Kavecansky J, Joiner C H, Schroeder F
Department of Pharmacology and Cell Biophysics, University of Cincinnati Medical Center, Ohio 45267-0004.
Biochemistry. 1994 Mar 15;33(10):2880-90. doi: 10.1021/bi00176a018.
Structural domains of cholesterol and their regulation in the erythrocyte membrane are poorly understood. Dehydroergosterol fluorescence polarization change was used to continuously monitor the kinetics of sterol exchange and sterol domain size in erythrocyte ghost membranes. Direct correlation between molecular sterol exchange and steady-state dehydroergosterol fluorescence polarization measurements was obtained without separation of donor and acceptor membranes. Three important observations were made. First, sterol exchange between small unilamellar vesicles (SUV) with the same cholesterol/phospholipid ratio as the erythrocyte membrane (1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol = 1:1) was resolved into three kinetic cholesterol domains: 23 +/- 9% of total sterol was rapidly exchangeable, with t1/2 = 23 +/- 6 min; 59 +/- 9% of total sterol was slowly exchangeable, with t1/2 = 135 +/- 3 min; and 19 +/- 9% of total sterol was essentially nonexchangeable, with a t1/2 of days. Second, the substitution of erythrocyte ghosts for SUV as an acceptor significantly altered the kinetic parameters of sterol exchange from donor SUV, graphically showing that both the properties of the acceptor and spontaneous desorption of cholesterol from the donor SUV influenced spontaneous cholesterol transfer. Third, studies of exchange between erythrocyte ghosts revealed multiple kinetic pools of sterol differing from those in the SUV: 4 +/- 2% of total sterol was rapidly exchangeable, with t1/2 = 32 +/- 9 min; 29 +/- 3% of total sterol was very slowly exchangeable, with t1/2 = 23 +/- 7 h; and a surprisingly large 67 +/- 2% of total sterol was nonexchangeable, with a t1/2 of days.
人们对红细胞膜中胆固醇的结构域及其调控了解甚少。脱氢麦角固醇荧光偏振变化被用于连续监测红细胞血影膜中甾醇交换动力学和甾醇结构域大小。在不分离供体膜和受体膜的情况下，获得了分子甾醇交换与稳态脱氢麦角固醇荧光偏振测量之间的直接相关性。有三项重要发现。第一，与红细胞膜胆固醇/磷脂比例相同（1-棕榈酰-2-油酰磷脂酰胆碱/胆固醇 = 1:1）的小单层囊泡（SUV）之间的甾醇交换可分为三个动力学胆固醇结构域：总甾醇的23±9%可快速交换，t1/2 = 23±6分钟；总甾醇的59±9%可缓慢交换，t1/2 = 135±3分钟；总甾醇的19±9%基本不可交换，t1/2为数天。第二，用红细胞血影替代SUV作为受体，显著改变了供体SUV甾醇交换的动力学参数，直观地表明受体的性质和胆固醇从供体SUV的自发解吸都影响了胆固醇的自发转移。第三，红细胞血影之间的交换研究揭示了甾醇的多个动力学池，与SUV中的不同：总甾醇的4±2%可快速交换，t1/2 = 32±9分钟；总甾醇的29±3%可非常缓慢地交换，t1/2 = 23±7小时；令人惊讶的是，总甾醇的67±2%不可交换，t1/2为数天。
