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) metabolism is whether physiologic transport of FA across cell membranes requires protein mediation or can be satisfied by the rate of spontaneous movement through the lipid phase. For this reason, considerable effort has been devoted to determining the rate-limiting steps for transport of FA across pure lipid bilayer membranes. Previously, we found that transbilayer flip-flop was the rate-limiting step for transport of long chain anthroyloxy FA (AOFA) across lipid bilayers and that the times for long chain AOFA flip-flop were > or = 100 s, yielding rate constants for flip-flop (k(ff)) that were < or = 0.01 s(-1) [Storch, J., & Kleinfeld, A. M. (1986) Biochemistry 25, 1717-1726; Kleinfeld, A. M., & Storch, J. (1993) Biochemistry 32, 2053-2061]. In those studies, k(ff) values were inferred from the time 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 vesicles 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 are not rate limiting for transbilayer transport. Because no defect was apparent in our previous measurements, we have extended, for AOFA, the pyranine method of Kamp et al. (1995) by using stopped-flow fluorometry to resolve flip-flop rates of both short and long chain AOFA in vesicles. 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 fluorescence. The differential quenching of AOFA fluorescence in the outer and inner leaflets of the bilayer allows flip-flop to be separated from the time course of AOFA binding to the vesicles. Results obtained from both the pyranine and CF methods indicate, in agreement with our previous 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 particular, 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 RET in CF vesicles was about 0.003 s(-1). Also, in contrast to Kamp et al. (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 within 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 for the longer chain AOFA. We conclude, therefore, that transbilayer transport of the AOFA is limited by the rate of flip-flop and that this rate is a sensitive function of the AOFA structure.
Kleinfeld
A M
AM
Medical Biology Institute, La Jolla, California 92037, USA.
Chu
P
P
Storch
J
J
D001190:Arylsulfonates; D001692:Biological Transport; D000431:Ethanol; D005227:Fatty Acids / Q000378:metabolism*; D005232:Fatty Acids, Volatile / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D014677:Pharmaceutical Vehicles; D010713:Phosphatidylcholines / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001190:Arylsulfonates; D005227:Fatty Acids; D005232:Fatty Acids, Volatile; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D014677:Pharmaceutical Vehicles; D010713:Phosphatidylcholines; C024098:6-carboxyfluorescein; D000431:Ethanol; C005047:pyranine