Freely suspended liposomes are widely used as model membranes for studying lipid-lipid and protein-lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical 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. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor and acceptor pools by centrifugation because of differences in vesicle size and density. We present two complementary assays allowing quantification of each leaflet's lipid composition: the overall lipid composition is determined by gas chromatography-mass spectrometry, whereas the lipid distribution between the two leaflets is determined by NMR, using the lanthanide shift reagent Pr³⁺. The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, whereas the NMR assay is specific to lipids with choline-containing headgroups, such as phosphatidylcholine and sphingomyelin. In ~12 h, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies.

中文翻译：

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用作细胞膜模型的不对称磷脂囊泡的制备。

自由悬浮的脂质体被广泛用作研究脂质-脂质和蛋白质-脂质相互作用的模型膜。通过常规方法制备的脂质体具有化学相同的双层小叶。相比之下，活细胞在质膜的两个小叶中主动维持不同的脂质成分，从而导致对正常细胞功能至关重要的不对称膜特性。在这里，我们提出了一种制备更好地模拟生物膜的单层不对称磷脂囊泡的方案。不对称性是由甲基-β-环糊精催化的不同脂质成分的囊泡池之间的外叶脂质交换产生的。用于不对称囊泡外叶的脂质由含有致密蔗糖核心的重供体多层囊泡提供。供体脂质被交换成缺乏蔗糖核心的挤出单层受体囊泡，由于囊泡大小和密度的差异，通过离心促进了供体和受体池的交换后分离。我们提出了两种互补的分析方法，可以量化每个小叶的脂质成分：整体脂质成分由气相色谱-质谱法确定，而两个小叶之间的脂质分布由 NMR 确定，使用镧系元素位移试剂 Pr³⁺。制备方案和色谱分析可适用于任何类型的磷脂双层，而 NMR 分析特定于具有含胆碱头基的脂质，例如磷脂酰胆碱和鞘磷脂。在 ~ 12 小时内，该协议可以产生大量的不对称囊泡（高达 20 毫克），适用于广泛的生物物理研究。