Cholesterol In Bilayers Research Articles

AFM Studies of the Effect of Temperature and Electric Field on the Structure of a DMPC−Cholesterol Bilayer Supported on a Au(111) Electrode Surface

Atomic force microscopy (AFM) was used to characterize a phospholipid bilayer composed of 70 mol % 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 30 mol % cholesterol, at a Au(111) electrode surface. Results indicate that addition of cholesterol relaxes membrane elastic stress, increases membrane thickness, and reduces defect density. The thickness and thermotropic properties of the mixed DMPC-cholesterol bilayer supported at the gold electrode surface are quite similar to the properties of the mixed membrane in unilamellar vesicles. The stability of the supported membrane at potentials negative to the potential of zero charge E(pzc) was investigated. This study demonstrates that the bilayer supported at the gold electrode surface is stable provided the applied potential (E - E(pzc)) is less than -0.3 V. At larger polarizations, swelling of the membrane is observed. Polarizations larger than -1 V cause electrodewetting of the bilayer from the gold surface. At these negative potentials, the bilayer remains in close proximity to the metal surface, separated from it by a approximately 2 nm thick layer of electrolyte.

Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells.

Much evidence has accumulated to show that cellular membranes such as the plasma membrane, contain multiple "microdomains" of differing lipid and protein composition and function. These domains are sometimes enriched in cholesterol and sphingolipids and are believed to be important structures for the regulation of many biological and pathological processes. This review focuses on the use of fluorescent (Bodipy) labeled analogs of sphingolipids and cholesterol to study such domains. We discuss the similarities between the behavior of Bodipy-cholesterol and natural cholesterol in artificial bilayers and in cultured cells, and the use of Bodipy-sphingolipid analogs to visualize membrane domains in living cells based on the concentration-dependent monomer-excimer fluorescence properties of the Bodipy-fluorophore. The use of Bodipy-D-erythro-lactosylceramide is highlighted for detection of domains on the plasma membrane and endosome membranes, and the importance of the sphingolipid stereochemistry in modulating domain formation is discussed. Finally, we suggest that Bodipy-sphingolipids may be useful in future studies to examine the relationship between membrane domains at the cell surface and domains enriched in other lipids and proteins on the inner leaflet of the plasma membrane.

Ultrastructural Characterization of Liposomes Using Transmission Electron Microscope

A liposome is a spherical vesicle composed of phospholipids and cholesterol bilayer membrane and contains a core of aqueous solution. Liposomes are polymeric nanoparticles used for drug delivery due to their unique properties. It can carry both hydrophobic and hydrophilic molecules. In this study, we showed the benefit of using transmission electron microscope (TEM) with negative staining technique to investigate the morphology of liposomes produced by thin film method. At the same magnification of micrograph results, we could see the multilamellar vesicles of liposomes in various figures and different sizes.

Plasma Membrane Cholesterol Content Affects Nitric Oxide Diffusion Dynamics and Signaling

Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO approximately 20-40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser(239) induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.

Cholesterol is a determinant of the structures of discoidal high density lipoproteins formed by the solubilization of phospholipid membranes by apolipoprotein A-I

