Niles W D, Eisenberg M
Biophys J. 1985 Aug;48(2):321-5. doi: 10.1016/S0006-3495(85)83785-1.
The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.
两个磷脂膜融合的第一步以两个脂质双层的聚集而告终。我们使用了一台定制的荧光计来检测含有与平面脂质双层（BLM）结合的荧光染料6-羧基荧光素（6-CF）的多层囊泡（脂质体）。将脂质体添加到BLM的一侧，未结合的囊泡被灌注出去。这使得BLM产生了残余荧光，但只有当膜中含有阴离子脂质时才会出现，而且只有在存在毫摩尔水平的钙时才会出现。只有在灌注过程中缓冲液中含有钙时，才能持续检测到这种残余荧光。这种残余荧光源自与BLM结合的脂质体。用蒸馏水破坏BLM或裂解吸附的囊泡会消除这种荧光。在没有脂质体的情况下，游离的6-CF和/或钙不会产生残余荧光。当脂质体和BLM都完全由两性离子脂质组成时，未检测到残余荧光。这被发现是由于荧光计对吸附在BLM上的少量脂质体不敏感所致。对于这个系统，我们得出结论，当膜含有带负电荷的脂质时，钙对于囊泡膜与平面双层结合状态的起始和维持都是必需的。这种附着比两性离子膜之间的相互作用更强。
