Oxidative-stress induced protein glutathione mixed-disulfide formation in the ocular lens.
The biochemistry of protein-glutathione mixed disulfide formation in the ocular lens was examined by 13C-NMR spectroscopic measurements of glutathione oxidative metabolism in intact rabbit lenses maintained in organ culture. Lenticular amino acid uptake and glutathione biosynthetic mechanisms were employed to facilitate the incorporation of L-[3-13C]cysteine from the incubation medium into the cysteinyl residue of glutathione. Subsequent exposure to increasing levels of oxidative stress induced by tert-butylhydroperoxide resulted in decreased levels of ([3-13C]cysteinyl)-glutathione and a loss of 13C NMR resonance intensity, a reflection of protein-glutathione mixed disulfide formation. The rate of ([3-13C]cysteinyl)-glutathione loss depended on the concentration of tert-butylhydroperoxide; 13C-labeled oxidized glutathione was observed only at the highest concentration (2 mM) of oxidant tested. Removal of the oxidative stress led to a partial recovery of ([3-13C]cysteinyl)-glutathione levels and 13C resonance intensity. Recovery was significantly enhanced by the addition of 2-mercaptoethanol. The mechanism of protein-glutathione adduct formation was further characterized by the in vitro monitoring of the reaction of oxidized glutathione with bovine lens gamma-II crystallin protein using proton NMR spectroscopy. These experiments provided insight into the role of the cellular glutathione redox-couple, [GSH]/[GSSG], in maintaining reduced protein thiol groups, and suggested that protein-glutathione adduct formation may function as a mechanism for modulating the glutathione redox buffer under conditions of oxidative stress in ocular tissue. In addition, the results demonstrate the feasibility of direct chemical reduction of protein-glutathione disulfide bonds in vivo which may reflect a mechanism for the inhibition of disulfide-linked light scattering protein aggregate formation.
Willis
J A
JA
Department of Chemistry and Biochemistry, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA.
Schleich
T
T
D000818:Animals; D003459:Crystallins / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D005978:Glutathione / Q000737:chemistry*; D005979:Glutathione Peroxidase / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009924:Organ Culture Techniques; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010545:Peroxides / Q000737:chemistry; D011817:Rabbits; D013312:Stress, Physiological / Q000378:metabolism; D020122:tert-Butylhydroperoxide
D003459:Crystallins; D004220:Disulfides; D010545:Peroxides; D020122:tert-Butylhydroperoxide; D005979:Glutathione Peroxidase; D005978:Glutathione; D003545:Cysteine