Abstract
In animal tissues, N-acyltransferase (NAT) catalyzes the first reaction in the biosynthetic pathway of bioactive N-acylethanolamines, in which an acyl chain is transferred from the sn-1 position of the donor phospholipid, such as phosphatidylcholine, to the amino group of phosphatidylethanolamine, resulting in the formation of N-acylphosphatidylethanolamine. NAT has long been known to be stimulated by Ca2+ and hence referred to as Ca2+-dependent NAT. Later, this enzyme was identified as cPLA2ε (also referred to as PLA2G4E). On the other hand, members of the phospholipase A/acyltransferase (PLAAT) family (also known as HRAS-like suppressor family) show Ca2+-independent NAT activity. In this chapter, we describe (1) partial purification of Ca2+-dependent NAT from rat brain, (2) purification of recombinant cPLA2ε and PLAAT-2, and (3) NAT assay using radiolabeled substrate.
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References
Ueda N, Tsuboi K, Uyama T (2013) Metabolism of endocannabinoids and related N-acylethanolamines: canonical and alternative pathways. FEBS J 280:1874–1894
Hussain Z, Uyama T, Tsuboi K, Ueda N (2017) Mammalian enzymes responsible for the biosynthesis of N-acylethanolamines. Biochim Biophys Acta 1862:1546–1561
Di Marzo V, Fontana A, Cadas H, Schinelli S, Cimino G, Schwartz J-C, Piomelli D (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372:686–691
Cadas H, di Tomaso E, Piomelli D (1997) Occurrence and biosynthesis of endogenous cannabinoid precursor, N-arachidonoyl phosphatidylethanolamine, in rat brain. J Neurosci 17:1226–1242
Hansen HS, Moesgaard B, Hansen HH, Petersen G (2000) N-Acylethanolamines and precursor phospholipids – relation to cell injury. Chem Phys Lipids 108:135–150
Ogura Y, Parsons WH, Kamat SS, Cravatt BF (2016) A calcium-dependent acyltransferase that produces N-acyl phosphatidylethanolamines. Nat Chem Biol 12:669–671
Jin X–H, Okamoto Y, Morishita J, Tsuboi K, Tonai T, Ueda N (2007) Discovery and characterization of a Ca2+-independent phosphatidylethanolamine N-acyltransferase generating the anandamide precursor and its congeners. J Biol Chem 282:3614–3623
Uyama T, Tsuboi K, Ueda N (2017) An involvement of phospholipase A/acyltransferase family proteins in peroxisome regulation and plasmalogen metabolism. FEBS Lett 591:2745–2760
Shinohara N, Uyama T, Jin X-H, Tsuboi K, Tonai T, Houchi H, Ueda N (2011) Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes. J Lipid Res 52:1927–1935
Jin X-H, Uyama T, Wang J, Okamoto Y, Tonai T, Ueda N (2009) cDNA cloning and characterization of human and mouse Ca2+-independent phosphatidylethanolamine N-acyltransferases. Biochim Biophys Acta 1791:32–38
Uyama T, Morishita J, Okamoto Y, Tsuboi K, Tonai T, Ueda N (2009) The tumor suppressor gene H-Rev107 functions as a novel Ca2+-independent cytosolic phospholipase A1/2 of the thiol hydrolase-type. J Lipid Res 50:685–693
Uyama T, Jin X–H, Tsuboi K, Tonai T, Ueda N (2009) Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipid-metabolizing enzymes. Biochim Biophys Acta 1791:1114–1124
Hussain Z, Uyama T, Kawai K, Binte Mustafiz SS, Tsuboi K, Araki N, Ueda N (2018) Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca2+-dependent N-acyltransferase. Biochim Biophys Acta 1863:493–502
Uyama T, Ikematsu N, Inoue M, Shinohara N, Jin X–H, Tsuboi K, Tonai T, Tokumura A, Ueda N (2012) Generation of N-acylphosphatidylethanolamine by members of the phospholipase A/acyltransferase (PLA/AT) family. J Biol Chem 287:31905–31919
Binte Mustafiz SS, Uyama T, Morito K, Takahashi N, Kawai K, Hussain Z, Tsuboi K, Araki N, Yamamoto K, Tanaka T, Ueda N (2019) Intracellular Ca2+-dependent formation of N-acyl-phosphatidylethanolamines by human cytosolic phospholipase A2ε. Biochim Biophys Acta 1864:158515
Golczak M, Kiser PD, Sears AE, Lodowski DT, Blaner WS, Palczewski K (2012) Structural basis for the acyltransferase activity of lecithin:retinol acyltransferase-like proteins. J Biol Chem 287:23790–23807
Natarajan V, Schmid PC, Schmid HHO (1986) N-Acylethanolamine phospholipid metabolism in normal and ischemic rat brain. Biochim Biophys Acta 878:32–41
Schmid PC, Reddy PV, Natarajan V, Schmid HHO (1983) Metabolism of N-acylethanolamine phospholipids by a mammalian phosphodiesterase of the phospholipase D type. J Biol Chem 258:9302–9306
Acknowledgments
We thank Iffat Ara Sonia Rahman, Zahir Hussain, and Smriti Sultana Binte Mustafiz for their careful reading of the manuscript and valuable suggestions.
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Uyama, T., Ueda, N. (2023). Assay of NAT Activity. In: Maccarrone, M. (eds) Endocannabinoid Signaling. Methods in Molecular Biology, vol 2576. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2728-0_17
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DOI: https://doi.org/10.1007/978-1-0716-2728-0_17
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