USE OF RESONANCE ENERGY-TRANSFER TO MONITOR MEMBRANE-FUSION
USE OF RESONANCE ENERGY-TRANSFER TO MONITOR MEMBRANE-FUSION
复制标题
DOI：
10.1021/bi00517a023
复制
发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
PAGANO, RE
中科院分区：
文献类型：
--
作者：
STRUCK, DK;HOEKSTRA, D;PAGANO, RE
建立了一种以N-（7-硝基-2，1，3-苯并恶二唑-4-基）为能量供体，罗丹明为能量受体的囊泡-囊泡融合共振能量转移检测方法。2个荧光团与磷脂酰乙醇胺的游离氨基偶联，以提供可掺入脂囊泡双层的类似物。当两种荧光脂质以适当的表面密度（荧光脂质与总脂质的比率）存在于磷脂酰丝氨酸囊泡中时，观察到有效的能量转移。当这样的囊泡通过添加Ca与纯磷脂酰丝氨酸囊泡的群体融合时，2个探针与存在的其他脂质混合以形成新的膜。这种混合降低了能量受体的表面密度，导致实验测量的共振能量转移效率降低。转移效率的这些变化允许融合过程的动力学和定量测量。使用该系统，研究了磷脂酰胆碱、磷脂酰丝氨酸和磷脂酰胆碱-磷脂酰丝氨酸（1：1）囊泡与培养的成纤维细胞[中国仓鼠V79]融合的能力。在所使用的条件下，囊泡脂质的大部分细胞摄取可归因于完整囊泡吸附到细胞表面，而不管囊泡双层的组成。
An assay for vesicle-vesicle fusion involving resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl), the energy donor, and rhodamine, the energy acceptor, was developed. The 2 fluorophores are coupled to the free amino group of phosphatidylethanolamine to provide analogs which can be incorporated into a lipid vesicle bilayer. When both fluorescent lipids are in phosphatidylserine vesicles at appropriate surface densities (ratio of fluorescent lipid to total lipid), efficient energy transfer is observed. When such vesicles are fused with a population of pure phosphatidylserine vesicles by the addition of Ca, the 2 probes mix with the other lipids present to form a new membrane. This mixing reduces the surface density of the energy acceptor resulting in a decreased efficiency of resonance energy transfer which is measured experimentally. These changes in transfer efficiency allow kinetic and quantitative measurements of the fusion process. Using this system, the ability of phosphatidylcholine, phosphatidylserine and phosphatidylcholine-phosphatidylserine (1:1) vesicles to fuse with cultured fibroblasts [Chinese hamster V79] was studied. Under the conditions used, the majority of the cellular uptake of vesicle lipid could be attributed to the adsorption of intact vesicles to the cell surface regardless of the composition of the vesicle bilayer.