Reversible hydrolysis and synthesis of anandamide demonstrated by recombinant rat fatty-acid amide hydrolase.
Previously we suggested that one porcine brain enzyme (anandamide amidohydrolase) catalyzed both the hydrolysis of anandamide and its synthesis from arachidonic acid and ethanolamine (Ueda et al., J. Biol. Chem. 270, 23823-23827, 1995). In the present study we investigated the reversibility of the enzyme reactions by the use of recombinant fatty-acid amide hydrolase of rat liver, which appears to be catalytically identical to porcine anandamide amidohydrolase. The particulate fraction of the COS-7 cells, in which the rat enzyme was overexpressed, hydrolyzed anandamide with a specific activity of 132 nmol/min/mg protein at 37 degrees C, and the Km value for anandamide was 18 microM. The enzyme also synthesized anandamide at a rate of 177 nmol/min/mg protein, and the Km values for arachidonic acid and ethanolamine as substrates were as high as 190 microM and 36 mM, respectively. The control cells transfected with the insert-free vector showed neither the hydrolase activity nor the synthase activity. Thus, the hydrolase and synthase are attributed to the same enzyme protein coded by one gene. However, the enzyme may act as a hydrolase rather than a synthase under physiological conditions judging from its high Km values for substrates in the synthase reactions. In addition, primary amides of fatty acids such as arachidonamide and oleamide and fatty acid ester like methyl arachidonate were hydrolyzed at considerable rates, and their reverse reactions occurred even if at lower rates.
Kurahashi
Y
Y
Department of Biochemistry, Tokushima University, School of Medicine, Japan.
Ueda
N
N
Suzuki
H
H
Suzuki
M
M
Yamamoto
S
S
D000581:Amidohydrolases / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D019556:COS Cells; D002186:Cannabinoids / Q000378:metabolism*; D017931:DNA Primers; D063388:Endocannabinoids; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D053284:Polyunsaturated Alkamides; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D002186:Cannabinoids; D017931:DNA Primers; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D011994:Recombinant Proteins; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide