Assessment of glutathione reductase inhibition

Abstract

Glutathione reductase is a crucial enzyme for maintaining of intracellular glutathione levels. This enzyme catalyzes the NADPH-dependent reduction of glutathione disulfide to the reduced form (GSH). The aim of our study was to estimate a possible inhibitory effect on glutathione reductase activity in the presence of two substances that cause glutathione depletion - ethacrynic acid and diethyl maleate. We also tested glutathione as a possible inhibitor. The experiments were performed with yeast glutathione reductase. GR activity was determined using spectrophotometric method based on measurement of absorbance decline (8 = 340 nm) due to oxidation of NADPH. We found that dose dependent inhibition of glutathione reductase occurred in the presence of ethacrynic acid; the enzyme activity was inhibited by 19 % and 29 % in the presence of 500 :M and 1000 :M ethacrynic acid, respectively. We also found that although diethyl maleate is able to induce deep glutathione depletion in the cell, it does not affect the GR activity. On the other hand, we found dose dependent inhibitory effect through reduced glutathione — the presence of 10 mM GSH caused a decrease in enzyme activity by 60 %. We conclude that our finding of inhibitory effect in the presence of glutathione is of great importance since the GSH levels are very high in the cells. It follows that glutathione reductase possesses a substantial reserve in enzyme activity that could be used in oxidative stress conditions.