Transport of long-chain native fatty acids across human erythrocyte ghost membranes

Evidence from a number of laboratories suggests that membrane proteins may meditate the transport of physiologic fatty acids (FA) across cell membranes. However, actual transport of unbound free fatty acids (unbound FFA) from the aqueous phase on one side of a cell membrane to the aqueous phase on the other side has not been measured previously. In this study, we have used the fluorescent probe of unbound FFA, 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 red cell ghosts. 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 red cell ghosts. Measurements done using palmitate, oleate, and linoleate at temperatures between 20 and 37 degreesC revealed that the overall transport times ranged from about 0.5 to more than 10 s, depending upon FA type and temperature. Analysis of these time courses yielded kff values between 0.3 and 3.0 s-1, and these values were consistent with those obtained using ghosts containing pyranine to detect intracellular acidification by the translocating FA. The measured koff values ranged from about 0.3 to 5 s-1, while the rate of binding, for the ghost concentrations used in this study (>50 microM phospholipid), exceed both kff and koff. Thus, long-chain FA transport across red cell ghost membranes is rate-limited by a combination of flip-flop and dissociation rates. Binding of FA to ghost membranes was well described by simple, nonsaturable, aqueous/membrane partition, and that partition appears to be governed by the aqueous solubility of the FA. Transport rates did not reveal any evidence of saturation and were not affected by a variety of protein-specific reagents. These FA binding and transport characteristics are similar to those observed previously for lipid vesicles, although the rate constants are generally about 2-3 fold larger for ghosts as compared to the lipid vesicles. We suggest, therefore, that FA transport across red cell ghosts is reasonably well described by transport across the lipid phase of the membrane.