Equilibrium in the hydrolysis and synthesis of cannabimimetic anandamide demonstrated by a purified enzyme.
Anandamide, an endogenous ligand for cannabinoid receptors, loses its biological activities when it is hydrolyzed to arachidonic acid and ethanolamine by anandamide amidohydrolase. We overexpressed a recombinant rat enzyme with a hexahistidine tag in a baculovirus-insect cell expression system, and purified the enzyme with the aid of a Ni-charged resin to a specific activity as high as 5.7 micromol/min/mg protein. The purified recombinant enzyme catalyzed not only the hydrolysis of anandamide and palmitoylethanolamide, but also their reverse synthetic reactions. In order to attain an equilibrium of the anandamide hydrolysis and its reverse reaction within 10 min, we utilized a large amount of the purified enzyme. The equilibrium constant ([arachidonic acid][ethanolamine])/([anandamide][water]) was calculated as 4x10(-3) (37 degrees C, pH 9.0). These experimental results with a purified enzyme preparation quantitatively confirmed the reversibility of the enzyme reaction previously observed with crude enzyme preparations.
Katayama
K
K
Department of Cardiovascular Surgery, Tokushima University, School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan.
Ueda
N
N
Katoh
I
I
Yamamoto
S
S
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000737:chemistry; D001095:Arachidonic Acids / Q000737:chemistry*; D002186:Cannabinoids / Q000737:chemistry*; D063388:Endocannabinoids; D019856:Ethanolamine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D053284:Polyunsaturated Alkamides; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D014105:Tosyl Compounds / Q000494:pharmacology; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D016718:Arachidonic Acid; D001095:Arachidonic Acids; D002186:Cannabinoids; D063388:Endocannabinoids; D019856:Ethanolamine; D053284:Polyunsaturated Alkamides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014105:Tosyl Compounds; D000099252:Fatty Acid Amide Hydrolases; C003614:4-toluenesulfonyl fluoride; C078814:anandamide