Clejan S, Bittman R
J Biol Chem. 1984 Jan 10;259(1):441-8.
The kinetics of exchange of radiolabeled cholesterol and phospholipids between intact Mycoplasma gallisepticum cells and unilamellar lipid vesicles were investigated over a wide range of cholesterol/phospholipid molar ratio. The change in cholesterol/phospholipid molar ratio was achieved by adapting the sterol-requiring M. gallisepticum to grow in cholesterol-poor media, providing cells with decreased unesterified cholesterol content. At least 90% of the cholesterol molecules in unsealed M. gallisepticum membranes underwent exchange at 37 degrees C as a single kinetic pool in the presence of albumin (2%, w/v). However, we observed biphasic exchange kinetics with intact cells, indicating that cholesterol translocation from the inner to outer monolayers was rate-limiting in the exchange process. Approximately 50% of the cholesterol molecules were localized in each kinetic pool, independent of the cholesterol/phospholipid molar ratio in the cells and vesicles. A striking change in the kinetic parameters for cholesterol exchange occurred between 20 and 26 mol % cholesterol; for example, when the cholesterol/phospholipid molar ratio was decreased from 0.36 to 0.25, the half-time for equilibration of the two cholesterol pools at 37 degrees C decreased from 4.6 +/- 0.5 to 2.5 +/- 0.1 h. Phospholipid exchange rates were also enhanced on decreasing the membrane cholesterol content. The ability of cholesterol to modulate its own exchange rate, as well as that of phospholipids, is suggested to arise from the sterol's ability to regulate membrane lipid order. Extensive chemical modification of the membrane surface by cross-linking of some of the protein constituents with 1,4-phenylenedimaleimide decreased the cholesterol exchange rate. Depletion of membrane proteins by treatment of growing cultures with chloramphenicol increased the cholesterol exchange rate, possibly because of removal of some of the protein mass that may impede lipid translocation. The observations that phospholipid exchange was one order of magnitude slower than cholesterol exchange and that dimethyl sulfoxide, potassium thiocyanate, and potassium salicylate enhanced the cholesterol exchange rate are consistent with a mechanism involving lipid exchange by diffusion through the aqueous phase.
在很宽的胆固醇/磷脂摩尔比范围内，研究了完整鸡毒支原体细胞与单层脂质囊泡之间放射性标记胆固醇和磷脂的交换动力学。通过使需要甾醇的鸡毒支原体适应在胆固醇含量低的培养基中生长来改变胆固醇/磷脂摩尔比，从而使细胞中未酯化胆固醇含量降低。在37℃、存在白蛋白（2%，w/v）的情况下，未封闭的鸡毒支原体膜中至少90%的胆固醇分子作为一个单一动力学池进行交换。然而，我们观察到完整细胞的交换动力学呈双相性，这表明胆固醇从内膜层向外膜层的转运是交换过程中的限速步骤。大约50%的胆固醇分子位于每个动力学池中，与细胞和囊泡中的胆固醇/磷脂摩尔比无关。在胆固醇含量为20%至26%（摩尔）之间，胆固醇交换的动力学参数发生了显著变化；例如，当胆固醇/磷脂摩尔比从0.36降至0.25时，37℃下两个胆固醇池达到平衡的半衰期从4.6±0.5小时降至2.5±0.1小时。降低膜胆固醇含量时，磷脂交换速率也会提高。胆固醇调节自身以及磷脂交换速率的能力，被认为源于甾醇调节膜脂有序性的能力。用1,4-苯二马来酰亚胺使一些蛋白质成分交联对膜表面进行广泛化学修饰，会降低胆固醇交换速率。用氯霉素处理生长中的培养物使膜蛋白耗尽，会提高胆固醇交换速率，这可能是因为去除了一些可能阻碍脂质转运的蛋白质。磷脂交换比胆固醇交换慢一个数量级，以及二甲亚砜、硫氰酸钾和水杨酸钾提高胆固醇交换速率的观察结果，与通过水相扩散进行脂质交换的机制一致。
