Cholesterol Mole Research Articles

A New High-Temperature Transition of Crystalline Cholesterol in Mixtures with Phosphatidylserine

Phosphatidylserine and cholesterol are two major components of the cytoplasmic leaflet of the plasma membrane. The arrangement of cholesterol is markedly affected by the presence of phosphatidylserine in model membranes. At relatively low mol fractions of cholesterol in phosphatidylserine, compared with other phospholipids, cholesterol crystallites are formed that exhibit both thermotropic phase transitions as well as diffraction of x-rays. In the present study we have observed and characterized a novel thermotropic transition occurring in mixtures of phosphatidylserine and cholesterol. This new transition is observed at 96°C by differential scanning calorimetry (DSC), using a heating scan rate of 2°C/min. Observation of the transition requires that the hydrated lipid mixture be incubated for several days, depending on the temperature of incubation. The rate of formation of the material exhibiting a transition at 96°C is more rapid at higher incubation temperatures. At 37°C the half-time of conversion is ∼7 days. Concomitant with the appearance of the 96°C peak the previously known transitions of cholesterol, occurring at ∼38°C and 75°C on heating scans of freshly prepared suspensions, disappear. These two transitions correspond to the polymorphic transition of anhydrous cholesterol and to the dehydration of cholesterol monohydrate, respectively. The loss of the 75°C peak takes a longer time than that of the 38°C peak, indicating that anhydrous cholesterol first gets hydrated to the monohydrate form exhibiting a transition at 75°C and subsequently is converted by additional time of incubation to an altered form of the monohydrate, showing a phase transition at 96°C. After several weeks of incubation at 37°C, only the form with a phase transition at 96°C remains. If such a sample undergoes several successive heating and cooling cycles, the 96°C peak disappears and the 38°C transition reappears on heating. For samples of 1-palmitoyl-2-oleoyl phosphatidylserine or of 1-stearoyl-2-oleoyl phosphatidylserine having mol fractions of cholesterol between 0.4 and 0.7, the 38°C transition that reappears after the melting of the 96°C component generally has the same enthalpy as do freshly prepared samples. This demonstrates that, at least for these samples, the amount of anhydrous cholesterol crystallites formed is indeed a property of the lipid mixture. We have also examined variations in the method of preparation of the sample and find similar behavior in all cases, although there are quantitative differences. The 96°C transition is partially reversible on cooling and reheating. This transition is also scan rate dependent, indicating that it is, at least in part, kinetically determined. The enthalpy of the 96°C transition, after incubation of the sample for 3 weeks at 37°C is dependent on the ratio of cholesterol to 1-palmitoyl-2-oleoyl phosphatidylserine or to 1-stearoyl-2-oleoyl phosphatidylserine, with the enthalpy per mole cholesterol increasing between cholesterol mol fractions of 0.2 and 0.5. Dimyristoyl phosphatidylserine at a 1:1 molar ratio with cholesterol, after incubation at 37°C, exhibits a transition at 95°C that reverses on cooling at 44°C, instead of 60°C, as observed with either 1-palmitoyl-2-oleoyl phosphatidylserine or 1-stearoyl-2-oleoyl phosphatidylserine. These findings along with the essential absence of the 96°C transition in pure cholesterol or in cholesterol/phosphatidylcholine mixtures, indicates that the phospholipid affects the characteristics of the transition, and therefore the cholesterol crystallites must be in direct contact with the phospholipid and are not simply in the form of pure crystals of cholesterol. These observations are particularly important in view of recent observations of the presence of cholesterol crystals in biological systems.

Biochemical analysis of cell-derived apoE3 particles active in stimulating neurite outgrowth

Susceptibility to the development of late-onset Alzheimer's disease is increased for individuals harboring one or more apolipoprotein E4 (apoE4) alleles. Although several isoform-specific effects of apoE have been identified, the relationship between biochemical function and risk factor assessment is unknown. Our previous studies showed that a physiologically relevant cell-derived apoE3 particle stimulates neurite outgrowth in an isoform-specific manner. In an attempt to delineate the biochemical mechanism responsible for the stimulatory effects of apoE3 on neurite outgrowth, we performed a detailed physical characterization of cell-derived apoE3 and apoE4 particles. Immunoaffinity chromatography followed by SDS-PAGE illustrated homogeneity in protein content (apoE >95%). The affinity-purified particles contained phospholipid and 1 mol of cholesterol per mole of apoE but no core lipids. Nondenaturing gradient gel electrophoresis identified two major particle populations with hydrated diameters of 8.0 and 9.2 nm. Neurite outgrowth assays performed with the affinity-purified particles resulted in similar isoform-specific differences as seen previously, apoE3 stimulatory and apoE4 neutral. Interestingly, we did not observe a reduction in apoE medium concentrations over the duration of the neurite outgrowth assays, suggesting little or no endocytic uptake. Ligand blot analysis demonstrated that the affinity-purified apoE particles bind to several Neuro-2a membrane proteins. Western blots of the Neuro-2a membrane proteins indicated that the LDL receptor, gp330, and LR8B might be involved in the apoE-binding event. These results discriminate against the lipid delivery hypothesis and suggest that the biological activity of the phospholipid apoE3 particles may be due to cell surface signaling.—DeMattos, R. B., L. L. Rudel, and D. L. Williams. Biochemical analysis of cell-derived apoE3 particles active in stimulating neurite outgrowth.

Ternary Phase Diagram of Dipalmitoyl-PC/Dilauroyl-PC/Cholesterol: Nanoscopic Domain Formation Driven by Cholesterol

A ternary phase diagram is proposed for the hydrated lamellar lipid mixture dipalmitoylphosphatidylcholine/dilauroylphosphatidylcholine/cholesterol (DPPC/DLPC/cholesterol) at room temperature. The entire composition space has been thoroughly mapped by complementary experimental techniques, revealing interesting phase behavior that has not been previously described. Confocal fluorescence microscopy shows a regime of coexisting DPPC-rich ordered and DLPC-rich fluid lamellar phases, having an upper boundary at apparently constant cholesterol mole fraction χ chol ∼ 0.16. Fluorescence resonance energy transfer experiments confirm the identification and extent of this two-phase regime and, furthermore, reveal a 1-phase regime between χ chol ∼ 0.16 and 0.25, consisting of ordered and fluid nanoscopic domains. Dipyrene-PC excimer/monomer measurements confirm the new regime between χ chol ∼ 0.16 and 0.25 and also show that rigidly ordered phases seem to disappear around χ chol ∼ 0.25. This study should be considered as a step toward a more complete understanding of lateral heterogeneity within biomembranes. Cholesterol may play a role in domain separation on the nanometer scale.

Fluid–Fluid Membrane Microheterogeneity: A Fluorescence Resonance Energy Transfer Study

Large unilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol mixtures were studied using fluorescence techniques (steady-state fluorescence intensity and anisotropy, fluorescence lifetime, and fluorescence resonance energy transfer (FRET)). Three compositions (cholesterol mole fraction 0.15, 0.20, and 0.25) and two temperatures (30 and 40°C) inside the coexistence range of liquid-ordered ( l o) and liquid-disordered ( l d) phases were investigated. Two common membrane probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dimyristoylphosphatidylethanolamine (NBD-DMPE) and N-(lissamine™-rhodamine B)-dimyristoylphosphatidylethanolamine (Rh-DMPE), which form a FRET pair, were used. The l o /l d partition coefficients of the probes were determined by individual photophysical measurements and global analysis of time-resolved FRET decays. Although the acceptor, Rh-DMPE, prefers the l d phase, the opposite is observed for the donor, NBD-DMPE. Accordingly, FRET efficiency decreases as a consequence of phase separation. Comparing the independent measurements of partition coefficient, it was possible to detect very small domains (<20 nm) of l o in the cholesterol-poor end of the phase coexistence range. In contrast, domains of l d in the cholesterol-rich end of the coexistence range have comparatively large size. These observations are probably related to different processes of phase separation, nucleation being preferred in formation of l o phase from initially pure l d, and domain growth being faster in formation of l d phase from initially pure l o.

