Characteristics of self-quenching of the fluorescence of lipid-conjugated rhodamine in membranes

Self- or concentration quenching of octadecylrhodamine B (C18-Rh) fluorescence increases linearly in egg phosphatidylcholine (PC) vesicles but exponentially in vesicles composed of egg PC:cholesterol, 1:1, as the probe concentration is raised to 10 mol%. Cholesterol-dependent enhancement of self-quenching also occurs when N-(lissamine-rhodamine-B-sulfonyl)dioleoylphosphatidylethanolamine is substituted for C18-Rh and resembles that in dipalmitoylphosphatidylcholine vesicles below, as opposed to above, the phase transition. These effects are not due to changes in dimer:monomer absorbance. Stern-Volmer plots indicate a dependence of quenching on nonfluorescent dimers both in the presence and absence of cholesterol. Decreases in fluorescence lifetimes with increasing probe concentration parallel decreases in residual fluorescence of C18-Rh with increasing probe concentration in PC and PC + cholesterol membranes, respectively. Decreases in the steady-state polarization of C18-Rh fluorescence as its concentration is raised to 10 mol% indicate energy transfer with emission between probe molecules in PC and to a lesser extent in PC + cholesterol membranes. The calculated R0 for 50% efficiency of energy transfer from excited state probe to monomer was 55-58 A and to dimer was 27 A. Since lateral diffusion of C18-Rh is probably too slow to permit collisional quenching during the lifetime of the probe, even if C18-Rh were concentrated in a separate phase, C18-Rh self-quenching appears to be due mainly to energy transfer without emission to nonfluorescent dimers.     

Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression

Stabilized plasmid lipid particles (SPLP) consist of a single copy of DNA surrounded by a lipid bilayer. The particles are small ( approximately 100 nm), stable, monodisperse and have a low surface charge. A diffusible polyethylene glycol (PEG) coating attached to a lipid anchor is critical to the SPLP's functionality. The PEG-lipid exchanges out of the bilayer at a rate determined by the size of the lipid anchor. Here we show that SPLP can be prepared using a series of PEG-diacylglycerol lipids (PEG-S-DAGs). SPLP were prepared incorporating PEG-dimyristoylglycerol (C14), PEG-dipalmitoylglycerol (C16) or PEG-distearoylglycerol (C18) and the rate of PEG-lipid diffusion from the bi-layer determined using a FRET assay. SPLP pharmacokinetics confirm a correlation between the stability of the PEG-lipid component and circulation lifetime. PEG-S-DAGs with longer lipid anchors yield more stable SPLP particles with longer circulation half-lives yielding an increase in tumor delivery and gene expression. PEG-distearoylglycerol (C18) containing SPLP bypass so-called 'first pass' organs, including the lung, and elicit levels of gene expression in distal tumor tissue 100- to 1000-fold greater than that observed in any other tissue. The incorporation of PEG-S-DAG in SPLP confirms that small size, low surface charge and extended circulation lifetimes are prerequisite to the accumulation and tumor selective expression of plasmid DNA following systemic administration.     

Long Open Amphotericin Channels Revealed in Cholesterol-Containing Phospholipid Membranes Are Blocked by Thiazole Derivative

