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.
Kiss
Z
Z
Hormel Institute, University of Minnesota, Austin 55912.
D000818:Animals; D002460:Cell Line; D019856:Ethanolamine; D004983:Ethanolamines / Q000302:isolation & purification; D005347:Fibroblasts / Q000201:enzymology; D007700:Kinetics; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004983:Ethanolamines; D010714:Phosphatidylethanolamines; D019856:Ethanolamine; C005448:phosphorylethanolamine; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate