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
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.