PH GRADIENTS ACROSS PHOSPHOLIPID-MEMBRANES CAUSED BY FAST FLIP-FLOP OF UNIONIZED FATTY-ACIDS
PH GRADIENTS ACROSS PHOSPHOLIPID-MEMBRANES CAUSED BY FAST FLIP-FLOP OF UNIONIZED FATTY-ACIDS
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DOI：
10.1073/pnas.89.23.11367
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发表时间：
1992-12-01
影响因子：
11.1
通讯作者：
HAMILTON, JA
中科院分区：
文献类型：
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作者：
KAMP, F;HAMILTON, JA
细胞生理学中一个尚未解决的中心问题是脂肪酸如何穿过细胞膜，以及是否需要蛋白质来促进跨双层运动。我们已经开发了一种方法，用于监测运动的脂肪酸跨蛋白质的模型膜（磷脂双层）。在Hepes缓冲液（pH 7.4）中，将水溶性、pH敏感的荧光分子吡喃素捕获在密封良好的磷脂酰胆碱囊泡（含或不含胆固醇）内。在加入长链脂肪酸（例如，油酸）加入到外部缓冲液（也是Hepes，pH 7.4）中，立即观察到吡喃苷荧光的降低（在10秒内）。这种内部体积的酸化是非离子化脂肪酸"翻转"到内部小叶的结果，随后是几乎等于50%的这些脂肪酸分子释放质子（双层中的表观pK（a）= 7.6）。由此产生的质子梯度消散缓慢，因为缓慢的循环质子转移脂肪酸。将牛血清白蛋白添加到具有脂肪酸的囊泡中立即去除pH梯度，表明脂肪酸的完全去除，这需要脂肪酸从内单层到外单层的快速"翻转"。使用膜中脂肪酸的四态动力学图，我们得出结论，非电离脂肪酸触发器迅速（t1/2小于或等于2秒），而电离脂肪酸触发器缓慢（t1/2分钟）。由于脂肪酸自发和快速地穿过磷脂酰胆碱双层，因此复杂的机制（例如，转运蛋白）可能不是生物膜中脂肪酸转运所必需的。伴随脂肪酸翻转的质子运动是正常生理学和疾病状态如心脏缺血中脂肪酸代谢的重要考虑因素。
A central, unresolved question in cell physiology is how fatty acids move across cell membranes and whether protein(s) are required to facilitate transbilayer movement. We have developed a method for monitoring movement of fatty acids across protein-free model membranes (phospholipid bilayers). Pyranin, a water-soluble, pH-sensitive fluorescent molecule, was trapped inside well-sealed phosphatidylcholine vesicles (with or without cholesterol) in Hepes buffer (pH 7.4). Upon addition of a long-chain fatty acid (e.g., oleic acid) to the external buffer (also Hepes, pH 7.4), a decrease in fluorescence of pyranin was observed immediately (within 10 sec). This acidification of the internal volume was the result of the "flip" of un-ionized fatty acids to the inner leaflet, followed by a release of protons from almost-equal-to 50% of these fatty acid molecules (apparent pK(a) in the bilayer = 7.6). The proton gradient thus generated dissipated slowly because of slow cyclic proton transfer by fatty acids. Addition of bovine serum albumin to vesicles with fatty acids instantly removed the pH gradient, indicating complete removal of fatty acids, which requires rapid "flop" of fatty acids from the inner to the outer monolayer layer. Using a four-state kinetic diagram of fatty acids in membranes, we conclude that un-ionized fatty acid flip-flops rapidly (t1/2 less-than-or-equal-to 2 sec) whereas ionized fatty acid flip-flops slowly (t1/2 of minutes). Since fatty acids move across phosphatidylcholine bilayers spontaneously and rapidly, complex mechanisms (e.g., transport proteins) may not be required for translocation of fatty acids in biological membranes. The proton movement accompanying fatty acid flip-flop is an important consideration for fatty acid metabolism in normal physiology and in disease states such as cardiac ischemia.