Faculty of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
Biochim Biophys Acta Biomembr. 2018 Feb;1860(2):245-249. doi: 10.1016/j.bbamem.2017.10.011. Epub 2017 Oct 13.
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.
大多数生物膜的磷脂分布在脂质双层的内外叶之间具有不对称结构。不对称分布的控制在细胞内膜融合和细胞分裂等几种细胞功能中起着关键作用。膜不对称性及其在这些转化过程中的变化如何发挥作用的机制尚不清楚。为了了解膜不对称性在细胞内囊泡成分运输和代谢中的意义，需要有一种实验再现生物膜不对称性的系统。在这里，我们成功地通过磷脂酶 D（PLD）的转磷酸化反应获得了不对称囊泡，PLD 仅作用于囊泡外叶中的磷脂酰胆碱（PC）。通过在 1.7M 丝氨酸和 0.3M 乙醇胺存在下用 PLD 处理 PC 囊泡，我们获得了拓扑上类似于含有细胞质叶中磷脂酰丝氨酸和磷脂酰乙醇胺的细胞内囊泡的不对称囊泡。PLD 和其他不需要的化合物可以通过胰蛋白酶消化和透析去除。与传统方法相比，我们建立的技术具有很大的优势，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所必需的。
