Phospholipid lateral phase separation and the partition of cis-parinaric acid and trans-parinaric acid among aqueous, solid lipid, and fluid lipid phases.

The partition of cis-parinaric acid (9,11,13,15-cis, trans, trans,cis-octadecatetraenoic acid, cis-PnA) and trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid, trans-PnA) among aqueous, solid lipid, and fluid lipid phases has been measured by three spectroscopic parameters: absorption spectral shifts, fluorescence quantum yield, and fluorescence polarization. The solid lipid was dipalmitoylphosphatidylcholine (DPPC); the fluid lipid was palmitoyldocosahexaenoylphosphatidylcholine (PDPC). Mole fraction partition coefficients between lipid and water were determined by absorption spectroscopy to be for ci--PnA, 5.3 X 10(5) with a solid lipid and 9 X 10(5) with fluid lipid and, for trans-PnA, 5 X 10(6) with solid lipid and 1.7 X 10(6) with fluid lipid. Ratios of the solid to the fluid partition coefficients (Kps/f) are 0.6 +/- 0.2 for cis-PnA and 3 +/- 1 for trans-PnA. A phase diagram for codispersions of DPPC and PDPC has been constructed from the measurements of the temperature dependence of the fluorescence quantum yield and polarization of cis-PnA and trans-PnA and their methyl ester derivatives. A simple analysis based on the phase diagram and fluorescence data allows additional calculations of Kps/f's which are determined to be 0.7 +/- 0.2 for the cis probes and 4 +/- 1 for the trans probes. The relative preference of trans-PnA for solid phase lipids and its enhanced quantum yield in solid phase lipids make it sensitive to a few percent solid. The trans probes provide evidence that structural order may persist in dispersions of these phospholipids 10 degrees C or more above their transition temperature. It is concluded that measurements of PnA fluorescence polarization vs. temperature are better suited than measurements of quantum yield vs. temperature for determining phospholipid phase separation.     

Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use or paramagnetic quenching.

Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short range interaction we developed a spectroscopic method to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase. This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.     

Spectroscopic characterization of 2-amino-N-hexadecyl-benzamide (AHBA), a new fluorescence probe for membranes

We report the results of investigation on the spectroscopic properties of a new fluorescent lipophylic probe. The fluorophore o-aminobenzoic acid was covalently bound to the acyl chain hexadecylamine, producing the compound 2-amino-N-hexadecyl-benzamide. The behavior of the probe was dependent on the polarity of the medium: absorption and emission spectral position, quantum yield and lifetime decay indicate distinct behavior in water compared to ethanol and cyclohexane. The probe dissolves in the organic solvents, as indicated by the very low value of steady state fluorescence anisotropy and the short rotational correlation times obtained from fluorescence anisotropy decay measurements. On the other hand, the probe has low solubility in water, leading to the formation of aggregates in aqueous medium. The complex absorption spectrum in water was interpreted as originating from different forms of aggregation, as deduced from the wavelength dependence of anisotropy parameters. The probe interacts with surfactants in pre-micellar and micellar forms, as observed in experiments in the presence of sodium n-dodecylsulphate (SDS), n-cetyltrimethylammonium bromide (CTAB); 3-(dodecyl-dimethylammonium) propane-1-sulphonate (DPS) and 3-(hexadecyl-dimethylammonium) propane-1-sulphonate (HPS), and with vesicles of the phospholipid dimiristoyl-phosphatidylcholine (DMPC). The results demonstrate that AHBA is able to monitor properties like surface electric potential and phase transition of micelles and vesicles.     

