Flip-flop is the rate-limiting step for transport of free fatty acids across lipid vesicle membranes.
The mechanism of transport of free fatty acids (FFA) across lipid bilayer membranes remains a subject of debate. The debate is whether the rate-limiting step for transport is flip-flop across the membrane or dissociation into the aqueous phase. Recently, a new method for assessing dissociation was described in which fluorescein phosphatidylethanolamine (FPE) introduced into the outer leaflet of lipid vesicles was used to monitor FFA dissociation. Transport of FFA into vesicles containing both FPE in the outer leaflet and pyranine trapped in the inside aqueous phase revealed identical rate constants for quenching of FPE and pyranine fluorescence. Because no difference was observed in the time for FFA binding to the outer surface and flip-flop across the bilayer, it was concluded that dissociation was slower than flip-flop. Here, we used FPE and BSA to assess dissociation of oleate from lipid vesicles. In separate pyranine- or ADIFAB-containing vesicles, we assessed flip-flop. We found that the FPE and BSA transfer methods yielded equivalent rate constants for dissociation, which were 3-10-fold faster than that of flip-flop. We found that in vesicles containing both FPE and pyranine, pyranine fluorescence cannot be separated from FPE fluorescence. The identical rate constants for FPE and pyranine observed with vesicles containing both fluorophores reflected the dominance (20-fold) of FPE fluorescence at pyranine excitation and emission wavelengths. Because the dissociation rate constants are 3-10 times faster than the rate constants for flip-flop, flip-flop must be the rate-limiting step for the transport of FFA across lipid vesicles.
Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121, USA.
Andrew N
AN
Carley
Alan M
AM
Kleinfeld
D001692:Biological Transport / Q000502:physiology*; D005230:Fatty Acids, Nonesterified / Q000737:chemistry* / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism
D005230:Fatty Acids, Nonesterified; D008051:Lipid Bilayers; D010712:Phosphatidic Acids; D012710:Serum Albumin, Bovine