicidin-containing liposomal membranes

Fluorescence steady-state anisotropy and phase-modulation lifetime techniques have been utilized to study the interactions of pyrethroid compounds with fluid-phase phosphatidylcholine membranes containing the polypeptide gramicidin. This polypeptide is considered to be a model of hydrophobic regions of cellular integral membrane proteins. The pyrethroids disorder lipid packing in cellular membranes and gel-phase liposomes but do not disorder lipid packing in fluid-phase lipid (Stelzer, K.J. and Gordon, M.A. (1984) J. Immunopharmacol. 6, 381-410; (1985) Biochim. Biophys. Acta 812, 361-368) Irrespective of liposomal size, gramicidin incorporation resulted in a substantial increase in anisotropy of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), in fluid phase lipid. In the absence of gramicidin, permethrin and three other pyrethroids, allethrin, cypermethrin and fenpropathrin, increased DPH anisotropy. In these fluid phase systems, as the protein:lipid ratio was increased, the extent of the pyrethroid-mediated increase in fluorescence anisotropy diminished. Also, the pyrethroids shortened DPH fluorescence lifetimes. At high gramicidin:lipid ratios, permethrin substantially lowered anisotropy in the fluid phase lipid, relative to controls. The data suggest that pyrethroids disturb fluid-phase lipids which have been promoted to a relative state of order by proximity to an integral membrane protein. This type of order is one which is represented by DPH fluorescence anisotropy. A model based on these results is proposed to explain the effects of pyrethroids on lipid packing order in cellular membranes, as determined by DPH fluorescence anisotropy.     

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.