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• W3125853620 abstract "A methyl-β-cyclodextrin-induced lipid exchange technique was devised to prepare small unilamellar vesicles with stable asymmetric lipid compositions. Asymmetric vesicles that mimic biological membranes were prepared with sphingomyelin (SM) or SM mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) as the predominant lipids in the outer leaflet and dioleoylphosphatidylcholine (DOPC), POPC, 1-palmitoyl-2-oleoyl-phosphatidyl-l-serine (POPS), or POPS mixed with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) in the inner leaflet. Fluorescence-based assays were developed to confirm lipid asymmetry. Cholesterol was introduced into these vesicles using a second methyl-β-cyclodextrin exchange step. In asymmetric vesicles composed of SM outside, DOPC inside (SMo/DOPCi) or SM outside, 2:1 mol:mol POPE:POPS inside (SMo/2:1 POPE:POPSi) the outer leaflet SM formed an ordered state with a thermal stability similar to that in pure SM vesicles and significantly greater than that in symmetric vesicles with the same overall lipid composition. Analogous behavior was observed in vesicles containing cholesterol. This shows that an asymmetric lipid distribution like that in eukaryotic plasma membranes can be conducive to ordered domain (raft) formation. Furthermore asymmetric vesicles containing ∼25 mol % cholesterol formed ordered domains more thermally stable than those in asymmetric vesicles lacking cholesterol, showing that the crucial ability of cholesterol to stabilize ordered domain formation is likely to contribute to ordered domain formation in cell membranes. Additional studies demonstrated that hydrophobic helix orientation is affected by lipid asymmetry with asymmetry favoring formation of the transmembrane configuration. The ability to form asymmetric vesicles represents an important improvement in model membrane studies and should find many applications in the future. A methyl-β-cyclodextrin-induced lipid exchange technique was devised to prepare small unilamellar vesicles with stable asymmetric lipid compositions. Asymmetric vesicles that mimic biological membranes were prepared with sphingomyelin (SM) or SM mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) as the predominant lipids in the outer leaflet and dioleoylphosphatidylcholine (DOPC), POPC, 1-palmitoyl-2-oleoyl-phosphatidyl-l-serine (POPS), or POPS mixed with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) in the inner leaflet. Fluorescence-based assays were developed to confirm lipid asymmetry. Cholesterol was introduced into these vesicles using a second methyl-β-cyclodextrin exchange step. In asymmetric vesicles composed of SM outside, DOPC inside (SMo/DOPCi) or SM outside, 2:1 mol:mol POPE:POPS inside (SMo/2:1 POPE:POPSi) the outer leaflet SM formed an ordered state with a thermal stability similar to that in pure SM vesicles and significantly greater than that in symmetric vesicles with the same overall lipid composition. Analogous behavior was observed in vesicles containing cholesterol. This shows that an asymmetric lipid distribution like that in eukaryotic plasma membranes can be conducive to ordered domain (raft) formation. Furthermore asymmetric vesicles containing ∼25 mol % cholesterol formed ordered domains more thermally stable than those in asymmetric vesicles lacking cholesterol, showing that the crucial ability of cholesterol to stabilize ordered domain formation is likely to contribute to ordered domain formation in cell membranes. Additional studies demonstrated that hydrophobic helix orientation is affected by lipid asymmetry with asymmetry favoring formation of the transmembrane configuration. The ability to form asymmetric vesicles represents an important improvement in model membrane studies and should find many applications in the future. Over the last few decades artificial lipid bilayers of various types have been successfully used as models for biological membranes, yielding