Plasma membrane aminophospholipid distribution in transformed murine fibroblasts

Abstract

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 methyl acetimidate 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.