IMMUNOCHEMICAL CHARACTERIZATION AND TISSUE DISTRIBUTION OF GLUTAREDOXIN (THIOLTRANSFERASE) FROM CALF

Glutaredoxin catalyzes glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH and glutathione reductas e and has an active site disulfide/dithiol with the sequence -Cys-Pro- Tyr-Cys-. Calf thymus glutaredoxin (thiol-transferase), which contains two additional structural half-cystine residues, was purified to homo geneity, using a modification of the previously described isolation pr ocedure. This method involved a pI-shift of glutaredoxin, obtained aft er oxidation of the fully reduced form with hydroxyethyl-disulfide, fo llowed by CM-Sepharose chromatography. On both SDS- and IEF-gels the p rotein migrated as one band (M(r) 12000). The pure protein was used to affinity-purify rabbit antiglutaredoxin antibodies obtained by immuni zation with the oxidized form of glutaredoxin. Using these antibodies the distribution of glutaredoxin was mapped in calf organs and tissues by Western blots and by immunohistochemistry. Glutaredoxin was demons trated in all organs investigated. Western blots showed the presence o f weak additional high molecular weight bands of unknown identity in c ertain organs. The immunohistochemical analyses revealed that glutared oxin is highly expressed in a wide variety of cell types, both epithel ial and mesenchymal. The distribution and occurrence in the calf organ s was similar to that previously described for thioredoxin in the rat. There were some exceptions: e.g., follicular cells in the ovary did n ot contain immunohistochemically demonstrable glutaredoxin but express ed thioredoxin. Particularly striking were observations of strong glut aredoxin immunoreactivity in oocytes in the ovary and the pattern of g lutaredoxin in epithelial tissue of the skin and tongue reflecting dif ferential expression during cell differentiation. The distribution dem onstrated that glutaredoxin serves functions apart from the originally described role as hydrogen donor for ribonucleotide reductase which o nly occurs in replicating cells. Such functions should relate particul arly to glutathione-catalyzed protein disulfide oxidoreductions and ce llular signalling by redox regulating mechanisms.