many important insights into the architecture of cell membranes. Vesicle dispersions (liposomes) have perhaps been the most useful model membrane system. However, commonly used preparation procedures do not provide control over differences in lipid composition between inner and outer leaflets (lipid asymmetry). This is a troubling limitation because biological membranes are highly asymmetric. In mammalian cells the plasma membrane outer leaflet (exofacial monolayer) is enriched in sphingolipids and phosphatidylcholine (PC), 2The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; Lo, liquid-ordered; Ld, liquid-disordered; MβCD, methyl-β-cyclodextrin; SM, sphingomyelin; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; DOPC, 1,2-dioleoylphosphatidylcholine; POPS, 1-palmitoyl-2-oleoyl-phosphatidyl-l-serine; POPE, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; o, outside; i, inside; DPPC, 1,2-dipalmitoylphosphatidylcholine; BrPC, 1,2-[9,10-dibromo]stearoylphosphatidylcholine; CHOL, cholesterol; DPH, 1,6-diphenyl-1,3,5-hexatriene; TMADPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate; rhodamine-PE, N-(Rhodamine Red-X)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; NBD-PE, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; HPLC, high pressure liquid chromatography; HP-TLC, high performance thin layer chromatography; MLV, multilamellar vesicle; SUV, small unilamellar vesicle; CLC, cholesterol-loaded MβCD; TM, transmembrane. whereas the inner leaflet (cytofacial monolayer) is enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS) (1Verkleij A.J. Zwaal R.F. Roelofsen B. Comfurius P. Kastelijn D. van Deenen L.L. Biochim. Biophys. Acta. 1973; 323: 178-193Crossref PubMed Scopus (819) Google Scholar).The subject of lipid asymmetry has become all the more important because of its potential role in the structure and function of lipid rafts. Lipid rafts are defined as sphingolipid and sterol-rich lipid domains that exist in the liquid-ordered (Lo) state. Rafts are thought to co-exist in many eukaryotic cell membranes with liquid-disordered (Ld) state domains rich in lipids having unsaturated acyl chains (2Brown D.A. London E. Biochem. Biophys. Res. Commun. 1997; 240: 1-7Crossref PubMed Scopus (458) Google Scholar, 3Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2542) Google Scholar) and have been proposed to be important for numerous cellular processes.The physical properties of Lo domains and the lipid structure dependence of domain formation have been extensively characterized in model membrane bilayers with a symmetric lipid distribution (4Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (609) Google Scholar, 5Feigenson G.W. Buboltz J.T. Biophys. J. 2001; 80: 2775-2788Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 6Megha Bakht O. London E. J. Biol. Chem. 2006; 281: 21903-21913Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 8Nybond S. Bjorkqvist Y.J. Ramstedt B. Slotte J.P. Biochim. Biophys. Acta. 2005; 1718: 61-66Crossref PubMed Scopus (60) Google Scholar, 9Schroeder R. London E. Brown D. 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Ries J. Schwille P. Biophys. J. 2006; 90: 4500-4508Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 25Fidorra M. Duelund L. Leidy C. Simonsen A.C. Bagatolli L.A. Biophys. J. 2006; 90: 4437-4451Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 26Sot J. Bagatolli L.A. Goni F.M. Alonso A. Biophys. J. 2006; 90: 903-914Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 27Veatch S.L. Keller S.L. Biophys. J. 2003; 85: 3074-3083Abstract Full Text Full Text PDF PubMed Scopus (1099) Google Scholar, 28Veatch S.L. Soubias O. Keller S.L. Gawrisch K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 17650-17655Crossref PubMed Scopus (336) Google Scholar, 29Ayuyan A.G. Cohen F.S. Biophys. J. 2008; 94: 2654-2666Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 30Ira Johnston L.J. Biochim. Biophys. Acta. 2008; 1778: 185-197Crossref PubMed Scopus (79) Google Scholar). The ability to prepare asymmetric vesicles would allow more direct comparison of raft-forming model membranes with cell membranes. Some important progress has been made in preparing asymmetric planar bilayers (31Collins M.D. Keller S.L. