TRANSPORT OF LONG-CHAIN NATIVE FATTY-ACIDS ACROSS LIPID BILAYER-MEMBRANES INDICATES THAT TRANSBILAYER FLIP-FLOP IS RATE-LIMITING

Evidence from a number of laboratories suggests that membrane proteins may meditate the transport of physiologic fatty acids (FA) across cel l membranes, However, studies using lipid membranes indicate that FA a re capable of spontaneous flip-flip, raising the possibility that rapi d transport through the lipid phase obviates the need for a transport protein. Determining the rate-limiting steps for transport of FA acros s lipid membranes, therefore, is central to understanding FA transport across cell membranes. The transport of long-chain FA across lipid me mbranes, from the aqueous compartment on one side of the lipid bilayer to the aqueous phase on the other side, has not been measured previou sly, In this study, we have used the fluorescent probe ADIFAB to monit or the time course of FA movement from the outer to the inner aqueous compartments and from the lipid membrane to the outer aqueous compartm ent of lipid vesicles. These two measurements, together with measureme nts of the lipid:aqueous partition coefficients, allowed the determina tion of the rate constants for binding (k(on)), flip-flop (k(ff)), and dissociation (K-off) for the transport of long-chain natural FA acros s lipid vesicles, These rates were determined using large unilamellar vesicles (LUV) of approximately 1000 Angstrom diameter, prepared by ex trusion and giant unilamellar vesicles (GUV), prepared by detergent di alysis, that an greater than or equal to 2000 Angstrom diameter. The r esults of these studies for vesicles composed of egg phosphatidylcholi ne (EPC) and cholesterol reveal k(ff) values that range from 3 to 15 s (-1) for LUV and from 0.1 to 1.0 s(-1) for GUV, depending upon tempera ture and FA type. For these same vesicles, dissociation rate constants range from 3 to 40 s(-1) for LUV and from 0.3 to 2.5 s(-1) for GUV. I n all instances, the rate constant for flip-flop is smaller than k(off ), and because the rate of binding is greater than the rate of transpo rt, we conclude that flip-flop is the rate-limiting step for transport . These results demonstrate that (1) k(ff) and F-off are smaller for G UV than for LUV, (2) the rate constants increase with FA type accordin g to oleate (18:1) < palmitate (16:0) < linoleate (18:2), and (3) the barrier for flip-flop has a significant enthalpic component. Compariso n of the flip-flop rates determined for GUV with values estimated from previously reported metabolic rates for cardiac myocytes, raises the possibility that flip-flop across the lipid phase alone may not be abl e to support metabolic requirements.