Purification and Characterization of an Acid Amidase Selective for N-Palmitoylethanolamine, a Putative Endogenous Anti-inflammatory Substance

Natsuo Ueda,Kenji Yamanaka,Shozo Yamamoto

doi:10.1074/jbc.m106261200

Natsuo Ueda, Kenji Yamanaka + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m106261200

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Abstract

N-Arachidonoylethanolamine (anandamide) is cannabimimetic, and N-palmitoylethanolamine is anti-inflammatory and immunosuppressive. We found an amidase that is more active with the latter than the former in contrast to the previously known anandamide amidohydrolase for which N-palmitoylethanolamine is a poor substrate. Proteins solubilized by freezing and thawing from the 12,000 x g pellet of various rat organs hydrolyzed [(14)C]N-palmitoylethanolamine to palmitic acid and ethanolamine. The specific enzyme activity was higher in the order of lung > spleen > small intestine > thymus > cecum, and high activity was found in peritoneal and alveolar macrophages. The enzyme with a molecular mass of 31 kDa was purified from rat lung to a specific activity of 1.8 micromol/min/mg protein. Relative reactivities of the enzyme with various N-acylethanolamines (100 microm) were as follows: N-palmitoylethanolamine, 100%; N-myristoylethanolamine, 48%; N-stearoylethanolamine, 21%; N-oleoylethanolamine, 20%; N-linoleoylethanolamine, 13%; anandamide, 8%. The enzyme was the most active at pH 5 and was activated 7-fold by Triton X-100. The enzyme was almost insensitive to methyl arachidonyl fluorophosphonate, which inhibited anandamide amidohydrolase potently. Thus, the new enzyme referred to as N-palmitoylethanolamine hydrolase was clearly distinguishable from anandamide amidohydrolase.

Highlights

N-Arachidonoylethanolamine is cannabimimetic, and N-palmitoylethanolamine is anti-inflammatory and immunosuppressive
Very recently we found an anandamide-hydrolyzing enzyme, which was catalytically distinct from the previously known anandamide amidohydrolase, in a human megakaryoblastic leukemia cell line (CMK) [36]
Organ Distribution of “N-Palmitoylethanolamine Hydrolase”—The soluble fractions prepared by freezing and thawing from the 12,000 ϫ g pellet of various rat organs were assayed for the N-palmitoylethanolamine-hydrolyzing activity

Summary

EXPERIMENTAL PROCEDURES

Materials—[1-14C]Arachidonic, [1-14C]linoleic, and [1-14C]oleic acids, Phenyl-Sepharose CL-4B, HiTrap Heparin HP, and HiTrap Butyl FF were purchased from Amersham Pharmacia Biotech, and [1-14C]palmitic, [1-14C]stearic, and [1-14C]myristic acids were from PerkinElmer Life Sciences. For the ceramidase assay [40], the enzyme was incubated with 100 ␮M [14C]ceramide (10,000 cpm) at 37 °C for 30 min in a 100-␮l reaction mixture containing 50 mM citrate-sodium phosphate (pH 5.0), 0.1% sodium cholate, and 0.05% Triton X-100. After the column was washed with 10 ml of 20 mM Tris-HCl (pH 7.4), adsorbed proteins were eluted in 1.0-ml fractions with 1% octyl glucoside This chromatography was performed 10 times using 100 ml of the sample in total, and all the active fractions were pooled. The sample was loaded on a HiTrap Heparin column (1-ml bed volume) pre-equilibrated with 20 mM Tris-HCl (pH 7.4) containing 1% octyl glucoside. The enzyme was loaded onto a HiTrap Butyl FF (1-ml bed volume) pre-equilibrated with 20 mM Tris-HCl (pH 7.4) containing 1 M ammonium sulfate. Of 20 mM Tris-HCl (pH 7.4) containing 0.1 M ammonium sulfate and 1 ml of 20 mM Tris-HCl (pH 7.4), the enzyme was eluted with 20 mM Tris-HCl (pH 7.4) containing 1% octyl glucoside

RESULTS

Specific activity

DISCUSSION