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 124-128Crossref PubMed Scopus (219) Google Scholar, 32Kiessling V. Crane J.M. Tamm L.K. Biophys. J. 2006; 91: 3313-3326Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 33Crane J.M. Kiessling V. Tamm L.K. Langmuir. 2005; 21: 1377-1388Crossref PubMed Scopus (118) Google Scholar). However, asymmetric lipid vesicles would be of wider utility. Ordinary vesicle preparation procedures (e.g. sonication) can yield some degree of asymmetry in some cases (34Malewicz B. Valiyaveettil J.T. Jacob K. Byun H.S. Mattjus P. Baumann W.J. Bittman R. Brown R.E. Biophys. J. 2005; 88: 2670-2680Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 35Barsukov L.I. Victorov A.V. Vasilenko I.A. Evstigneeva R.P. Bergelson L.D. Biochim. Biophys. Acta. 1980; 598: 153-168Crossref PubMed Scopus (76) Google Scholar), but it can be hard to control. Using pH gradients, asymmetry of small amounts of anionic lipids has been achieved (36Eastman S.J. Hope M.J. Cullis P.R. Biochemistry. 1991; 30: 1740-1745Crossref PubMed Scopus (71) Google Scholar, 37Hope M.J. Redelmeier T.E. Wong K.F. Rodrigueza W. Cullis P.R. Biochemistry. 1989; 28: 4181-4187Crossref PubMed Scopus (99) Google Scholar). The ability to exchange lipids in one leaflet of the bilayer can provide a method to prepare asymmetric vesicles with controlled asymmetry (38Bloj B. Zilversmit D.B. Mol. Cell. Biochem. 1981; 40: 163-172Crossref PubMed Scopus (20) Google Scholar, 39Everett J. Zlotnick A. Tennyson J. Holloway P.W. J. Biol. Chem. 1986; 261: 6725-6729Abstract Full Text PDF PubMed Google Scholar). In one study, a phospholipid exchange protein was used to effectively deliver labeled phosphatidylcholines to the outer leaflet of model membranes (39Everett J. Zlotnick A. Tennyson J. Holloway P.W. J. Biol. Chem. 1986; 261: 6725-6729Abstract Full Text PDF PubMed Google Scholar). In addition, a monolayer-by-monolayer assembly method for preparation of asymmetric vesicles has been reported (40Pautot S. Frisken B.J. Weitz D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10718-10721Crossref PubMed Scopus (356) Google Scholar). γ-Cyclodextrins have been used to deliver small amounts of labeled phospholipids into the outer leaflet of membranes (41Tanhuanpaa K. Somerharju P. J. Biol. Chem. 1999; 274: 35359-35366Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar).Nevertheless a facile and widely applicable method to prepare asymmetric vesicles with a wide variety of lipid compositions, including compositions that mimic cell membranes, has not been described. In this report, we introduce such a method. This procedure is based on the observation that methyl-β-cyclodextrin (MβCD) binds phospholipids at very high MβCD concentrations (42Ottico E. Prinetti A. Prioni S. Giannotta C. Basso L. Chigorno V. Sonnino S. J. Lipid Res. 2003; 44: 2142-2151Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 43Anderson T.G. Tan A. Ganz P. Seelig J. Biochemistry. 2004; 43: 2251-2261Crossref PubMed Scopus (71) Google Scholar). Using this method asymmetric vesicles were prepared with an external leaflet rich in sphingomyelin (SM) and an internal leaflet rich in PE and PS, similar to eukaryotic plasma membranes. Furthermore cholesterol was introduced into the asymmetric vesicles by exposure of the asymmetric vesicles to cholesterol-loaded MβCD (using lower MβCD concentrations).The physical properties of these vesicles reveal some important differences and similarities between symmetric and asymmetric bilayers. Overall it appears that the type of asymmetry found in eukaryotic cell membranes is not a barrier to raft formation and, even more importantly, that the stabilizing effects of cholesterol upon raft formation are not restricted to symmetric membranes. Furthermore we found that lipid asymmetry influences hydrophobic helix topography. Asymmetric vesicles prepared by this method should aid many studies of the role of lipid asymmetry in membrane structure and function.EXPERIMENTAL PROCEDURESMaterials-1,2-Dipalmitoylphosphatidylcholine (DPPC), porcine brain SM, 1,2-dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidyl-l-serine (POPS), 1,2-[9,10-dibromo] stearoylphosphatidylcholine (BrPC), and cholesterol (CHOL) were purchased from Avanti Polar Lipids (Alabaster, AL). 