| Abstract: | Transmembrane movement of phospholipids is a fundamental step in the process of biological membrane assembly and intracellular lipid sorting. To facilitate study of transmembrane movement, we have synthesized analogues of phosphatidylglycerol and diacylglycerol in which a sulfhydryl group replaces a hydroxyl group in the polar head group. A rapid, continuous assay for the movement of phospholipids across single-walled lipid vesicles was developed that exploits the reactivity of these analogues toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a nonpenetrating, colorimetric, sulfhydryl reagent. In the reaction of DTNB with vesicles containing phosphatidylthioglycerol, a phosphatidylglycerol analogue, two kinetic phases were seen, which represent the reaction of DTNB with phosphatidylthioglycerol in the outer and inner leaflets of the bilayer. Analysis of the slow second phase indicated that the half-time for phosphatidylthioglycerol transbilayer movement was in excess of 8 days. In a similar experiment using dioleoylthioglycerol, a diacylglycerol analogue, the reaction was complete within 15 s. The large difference in translocation rates between these two lipids indicates that the primary barrier to transmembrane movement is the polar head group and implies that phospholipid translocation events in biological membranes may not be unlike those for molecules similar to the polar head groups alone. |
