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Recent Progress in <i>N</i>-Acylethanolamine Research: Biological Functions and Metabolism Regulated by Two Distinct <i>N</i>-Acyltransferases: cPLA<sub>2</sub>ε and PLAAT Enzymes.

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PLAAT1 expression triggers fragmentation of mitochondria in an enzyme activity-dependent manner.

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PLAAT1 deficiency alleviates high-fat diet-induced hepatic lipid accumulation in mice.

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Assay of NAT Activity.

Toru Uyama1, Natsuo Ueda2

  • 1Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa, 761-0793, Japan.

Methods in Molecular Biology (Clifton, N.J.)
|June 2, 2016
PubMed
Summary

N-acyltransferase (NAT) enzymes are crucial for synthesizing bioactive N-acylethanolamines. This study details methods for purifying calcium-dependent NAT and recombinant PLA/AT-2, alongside an N-acyltransferase assay.

Keywords:
COS-7 cellCa-NATHRASLS familyN-acylphosphatidylethanolamineN-acyltransferaseNAPEPLA/AT familyRadioisotopeRat brainThin-layer chromatography

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • N-acyltransferase (NAT) initiates the biosynthesis of N-acylethanolamines.
  • Calcium-dependent NAT and calcium-independent PLA/AT family enzymes exhibit NAT activity.
  • N-acylethanolamines are bioactive lipids with diverse physiological roles.

Purpose of the Study:

  • To describe methods for the partial purification of Ca(2+)-dependent NAT from rat brain.
  • To detail the purification of recombinant phospholipase A/acyltransferase-2 (PLA/AT-2).
  • To outline a robust N-acyltransferase (NAT) assay utilizing radiolabeled substrates.

Main Methods:

  • Partial purification of Ca(2+)-dependent NAT from rat brain tissue.
  • Recombinant expression and purification of PLA/AT-2 enzyme.
  • Enzyme activity assay for NAT using radiolabeled acyl-donor substrates.

Main Results:

  • Successful partial purification of Ca(2+)-dependent NAT was achieved.
  • Recombinant PLA/AT-2 was purified to homogeneity.
  • The described NAT assay is effective for enzyme characterization.

Conclusions:

  • The study provides essential methodological details for investigating NAT enzymes.
  • Understanding NAT enzyme mechanisms is key to elucidating N-acylethanolamine biosynthesis.
  • These methods facilitate further research into the roles of N-acylethanolamines.