1,6-Diphenyl-1,3,5-hexatriene (DPH) and MβCD were purchased from Sigma-Aldrich. 1-(4-Trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMADPH), N-(Rhodamine Red-X)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine-PE), and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) were purchased from the Molecular Probes division of Invitrogen. [3H]cholesterol was purchased from PerkinElmer Life Sciences. Lipids were dissolved in chloroform and stored at –20 °C. DPH and TMADPH were dissolved in ethanol. Concentrations were determined by dry weight or, in the case of DPH and TMADPH, by absorbance using ∈ = 84,800 cm–1 m–1 at 353 nm in ethanol (44Du H. Fuh R.A. Li J. Corkan A. Lindsey J.S. Photochem. Photobiol. 1998; 68: 141-142Google Scholar). LW peptide (acetyl-K2W2L8AL8W2K2-amide) and pL4A18 peptide (acetyl-K2LA9LWLA9LK2-amide) were purchased from Anaspec (San Jose, CA). LW peptide was used without further purification, and pL4A18 was purified via reverse-phase HPLC (see below). Sephacryl S-200 and Sepharose CL-4B were purchased from Amersham Biosciences. High performance thin layer chromatography (HP-TLC) plates (Silica Gel 60) were purchased from VWR International. (Batavia, IL).Ordinary Vesicle Preparation Procedures-All steps in this and the following procedures were carried out at room temperature except where otherwise noted. Multilamellar vesicles (MLVs) and ethanol dilution small unilamellar vesicles (SUVs) were prepared in glass tubes as described previously (6Megha Bakht O. London E. J. Biol. Chem. 2006; 281: 21903-21913Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 45Bakht O. London E. McIntosh T.J. Lipid Rafts. Humana Press, Totowa, NJ2007: 29-40Google Scholar). For MLVs, lipid mixtures were mixed and dried under nitrogen followed by high vacuum for at least 1 h, dispersed at 70 °C in PBS (1.8 mm KH2PO4, 10 mm Na2HPO4, 137 mm NaCl, and 2.7 mm KCl at pH 7.4), and vortexed in a multitube vortexer (VWR International) at 55 °C for 15 min. In the case of SM MLVs, to remove any small vesicles present prior to lipid exchange, the preparation was centrifuged at 11,000 × g for 5 min at room temperature. The supernatant was discarded, and the pellet obtained was resuspended to the original volume with PBS and used for further experiments. In some cases, MLVs were prepared with 0.01 mol % rhodamine-PE. To prepare sonicated SUVs, MLVs containing 8 or 16 mm unsaturated glycerophospholipids dispersed in PBS (unless otherwise noted) were sonicated in a bath sonicator (Special Ultrasonic Cleaner Model G1112SP1, Laboratory Supplies Co., Hicksville, NY) at room temperature for at least 45 min (until the solution became nearly transparent) and then diluted to the desired concentration with PBS. As judged by their elution position on Sepharose CL-4B chromatography (see below) we found that the small size of the sonicated vesicles was stable for days. For SUVs prepared by ethanol dilution, the desired lipid mixtures were dried, dissolved in 20 μl of ethanol, dispersed in 980 μl of PBS at 70 °C, and then cooled to room temperature. In some cases, the sonicated SUVs contained 0.5 mol % LW peptide and/or 0.01 mol % NBD-PE.Cholesterol-loaded MβCD (CLC) Preparation-Generally 100 μmol of MβCD were dissolved in 600 μl of methanol and mixed with 30.8 μmol of cholesterol while vortexing at room temperature. The mixture was dried by nitrogen followed by high vacuum for at least 1 h and then dispersed in 2 ml