The action of antifungal drug, amphotericin B (AmB), on solvent-containing planar lipid bilayers made of sterols (cholesterol, ergosterol) and synthetic C14–C18 tail phospholipids (PCs) or egg PC has been investigated in a voltage-clamp mode. Within the range of PCs tested, a similar increase was achieved in the lifetime of one-sided AmB channels in cholesterol- and ergosterol-containing membranes with the C16 tail PC, DPhPC at sterol/DPhPC molar ratio ≤1. The AmB channel lifetimes decreased only at sterol/DPhPC molar ratio >1 that occurred with sterol/PC molar ratio of target cell membranes at a pathological state. These data obtained on bilayer membranes two times thicker than one-sided AmB channel length are consistent with the accepted AmB pore-forming mechanism, which is associated with membrane thinning around AmB–sterol complex in the lipid rafts. Our results show that AmB can create cytotoxic (long open) channels in cholesterol membrane with C14–C16 tail PCs and nontoxic (short open) channels with C17–C18 tail PCs as the lifetime of one-sided AmB channel depends on ~2–5 Å difference in the thickness of sterol-containing C16 and C18 tail PC membranes. The reduction in toxic AmB channels efficacy can be required at the drug administration because C16 tails in native membrane PCs occur almost as often as C18 tails. The comparative analysis of AmB channel blocking by tetraethylammonium chloride, tetramethylammonium chloride and thiazole derivative of vitamin B₁, 3-decyloxycarbonylmethyl-4-methyl-5-(2-hydroxyethyl) thiazole chloride (DMHT), has proved that DMHT is a comparable substitute for both tetraalkylammonia that exhibits a much higher affinity.     

Polarity of lipid bilayers. A fluorescence investigation.

Through steady-state and time-resolved fluorescence experiments, the polarity of the bilayers of egg phosphatidylcholine vesicles was studied by means of the solvatochromic 2-anthroyl fluorophore which we have recently introduced for investigating the environmental micropolarity of membranes and which was incorporated synthetically in phosphatidylcholine molecules (anthroyl-PC) in the form of 8-(2-anthroyl)octanoic acid. Fluorescence quenching experiments carried out with N,N-dimethylaniline and 12-doxylstearic acid as quenchers showed that the 2-anthroyl chromophore was located in depth in the hydrophobic region of the lipid bilayer corresponding to the C9-C16 segment of the acyl chains. Steady-state fluorescence spectroscopy revealed a nonstructured and red-shifted (lambda em(max) = 464 nm) spectrum for the probe in egg-PC bilayers, which greatly differed from the structured and blue (lambda em(max) = 404 nm) spectrum the fluorophore was shown to display in n-hexane. While the fluorescence decays of the fluorophore in organic solvents were monoexponential, three exponentials were required to account for the fluorescence decays of anthroyl-PC in egg-PC vesicles, with average characteristic times of 1.5 ns, 5.5 ns, and 20 ns. These lifetime values were independent of the emission wavelength used. Addition of cholesterol to the lipid did not alter these tau values. One just observed an increase in the fractional population of the 1.5-ns short-living species detrimental to the population of the 20-ns long-living ones. These observations enabled time-resolved fluorescence spectroscopy measurements to be achieved in the case of the 1/1 (mol/mol) egg-PC/cholesterol mixture. Three distinct decay associated spectra (DAS) were recorded, with maximum emission wavelengths, respectively, of 410 nm, 440 nm, and 477 nm for the 1.5-ns, 6-ns, and 20-ns lifetimes found in this system. On account of the properties and the polarity scale previously established for the 2-anthroyl chromophore in organic solvents, these data strongly suggest the occurrence of three distinct excited states for anthroyl-PC in egg-PC bilayers, corresponding to three environments for the 2-anthroyl chromophore, differing in polarity. The lifetime of 1.5 ns and the corresponding structured and blue (lambda em(max) = 410 nm) DAS account for a hydrophobic environment, with an apparent dielectric constant of 2, which is that expected for the hydrophobic core of the lipid bilayer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of cholesterol with the membrane lipid matrix. A solid state NMR approach.

