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 cel l membranes. However, actual transport of unbound free fatty acids (un bound FFA) from the aqueous phase on one side of a cell membrane to th e 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 inne r aqueous compartments, and from the lipid membrane to the outer aqueo us compartment of red cell ghosts. These two measurements, together wi th measurements of the lipid/aqueous partition coefficients, allowed t he determination of the rate constants for binding (k(on)), flip-flop (k(ff)), and dissociation (k(off)) for the transport of long-chain nat ural FA across red cell ghosts. Measurements done using palmitate, ole ate, and linoleate at temperatures between 20 and 37 degrees C reveale d 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 c ourses yielded k(ff) values between 0.3 and 3.0 s(-1), and these value s were consistent with those obtained using ghosts containing pyranine to detect intracellular acidification by the translocating FA. The me asured k(off) values ranged from about 0.3 to 5 s(-1), while the rate of binding, for the ghost concentrations used in this study (> 50 mu M phospholipid), exceed both k(ff) and k(off). Thus, long-chain FA tran sport 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 partitio n, and that partition appears to be governed by the: aqueous solubilit y of the FA. Transport rates did not reveal any evidence of saturation and were not affected by a variety of protein-specific reagents. Thes e FA binding and transport characteristics are similar to those observ ed previously for lipid vesicles, although the rate constants are gene rally about 2-3 fold larger for ghosts as compared to the lipid vesicl es. We suggest, therefore, that FA transport across red cell ghosts is reasonably well described by transport across the lipid phase of the membrane.