New fluorescent lysolipids: preparation and selective labeling of inner liposome leaflet.
New fluorescent lysolipids: preparation and selective labeling of inner liposome leaflet.
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新型荧光溶血脂：内脂质体小叶的制备和选择性标记。
DOI：
10.1016/s0005-2736(97)00103-x
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Molotkovsky,JG
中科院分区：
文献类型：
--
作者：
Razinkov,VI;Hernandez-Jimenez,EI;Mikhalyov,II;Cohen,FS;Molotkovsky,JG
合成了两种新的溶血磷脂酰胆碱荧光探针，作为荧光共振能量转移（FRET）的供体-受体对：9-蒽乙烯基（LAPC）为供体，3-芘酰（LPPC）为受体。LAPC和LPPC在膜与水相之间的分配系数分别为8.3× 105和10.5× 105。用这些探针标记单层脂质囊泡的内部小叶，以评估膜融合后膜侧性的保护。在两个小叶中用探针制备中等大小的单层囊泡（MUV）后，通过用过量的未标记的巨型单层囊泡（GUV）反复洗涤除去外小叶中的探针。通过离心分离MUV和GUV。探针在25°C下至少12小时内不会在双层上翻转。含有神经节苷脂GT 1b的MUV在内小叶中用LAPC/LPPC对标记，并在中性pH下与流感病毒孵育30分钟。通过酸化至pH 5.0来触发融合，并通过FRET测定中供体荧光的增加来监测融合。当用LAPC单独标记MUV的内小叶时，融合后其荧光没有变化。然而，当通过用GUV反复洗涤从融合膜的外小叶去除LAPC时，荧光降低了60%。我们得出结论，脂质的内部和外部小叶的融合MUV/病毒复合物相互混合。
Two new fluorescent lysophosphatidylcholine probes have been synthesized for use as a donor–acceptor pair in fluorescence resonance energy transfer (FRET): 9-anthrylvinyl (LAPC) as donor and 3-perylenoyl (LPPC) as acceptor. The partition coefficients between membrane and aqueous phases were 8.3×105and 10.5×105for LAPC and LPPC, respectively. The inner leaflets of unilamellar lipid vesicles were labeled with these probes to assess conservation of membrane sidedness after membrane fusion. After medium-sized unilamellar vesicles (MUV) were prepared with a probe in both leaflets, probe in the outer leaflet was removed by repeatedly washing with an excess of unlabeled giant unilamellar vesicles (GUV). MUV and GUV were separated by centrifugation. The probes did not flip-flop across bilayers at 25°C for at least 12 h. MUV containing the ganglioside GT1bwere labeled with the LAPC/LPPC pair in the inner leaflet and incubated for 30 min at neutral pH with influenza virus. Fusion was triggered by acidification to pH 5.0 and was monitored by an increase in donor fluorescence in a FRET assay. When the inner leaflets of MUV were labeled by LAPC only, its fluorescence did not change after fusion. However, the fluorescence decreased by 60% when the LAPC was removed from the outer leaflets of the fused membranes by repeated washings with GUV. We conclude that the lipids of the inner and outer leaflets of the fused MUV/virus complexes intermixed.