| Autor: |
Doktorova, Milka, Heberle, Frederick A., Eicher, Barbara, Standaert, Robert F., Katsaras, John, London, Erwin, Pabst, Georg, Marquardt, Drew |
| Jazyk: |
angličtina |
| Rok vydání: |
2018 |
| Předmět: |
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| Popis: |
Model membranes in the form of freely suspended liposomes are widely used as biophysical tools for studying lipid-lipid and protein-lipid interactions. Liposomes prepared by conventional methods have bilayer leaflets that are chemically identical. In contrast, living cells actively maintain a different lipid composition in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are crucial for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-β-cyclodextrin catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition, followed by separation of these pools by centrifugation. We further present two complementary assays for the quantification of each leaflet’s lipid composition: the overall lipid composition is determined by chromatography, while the lipid distribution between the two leaflets is determined by NMR using the lanthanide shift reagent Pr(3+). The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, while the NMR assay is specific to lipids with choline headgroups, such as phosphatidylcholine and sphingomyelin. In approximately 12 hours, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies. We discuss possible extensions of the protocol for the preparation of asymmetric proteoliposomes and cholesterol-containing vesicles, as well as alternative assays for quantifying the transbilayer lipid distribution of lipids that do not possess a choline headgroup. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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