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J. Biol. Chem., Vol. 281, Issue 30, 21566-21574, July 28, 2006

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Different Mechanisms of Free Fatty Acid Flip-Flop and Dissociation Revealed by Temperature and Molecular Species Dependence of Transport across Lipid Vesicles^*

From the Torrey Pines Institute for Molecular Studies, San Diego, California 92121

The mechanism of free fatty acid (FFA) transport across membranes is a subject of intense investigation. We have demonstrated recently that flip-flop is the rate-limiting step for transport of oleic acid across phospholipid vesicles (Cupp, D., Kampf, J. P., and Kleinfeld, A. M. (2004) Biochemistry 43, 4473-4481). To better understand the nature of the flip-flop barrier, we measured the temperature dependence of a series of saturated and monounsaturated FFA. We determined the rate constants for flip-flop and dissociation for small (SUV), large (LUV), and giant (GUV) unilamellar vesicles composed of egg phosphatidylcholine. For all FFA and vesicle types, dissociation was faster than flip-flop, and for all FFA, flip-flop and dissociation were faster in SUV than in LUV or GUV. Rate constants for both flip-flop and dissociation decreased exponentially with increasing FFA size. However, only the flip-flop rate constants increased significantly with temperature; the barrier to flip-flop was virtually entirely due to an enthalpic activation free energy. The barrier to dissociation was primarily entropic. Analysis in terms of a simple free volume (V_f) model revealed V_f values for flip-flop that ranged between 12 and 15ų, with larger values for SUV than for LUV or GUV. V_f values increased with temperature, and this temperature dependence generated the enthalpic barrier to flip-flop. The barrier for dissociation and its size dependence primarily reflect the aqueous solubility of FFA. These are the first results to distinguish the energetics of flipflop and dissociation. This should lead to a better understanding of the mechanisms governing FFA transport across biological membranes.

Received for publication, March 6, 2006 , and in revised form, May 29, 2006.

^* This work was supported by NIDDK Grant DK058762 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Torrey Pines Inst. for Molecular Studies, 3550 General Atomics Ct., San Diego, CA 92121. Tel.: 858-455-3724; Fax: 858-455-3792; E-mail: akleinfeld{at}tpims.org.

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