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2006 Fiscal Year Final Research Report Summary

Functional analysis of a novel enzyme of the phospholipase D type involved in the endocannabinoid

Research Project

Project/Area Number 17590251
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field General medical chemistry
Research InstitutionKagawa University

Principal Investigator

UEDA Natsuo  Kagawa University, Faculty of Medicine, Professor, 医学部, 教授 (20193807)

Co-Investigator(Kenkyū-buntansha) OKAMOTO Yasuo  Kagawa University, Faculty of Medicine, Assistant, 医学部, 助手 (80293877)
TSUBOI Kazuhito  Kagawa University, Faculty of Medicine, Assistant, 医学部, 助手 (80346642)
Project Period (FY) 2005 – 2006
KeywordsN-acylethanolamine / anandamide / endocannabinoid / enzyme / lipid mediator / phospholipase D / metallo-□-lactamase family / phospholipid
Research Abstract

Anandamide (N-arachidonoylethanolamine), which was found as an endogenous ligand for cannabinoid receptors (an endocannabinoid), is formed from membrane glycerophospholipids by two-step enzyme reactions in animal tissues. However, the responsible enzymes remained poorly characterized, and it has been difficult to study the anandamide biosynthesis by molecular biological approaches. Recently we succeeded for the first time in cDNA cloning and functional expression of a novel mammalian enzyme of the phospholipase D type (NAPE-PLD) that generates anandamide and other N-acylethanolamines from their corresponding N-acylphosphatidylethanolamines (NAPEs) (Okamoto et al. J. Biol. Chem. 279, 5298-305, 2004). In the present study, we principally characterized recombinant rat NAPE-PLD.
The recombinant NAPE-PLD was expressed in Escherichia coli as a GST-fusion protein together with molecular chaperone. The enzyme was then solubilized with CHAPS, followed by purification to apparent homogeneity by glutathione affinity chromatography and hydroxyapatite chromatography. The purified enzyme was highly active with NAPEs, and did not discriminate various N-acyl species with C_4-C_<20>. In contrast, the enzyme was almost inactive with major membrane glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol). These results suggested the ability of NAPE-PLD to specifically degrade different NAPEs without damaging other membrane phospholipids. The purified enzyme was remarkably activated in a dose-dependent manner by millimolar concentrations of Mg^<2+> as well as Ca^<2+>. Atomic absorption spectrometry exhibited the presence of catalytically important zinc in NAPE-PLD. In addition, site-directed mutagenesis studies revealed that Asp-147, His-185, His-187, Asp-189, His-190, His-253, Asp-284, and His-321 of NAPE-PLD, that are highly conserved within the metallo-□-lactamase family, play crucial roles in the catalytic activity.

  • Research Products

    (10 results)

All 2007 2006 Other

All Journal Article (10 results)

  • [Journal Article] Discovery and characterization of a Ca^<2+> -independent phosphatidylethanolamine N-acyltransferase generating anandamide precursor and its congeners2007

    • Author(s)
      Jin, X.-H.et al.
    • Journal Title

      J. Biol. Chem. 282(6)

      Pages: 3614-3623

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Predominant expression of lysosomal N-acylethanolamine-hydrolyzing acid amidase in macrophages revealed by immunochemical studies2007

    • Author(s)
      Tsuboi, K. et al.
    • Journal Title

      Biochim. Biophys. Acta - Mol. Cell Biol. Lipids 1771(5)

      Pages: 623-632

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Biosynthetic pathways of the endocannabinoid anandamide2007

    • Author(s)
      Okamoto, Y. et al.
    • Journal Title

      Chem. Biodivers. (in press)

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] The N-acylethanolamine-hydrolyzing acid amidase (NAAA)2007

    • Author(s)
      Tsuboi, K. et al.
    • Journal Title

      Chem. Biodivers. (in press)

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Discovery and characterization of a Ca^<2+>-independent phosphatidylethanolamine N-acyltransferase generating anandamide precursor and its congeners2007

    • Author(s)
      Jin, X.-H.et al.
    • Journal Title

      J.Biol.Chem. 282(6)

      Pages: 3614-3623

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Predominant expression of lysosomal N-acylethanolamine-hydrolyzing acid amidase in macrophages revealed by immunochemical studies2007

    • Author(s)
      Tsuboi, K.et al.
    • Journal Title

      Biochim.Biophys.Acta-Mol.Cell Biol.Lipids 1771(5)

      Pages: 623-632

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Functional analysis of the purified anandamide-generating phospholipase D as a member of the metallo-β-lactamase family2006

    • Author(s)
      Wang, J. et al.
    • Journal Title

      J. Biol. Chem. 281(18)

      Pages: 12325-12335

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Functional analysis of the purified anandamide-generating phospholipase D as a member of the metallo-□-lactamase family2006

    • Author(s)
      Wang, J.et al.
    • Journal Title

      J.Biol.Chem. 281 (18)

      Pages: 12325-12335

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Biosynthetic pathways of the endocannabinoid anandamide

    • Author(s)
      Okamoto, Y.et al.
    • Journal Title

      Chem.Biodivers. (in press)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] The N-acylethanolamine-hydrolyzing acid amidase (NAAA)

    • Author(s)
      Tsuboi, K.et al.
    • Journal Title

      Chem.Biodivers. (in press)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2008-05-27  

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