Oxidative-stress induced protein glutathione mixed-disulfide formation in the ocular lens

Autor: James A. Willis, Thomas Schleich
Rok vydání: 1996
Předmět:
2ME
2-mercaptoethanol
TSP
sodium 3-(trimethylsily)propionate-2
2
3
3-d4
Magnetic Resonance Spectroscopy
DTE
dithioerythritol
GSH
reduced glutathione
Glutathione reductase
Ocular lens
Protein aggregation
medicine.disease_cause
13C-GSH
([3-13C]cysteinyl)glutathione
Adduct
chemistry.chemical_compound
Organ Culture Techniques
tert-Butylhydroperoxide
Crystallin
Stress
Physiological
NMR
13C
Lens
Crystalline
medicine
Animals
Cysteine
Disulfides
IEF
isoelectric focussing
RF
radiofrequency
Molecular Biology
Glutathione Peroxidase
Chemistry
GSSG
oxidized glutathione
PSSG
protein-glutathione adduct
Glutathione
Cell Biology
Carbon-13 NMR
Mixed-disulfide formation
Crystallins
t-BHP
Peroxides
PSSP
protein-protein disulfide linked species
Oxidative Stress
Biochemistry
mS
milliSiemen ( = 1 mOhm−1)
FID
free induction decay
ppm
parts per million
Rabbits
Oxidation-Reduction
Oxidative stress
Zdroj: Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1313(1):20-28
ISSN: 0167-4889
DOI: 10.1016/0167-4889(96)00049-3
Popis: The biochemistry of protein-glutathione mixed disulfide formation in the ocular lens was examined by 13 C-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- 13 C]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- 13 C]cysteinyl)-glutathione and a loss of 13 C NMR resonance intensity, a reflection of protein-glutathione mixed disulfide formation. The rate of ([3- 13 C]cysteinyl)-glutathione loss depended on the concentration of tert -butylhydroperoxide; 13 C-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- 13 C]cysteinyl)-glutathione levels and 13 C 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 γ-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.
Databáze: OpenAIRE
Externí odkaz: