Further characterization and purification of the flavin-dependent S-benzyl-L-cysteine S-oxidase activities of rat liver and kidney microsomes.

Previously, we provided evidence that cysteine conjugate S-oxidase (S-oxidase) activities of rat liver and kidney microsomes may be associated with flavin-containing monooxygenases (FMOs). In this study, the biochemical properties of these activities were further investigated. When NADPH was replaced by NADH, the S-oxidase activities were reduced significantly. Removal of the flavin moiety from microsomes significantly reduced the S-oxidase activities; however, addition of exogenous FAD or FMN restored the activities of the flavin-depleted microsomes. Solubilization of hepatic or renal microsomes with Emulgen 911, Nonidet P-40, Triton X-100, or 3-[(3-cholamidopropyl)dimethyl-ammonio]-1- propane sulfate or inclusion of the sulfhydryl-reactive agents Hg2+, N-ethylmaleimide, or iodoacetamide did not affect the S-oxidase activities, whereas solubilization of either hepatic or renal microsomes by cholate or heating of renal microsomes in the absence of NADPH significantly reduced the S-oxidase activities. In addition to male rat hepatic and renal microsomes, the S-oxidase activities were detected in lung microsomes of male rats and hepatic and renal microsomes of male mice and female rats and rabbits. The male rat kidney maintained the highest S-oxidase activity of all species and tissues examined. Whereas the aforementioned results provided further evidence for the S-oxidase activities being associated with FMOs, unambiguous evidence for this hypothesis was provided by the purification of the activities from rat liver (580-fold) and kidney (700-fold) microsomes and by the use of the isolated proteins in polyacrylamide gel electrophoresis, flavin content determinations, amino-terminal amino acid sequence analysis, amino acid composition analysis, and substrate kinetic studies. The findings that the S-oxidases were immunoreactive with antibodies raised against the pig liver 1A1 isozyme but not with antibodies raised against the rabbit lung 1B1 isozyme and that the liver S-oxidase amino-terminal amino acid sequence was more comparable to the amino-terminal amino acid sequences of pig and rabbit liver 1A1 isozymes than to those of rabbit lung 1B1 and liver 1D1 isozymes provide evidence that the S-oxidases are related to the known FMO 1A1 isozymes.