The effects of cholesterol on the structure and dynamics of dimyristoylphosphatidylcholine (DMPC) model membranes have been monitored as functions of temperature and cholesterol concentration in the membrane. The use of deuterium labels both on the cholesterol fused ring system and on the lipid chains in conjunction with solid state deuterium nuclear magnetic resonance (2H-NMR) afforded to monitor the degree of ordering of both molecules in a mixed system. The degree of ordering of the lipid head group was followed by phosphorus-31 (31P)-NMR. New findings on the effect of cholesterol on DMPC may be summarized as follows: i) cholesterol disorders the lipid chains below temperature of the DMPC gel-to-fluid transition (Tc) and orders them above; the effect is linear with cholesterol concentration at 0 and 60 degrees C but for intermediate temperatures, a saturation effect is observed at 20-30 mol %; ii) the ordering-disordering effects are perceived similarly by all chain segments with, however, a greater sensitivity for positions near the bilayer center; iii) below Tc, the lipid head group is considerably disordered by increasing amounts of cholesterol but slightly affected above; iv) the degree of ordering of cholesterol is quasi temperature independent for fractions greater than or equal to 30%; v) the average orientation of the cholesterol rigid body is perpendicular to the bilayer surface and exhibits little variations with temperature and cholesterol concentration. Variations in membrane dynamics are interpreted in terms of cholesterol-induced changes in bilayer thickness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alamethicin channels incorporated into frog node of ranvier: calcium-induced inactivation and membrane surface charges

Alamethicin, a peptide antibiotic, partitions into artificial lipid bilayer membranes and into frog myelinated nerve membranes, inducing a voltage-dependent conductance. Discrete changes in conductance representing single-channel events with multiple open states can be detected in either frog node or lipid bilayer membranes. In 120 mM salt solution, the average conductance of a single channel is approximately 600 pS. The channel lifetimes are roughly two times longer in the node membrane than in a phosphatidylethanolamine bilayer at the same membrane potential. With 2 or 20 mM external Ca and internal CsCl, the alamethicin-induced conductance of frog nodal membrane inactivates. Inactivation is abolished by internal EGTA, suggesting that internal accumulation of calcium ions is responsible for the inactivation, through binding of Ca to negative internal surface charges. As a probe for both external and internal surface charges, alamethicin indicates a surface potential difference of approximately -20 to -30 mV, with the inner surface more negative. This surface charge asymmetry is opposite to the surface potential distribution near sodium channels.     

Cell membrane orientation visualized by polarized total internal reflection fluorescence.

In living cells, variations in membrane orientation occur both in easily imaged large-scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method is introduced here to visualize such regions. The method is based on fluorescence of an oriented membrane probe excited by a polarized evanescent field created by total internal reflection (TIR) illumination. The fluorescent carbocyanine dye diI-C(18)-(3) (diI) has previously been shown to embed in the lipid bilayer of cell membranes with its transition dipoles oriented nearly in the plane of the membrane. The membrane-embedded diI near the cell-substrate interface can be fluorescently excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane, and also gives information regarding the fraction of unoriented diI in the membrane. Both a theoretical background and experimental verification of the technique is presented for samples of 1) oriented diI in model lipid bilayer membranes, erythrocytes, and macrophages; and 2) randomly oriented fluorophores in rhodamine-labeled serum albumin adsorbed to glass, in rhodamine dextran solution, and in rhodamine dextran-loaded macrophages. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time-course maps of membrane orientations on diI-labeled macrophages from which low visibility membrane structures can be identified and quantified. To sharpen and contrast-enhance the TIR images, we deconvoluted them with an experimentally measured point spread function. Image deconvolution is especially effective and fast in our application because fluorescence in TIR emanates from a single focal plane.     

Lipid phase of transverse tubule membranes from skeletal muscle. An electron paramagnetic resonance study.

The lipid phase of transverse tubule membrane was probed with a variety of fatty acid spin labels. The motion of the probe increased as the distance between the spin label and polar head group increased, in agreement with results reported in other membranes. The value of the order parameter at 37 degrees C for a fatty acid spin label containing the label attached to its fifth carbon atom was closer to values reported for bacterial membranes than to the lower values reported for other mammalian membranes. Order parameters for spin labels containing the label nearer to the center of the bilayer were closer to the values reported in other mammalian membranes than to values reported for bacterial membranes. These results indicate that the lipid segments in the vicinity of the polar head group, and less so those near the center of the bilayer, are motionally more restricted in transverse tubules than in other mammalian membranes. In particular, the lipid phase of the transverse tubule membrane is less fluid than that of the sarcoplasmic reticulum membrane. A possible role of the high cholesterol content of transverse tubules in generating the lower fluidity of its lipid phase is discussed.     

