FORMATION OF A NOVEL ENZYMATIC METABOLITE OF LEUKOTRIENE A(4) IN TISSUES OF XENOPUS-LAEVIS

Leukotriene-A(4) hydrolase catalyzes the final step in the biosynthesi s of the potent proinflammatory mediator leukotriene B-4. Previously, leukotrienes hydrolase has been characterized from human, mouse and ra t sources, i.e. only from mammalian species. In the present investigat ion, expression of leukotriene-A(4) hydrolase was studied in organs of Xenopus laevis. Enzyme activity was found in all nine organs tested w ith the highest levels in the intestine and the reproductive organs, i .e. oocytes and testes, previously unrecognized rich sources of the en zyme. No immunoreactive leukotriene-A(4) hydrolase was detected in Wes tern blots of 10 000 x g supernatants of X. laevis organ homogenates, using a polyclonal antiserum raised against human leukotriene-A(4) hyd rolase. Likewise, Northern blot analysis of liver total RNA did not de tect Xenopus leukotriene-A(4) hydrolase mRNA using a human cDNA probe. These results indicate significant structural differences between the human and toad enzymes. Incubations of 10 000 x g supernatants of org an homogenates with leukotriene A(4) revealed the formation of a novel metabolite, denoted compound X. Conversion of leukotriene A(4) into c ompound X was due to an enzymatic activity as judged by its protein de pendence, heat sensitivity, and resistance to ultrafiltration, and thi s activity appeared to be linked, directly or indirectly, to leukotrie ne-A(4) hydrolase. From data obtained by ultraviolet spectrophotometry , gas chromatography coupled to mass spectrometry, ultraviolet-induced isomerization, and comparison with a synthetic standard, compound X w as assigned the structure 12R-dihydroxy-6,10-trans-8,14-cis-eicosatetr aenoic acid. Finally, compound X was found to exhibit contractile acti vity in guinea-pig lung parenchyma, apparently elicited via a leukotri ene B receptor.