Asymmetric exchange of vesicle phospholipids catalyzed by the phosphatidylcholine exhange protein. Measurement of inside--outside transitions.
Asymmetric exchange of vesicle phospholipids catalyzed by the phosphatidylcholine exhange protein. Measurement of inside--outside transitions.
复制标题
由磷脂酰胆碱交换蛋白催化的囊泡磷脂的不对称交换。
作者：
J. Rothman;E. Dawidowicz
用纯化的磷脂酰胆碱交换蛋白在均一的单壁磷脂酰胆碱囊泡和人红细胞膜之间交换磷脂酰胆碱。当过量的幽灵存在时，发现只有70%的囊泡磷脂酰胆碱可用于交换。这一组分与这些小泡外单层中的磷脂酰胆碱的量密切相关，表明只有小泡的外表面可以被交换蛋白访问。此外，还发现所有由交换蛋白引入囊泡的磷脂酰胆碱都可用于随后的交换。利用交换蛋白，制备了不对称的囊泡，与内单分子层相比，外单分子层中的放射性磷脂酰胆碱要么被富含，要么被耗尽。即使在37度下5天后，也不能检测到囊泡(触发器)两个表面之间的放射性重新平衡。据估计，在37度的温度下，触发器的半衰期超过11天。这些结果表明，在不改变膜结构的情况下，可以利用交换蛋白的性质来测量生物膜和模型膜中磷脂酰胆碱的翻转率和可能的不对称性。
Purified phosphatidylcholine exchange protein was used to exchange phosphatidylcholine between homogeneous single-walled phosphatidylcholine vesicles and human erythrocyte ghosts. When excess ghosts were present, it was found that only 70% of the vesicle phosphatidylcholine was available for exchange. This fraction corresponds closely to the amount of phosphatidycholine in the outer monolayer of these vesicles, indicating that only the outer surface of the vesicle is accessible to the exchange protein. Also, it was found that all phosphatidylcholine introduced into vesicles by the exchange protein was available for subsequent exchange. Using the exchange protein, asymmetrical vesicles were prepared in which the outer monolayer was either enriched or depleted in radioactive phosphatidylcholine as compared to the inner monolayer. Re-equilibration of the radioactivity between the two surfaces of the vesicle (flip-flop) could not be detected, even after 5 days at 37degrees. It is estimated that the half-time for flip-flop is in excess of 11 days at 37degrees. These results indicate that the properties of the exchange protein can be expolited to measure phosphatidylcholine flip-flop rates and possible phosphatidylcholine asymmetry in biological and model membranes, without altering the structure of the membrane.