Anandamide metabolism by Tetrahymena pyriformis in vitro. Characterization and identification of a 66 kDa fatty acid amidohydrolase

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• 作者：Vivi Karava ; Patapia-Maria Zafiriou ; Lambrini Fasia ; Dimitris Anagnostopoulos ; Effrossini Boutou ; Constantinos E. Vorgias ; Mauro Maccarrone and Athanassia Siafaka-Kapadai
• 刊名：Biochimie
• 出版年：2005
• 出版时间：2005
• 年：2005
• 卷：87
• 期：11
• 页码：967-974
• 全文大小：228 K

文摘

Fatty acid amidohydrolase, a membrane-bound enzyme found in a variety of mammalian cells, is responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide. In an earlier study we reported that Tetrahymena pyriformis was able to secrete a FAAH-like activity in starvation medium (Karava V., Fasia L., Siafaka-Kapadai A., FEBS Lett. 508 (2001) 327–331). In this study the endocannabinoid anandamide, was found to be metabolized by T. pyriformis homogenate by the action of a FAAH-like enzyme, in a time- and concentration-dependent manner. The main metabolic products of [³H]anandamide hydrolysis were [³H]arachidonic acid and ethanolamine. Amidohydrolase activity was maximal at pH 9–10, it was inhibited by phenylmethylsulfonyl fluoride and arachidonyltrifluoromethyl ketone and was Ca²⁺ and Mg²⁺-independent. Kinetic experiments demonstrated that the enzyme had an apparent K_m of 2.5 μM and V_max of 20.6 nmol/min mg. Subcellular fractionation of T. pyriformis homogenate showed that the activity was present in every subcellular fraction with highest specific activity in the microsomal as well as in non-microsomal membrane fraction. Immunoblot analysis of selected subcellular fractions, using an anti-FAAH polyclonal antibody, revealed the presence of an immunoreactive protein with a molecular mass ~66 kDa similar to the molecular mass of the mammalian enzyme. In conclusion, this study demonstrates that a FAAH similar to the mammalian enzyme is present in a unicellular eukaryote, indicating the importance of FAAH activity throughout evolution. It also supports the notion that Tetrahymena species may be a suitable model for metabolic studies on endocannabinoids, as well as for the study of drugs targeted towards FAAH.