Formation of discoidal high density lipoproteins (rHDL) by apolipoprotein A-I (apoA-I) mediated solubilization of dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles (MLV) was dramatically affected by bilayer cholesterol concentration. At a low ratio of DMPC/apoA-I (2 mg DMPC/mg apoA-I, 84/1 mol/mol), sterols (cholesterol, lathosterol, and β-sitosterol) that form ordered lipid phases increase the rate of solubilization similarly, yielding rHDL with similar structures. By changing the temperature and sterol concentration, the rates of solubilization varied almost 3 orders of magnitude; however, the sizes of the rHDL were independent of the rate of their formation and dependent upon the bilayer sterol concentration. At a high ratio of DMPC/apoA-I (10/1 mg DMPC/mg apoA-I, 420/1 mol/mol), changing the temperature and cholesterol concentration yielded rHDL that varied greatly in size, phospholipid/protein ratio, mol% cholesterol, and number of apoA-I molecules per particle. rHDL were isolated that had 2, 4, 6, and 8 molecules of apoA-I per particle, mean diameters of 117, 200, 303, and 396 Å, and a mol% cholesterol that was similar to the original MLV. Kinetic studies demonstrated that the different sized rHDL are formed independently and concurrently. The rate of formation, lipid composition, and three-dimensional structures of cholesterol-rich rHDL is dictated primarily by the original membrane phase properties and cholesterol content. The size speciation of rHDL and probably nascent HDL formed via the activity of the ABCA1 lipid transporter is mechanistically linked to the cholesterol content of the membranes from which they were formed.

Orientation of Fluorinated Cholesterol in Lipid Bilayers Analyzed by 19F Tensor Calculation and Solid-State NMR

6-F-cholesterol was reported to exhibit biological and interfacial properties similar to unmodified cholesterol. We have also found that 6-F-cholesterol mimicked the cholesterol activity observed in the systems of amphotericin B and lipid rafts. However, to use 6-F-cholesterol as a molecular probe to explore molecular recognition in membranes, it is indispensable to have detailed knowledge of the dynamic and orientation properties of the molecule in membrane environments. In this paper, we present the molecular orientation of 6-F-cholesterol (30 mol %) in dimyristoylphosphatidylcholine (DMPC) bilayers revealed by combined use of 19F chemical shift anisotropy (CSA), 2H NMR, and C-F rotational echo double resonance (REDOR) experiments. The axis of rotation of 6-F-cholesterol was shown to be in a similar direction to that of cholesterol in DMPC bilayers, which is almost parallel to the long axis of the molecular frame. The molecular order parameter of 6-F-cholesterol was determined to be ca. 0.85, which is within the range of reported values of cholesterol. These findings suggest that the dynamic properties of 6-F-cholesterol in DMPC are quite similar to those of unmodified cholesterol; therefore, the introduction of a fluorine atom at C6 has virtually no effect on cholesterol dynamics in membranes. In addition, this study demonstrates the practical utility of theoretical calculations for determining the 19F CSA principal axes, which would be extremely difficult to obtain experimentally. The combined use of quantum calculations and solid-state 19F NMR will make it possible to apply the orientation information of 19F CSA tensors to membrane systems.

Interleaflet Coupling Mechanisms in Bilayers of Lipids and Cholesterol

Whereas it appears to be generally believed that the leaflets of a phospholipid/cholesterol bilayer interact with each other in some way, the exact mechanism remains undetermined. Various suggestions have been invoked, including chain interdigitation and rapid translocation of cholesterol. There is little, if any, direct evidence supporting or excluding these hypotheses. In this letter, I examine a few different possibilities. Chain interdigitation is unlikely to be significant. Cholesterol translocation meets some, though not all, of the relevant criteria, and probably plays an important role. The simplest explanation is that the layers interact at the midplane in the same way that the ordered and disordered liquid phases common in these systems interact at their interfaces. A quick estimate of that interfacial energy shows that this is a very likely candidate. The consequences of such an energy in biological systems are briefly considered.

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55–1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid–cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid–cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid–cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 °C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.

Partitioning of Anti-inflammatory Steroid Drugs into Phosphatidylcholine and Phosphatidylcholine-Cholesterol Small Unilamellar Vesicles as Studied by Second-Derivative Spectrophotometry

