Sasaki T, Sakagami T
Biochim Biophys Acta. 1978 Oct 4;512(3):461-71. doi: 10.1016/0005-2736(78)90156-6.
A new assay system of phospholipid exchange activities is described. The exchange activities were quantitated by measuring the stimulation of phospholipid transfer between two separate populations of liposomes, which contained, as the major constituents, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and cholesterol in molar ratios of 6 :2 : 1: 1: 5. One population of the liposomes was made reactive to concanavalin A by the incorporation of 1.8 mol% alpha-D-mannosyl-(1 leads to 3)-alpha-D-mannosyl-sn-1, 2-diglyceride from Micrococcus lysodeikticus. The concanavalin A-reactive liposomes, a phospholipid donor, were doubly labelled with [6-3H] galactosylglucosyl ceramide and that class of 32P-labelled phospholipids whose exchange was being measured. The 3H-labelled glycolipid served as a non-exchangeable reference marker. The other population of the liposomes, a phospholipid acceptor, was concanavalin A nonreactive. These two populations of liposomes were incubated with the cytosol protein of rat liver in a total volume of 0.2 ml. After the incubation, two different procedures were used to separate the two liposomal populations. In one procedure concanavalin A was added to agglutinate the reactive liposomes; the flocculated lectin . liposome complex was separated from the non-reactive liposomes by brief centrifugation. In the other procedure the reactive liposomes were trapped by binding to concanavalin A covalently coupled to Sepharose 2B; the complex was separated from the non-reactive liposomes by filtration through a filter paper under suction. In both assay procedures the amount of phospholipid transferred from the donor to the acceptor liposomes was calculated from the decrease of 32P/3H ratio of the concanavalin A-reactive liposomes during the incubation. By the assasy system it is possible to determine phosphatidylcholine and phosphatidylinositol exchange activities in 100 micrograms of rat liver cytosol protein.
本文描述了一种新的磷脂交换活性检测系统。通过测量两个独立脂质体群体之间磷脂转移的刺激来定量交换活性，这两个脂质体群体的主要成分是磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇、鞘磷脂和胆固醇，其摩尔比为6:2:1:1:5。通过掺入来自溶壁微球菌的1.8摩尔%α-D-甘露糖基-(1→3)-α-D-甘露糖基-sn-1,2-甘油二酯，使一组脂质体对伴刀豆球蛋白A具有反应性。伴刀豆球蛋白A反应性脂质体作为磷脂供体，用[6-³H]半乳糖基葡糖神经酰胺和正在测量其交换的那类³²P标记的磷脂进行双重标记。³H标记的糖脂用作不可交换的参考标记。另一组脂质体作为磷脂受体，对伴刀豆球蛋白A无反应性。将这两组脂质体与大鼠肝脏的胞质溶胶蛋白在总体积为0.2毫升的条件下孵育。孵育后，使用两种不同的方法分离这两组脂质体。在一种方法中，加入伴刀豆球蛋白A使反应性脂质体凝集；通过短暂离心将絮凝的凝集素-脂质体复合物与无反应性脂质体分离。在另一种方法中，反应性脂质体通过与共价偶联到琼脂糖2B上的伴刀豆球蛋白A结合而被捕获；通过在吸力下通过滤纸过滤将复合物与无反应性脂质体分离。在两种检测方法中，根据孵育过程中伴刀豆球蛋白A反应性脂质体的³²P/³H比值的降低来计算从供体脂质体转移到受体脂质体的磷脂量。通过该检测系统，可以在100微克大鼠肝脏胞质溶胶蛋白中测定磷脂酰胆碱和磷脂酰肌醇的交换活性。