Liquid-crystalline phases of cholesterol/lipid bilayers as revealed by the fluorescence of trans-parinaric acid.

The presence of two liquid-crystalline phases, alpha and beta, in mixed bilayers of dimyristoylphosphatidylcholine/cholesterol was detected by the changes in the distribution of the fluorescence lifetimes of t-PnA, as analyzed by the Maximum Entropy Method. The formation of the liquid-ordered beta-phase, in the 30-40 degrees C temperature range as a function of cholesterol concentration (0-40 mol%), could be related quantitatively to the relative amplitude of a long lifetime component of the probe (10-14 ns). Based on this evidence, the phase behavior of mixtures of the unsaturated lipid palmitoyloleoylphosphatidylcholine and cholesterol was determined using the same technique, for cholesterol concentrations in the 0-50 mol% range, between 10 and 40 degrees C. It was found that two liquid-crystalline phases are also formed in this system, with physical properties reminiscent of the alpha- and beta-phases formed with saturated lipids. However, in this case it was determined that, for temperatures in the physiological range, the alpha- and beta-phases coexist up to 40 mol% cholesterol. This finding may be of significant biological relevance, because it supports the long held notion that cholesterol is responsible for the lipid packing heterogeneity of several natural membranes rich in unsaturated lipid components.     

Liquid-ordered microdomains in lipid rafts and plasma membrane of U-87 MG cells: a time-resolved fluorescence study

Lipid rafts, the functional microdomains in the cell membrane, are believed to exist as liquid-ordered (Lo) phase domains along with the liquid-disordered (Ld) phase of the bulk of the cell membranes. We have examined the lipid order in model and natural membranes by time-resolved fluorescence of trimethylammonium-1,6-diphenylhexatriene incorporated into the membranes. The lipid phases were discerned by the limiting anisotropy, rotational diffusion rate and distribution of the fluorescence lifetime. In dipalmitoylphosphatidylcholine (DPPC)-cholesterol mixtures the gel phase exhibited higher anisotropy and a two-fold slower rotational diffusion rate of the probe as compared to the Ld phase. On the other hand, the Lo phase exhibited higher limiting anisotropy but a rotational diffusion rate comparable to the Ld phase. The Ld and Lo phases elicited unimodal distribution of lifetimes with distinct mean values and their co-existence in phospholipid-cholesterol mixtures was reflected as a biphasic change in the width of the lifetime distribution. Global analysis of the lifetimes yielded a best fit with two lifetimes which were identical to those observed in single Lo or Ld phases, but their fractional contribution varied with cholesterol concentration. Attributing the shorter and longer lifetime components to the Ld and Lo phases, respectively, the extent of the Lo/Ld phase domains in the membranes was estimated by their fractional contribution to the fluorescence decay. In ternary mixtures of egg PC-gangliosides-cholesterol, the gangliosides induced heterogeneity in the membrane but the Ld phase prevailed. The Lo phase properties were observed only in the presence of cholesterol. Results obtained in the plasma membrane and detergent-resistant membrane fractions (DRMs) isolated from U-87 MG cells revealed that DRMs mainly possess the Lo phase; however, a substantially large proportion of plasma membrane also exists in the Lo phase. Our data show that, besides cholesterol, the membrane proteins play a significant role in the organization of lipid rafts and, furthermore, a considerable amount of heterogeneity is present among the lipid rafts.     