Cholesterol Superlattice Model Is Compatible with the Calorimetric Behavior of Cholesterol/Phosphatidylcholine Bilayers

Previous studies have demonstrated that addition of cholesterol to bilayers of saturated phosphatidylcholine (PC) has dramatic effects on the gel-to-liquid phase-transition characteristics of the bilayer. In particular, the enthalpy of the original sharp transition diminishes strongly with increasing cholesterol concentration and a new, broad transition component appears. The enthalpy of the sharp component becomes essentially zero close to the cholesterol mole fraction of 0.25, while the enthalpy of the broad component achieves a maximum near this mole fraction. The broad component typically becomes undetectable when the cholesterol mole fraction is increased to 0.5. In this study we show that these previous experimental findings are fully compatible with a simple model proposing that (i) the cholesterol molecules tend to adopt a regular, superlattice-like lateral distribution within the PC matrix and (ii) the contribution of an acyl chain to the sharp and broad components is critically dependent on its ...

Two moles of O2 consumption and one mole of H2O2 formation during cholesterol peroxidation with cholesterol oxidase from Pseudomonas sp. strain ST-200.

Cholesterol oxidase from Pseudomonas sp. strain ST-200 oxidized cholesterol and cholestanol to 6beta-hydroperoxycholest-4-en-3-one and 5alpha-cholestan-3-one respectively. The former was converted spontaneously to several oxysteroids such as 6-hydroxycholest-4-en-3-one and cholest-4-ene-3,6-dione, with the consumption of 2 mol of O(2) and the formation of 1 mol of H(2)O(2) for each mole of cholesterol oxidized. An oxidized form of the cholesterol oxidase dehydrogenates cholesterol, probably to the 5-en-3-one derivative. A reduced form of the enzyme, yielded from the cholesterol dehydrogenation reaction, dioxygenated cholest-5-en-3-one to 6beta-hydroperoxycholest-4-en-3-one.

Two moles of O2 consumption and one mole of H2O2 formation during cholesterol peroxidation with cholesterol oxidase from Pseudomonas sp. strain ST-200

Cholesterol oxidase from Pseudomonas sp. strain ST-200 oxidized cholesterol and cholestanol to 6β-hydroperoxycholest-4-en-3-one and 5α-cholestan-3-one respectively. The former was converted spontaneously to several oxysteroids such as 6-hydroxycholest-4-en-3-one and cholest-4-ene-3,6-dione, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for each mole of cholesterol oxidized. An oxidized form of the cholesterol oxidase dehydrogenates cholesterol, probably to the 5-en-3-one derivative. A reduced form of the enzyme, yielded from the cholesterol dehydrogenation reaction, dioxygenated cholest-5-en-3-one to 6β-hydroperoxycholest-4-en-3-one.

The pentaene macrolide antibiotic filipin prefers more rigid DPPC bilayers: a fluorescence pressure dependence study

Filipin is a pentaene macrolide antibiotic which was previously shown to incorporate more extensively into DPPC bilayers below the main phase transition temperature than above this temperature. This result was extremely unusual because drugs tend to be expelled from ordered gel phases. However, such results could not be safely attributed to the phase change of the bilayer itself because the temperature was changing concomitantly. In this work we changed the bilayer phase isothermally (53°C) by hydrostatic pressure variation and discovered that filipin has a slightly more extensive incorporation in the pure DPPC gel phase ( P>ca. 54.4 MPa): K p,lc≈3×10 3 vs. K p,gel≈6×10 3. The presence of sterols (45% molar ergosterol or cholesterol) caused an increase in the partition coefficients, regardless of pressure, ergosterol having a more pronounced effect ( K p≈2×10 4−6×10 4). K p was pressure dependent in both cases, but mainly with cholesterol ( K p≈2×10 3−2×10 4). At variance with cholesterol, when ergosterol was used, no phase transition was detected. This difference cannot be due to a more extended uptake of filipin by cholesterol-containing membranes, and so must be due to specific interactions with cholesterol. In agreement with this finding, we discovered that filipin is more tightly packed (lower partial molar volume) in the cholesterol-rich phase than in the ergosterol-rich phase. Our results also point to a 2:1 DPPC:cholesterol stoichiometry in the cholesterol-rich phase (17% molar cholesterol). All partition coefficients were calculated from steady-state fluorescence anisotropy measurements.

