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.

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The present study was designed to determine whether there are beneficial effects of intake of Omega-3E (containing 70% pure omega-3 and 2% natural vitamin E) in cardiac dysfunction of diabetic rats. We also examined whether there are gender-related differences in the responses to the intake of Omega-3E on the heart dysfunction. Experiments were performed by using Langendorff-perfused hearts from normal, diabetic (with 50 mg/kg streptozotocin), and Omega-3E (50 mg/kg body weight/day) treated diabetic 3-month-old Wistar rats. Omega-3E treatment of the diabetics caused small, but significant decrease (13% and 14% female versus male) in the blood glucose level. Omega-3E treatment of the diabetic female rats did not prevent diabetes-induced decrease in left ventricular developed pressure (LVDP) and increase in left ventricular end-diastolic pressure (LVEDP) with respect to the control female rats. On the other hand, the treatment of diabetic male rats caused significant recovery in depressed LVDP. Furthermore, such treatment of diabetic female and male rats caused significant recovery in depressed rates of changes of developed pressure. This effect was more significant in males. Besides, Omega-3E caused significant further lengthening in the diabetes-induced increased time to the peak of the developed pressure in females, while it normalized the lengthening in the relaxation of the developed pressure in diabetic males. In addition, Omega-3E treatment caused significant restorations in the diabetes-induced altered activities of antioxidant enzymes without any significant gender discrepancy. Present data show that there are gender related differences in diabetic heart dysfunction and the response to antioxidant treatment.

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Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn2+ (up to 2 mM) and activated above this concentration. Ca2+ inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.

Glutathione reductase was purified 34806-fold with a final yield of 85 % from the bovine kidney cortex. Some molecular and kinetic properties of purified enzyme are investigated. Product inhibition studies showed that the enzyme obeys 'branched" mechanism: Km(NADPH) 18 +/- 3 microM and Km(GSSG) 65 +/- 5 microM were determined.

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