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 [H-3]anandamide hydrolysis were [H-3]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 mu M 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 similar to 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. (C) 2005 Published by Elsevier SAS.

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

Maccarrone M;
2005

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 [H-3]anandamide hydrolysis were [H-3]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 mu M 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 similar to 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. (C) 2005 Published by Elsevier SAS.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11697/155762
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