A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol

A porcine homolog of the major secretory protein of human epididymis, HE1, was for the first time purified from the porcine cauda epididymal fluid. The HE1 homolog was secreted into the epididymal fluid as a 19-kDa glycoprotein, whose sugar moiety was gradually processed to form a 16-kDa protein during transit through the epididymis. The HE1 homolog mRNA was detected only in the caput and corpus epididymis among the porcine tissues examined. The purified HE1 homolog specifically bound cholesterol with high affinity ( K d=2.3 μM). The binding stoichiometry was determined to be 0.94 mol/mol, suggesting that 1 mol of cholesterol binds to 1 mol of the protein. It was also found that the HE1 homolog is a major cholesterol-binding protein in the porcine epididymal fluid. The possibility that the HE1 homolog is involved in the regulation of the lipid composition of the sperm membranes during the maturation in epididymis is discussed.

A Microscopic Interaction Model of Maximum Solubility of Cholesterol in Lipid Bilayers

We recently reported the equilibrium maximum solubility of cholesterol in a lipid bilayer, χ * chol, to be 0.66 in four different phosphatidylcholines, and 0.51 in a phosphatidylethanolamine (Huang, J., J. T. Buboltz, and G. W. Feigenson. 1999. Biochim. Biophys. Acta. in press). Here we present a model of cholesterol-phospholipid mixing that explains these observed values of χ * chol. Monte Carlo simulations show that pairwise-additivity of nearest-neighbor interactions is inadequate to describe all the χ * chol values. Instead, if cholesterol multibody interactions are assigned highly unfavorable energy, then jumps occur in cholesterol chemical potential that lead to its precipitation from the bilayer. Cholesterol precipitation is most likely to occur near three discrete values of cholesterol mole fraction, 0.50, 0.57, and 0.67, which correspond to cholesterol/phospholipid mole ratios of 1/1, 4/3, and 2/1, respectively. At these solubility limits, where cholesterol chemical potential jumps, the cholesterol-phospholipid bilayer mixture forms highly regular lipid distributions in order to minimize cholesterol-cholesterol contacts. This treatment shows that dramatic structural and thermodynamic changes can occur at particular cholesterol mole fractions without any stoichiometric complex formation. The physical origin of the unfavorable cholesterol multibody interaction is explained by an “umbrella model”: in a bilayer, nonpolar cholesterol relies on polar phospholipid headgroup coverage to avoid the unfavorable free energy of cholesterol contact with water. Thus, at high cholesterol mole fraction, this unfavorable free energy, not any favorable cholesterol-phospholipid interaction, dominates the mixing behavior. This physical origin also explains the “cholesterol condensing effect” and the increase in acyl chain order parameter in cholesterol-phospholipid mixtures.

Activation of acyl-CoA cholesterol acyltransferase: redistribution in microsomal fragments of cholesterol and its facilitated movement by methyl-beta-cyclodextrin.

Acyl-CoA cholesterol acyltransferase (ACAT) (EC 2.3.1.26) in the yolk sac membrane of chicken eggs plays an important role in the transport of lipids, which serve as both structural components and as an energy source during embryogenesis. ACAT from the yolk sac membrane of chicken eggs 16 d after fertilization has higher activity and better stability than its mammalian liver counterpart. During our study of the avian enzyme, ACAT was found to be activated up to twofold during storage at 4 degrees C. The activation was investigated, and data suggest that redistribution of cholesterol within microsomal vesicles leads to the increase. Methyl-beta-cyclodextrin (MbetaCD) increases activation an additional twofold, possibly by facilitating the movement of cholesterol within microsomal fragments and allowing redistribution of cholesterol in lipid bilayers to a greater extent. Treatment of microsomes with MbetaCD removes cholesterol from the membranes. Controlled amounts of cholesterol can be restored to the membranes by mixing them with cholesterol-phosphatidylcholine liposomes in the presence of MbetaCD. Under these conditions, the plot of ACAT vs. cholesterol mole fraction in the liposomes is sigmoidal. The finding that MbetaCD can enhance cholesterol transfer between liposomes and microsomes and reduce the limitation of slow movement of nonpolar molecules in aqueous media should make cyclodextrins more useful in in vitro studies of apolar molecule transport between membrane vesicles.

Evidence for a regulatory role of cholesterol superlattices in the hydrolytic activity of secretory phospholipase A2 in lipid membranes.

We have conducted a detailed study of the effect of membrane cholesterol content on the initial hydrolytic activity of Crotalus durissus terrificus venom phospholipase A2 (sPLA2) in large unilamellar vesicles of cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and cholesterol/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC) at 37 degrees C. The activity was monitored by using the acrylodan-labeled intestinal fatty acid binding protein and HPLC. In contrast to conventional approaches, we have used small cholesterol concentration increments ( approximately 0.3-1.0 mol %) over a wide concentration range (e.g., 13-54 mol % cholesterol). In both membrane systems examined, the initial hydrolytic activity of sPLA2 is found to change with cholesterol content in an alternating manner. The activity reaches a local minimum when the membrane cholesterol content is at or near the critical cholesterol mole fractions (e.g., 14.3, 15.4, 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % cholesterol) predicted for cholesterol regularly distributed in either hexagonal or centered rectangular superlattices. According to the sterol regular distribution model [Chong, P. L.-G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10069-10073; Liu et al. (1997) Biophys. J. 72, 2243-2254], the extent of lipid superlattices is maximal at the critical cholesterol mole fractions, at which the membrane free volume is minimal. Thus, our present data can be taken to indicate that the initial hydrolytic activity of sPLA2 is governed by the extent of cholesterol superlattice. These data provide the first functional evidence for the formation of cholesterol superlattices in both saturated (e.g., DMPC) and unsaturated (e.g., POPC) liquid-crystalline phospholipid bilayers. The data also illustrate the functional importance of cholesterol superlattice and demonstrate a new type of regulation of sPLA2. Furthermore, upon binding to cholesterol/POPC large unilamellar vesicles, the intrinsic fluorescence intensity of sPLA2 shows an alternating variation with cholesterol content, exhibiting a minimum at the critical cholesterol mole fractions. This result suggests that either the number of sPLA2 bound to lipid vesicles or the conformation of membrane-bound sPLA2 or both vary with the extent of the cholesterol superlattice in the plane of the membrane.

Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol

The effect of cholesterol on the interfacial elastic packing interactions of various molecular species of phosphatidylcholines (PCs) has been investigated by using a Langmuir-type film balance and analyzing the elastic area compressibility moduli (Cs(-1)) as a function of average cross-sectional molecular area. Emphasis was on the high surface pressure regions (pi > or = 30 mN/m) which are thought to mimic biomembrane conditions. Increasing levels of cholesterol generally caused the in-plane elasticity of the mixed monolayers to decrease. Yet, the magnitude of the cholesterol-induced changes was markedly dependent upon PC hydrocarbon structure. Among PC species with a saturated sn-1 chain but different sn-2 chain cis unsaturation levels [e.g., myristate (14:0), oleate (18:1delta9(c), linoleate (18:2delta9,12(c), arachidonate (20:4delta5,8,11,14(c), or docosahexenoate (22:6delta4,7,10,13,16,19(c)], the in-plane elasticity moduli of PC species with higher sn-2 unsaturation levels were less affected by high cholesterol mol fractions (e.g., >30 mol %) than were the more saturated PC species. The largest cholesterol-induced decreases in the in-plane elasticity were observed when both chains of PC were saturated (e.g., di-14:0 PC). When both acyl chains were identically unsaturated, the resulting PCs were 20-25% more elastic in the presence of cholesterol than when their sn-1 chains were long and saturated (e.g., palmitate). The mixing of cholesterol with PC was found to diminish the in-plane elasticity of the films beyond what was predicted from the additive behavior of the individual lipid components apportioned by mole and area fraction. Deviations from additivity were greatest for di-14:0 PC and were least for diarachidonoyl PC and didocosahexenoyl PC. In contrast to Cs(-1) analyses, sterol-induced area condensations were relatively unresponsive to subtle structural differences in the PCs at high surface pressures. Cs(-1) versus average area plots also indicated the presence of cholesterol concentration-dependent, low-pressure (<14 mN/m) phase boundaries that became more prominent as PC acyl chain unsaturation increased. Hence, area condensations measured at low surface pressures often do not accurately portray which lipid structural features are important in the lipid-sterol interactions that occur at high membrane-like surface pressures.

