GLUTATHIONE REDOX STATUS ALTERS THE DIMERIC-MONOMERIC EQUILIBRIUM OF HUMAN GLUTATHIONE REDUCTASE. + 1228

The major intracellular redox buffer in most eukaryotic cells is glutathione (GSH) and glutathione disulfide (GSSG). The NADPH-dependent conversion of GSSG to GSH is catalyzed by the flavoenzyme glutathione reductase (GR). When active, GR functions as a homodimer; the GR equilibrium greatly favors GSH formation at cellular NADPH/NADP* ratios. Steady-state levels of GSH and GSSG in cells are not constant, but may vary considerably in response to nutritional status, hormones, drugs and oxidative stress. Normal ratios of GSH/GSSG range from 100-400; however, under oxidative stress ratios may fall to as low as 1-10. Alterations in glutathione redox status due to a change in either the concentration of GSH, GSSG, or both, if coupled to the redox status of thiols and disulfides in specific proteins, could provide a unique regulatory mechanism affecting the biological activity of receptors, transporters, and transcription factors, as well as enzymes including GR. Studies have suggested that the inactivation/reactivation of GR involves a monomer-dimer equilibrium. Possibly, intramolecular disulfide bond formation between cysteine residues in the catalytic center of GR provide increased dimer stability. To determine whether alterations in the glutathione redox status might regulate the monomerdimer state of GR, equilibrium experiments were performed using purified recombinant human GR. Enzyme was incubated with varying ratios of GSH/GSSG at 37°C for 1 hr in phosphate buffer [0.1 M KPO₄ + 5 mM EDTA, pH =7.4]. Samples were quenched with 200 mM N-ethylmaleimide, electrophoresed on a 8% SDS-polyacrylamide gel, transferred to immobolin PVDF membrane and analyzed by Western blot. Results show that normal physiological GSH/GSSG ratios favor the monometric form, while ratios below normal result in the enzyme being predominantly in its homodimeric state. This study suggests the possibility of an autoregulatory mechanism for the inactivation/reactivation of GR via reversible disulfide bond formation and supports the importance of reversible thiol/disulfide redox equilibria in biological processes.