Glutathione-dependent enzymes in intact rod outer segments

We have compared the specific activities of glutathione-dependent enzymes in rod outer segments (ROS) and in whole retina of rabbits and rats, using three different assays. In the first, glutathione peroxidase (GSH-Px) activity was measured as the combined activities of the Se-dependent and Se-independent forms using cumene hydroxide as a substrate. In the second, Se-dependent GSH-Px alone was measured using hydrogen peroxide (H2O2) as a substrate. In the third, the combined activities of several enzymes, collectively known as GSH-S-transferase, were measured. The latter includes the activity of the Se-independent GSH-Px. GSH-Px activity (Se-dependent and Se-independent combined) in ROS of rat and rabbit were found to be 47.7 +/- 8.2 and 72.9 +/- 11.9 nmol of GSH oxidized min-1 mg-1 soluble protein, respectively. From whole retina, values were 67.3 +/- 6.0 and 128.8 +/- 12.3, respectively. Se-dependent GSH-Px specific activities from the above tissues were 43.5 +/- 2.9 (rat ROS). 70.6 +/- 11.3 (rabbit ROS), 30.6 +/- 9.6 (rat whole retina), and 113.2 +/- 12.2 (rabbit whole retina). GSH-S-transferase activity was negligible in rabbit ROS, whereas, in rat ROS, it was 40.4 +/- 8.0, expressed as nmol of S-2,4-dinitrophenylglutathione produced min-1 mg-1 soluble protein. In contrast, the GSH-S-transferase specific activity in whole rabbit retinas was about eight times that found in the rat retina (101.5 +/- 12.3 for rat retina and 885.3 +/- 60.0 for rabbit retina). These results demonstrate that ROS contain glutathione enzymes which are important in protecting membranes from oxidative stresses by reducing hydrogen peroxide and lipid hydroperoxides at the site of their formation.