Precipitation by Concanavalin A of Liposomes Containing Glycolipids with Terminal α-Linked Mannose and Assay of Phospholipid Exchange Activities

Abstract

A new separation method of two populations of liposomes and the use of the method in an assay of phospholipid exchange activities are described. The major constituents of these liposomes were rat liver total phospholipids and cholesterol in molar ratio of 2:1. One population of the liposomes was made reactive to concanavalin A by the incorporation of either 1.8 mole% α-D-mannosyl-(1→3)-α-D-mannosyl-sn-1, 2-diglyceride from Micrococcus lysodeikticus or 0.77 mole% α-D-mannosyl-(1→4)-β-D-mannosyl-(1→4)-D-glucosyl-ceramide from Corbicula japonica. The other population of the liposomes was concanavalin A-nonreactive. The two liposomal populations were separated by addition of concanavalin A to agglutinate the reactive liposomes. In the assay of phospholipid exchange activities, these two populations of the liposomes were incubated with either the cytosol protein of rat liver or partially purified phospholipid exchange proteins from rat liver in a total volume of 0.2 ml. Either the concanavalin A-reactive liposomes or the nonreactive liposomes were used as a phospholipid donor. The phospholipid donor was doubly labeled with [³H]lactosyl ceramide and that class of [³²P] phospholipids whose exchange was being measured. The [³H]glycolipid served as a nonexchangeable reference marker. After the incubation, the two liposomal populations were separated. The amount of phospholipid transferred from the donor to the acceptor liposomes was calculated from the decrease of ³²P to ³H ratio of the donor liposomes during the incubation.