The partition coefficients (Kps) of six anti-inflammatory steroid drugs, dexamethasone (DMS), betamethasone (BMS), triamcinolone acetonide (TCLA), fluocinolone acetonide (FCLA), betamethasone 17,21-dipropionate (BMSDP), and clobetasole propionate (CBSP), for phosphatidylcholine (PC), and PC-cholesterol small unilamellar vesicles (SUVs) were determined by a second-derivative spectrophotometric method. The Kp values were obtained with a relative standard deviation of below 10% and the following order was observed: BMS< or =DMS<TCLA<FCLA<<BMSDP<CBSP. BMSDP which has a structure that the two hydroxyl groups of BMS are esterified with propionic acid showed a largely enhanced Kp value of 10.5 times that of BMS. Further, replacement of a propionate group in BMSDP with a chlorine atom resulted in the highest Kp value (CBSP) within the drugs examined, i.e., the Kp value of CBSP was 1.2 times that of BMSDP. The presence of 30 mol% cholesterol in the SUV bilayers reduced these Kp values to approximately 35-50% of those values for the PC SUVs, although the order of the Kp values remained unchanged. The order of the Kp values agreed with that of the reported dermatological therapeutic potency of these drugs, although the order of their log P values for n-octanol/water systems showed a discrepancy. Our results indicate that the potency of steroid drugs in dermatological treatments depends to some extent on the Kp values of the drug, that is, the affinity of steroid drugs for PC bilayers influences their clinical potency, since potency is related to transdermal absorption.

Electrochemical and PM-IRRAS characterization of DMPC + cholesterol bilayers prepared using Langmuir–Blodgett/Langmuir–Schaefer deposition

Differential capacitance, chronocoulometry and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were used to characterize the structure and orientation of DMPC + cholesterol (70:30 mol%) phospholipid bilayers prepared using combined Langmuir–Blodgett/Langmuir–Schaefer (LB/LS) deposition. The electrochemical measurements indicate that incorporation of cholesterol into the membrane improves the quality of the bilayer if both leaflets contain cholesterol. Phospholipid bilayers containing DMPC and cholesterol prepared using LB/LS deposition were found to have smaller tilt angles (∼27°) than bilayers prepared by vesicle spreading (∼52°), indicating that LB/LS deposition is a superior method for preparing supported phospholipid bilayers at a gold electrode surface.

Characterization of the interactions between synthetic nematic LCs and model cell membranes

Differential scanning calorimetry (DSC) was used to characterize interactions of synthetic LCs, 4‐pentyl‐4′‐cyanobiphenyl (5CB) and TL205 (a mixture of cyclohexane‐fluorinated biphenyls and fluorinated terphenyls) with simple mimics of cell membranes. The investigation was motivated by reports that living cells can be placed into contact with TL205 without apparent toxicity, whereas contact of cells with 5CB leads to cell death. The tendency was examined for 5CB and TL205 to spontaneously partition into and influence the organization for model cell membranes composed of phospholipids. Upon contact of an aqueous dispersion of DPPC liposomes with neat LC for 4 h, 5CB partitioned into the liposomes at a weight ratio of 5:1 DPPC:5CB, whereas TL205 partitioned at a ratio of 310:1 DPPC:TL205. DSC endotherms indicated that the 5CB spontaneously partitioned into the liposomes was far more perturbing than TL205. DSC endotherms of DPPC bilayers containing the same concentration of either 5CB or TL205 also revealed 5CB to be more perturbing than TL205. The effect of up to 7.8 wt % of TL205 was small, resulting in a shift in the melting transition from 41.4°C to 40.1°C and a minor change in peak width, indicating only minor effects on the organization of the bilayer. These effects are similar to those caused by cholesterol in DPPC bilayers. In contrast, 5CB shifted the DPPC melting transition from 41.4°C to ∼36°C and increased the width of the transition peak by a factor of ten, indicating a destabilization of the ordered phase in the bilayer and a disruption of the cooperative nature of the gel‐to‐LC transition of the phospholipid bilayer. Taken together, the results demonstrate that 5CB and TL205 differ significantly in their interactions with model cell membranes, which suggests one possible origin of their different toxicities toward cells. *Current affiliation: SurModics, Inc., Eden Prairie, MN 55344, USA.

