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Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma ‘anandamide amidohydrolase’
Author(s) -
Maurelli Stefano,
Bisogno Tiziana,
De Petrocellis Luciano,
Di Luccia Aldo,
Marino Gennaro,
Di Marzo Vincenzo
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)01311-3
Subject(s) - anandamide , fatty acid amide hydrolase , chemistry , amidohydrolase , palmitoylethanolamide , amidase , biochemistry , amide , stereochemistry , enzyme , cannabinoid receptor , agonist , receptor
The endogenous cannabimimetic substance, anandamide ( N ‐arachidonoyl‐ethanolamine) and the recently isolated sleep‐inducing factor, oleoyl‐amide ( cis ‐9,10‐octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of ‘anandamide amidohydrolase’ from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [ 14 C]anandamide was found in both microsomal and 10,000 × g pellet fractions. The latter fractions, which displayed the highest V max for anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [ 14 C]oleoyl‐amide, with an apparent K m of 9.0 ± 2.2 μ M. [ 14 C]anandamide‐ and [ 14 C]oleoyl‐amide‐hydrolysing activities: (i) exhibited identical pH‐ and temperature‐dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A 2 inhibitor arachidonyl‐trifluoromethyl‐ketone with the same IC 50 (3 μM); (iv) were competitively inhibited by both anandamide (or other polyunsaturated fatty acid‐ethanolamides) and oleoyl‐amide. Proteins solubilised from 10,000 × g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [ 14 C]anandamide and [ 14 C]oleoyl‐amide. These data suggest that ‘anandamide amidohydrolase’ enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep‐inducing factor oleoyl‐amide.