A simple technique to determine glutathione (GSH) levels and synthesis in ocular tissues as GSH-bimane adduct: application to normal and galactosemic guinea-pigs.
A fluorimetric technique previously described for other tissues has been applied to determine levels of glutathione and its synthetic rates in ocular tissues of Hartley guinea-pigs. Monochlorobimane forms a stable, fluorescent adduct with glutathione in a reaction catalyzed by glutathione-S-transferase. The fluorescent signal recorded over time is directly proportional to the synthetic rate of glutathione. Lens, cornea and retina were homogenized and cytosolic fractions dialyzed overnight to deplete endogeneous glutathione. Glutathione synthetic rates were determined from a mixture of glutathione precursors and co-factors, viz. cysteine+dithiothreitol, glutamate+glycine, ATP and Mg++ in the presence of monochlorobimane. The mixture was supplemented with glutathione-S-transferase to catalyze the formation of the fluorescent adduct. Glutathione synthetic rates were determined in the absence and presence of buthionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase. The difference in fluorescence change over time in the presence and absence of buthionine sulfoximine was used to estimate glutathione synthesis. Basal levels of glutathione in pre-dialyzed cytosolic fractions of the lens, cornea, and retina were 21.8 +/- 2.2, 36.5 +/- 4.1 and 38.6 +/- 2.8 nmol mg-1 protein, respectively. The maximal glutathione synthetic rates in these tissues were 0.52 +/- 0.04, 2.25 +/- 0.67 and 3.35 +/- 0.65 nmol min-1 mg-1 protein, respectively. When gamma-glutamyl cysteine is used as a precursor instead of cysteine, the glutathione synthetase activities from lenses and retinas were 0.19 +/- 0.08 and 1.54 +/- 0.76 nmol-1 min mg-1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Kannan
R
R
Division of Gastrointestinal and Liver Diseases, Children's Hospital of Los Angeles, University of Southern California School of Medicine.
Tang
D
D
Mackic
J B
JB
Zlokovic
B V
BV
Fernandez-Checa
J C
JC
D000818:Animals; D003315:Cornea / Q000378:metabolism; D005136:Eye Proteins / Q000096:biosynthesis*; D005470:Fluorometry / Q000379:methods; D005693:Galactosemias / Q000378:metabolism*; D005978:Glutathione / Q000096:biosynthesis*; D006168:Guinea Pigs; D007908:Lens, Crystalline / Q000378:metabolism; D011720:Pyrazoles; D012160:Retina / Q000378:metabolism; D013997:Time Factors
D005136:Eye Proteins; D011720:Pyrazoles; C059597:monochlorobimane; D005978:Glutathione