Formation of a novel enzymatic metabolite of leukotriene A4 in tissues of Xenopus laevis.
Leukotriene-A4, hydrolase catalyzes the final step in the biosynthesis of the potent proinflammatory mediator leukotriene B4. Previously, leukotriene-A4 hydrolase has been characterized from human, mouse and rat sources, i.e. only from mammalian species. In the present investigation, expression of leukotriene-A4, hydrolase was studied in organs of Xenopus laevis. Enzyme activity was found in all nine organs tested with the highest levels in the intestine and the reproductive organs, i.e. oocytes and testes, previously unrecognized rich sources of the enzyme. No immunoreactive leukotriene-A4 hydrolase was detected in Western blots of 10000Xg supernatants of X. laevis organ homogenates, using a polyclonal antiserum raised against human leukotriene-A4 hydrolase. Likewise, Northern blot analysis of liver total RNA did not detect Xenopus leukotriene-A4 hydrolase mRNA using a human CDNA probe. These results indicate significant structural differences between the human and toad enzymes. Incubations of 10000Xg supernatants of organ homogenates with leukotriene A4 revealed the formation of a novel metabolite, denoted compound X. Conversion of leukotriene A4 into compound X was due to an enzymatic activity as judged by its protein dependence, heat sensitivity, and resistance to ultrafiltration, and this activity appeared to be linked, directly or indirectly,, to leukotriene A4 hydrolase. From data obtained by ultraviolet spectrophotometry, gas chromatography coupled to mass spectrometry, ultraviolet-induced isomerization, and comparison with a synthetic standard, compound X was assigned the structure 5S,12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid. Finally, compound X was found to exhibit contractile activity in guinea-pig lung parenchyma, apparently elicited via a leukotriene B receptor.
Strömberg
F
F
Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, Stockholm, Sweden.
Hamberg
M
M
Rosenzvist
U
U
Dahlén
S E
SE
Haeggström
J Z
JZ
D000818:Animals; D004851:Epoxide Hydrolases / Q000378:metabolism*; D005260:Female; D006168:Guinea Pigs; D017572:Leukotriene A4 / Q000378:metabolism*; D007975:Leukotriene B4 / Q000378:metabolism; D008168:Lung / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D015394:Molecular Structure; D014018:Tissue Distribution; D014020:Tissue Extracts; D014982:Xenopus laevis / Q000378:metabolism*
D017572:Leukotriene A4; D014020:Tissue Extracts; D007975:Leukotriene B4; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase