Plasma membrane aminophospholipid distribution in transformed murine fibroblasts

The possibility that the asymmetric distribution of aminophospholipids may be an intrinsic property of mammalian plasma membranes was examined in LM cells, a transformed murine fibroblast cell line. The cells were grown in suspension culture in a chemically defined medium without lipid, protein, or serum and then treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). A maximum of 4% of LM cell plasma membrane phosphatidylethanolamine and 5% of the phosphatidylserine was labelled with TNBS. Furthermore, long chain and unsaturated fatty acids were preferentially esterified to the non-derivatized phosphatidylethanolamine (inner monolayer) as compared to phosphatidylethanolamine derivatized with TNBS (outer monolayer). Isethionyl acetimidate, an alternative non-penetrating reagent, confirmed the results obtained with TNBS and provided supportive evidence for the highly asymmetric distribution of phosphatidylethanolamine; 6% of the phosphatidylethanolamine was labelled with isethionyl acetimidate. When the penetrating reagent methylacetimidate was used, more than 80% of the phosphatidylethanolamine was derivatized. Although the growing of the LM cells in 10% calf serum significantly increased plasma membrane phosphatidylcholine while decreasing phosphatidylethanolamine, calf serum had no significant effect on phosphatidylethanolamine or phosphatidylserine asymmetry.