| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Anandamide amidohydrolase is an amidase hydrolyzing anandamide (N-arachidonoyl-ethanolamine), which was identified as an endogenous ligand of cannabinoid receptors, to arachidonic acid and ethanolamine. Anandamide loses its cannabimimetic activities by this hydrolysis. In this study we overexpressed large amounts of the enzyme of rat liver in a baculovirus-insect cell system, and highly purified the enzyme. With the purified enzyme we demonstrated the reversibility of the enzymatic hydrolysis of anandamide and determined the equilibrium constant of the reaction. We also cloned a cDNA of the enzyme from porcine brain. When we changed several amino acid residues of the enzyme, which are highly conserved in an amidase family including this enzyme, to alanine or other amino acids by site-directed mutagenesis, the enzymatic activities of all the mutants were very low or hardly detectable. Furthermore, we discovered an anandamide hydrolyzing activity in a human megakaryoblastic cell line (CMK). The enzyme catalyzing this reaction could be solubilized from the membrane without detergent, had an optimal pH around 5, showed low sensitivity to serine hydrolase inhibitors, was activated by dithiothreitol, and was more active with N-palmitoylethanolamine than with anandamide. Based on these findings the enzyme was thought to be an isozyme distinct from the known anandamide amidohydrolase. When we screened for such an enzyme activity in various rat organs, the highest activity was detected in lung, followed by spleen, thymus, small intestine and other organs. The enzyme was potently inhibited by Tween 20, but was activated around seven fold by Triton X-100. As analyzed by SDS-polyacrylamide gel electrophoresis, the highly purified enzyme of lung showed a major band around 31 kDa. The purified enzyme was the most active with N-palmitoylethanolamine among various N-acylethanolamines.
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