Razinkov V I, Hernandez-Jimenez E I, Mikhalyov I I, Cohen F S, Molotkovsky J G
Frumkin Institute of Electrochemistry, Russian Academy of Sciences, Moscow.
Biochim Biophys Acta. 1997 Oct 2;1329(1):149-58. doi: 10.1016/s0005-2736(97)00103-x.
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 x 10(5) and 10.5 x 10(5) for 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 degrees C for at least 12 h. MUV containing the ganglioside GT1b were 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.
已合成两种新型荧光溶血磷脂酰胆碱探针，用作荧光共振能量转移（FRET）中的供体-受体对：9-蒽乙烯基（LAPC）作为供体，3-苝酰基（LPPC）作为受体。LAPC和LPPC在膜相和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小叶，以评估膜融合后膜不对称性的保持情况。在用探针标记两个小叶制备中型单层囊泡（MUV）后，通过用过量未标记的巨型单层囊泡（GUV）反复洗涤去除外小叶中的探针。通过离心分离MUV和GUV。在25℃下，探针至少12小时内不会跨双层翻转。含有神经节苷脂GT1b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触发融合，并在FRET测定中通过供体荧光的增加进行监测。当仅用LAPC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中去除LAPC时，荧光下降了60%。我们得出结论，融合的MUV/病毒复合物的内小叶和外小叶的脂质混合在一起。
