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