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

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

Perylenoyl- and anthrylvinyl-labeled lipids as membrane probes

The properties of a new family of lipid-specific fluorescent probes, a fatty acid, a phosphatidylcholine and a sphingomyelin, bearing a 3-perylenoyl-labeled hydrophobic chain, are described. Perylenoyl-labeled lipids readily enter the lipid bilayer, the fluorophore being localized in the apolar region of the membrane. The perylenoyl fluorophore is characterized by a high quantum yield, its fluorescence parameters (λ_ex 446 nm, λ_em 479–545 nm) permit to apply it as an acceptor of excitation energy from the 9-anthrylvinyl fluorophore used earlier for phospholipid labeling (Molotkovsky, Jul. G.; Manevich, Y.M., Gerasimova, E.N., Molotkovskaya, I.M., Polessky, V.A. and Bergelson, L.D. (1982) Eur. J. Biochem. 122, 573–579). The anthrylvinyl-labeled lipids were shown to be capable to report phase segregation between the corresponding prototype lipids in model systems. The combined use of anthrylvinyl- and perylenoyl-labeled lipids opens additional possibilities for investigation of lipid-lipid and lipid-protein interactions in artificial and biological membranes. Perylenoyl-labeled lipids appeared also to be useful as fluorescent dyes in cytological studies.     

Bimodal distribution and fluorescence response of environment-sensitive probes in lipid bilayers

A remarkable heterogeneity is often observed in the spectroscopic properties of environment-sensitive fluorescence probes in phospholipid bilayers. To explain its origin, we provided a detailed investigation of the fluorescence excitation and emission spectra of 4'-dimethylamino-3-hydroxyflavone (probe F) in bilayer vesicles with the variations of fatty acid composition, polar heads, temperature, and cholesterol content. Probe F, due to excited-state intramolecular proton transfer, exhibits two bands in emission that are differently sensitive to intermolecular interactions-thereby allowing us to distinguish universal (dipole-dipole) and specific (H-bonding) interactions within the bilayer. Spectroscopic, quenching, and anisotropy data suggest the presence of two forms of probe F at different locations in the bilayer: an H-bond free form located below sn(1)-carbonyls and an H-bonded form located at the polar membrane interface. We provide a quantitative analysis of the distribution of the probe between these two locations as well as the polarity of these locations, and show that both the distribution and the polarity contribute to the probe response. Moreover, analysis of literature data on other environment-sensitive probes (Prodan, Laurdan, Nile Red, NBD lipids, etc.) in lipid bilayers allows us to suggest that the bimodal distribution in the lipid bilayer is probably a general feature of low-polar molecules with polar groups capable of H-bonding interactions.     

Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

Binding of a cationic phenazinium dye in anionic liposomal membrane: a spectacular modification in the photophysics

Interaction of a cationic phenazinium dye, phenosafranin (PSF), with the anionic liposomal vesicle/bilayer of dimyristoyl-l-α-phosphatidylglycerol (DMPG) has been demonstrated using steady state and time resolved fluorescence and fluorescence anisotropy techniques. The charge transfer emission spectrum of PSF shows a dramatic modification in terms of fluorescence yield together with an appreciable hypsochromic shift in the lipid environment. The blue shift indicates a lowering in polarity inside the vesicle as compared to that in bulk water. The fluorescence and fluorescence quenching studies and micropolarity determination reveal that the cationic fluorophore has a profound binding interaction with the anionic DMPG membrane. Anisotropy study indicates the imposition of a motional restriction on the probe inside the bilayer. The electrostatic interaction between the cationic dye and the anionic lipid membrane has been argued to be the reason behind all these observations. The results could be useful in analyzing membrane organization and heterogeneity in natural membranes exploiting PSF or alike compounds as fluorescent probes.     

Interaction of fatty acids with lipid bilayers

We present a method by which it is possible to describe the binding of fatty acids to phospholipid bilayers. Binding constants for oleic acid and a number of fatty acids used as spectroscopic probes are deduced from electrophoresis measurements. There is a large shift in $\text{p}\text{K}$ value for the fatty acids on binding to the phospholipid bilayers, consistent with stronger binding of the uncharged form of the fatty acid. For dansylundecanoic acid, fluorescence titrations are consistent with the binding constants derived from the electrophoresis experiments. For 12-(9-anthroyloxy)stearic acid, fluorescence and electrophoresis data are inconsistent, and we attribute this to quenching of fluorescence at high molar ratios of 12-anthroylstearic acid to phospholipid in the bilayer.     

Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of n-(9-anthroyloxy) fatty acids to determine fluidity and polarity gradients in phospholipid bilayers

A set of n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12) have been used to examine gradients in fluorescence polarization, lifetime (tau F), relative quantum yield (phi rel) and positions of emission maxima (lambda max) through bilayers composed of synthetic phospholipids. The fluorophores of these probes report the environment at a graded series of depths from the surface to the centre of the bilayer structure. 1. Polarizations decrease as the fluorophore is moved deeper into the bilayer indicating greater rotational motion of the fluorophore in the hydrocarbon core of the bilayer. 2. The different responses of the probe diphenylhexatriene and the anthroyloxy fatty acids to the action of cholesterol on lipid bilayers are discussed in terms of the orientation of these probes in the bilayer and the types of anisotropic rotational motions which result in depolarization of fluorescence. 3. Stearic acid derivatives which have the fluorophore in the 6-, 9- and 12-positions along the acyl chain have a similar response to solvent polarity as measured by values of lambda max and phi rel in a variety of organic solvents. 4. The position of the emission maximum has little dependence on solvent viscosity, but viscosity does change the degree of vibrational structure seen in the emission spectrum. The vibrational structure itself may be used as an indication of the 'mciroviscosity' gradient in the transverse plane of the bilayer. 5. Values of lambda max, tau F and phi rel indicate that a gradient of polarity exists from the surface to the centre of the bilayer. For dipalmitoyl phosphatidylcholine in the crystalline phase, cholesterol acts to make this polarity gradient shallower.     

Interfacial properties of model membranes and plasma lipoproteins containing ether lipids

The interfacial properties of synthetic ester and ether phosphatidylcholines (PCs) were investigated by using the polarity-sensitive fluorescent probes 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and pyrene. The physical state of the phospholipid matrix was determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Single-bilayer phospholipid vesicles formed by sonication and model high-density lipoproteins were studied. On the basis of a number of spectroscopic and thermodynamic criteria, the interfacial regions of PCs and their ether analogues are similar. The fluorescence properties of Prodan in model lipoproteins or single-bilayer vesicles were independent of the phospholipid fatty acyl chain length and polar head group, as well as the substitution of ether linkage for ester bonds in the phospholipid. The spectral shifts correlated mainly with the physical state of the phospholipid. The emission spectrum of Prodan appeared at shorter wavelengths upon transfer from water to liquid-crystalline phospholipid and blue shifted further when the lipid was cooled to its gel phase. The effect of cholesterol in model high-density lipoproteins on the emission spectrum of Prodan was dose dependent and, at 18 mol % cholesterol, the spectrum was similar to that observed in a pure gel-phase lipid and was independent of temperature. The quantum yield of Prodan fluorescence in an ether-PC matrix was similar to that observed in water, whereas in an ester-PC matrix it was enhanced by a factor of about 5. Phospholipid-water partition coefficients of Prodan were independent of the physical state of 1,2-dimyristoyl-sn-glycero-3-phosphocholine or 1,2-tetradecyl-sn-glycero-3-phosphocholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biophysical properties of ergosterol-enriched lipid rafts in yeast and tools for their study: characterization of ergosterol/phosphatidylcholine membranes with three fluorescent membrane probes

In this work, binary mixtures of phospholipid/ergosterol (erg) were studied using three fluorescent membrane probes. The phospholipid was either saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) or monounsaturated (1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphocholine, POPC) phosphatidylcholine, to evaluate the fluorescence properties of the probes in gel, liquid ordered (l(o)) and liquid disordered (l(d)) phases. The probes have been used previously to study cholesterol-enriched domains, but their photophysical properties in erg-enriched membranes have not been characterized. N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-DPPE) presents modest blue-shifts upon erg addition, and the changes in the fluorescence lifetime are mainly due to differences in the efficiency of its fluorescence dynamic self-quenching. However, the steady-state fluorescence anisotropy of NBD-DPPE presents well-defined values in each lipid phase. N-(lissamine rhodamine B sulfonyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Rhod-DOPE) presents a close to random distribution in erg-rich membranes. There are no appreciable spectral shifts and the steady-state fluorescence anisotropy presents complex behavior, as a result of different photophysical processes. The probe is mostly useful to label l(d) domains in yeast membranes. 4-(2-(6-(Dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium (di-4-ANEPPS) is an electrochromic dye with excitation spectra largely insensitive to the presence of erg, but presenting a strong blue-shift of its emission with increasing concentrations of this sterol. Its partition coefficient is favorable to l(o) domains in POPC/erg mixtures. Although the fluorescence properties of di-4-ANEPPS are less sensitive to erg than to chol, in both cases the fluorescence lifetime responds monotonically to sterol mole fraction, becoming significantly longer in the presence of sterol as compared to pure POPC or DPPC bilayers. The probe displays a unique sensitivity to sterol-lipid interaction due to the influence of hydration and H-bonding patterns at the membrane/water interface on its fluorescence properties. This makes di-4-ANEPPS (and possibly similar probes) potentially useful in the study of erg-enriched domains in more complex lipid mixtures and in the membranes of living yeast cells.     

