FLIP-FLOP IS SLOW AND RATE-LIMITING FOR THE MOVEMENT OF LONG-CHAIN ANTHROYLOXY FATTY-ACIDS ACROSS LIPID VESICLES

An issue that is central to understanding cellular fatty acid (FA) met abolism is whether physiologic transport of FA across cell membranes r equires protein mediation or can be satisfied by the rate of spontaneo us movement through the lipid phase. For this reason, considerable eff ort has been devoted to determining the rate-limiting steps for transp ort of FA across pure lipid bilayer membranes. Previously, we found th at transbilayer flip-flop was the rate-limiting step for transport of long chain anthroyloxy FA (AOFA) across lipid bilayers and that the ti mes for long chain AOFA flip-flop were greater than or equal to 100 s, yielding rate constants for flip-flop (k(ff)) that were less than or equal to 0.01 s(-1) [Storch, J., & Kleinfeld, A. M. (1986) Biochemistr y, 25, 1717-1726; Kleinfeld, A. M., & Storch, J. (1993) Biochemistry 3 2, 2053-2061]. In those studies, k(ff) values were inferred from the t ime course of AOFA transfer between lipid vesicles. Recently, Kamp et al. [Kamp, F., Zakim, D., Zhang, F., Noy, N., & Hamilton, J. A. (1995) Biochemistry 34, 11928-11937], using pyranine trapped within lipid ve sicles to detect flip-flop more directly, have reported that flip-flop rates of long chain AOFA are extremely rapid (k(ff) > 10 s(-1)) and a re not rate limiting for transbilayer transport. Because no defect was apparent in our previous measurements, we have extended, for AOFA, th e pyranine method of Kamp et al. (1995) by using stopped-flow fluorome try to resolve flip-flop rates of bath short and long chain AOFA in ve sicles. In addition, we have monitored the time course of transbilayer AOFA flip-flop using carboxyfluorescein (CF) trapped within the lipid vesicles as a resonance energy transfer (RET) acceptor of AO fluoresc ence. The differential quenching of AOFA fluorescence in the outer and inner leaflets of the bilayer allows flip-flop to be separated from t he time course of AOFA binding to the vesicles. Results obtained from both the pyranine and CF methods indicate, in agreement with our previ ous results, that flip-flop of the long chain AOFA is slow relative to either the binding or the rate of dissociation from the vesicle. In p articular, we find that the time constant (tau) for pyranine quenching by 2-AO-palmitate (2-AOPA) was > 40 s and that k(ff) obtained from RE T in CF vesicles was about 0.003 s(-1). Also, in contrast to Kamp et a l. (1995) who reported that k(ff) values were independent of FA chain length or structure for the C-12 to C-18 native and the C-18 AOFA with in a factor of 2, we find that the rate of pyranine quenching for the shorter chain 11-AO-undecanoic acid is more than 50-fold faster than f or the longer chain AOFA. We conclude, therefore, that transbilayer tr ansport of the AOFA is limited by the rate of flip-flop and that this rate is a sensitive function of the AOFA structure.