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 (approximately 56 kDa) as isolated rat liver FMO3 and cDNA-expressed 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.

Figure 1. Purification of FMO3 from rat kidney microsomes

Lane A, 1.5 μg of cDNA-expressed human FMO3 standard; Lane B, 20 μg total rat kidney microsomes; Lane C, 10 μg solubilized rat kidney microsomes; Lane D, 2 μg Blue Pool I; Lane E, 1 μg Blue Pool II; Lane F, 0.7 μg ADP pool.

Figure 2. Representative immunoblots of ADP pools with anti-human FMO3 antibody

Lane A, 0.75 μg of cDNA-expressed human FMO3 standard; Lane B, 0.5 μg of rat liver ADP pool; Lane C, 0.5 μg of rat kidney ADP pool. Positions of molecular weight markers are shown.

Figure 3. Kinetics for rat renal FMO3

K_m and V_max were determined by non-linear regression in SigmaPlot. Oxidation products were measured and quantified as described in Materials and Methods.