DIACYLGLYCEROL AND HEXADECANE INCREASE DIVALENT CATION-INDUCED LIPID MIXING RATES BETWEEN PHOSPHATIDYLSERINE LARGE UNILAMELLAR VESICLES

Bovine brain phosphatidylserine (BBPS) vesicles were prepared with traces of dioleoylglycerol (18:1,18:1 DAG) or hexadecane (HD) to determine the influence of changes in headgroup or acyl chain packing on divalent cation-induced lipid mixing rates. A stopped-flow apparatus was used to combine vesicles with 3 mM Ca2+ or Ba2+. Aggregation was monitored by light scattering and lipid mixing by lipid probe dilution. Neither 3-6 mol % 18:1,18:1 DAG nor up to 10 mol % HD significantly altered the BBPS chain melting temperature, vesicle diameter, or vesicle aggregation rates. Lipid mixing rates doubled by adding either 3 mol % 18:1,18:1 DAG or 6 mot % HD to BBPS with no change in the Ca2+ concentration threshold. The Arrhenius slopes of the lipid mixing rates for control, 3 mol % 18:1,18:1 DAG, and 6 mol % HD vesicles were identical. H-2-nuclear magnetic resonance spectra of perdeuterated dipalmitoylglycerol and HD in BBPS in the absence and presence of Ca2+ and Ba2+ showed that the solutes occupied different time-averaged positions in the bilayer under each condition. These data suggest that: 1) the enhanced lipid mixing rate is related to the volume of the added alkyl chains; 2) 18:1,18:1 DAG and HD may alter the activation entropy or the attempt frequency at one or more steps in the lipid mixing process; 3) 18:1,18:1 DAG and MD are likely to act at a different spatial or temporal point than the divalent cation; and 4) it is unlikely that the effect of these solutes on lipid mixing is due to their equilibrium time-averaged positions in the bilayer. Others have shown that apolar lipids accelerate fusion in nonbilayer phase-forming systems, but BBPS does not form these phases under these conditions. Therefore, we propose that the effect of very small amounts of apolar substances may be very general, e,g., stabilizing the hydrophobic interstices associated with a variety of proposed intermediate structures.