Surfactant protein SP-B induces ordering at the surface of model membrane bilayers

The effects of bovine pulmonary surfactant-associated protein B (SP-B) on molecular packing of model membrane lipids (7:1 DPPC/DPPG) were studied by fluorescence anisotropy. The bilayer surface was markedly ordered by SP-B below the gel to fluid phase transition temperature (Tc) while it was only slightly ordered above this temperature as indicated by surface-sensitive probes 6-NBD-PC and 6-NBD-PG. The effects of SP-B on fluorescence anisotropy were concentration dependent, reaching maximal activity at 1-2% protein to phospholipid by weight. Anisotropy measurements of interior-selective fluorescent probes (cis-parinaric acid and DPH) imply that addition of SP-B into the phospholipid shifted the Tc of the model membrane but did not alter lipid order at the membrane interior. Since fluorescence anisotropy studies with trans-parinaric acid, an interior-sensitive probe with high affinity for gel-phase lipids, did not detect any changes in lipid packing or Tc, it is likely that SP-B resides primarily in fluid-phase domains. Fluorescence lifetime measurements indicated that two conformers of the NBD-PC probe exist in this DPPC/DPPG model membrane system. Fluorescence intensity measurements generated with NBD-PC and NBD-PG, in conjunction with information from lifetime measurements, support the concept that SP-B increases the distribution of the short-lifetime conformer in the gel phase. In addition, the anisotropy and intensity profiles of NBD-PG in the model membrane indicate that bovine SP-B interacts selectively with phosphatidylglycerol.     

A novel fluorescent coronenyl-phospholipid analogue for investigations of submicrosecond lipid fluctuations

A fluorescent phospholipid derivative, the 2'-(4-coronenylbutyric) ester of lyso-egg phosphatidylcholine, has been synthesized for use in studies of submicrosecond lipid dynamics. Synthesis of the phospholipid derivative involves Friedel-Crafts acylation of free coronene, followed by a Huang-Minlon reduction to yield the fatty-acyl derivative, 4-coronenylbutyric acid. Esterification of the carboxylic acid with lyso-phosphatidylcholine is achieved through a mixed anhydride intermediate. The resultant coronenyl-phospholipid adduct (Cor-PC) has been incorporated into sonicated unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) for dynamic lipid studies. Fluorescence quenching studies using potassium iodide, together with steady-state emission anisotropy (EA) measurements, confirm that the coronene moiety of the phospholipid adduct resides towards the head group interfacial region of the lipid bilayer. Unique properties of this new fluorescent phospholipid adduct are its long mean fluorescence lifetime (tau av approximately 112 ns at 14 degrees C), the planar symmetry of the fluorophore and its defined bilayer location. As a consequence, depolarizing motions of the coronene moiety target submicrosecond 'gel-fluid' lipid dynamics arising from a relatively narrow bilayer distribution. Our data suggest that the sensitivity of this new long-lived fluorescent phospholipid analogue to localized transverse submicrosecond lipid dynamics can provide important biological insights into varied processes including lipid-peptide interactions, bilayer fluidity gradients and passive ion transport.     

