AN ORIGINAL METHOD FOR RAPID SERIAL DETERMINATION OF PHOSPHOLIPID BIOSYNTHESIS - APPLICATIONS TO MAMMALIAN LYMPHOCYTIC CELLS AND A LOWER EUKARYOTE, PLASMODIUM-FALCIPARUM

被引:5
作者
ANCELIN, ML
VIALETTES, F
VIAL, HJ
机构
[1] CNRS U.A. 530, Département Biologie-Santé, USTL Bt 24, 34095 Montpellier Cedex 5, Case postale, 107, Place Eugene Bataillon
关键词
D O I
10.1016/0003-2697(91)90090-G
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A rapid, convenient, and efficient method is presented to measure phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol biosynthesis from [3H]choline, [3H]ethanolamine, and [3H]inositol, respectively. After incubation of the cells in 96-multiwell dishes with the appropriate radioactive precursor, cells were lysed with water and the water-insoluble materials, particularly cellular membranes which contain the bulk of phospholipids, were serially collected on glass-fiber papers using a cell harvester. The method was first applied to human lymphocytic cell lines then adapted to Plasmodium falciparum-infected human erythrocytes which in both cases allowed recovery of more than 90% of the newly biosynthesized phospholipids. With this quick method, adapted to short incubation periods (less than 5 h), we were able to determine optimal conditions such as the best medium (RPMI devoid of serum, thus avoiding interference from endogenous precursors, notably choline present in significant quantities in serum) and the lowest specific activity to be used for each radioactive precursor and the minimum quantity of cells. This method could be adapted to other cell systems, provided that the precursors are specific to phospholipids and that the bulk of biosynthesized phospholipids are present as membrane components. Finally, by this method the activity of effectors of phospholipid metabolism can be tested on a large scale, thus allowing rapid screening of original molecules specifically affecting cellular phospholipid metabolism. © 1991.
引用
收藏
页码:203 / 209
页数:7
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