Cholesterol hydroxyl group is found to reside in the center of a polyunsaturated lipid membrane

Cholesterol and saturated lipid species preferentially partition into liquid ordered microdomains, such as lipid rafts, away from unsaturated lipid species for which the sterol has less affinity in the surrounding liquid-disordered membrane. To observe how cholesterol interacts with unsaturated phospholipids, we have determined, from one-dimensional neutron scattering length density profiles, the depth of cholesterol in phosphatidylcholine (PC) bilayers with varying amounts of acyl chain unsaturation. Through the use of [2,2,3,4,4,6-(2)H(6)]-labeled cholesterol, we show that in 1-palmitoyl-2-oleoylphosphatidylcholine (16:0-18:1 PC), 1,2-dioleoylphosphatidylcholine (18:1-18:1 PC), and 1-stearoyl-2-arachidonylphosphatidylcholine (18:0-20:4 PC) bilayers the center of mass of the deuterated sites is approximately 16 A from the bilayer center. This location places the hydroxyl group of the sterol moiety at the hydrophobic/hydrophilic bilayer interface, which is the generally accepted position. In dramatic contrast, for 20:4-20:4 PC membranes the hydroxyl group is found, unequivocally, sequestered in the bilayer center. We attribute the change in location to the high disorder of polyunsaturated fatty acids (PUFA) that is incompatible with close proximity to the steroid moiety in its usual "upright" orientation.     

Rimantadine effects on the elasticity of bilayer lipid membranes and on ion transport through gramicidin D channels.

The effect of the antiviral preparation rimantadine on lipid bilayer membranes (BLM) was studied by measuring the modulus of elasticity in the direction normal to the surface (E perpendicular) and by estimating the conductance lambda, the lifetime tau of single gramicidin D channels (GRD), and the coefficient of nonlinearity beta of current voltage characteristics (IVC) of GRD-modified BLM. Rimantadine induced a nonmonotonic change in E perpendicular of BLM prepared from a mixture of egg lecithin with cholesterol: at relatively low rimantadine concentrations (0-40 micrograms/ml) E perpendicular first increased, reached a maximum and started to decrease. The effectivity of rimantadine was dependent on the cholesterol concentration in the BLM. Changes in E perpendicular suggest an increased ordering of the lipid bilayer at low rimantadine concentrations and formation of clusters of the preparation at concentrations exceeding those necessary to obtain maximal values of E perpendicular for the given BLM lipid composition. Rimantadine concentrations lifetime by approximately 20 percent, affected the degree of IVC nonlinearity and superlinearity of GRD-modified membranes, which suggests some effect on the height of the barrier at the ionic channel mouth and in its centre.     

Local anesthetics can interact electrostatically with membrane proteins

The fluorescent probe 1-anilinonaphthalene 8-sulfonate was used to examine the binding of spin-labeled local anesthetics to lipid model systems, to the membranes of human red blood cells, and rabbit sarcoplasmic reticulum. 1-Anilinonaphthalene 8-sulfonate exhibits two distinct fluorescent lifetimes when bound to these biological membranes. The shorter lifetime represents the probe associated with the purely lipid region while the longer lifetime is associated with the protein region. The spin-labeled local anesthetic quenches the fluorescence of both of these components as indicated by the decrease in the lifetimes. Since nitroxide free radicals are known to quench fluorophores upon 'contract', the results reflect the relative interaction of local anesthetics with membrane lipids and proteins. The evidence is consistent with the concept of multiple binding sites for local anesthetics in membranes. Local anesthetics, once intercalated into the bilayer, may diffuse laterally and interact with membrane components, lipid as well as proteins. In biological membranes, however, positively charged local anesthetics are better able to quench 1-anilinonaphthalene 8-sulfonate in protein regions, suggesting that the interaction between local anesthetics and membrane proteins can be electrostatic in nature.     

Reduced lateral mobility of a fluorescent lipid probe in cholesterol-depleted erythrocyte membrane

The effect of cholesterol depletion of the human erythrocyte membrane on the lateral diffusion rate of a fluorescent lipid probe is reported. At low temperatures (?5 to 5°C), the diffusion of the probe is 50% slower in the cholesterol-depleted membrane than in non-depleted membrane. At high temperatures (30 to 40° C), probe mobility is not affected by cholesterol depletion. These results suggest that cholesterol suppresses aspects of phospholipid phase changes in animal cells in a manner consistent with its behavior in artificial bilayers and multilayers.Whole erythrocytes were depleted of 30–50% of their cholesterol by incubation with a sonicated dispersion of dipalmitoyl phosphatidylcholine. Cells were then labeled with 3,3′-dioctadecylindocarbocyanine (diI), a phospholipid-like fluorescent dye, and hemolyzed into spherical ghosts. The rate of lateral motion of diI was measured by observing the fluorescence recovery after local photobleaching with a focused laser spot.The diffusion rate of the lipid probe in both control and cholesterol-depleted erythrocyte membrane is substantially smaller than in any cell or model membrane previously measured.     

