La3+-induced fusion of phosphatidylserine liposomes. Close approach, intermembrane intermediates, and the electrostatic surface potential.
La3+-induced fusion of phosphatidylserine liposomes. Close approach, intermembrane intermediates, and the electrostatic surface potential.
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La3 诱导的磷脂酰丝氨酸脂质体融合。
DOI：
10.1016/s0006-3495(88)83138-2
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发表时间：
1988
影响因子：
3.4
通讯作者：
Duzgunes,N
中科院分区：
文献类型：
--
作者：
Bentz,J;Alford,D;Cohen,J;Duzgunes,N
用1-氨基-3，6，8-三磺酸/对二甲双吡啶(ANTS/DPX)荧光混合水溶液监测了La3+诱导的大分子单层磷脂酰丝氨酸脂质体(PS LUV)的熔融过程。融合事件广泛且无泄漏，融合脂质体中的内容物混合高达95%。然而，过量的EDTA的加入会导致融合产物的破坏，这意味着存在亚稳定的膜间接触位点。最大的融合活性发生在10到100微米的La3+之间，并且通过添加过量的La3+，例如在pH 7.4时加入1 mM的La3+，可以快速终止融合，而不损失内容物。用微电泳法测得La~(3+)与PS头基团的结合常数非常大(约10(5)M~(-1))，从而解释了这种现象。添加1 mM的La3+会导致膜电荷反转和较大的正表面电位。La3+与PS的结合导致质子的释放。这些数据可以解释如果La3+可以在两个位置与PS络合，其中一个位置是伯氨基。这个结合模型成功地预测了在pH 4.5时，由于低pH时La3+结合减少，发生了高达2 mM的La3+融合。我们认为，膜融合的一般机理包括三个动力学步骤。除了(A)聚集，还有(B)表面的接近或水化层变薄，以及(C)膜间中间体的形成，这决定了膜失稳导致融合(含水内容物的混合)的程度，而不是溶解。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。
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.