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Catalytic properties of purified recombinant anandamide amidohydrolase
Abstracts / Prostaglandins & other Lipid Mediators 59 (1999) 1-235
CATALYTIC PROPERTIES AMIDOHYDROLASE
OF
PURIFIED
RECOMBINANT
49
ANANDAMIDE
NATSUO UEDA’, Kazuhisa Katayama *, Sravan Kumar Goparajul, Yuko Kurahashi’, Kenji Yamanaka’. Hiroshi Suzuki’ and Shozo Yamamoto’ Departments of ’Biochemistry and 2 Cardiovascular Surgery, Tokushima University, School of Medicine, Tokushima 770-8503, Japan The cannabimimetic activities of anandamide (arachidonoylethanolamide) are lost when the compound is hydrolyzed to arachidonic acid and ethanolamine by an enzyme referred to as anandamide amidohydrolase or fatty acid amide hydrolase. We overexpressed the rat enzyme bearing a hexahistidine tag in a baculovirus-Sf9 insect cel1 system, and highly purified the recombinant enzyme with the aid of Ni-chelating resin. The purified enzyme hydrolyzed anandamide with a specific activity of 5.7 pmol/min/mg protein at 37 “C, and Km for anandamide was 30 PM. In addition, the enzyme catalyzed the reverse reaction synthesizing anandamide from arachidonic acid (Km, 70 pM) and ethanolamine (Km, 40 mM) with a specific activity of 6.8 pmol/min/mg protein. As examined by the use of a large amount of the enzyme, the anandamide hydrolysis and its reverse reaction reached an equilibrium within 10 min. The equilibrium constant ([arachidonic acid] [ethanolamine]) / ([anandamide] [water]) was calculated as 4 x 10w3(37 “C , pH 9.0). We also cloned a cDNA of the enzyme of porcine bram, which encoded a protein of 579 amino acids with a molecular mass of 62.9 kDa. The amino acid sequence was 81,80 and 85% identical with the enzymes cloned by Cravatt er al. from the liver of rat, mouse, and human, respectively. Like the rat liver enzyme, the porcine bram enzyme expressed in COS-7 cells exhibited a wide substrate specificity hydrolyzing oleamide, 2arachidonoylglycerol and methyl arachidonate. The point mutation of Ser-217, Asp-237, Ser24 1, or Cys-249 abolished the hydrolyses of al1 these substrates as wel1 as the synthesis of anandamide in the reverse reaction.
CATALYTIC PROPERTIES AMIDOHYDROLASE
OF
PURIFIED
RECOMBINANT
49
ANANDAMIDE
NATSUO UEDA’, Kazuhisa Katayama *, Sravan Kumar Goparajul, Yuko Kurahashi’, Kenji Yamanaka’. Hiroshi Suzuki’ and Shozo Yamamoto’ Departments of ’Biochemistry and 2 Cardiovascular Surgery, Tokushima University, School of Medicine, Tokushima 770-8503, Japan The cannabimimetic activities of anandamide (arachidonoylethanolamide) are lost when the compound is hydrolyzed to arachidonic acid and ethanolamine by an enzyme referred to as anandamide amidohydrolase or fatty acid amide hydrolase. We overexpressed the rat enzyme bearing a hexahistidine tag in a baculovirus-Sf9 insect cel1 system, and highly purified the recombinant enzyme with the aid of Ni-chelating resin. The purified enzyme hydrolyzed anandamide with a specific activity of 5.7 pmol/min/mg protein at 37 “C, and Km for anandamide was 30 PM. In addition, the enzyme catalyzed the reverse reaction synthesizing anandamide from arachidonic acid (Km, 70 pM) and ethanolamine (Km, 40 mM) with a specific activity of 6.8 pmol/min/mg protein. As examined by the use of a large amount of the enzyme, the anandamide hydrolysis and its reverse reaction reached an equilibrium within 10 min. The equilibrium constant ([arachidonic acid] [ethanolamine]) / ([anandamide] [water]) was calculated as 4 x 10w3(37 “C , pH 9.0). We also cloned a cDNA of the enzyme of porcine bram, which encoded a protein of 579 amino acids with a molecular mass of 62.9 kDa. The amino acid sequence was 81,80 and 85% identical with the enzymes cloned by Cravatt er al. from the liver of rat, mouse, and human, respectively. Like the rat liver enzyme, the porcine bram enzyme expressed in COS-7 cells exhibited a wide substrate specificity hydrolyzing oleamide, 2arachidonoylglycerol and methyl arachidonate. The point mutation of Ser-217, Asp-237, Ser24 1, or Cys-249 abolished the hydrolyses of al1 these substrates as wel1 as the synthesis of anandamide in the reverse reaction.