MECHANISMS OF MEMBRANE-PROTEIN INSERTION INTO LIPOSOMES DURING RECONSTITUTION PROCEDURES INVOLVING THE USE OF DETERGENTS .1. SOLUBILIZATION OF LARGE UNILAMELLAR LIPOSOMES (PREPARED BY REVERSE-PHASE EVAPORATION) BY TRITON X-100, OCTYL GLUCOSIDE, AND SODIUM CHOLATE
MECHANISMS OF MEMBRANE-PROTEIN INSERTION INTO LIPOSOMES DURING RECONSTITUTION PROCEDURES INVOLVING THE USE OF DETERGENTS .1. SOLUBILIZATION OF LARGE UNILAMELLAR LIPOSOMES (PREPARED BY REVERSE-PHASE EVAPORATION) BY TRITON X-100, OCTYL GLUCOSIDE, AND SODIUM CHOLATE
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DOI：
10.1021/bi00408a006
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发表时间：
1988-04-19
期刊：
影响因子：
2.9
通讯作者：
RIGAUD, JL
中科院分区：
文献类型：
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作者：
PATERNOSTRE, MT;ROUX, M;RIGAUD, JL
研究了Triton X-100、辛基葡萄糖苷和胆酸钠对反相蒸发法制备的大单层脂质体的增溶作用机理。溶解过程由先前提出的三阶段模型描述[Lictenberg， D., Robson， R. J., and Dennis， E. A . (1983) Biochim]。Biophys。[j].中国科学：自然科学。在第一阶段，洗涤剂单体被掺入磷脂双分子层，直到它们使脂质体饱和。在这一点上，即阶段II，混合磷脂-洗涤剂胶束开始形成。到第三阶段，片层到胶束的转变完成，所有磷脂都以混合胶束的形式存在。系统地测量了脂质体制剂的浊度，作为在广泛的磷脂浓度范围内（从0.25到20 mM磷脂）添加洗涤剂量的函数。结果允许定量测定RSAt，饱和脂质体中有效清洁剂与脂质的摩尔比，Triton X-100，辛基葡萄糖苷和胆酸钠的摩尔比分别为0.64,1.3和0.30。在混合胶束RSol中，相应的比例分别为2.5、3.8和0.9 mol洗涤剂/mol磷脂。三种洗涤剂在水相中的单体浓度也在片层到胶束的转变（分别为0.18、17和2.8 mM）处进行了测定。这些转变也被31P核磁共振波谱研究，并与浊度测量结果完全一致。冷冻断裂电镜和亚溶解范围内洗涤剂浓度的渗透性研究表明，在溶解的第一阶段，洗涤剂在水相和脂质双分子层之间的分配极大地影响了脂质体的基本渗透性，而没有显著改变制备的形态。根据浊度和渗透率数据（Triton X-100、辛基葡萄糖苷和胆酸钠的K分别为3.5、0.09和0.11 mM-1），得出了分配系数的粗略近似。结论是，当系统地进行浊度测量时，浊度测量是一种非常方便和有效的定量研究脂质体被洗涤剂溶解过程的技术。
The mechanisms governing the solubilization by Triton X-100, octyl glucoside, and sodium cholate of large unilamellar liposomes prepared by reverse-phase evaporation were investigated. The solubilization process is described by the three-stage model previously proposed for these detergents [Lictenberg, D., Robson, R. J., and Dennis, E. A (1983) Biochim. Biophys. Acta 737, 285-304]. In stage I, detergent monomers are incorporated into the phospholipid bilayers until they saturate the liposomes. At that point, i.e., stage II, mixed phospholipid-detergent micelles begin to form. By stage III, the lamellar to micellar transition is complete and all the phospholipids are present as mixed micelles. The turbidity of liposome preparations was systematically measured as a function of the amount of detergent added for a wide range of phospholipid concentrations (from 0.25 to 20 mM phospholipid). The results allowed a quantitative determination of RSAt, the effective detergent to lipid molar ratios in the saturated liposomes, which were 0.64, 1.3, and 0.30 for Triton X-100, octyl glucoside, and sodium cholate, respectively. The corresponding ratios in the mixed micelles, RSol, were 2.5, 3.8, and 0.9 mol of detergent/mol of phospholipid. The monomer concentrations of three detergents in the aqueous phase were also determined at the lamellar to micellar transitions (0.18, 17, and 2.8 mM, respectively). These transitions were also investigated by 31P NMR spectroscopy, and complete agreement was found with turbidity measurements. Freeze-fracture electron microscopy and permeability studies in the sublytic range of detergent concentrations indicated that during stage I of solubilization detergent partitioning between the aqueous phase and the lipid bilayer greatly affects the basic permeability of the liposomes without significantly changing the morphology of the preparation. A rough approximation of the partition coefficients was derived from the turbidity and permeability data (K = 3.5, 0.09, and 0.11 mM-1 for Triton X-100, octyl glucoside, and sodium cholate, respectively). It is concluded that when performed systematically, turbidity measurements constitute a very convenient and powerful technique for the quantitative study of the liposome solubilization process by detergents.