Distribution of lysophosphatidylcholine in single bilayer vesicles prepared without sonication

The cholate method originally introduced by Kagawa et al. (J. Biol. Chem. (1973) 248, 676-684) and further developed by Brunner et al. (Biochim. Biophys. Acta (1976) 455, 322-331) has been used to prepare single bilayer vesicles containing 5 mol% lysophosphatidylcholine embedded in a matrix of phosphatidylcholine. The distribution of lysophosphatidylcholine over outer and inner monolayer was found to be highly asymmetric (ratio 9:1), as determined by lysophospholipase treatment of the vesicles. This distribution is similar to the value found in sonicated vesicles. Up to 20 mol% cholesterol could be incorporated in the vesicles by the cholate method. The method was succesfully used also for the preparation of single bilayer vesicles from total rat liver microsomal lipids, to which 5 mol% of 1-[1-14C]palmitoyl lysophosphatidylcholine had been added. Surprisingly, almost 100% of lysophosphatidylcholine in the latter vesicles was directly available for hydrolysis by sophospholipase. In contrast, only 70% of the lysophosphatidylcholine is sonicated vesicles of similar composition could be hydrolyzed by lysophospholipase.