RESUMEN
The enzyme 15-lipoxygenase-1 (15-LO-1) possesses mainly 15-LO activity and has so far only been described in human cells and rabbit reticulocytes. The animal ortholog, except rabbit reticulocytes, is an enzyme with predominantly a 12-lipoxygenase activity, commonly referred to as 12/15-LO. We describe herein the characterization of the 12/15-LOs in Macaca mulatta (rhesus monkey) and in Pongo pygmaeus (orang-utan). The rhesus and the orang-utan enzymes have mainly 12-lipoxygenase and 15-lipoxygenase activity, respectively, and they display 94% and 98% identity to the human 15-LO-1 protein. The rhesus enzyme was functionally different from the human enzyme with respect to substrate utilization in that anandamide was used differently and that the rhesus enzymes positional specificity could be affected by the substrate concentration. Furthermore, genomic data indicate that chimpanzees express an enzyme with mainly 15-lipoxygenase activity whereas marmosets express an enzyme with mainly 12-LO activity. Taken together, the switch during evolution from a 12-lipoxygenating enzyme in lower primates to a 15-lipoxygenating enzyme in higher primates and man might be of importance for the biological function of this enzyme.
Asunto(s)
Araquidonato 12-Lipooxigenasa/química , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/química , Araquidonato 15-Lipooxigenasa/metabolismo , Primates/metabolismo , Secuencia de Aminoácidos , Animales , Araquidonato 12-Lipooxigenasa/clasificación , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/clasificación , Araquidonato 15-Lipooxigenasa/genética , Ácido Araquidónico/farmacología , Ácidos Araquidónicos/farmacología , Línea Celular , Cromatografía Líquida de Alta Presión , Clonación Molecular , Endocannabinoides , Activación Enzimática/efectos de los fármacos , Humanos , Técnicas In Vitro , Pulmón/metabolismo , Macaca mulatta/metabolismo , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Alcamidas Poliinsaturadas/farmacología , Pongo pygmaeus/metabolismo , Homología de Secuencia de AminoácidoAsunto(s)
Araquidonato 15-Lipooxigenasa/genética , Araquidonato Lipooxigenasas/genética , Secuencia de Aminoácidos , Animales , Araquidonato 12-Lipooxigenasa/clasificación , Araquidonato 15-Lipooxigenasa/clasificación , Araquidonato 5-Lipooxigenasa/clasificación , Araquidonato Lipooxigenasas/clasificación , Clonación Molecular , Humanos , Mamíferos , Ratones , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa/métodosAsunto(s)
Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Animales , Araquidonato 15-Lipooxigenasa/química , Araquidonato 15-Lipooxigenasa/clasificación , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Regulación Enzimológica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/genética , Especificidad por SustratoRESUMEN
5-Lipoxygenase is the first committed enzyme in the leukotriene biosynthetic pathway and is known to catalyze not only the first oxygenation of arachidonate to form 5(S)-hydroperoxyeicosatetraenoic acid (5(S)-HPETE), but also dehydration of this intermediate into leukotriene A4 (LTA4) by an activity termed leukotriene A4 synthase. Inhibition of cytosolic 5-lipoxygenase prepared from human blood granulocytes with zileuton (100 microM) was virtually complete, but LTA4 synthase activity was only inhibited by 47%. Structural characterization of eicosanoids synthesized in these preparations revealed an abundance of 15-lipoxygenase metabolites including 15-HETE when arachidonate was used as substrate and 5(S),15(S)-dihydroxy-6,8,11,13(E,E,Z,Z)-eicosatetraenoic acid when 5(S)-HPETE was used as substrate. When neutrophils were prepared that contained less than 1% eosinophil contamination, zileuton was found to almost completely inhibit all 5-lipoxygenase, as well as LTA4 synthase products. Immunochemical analysis of the supernatants from purified neutrophils and eosinophils confirmed the previous observation that neutrophils do not express 15-lipoxygenase. Incubation of 5(S)-HPETE with recombinant mammalian 15-lipoxygenase resulted in the formation of 6-trans-LTB4 and 6-trans-12-epi-LTB4 as LTA4 products, as well as the 12-lipoxygenase product 5(S),12(S)-diHPETE. The mechanism of action of 15-lipoxygenase acting as an LTA4 synthase is proposed to involve removing the pro-R hydrogen atom at carbon-10 of 5(S)-HPETE, which is antarafacial to the hydroperoxy group to yield LTA4.