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Determination of phospholipase D‐mediated hydrolysis of phosphatidylethanolamine
Author(s) -
Kiss Zoltan
Publication year - 1991
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02537144
Subject(s) - ethanolamine , phosphatidylethanolamine , chemistry , hydrolysis , phosphatidylcholine , phospholipase c , phospholipid , phospholipase d , biochemistry , phospholipase , chromatography , enzyme , membrane
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 [ 14 C]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 [ 14 C]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 unincorporated 14 C‐labeled ethanolamine metabolites and improves the signal‐to‐noise ratio.