Inhibition of membrane fusion by lysophosphatidylcholine.
Inhibition of membrane fusion by lysophosphatidylcholine.
复制标题
溶血磷脂酰胆碱抑制膜融合。
DOI：
10.1021/bi00173a027
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Flanagan,TD
中科院分区：
文献类型：
--
作者：
Yeagle,PL;Smith,FT;Young,JE;Flanagan,TD
材料和METHODSTV-Methyldioleoylphosphatidylethanolamine(TV-甲基-DOPE)和溶血磷脂酰胆碱(来自卵磷脂)是从阿凡提极脂，比尔明翰，AL。十八烷基罗丹明B氯化物(RJG)、L-氨基-3，6，8-三磺酸(二钠盐)(ANTS)和对二甲苯双(吡啶溴)(DPX)来自分子探针公司，或。大的单层囊泡包裹ANT或DPX是按照前面描述的方法(Szoka等人，1980)和一些修饰(Elens等人，1989)制备的。在25 mM蚂蚁、45 mM氯化钠和10 mM甘氨酸(pH 9.5)或90 mM DPX/10 mM甘氨酸(pH 9.5)中，将TV-甲基涂料在冰上、氮气下水合3小时。脂类悬浮液接下来经历5次冻融循环，然后通过具有0.1-(Im)孔的聚碳酸酯膜挤压10次(Nucleopore Corp.，Pleasanton，CA)。在Sephadex G-50色谱柱(Pharmacia)上，以100 mM氯化钠，10 mM甘氨酸，0.1 mM EDTA，pH 9.5为洗脱缓冲液，将包封物与非包埋物分离。囊泡在冰上、氮气中储存，并在1天内使用。用负染透射电子显微镜和凝胶层析法表征了囊泡与挤出次数之间的关系。挤出10次后，囊泡大小的均匀性没有进一步改善。此外，也没有观察到多层囊泡的证据。根据电子显微镜的测量，LUV的尺寸在200-900 nm之间，大部分LUV在400 nm附近。
MATERIALS AND METHODSTV-Methyldioleoylphosphatidylethanolamine (TV-methyl-DOPE) and lysophosphatidylcholine (from egg phosphatidylcholine) were obtained from Avanti Polar Lipids, Bir-mingham, AL. Octadecylrhodamine B chloride (Rjg), l-aminonaphthalene-3, 6, 8-trisulfonic acid (disodium salt)(ANTS), andp-xylylenebis (pyridiniumbromide)(DPX) were from Molecular Probes, Inc., Junction City, OR.Vesicle Preparation. Large unilamellar vesicles encapsu-lating ANTS or DPX were prepared according to methods described previously (Szoka et al., 1980) with some modifi-cations (Ellens et al., 1989). TV-Methyl-DOPE was hydrated for 3 h on ice, under N2, either in 25 mM ANTS, 45 mM NaCl, and 10 mM glycine, pH 9.5, or in 90 mM DPX/10 mM glycine, pH 9.5. The lipid suspension was next subjected to 5 freeze-thaw cycles followed by 10 extrusions through a polycarbonate membrane with 0.1-(im pores (Nucleopore Corp., Pleasanton, CA). Encapsulated material was separated from unencapsulated material on a Sephadex G-50 column (Pharmacia) with 100 mM NaCl, 10 mM glycine, and 0.1 mM EDTA, pH 9.5, used as the elution buffer. Vesicles were stored on ice, under N2, and were used within 1 day. Vesicles were characterized by negative-stain transmission electron microscopy and by gel chromatography as a function of the number of extrusions. After 10 extrusions, no further improvement in homogeneity of vesicle size was seen. Also, no evidence of multilamellar vesicles was observed. According to measurements of the electron micrographs, the LUV ranged in size from 200 to 900 nm, with most LUV near 400 nm.