Molecular Dynamics Studies of the Molecular Structure and Interactions of Cholesterol Superlattices and Random Domains in an Unsaturated Phosphatidylcholine Bilayer Membrane

The effect of the molecular organization of lipid components on the properties of the bilayer membrane has been a topic of increasing interest. Several experimental and theoretical studies have suggested that cholesterol is not randomly distributed in the fluid-state lipid bilayer but forms nanoscale domains. Several cholesterol-enriched nanodomain structures have been proposed, including rafts, regular or maze arrays, complexes, and superlattices. At present, the molecular mechanisms by which lipid composition influences the formation and stability of lipid nanodomains remain unclear. In this study, we have used molecular dynamics (MD) simulations to investigate the effects of the molecular organization of cholesterol--superlattice versus random--on the structure of and interactions between lipids and water in lipid bilayers of cholesterol and 1-palmitoyl-2-oleoylphosphatidylcholine (cholesterol/POPC) at a fixed cholesterol mole fraction of 0.40. On the basis of four independent replicates of 200-ns MD simulations for a superlattice or random bilayer, statistically significant differences were observed in the lipid structural parameters, area per lipid, density profile, and acyl chain order profile, as well as the hydrogen bonding between various pairs (POPC and water, cholesterol and water, and POPC and cholesterol). The time evolution of the radial distribution of the cholesterol hydroxy oxygen suggests that the lateral distribution of cholesterol in the superlattice bilayer is more stable than that in the random bilayer. Furthermore, the results indicate that a relatively long simulation time, more than 100 ns, is required for these two-component bilayers to reach equilibrium and that this time is influenced by the initial lateral distribution of lipid components.

Membrane interactions of the hydrophobic segment of diacylglycerol kinase epsilon

Diacylglycerol kinase epsilon (DGKε) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKε of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH 2. Flanking Lys residues mimic the natural setting of this peptide in DGKε, while facilitating peptide synthesis and characterization. Circular dichroism and fluorescence spectroscopic analysis demonstrated that the peptide has increased helical content and significant blue shifts in the presence of anionic – but not zwitterionic – bilayer membranes. When labeled with fluorophores that can undergo fluorescence resonance energy transfer, the peptide was found to dimerize – a result also observed from migration rates on SDS-PAGE gels under both reducing and non-reducing disulfide bridge conditions. The peptide was shown to preferentially interact with cholesterol in lipid films comprised of homogeneous mixtures of cholesterol and phosphatidylcholine, yet the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. The peptide was also able to inhibit the activity of DGKε protein in vitro. Our overall findings suggest that the peptide ultimately cannot leave the bulk water for attachment/insertion into the outer leaflet of an erythrocyte-like bilayer, yet its core sequence is sufficiently hydrophobic to insert into membrane core regions when membrane attachment is promoted by electrostatic attraction to anionic lipid head groups of the inner leaflet of an erythrocyte-like bilayer.

The Condensing Effect of Cholesterol in Lipid Bilayers

The condensing effect of cholesterol on phospholipid bilayers was systematically investigated for saturated and unsaturated chains, as a function of cholesterol concentration. X-ray lamellar diffraction was used to measure the phosphate-to-phosphate distances, PtP, across the bilayers. The measured PtP increases nonlinearly with the cholesterol concentration until it reaches a maximum. With further increase of cholesterol concentration, the PtP remains at the maximum level until the cholesterol content reaches the solubility limit. The data in all cases can be quantitatively explained with a simple model that cholesterol forms complexes with phospholipids in the bilayers. The phospholipid molecules complexed with cholesterol are lengthened and this lengthening effect extends into the uncomplexed phospholipids surrounding the cholesterol complexes. This long-range thickening effect is similar to the effect of gramicidin on the thickness of lipid bilayers due to hydrophobic matching.