Modulation of pig kidney Na+/K+-ATPase activity by cholesterol: role of hydration

Cholesterol is known to affect the activity of membrane-bound enzymes, including Na(+)/K(+)-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na(+)/K(+)-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at the native cholesterol concentration, whereas the other derivatives showed a maximum value for the lifetime center at that cholesterol concentration. DPH-PC is known to sense the protein-lipid interface, whereas the other derivatives sense the bulk lipid phase. These data suggest that hydration at the protein-lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. Hydration at the protein-lipid interface is therefore proposed to be required for activity. These results are in agreement with current models of membrane dynamics and thermodynamics of protein function.     

Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy of fluorescent lipid analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate lipid domains in plasma membranes of mammalian cells as short lived and in the submicron range. Measuring the fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in giant unilamellar vesicles (GUVs) and the plasma membrane of mammalian cells by fluorescence lifetime imaging (FLIM). Fluorescence decay of C6-NBD-phosphatidylcholine is characterized by a short and long lifetime. For GUVs forming microscopically visible lipid domains the longer lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct, being approximately 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain-forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM as demonstrated for GUVs of palmitoyloleoyl phosphatidylcholine/N-palmitoyl-d-sphingomyelin/cholesterol mixtures. A broad distribution of the long lifetime was found for C6-NBD-phosphatidylcholine inserted in the plasma membrane of HepG2 and HeLa cells centered around 11 ns. FLIM studies on lipid domains forming giant vesicles derived from the plasma membrane of HeLa cells may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact cells.     

Interaction of the fluorescent probe diS-C3-(5) with lecithin-cholesterol membranes

Behaviour of fluorescent carbocyanine probe disS-C3(5) in the egg lecithin-cholesterol membrane suspension was studied in relation to the lecithin/cholesterol ratio. The partition coefficient of the probe between aqueous and lipid phases decreases unlinearly with increase of cholesterol molar part in a bilayer. This parameter over molar part units was estimated to be (2.4 +/- 0.1) X 10(6) for egg lecithin membranes and (1.8 +/- 0.2) X 10(6) for 10 mol% cholesterol, (1.2 +/- 0.1) X 10(6) for 20, (0.8 +/- 0.1) X 10(6) for 30, and (0.48 +/- 0.02) X 10(6) for 50 mol% cholesterol. It is suggested that the probe partition coefficient value consists of two components: one caused by pure lecithin bilayer regions and another by local lecithin concentration fluctuations in the mixed lecithin-cholesterol regions.     

Effect of hydrostatic pressure on water penetration and rotational dynamics in phospholipid-cholesterol bilayers.

