Evidence from a number of laboratories suggests that membrane proteins may meditate the transport of physiologic fatty acids (FA) across cell membranes. However, studies using lipid membranes indicate that FA are capable of spontaneous flip-flip, raising the possibility that rapid transport through the lipid phase obviates the need for a transport protein. Determining the rate-limiting steps for transport of FA across lipid membranes, therefore, is central to understanding FA transport across cell membranes. The transport of long-chain FA across lipid membranes, from the aqueous compartment on one side of the lipid bilayer to the aqueous phase on the other side, has not been measured previously. In this study, we have used the fluorescent probe 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 lipid vesicles. 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 lipid vesicles. These rates were determined using large unilamellar vesicles (LUV) of approximately 1000 A diameter, prepared by extrusion and giant unilamellar vesicles (GUV), prepared by detergent dialysis, that are >/=2000 A diameter. The results of these studies for vesicles composed of egg phosphatidylcholine (EPC) and cholesterol reveal kff values that range from 3 to 15 s-1 for LUV and from 0.1 to 1.0 s-1 for GUV, depending upon temperature and FA type. For these same vesicles, dissociation rate constants range from 4 to 40 s-1 for LUV and from 0.3 to 2.5 s-1 for GUV. In all instances, the rate constant for flip-flop is smaller than koff, and because the rate of binding is greater than the rate of transport, we conclude that flip-flop is the rate-limiting step for transport. These results demonstrate that (1) kff and koff are smaller for GUV than for LUV, (2) the rate constants increase with FA type according to oleate (18:1) < palmitate (16:0) < linoleate (18:2), and (3) the barrier for flip-flop has a significant enthalpic component. Comparison 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 able to support metabolic requirements.

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.

All 5 enzymes of the urea cycle have been determined on liver samples obtained from rats during late foetal and neonatal life. Since no argininosuccinate synthetase activity could be detected before birth it is suggested that the urea cycle does not function in the foetal rat. After birth, the activities of all the enzymes increase dramatically.

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