The Formation of Amphotericin B Ion Channels in Lipid Bilayers

The ability of amphotericin B (AmB) to form ion-permeable channels in cholesterol containing lipid bilayers was studied by UV/visible absorbance, circular dichroism, and fluorescence spectroscopy. Stable liposomes composed of distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylglycerol, and AmB were prepared so that a wide range of AmB concentrations in the bilayer could be studied. Singular value decomposition analysis (Henry & Hofrichter, 1992) of the circular dichroism spectra of AmB at different AmB/lipid ratios suggests that AmB exists primarily in only two states in the bilayer, a "monomeric" state and an "aggregated" state. The transition from the "monomeric" to the "aggregated" state begins to occur at a critical concentration of 1 AmB per 1000 lipids in the membrane and coincides with the appearance of channel activity. The data support the recent theoretical conclusions of Weakliem et al. (1995) which predict that pore formation in the lipid bilayer will occur when the drug molecule concentration exceeds a critical value. At this critical concentration, it is calculated that a minimum number of 16 AmB molecules per liposome are required to observe channel activity. The results are consistent with the sterol-dependent AmB channel models proposed by de Kruijff and Demel (1974), Andreoli (1974), and Khutorsky (1992). To further elucidate the effects of sterol on AmB-mediated channel formation, liposomes were prepared with varying ratios of cholesterol and AmB. At cholesterol mole percentages greater than 1, channel activity was observed to occur at AmB concentrations just above the critical value. Previous reports show that cholesterol forms "tail-to-tail" dimers at mole percentages greater than 2 (Harris et al., 1995). This suggests that formation of the bilayer-spanning channels by AmB is initiated most efficiently when the tail-to-tail dimer of cholesterol is present. Although the structural nature of the AmB channel could not be unambiguously determined, these experiments provide further evidence in support of the widely held view that AmB's primary mechanism of killing fungal cells occurs by forming ion-permeable channels.

Physical and kinetic characterization of recombinant human cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) mediates the exchange of triglycerides (TGs), cholesteryl esters (CEs) and phospholipids (PLs) between lipoproteins in the plasma. In order to better understand the lipid transfer process, we have used recombinant human CETP expressed in cultured mammalian cells, purified to homogeneity by immunoaffinity chromatography. Purified recombinant CETP had a weight-average relative molecular mass (MW) of 69561, determined by sedimentation equilibrium, and a specific absorption coefficient of 0.83 litre.g-1.cm-1. The corresponding hydrodynamic diameter (Dh) of the protein, determined by dynamic light scattering, was 14 nm, which is nearly twice the expected value for a spheroidal protein of this molecular mass. These data suggest that CETP has a non-spheroidal shape in solution. The secondary structure of CETP was estimated by CD to contain 32% alpha-helix, 35% beta-sheet, 17% turn and 16% random coil. Like the natural protein from plasma, the recombinant protein consisted of several glycoforms that could be only partially deglycosylated using N-glycosidase F. Organic extraction of CETP followed by TLC showed that CE, unesterified cholesterol (UC), PL, TG and fatty acids (FA) were associated with the pure protein. Quantitative analyses verified that each mol of CETP contained 1.0 mol of cholesterol, 0.5 mol of TG and 1.3 mol of PL. CETP mediated the transfer of CE, TG, PL, and UC between lipoproteins, or between protein-free liposomes. In dual-label transfer experiments, the transfer rates for CE or TG from HDL to LDL were found to be proportional to the initial concentrations of the respective ligands in the donor HDL particles. Kinetic analysis of CE transfer was consistent with a carrier mechanism, having a Km of 700 nM for LDL particles and of 2000 nM for HDL particles, and a kcat of 2 s-1. The Km values were thus in the low range of the normal physiological concentration for each substrate. The carrier mechanism was verified independently for CE, TG, PL and UC in 'half-reaction' experiments.

