EFFECT OF PHOSPHOLIPID HEADGROUP COMPOSITION ON THE TRANSFER OF FLUORESCENT LONG-CHAIN FREE FATTY-ACIDS BETWEEN MEMBRANES

The transfer of long-chain anthroyloxy-labeled-free fatty acids (AOffa ) between small unilamellar vesicles (SUV) was studied using a fluores cence energy transfer assay. Donor SUV were labeled with AOffa, and ac ceptor SUV contained the nonexchangeable quencher NBD-phosphatidyletha nolamine. Donor and acceptor membranes were mixed using a stopped-flow apparatus, and intermembrane transfer was monitored by the decrease i n AO fluorescence with time. The effect of donor membrane phospholipid headgroup composition on AOffa transfer was examined by incorporating phosphatidylethanolamine (PE), phosphatidic acid (PA), or phosphatidy lglycerol (PG) into donor SUV otherwise composed of phosphatidylcholin e (PC). Addition of 25 mol% of either of the negatively charged phosph olipids (PA or PG) resulted in an increase in the rate of AOffa transf er, whereas addition of zwitterionic PE had no effect on transfer rate . The transfer kinetics were in all cases best described by a biexpone ntial process, and it was found that the addition of PA caused an incr ease in the fraction of AOffa which transfer at the fast rate. This wa s likely due in large part to the asymmetric distribution of AOffa in these vesicles, with more fatty acid in the outer hemileaflet. This in turn may be due to the asymmetric distribution of PA between the inne r and outer hemileaflets. Thus the increased AOffa transfer rate from negatively charged vesicles may be caused by charge repulsion between ffa and negatively charged headgroups. This increase in transfer rate was maximized at pH 9 as compared to pH 7, further suggesting that the increased rate of intermembrane transfer may arise because of charge repulsion. Finally, it was shown that decreasing the membrane surface potential by increasing the ionic strength caused the rate of AOffa tr ansfer from PA-containing vesicles and PC vesicles to become identical . The results demonstrate that the ionic character of the donor membra ne bilayer is an important determinant of the transfer rate of long-ch ain fatty acids between membranes.