Kleinfeld A M, Chu P, Romero C
Medical Biology Institute, 11077 North Torrey Pines Rd., La Jolla, California 92037, USA.
Biochemistry. 1997 Nov 18;36(46):14146-58. doi: 10.1021/bi971440e.
Evidence from a number of laboratories suggests that membrane proteins may meditate the transport of physiologic fatty acids (FA) across cell membranes. However, studies using lipid membranes indicate that FA are capable of spontaneous flip-flip, raising the possibility that rapid transport through the lipid phase obviates the need for a transport protein. Determining the rate-limiting steps for transport of FA across lipid membranes, therefore, is central to understanding FA transport across cell membranes. The transport of long-chain FA across lipid membranes, from the aqueous compartment on one side of the lipid bilayer to the aqueous phase on the other side, has not been measured previously. In this study, we have used the fluorescent probe ADIFAB to monitor the time course of FA movement from the outer to the inner aqueous compartments and from the lipid membrane to the outer aqueous compartment of lipid vesicles. These two measurements, together with measurements of the lipid:aqueous partition coefficients, allowed the determination of the rate constants for binding (kon), flip-flop (kff), and dissociation (koff) for the transport of long-chain natural FA across lipid vesicles. These rates were determined using large unilamellar vesicles (LUV) of approximately 1000 A diameter, prepared by extrusion and giant unilamellar vesicles (GUV), prepared by detergent dialysis, that are >/=2000 A diameter. The results of these studies for vesicles composed of egg phosphatidylcholine (EPC) and cholesterol reveal kff values that range from 3 to 15 s-1 for LUV and from 0.1 to 1.0 s-1 for GUV, depending upon temperature and FA type. For these same vesicles, dissociation rate constants range from 4 to 40 s-1 for LUV and from 0.3 to 2.5 s-1 for GUV. In all instances, the rate constant for flip-flop is smaller than koff, and because the rate of binding is greater than the rate of transport, we conclude that flip-flop is the rate-limiting step for transport. These results demonstrate that (1) kff and koff are smaller for GUV than for LUV, (2) the rate constants increase with FA type according to oleate (18:1) < palmitate (16:0) < linoleate (18:2), and (3) the barrier for flip-flop has a significant enthalpic component. Comparison of the flip-flop rates determined for GUV with values estimated from previously reported metabolic rates for cardiac myocytes, raises the possibility that flip-flop across the lipid phase alone may not be able to support metabolic requirements.
多个实验室提供的证据表明，膜蛋白可能介导生理性脂肪酸（FA）跨细胞膜的转运。然而，利用脂质膜进行的研究表明，FA能够自发翻转，这增加了一种可能性，即通过脂质相的快速转运使得不需要转运蛋白。因此，确定FA跨脂质膜转运的限速步骤，对于理解FA跨细胞膜的转运至关重要。此前尚未测量过长链FA从脂质双层一侧的水相区室跨脂质膜转运到另一侧水相的过程。在本研究中，我们使用荧光探针ADIFAB来监测FA从脂质囊泡的外部水相区室移动到内部水相区室以及从脂质膜移动到外部水相区室的时间进程。这两项测量，连同脂质与水的分配系数测量，使得能够确定长链天然FA跨脂质囊泡转运的结合速率常数（kon）、翻转速率常数（kff）和解离速率常数（koff）。这些速率是使用通过挤压制备的直径约为1000 Å的大单层囊泡（LUV）以及通过去污剂透析制备的直径≥2000 Å的巨型单层囊泡（GUV）来测定的。这些针对由鸡蛋磷脂酰胆碱（EPC）和胆固醇组成的囊泡的研究结果表明，LUV的kff值范围为3至15 s-1，GUV的kff值范围为0.1至1.0 s-1，具体取决于温度和FA类型。对于这些相同的囊泡，LUV的解离速率常数范围为4至40 s-1，GUV的解离速率常数范围为0.3至2.5 s-1。在所有情况下，翻转速率常数均小于koff，并且由于结合速率大于转运速率，我们得出结论，翻转是转运的限速步骤。这些结果表明：（1）GUV的kff和koff小于LUV；（2）速率常数根据油酸（18:1）＜棕榈酸（16:0）＜亚油酸（18:2）的顺序随FA类型增加；（3）翻转的屏障具有显著的焓成分。将GUV测定的翻转速率与根据先前报道的心肌细胞代谢速率估算的值进行比较，增加了仅通过脂质相的翻转可能无法满足代谢需求的可能性。
