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.
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Abbreviations
- DMEM:
-
Dulbecco's modified Eagle medium
- PtdCho:
-
phosphatidylcholine
- PtdEtn:
-
phosphatidylethanolamine
- TPA:
-
12-O-tetradecanolylphorbol 13-acetate
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Kiss, Z. Determination of phospholipase D-mediated hydrolysis of phosphatidylethanolamine. Lipids 26, 321–323 (1991). https://doi.org/10.1007/BF02537144
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DOI: https://doi.org/10.1007/BF02537144