Kamp Frits, Guo Wen, Souto Ricardo, Pilch Paul F, Corkey Barbara E, Hamilton James A
Obesity Research Center, Boston Medical Center, Massachusetts 02118, USA.
J Biol Chem. 2003 Mar 7;278(10):7988-95. doi: 10.1074/jbc.M206648200. Epub 2002 Dec 23.
Nonesterified long-chain fatty acids may enter cells by free diffusion or by membrane protein transporters. A requirement for proteins to transport fatty acids across the plasma membrane would imply low partitioning of fatty acids into the membrane lipids, and/or a slower rate of diffusion (flip-flop) through the lipid domains compared to the rates of intracellular metabolism of fatty acids. We used both vesicles of the plasma membrane of adipocytes and intact adipocytes to study transmembrane fluxes of externally added oleic acid at concentrations below its solubility limit at pH 7.4. Binding of oleic acid to the plasma membrane was determined by measuring the fluorescent fatty acid-binding protein ADIFAB added to the external medium. Changes in internal pH caused by flip-flop and metabolism were measured by trapping a fluorescent pH indicator in the cells. The metabolic end products of oleic acid were evaluated over the time interval required for the return of intracellular pH to its initial value. The primary findings were that (i) oleic acid rapidly binds with high avidity in the lipid domains of the plasma membrane with an apparent partition coefficient similar to that of protein-free phospholipid bilayers; (ii) oleic acid rapidly crosses the plasma membrane by the flip-flop mechanism (both events occur within 5 s); and (iii) the kinetics of esterification of oleic acid closely follow the time dependence of the recovery of intracellular pH. Any postulated transport mechanism for facilitating translocation of fatty acid across the plasma membrane of adipocytes, including a protein transporter, would have to compete with the highly effective flip-flop mechanism.
非酯化长链脂肪酸可通过自由扩散或膜蛋白转运体进入细胞。脂肪酸跨质膜转运需要蛋白质这一点意味着脂肪酸在膜脂中的分配率较低，和/或与脂肪酸的细胞内代谢速率相比，其通过脂域的扩散（翻转）速率较慢。我们使用脂肪细胞质膜囊泡和完整脂肪细胞来研究在pH 7.4时外部添加的油酸浓度低于其溶解度极限时的跨膜通量。通过测量添加到外部培养基中的荧光脂肪酸结合蛋白ADIFAB来确定油酸与质膜的结合。通过在细胞中捕获荧光pH指示剂来测量由翻转和代谢引起的内部pH变化。在细胞内pH恢复到其初始值所需的时间间隔内评估油酸的代谢终产物。主要发现是：（i）油酸以高亲和力迅速结合在质膜的脂域中，其表观分配系数与无蛋白磷脂双层相似；（ii）油酸通过翻转机制迅速穿过质膜（这两个事件都在5秒内发生）；（iii）油酸酯化的动力学密切遵循细胞内pH恢复的时间依赖性。任何假定的促进脂肪酸跨脂肪细胞质膜转运的机制，包括蛋白质转运体，都必须与高效的翻转机制竞争。