of PBS. The resulting solution (which is turbid due to excess cholesterol) was sonicated in the bath sonicator for 3 min and then incubated in a shaker at 37 °C overnight. The CLC-containing solution was then filtered with a 0.22-μm-pore size syringe filter, and the filtrate was used in subsequent experiments.Exchange (Asymmetric) Vesicle Preparation-First 500 μl of the resuspended pellet from a 16 mm SM MLV preparation (see above) and 95 μl of 625 mm MβCD dissolved in PBS were vortexed in the multitube vortexer at 55 °C for 2 h. Then 500 μl of 4 mm sonicated SUVs containing unsaturated glycerophospholipid were added to the MLV-MβCD mixture and vortexed at 55 °C for 30 min. After cooling for 5 min, samples were centrifuged at 11,000 × g for 5 min, and the resulting supernatant was centrifuged at 49,000 × g for another 5 min using an air-driven microultracentrifuge (Beckman Airfuge). For asymmetric SUV preparations without cholesterol, the supernatant was chromatographed on a Sepharose CL-4B column (dimensions, 25-cm length and 1-cm diameter). Fractions of 1 ml were collected with asymmetric SUVs mainly eluting in fractions 10–14. Unless otherwise noted, fraction 12 was used for further analysis. Generally the approximate lipid concentration in the peak SUV fractions was about or somewhat greater than 200 μm as estimated by the recovery of peptide using fluorescence (by comparing it with that in the vesicles prior to exchange) or the recovery of lipid using HP-TLC. In cases in which lipid concentration was not explicitly measured, 200 μm was assumed unless otherwise noted.For asymmetric SUVs containing ∼25 mol % cholesterol, the supernatant from the centrifugation at 49,000 × g was chromatographed on a Sephacryl S-200 column (dimensions, 7-cm length and 1-cm diameter), and 1-ml fractions were collected (although we later realized 0.5 ml fractions would be better to avoid dilution of lipid). To prepare cholesterol-containing SM outside, DOPC inside SUVs (SMo/DOPCi/CHOL SUVs), 950 μl of fraction 4 from this column were transferred to a new glass tube and mixed with 50 μl of a CLC preparation at 55 °C for 30 min. To prepare SMo/2:1 POPE:POPSi/CHOL SUVs, 850 μl of fraction 4 were transferred to a new glass tube and mixed with 150 μl of the CLC preparation at 55 °C for 30 min. The samples were then chromatographed on Sepharose CL-4B as described above. Fractions 12 and 13 from the Sepharose CL-4B column were used for further analysis of cholesterol-containing asymmetric vesicles. Because of losses during this procedure, lipid concentration in the Sepharose CL-4B fractions was lower than that without cholesterol with fractions 12 and 13 each having ∼100 μm lipid as estimated by the recovery of peptide using fluorescence or lipid using HP-TLC. Although lipid recovery was occasionally even lower a 100 μm concentration in these fractions was assumed when the lipid concentration was not explicitly measured.Fluorescence Measurements-Fluorescence was measured by a SPEX FluoroLog 3 spectrofluorometer (Jobin-Yvon, Edison, NJ) using quartz semimicrocuvettes (excitation pathlength, 10 mm; emission, 4 mm). DPH and TMADPH fluorescence was measured at an excitation wavelength of 364 nm and emission wavelength of 426 nm. Trp fluorescence was measured at an excitation wavelength of 280 nm and emission wavelength of 340 nm. Rhodamine-PE fluorescence was measured at an excitation wavelength of 560 nm and emission wavelength of 580 nm. NBD-PE fluorescence was measured at an excitation wavelength of 465 nm and emission wavelength of 534 nm. The slit bandwidths for fluorescence measurements were generally set to 4.2 nm (2-mm physical size) for excitation and 4.2 nm (2-mm physical size) for emission. Background intensities in samples lacking fluorescent probe were negligible (<1–2%) and were generally not subtracted from the reported values. An exception to this was for measurements of TMADPH fluorescence anisotropy (see below).Steady-state Fluorescence Anisotropy