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

Vivi Karava, Patapia Maria Zafiriou, Lambrini Fasia, Dimitris Anagnostopoulos, Effrossini Boutou, Constantinos E. Vorgias, Mauro Maccarrone, Athanassia Siafaka-Kapadai

Research output: Contribution to journalArticle

Abstract

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 Km of 2.5 μM and Vmax 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.

Original languageEnglish
Pages (from-to)967-974
Number of pages8
JournalBiochimie
Volume87
Issue number11
DOIs
Publication statusPublished - Nov 2005

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Keywords

  • Anandamide
  • Fatty acid amidohydrolase
  • Tetrahymena pyriformis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Karava, V., Zafiriou, P. M., Fasia, L., Anagnostopoulos, D., Boutou, E., Vorgias, C. E., Maccarrone, M., & Siafaka-Kapadai, A. (2005). Anandamide metabolism by Tetrahymena pyriformis in vitro. Characterization and identification of a 66 kDa fatty acid amidohydrolase. Biochimie, 87(11), 967-974. https://doi.org/10.1016/j.biochi.2005.04.011