An acid amidase hydrolyzing anandamide as an endogenous ligand for cannabinoid receptors

Anandamide loses its cannabimimetic activities upon hydrolysis to arachidon ic acid and ethanolamine. So far the anandamide hydrolyzing activity widely distributed in mammalian organs has been attributed exclusively to an enzy me referred to as anandamide amidohydrolase with an optimum pH around 9. We found another enzyme hydrolyzing anandamide in a human megakaryoblastic ce ll line (CMK). The enzyme present in the 12 000 x g pellet of the cell homo genate was solubilized by freeze-thaw, The solubilized enzyme showed an opt imal pH around 5, and was almost inactive at alkaline pH, The enzyme activi ty was increased by the addition of dithiothreitol, In contrast, anandamide amidohydrolase of RBL-1 cells was mostly insoluble even after freeze-thaw, showed an optimal pH at 9, and was not affected by dithiothreitol, Further more, the enzyme of CMK cells was much less sensitive to phenylmethylsulfon yl fluoride and methyl arachidonoyl fluorophosphonate potently inhibiting a nandamide amidohydrolase, and effectively hydrolyzed palmitoylethanolamide, which was a poor substrate for anandamide amidohydrolase. Thus, the enzyme of CMK cells is distinguishable from anandamide amidohydrolase. (C) 1999 F ederation of European Biochemical Societies.