Glutathione reductase of calf trabecular meshwork.
Hydrogen peroxide has been found in both calf and human aqueous humor at a level of 25 microM. It is likely, therefore, that trabecular meshwork possesses mechanisms for detoxifying H2O2, both to protect itself and other more distal structures of the outflow pathway from oxidative damage. We have recently demonstrated an active glutathione peroxidase in calf trabecular meshwork. In this study, we have characterized the complementary enzyme, glutathione reductase. The activity was present at a level of 0.120 units/min/g wet of tissue (0.005 units/min/mg soluble protein). The enzyme quickly lost activity in crude extracts but could be stabilized by heating at 60 degrees C for 30 min. Denatured protein was removed by centrifuging at 43,000 X g. Heating at 80 degrees C for 10 min destroyed all enzyme activity. Addition of 1 mM GSSG protected the enzyme completely from heat denaturation; NADP+ and GSH offered some protection but NADPH provided none. The supernatant from the 60 degrees C heat treatment was further purified by affinity chromatography on 2',5'-ADP-agarose. Overall purification was 200-fold with a yield of 80%. The pH optimum of the purified enzyme was 7.0. The KmS for NADPH and GSSG were 19 microM and 78 microM, respectively. The heat inactivation properties of the purified enzyme were identical to those in the crude extract. An enzyme activity stain on disc gel electrophoresis showed that the enzyme exists in only one form.
D000818:Animals; D000867:Anterior Chamber / Q000201:enzymology / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D004589:Electrophoresis, Disc; D005980:Glutathione Reductase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D014129:Trabecular Meshwork / Q000201:enzymology* / Q000378:metabolism
D006861:Hydrogen Peroxide; D005980:Glutathione Reductase