RAPID TRANSBILAYER MOVEMENT OF PHOSPHATIDYLETHANOL IN UNILAMELLAR PHOSPHATIDYLCHOLINE VESICLES

Phosphatidylethanol (PtdEth) is a rare anionic phospholipid formed as a metabolite of ethanol through a transphosphatidylation reaction whic h is catalyzed by phospholipase D (PLD). The transbilayer distribution of dilute (0.5-2%) levels of PtdEth in small and large unilamellar ph osphatidylcholine vesicles (SUVs and LUVs, respectively) was monitored by C-13 nuclear magnetic resonance spectroscopy of PtdEth specificall y C-13-labeled at its headgroup methylene. Discrimination of interleaf let sites was based on shifts in the resonance frequencies of the head group methylene induced by differential packing in SUVs and induced by the shift reagent Pr3+ in LUVs. PtdEth is shown to undergo a rapid an d reversible transbilayer redistribution (t(0.5) less than or equal to 1 h at 26 C-degrees) in response to mM concentrations of added extern al multivalent cations (Ca2+, Pr3+, Mn2+, and Yb3+). The pH of the ves icle suspension vastly exceeds the pK of PtdEth (ca. 1.43), based on t he pH dependence of the labeled site's chemical shift in LUVs. The rat e of transbilayer transfer greatly exceeds, by 1-3 orders of magnitude , the transfer rates reported for other naturally occurring phospholip ids near physiological pH.