A novel mechanism of glutathione conjugate formation by lipoxygenase: a study with ethacrynic acid.
Ethacrynic acid (EA), a diuretic drug, is known to interact with glutathione transferases in the presence of reduced glutathione (GSH) to yield an EA-SG conjugate. Here we present evidence for a new mechanism for the formation of EA-SG conjugate by a soybean lipoxygenase (SLO)-mediated reaction involving oxidation of GSH to a GS.. Similar to the glutathione transferase-mediated reaction, EA-SG conjugate generated by SLO exhibited an absorbance maximum at 270 nm. The conjugate formation was dependent on the concentration of linoleic acid, EA, GSH, and SLO. The optimal assay conditions to observe a maximal rate of EA-SG formation required the presence of 0.4 mM linoleic acid, 1 mM GSH, 50 nM SLO, and 0.2 mM EA at pH 9.0. Classical inhibitors of lipoxygenase, e.g., nordihydroguaiaretic acid, gossypol, and 5,8,11-eicosatriynoic acid, significantly inhibited EA-SG conjugation. The SLO-generated EA-SG was isolated as a single peak by HPLC. Quantitation of EA-SG by HPLC-coupled radiometry using [3H]GSH yielded a rate of 16.5 mumol/min/mg SLO protein. This rate is up to 1650-fold greater than that reported for different purified isozymes of mammalian glutathione transferase. The structure of EA-SG isolated from HPLC column was confirmed by matrix-assisted laser desorption mass spectroscopy. These results suggest that lipoxygenase, which is primarily known for xenobiotic oxidation, may represent yet another important pathway for GSH conjugate formation that could lead to detoxification of certain chemicals.
Kulkarni
A P
AP
Florida Toxicology Research Center, Department of Environmental and Occupational Health, College of Public Health, University of South Florida, Tampa 33612-3805, USA.
Sajan
M P
MP
D000818:Animals; D002855:Chromatography, Thin Layer; D004232:Diuretics / Q000493:pharmacokinetics*; D004976:Ethacrynic Acid / Q000493:pharmacokinetics*; D005260:Female; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008658:Inactivation, Metabolic; D008084:Lipoxygenase / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D011247:Pregnancy; D013025:Glycine max; D013053:Spectrophotometry; D013481:Superoxides / Q000378:metabolism
D004232:Diuretics; D013481:Superoxides; D008084:Lipoxygenase; D005978:Glutathione; D004976:Ethacrynic Acid