2 NOVEL CLASSES OF NEUROACTIVE FATTY-ACID AMIDES ARE SUBSTRATES FOR MOUSE NEUROBLASTOMA ANANDAMIDE AMIDOHYDROLASE

Citation
S. Maurelli et al., 2 NOVEL CLASSES OF NEUROACTIVE FATTY-ACID AMIDES ARE SUBSTRATES FOR MOUSE NEUROBLASTOMA ANANDAMIDE AMIDOHYDROLASE, FEBS letters, 377(1), 1995, pp. 82-86
Citations number
19
Categorie Soggetti
Biophysics,Biology
Journal title
ISSN journal
00145793
Volume
377
Issue
1
Year of publication
1995
Pages
82 - 86
Database
ISI
SICI code
0014-5793(1995)377:1<82:2NCONF>2.0.ZU;2-P
Abstract
The endogenous cannabimimetic substance, anandamide (N-arachidonoyl-et hanolamine) and the recently isolated sleep-inducing factor, oleoyl-am ide (cis-9,10-octadecenoamide), belong to two neuroactive fatty acid a mide classes whose action in mammals has been shown to be controlled b y enzymatic amide bond hydrolysis. Here me report the partial characte risation and purification of 'anandamide amidohydrolase' from membrane fractions of N18 neuroblastoma cells, and provide evidence for a furt her and previously unsuspected role of this enzyme, An enzymatic activ ity catalysing the hydrolysis of [C-14]anandamide was found in both mi crosomal and 10,000 x g pellet fractions, The latter fractions, which displayed the highest V-max for anandamide, were used for further char acterisation of the enzyme, and were found to catalyse the hydrolysis also of [C-14]oleoyl-amide, with an apparent K-m of 9.0 +/- 2.2 mu M. [C-14]anandamide- and [C-14]oleoyl-amide-hydrolysing activities: (i) e xhibited 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 wi th the same IC50 (3 mu M); (iv) were competitively inhibited by both a nandamide (or other polyunsaturated fatty acid-ethanolamides) and oleo yl-amide, Proteins solubilised from 10,000 x g pellets were directly a nalysed by isoelectric focusing, yielding purified fractions capable o f catalysing the hydrolysis of both [C-14]anandamide and [C-14]oleoyl- amide. These data suggest that 'anandamide amidohydrolase' enzymes, su ch as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep-inducing factor oleoyl-amide.