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Oxidoreductases such as glutaredoxin are a major class of enzymes that reversibly catalyze thiol-disulfide exchange reactions. Transfection experiments using geneticin (G418) selection to identify the specific protein S-thiolated substrates of glutaredoxin-1 (Grx-1) noted the curious phenomenon that nontransfected control cells treated with G418 had increased levels of Grx-1 expression. Varied concentrations of gentamicin, kanamycin, and hygromycin increased Grx-1 expression in a time- and dose-dependent fashion in human cultured retinal pigment epithelial cells. Reactive oxygen species formation after aminoglycoside exposure correlated directly to aminoglycoside treatment. Further indication that oxidation regulates Grx-1 expression was noted by the positive effect of phorbol 12-myristate 13-acetate, a known inducer of redox-sensitive AP-1 transcription factor. In agreement with this hypothesis was the finding that the physiologic reductant N-acetylcysteine decreased Grx-1 expression whereas tert-butyl hydroperoxide increased Grx-1 expression. Our data suggest that aminoglycosides increased Grx-1 expression in response to oxidative stress.

S-thiolation of carbonic anhydrase III (CA III) in cultured rat hepatocytes under oxidative stress was studied by immunodetection on nitrocellulose blots of isoelectrofocusing gels. In cells treated with menadione, three S-thiolated forms of CA III were detected, whereas only two forms were observed in hepatocytes treated with t-butyl hydroperoxide. Two "nonreducible" oxidized forms of CA III were also detected on nitrocellulose blots. These forms increased with the amount of stress and were the only modified forms of CA III in buthionine sulfoxide-treated hepatocytes containing 10-fold less glutathione than control hepatocytes. These experiments support the concept that S-thiolation protects CA III from irreversible oxidation during oxidative stress. Partly and fully S-thiolated forms of CA III were easily detected in both male and female hepatocytes by the immunoblotting method, although female cells contained 15-fold less CA III than did male liver. S-thiolated forms of CA III were also detected in rat skeletal muscle and heart showing the utility of this method for determining the effect of oxidative stress on specific S-thiolatable protein in several tissues in vivo.

In order to examine potentially important S-thiolated proteins, 35 S-labeled hepatocytes were exposed to oxidative stress. A similar group of S-thiolated proteins including carbonic anhydrase III was observed in cells treated with t -butyl hydroperoxide, menadione, or stimulated neutrophils. The radioactive thiols bound to hepatocyte proteins were identified by HPLC and more than 85% was glutathione. In menadione-treated hepatocytes, proteins were gradually S-thiolated over 30 min and 25% of the cellular glutathione pool became protein-bound. In t -butyl hydroperoxide-treated cells, S-thiolation was more transient and 11% of the glutathione was protein-bound. Neutrophil-treated hepatocytes had nearly the same amount of protein S-thiolation (8% after 25 min). Two major proteins that were S-thiolated in untreated hepatocytes did not increase during any form of oxidative stress. In neutrophil-treated hepatocytes protein S-thiolation was not accompanied by either formation of glutathione disulfide or a measureable change in the total amount of glutathione. In both t -butyl hydroperoxide- and menadione-treated cells there was extensive formation of glutathione disulfide and in menadione-treated cells a significant increase in the total hepatocyte glutathione pool was observed. This result suggests that protein S-thiolation may occur by mechanisms that do not result from thiol/disulfide exchange between glutathione disulfide and protein sulfhydryls. It is suggested that a thiyl radical intermediate is important in neutrophil-mediated protein S-thiolation.

Protein S-thiolation, a reversible modification of protein sulfhydryls resulting in formation of mixed-disulfides, was studied in human neutrophils stimulated with phorbol diester to produce superoxide anion. Rapid S-thiolation of several proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glutathione was identified as the primary protein-bound thio by HPLC chromatography, contributing considerably more than 85% of the total. Minor amounts of homocysteine and/or cysteine were also detected as protein-bound thiols. During the first 30 min after stimulation, 10% of the cellular glutathione became protein bound (2 nmol/mg of protein). There was no increase in glutathione disulfide suggesting that S-thiolation of the proteins did not occur by thiol/disulfide exchange. Approximately 10 mol% of one heavily modified band (29 kDa) was S-thiolated after 30 min. A second major band of 42 kDa was identified as actin. It contained 1/10th of the total protein-bound glutathione and approximately 5 mol% was S-thiolated after 30 min. These experiments identify a subset of S-thiolated neutrophil proteins, including actin, whose modification is related to the phorbol diester stimulation of superoxide anion production in human neutrophils. Ten percent of the total glutathione pool became protein-bound without an appreciable change in non-bound concentration of glutathione or glutathione disulfide. These results suggest that glutathione was synthesized during initial phases of the respiratory burst, compensating for the amount of glutathione that became protein-bound. Since there was no significant increase in glutathione disulfide, it was probably not important in the observed protein S-thiolation.