Fluorescence depolarization study of lamellar liquid crystalline to inverted cylindrical micellar phase transition of phosphatidylethanolamine.

The orientational order and rotational dynamics of 2-[3-(diphenyl-hexatrienyl) propanoyl]-3-palmitoyl-L-alpha- phosphatidylcholine (DPH-PC) embedded in dioleoylphosphatidyl-ethanolamine (DOPE) were studied by fluorescence depolarization technique. Upon increasing the temperature, the calculated wobbling diffusion constant D perpendicular of the fluorescent probe was found to decrease at the lamellar (L alpha) to inverted cylindrical (H II) phase transition (10 degrees C). This suggested that the increased gauche rotamers of the alkene chains in the HII phase imposes a constraint in the wobbling motion of the fluorophore. The calculated ratio of order parameter in the L alpha phase to that in the HII phase was 1.7 and different from the theoretical value of 2.0 as predicted from the change in packing symmetry. This result can be explained by a slightly higher local order parameter of the fluorophore or by the fast rotational diffusion motion of the fluorophore around the symmetry axis of the cylindrical tubes in the HII phase.     

A new analysis method for the membrane viscosity from steady-state fluorescence depolarization

The absolute value of the viscosity in membrane lipid bilayers, which is different from the microviscosity advocated by Shinitzky, could be calculated from steady-state fluorescence depolarization of a hydrocarbon fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). This method was based on the theory of time-resolved fluorescence anisotropy and empirical relationships between fluorescence life time and the anisotropy parameters such as half cone angle in wobbling motion and wobbling diffusion rate of the fluorescent probe. Obtained viscosity values of various membranes from this method were consistent with those from time resolved method within experimental error.     

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. II. An experimental study.

The polymorphic phase behavior of unsaturated phosphatidylethanolamine (PE)/diacylglycerol (DG) binary lipid mixtures was investigated by the use of time-resolved fluorescence anisotropy. Using a fluorescent lipid, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenylethyl] carbonyl]3-sn-phosphatidyl-choline (DPH-PC), the orientational order and rotational dynamics of the above lipid mixtures in the liquid crystalline lamellar (L alpha) and inverted hexagonal (HII) phases were studied. By employing a one-exponential model (Cheng, K.H. 1989: Biophys. J. 55:1025-1031) to fit the anisotropy decay data, abrupt decreases in the normalized initial anisotropy decay slope and the residual anisotropy of DPH-PC were observed at approximately 6-8% DG, signifying a L alpha/HII phase transition. Using our new theoretical WOBHOP and P2P4HOP models as described in a preceding paper (Van Der Meer, B.W., K.H. Cheng, and S.Y. Chen. 1990. Biophys. J. 58:000-000), two or more rotational correlation times were required to describe the anisotropy decay behavior of DPH-PC in the HII phase. These rotation correlation times were further related to the second and fourth rank order parameters, and the wobbling and hopping diffusion constants of the fluorescent probe in the highly curved lipid cylindrical tubes of the HII phase. The hopping diffusion constant (DH) equals the lateral diffusion constant (DL) divided by R2 (R = radius of the lipid cylindrical tubes). The value of DL was estimated by measuring the excimer formation rate of 1-palmitoyl-2-[10-(1-pyrenl)decanoyl] phosphatidyl choline (py-PC) in the same PE/DG mixtures. Upon comparing the values of DH and DL, the value of R was determined to be approximately 10-15 A, and agreed with that derived from x-ray diffraction (Tate, M.W., and S.M. Gruner, 1989, Biochemistry. 28:4245-4253; Rand, R.P., N.L. Fuller, S.M. Gruner, and V.A. Parsegian. 1990. Biochemistry. 29:76-87).     

Dynamic motions of 1,6-diphenyl-1,3,5-hexatriene in interdigitated C(18):C(10)phosphatidylcholine bilayers.