Assess the nature of cholesterol–lipid interactions through the chemical potential of cholesterol in phosphatidylcholine bilayers

Cholesterol plays a vital role in determining the physiochemical properties of cell membranes. However, the detailed nature of cholesterol-lipid interactions is a subject of ongoing debate. Existing conceptual models, including the Condensed Complex Model, the Superlattice Model, and the Umbrella Model, identify different molecular mechanisms as the key to cholesterol-lipid interactions. In this work, the compositional dependence of the chemical potential of cholesterol in cholesterol/phosphatidylcholine mixtures was systematically measured at high resolution at 37 degrees C by using an improved cholesterol oxidase (COD) activity assay. The chemical potential of cholesterol was found to be much higher in di18:1-PC bilayers than in di16:0-PC bilayers, indicating a more favorable interaction between cholesterol and saturated chains. More significantly, in 16:0,18:1-PC and di18:1-PC bilayers, the COD initial-reaction rate displays a series of distinct jumps near the cholesterol mole fractions (chi(C)) of 0.15, 0.25, 0.40, 0.50, and 0.57 and a peak at the cholesterol maximum solubility limit of 0.67. These jumps have been identified as the thermodynamic signatures of stable cholesterol regular distributions. In contrast, no such jumps were evident in di16:0-PC bilayers below chi(C) of 0.57. The observed chemical potential profile is in excellent agreement with previous Monte Carlo simulations based on the Umbrella Model but not with the predictions from the other models. The data further indicate that the cholesterol regular distribution domains (superlattices) are not the hypothesized condensed complexes. Those complexes were mainly implicated from studies on lipid monolayer that may not be relevant to the lipid bilayer in cell membranes.

2P251 固体NMRによるβ-endorphinとコレステロール含有不飽和脂質膜の相互作用解析(生体膜・人工膜(構造・物性),ポスター発表,第45回日本生物物理学会年会)

2P251 固体NMRによるβ-endorphinとコレステロール含有不飽和脂質膜の相互作用解析(生体膜・人工膜(構造・物性),ポスター発表,第45回日本生物物理学会年会)

Retinal Counterion Switch Mechanism in Vision Evaluated by Molecular Simulations

Photoisomerization of the retinylidene chromophore of rhodopsin is the starting point in the vision cascade. A counterion switch mechanism that stabilizes the retinal protonated Schiff base (PSB) has been proposed to be an essential step in rhodopsin activation. On the basis of vibrational and UV-visible spectroscopy, two counterion switch models have emerged. In the first model, the PSB is stabilized by Glu181 in the meta I state, while in the most recent proposal, it is stabilized by Glu113 as well as Glu181. We assess these models by conducting a pair of microsecond scale, all-atom molecular dynamics simulations of rhodopsin embedded in a 99-lipid bilayer of SDPC, SDPE, and cholesterol (2:2:1 ratio) varying the starting protonation state of Glu181. Theoretical simulations gave different orientations of retinal for the two counterion switch mechanisms, which were used to simulate experimental 2H NMR spectra for the C5, C9, and C13 methyl groups. Comparison of the simulated 2H NMR spectra with experimental data supports the complex-counterion mechanism. Hence, our results indicate that Glu113 and Glu181 stabilize the retinal PSB in the meta I state prior to activation of rhodopsin.

Membrane-dependent oligomeric structure and pore formation of a β-hairpin antimicrobial peptide in lipid bilayers from solid-state NMR

