Transfer of Phosphatidylcholine between Liposomes

Abstract

Liposomes of sufficiently different surface charge can be separated on a DEAE-cellulose column. This made possible the present study on the transfer of phosphatidylcholine between liposomes consisting of phosphatidylcholine and various amounts of phosphatidic acid. It was demonstrated that the redistribution of ['4C]phosphatidylcholine between liposomes as catalyzed by the phosphatidylcholine exchange protein, followed the kinetics of an isotope-exchange reaction. Furthermore, it was shown that the phospholipid composition of the liposomes affected the activity of the exchange protein.It has been demonstrated for a number of tissues that soluble proteins are present which catalyze the transfer of phospholipids between membrane structures [l -41. Recently, one of these proteins denoted as phosphatidylcholine exchange protein, has been purified from beef liver and partially characterized [5]. It was shown that one mole of phosphatidylcholine was bound per mole of protein. The exchange protein exerted its action as a carrier of phosphatidylcholine between membranes by the exchange of its bound lipid molecule for one present in the membrane [6]. Recently, Ehnholm and Zilversmit [7] provided evidence for the catalyzed transfer of phosphatidylcholine between antigen-sensitized and non-sensitized liposomes after separation of the liposomes by immunoprecipitation. A drawback of this technique according to the authors was the relatively long time required to separate the liposomes.The present paper demonstrates the catalyzed transfer of phosphatidylcholine between two populations of liposomes one of which contains [14C]phosphatidylcholine. Differences of surface charge made possible the rapid separation of these liposomes on an ion-exchange column. It is shown that the redistribution of [14C]phosphatidylcholine between the liposomes as catalyzed by the exchange protein follows the kinetics of an isotope-exchange reaction. It is also shown that particularly the incorporation of negatively charged phospholipids into the liposomes has a pronounced inhibitory effect on the transfer. MATERIALS AND METHODSPhosphatidylcholine from egg yolk, phosphatidylinositol from rat liver and sphingomyelin from bovine erythrocytes were isolated according to established procedures [8 -101. Phosphatidic acid was prepared from phosphatidylcholine by degradation with phospholipase D [ll]. A [14C]methyl group was introduced into the polar head group of phosphatidylcholine by the method of Stoffel et al. [12]. [7cl-3H]Cholesteryloleate was purchased from New England Nuclear. Phosphatidylcholine exchange protein was isolated from beef liver according to the procedure of Kamp et al. [5].Liposomes were prepared in 0.05 M KCl-0.01 M glycine-KOH (pH 8.6) by sonication of a phospholipid dispersion for 30 min following the procedure of Huang [13]. Chromatography on a Sepharose-4B column indicated that more than 95 o/, of the phospholipid dispersed was present in single bilayer liposomes.Exchange of phosphatidylcholine between liposomes was determined b...

The Protein‐Mediated Transfer of Phosphatidylcholine between Membranes

“…3). A similar observation has been made for sphingomyelin [2]. This demonstrates that in addition to the membrane surface charge other factors may regulate the activity of the exchange protein.…”

“…It is presumably by this mechanism that in the present study the protein transfers ['4C]phosphatidylcholine from the 14C-labelled mitochondria to the liposomes. Previously in studying the transfer of phosphatidylcholine between single bilayer liposomes, it was demonstrated that the transfer decreased with an increase of negatively charged phosphatidic acid in the liposomes [2,3]. An analysis of the kinetic data indicated that the apparent dissociation constant of the exchange protein-liposome complex decreased in a parallel fashion resulting in an inhibition of transfer [3].…”

“…Recently it was demonstrated that the protein-mediated transfer of phosphatidylcholine between phosphatidylcholine liposomes was inhibited by incorporation of phosphatidic acid or phosphatidylinositol into these liposomes [2]. Steady state analysis of the kinetic data indicated that the apparent dissociation constant of the exchange protein-liposome complex decreased with an increasing phosphatidic acid content of the liposomes [3].…”

Hemolysis of Rat Erythrocytes by Replacement of the Natural Phosphatidylcholine by Various Phosphatidylcholines

“…Incubations of erythrocytes with lipid vesicles in the absence of exchange protein and incubations with exchange protein but without lipid vesicles showed that under these conditions erythrocytes stayed intact, maintained their original shape and appeared to have unaltered phospholipid compositions. In addition extensive studies on the properties of the purified exchange protein as used in our experiments have shown that this protein has no detectable proteolytic or other enzymatic functions but act solely as a phosphatidylcholinespecificcarrier [7][8][9][10][11][12][13]15,17,18]. Analysis of erythrocyte phosphatidylcholine at this extent of exchange (25 % replacement) was performed by both thin-layer and gas-liquid chromatography (Table 1).…”

“…In this regard the introduction of exchange proteins [l-41 offers the opportunity to conservatively alter the membrane structure in a systematic fashion. Thus far, phosphatidylcholineexchange proteins have been shown to function between various donor-acceptor pairs ; mitochondria with microsomes [5 -71, microsomes with liposomes [8], liposomes with liposomes [9] and erythrocyte ghosts with vesicles [lo] and liposomes [ll]. Recently van Meer et al [12,13] demonstrated the exchange of phosphatidylcholine between intact human and rat erythrocytes and various donor systems such as microsomes, liposomes, vesicles and etherosomes.…”