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首页> 外文期刊>Biochimica et biophysica acta. Biomembranes >Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation
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Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation

机译：通过磷脂酶D介导的转膦酰化形成不对称囊泡

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Abstract Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7M serine and 0.3M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays. Graphical abstract Display Omitted Highlights ? Novel method to produce asymmetric vesicles with phospholipase D is executed. ? PC in the outer leaflet is enzymatically converted to PE or PS. ? Incorporation of cholesterol is effective in long-lasting membrane asymmetry.
机译：摘要大多数Biomembranes在脂质双层的内部和外叶之间的磷脂分布方面具有不对称的结构。对非对称分布的控制在诸如细胞内膜融合和细胞分裂的几种细胞功能中起枢转作用。膜不对称及其在这些转化过程中的改变功能的机制尚不清楚。为了了解膜不对称对细胞内囊泡组分的贩运和代谢的重要性，实验再现生物膜的不对称性的系统是必不可少的。这里，我们通过与磷脂酶D（PLD）的转膦酰亚胺化反应成功地获得非对称囊泡，其专门用于存在于囊泡外部鳞片中的磷脂酰胆碱（PC）上。通过在1.7M丝氨酸和0.3M乙醇胺存在下用PLD处理PC囊泡，我们获得了拓扑囊泡的不对称囊泡，其与含有磷脂酰丝氨酸的细胞内囊泡和磷脂酰乙醇胺在细胞溶胶叶片中的细胞内囊泡。 PLD和其他不需要的化合物可以通过胰蛋白酶消化除去，然后通过透析除去。我们所建立的技术对常规方法具有很大的优势，因为可以以高产率和高效率提供不对称囊泡，这是大多数物理化学测定的必要条件。图形抽象显示省略了亮点？执行具有磷脂酶D的非对称囊泡的新方法。还外叶片中的PC酶促转化为PE或PS。还胆固醇的掺入在持久的膜不对称中是有效的。

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