REASSEMBLY OF PROTEIN-LIPID COMPLEXES INTO LARGE BILAYER VESICLES - PERSPECTIVES FOR MEMBRANE RECONSTITUTION
REASSEMBLY OF PROTEIN-LIPID COMPLEXES INTO LARGE BILAYER VESICLES - PERSPECTIVES FOR MEMBRANE RECONSTITUTION
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DOI：
10.1073/pnas.77.1.239
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发表时间：
1980-01-01
期刊：
影响因子：
--
通讯作者：
MONTAL, M
中科院分区：
文献类型：
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作者：
DARSZON, A;VANDENBERG, CA;MONTAL, M
蛋白质-脂质复合物在非极性溶剂中重新组装成直径为几微米的大的双层蛋白质-脂质囊泡（PLV）。除去溶剂后，蛋白质-脂质复合物残留物水合后，PLV自发形成。此程序适用于以下膜蛋白：牛和鱿鱼视紫红质，反应中心，从红球藻sphaeroides，牛心细胞色素c氧化酶和乙酰胆碱受体，从加利福尼亚鱼雷。PLV具有大的内部水空间（例如，对于牛视紫红质囊泡，为790 μ l/mg脂质）。PLV的冷冻断裂复制品揭示了内部和外部小叶都含有许多直径在80-120埃之间的膜内颗粒，这取决于结合在膜中的特定蛋白质。PLV中的视紫红质和反应中心的光谱特性与相应的天然膜中记录的那些相似。PLV中的牛视紫红质可与9-顺式-视黄醛化学再生。光化照明引起的质子流出反应中心囊泡被废除的质子离子载体。因此，该方法适用于掺入功能状态下的某些膜蛋白。用微电极穿透PLV，并通过注射荧光染料进行可视化。通过将PLV密封到分隔2个水室的隔膜中的孔来获得初步电记录。通过该程序组装的PLV允许通过化学、光学和电学技术同时分析重构膜。
Protein-lipid complexes in apolar solvents reassemble into large bilayer protein-lipid vesicles (PLV) with diameters of several micrometers. PLV form spontaneously upon hydration of the protein-lipid complex residue after solvent removal. This procedure was applied to the following membrane proteins: bovine and squid rhodopsin, reaction centers from Rhodopseudomonas sphaeroides, beef heart cytochrome c oxidase and acetylcholine receptors from Torpedo californica. PLV have a large internal aqueous space (e.g., 790 .mu.l/mg of lipid for cattle rhodopsin vesicles). Freeze-fracture replicas of PLV revealed that both internal and external leaflets contained numerous intramembranous particles with diameters between 80-120 .ANG., depending on the specific protein incorporated in the membrane. The optical spectral properties of rhodopsin and reaction centers in PLV were similar to those recorded in the respective natural membrane. Bovine rhodopsin in PLV was chemically regenerable with 9-cis-retinal. Actinic illumination induced proton efflux from reaction center vesicles that was abolished by proton ionophores. Therefore, this method is suitable for the incorporation of some membrane proteins in their functional state. PLV were penetrated with microelectrodes and visualized by the injection of a fluorescent dye. Preliminary electrical recordings were obtained by sealing PLV to a hole in a septum separating 2 aqueous compartments. PLV assembled by this procedure permit the simultaneous analysis of reconstituted membranes by chemical, optical and electrical techniques.