Determination of phospholipase D‐mediated hydrolysis of phosphatidylethanolamine

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Abstract
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‐tetradecanoylphorbor 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 reduces the cellular content of unincorporated14C‐labeled ethanolamine metabolites and improves the signal‐to‐noise ratio.