Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes

Treatment of phosphatidyl (14C) choline liposomes with phospholipase D showed that labeledospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholIPase, but that the labeled phosphoipid left after the rapid Exchange was completed cound not by hydrolyzed by phosphol Pipase D.

Abstract

The exchange of phosphatidylcholine between (32P)phosphatidylcholine lipososomes and unlabeled mitochondria was catalyzed by a purified phospholipid exchange protein from bovine heart cytosol. The loss of (23P)phosphatidylcholine from the liposomes appeared to proceed in two stages: with 100 units of phospholipid exchange protein per ml the half-time of the initial stage was about 10 min and that of the final stage 4 days or greater. Agarose-gel chromatography of the liposomes showed an elution compatible with a homofwnwoua pool od amLL single walled vesicles. Treatment of phosphatidyl (14C) choline liposomes with phospholipase D (phosphatidylcholine phosphantidohydrolase) showed that labeled phospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholipase, but that the labeled phospholipid left after the rapid exchange was completed cound not by hydrolyzed by phospholipase D. It is proposed that the rapidly exchanging phosphatidylcholine constitutes the outer layer of the liposome bilayer. The long half-lives of 4 days or more probably represent the transposition of phosphatidylcholine from the inner to the outer layer of the liposome bilayer.