Purification and characterization of an acid amidase selective for N-palmitoylethanolamine, a putative endogenous anti-inflammatory substance.
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.
Ueda
N
N
Department of Biochemistry, Kagawa Medical University, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan. nueda@kms.ac.jp
Yamanaka
K
K
Yamamoto
S
S
D000577:Amides; D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000378:metabolism*; D055572:Ceramidases; D063388:Endocannabinoids; D004983:Ethanolamines; D008168:Lung / Q000201:enzymology; D008297:Male; D010169:Palmitic Acids / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity
D000577:Amides; D000893:Anti-Inflammatory Agents; D063388:Endocannabinoids; D004983:Ethanolamines; D010169:Palmitic Acids; C005958:palmidrol; D000581:Amidohydrolases; D055572:Ceramidases