Anandamide amidohydrolase of porcine brain: cDNA cloning, functional expression and site-directed mutagenesis(1).
Biochim Biophys Acta
; 1441(1): 77-84, 1999 Oct 18.
Article
em En
| MEDLINE
| ID: mdl-10526230
ABSTRACT
Anandamide (arachidonoylethanolamide) is an endogenous ligand for cannabinoid receptors, and its cannabimimetic activities are lost when the compound is hydrolyzed to arachidonic acid and ethanolamine by an enzyme referred to as anandamide amidohydrolase. We cloned a cDNA for the enzyme of porcine brain, and the cDNA 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 previously cloned from the liver of rat, mouse, and human, respectively. When the enzyme protein was overexpressed in COS-7 cells, the particulate fraction of the cells showed an anandamide hydrolyzing activity and also catalyzed the reverse reaction synthesizing anandamide from arachidonic acid and ethanolamine both with a specific activity of 0. 2-0.3 micromol/min/mg protein at 37 degrees C. The brain enzyme exhibited a wide substrate specificity hydrolyzing oleamide, 2-arachidonoylglycerol, and methyl arachidonate. The point mutation of Ser-217, Asp-237, Ser-241, or Cys-249 completely abolished the hydrolyses of all the above-mentioned substrates as well as the synthesis of anandamide in the reverse reaction.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Encéfalo
/
Amidoidrolases
Limite:
Animals
Idioma:
En
Revista:
Biochim Biophys Acta
Ano de publicação:
1999
Tipo de documento:
Article
País de afiliação:
Japão