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 [14C]
anandamide was found in both microsomal and 10,000 x g pellet fractions. The latter fractions, which displayed the highest Vmax for
anandamide, were used for further characterisation of the
enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl-
amide, with an apparent Km of 9.0 +/- 2.2 microM. [14C]
anandamide- and [14C]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 A2 inhibitor arachidonyl-trifluoromethyl-ketone with the same IC50 (3 microM); (iv) were competitively inhibited by both
anandamide (or other
polyunsaturated fatty acid-ethanolamides) and oleoyl-
amide.
Proteins solubilised from 10,000 x g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]
anandamide and [14C]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.