In animal tissues, NAEs (N-acylethanolamines), including N-arachidonoylethanolamine (anandamide), are primarily formed from their corresponding NAPEs (N-acylphosphatidylethanolamines) by a phosphodiesterase of the PLD (phospholipase D) type (NAPE-PLD). Recently, we cloned cDNAs of NAPE-PLD from mouse, rat and human [Okamoto, Morishita, Tsuboi, Tonai and Ueda (2004) J. Biol. Chem. 279, 5298–5305]. However, it remained unclear whether NAPE-PLD acts on endogenous NAPEs contained in the membrane of living cells. To address this question, we stably transfected two mammalian cell lines (HEK-293 and CHO-K1) with mouse NAPE-PLD cDNA, and investigated the endogenous levels and compositions of NAPEs and NAEs in these cells, compared with mock-transfected cells, with the aid of GC-MS. The overexpression of NAPE-PLD caused a decrease in the total amount of NAPEs by 50–90% with a 1.5-fold increase in the total amount of NAEs, suggesting that the recombinant NAPE-PLD utilizes endogenous NAPE as a substrate in the cell. Since the compositions of NAEs and NAPEs of NAPE-PLD-overexpressing cells and mock-transfected cells were very similar, the enzyme did not appear to discriminate among the N-acyl groups of endogenous NAPEs. These results confirm that overexpressed NAPE-PLD is capable of forming NAEs, including anandamide, in living cells.
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Research Article|
June 21 2005
Mammalian cells stably overexpressing N-acylphosphatidylethanolamine-hydrolysing phospholipase D exhibit significantly decreased levels of N-acylphosphatidylethanolamines
Yasuo OKAMOTO;
Yasuo OKAMOTO
*Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan
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Jun MORISHITA;
Jun MORISHITA
*Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan
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Jun WANG;
Jun WANG
*Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan
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Patricia C. SCHMID;
Patricia C. SCHMID
†The Hormel Institute, University of Minnesota, Austin, MN 55912, U.S.A.
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Randy J. KREBSBACH;
Randy J. KREBSBACH
†The Hormel Institute, University of Minnesota, Austin, MN 55912, U.S.A.
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Harald H. O. SCHMID;
Harald H. O. SCHMID
†The Hormel Institute, University of Minnesota, Austin, MN 55912, U.S.A.
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Natsuo UEDA
Natsuo UEDA
1
*Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan
1To whom correspondence should be addressed (email nueda@med.kagawa-u.ac.jp).
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Publisher: Portland Press Ltd
Received:
October 25 2004
Revision Received:
February 28 2005
Accepted:
March 10 2005
Accepted Manuscript online:
March 10 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 389 (1): 241–247.
Article history
Received:
October 25 2004
Revision Received:
February 28 2005
Accepted:
March 10 2005
Accepted Manuscript online:
March 10 2005
Citation
Yasuo OKAMOTO, Jun MORISHITA, Jun WANG, Patricia C. SCHMID, Randy J. KREBSBACH, Harald H. O. SCHMID, Natsuo UEDA; Mammalian cells stably overexpressing N-acylphosphatidylethanolamine-hydrolysing phospholipase D exhibit significantly decreased levels of N-acylphosphatidylethanolamines. Biochem J 1 July 2005; 389 (1): 241–247. doi: https://doi.org/10.1042/BJ20041790
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