Measurements-Anisotropy measurements, unless otherwise noted, were made at room temperature using a SPEX automated Glan-Thompson polarizer accessory. DPH and TMADPH anisotropy values were calculated from the fluorescence intensities with polarizing filters set at all combinations of horizontal and vertical orientations. For TMADPH experiments anisotropy was calculated after subtraction of fluorescence intensity in background samples lacking fluorophore. Anisotropy was calculated from the following equation: A = [((Ivv × Ihh)/(Ivh × Ihv)) – 1]/[((Ivv × Ihh)/(Ivh × Ihv)) + 2] where A is anisotropy and Ivv, Ihh, Ivh, and Ihv are the fluorescence intensities with the excitation and emission polarization filters, respectively, set in the vertical (v) and horizontal (h) orientations (46Lacowicz J. Principles of Fluorescence Spectroscopy.2nd Ed. Kluwer Academic/Plenum Publishers, New York1999: 298-299Google Scholar). For these and all the following experiments in which DPH and TMADPH were used, the fluorescence probe was added (from a ∼100 μM stock solution dissolved in ethanol) to preformed ordinary or preformed exchange vesicles to a concentration of ∼0.1 mol % of the total lipid concentration, and the samples were incubated for at least 5 min before fluorescence was measured. This was sufficient time for the fluorescence of the probe, which increases upon binding to lipid vesicles, to reach nearly maximal intensity (Ref. 47London E. Feligenson G.W. Anal. Biochem. 1978; 88: 203-211Crossref PubMed Scopus (77) Google Scholar and data not shown).Measurement of the Temperature Dependence of Fluorescence Anisotropy-To measure the temperature dependence of DPH anisotropy, samples containing (unless otherwise noted) about 50 μm lipid and 0.1 mol % DPH added as described above were cooled to about 16 °C, and anisotropy was measured. Samples were then heated in steps of about 4 °C, measuring anisotropy at each step once temperature stabilized (as measured with a probe thermometer placed in the cuvette (Fisherbrand traceable digital thermometer with a YSI microprobe, Fisher Scientific)). This process was repeated up to 60–70 °C. The ordered domain melting temperature was defined by the midpoint of a sigmoid fit to the anisotropy versus temperature curve using SlideWrite Plus software (Advanced Graphics Software, Inc., Encinitas, CA).Re-reconstitution Experiments-Fraction 12 of a preparation of SMo/DOPCi SUVs, SMo/2:1 POPE:POPSi SUVs, or DPPCo/DOPCi SUVs was divided into four tubes (250 μl/tube), and then 750 μl of PBS were added to each aliquot. To two aliquots, either DPH (0.5 μl of 100 μm dissolved in ethanol) or TMADPH (0.57 μl of 87.7 μm dissolved in ethanol) was added to give a final membrane composition containing ∼0.1 mol % fluorescent probe. The other two aliquots were subjected to re-reconstitution. They were dried by a nitrogen stream, dissolved in 20 μl of ethanol, and then dispersed at 70 °C in 980 μl of distilled water (which should reconstitute the PBS as well as the lipid vesicles). After cooling to room temperature, DPH or TMADPH was added as described above. Ordinary vesicles were dried and then re-reconstituted by an analogous procedure. For cholesterol-containing asymmetric SUVs fractions 12 and 13 were combined, and then the same procedure was used except that 500 μl of the combined fractions were used per tube so that the lipid concentration would be similar to that in the samples lacking cholesterol.Extraction of TMADPH from vesicles by MβCD-To measure the MβCD concentration dependence of TMADPH extraction from the outer leaflet of vesicles at room temperature, TMADPH was added to preformed vesicles at a concentration of about 0.1 mol % of the lipid concentration. After a 5-min incubation, the initial TMADPH fluorescence of each sample was measured. Next an aliquot of MβCD from a 625 mm stock solution dissolved in PBS was added, and after incubation for 5 min TMADPH fluorescence intensity was remeasured. This was repeated for a series of aliquots of MβCD. Controls at both 0.5 and 1 mm MβCD showed that extraction by MβCD reached equilibrium within 3 min for vesicles of various lipid compositions.High Performance Thin Layer Chromatography-HP-TLC plates were preheated at 100 °C for 30 min and cooled to room temperature, and samples were then loaded. For asymmetric SUV samples, 200–500 μl of fraction 12 from the Sepharose CL-4B column were dried by an N2 stream and dissolved in 20 μl of 1:1 chloroform:methanol (v/v) (excess salt was present as a solid). Then 5 μl of the dissolved lipid were loaded onto the plate. For each lipid standard, the desired lipid was first dried by an N2 stream, dissolved in 20 μl of ethanol, and then dispersed in the same volume of PBS as present in the asymmetric SUV sample (so that the stock solution of the standards would have the same concentration of salt as the vesicle samples). The lipid standards were redried in N2 and dissolved in 50–100 μl of 1:1 chloroform:methanol, and then desired amounts of each lipid were spotted onto the plates (generally loading a total of 8–9 μl for each spot).For samples without cholesterol, 65:25:4 chloroform:methanol:water (v/v) was used to separate each lipid. Samples with cholesterol were generally chromatographed in two solvents. The first solvent system was 50:38:8:4 chloroform:methanol: water:acetic acid (v/v). After the solvent front migrated about halfway up the plate, the plate was air-dried for 5 min. Then the plate was rechromatographed in 1:1 hexane:ethyl acetate (v/v) until the solvent front migrated to near the top of the plate. After air drying for 10 min, the plate was evenly sprayed with a 3% (w/v) cupric acetate, 8% (v/v) phosphoric acid solution, dried for 45 min, and charred at 180 °C for 2–5 min.Charred HP-TLC plates were scanned using an Epson 1640XL scanner (Epson America Inc., Long Beach, CA), and charred band intensity was measured by Scion Image software (Scion Corp., Frederick, MD). Lipid in samples was quantitated by comparing band intensity with that of the standards fit to an exponential intensity versus concentration curve (SlideWrite Plus software).Sucrose Density Gradient Centrifugation-Sucrose gradient centrifugation was carried out in a Beckman L8-55M ultracentrifuge using an SW-60 rotor. Gradients for samples lacking cholesterol were prepared by freeze-thawing 3.4 ml of 25% (w/v) sucrose overnight at –20 °C in the (Beckman ultraclear) tubes used for centrifugation. Gradients for samples containing cholesterol were prepared by freeze-thawing 3.4 ml of 20% (w/v) sucrose. Next 400 μl of vesicle samples were loaded on top of the gradients, and the gradients were then centrifuged for 17 h at 38,000 rpm (average g, 148,305). After centrifugation the gradients were fractionated by pipetting into 200-μl aliquots. (The bottom, highest density fraction (fraction 18) contained 200–400 μl.) Lipids were extracted from each fraction with 2.5 ml of 2:2:1 (v/v) chloroform:methanol:water. After 5 min of low speed centrifugation the upper aqueous phase was discarded. Comparison of a control sample before and after extraction indicated that lipid was nearly fully recovered in the lower phase. The extract in the lower phase was then dried with N2 and redissolved in 15 μl of 1:1 (v/v) chloroform:methanol. Five microliters were spotted on an HP-TLC plate and chromatographed in 50:38:8:4 (v/v) chloroform:methanol:water:glacial acetic aci" @default.
• W3125853620 created "2021-02-01" @default.
• W3125853620 creator A5038894089 @default.
• W3125853620 creator A5040133867 @default.
• W3125853620 creator A5075111768 @default.
• W3125853620 date "2009-03-01" @default.
• W3125853620 modified "2023-10-11" @default.
• W3125853620 title "Preparation and Properties of Asymmetric Vesicles That Mimic Cell Membranes" @default.
• W3125853620 cites W1495577076 @default.
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• W3125853620 cites W1524508279 @default.
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