This study investigates the dynamic behavior of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C(18):C(10)phosphatidylcholine [C(18):C(10)PC] bilayers. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form mixed-interdigitated structures below the transition temperature and form partially interdigitated bilayers above the transition temperature. The rotation of DPH in C(18):C(10)PC has been described in terms of the thermal coefficient of rotation using the modified Y-plot method which takes into account the limiting anisotropy value. During the phase transition of C(18):C(10)PC, DPH exhibits a thermal coefficient b2M = 0.41 - 0.51 degrees C-1 which is similar to the b2M values obtained with noninterdigitated phosphatidylcholine bilayers. Differential polarized phase-modulation fluorometry has also been employed to study the dynamic behavior of DPH in C(18):C(10)PC in real time. The data show that DPH contains considerable motion in the highly ordered mixed interdigitated bilayers. The DPH motion steadily increases with an increase in temperature as shown by the rotational correlation time, and the wobbling diffusion constant. However, the limiting anisotropy, the order parameter, and the width of the lifetime distribution undergo an abrupt decrease, and a corresponding abrupt increase in the cone angle, at approximately 16 degrees C. This temperature range is near the onset temperature of the phase transition as determined by differential scanning calorimetry. The rotational parameters show strong hysteresis on heating and cooling. All the rotational parameters derived from DPH fluorescence in mixed interdigitated C(18):C(10)PC exhibit magnitudes similar to those obtained from noninterdigitated gel phases of symmetric diacylphosphatidylcholines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Order and dynamics in the lamellar L alpha and in the hexagonal HII phase. Dioleoylphosphatidylethanolamine studied with angle-resolved fluorescence depolarization.

Fluorescence depolarization techniques are used to determine the molecular order and reorientational dynamics of the probe molecule TMA-DPH embedded in the lamellar L alpha and the hexagonal HII phases of lipid/water mixtures. The thermotropically induced L alpha----HII phase transition of the lipid DOPE is used to obtain macroscopically aligned samples in the hexagonal HII phase at 45 degrees C from samples prepared in the lamellar L alpha phase at 7 degrees C. The interpretation of angle-resolved fluorescence depolarization experiments on these phases, within the framework of the rotational diffusion model, yields the order parameters (P2) and (P4), and the diffusion constants for the reorientational motions. The reorientational motion rates of the TMA-DPH molecules in the hexagonal HII phase are comparable with those in the lamellar L alpha phase. Furthermore, the lateral diffusion of the probe molecule on the surface of the lipid/water cylinder in the hexagonal phase is found to be considerably slower than the reorientational motion.     

The effect of cytochrome oxidase on lipid chain dynamics A nanosecond fluorescence depolarization study

Molecular motions in membranes composed of purified cytochrome oxidase (EC 1.9.3.1) and synthetic lipid (l-α-dimyristoylphosphatidylcholine or l-α-dioleoylphosphatidylcholine) at various ratios were investigated with a lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Nanosecond fluorescence depolarization kinetics of the probe showed that the rod-shaped probe molecules perform a fast wobbling motion (restricted rotation) in all membranes studied, presumably reflecting the motion of lipid acyl chains. At temperatures where the pure lipid was in the liquid-crystalline phase, presence of cytochrome oxidase reduced the angular range of the wobbling motion, whereas its rate; the wobbling diffusion constant, was unaffected. On the other hand, incorporation of the protein into lipid in the gel phase resulted in the increase in the wobbling diffusion constant while the range of the wobbling motion remained the same. A time-dependent view of lipid dynamics that accounts for the above findings, as well as the results of recent electron spin resonance and nuclear spin resonance studies of protein-lipid interactions, is proposed.     

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

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

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.     

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.     

Intramolecular excimer kinetics of fluorescent dipyrenyl lipids: 2. DOPE/DOPC membranes.

The intramolecular dynamics of the excimer-forming dipyrenyl lipids (DipynPE) of different chain lengths (n) in fully hydrated dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) binary mixtures was investigated by the use of frequency-domain fluorescence intensity dcay technique. Using a 3-state model (see companion paper), the extent of aggregation and rotational rate of the two covalently attached pyrene moieties in DipynPE were estimated from the frequency-domain data. At 1 degrees C, the rotational rate and aggregation for Dipy4PE and Dipy10PE were insensitive to DOPE% of the lipid bilayer. At 27 degrees C, the rotational rate decreased, whereas the aggregation increased steadily for Dipy10PE as the DOPE% of the bilayer increased from 0 to 80. However, an abrupt increase in the rotational rate and a decrease in the aggregation for Dipy10PE were detected as the DOPE% reached 100, at which point the membranes are in the inverted hexagonal (HII) phase. No similar changes were found for Dipy4PE. These results indicate that the presence of PE with large intrinsic-curvature increases the lateral stress at the region near the center of the bilayer, and that this stress can be relieved as the membranes enter the highly curved HII phase.     

