Lipoxygenase-catalyzed oxygenation of arachidonylethanolamide, a cannabinoid receptor agonist.
Various purified lipoxygenases were incubated with [14C]arachidonylethanolamide which is an endogenous ligand for cannabinoid receptors. When radioactive products were analyzed by thin-layer chromatography, porcine leukocyte 12-lipoxygenase and rabbit reticulocyte and soybean 15-lipoxygenases produced polar compounds at about the same reaction rates as that of oxygenation of free arachidonic acid. In contrast, the reaction of human platelet 12-lipoxygenase proceeded at a much lower rate, and porcine leukocyte 5-lipoxygenase was totally inactive. The result indicated that the lipoxygenases, which had been shown previously to be capable of oxygenating esterified polyunsaturated fatty acids, were also active with the arachidonylethanolamide. High-performance liquid chromatography, ultraviolet and mass spectrometry and nuclear magnetic resonance spectroscopy identified the major product by leukocyte 12-lipoxygenase as 12-hydroperoxy-5,8,10,14-eicosatetraenoylethanolamide and that by 15-lipoxygenases as 15-hydroperoxy-5,8,11,13-eicosatetraenoylethanolamide. The 15-hydroxy derivative inhibited electrically-evoked contraction of mouse vas deferens with an IC50 of 0.63 microM as well as arachidonylethanolamide (0.17 microM), but the 12-hydroxy derivative was much less effective.
Ueda
N
N
Department of Biochemistry, Tokushima University, School of Medicine, Japan.
Yamamoto
K
K
Yamamoto
S
S
Tokunaga
T
T
Shirakawa
E
E
Shinkai
H
H
Ogawa
M
M
Sato
T
T
Kudo
I
I
Inoue
K
K
D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism; D001095:Arachidonic Acids / Q000378:metabolism*; D001792:Blood Platelets / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D063388:Endocannabinoids; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology; D008084:Lipoxygenase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D053284:Polyunsaturated Alkamides; D011817:Rabbits; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000819:agonists* / Q000378:metabolism; D012156:Reticulocytes / Q000201:enzymology; D013025:Glycine max / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D001095:Arachidonic Acids; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D008084:Lipoxygenase; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; C078814:anandamide