Simard Jeffrey R, Pillai Biju K, Hamilton James A
Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA.
Biochemistry. 2008 Sep 2;47(35):9081-9. doi: 10.1021/bi800697q. Epub 2008 Aug 12.
Fatty acids (FA) are known to diffuse (flip-flop) rapidly across protein-free phospholipid bilayers in their un-ionized form. However, whether flip-flop through the hydrophobic core of the bilayer or desorption from the membrane into the aqueous phase is the rate-limiting step in FA transport through membranes is still debated. The issue has remained unresolved in part by disagreements over whether some methods of adding FA create artifacts that lead to erroneous conclusions and in part by the lack of fluorescence methods to monitor each individual step. Here we study the kinetics of FA transfer from donors to phospholipid vesicles (small and large unilamellar vesicles) by a dual fluorescence approach that utilizes the probes fluorescein phosphatidylethanolamine (FPE) and pyranine. FPE detects the concentration of FA anions in the outer membrane leaflet, allowing a precise measurement of kinetics of FA adsorption or desorption. Our results showed that as soon as FPE detects adsorption of FA into the outer leaflet, pyranine detects its movement to the inner leaflet. We further demonstrated that (i) flip-flop for FA with 14-22 carbons is much faster than the rates of desorption and therefore cannot be the rate-limiting step of FA translocation across membranes; (ii) fluorescence changes detected by probes located on or in acceptor vesicles are dependent upon the method used to deliver the FA (i.e., uncomplexed, or complexed to albumin or phospholipid bilayers); however, (iii) transfer kinetics observed in the presence of different donors is rate-limited by the desorption of FA from the donor into the aqueous phase rather than by flip-flop.
已知脂肪酸（FA）以非离子化形式在无蛋白的磷脂双分子层中快速扩散（翻转）。然而，FA通过双分子层疏水核心的翻转还是从膜解吸到水相是FA跨膜转运的限速步骤仍存在争议。部分原因是关于添加FA的一些方法是否会产生导致错误结论的假象存在分歧，部分原因是缺乏监测每个单独步骤的荧光方法，这个问题一直未得到解决。在这里，我们通过一种利用荧光素磷脂酰乙醇胺（FPE）和吡喃染料的双荧光方法研究了FA从供体转移到磷脂囊泡（小单层囊泡和大单层囊泡）的动力学。FPE检测外膜小叶中FA阴离子的浓度，从而能够精确测量FA吸附或解吸的动力学。我们的结果表明，一旦FPE检测到FA吸附到外小叶，吡喃染料就会检测到其向内小叶的移动。我们进一步证明：（i）含有14 - 22个碳的FA的翻转比解吸速率快得多，因此不可能是FA跨膜转运的限速步骤；（ii）位于受体囊泡上或内部的探针检测到的荧光变化取决于递送FA的方法（即未复合状态、与白蛋白复合或与磷脂双分子层复合）；然而，（iii）在不同供体存在下观察到的转移动力学受FA从供体解吸到水相的限制，而不是受翻转的限制。
