title: Lipoxygenase-another pathway for glutathione conjugation of xenobiotics: A study with human term placental lipoxygenase and ethacrynic acid.
abstract: In this study, we examined the ability of human term placental lipoxygenase (HTPLO) to catalyze glutathione (GSH) conjugate formation from ethacrynic acid (EA) in the presence of linoleic acid (LA) and GSH. HTPLO purified by affinity chromatography was used in all the experiments. The results indicate that the process of EA-SG is enzymatic in nature. The reaction shows dependence on pH, the enzyme, and the concentration of GSH, LA, and EA. The optimal assay conditions to observe a maximal rate of EA-SG formation required the presence of 0.3 mM LA, 0.2 mM EA, 2.0 mM GSH, and approximately 300 microg HTPLO in the reaction medium buffered at pH 9.0. Under the experimental conditions employed, the reaction exhibited K(m) values of 1.1 mM, 200 microM, and 130 microM for GSH, LA, and EA, respectively. The estimated specific activity of HTPLO-catalyzed EA-GS formation was approximately 4.4 +/- 0.4 micromol/min/mg protein. This rate is more than twofold greater than the rate noted for the reaction mediated by the purified human term placental glutathione transferase. Under physiologically relevant conditions (20 microM LA, 2.0 mM GSH, at pH 7.4), HTPLO produced EA-SG at 56% of the maximal rate noted under optimal assay conditions. Nordihydroguaiaretic acid, the classical inhibitor of different lipoxygenases, significantly blocked the reaction. It is proposed that free radicals are involved in the process of EA-SG formation by HTPLO. The evidence gathered in this in vitro study suggests for the first time that lipoxygenase present in the human term placenta is capable of EA-SG formation.
authors.affiliation: Department of Environmental and Occupational Health, University of South Florida, Tampa, Florida 33612-3805, USA. akulkarn@com1.med.usf.edu
authors.forename: A P
authors.initials: AP
authors.lastname: Kulkarni
authors.forename: M
authors.initials: M
authors.lastname: Sajan
mesh_terms: D002846:Chromatography, Affinity; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004976:Ethacrynic Acid / Q000378:metabolism*; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D019787:Linoleic Acid / Q000378:metabolism; D008084:Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D015262:Xenobiotics / Q000378:metabolism*
chemical_list: D004137:Dinitrochlorobenzene; D015262:Xenobiotics; D019787:Linoleic Acid; D008084:Lipoxygenase; D005978:Glutathione; D004976:Ethacrynic Acid