Purification and Characterization of Flavin-Containing Monooxygenase Isoform 3 from Rat Kidney Microsomes

ABSTRACT: Rats are a common animal model for metabolism and toxicity studies. Previously, the enzymatic properties of rat flavin-containing monooxygenase (FMO) 1 purified from hepatic and renal microsomes and that of FMO3 purified from hepatic microsomes were characterized. This study investigated the physical, immunological, and enzymatic properties of FMO3 purified from male rat kidney microsomes and compared the results with those obtained with isolated rat liver FMO3. Renal FMO3 was purified via affinity columns based on the elution of L-methionine (Met) S-oxidase activity and reactivity of the eluted proteins with human FMO3 antibody. In general, Met S-oxidase-specific activity was increased 100-fold through the purification steps. The resulting protein had similar mobility (ϳ56 kDa) as isolated rat liver FMO3 and cDNAexpressed human FMO3 by SDS-polyacrylamide gel electrophoresis. When the isolated kidney protein band was subjected to trypsin digestion and matrix-assisted laser desorption ionization/time of flight mass spectral analysis, 34% of the sequence of rat FMO3 was detected. The apparent K m and V max values for rat kidney FMO3 were determined using the known FMO substrates Met, seleno-L-methionine, S-allyl-L-cysteine (SAC), and methimazole (N-methyl-2-mercaptoimidazole). The stereoselectivity of the reactions with Met and SAC were also examined using high-performance liquid chromatography. The obtained kinetic and stereoselectivity results were similar to those we obtained in the present study, or those previously reported, for rat liver FMO3. Taken together, the results demonstrate many similar properties between rat hepatic and renal FMO3 forms and suggest that renal FMO3 may play an important role in kidney metabolism of xenobiotics containing sulfur and selenium atoms. Flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze NADPH-and O 2 -dependent oxidation of compounds with a nucleophilic sulfur, nitrogen, phosphorus, or selenium atom. FMOs have a broad substrate range that includes pharmaceutical drugs, pesticides, industrial chemicals, and endogenous compounds. In general, FMO-mediated metabolites are more readily excreted and are less harmful than the parent compounds; however, with some chemicals, toxic metabolites are formed. Five expressed FMO isoforms (FMO1-FMO5) and six nonexpressed pseudogenes (FMO6P-FMO11P) have been characterized in human

Metabolism of butadiene monoxide by freshly isolated hepatocytes from mice and rats: different partitioning between oxidative, hydrolytic, and conjugation pathways. Drug Metab. Dispos

This paper is available online at http://dmd.aspetjournals.org ABSTRACT: 1,3-Butadiene (BD) is a multisite carcinogen in rodents, with mice being much more susceptible than rats. This species difference in carcinogenicity has been attributed to differences in metabolism. In this study, coordinated metabolism of butadiene monoxide (BMO, 5, 25, and 250 M), the primary reactive metabolite of BD, was investigated in freshly isolated male B6C3F1 mouse and Sprague-Dawley rat hepatocytes. The hepatocytes from both species catalyzed BMO oxidation to meso-and (؎)-diepoxybutane (DEB), BMO hydrolysis to 3-butene-1,2-diol (BDD), and BMO conjugation with glutathione (GSH) to form GSH conjugates (GSBMO). Metabolite area under the curve (AUC) exhibited dependence on the BMO concentration and incubation time (0-45 min). However, the observed BMO activation/detoxication ratios (obtained by dividing the AUC for total DEB by the summed AUC values for BDD and GSBMO) with mouse hepatocytes were approximately 15-to 40-fold higher than the corresponding ratios observed with rat hepatocytes. At 5 M BMO, bioactivation in the mouse exceeded detoxication by approximately 2-fold, whereas at the 250 M concentration, activation was only about 31% of total detoxication. In rat hepatocytes, the activation-detoxication ratio was relatively independent of the initial BMO concentration, with flux through the oxidative pathway at approximately 2 to 5% of the total detoxication. These results, which are more consistent with in vivo mouse and rat toxicity data than the metabolic rates obtained with subcellular fractions, illustrate the potential utility of the isolated hepatocyte model for estimating flux through competing metabolic pathways and predicting in vivo metabolism of BMO and its parent compound, BD

Send IN WVO METABOLITES OF S-(2-BENZOTHIAZOLYL)-L-CYSTEINE AS MARKERS OF IN VIVO CYSTEINE CONJUGATE j9-LYASE AND THIOL GLUCURONOSYL TRANSFERASE ACTIVITIES

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