We used solid-state NMR spectroscopy to investigate the oligomeric structure and insertion of protegrin-1 (PG-1), a beta-hairpin antimicrobial peptide, in lipid bilayers that mimic either the bacterial inner membrane [palmitoyloleoylphosphatidyl ethanolamine and palmitoyloleoylphosphatidylglycerol (POPE/POPG) bilayers] or the red blood cell membrane [neutral palmitoyloleoylphosphatidylcholine (POPC)/cholesterol bilayers]. (1)H spin diffusion from lipids to the peptide indicates that PG-1 contacts both the lipid acyl chains and the headgroups in the anionic membrane but resides far from the lipid chains in the POPC/cholesterol bilayer. (19)F spin diffusion data indicates that 75% of the beta-hairpins have homodimerized N strands and C strands in the anionic membrane. The resulting (NCCN)(n) multimer suggests a membrane-inserted beta-barrel enclosing a water pore. The lipids surrounding the beta-barrel have high orientational disorder and chain upturns, thus they may act as fillers for the pore. These results revise several features of the toroidal pore model, first proposed for magainin and subsequently applied to PG-1. In the POPC/cholesterol membrane, the N and C strands of PG-1 cluster into tetramers, suggesting the formation of beta-sheets on the membrane surface. Thus, the membrane composition plays a decisive role in defining the assembly and insertion of PG-1. The different oligomeric structures of PG-1 help to explain its greater toxicity for bacteria than for eukaryotic cells.

Low cholesterol solubility in DODAB liposomes

Through the analysis of the ESR spectra of spin labels, we investigated the thermotropic properties of dioctadecyl dimethylammonium bromide (DODAB) liposomes, in low and high ionic strength, with different cholesterol contents. The cationic lipid gel phase is stabilized by the presence of ions, the bilayer having a higher gel/fluid transition temperature ( T m) in high ionic strength. As found for low ionic strength [Benatti, C.R., Feitosa, E., Fernandez, R.M., Lamy-Freund, M.T., 2001. Structural and thermal characterization of dioctadecyldimethylammonium bromide dispersions by spin labels. Chem. Phys. Lipids, 111, 93–104], high salt DODAB membranes also present a clear coexistence of the two phases around T m. Cholesterol solubility in DODAB bilayers seems to be rather low, as the coexistence of DODAB and cholesterol-rich domains can be clearly detected by spin labels, for cholesterol concentration as low as 15 mol% of the total lipid. For lower cholesterol concentrations, the effect of cholesterol in DODAB bilayers is similar to that in phospholipids. For concentrations at or above 45 mol% of cholesterol, spin labels do not detect the coexistence of structurally different domains.

Lipid Headgroup Superlattice Modulates the Activity of Surface-Acting Cholesterol Oxidase in Ternary Phospholipid/Cholesterol Bilayers

The relationship between the molecular organization of lipid headgroups and the activity of surface-acting enzyme was examined using a bacterial cholesterol oxidase (COD) as a model. The initial rate of cholesterol oxidation by COD in fluid state 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers was measured as a function of POPE-to-phospholipid mole ratio (X(PE)) and cholesterol-to-lipid mole ratio (X(CHOL)) at 37 degrees C. At X(PE) = 0, the COD activity changed abruptly at X(CHOL) approximately 0.40, whereas major activity peaks were detected at X(PE) approximately 0.18, 0.32, 0.50, 0.64, and 0.73 when X(CHOL) was fixed to 0.33 or 0.40. At a fixed X(CHOL) of 0.50, the COD activity increased progressively with PE content and exhibited small peaks or kinks at X(PE) approximately 0.40, 0.50, 0.58, 0.69, and 0.81. When X(PE) and X(CHOL) were systematically varied within a narrow 2-D lipid composition window, an onset of COD activity at X(CHOL) approximately 0.40 and the elimination of the activity peak at X(PE) approximately 0.64 for X(CHOL) >0.40 were clearly observed. Except for X(PE) approximately 0.40 and 0.58, the observed critical PE mole ratios agree closely (+/-0.03) with those predicted by a headgroup superlattice model (Virtanen, J.A., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4964-4969; Cannon, B., et al. (2006) J. Phys. Chem. B 110, 6339-6350), which proposes that lipids with headgroups of different sizes tend to adopt regular, superlattice-like distributions at discrete and predictable compositions in fluid lipid bilayers. Our results indicate that headgroup superlattice domains exist in lipid bilayers and that they may play a crucial role in modulating the activity of enzymes acting on the cell membrane surface.