Membrane structural dynamics of plasma membranes of living human prostatic carcinoma cells differing in metastatic potential

Rigidity of the outer hemileaflet of the plasma membrane of two prostatic carcinoma cell lines with different metastatic potential, 1-LN and 1-LN-EMS-10, was assessed by steady-state anisotropy, using a battery of fluorescent probes. The "bulk" membrane rigidity sensed by diphenylhexatriene, trimethylammonio-DPH, 1-palmitoyl-2-[DPH-ethylcarbonyl]-phosphatidylcholine, and 10-pyrenedecanoic acid indicated slightly higher rigidity in the membrane of the highly metastatic line (1-LN). This was accompanied by 26% greater mole fraction of cholesterol and 9% lower phospholipid, resulting in 40% greater cholesterol/phospholipid ratio. Phosphatidylethanolamine was increased 12%, but corresponding decreases in phosphatidylserine and phosphatidylinositol resulted in no significant change in molar ratio of choline/noncholine phospholipids. Whereas unsaturation index was slightly higher in 1-LN, fatty acids of 1-LN plasma membranes contained 15% more 18:1, 43% more 20:4, 26% more 22:4, and 38% less 18:2. Anisotropy gradients were determined for the two cell lines using a series of n-(9-anthroyloxy) fatty acid probes with n = 2, 3, 6, 7, 9, 12, and 16. Gradients differed only in position of anisotropy maxima, which occurred with n = 6, in 1-LN, and n = 7, in 1-LN-EMS-10. Possible relationships between observed anisotropy gradients and differences in membrane cholesterol and fatty acid composition are discussed.     

Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use of paramagnetic quenching

Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short fange interaction we developed a spectroscopic method to characterize the molecular arrangement within the lipid membrane. Applied to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase.This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.     

Influence of the calcium-induced gel phase on the behavior of small molecules in phosphatidylserine and phosphatidylserine-phosphatidylcholine multilamellar vesicles

The behavior of fluorescent and spin-label probes is examined in several fluid and gel phospholipid phases, with particular focus on the Ca2+-induced gel phase in phosphatidylserine (PS). These probes have behavior characteristic of the type of probe and of the type of lipid environment. Anthroyloxy- and doxyl-labeled PS [12-AS-PS and (7,6)PS, respectively] exhibit greatly restricted and/or slow probe motion in Ca(PS)2, even compared to thermotropic gel-phase lipid at the same temperature. In contrast, anthroyloxy- and doxyl-labeled phosphatidylcholine (PC), as well as fluorescent-labeled and spin-labeled fatty acid derivatives, show no apparent change in probe motion in Ca(PS)2 compared to fluid lamellar lipid. Doxyl-labeled phosphatidic acid, phosphatidylethanolamine, and phosphatidylglycerol show restricted motion in Ca(PS)2 relative to fluid-phase lipid, but the electron paramagnetic resonance (EPR) spectra could not be interpreted in terms of simple models for probe ordering. The fluorescent probes diphenylhexatriene (DPH) and trans-parinaric acid methyl ester (tPNA-Me) show motional behavior in Ca(PS)2 that is intermediate between that observed in fluid and in thermotropic gel-phase lipid. When Ca(PS)2 and fluid PS/PC phases coexist, probe molecules distribute between the two phases. Experiments using fluorescence quenching by spin-labeled PC in PS/PC in excess Ca2+ yield the distribution of several fluorophore probes between fluid liquid-crystal and Ca(PS)2 gel phases, expressed as a concentration ratio, RLC/G. The value of RLC/G = 100 in favor of the fluid phase is obtained for 12-AS-PC, 18 for 12-AS-Me, 12 for DPH, 3 for tPnA-Me, and 1 for 12-AS-PS.(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.     

Does diabetes mellitus affect diphenylhexatriene penetration into erythrocyte membrane ghosts?

Diphenylhexatriene transverse distribution has been studied in normal and diabetic erythrocyte membrane ghosts using fluorescence polarization and fluorescence quenching methods. Acrylamide quenched the fluorescence of diphenylhexatriene according to a dynamic mechanism in agreement with Stern-Volmer equation. Nonlinear least-squares analysis based on quenching results has shown greater accessibility of fluorophore to quencher molecules in diabetic ghosts (37.2 +/- 3.2% in normal vs. 67.5 +/- 6.4% in diabetic membranes). Steady-state fluorescence anisotropy measurements evidenced the lowered membrane lipid fluidity in diabetics (anisotropy values: 0.166 +/- 0.011 in normal subjects vs. 0.193 +/- 0.018 in diabetics). A model mechanism is proposed which attributes the lowered capacity of lipid bilayer in diabetes to the increased ordering and more compact structure of membrane phospholipids. The implications of the results for the resolving of steady-state anisotropy data are discussed.