Abstract
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 approximately 12 and 15 Angstroms(3), 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.
Highlights
Protein-mediated mechanism would necessitate slow diffusion across the lipid phase, and understanding the mechanism of transport across simple lipid membranes has received considerable attention [5,6,7,8,9,10,11,12,13]
Stopped-flow measurements were carried out to determine the three rate constants that define free fatty acid (FFA) transport across lipid vesicles: 1) transfer of FFA from bovine serum albumin (BSA) to the vesicle while monitoring trapped pyranine or Acrylodan-labeled rat intestinal fatty acid-binding protein (ADIFAB), 2) transfer of FFA from the vesicle to BSA while monitoring trapped pyranine or ADIFAB, and 3) transfer of FFA from the vesicle to BSA while monitoring BSA tryptophan fluorescence. This approach was used in our previous study of oleate at 22 °C [13] and has been extended to determine rate constants for 13 FFA using SUV, LUV, and GUV at temperatures between 15 and 37 °C
The results reveal a monotonic increase in all three rate constants with decreasing FFA chain length
Summary
Protein-mediated mechanism would necessitate slow diffusion across the lipid phase, and understanding the mechanism of transport across simple lipid membranes has received considerable attention [5,6,7,8,9,10,11,12,13]. Previous conclusions [7, 11] that flip-flop was rapid and that dissociation was rate-limiting were based on incorrect interpretations of the results, primarily measurements of oleate influx into vesicles using oleate that was not complexed with serum albumin. The studies of Cupp et al [13] revealed that flip-flop represents a major barrier to transport of oleate across the lipid bilayer and that this barrier increases with increasing vesicle diameter from SUV (ϳ250 Å) to LUV and GUV (Ͼ1000 Å). The lipid phase barrier to FFA flip-flop, at least in certain cell membranes, might be large enough so that the cell’s FFA metabolic requirements would necessitate a membrane protein transporter. If the polymer-like nature of the bilayer affects FFA flip-flop, both the FFA size and temperature dependence of transport should be significantly different from those expected for a simple organic fluid
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