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

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 ؋ 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 mol/min/mg protein. Relative reactivities of the enzyme with various N-acylethanolamines (100 M) 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.

Fatty Acid Amide Hydrolase Inhibition by JNJ‐42165279: A Multiple‐Ascending Dose and a Positron Emission Tomography Study in Healthy Volunteers

“…These data differ somewhat from observations in rat, and may be related to the much higher affinity of JNJ‐42165279 for human over rat FAAH. 4 Moreover, inactivation of OEA and PEA may be mediated by other mechanisms in addition to FAAH 12, 13, 14. Effects on FAA turnover and leukocyte FAAH activity consistent with this report have been observed previously with PF‐04457845.…”

PUFA-derived endocannabinoids: an overview

“…Other enzymes involved in anandamide hydrolysis are N-acylethanolamine acid amidase (71) and FAAH-2 (70) , this latter being an isozyme of FAAH-1 with about 20% sequence identity at the amino acid level, mainly expressed in human subjects, but not in rodents (70) . N-acylethanolamine acid amidase is an N-glycosylated protein, localised in the lysosomes or the Golgi apparatus with an optimal pH of 4·5-5 (71)(72)(73)(74) . FAAH-2 is more effective at metabolizing oleamide than anandamide or other N-acyl-ethanolamines.…”

Regulation of food intake by oleoylethanolamide

“…Two enzymes have been identified which may catalyze this reaction: fatty-acid amide hydrolase (FAAH) [13,14] and PEA-preferring acid amidase (PAA) [15]. FAAH is a membrane-bound intracellular serine hydrolase that catalyzes the hydrolysis of all FAEs, including OEA ( fig.…”

“…In fact, mice lacking the faah gene have dramatically reduced OEA hydrolysis and increased OEA levels in brain and liver tissues [20,21]. Unlike FAAH, PAA activity is most abundant in the rodent lung, spleen and small intestine [15]. In the presence of detergent this activity displays a marked preference for PEA as a substrate; however, when detergents are omitted from the assay PAA recognizes all FAEs, suggesting that it may play a broad role in the deactivation of these compounds by intact cells [15].…”