Victorov A V, Janes N, Taraschi T F, Hoek J B
Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Biophys J. 1997 Jun;72(6):2588-98. doi: 10.1016/S0006-3495(97)78902-1.
The energetic determinants of the distribution of anionic phospholipids across a phosphatidylcholine (PtdCho) bilayer with different packing constraints in the two leaflets were studied, using (13)CH2-ethyl-labeled phosphatidylethanol (PtdEth) as a (13)C NMR membrane probe. PtdEth is unique in exhibiting a split (13)CH2-ethyl resonance in sonicated vesicles, the two components originating from the inner and outer leaflets, thus permitting the determination of the PtdEth concentration in each leaflet. Small and large unilamellar PtdEth-PtdCho vesicles were prepared in solutions of different ionic strengths. A quantitative expression for the transbilayer distribution of PtdEth, based on the balance between steric and electrostatic factors, was derived. The transbilayer difference in packing constraints was obtained from the magnitude of the PtdEth signal splitting. The electrostatic contribution could be satisfactorily described by the transmembrane difference in Gouy-Chapman surface potentials. At low (0.1-0.25%) PtdEth levels and high (up to 500 mM) salt concentrations, PtdEth had a marked fivefold preference for the inner leaflet, presumably because of its small headgroup, which favors tighter packing. At higher PtdEth content (4.8-9.1%) and low salt concentrations, where electrostatic repulsion becomes a dominant factor, the asymmetry was markedly reduced and an almost even distribution across the bilayer was obtained. In less curved, large vesicles, where packing constraints in the two leaflets are approximately the same, the PtdEth distribution was almost symmetrical. This study is the first quantitative analysis of the balance between steric and electrostatic factors that determines the equilibrium transbilayer distribution of charged membrane constituents.
使用(13)CH2 - 乙基标记的磷脂酰乙醇（PtdEth）作为(13)C NMR膜探针，研究了阴离子磷脂在磷脂酰胆碱（PtdCho）双层膜中跨膜分布的能量决定因素，该双层膜的两个小叶具有不同的堆积限制。PtdEth在超声处理的囊泡中呈现分裂的(13)CH2 - 乙基共振峰，这一独特现象使得两个组分分别源自内小叶和外小叶，从而能够确定每个小叶中的PtdEth浓度。在不同离子强度的溶液中制备了大小不同的单层PtdEth - PtdCho囊泡。基于空间和静电因素之间的平衡，推导了PtdEth跨膜分布的定量表达式。从PtdEth信号分裂的幅度获得跨膜堆积限制的差异。静电贡献可以通过Gouy - Chapman表面电位的跨膜差异得到令人满意的描述。在低（0.1 - 0.25%）PtdEth水平和高（高达500 mM）盐浓度下，PtdEth对内小叶有明显的五倍偏好，这可能是因为其较小的头部基团有利于更紧密的堆积。在较高的PtdEth含量（4.8 - 9.1%）和低盐浓度下，静电排斥成为主导因素，不对称性明显降低，在双层膜中获得了几乎均匀的分布。在曲率较小的大囊泡中，两个小叶的堆积限制大致相同，PtdEth的分布几乎是对称的。这项研究首次对决定带电膜成分平衡跨膜分布的空间和静电因素之间的平衡进行了定量分析。
