Bentz J, Alford D, Cohen J, Düzgüneş N
Department of Pharmacy, School of Pharmacy, University of California, San Francisco 94143.
Biophys J. 1988 Apr;53(4):593-607. doi: 10.1016/S0006-3495(88)83138-2.
The fusion of large unilamellar phosphatidylserine liposomes (PS LUV) induced by La3+ has been monitored using the 1-aminoapthalene-3,6,8-trisulfonic acid/p-xylenebis(pyridinium bromide) (ANTS/DPX) fluorescence assay for the mixing of aqueous contents. The fusion event is extensive and nonleaky, with up to 95% mixing of contents in the fused liposomes. However, addition of excess EDTA leads to disruption of the fusion products in a way that implies the existence of metastable intermembrane contact sites. The maximal fusion activity occurs between 10 and 100 microM La3+ and fusion can be terminated rapidly, without loss of contents, by the addition of excess La3+, e.g., 1 mM La3+ at pH 7.4. This observation is explained by the very large intrinsic binding constant (approximately 10(5) M-1) of La3+ to the PS headgroup, as measured by microelectrophoresis. Addition of 1 mM La3+ causes charge reversal of the membrane and a large positive surface potential. La3+ binding to PS causes the release of a proton. These data can be explained if La3+ can chelate to PS at two sites, with one of the sites being the primary amino group. This binding model successfully predicts that at pH 4.5 fusion occurs up to 2 mM La3+, due to reduced La3+ binding at low pH. We conclude that the general mechanism of membrane fusion includes three kinetic steps. In addition to (a) aggregation, there is (b) the close approach of the surfaces, or thinning of the hydration layer, and (c) the formation of intermembrane intermediates which determine the extent to which membrane destabilization leads to fusion (mixing of aqueous contents), as opposed to lysis. The lifetime of these intermembrane intermediates appears to depend upon La3+ binding to both PS sites.
利用1-氨基萘-3,6,8-三磺酸/对二甲苯双（溴化吡啶）（ANTS/DPX）荧光分析法监测水相内容物的混合情况，对La3+诱导的大单层磷脂酰丝氨酸脂质体（PS LUV）融合进行了研究。融合过程广泛且无泄漏，融合脂质体中内容物的混合率高达95%。然而，加入过量的EDTA会导致融合产物的破坏，这意味着存在亚稳态的膜间接触位点。最大融合活性出现在10至100 microM的La3+之间，通过加入过量的La3+（例如在pH 7.4时加入1 mM La3+），融合可以迅速终止，且内容物不会损失。通过微电泳测量发现，La3+与PS头部基团的内在结合常数非常大（约为10(5) M-1），这一观察结果可以解释上述现象。加入1 mM La^3+会导致膜电荷反转和较大的正表面电位。La3+与PS结合会导致质子释放。如果La3+可以在两个位点与PS螯合，其中一个位点是伯氨基，那么这些数据就可以得到解释。该结合模型成功预测，在pH 4.5时，融合会发生至2 mM La3+，这是由于低pH下La3+结合减少所致。我们得出结论，膜融合的一般机制包括三个动力学步骤。除了（a）聚集外，还有（b）表面的紧密靠近或水化层的变薄，以及（c）膜间中间体的形成，这些中间体决定了膜不稳定导致融合（水相内容物混合）而非裂解的程度。这些膜间中间体的寿命似乎取决于La3+与两个PS位点的结合。
