Purification and kinetic properties of glutathione reductase from bovine liver

Abstract

Glutathione reductase (GR, NADPH: oxidized glutathione oxidoreductase, EC 1.6.4.2) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as reducing cofactor. The aim of the present work was to purify and characterize GR from bovine liver. GR was purified using 2', 5' ADP-Sepharose 4B and DEAE-Sepharose Fast Flow columns. The enzyme has been purified 5456-fold and with a yield of 38.4%. The molecular and catalytic properties of bovine liver GR have been studied. Optimum temperature and pH was found to be 50 degrees C and 7, respectively. The activation energy of the reaction catalyzed by the enzyme was 9.065 kcal/mole. The molecular weight of the enzyme was found to be 55 kDa by SDS-PAGE. Kinetic characterization of bovine liver GR was also investigated, Km(NADPH) 0.063 +/- 0.008 mM and Km(GSSG) 0.154 +/- 0.015 mM were determined. It is accepted that parallel lines observed in these double reciprocal plots obeys Ping Pong mechanism and we have showed this in our steady state study. According to our results of statistical analysis, the Ping Pong mechanism is a suitable model since the loss function is less than the other mechanisms. However, competitive inhibition by a product could be accepted in sequential mechanisms but not in a Ping Pong mechanism. In this study, kinetic data are consistent with a branching reaction mechanism previously proposed for GR from other sources by other studies.

Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics

“…Thus, the affinity of NADPH to the enzyme is greater than GSSG. Similar values were reported for GR from human erythrocyte and bovine liver 56,57 . NADP + has a higher rate of inhibition on the enzyme activities than GSH.…”

In vitroeffects of rosmarinic acid on glutathione reductase and glucose 6-phosphate dehydrogenase

“…We have purified GR from bovine liver and kidney cortex and then analyzed the effect of RA on the purified enzyme (Ulusu & Tandogan, 2007;Tandogan & Ulusu, 2010). Baker's yeast GR is obtained from Sigma and the specific activity of the enzyme is 1.25 U/mg.…”

“…Bovine kidney cortex and liver GR is purified by chromatography consisting of two steps after ultracentrifugation by Beckman L7-80 ultracentrifuge and heat denaturation: 2′,5′-ADP Sepharose 4B affinity and DEAE Sepharose Fast Flow anion exchange chromatography (Ulusu & Tandogan, 2007;Tandogan & Ulusu, 2010). Bovine liver GR was purified 28,028-fold with a final yield of 43%.…”

Effects of some antibiotics on human erythrocyte glutathione reductase: anin vitrostudy

“…Enzymatic defense is provided by many enzyme systems such as glutathione reductase, glutathione peroxidase, glutathione Stransferase, superoxide dismutase, catalase, aldoketoreductase and DNA repair enzymes [1,23]. Particularly, GR is essential for the maintenance of cellular glutathione in its reduced form, which is highly nucleophilic for many reactive electrophiles [24].…”