MOVEMENT OF FATTY-ACIDS, FATTY-ACID ANALOGS, AND BILE-ACIDS ACROSS PHOSPHOLIPID-BILAYERS - KINETICS OF FATTY ACID-MEDIATED PROTON MOVEMENT ACROSS SMALL UNILAMELLAR VESICLES

How lipophilic acids move across membranes, either model or biological , is the subject of controversy. We describe experiments which better define the mechanism and rates in protein-free phospholipid bilayers. The transbilayer movement of lipophilic acids [fatty acids (FA), coval ently-labeled FA, bile acids, and retinoic acid] was monitored by entr apping pyranin, a water-soluble, pH-sensitive fluorescent molecule to measure pH inside unilamellar vesicles [Kamp, F., & Hamilton, J. A. (1 992) Proc. Natl. Acad. Sci. U.S.A. 89, 11367-11370]. Equations for the pseudo-unimolecular rate constants for transbilayer movement of un-io nized (k(FAH)) and ionized (k(FA-)) acids are derived. All FA studied (octanoic, lauric, myristic, palmitic, stearic, oleic, elaidic, linole ic, linolelaidic, and arachidonic) and retinoic acid exhibited rapid t ransbilayer movement (t1/2 < 1 s) via the un-ionized form across small unilamellar egg phosphatidylcholine (PC) vesicles. FA produced by pho spholipase A2 in the outer leaflet of PC vesicles equilibrated rapidly to the inner leaflet. Ionized FA showed enhanced transbilayer movemen t (k(FA-) = 0.029 s-1) in the presence of equimolar valinomycin. The t hree FA analogues [12-(9-anthroyloxy)stearic acid, 5-doxylstearic acid , and 1-pyrenenonanoic acid] moved across PC bilayers via the un-ioniz ed form; except for the anthroyloxy FA (k(FAH) = 4.8 x 10(-3) s-1), th e rates were too fast to measure (t1/2 < 1 s). The rate for cholic aci d (CA) transbilayer movement was slow (k(CAH) = 0.056 s-1) compared to that of the more hydrophobic bile acids, deoxy- and chenodeoxycholic acid (t1/2 < 1 s). The taurine conjugates of the three bile acids did not cross the bilayer (t1/2 > 1 h). A further application of the pyran in method was to measure the partitioning of FA and bile acids among w ater, albumin, and PC vesicles. Our results show that the ability of l ipophilic acids to permeate a PC bilayer rapidly is dependent on the p resence of the un-ionized acid in the membrane interface. Considering the fast unfacilitated movement of FA across protein-free phospholipid bilayers, it is unlikely that there is a universal need for a transpo rt protein to enhance movement of FA across membrane bilayers. Physiol ogical implications of proton movement accompanying fast movement of u n-ionized lipophilic acids (and the consequent generation of a pH grad ient) are discussed.