Headgroup hydration and motional order of lipids in lamellar liquid crystalline and inverted hexagonal phases of unsaturated phosphatidylethanolamine--a time-resolved fluorescence study

By the use of frequency domain cross-correlation fluorometry, the fluorescence lifetime of the water soluble probe 8,1-anilinonapthalene sulfonic acid (ANS) in aqueous dispersions of dioleoylphosphatidylethanolamine (DOPE) and phosphatidylethanolamine transphosphatidylated from egg phosphatidylcholine (TPE) was measured. The orientational order parameter and rotational diffusion constant of the lipophilic probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) were also determined in TPE dispersions. In agreement with a previous study on DOPE (Cheng (1989) Biophys. J. 55, 1025-1031), abrupt changes in both the order packing and rotational diffusion constant were found at the lamellar liquid crystalline (L alpha) to inverted hexagonal (HII) phase transition of TPE. Owing to the subnanosecond resolution capability of this frequency domain fluorometric technique, the heterogeneous fluorescence decay of ANS was resolved into three distinct components with different decay lifetimes (tau's). They were 0 less than tau less than 0.5 ns, 2 less than tau less than 9 ns and tau greater than 15 ns. These lifetime regions were attributed to the partitioning of ANS into the bulk aqueous medium, the lipid/water interface and the lipid hydrocarbon region, respectively. These classifications of lifetime regions were further supported by the sensitivity of those lifetime components with the solvent isotopic shift of D2O. Similar to the changes of orientational order and rotational diffusion of lipophilic probe, the lifetime and intensity fraction of ANS associated with the lipid/water interfacial region declined abruptly at the L alpha-HII transition of both DOPE and TPE. This observation suggested that a dehydration of the lipid headgroup surface occurs at the L alpha-HII transition. This study provided evidence that both the lipid headgroup surface hydration and the lipid dynamics change drastically as a result of the macroscopic rearrangement of lipids at the L alpha-HII transition.     

Calcium-induced phase separation phenomena in multicomponent unsaturated lipid mixtures

The ability of calcium to induce phase separation in multicomponent lipid mixtures containing various unsaturated species of acidic and neutral phospholipids has been investigated by 31P NMR, 3H NMR, and small-angle X-ray diffraction techniques. It is shown that, in unsaturated (dioleoyl-) phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) (1:1) and phosphatidic acid (PA)/phosphatidylcholine (PC) (1:1) mixtures, calcium is unable to induce lateral phase separation of the acidic and neutral lipids and that all the lipids adopt a hexagonal (HII) phase in the presence of calcium. In multicomponent mixtures containing one or more acidic species the presence of cholesterol either facilitates calcium-induced lamellar to hexagonal (HII) transitions for all the lipid components or, in systems already in a hexagonal (HII) phase, mitigates against calcium-induced lateral phase separations. Further, cholesterol is shown to exhibit no preferential interaction on the NMR time scale with either PC, PE, or phosphatidylserine (PS) when the lipids are in the liquid-crystal state. The ability of cholesterol to directly induce HII phase formation in PC/PE mixtures is also shown to be common to various other sterols including ergosterol, stigmasterol, coprostanol, epicoprostanol, and androstanol.     

Dynamic structure of lipid bilayers studied by nanosecond fluorescence techniques

Molecular motions in liposomes of dipalmitoyl-phosphatidylcholine (DPPC) were studied by nanosecond fluorescence techniques. As a fluorescent probe for the hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used. Time courses of fluorescence intensity IT(t) and emission anisotropy r(t) of DPH embedded in DPPC liposomes were measured at various temperatures. The value of the fluorescence lifetime tau obtained froma single exponential decay of IT(t) was somewhat higher than that in liquid paraffin below the transition temperature Tt and decreased above Tt. Higher values of tau below Tt indicate the almost complete hydrophobic environment. The decay curves of r(t) were separated into two phases: an initial fast decreasing phase of the order of one nanosecond and a second almost constant phase. This indicates that the orientational motion of DPH in the hydrocarbon region is described by a wobbling diffusion restricted by a certain anisotropic potential. The results were analyzed on the model that the wobbling diffusion is confined in a cone with a uniform diffusion constant. Though temperature dependence of the cone angle was sigmoidal, that of the wobbling diffusion constant was like the exponential function. The change in the cone angle at Tt was sharper than that in the wobbling diffusion constant at Tt. Estimated values of the viscosity in the cone were an order of magnitude smaller than the values of "microviscosity" which were estimated from the steady-state emission anisotropy without considering the restrictions on the rotational motion.     

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. I. Theory.

