FLUORESCENCE ASSAY FOR PHOSPHOLIPID MEMBRANE ASYMMETRY
FLUORESCENCE ASSAY FOR PHOSPHOLIPID MEMBRANE ASYMMETRY
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DOI：
10.1021/bi00115a012
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发表时间：
1991-12-24
期刊：
影响因子：
2.9
通讯作者：
SLEIGHT, RG
中科院分区：
文献类型：
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作者：
MCINTYRE, JC;SLEIGHT, RG
高荧光7-硝基-2，1，3-苯并恶二唑-4-基-脂质（NBD-脂质）类似物被广泛用于检查脂质转运和膜结构。我们已经开发了一种方法，用于化学修饰NBD标记的脂质在人工和生物膜。这是通过用连二亚硫酸盐（S2 O 4 -2）处理荧光标记的膜来实现的。当含有NBD标记的磷脂的小单层囊泡与连二亚硫酸盐反应时，仅位于囊泡双层的外小叶上的荧光脂质被还原。七种不同的NBD-脂质类似物，包括荧光甾醇，减少了与连二亚硫酸盐的非荧光7-氨基-2，1，3-苯并恶二唑-4-基-脂质衍生物的处理。为了评估在生物系统中使用该试剂的可行性，将N-（7-硝基-2，1，3-苯并恶二唑-4-基）二油酰基-磷脂酰乙醇胺插入CHO-KI细胞质膜的外小叶中。随后将这些细胞与无毒浓度的连二亚硫酸盐孵育，导致质膜荧光完全丧失。相反，当细胞被允许内吞一些荧光标记的质膜，然后用连二亚硫酸盐处理，荧光在质膜被消除，而细胞内标记不受影响。这些数据表明，连二亚硫酸盐反应与NBD标记的脂质在膜双层的外小叶，产生非荧光衍生物。我们演示了如何减少NBD-脂质连二亚硫酸盐可以用来制备不对称标记的脂质体和测量囊泡中的横膜不对称性。这种方法应该是有用的许多生物化学研究，包括磷脂转位酶活性的测量。
Highly fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl-lipid (NBD-lipid) analogues are widely used to examine lipid transport and membrane structure. We have developed a method for chemically modifying NBD-labeled lipids in both artificial and biological membranes. This was achieved by treating fluorescently labeled membranes with dithionite (S2O4-2). When small unilamellar vesicles containing NBD-labeled phospholipids were reacted with dithionite, only the fluorescent lipid located on the outer leaflet of the vesicles' bilayer was reduced. Seven different NBD-lipid analogues, including a fluorescent sterol, were reduced by treatment with dithionite to nonfluorescent 7-amino-2,1,3-benzoxadiazol-4-yl-lipid derivatives. To assess the feasibility of using this reagent in biological systems, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoyl-phosphatidylethanolamine was inserted into the outer leaflet of the plasma membrane of CHO-KI cells. Subsequent incubation of these cells with a nontoxic concentration of dithionite resulted in the complete loss of fluorescence from the plasma membrane. In contrast, when cells were permitted to endocytose some of their fluorescently labeled plasma membrane and then treated with dithionite, fluorescence at the plasma membrane was eliminated, while intracellular labeling was not affected. These data suggest that dithionite reacts with NBD-labeled lipids in the outer leaflet of membrane bilayers, producing nonfluorescent derivatives. We demonstrate how reduction of NBD-lipids with dithionite can be used to prepare asymmetrically labeled liposomes and to measure transverse-membrane asymmetry in vesicles. This method should be useful in many biochemical investigations, including the measurement of phospholipid translocase activity.