Reconstitution of membrane proteins. Spontaneous association of integral membrane proteins with preformed unilamellar lipid bilayers.
Reconstitution of membrane proteins. Spontaneous association of integral membrane proteins with preformed unilamellar lipid bilayers.
复制标题
膜蛋白的重建。
作者：
A. Scotto;D. Zakim
我们开发了一种简单的方法，将纯净的、完整的膜蛋白重组为磷脂蛋白囊泡。该方法不依赖于洗涤剂或超声波的使用。它已成功地与三种不同类型的膜蛋白结合：来自猪肝微粒体的UDP葡萄糖醛酸基转移酶(EC 2.4.1.17)、来自猪心的细胞色素氧化酶(EC 1.9.3.1)和来自嗜盐杆菌的细菌视紫红质。该方法依赖于制备含有少量肉豆蔻酸酯的双肉豆蔻基磷脂酰胆碱(DMPC)单层囊泡。在DMPC的囊泡具有融合性的条件下，上述蛋白均被整合成双层。磷脂-蛋白质复合体的形成似乎涉及两个事件。第一种方法是将所有蛋白质快速插入一小部分囊泡中。第二种是较慢但持续的剩余磷脂蛋白囊泡或蛋白脂质体与DMPC的小单层囊泡的融合。后一种过程被DMPC的囊泡本身不融合的条件所抑制。在细菌视紫红质质子泵和阴性染色的基础上，小泡为单层大泡。这些数据表明，上述完整的膜蛋白插入囊泡是独立于囊泡之间的融合发生的。
We have developed a simple method for reconstituting pure, integral membrane proteins into phospholipid-protein vesicles. The method does not depend on use of detergents or sonication. It has been used successfully with three different types of integral membrane proteins: UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase (EC 1.9.3.1) from pig heart, and bacteriorhodopsin from Halobacterium halobium. The method depends on preparing unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) that contain a small amount of myristate as fusogen. Under conditions that the vesicles of DMPC have the property of fusing, all of the above proteins incorporated into bilayers. Two events appear to be involved in forming the phospholipid-protein complexes. The first is a rapid insertion of all proteins into a small percentage of total vesicles. The second is slower but continued fusion of the remaining phospholipid-protein vesicles, or proteoliposomes, with small unilamellar vesicles of DMPC. This latter process was inhibited by conditions under which vesicles of DMPC themselves would not fuse. On the basis of proton pumping by bacteriorhodopsin and negative staining, the vesicles were unilamellar and large. The data suggest that insertion of the above integral membrane proteins into vesicles occurred independently of fusion between vesicles.
影响因子：
2.9
作者：
WONG, M;ANTHONY, FH;THOMPSON, TE
通讯作者：
THOMPSON, TE