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 fluorescence energy transfer assay. Donor SUV were labeled with AOffa, and acceptor SUV contained the nonexchangeable quencher NBD-phosphatidylethanolamine. Donor and acceptor membranes were mixed using a stopped-flow apparatus, and intermembrane transfer was monitored by the decrease in 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 phosphatidylglycerol (PG) into donor SUV otherwise composed of phosphatidylcholine (PC). Addition of 25 mol% of either of the negatively charged phospholipids (PA or PG) resulted in an increase in the rate of AOffa transfer, whereas addition of zwitterionic PE had no effect on transfer rate. The transfer kinetics were in all cases best described by a biexponential process, and it was found that the addition of PA caused an increase in the fraction of AOffa which transfer at the fast rate. This was 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 inner 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 transfer from PA-containing vesicles and PC vesicles to become identical. The results demonstrate that the ionic character of the donor membrane bilayer is an important determinant of the transfer rate of long-chain fatty acids between membranes.
Sunderland
J E
JE
Department of Nutrition, Harvard School of Public Health, Boston, MA.
Storch
J
J
D005230:Fatty Acids, Nonesterified / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008566:Membranes / Q000378:metabolism*; D009994:Osmolar Concentration; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D005230:Fatty Acids, Nonesterified; D008563:Membrane Lipids; D010743:Phospholipids