Hydroperoxide specificity of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines.
Since hydroperoxide specificity of lipoxygenase (LO) is poorly understood at present, we investigated the ability of cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) to support cooxidase activity of the enzyme toward the selected xenobiotics. Considering the fact that in the past, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases, in this study, we investigated the ability of non-heme iron proteins, namely soybean LO (SLO) and human term placental LO (HTPLO) to mediate N-demethylation of phenothiazines. In addition to being dependent on peroxide concentration, the reaction was dependent on enzyme concentration, substrate concentration, incubation time, and pH of the medium. Using Nash reagent to estimate formaldehyde production, the specific activity under optimal assay conditions for the SLO mediated N-demethylation of chlorpromazine (CPZ), a prototypic phenothiazine, in the presence of TBHP, was determined to be 117+/-12 nmol HCHO/min/mg protein, while that of HTPLO was 3.9+/-0.40 nmol HCHO/min/mg protein. Similar experiments in the presence of CHP yielded specific activities of 106+/-11 nmol HCHO/min/mg SLO, and 3.2+/-0.35 nmol HCHO/min/mg HTPLO. As expected, nordihydroguaiaretic acid and gossypol, the classical inhibitors of LOs, as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of formaldehyde production from CPZ by SLO in the reaction media fortified with either CHP or TBHP. Besides chlorpromazine, both SLO and HTPLO also mediated the N-demethylation of other phenothiazines in the presence of these organic hydroperoxides.
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Florida Toxicology Research Center, Department of Environmental and Occupational Health, College of Public Health, MDC-56, University of South Florida, 13201 Bruce B. Downs Boulevard, Tampa, FL 33612-3805, USA.
Kulkarni
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D001555:Benzene Derivatives / Q000494:pharmacology; D002746:Chlorpromazine / Q000378:metabolism; D005557:Formaldehyde / Q000032:analysis; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008084:Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D018384:Oxidative Stress; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D010640:Phenothiazines / Q000378:metabolism*; D010865:Pilot Projects; D010920:Placenta / Q000201:enzymology; D013025:Glycine max; D013057:Spectrum Analysis; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D001555:Benzene Derivatives; D016859:Lipoxygenase Inhibitors; D010640:Phenothiazines; D005557:Formaldehyde; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D008084:Lipoxygenase; D010089:Oxidoreductases, N-Demethylating; C007164:cumene hydroperoxide; D002746:Chlorpromazine