Determination of phospholipase D-mediated hydrolysis of phosphatidylethanolamine.

While phospholipase D-mediated hydrolysis of phosphatidylcholine is well documented, we have recently shown that phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) [Kiss, Z., and Anderson, W.B., J. Biol. Chem. 264, 1483-1487 (1989); J. Biol. Chem. 265, 7345-7350 (1990)] is equally prominent. This made it necessary to define in detail the conditions required for the detection of agonist-stimulated PtdEtn hydrolysis. Using the [14C]ethanolamine-prelabeled rat-1 fibroblast model and 12-O-tetradecanoylphorbol 13-acetate (TPA) as a model compound with the known ability to stimulate phospholipase D, we demonstrated that optimal detection of TPA-induced ethanolamine release requires i) fractionation of water-soluble ethanolamine products; ii) addition of unlabeled ethanolamine to quench the phosphorylation of newly formed [14C]ethanolamine; and/or iii) prolonged preincubation of prelabeled cells in an isotope-free medium before the addition of TPA. This preincubation step reduced the cellular content of unincorporated 14C-labeled ethanolamine metabolites and improves the signal-to-noise ratio.