Flip-flop of phospholipids in vesicles: kinetic analysis with time-resolved small-angle neutron scattering.

We applied a time-resolved small-angle neutron scattering technique to vesicle systems to determine interparticle transfer and flip-flop of phospholipids. Measurements were performed for large unilamellar vesicles, consisting of dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), or 1-palmitoyl-2-oleoylphosphatidic acid (POPA), which differ either in their acyl chains or headgroup. POPC, which is analogous to naturally occurring phosphatidylcholines, exhibited no transbilayer transfer and very slow interbilayer migration. POPC on the inner leaflet of vesicles did not flop even when phospholipase D converted all POPC molecules on the outer leaflet into POPA, which was shown to exhibit fast flip-flop. From these results, together with the observation that the flip-flop of DMPC was entirely inhibited in the presence of cholesterol, it is deduced that the flip-flop of phosphatidylcholines does not take place spontaneously in cellular plasma membranes rich in cholesterol and that it requires enzymatic activities of energy-dependent and/or -independent flippases/floppases.