Electrochemical studies of lipophilic ion transport through BLM. The influence of sterols on its transport

AbstractCyclic voltammetry was employed to study the influence of sterols on the lipophilic ion transport through the BLM. The mole fraction of the sterols (cholesterol, oxidized cholesterol), as referred to total lipid, was varied in a range of 0–0.8. Data demonstrate that a thin‐layer model is suitable to this BLM system, By this model, the number of charges transported per lipophilic ion, the concentration of the ion in the membrane hulk phase and the aqueous/membrane phase partition coefficient could be calculated. These parameters proved that sterols had an obvious influence on the lipophilic ion transport. Cholesterol had a stronger influence on the ion transport than oxidized cholesterol. Its incorporation into egg lecithin membranes increased the partition coefficientβ of the ion up to more than 3‐fold. Yet, oxidized cholesterol incorporated into egg lecithin membranes only increased the β up to less than 2‐fold, and the β had no great variation at different oxidized cholesterol mole fractions. The higher β obtained was partly due to the trace amount of solvent existing in the core of the lipid bilayers. At the different sterol mole fractions, combining the change of β with the change of peak current, we also concluded that sterols had somewhat inhibiting effect on the ion transport at the higher sterols mole fraction (&gt; 0.4). These results are explained in terms of the possible change of dipole potential of the membrane produced by sterols and the decrease of the membrane fluidity caused by the condensation effect of sterols and the thinning effect caused by sterols. The substituting group (in the oxidized cholesterol) had some inhibiting effects on the ion transport at higher mole fractions (oxidized cholesterol mole fraction &gt; 0.4).

Phospholipid surface density determines the partitioning and permeability of acetic acid in DMPC:cholesterol bilayers.

Relationships between the permeability coefficient (PHA) and partition coefficient (K m/w) of acetic acid and the surface density of DMPC:cholesterol bilayers have been investigated. Permeability coefficients were measured in large unilamellar vesicles by NMR line broadening. Bilayer surface density, sigma, was varied over a range of 0.5-0.9 by changing cholesterol concentration and temperature. The temperature dependence of PHA for acetic acid exhibits Arrhenius behavior with an average apparent activation energy (Ea) of 22 +/- 3 kcal/mole over a cholesterol mole fraction range of 0.00-0.40. This value is much greater than the enthalpy change for acetic acid partitioning between bulk decane and water (delta H degree = 4.8 +/- 0.8 kcal/mole) and the calculated Ea (= 8.0 kcal/mole) assuming a "bulk phase" permeability model which includes the enthalpy of transfer from water to decane and the temperature dependence of acetic acid's diffusion coefficient in decane. These results suggest that dehydration, previously considered to be a dominant component, is a minor factor in determining Ea. Values of 1n PHA decrease linearly with the normalized phospholipid surface density with a slope of kappa = -12.4 +/- 1.1 (r = 0.90). Correction of PHA for those temperature effects considered to be independent of lipid chain order (i.e., enthalpy of transfer from water to decane and activation energy for diffusion in bulk hydrocarbon) yielded an improved correlation (kappa = -11.7 +/- 0.5 (r = 0.96)). The temperature dependence of Km/w is substantially smaller than that for PHA and dependent on cholesterol composition. Values of 1n K m/w decrease linearly with the surface density with a slope of kappa = -4.6 +/- 0.3 (r = 0.95), which is 2.7-fold smaller than the slope of the plot of 1n PHA vs. sigma. Thus, chain ordering is a major determinant for molecular partitioning into and transport across lipid bilayers, regardless of whether it is varied by lipid composition or temperature.

VIP21/caveolin is a cholesterol-binding protein.

VIP21/caveolin is localized to both caveolae and apical transport vesicles and presumably cycles between the cell surface and the Golgi complex. We have studied the lipid interactions of this protein by reconstituting Escherichia coli-expressed VIP21/caveolin into liposomes. Surprisingly, the protein reconstituted only with cholesterol-containing lipid mixtures. We demonstrated that the protein binds at least 1 mol of cholesterol per mole of protein and that this binding promotes formation of protein oligomers. These findings suggest that VIP21/caveolin, through its cholesterol-binding properties, serves a specific function in microdomain formation during membrane trafficking.

