Anandamide metabolism by Tetrahymena pyriformis in vitro. Characterization and identification of a 66 kDa fatty acid amidohydrolase.
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 Mg(2+)-independent. Kinetic experiments demonstrated that the enzyme had an apparent K(m) of 2.5 microM 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 approximately 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.
Karava
Vivi
V
Department of Chemistry, University of Athens, Panepistimioupolis 15771, Athens, Greece.
Zafiriou
Patapia-Maria
PM
Fasia
Lambrini
L
Anagnostopoulos
Dimitris
D
Boutou
Effrossini
E
Vorgias
Constantinos E
CE
Maccarrone
Mauro
M
Siafaka-Kapadai
Athanassia
A
D000581:Amidohydrolases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D063388:Endocannabinoids; D008970:Molecular Weight; D053284:Polyunsaturated Alkamides; D013769:Tetrahymena pyriformis / Q000378:metabolism*; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide