McLean L R, Phillips M C
Biochemistry. 1981 May 12;20(10):2893-900. doi: 10.1021/bi00513a028.
The mechanism of cholesterol and phosphatidylcholine exchange has been investigated by following the transfer of radiolabeled cholesterol and phosphatidylcholine from negatively charged, unilamellar cholesterol-egg yolk phosphatidylcholine donor vesicles to neutral acceptor vesicles of similar composition. Vesicles were incubated in the absence of protein and were stable to fusion over the course of the experiment. At intervals, donor and acceptor vesicles were separated by passage through a column of DEAE-Sepharose; less than 1% of the charged and 80-95% of the neutral vesicles were recovered in the eluate. Over 12 h at 37 degrees C, 90% of the donor vesicle [4-14C]cholesterol was transferred to the acceptor vesicles in a first-order process whose half-time was 2.3 +/- 0.3 h. This indicates that transfer of cholesterol molecules from the inner to outer monolayer of the vesicle bilayer is not rate limiting in exchange. In contrast to cholesterol exchange, the half-time for 1-palmitoyl-2-oleoyl[1-14C]phosphatidylcholine exchange was 48 +/- 5 h so that more than six molecules of cholesterol were transferred for each molecule of phosphatidylcholine. The interfacial flux of cholesterol from the donor bilayer is 5.3 x 10(-15) mol cm-2 s-1 (approximately 3 molecules/min for an average vesicle) and is similar to fluxes observed in other systems where phosphatidylcholine or cholesterol ester exchange is catalyzed by an exchange protein. When the acceptor vesicle concentration was increased 20-fold in cholesterol exchange experiments or 9-fold in phosphatidylcholine exchange experiments, the rate of label transfer was not affected. The activation energy of cholesterol exchange between 15 and 37 degrees C was 73 +/- 5 kJ mol-1. Transfer of cholesterol across a dialysis membrane is shown to be a slow process whose rate may be predicted by application of Fick's first law of diffusion. These results are only consistent with a mechanism of lipid exchange in which cholesterol and phosphatidylcholine diffuse through the aqueous phase; the experimental activation energy is associated with desorption of lipid from the donor bilayer into the aqueous phase.
通过追踪放射性标记的胆固醇和磷脂酰胆碱从带负电荷的单层胆固醇 - 蛋黄磷脂酰胆碱供体囊泡转移到组成相似的中性受体囊泡，对胆固醇和磷脂酰胆碱的交换机制进行了研究。囊泡在无蛋白质的情况下孵育，并且在实验过程中对融合稳定。每隔一段时间，通过DEAE - 琼脂糖柱将供体和受体囊泡分离；洗脱液中回收的带电荷囊泡少于1%，中性囊泡为80 - 95%。在37℃下12小时内，90%的供体囊泡[4 - 14C]胆固醇以一级过程转移到受体囊泡，其半衰期为2.3±0.3小时。这表明胆固醇分子从囊泡双层的内层向外层的转移在交换过程中不是限速步骤。与胆固醇交换相反，1 - 棕榈酰 - 2 - 油酰[1 - 14C]磷脂酰胆碱交换的半衰期为48±5小时，因此每分子磷脂酰胆碱转移的胆固醇分子超过六个。来自供体双层的胆固醇界面通量为5.3×10(-15) mol cm-2 s-1（对于平均囊泡约为3个分子/分钟），并且与在其他由交换蛋白催化磷脂酰胆碱或胆固醇酯交换的系统中观察到的通量相似。当在胆固醇交换实验中受体囊泡浓度增加20倍或在磷脂酰胆碱交换实验中增加9倍时，标记转移速率不受影响。15℃至37℃之间胆固醇交换的活化能为73±5 kJ mol-1。胆固醇跨透析膜的转移是一个缓慢的过程，其速率可以通过应用菲克第一扩散定律来预测。这些结果仅与脂质交换机制一致，即胆固醇和磷脂酰胆碱通过水相扩散；实验活化能与脂质从供体双层解吸到水相有关。