Lateral Organization of Liquid-Crystalline Cholesterol-Dimyristoylphosphatidylcholine Bilayers. Evidence for Domains with Hexagonal and Centered Rectangular Cholesterol Superlattices

The lateral organization of fluid cholesterol-dimyristoylphosphatidylcholine (DMPC) bilayers was studied by measuring the response of fluorescent membrane probes, dipyrenylphosphatidylcholines (diPyrxPCs) or merocyanine 540, to the variation of cholesterol concentration. Parallel absorbance and light-scattering measurements were also carried out. The excimer-to-monomer ratio of diPyrxPCs displayed abrupt deviations at particular cholesterol mole fractions (CMFs). The most notable of these occurred at CMFs of 0.15, 0.33, and 0.67. Deviations were also frequently observed at CMFs of 0.12, 0.20, 0.25, and 0.40. Merocyanine 540 reproducibly reported deviations at CMFs of 0.15 and 0.33 and frequently reported values close to 0.12, 0.20, and 0.25. In absorbance (turbidity) and light scattering versus CMF plots, well-defined kinks were observed at CMFs of 0.16, 0.33, 0.52, and 0.67. The occurrence of kinks or other deviations at those particular CMFs is most readily explained in terms of a superlattice model previously developed to explain the lateral distribution of pyrenylphospholipids in bilayers [Somerharju, et al. (1985) Biochemistry 24, 2773-2781; Virtanen, J. A., et al. (1988) J. Mol. Electron. 4, 233-236]. This model is based on the assumptions that (i) each cholesterol molecule replaces a single acyl chain in a hexagonal lattice, (ii) cholesterol molecules, because of their larger size, perturb the lattice, (iii) this perturbation is minimized when the cholesterol molecules are maximally separated from each other, and (iv) the maximal separation is achieved when the cholesterol molecules form a hexagonal or centered rectangular superlattice. All detected critical CMFs, except that at CMF 0.67, are predicted by the model, thus strongly supporting its validity. The critical CMF at 0.67 is a limiting case, which can be accounted for by assuming that cholesterol and phospholipid molecules form alternating rows, i.e., formation of a cholesterol superlattice with rectangular symmetry. As predicted by the superlattice model, composition-driven order-to-disorder transitions occur between the critical CMFs, as indicated by increased data scatter and sample fluctuations in those regions. Another important prediction of the superlattice model is that domains with different cholesterol superlattices should coexist at most cholesterol concentrations. Such domains do not have to be extensive to account for the critical events observed here; rather, they are expected to be dynamic entities of limited size. It is very likely that such microscopic domains with distinct cholesterol superlattices also coexist in biological membranes. This is expected to have remarkable effects on both the structure and functions of these membranes.

Lipoprotein phospholipid composition and LCAT activity in nephrotic and analbuminemic rats

Albumin is an acceptor of lysophosphatidylcholine (LPC), product of the lecithin:cholesterol acyl transferase (LCAT) reaction, and it has been suggested that low LCAT activity and reduced cholesterol esterification rate in patients with the nephrotic syndrome may be linked to depletion of albumin. Effects of low plasma albumin levels on LCAT activity, cholesterol esterification rates and LPC-binding were therefore studied in hyperlipidemic nephrotic (NS) and analbuminemic (NAR) rats. LPC-binding was also measured in normoalbuminemic rats with dietary hypercholesterolemia. Remarkably, LCAT activity, measured with excess exogenous substrate, was not decreased but increased in both NAR and NS rats. Molar esterification rates with endogenous substrate were increased in NAR but normal in NS rats. In normoalbuminemic rats, with or without hypercholesterolemia, LPC was primarily found in the lipoprotein-deficient plasma and the HDL3 fraction. In NAR and NS rats LPC levels were increased in lipoproteins (notably in LDL and HDL2), but, in marked contrast to normoalbuminemic rats, decreased in lipoprotein-deficient plasma. Phosphatidylcholine, quantitatively the major phospholipid, was distributed proportionally over the lipoproteins in NS, NAR and control rats. Therefore, in hypoalbuminemia and analbuminemia LPC is mainly bound to lipoproteins, which is in contrast to the paucity of LPC in these particles in normoalbuminemic rats. Cholesterol esterification in nephrotic plasma is thus not impaired by lack of an acceptor for LPC-binding. The absence of an increase in molar cholesterol esterification in conjunction with increased LCAT activity points to a possible defect of the substrate for this reaction in nephrotic plasma. Increased LPC levels in LDL, a characteristic of oxidized LDL, may be a hitherto unrecognized atherosclerotic risk factor in the nephrotic syndrome.