Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma 'anandamide amidohydrolase'.
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.
Istituto per la Chimica di Molecole di Interesse Biologico, Arco Felice, Naples, Italy.
S
S
Maurelli
T
T
Bisogno
L
L
De Petrocellis
A
A
Di Luccia
G
G
Marino
V
V
Di Marzo
D000577:Amides / Q000378:metabolism*; D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D001667:Binding, Competitive; D063388:Endocannabinoids; D004795:Enzyme Stability; D005227:Fatty Acids / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D051379:Mice; D009447:Neuroblastoma / Q000201:enzymology*; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D000099252:Fatty Acid Amide Hydrolases
D000577:Amides; D000581:Amidohydrolases; D001095:Arachidonic Acids; D063388:Endocannabinoids; D005227:Fatty Acids; D019301:Oleic Acid; D009829:Oleic Acids; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide