Stimulation by serum of 32P incorporation into the phospholipids of Ehrlich ascites cells.

Ehrlich ascites cells adapted for suspension culture were incubated with 32PO43- in a Krebs-Ringer-HEPES (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonate) buffer with or without 5% calf serum. Specific activity was determined for cellular [gamma-32P]ATP, total phospholipids, and the individual phospholipids fractionated by two-dimensional thin-layer chromatography. Serum stimulated the incorporation of 32P into the phosphatides with a 2-3-fold increase in specific activity of phosphatidylcholine and phosphatidylethanolamine. Phosphatidylinositol showed a moderate increase. Phosphatidic acid showed an increase that could be accounted for by a parallel small increase in the specific activity of precursor [gamma-32P]-ATP. The effect os serum could be discerned as early as 10 min after its addition. Complete inhibition of protein synthesis by cycloheximide or puromycin did not interfere with the serum stimulation of 32P incorporation into lipids, indicating that the serum effect is not dependent on synthesis of new protein. Incubation with palmitic acid caused a marked increase of phosphatidylinositol specific activity, little change in specific activity of phosphatidic acid but a tripling of its total amount, and no changes in the other phosphatides. Oleic acid caused only a small increase in phosphatidylinositol specific activity and no significant changes in the other lipids.