Clejan S, Bittman R
J Biol Chem. 1984 Jan 10;259(1):449-55.
Mycoplasma gallisepticum was adapted to grow with delta 5-sterols modified in the aliphatic side chain, and stopped-flow kinetic measurements of filipin association were made to estimate the sterol distribution between the two leaflets of the membrane. Cholesterol derivatives with unsaturated side chains (desmosterol, cis- and trans-22-dehydrocholesterol, and cholesta-5,22E,24-trien-3 beta-ol) or an alkyl substituent (beta-sitosterol) were predominantly (86-94%) localized in the outer leaflet of the bilayer. However, cholesterol, 20-isocholesterol, and sterols with side chains of varying lengths (in the 20(R)-n-alkylpregn-5-en-3 beta-ol series where the alkyl group ranged from ethyl to undecyl) were distributed nearly symmetrically between the two halves of the bilayer. Kinetic measurements of beta-[14C]sitosterol and [14C]desmosterol exchange between M. gallisepticum cells and an excess of sonicated sterol/phosphatidylcholine vesicles confirmed the filipin-binding studies. More than 90% of these radiolabeled sterols underwent exchange at 37 degrees C with unlabeled sterols in vesicles over a period of 12-14 h in the presence of 2% (w/v) albumin. beta-[14C]Sitosterol exchange was characterized by biphasic exchange kinetics, indicative of two pools of sitosterol molecules in the cell membrane. Only a single kinetic pool was detected for [14C]desmosterol exchange. Stopped flow measurements of filipin binding to beta-sitosterol and stigmasterol also revealed an asymmetrical localization of these sterols in membranes of growing Mycoplasma. capricolum cells. When an early exponential culture of beta-sitosterol- or stigmasterol-adapted M. capricolum was transferred to a sterol-rich medium at 37 degrees C, approximately three-quarters of the beta-sitosterol or stigmasterol was localized in the outer leaflet after growth was continued for 6 h; in contrast, cholesterol was distributed symmetrically after about 1 h. The asymmetric localization of sterols with alkylated or unsaturated side chains suggests that growth-supporting sterols need not be translocated extensively into the inner leaflet of the bilayers of M. gallisepticum and M. capricolum.
鸡毒支原体被驯化以在脂肪族侧链修饰的δ5 - 甾醇存在下生长，并进行了制霉菌素结合的停流动力学测量，以估计甾醇在膜的两个小叶之间的分布。具有不饱和侧链的胆固醇衍生物（脱氢胆固醇、顺式和反式22 - 脱氢胆固醇以及胆甾 - 5,22E,24 - 三烯 - 3β - 醇）或具有烷基取代基的（β - 谷甾醇）主要（86 - 94%）定位在双层膜的外层小叶中。然而，胆固醇、20 - 异胆固醇以及具有不同长度侧链的甾醇（在20(R) - n - 烷基孕甾 - 5 - 烯 - 3β - 醇系列中，烷基范围从乙基到十一烷基）在双层膜的两半之间几乎对称分布。鸡毒支原体细胞与过量的超声处理的甾醇/磷脂酰胆碱囊泡之间β - [14C]谷甾醇和[14C]脱氢胆固醇交换的动力学测量证实了制霉菌素结合研究。在2%（w/v）白蛋白存在下，超过90%的这些放射性标记的甾醇在37℃下与囊泡中的未标记甾醇在12 - 14小时内进行了交换。β - [14C]谷甾醇交换的特征是双相交换动力学，表明细胞膜中有两个谷甾醇分子池。对于[14C]脱氢胆固醇交换仅检测到一个动力学池。制霉菌素与β - 谷甾醇和豆甾醇结合的停流测量也揭示了这些甾醇在生长的山羊支原体细胞膜中的不对称定位。当将适应β - 谷甾醇或豆甾醇的山羊支原体早期指数培养物在37℃转移到富含甾醇的培养基中时，继续生长6小时后，约四分之三的β - 谷甾醇或豆甾醇定位在外层小叶中；相比之下，胆固醇在约1小时后对称分布。具有烷基化或不饱和侧链的甾醇的不对称定位表明，支持生长的甾醇无需大量转运到鸡毒支原体和山羊支原体双层膜的内层小叶中。
