Received for publication March 12, 2008.
Revised August 12, 2008.
Accepted for publication September 4, 2008.

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 FMO1 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 to 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. Typically, Met S-oxidase specific activity was increased 100-fold through the purification steps. The resulting protein had similar mobility (~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 MALDI-TOF mass spectral analysis, 34% of the sequence of rat FMO3 was detected. The apparent Km and Vmax values for rat kidney FMO3 were determined using the known FMO substrates Met, seleno-L-methionine (SeMet), S-allyl-L-cysteine (SAC) and methimazole. The stereoselectivity of the reactions with Met and SAC were also examined using HPLC. The obtained kinetic and stereoselectivity results were similar to those we obtained in the present study, or those previously reported, for rat liver FMO3. Collectively, 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.

Key words: extrahepatic drug metabolism, flavin-containing monooxygenase, renal toxicity