The effect of high hydrostatic pressure on the lipid bilayer hydration, the mean order parameter, and rotational dynamics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) cholesterol vesicles has been studied by time-resolved fluorescence spectroscopy up to 1500 bar. Whereas the degree of hydration in the lipid headgroup and interfacial region was assessed from fluorescence lifetime data using the probe 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), the corresponding information in the upper acyl chain region was estimated from its effect on the fluorescence lifetime of and 3-(diphenylhexatrienyl)propyl-trimethylammonium (TMAP-DPH). The lifetime data indicate a greater level of interfacial hydration for DPPC bilayers than for POPC bilayers, but there is no marked difference in interchain hydration of the two bilayer systems. The addition of cholesterol at levels from 30 to 50 mol% to DPPC has a greater effect on the increase of hydrophobicity in the interfacial region of the bilayer than the application of hydrostatic pressure of several hundred to 1000 bar. Although the same trend is observed in the corresponding system, POPC/30 mol% cholesterol, the observed effects are markedly less pronounced. Whereas the rotational correlation times of the fluorophores decrease in passing the pressure-induced liquid-crystalline to gel phase transition of DPPC, the wobbling diffusion coefficient remains essentially unchanged. The wobbling diffusion constant of the two fluorophores changes markedly upon incorporation of 30 mol% cholesterol, and increases at higher pressures, also in the case of POPC/30 mol% cholesterol. The observed effects are discussed in terms of changes in the rotational characteristics of the fluorophores and the phase-state of the lipid mixture. The results demonstrate the ability of cholesterol to adjust the structural and dynamic properties of membranes composed of different phospholipid components, and to efficiently regulate the motional freedom and hydrophobicity of membranes, so that they can withstand even drastic changes in environmental conditions, such as high external hydrostatic pressure.     

Reconstitution of transferrin receptor in mixed lipid vesicles. An example of the role of elastic and electrostatic forces for protein/lipid assembly

We studied the interaction of transferrin receptors (of cell line Molt-4) with mixed model membranes as a function of lipid chain length (phospholipids with C14:0 and C18:1 hydrocarbon chains) and of the surface charge of the membrane using mixtures of C14:0 lecithin (DMPC) with C14:0 phosphatidylglycerol (DMPG) and C14:0 phosphatidylserine (DMPS). Spontaneous self-assembly of receptors and lipids was achieved by freeze-thaw cycles of a codispersion of mixed vesicles and receptors in buffer and subsequent separation of receptor-loaded and receptor-free vesicles by density gradient centrifugation. Information on specific lipid/protein interaction mechanisms was obtained by evaluation of protein-induced shifts of phase boundaries of lipid mixtures by calorimetry and by FTIR spectroscopy of partially deuterated lipid mixtures. The important role (1) of minimizing the elastic forces caused by the mismatch of the lengths of hydrophobic cores of the protein (lp) and the bilayer (lL) and (2) of the electrostatic coupling of protein head groups with the charged membrane/water interface for the lipid/protein self-assembly is established. The electrostatic interaction energy per receptor is about 10(3) kBT (by coupling to about 1000 charged lipids) which is sufficient to overcompensate the elastic energy associated with a mismatch of lp - lL approximately 1.0 nm. The maximum receptor concentration incorporated was measured as a function of membrane surface charge and lipid chain length. The maximum receptor molar fraction varied from xpmax = 5 x 10(-5) for DMPC to xpmax = 4 x 10(-4) for 1:1 DMPC/DMPG; moreover xpmax is higher for DMPS than for DMPG as charged component. For the long-chain lipids, xpmax is higher for a 9:1 DEPE/DEPC mixture [(4.2-9) x 10(-4)] than for pure DEPC (ca. 3.5 x 10(-4)). By decomposition of reconstituted receptors with proteases, we demonstrated the homogeneous orientation of the receptor with its extracellular head group pointing to the convex side of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase.

A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity. The various ligand-stimulated adenylate cyclase activities exhibit different sensitivities to inhibition by cholesterol, with inhibition of glucagon-stimulated greater than fluoride-stimulated greater than basal activity. The bilayer-fluidizing agent benzyl alcohol is able to reverse the inhibitory effect of cholesterol on adenylate cyclase activity in full. The thermostability of fluoride-stimulated cyclase is increased in the cholesterol-rich membranes. Elevated cholesterol concentrations abolish the lipid-phase separation occurring at 28 degrees C in native membranes as detected by an incorporated fatty acid spin probe. This causes Arrhenius plots of glucagon-stimulated adenylate cyclase activity to become linear, rather than exhibiting a break at 28 degrees C. It is suggested that the cholesterol contents of both halves of the bilayer are increased by the method used and that inhibition of adenylate cyclase ensues, owing to the increase in lipid order and promotion of protein-protein and specific cholesterol-phospholipid interactions.