It is shown that fluorescence anisotropy from lipidlike probes in the hexagonal HII phase gives information of (a) orientational order parameters, (b) the wobbling diffusion constant, and (c) the hopping diffusion constant of the probe, DH, equals DL/R2, the lateral diffusion constant over the square of the radius of the hexagonal tubes. Here we consider only lipidlike probes having the absorption transition movement and/or the emission transition moment along the long axis of the molecule. Three models are introduced for analysis of time-resolved data: the "WOBHOP," the "reduced WOBHOP," and the "P2P4HOP" model. The fluorescence anisotropy in response to a very short excitation pulse in each of the three models is a constant plus a number of exponentials. The WOBHOP and reduced WOBHOP models have 3 and 2 exponentials, respectively, and both contain four fitting parameters: r0 (the fundamental anisotropy), (P2) (the second rank orientational order parameter), DW (the wobbling diffusion constant), and DH (the hopping diffusion constant). The P2P4HOP model has eight exponentials and five fitting parameters: the four parameters listed above and (P4) (the fourth rank orientational order parameter). Analysis of fluorescence anisotropy data in the hexagonal HII phase using one of these models allows for obtaining the hopping diffusion constant, and, if the lateral diffusion constant is known, the radius of the hexagonal tubes. Substitution of DH = 0 in each of the three models yields an expression for the fluorescence anisotropy that is used in the literature for lamellar (L alpha or L beta) phases. The fluorescence anisotropy in coexisting L alpha/HII phases is discussed.     

Dynamics of membrane structure of frog erythrocyte ghosts measured with a nanosecond fluorometer

The dynamics of membrane microstructure was studied as molecular motions of phospholipids for bullfrog erythrocyte ghosts by the DPH fluorescence depolarization technique with a nanosecond fluorometer. The bullfrog erythrocyte ghosts were obtained by hypotonic lysis and collagenase treatment. The constituents of membrane proteins were confirmed by the disk gel electrophoresis. The viscosity of erythrocyte membrane ghosts was estimated to be 3.3 +/- 1.0 at 10 degrees C, and 2.1 +/- 0.1 at 20 degrees C and 1.3 +/- 0.2 at 30 degrees C in the unit of poise and the wobbling angle of lipid molecule was 35 +/- 1, 41 +/- 1 and 43 +/- 1 degree at the respective temperatures on an average and +/- S.D. The viscosity is lower than that of human erythrocytes. The relatively low viscous phospholipid bilayer may be one of the factors for the deformability of bullfrog erythrocytes.     

Modulation of dolichyl-phosphomannose synthase activity by changes in the lipid environment of the enzyme

Rat liver dolichyl-phosphomannose synthase (GDP mannose-dolicholphosphate mannosyltransferase; EC 2.4.1.83) was previously shown to catalyze optimal rates of mannosyl transfer to dolichyl-P when the polyprenol acceptor was incorporated into a phosphatidylethanolamine (PE) matrix that has a tendency to adopt a nonbilayer (hexagonal HII) phase [Jensen, J. W., & Schutzbach, J. S. (1985) Eur. J. Biochem. 153, 41-48]. The present investigations now further define the properties of the lipid environment that are essential for mannosyltransferase activity. Monogalactosyl diglyceride (MGDG), a glycoglycerolipid that prefers a nonbilayer-phase organization in isolation, was shown to provide a suitable lipid matrix for synthase activity. By comparison, the enzyme was not activated by digalactosyl diglyceride (DGDG), which forms stable bilayer structures upon hydration. Enzyme activity in MGDG/DGDG mixtures decreased as the proportion of DGDG in the dispersion was increased. Although bilayer-forming phospholipids supported low rates of mannosyl transfer, enzyme activity was stimulated by the addition of MGDG to either phosphatidylcholine (PC) or PE/PC (1:1) membranes. The incorporation of agents known to destabilize bilayer structures including dolichols, ubiquinone, dodecane, and cholesterol into PE/PC (1:1) membranes also increased the rate of mannosyl transfer. Enzyme activity in PC membranes was stimulated by the presence of gramicidin and also by greatly increased concentrations of the substrate, dolichyl-P. The results demonstrate that the enzyme does not have a requirement for PE and suggest that the physical state of the lipid matrix is an important determinant for reconstitution of the synthase and polyprenol phosphate substrate in a productive complex. The formation of an enzyme/lipid complex was demonstrated by sucrose density gradient centrifugation and could be correlated with the lipid requirements for enzyme activity.     

