Xie X S, Tsai S J, Stone D K
Proc Natl Acad Sci U S A. 1986 Dec;83(23):8913-7. doi: 10.1073/pnas.83.23.8913.
We have recently reported the reconstitution of the clathrin-coated vesicle proton-translocating complex with liposomes prepared from ethanol-extracted crude bovine brain lipids. Reconstitution of proton pumping by the isolated proton ATPase has now been achieved with liposomes prepared from pure lipids. Optimal proton pumping, as assessed by ATP-generated acridine orange quenching and 32Pi-ATP exchange, is achieved with liposomes prepared from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol at a weight ratio of 40:26.5:7.5:26, and a lipid-to-protein weight ratio of 200:1. Under such conditions, the extent of decrease of ATP-generated acridine orange quenching is 150-fold greater than that of native clathrin-coated vesicles. Cholesterol is required to stabilize the proteoliposome. Phosphatidylserine, which is most effective in activating ATP-hydrolytic activity of the solubilized enzyme, is not obligatory for reconstitution of proton pumping.
我们最近报道了用从乙醇提取的粗制牛脑脂质制备的脂质体对网格蛋白包被囊泡质子转运复合物进行了重组。现在，通过用纯脂质制备的脂质体已实现了由分离的质子ATP酶进行的质子泵浦重组。通过ATP产生的吖啶橙淬灭和32Pi-ATP交换评估，当由磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸和胆固醇以40:26.5:7.5:26的重量比以及脂质与蛋白质200:1的重量比制备脂质体时，可实现最佳质子泵浦。在这种条件下，ATP产生的吖啶橙淬灭的降低程度比天然网格蛋白包被囊泡大150倍。胆固醇是稳定蛋白脂质体所必需的。磷脂酰丝氨酸在激活溶解酶的ATP水解活性方面最有效，但对于质子泵浦的重组不是必需的。
