Cupp David, Kampf J Patrick, Kleinfeld Alan M
Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121, USA.
Biochemistry. 2004 Apr 20;43(15):4473-81. doi: 10.1021/bi036335l.
Understanding the mechanism that governs the transport of long chain free fatty acids (FFA) across lipid bilayers is critical for understanding transport across cell membranes. Conflicting results have been reported for lipid vesicles; most investigators report that flip-flop occurs within the resolution time of the method (<5 ms) and that dissociation from the membrane is rate limiting, while other studies find that flip-flop is rate limiting and on the order of seconds. We have reinvestigated this problem and find that the methods used in studies reporting rapid flip-flop have not been interpreted correctly. We find that accurate information about transport of FFA across lipid vesicles requires that FFA be delivered to the vesicles as complexes with albumin (BSA). For example, we find that stopped-flow mixing of uncomplexed FFA with small unilamellar vesicles (SUV) containing pyranine yields the very fast influx rates reported previously (>100 s(-1)). However, these influx rates increase linearly with lipid vesicle concentration and can therefore not, as previously interpreted, represent flip-flop. In contrast, measurements of influx rates in SUV and giant unilamellar vesicles performed with oleate-BSA complexes reveal no dependence on vesicle concentration and yield influx rate constants of approximately 4 and approximately 0.5 s(-1), respectively. Rate constants for efflux and dissociation were determined from the transfer of oleate from vesicles to BSA and reveal similar influx and efflux but dissociation rate constants that are approximately 5-10-fold greater. We conclude that flip-flop is rate limiting for transport of FFA across lipid vesicles and slows with an increasing radius of curvature. These results, in contrast to those reporting that flip-flop is extremely fast, indicate that the lipid bilayer portion of biological membranes may present a significant barrier to transport of FFA across cell membranes.
了解长链游离脂肪酸（FFA）跨脂质双层转运的机制对于理解跨细胞膜的转运至关重要。关于脂质囊泡的研究结果相互矛盾；大多数研究者报告称，翻转发生在该方法的分辨时间内（<5毫秒），且从膜上解离是限速步骤，而其他研究发现翻转是限速步骤，且在秒的量级。我们重新研究了这个问题，发现报告快速翻转的研究中所使用的方法未得到正确解读。我们发现，要获得关于FFA跨脂质囊泡转运的准确信息，需要将FFA作为与白蛋白（BSA）的复合物递送至囊泡。例如，我们发现将未复合的FFA与含有吡喃荧光素的小单层囊泡（SUV）进行停流混合，会产生先前报道的非常快的内流速率（>100 s⁻¹）。然而，这些内流速率随脂质囊泡浓度呈线性增加，因此，如先前所解读的那样，它们并不能代表翻转。相比之下，用油酸 - BSA复合物对SUV和大单层囊泡进行的内流速率测量显示，其与囊泡浓度无关，且内流速率常数分别约为4和约0.5 s⁻¹。通过油酸从囊泡向BSA的转移确定了外流和解离的速率常数，结果显示内流和外流速率相似，但解离速率常数大约大5 - 10倍。我们得出结论，翻转是FFA跨脂质囊泡转运的限速步骤，并且随着曲率半径的增加而减慢。这些结果与那些报告翻转极其快速的结果相反，表明生物膜的脂质双层部分可能对FFA跨细胞膜的转运构成重大障碍。
