Cysteine conjugate S-oxidase. Characterization of a novel enzymatic activity in rat hepatic and renal microsomes.

P J Sausen,A A Elfarra

doi:10.1016/s0021-9258(19)39303-2

Abstract

Cysteine conjugate S-oxidase activity, with S-benzyl-L-cysteine as substrate, was found mostly in the microsomal fractions of rat liver and kidney. In the presence of oxygen and NADPH, S-benzyl-L-cysteine is converted to S-benzyl-L-cysteine sulfoxide; no S-benzyl-L-cysteine sulfone was detected. The Vmax for S-benzyl-L-cysteine sulfoxide formation by kidney microsomes was nearly 3-fold greater than the rate measured with liver microsomes. Inclusion of catalase, superoxide dismutase, glutathione, butylated hydroxyanisole, the peroxidase inhibitor, potassium cyanide, the cytochrome P-450 inhibitors, 1-benzylimidazole and metyrapone, or a monoclonal antibody to cytochrome P-450 reductase did not inhibit the metabolic reaction. Flavin-containing monooxygenase alternate substrates, N,N-dimethylaniline, n-octylamine, and methimazole inhibited the S-oxidase activities. Analogues of S-benzyl-L-cysteine, S-methyl-L-cysteine, and S-(1,2-dichlorovinyl)-L-cysteine inhibited the S-benzyl-L-cysteine S-oxidase activities, whereas S-carboxymethyl-L-cysteine and S-benzyl-L-cysteine methyl ester had no effect. These results provide clear evidence against the involvement of reactive oxygen intermediates or cytochrome P-450 in the sulfoxidation of S-benzyl-L-cysteine and indicate that the S-oxidase activities may be associated with flavin-containing monooxygenases which exhibit selectivity in the interaction with cysteine S-conjugates.

Highlights

Toxicology Center, Cysteine conjugate S-oxidase activity, with S-benzyl-L-cysteine as substrate, was found mostly in the microsomal fractions of rat liver and kidney
S-oxidase activities, whereas S-carboxymethyl-L-cysteine and S-benzyl-L-cysteine methyl ester had no effect. These results provide clear evidence against the involvement of reactive oxygen intermediates or cytochrome P-450 in the sulfoxidation of S-benzyl-L-cysteine and indicate that the S-oxidase activities may be associated with flavin-containing monooxygenases which exhibit selectivity in the interaction with cysteine S-conjugates
This metabolic reaction is catalyzed by glutathione S-transferases which are present in several tissues, with activity in the liver being much higher than in the kidney

Summary

PROCEDURES

Materials-NADPH, catalase, superoxide dismutase, horse heart cytochrome c, S-methyl-L-cysteine, S-carboxymethyl-L-cysteine, S-. Microsomes were incubated for 5 min at 37 “C before SBC was added to begin the enzymatic reaction, After 15 min ice-cold ethanol (0.5 ml) was added, samples were vortexed, and placed on ice until they were centrifuged for 30 min at 4 “C in a Beckman model TJ-6R benchtop centrifuge to remove precipitated proteins. All incubations conducted to characterize SBC S-oxidase dependence on protein concentration, incubation time, temperature, pH (6.8-8.0 potassium phosphate buffer) and the effects of various agents added to the incubation mixture were carried out using equimolar concentrations (2.5 mM) of SBC and NADPH, unless otherwise noted. The microsomal protein was mixed with the antibody and kept on ice for 15 min prior to the addition of SBC (2.5 mM, liver; 5.0 mM, kidney) and NADPH (2 mM) and incubating at 31 “C for 20 min.

RESULTS

DISCUSSION

Methods