Viscosity and order in erythrocyte membranes studied with nanosecond fluorometry

The viscosity and the order in the interior of human erythrocyte membranes were investigated by the fluorescence depolarization technique in the nanosecond region with 1,6-diphenyl-1,3,5-hexatriene (DPH). After pulsed excitation with a polarized light, the fluorescence anisotropy ratio of DPH in membranes rapidly decreased and gave a final value (r infinity). The rate of initial decrease and the value of r infinity related to the viscosity in the interior of the membranes and a wobbling angle of DPH which reflects a size of range for the phospholipid motion relating to the order of membrane structure. For normal human erythrocyte membranes the viscosity and the wobbling angle were obtained to be 0.82 poise and 42 degrees, at 37 degrees C. Similar values were obtained for spectrin-free membranes. Hardened membranes by the cross-linking of the cytoskeletal proteins with glutaraldehyde showed a small wobbling angle of 37 degrees, but the viscosity of them was unchanged.     

Quantitation of lateral stress in lipid layer containing nonbilayer phase preferring lipids by frequency-domain fluorescence spectroscopy.

Frequency-resolved fluorescence measurements have been performed to quantitate the lateral stress of the lipid layer containing nonbilayer phase preferring dioleoylphosphatidylethanolamine (DOPE). On the basis of a new rotational diffusion model, the wobbling diffusion constant (Dw), the curvature-related hopping diffusion constant (DH), and the two local orientational order parameters ([P2] and [P4]) of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] carbonyl]-3-sn-phosphatidylcholine (DPH-PC) in fully hydrated DOPE and DOPE/dioleoylphosphatidylcholine (DOPC) mixtures were calculated from the frequency-domain anisotropy data. The values of [P2], [P4], and DH for DOPE were found to increase significantly at approximately 12 degrees C, the known lamellar liquid crystalline (L alpha) to inverted hexagonal (HII) phase transition temperature of DOPE. Similar features as well as a decline of Dw were detected in the DOPE/DOPC mixtures as the DOPE content was increased from 85% to 90% at 23 degrees C, corresponding to the known lyotropic phase transition of the DOPE/DOPC. In contrast, for DOPC (0-40 degrees C) and DOPE/DOPC (0-100% DOPE at 3 degrees C), which remained in the L alpha phase, these changes were not detected. The most probable local orientation of DPH-PC in the DOPE/DOPC mixtures shifted progressively toward the normal of the lipid/water interface as the content of DOPE increased. We concluded that the curvature-related lateral stress in the lipid layer increases with the content of the nonbilayer phase preferring lipids.     

Gangliosides asymmetrically alter the membrane order in cultured PC-12 cells

Exogenous gangliosides readily associate with the cell membranes and produce marked effects on cell growth and differentiation. We have studied the effect of bovine brain gangliosides (BBG) on the membrane dynamics of intact cells. The structural and dynamic changes in the cell membrane were monitored by the fluorescence probes DPH, TMA-DPH and laurdan. Incorporation of BBG into the cell membrane decreased the fluorescence intensity, lifetime and the steady state anisotropy of TMA-DPH. Analysis of the time resolved anisotropy decay by wobbling in the cone model revealed that BBG decreased the order parameter, and increased the cone angle without altering the rotational relaxation rate. The fluorescence intensity and lifetime of DPH were unaffected by BBG incorporation, however, a modest increase was observed in the steady state anisotropy. BBG incorporation reduced the total fluorescence intensity of laurdan with pronounced quenching of the 440-nm band. The wavelength sensitivity of generalized polarization of laurdan manifested an ordered liquid crystalline environment of the probe in the cell membrane. BBG incorporation reduced the GP values and augmented the liquid crystalline behavior of the cell membrane. BBG incorporation also influenced the permeability of cell membranes to cations. An influx of Na+ and Ca2+ and an efflux of K+ was observed. The data demonstrate that incorporation of gangliosides into the cell membrane substantially enhances the disorder and hydration of the lipid bilayer region near the exoplasmic surface. The inner core region near the center of the bilayer becomes slightly more ordered and remains highly hydrophobic. Such changes in the structure and dynamics of the membrane could play an important role in modulation of transmembrane signaling events by the gangliosides.     

Limited rotational diffusion of DPH in human erythrocyte membranes

The rotational diffusion of diphenylhexatriene (DPH) determines its fluorescence depolarization. Time-resolved polarization measurements were used to calculate the coefficient of diffusion of this probe in human crythrocyte ghost membranes on the basis of a diffusion theory of limited rotation. The diffusion coefficient is 5.9 × 10⁷ sec^?1 at 37°C; this was compared with the diffusion coefficient of DPH in liquid paraffin for an estimation of the microviscosity of the membrane bilayer.